Biosensors

Preface to the course contents

The course on Biosensors is designed to cater the undergraduate and postgraduate

students of Biological sciences, medical sciences, computational sciences,
Bioinformatics, Bioengineering, Biophysics, Biotechnology and electronic engineers
dealing with the designing and fabrication of Biosensors. The course is equally good for
the researchers, who wants to initiate their career in the advance field of biosensor
Technology. To compete with the internationally offered online courses, exceptionally
novel contents from the basic and applied from the core subjects like Chemistry,
Biochemistry, Physics, Bioengineering, Genetic Engineering, Computational sciences is
included to rationalize the subject. The latest and advance knowledge on the designing
and fabrication of novel Biosensors in included in the course contents. The course is
comprised of 185 modules, that can be divided into three main Sections, covering the
basic, advance and application content of the subject. The syllabi covers approximately
47% basic content, 37% advance content and 16% content contains the applications
of the subject. Thus is syllabi is designed to produce graduate with strong foundation
of the subject, built on basic concepts with a combination of advancement and direct
applications.

Section I is comprised of chapter 1-7, constituting module 1- 85, focused on the basic
concepts that are involved in the multidisciplinary subject of Biosensor.

Section II comprised of Chapters 8- 14 constituting modules 86- 155 (total 70 modules)

, based on the advancement of the multi disciplinary subject of Biosensor.

Third section of the contents is formed on chapter 15-18 comprising of module No.
156-185 (30 modules).

In the preparation of the course the reference book, text books, latest research articles
and internet resources are used. The course is furnished with almost all the requisite
that make it popular among the students and researchers.
 Chapter 1. Introduction to Biosensors (Modules 1-13)

Module No. Title

1. Definition, Explanation with an example

2. Background Analyte

3. Components of Biosensor

4. Working Principle

5. Characteristics

6. Economics and Impact

7. Applications of Biosensors

8. Bioreceptors and their Types in a living system

9. Proteins ( Enzyme and Antibody)as Bioreceptors

10. Mechanism of Action of Enzyme

11. Antibody as Bioreceptor

12. Nucleic Acids as Bioreceptors

13. Cells, Virus and Tissues as Bioreceptors

The learning outcomes

1. Students will be able to understand the basic concept of the development of Biosensor
and working.
2. Students will be able to understand the definition, as per IUPAC union.
3. Students will be able to identify the role of Physical and Biological Components of a
Biosensor.
4. Students will be able to explain the types and significance of Biological component.
5. Students will be able to define the role of core sciences in the development of
Biosensor Technology.
6. Students will be able to identify the impact of Biosensor Technology on Economy.
 Chapter 1.

1.Definition, Explanation with an example

An introduction to Biosensors

Biosensor is a device used for the detection of a target molecule that binds with a
biological component, attached with a detector and a display unit. Biosensors offer
high quality output due to their accuracy, precision, specificity and sensitivity within
affordable prices. They are ideal for providing low cost, easy, quick and portable
solutions.

Definition of Biosensors

As IUPAC, a self-contained integrated device which is capable of providing specific

quantitative or semi quantitative and analytical information using a biological
recognition element (BRE) which is in direct special contact with a transducer element.
According to the definition, there are five qualities of a biosensor:

1. Self-contained integrated device

2. Can provide specific analytical information
3. Using a biological molecule (BRE)
4. Direct attachment
5. Transducer element (Detector + Display unit)

Biosensor = Bioreceptor + transducer

Science of biosensor is multidisciplinary as it involves chemistry, biochemistry,

physics and engineering. It deals with the translation of lab based technologies into
commercially viable products for benefit of human being. The methods of biosensors
are based on basic chemistry method for the detection / estimation of specific
molecules. Common example of a biosensor is a glucometer. This is a device for
detecting glucose in a drop of blood for diabetics. The glucometer contain enzyme
glucose oxidase. The enzyme attached with the glucose in the blood to release
electrons. These electrons are converted into electric current; the current generated is
proportional to the amount of glucose present. It is extremely sensitive and can
measure the glucose concentration in single drop of blood and display result within 20
seconds. Biosensor works on the same principle as in the lab including colorimetric,
calorimetric, potentiometric, volumetric and spectrophotometric. There are two
working platforms in biosensors, external and internal. External platform is wet
whereas internal platform is dry processing.
 External platform is wet platform that is for sample application that is wet
Biorecognition of Analyte. Internal plat is dry platform that is for transducer and data
processing.

Advantages:

Following are the main advantages of the Biosensors. Biosensor devices are

1. Sensitive
2. Small
3. Rapid response
4. Safe to use
5. Provide accurate results and
6. Can be mass produced.

Biosensors are first produced in laboratory and later they are integrated for
miniaturization.

Etymology

The term biosensor is the short for the term biological sensor. The word
biological attributes to biological molecules such as enzymes, proteins, DNA, RNA,
antibody, cell and virus etc. The term sensor is a combination of transducer and a
display unit. Transducer is a physical device which is used to convert signal from one
form of energy to another form of energy and transable is a readable source. The signal
is then displayed on a display unit for the end user which can be easily understood.

Example.

A blood glucose meter is a small, portable machine that's used to measure how
much glucose (a type of sugar) is in the blood (also known as the blood glucose level).
People with diabetes often use a blood glucose meter to help them manage their
condition. It has a strip on which blood sample is applied and then it is inserted into a
small box that contains a complete system for the estimation of blood glucose. The
strip contains the biological component that is an enzyme of the Biosensor and a part
of physical component that is an electrode. The hand held box contains different
physical components and a display unit.
 Fig shows a glucometer complete assembly with a strip and a box. Insertion of strip in
the glucometer for blood glucose estimation. A test strip os shown with
electrodes.

Module No.2. Analyte Background

Analyte

The term analyte is used for a target molecule that is required to be detected at
analytical level. For example, in a glucometer, glucose molecule is an analyte. For the
test of alcoholrythem, the molecule alcohol is an analyte. The type of analyte is
dependent on the nature of the biosensor. The nature of interaction between the
analyte and the biological molecules used in biosensor is of two types.

1. The analyte may be converted into a new chemical molecule by enzymes such
biosensors are called catalytic biosensors.
2. The analyte may simply bind to the biological molecule (antibody nucleic acid). The
biosensors are known as affinity sensors.

Analyte Sample
Glucose Blood

Lactic Acid Sweat

Uric Acid Mouth

Cortisol Foot

Bacteria Teeth

3.
Science of Biosensors
Science of biosensor is multidisciplinary as it involves chemistry, biochemistry, physics
and engineering. It deals with the translation of lab based technologies into
commercially viable products for benefit of human being. The methods of biosensors
are based on basic chemistry method for the detection / estimation of specific
molecules. Biosensors are designed with an objective to provide accurate and rapid
 response. An ideally designed Biosensor is Sensitive, Portable, Rapid, Safe to use,
Provide accurate results and Can be produced in bulk.

Biosensor technology is based on the translation of laboratory based research

methods into via able commercial products for the benefit of mankind. Biosensors are
designed with an objective to provide accurate, sensitive and provide rapid response.
Biosensors are Safe to use and Can be mass produced. The Biosensor technology is a
multidisciplinary science. Biosensors offer high quality output due to their accuracy,
precision, specificity and sensitivity within affordable prices. Biosensors involve cross
functional interaction among disciplines like electronics, electrical engineering etc.

Chemistry

It is the basic subject that is involved in the development of Biosensor

technology. All the methods employed for the detection of an analyte (target
molecule) are standard laboratory methods for the detection of the molecule. A
combination of different branches of chemistry named as Dry chemistry are being
used in clinical chemistry for more than fifty years now. Application of lab based test
method in a Biosensor is by the virtue of Dry chemistry, where we do not have to use
wet reagents separately. The principle of dry chemistry estimations/detections is
based on spectrophotometry (colour change).

Glucose molecule shown in Fir It is estimated by a glucometer.

Dry chemistry systems are widely used for routine blood chemistry and
microbial testing etc. The advantage of dry chemistry technology is that it eliminates
the need for reagent preparation and the preceding steps. Each test unit contains all

the reagents and reactants necessary to perform analysis. Dipstick is a good example of dry
chemistry application. The support material for the dipstick is based on plastic and cellulose.
Physics

The subject of Physics is essential for the detection of signals generated by the
Biosensor for the detection of target molecule. Physics provide three essential metrics
for biosensors; detection time, sensitivity and selectivity. Physics of detection time
allows us to organize a biosensor in a systematic way. It also allows to design principle,
interpretation and measuring of results.

Biochemistry

Biochemistry is the Chemistry of Biological Molecules and in a Biosensor Biological

molecules are employed as Bio recognition elements. The molecules are selected on
the binding characteristics of the biomolecules such as proteins.

Three-D structure of an enzyme acetyl choline esterase binding with thw target
molecule. The target molecule is shown in green color.

Engineering and Microelectronics

Engineering is the application of knowledge in the form of science, mathematics and

empirical evidence to the innovation and design. Engineering skills are required to
develop a biosensor by combining all the relevant knowledge gathered from the
relevant subjects such as Chemistry, Biochemistry, Physics etc. Microelctronics is the
branch of electronics which involves with the micro process technology. This is the
manufacturing of smaller electronic devices with full/ improved functionality.
Manufacturing offhand held set to replace old fashioned telephone set is a good
example of miniaturization, to begin with.
Fig shows an old fashioned telephone set and a hand held set. Small size hand hels phoneset
has more functions and performs better then first generation phone sets.

Module No. 3 Components of Biosensors

Biological Recognition Element (Bioreceptor)

Any biological molecule could be used in a biosensor which has strong / specific binding affinity
for the analyte. Such as DNA, enzyme, antibody whole cell.For example, for glucose, any
enzyme using glucose substrate is an ideal bioreceptor. When bioreceptor combined with a
target molecule, it produces a signal that may be in the form of production of new chemical,
a flow of electron, a change in pH, a change in Mass, emission of light or release of heat. The
signal is converted by an appropriate transducer to detect and respond the change.

Fig shows a DNA molecule, Antibody and a bacterial cell, that can be used as a bioreceptor
element.

Transducer

The transducer is an element that converts one form of energy into another. In a biosensor,
the role of transducer is to convert the biorecognition event (formation of new molecules,
release of electron, change in pH, release of heat, change in mass, change in colour etc.) into
a measurable signal. This process of energy conversion is known as signalization. Most
transducer produce either optical or electrical signals that are usually proportional to the
amount of analyte that bioreceptor interaction.

Electronics

This is a part of biosensor that processes the transduce signals and prepare is for display. It is
consists of complex electronic circuits that performs signal conditioning such as amplifications
and conversion of the signals from analogue into the digital form. The processed signals are
then quantified by the display units of the biosensors.

Display

The display unit often consists of combination of hardware and software that generates results
of the biosensors in a user friendly manner. The display unit exhibits the output signals. The
display consists of a user interpretation system such as liquid crystal display (LCD) of a
computer or a direct printer that generates number or curves understandable by the user.
Biosensors are designed using the standard laboratory technique and methods which provide
accurate results. A laboratory biosensor is an expensive, needs specialize user and processing
time. These laboratory biosensors are designed for translation into commercial biosensors.

Module No. 4 Working Principle

Thereare five steps in the working of Biosensor. There are five main steps

1. Biorecognition
2. Signal Generation
3. Signal Recognition
4. Signal Conversion
5. Display of Information

When a bioreceptor combines with a target molecule, it produces a signal. The signal may be
in the form of production of new chemical, a flow of electron, a change in pH, a change in
Mass, emission of light or release of heat. In a Biosensor signal is being converted by an
appropriate transducer to detect and respond the change.
The working of Biosensor is based on the working of living systems. For example eye is a good
example of Biosensing applications. The lens is formed from two protein superfamilies, the α-
and βγ-crystallins that act as a Bioreceptor element. The light and the rods and cons in the
retina at the back of the eye are transducer. They respond to light by producing the impulse,
which is an electrical event. The electrical signal is then readout by our brain. Another
example, taste buds on our tongue is another good example of a living system biosensor.
These taste buds detect different tastes including sweat, salt, bitter and salty at different
positions of the tongue

Example Glucometer

Blood sample.

Analyte is a glucose molecule.

B.R. E is an Enzyme.

Biorecognition signal is the Generation of electrons

Detector is an Electrode

Physical units composed of software and hardware with a powerful

processor.

Biological recognition element. Glucose oxidase (Enzyme Commission No. 1.1.3.4)

1. It is obtained from the fungus, Aspergillus niger.

2. Molecular weight of about 160kD. It is a dimer
3. Enzyme binds with glucose.
 Fig shows Catalysis of glucose molecule by the enzyme glucose oxidase.

1. Wet Platform
2. The disposable Test strip is a wet platform.
3. Blood travels through Microfluidic channel.
4. Glucose oxidase is immobilized on the test strip.
5. Electrodes are fixed on strip and in contact with the enzyme .

Dry Platform
The Dry platform consists of
a software and a hardware .
It has a powerful processing unit with a
1) Voltage converter
2) Amplifier
3) Filter
4) Display unit

Working of Glucometer

When blood is placed on the test strip, a chemical reaction takes place on working electrode.

a) An electrical current is generated.

b) Current is directly proportional to the concentration of glucose.
c) Information is displayed in mg/dl mmol/l.

Example. Working of Glucometer.

There are two platforms in Glucometer. Glucose strip is a wet platform. There are three
electrodes Impregnated with enzyme a) Counter electrode b) Working electrode c)
Reference Electrode. Blood sample when placed on test strip it travels through Microfluidic
channel electrode. A simple chemical reaction takes place and an electrical current is
generated, which is directly proportional to the concentration of glucose. Chemical reactions
take place and on the working electrode. To pick the signal Strip is immediately inserted into
the glucometer.
The dry platform is equipped with powerful processor, Current to voltage inverter, an
Amplifier, a filter and a display Unit. The result is displayed accurately, Within 5-60 seconds in
the required units. There is no pretreatment of blood sample needed.

Module 5. Charecteristics of a Biosensor

A successful commercially available biosensor is fabricated after passing
through the following steps including Bioreceptor engineering, transducer nano
engineering, reduction invariability, lowering manufacturing cost and proper packing
of a biosensor. A successfully designed biosensor must have the following properties:

Sensitivity:

It is the most important performance element in a biosensor. The measurement of

target analyte and conversion into usable signal in a real time detection manner. The
minimum amount of analyte that can be detected by a biosensor is defined as its limits
of detection (LOD) or sensitivity.

Fig shows the sensitivity of the Coomassie blue dye for protein test. An increase in the
blue co;our is observed with the increase in protein concentration.

In a number of medical and environmental monitoring applications, a biosensor is

required to detect analyte concentration as lows ng/ml or even fg/ml to confirm the
presence of the traces of Analyte in a sample. For example , a prostate-specific antigen
(PSA) concentration of 4ng/ml in blood is associated with prostate cancer for which
doctor suggest biopsy test. Therefore, sensitivity is consider to be an important
property of a biosensor.
Linearity

Linearity in the attribute that show the accuracy of the measured response (For
a set of measurement with different concentration of analyte) to a straight line,
Mathematically represented as y=mc. C is the concentration of analyte y is the output
signal and m is the sensitivity of the bioreceptor. Linearity of the biosensor can be
associated resolution of the biosenors and range of anlayte concentration under test.

The resolution of biosensor is defined as the smallest change in the concentration of

the analyte that is required to bring a change in the response of biosensor. Depending
upon the application, a good resolution is required because most of the biosensor can
detect analyte but also measures the concentration of the analyte over a wide working
range.

Graph shows the linearity for protein concentration in the range of 0-15 microgram of
protein in the sample. Range of the sensor is the maximum and minimum values of
applied parameter that can be measured.

Example

A Glucometer can measure 20-400 mg/dl. Normal physiological range of blood

glucose level is 70-140 mg/dl. Another term associated with linearity is linear range
which defined as the range of analyte concentration for which the biosensors response
changes linearly with the concentration

Response times
 The times a Biosensor takes to produce a result. A glucometer takes 3 to 60 seconds
to display the results for different models..

Selectivity

Selectivity is the most important feature of the biosensors. It is an ability of a

bioreceptor to detect a specific analyte in a sample containing other compounds and
contaminants as well. The best example of selectivity is depicted by the interaction of
a antigen with the antibody. Classically, antibody act as a bioreceptor and are
immobilize on the surface of transducer. A solution (usually buffer containing salt)
containing the antigen is then expose to the transducer, where antibodies interact only
the with specific antigen.

To construct a biosensor, selectivity is the main consideration when choosing a

bioreceptor.

1. Long term stability (Not subject to fowling, poisoning or oxide formation that interfare
with signals.
2. Dynamic response (Rapid response to variations in analyte concentration)
3. Biocompatibility
4. Commercial demand

Stability

Stability in the degree of susceptibility to ambient disturbances in an around the

biosensoring system. These disturbances can call a drift in the output signals of
biosensor under measurement. It can cause an error in the measured concentration
and can affect the precision and accuracy of the biosensor. Stability is the most feature
application where a biosensor require a long incubation steps or continuous
monitoring. Factors effecting stability

Fouling is accumulation of living or non living materials.

1) Temperature
2) Humidity
3) pH
4) Altitude, others .
 The response of transducer and electronics can be temperature-sensitive, which
may influence the stability of a biosensor. Therefore, appropriate turning of electronics
is required to ensure a stable response of the sensor.

Reproducibility

Reproducibility is the ability of the biosensors to generate identical response for

the duplicate experimental setup. The reproducibility is the characterize by the
precision an accuracy of the transducer an electronics in a biosensor. Precision and
accuracy are related to reproducibility.

Precision is the ability of the sensor to provide alike results every time a sample is
measured. Accuracy indicates the sensor’s capacity to provide a mean value close to
the true value when a sample is measured more than once. Reproduce able signals
provide high reliability and robustness to the inference made on the response of a
biosensor.

5. Economics and Impact

Biosensors are commercial Products

1. The biosensors market is categorized as a growth market.

2. Currently, biosensors are used in more than 47 different end-user application.
3. The global biosensors market is anticipated to reach USD 21.2 billion by 2020/

Biosensors are highly popular commercial Products because they are

1. High Sensitive
2. Specificity
3. Fast readout times
4. No or simple sample pretreatment
5. User friendly
6. Portable
7. Economical
8. Selfonfiguring biosensor

Two extremely popular products of Biosensor Technology are

1. Blood Glucose meterIt estimates Blood Glucose level Quantitatively and is very
useful for a diabetic patient
2. Pregnancy test kit it gives a clear Yes/No answer for pregnancy test.
1. Biosensor market is mainly a health care market. As 90% of market share belongs
to health care.
2. The aim of healthcare market is to benefit the consumers that is Public, current, or
future, patients.
3. The objective is to improve the population's health by encouraging the value of a
person's health.

Fig shows the main interest of the Biosensor technology. And its growth.

Biosensor Market for Self Monitoring Glucometer

1. First commercial glucose biosensor was introduced in 1973.
2. It was not accepted initially by patients and physicians.
3. Growth 2013-2018 0.7%.
4. Total Revenue in 2018 more than $ 4 Billion
Demand from a growing number of diabetics has sustained growth
Graph shows the growth of Biosensor market.

Drivers for the market

A major growth area for biosensors is
i. point-of-care (POC) testing
Ii diagnostics
The home diagnostic market continues to grow.
Home diagnostic market (2009–2016) revenues are reported growth rate for more
than 12.4%.
POC testing
1. Point-of-care testing is undertaken close to the patient.
2. The specimen is obtained, the test performed, and the result reviewed where care
is provided.
3. It involves testing by non-laboratory personnel using specialized technologies
adapted for near-patient bedside applications.
Fig shows the point of care testing in Biosensor technology.

Market Pull
The term 'Market Pull', refers to the need/requirement for a new product or a solution
to a problem, which comes from the market place. The need is identified by potential
customers or market research. A product or a range of products are developed, to solve
the original need.
Following are drivers for the market pull of Biosensor products.
1) Glucose sensors
2) Pathogen detection
3) EKG Sensor measures cardiac electrical potential waveforms (voltages
produced during the contraction of the heart).
This is currently the largest and most profitable area
 for the development and commercialization of biosensors
Biosensor technology has a huge impact on academia. There ia a huge increase in the
publications on Biosensor starting from 1980- 2016.

7. Applications of Biosensors
Applications of Biosensors

Biosensors have wide range of applications that aim to improve the quality of life.
The largest application within the field of medicines. Biosensors are particularly used
for clinical diagnosis. Use of biosensors reduces the risk of errors of diagnosis and time
and cost as well. Less time and minimum expertise are required. As the results are
obtained in minimum time, treatment can be started immediately. There is also less
chance of sample of being mishandled, lost or contaminated. Home diagnosis kits are
available for people who like to have this facility at home (Glucometer, pregnancy test
kits etc.). The main application of biosensors are

1. They acts as an Indicators for a disease. There are several cancer biomarkers are
identified by biosensors.
2. In Industry Process monitoring and control could be checked by using biosensors.
3. In battle field, monitoring of poisonous gases and nerve agents.
4. Detection of number of chemicals that are biological agents and are considered to be
toxic materials for defense interests and security.
5. Biosensors are used for the environmental monitoring including epidemiology of
diseases or pollution. Advantages of Environmental Biosensor
6. Environmental Biosensor provide benefits over the conventional analytical techniques.
1. Portability
2. Work on-site
3. Measuring pollutants
in complex matrices
4. with minimal sample
5. No pretreatment

Fig shows a the application of Environmental Biosensor in field.

