Hand book on “Emerging applications of biosensors” (22ETC152)

Hand Book on

Emerging Applications
of Biosensors
22ETC152

For First Year BE, VTU, Belgaum

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 Hand book on “Emerging applications of biosensors” (22ETC152)

Course Title: Emerging applications of biosensors

Course Code: 22ETC152 CIE Marks 50
Course Type (Theory/Practical Theory SEE Marks 50
/Integrated ) Total Marks 100
Teaching Hours/Week (L:T:P: S) 3:0:0:0 Exam Hours 3 hrs of Theory
Total Hours of Pedagogy 40 hours Credits 03
Course objectives
1. To learn the Fundamentals of biosensors.
2. To acquaint the student with design and construction of biosensors.
3. To expose the students to recent advances in application of biosensors in health, environment, agriculture and food
industry.
Module-1 (8)
INTRODUCTION TO BIOSENSORS
Introduction to biosensor, General components of biosensor, Biomolecules in biosensors such as enzyme, DNA,
antigen antibody, protein, Classification of biosensors based on principle: amperometric, potentiometric
biosensors, optical, acoustic, piezoelectric, and calorimetric biosensors, scope of biosensors and its limitations.
Module-2 (8)
BASIC DESIGN AND TRANSDUCER
Design Considerations: calibration, dynamic Range, signal to noise, sensitivity, selectivity, Interference recognition.
Transduction membrane protein sensors: ion channels, Types of Transducer, Optical; Fiber Optic, ECL, Surface
Plasmon Resonance, Electro chemical; FET, Impedance, Piezoelectric; Cantileaver,

Module-3(8)
APPLICATIONS OF BIOSENSORS IN HEALTH AND ENVIRONMENT
Biosensors and diabetes management, Microfabricated biosensors and point-of-care diagnostics systems,
Noninvasive biosensors in clinical analysis; Surface plasmon resonance and evanescent wave biosensors, Biosensor
in cancer and HIV early diagnosis.
Module-4(8)
APPLICATIONS OF BIOSENSORS IN FOOD AND AGRICULTURE INDUSTRY
Detection of product content, allergic components, pathogens, pesticide residues. Monitoring of raw material
conversions. Detection of crop diseases, pathogens in plants, Detection of soil nutrients, pesticide and its residual
detection.
Module-5 (8)
APPLICATIONS OF NANOMATERIALS IN BIOSENSORS
Nano Materials in biosensors; Carbon based Nano Material, Metal oxide and nano particle, Quantum dots, Role of
nano material in Signal Amplifications, Detection and Transducer Fabrication
Course outcome (Course Skill Set)

At the end of the course the student will be able to:

CO1 Classify types of biosensors based on principle
CO2 Able to differentiate different types of transducers based on their physicochemical characteristics
CO3 Apply bio sensing techniques in health, environment, and agriculture and food industry.
CO4 Use biomaterial and nanomaterials in biosensors for signal amplification, Detection and Transducer
Fabrication

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 Hand book on “Emerging applications of biosensors” (22ETC152)

Module 1

INTRODUCTION TO BIOSENSORS
1.1 Introduction to biosensor,
1.2 General components of biosensor,
1.3 Biomolecules in biosensors such as enzyme, DNA, antigen antibody, protein,
1.4 Classification of biosensors based on principle: amperometric, potentiometric biosensors, optical,
acoustic, piezoelectric, and calorimetric biosensors,
1.5 Scope of biosensors and its limitations.

1.1 Introduction to Biosensor

 Sensor is a device which detects changes in a physical quantity like temperature, humidity, water
flow, intensity of light etc. and converts it into a quantity that can be measured and/or analysed.
 Biosensor is an analytical device that converts a biological response into a more useful electrical
signal using biological responsive material or bio recognition element such as microorganisms,
cells, enzyme, antibody or nucleic acid.
 Biosensor is a combination of a Biological sensing element and a transducer, which converts the
data into electrical signals. Additionally, there will be an electronic circuit which consists of a
Signal Conditioning Unit, a Processor or Microcontroller and a Display Unit.
 Biosensors are self-sufficient integrated devices that has capacity to provide specific qualitative or
semi-quantitative analytical information using a biological recognition element which is in direct-
spatial contact with a transductional element.
 In simple words, biosensors are analytical devices that detects changes in biological processes and
transform the biological data into electrical signal.
 The main features of biosensors are, Stability, Economical, Sensitivity and Reproducibility.
 Biorecognition elements should be highly specific for the analyte.
 Biorecognition elements should be stable under assay conditions over a very large number of
assays
 The reaction should be independent of physical parameters such as stirring, pH and temperature
 Response should accurate, precise, reproducible, and linear over useful analytical range.
 Invasive biosensors should be tiny, biocompatible and sterelizable, if required.
 Complete biosensor should be cheap, small, portable and easy to operate.
 The desired biological material is usually in the form of an enzyme due to operate.

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 One of the commonly used biorecognition element is enzyme due to its advantages such as
specificity, rate enhancement, regulation, reuse, stability, immobilisation etc.
 General components of biosensors are bio recognition element, transducer, amplifier, signal
processing unit and display unit.
 Biosensors have become very important in the fields of medicine, clinical analysis and in general
health monitoring.
 The advantages of biosensors over lab based equipment are their small size, low cost, quick results,
and very easy to use.
 Biosensors are used in Medicine, Clinical and Diagnostic Applications, Environmental
Monitoring, Industrial Applications, Food Industry and Agriculture Industry
 Apart from the desired medicine and health based applications, Biosensors have also found critical
applications in several other fields like industrial processing, agriculture, food processing,
pollution control etc.
 Commercial Biosensor in the field of personal health care are becoming quite popular, especially,
self-monitoring of blood glucose.

1.2 General components of biosensor

 Generally, biosensors are composed of three main components as depicted in Figure 1.
 These include a biological sensing element, transducer, and signal processing unit.
 The block diagram of the biosensor consists of three segments namely, biological sensing
element, transducer, and signal processing unit.
1.2.1 Biological sensing element:
 This component is also known as a sensor or detector element and is responsible for sensing or
detecting the presence and/or the concentration of the target analyte or substance.
 This is a biological component, which serves as a biochemical receptor that specifically recognizes
the target analyte.
 When the biological receptor interacts with a target analyte, it generates a signal in the form of
light, heat, pH, charge or mass change.
 This material should be highly specific, stable under storage conditions and must be immobilized.
 Furthermore, the biological receptor should be capable of selectively detecting the target
compound or analyte in the test sample.
 Biological receptor determines the sensitivity of the entire device through the generation of the
physicochemical signal that is monitored by the transducer.

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 This component can be a tissue, microorganism, organelle, cell receptor, enzyme, antibody or
nucleic acid etc. These can be grouped into two categories, namely catalytic and non-catalytic
receptors.
 The catalytic group of biological receptors are used in devices intended for continuous monitoring
of substances at millimolar or micromolar concentrations.
 These include enzymes, tissues and microorganisms.
 The non-catalytic group is used mainly in biosensor devices that measure analytes such as steroids,
drugs, and toxins etc. which usually occur at very low concentrations (micro to picomollar range)
 These are non-reusable devices which can only be used once and discarded thereafter. Such
receptors include antibodies, antigens, nucleic acids etc.
1.2.2 Transducer:
 The second segment of the biosensor is the transducer and it is a physical component.
 Transducer converts the biochemical signal into proportional electrical signals.
 Generally, a transducer is a material that is capable of converting one form of energy to another.
 In a biosensor, a transducer is responsible for converting the biochemical signal received from the
biological receptor, which is a result of the interaction between the target analyte and the biological
receptor, into a measurable and quantifiable signal which can be piezo-electrical, optical,
electrochemical etc.
 The transducer detects and measures the change that occurs during biological receptor – analyte
interaction.
 An example of a transducer is a pH sensor in a glucose biosensor.
 An enzyme, known as glucose oxidase, is used as a biological receptor which binds glucose and
converts it to gluconic acid in the presence of oxygen.
 The pH sensor (transducer) then detects the change in pH (due to production of gluconic acid) and
converts it into a voltage change.
 The following features are recommended when a transducer is designed; specificity to the target
analyte, analyte concentration range, response time and suitability for practical applications.
 Ideally, a transducer should be highly specific to the analyte, give measurement at the lowest
analyte concentration within the shortest time possible.
1.2.3 Signal processing Unit:
 The output of the transducer will be either current or voltage relying on the type of enzyme.
 If the output is voltage, then it is fine.

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 Hand book on “Emerging applications of biosensors” (22ETC152)

 But if the output is current, then this current needs to be converted into equivalent voltage (using
an Op-Amp based current to voltage converter) before proceeding further.
 The output voltage signal is generally very low in amplitude and is superimposed on a high
frequency noise signal.
 Thus, the signal is amplified (using an Op-Amp based Amplifier) and then it is passed through a
Low Pass RC Filter.
 Signal Processing Unit or a Signal Conditioning Unit is accountable for performing the process of
amplifying and filtering the signal. The output of the signal processing unit is termed as an analog
signal.
 This output is equivalent to the biological quantity being measured.
 The analog signal can be exhibited directly on an LCD display but usually, this analog signal is
passed to a Microcontroller, where the analog signal is converted into digital signal.
 This is done since it is easy to analyse, process or store a digital signal.
 Signal processing unit is the associated part which consists of Signal Conditioning Unit, a
Processor or Micro-controller and a Display Unit.

Signal
Biological Conditioning Processor Display Unit
recognitio Transducer Unit
n element
Signal Processing Unit

Fig [Link] representation of Biosensors

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 Hand book on “Emerging applications of biosensors” (22ETC152)

1.3 Biomolecules in biosensors such as enzyme, DNA, Antigen-

Antibody, Protein.
1.3.1 Enzyme based biosensors
• Enzyme based biosensors are most popular biosensors due to its specialist features such as
specificity, rate enhancement, regulation and mild working condition.
• Enzyme biosensors are useful tools for monitoring rapid changes in metabolite levels in real-
time, include pure enzyme preparations or biological processes.
• They have been derived on immobilization processes such as van der Waals forces, ionic or
covalent bonding.
• The well-known enzymatic biosensors today are glucose and urea biosensors.
• However, glucose biosensors are most popular among researchers and are reportedly the mostly
commercialized biosensors.
• The glucose biosensor, which was developed by Clark, is made up of glucose oxidase
immobilized within a dialysis membrane which is integrated inside oxygen electrodes.
• Enzymatic biosensors are known for their prolonged use and reusability due to the fact that
enzymes used as biological receptors cannot be consumed.
• Thus, the detection limit and the lifetime of enzyme based biosensors is greatly enhanced by the
stability of the enzyme.

Fig 2 Enzyme based biosensors

1.3.2 DNA based biosensors
• Another group of biosensors based on a biological receptor is DNA biosensors.
• The most attractive feature of biosensors is the high selectivity of biosensors for their target
analytes in a matrix of chemical or biological elements.

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 Hand book on “Emerging applications of biosensors” (22ETC152)

• DNA biosensors, which use nucleic acids as their biological receptors, detect proteins and non-
macromolecular compounds that interact with certain DNA fragments known as DNA probes or
DNA primers.
• The interaction observed stems from the formation of stable hydrogen bonds between the double
helix nucleic acid strands.
• To develop DNA biosensors, immobilization of the probe becomes the most crucial step.
• The strong pairing of lined up nucleotide strands between bases in their complementary parts
influences biosensors based on DNA, RNA, and peptide nucleotide acids to be the most sensitive
tool.
• That probes, which are short oligonucleotides capable of hybridization with individual areas of
the target nucleotide sequence, together with various chemical composition and conformational
arrangements, were employed in the development of DNA biosensors.
• Extremely high sensibility and selectivity is needed to maximize the hybridization efficiency and
minimize non-specific binding.

Fig 3 DNA based biosensors

1.3.3 Antibody based sensors
• Antibody-based biosensors or immunosensors have revolutionized diagnostics for the detection
of a plethora of analytes such as disease markers, food and environmental contaminants,
biological warfare agents and illicit drugs.

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 Hand book on “Emerging applications of biosensors” (22ETC152)

• Antibodies are ideal biorecognition elements that provide sensors with high specificity and
sensitivity.
• Antibodies are large Y-shaped proteins produced by plasma cells that are utilized by the immune
system to identify and target pathogens such as bacteria and viruses.
• Their small size, high stability and easy genetic manipulation make recombinant antibody
fragments valuable and robust tools for the fabrication of immunosensors.
• Antibody-based biosensors have revolutionized diagnostics for the detection of a plethora of
analytes such as food and environmental contaminants, biological warfare agents, illicit drugs
and disease markers.
• Immobilization of antibodies on to a sensor surface without altering their specificity and
immunological activity is one of the most crucial steps in the fabrication of a successful
immunosensor.
• The immobilization step affects the detection limit, sensitivity and overall performance of the
immunosensor.
• Orientation of antibodies on sensor surfaces can be controlled by the interaction between specific
reactive groups on the surface and on the antibody.

Fig 4 Antibody based biosensors

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 Hand book on “Emerging applications of biosensors” (22ETC152)

1.3.4 Protein based sensors

• Peptides/peptides have been used as components in biological analysis and fabrication of novel
biosensors for a number of reasons, including mature synthesis protocols, diverse structures and
as highly selective substrates for enzymes.
• Bio-conjugation strategies can provide an efficient way to convert interaction information
between peptides and analytes into a measurable signal, which can be used for fabrication of
novel peptide/protein-based biosensors.
• Many sensitive fluorophores can respond rapidly to environmental changes and stimuli manifest
as a change in spectral characteristics, hence environmentally-sensitive fluorophores have been
widely used as signal markers to conjugate to peptides to construct peptide-based molecular
sensors.
• Additionally, nanoparticles, fluorescent polymers, graphene and near infrared dyes are also used
as peptide-conjugated signal markers.
• On the other hand, peptides/proteins may play a generalist role in peptide-based biosensors.
• Peptides/proteins have been utilized as biorecognition elements to bind various analytes
including proteins, nucleic acid, bacteria, metal ions, enzymes and antibodies in biosensors.
• The selectivity of peptides/proteins as an enzymatic substrate has thus been utilized to construct
enzyme sensors or enzyme-activity sensors.
• In addition, progress on immobilization and microarray techniques of peptides has facilitated the
progress and commercial application of chip-based peptide biosensors in clinical diagnosis.

Fig 5 Antibody based biosensors

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 Hand book on “Emerging applications of biosensors” (22ETC152)

1.4 Classification of biosensors based on principle: Amperometric,

Potentiometric, Optical, Acoustic, Piezoelectric, and Calorimetric
Biosensors
• The most commonly applied classification of biosensors is based on the type of transduction
element used in the sensor.
• These biosensors are grouped into three main categories, amperometric, potentiometric, optical,
acoustic, piezoelectric, and calorimetric biosensors.
• The working principles of each of the three biosensors are different and can thus be implemented
in a variety of applications.
Based on the working principles of Transducer of the biosensor, classification is done as
follows
 Amperometric Biosensor: Based on the movement of electrons produced in the redox reaction.
 Potentiometric Biosensor: Based on the changes in the distribution of charge causing an
electrical potential to be produced.
 Optical Biosensor: Light output during the reaction or light absorption difference between the
reactants and products.
 Acoustic Biosensor: Utilizes acoustic or mechanical waves as a detection mechanism to obtain
medical, biochemical, and biophysical information about the analyte of interest
 Piezoelectric Biosensor: Piezoelectric effect due to the mass of the reactants
 Calorimetric Biosensors: Heat output input by the reaction
1.4.1 Amperometric biosensors
• Amperometric biosensors function by the production of a current when a potential is applied
between two electrodes.
• The simplest amperometric biosensors in common usage involve the Clark oxygen electrode.
• This consists of a platinum cathode at which oxygen is reduced and a silver/silver chloride
reference electrode.
• When a potential of -0.6 V, relative to the Ag/AgCl electrode is applied to the platinum cathode,
a current proportional to the oxygen concentration is produced.
• Normally both electrodes are bathed in a solution of saturated potassium chloride and separated
from the bulk solution by an oxygen-permeable plastic membrane (e.g., Teflon,
polytetrafluoroethylene).
• The following reactions occur:
Ag anode

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 Hand book on “Emerging applications of biosensors” (22ETC152)

4Ag +
+ 4e−
4Ag+ + 4Cl− 4AgCl
4Ag+ + 4Cl− 4AgCl + 4e−
Pt cathode
O2 + 2H2O + 2e− 2H2O+ 2OH-
2H2O + 2e− 2OH-
O2 + 4H+ + 4e− 2H2O
• The efficient reduction of oxygen at the surface of the cathode causes the oxygen concentration
there to be effectively zero.
• The rate of this electrochemical reduction therefore depends on the rate of diffusion of the
oxygen from the bulk solution, which is dependent on the concentration gradient and hence the
bulk oxygen concentration.
• It is clear that a small, but significant, proportion of the oxygen present in the bulk is consumed
by this process; the oxygen electrode measuring the rate of a process which is far from
equilibrium, whereas ion-selective electrodes are used close to equilibrium conditions.
• This causes the oxygen electrode to be much more sensitive to changes in the temperature than
potentiometric sensors.
• A typical application for this simple type of biosensor is the determination of glucose
concentrations by the use of an immobilised glucose oxidase membrane.
• The reaction results in a reduction of the oxygen concentration as it diffuses through the
biocatalytic membrane to the cathode, this being detected by a reduction in the current between
the electrodes.
• Other oxidases may be used in a similar manner for the analysis of their substrates (e.g., alcohol
oxidase, D- and L-amino acid oxidases, cholesterol oxidase, galactose oxidase, and urate
oxidase).

