Biosensors in Agriculture

Concepts and Strategies in Plant Sciences
Series Editor: Chittaranjan Kole
Ramesh Namdeo Pudake
Utkarsh Jain
Chittaranjan Kole Editors
Biosensors in
Agriculture:
Recent Trends
and Future
Perspectives
Series Editor
Chittaranjan Kole, Raja Ramanna Fellow, Government of India, ICAR-National
Institute for Plant Biotechnology, Pusa, Delhi, India
This book series highlights the spectacular advances in the concepts, techniques and
tools in various areas of plant science. Individual volumes may cover topics like
genome editing, phenotyping, molecular pharming, bioremediation, miRNA, fast-
track breeding, crop evolution, IPR and farmers’ rights, to name just a few. The
books will demonstrate how advanced strategies in plant science can be utilized to
develop and improve agriculture, ecology and the environment. The series will be
of interest to students, scientists and professionals working in the fields of plant
genetics, genomics, breeding, biotechnology, and in the related disciplines of plant
production, improvement and protection.
Interested in editing a volume? Please contact Prof. Chittaranjan Kole, Series
Editor, at [email protected]
More information about this series at http://www.springer.com/series/16076

Ramesh Namdeo Pudake · Utkarsh Jain ·
Chittaranjan Kole
Editors
Biosensors in Agriculture:
Recent Trends and Future
Perspectives
Editors
Ramesh Namdeo Pudake Utkarsh Jain
Amity Institute of Nanotechnology Amity Institute of Nanotechnology
Amity University Uttar Pradesh Amity University Uttar Pradesh
Noida, UP, India Noida, UP, India
Chittaranjan Kole
Department of Atomic Energy
ICAR - National Institute for Plant
Biotechnology
New Delhi, Delhi, India
ISSN 2662-3188 ISSN 2662-3196 (electronic)

ISBN 978-3-030-66164-9 ISBN 978-3-030-66165-6 (eBook)
https://doi.org/10.1007/978-3-030-66165-6
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Preface
Agriculture and its allied industries are the backbone of global economies and need
to be continuously developed to achieve higher production and sustainability. The
industrial development and growing human population are among the major factors
that are having adverse effects on various aspects of food, meat, and milk produc-
tion. Augmentation of production but with minimum environmental impacts from
agricultural inputs requires innovation and utilization of advanced technologies. The
reduction in the quantity of agricultural inputs required for optimum production
will benefit farmers by lowering production costs. Along with this, we also need to
develop technologies which can reduce the wastage of agricultural products until it
reaches the consumer. For this conventional tools will be of limited use, and tech-
nologies like precision farming with help of various sensors and electronics support
have the potential to become an alternative strategy to achieve the above-mentioned
goals.
A biosensor can be defined as an analytical tool that combines a biological sensing
element and a physicochemical transducer; which can detect the analyte by trans-
forming bio-molecular interactions into identifiable signal. The role of nanomaterial-
based biosensors in agriculture is now a key research area of interest among scientists.
They can used to monitor the problems associated with food security, water and soil
quality, integrated pest management for crops and livestock. Currently the focus of
research is targeted for search of novel nanomaterials for improving the effectiveness
of biosensors by higher selectivity and sensitivity. Nanomaterials of different types,
shapes and sizes are being used presently in the development of different sensors for
precision farming, food processing industries and water quality monitoring use. The
research in this area has the potential to increase and maintain the food safely for the
consumption of the increasing global population. Despite potential advantages, the
application and commercialization of nanosensor technology in agricultural sector is
still comparably trivial. Like other technologies it also has some issues like interfer-
ence from various biomolecules, portability, stability of sensing device, etc. that still
need to be addressed. So, there is a need of continued research that will revolutionize
the commercial utilization. In the near future the upcoming technologies such as
microfluidics, robotics and artificial intelligence will further add additional features
to biosensors.
v
vi Preface
The aim of this book is addressing the issue of knowledge gap by bringing together
the acheievements from recent research and development pertaining to biosensors
and their uses in agriculture. We strongly believe that this book will be useful to
various stakeholders including students, researchers and manufacturers interested on
the use of nanosensors in field of agriculture and allied sciences. This book comprises
22 chapters, authored by 68 eminent scientists, covering the diverse applications of
nanosensors in a wide range of agricultural tasks.
We are indebted to the authors of the book chapters for their contributions. We
thank Mrs. Zuzana Bernhart, and Mrs. Banu Dhayalan from Springer Nature for their
immense help and patience in making this journey very comfortable. We also like to
thank our respective organizations for the support and encouragement provided for
academic endeavors. The encouragement and inspiration received from Dr. Ashok
K. Chauhan (Founder President, Ritnand Balved Education Foundation, New Delhi)
need special mention. We also like to thank our beloved families, friends, and
colleagues for their support during all the activities done for the duration of penning
of this book.
Noida, India Dr. Ramesh Namdeo Pudake

Noida, India Dr. Utkarsh Jain
New Delhi, India Dr. Chittaranjan Kole
Contents
Part I Biosensors in Crop Science

1 Recent Trends, Prospects, and Challenges of Nanobiosensors
in Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Ravindra Pratap Singh
2 Nanostructured Platforms Integrated to Biosensors: Recent
Applications in Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Sofía V. Piguillem Palacios, Nicolás Hoffmann,
Matías Regiart, Olga Rubilar, Gonzalo Tortella, Julio Raba,
and Martín A. Fernández-Baldo
3 Advances in Nanotechnology for Bio-Sensing in Agriculture
and Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Theivasanthi Thirugnanasambandan
4 Nanomaterial-Based Gas Sensors for Agriculture Sector . . . . . . . . . . 51
Robin Kumar, Monica Jaiswal, Neelam Kushwaha,
Shivansh Bansal, Neha Mazumder, and Jagjiwan Mittal
5 Volatile Organic Compounds (VOCs) Sensors for Stress
Management in Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Vartika Rohatgi, Navakanth Vijay Challagulla,
and Ramesh Namdeo Pudake
6 Current Trends of Plasmonic Nanosensors Use in Agriculture . . . . . 97
Tahira Qureshi, Deniz Türkmen, and Adil Denizli
7 Relevance of Biosensor in Climate Smart Organic Agriculture
and Their Role in Environmental Sustainability: What Has
Been Done and What We Need to Do? . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Kingsley Eghonghon Ukhurebor and Charles Oluwaseun Adetunji
8 New Trends in Biosensor Development for Pesticide Detection . . . . . 137
Narlawar Sagar Shrikrishna, Subhasis Mahari, Naina Abbineni,
S. A. Eremin, and Sonu Gandhi
vii
viii Contents
9 Application of Biosensor for the Identification of Various

Pathogens and Pests Mitigating Against the Agricultural
Production: Recent Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Charles Oluwaseun Adetunji, Wilson Nwankwo,
Kingsley Eghonghon Ukhurebor, Akinola Samson Olayinka,
and Ayodeji Samuel Makinde
10 Gold Nanoparticles-Based Point-of-Care Colorimetric
Diagnostic for Plant Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Ravi Mani Tripathi and Prashant Sharma
11 Advancements in Biosensors for Fungal Pathogen Detection
in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Utkarsh Jain, Ramesh Namdeo Pudake, Nidhi Chauhan,
and Sakshi Pareek
12 Journey of Agricultural Sensors—From Conventional
to Ultra-Modern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Ashish Mathur, Shikha Wadhwa, Shalini Nagabooshanam,
and Souradeep Roy
Part II Biosensors in Food Science

13 Advances in Biosensors Based on Electrospun
Micro/Nanomaterials for Food Quality Control
and Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Aylin Altan and Meryem Yılmaz
14 Current Trends of Electrochemical Sensing for Mycotoxins . . . . . . . 275
Ruchika Chauhan, Rashi Bhardwaj, Sheetal K. Bharadwaj,
Ajit Kaushik, Rajshekhar Karpoormath, and Tinku Basu
15 Biosensors for Fruit Quality Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . 325
Vinita Hooda, Nidhi Chauhan, and Shringika Soni
16 Lateral Flow Assays for Food Authentication . . . . . . . . . . . . . . . . . . . . 343
Despina P. Kalogianni
17 Nanobiosensors in Agriculture and Foods: A Scientometric
Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Ozcan Konur
Part III Biosensors in Animal and Fishery Sciences

18 Biosensors: Modern Tools for Disease Diagnosis and Animal
Health Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Anuj Tewari, Beenu Jain, Basanti Brar, Gaya Prasad,
and Minakshi Prasad
Contents ix
19 Nano-Biosensing Devices Detecting Biomarkers

of Communicable and Non-communicable Diseases of Animals . . . . 415
Utkarsh Jain, Saurabh Shakya, and Kirti Saxena
20 Recent Advances in Biosensor Development for Poultry
Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
Nidhi Chauhan, Ramesh Namdeo Pudake, Utkarsh Jain,
and Sapna Balayan
21 Smart Aquaculture: Integration of Sensors, Biosensors,
and Artificial Intelligence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
Dolly Sharma and Ranjit Kumar
22 Biosensor as a Potential Tool for On-Site Detection of Insect
Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
Mudasir Gani, Taskeena Hassan, Pawan Saini,
Khalid Hussain Bhat, Rakesh Kumar Gupta, and Kamlesh Bali
Contributors
Naina Abbineni DBT-National Institute of Animal Biotechnology, Hyderabad,

India
Charles Oluwaseun Adetunji Applied Microbiology, Biotechnology and
Nanotechnology Laboratory, Department of Microbiology, Edo University Iyamho,
Auchi, Edo State, Nigeria
Aylin Altan Department of Food Engineering, Mersin University, Mersin, Turkey
Sapna Balayan Amity Institute of Nanotechnology, Amity University Uttar
Pradesh, Noida, UP, India
Kamlesh Bali Division of Entomology, Sher-e-Kashmir University of Agricultural
Sciences and Technology, Chatha, J&K, India
Shivansh Bansal Amity Institute of Nanotechnology, Amity University Uttar
Tinku Basu Amity Centre for Nanomedicine, Amity University Uttar Pradesh,
Noida, UP, India
Sheetal K. Bharadwaj University of Amsterdam, Amsterdam, The Netherlands
Rashi Bhardwaj Amity Centre for Nanomedicine, Amity University Uttar Pradesh,
Noida, UP, India
Khalid Hussain Bhat Division of Basic Sciences and Humanities, Faculty of
Agriculture, Sher-e-Kashmir University of Agricultural Sciences and Technology,
Kashmir, J&K, India
Basanti Brar Department of Animal Biotechnology, College of Veterinary
Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar,
Haryana, India
Navakanth Vijay Challagulla Amity Institute of Nanotechnology, Amity Univer-
sity Uttar Pradesh, Noida, UP, India
xi
xii Contributors
Nidhi Chauhan Amity Institute of Nanotechnology, Amity University Uttar

Ruchika Chauhan Amity Centre for Nanomedicine, Amity University Uttar
Pradesh, Noida, UP, India;
Department of Pharmaceutical Chemistry, College of Health Sciences, University of
KwaZulu-Natal, Westville Campus, Durban, South Africa
Adil Denizli Department of Chemistry, Biochemistry Division, Hacettepe Univer-
sity, Ankara, Turkey
S. A. Eremin Faculty of Chemistry, M. V. Lomonosov, Moscow State University,
Moscow, Russia
Martín A. Fernández-Baldo Instituto de Química de San Luis (INQUISAL),
Consejo Nacional de Investigaciones Científicas Y Técnicas (CONICET), Univer-
sidad Nacional de San Luis (UNSL), San Luis, Argentina
Sonu Gandhi DBT-National Institute of Animal Biotechnology, Hyderabad, India
Mudasir Gani Division of Entomology, Faculty of Agriculture, Sher-e-Kashmir
University of Agricultural Sciences and Technology, Kashmir, J&K, India
Rakesh Kumar Gupta Division of Entomology, Sher-e-Kashmir University of
Agricultural Sciences and Technology, Chatha, J&K, India
Taskeena Hassan Department of Zoology, Aligarh Muslim University, Aligarh,
Uttar Pradesh, India
Nicolás Hoffmann Departamento de Ingeniería Química, Centro de Excelencia En
Investigación Biotecnológica, Aplicada al Medio Ambiente (CIBAMA), Universidad
de La Frontera, Temuco, Chile
Vinita Hooda Department of Botany, Maharshi Dayanand University, Rohtak,
Haryana, India
Beenu Jain Department of Animal Husbandry, Lucknow, Uttar Pradesh, India
Utkarsh Jain Amity Institute of Nanotechnology, Amity University Uttar Pradesh,
Noida, UP, India
Monica Jaiswal Amity Institute of Nanotechnology, Amity University Uttar
Despina P. Kalogianni Department of Chemistry, University of Patras, Rio, Patras,
Greece
Rajshekhar Karpoormath Department of Pharmaceutical Chemistry, College of
Health Sciences, University of KwaZulu-Natal, Durban, South Africa
Ajit Kaushik Florida Polytechnique University, Lakeland, FL, USA
Ozcan Konur Formerly Ankara Yildirim Beyazit University, Ankara, Turkey
Contributors xiii
Ranjit Kumar Department of Chemistry, School of Engineering, University of

Petroleum and Energy Studies (UPES), Bidholi, Dehradun, Uttarakhand, India
Robin Kumar Amity Institute of Nanotechnology, Amity University Uttar Pradesh,
Noida, UP, India
Neelam Kushwaha Amity Institute of Nanotechnology, Amity University Uttar
Subhasis Mahari DBT-National Institute of Animal Biotechnology, Hyderabad,
India
Ayodeji Samuel Makinde Climatic/Environmental/Telecommunication Physics
Unit, Department of Physics, Edo University Iyamho, Auchi, Edo State, Nigeria
Ashish Mathur Department of Physics, School of Engineering, University of
Petroleum and Energy Studies (UPES), Bidholi Campus, Dehradun, India
Neha Mazumder Amity Institute of Nanotechnology, Amity University Uttar
Jagjiwan Mittal Amity Institute of Nanotechnology, Amity University Uttar
Shalini Nagabooshanam Amity Institute of Nanotechnology, Amity University
Uttar Pradesh, Noida, UP, India
Wilson Nwankwo Climatic/Environmental/Telecommunication Physics Unit,
Department of Physics, Edo University Iyamho, Auchi, Edo State, Nigeria
Akinola Samson Olayinka Department of Physics, Edo University Iyamho, Auchi,
Edo State, Nigeria
Sakshi Pareek Amity Institute of Nanotechnology, Amity University Uttar
Pradesh, Noida, India
Sofía V. Piguillem Palacios Instituto de Química de San Luis (INQUISAL),
Consejo Nacional de Investigaciones Científicas Y Técnicas (CONICET), Univer-
sidad Nacional de San Luis (UNSL), San Luis, Argentina
Gaya Prasad International Institute of Veterinary Education and Research, Rohtak,
Haryana, India
Minakshi Prasad Department of Animal Biotechnology, College of Veterinary
Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar,
Haryana, India
Ramesh Namdeo Pudake Amity Institute of Nanotechnology, Amity University
Uttar Pradesh, Noida, UP, India
Tahira Qureshi Department of Chemistry, Biochemistry Division, Hacettepe
University, Ankara, Turkey
xiv Contributors
Julio Raba Instituto de Química de San Luis (INQUISAL), Consejo Nacional de

Investigaciones Científicas Y Técnicas (CONICET), Universidad Nacional de San
Luis (UNSL), San Luis, Argentina
Matías Regiart Instituto de Química de San Luis (INQUISAL), Consejo Nacional
de Investigaciones Científicas Y Técnicas (CONICET), Universidad Nacional de San
Luis (UNSL), San Luis, Argentina
Vartika Rohatgi Amity Institute of Nanotechnology, Amity University Uttar
Pradesh, Noida, Uttar Pradesh, India
Souradeep Roy Amity Institute of Nanotechnology, Amity University Uttar
Olga Rubilar Departamento de Ingeniería Química, Centro de Excelencia En
Pawan Saini Central Sericultural Research and Training Institute, Central Silk
Board, Ministry of Textiles, Govt. of India, Pampore, J&K, India
Kirti Saxena Amity Institute of Nanotechnology, Amity University Uttar Pradesh,
Noida, UP, India
Saurabh Shakya Viral Recombination Section, HIV Dynamics and Replication
Programme, National Cancer Institute, Frederick, MD, USA
Dolly Sharma Computer Science and Engineering, Shiv Nadar University, Noida,
Uttar Pradesh, India
Prashant Sharma Department of Pediatrics, Division of Pediatric Hematology,
Oncology & Bone Marrow Transplant, University of Wisconsin–Madison, Madison,
WI, USA
Narlawar Sagar Shrikrishna DBT-National Institute of Animal Biotechnology,
Hyderabad, India
Ravindra Pratap Singh Department of Biotechnology, Indira Gandhi National
Tribal University (Central University), Amarkantak, Anuppur (M.P.), India
Shringika Soni Amity Institute of Nanotechnology, Amity University Uttar
Anuj Tewari Department of Veterinary Microbiology, College of Veterinary and
Animal Sciences, Govind Ballabh Pant University of Agriculture and Technology,
Pantnagar, Uttarakhand, India
Theivasanthi Thirugnanasambandan International Research Centre,
Kalasalingam Academy of Research and Education (Deemed University),
Krishnankoil, Tamilnadu, India
Contributors xv
Gonzalo Tortella Departamento de Ingeniería Química, Centro de Excelencia En

Ravi Mani Tripathi Amity Institute of Nanotechnology, Amity University Uttar
Deniz Türkmen Department of Chemistry, Biochemistry Division, Hacettepe
University, Ankara, Turkey
Kingsley Eghonghon Ukhurebor Climatic/Environmental/Telecommunication
Unit, Department of Physics, Edo University Iyamho, Auchi, Edo State, Nigeria;
Informatics and CyberPhysical Systems Laboratory, Department of Computer
Science, Edo University Iyamho, Auchi, Edo State, Nigeria
Shikha Wadhwa Department of Chemistry, School of Engineering, University of
Petroleum and Energy Studies (UPES), Dehradun, India
Meryem Yılmaz Department of Food Engineering, Mersin University, Mersin,
Turkey
Part I
Biosensors in Crop Science
Chapter 1
Recent Trends, Prospects, and Challenges
of Nanobiosensors in Agriculture
Ravindra Pratap Singh
Abstract Nanotechnology (NT) is an interdisciplinary scientific approach at the

nanoscale which revolutionized nanobiosensors’ usage in agriculture. Agriculture is
a diversified field that plays a self-sustaining role to promote the economic develop-
ment of any country toward mankind. Agriculture requires nanotechnical interven-
tions toward food processing, food safety, food quality or quality assurance, food
security, disaster risk management, diagnosis, and prevention at the local and global
levels. Nanobiosensors have immense potential to solve severe problems pertaining
to agriculture. Nanobiotechnology (NBT) is a branch of NT at nano dimensions to
create tools and techniques, and functional structures which open new applications
toward agriculture and health. Agriculture is the major sector of national economy
in developing countries. A recent advance in biosensor technology toward agricul-
ture challenges is very challenging with the intervention of nanotechnology which
would emerge to enhance crop productivity and sustainability and also improve
real-time quality and food safety of livestock, and natural agricultural resources. In
this book chapter, the commercial and futuristic nanobiosensors are highlighted to
defend the challenges in the major sector of agriculture and bring out the implica-
tions of nanobiosensors design and development toward the improvement of crop
productivity. Thus, nanobiosensors are playing a crucial role in sensing insecticides,
herbicides, fertilizers, and pathogens.
Keywords Nanotechnology, Nanobiotechnology · Nanobiosensors · Agriculture
1.1 Introduction
Agriculture is proving to be the main source of raw materials to food industries

and also is the backbone of developing countries for their economic growth and
development. Nowadays, this sector is facing huge problems such as urbanization,
R. P. Singh (B)
Department of Biotechnology, Indira Gandhi National Tribal University (Central University),
Amarkantak, Anuppur (M.P.) Pin-484887, India
e-mail: [email protected]
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 3

R. N. Pudake et al. (eds.), Biosensors in Agriculture: Recent Trends and Future
Perspectives, Concepts and Strategies in Plant Sciences,
https://doi.org/10.1007/978-3-030-66165-6_1
4 R. P. Singh
harnessing bad use of cultivating soil, land, water, and runoff, and misutilization
and management of pesticides and fertilizers. Natural resources are critical factors
for economic growth and development phenomena pertaining to poverty and hunger
of large populations locally and globally. Agriculture is the science of soil, crops,
and livestock and an economic boon of any country for the source of livelihood
to improve social welfare in both urban and rural areas. However, accessibility of
resources such as quality of soil and water is declining for agriculture and created a
huge economical loss. This continued stress on agricultural resources is increasing
due to over population and high demand for food, constant increase of utilization
of pesticides, insecticides, herbicides, and heavy metals in soil. All these challenges
or issues can be solved by a new technology known as nanobiosensors to change
agriculture-based food systems to not only improve the quality of the agricultural
products but also boosts the national economy toward sustainable agriculture with
quality products and its less cost. A biosensor is an analytical device that combines
a biological entity with physico-chemical transducer to measure electrical signal
when interacting with the desired analyte of interest. Fig. 1.1 shows a schematic
representation of a biosensor.
An advanced version of biosensor known as nanobiosensor is very specific and
selective for the detection of target analytes of interest such as pesticides, insec-
ticides, herbicides, and various microbial pathogens (Singh 2017). Biosensor tech-
nology is playing a major role to utilize the physical-chemical properties of nanoscale
materials which provide ultra sensitivity and performance through new signal trans-
duction technologies. The advanced version of biosensor utilizing nanomaterial for
enhancing sensitivity and performance of the system is known as nanobiosensor.
Nanobiosensors are highly demanding technology at the early stage of development
Fig. 1.1 Schematic representation of biosensor

1 Recent Trends, Prospects, and Challenges … 5
for easy, rapid, ultrasensitive, and cost-effective systems which are very vital not
only to agriculture, food, and drink, food process industries, food safety, food secu-
rity but also in health care, environmental monitoring, and even for genome analysis.
The nanomaterials such as metals and metal oxides nanoparticles (NPs) (Au, Ag,
Cu, Co, ZnO, TiO2 , Fe3 O4 , MgO, etc.), magnetic NPs, CNT, graphene, dendrimers,
polymeric, and QDs nanoparticles were used in nanobiosensors development for the
detection of analytes of interests.
A nanobiosensor consists of 3 components: biological probe elements, trans-
ducer, and detector. The biological probe elements are several such as antibodies,
receptors, enzymes, DNA/RNA, Peptide Nucleic Acid (PNA), Locked nucleic acid
(LNA), cells, microorganisms, organelles, etc. The transducer is a physical interface
to recognize signal events into a digital signal. The detector detects the signals from
the transducer, then passes it to a microprocessor for amplification and analysis in
terms of data, and finally displayed in the output device. Data recording and display
unit consist of an amplifier, signal processor, and display that are responsible for
data transferred and results displayed (Wang 2005; Singh and Choi 2010; Rai et al.
2012). The characteristic features of nanobiosensor are miniaturized, cheap, biocom-
patible, non-toxic, portable, specific, and stable, less reaction time, zero electrical
noise, accurate, precise, and reproducible at optimum pH and temperature (Singh
et al. 2014).
Nanotechnology has the potential to make an impact in the field of health, the
environment, and agriculture (Singh 2017). The nanomaterials-based biosensors
have offered high sensitivity, specificity, and detection time being user-friendly and
detect an analyte of interest. In agriculture, nanobiosensors detect direct and indi-
rect foodborne pathogenic microorganisms, pesticides, veterinary drugs, toxicants,
contaminants, and heavy metals in foods. In addition to that nanobiosensors can also
monitor soil quality, food quality, crop stress, and antibiotic resistance (Singh 2016).
Keeping the above in view, nanobiosensors are helpful for healthy agriculture to
enhance crops, soil, and livestock productivity which feed our populations free from
any implications.
1.2 Use of Nanobiosensor in Agriculture
Nanomaterial-based biosensors are most commonly known as nanobiosensors with

enhanced detection specificity, selectivity, sensitivity, and possess potential applica-
tions in clinical, environmental, and agriculture. However, in this book chapter, we
are focusing on trends, prospects, and challenges of nanobiosensor in agriculture.
Recent progress in the development of nanobiosensors was to improve crop health by
the detection of plant pathogens, pesticides, herbicides, and soil testing. Nanoparti-
cles are utilized in the diagnostic tool to detect plant pathogens (Singh 2019). Fig. 1.2
shows a broad spectrum of potential applications of nanobiosensors in a variety of
scientific domains, and some applications in agriculture are described below.
6 R. P. Singh
Fig. 1.2 Broad spectrum of potential applications of nanobiosensors
1.2.1 Nanosensors in pathogens detection
In developing countries consumption of foods contaminated with pathogenic food-

borne bacteria is the main cause of concern posing public illness health locally and
globally. In the food industry, the first line of defense against foodborne illness is
laboratory-based methods to detect food pathogens with high selectivity and sensi-
tivity before outbreaks arise. It may be possible by using nanomaterials to design
nanobiosensor to detect food contaminants, pathogens, banned dyes, adulterants,
antibiotics, hormones, and allergens.
Foodborne microorganism pathogens or bacteria are a serious threat to the
animal’s production and health to causes food poisoning, gastroenteritis, etc., due
to Salmonella sps, Clostriudium spp, Vibrio cholera, Escherichia coli, and Campy-
lobacter spp. Salmonellosis in humans is a global problem due to foodborne bacteria
eaten contaminated meat and poultry (Nowak et al. 2007). There are no analyt-
ical devices to ascertain food samples. To overcome this problem, nanobiosensor
is very promising for rapid detection in the food chain during storage, distribu-
tion, and food processing. Viswanathan et al. (2006) reported the detection of
cholera toxin by an electrochemical immunosensor based on liposomal poly (3,4-
ethylenedioxythiophene)-coated carbon nanotubes. Kim et al. (2013) reported the
detection of Salmonella spp. in food by nanobiosensor immobilized anti-Salmonella
polyclonal antibodies on streptavidin-biotin onto the quantum dot surface. Afonso
et al. (2013) reported detection of Salmonella by nanobiosensor-based AuNPs in
skimmed milk. Wang and Alocijia (2015) reported the detection of Escherichia
coli O157:H7 by nanobiosensor based on functionalized Fe3 O4 NPs and AuNPs
conjugated monoclonal antibodies.
Lopez et al (2009) reported fluorescent oligo capture probe onto nano-chips for
detection of single nucleotide of plant pathogenic bacteria and viruses. Yao et al.
(2009) reported nano-gold-based immunosensor to detect the pathogen of Karnal
bunt disease in wheat. Brock et al. (2011) reported nanosensors for the detection of
plant pathogens, viruses, and soil nutrients.
1.2.2 Mycotoxins detection
Nanobiosensor promises rapid detection for the trace quantity of mycotoxins. The
mycotoxins are a threat to human health which influence the crops, feed, food prod-
ucts, especially in rainy seasons, and in turn leads to great economic losses glob-
ally. However, Masikini et al. (2015) reported the trace detection of fumonisins by
nanobiosensor fabricated by using PANI-CNT doped palladium telluride quantum
dots. Nanobiosensor is useful for maintaining food quality and safety for example
to detect mycotoxins derived from food. There are several nanostructures materials
such as QDs, nanoparticles (metal/metal oxide, polymer), nanowires, nanotubes or
nanorods, and graphene, which have the ability to be functionalized and immobi-
lized by various biomolecules such as antibodies, enzymes, DNA/RNA aptamers,
receptors to detect food toxicants, adulterants, and pathogen.
Mycotoxins are fungi/molds derived toxic chemicals, i.e. natural contaminants
found in foodstuffs and animal feed products which pose threat to human health and
most commonly known as hepatotoxic, nephrotoxic, carcinogenic, mutagenic, for
example, aflatoxins, ochratoxin, and zearalenone. Due to these causes of concern,
there is an urgent need to detect or monitor by a sophisticated device known as
nanobiosensor, an analytic instrument in contaminated foods and animal feeds. Parker
and Tothill (2009) reported the detection of aflatoxin M1 contaminates in the milk by
an electrochemical immunosensor. Xu et al. (2013) reported detection of aflatoxin
B1 in peanut by an optical biosensor constructed with gold nanorod conjugated
antibody. Eldin et al. (2014) reported detection of Aflatoxin B1 in peanuts, chillies,
maize, and rice by immunosensor fabricated AuNPs conjugation with anti-aflatoxin
B1 polyclonal antibody. Bonel et al. (2010) reported the detection of ochratoxin
A by an electrochemical immunosensor constructed AuNPs conjugated polyclonal
antibodies. Furthermore, Turan and Şahin (2016) reported the detection of ochratoxin
A by MIP-Fe3 O4 NPs.
1.2.3 Pesticides/ Insecticides/Herbicides detection
Pesticides are the chemicals to protect plants and animals in limited extent such as
herbicide to control weeds, a fungicide to control fungus and insecticide to control
insects but their application of heavy dose cause toxicity to human/animals. The tradi-
tional methods to detect pesticides are not good enough because of many disadvan-
tages, to overcome this problem nanobiosensor is used to detect pesticides (Vimala
et al. 2016; Zhao et al. 2015; McGrath et al. 2012).
Zhang et al. (2008) reported the detection of pesticide content in food by acetyl-
cholinesterase nanobiosensor. In addition to that Norouzi et al. (2010) reported
detection of monocrotophos and organophosphate pesticide by an electrochemical
8 R. P. Singh
biosensor. Zheng et al. (2011) reported the detection of paraoxon and parathion pesti-
cides by optical nanobiosensor using acetylcholinesterase and CdTe QDs. Further-
more, Guan et al. (2012) reported detection of dichlorvos pesticides by acetyl-
cholinesterase biosensors. Song et al. (2015) reported the detection of carbamate
pesticide by an electrochemical biosensor fabricated AuNPs/(3-mercaptopropyl)-
trimethoxysilane/Au electrode sensing surface. Apart from this, Songa et al. (2009a)
reported the detection of glyphosate, glufosinate herbicide in corn by biosensor using
horseradish peroxidase (HRP). Further, Songa et al. (2009b) reported the detec-
tion of glyphosate herbicide and its metabolite by nanobiosensor. In another study,
Songa et al. (2009c) reported detection of glyphosate herbicide by biosensor immo-
bilized HRP on sulfonated polymer matrix. Haddaoui and Raouafi (2015) reported
the detection of chlortoluron herbicide by nanobiosensor (inhibition of tyrosinase).
Mousavi and Rezaei (2011) reported the detection of environmental pollutants and
food contaminants by nanosmart dust. Sharon et al. (2010) reported that the intelli-
gent system monitors and minimizes the use of pesticides and antibiotics with the
help of a microelectronic circuit.
1.2.4 Veterinary drug and residues
Nanobiosensors are able to not only detect food contaminants and chemicals but also
biological contaminants like veterinary drug residues in food. They are being used
for the assessment and management of food safety and quality. Veterinary drugs are
mostly antibiotics frequently used in farming animals to treat animal diseases and also
enhance animal growth (McEwen and Fedorka-Cray 2002). However, antibiotic use
in animals in some extent cause serious risks not only in animal but also in human as
antibiotic resistance against microbial pathogens leads into outbreak among animals
(Levy and Marshall 2004; Courvalin 2008). Furthermore, antibiotic-treated meat
causes risk of resistance of flora in humans (Landers et al. 2012). Veterinary drugs
are frequently present in the black market, one can procure very easily to use in poultry
industry without consultation with a veterinary doctor to violate the guidelines of the
veterinary drug recommendation (Idowu et al. 2010). In this context, nanobiosensors
are able to detect the veterinary drug residues in food (Wu et al. 2014). In addition
to that same group, i.e. Wu et al. (2015) reported to detect chloramphenicol by
using aptamer-based fluorescence biosensor. In the past, Mungroo and Neethirajan
(2014) had reviewed the nano-based biosensors to detect veterinary drug residues
in food such as meat, milk, and poultry products. Hou et al. (2013) reported the
detection of oxytetracycline by aptamer-based cantilever array sensors. Furthermore,
Song et al. (2012) reported the detection of ampicillin in milk by using an aptamer
biosensor.
1.3 Nanobiosensors for food safety and security
Recent challenges like food safety and security and climate changes are very impor-
tant and need advanced scientific interventions such as nanotechnology to resolve
human health, growth, and development. In this regard, nutritious food is very impor-
tant in the diet with economical, safe, and sufficient. However, our diet is contami-
nated, i.e. not safe and secure so it requires food safety and food security measures.
To achieve this goal advance emerging technology, i.e. nanotechnology is used as a
convergent technology utilizing nanobiosensor toward food availability, food acces-
sibility, and food utilization (Sastry et al. 2013) to detect highly toxic natural food
contaminants. However, traditional methods (TLC, HPLC, LC-MS, etc.) are available
but very costly instrumentation, need manpower, and complicated (Agriopoulou et al,
2020). In the recent past, Parisi et al. (2015) has reported the easiness and affordability
of nanobiosensors more or less related to the crop production agriculture domain.
The nutrients not only provide nourishment but also give energy to our body to
do routine activities with good health. The food at room temperature for a long time
undergoes rapid bacterial growth and also chemical changes and becomes unhealthy
food. Food contamination is possible because of storage or chemical changes or
several bacteria and fungi that causes foodborne diseases, i.e. food poisoning globally.
In addition to that, food preservation using chemicals to increase the time span of
food is also a cause of illness. In this context, it is mandatory to develop a system
to identify the fresh or good quality of food. A smart system like biosensor and
electrical sensors can detect the freshness of food like dairy items, meat, and fruits. In
addition to that pH sensor, moisture sensor, and gas sensor are also able to detect food
freshness. Checking fruits and food by color and smell are possible but expensive,
time consuming, and less efficient. Furthermore, nanobiosensors are able to detect
vitamins, antibiotics, food spoilage, and microbial contaminants which are frequently
possible.
Biological systems have used nanoscale materials such as nucleic acid, enzymes,
antibodies, proteins, carbohydrates, fats, etc., whereas engineered nanomaterial use
has raised potential concerns pertaining to latent toxicity risks for agriculture, envi-
ronment, and biomedical or human health. The use of nanobiosensors has insufficient
research pertaining to toxicity. Therefore, nanomaterials used in nanobiosensors must
be characterized and tested by investigators, academics, and governmental ethical
committees globally (Vamvakaki and Chaniotakis 2007).
In addition to that, nanobiosensors are monitoring soil quality, water quality, and
protection against pests and microbial diseases to maintain the health of agricultural
crops. The challenges related to sustainability, food safety, and security and climate
changes as well are the main potential topic for the improvement of agriculture.
Inbaraj and Chen (2016) reported the detection of bacterial pathogen in meat by
using nanobiosensor. Detection of melamine, malathion, and foodborne contami-
nants in food such as milk, vegetable, and fruit are mandatory to use metal-based
nanobiosensor. However, nanostructured material such as metallic nanoparticles,
conjugated polymers, carbon nanotubes, and nanofibers can be utilized in variety of
10 R. P. Singh
nanobiosensors to detect food products during early stage of manufacture, storage,

and transportation if improper. In addition to that moisture, oxygen, carbon dioxide,
amines, and microorganisms in food industry are important aspects to ensure safe
food for the sake of human health. Toxins, pathogens, and chemicals are the main
cause of concern of human/animal diseases. For example, melamine is a nitrogen-
rich chemical used in food products such as in pet foods, infant milk powder foods,
which is added in foods to increase protein content and achieve high price or benefit
from the products. However, the high content of melamine in food products causes
very detrimental cause of concern even in pet and infant death. Several methods have
been established but most of the methods require manpower for the preconcentra-
tion of sample, time-taking, and using sophisticated instruments. To overcome this
problem, there are increasing demands to develop a fast, simple, convenient, and
sensitive method to detect melamine based on sensors as an alternative method.
Nanobiosensors have wide applications in the food industry for example QDs-
based nanobiosensor is for the detection of heavy metal and organophosphate pesti-
cides, infectious and foodborne pathogens. Nanobiosensors, e.g. electronic nose and
electronic tongue are able to detect odor and taste of food products.
Adulteration of the food products such as edible oil, wine, coffee, tea, chocolate,
cheese, and milk are well known. In this context food contaminants detection for
food safety and security is mandatory to use nanobiosensors. Chen and Park (2016)
reported identification and detection of microbial contaminants present in food and
suggested the prevention and control of the outbreak.
The electrospun nanofibers have been used in various fields such as wound
dressing, drug delivery, and filtration. Electrospun nanofibers’ use in agriculture is in a
very infancy phase. Few most common examples are reported including pheromone-
loaded nanofibers, encapsulation of biocontrol agents, protective clothes, food pack-
aging materials, nanobiosensors for pesticide detection, and filtration of beverage
products (Noruzi 2016). Nehra and Singh (2015) reported the use of nanostructures
in biosensing for the desired detection of analytes of interest. Nanotechnology (NT)
utilization in modern agriculture is known as agrifood nanotechnology which can
boost agricultural production using nanoformulated agrochemicals pesticides and
fertilizers (Sekhon 2014).
1.4 Conclusion and future prospects
The agrifood nanotechnology gives benefits to our farmers through food production
and food industry via food processing, preservation, and packaging. Nanomaterials-
based nanosensors/nanobiosensors are being developed to detect plant pathogens and
testing soil quality to improve plant health. It is well established that nanobiosensors
can be used for supporting sustainable agriculture by enhancing crop productivity.
Also, improved detection of food pathogens, pesticides, antibiotics, and food contam-
inants have to be done by using nanobiosensors toward food safety otherwise they
will pose a threat to human health. Nanobiosensors in agriculture not only detect
biological analyte present in agricultural food but also produce quality food to meet
the local and global demands.
Acknowledgment Dr. Ravindra Pratap Singh thanks IGNTU, Amarkantak, M.P. India for
providing facilities to prepare this book chapter.
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Chapter 2
Nanostructured Platforms Integrated
to Biosensors: Recent Applications
in Agriculture
Sofía V. Piguillem Palacios, Nicolás Hoffmann, Matías Regiart,

Olga Rubilar, Gonzalo Tortella, Julio Raba, and Martín A. Fernández-Baldo
Abstract Recently, various nanomaterials, such as nanostructured platforms, are

being used for the development of biosensors. This chapter provides a brief explana-
tion of the classification of biosensors according to their detection methods, and then
it talks about the materials that are commonly used as nanostructured platforms in the
manufacturing of biosensors. The field of nanotechnology continues to be a rapidly
growing and developing field, and researchers are very actively inventing and discov-
ering new nanomaterials. In this chapter, the most used sensors in the agricultural
sector are cataloged and then presented in detail. The nanomaterials selected for this
purpose ranges from metallic nanoparticles, magnetic nanoparticles, different types
of nanomaterials derived from carbon, silicon, nanoMOF, and quantum dots. Finally,
some recent works related to the subject are detailed, which have combined fast and
efficient detection methods with the incorporation of nanomaterials that improve the
characteristics and improve the biosensors in search of a more sensitive, selective,
low cost, and reliable determination.
Keywords Nanotechnology · Nanomaterials · Platforms · Biosensors · Agriculture
S. V. Piguillem Palacios · M. Regiart · J. Raba · M. A. Fernández-Baldo (B)

Instituto de Química de San Luis (INQUISAL), Consejo Nacional de Investigaciones Científicas
Y Técnicas (CONICET), Universidad Nacional de San Luis (UNSL), D5700BWS San Luis,
Argentina
N. Hoffmann · O. Rubilar · G. Tortella
Departamento de Ingeniería Química, Centro de Excelencia En Investigación Biotecnológica,
Aplicada al Medio Ambiente (CIBAMA), Universidad de La Frontera, Temuco, Chile

16 S. V. Piguillem Palacios et al.
2.1 Introduction
In recent years, several nanomaterials as nanostructured platforms were being used

for the biosensor’s development. A biosensor is a device composed of two funda-
mental elements: a biological receptor (i.e., some cellular macromolecules that can
bind to a compound) prepared to specifically detect a substance taking advantage
of the excellent specificity of biomolecular interactions, and a transducer or better
called a sensor, capable of sensing the biological recognition reaction and “translates
it” into a quantifiable signal (Fig. 2.1). The biosensors can be classified according to
the detection method in optical, electrochemical, and piezoelectric. These biosensors
have the advantage of rapid detection; the device can also be portable and, above all,
sensitive; however, they are still in the laboratory research stage (Hou et al. 2018;
Giorgini et al. 2018; Pohanka 2018).
A device should be called an immunosensor if the biological element is an antigen
or antibody fragment (Piguillem et al. 2018; Bravo et al. 2017). They can be classi-
fied into biosensors: biocatalytic biosensors and those based on bio-affinity (Regiart
et al. 2017a). The former use enzymes as recognition elements, and combine the
immanent specificity of enzymes with the excellent benefits of biosensors. This type
of biosensor currently attracts researchers to carry out various studies due to its enor-
mous potential for future bioassays due to its high sensitivity and specificity. These
biosensors are based on enzymes that are biocatalytic compounds, and biocatalytic
Fig. 2.1 Schematic representation of the biosensor. This device comprises three main components:
a sensitive biological recognition element, a transducer, and a signal processor
2 Nanostructured Platforms Integrated … 17
reactions are generally transduced by optical and electrochemical detection methods

(Zhu et al. 2019).
On the other hand, bio-affinity-based biosensors rely on specific binding proteins,
nucleic acids, cells, or antibodies for biomolecular recognition. This type of biosensor
is specifically developed to measure the biomolecular reaction (Campuzano et al.
2017).
Many of these biosensors are nanostructured, that is, they are modified with
nanomaterials in order to obtain nanoplatforms used for biomolecules (enzymes,
antigens, or antibodies) modification according to the final application (Fernández-
Baldo et al. 2009). A nanomaterial is one that has at least one of its dimensions size
between 0–100 nm. This makes its nanoscale properties very different from those
exhibited by the same macroscale material (López-Sanz et al. 2019), and there are
many techniques to determine its shape and size. There are two main approaches
used for nanomaterial synthesis: bottom-up and top-down (Fig. 2.2). Bottom-up
is an approach in which material elements are used at the atomic level and are
then followed by self-assembly reactions between them that result in the synthesis
of nanostructures (Henarava et al. 2019), while top-down is an approach where a
macroscopic structure is manipulated externally until the desired nanomaterials are
Fig. 2.2 Scheme of different methods that can be used for the nanomaterial’s synthesis
obtained (Chepurov et al. 2018). That is why the first approach is known as the
builder’s approach and the second the sculptor’s approach.
In general, the controlled synthesis of nanomaterials is performed using chem-
ical methods such as hydrothermal sol-gel (Chukova et al. 2019), co-precipitation
(Regiart et al. 2015), chemical reduction (Piguillem et al. 2018), since with these
methods the shape, size, and composition of the particles can be appropriately
adjusted to suit a specific catalytic activity. Another nanomaterial synthesis widely
used in recent years consists in the use of electrochemical methods, where we can
find methods such as nanoparticles electrodeposition (Regiart et al. 2013), dealloying
(Zhu et al. 2013), and dynamic hydrogen bubble template (DHBT) (Plowman et al.
2015) that have gained so much attention. DHBT-assisted growth involves hydrogen
synthesis, and the metal ions reduction/deposition on the surface of the working
electrode simultaneously. The rate/size of hydrogen bubbles evolution provides a
dynamic template for metal electrodeposition, creating three-dimensional nanoar-
chitectures porous materials. In these electrochemical methods, parameters like
electrodeposition time and potential strongly influence the grain size, porosity, and
density of the films (Sukeri and Bertotti 2017). Another way to produce nanomate-
rials is to combine methods, one of them uses ultrasound with electrochemistry, and
this combination is called sonoelectrochemistry (Islam et al. 2019).
The present chapter focuses on different nanostructured platforms used in the
biosensor’s fabrication, their recent applications in agriculture, conclusions, and
future perspectives.
2.2 Nanomaterials Used in Biosensors Methodologies
In recent years several interdisciplinary research groups are involved in the nanoma-
terial’s development for biosensor applications. When synthesizing a nanomaterial,
concerns are nucleation and crystal growth, composition without too many impuri-
ties, controlled size, interfaces, stability, and various assembly strategies to achieve
low-cost, large-scale production. Various nanomaterials have been used for agricul-
tural biosensors applications due to their exceptional characteristics, such as high
(bio) compatibility, easy to operate, inertia, high surface/area ratio, and eminent
thermal, chemical, and mechanical resistance (Maduraiveeran and Jin 2017).
The nanomaterials have two important properties to be outstanding. The first one
is the surface area, that allows the nanomaterials to interact with the analyte in the
sample strongly compared with bulk materials (Malhotra and Ali 2018). The material
at the macroscale level has atoms in its interior that are more coordinated since there
are many more links between them than the atoms that are on the surface of the
material, in fact in the corners and edges, the atoms have less coordination; therefore
they are less stable than interior atoms. If we now think at the nanoscale level, the
surface of a nanomaterial becomes quite reactive since there are very few atoms that
make it up, and therefore, it shows extraordinary catalytic and absorbance activity
(Malhotra and Ali 2018).
The other property has to do with the quantum well, that is, two-dimensional
confinement of particles. When the width or thickness of this well is similar to the
de Broglie wavelength, the particles that are generally electrons, those particles that
have energy, can now only take discrete energy values (Malhotra and Ali 2018). So,
this quantum confinement effect produces an effect that when the particle size is too
small can be compared with the wavelength of the electron, that is why if a particle
has nanoscale dimensions, the limiting dimensions make the levels of energy discrete
and this increases the band energy. In other words, if we have a nanoparticle whose
size is similar to the Bohr excitation radius, the excitonic transition energy, the blue
change in absorption, and the energy of the luminescence band gap increases, all as
a result of quantum confinement (Malhotra and Ali 2018).
There are several ways to classify nanomaterials, but we will focus on their classi-
fication by size and dimensions, and according to this, there are four types of nanoma-
terials. Zero-dimensional Nanomaterial (0D) is when the three dimensions are at the
nanoscale, such as nanoparticles (NP) of silver, gold, copper, or quantum dots. These
NPs are generally spherical with a diameter of 1–50 nm, although cube and polygon
shapes have also been found at 0D. One-dimensional (1D) nanomaterial is with one of
its dimensions with a range between 1–100 nm, and the others can be on a macroscale.
Typical examples of these are nanowires, nanofibers, nanorods, and nanotubes. Two-
dimensional (2D) nanomaterials are when two of its dimensions are in the nanoscale
and one at the macroscale. Such is the case of nanolayers or nanowalls. A clear
example of this is graphene. In three-dimensional (3D) nanomaterials, there are no
dimensions at the nanoscale, and all dimensions are at the macroscale. This category
involves materials whose internal structure is nanostructured.
2.2.1 Metal Nanoparticles
Metallic NPs are often used for the development of electrochemical sensors and
biosensors platforms due to interesting properties, either because synthesizing them
does not present drawbacks with suitable methods, they are easy to functionalize
since they can be covalently linked to functional groups, they are capable catalyze
electrochemical reactions and greatly facilitate the electron transfer process. The
oldest and most used noble metals in terms of nanomaterials can be found in gold,
platinum, and silver (Jamkhande et al. 2019).
It can be affirmed that AuNPs have been used successfully as an effective elec-
trocatalyst in innumerable electrochemical reactions, mainly because they are very
stable and manage to recover completely in redox chemical processes. On the other
hand, when using AuNP-modified electrodes, a higher signal/control (S/C), good
catalytic activity, and diffusion of electroactive species are observed (Regiart et al.
2013). Due to the physical property of gold as a conductive material, its nanoparti-
cles have surface plasmon resonance (SPR), so when they are irradiated with light of
the same plasma frequency, an oscillation is generated. This phenomenon is respon-

sible because, depending on the size of the nanoparticles, they present a colloidal
suspension of different colors (Saha et al. 2012).
2.2.2 Magnetic Nanoparticles
There are several forms of magnetic NP depending on the metals involved in its
synthesis, such as Fe3 O4 (magnetite), c-Fe2 O3 (maghemite), NiFe2 O4 (nickel ferrite),
and CoFe2 O4 (cobalt ferrite). As they have paramagnetic properties, they can be
manipulated by using an external magnetic field, making them extremely easy to use
or incorporate into biosensing platforms, and that is why they have many applications,
especially in medicine, as therapeutic drug carriers as well as labeled cell separators
(Knezevic et al. 2019). Moreover, the magnetic property was used in conjunction with
noble metal NPs like AuNPs, as a result giving core@shell NPs, thus the benefits of
both materials can be utilized (Azharuddin et al. 2019).
2.2.3 Carbon Nanomaterials
If it refers to carbon, many nanomaterials can be included incorporating this element

in different shapes and nanometric dimensions. There are nanotubes or nanofibers
of carbon, graphene, fullerene, and carbon materials with micro/meso/macropores
(Regiart et al. 2016). These materials have great application potential in agricul-
ture (Mittal et al. 2019). The most used for the development and incorporation of
biosensors are carbon nanotubes, porous carbon, and graphene.
2.2.3.1 Carbon Nanotubes
Carbon nanotubes (CNT) are tubular or cylindrical structures whose internal diameter
must not exceed the nanoscale. Some roll with a single layer called single-walled
or monolayer nanotubes (SWCNT), or those that roll concentrically called multiple-
walled nanotubes (MWCNT). Before its use in biosensors, an acid pretreatment
is performed to remove the tube caps, leaving it open and with functional oxygen
groups, which makes the nanotube very versatile for later use (Yang et al. 2015).
2.2.3.2 Graphene
Graphene is a 2D structure of sp2 carbon atoms that forms a hexagonal network. The
bonds between hexagons in the same plane are powerful (higher than the resistance
in steel), while the bonds between the upper or lower layers are weaker, hence
the allotropic property of carbon to be able to exfoliate and obtain monolayers. Its
applications in electrochemical sensors are derived more specifically from its use
in oxidized form (GO) or in its reduced oxide form (rGO); this is because it has
excellent electrical conductivity, and its surface is easy to modify (Wang et al. 2016).
2.2.3.3 Porous Carbon
Porous carbon has a high surface area since its internal structure that contains micro
pores (less than 2 nm) and meso (between 2 and 50 nm) added to the short path
for the transfer of mass and electrons has particular advantages to incorporate them
in electrochemical biosensors. The energy is stored in the double layer, which is
why the porous carbon can be used as a structural material and as electrochemical
energy storage. Without a doubt, its applications are innumerable as mostly due to
its adsorption properties, which are used for water and air purification filters, or
as a support for catalysts. At present, nanocasting templates are used which create
precisely ordered mesoporous carbon that may include the incorporation of other
atoms in your invention depending on the purpose of their use (Xu et al. 2017).
2.2.4 Silica Nanomaterials
Intelligently synthesized silica nanomaterials (SNMs) achieve a highly useful meso-

porous structure for many areas of science. For example in medicine, they are used
to administer medicines because they can contain, transport and release biologi-
cally active substances; or in biosensors to detect pathogens, aflatoxins in food,
biomarkers of different types of cancer (Eivazzadeh-Keihan et al. 2020; Wang et al.
2015). In addition to all its advantages that make nanostructured silica a very inter-
esting nanomaterial, it also exhibits low cytotoxicity, which is biocompatible and
inexpensive (Regiart et al. 2017b). One of the methods for SNM synthesis involves
high-temperature heat treatment, responsible for the formation of siloxane rings.
However, a low-temperature heat treatment reduces its crystallinity, which makes
it behave as a material with more exceptional biocompatibility, an objective that is
very interesting in order to immobilize biomolecule’s surface (Regiart et al. 2018).
2.2.5 NanoMOFs
Organometallic frameworks (MOFs) are organic–inorganic hybrid porous coordi-

nation polymers, the matrix of which is made up of metal ions bound by organic
molecules. Its internal structure exhibits a large cavity that depends on the ions
involved and the synthesis methods and solvents used for them. NanoMOFs are a
novel class of nanomaterials whose reduction in the size of said pores or cavities
improves the already existing benefits of traditional MOFs since they are currently
being applied in fields such as biomedicine and agriculture (Chansi et al. 2019; Zhang
et al. 2019).
2.2.6 Quantum Dots
Importantly, it is not an easy task to discern when a nanomaterial can be classified as

a quantum dot (QD) since it depends not only on the size but also on the material that
makes it up. QD are those nanomaterials that must be greater than 2 nm and less than
20 nm, and are produced with a limiting effect. As explained above, this depends
on the size of the Bohr radius (size that varies according to the article); therefore, it
depends on both criteria, the item, and the size to consider whether or not it is a true
QD. Due to this energy confinement, these fluorescent nanomaterials have unique
optical properties, such as CdS QD emits from blue to ultraviolet or CdSe QD that
cover the entire visible spectrum (Marcelo et al. 2020).
2.3 Recent Applications of Nanostructured Biosensors

in Agriculture
Agriculture is a vast monetary source for populations around the world that do all
kinds of jobs related to cultivation and processing. And according to the report
by The Food and Agriculture Organization of the United Nations (FAO), there are
around 2 billion farmers. Nevertheless, also, a more modern definition of agriculture
involves tasks that go from working on the land to reaching consumption in homes.
Although in agricultural jargon there are two main stages: pre-harvest, where condi-
tions are significant for cultivation, such as climatic conditions, minerals, nutrients,
fungal contaminants such as aflatoxins, and post-harvest (Kundu et al. 2019). It is
imperative to detect microbial contamination in the crop since it has a direct impact
on the quality of the final product and can cause massive losses in many batches.
Fortunately, there are currently various techniques to detect contamination, which
generates stress in plants. Those methods are techniques to assess and analyze agri-
culture problems in the laboratory. In order to avoid this, biosensors have contributed
to achieving new in situ methods that promise different tools for the early diagnosis
of microbial contamination. In this context, the evidenced works improve the diag-
nostic method for some specific microorganisms that can produce plant diseases.
Regiart et al. (2017a) have developed a microfluidic electrochemical immunosensor
for the detection of Xanthomonas arboricola (XA). In this work, a monoclonal anti-
XA antibody was covalently immobilized in the central channel of the chip in silica
called amino-SBA-15. As is well known, standard methodologies for characterizing
mesoporous materials correspond to N2 adsorption–desorption isotherm to assess
its porous capacity, scanning electron microscopy (SEM) to assess its morphology,
infrared spectroscopy (FTIR) to assess junctions and composition of silica groups.
Once the nanomaterial was characterized, and the immobilized antibody was quan-
tified XA using a direct sandwich immunoassay using an enzyme conjugate anti-XA
labeled with the enzyme alkaline phosphatase (AP) and finally when the oxidized
enzyme product is obtained the current measured in this process is directly propor-
tional to the amount of XA in the samples from walnut. Another successful appli-
cation of inmunosensors on Botrytis cinerea (BA) detection was carried out by
Fernández-Baldo et al. (2009). BA is a phytopathogenic fungus (a fungus that uses
plants as a host and its spores use soil or plant residues) that produces gray mold,
which primarily attacks agricultural hosts. In this case, the researchers developed a
competitive immunoassay on a previously modified spinning disk and immobilized
BA antigens for plant tissue or BA monoclonal antibodies to react there. The quan-
tification was done through a second anti-IgG-labeled horseradish peroxidase (HRP)
enzyme, and when the reaction product is obtained, a direct proportion was obtained
between the measured current and the amount of antigens present in the samples.
Biosensor’s development has proved to be useful as a fast detection tool to prevent
crop deficiencies or microbial contamination, but the biosensor’s application field
does not end in those aspects and is also used as pesticide detection. Organophosphate
insecticides (IOPs) highly toxic chemical compounds used to combat pests and weeds
(Dong et al. 2016). However, this high toxicity rate has caused its residues to generate
contamination not only for the environment but also for the health of the humans
who consume the crop product, as well as those who live in areas close to the treated
fields (Wang et al. 2017). To avoid this, many acetylcholinesterase biosensors are
used for the analysis of pesticides; however, these enzyme molecules have limited
conductivity, sensitivity, stability, and biosensor performance. Below, we present
two successful cases of improving the electrochemical signal through the design of
nanomaterials.
First, we quote Bao et al. (2019), who developed an electrochemical acetyl-
cholinesterase biosensor to detect organophosphate pesticides using graphene-copper
oxide nanoflowers with excellent sensitivity. In this case, the main design is based on
graphene, given that it has many properties mentioned above in the section on carbon
materials. However, in addition to all these abilities, the advance in the development of
new materials from graphene has led to the invention of three-dimensional graphene
(3DG), which until now is the hardest material in the world with high porosity and
lighter than air. Because acetylcholinesterase finds a favorable environment in these
3DG structures and due to the nature of this new nanomaterial, the surface area
increases considerably, this biosensor has been able to detect pesticides successfully
and with a low detection limit without altering the good sensitivity of the method.
As a second successful case, Vaghela et al. (2018) worked on a biosensor to detect
glyphosate using gold nanoparticles. Its determination methodology was potentio-
metric based on a urease bioconjugate. Glyphosate (N- (phosphonomethyl) glycine)
used worldwide to eliminate weeds, and for decades its use is considered highly
controversial. It has been shown to be harmful to the health of humans, animals,
and the environment since they cause serious diseases such as congenital disabilities
and neurological effects. On the other hand, the International Agency for Research
on Cancer cataloged glyphosate within the group as a possible human carcinogen.
All this makes it extremely necessary to measure and detect glyphosate levels in all
agricultural products, as well as in water for consumption and irrigation. The devel-
opment of biosensors based on gold nanoparticles is a perfect alternative to expensive
analyzes since it proposes a follow-up and monitoring of the levels of herbicide in the
same place where the agricultural work is carried out, but it is also cheap (Vaghela
et al. 2018). The biosensor is capable of detecting 0.5 ppm of glyphosate, which is
the limit that, according to the World Health Organization, must contain drinking
water. These researchers can be said to have developed a simple and inexpensive
method of detecting glyphosate by urease inhibition.
2.4 Conclusions and Future Perspectives
The different types of nanomaterials used as an integrated platform for the manufac-
ture of biosensors and their recent applications in agriculture are discussed in this
chapter. Nanomaterials have shown enormous potential to immobilize biomolecules
(enzymes, antigens, or antibodies) through various strategies on the surface of a
biosensor, capable of effectively conserving the bioactivity of its biological recep-
tors. Future research on new nanomaterials can be a relevant tool in the development
of new biosensors for various purposes, always to improve the characteristics that
make them as attractive as their low cost and time of analysis, outstanding sensitivity
and selectivity, as well as its ability to be transportable.
Acknowledgements The authors thank the financing of the National University of San
Luis (PROICO-1512-22/Q232), National Council for Scientific and Technical Research (PIP-
11220150100004CO), National Agency for Scientific and Technological Promotion (PICT-
2015-2246, PICT -2015-1575, PICT-2014-1184, PICT-2014-0375 and PICT-2013-3092) and the
University of La Frontera (REDES 180003).
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Chapter 3
Advances in Nanotechnology
for Bio-Sensing in Agriculture and Food
Theivasanthi Thirugnanasambandan
Abstract Biosensors are applied in biological related fields such as clinical, envi-
ronmental, agricultural, and food analysis. They are used to measure the concen-
tration of a biological analyte with the help of various transducers like electro-
chemical, optical, electronic, and piezoelectric biosensors. The performance of a
biosensor is well defined by its stability, cost, sensitivity, and reproducibility. Most
importantly, early detection of diseases becomes possible with biosensors. These
analytical devices make the binding between targets and probes more specifically
which is then converted to an electrical signal. Using nanomaterials in biosen-
sors becomes more advantageous for pathogen detection particularly in fields like
healthcare, food industry, and agriculture. Nanomaterials offer a low loading of
bioreceptor units at reduced volumes and act as transduction elements in biosen-
sors. Their outstanding chemical and electrical properties lead to more advanced
sensor technologies like FET transistors, screen-printed electrodes, and lateral flow
immunoassay. This chapter reviews the use of advanced nanomaterials like gold
nanoparticles, graphene, and conducting polymers that can support these technolo-
gies. The performance of a nano biosensor is also reviewed, that is decided by the
size, shape, conductivity, and morphology of the nanoparticles.
Keywords Nanomaterials · Nanocomposites · Biosensors · Soil moisture/water

uptake · Pesticides · Pathogens · Phytohormones · Transgenic plants · Raman
spectroscopy · Lateral flow immunoassay
Abbreviations
PCR Polymerase chain reaction

ELISA Enzyme linked immunosorbent assay
LED Light emitting diode
T. Thirugnanasambandan (B)
International Research Centre, Kalasalingam Academy of Research and Education (Deemed
University), Krishnankoil 626126, Tamilnadu, India

28 T. Thirugnanasambandan
NPs Nanoparticles
TiO2 Titanium oxide
AuNPs Gold nanoparticles
LCR Inductance (L), capacitance (C), and resistance (R)
MEMS Micro-electromechanical system
SERS Surface-enhanced Raman spectroscopy
LFIA Lateral flow immunoassay
RPA Recombinase polymerase amplification
EIS Electrochemical impedance spectroscopy
In2 O3 Indium oxide
IAA Indole-3-acetic acid
3.1 Introduction
The components of biosensors are bio-analyte (which is to be analyzed), bio-

recognition elements (like enzymes, antibodies, and aptamers or nucleic acid
sequences), and the transducers (like electrochemical and optical). There are two
types of biosensors viz enzymatic and nonenzymatic biosensors. Nonenzymatic has
become possible with the advancement of nanomaterials with its superior sensitivity.
Aptamers are single stranded DNA- and RNA-based oligonucleotides used as bio-
recognition elements in biosensors. They can bind to the targets with high affinity and
specificity. The properties of conformation make them suitable for making label-free
and portable devices for diagnostic applications (Sekhon et al. 2018).
New advanced technologies like electrochemical nanosensors, optical nanosen-
sors, and some other nanosensors are emerging. They offer immediate detection
within a short span of time. High sensitivity, low limit of detection, good selectivity
(with the help of bio-probes), and expected reliability are some of the parameters
that can be achieved through these kinds of sensors. These sensors can also be
handled by people who are not having technical knowledge or with limited tech-
nical knowledge. Nanoscale materials such as metal nanoparticles or nanoclusters,
metal oxide nanoparticles, carbon nanomaterials (carbon quantum dots, graphene,
carbon nanotubes), and nanocomposites have high sensitivity. These materials inte-
grate transduction principles (electrochemical, optical, Raman, catalysis, and super-
paramagnetic properties) into the nanosensors through signal amplification. Elec-
trochemical/optical nanosensors, electronic nose/tongue, nanobarcode, and wireless
nanosensors have multiplex and real-time sensing capabilities. They are applied in
the food and agriculture sector for the detection of food contaminants, preservatives,
microbes, pathogens antibiotics, heavy metal ions, and toxins. Also, they are utilized
in the monitoring of temperature, humidity, and gas (Srivastava et al. 2018).
Conventional tools like PCR and ELISA need more time to get the results. They
also need trained professionals to work with the analysis. Spectroscopic techniques
like Raman spectroscopy is widely used nowadays for sensing biological molecules.
3 Advances in Nanotechnology for Bio-Sensing … 29
This kind of detection is in the infant stage where interpretation of the spectrum
is difficult. Even though they provide more chemical information, the cost of the
instrument is very high that makes it beyond the reach of common people. In agri-
culture, sensors are needed to measure the moisture and pH level of soil, the amount
of fertilizers/nutrients applied in the soil and pesticide detection in plants.
Nano-inspired biosensors improve the quality of life through clinical, agricul-
tural, and environmental applications. They are utilized in agriculture for applications
such as detection of plant infections (fungal, viral, and bacterial), plant physiological
activities (abiotic stress), metabolic content (Phytohormones), mi RNAs, genetically
modified plants, and genetically encoded biosensors (Kumar and Arora 2020). Agri-
culture becomes smart when sensors are applied to increase crop production. Optical
crop sensors evaluate crop conditions by shining the light with specific wavelengths
on the crop leaves and measuring the light wavelengths reflected back to the sensors.
In an NPK sensor optical transducer is used to measure the presence of Nitrogen (N),
Phosphorus (P), and Potassium (K) in soil. The wavelength of LEDs is selected in
such a way as to fit the absorption band of each nutrient.
Crop loss (due to environmental and pathogen-related stresses) reduction and effi-
cient utilization of resources are some of the challenges in plant agriculture. Smart
plant sensors improve plant productivity by optimizing the water and agrochemical
allocation. New technologies are essential for real-time monitoring of plant physio-
logical and developmental responses. Nanomaterials are utilized in such new tech-
nologies for the conversion of the plant chemical signals into digital information that
can be monitored through electronic devices like biosensors (Giraldo et al. 2019).
Modern ICT (Information and Communication Technologies) can be applied in
agriculture by IoT (Internet of things)-based smart farming system. Here the crop
field is continuously monitored with the help of sensors (light, humidity, temperature,
soil moisture, etc.) and automating the irrigation system. Smart farming utilizes
agriculture sensors that can provide data to the farmers to monitor and optimize
crops by adapting to changes in the environmental conditions. Installation of sensors
can be done over weather stations, drones, and robots.
3.2 Nanoparticles for Sensing
Nanoparticles like gold NPs, Zinc oxide NPs, graphene, and carbon nanotubes
possess high sensitivity. Farmers can perform field analysis in fast, accurate, and
cost-effective ways using biosensors with nanoparticles. The performance of nanos-
tructured biosensors can be improved by the functionalization of the sensor substrates
and the immobilization of the bio-probes on a transducer. Since nanoparticles possess
high surface-to-volume ratio, nano biosensors offer high sensitivity, fast response
time, mediate fast electron-transfer kinetics, high stability, and longer lifetime
(Antonacci et al. 2018).
Nano biosensors are highly helpful to the farmers. For example, nutrition sensors
and soil moisture sensors are useful to measure the levels of fertilizers and water,
respectively. After getting accurate information from these sensors, farmers can apply
the fertilizers and water properly. Chlorophyll in plants reflects infrared light. So,
cameras carried by drones are used to take near infrared images of crops (Preci-
sion Farming Market 2020). Nowadays biosensors based on aptamers have become
more sensitive because of the use of nucleic acid amplification techniques. Detec-
tion of plant pathogen before appearance of disease symptoms is possible with this
technology (Khater et al. 2017).
Quantum dots are semiconductor nanocrystals having a particle size from 1 to
5 nm. In agriculture, quantum dots (QDs) are used in the detection of pesticides
and veterinary drug residues. Since they are available in colloidal form and are
highly stable, they can easily be assembled into sensors with bio-recognition elements
such as enzymes, antibodies, and aptamers. They possess very good optical proper-
ties and so signal can be received in the form of fluorescence, chemiluminescence,
electrochemical luminescence, photo electrochemistry, etc.
Nanoparticles such as gold nanoparticles, magnetic nanoparticles, and quantum
dots are widely used for molecular detection purposes. Gold nanoparticles are partic-
ularly well known for rapid immune diagnosis. They have unique physicochemical
properties, including small-sized effect, surface plasmon resonance behavior, and
catalytic effect. They react with nucleic acid through an Au-S covalent bond which
is used for DNA identification. Since gold nanoparticles can produce visible color
change because of their high surface area to volume ratio, they are ideal for rapid bio-
sensing. AuNPs also have the advantage of facile surface modification for effective
targeting of biomolecules.
Several parameters such as fertilizers, nutrients, herbicides, insecticides, pesti-
cides‚ pathogens‚ moisture‚ and soil pH can be effectively analyzed by nanobiosen-
sors. In addition to the analysis-related activities, these nanobiosensors support
sustainable agriculture to improve crop production. Verma et al. have reported that
gold nanoparticles give a simple output because of their superior optical properties.
These sensors must be developed into point-of-care devices for that they need high
sensitivity in complex media. With the utilization of gold nanoparticles, biosensors
can be implemented in diverse environments to prevent the spread of infectious
diseases (Verma et al. 2015). Silicon nanoparticles have many scopes in agricultural
applications. Si-NP is utilized as the growth regulator, soil quality improving agent,
pesticides, nano-carriers (delivery agents for proteins, fertilizers, nucleotides, and
other chemicals) and nanosensors. Figure 3.1 exhibits its agricultural applications
(Rastogi et al. 2019).
3.3 Pesticides Detection
TiO2 nanotubes are well known for their sensing performance. In agriculture, they are
used for the detection of free organophosphate pesticides, dichlofenthion, and chlor-
pyrifos pesticides. The nanocomposite like graphene modified with TiO2 nanotubes
Fig. 3.1 Schematic diagram explains the various applications of silicon nanoparticles in agriculture
(from Rastogi et al. 2019)
is applied for the detection of carbamate pesticides including metolcarb, carbaryl,

isoprocarb, and diethofencarb with the detection limit from 2.27 to 3.26 μgL−1 (Wang
et al. 2016). Even though the use of pesticides like pyrethorids improves crop yield in
vegetable farming, the long-term consumption of fruits and vegetables (obtained from
the pesticides applied farm) could lead to adverse health effects. As per the report
of Yu et al., polystyrene-coated magnetic nanoparticles could detect pyrethroids at a
concentration level as low as 0.02 ng g−1 of vegetables. The duration of the analysis
is less than two hours. The nanoparticles can be reused 30 times thus making the
analysis both time and cost efficient. The nanoparticles also showed strong affinity
for beta-cyhalothrin, bifenthrin, fenvalerate, permethrin, and decamethrin (Yu and
Yang 2017).
Simple paper-based sensors are made by incorporating quantum dots (QDs) in
cellulose papers for pesticide detection. The sensing material is silicon quantum dots
to detect the pesticides paraoxon and parathion by colorimetric analysis. The sensors
are built by combining a green fluorescent protein with silicon quantum dots that
emit red light. The paper turned yellow or green depending on the amount of toxin
present (Zor et al. 2015). In paper-based techniques, while using radio frequency
identification (RFID) tags, the results can be obtained in a smartphone. It is a simpler
way for the farmers. There is no need for highly trained technicians for this technique
(Robidillo et al. 2019). Rapid detection of organophosphorus pesticides was achieved
through using gold nanoparticles and organophosphorus pesticide aptamers. The
color changes from red to purple blue since the aptamers binding to the pesticides
were detached from the AuNPs, resulting in aggregation of AuNPs (Bai et al. 2015).
Chemiluminescence (CL) is the production of light when a chemical reaction
occurs. Luminol is a white-to-pale-yellow crystalline solid that exhibits chemilumi-
nescence with a blue glow. Organophosphate and carbamate pesticides are detected
using luminol-functionalized silver nanoparticles (Lum-Ag NP). The distinct CL
response patterns are produced as “fingerprints” related to each specific pesticide
after interaction with pesticides. A concentration of 24 μg mL−1 of dimethoate,
dipterex, carbaryl, chlorpyrifos, and carbofuran has been measured (He et al. 2015).
The analysis of organophosphorus pesticides was performed with Au nanoparti-
cles and acetylcholinesterase. The color changes from claret-red to purple or even
grey. The colorimetric assay gives a high reproducibility and the detection was
rapid (Li et al. 2011). Plant esterase–chitosan/gold nanoparticles–graphene nanosheet
(PLaE-CS/AuNPs-GNs) nanocomposites were used for the detection of methyl
parathion and malathion. Highly pure plant esterase produced from plants was used
as the probe. The sensing ability of PLaE-CS/AuNPs-GNs composite is as low as 50
ppt (0.19 nM) of methyl parathion and 0.5 ppb (1.51 nM) of malathion (S/N = 3).
The system possessed high selectivity. Hence, there was no interference from metal
ions, inorganic ions, glucose, and citric acid (Bao et al. 2015).
Glyphosate (Gly) on the surface of spinach, apple, and corn leaves was detected
in situ using cysteamine-modified gold nanoparticles. Gly changed the color from
red to blue on the surface of plant tissues. Gly distribution on plant tissues can
facilitate the development of precision agriculture (Tu et al. 2019). Silver nanoparti-
cles (AgNPs) were prepared by green synthesis using clove (Syzygium aromaticum)
extract as a reducing agent. Vinclozolin (fungicide) was detected in trace amounts
by UV–Vis spectroscopy with Ag NPs. Figure 3.2 shows the UV–Vis spectra and
HRTEM images of the silver nanoparticles. AgNPs showed a stable LSPR band at
397 nm. The clove-AgNPs were highly selective and extremely sensitive for colori-
metric sensing of vinclozolin with a lower detection limit of 21 nM (Hussain et al.
2019).
3.4 Soil Moisture Detection
It is important to measure the soil moisture to study the evaporation and transpiration
in plants. There is always a change in humidity levels because of the change in
air temperature and transpiration of water vapor (added by plants) to the air. The
Fig. 3.2 Silver nanoparticles synthesized using aqueous solution of clove (Syzygium aromaticum)
for vinclozolin detection. (a and c) and (b and d) exhibit the UV–Vis spectra and HRTEM images
of clove-AgNPs and clove-AgNPs + VIN (16 μM), respectively. The size range = 2–20 nm and
average size = 14.4 nm of AgNPs are calculated from the (c) (from Hussain et al. 2019)
humidity level (high or low) affects the quality of crop production, and the loss of
quality reduces the selling price of crops and increases production costs which in
turn reduces profits. In the case of a soil moisture sensor, the sensing material causes
a change in the capacitance according to the amount of moisture in the soil which
in turn is used to measure the dielectric permittivity of the surrounding medium.
The applications of soil moisture sensors in agriculture include irrigation planning,
climate research, solute transport studies, and soil respiration measurements.
Zinc oxide (ZnO) nanoparticles were coated as thinfilm on electrodes as the
sensing material. Since ZnO nanoparticles possess oxygen vacancy dipoles, they give
blue emission in the photoluminescence spectrum. So, they have enhanced dielectric
properties. Moisture content from 7 to 25% was measured for wheat grains. The
dielectric constant was determined using LCR meter. Because of the interaction of
ZnO nanoparticles with water vapor, high sensitivity of 36.4% at 1 MHz and 97.4%
at 500 Hz had been achieved. The actual sensing mechanism is the chemisorption of
water molecules on activated sites of the ZnO grains. The dielectric-based sensor is
becoming more popular for soil moisture measurement because this technique needs
small sampling volumes, provides higher accuracy, and is of low cost (Singh et al.
2018).
Simple paper-based sensors were used to measure the humidity of 20–70% relative
humidity (RH) range. The device was made by spin coating of ZnO nanoparticles
on commercial printing papers as substrate and patterning interdigitated electrodes.
After spin coating, the paper was annealed at 100°C for 10 min on a hotplate for
binding of ZnO nanoparticles with the paper. The change in resistivity was recorded
with respect to various humidity levels. Paper is a valuable substrate since it is
available at low cost, is disposable, and has the capacity to absorb water vapor.
Figure 3.3 exhibits the microdevice fabricated on a paper substrate and sensing
activities. Figure 3.3 (a), (b), and (d) exhibit the paper substrate, gold layer, and ZnO
nanoparticles deposited paper substrate, respectively. Figure 3.3 (c) and (e) exhibit
the interdigitated electrodes and two fabricated devices, respectively. Figure 3.3 (f)
shows the SEM image of the ZnO nanoparticles on the paper substrate. Humidity
response and sensing response graph are also shown (Niarchos et al. 2017).
In another study, the soil moisture sensor was constructed using a low-cost thermo
electric generator (TEG) with high sensitivity. The nanostructured thermosensitive
resistors were fabricated on the same ceramic substrate of the TEG, i.e. lead sulphide
(PbS) quantum dots were printed on the TEG substrate (Dias et al. 2016). Graphene
oxide coated interdigitated electrode was fabricated by MEMS technique to be used
as a capacitive sensor. The sensor possesses high sensitivity up to 340 and 370% for
soil moisture changes from 1 to 55% for red and black soil, respectively. The diurnal
temperature and salt concentration were the other parameters that can be measured
based on soil conductivity. The salt concentration varies from 0 mol to 0.35 mol in
the soil (Palaparthy et al. 2018).
Fig. 3.3 Microdevice fabrication process. (a) Paper substrate. (b) Gold layer deposition. (c) Laser
micromachining of the interdigitated electrodes. (d) Spin-coated ZnO-nanoparticle layers. (e) fabri-
cated devices on paper (2 devices on a coin of 1 cent), (f) SEM image of the nanotextured ZnO
film. Humidity response at controlled RH levels ranging from 20 to 70%, (b) sensing response for
both papers as function of the RH (from Niarchos et al. 2017)
3.5 Pathogen Detection
Plant diseases cause more economic losses for the agricultural industry. In agriculture
infections to crops arise from pathogens such as fungi, bacteria, mollicutes, para-
sitic higher plants, parasitic green algae, nematodes, protozoa, and viruses. These
pathogens can affect the plants during growth, harvest, and postharvest processing. To
attain maximum productivity and ensure agricultural sustainability, advanced disease
detection and prevention in crops become more important. Conventional methods
like polymerase chain reaction (PCR), immunofluorescence (IF), fluorescence in situ
hybridization (FISH), enzyme-linked immunosorbent assay (ELISA), flow cytom-
etry (FCM), and gas chromatography-mass spectrometry (GC-MS) are available for
the detection of pathogens. Early detection of crop diseases is nowadays possible
with bio-recognition elements such as enzyme, antibody, DNA/RNA, and bacterio-
phage with high selectivity (Fang and Ramasamy 2015). Nanobiosensors become
more important for the proper identification of the disease and the disease-causing
agent. Otherwise, disease control measures can be a waste of time and money and
can lead to loss of plant production.
Early detection of infections in Arabidopsis thaliana by Pseudomonas syringae
was performed through the rapid isothermal amplification of target pathogen DNA
sequences by recombinase polymerase amplification (RPA) and gold nanoparticle-
based electrochemical assessment with differential pulse voltammetry. This anal-
ysis takes one hour and this method is found to be 10,000 times more sensitive
than conventional polymerase chain reaction (PCR)/gel electrophoresis. Figure 3.4
illustrates the electrochemical bioassay of plant pathogen (Lau et al. 2017).
Fig. 3.4 Schematic diagram shows the electrochemical bioassay for the detection of plant pathogen
DNA (from Lau et al. 2017)
Fluorescent silica nanoparticles when conjugated with antibodies are used to

detect plant pathogens such as Xanthomonas axonopodis pv. vesicatoria which
causes bacterial spot disease in Solanaceae plant. Yao et al., have reported that silica
nanoparticles are incorporated with the organic dye, tris-2,2 -bipyridyl dichlororuthe-
nium (II) hexahydrate (Rubpy) and conjugated with the secondary antibody of goat
anti-rabbit immunoglobulin G (IgG). The fluorescence silica nano biomarker has
the potential for the diagnosis of plant diseases (Yao et al. 2009). Detection of plant
pathogen Xanthomonas campestris was performed with gold nanoparticles and bacte-
riophage M13 to display the receptor-binding protein from a phage. The analysis can
be performed in less than one hour and can detect ~100 cells (Peng and Chen 2018).
Citrus tristeza virus (CTV) causes Tristeza disease in citrus plants. The nucleic acid
of CTV was measured using electrochemical sensing. A screen-printed carbon elec-
trode was modified by electrodepositing gold nanoparticles and thiolated ssDNA
was used as a probe. EIS measurements were performed in Fe (CN6 )4− /Fe (CN6 )3−
with the limit of detection 0.1–10 μM and selectivity was also good in this system
(Khater et al. 2019).
3.6 Transgenic Plant Detection
Transgenic tobacco plants carrying a Streptococcus mutans antigen were identi-

fied using the surface resonance property of gold nanoparticles. The genomic DNA
(isolated from the various parts like leaves, stems, and roots of the transgenic
tobacco), as well as the biotinylated oligonucleotide probes, are immobilized onto
a streptavidin (SA) sensor chip. The detection limit of this sensor was very low,
i.e. 1 pM. Transgenic plant detection is shown in Fig. 3.5 (a). Figure 3.5 (b) exhibits
transgenic DNA detection using SA SPR sensor and gold nanoparticles (Grześkowiak
et al. 2019).
The genetically modified maize (MON 810 corn) is combating crop loss (created
mainly by insects). Highly monodisperse Fe3 O4 @Au nanoparticles were used to
detect them by the electrochemical sensing method. Usually, these monodisperse
NPs are prepared by the thermal decomposition method. Freitas et al. have reported
about the coating of Fe3 O4 @Au nanoparticles on the screen-printed electrodes. DNA
covalently linked to a carboxylated self-assembled monolayer and a fluorescein isoth-
iocyanate (FITC) signaling are used as probes. Chronoamperometric measurements
were made after PCR amplification for the genoassay range from 0.25 to 2.5 nM
(Freitas et al. 2016).
3.7 Raman Spectroscopy in Sensing
Surface-enhanced Raman spectroscopy (SERS) is a powerful technique for the quan-

titative measurement of pesticides without pre-treating the sample. Tognaccini et al.
Fig. 3.5 Schematic diagram of the transgenic plant detection experimental. (a): a general proce-
dure of the transgenic plant detection using gold nanoparticles (AuNPs)-based SPR biosensor,
(b): schematic illustration of transgenic DNA detection using SA SPR sensor and AuNPs (from
Grzeskowiak et al. 2019)
have reported that dimethoate (DMT) is an organophosphate insecticide applied to

protect olive trees. DMT rapidly degrades than omethoate (OMT) and both disappear
within two months. So, in-field determination of dimethoate becomes more impor-
tant. The maximum limit of DMT permitted on olives for oil production is 3 mg
Kg−1 and for OMT 1.5 mg Kg−1 . If the concentration of DMT in water is below
0.1 mg L−1 , such water will be sent to the sewage system (Tognaccini et al. 2019).
Simple and effective methods are essential for its in-field identification for public
health and for the proper certification of organic produce. Raman scattering intensity
can increase enormously with its surface coated with nanostructured silver or gold.
Detection of DMT and OMT in water and on olive leaves is performed by surface-
enhanced Raman spectroscopy (SERS) using portable instrumentations. Figure 3.6
shows the variation in the SERS spectra depending upon the DMT concentration
Fig. 3.6 SERS spectra

related to the DMT at
different concentrations. (a)
0.0 (only AgNPs in water).
(b) 1 × 10−6 . (c) 5 × 10−6 .
(d) 1 × 10−5 M. Vertical
shifting in the spectra
improves data readability.
The arrow point at 406 cm−1
band can be used to
differentiate in the DMT
quantification (from
Tognaccini et al. 2019)
(Tognaccini et al. 2019). When compared to the conventional methods like gas
or liquid chromatography combined with mass spectroscopy, SERS possess ultra-
sensitivity and simpler protocols for the detection of pesticide residues. Since SERS
can be used for the direct detection of pesticides at trace levels in liquid samples or
on the surface of solid samples. SERS provides vibrational fingerprints of molecules
with non-destructive testing (Xu et al. 2017).
Trace level detection of pesticides at parts per million (ppm) or parts per billion
(ppb) is possible with SERS. SERS utilizes noble metal nanostructures like gold and
silver nanoparticles to increase the weak Raman signals. Chemically synthesized gold
nanoparticles enhance Raman scattering in the detection of suspended pesticides
(fungicides) and insecticides (neonicotinoids and organothiophosphates) with the
detection limits from 0.001 to 10 parts per million (ppm).
Pesticides like phosmet and thiram on apple skin were analyzed using SERS anal-
ysis. The results showed that SERS with colloidal gold nanoparticles is a potential
tool for identifying pesticides at trace levels for food safety applications. The surface
of apple skin was cleaned and added to the colloidal gold nanoparticle. The suspen-
sion was then subjected to 785 nm laser excitation to observe the Raman spectral
signatures of these pesticides. In this technique, pesticides less than 1 part per million
can be measured. The pesticide tolerance level on apples given by the 2018 Code of
Federal Regulations for the pesticides thiram, malathion, acetamiprid, and phosmet
are 5 ppm, 8 ppm, 1 ppm, and 10 ppm, respectively (Dowgiallo and Guenther 2019).
In another study, silver-coated gold nanoparticles (Au@Ag NPs) were applied for
the detection of pesticide residues in various fruit peels such as apple, grape, mango,
pear, and peach using SERS. The Raman enhancement of Au@Ag NPs for sulfur-
containing pesticides is stronger than those of bare Au and Ag NPs. The enhancement
depends on the Ag shell thickness. The particle sensors can be cast onto fruit peels
to measure pesticide residues like thiocarbamate and organophosphorus compounds.
The detection was highly reliable and rapid for example, 1.5 nanograms of thiram
per square centimeter at apple peel was measured (Liu et al. 2012).
The extraction of targets from complex surfaces is difficult in the analysis of
pesticide residues. Surface-enhanced Raman scattering active substrate is an excel-
lent tool for the detection of target molecules. A novel substrate is constructed by
decorating the commercial tape with colloidal gold nanoparticles (Au NPs) which
is able to provide SERS activity. The SERS tape was pasted and peeled off on
fruits and vegetables. The strong SERS signals were utilized in the detection of
different pesticide residues (like parathion-methyl, thiram, and chlorpyrifos) present
in the real samples having complex surfaces including green vegetable, cucumber,
orange, and apple (Chen et al. 2016). The detection of the organochlorine pesticides
aldrin, dieldrin, lindane, and α-endosulfan with a limit of detection reaching 10−8 M
was achieved using high sensitivity of SERS. To improve the affinity of the pesti-
cides with SERS substrate, the metal surface was functionalized with alkyl dithiols
(Kubackova et al. 2015). Label-free detection of pathogens is possible with Raman
optical spectroscopy. But it remains challenging to achieve clinically relevant speeds
and accuracies due to weak Raman signal from bacterial cells.
3.8 Lateral Flow Immunoassay
Ralstonia solanacearum is a bacterium that causes potato brown rot. This pathogen
was detected with Lateral flow immunoassay (LFIA) where the sensing material
is gold nanoparticles conjugated with antibodies specific to R. solanacearum. This
assay was able to detect up to 3–104 cells mL−1 in the potato tuber extract. Lateral
flow test strips can be developed for making sensors for plants which can be highly
useful for the farmers for immediate detection without a high-level scientific knowl-
edge. Figure 3.7 shows the potato tuber extract analysis done through LFIA method
(Razo et al. 2019).
Gold nanoparticles antibody conjugation, immobilization of the antibody specific
to R. solanacearum, immobilization of protein A on the control zone and potato tuber
extract with R. solanacearum are shown in the part (1), (2), (3), and (4) of Fig. 3.7
(a), respectively. Test zone without color band after analysis (negative result), red
color band in the control zone and formation of the immune complex (immobilized
specific antibodies to R. solanacearum, GNP conjugate with specific antibody) after
completion of conventional LFIA can be seen in the (5), (6), and (7) of Fig. 3.7 (b),
respectively. Figure 3.7 (c) shows the results of adding the amplification solution.
After signal amplification of test zone, control zone and size enlarged gold nanopar-
ticles are shown in the part (8), (9), and (10) of Fig. 3.7 (c), respectively (Razo et al.
2019).
Also, the rapid and in-field detection of pathogenic viruses is important in modern
agricultural technologies. Potato leafroll virus was detected with a highly sensitive
lateral flow immunoassay. The sensing material was gold nanoparticles with silver
Fig. 3.7 Schematic diagram shows the LFIA analysis of pre and post signal amplification: (a) Inser-
tion of the test strip into the potato tuber extract; (b) conventional LFIA indicates negative result;
color band did not appear in the test zone; (c) after adding the amplification solution to the test strip
color band appeared (positive result) due to the enlarged size of the gold nanoparticle (from Razo
et al. 2019)
enhancement. The enhanced silver causes 15 times more sensitivity (detection limit
0.2 ng mL−1 ; 15 min.) compared with conventional LFIA (detection limit 3 ng mL−1 ;
10 min.) (Panferov et al. 2018).
The bacterial spot disease of stone fruits and almond is caused by the pathogen
Xanthomonas arboricola pv. pruni. In one study, Lateral flow immunoassay (LFIA)
was designed with polyclonal antibodies and carbon nanoparticles assembled on
nitrocellulose strips. The sensitivity of the LFIA was analyzed for the plants like
almond, apricot, Japanese plum, and peach using the suspensions obtained from the
pure cultures of three X. arboricola pv. pruni strains and spiked leaf extracts of the
plants (inoculated with this pathogen). The detection limit observed with both pure
cultures and spiked samples is 104 CFU ml−1 (Lopez-Soriano et al. 2017).
3.9 Screen-Printed Electrodes
Commercial electrochemical sensors are fabricated from screen printing technology.

So, miniaturized, sensitive, and portable devices can be made available lab-to-market
(Hayat and Marty 2014). Figure 3.8 shows the schematic diagram of a portable sensor
design using screen-printed electrode. This is a disposable sensor.
Solid-phase isothermal recombinase polymerase amplification (RPA) followed
by electrochemical detection is applied for the detection of Citrus tristeza virus.
RPA devices are limited by the need for heating sources to reach sensitive detection.
This limitation can be avoided with gold nanoparticle (AuNP)-modified sensing
substrates. Plant disease RPA assay for amplification of the P20 gene (387-bp) of
Fig. 3.8 Schematic diagram shows the screen-printed electrode. In this model, reference, working,
and auxiliary electrodes are present in the same substrate. This is a disposable and portable sensor
design
CTV was designed and tested by standard gel electrophoresis analysis. The Electro-
chemical impedance spectroscopy analysis (EIS) is performed in a Fe (CN6 )4− /Fe
(CN6 )3− electrolyte and a high limit of detection of up to 1000 fg μL−1 of nucleic
acid was achieved (Khater et al. 2019).
Screen-printed electrode was modified with Ag nanoseeds and Ag nanoprisms
and three different carbon substrates, graphite, graphene, and carbon nanofibers for
the determination of Pb (II) and Cu (II). Figure 3.9 Voltammetric measurements of
Pb (II) and Cu (II). Calibration curves are shown in the insets. The results confirm
that the Ag-nanoseeds-SPCNFE has higher sensitivities. It can be applied for the
simultaneous determination of Cu (II) and Pb (II) present in the natural samples
(Pérez-Ràfols et al. 2017).
Nitrites are used as a preservative in dairy and meat products. They are poten-
tial carcinogens and cause detrimental health effects; if they are utilized beyond
safe limits that will be harmful to public health and environment. Talbi et al. have
reported that the electrochemical sensing of nitrite was performed using graphite
screen-printed electrodes (GSPE) functionalized with gold nanoparticles stabilized
by branched polyethyleneimine (AuNPs-PEI) with a concentration of 300 μg mL−1 .
The sensor performs well in the linear range 1–10 μM and the limit of detection
1 μM (Talbi et al. 2019). Graphite screen-printed sensor developed using silver metal
nanoparticle embedded chitosan composite was also employed in the quantification
of nitrite at trace level (Patri et al. 2019).
Fig. 3.9 Stripping voltammetric measurements and calibration curves (insets). (a, b) and (c, d)
calibration of Pb (II) and Cu(II), respectively, in acetate buffer pH 4.5 at an Ed of −1.1 V and a td of
120 s. In Fig. 3.9 (a, c) and (b, d) Ag-nanoseeds-SPCNFE and Ag-nanoprisms-SPCNFE electrodes
are used. SPCNFE: carbon nanofiber modified screen-printed electrode (Pérez-Ràfols et al. 2017)
3.10 Detection of Phytohormones
Salicylic acid (SA) acts as a phytohormone in plants. SA concentration changes when

a plant is infected by pathogens. Electrochemical sensing of SA was performed by
coating the gold electrode with copper nanoparticles. The electrocatalytic oxidation
of salicylic acid in oilseed rape infected with the fungal pathogen Sclerotinia scle-
rotiorum was studied and the sensitivity was high with gold electrode modified with
copper nanoparticles than the bare gold electrode (Wang et al. 2010).
NaLuF4 nanoparticles are upconversion nanoparticles that can emit multicolor
visible light under the excitation of 980 nm near-infrared (NIR) photons. Rhodamine-
B dye in plant cells was measured effectively with a detection limit of 0.25 μg cm−3
in plant cell. RB absorbs the green fluorescence from nanoparticles and emits red
light. The phenomenon is called luminescence resonance energy transfer (LRET)
(Xiaofeng et al. 2016).
Neoglycoprotein functionalized with fluorescent gold nanoclusters (AuNCs),
contains biantennary N-glycan (G0) as targeting molecule, ovalbumin (OVA) as
carrier/model antigen. This fluorescent gold core (G0-OVA-AuNCs) can be utilized
as imaging probe for the detection of plant lectins and in vitro imaging of dendritic
cells (Brzezicka et al. 2018). Indole-3-acetic acid (IAA) was detected by electro-
chemical method using gold nanoparticles (AuNPs) functionalized with horseradish
peroxidase labeled immunoglobulins G (AuNPs-HRP-IgG) as signal amplification
probe. Studies were taken by differential pulse voltammetry (DPV) in 0.1 M PBS
(pH 7.4) containing Fe (CN)63 −/4 − electrolyte and the limit of detection was
found to be 5.5 × 10−10 M (S/N = 3). The immunosensor possesses high selectivity
toward indole-3-acetic acid and can differentiate IAA from other phytohormones,
such as gibberellin, abscisic acid, and salicylic acid (Zhou et al. 2013). In another
study, gold nanoparticles/polyaniline/multiwall carbon nanotubes (AuNPs/TPANI-
MWCNTs) nanocomposite was used to construct an amperometric immunosensor
for the detection of indole-3-acetic acid. The thiolated bionanocomposite film was
made from the chemical reaction between boronic acid functionalized AuNPs and
the vicinal diol functionalized AuNP labeled immunoglobulin G (IgG–AuNPs). It
was utilized to get more amplification of the signal of the immunosensor. The limit
of detection of the sensor was 0.97 pg mL−1 with good reproducibility and stability
(Su et al. 2019).
Abscisic acid (ABA) is a plant hormone which participates in seed, bud dormancy,
the control of organ size and stomatal closure. Wang et al. have reported that ABA
from fresh leaves of rice was detected by localized surface plasmon resonance (LSPR)
using aptamer-functionalized gold nanoparticles (AuNPs) without using expensive
instrument and antibody. Abscisic acid involves in abiotic stress response and plays
an indispensable role in plant physiological activities. The sensor shows a linear
range from 5 × 10−7 M to 5 × 10−5 M with a detection limit of 0.33 μM (Wang
et al. 2017).
Glassy carbon paste electrode modified with In2 O3 nanoparticles is used for the
electrochemical determination of luteolin (LU) in thyme. The analytical performance
of the sensor shows a wide response to LU from 9.98 × 10−9 M to 8.84 × 10−8 M LU
and a low detection limit of 1.99 × 10−10 M of LU was achieved (Ibrahim and Temerk
2015). A simple electrochemical sensor was constructed by electrodeposition of
glassy carbon electrode with graphene quantum dots (GQDs) and gold nanoparticles
(GNPs) for the determination of luteolin peanut hull samples. The nanocomposite
resulted in a 16-fold increase in the oxidation current of luteolin in phosphate buffer
at pH 5.0 with the detection limit of luteolin 1.0 nM (S/N = 3) (Tang et al. 2019).
3.11 Detection of Water Uptake
Direct visualization of water-conducting pathways and analysis of sap flows in xylem

vessels can be done through AuNPs (gold nanoparticles) combined synchrotron X-
ray imaging technique. AuNPs were used as flow tracers in the xylem vessels in the
first 20–30 min without any physiological barrier. So, fluid dynamic phenomena can
be visualized using gold nanoparticles in vascular plants (Hwang et al. 2014). In
another study, TiO2 nano-flowers were coated on fluorine-doped tin oxide substrate
and used as a pH sensor which can measure pH range of 2–12. TiO2 nano-flowers
pH sensor had high sensitivity value, i.e. 2.7 (μA)1/2 /pH and a linear relationship
between IDS and pH (regression of 0.9991). The voltage reference and pH relationship
showed the sensitivity value 46 mV/pH and a linear regression of 0.9989. The results
confirm that a flower-like TiO2 nanostructure extended gate field effect transistor pH
sensor can be utilized in the detection of the pH value effectively (Yang et al. 2019)
(Fig. 3.10).
3.12 Conclusion
Biosensors can help farmers to increase the crop yield by detecting various influ-
encing factors. In agriculture, many parameters like fertilizers, herbicides‚ pesti-
cides‚ insecticides‚ pathogens, moisture, and soil pH are analyzed by nanobiosen-
sors. The challenges for the researchers in this area lie in the performance of the
sensors, sampling, detection in open areas and scaling up measurements. Spectro-
scopic techniques like Raman spectroscopy, Fourier transform infrared spectroscopy,
and fluorescence spectroscopy are the best alternatives for PCR technology and offer
immediate detection. The achievement of a low limit of detection by various trans-
duction techniques with the help of nanomaterials is discussed in this chapter. These
transducers can transform plant chemical signals to digital form which can be easily
measured. Wearable sensors can be produced to enhance crop productivity in order
to meet the demand for food. These sensors are also useful to control the utilization
of costly chemicals like pesticides and herbicides.
Slow technology transfer to the marketplace is the major drawback of the biosen-
sors. However, biosensors technology will replace conventional analytical techniques
with the help of highly sensitive nanoparticles. Nanotechnology enables the smart
plant sensors for monitoring and optimizing the plant productivity and proper utiliza-
tion of resources. These sensing techniques can be highly reliable for continuous
monitoring of plant health which will be more helpful in modern agriculture.
Fig. 3.10 Application of TiO2 in the extended gate field effect transistor (EGFET) pH sensor.
(a) Shows the schematic diagram of the TiO2 EGFET pH sensor. (b) HR-SEM image of TiO2
nano-flowers (Top view). Randomly oriented flower-like nanostructures can be seen. (c) The same
image (TiO2 nano-flowers) is shown with high-magnification (Yang et al. 2019)
Acknowledgements The author expresses thanks to her husband Mr. G. Sankar for his assistance
in this work. Also, she acknowledges the assistance of International Research Center, Kalasalingam
Academy of Research and Education (Deemed University), Krishnankoil – 626 126, Tamilnadu,
(India).
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Chapter 4
Nanomaterial-Based Gas Sensors
for Agriculture Sector
Robin Kumar, Monica Jaiswal, Neelam Kushwaha, Shivansh Bansal,

Neha Mazumder, and Jagjiwan Mittal
Abstract Discovery of nanomaterials in the last three decades and their unique prop-
erties led the boom in their applications in various areas. Use of these materials in
engineering, medical and environment also increased their attraction for the potential
applications in agriculture. The current focus of agriculture research is the sustain-
able increase in crop production and protection. For achieving the goals, constant
checking of parameters such as moisture content, soil fertility, temperature, crop
nutrient capacity, pathogens, plant diseases, etc. are highly required. Use of nano
sensors for monitoring these aspects are found to be very effective in increasing the
healthy crop production. This chapter describes the applications of different nano-
materials in sensing of gases during different phases of agricultural practices. The
objective is to evaluate the current literature using the gas sensors in agriculture and
meat production, and their storage and transport applications.
Keywords Sensors · Gas sensors · Nanomaterials · Agriculture · Meat industry
4.1 Introduction
For many decades, agriculture has been mainly associated with the production of
essential food crops for human survival. At present, current agriculture acknowledge
not only farming but processing, distribution of crops and livestock products and
their marketing. Thus, the word agriculture encompasses promotion of processing
production, and distribution of agricultural products and derivatives. Agriculture
plays most significant role in the economic well-being and growth for any given
country. Advanced technology and ideas have helped us to be able to grow as well as
store crops according to our needs, even if climate is not favourable for some crops.
We can make artificial atmosphere to facilitate the growth and healthy storage of
various regular as well as exotic vegetables, fruits, herbs including hybrids. Besides
providing all the required conditions for favourable growth and storage of crops,
R. Kumar (B) · M. Jaiswal · N. Kushwaha · S. Bansal · N. Mazumder · J. Mittal

Amity Institute of Nanotechnology, Amity University Uttar Pradesh, Noida, UP, India

52 R. Kumar et al.
several technologies combine to give expected results. Several studies are going on
to improvise the agricultural standards in many aspects. Different regions of the world
with varying climates require customised conditions for crop growth. These include
fertilizers, pesticides and various types of sensors to keep a check on all conditions
required, for example, humidity sensor, temperature sensor and gas sensors. Precision
agriculture model consists sensors for collection of data, providing information to
farmers by means of irrigation control model arrangement, variable-rate technology
model and control system using parameter of green house (Chaudhary et al. 2011).
The demand for huge supply of food by the increasing global population motivates
scientists to explore new methods and materials to accelerate agricultural produc-
tion. As the natural resources such as land and water are meagre, the agricultural
productivity can only be enhanced by better agronomy with the support of effec-
tively using advanced technology (Pudake et al. 2019). Sustainable agricultural esca-
lation is a concept designed for increasing food production from the same farmland
without pernicious impact on the environment. Besides production, prime concerns
of agricultural activities are food safety, storage as well as transport. With the surge
in globalization of food, this suggests the importance of food quality assessment
during all steps of the agri-food chain supply. Therefore, primary focus of agricul-
tural research is on the improvement of efficiency of the crop, food processing and
safety, food supplements and environmental effect on the production, storage and
distribution of food (Pudake et al. 2019). Food quality assessment can be achieved
qualitatively at the laboratory level by the number of methods including cell culture
and instrumental analysis. These methods are time-consuming i.e. they can take
several hours to days, including different pre-treatment steps of the sample as well
as instrumentation. Safety of food is also vulnerable to some contaminants which
cannot be noticed and sensed with standard analytical techniques e.g. plant pathogens,
formaldehyde, excessive residues of pesticides (Kannan and Guo 2020). The inability
of conventional techniques to assess food safety gives way to the development of
novel, miniaturized, fast and handy analytical methods having low range detection
limits. Nanotechnology offers one key solution for these requirements of analytical
tools.
Nanotechnology consists of the power to contribute to every step of agricultural
advancements by exploiting novel properties of nanomaterials. Understanding and
manipulating the nanometric structure of the material provides the potential to bring
significant change in the agricultural field. Due to the unique properties of nanomate-
rials, it helps to boost agricultural production and food processing. To use inputs more
efficiently, and to head towards sustainable agriculture, a farmer needs to monitor
the crop production. Therefore, a constant check on parameters such as moisture
content, soil fertility, temperature, crop nutrient capacity, pathogens, plant diseases,
etc. is highly required. The use of nano sensors is one way for increasing healthy
crop production. Nano sensors assist in the detection of microbes, humidity and toxic
pollutants at tiny levels (Kaphle et al. 2018). This is done by accurately measuring
the pH of soil, nutrients, residual pesticides and moisture in the soil, examining
pathogens, assessment of nitrogen uptake, etc.
4 Nanomaterial-Based Gas Sensors for Agriculture Sector 53
4.2 Sensors
Sensor is a device which can sense a property like temperature, pressure, force and
convert it into preferred output like colour, electrical and thermal signal. Measuring
of these signals helps in the detecting the desired property. Occasionally, signal
conditioning unit must be used with the sensor as it may not be able to analyse the
obtained signal alone. Signal processing unit maintains the output voltage levels of
sensor in the anticipated range with respect to the end device.
Main objective in the agriculture is to regularly monitor and adjust the environ-
ments with respect to the requirement of the crop. For this, environmental parameter
sensors for measuring the temperature, humidity, amount of carbon dioxide, etc. are
required (Chaudhary et al. 2011). Therefore, a network of sensors is used which
consists of tiny autonomous devices known as sensor nodes in large numbers. The
sensors are designed for collecting information about the climate like light, pressure,
temperature, humidity, carbon dioxide, speed and direction of wind.
4.2.1 Advantages of Nanomaterials
The improvement of sensitivity and the increase of selectivity are quite easily possible
with a decrease in components size, which in turn gives more rapid response. Nano-
materials are characterized by having unique physicochemical properties, including
high electrical and thermal conductivity, extremely high surface area/volume ratio,
high mechanical strength and even excellent catalytic properties. Nanomaterials can
become more efficient by decreasing the sensor and sample sizes, maximizing the
number of sensors and reducing the power consumption
Nanotechnology has the potential to increase the productivity in agriculture
by improved techniques and sensors being identified for precise detection of
pathogens and pollutants in food, convenient management of natural resources, effi-
cient and more reliable delivery system for agro-chemicals (fertilizers and pesti-
cides), enhanced surveillance technique for food safety and agricultural activities,
processing and packaging (Pudake et al. 2019).
Nano sensors are beneficial for detecting and updating the real-time information
about the product from production site to delivery site of consumers (Patel et al.
2020). Nano sensors are replacing traditional sensors as they are portable, sensitive
and specific (Kaushal and Wani 2017). These sensors are extremely sensitive and can
detect the desired property very minutely without using sophisticated instruments and
skilled workers. Development of nano sensors exclusively for the agriculture (agro-
sensors) is an emerging field which helps the farmers for monitoring the diseases and
contaminants at early stage. This can prevent their economic losses by controlling
the diseases and containments (Saini et al. 2017).
The ideal sensor should possess the following characteristics (Campbell and
Compton 2010):
54 R. Kumar et al.
1. Specificity: Selectivity for the target species,

