sensors

Review
A Review on Biosensors and Recent Development of
Nanostructured Materials-Enabled Biosensors
Varnakavi. Naresh * and Nohyun Lee *

School of Advanced Materials Engineering, Kookmin University, Seoul 02707, Korea

* Correspondence: [email protected] (V.N.); [email protected] (N.L.);
Tel.: +82-2-910-5574 (V.N.); +82-2-910-4227 (N.L.)

Abstract: A biosensor is an integrated receptor-transducer device, which can convert a biological

response into an electrical signal. The design and development of biosensors have taken a center stage
for researchers or scientists in the recent decade owing to the wide range of biosensor applications,
such as health care and disease diagnosis, environmental monitoring, water and food quality moni-
toring, and drug delivery. The main challenges involved in the biosensor progress are (i) the efficient
capturing of biorecognition signals and the transformation of these signals into electrochemical,
electrical, optical, gravimetric, or acoustic signals (transduction process), (ii) enhancing transducer
performance i.e., increasing sensitivity, shorter response time, reproducibility, and low detection
limits even to detect individual molecules, and (iii) miniaturization of the biosensing devices using
micro-and nano-fabrication technologies. Those challenges can be met through the integration of
sensing technology with nanomaterials, which range from zero- to three-dimensional, possessing
a high surface-to-volume ratio, good conductivities, shock-bearing abilities, and color tunability.
Nanomaterials (NMs) employed in the fabrication and nanobiosensors include nanoparticles (NPs)
(high stability and high carrier capacity), nanowires (NWs) and nanorods (NRs) (capable of high
detection sensitivity), carbon nanotubes (CNTs) (large surface area, high electrical and thermal



conductivity), and quantum dots (QDs) (color tunability). Furthermore, these nanomaterials can


themselves act as transduction elements. This review summarizes the evolution of biosensors, the
Citation: Naresh, V..; Lee, N. A types of biosensors based on their receptors, transducers, and modern approaches employed in
Review on Biosensors and Recent biosensors using nanomaterials such as NPs (e.g., noble metal NPs and metal oxide NPs), NWs,
Development of Nanostructured NRs, CNTs, QDs, and dendrimers and their recent advancement in biosensing technology with the
Materials-Enabled Biosensors. Sensors expansion of nanotechnology.
2021, 21, 1109. https://doi.org/
10.3390/s21041109
Keywords: biosensors; nanomaterials; nanobiosensing; gold nanoparticles; carbon nanotubes; quan-
tum dots
Received: 31 December 2020
Accepted: 2 February 2021
Published: 5 February 2021

1. Introduction
Publisher’s Note: MDPI stays neutral
1.1. Sensors
with regard to jurisdictional claims in
published maps and institutional affil- Nowadays, we enjoy the results of science and technology for the smoothly running
iations. lives. We frequently rely on various types of appliances or gadgets, such as computers,
copy machines, mobile phones, microwave ovens, refrigerators, air conditioning and
television remotes, smoke detectors, infrared (IR) thermometers, turning on and off lamps
and fans, which help us interact with the physical environment. Many of these applications
Copyright: © 2021 by the authors.
perform with the help of sensors. A sensor is defined as a device or module that aids
Licensee MDPI, Basel, Switzerland.
in detecting changes in physical quantities, such as pressure, heat, humidity, movement,
This article is an open access article
force, and an electrical quantity like current, and thereby converts these to signals that
distributed under the terms and can be detected and analyzed [1,2]. A transducer is defined as a device that can convert
conditions of the Creative Commons energy from one form to another. The sensor is the heart of a measurement system.
Attribution (CC BY) license (https:// An ideal sensor should possess certain characteristics, such as range, drift, calibration,
creativecommons.org/licenses/by/ sensitivity, selectivity, linearity, high resolution, reproducibility, repeatability, and response
4.0/). time [3,4]. The progress of sensor technology has become increasingly important, owing

Sensors 2021, 21, 1109. https://doi.org/10.3390/s21041109 https://www.mdpi.com/journal/sensors

Sensors 2021, 21, 1109 2 of 35

to various applications, such as environmental and food quality monitoring, medical

diagnosis and health care, automotive and industrial manufacturing, as well as space,
defense, and security.

1.2. Classification of Sensors

Sensors are broadly classified into various categories (Figure 1) depending on the
physical quantity (substance) or analyte to be measured, such as (a) energy source (active
and passive sensors), (b) physical contact (contact and non-contact sensors), (c) com-
parability (absolute and relative sensors), (d) analog and digital sensors, and (e) signal
detection (physical, chemical, thermal, and biological) [5,6]. Details of each classification
are as follows.

Figure 1. Classification of sensors based on measurand, energy/power, physical contact, signal

conversion, output signal, comparability, sensor material, specification, and applications (reproduced
from White et al. Ref. [6]).

(a) Active and passive sensors: Active sensors need an external energy source to operate,
for example, microphones, thermistors, strain gauges, and capacitive and inductive
sensors. These types of sensors are called parametric sensors (output is a function
of the parameter). Passive sensors generate their signals but do not require external
energy, for example, thermocouples, piezoelectric sensors, photodiodes. These types
of sensors are called self-generating sensors.
(b) Contact and noncontact sensors: Contact sensors require physical contact with a
stimulus, for example, temperature sensors, while non-contact sensors require no
physical contact, such as optical and magnetic sensors and IR thermometers.
(c) Absolute and relative sensors: Absolute sensors, such as thermistor and strain gauge,
react to a stimulus on an absolute scale. Relative sensors sense the stimulus relative
to a fixed or variable reference, like a thermocouple that measures the temperature
difference and the pressure that is measured relative to atmospheric pressure.
(d) Analog and digital sensor: An analog sensor transforms a measured physical quantity
to an analog form (continuous in time). Thermocouples, resistance temperature detec-
tors (RTD), and strain gauge belong to this class of analog sensors. A digital sensor
generates output in the form of a pulse. Encoders belong to the digital sensor category.
Sensors 2021, 21, 1109 3 of 35

(e) Signal detection: Based on the form of signal detection, sensors can be divided as (i)
physical, (ii) chemical, (iii) thermal, and (iv) biological sensors.
(i) Physical sensors: Physical sensors measure a physical quantity and convert
it into a signal, which can be identified by the user. These sensors can detect
environmental changes, such as force, acceleration, rate of flow, mass, volume,
density, and pressure. Physical sensors have been largely employed in the
biomedical field, particularly with the advancement of microelectromechanical
system (MEMS) technology for developing more precise and compact sensors,
along with the development of novel measuring technology.
(ii) Chemical sensors: According to the international union of pure and applied
chemistry (IUPAC), a chemical sensor is defined as, “a device that converts
chemical information into an analytically useful signal ranging from the con-
centration of a particular sample component to total composition analysis.”
Chemical sensor is employed to monitor the activity or concentration of the
respective chemical species in the gas or liquid phase. They are also used for
environmental pollution monitoring, food and drug analysis, and assay moni-
toring of organophosphorus compounds. They can also be used for clinical
diagnostic purposes.
(iii) Thermal sensors: A thermal sensor is a device that is used to measure the tem-
perature of an environment and transforms the input data into electronic data
to record or monitor signal of temperature changes. Examples of temperature
sensors include thermocouples, thermistors, and RTDs.
(iv) Biological sensors: Biological sensors monitor biomolecular processes, such
as antibody/antigen interactions, DNA interactions, enzymatic interactions,
or cellular communication processes. Biological sensors can be referred to as
biosensors in short form.

2. Biosensor
2.1. Design and Principle
A biosensor is a device or probe that integrates a biological element, such as an enzyme
or antibody, with an electronic component to generate a measurable signal. The electronic
component detects, records, and transmits information regarding a physiological change
or the presence of various chemical or biological materials in the environment. Biosensors
come in different sizes and shapes and can detect and measure even low concentrations of
specific pathogens, or toxic chemicals, and pH levels. A typical biosensor comprises (a) an
analyte, (b) bioreceptor, (c) transducer, (d) electronics, and (e) display (Figure 2) [7].
(a) Analyte: A substance of interest whose constituents are being identified or detected
(e.g., glucose, ammonia, alcohol, and lactose).
(b) Bioreceptor: A biomolecule (molecule) or a biological element that can recognize
the target substrate (i.e., an analyte) is known as bioreceptor (e.g., enzymes, cells,
aptamers, deoxyribonucleic acid (DNA or RNA), and antibodies). The process of
signal production (in the form of light, heat, pH, charge or mass change, plant or
animal tissue, and microbial products) during the interaction between bioreceptor
and analyte is called biorecognition.
(c) Transducer: A device that transforms energy from one form to another. The trans-
ducer is a key element in a biosensor. It converts the biorecognition event into a
measurable signal (electrical) that connects with the quantity or in the presence of
a chemical or biological target. This process of energy conversion is known as sig-
nalization. Transducers generate either optical or electrical signals proportional to
the number of analyte–bioreceptor interactions. According to the operating principle,
transducers are broadly categorized as electrochemical, optical, thermal, electronic,
and gravimetric transducers
Sensors 2021, 21, 1109 4 of 35

(d) Electronics: The transduced signal is processed and prepared for the display. The
electrical signals obtained from the transducer are amplified and converted into digital
form. The processed signals are quantified by the display unit.
(e) Display: The display unit is composed of a user interpretation system, such as a
computer or a printer that generates the output so that the corresponding response can
be readable and understandable by the user. Depending on the end-user prerequisite,
the output can be in the form of a numerical, graphical, or tabular value, or a figure.

Figure 2. Schematic diagram of typical biosensor consisting of bioreceptor, transducer, electronic

system (amplifier and processor), and display (PC or printer) and various types of bioreceptors and
transducers used in the biosensors are also shown.

