Introduction

The term “ biosensor ” was introduced By Clark and

Lyos in 1962.

Self-contained integrated device that is capable of

providing specific qualitative or semi-quantitative
analytical information using a biological recognition
element which is in direct-spatial contact with a
transduction element. (IUPAC,1998)
CURRENT DEFINITION
A sensor that integrates a biological element with a
physiochemical transducer to produce an
electronic signal proportional to a single analyte
which is then conveyed to a detector.
 Any device that has specific biochemical
reactions to detect chemical compounds in
biological samples.

FATHER OF BIOSENSOR.
Professor Leland C Clark Jnr
(1918–2005)
 BASIC COMPONENTS
 Bio-element

 Transducer component
RESPONSE FROM BIO-ELEMENT
 Heat absorbed (or liberated ) during the interaction.

 Movement of electrons produced in a redox reaction.

 Light absorbed (or liberated ) during the interaction.

 Effect due to mass of reactants or products.

TYPES OF BIOSENSOR

 Electrochemical biosensor
 Optical biosensor
 Thermal biosensor
 Resonant biosensor
 Ion-sensitive biosensor
COMPONENTS

Detector
 THE ANALYTE.
(What do you want to detect?)
Molecule
Protein, toxin, peptide, vitamin, sugar, metal ion

Cholera toxin Glucose

 DETECTION/RECOGNITION.
(How do you specifically recognise the analyte?)
Fab

Active site Membrane receptors

Competitive binding

Fc
Antibody Enzyme Cell Polymer/Hydrogel
 SIGNAL
(How do you know there was a detection?)
Specific recognition?
Common Signalling Principles
● Optical(SPR,ELM,IR).
● Electrical(Voltametry,Potentiometry,
Conductivity).
● Electromechanical(QCM).
● Thermal.
● Magnetic.
● Pressure.

Often the detector is immobilized on a solid support/sensor.

(The immobilisation permits repeated use of the costly Biological
Molecule.)
 WORKING PRINCIPLE
Analyte diffuses from the solution to the surface of
the Biosensor.
Analyte reacts specifically & efficiently with the
Biological Component of the Biosensor.
This reaction changes the physicochmical properties
of the Transducer surface.
This leads to a change in the optical/electronic
properties of the Transducer Surface.
The change in the optical/electronic properties is
measured/converted into electrical signal, which is
detected.
BASIC CHARACTERESTICS

 LINEARITY - Should be High – For the

detection of High Substrate Concentration.
 SENSITIVITY - Value of Electrode Response
per Substrate Concentration.
 SELECTIVITY - Chemical Interference must be
minimised for obtaining Correct Result.
 RESPONSE TIME – Time necessary for having
95% of the Response.
 ADVANTAGES

Highly Specific.
Independent of Factors like stirring, pH, etc.
Linear response, Tiny & Biocompatible.
Easy to Use, Durable.
Require only Small Sample Volume.
Rapid, Accurate, Stable & Sterilizable.
TYPICAL SENSING TECHNIQUES
 Fluorescence.
 DNA Microarray.
 SPR (Surface Plasma Resistance).
 Impedance Spectroscopy.
 SPM (Scanning Probe Microscopy, AFM, STM).
 QCM (Quartz Crystal Microbalance).
 SERS (Surface Enhanced Raman Spectroscopy).
 Electrochemical.
 TYPES
 Calorimetric/Thermal Detection Biosensors.
 Optical Biosensors.
 Resonant Biosensors.
 Piezoelectric Biosensors.
 Ion Sensitive Biosensors.
 Electrochemical Biosensors.
 Conductimetric Sensors.
 Amperometric Sensors.
 Potentiometric Sensors.
 Calorimetric / Thermal Detection Biosensors.
 Uses Absorption / Production of Heat.
 Total heat produced/absorbed is ᾶ Molar
Enthalpy/Total No. of molecules in the rn.
 Temp. measured by Enzyme Thermistors.
Advantages:
• No need of Frequent recalibration.
• Insensitive to the Optical & Electrochemical
Properties of the sample.
Uses:
Detection of: (1) Pesticides .
(2) Pathogenic Bacteria.
 Optical Biosensors.
 Colorimetric for colour - Measures change in
Light Adsorption.
 Photometric for Light Intensity - Detects the
Photon output.