2. Sensitivity: Detecting changes in target species concentrations,
3. Fast response time,
4. Stability: Extended lifetime of at least several months, and
5. Miniaturization: Small size with the possibility of low-cost manufacture as well
as increasing number of sensors in array.
4.2.2 Types of Sensors
The sensors are classified based on attributes such as stimulus, working principle,
properties (attributes of the characteristic) and application.
Main type of sensors based on stimulus are listed below:
a. Electrical Sensor: These sensors detect the existence of electrical signals in an
environmental input. e.g. metal detectors, RADAR systems.
b. Magnetic Sensor: These sensors notice the variation in magnetic signals
(magnetic flux) in an environmental contribution.
c. Optical Sensor: These sensors use light to quantify the characteristics of any
object. They are applied to sense objects lying outside the visible spectrum i.e.
ultraviolet and infrared. Electric eye is the commonly known optical sensor. This
applies a light beam for detecting the existence of an object. Fibre optic sensors
use the principle of total internal refection and travelling through a glass fibre
by light. This measures a various characteristic, such as temperature and strain.
Motion and photo detectors are used as switches for turning on and off system
of lighting.
d. Chemical Sensor: Chemical sensors detect the existence of a certain molecule in
a location. pH, oxygen, carbon monoxide sensors are some example of chemical
sensors
e. Thermal/Radiation Sensor: These sensor measures the change/presence of
radiation or temperature of the environment.
f. Mechanical Sensor: These sensors notice the variation in the mechanical proper-
ties of a system or object. In this setup, strain gauge is the primary mechan-
ical sensor. This forms the base of many mechanical sensors such as load
cells, humidity sensors and pressure transducers. A strain gauge comprises of
adjustable resistor which measures deformation amount in a part during use of
force. Potentiometer is another mechanical sensor for measuring the linear or
angular displacement.
Various types of sensors employed according to parameters that are required to
measure
a. Light Sensors: Infrared sensor using as Transmitter or LED, IR receiver sensor
for example photodiode, register depends on light
b. Gas sensors: Humidity sensor, sensor for toxic gases
c. Pressure/Force/Weight Sensors: like strain gauge, and load cells
d. Position Sensor: Encoder, potentiometer

e. Hall Sensor: To measure the variation in magnetic field
f. Temperature Sensor: Thermistor, thermocouple.
Among all the above sensors, present chapter describes the detailed studies on the
gas sensors used in the various areas of agriculture. The objective is to summarize
the current literature on usage of gas sensors in agriculture and meat production, and
their storage and transport applications.
4.3 Gas Sensors
Changing climate and weather conditions for growing crops and to preserve harvested
food require highly reliable sensor technologies. There are three major factors on
which shelf lives of highly putrescible agricultural foodstuffs for instance fruits,
vegetables, baked goods and livestock products depends (Campbell and Compton
2010). They are rate of oxidation, growth of aerobic spoilage microorganisms and
attack of insects and pests. Early detection and assessment of the various gases
are very important in monitoring of environment and chemical processes. For this
purpose, gas sensors/electronic noses are used.
With recent development in nanotechnology, it had created a huge scope for the
development of highly specific and sensitive, affordable, small sized and low power
consumable portable sensors. High surface area to volume ratio and hollow struc-
tures makes nanomaterials perfect for absorption of gas molecules. Thus, gas sensors
based on nanomaterials have been widely studied (Jian et al. 2020; Meng et al. 2019).
Substantially smaller size, lower mass, more modest power requirements, greater
sensitivity, and better specificity are the potential benefits of exploiting microelec-
tronics and nanotechnology in sensor fabrication (Neethirajan et al. 2009). Nano-
materials such as graphene, CNT and MWCNT have been found to be promising
materials for gas detection. Their special electronic properties (Mittal and Lin 2020),
are strongly affected by the adsorption of gases on their surfaces. These materials have
advantage of high stability and show high sensitivity and good reversibility during
gas sensing, and stability. Metal, metal oxides and doped metal oxide nanoparticles
(Kumar et al. 2017a, 2019) have also been studied in detail for gas sensing.
Nano sensors with functionalized and decorated nanoparticles such as SWCNTs,
nanowires, nanofibers to detect different gases like NH3 , NO2 , SO2 and VOCs, have
the incredible scope in monitoring pollutants in agriculture (Wanekaya et al. 2006).
For quantifying the environmental pollution, nano-smart dust consisting small wire-
less sensors and transponders and gas sensors can be used (Mousavi and Rezaei
2011). In this regard, some of our studies had reported graphene modified or inter-
calated with metal oxides for the detection of NH3 gases (Jaiswal et al. 2020; Kumar
et al. 2017a), also metal nanoparticles and modified OMS-2 material was used for
detection of various gases (Kumar et al. 2016, 2017b, 2018).
56 R. Kumar et al.
4.3.1 Type of Gas Sensors
The gas sensors are divided into four major categories
4.3.1.1 Solid-State Gas Sensor
A solid-state sensor consists of one or more metal oxides from the transition metals,
such as tin oxide, aluminium oxide, etc. A thick or thin film-chip sensor are made by
the metal oxides film deposited onto a substrate with an integrated heating element
to regulate the sensor temperature, since the finished sensors exhibit different gas
response characteristics at different temperature ranges. In the presence of gas, the
metal oxide causes the gas to dissociate into charged ions or complexes which results
in the transfer of electrons. Solid-state gas sensors have numerous advantages which
cause great interest in them. Some of these advantages are, small sizes, high sensi-
tivities in detecting very low concentrations, ability in detecting a wide range of
gaseous chemical compounds, possibility of online operation and low cost.
4.3.1.2 Semiconductor Gas Sensors
Semiconductor gas sensors are based on metal oxides such as SnO2 , TiO2 , InO3 , etc.
and known as chemo-resistive gas sensors. The gas-sensing mechanisms is based
on gas/semiconductor surface interactions such as reduction/oxidation processes,
adsorption of the chemical species on the semiconductor or adsorption with surface,
chemical reactions between different adsorbed chemical species, etc. These interac-
tions occur at the grain boundaries of the polycrystalline oxide film. These surface
phenomena cause changes in electrical resistance (Warneck 1999). The change in
electrical resistivity could be perceived and used to detect chemical species. The basic
phenomenon involved in gas detection by a film semiconducting metal oxide film
using change in its electrical resistance (Jonda et al. 1996). During process, oxygen
concentration change caused by the adsorption and oxidation and reduction reaction
of oxidized and reduced gaseous species at the surface of metal oxides, is detected.
Here, electrical conductivity of sensing material is a function of temperature and the
concentration of exposed gas (Eranna et al. 2004). Figure 4.1 shows set up of device
using semiconductors for gas-sensing measurements.
4.3.1.3 Optical Gas Sensors
The measurement of changes in absorption spectrum was the first method to detect
the desired quantity of the gas molecules. But newest optical gas sensors use other
methods including spectroscopy, surface plasmon resonance and interferometry. As
shown in Fig. 4.2, a setup using optical method for gas-sensing measurements detects
Fig. 4.1 A setup for gas sensing using semiconductors
Fig. 4.2 A schematic of setup for gas sensing using optical method
changes in light intensity in sample thickness due to gas concentrations. The optical
gas sensors could be used for measuring the chemical and biological quantities.
Features of these sensors including safety, low power consumption, remote sensing
and multiplexing of sensor arrays are advantages of theses sensors over the other
sensors such as electrical gas sensors (Penza et al. 2007). The optical gas sensors are
more practical than the other gas sensors.
58 R. Kumar et al.
4.3.1.4 Electrochemical Gas Sensors
Electrochemical gas sensors are based on the fact that chemical reactions at an elec-
tronic conductor interface exchange electric charge. Oxidization takes place at anode
and reduction happens at the anode. This leads to the generation of current by the
flow of positive ions to the cathode, and the negative ions to the anode. Therefore,
reducible gases like O2 , and NO are detected at the cathode whereas oxidizable gases
like CO, NO2 and H2 S are sensed at the anode. The gas sensing using electrochemical
method is either potentiometric or amperometric which depends on the electromotive
force or electrical current output. Potentiometric measurements are carried out under
settings of near-zero current. On the other hand, amperometric sensors are usually
carried out by using high voltage by external cell to keep a nil-oxygen concentra-
tion at the surface of cathodic; here, the response of sensor is diffusion controlled.
An important application of electrochemical sensors in agriculture is in the direct
measurement of soil chemistry through tests such as pH or nutrient content (Li et al.
2010). Figure 4.3 shows one representative setup using electrochemical method for
gas-sensing measurements.
Fig. 4.3 A setup for gas sensing using electrochemical method

Fig. 4.4 A setup for gas

sensing using
electrochemical method
4.3.1.5 Chemiresistor/FET Sensors
These sensors have gained recommendable attention in the last couple of decades
due to their advantages of quick response time, possible integration in manufacturing
processes, miniaturization potential and parallel sensing (Bandodkar et al. 2016). As
shown by a set up used for gas-sensing measurements using chemiresistor/FET in
Fig. 4.4, sensor material is used between two electrodes. For chemiresistor/FET nano
sensor configuration, carbon nanomaterials have been observed to be advantageous
as the functional channel. Both carbon nanotubes having tubular and graphene with
planar geometry uses maximum exposure of surface atoms to bind the target analyte
molecules with the material of electrode. Label-free sensing of analytes with rela-
tively high sensitivities can be carried out using carbon nanostructures (Nehra et al.
2019)
4.3.2 Development of Sensor Materials and Technologies

for Gases/Vapours
In agriculture, moisture content as well as the main gases required to be detected,

monitor and estimated are carbon monoxide (CO), ammonia (NH3 ), oxygen (O2 ),
nitrogen (N2 ) and volatile organic compounds (VOC). The various type of sensor
materials and technologies, developed for these gases/vapours are:
60 R. Kumar et al.
4.3.2.1 Humidity Sensors
Humidity is the amount of water vapour in the air. Measurement for air humidity is
carried out by the ratio of partial water vapour pressure to saturation vapour pres-
sure and known as relative humidity (RH). Relative humidity shows the probability
of occurring fog, dew, rain and precipitation. Most of the crops depend upon these
natural factors for water. Therefore, sensitive, and accurate humidity sensors are
needed (Srbinovska et al. 2015). As water is the most significant resource in agri-
culture, irrigation management systems should be installed with more accurate and
reliable sensors to get ordered updates regarding moisture in soil at the roots of crops.
Widespread techniques of detections are not capable of providing profiles of
accurate temperature and moisture in soil. They are also cumbersome and expen-
sive. Recent advancements in sensor technology have led to the development of
affordable, low power, multifunctional sensor nodes. Sensor nodes enable environ-
ment sensing together with data processing (Imam et al. 2015). Instrumented with
a variety of sensors, such as temperature, humidity and volatile organic (VOCs)
compound detection, allow monitoring of different environments. They can network
with other sensor systems and exchange data with external users (Ruiz-Garcia et al.
2009).
Electric humidity sensors are divided into three groupings based on their technique
for sensing:
1. Humidity sensor based on Thermal conductivity: This sensor utilizes two metallic
resistors, out of which one sealed in the closed chamber and other kept in open
environment. Using same electrical biasing, the resistors are heated. The resistor
which is exposed cools by the humidity of environment. Therefore, in this case,
thermal conductivity is a function of humidity. Thermal conductivity difference
of two resistors is measure of humidity (Sohraby et al. 2007).
2. Humidity sensor based on resistivity: This sensor utilizes conductive film (may
be thin or thick). This film is coated by polymers like polyvinyl alcohol (PVA).
Here, number of movable ions move through the film are dependent on humidity
of atmosphere. So, the change in impedance depends on relative humidity (Okcan
and Akin 2007).
3. Humidity sensor based on capacitance: In this sensor, a capacitor having printed
electrodes on polyimide thin films is used. Film’s electric constant is related with
humidity (Farahani et al. 2014).
Chang and colleagues developed a new type of antenna with an RH (relative humidity)
sensing function using a modified polyimide for passive Radio Frequency Identifi-
cation (RFID) sensing (Chang et al. 2007). Designed to operate at a frequency that
depends on the relative humidity level, the proposed antenna is a passive device that
physically and functionally combines an antenna with an RH sensor. The compact-
ness and cost-efficiency of the antenna enables it to realize a passive tag of the RFID
sensing without an additional sensor component (Pelegrí-Sebastiá et al. 2011).
4.3.2.2 Carbon Monooxide Gas Sensor
Carbon monoxide (CO), a gaseous second messenger in animals, arises in biological

systems mainly during the oxidative catabolism of heme by the heme oxygenase (HO)
enzymes (Raimundo Jr and Narayanaswamy 2001). Resolution of the response of CO
fluxes to land use is essential since changes in the CO budget due to changes in land
use could either ameliorate or exacerbate the well-known, greenhouse-reinforcing
impacts of agriculture on methane and nitrogen oxides (King 2000). CO dynamics
may play a significant role in strategies to mitigate greenhouse warming since CO
affects the fate of methane (King 2000). Net CO consumption for a given soil type
increased with decreasing organic matter content associated with forest to agriculture
transitions in land use. Although interactions among soil organic matter and various
microbiological, physical and chemical parameters in soils are complex, changes in
organic matter at the sites described here appear to affect net CO fluxes primarily by
reducing the relative rate of a biological CO production (King 2000).
Semiconducting metal-oxide sensors have been studied using metal oxides like
SnO2 , ZnO, TiO2 , MoO3 and Fe2 O3 to produce sensors with low cost, highly sensitive
and fast response time (Menil et al. 2000; Seal and Shukla 2002; Yamazoe and Miura
1995). ZnO is an interesting chemically and thermally stable n-type semiconductor
with exciton binding energy and bandgap energy of 60 meV 3.37 eV, respectively, at
room temperature. With these properties, ZnO nanowire in presence of noble metal
elements (Pt, Pd, Au, etc.) on the surface of metal oxides can enhance the interaction
of reducing gas with the absorbed oxygen on the surface (Menil et al. 2000). Recently
our team had developed the Cu-doped OMS-2 and Nb-doped OMS-2 nanofibers for
the detection of CO gas (Kumar et al. 2018, 2019). Table 4.1 list the response of
some nanosensor material and their operating temperature studied for the sensing of
different concentration of CO gas.
Table 4.1 List of some nanomaterials used for CO gas sensing

CO -Sensors Operating Response time, Response (%) Reference
temperature (K) gas concentration
SnO2 nanosheets 673 6 s, 500 ppm 1.5 Li et al. (2019),
Moon et al. (2008)
Stabilized 873 10 s, 100 ppm 90% Liu et al. (2015)
Zirconia
Au-decorated 523–673 30 s, 10 ppm 90% Qian et al. (2006)
SnO2 nanobelts
Pd-decorated 300 50 s, 100 ppm 85% Lai and Chen
In2 O3 (2012)
Cu-doped OMS2 296 60 s, 100 ppm 20% Kumar et al. (2018)
ZnO nanowires 593 100 s, 500 ppm 60% Choi and Kim
(2012)
62 R. Kumar et al.
4.3.2.3 Ammonia Gas Sensors
Ammonia pollution also impacts species composition through soil acidification,

direct toxic damage to leaves and by altering the susceptibility of plants to frost,
drought and pathogens (including insect pests and invasive species) (Guthrie et al.
2018). The volatilization of ammonia (NH3 ) from livestock manure has been the
focus of many studies because it is a loss of nitrogen for crop production and can
also have an adverse impact on the environment. Above threshold levels, NH3 can
damage vegetation. Ammonia affects freshwater ecosystems through direct agricul-
tural run-off leading to eutrophication (accumulation of nutrients, leading to algal
growth and oxygen depletion) and also has toxic effects on aquatic animals that often
have thin and permeable skin surfaces (Guthrie et al. 2018).
Ammonia gas sensor based on chemically reduced graphene oxide (rGO) sheets
by self-assembly technique to create conductive networks between parallel Au elec-
trodes was studied (Wang et al. 2014). This new gas sensor showed excellent respon-
sive repeatability to NH3 and possess low-cost portable characteristics. Zhang and
co-workers observed that upon exposure to NH3 , the conductance of the semicon-
ducting SWNT changed dramatically (Zhang et al. 2006). An increase in the conduc-
tance by three orders of magnitude was observed within several seconds. The change
is irreversible (Kong et al. 2000). When polyethyleneimine (PEI), Nafion (a poly-
meric perfluorinated sulfonic acid ionomer) is used for sensor studies with SWNTs
for NO2 and NH3 sensing up to 100 ppm shows reversible response with response
time of 2 min (Qi et al. 2003). Au, Pt MWNTs based sensors also studied for NH3
sensing (Penza et al. 2007). Renganathan and his team proposed a nanocrystalline
ZnO coated modified fibre optic sensor for NH3 gas detection (Renganathan et al.
2011).
Ammonia gas sensors using metal oxides such as SnO2 , ZnO and TiO2 are also
reported. These sensors are easily to construct and shows better sensitivity towards
NH3 (Karunagaran et al. 2007; Patil and Patil 2007; Renganathan et al. 2010; Van
Hieu et al. 2008; Wagh et al. 2006; Wang et al. 2001). Resistance of these sensor
changes with the exposure to a particular gas and the sensors are described as elec-
trical resistive type. But high sensitivity of these sensors works only at higher temper-
atures (>200 °C) (de Lacy Costello et al. 2008; Moon et al. 2004; Pourfayaz et al.
2005; Tang et al. 2006). Selectivity of these sensors is also a problem as they respond
to many gases such as carbon monoxides, methanol, ammonia and methane (Wagh
et al. 2006). Doping (Epifani et al. 2008; Esfandyarpour et al. 2004; Ruiz et al. 2005;
Srivastava and Jain 2007), or variation in size (Lee and Park 2004; Nunes et al. 1999)
or annealing (Dikovska et al. 2010; Hongsith et al. 2008; Vaezi and Sadrnezhaad
2007) of these sensors changes their physical properties so that they can work at
lower temperatures with improved the gas sensitivity and selectivity.
Most frequently used detectors, for ammonia gas use conducting polymer, metal-
oxide. Polymer ammonia sensors use thin film coating of polyaniline (Imamura
and Yumoto 2008; Kohl 2001; Timmer et al. 2005) Nafion (Raimundo and
Narayanaswamy 2001) and polypyrrole (Lähdesmäki et al. 1996). The lowest
ammonia detection limit found in literature is 1 ppm and response time is ~1 min
(Coutinho et al. 2007). In conducting polymer, gas detection involves an irreversible
reaction between ammonia and the polymer. The irreversible nature of the reaction
deteriorates the sensitivity over time when exposed to ammonia. These sensors have
poor selectivity, sensitivity and are generally slower. Further, in catalytic ammonia
sensors the charge carrier concentration in catalytic metal is altered by change in
concentration of the gas of influence. This change in charge carries can be quantified
using a field effect device. The selectivity of catalytic ammonia sensors varies with
the variation of catalytic metal used in device and operating temperature.
Our group used various nanomaterials such as Sn nanoparticles (Kumar et al.
2016), metal oxides modified graphene (Jaiswal et al. 2020; Kumar et al. 2017a) and
metal doped OMS-2 (Kumar et al. 2017b) for the sensing of ppm level of ammonia.
Chemisorption based, MOS sensors are an economical type of gas sensors that are
stable during long time exposure to NH3 gas. These sensors can detect ammonia
from small concentrations (<30 ppm) to very high concentrations (27% of air).
However, for sensing the gas in correct range, calibration of attenuated output
signal must be carried out in the desired range. But this reduces accuracy and resolu-
tion at low concentrations. Another concern with chemisorption sensors is the hassle
in interpretation of readings, the possible incorrect alarms, and effect of humidity on
the sensor. With the variation of humidity, the sensor characteristics, and the output
changes, it increases with an increase in humidity and decreases with the decrease
in humidity. Even it can fall to null due to the presence of measured contaminants.
Lifespan of the sensor is directly related to its exposure to NH3 . So, these are useful
only in low concentration measurement applications. Table 4.2 lists the response
of some sensor materials and their operating temperature studied for the sensing of
different concentration of NH3 gas.
Table 4.2 List of some graphene-based sensor materials studied for the sensing of NH3 gas
Sensor Response Operating Response Recovery Reference
(%) temperature (Sec) (sec)
SnO2 -graphene 7 15 <60 <60 Feng et al.
(2017)
Chemically rGO 14 27 ~500 125 Wang et al.
sheets on Au (2014)
electrodes
RGO-SnO2 9 27 ~60 180 Feng et al.
10:8 (2017)
SnO-graphene 21 27 15 30 Kumar et al.
(2017a)
SnO2 /MWCNTs 20 27 100 210 Van Hieu et al.
(2008)
64 R. Kumar et al.
4.3.2.4 Carbon Dioxide Sensors
Commonly, CO2 gas is ejected into the food package as the excessive level of CO2
levels in the headspace leads to reduction of the rates of metabolism, even during
the existence of O2 . The reduction of carbon dioxide gas levels from the original in
the headspace symbolizes the sign of leakage in the package. Besides this headspace
CO2 level may also help in spot ripeness or spoilage of agricultural products. As there
is a continuous generation of CO2 in the headspace due to growth of microorganisms
and any other biological activity (Meng et al. 2014)
Severing-Haus reported the electrochemical principle based first carbon dioxide
gas sensor. Using potentiometric sensors for gas detection, the potential gradient is
calculated by using a reference and working electrodes (Holzinger et al. 1997). A
glass pH-electrode is used to determine the pH of the thin electrolyte layer, which in
last depends upon the amount of CO2 diffused through the permeable membrane (e.g.
rubber, Teflon or polyethylene). At last, the partial pressure of the gas is indirectly
helping to determine the pH of the potentiometric sensor.
For ideal optical detection of carbon dioxide, the indicators are prepared by
pigments (commercially available) (Lv et al. 2014; Schutting et al. 2013)
Von Bültzingslöwen and colleagues had developed an optical sol–gel-based CO2
sensor strip using a fluorescent pH indicator. The sensor membrane comprises a
ruthenium dye, filled in polymer nanobeads, and utilizing the ratiometric method,
the fluorescence intensity is measured using the analyte. This sensor strip can be
located inside a food package or can be incorporated by a handheld scanning device
from the outside (von Bültzingslöwen et al. 2002).
4.3.2.5 Oxygen Sensors
The concentration of O2 of the headspace impacts the shelf life and quality of agro
related products which weakens by excessive O2 quantity. Thus, O2 gas sensors
are used in agro-industry to show the presence of O2 and measure the gas into the
dissolved state (Meng et al. 2014).
As plants are immobile organisms; thus, for essential nutrients supply, plants
depend on the direct environment. In the night, photosynthetic process of the plants
is inactive, therefore, plants including their non green tissue like roots depends on
environmental oxygen. Additionally, due to unavailability of system of O2 distribu-
tion, plants use tissue for O2 diffusion. As consumption rate of oxygen increases in
comparison to the imparting environment O2 , the internal oxygen concentration of
plant may reduce lower than ambient. According to investigation, the O2 gas amount
of cells in the tissue of plant can withstand 5 times lesser concentration of O2 to the
surface of the plant (Licausi et al. 2011; van Dongen et al. 2003). The root system can
be damaged by deficiency of O2 , which restrains nutriment and ultimately growth of
the plant. O2 is a main metabolite in plants. Its deficiency induces rearrangements in
metabolic and morphology of plants (Miura et al. 2011).
With the recent development of oxygen sensors, it has enhanced the understanding
of oxygen distribution and consumption within the plant tissue. Still, the quantifica-
tion of oxygen in the tissue of plant encircles difficulties that don’t happen with the
animal cells (Ast et al. 2012). Organometallic compounds have been widely used in
dye structure due to their extended lifetime and intense luminescence makes them
a best-suited candidate for oxygen indicator dyes in oxygen nanosensors (Ast et al.
2012). For carrying out the intracellular studies for various analytes including O2 in
macrophages, Clark and colleagues have developed sensors (Clark et al. 1998).
Gases such as oxygen, chlorine and nitrogen dioxide exhibit an intense paramag-
netic effect (Kocache 1986). The gases which are attracted by the magnetic field are
commonly known as paramagnetic gases. The basic principle of the thermomagnetic
oxygen sensor is magnetic susceptibility. The molecular oxygen is forced to flow
through an intense and heterogeneous magnetic field of the sensor, the heating of
wire is used for increasing the temperature. This leads to decline of magnetic field
(Uetake et al. 2000). Oxygen sensing by TiO2 , Nb2 O5 , and CeO2 semiconductor like
uses the change in resistance. The change of oxide semiconductor through the redox
reaction occurs because of partial pressure of oxygen in the surroundings (Takeuchi
and Igarashi 1988).
Membrane oxygen electrodes, also known as Clark electrodes, is the most
commonly known oxygen electrodes. Comprising Ag as an anode, Pt as cathode and
a circular ring is used as a reference electrode. Polytetrafluoroethylene/polyethylene
film with a thickness of 15–25 µm is regarded as the most suitable permeable
membrane for O2 , it also covers electrodes surface. KCl acts as a supporting elec-
trolyte. This electrolyte helps in the formation of the permeable membrane for O2
and impenetrable for all others (Nei and Compton 1996). Tracking of the dynamic
change is possible with continuous measurement (Koudelka 1986). Generally, optical
sensors for O2 provides luminescence’s intensity change from a probe molecule and
show the change in concentration of O2 . Since molecular O2 can reversibly quench
the luminescence, this is a reversible luminescence effect (Coutinho et al. 2007).
4.3.2.6 Nitrogen Sensors
Nitrogen (N) element is considered as the most important most concerning nutrient
element for maintaining healthy environment for the growing crops (Lee and Searcy
2000). Its accurate assessment in plants is a key to nutrient management for the
crop. N2 O is produced and consumed by the soil microbes during nitrification and
denitrification (Williams et al. 1992). The interplay between the amount of nitrogen
cycling in soils and soil environmental conditions (mainly soil moisture, tempera-
ture, pH, oxygen and organic carbon concentration) governs microbial processes and
thereby gaseous nitrogen production and consumption (Weitz et al. 2001). The reac-
tive nitrogen escaping from agricultural soils, including groundwater contamination,
eutrophication of freshwater and estuarine ecosystems, tropospheric pollution due
to nitrogen oxides and NH3 emissions, and accumulation of the greenhouse gas and
66 R. Kumar et al.
stratospheric ozone depleting substance, nitrous oxide (Aber et al. 2003; Galloway
et al. 2008; Reay et al. 2012) are the unintended adverse environmental and human
health impact.
Emission growth in N2 O is also enhanced by the widespread use of nitrogenous
fertilizers and manure. Need for more food production as consequence of increasing
population, both agricultural land area and N2 O emissions are likely to continue
to rise (Reay et al. 2012). These facts indicates that, lack of nitrogen means lower
crop productivity, poor human nutrition and soil degradation (Sanchez and Swami-
nathan 2005), but excess N leads to environmental pollution and its concomitant
threats to agricultural productivity, food security, ecosystem health, human health and
economic prosperity (Zhang et al. 2015). So, to assess nitrogen stress, the requisite
is to implement a real-time nitrogen sensor system.
4.3.2.7 Sensors for Volatile Organic Compounds
Plants and trees normally release volatile organic compounds (VOCs) as a by-product
of everyday physiological processes. VOCs released by the plant are signature of
crop and filed condition (Li et al. 2010). This emission become higher in amount
and variety when plants are attacked by pests and diseases than non- attacked plants
(Dudareva et al. 2006). The emission of these VOCs is not limited to the sites of
injury, but also from elsewhere on the plants (Dudareva et al. 2006). Moreover,
the VOC profiles emitted by plants during attack display a degree of specificity
corresponding to the type of attackers. Further these profiles are markedly distinct
from each other. This change in emission of VOCs can help determine the spread of
disease in plants. The VOCs released by plants contribute around two-third of total
emission of VOCs in environment (Guenther 1997). The range of VOCs released due
to biotic and abiotic interactions were compiled earlier reports. Commonly found
secondary plant volatiles are fatty acids, phenylpropanoids, amino acid volatiles and
benzenoids (Dudareva et al. 2006).
The electronic nose is a highly complex device consisting of an array of various
chemical sensors that are coupled to a state-of-the-art pattern-recognition program
that emulates the human olfactory system. The individual sensors which have a
defined electrically conductive adsorptive surfaces for VOC detection that, on inter-
acting with volatile chemicals, display a change of electrical resistance. Even though
each individual sensor responds selectively to a certain group of chemicals, they
all show an overlapping response pattern called cross-selectivity. For each complex
aroma, the array of sensors produces a unique response pattern called a “fingerprint”
(Pinheiro et al. 2002).
VOC bouquets emitted by cucumber, pepper and tomato leaves subjected to
mechanical damage or pest and disease attacks compared with undamaged control
leaves. e-nose can discriminate among VOCs from undamaged leaves. It can also
discriminate undamaged and artificially damaged leaves of the same plant species. In
cucumber, the e-nose can discriminate among VOCs emitted from control, artificially
damaged, and spider-mite-infested leaves (Laothawornkitkul et al. 2008). In another
study, a conducting polymer sensor-based electronic nose was applied successfully to
detect the presence and differentiate among three postharvest diseases in blueberries
(Li et al. 2010).
Electronic nose (e-nose), also known as artificial olfactory, is a simulation of
biological functions to identify some simple or complex odour (Gardner and Bartlett
1994). The electronic nose has derived its name because it in several aspects tries to
resemble the human nose. The electronic nose is a device that detects the smell more
effectively then the human sense of smell. A typical e-nose system contains a selective
chemical sensor array, a signal processing and a pattern recognition subsystem is
shown in Fig. 4.5. The sensors in the sensor array are sensitive to different substances,
therefore an e-nose can extract the whole information of substances for identification
(Wang and Li 2010).
E-nose is described as an array of chemical gas sensors with a broad and partly
overlapping selectivity for measurement of volatile compounds within the headspace
over a sample combined with computerized multivariate statistical data processing
tools. An electronic nose consists of a mechanism for chemical detection. The elec-
tronic nose is an intelligent sensing device that uses an array of gas sensors which
Fig. 4.5 E-nose system contains a selective chemical sensor array, a signal processing, and a pattern
recognition subsystem
68 R. Kumar et al.
are overlapping selectively along with a pattern reorganization component. The elec-
tronic nose is primarily composed of gas sensor array, signal pre-processing, and
pattern recognition. Gas molecules are presented in front of a sensor of an active
material that converts the chemical input into an electrical signal. The response of a
plurality of sensors to an odour constitutes the response spectrum of the sensor array
to the odour. To achieve qualitative or quantitative analysis of the odour, the sensor
signal must be properly pre-treated and processed by appropriate pattern recognition
analysis (O’Farrell et al. 2007). The electronic nose is used to analyse and evaluate
the odour quality according to the principle that each of the gas sensors has a response
to complex component gases but different from each other.
Emission of ethylene increases with tissue senescing and ripening of fruits. A
hybrid electronic nose was developed to detect the onset of biotic and abiotic stresses
(Baratto et al. 2005). During the ripening of apples, trans-2-hexenal can serve as an
indicator compound, because its concentration increases significantly. Accordingly,
monitoring the development of this aldehyde enables monitoring of the postharvest
ripening of apples (Herrmann et al. 2002). Henderson and colleagues developed an
e-nose to detect stink bugs presence and to detect the damage in cotton by detecting
VOCs emitted by stink bugs (Henderson et al. 2010).
4.4 Sensors for Meat Industry
Odour is one of the most important parameters for evaluating the freshness of meat.
The characteristics of volatile compounds in each meat product is different therefore
each meat has its own characteristic odour. Studies show that the biogenic amines,
such as tyramine, tryptamine, putrescine, cadaverine, are significantly related to
traditional quality indices (total bacterial counts, pH, and TVBN1) in meat products
(Xiao et al. 2014). Sensing the gaseous metabolites can give information on the status
of the stored meat products. This provide the platform of developing electronic nose
sensing systems, which provide rapid and non-destructive sensors for meat safety
applications (Shi et al. 2018).
4.4.1 Electronic Nose for Meat Quality
Main factors that are considered by consumers when choosing meat products
are colour and aroma, of which the latter is a more reliable indicator of quality
(Wojnowski et al. 2017). The biotic factors, ambient temperature, humidity, and
transportation are causative factors of meat quality degradation (Nychas et al. 2008).
The spoiled meat products consumption can trigger serious health hazards. So, it is
very important to develop the mechanism to monitor and assess the quality of meat
products (Wijaya et al. 2017). Volatile organic compounds (VOCs) (alcohols, alde-
hydes, ketones, fatty acids, esters, and sulphur compounds) also generated during
meat storage and which can have an olfactory impact as well (Casaburi et al. 2015;
Nychas et al. 2008).
The main applications of e-noses in meat industry are in spoilage detection
of off-flavours, classification, and estimation of shelf life. There are three prin-
cipal mechanisms of meat spoilage, namely microbial spoilage, lipid oxidation and
autolytic enzymatic spoilage (Dave and Ghaly 2011). This can lead to a change
in the composition of the volatile fraction of a meat sample, which in turn can
lead to the identification of potential meat spoilage markers (Nychas et al. 2008).
The main ingredients like carbohydrates, protein and fat will be decomposed by
enzymes and bacteria, producing many volatile organic compounds (VOCs): the
carbohydrates will be decomposed into hydrocarbons, alcohols, ketones, aldehydes
and carboxylic acid gases; the protein will be decomposed into ammonia, hydrogen
sulphide, ethyl mercaptan, etc. the fat will be decomposed into aldehydes and alde-
hyde acids. Total volatile basic nitrogen (TVB-N) content is an important reference
index for evaluating pork freshness. The sensors array is chosen on the basis of
their sensitivity towards the VOCs in pork meat, and they are composed of eleven
different metal-oxide semiconductor (MOS) sensors positioned in a small chamber
(Huang et al. 2014). The E-nose real-time responses were recorded by the ratio of
conductance.
The development of Mobile e-nose for beef quality monitoring and detection has
been studied (Wijaya et al. 2017). The performance of a portable quartz microbalance
based electronic nose has been evaluated in monitoring aerobically packaged beef
fillet spoilage at different storage temperatures. The collected signal responses could
be considered as a volatile fingerprint of an active biological system, containing
information for discrimination of meat samples in sensory classes corresponding to
different spoilage levels (Papadopoulou et al. 2013).
Conducting polymer electronic nose was evaluated for the capability of detecting
the presence of off-flavour malodorous compounds in catfish meat fillets to assess
meat quality for potential merchantability. Sensors responses were measured as a
percentage of electrical resistance changes to current flow in the sensors relative to
baseline resistance (%R/Rbase). The sorption of headspace volatiles, composed of
specific VOC mixtures, to the conducting polymer sensor surfaces induce a change in
the electrical resistance to current flow which is detected by a transducer to produce
the output from the sensor array (Wilson et al. 2013). A study on the use of a
MOS-based electronic nose prototype for spoilage classification of red meat, namely
beef and mutton was carried out. Using the sensor array system, it was possible to
discriminate between spoiled and unspoiled meat with a success rate of 98.87% in
the case of beef, and 96.43% in the case of mutton (El Barbri et al. 2008).
70 R. Kumar et al.
4.4.2 Optical Sensor for Meat Quality Monitoring
An optoelectronic array of sensors is developed for monitoring fresh pork inside

MAP. The array is based on the combination of pH indicators and selective chro-
mogenic reagents supported on inorganic materials with diverse acidities and topolo-
gies. This approach results in a final array containing seven materials as a suitable
system capable of monitoring meat spoilage of fresh pork under MAP atmosphere
via simple colour changes. It is based on 16 pigments prepared by the incorporation
of different dyes (pH indicators, Lewis acids, hydrogen-bonding derivatives, selec-
tive probes and natural dyes) into inorganic materials (UVM-7, silica and alumina).
The colour changes of the sensor array were characteristic of chicken aging in a
modified packaging atmosphere (30% CO2–7 0% N2 ). The chromogenic array data
were processed with qualitative (PCA) and quantitative (PLS) tools (Salinas et al.
2014).
4.4.3 Colorimetric Sensors
The colorimetric sensor array is designed based on two fundamental requirements.

Chemo responsive pigment, which must contain centre (functional group) to interact
strongly with analytes, and then this interaction centre must be strongly coupled
to an intense chromophore. The first requirement implies that the interaction must
not be simple physical adsorption, but rather must involve other, stronger chemical
interactions (Xiao et al. 2014).
For instance, the freshness of beef degrades because microbial spoilage and
biochemical reactions occur during storage. The main ingredients like protein, fat
and carbohydrates are decomposed by enzymes and bacteria, producing odour: the
protein will be decomposed into ammonia, hydrogen sulphide, ethyl mercaptan; the
fat will be decomposed into aldehydes and aldehyde acids; the carbohydrates will be
decomposed into alcohols, ketones, aldehydes and carboxylic acid gases (Smolander
2003).
Chemo responsive pigments are those pigments that change colour in either
reflected or absorbed light, upon changes in their chemical environment. Image
processing is used to assess the meat freshness. Images are taken from meat to
identify regions of interest. Based on the colour of the meat, these regions processed
to produce a RGB colour index (Dini et al. 2010; Zaragozá et al. 2012). One of the
drawbacks of this method is that the relationship between colour processing and the
degree of freshness cannot be determined accurately
The employment of natural pigments in colorimetric sensors is advantageous,
because such sensors do not have chemical effect on the packaged meat. Xiao and
colleagues developed a colorimetric sensor array based on four natural pigments, that
are extracted from spinach, red radish, winter jasmine and black rice for the sensitive
detection of amines generated in pork meat during its gradual abiotic decomposition
(Xiao et al. 2014). Based on the investigation into the structure and properties of
pigment molecules, they discover that black rice extract is the most sensitive of the
extracts. This is due to the better interaction between the carbonyl and hydroxyl
groups of anthocyanin molecules in the black rice extract with the amines generated
during spoilage.
4.4.4 Gas Sensor for Meat Industry
Gas sensors are devices that respond reversibly and quantitatively to the presence of a
gaseous analyte by changing the physical parameters of the sensor and are monitored
by an external device (Kerry et al. 2006). An opto chemical CO2 sensor (Mohebi
and Marquez 2015) which uses a phosphorescent reporter dye and a colorimetric pH
indicator showed robust optical responses to CO2 . The sensor is designed as a film
coating to be applicable in meat packaging. The results show that the sensor is stable
for at least 14 days at 4 °C and 50 days at 20 °C, however, they deteriorate within
7 days by losing colour and sensitivity to CO2 at room temperature. Most of the well-
known gas sensors such as carbon dioxide sensors are susceptible to consumption
of analyte (oxygen) (Kolle et al. 1997). Due to this fact, optical oxygen sensors are
superior to conventional electrochemical sensors, such as metal oxide semiconductor
field effect transistors, organic conducting polymers and piezoelectric crystal sensors.
Oxygen sensors are less complicated than e-noses and easy to be adopted into the
meat packaging industry. These sensors are employed to compare the oxygen content
in MAP and vacuum-packed beef as a non-destructive method to check the impact
of oxygen content on lipid oxidation (Klimant et al. 1997). The oxygen sensors
monitor the changes in oxygen levels in all samples, which have been stored at 4 °C
for 15–35 days. The initial oxygen content of 1.15% in MAP and 0.07% in vacuum
beef samples is reported, which increased to 1.26% in MAP and 0.55% in vacuum
beef samples. The results demonstrate that the samples containing greatest levels of
oxygen are the most oxidized. Moreover, it suggests that a certain level of oxygen
is required for lipid oxidation and the extra oxygen has no effect on further lipid
oxidation. The commercialized version of oxygen sensors is cheap and suitable for
large scale production to be used in each individual meat package.
4.4.5 Meat Quality Control Technologies
As discussed above, various sensors are being used for controlling the quality of
meat. Advantages and disadvantages of these sensors of these technologies are listed
in Table 4.3.
72 R. Kumar et al.
Table 4.3 Meat quality control technologies

Sensor/Indicator Advantages Disadvantages
Time temperature indicator Easy integration into No information about quality
packaging system, cheap and of food, must be conditioned
economical, can be checked by before use
naked eye or measured by
electronic devices
Gas indicator Easy integration into No information about gas
packaging system, cheap and concentration and its chemical
economical, can be checked by dye may interfere in food
naked eye or measured by quality
electronic devices
Freshness indicator Sensitive, can be checked by Not reliable attached inside the
naked eye, cheap and package which may interfere
economical, can be measured in food quality
by electronic devices
Biosensors Easy integration into Cannot detect low concentrated
packaging system, cheap and contamination, may have
economical, can be checked by chemical effect on the food
naked eye or measured by
electronic devices pathogen
and microbial detection
Gas sensors Sensitive, can be integrated Nil
into the packaging films, high
spatial resolution, can be
checked by naked eye and
optical devices not affected by
heat, electromagnetic and
stirring
E (electronic)-nose Accurate since uses an array of Expensive for packaging,
and sensors, can detect many fabrication, and commercial
kinds of volatile compounds, purpose
can be integrated into pattern
recognition and
decision-making systems
Fluorescence-based (oxygen) Sensitive, concentration of can Cannot be checked by naked
be checked by optical device, eye
can be used in headspace or in
liquid, fast and reliable
RFID tags Accurate, can be integrated Expensive
into barcodes, wireless
technology, and fast
4.5 Sensors for Storage and Transport of Food
The agriculture and food industry have witnessed rapid and significant development
in packaging technology in the last several decades, including particularly pack-
aging materials, packaging methods, packaging equipment etc. (Moon et al. 2004).
Nanosensors are far from being simply a passive, information-receiving device. They
can get information from immediate and remote contexts and can analyse, record
and report data (Patel et al. 2020). The part of food production where the need for
the nanosensors is the most visible is food packaging and food transport (Patel et al.
2020). Food packaging prevents sensory exposure from the foods thus consumers
must rely on expiry dates provided by producers based on a set of idealized assump-
tions about the way that the food is stored or transported. If the transport or storage
conditions are violated for any period of time, the quality of food might be deterio-
rated which might not be known to the consumer unless the food package is opened,
or even consumed (Sun and Fung 2006). Nanosensors are able to detect the presence
of gasses, aromas, chemical contaminants, pathogens and even changes in environ-
mental conditions, hence ensure that consumers purchase fresh and tasty products
and reduce the frequency of food-borne illnesses which improve food safety (Patel
et al. 2020).
Due to its small size this type of sensor can also be designed to detect multiple
toxins and microorganisms by putting a series of cantilevers with different molecular
recognition elements together in an array mounted on a single chip (Lange et al.
2002; Ziegler 2004). There are a number of nanosensors being developed that have
applications in food, either for rapid detection or as nano tracers to show the history
of the food product and whether it is of acceptable quality at any given time (Joyner
and Kumar 2015).
Nanotechnology derived food packaging materials are the largest category of
current nanotechnology applications for the food sector. Following main applications
for food contact materials (FCMs):
• FCMs incorporating nanomaterials to improve packaging properties (flexibility,
gas barrier properties, temperature/moisture stability).
• “Active” FCMs that incorporate nanoparticles with antimicrobial or oxygen
scavenging properties.
• “Intelligent” food packaging incorporating nanosensors to monitor and report the
condition of the food.
• Biodegradable polymer–nanomaterial composites (Mabeck and Malliaras 2006).
Due to very large aspect ratios, a relatively low level of nanoparticle is sufficient to
change the properties of packaging materials without significant changes in density,
transparency and processing characteristics (Chaudhry et al. 2008).
For example, nanocomposites have been reported to have improved properties
with regard to durability (higher modulus, tensile strength, and elongation properties,
rephrase) (Lei et al. 2006), temperature resistance, barrier properties (Lei et al. 2006),
74 R. Kumar et al.
optical properties (mechanical, optical: The stiffness and impact strength) (Alexandre
and Dubois 2000), processability due to lower viscosity (Wan et al. 2003).
Electronic noses are thought to emerge as a possibility for aroma profile analysis
of wines and various food items with aroma as significant signature. The electronic
nose consists of an array of gas sensors with different selectivity, a signal-collecting
unit and pattern recognition software. Garcıa and colleagues, studied in same area for
differentiating various types of wines electronic nose based on metal-oxide semicon-
ductor sensors. The development of ultra-low-cost humidity sensors for large-scale
packaging or storage of food and for numerous household applications has created an
urgent demand for electrical-circuit free, consumer goods-integrated sensor systems
(García et al. 2006). Luechinger and colleagues demonstrated the use of ultra-low-
cost carbon-coated copper nanoparticles for the preparation of porous, tensile- and
wetting-agent-loaded thin films for optical humidity sensing. Nanosensors based on
nanoparticles have also been developed to detect the presence of moisture content
inside a food packaging (Luechinger et al. 2007).
4.6 Conclusion
Studies show that nanomaterial-based gas sensors can contribute to crop production
and protection in a big way. They can help in constant checking of parameters such
as moisture content, soil fertility, temperature, crop nutrient capacity, pathogens,
plant diseases, etc. Use of these sensors are found to be very effective in increasing
the healthy crop production. Beside crops, nanomaterial-based gas sensors help in
increasing the quality and quantity of meat production. These sensors also provide
great help in storage and transport applications of food and meat. As a major challenge
for the future, comprehensive studies on using the nano gas sensors in fields will be
important for increasing the crop production for feeding the vast and increasing
population.
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Chapter 5
Volatile Organic Compounds (VOCs)
Sensors for Stress Management in Crops
Vartika Rohatgi, Navakanth Vijay Challagulla,

and Ramesh Namdeo Pudake
Abstract Due to current environmental challenges, crop production all over the
world is in urgent need for new sustainable solutions. Nanotechnology has a poten-
tial to provide answers to various problems associated with productivity and food
security. The agronomics importance of volatile organic compounds (VOCs) from
plant is well established in processes associated with plant defence against the biotic
and abiotic stresses. They have been playing important role in enhanced stress resis-
tance through maintaining reactive oxygen species (ROS) homeostasis, regulator
of plant growth and development, and as a potential antimicrobial agent. Current
limitations associated with its real-time detection in open field is hampering its
better exploitation in future sustainable agriculture. Nano-based sensors are being
developed as emerging devices for early detection of VOCs as plant stress signal
molecules/reporters for real-time monitoring of plant health. This chapter analyses
the current research done in this field for improving our understanding of plant stress
communication. The development of novel tools for precision agriculture through
VOCs sensors is also envisioned.
Keywords Volatile organic compounds · Plant stress · Sensors · Precision

agriculture · Non-invasive diagnostics
5.1 Introduction
Almost every single sector is influenced directly or indirectly by agriculture. The

increasing population has put a lot of pressure on fulfilling agricultural demands. So,
it is very important to meet the challenges faced by the agriculture sector. Moreover,
V. Rohatgi · N. V. Challagulla · R. N. Pudake (B)

Amity Institute of Nanotechnology, Amity University Uttar Pradesh, Noida, UP 201313, India

82 V. Rohatgi et al.
plant diseases and environmental stresses can cause huge economic losses in the
agriculture sector by reducing crop yield (Chakraborty and Newton 2011; Velásquez
et al. 2018). It is very essential to manage and look after plant health with sustainable
methods. New innovating strategies for plant protection should decrease the losses
in food production. Rapid technologies with low cost and less time consuming need
to be developed for plant protection.
Volatile organic compounds (VOCs) are chemical substances produced and
emitted by plants and other organisms in gaseous form. Plants emit these volatile
organic compounds through flowers, leaves, roots, or other tissues with complex
processes. The emission of VOCs is significantly affected by the surrounding plant
atmosphere. Every part of the plant or tissue releases a different kind of volatile
organic compound. Particularly leaves have many specialized cells that produce a
variety of organic compounds. During the process of biosynthetic conversion of
solar energy to chemical energy, multiple organic compounds are produced. Few
of these smaller organic molecules are used to build various important biological
macromolecules like lipids, carbohydrates, proteins, or even nucleic acids. These
macromolecules have generally lower volatile nature due to their massive molec-
ular weight. While there are intermediates of these biochemical processes that form
various metabolites of primary and secondary nature. Then there are few lower than
350 Daltons compounds having functional groups containing non-polar and polar
nature. This polarity also affects the volatility of the compound. Thus, bringing us to
the compounds that have a low boiling point (high vapour pressure) and are easily
vapourized.
These released VOCs from plants can interact with other organisms or neigh-
bouring plants, during signalling and protecting plant from biotic or abiotic stress
that are affecting our crops globally (Fig. 5.1) (Brilli et al. 2019). VOCs released
from plants are involved in different functions like attracting pollinators, defending
against herbivore insects and pathogens, and serve as signals to other plants. These
molecules also help plant in production of defence proteins, metabolites, and promo-
tion of defence related gene expression, thereby improving tolerance to stress. There
are hundreds of these (such) volatile compounds and their compositions that can
be correlated with the biological information of the plant and their processes. Plant
volatile organic compounds consist of isoprene, mono-, sesquiterpenes, cis-jasmone
(CJ), α-pinene, limonene, γ-terpinene. The release of VOCs constitutes a significant
amount of reactive carbon released by plants into the atmosphere. The most common
type of VOCs is terpenes, that is present in resin, aromatic plant tissues like flower.
It helps in communication between plants and insects, and may be attractants or
repellents (Brilli et al. 2019; Maleknia et al. 2007).
As discussed above, the emission rate of plant VOCs largely depends on the
environmental conditions or due to stresses from bio-agents. In this chapter, we have
summarized the roles of the VOCs in plants and the role of nanosensors in their
detection and monitoring of plant health.
5 Volatile Organic Compounds (VOCs) Sensors … 83
Fig. 5.1 Schematics showing the possible role of VOCs in various plant processes ranging from
plant growth regulator, abiotic stress tolerance, inhibition of pathogen growth, herbivore repellence,
and activation of plant defence mechanism. [Reprinted with permission from (Brilli et al. 2019)
Copyright (2019) Brilli, Loreto and Baccelli]
5.2 Role of VOCs in Plant Health
Organisms have a keen way of sensing the environment around them or coordinating
with other organisms that are present in their vicinity. The organisms perform this by
employing series of signalling pathways. These signals allow the organism to have
proper growth, homeostasis, and control over the organism’s behaviour. Most of
the organisms sense environment via recognition of certain molecules that are often
proteins or metabolites secreted by organisms in the vicinity. Since VOCs are known
for their volatile nature and can easily permeate through soil and fluids, therefore,
they serve as the ideal candidate as a signalling molecule for mid-range to long-range
communications for intercellular and for the whole organism (Bednarek et al. 2010;
Bonfante and Anca 2009; Brilli et al. 2019).
Biological VOCs have a few common properties (Schulz and Dickschat 2007).
These VOCs often comprise esters, alcohols, terpenoids, derivatives of fatty acid,
aldehydes, thiols. They show high lipophilicity, and often have high vapor pressure
and low molecular weight. They are easily evaporated at room temperature. It has
been recorded that plants use a high amount of fixed CO2 (20%) just to produce
VOCs (Schulz and Dickschat 2007). VOCs produced by plants may be secondary
or specialized metabolites and are of low molecular weight. They help the plant
by creating an aroma or by releasing necessary chemicals that steers the insects
and pathogens away from the plant, while attracting the beneficial organisms like
the pollinators or the seed dispersers, and also promote symbiosis by attracting the
necessary microorganisms (Fig. 5.1).
Some VOCs also participate in moderating the abiotic stresses such as better resis-
tance to extreme temperatures, high light intensity, droughts, saline soils, or even
some soil pollutants. Ethylene, methyl salicylate, methyl jasmonate (or their deriva-
tive) are few volatile hormones that help in controlling plant’s defence mechanism
(Robert-Seilaniantz et al. 2011). The VOCs signal the adjacent tissues and ultimately
to the whole plant to be more prepared in case they are attacked by pathogens. For
instance, VOCs from tomato leaves inhibit the growth of pathogenic fungi (Zhang
and Chen 2009). A similar effect is seen in lima bean as VOCs from the neighbouring
resistance plants primed the expression of resistance marker genes in the uninfected
plant (Yi et al. 2010).
Also the symbiotic microorganism or plant growth promoting rhizobacteria
(PGPR) release VOCs that are involved in induced systemic resistance against plant
diseases (Han et al. 2006). It has been reported that 2R and 3R-Butanediol from plant-
beneficial rhizobacterium, Pseudomonas chlororaphis O6, increases the tobacco’s
resistance in Erwinia carotovora, but was not effective against P. syringae, pv. Tabaci
(Han et al. 2006). 2R, 3R-butanediol was also found to be effective in improving
drought tolerance in Arabidopsis thaliana (Cho et al. 2008). It has been reported
that PGPR strain Paenibacillus polymyxa E681, increases the growth and induced
systemic resistance of A. thaliana, and the presence of tridecane, a C13 hydro-
carbon was responsible for both (Lee et al. 2012). In another study, it was found that
Burkholderia cepacia (rhizobacteria) produce VOCs that can increase the growth
rate of A. thaliana while the identity of the specific compound is still unknown
(Vespermann et al. 2007). But certain microbes may release compounds that reduce
the productivity or growth of the plants. Few of those microbes include bacteria such
as Pseudomonas, Stenotrophomonas, Serratia, Burkholderia, and Chromobacterium
(Bailly and Weisskopf 2012; Kai and Piechulla 2009). Few bacteria in soil can hinder
the defensive response of plants by using VOC as an effector (Blom et al. 2011).
These findings suggest that plant VOCs have great agronomic potential in devel-
oping some modern and eco-friendly solutions for plant stress management and
increased crop production. The current research in this area is about designing and
validating a VOC sensing platform, and many innovative solutions have been devel-
oped to use VOCs in crop stress management. The sole objective behind the recent
studies is to detect plant volatility that helps in monitoring the plant’s health and
diseases which decreases the agricultural losses.
5.3 Limitations of Current Methods VOCs Detection
A conventional gas chromatography-mass spectrometry (GS-MS) plays a major role

in the analysis of VOCs in plants (Borges et al. 2018; Chang et al. 2014; Cui et al.
2018) that are exposed to various stresses. The characteristic features of analysis of
VOC using MS include detection limits, automated and detailed structural character-
ization of different types of compounds (Maleknia et al. 2007). However, this routine
technique is costly and time consuming, and not suitable for real-time monitoring in
field conditions. So, to take full advantage of the real-time VOC pattern, generated in
plant species for various stress management, there is a need to develop convenient,
easy to operate, real-time detection devices.
The real-time analytical techniques can be used to measure the VOCs to provide
information about the health of plants at any given time. Thus, evaluating and
measuring the VOCs is an engaging avenue as a non-invasive and quick approach in
supervising the structure and physiological health of plants (Materić et al. 2015). It
is possible to collect the sample of plant VOC by in situ approach from whole plant
or organs (like fruits, flowers, fruits) or precisely from the removed or isolated plant
tissues.
VOC patterns emitted by plants are extremely complex and thus it becomes chal-
lenging to directly correlate its production with a definite cause. There are four main
aspects of this analysis that are necessarily and meticulously considered before we
can understand changes in VOC emission pattern and then correlate them to possible
reasons. First and foremost is the recognition of the variations in VOC due to the
biological state of the plant and attributing it to a cause as many factors like biotic
and abiotic stress may cause VOC change at the same time. Also, the age of the
plant parts may also show the differential pattern of VOC release, like older foliage
is expected to be less metabolically active than the young foliage resulting in more
production of VOCs. So, it is very important that we should consider all these aspects
while designing the experiments, so that correlation of VOC production can be accu-
rately correlated to the factor responsible. The second most important aspect is VOC
sampling, as for practical field use purpose the sampling needs to be done through
non-invasive tools. The use of improper sampling techniques can introduce large
errors. Also, a sampling technique can be bias towards certain compounds, which
may or may not be essential in that specific plant system. The third essential aspect
is to select an appropriate analytical chemistry technique used in the VOC study.
The technique chosen should enable the identification of sampled chemical differ-
ences. It helps in identifying the biomarker VOCs of interest. Finally, the information
collected during the analysis needs to be processed, interpreted, and correlated to
the set of conditions being studied through various mathematical models/algorithms

(Aksenov et al. 2013). If the issues discussed above are not addressed suitably may
lead to negative results or some cause may be ignored during the establishment
cause–effect relationship between VOCs and stress.
5.4 Nanosensors for Detection of Plant VOCs
For the site-specific crop management needs the knowledge of variability in various
field attributes like soil and plant characteristics. The agriculture industry in many
countries is now heavily dependent on sensor technology that can provide precise data
for crop health. Nanotechnology has the potential to provide a promising solution
for existing problems in agriculture. The nanosensors can provide high specificity
and selectivity to the components that are related to plant protection. It is a rapid
technology with low cost and less time-consuming. Agricultural industries have
recognized the benefits of these technologies in determining the presence of specific
gas mixtures or to find the specific source or even to monitor the release of specific
gases during the storage and production period. It helps in monitoring plant health
too. This will ultimately increase productivity and improves the quality of food
production. The sensor technology has evolved into different spectrums which can
help agriculture that ranges from aerial-based remote sensing, environment-based
greenhouse sensor, electrochemical sensor, e-nose (electronic nose) sensors, optical
sensors, and many others (Li et al. 2010). The various nanomaterial-based tools
like e-nose that mimic the functions of the human nose can be used as a real-time
approach to detect VOCs. Other tools like plant wearable and nanoparticle sensors
coupled with data acquisition systems and analytical algorithms are widely being
used in recent decades to monitor plant health. Few of the studies are reviewed in
the following sections of this chapter.
5.4.1 Use of E-Nose for VOCs Analysis
E-nose has been playing a major role in real-time monitoring of crops, as they gather
information on plant’s health, the interaction of plants with animal/insects, pesticide,
and other factors influencing the plants. The different principle and mechanism of
Electronic Aroma Detection (EAD) has led to the development of e-nose sensor
for diverse application in agriculture. Analysis of animal and plant-based VOCs
can be performed on the simple to complex mixture of organic compounds that are
volatilized from living tissues or processed products of plant or animal cells. Most
often the e-nose devices are used to identify the unique mixture of VOCs in the
samples derived from plant, animal, or their products. They are not generally used
to detect the specific compound in the sample. With the help of e-nose, one can find
the age, product consistency, freshness of the fruit, ripeness, quality of the product,
purity, and one can even estimate the shelf life. These are highly beneficial when
evaluating batches of wine, and find which can be considered vintage and which is
recently produced. In wines, it also plays a critical role in determining the uniformity
of the wine, aroma, age, taste, etc., which are important characteristics to decide its
market value (Wilson 2013).
Olfactory senses of human are able to detect food degradation at much later
stages and that time the food is not suitable for eating. This degradation can be due
to fungal or bacterial growth that produces a stale smell as a by-product of their
metabolic activity. Therefore, it needs to be detected in early stage to preserve the
edible nature of the food, or else the nutritional as well as the economic value of the
food deteriorates. E-nose sensors are used to constantly check the quality as well
as the freshness of the agriculture products. E-nose alarms us by letting us know
the deterioration of food and the presence of contaminants near the product that may
spoil it (Wilson 2013). The e-nose can have different sensor platform that contributes
to EAD, and few of the most commonly used are, conducting polymer-based (CP),
complementary metal-oxide-semiconductor (CMOS), metal oxide semiconductor
(MOS), quartz crystal microbalance (QCM), nano-electrochemical, and surface or
bulk acoustic wave-based sensors. The advantages and limitations of these various
e-nose sensor platforms have been reviewed earlier (Wilson and Baietto 2009).
From the literature, it was found that MOS-based sensors are the most used sensors
as they have low production cost, response time is fast, require little time to recovery,
and their reproducibility result is high (de Julián Fernández et al. 2008; Ge et al.
2007; Itoh et al. 2008; Kim 2009; Zhu et al. 2006). But these MOS-based sensors
are not too selective with the types of VOCs with similar structures and so, cannot
be used indoors to monitor air quality. MOS has shown rather poor distinguishing
ability towards pollutants such as benzene from toluene, xylene, or formaldehyde.
By using a mixture of multiple metal oxides-based semiconductor sensors showed
a differential selectivity towards different gases at defined operating conditions (Xu
et al. 2007). This shows that sensor selection should be done appropriately depending
on the conditions in which the sensor has to operate. The other thing to keep in mind
while selecting the sensor is to find the range in which the sensor works and that
whether the sensor shows cross-sensitivity and how much of zero-error the sensor
has, as this may affect the analyte recognition capabilities of it (Xu et al. 2007).
The concept of nano e-nose comes from the olfactory sensors that are present in the
biological organisms and so, they are known to be acting as biomimicking the systems
that are present in nature (Harun et al. 2009). Some of the key important details
captured by e-noses related to plant health are rate of production of certain VOCs
upon catching an infection, or determination of plant physiological processes with
change in conditions of the field, weather/humidity, light, surrounding temperature
(Komaraiah et al. 2004; Su 2008). A multisensor array with 20 different MOS sensor
was used to monitor cell culture of Morinda citrifolia and Nicotiana tabacum that
are cultivated under batch conditions. The data generated was able to predict the
biomass concentration and secondary metabolite quantity in cell culture (Komaraiah
et al. 2004). In another study, VOCs emitted by tomato plant under aphid attack was
measured by e-nose and able to correlate the VOCs generated to the extent of damage
(Cui et al. 2019).
In order to study the possibility of using Metal-OXide (MOX) gas sensors in water
management in tomato and maize, Fabbri and colleagues have analysed in situ data
gathered from portable sensing units, and compared this with meteo-sat data and
relevant information related to irrigations, rainfalls, or pesticide-based treatments in
the farm. The results had confirmed the correlation between the VOCs emissions and
the hydric/metabolic status of the plants (Fabbri et al. 2020).
Several e-nose platforms are readily available that can be used to monitor plant
VOCs as indicators related to stress. By using them one can easily monitor the plant’s
growth and health. An e-nose system, Alpha MOS Fox 3000 was used for the detec-
tion of a Citrus Tristeza Virus (CTV) in Khasi Mandarin Orange with a precision of
95.30% (Hazarika et al. 2020). Wireless sensor networks (WSNs) are an ideal solution
to simultaneously and remotely observing many plants. The total VOCs produced
by European privet (Ligustrum jonandrum) that was kept under different conditions
was monitored by Sensirion device (SGP30) containing metal oxide semiconductor
sensors. The networked nodes confirmed the measurable correlation of VOCs with
environmental conditions that are prevailing near the plant (Catini et al. 2019).
5.4.2 Plant Wearable Sensors for Monitoring Plant Stress
Recent studies are focusing on one of the future applications of nanosensor networks
that can obtain real-time information with a non-invasive method from difficult-to-
reach areas. Due to the small dimensions of nanosensors, they can be embedded
in small gadgets and have the ability to execute auxiliary in-network processing.
These properties open horizons of its applications in the fields of agricultural,
industrial, and biomedical settings; that are not possible by the standard network
of the sensors. Micro-electro-mechanical systems (MEMS) technology is rapidly
advancing in recent days, and the number of implantable and wearable diagnostic
devices based on conductive nanomaterials are being manufactured for monitoring
human health issues. This technology has great potential in long term and on-demand
measuring plant VOC and other biomarkers and can be a new tool for crop health
diagnostics in near future. Here, a flexible sensor that is very thin and lightweight
nanosensors can be directly attached to plant tissues such as leaves for continuous
observation of VOCs that are induced by stresses.
In one study, in situ synthesis of monolithically integrated electronic devices based
on single-walled carbon nanotube (SWCNT) was achieved. This flexible device was
then precisely placed onto various bio surfaces. In one experiment, it was placed
on the leaf surface of lucky bamboo (Dracaena sanderiana cv. Virens) plant to
detect the simulants of sarin nerve agent (Lee et al. 2014). Recently, a vapour-
printing technique was used to prepare a plant tissue mounted polymer electrode
that allowed long-term plant health monitoring using bioimpedance measurements.
Fig. 5.2 Schematic showing the printing of ProDOT electrode and impedance-based detection of
ozone damage in plants. Reprinted with permission from (Kim et al. 2020) Copyright (2020) AAAS
With this plant mounted electrode, they were able to monitor drought and photo-
damage. The monomer 3,4-propylenedioxythiophene (ProDOT) was directly poly-
merized in a specially designed hot wall reactor that was used to print electrode on
14 plant species. Oxidative polymerization mediated by FeCl3 progressed inside the
reactor tube where monomer and oxidant vapours met. They also found that, printed
polymer doesn’t significantly influence the plant growth pattern (Kim et al. 2019).
The same team has also used these polymer patches on the plant leaves to monitor
the ozone damage to grapes and apple plant (Kim et al. 2020) (Fig. 5.2). The plant
wearable sensor technology has the capability to real-time monitoring of plant health
remotely with wireless network (Coppedè et al. 2017; Koman et al. 2017; Nassar
et al. 2018). These studies have not used VOCs to monitor plant health, but the results
will be helpful in designing plant wearable VOC sensors. The smart and portable
electronic devices will be available in future with specificity in detection of plant
molecules due to the development of flexible and biocompatible materials, and low
power requirement.
5.4.3 Nanoparticle-Based Smart Sensors for Detection

of Plant Stress
5.4.3.1 Electrochemical Sensors
As compared to other techniques, there are unique advantages in using electrochem-

ical sensing for VOC profiling. Selectivity for multi-species of VOCs, real-time
monitoring with rapid detection, and portability suitable for field applications are
some of them. They can be used in real field conditions and can be connected wire-
lessly or on some moving vehicles. There are some studies with above said goals to
monitor the key chemicals released by plants during infection or other stresses, and
they are reviewed below.
The gold nanoparticles modified screen-printed carbon electrode (SPCE) was
developed for detecting methyl salicylate in soybean plants (Umasankar and
Ramasamy 2013). Methyl salicylate is a vital plant volatile and is involved in
pathogen and pest attack in soybean. The AuNP–SPCE has shown to be highly stable,
and sensitive towards the methyl salicylate sensing. The electrodes were prepared in
a way so that they can easily detect methyl salicylate even in the existence of other
compounds. This electrochemical sensor was able to detect the hydrolysis of methyl
salicylate and the oxidation of negative species, correlate the characteristic signals to
quantify the volatile present in the soybean pod extract (Umasankar and Ramasamy
2013).
In another study, NPs of metal oxide namely TiO2 and SnO2 with screen-printed
carbon (SP) electrodes have been used to develop a potential device for sensing
p-ethylguaiacol volatiles in strawberry fruits and plants. p-ethylguaiacol is one of
the VOCs released from fruits and plants when it is infected with Phytophthora
cactorum—a pathogenic fungus. The electrochemical analysis with the electrode
developed in this study had shown high sensitivity (174–188 μA cm−2 mM−1 ) and
low detection limit (35–62 nM) for target analyte with high repeatability. The authors
had suggested that their findings support the metal oxides as a cheaper alternative to
expensive gold or platinum electrodes for amperometric sensor applications (Fang
et al. 2014a, b).
In later studies, the same group had developed a bi-enzyme (alcohol oxidase and
horseradish peroxidase) modified CNT sensor for selective and sensitive determina-
tion of methyl salicylate (Fang et al. 2016). On this novel electrode, the hydrolysis of
methyl salicylate produced methanol. Then the two enzymes came into action, where
alcohol oxidase produced formaldehyde while simultaneously reducing oxygen into
H2 O2. This H2 O2 was further reduced to water by horseradish peroxidase, that
resulted in an amperometric signal via direct electron transfer. A rapid method
developed by them can be used for crop disease detection based on VOC production.
5.4.3.2 Optical Sensors
The optical nanosensors have the potential to detect the beginning of stress in plant
with the help of nIR imaging devices. They can be used as new and rapid monitoring
tools for plant health and under stressed growth conditions. H2 O2 and NO signalling
pathways have been reported to play a very important role in plant defence responses
(Giraldo et al. 2015; Lew et al. 2020a). The functionalized SWCNT has been reported
to produce unique optical signals and was used in ratiometric sensing platform to
detect H2 O2 and NO in living plant tissue. Recently new approach had been reported
to detect how plants respond or communicate to stress such as injury, infection, and
light damage by using sensors made of carbon nanotubes (Lew et al. 2020b). This
sensor tries to track the hydrogen peroxide which is used by the plants for repairing
damage and defend themselves from predators. The plants used hydrogen peroxide
for sending signals to leaf cells. This new sensor with SWCNTs is exposed on the
leaves of the plants. The SWCNTs were wrapped in the single stranded (GT)15
oligonucleotides, and it was found to be specific to H2 O2 . The binding of H2 O2 to
functionalized SWCNTs resulted in quenching of the fluorescence intensity, making
it a suitable probe for active sensor. The hydrogen peroxide signals used by this sensor
helped in identifying and tracking light, heat, and wound stress responsive changes
in 6 plant species. The functionalized SWCNTs were infiltered in test plant species,
and the images were taken before and after the stress treatment. Ratiometric sensor
responses were correlated with H2 O2 responses. This advancement in technology
can help the farmers in monitoring crop as the device was species-independent and
capable of real-time, spatial and temporal biochemical measurements in plants (Lew
et al. 2020a, b). Similarly, near-infrared (nIR) fluorescent SWCNTs functionalized
with DNA aptamer that attaches to hemin (HeAptDNA-SWCNT) were designed for
developing an optical sensor for H2 O2 detection (Fig. 5.3). These kinds of optical
nanobiotechnology sensors allow the detection of both environmental and pathogen
related stresses, will increase our understanding of plant physiology, and provide a
sustainable long-term solution in crop management (Wu et al. 2020).
Localized surface plasmon resonance (LSPR) phenomenon has been used as a
valuable tool in biosensor, that can detect the collective electron charge oscillations
Fig. 5.3 In planta monitoring of H2 O2 by HeAptDNA-SWCNT sensor. The binding of analyte

resulted into quenching of nIR fluorescence of HeAptDNA-SWCNT, that was remotely measured
by nIR camera. Reprinted with permission from (Wu et al. 2020) Copyright (2020) American
Chemical Society
at surfaces of NPs of noble metal (Au, Pt) by converting changes in refractive index
(RI) into spectral shifts. In one study, LSPR sensor coated with gold nanoparticles
(AuNPs) doped in molecularly imprinted sol-gel (MISG) used for determination of
plant volatile. The gold nanoparticles provide more specificity, stability, and sensi-
tivity to the LSPR sensor. A five-channel AuNPs@MISG LSPR sensor showed more
effective results as 12.33 times higher than the sensor without AuNPs under optimum
conditions. The size of AuNP was 30 nm and the amount of AuNPs doped in the MISG
was 20 μL. This technology helps in the detection and identification of VOCs which
ultimately provides various benefits in the agricultural sector (Shang et al. 2018).
The results of the above studies have demonstrated that optical sensors combined
with other electronic devices can be used for plant VOC detection and identification,
which may become a useful technology for future agricultural applications.
5.5 Conclusion and Future Perspective
The volatile organic compounds (VOCs) emitted by a plant are a notable and vital
part of the plant’s life cycle and are introspective of the physiological status of the
plant. The release of VOCs may occur as a side effect of continuous metabolic
processes or some chemicals could be emitted for a specific reason, such as defence
or signalling. The detection and identification of these VOCs help in monitoring
and tracking plant health status. The tracking and identification of VOCs will also
help in developing new technologies for the plant betterment. Researchers have
tried to use nanotechnology for reducing agriculture losses by providing various
new technologies like nanosensors. Agriculture and nanotechnology both are always
evolving fields. Scientists are trying to develop new technologies that can be used to
protect crops for better food security and reduce the various agricultural losses. VOCs
detection with nanosensors has the potential to become a future tool in the agriculture
sector for tracking, identification, and monitoring plant health status remotely.
Few of the most anticipated improvements by incorporating nanomaterials will
be in, more specific diagnosis of VOCs and sensitivity, faster detection rate, minia-
turization for cheaper production. More and more VOC sensors will be employed to
monitor the plant’s health. Thus, reducing a large portion of losses for farmers and
agricultural sectors. This technology not only helps in monitoring the plant health
but also provides information about the behaviour and metabolism of plants. It also
gives valuable insight about how plants communicate or interact. Upcoming research
will be more focused on the identification of the new plant volatile which in turn
will help in giving an understanding of the plant’s metabolism and mechanism. More
focus will be there on developing various technologies for sensing different types of
plant stresses which will help us in understanding their behaviour. This will provide
us with more understanding of the pathogen infestation over the crop and allow us
to take actions before the crop is destroyed. In future, it is expected that new sensor
technology will be developed that is capable of tracking various kinds of plants VOCs
with low price and take less time in sensing.
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Chapter 6
Current Trends of Plasmonic
Nanosensors Use in Agriculture
Tahira Qureshi, Deniz Türkmen, and Adil Denizli
Abstract Plasmonic sensors based on the surface plasmon resonance (SPR) have
shown prosperous growth in recent years due to their flexibility in designing it
at smaller scale devices. The sensing techniques predominantly include surface-
enhanced spectroscopic sensors such as surface-enhanced Raman scattering (SERS),
surface-enhanced fluorescence (SEF) and surface-enhanced infrared absorption
(SEIR), and SPR sensors which already have good commercial options. This chapter
overviews and discusses the plasmonic nanosensors advancement and the most rele-
vant applications in the field of agriculture. We focus on the effects that distinguish
plasmonic nanosensors and give them their particular conduct for agrotechnology.
We also evaluated the main applications of plasmonic nanosensors developed within
the last five years for agriculture sector benefit.
Keywords Plasmonic nanosensors · Surface plasmon resonance ·

Surface-enhanced Raman scattering · Agricultural sensors · Nanotechnology in
agriculture
6.1 Introduction
Agriculture sector plays a major role in the ineradicable development and financial
flourishing of a country. In current times with growing populations, the agriculture
sector also requires fast and advance approaches for its productive outcome. The
advance agricultural productivity has some adverse effects on soil quality, water
contamination, hypertrophication of water bodies and imbalance generated in water-
ways consequently disturbing the environment (Bezemer and Headey 2008; Milder
et al. 2014). In recent times, food and nutritional safety measures are monitored
by authorities and published for consumers knowledge and betterment. The modi-
fied agricultural product directly impacts the consumers health, climate changes,
T. Qureshi · D. Türkmen · A. Denizli (B)

Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey

98 T. Qureshi et al.
energy, ecosystem processes, natural resources, etc. Therefore, sustainable agricul-

tural reinforcing helps in the practical approach toward better environment outcomes.
Advancement in agriculture sector for sustainable production and efficient yield is
also required for the improvement of agrochemicals (pesticides, fertilizers, etc.) too
(Hobbs et al. 2008; Pretty 1995). Although, all agrochemicals are to some extent
source of contamination in water bodies or residues on yielded crops. This issue
leads to adverse effect on human health as well as on environment, which needs to
resolve by smart and advance management (Almasri and Kaluarachchi 2004; Burney
and Ramanathan 2014; Smith and Gilbertson 2018). It is sure that for the sustainable
growth of agriculture also needed innovative technologies such as nanotechnology.
This budding technology controls several exclusive electronic associations, plas-
monic and optical properties which are related with the quantum confinement effects,
the alteration of the electronic energy levels that may appear due to the surface area in
relation to volume ratio. The merge of nanotechnology in agrotechnology has offered
an advancement and beginning of new prospects in traditional agriculture practices.
The nanotechnology approach has improved irrigation and fertilizer utilization, and
enhanced food production and processing, packaging and storage. In addition, the
nanotechnology-based sensing gained enormous momentum and provided broad-
spectrum application in the field of agriculture (Kumari and Yadav 2014; Mousavi
and Rezaei 2011; Prasad et al. 2014).
Scientific inventions in agriculture have been the main requirement to ease world-
wide load of population growth, climate change and limited food availability. Sustain-
able agriculture has the power of strengthening a notion targeted at increasing yield of
crops from the same agricultural fields without harmful impacts on environment. The
utilization of nanotechnology approach in agriculture sector is an efficient tool for
sustainable agrotechnology. Nanotechnology is efficiently helping in plant protec-
tion, reduces nutrient deficiency, helps fast pest detection and enriches yields through
improved nutrient management (Poddar et al. 2018; Pudake et al. 2019).
The wide spectrum applications of nanotechnology in agriculture contain (1) soil
efficiency enhancement such as use of nanoclays and nanozeolites which improves
the water-holding ability of soil; (2) crop production enhancement by use of nanopar-
ticles (NPs) in insecticides and pesticides; (3) crop improvement by using nanoma-
terials, which enabled the delivery of genetic material for crop development; (4)
precision farming achieved by utilizing nanosensors for soil analysis, water manage-
ment and transmission, pesticide, nutrient deficiency, nanomagnets for removal of
soil contaminants. The precision farming in agriculture sector has helped in enor-
mous need for rapid, consistent and cost-effective system for monitoring, detection
and analysis of agricultural products. Precision farming applies nanosensors and
nano-based smart delivery systems which help out farmers to employ agricultural
resources such as water, nutrients and agrochemicals to increase crop productivity
with improved fertilization supervision and reducing inputs (Chen and Yada 2011;
Chhipa and Joshi 2016; Ditta 2012; Handford et al. 2014; Sekhon 2014).
Real-time monitoring of crop progress and farm condition such as moisture
content, soil fertility, temperature, crop nutrient capacity, pathogens, plant diseases,
6 Current Trends of Plasmonic Nanosensors Use in Agriculture 99
etc., can be done through the advancement of nanotechnology. Utilization of nanosen-

sors to accurately measure the soil condition (pH, nutrients, residual pesticides and
soil moisture), checking of pathogens helps farmers to utilize inputs more profi-
ciently. The current advancement in plasmon-based nanosensors has significantly
overcome the shortcomings of traditional optical sensors, in terms of sensitivity,
adjustability, photostability and in vivo applicability, by utilizing nanostructured
materials. The aim of this chapter is to summarize the most relevant contributions in
the agriculture sector of nanosensors under the plasmonic system (Rameshaiah et al.
2015; Sanchez-Cortes et al. 2001; Yang et al. 2014; Zeng et al. 2014).
6.2 Plasmonic Nanosensors Employed Techniques

in Agriculture
Plasmonic sensors are availing SPR technique more often, which have shown
prosperous development in recent years. In the agriculture field the sensing tech-
niques predominantly comprise: surface-enhanced spectroscopic sensors such as
surface-enhanced Raman scattering (SERS), surface-enhanced fluorescence (SEF)
and surface-enhanced infrared absorption (SEIR), and SPR sensors.
6.2.1 Surface Plasmon Resonance (SPR)
Plasmonic tools and systems created on bottom-up synthesized or top-down fabri-

cated metal nanostructures can be used to enhance light-matter interactions at the
nanoscale in physical, chemical, and biological systems. Plasmonic is one main area
of nano-optics. Due to its ability to generate nanoscale hot spots which are close to the
size of bioparticles, it has been applied in biosensing mostly with enhanced sensitivity
for refractive index (RI) changes and enhanced light/matter interactions. Plasmonic
systems work under three modes such as propagating surface plasmon polariton (SPP)
modes at extended continuous metal-dielectric interfaces, localized surface plasmon
(LSP) modes at confined nanoscale metal-dielectric interfaces and delocalized Bloch
plasmonic modes (or lattice plasmon modes) in periodic metal-dielectric nanostruc-
tures (Fig. 6.1). In some reports by connecting different modes of plasmonic building
blocks into a composite plasmonic system, it is possible for their associated elemen-
tary plasmonic modes to strongly interact with each other and consequently produce
multiple new hybridized modes at different resonant wavelengths with spatial mode
overlap. From these plasmonic modes LSP is of our interest, which plays a vital role
in the analyte detection or monitoring at the molecular level. LSPR mode is primarily
used for plasmonics assembled on nanomaterials (Homola and Piliarik 2006; Piliarik
and Homola 2009; Simsek 2010).
Fig. 6.1 Basic plasmonic

system modes
Surface plasmon polariton
Plasmonic modes
Localized surface plasmon
Delocalized Bloch plasmonic
LSPR is based on metallic nanoparticles, and the plasmonic nanosensors using it

appeared as a strong tool for biosensing applications. Many detection schemes have
been developed and the field is rapidly growing to incorporate new methodologies and
applications. Propagating SPR is performed on a noble metal thin film, which shows
even the slightest change in refractive index near the metal surface and produces a
resonance angle shift. Hence, SPR sensors have been used practically as biosensors
and chemical sensors in many scientific reports. However, LSPR of a noble metal
nanoparticle (NP) can be utilized as sensors too because of their shift in resonant
wavelength in response to a refractive index change. LSPR sensors basically calculate
an extinction (absorption + scattering) peak shift or change in extinction energy,
it simply works via configuration without a prism which makes it cost-efficient
and appropriate for miniaturize setup. This characteristic helps in real-time and on-
site environmental, food, and medical investigations. LSPR sensors also have some
shortcomings like for bulk refractive index changes its response is less sensitive as
compare to SPR sensors. SPR is sensitive to refractive index changes in the close
vicinity of the NP surface due to a closely confined optical near field (Hong et al.
2012; Sari et al. 2018).
Propagating SPR is performed on a noble metal thin film, which shows even
slightest change in refractive index near the metal surface and produces a resonance
angle shift. Hence, SPR sensors have been used practically as biosensors and chem-
ical sensors in many scientific reports. However, LSPR of a noble metal nanoparticle
(NP) can be utilized as sensors too because of their shift in resonant wavelength in
response to a refractive index change. LSPR sensors basically calculate an extinction
(absorption + scattering) peak shift or change in extinction energy, it simply works
via configuration without a prism which makes it cost-efficient and appropriate for
miniaturize setup. This characteristic helps in real-time and on-site environmental,
food and medical investigations. LSPR sensors also have some shortcomings like
for bulk refractive index changes its response is less sensitive as compare to SPR
sensors. SPR is sensitive to refractive index changes in the close vicinity of the NP
surface due to a closely confined optical near field (Gupta and Kondoh 2007; Sherry
et al. 2005).
Guo and coworkers have reviewed recent improvements for LSPR and described
them in schematic form as follows: (1) refractive index nanosensors, (2) colori-
metric sensing based on plasmon coupling and (3) amplified sensitivity based on
nanoparticle growth (Fig. 6.2). They suggested to impact the local dielectric proper-
ties surrounding plasmonic nanoparticles that are motivated by several factors such
as nanoparticle characteristics, amplification schemes and plasmon coupling. They
also give distinct approaches for plasmonic nanosensors response enhancement with
outcome prediction (Table 6.1) (Guo et al. 2015a).
Fig. 6.2 Illustration of different strategies to improve the sensitivity of plasmonic nanosensors.
Reprinted with permission (Gupta and Kondoh 2007)
Table 6.1 Strategies used for enhancing the sensitivity of plasmonic nanosensors (Reprinted with
permission from Guo et al. 2015a)
Category Strategies for enhancing Comments
sensitivity
Refractive index-based sensing Composition Ag is superior to Au. However,
the high reactivity of Ag
nanoparticles must be considered
Size Small size nanoparticles have
high absorption ratios, while big
size nanoparticles have high
scattering ratios
Shape Anisotropic nanostructures
exhibit higher refractive index
sensitivity than spherical
nanoparticles
Increased molecular mass An antibody or a nanoparticle
labelled antibody could
effectively increase the
molecular mass of a target
3D assembled nanostructures 3D assembly may enhance the
intensity of sensing signal but
the interparticle distance should
be carefully controlled to avoid
peak broadening
Plasmonic-molecular resonance An effective way to detect
coupling small-molecular-weight
chromophores
Sensing with single nanoparticles Single nanoparticle sensing
greatly improves the molecular
detection limit and the spatial
resolution
Sensing based on plasmon Increased aggregate size Enzyme-amplified gold
coupling nanoparticle aggregation may
increase the size of aggregates,
enhancing the detection
sensitivity
Minimized interparticle distance Decreasing the interparticle
distance may greatly enhance
the plasmon coupling so that
effectively improves the
detection sensitivity
To detect the scattering signals of The detection of scattering signal
plasmon coupling offers greatly enhanced detection
sensitivity compared with
detection based on absorbance
Sensing based on nanoparticle Scanometric array detection The silver enhancement
growth coupled with silver enhancement significantly improves the
detection sensitivity
(continued)
Table 6.1 (continued)

Category Strategies for enhancing Comments
sensitivity
Biocatalytic enlargement of Any compounds involved in the
AuNP biocatalytic reaction can be
detected
6.2.2 Surface-Enhanced Raman Scattering
Raman spectroscopy in early times was considered as weak scattering process, till
the advancement of Surface Enhanced Raman Scattering (SERS) in the early 1970s.
Here, LSPs have coupled with Raman scattered light, which generates the intense
signal in response on the molecular level (Chang 2013). The SERS boosted signal has
resulted due to the Raman signals acquired from a given number of molecules in the
presence of metal nanostructure, in this size and morphology of the nanostructures
play a major role in the signals response of the analyte. The SERS system is competent
to detect and differentiate in a broad range of analytes within a minute time scale and
with negligible interference from environmental contaminants (Meng et al. 2014;
Song et al. 2015; Zhu et al. 2016).
Also, in the study of pathogens, the SERS-based detection for slow growing
bacteria is an effective tool as compared to basic laboratory testing which on average
takes a few weeks for completion of analysis. Although the response obtained by
SERS on quantification of bacteria shows lesser molecular specificity. SERS-based
analysis does not have direct application for pure bacterial culture but for clinical
samples can be detected directly. The multi-disciplinary applications of SERS have
been enhanced via advanced instrumentation of RAMAN along with nanotechnology
innovation makes it a valuable tool (Cheng et al. 2010; Jarvis et al. 2006).
6.2.3 Colorimetric Nanosensors
Plasmonic colorimetric sensors have appeared as a predominant device in chemical

and biological sensing devices as compare to LSPR because of its lack of response in
the visible range. The most prominent plasmonic colorimetric sensor has an efficient
working response at plasmon coupling anticipated by aggregation of nanoparticles.
The colorimetric nanosensors have applied to detect a particular analyte on color
changing, which can be observed by naked eyes or portable optical detectors for
quantitative measurement (Tang and Li 2017; Xiao et al. 2014).
6.2.4 Miscellaneous Coupled Techniques
The sensitivity improvement has been achieved by coupling between plasmonic-

based materials detection of various approaches. For instance, LSPR combined with
SERS allows sensitive molecular identification by overcoming LSPR limitations.
The electromagnetic improvement in LSPR mode of nanoparticles stimulates the
enhanced Raman excitation and emission in SERS mode for sensitive identification
of analyte (Camden et al. 2008; Singh et al. 2009). Although, these multidisciplinary
approaches have broad room for investigating in detail to conclude its characteristics.
There is a need for improvement required to make beneficial research outcomes
at cost-effective and reliable analysis. Enhancing the detection limit of plasmonic
nanosensing facilitating a variety of sample matrices (water, soil, serum, etc.) will
allow the detection of compounds that were previously not detected.
6.3 Application of Plasmonic Nanosensors in Agriculture
Nanotechnology has broadened the unconventional agricultural practices by

producing safer and better quality of agricultural products, additionally has helped
in generating new agricultural products, improved storage options and provided
better treatment options for its directly associated field such as water and soil
quality. Agro-nanotechnology has helped in various domains, like for better yield of
crops with nanomaterial-based fertilizers, pesticides, herbicides, advanced genetic
material agent for transfer, nutrient improvement of soils or crops and sensors for
directly or indirectly linked compounds. To investigate and further improve in agro-
nanotechnology, both prospects are required specific and rapid analysis in these
domains. To achieve accurate, reliable and fast analysis in this area plasmonic
nanosensors are playing a key role. With the minimum requirement of sample
volume, real time analysis and sophistication of nanomaterials improve the outcome.
Various applications of these plasmonic nanosensors have reported, which are shown
in Table 6.2 from 2015 to 2019.
Kubackova and coworkers have reported the detection of the organochlorine pesti-
cides (OPs) aldrin, dieldrin, lindane, and α-endosulfan by using SERS. To overcome
the problem associated with the binding ability of these pesticides, they functional-
ized the metal surfaces with alkyl dithiols. By doing this they have achieved increased
nanoparticle linkages and created interparticle junctions that can act as sensitive hot
spots that were needed for SERS enhancement. Also, they were able to create suitable
nanogaps between the metal nanoparticles, which helped in assembly and detection
of pesticides. In experiments the metal surface was treated with dithiol, which forms
aliphatic chain layers on the metallic surface and the interparticle cavity, this makes
the reported system efficient for OPs detection (Kubackova et al. 2015). Thiram
(tetramethylthiuram disulfide) has an important status among dithiocarbamate (DTC)
pesticide due to its frequent usage in fungal affected crops, vegetables, seeds, etc.
Table 6.2 Plasmonic nanosensors utilized in agriculture (2015–2019)
Nanosensor/Sensor Analyte Linear range Detection limit Analytical Ref.
technique
Au Nanostars Thiram 10−5 –10−10 M 1 × 10−10 M SERS Zhu et al.
(2018)
Au NPs and Ag NPs colloidal suspension (a) aldrin, (a) 0.1 × 10−7 –5 × (a) 45 μg L−1 , SERS Kubackova
(b) endosulfan, 10−7 M (b) 170 μg L−1 et al. (2015)
(c) lindane, and (b) 0.1 × 10−7 –10 × (c) 1028 μg L−1 ,
(d) dieldrin 10−7 M (d) 316 μg L−1
(c) 0.2 × 10−6 –7 ×
10−6 M
(d) 0.1 × 10−7 –10 ×
10−7 M
(a) Au@Ag core–shell NCs Thiram 1 × 10−6 –1 × 10−10 M (a) 100 pM SERS Guo et al.
(b) Au@Ag NBs (b) 80 pM (2015)
Au nanoisland films Thiram 5−250 ppb 20 fM SERS Khlebtsov
et al. (2015)
AgNP/TiO2 /SiO Parathion NA 10−5 M RS Zhang et al.
whispering-gallery (2015)
6 Current Trends of Plasmonic Nanosensors Use in Agriculture
modes
Imprinted film Parathion methyl 10−13 –10−10 mol/L 10−13 mol L−1 SPR Tan et al.
(2015)
Ag-NC@PE (a) Thiram, 10 nM to 1-pM (a) 10 nM SERS Zhou et al.
(b)4- Polychlorinated (b) 1 μM (2016)
biphenyl, (c) 10 nM
(c) Methyl parathion
Ag Nanoslits Imidacloprid 10 ppm–10 ppb 1 ppb SPR Lee et al.
(2016)
(continued)
105
106

technique
AuNPs/DW (a) Thiram 10−4 M–10−8 M 10−7 M SERS Wei et al.
(b) Carbaryl (2016)
L-cys-Ag NPs (a) Cypermethrin 0–100 ppm 100 ppm Colorimetric Kodir et al.
(b) Monosultap sensing (2016)
MIP/PHEMA NPs Atrazine 0.5–15 ng mL−1 0.7134 ng mL−1 SPR Yılmaz et al.
(2017)
Au nanopillars Thiram 10−9 M–10−5 M 62 pg/mL LSPR Rippa et al.
(2017)
3D Thiram 10 ppb–1 ppm 42 nM SERS Liang et al.
MoS2 -NS@Ag-NP/MoS2 -NS@Au-seeds (2017)
Au NPs (a) Parathion-methyl 10−5 –10−10 M (a) 1 × 10−3 M SERS Wang et al.
(b) chlorpyrifos (b) 1 × 10−4 M (2017)
Au NPs Boric acid 0.01–100 mM 0.01 mM LSPR Morsin et al.
(2017)
Ag-Au NPs (a) Ethion, NA (a) 7.5 × 10−4 Colorimetric Dissanayake
(b) Fenthion, (b) 8.3 × 10−4 sensing et al. (2019)
(c) Malathion, (c) 3.6 × 10−3
(d) Parathion (d) 6.3 × 10−3
(e) Paraoxon-ethyl (e) 0
Au nano-chestnuts Thiram 10−4 M–10−12 M 1 × 10−14 M SERS Geng et al.
(2018)
Fe3 O4 @SiO2@ZnO@Ag Thiram 10−5 –10−9 M. 1 × 10−9 M SERS Han et al.
(2019)
AuAg nanochains Thiram 10−3 –10−7 M 1 × 10−7 M SERS Jiao et al.
(2019)
T. Qureshi et al.
(continued)
technique
AuNPs Acetamiprid 0–5.0 μM 0.5 μM Colorimetric Tian et al.
sensing (2016)
* Nanoparticles = NPs; Nanocubes = NCs; Nanocuboids = NBs; PE = Polyethylene; DW = dragonfly wing; L-cysteine = L-cys
6 Current Trends of Plasmonic Nanosensors Use in Agriculture
107
Furthermore, its contamination in edible stuff such as fruits and vegetables is often
reported. Zhu and coworkers detected Thiram from apple peels by SERS technique.
They synthesized gold nano stars via the seed growth method. The linear range was
observed from 10−5 to 10−10 M, and the method detection limit was 1 × 10−10 M.
However, the detection limit of thiram on apple peels was obtained as 0.24 ng/cm2
(Zhu et al. 2018). In another report, thiram was detected by SERS technique via
plasmonic core-shell nanoparticle, with respect to change in dimension of analyte.
They had reported the synthesis of monodisperse Au@Ag nanocubes (NCs) and
Au@Ag nanocuboids (NBs). The detection limits were observed for NCs and NBs
of thiram as 100 pM and 80 pM, respectively. Khlebtsov et al. (2015) used a wet
chemical approach for fabrication of gold nanoisland films (NIFs) to detect thiram
from apple peels. For synthesis, they used a uniform seeding method by using glass
or silicon substrate to achieve manageable growth of gold nanoisland films. The
reported SERS-based NIFs detected thiram from apple peels was found 5 ng/cm2
(Guo et al. 2015b; Khlebtsov et al. 2015).
Parathion is a broad-spectrum organophosphorus insecticide. Parathion is clas-
sified in Restricted Use Pesticide (RUP) due to its toxicity. Zhang et al. (2015)
synthesized twisted tubular AgNP/TiO2/SiO multiple nanomembranes to detect
parathion. They pumped the pesticide parathion solution in the nanocavity by the
capillary effect. The obtained detection limit was 10−5 M parathion. Another study
had reported the development of an imprinted films (MIF) SPR chip for parathion
methyl (PM) detection. The MIF was prepared by thermally initiated polymeriza-
tion reactions (Tan et al. 2015; Zhang et al. 2015). Zhou et al. also detected PM
along with Thiram and 4-polychlorinated biphenyl contamination by SERS by in situ
approach. They synthesized Ag-nanocuboids (NC), which was placed on transparent
polyethylene (PE) film to obtain Ag-NC@PE composite film. The flexible transparent
AgNCs@PE composite film was used to detect thiram, polychlorinated biphenyl and
methyl parathion from orange peels by in situ SERS method (Zhou et al. 2016). Imida-
cloprid is a well-renowned and widely used neonicotinoid insecticide, it is a broad-
spectrum neonicotinoid insecticide. Imidacloprid is required to be detected from
various matrices due to its extensive use. Lee et al. stated a portable sensing platform
for label-free detection of imidacloprid pesticides. In this study, SPR biochips were
used for simultaneous detection of four different concentrations of imidacloprid pesti-
cides. The visual detection limit was found around 1 ppb (Lee et al. 2016). Wei et al.
reported detecting the pesticide residues by using Au nanoparticles/dragonfly wing
(AuNPs/DW) arrays as SERS substrate. They have functionalized SERS substrate for
thiram and carbaryl, and the result has shown that the detection limit of both pesticides
was obtained at 10−7 M (Wei et al. 2016). Kodir et al. have synthesized colorimetric
sensor based on L-cysteine modified silver nanoparticles. In this study, Ag NPs were
modified by L-cysteine (L-cys-AgNPs). The monosultap and cypermethrin were
detected by L-cys-AgNPs substrate (Kodir et al. 2016). Atrazine is widely used in
corn production due to its effectiveness, application flexibility and cost-effectiveness.
After long-term exposure disrupts regular hormone function, initiating birth defects,
reproductive tumors and weight loss in mothers and embryos. Atrazine may cause
skin sensitization and allergy. Yilmaz et al. synthesized affinity-based SPR sensor
Fig. 6.3 Preparation of SPR sensor (Kodir et al. 2016)
system by molecular imprinting. Figure 6.3 shows the schematic representation of

the SPR sensor preparation process. They combined SPR and MIP techniques for
selective sensing and the sensor had long shelf-life too. The LOD and LOQ were
determined as 0.7134 ng mL−1 and 2.378 ng mL−1 , respectively (Yılmaz et al. 2017).
Rippa and group mates analyzed the characteristics of the LSPR of aperiodic
nanostructures with the arrangement in Thue-Morse (ThMo) NPs as a function of
both their shapes and interparticle distances, comparing experimental and numerical
results. They obtained a detection limit of 260 pM (62 pg mL−1 ) of thiram pesti-
cide was achieved using an LSPR nanosensor based on an optimized ThMo pattern
with triangular NPs employing a phase-sensitive setup to increase the figure-of-merit
(FOM) of the sensor (Rippa et al. 2017). In one more report, thiram was detected
by uniform and well-dispersed 3D MoS2-NS@Ag-NP hybrids, which were fabri-
cated by interfacial synthesis via a seeded growth route from MoS2-NS@Au seeds,
which provide well-defined and simple platforms to attain a composite nanostruc-
ture (Liang et al. 2017). Wang et al. proposed sandwich configuration, which was
accomplished by post-assembling a flexible transparent gel tape embedded with plas-
monic nanoparticles onto an Au film decorated with desired analyte molecules. The
main characteristic response obtained for pesticides, indicating that a post-assembly
strategy is an effective approach for on-site detection of pesticide molecules (Wang
et al. 2017). Morsin and the team studied the LSPR response of Au nanoplates to the
presence of boric acid in water has been investigated. This study has shown that the
LSPR of Au nanoplates is sensitive toward the presence of boric acid in concentra-
tions as low as 0.01 mM (Morsin et al. 2017). Dissanayake et al. demonstrate the use
of plasmonic Ag, Au, and bimetallic Ag-Au nanoparticles to detect and distinguish
between organophosphorus (OP) pesticides. All pesticides showed nearly the same
trend in their sedimentation with the plasmonic NPs (Dissanayake et al. 2019). Geng
et al. fabricated gold nanochestnuts (GNCs) as ultra-sensitive SERS substrate for
detecting trace pesticide residues and have been developed based on anodic aluminum
oxide (AAO) templates. Then SERS measurement of thiram indicates a remarkable
SERS-active sensitivity of the as-prepared GNCs with a detection limit of thiram
up to 10−14 M (Geng et al. 2018). Han et al. also detected thiram by synthesized,
Fe3 O4 /SiO2 /ZnO/Ag (FSZA), which consisted of Fe3 O4 core with strong magnetic
field response and an intermediate SiO2 layer as an electronic barrier to keep the
stability of magnetite particles and outer ZnO and Ag as the effective layers for
detecting pesticide (Han et al. 2019). Jiao et al. detected pesticide residues on agri-
cultural products using SERS. They synthesized worm-like AuAg nanochains with
highly interconnected ultrafine (~6.2 nm) bimetallic particles for SERS nanosensor
via laser-assisted strategy. The SERS detection limit of thiram molecules on apple
surfaces was about 10−7 M (Jiao et al. 2019). Tian et al. developed a colorimetric
assay for the detection of acetamiprid. The flanking nucleotides were truncated to
generation a 37-mer and 25-mer aptamers used as recognition elements in the sensing
assays. The sensitivity was enhanced by removing flanking nucleotides outside of the
binding region. This enhancement was attributed to complete dissociation of shorter
target-binding aptamer without excess bases adhered to AuNPs (Tian et al. 2016).
6.4 Summary
In summary, a plasmon nanosensor has basically responded in two manners in accor-

dance with substrates: (1) individual nanoparticles and their random aggregates, or
(2) chip-based two-dimensional (2D) or three-dimensional (3D) nano-array patterns.
The accessibility of fabrication methods along with the invention and knowledge
of plasmonic modes have played role in frequent use of plasmon approach for
research. Innovative approaches of fabrication process have made it easy in compar-
ison to previous conventional photolithography. Although, nano-arrays fabrication
at nano level is cost-efficient and possesses elasticity for adjusting optical charac-
teristics. There are yet technological hurdles for the fabrication of large-area, long-
range-ordered periodic nano-array models. The optical probes like colorimetric have
performed the qualitative detection of pesticide residues, heavy metal and some
hazardous organic molecules in various edible crops, fruits and vegetables by using
plasmonic nanomaterials substrate. With plasmonic nanosensors colorimetric tech-
nique performs sensitive and rapid analysis. Above all plasmonic nanosensors facil-
itate for on-site monitoring of the pollutants, either from agricultural soil, crops or
water assessment. From this point forward, the expansion of various morphological
plasmonic nanosensors will increase the sensitivity and rapid detection of pesticides
residues, and also their delivery mechanisms for enhancement of the yield. Further,
the NPs clustered evenly on the surfaces like papers or chips is broadly emerging
trend too. The progress of plasmonic nanosensors have the potential for inexpensive,
competent, durable, and eco-friendly substrates with the potential in the direction
of onsite applications. Although, to employ agro-nanotechnology has critical aspect
too, the approach of synthesis needs to be improved such as biocompatible and
ecofriendly in nature to gain better outcomes.
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Chapter 7
Relevance of Biosensor in Climate Smart
Organic Agriculture and Their Role
in Environmental Sustainability: What
Has Been Done and What We Need to
Do?
Kingsley Eghonghon Ukhurebor and Charles Oluwaseun Adetunji
Abstract The application of climate smart agriculture (CSA) procedure has been
recognized as a significant part in offering resolutions to the challenging issues of
climate change (CC) and its extenuation for agricultural and environmental sustain-
ability (AES). Moreover, it has been highlighted that several agricultural activities in
several countries could be linked to the high amount of greenhouse gas emissions. The
application of CSA technologies would play a significant role in the aspect of capacity
building, skill and managerial future adoptions as well as intelligent management
of natural resources for AES. Numerous biosensors and biosensing technologies
(BSTs) like nanoparticles (NPs) and polymers along with their enormous benefits
are presently being applied globally for resolving most of the challenging issues in
AES. Nevertheless, it is important to integrate multi-faceted procedures for AES, so
as to effectively apply biosensors that could conceivably be employed for various
applications particularly in the area of CSA for AES. Hence, this chapter provides an
assessment of BSTs and their wide-ranging benefits in CSA along with their role in
AES. The restrictions confronted by some of the projecting BSTs particularly in rela-
tionship with CSA will be emphasized, and as well offer suitable recommendations
for the improvement in AES.
Keywords Sustainability · Food · Environment · Biosensors · Agriculture
K. E. Ukhurebor (B)
Climatic/Environmental/Telecommunication Unit, Department of Physics, Edo University
Iyamho, P.M.B. 04, Auchi 312101, Edo State, Nigeria
C. O. Adetunji
Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of
Microbiology, Edo University Iyamho, P.M.B. 04, Auchi 312101, Edo State, Nigeria
116 K. E. Ukhurebor and C. O. Adetunji
7.1 Introduction
Agriculture is a domineering and steady part for the survival of human life. Engaging
in activities of agricultural has hypothetically and incessantly been of huge assistance
in producing food, as well as the production of several natural resources for both
human consumption and industrial benefits (Garnett 2013; Karimi et al. 2018). The
agricultural segment as described by the Food and Agriculture Organization of the
United Nations (FAO) is faced with several problems such as failures in the market
system and barriers in the trading system, uneven and futile socio-economic strate-
gies, insufficient information, availability of finance and infrastructures, pressure at
a result of an upsurge in the population and insufficiency resources, agronomic prac-
tices unsustainability and dilapidation in the environment, etc. (FAO 2015). These
complications are further challenged by the effects of CC, since agriculture is mainly
dependent on the variables or parameters of weather (Field et al. 2014; FAO 2015;
Akrofi-Atitianti et al. 2018; Ukhurebor and Abiodun 2018). According to Akrofi-
Atitianti et al. (2018), the agricultural segment in Africa and most of the other devel-
oping countries, are the greatest vulnerable segments to these complications that are
resulting from CC.
The problem of food safety and security (FSS) along with the shortage in AES
techniques as well as the problem of CC have great connexion (Karimi et al. 2018).
Therefore, there is a need to lay more emphasis on their synergetic influence. In view
of this, the United Nations Framework Convention on Climate Change (UNFCCC);
as well as the Intergovernmental Panel on Climate Change (IPCC) have continuously
emphasized the significance of AES, as they continually placed great precedence
on AES (Field et al. 2014; FAO 2015). Both Karimi et al. (2018) and Ukhurebor
et al. (2020a) reported that the effects of CC on AES are still insulated with some
reservations and uncertainties. Hence, CC is expected to hostilely affect AES as well
as other aspects of human doings universally. This could be as a result of the resultant
effects of changes in most climatic variables or parameters such as temperature,
rainfall, carbon dioxide pollination, etc. (Ukhurebor and Abiodun 2018; Herndon
and Whiteside 2019; Nwankwo and Ukhurebor 2019; Ukhurebor and Nwankwo
2020; Nwankwo et al. 2020a, b). Accordingly, climate adaptation procedures are
eventually critical for the mitigation and extenuation of these incessant climatic
activities particularly in our atmospheric environment (Ukhurebor and Azi 2019;
Ukhurebor et al. 2019; Ukhurebor and Umukoro 2018).
As reported by Abegunde et al. (2019), CSA has been identified as sustainable
technique that is necessary for resolving both CC mitigation and AES, respec-
tively. According to them the following are ways by which agricultural actions could
contribute suggestively to CC mitigation:
• The prevention of more deforestation and adaptation or/and modification of the
wetlands.
• The strengthening and escalation in the storage of carbon content of the soil and
environment.
7 Relevance of Biosensor in Climate Smart Organic Agriculture … 117
• The decrease and avoidance of present and impending surges in greenhouse gases
(GHGs) emissions.
Hence, genuine efforts should be gear towards the lessening of GHG emissions,
especially from agricultural activities. As reported by Fanen and Olalekan (2014),
some of the utmost significant produce from agriculture have the budding of shading
the gaps between innovative agricultural produces and some of these innovative
produces have the potential for increasing contributions and management of abridged
GHS emission opportunities.
CSA has some exceptional opportunities in handling the issues of food safety,
adaptation, and control determinations (Fanen and Olalekan 2014; Abegunde et al.
2019). Undoubtedly, CSA is a reliable alternative that could be of assistance in the
management of food insecurity problems, which are allegedly been instigated by
CC (Fanen and Olalekan 2014; Abegunde et al. 2019). However, most unindustri-
alized countries have comprehended that some of these perceptions of CSA which
have been recommended as a possible solution for solving the prevailing issues
resulting from CC are by some means not too appropriate in their backgrounds due
to some environmental unpredictability (Fanen and Olalekan 2014; Karimi et al.
2018; Abegunde et al. 2019). In addition, agriculture occupies a fundamental aspect
in the assuagement of poverty and in enacting some core objectionable effects that
CC is anticipated to have on quite a lot of regions universally.
Reportedly, initial achievement in CSA has been recognized as an essential
means of capacity development along with skill and guide for imminent prospects
(Abegunde et al. 2019). Nevertheless, it is advisable to have an appropriate meaning
of “smart system (SS) or smart product” before reconnoitering to CSA practice.
Abegunde et al. (2019) describe a SS as “that which facilitates the interface of
a system with persons/users and is able to acclimate the framework of the user
without compelling the user to acclimate to it”. According to Karimi et al. (2018)
and Abegunde et al. (2019), a SS will possibly encompass the following features:
• Competence or ability to work together with other mechanisms and devices.
• Flexibility in acquiring and improving the compatibility among its operational
environment.
• Self-adequacy, specifies that SS can work without interference from the operator.
• Competence in networking with a person by means of a natural or ordinary
interface.
• Multi-determined, specifies that a sole product of a SS is proficient in imple-
menting several roles.
• Personality, specifies that a SS is capable of dynamic and accomplishing the
structures of reliable performance.
• Reactivity, this specifies that a SS could respond to its surroundings in a distinct
technique.
A SS has been identified as a crucial method in sensing, acting, and performing
optimum operational control for routine or product eminence via what is known
as “smart sensing techniques”. The development of biosensors has significantly

contributed to the improvement of both SS and CSA. Biosensors technology has been
identified as a potential technique that could lead to increase in agricultural efficiency
and the improvement in food production. These techniques could be utilized for
controlling and management of immediate diagnosis of infectious diseases, improve-
ment of plants capacity for nutrients fascination, the enhancement of the capability
for animal production, etc. (Fanen and Olalekan 2014).
In addressing the influence of agricultural actions to these complications of
ensuing from CC as a result of the modifications in the climate scheme, CSA is
increasingly being endorsed in several regions globally, to contribution in the incorpo-
ration of the economy, societal and ecological extents of development sustainability
in the development of the three main aspects; “sustainably increasing agricultural
productivity and revenues; acclimating and building resilience to climate change and;
reducing and/or removing GHGs emissions relative to conventional practices” (FAO
2015).
Moreover, the application of CSA has not yet been completely espoused in several
developing regions, particularly in Africa. It is however alleged that the inadequate
understanding on how to efficiently implement the adaptation procedures faced by
those involved in agricultural actions across these regions are some of the limitations
(Antwi-Agyei et al. 2014; Akrofi-Atitianti et al. 2018; Ukhurebor 2020). Neverthe-
less, several industrialized regions that are alleged to have the sufficient understanding
that has started adopting and applying CSA, still have enormous arrangement to be
put in place for its enhancement and development. Hence, biosensor in CSA are now
been incorporated, and this would absolutely play an important role in AES.
This chapter will therefore attempt to present an appraisal of BSTs for the devel-
opment of CSA drawn from previous studies as well as their role in AES as it relates
to food security and CC, in the area of BST, for the enhancement and development of
the agricultural sector. The constraints and restrictions that are confronted with most
of the projecting procedures particularly as it recounts to CSA will be emphasized. It
is believed that this be of great assistance in proffering beneficial recommendations
for imminent research studies for the improvement and development of AES.
7.2 Benefits of Biosensors in CSA
According to Ukhurebor (2020), biosensors are known as investigative devices

that combined biological ingredients and transducers for the detection of samples
such as medications, metabolites, pollutants, infectious load, control variables,
etc. They accomplish the task via the translation of “biochemical reactions” into
computable physiochemical signals, which then quantify the quantity of sample that
are employed for detecting the analyte concentration (Borgmann et al. 2011; Arora
2013; Vigneshvar et al. 2016; Salek-Maghsoudi et al. 2018). Biosensors have been
reported to have numerous applications in the various aspects of human endeavours
such as processing and safety of food, medical detection, and diagnosis, security and
defence, AES, etc. (El-Said et al. 2020; Ukhurebor 2020).
There are numerous categories of biosensors employed for environmental sciences
as it recounts to water, soil, and air in the area of CSA. These biosensors rely on
detecting transducers or rudiments. Since the first discovering of biosensors which
was known as the glucose one in 1956, by Clark, L.C Jnr, that came into public
interest in 1975 (Turner et al. 1987), there are presently numerous biosensors that
are now established for several commercial tenacities. These present-day biosensors
have a widespread range of benefits that proposes supplementary sensitive, profligate,
specific, perceptible, and multiplicative consequences as against the preceding ones
(Borgmann et al. 2011; Arora 2013; El-Said et al. 2020; Ukhurebor 2020).
At the moment, with the development and improvement of nanotechnology,
advanced NPs are now being developed and their advanced structures as well
as their numerous applications that are present in biosensors (Rodriguez-Mozaz
et al. 2005; El-Said et al. 2020; Ukhurebor 2020). NPs-built biosensors, comprise
the combination of molecular engineering, biotechnology, chemistry, environ-
mental science, physics, and material science. These diverse fields of study have
been of boundless assistance in evolving the comprehension and specificity of
“bioparticle/biomolecule” detection, the capability of sensing or operating particles
(molecules/atoms), analysis and diagnosis of pathogenic, biomolecular recognition,
along with the controlling and management of agricultural and the environmental
actions (Rodriguez-Mozaz et al. 2005; El-Said et al. 2020). The benefits of the innu-
merable biosensors such as NPs, microorganisms built biosensors, and polymers
for environmental and agricultural dealings, have helped in reducing the amount of
substances spread, reduction in the losses of nutritional substances for insemination,
and increase in the yields through pest and nutrient (Antonacci et al. 2016).
Biosensors are generally characterized into two groups which are instituted on
sensing mechanisms and transduction procedures. The detecting/sensing mech-
anisms comprise “enzymes, antibodies (immunosensors), micro-organisms (cell
biosensors), biological tissues and organelles”. Whereas, the transduction approaches
are centered on the physiochemical difference ensuing from the sensing mechanisms
(Ukhurebor 2020). Consequently, unlike transducer biosensors could be either piezo-
electric, electrochemical, calorimetric, or optical (Borgmann et al. 2011; Reyes De
Corcuera and Cavalieri 2003). Reyes De Corcuera and Cavalieri (2003), reported
that some of the utmost categories of piezoelectric transducer biosensors are acoustic
and ultrasonic; some of the utmost categories of electrochemical transducer biosen-
sors are amperometric, conductometric, and potentiometric; whereas, some of the
utmost categories of optical transducer biosensors are absorbance, fluorescence, and
chemiluminescence.
Arora, (2013) also reported that biosensors could be characterized on the basis
of the period or order that they were established. Consequently, in these groupings
we could identify; first-generation biosensors (FGBs), second-generation biosensors
(SGBs), and third-generation biosensors (TGBs).
The first-generation biosensors are the biosensors that are reportedly the modest
procedure concerning the unwavering discovery of either upsurge of an enzymatically
produced product or decline of a substratum of redox enzymes employing ordinary

mediator for transferring particles or electrons. Examples of FGBs are the glucose
biosensor that makes use of the enzyme glucose oxidase and oxygen for detecting a
decline in the range of oxygen or upsurge in hydrogen peroxide conforming to the
range of the glucose. The second-generation biosensors are biosensors which make
use of non-ordinary redox mediators such as ferricyanide, quinones, and ferrocene for
transferring particles or electrons, which upsurges the sensitivity and reproducibility.
Examples of SGBs are self-monitoring amperometric glucose biosensors. The third-
generation biosensors are those that fall wherein the redox enzymes, controlled on
the surface electrode, in a manner that unwavering particles or electrons transference
are imaginable among the transducer and enzyme.
At the moment, in relation to the development, and improvement of nanotech-
nology, state-of-the-art NPs are presently being developed and their advanced struc-
tures along with their benefits occur in biosensors (Rodriguez-Mozaz et al. 2005;
Ukhurebor 2020). Implausible enhancement has been accomplished together with
the expertise involved and the uses of biosensors by means of innovative strategies,
encompassing electrochemistry, nanotechnology, and bioelectronics (Borgmann
et al. 2011; Clark and Lyons 1962; Fracchiolla et al. 2013; Turner 2013; Vigneshvar
et al. 2016; El-Said et al. 2020; Ukhurebor 2020).
The development of biosensors as a dominant and ground-breaking analytical
mechanism (that entail biological identifying constituents with numerous benefits)
has indisputably adopted foremost reputation in several fields of study. Its exploita-
tion has achieved some momentous benefits in the field of biomedicine, pharma-
cology, environmental science, food safety, and dealings (as well as agriculture in
general). The development of biosensors has resulted in the growth and advancement
of precise and dominant analytical mechanisms via biological identifying constituent
as biosensors (Vigneshvar et al. 2016; Salek-Maghsoudi et al. 2018; Ukhurebor
2020).
According to Ukhurebor (2020) and Turner (2013), the methodological proce-
dures that are employed in biosensor mechanisms are based on the label-assembled
and label-unlimited discovery. Label-assembled discovery is mostly hooked on the
obvious structures of label compounds for detection targeting. However, these groups
of biosensors are reliable; but are however consistently encompass in bringing
together obvious sensing mechanisms, invented with the controlled target protein.
Whereas, label-unlimited procedure tolerates the recognition of the target substances
that are not characterized or difficult in tagging (Sang et al. 2015; Citartan et al. 2013;
Ukhurebor 2020). Contemporary interdisciplinary methods of electronics mecha-
nisms and biotechnology brought about the evolution and advancement of label-
unlimited biosensors for numerous discovery procedures with several benefits in
environmental and medical sciences.
Also, BSTs are been applied in AES. According to Ukhurebor (2020) and Verma
and Bhardwaj (2015), this is an additional beneficial aspect in which BST is presently
gaining ground. These BSTs in AES will indisputably be of assistance in the
instantaneous identification of pesticidal deposits that would have resulted in some

averted health menaces in form of CSA (Long et al. 2013a; Fanen and Olalekan 2014;
Marrazza 2014; Verma and Bhardwaj 2015; Verma and Bhardwaj 2015; Antonacci
et al. 2016; Adesina et al. 2017; Neethirajan et al. 2018; Ukhurebor 2020).
Verma and Bhardwaj (2015) reported that the traditional or conventional approach,
such as high-performance liquid chromatography, capillary electrophoresis, and mass
spectrometry are effective in the inquiry of environmental pesticides. Yet, there
are some limitations such as difficulty, time-intense procedures, need of high-end
mechanisms, and operational proficiencies.
In light of all these, even if it is supposed that the modest biosensors have enormous
benefits; yet, it is somehow hard to formulate integrated biosensors which can inves-
tigate numerous groups of pesticides. Therefore, stable enzyme-built biosensors have
been developed for understanding the physiological (such as physical and biolog-
ical) effect ensuing from the use of pesticides in the environment, food safekeeping,
and quality monitoring and management (Marrazza 2014; Ukhurebor 2020; Verma
and Bhardwaj 2015). Pundir and Chauhan (2012) reported that acetylcholinesterase
inhibition-built biosensors that been invented still require some advancement to
effectively deal with these limitations. For quite some time now, in driving for
instantaneous analysis, this technique has received extreme enhancement with
further contemporary developments in acetylcholinesterase inhibition-built biosen-
sors together with immobilization mechanisms and other diverse procedures for their
fabrication (Vigneshvar et al. 2016; Ukhurebor 2020).
Similarly, piezoelectric biosensors have been developed for identifying the
organophosphate and carbamate on the environmental effect of pesticides (Marrazza
2014). According to Senthilkumaran (2015), organochlorine pesticides are known
for disturbing the ecology of pesticides, like Endosulfan which could cause consid-
erable environmental damages. Undoubtedly, organochlorine pesticides have been
described to be one of the utmost causes of modification in the reproductive system
of some fishes (Senthilkumaran 2015). Consequently, the development of biosen-
sors for sensing, detecting, or noticing the aquatic ecology will have further conse-
quences due to biomagnification (Senthilkumaran 2015; Vigneshvar et al. 2016). So,
to deal with these issues, the electrochemical biosensors have qualified revolution
with instantaneous advances in its fabrication and the use of ingredients such as NPs
(Turner 2013; Senthilkumaran 2015; Verma and Bhardwaj 2015).
Hence, it is of enormous importance to place discrete prominence in selecting
or collecting receptors for biosensor improvement, as well as the application of the
various transduction actions and instantaneous screening methods for the use of
biosensor in agricultural events such as the production of food and food safety along
with dealing with environmental issues. To support this, biosensor manufacturing
seems to be dynamic and the enhancements in this aspect have been categorically
explained by numerous scholars and scientists.
7.3 Advantages and Technological Comparison of Some