2.2. Evolution of Biosensors

The evolution of biosensors has been classified into three generations based on the
attachment of the components, that is, according to the method of integration of the bio-
recognition element (bioreceptor) to the transducer. In the first generation (Ist gen), the
biosensors measure the content of the analytes and products of the bioreceptor reactions,
which diffuse to the surface of the transducer and produce an electric response. This type
of sensor is also called mediator-less amperometric biosensors. Leland Charles Clark Jr.,
the father of biosensors, described components of a biosensor in his first report. This report,
published in 1956, was about an electrode that can measure the oxygen concentration in
blood [8]. In 1962, Clark experimentally described the employment of an amperometric
enzyme electrode for glucose detection [9]. In 1967, Clark’s work was modified by Updike
and Hicks, who realized the first functional enzyme electrode-based on glucose oxidase
immobilized on an oxygen sensor [10].
In 1969, Guilbault and Montalvo demonstrated and reported the first potentiometric
enzyme electrode-based sensor for detecting urea [11]. In 1973, Guilbault and Lubrano
described glucose and a lactate enzyme sensor based on hydrogen peroxide detection at
a platinum electrode [12]. A heat-sensitive enzyme sensor known as ”thermistor” was
developed by the Klaus Mosbach group in 1974 [13]. In 1975, Lubbers and Opitz extended
the concept to make an optical biosensor for alcohol [14]. In the second generation (IInd
gen), individual components such as auxiliary enzymes and co-reactants (artificial or
partially toxic mediators or nanomaterials), are integrated into the biological component
layer of the biosensor with the view of enhancing analytical efficiency. These types of
sensors are called mediator amperometric biosensors. In 1976, Clemens et al. incorporated
an electrochemical glucose biosensor in a “bedside artificial pancreas” [15,16]. VIA Medical
introduced a novel semi-continuous catheter-based blood glucose analyzer and, later
in 1976, La Roche presented the lactate analyzer LA 640, which was used for electron
transport from lactate dehydrogenase to an electrode [17]. In the third generation (IIIrd
Sensors 2021, 21, 1109 5 of 35

gen), the bioreceptor molecule becomes an integral part of the base sensing element,
that is, biosensors progressed toward employing enzymes and mediators on the same
electrode rather than freely diffusing mediators in the electrolyte. A direct interaction was
established between the enzymes and electrode through the transfer of electrons, without
any requirement of intermediate stages like in nanomaterials. Besides the interaction,
low design cost and feasibility of having repeated measurements are the advantages of
this generation of biosensor [18]. In 1983, Liedberg identified dependency reactions in
real-time using the surface plasmon resonance (SPR) technique [19]. The blood glucose
level was measured in 1987 with a pen-sized detector by Cambridge, USA. Figure 3 shows
the three generations of the biosensors and Table 1 presents the timeline for the historical
development of biosensors.

Table 1. Development of biosensors in different timelines.

Year Development of Biosensor

1906 M. Cramer observed electric potential arising between parts of the fluid [20]
1909 Soren Sorensen developed the concept of pH and pH scale [21]
Griffin and Nelson were the first to demonstrate the immobilization of the enzyme invertase on aluminum
1909–1922
hydroxide and charcoal [22,23]
1922 W.S. Hughes discovered a pH measurement electrode [24]
1956 Leland C. Clark, Jr invented the first oxygen electrode [8]
1962 Leland C. Clark, Jr et al. experimentally demonstrated an amperometric enzyme electrode for detecting glucose [9]
Updike and Hicks and realized the first functional enzyme electrode based on glucose oxidase immobilized onto
1967
an oxygen sensor [10]
Guilbault and Montalvo demonstrated and reported the first potentiometric enzyme electrode-based sensor for
1969
the detecting urea [11]
1970 Discovery of ion-sensitive field-effect transistor (ISFET) by Bergveld [25]
Guilbault and Lubrano defined glucose and a lactate enzyme sensor based on hydrogen peroxide detection at a
1973
platinum electrode [12]
1974 K. Mosbach and B. Danielsson developed enzyme thermistor [13]
1975 D.W. Lubbers and N. Opitz demonstrated fiber-optic biosensor for carbon dioxide and oxygen detection [14]
1975 First commercial biosensor for glucose detection by YSI [26,27]
1975 Suzuki et al. First demonstrated microbe-based immunosensor [28]
1976 Clemens et al. demonstrated first bedside artificial pancreas [15]
1980 Peterson demonstrated the first fiber-optic pH sensor for in vivo blood gases [29]
1982 Fiber-optic biosensor for glucose detection by Schultz [30]
1983 Liedberg et al. observed surface plasmon resonance (SPR) immunosensor [19]
1983 Roederer and Bastiaans developed the first immunosensor based on piezoelectric detection [31]
1984 First mediated amperometric biosensor: ferrocene used with a glucose oxidase for glucose detection [18]
1990 SPR-based biosensor by Pharmacia Biacore [32]
1992 Handheld blood biosensor by i-STAT [32]
1999 Poncharal et al. demonstrated the first nanobiosensor [33]
2018 S. Girbi et al. demonstrated nerve-on-chip type biosensor for assessment of nerve impulse conduction [34]
Sensors 2021, 21, 1109 6 of 35

Figure 3. Three generations of the biosensor construction (MOX : Oxidized mediator; MRed : Re-
duced mediator).

2.3. Characteristics of Biosensors

To develop a highly effective and capable biosensor system, certain static and dynamic
requirements are necessary. Based on these specifications, the performance of the biosensors
can be optimized for commercial uses [3,4,35].
(a) Selectivity: Selectivity is a crucial feature to consider when selecting a bioreceptor for
a biosensor. A bioreceptor can detect a particular target analyte molecule in a sample
comprised of admixture spices and unwanted contaminants.
(b) Sensitivity: The minimum amount of analyte that can be correctly detected/identified
in a minimum number of steps and in low concentrations (ng/mL or fg/mL) to verify
the existence of analyte traces in the sample.
(c) Linearity: Linearity contributes to the accuracy of the measured results. The higher
the linearity (straight line), the higher the substrate concentration detection.
(d) Response time: The time is taken for obtaining 95% of the results.
(e) Reproducibility: Reproducibility is characterized by precision (similar output when
the sample is measured more than once) and accuracy (capability of a sensor to
generate a mean value closer to the actual value when the sample is measured every
time). It is the ability of the biosensor to produce identical results whenever the same
sample is measured more than once.
(f) Stability: Stability is one of the key characteristics in biosensor applications where
continuous monitoring is required. Stability is the extent of vulnerability to environ-
mental disturbances inside and outside the biosensing device. The factors that affect
stability are the affinity of the bioreceptor (the extent of binding of the analyte to the
bioreceptor) and the degradation of the bioreceptor over time.

2.4. Classification of Biosensors

Classification of biosensors is a diverse and multidisciplinary field. Various criteria are
involved in the classification of biosensors and the outline classification scheme is shown
in Figure 4.
As discussed earlier, bioreceptors are considered as the primary component in biosen-
sor construction. Based on the bioreceptor, biosensors are classified as enzymatic biosensors
(most common biosensor class), immunosensors (possess high specificity and sensitivity
and are specifically useful in diagnosis), aptamer or nucleic acid-based biosensors (possess
high specificity for microbial strains and nucleic acid-containing analyte), and microbial
or whole-cell biosensors. The second classification is made on the basis of the transducer
and sensors are categorized as electrochemical (which is further grouped as potentiometric,
amperometric, impedance and conductometric), electronic biosensor, thermal biosensor,
optical, and mass-based or gravimetric. Another classification includes bioreceptor-analyte
combinations, which are limited. Some classifications are made depending on the detec-
Sensors 2021, 21, 1109 7 of 35

tion system (optical, electrical, electronic, thermal, mechanical, and magnetic) and the
technology (nano, surface plasmon resonance (SPR), biosensors-on-chip (lab-on-chip),
electrometers, and deployable).

Figure 4. Classification of biosensors based on various bioreceptors and transducers used.

2.5. Classification of Biosensors Based on Bioreceptors

According to the biorecognition principle, biosensors are classified as catalytic biosen-
sors and affinity or non-catalytic biosensors [36]. In a catalytic biosensor, analyte–bioreceptor
interaction results in the development of a new biochemical reaction product. This biosen-
sor includes enzymes, microorganisms, tissues, and whole cells. In the case of affinity
(non-catalytic) biosensor, the analyte is bound to the receptor irreversibly, and during the
interaction no new biochemical reaction product is formed. This type of sensor comprises
antibodies, cell receptors, and nucleic acids as the target for detection [37].

2.5.1. Enzyme-Based Biosensors

Enzymes are common biocatalysts, which are efficient at increasing the biological
reaction rate. The working principle of an enzyme-based biosensor depends on the catalytic
reaction and binding capabilities for the target analyte detection [38]. Various possible
mechanisms are involved in the analyte recognition process: (i) The analyte is metabolized
by the enzyme, so the enzyme concentration is estimated by measuring the catalytic
transformation of the analyte by the enzyme, (ii) an enzyme inhibited or activated by
analyte, so the analyte concentration is related to decreased enzymatic product formation,
and (iii) tracking of the alteration of enzyme characteristics [39–42]. Owing to the long
history of enzyme-based biosensors, various biosensors can be produced on the basis of
enzyme specificity. However, the enzyme structure is extremely sensitive, which makes
it expensive and complicated to improve its sensitivity, stability, and adaptability [42].
Electrochemical transducers are most commonly used for enzyme-based biosensors. The
most common enzyme-based biosensors are glucose and urea biosensors. Cordeiro et al.
developed and characterized W-Au based amperometric enzyme-based glucose biosensors
for real-time monitoring of glucose in the brain in vitro. Their experiments revealed
that developed W-Au-based sensor can monitor changes in brain glucose in response
to relevant pharmacological challenges [43]. Uygun et al. developed a highly stable
potentiometric urea biosensor using nanoparticles. The response time and detection limit
of their developed sensor were 30 s and 0.77 µM, respectively [44]. Integrating enzymes
with nanomaterials has resulted in increased use of enzymes as recognition elements in
biosensors [45].

2.5.2. Antibody-Based Biosensors

Antibodies are affinity biorecognition elements, which have been used over two
decades because of their wide application range and strong antigen–antibody interactions.
Sensors 2021, 21, 1109 8 of 35

Antibodies possess the structure of immunoglobulins (Ig) in the form of “Y” shape, which
consists of two heavy and two light polypeptidic chains connected by disulfide bonds. Five
classes of antibodies have been defined based on differences in heavy chains: IgG, IgM, IgA,
IgD, and IgE [45]. Biosensors that have an embedded antibody as ligands or function on the
antibody–antigen interaction are called immunosensors. Immunosensors are classified as
(i) non-labeled and (ii) labeled. Non-labeled immunosensors are constructed to specifically
determine the antigen–antibody complex by estimating the physical changes caused by the
development of the complex. In the case of labeled immunosensor, a sensitively detectable
label is introduced. The antigen–antibody complex is sensitively assessed through label
measurement [46,47]. Madurro et al. constructed a label-free immunosensor to detect
ovarium cancer. The system has a linear relationship of anti-CA125 concentration in the
range of 5 to 80 U mL−1 , exhibiting a limit of detection of 1.45 U mL−1 [48]. Marquette
et al. developed electrical and optical biosensing for label-free detection of Aflatoxin B1
(AFB1) using gold (Au) nanobipyramids (NBPs). SPR-based AFB1 detection was found to
be in the linear range of 0.1–500 nM with a detection limit of 0.4 nM, while impedimetric
AFB1 detection was exhibited in the linear range of 0.1–25 nM, having a detection limit of
0.1 nM [49].