Figure 6. Schematic diagram of a simple amperometric biosensor. A potential is applied

between the central platinum cathode and the annular silver anode.

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 This generates a current (I) which is carried between the electrodes by means of a saturated solution
of KCl.
 This electrode compartment is separated from the biocatalyst (here shown glucose oxidase, GOD)
by a thin plastic membrane, permeable only to oxygen. T
 he analyte solution is separated from the biocatalyst by another membrane, permeable to the
substrate(s) and product(s).
 This biosensor is normally about 1 cm in diameter but has been scaled down to 0.25 mm diameter
using a Pt wire cathode within a silver plated steel needle anode and utilising dip-coated
membranes.

1.4.2 Potentiometric biosensors

• Potentiometric biosensors make use of ion-selective electrodes in order to transduce the biological
reaction into an electrical signal.
• It consists of an immobilised enzyme membrane surrounding the probe from a pH-meter (, where
the catalysed reaction generates or absorbs hydrogen ions.
• The reaction occurring next to the thin sensing glass membrane causes a change in pH which may
be read directly from the pH-meter's display.
• Here electrical potential is determined at very high impedance allowing effectively zero current
flow and causing no interference with the reaction.

Hg ↔ Hg+ + e−
Ag ↔ Ag+ + e−
HgCl2 ↔ 2 Hg+ + 2Cl−
AgCl ↔ Ag+ + Cl−
KCl ↔ K+ + Cl−
HCl ↔ H+ + Cl−

Eg: Any change in H+,

K for each of the
reaction gets effected to
keep the equilibrium.
[ H+][Cl−]
Kequ =
[HCl]

Fig 7. A simple potentiometric biosensor. A semi-permeable membrane surrounds the biocatalyst

entrapped next to the active glass membrane of a pH probe.

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The electrical potential is generated between the internal Ag/AgCl electrode bathed in dilute HCl
and an external reference electrode. Semipermeable membrane surrounds the enzyme allows the
analyte to move in. Potentials at reference electrode are unaffected by changes in H+. Reaction
generates or absorbs H+ at glass electrodes. So, equilibrium of the reaction alters to keep the K
constant for each of the reaction. Reaction. Changes in H+ glass electrodes ultimately changes the
electron concentration of the reaction. Measure of the current between two electrodes will be
measure of H+ of the solution.

1.4.3 Optical biosensors

 An optical biosensor is a compact analytical device containing a biorecognition sensing element
integrated with an optical transducer system (Figure 8).
 The basic objective of an optical biosensor is to produce a signal which is proportionate to the
concentration of a measured substance (analyte).
 The optical biosensor can use various biological materials, including enzymes, antibodies,
antigens, receptors, nucleic acids, whole cells and tissues as biorecognition elements.
 Surface plasmon resonance (SPR), evanescent wave fluorescence and optical waveguide
interferometry utilize the evanescent field in close proximity to the biosensor surface to detect the
interaction of the biorecognition element with the analyte.
 There are a huge number of variations in the construction of optical biosensors.

Figure 8 Generalised representation of optical biosensors.

Surface plasmon resonance biosensors (SPR)
 The SPR phenomenon occurs on the surface of metal (or other conducting materials) at the
interface of two media (usually glass and liquid) when it is illuminated by polarized light at a
specific angle.
 This generates surface plasmons and consequently a reduction of the intensity of reflected light at
a specific angle known as the resonance angle.
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 This effect is proportionate to the mass on the surface.

 A sensorgram can be obtained by measuring the shift of reflectivity, angle or wavelengths against
time.
 In all configurations, the SPR phenomenon enables direct, label-free and real-time changes of
refractive index at the sensor surface, which is proportionate to the biomolecule concentration.
 To measure a ligand–analyte interaction, one interacting molecule must be immobilized on the
sensor surface.
 A practical SPR instrument combines an optical detector part, usually measuring intensity shift, a
sensor chip with a gold surface and a layer enabling ligand immobilization, which is integrated
with a fluidics system enabling a flow-through operation.
 The operating principle of a typical SPR instrument is presented in Figure 9

Figure 9: The operating principle of a typical SPR instrument

Surface plasmon resonance imaging (SPRi)

 SPR imaging (SPRi) takes the SPR analysis a step further by merging the sensitivity of SPR and
spatial imaging in a microarray format allowing the simultaneous study of multiple different
interactions.
 SPRi allows simultaneously studying multiple different interactions on an array of precisely
patterned molecules (Figure 10).
 High throughput, sensitivity and obtaining the spatially resolved images of biointeractions open
up a great future for SPRi to be applied in clinical chemistry and medicine for the screening of
biomarkers and therapeutic targets.
 For example, a successful application of this method was the kinetic study of the binding between
an immunosuppressive drug (FK506) and its target protein (FK506-binding protein 12 (FKBP12))
in a high-throughput SPRi format with a detection limit of 0.5 nM.

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Figure 10: The operating principle of a typical SPRi

Localized surface plasmon resonance
 Localized SPR (LSPR) is based on metallic nanostructures (MNPs) (Au, Ag, etc.) having unique
optical properties which are not seen in larger metal structures.
 A particularly striking example of such phenomenon is the red colour of aqueous dispersions of
colloidal gold particles, which is a manifestation of LSPR.
 The optical phenomenon of LSPR occurs when incident light interacts with MNPs, the
electromagnetic field of the light induces collective electron charge oscillations confined in MNPs
and the subsequent absorbance of light within the ultraviolet–visible (UV-VIS) band (Figure 4).
 Thus, the major difference between SPR and LSPR is that induced p plasmons oscillate locally on
thenanostructure rather than along the metal/dielectric interface (Figure 11).

Fig 11 Localized surface plasmon resonance

Evanescent wave fluorescence biosensors
 In these biosensors, the biological recognition and the consequent binding event occur within the
confines of an evanescent wave.
 The evanescent wave arises from the manner in which light behaves when confined in an optical
waveguide or fibres.

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 Guided light is totally internally reflected when it meets the interface of the waveguide/fibre and
a surrounding medium with a lower index of refraction, as a result an electromagnetic field called
an evanescent wave extends out from the interface into the lower index medium.
 The evanescent wave decays exponentially with distance from the surface, generally over the
distance of 100 nm to approximately a wavelength.
 Since the evanescent wave is such a near-surface phenomena, detection employing evanescent
wave excitation to generate the fluorescent signal is surface-sensitive, meaning that only
fluorescent molecules near the surface are excited (Figure 12).

Fig 12 Evanescent wave fluorescence biosensors

 Bioluminescent optical fibre biosensors: This technique uses recombinant bioluminescent cells
and the bioluminescent signal is transferred from the analyte by an optical fibre.
 Optical waveguide interferometric biosensors: An integrated planar optical waveguide
interferometric biosensor is a combination of evanescent field sensing and optical phase difference
measurement methods. By probing the near-surface region of a grating sensor area with the
evanescent field, any change of the refractive index of the probed volume induces a phase shift of
the guided mode compared with a reference field, typically of a mode propagating through the
reference arm of the same waveguide structure. The interfering fields of these modes produce an
interference signal detected at the sensor’s output, whose alteration is proportional to the refractive
index change and the signal is related to the concentration of the analyte.
 Ellipsometric biosensors: An ellipsometric biosensor measures changes in the polarization of
light when it is reflected from a surface. This platform was applied in detecting the binding of
influenza A virus strains with a panel of glycans of diverse structures.
 Reflectometric interference spectroscopy biosensors: Reflectometric interference spectroscopy
(RIfS) is a label-free and time-resolved method where the simple optical setup is based on white
light interference at thin layers. Changes in the phase and amplitude of polarized light provides
information about the thickness and refractive index of the adsorbed protein layer.

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 Surface-enhanced Raman scattering biosensors: Surface-enhanced Raman scattering (SERS)

is a biosensing technique which enhances the intensity of the vibration spectra of a molecule by
several orders of magnitude when it is in close proximity to nano-roughened metallic surfaces or
nanoparticles made of gold or silver

1.4.4 Acoustic biosensors

 Bulk acoustic wave (BAW) biosensors employ either longitudinal or shear waves, although the
latter is often preferred to reduce acoustic radiation in the medium of interest.
 They are the oldest and the simplest acoustic wave devices.
 BAW devices consist of a parallel electrode placed on both sides of the thin piece of crystal. BAW
sensor can technically employ any piezoelectric element, and typically quartz is used, as it is an
inexpensive material readily available in nature and easily synthesizable in abundant quantities.
 In addition, thin disks of quartz are more stable at high temperatures than other piezoelectric
elements.
 When an alternating electric field is applied, it results in a potential difference between the two
electrodes and the shear deformation of the crystal.
 As a result, there is mechanical oscillation of a standing wave across the bulk of the quartz.
 The frequency of the vibrations is dependent on quartz properties such as density, size, and phase
in contact with the crystal surface.
 Currently, thickness shear mode (TSM) resonator and shear horizontal acoustic plate mode (SH-
APM) sensors remain the most widespread BAW sensors (Fig 13).
 The other two common BAW sensors that lie beyond the scope of this entry are the thin rod
acoustic wave sensors and the flexural plate wave devices.

Fig 13 Modes of BAW. Thickness shear mode (TSM) and longitudinal mode
 Thickness Shear Mode (TSM) Resonator: TSM resonator, also known as quartz crystal
microbalance (QCM), is the simplest and most widespread acoustic wave device today. TSM
typically composes of a quartz plate sandwiched by electrodes on opposite faces. Electric field

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crosses through this plate when voltage is applied to the electrodes, resulting in a shear mechanical
strain or displacement in the quartz. By oscillating the voltage frequency, a mechanical resonance
can be generated, where the maximum displacement of crystal occurs at the surfaces.
 Shear Horizontal Acoustic Plate Mode (SH-APM) Sensor:
SH-APM sensors use a thin piezoelectric substrate, or a plate, to guide the acoustic wave and to
confine its energy within the plate’s top and bottom surfaces. Most of the production and analysis
principles employed in SH-APM sensors are used in a TSM resonator. Their most striking
difference is that SH-APM sensors employ inter digital transducers (IDT) rather than electrode
plates. IDTs are deposited on opposite ends of a surface, where one IDT generates displacement
waves through application of an oscillating voltage and the other receives it. The surface without
IDT is immersed in the targeted liquid and acts as the sensor, so the device will not suffer from
corrosion problems as electrode plates do in biological solutions.

1.4.5 Piezoelectric biosensors

 Anisotropic crystals i.e. crystals without centre of symmetry can generate electric dipole when
mechanically squeezed.
 The electric dipole is also called piezoelectricity.
 The described effect can work in oppose way when an anisotropic crystal become deformed due
to voltage imposed on it.
 The mechanical deformation is, however, a simple situation and oscillation is rather chosen in
the common applications like here described analytical devices.
 In the case of oscillation, an alternating voltage is imposed on the crystal and mechanical
oscillation then occurs

a b
Figure 14. Piezoelectric effect when voltage is generated because of mechanical deformation (a),
when mechanical deformation is initiated by an applied voltage (b)

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 Hand book on “Emerging applications of biosensors” (22ETC152)

 The oscillations can have many appearances depending upon material and other conditions like
electrical contacts, shape of the crystals etc.
 The oscillations occur in adiabatic waves which are typically spread over the mass like the acoustic
one.
 In the oscillating crystals, the both surface acoustic waves spreading on the material and bulk
acoustic waves occurring in deep matter can take place.
 In standard analytical applications, frequencies of oscillations are measured and interaction with
either crystal alone or electrode leading electricity impulse on the crystal surface can serve for the
determination of analyte.
 Piezoelectric biosensors are a group of analytical devices working on a principle of affinity
interaction recording.
 A piezoelectric platform or piezoelectric crystal is a sensor part working on the principle of
oscillations change due to a mass bound on the piezoelectric crystal surface.
 Biosensors having their surface modified with an antibody or antigen, with a molecularly
imprinted polymer, with genetic information like single stranded DNA, and biosensors with bound
receptors of organic of biochemical origin, are presented and discussed.
 Piezoelectric Immunosensors: The piezoelectric immunosensors are biosensors which contains
an antibody as a biorecognition element and specificity of the antibody significantly influences
specificity of the whole immunosensor.

Figure 15. Piezoelectric immunosensors for the determination of an antigen (a) or an antibody(b)

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 Molecularly Imprinted Polymers on Piezoelectric Platform: Molecularly Imprinted Polymers

are specific artificial materials that can substitute antibodies or antigens as a biorecognition part
in a biosensor.

Figure16. Covering of crystal with a Molecularly Imprinted Polymer and following assay of an
analyte chemically identical or close to the template.
 Genetic Information Using Piezoelectric Biosensors: Genetic information can be employed as
a biorecognition part of various biosensors. Single-strand short strains of DNA or RNA can by
written down as typical examples of genetic information forms that are suitable for biosensors
construction.

Figure 17. DNA piezoelectric biosensor.

1.4.6 Calorimetric biosensors

 Many enzyme catalysed reactions are exothermic, generating heat which may be used as a basis
for measuring the rate of reaction and, hence, the analyte concentration.

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 This represents the most generally applicable type of biosensor.

 The temperature changes are usually determined by means of thermistors at the entrance and exit
of small packed bed columns containing immobilised enzymes within a constant temperature
environment (Figure 18).
 Under such closely controlled conditions, up to 80% of the heat generated in the reaction may be
registered as a temperature change in the sample stream.
 This may be simply calculated from the enthalpy change and the amount reacted.
 Calorimeter sensors are widely used in different areas such as biochemical, clinical, and
pharmaceutical industries, and many more.
 In the calorimetry sensor, the energy released during a biochemical reaction is calculated as the
measure of the interaction of the tested molecules.
 Two processes are used with the calorimetry sensor: (1) Adiabatic calorimetry, where there is no
heat exchange between the external environment and the reaction vessel, and (2) Heat conduction
calorimetry, which involves the heat transfer from a vessel to the surrounding heat sink.

Figure 18. Calorimetric biosensors. Schematic diagram of a calorimetric biosensor. The sample stream
passes through the outer insulated box to the heat exchanger within an aluminium block. From
there, it flows past the reference thermistor and into the packed bed bioreactor, containing the
biocatalyst, where the reaction occurs. The change in temperature is determined by the thermistor
and the solution passed to waste. External electronics (l) determines the difference in the resistance,
and hence temperature, between the thermistors.

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1.5 Scope of biosensors and its limitations.

 There is a huge application of biosensor in the medical industry for testing purpose.
 The biosensor is used for pathogens detection purpose.
 Biosensors are also used for water treatment purpose.
 Biosensors are used for environmental monitoring purposes such as gas detection or tracing.
 They used for toxic metabolites detection.
 The biosensor is used to identify the contaminants in water such as heavy metal ions. This process
is generally used when river water is treated for drinking.
 Biosensors are used in the security system and biodefense technology.
 Blood Glucose biosensor is used for glucose monitoring inside the human body.
 The biosensor also used for testing of cholesterol.
 Biosensors are used for the measurement of vitamins, biotin, folic acid, etc.