Agriculture
Biosensors are widely used for
1. Testing the pesticides
2. Crop deterioration
3. Gases detection
4. Ripening of fruits
5. pH of Soil
 Fig shows a small size device is hooked up to a smartphone app that can allow to scan
a piece of fruit or a vegetable to determine the pesticide levels.
Food Industry
is an increasing demand for biosensors in the food industry to examine food quality
and Detection of pathogens.
Biosensor that can accurately and quickly determine if food is contaminated or not.,
such as salmonella and listeria.

Fig shows a biosensor for testing the pathogen in food.

Enzyme based biosensor for security surveillance

A system that rapidly detects both explosives and nerve agents,is developed, providing a
simple yes-no response. The technique could replace two time-consuming tests that are
currently used to assess these threats. The explosives and nerve gases are fed through an
enzyme-based logic gate system, in which the depletion of hydrogen peroxide detects their
presence.

Fig shows an enzyme based biosensor for the detection of security survillence using an
enzyme based biosensor.
 Module No, 8.Bioreceptors and their Types in a living system
There are five general senses. Receptors are located on sense organs. Receptors are specific
to stimuli. A sensory receptor is a specialized cell Ionic movement is involved in generating
a CNS response .

Olfaction sense of smell

Olfactory receptor, also called smell receptor, protein capable of binding odour molecules
that plays a central role in the sense of smell (olfaction). These receptors are common to
arthropods, terrestrial vertebrates, fish, and other animals. The sense of smell is closely
linked with memory, probably more so than any of our other senses. Those with full olfactory
function may be able to think of smells that evoke particular memories; the scent of an
orchard in blossom conjuring up recollections of a childhood picnic, for example. Sense of
smell is provided by paired olfactory organs on either side of the nasal septum. 10 – 20
million olfactory receptors containing cilia are packed in nasal area.

Fig shows the mechanism of olfaction and its components.

Odorant + receptor leads to activation of enzymatic mechanism involving ATP, for opening
of sodium calcium gate channels leading to Na+ influx. During the process of smell detection
influx of cations, including both Na+ and Ca2+, lead to depolarization of the cell membrane
strongest and most dominant sense is the sense of smell. Olfactory neurons maintain a very
high intracellular Cl− concentration, perhaps as high as 125 mm, so that the driving force for
Cl− ions is outward and, therefore, further depolarizes the cell. Olfaction is a
chemoreception that, through the sensory olfactory system, forms the perception of smell.
Olfaction has many purposes, such as the detection of hazards, pheromones, and food.
Olfaction occurs when odorants bind to specific sites on olfactory receptors located in the
nasal cavity. In terrestrial vertebrates, including humans, the receptors are located on
olfactory receptor cells, which are present in very large numbers (millions) and are clustered
within a small area in the back of the nasal cavity, forming an olfactory epithelium.

Gustation (Sense of taste)

It Provides information about the taste foods and liquids. Gustatory receptors are called
taste buds distributed over Tongue superior surface ,pharynx and larynx. There are about
3000 taste buds in an human adult.

Taste Buds - The taste bud is composed of about 50 modified epithelial cells, some of which
are supporting cells called sustentacular cells and others of which are taste cells. - The taste
cells are continually being replaced by mitotic division of surrounding epithelial cells. - The
outer tips of the taste cells are arranged around a minute taste pore.
Fig shows the gustation and its components and mechanism.

Adults have 3000 to 10,000 taste buds, and children have a few more. - Beyond the age of
45 years, many taste buds degenerate, causing the taste sensation to become progressively
decreased in old age. A single taste buds show that each taste bud usually responds mostly
to one of the five primary taste stimuli when the taste substance is in low concentration. It
may be noted that at high concentration, most buds can be excited by two or more of the
primary taste stimuli. Mechanism of gustation for different tastes is discussed in lecture.

Vision
The sense through which a person or animal becomes aware of light Color, etc using the
eyes. Eye Receptors (Rods and Cones) transfer light energy into nerve impulses and transmit
the information to the cortex of the brain's occipital lobe. Flow of Na ions is essential for
generation of the response.
Fig shows the eye, cone cells and rods.

Transduction is the process through which energy from environmental stimuli is converted
to neural activity. The retina contains three different cell layers: photoreceptor layer, bipolar
cell layer and ganglion cell layer. The photoreceptor layer where transduction occurs is
farthest from the lens. It contains photoreceptors with different sensitivities call rods and
cones . The cones are responsible for color perception and are of three distinct types labelled
red, green and blue. Rods, are responsible for the perception of objects in low
light.Photoreceptors contain within them a special chemical called a photopigment, which
is embedded in the membrane of the lamellae; a single human rod contains approximately
10 million of them. The photopigment molecules consist of two parts: an opsin (a protein)
and retinal (a lipid).

Hearing

Hearing, or auditory perception, is the ability to perceive sounds by detecting vibrations,

changes in the Hair cells are the sensory receptors of the auditory system in ear. The
receptor neurons are topped by hair bundles that move in response to the vibrations of
sound. Hair cells transform the mechanical forces of sound into the electrical impulses of
the surrounding medium through time, through an organ such as the ear. During the
phenomenon of hearing The back and forth mechanical movements of the hair alternately
opens ans closes ion channels for potassium and calcium ions in the hair which resulted in
the alternate depolarization hyperpolarization leading to potential changes, at the same
frequency as the original sound

Fig shows the hearing process in ear. Movement of hair and ions inside ear is also shown

Module No. 9. Enzyme

Bioreceptors

Proteins are polymers of amino acids join through peptide bonds. Enzymes and
antibodies are functional proteins. Enzymes have molecular structure while antigens
have a unique Y shape architect. The enzymes have marvelous binding affinity for the
substrate. Biomolecules are chosen depending upon the analyte to detect with strong
stability at high temperature, pH, hydrophobicity and oxidizing environment. Enzymes
as Bioreceptor

Enzymes can be divided into three different types, depending on the detection of
molecules, as signal.

a) End product.
b) Side product
c) Detection of electron
d) Inhibition of enzyme

Enzyme immobilization on different interfaces and possible effects on the enzyme

conformation.

1. High charge density or a hydrophobic surface are possible causes of enzyme

conformational changes and inactivation.

2. Enzyme co-immobilized with hydrophilic polymers or tethered can reduce unfolding

and inactivating support–enzyme interactions.
3. Incorrect orientation and multilayer formation may cause reduction of specific
activity.

The high specificity allows to at the larger amount of enzyme on small surface area.
Generally, purified enzymes are required to be loaded on a biosensor. While designing
a biosensor, the optimal conditions required by enzymes are to be considered. Purified
enzymes are expensive and are easily denatured. To overcome this whole cell or tissues
containing the enzymes are also loaded on biosensors. Viruses are also used as
biological recognition element due to their stability at wide range of pH and
temperature. Similarly, antibody – antigen are frequently used molecule due to their
excellent specificity. The antibody bioreceptors are used on one due to strong binding
nature.

Detection of Pesticides by Enzymes

There are several enzymes used for the detection of Pesticides by Biosensor including.

1) Peroxidase
2) Alkaline Phosphatase
3) Acetylcholine esterase

Using a variety of sensors

i. Optical
ii. Potentiometric and others

Enzymes Used in Glucometer

There are several enzymes that use glucose as a substrate. There are two types of
enzymes that are used in Glucometer for the detection of glucose.

a) Glucose Oxidase
b) Glucose Dehydrogenase

Enzymes Mechanism/Classification

Enzymes mechanism and Biosensors

Reaction depends on the physical and chemical structures of the enzyme and the
substrate. Different classes of Enzymes display specific mechanism of action. Designing
of biosensor is dependent on the mechanism of enzyme action. Enzymes can be
classified by the kind of chemical reaction catalyzed.

I. Addition or removal of water A. Hydrolases - these include esterases,

carbohydrases, nucleases, deaminases, amidases, and proteases B. Hydrases such
as fumarase, enolase, aconitase and carbonic anhydrase

Fig shows Mechanism Hydrolase

Fig shows Mechanism of Hydrase.

II. Transfer of electrons A. Oxidases B. Dehydrogenases.

Enzyme catalyze oxidation-reduction reactions where electrons are transferred.
Examples of their sub classes are

1. Oxidases 2. Dehydrogenase 3. Oxygenase 4. Peroxidase

Application in Biosensor:, Release of electrons by glucose oxidase is detected by the

Biosensor that is a step in mechanism of action

III. Transfer of a radical A. Transglycosidases - of monosaccharides B.

Transphosphorylases and phosphomutases - of a phosphate group C. Transaminases -
of amino group D. Transmethylases - of a methyl group E. Transacetylases - of an acetyl
group

IV. Splitting or forming a C-C bond A. Desmolases

V. Changing geometry or structure of a molecule A. Isomerases

Isomerase enzymes catalyze reactions that transfer functional groups within a

molecule so that isomeric forms are produced.

Example.
VI. Joining two molecules through hydrolysis of pyrophosphate bond in ATP or other
tri-phosphate A. Ligases

The enzyme – substrate binding is highly specific to shape and similar to lock and key
mechanism. In a glucose sensor, and an enzyme glucose oxidase is used to capture and
detect the glucose molecule. Glucose oxidase enzymes binds to glucose only and
oxidizes into another chemical called glucono lactone and eventually gluconic acid.

Module No. 11. Antibody as Bioreceptor

An antibody is also very popular by receptor when a foreign molecules invades the
body, the immune system recognizes and memorizes the molecules shape by creating
an antibody molecule that is a perfectly engineered to be invaders. In is Y shape
molecules and called immunoglobulin. It consists of two heavy chain (H-chain) and light
chain. The antibody has a constant and a variable part, the variable part sometimes act
like a key which specifically fits into a lock. Human body can produce the estimated
100 million different antibodies, recognizing all kinds of foreign substances. It does this
by shuffling different sections of part of the genes which produce the variable reagent
(Like building different models from basic set of shapes). An antibody can also
recognize and viral particle or a bacterial by recognizing the proteins on the surface.
Therefore, antibodies can be used as excellent bioreceptor for wide variety of protein
molecules as well as viruses and bacteria. Antibody as Bioreceptor

There are two types of antibodies. Monoclonal and Polyclonal.Monoclonal antibody

are produced by the progeny of a single B cell against single epitope.An epitope is five
to six amino acid long region.Polyclonal antibody are generated against multiple
epitope.

Fig shows growth hormone molecule and antibody epitopes for antibody binding on
Immobilization of Antibody

Orientation of antibodies on the surface is important for sensitivity.

Covalent cross linking or oxidation of carbohydrate groups can be used to control the
interaction between specific reactive groups on the surface and on the antibody.

Fig shows the different orientations of antibody after immobilization. Most suitable
orientation is shown with green face.

Immuno biosesor with ELISA Assay

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique. It is
designed to detect and quantify substances like peptides, proteins, antibodies and
hormones. It is also known as enzyme immunoassay (EIA). ELISA immunosensors

In assay, the antigen is immobilized on to a solid surface. An antibody (grey) is

immobilized on multi well plate, to detect the antigen of interest. The antigen
(turquoise) recognizes the detection antibody (green)that is conjugated to biotin and
streptavidin-HRP, for signals.

Fig shows scheme for EIA.

Recombinant DNA Technology For Antibody Engineering

A library generate a library by PCR using spleen DNA can be used in recombinant DNA
technology based production. The DNA could be used for PCR The resultant PCR
products for one heavy chain and one light chain are cloned separately and expressed.
It is a protein with antibody like activity.

Module No. 12 Nucleic Acids as Bioreceptor

DNA or RNA are the Biorecognition elements in Nucleic acid Biosensors.The detection
methods are based on recognition of complementary nucleic acid sequence

a) Rapid
b) Simple and
c) Economical testing

for clinical, environmental, food analysis and other fields Nucleic can also serve as
good bioreceptors. These include specific sequences of deoxiribo (DNA) nucleic acid.
The genetic information is told in a huge storage of DNA or RNA in other words, it is
like a hard disc drive of a computer with lots of programmers stored in many different
places. The main difference is that genetic information is stored in four base system
consisting of adenine (A, adenine, T thiamine and C cytosine for DNA). For RNA it is
adnine A, U uracil, G Guanine and C cytosine. A always binds to T and G always binds
to C (for DNA). In case of RNA, A always binds to U and G always binds with C.
Therefore, a specific RNA sequence of these basis can be used to detect the existence
of their complimentary sequence.

Fig shows the working of DNA biosensor.

Nucleic Acids immobilization

The DNA fragments have to be immobilized in a way that retains their

1) Stability
2) Reactivity
3) Accessibility
4) to target analyte for hybridization.
5) Adsorption

DNA probes are immobilized via electrostatic adsorption between a negatively charged
phosphate group of DNAs on the positively charged surface
 ..

Fig shows the immobilization of DNA.

Hybridization and Biorecognition

1. DNA hybridization is the process of joining two single strands of complementary

DNA sequence and forming dsDNA.

2. Hybridization of complementary DNA base pairing is the basis for the biorecognition
process.

3. An electrical signal is produced when target DNA binds to the complementary

sequence.

. DNA molecules are sensed through hybridization because of the affinity of the single
stranded DNA complementary stand hybridization DNA/RNA based oligonucleotides
are known as probes. Probes are short nucleic acid sequences that recognize and
specifically binds to target nucleic acid.. The use of labeled DNA is away to detect target
molecule. The main disadvantage of using DNA is its susceptibility toward nucleases.

Hybridization of DNA in a biosensor.

Sensing Methods

Label based method

specific organic dyes, metal complexes and enzymes are used for hybridization
detection.

Examples

1. Methylene blue binds at minor groove of DNA.

2. Horseradish Peroxidase and alkaline phosphatase

3. Ferrocenyl naphthalene diimide (FND)

DNA Microarray:

A DNA microarray is a collection of microscopic DNA spots attached to a solid surface.

DNA microchips detect the presence of specific DNA sequences. It can be used as a
rapid indicator in clinical diagnostics, research and food safety testing.

DNA microarray in a Biosensor.

Moduel 13. Cells, Virus and Tissues as Bioreceptors

1. Cell-based biosensors use cells as Biological Receptor element

1) Prokaryotic (bacteria)
2) Eukaryotic
3) yeast
4) invertebrate
5) vertebrate.
 Certain cells that possesses strong affinity for a specific target can also be used as a
bioreceptor. Cells as Bioreceptor Cell-based biosensors use cells as BRE. They apply
the principles of cell-based assays by employing living cells for detection of different
analytes from

a) Environment
b) Food
c) Clinical
d) or other sources.

Immobilization of Cells

Entrapment is the ideal method to immobilize the cells/ tissues on support material. For
example

a) Cellulose based polymer

b) Chitosan
c) Polyacrylamide
d) Porous silica
e) Polyvinyl chloride based polymers

Application for detecting Toxins Bio-toxins

Cells report the cellular responses upon exposures to toxins under Physiological
conditions.

It reveals functional information about

i. mode of action
 ii. toxic potency

iii. bioavailability

iv. Target tissue or organ

Chapter 02
 Miniaturization and microsystems including sensing by optical
techniques, Field effect transistor, Ion Selective and Enzyme
sensitive Electrodes

Module No. Title

14. An introduction to miniaturization

15. Microsystems

16. Sensing techniques

17. Optical techniques

18. Transistors

19. Electrodes

20. Enzyme sensitive electrodes

21. Field effect transistor

22. Ion Selective Electrodes

23. Latest Research and Current Trends

Learning Outcome.

1. Students will be able to understand and explain the concept of miniaturization

2. Students will be able to rationalize the concept of Microsystems in the development of
Biosensors.
3. Students will have a clear idea about the Sensing techniques that are required in Biosensor
Technology.
4. Students will be able to learn about the basic concept required in developing an Optical
Biosensor.
5. Students are required to learn about the Transistors, that are among the basic tools in
Biosensor technology. Students will be able understand and explain the concept of
Transistor.
 6. Students will be able to revise the science/concept of electrodes in the light of Biosensor
Technology.
7. Students will learn and will be able to explain the principle of fabrication of Enzyme
sensitive electrodes and their types.
8. Students will be able to explain the design and working of the Field effect transistor
9. Students will be able to explain different types on Ion Selective Electrodes and their
applications
10. Students will learn the Latest Research and Current Trends in the miniaturization of
Biosensor and applications.

Module No. 14. Miniaturization

The process of making some thing very small using modern technology. All the objects have size
dependent properties. These physical properties change when the size of an object changes.
Examples of size dependent properties are Length, Width, Height, Volume, Mass.

Advantage.

Smaller objects perform faster simply because they have, less mass which leads to less inertia
(tendency of a body to resist acceleration). leads to products that perform quicker. smaller objects
need less energy to function. They are economical.

Scaling

Study involve in the operation of sizing is known as scaling. Some functional physical systems
either cannot be scaled down favorably, or cannot be scaled down at all. As the sizes reduce
different physical forces become more important less important depending on their nature.
These changes dictate how the device must be built. what forces it will use to operate. This is
called scaling.

Scaling Forces There are four types scaling of forces

S1. The force due to surface tension scales.

S2 The force due to electrostatics with constant field scales

S3 The force due to certain magnetic forces scales

S4 Gravitational forces scale.

Scaling Factors
 There are many factors involve in down sizing

1. Gravitational force 2. Strength 3. Speed 4. Heat capacity

5. Resistance 6. Surface tension 7. Inertia Others

Scaling laws

1. Scaling law are the laws that describes the variations of physical quantities with the size of the
system.
2. It uses use of dimensional analysis.
3. Scaling Laws allow to determine whether physical phenomena will be favorable or not.
4. These are simple observations about how physics works on different sizes.
5. These laws are valuable guide to what may work and what may not work when we start to design
the world of micro.

Types of Scaling Laws

There are two types of scaling laws

1. Scaling in Geometry: 2. Scaling of Phenomenological Behavior

Scaling of physical size of objects

● Volume (V) and surface (S) are two physical parameters that are frequently involved in machine
design.

● Volume leads to the mass and weight of device components.

● Volume relates to both mechanical and thermal inertia. Thermal inertia is a measure on how
fast we can heat or cool a solid. It is an important parameter in the design of a thermally actuated
device.

● Surface is related to pressure and fluid mechanics, heat absorption dissipation by a solid in
convective heat transfer.

When the physical quantity is to be miniaturized, the design engineer must weigh the magnitudes
of the possible consequences from the reduction on both the volume and surface of the particular
device.

That is Size reduction with Isometry (Geometry Similarity).

1. length dimension (l),

2. length dimension square (l2),
3. length dimension cube (l3)
2. Scaling Law based on Scaling of Phenomenological Behavior

Scaling of both size and material Characterizations. The second type: involves both the size and
material properties of the system..

Material behavior and microstructure geometries at small scales strongly influence the physical
behavior at higher scales. For example, defects like cracks and dislocations evolve at lower scales
and will strongly impact the material properties (mechanical, electrical, thermal, and chemical) at
the macro scale.

Objective of the scaling law is to develop a systematic approach about the likely behavior of
downsized systems .

Impact of size on Function.

As the size decreases, its S/V ratio increases. - Examples ¾ S/V ratio of an elephant (10-4) vs. of a
dragonfly (10-1)

Since volume, V relates to mass and surface area, S relates to buoyancy force:

S/V ≈ 10-4/mm S/V ≈ 10-1/mm

So, an elephant can never fly as easily as a dragonfly!!

An elephant and a flea have cells of about the same size. Too large a cell will not have enough
surface for substance exchanges with its surroundings to support the active metabolism within,
unless it is highly elongated like a vertebrate nerve cell, increasing the S/V ratio. (Biochemistry by
Mathews et al.) There is a cell size limit in living cell. If surface area to volume ratio is too small it
will decrease the rate of chemical reaction and cell will die. Therefore in order to built larger
organisms, they must be built from small cell sub units

There is a cell size limit in living cell. If surface area to volume ratio is too small it will decrease the
rate of chemical reaction and cell will die. Therefore in order to built larger organisms, they must
be built from small cell sub units

Effect of Miniaturization
 Generally, smaller things are less effected by volume dependent phenomena such as mass and
inertia, and are more effected by surface area dependent phenomena such as contact forces or
heat transfer.

1. A 50% reduction in size would result in a factor of 32 times reduction in torque.

2. A 10 times reduction in size100 time reduction in time to heat the solid.
3. A 10 times reduction of linear size of electrodes 1000 times reduction in Potential energy.
4. A 10 times reduction of length leads to 100 times greater power loss due to the resistance
increase. Small object will loose heat rapidly.
5. A reduction of 10 in radius 10000 times reduction in volumetric flow.
6. If 10 times reduction in size there will be 10,000 times reduction Electromagnetic force:
7. If 10 times reduction in size (l) there will be only 100 times reduction in Electrostatic force:

Size change in living bodies has numerous effects and profound biological significance. Dependent
height or length (e.g. Height in trees, leg length in running animals)

Generally, smaller things are less effected by volume dependent phenomena such as mass and
inertia, and are more effected by surface area dependent phenomena such as contact forces or
heat transfer.