2.5.3. Aptamer-Based Biosensors

Aptamers are synthetic single-stranded nucleic acids (sequences of DNA or RNA)
that bind to target molecules selectively and can be folded into two-dimensional (2D) and
three-dimensional (3D) structures. In 2D or 3D structures, the targets have high-binding
performance due to greater surface density and less spatial blocking [50–52]. Due to the
nucleic acid character of aptamers, they are structurally and functionally stable over a
wide range of temperatures and storage conditions. Unlike antibodies that require bio-
logical systems to be generated, aptamers can be chemically synthesized, are stable in a
pH range of 2–12, and have certain thermal refolding capabilities. A further benefit of
aptamers is that they can be chemically modified according to the detection criteria for the
target molecule [53]. By an in vitro selection mechanism, SELEX (Systematic Evolution
of Ligands by EXponential enrichment), aptamers can be isolated from oligonucleotides
libraries [54,55]. Several SELEX variants have recently been established, including cell-
SELEX, capillary electrophoresis-based SELEX, microfluidics-SELEX, FACS-based SELEX,
microtiter plate-SELEX, magnetic bead SELEX, and in vivo SELEX [56]. Optical, electro-
chemical, and piezoelectric techniques are the most frequently used in biosensors. De-
pending on different transduction techniques, these biosensors are further categorized as
labeled or label-free aptasensors. Surface plasmon resonance (SPR) is the most commonly
used method for the label-free optical sensors, whereas fluorescent dyes (fluorescein) are
used for label-based optical aptasensors [57]. For tracking biological systems in real-time,
fluorescent NPs, such as QDs, provides many benefits over regular fluorescent dyes. To
identify targets, such as cancer cells, bacterial spores, and proteins, aptamer-QD conjugates
were used [58,59]. Starno et al. demonstrated aptamer capped NIR PbS QDs to detect
thrombin protein, based on selective charge transfer, within 1 min and with a detection
limit of ~1 nM [60]. Gold nanoparticles (GNPs) have demonstrated interesting absorption
characteristics that vary depending upon their aggregation state. Furthermore, GNPs
are more biocompatible, easier to bioconjugate, and less toxic than QDs [61]. Feng et al.
fabricated an electrochemical impedance spectroscopy (EIS)-based biosensor for L-Arg
detection, which exhibited acceptable performance in the linear range of 0.1 pM–0.1 mM
with a detection limit of 0.01 pM [62].

2.5.4. Whole-Cell-Based Biosensors

Whole-cell-based biosensors utilize microbes (bacterial, fungi (yeasts and molds),
algae, protozoa, and viruses), since they possess potential biorecognition elements, in the
construction of whole-cell-based biosensors. They are self-replicating and can produce
recognition elements, such as antibodies, without the need for extraction and purifica-
Sensors 2021, 21, 1109 9 of 35

tion [63,64]. Compared with animal or plant cells, whole-cell-based biosensors are easy
to handle and rapidly proliferating. The cells can interact with a wide variety of ana-
lytes, display the electrochemical response that a transducer can register, and can transmit
(whole-cell-based biosensor principle) [65]. Owing to their good sensitivity, high selectivity,
and capability of detection, these biosensors were successfully employed in environmental
monitoring, food analysis, pharmacology, heavy metals, pesticides, detection of organic
contaminants, and drug screening [66]. Polizzi et al. developed label-free optical whole-cell
Escherichia coli biosensors to detect pyrethroid insecticide exposure with a detection limit of
3 ng mL−1 3-PBA in the linear range of 0.01–2 ng mL−1 [67].

2.5.5. Nanoparticle-Based Biosensors

Apart from the above class of bioreceptors, NMs have recently been considered as a
new category of bioreceptors. With the advancement of nanotechnology and nanoscience,
different NMs have been used as bioreceptors [68]. NPs deliver a more vast range of
applications in biosensing technology. NMs can act as bioreceptors as well as transducers.
For example, cerium oxide-based NMs exhibit biomimetic catalytic activity favorable
for bioreceptors [69]. Due to their effective transduction capabilities, different inorganic
materials, such as graphene and CNTs-based NMs, noble metal NPs, and QDs, have
successfully been used as transducers.

2.6. Immobilization Techniques in Biosensors

To design the biorecognition part of a biosensor, immobilization of biological ele-
ments on the surface of the transducer is necessary. One of the key aspects of sensor
preparation is the selection of the appropriate immobilization technique. This is because
after immobilization there will be chances for the biorecognition element/molecule to
become inactivated or leached away. The biomolecules should be capable to preserve their
structure, function, and biological activity during biosensor use. Enzyme immobilization
techniques in biosensors are shown in Figure 5. Two important immobilization methods
are widely familiar: Physical (reversible) and chemical (irreversible). The selection of a
suitable technique relies on the biorecognition element chosen, the transducer, and the
physical-chemical environment and on the characteristics of the analyte [61,70].

Figure 5. Enzyme immobilization techniques.

Sensors 2021, 21, 1109 10 of 35

2.6.1. Physical or Reversible Immobilization

This technique is based on attaching enzymes to the surface of the transducer without
the creation of chemical bonds. These immobilizations include (a) physical adsorption, and
(b) physical entrapment (electropolymerization, sol-gel technique, and microencapsula-
tion) [36,70–73].
(a) Physical adsorption
In the adsorption technique, a biorecognition element immobilized on the outer
surface of the inert solid material by weak attractive forces, such as van der waals force,
the electrostatic force, ionic bonding, or hydrogen bonding. This method is seen in the
case of enzyme biosensors. Advantages of physical adsorption method include its simple
and economical process that it is non-destructive toward bioreceptor activity, and requires
no modification of biological elements and generation of a matrix. The disadvantages of
this method include that immobilization is susceptible to changes in pH, temperature, and
ionic strength, is connected by a feeble interactions, and has poor operational and storage
stability [61,70–73].
(b) Physical entrapment
In this process, biorecognition elements are physically entrapped within the 3D matri-
ces via covalent or non-covalent bonds. The entrapment immobilization process can occur
via two paths: The enzyme is mixed into a monomer solution, followed by polymerization
of monomer solution by a chemical reaction, or by changing experimental conditions. The
biorecognition elements are attached inside the 3D network of organic or inorganic materi-
als. The organic materials used are polydimethylsiloxane, photopolymer, gelatin, alginate,
cellulose, acetate phthalate, modified polypropylene, and polyacrylamide, whereas the
inorganic materials are activated carbon and porous ceramic materials. Frequently used
techniques in this process are (i) electropolymerization, (ii) the sol-gel process, and (iii)
microencapsulation [61,70–73].
(i) Electropolymerization is a simple process in which a current or potential is applied
to an aqueous solution or electrolyte containing both biomolecules and monomer molecules.
Either reduction or oxidation of the monomer occurs on the surface of the electrode in the
electrolyte, producing reactive radical species that join together and result in the formation
of a polymer, which traps the biorecognition elements (enzymes) that are near the electrode
in the aqueous solution. The electropolymerized films used for enzyme immobilization are
aniline, pyrrole, and thiophene. [61,70–73].
(ii) The Sol-gel process is the most frequently used technique maintained at low-
temperatures for enzyme trapping. A nanoporous material is formed via hydrolysis and
condensation of metal alkoxides. As the network grows with time and temperature, a
3D matrix is formed in which the bio-elements are encapsulated. The advantages of this
technique are thermal and chemical stability, easier way of synthesis, and the ability to en-
capsulate high concentrations of biomolecules at mild immobilizing conditions [61,70–73].
(iii) The Microencapsulation technique is a simple and inexpensive process where
biorecognition elements (enzymes) are enclosed in a spherical semi-permeable membrane.
The membrane can be of polymeric, lipoidal, lipoprotein based, or non-ionic. In microen-
capsulation, two methods are preferred: (i) Phase separation (coacervation) of enzyme
micro-droplets in water-immiscible liquid phases and (ii) polymerization of a monomer at
the interface of two immiscible substances (interfacial polymerization). This process results
in the obscuration of the enzyme within the polymeric membrane [61,70–73].

2.6.2. Chemical or Irreversible Immobilization

This method deals with the formation of strong chemical bonds, like covalent binding
or covalent linking, between biorecognition element functional groups and the transducer
surface. Based on the chemical bonding, chemical immobilization methods are categorized
as (a) direct covalent binding and (b) covalent cross-linking [61,70–73].
Sensors 2021, 21, 1109 11 of 35

(a) Covalent binding

Direct covalent binding is the most widely used enzyme immobilization technique,
in which the biorecognition element is firmly bonded either to the electrode/transducer
surface or to the inert matrix of the membrane. The immobilization process through the
membrane matrix involves two steps: Synthesis of the functional polymer and covalent
immobilization. The binding mechanism depends on the interaction between biorecog-
nition element and functional protein groups (usually side chains of amino acids) and
reactive groups of the transducer/membrane matrix surface. The advantages of direct
covalent binding include strong resistance to environmental changes, little leakage of
biorecognition element (enzyme), and strong bond formation between the biorecognition
element (enzyme) and matrix. The main disadvantages of this method are the use of harsh
chemicals and that the developed matrix cannot be regenerated once used [61,70–73].
(b) Cross-linking
The mechanism of cross-linking occurs via the creation of intermolecular covalent
cross-linkages between biorecognition elements (enzymes) or between biorecognition
elements and functionally inert proteins (for example bovine serum albumin). This process
is performed with the help of multi-functional reagents that act as a linker to connect
enzyme molecules in 3D cross-linked aggregates to the transducer surface. The optimal
conditions required for cross-linking are pH, temperature, and ionic strength. It allows
shorter response time, stronger attachment, and higher catalytic activity of enzymes. The
advantages are less leakage of enzymes, stronger chemical binding, and the possibility to
adjust the environment for the biorecognition element using appropriate stabilizing agents,
The disadvantages are the formation of covalent cross-links between protein molecules
instead of the matrix and protein and that partial denaturation of protein structure limit
the application of cross-linking immobilization [61,70–73].

2.7. Classification Based on Transducers

According to their operating principle, transducers are broadly categorized as electro-
chemical, optical, thermal, electronic, and gravimetric (shown in Figure 4).