 Biosensors are used in Agriculture, Biotech industries to continuously monitor types of chemical,
their properties in a specific system.

Table 1: Scope of biosensors and its limitations

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Beneficial Features: A successful biosensor must possess the following features:

 The biocatalyst must be highly specific for the purpose of the analyses be stable under normal
storage conditions except in the case of calorimetric enzyme strips and show good stability over a
large number of assays (i.e. much greater than 100).
 The reaction should be as independent of physical parameters as like stirring, pH and temperature.
 The response should be accurate, precise, reproducible and linear over the useful analytical range
without dilution or concentration. It should also be free from electrical noise.
 If the biosensor is to be used for invasive monitoring in clinical situations the probe must be tiny
and biocompatible having no toxic or antigenic effects. If it is to be used in fermenters it should
be sterilisable. This is preferably performed by autoclaving but no biosensor enzymes can
presently withstand such drastic wet-heat treatment. In either case the biosensor should not be
prone to fouling or proteolysis.
 The complete biosensor should be cheap, small, portable and capable of being used by semi-skilled
operators.
 The biosensors have been considered to be superior and more sensitive, in comparison to physical
instruments due to the following reasons:
o In a biosensor the immobilized biological material is present in intimate contact of a suitable
transducer so that the biochemical signal is quickly converted into an electrical signal.
o The immobilization of biomolecules permits reuse of these molecules (which are expensive) and
allows simplification of the entire apparatus.
o The biological sensing element is present in a small area and is very sensitive, thus facilitating
analysis of substances in small quantities.
o Biosensors may be developed according to specific needs and can be highly specific or show broad
spectrum.
Disadvantages:
 Heat sterilization is not possible as this would denature the biological part of the biosensor.
 The membrane that separates the reactor media from the immobilized cells of the sensor can
become fouled by deposits.
 The cells in the biosensor can become intoxicated by other molecules that are capable of diffusing
through the membrane.
 Changes in the reactor broth (i.e., pH) can put chemical and mechanical stress on the biosensor
that might eventually impair it.

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Module 2

BASIC DESIGN AND TRANSDUCER

2.1 Design Considerations: calibration, dynamic Range, signal to noise, sensitivity, selectivity,
Interference recognition.

2.2 Transduction membrane protein sensors: ion channels,

2.3 Types of Transducer, Optical; Fiber Optic, ECL, Surface Plasmon Resonance, Electro
chemical; FET, Impedance, Piezoelectric; Cantileaver,

2.1 Design Considerations: Calibration, Dynamic Range, Signal to

Noise, Sensitivity, Selectivity, Interference Recognition.
 A successful biosensor is composed of two main components, mainly a biological receptor or
sensor element and a transducer.
 The first step in developing a biosensing device involves investigating the target analyte and
understanding how this analyte interacts with certain biological molecules.
 Once this has been established, the following tasks are critical: Selection of a biological receptor:
the specificity and selectivity of a biosensor to the analyte of interest is dependent upon the
biological receptor used.
 A suitable receptor with high affinity for the analyte is thus recommended.
 Having knowledge of the advantages and disadvantages of various biological receptors in different
biosensor applications is very important in selecting a suitable receptor.
 Selection of a suitable immobilization method: for any biological molecule to operate reliably
as a biological receptor, it requires attachment onto the surface of a transducer, the process is
known as immobilization.
 Various methods have been used for this task and include adsorption, entrapment, covalent
attachment, micro encapsulation and cross linking.
 Selection of a transducer element: the transducer element greatly influences the sensitivity of the
biosensor device.
 Employing the right transducer will result in a device with increased sensitivity while the
sensitivity is more likely to be compromised by the use of an ineffective transducer

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2.1.1 Calibration
 Sensor Calibration in simple terms can be defined as the comparison between the desired output
and the measured output.
 On-site monitoring requires enhanced sensitivity, selectivity, rapidity, and ease of operation of the
analytical equipment, which should provide reliable continuous information in real-time and
demonstrate sufficient stability of action.
 We use different systems and types of equipment for measuring various physical quantities.
 The accuracy of the measurement depends upon various factors.
 The equipment used for measurements can lose their precision when used at higher temperatures,
high moisture or humidity conditions, subjected to degradation, subjected to external shocks,
[Link] can be observed as the error in the measurement.
 To tackle this error and make necessary changes to the equipment calibration methods are used.
Today sensors are being used for making various measurements.
 There are sensors to measure temperature, colour, humidity, etc…Sensor Calibration plays a
crucial role in removing the errors in sensor measurements.
 Sensors are electronic devices. They are sensitive to the changes in their working environment.
Undesirable and sudden changes in the working environments of the sensors give undesired output
values. Thus, the expected output differs from the measured output. This comparison between the
Expected output and measured output is called Sensor Calibration.
 Sensor calibration plays a crucial role in increasing the performance of the sensor. It is used to
measure the Structural errors caused by sensors. The difference between the expected value and
the measured value of the sensor is known as the Structural Error.
 Sensor calibration helps in improving the performance and accuracy of the sensors.
 There are two well-known processes in which sensor calibration is done by industries.
 In the first method companies add an In-house calibration process to their manufacturing unit
to perform individual calibration of the sensors. Here the company also adds necessary hardware
to their design for sensor output correction. By this process, the sensor calibration can be changed
to match the application-specific requirements. But this process increases the time to market.
 The alternative of this In-house calibration process, several manufacturing companies provides
sensor packages with a high-quality automotive-grade MEMS sensor along with complete system-
level calibration. In this process, the companies include an on-board digital circuitry and software
to help designers to improve the functionality and performance of the sensors.
 To reduce the product design time and component count, digital circuitry such as voltage
regulation and Analog signal filtering techniques are included.

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 To improve the overall performance and functionality, the on-board processor is provided with
sophisticated sensor fusion algorithms.
 Some of the sophisticated on-board signal processing algorithms also help in reducing the
manufacturing time enabling the faster time to market.
Standard Reference Method
 Here the sensor output is compared with a standard physical reference to know the error in some
sensors. Examples of sensor calibration are rulers and meter sticks, For temperature sensors-
Boiling water at 100C, Triple point of water, For Accelerometers- ”gravity is constant 1G on the
surface of the earth”.
Calibration Methods:
 There are three standard calibration methods used for sensors. They are-One point calibration,
Two-point calibration, and Multi-Point Curve Fitting.
 Before knowing these methods we have to know the concept of Characteristic curve.
 Every sensor has a characteristic curve that shows the response of the senor to the given input
value. In the calibration process, this characteristic curve of the sensor is compared with its ideal
linear response.
 Some of the terms used with the characteristic curve are-
 Offset – This value tells us whether the sensor output is higher or lower than the ideal linear
response.
 Sensitivity or Slope – This gives the rate of change of sensor output. A difference in slope shows
that the sensor output changes at a different rate than the ideal response.
 Linearity – Not all sensors have a linear characteristic curve over the given measurement range.
 One point calibration is used to correct the sensor offset errors when accurate measurement of only
a single level is required and the sensor is linear.
 Temperature sensors are usually one point calibrated.

Measured parameter
Fig 19: One-Point-Calibration
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 Two-point calibration is used to correct both slope and off-set errors. This calibration is used in
the cases when the sensor we know that the sensor output is reasonably linear over a measurement
range. Here two reference values are needed- reference high, reference Low.

Measured parameter
Fig 20 Two-Point-Calibration
 Multi-point Curve fitting is used for sensors that are not linear over the measurement range and
require some curve-fitting to get the accurate measurements.
 Multi-point curve fitting is usually done for thermocouples when used in extremely hot or
extremely cold conditions.
 For all the above calibration process, the characteristic curves of the sensors are drawn and
compared with the linear response and error is known.
 The calibration process helps us to determine the following results-No error noted on the DUT,
 An error is noted and no adjustment is made.
 An adjustment is made to remove the error and the error is corrected to the desired level.
 For sensor calibration sensor models are used. Sensor calibration is applied in Control systems to
monitor and adjust the control processes. Automatic systems also apply the sensor calibration to
get error-free results.
 Use of Sensor Calibration: The calibration process is used to increase the performance and
functionality of the system. It helps in reducing errors in the system. A calibrated sensor provides
accurate results and can be used as a reference reading for comparison.
2.1.2 Dynamic range
 Dynamic range is one of the design parameter, when digital sensors are component of sensor
device.

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 Here, dynamic range of measurement will be also related to the number of binary digits (bits) used
in a digital numeric representation in which the measured value is linearly related to the digital
number.
 Often this dynamic range of measurement is limited at one end of the range by saturation of a
sensing signal sensor or by physical limits that exist on the motion or other response capability of
a mechanical indicator.
 The other end of the dynamic range of measurement is often limited by one or more sources of
random noise or uncertainty in signal levels that may be described as defining the sensitivity of
the sensor or metrology device.
 The range of concentrations over which the biosensor exhibits a change in output is the operational
range of the biosensor.
 Operational range is determined by measuring the concentrations over which the biosensor shows
a graded, concentration-dependent change in response (Figure 21).

Fig 21 Biosensor transfer function

 Defining engineering parameters for biosensors is a prerequisite for biosensor-based
measurements.
 The relationship between biosensor output and product concentration is the biosensor transfer
function.
 The range of concentrations over which the biosensor functions is the operational range. The
intensity of the biosensor response is the dynamic range.
 Sensor dynamic range quantifies the ability of a sensor to acquire both high and low signals.
 It is defined as the ratio of the largest no saturating input signal to the smallest detectable input
signal.
 The false positive rate of a biosensor system determines the maximum number of designs that can
be evaluated in a given experiment.

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 The signal-to-noise of a biosensor, also referred to as the dynamic range of the system, can be
quantified as the ratio of the highest measured output of the biosensor to the lowest measured
output of the biosensor.
 Dynamic range is the ratio between the largest and smallest values that a certain quantity can
assume.
 Various strategies are used to tune, extend, and narrow the dynamic range of Biosensors that use
either optical or electrochemical readouts.
 These strategies enable one to tune the affinity of biosensors that detect nucleic acids, small
molecules, heavy metal ions, pH, and temperature.
 In addition, environmental changes and material sizes are also used to adjust the dynamic range of
Biosensors.
 However, biosensors with extended dynamic range display reduced precision, while highly precise
sensors display narrowed dynamic range.
 Biosensor design strategy should be to develop dual-signalling biosensor architecture that
simultaneously provides both a highly sensitive “signal-on” readout over a small fixed dynamic
range as well as a “signal-off” readout, enabling quantification over a large, extended dynamic
range.
2.1.3 Signal-to-noise ratio

 In quite many applications of sensors, noise adds to the useful signal, while in others, noise is
proportional to signal.
 Whatever the case, the larger the noise fluctuations, the more difficult it is to detect or measure the
useful signal.
 Figure 22 shows a typical detector output with respect to time shows that the performance of an
electro-optical sensor does not depend on its signal alone but on its signal to noise ratio.

Figure 22: Signal to noise ratio

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 Since the sensor output is noisy, it fluctuates above and below its average value by an instantaneous
amount, for example ib (t) if one is concerned with the output current. The corresponding current
or voltage variances, σ2i or σ2v, inside the electronic bandpass of the sensor generate the following
electrical noise power Pn across the load resistor RL:
 If it is the instantaneous pertinent output from the detector, the corresponding electric power of the
signal is :
 By definition, the power signal to noise ratio of the sensor at that corresponding instant is the ratio
between the electrical pertinent signal power and that of the noise, both being evaluated inside the
sensor bandpass:
 Spatial (geometric) and spectral filtering are aimed at minimizing shot noise, due for example to
stray light, and at maximizing lens transmittance for the pertinent signal.
 Spatial filtering eliminate stray light from intense sources of light outside the field of view, by
means of diaphragms, baffles, or protective screens.
 Spectral filtering separates useful from parasitic radiations.
 In Electronic filtering after detection, if the variation in time of the expected signal is known, and
this signal is band limited and if the noise spectrum is white, signal processing techniques such as
matched filtering are a good choice.

2.1.4 Sensitivity
 The sensitivity of the sensor is defined as the slope of the output characteristic curve (DY/DX in
Figure 23) or, more generally, the minimum input of physical parameter that will create a
detectable output change.
 In some sensors, the sensitivity is defined as the input parameter change required to produce a
standardized output change.
 In others, it is defined as an output voltage change for a given change in input parameter. For
example, a typical blood pressure transducer may have a sensitivity rating of 10 mV/V/mm Hg;
that is, there will be a 10-mV output voltage for each volt of excitation potential and each mm Hg
of applied pressure.
 The sensitivity is the derivative of the output with respect to the stimulus. For a transfer function,
A, the sensitivity, b, for a particular input value B, so, is given by;
dA
 b
dB so

 Therefore, in basic terms, the sensitivity is simply the smallest fractional change in a device that
can be measured.

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 Sensitivity is considered as the most important characteristic of a biosensor.

 The sensitivity of a biosensor is defined as the relationship between the change in analyte
concentration and the intensity of the signal generated from the transducer.
 Ideally, a biosensor should generate a signal in response to small fluctuations in the concentration
of the target analyte.
 Depending on the application, biosensors are required to detect analytes in the ng/ml or fg/ml
concentration ranges.
 This is usually important for medical applications and environmental monitoring purposes.

Figure 23: Illustration of sensitivity in biosensor

2.1.5 Selectivity
 This refers to the ability of the biosensor to selectively bind and respond only to the desired analyte,
in the presence of other molecules or substances.
 When a signal or response is generated from interactions with an analyte that is different from the
target analyte such is termed a false positive result.
 This is common in biosensors with poor selectivity, thus failing in clinical applications.
 Selectivity is a very important feature especially in medical applications where the test sample or
sample matrix, usually blood or urine, contains numerous molecules that are quite similar to the
target analyte and compete for binding to the biological receptor.
 The selectivity of a sensor is the ability to discriminate the target from the interference molecules
and display a target-specific sensor response.
 It is a critical trait for chemical sensors that are used in real-time air pollution control, hazardous
materials detection, food quality inspection and personal health monitoring.
 Attaining high target selectivity ensures that sensors will exhibit accurate information about the
existence and concentration of a target gas, which is essential for reliable sensor response.

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 To obtain target selectivity, it is critical to determine the optimum modification technique and
receptor materials as well as to understand how each method works and how it could be designed
for a specific target.

Non selective sensor Selective sensor

6 10

4
5
2

0 0
Selectivity of the biosensor to biomolecules Selectivity of the biosensor to biomolecules

Glucose Urea Galactose Glucose Urea Galactose

Figure 24: Selectivity test of sensor with respect to different biomolecules

2.1.6 Interference recognition

 Sensor systems are used every time a microcontroller needs to interact with the physical world.
 They are abundant in home automation, factory control systems, critical infrastructure, transport
systems and many, many other things.
 In a sensor system, a sensor transforms a physical quantity into an analog signal which is sent to
an ADC and a microcontroller for digitization and further processing.
 Once the measurement is in digital form, the microcontroller can execute tasks according to the
measurement.
 Electromagnetic interference (EMI) can affect a measurement as it is transferred to the
microcontroller.
 An attacker can manipulate the sensor output by intentionally inducing EMI in the wire between
the sensor and the microcontroller.
 The nature of the analog channel between the sensor and the microcontroller means that the
microcontroller cannot authenticate whether the measurement is from the sensor or the attacker.
 If the microcontroller includes incorrect measurements in its control decisions, it could have
disastrous consequences
 The active sensors communicate by transmitting probe signals.
 The communication of probe signals may result in cross-interference which may vary in time.
 Cross-interference is detected, and can later be avoided, by determining a difference between
signals received in a first part of a timeslot and signals received in a second part of the timeslot.

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 In order to do so probe signals comprising two non-zero pulses are transmitted in respective parts
of the timeslot.
 Applications are, for example, active presence sensors in lighting control applications in indoor as
well as outdoor environments.
 It has been discovered that cross-interference across active sensors (such as sensors based on
ultrasound, or radio frequency) is a problem in indoor as well as outdoor sensing applications.
 Cross-interference across active sensors generally depends on the dimensions of the monitored
space and presence/absence of objects therein.
 For instance, when an object is moved (or added/removed) the cross-interference pattern across
sensors tends to vary.
 This affects proper operation of the presence sensing systems.