Biosensors
The different kinds of biosensors have their applications and machinery variance.
Hence, this section will discuss the technological comparison and specificity as well
as the detection extent, linear extent, investigation time, cost, and compactness of
some of these biosensors.
Advancements in electrochemical sensors with substantial approaches concen-
trating on discovery extent, investigation time, and compactness afforded huge-scale
user markets for low-cost biosensors designed for glucose and prenatal period assess-
ments via anti-human chorionic gonadotropin immobilization strip with lateral-flow
technique (Turner 2013; Ukhurebor 2020). According to Vigneshvar et al. (2016), the
control of analytes by means of polymers and nanoparticles/nanomaterials are vital
constituents for the improvement of the sensitivity and detection extent. As a result of
this, lateral-flow technique permits the direct conveyance of samples to anticipated
spots so as to generate precise interactions as an alternative to random interactions.
Some of the above-mentioned biosensors have applied this technique; it has also
given prospects for bio-fabrication by means of either contact or non-contact-built
modeling (Vigneshvar et al. 2016).
The use of NPs such as gold, silver, and silicon-built bio-fabrication produced
innovative procedures. Also, coating/covering of polymers on these NPs brought
enormous advancements in contact-built electrochemical sensing. The foremost
benefit of these categories of electrochemical sensors is sensitivity and specific nature
with the real-time investigation. Though, there are some limitations such as the
recovering capability or long-term procedure of polymers or/and other constituents;
hitherto, the inexpensive nature of such electrochemical sensors make them more
affordable (Vigneshvar et al. 2016).
Single or sole analyte discovery by means of contact-built sensing has great bene-
fits, for instance, real-time quantification of particles with lofty specificity. To support
this, Fluorescence Resonance Energy Transfer (FRET), bioluminescent resonance
energy transmission, fluorescent-built as well as surface plasmon resonance-built
transducers were developed for improving the sensitive and specific nature in terms
of sole or single molecule/particle discovery (Dias et al. 2014; Vigneshvar et al. 2016).
These procedures also have some limitations in terms of multiple analyte discovery
as a result of signal emission intersection/overlap; hitherto, resonance energy trans-
mission procedures habitually established for numerous analyte discoveries, that
are extremely rewarding in medication diagnosis as a result of transformation in
bio-markers between patients and interrelated infections (Vigneshvar et al. 2016).
The application of microcantilevers or nanocantilevers by way of transducers
in electrochemical sensor bio-fabrication is similarly having widespread potential
in numerous analytes discovery (Vigneshvar et al. 2016). Also, non-contact-built
sensors by means of three-dimensional (3D) bioprinting applying inkjet or laser
direct inscription afforded improved consequences. However, the expensive and
customizable capability has great implications in these procedures.
Remarkably, some of these substantial biosensors have been joint with electro-
chemical sensing for precise purposes. Most of the prominent real-time and sensitive,
as well as portable amperometric electrochemical biosensors, have been established
for infection/disease diagnosis by means of the fluid content of the body (body fluids).
Generally, electrochemical biosensors combined with bio-fabrication have truncated
discovery extent for sole or single analyte discovery specific nature by means of
real-time investigation and at an inexpensive price in view of the portable nature of
the device (Kim et al. 2015a; Vigneshvar et al. 2016; El-Said et al. 2020).
Optic-built biosensors are also now leading technology in biosensing that has to
do with fiber-optic chemistry. Single or sole particle/molecule discovery or deten-
tion, such as Deoxyribonucleic acid (DNA) or peptide, is done appropriately by
means of hydrogel-built cross-linking as a result of the benefit of having great
packing capability and hydrophilic nature. More so, optical biosensors which had
widespread applications in biomedical and forensic science for DNA measurement
and quantification was developed (Kwon and Bard 2012). According to Vigneshvar
et al. (2016), the combination of organic or biological constituents, for example,
“enzyme/substrate, antibody/antigen, and nucleic acids”, resulted in a great version
of optical BST. Also, it is now been conceivable to integrate microbes, other living
organism cells, and tissue segments in the biosensing mechanisms.
The advancements in molecular optoelectronics brought forth optical biometric
recognition systems. Combined optics machinery gave room for the integration of
both inert and dynamic optical mechanisms on the similar substrates for evolving
abated or curtailed dense sensing strategies by means of fabrication of numerous
sensors on a sole chip. In this situation, top class polymers brought forth hybrid
systems for optical BSTs. In effect, optic-built BSTs were enhanced as a result
of recent innovations in surface morphology investigation by means of advanced
electron and atomic force microscopy.
Nevertheless, the discovery extent of optical biosensors at no time would have
been near femto-level in view of the instrumentation cost and device non-portability.
As a result of this, recent optic machinery that has to do with nano-mechanical
biosensors built on microcantilevers and surface resonance machinery assisted in
the provision for advanced DNA chips at best for instantaneous precise and delicate
investigation (Scheller et al. 2014; Ukhurebor 2020; Wang et al. 2015; El-Said et al.
2020; Zhang et al. 2015).
Basically, the benefits of optical biosensors comprise of a fast instant investigation
by means of resistance of signal to electrical/magnetic intrusion in addition with the
budding for spectrum of data (Vigneshvar et al. 2016). However, the main disad-
vantage is the expensive nature as a result of certain instrumentation necessities.
Further procedural complications include complexity in immobilization particularly
for bio-fabrication in addition with the need to disparagingly resolved decontaminate
the environment in order to take complete benefit of optical biosensors (Vigneshvar
et al. 2016).
Bio-fabrication of mechanical devices needs improved outcomes for mass-built

biosensors. Irrefutably, electrochemical and optical biosensors together make one
of the furthermost of this machinery for devising higher biosensors (Vigneshvar
et al. 2016; El-Said et al. 2020). According to Arlett et al. (2011), the major devel-
opments in micro-fabrication and nano-fabrication machineries made it possible to
produce mechanical devices with nano-sized stirring fragments. The capability to
produce such devices, by means of semiconductor processing measures are linked
to biophysics and bioengineering moralities in the direction of the advancement
of applied micro-electromechanical and nano-electromechanical biosensors which
were produced in huge quantities (Arlett et al. 2011; Vigneshvar et al. 2016).
Glass, silicon as well as quartz constituents have been reportedly applied
whichever after tagging with fluorescence or gold NPs efficaciously. Although
they have superior precision in terms of sole particle discovery, cost-effectiveness,
multiple productions is hardly achievable (Shen et al. 2014; Peng et al. 2014;
Vigneshvar et al. 2016). Several challenges still are however still noticed in mass-
built sensors especially in aspects of improved capture agents to produce at nanoscale
by means of microelectronic fabrication for top-throughput investigation. As such,
the great application possibilities of semiconductor materials and quantum dot
machineries are worth mentioning (Vigneshvar et al. 2016).
However, there are presently technologies/machineries in biosensors that are
capable of simultaneously executing real-time and quantitative analyzes for bulky
arrays. It is the use of micro-scale and nanoscale cantilevers fabrication that has made
this achievable (Dias et al. 2014; Vigneshvar et al. 2016).
Further vital mechanical uprising in biosensors machinery is the detection of
genetically encoded or synthetic fluorescent biosensors for the analysis of molecular
devices by means of biological or organic procedures (Randriamampita and Lellouch
2014; Oldach and Zhang 2014; Kunzelmann et al. 2014). However, these categories
sensors have great vision for sole or single particle discovery with precise analyte
quantification, the procedure and investigative preparation and discovery are hard,
and similarly need advanced instrumentation (Vigneshvar et al. 2016).
Biomaterials/bioparticles and microbial fuel-built biosensors also have discrep-
ancy as a result of the highly sensitive and selective nature. Yet, multiple productions
and genetic engineering procedures for developing microbial strain, need intricate
measures and are expensive. But, the foremost benefit of microbial biosensors is
the capability to employ them as a means for bioremediation, that have a superior
consequence for agriculture as well as environmental management and monitoring.
Nevertheless, the advancement of such improved genetic strain needs to be sternly
analyzed by means of appropriate governing rules and regulations as well as ethical
clearance and production cost management. Hence, Scheller et al. (2014) reported
that the advancement of diverse micro-biosensor and nano-biosensor frameworks
that as to do with combined technologies that utilize electrochemical or optic bio-
electronic principles through the grouping of bioparticles or biological constituents,
polymers and NPs are needed so as to attain exceedingly delicate and miniaturized
mechanisms.
As the quest and need for applying biosensors for swift investigation with cost-
efficiency, entail bio-fabrication that now pave way for the identification of cellular to
complete instinctive action with a discovery extent of top precision for sole particles.
According to Dias et al. (2014), biosensors ought to be designed to function under
multiplex and complex circumstances. In that condition, both “three-dimensional
(3D) and two-dimensional (2D) discovery” are needed by means of elegant trans-
ducers for detecting and measuring minor analytes of interest. With this in mind,
some breakthrough discoveries have been achieved with contact or non-contact-built
modeling at diverse stages.
We now have biosensors that are developed for discovering additional robust
recovering long-term usage. These diagnostic biosensors are developed by means of
therapeutics, which assist both the medical staff/team and their patients for additional
assimilative consideration of infections and the treatment procedures (El-Said et al.
2020). Hence, Fracchiolla et al. (2013) reported that fluorescence resonance energy
transfer-built biosensor provides an exceptional analytical technique for evaluating
the effectiveness of “imatinib” management and handling of continuing or chronic
myeloid leukemia. The present use of affibodies, aptamers, peptide arrays as well as
imprinted molecular polymers are conventional illustrations for potential research
methods in this aspect (Verma and Bhardwaj 2015; Vigneshvar et al. 2016; Citartan
et al. 2013; El-Said et al. 2020; Abe et al. 2014).
However, little achievement has also been reported with limited possible parti-
cles for advanced therapeutic, antimicrobial, and medication delivery. According to
Grabowska et al. (2014), the development in this aspect resulted in the discovery
of electrochemical biosensors as consistent and dependable diagnostic means for
pathogen discovery of avian influenza virus in intricate conditions. However, Mazzei
et al. (2014) reported that an additional new report showed possible benefits of
affinity-built biosensors in medication for sports and doping monitored investigation.
Also, Bandodkar and Wang (2014), reported that there is now a multiplicity of even
or wearable electrochemical biosensors for instantaneous non-hostile transmission
of electrolytes and metabolites in the fluid content as indicators or determinants of a
wearer’s medical situations. According to Lawal and Adeloju (2012), there are also
additional attractive applications in the assessment of meat and fish value by means of
hypoxanthine biosensors through fabrication. According to Kumar and Rani (2013),
the advancement in biosensors for the discovery of biological or organic warfare
agents such as virus and bacteria as well as contaminants, are now sometimes use
for various means of biosensors such as nucleic acid, electrochemical, piezoelectric
and optical, which now have massive benefits in military (defence, protection, safety
and security) and medical science.
The alliance of NPs with polymers of different categories of biosensors has
assisted in the provision of hybrid devices that have now improved the use for the
above-mentioned applications (Vigneshvar et al. 2016; Citartan et al. 2013; El-Said
et al. 2020; Turner 2013). Moreover, scientific advancement has been of great assis-
tance in the development of microbial biosensors by means of biological or organic
synthesis approaches that are now contributing to energy and environmental manage-
ment, monitoring, and sustainability (Sun et al. 2015; Vigneshvar et al. 2016; El-Said
et al. 2020).
There are presently several other reports that have shown the significance of using
microbial fuel cells for evolving techniques for the treatment/remediation of water
as power sources for environmental sensors (Vigneshvar et al. 2016).
7.4 Application of Biosensors for the Detection of Pesticides

Available in the Environment
It has been observed that toxic and poisonous herbicides, insecticides, and pesti-
cides are usually applied in industries that are agriculturally based (Adetunji et al.
2018a, b, c, 2019, 2020; Alavanja et al. 2004). The application of pesticides has
been documented for improving the increase in agricultural crops. But it has been
observed that there are several health and environmental hazards that are involved in
whenever pesticides are applied in an uncontrolled manner (Ukhurebor et al. 2020b).
This can lead to the incidence of cancer, allergies, and several breathing challenges
(Criswell et al. 2013; Givaudan et al. 2014). The application of high-performance
liquid chromatography and gas chromatography have been discovered that not to be
economical and requires great expertise as well as consume lot of times.
Therefore, the application of a biosensor might be an alternative technique that
could determine the level of pesticides available in the environment.
Organophosphorus has been identified as a pesticide that is commonly utilized in
most parts of the globe irrespective of its negative effect on the ecosystem and human
health when applied. Hence, it is very important to develop a technique that could be
used for the effective determination of these pesticides. The utilization of enzymatic
biosensors has been identified in this regard which might be linked to the following
facts such as their accuracy, stability, and their sensitivity. The utilization of piezo-
electric, optical, electrochemical, and calorimetric have been manufactured based on
the enzyme inhibition to determine the level of pesticides (Arduini and Amine 2014).
Moreover, ureases and cholinesterases, organophosphorus hydrolase, are some of the
different types of enzymes that have been utilized in the fabrication of biosensors for
effective detection of pesticides (Zhang et al. 2014). Some of the typical examples of
these biosensors which are enzyme based includes a screen-printed electrode, cetyl-
cholinesterase, butyryl cholinesterase, organophosphorus hydrolyase, organophos-
phorus while others were multi-walled carbon nanotubes, screen-printed electrode,
mesoporous carbon/carbon black, reduced graphene oxide and carbon nanotubes,
respectively (Yada 2015).
7.5 Utilization of Biosensors for the Detection of Heavy

Metals in the Environment
It has been discovered that the presence of heavy metals in the environment have
several detrimental effects on animal and human health. They could build up in
organism which might later result in metabolic changes in animals, most especially
those that feeds on the industrial area and highly polluted environment. These might
result in adverse effects through a decrease in hormonal action, malfunctioning of
principal organs, cardiovascular and respiratory challenges, high level of infertility,
and irritation and eventual death.
The movement of heavy metal has been identified majorly from the industrial envi-
ronment to constitute a heavy challenge to human health. Some of them have been
discovered to be non-biodegradable in nature such as lead, mercury, and cadmium.
Most of these heavy metals are derived from lead-acid batteries, vehicle emissions,
and chemical fertilizers (Gammoudi et al. 2014). Therefore, there is a need to protect
the environment and human health from constant exposure to these heavy metals.
However, it has been discovered that the detection of these heavy metals using chro-
matography, and spectrometry are time consuming, non-economical and has a greater
demand for well knowledgeable experts (Bagal-Kestwal et al. 2008).
Therefore, the application of inexpensive, portable biosensors for the detection of
these heavy metals will go a long way in mitigating against all these aforementioned
challenges. The application of microbial sensors has been identified for the identifi-
cation of heavy metals ions when applied at very low cost. A typical example of this
includes microbial fluorescence-based biosensor devices (Amaro et al. 2014). This
was design based on the application of reporter genes that have a greater tendency
to react whenever there is a biochemical reaction between inducer molecules and
cellular reporters. Kim et al. (2015b) stated the application of microbial biosensors
and chemostat-like microfluidic platform that could enhance the identification of
molecular analyte through the uses of a chip. Moreover, for effective determination
of heavy metal ion the application of DNA optical biosensor together with evanes-
cent wave evaluation could be applied in the identification of heavy metal in an
in situ detection (Long et al. 2013b). He et al. (2013) fabricated a luminescent G-
quadruplex-based sensing device that could be identify sub-nanomolar Pb2+ ions in
liquefied solutions (He et al. 2013). Also, the detection of heavy metal ion present in
the environment have been carried out using several techniques such as inhibition-
based biosensors, amperometric and electrochemical sensors (Amine et al. 2014;
Wang et al. 2013; Ghica et al. 2013; Sbartai et al. 2012; Gammoudi et al. 2014).
It has been discovered that graphene oxide is eco-friendly, sensitive and it is
cost feasible. The application of graphene oxide-based fluorescent sensors has been
employed for the detection of Hg2+ ions while graphene oxide plays a crucial role of
fluorophore. It was observed that the molecular recognition probe joins to the Hg2+
ions through a DNA aptamer (Li et al. 2013).
Furthermore, β-Carotene available in the palm kernels could be utilized as a

source of biological reporter most especially in the biosensor that are biopolymer-
based utilized for detection of heavy metals such as aluminum (Wong and Wong
2015). Also, the application of an on-chip label-free sensing device was fabricated
for high-throughput identification of doses as minimal as five parts per billion of
heavy metal such as cadmium-chelate conjugates (Yan et al. 2016).
7.6 Application of Biosensor in CSA
According to Neethirajan et al. (2018) and Ukhurebor (2020), some of the utmost
challenges confronting agriculture in relation to food protection and sustainability
are contain directly or indirectly on following three fundamental aspects:
• NPs and their benefit for agricultural sustainability issues.
• Energy sustainability issues.
• Industrialization of maintainable mechanism issues.
Nanotechnology (NT) is one of the utmost benefits in the monitoring and manage-
ment of AES. It has some valuable features and applicability (Ukhurebor 2020;
Neethirajan et al. 2018). Prasad et al. (2017) reported that NT has the required
potentials to advance food security and eminence, develop and improve the capability
absorption of the nutritional content of the soil, decrease agricultural contributions
and ensure the possibilities in the shrunk mechanism quantifications.
Reportedly, NT has been applied efficiently for/in the following; “precision in
farming machinery (agricultural precision), smart feed management, food waste
management, production of agro-chemicals/agro-materials such as nano-pesticides,
nano-herbicides and nano-fertilizers, labelling and packaging of agricultural products
and multiple other agricultural fields” (Neethirajan et al. 2018; Ukhurebor 2020).
Also, Neethirajan et al. (2018) reported that the use of NT for agricultural deal-
ings in relation to food sustainability is expected to result in some issues in the
forthcoming years. This affirmed the work of Dasgupta et al. (2015), that neverthe-
less the usefulness from the present combination of NPs and animated charcoal for
the improvement of antimicrobial features, food grade nano-emulsion (used for fruit
juice), integrated nano-microbials employed as water sterilizers, active nutraceutical
nano-delivery and improved plant extracts conjugated via nano-packaging could have
some defects also.
A key prominence in NT is in its usefulness in agricultural precision also known
as precision in farming machinery (Ukhurebor 2020), such that plant excerpts from
its core parts like leaves, flowers, stems and roots, from several species have been
efficiently combined into NPs (Duhan et al. 2017; Dasgupta et al. 2015).
NPs have the potential to advance green synthesis (GS) in a one-step via ion
along with metal diminishing consequences. This according to Ukhurebor (2020) and
Prasad et al. (2017), is favourable for use at room temperature, easy-use, adaptable,
and scalable as well as ecological sustainability. During GS, co-enzymes and soluble
metabolites such as phenolic composites, alkaloids and terpenoids are absolutely

condensed to NPs. Essentially, NPs are known as magic bullets ensuing in enhanced
plant growth, location-specific delivery of nutritional values, and improved resistance
of plants to contagions or diseases.
According to Ukhurebor (2020), one of the greatest considerable issues with NT
is in the advancement of dependable risk-benefit assessments via standardized evalu-
ation and measures. Hence, the formation of dependable and standardized measures
in NPs quantifications, arrangements, and evaluation of their environmental conse-
quence on living creatures as well as the participation of all pertinent stakeholders
such as agriculturalists, food industrialists, etc. in a critical debate on the way further
would be of great assistance (Fraceto et al. 2016; Prasad et al. 2017; Ukhurebor
2020). The ensuing issues in sustainable energy could be efficiently taken care of
via the application of organic/biological resolutions. Consequently, Adesina et al.
(2017), reported that one of the foremost benefits in this regard has been established
in applied organic/biological for energy generation.
Also, biofuels could be dumped, generated, rehabilitated, and changed in elec-
tricity (bio-electricity) to meaningfully moderate the cost in fabricating or gener-
ating solar electricity. This according to Dasgupta et al. (2015), could be proficient
via leveraging by means of the ingestion of H2 or electron lashing carbon fixing
metabolism, for the simplification of the their combination via the use of photo-
voltaics efficiency in a process recognized as electro-photosynthesis (Dasgupta et al.
2015; Ukhurebor 2020).
According to Neethirajan et al. (2018), hydrogen-built electrosynthesis is one
of the furthermost efficacious bioengineering energy creation systems. It displays
some excellent physiognomies like; top effective bioenergy storage ability for elec-
trical energy of approximately 80%, extensive length transportability with minimum
energy forfeit, hydrogen oxidation in microbes involving Nicotinamide Adenine
Dinucleotide (NAD+ ; C21 H27 N7 O14 P2 ) reduction of the potential inconsistency and
affordability due to the lesser cell-protein necessities of hydrogen oxidation (Neethi-
rajan et al. 2018). The transmission of electrons according to Neethirajan et al. (2018)
could extracellularly mediate electro-synthesis proficiently. This could be carried
out via reproducible through a nanostructured surface to abridge the of generation
or production of biofilm. It could cavort the need for prolonged surface area and
advance the transmission of hydrogen electrons (Neethirajan et al. 2018).
Advanced organic/biological energy generation according to Neethirajan et al.
(2018), can be meaningfully enhanced via the development of some other technolo-
gies. Such state-of-the-art technologies are gene engineering, whole genome engi-
neering, protein engineering, and biosensing. These state-of-the-art technologies can
interestedly improve the development and generation of biofuel. Reportedly, since it
is one of the utmost prominent benefits of advanced biological science in the aspect of
energy sustainability; biofuel supposedly is to act as one of the utmost sited measures
for capturing and storage of solar energy with little costs (Neethirajan et al. 2018).
Presently, some of the challenges confronting the production, advancement, and
generation of biofuel according to Neethirajan et al. (2018) and Ukhurebor (2020)
are; the energy generation effectiveness and scale, competence investigation in cell
self-assembly as well as duplication monitoring and management, and antagonistic
environmental consequences. Expectedly, it is projected that in the future, biofuel
will develop, advance, and encompass sources of conventional energy for reuti-
lization and replication for the generation of constituents of energy and for the
improvement and advancement of hybrid energy photosynthesis (Neethirajan et al.
2018). Nevertheless, some of the commercialization/industrialization of sustain-
able technologies in the agronomy aspect is enduring via some essential promi-
nences such as; biosensor commercialization/industrialization, sensing technology
commercialization/industrialization and intelligent agricultural/food commercializa-
tion/industrialization, such as CSA (Ukhurebor 2020).
In the commercialization/industrialization of biosensors, some significant aspects
for the purpose of its commercialization/industrialization are; modest sample pre-
treatment, bioreceptor steadiness, multi-detecting/multi-sensing features, impover-
ishment/miniaturization, quicker testing period, wireless accessibility, and afford-
ability (Bahadır and Sezgintürk 2015; Ukhurebor 2020). Some of the present-day
technologically advanced biosensors industries for food safety, security, monitoring,
management, and quality control, their merchants as well as their anticipated analytes
are summarily enumerated in Table 7.1 as revised from Neethirajan et al. (2018).
Antonacci et al. (2016) and Neethirajan et al. (2018) also accounted for some
of the utmost possessions of commercialized available recognized biosensors estab-
lishments as their simple structure, reduced sizes, and ideal potentials for point of
care benefits. They targeted food configuration, progression control, monitoring,
and management in addition to food security such as allergens, pathogens, toxins,
Table 7.1 Some technologically advanced biosensors industries for food safety, security,
monitoring, management and quality
Industries Anticipated analytes
Analox Instruments; in the UK and USA Ethanol, methanol, glucose, lactate, glycerol
Biacore AB; in Sweden Vitamins that are soluble in water, veterinary
deposits (chemical) and mycotoxins
Biomerieux; in France Microorganisms
Biosensores S.L.; in Spain Microorganisms, biochemical oxygen demand
Biosentec; in France Alcohol, allergen, acids, sulphites
Biotech-IgG; in Sweden Allergens, Vitamins, Microorganisms
Eurofins; in Luxembourg Microorganisms, drug residue
Gwent Sensors; in UK Glucose, lactate, ammonia, pyruvate
Nova Biomedical; in USA Bio profile chemistry analyser
Tectronik, in Italy Ethanol, glucose, malate, D-lactate, L-lactate,
fructose
Trace Analytics; in Germany Glucose, L-glutamate, L-glutamine
Yellow Springs Instrument; in USA Glucose, sucrose, galactose, ethanol, lactose,
L-lactate, L-glutamate, H2 O2 , glutamine, choline
pollutants/contaminants, and additives. Neethirajan et al. (2018), stated that some

of the utmost establishments for food quality biosensors is principally from the
following metabolites like glucose, sucrose, glycerol, cholesterol, creatinine, alcohol,
methanol, lactate, lactose, glutamate, malate, and ascorbic acid.
Bahadır and Sezgintürk (2015) reported that likened to the earlier recognized
biosensors in scientific laboratories, the contemporary biosensors which are habit-
ually commercialized are faraway scarcer indicatory of the abridged accomplish-
ment extents in agronomy-associated biosensor development. The issues burdening
biosensor advancement in the agronomy sector are considerable impairments. Such
considerable impairments according to Neethirajan et al. (2018) are mass production,
sensor lifetime, component integration, and handling practicability.
The motivations behind these limitations are that the utmost technologies
employed in present and imminent agronomy-associated biosensor technologies
are in their early phases and they include NT, agriculture/food material science,
biomimetic chemistry, and microengineering (Neethirajan et al. 2018; Ukhurebor
2020). According to Neethirajan et al. (2018), these basic issues can be of assistance
for the purpose of imminent biosensors industries as well as its safety to human
comfort, which infers that it is those with inadequate or no human comfort conse-
quence will have their scientific improvement in the near future (Ukhurebor 2020).
The scientific improvement of intelligent agriculture/food industry stipulates crucial
requirements in innovative and operative measures to guarantee AES vis-à-vis food
security, protection, safety, and quality, as well as to reduce production techniques
and diminishing losses in agronomy (Vanderroost et al. 2014; Ukhurebor 2020).
7.7 Conclusion and Future Contribution to Knowledge
CSA purposes excellent prospects for dealing with the issues of AES vis-à-vis food
security, protection, safety, and quality in addition to enabling the adaptation and
extenuation succours for AES. Consequently, CSA has been of enormous assistance
in this aspect, particularly to several industrialized nations. The implementation of
CSA as a proficient and instantaneous CC response is tremendously critical for the
development of capacity and accomplishing food protection and safety as well as
AES universally. In unindustrialized nations particularly those of Sub-Sahara Africa,
considering the vulnerability to CC, their considerable reliance on agriculture for
livelihoods and the vital role agricultural dealings play in their pecuniary sector;
they would largely benefit from CSA. However, there is a need for modification
procedures in inspiring the acceptance and improvement of CSA.
The minor agricultural section in some of these unindustrialized nations is char-
acterized by various inhabitant. Accordingly, a solitary uniform technique would
hardly be appropriate in advancing CSA dealings among these sets of agricultural-
ists. The consequence ensuing from this is that methods in supporting CSA execution
should factor in explicit and specific communal dealings as an addition for main-
streaming procedures universally. Therefore, all relevant stakeholders must antici-
pate applying modalities that could accommodate the various structures and physiog-
nomies of CSA, and avoid the possible issues that could otherwise arise. Furthermore,
since CSA development in most unindustrialized regions of the world relies on the
preparedness of those involved in agricultural dealings; consequently, it is necessary
for all relevant stakeholders to comprehend and recognize the multi-dimensional
CC issues and the succeeding self-mobilization for developing and implementing
approaches that will react to the issues at suitable scales.
Irrefutably, despite the several benefits of biosensors and BSTs such as poly-
mers, NPs and microorganisms built biosensors in resolving some of the problems in
AES; there is still the necessity to expressively integrate multi-faceted approaches in
evolving biosensors that could hypothetically be employed for various applications in
CSA for AES. Consequently, it is recommended that suitable combination of BSTs
along with bio-fabrication with non-natural/synthetic biology approaches via the
application of either/both electrochemical, optical, bio-electronic moralities would
be critical for the effective enhancement of wide-ranging and dominant biosensors
for present-day and forthcoming involvement to knowledge in the aspect of BSTs in
CSA for AES.
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Chapter 8
New Trends in Biosensor Development
for Pesticide Detection
Narlawar Sagar Shrikrishna, Subhasis Mahari, Naina Abbineni,

S. A. Eremin, and Sonu Gandhi
Abstract Increased use of pesticides globally has led to harmful consequences

on the environment, livestock, animals, as well as humans. Hence, its detection
in animal feed, vegetables, agricultural crops, milk, eggs, and meat has become
a pre-requisite. Conventional techniques such as High-Performance Liquid Chro-
matography (HPLC), Gas Chromatography (GC), Fourier transform Infrared Spec-
troscopy (FTIR), Mass Spectrometry (MS), Spectrophotometry, Polarography, etc.,
are generally being used in detection of pesticides. Alternatively, biosensors can be
employed which are cost-effective and an easy way to detect pesticides. Biosensors
can be differentiated based upon its biorecognition elements ranging from antibodies
to cells. This chapter gives an overview of recent advances in biosensor development
for the detection of pesticides with major emphasis on electrochemical biosensors.
Keywords Pesticides · Biosensors · Aptamers · Antibodies · Nanocomposites
8.1 Introduction
Pesticides, as the name suggest, are organic toxic compounds (chemicals) that are
used to manage variety of insects, insect’s larva, nematodes, rodents, fungi, weeds,
and numerous other pests. Farmers use various chemical compounds in agriculture,
such as pesticides, fumigants, plant growth regulators, and fertilizers, to protect their
crops from hazardous pests (Sassolas et al. 2012). Residues of pesticides may come
into the food chain by means of water, air, and soil (Kumar et al. 2019). It impacts
the ecosystem and induces a variety of human and animal health issues (Özkara
et al. 2016). They can cause diseases of the respiratory system, bone marrow, and
nerves, infertility, immunological illness (Tsatsakis et al. 2019). Many pesticides
N. S. Shrikrishna · S. Mahari · N. Abbineni · S. Gandhi (B)

DBT-National Institute of Animal Biotechnology, Hyderabad 500032, India
S. A. Eremin
Faculty of Chemistry, M. V. Lomonosov,
Moscow State University, Leninsky Gory, 1, Moscow, 119991, Russia
138 N. S. Shrikrishna et al.
are persistent organic pollutants (POPs), primarily pesticides of the organochlorine

family which are noted for their bioaccumulating effects. Pesticides that are used for
vector management bioaccumulate in conserved areas. There is a substantial chance
of human cancer correlated with the ingestion of such fish from the rivers (Gerber
et al. 2016). POPs are lipophilic in nature, so they are found in the milk of animals
(Hussain 2018) and their effective detection is the need of the hour.
Detection of pesticides at standards set by the Environmental Protection Agency
(EPA) remains a challenge. Chromatographic approaches coupled with specific
detectors have historically been used to evaluate pesticides because of their safety,
durability, and performance. Nonetheless, they are time consuming and laborious,
and require highly skilled technicians and costly machinery. Hence the develop-
ment of biosensors for the sensitive detection of pesticides has been given signif-
icant attention in recent years as a promising tool. A biosensor is a self-contained
system that incorporates an immobilized biological entity (e.g., whole cell, DNA frag-
ment (Kasoju et al. 2020a, b), enzyme, biomembranes, antibody (Talan et al. 2018))
that detects the analyte (e.g., antigen, DNA, enzyme substrate, enzyme inhibitors
such as some pesticides (Islam et al. 2019; Thakur et al. 2011; Mahendra et al. 2010;
Tey et al. 2010; Raman et al. 2009)) and a transduction elements used to trans-
form the biochemical signal arising from the analyte’s contact with the bioreceptor
into an electrical signal (Sassolas et al. 2012). Biosensors are categorized as elec-
trochemical, electrical, piezoelectric, and mechanical biosensors, depending on the
signal transduction technique (Raman et al. 2009). Because of their high sensitivity,
electrochemical transductors have been commonly used in biosensors for pesticide
detection. It is a low cost, basic design and compact size also make them excellent
candidates for the construction of portable biosensors. Based on the biorecognition
element biosensors are classified as enzymatic, immunochemical, whole cell, and
DNA biosensors.
Classical methods such as chromatography, Mass spectroscopy, UV-Visible
spectroscopy, Nuclear Magnetic Resonance (NMR), matrix-assisted laser desorp-
tion/ionization (MALDI-TOF) can be performed to detect those pesticides. However,
these techniques are laborious, costly, time consuming, and require skilled personnel.
In order to overcome these difficulties, certain new techniques have been developed
that are based on bio recognition of molecules using biosensors.
8.2 Classical Methods of Pesticide Detection
Food and Drug Administration (FDA) and Food Safety Inspection Service (FSIS)
monitor the presence of pesticides in different food commodities. There have been
several advancements in technologies for the identification and quantification of
pesticides in different products. FSIS provides suitable criteria for pesticide detection
for example the detection should not take more than 24 hours, should require easily
available equipment and reagents, and should be able to detect below the tolerance
level (Pesticide Residues in Food: Technologies for Detection - U.S Congress 1988).
8 New Trends in Biosensor Development for Pesticide Detection 139
Different chromatogenic methods specifically thin layer chromatography (TLC), gas

chromatography (GC), liquid chromatography, gel permeation chromatography, and
high-performance chromatography helps in the separation of different products to
check the presence of pesticides in it. These methods were developed in the early and
mid-twentieth century and are hence classified under a classical method of pesticide
detection.
8.2.1 Chromatographic Methods
8.2.1.1 Selective Gas Chromatography Detectors
Selective gas chromatography detectors depend upon different chemical actions on

the sample before the actual detection. Selective detectors are usually liable to
changes in the operating framework apart from the two main universal detectors,
i.e., katharometer and the Flame ionization detector (FID). These detectors require
calibration for the linearity of the data. To improve on this flaw, advancements have
been made on the detectors in the last few years. Selective gas chromatography detec-
tors include pulsed flamed photometric detector (PFPD), Halogen specific detector
(XSDTM , manufactured by OI Analytical), Tandem GC detectors, and Electrolyte
conductivity detector (ELCD) (Adlard and Juvet 1975).
8.2.1.2 Pulsed Flame Photometric Detectors (PFPD)
PFPD is considered as a new tool in the hands of an analytical chemist. It was

developed 30 years ago and uses a flame for high sensitivity and better selectivity
for sulphur and phosphorus (Amirav and Jing 1995). In PFPD, the factors affecting
sensitivity are analysed with a specific detector without interference of hydrocar-
bons. PFPD is coupled with a pulsed flame ionization detector (PFID) to produce
combined pulse flamed photometric ionization detector (PFPID) (Fan 2005). PFPD
also features light emission time having the ability to separate in time the emission of
carbon species from sulphur and phosphorus which leads to enhancement in detection
selectivity. It also has characteristics of reduced background noise that gets filtered
in time, increased signal because of higher brightness of pulsed flame, less flow of
combustible gas, and the using broad band filters. Again, air and hydrogen consump-
tion is reduced to a great extent for increased selectivity and sensitivity in selective
detection of nitrogen using Nitric acid flame chemiluminiscence (Qian and Burbank
2007). PFPD consists of a main body mounted on GC detector base, two hydrogen
gas mixtures, a gas mixture combustor, a quartz light guide rod, a colored glass
filter, and a photomultiplier for detection (Amirav and Jing 1995). A combustible
gas mixture is eluted and at the same time, the combustor gets filled with that gas
which is surrounded by a separate air-richer combustible gas mixture which bypasses
the combustor but mixes with other gases from the previous pulse and helps in filling
the complete igniter light shield element. A heated wire igniter ignites the pulsed
flame and disseminates via an igniter light shield, passing through the combustor
and terminating at the combustor holder. A hole of around 0.9 mm does not allow
further movement (Qian and Burbank 2007).
Jing and Amirav (1997) analysed methyl parathion in eggplant extract where
PFPD had superior sensitivity of detection that could be used in achieving lower
detection limits and increased stability. Further, M. Salvador and his group verified
the presence of organophosphorus in vegetables finding 24 pesticides with 0.01 mg/kg
of limit of detection (Martínez Salvador et al. 2006).
8.2.1.3 Halogen Specific Detectors (XSDTM )
Halogen specific detector (XSDTM ) detects halogen-containing compounds from the

elution of GC capillary column. It neither uses any organic solvent nor has any
radioactive source. It does not require any catalyst tube, solvent, resin cartridges,
pumps, or transfer lines. XSDTM consists of a high detector that allows low-level
selective analysis of halogens and a unique jet design that reduces the peak tailing
and requires only air for operation. It has a very low range of detection of around
2 pg chlorine and has a high selectivity of 106 molecules of chlorinated fatty acids.
Compounds with concentrations larger than 1 ng μL−1 has a specific peak broad-
ening and peak trailing shows inappropriate results is a major drawback of XSDTM
(Marquardt et al. 2019; Zhuang et al. 2005). XSDTM consists of an alkali glass-
ceramic rod, a cathodic platinum bead attached to the bottom of the rod, an anodic
platinum coil wound around the rod above the bead, and a reactor core that helps
in maintaining a working temperature for the electrodes (Zhuang et al. 2005). The
current that is emitted from XSDTM consists of negative, positive, and free electrons.
The background current is used for thermal electron emission of the cathode and
when halogen atoms are produced from the combustion of halogenated analytes,
they get adsorbed onto the surface of the cathode sensitized by alkali metal (Nilsson
et al. 2001).
Pesticides such as chlorothalonil was detected by Brown and colleagues from
fruit and vegetable samples including Tomato, Potato, and Cabbage using Halogen
specific detectors with a limit of detection of 4 mg mL−1 (Brown et al. 2011).
8.2.1.4 Electrolyte Conductivity Detector (ELCD)
Electrolyte conductivity detector (ELCD) was first described by Piringer and

Pascalau in 1962 for the detection of organic compounds (Piringer and Paşcalǎu
1962). Here, the compounds eluted from the column are collected in a quartz capil-
lary producing carbon dioxide. The carrier gas in a long capillary tube is mixed with
carbon dioxide and the conductivity of the aqueous solution is measured (De Vos
et al. 2011). In 1962, Sternberg explained an ELCD coupled with a flame ionization
detector (De Vos et al. 2011) where water flowing on the surface of reactants was posi-
tioned above the burner of the flame ionization detector. The products were entrapped
in a liquid film and the change in conductivity was measured. With time, different
models enhanced from the initials. In 1974, a micro detector (ELCD) was first
described by Hall (Pape et al. 1977). The size was significantly small, and it showed
increased sensitivity. It also carried space for liquid and gaseous phase distinction.
Here, it contained a temperature regulating system and the solvent could be non-
aqueous. With further improvement, an increased sensitivity was achieved using a
bipolar pulse differential detector, where the conductivity of the solvent was measured
in the first detector cell and solvent with products in the second cell (Dressler 1986).
This enhanced the capability of the detector in solvent purity and temperature vari-
ations. Recently, a further improved design of ELCD was described as suitable for
high resolution capillary gas chromatography (Verhaar et al. 1984). In this detector,
no phase separator was present leading to liquid and gas mixture passing directly
through the conductivity cell. The ELCD could be used for selective detection of
halogens, sulphur, and nitrogen containing compounds. Dolan and Seiber (1977)
used electrolyte conductivity detector for detection of different pesticides including
Mirex, Heptachlor, Aldrin, Dieldrin, Lindane, and Endrin, with detection limits in
the range of nanograms.
8.2.1.5 Tandem GC Detectors
Tandem GC detectors have always been tools for clinical assessment of steroids since
the last 3 decades (Nyiredy 2004). Production of simultaneous chromatograms for
aromatics and aliphatics is possible using the detectors that eliminate the need for
separate analysis. Tandem GC detectors can be used as packed or capillary columns.
The stream that elutes samples from GC column to the reaction chamber provides
a continuous irradiation of high energy ultraviolet light, increasing the ionization
potential of the samples. An electric field inside it collects the ions formed, leading
to the production of ion current which can be amplified, and output could be checked
by the GC’s electrometer. Tandem GC detectors are non-destructive detectors that
do not destroy the structural and elemental characteristics of the compounds. Next,
the samples are passed inside a chamber where the analytes are combusted in a
hydrogen-air flame, after which the ionized combustion products pass through a
negatively biased annular electrode, producing an ion current proportional to the
sample concentration in the flame. Tandem GC detectors can acquire two simulta-
neous chromatograms from a single injection leading to the elimination of transfer
lines between detectors which improves the shape and performance of the peak.
8.2.1.6 Thin Layer Chromatography (TLC)
It is a separation technique used mainly for the separation of non-volatile mixtures

and is used on a glass or plastic coated with an absorbent material usually silica gel,
aluminium oxide, or cellulose. TLC has always been an important aspect in pesticide
analysis like the determination of quantitative structure-activity relations (QSAR),
molecular structure, and the biological activity of the compound. Analyses using
TLC have been done on food samples, pesticides, fungicides, herbicides, and many
more (Sherma and Fried 2005). It has also played an important role in pesticide
separation, detection, identification, and quantification (Sherma and Fried 2005).
Analysis of pesticides has always been one of the major applications of TLC for
both qualitative and quantitative analysis. The idea behind TLC came from Izmailov
and Shraiber (1938) when they used a powdered absorbent, like calcium chloride,
magnesium oxide, or alumina to acquire a chromatographic zone from a single drop
of analyte (Poole et al. 1986). Modifying the set-up, Williams (1946) used circular
chromatograms to produce different layers of adsorbent in between two glass plates.
Later, in 1952, researchers initiated a spreading technique to prepare alumina on glass
plates (Grinberg 1990). Finally, the modern-day TLC was generated by Meinhard
and Hall in 1949 following the research by Williams (1946) who used starch as the
binding agent for mechanical stability (Abbott and Thomson 1965). The use of TLC
in detection of pesticides started around the 1960s for pesticide residue analysis but
its application reduced by a great extent due to the availability of gas-liquid chro-
matography (GLC) and High-performance liquid chromatography (HPLC) easily. In
recent years, improvisation has been observed in the coating of materials, detection
systems, and extraction of pesticides which has once again increased the use of TLC
globally (Aktar et al. 2009; Institute of Medicine 1997). In addition to a separation
technique, TLC is also used in the cleanup procedures prior to gas chromatography
(GC) or HPLC (Sherma 1982).
Procedure of TLC: In the primary step, the sample is applied on the coated plate
and then a solvent is drawn upward by capillary action. Commercially, pre-coated
organic-bound, layers of silica are more favorable than laboratory made silica gel
due to their stability and uniformity. Use of silica gel is usually preferred but there
is an increasing shift toward high-performance silica gel and chemically bonded
C18 reversed phase (Gabriëls and Plaizier-Vercammen 2004). Certificate of Analysis
program recommends that silica gel, High-performance silica gel, and reversed phase
plates with preadsorbent spotting areas are better suitable for pesticide analysis by
TLC due to uniform spotting of samples and control (Lau and Dunn 2018; Kogan
et al. 1989; Sherma and Fried 1987). Selection of the ideal mobile phase is quite
necessary for the movement of solutes. Mobile phase is usually made up of pure
organic or mixed aqueous—organic solvent systems differing in ratio of the solvent
mixture (Sherma 1982). Mobile phase is usually carried out in rectangular chambers
(usually 10 × 10 cm) but results vary when the size changes (Nyiredy 2004). The
analytes in the sample move at different rates which helps in its separation (Tong
et al. 1985). The properties of stationary phase are different from mobile phase,
i.e., if the mobile phase is polar then the stationary phase is nonpolar, helping in
proper movement of the molecules. This flow of molecules through the capillary
action leads to separation of analytes. Components of pesticides are detected on the
layers because of their natural color, fluorescence, or ultraviolet light by fluorescence
quenching or fluorescence or color zone. A gaseous electrical discharge technique
for fluorescence is used in some organophosphate insecticide zones on TLC plates

can be produced (Hauck and Halpaap 1980). For identification of zones of pesticides
on TLC plate, few drops of a reducing agent were added to the spots and the plate
was heated at 100 °C for 10 min. The compound could be identified on the plate after
cooling (Sherma 1982).
Applications of TLC in detection of pesticides: Use of TLC in forensic sciences
is possible for estimation of the distribution of carbamate pesticides (Tewari 1980),
organophosphate pesticides, urea-related pesticides, and triazine herbicides as well
as different fungicides. Abbott and Thomson (1966) checked water samples were
also analysed using TLC to check the presence of pesticides.
8.2.1.7 Gas Chromatography Coupled with Mass Spectrometry

(GC-MS)
GC-MS is a technique that collaborates with the work of gas chromatography and
mass spectrometry for sample analysis (Sparkman et al. 2011). There are several
applications of GC-MS including drug detection, environmental analysis, fire and
explosives investigation, identification of trace elements from materials, etc. GC–
MS is made up of two blocks, i.e., gas chromatograph (GC) and mass spectrometer
(MS). GC consists of a capillary column which is relative to the phase properties and
column’s dimensions. The diversity in chemical properties in molecules and their
affinity toward the stationary phase enhances the separation of molecules during
their travel in the column. Then the molecules are eluted at different time intervals
from the column. This helps MS in capturing, analysing, and detecting the ionized
molecules separately. MS breaks each ionized molecule and measures the fragments
in mass to charge ratio. The two components connected together provide a greater
degree of substance identification than when used individually. The only drawback is
that MS cannot differentiate multiple samples having same travel time in the column
as they elute at the same time and hence, combining the two methods ceases this
chance of error as it is very rare that more than one molecule will show the same
characteristics in both GC and MS.
In the year 1950, Fred McLafferty and Roland Gohlke, two researchers improved
the analytical power of GC by coupling it with MS (https://www.acs.org/content/
acs/en/education/whatischemistry/landmarks/gas-chromatography-mass-spectrome
try.html). Coupling both the instruments lead to the determination of unambiguous
identification of each component. Taking an unknown mixture of samples, the mass
spectrum of each peak can reduce the possible identity of each component of the
mixture. Having the known standard can help in understanding the retention time
and a mass spectra matching with the. Components of GC–MS include capillary
GC system, mass selective detector which constitutes an ionization source, mass
separator, and ion detector. Two common mass analyzers or separators that are
available for GC–MS is quadrapole and the ion trap.
Analysis of sample in GC–MS can be of two types, i.e., full scan or selective ion
monitoring. The main goal of the instrument is to quantify the amount of substance
that is possible by comparing different concentrations of the molecules in the analyte.

Two kinds of analysis are usually done which are comparative and original. A compar-
ative analysis compares the given spectrum with the spectrum library to correlate
the characteristics while the original analysis is a simple analysis of the analytes.
A full spectrum analysis checks all the peaks in the spectrum while selective ion
monitoring only monitors specific ions linked with a specific substance. It is a
fast mode of analysis based upon the previous data about the sample (Alves et al.
2018).
8.2.1.8 HPLC Coupled with Mass Spectrometry (HPLC–MS)
It is a technique that combines liquid chromatography with mass spectrometry to

provide a proper analysis of a mixture of samples. Liquid chromatography helps in
the separation of the molecules and mass spectrometry helps in the identification of
the molecules. This technique helps in analysing biochemical, organic, and inorganic
compounds commonly found in complex samples present in the environment (Jacob
et al. 2014; Dass 2007). HPLC and MS are not compatible with each other as the
mobile phase of liquid chromatography is a highly pressurized liquid while the MS
works under high vacuum conditions. So, it is not possible to elute samples from
HPLC system to MS source and hence an interface is required between the two. The
interface could be done by transferring the maximum amount of elute from the
mobile phase of the HPLC column while making sure that the interface should not
interfere with the ionizing efficiency and vacuum conditions of the MS system (Dass
2007). Setting up HPLC–MS for analysis requires specific conditions and parameters.
Each analyte requires specific optimization even when the standardized conditions
are present. Analyte’s nature determines the optical ionization and polarity, and the
detection depends upon derivatisation (Gao et al. 2005; Zaikin and Halket 2006;
Johnson 2007).
HPLC coupling with MS started around the 1950s. MS was originated in 1952
by A.J.P. Martin and A.T. James who tried to separate molecules based on analysis
of mass (James and Martin 1952). Commercially available HPLC–MS was made
available during the 1970s when capillaries were used to connect LC columns with
MS ion sources (Niessen 2006). The preliminary interface between HPLC–MS was
limited to volatile substances and nonpolar analytes with a less molecular mass.
The interface between HPLC in the liquid phase and MS in a vacuum took a long
time. The innovation toward electrospray ionization was a major step in this. Nowa-
days, almost all HPLC–MS utilizes electrospray ionization (ESI), atmospheric pres-
sure photo-ionization (APPI), and atmospheric pressure chemical ionization (APCI)
as the interface (Arpino 1992; Pitt 2009). Recently, various drying and deposition
techniques have evolved like MALDI deposition (Arpino 1989; Murray 1997). An
approach called direct—EI LC–MS interface coupled with nano HPLC and elec-
tron ionization attached to mass spectrometer is under development (Cappiello et al.
2008; James and Martin 1952). Barrek and colleagues analysed pesticide residues in
olive oil using HPLC–MS. The group could separate 11 pesticides from the mixture
in olive oil (Barrek et al. 2003).
8.2.1.9 Gel Permeation Chromatography (GPC)
This is a separation technique that helps in the separation of analytes based upon
weight or size. It was first developed by Lathe and Ruthven (1956). In 1973, Jim
Waters was the first person to construct the first commercial GPC based upon John
Moore’s design. It works on the basis of the hydrodynamic volume of the analytes.
The analytes gets separated depending upon their physical and chemical interactions
(Skoog et al. 1998). Porous beads packed in the column help in separation. If the size
of the analytes is small, they enter into the beads in the column leading to an increase
in retention time while larger-sized analytes do not get trapped in the column and
are eluted first. But if the analytes are too large, they get eluted immediately and if
the analytes are too small, they do not get eluted at all. This method of separation
leads to differentiation of a broad size range of analytes. GPC consists of two phases-
mobile phase and the stationary phase. The stationary phase constitutes of polymer
matrix having pores completely filled with the solvent required for the mobile phase.
Determination of the size of the pore is essential as the basis of separation of the
molecules greater than a certain size do not enter the pores, and the interior of the
pores are available, partly or completely to molecules having a smaller size. The
theory of separation is based upon an equation:
Vt = V0 + Vi + Vm
where,
Vt = The column’s maximum capacity
V0 = Liquid present outside the gel matrix
Vi = Liquid present inside the matrix
Vm = Volume of the gel matrix
GPC consists of a stationary phase composed of porous polymer gel beads, semi-
permeable in nature having specific pore sizes. These gel beads are mechanically
stable, chemically inert, and have an ideal homogenous pore structure and size.
Mobile phase of GPC consists of a liquid that helps in dissolving the biomolecules
allowing high and accurate detection. Pump used in GPC is either a syringe pump
or a reciprocating pump with a constant rate of flow. The detectors can be of three
types, i.e., refractive index detector, ultraviolet absorption detector, and evaporating
light scattering detector. There are certain advantages of GPC including, it takes a
short time for sample analysis, provides narrow bands showing high sensitivity, low
or no loss of samples, requires less amount of mobile phase and the flow rate can
be set. Along with all these GPC has some disadvantages like a limited number of
peaks that can be resolved in a small GPC run. It requires filtration of samples before
use which may otherwise lead to biased results. It also provides no conclusive data
when the molecular masses of the analytes are very close. Apart from these disad-
vantages, GPC is used in protein fractionation, purification, and molecular weight
determination. Along with that GPC is used in separation of sugars, proteins, peptides
basing upon their size and even helpful in determination of the quaternary structure
of the purified proteins. Jongenotter and his group analysed the concentration of
organophosphates in olive oil. Pesticides with a limit of detection of 0.0002 mg kg−1
could be detected using gel permeation chromatography (Jongenotter et al. 1999).
8.2.2 Matrix-Assisted Laser Desorption/Ionization–Time

of Flight (MALDI–TOF MS)
It is a tool for pesticide analysis with advantages like simplicity of obtained spectra,
broad applicability, a better tolerance toward contaminants, and an ability to analyse
complex mixtures without separating the analytes even before prior analysis (Madla
et al. 2012). It was first termed by Franz Hillenkemp, Michael Karas, and their
colleagues in 1985 (Karas et al. 1985). It must be noted that, no specific protocol
has been designed for sample preparation in MALDI-TOF MS but the spectra
depend upon the matrices. However, commercially available matrices are avail-
able which are analyte specific (Ayorinde et al. 1999). Mostly, organic matrices
are used for MALDI-TOF. Matrix of MALDI consists of crystallized molecules
like α-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and
2,5-dihydroxybenzoic acid (DHB). Selection of matrix is usually based upon charac-
teristics like low molecular weight and a strong optical absorption range either in the
infrared (IR) or ultraviolet (UV) range for efficient absorption of laser light (Zenobi
and Knochenmuss 1998). Sample preparation for MALDI undergoes the following
procedure the sample needs to be mixed with the matrix solution and around 1 μL
of the mixture is to be spotted on the plate and dried before taking the readings by
MALDI–TOF MS. Formation of MALDI includes laser, time of flight, and atmo-
spheric pressure. UV lasers are nitrogen lasers of 337 nm and frequency-tripled and
quadrupled neodymium-doped yttrium aluminium garnet (Nd:YAG) with 355 nm
and 266 nm (Dreisewerd 2014) and IR laser constitutes 2.94 μM Er:YAG laser,
mid-IR optical parametric oscillator, and 10.6 μM carbon dioxide laser (Bahr et al.
1992). Madla and colleagues determined pesticides including Atrazine, Azoxys-
trobin, Flutolanil, Tricyclazole, etc., using MALDI–TOF. They reported that detec-
tion of small molecular weight pesticides can be analysed using MALDI–TOF that
shows better sensitivity than other methods with a less amount of sample (Madla
et al. 2012).
8.3 New Methods of Pesticide Detection and Analysis
Pesticide detection at low concentrations, decided by Environmental Protection

Agency (EPA) has always been a challenge even after the development of
certain chromatogenic methods since the early 1900s. There have been certain
developments for pesticide detection which would be cost-effective, less laborious,

require less expensive equipment, and be less time consuming. A biosensor can
be a good alternative for pesticide detection which would contain an immobilized
bio-element converting the biological signal into an electrical signal. Basing upon
the transduction technique, they can be classified into electrochemical, piezoelec-
tric, optical, and mechanical biosensors. Classifying on the basis of biorecognition
elements, biosensors can be divided into enzymatic whole cell, immunochemical,
and DNA sensors. Recent reports provide data on the identification of pesticides
on the basis of molecular imprinted polymers (MIPs) and artificial neural networks
(ANNs) coupled with sensor arrays. Along with that, nanotubes and nanorods help
in improvising the efficiency of biosensors for pesticide detection. Further, different
kinds of biosensors available commercially are described in Table 8.1.
8.3.1 Enzyme-Based Biosensors
Conventional methods of pesticide detections are laborious, time consuming, have

a high operation cost, and require skilled personnel to operate machines, and hence
some new methods that are based on biosensors are being developed. Enzyme-based
biosensor is one such technique among the new methods.
Several pesticides have been engineered (designed) to inhibit the main enzymatic
mechanism, and therefore biosensors that are based on the principle of enzyme
inhibition are especially important as they accomplish quantitative detection of a
pesticide. So, these enzyme-based biosensors take advantage of an enzyme’s inhi-
bition and molecular affinity toward its substrate (pesticide) in order to quantify the
amount of pesticide present as shown in Fig. 8.1. Following are some of the enzymes
used in pesticide detection.
8.3.1.1 Detection of Cholinesterase Enzyme Inhibiting Neurotoxic

Insecticides
The application of insecticides on crops is stringently supervised and monitored

by various authorities. For e.g., organophosphorus insecticides in the EU (Euro-
pean Union) are either allowed (dimethoate, malathion, chlorpyrifos) or prohibited
(diazinon, coumaphos, parathion). A similar scenario exists for carbamate insecti-
cide as both approved (formatanate, methomyl, pirimicarb) or prohibited (carbofuran,
carbaryl, aldicarb) examples. (Commission, E. Eu Pesticides Database).
Because of its relevant toxicity, there is a need for rapid and flexible analytical
methods for the analysis of a vast number of environmental samples and food tainted
with small quantities of specific insecticides and their metabolites. The principle
mechanism of toxicity of theses insecticides are based on the inhibition of the enzyme
Acetylcholinesterase (King and Aaron 2015).
Table 8.1 Different types of biosensors developed for pesticide detection along with its limit of
detection
Transduction system (Analyte) Limit of detection (LOD) References
Optical biosensor
Isoproturon 0.046 ng mL−1 Rodriguez-Mozaz et al.
(2004)
Atrazine 0.155 ng mL−1 Rodriguez-Mozaz et al.
(2004)
Herbicide subclasses 3 nM Giardi et al. (2005)
Diuron 0.01 ng mL−1 Mallat et al. (2001)
Linuron 0.14 ng mL−1 Mallat et al. (2001)
DDT 15 pg mL−1 Mauriz et al. (2006)
Surface Plasmon resonance (SPR)
2-4 D 4 ng/mL−1 Kim et al. (2006)
Cyanazine 0.095 nM Saylan et al. (2017)
Atrazine 1 μg mL−1 Nakamura et al. (2003)
Simazine 0.031 nM Saylan et al. (2017)
Atrazine 0.091 nM Saylan et al. (2017)
Chloropyrifos 0.056 ng mL−1 Li, Q. et al. (2019a)
Carbendazim 0.44 ng mL−1 Li, Q. et al. (2019b)
Triazophos 0.096 ng mL−1 Guo et al. (2018)
Fluorescence/chemiluminescence
2,4-D, linuron and simazine 0.02 ng mL−1 Herranz et al. (2008)
2,4-D 4 ng mL−1 Marquette and Blum
(2000)
Carbaryl 26.8 × 10−12 mol L−1 Li et al. (2016)
Parathion-methyl 3.8 × 10−6 Yan et al. (2015)
Glyphosate 12 ng mL−1 Wang et al. (2016)
Dichlorvos 4.4 nM Meng et al. (2013)
Paraquat 0.0117 nM Durán et al. (2013)
Quartz crystal microbalance
Carbaryl 1 × 10−7 M Abad et al. (1998)
2,4-D 0.5 nM Skládal et al. (1994)
Paraoxon 5 × 10−8 M Abad et al. (1998)
Carbofuran 4.5 × 10−6 M Jia et al. (2013)
Carbamate and Organophosphate 1 ng mL−1 Karousos et al. (2002)
Endosulfan 5.59 μg L−1 Liu et al. (2013)
Pirimicarb 5× 10−7 M Sun and Fung (2006)
Metolcarb 0.019 mg L−1 Pan et al. (2013)
Parathion 4 μg L−1 Funari et al. (2013)
Electrochemical Biosensors
(continued)

Transduction system (Analyte) Limit of detection (LOD) References
2,4-D 50 ng mL−1 Dzantiev et al. (1996)
Simazine 5-175 ng mL−1 Starodub et al. (2000)
Monocrotophos 0.3 ng mL−1 Du et al. (2008)
Dichlorvos 7 × 10−12 Valdés-Ramírez et al.
(2008)
Acephate 5.45 × 10−14 Ishii et al. (2008)
Malathion 0.31 ppt (Parts per trillion) Bao et al. (2019)
Chlorpyrifos 10 fM Talan et al. (2018)
Micromechanical cantilever
Atrazine 0.1 ng mL−1 Raman et al. (2008)
Carbaryl 2× 10−10 M Chen et al. (2018)
Paraoxon 10−7 M Karnati et al. (2007)
Rapid screening (dipstick and
lateral flow assay biosensor)
Atrazine < 1.0 ng mL−1 Kaur et al. (2007)
Sulfamethazine 50–100 ng mL−1 Kandimalla et al. (2008)
Benzothiostrobin 25 μg L−1 Wu et al. (2019)
Imidacloprid 0.02 ng mL−1 Tan et al. (2020)
Profenophos 75 nM Kim et al. (2018)
Chlorpyrifos 3.3 μg mL−1 Fu et al. (2019)
AChE act as a biorecognition element for the analyte (Pesticide). When pesti-
cide molecules come in close proximity to enzyme, chemical changes take place
in reacting elements. These chemical changes lead to the formation of electroac-
tive species, consumption of oxygen, reduced forms of by-products, and so forth
(Wang 2008). These changes are detected and displayed on the controlling system
(Fig. 8.1). There are reports of various types of biosensors developed for the detec-
tion of organophosphate and carbamate insecticides based on the mechanism of
cholinesterase enzyme (Štěpánková et al. 2016).
8.3.1.2 Detection of Herbicides (Inhibiting the Process

of Photosynthesis)
Herbicides as the name suggest kills a variety of grass (weed) species in agricul-
tural practises, they are widely used in agriculture for the controlling of weeds.
Though the lists of permitted herbicides and their permissible levels of residues
accepted in food, feed, and water in different parts of the world are continuously
being updated, certain herbicides such as atrazine, which have been prohibited in the
European Union since 2000, are still permitted for use in many parts of the world, for
Fig. 8.1 Enzyme biosensor—A schematic illustration showing the enzyme-based detection of a
specific analyte (Pesticide molecule), it illustrates enzyme coated on conductive surface recognizes
the analyte by binding to it, transducer senses a subsequent change in electrochemical parameters
upon binding and resultant value displays on the screen
example, in Asia and North America. Existing herbicide detection methods depend
on Enzyme-linked immunosorbent assay (ELISA), Capillary electrophoresis liquid
or gas chromatography linked with mass spectrometry (LC–MS or GC–MS) (Giardi
et al. 2001). Herbicides pertaining to the class of phenylurea (e.g., chlorotoluron,
chlorbromuron, diuron, fenuron, etc.), class of diazine (lenacil, bromacil, etc.), class
of triazine (cyanazine, simazine, atrazine, etc.), and group of synthetic phenols
(bromoxynil dinoseb, ioxynil, etc.) inhibit photosynthetic diatoms, algae, cyanobac-
teria, and plants. Several biosensors for the detection of these herbicides have been
developed (Giardi et al. 2001). Table 8.1 shows the various biosensors which detect
herbicides along with their Limit of detection (LOD). In the process of photosyn-
thesis plants, diatoms, algae, and cyanobacteria transform light into chemical energy.
The first step in this process is to break water (photolysis) to produce protons and
oxygen as a byproduct. This takes place in Photosystem II complex where water, plas-
toquinone, oxidoreductase enzyme, and protein D1 present. Herbicide’s molecules
interact and bind to D1 in PSII, which blocks the electron transfer and therefore
inhibits the process of photosynthesis.
8.3.1.3 Detection of Organophosphorus Hydrolase (OPH) Inhibiting

Pesticides
Organophosphate hydrolase is an organophosphotriester hydrolyzing enzyme that

uses organophosphate pesticides as a substrate and degrades it. The enzyme has
a broad substrate specificity toward analyte and is able to hydrolyze monocro-
tophos, chlorpyrifos, parathion, dursban, diazinon, etc. OPH enzyme-based biosen-
sors have low sensitivity but high detection values as compared to cholinesterase
enzyme inhibition-based biosensors. Limit of detection of those biosensors is given
in Table 8.1.
8.3.1.4 Glutathione-S Transferase (GST)
GST primarily metabolizes organophosphorus. It metabolizes endogenous and

exogenous toxicants leading to the formation of Glutathione conjugates with the
compounds or with their reactive intermediates (Fujioka and Casida 2007). It is used
for the detection of atrazine by developing a fiber optic biosensor (Andreou and
Clonis 2002).GST is immobilized on the glass using an intermediate binder sol-gel
layer. Nucleophile attack of atrazine is catalyzed by GST helping in release of H+
ions. This leads to a change in pH which changes the color measured by bromocresol
green.
8.3.1.5 Electrochemical Biosensors
Biosensors based on amperometric detection of pesticides: In amperometric detection

technic a constant potential to the working electrode is provided to the working
electrode and resultant current is measured by the device as a function of time.
Such amperometric biosensors have been constructed to detect biodegradable organic
pollutants in aqueous samples by measuring biochemical oxygen demand (BOD).
These amperometric biosensors used microbes as biosensing elements, examples
of which includes Serratia marcescens, Bacillus subtilis, and Arxula adeninivorans
(Liu and Mattiasson 2002; Chan et al. 2000; Kim and kwon 1999; Riedel et al. 1988;
Jia et al. 2003; Hikuma et al. 1979; Liu et al. 2004).
Similar microbial amperometric biosensors were also developed for the detec-
tion of organophosphate pesticide residues (e.g., fenitrothion, parathion, methyl
parathion, and ethyl p-nitrophenol thiobenzenephosphonate (EPN) (Su et al.
2011). Those biosensors were developed by co-immobilizing microorganisms and
organophosphate hydrolase enzyme (OPH). OPH hydrolyses organophosphate pesti-
cide molecules and due to which p-nitrophenol releases. Released p-nitrophenol is
then oxidized by those microorganisms (e.g., Pseudomonas putida JS444). Oxida-
tion of p-nitrophenol by microorganisms generates electroactive species, which is in
turn detected by amperometric biosensors. (Lei et al. 2004). Some microbes consume
oxygen, e.g., Arthrobacter sp. JS443, decrease in oxygen concentration in a system
Fig. 8.2 Amperometric detection of pesticide—A schematic illustration showing the enzyme-
based recognition of Organophosphate pesticideand amperometric detection, it illustrates enzyme
Acetylcholinesterase (AChE) inhibited by organophosphate pesticide and resultant change in current
is measured by potentiostat and displayed on monitor
is measure by Clark oxygen electrode as shown in Fig. 8.2 (working electrode) (Lei
et al. 2005, 2006; Mulchandani et al. 2005).
Potentiometric detection: Ion-selective electrodes or gas selective electrodes
have been developed with an immobilized microbial layer and the bio-analytes
are converted into electrical signals and the change in current is measured. Poten-
tiometric sensors measure the difference between two electrodes as well as one
reference electrode having constant potential. The change in current is because of
the change in sample dropped on the positive electrode (working electrode) and the
counter electrode. Direct detection of paraoxon using potentiometric detection was
done based upon immobilizing recombinant E. coli on a glass electrode. Opd gene
containing OPH enzyme was inserted into E. coli. OPH enzyme activated bacteria
which hydrolyzed organophosophorus pesticides leading to the production of two
protons. Concentration of paraoxon was related to the quantity of hydrogen ions
produced (Rainina et al. 1996). Again, a potentiometric biosensor was developed for
the detection of terbuthylazine (TBA). Urea labeled antibody was fixed while free
TBA and TBA conjugated BSA competed among each other for binding. Addition
of urea (substrate) helped in the measurement of ammonia. Production of urea is
inversely proportional to the amount of TBA present in a sample (Sassolas et al.
2012).
Conductometric detection: It is associated with the measurement of conduc-

tivity at different frequencies. Conductometric sensors depend upon the electrical
conductivity of the material that affects the conductivity of the analytes. Advantages
including low cost, simplicity, and no requirement of electrodes make conducto-
metric sensors predominant (Stradiotto et al. 2003). Jardim et al. used conductometric
sensors coupled with flow injection for detection of CO2 in atmosphere (Almeida
et al. 2001). Further, Valera et al. utilized conductometric sensors for detection of
atrazine. Immobilization of Atrazine on interdigited μ-electrodes. Detection of free
atrazine could be verified after a competitive reaction of immobilized atrazine with
the antibody present in the solution. The method of detection used gold nanoparti-
cles for labeling. The presence of gold nanoparticles produced red wine color which
detected atrazine (Valera et al. 2010; Sassolas et al. 2012; Valera et al. 2008).
8.3.1.6 Piezoelectric Biosensors/QCM Biosensors
Piezoelectric biosensors are mass-based biosensors that uses quartz crystal as a trans-
ducer. Piezoelectric biosensors use quartz crystal as s principle sensing component
and being highly sensitive, it is called Quartz Crystal Microbalance (QCM). QCM
can sense or measure up to nanogram level of changes in mass per unit area. QCM’s
crystal is made of silicon dioxide (SiO2 ) which can be tuned to oscillate with desired
frequency by providing appropriate voltage to metal electrodes coupled to the crystal.
Upon addition or deletion of mass (analyte) on to the surface of crystal coupled
area, frequency of oscillation of quartz crystal alters. This alters in the frequency of
the oscillation can be monitored in real time to acquire important information about
interaction/reaction of analyte happening at the sensor surface, e.g., antigen-antibody
interaction, etc. QCM has been reported to use under vacuum and gas phase (Sauer-
brey et al. 1959) and this technique was shown to be pertinent in liquid media, as
well (Nomura and Hattori 1980).
Molecular adsorption of the analyte onto the sensor surface can be done in gas
or vacuum phase, the result of which is the formation of rigid films adhered to the
sensor surface. The change in mass of such films is linearly related to the change
in frequency of oscillation. The change in oscillation frequency of quartz crystal is
given by infamous Sauerbrey Equation. Quartz crystal microbalance (QCM) provide
accurate and useful information with respect to change in mass deposited or mass
removal (it actually provides the rate of deposition or rate of removal of rigid films)
(Sauerbrey et al. 1959).
When QCM is operated in a liquid medium, water molecules may additionally
impart its mass on rigid films by hydration or by entrapment of water molecules.
Because of this viscoelastic or soft films can generate. The mass of such resultant
films cannot be determined accurately and the frequency of oscillation of such films
are not accurate because of the dampening of frequency or energy loss. To overcome
this problem new technic was developed. It called Quartz Crystal Microbalance with
Dissipation (QCM-D) (Rodahl et al. 1996). Figure 8.3 shows the over-all design of
the QCM biosensor.
Fig. 8.3 QCM-based biosensor—A schematic illustration showing the QCM sensor chip-based
detection of specific analyte (Pesticide). QCM sensor chip is composed of gold-coated glass slide
on which analyte (Pesticide) specific antibody is adsorbed, this antibody recognizes the pesticide
molecule and bind with it. The change in mass in response to pesticide binding with the selective
antibody is detected as a change in frequency of QCM transducer
As the maximum mass sensitivity of QCM in the liquid is less than 1 ng/cm2 , it has
been used to detect a number of different pesticides. QCM has been used to analyse
pesticide contamination in water and traces of deltametrin, fenthion, methiocarb,
and triadimol were found (Erbahar et al. 2012). QCM has been also used to detect
organophosphate pesticides. The presence of paraoxone or carbaryl is detected by
decreasing in the signal, i.e., resulting from its inhibitory effects. The standard curve
gives the detection limit of pesticide by plotting the percent inhibition vs. pesticide
logarithm concentration. Detection limits of 5.0 × 10−8 and 1.0 × 10−7 M were
obtained for paraoxon and carbaryl, respectively (Abad et al. 1998).
8.3.1.7 Surface Plasmon Resonance (SPR)
SPR is a non-radiative electromagnetic surface wave that moves in parallel to the

negative permittivity of the interface of the material. SPR involves reflection of free
electrons and protons in a specific angle which is absorbed in the film, called as SPR
angle (Sassolas et al. 2012). SPR-based biosensors can have real time monitoring
values during the detection of pesticides and notably it does not require any labeling
of molecules (Zhao et al. 2015; Homola et al. 1999). Illumination of polarized light
in specific conditions of complete reflection, having a thin film in between two media
having different refractive indices causes the production of SPR as shown in Fig. 8.4.
For the first time, Farré and fellows used real samples for detection using SPR (Farré
et al. 2007). Pesticides were primarily conjugated with BSA and was assembled
Fig. 8.4 SPR biosensor—A schematic illustration showing the SPR-based detection of a pesticide
molecule conjugated to Bovine Serum Albumin (BSA) protein and adsorbed on sensor chip. The
binding of BSA-Pesticide complex to the sensor surface increase the refractive index, which induces
shift of the SPR angle. The shift is directly proportional to the mass increase
on a monolayer of gold electrode which is reusable. Change in concentration of

samples leads to change of refractive indices (Sassolas et al. 2012). Detection of pesti-
cides using SPR was a tremendous advancement as real samples could be detected
without pre-treatment modifications (Cortina-Puig et al. 2010; Zhang et al. 2009).
A low concentration of any molecule can also be detected using antigen-antibody
recognition using SPR biosensor.
8.3.1.8 Fluorescence Polarization (Total Internal Reflection

Fluorescence-TIRF)
TIRF uses properties of induced evanescent wave or field in a specific region asso-
ciated with the specimen that is present close to the interface of two media having
different refractive indices. TIRF is used to observe the composition, extent, and
position of the substrate (Weis et al. 1982). spectroscopy and visualization of single-
molecule fluorescence close to a surface can be done for single-molecule detection
as shown in Fig. 8.5 (Kuter et al. 2008; Khan et al. 2000, 1996; Dickson et al. 1998;
Sako et al. 2000; Ha et al. 1999). Apart from these, there are several applications of
TIRF including (a) measuring kinetic rates of intracellular and extracellular protein
binding to cell surface receptors and artificial membranes (Thompson et al. 1981;
Burghardt and Axelrod 1981; Axelrod 2001; Fulbright and Axelrod 1993; Stout and
Axelrod 1994; Mc Kiernan et al. 1997; Sund and Axelrod 2000; Nollert et al. 1995),
(b) Structural dynamics of living cells (Mathur et al. 2000), (c) Fluorescence of cells
during cultures (Wang and Axelrod 1994), (d) comparing membrane proximal ionic
transients and transients in the cytoplasm (Omann and Axelrod 1996). Recently,
europium-chelate-dyed polystyrene nano-composites are being produced for detec-
tion of atrazine while TIRF has been used for the development of immunosensors to
estimate water pollution (Sassolas et al. 2012; Barzen et al. 2002).
Fig. 8.5 Fluorescence Polarization: Fluorescence polarization—Excitation of fluorescent labels

leads to selective absorption of polarized light by appropriately oriented molecules. Polarized emis-
sion of these oriented molecules occurs when their rate of rotation is low relative to the rate of
fluorescence emission
8.3.1.9 Cantilever-Based Biosensors
Cantilever-based biosensors detect biomolecular interactions in a label-less proce-

dure with high efficiency. Cheap and reliable standard silicon technologies are used
in fabricating cantilevers. The working principle of cantilever-based biosensors is
based upon translating biochemical reactions into mechanical movement. Biomolec-
ular recognition shows a quantitative change in the cantilever beam, when there is
an interaction between an analyte and its receptor (Ziegler 2004). Fabrication of
cantilever can be possible in any shape like crystalline, polymer, silicon nitride or
silicon oxide. After fabrication, the preliminary setting of beam was quite compli-
cated, so newly developed cantilever biosensors are made up of single crystal silicon
as shown in Fig. 8.6. Applications of cantilever-based biosensors are observed in
environmental fields and in immunological detection. Biosensors are based upon
antigen-antibody interaction directly, by specific immobilization of haptens on the
surface and modulating the binding of antigen; or indirectly, by checking the inhibi-
tion proportions of the antibody with the antigen present on the immobilized surface
(Alvarez et al. 2009).
Fig. 8.6 Cantilever-based biosensor—A schematic illustration showing the nanomechanical detec-
tion of an anti-pesticide antibody-pesticide interactions on a cantilever. It illustrates the cantilevers
coated with anti-pesticide antibody on a gold surface. Pesticide in solution forms a binding event
with the antibody, causing the cantilever to bend downward due to compressive surface stress
Stimulation of Cantilever and Fabrication: z is called a cantilever deflection

that is generated by change in surface stress, s, that depends upon microcantilever’s
dimensions, i.e., length l, thickness h, and properties of the material. According to
Stroney’s equation, a greater change in surface stress will be higher for longer and
thinner cantilevers (Alvarez et al. 2009).
8.3.1.10 Lateral Flow Biosensors
Lateral flow biosensors are considered as improvisations in immunochromatogenic

assay (Mishra et al. 2018; Gandhi et al. 2009; Parolo and Merkoçi 2013). Lateral
flow biosensors have a low limit of detection, low sample required for analysis, high
sensitivity, better selectivity, low cost, and quick assay with single-step detection
(Quesada-González and Merkoçi 2015). Further, lateral flow biosensors can be used
in the detection of biomarkers that includes nucleic acids, proteins, and even whole
cells. Recent research shows evidence in the detection of metal ions and pesticides.
Along with all these advantages, lateral flow biosensors have a few disadvantages.
Results in lateral flow are qualitative but are not quantitative and solid or gaseous
samples cannot be analysed in these sensors. It consists of a nitrocellulose layer and
unspecific absorption on the pores can give biased results. However, commercially
available lateral flow biosensors are used for pregnancy detection and HIV test (Pesce
and Spitlnik 2007) as well as malaria (Cordray and Richards-Kortum 2012; Kersting
et al. 2014). Advancement in the field of nanotechnology has provided opportunity
in improvement of performance by these sensors.
Working of lateral flow biosensors: Manufacturing of lateral flow assays occurs
in the form of strips. It consists of four parts, (a) cellulose pad, where the sample
is placed, (b) a conjugate pad that helps in conjugation, usually made up of fiber,
(c) detection pad, and (d) nitrocellulose sheet consisting both control and test line
as shown in Fig. 8.7 (Ahmad et al. 2009). Furthermore, lateral flow biosensors that
could be connected to electrical devices by integrating carbon nanotubes on top of
them (Zhu et al. 2014).
8.4 Conclusion
Biosensors have always been a vital apparatus for pesticide detection. Develop-
ment of different sensors with increased efficiency and accuracy has remarkably
increased. Biological elements including antibodies, aptamers, enzymes, or whole
cells are being used for detection. In recent times, development of genetically modi-
fied enzymes like AChEs have a better attachment and efficiency in comparison
to normal enzymes and are being widely used in enzyme inhibition for pesticide
detection. Further, genetically engineered microorganisms are being used for detec-
tion of pesticides. Nanotechnology has been playing a significant role in developing
biosensors for pesticide detection. Nanomaterials such as nanorods and nanotubes
Fig. 8.7 Lateral flow assay (LFA)—(a) A schematic illustration showing design of LFA pad. (b) if
pesticide is absent in the test sample then only one line (Control) appears (c) if pesticide is present
in test sample then pesticide bind to capture antibody which is specific for that pesticide, after
pesticide captured on capture antibody, it is recognized by anti-pesticide antibody which is coated
on gold nanoparticle and test line appears along with control line
are being used for increasing the efficiency of the sensors. However, biosensors in
the field of pesticide detection are not as enhanced as in medical applications. Hence,
development of low detection limit, user-friendly, and cost-effective biosensors are
required.
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Chapter 9
Application of Biosensor
for the Identification of Various
Pathogens and Pests Mitigating Against
the Agricultural Production: Recent
Advances
Charles Oluwaseun Adetunji, Wilson Nwankwo,

Kingsley Eghonghon Ukhurebor, Akinola Samson Olayinka,
and Ayodeji Samuel Makinde
Abstract The growing global population which is estimated to hit 9 billion by

2050 is considered a time bomb considering the scarce resources at the disposal of
the nations. Hence, there is a need to identify a sustainable tool that could lead to
an increase in the production of food production globally. Smart monitoring using
biosensors ensure that chemical and other forms of contaminants are kept at bay
from compromising the quality and safety of the stored food as well as the pest and
pathogens that could affect fresh agricultural produce. Biosensors are also deployed
for the purpose of measuring carbohydrates, alcohol, acids. Though present tech-
niques in chromatography like high-performance liquid chromatography, capillary
electrophoresis, flow immunoanalysis, gas chromatography, and spectroscopic tech-
niques (Ultraviolet-visible spectroscopy, nuclear magnetic resonance spectroscopy,
fourier-transform infrared spectroscopy) are advantageous because they are highly
responsive, reliable, and efficient for pesticide residue analysis as well as the applica-
tion of immunoanalysis in plant pathogen detection, and utilization of electrophoresis
for plant nucleotide evaluation. However, these techniques are time-consuming,
C. O. Adetunji (B)
Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Department of
Microbiology, Edo University Iyamho, P.M.B. 04, Auchi, Edo State, Nigeria
W. Nwankwo · A. S. Makinde
Climatic/Environmental/Telecommunication Physics Unit, Department of Physics, Edo
University Iyamho, P.M.B. 04, Auchi, Edo State, Nigeria
K. E. Ukhurebor
Informatics and CyberPhysical Systems Laboratory, Department of Computer Science, Edo
University Iyamho, P.M.B. 04, Auchi, Edo State, Nigeria
A. S. Olayinka
Department of Physics, Edo University Iyamho, P.M.B. 04, Auchi, Edo State, Nigeria
170 C. O. Adetunji et al.
arduous, costly, complicated, and voluminous and involve the operation of quali-
fied technicians. Hence, the application of biosensors has been identified as a reli-
able technology for effective detection of pathogen and pest affecting the increase
in the agricultural production as well as utilized to reduce the impact of pesticides
on farmlands and on the health of the farmers and residents. Therefore, this chapter
intends to provide comprehensive detail about the application of biosensors for the
identification of pest and pathogens affecting the increase in agricultural produc-
tion. Numerous types of biosensors with high relevance in the detection of pest and
pathogen were also highlighted.
Keywords Pathogen · Pest · Agriculture · Food · Biosensor · Global population
9.1 Introduction
Food security and safety have been a major concern of researchers as the world popu-
lation is estimated to peak at 9.7 billion by the year 2050 from the current estimate of
7.7 billion. This is due to the fact that an increase in population will naturally translate
to a corresponding increasing in food demand. It has been stated that adequate insight
on the nutrition and the varying nature of malnutrition have enhanced curiosity in diet
quality. Most countries around the globe do not have an adequate sub-national level
and national data about diet and diet quality. Nevertheless, there is minimal insight
about dietary outlines, as well as little knowledge on health policy, food industry,
trade, and agriculture could be utilized for enhancement of diet quality (Vandevijvere
et al. 2013). At present, it has been validated that the implementation of up-to-date
and forthcoming technologies together with intelligent policy execution will be the
operative and workable path to deciphering food security trials (Barretto et al. 2013;
Charlebois 2015).
Also, pests and diseases have been identified as one of the biotic factors respon-
sible for numerous agricultural losses. So, there is a need for an all-encompassing
approach to address this devasting effect on food security and sustenance. Concerted
effort has been geared towards curtailing the spread of disease and pests across
borders by International Plant Protection Convention (IPPC), but the in situ pest and
disease remains a menace in agricultural production system. Since the invention of
biosensors, which is dated back to 1950, much attention has been drawn to its appli-
cations in various strata of human endeavours, medicine, drug discovery, ecological
pollution control, and agriculture. It has been successfully used in the food industry
as a quantifier for biological signals in quality control developments.
As discussed in other chapters, biosensors are bioelectronics devices used for
signal detection and processing of biological materials which may be classified based
on different sensing techniques. Piezoelectric biosensors, which are also branded as
acoustic biosensors work on the principle of sound vibrations. Electrical signals are
produced when vibrational force is sensed by a piezoelectric biosensor. In elec-
trochemical biosensors, the probing surface is coated with biological (sensing)
molecules and held in place with non-interfering membrane. Optical biosensors
9 Application of Biosensor for the Identification … 171
allow signal sensing using various properties of light like scattering, absorption, and
diffraction. Smart biosensors, these types of biosensors belong to recent advances in
biosensing technology. These emerging biosensors have been termed smart because
of its inherent intelligence derived from machine learning and other computational
techniques. Smart biosensors can be optical, piezoelectric, or electromechanical
based (Adetunji et al. 2018).
Hence, this chapter intends to provide comprehensive detail about the application
of biosensors for the identification of pests and pathogens affecting the increase in
Agricultural production. Numerous types of biosensors with high relevance in the
detection of pests and pathogens were also highlighted.
9.2 Principles of Biosensor Operation
The first biosensor was in the early 1950s by Leland Clark, a biochemist who wanted
a device that could effectively detect the amount of oxygen circulating in the blood
(Nastuk 2013; Clark et al. 1950). His biosensor was then called the Clark electrode.
The Clark electrode was purely an oxygen detector and nothing more largely because
only oxygen-permeable membranes could be utilized (Miniaev et al. 2013; Holt
2007). Following the creation of Clark electrode, there have been several develop-
ments thereafter. The first improvement on the oxygen detector was the overlaying of
a glucose-sensitive enzyme film over the existing electrode and this further enhanced
the functionality of the Clarke electrode towards acting as a blood sugar sensor. The
blood sugar sensor is used for detecting and measuring the level of blood glucose
(Ghosh et al. 2020; Zhang and Hoshino 2014). In furtherance to this development
was the use of a layer of urease enzyme which was able to detect urea in blood, urine,
etc. (Morales-Cruz et al. 2019; Tricoli and Neri 2018).
The concept of biosensor is derived from the two keywords that constitute the
word i.e. bio and sense. Bio connotes life or living whereas sense connotes detection
or recognition. In other words, biosensing implies the ability to recognize something
within or around a living environment or living organism. Thus, a biosensor is any
device designed to detect, recognize, measure, and relay information on a biophys-
ical or biochemical substance e.g. oxygen, glucose, urea, protein, neurotransmitter,
etc., around or within a living organism or environment, through electrical signals.
Invariably, a biosensor can work inside or outside a living organism however, the
target characteristic parameter to which the device is applied to detect/measure often
emanate from the living organism or ecosystem (Cinti et al. 2017; Ali et al. 2017;
Kaur et al. 2018).
Biosensors may be categorized according to the following characteristics:
a. Targeted analyte: is the target substance to be sensed e.g. glucose biosensor,
oxygen biosensor, nitrogen biosensor, DNA biosensor. Note that the analyte
must have an affinity for the sensing surface to which it may react chemically,
and electrochemically.
b. Bioreceptor: This is the deactivated medium (an immobilized hard or soft

biocomponent) often selectivity specific, that is to be exposed to the targeted
substance or analyte (Kahn and Plaxco 2010). The common bioreceptor
categories are enzymes (oxidoreductases, peroxidases, polyphenol oxidases
aminooxidases, etc.) (Mehrotra 2016), tissues, antibodies, DNA, microbes (Kaur
et al. 2018), biomimetics (Bharat 2009; Chauhan et al. 2020), nanoparticles. The
bioreceptor is often interfaced to the transducer.
c. Generation (the period during when it was launched; biosensors manufactured
and introduced into the market over a given period with similar characteristics
belong to a generation). There are first generation, second generation, third gener-
ation, etc. Like computers and other electronic devices, the later generation often
shows more sophistication than the earlier generation. The sophistication may be
expressly defined in terms of precision, energy consumption, multifunctionality,
accuracy, size, the scale of integration, and refinement.
d. Sensing or transduction strategy: The sensing strategy depicts the principle of
detection adopted and embedded in the biosensor for detection. Ordinarily, the
strategy is based on the behaviour of the transducer. Modern strategies include
electrical, optical, piezoelectric, photoelectric, thermometric, photoelectrochem-
ical, nano, and electrochemical sensing strategies (Monosik et al. 2012; Niu et al.
2018; Farzin et al. 2020; Long et al. 2020; Zhou and Tang 2020).
Biosensors are not just detectors; they also serve analytical functions depending
on the domain of application. For example, as an analytical tool, a biosensor may be
deployed to detect the specific existence of specific biomolecules, microorganisms,
biological structures, or any other biological analytes. Biosensors operate on the prin-
ciple of signal transduction (a process involving the relay of physical or biochemical
signals that reflects molecular events from a cell or substance in such a manner that
is measurable (Mehrotra 2016). Biosensors are more advanced regarding selectivity
and sensitivity if compared with any other currently existing diagnostic tool. Some
of the significant benefits of biosensors include fast and continuous measurement,
reduced use of reagents, elimination of calibration and re-calibration, high precision,
quick response time, ability to measure non-polar molecules that other traditional
devices cannot estimate.
9.3 Electronic Principles of Biosensor
As stated earlier, biosensors operate on signal transduction principle. The main

components of a typical biosensor include an element of biorecognition, a biotrans-
ducer, and an electronic component that may integrate, microprocessor, amplifier,
and display/monitor, respectively (Gupta and Kakkar 2020). The biorecognition or
sensing component is essentially a bioreceptor designed to detect or act on a partic-
ular analyte—the target substance whose relevant characteristics is to be detected or
measured. The transducer receives input from the bioreceptor and depending on the
transducer employed, a signal is relayed to the signal processor. The signal output
amplitude is proportional to an analyte’s concentration, and then amplified by the
electronic device and processed (Sode et al. 2016).
For instance, in an amperometric sensor, the bioreceptor comprises a deactivated
enzyme or specific biomaterial, and said deactivated biomaterial is in close contact
with the transducer. The analyte reacts with the biomaterial. In this way, a new analyte
may be formed which in effect gives the calculable electronic reaction. Sometimes,
the analyte is transferred to a system that can be attached to the gas, heat, electron
ions, or hydrogen ions discharge. In this, the transducer can adjust the device linked,
then converts to electrical signals that could be altered and computed.
Biosensors could be distinguished by way of the transduction sensing strategy
employed in its construction, based on the principle of translating biological signal
into an electronic signal. Common families are electrochemical, calorimetric, optical.
In some cases, there one family may dovetail into the other in which case there may
not be very clear boundaries of distinction. One remarkable feature of any biosensor is
specificity and selectivity to the analyte. A particular bioreceptor needs to be activated
towards its attachment to the transducer/electrode to ensure that only the specific
analyte is detected. Bioreceptors may be proteins (antibodies, enzymes) (Song et al.
2018), cellular material (cells, tissue culture, etc.) (Soleymani et al. 2018, 2020),
nucleic acids (DNA, RNA) (Mittal et al. 2017; Saadati et al. 2019), nanomaterial
(Zamora-Galvez et al. 2017), or biomimetic material (Moro et al. 2019).
Seven major types of biosensors based on sensing strategy utilized in the construc-
tion of the biosensor have been identified (Akgönüllü et al. 2020) There is also an
evolving category, nanobiosensors (Sharifi et al. 2020; Sung et al. 2019), which are
derived from nanomaterials. A nanobiosensor may be a hybrid device, that may utilize
different sensing strategies available in the other major types highlighted above.
9.4 Mode of Operation of Biosensors
The components of a biosensor are instrumental to its operation. Together with

physicochemical transducers, biosensor harness the exquisite sensitivity and speci-
ficity of the biology to deliver complex bioanalytical measurements in simple, and
easy-to-use formats (Turner 2013). First, every biosensor is designed to perform
specific functions. The way and manner a biosensor operates is dependent on the
bio receptor (enzyme, DNA, phage, antibody, and many more) and the sensing
strategy i.e. the workings of the transducer. The transducer’s signalling is always
small and overlays on a fairly high baseline. Usually, the processing of the signal
requires the deduction of a baseline location signal, collected from a similar trans-
ducer without covering any biocatalyst. The direct output is an analogue signal at
this point (Malhotra et al. 2017). However, the signal may be digitized and relayed
to the microprocessor unit where the information is processed, guided by preferred
units, and exported to a data store.
9.5 Biosensors in Agriculture
Agriculture is a major sector that pervades other sectors. It contributes to the

development of all other economic sectors (Veldhuizen et al. 2020). Like educa-
tion, agricultural innovations are desired for economic sustainability (Nwankwo and
Njoku 2020). Agriculture contributes to socioeconomic development through prod-
ucts and raw materials, foreign exchange, market, poverty alleviation, employment
(Nwankwo et al. 2019).
9.5.1 Applications of Biosensors in Agriculture
Summary of areas to which biosensors are deployed in agriculture is given in

Table 9.1. Some other applications are discussed herein.
9.5.2 Biotoxicity Detection
Annually, a significant percentage of the citizenry in developing societies suffers

from pesticide contamination. Various pesticides are commonly utilized in the fields
by farmers around the world to prevent damages due to pests and diseases during the
various phases of plant growth. However, the application or use of these chemicals
has some significant effect on the farmer, as the drift of sprayers contributes to air
pollution and the worker’s safety is severely affected by the inhalation of the spray
particles. The floating particles and spray droplets often contaminate the water bodies.
Biosensors are now used to reduce the impact of pesticides on farmlands and on the
health of the farmers and residents (Kundu et al. 2019). A good number of available
pesticides has carbamates and organophosphates as their main components. Biosen-
sors have been applied to enhance the monitoring of toxicity and risks that may ensue
from the use of pesticides on farmlands. Three things are achieved through pesticide
toxicity biosensors (Songa and Okonkwo 2016; Garg and Mehrotra 2017; Uniyal
and Sharma 2018), first, to prevent the excessive absorption of harmful pesticides
by crops, fruits, and stored products, second, to prevent soil toxicity and subsequent
destruction of useful soil flora; and third, to prevent local and remote health hazards
arising from toxic substances and environmental pollution.
Several methods have been reported recently in the development of biotoxicity
detection biosensor. Gao et al. (2017) fabricated a double-mediator based whole-cell
electrochemical biosensor for acute biotoxicity detection of wastewater. Biosensor
sensitivity was improved using ferricyanide-menadione double-mediator. Also, a
chitosan hydrogel polymer film with boron-doped nanocrystalline diamond particle
Table 9.1 Some biosensor applications in agriculture

Transduction type Electrode Analyte detected Benefits References
Electrochemical Magnetic Salmonella in milk No requirement of Liébana
magneto-immunosensing graphite-epoxy reagents and is et al. (2009),
composite rapid detection Brandão
(m-GEC) et al. (2015)
Electrochemical magnet Magnetic b-lactamase GEC products have Pividori and
immunosensing graphite-epoxy resistance in weak non-specific Alegret
composite Staphylococcus adsorption for either (2005),
(m-GEC) aureus DNA samples or Adetunji
enzymes labels. et al. (2018)
They do not require
blocking steps on
the transducer’s free
sites to mitigate
non-specific
adsorption
Gold nanoparticle-based graphite-epoxy Salmonella IS200 Gold Brasil de
composite nanocomposite is a Oliveira
good substrate for Marques
improved and et al. (2009),
directed Wang et al.
immobilisation of (2016)
biomolecules with
excellent
transductive
properties for the
construction of a
wide variety of
electrochemical
biosensors, such as
immunosensors,
genosensors, and
enzyme sensors
Amperometric Working and Staphylococcus The electrochemical Majumdar
electrochemical counter aureus in milk, assay technique has et al. (2012),
immunoassay electrode of cheese, and meat been shown to be Silva et al.
Platinum (Pt), fast, effective, and (2018)
and Ag/AgCl reproducible, and
reference can be used to
electrode detect certain
pathogenic
microorganisms
(Salmonella spp,
Lysteria
monocytohenes, or
Staphylococcus
aureus) by using
antibodies against
antigens
(continued)