2.7.1. Electrochemical Biosensors

Electrochemical biosensors are the most widely investigated and used biosensors
whose operating principles rely on the electrochemical properties of the analyte and trans-
ducer. Electrochemical biosensors exhibit high sensitivity, selectivity, and the capability of
detection. In this biosensor, an electrochemical reaction occurs on the transducer surface
between bioreceptor and analyte producing detectable electrochemical signals in terms of
voltage, current, impedance, and capacitance [74,75]. Based on the transduction principle,
electrochemical biosensors are categorized as: (a) Potentiometric, (b) amperometric, (c)
impedimetric, (d) conductometric, and (e) voltammetric [74–77]. Figure 6 illustrates the
schematic diagrams of (a) amperometric/voltammetric, (b) potentiometric, (c) conducto-
metric, and (d) impedimetric (showing the equivalent circuit) biosensors.
(a) Potentiometric biosensors: Potentiometric biosensors measure the charge accumu-
lated due to the analyte and bioreceptor interaction at the working electrode relative
to the reference electrode under zero current. To transform a biochemical reaction into
a potential signal, ion-selective electrodes and ion-sensitive field-effect transistors are
used [74–78].
(b) Amperometric biosensors: Amperometric biosensors operate in a two or three-
electrode configurations. These sensors measure the current produced due to elec-
trochemical oxidation or reduction of electroactive species at the working electrode
when a constant potential is applied to the working electrode with respect to the
reference electrode. The current produced on the surface of the working electrode is
proportional to the concentration of the analyte present in the solution [74–77]. Com-
pared with potentiometric biosensors, this method allows sensitive, fast, precise, and
Sensors 2021, 21, 1109 12 of 35

linear response, which makes it more suitable for mass production. However, poor
selectivity and interferences from other electroactive substances are the disadvantages
of these sensors [79,80].
(c) Conductometric biosensors: Conductometric biosensors quantify the change in the
conductance between the pair of electrodes because of an electrochemical reaction
(change in conductivity properties of the analyte). Conductometric and impedimet-
ric biosensors are usually used to monitor metabolic processes in living biological
systems [76,81].
(d) Impedimetric biosensors: Impedimetric biosensors measure the electrical impedance
produced at the electrode/electrolyte interface when a small sinusoidal excitation
signal is applied. It involves the application of low amplitude AC voltage at the sensor
electrode and then the in/out-of-phase current response is measured as a function of
frequency using an impedance analyzer [76,82].
(e) Voltammetric biosensors: Voltammetric biosensors detect analyte by measuring the
current during the controlled variation of the applied potential. Advantages of
these sensors include highly sensitive measurements and simultaneous detection of
multiple analytes [76].

Figure 6. Schematic diagram of (a) amperometric/voltammetric, (b) potentiometric, (c) conduc-

tometric biosensors, and (d) equivalent circuit of the impedimetric biosensor (Cdl = double-layer
capacitance of the electrodes, Rsol = resistance of the solution, Cde = capacitance of the electrode,
Zcell = impedance introduced by the bound nanoparticles, and Rcell and Ccell are the resistance and
capacitance in parallel).

2.7.2. Optical Biosensors

Optical biosensors are analytical devices consisting of a biorecognition element inte-
grated into an optical transducer system. The working principle of an optical biosensor is to
generate signals, which are proportional to the concentration of the analyte and to provide
label-free and real-time parallel detection. Optical biosensors utilize enzymes, antibodies,
aptamers, whole cells, and tissues, as biorecognition elements. In optical biosensors, the
transduction process induces a change in the absorption, transmission, reflection, refraction,
phase, amplitude, frequency, and-/or light polarization, in response to physical or chemical
Sensors 2021, 21, 1109 13 of 35

changes created by the biorecognition elements. According to the principle, optical biosen-
sors are categorized as label-free and label-based. In label-free sensing, the detected signal
is produced by the interaction of the analyte with the transducer. On the contrary, in label-
based sensing, the optical signal is generated by calorimetric, fluorescence, or luminescent
methods. Optical biosensors can be designed based on various optical principles, such as
SPR, evanescent wave (EW) fluorescence, optical waveguide interferometry, chemilumines-
cence, fluorescence, refractive index, and surfaced-enhanced Raman scattering. The most
commonly used optical-based biosensors are (a) fluorescence-based optical biosensors, (b)
chemiluminescence-based optical biosensors, (c) SPR-based optical biosensors, and (d)
optical fiber-based optical biosensors [70,83–85]. In Figure 7, schematic diagrams of (a)
chemiluminescence-based optical biosensors, (b) surface plasmon resonance (SPR)-based
optical biosensors, and (c) evanescent wave-based optical fiber biosensors are shown.

Figure 7. Schematic diagrams of (a) chemiluminescence biosensor, (b) surface plasmon resonance
(SPR) biosensor, and (c) evanescent wave-based optical fiber biosensor.

(a) Fluorescence-based optical biosensors: Fluorescence is the optical phenomenon that

includes labeling for the detecting analyte or molecules. This phenomenon has drawn
much attention to the design of fluorescence-based optical biosensors. This type of
biosensor is the most widely investigated for medical diagnosis, and environment
and food quality monitoring applications because of its high selectivity, sensitivity,
and short response time. Different kinds of fluorescent dyes, such as QDs, dyes, and
fluorescent proteins, are used in this biosensor. Fluorescence-based biosensors include
three approaches: (i) Fluorescent quenching (turn-off), (ii) fluorescent enhancement
(turn–on), and (iii) fluorescence resonance energy transfer (FRET). Recently, fluo-
rescence resonance energy transfer (FRET)-based optical biosensors have prevailed
in the study if the intercellular process owing to their higher sensitivity. The FRET
process involves nonradiative energy transfer from an excited donor molecule (D) to
the acceptor molecule (A) at the ground state via long-range multipole interactions.
As FRET-based optical sensors can detect changes in angstroms to nanometers, they
are widely employed in clinical applications, such as cancer therapy and aptamers
analysis [74,86]. Liu et al. constructed a carbon dots (CDs)/Au NR assembly-based
FRET sensor for detection of lead ions. The linear range was obtained from 0 to
155µM with a detection limit of 0.05µM [87].
(b) Chemiluminescence-based optical biosensors: Chemiluminescence is the phenomenon
in which light energy is released because of the chemical reaction. By virtue of its
simplicity, low detection limit, wide calibration limit, and affordable instrumentation,
chemiluminescence-based biosensors have received considerable interest. In recent
Sensors 2021, 21, 1109 14 of 35

times, chemiluminescence studies have been expanded to nanomaterials to improve

intrinsic sensitivity and extended to the new applications of detection [88]. He et al.
developed a chemiluminescence-based biosensor to detect DNA using graphene
oxide, which exhibited high sensitivity and selectivity and the linear range is 0.1–3 nm
with a limit of detection of 34 pM [89].
(c) SPR-based biosensors: SPR-based biosensors detect the change in the refractive index
caused by molecular interaction at a metal surface through surface plasmon waves.
This biosensor falls into the group of label-free biosensing technology and operates
on the principle of SPR. According to the SPR phenomenon, when polarized light
illuminates a metal surface at the interface between two media of different refractive
indices, at a certain angle it produces electron charge density waves called plasmons.
Based on the thickness of the layer at the metal surface, the SPR phenomenon results
in the declined intensity of the reflected light relative to the incident light at a specific
angle known as the resonance angle. The decrease in intensity is proportional to the
mass on the surface [90,91]. Moreover, the SPR method relies on refractive index
variations connected with the binding of the analyte to the biorecognition element
on the transducer or SPR sensor. SPR phenomenon offers various applications in
disease diagnosis and environmental and food quality monitoring. When the SPR
phenomenon is extended to metal-based nanomaterials, such as gold and silver, NPs
have given rise to a new phenomenon called localized surface plasmon resonance
(LSPR). The main difference between LSPR and SPR phenomena is that plasma
oscillations are governed by the total internal reflection locally at the surface of
the nanostructure instead of at the metal surface [85,90,91]. The SPR-based DNA
biosensor was developed by Rashidi et al. for detecting a donkey meat marker
using gold nanostars. Its low detection limit of DNA was 1.0 nM with a relative
standard deviation (RSD, n = 3) of 0.85% [92]. Hosseini et al. constructed a silver-
based LSPR biosensor for the detection of endotoxin E. coli. with a detection limit of
340 pg mL−1 [93].
(d) Optical fiber-based optical biosensors: Optical fiber biosensor is an optical fiber-
derived sensor system that employs an optical field to quantify biological species
such as whole-cells, proteins, and aptamers. Optical fiber-based biosensors are consid-
ered as a promising alternative to the traditional methods employed for biomolecule
assessment. The dependable optical fiber technique is an evanescent field sensing,
which is observed in the case of tapered optical fibers. An evanescent wave is pro-
duced at the sample interface when light passes through an optical fiber as a result
of total internal reflection. This field decays exponentially with distance from the
interface. The evanescent wave can be used to excite fluorescence in the proximity
of a sensing surface [94]. Tapered optical fibers are frequently used with different
optical transduction processes, such as variation in refractive index, absorption, flu-
orescence and SPR [95,96]. Huang et al. developed a fiber optic sensor based on
poly(N-isopropylacrylamide)-co-acrylamide)(P(NIPAAm-co-AAm))-magnetic immo-
bilized glucose oxidase(GOD) complex (PMIGC) and glucose oxidase (GOD) to detect
cholesterol and glucose. The sensor detected cholesterol (@ 38 ◦ C) and glucose concen-
tration (@ 25 ◦ C) with the detection ranges of 25–250 mg dL−1 and 50–700 mg dL−1 ,
respectively [97].

2.7.3. Gravimetric Biosensors

Gravimetric biosensors are mass-based biosensors, which respond to a small change
in the mass of binding material, such as proteins or antibodies on the surface, producing a
measurable signal. Gravimetric biosensors are employed with thin piezoelectric quartz
crystals, which vibrate at a specific frequency according to the applied current and the
mass of the detected material [98,99]. Figure 8 demonstrates schematic diagrams of (a)
piezoelectric, (b) quartz crystal microbalance, and (c) magnetoelastic-based biosensors.
Piezoelectric-based biosensors, magnetoelastic-based biosensors (MES), and quartz crys-
Sensors 2021, 21, 1109 15 of 35

tal microbalance (QCM) sensors are most commonly used for gravimetric transduction.
These transducers are also used to identify pathogens and antigens by means of binding
interactions. Piezoelectric biosensors are employed with a crystal that can undergo elastic
deformation when a current or potential is applied. The alternating electric field at a
specific frequency produces a wave in the crystal. If the analyte is absorbed or desorbed on
the surface of the crystal, which is covered with the biorecognition element, the resonant
frequency changes and suggests the occurrence of binding [100]. Yang et al. demonstrated
a piezoelectric biosensor using a lead titanate zirconate (PZT) ceramic resonator as the
transducer for label-free detection of cancer biomarkers. The developed device displayed
a high sensitivity of 0.25 ng mL−1 in 30 min detection time for a small amount of sample
(µL) [101]. QCM biosensors also work on the piezoelectric principle. A thin disk of quartz
crystal is sandwiched between two conducting electrodes and the resonance frequency
of crystal will change in response to the change in the mass of detected materials. Lee
et al. developed sensitive and selective detection technology for miR-21 molecules using a
QCM biosensor. The constructed QCM biosensor detected miR-21 with a detection limit of
0.87 pM in the linear range from 0.1 pM to 10 µM, with a correlation coefficient of 0.988. In
addition to these results, this sensor was also highly effective in the measurement of miR-21
in serum samples, which can be an excellent alternative for clinical diagnosis [102]. MES
has gained much attention as they are wireless and passive and can be used to determine
force, stress, pressure, and strain. An MES comprises of thick-film-like amorphous ferro-
magnetic ribbons with high mechanical tensile strength (1000–1700 MPa). MES work on
the principle of magnetostriction, in which mechanical deformation is developed as a result
of the applied magnetic field. Magnetoelastic vibrations are produced when time-varying
magnetic field is applied, causing the field-generated strain to change with time, which in
turn produces longitudinal elastic waves. The elastic waves within the magnetoelastic ma-
terial produce detectable magnetic flux. MES are ideal for biomedical applications owing to
their cost effectiveness, long lifetime, small size, passive and wireless characteristics [103].
Atalay et al. used a magnetoelastic sensor to detect Fe3 O4 magnetic nanoparticles. The
minimum number of MNPs was measured to be about 1.1 × 109, which corresponds to
0.025 mg or 1 µL of MNPs [104].