2.2 Transduction membrane protein sensors: ion channels

 Biological membranes are one of the essential components of living organisms, forming physical
boundaries in biological cells, such as the plasma membrane and the organelle membranes.
 The principal components of membranes are phospholipids and membrane proteins.
 Phospholipids are amphiphilic molecules consisting of a hydrophilic head group and hydrophobic
tails.
 They form a bilayer-membrane configuration in aqueous environments, which is attributed to the
hydrophobic interactions of their hydrocarbon chains.
 Lipid bilayer membranes function as hydrophobic barriers against soluble and ionic molecules and
prevent the entry of such molecules into the cytoplasm and organelles.
 Membrane proteins are incorporated in the lipid bilayer and allow signal transduction and transport
of ligand molecules across the membrane.
 Binding of an odorant molecule to the ionotropic receptor directly triggers the influx of cations
into the cell through the membrane; this influx stimulates the neurons.
 Since these protein receptors act as ligand-sensing elements, numerous studies have attempted to
use this sensing property of the highly specific membrane receptors for the development of
biosensors.
 Moreover, sensory systems often include a mechanism of input amplification that enhances the
output signal-to-noise ratio.
 Biosensors that embed sensory systems are based on two major platforms: the lipid bilayer-based
platform and the cell-based platform (figure 24).

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Figure25. Membrane protein-based biosensors

 Figure 1. Membrane protein-based biosensors on (a) a lipid-bilayer platform and (b,c) cell-based
platforms. On the lipid bilayer platform, the nanopore protein is incorporated in the lipid bilayer.
Single analyte molecules are detected based on the signatures of the current trace that translates
the interaction between the analytes and the nanopore. On the cell-based platforms, such as those
for odorant sensing, cell responses to odorants can be determined by (b) measuring the electrical
alterations of the cell using electric signal measuring systems, including ion-sensitive field effect
transistor (ISFET), (c) detection of fluorescence or luminescence changes initiated by olfactory
stimuli. In this case, utilized cells express G protein-coupled receptor (GPCR) or olfactory receptor
(OR) with OR co-receptor (Orco). (Online version in colour.)
Direct and indirect transduction

Fig: 26 Direct and indirect transduction

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 In direct approach, the electron transfers are close to the surface, whereas in the indirect one,
electron shuttles between the reaction site and the sensor surface.
 In indirect approach, The S-layer protein lattice constitutes an intermediate matrix. In the lipid-
based biosensor (left), electrons transfer from the outer membrane to the inner membrane and vice
versa via a channel protein.
 In the detection biosensor (middle and right), electrons transfer between the enzyme–substrate
complex and cell/antibody and electrode surface, respectively. The S-layer lattice provides an
immobilization matrix and ion reservoir.
 The pores of the S-layer lattice ensure no impact on the electron transfer. Fc: fragment
crystallizable; rSbpA/ZZ: recombinant S-layer protein from Lysinibacillus sphaericus CCM 2177
with fused Fc-binding Z-domain (synthetic analog of immunoglobulin G (IgG-binding B—
domain) of protein A of Staphylococcus aureus).

2.3 Types of Transducer, Optical; Fiber Optic, ECL, Surface

Plasmon Resonance, Electro chemical; FET, Impedance,
Piezoelectric; Cantilever,
2.3.1 Fibre optic Biosensors
 Optical fibers transmit light on the basis of the principle of total internal reflection (TIR).
 When this phenomenon occurs the light rays are guided through the core of the fiber with very
little loss to the surroundings.
 The optical fiber is formed by a core with a refractive index n1and a cladding with a refractive
index n2.
 For light propagation by TIR the refractive index of the core (n1) must be larger than that of the
cladding (n2), i.e. n1>n2.
 When a ray of light strikes the boundary interface between these transparent media of different
refractive index and the angle of incidence is larger than the critical angle, defined by the Snell’s
law (θc=sin–1[n2/n1]), it will be totally internally reflected and propagated through the fiber.
 When the incident light is totally internally reflected, its intensity does not abruptly decay to zero
at the inter-face.
 A small portion of light penetrates the reflecting medium by a fraction of wavelength, far enough
for recognition of the different refractive index.
 This electromagnetic field, called the evanescent wave, has an intensity that decays exponentially
with distance, starting at the interface and extending into the medium of lower refractive index.
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 The penetration depth (dp), defined as the distance required for the electric field amplitude to fall
to 1/e (0.37) of its value at the interface, increases with closer index matching and it is also a
function of the wavelength of the light and the angle of incidence.
 The evanescent wave can interact with molecules within the penetration depth, thereby producing
a net flow of energy across the reflecting surface in the surrounding medium (i.e. that with
refractive index n2) to maintain the evanescent field.
 This transfer of energy will lead to attenuation in reflectance which can be used to develop ab-
sorption sensors based on evanescent waves (attenuated total reflection (ATR) sensors).
 When the evanescent lights electively excites a fluorophore, the fluorescence emitted can be
directed back into the fiber and guided to the detector.

To

Fig: 27 Total Internal refraction [a] and electrical field amplified[b].

2.3.2 Electro chemiluminescence Biosensor (ECL)
 Electrochemiluminescence (ECL) is a chemiluminescence phenomenon resulting from the
electrochemical excitation of a luminescence system (luminophore) that emits light when it
returns to its fundamental state.
 The mechanisms associated with these phenomena are classified into two main types,
annihilation pathway and Co-reactant pathway (Fig 26).

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Fig 28: Mechanisms associated with Electrochemiluminescence

The annihilation pathway:
 A reduced specie and an oxidized specie (charged radical ions) are simultaneously generated at
the electrode surface by applying alternating pulse potentials.
 These two species react between them generating an excited form, which in the relaxation process
to the ground state emits a photon.
Co-reactant pathway:
 A co-reactant is a chemical specie that is reduced or oxidized at the electrode surface, generating
a very reactive intermediates that react with the reduced or oxidized luminophore (specie capable
of emit light) present in the solution to produce the excited state.
 Finally, the excited state returns to the ground state to cause chemiluminescence.
 Employing a co-reactant is especially useful when either radical charged ions are not stable enough
for the ECL annihilation reaction, or radical ions cannot both be formed because of the solvent has
a narrow potential window.
 With a co-reactant ECL can be generated by applying a potential in one direction.
 There are two reaction paths to produce the excited state of the ECL emitter, reductive-oxidation
or oxidative-reduction ECL.
 For instance, oxalate ion (C2O42−) [4, 5] and several amines [6, 7, 8, 9] can be used for oxidative-
reduction ECL where an oxidative step produces a strong reductant, whereas peroxidisulfate ion
(S2O82−) is frequently used for reductive-oxidation ECL.
 As described above, ECL reactions require a luminophore.

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 Few compounds and their derivatives primarily utilized for aqueous-based ECL bioanalytical
detection methods, are luminol (5-amino-2, 3-dihydrophthalazine-1, 4-Dione) and ruthenium (II)
chelates [RuL3]2+.
 Nowadays, new luminophores such as semiconductor nanomaterials are being widely used with
great results. This fact is one of the main reasons ECL sensor and biosensor are having a great and
successful advance.
2.3.4 Surface Plasmon Resonance Biosensor
 Surface Plasmon Resonance is a phenomenon that occurs when polarized light hits a metal film at
the interface of media with different refractive indices.
 SPR techniques excite and detect collective oscillations of free electrons (known as surface
plasmons) via the Kretschmann configuration, in which light is focused onto a metal film through
a glass prism and the subsequent reflection is detected (Fig. 1).

Fig 29: Basics of Surface Plasmon Resonance

 At a certain incident angle (or resonance angle), the plasmons are set to resonate with light,
resulting in absorption of light at that angle. This creates a dark line in the reflected beam that
contains a wealth of information.
 The resonance angle can be obtained by observing a dip in SPR reflection intensity.
 Comparing to the conventional diagnostic tools, SPR biosensors have multiple advantages such as
easy preparation, no requirement of labelling, real-time detection capability, cost- effectiveness,
and high specificity and sensitivity.
 However, for the label-free detection of low concentrations of analytes with small molecular
weight its sensitivity is not enough.
 Therefore, considerable efforts have been invested to overcome these challenges and improve the
sensitivity of the SPR biosensor such as ligands or functional nanomaterials with keeping all its
advantages.

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 The enhancement of the SPR biosensor needs modification of its surface with suitable ligands to
capture the target compound (the analyte) and neglect other molecules available in the sample as
shown in Figure 28.

Figure 30. Direct label free detection SPR biosensor.

 These ligands can be permanently or temporarily immobilized on the sensor surface.
 The analyte accumulation results in a RI change in the evanescent field detected.
 When the ligand captures the analyte, the measurable signal rises and this is called direct label free
detection.
 Following in time the resonance angle or wave length shift at which the dip is observed produces
the sensogram (Figure 29), then the amount of adsorbed species after injection of the original
baseline buffer can be determined, and a study of the kinetics of the biomolecular interaction can
be done.

Figure 31. Surface plasmon resonance (SPR) sensogram.

2.3.5 Field-effect transistor (FET) biosensors
 Among various kinds of biosensors, field-effect transistor biosensors (Bio-FETs), an integrated
between bio-receptors and ion-sensitive field-effect transistors (ISFET), emerged as the most
developed candidates because of several advantages.

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 In a typical FET system, the sensing elements are immobilized on the sensing channels
(semiconductor path), which are connected to source (S) and drain (D) electrodes, to capture the
targets (usually via high specificity and binding affinity).
 A bias potential is applied and modulated to a third electrode (gate).

Figure 32. Field-effect transistor based biosensors.

 The channel conductance, which is varied by detection of the targets, is recorded and further
processed by an electrical measurement system.
 There are two kinds of FETs: n-type with electrons as the main charge carriers and p-type with
holes as the primary charge carriers.
 In an n-type FET system, if the probes detect positively charged molecules, the charge carriers
(electrons) will accumulate on the sensing channels and increase the conductance.
 If negatively charged targets are recognized, the conductance will be decreased due to the depletion
of the electrons.
 Conversely, for a p-type FET system, binding with positive charges results in conductance decline
due to a reduction of the charge carriers (holes) and capturing negative charges raises the
conductance because of hole accumulation.
 Outburst of nanotechnology triggers combination between biosensors and nanomaterials for
sensing application with breakthrough designs in which biomolecules (antibodies, nucleotides and
so on) as receptors are immobilized on the surface of nanotransducers (nanowires, nanotubes,
nanoparticles, etc.).
2.3.6 Impedimetric biosensors
 Label-free electrochemical biosensors include impedimetric biosensors which measure the
impedance, i.e., the opposition presented to a current in an alternating current (AC) circuit when a
voltage is applied.
 The impedance is a complex quantity, and a common graphical representation is the Nyquist plot.

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 This is a frequency response plot, where the values of the real part are plotted on the x-axis and
those of the imaginary part on the y-axis.
 An ideal Nyquist plot shows a semicircle resulting from the dominating, kinetically limited charge
transfer through the electric double layer at the electrode.
 Binding of analyte molecules to the electrode will influence the charge transfer and, hence, result
in a shift of the Nyquist plot.
 As a consequence, the opportunity for charge transfer is an additional requirement for sensing
layers of impedance biosensors.
 At low frequencies, Nyquist plots may show straight lines with a slope of 45°. This is characteristic
for diffusion limited processes and described by the Warburg impedance.
 Electrodes of impedance biosensors are typically made of gold.
 A well-established procedure for the introduction of functional groups on this material is to use
suitably substituted thiols forming self-assembled monolayers (SAMs).
 Thiols with aliphatic hydrocarbon spacers of sufficient chain length lead to well-defined and stable
SAMs of high density.
 The brush-like structure of such layers makes it possible to effectively reduce nonspecific protein
adsorption on the underlying gold surface.
 However, such SAMs may result in insulating layers, hindering the charge transfer required for
the transduction principle of impedimetric biosensors.
 The use of aromatic hydrocarbons featuring delocalized π-electrons would be more beneficial for
charge transfer processes, but nonspecific protein adsorption in the subsequent measurements may
increase because of a reduced density of the layer.
 Conductive polymers would offer an alternative, but are often linked with coating procedures more
complex than wet chemistry.
 Thiolated single-strand DNA (ssDNA) oligomers, on the other hand, can be packed densely on the
gold surface by wet chemistry methods similar to those of thiolated hydrocarbons.
 Coimmobilization of thiolated ssDNA with thiolated hydrocarbons may be recommended to
improve the integrity of the brush-like structure.
 The negatively charged backbone of the DNA oligomers—resulting from the composition of
alternating sugar (deoxyribose) and phosphate groups—promises lower initial impedance values
and, hence, the possibility of charge transfer events.
 With impedance sensors, immobilized ssDNA or immobilized oligonucleotides has been used
directly as probe for DNA or protein detection, respectively.

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Fig 33 Impedimetric biosensors

2.3.7 Piezoelectric biosensor;
 Piezoelectric sensors utilize crystals which undergo an elastic deformation when an electrical
potential is applied to them.
 The applied alternating potential produces a standing wave in the crystal at a characteristic
frequency.
 This characteristic frequency is highly dependent on the elastic properties of the crystal.
 If a crystal is coated with a biological recognition element the binding of a (large) target analyte
to a receptor will produce a change in the resonance frequency, giving a binding signal.
 In a mode that uses surface waves (SAW), the sensitivity is greatly increased.
 This is a special application of the Quartz crystal microbalance in biosensor.
 The piezoelectric generator is the method of transforming high frequency electric oscillations
into mechanical oscillations.
 It is most frequently used at the present day for producing ultrasonic and it allows us to attain
the highest frequencies now possible.
 Two classes of bio-recognition processes-bio-affinity recognition-and bio-metabolic
recognition, offer different methods of detection.
 Both processes involve the binding of a chemical species with another, which has a
complementary structure.
 This is referred to as shape-specific binding. In bio-affinity recognition, the binding is very
strong, and the transducer detects the presence of the bound receptor-analyte pair.

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 The most common types of processes are receptor-ligand and antibody-antigen binding.
 In bio-metabolic recognition, the analyte and other co-reactants are chemically altered to form
the product molecules.
 The biomaterials that can be recognized by the bio-recognition elements are as varied as the
different reactions that occur in biological systems.
 Almost all types of biological reactions, (chemical or affinity), can be exploited for biosensors.
The concept of shape-specific recognition is commonly used to explain the high sensitivity and
selectivity of biological molecules, especially antigen-antibody systems.
 The analyte molecule has a complementary structure to the antibody, and the bound pair is in
a lower energy state than the two separate molecules. . The interaction of antibodies with their
corresponding antigens is an attractive reason for attempting to develop antibody-based
chemical biosensors, i.e. immunosensors
Cantilever Biosensor
 Cantilevers (springboard) are nanomechanical biosensors, microfabricated with the standard
silicon technology.
 Due to their intrinsic flexibility, together with the availability of techniques designed to
monitor bending, cantilevers have become versatile tools.
 This technology is a multifunctional and highly sensitive technique, and a real time method
useful for a variety of applications, such as plastic explosive detection using gas biosensors,
whole microorganism detection as part of liquid biosensors, or DNA and proteins studies.
 By incorporating a piezoresistor to each cantilever in a Wheatstone bridge type configuration,
it is possible to read resistance changes as voltage changes.
 The Wheatstone bridge configuration uses a pair of cantilevers; one of them will be used as
reference.
 The differential signal between both cantilevers will be the output of this configuration.
 The signal-noise relation is substantially improved with this configuration, and the noise
originated by unspecific binding, thermal fluctuation, or vibrations is eliminated.
 Non-specific binding to the surface is a general problem that must be minimized in all analyses.
 Although the complete elimination of this parameter is not possible, its influence on detection
could be controlled with the use of the reference cantilever.
 The immobilization of molecules on the cantilever surface is required for its use as a
nanomechanical sensor (Figures 32).
 The immobilized molecules provide the cantilever with specificity for the analyte.

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 The specific molecular interactions taking place at the flexible surface of cantilever increase
surface tension, forcing the cantilever to bend.
 This type of surface tension induced by molecular interactions is not generally observed on the
surface of common materials.
 The cantilever senses the tension and bends in response to the free energy changes taking place
at its surface.