Transduction type Electrode Analyte detected Benefits References
Multiplexing optical NA E. coli O157: H7, The total Karsunke
(luminescence) S. typhimurium and measurement and et al. (2009),
Legionella calibration assay Maas et al.
pneumophila time is 18 min, (2018)
enabling very rapid
analysis
High-density NA E. coli O157: H7 Field ready device Radke and
microelectrode array bacteria in food that is easy to use, Alocilja
materials compact, and (2005)
reagent-less. Can
provide rapid result
within minutes
Flow-type antibody quartz crystal E. coli in drinking The sensor Kim and
sensor microbalance water, beef, pork, measures changes Park (2003)
chip and dumpling in frequency due to
mass deposits that
are formed by
antigen-antibody
interaction
Acoustic-based biosensor NA DNA detection Device significantly Papadakis
(the Quartz Crystal reduces the et al. (2015)
Microbalance) processing of time
by avoiding gel
electrophoresis and
can be combined in
a diagnostic
laboratory or an
automated
lab-on-a-chip
device for plant
pathogen
diagnostics
NA Not available
was electrodeposited onto a glassy carbon (GC) electrode to immobilize Saccha-

romyces cerevisiae cells and the mediators. Zhang et al. (2019) developed a microflu-
idic paper-based analytical biosensor device with benzoquinone using E. coli as a
direct biotoxicity indicator through its respiratory inhibition induced by toxicants
to measure the biotoxicities of pollutants. Detection samples in the form of a solu-
tion, vegetable juice, and soil were also applicable to the proposed microfluidic
paper-based analytical biosensor device.
Qiu et al. (2018) demonstrate the feasibility of using an aptamer-invertase
biosensor integrated with a personal glucose meter (PGM) to quantify quinine detec-
tion in reclaimed wastewater. The limit of detection for quinine detection in pure
water and recycled sewer are 0.13 μM and 0.32 μM, respectively. Chen et al. (2020)
developed an Escherichia coli-based electrochemical biosensor for detecting myco-
toxin (aflatoxin B1 and zearalenone) toxicity. E. coli acts as a signal recognition
Table 9.2 Biotoxicity detection sensors

Biosensor Biotoxicity detected Mediator References
Double-mediator based Acute toxicity of real Menadione Gao et al. (2017)
whole-cell wastewater
electrochemical
Microfluidic paper-based Biotoxicities of Benzoquinone Zhang et al. (2019)
analytical pollutants
E. coli-based Mycotoxin p-benzoquinone Chen et al. (2020)
electrochemical
Aptamer-invertase Quinine NA Qiu et al. (2018)
biosensor integrated with a
personal glucose meter
(PGM)
Mediated microbial Toxicity detection in 1, 4-benzoquinone Yu et al. (2020)
electrochemical water
NA Not available
element, while p-benzoquinone acts as the mediator. A two-step reaction procedure

has been developed to separate the mediator from the mycotoxins. The various types
of a biosensor used in the detection of biotoxicity are listed in Table 9.2.
9.5.3 Sustainable Food Safety and Quality
Food quality and safety are important during the harvest and postharvest stages.
The farm produces such as grains, tubers, etc. should be protected from contami-
nation by chemicals. Smart monitoring using biosensors ensures that chemical and
other forms of contaminants are kept at bay from compromising the quality and
safety of the stored food. Biosensors are also deployed for the purpose of measuring
carbohydrates, alcohol, acids, etc. The use of these facilities helps food technolo-
gists and producers to conduct quality assurance in that the food content or mixture is
certified based on its measured quality parameters in respect of the various nutrients
e.g. carbohydrates, proteins, alcohols, vitamins, gases, etc.
9.5.3.1 Immunosensors
Immunosensors are deployed in food processing and packaging for quality assurance
and successful contamination detection purposes. One of its major applications is to
enable adequate interaction between antibodies and antibodies (Zhu et al. 2019). This
immunoassay functions similarly to the enzyme-linked immunosorbent enzyme-
linked assay (ELISA). Detection of antigens is achieved in food items through obser-
vation of the antibody-antigen binding that is connected to the substrate-bearing
Table 9.3 Immunosensors development for food quality monitoring

Immunosensor Test samples Detected Limit of detection References
Label-free Drinking Escherichia 94 CFU mL−1 Kaushik et al.
immunosensor water and coli (2019)
orange juice
Laser-Induced Graphene Chicken Salmonella 13 ± 7 CFU mL−1 Soares et al.
Electrochemical Broth enterica (2020)
Immunosensors
Electrochemical Food Escherichia ~30 CFU mL−1 Güner et al.
immunosensor coli O157: H7 (2017)
electrochemical gold Cookies and Ara h 1 3.8 ng mL−1 Alves et al.
nanoparticle-coated chocolate (2015)
screen-printed
immunosensor
electrochemical Paste, rice Gliadin 1.2 ng mL−1 Chekin et al.
label-free immunosensor flour, wheat (2016)
flour,
cornflour,
quaker oats
magnetoimmunosensing Peanut Ara h 1 6.3 ng mL−1 Montiel et al.
platform based (2015)
derivatives,
Hazelnuts
enzyme. The antigen quantity or volume is known to exhibit a proportional rela-

tionship with the fluorescence produced by the interaction of antigen and antibodies
(Sakamoto et al. 2018).
Several studies have been conducted recently on immunosensors development
for food quality monitoring. Seo et al. (2016) fabricated an immunosensor system
using CMOS image sensor (CIS) to pocket-size dimensions for detecting food-borne
pathogen (Vibrio parahaemolyticus). The CIS helps in resolving the detection issue
hindering monitoring via the internet of things (IoT). Information collected from
the sensor is uploaded as data to the server to be shared with the public. Kaushik
et al. (2019) proposed a fibre optic surface plasmon resonance immunosensor based
on biofunctionalized molybdenum disulphide (MoS2 ) nanosheets for rapid detec-
tion of E. coli. The immunosensor uses wavelength interrogation method with a
strong linear relationship of R2 = 0.994 was observed between the spectral response
of immunosensor and different concentrations of E. coli. It was recorded that the
method shows promising applications in regular water and food quality monitoring
for different pathogenic microorganisms.
Soares et al. (2020) reported the fabrication of highly sensitive label-free
laser-induced graphene electrochemical immunosensors to quantify the food-borne
pathogen Salmonella ecterica in chicken broth. The list of immunosensors utilized
for food quality monitoring is given in Table 9.3.
9.5.3.2 e-nose
This is commonly used for detecting, recognizing, and classifying volatile

compounds. They may be utilized to categorize odoriferous agricultural products.
The e-nose is a smart tool. It mimics the human olfactory organs. e-nose consists
of the sensing element, signal collection unit, and an effective algorithm for pattern
recognition. An e-nose was successfully implemented in agricultural applications for
the determination of fruit maturity, identification of soil-borne pathogens, inspection
of fish, and many more. The e-nose detection principle is that the sensor array iden-
tifies an odour composed of many different volatiles in the headspace of a sample.
It then produces an output that reflects a fingerprint of all the components for the
sample. The fingerprint identified by the e-nose sensors is used for detection of
possible sample information based on suitable algorithms provided (Qiu and Wang
2017).
Across many fields, e-nose has become an increasingly common detection device
with significant advantages such as high sensitivity, simple construction, high
response, and recovery time, operate at room temperature, and cost-effectiveness
(Deshmukh et al. 2015). Huang et al. (2017) forecast the days until peach fruit decay
using near-infrared spectroscopy and an e-nose. Their study shows that the integration
of near-infrared spectroscope and e-nose gives reliable and rapid forecasting of the
day before decay of peach fruit with 82.26% accuracy. Tan and Kerr (2019) employed
the use of e-nose to continuously monitor the volatile compounds of cocoa samples
going through refining and conching that differs in degree of roasting and mass.
The e-nose uses kernel distribution model to characterize volatiles profiles. Dong
et al. (2019) measured the quality properties effect of roasted coffee beans under
different drying process conditions (solar drying, hot-air drying, room temperature
drying, heat pump drying, and freeze-drying) using e-nose. It was recorded that the
drying process affected the total titratable acidity, pH, total soluble solids, and total
solids. Wen et al. (2019) developed a sweeping electronic nose system (SENS) for
detecting early infestation in citrus fruits. The e-nose based system utilized eigenvalue
extraction method for sensor signal, INV, and RSVA. The infestation citrus fruits are
classified using linear discriminate analysis (LDA) and principal component analysis
(PCA). Their research demonstrated the possible feasibility of the e-nose technology
under market conditions for in-filed detection of postharvest pest infestation in citrus
fruit. The numerous types of e-nose development for food quality monitoring is given
in Table 9.4.
9.5.3.3 Surface Plasmon Resonance (SPR) Sensor
SPR biosensors have been widely applied to calculate kinetics and bimolecular
binding affinity in real-time and label-free mode with low reagent consump-
tion. Furthermore, the SPR biosensor acts as an essential tool in multichannel or
array-imaging instrumental set-ups for the high-throughput study of food allergens
(Zhou et al. 2019a, b). However, conventional SPR technologies suffer from many
Table 9.4 e-nose development for food quality monitoring

e-nose sensor Test sample Application References
Metal oxide sensor Peach Fruit decay Huang et al. (2017)
Metal oxide gas sensor Potatoes Soft-rot infection Rutolo et al. (2018)
detection
Gas sensor Spinach Odour Classification Shen and Tao (2018)
Gas sensor Cocoa Monitoring volatile Tan and Kerr (2019)
compounds during the
refining.
MOS sensor Coffee Profiled volatile and Dong et al. (2019)
Taste properties.
MOS sensor Citrus Early detection of Wen et al. (2019)
infestation in citrus fruits.
MOS gas sensor Broccoli Freshness Analysis Chen et al. (2019)
MOS sensor Galangal Odour classification
performance limitations: (1) several allergens are challenging to examine in food

samples at the same time; (2) SPR equipment is too large and costly and thus cannot
be used to detect food allergen in a consumer-friendly way (Zhou et al. 2019a, b).
Farka et al. (2016) developed an SPR immunosensor based biocatalyzed precipi-
tation for the analysis of Salmonella in dairy products. The specific capture antibody
on the SPR chip was immobilized, which allowed a precipitation-enhanced detection
of Salmonella with a limit of detection ranging from 100 to 106 CFU mL−1 . The
total analytical time, including the binding and enhancement stage for the bacteria,
was less than 60 min. Zhou et al. (2019a, b) reviewed four types of surface plasmon
resonance (SPR) biosensors for allergens detection in milk, egg, peanut, and seafood.
The four studied SPR include localized surface plasmon resonance (LSPR), fiber-
optic surface plasmon resonance, transmission surface plasmon resonance (TSPR),
and surface plasmon resonance imaging (SPRI). The SPRI biosensor can be joined
with LSPR and TSPR with the advancement of technologies to provide an accurate,
secure, economical, and highly sensitive method for controlling hundreds of food
allergens in one or more food matrices. The typical examples of SPR sensor utilised
for food quality monitoring is listed in Table 9.5.
9.5.3.4 Quartz Crystalline Microbalance Biosensor
This is an acoustic biosensor used to detect bacterial phytopathogens of tomato,

pepper, etc. such as Pseudomonas syringae, Xanthomonas vesicatoria, Xanthomonas
campestris, and Ralstonia solanacearum (Papadakis et al. 2015). Compagnone
et al. (2015) carried out quality control test on two series of chocolate samples
having different formulated products over two sets of sensors including metal-
loporphyrins and nanoparticles peptide coated quartz crystal microbalances. The
Table 9.5 SPR development for food quality monitoring

SPR sensor Test sample Capturing ligand Limit of detection References
SPR based Powder Milk Antibody 100 - 106 CFU mL−1 Farka et al.
biocatalyzed (2016)
precipitation
SPR Milk Antibody 57.8 ng mL−1 Ashley et al.
(2017)
Gold-coated Water E. coli 1.5 × 103 CFU mL−1 Arcas et al.
U-shaped plastic (2018)
optical fiber (POF)
SPR Egg Antibody 0.66 nM Saylan et al.
(2017)
SPR Milk NanoMIPs 127 ± 97.6 ng mL−1 Ashley et al.
(2018)
SPR Shellfish Antibody 1.0 μg mL−1 Zhou et al.
(2019a, b)
Table 9.6 QCM sensor development for food quality monitoring

QCM sensor Test sample Detection method Limit of References
detection
QCM mango Beta-caryophyllene 0.32 ppm Ali et al. (2018)
Piezoelectric Banana, ethylene ethylene 0.42 ppm Tolentino et al.
pear, orange (2018)
QCM tomato trans-2-hexenal NA Debabhuti et al.
(2016)
QCM melon Ethyl butyrate or 0.2 mmol Sasaki et al. (2018)
limonene L−1
NA Not available
first series of chocolate was obtained under standard process conditions while the
second comprising sample with some volatile compounds associated with low-
quality raw materials. It was recorded that the best performance was obtained using
the gold nanoparticle peptide sensors. Cervera-Chiner et al. (2018) proposed a high
fundamental frequency quartz crystal microbalance (HFF-QCM) immunosensor for
detecting pesticide in honey. The HFF-QCM is known to have high sensitivity,
low cost, and fast detection. The HFF-QCM is based on piezoelectric sensors with
frequencies of 100 MHz. Liu and Zhang (2020) have reviewed the state-of-the-art
volatile organic compounds gas sensor based on quartz crystal microbalance for
detecting fruit freshness. The QCM-based gas sensor has been widely applied to
judge the freshness, shelf life, and maturity of fruits because of its high integration
level, low cost, and ease of access to changing the sensitive membrane material. The
QCM sensor development for food quality monitoring is listed in Table 9.6.
9.5.4 The Utilization of e-nose for Plant Diagnosis
The plant volatile organic compounds have been identified to play a crucial role in
their reaction against pest detection. The composition of volatile organic compounds
liberated by plants could be linked to types of damage. It has been stated that
volatile organic compounds are biologically generated due to numerous physio-
logical processes that occur in different types of plant tissue. Whenever agricultural
pathogen invades the plant defence mechanism after infection there is a tendency
that the composition of the volatile organic compounds might change. Plant needs
numerous ranges of defence mechanisms to fight against numerous mechanical and
herbivorous insects (Choudhary et al. 2008).
It has been discovered that the liberation of volatile organic compounds depends on
the response of the potential pathogen and pests whenever they attach plant which
area triggers in response to their attack. These constitute the changes of volatile
organic compounds which give reliable principles of pest discovery using e-nose
most especially from bacterial and fungi infections as well as insect infestations in
plants (Pare and Tumlinson 1999). Several scientists have validated the merits and
demerits on the profile of the volatile organic compounds when utilized as a biosensor
which includes their non-destructive, reliability and their application in the real-time
plant disease and insect management and monitoring (Rajendran et al. 2016; Cellini
et al. 2016; Fang et al. 2014; Fang and Ramasamy 2015).
9.5.4.1 Bacterial and Fungal Disease Infection
The detection of numerous agricultural diseases and plant infection before the onset
of all the symptom associated are very paramount management techniques and pest
control strategies for effective mitigation of the diseases (Sankaran et al. 2010; Shaikh
et al. 2017; Ray et al. 2017). e-noses have been identified as crucial in the identifica-
tion of bacterial and fungal infections. This technique involves the identification of
volatile organic compounds liberated from isolated microorganisms utilizing e-nose;
while the indirect techniques entail evaluating changes in volatile organic compounds
liberated from the infected plants that have been inoculated with particular bacteria
or fungus. The techniques using e-noses have been utilized for the quick identifica-
tion of blossom blight which is caused by Pseudomonas syringae PV. Syringae and
fire blight caused by Erwinia amylovor most especially on apple trees when carried
out in a field (Spinelli et al. 2010) and controlled laboratory environment (Fuentes
et al. 2018).
MOS sensors could be utilized for the detection of numerous types of compounds
which entails some reducing compounds and oxidizing compounds. Also, the MOS
sensors possess a very high sensitivity which could detect sub-part per billion ppb
levels for some gases to the oxidizing and some reducing compounds. Some other
merits include rapid rejoinder time to the analytes, the capability to operates at high
temperature and pressure and they could easily be fabricated (Mielle 1996). The
application of e-nose based on MOS sensors has been highlighted to detect fire-
blight-infected pear trees when they are infected at an early stage. The inoculation
of tumorigenic strains of Agrobacterium vitis has been shown to variation when
subjected to the uninoculated ones which display 83.3% effectiveness through the
application of portable e-nose system (Spinelli et al. 2010, 2012). Tomato plant has
been identified as one of the most important crops which has been utilized in the
greenhouse crop globally because it has drawn the attention of scientist for vali-
dating the VOC fingerprints for evaluating the status of plants health. Zhang and co-
workers utilized the influence of powdery mildew on the volatile organic compounds
of the tomato plant that has been affected most especially under greenhouse condi-
tion. The outcome indicated that these diseases had a significant influence on the
volatile organic compounds profile and the application of the e-nose could be able
to detect between healthy and infected ones most especially with the accuracy of
94% (Ghaffari et al. 2011; Zhang et al. 2011). While the rate of infection induced
by Ceratocystis fagacearum have been detected with 78.65% accuracy using e-nose
recognition (Wilson et al. 2004).
9.5.4.2 Insect Damage
The application of e-noses has been demonstrated for their practical application
for identification of infested plants as well as the insect population dynamics. The
utilization of e-nose technology has been affirmed in the effective differentiation of
Nilaparva talugens (brown planthopper) and Chilo suppressalis (striped rice stem
borer) that are responsible for the damage of rice plant. Interestingly, the number of
volatiles liberated from insects such as stink bugs and brown planthoppers could be
used in the numerous aspects of insect-infested plants. Xu et al. (2014) demonstrated
the possibility of estimating the age and number of brown planthoppers using an
e-nose with classification accuracies of 100% and 48.93%, respectively. Also, the
e-nose could be utilized for quick identification of the gender of an insect in a quick
and practical way, that is very paramount importance in the mating disruption (Lan
et al. 2008; Zhou and Wang 2011).
9.6 Conclusion and Future Recommendation
This chapter has provided a detailed information on the application of biosensor for
mitigating the pest and pathogens problems affecting the food and crop production.
The various types of biosensor that are being developed in this regard have been
discussed in detail. The application of e-nose as a typical sensor for the detection
of pest and pathogen were also highlighted. However, there is a need to perform
numerous experiments most especially on the field and compare the laboratory results
to validate its field application for the detection of pest and pathogen. This will go
a long way towards the development of sustainable technology, that is sensitive,

portable, eco-friendly, and economical. Moreover, there is a need to adopt and invest
more in futuristic approaches such as smartphone-based sensing or wireless sensing.
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68-5
Chapter 10
Gold Nanoparticles-Based Point-of-Care
Colorimetric Diagnostic for Plant
Diseases
Ravi Mani Tripathi and Prashant Sharma
Abstract Plant diseases caused by pathogens such as fungi, viruses, and bacteria
create a substantial loss worldwide that leads to a severe threat to food security
and human health. Therefore, disease management is a priority in agricultural-
based countries. The detection of plant-pathogens is the primary step in disease
management, which is crucial for plant health monitoring. Presently, few immuno-
logical methods are utilized to detect plant pathogens such as direct tissue blot
immunoassay, enzyme-linked immunosorbent assay, and fluorometric immunoassay.
DNA-based detection methods are also keenly applied for plant pathogen identifica-
tion and detection, such as polymerase chain reaction, real-time PCR, and dot blot
hybridization. However, these detection methods are inherited with some disadvan-
tages like costly antibodies, complicated procedures, challenge to produce specific
antibodies, expensive equipment, and susceptibility to contamination. This chapter
reviews an instrument-free, instant, highly selective, and sensitive colorimetric detec-
tion of plant pathogens. The colorimetric detection is working on localized surface
plasmon resonance properties of AuNPs, which causes the colour change of assay.
Keywords Plant pathogens · Colorimetric detection · Gold nanoparticles ·

Properties · Functionalization · Mechanism
R. M. Tripathi (B)
Amity Institute of Nanotechnology, Amity University Uttar Pradesh, Sector 125, Noida,
UP 201303, India
P. Sharma
Department of Pediatrics, Division of Pediatric Hematology, Oncology & Bone Marrow
Transplant, University of Wisconsin–Madison, Madison, WI 53706, USA
192 R. M. Tripathi and P. Sharma
10.1 Introduction
Plant pathogens like fungi, viruses, and bacteria cause significant agricultural product
loss in developing countries (Agrios 2005). The fungi, bacteria, and viruses infect
various plant parts like leaves, stems, tubers, fruits, with various agronomic impacts
and finally lead to severe symptoms such as etiolation, necrosis, and blight (Fox
and Mumford 2017; Khater et al. 2017; Martinelli et al. 2015). These plant diseases
can create a significant loss in crop production and lead to severe threats to food
security and human health. The control of diseases is challenging due to the lack of
efficient chemical treatment products under field conditions. Therefore, this problem
is making a priority for disease management in agriculture-based countries. The
detection of plant diseases is a crucial technique to prevent the loss of crop production.
Several techniques have been established to identify plant pathogens (Punja et al.
2007). Plant pathogens can be detected by skilled persons/farmers based on symp-
toms or by laboratory experts by microscopic, molecular, or culturing techniques
(Horsfall 2012). The physiological, biochemical, and pathological parameters are
also examined on the plant diseases by culturing pathogens on selective media. The
traditional techniques are time-consuming and need skilled plant pathologists to
analyse and confirm the particular disease symptoms and responsible pathogens.
Therefore, these shortcomings compelled us to develop detection methods with
cost-effective, higher sensitivity, specificity, and rapid process to identify plant
pathogen.
Extensive assays such as direct tissue blot immunoassay (DTBIA), enzyme-linked
immunosorbent assay (ELISA), and fluorometric immunoassay used to detect plant
pathogens (Fang and Ramasamy 2015; Martín et al. 2002; Shojaei et al. 2016). These
methods are inherited with some drawbacks like costly antibodies, complicated oper-
ation, and challenge to generate specific antibodies that restrict immunoassay appli-
cation for plant disease management (Toh et al. 2015). DNA-based approaches have
been applied to detect plant pathogens after the invention of polymerase chain reac-
tion (PCR) in the 1980s. Several PCR-based detection methods have reported for
the identification of plant pathogens (Vincelli and Tisserat 2008; Ward et al. 2004).
Issues related to specificity and sensitivity to the amplification of pathogen-specific
sequences were overcome by the coupling of PCR with other techniques (Brasileiro
et al. 2004; Carrasco-Ballesteros et al. 2007; Merighi et al. 2000). Another strategy,
immunocapture-PCR (IC-PCR), has been adopted for the detection of viruses by
fusing conventional PCR amplification with antibody capturing virus. This strategy
enhanced the sensitivity by 250-fold compared to PCR amplification and developed
a platform to detect the bacterial blight disease in Anthurium propagation material
(Khoodoo et al. 2005). However, PCR-based plant pathogen detection is associ-
ated with some limitations, such as expensive equipment and susceptibility to cross
contamination (Yang et al. 2016).
Nowadays, the nanomaterials-based approach for the detection of plant pathogens
is getting more popular among researchers. Researchers have used gold nanoparti-
cles (AuNPs) for various applications, such as catalysts, antibacterial, colorimetric
10 Gold Nanoparticles-Based Point-of-Care Colorimetric Diagnostic … 193
sensors, and also in drug delivery systems, because of their unique chemical and
physical properties (Mehrotra and Tripathi 2015; Tripathi and Chung 2019; Tripathi
et al. 2014b, 2015a, 2018b, 2019a). The colorimetric detections, non-biological, and
biological analytes using AuNPs by surface modification with ligands and biological
receptors (Tripathi et al. 2019a; Verma et al. 2015). The principle behind the colori-
metric detection is the change in the localized surface plasmon resonance prop-
erties of AuNPs, which causes the change in colour of the assay (Tripathi et al.
2019a). AuNPs based colorimetric detection is getting extensive exploration due to
its simplicity, rapid detection, and cost-effectiveness.
In this chapter, we discussed the relevant physical and chemical properties of
AuNPs for the detection of plant pathogens such as fungi, bacteria, and viruses. This
chapter reviews the colorimetric detection of various plant pathogens. The principle
behind the colorimetric detection is localized surface plasmon resonance proper-
ties of AuNPs. As per the requirement application, AuNPs are surface functional-
ized with different ligands, such as polymers and biomolecules. Nanoparticle-based
colorimetric detection techniques are instrument-free, instant, highly selective, and
sensitive for detecting plant pathogens.
10.2 AuNPs and Their Properties
AuNPs are most exploited as compared to other metallic nanoparticles for various
purposes, such as catalysis fields (Tripathi et al. 2018b), antibacterial agents (Tripathi
et al. 2018b), drug carriers (Tripathi et al. 2015a), colorimetric sensor (Tripathi et al.
2014b, 2019a), etc. AuNPs show several excellent characteristics like large surface-
to-volume ratio, shape-dependent optoelectronic properties, exceptional biocompat-
ibility, and low toxicity. The shape and physico-chemical properties of AuNPs are
associated with optical features, and their size affects the colour of the colloidal
solution. The most applicable optical properties of AuNPs are the surface plasmon
resonance (SPR) (Tripathi et al. 2014b) and fluorescence quenching (Xia et al. 2016).
SPR is a unique property of AuNPs, and it depends on the morphology and size
of nanoparticles. SPR phenomenon produces a strong characteristic absorption for
AuNPs in the UV-visible region. The appropriate term for the SPR in this condi-
tion is localized surface plasmon resonance (LSPR), which was recognized as Mie-
scattering in 1904 (Tripathi et al. 2019a). LSPR has theoretically anticipated for
the spherical metal particles with dimensions similar to the light wavelength. LSPR
is well-defined by the quantization of surface plasmons in different types of nano-
metal. The most exciting property of AuNPs is their LSPR, which is the critical
principle of colorimetric detection of a biological and chemical analytes. Generally,
the AuNPs colloidal solution is red or pink, but agglomeration caused the colour
change to purple blue (Fig. 10.1). These properties of AuNPs make them a suitable
candidate for applications in sensing various analytes.
Fig. 10.1 A representative example of calorimetric detection of an analyte by AuNPs. The graph
shows the UV–Vis spectra of the blank sample and the assay containing targets. The inset shows
the colour change of the assay in the presence of target (the figure was reproduced from Tripathi
et al. 2019 with the permission of Elsevier Science)
10.3 Synthesis of AuNPs
The nanoparticles are synthesized by two leading approaches: top-down and bottom-
up. The creation of particles at the nanoscale by crushing or cutting bulk materials is a
top-down approach, whereas the creation of nanoscale materials by assembling them
atom by atom, molecule by molecule, or cluster by cluster is bottom-up. Further, the
type of nanomaterials synthesis categorized into three methods: physical, chemical,
and biological. The physical methods required high energy and pressure, whereas
chemical methods are hazardous and create serious environmental problems.
We have synthesized AuNPs with a narrow size distribution ranging from 5 to
25 nm, and the average nanoparticle size was 10 nm using the Turkevich method
(Fig. 10.2). The synthesized nanoparticles have been used for instrument-free, instant,
highly selective, and sensitive colorimetric detection of lead (Tripathi et al. 2019a).
Nowadays, biosynthesis of the nanomaterial is getting more popular because of
the issues related to physical and chemical methods (Tripathi et al. 2013, 2015a, b,
2018a). Biologically originated nanomaterials have been shown excellent properties
for colorimetric detection and do not need special surface modification (Tripathi et al.
2014a, b). Typically, bacteria, fungi, and plants are used for the intra and extracellular
synthesis of various metallic and semiconductor nanomaterials (Agarwal et al. 2016;
Mahajan et al. 2016; Tripathi et al. 2014a, 2015a; Tripathi and Chung 2020).
Gold exists in three unique forms, gold (0), gold(I), and gold (III) in an aqueous
solution (El-Brolossy et al. 2008; Tripathi and Chung 2019). AuNPs show excep-
tional chemical and physical properties as compared to their bulk due to quantum
effects. AuNPs can be synthesized by using the biomass of fungi and bacteria. The
various morphologies of AuNPs have been synthesized aqueous extract of Rhizopus
Fig. 10.2 TEM images of the AuNPs at different magnifications: a sample large view and the
inset shows the TEM histogram; b focused on a few particles and the inset shows the SAED
pattern; c HR-TEM image; d polycrystalline nature and d-spacing; e EDX spectrum (figures were
reproduced from Tripathi et al. 2019 with the permission of Elsevier Science)
oryzae by changing the synthesis parameters, including reaction time, the concen-
tration of gold salt, and the pH of the reaction mixture (Das et al. 2010). Pseu-
domonas aeruginosa and Rhodopseudomonas capsulate biomass have been used
to synthesis of AuNPs (Singh and Kundu 2014). Coriolus versicolor (Sanghi and
Verma 2010) and Fusarium oxysporum (Thakker et al. 2012) have also been used
for the synthesis AuNPs, but these are pathogenic fungi. Therefore, in an earlier
study, AuNPs synthesis method using a non-pathogenic and agriculturally beneficial
fungus; Trichoderma harzianum has been developed (Tripathi et al. 2014b). Synthe-
sized nanoparticles using T. harzianum showed narrow size distributions (26–34 nm)
with spherical shape and having high stability were noticed (Tripathi et al. 2014b).
However, the microorganism mediated synthesis of nanoparticles is time-
consuming and needs to grow enough biomass. Therefore, plant extract mediated
synthesis gained advancement over microbial biomass assisted synthesis. The plant
extract is used as a reducing and capping agents for the synthesis of nanoparticles.
We have developed a method for the synthesis of AuNPs using Ficus benghalensis
leaf extract, and synthesized nanoparticles had a spherical shape with a size ranging
from 17 to 50 nm (Tripathi et al. 2012). The shape of nanoparticles can also be
changed according to their application. Recently we have synthesized hexagonal
and triangular AuNPs using Erigeron annuus leaf extract (Tripathi et al. 2019b).
For this, fresh leaves of E. annuus were taken and cut into small pieces. Thereafter,
8.5 g leaves were dispersed in 100 mL DI water and boiled at continuous stirring.
Finally, the obtained solution was filtered and stored at 4 °C for the synthesis of
AuNPs. The synthesized nanoparticles were characterized by transmission electron
microscopy and micrographs show triangular AuNPs synthesized (Fig. 10.3). These
studies make the biosynthesized nanoparticles a putative and robust candidate for
different calorimetric detection.
10.4 Functionalization of AuNPs for Pathogen Detection
Surface modification of AuNPs with a functional group is a crucial step in detecting

pathogens, which provides high selectivity to the method. Chemically anchor
nanoparticles conjugate functionalizing molecules, and another end remains free,
which binds with the target molecule of interest (Wang and Yu 2013). AuNPs can be
functionalized with a variety of ligands, such as polymers, biomolecules, etc. AuNPs
can easily be conjugated with DNA, peptides, and antibodies due to its strong binding
affinity with the thiols group. The surface can be modified by covalent bonding
(Bhamore et al. 2015; Zhang et al. 2016), electrostatic interaction (Liu et al. 2016),
or attachment of a molecule for precise identification of the interested target (Liu et al.
2018). The most widely adopted surface modification of nanoparticles is the cova-
lent form. The AuNPs are functionalized with the molecule comprising sulfhydryl
groups. The sulfhydryl group interaction is found between Au and S groups. Thiol
(Shahrivari et al. 2018), cysteamine (Gukowsky et al. 2018), glutathione (Zhang et al.
2012), thioglycolic acid (Tolessa et al. 2018), cysteine (Raj et al. 2015), and protein
Fig. 10.3 TEM micrographs of triangular AuNPs. a Large view of sample; b magnified view;
c thickness difference between hexagonal and triangular nanoparticles; d broad and sharp angles;
e high magnification of single triangular; f angle feature of triangle; g EDX of triangular (Tripathi
et al. 2019)
(Hu et al. 2017) are widely used sulfhydryl ligands. AuNPs prepared by NaBH4
reduction and stabilized by thiol surfactant can be easily substituted by other thiol-
terminated groups (Gupta et al. 2012). The reaction for ligand exchange is performed
in a toluene solvent under ambient temperature for a long time (Stobiecka et al. 2010).
AuNPs surface modification by ionic modifiers or ionic capping agents is providing
stability by electrostatic repulsion. The surface charge magnitude (SCM) influenced
repulsion strength and stability. SCM depends on the ionic strength and pH value
solution and the pKa value of immobilized ligands (Liu and Lämmerhofer 2019).
The electrostatic interactions are weak compared to the covalent approach, and elec-
trostatic modified nanoparticles have poor stability. The ligands are easily detached
under the high salt circumstances, which caused aggregation of AuNPs (Xu et al.
2017).
10.5 Mechanism of Colorimetric Detection
AuNPs possess unique chemical and physical properties, which make them
promising materials for pathogen detection. LSPR is an interesting property of
AuNPs show LSPR peaks in the visible wavelength region. The SPR peak position
of AuNPs shows a red-shift as the refractive index of the surrounding environment
increases (Nguyen et al. 2016). The working principle of colorimetric sensors is the
sensitivity of their LSPR to the refractive index of the surrounding environment.
The LSPR depends on the size and shape of AuNPs. It was found the spherical
nanoparticles having an average size ranging from 9 to 99 nm, which observed with
absorbance peaks from 517 to 575 nm, respectively (Daniel and Astruc 2004). The
surface modification of AuNPs is a critical factor which brought together through
the formation of linkages between the individual particles and causes aggregation
AuNPs. The synthesized AuNPs colloidal solution has a red colour, but after aggre-
gation, the colour turned into purple-blue which give impact on the absorbance and
size of nanoparticles (Fig. 10.4) (Assah et al. 2018). The intensity of colour change
can be quantified by UV-visible spectroscopy. In our previous study, the synthesized
AuNPs absorbance occurs at 528 nm, but after aggregation peak shifts ranging from
630 to 700 nm depending on the degree of aggregation was observed (Tripathi et al.
2019a).
10.6 Colorimetric Detection of Plant Pathogens
10.6.1 Fungi
Fungi are the most responsible disease-causing agents in crop plants worldwide.
They can survive for a longer time in diseased and dead plant tissues in a dormant
state and proliferate in favourable conditions. The spores of fungi can easily spread
in several ways, such as water, wind, insects, soil, and other invertebrates (Jain
et al. 2019). The crops and plant diseases are caused by more than 19,000 species
of fungi (Jain et al. 2019). A biosensor has been developed for the detection of a
plant pathogen fungus Phakopsora pachyrhizi, which causes Soybean rust (Mendes
et al. 2009). Potato late blight caused by Phytophthora infestans which is a most
destructive plant pathogen. Therefore, the detection of P. infestans is a critical concern
for crops and plant disease management. Researchers are attracted to colorimetric
detection because of rapid, instrument-free, visual detection, and fieldable estimation
of the plant pathogens. Chang and coworkers have reported colorimetric detection
Fig. 10.4 Representative example of colorimetric detection by using Au NPs. a Schematic diagram
showing working principle of the colorimetric assay. b UV-vis absorption spectra of RE-AuNP
probes (i) absence of p53 protein (black dashed line), (ii) wildtype p53 protein (p53-WT) (blue
line), and (iii) mutant p53 protein (p53-R273H) (red line), c aggregation ratio, d TEM micrographs
of samples (i) to (iii) and the insets show corresponding assay colour (figures were reproduced from
Assah et al. 2018 with the permission of Elsevier Science)
for the specific nucleotide sequences of P. infestans (Chang et al. 2019). They have
used AuNPs as optical probes and CRISPR/Cas9-triggered isothermal amplification.
Cas9/sgRNA complex has broken the recognized target DNA and used for isothermal
amplification. The oligonucleotide functionalized AuNPs were hybridized with a
specific amplified product, which caused aggregation of AuNPs and produces colour
change from red to purple.
10.6.2 Bacteria
Plant diseases caused by bacteria can be broadly classified into four major groups,
based on plant tissue damage and the symptoms that they cause, which may
comprise vascular wilt, soft rot, necrosis, and tumour. Bacteria are invading plant
tissues by wounds produced by adverse weather, human, insects, and nematodes.
The natural ways are also responsible for bacterial infection such as natural open-
ings such as stomata, hydathodes, lenticels, and nectar-producing glands. The
bacterial plant diseases caused by Pseudomonas, Agrobacterium, Xanthomonas,
Erwinia, Proteobacteria, etc. are responsible for a big loss in agricultural yields.
Nowadays, colorimetric, or visible detection methods are popular due to advance-
ments over conventional detection methods. Besides the AuNPs aggregation mech-
anism, bridging flocculation is a very well-recognized strategy in colloid chem-
istry (Ruehrwein and Ward 1952). Several detection methods have been developed
based on aggregation mechanism using an antibody, DNA probe modified- and
electrostatic-mediated aggregation of nanoparticles (Li and Rothberg 2004; Mirkin
et al. 1996; Xu et al. 2009). The rapid detection of plant pathogen has been reported
based on reversible adsorption to differentiate between long and short DNA polymers
(Wee et al. 2015).
10.6.3 Viruses
Lateral flow immunoassay techniques have been developed for the detection of
various plant pathogenic viruses such as Citrus Tristeza virus (Salomone et al.
2004), Prunus necrotic ringspot virus (Jarocka et al. 2013), Potato virus x (Drygin
et al. 2012). DNA-based biosensors for label-free detection have been developed for
rapid and sensitive detection of the pathogen, which offered qualitative and quanti-
tative investigation of pathogens. The colorimetric nucleic acid detection has been
developed using the AuNPs-labelled DNA probe, which is simple, rapid, and high
sensitivity (Khater et al. 2017). Unmodified AuNPs based probes have been used to
detect RT-PCR amplified Cucumber green mottle mosaic virus (Wang et al. 2017).
The limit of detection (LOD) 30 pg/μL of CGMMV RNA was reported in this
study. Dharanivasan and coworkers reported AuNPs conjugated bifunctional oligo
probe for the colorimetric detection of PCR amplified DNA of Tomato leaf curl New
Delhi virus (Dharanivasan et al. 2016). The LOD of this assay was about ~7.2 ng
dsDNA target, has potential for the development of ultra-sensitive nanoassay method.
The colorimetric detection method has been developed, based on LSPR activity of
AuNPs for analysis of unamplified Tomato yellow leaf curl virus (TYLCV) genome in
infected plants (Razmi et al. 2019). In this method, the AuNP-DNA probe hybridized
with specific complementary targets and caused the colour change of assay due to
LSPR, which can measure using UV-visible spectroscopy. This technique provides
advancement over the conventional detection method by eliminating the used of
PCR amplification and detection equipment. Similarly, the Maize chlorotic mottle
virus is a serious pathogen that can be transmitted via infected maize seeds. So, to
prevent its further spread a rapid and sensitive assay was needed, and to achieve this
Liu and colleagues explored the use of unmodified gold nanoparticles (AuNPs) to
visual detection (Liu et al. 2015). In their study, the interaction between the species-
specific probes and RT-PCR target products changed the colour of dispersed AuNPs
from red to grey blue. The LOD of their assay was as low as 30 pg μL−1 of viral
RNA.
10.7 Conclusion and Prospects
In this chapter, the colorimetric detection of plant pathogens using AuNPs have been
described. A variety of surface characteristics of AuNPs have been exploited for the
colorimetric detection of pathogens either by sensing nucleic acids, surface proteins,
or whole pathogens. The surface modification of AuNPs is essential in respect of
selective detection of the target. The most adopted nanoparticle surface modification
is the covalent approach. The AuNPs are functionalized with the molecule comprising
sulfhydryl groups, having interaction between Au and S groups. The mechanism
behind colorimetric detection is the sensitivity of AuNPs LSPR towards the refractive
index of the surrounding environment. The LSPR depends on the size and shape of
nanoparticles, and agglomeration cause sufficient signal for the detection of targets.
This technique is simple, cost-effective, and rapid for the detection of plant pathogens
such as fungi, bacteria, and viruses. In the future, this colorimetric method might
be used in field detection of plant pathogens and a promising technique for clinical
diagnosis of human and animal pathogens.
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Chapter 11
Advancements in Biosensors for Fungal
Pathogen Detection in Plants
Utkarsh Jain, Ramesh Namdeo Pudake, Nidhi Chauhan, and Sakshi Pareek
Abstract Globally, the pathogenic diseases caused by fungi in plants are the
major constraints for healthy harvesting and plantation. Therefore, to lessen these
constraints, it is required to monitor these fungal pathogens at an early phase.
Currently, ample methods to identify, detect, and quantify various fungal pathogens
in the infected plant incorporate ELISA, PCR, RT-PCR are available. But these have
their own limitations, for instance, lack of sensitivity, time-consuming, expensive,
labor-intensive, requires highly equipped instruments, and skilled personnel for their
handling. Thus, there is a strong demand for fabricating biosensing methods at early
stage detection with great sensitivity and specificity for different plant diseases.
In context, this chapter provides an outline of biosensors based on various bio-
recognition elements, like DNA, antibodies, and enzymes. They are emerging as a
powerful tool for the initial stage monitoring of fungal disease because of their beni-
ficial properties like specificity, sensitivity, rapidness, cost-effectiveness, easy to use
when compared to the available traditional methods. Thus, biosensors are considered
to be a reliable and promising tool for the diagnosis of these pathogens and increasing
the quantity as well as the quality of crop yield.
Keywords Fungi · Biosensors · Plant diseases · DNA-based biosensor ·

Antibody-based biosensor
11.1 Introduction
Diseases in plants occur due to the multiplex interaction of plant host, pathogen,
and the environmental conditions, causing the privation in yields as well as harming
the health of humans. Fungi, viruses, parasite plants, bacteria, and nematodes are the
causes of various plant diseases. The diversity in the variety of pathogenic interactions
between fungi and plants considered by the formation of a parasitic relation of about
400 million years back (Kumar et al. 2016). There is a vital loss of production
U. Jain (B) · R. N. Pudake · N. Chauhan · S. Pareek

Amity Institute of Nanotechnology, Amity University Uttar Pradesh, 201303 Noida, UP, India
206 U. Jain et al.
and revenue in the agricultural and forestry fields due to the diseases. For instance,
soybean rust, a fungal infection causes a huge economical loss and by preventing the
infection only by 20%, there may be a profit of 11 million dollars (Sankaran et al.
2010). Fungal plant pathogens are a great economical threat and it is evaluated that
agriculture production globally bears an average loss of 15% annually (Neeraja et al.
2010). With the developing results of environmental change, the loss is anticipated
to increase (Lo Presti et al. 2015). Many fungal diseases have come up from small
to large importance and were discovered recently. More than 40 fungi are causing
major disease problems globally (Grau et al. 2004).
Plants create a significant environment for microorganisms as they provide an
extensive range of domains for growth, including the aerial plant part, i.e., phyllo-
sphere, the root system i.e., rhizosphere, and the inner transport system i.e., endo-
sphere (Ferreira et al. 2006). Plant–fungus correlation described as vulnerable if the
fungus process characteristic effects and regenerates; or as resistant if a plant can
counter the disease to limit side effects of pathogen proliferation. Understanding
the mechanism of pathogenicity and the qualities that control them may permit the
utilization of recombinant DNA innovation to adjust the plant’s defense system to
encourage resistance to the attack. The characterization of fungal pathogenic genes
is a demanding area in plant pathology at present (Martínez-Medina et al. 2019).
The life cycle of the fungal pathogen is complex, together with sexual and asexual
reproduction and development of various vegetative and reproductive forms during
infection stages. The essential stages of infection development are colonization,
growth and propagation, dissemination, and endurance of the pathogen without the
involvement of the host. Although, reaching every stage varies from pathogen to
pathogen (González-Fernández et al. 2010).
Fungal plant pathogen harms the plants in the vegetative phase as well as in
the post-harvest infection form (Fig. 11.1). The main plant pathogen for economic
aspects are fungi which include Botrytis cinerea, and species of Fusarium, Magna-
porthe, and Rhizoctonia (Hua et al. 2018; Sharon and Shlezinger 2013). Ascomycete
genus, Cercospora members, Mycosphaerellaceae, Dothideomycetes, Capnodiales
appear universally causing leaf spots on dicot and plant families of monocot, ferns,
and gymnosperm ranking in the main cause of plant pathogens destruction (Cai
et al. 2011). Wheat harvests in middle age were usually ruined when the grains got
infected with spores of fungus known as bunt or stinking smut which was a dark
and dusty powder. During the 1870s, a pandemic of downy mildew, which occurred
by the organism Plasmopara viticola, struck the grape vineyards of central Europe,
effecting grape cultivators and wine producers. In the United States itself, a huge
number of bushels of wheat have been lost in epidemic for a very long time to stem
rust (Ellis et al. 2008).
Hence, the accessibility of rapid, sensitive, and precise techniques for the iden-
tification and detection of fungi is increasingly important to improve the decision-
making process for infection control. The traditional methods used for the identi-
fication of plant pathogens depend on culture-based structural methodologies. But
these methods are time utilizing, difficult, and require broad information of tradi-
tional groupings. Even these methods have other disadvantages including problems
11 Advancements in Biosensors for Fungal Pathogen … 207
Fig. 11.1 Fungi infection cycle in plants
to in vitro culture certain species and the failure to precisely measure the pathogen
(Capote et al. 2012) (Tsui et al. 2011). New innovations, for example, nanoscale plat-
forms, biosensors, miniaturization of detection devices, and nanoscale play a major
role in pathogen detection and management (Abd-Elsalam 2013). The requirement
for rapid, on-line, and precise detecting opens up doors for biosensors in a wide
range of agricultural fields in situ investigation of contaminations in yields and
soils, recognition and distinguishing proof of irresistible illnesses in harvests and
domesticated animals, on-line estimations of significant food handling boundaries,
checking creature ripeness and screening restorative medications in veterinary testing
(Velasco-Garcia and Mottram 2003).
By definition, a biosensor is a self-contained combined system, which recognizes
an analyte of biological element and quantitatively by utilizing a bioreceptor compo-
nent (called a biorecognition component) and a sign transduction component. The
analytes of benefits can be biological, such as DNA, protein, cell. When a biore-
ceptor component perceives a particular analyte and binds, a transducer component
gives quantitative or semi-quantitative data about the objective by changing over the
physicochemical signal produced during the biorecognition into a quantifiable elec-
trical signal (Ali et al. 2017; Chen and Shamsi 2017; Turner 2013). The term biosensor
has been connected to numerous innovations. They can be both subjective or quantita-
tive, encrypted, or solid physical devices, giving concurrent time, or time-fractioned
information, in vivo or ex vivo, and of numerous different structures (Sadanandom
and Napier 2010). Fundamentally, a biosensor couples to a recognition element or
a chemical response by a biological element with a transducer, which then converts
this response into a readable form (Singh and Verma 2019). Biosensors are getting
significant in a wide scope of analysis. Diverse biosensor designs have been created
for single target analytes and wide range monitoring (Tothill 2001). The materials
utilized in biosensors are distinguished into three categories depending on their
208 U. Jain et al.
mechanisms: biocatalytic consisting of enzymes, bio affinity group, with antibodies

and nucleic acids, and microbe-based microorganisms (Mehrotra 2016; Rodriguez-
Mozaz et al. 2006). The outstanding performance factors for biosensors enhancing
are sensitivity, limit of detection, and specificity (Bhatt and Bhattacharya 2019).
11.2 Fungal Disease Detection in Plants
11.2.1 Conventional Assays for Fungal Detection
Few fungal pathogens have various symptoms which are noticeable to the normal
eye, or at less amplification. These symptoms include stain, withering, and fissure
on the fungal pathogens. For example, soybean seeds decay is caused by Phomopsis
spp. as well as Cercospora kikuchii and the spots on peanut seeds that are infected
by Cylindrocladium parasiticum (Mancini et al. 2016).
Also, incubation methods are often utilized for pathogens, particularly for high-
frequency fungi that can happen on seed tests at a level >1%, additionally giving data
about the practicality of the inoculum on the seeds (Mancini et al. 2016). Despite
being inexpensive and convenient in use, the precision and reliability of these tech-
niques rely upon the experience and skill of the individual doing it (McCartney et al.
2003). Also, these conventional methods require much time as it takes days and weeks
for the verification, making them not suitable for instance and fast detection (Ray
et al. 2017). These traditional methods for the identification of plant pathogens are
dependent on culture-based methods. The main drawbacks of these approaches were
the ability of the organism to be cultured, contamination prone, requiring more time
and arduous nature, and the necessity for broad taxonomical information (Atkins and
Clark 2004; Lievens and Thomma 2005) (Table 11.1). Thus, there is an urge in the
search for some other diagnostic approaches (Atkins and Clark 2004).
Table 11.1 Some examples of fungal disease identification using different conventional techniques
Plant/Tree Pathogen Technique Reference
Soybean Phhakopsora pachayrhizi IF (Baysal-Gurel et al. 2008)
Timber Serpula lacrymans and GC–MS (Ewen et al. 2004)
Coniophora puteana
Tomato Lycopersicon esculentum RT-PCR (Lievens et al. 2006)
Apple Ventura ineaequalis Hyperspectral (Delalieux et al. 2007)
Onion Sclerotium cepivorum PCR (Haq et al. 2003)
11.2.2 Molecular Assays for Fungal Detection
Molecular Assay is classified into two categories—direct and indirect detection

methods. Various direct traditional assays for detection post-harvest fungi involve
polymerase chain reaction (PCR), real-time PCR (RT-PCR), ELISA, and magnetic
capture hybridization (MCH-PCR, which is accurate and sensitive. Having complex
protocols with more cost, these methods are less used. Moreover, they detect the
fungal infection in crops, but cannot evaluate their pathogenicity state (Kumar et al.
2016; Zamir et al. 2020). Also, a plant can be infected by different other pathogens,
therefore PCR and DNA arrays are used for multiplexing. Although the diagnostic
methods comprising molecular tools are reduced by removing cultured cultivation,
still this process of molecular validation of target genes is time utilizing and expen-
sive (Kumar et al. 2016). Although PCR is considered to be a sensitive important
method that helps with problems related to control, identify, and containment in plants
(Schena et al. 2004) but recurrent use of PCR inhibitors in the soil or plant tissues,
lessening the sensitivity of reactions resulting in false results. This false result even
occurs when DNA or PCR products toxin are present (Narayanasamy 2011; Schena
et al. 2004). Lately, quantitative PCR (qPCR) identification techniques have been
broadly used to identify phytopathogenic parasites and have incredibly added to the
progression of information on plant pathology. However, significant errors and limi-
tations, are still there. Errors at the whole procedure for the advancement of qPCR
strategies, at the degree of choice of proper DNA extraction and purification method,
distinguishing suitable target regions, decision of chemistry, design, and approval
of particular primers and probes, choice of an absolute quantification method, and
analysis of the risk of identifying target DNA from dead sources (Schena et al. 2013).
Fast detection assays emerged as an easy and effective qualitative test. In spite of their
great specificity, the need for reproducibility, reliability, and sensitivity are regular
constraints, also frequently false-positive outcomes have appeared and are related
to cross-responses among antibody and fungal metabolites (Oliveira et al. 2019).
Indirect strategies are basically imaging methods, they are sensitive and very much
utilized for huge information data, hyperspectral imaging is applied the most for
ailment location in plants (Madufor et al. 2017).
11.3 Biosensors for Fungi Detection in Plants
11.3.1 DNA-Based Biosensors
The DNA-based biosensors consists of hybridization, amplification, and recombi-

nation on the double helix DNA structure. Nucleic acid hybridization is the basic
principle of DNA biosensors. Arrangement hybridization is faster than hybridization
on strong support, yet, except if the measure is a homogeneous test, a separation
step is required before the final identification (Zhai et al. 1997) (Fig. 11.2). DNA
210 U. Jain et al.
Fig. 11.2 Scheme for DNA based biosensors
biosensors are promising in rapid, simple, and cost-effective detection of the target
molecules (Chao et al. 2016).
Basal Stem Rot and Upper Stem Rot infections are caused due to pathogenic
fungus Ganoderma boninense and are a major threat to the palm oil industry. There-
fore, an automatic high DNA output extraction method using a microfluidics device
for disposable and rapid, without label detection was done within 2 hrs. It was exam-
ined using current and potential measurements, UV visible, FTIR. With the help
of PCR, results were compared between extracted and commercial ssDNA of G.
boninense and appeared differently in relation to unknown fungus, demonstrating an
effective DNA extraction convention through microfluidics (Ayoib et al. 2020).
Alternaria panax Whetz is one of the most destructive diseases in ginseng and
may lead to major economic loss in ginseng production. Therefore, a fast and precise
atomic symptomatic technique, a single-tube nested PCR-lateral flow biosensor
assay (STNPCR-LFBA) was fabricated to detect the pathogen. The sensitivity of
STNPCR-LFBA was compared with conventional PCR-LFBA, and it was found to
be more effective. Other than that, the PCR sample was checked by lateral flow-
based biosensor assay, which gave the migration of the detection technology to a
point-of-care test (POCT) format. STNPCR-LFBA was specific to A. panax Whetz,
and no cross-response was seen in any other non-targeted samples with the limit of
detection 0.01 pg. STNPCR-LFBA could also be used for particular identification in
real samples (Wei et al. 2018).
Phytophthora infestans is the causal agent for late blight in potatoes and tomatoes,
with a global impact on agriculture. A visual detection method for P. infestans
has been designed by combining unique primer mediated asymmetric PCR with
gold nanoparticle modified lateral flow biosensor. Quantitative analysis reported the
optical intensity of the red band with a detection limit of 0.1 pg µL−1 . The specificity
of this biosensor was measured by three different Phytophthora species as well as
two pathogenic fungi. It was assumed that this method was possible for early stage
prediction of potato late blight disease and prompting of the administrator’s activities
to lessen the danger of scourge improvement (Zhan et al. 2018).
11.3.2 Antibody-Based Biosensors
The ways to deal with direct-acting immunosensors are the utilization of their anti-
bodies simply to give specific binding of analyte to a surface and property of that
surface detected by the transducing component (North 1985) (Fig. 11.3). Few studies
to detect the plant pathogens using the antibody-based sensors are reviewed below.
An artificial cellulose membrane, particularly deciding if a phytopathogenic
pythium is present in an aqueous mixture of Pythium (phytopathogenic and non-
pathogenic) was proposed in a recent study (Yamaguchi et al. 2017). Dark Sigatoka
is an infection that occurs in banana cultivation globally and is occurred by the
hemibiotrophic fungus Pseudocercospora fijiensis. An exceptionally sensitive SPR
immunosensor was fabricated to recognize P. fijiensis in real samples of leaf separates
in the beginning phase of the infection. A polyclonal antibody produced against HF1
was covalently immobilized on a gold-covered chip by mixing self-assembled mono-
layer (SAM) of alkanethiols with EDC/NHS method. The immunosensor obtained
a detection limit of 11.7 µg mL−1 , with sensitivity of 0.0021 units of reflectance per
ng mL−1 and linear range from 39.1 to 122 µg mL−1 (Luna-Moreno et al. 2019).
Pectobacterium atrosepticum (Pba) is an isolated and terrifying phytopathogen
known agent of blackleg and soft rot infection in potatoes in many areas. Current
Fig. 11.3 Scheme for antibody-based biosensors

212 U. Jain et al.
Table 11.2 Various biosensors for the detection of fungal pathogens

Biosensor Pathogen Detection limit Reference
Electrochemcial Ganoderma boninense NA (Ayoib et al. 2020)
Electrochemcial Alternaria panax 0.01 pg (Wei et al. 2018)
Electrochemcial Phytophthora species 0.1 pg µL−1 (Zhan et al. 2018)
Electrochemcial Phytopathogenicity NA* (Yamaguchi et al. 2017)
Immunosensor Pseudocercospora 1.7 µg mL−1 (Luna-Moreno et al.
fijiens 2019)
Immunosensor Pectobacterium 104 CFU mL−1 (Hashemi Tameh et al.
atrosepticum 2020)
Impedimetric Sclerotinia 7.8 × 104 ascospores (Shoute et al. 2018)
sclerotiorum mL−1
Optical Aspergillus niger NA* (Gambhir et al. 2018)
*NA Not available
conventional techniques, for example, ELISA and PCR are utilized for Pba identi-
fication; however, they are costly and time-consuming. Therefore, an electrochem-
ical impedance immunosensor was reported with a microfluidic module and micro-
electrodes array with the various advantages of cost-efficiency, usability, and porta-
bility (Hashemi Tameh et al. 2020). Sclerotinia stem decay, occurred by parasitic
pathogen Sclerotinia sclerotiorum, is a dangerous disease of canola and numerous
other broadleaf yields. Hence, advancement in impedimetric non-faradaic biosensor
dependent on hostile to S. sclerotiorum polyclonal antibodies as tests to specifically
catch the ascospores and sense their attachments by an impedance-based interdigi-
tated electrodes which were found in straightforward and unambiguously relate the
quantity of ascospores on the sensor surface with the impedance reaction (Shoute
et al. 2018).
The advancement of a smaller and label-free sensor dependent on a superficial
level adjustment of copper vapor laser manufactured long-period fiber gratings for
recognition of airborne Aspergillus niger contagious spores was introduced. In this
optical sensor IgG1 antibody with immobilized monoclonal glucose oxidase was
detected. Thus, optical sensors have solved many problems related to biochemical
sensing (Gambhir et al. 2018). Some of the biosensors reported for the detection of
fungal pathogens are listed in Table 11.2.
11.4 Significance of Biosensors for Plant Pathogen

Detection
In this chapter we have reviewed the conventional methods for detection of fungal
diseases caused, for instance, PCR, ELISA, immunoassays with their limitations to
monitor various fungal diseases. In addition, these methods do not provide real-time
detection making them less suitable for use. Thus, biosensors usage for these fungal
pathogens has gained importance as they help in increasing crop quality and economic
improvement. Early stage detection has gained individual importance in the detection
of fungi. With these biosensors potential to detect various fungal pathogens enhancing
the use of antibodies and DNA has been comprehensively reviewed with the present
approaches’ comparison.
11.5 Conclusion
The chapter outlines the early detection of various diseases caused by fungal
pathogens. The effect of these fungi harms the yields and economic largely. There-
fore, early monitoring plays an important role in disease management, leading to
lessen the harm that is caused by these pathogens worldwide. To estimate and
control the solemnity of fungal plant infections, it is required to design and fabricate
fast, precise, futuristic, and progressive detection approaches for these diseases. The
current strategies are not potentially abundant for detecting fungal pathogens as these
conventional detection techniques are time-consuming, related to special equipment,
and require skilled professionals with some experience. The main aspect of devel-
oping advanced methods is high sensitivity. To overcome all these difficulties, recent
increase and enhancement in micro and nanotechnology have opened paths for the
development of biosensors for determination of fungi infections. The use of biosensor
with additional efforts has gained significant importance in the development of highly
sensitive and selective tools for real-time monitoring of plant diseases in different
field conditions and infections. With these more than one fungal disease in infected
plant can be diagnosed reducing risk of false results. DNA-based biosensors and
antibody-based biosensors discussed here have gained much importance due to their
speed, sensitivity of even detecting samples at small amounts, and help in securing
the quality of plants. Moreover, biosensors for enormous fungus disease screening
assessment has paid attention to several aspects such as cost, accessibility of on-site
detection and pre- and post-test of risk caused by infection. Lastly, we conclude
that biosensing platform for these fungus pathogens provides an essential role in
overcoming the limitations of the present methods and enhancing crop harvesting.
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Chapter 12
Journey of Agricultural Sensors—From
Conventional to Ultra-Modern
Ashish Mathur, Shikha Wadhwa, Shalini Nagabooshanam,

and Souradeep Roy
Abstract Agriculture has a major contribution to the evolution of human civiliza-

tion. Owing to the rise of the human population, the demand for food increases
simultaneously which requires the development of advanced techniques to augment
food production. Sensors play a vital role in crop production and are used very
successfully in almost all main sectors of agriculture. From the production to the
consumers, sensors are used in every process. Monitoring the agronomic environ-
ment for several factors such as humidity, temperature, moisture, pH, salinity, and
other factors will aid sustainable crop production. As a conventional approach to
monitor these factors during farming, individuals are employed physically to check
them at regular time intervals. Currently, the conscious employment of advanced tech-
nologies for significant improvement in the agronomic environment has increased.
Collecting manual data for the desired environmental factors leads to irregular and
incorrect measurements which will deviate from the actual values. Thus, leading to
difficulties in controlling vital environmental factors. The best solution would be
to use wireless distinctive sensor nodules which can minimize the time utilization
and physical effort for monitoring the agronomical environment effectively. The
data logging ensures the safety and security of the sorted data. Also, a possibility of
engagement in dangerous situations without the need to put personnel would reduce
the hazardous health risk. Hence, monitoring systems ensure faster response times
to environmental factors, improved quality control of the produce, and a lower labor
cost. Various factors affecting food production include temperature, moisture, atmo-
spheric pressure, and level of ground water. Advanced technology would definitely
be a great support for remote measurement of these vital factors. The aim of this
A. Mathur (B) · S. Wadhwa

Department of Physics, School of Engineering, University of Petroleum and Energy Studies
(UPES), Bidholi Campus, Dehradun, India
S. Nagabooshanam · S. Roy
S. Wadhwa
Department of Chemistry, School of Engineering, University of Petroleum and Energy Studies
(UPES), Bidholi Campus, Dehradun, India
218 A. Mathur et al.
chapter is to cover the most advanced and commonly used sensors in agriculture in
all processes involved.
Keywords Agriculture sensors · Smart technologies · Agriculture · Precision

farming · MEMS · NEMS
12.1 Introduction
Agriculture plays a vital role in our economy and in the survival of the nation. People
in rural areas rely on agriculture for their nutrition, employment, income generation,
and trade. With rising population, food demand has risen abundantly. For increasing
population, economic, and well-managed agriculture is desirable. The global popu-
lation will rise to 9.55 billion by 2050 and 11.2 billion in 2100 as anticipated by the
United Nations. Therefore, agricultural methods will need to change dramatically to
meet associated food demand without triggering irreversible environmental damage.
At present agriculture face a number of important challenges which include a dearth
of labors, climatic variability, poor land fertility due to drought, erosion, urbaniza-
tion, and the ever-increasing communal anticipations for sustainable practices like
marginal use of water and agrochemicals.
Agricultural growth is necessary for country’s development and conversion from
an old-fashioned to a contemporary economy. The technical advancement in methods
incorporated by farmers not only raises the value and scope of agri-production but
also enhances the standard of the economy and the livelihood of farmers. Sensor
technology is one such method used in varied fields. Sensors have wide scope
in agriculture. They can maneuver in a wide range of environments such as bare
fields, vineyards, orchards, and from flat to complex topography and varied weather
conditions. Though the maintenance of sensors and their usage is complex but once
installed, this functionality is always beneficial.
Agriculture sensors include sensors which operate in smart farming providing
monitoring of the produce, soil and moisture conditions, plant diseases, and other
important environmental factors. The data provided by sensors guide farmers to
effectively monitor and grow crops by regulating according to the environmental
variations. Sensors can be mounted over the weather stations, drones, and robots
which are employed in the agricultural industry. Specifically developed mobile apps
are used to control these sensors using wifi connections or cellular towers. Some of
the basic usages of sensors in agriculture include, the installation of them at weather
stations aids to provide vital data such as temperature of soil/air, moisture/humidity
of soil, chlorophyll content, climate/wind predictions, atmospheric pressure, etc.
They can also be integrated to several agricultural equipment’s like dendrometer
which is utilized to measure the diameter of tree or plant trunk, leaf wetness, etc.,
12 Journey of Agricultural Sensors—From Conventional … 219
agricultural drones to spray insecticides and pesticides and so on. Utilization of

solar pumps which are mobile operated is quite prevalent in agriculture for the sake
of electricity cost minimization. Also, e-fences can be beneficial for rural Indian
farmers which can saves crops from wild animals such as elephants (Fig. 12.1 and
Table 12.1).
Fig. 12.1 Schematic of interconnected sensors in the field and collection of data through cloud
computing (RF Wireless World 2012)
Table 12.1 List of agricultural sensors utilized for various purposes in agricultural industry
Agricultural Sensors Functional depiction
Locality Sensors To find out the latitude, longitude, and the altitude of any
position in a specific region. GPS satellites are used for
assistance
Optical Sensors To monitor various soil properties and they can be easily
installed on satellites, drones, or robots to regulate the
properties of the soil
Electrochemical Sensors To gather chemical data of the soil by detecting specific ions
in the soil. The information is obtained in the form of pH and
soil nutrient levels
Mechanical Sensors To monitor soil compaction
Dielectric Soil Moisture Sensors To monitor moisture levels
Air Flow Sensors To check air permeability
Fig. 12.2 Schematic of integrated wireless sensors for water content in agri-field (Damas et al.
2001)
12.2 Few Applications of Sensors in Agricultural Sector
12.2.1 Irrigation
Irrigation refers to a man-made application of water in agricultural land and forms a

key resource in agriculture. Due to the global scarcity of water, the use of it in agri-
culture must be monitored properly for its judicious use. Several means of irrigation
are followed such as drip irrigation, sprinkler irrigation, etc, which aids in solving
water-related problems during flood and drought. Spanish researchers worked on
developing a device controlled, automatic irrigation method for a specific region
(Damas et al. 2001). The region was further divided equally into seven small regions.
Wireless Local Access Network (WLAN) (Fig. 12.2) was connected to a controller in
which all the control sectors are interlinked to one another. The results demonstrated
good water conservation up to 30–60%.
A number of sensor projects have been formulated, keeping in view the require-
ment of controlling irrigated water usage for improved yield production. Another
researcher (Basu et al. 2006) developed an advanced computer-controlled drip irri-
gation system which has the ability to acquire data remotely. The saved sensor data
is utilized for the statistical analysis which aids in determining the irrigation method
required for a wide variety of crops. Automated irrigation integrated to Internet-
of-Things (IoT) provides the best solution to conserve water in agriculture and also
cost-effective.
12.2.2 Fertilizers
Soil fertility is an important factor for produce as it varies the rate of plant growth and
essence of food. Soil fertility can be affected to a desirable level by the application
of fertilizers. Broadcasting, manual spreading, and spraying are some of the various
ways of the use of fertilizers. The supply of fertilizer to the necessary place requires
sensing ability to make it more effective. Many researchers have demonstrated a
variety of solutions for optimal fertilization.
A mechanical sensor developed for an area specific fertilization (Pendulum meter)
by a German research team (Ehlert et al. 2004). This pendulum meter was used to
analyze the density of plantations and was mounted in the forepart of the tractor. A
fertilizer spreader and onboard computer were used along with the pendulum meter
to determine the level of N applied in the region. Another agri-researcher (He et al.
2011) developed an integrated support system to make decisions related to the use of
fertilizers using wireless sensors LAN with IEEE entente and GPS server analysis.
Real time soil moisture, temperature, conductivity, air moisture, pH value, radiance,
etc., can be monitored using this sensor system.
12.2.3 Pest Control
All over the globe the average crop production loss accounted due to pests and
insects during pre-harvest is about 35% (Oerke 2006) and an additional loss of 35%
is accounted for loss during transportation, food processing, storage, packaging, and
marketing (Popp 2011). The global demand for the increase in food production, crop
yield, and quality raw agricultural commodities intended the utilization of pesticides
in agricultural sector. The judicious usage of pesticides in agricultural practice helps
in protecting crops from pests and insects with unambiguous multi-fold increase in
the crop yield (Hassani et al. 2017). Controversially, the rampant usage of pesticides
has created havoc for the environment and human beings with serious health hazards.
The prolonged utilization of pesticides in agricultural fields affects soil fertility and
soil organisms over a longer period.
The main area of interest in “pest control” methods and procedures remains on
improving the products which help in better yield and advanced production. With the
advancement in more compatible and ecological newer technology, the pest control
techniques have become most optimal and best suited.
The overuse of pesticides contaminates the environment which causes various
issues related to health. In addition to it, the pesticides reach non-targeted species
and get accumulated and cause deleterious effects (Damalas and Koutroubas 2016).
Pesticides get drifted from the sprayed field to the environment through air, water,
soil, crops (fruits, vegetables, grains, pulses, and animal products), and human or
living beings as depicted in Fig. 12.1. When pesticides are sprayed in agricultural
fields or crops, the excessive amounts leach out into soil. They also get transported
into soil by the plat residues (Fenik et al. 2011). The consumption of pesticides by
human or living beings also occurs through residues present in fruits and vegetables.
In addition, the toxic pesticides enter humans when it is inhaled, orally in taken
and dermal exposure. By means of wind, dust, rain, volatilization, and evaporation
the harmful pesticides enter the water bodies and also contaminates air. Despite the
regulations formulated for the proper usage of pesticides in agricultural lands, the
Fig. 12.3 Life cycle of pesticides in the environment
overuse or misuse of pesticides still remains a menace. The exposure or persistence of

pesticides in the environment poses a high threat and therefore, proper management
on the usage of pesticides is more important for sustainable agriculture (Fig. 12.3).
From the literature, it is seen that the generally used techniques for detection
of pesticides were chromatographic techniques and other techniques include spec-
trophotometric sensors and immunochemical sensors (Stoytcheva and Zlatev 2011).
Chromatography-based techniques such as Gas Chromatography (GC), Liquid
Chromatography (LC), Mass Spectrometry (MS), Capillary Electrophoresis (CE),
Ion Mobility Spectrometry (IMS), Gas Chromatography-Mass Spectrometry (GC-
MS), Liquid Chromatography-Mass Spectrometry (LC-MS), and High Performance
Liquid Chromatography (HPLC) are the extensively utilized among the traditional
techniques for the determination and detection of pesticides (Stoytcheva and Zlatev
2011). The analytical techniques such as GC, LC, MS, CE, IMS are two-dimensional
sensors and based on separation methods. Due to the complex chemical nature and
their matrices of pesticides, the analytical separation methods which are integrated
with an analytical detector provides a better specificity and broad analysis range
(Hill and Martin 2002). Despite better specificity and broad analysis range, they fail
to determine the unknown samples. In order to overcome these three-dimensional
sensors which are upgraded versions and can detect unknown analytes are GC-MS,
LC-MS, and HPLC. For vapor phase samples GC is utilized which has the separa-
tion time of about ~ 30 minutes and this time can be minimized by wisely choosing
the selective detectors. However, the high-pressure requirement for the detection of
complex compounds and the small particle size of samples make GC less interesting.
The LC can be utilized for compounds which are non-volatile and thermally stable.
In LC, the slow diffusion rates lead to a longer detection time of about > 20 minutes.
For ionic and ionizable substances CE is preferred which has faster analysis time < 10
minutes with the use of specific detector named flame photometric detector. HPLC
and LC are similar in the process, but the difference is LC uses the gravitational force
for solvent movement in the column, whereas HPLC uses a high-pressure pump for
solvent movement in the column.
For field analysis, IMS are widely used which uses gas phase ions for sensing
pesticides and nerve agents within 1-minute exposure time of radioactive ionization
source. However, the use of radioactive ionization sources and low resolving power
of these devices make them less effective. Compared to IMS, MS uses the same
gas phase ions, but they measure the mass difference to the charge ratio traveling
through the vacuum column. Despite of having better resolving power, MS needs
more crucial separation process which makes it very tedious during sample analysis.
The coupling of GC and LC with MS obviously improves the performance with more
reliable data analysis. LC-MS can be employed for detecting unknown samples in the
range of ppb and yet it has the disadvantages such as the tedious sample preparation
process with slow diffusion rates.
There are few reports which utilize spectrophotometric techniques and immuno-
chemical assays. The general colorimetric test involves the detection of pesticides,
simply based on color change. In brief, the pre-treated spots are developed and when
the analyte gets absorbed on the pre-treated spots there will be a noticeable change in
color, representing the presence of OPs. This approach is less sensitive and not a reli-
able way for the practical utilization of spectrophotometers. It is based on luminance
and fluorescence, in which a change in signal is detected upon the absorption of OPs.
The advantage of spectrophotometric techniques is good sensitivity, relatively simple,
and low detection limits in the range of ppb. The disadvantage of this technique is
the detection time of about ~ 10–20 minutes, less selectivity, and unsatisfactory
quantitative detection. The immunological assays, for example, the Enzyme-Linked
Immuno-Sorbent Assay (ELISA), overcome the limitations of these techniques. It
is based on the principle of immune-assays which utilizes antigen-antibody specific
interaction. With the help of ELISA, we can determine the measure of antigen or
antibody concentration. The advantages of ELISA are that they are highly selective,
sensitive, accurate, and do not use any radioactive agents or sophisticated devices. The
recent interest is on developing a bio-sensing platform which provides several benefits
such as extraordinary selectivity, sensitivity, lower detection limits, a wider detec-
tion range, cost-effectiveness, user-friendly, and non-requirement of a sophisticated
analytical laboratory. Nagabooshanam and colleagues fabricated an electrochemical
sensing platform to detect organophosphates utilizing functional materials such as
carbon nanocomposites and metal organic frameworks (Nagabooshanam et al. 2019,
2020). Other types of pesticide sensors are based on an economical paper-based 3D
origami electrochemical sensor developed in recent studies (Arjmand et al. 2017;
Arduini et al. 2019).
12.2.4 Use of Sensors in Horticulture
The practice of cultivation, production, distribution, and use of flower, fruit, green-
house, ornamental crops are considered as horticulture. Horticulture represents low
scale or low levered farming. Greenhouse is a key area under horticulture as the
research community has shown core concern toward this. A wireless network devel-
oped by Zhang et al. (2004) tracks various key parameters such as temperature,
moisture/humidity of the soil, ambient light, etc., which helps in crop production
and yield.
12.2.5 Soil and Crop Sensor
Essential soil properties can be determined using agriculture sensors. Such sensors
are utilized to create field maps to investigate the properties of soil which can be done
by using control equipment in a real-time basis or along with Global Positioning
System (GPS). Also, determines the factors such as the frequency of sampling or
measurement, the space in between the passes, and the travel speed, and the total
measurement of points per acre. This provides a cost-effective route for field mapping.
The efforts had been made by scientists and equipment manufacturers in order to
modify the existing laboratory and other indirect monitoring techniques for on-the-
go soil mapping. But, as of now, very few types of sensors have been developed
and investigated which includes electrochemical, electromagnetic, optical, acoustic,
mechanical, and airflow sensors to name a few.
Electrochemical sensors detect the soil pH and soil nutrients. The soil samples
testing at the laboratory involves various standard laboratory procedures, sample
preparation methods, and measurement techniques. Some of the techniques utilize
ion-selective electrodes which measure the activity of specific ions. Determination of
soil pH, nitrogen, and potassium can be done using an ion-selective electrode. Several
traditional soil preparation and measurement procedures are adapted to conduct a
laboratory test on-the-go which eventually increases the overall accuracy of soil
nutrient or pH testing.
Electromagnetic sensors utilize electromagnetic circuits to monitor the conduc-
tivity or accumulated electrical charge of the soil particles. In these types of sensors,
the soil becomes part of an electromagnetic circuit itself and the small variations in
the local soil conditions can be readily measured. Several commercial sensors that
available in the market. One such is the Soil EC 3100 which is an on-the-go soil
mapping sensor developed by Veris Technologies, Salina, Kansas (https://www.ver
istech.com/the-sensors/v3100). Another commercial device is EM-38 developed by
Geonics Ltd, Ontario, Canada which maps the electromagnetic response to inves-
tigate soil properties. The electromagnetic properties of soil alter according to its
texture, moisture/humidity, salinity, and organic matter constituents. Electromagnetic
sensors can also be utilized to monitor soil pH and nutrients.
Optical sensors utilize light, near/mid-infrared light, and polarized light

reflectance to measure soil properties such as moisture, nutrients, and organic
constituents. The light reflectance varies in between the healthy or deficient soil
and once the data is procured it is analyzed by mapping method to measure various
soil properties such as moisture, nutrient level, etc. Few companies offer a remote
sensing facility which aids to determine soil reflectance by engaging a satellite or
airplane platform. Despite, the advantages of these platforms there are several factors
such as the cost, timing, clouds, and ground covered plant residues limit the usage
of rare soil imagery. Nearby-range, subsurface, and on-the-go vehicle-based optical
sensors have the potential to measure the data points or information certainly at more
than one portion of the spectrum at a time.
Acoustic sensors measure the change in acoustic pulse transmission which
happens due to a change in the soil properties. It helps to monitor soil texture,
moisture, nutrient constituents, etc. The major limitation of acoustic sensors is that
the emitted acoustic signal is very weak and noisy. Mechanical sensors measure the
mechanical resistance or compaction of the soil. Due to the force exerted by strain
gauges or load cells at the time of measurement a cut/penetration which propagates
through the soil is measured. Several device prototypes have been developed which
clearly demonstrates the applicability of uninterrupted mapping of soil resistance.
Still, these devices are not yet available commercially.
Airflow sensors measure the soil air permeability. In these types of sensors, the
pressure required to squeeze a certain volume of air into the soil at a fixed depth is
associated with various soil properties. Airflow sensors can be utilized for analyzing
varied soil types, moisture levels, and soil structure/compaction.
12.3 Gas Sensors Used in Agriculture
Gas sensors provide an important platform for measurements within the agricultural
industry for various applications. Temperature and Carbon Dioxide (CO2 ) concen-
tration can be monitored using commercial greenhouse control systems for better
crop growth. Low range CO2 Guardian NG DC developed a device for continuous
monitoring of CO2 which provides an economical solution to increase crop growth
(https://edinburghsensors.com/). In pig and poultry farming the monitoring CO2 is
of utmost importance during the process of gas stunning. For this, the Gascard NG
is utilized which can monitor the high concentrations of CO2 during poultry food
processing. CO2 measurement is also vital to detect the spoilage of stored grains
wherein gas sensors are employed. The example of commercial gas sensors for the
measurement of CO2 is shown in Fig. 12.4.
Fig. 12.4 Commercial gas sensors available for monitoring CO2 (https://edinburghsensors.com/)
12.4 Sensors in Food Packaging, Transportation,

and Inspection
Economical RFID biosensor tags were generally utilized to maintain inventory

and examine food products. Few reports demonstrated a prospective of RFID
tags for sophisticated appliances, recycling, packaging, automatic checkout, and
marketing/furthering opportunities (Kumar et al. 2009; Smits et al. 2012). The RFID
technology aids to provide safe, productive, and traceable produce which leads to
operational savings and increased capital. Geers et al. (1998) developed a monitoring
system that functions on the road for animals during transportation and handling. This
technology involved the installation of sensors in the animal section in order to detect
the animal variety, to check on the air-quality and animal behavior. The location of
the vehicle during the transportation is provided by GPS. Regular merits are sent
to service centers through GSM network by a data transfer unit regularly. Najjar
et al. (1997) developed a portable PC for the quality check (QC) inspectors working
in food-processing plants. This type of system helps the QC inspectors to select
a suitable for filling the data and send it to the plant manager through full duplex
audio and wireless data communication. Recently, wireless sensors are being used
to monitor and for quality control purposes in the food processing industry. The data
of the sensor can later be transferred to the central data controller wirelessly. Marra
and Romano (2003) designed a systemic mathematical model in order to investigate
the effects of various methods which interposes wireless temperature sensors into
conductive tinned food for inspecting thermal sterilization.
12.5 Smartphone-Based Sensors in Agriculture
Farming activities are more inclined toward the growth of plants, killing weeds/pests,
identifying and correcting plant diseases, applying fertilizers, and estimating
growth/yield of crops. Recent progress in smartphone application development and

increasing availability of smartphones have been very helpful in lifting some of these
agricultural burdens and provide necessary guidance. For instance, smartphone appli-
cations can be used to examine color of crop leaves and farmers can calculate the
amounts of fertilizers to be applied to the crop fields. Many tasks in the field such
as seeding, weeding, fertilizing, and watering, appear repetitive, mundane, and labo-
rious. However, these tasks usually require precision and involve decision-making
steps prior to the actual activities so that the farming cycle becomes effective (Yang
and Li 2008). Prior knowledge on identifying crop diseases, the location of the disease
in crops, and their prevention and cure is necessary to save the time of farmers and
costs in the practice of farming. This can be made possible by smartphone appli-
cations in farming. The subcategories of smartphone applications in farming are
discussed below:
Disease Detection and Diagnosis: Smartphones enabled with sensors can be
utilized for disease detection/diagnosis in farms. Prasad et al. (2014) developed a
mobile vision system for plant disease identification. With this system, images of
diseased plant leaves are captured and preprocessed. Then the images are transmitted
and sent to remote laboratories. The image preprocessing step saves the transmis-
sion cost of sending diseased leaf images to plant pathologists in remote laboratories.
Leaf images are further segmented into three areas: background, non-diseased and
diseased portions of the leaf. Suggestions on cure and prevention methods for plant
diseases are then provided to farmers. A mobile application Magri was suggested by
Wu and Chang (2013), which uses pest/disease information previously self-reported
by farmers. Magri’s report module facilitated the generation of advice for the cure
of pests and pest alerts in many nearby areas using GPS.
Fertilizer Calculator: Application of fertilizers on crops is an important farming
activity which has a great potential to affect farm productivity. Farmers make a
key decision about the chemicals to apply and their crop-specific suitable amounts
needed. Sumriddetchkajorn (2013a) reviewed a few mobile device-based optical
applications for agriculture. It has mentioned a smartphone-based color estimator
named BaiKhao, dedicated for rice leaves’ chlorophyll evaluation. The newer
released version BaiKhaoNK could evaluate the color level of rice leaves and
suggested the amounts of nitrogen fertilizer needed onto the rice field (Intaravanne
and Sumriddetchkajorn 2012; Sumriddetchkajorn 2013a, b).
Soil Study: Soil forms a key component in farming that has a huge effect on the
agricultural output. Soil data supplied to farmers gives them an advantage in farming,
especially in precision agriculture. A large variety of smartphone applications are
exemplified in the literature that uses smartphone sensors to study soil properties
and collects soil data for agricultural purpose. Mobile phones were used as soil
color sensors (Gómez-Robledo et al. 2013) which could read soil color informa-
tion based on the images captured by built-in digital cameras on smartphones and
processed using image-processing techniques. One more smartphone application is
‘Soil Indicators for Scottish Soils’, SIFSS, has demonstrated the capability to study
soil (Aitkenhead et al. 2013). Farmers were able to receive soil’s detailed information,
such as pH, soil carbon, N, P, and K, based on their locations in Scotland. SOCiT,
another application is aimed at users in Scotland, provided information about soil’s

carbon content based on users’ geographical positions (Aitkenhead et al. 2013).
Water Study: The quality of water is an important parameter affecting farming and
agriculture in various regions. A smartphone application has been developed under
a project in Scotland, iDee, which is devoted to inspire users to provide information
of water conditions, such as water level, clarity, obstruction in river, algae cover,
temperature, and nonnative plants in water, and accompanying photographs of the
River Dee (Aitkenhead et al. 2013).
Crop Water Needs Estimation: Crop water requirements of an agricultural land
rely on numerous conditions including crop types, season, climate, and growth stages
of crops (Allen et al. 1998). Crops lose water through transpiration and nearby soil,
water, and canopy lose water through evaporation. Collectively, the loss of water
through both these processes is termed as evapotranspiration (Allen et al. 1998).
Crop water requirements are verified to supplement water loss so that crops grow
according to farmers’ needs. PocketLAI, a smartphone application is developed to
help farmers to determine Leaf Area Index (LAI), one of the key factors to evaluate
crop water needs (Confalonieri et al. 2013). The application PocketLAI evaluated LAI
by an indirect method based on sensors equipped on modern smartphones. Image
sensors (cameras) captures the pictures of leaf canopy to evaluate Leaf Area Index
while accelerometers obtains the angle of smartphones as the devices are rotated
to evaluate LAI and the gap fraction was measured at 57.5° (Confalonieri et al.
2013). Farmers can regulate their watering/irrigation tools accordingly after water
requirements were estimated from Leaf Area Index. Sumriddetchkajorn et al. (2013a,
b) devised an application to evaluate brightness smartphone cameras. Another mobile
application RaGPS was developed by Molina-Martínez et al. (2011), for devices with
Windows Mobile operating system to determine the extraterrestrial solar radiation
and similar other parameters in real time. Such radiation parameters offer a useful
tool to estimate crop water requirements and validating weather data provided by the
agricultural weather stations. The experiments demonstrated that RaGPS was able
to estimate radiation parameters quite similar to those obtained from the weather
station in clear-sky days.
Crop Produce Readiness Analysis: Another novel application of smartphone appli-
cation is to determine the ripeness of fruits (Sumriddetchkajorn 2013a, b). Various
images of fruits under white and UV-A light sources were captured to evaluate
ripeness levels of green fruits. The system could be integrated by farmers into their
farms to determine different ripeness levels of various fruits before sending them to
markets. The computer vision techniques helped farmers to complete the process at
a bulk scale rather than manual inspection of each fruit by farmers.
Smartphone applications are useful in sensing the environment using a number of
sensors under various farming categories and processes of planting crop. Observed
data may be in the form of snapped pictures, specific locations in the farm, colors of
soil, water, and plant leaves, and light. Farmers are the key target users of smartphone
applications as they are involved in mundane and laborious farming activities. Such
applications aim at enhancing farm productivity, by evaluating agricultural speci-

mens, supporting agricultural decisions, and solving task-specific problems. (Intar-
avanne and Sumriddetchkajorn 2012; Confalonieri et al. 2013; Sumriddetchkajorn
2013a, b; Gómez-Robledo et al. 2013; Prasad et al. 2014).
12.6 Conventional Sensors in Agriculture
12.6.1 Soil Quality Sensors
Soil properties play a key role in promoting crop growth and yield. Hence the prop-
erties need to be continuously monitored for precision agricultural practice. For a
higher yield and quality production, NPK as fertilizers are utilized (Prativa and Bhat-
tarai 2012). Despite the availability of NPK naturally in the soil to further enhance
the growth of crops man-made formulations are incorporated (Mengistu et al. 2017).
These formulations help to improve the soil quality in the nutrient scarcity areas.
The blend of organic manures and the man-made NPK formulations are known to
increase the yield but, as a controversy the injudicious use of NPK is a major threat
to soil quality and fertility (Bhati et al. 2018). Therefore, soil nutrient management is
of utmost importance and there are some traditional techniques to monitor the NPK
values in the soil for sustainable agriculture one such is the random grid sampling
technique (Wollenhaupt and Wolkowski 1994). In the random grid technique, the soil
samples were collected in the random grid area and the samples were sent to analyt-
ical laboratory. Here, the samples go through the extraction process and extensive
chemical analysis for the determination of NPK values. The other conventional tech-
nics utilized for the NPK sensing are chromatographic techniques, spectral imaging
analysis and ion-selective field effect transistors (Adamchuk et al. 2005; Chen et al.
2014; de Castro Vasconcellos et al. 2015). The aforementioned techniques are selec-
tive, and sensitive but they are time consuming, expensive, and require sophisticated
laboratory equipment’s. In order to overcome above limitations, various sensor tech-
niques such as optical, electrochemical, spectroscopic, etc., are explored which are
economical, portable, and user-friendly. Lanfranco and colleagues utilized Raman
spectroscopic technique to detect the mixed-metal phosphates in soils at low concen-
trations (Lanfranco et al. 2003). Raman spectroscopy involves a simple detection
technique depends on Raman scattering upon the interaction with various samples.
Other than spectroscopic detection electrochemical sensors are used for NPK detec-
tion. Chauhan and Urooj (2019) developed an ion-selective field effect transistor
(ISFET)-based electrochemical sensor for the analysis of NPK values in real time.
Apart from the soil nutrients as mentioned above other parameters of soil such
as pH, moisture, salinity, etc., are also of equal importance for the overall crop
production and sustainable agriculture. The pH values of the soil influence the crop
growth, i.e., the growth is good at higher values of pH and the growth is declined
when the pH values are lower (Kumar et al. 2015). The optimum pH value is between
6–7.5 and it varies for different types of crops. Rani et al. (2008) reported a sensitive
pH sensor based on multi-finger ISFET sensor. In this ISFET the change in the H+
ion concentration is detected between the electrode and electrolyte which influences
the change in potential at the gate.
12.6.2 Monitoring Moisture Content and Temperature of Soil
Soil moisture is also important along with the soil nutrients, pH, salinity, etc., and
hence it is necessary to monitor the soil moisture levels regularly (Chadha et al.
2019). The conventional laboratory method to test the soil moisture is gravimetric
water content and volumetric water content analysis (Almaw et al. 2018). In former,
it measures the mass of water per mass of soil sample and the later measures the
volume. The exchange of thermal and hydro energy between the surface of land and
atmosphere is controlled by soil temperature and moisture content by means of evap-
oration and water/nutrient transportation in plants. Consequently, moisture content
and soil temperature play a vital role in the regulation of weather conditions, the
latter being a key player in crop production and growth. Micro-machined cantilever
beams consisting of a water responsive nano-polymer integrated with piezo-resistive
temperature sensor form the basic components of MEMS-based temperature and
moisture sensing devices.
Soil moisture levels and corresponding electrical conductivity are the two most
important parameters employed in agriculture. However, recently soil pH is often
employed as a criterion for assessing crop productivity as well as being used as a
tool to scrutinize areas where adequate fertilization has occurred. Few other param-
eters, namely, water flux and thermal properties of soil (thermal diffusivity, thermal
conductivity, and specific heat capacity) are also employed for ensuring enhanced
crop productivity (Heitman et al. 2007; Bristow et al. 1994; Ren et al. 1999; Ham
and Benson 2004). Electrical conductivity readings can also be monitored using the
same conducting needle-like probe (Mortensen et al. 2005; Valente et al. 2006). Ion-
Sensitive Field Effect Transistors (ISFETs) are normally used for measuring the soil
pH as shown in Fig. 12.5.
12.7 MEMS Devices Used in Agriculture
In 1986, the concept of Micro Electro Mechanical System (MEMS) was first
presented. Since then, numerous devices were commercialized throughout the globe.
Nowadays, multiple devices incorporate MEMS technology, namely, inkjet printing
heads, accelerometers, gyroscopes as well as modern surgical tools and Lab-on-
Chips (LoCs) for healthcare applications. Some other applications of MEMS-based
devices include modern car airbags, touch screen-based smart phones, drones, or
UAVs (Unmanned Arial Vehicles), gaming consoles, digital cameras to name a few.
Fig. 12.5 A typical MEMS-based ISFET employed for monitoring soil temperature, nutrient
content, moisture levels, and pH toward smart agro-applications (https://www.davisinstruments.
com/)
It can therefore be concluded that consumer goods, electronics, and healthcare are
the prime areas of research for MEMS-based device applications. In agriculture
sector, MEMS is used in drone technology for monitoring crop quality and growth.
However, when it comes to Precision Agriculture (PA) which states that farmlands
should be categorized into zones rather than whole fields in order to increase produc-
tion efficiency, Internet-of-Things (IoT) technology has the capability to be inte-
grated with MEMS devices for ensuring enhanced crop quality monitoring. MEMS
market is expected to rise drastically from $11 billion in 2014 to $21 billion in 2020
(Bariáin et al. 2000). This is further supplemented due to the availability of highly
efficient biomedical, biochemical sensors, and energy harvesters which can impart a
huge impact on the agriculture sector. Owing to high-market value of MEMS-based
devices facilitated by multitude of applications and advantages, such technologies
find huge potential to be implemented in agriculture sector for enhanced outcomes.
12.7.1 MEMS for Monitoring Crop Root Growth
In order to quantitatively assess physical interaction between crop root and the soil,
a silicon microchannel device integrated with motion sensors has been recently
developed which helps in the analysis of certain barriers which may impede the
uptake of minerals and nutrients from soil to crop roots.
12.7.2 MEMS Microchip-Based Capillary Electrophoresis

Sensor
Plants generate a defense mechanism against numerous pathogens or biotic stress

through different strategies. Plants respond to fungal attack by activating defensive
responses associated with the accumulation of pathogenesis-related proteins (PRs),
which prevent the occurrence of pathogen infection. Capillary electrophoresis is an
important technique which aids in the detection as well as the separation of variety
of species—from heavy metals to phenolic acid in plant extracts. Analysis of PRs
by means of MEMS-based capillary electrophoresis tool in Eruca sativa cultivation
to detect pathogen presence was investigated in 15 and 25 days old plants (Sharma
et al. 2018, 2019).
12.8 3-D Imaging Systems for Agricultural Applications
Currently, 3-D sensors are becoming smaller, smarter, and highly affordable. There-
fore, technological breakthroughs are viable provided sufficient research leads to
commercialization since agricultural environments are substantially complex. In such
scenario, 3-D vision can serve an important role in improved perception which can
positively impact a huge number of applications in every agricultural aspect. The
ultimate efficacy of 3-D data depends on the excellent sensing capabilities compared
to 2-D strategies. Several market predictions have recently surfaced around topics
such as 3D sensors, LED lighting, UAVs, and agricultural automation. Almost all of
them predict a profitable market value for these interconnected topics by the end of
the decade. Few 3-D techniques have either not been tested (i.e., shape polarization,
photometric stereo, shape-from-zooming) or more research is yet to be performed
with the rich variety of 3-D imaging techniques for agricultural applications (i.e.,
interferometry, light field, CTCs). 2-D and 3-D fusion are highly promising since
it exploits the advantages of both the former and latter and has proved to be useful
for either obtaining detailed information about the object’s surface or facilitating
the image segmentation process. All 3-D sensors are sensitive to sunlight; however,
further research must be performed to reduce the latter’s effects and reduce depen-
dence on shading devices. A positive outcome of this disadvantage is enabling of
a thorough investigation of night farming since 3-D sensors behave properly in this
environment.
12.9 Use of Smart Cameras in Agriculture
While smart camera technology happens to be far from a typical analog sensor, it
has been increasingly implemented in a variety of smart agro-applications. Indus-
trial firms such as Blue River Technology, USA have incorporated smart camera
technologies to monitor the presence of weeds and plants to release herbicides and
fertilizer automatically and precisely. This process increases the overall productivity
by decreasing chemical usage, the latter at high quantities might often damage crops
and plants. Moreover, pest control has always been one of the most prolific chal-
lenges in agriculture. Nowadays, farmers employ smart cameras for real-time pest
monitoring and to effectively minimize/eliminate pest presence without harming the
crops. Smart cameras also possess the potential to replace sensing devices, namely,
ambient light monitoring, which enables system simplification and a reduction in the
component count.
12.10 Some Challenges
For a practical application, a sensor must be positioned in direct contact with, or in

close proximity to the ground and coupled to a “black box” in order to analyze the
sensor response with further processing of the data. A sensor must be able to provide
real-time data to improve the total economic or agronomic effect of the production
input. Nevertheless, the following difficulties observed in the “real world” are not
considered while considering this approach:
• Many agronomic sensors and applicator controllers need a definite time for
measurement, integration, and analysis. This lessens the operational speed or
measurement density.
• Additional information is required by variable rate fertilizer and pesticide (such
as yield potential) to design prescription algorithms.
• Presently, no site-specific management prescription algorithm is available that
demonstrates compatibility for all variables involved in crop production.
The most appropriate approach, rather than utilizing real-time sensors, would be
to use on-the-go sensors equipped with controllers and a map-based method. This
will allow the possibility of data collection and analysis, creating the prescription,
and carry out the variable rate application in minimal steps.
12.11 Limitations of Agriculture Sensors
Some of the drawbacks of agriculture sensors comprise the following:

• Continuous internet connectivity is required for smart farming and IoT technology
which is more challenging in developing countries and other parts of the world.
• There is some resistance from the consumer’s side to acknowledge and accept the
latest IoT-based devices equipped with smart agricultural sensors.
• Some basic infrastructure necessities including smart grids, traffic systems,
and cellular towers are unavailable at vast places. Therefore, further growth is
hampered.
12.12 Future of Agriculture Sensor Technology
The smart agriculture industry is continuously growing, along with almost daily
arrival of new innovative smart solutions to the market. Devices which accumulates
sensor data, relay critical information to farmers and ranchers, which in-turn helps
to optimize vast agricultural processes are continuously increasing in capability and
importance. Smart sensors are used for precision agriculture to import critical data
that helps farmers to monitor and optimize crop yield, and also to keep up with
changing environmental conditions. By employing smart sensors, farmers can assess
their crops at a micro scale, sustain resources, and reduce environmental impacts.
Smart agriculture dates back to the 1980s when GPS became accessible for civic
use. At that point, farmers were able to accurately examine their fields, as well as
monitor and apply fertilizers accurately at desired areas.
Attaching a sensor and tracking device to a cow will facilitate the ability to track
the cow’s health and other critical behaviors (Fig. 12.6). A smart sensor will thus
help with easy animal examination such as body temperature detection and health
monitoring to separate and treat sick cows within the herd. A smart sensing device
will further help with early mastitis detection to lower risk of milk loss.
Due to shortages in labor and the urgent need to feed an increasing global popu-
lation, agricultural robots are now commonly implemented by farmers. In the USA,
Fig. 12.6 Representative

pics showing applications of
sensor with IoT technology
for monitoring cow health
and activity for enhanced
production (Calderone 2019)
crop production has decreased by an estimated $3 billion a year due to labor shortages.
At the same time, the world’s population is expected to grow by 2.3 billion within
the next 3 decades. Vision and machine learning now allows robots to watch and
train using their surroundings, and due to the cost-effective nature of smart sensors,
they are being deployed in an increased rate. Yet, we are in the early stages of an
agriculture robotics revolution with most of the smart devices in the early phases of
trial process and R&D mode.
Emerging smart sensing technologies allow farmers to remotely assess their
fields’ pest population in real-time and take immediate action to protect their crops,
facilitated by online cloud services and a dashboard acting as the central hub
(Fig. 12.7).
A trap employs a kind of lure (pheromone) to attract and imprison a particular
pest. Information regarding pests is wirelessly transmitted to the central hub and
eventually to farmer’s smartphone or computer. The farmer will then be able to view
a satellite image of their field with a quantitative measure of number of pests captured
Fig. 12.7 Representation of remote-controlled access of pest population in specific spots in

farmlands in real-time facilitated by state-of-art cloud computing techniques (Calderone 2019)
in each trap as well as details on historic trends and pesticide use. This facilitates
the process of quantitatively checking insect traps and figuring out where and how
much pesticide to be used in a particular spot in the field, thereby eliminating billion
dollars’ worth of pest damage.
Optical Sensors employ light to analyze soil properties, by recording various
frequencies of light in near-infrared and mid-infrared spectrums. These sensors can
be placed on vehicles or drones which allows soil reflectivity and plant color data to
be recorded and processed. Nature of clay, organic matter, and soil moisture can be
determined by the implementation of optical sensors.
Electrochemical Sensors mounted on specially designed spots help collect,
process, and map the chemical nature of soil. Electroanalytical sensors provide
the information required for precision agriculture, that is facilitating the monitoring
of soil pH and nutrient levels. Soil samples are sent to a soil-analyzing laboratory
and an array of standard protocols are carried out. Certain measurements, especially
the identification of pH, are performed by utilizing an ion-selective electrode. These
electrodes monitor the activity of certain ionic species such as potassium, nitrate, or
hydrogen.
Mechanical sensors are used to assess soil compactness which is another key
parameter which affects the overall crop yield. These sensors employ a mechanism
that penetrates the soil and quantifies the force monitored by load cells or strain
gauges. When such a sensor probe penetrates the soil, it records the resistive forces
resulting from the cutting, breaking, and displacing of soil. The soil mechanical
resistance is then measured in pressure units and signifies the ratio of the force
required for penetrating the soil medium, to the front surface area of the tool engaged
with the soil.
Dielectric soil moisture detectors monitor humidity levels by measuring the dielec-
tric constant, which varies depending on the soil moisture content. The moisture
sensors are used in conjunction with rain gauge stations throughout the farm. This
allows for the observation of soil moisture conditions when vegetation levels are low.
Agricultural weather stations primarily are self-contained sensors that are situ-
ated at different locations in particular farmland. Such stations consist of a variety of
sensors that are suitable for examination of local climatic conditions, thereby leading
to improved assessment of crop quality. Parameters such as soil and air temperature
at various depths, leaf wetness, rainfall, chlorophyll content, dew point temperature,
wind speed, wind direction, solar radiative power, relative humidity, and atmospheric
pressure are determined and recorded at programmed intervals. Due to the minia-
turized nature of such devices, the acquired data are then transmitted wirelessly to
a central hub followed by transfer to nearby control stations for facilitating faster
necessary actions.
There could be a loss of time and production should a farm vehicle break down.
Farmers now have the ability to remotely gather and manage information from their
field equipment. Equipment telemetry lets mechanical devices such as tractors to
Fig. 12.8 Representation for emerging smart sensing technologies employed to transmit soil-nature
data to far away field supervisors by means of satellite communication (Calderone 2019)
notify mechanics that a failure is likely to happen soon. Most of the agricultural
equipment companies are building telematics systems.
Electronic sensors are often mounted on tractors and other field equipment in
order to monitor the former’s operating conditions. Meanwhile, cellular and satellite
communication systems are also used to transmit the data immediately to computers
or e-mail it to certain individuals as represented in Fig. 12.8. The field supervisor
can then retrieve the information on their office computers or cell phone and take
necessary action.
Airflow sensors measure the permeability of air in the soil. Measurements can be
made at one particular location or at multiple spots. The desired output is the pressure
required to push a predetermined amount of air into the ground at a pre-programmed
depth. Various types of soil properties, including compactness, chemical nature, and
moisture level produce unique signatures.
The use of smart sensors, software, and eventual integration with IoT as well
as artificial intelligence-based platforms possess huge potential to transform the
agricultural market. Combinational sensors along with sensor fusion hardware
possess tremendous potential to generate the required plant data in order to enhance
crop production rate and implement disease-free high yield crops to maintain the
synchronization with the increasing demand for food proportionally with the global
population.
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Part II
Biosensors in Food Science
Chapter 13
Advances in Biosensors Based
on Electrospun Micro/Nanomaterials
for Food Quality Control and Safety
Aylin Altan and Meryem Yılmaz
Abstract The food industry needs fast, sensitive, simple, and low-cost techniques to
ensure the safety and quality of food products throughout all the stages from produc-
tion to dispatch. The application of different types of biosensors to monitor safety
and quality of food has an increasing importance to meet these requirements. Elec-
trospun nanomaterials produced by electrospinning technology have high potential
for use in the development of biosensors due to their characteristic features such
as a high surface area-to-volume ratio, controllable porosity, and interconnectivity.
The large surface area of electrospun nanofibers provides the abundant active sites
for immobilization of biomolecules and active species. Furthermore, electrospun
nanofibers can be further functionalized by incorporation of nanomaterials within
polymer solution or after electrospinning process to improve sensitivity, stability, and
selectivity of biosensors. Recent studies show that electrospun fibers are promising
for the design of the biosensor platform in many areas such as clinical, biomedical,
and environmental fields, but there are limited number of studies for their use in food
applications. This chapter aims to present the recent progress regarding the devel-
opment of electrospun nanofibers-based electrochemical biosensors in monitoring
food safety and quality, such as detection of pesticides, pathogens, and toxins, both
detection and quantification of food components, and process control.
Keywords Biosensors · Electrospinning · Nanofiber · Food safety · Quality

control
13.1 Introduction
Quality assurance and safety of food during manufacturing process are the essential
requirements to preserve the quality of foods while preventing contamination and
spread of foodborne illness. Food safety and quality relates to the microbiological,
toxicological, chemical, physical, and organoleptic characteristics of foods, which
A. Altan (B) · M. Yılmaz

Department of Food Engineering, Mersin University, Mersin, Turkey
244 A. Altan and M. Yılmaz
have to be ensured to guarantee in an acceptable level for adequate protection to

consumers. Conventional methods have an important role in evaluating the safety
and quality of foods, but they require highly trained staff and long procedures that
limit their use for routine analysis. The demand for rapid, sensitive, and accurate
measurements has been continuously rising in evaluating of food quality and safety
during and after food processing. Nanotechnology has great potential to meet these
demands of food industry. Recent studies have focused on the potential applications
of nanotechnology in food and agriculture as encapsulation of active compounds,
controlled release of active ingredients, smart packaging, and sensors. Sensors are
devices used for the detection or measurement of the physical or chemical properties
of the target analyte to which the device responds by giving a signal (Kress-Rogers
1998). These devices contain two main components which are a receptor and a trans-
ducer. The physical and chemical information is transformed into a form of energy by
receptors, while this energy is converted into electrical signal by transducers (Caon
et al. 2017). In biosensors, bioreceptors recognize a target analyte, while transducers
convert biochemical signals into a measurable signal. The bioreceptor or biorecog-
nition element in biosensors can be an organic or biological material (e.g., enzyme,
antigen, microbe, hormone, or nucleic acid) (Yam et al. 2005). Biosensors devel-
oped based on nanomaterials are also called as nanobiosensors. Low detection limits
can be achieved by nanobiosensors, leading to significant improvements in food
quality control and safety (Pérez-López and Merkoçi 2011). Nanotechnology allows
development of nanomaterials such as nanocomposites, carbon nanotubes, metal
nanoparticles, and nanowires. These materials play a major role in the design of
biosensing systems because they exhibit unique surface chemistry, thermal and elec-
trical properties, and high surface area per unit mass (Asefa et al. 2009; Pérez-López
and Merkoçi 2011; D’Souza et al. 2017). The unique properties of nanomaterials can
help improve response times and the sensitivity of biosensors.
The versatility of electrospinning technology allows spinning of various polymers
for the production of micro/nanofibers with large surface area, high porosity, and high
molecular orientation (Zhao et al. 2020). Such submicron and nanofiber electro-
spun materials have many structural and functional advantages such as tunable fiber
diameters and tailored morphology, efficient encapsulation, sustained and controlled
release of active compound (Wen et al. 2017). These materials have been applied
in a wide range of fields such as biomaterials, drug delivery, energy storage and
conversion, environmental protection but the application of nanofibers in agriculture
and foods is relatively new. Recent studies have demonstrated the great potential of
electrospun fibers in encapsulation of bioactive compounds with enhanced stability
and high encapsulation efficiency and active and intelligent food packaging appli-
cations (Fig. 13.1) (Echegoyen et al. 2017; Wen et al. 2017; Topuz and Uyar 2020).
However, many structural and functional advantages of electrospun fibers make them
potential candidates for biosensor applications (Yılmaz and Altan 2017). The large
surface area of nanofibers can provide an increase in the availability of sites for the
interaction or loading of catalysts or other reactive materials and this can increase the
reactivity of the material and fast adsorption or release mechanism in the develop-
ment of biosensors (Mercante et al. 2017). This chapter presents the electrospinning
13 Advances in Biosensors Based on Electrospun … 245
Cells and enzymes immobilization