Module 15. Micro electro mechanical System (MEMS)

Micro electro mechanical System (MEMS) is the making and combining of miniaturized mechanical
and electrical components on a common silicon substrate through micro fabrication technology.
MEMS consist of mechanical microstructures, microsensors, microactuators and microelectronics,
all integrated onto the same silicon chip.Microsensors detect changes in the system’s environment
by measuring mechanical, thermal, magnetic, chemical or electromagnetic information or
phenomena.

In the first place, the interdisciplinary nature of MEMS technology and its micromachining
techniques, as well as its diversity of applications has resulted in an unprecedented range of devices
and synergies across previously unrelated fields (for example biology and microelectronics).

Secondly, MEMS with its batch fabrication techniques enables components and devices to be
manufactured with increased performance and reliability, combined with the obvious advantages
of reduced physical size, volume, weight and cost.

Thirdly, MEMS provides the basis for the manufacture of products that cannot be made by other
methods. MEMS devices generally range in size from 20 micrometres to a millimetre (i.e., 0.02 to
1.0 mm), components arranged in arrays (e.g., digital micromirror devices) can be more than 1000
mm

MEMS stands for Micro-electromechanical systems, a manufacturing technology that enables the
development of electromechanical systems using batch fabrication techniques similar to those used
in integrated circuit (IC) design.

They can range in size from micrometers to millimeters. MEMS devices are very small; their
components are usually microscopic. Levers, gears, pistons, as well as motors and even steam
engines have all been fabricated by MEMS .

MEMS devices has large surface area to volume ratio and forces produced by

1. electrostatic charges , 2. magnetic moments, 3. surface tension 4. and viscosity

are important in design considerations.

The fabrication of MEMS evolved from the process technology in semiconductor device fabrication,
i.e. the basic techniques are

1. Deposition of material layers. It is an operation by which a film is placed on a surface of the chip.

2. Patterning by lithography. A process in which the desired circuit pattern is projected onto a
photoresist coating covering on a silicon wafer. When a pattern is developed, portions of the
wafer can be selectively removed with a solvent, exposing parts of the wafer for etching and
diffusion.

3.Etching to produce the required shapes. A process in which chemicals are used to remove
previously defined portions of the silicon oxide layer covering the wafer to expose the silicon
underneath..

Materials used for the Fabrication of MEMS.

1. Silicon

Silicon symbol Si and atomic number 14 is a non metal and a semi conductor.It is a hard and brittle
crystalline solid with a blue-grey metallic luster. Si is a tetravalent metalloid and a semiconductor.
It has a melting point of 1414 °C and Boiling point is 3265 °C. Silicon can make for highly repeatable
motion. Silicon suffers very little fatigue in the range of billions to trillions of cycles without
breaking.. It can have service lifetimes.

Crystalline Silicon
Each silicon atom is bonded to four neighbouring atoms. Tetrahedral crystal structure like Diamond.
Crystalline Silicon (c-Si) is the crystalline forms of silicon, multi-crystalline Silicon, or mono-
crystalline silicon . Silicon wafers Silicon wafers are made up of very thin slices of c-silicon (several
hundred microns).

Fig shows atomic arrangement of crystalline silicone and silicon wafer

Integrated Circuit.

An integrated circuit, or IC, is small chip that can function as an amplifier, oscillator, timer,
microprocessor, or even computer memory. An IC is a small wafer, usually made of silicon, that can
hold anywhere from hundreds to millions of transistors, resistors, and capacitors.

Intergrated circuit

Integrated circuit (IC), sometimes called as a chip or microchip, is a semiconductor wafer on which
a thousand or millions of tiny resistors, capacitors, and transistors are fabricated. An IC can be a
function as an amplifier, oscillator, timer, counter, computer memory, or microprocessor.
Integrated circuits are the building blocks of most electronic devices and equipment. They are
between 1 and 100 micrometers in size (i.e., 0.001 to 0.1 mm). An integrated circuit (IC), sometimes
called a Chip or microchip is a semiconductor wafer on which thousands or millions of tiny resistors,
capacitors, and transistors are fabricated.

Metals and ceramics in Process Technology

 Metals are used in processed technology. Commonly used metals include Gold
Nickel Aluminium Copper Chromium Titanium Tungsten Platinum and Silver,

A ceramic is an inorganic non-metallic solid made up of either metal or non-metal compounds. They
are hard, corrosion-resistant and brittle.

Applications

inkjet-printer cartridges miniature robots micro engines.

inertial sensors micro actuator optical scanners fluid pump,chemical, pressure and flow sensors

Issues associated with Micro level Fabrication

There are a number of issues at the microlevel fabrication. Such as

1. Friction is greater than inertia. Capillary, electrostatic and atomic forces at a micro-level can be
significant.
2. Heat dissipation is greater than heat storage and consequently thermal transport properties could
be a problem or, conversely, a great benefit.
3. Fluidic or mass transport properties are extremely important. Tiny flow spaces are prone to
blockages but can conversely regulate fluid movement.
4. Material properties (Young’s modulus, Poisson’s ratio, grain structure) and mechanical theory
(residual stress, wear and fatigue etc.) may be size dependent.
5. Integration with on-chip circuitry is complex and device/domain specific. Lab-on-achip systems
components may not scale down comparably.
6. Miniature device packaging and testing is not straightforward. Certain MEMS sensors require
environmental access as well as protection from other external influences. Testing is not rapid and
is expensive in comparison with conventional IC devices.

Module No. 16 Sensing Techniques in Biosensors

The sensing techniques used in Bisensors could be classified into label-free and label based
on the detection technology. It can also be further classified as shown above. Label-based
techniques rely on the specific properties of labels like fluorescence, chemiluminescence etc. for
detecting a particular target. The process of labeling and purification processes is associated with
sample losses, which is critical when sample quantity is limited. Labeling processes can also have a
detrimental effect on the functionality and stability of molecules like proteins.

Fluorescence
 Fluorescence is the short-time (< 1 µs) category of luminescence, which is mostly exploited as an
optical phenomenon in cold bodies. In the process molecular component used absorbs a photon
and can consecutively emit a photon with a longer (less energetic) wave‐length. Molecular
rotations, vibrations or heat can be produced because the absorbed photons have different
energy; with the emitted photons, for example, the emitted light can be in the visible range even if
the absorbed photon is in the ultraviolet range. Fluorescent Probes

A fluorophore is a chemical compound that can reemit light upon light excitation. It contain
several combined aromatic groups, or planar or cyclic molecules with several π bonds. producing a
detectable signal.

Binding of Rhodamine molecule to DNA molecule as a fluorescent probe.

Chemiluminescence

Chemiluminescence or chemoluminescence is the phenomenon of light emission as the result of a

chemical reaction, which leads to limited emission of heat (luminescence). Chemiluminescence
(CL) analysis promises high sensitivity with simple instruments and without any light source, so
chemiluminescence has become an attractive detection method.

Biotinylation

Biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other
molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the
molecule due to the small size of biotin (MW = 244.31 g/mol). Biotin, a member of the water-
soluble B-complex group of vitamins, is synthesized by higher plants and most fungi and bacteria.
Humans, other mammals, and birds cannot synthesize biotin de novo and therefore must obtain
this essential micronutrient from material synthesized by intestinal microflora and from dietary
sources.
Biotin molecule and Biotin derivatives are shown in Fig.

Biotin can binds to two different proteins with very high affinity, that arestreptavidin and avidin.
Biotin moiety can covalently bound to the epsilon amino group of a Lysine residues of the
proteins.

Streptavidin is a 52.8 (tetramer) kDa protein purified from the bacterium Streptomyces avidinii.
Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin
B7 or vitamin H). Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds,
reptiles and amphibians Avidin is deposited in the whites of their eggs. Dimeric members of the
avidin family are also found in some bacteria. In chicken egg white, avidin makes up approximately
0.05% of total protein.

Biotin with an extremely high affinity, fast on-rate, and high specificity, and these interactions are
exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-
binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making
capture of biotinylated molecules possible in a variety of environments. Also, multiple biotin
molecules can be conjugated to a protein of interest, which allows binding of multiple
streptavidin, avidin or neutravidin protein molecules and increases the sensitivity of detection of
the protein of interest. The biotin–STV.Adv complexes can be chemisorbed and could be used for
sensing.

Enzyme Label based Sensing Techniques

Enzymes can function as highly sensitive probes ideal for the detection of proteins in whole cells,
tissues and/or lysates such as

1. Alkaline phosphatase
2. Glucose oxidase
3. Beta galactosidase
4. Horseradish peroxidase
These enzymes are discussed in detail in next modules.
Labelling of antibody using enzyme horse reddish peroxidase.

Module 17. Optical detection

Detection by optical means is the most inexpensive and simplest method in Biosensor Technology
Working of Optical sensor. White light is composed of seven colors, they are visible after being
dispersed by a prism. Monochromatic light is used in optical techniques.

Example Computer Mouse.

The basic units of computer mouse are

1. Light source

2. Len s
3. Camera
4. Mounted Wheel

LASER Light

I. Light Amplification by Stimulated Emission of Radiation.

II. Lasers produce a narrow beam of light with very similar wavelengths.
III. It is coherence, monochromatic, unidirectional with high intensity.
 Optical Techniques

1. Mass Spectrometry
2. Surface plasmon resonance (SPR)
3. Fiber-optics sensing technique
4. Reflectometric interference spectroscopy technique

1. Basic Principle of Mass Spectrometry. “The basic principle of mass spectrometry (MS) is to
generate ions from either inorganic or organic compounds by any suitable method, to separate
these ions by their mass-to-charge ratio (m/z) and to detect them qualitatively and quantitatively
by their respective m/z and abundance..

Working.

There are four stages in a mass spectrometer which we need to consider, these are – ionisation,
acceleration, deflection, and detection. Let's go through these in order. The sample needs to be
vapourised first, before being passed into the ionisation chamber.It turns the atoms into ions
(electrically charged atoms with either too few or too many electrons). Then it separates the ions
by passing them first through an electric field, then through a magnetic field, so they fan out into a
spectrum.
 Scheme for mass spectrometry

Mass spectrometry is an analytical tool useful for measuring the mass-to-charge ratio (m/z) of one
or more molecules present in a sample. These measurements can often be used to calculate the
exact molecular weight of the sample components as well.It is used to identy the substances. The
substance is bombarded with a beam of electrons so the atoms or molecules it contains are
turned into ions. .A computerized, electrical detector records a spectrum pattern showing how
many ions arrive for each mass/charge. This can be used to identify the atoms or molecules in the
original sample.

2. Fiber Optic Sensors.

The fiber optic sensors also called as optical fiber sensors use optical fiber or sensing element.
These sensors are used to sense some quantities like temperature, pressure, vibrations,
displacements, rotations or concentration of chemical species. Optical Sensors are used for
contact-less detection, counting or positioning of parts. Optical sensors can be either internal or
external. External sensors gather and transmit a required quantity of light, while internal sensors
are most often used to measure the bends and other small changes in direction.

Fig shows the transverse section of fiber optic. Fiber optic used in a Biosensor,

3. Surface Plasmon Resonance

The surface plasmon polariton is a non-radiative electromagnetic surface wave that propagates in
a direction parallel to the negative permittivity/dielectric material interface. Since the wave is on
the boundary of the conductor and the external medium (air, water or vacuum for example), these
oscillations are very sensitive to any change of this boundary, such as the adsorption of molecules
to the conducting surface.
 Schematic diagrams for Surfaceplasmonresonance in a Biosensor.

4. Reflectometric interferencespectroscopy (RIfS)

Reflectometric interference spectroscopy (RIfS) is a physical method based on the interference of

white light at thin films, which is used to investigate molecular interaction. Reflectometric
interference spectroscopy is an optical method suitable for thin film characterization .

The RIfS technique is based on the spectral shift of the interference pattern reflected from thin
films with a few hundred nanometers of layer thickness. The wavelength shift is caused by the
adsorption or adhesion of molecules or nanoparticles, and thus it is applicable as a biosensor. The
advantage of this technique is the extensive detection layer: it can be upto a few hundred
nanometers, making it capable of the detection of bacteria or cells. the drawback of the method
is that a relatively high film thickness (200–600 nm) is required.
Diagram showing RIFS in a biosensor.

Module No. 18 Transistor

Transistor is an electronic device that can be used to switch or amplify electronic signals.
Transistors revolutionized the field of electronics, and paved the way for smaller and cheaper
radios, calculators, and computers, among other things. Transistor is the fundamental building
block of modern electronic devices, and is considered essential in modern electronic systems. A
transistor is a semiconductor device used to amplify or switch electronic signals and electrical
power. Transistor's are low cost, flexible, and reliable devices.
Fig shows Structure of a transistor.

It is composed of semiconductor material usually with at least three terminals for connection to
an external circuit. Most transistors are made from very pure silicon or germanium, but certain
other semiconductor materials can also be used. A transistor may have only one kind of charge
carrier, in a field effect transistor, or may have two kinds of charge carriers in bipolar junction
transistor devices. The fundamental principle behind all transistors is simple: Current flow
between two terminals is prevented by an energy barrier that has been set up between them. To
operate the transistor, a third terminal is provided that allows you to lower the energy barrier. The
main differences between emitter and collector are doping concentration and size. The emitter is
heavily doped, while the collector is lightly doped.

A voltage or current applied to one pair of the transistor's terminals controls the current through
another pair of terminals.

Because the controlled (output) power can be higher than the controlling (input) power, a transistor
can amplify a signal.

Example. Hearing Aid device. Hearing aid

Hearing Aid was devised by using transistors. It is a tiny microphone. it that picks up sounds from
the world around and turns them into fluctuating electric currents. The fluctuating current is fed
into a transistor. The signals are amplified and send to a tiny loudspeaker.

A hearing Aid outside view and Inside view.

Doping

Doping is the addition of an impurity (called dopant) into a pure semiconductor

If the dopant is from 5th group of periodic table we gets p-type extrinsic semiconductor Ar, P, Sb.

If the (dopant) is from 3rd group of periodic table we gets n-type extrinsic semiconductorB ,Ga, Al.
Fig shows the N-type and P-type channels ina transistor.

Band Theory of Electrical Conductivity

States that delocalized (free) electrons. They move freely through"bands" formed by
overlapping molecular orbitals.Free electrons are the ones that can freely move about and help to
carry an Summary

Transistors are the building blocks of the modern electronics

They are composed of three parts called Emitter, Base and collector 'collects' the current and
the emitter 'emits' the current, while the base is the region between them.

The pure elements of group 4 (Silicon and Gallium) are used as semi conductors and doped with
the elements of group 3 and 5.

The process of doping is used improve the electrical property of the semiconductor. There are two
types of transistors P-type and N-Type.

There are millions of transistors in a computer.electric current.

Module No. 19. Electrodes

An electrode is an electrical conductor used to make contact with a nonmetallic part of a circuit (e.g.
a semiconductor, an electrolyte, a vacuum or air).
 An electrode in an electrochemical cell is referred to as either an anode or a cathode The anode is
now defined as the electrode at which electrons leave the cell and oxidation occurs (indicated by a
minus symbol, "−"), and the cathode as the electrode at which electrons enter the cell and reduction
occurs (indicated by a plus symbol, "+"). Each electrode may become either the anode or the
cathode depending on the direction of current through the cell. A bipolar electrode is an electrode
that functions as the anode of one cell and the cathode of another cell. A primary cell is a special
type of electrochemical cell in which the reaction cannot be reversed, and the identities of the
anode and cathode are therefore fixed. The anode is always the negative electrode. The cell can be
discharged but not recharged, as in case of dry battery cell.

With reference to Biosensors different types of electrodes are discussed.

1. Carbon di oxide sensitive electrode. The Severinghaus electrode is an electrode that measures
carbon dioxide (CO2). The carbon dioxide pressure of a sample gas or liquid equilibrates through
the membrane and the glass electrode measures the resulting pH of the bicarbonate solution.

Fig shows a carbon di oxide electrode.

2. ECG electrode

ECG (electrocardiography) is a method of collecting electrical signals generated by the heart.

This allows us to understand the level of physiological arousal that someone is experiencing, but it
can also be used to better understand someone’s psychological state.ECG records the electrical
activity generated by heart muscle depolarizations, which propagate in pulsating electrical waves
 towards the skin. Although the electricity amount is in fact very small, it can be picked up reliably
with ECG electrodes attached to the skin (in microvolts, or uV).

The full ECG setup comprises at least four electrodes which are placed on the chest or at the four
extremities according to standard nomenclature (RA = right arm; LA = left arm; RL = right leg; LL =
left leg). ECG electrodes are typically wet sensors, requiring the use of a conductive gel to increase
conductivity between skin and electrodes.

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3. Microelectrode

A microelectrode is an electrode used in electrophysiology either for recording neural signals or for
the electrical stimulation of nervous tissue. Pulled glass pipettes with tip diameters of 0.5 μm or less
are usually filled with 3 molars potassium chloride solution as the electrical conductor

Fig shows a micro electrode.

Module No. 20. Enzyme Sensitive Electrodes

1. Enzyme electrodes

Enzyme electrodes has a "double reaction" mechanism - an enzyme reacts with a specific substance,
and the product of this reaction (usually H+ or OH−) is detected by a true ion-selective electrode,
such as a pH-selective electrodes. All these reactions occur inside a special membrane which covers
the true ion-selective electrode, which is why enzyme electrodes sometimes are considered as ion-
selective. An example is glucose selective electrodes.

Clark enzyme sensitive electrode.

2. Clark enzyme electrode.

the most typical example of first-generation biosensor is the Clark electrode covered with GLUCOSE
OXIDASE -soaked membrane. The measured compound here is oxygen, the concentration of which
changes with changed enzyme activity depending, in turn, on glucose (analyte) concentration.

There are different types of enzyme electrodes such as Ampermetric enzyme electrode, Enzyme
electrode with a mediator, Three electrode system, Screen printed electrode and enzyme printed
electrodes. All the enzyme electrode are based on the working principle that the membranes or ink
used in the fabrication of printed electrodes is impregnated with the enzyme. Different types of
enzyme electrodes are discussed in lecture.

Three Electrode System

In biosensors 3 electrode systems are used.

a) Working electrode contains the sample which is being studied.

b) Reference Electrodes sets the potential of the system.
c) Counter electrode is a current source.
Three electrode system in a biosensor.

Screen printed Electrodes

Screen printed electrode methodology is used for the fabrication of electrodes.

Screen-printed electrodes are based on carbon, gold, platinum, silver inks.

Ink paste is used to print, a previously marked plastic sheet substrate using a printing nozzle.

.Screen printing of electrodes.

Enzyme Printed Electrode

In Biosensors enzyme printed electrodes are used.

The enzyme layer is place between the carbon electrode (being inert) and mediator layer.
A mediator dielectric layer is placed for the proper functioning of the electrode.

Urease enzyme Electrode

Urease, an enzyme that catalyzes the hydrolysis of urea, forming ammonia and carbon dioxide.

Urease biosensor is used to determine human serum urea amount.

A liquid layer of urease trapped in a cellophane layer over a cation electrode, reacts with urea and
increase the pH.

Urease enzyme electrode

21. Field Effect Transistors (FET)

The field-effect transistor (FET) is an electronic device that can control the flow of current. There
is a terminal, turn alters the conductivity between the drain and source terminals.

FETs are also known as unipolar transistors because of the single-carrier-type operation. Field
effect transistors generally display very high input impedance at low frequencies. . The device
consists of an active channel through which charge carriers, electrons or holes, flow from the
source to the drain. Source and drain terminal conductors are connected to the semiconductor.
FET is three-terminal semiconductor devices, with source, drain, and gate terminals. The charge
carries are electrons or holes, which flow from the source to drain through an active channel. This
flow of electrons from source to drain is controlled by the voltage applied across the gate and source
terminal. The names of the terminals related to their functions.

The gate is referred to as the controlling the opening and closing of a physical gate. This gate permits
electrons to flow through or blocks their passage by either creating or eliminating a channel
between the source and drain. Electron-flow from the source terminal towards the drain terminal
is influenced by an applied voltage.

The body refers to the bulk of the semiconductor in which the gate, source and drain are packed.
Usually the body terminal is connected to the highest or lowest voltage within the circuit, depending
on the type of the FET. The body terminal and the source terminal are sometimes connected
together since the source is often connected to the highest or lowest voltage within the circuit.

Classification

Bio-FETs are classified based on the bio recognition element used for detection:

1) En-FET
2) Immuno-FET
3) DNA-FET
4) CPFET
5) Beetle/chip FET
6) Artificial BioFET-based.

Working of Bio-FET

In a BioFET an Analyte binds with the BRE, on a bio-functionalized surface.

This introduce changes in the electrostatic potential of the surface .

The measurable change in current between electrodes is recorded, as a detecting signal.

DNA field effect Transistor

A DNAFET is a field-effect transistor which uses the field-effect due to the partial charges of DNA
molecules.

In DNAFETs, a layer of immobilized ssDNA acts as surface receptors.