Figures 34 Nanomechanical cantilever sensor

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Module 3
APPLICATIONS OF BIOSENSORS IN HEALTH AND
ENVIRONMENT
3.1 Biosensors and diabetes management:
 Several factors have combined over the past few years to make glucose biosensor one of the
most financially attractive areas in medical diagnosis.
 Generally, glucose measurements are based on interactions with one of three enzymes:
hexokinase, glucose oxidase (GOx) or glucose-1-dehydrogenase (GDH).
 The hexokinase assay is the reference method for measuring glucose using spectrophotometry
in many clinical laboratories.
 Successful glucose biosensor must also meet the diabetic’s expectations.
 There are many theories of operations of biosensors such as Reflectance based method,
Electrochemical methods, Enzymes and reagents, Hexokinase methods, Glucose oxidase
based- Peroxidase optical method, Organic mediator optical methods, Perssian Blue method,
Ferricyanide electrochemical method, Ferrocene Electrochemical method, reusable sensors
etc.
 Electrochemical sensors may be subdivided into potentiometric, amperometric, or
conductometric types.
 Enzymatic amperometric glucose biosensors are the most common devices commercially
available, and have been widely studied over the last few decades.
 Amperometric sensors monitor currents generated when electrons are exchanged either
directly or indirectly between a biological system and an electrode

Fig. 35. Classification of blood glucose sensors in this review.

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 Electrochemical methods are popular, and over the years, enzymatic amperometric glucose
sensors were the first and widespread glucose sensors available
 Electrochemical glucose sensors can be divided into potentiometric (employed to detect
variations of surface charge onto a counter electrode), amperometric (charge flow between the
counter electrode and the bio-system), or conductometric (variations in ionic conductance
between electrodes.
 They are generally fabricated by using two families of enzymes, the glucose oxidase and the
glucose dehydrogenase (GDH). The reaction, catalyzed is as follows:

Figures 36 Glucose sensor reaction scheme

 GOx is the standard enzyme for biosensors; it has a relatively higher selectivity for glucose.
GOx is easy to obtain, cheap, and can withstand greater extremes of pH, ionic strength, and
temperature than many other enzymes, thus allowing less stringent conditions during the
manufacturing process and relatively relaxed storage norms for use by lay biosensor users
 The concept behind a glucose biosensor is based on the fact that the immobilized GOx
catalyzes the oxidation of D-glucose by molecular oxygen producing gluconic acid and
hydrogen peroxide.
 To function as a catalyst, GOx requires a flavin adenine dinucleotide (FAD) redox cofactor.
FAD functions as an initial electron acceptor and is reduced to FADH2.
 This enzymatic electrode evaluates the glucose level by the amperometric tracking of the
released hydrogen peroxide.

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 Glucose dehydrogenases are instead defined as oxidoreductases which are unable to use
oxygen as an electron acceptor and therefore transfer electrons to other natural and artificial
acceptors.
 GDHs also need a cofactor. These are mainly nicotine adenine dinucleotide (NAD+ or NADH
depending on the oxidation state) or pyrroloquinoline quinone (PQQ).
 FAD, NAD+ and PQQ remove hydrogen, H+ and e-, from glucose.
 GDH-PQQ is a particularly efficient enzyme system, with a fast electron transfer rate, but it is
relatively expensive.
 GDH with NAD+ as a cofactor produces NADH rather than H2O2.
 Nicotine adenine dinucleotide is an important electron acceptor in glucose oxidation, during
which NAD's nicotinamide ring accepts one hydrogen ion and two electrons, equivalent to one
hydride ion.
 In this reaction, the generated reduced form of this cofactor is NADH, which can be
electrochemically oxidized.

Figures 37 Glucose sensor designs

 1st Generation: Based on the sensor designed by Clark and Lyons
Formation of hydrogen peroxide
Oxygen as an electron acceptor
Errors due to interference from other electroactive species
 2nd Generation Replacement of oxygen as an electron acceptor
Introduction of the non-physiological mediator

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Limitations in the transfer from the enzymatic active site to the electrode
3rd Generation Absence of mediator
Direct transfer between enzyme and electrode
Low operating potential, higher selectivity, less interference
 An electrochemical biosensor is composed by working electrodes (on which the reaction of
interest, responsible for the measurement, takes place), reference electrodes and auxiliary
electrodes (to ensure that the current does not circulate through the electrode).
 Glucose concentration is mostly evaluated using the amperometric method that monitors the
current flowing between the working electrode and the reference electrode.
 The basic concept of the glucose biosensor is based on the fact that the immobilized GOx
catalyzes the oxidation of β-D-glucose by molecular oxygen producing gluconic acid and
hydrogen peroxide. In order to work as a catalyst, GOx requires a redox cofactor—flavin
adenine dinucleotide (FAD). FAD works as the initial electron acceptor and is reduced to
FADH2.
Glucose + GOx − FAD+ → Glucolactone + GOx − FADH2
 The cofactor is regenerated by reacting with oxygen, leading to the formation of hydrogen
peroxides.
GOx − FADH2 + O2 → GOx − FAD + H2 O2
 Hydrogen peroxide is oxidized at a catalytic, classically platinum (Pt) anode. The electrode
easily recognizes the number of electron transfers, and this electron flow is proportional to the
number of glucose molecules present in blood [36].
H2O2 → 2H+ + O2 + 2e
 Three general strategies are used for the electrochemical sensing of glucose; by measuring
oxygen consumption, by measuring the amount of hydrogen peroxide produced by the enzyme
reaction or by using a diffusible or immobilized mediator to transfer the electrons from the
GOx to the electrode.
 The number and types of GDH-based amperometric biosensors have been increasing recently.
The GDH family includes GDH-pyrroquinolinequinone (PQQ) and GDH-nicotinamide-
adenine dinucleotide (NAD). The enzymatic reaction of GDH is independent of the dissolved
oxygen. The quinoprotein GDH recognition element uses PQQ as a cofactor.
Glucose + PQQ(ox) → gluconolactone + PQQ(red)
 This mechanism requires neither oxygen nor NAD+. GDH-PQQ is a particularly efficient
enzyme system, with a rapid electron transfer rate, but it is relatively expensive [17].

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 GDH with NAD as a cofactor produces NADH rather than H2O2. NAD is a major electron
acceptor in the oxidation of glucose, during which the nicotinamide ring of NAD+ accepts a
hydrogen ion and two electrons, equivalent to a hydride ion. The reduced form of this carrier
generated in this reaction is called NADH, which can be electrochemically oxidized.
Glucose+NAD+→gluconolactone+NADHNADH→NAD++H++2e

3.2 Microfabricated biosensors and point-of-care diagnostics

systems
 Even with tremendous growth in hospital sector, providing centralized clinical testing facility
is inadequate.
 Patient admitted to the hospital with number of possible life-threatening causes for symptoms
during initial assessment may get unattended in initial and comprehensive qualitative and
quantitative assessment to get information that is crucial in managing the disease by any doctor.
 Without clinical chemistry or microbiological parameters physician is often unable to make
any reliable diagnosis.
 To do this clinical samples need to be draw from the patient, labelled, transported to laboratory,
segregated, tested, analysed, certified and reported back that often located at central place that
takes several hours due to remote locations of the laboratory and its expensive equipment.
 So, such delay is major concern as physician is unable to initiate timely effective treatment.
 Hence, approach to solve such issues can only be solved by addressing laboratory-based steps,
and not through transportation logistic problem.
 However, approached in delaying the testing time by automation of the analysis lead to
increase in the coast of testing due to expensive instruments.
 Hence, “Point-of-Care” clinical sample analysis system incorporates simple, safe,
maintenance-free home-use monitoring system works with whole clinical samples rather than
clinical samples that requires preclinical treatments, so precludes all the laboratory instruments
such as centrifuges, filters etc. that requires money and time.
 Such “Point-of-Care” blood analysis system should requires design that (a) replace in-line
calibration with factory calibration, (b) replace wash step with single-use analytical
component, (c) eliminate need for metered samples, (d) simple reusable reading device to
display results.

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 “Point-of-Care system” that can be utilised along the patient’s “bed-side” rather than the
laboratory’s “table-top” with the same analytical quality, along emergency room, intensive
care unit, critical care unit etc.
 “Point-of-Care system” is crucial for estimating clinical parameters such as blood gases,
electrolytes, metabolites that covers many conditions such as arrhythmia, dehydration,
anemoia, respiratory problems, and diabetes.
 A successful hospital “Point-of-Care system” would, at minimum, need to perform all of these.
 The microfabrication technology focuses on the miniaturization of engineering systems and
has evolved from the mature process technology in semiconductor device fabrication.
 The main techniques of microfabrication are thin films deposition, layers doping, patterning
via photolithography, etching to obtain the required design, polishing and bonding.
 Microfabrication technology has been widely used for the development of complex electronic
components, integrated micro-electromechanical systems (MEMS), and different types of
sensors for a wide range of applications.
 The benefits of developing sensors with such technological processes include low costs, large
scale fabrication of nominally identical structures, the possibility of integration with other
devices and the compatibility with a numerous of technologies, such as complimentary metal-
oxide semiconductor technology (CMOS) for the manufacturing of integrated circuits or
silicon on insulator (SOI) technology for semiconductor engineering.
 The most common assembly of electrodes for electrochemical applications is the three-
electrode system.
 A three-electrode structure consists in a working electrode (WE), a reference electrode (RE)
and a counter electrode (CE).
 The working electrode serves as the transduction element in the (bio) chemical reaction, while
the counter electrode establishes a connection to the electrolyte solution so that a current can
be applied to the working electrode.
 This system is also beneficial because it averts the RE from pushing the current which could
modify its potential.
 The potential is applied between the WE and the RE and the CE provides the mandatory current
to sustain electrolysis at the WE.
Fundamental of POC biosensors
 Biosensors are analytical devices used for the detection of a biological substance. In general,
biosensors consist of three components: a receptor (specific for a disease) that recognizes the

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analyte, a transducer that converts the bio-recognition event into a measurable signal, and a
reader.
 This technology allows precise control and manipulation of fluids, which typically requires
much less sample volume than that of conventional assays.
 The efficient liquid mixing in biosensors also enhances the interaction between assay reagents
and target biomarkers, which shortens the assay duration and provides fast readout.
 Moreover, the portability of biosensors makes them ideal candidates for POC field settings. To
date, researchers around the globe have developed various types of biosensors for wide
applications in POC settings such as the diagnosis of infectious diseases, food safety analysis,
and environmental monitoring.
 These biosensors include chip-based, paper-based, and other biosensors (textile-based or
nanomaterial-based biosensors), which will be briefly discussed in the following sections.
 For POC testing of chronic and infectious diseases, there is always an increasing demand for
low-cost, portable, and integrated biosensors, which can provide rapid results with low sample
consumption.
 Chip-based biosensors are one of the POC biosensors used for POC diagnosis of many
infectious diseases.
 They are prepared either by miniaturizing conventional biochemical assays on a microchip
scale or by integrating novel detection principles with microfluidic chips, referred as “Lab-on-
chip”.
 These biosensors are mainly made of polymethyl methacrylate (PMMA),
polytetrafluoroethylene (PTEE), or polydimethylsiloxane (PDMS).
 PDMS biosensors are commonly used due to their cost-effectiveness, high specificity, and
minimal reagent consumption.
 Specifically, they consist of multiple channels which enable nucleic acid testing steps,
including nucleic acid extraction, amplification, and amplicon detection, to be performed in an
automated manner.
 In recent years, smartphone has also been integrated into chip-based biosensors for imaging
and signal analysis.
 For instance, chip-based biosensors coupled with a smartphone have been used to rapidly
detect amplicon signals within an hour for the diagnosis of H1N1 and Zika virus infections.
 Paper-based biosensors have been broadly used for rapid testing of infectious diseases, which
show potential to substitute the conventional laboratory tests and chip-based biosensors.

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 Paper is inexpensive, readily available, and biodegradable, showing a promising tool for onsite
rapid diagnosis.
 It allows the diffusion of a biological sample through a capillary effect, eliminating the need
for external power sources.
 Earlier studies have introduced lateral flow test strips and microfluidic paper-based analytical
devices (μPAD) for POC testing.
 The assay usually involves hybridization of single-stranded DNA or RNA with a
complementary probe to produce double-stranded nucleic acids or interaction between antigen
and antibody to produce an Ag-Ab complex, generating signals such as colorimetric,
fluorescence, or chemiluminescence signals.
 Their special characteristics such as simple, affordable, and ease of fabrication, modification,
and functionalization have made them possible to achieve rapid, onsite POC testing.
 Other biosensors like film-based, textile-based, and nanomaterial-based biosensors have also
been used for the diagnosis of infectious diseases.
 For instance, film-based biosensors which are made of transparent polyester substrate film
have been used for the detection of pathogens.
 This material has the ability to withstand thermal cycling and amplification process.
 Textile-based biosensors are biosensors which are typically made of thread, fabric, or clothes
which are inexpensive and readily available with low sample consumption. For example,
textile-based biosensors are cheaper and require a smaller sample volume (optical
 Emerging point-of-care biosensors for rapid diagnosis of COVID-19 have exploited these
biosensors for POC diagnoses, especially by integrating with mobile phones.
 The device packaged in a compact portable assembly shows a bright potential for performing
fast and accurate cytokine assays for COVID-19 in clinics and POC settings for responsive
disease management.

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Figure 38. Presentation of (A) the layout of MFBS used for glucose colorimetric assays
and (B) a simplified view of the enzymatic reaction involved in the presence of
chromogenic reagent (TMB) for glucose detection.

3.4 Noninvasive biosensors in clinical analysis:

 Neonatal, elderly and diabetic patient who have to undergo daily metabolite measurement have
problem in giving samples demand non-invasive method,
 Noninvasive method does not require invasion of the body to collect sample, as these method
can efficiently detect metabolites directly in urine, swat, saliva, or skin.
3.4.1 Non Invasive Glucose Monitoring
 Multitude principles and techniques of non-invasive glucose monitoring have been pursued in
academic research as well as in industry in recent times.
 Among them, broadly four particular non-invasing glucose monitoring principles are widely
investigated and reported.
 These four principles are differentiated based on the principles of detection of glucose. They
are as follows:
• Optical Spectroscopy (Optical Detection)
• Photoacoustic Spectroscopy (Acoustic Detection)
• Electromagnetic Sensing (Electromagnetic Detection)
• Nanomaterial Based Sensing (Electrochemical Detection)
 Optical spectroscopy: The detection principle of the Optical Spectroscopy is based on the fact
that glucose influences the optical signal passing through it by absorption of light at some
particular overtones and combination band wavelengths in the mid infrared (mid-IR) and
nearinfrared (NIR) spectrum regions.