Figure 8. Schematic diagrams of (a) piezoelectric-based biosensor, (b) quartz crystal microbalance-
based biosensor, and (c) magnetoelastic-based biosensor.
Sensors 2021, 21, 1109 16 of 35

2.7.4. Thermal Biosensors

Thermal biosensor exploits the basic characteristics of the biological reactions (exother-
mic or endothermic), i.e., measurement of heat energy absorbed or released during the
reaction. The total heat energy absorbed/evolved or the temperature change (∆T) mea-
sured by thermal biosensor is proportional to the enthalpy (∆H) and to the total number of
product molecules (np ) created in the biochemical reaction and inversely proportional to
heat capacity (Cp ) of the reaction, which can be shown as ∆T = −(np ∆H)/Cp [105–109].
Calorimetric-based biosensors measure the change in heat, which is directly monitored to
calculate the extent of reaction (for catalyst) or structural dynamics of biomolecules in the
dissolved state [108,110]. Calorimetric devices are limited because of the relatively long
experimental procedures and lack of specificity in the temperature measurement; there is
no direct way to discriminate between specific and non-specific heat changes. However,
the invention of the enzyme thermistor-based on flow injection analysis in combination
with an immobilized biocatalyst and heat-sensing element circumvented several of these
shortcomings [107]. Usually, thermal biosensors employ a flow injection analysis method
utilizing an immobilized enzyme reactor, together with a differential temperature measure-
ment across the enzyme reactor. The configuration involves a pair of thermal transducers,
such as thermistors or thermopiles, positioned across the enzyme column packed with
immobilized enzymes for the conversion of given substrate to product. This differential
measurement system generates a high common-mode-rejection ratio that greatly reduces
the effects of ambient temperature fluctuations, allowing the specific measurement of
enzyme catalysis. The thermal signals generated during the catalytic reaction sensed
by the thermistor are proportional to the concentration of the substrate. Generally, the
enthalpy changes for enzymatic catalysis are around −10 to −200 kJ mol−1 , which is
adequate to determine the substrate concentrations at clinically interesting levels for a
range of metabolites [106–110]. Thermometric detection is advantageous when multiple
reactions are involved since it is the sum of all enthalpies that determines the sensitivity
of the assay. Thermistors or thermopiles are the two most commonly used temperature
sensors. The thermistor is a sensitive temperature transducer, which depends on the
changes in the electric resistance with temperature from which the absolute temperature
can be determined but have limited sensitivity. Thermopiles measure the temperature
difference between the two regions. Thermopiles are the set of thermocouple junctions
in series fabricated from metals, semiconductors, and various substrate semiconductor
components [108,109]. An enzyme thermistor-based biosensor is shown in Figure 9a. In
addition to thermistors, bimetallic strips, liquid gas expansion, and pyroelectric systems
(according to the production of an electrical signal because of a change in the temperature),
metal resistance and microelectromechanical systems (MEMS) are being used as thermal
transducers. Thermocouples with high sensitivity are also an excellent alternative for
detecting changes in temperature [61]. Recently, microelectromechanical (MEMS) thermal
sensors are being used to monitor metabolic applications on the basis of temperature
detection. Low-cost integration of miniaturized devices and low-cost batch fabrication are
the advantages of MEMS technology. MEMS thermal sensors exhibited improved thermal
isolation, low thermal mass, and sample volume of the MEMS thermal biosensor provides
linear range and high sensitivity, low measurement time, and low power consumption. It
is possible to measure multiple samples in parallel [111].

2.7.5. Electronic Biosensors

Figure 9b displays the Si NWs-based FET biosensors. The working principle of an
electronic biosensor relies on field-effect transistors (FETs). The FET is a three-terminal
device that regulates the current flowing through it using an electric field. They operate
between the source and drain terminals on a semiconductor, whose impedance changes
through the gate terminal [112–114]. Recently, FET-based transducers have gained much
attention due to their capacity to directly translate the interactions between the analyte and
FET surface [61]. If a biomolecule binds to the bioreceptor surface, it changes the surface
Sensors 2021, 21, 1109 17 of 35

potential. The corresponding change in channel width alters the current between the
source and drain terminals [114]. The high input impedance of these semiconductor-based
transducers is used to detect chemical changes from analyte and bioreceptor reactions. FET-
based biosensors possess advantages compared to the other biosensing methods because
of their high sensitivity and high spatial resolution, however they suffer some limitations
when employed in vitro applications [115,116]. Frequently used transistor-based sensing
platforms in biological applications are ion-sensitive field-effect transistors (ISFETs) and
metal-oxide-semiconductor field-effect transistors (MOSFETs), depending on the technique
of applying the gate voltage, design, and material of the gate and the channel region.
Zhu et al. reported a ZnO NRs-based FET glucose sensor using AC frequency mixing
detection rather than traditional three-electrode measurement in DC mode. The fabricated
senor achieved a high sensitivity of 1.6 mA (µm-cm2 )−1 with a detection limit of 1.0 µM
glucose concentration. It also exhibited long-term stability of 38 h [117].

Figure 9. Schematic diagrams of (a) enzyme thermistor-based biosensor, (b) Si nanowire-based

field-effect transistor (FET) (D is drain and S is the source) biosensor, and (c) surface acoustic wave
(SAW)-based biosensor.

2.7.6. Acoustic Biosensors

Acoustic biosensors operate based on the change in the physical properties of an
acoustic wave in response that can be correlated to the amount of analyte absorbed [118].
Piezoelectric materials are commonly used for sensor transducers, because of their ability
to produce and transmit acoustic waves in a frequency-dependent manner. For acoustic-
wave propagation, the optimum resonant frequency is highly dependent on the physical
dimensions and properties of the piezoelectric crystal. The changes in material mass
on the surface of the crystal can induce measurable variations in the natural resonant
frequency of crystal [100,101]. There are two classes of mass-balance acoustic transducers:
Bulk-acoustic wave (BAW) and surface-acoustic wave (SAW) devices. BAW devices can
transmit an acoustic wave from one crystal surface to another whereas SAW devices can
transmit an acoustic wave along a single crystal face from one location to another [119,120].
The operation of these devices in the gas phase is well understood, but in liquid media,
it is not that well understood. For many years the piezoelectric mechanism has been
established and can be an alternative for the transduction process in affinity biosensors if
Sensors 2021, 21, 1109 18 of 35

the challenges of non-specific binding and poor sensitivity are solved [72,83]. The surface
acoustic wave-based biosensor is shown in Figure 9c.

3. Advancement of Nanotechnology
To meet the increasing demands of various fields, new approaches to sensor tech-
nology have been employed. Sensor technology has further flourished with progress in
nanotechnology and nanoscience. Nanotechnology has stretched across various fields of
science and industry, such as physics, chemistry, biotechnology, bioscience, bioinformatics,
medical science, healthcare, food engineering or processing, aerospace, and electronics, the
energy sector, and environmental studies. The ability to manipulate and control materials
at an atomic and molecular level (nanometer range) and subsequent understanding of the
fundamental processes at the nanoscale have led to new avenues of biosensor development.
More importantly, dimensionality plays a major role in determining the characteristics
of nanomaterials, including physical, chemical, biological, electrical, and optical charac-
teristics. Nanomaterials are broadly classified, based on their nanoscopic dimensions,
such as 0D, 1D, 2D, and 3D materials (Figure 10). If all three dimensions of a material are
nano-sized, it would be called a 0D NM (NPs and QDs),). If two dimensions of a material
are nanosized, with the other dimension being much larger, then it is 1D NM (NWs, NRs,
NTs, nanobelts, and nanoribbons). If only one dimension is nanosized, it would be a 2D
NM (nanoprisms, nanoplates, nanocoatings, nanolayers, nanosheets, nanowalls, nanodisks,
and CNTs). Bulk nanomaterials are materials that are not confined to the nanoscale in
any dimension (≥100 nm) and are referred as 3D NMs (nanoballs, dendritic structures,
nanocoils, nanocones, nanopillars, multi-nanolayers, and nanoflowers) [121,122]. Synthesis
of materials in the nanoscale range enables the unique physical, chemical, and biological
properties and plays a pivotal role in the success of nanotechnology. Various approaches
have been employed for the synthesis of NMs, categorized as a top-down approach (a bulk
material is restructured to form nanosized materials) and a bottom-up approach (materials
of nanodimension are formed by assembling molecule by molecule or atom by atom). The
top-down approach includes various techniques, such as lithography, laser ablation, ion
milling, and chemical etching. The commonly used techniques in the bottom-up approach
are molecular beam epitaxy, physical or chemical vapor deposition and evaporation, and
bio/chemical processes for the production of supra-molecular complexes, self-assembled
monolayers, and protein-polymer nanocomposites [121,122].

Figure 10. Classification of nanomaterials according to their dimensionality.

Sensors 2021, 21, 1109 19 of 35

4. Nanomaterial-Based Biosensors (Nanobiosensors)

With advances in nanotechnology, research and development in the field of biosen-
sors have become open and multidisciplinary. Exploring NMs, such as NPs (metal- and
oxide-based), NWs, NRs, CNTs, QDs, and nanocomposites (dendrimers), for different
characteristics provides the possibility of improving the performance of biosensors and
increase the power of detection through size and morphology control. Different types of
NMs-based biosensors (nanobiosensors) are shown in Figure 11.

Figure 11. Types of nanomaterials-based biosensors (nanobiosensors).

The basic working principle of nanobiosensors is along the same lines of conventional
macro- and micro-counterparts, but they are constructed using nanoscale components
for signal or data transformation [123]. Nanobiosensors have an edge over their conven-
tional macro- and micro-counterparts because of their multidisciplinary applications due
to dimensionality. Nanobiosensors are instrumental in the field of nanotechnology for (a)
monitoring physical and chemical phenomena in regions difficult to reach, (b) detecting
biochemicals in cellular organelles and medical diagnosis, (c) measuring nanoscopic parti-
cles in industrial areas and the environment, and (d) detecting ultra-low concentrations of
potentially harmful substances [123]. Based on the classification of the NMs, their involve-
ment in the enhancement of biosensing mechanisms has been broadly investigated. For
instance, NPs-based biosensors include all sensors that employ metallic NPs as enhancers
of biochemical signals. Similarly, nanotube-based biosensors, if they involve CNTs, are
used as enhancers of reaction specificity and efficiency, while biosensors using NWs as
charge transport and carriers are termed as NW biosensors. Likewise, QD-based sensors
employ QDs as contrast agents for improving optical responses.