 Resonant Biosensors.
 An Acoustic Wave Transducer is coupled with
Bioelement.
 Measures the change in Resonant Frequency.
 Piezoelectric Biosensors.
 Uses Gold - To detect specific angle at which ȇ waves
are emitted when the substance is exposed to laser
light/crystals like quartz, which vibrates under the
influence of an electric field.
 Change in Frequency ᾶ Mass of Absorbed
material.
 Ion Sensitive Biosensors.
 Are semiconductor FETs with ion-sensitive surface.
 Surface Electrical Potential changes when the ions &
semiconductors interact.
 Measures the Change in Potential.
Uses:
o pH Detection.
 Electrochemical Biosensors.
Underlying Principle – Many chem.rns produce or
consume ions or ȇs causing some change in the
elctrical properties of the solution that can be sensed
out & used as a measuring parameter.
Uses:
Detection of :
o Hybridized DNA
o DNA- binding Drugs &
o Glucose Concentration.
 Conductimetric Sensors.
 Measures Electrical Conductance/Resistance of the
solution.
  Conductance Measurements have relatively Low
Sensitivity.
 Electrical Field is generated using sinusoidal(ac)
voltage, which helps in minimizing undesirable
effects like:
i. Faradaic processes.
ii. Double layer charging &
iii. Concentration polarization.

 Amperometric Biosensors.
 High Sensitivity Biosensor.
 Detects electroactive species present in the
biological test samples.
 Measured Parameter – Current.
 Potentiometric Sensors.
 Working Principle – When ramp voltage is
applied to an electrode in solution, a current flow
occurs because of electrochemical reactions.
 Measured Parameter – Oxidation / reduction
Potential of an Electrochemical rn.
APPLICATIONS
Food Analysis.
Study of Biomolecules & their Interaction.
Drug Development.
Crime Detection.
Medical Diagnosis (Clin&Lab).
Environmental Field Monitoring.
Quality Control.
Industrial Process Control.
Detection Systems for Biological Warfare Agents.
Manf. Of Pharmaceuticals & Replacement organs.
BIOSENSOR FOR AGRICULTURAL
& FOOD INDUSTRY.
o Detection of viral, fungal, bacterial diseases of
plants.
o In food industry, detection of total microbes &
food quantification in soft drinks.
o To determine the freshness of other fish, beef &
other food items.
o Makes Bacteria GLOW by OPTICAL Biosensor
Pregnancy Test. Infectious Disease Biosensor.
•Detects the hCG protein in urine. •Data analysis and interpretation
• Interpretation and data analysis performed by a microprocessor.
performed by the user.
 Components of biosensor

 Biologically active material or biological recognition

element.

 Detector element or transducer.

 Signal processor.
 the sensitive biological element (biological material
(e.g. tissue, microorganisms, organelles, cell
receptors, enzymes, antibodies, nucleic acids, etc.), a
biologically derived material that interacts (binds or
recognises) the analyte under study. The biologically
sensitive elements can also be created by biological
engineering.
 the transducer or the detector element (works in a
physicochemical way; optical, piezoelectric,
electrochemical, etc.) that transforms the signal
resulting from the interaction of the analyte with the
biological element into another signal (i.e.,
transducers) that can be more easily measured and
quantified;
 Principle of biosensor
Immobilization of biological material on the
immobilization support, the permeable membrane, in the
direct vicinity of a sensor.

The substances to be measured pass through the

membrane and interact with the immobilized material and
yield the product.
 The product passes through another membrane to the
transducer.