After analyte/BRE hybridization, the charge distribution changes and signals are generated.

DNA field effect transistor in a Biosensor

Cell-Transistor-Hybrid

A Biological cells are able to receive, process, and transmit information.

The platform based on the bio-FET would provide substantial information about different
biological phenomenon.

Bio-FET is used in the shown slide to detect the molecular charges at cell membrane in an
induced system.

BioFET using Beetle antenna to detect Crop Damage

The antenna is highly sensitive towards different odours.

The antenna of a beetle is coupled via an electrolyte to bioelectronic field-effect sensor.

Plant damages by the attack can be identified, due to the presence of odour.
Module No. 22. Ion Selective electrodes
The basic structure of the ion-selective electrode requires a membrane which excludes all ions
except for the ion of interest. In this fashion, the potential generated across the membrane is
generated only because that specific ion species is migrating across it.

An ion-selective electrode (ISE), or a specific ion electrode (SIE), is a transducer that converts the
activity of a specific ion dissolved in a solution into an electrical potential. Ion-selective electrodes
are used in analytical chemistry and biochemical/biophysical research, where measurements of
ionic concentration in an aqueous solution are required.

Principle of ion-selective electrode (I.S.E.) An ideal I.S.E. consists of a thin membrane across which
only the intended ion can be transported. The transport of ions from a high conc. to a low one
through a selective binding with some sites within the membrane creates a potential difference.

Types of ion-selective membrane

There are four main types of ion-selective membrane used in ion-selective electrodes (ISEs): glass,
solid state, liquid based, and compound electrode. For Ion Selective Membranes high
permeability is very important for a good ISE membrane. Ionophores are molecules which act as
ion-binding receptors, which are selective for specific ions. To "dope" an ion-impermeable
membranes ioophores are used, resulting a selectively permeability.
 Different types of Ion selective electrodes are shown in Fig.

Working of Ion selective electrodes are membrane electrode.

The transport of the ions from high conc. to low conc. within the membrane. Produces a produce
a potential. That can be measured using a pH meter or voltmeter.

Fig shows Working of ion selective electrode.

1. Glass electrode

Glass membranes are made from an ion-exchange type of glass (silicate or chalcogenide). This
type of ISE has good selectivity, but only for several single-charged cations; mainly H+, Na+, and
Ag+. Chalcogenide glass also has selectivity for double-charged metal ions, such as Pb2+, and
Cd2+. The glass membrane has excellent chemical durability and can work in very aggressive
media. A very common example of this type of electrode is the pH glass electrode.
 Structure of Glass electrode is shown in Fig.

Ions and Glucose sensitive Electrode

The ion sensitive electrodes in the ABG machine measure the concentrations of potassium,
sodium, calcium, chloride and lactatem as well as non-ionised glucose. Similar principles of
operation apply to each electrode, and they are coupled into a "electrode chain" in the blood gas
analyser.

2. Crystalline membranes

Crystalline membranes are made from mono- or polycrystallites of a single substance. They have
good selectivity, because only ions which can introduce themselves into the crystal structure can
interfere with the electrode response. This is the major difference between this type of electrodes
and the glass membrane electrodes. The lack of internal solution reduces the potential junctions.
 Selectivity of crystalline membranes can be for both cation and anion of the membrane-forming
substance. An example is the fluoride selective electrode based on LaF3 crystals.

3. Ion-exchange resin membranes

Ion-exchange resins are based on special organic polymer membranes which contain a specific
ion-exchange substance (resin). This is the most widespread type of ion-specific electrode. Usage
of specific resins allows preparation of selective electrodes for tens of different ions, both single-
atom or multi-atom.

4. Alkali metal ISE (Applications)

Valinomycin based lectrode.

Electrodes specific for each alkali metal ion, Li+, Na+, K+, Rb+ and Cs+ have been developed. The
principle on which these electrodes are based is that the alkali metal ion is encapsulated in a
molecular cavity whose size is matched to the size of the ion. For example, an electrode based on
Valinomycin may be used for the determination of potassium ion concentration. A potassium-
sensitive electrode is made up of a ionophore- (Valinomycin) impregnated PVC membrane. The
protective cellophane membrane is semipermeable, and it keeps the larger molecules from
befouling the delicate PVC inside. Water and ions get through the PVC membrane and a potential
difference is created.
Module No.23 Latest trends
A few examples of Biosensors are discussed to elaborate the process of MEMS technology and its
applications in the field. Latest trends in Miniaturization

The latest trends in miniaturization of devices are noted in the health sector. The small devices
are produced for diagnostics and wearable Biosensors.A huge variety of products including smart
watch and tattoos are available in market

Microbial Detection Array

1. is capable of identifying microbes in a sample devices.

2. 3-inch long can identify approximately 2,000 viruses and 900 bacteria.

3. The can identify thousands of pathogens in just 24 hours.

A micro array for the detection of pathogens.

HIV Detection Chip

Microfluidics-based detection chip which functions to not only detect HIV

I. It also determine viral load in patients.

II. It could test diagnose 100 samples at a time.
III. The chip can determine their viral load within 4 hours.

A microchip for HIV detection.

Wearable Tattoo
First the sketch of the circuit is design with a graphic design software.
It is fabricated on conductive gold leaf on top with spray adhesives.
The next step is mounting the electronics. The DuoSkin device is planted on the user’s
skin like a regular temporary tattoo.

Fig shows different Wearable Tattoo.

Chapter 03 Biosignals

Modules 24-33

24. Definition

25. Classification

26. Electrical Biosignal

27. Magnetic Biosignal

28. Mechanical Biosignal

29. Optic Biosignal

30. Acoustic Biosignal

31. Chemical Biosignal

32. Thermal Biosignal

33. Latest Research and Current Trends

Learning Outcome of the course

1. Students will be able to clearly define biosignals with examples.

2. Students will be able to classify Biosignals on different bases.
3. Students will be able to identify Electrical Biosignals withthier significance.
4. Students will learn about the Magnetic Biosignals in human and other organism with
their applications.
5. Students will have a clear concept and importance of Mechanical Biosignals.
6. Students will learn about the basics and importance of optic biosignals with their
applications in Biotechnology.
7. Students will learn able to identify Acoustic Biosignal with their importance.
 8. Students will be able to learn about the chemical biosignals and their importance.
9. Students will be given an idea about Thermal Biosignals and their importance
10. Students will learn Latest Research and Current Trends in the field of Biosignals.

Module No 24. Definition Introduction

A biosignal can be defined as a physiological phenomenom, a body variable that can be measured
and monitored Biological signals, or biosignals, are space, time, or space-time records of a
biological event such as a beating heart or a contracting muscle. The electrical, chemical, and
mechanical activity that occurs during this biological event often produces signals that can be
measured and analyzed. Biosignals, therefore, contain useful information that can be used to
understand the underlying physiological mechanisms of a specific biological event or system and
that may be useful for medical diagnosis.

Biological signal is a summarizing term for all kinds of signals that can be (continually) measured
and monitored from biological beings. It shows chemical or physical quantities that characterize
the property or state of human biological condition. The term biosignal is often used to mean
bio-electrical signal but in fact, biosignal refers to both electrical* and non-electrical
signals.Biosignal can be defined as a description of a physiological phenomenon.

Biological Module signals can be acquired in a variety of ways—for example, by a physician who
uses a stethoscope to listen to a patient's heart sounds or with the aid of technologically
advanced biomedical instruments. Following data acquisition, biological signals are analyzed in
order to retrieve useful information. Basic methods of signal analysis, such as amplification,
filtering, digitization, processing, and storage, can be applied to many biological signals. These
techniques are generally accomplished with simple electronic circuits or with digital computers.
In addition to these common procedures, sophisticated digital processing methods are quite
common and can significantly improve the quality of the retrieved data.

Module No. 25. Classification.

As the number of physiological mechanisms is nearly unlimited, the diversity of biosignals is huge.
This can also be justified by the fact that there are many ways to classify the biosignals:
 1. PERMANENT/INDUCED (INTRINSIC/EXTRINSIC TO BODY)
2. Static/Dynamic
3. Origin

As referred the variety of biosignals is nearly unlimited, this makes a unique classification of
biosignals impossible. Their classification is based on their characteristics.

Permanent Biosignals

This kind of Biosignals exist without any excitation from outside body and are always present in
the Human Body because source is inside the body. One example is the electrocardiographic
signal (ECG) induced by electrical heart muscle excitation with the peaks P-Q-R-T-S.

Induced Biosignals

This group of biosignals includes biosignals that are artificially induced. In contrast with the
permanent biosignals this ones exist only during the excitation. It means that, when the artificial
induction is over the induced biosignal decays with a time constant determined by the body
properties. One example is electric plethsysmography, here an artificial current is induced in the
tissue.

Static Biosignal

Static biosignals carry information during their steady-state lever which may show slow changes
over the time. For example the body temperature, which shows slightly changes during the day,
that's why we can consider it a static biosignal.

Dynamic Biosignal

Dynamic biosignals show big changes during time, for example the heart rate. The course of the
heart rate represents a highly dynamic biosignal.

Origin of Biosignals
The last method is using the origin of the biosignal as a basis for their classification, here are
some examples:

1. Electric Biosignals
2. Magnetic Biosignals
3. Mechanic Biosignal
4. Optic Biosignals
5. Acoustic Biosignals
6. Chemical Biosignals
7. Thermal Biosignal

Active and Passive Biosignals

Biosignals can also be divided in two main groups according to their source for measurement:
Active and Passive.

Active Biosignals

These are biosignals where the energy source for measurement is the patient himself. Here we
have two types of "sub" biosignals:

Electrical Biosignals: ECG; EEG; EMG.

Non-electrical biosignals: Thermography and pH for example.

Passive Biosignals

These ones, the energy source for measurement is not the patient e.g. wrist oximeter.

Modules No. 26. Bioelectrical Signals

.Electrical biosignals, or bioelectrical time signals, usually refers to the change in electric current
produced by the sum of an electrical potential difference across a specialized tissue, organ or cell
system like the nervous system. Thus, among the best-known bioelectrical signals are:
1. Electroencephalogram (EEG)

2. Electrocardiogram (ECG)

3. Electromyogram (EMG)

4. Mechanomyogram (MMG)

5. Electrooculography (EOG)

6. Galvanic skin response (GSR)

7. Magnetoencephalogram (MEG)Electric field is generated in cells (nerve and muscle) and

organs because of intra- and extracellular ionic currents. Typical values of membrane potential
are in the range –40 mV to –70 mV. Bioelectrical signals are generated from the complex self-
regulatory system in human. These signals are very low in frequency and can be measured.
Differences in the concentrations of ions on opposite sides of a cellular membrane lead to a
voltage called the membrane potential. Our body generates electrical signals and control the
body functions. Different cells produce different types of signals for example, the signals
generated by Neural cells can be measured by the techniques like.

Electroneurogram ENG

Electroencephalogram EEG

Electroretinogram ERG

2. Signals generated by muscle cells can be measured by

Electrocardiogram ECG

Electromyogram EMG

3. Other cells signals can be measured by Electrooculorgam EOG and Galvanic skin response
GSR
EEG, ECG, EOG and EMG are measured with a differential amplifier which registers the difference
between two electrodes attached to the skin. However, the galvanic skin response measures
electrical resistance and the MEG measures the magnetic field induced by electrical currents
(electroencephalogram) of the brain.

With the development of methods for remote measurement of electric fields using new sensor
technology, electric biosignals such as EEG and ECG can be measured without electric contact
with the skin. This can be applied for example for remote monitoring of brain waves and heart
beat of patients who must not be touched, in particular patients with serious burns.

Electric biosignals can be defined as a change in the electric current across a specialized tissue,
organ or cell like the nervous system for example. Some examples of eletric biosignals are:

Electrocardiogram ECG

The electrocardiogram is a graphic which is produced by an electrocardigraph. This device

records the heart activity over the time. In a typical 12 lead ECG, ten electrodes are placed on the
patient's body. The different positions of the electrodes provide more information about the
heart allowing the heart to be viewed from different angles. When a current flow is towards an
electrode it causes an upward deflection on the ECG while a current flow away form the electrode
produces a downward deflection on the ECG. The current's flow in the heart is divided into
different parts. At the beginning of one cardiac cycle the sinus node gives an impulse which
activates the atria (P-Wave). The current travels down to the ventricles (QRS Complex) over the
atrioventricular node and then spreads back over the ventricles which is known as the recovery
wave (T-Wave). When the electrical waves which cause the heart muscles to contract
passthrough the body, they can be measured by the electrodes placed on the patient skin,
providing a view of the hearth muscle activity. A typical ECG tracing is a cycle of three entities:

i. P wave (atrial depolarization)

ii. QRS (ventricular depolarization)

iii. T wave (ventricular repolarization)

EEG

Electroencephalography is the measurement of the brain electrical activity, recorded from

electrodes placed on the scalp. When these signals are analyzed they are used as a diagnostic
tool to detect pathologies associated with strange electrical behavior. Electroencephalography
on the other hand records spontaneous electrical activities of the brain. The EEG is a procedure
in which electrodes are placed on the skin and are connected through wires to a computer.
Billions of nerve cells in the brain produce tiny electrical signals which are called brain waves. The
electrodes detect these signals, they are being amplified and shown as waves on the screen of
the computer.

Few examples are discussed in lecture.

Module No. 27. Magnetic Signals

Magnetic biosignals Weak magnetic fields are generated by different organs and cells. Fir
example Neural cells signals can be tested by Magnetoneurogram (MNG) and
Magnetoencephalogram (MEG). The signals of Muscle cells can be tested by Magnetocardiogram
(MCG) and Magnetomyogram (MMG).

Magnetoencephalogram (MEG).

MEG is based on measuring the magnetic field outside the head using an array of very sensitive
magnetic field detectors (magnetometers). Because the magnetic filed in in the human body is
generated under the influence of electric field, the magnetic biosignals are also considered under
electric biosignals.

Magnetocardiography (MCG)

Magnetocardiography (MCG) is a technique to measure the magnetic fields produced by

electricalcurrents in the heart.It uses extremely sensitive devices such as the superconducting
quantum interference device (SQUID).

Magnetic signal in birds

Birds use Earth's magnetic field as sort of a heads-up display to help them navigate the globe.
The Earth's magnetic field is a result of the movement or convection of liquid iron in the outer
core. Iron-rich structures have been found in the beak of homing pigeons, chickens and several
species of migratory birds and interpreted as magnetoreceptors. As the liquid metal in the outer
core moves, it generates electric currents, which lead to a magnetic field.

Cryptochrome protein helps birds navigate via magnetic field. Researchers have found one one
possible explanation for some birds' ability to sense the earth's magnetic field and use it to orient
themselves: a magnetically sensitive protein called cryptochrome that mediates circadian
rhythms in plants and animals. Cryptochromes are blue-light absorbing flavoproteins that in
animals have an important function in the circadian clock. It act as receptor molecules with
magnetically sensitive radical pair reactions in the light-dependent magnetic compass sense of
birds. The presence of aromatic amino acid residues in the active site of the protein enable this
protein to absorb light. This protein acts as a iron free compass. Cryptochrome is also expressed
in human retina.

Module No. 28 Mechanical Biosignals

produced by the mechanical functions of biological signals such as motion and displacement ,
pressure.

They could be of two types internal and external. Blood pressure is an example of an internal
mechanical signal which will be discussed in other module. In human external mechanical signals
are under the influence of certain hormones or emotions. In this lecture external mechanical
signals are discussed with reference to other organisms.

Honey bee

Honey bees (Apis sp.) are the only known bee genus that uses nest-based communication to
provide nest-mates with information about the location of resources., the so-called “dance
language.” Successful foragers perform waggle dances for high quality food sources and, when
swarming, suitable nest-site. When a worker discovers a good source of nectar or pollen (note
the pollen spores dusting this bee's back), she will return to the hive to perform a waggle dance
to let her nest mates know where it lies. A bee performs the waggle dance when she wants to
inform other bees of a nectar source she has found. A round dance is the communicative behavior
of a foraging honey bee (Apis mellifera), in which she moves on the comb in close circles,
alternating right and then left. Elements of the round dance also provide information regarding
the forager's subjective evaluation of the food source's profitability.

Birds

Birds make a unique "whistling" sound with their wings when they flee a predator in order to
alert other members of the flock to the danger, new research shows. A study of crested pigeons
found their flapping wings make a whooshing sound if they take-off in alarm that is different to
that produced in routine flight.

A peacock's body is covered with feathers which are attractive in colour. The male peacock open
its feathers before rain. They dance or just open up their feathers to attract female peahens to
mate. This is the reason why peacocks mate mainly in the period April to September.

Buzzing sound by Mosquito

The buzzing sound is made by the beating of the mosquitoes wings. The wings of both males and
females make a buzzing sound. Females make a higher pitched sound than males.

Cricket chirping

Male crickets produce sounds by rubbing their leathery front wings together, i.e., file-like
serrations on the wings' edges rub against a sharp edge (scraper). This is called “stridulation” and
is used to attract female crickets as mates. When this sound is being produced, the cricket's wings
are elevated.

Mechanical Biosignals in plants

Sunflower Flowers follow a circadian rhythm meaning that they are attuned to sense changes in
light and temperature as the day progresses. It is related to their growth.

Night blooming flowers are heavily scented. They use their fragrance as signals to attract the
pollinator at night.

Module No. 29. Optic biosignals

are the result of optical functions of the biologic system, occurring naturally or induced by the
measurement.

Bio luminance

Bioluminescence is the production and emission of light by a living organism. It is a form of

chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as
well as in some fungi, microorganisms including some bioluminescent bacteria and terrestrial
arthropods such as fireflies. Bioluminescence occurs through a chemical reaction that produces
light energy within an organism's body. For a reaction to occur, a species must contain luciferin,
a molecule that, when it reacts with oxygen, produces light. In firefly. The main reason fireflies
glow is to find a mate, though they can also light up under other situations as a warning to others.
Usually male fireflies fly around and flash light to advertise to the females. Fireflies only live a
couple of weeks as adults. But, accounting for each stage of development from egg to adult,
fireflies typically live for about a year. Throughout that time, they are only capable of flying and
laying eggs for approximately two months. Chemistry of the Bioluminance in the firefly is
summarized below.
Bio luminance in Bacteria

All bacterial luciferases are approximately 80 KDa heterodimers containing two subunits: α and
β. The α subunit is responsible for light emission. The luxA and luxB genes encode for the α and
β subunits, respectively. The biochemical reaction involves the oxidation of an aliphatic aldehyde
by a reduced flavin mononucleotide. The products of this oxidation reaction include an oxidized
flavin mononucleotide, a fatty acid chain, and energy in the form of a blue-green visible light.

The bioluminescent reaction is as follows:

FMNH2 + O2 + R-CHO -> FMN + H2O + R-COOH + Light (~ 495 nm)

Bioluminescence in bacteria can be regulated through a phenomenon known as quorum sensing.

Quorom sensing is a form of cell-to-cell communication that alters gene expression in response
to cell density. An autoinducer is produced to regulate the process by the bacteria. Autoinducer
is a diffusible pheromone. It serves as an extracellular signalling molecule. When the
concentration of autoinducer secreted by bioluminescent cells in the environment reaches a
threshold (above 107 cells per mL), it induces the expression of luciferase and other enzymes
involved in bioluminescence. Bacteria are able to estimate their density by sensing the level of
autoinducer in the environment and regulate their bioluminescence such that it is expressed only
when there is a sufficiently high cell population. A sufficiently high cell population ensures that
the bioluminescence produced by the cells will be visible in the environment for example in sea.
Module No. 30. Acoustic biosignals

Many physiological phenomena create noise like the flow of blood in the heart or throuhg blood
vessels also the flow or air through the airways creates acoustic sounds.

Heart Sound

There are four heart sounds, named as lub dub. The “ lub” is the first heart sound, commonly
termed S1, and is caused by turbulence caused by the closure of mitral and tricuspid valves at
the start of systole. The second sound,” dub” or S2, is caused by the closure of aortic and
pulmonic valves, marking the end of systole. The third heart sound, also known as the
“ventricular gallop,” occurs just after S2 when the mitral valve opens, allowing passive filling of
the left ventricle. ... A S3 can be a normal finding in children, pregnant females and well-trained
athletes; however, a S4 heart sound is almost always abnormal.

Lung Sound

Lung sounds, also called breath sounds, can be auscultated across the anterior and posterior
chest walls with a stethoscope. Adventitious lung sounds are referenced as crackles (rales),
wheezes (rhonchi), stridor and pleural rubs as well as voiced sounds that include egophony,
bronchophony and whispered pectoriloquy. In pneumonia there is a change in the sound,
signaling a pathological condition/infection. Rhonchi sounds occur when air tries to pass through
bronchial tubes that contain fluid or mucus. Crackles occur if the small air sacs in the lungs fill
with fluid and there's any air movement in the sacs, during breathing. The air sacs fill with fluid
when a person has pneumonia or heart failure.

Snoring

Snoring is a common but treatable condition. It happens when turbulent air flows through the
airway, causing the uvula and soft palate to vibrate. Snoring may be related to sleep apnea, which
is a symptom of high blood pressure and other conditions. It is reported that 44% of men and
28% of women snore. Snoring is often the sign of a condition called obstructive sleep apnea,
which raises the risk for diabetes, obesity, hypertension, stroke, heart attack and other
cardiovascular problems.