4.1. Nanoparticle-Based Biosensors

NPs have been widely used in various biomedical applications, like in the develop-
ment of biosensors for health diagnosis, imaging, drug delivery, and therapy, owing to
their unique properties. Because of their small size and shape, their physical and chemi-
cal properties are strongly influenced by the binding of target biomolecules [124]. These
properties of NPs enable them to be exploited for various bioanalytical applications. They
are considered suitable for electrode modification in which they increase the sensitivity
and specificity of electrochemical catalysis [70]. Moreover, catalytic active NMs, such as
transition metal oxides, have been developed as nanoenzymes, which allow providing
catalysis of biochemical reactions on biosensors. The NPs include metal and noble metal
NPs, such as gold (Au), silver (Ag), platinum (Pt), palladium (Pd), cobalt (Co), iron (Fe),
and copper (Cu), and metal oxide NPs (ZnO, TiO2 , SnO2 and MnO2 ), which exhibit ex-
Sensors 2021, 21, 1109 20 of 35

cellent optical, electronic, magnetic, chemical, mechanical, and catalytic properties. NP

biosensing performance is tailored by coating with various matrices, such as metal oxides,
silica network, polymers, graphene, fibers, and dendrimers [125].
Gold NPs, which fall into the class of noble metal NPs, are extensively investigated
and used owing to their unique optical, electronic, and physicochemical properties. They
are widely used in biomedical research because of the following advantages: Simple
synthesis techniques, easier fabrication procedures, greater chemical stability, biocompati-
bility, vast electrochemical potential range, high catalytic activity, and their nanocomposite
forms [126,127]. Wu et al. demonstrated gold NP-based electrochemical sensors for sen-
sitive detection of uranyl in natural water. The developed sensor determined uranyl in
the range of 2.4 to 480 µg L−1 , and a detection limit of 0.3 µg L−1 was obtained by anodic
stripping voltammetry [128]. Luo et al. established a novel “turn–on” fluorescent sensor for
detecting Pb2+ , based on graphene quantum dots (GQDs) and gold nanoparticles (AuNPs).
The designed sensor showed an extremely broad detection range of Pb2+ from 50 nm
to 4 µm, with a detection limit of 16.7 nm [129]. Ghasemi et al. demonstrated a novel
non-enzymatic glucose sensor based on gold-nickel bimetallic NPs doped aluminosili-
cate framework prepared from agro-waste material that exhibited a wide linear range for
glucose (1–1900 µM) and low limit of detection (0.063 µM) [130].
Silver nanoparticles (Ag NPs) have gained much research interest in biomedical
applications due to excellent surface-enhanced Raman scattering (SERS), biocompatibility,
high conductivity, amplified electrochemical signals, and catalytic activity [131–133]. Rivero
et al. demonstrated an optical fiber sensor based on both localized surface plasmon
resonance (LSPR) and lossy-mode resonance (LMR) using Ag NPs. The devices showed
high sensitivity (0.943 nm per RH %), a large dynamic range (42.4 nm for RH changes
between 25% and 70%), and a fast response time (476 ms and 447 ms for rise and fall,
respectively). This device can be used to monitor human breathing [134]. Mehdinia et al.
developed a multi-functional colorimetric probe for Fe2+ , H2 O2 , and glucose detection
based on the Fenton reaction and biosynthesized AgNPs. The low detection limit for Fe2+
was 0.54 µM, for H2 O2 was 0.032 µM, and for glucose was 0.29 µM [135].
Over the past decade, owing to their unique electronic and electro-catalytic char-
acteristics, platinum nanoparticles (Pt NPs) have gained much interest in the field of
electrochemical biosensors for biomedical applications. The electron transfer process in Pt
NPs is affected by material composition, surface reactive environment, crystalline plane,
and orientation [133]. Liu et al. fabricated a new Pt NP/a-IGZO-based ammonia sensor
showing high sensing response (SR ) of 1467 (at 1000 ppm NH3 /air, 250 ◦ C and exhibiting
fast sensing speed [136]. Dharuman et al. developed a graphitic carbon nitride modified
with Pt and zinc oxide NPs for non-enzymatic glucose sensing with a wide linear detection
range of 250 µM to 110 mM. It can be reusable four times in whole blood and eight times in
blood serum [137].
Palladium nanoparticles (Pd NPs) are another fascinating NP for biomedical applica-
tions because of their high catalytic and sensing activities. In addition, palladium (Pd) is
much more abundant than Au and Pt metals making it employable in various sensors for
sensing applications in a cost-effective way. Pd NPs with controllable size and shape ex-
hibit high electro-catalytic and sensing characteristics for different chemical and biological
analytes [135,138]. Ye et al. prepared a Pd/Co-NCNTs exhibited excellent electrocatalytic
ability for hydrazine oxidation and showed a high sensitivity of 343.909 µA mM−1 with a
low detection limit of 0.007 µM for hydrazine [139]. Swihart et al. developed a unique 3D
Pd-decorated crumple reduced graphene oxide ball (Pd-CGB) nanocomposite for hydrogen
(H2 ) detection in air at room temperature. The sensitivity of the sensor is measured for the
H2 concentrations (0.0025–2%) with response value, response time, and recovery time of
14.8%, 73 s, and 126 s, respectively, at 2% H2 [140]. Afzali et al. developed a novel sensor
based on Pd/CNF/[M3OA]+ [NTF2]− modified CPE through a sensitive square-wave
voltammetric procedure for the determination of the anticancer drug pemetrexed. A linear
Sensors 2021, 21, 1109 21 of 35

concentration was detected in the range of 1.00–35.0 nM with a detection limit of 0.33 nM
by square wave voltammetry (SWV) technique [141].
Copper (Cu) has gathered much research interest as a unique sensing material owing
to its excellent electrical conductivity, stability, electrocatalytic properties, and low cost
compared to noble metals. Recently, Huang et al. prepared and studied the performance of
electrochemical glucose sensors based on copper nanoparticles (Cu NPs) loaded on a flexi-
ble graphite sheet. The developed sensor exhibited a low detection limit of 1.05 µmol L−1
and high sensitivity of 7254.1 µA mM−1 cm−2 , with R2 = 0.9961 from 0.1 to 3.4 mmol L−1
and 3804.5 µA mM−1 cm−2 (R2 = 0.9995) from 3.4 to 5.6 mmol L−1 . Cu NPs also exhibited
excellent anti-interference properties against sodium chloride, acetaminophenol, ascorbic
acid, dopamine, and uric acid, with good reproducibility [142]. Roushani et al. developed
a novel sensor for analytical detection of H2 O2 based on the incorporation of CuNPs onto
an MWCNTs/IL/Chit/Rutin nanocomposite film. The electrochemical performance of the
sensor for detecting H2 O2 was investigated by cyclic voltammetry and chronoamperometry
techniques. The response to H2 O2 was linear, in the range of 0.35 µM to 2500 µM, with
a detection limit of 0.11 µM [143]. Zhao et al. fabricated a Cu/rGO decorated buckypa-
per electrode for glucose detection. The constructed device exhibited a linear range of
0.1–2 mM, with a detection limit of 11 µM [144].

Metal Oxide-Based Nanoparticles

Over the last decade, metal oxide-based nanomaterials have been vividly employed in
various fields, such as electrochemistry, magnetism, catalysis, and sensors, because of their
broad range of electrical, chemical, and physical properties. These oxide-based materials
are used as an effective electrocatalyst for sensing various analytes in the field of biology
and biomedicine because of their strong electrocatalytic activity, low cost, and high organic
capture ability. The most often employed metal oxide nanoparticles include copper oxide
(CuO), nickel oxide (NiO), iron oxide (Fe2 O3 ), cobalt oxide (Co3 O4 ), manganese oxide
(MnO2 ), zinc oxide (ZnO), titanium oxide (TiO2 ), tin oxide (SnO2 ), cadmium oxide (CdO),
molybdenum oxide (MoO3 ), and cerium oxide (CeO2 ) [145].
Nickel oxide-based nanoparticles (NiO NPs) exhibit superior electrical, magnetic,
optical, thermal, catalytic, and mechanical properties. NiO-based nanostructures have
been used as catalysts, thermistors, sensors, and additives, for gases and ceramics. NiO
NPs are also p-type semiconductors with a direct bandgap of (3.56 eV) that can exhibit
super-paramagnetic, as well as superanti-ferromagnetic properties, depending on their size
and oxidation states [146]. Recently, Recently, Duan et al. fabricated high performance FET
glucose biosensors based on bimetallic Ni/Cu metal organic frame works. The fabricated
device exhibited a low detection limit of 0.51 µM with a linear range of 1 µM–20 mM [147].
Kamyabi et al. fabricated novel electroluminescence (ECL) glucose biosensors based on
immobilized glucose oxidase in the cavity of nickel foam modified with NiO NPs. The
proposed ECL biosensor showed superior performance toward glucose in 0.1 M phosphate
buffer solution (pH 7.4) with a wide linear range (2.7 × 10−9 to 4.5 × 10−3 M) and a low
detection limit (5.0 × 10−10 M) [148].
Cobalt oxide-based nanoparticles (Co3 O4 NPS) have attracted considerable interest
due to their exceptional physical, magnetic, optical, electronic, and chemical properties.
They possess promising applications, such as catalysts, solar selective absorber, gas sensors,
lithium-ion batteries, supercapacitors, and pigment for glasses, and ceramics, photocatal-
ysis, magnetic material, and chemical sensors. It is a p-type semiconductor with a direct
optical band gap of 1.66 and 2.19 eV [149]. Chu et al. developed a screen-printed glutamate
biosensor chip using porous Co3 O4 nanocubic crystals. The developed biosensor chip
exhibited a high sensitivity of 20.12 µA mM−1 cm−2 , as well as a wide linear range from
10 to 600 µM and a detection limit of 10 µM [150]. Wazir et al. developed a potentiomet-
ric urea biosensor fabricated on glass filter paper through the immobilization of urease
enzyme onto cobalt oxide- chitosan nanocomposite. The sensitivity was measured over
the concentration range between 1 × 10−4 and 8 × 10−2 M of the urea electrolyte solution,
Sensors 2021, 21, 1109 22 of 35