 The transducer converts product into an electric signal which is

amplified.

 The signal processing equipment converts the amplified

signals into a display most commonly the electric signal which
can be read out and recorded.
 Types of biosensor
 Biosensor is broadly classified into two classes:
 I) On the basis of biological element

 a) Enzyme Biosensor
 b) Microbial Biosensor
 c) Antibody Based.
 II) On the basis of transducing element

 a)Electrochemical transducers: The two common types of

electrochemical transducers are:

 Amperometric and Potentiometric transducer.

 Amperometric transducers measure the potential between the two

electrodes is set and current produced by oxidation –reduction of electro
active species is measured and correlated with the concentration of the
analyte of the interest.
 Potentiometric measures the potential of electrochemical cell
with very low current value.

 b) Optical Transducer: They are used for determining the

concentration of analyte on the basis of change in optical
density at appropriate wavelength. I.e. Total Internal Reflection
is measured with a Photodetector as a function of incident
angle.

 c) Calorimetric Transducers: It is used for calculating the

heat of biochemical reactions by measuring the temperature
difference between the reaction vessel and isothermal heat sink
surrounding
 The advantages of employing enzymes in biosensor
architecture
 Enzymes have a high selectivity for their substrates.

 They exhibit a very high catalytic activity.

 An enzyme-based electrochemical sensor is formulated by

immobilizing a thin layer of enzyme on the surface of the membrane of
an electrode. The analyte to be monitored diffuses into the enzyme
layer where the catalytic reaction occurs, either consuming a substrate
or generating a product that can be detected electrochemically.
 Enzyme Biosensor: Enzyme because of its high
selectivity and activity towards substrate are the best
candidate to be used as biologically active materials.

 Enzymatic biosensors measure the selective

inhibition or the catalysis of enzymes by a specific
target. Most of the enzymes used in biosensor are
Oxidizes but there are certain limitations as their
activity is susceptible to pH, temperature, ionic
strength etc.
 Enzyme-based biosensors represent a major application of
immobilized enzymes in medicine.

 The high specificity and reactivity of an enzyme towards

its substrate are properties being exploited in biosensor
technology.
 Enzymes that are used in biosensors often belong to the
classes of oxidoreductases and hydrolases.

 Thus, they are particularly suitable for their coupling with

electrochemical transducers.
 Biosensor reader device with the associated electronics
or signal processors is primarily responsible for the
display of results

 This sometimes accounts for the most expensive part

of the sensor device.

 The readers are usually custom designed and

manufactured to suit the different working principles
of biosensors.
 First generation: the two components (biocatalyst &
tranducer) may be easily separated & both may remain
functional in the absence of the other
 Second generation :the two components interact in a
more intimate fashion & removal of one of the two
components affects the usual functioning of the other
 Third generation : the biochemistry & where the
electrochemistry occurs at a semiconductor ,the term
biochip may be applied to describe such instruments.
First generation instruments
 Glucose+o2-----------gluconic acid+H2o2
 The rate of consumption of the substrate o2 can be
measured by its reduction at a platinum cathode
 The rate of production of the product H2O2 can be
measured by its oxidation at a platinum anode
 The rate of production of the product gluconic acid
can be measured using a pH electrode
YSI MODEL 23
Second generation instruments
 SECOND GENERATION INSTRUMENTS can be
constructed by designing an electrode surface that is
capable of capturing electrons which are usually
transferred in the oxidation reduction reactions.
 Glucose + GO/FAD Gluconic acid+ GO/FADH2