Burps

burp. A burp is a belch, or the loud release of trapped air loudly through your mouth. A burp is a
belch, or the loud release of trapped air loudly through your mouth. A reflex is initiated, leading
to relaxation of the lower esophageal sphincter, upward movement of the air through the
esophagus. It passes through the upper esophageal sphincter, during which an audible belch can
sometimes be heard.

Hiccups

Hiccups are sudden, involuntary contractions of the diaphragm muscle. As the muscle contracts
repeatedly, the opening between the vocal cords snaps shut to check the inflow of air and makes
the hiccup sound. Irritation of the nerves that extend from the neck to the chest can cause
hiccups.

Module No. 31. Chemical biosignals

reflect chemical composition and its temporal changes in body solids, liquids, and gases.
Examples are measuring the concentration of various ions and vicinity of a cell by means of
specific ion electrodes. These signals could be detected by testing a sample. For example urine
sample could be chemically tested for pregnancy, diabetes, urinary tract infection or other
diseases. These chemical signals are accepted as authenticated tests. The subject of Dipstik is
discussed in other sections.

Chemical signals could be divided into two types. Chemical signals produced inside the cells in
response to a an internal stimuli. For example Level of glucose – Blood, oxygen level (heart and
kidney failure). Gases in blood and breathing air flow and pH.
Second types of Biosignals are Chemical signals produced inside the cells in response to an
external stimuli. These chemical are volatile compounds and are released in the environment.
These signals are produced by all type of organisms bacterial, fungal, plants, animals. When a
plant is damaged by herbivorous insects, volatiles are released in defense. Different types of
chemicals are produced by the living organisms, under different environmental and physiological
conditions.

These molecules includes

1. Pheromones

2. Volatile organic compounds

3. Ink

These volatiles attack both parasitic and predatory insects that are natural enemies of the
herbivores. They may also induce defense responses in neighboring plants. In response to insect
feeding, volatile compounds are released by plants. Jasmonic acid and its derivatives are
released under stress and activate the genetic expression of protease inhibitors in the plants.
Jasmonic acid is also released into the environment.

The cephalopods release ink to create floating glowing blobs to create a distraction to predators.

It is Mucus-rich ink is a dangerous or annoying substance that interferes with fish gills.

The blue-ringed octopus has deadly toxin named tetrodotoxin, is present in its ink. Sea hares
are known for the colorful, sticky ink they let loose when knocked around by hungry predators.

Pheromones The substance is a mixture that prevents the odor molecules from getting to the
sensory receptors. They are secreted outside the body, and they influence the behavior of
another individual. Pheromones may be present in all bodily secretions but most attention has
been geared toward axillary sweat which contains the odorous 16-androstenes. Pheromones are
similar to hormones but work outside of the body.
Module No. 32 Thermal Biosignals

Temperature measurement shows physical and biochemical processes proceeded in

organism.(Heat loss, heat absorption)Our body has a fixed range of temperature. If there is a
change in the temperature it is a thermal signal for a change in the normal physiology or an
indication of a disease. The average normal body temperature is generally accepted as 98.6°F
(37°C). Some studies have shown that the "normal" body temperature can have a wide range,
from 97°F (36.1°C) to 99°F (37.2°C). A temperature over 100.4°F (38°C) most often means you
have a fever caused by an infection or illness.

There are various types of medical thermometers, as well as sites used for measurement,
including:

1. In the rectum (rectal temperature)

2. In the mouth (oral temperature)
3. Under the arm (axillary temperature)
4. In the ear (tympanic temperature)
5. On the skin of the forehead over the temporal artery Variations

Temperature control (thermoregulation) is part of a homeostatic mechanism that keeps

the organism at optimum operating temperature, as the temperature affects the rate of
chemical reactions. In humans, the average internal temperature is 37.0 °C (98.6 °F),
though it varies around this point by 0.5 to 1 C. Body temperature normally fluctuates
over the day following circadian rhythms, with the lowest levels around 4 a.m. and the
highest in the late afternoon, between 4:00 and 6:00 p.m. (assuming the person sleeps at
night and stays awake during the day). Body temperature is sensitive to many hormones,
so women have a temperature rhythm that varies with the menstrual cycle, called a
circamensal rhythm. A woman's basal body temperature rises sharply after ovulation, as
estrogen production decreases and progesterone increases. The information about the
biological signal is used Fertility awareness programs. The schange in temperature is a
 thermal signal to identify when a woman has ovulated in order to achieve or avoid
pregnancy.

Hypothermia
In hypothermia, body temperature drops below that required for normal metabolism and
bodily functions. In humans, this is usually due to excessive exposure to cold air or water,
but it can be deliberately induced as a medical treatment. Symptoms usually appear when
the body's core temperature drops by 1–2 °C (1.8–3.6 °F) below normal temperature.

Hyperthermia
Hyperthermia occurs when the body produces or absorbs more heat than it can dissipate.
It is usually caused by prolonged exposure to high temperatures. The heat-regulating
mechanisms of the body eventually become overwhelmed and unable to deal effectively
with the heat, causing the body temperature to climb uncontrollably. Hyperthermia at or
above about 40 °C (104 °F) is a life-threatening medical emergency that requires
immediate treatment. Onion is capable o increasing the body temperature when
uncooked piece of onion is put under the armpit, it increases the body temperature.

Death Chill
After the heart stops beating, the body immediately starts turning cold. This phase is
known as algor mortis, or the death chill. Each hour, the body temperature falls about 1.5
degrees Fahrenheit (0.83 degrees Celsius) until it reaches room temperature.

Module No 33. The latest techniques that are mentioned the modules 24-32 are discussed as
examples.

Biosensors Chapter 04

Support materials and Biomolecule Immobilization methods

Reading material and Notes
Module No.

34. An introduction to support materials Types

35. Natural Support materials

36. Synthetic support materials

37. Hydrogels

38. Immobilization , introduction types

39. Reversible Immobilization

40. Irreversible methods

41. Applications of immobilization in Biosensors

42. Latest Research and Current Trends

Learning Outcome.

01. Students will learn about the support materials and their types used in
Biosensors.Types
02. Different types of support materials including natural and synthetic materials will be
explained. Students will be able to describe the advantages of different materials
that are being used.
03. Students will get a concept of Hydrogels and their uses in Biosenso technology.
04. Students will learn reversible and irreversible methods of immobilization and their
applications.

An introduction to support materials Types

The term support material generally referred to be insoluble metrics where biorecognition
elements can be attached. The support material could be natural, synthetic or hybrid of
both.
Fig shows the components of a biosensor alongwith a sensory layer.

The support material is a part of Bioactive or bio sensitized surface of the Biosensor.
Support materials are widely implemented in an attempt to conserve the binding ability
of the biorecognition element. Support material must display good chemical and
mechanical stability as they can alter the properties of immobilize molecules. The
material must be carefully screened and selected to avoid interference with chemistry of
bioreceptor elements. Selection of Support material

In Biosensors the BRE is required to be attached on a support material Carrier matrixes for
enzyme immobilization must be chosen with care. The surface density of the binding side
together with volumetric surface area strictly available to the enzymes determine the
maximum binding capacity. The actual capacity will be effective by number of potential
coupling sides in the biomolecule are used and electrostatic charge distribution and
polarity. The form shape, density, curiosity, pore size distribution, operational stability and
particles size distribution will influence the reaction configuration in which the immobilize
molecules will be used. The ideal support is cheap, inert, physical strong and stable. It will
increase the biomolecules specificity while reducing product inhibition, shift the pH
optimum to the desired value for the process and discourage microbial growth and non-
specific absorption. Some matrices possess other properties which are useful for particular
purposes such as ferromagnetism (magnetic iron-oxide enable transfer of the biocatalyst
by means of magnetic field).

General Properties

The properties of material including composition solubility, viscosity, solid contents,

surfactants, residual initiatives, film forming temperature, glass forming temperature and
particle size should be carefully considered.

Mean particle size: It is the parameter which decides the porosity of a carrier.

Hydrophilic character: It determines the level of activity of an immobilized enzyme.

Mechanical strength:

A mechanically strong material is required as a support material for immobilization.

Generally, the material should be good formal, with good addition to support the substrate
and should have minimal or no track for handling purposes. The metrics should have the
desired swelling characteristics and surface energetic. An optimum support is expected to
be inert against the biological recognition elements and reaction mixture components. It
is expected to be cheap (economical) accessible, biocompatible and non-toxic. An ideal
support material needs to be mesoporous materials to provide wide surface area and high
porosity to increase the amount of the immobilize molecules to be loaded on the material.
 As biomolecules bind to the functional group, the support material should also contain
goods that can easily modified.

Classification

Schematic classification of support material.

There are at least four types of materials that are being used for the platform for
biosensors. 1. Plant derived materials, 2. Animal derived materials, 3. Carbon Based
materials and 4. Silicone based materials.The support material could be classified as
Synthetic material, Inorganic matrix. Further they could be classified as natural and
processed materials. Synthetic polymers show high chemical and mechanical stability.
Here are few examples of the organic support materials:

Natural Support Materials

Different type of materials are present in nature from biological and Inorganic sources. The
biological compounds include cellulose, nano cellulose, collagen. The inorganic and
processed materials include silicon based and nano carbon based materials. A number of
inorganic materials are used for the matrix to immobilize BRE. These materials are porous

1. Zeolite
2. Ceramic
3. Silica
4. Glass
5. Activated Charcoal

Carbon based nano materials. Carbon has an ability to polymerize at the atomic level,
thus can form long carbon chains. Carbon has four electrons in the outer electron and
can be linked via single, double or triple covalent bonds, or also with other elements.
Carbon has an ability to polymerize at the atomic level, thus can form long carbon
chains. Carbon paste has successfully used in Biosensor as a platform. It requires simple
construction procedures. It exhibits low background current. It has an ability for surface
regeneration. It has a very low cost.

Support matrix in a Biosensor is a sensitizing platform on which the Biorecognition

process take place. It is required to be, stable, semi conductor and inert.

There is a huge variety of organic materials is available including plant based, animal
based, microbial based .Synthetic and inorganic and allotropic forms of Carbon based
materials are also used.

Module 35. Types of Natural Material Organic and Inorganic

Different type of materials are present in nature from biological and Inorganic sources.

The biological compounds include cellulose, nano cellulose, collagen.

The inorganic and processed materials include silicon based and nano carbon based
materials. There are at least four types of materials that are being used for the platform
for biosensors.

• Plant derived materials

• Animal derived materials
• Carbon Based materials
• Silicone based materials
Cellulose

Cellulose is a plant based polymer. It is composed of sub units of glucose joined through
beta linkages. It is an important structural component of the primary cell wall of green
plants, many forms of algae. Cellulose is the most abundant organic polymer on Earth.

Fig shows cellulose structure and fiber in pant tissue.

Another important material is Nano Cellulose. It is Light weight, stiff, electrically

conductive and non-toxic. The crystalline form is transparent, and gas impermeable. It has
a very high tensile strength - 8 times that of steel. It is highly absorbent.

A Transmission Electron Micrograph image of a nanocellulose membrane shows the

individual cellulose nanofibers..

Alginate

It is an un-branched polyscitorite derived from brown algae. Alginate is a naturally

occurring anionic gelatinous polysaccharide. It is present in cell wall of algae and seaweed.
Alginate is an anionic linear polymer of linear copolymers of β-(1–4) linked D-mannuronic
acid and β-(1–4)-linked L-guluronic acid units. Alginate is biocompatible and can keep the
entrapped biomolecules stable.
Fig. Shows linear polymer of linear copolymers of β-(1–4) linked D-mannuronic acid and β-
(1–4)-linked L-guluronic acid units.

Diatomaceous earth

Fig. Highly magnified diatomaceous earth shows the intricate fossils that make up the
substance

Diatomaceous earth is a naturally occurring, soft, siliceous sedimentary rock forms a fine
white to off-white powder. It has a particle size ranging from 10 to 200 μm. Diatomite
forms by the accumulation of the amorphous silica (opal, SiO2•nH2O) from the remains of
dead diatoms.

Chitosan

Fig.

It is a non-toxic biocompatible polymer shows great affinity to protein. Chitosan is a linear

polysaccharide. It is composed of randomly distributed β-(1→4)-linked D-glucosamine
(deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is made by treating
the chitin shells of shrimp and other crustaceans with an alkaline solution. It possesses
highly number of reactive hydroxyl and amino group which facilitate protein entrapment.
It has high fill forming ability and high adhesion characteristics.

Collagen

It is a structural protein nature with high water holding potential, high porosity,
economical. Collagen is the main structural protein in the extracellular space in the various
connective tissues in the body. Collagen consists of amino acids bound together

Fig shows the structure of a collagen fibril.

to form triple-helices of elongated fibrils. It is mostly found in fibrous tissues such as

tendons, ligaments, and skin.

Module No. 36 Synthetic Material

n number of synthetic materials are used as platform for the immobilization of

Biorecognition element. A number of novel materials including Glassy carbon, nano carbon
, silicone based polymers are used. For example Ion exchange resins, Aniline based
polymers, Carbon based materials Silicone based materials are used as platform for
immobilization.

Fig shows cation and anion beads.

Ion Exchange resins are small beads that are composed of organic polymer chains. Cationic
resin has a negative functional group and therefore attracts positively charged ions.
Anionic resin has a positive functional group and attracts negatively charged ions

Polyanailine (PANI). The basic subunit PANI is aniline. It forms discrete long threads with
an average diameter in the range of 30 nm - 100 nm. PANI is a semi-flexible. It has different
colors in oxidized and reduced forms. Polyaniline is typically produced in the form of long-
chain polymer aggregates, surfactant (or dopant) stabilized nanoparticle dispersions, or
stabilizer-free nanofiber dispersions depending on the supplier and synthetic route.
Surfactant or dopant stabilized polyaniline dispersions

Fig. shows Microscopic and molecular structure of PANI.

Polyvinyl alcohol. It has ability to form membrane and fiber is biocompatible, can stabilize
enzyme activity, freeze thawed PVA used to immobilize cells PVA cryogels can be employed
for enzyme immobilization.

Glassy Carbon

Glass-like carbon, (non-graphitizing), combines glassy and ceramic properties with those
of graphite. Glass-like carbon, often called glassy carbon or vitreous carbon, is a non-
graphitizing, or nongraphitizable, carbon which combines glassy and ceramic properties
with those of graphite. The most important properties are high temperature resistance,
hardness (7 Mohs), low density, low electrical resistance, low friction, low thermal
resistance, extreme resistance to chemical attack and impermeability to gases and
liquids. Glassy carbon is widely used as an electrode material in electrochemistry, as well
as for high temperature crucibles and as a component of some prosthetic devices, and
can be fabricated as different shapes, sizes and sections. The structure of glassy carbon
has long been a subject of debate. Early structural models assumed that both sp2- and
sp3-bonded atoms were present, but it is now known that glassy carbon is 100% sp2.
Fig. Glassy carbon.

1. Mechanically stable, Impermeable to gases and fluids

2. chemically resistant
3. high porosity
4. low background current.
5. its low sensitivity to peroxide, as well as to other mediators

Module No. 37. Hydrogels

Hydrogels are regarded as the three dimensional network of natural or synthetic

polymers.They have an ability to absorb a large amount of water or biological fluids.
Hydrogels do not dribble water due to the presence of a novel exclusive zone (about
100micrometer).
Hydrogels are considered as water insoluble, cross-linked, three-dimensional networks of
polymer chains plus water that fills the voids between polymer chains. Crosslinking
facilitates insolubility in water and provides required mechanical strength and physical
integrity. Hydrogel is mostly water (the mass fraction of water is much greater than that
of polymer). The ability of a hydrogel to hold significant amount of water implies that the
polymer chains must have at least moderate hydrophilic character. Alternatively three-
dimensional lattices (hydrogel/exrogel) can be used to chemically or physically entrap
these (where by chemically entrapped. Hydrogels do not flow, which means that the
biological element is kept in place by a strong bond, while physically they are kept in
place being unable to pass through the pores of the gel matrix).

Types

Hydrogels can be produced from natural polymers include proteins such as collagen and
gelatin.

i. The polysaccharides include starch, alginate, and agarose.

ii. Synthetic polymers that form hydrogels are prepared using chemical
polymerization methods.

Gelatin is a translucent, colorless, flavorless food ingredient, derived from collagen taken
from animal body parts. It is brittle when dry and gummy when moist. It may also be
referred to as hydrolyzed collagen, collagen hydrolysate, gelatine hydrolysate, hydrolyzed
gelatine, and collagen peptides after it has undergone hydrolysis. It is commonly used as
a gelling agent in food, medications, drug and vitamin capsules, photographic films and
papers, and cosmetics.

Gelatin is a heterogeneous mixture of single or multi-stranded polypeptides. Glycine,

proline, and valine most abundant amino acids. Each strand with extended left-handed
helix conformations. A helix contains between 50 - 1000 amino acids.

Hydrogels are used in eye lens. The most commonly used hydrogel is sol-gel, a glassy silica
generated by polymerization of silicate monomers (added as tetra alkyl orthosilicates
tetramethoxysilane (TMOS) and tetraethoxysilane (TEOS)) in the presence of the biological
elements (along with other stabilizing polymers, such as PEG Poly ethylene glycol) in the
case of physical entrapment.

Fig shows the structure of synthetic monomers used in the preparation of cosmetic for
eye lens.

PVA polyvinyl alcohol. Artificial or synthetic eye lens are made up of hydrogels. Silicone
hydrogel contact lenses are advanced soft lenses that allow more oxygen to pass through
the lens to the cornea. Silicon based eye lens are made up of N- Vinyl-pyrolidone and
polymethyl methacrylate (PMMA) wearer

Hydrogels are also used in diapers. Sodium polyacrylate is used in the production of
disposable diapers. When exposed to water, the sodium polyacrylate swells and adsorbs
up to 1000 times its own weight in water. The dipers and sanitary napkins absorbs liquid
by osmosis. Hydrogels can be regarded as highly valuable biomaterials for human-beings.
There are numerous applications of hydrogels in modern day life.

Fig shows Swelling of Sodium polyacrylate in the presence of water.

Applications

Hydrogels can be regarded as highly valuable biomaterials for human-beings. There are
numerous applications of hydrogels in modern day life.

i. Tissue engineering
ii. Dressing of wounds
iii. Diapers
iv. Cosmetic products skin and hair gel
v. Biosensing.

Fig shows theapplications of hydrogel in the bone regeneration therapy.

In summary Hydrogels are polymers and are able to retain maximum quantity of water.
Hydrogels could be made from natural and synthetic materials. There are several
applications of hydrogels in different fields including medicine, drug delivery, agriculture,
environment.

38. Immobilization Introduction Types

Immobilization is the binding of biomolecules on to a solid support with functional activity.
It gives a stable environment to the biomolecules.In Biosensors a close proximity of the
biomolecules with the transducer is required. The main components of an enzyme
immobilization include a Biological recognition element , a support matrix and mode of
attachment of a catalyst to the carrier. Support matrix is a material that uses for the
imprisonment of an enzyme. There are two methods of for immobilization.

Components of Immobilization

The main components of an enzyme immobilization include a Biological recognition

element , a support matrix and mode of attachment of a catalyst to the carrier.Support
matrix is a material that uses for the imprisonment of an enzyme.

Fig shows components of immobilization.

Types of Immobilization.

Fig summarizes the methods for immobilization.

Chemical Methods
 i. Covalent Binding
ii. Copolymerization

Physical methods.

i. Adsorption
ii. Entrapment
iii. Encapsulation

Ionic binding
This is a simple reversible mode of immobilization of proteins, which involves ionic
interaction between the enzyme and the support.

Principle: The support used is generally charged, such that the protein to be bound has an
opposite charge. The enzyme is therefore bound to the support via ionic interactions. It
can be easily reversed by altering the pH or ‘salting out’ of the enzyme.

Matrix used: Common polymers used for enzyme entrapment

include alginate, carrageenan, collagen, polyacrylamide, gelatin, silicon rubber and
polyurethane

Advantage: It is very easy, inexpensive and requires simple inputsfor reversal of the
binding. To maintain an optimum pH during the reaction tenure, easy manipulation of the
acidity or alkalinity in the reaction mixture can be performed, as the matrix which
immobilizesthe enzyme is stably charged.
Disadvantage: The presence of the charged support causes several problems like enzyme
structure distortion and alterations in enzyme kinetics. High charge has the potential to
disrupt the enzyme catalysis. As a result, maximum yield is hindered
molecules diffuse into the mesh, the reaction will not be initiated and according to Le
Chatelier’s principle, the reaction rate does not reach a peak unless the products sieve out.

Covalent Binding

Covalent bonds are highly stable and hence, covalent binding ensures that the enzyme is
strongly bound to the support . It has been used in a number of industries. Chemical
binding between functional groups of the enzyme and the support. Cyanogen bromide
can be used.
1. No diffusion barrier
2. Stable
 3. Short response time
4. High enzyme activity loss
5. Disadvantage
6. Matrix not regenertable
7. Coupling with toxic product.