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 Photoacoustic Spectroscopy: The photoacoustic glucose monitoring is a hybrid approach,

which combines optical excitation and acoustic detection in determining the glucose
concentration. Here, the optical energy from the excitation is converted into an acoustic energy
by a multistage energy conversion process.
 Electromagnetic sensing: Human blood consists of 55% of plasma. The blood plasma consists
of around 90% of water. Water being a dipolar compound, the polarization is high. This makes
the relative permittivity to be high as well. In contrast, glucose has a smaller relative
permittivity, being less polarized. Thus, the overall increase or decrease in glucose
concentration in the same volume of blood sample reduces or elevates the relative permittivity
of blood plasma, respectively. The measure of the relative permittivity by multiple techniques
of electromagnetic sensing at a particular microwave/mm-wave frequency gives the value of
the glucose concentration in blood in the form of measured electrical power in a very effective
manner which helps in the measurement of blood glucose. It is important to note that the
dielectric properties of blood are modified because the change in blood glucose levels is of
much greater extent than due to changes of other compounds present in the blood.
 Nanomaterial based sensing: The emergence of nanomaterials, starting from metallic gold,
silver, copper oxide, iron oxide to polymer composites, carbon nanotubes and graphene, as
primary components in sensing technologies, has significantly upgraded the modern-day
biosensors, extracting valuable physiological data and useful information from essential body
fluids like urine, saliva, sweat and tears. The impact of nanomaterials in sensing applications
is remarkable as they exhibit large surface area, enhanced sensitivity and selectivity, improved
catalytic activities which are essential prerequisites for an accurate and precise estimation of
glucose levels in humans.
3.4.2 Alcohol based sensor.
 Measurement of alcohol in saliva is one of the frequently performed test in legal case to
establish the degree of alcohol intoxication, which is one of the major cause of traffic accidents.
 Impairment of driving ability starts as low as 50 mg of ethanol per 100 mL of blood.
 Measurement of ethanol in saliva, urine and blood are of great practical importance in forensic
purpose.
 Measurement of ethanol is used in industry to estimate the levels of ethanol produced during
fermentation.
 Saliva alcohol device requires a specific, sensitive, reliable alcohol sensor,
 The two most appropriate base sensor for alcohol electrode are oxygen electrode and hydrogen
peroxide electrode

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 When these sensors are coupled with alcohol oxidase, response to alcohol is achieved based
on reaction.
Alcohol oxidase
Alcohol + O2 ---------------------------- H2O2 + Acetaldehyde
 Assay of the alcohol is achieved by measuring the decrease in oxygen with an oxygen electrode
or by measuring the increase in hydrogen peroxide with hydrogen peroxide electrode during
initial rate of reaction of 12 s or steady state current at 1-2 min after injection of ethanol.
(Refer Amperometic biosensor principle in Module 1, Page 11).
3.4.3 Lactate based sensor
 Lactate is an important metabolite that need to be monitored in critical care patients, diabetic
control, food analysis and orts medicine.
 Electrochemical Amperometric acetate probe operates in following way.
Lactate oxidase
Lactate + O2 + H2O---------------------------- Pyruvate+ Acetaldehyde
 Assay of the lactate is achieved by measuring the increase in hydrogen peroxide with platinum
working electrode and silver/silver chloride reference-counter electrode. Current change due
to oxidation of hydrogen peroxide is proportional to the lactate concentration in the sample.
(Refer Amperometic biosensor principle in Module 1, Page 11).
3.4.4 Transcutaneous arterial oxygen tension sensor
 A Clark amperometric electrode is used in a sensor unit that is placed in contact with the skin.
 Three Glass- glass sealed platinum cathode separately connected via current amplifier to an
Ag/Agcl anode ring, and are dipped in KCl electrolyte where chemical reaction takes place.
 Under normal condition partial pressure of oxygen on skin surface is that of atmospheric
regardless of the pressure beneath the skin.
 Hyperemia of the skin causes partial pressure of oxygen to approach to that of arterial partial
pressure of oxygen, when induced by drugs administration, applying nicotinic acid, heating or
abrasion of the skin during measurement.
 Heating elements are integrated in the sensor with thermistor sensor to heat the skin between
43-44oC.
 Oxygen permeable membrane separates the inner electrodes and outer environment.
(Refer Amperometic biosensor(Oxygen Electrode) principle in Module 1, Page 11).
3.4.5 Transcutaneous arterial carbon dioxide tension sensor
 Transcutaneous arterial carbon dioxide tension sensor is similar to transcutaneous arterial
oxygen tension sensor except that sensor is potentiometric sensor.

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 Glass pH electrode with concentric Ag/AgCl reference electrode.

 Concentric Heating elements are integrated in the sensor with thermistor sensor to heat the skin
between 43-44oC.
 Carbon dioxide permeable Teflon membrane separates the inner electrodes and outer
environment.
(Refer Potentiometric biosensor (pH electrode based) principle in page number 13)

3.5 Surface plasmon resonance and evanescent wave biosensors

 Surface plasmon resonance (SPR) is a unique optical surface sensing technique with numerous
applications in variety of discipline.
 SPR can be used to probe refractive index changes that occur within the vicinity of a sensor
surface.
 Thus any physical phenomenon at the surface that alters the refractive index will elicit a
response.
 The basic SPR apparatus is generally referred to as the Kretschmann prism arrangement.
 Thin film of metal is coated on one face of a prism, or alternatively, the metal film can be
deposited onto a glass slide that is brought into optical contact with the prism using refractive
index matching fluid.
 This metal film forms the sensor surface on which the bio system sample is placed.
 Light is launched into prism, where it is both coupled into the plasma mode of the metal film
as well as being partially reflected off the metal film to an optical photometer.
 The changes in the amount of light striking the detector represent the sensor output.
 This output is affected by changes in the bio system layer on the metal film on the metal film,
which in turn affects the refractive index of the metal film-bio system pair, which in turn affects
amount of light that couples that coupled into the surface plasmon mode of the metal film.
 Evanescent wave sensor concepts is composed of three major concepts, a bilayer on surface
which specifically and directly or indirectly binds the analyte of interest, and a transducer
which forms a binding event between the bilayer and the analyte into an electric signal that
serves as a quantifiable output for simple analysis.
 This is generally achieved by immobilizing one of the receptors on the surface of the
transducer.
 For direct assay binding event itself is detected, and since this event does not provide
significant signals, physical even is detected via changes in local density, dielctic constant or
refractive index using electrical, gravimetric, and optical transducers.

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Fig 39. Basic components of a surface plasmon resonance (SPR) system

 SPR-based biosensors demonstrate interesting potential for environmental applications, and a
substantial number of studies have been reported for heavy metals, dioxins, pesticides,
aromatic hydrocarbons, phenol, polychlorinated biphenyls and detection.
 Biosensors can be used to monitor environmental pollution in air, soil, water, etc.; toxic
elements in food and quality control; biohazardous bacteria or virus, and biomolecules for
clinical diagnostics, etc.; and to necessitate sensitive, fast, and selective equipments or tools.

3.6 Biosensor in cancer and HIV early diagnosis.

 The timely diagnosis of cancer represents the best chance to increase treatment success and
To reduce cancer deaths.
 Nanomaterials-based biosensors containing graphene quantum dots (GQDs) as a sensing
platform show great promise in the early and sensitive detection of cancer biomarkers, due to
their unique chemical and physical properties, large surface area and ease of functionalization
with different biomolecules able to recognize relevant cancer biomarkers.
 The developed optical, electrochemical and chemiluminescent biosensors based on GQDs have
been shown to ensure the effective diagnosis of several cancer diseases as well as the
possibility to evaluate the effectiveness of anticancer therapy.
 The wide linear range of detection and low detection limits recorded for most of the reported
biosensors highlight their great potential in clinics for the diagnosis and management of cancer.

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Fig 40 Representation of Graphene quantum dots for cancer detection

 GQD is new class of fluorescent materials from carbon nanomaterials family, possess ideal
chemical and physical properties to be used and integrated in sensors for biological and medical
applications.
 From a morphological point of view, GQDs show the peculiar features of both graphene and
carbon dots (CDs).
 CDs and GQDs are zero dimensional carbon based materials, both endowed with unique
physicochemical properties associated with quantum size and edge effects.
 While CDs are mainly synthesized by bottom-up strategies and show spherical shape up to 10
nm,
 GQDs are typically derived from materials where the sp2 carbon atoms are organized into a
graphene structure.
 The presence of a graphene structure and the large surface to volume ratio allow the ease
functionalization to a large number of sites of graphene surface.
 Antibodies, proteins, nucleic acids and polymers have been covalently linked and/or
conjugated via π−π interaction to the graphene surface of GQDs, affording high selective and
sensitive biosensors able to recognize biomarkers associated with various types of cancers.
 The electronic and optical properties of GQDs make these nanomaterials particularly attractive
in optoelectronics.
 These properties depend on their band gap because of quantum confinement, and are
influenced by their size, number of layers, shape and edge configuration.

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 As a consequence, these properties can be modulated by using optimal carbon sources and
size-controlling synthetic methodologies.
 Human immunodeficiency virus (HIV) is a global epidemic; however, many individuals are
able to obtain treatment and manage their condition.
 Progression to acquired immunodeficiency syndrome (AIDS) occurs during late-stage HIV
infection, which compromises the immune system, making it susceptible to infections. While
there is no cure, antiretroviral therapy can be used provided that detection occurs, preferably
during the early phase.
 However, the detection of HIV is expensive and resource-intensive when tested with
conventional methods, such as flow cytometry, polymerase chain reaction (PCR), or enzyme-
linked immunosorbent assays (ELISA).
 Improving disease detection in resource-constrained areas requires equipment that is
affordable, portable, and can deliver rapid results.
 Microfluidic devices have transformed many bench top techniques to on-chip detection for
portable and rapid point-of-care (POC) testing.
 These devices are cost-effective, sensitive, and rapid and can be used in areas lacking
resources.
 Moreover, their functionality can rival their bench top counterparts, making them efficient for
disease detection.

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Chapter 4
Applications of Biosensors in Food and Agriculture Industry
 Detection of product content, allergic components, pathogens, pesticide residues.
 Monitoring of raw material conversions.
 Detection of crop diseases, pathogens in plants,
 Detection of soil nutrients, pesticide and its residual detection.

4.1 Detection of product content, allergic components, pathogens,

pesticide residues.
Detection of food content
 Several changes can take place in packaged food as a result of metabolism or microbial growth
over time.
For example, changes in gas evolution or microbial accumulation can be used to obtain
information about the status of food, e.g., freshness or degradation.
 Sensors that can measure such changes could provide an overall estimation of food quality.
Examples include “on-package” pH indicators that change color when food decays as a result
of pH changes associated with the release of volatile amines generated during meat or fish
spoilage.
 Commercially available freshness indicators for different types of food including fish, meat,
and poultry, cereal grains, fruits, and vegetables are as follows.

Fig 41. Representation of the concept of smart and active packaging technology
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Fish, Meat, and Poultry

 When meat, fish, or poultry undergo degradation, different spoilage indicators can be found
indicating lipid decay, protein breakdown, and adenosine triphosphate (ATP) decay.
 The speed of degradation is dependent on the type of product, storage temperature, feeding
habits, and harvesting methods.
 Traditional methods to assess freshness rely on human senses; although they are essential, they
provide no quantitative data of spoiled food.
 Methods that can quantitatively measure markers of degradation through chemical or
biological reactions can provide the means to more precisely assess the status and quality of
food.
 In fish products, for example, one of the main freshness indicators is hypoxanthine, which is
produced by the metabolic degradation of ATP.
Xanthine oxidase
Hypoxanthine + O2 --------------------- Xanthine + H2O2

Xanthine oxidase
Xanthine + O2 ----------------------Uric acid + H2O2

Fog 42 colorimetric freshness sensor for fish via hypoxanthine detection

Cereal Grains
 One of the indicators of grain spoilage during storage is the emission of CO2 as a result of
insect infestations, and mold spoilage causing grain deterioration or the production of harmful
mycotoxins.
 Developing CO2 sensors for early spoilage detection has been reported.
 Sensor developed based on polyaniline boronic acid (PABA) conducting polymer for
measuring CO2 levels in the range of 380–2400 ppm in grain.
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 The sensing mechanism is based on the conversion between the emeraldine salt form and the
insulating emeraldine base form of polyaniline and PABA through protonation and
deprotonation.
 When gaseous CO2 reacts with water, it creates carbonic acid that protonates the polyaniline
and further increases conductivity as CO2 partial pressure increases.
 Gluten is another component of interest for grain analysis, as certain individuals can develop
gluten intolerance that can cause serious disorders of the digestive system.
 The most common method for gluten analysis is by the conventional enzyme-linked
immunosorbent assay (ELISA).
 Analysis of toxicity caused by mycotoxin contamination in cereals is also of special interest.
 Mycotoxins, such as o chratoxin A (OTA), aflatoxins, trichothecenes, fumonisins, zearalenone,
and ergot alkaloids, are produced by fungi.
 Several biosensor types for mycotoxin detection have been reported such as electrochemical
biosensors were developed for the detection of OTA using a competitive mechanism using
OTA-specific aptamers and horseradish peroxidase (HRP) enzyme [70].
Fruits and Vegetables
 Technology for monitoring and preserving fruits and vegetables is necessary to decrease food
loss during transportation and storage.
 Many fruits and vegetables produce ethylene due to environmental stress after being harvested.
 Ethylene can enhance ripening even at extremely low concentrations.
 The presence of aging fruits and vegetables close to fresh ones can also cause aging and
ripening as ethylene is emitted.
 Ethylene can be removed by using ethylene absorbers or oxidizers (scavengers).
 Scavenging systems facilitate removal, thus lowering the loss of other products due to
overproduced ethylene.
 The most available ethylene scavenger is potassium permanganate (KMnO4), which oxides
ethylene to ethylene glycol and can be further oxidized to CO2 and H2O, producing dark brown
MnO2.
 Several commercial scavengers have been developed based on ethylene chemisorption by
KMnO4 granules over clays or activated carbon.
 Volatile organic compounds (VOC) accumulate in the presence of fruits and vegetables in
closed containers or packages.

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Fig 43: Dual-channel catalytic-biosensor. Acetaldehyde (AcAl) generated by fruit diffuses

through the gas phase to biosensor cells (AIRCHO-SEAP) genetically engineered to express
the Aspergillus nidulans-derived transactivator AlcR that, in the presence of acetaldehyde,
activates its cognate promoter PAIR, driving expression of the reporter gene SEAP (AIRCHO-
SEAP cells). Ethylene is oxidized to acetaldehyde on PdCl2 with Cu+ based on the Wacker
process. The generated acetaldehyde is captured by AIRCHO-SEAP and converted into SEAP
expression, measured as a colorimetric signal .Indicators for the detection of these compounds,
such as terpenes, carboxylic acids, alcohols, aldehydes, sulfur compounds, ammonia, and
jasmonates, have been reported.
 A color-based pH indicator, developed using bromophenol blue immobilized on a cellulose
membrane, enabled the detection of VOC (e.g., acetic acid) evolution in the headspace of guava
packaging. This label provides the consumer with the freshness status of guava.
Biosensors for the detection of pathogens, allergens, and other toxicants, such as
pesticides
 The need for simple, rapid, and field-portable analytical methods has boosted development of
biosensors for food analysis.
 The integration of biomolecules, such as enzymes, immunosystems, tissues, organelles, or
whole cells, with a variety of transduction methods, such as electrical, thermal, or optical
signals, has enabled the development of a wide array of biosensing devices.
 Their selectivity and relative ease of analysis make them advantageous for use in food analysis.
 The development of biosensors in this field is described with biosensor examples for the
detection of pathogens, allergens, and other toxicants, such as pesticides and mycotoxins.
 In this section, we briefly highlight the application of biosensors for allergens, toxicants, and
pathogen detection.

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Biosensors for Food-Allergen Detection

 The presence of allergens in food products such as milk, soybeans, crustaceans, eggs, gluten-
containing cereals, peanuts, and nuts (e.g., almonds, Brazil nuts, cashews, walnuts) is an
increased safety concern, as prevalence of food allergies due to even trace amounts of allergens
is increasing.
 About 10% of preschool children in industrial countries suffer from clinical food allergies.
 A variety of DNA or immune-based biosensors have been developed for allergen detection,
but in many cases sample preparation and purification are laborious and time-consuming.
 Employing antibody-based detection and magnetite beads, has commercially developed a
sensor to detect peanut allergens in ppm.
 Peanut allergens were detected by surface plasmon resonance (SPR)-immune-based biosensor
in chocolate candy bars.
 Colorimetric, silicon-based, optical thin-film biosensor chip with PCR amplification was
developed with the ability to simultaneously identify eight food allergens found in soybeans,
wheat, peanuts, cashews, shrimp, fish, beef, and chicken.

Figure 44. Allergen-detection chip, detection of corresponding allergens: 1, H2O; 2, cashews;

3, peanuts; 4, wheat; 5, soybeans; 6, chicken; 7, fish; 8, shrimp; 9, beef
 The presence of gluten in food such as wheat, barley, and rye causes celiac disease for
individuals who are unable to digest gluten.
 A label-free electrochemical immunological sensor for β-lactoglobulin, an allergen usually
found in milk was developed.
 Allergy-causing proteins, such as casein and β-lactoglobulin (β-LG), in milk have also been
detected based on aptamer recognition with the aptamer immobilized on a graphene-modified
screen-printed electrode with voltammetric detection.