Sustained or controlled releaseof
bioactive compounds
Micro/nano
encapsulation
Food
packaging Detecting pesticides
Bioprocess monitoring
Sensor Detecting food components
Active food packaging Electrospinning and contaminants
Intelligent food packaging
Edible films
Filtration
Filtration of beverage products
Filtration of yeast and bacteria
Fig. 13.1 The potential use of electrospun nanofibers in food applications
process and the recent applications of electrospun nanofibers in the development of

electrochemical biosensors for food quality control and safety.
13.2 Electrospinning Process
Electrospinning is an effective and versatile process that produces fibers from a

variety of polymers in submicron to nanoscale range. A power supply with high
voltage, spinneret, syringe pump, and collector are basic parts of the electrospinning
system (Fig. 13.2). During processing, the polymer solution is first pumped at a
constant rate, polymer droplet is formed at the tip of the needle or nozzle. The
droplet at the nozzle tip is subjected to two forces that are the Coulombic force
of the external electric field and the electrostatic repulsion of similar charges, and
hence the droplet shape changes from hemispherical to conical shape, known as the
Taylor cone. After reaching a critical electric field value where the electrostatic force
exceeds the surface tension, the charged liquid jet of polymer solution is ejected
from the tip of the cone. The jet is elongated by whipping and bending motion as
it travels to the grounded collector (Bhushani and Anandharamakrishnan 2014). As
the jet moves toward the collector, the charged jet becomes thinner and solvent is
evaporated rapidly. Consequently, the solidified ultrafine fibers are collected on the
collector. The forces that are electrostatic, drag, gravitational, Coulombic repulsion,
viscoelastic and surface tension act on the charged droplet (Mit-uppatham et al.
2004). The electrostatic force is responsible for carrying the charged jet from tip
to the grounded collector, while the repulsive Coulombic force repels the adjacent
charged species present in the jet and therefore stretches the charged jet. Both the
surface tension and viscoelastic forces work against the stretching of charged jet.
Fig. 13.2 Schematic illustration of the electrospinning process with different collectors and
spinnerets
The jet is also subject to drag force due to friction between the charged jet and the
surrounding air (Mit-uppatham et al. 2004).
The solution electrospinning and melt electrospinning are two major categories
of the electrospinning systems (Topuz and Uyar 2020). The solution electrospin-
ning has been commonly employed in the development of electrospun based
micro/nanostructured materials for food applications due to feasibility, versatility,
and absence of heat during process. The morphology and structure of electrospun
fibers can be modified using different electrospinning designs (Fig. 13.2). These are
blending electrospinning, coaxial electrospinning, emulsion electrospinning, edge
electrospinning, and magnetic electrospinning (Zhang et al. 2020). In blending elec-
trospinning, all ingredients are co-dissolved in one solution and processed using
a single nozzle. Solution and processing parameters determine the morphology of
the electrospun fibers, and the resulting fiber can be beaded, porous, or smooth
depending on these properties (Fig. 13.3). There are two needles arranged concentri-
cally in coaxial electrospinning to obtain the core-shell nanofibers. Emulsion elec-
trospinning using a single nozzle is also another way to produce core-shell structured
fibers. During the electrospinning of the emulsion, the continuous phase of emulsion
produces the shell structure of fibers, while the core part of fibers is obtained from
the droplet phase.
A typical electrospinning system has a lower throughput because a single needle
can only produce one polymer jet. Mass production of nanofibers can be achieved
PVA PLA PS
(a) (b) (c)

Fig. 13.3 Scanning electron microscopy (SEM) images of electrospun fibers with different
morphologies (a) beaded; (b) porous, and (c) smooth (PVA: polyvinyl alcohol; PLA: polylactic
acid; PS: polystyrene)
by the formation of multiple jet system with multiplying needles or multiple jets
accomplished with various needless electrospinning methods (Wei et al. 2019). In
edge electrospinning set up, the source of electrode is either the edge of flat plate
or the edge of a bowl filled with the solution (Echegoyen et al. 2017). The polymer
flow is gravity assisted in edge-plate configuration and dependent on the angle of
plates and the volume of the solution in the reservoir and the size of pipette aperture
(Thoppey et al. 2010). For bowl configuration, spinning takes place directly from the
polymer solution surface within the bowl rather than utilizing falling or elongated
droplets as in the edge-plate geometry (Thoppey et al. 2011). Highly aligned fibers
can be obtained using rotating mandrel, drum and disk collectors that allow scalability
and improved conformability (Quirós et al. 2016; Zhang et al. 2020). The electro-
spinning technique has many advantages over other fiber production methods such
as cost effectiveness, easy operation and function, a wide range of polymer mate-
rials, mild processing conditions and easy incorporation of bioactive compounds
into nanofibers (Wen et al. 2017; Zhao et al. 2020). The process parameters can be
adjusted to prepare electrospun nanofibers with custom shapes and various orien-
tations for requirements in various research fields (Zhang et al. 2020). The main
parameters that affect the spinnability, fiber morphology, and diameter distribution
are polymer solution properties, processing parameters, and ambient conditions.
13.2.1 Polymer Solution Properties
In the electrospinning process, solution parameters influencing the electro-

spinnability of polymer solution are viscosity, polymer concentration, polymer
molecular weight, electrical conductivity, and surface tension. Solution viscosity
which depends on the polymer concentration and the molecular weight of the polymer
is an important parameter in electrospinning (Neo et al. 2012). The higher the molec-
ular weight of the polymer, the more entangled polymer chains in the solution and
hence the viscosity of polymer solution increases (Tan et al. 2005). Higher levels
of chain entanglements as a result of both higher molecular weights of the polymer
and increased polymer concentrations favor fiber formation. Therefore, there is a
minimum concentration for a particular polymer solution that provides critical chain
entanglements to obtain fiber during electrospinning (Shenoy et al. 2005). Beaded
fibers are obtained at low concentration with low solution viscosity, but further
increase in concentration or solution viscosity results in uniform fibers with increased
diameter (Deitzel et al. 2001). The size and geometry of the fiber are influenced by
the physical instabilities acting on the polymer solution jet. When the viscosity of
the solution is not high enough or when the electric field strength is low, the axisym-
metric instability, known as the Rayleigh instability, occurs due to the surface tension
of solution. The charged polymer jet breaks up into droplets and leads to the beaded
fibers morphology (Baji et al. 2010). Adding a surfactant to the solution can reduce
the surface tension, and this facilitates the formation of fibers at a lower electric field
(Shenoy et al. 2005).
The solution conductivity is another important parameter affecting the process and
morphology of the resulting electrospun fibers. The polymer type, solvent and the
availability of ionisable salts affect the solution conductivity (Bhardwaj and Kundu
2010). It determines the elongation level of a jet by an electrical force because it
reflects a charge density on a jet (Tan et al. 2005). Solutions having higher conduc-
tivity results in higher elongation of jet due to high surface charge density. The
self-repulsion between the excess charges present in the jet causes the bending and
whipping instabilities. These instabilities promote the thinning and elongation of the
jet and thus lead to smaller diameter fibers (Baji et al. 2010).
13.2.2 Electrospinning Process Parameters
The main processing parameters that affect electrospinning are solution feeding rate,
applied voltage, distance from needle tip to collector. The critical voltage is required
to induce necessary charge on the droplet and the electrospinning process begins
when the electrostatic force exceeds the surface tension (Mazoochi and Jabbari 2011).
The higher stretching of the jet occurs due to the greater Coulombic forces in the
jet at high voltage and the resulting electric field, leading to a decrease in the diam-
eter of fiber as well as rapid evaporation of the solvent from the fibers (Bhardwaj
and Kundu 2010; Mazoochi and Jabbari 2011). Shenoy et al. (2005) suggested that
the morphology varies from beads to beaded fibers to only fibers as the voltage is
increased. However, any further increase in voltage may result in relatively large
diameter fibers due to rapid removal of the solution from the needle tip (Haghi and
Akbari 2007). Meanwhile, the tendency to bead formation increases at higher volt-
ages, possibly due to jet instability in the high voltage electrical field (Deitzel et al.
2001; Shenoy et al. 2005).
The flow rate controls polymer solution available for electrospinning. A minimum
solution flow rate is required to keep a stable Taylor cone (Zong et al. 2002). Megelski
et al. (2002) reported that an increasing flow rate with a decrease in spinning voltage
resulted in increased fiber size. Yuan et al. (2004) suggested that the lower flow rate
gives the solvent much more time to evaporate so that the formation of beads in the
electrospun fibers can be avoided.
The polymer jet behavior and the morphology of the resultant fibers are affected by
changing the distance from the nozzle tip to the collector. For instance, if the distance
is short, the electric field strength increases and speeds up the electrospinning process
(Zaikov 2017). However, the time required for evaporation of the solvent cannot be
provided. Consequently, wet electrospun fibers can be obtained due to insufficient
evaporation of solvent with the short distance between the nozzle tip and the collector
(Ghorani and Tucker 2015). Zhao et al. (2004) reported that increasing the distance
causes a decrease in the flight speed of the jet and the jet flight takes longer time
from the nozzle tip to the collector. As a result, the charged jet elongates more and
thin fibers with a narrow diameter range are formed.
Other processing parameters such as needle or nozzle inner diameter can also
influence the electrospinning process (Ghorani and Tucker 2015). Mo et al. (2004)
observed clogging at the tip of nozzle when the nozzle diameter was increased. They
found that using smaller diameter needle led to bead free fibers without clogging
the needle tip. Zhao et al. (2004) suggested that decreasing the diameter of nozzle
increased surface tension of the droplet. The flight time for the jet from the nozzle
to the collector is increased due to the increased surface tension and small diameter
can be obtained.
13.2.3 Environmental Conditions
Electrospinning process and fiber properties are affected by environmental condi-

tions such as ambient temperature and relative humidity since these parameters may
influence the properties of electrified jets (Quirós et al. 2016). De Vrieze et al. (2008)
found that increasing ambient temperature causes a decrease in fiber diameter due
to the effect of temperature on solvent evaporation rate and viscosity of the polymer
solution. Chen and Yu (2010) investigated the effect of the temperature of the polymer
solution on the fiber diameter. It has been reported that the electrospinning ability
of the polymer solutions is improved by increasing the temperature. This is because
the viscosity and surface tension decrease as the temperature increases (Chen and
Yu 2010). Casper et al. (2004) showed that raising the relative humidity causes an
increased number of pores on the surface of fibers. The volatile solvent evaporation
can be fast at very low humidity. This can create a problem such as clogging of
needle tip during electrospinning process. The discharge of the electrospun fibers
may increase in a more humid environment (Bhardwaj and Kundu 2010).
13.3 Biosensors Based on Electrospun

Micro/Nanomaterials
Biosensors can be defined as devices that can detect the presence of a partic-
ular biochemical analyte and convert a biological response into a readable elec-
trical/optical signal. Biosensors basically consist of three components, a bioreceptor
or biorecognition element, a transducer, and a detector. The function of biorecep-
tors is to recognize the target analyte, while transducers are used to convert the
recognized event into a measurable electrical signal (Maftoonazad and Ramaswamy
2018). The steps required for the fabrication of a biosensor have been described by
De Cesare and Macagnano (2015). These steps are as follows: (i) choosing a suit-
able bioreceptor that targets biological identification; (ii) immobilizing the receptor
on the transducer; (iii) selecting the transduction mechanism; and (iv) using a suit-
able detector to obtain reproducible responses (De Cesare and Macagnano 2015).
Biosensors can be categorized either on the basis of their transduction mechanisms
or their bioreceptors used. The types of transduction mechanism include electro-
chemical, optoelectronic, piezoelectric, bioluminescent, resonant mirror, and ther-
mistor. A variety of biomolecules can be used as a biorecognition element depending
on the sensing mechanism. They include antibodies, aptamers, carbohydrates, and
antimicrobial peptides (Gunasekaran 2014). Biosensors designed using nanomate-
rials are called nanobiosensors. Nanobiosensors can have very low detection limits
and high stability due to the incorporation of nanomaterials because these nano-
materials can provide improved electrical conductivity and chemical reactivity with
increased interacting surface area. Electrospinning technology can be one of the
most effective routes to significantly increase the sensitivity of biosensors by taking
advantage of the extremely large surface area provided by nanofibers (De Cesare
and Macagnano 2015). It is possible to produce nanofibers with diameter ranging
from micro to nanoscale, which leads to large surface area per unit mass. Elec-
trospinning also allows to produce different structures such as hollow, porous, and
core-sheath by adjusting process parameters and solution properties. Therefore, elec-
trospun nanofiber membranes with such outstanding properties make these materials
highly attractive to incorporate them within sensors designed for different applica-
tions (Ding et al. 2010). The use of nanofibers can remarkably increase the avail-
ability of sites for immobilization within biosensors due to their large surface area and
porosity. The number of biorecognition sites also increases for biosensing through
the immobilization of bioreceptors into or onto electrospun nanofibers (Matlock-
Colangelo and Baeumner 2012; De Cesare and Macagnano 2015). Figure 13.4 shows
a schematic representation of the electrochemical biosensor based on the electrospun
fiber. Electrospinning technology allows to the use of various polymer materials to
obtain fibers with controlled morphologies, sizes, and compositions.
Electrospun nanofibers can further be functionalized through the incorporation
of nanoscale materials (e.g., magnetic nanoparticles, quantum dots) within spinning
solutions or on the surface of nanofibers after spinning (Zhang et al. 2017). There-
fore, the incorporation of these material in the biosensing platform can enable to
Fig. 13.4 Schematic representation of the electrochemical biosensor assembly
achieve low detection limits with high sensitivity of the resulting biosensors. Appli-
cations related to the use of nanobiosensors are mostly in diagnostic, biomedical,
and environmental but they can also be powerful tools in food safety, quality control,
and traceability by providing fast, high sensitivity and specificity in measurements
(De Cesare and Macagnano 2015). Electrospun nanofibers have many applications
in various fields such as material science, tissue engineering, biomedicine, environ-
mental science, and sensing (Zhang et al. 2019). However, the use of nanofibers in
food applications is very recent, especially in biosensors. All these unique features
of electrospun nanomaterials make electrospinning technology highly attractive to
nanotechnological applications in food field. This review provides the main recent
electrospun nanobiosensors studies and applications related to the food safety and
quality control.
13.3.1 Electrochemical Biosensors
By definition of the International Union of Pure and Applied Chemistry (IUPAC),

an electrochemical biosensor is a self-contained device that can selectively provide
quantitative or semi-quantitative analytic information using a bioreceptor being in
direct spatial contact with an electrochemical transduction element (Thévenot et al.
1999). Electrochemical biosensors offer great promise for a wide variety of appli-
cations in clinical diagnosis, food and environmental analysis because they have
many advantages such as high selectivity, wide detection range, low cost, and easy
miniaturization (Viswanathan et al. 2009; Pilolli et al. 2013). Depending on the
biological recognition process, affinity sensors and biocatalytic devices are the two
main categories of electrochemical biosensors. In an affinity biosensor, a binding
reaction occurs among biomolecules such as an antibody and targeted analyte. On
the other hand, a biocatalytic device uses biological elements such as enzymes,
whole cells, or tissue that can recognize the target analyte and produce electroactive
species (Mondal and Sharma 2016). In a typical electrochemical sensing, there are
three electrodes which are the counter or auxiliary electrode, reference electrode,
and working electrodes. The counter electrode is used to complete the current circuit
in the electrochemical cell, while the reference electrode provides a stable poten-
tial control. The counter electrode can be made of inert materials such as platinum,
indium tin oxide, gold, etc. A silver (Ag)/silver chloride (AgCl) or glassy carbon
material is commonly used for the reference electrode. The working electrode is
the main electrode of an electrochemical biosensors. The working electrode acts
as a transduction element in the biochemical reaction. The working electrode can
be made of gold, platinum, indium tin oxide, and glassy carbon (Malhotra and Ali
2018). Electrochemical biosensors produce electrical outputs as a function of target
concentration. They are classified as potentiometric, amperometric, voltammetric,
and impedimetric (Thévenot et al. 1999; Pilolli et al. 2013).
13.3.1.1 Potentiometric Biosensors
Potentiometric sensors measure potential difference between two reference elec-

trodes at near zero current, separated by a membrane (Pilolli et al. 2013). For
biosensing, a biological element (enzyme, antibody, aptamers) within the sensor
catalyzes a reaction, an ion formed can be detected using specific electrode. An ion-
selective electrode is used to determine the change in ionic concentrations in these
biosensors because the release and uptake of hydrogen ions occurs in many enzy-
matic reactions. In potentiometric biosensors, the potential difference between the
potentiometric electrode and reference electrode is measured because this measured
difference is proportional to the concentration of the substrate. There are three types
of ion-selective electrodes (ISE) which are based on glass electrodes for cations,
glass pH electrode layered with a gas-permeable membrane and solid-state elec-
trode (Sahota et al. 2017). The ion-selective field effect transistors (ISFET) and light
addressable potentiometric sensor (LAPS) are two other important types of poten-
tiometric biosensor. They have basically adopted a similar operational principle and
both systems consist of electrolyte/insulator/semiconductor structure. The potential
is generated by the effect of surface ion concentration from a solution in the ISFET
but, in the case of LAPS, the area of interest is illuminated by the light that generates
photocurrent, which is correlated with surface ion concentration (Zaid et al. 2019).
13.3.1.2 Voltammetric/Amperometric Biosensors
Both voltammetric and amperometric techniques measure the electric current as

a result of the electrochemical oxidation or reduction of an electroactive species
(Thévenot et al. 1999). Amperometric biosensors are a subclass of the voltammetric

sensors (Viswanathan et al. 2009). The amperometric biosensor is based on moni-
toring the current as a function of time while keeping the working electrode at constant
potential, but in voltammetric technique, the current is measured at varying potential
(Pilolli et al. 2013). The continuous alteration of the potential is applied to the solution
and the resulting current is measured. Cyclic voltammetry (CV) is the most popular
technique, which is used to determine electrochemical behavior of analyte. The
most commonly used voltammetric techniques are differential pulse (DPV), normal
pulse (NPV), and square wave (SWV). In these techniques, the applied potential
changes through pulsing from one potential to another rather than sweeping through
varying potentials (Chen and Shah 2013). Amperometric measurements are usually
performed on a platinum, gold, titanium, indium tin oxide, or carbon-based working
electrode or an array of electrodes with respect to a reference electrode (Thévenot
et al. 1999). The detection mechanism of amperometric biosensors is the movement
of electrons due to enzyme-catalyzed redox reactions and the current flow resulting
from the transferred electron by the enzymatic reaction substrate is measured (Sahota
et al. 2017). The improvement of sensitivity in enzymatic biosensors can be provided
by increasing the electrode surface area. The electrode surface area can be increased
by using nanomaterials and thus this allows a higher amount of enzyme loading in
the recognition layer (Rathee et al. 2016).
13.3.1.3 Impedimetric Biosensors
In impedimetric biosensors, the electrical impedance of the electrode-electrolyte

interface is measured by applying a sinusoidal voltage to a sample and by simultane-
ously measuring the current that flows through it (Pilolli et al. 2013). The electrical
impedance is equal to the ratio between the voltage and the corresponding current in
a given frequency domain (Malhotra and Ali 2018). The impedimetric technique is
generally referred to as electrochemical impedance spectroscopy (EIS), which is a
very powerful technique for analysis of both resistive and capacitive properties arising
from the biorecognition events at the interface between electrode and analyte (Tran
et al. 2016). Biomolecular interactions such as DNA hybridization, antigen-antibody,
enzymatic and protein-protein interactions can be quantified by using impedimetric
biosensors (Malhotra and Ali 2018). The impedimetric biosensing is based on moni-
toring changes in interfacial properties such as electric current flow, charge transfer
resistance, or double layer capacitance when the target analyte reacts with a specific
bioreceptor on the surface of the sensor. The impedance value varies in proportion
to both biochemical reaction rate and the related analyte concentration (Tran et al.
2016).
13.3.2 Electrochemical Biosensors Through Electrospinning
Electrochemical nanobiosensors with high efficiency (e.g., rapid response, low detec-
tion limit, high sensitivity) are nowadays being developed by the incorporation of
nanomaterials (Hammond et al. 2016). The unique features of nanofibers make them
an ideal candidate for incorporation within sensors designed for food analysis and
food processing control as well as the tracing of contaminants. There is an increasing
demand in the food industry for fast, real-time, simple, and low-cost techniques to
ensure safety and quality of food products. Some of these advantages and potentials
of electrospun materials open doors to the development of different biosensing tools
for possible applications in the food industry. Table 13.1 summarizes some of the
recent electrospun based electrochemical biosensor studies and applications for food
safety and quality control.
Contamination of food products with physical, chemical, or biological hazards
can happen at any point in the food production and processing chain. The source of
chemical hazards can be ingredient-related (e.g., pesticides, mycotoxins and other
natural toxins, animal drug residues, heavy metals, environmental contaminants),
process-related, and facility-related (FDA 2018). These chemical contaminants may
show adverse effects on human and animal health. Pesticides are generally used for
pest control, especially in plant protection. Among pesticides, pirimiphos-methyl is
an organophosphorus insecticide with both contact and fumigant action. It can be
applied to a variety of plants such as citrus, cereals, olives, sugar canes, and vegetables
(El-Moghazy et al. 2016). El-Moghazy et al. (2016) fabricated an enzyme-based
electrochemical biosensor using electrospun nanofibers for detection of pirimiphos-
methyl in olive oil. Electrospun fibers were prepared from a polymer blend of chitosan
and poly (vinyl alcohol) and collected on the surface of screen-printed electrodes
to immobilize genetically modified acetylcholinesterase (AChE). The electrospun
based biosensor prototype was tested to determine pirimiphos-methyl concentration
in olive oil. This study showed that the response of developed biosensor could be
improved by twofolds compared to casted electrodes due to the use of nanofibers.
The detection limit achieved for primiphos-methyl was 0.2 nM, which is well below
the limit permitted by international regulations (164 nM).
In another study, a novel biosensor has been developed for detecting organophos-
phate pesticides (OPs) in aqueous system (Bao et al. 2016). The proposed biosensor
was constructed based on nanocomposites of titanium dioxide nanofibers (TiO2 NFs)
and carboxylic acid functionalized multi-walled carbon nanotubes (c-MWCNTs).
The suspension of both TiO2 NFs and c-MWCNTs was casted onto the glassy carbon
electrode surface. The mixture of elastin-like-polypeptide-organophosphate hydro-
lase (ELP-OPH) and bovine serum albumin (BSA) was coated on the surface of the
prepared electrode to immobilize OPH enzyme. The ELP-OPH, acted as biocata-
lyst for OPs, was obtained by purification from genetically engineered Escherichia
coli based on the unique thermal triggered phase transition of ELP. TiO2 NFs were
produced by electrospinning technique and subsequent calcination process. They
were preferred because of their affinity to the phosphoric group in organophosphates.
Table 13.1 Electrospun nanofiber-based electrodes in electrochemical biosensing and potential applications for food safety and quality control
Working electrode material Transducer type Analyte Application Detection Linearity Reference
limit
AChE/CS-PVA NFM/SPE Amperometric Pirimisphos-methyl Pesticide 0.2 nM 1 × 10−10 –8 × El-Moghazy
detection in 10−8 M et al. (2016)
olive oil
ELP-OPH/BSA/TiO2 NFs/c-MWCNTs Amperometric Methyl parathion Pesticide 12 nM Up to 36.4 μM Bao et al.
detection in (2016)
lake water
BCL@MOF nanofibers/chitosan/GCE Voltammetric Methyl parathion Pesticide 0.067 μM 0.1–38 μM Wang et al.
detection in (2019a)
vegetables
MWCNT-ZnO nanofibers/GCE Impedimetric Atrazine Pesticide 5.368 zM 10 zM–1 μM Supraja et al.
detection (2020)
Aptamer modified CMCNFs FET Potentiometric Bisphenol A Bisphenol A 1 fM 100 –104 fM Kim et al.
detection in (2016)
13 Advances in Biosensors Based on Electrospun …
bottled water
PVA/GQD/GCE Amperometric H2 O2 – 0.53 μM 0.1–200 mM Zhang et al.
(2015)
Gelatin/chitosan nanofibers/HRP Voltammetric H2 O2 Disinfectant 0.05 mM 0.1–1.7 mM Teepoo et al.
(2017)
CuO/PANI nanofibers/GCE Amperometric H2 O2 – 0.11 × 10−6 5 μM–9.25 mM Liu et al.
M (2019)
CNFs/MnCo2 O4.5 -TMB Colorimetric Sulfite, Ascorbic acid – 19 nM 0–1 μM Gao et al.
0M 0–40 μM (2017)
(continued)
255
256
Working electrode material Transducer type Analyte Application Detection Linearity Reference
limit
AChE: acetylcholinesterase, CS-PVA NFM: chitosan-poly (vinyl alcohol) nanofibrous membranes, SPE: screen-printed electrodes, ELP-OPH:
elastin-like-polypeptide-organophosphate hydrolase, BSA: bovine serum albumin, TiO2NFs: titanium dioxide nanofibers, c-MWCNTs: carboxylic acid
functionalized multi-walled carbon nanotubes, BCL@MOF: Burkholderia cepacia lipase@Metal organic framework, GCE: glassy carbon electrode,
MWCNT-ZnO: multi-walled carbon nanotube-Zinc oxide, CMCNFs: carboxyl-functionalized modified multichannel carbon nanofibers, FET: field-effect
transistor, GQD: graphene quantum dots, HRP: Horseradish peroxidase, CuO/PANI: cupric oxide/polyaniline, CNFs: carbon nanofibers, TMB: 3,3’,5,5’
Tetramethylbenzi-dine
FTO/PA6/PPy/ZnO/urease Impedimetric Urea Urea detection in 0.011 mg/dL 0.1–250 mg/dL Migliorini
milk et al. (2018)
Laccase/A-CZUF/GCE Voltammetric Catechol – 0.166 μM 0.166–7 μM Chen et al.
(2015)
BSA/anti-AFB1/μchannel/carbon-Pt Impedimetric Aflatoxin B1 – 1 pg/mL 1 pg/mL-μg/L Mondal et al.
(2016)
PAN nanofiber modified electrode Voltammetric Zearalenone Mycotoxin 5 nM 5–30 nM Moradi et al.
detection in food (2020)
simulant
PANi/PEO composite nanofiber-based Chemiresistive DENVCP, DNA 1.9 fM 10 fM–1 μM Tripathy et al.
interdigitated electrode Staphylococcus hybridization 0.31 pM, 1 pM–1 μM (2019)
aureus specific detection 0.42 pM,
genes: nuc, mecA, 0.67 pM
vanA, protein A 0.17 pM
PVA/PAA hydrogel NF-LAPS Potentiometric Escherichia coli Escherichia coli 102 CFU/mL 102 –106 CFU/mL Shaibani
detection in et al. (2018)
orange juice
GN/CN/GelMA/RBL mast cell/SPE Voltammetric Milk allergen casein Casein detection 3.2 × 10−8 1 × 10−7 –1 × 10−6 Jiang et al.
in milk g/mL g/mL (2019)
(continued)
A. Altan and M. Yılmaz
FTO: fluorine doped tin oxide, PA6: polyamide 6, PPy: polypyrrole, A-CZUF:gold-crosslinked zein ultrafine fibers, AFB1: Aflatoxin B1, Pt: platinum,
PAN: Polyacrylonitrile, PANi/PEO: polyaniline/polyethylene oxide, DENVCP: dengue virus specific consensus primer, PAA: poly(acrylic acid), NF-LAPS:
nanofiber-light addressable potentiometric sensor, GN/CN/GelMA/RBL: graphene/carbon nanofiber/gelatin methacryloyl/rat basophilic leukemia, SPE:
Screen Printed Electrode
XOD@Cu-MOF/SA/GCE Amperometric Hypoxanthine Hypoxanthine and 0.0023 μM 0.01–10 μM Wang et al.
Xanthine xanthine detection in 0.0064 μM 0.01–10 μM (2019b)
seafood
GOX/CNT/NFM/GCE Amperometric Glucose Glucose detection in 20 μM 1–3 mM Mason et al.
commercial beverage (2016)
and monitoring
brewing process
Ni-Co3O4/CPE Amperometric Maltose Maltose detection in 32.5 nM 0.2–100 μM Xu et al.
malt syrup (2016)
GOD/TCNFs/GCE Amperometric Glucose Glucose detection in 3.7 μM 0.013–10.5 mM Guo et al.
human blood serum (2018)
13 Advances in Biosensors Based on Electrospun …
Ni-CoO/CNF/GCE Voltammetric Glucose 0.03 μM 0.25–600 μM Mei et al.

(2019)
Cu-nanoflower @AuNPs-GO NFs Voltammetric Glucose – 0.018 μM 0.001–0.1 mM Baek et al.
(2020)
BSA/Anti-VD/Fe3O4-PANnFs/ITO DPV Vitamin-D3 – 12 ng/mL 10–100 ng/mL Chauhan et al.
(2018)
PtNps/GCNF/LOx-SPCEs Amperometric Lactate Lactate detection in 6 μM 10–2000 μM Loaiza et al.
wines and ciders (2015)
(continued)
257
258
XOD@Cu-MOF/SA: Xanthine oxidase@copper-based metal organic framework nanofibers/sodium alginate, GOX/CNT/NFM: Glucose oxidase/carbon
nanotubes/nylon nanofibrous membrane, Ni-Co3 O4 /CPE: Nickel doped cobalt (II, III)/carbon paste electrode, AuNPs: Gold nanoparticles, GO: graphene
oxide, Anti-VD: antibody specific to Vitamin-D3 , Fe3 O4 -PANnFs/ITO: Magnetite nanoparticles-polyacrylonitrile nanofibers/indium tin oxide,
PtNps/GCNF/LOx-SPCEs: platinum nanoparticles/graphitized carbon nanofibers/lactate oxidase-screen-printed carbon electrodes
A. Altan and M. Yılmaz
The functions of BSA and c-MWCNTs on the biosensor platform were stabiliza-
tion of OPH activity, immobilization of ELP-OPH, and enhancement of the elec-
tron transfer in amperometric detection. The developed sensor exhibited excellent
performance for methyl parathion and parathion detection in terms of response time
(5 s), detection limits (12 nM and 10 nM), and reliability (94.67 ± 2.82%–106.73 ±
2.61%). The high performance of sensor has been attributed to the strong adsorption
between organophosphates and nanofibers, and the improvement of electron transfer
achieved by using both nanofibers and carbon nanotubes in the biosensor.
Wang et al. (2019a) fabricated enzyme-based biosensor for rapid determination of
methyl parathion residues in vegetable samples. Metal-organic framework nanofibers
with lipase from Burkholderia cepacia and chitosan were used for the fabrication of
biosensor. The mixture of nanofibers, lipase, and chitosan was coated on the glassy
carbon electrode surface. The prepared electrode was used as working electrode.
The developed biosensor was tested with vegetable samples (lettuce and cabbage)
spiked with methyl parathion solutions at different concentrations. The samples were
immersed in methanol to dissolve residual methyl parathion and the methanolic
extracts were used in differential pulse voltammetry measurement. The prepared
biosensor responded within concentration range of 0.1–38 μm and a detection limit
of 0.067 μm. In addition, the developed biosensor maintained at least 80% of its
initial response after 3 weeks of storage at 4 °C.
Recently, Supraja et al. (2020) developed an electrochemical biosensor based on
the multi-walled carbon nanotube (MWCNT)-ZnO composite nanofibers for atrazine
(ATZ) detection. The composite of MWCNT-ZnO nanofibers were produced by elec-
trospinning technique. The polymer solution containing polyacrylonitrile (PAN),
MWCNTs, and zinc acetate dehydrate was prepared and electrospun via electrospin-
ning. The resulting fibers were calcinated to remove PAN and also to obtain ZnO
from zinc acetate dehydrate without decomposition of MWCNTs. The electrode was
prepared by immobilizing the anti-ATZ antibody onto the glassy carbon working
electrode (GCE). Before immobilization of antibody, MWCNT-ZnO nanofibers were
used to modify the electrode and then carboxylic functionalization was applied by
dipping in BSA. The concentration range of MWCNT-ZnO nanofiber-based electro-
chemical biosensor was 100 zM–1 μM, while the detection limit was found to be
5.368 zM.
Chemical contaminants must be properly controlled during processing and
storage, because if they are above limits, they can be extremely harmful to the
human body. Chemicals can become part of food products without intentionally
added such as the inevitable migration of chemical compounds from packaging mate-
rial into food and beverages. For example, there are major concerns about bisphenol
A (BPA) because the migration of BPA into food can occur at high temperatures
(Kim et al. 2016). Therefore, it is essential to develop rapid, reliable, and sensitive
methods for detection of BPA. Kim et al. (2016) developed a field-effect transistor
(FET) sensor for BPA detection. The sensor was designed using multichannel carbon
nanofibers (MCNFs) and BPA-binding aptamers. The MCNFs were produced from
polymer solutions of poly(acrylonitrile) and poly(methyl methacrylate) by a single-
nozzle electrospinning technique and carboxyl-functionalized by oxidative treatment
(CMCNFs). They were served as a conductive signal transducer. The performance of

sensor was tested on the real samples which are thermal printing paper and polycar-
bonate bottle filled with distilled water. Hot water and ethanol extracts of BPA from
polycarbonate bottle and paper were used. Figure 13.5 shows the schematic diagram
and FE-SEM and TEM images of functionalized multichannel carbon nanofibers
(CMCNFs). The MCNFs increased the affinity of aptamer to the BPA molecules
since they were covalently bonded between the electrode substrate and the aptamer.
A large specific surface area offered by the multichannel structure increased the
quantity of conjugated aptamers. Therefore, improved performance of the proposed
sensor in terms of sensitivity and specificity has been achieved with a detection limit
of 1 fM for BPA detection.
Hydrogen peroxide (H2 O2 ) is one of the most widely used chemical sterilization
agents in food industry for food packaging. After applying H2 O2 , a rinsing step with
sterile water and/or blowing with sterile air is required to remove residual H2 O2 . The
residual levels permitted in products are legally regulated. Additionally, some milk
processors intentionally add H2 O2 to milk to reduce microbial load. Therefore, the
determination of H2 O2 residues is of practical importance in the food industry and
other applications such as clinical, biological, and chemical (Hsu et al. 2008). Teepoo
et al. (2017) developed hydrogen peroxide biosensor using composite nanofibers
of chitosan and gelatin biopolymers. The composite nanofibers were produced
using electrospinning and deposited onto graphite electrode surface. The horseradish
Fig. 13.5 a Schematic diagram of carboxyl-functionalized multichannel carbon nanofibers

(CMCNFs). b FE-SEM and TEM images of PAN-PMMA nanofibers. c FE-SEM image of the
multichannel carbon nanofibers (MCNFs) and a high magnification FE-SEM image of MCNF cross-
section. d TEM image of CMCNF (Reprinted from Kim et al. 2016. Copyright with permission
from American Chemical Society)
peroxidase enzyme was immobilized on the nanofibers to prepare the biosensor.

The prepared electrodes were characterized by cyclic voltammetry. The hydrogen
peroxide biosensor had a detection limit of 0.05 mM with a linear calibration range of
0.1–1.7 mM. The immobilized enzyme on the composite nanofibers possessed good
bioactivity after two months of use. Recently, the CuO/polyaniline (CuO/PANI)
nanocomposite-based enzyme-free electrochemical biosensor has been fabricated
for H2 O2 detection (Liu et al. 2019). To fabricate sensor template, polyamide acid
(PAA) nanofibers were produced by electrospinning technique. The hollow PANI
nanofibers were prepared by in situ polymerization of aniline monomers followed
by removing of the PAA template via dissolving in N, N-dimethylacetamide solu-
tion. CuO was coated on the surface of hollow PANI nanofibers to improve the
electrochemical properties of PANI fibers. The developed sensor detected H2 O2 in
the range of 5 μM–9.255 mM with a low detection limit of 0.110 × 10−6 M. It
has been shown that the prepared sensor maintains its stability when stored at 4 °C
for 10 days. These results suggested that the developed sensor can be used for food
safety and environmental applications.
The detection of urea is of great importance in many areas, including clinical
diagnosis, environmental analysis, and food. Urea is added to milk as an adulterant
to increase solids-not-fat value and nitrogen content. Serious health problems such
as indigestion, urinary tract obstruction, gastrointestinal bleeding, and cancer may
arise when the urea concentration in milk exceeds 70 mg dL−1 (Nikoleli et al. 2010).
Therefore, rapid and sensitive detection of urea in milk and other food products
is essential to ensure food safety. Recently, an impedimetric biosensor based on the
electrospun nanofibers has been developed for urea detection (Migliorini et al. 2018).
Polymer solutions of polyamide 6 (PA6) and polypyrrole (PPy) were used to obtain
electrospun nanofibers by electrospinning and the resulting fibers were collected onto
fluorine doped tin oxide (FTO) electrodes. The prepared electrode was modified by
the adsorption of zinc oxide nanoparticles (ZnO) on the surface of fibers. The urease
immobilized FTO/PA6/PPy/ZnO electrode was used as the working electrode. Milk
sample was used to test the fabricated biosensor. The results of this study showed
that the urea biosensor based on electrospun nanofibers having a low detection limit
(0.011 mg dL−1 ) with a wide concentration range (0.1–250 mg dL−1 ) can be used
for monitoring urea in dairy products.
Phenolic compounds are important because of their environmental significance
and biological roles. The concentration levels of phenolic compounds in community
water need to be strictly monitored due to their toxicity (Sarika et al. 2017). So,
an effective detection method is needed to monitor phenolic compounds. A novel
laccase biosensor for catechol detection has been proposed by Chen et al. (2015). The
ultrafine zein fibers cross-linked with glyoxal (CZUF) were produced by electrospin-
ning and coated with gold nanoparticles (Au NPs) by in situ polymerization using
poly(ethyleneimine) (PEI). The electrochemical performance of the proposed sensor
was measured by square wave voltammetry and cyclic voltammetry. The selectivity
of proposed sensor was determined in the presence of other phenolic compounds such
as guaiacol, epicatechin, aminophenol, and phenol. The small interference of these
phenolic compounds (≤3%) toward the biosensor response demonstrated favorable
selectivity of the biosensor in this study. The detection limit of catechol was 0.166
μM and a linear range of 0.166–7 μM. Catechol is a natural polyphenolic compound
found in some plant products such as vegetables, fruits, and teas. The quantification
of catechol is important because of biological roles such as antioxidation and antiviral
properties (Wahab and Darain 2017). Therefore, the rapid estimation of polyphenol
levels with a catechol biosensor can be used to evaluate the freshness, antioxidant
content, and quality control of foods (Sarika et al. 2017; Wahab and Darain 2017).
Foods and feeds can be contaminated with mycotoxins at any stage of their
handling or storage. Aflatoxins are mycotoxins that are produced by strains from
the Aspergillus family. Aflatoxin B1 is one of the highly toxic food contaminants
and known to be carcinogenic in human (Xu et al. 2016). The fast, sensitive,
specific, and reliable methods for their detection and quantification are important
to ensure food safety. Mondal et al. (2016) fabricated an electrochemical immuno-
biosensor for aflatoxin B1 (AFB1) detection. The biosensor was fabricated based
on the micro/nanochannels created by poly(methyl methacrylate) (PMMA) fibers
that embedded in porous carbon electrodes. To fabricate microchannels, the PMMA
fibers deposited on the Si wafer were spin coated with polyacrylonitrile (PAN) films.
Carbonization was applied to the composite film to remove the PMMA fibers and
produce amorphous macroporous carbon films. The channels created were doped
with Pt nanoparticles (Pt NPs) to improve the performance of the carbon electrode.
The widths of channels were in the range of 200 nm to tens of micrometers. Oxygen
plasma treatment was applied to fabricated electrodes with and without microchan-
nels to form −COOH groups on their surfaces and activated using 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The
−COOH groups created on the surface covalently interacted with the −NH2 groups
of the anti-AFB1 to form strong amide bond on the transducer surface. The AFB1
concentration was determined by an electrochemical impedance spectroscopy. The
concentration of aflatoxin B1 was detected in the range of 10−12 –10−7 g mL−1 with
a low detection limit of 1 pg mL−1 . The improved performance of biosensor due to
the aligned channels in the porous carbon film was shown in this study. It has been
reported that the channels provided by electrospun fibers enhanced antigen-antibody
interactions and electron transport between the electrode and the electrolyte. Moradi
et al. (2020) fabricated electrospun nanofibers-based sensor for zearalenone (ZEN)
mycotoxin detection in the food simulant. Polyacrylonitrile (PAN) was electrospun
to produce a layer of nanofiber using electrospinning and deposited on the graphite
electrode surface. This modified electrode was used as a working electrode. The limit
of quantification and limit of detection were found to be 5 and 1.66 nM, respectively.
Recently, electrospun nanofibers have been used to develop a chemiresistive
DNA hybridization sensor for applications in healthcare and food analysis (Tripathy
et al. 2019). In this chemiresistive biosensing, interdigitated metal electrodes were
prepared using Si wafers and glass substrates. They were then subjected to a metal
deposition followed by an optical photolithography process and metal etching. The
composite conductive nanofibers were prepared from polymer blend of polyani-
line (PANi) and polyethylene oxide (PEO) and used to modify metal electrodes.
Graphene doped Mn2 O3 nanofibers were drop casted onto surface of the electrodes
as a second approach in this study. The surface modified nanofibers incubated with
probe DNA were used as the capturing agent for the corresponding target DNA.
Multiple target DNAs include dengue virus specific consensus primer (DENVCP)
and specific genes of Staphylococcus aureus (nuc, mecA, vanA, and protein A). The
concentration range of DENVCP detection was 10 fM–1 μM with a detection limit
of 1.9 fM. The detection of four DNA targets was ranged between 1 pM and 1 μM.
Shaibani et al. (2018) developed a potentiometric sensor for the detection of
Escherichia coli in orange juice. The light addressable sensor was fabricated based
on a combination of hydrogel nanofibers as a pH sensitive layer and illumination
with a red LED. The solution of poly(vinyl alcohol) and poly(acrylic acid) polymers
was electrospun and collected on the Si substrates with SiO2 layer. Sensor chips were
annealed and functionalized with D-mannose. The working electrode was connected
to the potentiostat, displaying the rectified values of the current at any given applied
voltage. The developed sensor was tested with orange juice spiked with Escherichia
coli at concentrations of 102 –106 CFU mL−1 . The pH change was adjusted by adding
glucose at a concentration of 80 μM. The selectivity of the sensor toward Escherichia
coli was tested using Salmonella typhimurium as a sugar fermenting bacterium. The
presence of E. coli was detected in less than 1 h with the developed sensor. The
detection limit was 102 CFU mL−1 with a high selectivity. This study proposed that
the sensor can be used for monitoring to ensure the safety of orange juice at various
stages from production to consumption.
Certain food components can cause allergic reactions when foods are consumed.
The severity of allergic reactions can range from minor to life threatening. The
major potential allergenic foods are peanuts, wheat protein gluten, milk, egg, nuts,
shellfish, and soybeans. Food allergies affect the quality of life of a significant part of
the population (Neethirajan et al. 2018). Therefore, it is necessary to develop rapid
and simple analysis tools for monitoring and detection of food allergens (Neethirajan
et al. 2018; Jiang et al. 2019). An electrochemical paper biosensor has been developed
to detect milk allergen casein (Jiang et al. 2019). The sensor platform was fabricated
on wax-patterned cellulose paper. The composite hydrogel of carbon nanofibers,
gelatin methacryloyl (GelMA), and graphene were prepared and coated on the screen-
printed electrode. This modified electrode provided an effective layer to immobilize
mast cells of rat basophilic leukemia (RBL-2H3) due to the biological affinity of
the GelMA hydrogel. The performance of the sensor was tested by exposing RBL
mast cells to casein. In the study, an inverse relationship was found between the
casein concentration and the current signal. The paper-based sensor showed a casein
detection limit of 3.2 × 10−8 g mL−1 with a linear concentration range of 1 × 10−7 –1
× 10−6 g mL−1 . The results of this study proposed that various food allergens can
be monitored on-site and determined by the developed paper-based sensor.
In a recent study, an electrochemical biosensor based on a copper-based metal
organic framework (Cu-MOF) nanofibers film has been developed for the evalu-
ation of the freshness of chilled seafood (Wang et al. 2019b). Hypoxanthine and
xanthine can be used as an indicator of the freshness of chilled seafood because these
compounds occur during adenosine 5’-triphosphate (ATP) degradation process. Cu-

MOF nanofibers were used to immobilize xanthine oxidase (XOD) and their suspen-
sion with sodium alginate was drop cast onto a glassy carbon electrode (GCE). The
developed XOD-electrochemical biosensor had a wide linear range from 0.01 to
10 μM and low limit of detections (0.0023 and 0.0064 μM) for hypoxanthine and
xanthine. The hypoxanthine and xanthine in chilled squid and large yellow croaker
were detected sensitively with good recovery rates using the proposed biosensor.
Glucose biosensors are some of the most used electrochemical biosensors in the
market (Hammond et al. 2016). Their practical applications from biomedical to the
pharmaceutical field have been well developed (Mason et al. 2016). However, glucose
biosensors also have many potential applications in food industry. For instance, real-
time monitoring of carbohydrate levels can be useful for controlling the fermenta-
tion process. Mason et al. (2016) designed an electrochemical biosensor based on
naylon nanofibers (NFM) and multi-walled carbon nanotubes (CNT) composites. A
glassy carbon electrode was coated with a composite of nylon fibrous membrane
and CNT. Glucose oxidase (GOX) was immobilized on the surface of electrode. The
resulting electrode was used as a working electrode. Electrochemical characteriza-
tion was performed by cyclic voltammetry and chronoamperometry. The developed
sensor was used to determine glucose during the beer brewing process. It has been
reported that the binding of GOX around the functionalized NFM greatly enhanced
the electron transfer and the resulting current response of biosensor increased five-
fold compared to NFM without CNT. The limit of quantification was 20 μM with the
sensitivity of 1.2 μAmM−1 . The results of this study showed that the applicability
of the biosensor may be limited only when the concentrations of other redox species
such as ascorbic acid are high.
A maltose sensor based on the carbon paste electrode (CPE) modified with an
electrospun nickel doped cobalt (II, III) nanofibers (Ni-Co3O4) has been developed
(Xu et al. 2015). The composite nanofibers were produced by electrospinning of
nickel acetate, cobalt acetate, and polyvinyl pyrolidone solutions. The calcination
process was applied to the resulting fibers to obtain Ni-Co3O4 electrospun fibers. The
suspension of Ni-Co3O4 electrospun fibers was casted on the surface carbon paste
electrode and dried with an infrared lamp. Cyclic voltammetry and amperometry were
employed in maltose determination. Malt syrup was used to test the proposed sensor.
The sensor responded to maltose over a wide concentration range (0.2–100 μM) with
a low detection limit (32.5 nM). The results demonstrated that the sensor can be used
for practical analysis. In a recent study, titanium carbide (TiC)-carbon nanofibers
(CNFs) film (TCNFs) has been used to improve the performance of the glucose
biosensor (Guo et al. 2018). TCNFs were produced by electrospinning followed by
carbothermal reduction. A glassy carbon electrode was coated with the suspension
of the TCNFs with glucose oxidase (GOD) and used as a working electrode. The
detection range of glucose sensor was from 0.013 to 10.5 mM with a detection limit
of 3.7 μM. The use of both CNFs and TiC nanoparticles resulted in an increase in
surface area and an improvement of electrical conductivity with an enhancement of
electrocatalytic activity of TCNFs.
Zhang et al. (2015) fabricated a dual-purpose fluorescent and electrochemical

biosensor based on the graphene quantum dots (GQDs) with polyvinyl alcohol (PVA)
nanofibers for H2 O2 and glucose determination. The PVA/GQD nanofibers were
produced by electrospinning and collected on the glass slide coated with indium tin
oxide (ITO) for fluorescent biosensing of H2 O2 and glucose but coated on the glass
carbon electrode (GCE) for electrochemical biosensing of H2 O2 . The immobilization
of glucose oxidase on the surface of the PVA/GQD fibrous membrane was based on
the physical adsorption. The fluorescent biosensor responded in wide concentration
ranges of 0.05–35 and 0.25–24 mM and detection limits of 1.0 and 10.0 μM for both
H2 O2 and glucose. In the case of the electrochemical biosensor, the H2 O2 detection
range was 0.1–200 mM and a detection limit of 0.53 μM.
An electrochemical non-enzymatic glucose biosensor based on Ni/CoO loaded
carbon nanofiber (Ni-CoO/CNF) has been developed by Mei et al. (2019). The
electrospun fibers were prepared by electrospinning of polyacrylonitrile solutions
containing nickel acetate and cobaltous acetate with different ratios of sodium
dodecyl sulfate (SDS). The carbon nanofibers with Ni-CoO were obtained by the
pyrolysis of the resulting electrospun fibers. SDS was used as a surfactant to improve
the dispersion of metals (Ni, Co) on the carbon nanofibers surface to form unique
shape (Fig. 13.6). Carbon nanofibers with acicular and spherical shaped materials
grown on the surface were obtained when the amount of SDS was at 3% (Fig. 13.6C).
Glass carbon electrode (GCE) with Ni-CoO/CNF was used as a working electrode.
The glucose concentration ranged from 0.25 to 600 μM with a low limit of detection
at 0.03 μM. The glucose concentration in human serum was determined to test the
applicability of the developed sensor. The results demonstrated that the developed
sensor can be used for glucose detection in real sample.
Recently, Baek et al. (2020) fabricated an electrochemical biosensor based on
graphene oxide nanofibers (GO NFs) modified with gold nanoparticles (AuNPs) and
copper (Cu)-nanoflowers for glucose detection. The mixture of poly(vinyl alcohol)
(PVA) and GO solution was used to obtain GO NFs by electrospinning and the
resulting electrospun nanofibers were deposited on the surface of the gold chip
(Fig. 13.7a). After calcination of nanofibers, GO NFs were modified with gold
nanoparticles (AuNPs) by electrostatic interaction to enhance the conductivity of NFs
(Fig. 13.7b). Cu-nanoflowers (Fig. 13.7c) were synthesized using glucose oxidase
(GOx), horseradish peroxidase (HRP), and CuSO4 and they were casted on AuNPs-
GO NFs (Cu-nanoflower@AuNPs-GO NFs). The prepared electrode was used as
working electrode for glucose detection. The selectivity of developed glucose sensor
was tested in the presence of some interfering substances (such as ascorbic acid,
saccharose, urea, etc.). The results showed that the use of GO, AuNPs, and Cu-
nanoflowers with nanofibers improved the electrochemical properties of the devel-
oped biosensor. The glucose detection limit of biosensor was found to be 0.018 μM
with a linear range of 0.001–0.1 mM. These results suggested that the fabricated
biosensor could be used for determining glucose in biofluids for clinical application.
Chauhan et al. (2018) developed an electrochemical immunosensor for Vitamin-
D3 detection. Electrospun polyacrylonitrile nanofibers (PANnFs) incorporated with
Fig. 13.6 SEM images of Ni-CoO/CNF with different mass ratio of SDS. (A) 1%, (B) 2%, (C)
3%, (D) 4% (Reprinted from Mei et al. 2019. Copyright with permission from Elsevier)
magnetite nanoparticles (Fe3 O4 NPs) were fabricated using electrospinning tech-

nique (Fig. 13.8). The composite nanofibers (Fe3 O4 -PANnFs) were collected directly
onto glass electrodes coated with indium tin oxide (ITO). Nafion solution was casted
on the surface of electrode to ensure the adhesion of nanofibers. The fabricated
electrode was hydrolyzed with NaOH solution to partially convert the nitrile group
to carboxyl and amine functional groups. The electrode was further modified with
crosslinking agents 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) and N-
hydroxysuccinimide (NHS) for immobilization of monoclonal antibody specific to
Vitamin-D3 (Anti-VD). Nonspecific binding sites on the surface of the immunoelec-
trode were blocked using BSA. The fabricated biosensor detected Vit-D3 at a concen-
tration range from 10 to 100 ng mL−1 with a low detection limit of 0.12 ng mL−1 .
The developed immunosensor has been proposed to potential use in clinical appli-
cations for rapid detection of Vit-D3 . However, the application of this sensor can be
extended to other fields such as food industry. The rapid and sensitive detections of
vitamins are important for the purpose of labelling food products for their vitamin
content present.
Recently, electrospun nanofibers have been used to produce artificial enzyme,
which have shown many applications in various fields such as biosensing,
10 min 30 min 60 min 0.3 mg/mL 0.5 mg/mL
90 min 120 min B 150 min 0.7 mg/mL

C 1.0 mg/mL
Fig. 13.7 a Schematic illustration for the fabrication of Cu-nanoflower@AuNPs-GO NFs-based

electrochemical glucose nanobiosensor. b FE-SEM images of AuNPs-GO NFs at different reac-
tion times (10–150 min). (C) Cu-nanoflowers at different GOx concentrations (0.3–1.0 mg/mL)
(Reprinted from Baek et al. 2020. Copyright with permission from Elsevier)
immunoassays, and environmental monitoring (Gao et al. 2017). To fabricate

oxidase mimics, carbon nanofibers (CNFs) were first produced by calcina-
tion of electrospun polyacrylonitrile nanofibers. The composite of CNFs with
manganese cobalt oxides was prepared by electrochemical deposition of MnCo2 O4.5
nanosheets on the surface of CNFs followed by annealing. The oxidase-like
activities of CNFs/MnCo2 O4.5 nanofibers were explored against to oxidation
of 3,3’,5,5’-tetramethylbenzidine (TMB), 2,2’-azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium (ABTS), and o-pheynlenediamine (OPD). The prepared
oxidase mimics were used to detect sulfite and ascorbic acid. The results demon-
strated that an improved intrinsic oxidase-like activity was obtained with the metal
oxide-carbon nanofibers compared to CNFs and MnCo2 O4.5 alone. The prepared
sensing platform had a sulfite detection limit of 15.9 nM and a linear concentra-
tion range of 0–1 μM, while the detection limit of ascorbic acid was 50 nM with a
concentration range of 0–40 μM.
Adabi et al. (2015) fabricated carbon nanofibers (CNFs)-based electrodes
produced from polyacrylonitrile nanofibers for potential use in electrochemical
sensors and biosensors applications. Electrospinning technique was used to obtain
electrospun PAN nanofibers. The effects of process parameters such as applied
voltage, flow rate, and distance between needle tip and collector on morphology
and diameter of PAN fibers were investigated. PAN nanofibers were carbonized to
Fig. 13.8 Schematic illustration of the stepwise fabrication of electrospun nanofibers-based

BSA/Anti-VD/Fe3O4-PANnFs/ITO immunoelectrode (Reprinted from Chauhan et al. 2018.
Copyright with permission from Elsevier)
obtain CNFs and the resulting carbon nanofibers were used as electrode. Electro-
chemical characterization of electrode was evaluated by cyclic voltammetry. The
diameter of PAN nanofibers was significantly affected by polymer concentration and
electrospinning processing parameters except for feed flow rate. It was shown that
the diameter of electrospun CNFs used as nanoelectrode ranged from 75 to 80 nm.
The cyclic voltammetric response in CNFs electrode decreased as the diameter of
the CNFs or the presence of beads increased.
The rapid and accurate determination of lactic acid or lactate plays an important
role in monitoring product quality and controlling manufacturing processes because
many foods, beverages, and fermented products contain lactic acid. Lactate levels are
measured as an indicator during the fermentation process and provide information
about freshness, stability, and storage quality of food products. Therefore, food and
beverage industries need fast, easy, and affordable methods for the determination of
lactate. Loaiza et al. (2015) fabricated a new lactate biosensor aimed at detecting
lactate in wines and ciders. A composite nanomaterial composed of graphitized
carbon nanofibers (GCNF) and platinum nanoparticles (PtNps) was used to prepare
the biosensor. The dispersion of the PtNps/GCNFs was casted on the screen-printed
carbon electrode and lactate oxidase (LOx) was immobilized on the modified elec-
trode. The proposed sensor was used to determine lactate in Basque ciders and
certified wines. The lactate biosensor concentration ranged from 10 to 2000 μM,
while the limit of detection was 6.9 μM. The developed sensor exhibited long term
stability, maintaining 90 and 95% of the initial response after storage at room temper-
ature for 3 months and 18 months of storage at −20 °C. The use of the lactate sensor
for L-lactate detection in wines and cider has been shown to be suitable when sample
is sufficiently diluted for analysis.
13.4 Concluding Remarks and Future Perspectives
The versatility of electrospinning allows the production of high-performance and

functional nanofibers from various polymers that can be used in different applica-
tions. Electrospun materials have many advantages such as large specific surface
area, suitable porosity, tunable fiber diameters, and tailored morphology. Such char-
acteristic features of electrospun nanofibers make them an excellent candidate for
their use in biosensors. They can provide multiple sites for interaction or attachment
of enzymes and the more efficient transport of analytes to the sensor receptor via
nanofibers, which is important for efficiency, sensitivity, and rapid response of biosen-
sors. Even though the particular recent interest in electrospun fibers has focused on
encapsulation and food packaging, another important application area where electro-
spun nanofibers can be used in the future is in biosensors. Recent studies show that
electrospun fibers have high potential for use in the biosensor platform in many fields
such as clinical, biomedical, and environmental applications, but there are limited
number of studies for their use in food applications. This chapter summarizes the
recent developments within past five years regarding the application of electrospun
based electrochemical biosensors for detection of various compounds with interest in
food industry. In addition, some applications in other fields are also included because
the detected analytes are also important in food analysis, process, and quality control.
Nanofibers fabricated from a variety of materials, including polymers only or doped
with nanomaterials (e.g., metal nanoparticles, carbon nanotubes, and quantum dots),
metal oxides, carbon or conducting polymers (e.g., polyaniline and polypyrrole),
have been explored to fabricate biosensing platforms. Biosensors developed based
on these electrospun nanofibers have demonstrated a good analytic performance such
as high selectivity, fast response time, and low analyte detection limits. Although the
utilization of electrospun nanofibers in biosensing is very promising, there are some
limitations and challenges that need to be solved for further development of nanofiber-
based biosensors. Conventional electrospinning mostly produces randomly oriented
electrospun nanofibers which limit the repeatability of the final structures and there-
fore a better control of the nanofiber production and arrangement on the transducers
is needed. Electrospun nanofibers have been functionalized by dispersing nanoma-
terials in the nanofibers or on the surface of nanofibrous membrane to enhance
the performance of biosensors. While dispersing nanomaterials in the nanofibers,
the uniform distribution of nanomaterials without aggregation with the spinnable
polymer solution is crucial for the electrospinning process. On the other hand, the
binding and assembling of nanomaterials onto electrospun nanofibers should be
optimized in post-processing. In addition, the composite nanofibers with different
structures such as hollow, porous, and core-shell can be used to increase surface
area and immobilization sites for target biomolecules. The increasing demand on
rapid, simple, low-cost, and accurate monitoring of food quality and safety will push
forward the development of biosensors. Electrospun-based biosensors are of great
potential, but studies on food applications are limited and more research is needed to
develop nanofibers-based biosensors applied to real sample analysis. In the future,
it is expected that the development of electrospun nanofibers-based platforms will
result in the production of highly sensitive and selective biosensors for fulfilling the
needs of food industry.
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Chapter 14
Current Trends of Electrochemical
Sensing for Mycotoxins
Ruchika Chauhan, Rashi Bhardwaj, Sheetal K. Bharadwaj, Ajit Kaushik,

Rajshekhar Karpoormath, and Tinku Basu
Abstract The presence of mycotoxins in food materials is difficult to control, and

some mycotoxins are carcinogenic even at very low concentration. Therefore, there
is a demand for fast, sensitive, user friendly, and stable method for the detection
of mycotoxins. A portable smart biosensor is in a recent appeal in industries and
daily life in place of tedious and time taking conventional techniques. In the present
chapter, we analyze the current scenario for mycotoxin detection by immunosensors
based on labeled and label-free methods, aptasensors. Some other recent trends such
as mimotope and nanobody as a recognition agent for mycotoxin sensing are also
discussed. We also discussed the new trends in nanomaterials, or matrix of electrode
and transuding techniques. The current challenges, and technological aspects which
experience during the development of biosensors are also discussed, at the end future
research aspects mentioned.
Keywords Labeled and label-free immunosensor · Aptamers · Mycotoxins ·

Mimotopes · Molecular imprinting polymers · Nanobodies
R. Chauhan · R. Bhardwaj · T. Basu (B)

Amity Centre for Nanomedicine, Amity University Uttar Pradesh, Noida 201303, UP, India
R. Chauhan · R. Karpoormath
Department of Pharmaceutical Chemistry, College of Health Sciences, University of
KwaZulu-Natal, Westville Campus, Durban 4000, South Africa
S. K. Bharadwaj
University of Amsterdam, Amsterdam, The Netherlands
A. Kaushik
Florida Polytechnique University, Lakeland, FL, USA
276 R. Chauhan et al.
14.1 Introduction
The contamination of food materials by mycotoxins is becoming a serious problem

worldwide. These mycotoxins are secondary metabolites of fungi, particularly from
Aspergillus spp., Fusarium spp., and Penicillium spp. These species can contami-
nate during all stages of the crop, pre- and post-harvesting, or throughout the storage
(Robbins et al. 2000). About 400 specific mycotoxins are known till now; among
these mycotoxins, predominant are; aflatoxins (AFB), ochratoxin A (OTA), and zear-
alenone (ZEA) (Liew and Mohd-Redzwan 2018), which occurs in wheat, coffee,
fruit juices, fermented food products, and even in animal feed (Reddy et al. 2010).
Owing to low molecular weight, and heat resistance, they are capable to persist in
the food chain. Among all mycotoxins, aflatoxin B1 has been categorized as a potent
carcinogen (in Group 1 by International Agency for Research on Cancer [IARC])
and cause liver cancer in human (Henry et al. 1999). Also, ochratoxin A (OTA), and
fumonisins are considered possible carcinogen (Group 2B) (IARC 2002). Conse-
quently, the European Commission set the level of maximum acceptance of these
Group A and Group B mycotoxins in food materials (Table 14.1). These mycotoxins
are extremely hazardous than food additives and residues of pesticides present in
foods (van Egmond et al. 2007). Therefore, mycotoxin-contaminated food consump-
tion causes a harsh effect on human as well as animal health (Zheng et al. 2006;
Rodrigues et al. 2011). The lowermost limits of mycotoxins in food materials are
in part per billion (ppb) while for baby foods are below (0.05 ppb) which has been
set authorized by the European Union (EU). The analysis of mycotoxins (molecular
weight ~300–400 g/mol) in very small concentrations is the root of, major restric-
tion to develop a standard method for analysis (Tamerler et al. 2006). Internationally,
major research has been done by different research groups is designed to identify and
quantify the mycotoxins and assessment of their effects on the health of live stocks
(Zain 2011). The major concern group of fungi and their mycotoxins are listed in
Table 14.1.
14.2 Traditional Methods for Mycotoxins Detection
Several traditional analytical methods including high-performance liquid chromatog-

raphy (HPLC) (Lippolis et al. 2008), thin-layer chromatography (TLC), gas chro-
matography (GC) (Visconti et al. 2005), and enzyme-linked immunoassay (ELISA)
(Zheng et al. 2005) are commonly exploited for mycotoxins detection. However, these
methods are acknowledged for their accuracy and precision in the field of mycotoxin
detection in food samples, despite the costly equipment, needed skilled personal,
sample pretreatment, and time taking (Goryacheva et al. 2007). Consequently, a fast,
highly specific, and sensitive technique is required for the rapid and accurate detec-
tion of mycotoxins. In this chapter, we would like to cover major research carried out
in process of development of labeled and label-free immunosensors and aptasensors
14 Current Trends of Electrochemical Sensing for Mycotoxins 277
Table 14.1 Occurrence, restricted limit as defined by the European Commission along with IARC
classification of some hazardous mycotoxins
Sl. Name of Mycotoxin Occurrence in food and Restricted IARC
No. Fungi feeds limit (μg classification
Kg-1)
1 Aspergillus Aflatoxins Groundnuts, nuts, spices 2 1

spp. maize, rice, figs, cocoa,
B1
crude vegetable oils,
B2 (peanut, soybean,
G1 sunflower, cotton, chili 5-10
pepper, coriander,
G2 2B
turmeric, ginger)
Milk and milk products

M1
M2 0.05
2 Aspergillus Ochratoxins Dried vine fruit 0.5-10 2B

spp., (currants, raisins, and
A
Penicillium sultanas), Wine, Grape
spp. B juice, Wheat, Coffee,
C beans, Processed cereal
3 Fusarium Fumonisins Mainly in maize, other 50-1000 2B

spp. B1 cereals also wheat,
barley
B2
4 Fusarium Zearalenone In wheat, maize, 100-400 -

spp. sorghum, and barley
5 Fusarium HT-2, T-2 Oats and oat products, 50-100 -

spp. barley, rice, maize etc.
6 Fusarium Deoxynivalenol Cereal crops such as 200-2000 -

spp. wheat, maize, or barley
and additionally recent technology for rapid quantification of mycotoxins in daily

consumable food items.
14.3 Current Trends of Mycotoxins Detection
Compared to conventional mycotoxin detection methods, biosensors attain more

attention due to their simplicity, low cost, faster in response, easy-to-handle, and
portability. Biosensor converts biological responses into the signal, in this, an analyte
interacts with their bioreceptor, and this interaction measures by transducer detector.
The basic components of biosensors include biorecognition agents (bioreceptor),
transducer, and display. The biorecognition agent identified for mycotoxin sensing
are antibodies, DNA, cell receptors, enzymes, etc. In addition, few engineered chemi-
cals such as aptamers, MIP (molecularly impregnated polymer, mimotopes, etc.) are
also exploited as a recognition agent. Mycotoxins are small and neutral organic
molecules, so direct sensing through bio affinity-based receptor develops too weak
signal to be detected. Reliable sensing of mycotoxin demands signal amplifica-
tion through appropriate labeling like nanoparticles, enzymes, redox moieties, fluo-
rophores, etc. As a result, the mycotoxin biosensors are broadly classified into two
categories, first is labeled while second is label-free biosensors based on detec-
tion strategy, and those are further subdivided into two, (i) competitive (ii) non-
competitive. Labeled sensor majorly dependent on label moiety and the response
received by virtue of labels (like enzymes, Quantum Dots, Fluorescence nanoma-
terials, etc.), while label-free sensors are independent, and response receive due to
direct interaction of antibody and analyte. Significant research efforts are devoted to
developing a suitable mycotoxin biosensor for rapid and reliable sensing with respect
to transducing techniques (electrochemical, optical, and piezoelectric), immobilizing
matrix, bioreceptor, and label selection. The present chapter will discuss about the
material composites, the function of labeled moieties, and the principle of both
labeled and label-free sensor. A schematic representation of labeled and label-free
biosensors, i.e., Fig. 14.1a, b, respectively, are given below:
Fig. 14.1 a Schematic representation of labeled mycotoxin biosensor. b Schematic representation

of label-free mycotoxin biosensor
14.3.1 Labeled Mycotoxin Biosensor
14.3.1.1 Immunosensor for Detection of Mycotoxins
For small molecules like mycotoxin, the competitive immunoassay is more reli-
able than a non-competitive one. In the competitive mechanism, competition occurs
between the free mycotoxin and labeled mycotoxin or protein conjugated mycotoxin
for limited antibodies present in the reaction mixture or surface of the electrode. When
concentration of mycotoxin increases in the reaction mixture, more labeled myco-
toxin or protein-mycotoxin conjugates will be displaced from the surface of trans-
ducer (Pemberton et al. 2006; Prieto-Simón et al. 2012; Piermarini et al. 2007). In a
competitive mechanism, there are two types of approaches. In the first approach, the
optimum concentration of protein conjugated or labeled mycotoxin was tagged with
the optimized antibody concentration. When the analyte (mycotoxin) is added over
the electrode surface, competition occurs between loaded mycotoxin and the sample
mycotoxin for the optimized antibody, this method is named as “indirect method.”
The BSA-mycotoxin conjugate is extensively used. Sometimes, this competition
causes a steric effect. To avoid steric effect, spacer molecules (like polyethylene
glycol [PEG]) were introduced (Yuan et al. 2009) over the electrode in between
the mycotoxin and protein conjugate. Alkaline phosphatase (AP) was studied as
label in an indirect competitive immunosensing of Aflatoxin B1(AFB1), and the
electrochemical signal produced by AP catalysis in which α naphthyl phosphate is
hydrolyzed (Zhang et al. 2016).
High electron transfer rate (ETR) of the nanomaterial modified electrode surface
amplifies the response signal of interaction between mycotoxin and their antibody
(Bonel et al. 2010; Liu et al. 2009). The sandwiched immunosensor of AuNPs
labeled secondary antibody amplified the surface plasmon resonance (SPR) signal
for OTA detection by 10-fold with an extremely low sensing limit of (60 pg mL−1 ),
in comparison to AuNPs labeled primary anti-OTA (Urusov et al. 2011).
The cadmium telluride labeled antibody over the Fe-GO matrix was used for the
detection of aflatoxin M1 with the 0.3 pg mL−1 of LOD (Gan et al. 2013). For concur-
rent detection of two or more mycotoxins in foods, upconversion nanoparticles (Wu
et al. 2011) were successfully tried. Feng et al. have fabricated (Feng et al. 2018)
a photo electrotypes competitive immunosensor for quantitative detection of AFB1 .
The silver ion modified BiVO4 nano-polyhedron (BiVO4 @Ag+ ) was employed as a
label of anti-AFB1. A nonenzymatic immunosensor was developed with photoactive
material (Mn2+ incorporated zinc complex) for AFB1 detection in foodstuff (Lin et al.
2017). The proposed photo active electrochemical immunosensor attained satisfac-
tory detection range 0.5 pg mL−1 –10 ng mL−1 , and LOD as 0.3 pg mL−1 by using a
competitive strategy. Here, dopamine loaded liposome was exploited as an electron
donor, which significantly enhanced the photocurrent response of the photoelectrode.
Another phenomenon is called “direct method,” in this format, competition occurs
between the free analyte (mycotoxin) and the absorbed labeled mycotoxin for the
optimized concentration of antibody loaded over the electrode surface. Initially, the
signal is obtained from saturated concentration of labeled mycotoxin, later, alteration

in transducer signal in the presence of sample mycotoxin reflects the interaction of
analyte (mycotoxin) with their specific antibodies. It was found that the indirect
method is less effective in comparison to the direct method (Alarcón et al. 2006).
The quartz crystal microbalance (QCM) is a highly sensitive technique to analyze
the small molecule at the scale of the nano dimension. For mycotoxin, researchers
used a sandwiched format for the more precision in results. For the signal amplifica-
tion, relied on QCM plinth, HRP labeled secondary antibodies for the oxidation of
4-chloro-1-naphthol into insoluble benzo -4-chlorohexadienone and AuNP decorated
secondary antibody were used as a mass enhancer on QCM surface which enhance
the sensitivity of the QCM electrode (Jin et al. 2009a, b). Unlike the conventional
QCM technique, these formats attained a low detection range of 0.01–10.0 ng mL−1 .
The magnetic nanoparticles have great advantages because of their inherent prop-
erty of magnetism therefore these magnetic nanoparticles were used for regeneration
procedure (Aguilar-Arteaga et al. 2010; Vidal et al. 2012). The almost ~100% effi-
ciency of magnetic nanoparticles were reported for the separation of interfering
substances from the reaction mixture (Prieto-Simón et al. 2008). By following this
Perrotta et al. (2012) determined the OTA in red wine and achieved 0.008 ppb LOD.
The simultaneous detection of mycotoxins was accomplished with a combination of
competitive and sandwich immunoassay on a single array biosensor (Sapsford et al.
2006).
The non-competitive labeled immunoassays are mostly based on sandwiched
format, in this, signal obtained through labeled secondary antibodies, as the number
of labeled antibodies increases over the electrode surface, increase in signal is
observed. A sandwiched immunosensor was designed by using catechol as a label
for the detection of AFB1 and in this study Au–Fe nanoparticle played a role in the
regeneration of sensor. The external horse magnet was used for the regeneration of
immunosensor. When catechol oxidized into o-quinone signal read via a transducer
and broad response from 0.6 to 110 ng mL−1 for AFB1 detection with a low detection
limit of 0.2 ng mL−1 was observed (Masoomi et al. 2013), though the whole system
of the biosensor was typical and chances of false signals. Another study of multiple
labels was carried out for zearalenone detection with HRP tagged secondary anti-
bodies (Feng et al. 2013a). They used sodium montmorillonites NPs and thionine
for achieving LOD of 3 pg mL−1 for zearalenone detection in real samples.
14.3.1.2 Labeled Aptasensor for Detection of Mycotoxin
In aptasensor, aptamer is an important component, which is synthesized from 10 or

more variable bases. These synthetic sequences of oligonucleotides (DNA/RNA) are
specific for the target because of their specific binding affinity (Willner and Zayats
2007; Cho et al. 2009). Aptamers have plenty of advantages over the antibodies, as the
synthesis and handling of aptamers are easy and other factors like cost effect, stability,
and ability for further functionalization are enough to take over the antibody (Yang
et al. 2013b; Schmidt et al. 2004). Aptamers are single-stranded oligonucleotides
with three-dimensional structures, which can help ligands bind strongly with their
complementary strands (Hermann and Patel 2000; Cruz-Aguado and Penner 2008a).
The aptamers are highly stable, it can resist denaturation and active toward their
targets (like peptides, proteins, toxins, drugs, whole cells, etc.) with strong affinity.
Thus, aptamers are deliberated as a recovering substitute in many research studies
instead of antibodies. Some reported sequences of aptamers for mycotoxins are listed
in Table 14.2.
The mechanism of aptasensors is orchestrated on the interaction of aptamer with
the analyte (mycotoxins) in the reaction mixture. After the interaction, the signal
received by the transducer, which may be due to the enhancement of signal or decre-
ment in signal depends upon the assay format called as “signal on” and “signal
off,” respectively. In labeled aptasensor signal receives to the transducer via labels
(nanoparticles, enzyme, fluorescence material, etc.) either on the aptamer or on
complementary DNA (Loo et al. 2015).
Labeled Signal On
“Signal On” reaction mechanism is shown in Fig. 14.2a. In this phenomenon, the
signal increases after the interaction of aptamer and analyte because labeled moiety
attached to the sequence aptamer of the analyte. For this phenomenon, mostly fluo-
rescence materials have been used with the combination of quencher, as carbon-based
nanomaterials are good quencher (CNTs, graphene) (Guo et al. 2011; Cruz-Aguado
and Penner 2008b). Chen and coauthors achieved dynamic range for the OTA detec-
tion from 1 to 100 ng mL−1 via fluorescence-quencher mechanism (Chen et al. 2012).
In fluorescence-quencher mechanism, the fluorescence material of aptamer interacts
or quenched via “π– π” interaction between fluorescence material and carbon mate-
rial. As the analyte (mycotoxin) enters, the aptamers change to their conformation
due to more affinity toward the analyte (Zhu et al. 2010). The polymeric coating on
carbon material like polyvinyl pyrrolidone coated on graphene oxide prevents the
nonspecific adsorption over the electrode, which helps in enhancement in sensitivity
(Sheng et al. 2011).
The upconversion nanoparticle (UCNP) in combination with rare earth metal
(Tm, Er, Ho) gives multicolor. This property of UCNP has advantages over the
other nanoparticles for the multi detection of analyte at the single electrode. Forster
resonance energy transfer (FRET) is the best tool for multiple analyte detection using
UCNP. The simultaneous detection of OTA and FB1 (Fumonisen B1) was reported by
Wu et al. (Wu et al. 2012b) via “signal on” format. The AuNPs modified with luminol
derivative-based aptasensor produced a 10-fold low detection limit (0.06 ng mL−1 )
and linear range (0.2–5.0 ng mL−1 ) for OTA (Wang et al. 2010b) in comparison to
bare Au electrode.
The nucleic acids act as catalysts and receptors called as functional nuclic acid
(DNAzyme or RNAzyme) (Li and Lu 2009). In aptasensor, DNAzyme plays an
important catalytic role in HRP mimicking caused by hemin and G-quadruplexes
interaction (Li et al. 2009; Cheng et al. 2009). Yang et al. (2013a) have designed
colorimetric aptasensor for OTA detection, the “signal ON” phenomenon occurs
Table 14.2 Aptamer sequences for different mycotoxin detection in aptasensor
282
Sl. Aptamer sequence Toxin Ref.

No.
1 5 -ATGCTCCCTTTACGCCACGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3 OTA Chen et al.
(2014)
2 5 -GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-C6-biotin-3 OTA Zhang et al.
(2013b)
3 5 -TGGGTAGGGCGGGTTGGGAAAGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3 OTA Yang et al.
(2013a)
4 5 -MB-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-SH-3 OTA Wu et al.
(2012b)
5 5 - AAAGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3 OTA Tong et al.
(2011)
6 5 -GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-FAM-3 OTA Chen et al.
(2012)
7 5 -AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3 ZEN Chen et al.
(2013)
8 5 end-labeled by fluorescein (F-Apt05 -F-GATCGGGTGTGGGTGGCGTAAAGGGAGCATGGACA-3 ) OTA Galarreta
et al. (2013)
(continued)
R. Chauhan et al.
Sl. Aptamer sequence Toxin Ref.
No.
9 5 -GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3 OTA Kuang et al.
(2010), De
Girolamo
et al. (2011),
Wu et al.
(2012a),
Rhouati et al.
(2013), Hun
et al. (2013),
Lv et al.
(2014)
10 OTA’s aptamer (5 -GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-FAM-3 ) OTA Guo et al.
(2011)
11 5-FAM-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3 OTA Sheng et al.
(2011)
12 5 -SH-C6-ATCTACGGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCC-3 OTA Wang et al.
(2009, 2011)
13 5 -biotin-(CH2 ) Fumonisin Wu et al.
6-ATACCAGCTTATTCAATTAATCGCATTACCTTATACCAGCTTATTCAATTACGTCTGCACATACCAGCTTATTCAATTAGATAGTAAGTGCAATCT-3 (2012b)
14 Current Trends of Electrochemical Sensing for Mycotoxins
283
Fig. 14.2 a Schematic diagram for “signal on” aptasensor. b Scheme for rolling cycling amplifi-
cation (RCA) for the detection of OTA (Tong et al. 2012). c Schematic diagram for labeled “signal
off” mode for aptasensor
when OTA interacts with complex and hemin was displaced from G-quadruplex. The
oxidation reaction was completed through H2 O2 and tetramethylbenzidine (TMB).
The multiple HRP-DNAzymes attached with AFB1 aptamer was used for signal
amplification in chemiluminescence-based aptamers (Shim et al. 2014). Chen and
group designed DNA probes based on biotinylated hairpin and labeled with strepta-
vidin coated AuNPs for enzyme-free catalytic detection of AFB1 (Chen et al. 2016).
In this AuNPs(red) aggregated into a cross-linked network via biotin-streptavidin
coupling, on aggregation color changed from red to blue.
Based on peroxidase mimicking DNAzyme activity, Seok et al. (2015) have recog-
nized AFB1 in ground corn samples. In this the aptamer-DNAzyme complex distorted
and the DNAzyme halves split and generated a homogeneous colorimetric sensor
with LOD of 0.1 ng mL−1 .
Aptasensor has limitations due to the equal ratio of receptor and analyte which
causes the major issue of sensitivity (Tong et al. 2011). There are several signal
amplification methods which expand the sensitivity of aptasensor, like rolling circle
amplification (RCA) (Li et al. 2010), polymerase chain reaction (PCR) (Schmittgen
and Livak 2008), nicking endonuclease amplification (Hun et al. 2010), and loop-
mediated isothermal amplification (LAMP) (Wang et al. 2010a). A sandwiched
aptasensor designed for OTA, in which DNAzyme quenched the chemiluminescent
light from the O2 and RecJf endonuclease recycle the assembly which enhanced the
sensitivity up to 75 fg mL−1 (Chen et al. 2014).
Tong and group have electrochemically detected OTA using rolling cycling ampli-
fication, which amplified the signal. On the addition of T4 ligase capture DNA and
padlock DNA formed circular template. Later this template DNA initiate cycling
amplification reaction in the presence of phi2 DNA polymerase and dNTPs and gener-
ated a long tandem repeated sequence (Tong et al. 2012). Another circular amplifi-
cation enzyme—nicking endonuclease, was used for the sensitive detection of OTA
mycotoxins for sensitive detection that cleaved and released the OTA template strand
(Hun et al. 2013). Hetero-enzyme-based two-round signal amplification strategy has
been used for trace detection of aflatoxin B1 using an electrochemical aptasensor
(Zheng et al. 2016).
Labeled Signal Off
The labeled signal off aptasensor based on a decrease in signal due to the forma-
tion of complex in between the aptamer and mycotoxin (Fig. 14.2c). Or in other
words, the presence of labels (like enzyme, fluorophore QDs) in sensor show the
maximum signal which suppose to reduce or off after the target interaction with their
complementary aptamer, and aptamer with label moiety removes from the electrode.
In signal off mode, the effect of nonspecific DNA adsorption reduces therefore, it
has been found that the signal off mode is more sensitive and gives positive results
rather than the signal on. The redox probe of AuNP/methylene blue used for the
detection of OTA, on interaction with OTA to the probe, the transducer signal was
reduced (Kuang et al. 2010).
Fig. 14.3 Calibration curves

for electrochemical
aptasensor for detection of
OTA (Wu et al. 2012a)
Those aptamers functionalized with magnetic bead have benefits of regeneration

of aptasensor and easy to immobilize over the electrode with an external magnetic
field (Rhouati et al. 2013). The multiple steps of processing of aptasensor produced
the false results with real sample analysis. Though one step Au electrode-based
aptasensor was designed with a dual label (thiol- and methylene blue) for the detection
of OTA. The broad response range (0.1 pg mL−1 –1000 pg mL−1 ) and 0.095 pg mL−1
LOD was obtained (Wu et al. 2012a) (Fig. 14.3).
14.3.2 Label-Free Mycotoxin Biosensor
14.3.2.1 Immunosensor for Label-Free Detection of Mycotoxin
The label-free immunosensors had advantages over the labeled immunosensor

because of their simplicity and one step analysis. Like the labeled immunosensor
it was also categorized into two (i) non-competitive and (ii) competitive. Sometimes
label-free immunosensor are unable to reach the required sensitivity specially for
small molecules like mycotoxin. The non-competitive immunosensors are fast and
simple in comparison to competitive immunosensor, though they are less sensitive
because of nonspecific binding and receive false positive results.
The mechanism of label-free non-competitive immunosensor is based on direct
interaction between immobilized antibodies and mycotoxin. Researchers used
different transducers and varying the matrix of electrode (carbon nanocomposites,
CNTs, graphene oxide) or conducting polymer or metal, and metal oxide nanoparti-
cles (Fe3 O4 , ZnO, TiO2, etc.) to enhance the sensitivity of label-free immunosensor
(Sharma et al. 2010; Solanki et al. 2009; Zhang et al. 2016; Díaz Nieto et al. 2019;
Srivastava et al. 2013). An immunoassay based on lateral flow was developed by
controlling the concentration of antibody and size of magnetic beads. It exhibited a
cutoff value of 0.02 mg L−1 of AFM1 in milk samples (Liu et al. 2015a). Using
electro-spinning technique, a polyacrylonitrile (PAN) nanofiber-modified pencil-
graphite electrode was developed for Zearalenone (ZEN) detection in the liquid
foods, including dairy, juices, and other liquid foods (Moradi et al. 2020).
Among the electrochemical spectroscopic studies impedance spectroscopy is
more sensitive and reliable because in this interface of electrode studies with electrode
surface reactions (Radi et al. 2009; Zamfir et al. 2011). A label free impedemetric
immunosensor for OTA has been developed (Malvano et al. 2016). Yu and group
developed an impedimetric immunosensor for the determination of AFB1 based on
MWCNTs-ionic liquid modified electrode and acquired the LOD 0.03 ng mL−1 . The
MWCNTs in ionic liquid exists as a three-dimensional (3D) network and cross-linked
due to cation π and π–π interactions between the imidazolium cations and CNTs.
Here the ionic liquid delivers excellent biocompatible microenvironment to uphold
the activity of antibody and used as an exceptional conductive binder to immobilize
antibodies and MWCNTs onto the electrode (Yu et al. 2015).
The self-assembled monolayer of 11-mercaptoundacanoic acid was assembled on
the silver wire for dual detection of AFB1 and AFM1 in nuts and milk, respectively,
using EIS immunosensing technique (Bacher et al. 2012). The online flow system
with EIS technique is more persuasive then lateral flow system and shows enhanced
sensitivity and lower LOD. On the coupling of these two systems Kanungo and group
have detected AFM1 and AFM2 in beverages with the LOD of 1 pg mL−1 (Kanungo
et al. 2014).
The fragment antibody of AFB1 showed 10-fold higher sensitivity than the whole
antibody. Li and coauthors used fluorescence resonance energy transfer technique
with tryptophan (Trp) residues-based fragment antibody, they followed a label-free
non-competitive format, where tryptophan (Trp) residues played a role of donor
wherein fragment comes in contact of AFB1 mycotoxin (Li et al. 2013). The
optical sensing technique was found more compatible with label-free immunosensing
because the reflection of light can read minor changes on the surface of the electrode
(Nabok et al. 2005, 2007). The SPR technique based on the reflection of light from the
gold surface of the electrode, therefore gold-coated protein G were more compatible
rather than thiol or ProG modified electrode surface (Tamerler et al. 2006; Park et al.
2014). The simultaneous detection of AFB1 and OTA was done by optical waveg-
uide light spectroscopy (OWLS). The OWLS technique compatible with competitive
immunosensor and show highly sensitive response in compare to non-competitive
immunosensor (Adanyi et al. 2007). Later study applied OWLS for the detection
of AFB1 in competitive immunoassay with AuNPs of different sizes and origins
(obtained by chemical or biotechnological synthesis) (Adányi et al. 2018). The addi-
tion of nanomaterial to the optical label-free immunosensor may reduce the challenge
related to the sensitivity (Ah et al. 2012). The gold nanorods were applied for AFB1
detection on an optical electrode which followed the label-free competitive format
and obtained the LOD of 0.16 ng mL−1 for AFB1 (Xu et al. 2013). Though optical
immunosensors are sensitive they have some drawbacks like, expensive equipment,
regeneration of sensor chip, and nonspecific adsorption. Over the other optical tech-
nologies, White Light Reflectance Spectroscopy (WLRS) has certain advantages like
free-space interference concepts, the limited effect from the change in the refractive
index of the sample under analysis and provides immunity to temperature varia-
tions. Moreover, the illumination of the sensing area is straightforward during the
bioreaction monitoring. Additionally, biochips are easy to be fabricated and of low
cost. The principle of WLRS depends on the change in the interference spectrum,
which occurs after the interaction of analyte over the SiO2 fabricated silicon chip.
With a slight modification a new approach, applied by Anastasiadis and coworkers
(Anastasiadis et al. 2020) in which they have designed the WLRS electrode surface
for multiple detections of mycotoxins using the variable thickness of SiO2 .
The QCM transducer use is not much widespread for the detection of mycotoxins,
because of its small size (Tsai and Hsieh 2007; Cheng et al. 2012; Vidal et al. 2009).
However, label-free detection of an analyte using the QCM transducer has advan-
tages over the other transducer. The mass-based cantilever was used for multiple
detections of mycotoxins (Ricciardi et al. 2013). The coupling of QCM with electro-
chemical transducer showed a high sensitivity for label-free detection of mycotoxins.
The electrochemical quartz crystal microbalance (EQCM)-based immunosensor was
developed, in this thiol self-assembled monolayer modified with AuNPs (Fig. 14.4a)
for the detection of AFB1 (Chauhan et al. 2015b; Chauhan et al. 2016). They devel-
oped a reusable label-free immunosensor (Fig. 14.4b). This immunosensor format
was competitive and sandwiched (Au coated Fe3 O4 nanocomposites with secondary
antibodies) and detect AFB1 in wine samples (Chauhan et al. 2015a). A list of
developed label-free and labeled immunosensors for mycotoxin given in Table 14.3.
A competitive multiplexed imaging surface plasmon resonance (iSPR) biosensor
assays were corroborated for DON, ZEA, and T-2 toxin (Hossain and Maragos 2018).
In this, AuNP was used for signal amplification conjugated with secondary anti-
bodies. One of the advantages of iSPR is the ability to use the sensor multiple
times, however, it is a challenging task to regulate the optimal conditions at the
time of binding and regeneration for multiple antigens, and antibodies. Lu et al.
(2019) designed a microfluidic immunosensor for simultaneous detection of FB1
and DON. This capillary-driven microfluidic system achieved LODs of 97 pg mL−1
and 35 pg mL−1 , and their corresponding linear ranges of detection are 0.3–140 ppb
and 0.2–60 ppb, respectively, for FB1 and DON (Lu et al. 2019). Liu and group devel-
oped a label-free immunosensor based on magnetic CdSeFe3 O4 nanocomposites for
AFB1 detection (Liu et al. 2015b). Anti-AFB1 was immobilized on the CdSeFe3 O4
modified screen-printed electrode (SPE). The determination of AFB1 concentration
was measured by a decrease in photocurrent, caused by the steric hindrances due
to the immunocomplex. Bhardwaj et al. develop an AuNPs-based microfluidic SPR
biosensor for AFB1 detection relied on modified AuNPs grafted over a SAM-linked
Au chip surface via an amine linkage (Bhardwaj et al. 2020).
Xia and coworkers designed a cell-based impedance sensor for DON, ZEA, and
AFB1 detection (Xia et al. 2017). A luminescent bacterium, Photobacterium phos-
phoreum was employed used for the multi mycotoxin detection under discrete and
flow-through analysis (Senko et al. 2019). Though the method is simple, convenient,
and exhibits a quick response rate incubation time was found to be more than 12 h
Fig. 14.4 a A diagrammatic representation of label-free detection of AFB1. b A pictorial

representation of the regeneration of immuno-electrode
Table 14.3 The list of label-free and labeled immunosensors developed for detection of mycotoxins
290
Sl. No. Matrix Electrode Type of Ab Mode of detection Label Technique

1 BSA/ab/CeO2 –CS/GO–CS/SPE CeO2 –CS/GO-CS/SPE. Ab Non-competitive Label-free DPV
2 AFB1/BSA/aAFB1/4-ATP/Au – mAb Competitive – EQCM
3 CS–PtCo–Ab/Graphene-Thionine/GCE GCE mAb NC – Electrochemical
4 Graphite/ SPCE mAb Competitive OTA-ALP DPV
silver
5 Graphite based ink SPCE pAb Competitive Au-labeled ALP-Sec Ab DPV
6 1,6-Hexanedithiol Au mAb Competitive ALP Electrochemical
(DPV)
7 Magnetic beads with Protein G SPCE mAb Competitive HRP SWV
8 – SPE mAb Competitive OTA –
(continued)
R. Chauhan et al.
9 GNRs – mAb Competitive – Optical
10 16-MHDA Au pAb Competitive Au NPs QCM
11 Magnetic Beads SPCE mAb Competitive HRP DPV
12 11-MUOH Au mAb Competitive Au NPs SPR
13 mAb Competitive – EIS
14 MPA-BSA mAb Competitive HRP QCM
15 1-dodecanethiol (DDT) Au pAb NC – Electrochemical
16 Au NPs – – Competitive – SPR
17 GCE/PTH/AuNP AFB1- BSA-polythionine (PTH)/gold GCE pAb Competitive HRP Electrochemical
nanoparticles
18 pAb Competitive ALP Intermittent pulse
amperometry (IPA)
(continued)
291
292

19 magnetic nanoparticles (MNPs) – mAb Competitive – Fluorescent
20 Au/3-Mercaptopropionic acid (MPA) mAb Competitive Au NPs Piezoelectric
21 Au/DDT SAM Polycrystaline gold electrode mAb NC – TIRE and QCM
22 DSP Au mAb NC – QCM
(3,3 -Dithiodipropionic-acid-di-N-hydroxysuccinimide
ester)
23 Fe3O4/SiO2 composite nanoparticles Au mAb NC – QCM
24 Fe3O4-graphene oxides SPCE mAb Non-competitive CdTe-CNT QDs Conjugate ECL
25 AET- Au NPs Au interdigitated electrodes mAb Non-competitive HRP conductometric
(150 nm thick)
26 GCE/chitosan/Au nanoparticle (AuNP)/anti-Aflatoxin GCE pAb Non-competitive catechol–Au–Fe3 O4 NPs Electrochemical
B1 (a AFB1),
27 nanoporous gold films (NPG) GCE mAb Non-competitive Na-Mont-TH-HRP-Ab2 Electrochemical
(NC)
28 Chitosan-iron oxide ITO pAb NC – DPV
(continued)
R. Chauhan et al.
29 Nano CeO2 – CH ITO pAb NC – DPV
30 Chitosan- PANI ITO pAb NC EIS
31 4-carboxyphenyl SAM Au – NC – EIS
32 BSA/AO-IgG/AUT/Au Au pAb NC – EIS
(SAM of AUT)
33 16-MHDA Au pAb NC – QCM
34 Au/TA/Glu/BSA/MNP-Ab Au mAb NC – EIS, CV
35 BSA/aAFB1-C-AuNP/MBA/Au Au mAb NC – Volta metric
36 anti- AFB1/MWCNTs/ITO ITO mAb Non-competitive – Volta metric
37 SAM of Silver Wire mAb Non-competitive – Impedimetric
11-MUA
38 BSA/AbAFB1/n-Sm2 O3 /ITO ITO mAb Non-competitive – Volta metric
(continued)
293
294

39 – – Fragment Ab Non-competitive – FRET based
Immunoassay
40 Nano-sized gold hollow balls (NGB) with dendritic Au NC – QCM
surface
41 Silica gel-ionic liquid-antibody GCE pAb NC – EIS
42 APTES, Silane, Protein G – mAb NC – Microcantilever
43 RGO/ITO ITO mAb NC – Electrochemical
44 MWCNTs/RTIL/Ab glassy carbon electrodes mAb Competitive EIS
46 Au/TGA/BSA-Ag/anti-OTA McAb Au Ab competitive – DPV
47 anti-aflatoxin antibody/Au/rGO/ITO AU/RGO/ITO Ab – – CV
Sl. No. Toxin Response range LOD Sensitivity Relative Standard Coefficient of Ref.
Deviation (RSD)in variation (CV)in %
%
1 AFM1 0.01–1 μgL−1 0.009 μgL−1 – 2.7–3.5 – An et al. (2020)
2 AFB 1 0.1–4.0 ng mL − 1 0.16 ng mL−1 , 532.7 mA ng − 1 mL cm − 5.13 – Chauhan et al.
2 (2015b)
3 ZON 0.05–5.0 ng mL−1 13 pg mL−1 – 4.5 – Feng et al. (2013b)
4 OTA 0.05–2.5 ng mL−1 60 ng mL−1 – 8–16 7 Alarcón et al.
0.1–7.5 ng mL−1 0.4 ng mL−1 (2006)
100 ng mL−1
5 OTA 0.9–9 ng mL−1 0.86 ng mL−1 – 8, 10 6 Bonel et al. (2010)
0.3–8.5 ng mL−1 0.20 ng mL−1
6 OTA 10 pg mL−1 - 100 ng mL−1 8.2 pg mL−1 – – – Liu et al. (2009)
7 OTA 0.01–20 ppb 0.008 ppb – 5.56 – Perrotta et al.
(2012)
8 OTA 0.3 ngL−1 - μgL−1 – – 20, 36 – Prieto-Simón et al.
(2012)
9 AFB 1 0.5–20 ng mL−1 0.16 ng mL−1 , – – – Xu et al. (2013)
10 OTA 10–128 ng mL−1 ng mL−1 8 ng mL−1 – – 8.96 Vidal et al. (2009)
R. Chauhan et al.
11 OTA 0.1 pg mL−1 –1 μg mL−1 10 pg L−1 – 9 – Vidal et al. (2012)

(continued)
%
12 OTA – 1.5 ng mL−1 – – – Yuan et al. (2009)
Au–0.042 ng mL−1
13 AFM 1 1–100 pg mL−1 1pgmL−1 – – Kanungo et al.
(2014)
14 AFB 1 0.01–10.0 ng mL−1 0.01 ng mL−1 – 4–7 – Jin et al. (2009b)
15 altertoxin I (ATX-I) – 4 × 10−8 moldm−3 – 7 – Moressi et al.
(14 ppb) (2007)
16 OTA 0.1 ng mL−1 –0.1 μg mL−1 60 pg mL−1 – – – Urusov et al. (2011)
17 AFB 1 0.6–2.4 ng mL−1 0.07 ng mL−1 – 2–5 – Owino et al. (2008)
18 AFB 1 0.05 and 2 ng mL−1 – – 7–10 – Piermarini et al.
(2007)
19 AFB 1 0.01–10 ng mL−1 0.01ngmL−1 – 5.19 – Wu et al. (2011)
OTA 2.14
20 AFB 1 0.1–100ngmL−1 0.01 ng mL−1 – 4–7 – Jin et al. (2009a)
21 AFB1 100 μg mL-1 to – – – Nabok et al. (2007)
0.15 ng mL−1
(continued)
295
296
%
22 AFB 1 0.5–10 ppb – – – – Spinella (2013)
23 AFB 1 0.3–7.0 ng mL−1 0.3 ng mL−1 – 8.0 6.3 Wang et al. (2009)
24 AFM 1 1.0–1.0 × 105 pg mL−1 0.3 pg mL−1 – 3.5 – Gan et al. (2013)
25 AFB 1 0.5–10 ng mL−1 0.1 ng mL−1 at 3δ. 7 7.1 Liu et al. (2006)
26 AFB 1 0.6–110 ng mL−1 0.2 ng mL−1 – – 6.2, 5.7 Masoomi et al.
(2013)
27 ZON 0.01–12 ng mL−1 3 pg mL−1 – 2.46–5.17 4.2–5.2 Feng et al. (2013a)
28 OTA 0.5 ng dL−1 36μAng−1 dL−1 – – Kaushik et al.
(2008)
29 OTA 0.5–6 ng dL−1 0.25 ng dL−1 1.27 μA ng−1 dl−1 cm−2 – – Kaushik et al.
(2009)
30 OTA Up to 10 ng mL−1 1.0 ng mL−1 – – – Khan and Dhayal.
(2009)
31 OTA 1–20 ng mL−1 0.5 ng mL−1 – 3.7–5.5 – Radi et al. (2009)
(continued)
R. Chauhan et al.
%
32 OTA 0.5–6 ng dL−1 0.08 ng dL-1 36.86 O ng-1 dL-1 – – Solanki et al. (2010)
33 OTA 50–1000 ng mL−1 16.1 ng mL−1 – 64 Tsai and Hsieh
(2007)
34 OTA 0.01–5 ng mL−1 0.01 ng mL−1 – 4.7 – Zamfir et al. (2011)
35 AFM 1 10–100 ng dL-1 17.90 ng dL-1 0.45 μAng− 1 dL – – Sharma et al. (2010)
36 AFB 1 0.25–1.375 ng mL−1 0.08 ng mL−1 (95.2μAng−1 mLcm−2 ) – – Singh et al. (2013a)
37 AFM 1 6.25–100pgmL−1 1 pg mL−1 6.25 pg mL−1 – – Bacher et al. (2012)
38 AFB 1 10–700 pg mL_1 57.82 pg mL_1 cm_2 , 48.39 μApg-1 mL cm_2 0.39 – Singh et al. (2013b)
39 AFB 1 – 0.85 and 0.09 ng mL-1 – – – Li et al. (2013)
40 AFB 1 0.05 ng mL−1 2.3, 5.8 3.8–5.1 Liao (2007)
41 AFB1 0.1–10 ng mL−1 0.01 ng mL−1 1.7 – Zaijun et al. (2010)
42 Aflatoxins Aflatoxins; 3 ngmL-1 – – – – Ricciardi et al.
OTA 1 OTA 1: 6 ng mL−1 (2013)
43 AFB 1 – 0.12 ng mL−1 68 μAng-1 mL cm-2 – – Srivastava et al.
(2013)
44 AFB 1 0.1–10 ng mL−1 0.03 ng mL−1 – 2.7–4.3 Yu et al. (2015)
46 OTA 0.1–1.0 ng mL−1 0.08 ng mL−1 – <4.67 – Sun et al. (2020)

47 AFB1 5 ng mL−1 to 1 pg mL−1 6.9 pg mL−1 – – – Althagafi et al.
(2019)
297
and the Hep G2 cell or any whole cell-based sensors are not specific for the targeted
analyte, therefore there are more probabilities of false results.
The detection of mycotoxins by aptamers without using any label is called label-
free aptasensor, where the change in signal in transducer for aptamer and mycotoxin
interaction is read without a label (Schöning and Poghossian 2002). Researchers
have modified the electrode surface via nanoparticles, polymers, oxides of metal
nanoparticles, etc., for electrochemical detection of OTA (Cai et al. 2018; Khan
et al. 2011). Some research group used click chemistry for uniform immobilization
of aptamer, which provides the better surface to the interaction of mycotoxins, and
trigger to enhance the sensitivity (Hayat et al. 2013b; Ran et al. 2011). The alteration
in aptamer’s conformation during the mycotoxin interaction reduces the background
current in aptasensor. Hayat and group designed polyethylene glycol–aptamer-
based screen-printed carbon electrode (SPCE). Consequently, macromolecules were
formed through long tunnels, at the tunnel OTA aptamer was present for detection of
OTA (Hayat et al. 2013a). The signal on label-free aptasensor formed G-quadruplex
for the detection of OTA and Tb3+ introduce to enhance the sensitivity (Fig. 14.5). The
LOD was noticed to be as 20 pg mL−1 (Zhang et al. 2013a). Castillo and coworkers
have proposed an electrochemical aptasensor for detection of AFB1 using cystamine-
dendrimers (Cys-PAMAM) layers on gold electrode. The limit of detection attained
by this sensor is 0.40 ± 0.03 nM, in this dendrimer enhances the sensitivity. The
dendrimers were positively charged due to this property of dendrimer the aptasensor
possesses less negative charge over the electrode surface. This effect of dendrimer on
the electrode surface helps in electron transfer and also effective for redox reaction
(Castillo et al. 2015).
The reversible ligand grafted aptasensor was developed for wave optical detection
of OTA. The real samples of food were analyzed, where the electrode contained
glutaraldehyde and ethylenediamine as linker and the OTA attached with covalent
bonding over optical fiber. The binding energy was measured on the bases of tied
ligand and free OTA aptamer in the reaction mixture. Using a competitive detection
Fig. 14.5 The response

analysis of aptasensor for
OTA (from 0 to 1 ngmL−1 )
in fluorimeter. Inset: shown
calibration curve between
different concentrations of
OTA and fluorescence
intensity (Zhang et al. 2013b)
mode, they attained the linear range from 0.73 μg L−1 to 12.50 μg L−1 and LOD of
0.39 μg L−1 (Liu et al. 2015a).
In optical resonance techniques, surface plasmon resonance spectroscopy(SPR) is
also valuable for the detection of low molecular weight molecules. In some studies, it
was observed that in SPR the signal obtained with the change of mycotoxin concentra-
tion was very low in respective of the antibody (Wang et al. 2009). The acoustic tech-
nique for label-free aptasensor was applied for the detection of OTA in real sample,
the thickness-shear mode (TSM) transducer was applied (Lamberti et al. 2011). The
other technique like Raman spectroscopy (SERS) was also reported (Galarreta et al.
2011). A Canada based Biotech company first time patented specific aptamers for
AFB1 and ZAE in 2010. Guo and coworkers reported real time for PCR-based detec-
tion of AFB1. The real-time PCR is highly sensitive, quantitative technique, which
can detect AFB1 at femtogram level. The biotinylated functionalized aptamer was
used and obtained the LOD of 25 fg mL−1 for AFB1 (Guo et al. 2014). The list of
developed aptasensor for mycotoxins is given in Table 14.4.
14.3.3 Molecular Imprinting Polymer
The synthetically produced polymer which possesses affinity toward the target
molecule or has a frame to fix the target molecule is called molecularly imprinted
polymers (MIPs). These are synthesized by polymerization of monomer around
the target molecule and link via cross-linker to make solid frame of a respective
target. On the complete formation of MIP there is a cavity in replacement of target
molecule, which provides complimentary outline and functionality to the MIPS.
However, very few works were reported on MIP-based detection of mycotoxins
(Piletsky et al. 2001). Yu and team reported OTA detection based on MIPs, they elec-
trochemically polymerized polypyrrole with chloride (PPy-Cl) on the gold surface
(Yu and Lai 2005). The metal ions in MIP enhanced the chelation property. Gao and
coworkers detected T-2 mycotoxin using Fe3+ introduced MIPs and attained selective
and sensitive MIP-based detection (Gao et al. 2014).
14.3.4 Enzymatic Inhibitor
Mycotoxins inhibit enzymatic activity which includes cholinesterase, acetyl-

cholinesterase (AChE), glucose oxidase, urease, etc. The AChE enzyme is sensi-
tive toward AFB1 detection; therefore, reversible SPR sensor was used for real-
time detection of AFB1(Puiu et al. 2012). Further, because of the inherent property
of AChE toward AFB1, researcher exploited AChE for the detection of AFB1in
real samples (Moscone et al. 2011; Ben Rejeb et al. 2009). The mycotoxins bound
non-covalently and reversibly with an enzyme (Stepurska et al. 2015). Among the
mycotoxins, AFB1 has maximum sensitivity (Soldatkin et al. 2013).
Table 14.4 The list of developed label-free and labeled aptasensor for mycotoxins
300
Sl. No. Matrix Electrode Type of Aptamer Mode of detection Label Technique
1 Magnetic Beads – Aptamer Competitive ALP DPV
2 Paramagnetic microparticle beads SPCE Aptamer Competitive HRP DPV
3 SH-Apta Au Aptamer Competitive Fluoresceinaptamer Microfluidic channel
SERS
4 Magnetic beads-based DNA (ssDNA) aptamers DNA (ssDNA) aptamers Non-competitive – Fluorometer
5 6-mercapto-1-hexanol/captured probe (MCH)/(CP)/AuE Au Aptamer Competitive – Electrochemiluminescent
6 Carboxyfluorescein SWCNT Aptamer Competitive – Fluorescence
7 magnetic bead (MB). sensing DNA ABEI-labeled chemiluminescence,
carboxylic silica
nanoparticles
8 – – Competitive Au NP -DNA Electrochemical
9 – – Fluorescent
10 Magnetic beads SPCE Competitive Biotin-labeled OTA, Automated flow
ALP electrochemical aptasensor
11 Graphene oxide Competitive FAM
(carboxyfluorescein)
12 Graphene UCNPs FRET
13 Phi 29 DNA Magnetic bead Aptamer – QDs Electrochemical
14 Au NPs Au Aptamer – DNA 1–Au NPs Electrochemi-luminescence
DNA 2–ABEI
15 PGE/DNA PGE fsDNA – Label-free EIS
16 OTA chemisorbed on Au – Aptamer – – EIS
4 sequences
with one Dimer
17 SA–Au NPs@Cd-MOF-74/S1/MCH/S2/Au NPs/Chit/GCE MOF/GCE cDNA – labeled DPV
(continued)
R. Chauhan et al.
18 OTA-APT:OTA-cAPT Aptamer RNA-based aptasensor – labeled OTA-RNA Fluorescence
19 GCE/modified/aptamer/AFB1/rGO GCE/rGO Apt – Label-free DPV, CV, EIS
20 Ab/BSA/Au-PANI/ITO ITO Ab – Label-free EIS
21 AuNP/GQD/GSPE GSPE direct Label-free LSV
22 GO modified PAA/AFB1/Apt GO/PAA Apt – – Amperometry
23 GO/AuNW/dsDNA/SPCE SPE/GO Aptamer – Label-free DPV
24 BSA/anti-OTA/PdNPs/CF PdNPs/CF Aptamer Non-competitive Label-free DPV
25 Aptamer Electrochemical
26 Tb3þ, structure-switching aptamer, and magnetic beads (MBs) Signal on Fluorescent
27 label-free for rapid and high-throughput detection of DNAzyme–aptamer conjugate colorimetric
OchratoxinA
28 Ferrocene-labeled DNA probe Au Aptamer – – DPV
29 Au NPs and DNA probe Gold ssHSDNA Non-competitive – Impedimetric
30 Biotin-Streptavidin – AFB1 Aptamer Non-competitive – Electrochemical
31 (Fe3 O4 /PANI) film Micro- IDE of Pt 21-mer aptamer Non-competitive – Electrochemical-based
aptasensor
32 GCE/rMoS2 -Au/APs/BSA/L-CPs GCE Aptamer – Label DPV
33 Zn-SnS2 /N-GQDs ITO Aptamer Photocurrent response
34 Au/PS/DNAapt/MB Au/PS Aptamer – Label-free CV, DPV