revealing that the fabricated biosensor exhibited good sensing ability with a linear slope
curve of 45 mV/decade [151]. Ge et al. constructed a Co3 O4 -Au polyhedron-based photo-
electrochemical (PEC) biosensor for detecting miRNA-141 detection with a linear range of
1 pM to 50 nM, and a detection limit of 0.2 pM [152].
Iron oxide (Fe2 O3 ) and manganese oxide (MnO2 )-based NPs are considered the best
known magnetic NMs because of their bioanalytical applications and higher electron
transfer rates. They are also considered to be promising materials for electrochemical
biosensors [153,154]. Phan et al. demonstrated the possibility of using the magneto-
reactance effect of a soft ferromagnetic amorphous ribbon with a nanohole-patterned
surface to develop a highly sensitive magnetic biosensor for detection and quantification
of anticancer drugs tagged to super-paramagnetic NPs [155]. Zhang et al. developed a
fast and highly specific LF-NMR biosensor that can directly detect Salmonella, without
sample pretreatment, with a detection limit of 2.6 × 104 CFU mL−1 [156]. Stankovic et al.
developed a disposable biosensor based on graphene nanoribbons supported with MnO2
NPs. The sensor displayed a detection limit of 0.05 mmol L−1 and a high sensitivity of
56.32 µA mmol−1 cm−2 [157].
Other metal oxide-based NPs, such as ZnO, TiO2 , SnO2 , and MoO3 , have recently
gained much attention. ZnO based nanoparticles possess good electron transfer rate and
thermal/chemical stability, oxidation resistance, biocompatibility, and high conductivity.
ZnO is an n-type semiconductor with a wide bandgap energy of 3.37 eV [158,159]. Hjiri
et al. prepared a carbon monoxide sensor using ZnO NPs synthesized by the sol-gel
technique. The developed gas sensor exhibited a response of 74% toward 80 ppm of CO gas
with a response/recovery time of 21 and 70 s, respectively, at 250 ◦ C and high stability with
time [160]. SnO2 -based NPs have also been used for detecting toxic gases and pesticide
sensing applications [161,162]. TiO2 -based NPs can be used in electrochemical sensors for
medical, biomedical, and pharmaceutical applications. Tereshchenko et al. introduced a
novel and simple optical immunosensor to determine Salmonella typhimurium based on TiO2
NPs deposited on a glass substrate with a sensitivity in the range of 103 –105 cL mL−1 [163].
Recently, Ravikumar et al. reported on rapid and facile method for detecting H2 O2 in
chemical reactions using molybdenum oxide (MoO3 ) NPs [164].

4.2. Quantum Dot-Based Biosensors

QDs are inorganic nanocrystals (NCs), which belong to 0D NMs displaying unique
optical properties of broad excitation, narrow size-tunable emission spectra, high pho-
tochemical stability, and negligible photo-bleaching [165]. They have been widely used,
mainly as alternatives to fluorophores, for developing optical biosensors to detect organic
compounds, pharmaceutical analytes, and biomolecules, such as nucleic acids, proteins,
amino acids, enzymes, carbohydrates, and neurotransmitters [166]. They have also been
employed for the in vivo detection of cancer. They are ideal candidates for multiplexed
optical bioanalysis due to their ultra-high sensitivity, high specificity, cost-effectiveness,
miniaturized size, size-dependent emission wavelength, and rapid analyte detection [167].
Cui et al. proposed a simple and efficient electrochemical sensor for Cu (II) based on GQDs
and graphene. The combination of GQDs and graphene-enhanced the performance, with
a low detection limit of 1.34 nM in a wide linear range of 0.015 µM to 8.775 µM for Cu
(II) [168]. Packirisamy et al. demonstrated the fabrication and application of fluorescent
turn-on biosensors for ultrasensitive detection of small cell lung cancer biomarkers using
biofunctionalized GQDs as the energy donor and AuNPs as energy acceptor. The fluores-
cent biosensor exhibited a fast response time (16 min), and broader linear detection range
(0.1 pg mL−1 to 1000 ng mL−1 ), and low detection limit of 0.09 pg mL−1 [169]. Xiao et al.
demonstrated CdTe/CdS/ZnS core/shell/shell QDs-based fluorescent biosensors for the
determination of L-ascorbic acid. The concentration was detected in the linear range of
8.0 × 10−9 to 1.0 × 10−7 M with a detection limit of 1.8 × 10−9 M [170]. Sun et al. described
a “turn-on” magnetic fluorescent biosensor based on GQDs, Fe3 O4 , and molybdenum
disulfide (MoS2 ) nanosheets for detecting EpCAM in the linear range between 2 and 64 nM
Sensors 2021, 21, 1109 23 of 35

with a detection limit of 1.19 nM. It is used for rapid, efficient, and sensitive separation and
detection of circulating tumor cells (CTCs) [171].

4.3. Nanowire-Based Biosensors

NWs provide favorable conditions for creating robust, sensitive, and selective electrical
detectors of biological binding events. The NWs exhibit highly reproducible optical and
electrical characteristics. Current flow in any 1D systems, such as NWs and NTs is extremely
sensitive to minor perturbations because, in such systems, flow is extremely close to the
surface [172]. The diameter of the NWs are comparable to the biological and chemical
species that are being sensed. They offer excellent transduction generating signals, which
ultimately interface to macroscopic instruments. The combination of tunable conducting
properties of semiconducting NWs and the ability to bind analytes on their surface yields a
direct, label-free electrical readout [173]. NWs-based sensors operate on the principle of
ion-selective FETs and rely on the interaction of external charges with carriers in the nearby
semiconductor, which results in enhanced sensitivity at low ionic strength. Park et al.
fabricated fiber optic sensor using ZnO NWs and AuNPs for highly sensitive plasmonic
biosensing [174]. Priolo et al. demonstrated label-free and PCR-free silicon NWs-based
optical biosensor for direct genome detection. They exhibited a detection limit of 2 copies
per reaction for the synthetic genome and 20 copies per reaction for the genome extracted
from human blood [175]. Nuzaihan et al. prepared a silicon NW-based biosensor with
novel molecular gate control for electrical detection of Dengue virus (DENV) DNA. The
developed sensor had a low detection limit of 2.0 fM concentration with high sensitivity of
45.0 µA M−1 [176].

4.4. Nanorods-Based Biosensors

Nanorods are often used as simple electrochemical modifiers providing a highly
specific process. They are usually prepared from gold, graphene, manganese, zinc, or
iron oxide, or the combination of these materials [177,178]. The detection of nucleic acid
or basic biochemical markers, such as glucose and hydrogen peroxide, are their most
common applications. Liu et al. constructed a new CDs/Au NR assembly-based FRET
sensor for detecting lead ions. A linear range from 0 to 155 µM, with a detection limit of
0.05 µM [87]. Zhu et al. demonstrated a ZnO NRs-based FET biosensor for continuous
glucose monitoring using AC frequency mixing. The fabricated sensor achieved a high
sensitivity of 1.6 mA (µM−1 cm−2 ) with a concentration detection limit of 1 µM, and
exhibited an excellent long-term stability on continuous monitoring for 38 h [117]. Sun et al.
used GNRs and graphene oxide (GO) to enhance the sensitivity of a wavelength modulation
SPR biosensor to detect bovine IgG. The developed biosensor based on GNRs/GO sensing
had detected bovine IgG in the concentration range of 0.075–40.0 µg m L−1 [179]. Hahn
et al. fabricated a vertically grown ZnO NRs-based FET biosensor to detect phosphate with
high sensitivity (80.57 µA mM−1 cm−2 ) in a wide linear range (0.1 µM–7.0 mM) [180].

4.5. Carbon Nanotubes-Based Biosensors

CNTs are exciting 1D NMs and are the most extensively investigated nanotubes class
of materials in biosensors, diagnostics, tissue engineering, cell tracking and labeling, drug
delivery, and biomolecules. They are hollow cylindrical tubes composed of one, two, or sev-
eral concentric graphite layers capped by fullerenic hemispheres, which are referred to as
single-, double-, and multi-walled CNTs, respectively. They have unique structures, excel-
lent electrical and mechanical properties, high thermal conductivity, high chemical stability,
remarkable electrocatalytic activity, minimal surface fouling, low over-voltage, and high as-
pect ratio (surface-to-volume) [181–183]. Because of their high surface-to-volume ratio and
novel electron transport properties, the electronic conductance of theses nanostructures is
strongly influenced by minor surface perturbations, such as those associated with the bind-
ing of macromolecules. CNT-based biosensors and diagnostics have been employed for the
highly sensitive detection of analytes in healthcare, industries, environmental monitoring,
Sensors 2021, 21, 1109 24 of 35

and food quality analysis. They have been predominantly used in electrochemical sensing,
for glucose monitoring, but also for detecting fructose, galactose, neurotransmitters, neuro-
chemicals, amino acids, immunoglobulin, albumin, streptavidin, insulin, human chorionic
gonadotropin, C-reactive protein, cancer biomarkers, cells, microorganisms, DNA, and
other biomolecules. Multi-wall-carbon nanotubes (MWCNTs) are represented in all appli-
cations of nanotubes in biosensing. Such 1D nanomaterials provide real-time and sensitive
label-free bioelectronic detection and massive redundancy in nanosensor arrays [165]. Cui
et al. developed a wearable-based amperometric biosensor painted onto gloves as a new
sensing platform used to determine lactate [184]. Janssen et al. demonstrated a CNT-based
biosensor to sense a standard protein, bovine serum albumin (BSA), as a proof-of-concept.
The developed sensor had a detection limit of 2.89 ng mL−1 [185]. Tang et al. fabricated
a single-walled carbon nanotube (SWNT)-based DNA sensors and described the sensing
mechanism. This work demonstrated clear experimental evidence on SWNT-DNA binding
on DNA functionality, which paved a path for the future design of SWNT biocomplexes for
applications in biotechnology and DNA-based nanotube manipulation techniques [186].
Hong et al. constructed metallic floating electrode-based DNA sensors with controllable re-
sponses. They showed the enhancement in the sensor response on increasing the number of
floating electrodes [187]. Park et al. demonstrated a CNT-based biosensor system-on-a-chip
for detecting a neurotransmitter. Here, CNT-based sensors were integrated with CMOS
chips, which is useful in various biomedical applications, such as sensing components in
LoC (lab-on-a-chip) systems for neuronal culture [188].

4.6. Dendrimer-Based Biosensors

Dendrimers are nanometer-scale 3D macromolecules in the size of an average protein-,
and are hyper-branched, mono-dispersed, and star-shaped, with a high density of surface
functional groups. The shape of dendrimers provides a vast surface area for the conjugation
of biologically active molecules. They are composed of three distinct components: The core,
the interior dendron, and the exterior surface with terminal functional groups [189,190].
They have been used extensively in various biosensors, diagnostics, and drug delivery
based on electrochemistry, fluorescence, SERC, impedimentary, and SPR. Dendrimer-
based biosensors increase analytical sensitivity, stability, and reproducibility but reduce no
specific interactions [191–193]. Bakar et al. detected dengue using a PAMAM dendrimer
integrated tapered optical fiber sensor. The resolution and detection limit of the sensor
were 19.53 nM−1 and 1 pM, respectively, in the concentration range of 0.1 pM to 1 µM [194].
Fen et al. developed an SPR sensor based on self-assembled monolayer/reduced graphene
oxide-polyamidoamine dendrimer (SAM/NH2 rGO/PAMAM) thin films to detect DENV-2
E-proteins. Their SPR sensor exhibited a detection limit of 0.08 pM DENV-2E-proteins in
the range of 0.08 pM–0.5 pM [195]. Table 2 represents the list of various nanomaterials
employed in the development of biosensors.