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 A casein immunosensor with Localized Surface Plasmon Resonance (LSPR) detection and
immobilized casein antibodies was reported to detect casein in raw milk with.
 Several types of electrochemical biosensors have been designed for lactose quantification
utilizing coimmobilized β-galactosidase and glucose oxidase enzymes.
 Several enzymatic lactose sensors are also available for the detection of trace amounts of
lactose in lactose-free milk products.
Biosensors for Bacterial-Pathogen Detection
 The main causes of these million foodborne illnesses diseases are pathogenic bacteria
Escherichia coli and Salmonella spp.
 Rapid detection of pathogenic bacteria plays an important role in food analysis.
 The main methods for pathogen detection are based on Polymerase Chain Reaction (PCR) or
plate counting, which require sample enrichment and long analysis time.
 Most biosensors for bacterial-pathogen detection are those based on immune and DNA
recognition, but these require extensive preparation procedures, involve labeling, multiple
washing steps, and specialized facilities.
 Alternatively, synthetic antimicrobial peptides have been proposed as recognition agents,
enabling detection and quantification of four bacterial strains, Escherichia coli, Pseudomonas
aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis.
 Colorimetric biosensor strips fabricated with peptides immobilized on a gold chip were also
reported for the detection of Listeria monocytogenes in milk and meat samples.
 Fluorescent DNAzyme probe that specifically binds E. coli was developed and printed on a
cyclo-olefin polymer transparentv package.

Figure 45. Fluorescent DNAzyme probe with specific binding characteristics for E. coli.

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Biosensors for pesticide residues:

 The widespread use of pesticides in agriculture leads to their accumulation in soil, ground
water, and crops.
 Due to their inherent toxicity, a control of pesticide levels in food products is necessary.
 Biosensors based on acetylcholinesterase (AChE) inhibition have been reported for the
detection of carbamates and organophosphate used in insecticides.
 AChE immobilization approaches on screen-printed electrodes by was tested on
bioincapsulation in sol-gel composites, metal-chelate affinity, and entrapment in a
photopolymerizable polymer.
 Biosensors based on organ phosphorus hydrolase, which catalyzes the hydrolysis of
organophosphorous pesticides, were also developed.
 Insecticides such as Organophosphates, Carbamates, Neonicotinoids, Pyrethroids,
Organochlorines were tested by various optical sensors.

Figure 46. Basic principle of OPs biosensor by AChE enzyme

4.2 Monitoring of raw material conversions.

 To take up the challenge of world hunger, increased demand for food and biodiversity, new
interdisciplinary strategies should be implemented to boost the current food production
systems.
 From a technological point of view, such strategies should imply the development of more
efficient and sustainable food production processes, with low environmental impact and
ensuring high food quality standards.

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 Since ancient times, microorganisms and active biomolecules have been employed for food
and food additives production.
 Nowadays, food industry comprises a large variety of bioprocess technologies such as
fermentations, biotransformations and downstream processes.
 These biotechnological processes are carried out in a well-controlled environment, with a high
degree of automatization, and under continuous monitoring.
 Physical and chemical sensors are the most commonly used for food bioprocess monitoring to
monitor raw material conversion.
 However, the detection of complex chemical compounds, relevant biomolecules or cells
requires the use of more selective and specific analytical methods.
 In this context, biosensors constitute the most promised sensing technology for food bioprocess
control and monitoring to optimize the conversion of raw material into final food product.
 Electrochemical biosensors are self-contained, integrated analytical devices, in which a
biological recognition element is in intimate contact or incorporated with an electrochemical
transducer, allowing the use of an electroanalytical technique (potentiometric, amperometric,
conductometric, impedimetric, field effect, etc.) to measure the analytical response.
 These devices can detect target analytes by using catalytic (enzymes, cell, tissues, etc.) or
affinity (antibodies, aptamer, lectins, DNA, etc.) bioreceptors.
 Electrochemical biosensors technology allows the design of miniaturized, portable and cost-
effective sensors that exhibit the inherent specificity provided by the bioreceptor, combined
with the high sensitivity and low detection limit of the physicochemical transducer.
 These biosensors can also provide rapid, on-site measurements with minimal or without sample
preparation. Because of these relevant properties, electrochemical biosensors are promising
alternatives to traditional methods for food safety and food processing analysis.

Figure 47. Bioprocess monitoring through biosensors

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 In the last years, electrochemical biosensors have been mainly based on nanomaterials-
modified electrodes due to the relevant properties of these materials for transduction.
 Nanomaterials have high surface area-to-volume ratio, allowing to immobilize bioreceptors in
large yield with the consequent increase in the analytical sensitivity.
 These materials can be also chemically modified with a variety of ligands to allow easy
immobilization of bioreceptors through covalent and non-covalent linkages.
 In addition, several nanomaterials have excellent electroconductive and electrocatalytic
properties, allowing to construct biosensor devices with improved analytical characteristics.
 Enzymes are, by far, the biological receptors more commonly employed to construct
electrochemical biosensors for food analysis.
 These biosensors have been mainly designed for food safety and food composition analysis as
explained in the module 1.
 However, much of them can be easily adapted to monitoring food bioprocesses, such as
fermentation, in which microorganisms are employed to produce and modify foods.
 These bioprocesses require the accurate control and monitoring of the concentrations of
substrates and products,
 Electrochemical biosensors based on affinity recognition mechanisms have been proposed as
alternatives to detect microbiological contamination and spoilage in food, as well as the
presence of microbial toxins.
 Although these biosensors have been mainly conceived for food quality control, they can be
also potentially employed for food bioprocess monitoring to keep check on raw material yield.
 Electrochemical biosensors are powerful alternatives to conventional laboratory techniques for
food analysis, with potential application in food bioprocess monitoring.

4.3 Detection of crop diseases, pathogens in plants,

 When plants are exposed to pathogens they initiate protection reactions whose molecular
mechanisms are very complex.
 At the early stages, when visual symptoms such as injuries on the leaf surface are absent, plants
respond to the presence of a pathogen with physiological component such as the decrease of
the photosynthesis rate, which induces an increase of fluorescence and heat emission.
 The possibility to distinguish distinctive contaminations in a similar plant is attractive, since
plants can be influenced at the same time by numerous pathogens, for example, nematodes,
fungi, bacteria, phytoplasmas, viruses and viroids that conventional strategies identify at a late
symptomatic stage.

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 Detection of plant infection utilizing electrochemical techniques has pulled in much intrigue
in light of their basic instrumentation, high specificity, affectability, quick, and is economical
with potential for applications in sub-atomic sensing instrument.
 Recently, nanomaterial-based electrochemical sensors have been reported for plant disease
detection.
 The utilization of gold nanoparticle (AuNP) modified cathode for the electrochemical detection
of methyl salicylate, a key plant volatile organic compound released by plants during
infections.
 In case of DNA based electrochemical strategies, voltammetric examinations have been
utilized as a basic device for a discriminative investigation of nucleic acid conformation and
modification with the synchronous identification of all bases of the DNA without the need of
a hydrolysis step.

Figure 48. Schematic exemplum of the DNA-based electrochemical bioassay for plant
pathogen detection

 The detection generally includes the redox labels physically or covalently linked to specific
target DNA allocated on the active electrode surface.
 Current advance in DNA based biosensors for improvement of parallel microarrays and high-
throughput outlines can be connected to DNA sequencing innovations.
 Although much research has been done on electrochemical biosensors for other areas like food
quality but its practical application for plant disease detection is in pipeline for detailed
investigation.
 Colorimetric biosensors are an appealing optical biosensor since one can undoubtedly and
immediately see with the bare eye the presence of pathogenic microorganisms in the specimen

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through a colour change without the requirement for any expository instrument or chemical
reagent.

Figure 49. Biosensor concepts for plat disease detection

 Optical biosensors measure light assimilated or radiated as the after effect of a biological or
synthetic response, while electrochemical biosensors depend on biochemical responses that
cause electron exchange between a functionalized electrode and an analyte in solution, and can
make amperometric, voltammetric, or impedimetric estimations.
 Another approach, nanotechnology has real way against numerous agricultural problems
including plant disease identification as well as control.
 Nanoparticles show interesting electronic and optical properties and can be incorporated
utilizing various kinds of materials for electronics and detecting applications.
 The prevalence of nanomaterials for sensor improvement could be credited to the friendly
platform it facilitate the gathering of bio-recognition component, the high surface region, high
electronic conductivity and plasmonic properties of nanomaterials that upgrade the constraint
of detection.
 Now a day’s nano-based materials is introduced which enhance the effectiveness of fungicides
and pesticides, enabling minor dosages to be utilized.
 Additionally, nanodiagnostic and microfluidics offer novel tools to improve the sample
preparation step that remains difficult to incorporate in a miniaturized platform.
 The signal intensification methodologies may perhaps challenge those of target enhancement.
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 Fast nearby recognition of plant pathogens utilizing nanosensor, nanobased kits, nanobarcodes,
nanobiosensors and other portable diagnostic systems can help agricultural and food industry
to manage different plant diseases.
 The Cucumber Mosaic Virus (CMV) and Papaya Ring Spot Virus (PRSV) is a highly
aggressive disease that can reduce yield and quality of the vegetable and fruit.
 Enzyme-based biosensors are based on using enzymes that are specific to the biomolecules
under recognition to catalyze the generation of a product that can be evaluated by a transducer.
 A large portion of the enzyme utilized in biosensors is oxidases that respond with dissolved
oxygen to produce hydrogen peroxide.
 Antibody-based biosensors with a range of transducing methods have been developed.
 Detection of phytopathogenic organism by immunological techniques depends on availability
and affinity of selective antibody binding to the target bio-molecule.
 In case of complex plant material, sample is prepared by separation and accumulation of target
molecules to facilitate effective pathogen sensing.
 Antibody-based sensors, also known as immunosensors involve the use of both polyclonal and
monoclonal antibodies.
 Antibodies can be directly immobilized on the exterior of the transducer or attached to the
surface of magnetic beads to perform immunomagnetic separation and detection.
 Utilizing biosensors and other compact demonstrative frameworks can definitely help the
agriculture business.
 However several drawbacks of biosensor technology for on-site diagnosis of multiple
pathogens includes sample preparation, limited life span of biological entity, weak selectivity
in complex sample matrices, complexity of manipulations and obviously the high cost.

4.4 Detection of soil nutrients, pesticide and its residual detection.

 The soil N (nitrogen), P (phosphorus) and K (potassium) sensor is suitable for detecting the
content of nitrogen, phosphorus, and potassium in the soil, and judging the fertility of the soil
thereby facilitating the systematic evaluation of the soil condition.
 Can be buried in the soil for a long time, resistant to long-term electrolysis, corrosion
resistance, vacuum potting, and completely waterproof.
 Soil NPK sensors are widely used in soil nitrogen, phosphorus and potassium detection,
precision agriculture, forestry, soil research, geological prospecting, plant cultivation and other
fields.
 Commercially available sensors has following features.

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 Simple to use, few operation steps, fast measurement, no reagents, unlimited detection times.
 High measurement accuracy, fast response speed, and good interchangeability.
 The electrode is made of specially treated alloy material, which can withstand strong external
impact and is not easy to damage.
 Completely sealed, resistant to acid and alkali corrosion, and can be buried in soil for long-
term dynamic testing.
 The probe plug-in design ensures accurate measurement and reliable performance.
 Measurement of NPK contents of soil is necessary to decide how much extra contents of these
nutrients are to be added in the soil to increase crop fertility.
 This improves the quality of the soil which in turn yields a good quality crop.
 The color sensor is based on the principle of absorption of color by solution.
 It helps in determining the N, P, K amounts as high, medium, low, or none.
 The sensor probe along with proper signal conditioning circuits is built to detect the deficient
component of the soil.
 It is useful in dispensing only required amount of fertilizers in the soil

Fig 50 Working principle of soil NPK fiber optic sensor

 Plenty of electrochemical sensors, such as potentiometric ion-selective electrodes (ISEs), and
ion-selective field-effect transistors (ISFETS); impedimetric sensors with solvent polymeric
membranes; and optical sensors, have been developed for nitrogen’s ionic forms in soils.
 The solubility of phosphates depends on the pH of the soil: below pH 7, H2PO4− is prevalent,
and therefore, the majority of potentiometric ISEs have been developed for detection of this
ion in soils, even if the development of highly selective potentiometric sensors for detection of
the hydrophilic phosphates is a rather challenging task.
 Novel ionophores and sensing ligands, permitting selective binding of H2PO4− and HPO42−
ions, should be employed inside sensing membranes in order to replace the classical anion-
exchangers having the lowest selectivity to these hydrophilic ions according to the Hoffmeister
selectivity sequence determined by the free energy of ion solvation.

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 Electrochemical and optical sensors have been developed previously for potassium
assessments in soils.
 Among the soil moisture sensors, capacitive sensors are the most exploited.
 Electrochemical microfluidic paper-based analytical device (μPAD) used to monitor the
inhibition of the respiratory activity of Escherichia coli following exposure to heavy metals,
pesticides, and penicillin sodium.

Figure 51. Electrochemical μPAD used to monitor the inhibition of the respiratory activity of E.
coli following exposure to heavy metals, pesticides, and penicillin sodium.
 Electrochemical biosensors reported for pesticide sensing applications include those based on
(1) enzymes, (2) whole cells, and (3) antibody–antigen interaction (immunosensors)
 A direct mode enzymatic biosensor measures analyte concentration or product formation
during enzymatic reactions, whereas indirect mode biosensors monitor enzyme inhibition due
to contact with the analyte of interest.
 Acetylcholine esterase (AChE) is an enzyme mainly found in cholinergic neurons and
catalyzes the hydrolysis of the neurotransmitter acetylcholine to choline and acetate, as di
Organophosphates inhibit AChE activity through phosphorylation of the serine residue of the
enzyme’s active center, preventing the hydrolysis of acetylcholine discussed earlier.
 The biosensor designed by (1) cross-linking AChE with glutaraldehyde, (2) immobilization of
the cross-linked enzyme on the surface of a glassy carbon electrode modified with
semiconducting single-walled carbon nanotubes (s-SWCNTs), and (3) treatment with bovine
serum albumin (BSA) to prevent nonspecific binding.
 In addition to AChE, other enzymes such as butyrylcholinesterase (BChe), choline oxidase
(ChOx), phosphotriesterase (PTE), and organophosphorus hydrolase (OPH) have been
successfully utilized for electrochemical biosensing of organophosphate pesticides.

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 The biosensor had the ability to detect different classes of pesticides, including carbendazim
(benzimidazole), chlorpyrifos (organophosphate), DDT (organochlorine), dinocap (dinitro-
octylophenyl crotonate), and ethion (organophosphate).
 Microbial biosensors have been reported for the detection of several classes of pesticides,
including organochlorines, triazines, and organophosphates.
 Microbial biosensors developed based on the inhibition of the photocurrent generated by the
cyanobacterial species Anabaena variabilis.
 In addition to microbial cells, mammalian cells have been successfully utilized in biosensing
applications.

Figure 52. Cells adhered to the electrode surface are exposed to a high-frequency, low-voltage,
and amperage ac signal with the monolayer of cells impeding the flow of electrons. Upon
exposure to toxicants, the cells detach from the electrode surface, resulting in a decrease in
measured impedance

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Module-5
Applications of Nanomaterials in Biosensors
5.1 Nano Materials in biosensors; Carbon based Nano Material,
Metal oxide and nano particle, Quantum dots,
 The nanomaterials have aroused much interest soon after the discovery of nanostructures in
the early meteorites. Synthesis of gold NPs was the first
 Nanomaterials can be defined as a set of materials having at least one dimension less than
w100 nanometer (nm or 109 m) or 1-00 nm.
 1 nm is one-millionth of a millimeter or w 1, 00,000 times smaller as compared to the diameter
of a human hair. Some nanomaterials can be found naturally.
 Nanomaterials can be designed with specific applications and are being used already for
various commercial products and processes.
 Because of their small size, nanomaterials exhibit unique or novel properties.
 The length scale indicating the size of nanomaterials in comparison to various biological
components is shown below.

Figure 53. Length scale of nanomaterials

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 Atoms are the basic building blocks of nature.