35 cystamine, PAMAM G4 dendrimers, and DNA Au DNA Competitive EIS, CV
36 aptamer/G-quadruplex DNAzyme and Fe3 O4 @oMWCNTs Fe3 O4 @MWCNTs Aptamer – Fluorescence Fluorescence spectra
37 SPCE/FGO/HMDA/Apt SPCE MB-tagged aptamer Competitive Labeled DPV
38 Aptamer and DNA – HRP mimicking DNAzyme – Label-free HCR, Gel electrophoresis
(continued)
301
302
39 SPCE/4-CP/act/Apt/OTA SPCE – Label-free EIS
40 PPy/Au Au Aptamer competitive Label-free SPR, EIS
41 PEI-MoS2 -MWCNTs and Tb Au ZEN – Label-free
Aptamer
42 SDA and DNA G-quadruplex – cDNA competitive Fluoresce AIE assay
43 Janus/COOH-GN/GCE GCE OTA aptamer Non-competitive Label-free DPV
44 Gold nanoparticles decorated MoS2 nanosheets Au MB-Aptamer – label-free
45 SPCE/4-CP/aptamer SPCE Aptamer – – EIS
46 HRP/BSA/AFB1 Microplates HRP-linked aptamer Competitive label Chemiluminescence
47 anti-M13-HRP/phage/BSA/anti- GCE mcAb Competitive label CV, EIS and SWV
OTA-McAb/Mal-PEG-NH2/L-Cysteine/MPA/AuNps/GCE.
48 Apt-PDMS-AuNPs-SPCE SPCE Aptamer – label-free EIS, CV
49 Au/cDNA Au dsDNA Label-free CV
50 MnO2/ACP MnO2 biotin-labeled OTA aptamer Competitive labeled colorimetric
51 MB-cDNA/apt-SiO2@CdTe and MB-cDNA/apt-SiO2@PbS SiO2 Aptamer – label SWV
52 Streptavidin/AFM1 aptamer – ssDNA – fluorescence RT-qPCR
53 dendritic-DNA/aptamer/AMNPs Amino modified magnetic DNA – Fluorescence probe PAPDI-HCR amplification
nanoparticles strategy
54 SPCE/PTH/IrO2NPs/aptamer/EA SPCE Aptamer Label-Free EIS
55 AFB1–QD–GO GO Aptamer Competitive Labeled PL spectroscopy
Quencher
56 LMOF-241 MOF – – Photoluminescence UV spectroscopy
57 Exo I enzyme/SYBR Gold/aptamer SYBRAu fluorescence – label-free fluorescence measurement
(continued)
R. Chauhan et al.
58 TS-AuNP-c-DNA Au cDNA Non-Competitive label-free SWV
59 SPCE/4-CP/Apt/AFB1 SPCE Aptamer Non-Competitive label-free EIS
60 CAs-cDNA/apt/AuE Au Aptamer Non-Competitive label-free DPV
61 3DOM MoS2 − AuNPs Au H1/HRP/AuNPs-SiO@Fe3 O4 – – DPV
62 poly-NR/GCE GCE Aptamer – mNR EIS and DC voltammetry
63 pβ-CD/AuNPs/GC GCE Fc-cDNA – label DPV and EIS
64 Anti-AFB1/MB/Au Au MB-labeled aptamer Competitive label SWV
65 SiO2 @PbS/Fe3 O4 @SiO2 /CdTe CdTe and PbS QDs DNA – label SWV
66 mSiO2 @Au-DNA1/apt rGO/AuNP/CdTe/DNA2 – – label-free SWV
67 RGO/MoS2 /PANI@AuNPs/Apt Au Aptamer – label-free DPV
68 OTA-aptamer-ZnPPIX ZnPPIX Fluorescence – label-free Fluorescence spectra
69 Apt-modified SPGE with CS-modified AuNPs SPGE cDNA Non-Competitive – DPV
70 Fe3O4@Au-Apt Fe3O4@Au Aptamer – label-free EIS
71 2D–MoS2 NPs Au cDNA Competitive label-free chronoamperometry
72 Au@Au- Ag NNSs coupled Fe3O4 MNPs Au@Au–Ag NNSs cDNA – – Surface-enhanced Raman
73 SPCE/PAM/PA/PDA/Apt SPCE Aptamer – – DPV
74 SPCE/Apt SPE cApt – label DPV
75 DNA-AuNPs-HRP nanoprobes GCE cDNA – – DPV

76 AgNPs/PDANS Au cDNA competitive label LSV
77 AuNP@CuCoPBA Au Aptamer Label-free EIS
78 Apt2-AuNRs-Fc/Apt1-AuNRs-Th/cDNA/Au Au Y-shaped DNA and AuNRs – – DPV
(continued)
303
304
79 PANI-aptamer-PANI/GCE GCE Aptamer Non-competitive Label-free CV, EIS
80 Apt/PtNP/MIL–101(Fe)/GCE GCE Aptamer – label–free EIS
Sl. No. Toxin Response range LOD Sensitivity Relative Standard Deviation Coefficient of variation (CV) in Reference
(RSD)in % %
1 OTA – 0.11 ng mL−1 – – – Barthelmebs et al. (2011)
2 OTA 0.78–8.74 ng mL−1 0.07 ng mL−1 – – – (Bonel et al. (2011)
3 OTA 0.05–4 μM. – – Galarreta et al. (2013)
4 ZON 3.14 × 10−9 to 3.14 × 10−5 M 7.85 × 10−10 M. – – – Chen et al. (2013)
5 OTA 0.2 pg mL−1 to 1 ng mL−1 75 fg mL−1 9.1, 6.0 – Chen et al. (2014)
6 OTA 25 nM to 200 nM 24.1 nM – – Guo et al. (2011)
7 OTA 1.0 × 10−12 to 5.0 × 10−8 3.0 × 10−13 g mL−1 Hun et al. (2013)
g mL−1
8 OTA 0.1–20 ng mL−1 . 30 pg mL−1 , – 10 Kuang et al. (2010)
9 OTA 1 ng mL−1 –100 mg mL−1 1 ng mL−1 – – Lv et al. (2014)
10 OTA 1 μL to 3μL 0.05 μgL−1 4–5 5 Rhouati et al. (2013)
11 OTA 2 μM to 35 μM. 21.8 nM – – Sheng et al. (2011)
(continued)
R. Chauhan et al.
(RSD)in % %
12 OTA, FB1 0.05–100 ng·mL−1 for OTA and OTA 0.02 ng·mL−1 and FB1 4.74, – Wu et al. (2012b)
0.1–500 ng·mL−1 for FB1 0.1 ng·mL−1 . 5.86
13 OTA 0.5 pg mL−1 –10 ng mL−1 0.2 pg mL−1 – – – Tong et al. (2012)
14 OTA 0.02–3 ng mL−1 0.007 ng mL−1 – 3.8 – Wang et al. (2010b)
15 FB1 2.5 μgmL−1 to 12.5 gmL−1 3.69 ng mL−1 – – – Kesici and Erdem (2019)
16 OTA 0.1–100 nM 0.12–0.4 nM – 0.2–3.5 – Castillo et al. (2012)
17 OTA 0.05–100 ng mL−1 10 pg mL−1 – 1.4–7.4 – Li et al. (2018)
18 OTA 0.4–20 ng mL−1 0.08 ng mL−1 – – – Wu et al. (2019a)
19 AFB1 0.5 nM-4 μM 0.07 nM – 2.9 – Beheshti-Marnani et al.
(2019)
20 AFB1 0.1–100 ng mL−1 0.05 ng mL−1 – 3.6 to 5.3 – Yagati et al. (2018)
21 AFB1 0.10–100 nmol L−1 0.47 nmolL−1 92.4 A mol L−1 3.1 – Gevaerd et al. (2020)
22 AFB1 1–20 ng mL−1 0.13 ng mL−1 – – – Mo et al. (2018)
23 AFB1 5–750 pM 1.4 pM – 5.2 – Nodoushan et al. (2019)
24 OTA 0.5 − 20 ng mL−1 0.096 ng mL−1 – 5.6 – Kunene et al. (2020)
25 OTA 0.1pgmL−1 –1000pgmL−1 0.095 pg mL−1 – 4.9 – Wu et al. (2012a)
26 OTA 20 pg mL−1 OTA with high – 1.67 – Zhang et al. (2013b)
specificity
27 OTA upto30nM 4 nM – – – Yang et al. (2013a)

28 OTA 5pgmL−1 –10 ng mL−1 1 pg mL−1 – – – Tong et al. (2011)
(continued)
305
306
(RSD)in % %
29 AFM 1 1–14 ng mL−1 – – – 6.2 Dinckaya et al. (2011)
30 AFB 1 5.0 × 10−5 to 5.0 ng mL−1 25 fg mL−1 – 2 – Guo et al. (2014)
31 AFM 1 6–60 ngL−1 1.98 ngL−1 . – 5 – Nguyen et al. (2013)
32 ZEN and FB1 1 × 10−3 –10 ng mL−1 (ZEN) 5 × 10−4 ng mL−1 – 1.6–3.8 – Han et al. (2020)
1 × 10−3 –1 × 102 ng mL−1
(FB1)
33 AFB1 0.01 ng mL−1 –20 ng mL−1 3 pg mL−1 – 2.0 Kong et al. (2019)
34 OTA 0.1–300 ng mL−1 78.3 pg mL−1 – – – Mazaafrianto et al. (2018)
35 AFB 1 0.1–10 nM 0.40 ± 0.03 nM – 1.73 – Castillo et al. (2015)
36 AFB1 0.5–15 ng mL−1 0.02 ng mL−1 – 2.8–4.5 – Wang et al. (2019c)
37 AFB 1 0.05– 0.05 ng mL−1 – 3.7 – Goud et al. (2017)
6.0 ng mL−1
38 OTA 0.01–0.32 nM 0.01 nM – – – Wang et al. (2015)
39 Ochratoxin A 0.15–2.5 ng mL−1 0.15 ng mL−1 4.8 Mishra et al. (2015)
40 OTA up to 5 mgL−1 2 mgL−1 0.1 k/ng – – Mejri-Omrani et al. (2016)
41 zearalenone 5.0 × 10−4 ~ 50 ng mL−1 1.7 × 10−4 – 3.23 and 9.96 – Ma et al. (2019)
42 patulin 0.001–100 ng mL−1 0.042 pg mL−1 1.1–4.4 – Zhang et al. (2020)
43 OTA 1 × 10−5 nM to 10 nM 3.3 × 10−3 pM – – – Yang et al. (2019)
(continued)
R. Chauhan et al.
(RSD)in % %
44 OTA 0.1–50.0 nM 0.06 nM – 6.5 – Wang et al. (2018)
45 AFM1 2–150 ngL−1 1.15 ngL−1 – – – Istamboulié et al. (2016)
46 AFB1 0.01–0.2 nM 0.01 nM – – – Sun and Zhao (2018)
47 OTA 7.17–548.76 fgmL−1 2.04 fgmL−1 – – – Hou et al. (2019)
48 fumonisin B1 10 pg mL−1 to 50 ng mL−1 3.4 pg mL−1 4.9 – Lu et al. (2016)
49 OTA 0.2–500 nM 0.073 nM 2.8 – Zhu et al. (2018)
50 OTA 1.25–250 nM 0.069 nM – – – Tian et al. (2019)
51 OTA and FB1 0.05 − 50 ng mL−1 (FB1) and 20 pg mL (FB1) and 5 pg/mL Less than 3 pg/mL 6.1 for OTA and 5.3 for FB1 – Wang et al. (2017)
0.01 − 10 ng mL−1 (OTA) (OTA)
52 OTA 1.0 × 10−4 –2 × 10−2 nM 0.03 ngL−1 – 5 – Guo et al. (2016)
53 OTA 1.0–20 pM 0.10 pM – 2.9–4.3 – Wang et al. (2016)
54 OTA 0.01 − 100 nM 14 pM – 17 – Rivas et al. (2015)
55 AFB1 6 nM–600 mM 4 ngL−1 – – – Kumar et al. (2018)
56 AFB1 1–16.7 nM 46 ppb – – – Hu et al. (2015)
57 OTA 20–500 nM 16.5 nM – 2.5 – Lv et al. (2017)
58 AFB1 0.001 ppt to 100 ppt 0.6 × 10−4 ppt – – – Zhang et al. (2016)
59 AFB1 0.125–16.0 ng mL−1 0.125 ng mL−1 0.271 ng mL−1 2.72–4.83 0.25 ng mL−1 Yugender Goud et al. (2016)
60 OTA 1.0 × 10−4 –500 ng mL−1 1.0 × 10−4 ng mL−1 – 6.7 – Wei and Zhang (2018)
61 AFB1 0.1 fgmL−1 −0.1 μgmL−1 0.01 fgmL−1 – 4.2–6.5 – Peng et al. (2018)
(continued)
307
308
(RSD)in % %
62 AFM1 5–120 ngL−1 0.5–1 ngL−1 – 2.3 – Smolko et al. (2018)
63 AFB1 0.1 pg mL−1 –10 ng mL−1 0.0491 pg mL−1 – lower than 11.51% – Wu et al. (2019b)
64 AFB1 8 nM to 4 μM 2 nM – less than 4 – Wang et al. (2019a)
65 AFB1 5 pg mL−1 –50 ng mL−1 4.5 pg mL−1 – 4.7 – Wang et al. (2019b)
66 OTA 0.2 pg mL−1−4 ng mL−1 0.07 pg mL−1 – – – Hao et al. (2016)
67 AFB1 0.01–1.0 fgmL−1 0.002 fgmL−1 – 5.3 – Geleta et al. (2018a)
68 OTA 0.1 ~ 1.2 nM 0.03 nM – 6.1 – Liu et al. (2018)
69 AFM1 2–600 ngL−1 0.9 ngL−1 – 3.9–8.1 – Jalalian et al. (2018)
70 AFB1 20 pg mL−1−5 0 ng mL−1 15 pg mL−1 – 6.2 – Wang et al. (2018)
71 OTA 0.0005–10 ng mL−1 0.23 pg mL−1 – 8.8–9.0 – Tang et al. (2018a)
72 OTA 0.01–50 ng mL−1 0.004 ng mL−1 – – – Shao et al. (2018)
73 AFB2 0.1 pg mL−1 to 100 ng mL−1 0.10 pg mL−1 3.9 – Geleta et al. (2018b)
74 OTA 0.15–5 ng mL−1 0.07 ng mL−1 – 3.7 – Mishra et al. (2016)
75 AFB1 10−3 to 200 ng mL−1 3.3 × 10 − 4 ng mL−1 – 3.07 – Hui et al. (2020)
76 1.0 × 10−6 –1.0 × 10−2 μM 0.57 pM – 2.9 – Zhang et al. (2019)
77 OTA 50 fgmL−1 to 10 ng mL−1 5.2 fg mL−1 – <4.8 – Gu et al. (2019)
78 OTA and FB1 1 × 10−3 –100 ng mL−1 0.47 × 10−3 ng mL−1 (OTA) – 6.17 and 5.38 Wei et al. (2020)
0.26 × 10−3 ng mL−1 (FB1)
79 AFM1 3–90 ngL−1 1–5 ngL−1 – – – Kulikova et al. (2019)
(continued)
R. Chauhan et al.
(RSD)in % %
80 AFM1 1.0 × 10−2 to 80.0 ng mL−1 2.0 × 10 − 3 ng mL−1 – 6.4 – Jahangiri-Dehaghani et al.
(2020)
309
14.3.5 Mimotope for Mycotoxin
The mycotoxins conjugates need pure mycotoxin, which is highly risky to the manu-
facturer due to its carcinogenic nature, and these are hazardous to the environment,
due to its persistence at high temperature. Therefore, for the environment and labo-
ratory safety purpose, it is important to avoid the use of mycotoxins and choose
the alternate. The formation of a nontoxic immunochemical compound, i.e., mimo-
tope has advantages in the replacement of mycotoxin conjugate. The mimotopes are
synthesized from peptides with the respective sequences of the peptide that have a
good affinity toward mycotoxin. The peptide sequence selected from random phage-
displayed peptide libraries. The mimotopes of OTA, ZEA, and DON were developed
and applied for their respective mycotoxin’s detection (Bazin et al. 2013; Lai et al.
2009; He et al. 2013)
14.3.6 Nanobody (VHHs Antibody)
All types of antibodies and labeled antibodies have their distinctive possessions and
affinity toward the target (mycotoxins) on biosensor’s surface (Regiart et al. 2018;
Solanki et al. 2017; Karczmarczyk et al. 2017). The whole process of synthesis of
antibodies and their conjugates with labels is expensive, time taking and depends on
vaccination. Sometimes the enzyme chemically linked with antibodies which may
cause false results because of instability and arbitrarily link in between the molecules.
The aforesaid problems are not inconsequential, therefore, researchers have worked
on synthetically engineered antibodies like (recombinant antibody, single domain
antibody or nanobodies, and single chain fragment variable (scFv), to solve these
problems. The nanobodies are designed from a variable part of the heavy chain of
antibodies. The llamas, camels, and dromedaries’ animals’ antibodies have been used
for the fragment and the cartilaginous fishes VNAR fragment was used (De Groof
et al. 2019; De Meyer et al. 2014). Nbs have discrete advantages over the conventional
antibodies, as they are high in yield and stability, easy to modify genetically, and
refolding tendency (Steeland et al. 2016; Könning et al. 2017). Liu and their group
have synthesized Nbs for the detection of OTA. The Nbs were derived from the alpaca
VHH library and were analyzed OTA from PCR and ELISA methods (Liu et al. 2017).
A one-step qualitative detection of OTA using Nbs has been conducted. Alkaline
phosphatase linked Nbs were used on polyvinylidene fluoride (PVDF) membrane
immunoassay. And this immunoassay reached up to the LOD of 0.3125 ng/mL−1
in 6 min for OTA in cereals (Tang et al. 2018b). Pan et al. (2018) fabricated direct
immunosensor for AFB1, the signal received by the interaction of Nbs and carbon
materials. The proposed immunosensor showed LOD of 3.3 pg mL−1 (S/N = 3).
The immunosensor with Nbs was 10-fold more sensitive for the detection of AFB1
than the immunosensor with complete monoclonal antibody. Though the selection
of Nbs which will interact with mycotoxins is very complexing, because each has
their specific epitopes from the phage display Nbs range, and this is one of the
disadvantages of wide applications of Nbs in mycotoxin.
14.4 Conclusions
The concept of the biosensor is demonstrating a compatible alternative to replace

previous techniques which are tedious, costly, and time taking. In this regard there
is an immense revolution in field of transducers (electrochemical, optical, and
cantilever), nanomaterials synthesis (quantum dots, metal oxides, and metal nanopar-
ticles), and in the selection of receptors (peptides, MIP, nucleotides, nanobody, etc.).
These changes lead to enhancement in the linear range, shelf life, and LOD for the
developed biosensor. The developed sensor can apply to detect the contaminated
food sample. However, false-positive results observed due to the following reasons
(i) cross-reactions between the receptors and mimic molecules (ii) nonspecific reac-
tion on the electrode surface (iii) physical parameters temperature, pH, viscosity, and
ionic strength, etc. Though everyday research steps on the pathway of new science
and technology, innovate new materials and techniques, still they are far away from
the practical applicability of these techniques in daily life. Therefore, there is a need
of enormous advancements in mycotoxin detection available to the public and field
sector.
14.5 Future Trends
The invention of nanotechnology is boon to the science and technology field. The
researchers are paying attention to the advancement in technology for the develop-
ment of mycotoxin detection devices. The success rate of the development of a smart,
fast, reliable sensing device is very slow. The mycotoxin detection device was not
developed for the field trial. The contamination of mycotoxins in food is not uniform
and therefore it needs a huge number of samples for detection, researchers follow the
set levels established by the European Commission and other international agencies.
The conventional techniques (like PCR, chromatography, and ELISA techniques)
are time consuming, costly, skilled technicians required for the detection of myco-
toxins. The biosensor for mycotoxin detection is promising tool though there are
some challenges affecting it, can be addressed judiciously such as (i) replacement of
monoclonal antibody to the synthetically prepared receptors like aptamers, MIP, etc.
(ii) label-free system adaptation for detection (iii) incorporation of contemporary
technologies, e.g., microfluidic (iv) finally the adaptation of innovation to practical
application. Future research can be detecting multi-analyte on a chip platform at a
reasonable cost.
Acknowledgments Prof. Tinku Basu, Director, Centre for Nanomedicine is thankful to Funding
Agency, BIRAC for financial support (BT/AIR0416/PACE-I 4/18). R. Chauhan acknowledges the
postdoctoral fellowship from the University of KwaZulu Natal, Westville, South Africa.
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Chapter 15
Biosensors for Fruit Quality Monitoring
Vinita Hooda, Nidhi Chauhan, and Shringika Soni
Abstract The growing interest in development of reliable and sensitive detection

methods for fruit quality and safety has been observed in scientific communities
and food industry. For better management of existing analytical technologies and
to bring next-generation sensing platform in realistic market, updated and accurate
information is necessary for proper planning and decision-making. This chapter
describes the current status of chemical and biological sensors available for fruit
quality monitoring and smart packaging. These sensing designs should have features
for measuring adulterants, freshness, allergens, microorganisms, and toxicants. On
the other hand potential applications of smart packaging and e-nose are also explained
in fruit quality and spoilage monitoring in logistic applications. The last section of the
chapter also summarized commercially available sensing platform for fruit quality
assessment and opportunities for future research discussed in brief.
Keywords Fruit quality · Sensors · Indicator sensing · RFID tag · Smart and
intelligent packaging · Nanotechnology
15.1 Introduction
According to literature, approximately one-third of food produced across the globe is

never consumed and from the last three decades quality and safety of food products
are compromised (Huang et al. 2008; Jedermann et al. 2014). The World Health
Organization (WHO) based report also evaluated contaminated food induced 600
million cases of illness and 420,000 deaths each year (WHO 2017). In addition,
food poisoning, diarrhea, and various infections are major human diseases caused
by consuming spoiled fruit products. The major share of these losses is linked with
V. Hooda (B)
Department of Botany, Maharshi Dayanand University, Rohtak, Haryana, India
N. Chauhan · S. Soni
326 V. Hooda et al.
improper handling and mismanagement during pre- and post-harvesting operations.

Besides, economic development and improvement in living standards increased fresh
and nutritious fruit consumption and also the availability of fruits in all seasons.
Yet fruit damage during transportation is an important factor of concern, because
of mismanagement of controlled environmental variables and longer time taken in
transportation.
The fruit quality measurement is defined by different variables such as, fruit
health, nutritional values, consumer standards, stability, and various other country-
wise factors (Yousefi et al. 2019), though consumers’ primary perception of packaged
fruit products depends upon visual appeal, aroma, flavor, ripeness, and date of pack-
aging mentioned on the label. Conventionally spectroscopy, chromatography, and
culture-based laboratory methods were applied in agricultural sector for qualitative
evaluation of food products. Imaging and spectroscopy are important conventional
optical technology where they provide spatial, chemical, and physical information
about fruit products (Wang et al. 2015). But these techniques are time-consuming,
expensive, laboratory-based, and requires heavy equipment and expertise for result
analysis. Therefore, we require a rapid and reliable on-field and real-time monitoring
system of food products.
In recent years, several new portable and remote wireless sensing technologies
have come into the worldwide market. On-field sensors monitor crops, soil, and water
samples for potential disease or pesticide contaminations, whereas smart packaging
and wireless sensor systems show satisfactory solutions for real-time monitoring
in food. On-field electrochemical or optical sensors are direct detection method to
capture analyte and know the current status of food product, but smart packaging
and wireless sensing applied for real-time monitoring on the basis of changes in pH,
temperature, moisture, ripening, and sugar content.
15.2 Biosensor: A Miniaturized Tool for Detection
The term “Biosensor” is a very powerful and innovative analytical platform that
involves biological sensing element as a recognition template with a broad range of
applications in diagnosis, food safety, healthcare, defense, biomedicine, and environ-
mental monitoring (Chauhan et al. 2020; Gu and Liu 2020). It works on the principle
of biological and chemical reactions and output signals are directly proportional to
the concentration of analyte (Chauhan et al. 2019; Gu and Liu 2020). Their purpose
is to provide quick, reliable, real time, and accurate detection of analyte of interro-
gation, yet ideally biosensors are capable of continuous and reversible monitoring.
After the first enzyme-based biosensor by Clark in 60’s, huge progress has been made
in sensing technologies by development in biorecognition of elements and artificial
transducers (Liu et al. 2019; Coulet and Blum 2019). The most typical components of
biosensors include suitable receptors such as enzymes, antibodies, oligonucleotides,
cells, nanoparticles, and imprinting polymers along with pH, temperature, change
in electrical and optical signals-based physiochemical transducers (Mehrotra 2016;
15 Biosensors for Fruit Quality Monitoring 327
Fig. 15.1 Components and signaling of electrochemical and optical sensors
Davis and Altintas 2018). The basic configurations of biosensors are explained in
Fig. 15.1. On the basis of biorecognition elements, the biosensors are classified into
immunosensors, DNA or aptamer sensors, microbial sensors, enzymatic sensors, and
colorimetric sensors. These sensors have benefits of higher selectivity, sensitivity,
easy portability, quick response time, and no sample preprocessing requirements
(Zhu et al. 2019; Cui et al. 2019; Jain et al. 2020). Further improvement and genera-
tion of next-generation sensing technology, nanotechnology-based sensing platform
recently has been reported for application in analytical chemistry.
Recently electronic nose (e-nose) and radio frequency-based sensing techniques
have been used in most of the agriculture sector, where nanomaterials assisted
sensing improved detection capacity of biosensor. The e-nose is artificial olfactory
sensor technology, which is a combination of biochemistry, electronics, and artificial
intelligence to use in numerous applications (Burlachenko et al. 2016; Kodogiannis
2017; Wasilewski et al. 2019). The e-nose is basically a gas multisensor array that is
designed to measure gas mixture as a whole instead of targeting individual chemical
species. The sensing array-like electrochemical and optical sensors, data acquisition
system as signal processing unit along with multivariate data processing tools, and a
pattern recognition unit to identify and classify target on the basis of specific odors
are the major components of e-nose, as shown in Fig. 15.2 (Karakaya et al. 2020;
Ramgir 2013). Volatile chemicals originated from adulteration, microbial contamina-
tion, maturity, and other quality parameters of post-harvested or packaged food prod-
ucts, reacts with biorecognition molecules on e-nose multisensory array and pattern
recognition unit identify the chemicals according to their database (Gliszczyńska-
Świgło and Chmielewski 2017; Karakaya et al. 2020; Shi et al. 2018). Recently,
nanoparticles namely, tin oxide, carbon nanotubes, silicon and titanium nanowires,
and many more, were reported in e-nose based sensing due to high surface to volume
ratio, microelectronic processing, and sensitivity toward target molecule (Chauhan
et al. 2020; Soni et al. 2016).
328 V. Hooda et al.
Fig. 15.2 Working mechanism of e-nose
15.3 Recent Trends in Fruit Quality Monitoring
In the last decades, farmers and manufacturers faced huge economical losses due to
inaccessibility of cultivable land, depleting water resources, use of excessive pesti-
cides, pathogen infection, and post-market commercialization in agricultural indus-
tries. For assurance of good quality agricultural output, on-site detection is very much
required for quality control in the agricultural food sector. Due to increasing number
of pollutants and hazardous chemicals in soil and water resources, scientists are
looking forward to develop new methods in monitoring and controlling the quality
of the food. Therefore, the diagnostic industry is flourishing in the agricultural sector
after playing an important role in medical diagnosis. In recent years, countless sensing
platforms have been developed for detection of fruit ripening, contamination, and
real-time monitoring for packaged fruit quality (Satpute and Jagdale 2016; Mustafa
and Andreescu 2018). Markets-based sensing platforms for agro-food-diagnostic are
generally multianalyzer, bench-top portable equipment, and single-time use dispos-
able sensors (Tothill 2001). In this chapter, we will be elaborating on emerging
sensing technologies and recent trends in biosensor development for fruit quality
monitoring.
15.3.1 Gas Detector for Fruit Quality Assessment
To avoid fruit spoilage during transport and maintain the quality in the market,
fruit products are shipped in preservation chambers to delay the ripening process.
These transported fruits are exposed to 2–3% oxygen, ~ 0 °C temperature, high
humidity, and high CO2 level (Jochum et al. 2016). Also, the application of ethylene
(C2 H4 ) in fruit ripening increases respiration rise, change in texture, aroma, color,
and flavor along with autocatalytic production of C2 H2 (Pech et al. 2018; Gao et al.
2020). Therefore, C2 H4 concentration should be kept as low as possible. Additionally,
ammonia (NH3 ) monitoring is of particular interest because it is the commonly used
coolant in fruit transportation yet leakage of NH3 can endanger the ozone layer, effect
human health at a bad scale, and spoil stored fruits (Jochum et al. 2016; Tzortzakis
and Chrysargyris 2017; Yang and Jiao 2017).
Since last decades, lots of sensing platforms and electronic noses have been
designed for C2 H2 detection. Sklorz and colleagues designed an intelligent container
for C2 H4 measurement in fruit logistic applications (Sklorz et al. 2012a). They exhib-
ited selective detection of C2 H4 gas detection via miniaturized gas chromatograph
(μGC) coupled with miniaturized preconcentrator device (μPCD) with absorbing
material, Carbosieve S-II. The carbosieve S-II absorbed C2 H4 and 16 times higher
peak in chromatogram was achieved as compared to empty μPCD. Thus, this inno-
vative technology could be very beneficial for fruit logistic applications. Similarly,
another preconcentrator microfluidic sensing platform has been previously devel-
oped by Germany-based research team, where they used Carboxen 1000 fabricated
microchannel to detect C2 H4 and 100-fold enhancement was achieved (Dow et al.
2011). The Carbosieve S-II and Carboxen 1000 are carbon molecular sieve absorbent
materials with large surface area and small pore size therefore ideal for trapping small
and volatile organics.
A recent study reported infrared (IR) thermal emission-based sensing platform for
C2 H4 detection in banana fruit samples (Kathirvelan and Vijayaraghavan 2017). This
efficient alternate approach, combined IR thermal emitter with tunable wavelength
with silicon temperature detector and enclosed the system in a gas test cell. When
released C2 H4 introduced in the test cell, the analyte absorbed IR radiation and
decreased the temperature of the system. Though this method has C2 H4 detectability
of 3.3% during fruit ripening process that was higher than previously developed IR-
based thermopile, 0.2% (Sklorz et al. 2012b) and fiber optic sensing, 0.25% (Wei et al.
2015) methods. But due to limitation of silicon temperature detector, the sensitivity
was achieved at 5 ppm, while sensing ppb range is desirable for higher accuracy in
long-term preserved fruit samples.
For further advancement in fruit ripening detection, Agarwal and colleagues have
used dielectric property of SnO2 nanoparticles (NPs) for the detection of C2 H4 in
fruit samples (Agarwal et al. 2012). The n-type SnO2 with Pd/Pt NPs exhibited
higher conductivity in the presence of target and change in depletion region also
presented by reaction between O− and Pd /Pt nanoparticle (NPs). The developed
sensing platform showed decrease in capacitance with increasing C2 H4 concentra-
tion from 0 to 100 ppm, which has the potential for usage in RFID tags for fruit
quality monitoring. Similarly, USA-based research team also used highly resistive
copper (I) complex 1 conjugated single-walled carbon nanotubes (SWNTs) chemo-
resistive sensing of C2 H4 in banana, orange, apple, pear, and avocado fruit samples
(Esser et al. 2012). When 1-SWNT fabricated field-effect transistor (FET) exposed to
target C2 H4 gas, the target bound to 1-SWNT in associative fashion without breaking
up compound 1 with SWNT and a linear change in resistivity was observed in the
range of 0.5–50 ppm C2 H4 . Similarly, a recent study utilized MoS2 , SWNTs, and
Cu(I)–tris (mercaptoimidazolyl) borate complexes (Cu-Tm) fabricated sensing plat-
form for direct detection of C2 H4 in fruit samples (Chen, et al. 2020). The conduc-
tometric property of MoS2 and Cu(I) compounds as C2 H4 receptors followed the
similar concept as in the previous study, but in the presence of analyte charge transfer
330 V. Hooda et al.
coupling between MoS2 and Cu-TM lowered the conductance and output recorded
in the range of 0.1–1.0 ppm. In continuation of nanotechnology application in C2 H4
detection in fruit ripening, Kathirvelan and colleagues have also designed TiO2 -
WO3 nanocomposite-based chemo-resistive sensor which showed detectability of
46.2% in 200 ppm C2 H4 and reported limit of detection (LOD) of 8 ppm in real fruit
samples (Kathirvelan et al. 2017). Similarly, AgBF4 /polyvinylpyrrolidone polymer
composite-based quartz crystal microbalance (QCM) (Tolentino et al. 2018), ZnO
nanosheets (Wang et al. 2019), and MnO2 NPs (Bigiani et al., 2020)-based sensing
platforms are recently developed for effective quantification of C2 H4 released during
fruit ripening.
In another study, poly(3-hexylthiophene) (P3HT) and polystyrene (PS) fabricated
organic field-effect transistor (OFET) were developed for ammonia (NH3 ) detection
(Han et al. 2016). The dual-sensing possibility of the platform worked on the basis
of reduced subthreshold slope and negative threshold voltage due to positivity of
trapped NH3 . On the other hand, when lone pair of diffused NH3 interacted with
P3HT/PS layer there was decrement in drain current and reduced net positive charge
was observed, therefore, decrement in current was obtained with increasing NH3
concentration in the range of 0–50 ppm. This poly/insulator blended OFET device
stable for 40 days, thus they can be utilized in transportation and logistics appli-
cations. Similarly, USA-based research team recently developed N-(tert-Butoxy-
carbonyloxy)-phthalimide and palladium particles blended P3HT fabricated OFET
sensor for direct detection of C2 H4 (Besar et al. 2017). When analyte exposed to
sensing platform palladium particles acted as C2 H4 receptor and detection principle
was similar as for ammonia in the previous study with sensitivity of 25 ppm V/V.
The catalytic property of Pd was also used by Li and colleagues where they used
Pd NPs and rGO modified porous α-Fe2 O3 in C2 H4 detection in fruit samples (Li
et al. 2019). The Pd NPs assisted catalysis of O2 molecule not only formed deple-
tion layer onto α-Fe2 O3 to increase air resistance, it also promoted C = C bond
cleavage to allow interaction between C2 H4 and absorbed O− to produce ethylene
oxide and free electrons. These free electrons caused resistance drop while rGO acted
as an excellent electrical site for rapid transmission of electrons between O− and α-
Fe2 O3 . Therefore, sensitivity of the developed platform significantly increased with
increasing C2 H4 concentration in the range of 100–1000 ppm and LOD of 10 ppb
were reported in 18 s.
In commercially developed portable electronic nose (PEN 2) e-nose-based sensing
technology for C2 H4 detection, a principal component analysis (PCA) was performed
on sensor data (Benedetti et al. 2008). This system discriminated quality of peach
fruit on the basis of increasing days of shelf life and avoided aroma-based fruit
quality monitoring, which means consistent low presence of C2 H4 for a long time
period in stored fruit. Similarly, China-based research team also designed FOX 4000
e-nose-based sensing system to discriminate a variety of peaches on the basis of
respiration rate (CO2 ) and C2 H4 (Su et al. 2017). Whereas India-based scientists
reported enhancement in conductivity when ripped fruits release C2 H4 and reacted
with GO (Mallick and Das 2017; Mallick et al. 2018), thus low cost and easily
synthesized GO can be used in smart packaging or e-nose for fruit monitoring. In
another study, nano zeolite-molybdate was also used as a pH indicator for avocado
ripening where C2 H4 reacted with nanosystem and change in color from yellow to
blue was stable till 10 days in storage condition (Putri et al. 2019).
In continuation of this, scientists incorporated more and more artificial intelli-
gence (AI) in prediction modeling and pattern recognition. In this scenario, Du and
colleagues designed metal oxides-based gas sensors to predict different ripening
stages of kiwifruit (Du et al. 2019). They incorporated different chemometrics
pattern recognition algorithms, such as linear discriminant analysis (LDA) to recog-
nize ripeness of kiwifruit at different time points, whereas compared partial least
squares regression (PLSR), random forest (RF), and support vector machine (SVM)
determined complete ripeness, firmness, and soluble solids content. The regres-
sion outcome supported RF-based algorithm for predicting ripeness indexes in
post-harvested kiwifruits. In another study, China-based research team used similar
chemometrics algorithms to predict the quality of peaches where they studied the
after-damage quality of packaged peaches and confirmed accuracy of 93.33% after
24 h of fruit spoilage (Yang et al. 2020).
15.3.2 pH-Based Detection in Fruit Quality Measurement
The pH and titratable acidity (TA) are considered as important quality attributes for
fruit processing and ripening, where pH gradually increases with increasing fruit
ripening and TA is significantly high at the beginning of fruit ripening (Anthon et al.
2011). This acidity of fruit is maintained by oligosaccharides such as glucose and
fructose and organic acids, namely, citrate, malate, and tartaric acids (Batista-Silva
et al. 2018). Malic acid is dominant in apple, pear, and loquat whereas citric acid is
generally found in citrus fruits such as pineapple (Etienne et al. 2013). The metabolic
tri-citric acid (TCA) cycle intermediates play important role in fruit ripening and
changes organic acids or oligosaccharides level in fruits, thus enzyme-based sensing
platform can be used to determine the qualitative changes in fruit product.
In recent years, several dye and optical nanoparticles-based colorimetric sensing
technology came into market for fruit quality assessment. Jawaheer and colleagues
have developed an enzymatic electrochemical sensing platform for detection of β-
D-glucose, ascorbic acid, total D-glucose, and sucrose in fruit samples (Jawaheer
et al. 2003). They further used cellulose acetate (CA) membrane onto developed
enzymatic sensor where enzymatic reaction generates H2 O2 and CA improved the
sensitivity by avoiding binding of other interfering agents of sensing platform. On the
other hand, Malaysia-based research team developed optical fiber sensor for quality
detection of Averrhoa carambola (star fruit) on the basis of pH and firmness (Omar
and Matjafri 2013). At the peak of 635 nm, the optical fiber red system (OF-RS)
obtained excellent accuracy in pH and firmness determination. For further improve-
ment in sensing platform, a paper-based microfluidic device (μPAD) was designed
for glucose determination in fruit samples (Li et al. 2017). When horseradish perox-
idase/glucose oxidase fabricated microchannel were exposed to fruits pulp samples,
332 V. Hooda et al.
the color of the working electrode gradually changed to dark brown due to HRP
assisted metabolic reaction which resulted into I2 (brown color). To incorporate the
nanotechnology in fruit quality sensing system, date fruit was exposed to polyvinyl
alcohol (PVA) matrix-based nanomat in a study, where anthocyanin (AC) induced
pH changes was determined by variable colorimetric images and good quality fruit
further consumed (Maftoonazad and Ramaswamy 2019).
For the pH-based e-nose or smart packaging development, several studies have
been performed. In this direction Kantor and colleagues have designed an electronic
tongue (ET) for suitable apricot measurement (Kantor et al. 2008). They studied the
effect of 1-methylcyclopropene (1-MCP) on stored apricot ripening, which confirmed
consistent pH and increment in glucose content throughout the four weeks of storage
at 1 °C and 95% relative humidity. Thus, 1-MCP treated apricot showed reduced
ripening and sensing can be further used in long-term fruit storage. On other hand,
China-based research team established a quality model index to analyze peach quality
in controlled conditions (Zhang and Wang 2009). They developed PCA and LDA-
based algorithms determined ripening monitoring whereas multiple linear regression
(MLR) and artificial neural network (ANN) prediction models helped in differenti-
ating the right quality of peaches. The sensing system reported an increase in pH,
sugar content, and at last 9.91% growth of peaches within 14-days time period.
Song and colleagues designed MLR, partial least square regression (PLS), and back-
propagation (BP) network-based e-nose system for quality prediction of kiwifruit
(Song et al. 2014). According to the study, when the firmness, soluble solid content,
and pH of the kiwifruit in cold storage were reduced, the change in volatile aromatic
compounds was also observed. In continuation of this, China-based scientists team
used PEN2 based e-nose and integrated it with metal oxide sensor to detect five
different quality parameters in strawberry juice (Qiu et al. 2015). They also used
e-tongue to check the acidity and basicity on the basis of glucose, citrate, caffeine,
and salt presence and significant accuracy of 98% and 99% for e-tongue and e-nose
were obtained. Similarly, metal oxide sensing platform integrated LDA system was
used to established quality indices for stored banana fruit (Sanaeifar et al. 2016).
They discriminate the banana fruit on the basis of TA, pH, and total soluble solids at
ripening and senescence stage and demonstrated decrease and increase in pH in both
stages respectively and correlate the pH change with respect to malic acid. Zhang
and colleagues designed extreme learning machine (ELM) and partial least squares
(PLS) based e-nose to determine firmness, pH, and total soluble solids in stored
cherry tomato fruit (Feng et al. 2018). The ELM model system showed significant
discrimination property in which firmness of the fruits steadily decreased till 25 days,
whereas pH and total soluble solids increase till 5 days then gradually decreased.
Thus, eatable, and non-eatable quality of stored cherry tomato fruit can be easily
determined.
For the real-time monitoring of grape quality in logistics chain, Indonesia-based
research team developed a colorimetric pH sensor-based package indicator label
(Kuswandi and Murdyaningsih 2017). When grapes exposed to chlorophenol red
(CPR) fabricated sensing film, the sensitive CPR confirmed the changes in pH, TA,
and total soluble solids in ripening grapefruit and change in color directly observed
by naked eyes. Recently, Kurnianto and colleagues used betacyanin extracts from
dragon fruit’s (Caesalpina sappan L.) peel as a pH indicator to monitor banana
deterioration (Kurnianto et al. 2020). The sensing platform was sensitive toward
pH, texture and respiration rate, CO2 , and colorimetric change from yellow to dark
red determined the spoilage of banana. Although, Indonesia-based research team
has previously designed bromophenol blue (BPB) tagged bacterial cellulose film-
based smart packaging to monitor mangoes Arummanis (Mangifera indica L. var.
Arummanisa) (Dirpan et al. 2018). The study evaluated changes in pH, TA, and
total soluble solids and colorimetric changes of tag from blue to green confirmed the
gradual ripening of mangoes.
15.3.3 Aroma or Flavor Sensing in Fruit Quality Assessment
Aroma or flavor is the perception of aromatic volatile compounds by olfactory system

or taste buds in which phenolic, few aldehydes, and triterpenes compounds provide
bitterness whereas soluble sugar and organic acids are responsible for sweet and
sour taste (Sánchez-Rodríguez et al. 2019; Zhu et al. 2020). Been as a precursor
of volatile compounds (VOC), fatty acids are indirectly linked to fruit quality traits
(Zhu et al. 2020), whereas amino acids induced polyphenol oxidases reaction causes
fruit browning (Mertens et al. 2019). Despite several analytical advantages, aroma-
based sensing is difficult to study because of the mixture of volatile compounds and
chemical sensations. Yet recent studies on new sensor or e-nose development have
improved the fruit quality monitoring.
For on-field sensing, Malaysia-based research team designed MIP-based QCM
sensor for detection of α-pinene volatile compound for mango ripening confirmation
(Hawari et al. 2013). They evaluated the freshness of mango fruit in 27 ± 2 °C
and 55 ± 10% humidity for seven weeks and significant results were obtained with
higher sensitivity and selectivity toward target volatile molecules. Similarly, Ghatak
and colleagues have developed molecularly imprinted polymer (MIP) fabricated
QCM sensing platform and reported 0.16 Hz/ppm sensitivity of the sensor toward
d-limonene from ripped mango (Ghatak et al. 2019a). This research team has also
designed MIP-based (3-Aminopropyl) trimethoxysilane (APTMS) fabricated QCM
sensing platform for selective detection of furaneol from stored mango (Ghatak et al.
2019b). This sensor exhibited 79.70% selectivity and 1.23 ppm resolution in the
range of 100–1000 ppm concentration of furaneol.
In another study, China-based research team developed four different enzymatic
sensors for volatile compound detection in real fruit samples (Hou et al. 2014).
They fabricated QCM sensing platform with ethyl cellulose, cellulose acetate, 1,2-
dioleoyl-sn-glycero-3-[phosphor-L-serine], and galactosylceramide for detection of
isobutyl alcohol, ethyl acetate, and ethylene VOCs. The result indicated linear change
in singling in the range of 5–25 ppm. In another study, Ali and colleagues have
also designed QCM sensing platform for efficient detection of β- Caryophyllene
334 V. Hooda et al.
(β-CF) for mango quality evaluation (Ali et al. 2018). They fabricated QCM plat-
form with embedded 2-mercaptobenzothiazole as receptor for β-CF and interaction
between ring structures of thiolated sensing matrix and terpene caused change in
resonant frequency in the range of 5–1000 ppm analyte concentration and LOD of
0.32 ppm. This research team has previously designed PDMS fabricated QCM sensor
3-carene gas detection from ripped mango fruit (Ali et al. 2016). The hydrophilic
property of CH3 group on -OSi(CH3 )2 - of PDMS and hydrophobic nature of 3-carene
allows the absorption-based sensing in the range of 10–1000 ppm with sensitivity
of 0.06 Hz/ppm. For further improvement, they also studied absorptive binding of
ocimene (OCM) onto large molecular voids containing mustard-based QCM sensing
platform (Ghatak et al. 2019c). The developed sensor showed excellent sensitivity
of 0.276 Hz/ppm with LOD of 1.04 ppm in mango samples. Not stopping these
studies, Ali and colleagues developed ethyl cellulose fabricated QCM sensor for
β-myrcene VOC detection in mango quality monitoring (Ali et al. 2019). They eval-
uated sensitivity of 0.1 Hz/ppm due to the strong binding of ethyl ether groups from
ethyl cellulose with mango specific β-myrcene in fruit quality monitoring.
To develop smart packaging material, Brazil-based research team used low-cost
polyaniline film as a sensing material gas sensor to detect different aromas from
apples, grapes, and strawberry fruit samples (Tiggemann et al. 2016). The HCl doped
polyaniline layer allowed transferring from or to the active species of VOC with sensi-
tivities in 75–125% range. The sensing film reported LOD of 0.081 ppm, 0.46 ppm,
and 0.621 ppm for strawberries, grapes, and apples in the linear range of 40–200 ppm.
Till date several researches have been conducted on developing e-nose and smart
packaging for real-time fruit quality monitoring and aroma-based sensing is proved
as excellent target analyte. In this direction Chile-based research team designed
“single point” algorithm (SACMI)-based e-nose for evaluation of peaches stored in
cold storage (Infante et al. 2008). They compared four types of peaches on the basis
of TA, total soluble solid, and aroma at 0 °C and 90% RH; and reported decline
in TA/total soluble solid ratio and aroma till 42 days. In another study, Lu and
colleagues have generated sensitive e-nose for 1-MCP and calcium coated apple
fruit (Lu et al. 2018). The combination of 1-MCP and calcium are in use to improve
volatile aroma and lowers fruit ripening, therefore scientists used PEN3 portable
e-nose consisting 10 different sensing arrays and resulted in high-quality aroma
detection till ~ 90 days. Similarly, a study was conducted in which several pattern
recognition system modeling was combined to discriminate good quality saffron,
obtained from Crocus sativus L. (Iridaceae) (Kiani et al. 2016). The PCA model
revealed 11 different groups of aroma associated with 11 distinct saffron samples and
this further classified into five quality classes on the basis of hierarchical clustering
analysis (HCA) and MLP model system. Thus, the system was 100% successful
in differentiating saffron samples and proposed to utilize as aroma quality control
system.
Recent studies also combined e-nose with sensors to improve sensitivity and
reliability toward aroma detection. In this direction, China-based research inte-
grated e-nose with surface acoustic wave resonator (SAWR) for quality evaluation
of kiwifruit (Actinidia chinensis) (Wei and Guohua 2015). The PCA-based method
discriminated kiwifruits with different storage time on the basis of aroma-induced

quality and SAWR frequency analysis modeling system exhibited excellent qualita-
tive discrimination from all time zoned groups with high accuracy and high regression
coefficient.
15.4 Commercially Available Sensors in Fruit Monitoring
Biosensors have been grown from a tiny niche laboratory discipline since the 70’s
to multidollar industrial market in today’s world. However, this growth represents in
a very small fraction of agricultural and industries level, yet there are few effective
seining platforms available in market. Few smartphone-based techniques, such as
SCiO was developed by Consumer Physics Ltd., Israel, which work on the basis
of infrared technology and provide ripeness and nutritional values of fruits (“This
Pocket-Sized Sensor Will Tell You When Fruit Is Ripe” 2014; “What Happened
When We Took the SCiO Food Analyzer Grocery Shopping” 2017). The SCiO is
car-clicker-sized tiny optical sensor that captures the molecular footprint when the
fruit was scanned and SCiO’s in-house mobile app provides barcode-like readout.
Clarifruit and Fresh Fruit Detector—Check Fruits Quality by Dragster Production
are the other smartphone-based application for real-time fruit quality assessment
on the basis of texture and soluble solid contents. A smart ripening label, namely,
RipeSense® is also proposed by RipeSense, Auckland, New Zealand that worked on
ethylene sensing to communicate the degree of ripening by change in color of label.
15.5 Outlook and Conclusions
With current economic status and improved lifestyle, consumers are aware of hands-
on fruit quality monitoring and prefer nutritional values and healthy fruit consump-
tion. The next-generation trends in commercialization also entered daily life for effec-
tive and quick quality assessment system. Unfortunately, effective portable sensors
and smart packaging or e-nose are very limited in market and compromise consumer
goods. The major reasons for this shortcoming can be additional costs and acceptance
of sensing tags by dealers or manufacturer. Though, nobody can deny the advantages
of these sensing systems in fruit quality monitoring.
The success of fruit quality assessment approaches critically relies on sensitivity
and selectivity of the sensing platform. In this scenario, e-noses have futuristic posi-
tion for real time monitoring compared to on-field static sensing technology. Conven-
tionally used sensing approaches measure the quality of fruits in bulk therefore
intrinsic variability of fruit will be negotiable for quality assessment, hence individual
quality measurement can be performed with e-nose and smart/intelligent packaging.
336 V. Hooda et al.
In order to fulfill the requirement, next-generation RFID tags and nanotechnology-

based sensing platform are generated which are linked with multimodel pattern
recognition models.
The practical implementation of reliable sensors and indicator labels in active
and intelligent packaging, along with the development of next-generation sensing
methods for freshness investigation of fruits, is growing field of study. As we
discussed in this chapter, significant progresses have been made till date including
time–temperature indicator (TTI), pH, and aroma-based indictors from on-site to
consumer end and on-field fruit quality assessment. But there are still major efforts
need to make to apply the functionality of these sensing devices in realistic settings.
The major challenges in this scenario are the molecular complexity of fruits and diffi-
culty to identify accurate markers for fruit ripening or spoilage. On other hand, on-
field fruit quality monitoring still requires fruit sample pre-treatment. Most successful
approach of fruit quality monitoring till now is the assessment of VOCs such as ethy-
lene and amines, however no effective and hurdle-free approach has been selected
for on-field assessment.
Future development in fruit quality monitoring needs to be directed toward
lowering detection limit, for say ppm and improving the possibility of accurate marker
detection upon simple contact with fruit sample. The futuristic benefits of polyani-
line due to higher stability as sensing element also need to be promoted in e-nose
and smart packaging material. The higher stability and low reactivity with fruits of
packaging material will be very helpful in fruit quality assessment in stored packaged
fruit for long time. Maintaining variable environmental parameters in fruit logistics
application is another challenge that needed to be addressed. Recently, the use of
antimicrobial agents and nanomaterials in sensing and packaging technology geared
up the fruit quality assessment approaches and positive impact has been achieved.
However, toxicity and safety concern of nanomaterials limit their application in food
industry. These sensing technologies are still in initial proof of concept stage and
significant improvement and efforts should be made for worldwide launch and accept-
ability of sensing packaging material and indicator tags into a marketable product to
meet positive health concerns. Additionally, effective safety regulations need to be
issued by governmental health authorities and manufacturers should strictly follow
the use of these sensing platforms in their fruit products.
Acknowledgements Dr. Vinita Hooda is thankful to the Department of Science and Technology
(DST), Ministry of Science and Technology, Government of India grant under its “Fund for Improve-
ment of Science & Technology Infrastructure (FIST)” scheme (No. SR/FST/LS1-529/2012(C))
(http://www.fist-dst.org/) and Haryana State Council for Science, Innovation, and Technology
(HSCSIT, Endst. No. HSCSIT/R&D/2020/474) for providing financial support to work in the field
of Agriculture Nanotechnology.
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Chapter 16
Lateral Flow Assays for Food
Authentication
Despina P. Kalogianni
Abstract Foodstuffs with a particularly high nutritional and economic value are
often adulterated with lower value products. Products of protected geographical
origin are also included in the list of the most prone to fraud products, having a
negative impact on the national economy. This contravenes the rights of consumers
and producers, while it often jeopardizes public health. Due to these reasons, rapid
and reliable tests, as well as state control mechanisms, should be developed to detect
food adulteration and protect consumers and local producers from fraud. Lateral flow
assays (strips) have played an important role in this direction and for on-site appli-
cations. This chapter reports all the DNA and protein-based lateral flow assays that
have been constructed so far for food adulteration detection. The advantages of these
assays are the rapid and one-step analysis (within a few minutes), the simplicity, the
high detectability and reproducibility and the portability, maintaining also a low-cost
per sample analysis.
Keywords Strip · Biosensor · Dipstick · Adulteration · Authenticity ·

Nanoparticles
16.1 Introduction
Food adulteration has been a global concern in current times for all the stakeholders
involved in the food industry (Zhao et al. 2020a; Hong et al. 2017). The term adul-
teration refers mostly (95%) to the substitution of a food ingredient with another
ingredient of a much lower nutritional or quality value and of lower cost, for econom-
ical profit. Usually the substitution is difficult to be recognized by the consumers or
routine analytical techniques. Apart from the financial fraud, food adulteration may
have a great impact on public health, such as severe allergic reactions (Magiati et al.
2019; Böhme et al. 2019; Abbas et al. 2018). For example, substitution of mustard
oil with that of argemone causes oxidative stress and ultimate death of red blood
D. P. Kalogianni (B)
Department of Chemistry, University of Patras, Rio, Patras 26504, Greece
344 D. P. Kalogianni
cells, resulting in serious health problems (Lo and Shaw 2018). For these reasons,
people have the right to be protected from fraud, health risks, and producers from
illicit antagonism.
The most vulnerable to adulteration are products of high nutritional and economic
value such as meat and fish, olive oil, dairy products, honey, spices and several herbs,
grain-based foods and organic foods, fruit juices, wine and other alcoholic beverages,
coffee and special varieties of tea (Hong et al. 2017).
Sensitive and robust analytical methods as well as biosensors have to be developed
for food authenticity testing of varieties of food products checking safety, quality,
and religious issues. Moreover, reducing unfair competition is also a concern, while
identification of the geographical origin of special food products has also gained
the interest of the consumers (Wadood et al. 2020). Traditional methods used for
food adulteration detection include gas/liquid chromatography, single or in combi-
nation with mass spectrometry, nuclear magnetic resonance spectroscopy, infrared
and mid-infrared spectroscopy, Raman spectroscopy, inductively coupled plasma-
atomic emission (elemental analysis) and fluorescence spectroscopy, electrophoresis,
isotope ratio analysis, and immunoassays. Mass spectrometry has been extensively
used in most of the categories of adulterated products, while the advantages of some
spectroscopic techniques are the fast and non-invasive analysis with no sample prepa-
ration (Hong et al. 2017; Wadood et al. 2020). The drawbacks of these techniques are
mainly the long analysis time, the extensive and laborious protocols, and the expen-
sive instruments required (Bansal et al. 2017). Some of these techniques use different
chemical or metabolic profiles for species discrimination. However, chemical profiles
are strongly affected by environmental changes, harvest time, storage conditions, and
different processing procedures (Woolfe and Primrose 2004). Moreover, proteins are
not stable during thermal food processing or in case of plants, different proteins are
secreted in different parts of the plant. For these reasons, DNA-based techniques
are preferred, because DNA has high thermal stability, it is present in all cells of the
organisms and not affected by environmental conditions, therefore it overcomes many
limitations of the aforementioned techniques (Zhao et al. 2020a; Böhme et al. 2019).
Molecular authentication is also a powerful tool for the most specific discrimina-
tion of closed relative species or cultivars of different geographical origin. However,
DNA-based techniques are also affected by some factors including DNA degrada-
tion and PCR inhibitors. The researchers are trying to overcome these limitations
by introducing more effective DNA isolation protocols, while amplification targets
small-size DNA sequences (Lo and Shaw 2018).
16.2 Lateral Low Assays Used in Food Authentication

(LFAs)
Among other methods used for food authenticity testing, lateral flow assays (LFAs)
have been constructed for the identification and authentication of various food
16 Lateral Flow Assays for Food Authentication 345
products, providing simplicity, rapidness, one-step analysis, user-friendly format,

long-term stability, high specificity, and detectability. LFAs are also cost-effective,
as they do not require expensive instruments, disposable, and portable in some
cases (Sajid et al. 2015). Some DNA-based LFAs are also universal and can be
applied for the detection of any DNA sequence‚ because the molecular recognition
is performed prior to the application to the LFA. Both DNA-based LFAs and lateral
flow immunoassays have been reported for food authentication (Fig. 16.1). LFAs are
divided into DNA-based and protein-based analytical systems, whereas the former
possessing the greater part of the applications.
Fig. 16.1 The lateral flow

assays developed for the
detection of adulteration in
specific food products
16.2.1 Principle of the Lateral Flow Assays
Lateral flow assays are constructed in a strip-based format and consist of four discrete
parts: an immersion pad, a conjugate pad, a diagnostic membrane, and an adsorbent
pad (Magiati et al. 2018; Spyrou et al. 2016; Sajid et al. 2015). A schematic illustration
of a lateral flow assay is presented in Fig. 16.2. The four parts of the strip are
summarized below:
Immersion pad: It is usually made of cellulose and dipped into the appropriate
developing solution. It enables the smooth, continuous, and homogeneous flow of
the developing solution.
Conjugate pad: It is made of glass fibers, where the reporters (usually colored
nanoparticles conjugated to biorecognition molecules) and the sample are deposited
on. This material must have no interaction with the sample and the reporters, while
it should be immediately releasing the sample and reagents upon contact with the
developing solution. For the preparation of dry-reagent strip format, reporters are
Fig. 16.2 Lateral flow assay schematic configuration in case of a negative (left) and a positive
(right) sample. IP: immersion pad, CP: conjugate pad, M: membrane, AP: adsorbent pad, TZ: test
zone, and CZ: control zone
dispensed and let to dry on the conjugate pad. The reagents must have long-term
stability on the strip. Other materials used for conjugate pad are cellulose and
polyesters.
Diagnostic membrane: It is usually made of nitrocellulose, available in different
pore sizes. It is crucial for the sensitivity and detectability of the assay, providing good
binding ability for the immobilization of biomolecules and minimizing non-specific
binding. A colored test line and a control line are formed onto the membrane by immo-
bilizing specific biorecognition molecules at these two areas. Again, biomolecules
are sprayed and dried onto the membrane to construct the test and the control zone
of the membrane.
Adsorbent pad: It consists of an absorbent material and enables the continuous
flow of the developing solution and reagents, avoiding the backflow of the sample.
The four parts are attached on a plastic backing pad that serves as a solid support,
with overlapping ends to enable the continuous flow on the strip. The reporters are
usually conjugated with specific recognition biomolecules and deposited onto the
conjugate pad of the strip. The sample is also added on the conjugate pad of the
strip just above the reporters. The strip is then dipped into an appropriate developing
solution. The solution is moving upwards due to capillary forces and removes the
reporters and the sample in the same direction. The reporters interact with the sample
and the complex is captured at the test line of the strip by other immobilized recog-
nition molecules, through a label carried by the sample, forming a visual (colored)
line, due to the accumulation of colored nanoparticles. The excess of the reporters
is finally captured by specific immobilized molecules at the control line of the strip
forming a second visual line and ensuring that the lateral flow assay works perfectly.
So, in case of a positive result, both visual lines are formed and in the absence of the
analyte, only the control line of the strip is shaped (Fig. 16.2). The concentration of
all reagents, the constitution of the developing solution, as well as the molar ratio
of coupling agents have to be carefully optimized in order to get the highest sensi-
tivity and specificity of the assay, along with reliable and accurate results. Surface
surfactants and denaturing agents are also used in the developing solution to minimize
cross-reactivities, and to avoid false-positive outcomes. The developing solution must
contain also the optimum salt concentration to enable hybridization in DNA-based
LFAs and liquids with high viscosity, such as glycerol and sucrose, in order to gain
the optimum mobility through the strip and to provide enough time for successful
interaction between the reagents. Finally, diagnostic membranes with different pore
sizes provide different liquid mobilities and have to be carefully selected.
LFAs provide qualitative or semi-quantitative analysis. The visualization of the
lines is accomplished either by the naked eye when colored nanoparticles are used or
by a digital camera or a smartphone in case of a fluorescent or a chemiluminescent
signal. The images of the strips with the colored lines can be also obtained using a
regular camera or a smartphone, while all captured images can be further processed
by an image processing software. The intensity of the color of the test line is measured
and compared to standards to get semi-quantitative results. Optical strip-readers are
also available to get more accurate quantitative results, by also measuring the intensity
of the colored line and using suitable image processing software (Kalogianni et al.
2006; Sajid et al. 2015; Dzantiev et al. 2014).
16.2.2 Sensing Systems and Labels
LFAs are available in different formats using different biorecognition molecules,

labels, and detection systems. Sensitive and selective sensing systems have been
exploited in LFAs for the detection of specific analytes. These systems are
based on the strong and extremely specific and stable interaction between the
following molecules: antigen (analyte)–antibody, streptavidin–biotin, avidin–biotin,
anti-biotin antibody–biotin, anti-FITC antibody–FITC (fluorescein isothiocyanate),
anti-fluorescein antibody–fluorescein, anti-digoxigenin antibody–digoxigenin, and
anti-Cy5 antibody–Cy5 dye.
16.2.3 Nanoparticles and Nanoparticles Conjugates Used

as Reporters in LFAs for Food Authentication
Applications
Metallic nanoparticles, in general, have gained the great interest of the researchers
because of their visual and electrical properties for applications in food analysis. Their
characteristic properties are mostly dependent on size and shape. As their synthesis
has become easier and of lower cost, they have been further used as a new analytical
tool and applied to food authenticity testing. Gold nanoparticles (AuNPs) are the main
nanoparticles used in LFAs developed for food authentication, as they are considered
the key tool for visual detection. Gold nanoparticles are well known for their unique
optical characteristics, having a bright red color that enables their visual detection
even at low concentrations, as well as for their good stability, large surface to volume
ratio, and capability of facile conjugation to several different biomolecules. The
optical properties of gold nanoparticles have increased the detectability of LFAs,
due to their extremely high molar absorption coefficient due to surface plasmon
resonance. They have been extensively used in numerous applications, providing
high sensitivity and detectability (Subara and Jaswir 2018; Sajid et al. 2015).
Gold nanoparticles have been conjugated to specific biorecognition molecules, in
order to be applied for the detection of specific adulterants by LFAs. The substances
that have been used for bioconjugation to the gold nanoparticles in LFAs for
authentication purposes are the following:
• Streptavidin
• Anti-biotin antibody
• Anti- digoxigenin antibody
• Anti-fluorescein (FAM) antibody
• Anti-fluorescein isothiocyanate (FITC) antibody

• Species-specific antibodies
• Species-specific DNA probes or other oligonucleotides.
16.3 DNA-Based Lateral Flow Assays
As discussed above, DNA-based LFAs are the most preferred one especially because
of their higher discrimination between close-related species compared to protein-
based analysis. Most of the DNA-based techniques applied for food adulteration
detection employ the amplification of genomic material using species-specific
primers. Apart from real-time PCR that enables the detection and quantification
of the PCR products in real time during each cycle, all other methods need the
post-PCR identification of the amplified products. However, real-time PCR requires
special and expensive instrumentation. LFAs are considered one of the most suitable
alternatives for post-PCR analysis and have successfully integrated with amplifica-
tion techniques for food authenticity assessments (Zhao et al. 2020a). DNA-based
LFAs have been applied for the authenticity assessment of meat, fish, milk, saffron,
and coffee.
16.3.1 Meat Authentication
Most of the developed DNA-based assays have been applied for the detection of meat
adulteration, as meat is largely consumed and is one of the most susceptible to adul-
teration food products worldwide (Magiati et al. 2019; Rahmati et al. 2016). Meat
adulteration mainly includes the adulteration of meat with other meat species of lower
value and quality, meat from other geographic origin, processed meat, or non-meat
ingredients, such as water or vegetable proteins (Hong et al. 2017). Meat adulteration
arises substantial threats to public health and ethical issues in some cases (Böhme
et al. 2019). Recently, Zhao and coworkers developed a lateral flow assay for the
identification of turkey meat in meat products. Turkey-specific DNA sequences were
amplified using specific primers, while the PCR products were labeled with biotin
at one end and with fluorescein at the other end. The PCR products were directly
applied onto the conjugate pad, along with gold nanoparticles as reporters that were
conjugated to anti-fluorescein polyclonal antibodies. The strip was then immersed
into the developing solution. A red line was formed at the test zone of the strip due
to the immobilized streptavidin at this zone via biotin–streptavidin interaction, while
the gold nanoparticles conjugates were attached to the PCR products via the inter-
action between the anti-fluorescein antibody and the fluorescein label. The excess
of the gold nanoparticles conjugates was captured by a second antibody specific
to anti-fluorescein antibody immobilized at the control zone of the strip forming a
second line and ensuring the right strip function (Fig. 16.3). The LFA was specific
Fig. 16.3 The detection of species-specific meat products using PCR-amplified DNA sequences
labeled with biotin and fluorescein (FAM) or fluorescein isothiocyanate (FITC) and gold nanopar-
ticles coupled to anti-FAM or anti-FITC antibodies as reporters. AuNPs: gold nanoparticles, SA:
streptavidin, B: biotin, F: fluorescein or fluorescein isothiocyanate, TZ: test zone, and CZ: control
zone
to turkey meat among nineteen other meat samples and two plant species, while the
detectability of the method was set at 0.1% of turkey meat in mixtures with beef meat
samples. The detectability of the method was not affected by heat treatment of the
meat mixtures in the same concentration. The analysis was completed in 5 min with
good reproducibility evaluated by both intra- and inter-laboratory tests (Zhao et al.
2020a). The same configuration was also used for the identification of donkey meat
in food samples with a limit of detection (LOD) of 0.001% w/w for raw and 0.01%
w/w for cooked donkey meat in beef meat (Zhao et al. 2019a). The same assay was
exploited as well for camel species identification with a limit of detection of 0.1%
w/w of camel meat in beef for both the raw and cooked meat samples (Zhao et al.
2020b) and for 0.01% w/w of goose meat in uncooked binary mixtures and 0.1%
w/w in cooked binary mixtures (Zhao et al. 2020c). In some of these approaches,
fluorescein isothiocyanate (FITC) was used as label instead of fluorescein (FAM)

along with the corresponding antibodies.
Another report involves the detection of pork meat in meat mixtures using PCR
combined with LFA. Pork-specific sequences were amplified by PCR using a primer
labeled with fluorescein. The PCR products were then heat-denatured and let to
hybridize with a complementary probe labeled with biotin. The hybrids were loaded
onto the conjugate pad along with streptavidin-gold nanoparticles conjugates. The
strip was then dipped into the developing solution. A first red line was formed as
the hybrids are captured by immobilized anti-fluorescein antibody at the test zone
of the strip, while gold nanoparticles were accumulated there through streptavidin–
biotin interaction. The excess of the gold nanoparticles conjugates was captured by
biotinylated bovine serum albumin (BSA), producing a second red line at the control
zone of the strip. The authors achieved a limit of detection of 10 fg of pork-specific
DNA sequence and 0.01 of pork meat in meat mixtures of mutton, beef, chicken,
goose, duck, horsemeat, and rabbit meats in equal quantities. The result was obtained
within 3 min (Yin et al. 2020).
An LFA was developed for the on-site detection of horse and donkey meat. This
LFA involved gold nanoparticles and tag-labeled multiplex loop-mediated isothermal
amplification (LAMP) in order to simplify amplification and detection. The advan-
tages of the LAMP technique were the rapid amplification of the DNA sequences,
along with high specificity and sensitivity, performed in one step and at a constant
temperature. Three LAMP products were produced carrying one biotin moiety at
their 5’ end. The three products were specific to donkey meat, to horse meat, and to
an endogenous reference gene present in all mammals. The donkey-specific products
carried a digoxigenin label, the horse-specific products a fluorescein isothiocyanate
(FITC) label, and the endogenous gene a Cy5 label at their 3’ end to allow discrim-
ination with the LFA. The LAMP products were added onto the conjugate pad of
the strip after amplification, and the strips were immersed again into the appropriate
solution. Then, three red test lines were formed on the test zone of three single strips
by immobilizing antibodies specific to digoxigenin, FITC, and Cy5, respectively,
while the control zone was constructed in all strips by immobilized goat anti-mouse
IgG. The captured LAMP products were detected by gold nanoparticles attached
to mouse anti-biotin antibodies through their biotin moieties, while the excess of
the gold nanoparticles was captured at the control line of the strip through interac-
tion with the IgG molecules to ensure the proper function of the assay. The authors
could detect as low as 40 pg or 15 copies of DNA sequences corresponding to horse
and donkey species. The method was finally applied to various processed foods
and mixed meat products. The whole procedure was very fast, and the final result
was obtained within 40 min (Zhang et al. 2019a). A similar approach was used by
Xu et al. using the same endogenous gene for the on-site detection of mammalian-
derived meat samples. The PCR products were again labeled with biotin and FITC
and detected at the test zone by anti-FITC antibody and anti-biotin antibody—gold
nanoparticles conjugates. The control line was formed when the excess of the gold
nanoparticles conjugates was captivated by a secondary antibody. The LOD of the
method was 10 pg, which was equivalent to 3 ~ 5 copies in mammals. Finally, the
method was successfully applied to various meat materials and processed foods (Xu
et al. 2017). Again, LAMP was combined with LFA for the detection of duck-derived
ingredients in adulterated meat. LAMP primers were labeled with FITC and biotin
and detected using LFA and gold nanoparticles. An amount of 3 pg of duck DNA, as
well as 0.01% of duck meat in beef meat were detectd by this approach. The LOD
of 3 pg is 10 times lower than that of quantitative real-time PCR (Shi et al. 2017).
PCR products specific to duck species were labeled, as above, with biotin and
FITC and applied onto a new LFA. The amplified products were detected by anti-
FITC antibody immobilized to the test zone and visualized by streptavidin-gold
nanoparticles conjugates, in order to check the adulteration of beef meat samples.
Beef species were also amplified in the same reaction but were dually labeled with
biotin. The beef-specific DNA sequences were then captured at the control line of the
strip by immobilized streptavidin to give a positive control signal, in the presence or
absence of duck meat in the sample. The LFA was finally applied for the detection of
adulteration in raw and processed meat samples. The whole protocol had a duration
of 2 h (Qin et al. 2019).
In another study, an LFA was combined with recombinase polymerase ampli-
fication (RPA) for authentication of mutton slices or heat-processed shish kebabs.
RPA is an isothermal amplification technology that can be completed within 20 min,
and is the method of choice for on-site detection compared with other isothermal
amplification methods, such as loop-mediated isothermal amplification (LAMP),
strand displacement amplification (SDA), and rolling circle amplification (RCA).
The RPA reaction was performed in low temperature (37–42 °C) and finished within
15 min, while the DNA extraction step and the subsequent detection with the LFA
both ended up in 5 min. The sheep-specific PCR products were labeled with biotin
at one end and digoxigenin at the other end and were immediately applied onto the
strip. The amplified products were detected at the test zone of the strip by immobi-
lized avidin and gold nanoparticles conjugated to anti-digoxigenin antibody through
avidin–biotin and anti-digoxigenin antibody–digoxigenin interactions. The excess
of the gold nanoparticles conjugates was captured finally by goat anti-mouse anti-
bodies to form the control line of the strip. The LOD of this LFA was assessed to
200 fg of sheep DNA (Li et al. 2019). Mutton has also been detected by an LFA
combined this time with a cross-priming amplification reaction (CPA). This reaction
was performed at 63 °C for 60 min. The products were labeled at both ends with
biotin and fluorescein instead of digoxigenin and detected as described above using
anti-fluorescein antibody—gold nanoparticles conjugates. The LOD of the method
was 1% of mutton in thermal treated meat mixtures, while the detection with the LFA
was completed within 5 min (Feng et al. 2018). Sheep-specific PCR products were
also detected by another LFA for the authentic identification of raw and heat-treated
mutton. The strip format was the same as the one described by Zhao et al. (2020a).
This LFA had an LOD of 0.01% of adulterated meat in mixtures with six other meats,
as well as 0.01 pg of sheep DNA (Yin et al. 2016).
Magiati et al. developed an LFA for the detection of 0.01% of adulteration of
beef and horse meat with pork and horse meat, respectively, in binary mixtures. Four
species-specific DNA sequences were PCR-amplified. The PCR products carried
biotin at one end and allowed to hybridize to species-specific oligonucleotide probes

that had a poly(dA) tail at their 5’ end. The hybrids were then applied to the conju-
gate pad of the strip, next to gold nanoparticles coupled to streptavidin. A first red
line was formed at the test zone of the strip when the hybrids were captured by
immobilized poly(dT) sequences through dA/dT hybridization and gold nanopar-
ticles conjugates were accumulated there via streptavidin–biotin interaction. The
excess of gold nanoparticles was captured finally at the control zone of the strip
to form a second red line to ensure that the strip functions perfectly. The LFA was
completed within 25–30 min (Magiati et al. 2019).
Finally, a multiplex DNA LFA kit has been commercially produced. Ha et al. have
used this kit for the simultaneous detection of as low as 0.1% of adulteration of seven
meat species: beef, pork, lamb, goat, horse, chicken, and turkey. Species-specific
DNA sequences were amplified by a multiplex PCR. The PCR products were then
detected simultaneously on the strip by immobilized species-specific oligonucleotide
probes and gold nanoparticles (Ha et al. 2019).
16.3.2 Fish Authentication
Four fish species were also detected by a PCR-based LFA. Four sets of specific
primers were used for the amplification of each species. The PCR products were
dually labeled with biotin and fluorescein that allowed their visual detection with
the LFA through immobilized biotin ligands at the test zone of the strip and anti-
fluorescein gold nanoparticles. The excess of the gold nanoparticles was captured
by immobilized anti-rabbit antibodies at the control zone of the strip. The LOD of
this assay was 50 pg for Gadus morhua and Molva molva species and 500 pg for
Gadus chalcogrammus and Gadus macrocephalus fish species, respectively. Finally,
the LFA was successfully applied for the identification of the four species in 31
commercial fish-derived samples (Taboada et al. 2017).
16.3.3 Milk Authentication
The adulteration of milk-based products derived from goat, sheep, buffalos, and other
animal species with cow milk that has lower quality and cost is a serious concern for
many years now. Apart from health risks that are discussed above, many Protected
Designation of Origin (PDO) councils have made great efforts in developing specific
and reliable tests, to assure the quality and the genuineness of such dairy products
(Galan-Malo et al. 2018).
Lateral flow assays were also exploited for milk authentication in dairy products.
An LFA was developed for the detection of yak milk. This LFA was based on rapid
isothermal recombinant polymerase amplification (RPA). Double-stranded amplified
products were obtained containing a DNA tail at both ends that was attached to the
primers through a special abasic C3 spacer. One DNA tail was used for the direct
binding to gold nanoparticles labeled with complementary detection probes, while the
final complex was captured by immobilized specific DNA sequences at the test zone
of the strip through hybridization to the second DNA tail. The assay was completed
within 40 min, allowing detection down to 5% yak milk in mixed cow milk samples
(Wang et al. 2020).
Another LFA was developed for the detection of adulteration of sheep and goat
milk with cow milk in yogurt samples. Three animal-specific DNA sequences were
amplified by PCR. The products‚ biotinylated at one end‚ were let to hybridize to
species-specific probes that contained a poly(dA) tail and applied to the strip next to
gold nanoparticles conjugated to streptavidin. Then a red spot formed at the test area
of the strip when the hybrids were captured by immobilized carboxylated beads, 2 μm
in diameter, attached to an amino-poly(dT) probe through dA/dT hybridization, while
the detection was accomplished through the streptavidin-gold nanoparticles conju-
gates and streptavidin–biotin interaction. The excess of the nanoparticle conjugates
was captured at the control area of the strips by carboxylated beads that coupled to
biotinylated DNA probe (Fig. 16.4). The authors could detect as low as 1.6 fmol of
cow and goat, and 3.1 fmol of sheep PCR product, respectively. Also, adulteration
of 0.01% of cow species in binary yogurt samples could be detected by this LFA
(Bougadi and Kalogianni 2020).
16.3.4 Crocus Sativus (Saffron) Adulteration
Saffron is derived from the dried stigmas of the flowers of the plant Crocus sativus
and is one of the most expensive herbs cultivated mainly in India, China, Greece,
Spain, Italy, France, and Turkey. Its special flavor, along with some nutritional char-
acteristics, makes saffron one of the most valuable spices, and at the same time
vulnerable to adulteration. Some adulterants used are carrot, marigold, safflower,
and others with a similar appearance that makes the discrimination by the consumers
extremely difficult.
Recombinase polymerase amplification (RPA) in combination with a universal
LFA was reported for the adulteration of saffron. The RPA products were labeled with
fluorescein and biotin and were detected by the LFA through immobilized anti-biotin
substances at the test zone of the strip, while the visualization was accomplished
through conjugates of gold nanoparticles with anti-fluorescein antibody. The whole
assay was ended up within 15 min at 37 °C and the authors could detect as low as 1 pg
of genomic DNA with high specificity among common saffron adulterants. (Zhao
et al. 2019b).
Fig. 16.4 Detection of adulteration of goat- and sheep-derived milk samples with cow milk using an
LFA. The visual detection is based on species-specific PCR products hybridized to species-specific
DNA probes carrying a poly(dA) tail and gold nanoparticles conjugated to streptavidin. AuNPs:
gold nanoparticles, SA: streptavidin, B: biotin, TZ: test zone, CZ: control zone
16.3.5 Coffee Adulteration
Coffee authenticity assessment was accomplished through another LFA that used
gold nanoparticles as reporters. Coffee is one of the most used food products
daily with increasing global consumption. Two different coffee varieties-species are
commercially available worldwide: Coffea arabica and Coffea robusta. C. arabica
is preferred by the consumers, because it has a better and more intense taste, better
quality, and lower caffeine content, therefore it is two–three times more expensive
than C. robusta. The discrimination of the two coffee species was based on the
detection of a unique single-nucleotide polymorphisms (SNP) in the chloroplastic
trnL(UAA)—trnF(GAA) intraspacer region of the plant genome. DNA was isolated
from coffee beans from the two species and amplified by PCR using a common primer
pair. Then, the discrimination of the two alleles that correspond to the two coffee
species was accomplished through a 15-min primer extension reaction (PEXT). Two
allele-specific primers were used that have the same sequence, containing a poly(dA)
tail at 5’ end and differ at the 3’ end that contained the analyzed SNP. The primers were
extended, afterwards, by the DNA polymerase only if they were perfectly comple-
mentary to the target DNA. During the extension, biotin molecules were incorporated
to enable the capturing of the PEXT products by immobilized streptavidin at the test
zone of the strip. The PEXT products were finally detected by gold nanoparticles
conjugated to poly(dT) sequences through the poly(dA) tail of the primer. The detec-
tion was finished in a few minutes, and Robusta species could be detected as low as
5% of by this LFA, in the presence of Arabica species (Trantakis et al. 2012)
16.4 Lateral Flow Immunoassays
Lateral flow immunoassays exhibit similar or better detectability than common

enzyme-linked immunosorbent assay (ELISA) methods. The most widespread
immunochromatographic assembly is the immunochromatographic strip test that
has been also used for food authentication purposes. Immune complexes are formed
at the test and control zone of the strip and are detectable by gold nanoparticles, that
are covalently coupled to specific recognition antibodies. Immunochromatographic
strips are easy to use, simple and rapid, allowing on-site testing. The sensitivity and
specificity of the immunoassay is mainly based on the immunoreagents (antibodies)
used. These reagents must have high affinity for the target (protein or antibody),
along with low cross-reactivity with similar target molecules. The kinetic binding
constant is also a factor that affects the detectability of the strip assay. Moreover, these
strip tests usually include no sample pre-treatment, so interfering substances are not
removed from the sample leading to high matrix effect and resulting in lower detec-
tion limit. In some cases, however, purification of analytes is required. Finally, the
gold nanoparticles are again the most popular reporters in lateral flow immunoassays,
due to their unique optical characteristics (Dzantiev et al. 2014).
16.4.1 Meat Authentication
Horse meat residues were detected in raw and cooked samples by an LFA, based on
both a competitive and a sandwich-type immunoassay. Horse serum albumin (HSA)
was used as the target in raw meat, while the horse thermal-stable meat protein
(H-TSMP) was used as the target in cooked meat. Anti-HSA and anti-H-TSMP
polyclonal antibodies produced by immunization protocols were conjugated to gold
nanoparticles and used for the preparation of the sandwich-type test zones of the strip.
Standard HSA and H-TSMP targets were used for the formation of the competitive
format test lines. Chicken anti-goat antibodies were immobilized at the control line
of the strip to capture the excess of the gold nanoparticles conjugates, ensuring the
validity of the LFA. This LFA had a detection limit (LOD) of 0.01% and 1.0% for raw
and cooked horse meat in beef meat, respectively. The final results were observed
within 35 min with very good specificity and reproducibility (Masiri et al. 2017). A
similar approach was developed by Masiri et al. (2016) for pork identification. The
results were obtained after 15 min with an LOD of 0.01% of pork meat in beef meat
(Masiri et al. 2016).
A rapid immunochromatographic strip was also developed for the identification
of pork adulteration on beef cooked meatballs. Gold nanoparticles were coated with
anti-swine IgG polyclonal antibody, while the same polyclonal antibodies were used
to construct the test zone of the strip and a sandwich format immunoassay. The excess
of gold nanoparticles was captured by goat anti-mouse IgG at the control zone of the
strip (Fig. 16.5). The detection limit of the immunoassay was 0.1% (w/w) of pork
meat, while the response was visualized within 5 min (Kuswandi et al. 2017).
Moreover, bovine authentication was based on the detection of bovine fat by a
lateral flow immunoassay. This assay was based on the detection of heat-stable muscle
protein. The protein was extracted from meat samples and the protein extracts were
deposited onto the conjugate pad of the strip along with gold nanoparticles conjugated
to anti-muscle protein antibodies, forming an immunocomplex. The strip was then
dipped into the appropriate solution. A first red line formed at the test zone of the
Fig. 16.5 A sandwich-type immunochromatographic strip for the detection of pork meat. AuNPs:
gold nanoparticles, TZ: test zone, and CZ: control zone
strip, as the immunocomplex was captured by immobilized secondary antibodies

specific to muscle protein. A second visual line formed, when the excess of the gold
nanoparticles conjugates was accumulated by a third antibody immobilized at this
area. This LFA could detect 2% bovine fat-in-pork fat, 1% bovine fat-in-porcine
meat-and-bone meal, and 0.5% bovine fat-in-soy meal mixtures. Bovine fat could
also be detected at high temperatures up to 213 °C (Hsieh and Gajewski 2016). The
same format was used by another research team in 2007 for the detection of beef
or ovine meat in beef-in-chicken, beef-in-turkey, and lamb-in-pork meat mixtures,
with an LOD of 0.5%. The method was successfully applied in raw and cooked meat
mixtures (Rao and Hsieh 2007).
16.4.2 Milk Authentication
Lateral flow immunoassays have been also developed for the identification of animal-
specific proteins for the authentication of milk and milk-based products. A new
competitive lateral flow immunoassay was reported for the detection of adulter-
ation of goat milk with cow milk. Gold nanoparticles were coupled to monoclonal
antibodies specific to the cow milk protein casein and used as reporters for visual
detection. The limit of detection of this immunoassay was as low as 0.07% of cow
milk in milk mixtures with goat milk. The assay offered equal specificity and higher
detectability than commercial ELISA assay (Liu et al. 2019).
Another rapid immunochromatographic strip test was developed for the adulter-
ation of milk from goat, sheep, and buffalo with cow milk based on the detection
of bovine immunoglobulins (IgG). A validation study was also conducted including
evaluation of the specificity, analyzing 146 negative samples, detectability, repro-
ducibility, and robustness by studying the effect of several factors such as different
batches of the strip, sample dilution, assay time, high level of fat, protein, and
somatic cell counts. The detectability of the assay was as low as 0.5% of cow milk
in milk mixtures, while a strip reader could be useful for interpretation of the results
(Galan-Malo et al. 2018).
Along with these, a competitive lateral flow immunoassay was developed for
the detection of soy milk in cow’s milk. The strip test was based on the detection
of soy milk protein through rabbit anti-soy protein antibodies conjugated to gold
nanoparticles. Standard soy milk protein was immobilized at the test line of the strip
competing the protein present in soy milk sample. At the control line of the strip,
anti-rabbit immunoglobulin was immobilized to ensure the properties of antibody—
conjugated gold nanoparticles (Fig. 16.6). The detection limit of soy milk was 1.75%
in whole bovine milk, while the results were available in 5 min (Gautam et al. 2017).
Fig. 16.6 Detection of adulteration of bovine milk with soy milk by a lateral flow competitive
immunoassay targeting the soy milk protein. AuNPs: gold nanoparticles, Ab: antibody, TZ: test
zone, and CZ: control zone
16.4.3 Honey Authentication
Honey is a natural sweet source that is considered to have health-promoting and

nutritional characteristics, such as antioxidant, antibacterial, antimutagenic, and anti-
inflammatory properties, as well as healing properties. Honey is rich in enzymatic
and non-enzymatic substances, including polyphenols, ascorbic acid, α-tocopherol,
carotenoids, nucleic acids, proteins, and others. Its antibacterial activity is mainly
attributed to its high osmosis phenomena in combination with its low pH and the
presence of hydrogen peroxide, bee defensin-1, and methylglyoxal. For the above
reasons, honey is one of the favorite natural products worldwide. Its composition
includes mainly carbohydrates and water that makes its adulteration with cheaper
sugar syrups with similar composition very easily. Many analytical methods have
been used for the detection of honey adulteration, but they use expensive instruments
and require high analysis time (Zhang et al. 2019b; Pita-Calvo and Vázquez 2018).
An immunochromatographic strip test was constructed for the detection of the
major royal jelly protein 1 (MRJP1) in honey. The assay was based on the use of
novel polyclonal antibodies, a sandwich-type ELISA format, and gold nanoparticles
conjugated to the polyclonal antibodies as reporters. For quality control of the strip
test, goat anti-rabbit IgG was immobilized on the control zone of the strip to capture
the excess of gold nanoparticles conjugates with the specific antibodies. The LOD of
the method was 2 μg mL−1 for the analyzed protein. Also, the test line formed in case
of a positive sample remained constant for adulterant mixtures that contained above
30% honey. The adulterants tested were rice and corn syrup (Zhang et al. 2019b).
16.4.4 Edible Bird’s Nest Authentication
Edible bird’s nest is produced by the saliva secretion of several kinds of birds and
is considered as a health food, containing glycoproteins, carbohydrates, essential
amino acids, minerals, and other elements. It is commercially available at high price;
therefore, it is adulterated with substances of lower cost. These adulterants may are
plant-exude, gum karaya, pork and fish skin, algae and the white part of the egg. A
lateral flow sandwich-type immunoassay has been developed for the identification of
bird’s nest based on the detection of its glycoprotein. Polyclonal antibodies specific
to the glycoprotein were produced and used to construct the test line of the strip and
prepare conjugates with gold-silica (SiO2 ) nanoparticles. The detection limit of the
LFA was 7.02 ng mL−1 of purified glycoprotein, from nest, in buffer (Xu et al. 2019).
16.5 Conclusions and Future Perspectives
Food products with an extra added value are more and more often vulnerable to fraud.
Food safety is a severe issue continuously investigated by the research community to
prevent food fraud and counteract new “sophisticated” types of adulteration. Lateral
flow assays have gained the interest of researches for a rapid and cheap alternative for
food authenticity testing. DNA- and protein-based techniques in combination with
LFAs have successfully applied for the detection of food adulteration and species
identification in various food products such as meat, fish, and milk. DNA-based tech-
niques are preferred due to higher DNA stability especially in processed food, higher
sensitivity, and specificity among closely related species. Moreover, the integration
of the nanotechnology (nanoparticles) in LFAs have increased the flexibility in the
strip format configuration, which also provide very low detection limits, even in food
mixtures and processed foodstuffs.
On the other hand, some issues have to be further addressed in order to improve the
analytical performance of LFAs. New and different labels, as well as new detection
systems are needed in order to increase the detectability and the multiplicity of these
assays. The combination of LFAs with easy amplification systems, in case of DNA
targets, could enable the on-site detection of food adulteration. Moreover, the use of
portable recording devices along with the test strips could increase the possibility
for more accurate quantitative results, enabling the data processing outside of the
laboratory. This would increase the portability of these rapid tests that would greatly
outperform modern technologies of food analysis in terms of speed, cost, simplicity,
and efficacy. Finally, a strong effort has to be made in order to construct and produce
universal strip tests that are not dedicated only to the detection of a single analyte but
can be easily applied for the analysis of various different analytes. It is anticipated that
the recognition is performed outside the strip, while the final detection is achieved
with the strip test.
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Chapter 17
Nanobiosensors in Agriculture
and Foods: A Scientometric Review
Ozcan Konur
Abstract Nanobiosensors have been developed and increasingly used in agricul-

ture and food to ensure the safety of the agricultural produces and foods. Thus, these
advanced sensors have the utmost public importance. However, for the efficient
development and use of these nanobiosensors, it is necessary to develop efficient
incentive structures for the primary stakeholders and to inform these stakeholders
about the research on nanobiosensors in the agricultural and food context. Sciento-
metric analysis of the research offers ways to evaluate the research in a respective
field. This method has been used to evaluate research in several research fields. There
has been a limited number of pioneering scientometric studies on the various aspects
of the nanobiosensors in recent years. This book chapter presents a study on the
scientometric evaluation of the research on the nanobiosensing and nanobioanal-
ysis of agricultural and food analytes using two data sets. The first data set includes
100 most-cited papers whilst the second set includes over 22,000 papers published
between 1980 and 2019. The data on the indices, document types, authors, institu-
tions, funding bodies, source titles, ‘Web of Science’ subject categories, key words,
research fronts and citation impact are presented and discussed.
Keywords Nanobiosensors · Nanobiosensing · Nanobioanalysis · Scientometrics ·

Nanobiomaterials · Food · Agriculture · Analytes · Graphene · Nanoparticles
17.1 Introduction
Nanobiosensors are being developed and increasingly used in agriculture and food
to ensure the safety of the agricultural produces and foods (Nam et al. 2003; Song
et al. 2010; Dong et al. 2012; Shan et al. 2009; Srivastava et al. 2018; Lee et al. 2007).
Thus, these advanced sensors have the utmost public importance (Carvalho 2006;
Franz et al. 1999; Grunert 2005; McLaughlin et al. 1999; Shalaby 1996). However,
for the efficient development and use of these nanobiosensors, it is necessary to
O. Konur (B)
Formerly Ankara Yildirim Beyazit University, Ankara, Turkey
366 O. Konur
develop efficient incentive structures for the primary stakeholders and to inform
these stakeholders about the research on the nanobiosensors in the agricultural and
food context (North 1990, 1991; Konur 2000, 2002a, b, c, 2006a, b, 2007a, b).
Scientometric analysis of the research offers ways to evaluate the research in a
respective field (Garfield 1955, 1972). This method has been used to evaluate research
in a number of research fields (Konur 2011, 2012a, b, c, d, e, f, g, h, i, j, k, l, m, n,
2016a, c, d, e, f, 2017a, c, d, f, 2018a, b, 2019a). Although there have been a number
of studies on the nanotechnology in general (Porter et al. 2008; Schummer 2004),
there has been a limited number of pioneering scientometric studies on the various
aspects of the nanobiosensors in recent years (Konur 2016b, 2017b, e, 2019b).
This book chapter presents a study on the scientometric evaluation of the research
on the nanobiosensing and nanobioanalysis of agricultural and food analytes using
two data sets. The first data set includes 100 most-cited papers whilst the second
set includes over 22,000 papers published between 1980 and 2019. The data on the
indices, document types, authors, institutions, funding bodies, source titles, ‘Web
of Science’ subject categories, key words, research fronts, and citation impact are
presented and discussed.
17.2 Materials and Methodology
The search for the literature was carried out in the ‘Web of Science’ database
in January 2020. This database includes ‘Science Citation Index Expanded’
(SCI-E), ‘Social Sciences Citation Index’ (SSCI), ‘Book Citation Index–Science’
(BCI-S), ‘Conference Proceedings Citation Index-Science’ (CPCI-S), ‘Emerging
Sources Citation Index’ (ESCI), ‘Book Citation Index-Social Sciences and Human-
ities’ (BCI-SSH), ‘Conference Proceedings Citation Index-Social Sciences and
Humanities’(CPCI-SSH), and ‘Arts and Humanities Citation Index’ (A&HCI).
The keywords for the search of the literature are developed from the screening of
abstract pages for the first 1,000 highly cited papers. Three sets of keywords were
developed for food and agriculture, sensors and nanotechnology. These keyword sets
are provided in the appendix.
Two data sets are used for this study. The most-cited 100 papers form the first
data set (sample data set, n = 100 papers) whilst all the papers form the second data
set (population data set, n = over 22,400 papers). The data on the indices, document
types, publication years, institutions, funding bodies, source titles, countries, ‘Web
of Science’ subject categories, citation impact, keywords and research fronts are
collated from these data sets.
The key findings are provided in the relevant tables and one figure, supplemented
with explanatory notes in the text. These findings are discussed whilst a number of
conclusions are drawn and a number of recommendations for further study are made.
17 Nanobiosensors in Agriculture and Foods: A Scientometric Review 367
Table 17.1 Document types

Document type Sample data set % Population data set % Difference %
1 Article 95 97.9 −2.9
2 Review 5 1.8 3.2
3 Book chapter 0 0.8 −0.8
4 Proc. paper 0 1.8 −1.8
5 Editorial mat. 0 0.2 −0.2
6 Letter 0 0.1 −0.1
7 Book 0 0.0 0.0
8 Note 0 0.1 −0.1
17.3 Results
17.3.1 Indices and Documents
There are over 24,500 papers related to nanobiosensors in the ‘Web of Science’ as
of January 2020. This original population data set was refined for the document
type (article, review, book chapter, book, editorial material, note, and letter) and
language (English), resulting in over 22,400 papers comprising over 91% of the
original population data set.
The primary index is SCI-E with 97.9% of this refined population data set. The
papers on the social and humanitarian aspects of the nanobiosensors are relatively
negligible with only 3 papers indexed by the SSCI and A&HCI. The BCI-S, CPCI-S,
and ESCI form 0.8, 1.8, and 1.4% of this data set, respectively. On the other hand, the
sole index for the sample data set is the SCI-E. The brief information on the document
types for both data set is provided in Table 17.1. The key finding is that article and
review types of documents are the primary documents and they are under-represented
and over-represented in the sample papers by around 3%, respectively.
17.3.2 Authors
The brief information about the most-prolific 10 authors is provided in Table 17.2.
The sample and population papers are authored by over 450 and 41,600 researchers,
respectively. The most-prolific authors are ‘Chad A. Mirkin’ of Northwestern Univer-
sity of the US working primarily on protein, ion, and pathogen detection and ‘Yuehe
Lin’ of Pacific Northwest National Laboratory. of the US working primarily on the
glucose and pesticide detection with 4 sample papers, each.
The most-prolific institutions for these top authors are the ‘Chinese Academy of
Sciences’ and ‘National Taiwan University’ with 3 and 2 authors from these institutes,
respectively. In total, 7 institutions house these top authors. It is notable that 4 of
368 O. Konur
Table 17.2 Authors

Authors No. papers Inst. Country Research front HCR fields
1 Lin, Yuehe 4 Pacific NW US Glucose and Chem.
Natl. Lab. pesticide
detection
2 Mirkin, Chad 4 Northwestern US Protein, ion and Chem.
A. Univ. pathogen
detection
3 Chang, 3 Natl. Taiwan Taiwan Ion detection
Huan-Tsung Univ.
4 Huang, 3 Natl. Taiwan Taiwan Ion detection
Chih-Ching Univ.
5 Lei, Yu 3 Nanyang Singapore Glucose
Technol. Univ. detection
6 Li, Di 3 Chinese Acad. China Ion detection
Sci.
7 Liu, Guodong 3 New Mexico US Protein,
State Univ. pesticide and
DNA detection
8 Qu, Xiaogang 3 Chinese Acad. China Glucose, ion, Cross-field
Sci. dopamine and
thiol detection
9 Ren, Jinsong 3 Chinese Acad. China Ion, glucose, Cross-field
Sci. dopamine and
thiol detection
10 Wang, Ying 3 Tsinghua China Dopamine,
Univ. glucose and
thiol detection
*HCR: Highly Cited Researchers
these top researchers are listed in the ‘Highly Cited Researchers’ (HCR) list in 2019
(Docampo and Cram 2019).
The most-prolific countries for these top authors are China, the US, and Taiwan
with 4, 3, and 2 authors, respectively. The other country is Singapore. The most-
prolific research front for these top authors is ‘metal ion detection’ with 6 authors.
The other prolific research fronts are ‘glucose detection’, ‘dopamine detection’, ‘thiol
detection’, ‘pesticide detection’, and ‘protein detection’ with 5, 3, 3, 2 and 2 authors,
respectively. It is further notable that there is a significant gender deficit amongst
these top authors as only one researcher is female.
16
14
12
No. Papers (%)
10
Sample Population
Fig. 17.1 The research output between 1980 and 2019
17.3.3 Publication Years
The information about the publication years for both data sets is provided in Fig. 17.1.
This figure shows that whilst 57 and 41% of the sample papers are published in the
2000s and 2010s, respectively, over 90% of the population papers are published in
the 2010s. The research output in the 1980s and 1990s are relatively negligible for
both data sets. Similarly, the most-prolific publication years for the sample data set
are 2009 and 2010 with 12 and 15 sample papers, respectively. On the other hand,
the most-prolific publication years for the population data set are 2015, 2016, 2017,
2018 and 2019 with over 10% of the population papers each.
17.3.4 Institutions
The brief information on the top 8 institutions with at least 3 sample papers is provided
in Table 17.3. In total, 136 and over 6,300 institutions contribute to the sample and
population papers, respectively. These top institutions publish 48 and 12% of the
sample and population papers, respectively. The top institution is ‘Chinese Academy
of Sciences’ with 17 sample papers and with 10.5% publication surplus. All of these
8 institutions are significantly over-represented in the sample papers. The most-
prolific countries for these top institutions are the US and China with 3 institutions
each. Additionally, Taiwan and Singapore have 2 and one institutions, respectively.
370 O. Konur
Table 17.3 Institutions

Institutions Country Sample no. papers Population no. Difference %
% papers %
1 Chinese Acad. Sci. China 17 6.5 10.5
2 Northwestern Univ. US 7 0.8 6.2
3 Nanjing Univ. China 4 1.6 2.4
4 Pacific NW Natl. US 4 0.3 3.7
Lab.
5 Tsinghua Univ. China 4 0.7 3.3
6 Nanyang Technol. Singapore 3 0.8 2.2
Univ.
7 Natl. Hlth. Inst. US 3 0.3 2.7
8 Natl. Taiwan Univ. Taiwan 3 0.7 2.3
9 Natl. Taitung Univ. Taiwan 3 0.3 2.7
17.3.5 Funding Bodies
The brief info about top 11 funding bodies is provided in Table 17.4. It is significant
that only 58% of the sample papers declare any funding. This funding rate is less than
the funding rate, 67%, reported in a study (Wang and Shapira 2011). In total, 140
and over 14,900 funding bodies all over the world fund the sample and population
papers, respectively. The funding rate is higher for the population papers with 84%
funding rate.
The top funding body is the ‘National Natural Science Foundation’ of China,
funding 31 and 39.2% of the sample and population papers, respectively. The
‘National Basic Research Program of China’, ‘National Institutes of Health’, and
‘US Department of Health Human Services’ of the US further fund 12, 9, and 9%
of the sample papers, respectively. The most-prolific country for these top funding
bodies is China with 7 funding bodies. Three funding bodies are from the US whilst
there is one Japanese funding body.
17.3.6 Source Titles
The brief information about the top source titles is provided in Table 17.5. In total, 32
and over 1200 source titles publish the sample and population papers, respectively. On
the other hand, 11 top journals publish 79 and 12.8% of the sample and population
papers, respectively. They are over-represented in the sample papers with 66.2%
publication surplus.
The top journals are ‘Analytical Chemistry’ and ‘Journal of the American Chem-
ical Society’ publishing 16 and 15 sample papers, with 13.1 and 14.7% publica-
tion surplus, respectively. The other top journals are ‘Biosensors Bioelectronics’,
Table 17.4 Funding bodies

Institutions Country Sample no. papers % Population no. Difference %
papers %
1 National Natural China 31 39.2 −8.2
Science Foundation
of China
2 National Basic China 12 4.6 7.4
Research Program of
China
3 National Institutes of US 9 1.9 7.1
Health
4 US Department of US 9 1.9 7.1
Health Human
Services
5 Chinese Academy of China 4 1.5 2.5
Sciences
6 Ministry of Japan 4 1.0 3.0
Education Culture
Sports Science and
Technology Japan
7 National Science US 4 1.9 2.1
Foundation
8 Fundamental China 3 4.3 −1.3
Research Funds for
the Central
Universities
9 Ministry of Science China 3 1.1 1.9
and Technology
China
10 Program for China 3 1.0 2.0
Changjiang Scholars
Innovative Research
Team in University
11 Program for New China 3 1.2 1.8
Century Excellent
Talents in University
‘Chemical Communications’, ‘Nano Letters’, and ‘ACS Nano’. The sample papers
are indexed by 8 subject categories in total. The most-prolific subject categories
are ‘Chemistry Multidisciplinary’, and ‘Nanoscience Nanotechnology’ with 6 and
5 sample papers, respectively. The other prolific categories are ‘Materials Science
Multidisciplinary’, ‘Physics Applied’, ‘Physics Condensed Matter’ and ‘Chemistry
Analytics’.
372 O. Konur
Table 17.5 Source titles