Covalent linking of enzyme by using cyanogen bromide.

Advantage: The reaction is highly specific and no contaminants are present on the carrier.
If the antibody on the support is highly specific for the enzyme, the step of enzyme
purification can be bypassed. Enzymes from an impure solution can also specifically attach
to the matrix. Maximum activity of the enzyme is also ensured if the antibody is targeted
at an epitope away from the activity site.

Principle: It involves the formation of a covalent bond between the support and the side
chains of the amino acids of the enzyme, most commonly lysine (ε-amino group), cysteine
(thiol group), aspartic acid and glutamic acid (carboxylic group), hydroxyl group, imidazole
group, phenol groups, etc. . These groups are nucleophiles and tend to bind to electrophilic
groups of the support. A wide variety of reactions have been developed, depending on the
functional groups available on the matrix. For coupling of the enzyme to the support, it is
often necessary to ‘activate the support’, i.e., modify the support so as to make it bind to
the enzyme more efficiently. The activation methods in general can be divided into two
main classes,(i) addition of a reactive group to the support polymer to activate it, and (ii)
modification of the polymer backbone to produce an activated group. The activation
processes are generally designed to generate electrophilic groups on the support. This
allows the support to react with the strong nucleophiles on the proteins, allowing stable
immobilization .
Carriers used: The supports used are generally stable and easily available and are activated
by the appropriate reagents. The common supports used are cyanogen bromide (CNBr)-
activated Sephadex or CNBr-activated Sepharose. Other common carriers include
activatedforms of dextran, cellulose, agarose, etc. Artificial matrices include Polyvinyl
chloride, ion exchange resins and porous glass .

Advantage: The binding involves covalent interactions and is strong. Hence, leaking of the
enzyme into the reaction mixture is totally prevented. This prevents mixing of the enzyme
with the product, thereby reducing contamination and the cost of purification. The
covalent binding also stabilizes the enzyme in specific protein orientations, and may
promote higher specific activity .

Disadvantage: The covalent bond formed between the support and enzyme may involve
the amino acids of the active site of the enzyme, which may lead to significant loss in
activity. Since the method is irreversible, the support cannot be recycled, as the enzymatic
activity declines. The support along with the bound enzyme has to be discarded.

Crosslinking

It is an irreversible method of enzyme immobilization. It is different from other techniques

in the sense that it does not require a support for the immobilization.

The cross linking of Biological recognition element with the support material involves
bonding between the amino group of the matrix and amino group of an enzyme.
Example: Glutaraldehyde.
Advantages are Low or no enzyme leakage Higher enzyme stability.
Simple and cheap method, unlike the other systems of enzyme immobilization, the
immobilized enzyme is not bound to any matrix, but is present in the reaction mixture,
albeit in an immobilized form.
In summary In Biosensors the BRE (Protein, DNA, Cell) is required to be attached on a
support material. A solid, stable and inert support is required to entrap the protein
molecule.In Biosensing adsorption, entrapment, chemical binding, encapsulation and
crosslinking are used

Module No. 39. Reversible Immobilization

Reversible immobilized enzyme technology cannot fulfil the aim of using an immobilized
enzyme on a long term basis. Irreversibleimmobilization involves strong chemical bonds
and particularlyserves to maintain reasonable stability of the enzymes over a long period
of time. Most industries use enzymes immobilized by these methods, thereby allowing
continuous processing of the substrates without the need of replacing the enzyme very
 often. Different techniques classified as irreversible enzyme immobilization are discussed
in the following sections.
Reversible immobilization is the method in which the biological recognition element can
be removed from the matrix. It is temporarily fixed on support material.
Generally no drastic conditions are required for the immobilization.
Reversible immobilization techniques allow the multiple time use of BRE.
Methods of immobilization by adsorption
i. Static method: immobilization of enzyme and carrier molecule without
agitation.
ii. Dynamic process: The mixing of an enzyme with the carrier under constant
agitation.
iii. Reactor loading: transferring of both enzyme and carrier in the reactor with the
agitation Electro-deposition: Due to electric current the enzyme gets deposited
on the surface.

Fig. Immobilization of Biomolecule on matrix.

There are four methods for encapsulation.

1. Encapsulation in reaction vessel.
2. Encapsulation by hollow fibre membrane.
3. Microencapsulation by chemical polymerization, by using 1-6-diaminohexane.
4. Encapsulation by liposomes.

Encapsulation

Encapsulation can be regarded as a special type of entrapmentwhere the enzyme is

immobilized by entrapping it in a sphericalsemi-permeable membrane.
Principle: The basic underlying principle is the larger size of theenzyme as compared to
substrates or products. When the enzyme can be entrapped or occluded within a semi-
permeable membrane, it would allow the small substrate molecules to diffuse in and the
product molecules to diffuse out. However, the enzymes being much larger, cannot diffuse
through the membrane and remains with in it.

. The permeability of the membrane is controlled according to the enzyme being

immobilized .Encapsulation is achieved by one of the two methods,

Advantage: Encapsulation within a membrane maintains the enzyme structure in its native
form and prevents leakage of the enzyme, protecting it from the harsh conditions of the
medium. Multi-enzyme encapsulations can also be created by trapping more than one
enzyme within a membrane .

Disadvantage: As mentioned earlier with entrapment method, the diffusion of substrate and
product across the membrane controls the reaction rate. The pore size needs to be
maintained accurately to prohibit enzyme leakage (if the pore is too large) or poor loading
of the enzyme (if pores are very minute). This technique is non recommendable for
reactions involving substrate and enzyme molecules of similar diameters as an optimum
pore size cannot be selected in such cases.

Entrapment

Entrapment is another prominent technique of irreversible enzyme immobilization, where

the enzyme is immobilized by entrapping it within a support matrix or within fiber.

Principle: Enzymes, being large macromolecules, tend to be larger than the substrates or
products. Thus, the enzyme is immobilized within a matrix of appropriate pore size to allow
only the substrates and products of a diameter smaller than the matrix pore size to diffuse
in and out of the mesh respectively .
Matrix used: Common polymers used for enzyme entrapmentinclude alginate, carrageenan,
collagen, polyacrylamide, gelatin,silicon rubber and polyurethane

The enzyme size-to-pore size of support is a deciding factor in selecting the support. Smaller
the pores, lesser the enzyme entrapped, while larger the pores, more the leaking of the
enzyme. Hence, accurate pore size selection of the support is crucial .

Advantage: The method is fast, cheap and easily carried out under mild or physiological
conditions. As the enzyme remains confined within a matrix, it is protected from
contamination by microbes, proteases or other enzymes .

Disadvantage: The meshwork of the matrix cannot support a hugevolume of enzyme

molecules and can lead to enzyme inactivation. The process can be costly at times. The rate
of diffusion of the substrate and product dictate the reaction rate. This is because unless the
substrate molecules diffuse into the mesh, the reaction will not be initiated and according to
Le Chatelier’s principle, the reaction rate does not reach a peak unless the products sieve
out.

Adsorption

In adsorption Weak bonds are involved such as Hydrogen bond and Vander Wall forces.

The matrix particle size must be small. It is simple and easy process. There is limited loss of
enzyme activity.

Disadvantages include desorption and Non-specific adsorption Immobilization by

Adsorption

Module No. 40. Irreversible Immobilization

Covalent bonding

In covalent bonding , enzyme molecule binds to the carrier by a covalent bond. A complex
form through this bonding is stable. There is no enzyme loss during the process.Covalent
 binding occurs between the active part, the, i.e. functional group of an enzyme and carrier
molecule.

Chemical groups

There are several groups in protein structure which could participate in chemical bonding
with the support material.

The order of reactivity of these functional group to the carrier depends upon their charged
status:

-S– > -SH > -O– > -NH2 > -COO– > -OH >> -NH3

Carboxyl group at C term

Amino group at N terminal

1. Carboxyl group of Asp/Glu

2. Amino group of Lys/Arg
3. Phenol ring of Tyrosine
4. Thiol group of Cysteine
5. OH group of Ser/Thre
6. Imdazole group of His
7. Indole group of Tryp

Lysine

Lysine residues are found to be the most useful groups for covalent bonding of enzymes to
insoluble supports due to their widespread surface exposure and high reactivity, especially in
slightly alkaline solutions.Epsilon amino group of lysine take part in the reaction.Lysine residues
appear to be only very rarely involved in the active sites of enzymes.
 Structure of Lysine molecule

Supports Used for Covalent Binding

A huge variety of support materials are used for immobilization, including.

1. Cellulose
2. Polyacrylamide
3. Collagen
4. Gelatin
5. Porus glass
6. Silica
7. CNBr- Agarose

Methods for Irreversible Immobilization

Diazotization Reaction between a primary aromatic amine and nitrous acid to give a diazo
compound.
Peptide bond formation between carboxyl and amino group of carrier and protein.

Diazotization
The process of conversion of primary aromatic amines into its diazonium salt is called
diazotization. This process involves bonding between the amino group of the matrix and tyrosyl
or histidyl group of an enzyme on reaction with NaNO2 and HCl.
Cyanogen Bromide
Cyanogen bromide is used for the activation of Sepharose or Agarose hydroxyl groups.
The activate hydroxyl groups on carbohydrate creates reactive cyanate esters.
CNBr hydrolyzes peptide bonds at the C-terminus of Met residues. Met is the first amino acid
residue of a protein.

Cyanogen bromide can be used to activate a hydroxyl particle to a reactive cyanate ester, which
can then be used to couple
Glutaraldehyde
Glutaraldehyde reacts with the support material containing primary amino groups.
It requires the low-ionic strength.
It acts as a link between the protein and support material.
The amino group of glutaraldehyde reacts with the protein and support.
Summary
This is a permanent method for protein immobilization.
It involves formation of chemical bonds between the biological recognition element and the
support.
Different types of reagents such as Glutaraldehyde and CNBr acts as a linker between the
support material and the Biomolecule.

Module No. 41 Applications of Immobilization

Immobilization is the most important step in the fabrication of biosensor.

It forms the foundation of the Biosensor. Immobilization of BRE provides a a working platform
for the bio-recognition process. Several novel approaches are used for fabricate novel working
platform.
Common methods of immobilization used in biosensing: (A) covalent binding; (B) adsorption; (C)
cross-linking; (D) encapsulation; and, (E) entrapment.

Porous Carbon Material

Porous carbon materials (PCM) were synthesized by sucrose infiltration into template material.
Carbon particles from 125 to 200 μm were generated. Tailor-made porous carbon material is a
promising support for lipase immobilization, which is adaptable in shape and dimension.

Porous carbon material synthesis followed by Thermomyces lanuginosus lipase (TLL)

immobilization.

Immobilization on PANI
Polyaniline (PANI) is a conducting polymer of the semi-flexible rod polymer family.
PANI is used to develop mediator-less biosensors.It is used as support matrix and insulating
material. PANI is an efficient electrode material due to low redox potential and high
conductivity.
Immobilization of enzyme on PANI.

Immobilization on NPG
Nanoporous gold (NPG) provides a high surface area form of gold that is suitable for covalent
modification by self-assembled monolayers. The material can be used as a high surface area
electrode and with immobilized enzymes can be used for amperometric detection schemes.

Fig shows Hydrophobic and ionic interactions between antibody and gold.

Immobilization on NPG (nano particle gold)

NPG films with tunable pore sizes have been successfully developed for enzyme-modified
electrochemical biosensors. By immobilizing GOx on the surface of gold ligament via thiol linking
The bio-functionalized NPG exhibits remarkable glucose sensitivity, detection limit, selectivity
and response time.
Fig shows immobilization of enzyme on NPG with and without thiol.

Module No. 42. Latest trends in Immobilization

Photonic Immobilization Technique (PIT)

It is a novel approach to the functionalization of gold surface that results in antibodies spatially
oriented with their variable part side-up. It relies on the presence of few Trp-Cys-Cys triads in
the Fc region of the antibodies that interacts with high power UV source.

Photonic immobilization of enzyme on Gold electrode under UV light.

Immobilization on Reduced Graphene oxide nano-ribbon and GNP

Tuberculosis is one of the most dreadful diseases caused by Mycobacterium tuberculosis with
more than 9 million individuals suffering from it in 2014. Traditional methods of detection are
not efficient enough for its quick and reliable detection; therefore, it is imperative to develop
methods of its detection in the early stages. A gold nanoparticles (AuNPs, dia. ∼6 nm, 1.81 wt%
loading) are immobilized over reduced graphene oxide nanoribbons (RGONRs). An
ssDNA/Au/RGONR electrode is prepared by immobilizing Au nanoparticles followed by covalent
modification of Au nanoparticles with 5′SH-ssDNA. the ssDNA/Au/RGONR bioelectrode
displayed good linear response to different concentrations of target M. tuberculosis DNA. The
ssDNA/Au/RGONR has shown excellent specificity (92%) to Mycobacterium tuberculosis target
DNA as compared with non-complementary DNA. The Au/RGONR matrix has the potential to be
used as an immobilization platform for single-stranded probe DNAs of different diseases other
than tuberculosis reported here.

GNP(dia. ∼6 nm) are immobilized over reduced Graphene oxide nanoribbons (RGONRs).
An ssDNA/Au/RGONR electrode is prepared by immobilizing Au nanoparticles followed by
covalent modification of Au nanoparticles with 5′SH-ssDNA.

Efficient Direct Electron Transfer Immobilized Electrode

A bioanode with improved enzyme orientation was developed to achieve an efficient enzyme
reaction and Electron transfer on an electrode surface.
This is an application to practical bioelectric devices.Soluble glucose dehydrogenase (s-GDH; EC
1.1.99.17), also named pyrroloquinoline quinone glucose dehydrogenase (PQQGDH-B) from
Acinetobacter calcoaceticus. It is a classical quinoprotein which requires the cofactor
pyrroloquinoline quinone (PQQ) in its reaction with glucose. The enzyme oxidizes glucose to
 gluconolactone with the concomitant reduction of PQQ to PQQH2. A subsequent electron
acceptor is required to transfer the electrons from the reduced cofactor to regenerate the
enzyme. Although the actual physiological electron acceptor for this enzyme is unknown, a
number of artificial electron acceptors, such as phenazine methosulfate (PMS), have been found
effective.

Chapter 05

Nano particles and Nano biotechnology and Biotechnology

Modules 43-51

43. Definition of nano particles, Properties

44 Organic and Inorganic nanoparticles

45. Synthesis of Nano particles and sol gel

46. Nono gold particle

47. Quantum Dots

48. Zinc Oxide Nano Particle

49. Carbon Nano Tubes and Grephne Oxide

50. Nano Porous Gold

 51. Latest Research and Current Trends

Learning Outcome.

1. Students will learn about the nanoparticles and their classification.

2. Students will be able to define the properties of different classes of NPs.
3. Students will be able to explain the importance of gld in NPs and in the form of pours
gold.
4. Students will be able to identify the properties of carbon based nanoparticles and their
applications

43. Definition of nano particles, Properties

Nanoparticles are particles between 1 and 100 nanometres (nm) in size with a surrounding
interfacial layer. The interfacial layer is an integral part of nanoscale matter, fundamentally
affecting all of its properties. The interfacial layer typically consists of ions, inorganic and
organic molecules. Organic molecules coating inorganic nanoparticles are known as
stabilizers, capping and surface ligands, or passivating agents. In nanotechnology, a particle
is defined as a small object that behaves as a whole unit with respect to its transport and
properties. Particles are further classified according to diameter.

IUPAC definition

Particle of any shape with dimensions in the 1 × 10−9 and 1 × 10−7 m range The basis of the
100-nm limit is the fact that novel properties that differentiate particles from the bulk
material typically develop at a critical length scale of under 100 nm. According to ISO
Technical Specification 80004, a nanoparticle is defined as a nano-object with all three
external dimensions in the nanoscale, whose longest and shortest axes do not differ
significantly, with a significant difference typically being a factor of at least 3.

Source
Natural sources of nanoparticles include combustion products forest fires, volcanic ash,
ocean spray, and the radioactive decay of radon gas. Natural nanomaterials can also be
formed through weathering processes of metal- or anion-containing rocks, as well as at acid
mine drainage sites.

Fig shows the routes/ natural sources for production of nanoparticles

Classification

Nanoparticles can be classified into any of various types. Most reliable classification is based
on their Chemistry. Organic Nanoparticles includes dendrimers, liposomes, carbon based
nanoparticles and polymeric nanoparticles.

Fig shows different organic and inorganic Nanoparticles.

Inorganic Nanoparticles include Mesoporous silica, Gold nano particles, Iron oxide,
Quantum dots. A part from Organic and Inorganic NPS engineered particles has gained
much importance. An array of ENPs have been manufactured which include mainly metals,
non-metals, metal oxides, lipids, and polymers) as well as various nanocomposites.

Properties

The principal parameters of nanoparticles are their shape (including aspect ratios where
appropriate), size, and the morphological sub-structure of the substance. The high surface
area to volume ratio of nanoparticles provides a tremendous driving force for diffusion,
especially at elevated temperatures. Sintering ("when the moisture is removed from the
slurry the powder compact is sintered") can take place at lower temperatures, over shorter
time scales than for larger particles.

Shape of Nanoparticles. Nanoparticles are very heterogeneous in shape. Depending on the

production method and manufacturing conditions, nanoparticles exhibit different forms
and structures. Nps come in different shapes including spherical, Triangular, Cubical,
Hexagonal, Oval, Helical, Pyramidal and rod like.

Nanoparticles are presented as an aerosol (mostly solid or liquid phase in air), a suspension
(mostly solid in liquids) or an emulsion (two liquid phases). Clay nanoparticles when
incorporated into polymer matrices increase reinforcement, leading to stronger plastics,
verifiable by a higher glass transition temperature and other mechanical property tests.

These nanoparticles are hard, and impart their properties to the polymer (plastic).
Nanoparticles have also been attached to textile fibers in order to create smart and
functional clothing In the presence of chemical agents (surfactants), the surface and
interfacial properties may be modified. Indirectly such agents can stabilise against
coagulation or aggregation by conserving particle charge and by modifying the outmost
layer of the particle.

Engineered NPs

A Nanoparticles engineered for shape, size, and surface properties.They impart

1) Special functions catalytic behavior

 2) Improved strength
3) Enhanced thermal conductivity and
4) Electrical conductivity
5) controlled release of host molecules.

Fig shows engineered NPs

44. Organic and Inorganic nanoparticles

Organic Nanoparticles

Organic NPs are derived from biological material and processed materials such as lipids and
biological polymers. The processed material based NPs includes carbon NPs.The organic
NPs are biocompatible and are widely used in Biomedical applications.

Polymeric nanoparticles are nano sized solid particles. They are prepared from natural and
synthetic polymers and are used I n drug delivery. Natural material includes Chitosan,
Gelatin.

Synthetic materials include Polylactides, and Poly malic acid Polymeric NPs are solid
colloidal systems in which the therapeutic agent is dissolved, entrapped, encapsulated, or
adsorbed onto the constituent polymer matrix.
Depending upon the process of formation of NPs, the structure of resulting polymeric NPs
may vary from nanospheres (matrix systems in which the drug is dispersed throughout the
particles) to nanocapsules (vesicular reservoir systems in which the drug is confined to an
aqueous or oily cavity surrounded by a single polymeric membrane). Several polymers such
as poly(lactide-co-glycolide) (PLGA), polylactide (PLA), polyglycolide, polycaprolactone
(PCL), poly(d,l-lactide), chitosan, and PLGA–polyethylene glycol (PEG) have been developed
for passive and ligand-targeted delivery of therapeutic moieties.

Liposomes

A liposome is a spherical-shaped vesicle. Liposomes can be between a few nanometers to

even 10 microns in size. They consist of certain lipids (so-called phospholipids, e.g from soy)
together with other materials and form a hollow sphere consisting of one or more double
membranes. It is composed of one or more phospholipid bilayers, which closely resembles
the structure of cell membranes.
The ability of liposomes to encapsulate hydrophilic or lipophilic drugs have allowed these
vesicles to become useful drug delivery systems. Liposomes are intracellular vesicles that
can be used to transport substances such as nutrients and drugs (i) into the cell, (ii) out of
the cell, and (iii) between different parts of a cell. A drug preparation that contains the
active drug inside very tiny, fat-like particles. This form is easier for the body to absorb and
allows more drug to get to the target area of the body, such as a tumor.

Dendrimers are highly branched, star-shaped macromolecules with nanometer-scale

dimensions. Dendrimers are defined by three components: a central core, an interior
dendritic structure (the branches), and an exterior surface with functional surface groups.
Applications of dendrimers typically involve conjugating other chemical species to the
dendrimer surface that can function as detecting agents (such as a dye molecule), affinity
ligands, targeting components, radioligands, imaging agents, or pharmaceutically active
compounds.
Dendrimers is considered as a class of polymers in the nanotechnology for the delivery of
active pharmaceuticals. Hydrophobic drugs can be complexed within the hydrophobic
dendrimer interior to make them water-soluble or drugs can be covalently coupled onto
the surface of the dendrimer.

Inorganic Nano particles

Inorganic nanoparticles cover a broad range of substances including elemental metals,

metal oxides, and metal salts. Inorganic nanoparticles are non-toxic, hydrophilic,
biocompatible and highly stable compared to organic materials. They are widely used in
drug delivery systems.
 Fig shows calcium phosphate NPs.