 The size of an atom is measured in angstrom: 1010 m or 0.1 nm which is comparable with the
Bohr radius (0.5 nm).
 Two atoms provide a molecule, e.g., fullerene contains 100 atoms.
 The quantum effect is an important factor for the size of a semiconductor particle.
 The Bohr radius of a particle can be defined as ab= εa0. m/m*, ε is a dielectric constant of a
material, m is the rest mass of the electron, m* is the particle mass, and a0 is the Bohr radius.
 A 1 mm-sized cube crystal of salt can contain 1019 atoms or more, thus a bulk material can be
made by a number of atoms.
 Similarly, the building blocks of living organisms are cells (size 1 mm) that consist of
thousands of small and large molecules that are much larger than a NP.
Properties of nanomaterial
 The materials at the nanoscale have interesting properties as described below:
 Surface Area: Suppose a cube having a size of 1 cm x 1 cm x 1 cm is cut into several equal
pieces ending in cubes of size 0.1 mm x 0.1 mm x 0.1 mm each.
 If we cut the cube, the volume of all small cubes would be the same as that of the starting cube.
 The surfaces of all cubes have 100 times more area than that of the cube with which we started.
 And if we cut more cubes of the size of 1 nm x 1 nm x 1 nm, the area of the surfaces will
increase 10 million times as compared with that of the original cube.
 This is how nanomaterials have extremely high surface-to-volume ratio.
 This allows for nanomaterials to interact with the environment or other materials strongly
compared with bulk materials.
 In a material, the interior atoms are much more coordinated due to more bonds than surface
atoms, resulting in stable atoms.
 At corners and edges, the atoms have less coordination leading to lesser stability than the
interior atoms.
 The surface of a nanomaterial becomes quite reactive with nanoscale dimensions material and
shows extraordinary catalytic and absorbance activity.
 Quantum Confinement: In quantum mechanics, the characteristic radius of an electron is
defined as Bohr exciton radius.
 In bulk semiconductor materials, the radius of electron mobility is known to be higher than the
Bohr exciton radius, with the result that mobility is not disturbed or not confined.
 The quantum confinement effect can be found when the size of the particle is too small to be
comparable with the wavelength of the electron.
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 When a particle size of a material becomes too small or comparable with Bohr exciton radius
the electron mobility is confined.
 The electrons and holes are thus squeezed into small particles resulting in “quantum
confinement” of the electron-hole pairs.
 The confinement means restricting the motion of randomly moving electrons to specific energy
levels (discreteness).
 If a particle is of nanoscale dimensions the confining dimensions make energy levels discrete
and this will increase or widen the material band gap or energy gap.
 When the size of a particle becomes nearly Bohr exciton radius, the excitonic transition energy,
blue shift in the absorption, and luminescence band gap energy increases due to the quantum
confinement effect.
Classification of nanomaterials
 Nanomaterials can be classified as per the size and dimensions.
 There are four types of nanomaterials such as zero dimension, one dimension, two dimensions,
and three dimensions.
 Zero-dimensional: In zero-dimensional (0D) nanomaterials, all three dimensions of materials
exist in nanoscale, e.g, NPs such as gold,palladium, platinum, silver, or quantum dots.
 NPs can be spherical in size with a diameter of 1e50 nm. It has been found that some cube and
polygon shapes constitute 0D nanomaterials.
 One-dimensional: These nanomaterials having one dimension are in the range of 1e100 nm
and the other two dimensions can be in macroscale.
 Nanowires, nanofibers, nanorods, and nanotubes are examples of one-dimensional (1D)
nanomaterials.
 Some metals (Au, Ag, Si, etc.), metal oxides (ZnO, TiO2, CeO2, etc.), quantum dots, and
others can provide 1D nanostructures.
 Two-dimensional: In this class of nanomaterials, two dimensions are in nanoscale and one
dimension is in macroscale. Nano thin-films, thin-film multilayers, nanosheets, or nanowalls
are two-dimensional (2D) nanomaterials.
 The area of 2D nanomaterials can be several square micrometers keeping thickness always in
the nanoscale range. 4. Three-dimensional: In three-dimensional (3D) nanomaterials, there are
no dimensions in nanoscale, and all dimensions are in macroscale. Bulk materials are 3D
nanomaterials that are composed of individual blocks which may be in nanometer scale (1e100
nm) or more.

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Figure 54. Different types of nanomaterial

5.1.1 Carbon based nanomaterials
 In recent years, carbon-based nanomaterials derived from a range of forms, from C60, to CNTs
to graphene have become the most active research field in nanoscience, effectively promoting
the rapid development of nanotechnology.
 Carbon-based nanomaterials usually have good electrical conductivity and biocompatibility,
and can improve the active sites of electrochemical reactions.
 Due to the large specific surface area that increases the amount of immobilized biomolecule,
this kind of materials are of interest in the field of electrochemical research.
Carbon Nanotubes (CNTs)
 Carbon nanotubes (CNTs) are considered as a derivative of both carbon fibers and fullerene
with molecules composed of 60 atoms of carbons arranged in particular muffled tubes.
 Carbon nanotubes (CNTs) are considered as a derivative of both carbon fibers and fullerene
with molecules composed of 60 atoms of carbons arranged in particular muffled tubes Among
one-dimensional nanomaterials CNTs have both a special structure (radius: 2–20 nm, with
axial dimensions on the micron scale) and a large specific surface area.
 The carbon atoms in CNTs form a wide range of delocalized bonds with significant conjugation
effects, resulting in substantive conductivity.
 There are four different categories of CNTs, Categories of carbon nanotubes: (a) armchair, (b)
zigzag, and (c) chiral.

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 Based on these excellent features, CNTs have a very wide range of applications in
immunosensors.
 Among these, the use of CNTs as electrode modifying substrates or markers to build highly
sensitive sandwich-type immunosensors represent an important aspect of their study.

Figure 55. Different types of CNTs

 CNTs have good intermolecular electron transfer properties, when they are linked to
horseradish peroxidase (HRP) to modify the electrode.
 Sandwich electrochemical immunosensor developed using multi-walled CNTs includes the
following steps: (1) CNTs with specific surface area were used to immobilize the primary
antibody on the electrode surface; (2) a sandwich reaction was performed to capture enzyme-
labeled secondary antibodies loaded on CNTs on the electrode surface; (3) the addition of H2O2
to the substrate to reflect the concentration of the antigen.

Figure 56 Sandwich electrochemical immunosensor based on CNT

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 Graphene and Graphene Oxide Graphene exhibits unique physical and chemical properties, in
particular a monolithic structure, high conductivity, large specific surface area, no toxicity and
good electron mobility, and is widely used in the fields of electrochemical sensing and
biosensing.
 The graphene materials carries a high density of defects on the surface and demonstrates
particularly impressive positive electrochemical properties.
 To date, graphene-modified electrodes have been successfully applied for assessing H2O2,
NADH, dopamine, ascorbic acid, uric acid and acetaminophen.

Figure 57 Graphene base electrochemical sensors

 The application of graphene onto a variety of inorganic and organic electroactive materials is
lead its role as a promising new carbon substrate in the field of electrochemical analysis.
 Carbon nanofibers have a greater functional surface area and more surface active groups, and
thus are considered to be a more promising material.
 Carbon nanospheres have relatively richer functional groups with better biocompatibility,
dispersibility, and relative activity.

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Figure 58 Schematic presentation of CNT-based biosensors

Metal oxide nano particle
 Metal oxide nanoparticles (MONPs) are of particular interests and have received much
attention because of their unique physical, chemical and catalytic properties.
 We take an example of enzymatic biosensors based on (a) zinc oxide nanoparticle-based
enzymatic biosensors, (b) titanium oxide nanoparticle-based enzymatic biosensors; (c) iron
oxide nanoparticle-based enzymatic biosensors, and (d) on other metal oxide nanoparticle-
based enzymatic biosensors.
 Substantial advances have been made in MONPs-based enzymatic biosensors.

Figure 59. MONPs-based enzymatic biosensors

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 Metal oxide nanoparticles (MONPs) have attracted much attention for their optical, magnetic
and electronic properties.
 It is well known that MONPs not only have high surface area, good biocompatibility and
chemical stability, but also display fast electron transfer ability.
 All these features make MONPs ideal immobilization matrices as well as transduction platform
and/or mediators.
 Nanofabricated metal oxides also have been successfully applied in the field of biosensing
system.
 It is well known that enzymes and enzyme mimetics can offer great amplification power
through efficient catalytic turnover of substrates.
 The combination of enzymes with MONPs could provide a versatile platform for biosensing
with various signal readout strategies, such as electrochemical, colorimetric, fluorimetric,
chemiluminescence and so on.
 (a) zinc oxide nanoparticle-based enzymatic biosensors :As a biomimetic material, ZnO
provides a versatile platform for biomolecules loading. ZnO nanoparticles with high isoelectric
point are suitable for the adsorption of low-isoelectric point enzymes for biosensor fabrication.

Figure 60. Schematic illustration of fabricated electrode and glucose detection mechanism
over 3D porous ZnO–CuO HNCs surface
 (b) Titanium oxide nanoparticle-based enzymatic biosensors: TiO2 is one of the most
prominent materials in various applications related to catalysis, photovoltaic devices, sensors
and paintings.
 c) Iron oxide nanoparticle-based enzymatic biosensors: Well-established magnetic
nanoparticles with appropriate surface chemistry have been widely used in pollution
remediation, chemical or biological separation, bioimaging, targeted drug delivery, diseases
diagnostics and therapy, enzyme immobilization, biosensing, etc.

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Figure 61. Schematic representation of the configuration of the microfluidic biosensor with 3D
porous GF electrode modified with carbon-doped TiO2 NFs for the detection of breast cancer
biomarkers
 d) Other metal oxide nanoparticle-based enzymatic biosensors: zirconia nanoparticles and
their nanocomposites have been reported to be used for enzyme immobilization, such as HRP,
GOx, AChE, Hb and Bilirubin oxidase. An electrochemical quartz crystal microbalance
(EQCM) immunoassay was developed utilizing ZrO2 nanoparticles for selective capture
phosphorylated AChE (Phospho-AChE) due to their strong affinity for phosphoric groups.
Quantum Dots
 Quantum dots are tiny specks of material, so small that some people say they have no
dimensions. They exist as points of materials, typically 1/10,000 the size of a human hair.
 A variety of quantum dots not only have the characteristics of nanomaterials, but also can give
good, sharp and sensitive dissolution peaks through electrochemical dissolution voltammetry.
 Quantum dots are nanoparticles made from semiconducting materials.
 The dots show quantum effects because they are so little.
 This means that electrons inside the dot are trapped and can only occupy defined energy levels.
 With only confined, discrete energy levels available, quantum dots can have different optical
and electrical properties to a large quantity of the same material.
 This makes quantum dots useful in developing nanotechnology.
 Quantum dots can capture light and convert it into electricity.
 They can do this efficiently and require less space than larger, conventional materials.
 Changing the size of the quantum dot allows us to tune their ability to absorb and emit specific
light frequencies.

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 Within the chemical growth process, we can use flowing gases and cold temperatures to control
the size of the quantum dot.
 A bigger dot will emit light at a longer wavelength. In the visible range, larger dots glow red.
Smaller dots shine blue.
 Quantum dots confine the motion of electrons in all three spatial directions.
 This restriction leads us into the quantum world, and quantum dots' electrical and optical
properties.
 The QD-based Fluorescence Resonance Energy Transfer (FRET) genosensor, most widely
used in the biomolecule detection fields, and QD-based nanosensor for Rev-RRE interaction
assay are presented as examples.
 Fluorescence resonance energy transfer (FRET)* is a distance-dependent physical process by
which energy is transferred nonradiatively from an excited molecular fluorophore (the donor)
to another fluorophore (the acceptor) by means of intermolecular long-range dipole–dipole
coupling.
 FRET can be an accurate measurement of molecular proximity at angstrom distances (10–100
Å) and highly efficient if the donor and acceptor are positioned within the Förster radius (the
distance at which half the excitation energy of the donor is transferred to the acceptor, typically
3–6 nm).
 The efficiency of FRET is dependent on the inverse sixth power of intermolecular separation,
making it a sensitive technique for investigating a variety of biological phenomena that
produce changes in molecular proximity
 In recent years, QD-based biosensors have emerged as a new class of sensor and are expected
to open opportunities in plant virus detection, but as yet there have been very few practical
applications.

Figure 62. Competitive displacement of a quenching dye by the analyte removes the FRET
interaction, resulting in QD emission recovery.

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 Thus, quantum dot materials have good prospects in the related fields of nanomaterials and
electrochemical analysis.
 Different semiconductor nanomaterials (ZnS, CdS, PbS and CuS) were used for labeling
various proteins (α2-microglobulin, IgG, bovine serum albumin, C-reactive protein).
 According to the peak position of the marker and the tested peak current from the stripping
voltammetry, different antigens were identified and determined.
 Sandwich-type immunosensor fabricated on an ITO chip covered with a well-ordered AuNPs
monolayer applying CdTe quantum dots as electrochemical and fluorescent labels.

5.2 Role of nano material in Signal Amplifications, Detection and

Transducer Fabrication
 Nanomaterials have recently aroused much interest due to the increased need for control of
desired molecules present in the human body and environment.
 A nanomaterial comprises of nanoparticles (NPs) that are less than 100 nm at least in one
dimension.
 The term “nanotechnology” deals with small-sized materials when the size is down to
subnanometer or several hundred nanometers.
 The controlled synthesis and tuning properties of nanomaterials require knowledge of different
disciplines such as physics, chemistry, electronics, computer science, biology, engineering,
agriculture, etc. that may lead to the emergence of novel and multifunctional nanotechnologies.
 In this context, the exciting properties of nanomaterials have attracted the world scientific
community toward their application in various sectors such as health, food, security, transport,
and information technology, etc.

Fig. 63. The roles of nanomaterials in signal amplification.

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 The intelligent use of nanomaterials is predicted to enhance the performance of biomolecular

electronic devices with high sensitivities and detection limits.
 Diagnostics is clinically important both for identification of a disease and therapeutics.
 Early diagnostics plays an important role to detect a disease (prevention) or the outcome of a
disease (prognosis).
 A number of biodevices have been fabricated for studies of blood gas,
glucose/lactate/cholesterol, nucleic acid sequence analysis, proteins/peptides, combinatorial
synthesis, toxicity monitoring, immunoassays, environment, defense, and forensic analysis.
 A biosensor for blood glucose monitoring has been successfully commercialized.
 Efforts are being made to enhance the resolution, accuracy, and miniaturization of biosensors
for detection of biomolecules along with microfluidics and sample preprocessing.
 Because of their portability, the short time to obtain results, biosensors have been predicted to
fulfill a number of unmet needs in the diagnostics industry.
 Microfluidic biosensing devices offer important opportunities for research, especially for
clinical diagnosis, due to their numerous advantages.
 These miniaturized devices require minute volumes (10−9 to 10−18 L) in micron-sized
channels and containers leading to the development of a “lab-on-a-chip”.
 Nanomaterials are currently undergoing rapid development due to their potential applications
in the field of nanoelectronics, catalysis, magnetic data storage, structural components,
biomaterials, and biosensors.
 The use of NPs, nanotubes, and nanowires, etc. in biosensor diagnostic devices are being
explored.
 With the advancement in properties of nanomaterials, their dimensions at the nanoscale level,
new biodevices (smart biosensors) that can detect minute concentration of a desired analyte
are emerging.
 Nanomaterials are generally used as transducer materials that are an important part for
biosensor development.
 A biosensor consists of four parts namely (1) bioreceptor, (2) a transducer, (3) a signal
processor for converting electronic signal to a desired signal, and (4) an interface to display.
 A variety of samples such as body fluids, food samples, and cells culture can be explored to
analyze using biosensors.
 The engineered nanomaterials provide higher electrical conductivity, have nanoscale size, can
be used to amplify desired signals, and are compatible with biological molecules.

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 For example, carbon materials can be utilized for conjugation of biomolecules (enzyme,
antibody, DNA, cell, etc.).
 It has been found that the use of nanomaterials may lead to increased biosensor performance
including increased sensitivities and low limit-of-detection of several orders of magnitudes.
 Nanostructured materials show increased surface-to-volume ratio, chemical activity,
mechanical strength, electrocatalytic properties, and enhanced diffusivity.
 Nanomaterials have been predicted to play an important role toward the high performance of
a biosensor.
 To probe biomolecules such as bacteria, virus, DNA, etc. biocompatibility of nanomaterials is
an important factor for designing a biosensor.
 Nanomaterials with various applications for biosensor development are discussed in this
chapter.
 An important challenge is the standardization of immobilization procedure that can be utilized
to intimately conjugate a biomolecule onto a nanomaterial.
 Therefore, the technique used to immobilize a given enzyme is one of the key factors in
developing a reliable biosensor.
 A nanomatrix can be an excellent candidate to immobilize biomolecules on a transducer
surface that can efficiently maintain bioactivity of the biomolecules.
 There are still many challenges such as miniaturization, automation, and integration of the
nanostructured-based biosensors.
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