Source titles WOS subject cat. Sample no. Population no. Difference %
papers % papers %
1 Analytical Chem. Analy. 16 2.9 13.1
Chemistry
2 Journal of the Chem. Mult. 15 0.3 14.7
American
Chemical Society
3 Biosensors Biophys., Biot. 11 6.2 4.8
Bioelectronics Appl. Microb.,
Chem. Analy.,
Electrochem.,
Nanosci.
Nanotechnol.
4 Chemical Chem. Mult. 8 1.3 6.7
Communications
5 Nano Letters Chem. Mult., 7 0.3 6.7
Chem. Phys.,
Nanosci.
Nanotechnol.,
Mats. Sci. Mult.,
Phys. Appl., Phys.
Cond. Matt.
6 ACS Nano Chem. Mult., 6 0.5 5.5
Chem. Phys.,
Nanosci.
Nanotechnol.,
Mats. Sci. Mult.
7 Angewandte Chem. Mult. 5 0.3 4.7
Chemie
International
Edition
8 Proceedings of the Mult. Sci. 4 0.3 3.7
National Academy
of Sciences of the
United States of
America
9 Applied Physics Phys. Appl. 3 0.3 2.7
Letters
10 Advanced Chem. Mult., 2 0.3 1.7
Materials Chem. Phys.,
Nanosci.
Nanotechnol.,
Mats. Sci. Mult.,
Phys. Appl., Phys.
Cond. Matt.
11 Nature Nanosci. 2 0.1 1.9
Nanotechnology Nanotechnol.,
Mats. Sci. Mult.
(continued)

Source titles WOS subject cat. Sample no. Population no. Difference %
papers % papers %
79 12.8 66.2
17.3.7 Countries
The brief info about the top countries is provided in Table 17.6. In total 18 and 117
countries contribute to the sample and population papers, respectively. The top county
is China publishing 45 and 51% of the sample and population papers, respectively
with 6% publication deficit. The US follows China publishing 37 and 10% of the
sample and population papers with 27% publication surplus. The other prolific coun-
tries are Singapore, Germany, South Korea, and Taiwan. The European and Asian
countries represented in this table publish altogether 11 and 65% of the sample papers
whilst they publish 4.6 and 74.5% of the population papers, respectively.
It is notable that the publication surplus for the US, these European and Asian
countries is 27, 6.4 and −9.5%, respectively. It is further notable that the bulk of
the Asian publication deficit originates from China and India with −6 and −7.4%
publication deficit, respectively.
Table 17.6 Countries

Countries Sample no. papers % Population no. papers % Difference %
1 China 45 51.0 −6.0
2 US 37 10.0 27.0
3 Singapore 6 1.3 4.7
4 Germany 4 1.8 2.2
5 South Korea 4 5.8 −1.8
6 Taiwan 4 3.6 0.4
7 Unt. Kingd. 3 1.9 1.1
8 India 3 10.4 −7.4
9 Japan 3 2.3 0.7
10 Saudi Arabia 3 1.6 1.4
11 Finland 2 0.2 1.8
12 Israel 2 0.4 1.6
13 Sweden 2 0.8 1.2
14 Europe-4 11 4.6 6.4
15 Asia-5 65 74.5 −9.5
374 O. Konur
Table 17.7 Web of science subject categories

Subjects Sample no. papers % Population no. papers % Difference %
1 Chemistry 48 17.2 30.8
Multidisciplinary
2 Chemistry Analytical 34 42.7 −8.7
3 Nanoscience 30 19.4 10.6
Nanotechnology
4 Materials Science 21 16.4 4.6
Multidisciplinary
5 Chemistry Physical 19 8.6 10.4
6 Physics Applied 14 9.7 4.3
7 Electrochemistry 13 25.4 −12.4
8 Biotechnology Applied 12 7.7 4.3
Microbiology
9 Biophysics 11 7.3 3.7
10 Physics Condensed 11 4.8 6.2
Matter
11 Multidisciplinary 6 1.4 4.6
Sciences
17.3.8 Web of Science Subject Categories
The brief information about the top ‘Web of Science’ subject categories is provided
in Table 17.7. The sample and population papers are indexed by 20 and 133 subject
categories, respectively. The top subject category is ‘Chemistry Multidisciplinary’
publishing 48 and 17.2% of the sample and population papers, respectively with
30.8% publication surplus. Additionally, ‘Chemistry Analytical’ and ‘Nanoscience
Nanotechnology’ index 34 and 30% of the sample papers, with −8.7 and 10.6%
publication surpluses. The other prolific subject categories are ‘Materials Science
Multidisciplinary’, ‘Chemistry Physical’, ‘Physics Applied’, and ‘Electrochemistry’
with 21, 19, 14, and 13 sample papers, respectively.
The subject categories with the large publication surplus are ‘Chemistry Multi-
disciplinary’, ‘Nanoscience Nanotechnology’, ‘Chemistry Physical’ and ‘Physics
Condensed Matter’ with 30.8, 10.6, 10.4 and 6.2% publication surplus, respectively.
On the other hand, the subjects with least impact are ‘Chemistry Analytical’ and
‘Electrochemistry’ with −8.7 and −12.4% publication deficit, respectively.
17.3.9 Citation Impact
These sample papers receive over 51,000 citations as of January 2020. Thus, the
average number of citations per paper is over 500.
17.3.10 Keywords
Although several keywords are listed in the appendix for the data sets of food and
agriculture, sensors and nanotechnology, some of them are more significant. For the
data set of agriculture and food, the most-prolific keyword is ‘glucose’ with 30 occur-
rences. The other prolific keywords are ‘protein*’, ‘ion*’, ‘mercur*’, ‘dopamine’,
‘dna’, and ‘hg + 2’ with over 7 occurrences each at the first instance. The other
keywords are ‘hydrogen peroxide’, ‘ascorbic acid’, ‘virus*’, ‘food*’, ‘pathogen*’,
‘uric acid*’, ‘cysteine’, ‘pesticide’, and ‘nerve agent*’ with over 2 occurrences each.
For the data set of sensors, the most-prolific keywords are ‘detect*’ and
‘*sensor*’with 57 and 47 occurrences, respectively. The other keyword is ‘*sensing’
with 19 occurrences. For the data set of nanotechnologies, the most prolific keyword
is ‘*nano*’ with 93 occurrences. The other prolific keywords are ‘graphene’,
‘single-molecule*’ and ‘metal-organic framework*’ with 19, 2, and 2 occurrences,
respectively.
17.3.11 Research Fronts
The brief information about the key research fronts is provided in Table 17.8. The
most-prolific research fronts for the analytes are ‘glucose detection’, ‘protein detec-
tion’ and ‘toxic metal ion detection’ with 27, 27 and 23 sample papers, respectively.
The other prolific keywords for the analytes are ‘dopamine detection’, ‘hydrogen
peroxide detection’, ‘virus detection’, and ‘thiol detection’.
Table 17.8 Research fronts

Res. fronts-analytes Sample no. papers % Res. Sample papers %
fronts-nanotechnology
1 Glucose 27 Nanoparticles 30
2 Proteins 27 Graphene 19
3 Metal ions 23 Quantum dots 9
4 Dopamine 6 Nanotubes 9
5 Hydrogen peroxide 5 Nanorods 3
6 Viruses 4 Surface-enhanced 3
Raman scattering
7 Thiols 4 Nanoclusters 3
8 Vitamins 3 Metal-organic 3
framework
9 Uric acids 3 Nanocomposites 3
10 Pesticides 2 Nanowires 2
11 Melamine 2 Single molecules 2
376 O. Konur
The most-prolific research fronts for the nanomaterials and nanotechnology are
‘nanoparticles’ and ‘graphene’ with 30 and 19 sample papers, respectively. The other
prolific keywords for in this area are ‘quantum dots’ and ‘nanotubes’ with 9 sample
papers each.
17.4 Discussion
The number of papers on nanobiosensing of agricultural and food analytes has

increased to over 22,000 papers, forming over 1.4% of the research papers on the
nanotechnology. The research has developed more in the technological aspects of
this field, rather than the social and humanitarian pathways as there are only 3 papers
in these pathways in the ‘Web of Science’.
The articles and reviews are under-represented and over-represented by about
3% each in the sample papers, respectively (Table 17.1). Thus, the contribution of
reviews by 5% of the sample papers in this field is not highly exceptional (c.f. Konur
2016a, b, c, d, e, f, 2017a, b, c, d, e, 2019b).
Ten authors from 7 institutions have 3 or more sample papers (Table 17.2). These
prolific authors are mainly from China, the US, and Taiwan. These authors focus on
‘metal ion detection’, ‘glucose detection’, ‘dopamine detection’, ‘thiol detection’,
‘pesticide detection’, and ‘protein detection’. It is significant that there is ample
‘gender deficit’ amongst these top authors as only one researcher is female.
Whilst 57 and 41% of the sample papers are published in the 2000s and 2010s,
respectively, over 90% of the population papers are published in the 2010s (Fig. 17.1).
This finding suggests that the population papers have built up on the sample papers,
primarily published in the 2000s and 2010s, in the 2010s. These data suggest that
this field of research is an ‘emerging research’, booming in the 2010s (Rotolo et al.
2015; Wang 2018).
The engagement of the institutions on the research in the nanobiosensing of agri-
cultural and food analytes at the global scale is significant as 130 and over 6,300
institutions contribute to the sample and population papers, respectively. Eight top
institutions publish 48 and 12% of the sample and population papers, respectively
(Table 17.3). The top institutions are ‘Chinese Academy of Sciences’ ‘of China and
‘Northwestern University’ of the US with 17 and 7 sample papers, respectively as
they are significantly over-represented in sample papers with 10.5 and 6.2% publi-
cation surplus. As in the case of the top authors, most-prolific countries for these top
instructions are the US and China with 3 institutions each.
It is significant that only 58% of the sample papers declare any funding. The
most-prolific country for these top funding bodies is China with 7 funding bodies
(Table 17.4). The other 3 funding bodies are from the US. The top funding body
is the ‘National Natural Science Foundation of China’ (NSFC) of China, funding
31% and 39.2% of the sample and population papers, respectively, and with 8.2%
publication deficit. These findings are in line with the studies showing the heavy
research funding in China and the NSFC is the primary funding agency in China
(Wang et al. 2012).
The sample and population papers are published by 32 and over 1,200 journals,
respectively. It is significant that 11 top journals publish 79 and 12.8% of the sample
and population papers, respectively (Table 17.5). The top journals ‘Analytical Chem-
istry’, ‘Journal of the American Chemical Society’, and ‘Biosensors Bioelectronics’
publish together 42 sample papers with 32.6% publication surplus. It is significant
that there are no journals related to ‘Agriculture’ and ‘Food Science and Technology’
in this top journal table. On the contrary, there are 3 journals related to ‘Nanotech-
nology’ in this top journal table. The most-prolific subject categories are ‘Chemistry
Multidisciplinary’, and ‘Nanoscience Nanotechnology’.
In total, 18 and 117 countries contribute to the sample and population papers,
respectively. The top country is China publishing 45 and 51% of the sample and
population papers, respectively with 6% publication deficit (Table 17.6). The US
follows China publishing 37 and 10% of the sample and population papers with 27%
publication surplus. This finding is in line with the studies arguing that the US is not
losing ground completely in science and technology (Leydesdorff and Wagner 2009).
The other prolific countries are Singapore, Germany, South Korea, and Taiwan. These
findings are in contrast to the studies showing that European countries have superior
publication performance in nanotechnology (Youtie et al. 2008).
The European and Asian countries represented in this table publish altogether 11
and 65% of the sample papers whilst they publish 4.6 and 74.5% of the population
papers, respectively. It is notable that the publication surplus for the US, these Euro-
pean and Asian countries is 27, 6.4 and −9.5%, respectively. It is further notable that
the bulk of the Asian publication deficit originates from China and India with −6 and
−7.4% publication deficit, respectively. This finding is in contrast with China’s and
India’s efforts to be a leading nation in science and technology (Zhou and Leydes-
dorff 2006) but it is in line with the findings of Guan and Ma (2007), Youtie et al.
(2008), and Karpagam et al. (2011).
The sample and population papers are indexed by 20 and 133 subject categories,
respectively. For the sample papers, the top subject categories are ‘Chemistry Multi-
disciplinary’, ‘Chemistry Analytical’ and ‘Nanoscience Nanotechnology’ with 48,
34 and 30 papers, respectively (Table 17.7). The other prolific subject categories are
‘Materials Science Multidisciplinary’, ‘Chemistry Physical’, ‘Physics Applied’ and
‘Electrochemistry’. It is notable that the publication surplus is most significant for
‘Chemistry Multidisciplinary’ with 30.8% surplus. The other high-impact subjects
are ‘Nanoscience Nanotechnology’, ‘Chemistry Physical’, and ‘Physics Condensed
Matter’. On the other hand, the subjects with least impact are ‘Chemistry Analyt-
ical’ and ‘Electrochemistry’ with 8.7 and 12.4% publication deficit, respectively.
These sample papers receive over 51,000 citations as of January 2020. This is highly
exceptional (Konur 2016a, b, c, d, e, f, 2017a, b, c, d, e, 2019b).
Although several keywords are listed in the appendix for the data sets of data sets
of analytes, sensors and nanotechnology, some of them are more significant. For the
data set of agriculture and food, the most-prolific keyword is ‘glucose’ with 30 occur-
rences. The other prolific keywords are ‘protein*’, ‘ion*’, ‘mercur*’, ‘dopamine’,
378 O. Konur
‘dna’ and ‘hg + 2’ with over 7 occurrences each at the first instance. The other
keywords are ‘hydrogen peroxide’, ‘ascorbic acid’, ‘virus*’, ‘food*’, ‘pathogen*’,
‘uric acid*’, ‘cysteine’, ‘pesticide’ and ‘nerve agent*’ with over 2 occurrences each.
For the data set of sensors, the most-prolific keywords are ‘detect*’ and ‘*sensor*’
with 57 and 47 occurrences, respectively. The other keyword is ‘*sensing’ with 19
occurrences.
For the data set of nanotechnologies, the most prolific keyword is ‘*nano*’ with
93 occurrences. The other prolific keywords are ‘graphene’, ‘single-molecule*’, and
‘metal-organic framework*’ with 19, 2 and 2 occurrences, respectively.
As expected, these keywords provide valuable information about the pathways of
the research on the nanobiosensing of agricultural and food analytes. Pragmatically,
11 research fronts emerge from the examination of the sample papers for both the
analytes and nanomaterials (Table 17.8).
The most-prolific research fronts for the analytes are ‘glucose detection’, ‘protein
detection’, and ‘toxic metal ion detection’ with 27, 27 and 23 sample papers,
respectively. The other prolific keywords for the analytes are ‘dopamine detection’,
‘hydrogen peroxide detection’ ‘virus detection’ and ‘thiol detection’. The most-
prolific research fronts for the nanomaterials and nanotechnology are ‘nanoparti-
cles’ and ‘graphene’ with 30 and 19 sample papers, respectively. The other prolific
keywords in this area are ‘quantum dots’ and ‘nanotubes’ with 9 sample papers
each. The key emphasis in these research fronts is the exploration of the structure-
processing-property relationships for these nanobiosensors for nanobiosensing agri-
cultural and food analytes (Konur and Matthews 1989; Nam et al. 2003; Song et al.
2010; Dong et al. 2012; Shan et al. 2009; Lee et al. 2007).
17.5 Conclusion
This chapter maps the research on the nanobiosensing and nanobioanalysis of agri-
cultural and food analytes using a scientometric method. The significant size of
over 22,000 population papers shows the public importance of this interdisciplinary
research field. However, it is significant that the research has developed more in the
technological aspects of these issues, rather than the social and humanitarian path-
ways. It is also significant to note that the research in this field has peaked in the
2010s with over 90% of the population papers as an ‘emerging research’ field.
Articles and reviews dominate the sample papers, primarily published in the 2000s
and 2010s. This is also relevant for the population papers, mostly published in the
2010s. The data presented in the tables and in one figure show that a small number
of authors, institutions, funding bodies, journals, keywords, research fronts, subject
categories, and countries have shaped the research in this field.
It is notable that the authors, institutions and funding bodies from China dominate
the research in this field. Furthermore, China and other Asian countries represented
in the top country table are dominant in both sample and population papers. These
findings show the importance of the development of efficient incentive structures for
the development of the research in this field as in other fields. It seems that the US
and, to a lesser extent, European countries, have efficient incentive structures for the
development of the research in this field, contrary to the Asian countries. It is likely
that the key reason behind that surge in the number of population papers during the
last decade has been the development of efficient incentive structures in this field. It
is also likely that with the continuation of these incentive structures in this field, the
number of papers would continue to rise, reaching to over 50,000 papers at the end
of the 2020s.
However, it seems there is more to do to reduce significant gender deficit in this
field as in other field of the biomedical research (Xie and Shauman 1998). It further
seems that although the research funding is a significant element of these incentive
structures, it might not be a sole solution for increasing the incentives for the research
in this field as in the case of Asian countries.
The data on the research fronts, keywords, source titles and subject categories
provides valuable evidence for the interdisciplinary nature of the research in this
field. These findings are in line with studies arguing for the interdisciplinarity
of the research in nanomaterials (Schummer 2004; Meyer and Persson 1998).
As only 19.4% of the population papers are indexed by the subject category of
the ‘Nanoscience and Nanotechnology’, there is ample justification for the broad
search strategy employed in this study. It is also significant that the contribution of
agriculture-related subject categories to the research in this field is relatively negli-
gible with about 1% contribution. The contribution of ‘Food Science and Technology’
is 7.5%.
The search strategy employed in this study is in line with the search strategies
employed for nanotechnology research in the relevant studies (Porter et al. 2008;
Mogoutov and Kahane 2007; Arora et al. 2013). Pragmatically, 11 research fronts
for both analytes and nanomaterials emerge from the examination of the sample
papers. The most-prolific research fronts for the analytes are ‘glucose detection’,
‘protein detection’ and ‘toxic metal ion detection’ with 27, 27 and 23 sample papers,
respectively. The other prolific keywords for the analytes are ‘dopamine detection’,
‘hydrogen peroxide detection’, ‘virus detection’ and ‘thiol detection’.
The most-prolific research fronts for the nanomaterials and nanotechnology are
‘nanoparticles’ and ‘graphene’ with 30 and 19 sample papers, respectively. The other
prolific keywords for in this area are ‘quantum dots’ and ‘nanotubes’ with 9 sample
papers each.
It is recommended that further scientometric studies are carried out for each of
these research fronts building on the pioneering studies in these fields. Such studies
might also be carried out for special types of nanomaterials such as nanoparticles,
graphene and quantum dots.
Acknowledgements The contribution of the highly cited researchers in the field of nanobiosensing
and nanobioanalysis of agricultural and food analytes has been gratefully acknowledged.
380 O. Konur
Appendix
Syntax: I and II and III

I Food and agriculture-related keywords
(TI = (*food* or *agr* or *pathogen* or pesticid* or “nerve agent*” or *toxin* or
contaminant* or meat* or nutr* or dairy or adulteran* or milk* or melamine* or
feed* or plant* or escherichia or listeri* or salmonella or *bacter* or campylobacter
or insecticid* or organophosphat* or organophosphor* or (enzyme and (sensing or
detection)) or tyrosin* or (food* and (*toxic* or analysis or safety)) or “polybromi-
nated diphenyl ether*” or carbohydrate* or carcin* or staphylococc* or “hydrogen
peroxide” or *microb* or vibrio or h2o2 or bacillus or shigella or *carbamate* or
*bisphenol or *phenol* or antibiotic* or *virus* or poultry or vegetable* or fruit*
or protein* or chloramphenicol or sulfamethazine or sulfadimidine or “e-coli” or
sudan or chicken or tartrazine or colourant* or “ascorbic acid*” or lovastatin or
rice or chicken or glutathione or “sunset yellow” or colourant* or clenbuterol or
“crystal violet” or “*malachite green” or dye* or sulfonamide* or acetaminophen or
arabidopsis or streptomycin or “picric acid” or nitrophenol or tyramine or phosmet or
thiabendazole or concanavalin or carbofuran or mercury or “hg2+” or zearalenone
or enrofloxacin or estradiol or *dopamine or “uric acid” or *epinephrine or pork
or goat* or sheep or carbaryl or maize or acetamiprid or apple* or fungicide or
tocopherol or prion or corn or ellagic or gallic or punica or myrtus or itriphal
or anthelmintic* or rhizosphere or amlodipine or hydrochlorothiazide or fish* or
honey or *tetracycline or glyphosate or herb* or allerg* or *albumin or wheat or
gliadin or rutin or *flavonoid* or amaranth* or *cysteine* or biothiol* or salbu-
tamol or residue* or ions or phenylethanolamine or tomato* or cholesterol or “soft
drink*” or carmoisine or arsenic or *xanthine or glucose or pyrophosphate or “tert-
butylhydroquinone” or chlorpyrifos or theanine or “parathion-methyl” or chilli or
tricyclazole or ricin or sulfaquinoxaline or ciprofloxacin or gelatin* or methyldopa
or methamidophos or “lactic acid” or tocotrienol* or triclosan or diet* or orange or
patulin or pyrimethanil or fumonisin or deoxynivalenol or kanamycin or carbendazim
or “cr(iii)” or nitrofuran or juice or gallate or ponceat or dichlorvos or risperidone
or brucella or paracetamol or crop* or parathion or pear* or apple* or fusarium or
morphine or capsaicin or wine* or omethoate or peach* or thiabendazole or histamine
or beverage* or digoxin or parathion or eggs or freshness or bleomycin or nitrate
or nitrite or trimethylamine or “l-dopa” or acetylcysteine or lysine or honokiol or
magnolol or saccharide* or carmine or phenylenediamine or curcum* or “methy-
lene blue” or aurantifolia or tobramycin or benzimidazole or mucin or tryptophan
or polymyxa or phosmet or linezolid or bean* or hypochlorous or folic or sesam*or
iodide or sulphite or infant) or WC = (food* or agr* or nutr* or hort* or plant*)
NOT TI = (sensory or lithium*)
II Sensor-related keywords
TI = (*sensor* or *sensing or interferomet* or ellipsomet* or *detect*)
III Nanotechnology-related keywords
(TI = (*nano* or “quantum dot*” or “quantum-dot*” or plasmonic* or triboelec-

tric* or “carbon dot*” or skyrmion* or dendrimer* or “carbon onion*” or “exfoliated
graphite*” or “atomic crystal*” or “langmuir-blodgett” or “c-dot*” or electrospin-
ning or ((graphit* or carbon) and *tubule*) or semimetal* or “polymer dot”) OR TI =
(*fulleren* or pcbm or *c60* or *c61* or *c70* or *c120* or “*butyric acid methyl
ester*” or pc70bm or cnt or swnt or mwnt or swcnt or mwcnt or “c-60” or “c-61” or “c-
70” or “c-120” or fulleri* or fullero* or buckyball*) OR TI = (mos2 or *graphene*
or “transition metal dichalcogenide*” or mose2 or ws2 or wse2 or “black phos-
phorus” or phosphorene or “two-dimensional material*” or “low-dimensional mate-
rial*” or “molybdenum disulfide” or “boron nitride layer*” or “g-c3n4” or mxene*
or “2d *material*” or “atom thick” or “atomically thin” or graphane* or “graphitic
carbon nitride*” or “atomic layer*” or “two-dimensional carbon*” or tmdc* or “waals
heterostructure*” or “two-dimensional titan*” or “two-dimensional carbide*” or “2d
crystal*” or “two-dimensional crystal*”) OR TI = (“inorganic-organic hybrid*”
or “organic-inorganic hybrid*” or “metal-organic framework*” or mof* or meso-
porous or microporous or “hybrid inorganic-organic” or “hybrid organic-inorganic”
or “coordination polymer*” or “zeolitic imidazolate framework*” or “*molecular
sieve*” or “metal-organic material*” or “covalent organic framework*” or cofs or
mesopore* or “organometal halide perovskite*” or “hybrid halide perovskite*” or
mesocrystal*) OR TI = (“atomic force microscop*” or afm or “scanning tunneling
microscop*” or “fluorescence microscop*” or sted or “stimulated emission depletion
microscop*” or “localized surface plasmon resonance” or lspr or “surface-enhanced
ramanspectroscop*” or sers or “scanning probe microscop*” or “optical recon-
struction microscop*” or “dynamic force spectroscop*”) OR TI = (“*molecular
machin*” or “*molecular motor*” or “*molecular self-assembl*” or superlens
or “quantum wire*” or “quantum well*” or “molecular photov*” or “*molecular
assembl*” or “*molecular wire*” or “*molecular device*” or supramolecul* or
“*molecular engineering” or “single atom*” or “single molecul*” or “dna machine*”
or “molecular rotor*” or “molecular electronic device*” or “molecular biomimetic*”
or “*molecular junction*” or “atomic scale” or “sub nm” or “single layer*” or
“atomic sized” or nems) OR SO = (“ACS Nano” or “Advances in Nano*” or “Applied
Nanoscience” or “Beilstein Journal of Nano*” or “Cancer Nano*” or Chem Nanomat
or “Current Nano*” or “Environmental Science—Nano” or “Frontiers of Nano*” or
Fullerenes* or “IEEE Transactions on Nano*” or “IET Nano*” or “International
Journal of Nano*” or “Journal of Biomedical Nano*” or “Journal of Computa-
tional and Theoretical Nano*” or “Journal of Experimental Nano*” or “Journal of
Nano*” or “Microporous and Mesoporous*” or Nano* or “Nature Nano*” or “NPJ
2d” or “Recent Patents on Nano*” or “RSC Nano*” or Small* or “Wiley Inter-
disciplinary Reviews—Nano*”) or SO = (“ACS Applied Nano*” or “Advances in
Natural Sciences—Nano*” or “European Journal of Nano*” or “IEEE Nano*” or
“International Journal of Nano*” or “International Nano*” or “Journal of Nano*” or
Nano* or “Proceedings of the Institution of Mechanical Engineers Part N-Journal
of Nano*”)) NOT TI = (nanog or nanogram* or “nano-gram*” or nanosecond* or
382 O. Konur
“nano-second*” or nanoliter* or nanolitre* or nanokelvin or nanoamper* or “nano-

amper*” or nano2 or nano3 or nanoplan* or nanoflag* or nanoprot* or nanobacter*
or nanor or nanobody or nanostring or nanosatel*)
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Part III
Biosensors in Animal and Fishery Sciences
Chapter 18
Biosensors: Modern Tools for Disease
Diagnosis and Animal Health Monitoring
Anuj Tewari, Beenu Jain, Basanti Brar, Gaya Prasad, and Minakshi Prasad
Abstract Animals play a vital role in our lives as they provide milk, meat, and other
by-products for daily consumption. At the same time, their health directly/indirectly
affects human beings as we both share a common environment. The One Health
concept makes it imperative that the animals should remain healthy as it will have
an impact on our health as well as economy. Animal disease diagnosis, their health
monitoring, prevention, and control of diseases are important from this aspect. Tools
for rapid diagnosis of animal diseases are crucial for early diagnosis and imposing
control measures. Molecular techniques such as, genome sequencing, restriction
fragment length polymorphism (RFLP), DNA microarray, PCR, and real-time PCR
are some of the rapid techniques, but they need expertise and sophisticated labs.
Conventional methods such as isolation of the pathogen, serological techniques, are
laborious and time taking process. Therefore, to overcome the said limitations of
conventional and molecular tools, biosensors are the better alternatives as they are
rapid and can be used as pen-side diagnostic tests. They can provide test results in a
few minutes under field conditions. In this chapter, we give an elaborative description
of various types of biosensors and their utility in animal disease diagnostics and health
monitoring.
Keywords Biosensors · Animal disease diagnosis · Field-based tests · Rapid

tests · Animal health monitoring
A. Tewari (B)
Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Govind
Ballabh Pant University of Agriculture and Technology, Pantnagar 263145, Uttarakhand, India
B. Jain
Department of Animal Husbandry, Lucknow, Uttar Pradesh, India
G. Prasad
International Institute of Veterinary Education and Research, Rohtak, Haryana, India
B. Brar · M. Prasad (B)
Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University
of Veterinary and Animal Sciences, Hisar, Haryana, India
388 A. Tewari et al.
18.1 Introduction
Globally, infectious diseases are the leading cause of mortality in man and animals.
Infectious diseases of both domestic and wildlife pose a serious health risk to the
human race as exhibited by the current ongoing COVID-19 pandemic. These infec-
tious diseases can be of zoonotic origin affecting not only human health, but also,
animal health and production. Such diseases pose a serious threat to a country’s
growth, economy, and health perspectives. To prevent the spread of infectious
diseases it is imperative to develop rapid and sensitive tests for early detection of the
causative agents. It aids in adopting suitable preventive measures, compartmental-
ization of the infected zones, and designing strategies to prevent further spread of the
disease. The viral agents can be detected in the clinical samples by gold standard virus
isolation in cell culture system. This method has well-known merits and demerits
such as economic investment, expert manpower, and time-consuming lengthy proto-
cols (from days to weeks). On the other hand, molecular detection methods such as
conventional Polymerase Chain Reaction (PCR), Real-time PCR, although they are
both sensitive and specific, cannot be employed for pen-side diagnosis due to the
involvement of sophisticated instruments.
In such a scenario, the development of a rapid, sensitive, and specific pen-side test
is the need of the hour and is a challenge for the scientific community to combat the
emerging and re-emerging diseases. With the limited infrastructure for the diagnosis
of the emerging and re-emerging infection, biosensors can be an effective tool to
perform field-based diagnosis and animal health monitoring. Easy handling, user-
friendly, and minimum processing endows biosensor an effective tool for disease
diagnosis.
In the past few decades, a lot of work has been done on this aspect for the detec-
tion of both viral and bacterial pathogens. Various molecules like nucleic acids, anti-
bodies, antigens, and proteins of animal origin are detected in the clinical samples
in these biosensing elements.
18.2 Biosensors Principles and Types
According to The National Research Council (part of the US National Academy

of Science), a biosensor is defined as a detection device that incorporates a
living organism or product derived from a living system as the biorecognition
element (BRE) or a bioreceptor and a transducer (electrochemical, optical, or mass-
based) to convert a biological reaction into a quantifiable signal. The BRE can be
enzymes, nucleic acids, antibodies, aptamers, bacterial cells, bacteriophages. The
first biosensor was found by Clark and Lyon in 1962 for glucose detection.
The boost up in the field of biosensor came from the pioneering work done by a
group of scientists in the year 1962–1969 (Clark and Lyons 1962; Guilbault et al.
18 Biosensors: Modern Tools for Disease Diagnosis … 389
1962; Updike and Hicks 1967; Guilbault and Montalvo 1969). Later on, an elec-
trochemical biosensor had been described using ferrocene to detect electroactive
species (Di Gleria et al. 1986). This invention led to the commercialization of a
glucose pen by the company Medisens. After these reports, there has been a large
amount of work done in the domain of biosensors which has an extensive application
for environmental sensing and biological monitoring (Luong et al. 2008).
The principle of a biosensor is based on the detection of a biomarker molecule in
the clinical sample (Fig. 18.1). This biomarker can be any protein, antigen, or anti-
body of the infectious agent. The biomarker molecule is detected by the bioreceptor
which is immobilized on a chip or another base. This bioreceptor could be DNA,
RNA, monoclonal antibody, protein, or any cell. However, it is important to choose
the bioreceptor carefully as it is the deciding factor for the sensitivity and specificity
of a biosensor. Although there are several types of bioreceptors, the most common
types used are nucleic acids, enzymes, and antibodies. The interaction between the
biomarker and bioreceptor generates signals via transductor which are read and inter-
preted. The signals generated give information regarding the presence or absence of
the pathogen in the sample.
Biosensors can be classified on the basis of biorecognition elements or transducer,
more details are discussed below.
Fig. 18.1 Principle of biosensor for disease/pathogen detection

18.2.1 Biorecognition Element (BRE)
The type of BRE to be used depends on the target molecule to be recognized. Each
of the BRE has its own advantage and disadvantage, hence, its selection depends on
the application. Various types of BREs used are as follows:
18.2.1.1 Antibodies
Antibodies are the most commonly used BRE and their use does not require antigen
purification during sample addition (Chambers et al. 2008). Monoclonal, polyclonal,
and recombinant antibodies can be used. Monoclonal antibodies recognize a single
antigenic epitope hence detects the antigen with less sensitivity in comparison to the
polyclonal antibodies. Polyclonal antibodies recognize several epitopes and are able
to detect the antigen with more sensitivity than the monoclonal antibodies. However,
polyclonal antibodies show batch-to-batch variation in titre and reactivity in compar-
ison to monoclonal antibodies which show unique specificity in all batches (Nelson
et al. 2000). The third generation of antibodies known as recombinant antibodies are
less expensive and time-consuming in production (Sharon et al. 2005; Haurum 2006;
Chambers et al. 2008).
18.2.1.2 Nucleic Acids
Nucleic acids (NAs) can bind with the complementary sequence present in the sample
through hybridization (Wetmur and Fresco 1991). One of the class of NAs that is
used as BRE is Nucleic Acid Aptamers (NAAs) which are capable of binding two
proteins (Ni et al. 2011). Such kind of biosensors which employ the use of NAAs are
called as genosensors (Tosar et al. 2010; Martinkova et al. 2017). NAAs are high-
affinity short stretch of oligonucleotides (Chambers et al. 2008). NAAs are easy to
synthesize, are foldable, and easy to store, hence, they offer similar attributes as
antibodies. Disadvantages of NAAs are degradation, cross-reactivity, and labeling
costs (Chambers et al. 2008; Lakhin et al. 2013). Aptazymes are types of aptamers
which have catalytic properties like enzymes (Chambers et al. 2008). Other types
of aptamers are peptide nucleic acids (PNAs) which are synthetic DNA analogues
having polyamide backbone (Chambers et al. 2008; Bonifazi et al. 2012; Briones
and Moreno 2012; Gupta et al. 2017). Since, PNAs do not have any charge they
have higher hybridization characteristics than NAs (Bonifazi et al. 2012; Briones
and Moreno 2012).
18.2.1.3 Enzymes and Proteins
Enzymes are exclusively specific for their substrate hence are an excellent BRE for
biosensors. Enzymes consist of protein components, cofactors, or prosthetic groups
(Elnashar 2010). Enzymes are used extensively for biosensing purposes as they can
measure a variety of reactions and the products generated thereof.
18.2.1.4 Cells
In certain cases, the immobilization of the enzymes may be difficult, or the isolation
and purification of the enzymes may not be possible. In such a scenario, the whole
cells offer a better alternative than enzymes as the enzymes inside the cell are stable
for a long time. Also, the cells have multiple enzymes that can be used for several
analytes in a single testing. The immobilized cells can also be genetically modified
to express specific proteins to detect the analytes in the extracellular matrix (Fine
et al. 2006; Jung et al. 2014; Vopálenská et al. 2015).
18.2.1.5 Biomimetics
In these kinds of biosensors, the non-biological receptors are used as BREs. These
biosensors can mimic the original biological bioreceptor. The various forms of
biomimetics are molecular imprinted polymers (MIP), membrane mimics, self-
assembled monolayers (SAM), nanoparticles, nanostructured materials, and quantum
dots (QDs). Molecular imprints aim to design artificial receptors like artificial anti-
bodies. Nanoparticles and nanostructures along with other receptors can also act as
biomimetics.
18.2.2 Classification of Biosensors on the Basis

of Transducer
Transducers are the important components that determine the specificity of a

biosensor as they are responsible to translate the biorecognition signals to measur-
able signals. Biosensors are mainly classified as Electrochemical, Mass-based, and
Optical biosensors. These are further subclassified.
18.2.2.1 Electrochemical Biosensors
The electrochemical (EC) biosensors are the most common types of biosensors. They
are inexpensive and have good bio-interaction. Due to the biochemical interaction on
the sensor surface, the potential difference is measured as a recognizable signal. These
biosensors have been further classified into conductometric, impedimetric, potentio-

metric, and amperometric types which are based on the conductance, impedance,
potential, and current generation, respectively (Lazcka et al. 2007; Setterington and
Alocilja 2012).
The EC biosensors are the most successful biosensors which have been used
commercially. However, the disadvantage associated with the EC biosensor is the
random orientation and their immobilization without getting denatured (Jung et al.
2008). Potentiometric sensors measure the change in the potential at the electrode
happening after the recognition of the analyte. However, this method comes with a
limitation that for large molecules (bacteria) it cannot provide sensitive and specific
signals. Amperometric sensors are the kind of sensors in which current is generated
after oxidation or reduction due to analyte and bioreceptor interaction. The most
common example of this type of biosensor is the measurement of glucose which
is based on the amperometric sensing of glucose. These kinds of sensors impart
good sensitivity but low specificity. Impedimetric sensors are the kind of biosensors
which are good for detection of whole bacteria. The sensors are label-free, of low
cost, high sensitivity, and are not affected by other analytes present in the sample
matrix. Moreover, these can be miniaturized easily, hence, easily translated to the
point of care systems. Conductometric biosensors measure the ability of an analyte
to conduct current between electrodes or the reference electrodes. Several enzymatic
reactions are capable of changing the ionic strength of the sample which can then be
measured by a conductometric instrument.
With the recent advances in nanotechnology, the incorporation of nanoparti-
cles has improved the diagnostic performance of the electrochemical biosensors by
increasing the signal transduction. The signal transduction, which can be increased
by using the nanomaterials along with the BREs can increase the signal significantly
(Fig. 18.2). Nanoparticles provide increased surface area for more binding of the
analyte and also have high conductance which results in increased signal intensity.
The nanomaterials used are gold nanoparticles, iron oxide, graphene, and carbon
nanotubes.
18.2.2.2 Optical Biosensors
Optical biosensors are useful in attending high specificity, sensitivity, and rapidity. It
has been further divided into label-free and real-time detection. Based on the mech-
anism of the signal generation they have been further categorized as colorimetric
sensors, surface plasmon resonance (SPR), fluorescence-based sensors, surface-
enhanced Raman spectroscopy (SERS), and chemiluminescence-based sensors.
Fig. 18.2 Nanomaterials-based electrochemical biosensor
18.2.2.3 Mass-Based Biosensors
These biosensors are based on the principle of signal production which depend on
the mass of chemicals interacting with a vibrating piezoelectric quartz crystal. Mass-
based biosensors are label-free and offer increased sensitivity and specificity. The
signal transduction is based on the minute change in the mass occurring on the sensor
surface and is sensed by resonance phenomena (Turner and Zhang 2001). Various
categories of mass-based biosensors are microcantilever-based (MCL) biosensors,
surface acoustic wave (SAW) biosensors, and quartz crystal microbalances (QCM).
18.3 Applications of Biosensors in Animal Husbandry
Biosensors have a large application in the detection of pathogens, drug residues,

food quality monitoring, animal location monitoring, and animal health monitoring
(Fig. 18.3). Some of the details of the application of biosensors have been mentioned
below.
18.3.1 Biosensors for Detection of Bacterial Infection
18.3.1.1 Detection of Escherichia coli
Escherichia coli causes disease related to gastroenteritis, urinary tract infection,

peritonitis, and septicemia in cattle, sheep, goats, pigs, and poultry. E. coli is a
gram-negative rod-shaped bacterium. For example, in poultry, it causes colibacillosis
followed by the localization of this pathogen in the respiratory and urinary tract of
the birds. The virulent strains causing colibacillosis are O1: K1, O78: K80, and O2:
K1. In poultry, E. coli infection is characterized by reduced growth rate, decreases
egg production, and high mortality subsequently causing heavy economic loss to
the stakeholders. E. coli causes colibacillosis in other farm animals also like cattle,
goats, and pigs (Bélanger et al. 2011). In newborn calves it causes septicemia and
in milking cattle causes mastitis. Moreover, due to the excessive use of antibiotics,
there is an increased incidence of antibiotic resistance in E. coli. Thus, it becomes
Fig. 18.3 Probable applications of biosensor in animal husbandry

imperative to detect the virulent strains of E. coli and to differentiate between them
from the non-pathogenic strains.
The conventional and molecular methods used for the detection of E. coli infection
are isolation of bacteria from clinical samples, ELISA, and PCR. However, these
methods involve high cost, time, and manpower (Hobson et al. 1996; Fournier-Wirth
et al. 2006). To overcome these hurdles advanced methods involving the use of
biosensor have been explored. For detection of E. coli, the novel biosensors such as
surface plasmon resonance (Yazgan et al. 2014), quartz crystal microbalance system
(Jiang et al. 2011), chemiluminescence (Zhang et al. 2014), and electrochemistry
(Kim et al. 2015a; Guo et al. 2016; Idil et al. 2017) have been explored. The E. coli
cells are captured by specific antibodies directed against E. coli antigens like flagella.
The signals generated are subsequently amplified by using suitable detectable labels
such as fluorophores, enzymes, and biofunctionalized nanoparticles. The biosensors
based on sandwich format have been developed for the rapid detection of E. coli. In an
electrochemical biosensor (Jaffrezic-Renault et al. 2007) the E. coli cells are captured
by anti-LPS antibodies coupled with magnetic nanoparticles which are conducted
on the graphite ink electrode. The use of immunomagnetic beads aid in the detection
of analyte present in the test sample.
The conductometric method can detect E. coli from the culture from 1 to 103
CFU mL−1 . The immunosensors based on conductometric method have been used
successfully for detection of Pseudomonas aeruginosa and Acinetobacter baumannii
from 10 to 103 CFU mL−1 . These pathogens were found to be undetectable using
immunoblot methods. This test was also found to be specific as the gram-positive
bacteria Staphylococcus epidermidis was found to be undetectable (El Ichi et al.
2014).
A point of care test using stacked paper membranes has also been proposed (Eltzov
and Marks 2016, 2017) which can quantify E.coli in less than five minutes. When the
infected clinical sample is added to the bottom layer of the membrane, the bacterial
cells are pushed up to the top layer during which they bind with anti-E. coli anti-
bodies conjugated with horseradish peroxidase (HRPO) which allows measurement
of generated signals. In the case of the negative sample, the anti- E. coli antibodies
are prevented to move to the topmost membrane due to their binding with the immo-
bilized bacterial cells on the capture layer. HRPO specific substrate produces signals
only with E. coli cells bound with HRPO labeled antibodies. This test is 1000 times
more sensitive than ELISA and was able to quantify upto 100 cells mL−1 . Other
added advantages were its rapidity and portability.
18.3.1.2 Detection of Clostridium Perfringens
C. perfringens is an important enteric pathogen and all domestic animals and poultry
are infected by this pathogen. It is a gram-positive spore-forming bacteria with 17
different toxins (Gibert et al. 1997; Petit et al. 1999) and is also an important agent of
foodborne illness. Cell-based biosensors are one of the emerging technologies which
have mammalian cells as sensing elements to detect the pathogens or toxins in the
clinical and food samples (Yoo and Lee 2016).
For this purpose, the mammalian cells fixed on 96 well plates and modified elec-
trodes have been used (Banerjee and Bhunia 2010) to detect C. perfringens and their
toxins in the samples, as each type of C. perfringens carries a unique sequence of
virulence genes. In a microarray system, oligo probes against six toxin-producing
sequences of C. perfringens have been designed and used successfully (Sergeev et al.
2004). The fluorescently labeled PCR amplicon is hybridized with the oligoprobes
which is subsequently read to detect the presence of C. perfringens in the test sample.
This platform enables the simultaneous detection of several bacterial strains and their
toxins detected simultaneously (Volokhov et al. 2011).
Toxins of C. perfringens can also be detected by using specific antibodies in an
immunosensor. It has been used successfully for the detection of epsilon toxin from
C. perfringens (Palaniappan et al. 2013). The monoclonal antibody specific to the
epsilon toxin was fixed on the carbon nanotube and was able to detect toxins with
comparable sensitivity to ELISA.
18.3.1.3 Detection of Campylobacter
Campylobacter causes disease in wild animals, pets, domestic livestock, and human
beings. C. jejuni and C. coli mainly infect cattle sheep, dogs, cats, pigs, and turkeys.
C. fetus mainly infects cattle, goats, and sheep. Campylobacter causes enteritis with
diarrhea, fever, vomiting, abortion, and infertility. To detect Campylobacter, various
bioreceptor molecules like proteins, aptamers, nucleic acids, antibodies, and cells
have been used. Out of these, DNA-based biosensors are very useful due to their low
cost, specificity, and less time required to detect the pathogen.
An organic light-emitting diode (OLED) biosensor has been used for the detec-
tion of Campylobacter in meat samples (poultry) through capturing Campylobacter
DNA via a labeled DNA probe on a glass slide. This captured DNA is detected
by a secondary DNA probe labeled with a fluorophore. It allows detecting Campy-
lobacter in the sample with a sensitivity of 0.37 ngμL−1 DNA and 1.5 × 101 CFUg−1
(Manzano et al. 2015).
A biosensor based on surface plasmon resonance (SPR) has also been proposed
for the detection of C. jejuni (Wei et al. 2007); which can detect C. jejuni up to
103 CFU mL−1 . An optical biosensor based on SPR and diffraction optic technology
has also been developed (Gnanaprakasa et al. 2011) targeting the hippuricase gene
of C. jejuni. The complementary probe was thiolated and subsequently fixed on the
gold surface of the chip. After hybridization of probe and hippuricase gene, it led
to the transduction of an optical signal which could quantify C. jejuni up to 5 nM.
In another study (Wadl et al. 2009) a lateral flow device was developed to detect C.
jejuni or C. coli which can detect pathogen up to 7.3 log of CFU g−1 .
In recent study, functionalized magnetic particles with tripled silica layer and
coated with anti-C. jejuni antibodies have been found to detect C. jejuni in water
with a detection limit of 102 CFU mL−1 (Zhang et al. 2015).
18.3.1.4 Detection of Salmonella
Salmonella is a gram-negative rod-shaped bacterium which is associated with diar-

rhea and food poisoning in man and animals. An important reservoir of Salmonella
is poultry. Several serotypes of Salmonella are present which can infect both man
and animals (Alcaine et al. 2006). Infection arises through contaminated food and
drinking water. Several workers have tried to develop different forms of biosensors
for the detection of Salmonella. A wireless and mass sensitive biosensor for the
detection of S. typhimurium has also been developed which was able to detect 1.6 ×
102 CFU mL−1 (Chai et al. 2012). A lateral flow system developed by DuPont can
detect Salmonella in 10 min. In this test, the samples are initially pre-enriched before
sample addition to the strip which contains precoated antibodies against Salmonella.
A lateral flow immunochromatographic strip using the invA gene of the Salmonella
as a target has also been developed (Bulut 2014). An aptamer-based lateral flow
biosensor has been developed to detect Salmonella (Fang et al. 2014). It includes an
amplification step which forms a single-stranded DNA followed by its deposition on
a sensor membrane. This test has been found to have a sensitivity of 101 CFU mL−1 .
Further improvement in Salmonella detection has also been made by using
microfluidic devices where efficient binding of S. typhimurium bacterial cells has
been achieved by using anti-Salmonella antibodies. By using quantum dots conju-
gated to antibodies a portable fluorometer was also developed which could detect
Salmonella cells up to 103 CFU mL−1 in chicken meat extract (Kim et al. 2015b).
18.3.2 Biosensors for Detection of Viral Infection
18.3.2.1 Detection of Avian Influenza (AI) Virus
Avian influenza virus (AIV) poses a serious threat to mankind and has been divided
into highly pathogenic AIV and low pathogenic AIV. Highly pathogenic AIV tends
to rapidly spread which may cause high mortality. Infection by low pathogenic AIV
results in mild gastrointestinal and respiratory symptoms but may turn into a highly
pathogenic type after multiple infections (Morse et al. 2012). A variety of tests which
include haemagglutination inhibition test, haemagglutination test, virus neutraliza-
tion test, ELISA, virus isolation in cell culture, virus propagation in embryonated
chicken eggs are available to detect the virus infection. However, these processes
require large cost input, specific maneuvers, and infrastructure are required for their
conduction.
Moreover, certain strains of Avian influenza-like H5N1 kills the embryos very
quickly, thus, the amplification obtained in eggs is not enough to proceed further.
Similarly, molecular methods require RNA extraction and the requirement of high
Biosecurity Levels equipment’s. In such conditions, pen-side tests for detection
provide a window for an early and rapid on-site detection (Chang et al. 2009; Chiou
et al. 2010; Tseng et al. 2014). Some serological tests are also available to detect the
serological response against AI. However, they are endowed with low sensitivity and
false-negative results due to which it limits the application of serological tests.
The site of the binding of the influenza virus is different for both humans and
birds. In the case of humans, the virus binds to the α-2, 6 sialic acid glycan of the
upper respiratory tract. In the case of birds, it binds to the α-2, 3 sialic acid expressed
in the intestine. Hence, based on this differentiating feature, biosensors have been
developed to detect and differentiate between the human and avian influenza viruses.
A biosensor based on glycan-immobilized field-effect transistor has been demon-
strated to differentiate between the avian (H5) and human (H1) influenza viruses with
a sensitivity of up to 10−18 mol L−1 (Hideshima et al. 2013). Sialic acid bound to gold
nanoparticles can bind with the HA of the virus, thus producing the signals for virus
detection (Lee et al. 2013). The glycans have been printed in the glass slides to form
a microarray (Dinh et al. 2014) which can detect various strains of influenza virus up
to ten plaque-forming units. Portable lateral flow tests have also been developed for
the diagnosis of influenza virus (Sajid et al. 2015). In these kinds of lateral flow assay,
the sample to be tested is diluted in the detergent solution followed by its addition
into a sample pad. Thus, the components in the sample start moving in the pad. If
the sample is positive for the influenza virus, the virus particles bind with the anti-
influenza antibodies conjugated with gold nanoparticles which are pre-embedded in
the conjugate pad. The complex of virus-antibodies-GNP reaches the test lines where
anti-influenza A and anti-influenza B antibodies are pre-coated. These antibodies are
directed against the HA protein of influenza virus. In the case of positive samples, a
visible red line is formed. The unbound GNPs reach the control line and bind with
the secondary antibodies which subsequently form a red line. If the virus particles
are absent in the test sample, only the GNP labeled antibodies will move and bind
to the control line. However, these tests could not differentiate between the various
subtypes of LPAIV and HPAIV.
This shortcoming can be overcomed by using aptamers against AIV or RNA/DNA
probes on the test lines, which enables the differentiation of subtypes as well. These
lateral flow assays are simple and easy to use, thus, it makes them indispensable. To
amplify the signal generated silver nanoparticles have been used successfully and
amplification of 1000 times has been achieved (Huang et al. 2015). By using quantum
dots a limit of detection of 0.09 ng mL−1 was achieved for detection of AIV (Li et al.
2012) and a sensitivity of 100 times than ELISA was achieved.
For the quantitative detection of influenza A virus (subtypes H5 and H9) a
quantum-dot-based lateral flow test has been developed (Wu et al. 2016). An elec-
trochemical sensor employing the anti-M1 antibody has been found to detect all
serotypes of influenza A virus (Nidzworski et al. 2014). After conjugation of the
anti-M1 monoclonal antibodies with GNPs in a quartz crystal microbalance assay, a
limit of detection up to 1 × 103 PFU mL−1 has been achieved (Hewa et al. 2009).
An electrochemical immunosensor that detects and
quantifies PB1-F2 protein of the influenza virus
has been found to be sensitive for detection of influenza virus (Miodek et al.
2014a, b) with a limit of detection of 0.42 nM. A surface plasma wave biosensor has
also been developed for the detection of Influenza A virus (Su et al. 2012). Although
this method is sensitive, accurate, and fast, however, it comes with a limitation that
it can be used only for laboratory purposes.
Aptamers which are artificial nucleic acids with known 3D structures can differ-
entiate between various serotypes of the influenza virus. These aptamers are synthe-
sized by using systematic evolution of ligands by exponential enrichment (SELEX)
method. Aptamers-based biosensors open newer insights; because they do not require
any animal host for synthesis, are less expensive than antibodies, and consume less
time. Moreover, they bind the target molecules in the quantity of picomolar which
offers more sensitivity in the detection of the target molecule. Therefore, based on
this concept aptasensors are developed for the detection of AI virus. These aptamer-
based biosensors have been found to have target specificity to detect both the Avian
influenza virus as well as its subtypes (H5N1 and H1N1). Due to the binding of
aptamers with HA, it opens new opportunities for its use as an antiviral (Sung et al.
2004; Cheng et al. 2008).
For the multiplexed detection of influenza virus multiplexed RT-PCR microarray
coupled with DNA microarray has been used for subtyping the influenza virus (Li
et al. 2001). A microarray for rapid detection of various subtypes of influenza virus
(H1N1, H3N2 in man and H5N1 from poultry) has also been developed (Townsend
et al. 2006).
18.3.2.2 Detection of Bluetongue and Epizootic Hemorrhagic Disease

Viruses
Bluetongue (BT) is a non-contagious infectious disease of domestic and wild rumi-

nants caused by Bluetongue virus (BTV). It infects sheep, cattle, and deer. It is
transmitted by biting midges belonging to the family Ceratopogonidae. The cattle
may be subclinically infected and may act as reservoirs of the virus. Economic losses
are caused due to reduced milk and meat production. Epizootic hemorrhagic disease
is another disease that mainly affects sheep. It is caused by Epizootic hemorrhagic
disease virus of family Reoviridae and genus Orbivirus. This virus is also transferred
through the same arthropod that transfers BTV. However, this disease is exhibited by
high mortality in contrast to BT which has low mortality, both having similar clinical
signs and symptoms.
There is a wide array of tests like real-time PCR, DNA microarray, microsphere
bead-based tests, and genotyping methods to detect the blue tongue-virus (Weis et al.
2015; Wilson et al. 2015). A lateral flow assay to detect the BTV antibodies has been
employed and commercialized (Hanon et al. 2016). Magnetic modulation biosensing
has also been developed using FRET assay which allows fluorescent detection of the
virus using fluorescently labeled probes tagged to magnetic microspheres (Danielli
et al. 2009).
18.3.2.3 Detection of Foot-and-Mouth Disease Virus
Foot-and-mouth disease (FMD) is caused by foot-and-mouth disease virus (FMDV)

and is an economically important disease. It is a highly contagious disease of domestic
and wild ruminants. The FMDV can be detected virus isolation, enzyme-linked
immunosorbent assay (ELISA), and reverse-transcriptase polymerase chain reaction
(RT-PCR) (King et al. 2006).
A pen-side test for detection of FMDV antigen has been developed (Reid et al.
2001; Ferris et al. 2010). In a GNP-IPCR (Gold nanoparticle improved immune PCR)
the immune complex is formed by capturing the virus particles by polyclonal antibody
followed by addition of gold nanoparticle conjugated oligonucleotides and FMDV
specific monoclonal antibodies. The signal DNA is released by heating followed by
an analysis by PCR/real-time PCR (Ding et al. 2011). This assay was found to detect
10 fg mL−1 of purified FMD particles with high sensitivity in clinical samples. A
GNP biosensor using thiol-linked oligonucleotides has been designed for detection
of FMDV (Hamdy et al. 2018). Recently, a GNP-based dot-blot assay has been
developed for the detection of anti-FMDV antibodies (Jain et al. 2018).
18.3.2.4 Detection of Bovine Respiratory Syncytial Virus
Bovine respiratory syncytial virus (BRSV) is one of the major respiratory diseases
of cattle and is present worldwide. It is a single-stranded negative-sense RNA of the
family Paramyxoviridae. An electrochemical biosensor for the detection of mRNA
of RSV has been developed (Cai et al. 2013). This kind of sensor has been developed
by using molecular beacons which are oligonucleotides hybridization probes.
In another study enzymatic substrate reaction using HRPO and TMB have been
used for the detection of RSV antigens (Rochelet et al. 2012). It was an electrochem-
ical assay in which the RSV specific antibodies were immobilized on the polystyrene
slide, followed by the addition of the antigen and then enzyme-labeled antibody. The
signal generated by HRPO-TMB breakdown was transduced through an electrode.
This test was found to be rapid which can be conducted within 25 min. An immuno-
PCR has also been developed to detect the RSV particles which could detect virus
particles up to 4.1 PFU mL−1 and showed a 103 higher limit of detection than ELISA
and RT-PCR (Perez et al. 2011).
18.3.2.5 Detection of Bovine Viral Diarrhea Virus (BVDV)
An integrated nanowire-based immunosensor has been developed for the detection

of BVDV in serum (Montrose et al. 2015). For the detection of anti-BVDV anti-
bodies, an electrospun biosensor based on the principle of capillary separation and
conductometric immunoassay has been developed (Luo et al. 2010). This biosensor
has been found to have a detection limit of eight minutes and 103 CCID mL−1 (Luo
et al. 2010). A microparticle immunoagglutination assay on a microfluidic chip has
been used to detect BVDV particles (Heinze et al. 2009).
18.3.2.6 Detection of Porcine Reproductive and Respiratory Syndrome

(PRRS) Virus
Several immunodetection-based biosensors have been developed for the detection

of PRRSV. Infrared electrochemical luminescence biosensor (Shao et al. 2017),
platinum nanotube-based fluorescent immunoassay (Chen et al. 2015), enzyme-
linked aptamer antibody sandwich (Lee et al. 2013), Fluorescence Resonance
Energy Transfer (FRET) based optical biosensor (Stringer et al. 2008) using gold
nanoparticles and quantum dots have been developed for detection of PRRSV.
18.3.3 Biosensors for Detection of Mastitis Pathogens
Mastitis is the inflammation of the mammary gland and most of the time it is infec-
tious in origin. Milk from mastitic animals is not suitable for purpose of human
consumption because of the altered chemical composition, organoleptic properties
(Seegers et al. 2003; Adkins and Middleton 2018; Ashraf and Imran 2018), and shelf
life of the dairy products (Hogeveen et al. 2010). Mastitis can be classified as clinical
or subclinical. The clinical form is manifested by visible features like the presence of
clots, flakes, change in colour, swelling of the mammary gland. Subclinical mastitis
does not manifest any clinical sign, but the milk quality and production are declined.
Moreover, the excessive use of antibiotics to treat mastitis gives rise to the problem
of antimicrobial resistance (Pol and Ruegg 2007). Therefore, it becomes imperative
to detect mastitis at an early stage so that preventive measures can be applied as soon
as possible (More 2009).
In this regard, there is an increasing demand for pen-side tests to confirm the
infectious origin of mastitis (Adkins and Middleton 2018). Due to the persistence
of the pathogen in the mammary gland, and the immunological response is gener-
ated. Therefore, for the detection of subclinical mastitis, several approaches have
been suggested which are based on the testing for immunological modulators and
chemical properties of milk (Viguier et al. 2009; Adkins and Middleton 2018;
Ashraf and Imran 2018). Out of these increased somatic cell count (SCC), N-
acetyl-b-D-glucosaminidase (NAGase) and lactate dehydrogenase (LDH) are the
most commonly used markers (Viguier et al. 2009; Addis et al. 2016; Adkins and
Middleton 2018; Ashraf and Imran 2018).
Both colorimetric and fluorometric assays have been developed for the detection
of LDH and NAGase activity. A fluorometric assay has been developed for the
estimation of the catalytic activity of NAGase on the substrate 4-MUAG (Hovinen
et al. 2016). A portable spectrophotometer has also been developed to estimate the
LDH activity (Hiss et al. 2007).
Electroconductivity (EC) metres for the measurement of increased EC of mastitic
milk (due to increased sodium and chloride ions) has also been developed (Norberg
et al. 2004; Lam et al. 2009; Viguier et al. 2009). However, they are found to vary
between animals.
The pathogens associated with the causative agent of mastitis also need to be iden-
tified. Timely identification is essential to reduce the excessive use of administration.
Most of the pathogens causing udder infection are derived from the environment.
The most common pathogens associated with mastitis are Streptococcus agalactiae,
Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Streptococcus
uberis, Corynebacterium sp., Pseudomonas aeruginosa, Mycoplasma sp., and Kleb
siella. An electrochemical sensor based on a screen-printed carbon electrode has been
devised to detect the NAGase enzyme in the mastitic milk with a limit of detection
of 10 mU mL−1 (Pemberton et al. 2001a).
For the detection of Mycoplasma bovis, a single-stranded DNA aptamer has shown
high specificity and binding affinity towards P48 protein of M. bovis (Fu et al. 2014).
A biochip for the DNA amplification of genes specific for mastitis-causing pathogens
could detect six pathogens and M. bovis with a limit of detection up to 103 CFU mL−1
(Lee et al. 2008). An electrochemical sensor that is based on electrochemical spec-
troscopy has been developed for the detection of pathogenic Staphylococcus aureus
(Braiek et al. 2012), with a limit of detection of 10 CFU mL−1 . The assay was found
to be specific as it did not detect E. coli and S. epidermidis. In another recent study,
the electrochemical paper-based analytical device (EPAD) was developed. In this
paper-based genosensors they used graphene nanodots (GNDs) and zeolite (Zeo)
with the S. aureus specific DNA probe to detect the target (Mathur et al. 2018). This
limit of detection (LOD) of this sensor was found to be 0.1 nM of ss-DNA in test
samples.
18.3.4 Biosensors for Detection of Drug Residues in Meat

and Dairy Products
The drug residues in food can give rise to antimicrobial resistance (Du and Liu 2012;
Leibovici et al. 2016). There is a chance that via food (chicken, milk, egg, fish, meat,
and honey) these antimicrobial-resistant strains may be transferred to human beings.
A label-free amperometric immunosensor was developed for the detection of
kanamycin which showed a limit of detection of up to 5.74 pg mL−1 and was used to
test kanamycin residues in animal-derived food samples (Wei et al. 2012). A label-
free immunosensor designed using silver hybridized ferroferric oxide nanoparticles
was found to detect kanamycin with a detection limit up to 15 pg mL−1 in pork meat
samples (Yu et al. 2013).
Aptamer-based biosensors (Jiang and Yu 2008; Derbyshire et al. 2012; Citartan
et al. 2012; Bai et al. 2014; Niu et al. 2014), have also been developed for the
detection of kanamycin residues in food samples. For detection of chloramphenicol
electrochemical sensors have been developed which are based on multiwalled carbon
nanotubes (Yang and Zhao 2015). Similarly, an electrochemical aptasensor has also
been developed for chloramphenicol detection which showed a detection limit of
0.059 ng mL−1 (Zhang et al. 2011).
An electrochemical sensor using three-dimensional reduced graphene oxide archi-

tectures has also been developed (Zhang et al. 2016) which was able to detect chlo-
ramphenicol up to 48.45 ng mL−1 . A molecularly imprinted polymer (MIP) has been
synthesized for the electrochemical detection of streptomycin (Que et al. 2013). To
detect Streptomycin, magnetic molecularly imprinted polymer nanospheres (mMIP)
have been used (Liu et al. 2013) by assembling AUCl4 ions on the magnetic bead
surface. This was followed by the o-phenylenediamine polymerization on the surface
of magnetic beads.
For the detection of Streptomycin an electrochemical aptasensor based on gold
nanoparticle-functionalized magnetic multiwalled carbon nanotubes and nanoporous
PtTi alloy has been developed (Yin et al. 2016). An electrochemical arc-shaped
aptasensor has also been developed for the quantification of Streptomycin in milk
(Danesh et al. 2016).
18.3.5 Biosensors for Measuring Physiological, Metabolic,

and Biochemical Parameters of Livestock
Apart from disease diagnosis in livestock and poultry, biosensors have also been used
to monitor other physiological, metabolic, and biochemical parameters like animal
jaw movement, breath analysis, analysis of metabolites in perspiration, analyzing
tears for glucose monitoring, analysis of stress in fish, and detection of ovulation.
Jaw movements of animals are required to know the grazing behaviour of cattle. The
rumination behaviour has been recorded by using a pressure sensor as a noseband
along with a data logger which records the jaw movements via pressure (Braun et al.
2013). Acoustic analysis is used to estimate the biting and chewing movements for
the analysis of food intake by the cattle (Laca and WallisDeVries 2000). Microphones
have been used to record the jaw movement sounds of grazing animals (Ungar and
Rutter 2006). Machine learning algorithms are used to analyze the signal pattern for
the estimation of the intervals between the jaw movements, intensity, and duration
(Navon et al. 2013). Accelerometer sensors have also been used to estimate the jaw
movement and grazing behaviour of animals (Tani et al. 2013; Mattachini et al. 2016;
Giovanetti et al. 2017). These sensors can convert the acceleration generated from
movement or gravity into a suitable voltage output.
Some of the biosensors have also been developed to analyze the metabolites in
sweat. These are the electrochemical sensors used for the measurement of sodium
and lactate levels in sweat. They are also used to measure sweat as a measure of stress.
Biosensors have also been used to analyze the infection by M. bovis by estimating
the volatile organic compounds in breath (Ellis et al. 2014). A nanobiosensor and a
strip-based lateral flow biosensor has been developed to detect M. bovis and M. avium
subspecies paratuberculosis, respectively. An amperometric biosensor using immo-
bilized glucose oxidase has also been used to detect glucose in the tear (Iguchi et al.
2007). It gives a measure of glucose concentration in blood. Other self-monitored

biosensors are also in the stage of development and validation (La Belle et al. 2014).
Implanted biosensors inside the sclera of fish have also been used to monitor the
stress biomarker as an indicator of the health of fish as well as the pollution status of
the water (Hibi et al. 2012; Wu et al. 2015). Biosensors have also been implicated in
determining ovulation in cattle. An amperometric progesterone biosensor has been
developed to determine the ovulation in cattle. It consists of an anti-progesterone
monoclonal antibody immobilized on a screen-printed carbon electrode (Pemberton
et al. 2001b). Biosensors have also been developed to detect the progesterone in milk
with software which lists the insemination time (Mazeris 2010) with more advance-
ment integration of novel aptamer with a Surface Plasmon Resonance (SPR) imaging
sensor (Zeidan et al. 2016). Intravaginal probes that can detect the conductivity and
temperature inside the vagina have also been used to predict the ovulation of the
cattle (Andersson et al. 2015, 2016).
Testing of saliva for various metabolites is an extremely useful non-invasive
method (Bandodkar and Wang 2014) and is useful for disease diagnosis. The
biomarkers found in saliva are useful in the early detection and diagnosis of diseases
(Malon et al. 2014). Detection of corticosteroid hormones in saliva as a marker of
stress has been used successfully. A cortisol immunosensor has been developed for
the quantitative analysis of salivary cortisol (Yamaguchi et al. 2013). Similarly, α-
amylase in the saliva is an indicator of psychosocial stress. An α-amylase biosensor
has also been developed which is based on the hydrolysis of starch by α-amylase
(Wu et al. 2007). The resonance of the starch gel immobilized sensor increases on the
hydrolysis of starch by α-amylase. Salivary glucose biosensors have also been devel-
oped to measure glucose on the saliva of domestic pets and human beings (Stein and
Greco 2002; Reusch et al. 2006). Sensors have also been developed for monitoring
the physiological status of livestock. A livestock monitoring system integrated with
a wireless sensor network for the collection of data on breathing rate, heart rate, and
cattle movement has been developed (Park and Ha 2015).
Bioacoustics based system has been developed to detect the sex (Pereira et al.
2015), routine activities of poultry (Fontana et al. 2016), disease diagnosis like
necrotic enteritis (Sadeghi et al. 2015). A sound detection system has been devel-
oped to differentiate between New Castle Disease, Infectious Bronchitis, and Avian
Influenza (Banakar et al. 2016).
18.4 Conclusion
The Biosensors have a wide application in the field of veterinary sciences. Starting
from the disease diagnosis biosensors have been used successfully in monitoring the
metabolism and physiological parameters of animals and poultry. Animals’ health
plays an important role in any country’s economy as directly and indirectly it affects
the country’s economy as well as human health. In the past few decades, there has
been excessive use of antibiotics, the emergence of viral diseases, the emergence
of new antibiotic-resistant bacterial strains, antibiotic residues in milk and other

food commodities derived from animal sources. This imbalance is the outcome of
the indiscriminate use of the available resources like antibiotics in feed/treatment
of animal diseases, human activities like deforestation, mutation in viruses, and
overlapping of human niche with that of wildlife. Excessive antibiotic use leads
to antibiotic bacterial strains. Moreover, the infectious diseases in animals cause
heavy economic losses and most of them are important from a zoonotic point of
view. Therefore, with an increasing demand for rapid diagnosis of the viral/bacterial
diseases, antibiotic residues, and monitoring the health status of animals, rapid pen-
side tests need to be developed. The conventional diagnostic techniques take time,
demands labour and sophisticated instruments for their interpretation. Biosensors
can be the rapid pen-side tests that deliver the test results in a few minutes for early
diagnosis and preventive measures required for disease control. Moreover, there is a
requirement of multitargeting biosensors in the field to detect the target analyte with
more confidence to overcome the impediment of false positive and false negative
test outcomes. The nucleic acid-based biosensors are one of the biosensors for the
sensitive detection of analytes. Biosensors powered with nanotechnologies and next-
generation sequencing can offer more robust tools for the detection of pathogens and
animal surveillance.
The layers on which the bioreceptors are immobilized also play an important
role in the electrochemical signals and non-specific binding which depends on their
thickness and surface charge. Moreover, choosing novel bioreceptors like bacte-
riophages, single-chain antibodies, non-antibody-binding proteins, and half anti-
bodies can offer high specificity. Antibodies are the most widely used biorecep-
tors, however, their production and purification is costly. Moreover, the stability
of antibodies on the sensor surface is not long-lasting and the binding capacity
of these antibodies decreases with due time course. Hence, to counter these diffi-
culties, some advancements in bioengineering have been implicated like peptoid
nanosheets (Olivier et al. 2013). Such types of implications comprise thick sheets
of 3–5 mm thickness formed by self-assembly of antibody mimetic peptoids. These
thick sheets have surface loops expressing antigen-binding sites. The whole aggrega-
tion is stable and easy to produce. Some other alternatives for stable antibodies are,
DARPins (Stumpp and Amstutz 2007), camel-derived heavy variable chain (VHH)
(Hassanzadeh-Ghassabeh et al. 2013; Muyldermans 2013), single-chain antibodies
expressed via yeast surface display (Richman et al. 2009), single-chain variable frag-
ments (ScFv) (Ahmad et al. 2012), and adhirons (Tiede et al. 2014). These alterna-
tives can be synthesized easily, are small, and can be produced in bacterial expression
systems. Another important aspect is the regeneration of the sensor surface. Regener-
ation of the biosensor surface is cost-effective. Biosensor surface can be regenerated
successfully after using the above bioreceptor molecules. These bioreceptors can
withstand harsh buffers without affecting their binding capacity.
Nanomaterial-based electrochemical biosensors are also good biosensors due to
their low cost, good selectivity, sensitivity. They need a precise choice of the trans-
ducer surface. The incorporation of the nanomaterials accelerates the sensitivity and
specificity of the detection of the analyte. Electrochemical biosensors offer very
low detection of analytes like as observed while detection of antibiotic residues of

kanamycin, streptomycin, and tetracycline. Moreover, the portability and reusability
of electrochemical biosensors add to its other features of rapid on-site detection. The
sensitivity of the electrochemical biosensors can be increased further by replacing
the enzyme-based labels by the nano labels.
Antibody-based biosensors can also offer high sensitivity for the target molecule
depending on the selective binding of the target molecules with the immobilized
antibodies and the stability of the antibody molecules on the sensor surface. There
is also a need for international recognition and widespread use throughout the world
these biosensors must be validated via intra-lab and inter-lab validation.
From future perspective, development, and validation of nanobiosensors can
open new insights in animal disease diagnostics. With the recent advancements
in nanotechnology, nanobiosensors will be an excellent platform with increased
sensitivity for analyte detection.
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Chapter 19
Nano-Biosensing Devices Detecting
Biomarkers of Communicable
and Non-communicable Diseases
of Animals
Utkarsh Jain, Saurabh Shakya, and Kirti Saxena
Abstract Diseases are either communicable or non-communicable. Communicable

diseases or infectious diseases are caused by interactions between two living organ-
isms whereas non-communicable diseases are either hereditary or due to the living
environment of animals. Animals are very susceptible to various types of infections
and pathogens may live outside or inside their body very conveniently. Epidemic
diseases affect animal herd, livestock and the leading cause of animal mortality
which may result in significant losses to the agricultural economy. Animal diseases
are a matter of concern due to the direct effect on the economy as well as the possi-
bility of pathogen transmission in humans. The accurate detection of animal diseases
is an important factor to control diseases in livestock and in wild animals. Disease
diagnosis in animals is very challenging and based on the variety of examinations
and tests. In this context, nano-biosensors exhibit better disease detection capacity
than conventional methods. Nano-biosensors show high sensitivity, stability, repro-
ducibility with a lower limit of detection. This chapter emphasized the insight of
communicable and non-communicable animal diseases and their detection through
nano-biosensing devices. Some important animal diseases are discussed along with
a description of developed nano-biosensors for them. Nano-biosensor functional-
ized with disease-related biomarkers performed better than any other conventional
methods in various aspects.
Keywords Biosensor · Communicable diseases · Non-communicable diseases ·

Animal diseases · Nanotechnology
U. Jain (B) · K. Saxena

Amity Institute of Nanotechnology, Amity University Uttar Pradesh,
Noida, UP, India
S. Shakya
Viral Recombination Section, HIV Dynamics and Replication Programme,
National Cancer Institute, Frederick, MD, USA
416 U. Jain et al.
19.1 Introduction
19.1.1 Overview (History of Animal Diseases)
Animals are an important part of our ecosystem and their existence either directly or
indirectly affects the life of humans in various aspects and vice versa. Like humans,
animals also suffer with several types of communicable and non-communicable
diseases. Sometimes there are some common diseases also found in humans and
animals as well as they can play a role of a disease carrier for each other’s, for
example, anthrax, brucellosis, giardiasis, and swine flu. Health risk of animal popu-
lation may have an impact on food supply chains which is a threat to human health
with long lasting effects and to economy as well. For the animal disease control
which are communicable to man from livestock as well as wildlife animals, several
organization including WHO conduct various programmes. These diseases known
as zoonoses, comprised of existing infectious disease along with newly identified
infectious disease to monitor the impact on human health.
History related to animal diseases describes that human and animal diseases
developed parallelly. Aristotle, the father of Biology and Hippocrates, the father
of medicines, demonstrate animals and man’s diseases symptomatology as well as
therapy in their writing. Ancient literature are evident to animal and human diseases
which include Egyptian, Roman, and Greeks monographs. In history, agriculture,
domesticated animals were focused particularly. After the discovery of microor-
ganisms by Pasteur, animal diseases became a matter of concern for veterinarians
because of their direct effect on human health by consuming animal origin foods like
meat and dairy products. After that basic research and zoonoses have been started
for the detection and treatment of animal diseases (Charles et al. 2018).
19.1.2 Communicable and Non-Communicable Diseases
Any disease either in animals or in humans may be communicable and non-

communicable. The communicable term implies for interaction or communication
between two living things which are known as host and parasite. The parasite exists
in host cells and lives at the expense of their cellular machinery. Parasites are certain
microorganisms such as virus, bacteria, protozoans, arthropods, and fungi, which are
unable to survive on their own, so they depend on host organism for food, multiplica-
tion, or some other requirements. The disease-causing ability of a parasite is known
as pathogenicity whereas ability to cause infection is known as virulence. The viru-
lence property of a parasite is differed from one host to other, that it can be virulent
for one but not to other hosts. Most of the parasite occurred in the atmosphere as
an inactive form until they find appropriate conditions to establish themselves inside
host cells. For example, Clostridium and Bacillus bacteria occurred as an inactive
form known as spores for several years even in harsh environmental conditions.
19 Nano-Biosensing Devices Detecting Biomarkers … 417
Fig. 19.1 Representation of communicable diseases and their host animals
The host cell defense system i.e. immune system provides a variety of mechanical
and chemical barriers against infection, and to establish disease in the host body, it
is essential to cross all these barriers. In animals, communicable diseases can be
spread through direct contact of animals, waste of sick animals, unhygienic housing
of animals, and presence of microorganisms in feed and water. Certain types of
lice, mites, ticks, and fleas present inside or outside of body, also play a role in
spreading of diseases, for example, plague (Heymann 2008). Some common types
of communicable diseases and their host animals are represented in Fig. 19.1.
Non-communicable diseases, as the name suggested, not communicate from one
animal to another and do not involve any virulent pathogens. Non-communicable
diseases are caused by hereditary factors, poor feed, lack of nutrients, and environ-
mental factors in surroundings. Sometimes it also caused by cuts, burns, certain toxic
agents (insecticides, pesticides, fungicides, fluorine, and herbicides), or poisonous
plants, and broken bones. Sometimes humans are responsible for some metabolic
diseases in animals by injecting chemicals which causes the unsuitable alteration
in body environment such as oxytocin is very commonly used in dairy animals to
get more milk due to which contraction of uterus takes place and causes immense
pain to the animal. Any disturbance in normal physiological conditions to maintain
homeostasis and stability, leads to metabolic diseases. Non-communicable diseases
are chronic and can be acute as well which causes a huge loss in milk, meat, and wool
418 U. Jain et al.
production. The working capacity and rate of reproduction of livestock can also be
affected (Habib and Saha 2010).
19.1.3 Conventional Approaches for Detection of Animal

Diseases
Since it is impossible for a veterinarian to interrogate the animal, diagnosis of

the animal disease includes a number of tests and examinations based on inspec-
tion of behaviour, palpation percussion, smells, eye examination, appearance, etc.
Diseases like pullorum can be detected by agglutination test, tuberculin skin test for
tuberculosis, faeces and eggs examination can be done in lab for early detection of
diseases. Determination of chemicals, toxins in blood, or body fluids are performed
for several years for a diagnosis of variety of animal diseases.
19.1.4 Nano Biosensors and Its Applications
To control certain chronic communicable and non-communicable diseases, early

and fast detection is essential in a population of animals such as a herd of cattle.
In recent times, every year newly emerging diseases threaten animal health are
a matter of concern. For the early stage detection of diseases in the case of animals,
there is currently a lack of authentication and cost-effective diagnostics. Considering
biosensing technologies, as they have the potential to overcome these problems by
developing novel diagnostic tools because of their high sensitivity to detect disease-
related biomarkers with a low limit of detection. The available conventional sensors
are used in livestock monitoring and also as a tool for the assessment and monitoring
in health monitoring and disease detection. These sensors when integrated with wire-
less data transfer through a server or with cloud-based systems, enabling access to the
analyzed data from any internet enabled device. Applications of nanobiosensor not
only help in the reduction of the current costs for reagents, handling samples, time
of analysis, and cost of transportation but also helps in adapting and promoting
the handling of livestock. Biosensor is a simple, miniaturized analytical device
which is utilized for rapid and efficient diagnosis of infectious diseases and has
the ability for real time analysis (Sagadevan and Periasamy 2014).
19.1.5 Biomarkers and Their Utilization in Biosensors
Biomarkers are typical indicators for biological, pathogenic, or pharmacologic

processes which are used to identify an event or physiologic process of interest. For
animal medicine, these biomarkers help in the early detection of several cardiopul-
monary diseases, confirmation of doubtful processes (normal or abnormal), and
contribute to the establishment of prognosis to decide a specific treatment (Fry and
Dunbar 2007; Carretón et al. 2013). Also, they are used to detect the course of therapy
or to predict safety or drug response. Biosensor consists of analytes, bio-recognition
element (a biological element to detect analyte), and transducer surface (physio-
chemical detector component) to convert any biological signal to a measurable elec-
tric signal. Biomarkers play a key role in the fabrication of biosensors to detect
several important diseases like cancer, Alzheimer, organ health monitoring after a
transplant, diabetes, neurological disorders, etc. Biomarkers exploit in biosensors
could be antigen-antibody, dopamine, uric acid, hydrogen peroxide, ascorbic acid,
hormones, microRNA, enzymes, or whole cell (bacteria/viruses) (Zetterberg et al.
2019; Dahal 2019; Viitanen et al. 2014; Scherr et al. 2016; Pereira et al. 2016; Çolak
et al. 2013; Alcaraz et al. 2016; Fei et al. 2016; Peng et al. 2017). Some major diseases
of animals along with their biomarkers are given in Table 19.1.
19.1.6 How Can Animal Diseases Affect Human Health?
In most of the cases, human infectious diseases which include disease agents and
pathogens are acquired from animal species. Several diseases such as SARS, Nipah
virus, avian influenza, cat scratch disease, West Nile disease, mad cow disease etc.
are associated with animals. Recent pandemic Covid 19 is also acquired from bat
species. So, humans and animal’s diseases are directly proportional to each other in
about 60–75% cases (Wang and Anderson 2019).
19.2 Communicable and Non-Communicable Diseases

of Animals
19.2.1 Communicable Diseases of Animals
Aflatoxicosis: Aflatoxicosis is a widespread disease in animals and affects several

animal species including cattle, chicks, ducklings, turkeys, horses, and pigs. Disease
is caused by a mycotoxin which is produced by Aspergillus flavus, a most popular
form of fungus. The feed items like millets, maize, soybeans, and peanuts, contam-
inated with fungi are source of disease. Aflatoxin is a highly toxic metabolite and
mainly affects the liver through ingestion of contaminated feeds. The disease is char-
acterized by drop in egg production particularly in poultry birds, loss of weight,
and sometimes hepatocellular tumors appeared in severe conditions. Aflatoxicosis
is associated with great economic loss as it reduced productivity by many folds, for
Table 19.1 The major diseases of animals and their biomarkers
420
S. No Animal Causing pathogen Host Symptoms Associated Biomarkers

disease
1. Aflatoxicosis Aspergillus flavus cattle, chicks, drop in egg production, aflatoxin B1 , B2 , G1 , G2 ,
ducklings, weight loss, hepatocellular M1 , P1 , Q1 , and aflatoxin
turkeys, horses, tumors B1 -N7 -guanine
pigs (AFB1 -N7 -Gua)
2. African swine DNA virus of Asfarviridae family domestic and high fever, loss of appetite, sCD163 & E75CV (strain
fever wild pigs vomiting, diarrhoea, of virus)
haemorrhages, loss of antibodies, ASFV DNA
weight, respiratory signs,
chronic skin ulcers
3. Anthrax Bacillus anthracis Cattles fever, difficulty in Calcium dipicolinate,
breathing, loss of appetite, dipicolinic acid,
Crepitation swelling
4. Avian Avian influenza A viruses Birds, poultry cough, cold, fever, AIV H5-hemagglutinin,
influenza headache, shortness of IL-6, microRNA
breath, and sore throat
5. Bluetongue bluetongue virus (BTV) Ruminants eye nasal discharge, high VP-7
fever, drooling,
inflammation
6. Botulism Clostridium botulinum cattle and avian hindlimb weakness, botulinum neurotoxin
species paralysis, death serotype A
7. Brucellosis Brucella Goats, sheep, stillbirth or abortion S19 and RB51
cows, ruminants
like deer, bison
and elk
(continued)
U. Jain et al.
S. No Animal Causing pathogen Host Symptoms Associated Biomarkers
disease
8. Ehrlichiosis Ehrlichia, Anaplasma, and Neorickettsia of Cannines fever, Reticuloendothelial IgM and IgG antibodies,
Anaplasmataceae family hyperplasia, antigenic proteins P28-19,
lymphadenopathy, and Ehrlichia Hsp60
Splenomegaly, loss of
stamina, stiffness, and
edema of limbs
9. Hendra virus Hendra virus fruit bats, horses, fever, cough, headache, microRNA, physiological
disease humans tiredness, meningitis, coma biomarkers like glucose,
triglycerides, urea, protein
etc.
10. Hydatid Echinococcus granulosus tapeworm Dogs weight loss, weakness and interferon-gamma, IL-4,
loss of appetite, allergy IL-10, specific IgE, total
IgG, Glutathione
S-Transferase, microRNA
19 Nano-Biosensing Devices Detecting Biomarkers …
11. Listeriosis Listeria monocytogenes Animals, fishes, encephalitis, meningitis, invasion-associated protein,
crustacean, birds meningoencephalitis, hemolysin, BE-LisAll
and humans stillbirth, abortion
septicaemia, perinatal
infections
12. Newcastle Newcastle disease virus domestic poultry Respiratory disease, Nucleoprotein,
and other species diarrohea, depression, or glycoprotein, antibodies
of birds nervous manifestations
13. Nipah Nipah virus Fruit bat, Pigs, barking pig syndrome, Nipah virus G-protein
humans encephalitic syndrome
421
422 U. Jain et al.
example, milk production can be reduced by 15% or more (Dalvi 1986; Solis-Cruz
et al. 2019).
African swine fever: African swine fever (ASF) is a severely contagious viral
disease affecting domestic and wild pigs. It is caused by a DNA virus of Asfarviridae
family which also responsible for infection in ticks of Ornithodoros genus. ASF asso-
ciated with serious loss in production (pork, lard, leather, glue, fertilizer, and a variety
of medicines produces through pigs) and economy. The virus is transmitted through
a contaminated feed to animals and fomites such as shoes, knives, cloth, equipment,
and vehicles. i.e. non-living things. The virus shows high resistant capacity for harsh
environmental conditions. ASF has been reported in Europe, South America, Asia,
Africa, and Caribbean. Symptoms of ASF include acute and chronic forms. Acute
forms indicated by high fever, loss of appetite, vomiting, diarrhoea, haemorrhages in
the skin, and death within few days (6–20 days). Chronic forms are characterized by
loss of weight, respiratory signs, chronic skin ulcers, intermittent fever, and arthritis.
Chronic forms are less intense clinically and can persist for longer period with low
mortality rate (Dixon et al. 2019).
Anthrax: A large spore forming Bacillus anthracis bacteria is responsible for
anthrax disease. It is an extremely infectious and fatal disease that causes acute
mortality in cattles. The bacterial toxin is highly potent which immediately shows ill
effects and a high mortality rate. After exposure to this bacterial infection, symptoms
usually appear within 3–7 days and animal death occurred usually within 2 days.
Cattles usually get infections by grazing and inhaling the spores of bacteria which
are colourless, odourless, and tasteless. The infected animal usually expresses sudden
symptoms such as fever, difficulty in breathing, loss of appetite, Crepitation swelling
over the shoulder hip, and back, which is followed by death. This is a highly dangerous
disease and exhibit potential loss of economy (Doganay and Demiraslan 2015).
Avian influenza: Avian influenza or bird flu is caused by avian influenza A viruses
which naturally occur among wild water birds. The virus generally infects birds,
poultry, animals, and sometimes human beings may also get infected through contam-
inated poultry. Although humans rarely get infected with this virus, however virus
can change itself and gain the ability to infect humans and spread among them.
Influenza virus have the quality to change themselves time to time and certain
strains spread easily in human populations. Symptoms arise within 2–8 days and
include cough, cold, fever, headache, shortness of breath, and sore throat. The disease
refers to high mortality rate in humans also (Li et al. 2019). In recent times poultry
business is one of the major areas of interest because of high demand in food indus-
tries. But avian influenza is one of the big challenges as it may cause major economic
loss. Recent advancement makes possible to detect and continuously monitoring
of disease through biosensors, wearable sensors, real time monitoring, and data
management (Raj and Jayanthi 2018; Vidic et al. 2017) (Fig. 19.2).
Bluetongue: Bluetongue disease refers mainly the disease of ruminants including
both domestic and wild animals, however Sheep are badly infected with disease.
Cattle although frequently get infection than sheep but they lack symptoms most of
the times. It is normally an insect-borne disease caused by bluetongue virus (BTV)
and transmitted by different species of midges Culicoides. The infected animals
Fig. 19.2 Different methods for monitoring and detection of disease in poultry farms
usually suffer with eye, nasal discharge, high fever, drooling, and inflammation of the
coronary band, haemorrhages into or under skin, lethargy, swollen teats, respiratory
problems, and swelling in mouth, head, and neck (Van den Bergh et al. 2018).
Botulism: Botulism is a food borne disease caused by Clostridium botulinum
bacteria which produce highly potential neurotoxin commonly known as botulinum.
It is a rapid onset fatal disease of cattle and avian species. Botulism typically repre-
sents by hindlimb weakness followed by collapse, paralysis, and death. Animal
carcasses, poorly prepared silage and rotting organic material are the main sources of
bacterial infection. Although the vaccine is available for this disease but still it is not
completely eradicated. The disease outbreak can be seen in poultry and waterfowl
(Mariano et al. 2019).
Brucellosis: Brucellosis is a bacterium causing the disease which mainly affects
livestock including goats, sheep, cows, and some wild ruminants like deer, bison,
and elk. It is caused by bacteria of genus Brucella. The disease mainly characterized
by stillbirth or abortion in animals. The disease is spread through contaminated
milk, birthing fluids, the infected placenta of animals, or direct contact with other
animals or human beings. Canines can also infect with bacteria, but they are rarely
spread infection to human beings. Complications associated with brucellosis consist
of inflammation of the brain, infection of the heart’s inner lining, meningitis, and
lesions on the bones and joints (Shoukat et al. 2017).
Ehrlichiosis: Ticks are the carrier of this disease which commonly developed in
canine bitten by infected ticks. It is caused by intracellular obligate organisms that
belong to genus Ehrlichia, Anaplasma, and Neorickettsia of Anaplasmataceae family.
The parasite is primarily infecting the immune cells of canines and humans. Infected
424 U. Jain et al.
animals are suffered from fever, Reticuloendothelial hyperplasia, lymphadenopathy,

Splenomegaly, loss of stamina, stiffness, and edema of limbs (Mylonakis et al. 2019).
Hendra virus Disease: It is mainly a disease of fruit bats which can be infectious
to horses also. Hendra virus infection has infrequently been infectious to humans
who came into contact with an infected horse. Infected horses may possess a range
of symptoms like fever, respiratory, and neurological disorders and show rapid onset
illness. The host develops symptoms within 5–21 days after infection with Hendra
virus. At initial stage, some common symptoms appear as fever, cough, headache,
and tiredness whereas later, meningitis and coma have been seen in severe infection
conditions. Hendra virus disease can be fatal sometimes (Field et al. 2011).
Hydatid disease: Hydatidosis, echinococcosis, or hydatid disease, caused by cyst
consist of Echinococcus granulosus tapeworm (Dog Tapeworm) larval stage, is an
extremely serious condition and sometimes may be fatal. The disease is mainly
infecting dogs and other canines. Infected animals shed eggs in their faeces which
further infect other host by swallowing these eggs or by direct contact with infected
dogs. The cyst can be remained inside the liver and lungs for months to several years.
The disease show signs of weight loss, weakness, and loss of appetite. The rupture of
cyst may cause serious allergic reactions even death of animals. Although Hydatid
infection is potentially severe but may be treatable. The disease occurs worldwide,
commonly in grazing areas (Qingling et al. 2014).
Listeriosis: Animals, fishes, crustacean, birds, and even humans can be infected
with Listeriosis; which is a serious infection and become fatal sometimes. Listeria
monocytogenes is the causative agent of this food borne disease. It has the poten-
tial to transmit from one cell to another and can cross intestinal, placental, and
blood brain barriers. The disease is characterized by encephalitis, meningitis, menin-
goencephalitis, stillbirth, abortion septicaemia, perinatal infections. Listeriosis is a
sporadic disease which spread through soil, water, milk, meat, seafood, and vegeta-
bles. There is no vaccine available for this disease so it can be prevented only through
hygienic practices (Oevermann et al. 2010).
Newcastle disease: Newcastle disease virus (NDV) is a causative agent of
Newcastle disease of domestic poultry and other species of birds. It is primarily
represented by acute respiratory diseases, but diarrhoea, depression, or nervous mani-
festations can also be seen predominantly as a clinical form. Infected animals shed
infection through respiratory discharge, eggs, and faeces. In poultry disease, may
spread by ingesting water or food contaminated with virus. Movement of infected
poultry birds, contaminated equipment, or litter plays an important role for spreading
viruses. The signs of disease in an infected organism are appeared in 2–12 days of
exposure of virus (Brown and Bevins 2017).
Nipah virus disease: Nipah virus or NiV belongs to genus henipavirus and
paramyxovirus family causes a highly fatal disease Nipah. Natural host of nipah
virus is fruit bat and currently, it affects pigs and humans very severely with high
mortality rate. The virus is spread through bat urine, faeces, birthing fluids, and
saliva. It is assumed that bat excreta initiate infection in pigs. The virus mainly
affects the nervous system and respiratory system. The virus is highly contagious
to pigs and associated with barking pig syndrome (BPS) and encephalitic syndrome
(PRES). Nipah virus may also infect dogs naturally and causes a distemper-like
syndrome which is a highly fatal condition (Ang et al. 2018).
19.2.2 Non-Communicable Diseases
Ruminal tympany: It is also known as bloat. The disease mainly found in ruminant
animals and characterized by high amount of gas in rumen. It can be divided into two
phase, primary frothy bloat, and secondary free gas bloat. In a natural process, the
rumen is dedicated for fermentation of eaten food by gut microbes and the fermen-
tation process produces excessive amount of gas which expelled out from rumen by
burping continuously but sometimes gas becomes trapped inside the rumen. This
condition is remarked as bloat or ruminal tympany. In primary bloat, produced gas
trapped within fermenting material which causes a raised amount of foam and unable
to remove by burping. The disease is characterized by distension of abdomen on left
side, distress, and difficulty in breathing. This disease can be fatal if gas accumulation
process continues which also distended right side of abdomen. After visualization
of such symptoms, the cattle death may occur within 3–4 h. In the secondary bloat,
oesophagus becomes blocked so accumulated gas cannot be pass through it (Priyanka
and Dey 2018).
Nutritional deficiencies in animals: In the case of animals, this is very difficult
to find out the nutritional deficiencies. Most of the animals suffer from malnutrition.
There is a lack of signs related to most of the nutritional deficiencies because signs
are common and not specific such as loss of appetite, reduction in growth, and
thriftiness. Multiple types of nutritional deficiencies may be caused in many animals
due to starvation. Deficiency of some essential amino acids or suboptimal feed intake
may result into protein deficiencies which causes weight loss and fatter carcasses in
pigs. In lactating animals for example cows, protein deficiency directly affects milk
production. Some fatty acids like linoleic acid are found very essential in the diet
and utilized in production of another essential form i.e. longer chain fatty acids. The
lack of linoleic acid in the diet leads to scaly dermatitis, necrosis in skin, unthrifty
appearance, and hair loss in growing pigs. Deficiency of minerals like calcium and
phosphorous may outcome as a disease known as rickets in pigs, dogs, and others. The
disease is characterized by deformity, lameness, and bending of bones specifically
long bones. Consequences of severe conditions are fracture and paralysis at posterior
parts, generally in old ones. Salt deficiency also affects the growth of animals.
Iodine deficiency may result into hairless and week pig’s new-born. New-born
pigs may show enlarged thyroids and histological abnormalities. Enlarged thyroid
can also be seen in many animals and known as Goitre. Iodized salt may get rid of
this disease.
Iron and copper deficiencies efficiently develop anaemia in animals which is
caused by a reduction in haemoglobin synthesis. Anaemia may be recognized by
low RBC and haemoglobin count, enlarged heart, edema, and thumps. Deficiency
of zinc, selenium, and vitamin E also have an adverse effect on growing or younger
ones.
426 U. Jain et al.
Vitamins are very essential for the development and metabolic processes in
humans as well as in animals. In livestock and pets, if they provided with some
supplemented commercially available diets then chances are very low to face the
vitamin deficiency challenges because these are fortified with vitamins. Vitamin A
deficiency affects eyes, epithelial tissues of important organ systems like the nervous,
urinary, digestive, respiratory, and reproductive. Vitamin K deficiency is related with
lengthened blood clotting and sometimes animals may die due to haemorrhage.
Riboflavin deficiency in pigs may result into impaired reproduction. Vitamin B defi-
ciency also affects severely to neonatal pigs in terms of voice failure, hyperirritability,
and incoordination like symptoms. Biotin deficiency also characterized by excessive
hair loss, dermatitis, inflammation of mouth, and mucous membrane (Innis 2000;
Vieyra-Reyes et al. 2017; Shukla et al. 2016).
19.3 Development of Nano-Biosensing Devices

for Detection of Animal Diseases
Nano biosensing devices exploit a biorecognition element which can be DNA/RNA,

antigen/antibodies, enzymes, aptamers, and other polymers, etc. There are several
studies done to develop a nano-biosensing device by using various biomarkers. Here,
some types of biosensors description are given that are developed to detect several
serious diseases (Table 19.2).
19.3.1 DNA Sensors
DNA based sensors are developed for the detection of several biomarkers depend
upon the specific nucleic acid sequences. Various forms of nanomaterial are used for
signal amplification which generated through the hybridization of two complemen-
tary DNA. Anthrax disease can be detected through a label free nanopore real time
sensing device, in which a single-stranded DNA (ssDNA) was used as a molecular
probe for anthrax lethal factor (Wang et al. 2014). A unique nanostructure based
geno-sensor was fabricated to detect Brucella-specific probe. A gold nanoribbon
surrounded by gold nano blooms were further deposited on polycrystalline gold
surface by sonoelectrodeposition method. In this approach, methylene blue was used
as redox marker to detect complementary sequence, sequences with one-, two-, and
three bases mismatches (Rahi et al. 2015). Another DNA biosensor also studied for
Brucella species detection in animals which consists of a specific oligonucleotide
probe taken from S711 gene region and gold nanoparticle. This probe hybridized with
complementary DNA biomarker present in a clinical sample. The device was capable
to detect unamplified Brucella genomic DNA up to 7 pg µL−1 . The DNA biosensor
Table 19.2 List of different types of fabricated Nano-biosensors in past few years for some common
animal diseases
Nano-biosensor Disease Nanomaterial Detection limit Reference
DNA biosensor Brucellosis Gold 1.71 zmol dm−3 Rahi et al.
(nanoribbons/nanoblooms) 2015
Gold 7 pg µL−1 Sattarahmady
et al. 2016
ehrlichiosis QCM based 4.1x109 molecules Bunroddith
µL−1 et al. 2018
Newcastle Magnetic nanoparticles 75 fM Tian et al. 2016
Disease (MNPs)
Aptasensor Avian Gold 0.25 HAU Karash et al.
influenza 2016
Anthrax Multiwalled carbon 20 ng m L−1 Karimi and
nanotubes Dabbagh. 2019
bovine Gold 800 copies mL−1 Park et al. 2014
viral
diarrhea
Immunosensor Bluetongue Gold 10 − 2 fg mL−1 Yin et al. 2012
Hydatoid Silicon 300 fg mL−1 Lv et al. 2017
Enzootic Magnetic gold 0.95 mg mL−1 Baniukevic
bovine nanoparticles et al. 2013
leukemia
exhibited high sensitivity and selectivity to amplified clinical samples (Sattarah-

mady et al. 2016). In a study, gold nanorods are functionalized with specific ssDNA
probe of ‘cadF gene’ to detect campylobacter species. In this study about 283 stool
samples were analysed and showed 44 positive cases, that indicated sensitivity of
device, rapid in comparison to PCR, culture, and real time PCR (Shams et al. 2019).
Another study demonstrates the utilization of quartz crystal microbalance (QCM)
in combination with PCR assay for successful detection of Ehrlichia canis. The
QCM based DNA nano-biosensor device was reusable, simple and do not need any
critical equipment. The basic principle of this device was DNA-DNA hybridization
without any cross hybridization. The limit of detection of QCM was calculated as
low as 4.1×109 molecules/µl of 289 bp E. canis PCR product that represented to 22
copy numbers/µl of E. canis (Bunroddith et al. 2018). Newcastle disease virus RNA
detection was carried out by Tian and co-workers by combination of two highly effi-
cient techniques that are loop-mediated isothermal amplification (LAMP) and opto
magnetic nanoparticle-based readout system. The detection limit was 10 aM of target
sequence in just 30 min (Tian et al. 2016).
428 U. Jain et al.
19.3.2 Aptasensors
Aptasensors are fabricated by employing aptamers. Aptamers are naturally occur-

ring or synthesized short random oligonucleotides or peptides sequences. Generally,
they are developed through SELEX method. In the animal disease detection aptasen-
sors were also utilized for rapid and accurate detection of disease biomarkers. An
electrochemical graphene field-effect transistor based aptasensor has been devel-
oped for detection of anthrax toxin (Kim et al. 2013). The avian influenza detection
can be possible through aptamer-based nano-biosensor devices. Several aptasensors
work has been carried out for detection of avian influenza virus. For example, an
impedance aptasensor was developed for the detection of highly pathogenic avian
influenza (H5N1). To enhance impedance signal, a gold nanoparticle-based ampli-
fier was designed and functionalized with H5N1-aptamer and thiocyanuric acid. The
device exhibited a robust, specific, and rapid detection method for the H5N1 virus
with the detection limit of 0.25 haemagglutination unit (HAU) (Karash et al. 2016).
Aptasensors were also used for detection of Anthrax disease in animals. Recently,
a nano-biosensor device which was based on multi-walled carbon nanotubes and
fluorescence aptasensors was fabricated. In this study, researchers detected ‘recom-
binant protective antigen domain 4 (rPAD4)’ of Bacillus anthracis which plays an
important role in the development of disease. The aptamers were labelled with gel
green fluorescent dye and immobilized on carbon nanotubes to give amplified signals
after hybridization of complementary sequences. The device had the potential to be
used for diagnostic tests for anthrax in a fast, accurate, sensitive, and cost-effective
manners (Karimi and Dabbagh 2019). Another frequently occurred disease in animals
is bovine viral diarrhoea that could be also detected by aptasensors. In 2013 a
biosensor based on two different aptamers was fabricated for successful screening of
binding to bovine viral diarrhoea virus type 1 (BVDV type 1) in a clinical sample. The
aptamer had a high affinity and specificity to this virus biomarker. In this study, the
work was based upon basic principle of aptamer-aptamer sandwich type sensing plat-
form in which one aptamer immobilized with gold nanoparticle and other hybridized
with viral DNA BVDV type 1. The device was capable to detect 800 copies/ml (Park
et al. 2014).
19.3.3 Immunosensors
Immunosensors are basically worked on the principle of antigen and antibody reac-
tion. Several types of biosensors have been developed to detect animal diseases
in minimum time and cost-effective way. Immunosensors utilized different type
of transducer surface such as electrochemical, and optical (colorimetric, surface
plasmon resonance (SPR), fluorescent, chemiluminescence), etc. Bluetongue virus
associated biomarker VP7outer-core protein was detected by a nanobiosensor fabri-
cated by exploitation of gold nanoparticle probe-based assay and bio-barcode assay
technique. In this device anti-VP7 monoclonal antibodies and ssDNA are immobi-
lized on magnetic microparticle probes. The developed biosensor exhibited the detec-
tion limit of 10−2 fg mL−1 (Yin et al. 2012). Electrochemical immunosensor consists
of single gold nanowire that has been developed in 2015 for the detection of bovine
viral diarrhoea virus. In this device researchers immobilized captured biomarker
of virus on gold nanowire covalently by electrodeposition method. Immunosensing
device was applied for detection of serum antibodies in the serum of infected animals.
Team specified their device as a very sensitive with very low detection limit so could
be applied for on-farm diagnosis for animal health monitoring (Montrose et al. 2015).
BVDV monitoring could make possible through another microfluidic immunosensor
by Heinze and co-workers in 2009. In this study they demonstrated a microfluidic
based platform which consist of anti-BVDV monoclonal antibodies immobilized
on carboxylated polystyrene microparticles. The performance of device had been
compared with real time PCR where they found the very low detection limit in
microfluidic device (Heinze et al. 2009). Enzootic bovine leucosis is a blood borne
disease which affect mainly cattle and caused by BLV or bovine leukosis virus. The
virus resides in white blood cells and asymptomatic most of the time. SPR based
immunosensor was fabricated recently in 2019 for detection of BLV in blood serum
of sick animals (Klestova et al. 2019).
An optical immunosensor for the detection of BLV has been developed by
exploiting the UV and visible photoluminescence property of zinc oxide nanopar-
ticles. The device was suitable for field use. The device showed high sensitivity
and a wide range of leucosis antibody detection i.e. 0.001 mg mL−1 to 1 mg ml−1
(Ruban et al. 2017). Another surface-enhanced Raman scattering (SERS) based
immunosensor has been also developed for the detection of this disease in 2013. In
this study magnetic gold nanoparticles (MNP-Au) functionalized with fragmented
antibodies specific for gp51 biomarker were used. They were resulted out the limit
of detection as 0.95 µg mL−1 and limit of quantification as 3.14 µg mL−1 for gp51
(Baniukevic et al. 2013). A method for the diagnosis of foot and mouth disease was
also developed with the help of an electrochemical immunosensor. A non-structural
protein i.e. 3ABC of associated disease was detected through specific antibodies by
utilization of a screen-printed electrode (Longinotti et al. 2010). Hydatid disease is
associated with Echinococcus granulosis. Several studies have been carried out for
the development of immunosensor to detect the associated biomarker. In a study,
researcher developed silicon microcavities based nanosensor to perform antigen
antibody interaction on it. An associated protein named protein kinase P38 was
targeted for detection of hydatid diseases (Lv et al. 2017). Quantum dots based
immuno biosensor also demonstrated in a 2017 study. In this device they immo-
bilized Egp38 antigen on silicon pores which further functionalized with anti-p38
labelled CdSe/ZnS QDs. This was a fluorescence-based detection. They achieved
detection limit 300 fg mL−1 for Egp38 antigen (Li et al. 2017). Very recently, an
immunosensor has been developed for this disease using square wave voltammetry
technique. Cysteamine/phenylene isothiocyanate linkers have been coated on gold
electrodes followed by immobilization of either purified ‘rabbit polyclonal antibody’
or ‘recombinant antigen B’ (AgB) to detect hydatid antigen. Detection limits for
430 U. Jain et al.
echinococcus antigen and antibody were 0.4 pg mL−1 and 0.3 pg mL−1 , respectively.
The device is low of cost, sensitive and can be used as a point of care device (Eissa
et al. 2020). Newcastle disease is also a fatal disease which affect several animals
worldwide. A very simple electrochemical immunosensor was developed recently
for detection of NDV. In this device chicken egg yolk antibodies (IgY) was utilized as
a biorecognition element and were specific for NDV antigen. The sensor had exhib-
ited a sensitivity range of 106 to 102 EID50 mL−1 (Thinh et al. 2018). Listeriosis is
also a very harmful disease and can be detected through immunosensors. A 2013
work reported a Screen-printed carbon electrode modified with gold nanoparticle
exhibited great sensitivity for detection of Listeria species. Listeria monocytogenes
presence was monitored through conjugating secondary enzyme-labelled antibodies
(Davis et al. 2013). The biosensor found to be effective in detection of other enteric
bacterial species such as E. coli and Salmonella Typhimurium. Another 2017 study
revealed an immunosensor fabricated with a pair of monoclonal antibodies. These
pair of mAbs used to recognize P60 protein associated with Listeria species (Wang
et al. 2017).
19.3.4 Miscellaneous
CRISPR-Cas assay with a fluorescence-based point-of-care (POC) system has been

developed for the detection of African swine fever virus detection. The LOD was
found as 1 pM in 2 h (He et al. 2020). Recently, a metal oxide semiconductor (MOS)
based sensor was developed for detection of Campylobacter jejuni in a milk sample.
This microbial agent is responsible for several gastrointestinal related diseases in
humans. A MOS sensor has the ability to differentiate between contaminated and
control samples (Núñez-Carmona et al. 2020). Another advancement in nanobiosen-
sors can be represented by wearable biosensors. For continuous real time monitoring
of herd of animals and livestock; wearable biosensors show a great significance in
the health management of animals. They provide physiological information through
non-invasive, dynamic measurement of biomarkers in biofluids like saliva, sweat,
tears, and interstitial fluids (Neethirajan 2017).
19.4 Future Aspects
Nano-biosensing devices are known for their high sensitivity, simplicity, and selec-
tivity to detect animals or human diseases. They are able to provide rapid results
with low cost in comparison to conventional methods (Malik et al. 2013). Animals
are extremely susceptible to infection because of unhygienic living environment
therefore suffer with number of communicable diseases and their direct contact with
human beings make the situation more problematic. Animal communicable diseases
cause significant economic loss in various ways like loss in food production system,
and increased veterinary costs (Kivaria 2006; Ott et al. 1999; Dufour 1999; Neethi-
rajan 2019). We consider that nano-biosensors have capability to advance conven-
tional technologies for detection of disease-causing microorganisms and can improve
the surveillance system to prevent any disease outbreak. Wearable devices can play
an important role in future to manage animal diseases. Real time field monitoring,
data networking, sampling methods, and mobile analysis will provide remarkable
support through highly sensitive and selective nano-biosensors. The data through
device will help in disease control and prevention of outbreak in poultry, livestock
as well as wild animals.
19.5 Conclusion
Prevention of communicable diseases and non-communicable diseases is very essen-

tial for the health management of animals. Human beings are also affected with
animal disease directly or indirectly and have various common diseases. The first
step required for prevention of any disease is accurate diagnosis. It is important
to develop some reliable, cost effective, and less time-consuming methods. Fast
diagnosis is must in some disease for example anthrax which causes death within
2 days. Sometimes some diseases are remained asymptomatic like bovine leucosis, so
they also need accurate detection in very low concentration. Nanotechnology exhibit
several new opportunities to fabricate new devices for the successful determination of
diseases. With the help of biomarkers, these nano-biosensors have given promising
results in the diagnosis of animal diseases. Communicable and non-communicable
diseases affecting animals at a different level can now detect at an early stage,
monitored, and controlled with the help of nano-biosensor.
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Chapter 20
Recent Advances in Biosensor
Development for Poultry Industry
Nidhi Chauhan, Ramesh Namdeo Pudake, Utkarsh Jain, and Sapna Balayan
Abstract Poultry sector is becoming important for the sustainable food chain. It
is now rapidly growing across the world. Various biosensors for antibiotics and
pathogens identification have been developed in comparison to traditional methods
in poultry industry. Poultry market has already occupied a major share of world-
wide farmer’s income. To prevent bacterial growth in farm birds many antibiotics
are injected. Ultimately these antibiotic residues are found in them which results in a
huge life-threatening scenario for the person who is consuming these animal products.
The antibiotics that are widely used in poultry animals create antibiotic resistance in
humans. Several biosensors are developed for detecting antibiotics residue in poultry
birds. Biosensors are also used for the detection of infection-causing pathogens in
farm birds and they have proven to be very useful when compared to other tradi-
tional screenings. Because of the increasing demand for eggs and meat due to the
increasing population, it is expected that the poultry sector will expand in the future.
Emerging nanotechnology is a key factor development of biosensors. Therefore,
the chapter briefly explains the nanotechnology-based sensors for antibiotics and
pathogens detection in poultry birds.
Keywords Poultry · Antibiotics · Antibiotic detection · Pathogens · Biosensor
20.1 Introduction
Presently, the poultry sector is becoming one of the rapidly growing components of
the agriculture-based industry across the world for higher economic growth. But the
growing field of poultry farming in large-scale is resulting in the deterioration and,
higher rate destruction of environment, it may cause economic loss (Kabir 2009). It
has been predicted that till 2025 poultry industry will be the largest producer of meat
across the world (King et al. 2018). According to “The United States Department of
Agriculture” (USDA) globally the poultry production was around 111,000 thousand
N. Chauhan (B) · R. N. Pudake · U. Jain · S. Balayan