Metal nanoparticles (NPs) are widespread used in various scientific fields including
biochemistry, optics, and plasmonics, but specially employed in heterogeneous catalysis for
maximizing the exposed area of a metal catalyst, typically a rare, expensive late transition
metal. The metal NPs chemical reactivity is intimately linked to their elementary
composition, atomic arrangement, shape, and size, a variety of features whose role is often
intertwined. Physiochemical properties, high stability, high reactivity, photothermal and
plasmonic properties of metallic NPs make them potent carriers as a therapeutic agent.
Hence, plasmonic NPs like gold NPs have been used in photothermal therapy to kill brain
tumor cells. Quantum dots and magnetic iron NPs have been utilized for bioimaging
purposes . Indeed, some metallic NPs have been used in theranostics due to their
therapeutic and diagnostic applications.

Module No. 45 Synthesis of Nano particles and sol gel

Free nanoparticles are formed through either the breaking down of larger particles or by
controlled assembly processes. Natural phenomena and many human industrial and
domestic activities, such as cooking, manufacturing or road and air transport release
nanoparticles into the atmosphere.
Methods
 There are three main methods that are used to produce nanoparticles.

1. Biological method involved living organism for the synthesis.

2. Physical method employed different physical techniques.
3. Chemical methods used different chemical reagents and techniques.

Plasma source system

In a plasma source system, an inert gas, such as argon, flows into a chamber..
Fig shows a plasma system for NPs synthesis.

Gas carries macroscopic particles for nanoparticles production. The plasma is produced by
high-power radio frequency signal applied to the carrier gas. The ions then condense into
nanoparticles.

Green synthesis method; provides a faster metallic nanoparticle production by offering an

environmentally friendly, simple, economical and reproducible approach. Given the wide
range of applications of metallic nanoparticles produced, biological methods play a major
role in the synthesis of metallic nanoparticle.
Fig shows the fabrication of Silver nanoparticles.

Sol–gel

The sol–gel process is a wet-chemical technique widely used recently in the fields of
materials science and ceramic engineering. There are three main steps in the formation of
sol gel.
1) Partial hydrolysis of metal alkoxides to form reactive monomers,
2) Condensation of these monomers to form colloid-like oligomers (sol formation).
3) Additional hydrolysis to promote polymerization and cross-linking leading to a
three-dimensional matrix (gel formation).

Fig shows the method for the synthesis of sol gel from metal alkaloid.
Typical precursors are metal alkoxides and metal chlorides, which undergo hydrolysis and
polycondensation reactions to form either a network "elastic solid" or a colloidal
suspension (or dispersion) – a system composed of discrete (often amorphous)
submicrometer particles dispersed to various degrees in a host fluid. Formation of a metal
oxide involves connecting the metal centers with oxo (M-O-M) or hydroxo (M-OH-M)
bridges, therefore generating metal-oxo or metal-hydroxo polymers in solution. Thus, the
sol evolves toward the formation of a gel-like diphasic system containing both a liquid phase
and solid phase whose morphologies range from discrete particles to continuous polymer
 networks. Removal of the remaining liquid (solvent) phase requires a drying process, which
typically causes shrinkage and densification.
The sol–gel approach is a cheap and low-temperature technique that allows for the fine
control of the product’s chemical composition. Even small quantities of dopants, such as
organic dyes and rare earth metals, can be introduced in the sol and end up uniformly
dispersed in the final product. It can be used in ceramics processing and manufacturing as
an investment casting material, or as a means of producing very thin films of metal oxides
for various purposes. Sol–gel derived materials have diverse applications in optics,
electronics, energy, space, (bio)sensors, medicine (e.g., controlled drug release) and
separation (e.g., chromatography) technology.

45. Nono gold particle

Gold nanoparticles (AuNPs) are small gold particles with a diameter of 1 to 100 nm which,
once dispersed in water, are also known as colloidal gold.

Fig shows the gold bricks.

Colloidal gold nanoparticles have been utilized for centuries by artists due to the vibrant
colors produced by their interaction with visible light. More recently, these unique
optoelectronic properties have been researched and utilized in high technology
applications such as organic photovoltaics, sensory probes, therapeutic agents, drug
delivery in biological and medical applications, electronic conductors and catalysis. The
optical and electronic properties of gold nanoparticles are tunable by changing the size,
shape, surface chemistry, or aggregation state.
Shapes and color emission

AuNPs come in different shapes such as, star, rods, sphere and branch. depending upon
the mode of synthesis.For small (~30nm) mono-disperse gold NPs, there is an absorption of
light in the range ~450 nm.For larger AuNPs light is absorbed in the range of ~700 nm is
reflected, yielding a rich red color

Gold nanoparticles’ interaction with light is strongly dictated by their environment, size and
physical dimensions. Oscillating electric fields of a light ray propagating near a colloidal
nanoparticle interact with the free electrons causing a concerted oscillation of electron
charge that is in resonance with the frequency of visible light. These resonant oscillations
are known as surface plasmons. For small (~30nm) monodisperse gold nanoparticles, the
surface plasmon resonance phenomenon causes an absorption of light in the blue-green
portion of the spectrum (~450 nm) while red light (~700 nm) is reflected, yielding a rich red
color. As particle size increases, the wavelength of surface plasmon resonance related
absorption shifts to longer, redder wavelengths. Red light is then absorbed, and blue light
is reflected, yielding solutions with a pale blue or purple color. As particle size continues to
increase toward the bulk limit, surface plasmon resonance wavelengths move into the IR
portion of the spectrum and most visible wavelengths are reflected, giving the
nanoparticles clear or translucent color. The surface plasmon resonance can be tuned by
varying the size or shape of the nanoparticles, leading to particles with tailored optical
properties for different applications.

Fig shows color display by gold NP depending upon the size.

This phenomenon is also seen when excess salt is added to the gold solution. The surface
charge of the gold nanoparticle becomes neutral, causing nanoparticles to aggregate. As a
result, the solution color changes from red to blue. To minimize aggregation, the versatile
surface chemistry of gold nanoparticles allows them to be coated with polymers, small
molecules, and biological recognition molecules.

Synthesis

An other method for the synthesis of AuNPs is the reduction of gold salt by Sodium tetra
borate. This is a two step reaction. First step is the seeding in the presence of sodium tetra
borate. Second step is the separation of gold particles in the presence of Ascorbic Acid.
 Schematic synthesis if gold NPs.

Green Synthesis

A Green method for the isolation of AuNPs is also reported. A gold compound chloroauric
acid (HAuCl4) is reduced with plant extract. Size of Au nanoparticles was found to be around
20 nm and spherical in shape. This is a simple method.

Application

1) Electronics
2) Therapeutic Agent Delivery.
3) Sensors
4) Probes
5) Diagnostics
6) Biosensors
7) Catalysis
8) Tissue Engineering

A schematic diagram shows the applications of Gold Nps.

In summary
I. AuNPs are most suitable nano materials for Biosensor fabrication due to their
II. Stability
III. Biocompatibility,
IV. Conductive properties
V. Surface plasmon effect
VI. High surface area
VII. AuNPs can be prepared by chemical methods and Green method.
46. Quantum Dots

Introduction

. Quantum dots are tiny particles or nanocrystals of a semiconducting material with

diameters in the range of 2-10 nanometers (10-50 atoms). They were first discovered in
1980. They display unique electronic properties, intermediate between those of bulk
semiconductors and discrete molecules, that are partly the result of the unusually high
surface-to-volume ratios for these particles.2-4 The most apparent result of this is
fluorescence, wherein the nanocrystals can produce distinctive colors determined by the
size of the particles. The core of QDs is usually composed of elements from groups II–VI
such as CdSe, CdS or CdTe, groups III–V such as InP or InAs, or groups IV–VI such as PbSe.
QDs are in fluorescent in nature QDs are comprised of two semiconductor materials.
Example lead sulfide and lead selenide.

Quantum dots (QDs) are man-made nanoscale crystals that that can transport electrons.
When UV light hits these semiconducting nanoparticles, they can emit light of various
colors. These artificial semiconductor nanoparticles that have found applications in
composites, solar cells and fluorescent biological labels. Quantum dots are tiny particles of
a semiconducting material with diameters in the range of 2-10 nanometers (10-50 atoms).
They can emit any color of light from the same material simply by changing the dot size.
They have immense applications in the field of Biosensors. Color Emission

A small Quantum dots require more energy to excite it. Smaller dots with higher energy
produce higher frequencies and shorter wavelengths. The smaller emit blue light and
intermediate emit green light. The larger dots produce red color.

Synthesis

There are several ways to fabricate quantum dots.

1. Colloidal synthesis QDs are synthesized from solutions, like a chemical processes.
2. Plasma synthesis , is the gas-phase approaches for the production of QDs.
3. Green method plant derived material is reduced to produce QDs.
Quantum Confinement Effect

Color Emission

A small Quantum dots require more energy to excite it. Smaller dots with higher energy
produce higher frequencies and shorter wavelengths. The smaller size QDs emit blue light
and intermediate emit green light. The larger size QDs dots produce red color.

Due to their small size, the electrons in these particles are confined in a small space
(quantum box), and when the radii of the semiconductor nanocrystal is smaller than the
exciton Bohr radius (exciton Bohr radius is the average distance between the electron in
the conduction band and the hole it leaves behind in the valence band). Generally, as the
size of the crystal decreases, the difference in energy between the highest valence band
and the lowest conduction band increases. More energy is then needed to excite the dot,
and concurrently, more energy is released when the crystal returns to its ground state,
resulting in a color shift from red to blue in the emitted light. As a result of this
phenomenon, these nanomaterials can emit any color of light from the same material
simply by changing the dot size. Additionally, because of the high level of control possible
over the size of the nanocrystals produced, these semiconducting structures can be tuned
during manufacturing to emit any color of light.
 Classification

Quantum dots can be classified into different types based on their composition and
structure.

1. Core-Type Quantum Dots

2. Core-Shell Quantum Dots

3. Alloyed Quantum Dots

4. Quantum Dots Applications

Core-Type Quantum Dots

These nano dots can be single component materials with uniform internal compositions,
such as chalcogenides (selenides, sulfides or tellurides) of metals like cadmium, lead or zinc,
example, CdTe. The photo- and electroluminescence properties of core-type nanocrystals
can be fine-tuned by simply changing the crystallite size.

Core-Shell Quantum Dots

The luminescent properties of quantum dots arise from recombination of electron-hole

pairs (exciton decay) through radiative pathways. However, the exciton decay can also
occur through nonradiative methods, reducing the fluorescence quantum yield.
One of the methods used to improve efficiency and brightness of semiconductor
nanocrystals is growing shells of another higher band gap semiconducting material around
them. These particles with small regions of one material embedded in another with a wider
band gap are known as core-shell quantum dots (CSQDs) or core-shell semiconducting
nanocrystals (CSSNCs). For example, quantum dots with CdSe in the core and ZnS in the sh
exhibit greater than 50% quantum yield. Coating quantum dots with shells improves
quantum yield by passivizing nonradiative recombination sites and also makes them more
robust to processing conditions for various applications. This method has been widely
explored as a way to adjust the photophysical properties of quantum dots.8-10

Alloyed Quantum Dots

The ability to tune optical and electronic properties by changing the crystallite size has
become a hallmark of quantum dots. However, tuning the properties by changing the
crystallite size could cause problems in many applications with size restrictions.
Multicomponent dots offer an alternative method to tune properties without changing
crystallite size. Alloyed semiconductor nanodots with both homogeneous and gradient
internal structures allow tuning of the optical and electronic properties by merely changing
the composition and internal structure without changing the crystallite size. For example,
alloyed quantum dots of the compositions CdSxSe1-x/ZnS of 6nm diameter emits light of
different wavelengths by just changing the composition. Alloyed semiconductor quantum
dots formed by alloying together two semiconductors with different band gap energies
exhibited interesting properties distinct not only from the properties of their bulk
counterparts but also from those of their parent semiconductors. Thus, alloyed
nanocrystals possess novel and additional composition-tunable properties aside from the
properties that emerge due to quantum confinement effects.

The material emits different color of light by tuning the composition.

Quantum Dots Applications

The unique size and composition tunable electronic property of these very small,
semconducting quantum dots make them very appealing for a variety of applications and
new technologies.

Quantum dots ar particularly significant for optical applications owing to their bright, pure
colors along with their ability to emit rainbow of colors coupled with their high efficiencies,
longer lifetimes and high extinction coefficient. Examples include LEDs and solid state
lighting, displays and photovoltaics.

Being zero dimensional, quantum dots have a sharper density of states than higher-
dimensional structures. Their small size also means that electrons do not have to travel as
far as with larger particles, thus electronic devices can operate faster. Examples of
applications taking advantage of these unique electronic properties include transistors,
solar cells, ultrafast all-optical switches and logic gates, and quantum computing, among
many others.

The small size of dots allow them to go anywhere in the body making them suitable for
different bio-medical applications like medical imaging, biosensors, etc. At present,
fluorescence based biosensors depend on organic dyes with a broad spectral width, which
limits their effectiveness to a small number of colors and shorter lifetimes to tag the agents.
On the other hand, quantum dots can emit the whole spectrum, are brighter and have little
degradation over time thus proving them superior to traditional organic dyes used in
biomedical applications.

Qds are semiconductor, core-shell and alloyed quantum dots tuned to emit different colors
in the visible spectrum while exhibiting high quantum yield. Nanocrystals are available in
both aqueous and organic formulations suitable for use in different applications.
47. Zinc Oxide Nano Particle

Zinc oxide nanoparticles are nanoparticles of zinc oxide (ZnO) that have diameters less than
100 nanometers. They have a large surface area relative to their size and high catalytic
activity. ZnO is a wide-bandgap semiconductor with an energy gap of 3.37 eV at room
temperature. nanoparticle zinc oxide is safe and effective (all research has shown that
particles in the size range used in sunscreens (>30nm) do not penetrate the skin and are
completely safe to use in sunscreen creams and lotions),

Zinc oxide is a metal oxide. Zn is a chemical element with the symbol Zn and atomic number
30. Zinc is a slightly brittle metal at room temperature.It is the first element in group 12 of
the periodic table. Zinc oxide crystalizes in two main forms Hexagonal Wurtzite crystals and
cubic zinc blend crystals. The Wurtzite structure is most stable at ambient conditions. The
zinc oxide nano particles exist in wurtzite crystal form. The atomic position of crystal is same
as hexagonal diamond.

Electrical Properties

Zinc oxide has a relatively large direct band gap of around 3.3 eV at room temperature. The
advantages associated with large band gap include:

1. Higher breakdown voltage

2. Ability to sustain large electrical field
 3. Lower electronic noise.
4. High temperature operation

High power operation Zinc oxide is used for the synthesis of nanoparticles.. ZnO-NPs are
widely utilized in several industrial areas such as UV light-emitting devices, ethanol gas
sensors, photo-catalysts , pharmaceutical, and cosmetic industries . Properties including
non-toxic, self-cleansing , compatible with skin, antimicrobial, and dermatologic associate
degreed are employed as UV-blocker in sunscreens and lots of medical specialty
applications . Coated nano zinc oxide are renowned for its UV blocking capabilities.

Fig shows ZnU MPs.

Quantum properties and others

The UV-vis absorption spectrum shows an absorption band at 355 nm due to ZnO
nanoparticles. The photoluminescence spectrum exhibits two emission peaks First at
392 nm Second is located at 520 nm. Zinc oxide is a metal oxide. Zinc oxide nano particles
are within the range of 1-100 nano-meters.

1. They are chemically stable.

2. Thermal resistant
3. High catalytic activity
4. Long shelf life
5. Anti-bacterial activity
 6. Zn is an essential mineral

It is mostly used as a viable ingredient for developing sunscreen cosmetic products.

Stringent federal regulations and standards regarding the safety of cosmetic products could
improve coated nano zinc oxide market dynamics.. The zinc oxide nanoparticles disrupt
bacterial cell membrane integrity, reduce cell surface hydrophobicity, and down regulate
the transcription of oxidative stress-resistance genes in bacteria. They enhance intracellular
bacterial killing by inducing Reactive Oxygen Specie production ZnO seems to powerfully
resist microorganisms,

synthesis of ZnO nanoparticles

ZnO nanoparticles can be synthesized by direct precipitation method using zinc nitrate and
KOH as precursors Chemical and physical synthesis strategies are pricey and require in
depth labor and time. Moreover, giant quantities of secondary waste are generated,
ensuing from the addition of chemical agents for precipitation and reduction within the
processes.

Chemical synthesis strategies that are typical such as chemical precipitation result in the
presence of some poisonous chemical species which are adsorbate on the surface that will
have adverse effects in medical applications.

There are reactions that need heat and/or air mass to initiate, whereas some others need
inert atmosphere protection, and/or utilization poisonous matters similar to Hydrogen di
sulfide, poisonous model and stabilizer, and bimetallic precursors. Chemicals which are
used for nanoparticles synthesis and stabilization are poisonous and result in non-
ecofriendly byproducts. Chemical strategies result in the presence of some noxious
chemicals that are absorbable on the surface and have adverse effects in medical
application.

Several physical and chemical procedures are used to synthesize huge quantities of
nanoparticles in an exceedingly and comparatively short period of time . Among the most
generally used approaches for preparing of ZnO-NPs, solution-based routes are very wide
and simple such as chemical precipitation sol-gel solvothermal , hydrothermal and etc.

Green synthesis of nanoparticles makes use of environmental friendly, non-toxic and safe
reagents.

Applications

Applications

ZnO nano particles has application in many fields. Large surface area relative to their size

1. Medical Science
 2. Nano generator
3. Gas sensor
4. Biosensor
5. Solar cells
6. Photodetector
7. Biosensors

48. Carbon Nano Tubes and Graphene Oxide

Gtaphene the form of a single layer of atoms in a two-dimensional hexagonal lattice, in

which one atom forms each vertex. It is the basic structural element of other allotropes,
including graphite, charcoal, carbon nanotubes and fullerenes. It can also be considered as
an indefinitely large aromatic molecule, the ultimate case of the family of flat polycyclic
aromatic hydrocarbons.. Graphene's hexagonal lattice can be regarded as two interleaving
triangular lattices. This perspective was successfully used to calculate the band structure
for a single graphite layer using a tight-binding approximation.

Fig illustrates the structure of graphene.

Graphene's stability is due to its tightly packed carbon atoms and a sp2 orbital hybridization
– a combination of orbitals s, px and py that constitute the σ-bond. The final pz electron
makes up the π-bond. The π-bonds hybridize together to form the π-band and π∗-bands.
These bands are responsible for most of graphene's notable electronic properties, via the
half-filled band that permits free-moving electrons... Its sp2 hybridization & very thin
atomic thickness. These properties are enable graphene to break so many records in term
of strength, electricity & heat conduction.

Fullerene

Twisting and warping of the graphene sheet results in formation of 0-dim Fullerenes..
Fullerene is single and double bonds with fused rings of five to seven atoms. The molecule
may be a hollow sphere, ellipsoid, tube, or many other shapes and sizes.

Carbon Nanotubes

Rolling of the sheet along an axis results in formation of 1-dim CNTs. Carbon nanotubes
(CNTs) are cylindrical molecules that consist of rolled-up sheets. The tensile strength is
approximately 100 times greater than that of steel of the same diameter. Nanotubes are
elastic in nature. Carbon nanotubes are large molecules of pure carbon that are long and
thin and shaped like tubes, about 1-3 nanometers (1 nm = 1 billionth of a meter) in
diameter, and hundreds to thousands of nanometers long.

Structure and stability

1. The unique strength of carbon nanotubes is due to orbital hybridization.

2. It causes the bonds between adjacent carbon atoms to be of the sp2 type.
3. These bonds, which are similar to those of graphene, are stronger than the sp3
bonds in alkanes and diamond.

As individual molecules, nanotubes are 100 times stronger-than-steel and one-sixth its
weight. These three types of CNTs are armchair carbon nanotubes, zigzag carbon
nanotubes, and chiral carbon nanotubes. The difference in these types of carbon nanotubes
are created depending on how the graphite is “rolled up” during its creation process. Each
individual nanotube is only between 2 and 4 nanometers across, but each one is incredibly
strong and tough.

It's only 10% the weight of steel but has hundreds of times the strength. CNTs can act as
antennas for radios and other electromagnetic devices. Conductive CNTs are used in
brushes for commercial electric motors. They replace traditional carbon black. The
nanotubes improve electrical and thermal conductivity because they stretch through the
plastic matrix of the brush.

Single Walled CNTs

The structure of an ideal (infinitely long) single-walled carbon nanotube is a regular

hexagonal lattice.It is drawn on an infinite cylindrical surface, whose vertices are the
positions of the carbon atoms.

There are three types of CNTs. The zigzag, chiral and armchair configurations.