Table 2. Represents the list of various nanomaterials employed in the development of biosensors.

Nanomaterial Analyte Transducer Linear Range Detection Limit References

Aflatoxin B1
(AFB1) SPR 0.1–500 nM 0.4 nM
Au NBPs [48]
Aflatoxin B1 Impedimetric 0.1–25 nM 0.1 nM
(AFB1)
Au NPs Uranyl Electrochemical 2.4–480 µg L−1 0.3 µg L−1 [128]
Au NPs Pb2+ Fluorescent 50 nm–4 µm 16.7 nm [129]
Cholesterol Electrochemical 0.024–1.2 mM 0.012 µM
Au/CdS QDs/TNTs [196]
H2 O2 Electrochemical 18.73–355.87 µm 0.06 µM
Au NPs E.coli Electrochemical 10–106 CFU mL−1 15 CFU mL−1 [197]
Sensors 2021, 21, 1109 25 of 35

Table 2. Cont.

Nanomaterial Analyte Transducer Linear Range Detection Limit References

Carcinoembryonic
Au NP-MoS2 -rGO SAW 36.58 ng mL−1 0.084 ng mL−1 [198]
antigen (CEA)
Au/rGO miENA-122 Electrochemical 10 µm–10 pm 1.73 pM [199]
Au NPs/TiO2 H2 O2 Electrochemical 65–1600 µm 5 µm [200]
H2 O2 0.05–7.5 µm 0.032 µm
Ag NPs Glucose Colorimetric 1.5–3.0 µm 0.29 µm [135]
Fe2+ 1–90 µm 0.54 µm
Ractopamine 0.01–100 ng mL−1 1.52 pg mL−1
Ag/Pd NPs Clenbuterol Electrochemical 0.01–100 ng mL−1 1.44 pg mL−1 [201]
Salbutamol 0.01–100 ng mL−1 1.38 pg mL−1
Ag@CQDs-rGO Dopamine Electrochemical 0.1–300 µm 0.59 nm [202]
Ag NP-MWNT Glucose Electrochemical 0.025–1.0 mM 0.01 mM [203]
Electro- 1.135 fg mL−1
Ag NPs Mucin 1 0.37 fg mL−1 [204]
chemiluminescence –0.1135 ng mL−1
9.99 × 10−1 – 2.13 2.93 × 10−4
Pt NPs Adrenaline Voltammetric [205]
× 10−4 mol L−1 mol L−1
2.0 × 10−8 M
Pt NPs/RGO-CS-Fc H2 O2 Electrochemical 20 nm [206]
–3.0 × 10−8 M
Pt-Fe3 O4 @C Sarcosine Amperometric 0.5–60 µm 0.43 µm [207]
Cyclic
Pt NFs/PANi Urea 20 mM 10 µm [208]
Voltammetry
Pt@CeO2 NM Dopamine Electrochemical 2–180 nM 0.71 nM [209]
Pd/Co-NCNT Hydrazin Electrochemical 0.05–406.045 µm 0.007 µm [139]
Pd/CNF/[M3OA]+ [NTF2]− H2 1.00–35.0 nM 0.33 nM [141]
Cu
Cyclic
NPs/Rutin/MWCNTs/ H2 O2 0.35–2500 µM 0.11 µm [143]
Voltammetry
IL/Chit/GCE
Cu/rGO-BP Glucose Electrochemical 0.1–2 mM 11 µm [144]
0.0001–100.0 ng
Cu2 O@CeO2 -Au PSA Amperometric 0.03 pg mL−1 [210]
mL−1
Ni/Cu MOF Glucose FET 1 µM–20 mM 0.51 µM [147]
NiO/PANINS Glucose Amperometric 1–3000 µM 0.06 µM [211]
NiO@Au Lactic acid Electrochemical 100.0 µM–0.5 M 11.6 µM [212]
Electrochemical
Co3 O4 NCs Glutamate 10–600 µM 10 µM [150]
chip
Photo-
Co3 O4 -Au miRNA-141 1 pM–50 nM 0.2 pM [152]
electricalchemical
MnO-Mn3 O4 @rGO H2 O2 Impedimetric 0.004–17 mM 0.1 µM [213]
MnO2 NFs Salmonella Impedimetric 3.0 × 101 –3.0 × 106 19 CFU mL−1 [214]
Fe2 O3 /NiO/Mn2 O3
Folic acid Electrochemical 0.1 nM-0.01 mM 96.89 ± 4.85 pM [215]
NPs
Cyclic
ZnO-rGO Dopamine 0.1–1500 pM 8.75 ± 0.64 pM [216]
Voltammetric
ZnO NRs Phosphate FET 0.1 µM–7.0 mM 0.5 mM [180]
Sensors 2021, 21, 1109 26 of 35

Table 2. Cont.

Nanomaterial Analyte Transducer Linear Range Detection Limit References

ZnO NFs Amyloid Optical 2–20 µL 2.76 µg [217]
Ca/Al-ZnO NPs CO2 Semiconductor 0.25–5 RH% 200 ppm [218]
0.2 ×10−6 –
Cr doped SnO2 NPs Riboflavin Voltammetric 107 nM [219]
1.0 × 10−4 M
TiO2 /APTES glucose Impedimetric 50–1000 µmol 24 µmol [220]
TiO2 NTs Asulam photoelectrochemical 0.02–2.0 ng mL−1 4.1 pg mL−1 [221]
MoO3 @RGO Breast cancer Electrochemical 0.001–500 ng mL−1 0.001 ng mL−1 [222]
Graphene QDs Cu2+ Electrochemical 0.015–8.775 µM 1.34 nM [168]
0.1 pgmL−1 –1000
Graphene QDs Lung cancer+ Fluorescence 0.09 pg mL−1 [169]
ng mL−1
CdTe/CdS//ZnS
8.0 × 10−9 –
core/shell/shell l-ascorbic acid Fluorescence 1.8 × 10−9 M [170]
1.0 × 10−7 M
QDs
NSET
Tumor
amptamer@Fe3 O4 @GOD Magnetic fluorescence 2–64 nM 1.19 nM [171]
cell(EpCAM)
and MoS2
Au
NPs@PDA@CuInZnS P53 gene Electrochemiluminescenece 0.1–15 nmol L−1 0.03 nmol L−1 [223]
QDs
CaM/SiNW-FETs Protein FET 10−8 –10−6 M 7 nM [224]
Si NWs Dengue virus FET 1 µM–10 fM 2.0 fM [176]
ZnO NRs Phosphate FET 0.1 µM–7.0 mM 0.5 mM [180]
1.0 ×10−13 –1.0× 4.03 ×
G/Au NR/PT HPV DNA Electrochemical [225]
10−10 m L−1 10−14 m L−1
Amperometric 20–160 µM5–
Graphene-Au NRs NADHEthanol 6 µM1.5 µM [226]
Voltammetric 377 µM
LAC-CNTs-SPCE Para-cresol Electrochemical 0.2–25 ppm 0.05 ppm [227]
Co3 O4 -CNT/TiO2 Glucose Photoelectrochemical 0–4 mM 0.16 µM [228]
CNT thin-film Thin film transistor 1.6 ×10−4 –
DNA 0.88 µg L−1 [229]
transistor (TFT) (TFT) 5 µmol L−1
GQDs-MWCNTs Dopamine Electrochemical 0.005–100.0 µM 0.87 nM [230]
CNT/Au NPs Choline Amperometric 0.05–0.8 mM 15 µM [231]
PAMAM dendrimer DENV 2E Optical fiber 0.1 pM–1 µM 19.53 nm nM−1 [194]
SAM/NH2 rGO/PAMAM DENV 2E SPR 0.08 pM–0.5 pM 0.08 pM [195]

5. Conclusions
In this review article, we have discussed types and mechanisms of biosensors based
on receptors (enzymes, antibodies, whole-cell, and aptamers), transducers (electrochemical,
electronic, optical, gravimetric, and acoustic), and nanomaterials (gold NPs, Ag NPs, Pt
NPs, Pd NPs, NWs, NRs, CNTs, QDs, and dendrimers). Biosensors offer versatile applica-
tions in the fields of engineering and technology, medicine and biomedical, toxicology and
ecotoxicology, food safety monitoring, drug delivery, and disease progression. With the ap-
plication of NMs in biosensors, we have witnessed rapid growth in biosensing technology
is witnessed in the recent decade. This is because of the employment of new biorecogni-
tion elements and transducers, progress in miniaturization, design, and manufacture of
nanostructured devices at the micro-level, and new synthesis techniques of NMs, all of
Sensors 2021, 21, 1109 27 of 35

which bring together the life and physical scientists and engineering and technology. The
sensing technology has become more versatile, robust, and dynamic with the induction of
nanomaterials. The transduction mechanism has been improved significantly (like greater
sensitivity, faster detection, shorter response time, and reproducibility) by using different
nanomaterials (such as NPs, NRs, NWs, CNTs, QDs, and dendrimers) that each has differ-
ent characteristics within biosensors. Though there is considerable improvement in the use
of nanostructured materials in biosensors applications, there are few limitations, which hin-
der these applications for the next level. For instance, lack of selectivity remains a setback
for the CNT-based gas sensors, hampering its usage in CNT-based devices. However, this
hurdle can be overcome by coupling CNTs with other materials. The other issues in these
sensors include (i) the sustainability of nanostructures in sensor applications, which have
been insufficiently investigated, (ii) the fabrication of nanostructures, and (iii) the toxicity,
which changes according to the physical properties of the material type. These issues
should be investigated and addressed while expanding new nanostructured materials
for their use in biosensors. Most nanobiosensor devices used in biomedical applications
require a large sample for detection, which may lead to false-positive or false-negative
results. Very few biosensors have attained commercial success at the global level, apart
from electrochemical glucose sensors and lateral flow pregnancy tests. There is also a need
for making nanostructure-based biosensors at an affordable cost that give rapid results
with accuracy and are user-friendly. For example, nanomaterials should be integrated
with a tiny biochip (lab-on-chip) for sample handling and analysis for multiplexed clinical
diagnosis. More research should be done in this area and we expect the ongoing academic
research to be realized into commercially viable prototypes by industries in near future.

Author Contributions: V.N.: Conceptualization, investigation, writing the original draft. N.L.: Con-
ceptualization, supervision, writing, review and editing the original draft. Both authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by the National Research Foundation of Korea, 2017M3A9G5082642
and 2019R1A2C1008021.
Conflicts of Interest: The author declares no conflict of interest.

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