436 N. Chauhan et al.
metric tons during 2015. It is assumed that poultry production worldwide over the
next few decades will be projected to be raised by 24 percent that will surely become
around 131,255 thousand metric tons in 2025. Till 2050 the estimation for the global
population is awaited to be increased to 9 billion and also increase in the income
growth, which will be leading to the higher demand for animal-based protein diet.
This explains the important part played by the poultry industry for a sustainable food
supply (King et al. 2018).
Different factors play a role in the continuous growth of this sector including
rising population, industrialization, rising income, geographical distribution. All
these mentioned factors are believed to be responsible for the rapid expansion of
poultry industry worldwide. There are several small poultry dressing plants every-
where in the developed and developing countries for meat and egg processing. Around
every locality, we can find these poultry products in close proximity.
Also, more and more employment will be generated for the poor people in this
industry. Modernization too is an important factor in the rise of poultry sector. Poultry
meat especially chicken is highly consumed, due to its low-fat content, higher protein
level, and producing less greenhouse gas. Also, when compared to other birds’
chickens have a higher feed conversion ratio (FCR) and more meat is produced
with lesser feed input (King et al. 2018).
In both rural and urban areas poultry has proven to be a valuable thing especially
for small holders or shopkeepers. This industry may help them to earn their livelihood
and the easiest way to enter the business world. With the growing demand for poultry
products, there are many drawbacks associated with this sector too. The poultry sector
is having many disastrous challenges such as bird flu and other contagious diseases.
Pathogen detection in poultry animals has remained a big challenge for the poultry
industry in the last decades. Various pathogen detection methods have already been
developed which is discussed further in this chapter (Mottet and Tempio 2017).
Due to the increase in demand for poultry meat, the industry will explore different
methods and incorporate new technologies to increase their production, to continue
the sustainable food chain, and meet the demands for safe food at an affordable
prices (King et al. 2018). And the challenges faced by the poultry industry are briefly
discussed further.
20.2 Challenges Faced by the Poultry Sector
Along with the growing market of poultry sector, it has been indicated that this
industry is facing many challenges during pathogen attack on the animals. Apart
from spoiling human health, it has a significant effect on our environment and natural
resources too. Any change in the environmental factor directly results in climate
change through pollution of air and water and production of feeding materials (Hu
et al. 2017). The most important risk factor in poultry industries is represented in
Fig. 20.1 and discussed in brief.
20 Recent Advances in Biosensor Development … 437
Fig. 20.1 Important risk factor in poultry industry
20.2.1 Water
Water source which is used for various purposes in poultry farm must be clean enough
to ensure animal’s good health, as most of the contagious diseases are due to dirty
or unhygienic water (Sims 2008).
20.2.2 Work Force on the Farm
Not only visitors but caretakers on the farm can also be a disease carrier through their
dirty shoes or clothes. So, to avoid this proper cleaning of the shoes and wearing
protective gear is advisable. This personal protective equipment will also help in
preventing the disease transfer to the operator (Sims 2008).
20.2.3 Feed Storage
Storage of the feeding material or may be transporting it under unhygienic condition

can worsen the situation. The feed can be a breeding ground for vectors or may
become a favorable place for the infectious pathogen to grow. So, the farmers should
take care while storing the feed (Sims 2008).
20.2.4 Hygiene of Equipments Used in Farming
Farmers unintentionally or may be due to lack of knowledge use the same equipment
for the transfer of dead animals and then for feed handling. This may cause the
unintentional spreading of the pathogen to healthy animals, so this should be avoided.
Therefore, regular cleaning and sanitization of the equipment needs to be done (Sims
2008).
20.2.5 Stock Hygiene
Animals kept in unhygienic conditions may affect their health and can result in heavy
losses. Therefore, the maintenance of hygiene is very much important in maintaining
bird stocks. The growers must take utmost care while disposing of the dead birds.
This needs to be done as early as possible to prevent the transmission of infectious
disease to other birds (Sims 2008).
20.3 Concern Regarding the Use of Antibiotics

and Transmissible Diseases from Poultry
Antibiotics are being in use very commonly as veterinary drugs. The farm animals
receive antibiotic treatment for microbial resistance. But it is a big and serious concern
for public health as after treatment of animals with antibiotics, antibiotic residue
remains in the animals that have proven to be very harmful to the person who is
consuming it. The maximum acceptable amount of any medicinal drug or substance
and their intermediates in the edible products in food extracted from animals is
decided based on various safety studies and can be termed as acceptable daily intake
(ADI). Scientists have shown that medicine residues in the food that originated from
animals ultimately go through the human immune system. Several immunological
reactions occur in humans after exposure of a certain level of these antibiotic residues.
These residues may also interfere with our intestinal bacterium that helps to maintain
our immune system (Nel 2006; Kriebel et al. 2001). Some commonly used antibi-
otics for the antimicrobial treatment in animals are: Sulfonamides (Mubito et al.
2014), Tetracyclines (Doyle 2006), Quinolones (Er et al. 2013), and Streptomycin
(Pyun et al. 2008). There is a high association between the residues of antibiotics
given to poultry animals and health issues in people such as digestive disturbance,
neurological disease, hypersensitivity, and many more after consumption.
On the other hand, poultry industry displays an intimidate effect on the human
population, as poultry animals are a vector of many infectious diseases especially
bird flu that has already shown its mortal effects in the past recent years (Mangsi
et al. 2014; Babapour et al. 2012). Therefore, measures must be taken to reduce the
adverse effect of poultry infection on human beings. Nanotechnology can be used as

an essential tool for the development of a platform for pathogen detection in poultry
birds.
20.4 Nanoparticles in Poultry Sector
Recently, due to the potential advantages of nanoparticles, interest in utilizing them

in poultry production has significantly increased. In the future, it will play a vital role
in poultry industry with the integration of biotechnology. For maintaining hygienic
and more nutritious condition, nanomaterials are introduced in food produced with
chicken eggs that help in the blocking of cholesterol in the bloodstream. Also, in
the future for hygienic and nutritious meat, nanotechnology can be incorporated in
the poultry sector. Also, it can be used in the challenges like feed efficiency, drug
delivery, modification of content in the egg (Fig. 20.2). These and other potential
applications and various aspects have been reviewed earlier by some of the authors
(Anwar et al. 2019; Hassan et al. 2020; Kannaki and Verma 2006), and here we will
be focusing on the use of sensors in poultry industry.
Fig. 20.2 Nanomaterial for binding pathogen in the poultry birds (Anwar et al. 2019)
20.5 Biosensors for Poultry Industry
Integration of two-stream biology and physics results into biosensors and are the
platform for the detection of biological compounds. The biosensor consists of a
recognition element and a transducing device. On recognition element, biological
(enzyme/protein, antigen/antibody) components are immobilized, and the analyte
binds to them (Fig. 20.3). The transducer is used for the detection of electrons/ions
transmitted or generated on the surface (biological signals are converted into an elec-
trical signal for the detection) (Mungroo and Neethirajan 2014). Biosensors provide
higher sensitivity, selectivity, low-cost fabrication, durable, less time-consuming, and
are easy to handle. Biosensors are being developed for the identification of different
antibiotics and diseases causing pathogens in poultry (Astill et al. 2020; Kannaki and
Verma 2006). Many biosensors with different platforms that are recently developed
are further discussed in this chapter.
Fig. 20.3 Schematic diagram showing the working of antibiotic detection in poultry animals
through biosensor
20.5.1 Biosensors for Antibiotic Detection in Poultry
Various detection methods have been developed for the detection of antibiotics in
poultry animals such as LC-MS (liquid chromatography-mass spectroscopy), ELISA
(enzyme-linked immunosorbent assay), High-performance liquid chromatography
(HPLC), Thin-layer chromatography (TLC) (Okerman et al. 2001; Wang et al. 2009)
and tube or plate test. All the above screening procedures are proven to be useful for
their small volume sample preparation property, detection of multiple samples at a
time, and broad specificity. Apart from these advantages, it has many drawbacks too
such as it requires long hours of duration to be processed, no real-time detection and it
lacks quick and sensitive detection practically (Pikkemaat 2009). Due to the various
drawbacks in the above used analytical methods, there is a need for quick, sensitive,
and specific method. In this regard, a scientists have designed several biosensors that
fulfill almost all the properties in relation to an ideal detection like sensitive, accurate,
user-friendly, and low cost. A quick semi-quantitative mechanism-based approach
is the basic principle of a biosensor that made them a good and efficient idea toward
the detection of antibiotic residual volume present in poultry animals (Cháfer et al.
2010). Some biosensors for the detection of antibiotics in poultry field are listed in
Table 20.1 and are discussed below.
20.5.1.1 Molecular Imprinted Polymer (MIP) Based Sensor
The molecular imprinting polymer (MIP) has been a popular tool for drug and other
molecular detection since the beginning of molecularly imprinting technology (MIT)
in the 1970s. Many scientists have been focusing on using MIPs for the detection of
molecules in traces and achieved significant success in the quantification of drugs at
trace levels. Recently, the combination of MIP with magnetic particles have provided
additional benefits, like elevated adsorption capacity to template molecule, unique
selective recognition, and magnetic adsorption. The use of these materials for drug
analysis with their advantages and limitations has been reviewed recently (Ansari
and Karimi 2017). In the case of poultry, a magnetic electrochemical sensor fabri-
cated was based on the molecular imprinting technology with multiwalled carbon
nanotubes (MWCNTs) to carry out the quick determination of kanamycin traces in a
complex sample matrix. Also, PMMA (polymethyl methacrylate) is used as a poly-
meric film for the grafting of MWCNTs that enhances compatibility for the compact-
ness of the imprinted sensor structure. This MIP sensor has enhanced conductivity of
electrode that initiates transfer of electrons for the kanamycin detection in complex
samples (Long et al. 2015). The results had indicated that, under the test condition
the linear relationship between the kanamycin concentration (from 1.0 × 10−10 M
L−1 to 1.0 × 10−6 M L−1 ) and response current from the sensor. The sensor had a
detection limit of 2.3 × 10−11 M L−1 in the real samples.
Table 20.1 Antibiotic detection in poultry industry using various biosensors

S. No. Transducer Analyte Limit of References
detection
1 Luminescent Tetracycline 10 mg kg−1 (Pikkemaat et al.
2010)
2 Colorimetric Tetracycline 0.1 ng mL−1 (Weber et al. 2005)
Streptogramin 2.7 ng mL−1 (Weber et al. 2005)
Macrolide 1.7 ng mL−1 (Weber et al. 2005)
3 Electrochemical Penicillin 41.2 μA (Li et al. 2019)
biosensors μM−1 cm−1
Tetracycline 26.4 μA (Li et al. 2019)
μM−1 cm−1
Ceftazidime 0.01 μM (Sun et al. 2018)
Sulfadiazine 0.16 μM (Sun et al. 2019)
Acetaminophen 0.032 μM (Sun et al. 2019)
Ceftiofur 0.01 ng (Stevenson et al.
mL−1 2019)
Sulfadimethoxine 3.7 × (Mohammad-Razdari
10−16 M et al. 2019a)
4 Drop-Type Enrofloxacin 0.039 pM (Ahn and Lim 2015)
Chemiluminescence mL−1
Ciprofloxacin 0.022 pM (Ahn and Lim 2015)
mL−1
5 Magnetic-nanobead-based Oxytetracycline 0.88 ng (Lu et al. 2015)
biosensor mL−1
6 Montmorillonite Tetracycline 0.10 μM (Gan et al. 2014)
film-based sensor
7 Screen-printed gold Tetracycline 0.96 μM (Masawat and Slater
electrode-based biosensor L−1 2007)
Chlortetracycline 0.58 μM (Masawat and Slater
L−1 2007)
Oxytetracycline 0.35 μM (Masawat and Slater
L−1 2007)
8 Surface plasmon Chloramphenicol 0.005 μg (Ferguson et al. 2005)
resonance (SPR) kg−1
9 Optical-surface plasmon Quinolones 0.13 ng (Huet et al. 2009)
resonance (SPR) gm−1
10 Impedimetric biosensor Tetracycline 3 × 10−17 M (Mohammad-Razdari
et al. 2019b)
11 Electrochemical Tetracycline 3.7 × 10−17 (Benvidi et al. 2018)
aptasensor
12 Label-Free immunosensor Kanamycin 15 pg mL−1 (Yu et al. 2013)
13 Amperometric chloramphenicol 45 pg mL−1 (Kim et al. 2010)
immunosensor
20.5.1.2 Electrochemical Biosensor
Electrochemical biosensors are composed of the 3-electrode system with counter,

working, and reference electrodes. The electrodes are placed in an electrochemical
cell containing solution and different studies are performed for the measurements
like amperometric and potentiometry. The electrochemical biosensor works on the
principle of change in voltage and current during the reaction in an electrochemical
cell. Researchers fabricated an electrochemical biosensor with a multisegmented
nanowire/nanoparticle hybrid array, for the detection of penicillin and tetracycline
antibiotics simultaneously in meat samples (Li et al. 2019). The limit of detection
(LOD) of the sensor observed for penicillin was 41.2 μAμM−1 cm −2 and for tetra-
cycline was 26.4 μA μM −1 cm −2 . In another study, the fabrication of an electro-
chemical biosensor for the identification of ceftiofur residue in the meat samples
within 15 minutes has been reported (Stevenson et al. 2019), with the detection limit
of 0.01 ng mL−1 in test buffer and 10 ng mL−1 in spiked samples.
20.5.1.3 Piezoelectric Sensors
Piezoelectric sensors are the sensors that operate on piezoelectric effect to measure
quantities like temperature, pressure, etc. In a reported study, researchers have fabri-
cated a piezoelectric sensor based on the imprinting polymer technique (Karaseva
et al. 2016) for diagnosis of two different antibiotics penicillin and ampicillin in a
meat sample. The LOD of the sensor was reported as 0.04 μg mL−1 for penicillin
and 0.09 μg mL−1 ampicillin.
20.5.1.4 Aptamer Based Electrochemical Biosensor
An aptamer-based electrochemical biosensing platform for the identification of tetra-

cycline in milk, meat, chicken/egg, drinking water, and pharmaceutical was fabri-
cated by Kim and colleagues. The screen-printed gold electrode (SPE) was modi-
fied with streptavidin and the surface of streptavidin was immobilized with the
biotinylated ssDNA aptamer. For analyzing the binding of tetracycline with ssDNA
aptamer electrochemical measurements like cyclic voltammetry (CV) and square
wave voltammetry (SWV) were performed. The limit of detection for the sensor was
recorded in a range from 10 nM to micromolar (Kim et al. 2010). In another study,
Benvidi and coworkers described the fabrication of an electrochemical aptasensor
for identifying the concentration of tetracycline using multiwalled carbon nanotubes
and electropolymerized poly (LL-glutamic acid) for the immobilization of anti-
tetracycline aptamer. For analysis, electrochemical CV, differential pulse voltam-
metry (DPV), and Electrochemical impedance spectroscopy (EIS) measurements
were performed (Benvidi et al. 2018). Also, an electrochemical biosensing platform
for sulfadimethoxine (SDM) antibiotic identification in meat samples was fabricated
on a pencil graphite electrode improved with Au-RGo NPs by Mohammad-Razdari
and colleagues. The developed sensing platform shows excellent stability, selectivity,
and high reproducibility (Mohammad-Razdari et al. 2019a).
20.5.1.5 Surface Plasmon Resonance (SPR)
SPR biosensors based on optical biosensing technology, the working principle of SPR
is associated with modification in the refractive index with a binding analyte and bio
recognize molecule on the sensing platform (Piliarik et al. 2009). For detection of
antibiotics in poultry, SPR immunoassay was developed by Heut et al. (2008) for
antibiotics 13 (fluoro) quinolones and flumequine in animals which are used in food
production. The platform is based on the antigen-antibody combination. The SPR
biosensor was validated for screening (fluoro) quinolones in poultry, egg, and fish
(Heut et al. 2009)
20.5.1.6 Impedimetric Biosensor
Impedimetric aptasensor biosensor based on AuNPs/RGO NPs improved with pencil

graphite electrode was fabricated for the identification of tetracycline in meat, milk,
egg, shrimp, fish, chicken. The sensor shows that the combination of aptamer and EIS
helps in the quantitatively and qualitatively detection of tetracycline (Mohammad-
Razdari et al. 2019b).
20.5.1.7 Label-Free Immunosensor
A label-free immunosensing platform for the identification of kanamycin antibi-

otic was developed by using thionine mixed graphene sheet (TH-GS) modified
with silver hybridized mesoporous ferroferric oxide NPs (Ag@Fe3 O4 NPs). For the
electrochemical characterization, cyclic voltammetry and square wave voltammetry
measurements were applied. The sensor provides fast detection within 3 min and the
observed LOD was 15 pg mL−1 (Yu et al. 2013).
20.5.1.8 Amperometric Immunosensor
For the chloramphenicol antibiotic identification in meat sample, an amperometric

immunosensing platform was fabricated using CdS NPs modified-dendrimer which
were bonded with the conducting polymer (poly 5, 2 : 5 , 2 -terthiophene-3 -carboxyl
acid [poly-TTCA]) layer. To increase the sensitivity of the sensor the polymer was
modified with Au NPs, dendrimers, and CdS NPs. The LOD of the sensor was
recorded as 45 pg mL−1 (Kim et al. 2010).
20.5.2 Biosensors for Pathogen Detection
For pathogens identification in the poultry products various types of biosensing

platforms are developed and are discussed below (Table 20.2).
20.5.2.1 Wave Fiber Optic Biosensor
Salmonella enterica is a prime pathogen present in food and is most common in

poultry products. Therefore, for identification of S. enterica in chicken and shell egg
evanescent wave optical fiber assay was developed that provide results in 8 hours
with higher sensitivity and specificity. The LOD of the sensor was recorded as 104
CFU mL−1 (Valadez et al. 2009).
20.5.2.2 Microfluidic Device-Based Nano Biosensor
In this assay, S. typhimurium was identified with the help of a microfluidic nano
biosensor based on magnetic beads and quantum dots (QDs) as a fluorescent label.
Magnetic beads were used for coating anti-Salmonella to separate and concentrate
a small volume of samples of the bacterium. A sandwich complex was made using
antibody-conjugated QDs that label the cells. Fluorometer was used to measure the
signals for quantitative detection of S. typhimurium (Kim et al. 2015).
20.5.2.3 Impedance Biosensor
Different types of impedance-based biosensing platforms were fabricated for the

detection of pathogens in poultry animals. Jasim and his colleagues. developed
impedance-based MEMS (micro-electromechanical system) biosensing platform for
the screening of Salmonella serogroups (Jasim et al. 2017, 2019). In another study,
Liu and coworkers fabricated an integrated impedance biosensor for the identifica-
tion of Salmonella serotypes (B, D, and E) (Liu et al. 2018). Wang and the team
fabricated an impedance biosensor based on immunomagnetic separation to identify
the concentration of Salmonella (Wang et al. 2020).
20.5.2.4 Colorimetric Biosensor
Surface plasma resonance (SPR), Quartz crystal microbalance (QCM), and optical
waveguide biosensors were used to identify glycan based Avian Influenza virus
(AIV) (Hidari et al. 2007; Suenaga et al. 2012). HA (Influenza hemagglutinin surface
protein) binding to gold NPs attached sialic acid helped in the detection of influenza
virus without any amplification process or prior treatment in solution. The reaction
Table 20.2 Disease causing pathogen detection in poultry animals using biosensors
S. No. Transducer Pathogen detected Detection References
limit
1 Microfluidic biosensor Salmonella 103 CFU (Kim et al.
typhimurium mL−1 2015)
2 QCM biosensor Avian Influenza virus 1 × 104 PFU (Hewa et al.
mL−1 2009)
3 Surface plasmon Influenza virus 50 PFU (Lee et al.
biosensor mL−1 2015)
S. typhimurium 1 × 106 CFU (Lan et al.
mL−1 2008)
4 Electrochemical Influenza virus 80–100 virus (Nidzworski
biosensor particles et al. 2014)
μL−1
S. pullorum 89 CFU (Fei et al. 2016)
mL−1
Avian influenza virus 1.6 pg mL−1 (Huang et al.
H7 2016)
5 Electrochemical S. serogroups 7 Cell mL−1 (Jasim et al.
impedance biosensor 2019)
6 Optical fiber and light Salmonella 103 CFU (Abdelhaseib
scattering sensors mL−1 et al. 2016)
7 Multiplex optical fiber Listeria ~103 CFU (Ohk and
biosensor monocytogenes, E. coli mL−1 Bhunia 2013)
and Salmonella
8 Immunosensors E. coli 7.1 × 102 (Tokarskyy and
cells mL−1 Marshall 2008)
9 Wave Fiber Optic S. enterica 104 CFU (Valadez et al.
Biosensor mL−1 2009)
10 Microfluidic colorimetric E. coli 50 CFU (Zheng et al.
biosensor mL−1 2019)
11 Enzyme-Free biosensor S. typhimurium 50 CFU (Huang et al.
mL−1 2018)
12 Phage-based capacitive Salmonella spp 200 CFU (Niyomdecha
biosensor mL−1 et al. 2018)
13 nano-bead based S. typhimurium 10 CFU (Alamer et al.
colorimetric mL−1 2017)
immune-biosensors
14 Impedance biosensor with Salmonella 102 CFU (Wang et al.
immunomagnetic mL−1 2020)
16 Impedance biosensor Salmonella serogroups 7 cells mL−1 ( Jasim et al.
2019)
17 Integrated impedance Salmonella serotypes 8 cells mL−1 (Liu et al. 2018)
biosensor B and D
18 Impedance-based MEMS Salmonella serogroups 7 cells mL−1 (Jasim et al.
(micro-electromechanical 2017)
system) biosensor
(continued)

S. No. Transducer Pathogen detected Detection References
limit
19 Colorimetric S. enteritidis, S. 10 CFU (Alamer et al.
immunoassay typhimurium mL−1 2018)
Staphylococcus
aureus, and
Campylobacter jejuni
produced after binding of HA with the sialic acid released a signal in this colori-
metric assay which shows linear relation with the viral strain (Gopinath et al. 2013;
Takahashi et al. 2013; Lee et al. 2013). Alamar and group developed a colorimetric
immunoassay for the detection of S. enteritidis, S. Typhimurium, Campylobacter
jejuni, and Staph aureus, using a cotton swab and nanobead conjugated antibody
(Alamer et al. 2018)
20.5.2.5 Piezoelectric Biosensor
A quartz crystal microbalance assay was utilized for the identification of avian
influenza virus-based coupling of anti-M1 monoclonal antibody attached to Au NPs
and recorded LOD was 1 × 104 PFU mL−1 with a linear range of 103 –108 PFU
mL−1 (Hewa et al. 2009).
20.5.2.6 Surface Plasmon Biosensor
An immunosensor based on plasmon assisted fluorescence was designed to detect

influenza virus through anti-M1 antibody attached to Au NPs coated with carbon
nanotubes. When virus combines with the NPs, a fluorescent signal was released
following the addition of cadmium telluride quantum dots, which shows the lower
detection limit of the sensor at 50 PFU mL−1 (Lee et al. 2015).
20.5.2.7 Electrochemical Biosensor
Several other biosensors were also made for the detection of pathogens such as an
electrochemical sensor was employed based on an anti-M1 antibody specific to the
virus which detected all the influenzas serotypes. The detection limit was observed
as 80–100 virus particles μL−1 (Nidzworski et al. 2014).
Salmonella pullorum is a deadly organism that causes high mortality in chickens.
For the detection of S. pullorum, an immunomagnetic assay based on magnetic
beads and an electrochemical label comprising of gold-coated graphene oxide
(rGO/AuNPs) were designed. The developed electrochemical immunosensor was
based on the conjugation of antibody and magnetic beads, the process was carried
out on the surface of screen-printed electrode (SPCE), by immobilizing rGO/antibody
complex and using differential pulse voltammetry. The studies show that gold-coated
rGO initiates signal amplification more strongly. The LOD of the system was 89 CFU
mL−1 and the linear range for the experiment was 102 –106 CFU mL−1 (Fei et al.
2016). For E. coli K12 identification in frozen chicken, Helali and the team fabricated
a platform based on the antigen and antibody immobilization. The physisorption
technique was used for the immobilization of anti-E.coli on the gold surface. For
determining E. coli, the EIS and surface plasmon resonance studies were performed.
The lower limit of detection for the sensor was 103 CFU mL−1 (Helali et al. 2018).
20.5.2.8 Microfluidic Colorimetric Biosensor
A novel microfluidic colorimetric biosensor using gold nanoparticles (AuNPs) was

developed for the identification of E.coli pathogen in chicken samples. The sensor
is based on the capture and detection of antibodies, where capture antibodies were
used to modify magnetic nanoparticles and detection antibodies were used to modify
polystyrene microspheres (PSs). The lower detection limit of the biosensor was 50
CFU mL−1 (Zheng et al. 2019).
20.5.2.9 Enzyme-Free Biosensor
A study by Huang and colleagues describes the fabrication of a biosensor for identifi-
cation of Salmonella Typhimurium (S. Typhimurium), the developed platform was an
enzyme-free biosensor. The sensor was developed using signal reporter as curcumin
(CUR) and for signal amplification 1,2,4,5-tetrazine (Tz)-trans-cyclooctene (TCO)
was used. The LOD for the developed platform was calculated as 50 CFU mL−1
(Huang et al. 2018).
20.5.2.10 Phage-Based Capacitive Biosensor
For the identification of Salmonella spp., the biosensor fabricated was based on
a capacitive flow injection system. In this system, the gold surface was modified
with Salmonella-specific M13 bacteriophage using crosslinker (glutaraldehyde). The
sensor can produce the results after 40 min of the sample injection, the observed LOD
for the sensor was 200 CFU mL−1 (Niyomdecha et al. 2018).
20.6 Conclusion
For the rapidly growing poultry sector, it is necessary to provide hygienic food, so
that the effect of antibiotics may not be suffered by humans. Antibiotic resistance
in poultry industry which has become a crisis worldwide for the human population
has come out to the life-threatening event in recent past years. Mortality rates have
been increasing day by day due to the consumption of poultry animals or animal
products. Drug-resistance in humans arises when the drug-resistant bacteria enters
our immune system on the consumption of poultry products as they receive a high
amount of antibiotics for the prevention of bacterial diseases. Research study has
already shown that antibiotic residue in animals when transferred to the human
body there occurs an imbalance between our good microbial bacteria and harmful
disease-causing bacteria.
There should be some alternative ways to prevent diseases and control antibiotic
use in farm animals. Today many platforms are already available in the market for the
diagnosis of the antibiotics and pathogens present in the food. Hygiene practices must
be employed in the farms which are neglected generally, and strict rules should be
implemented to check the maximum residual limit in farm animals. Some safeguards
must be utilized for controlling the rate of infection in both animals and humans.
All the antibiotics are not dangerous but their use beyond Maximum Residue Limits
(MRL) is a frightening event. Along with these management practices, the use of
nanosensors can be of great help, and more research in this area is needed to reduce
the cost and to improve the reliability of these sensors. We firmly believe that, as the
field of sensors developments is supported by more and more research, there will be
many opportunities for the poultry industry to benefit from it.
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Chapter 21
Smart Aquaculture: Integration
of Sensors, Biosensors, and Artificial
Intelligence
Dolly Sharma and Ranjit Kumar
Abstract Aquaculture involves cultivating aquatic plants and a wide variety of

aquatic animals used for human consumption, medicines, and supplements. This
industry is consistently growing due to growing demands from people preferring
organic seafood and marine supplements, such as fish oil, marine collagen, seaweed,
algae, etc. This extremely complex task of setting up and maintaining the aquacul-
ture industry requires fine-tuning a large number of variables to produce an optimal
outcome. Technology integrated with efficient algorithms helps create a framework
for the intelligent aquaculture industry. Sensing and machine learning algorithms
play a key role here, as they enable automated real-time monitoring of the environ-
ment along with quick decision-making to mitigate risks. This chapter presents a
review of major tasks involved, namely technology used for collecting real-time data
such as physical sensors, biosensors, optical sensors, etc., the role of automation and
automated decision-making.
Keywords Machine learning · Artificial intelligence · Sensors · Aquaculture
21.1 Introduction
Aquaculture is synonymous with aquatic farming and has been in existence for a
very long time. The FAO (Food and Agriculture Organization) of United Nations
Organization (UNO) defined aquaculture as rearing or farming of aquatic organisms
such as fish, molluscs, crustaceans, aquatic plants, crocodiles, alligators, turtles, and
amphibians (FAO 2008). There are three kinds of culture environments: freshwater,
D. Sharma
Computer Science and Engineering, Shiv Nadar University, Gautam Buddha Nagar,
Noida, Uttar Pradesh, India
R. Kumar (B)
Department of Chemistry, School of Engineering, University of Petroleum and Energy Studies
(UPES), Bidholi, Dehradun, Uttarakhand, India
456 D. Sharma and R. Kumar
Fig. 21.1 Smart aquaculture model
brackishwater, and mariculture. The aquaculture units could be ponds, tanks, cages,
raceways, barrages, rice-cum-fish paddies, rafts, hatcheries, nurseries, etc. Setting up
the right environment is key to succeeding in this industry. Some of the components
that must be carefully thought through during the initial phase of setup are: a selection
of the species that you are planning to grow/breed, procurement, and transportation,
selecting an appropriate location and type of medium to set up the farm, and choosing
the right culture type.
Once the big picture is ready, every small aspect needs to be carefully planned and
executed. Technology is great support for designing a successful aquaculture model,
as an integrated smart environment makes real-time monitoring and decision-making
possible. There are many such models presented in the past, where data from the
sensors is collected and fed into a machine learning model for predictions and then
further into a decision support system. These models work well in small experimental
settings. However, with larger setups and the requirement of constant monitoring,
there is a need for a scalable big data and cloud computing-based smart aquaculture
system. This setup will also bring down the hardware cost. Some such efforts have
been initiated by (Rao et al. 2018; Dzulqornain et al. 2018), where data analytics is
integrated with cloud computing.
In this chapter, we discuss the current scenario of the aquaculture industry. We aim
to present the need to design a functional smart aquaculture setup, where operations
can be standardized for better management. A high-level model for smart aquaculture
is shown in Fig. 21.1.
21.2 Monitoring the Quality of Water
The monitoring of the quality of water in aquaculture tanks is essential in order to

monitor the health of fish. Various parameters such as temperature, pH, ammonia,
nitrite/nitrate, hardness, alkalinity, turbidity, gas concentration, and dissolved oxygen
level need to be monitored in aquaculture. Earlier traditional ways like potentiometry,
21 Smart Aquaculture: Integration of Sensors … 457
titration, and conductivity measurement methods were used to monitor these param-
eters (Amrita and Babiyola 2018). Modern methods use sensory data to analyze
the water conditions. Different types of sensors, including optical sensors, thermal
sensors, biosensors, electrochemical sensors, etc., are being used (Parra et al. 2018).
The temperature of the water has a direct impact on feeding patterns, growth of
fish, stress, and disease breakout. Warm water has less oxygen solubility than cold
water. The level of dissolved oxygen (DO) in water is related to the amount of oxygen
consumed, which in turn affects the size of fish, feeding rate, and activity level. The
optimal level of DO is essential for fish respiration, as well as for the survival of phyto-
plankton. Phytoplankton breaks down the toxic ammonia into harmless nitrogen.
Through this process, phytoplankton indirectly maintains the pH level within the
acceptable range of 6.5–9.0 for fish culture. When pH rises above 9, ammonium
ion (NH4 + ) in the water is converted to toxic ammonia (NH3 ), which is lethal for
fish. Acidic water with pH less than 5 leads to leaching of metals from rocks and
sediments. These metals concentration also needs to be monitored as it adversely
affects fishes’ metabolism rates and their ability to take in water (Su et al. 2020).
Li and coworkers proposed a hybrid model to predict dissolved oxygen level, based
on multi-scale features using ensemble empirical mode decomposition (EEMD) (Li
et al. 2018). Wireless sensor networks (WSN) have been used to sense and measure
pH, NH4 + , and temperature for a fish farm. Sensors based on a specially designed Ion
Sensitive Field Effect Transistor (ISFET) can be used to monitor different parameters
in aquaculture. The sensor module can collect data and transmit the data to the
wireless module by means of a 9600 b/s asynchronous wired communication. The
wireless node retransmits the data to the central unit via radio frequency (RF) (López
et al. 2009). Liu and coworkers proposed a real-value genetic algorithm support vector
regression (RGA-SVR), based on genetic algorithms and Support Vector Regression,
that predicts water quality (Liu et al. 2013).
21.3 Health Monitoring of Aquatic Animals
Customized food options and optimal feeding pattern is crucial to aquatic animal’s
survival. One such example is cottonseed, which is an inexpensive and very widely
available plant-based protein source and is liked by most aquatic animals in the diet
(Li and Robinson 2006). However, it is only used in limited quantities as it is not
easily digested by aquatic animals and is potentially toxic. Thus, depending upon the
species, a specific percentage of cottonseed can be incorporated into aquatic food in
combination with other components.
Other than taking care of deciding the quality and quantity of food, it is also essen-
tial to observe the feeding behavior of aquatic animals. Biosensors and other optical
sensors have been very helpful in monitoring the feeding patterns of aquatic animals.
This task cannot be accomplished manually because of low visibility underwater.
Image processing techniques have been used to analyze the uneaten food pellets that
pollute the water and cause sickness to animals. Continuously learning the feeding
patterns also leads to a cost-effective, sustainable, and environment-friendly aqua-

culture industry setup. Liu et al. (2015) used an underwater camera with LED lights
for this task. They used a linear-time component-labeling algorithm to figure out
the number of pellets left. Their algorithm has a counting error of less than 8%
and is suitable for practical applications. The complex task of identifying animal’s
food preferences and identifying their hunger is very important in maintaining good
animal health. Razman et al. (2019) designed a method to identify hunger state for
Lates calcarifer. Machine learning algorithms were used to analyze the 16 features
collected through time-series data using window size of 0.5, 1, 1.5, and 2 min from the
videos. The study demonstrated that they could identify the stage of hunger and use
of automated feeding resulted in growing heavier and longer fish. Nanobiosensors
are also used for monitoring and controlling microbes in water (Dar et al. 2019).
Other than maintaining healthy feeding protocols, it is also essential to monitor
the health of aquatic animals for diseases, epidemics, and stress. Biosensors are
very useful in health monitoring. Surface Plasmon Resonance (SPR)-based biosen-
sors detect poisoning in Paralytic Shellfish (Fonfría et al. 2007), and are essen-
tial for the safety of consumers. Based on molecular interaction detection by SPR
biosensors, anti-GTX2/3 (Gonyautoxin 2,3) antibody (GT13-A), and saxitoxin-CM5
chip; an inhibition assay was developed. Most countries set the Paralytic Shellfish
Poison (PSP) toxin limit of 80 µg per 100 g of meat and this process is capable
of detecting 15–400 µg per 100 g of shellfish meat. This method detects decar-
bamoyl saxitoxin-dcSTX, gonyautoxin 2,3 (GTX2/3), decarbamoyl gonyautoxin 2,3
(dcGTX2/3), gonyautoxin 5 (GTX5), C1/2, and saxitoxin (STX). The detection of
STX can be improved by improving the cross-reactivity among the analogues.
When a fish is exposed to an internal or external stressful situation, it activates
the hypothalamic-pituitary-interrenal (HPI) axis as a primary response followed by
secondary and tertiary responses (Sopinka et al. 2016). The authors presented a study
of various stress indicators and factors that are responsible. Understanding how stress
effects to fish and the ways by which it can be identified will be beneficial for fishes.
Biosensors also play a vital role in evaluating the freshness of fish. Table 21.1
enlists different types of biosensors used in aquaculture. Amperometric electrodes
based on oxygen or hydrogen peroxide detection, together with an oxidase system or
an aerobic microorganism are commonly used in food and drink industries because of
their simplicity and high selectivity (Luong et al. 1991). The important areas of appli-
cations of biosensors are proximate analysis, nutritional labeling, determination of
pesticide residues, naturally occurring toxins and antinutrients, processing changes,
microbial contamination, enzymatic inactivation, and Biochemical Oxygen Demand
(BOD) of wastes (Rajee and Alicia 2019; Suleiman and Guilbault 1994).
Biochemical Oxygen Demand (BOD) is an important parameter for the well-
being of aquatic animals. It can be monitored using biosensors based on bacteria
like Pseudomonas sp., Bacillus sp., and thermophilic bacteria. These are very fast
biosensors and are capable of determining BOD values as low as 1 ppm within 30 min
(Venugopal 2002; Rajee and Alicia 2019). Various biosensors have been developed
to detect pathogenic bacteria such as Salmonella sp., E. coli, etc., in the contaminated
water discharged from the aquaculture (Monzó et al. 2015).
Table 21.1 Types of biosensors used in aquaculture

Biosensor Description Application References
Optical biosensors Measures the change in Detection of (Narsaiah et al.
amplitude, phase, pathogens, pesticide 2012)
frequency, or and drug residues, (Zhao et al. 2020)
polarization of light hygiene monitoring,
heavy metals, and other
toxic substances in the
food to check whether
the food is safe for the
consumption
Detection of antibiotics
Aptamer-based High sensitivity and Disease diagnosis, (Chong and Low
biosensors selectivity of prophylactic, and 2019)
transducer-linked therapeutic
molecular recognition
element for biosensing
that are used in
electrochemical,
optical, or
mass-sensitive
analytical techniques
Enzyme-based Uses a combination of Detection of (Mitsubayashi et al.
biosensor an enzyme with a Trimethylamine in fish 2004)
transducer to generate a Antibiotics and (Rajee and Alicia
signal relative to antimicrobials 2019)
concentration of target detection, BOD,
analyte insecticides detection
Fluorometric Works on Nitrite detection in (Pires et al. 2019)
biosensor chemiluminescence and aquaculture water
fluorometric detection
Nanomaterial-based Carbon Detects microbes such (Dar et al. 2019)
biosensor nanotubes-based as bacteria, viruses,
biosensor and parasites and also
heavy metals in food
and water
Electrochemical Based on catalysis Detection of ostreid (Toldrà et al. 2020)
biosensors reaction by enzymes herpesvirus 1, which
that generates or poses threat to shellfish
consumes electrons aquaculture
Biosensors are also used for the detection of pesticides found in the food chain
of fish as reported. Campàs et al. (2007) reported an array of biosensors to detect
marine toxins. Biosensors based on the inhibition of urease can be developed for
the determination of Hg, Ag, and Cu in the ppb range (Majlesi et al. 2019). Detection
of antibiotics like Penicillin in the range 5–30 mmol was achieved using immobilized
E. coli with a response time of 8 min (Galindo et al. 1990). A highly sensitive
colorimetric biosensor for antibiotics like chloramphenicol (CAP) was reported by
incorporating ELISA onto surfaces of microporous and nanofibrous membranes with

a detection limit up to 0.3 ng/mL (Zhao et al. 2020).
Optical biosensors measure polarization as well as frequency, phase, and ampli-
tude of the light wave and are used to identify food borne pathogens. Some of such
optical biosensors are microarrays, optical fibers, and SPR (Narsaiah et al. 2012).
21.4 Role of Technology in Aquaculture Industry
Information and Communication Technology plays a huge role in the success of

the aquaculture industry, especially the large-scale setups where physical monitoring
at all times is almost impossible. The role of ICT starts from the very beginning, such
as the use of GIS-based methods for selecting the right location for setup (Kapetsky
et al. 1990; Gimpel et al. 2018; Falconer et al. 2019). The authors presented an
automated system that used a dataset composed of many features, including spatial
features; and analyzed the potential of development of aquaculture setup. They
proposed that some other features that could be helpful are the location of feed mills,
availability of workforce, land use, water use, level of groundwater, frequency of
floods, other factors to optimize logistics. Their experiment was set up in Louisiana,
where they studied double cropping for catfish and crawfish. This area has been of
interest to many researchers in the last three decades. GIS-based planning tools such
as AquaSpace, enable mapping of 30 indicators based on opportunities (Gimpel et al.
2018).
21.4.1 Study Growth Patterns
Other parameters that help keep a tab on fish health are growth pattern, fish count,
fish density, movement patterns, etc. Keeping an accurate tally of fish count majorly
involves tagging and vision-based methods. A fish counting method (Le and Xu 2017)
was proposed and they identified that underwater images are not very high quality
and have a lot of noise. Thus, they designed a threshold-based algorithm that was
able to identify the fishes using only the contour information and removing the noise.
The situations where two or more fishes were overlapping in the image were also
resolved using the skeletal endpoints. The experiments were carried out in a small
experimental setup, however, the performance in large complex real-world setups
would be more challenging. Another effective method to keep track of fish count is
Tagging (Pine et al. 2003). The studies integrate different types of tags such as fin
clips, telemetry tags, etc., with computer software so that the population size and the
mortality-related data can be analyzed. This data when integrated with data from other
sources gives significant insight into factors leading to an ideal breeding scenario.
The way multiple sensors integrated with computer vision techniques have been
incorporated into computational models, has been studied by many researchers and
in-depth reviews of the methods have also been studied (Saberioon et al. 2017; Hassan
et al. 2016). A latest trend in aquaculture is to create the feed using a lower percentage
of fish meal and higher percentage of several plant ingredients and nutrients. The
experiments involved creating a multi-choice feeding system in an aquaculture setup
(da Silva et al. 2016).
21.4.2 Real-Time Monitoring
Another important aspect where technology can support is real-time automated moni-
toring. An automated fish farm monitoring system (Chen et al. 2016) was proposed
where environmental monitoring could be done by a mobile device. They tested the
performance of the wireless sensor networks in a simulation setup using MSP430
chip and ZigBee wireless transmission, in addition to the feeder, inflator to manage
oxygen levels, heaters to maintain temperature, and RGB light modulation system.
The proposed system proved to be power efficient as well as cost efficient according
to the simulation settings. A system where real-time monitoring can be valuable,
if the corrective actions can also be taken on a timely basis without any human
intervention. An integrated model using environmental, biological, economical, and
technical data was simulated to implement a decision support system (DSS) using
Particle Swarm Optimization (Cobo et al. 2019). This DSS provided support in both
operational and strategic decision-making, and at both levels: day-to-day basis as
well as long-term planning. With the availability of high computational power with
lower cost, it is now possible to train deep learning models for smart fish farming and
can be used for fish counting, classification of species, analyze behavior patterns,
feeding patterns, size, analyze water (Yang et al. 2020).
Another DSS was proposed (Shahriar and McCulluch 2014), where two methods
were explored for decision-making in aquaculture closure. They used data such as
rain, river flow, salinity and temperature for time-series prediction and expert rules
and classification. The experiment was conducted on the dataset from a shellfish farm
from Tasmania, where they collected one-year data for training purpose and the next
two months data for testing.
Thus, we see that technology plays a huge role in optimal functioning of aquacul-
ture industry. Machine learning algorithms (Kang et al. 2017) built and trained specif-
ically for this industry, can reshape it and help in standardization across the globe.
These algorithms are capable of parameter tuning for optimal results, prediction, and
decision-making; thus, enabling the stakeholders to maximize profits.
21.5 Conclusion and Future Directions
Aquaculture industry is growing consistently as the demand is growing. It is important

that the industry brings profit, thus there is a lot of interest in the area of optimizing
the operations leading to low cost and low energy. There is an additional requirement
that all stakeholders must also address, that is, sustainability. The leading cause of
pollution caused by this industry is the leftover food pellets. Thus, the setups which
have recirculating aquacultures (Sönmez et al. 2016), need to make sure that the
water supply is not contaminated. Also, methods such as double cropping and inte-
gration of aquaculture systems to irrigation water should be encouraged. Futuristic
planning of aquaculture industry has to involve an efficient model that incorporates
all relevant physical sensors, biosensors, audio/video monitoring, and IoT devices,
which provides input to a big data and cloud computing environment, for real-time
monitoring of this large-scale data.
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Chapter 22
Biosensor as a Potential Tool for On-Site
Detection of Insect Pathogens
Mudasir Gani, Taskeena Hassan, Pawan Saini, Khalid Hussain Bhat,

Rakesh Kumar Gupta, and Kamlesh Bali
Abstract Insects constitute the largest and most diverse group of animals on the
earth and are potentially the largest pathogen reservoir. Several pathogens are known
to infect insects and cause lethal or sub-lethal effects. These entomopathogens are
widespread in the natural environment and invade and reproduce in an insect and
spread to infect other ones through horizontal and vertical transmission. Historically,
insect pathogens have been of interest due to their economic impact on beneficial
insects in terms of morbidity and mortality or their potential as specific biolog-
ical control agents of insect pests. The present methodologies for insect pathogen
detection viz. microscopic examination, the culture of pathogens, biochemical,
immunological, and molecular techniques are entirely laboratory-based and cannot
be performed under field conditions. Hence, there is a great scope and need for
the development of sensitive and specific techniques for early, rapid, and on-site
detection of insect pathogens. One of the useful emerging technology is the use
of a biosensor for on-site and point-of-care detection of pathogens of beneficial
insects for efficient disease management and for on-site detection and distribution
of native insect pathogens for commercial production of insect-specific biopesti-
cides. The biosensor technology has the potential to surpass conventional or present
M. Gani (B)
Division of Entomology, Faculty of Agriculture, Sher-e-Kashmir University of Agricultural
Sciences and Technology, Kashmir 193201, J&K, India
T. Hassan
Department of Zoology, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India
P. Saini
Central Sericultural Research and Training Institute, Central Silk Board, Ministry of Textiles,
Govt. of India, Pampore 192121, J&K, India
K. H. Bhat
Division of Basic Sciences and Humanities, Faculty of Agriculture, Sher-e-Kashmir University of
Agricultural Sciences and Technology, Kashmir 193201, J&K, India
R. K. Gupta · K. Bali
Division of Entomology, Sher-e-Kashmir University of Agricultural Sciences and Technology,
Chatha 180009, J&K, India
466 M. Gani et al.
detection techniques in time, accuracy and cost owing to recent developments in

antibody production, nanotechnology, and microfluidics along with the availability
of improved antibody immobilization strategies. This chapter provides a compre-
hensive review of the insect pathogen detection techniques developed and used so
far, as well as their limitations and the potential of biosensors for on-site detection
of insect pathogens.
Keywords Insect · Pathogen · Diagnosis · Biopesticide · Nanotechnology ·

Transducer · Immunosensor
22.1 Introduction
Insects represent the largest and most diverse group of animals on the Earth with an
estimated 5.5 million different species (Stork 2018). Like other animals, insects are
sensitive to environmental, nutritional, and microbial factors which result in various
types of diseases caused by noncellular agents (viruses), prokaryotes (bacteria),
eukaryotes (fungi and protists), and multicellular animals (nematodes) (Gani et al.
2019). The microbes exist everywhere in nature and have the capability to invade and
reproduce in an insect, spread, and infect other insects, and cause lethal or sub-lethal
effects. Historically, insect pathogens have been of interest due to their economic
impact on beneficial insects in terms of morbidity and mortality or their potential as
biological control agents of insect pests. Accordingly, the insect diseases are broadly
divided into two categories which include: (a) diseases of beneficial insects (produc-
tive insects, i.e. honey bee, silkworm, lac insect; pollinators such as honey bees,
syrphid flies, bumblebees, etc.; biological control agents, i.e. coccinellids, praying
mantis, predatory stink bugs, etc.) and (b) diseases of injurious or harmful insects
(insect pests of cultivated crop plants like cotton bollworm, tobacco caterpillar, etc.;
storage insect pests like rice weevil, pulse beetle, etc.). The protection of benefi-
cial insects from diseases is an important aspect of insect pathology as beneficial
insects provide useful products such as honey, silk, lac, etc., and act as pollinators
and biological control agents. Much of the research in this area concerns the causal
agents of insect diseases, prevalence, virulence, transmission, genetics, genomics,
and prevention and management of diseases (Chen et al. 2014; Gani et al. 2018).
On the other hand, insect pathogens can regulate insect pest populations (Anderson
and May 1980; Cory and Myers 2003; Elderd 2013) and have been developed as
biological insecticides for the management of the insect pests of agricultural and
forest crops (Moscardi 1999; Gupta et al. 2016). Here focus is on the exploitation of
insect pathogens for biological pest control, their mass production, formulation, and
application to insect pest populations in a manner analogous to chemical pesticides
(Del Rincón-Castro and Ibarra 2011; Gani et al. 2019).
The insect disease epidemics are determined by the quantity of initial inoculum
and the rate of transmission. Therefore, diagnostic techniques which allow early, and
rapid detection of initial inoculum are needed. The detection of insect pathogens is
being routinely conducted by traditional techniques based on symptomology, in vitro
22 Biosensor as a Potential Tool for On-Site Detection … 467
culturing, and microscopy (Evans and Shapiro 1997; Gani et al. 2017a). However, it
takes a long time for bacteria and fungus to grow on a culture media and produce diag-
nostic spores. Such a delay is not acceptable when trying to decide on a management
strategy for beneficial insects or when rapid detection is required to eradicate the
spread of a newly introduced pathogen. Further, there are pathogens such as viruses,
rickettsia, and microsporidia (obligate pathogens) which cannot be cultured on artifi-
cial media. The alternative methods for insect pathogen detection viz. immunoassays
and nucleic-acid amplification {polymerase chain reaction (PCR)} are also entirely
laboratory-based and require well-trained staff and equipments (Gani et al. 2017a).
These techniques also lack the convenience of “on-site” testing and require a complex
workflow from sample collection, sample labeling, sample storage, and transport to
appropriate facility and further followed by sample processing, assays, analysis, and
interpretation of results.
Normally, the disease is recognized by the presence of clear symptoms at later
stages of infection. Many diseases remain undetected (latent, sub-lethal) and are
recognized only once the disease has clear symptoms or causes lethal effects (Vogel
et al. 2019). Many insect pathogens are present in asymptomatic form and hence
the absence of pathogens from insect populations cannot be ruled out. Moreover,
the pathogen load or the initial rate of infection may often be low in insect popula-
tions which demonstrates that highly sensitive and specific detection techniques are
also needed. Therefore, it is imperative for insect pathologists to develop sensitive,
specific, and portable assays capable of early, rapid, and on-site diagnosis. In recent
years, major advances has been made in the field of nanotechnology which have
several potential applications in agriculture (Fig. 22.1). The biosensor for disease
Fig. 22.1 Potential applications of nanotechnology in agriculture

468 M. Gani et al.
diagnosis was developed by the integration of a powerful nanotechnology approach

with molecular biology. The biosensor is one of the useful emerging technology for
on-site detection of pathogens of beneficial insects for effective disease management
and for on-site detection and geographical distribution of native insect pathogens for
commercial production of insect-specific biopesticides as a component of biological
control. The native strains are always preferred in biological control owing to their
adaptability, sustainability, and efficacy under a given set of agro-ecosystem. The
identification and utilization of native strains hold ample scope for their widespread
multiplication and commercial use as novel biopesticides. The biosensors were devel-
oped and used for the detection of diverse pathogens (Ahmed et al. 2014), however,
this technology has not been widely applied to insect pathogen detection.
22.2 Current Approaches for Insect Pathogen Detection
The microscopic examination of insect pathogens has been well known as one of the
most available, easy to perform, and inexpensive techniques. However, microscopy
has inherent problems such as late detection, presence of secondary pathogens, and
inaccuracy as some pathogens can be overlooked under a microscope being very
small in size (Iwano and Ishihara 1981; Kawarabata and Ishihara 1984; Poláchová
et al. 2019). The culture of pathogens is done by the aseptic transfer of an inoculum
from a source (insect, soil, food, etc.) to a suitable growth media or host resulting
in amplification of cell numbers for quantitative determination. This propagation
of pathogens may be performed using antibiotics to suppress the growth of other
strains of pathogens that may also reside in the inoculum. The subsequent transfer
of pathogens to selective or differential media generates colonies that can be distin-
guished based on colony morphology by ocular inspection and identified by rigorous
biochemical, microscopic or nucleic acid-based assays. Koch’s postulates are used to
identify unknown pathogens in diseased samples (Koch 1884; Rivers 1937). Koch’s
postulates are based on four criteria to establish the identity the causal agent of a
disease: (1) the pathogen is always associated with a disease; (2) the pathogen must
be isolated from the diseased sample and grown in pure culture; (3) the pathogen
from the pure culture must be re-inoculated into a healthy host and should cause the
same disease symptoms; and (4) the pathogen must be re-isolated from the newly
inoculated host and proven to be the same as the original one. Electron microscopy
(EM) has also been used for insect pathogen identification, description, and discovery
(Adams et al. 1977; Van Iddekinge et al. 1983; Kumar et al. 2011). The problems in
the use of EM in insect pathogen diagnosis include sample transportation, processing,
testing conditions, variations induced by the personnel in sample fixation and time
of exposure, and use of high-end instruments.
Serological techniques including complement fixation, immunodiffusion,
radioimmune assays, western blotting, and enzyme-linked immunosorbent assay
(ELISA) have been used to detect and quantify insect pathogens (McCarthy and
Gettig 1986; Harlow and Lane’s 1988; Liu et al. 2011). These methods employ
antibodies for the detection of a given insect pathogen from analytical samples
isolated from infected insects. The latex agglutination test for the detection of
flacherie (Shimizu et al. 1983), cytoplasmic polyhedrosis (Shimizu and Arakawa
1986), and densonucleosis (Shimizu and Tauchi 1991) viruses in Bombyx mori
L. (Lepidoptera: Bombycidae) have been used. Arakawa (1989) introduced a
latex agglutination test for the detection of nucleopolyhedrovirus (NPV) infection
in B. mori. Arakawa and Shimizu (1990) developed a protein-A linked latex agglu-
tination test using polyclonal antibodies for the detection of NPV infection and the
test was more sensitive than the direct latex agglutination test. A colloidal textile
dye-based dipstick immunoassay for the detection of B. mori nucleopolyhedrovirus
(BmNPV) was developed by Nataraju et al. (1994), with comparable sensitivity and
cost-effectiveness. Shamim et al. (1994) produced murine monoclonal antibodies
(MCAs) against BmNPV which reacted well with polyhedra of BmNPV and to a
variable extent with the NPV of other species, namely Amsacta albistriga, Heli-
coverpa armigera, and Spodoptera litura. These anti-BmNPV MCAs showed no
cross-reactivity with Nosema bombycis, Serratia marcescens, group A Streptococci,
Staphylococcus aureus, Escherichia coli, and normal hemolymph proteins, except
Bacillus thuringiensis. The MCAs specifically recognized BmNPV polyhedra and
were found effective for the detection of BmNPV infection in B. mori at early stages.
Shamim et al. (1995) developed a protein-A linked monoclonal antibody latex agglu-
tination test (PALMAL) for BmNPV detection in B. mori. The latex beads were
precoated with protein-A and then sensitized with monoclonal antibody (MA-231)
(125pgml−1 ) directed against polyhedrin protein of BmNPV. PALMAL test has the
capability to detect l × 105 polyhedra/test and is more sensitive than the direct
agglutination test. The use of specific monoclonal antibody and the presence of free-
floating antigen-binding sites (Fab) of the immunoglobulin to react with antigen have
improved the sensitivity of the PALMAL test (Torrance 1980).
ELISA, Southern hybridization, and PCR assay were used to detect Orgyia pseu-
dotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth
larvae (Thorne et al. 2007). Kumar et al. (2007) developed a direct antigen coating
enzyme-linked immunosorbent assay (DAC-ELISA) to detect NPV infection in Heli-
coverpa armigera using polyhedrin-specific polyclonal antibodies of Helicoverpa
armigera nucleopolyhedrovirus (HaNPV). The polyclonal antibodies were highly
specific and useful to detect HaNPV at the early stages of infection. Monoclonal
antibodies were also generated against Autographa californica nucleopolyhedrovirus
(AcNPV) which reacted well with polyhedrin protein of AcNPV and with a wide
range of baculoviruses (Roberts and Naser 1982). The western blot was developed
and described as an extremely sensitive technique for the detection of traces of
AcNPV in very crude materials (impure polyhedra, infectious haemolymph, and
larval homogenates) (Naser and Miltenburger 1983). Poláchová et al. (2019) devel-
oped a sandwich upconversion-linked immunosorbent assay (ULISA) for M. pluto-
nius detection using rabbit polyclonal anti-Melissococcus antibody and successfully
demonstrated its practical applicability. But these assays are time-consuming and
are not suitable for rapid on-site diagnosis. Further, the selectivity and sensitivity of
470 M. Gani et al.
ELISA and western blotting techniques depend on the antigen–antibody interaction

and are well suited for samples without any contaminants or interfering molecules
such as proteins, DNA, and non-target cells.
Another method for insect pathogen detection is the amplification and subsequent
analysis of pathogen-specific nucleic acid by PCR and sequencing. The PCR tech-
nology is commonly used for the identification and detection of insect pathogens
owing to its sensitivity to detect specific DNA templates at nanogram and picogram
concentrations (Gani et al. 2017b; Dieffenbach and Dveksler 2003). An important
step in the PCR procedure is the extraction and purification of clean nucleic acid
as a template for the reactions. While it is relatively easy and simple to isolate the
pathogen from the insect cadaver or living infected insect, it is technically more
cumbersome to extract quality DNA from the stock solution of the pathogen for
PCR. There are also chances of getting false results due to the presence of contam-
inants in the sample. Further, simultaneous processing of large numbers of samples
increases the risk of contamination and false positive results. These methodologies
are now emphasized by real-time PCR and reverse transcriptase PCR. However, the
implementation of these techniques for insect pathogens detection can be limited by
external factors. For example, the original stock inoculum often contains high levels
of fats, carbohydrates, and tissue debris which requires a sample purification step
prior to analysis. Further, there are chances of non-specific DNA amplification due
to the presence of “naked” DNA in analytical samples that may act as a template for
the amplification of these superfluous products. Ravikumar et al. (2011) developed
a novel PCR-based assay for individual and simultaneous detection of three major
pathogens {N. bombycis, NPV, and densovirus (DNV)} infecting the silkworm, B.
mori. A multiplex real-time PCR assay for the simultaneous detection of three major
pathogens of B. mori (N. bombycis, NPV, and densovirus) was developed by Wu
et al. (2017). The Helicoverpa armigera Nucleopolyhedrovirus (HaNPV) isolate
from Heliothis peltigera was confirmed by tissue polymerase chain reaction and
sequence analysis by Eroglu et al. (2019).
The Next Generation Sequencing (NGS) technology has greatly helped in insect
pathogen discovery and detection (Liu et al. 2011; Cox-Foster et al. 2007). The most
common NGS platforms are Roche 454 pyrosequencing (454 Life Science), Illumina
(Solexa) sequencing, and SOLiD sequencing (ABI Biosystems). Huang et al. (2019)
determined the nucleotide sequence of the whole Troides aeacus NPV genome for
identification using NGS technology. However, this technology involves a series
of wet (sample processing, library preparation, and sequencing) and dry (bioinfor-
matics analysis and interpretation of data) laboratory steps. Besides, NGS-based
approaches are highly sophisticated and financially non-viable options. Another key
disadvantage is that microbial nucleic acids from infected samples are dominated by
insect host background which limits the overall analytical sensitivity of the genome
sequencing approach for pathogen detection (Liu et al. 2011). The current techniques
for insect pathogen detection are listed in Table 22.1. Due to the limitations of the
above-discussed techniques the development of new diagnostic techniques with high
sensitivity and specificity for on-site detection of insect pathogens is highly desirable.
Table 22.1 Summary of current techniques used for detection of the insect pathogens
Technique Detection limit Constraints Reference
Microscopy Neither specific, Late detection, Wu et al. (2017)
sensitive nor accurate Misdiagnosis
Immunoassays 850 baculovirus Cross-reactivity, Ndao (2009),
(Complement fixation, occlusion bodies possibility of false Thorne et al. (2007)
Immunodiffusion, positive/negative results
Radioimmune assays,
ELISA, Western
blotting, Latex
agglutination test)
LAMP 50 viral copies ng−1 Designing of primers, Chaivisuthangkura
genomic DNA sensitivity et al. (2009), Sahoo
(equivalent to 150 et al. (2016)
viral copies per
reaction)
Lateral Flow Dipstick 100 viral copies Suitable for primary Jaroenram et al.
immunoassay screening only, good (2009),
antibody preparation Koczula and
Gallotta (2016)
LAMP-LFD 0.2 pg DNA Designing of primers, Zhou et al. (2015)
sensitivity
Southern hybridization 6000 baculovirus Complex and labour Thorne et al. (2007)
occlusion bodies intensive;
time-consuming
PCR 8.0 baculovirus DNA extraction; use of Thorne et al. (2007)
occlusion bodies reagents, high-end
instruments, and skilled
manpower
Quantitative real-time 0.013 pg of viral DNA extraction; use of Krokene et al.
PCR (qPCR) DNA reagents, high-end (2013)
instruments, and skilled
manpower
Next Generation Varies with the Use of complicated Liu et al. (2011)
Sequencing sequencing platform data analysis methods,
Difficult to identify
novel viruses due to
non-homology to
known viruses
22.3 Biosensors
The biosensor technology is being developed as an alternative to laboratory tech-

niques for pathogen detection. It is one of the useful emerging techniques developed
by assembling complex analytical platforms into a single miniature device for on-site
and point-of-care detection of pathogens by monitoring a biological reaction at the
surface of transducers (Helali et al. 2006). Biosensors are portable, easy to use, and
472 M. Gani et al.
do not require skilled personnel, laboratory equipments, or reagents, thus, capable

of on-site diagnostics, cutting short the cumbersome procedures, and significantly
reducing the costs involved. A biosensor is actually a compact analytical device with
a ligand-specific biorecognition element or bioreceptor (antibody, enzyme, nucleic
acid, receptor, aptamers, peptide or protein, lectin, cells, tissue, or whole organism)
coated on a sensor surface integrated directly or indirectly with a signal conversion
unit called a transducer (Tiwari et al. 2017). The specific physiological interaction
between the target analyte and biorecognition element is translated, by the transducer,
into a measurable electric signal, which is further interpreted by a computer-aided
readout system for the user (Fig. 22.2) (Arora et al. 2010). The biorecognition element
can sense the presence of an analyte and determines the selectivity or specificity of the
biosensor. The transducer element has a major influence on sensitivity as it translates
the selective recognition of the analyte into a quantifiable signal. The miniaturization
achieved by utilization of nanostructures in biosensors for sensing or detection of
analyte results in the formation of a nanobiosensor (Girigoswami and Akhtar 2019).
Biosensors are mainly classified based on the biorecognition element and the
transducer type. The immunosensors employ antibody or antigen as the biorecog-
nition element and are well suited for the development of point-of-care and on-site
diagnostics (Fig. 22.3) (Conroy et al. 2009). Immunosensors represent a potential
tool for simple, label-free, sensitive, and rapid qualitative and quantitative detection
of pathogens. The result of the immunosensor depends on a reaction between an
antigen and an antibody. The high selectivity and sensitivity of immunosensors to
recognize a biomolecular component depends on the specific interactions and the
equilibrium association constants (1010 M−1 and greater) between an antigen and its
corresponding antibody (Farka et al. 2017). The sensing is carried out using compet-
itive and non-competitive assay format for the development of an immunosensor and
Fig. 22.2 Principle of biosensor. The bioreceptor is coated on a sensor surface and integrated
directly or indirectly with the transducer. The specific physiological interaction between the analyte
and bioreceptor is translated by the transducer into a measurable electric signal for interpretation
Fig. 22.3 A simple representation of an antibody-based biosensor (immunosensor) used for the
detection of insect pathogens. Here antibody is immobilized on a sensor surface for the capture of an
analyte and nanoparticles are used as signal amplification labels. The antigen–antibody interaction
produces a specific physicochemical change which is then converted (via a transducer) to a signal
which the user can interpret (Source Modified from Ayyar and Arora 2013; Farka et al. 2017)
the choice is made based on the molecular size and reactivity of the analyte (Farka
et al. 2017). The non-competitive approach is used for analytes holding two or more
epitopes and a competitive approach is generally preferred for smaller or less reac-
tive analytes. In recent years, several reports on biosensor development for sensitive,
specific, and on-site detection of human, animal, and plant pathogens were published
(Du and Zhou 2018; Devarakonda et al. 2017; Khater et al. 2017) (Table 22.2). Most
474 M. Gani et al.
Table 22.2 List of biosensors for sensitive, specific, and on-site detection of different pathogens
Virus Antibody type Transducer type Detection time Reference
Tobacco Mosaic mAb SPR Not specified Boltovets et al.
virus (2004)
Autographa mAb SPR Not specified Baac et al.
californica MNPV (2006)
West Nile virus mAb Voltametric 30 min Nguyen et al.
(2009)
Bovine viral pAb and mAb Conductometric 8 min Luo et al.
diarrhoea virus (2010)
Avian influenza virus mAb and pAb Voltametric 10 min Kamikawa
(subtype H5) et al. (2010)
Plum pox virus pAb Impedimetric Not specified Jarocka et al.
Immunosensor (2011)
Rabies virus mAb SPR Not specified Xu et al. (2012)
Influenza virus mAb Electrochemical 30 min Devarakonda
H1N1 immunosensor et al. (2017)
Newcastle disease chicken egg Electrochemical Not specified Tran et al.
virus yolk antibody immunosensor (2019)
(IgY)
European foulbrood, pAb Amperometric 2h Mikušová et al.
Melissococcus Immunosensor (2019)
Plutonius
Staphylococcus pAb Electrochemical 20 min Wang et al.
aureus immunosensor (2019)
Salmonella mAb and pAb Conductive 30 min Wonsawat
typhimurium immunosensor et al. (2020)
mAb: monoclonal antibody; pAb: polyclonal antibody, SPR: Surface Plasmon Resonance
of the reported applications used nanomaterials such as gold nanoparticles (Pingarron

et al. 2008; Kwon et al. 2017; Lu et al. 2019; Zheng et al. 2019; Alagappan et al.
2020), catalytic nanoparticles (Ramanavicius et al. 2005; Luo et al. 2006; Farka et al.
2018), quantum dots (Annio et al. 2018; Faridbod and Sanati 2019; Zhao et al. 2020),
or carbon-based materials (Sotiropoulou et al. 2003; Lin et al. 2017; Sharma et al.
2020) for enhanced assay properties.
The biosensor is of high potential interest for in-field applications and the rapid
diagnostic kit suitable for field use is represented by the lateral flow assay (LFA).
The LFA is a paper-based platform for the detection and quantification of analytes
in complex mixtures, where the sample is placed on a test device and the results
are displayed within minutes (Tomkies et al. 2009). The Lateral Flow Device (LFD)
is comprised of a release pad, a nitrocellulose test membrane, and an absorbent
pad and is used to detect biological antigens in infected tissue extracts (Danks and
Barker 2000). Due to their affordability, easy handling LFAs are being widely used for
disease detection (Koczula and Gallotta 2016). Moreover, they possess long shelf life
and do not need refrigerated storage, which makes them most suited for field applica-
tion. LFAs are classified into “lateral flow immunoassays” (LFIAs) when antibodies
are exclusively used as recognition elements, and Nucleic acid LFA (NALFA) that
detect PCR amplicons. LFDs have proved to be reliable and rapid diagnostic tools
for a variety of viruses, bacteria, and fungi (Danks et al. 2003). The gold nanopar-
ticles (Au NPs) are the most common labels for lateral flow immunoassays (Ge
et al. 2014). The biosensor technology holds a lot of potential for early disease diag-
nosis and timely initiation of appropriate treatment against the diseases of beneficial
insects and on-site detection and geographical distribution of native insect pathogens
for commercial production of insect-specific biopesticides.
A point-of-care device had been reported, when an amperometric immunosensor
based on a sandwich assay was used for in-field diagnosis of European foulbrood
(EFB) pathogen, Melissococcus plutonius (Mikušová et al. 2019). They have immo-
bilized anti-Melissococcus antibody on a gold surface of a screen-printed sensor via
a self-assembled monolayer of cysteamine activated with glutaraldehyde. The better
performance was obtained after the formation of the sandwich with the peroxidase-
labelled antibody in the amperometric mode. The limit of detection (LOD) of the
reported device was 6.6 × 104 CFU mL−1 with a wide linear range between 105
CFU mL−1 and 109 CFU mL−1 . The immunosensor was successfully employed in
the diagnosis of honeybee samples and helped in preventing M. plutonis infection
spread and losses of honeybee colonies.
A lateral flow immunoassay was developed as a rapid and sensitive test to diagnose
EFB in the field using a highly specific anti-Melissococcus plutonius monoclonal
antibody (Tomkies et al. 2009). The immunoassay was found to be very effective in
detecting M. plutonius in 96–100% of EFB-infected samples with no cross-reactivity
with other bee brood pathogens in laboratory trials. The field validation revealed
correct diagnoses in 96% of samples and the EFB LFDs are now used as a diagnostic
tool for routine confirmation of M. plutonius infection in the field, thus helping in
disease detection and control.
Surface Plasmon Resonance (SPR)-based sensor chip with three bio-functional
layers (amine-reactive crosslinker with a disulfide bond that chemisorbs to gold film,
protein-A, and a mouse IgG monoclonal antibody raised against a surface protein of
the target viral pathogen) was used to directly detect the intact Autographa califor-
nica multiple nucleopolyhedrovirus (AcMNPV) (Baac et al. 2006). Here AcMNPV
was introduced as a target pathogen to couple with a substrate-immobilized anti-
body while tobacco mosaic virus (TMV) was used as a non-binding control. The
consecutive exposure of the sensor chip to both viruses, i.e. AcMNPV and TMV
demonstrated the selective response to the AcMNPV with an SPR angular shift of
0.037o for 107 pfu mL−1 (plaque forming units per ml) virus concentration. The
improvement in electronic components and the use of nanoparticles as labels allow
for a significant reduction in the detection limit of SPR-based sensors.
476 M. Gani et al.
22.4 Transducers
A transducer is a device that converts the biorecognition event, i.e. the specific phys-
iological interaction between the antibodies immobilized on the sensor surface and
their target analyte into a measurable electric signal. The biosensors are classified
into electrochemical, optical, mass-based, and calorimetric based on the detection
method and transducer system (Velusamy et al. 2010; Monosik et al. 2012). The
most commonly used techniques for pathogen detection include electrochemical
and optical transducers.
22.5 Types of Biosensors
22.5.1 Electrochemical Biosensors
Electrochemical biosensors are those that measure the change in electrical properties
following biorecognition due to the consumption or production of the ions or elec-
trons (Mohanty and Kougianos 2006). They operate by reacting with the analyte of
interest and produce an electrical signal that is proportional to the analyte concen-
tration. The electrochemical sensors are the first scientifically proposed as well as
successfully commercialized biosensors. Electrochemical detection has been used
in conjunction with paper-based biosensors because of its high sensitivity, selec-
tivity, portability, cost-effectiveness, reproducibility, and compatibility with micro-
machining technology. The application of nanoparticles in electrochemical tech-
niques leads to major developments in electrochemical immunosensing. The nano-
materials (carbon nanotubes and graphene) lower the limit of detection (LOD) in
electrochemical biosensors.
Electrochemical impedance spectroscopy (EIS) has received considerable atten-
tion in the field of immunosensors, especially due to the possibility of ultrasensi-
tive, non-destructive, and rapid electrochemical sensing. EIS embodies a powerful
technique to investigate the physicochemical properties of the biorecognition events
connected to a respective transducer. The electrode is a key component of an elec-
trochemical biosensor that is employed as solid support for the immobilization of
biomolecules (antibody, enzyme, and nucleic acid) and movement of electrons. Typi-
cally, the electrochemical biosensor is comprised of two or three electrodes on the
same substrate. In the two-electrode configuration, one functions as the working elec-
trode while the other works as the reference electrode. The three-electrode config-
uration consists of the reference, working, and counter or auxiliary electrodes. The
working electrode also known as the sensing or redox electrode serves as the trans-
duction element in the biochemical reaction. The counter electrode in connection
with the electrolytic solution applies current to the working electrode. The reference
electrode is located at a distance from the reaction site to maintain a known and stable
potential. These electrodes should be both chemically stable and conductive. There-
fore, gold, platinum, carbon (e.g. graphite), and silicon compounds are commonly
used depending on the analyte. Electrochemical biosensors are further classified
into potentiometric, amperometric, voltammetric, and impedimetric/conductometric
(Velusamy et al. 2010). The most used is impedimetric followed by amperometric
immunosensors.
22.5.1.1 Potentiometric
The potentiometric biosensors monitor the potential of a system at a working elec-

trode with respect to the reference electrode under zero current flow. They are based
on ion-selective electrodes (ISE) and ion-sensitive field effect transistors (ISFET)
(Pohanka and Skladal 2008). The biorecognition event is converted into a change
in potential signal which is detected by a reference electrode. Here the biosensor
format typically consists of a perm-selective outer layer and a bioactive element,
such as urease for enhancement of the assay performance (Leonard et al. 2003). The
light-addressable potentiometric sensor (LAPS) which combines potentiometric and
optical detection has been applied for pathogen detection (Gehring et al. 1998; Dill
et al. 1999).
22.5.1.2 Amperometric
The enzymes in amperometric biosensors produce electroactive product due to oxida-

tion or reduction of analytes on the working electrode (carbon, gold, etc.). The resul-
tant current can then be detected. The amperometric sensors can be fabricated by
using disposable and customized screen-printed electrochemical electrodes (screen-
printed carbon electrodes) by depositing conducting inks (carbon, silver, etc.) in
a pre-determined arrangement and thickness (Palchetti and Mascini 2008). These
systems are robust, economical, and sensitive and can be used in conjunction with
mediators such as ferrocene dicarboxylic acid (FEDC) or iodine to improve selec-
tivity (Leonard et al. 2003). Besides, there is major potential to miniaturize these
systems that lead to smaller sample volume requirements. Amperometric biosensors
are more sensitive and well suited for mass production than the potentiometric ones
(Pohanka and Skladal 2008; Ghindilis et al. 1998).
22.5.1.3 Impedimetric
The impedimetric biosensor set-up follows either impedance (Z) or its components
resistance (R) and capacitance (C). The inverse value of resistance is known as
conductance and for this reason, these types of biosensors are also named as conduc-
tometric. Many strategies have been used to design impedimetric biosensors. Elec-
trochemical impedance spectroscopy (EIS) is a powerful tool and a non-destructive
478 M. Gani et al.
technique for investigating the interfacial properties related to biorecognition events

(Bahadır and Sezginturk 2016). EIS is the most common technique used in impedi-
metric biosensors, where the impedance is measured over a wide range of potential
frequencies (typically from 100 kHz to 1 MHz) (Hammond et al. 2016). Impedimetric
immunosensors have shown promising applications owing to sensitive, simultaneous,
and label-free detection of pathogens. In impedimetric immunosensors the antibodies
are immobilized on the electrodes, semiconductor chips, or optical fibre (Bahadır
and Sezginturk 2016). The impedimetric biosensors are less frequently developed
and used as compared to potentiometric and amperometric biosensors (Pohanka and
Skladal 2008).
22.5.2 Optical Biosensor
The transducers that measure changes in the intensity of light are known as optical
biosensors. Here the biorecognition sensing element is integrated with an optical
transducer system with the basic objective to produce a signal which is proportionate
to the concentration of a measured substance (analyte). The optical biosensors are
broadly divided into two general modes: label-free and label-based. The detected
signal is generated directly by the interaction of the analysed material with the trans-
ducer in a label-free mode. The label-based sensing involves the use of a label and
the optical signal is then generated by a colorimetric, fluorescent, or luminescent
method. The optical biosensors have high sensitivity and compatibility with several
well-established optical phenomena such as luminescence, fluorescence, phospho-
rescence, reflectance, colorimetry, light polarization and rotation, spectroscopy, inter-
ference, ellipsometry, and surface plasmon resonance (SPR) (Luong et al. 2008;
Velusamy et al. 2010; Huang et al. 2011). However, fluorescence and SPR-based
biosensors are most common (Velusamy et al. 2010).
Fluorescence is defined as “a short-lived type of luminescence created by elec-
tromagnetic excitation which is generated when a substance absorbs light energy at
a short (higher energy) wavelength and then emits light energy at a longer (lower
energy) wavelength” (Behlke et al. 2005). The time interval between absorption
and emission is very short, usually at the order of 10−9 to 10−8 s in fluorescence
(Serrano-Andres and Serrano-Perez 2012). The fluorescent biosensors have high
selectivity and sensitivity and profound applications in disease diagnosis. The bright-
ness of the fluorescent labels, packing density of the fluorophores, and the detection
optics parameters determine the sensitivity of a fluorescence-based biosensor. The
polymeric materials have made remarkable improvements in the design and func-
tioning of fluorescent-based biosensors (Liu and Tang 2013). The conjugated poly-
mers having light harvesting and electron delocalizing properties are also used in
fluorescent sensors (Girigoswami and Akhtar 2019). Recently, the Fluorescent Reso-
nance Energy Transfer (FRET) based ratiometric sensors have been designed with
the advantage that the ratio between two fluorescence intensities is independent of the
external factors such as fluctuation of the source of excitation and sensor concentra-
tion (Zhang et al. 2008; Jun et al. 2010). The different designs of fluorescent biosen-
sors can be developed depending on the nature of the sensing element. In a single
fluorophore-based biosensor, the change in fluorescence intensity of a single fluo-
rescent reporter is monitored. This change in fluorescence intensity is due to various
mechanisms such as accessibility of the solvent or surrounding environment polarity
or fluorophore protein interactions are changed. The fluorophores-based biosensors
are designed based on the principle of FRET. In such biosensors, fluorophore pairs,
particularly a FRET donor and an acceptor, are fused with the recognition element.
SPR is the excitation of an electromagnetic wave that propagates along with the
interface of two different media such as metal and sample buffer at a specific angle
of incident light beam (Damborsky et al. 2016; Guo 2012). The signal generated
is based on total internal reflection. The angle at which the resonance occurs is
sensitive to change in refractive index or formation of a nanoscale film thickness.
These changes can be quantified by monitoring the light intensity minimum shift
over time. SPR-based biosensors have demonstrated to be user friendly, versatile,
and are very powerful tools for biomolecular interaction studies in immunoassays.
SPR measurements mostly consist of direct detection (Nakamura et al. 2003), sand-
wich (Wei et al. 2003), and competitive inhibition (Gobi et al. 2004) assays. Direct
detection is appropriate where the direct binding of the analyte yields a sufficient
response and its detection limits can be enhanced using a sandwich or inhibition
assay. In a sandwich assay, the measurement is carried out in two steps viz. the
analyte is bound to the antibodies on the sensor surface and then the sensor surface
is incubated with a solution containing secondary antibodies (which can be labelled
by nanoparticles or enzymes), which binds to the previously captured analyte and
increase the sensor response. The sample is first mixed with labelled antibodies and
then the mixture is brought into contact with the sensor surface exhibiting immobi-
lized analyte molecules in an inhibition assay. The free antibodies remaining in the
solution can subsequently bind to the surface.
A practical SPR instrument consists of an optical detector part which measures
intensity shift, a sensor chip with a gold surface, and a layer enabling ligand immo-
bilization that is integrated with a fluidics system enabling a flow-through operation.
The interaction component (ligand) is permanently attached to the chip surface and
another interacting component (analyte) flows over the surface and binds to the ligand
in a practical experiment. There are three types of SPR analyses: kinetic, equilib-
rium, and concentration analysis for practical applications (Damborsky et al. 2016).
Kinetic and equilibrium analyses are used to characterize any molecular interaction
viz. ligand–analyte binding, receptor characterization, antibody–antigen interaction,
etc. The SPR technique has multiple applications in the concentration analysis of
any analyte where the availability of a specific ligand and its immobilization on the
SPR chip is important. The concentration is obtained by measuring direct binding or
from the rate of binding in a mass transport limited mode.
480 M. Gani et al.
22.5.3 Mass-Based Biosensor
Mass-based biosensors (also known as gravimetric biosensors) detect a change in

mass by using piezoelectric materials that change their resonant frequency thus,
generating acoustic waves (Holford et al. 2012). These waves propagate either along
(surface acoustic wave, SAW) or through (bulk acoustic wave, BAW) the substrate
surface (Rocha–Gaso et al. 2009). The piezoelectric quartz crystals can either be in
the form of resonating crystals (quartz crystal microbalance, QCM), or as surface
acoustic wave (SAW) devices. A QCM biosensor consists of a quartz crystal wafer
in the middle of two metal electrodes, and the resonant frequency of QCM changes
according to the mass change at the crystal’s surface. The change in frequency is
proportional to the mass of the sensor. The combining of QCM device with highly
specific antigen–antibody interaction enables the direct detection of micro-organisms
(Ngo et al. 2014). QCM based biosensors have been used in the rapid detection of
pathogens (Ozalp et al. 2015) and toxins (Neethirajan et al. 2018) due to their ease
of use, cost-effectiveness, shorter analysis time, and the ability to produce label-free
measurement.
SAW-based biosensors detect acoustic waves generated by mass loading on the
surface of the piezoelectric crystal via the interdigital transducers (IDT) (White and
Voltmer 1965). The IDT allows the acoustic energy to be strongly confined to the
surface irrespective of the thickness of the substrate. The analyte recognition by
the immobilized receptors changes the velocity of SAW and produces signal by the
driving electronics (Lange et al. 2008). The piezoelectric materials produce an electric
signal in response to mechanical forces and the commonly employed piezoelectric
materials in sensors are the quartz crystals (Holford et al. 2012). The crystals are
made to vibrate at a specific frequency by the application of an electric signal and
the oscillation frequency of the crystal depends on the applied frequency. A shear
wave is generated at a thin (5 μm) guiding layer deposited at the sensor surface
by applying an electrical field to quartz crystal (Gronewold 2007). The binding
events are detected by changes in the physical properties of the shear wave via the
angular phase shift, and changes in the oscillation amplitude. SAW sensors have
been developed as a powerful and promising system for detecting various biological
recognition events (Gronewold 2007; Lange et al. 2008). The bio-capture layer on
the surface of the crystal exhibits specific binding with the analyte. The mass-based
sensors have antibodies as the most common bio-capture molecules and such sensors
are known as piezoelectric immunosensors.
22.5.4 Calorimetric Biosensor
The calorimetric biosensors or Thermal biosensors measure the heat generated in an

enzymatic reaction. The calorimetric sensors have many advantages over ampero-
metric and optical sensors such as relatively easy fabrication, label-free detection,
and less complex detection (Kopparthy et al. 2015). These types of biosensors are
developed using either thermistors or thermopiles. The thermistors transform the heat
(generated or lost) during a reaction into an electrical signal. One thermistor is placed
at the inlet of the reactor containing the immobilized enzyme and the second ther-
mistor is placed at the exit of the reactor to measure the temperature after conversion
of the substrate to product. The thermistor records temperature changes by altering
its resistance. High temperature causes a reduction in resistance of the thermistor.
The difference in resistance between thermistors located at the entry and exit points
to the reactor is recorded as a measure of the temperature change produced as a result
of the passage of the sample. Thermopiles are widely used in calorimetric biosensors
due to their compatibility with miniaturized devices and high common mode thermal
noise rejection ratio. Reference junctions of the thermopiles are controlled either by
a constant heat source, heat sink, or by vacuum encapsulation.
The entire calorimetric sensing set-up is surrounded by an insulated jacket to
prevent the loss of heat generated during the reaction. The analyte acts as the substrate
for the enzyme. The enzyme is immobilized in a packed bed column which is placed
in the centre of the insulated chamber. The sample is passed through a coiled column
of aluminium that serves as a heat exchanger to maintain a uniform temperature of the
sample in the reaction chamber. When the analyte is exposed to such immobilized
enzymes or antibodies on the sensor active area, the biochemical reaction begins
and its evolution in terms of the total amount of heat generated and kinetics is
proportional to the concentration of the reactants and rate constants of the reaction.
Enzyme-catalysed reactions involve enthalpy change and show high selectivity and
are highly suitable as thermal biosensors for a broad range of applications.
The most common calorimetric technique, differential scanning calorimetry
(DSC) is used to measure the heat generated by the differential thermal activity of
the biomolecular sample and reference material in two calorimetric chambers whose
temperatures are scanned over time (Lerchner et al. 2008). However, the conventional
DSC systems are bulky, require large sample volume, and are not well suited for rapid
detection of analytes (MicroCal 2017). Therefore, microcalorimetric biosensing has
been developed using microsystem and microfabrication technologies to overcome
the weaknesses of the conventional calorimeters (Hohne et al. 2004). The lower back-
ground signal noise is a key performance parameter of microcalorimetric biosensors.
The technological advancement in microsystem and microfabrication technologies
has the potential to show great promise in the utilization of calorimetric biosensors
in disease diagnosis.
22.6 Antibodies as Biorecognition Elements
Biorecognition is an important aspect in biosensor design and the biorecognition

probe needs to be chosen carefully. The antibodies (immunoglobulins—Ig’s) are the
most popular class of biorecognition elements owing to their high binding affinity and
specificity towards the target (Connelly and Baeumner 2012). The Ig is comprised
482 M. Gani et al.
Fig. 22.4 Structure of antibodies comprised of four polypeptide chains, two identical shorter light
chains (VL ) and two identical larger heavy chains (VH ). A light chain is coupled to a heavy chain via
a disulfide bond and the heavy chains are connected with each other by covalent disulfide bridges and
noncovalent bonds which correspondingly form the typical Y-shaped antibody structure. The two
Fabs are antigen-binding fragments, and Fc is a crystallizable fragment. In the right-hand structure,
flexibility is outlined: Fab arm waving (violet), Fab rotation (blue), Fab elbow bend (orange), and
Fc wagging (green) (Source Farka et al. 2017)
of four polypeptide chains with two identical shorter light chains (VL) and two
identical larger heavy chains (VH). A light chain is connected to a heavy chain by
a disulfide bond and the heavy chains are connected with each other by covalent
disulfide bridges and noncovalent bonds which correspondingly form the typical Y-
shaped antibody structure (Fig. 22.4). The light and heavy chains consist of conserved
protein structures called domains corresponding to approximately 110 amino acids.
The light chains have variable (VL) and constant (CL) domains. The heavy chains
also have one variable domain (VH) and three (IgG, IgA, and IgD) or four (IgM and
IgE) constant domains (CH1 , CH2 , CH3 , CH4 ) depending on the type of antibody.
The arms of the “Y” consist of the N-termini of each heavy chain associated with one
of the light chains to create two antigen-binding domains termed as the “antigen-
binding fragment” (Fab fragment or domain). The tail of the “Y” is formed by a
combination of the C-termini of the two heavy chains and is termed the “crystallizable
fragment” (Fc fragment or domain). The hinge region located between the CH1 and
CH2 domains gives flexibility (arm rotation and waving) to the Fab fragment. Due to
the variability of the VL and VH domains, the Fab fragment determines the specificity
and ability to interact and bind the immunogen. Each antibody molecule has two
antigen-binding sites formed by the regions, located at the tip of each arm of the “Y”
shaped structure.
Antibodies are classified as polyclonal (PAb), monoclonal (MAb), and recom-
binant (RAb) antibodies. The predominant antibody form used in biosensors is
monoclonal and polyclonal. However, recombinant antibodies are now gaining
increasing significance for biosensor applications. MAbs are produced by hybridoma
technology and are monospecific and more homogeneous than PAbs. For the
use of monoclonal antibody the important considerations include biochem-
ical/physicochemical (class, subclass, molecular weight, light-chain composition,
either N- and C-terminal amino acid sequence, secondary and tertiary structure) and
biological/immunological properties (antigenic specificity, the ability for comple-
ment binding and activation, binding capacity, cytotoxic properties, ability to modify
relevant antigens, capacity to stimulate immunocompetent cells and to induce secre-
tion of cytokines or other mediators) of the antibody. Besides specificity (deter-
mination of unintentional reactivity with tissues distinct from the intended target)
and cross-reactivity (reactivity with a range of tissues by immunohistochemical
procedures) of the monoclonal antibody are also important. PAbs are produced by
animal immunization and are extracted from the living animal serum. The PAbs are
polyspecific in nature and recognize different epitopes on the same antigen. RAbs are
monoclonal antibodies produced in bacterial cells using phage display technology.
The nanobodies (Nbs) found in camelids (llamas, dromedaries, alpacas) or in non-
mammals (ratfish and sharks) are well suited for the development of immunosensor
as they can be easily modified by site-specific functional groups with a negligible loss
of specificity and affinity (Flajnik et al. 2011). The Nbs are single-domain antibodies
with molecular weights of 12–14 kDa and constitute the smallest domains of natural
antibodies with antigen-binding capacity. Nbs are encoded by a single gene segment
and can be produced in bacteria and yeast with less cost than conventional monoclonal
antibodies.
The immobilization procedure is crucial in the development of an immunosensor
and glass, gold, metal oxides, and carbon (i.e. graphite, graphene, glassy carbon, etc.)
are the most common materials of optical and electrochemical transducer surfaces.
The different procedures used for the immobilization of antibody or antigen on the
transducer surface include physical adsorption, covalent chemical binding, and the
use of ordered layers obtained by deposition of self-assembled layers (SAM). The
most common practice is the use of ordered layers on the transducer surface for
oriented immobilization and better presentation of the antibody to the target analyte.
22.6.1 Antibodies: Production, Purification, and Selection
The in vitro offers a high degree of control and standardization and is the preferred
method of antibody production. It is advantageous over in vivo production method
with respect to consistency of production, absence of contaminant immunoglobulins
in the crude harvest, and viral safety. Reduction of animal usage is another advantage.
However, for each in vitro production run, the microbial contamination in the culture
vessels needs to be thoroughly examined prior to harvesting. The bulk culture fluid
should be free from bacteria, mycoplasmal, and mycotic contamination and should
be tested for the presence of viruses using a general test involving inoculation into
suitable cell substrates. Simultaneously only one cell line should be cultivated in a
production area and records must be kept of the cell lines cultivated in parallel with
484 M. Gani et al.
an evidence of the absence of cross contamination between them. In vivo production

involves the exploitation of the immune system of leporine, murine, ovine, or avian
hosts. The strain, origin, genotype, and age of the animal used for production should
be specified. The animal used should be from a closed, specific pathogen-free (SPF)
colony which is routinely monitored for the diseases. During in vivo production, the
maximum permissible number of serial passages should be defined and restricted.
The purification is done to remove tissue debris, unwanted host cell derived
proteins, nucleic acids, carbohydrates, viruses, and other impurities. The purification
should be effective and not compromise the biological activity of the product. Further
purification procedures should not impair relevant immunobiological features of the
immunoglobulin. The choice of the nucleotide probe to detect DNA contamination
should be relevant to the system used. A reduction factor at each stage of purifi-
cation, and overall, should be established by using concentrations of viruses, host
cell proteins, other impurities, and DNA in excess of that expected during normal
production. Validation of the purification process should also include justification
of the working conditions such as column loading capacity, length of use of the
columns, column regeneration, and sanitisation.
When selecting antibodies for the detection of pathogens, the key parameters
include sensitivity, selectivity, stability, immobilization, labelling, antibody size, ease
of production, cost, commercial availability, and capacity for engineering affinity.
Many bacterial strains share homologues of surface-presented proteins leading to the
detection of multiple cell-types by a single antibody as such the selection of a highly
specific epitope on the pathogen is a key consideration. Therefore, a constitutively
expressed antigen which is species-specific is targeted. Where possible, the expres-
sion of this target antigen should not be highly dependent on the growth matrix of
the concerned pathogen. Finally, the antibody should bind with its cognate target
with sufficient strength to permit interrogation (high affinity). The identification of
an antibody that satisfies these requirements can be facilitated by ELISA to reduce
the number of potential antibodies to a smaller number followed by sensor-based
analysis to identify the candidate with the best affinity for the target epitope. This
antibody can further be used for incorporation on an immunosensor-based platform.
These factors are optimized by recombinant antibodies as genetic modification facil-
itates improvements in selectivity, stability, size, and in addition aid effective immo-
bilization (Townsend et al. 2006). Display libraries and high throughput screening
facilitate improvements in sensitivity due to the ability to screen much larger recom-
binant libraries. The use of genetic insertion of tags for immobilization and coupling
chemistries are useful methods to help in the orientation and immobilization of
novel recombinant antibodies. Besides antibody selection, the immobilization of
antibodies to the sensor surface is an important factor that influences biosensing
mechanism. The immobilization depends on the properties of the sensor interface.
For this purpose, gold, silver, glass, platinum, and silica type of surfaces have been
studied. The sensor surface should possess less signal-to-noise ratio, excellent elec-
trical and thermal conductivity, low diffusion rates, stability, and large surface area
for high density probe immobilization (Holford et al. 2012). It is difficult to get all
these characteristics on one sensor surface and further to keep the device small to be
portable. However, the development of new generation sensor platforms compatible

with the aforementioned sensor needs is possible with the improvement in matrices
such as carbon nanotubes, self-assembled monolayers (SAMs), fabricated nanopar-
ticles, and quantum dots (Holford et al. 2012). The most commonly used methods
for coupling the antibodies to the sensor surface include passive absorption, matrix
entrapment, encapsulation, covalent coupling, and affinity tags with their own pros
and cons.
22.7 Importance of Nanoparticles in Biosensors
The nanoparticles (NPs) have been used in biosensors for improvement of pathogen
detection efficiency, expeditiously, and cost effectively. The diagnostic nanoparti-
cles have the ability to amplify the signal, provide a more effective environment
for anchoring of the biological element and in the miniaturization. NPs improve
the reproducibility of nanoimmunosensors because a high and targeted surface area
allows faster and controlled charge transfer at lower overpotentials. The most impor-
tant properties of nanoparticles include particle size and charge, core and surface
properties, shape and flexibility, as well as multivalency and controlled synthesis.
Besides, the enormous surface to volume proportion, higher reactivity, stability, and
controlled particle size are important physicochemical characteristics superior to the
mass material. These properties determine the targeting ability, toxicity, stability, and
persistence of nanoparticles. The nanoparticles are classified into organic (proteins
and peptide nanoparticles), inorganic (gold, silver, iron oxide, and calcium phos-
phate), and hybrid nanoparticles (composed of more than one set of nanoparticles,
e.g. mixture of polymeric nanoparticles and liposomes) on the basis of their origin.
The nanoparticles that are used for diagnostic applications include metallic nanopar-
ticles (gold, silver), magnetic nanoparticles, carbon-based nanomaterials (carbon
nanotubes, graphene, fullerene, carbon nanohorns, and carbon dots), and luminescent
nanocrystals (quantum dots, photon-upconverting nanoparticles).
The gold (Au) NPs are the most common signal amplification labels for various
kinds of immunoassays and immunosensors due to their unique optical, chemical,
electrical, and catalytic properties (Fenzl et al. 2016). Besides, easy preparation and
modification, superior biocompatibility, and intensive colour of Au NPs have drawn
the focus of scientists on their use in the bioanalytical field (Jans and Huo 2012).
The excellent stability compared to that of other metals, pronounced colours and
sensitive plasmon changes makes Au NPs the first nanomaterial to be considered
for improving the performance of classic immunoassays. The silver (Ag) NPs are
the most common commercially produced and excellent candidates for detection
tags in electrochemical sensing as they offer improved electrochemical activity and
can be oxidized more easily as compared to Au NPs. However, the significantly
lower stability, limited biocompatibility compared to those of Au NPs and difficulty
to functionalize restricts the practical applications of Ag NPs in immunosensing
(Li and Xu 2014). The magnetic NPs-based analytical applications are represented
486 M. Gani et al.
mainly by three strategies viz. magnetic preconcentration of an analyte due to its

interaction with biofunctionalized nanoparticles, use of magnetic NPs as tags for the
visualization and sensitive detection of immunocomplexes with a specific analyte
and the integration of magnetic NPs into the transducer material or the modification
of the sensor surface to improve the analytical signal. The functionalized magnetic
NPs are referred as magnetic nanosensors by some authors.
Carbon nanotubes (CNTs) are divided into two main categories: single-walled
carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) and
are often used in immunosensing due to their unique physical and chemical proper-
ties (Oliveira et al. 2014; Jiang et al. 2015). The graphene is a good candidate for
the preparation of biosensors due to its outstanding mechanical, electrochemical,
and thermal properties, optical transparency, and flexibility (Ramnani et al. 2016).
Fullerene C60 is the smallest stable and most abundant member of the fullerene
group that is well suited for the development of electrochemical sensors (Pilehvar
and De Wael 2015). The fullerene C60 modified electrodes have an increased elec-
troactive surface area and high electric conductivity, however, insolubility in water
and hydrophobicity complicate the conjugation of biologically active molecules with
fullerene (Yanez-Sedeno et al. 2016). Carbon nanohorns (CNHs) are convenient for
amplification of the electrochemical signal and as a scaffold for immobilization of
the antibodies or antigens (Liu et al. 2014). The electrochemical properties of carbon
dots (carbon quantum dots and graphene quantum dots) have also been exploited to
develop electrochemical biosensors (Wang and Dai 2015). The graphene variants and
carbon nanotubes play an important role in the enhancement of the electron transfer
rate in electrochemical immunosensors.
Therefore, the sensitive and specific immunosensor will allow farmers or exten-
sion workers to detect pathogen at early stages resulting in more efficient disease
management in beneficial insects. The immunosensor will also be used for the on-
site detection and distribution of native isolates of insect pathogens for the commer-
cial production of insect-specific biopesticides. Moreover, long shelf life and no
refrigeration for storage make the immuno-diagnostic kits very well suited for field
use.
22.8 Conclusion
The present microbiological and molecular methods for pathogen detection are time-
consuming, expensive, relatively insensitive, and entirely laboratory-based. Hence,
cost-effective, sensitive, and specific detection techniques suitable for field diagnosis
are highly desired. We believe that the biosensor technology is of high potential
interest for in-field applications and exhibits great advantages over other techniques
in terms of time, simplicity, and quantitative analysis. Besides, the biosensor tech-
nology exhibits high sensitivity and specificity and thus, can serve as good detector
of pathogens with unprocessed or crude samples. The use of nanoparticles as signal
amplification labels has the ability to improve the performance of biosensor for real-
time monitoring of pathogens. A significant progress has been made in biosensor
technology for the detection of human and veterinary pathogens in recent years. This
technology is still in its infancy with respect to insect pathogen detection. However,
the commercialization of biosensors has not yet maintained pace with the research
activities carried out throughout the world due to cost and technical issues. It is
important to continue working on these technologies for a successful transition from
laboratory to field diagnosis. Therefore, to improve the development of nanodiagnos-
tics, an increased interdisciplinary collaboration and knowledge exchange between
scientists from various disciplines is necessary.
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Concepts and Strategies in Plant Sciences

Series Editor: Chittaranjan Kole

More information about this series at http://www.springer.com/series/16076

ISSN 2662-3188 ISSN 2662-3196 (electronic)

Noida, India Dr. Ramesh Namdeo Pudake

Part I Biosensors in Crop Science

9 Application of Biosensor for the Identification of Various

Part II Biosensors in Food Science

Part III Biosensors in Animal and Fishery Sciences

19 Nano-Biosensing Devices Detecting Biomarkers

Naina Abbineni DBT-National Institute of Animal Biotechnology, Hyderabad,

Nidhi Chauhan Amity Institute of Nanotechnology, Amity University Uttar

Ranjit Kumar Department of Chemistry, School of Engineering, University of

Julio Raba Instituto de Química de San Luis (INQUISAL), Consejo Nacional de

Gonzalo Tortella Departamento de Ingeniería Química, Centro de Excelencia En

Ravindra Pratap Singh

Abstract Nanotechnology (NT) is an interdisciplinary scientific approach at the

Keywords Nanotechnology, Nanobiotechnology · Nanobiosensors · Agriculture

Agriculture is proving to be the main source of raw materials to food industries

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 3

Fig. 1.1 Schematic representation of biosensor

1.2 Use of Nanobiosensor in Agriculture

Nanomaterial-based biosensors are most commonly known as nanobiosensors with

Fig. 1.2 Broad spectrum of potential applications of nanobiosensors

1.2.1 Nanosensors in pathogens detection

In developing countries consumption of foods contaminated with pathogenic food-

1.2.2 Mycotoxins detection

1.2.3 Pesticides/ Insecticides/Herbicides detection

1.2.4 Veterinary drug and residues

1.3 Nanobiosensors for food safety and security

nanobiosensors to detect food products during early stage of manufacture, storage,

1.4 Conclusion and future prospects

Agriopoulou S, Stamatelopoulou E, Varzakas T (2020) Advances in analysis and detection of major

Sofía V. Piguillem Palacios, Nicolás Hoffmann, Matías Regiart,

Abstract Recently, various nanomaterials, such as nanostructured platforms, are

Keywords Nanotechnology · Nanomaterials · Platforms · Biosensors · Agriculture

S. V. Piguillem Palacios · M. Regiart · J. Raba · M. A. Fernández-Baldo (B)

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 15

In recent years, several nanomaterials as nanostructured platforms were being used

reactions are generally transduced by optical and electrochemical detection methods

2.2 Nanomaterials Used in Biosensors Methodologies

2.2.1 Metal Nanoparticles

the same plasma frequency, an oscillation is generated. This phenomenon is respon-

2.2.2 Magnetic Nanoparticles

2.2.3 Carbon Nanomaterials

If it refers to carbon, many nanomaterials can be included incorporating this element

2.2.3.1 Carbon Nanotubes

2.2.3.3 Porous Carbon

2.2.4 Silica Nanomaterials

Intelligently synthesized silica nanomaterials (SNMs) achieve a highly useful meso-

Organometallic frameworks (MOFs) are organic–inorganic hybrid porous coordi-

2.2.6 Quantum Dots

Importantly, it is not an easy task to discern when a nanomaterial can be classified as

2.3 Recent Applications of Nanostructured Biosensors

2.4 Conclusions and Future Perspectives

Keywords Nanomaterials · Nanocomposites · Biosensors · Soil moisture/water

PCR Polymerase chain reaction

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 27