Graphene Oxide

Graphene oxide (GO) is a single monomolecular layer of graphite. It has various oxygen-
containing functionalities such as epoxide, carbonyl, carboxyl, and hydroxyl groups.
 GO is one of the suitable nanoparticles to enhance the hydrophilicity of the membrane. GO
is usually prepared by Hummer’s method. GO contains functional groups making it more
dispersed in the polymeric solution GO is an intriguing material. As a functionalized single
atomic layer of carbon, the thickness of GO is approximately 1 nm. On the other hand, the
lateral length of GO sheets can span to micrometres It has optical and electronic properties
enable graphene oxide to be used in many fields. structural model of GO shows that the
edges of a GO sheet consist predominantly of ionizable carboxylic acid groups, which are
hydrophilic. Meanwhile, the basal plane consists of both hydrophilic oxygen-containing
functional groups and hydrophobic aromatic domains.

49. Nano Porous Gold Nanoporous gold

Nano Porus Gold is a sponge like with is an interconnected three-dimensional network.The

network formed with ligament and pore sizes typically in the range of 20–50 nm.It has
emerged as a promising solid catalyst for reactions It allows to work at moderate
conditions.

Nanoporous gold is actually a Au-rich Ag–Au alloy which, specifically the Ag0.03Au0.97
composition, combines high reactivity and selectivity for a wide variety of oxidation
reactions, from simple CO oxidation to complex oxygen-assisted coupling reactions. Based
on a self-organisation process.
It is a 3 dimensional sponge like gold structure evolves with ligaments in the range of only
a few tens of nanometers. Due to its continuous porosity, the material can be penetrated
by gases which then adsorb and interact with the surface.

Fig shows the nanoporous gold. Au atoms are shown in orange wuile Ag atoms atr shown
in blue.

Nanoporous gold (np-Au) has many interesting and useful properties that make it a material
of interest for use in many technological applications. Its biocompatible nature and ability
to serve as a support for self-assembled monolayers of alkanethiols and their derivative
make it a suitable support for the immobilization of carbohydrates, enzymes, proteins, and
DNA. Unique properties

NPG is a heterogeneous catalyst due has unique structural properties, including

1. Open nanoporosity
2. High surface area
3. Excellent electrical conductivity,
4. Nontoxicity nature
5. Easy recyclability
6. tunable surface chemistry.
 Catalytic properties of NP fold ate shown inpic.NPG is a heterogeneous catalyst due has
unique structural properties, including

1. Open nanoporosity
2. High surface area
3. Excellent electrical conductivity,
4. Nontoxicity nature
5. Easy recyclability
6. tunable surface chemistry

Its chemically inert, physically robust and conductive high-surface area makes it useful for
the design of electrochemistry-based chemical/bio-sensors and reactors. Furthermore, it is
also used as solid support for organic molecular synthesis and biomolecules separation. Its
enhanced optical property has application in design of plasmonics-based sensitive
biosensors. In fact, np-Au is one of the few materials that can be used as a transducer for
both optical and electrochemical biosensing. Due to the presence of low-coordination
surface sites, np-Au shows remarkable catalytic activity for oxidation of molecules like
carbon monoxide and methanol. Owing to the importance of np-Au, in this chapter we will
highlight different strategies of fabrication of np-Au and its emerging applications based on
its unique properties. The catalytic performance of nanoporous gold

1. CO oxidation
2. Hydrogen oxidation and production
3. Oxidation of alcohols
4. Reactions in liquid phase
5. Reduction reaction or hydrogenation is more difficult because H‐H dissociation is not easy
on gold.

Fig shows shows catalytic properties of nanoporus gold.

The catalytic performance of nano particulate gold strongly depends on size and support.
CO oxidation has been the representative model reaction for investigating the catalytic
efficiency. It is found to be very effective at temperatures as low as 30°C when nanoporus
gold was used as catalylst.

Fig shows Aerobic Methanol Oxidation over Unsupported Nanoporous Gol

Selective oxidation of alcohol is one of the important catalytic conversions in petrochemical
industry

Aerobic Methanol Oxidation over Unsupported Nanoporous Gold

In Gold‐based catalysts molecular oxygen can be used as an oxidant for the selective
oxidations.It works under mild conditions , that is, at low temperature (<100°C) and under
an ambient pressure/

CO Oxidation

The catalytic performance of nano particulate gold strongly depends on size and support.
CO oxidation has been the representative model reaction for investigating the catalytic
efficiency.
 It is found to be very effective at temperatures as low as 30°C when nanoporus gold was
used as cataylst.

Fabrication process for the fabrication and functionalization of high-surface-area,

nanoporous gold films. The fabrication process involves the electrodeposition of a binary
gold-zinc alloy at gold wires, followed by subsequent electrochem. dealloying of the less
noble component zinc from the surface.

Module No 5. Latest trends and research

Nanoparticles for bone regeneration

1. Nano particles are ultra small particles within the range of nano scales.
2. They have high loading capacity due to large surface area;
3. They can easily travel into the target cell due to their small size, shape, and Bioactive
hydrogels for bone regeneration.

Carbon nanotubes (CNTs) are suitable scaffold materials that have proved to support osteoblast
proliferation in bone regeneration. CNTs possess exceptional mechanical, thermal, and electrical
properties, facilitating their use as reinforcements or, in combination with other biomaterials, to
improve and to support bone growth.

4. The human skeleton is the internal framework of the human body.

5. In elderly people bone regeneration is a problem due to decrease in level of certain
hormone ands growth factors.
6. To address the issue scientists has introduced a Bioactive hydrogel for bone
regeneration. crystallinity.
To mimic the natural bone nanocomposite architecture, novel biomaterials and nanofabrication
techniques are currently being employed and many different nanostructures have already been
designed and tested. Electrospinning has been extensively applied to create bone nanofiber
scaffolds and biomaterials typically used for this purpose, including synthetic organic polymers
such as PCL, PLGA, PLLA, Chitosan, and silk fibroin.

PLLA nanofibers are often functionalized to improve their biological performance with peptides
such as RGD (Arg-Gly-Asp); with osteogenic molecules such as hydroxyapatite; or with proteins
such as collagen and the growth factor bone morphogenic protein 2 (BMP-2). It was found that
direct incorporation of BMP-2 into PLLA nanofibers enhances the osteoinductivity of the
scaffolds.

Titanium, as a biocompatible material, has been used to enhance implant incorporation in bone
for dental, craniofacial, and orthopedic applications. Studies have demonstrated that
nanoporous titanium dioxide (TiO2) surface modification alters nanoscale topography improving
soft tissue attachment on titanium implants surface

The advantages of nanomaterials as therapeutic and diagnostic tools are vast, due to
design flexibility, small sizes, large surface-to-volume ratio, and ease of sur.face
modification. The potential of these bio-devices has shown promising results in vitro,
and some of them have also been successfully tested in vivo with animal models
Graphene Oxide for Textile wastewater Treatment

1) Textile waste water contained a considerable amount of carcinogenic dyes.

2) Graphene oxide is an oxidized form of Graphene.
3) GO is helpful in efficiently removing the organic dyes from wastewater.
 Fig shows the dye aabsortion by GO based hydrogel from water,

Biosensors

Chapter 06
Modules 52-68

TYPES OF SENSORS AND BIOSENSORS

1. 52. Electrochemical sensors Types

2. 53. Mass Sensitive Sensors
3. 54. Conducto metric sensor
4. 55. Calorimetric Sensors
5. 56. Amperometric sensors
6. 57. Potentiometric sensors
7. 58. Piezo electric sensors
8. 59, Surface Plasmon resonance sensors
9. 60. Optical sensors Introduction Principle
10. 61. Light sources for optical sensors
11. 62. Types of optical sensors
12. 63. Applications of Optical sensors
13. 64. Temperature sensors working and principle and applications
14. 65. Pressure sensors Definition Working
15. 66. Types and Applications of Physiological sensor
 16. 67. Types of Biosensors
17. 68. Latest Research and Current Trends

Learning Outcome.
Students will be able to differentiate between different types of sensors.
Students will be able to describe the role of a specific sensor in a Biosensor.
The mechanism of Sensor working will be explained to the students.
Students will be able to classify different types of Biosensors.

SENSORS

Sensor is a device that is used to sense a physical variable, which includes what is not limited to
temperature, strain, humidity, pressure, mass, light and voltage. To sense these variables we
need to convert them into a universal and easily assessable signal usually a voltage. This voltage
signal changes continuously with time, and is directly proportional to a corresponding physical
variable. A component responsible for this conversion is called transducer. The resulting voltage
signal is usually an analogue signal. The analog voltage signal is then transferred to a computer
or a microprocessor which recognizes digital signals only. An analog signal is converted into a
series of high and low voltages (for example, binary numbers). Such that a small fluctuation in
the analog signals, such as noise does not affect the over all digital signals. An analog digital
converter (A/D) performs this conversion. Today all in one type sensors have become very
popular. In corporate a transducer, A/D convertor, a micro processor and small crystal display
(LCD) panel. The signal can be sent to a computer universal series bus (USB) from a A/D convertor.
They have 4 types of sensors:

1. Electrochemical sensors
2. Mass Sensitive Sensors
i) Piezo electric sensors
ii) Surface plasmon resolence based sensors
3. Calorimetric Sensors

Electrochemical Sensors

Electrochemical sensors are based on the principle of Electrochemistry. Oxidation and reactions
are monitors inthe Electrochemical Sensor. Oxidation and reduction involved the transfer of
electrons between two electrodes, leading to the generation of current. A typical electrochemical
It is composed of following components.

1. Gas Permeable Membrane

 2. Sensing Electrodes
3. Reference Electrode
4. Counter electrode
5. Electrolyte
6. Filter

Working

Electrochemical sensors are operated based on the diffusion of gas of interest into the
sensor (Physical Process). The diffused gas in turn gets oxidized or reduced at the sensing
electrode. This results in the production of an electrical signal that is proportional to the
gas concentration.

Factors effecting

Pressure changes. The differential pressure within the sensor can cause sensor damage.

Temperature. Electrochemical sensors are also quite sensitive to temperature.

Humidity. As the sensor is dependent on REDOX reaction humidity can alter the catalytic
properties of the system.

These sensors employ REDOX reactions to quantifying the amount of an analyte. The current
flowing through the system or the potential difference between the electrode as a result of the
oxidation and reduction reactions involving the analyte are used for its quantification in the
sample. The electrochemical sensors do not suffer from the drawbacks of optical sensors. They
have stable output at high sensitivity, fast response and suffer from lesser interference.

Alcohol Sensor

Ethanol is oxidized to acetic acid in the presence of Oxygen. A layer of Titanium oxide on alumina
act as a catalyst and reaction occurs. The electric current produced by this reaction is measured
by a microcontroller, is proportional to blood alcohol content (BAC).

Reaction

When the user exhales into a breath analyzer, any ethanol present in their breath is oxidized to
acetic acid at the anode:

Type of Electrochemical

1. Conductro-metric Sensors
 A sensor which measure changes in the conductance of systems, due to presence of an
analyte.

2. Potentiometric Sensors

Which measures the potential difference between a working a reference electrode. The
reference electrode is one who potential remains in variant during entire duration of
measurement. The working electrode undergoes significant changes in its potential even
for small changes in the analyte concentration. Potentiometric measurements are also
carried out to monitor the accumulation of charge at zero current created by selective
binding of the analyte at the electrode surface. The electrode may be selective for certain
ions or gases including F-, I-, CN-, Na+, K+, Ca2+, H+, NH++, CO2, NH+ etc.

Amperometric Sensors

It involves measuring the current generated by electrochemical oxidation or reduction of

electronegative species, and constant applied potential. First measurement sensitivity
(ability to sense even 10-9 M concentration) and ability to perform measurements on
turbid/opaque solutions are its advantages over conventional optical sensors. However,
pH-sensing mechanisms require weakly buffered or non-buffered solutions and this is a
drawback for this category of sensors. Amperometry is one of anelectrochemical
methods. The potential applied to a sensing electrode is controlled instrumentally; As a
result current is generated due to REDOX reactions. The current occurring at electrode
surface is recorded as analytical signal.

Fick's laws of diffusion describe diffusion and were derived by Adolf Fick in 1855. They
can be used to solve for the diffusion coefficient, D. Fick's first law can be used to derive
his second law which in turn is identical to the diffusion equation. A diffusion process that
obeys Fick's laws is called normal diffusion (or Fickian diffusion); otherwise, it's called
Anomalous diffusion (or non-Fickian diffusion).

Types of Electrodes

Dropping Mercury Electrode

The dropping mercury electrode (DME) is a working electrode made of mercury and used in
polarography. These electrodes are used in electrochemical studies using three electrode
systems when investigating reaction mechanisms related to redox chemistry among other
chemical phenomena.

The platinum rotating electrode

This electrode is mounted in the shaft of the motor and rotated at a constant speed of 600
rotations per minute. .A rotating platinum micro electrode OR Dropping mercury electrode is
inserted into cell and this electrode is made as cathode. A saturated calomel electrode (SCE) is
made as reference electrode as anode.

Twin Polarized Micro Electrode

It is also known as dead stop end point or bi-amperometric method. This method is performed
by using two platinum electrodes. This technique is specifically applied when the oxidation
reduction exists both before as well as after the equivalent point.

Mass sensitive measurements sensors

PIEZO electro sensors

PIEZO electro sensors used piezo electric crystal for detection. Piezo electric material are those
that produce an electric signal in response to mechanical force. Such as quartz crystals. The
principal is that the electrical voltage can be generated when piezo crystal is compressed or
stressed. This is called piezo electric effect. This effect is reversible meaning that quarts can be
lengthen or shorten when an electric voltage is applied piezo electricity referred to the materials
ability to exhibit the piezo electric effect. Quartz is the most popular material used in the piezo
electric sensors. Quartz (SiO2) has a unique tetra hadron crystal structure. There are 4 oxygen
molecules in a single tetra hadron (SiO4) where all oxygen molecules are shared with nearby tetra
hydra thus, it becomes SiO2. These tetra hydra are stacked in a highly ordered manner to create
a much larger crystal. Like any other material, the distribution of polarity not uniform throughout
its crystal structure, creating dipoles. As typical quartz is mono crystal, indicating all tetra hydra
are order inward direction. Thus, the dipoles are also ordered in one direction. To use the mono
crystal quartz as a piezo electric sensor it is important to cut the crystal in a certain angle so that
diploes are aligned parallel to the electric voltage. The most widely used angle is 35 degree. Once
the dipoles are aligned parallel to the electrode piezo electric can be observed. When quartz
crystal is compressed, the dipole itself is also compressed. Creating an electrical voltage that has
the same directions of the dipole. When the quartz crystal is stressed the dipole is also stressed
creating a negative electrical voltage.

In these sensors, the crystal are made to vibrate at a specific frequency by the application of
electrical signals. The oscillation frequency of the crystal depends upon the applied frequency.
For sensing applications, a bio-capture layer is introduced on the surface of crystal. The bio-
capture layer consists of a biomolecules that will exhibit the specific binding with the analyte.
Generally, antibodies are the most commonly employed bio-capture molecules and such sensors
are referred as piezo electric amino sensors. The piezo electric sensors are among most sensitive
biosensors available. The mechanism on action is that upon the addition of the sample specific
binding occurs between the bioreceptor and the analyte and consequently and mass change
occurs leading to a change in oscillation frequency that leads to production of electric signals that
are detected. The piezo electric amino sensors have been successfully demonstrated for the
ultrasensitive detection of HIV (Human Immuno Deficiency Virus)

Surface Plasmon Resolence Based Sensors

Plasmon are describe as the vibration of the electron cloud in a molecule. These plasmon oscillate
at particular frequency characteristic the material. Surface plasmons are those species of
plasmon whose oscillations are confined to be surface of the material. Generally gold or silver
surfaces are preferred for use in SPR based sensors. Then electromagnetic variation is allowed to
fall on a metal surface (gold or silver) at a particular of angle of incidence the frequency of the
electromagnetic radiation matches the frequency of the vibration resulting in rasonance (and
hence the name, surface plasmon resonance) This resonant angle depends upon the refractive
index of medium, which in term determine by the local mass density of the metal surface. If the
surface the metal film is modified with the capture melcule (Antibody / receptor) There an
addition of the sample, specific binding occurs between the analyte and its ligand leading to a
change in the mass and further resulting in change in the angle of the resonance. This change can
be quantified for determining the concentration of the analyte. SPR based biosensors have been
used to understand the functional aspects of HIV, both qualitatively and quantitatively. The
receptor was incorporated into the retroviral particals and immobilize surface on the metal film
in an SPR based sensor.. One of the major disadvantages is that turbid solutions cannot be used
for detection. In some cases, small ligands may interfere with the binding .

Temperature sensor or Probe. Temperature probes are another term for thermocouples, which
are temperature measuring and monitoring devices that sense the electrical conductivity
produced by heat in gas or liquids to maintain an appropriate temperature in a wide variety of
different systems that use heat energy.

Working

A temperature sensor basically senses temperature, but it does it in a number of ways. A contact
temperature sensor will read the temperature of an object that it is attached to physically.
Contact sensors work in many ways. Thermocouples work using the Seebeck effect, which has to
do with the temperature change in electrical circuits. Thermocouple temperature probes are
devices that generate a temperature-dependent voltage. The wires are joined at one end to form
a measuring junction within a casing. To measure temperature, the probe is inserted into the
fluid or medium and a voltage is generated.

Temperature Sensor Types, An infrared thermometer is a sensor that

consists of a lens to focus the infrared (IR) energy on to a detector, which
converts the energy to an electrical signal that can be displayed in units of
temperature after being compensated for ambient temperature variation.

Calorimetric Sensors

Calorimetric Sensors involve the measurement of heat that is generated in an enziemetic

reaction. These sensors typically utilize thermistors that transform heat generated or lost during
a reaction in to an electric signal. The enzyme is immobilize in a packed bad column that is placed
in the centre of the insulated chamber. The sample is allowed to pass through a coiled column of
aluminum that served as a heat exchanger to ensure uniform temperature of the sample entering
the reaction chamber. A thermistor is placed at the inlet of the reactor containing the
immobilized enzyme and another thermistor is placed at the exit of the reactor to measure the
temperature after the conversion of the substrate into the product. A thermistor record
temperature changes by altering the resistance. Higher temperature will cause reduction in the
resistance of thermistor. The difference in resistance between the thermistor located at the entry
in the exit point to the reactor is recorded as a measure of the temperature change produce.

Disadvantages

The necessity of maintaining the insulated jacket to prevent loss of heat, make the system
bulky. The sensitivity depends upon accuracy of the thermistor employed generally the
sensitivity of the calorimetric sensor is very low. Large sample volumes are required for
detectable change. The list of enzyme shows that the following enzymes released an
amount of heat:

Enzyme Substrate Heat output□-∆H (kJ/mol)

Catalase Hydrogen peroxide 100
Glucose oxidase Glucose 80
Urease Urea 61
Trypsin Peptides 10-30
Amylase Starch 8
Optical Sensors/ Light Sensors

Light is a part of electromagnetic radiation that is visible to human eye, however, the word light
is also used for some electromagnetic radiation that are not visible to human eye. For example,
ultraviolet (UV) or infrared (IR). The light can be seen by human eye is called visible light.
Electromagnetic radiation can be categorized into several groups depending upon the
wavelength.

1. Photo resister

A photo resister is also known as photo conductor cells. It is extremely sensitive to

light radiation. It does not show linear response against light radiation.

2. Photo diode

A photo diode is a special type of diode that is very sensitive to photon.

3. Photo transistor

A photo transistor can produce high light output current than a PD. Despite this
superiority it has a disadvantage that it can be damaged easily during handling.

4. Light Emitting Diode

LED consume much less power as compared to other and can produce single colour
light. There is another type of LED that generates white light mimicking the natural
sunlight referred as white LED.

5. Laser diode

A laser diode is a special type of LED that generate laser which is a coherent beam of
light that is extremely mono chromatic. Laser diode are used in CD, DVD, laser printer
and barcode scanner. They are also become very important in biosensor application
specially when light needs to be irradiated to the small area of interest. Such as micro
capillary channels. Applications and types of optical sensors are discussed in detail.

Pressure Senor

A pressure sensor is a device for pressure measurement of gases or liquids. Pressure

is an expression of the force required to stop a fluid from expanding, and is usually
 stated in terms of force per unit area. A pressure sensor usually acts as a transducer;
it generates a signal as a function of the pressure imposed. For the purposes of this
article, such a signal is electrical.

or

A pressure sensor is a device that detects a force exerted on a surface (pressure) and
converts it to an electronic signal whose strength is relative to the strength of the
force. Pressure sensors can also be used to measure the force exerted. Amperometry
is one of an

Pressure sensing Elements and Basic Mechanism Pressure is sensed by mechanical elements such
as plates, shells, tube and diaphrams .These elements are designed and constructed to deflect
when pressure is applied. The basic mechanism in the pressure sensors is to convert the pressure
tophysical movement. Examples are discussed in detail in the lecture including of sensors in
diagnostics include A cuff less wearable sensor blood pressure. Sensor for IOC in eye Measurment
of Glucose.

Physiological sensors

Physiological sensing deals with the sensors and analyses of different biological signals.
Developed applications have mainly been for clinical and medical purposes in hospitals,
healthcare centers, and clinics

Types of Biosensors are discussed in the lecture. Biosensors are classified on the basis if
Bioreceptor or sensing techniques.