 GO FADH2+ 2M+ GO/FAD + 2M + 2H+

 2M 2M+ + 2e-
Exac tech glucometer
Third generation instruments
 These instruments involve the most intimate
interactions of the biocatalyst and transducer
 A glucose biosensor operating on the principle of Exac
Tech meter but in which the enzyme was directly
reduced at the electrode surface (obviating the need
for a mediator) is an example of such an instrument
Cell based biosensors
 Immobilised whole cells or tissues are used to produce
biosensors.
 More recent immobilisation techniques have intended
to use gentler physical methods such that cell viability
is retained
 The advantage of this is that such cells may be
involved in converting substrate into product via a
complex multi enzyme pathway
Without having to immobilise each of the enzymes &
then provide them with expensive coenzymes
 Eg.,Nocardia erythropolis immobilised in poly acrylamide
on an oxygen electrode
Chol.oxidase

 Cholesterol+ O2 cholest-4-en-3-one+ H2O2

N.erythropolis

 The oxygen electrode measures the rate of oxygen uptake &

this can be related to the cholesterol content of the
biological sample
Advantages Disadvantages
 Cheaper  Cells contain many
 No requirement for a enzymes.Hence, care has
complex biocatalyst to be taken to ensure
 They have longer
selectivity of response
response times than do  Time taken for cell based
enzyme based sensors biosensor to return to
base line potential after
use is more.
Enzyme immunosensors
 Several kinds of enzyme immunosensors have been
developed
 They combine the molecular recognition properties of
antibodies with the high sensitivity of enzyme based
analytical methods
 The enzyme is used as a marker as it reacts with its
substrate,giving changes that can be detected by a
transducer.
 There is similarity between such methods & ELISA
techniques.
 A similar assay can be carried out for hCG .
 Catalase is used to label hCG & oxygen evolution
noted by oxygen electrode
 In attempts to construct enzyme immunosensors,
bioluminiscence,chemiluminiscence & fluorescence
principles are exploited because of their great
sensitivity
 A luminiscent immunoassay with catalase has been
used to detect human serumalbumin at only 1 ng/cm3
 Enzyme based biosensors are used in different
analysers for quantification of glucose (PO2 electrode)
,urea, creatinine etc where the enzyme is immobilized
on the sensor.
 Affinity sensors have immobilized molecules with
specific high affinity binding properties like binding
proteins, antibodies, aptamers (DNA SENSORS)
Oxygen electrode
 O2+2e+2H2O--H2O2+2OH
 H2O2+2e------2OH
 -----------------------------------------
 Total O2+4e+2H2O--4OH
 The reaction occuring at the anode is 4Ag+4Cl---
4Agcl+4e
 The Overall electrochemical process is
4Ag+O2+4Cl+2H2O-4Agcl+4OH
Schematic diagram showing the
main components of biosensor
 The biocatalyst (a) converts the substrate to product.
 This reaction is determined by the transducer (b)
which converts it to an electrical signal.
 The output from the transducer is amplified (c),
processed (d) and displayed (e).
Principles of detection
 Photometric
 Electrochemical
 Ion channel switch
 Others…,like piezoelectric
thermometric etc
photometric
 Many optical biosensors based on the principle of surface
plasma resonance(SPR) are evanescent wave techniques
 This utilises a property of gold & other materials
specifically that a thin layer of gold on a high refractive
index glass surface can absorb light producing electron
waves (surface plasmons) on the glass surface.
 This occurs only at a specific angle & wave length of
incident light and is highly dependent on the surface of
gold , such that binding of a target analyte to a receptor on
the gold surface produces a measurable signal
Interferometric reflectance imaging
sensor(IRIS)
 The Interferometric Reflectance Imaging Sensor (IRIS)
was developed by the Unlu research group at Boston
University for the purpose of label-free biosensing.
 Using simple lenses and low-powered, coherent
LED’s, the device offers exquisite sensitivity and
reproducibility and is able to image with remarkable
resolution beyond the classical diffraction limit.
 This relatively cheap solution also presents minimal
hazards when compared to a laser illumination source.
Applications of biosensors
 Glucose monitoring in diabetes patients ←historical
market driver related targets

 Remote sensing of airborne bacteria e.g. in counter-

bioterrorist activities
Invivo biosensors
 Invivo miniaturized sensors are being developed for
measurement of saO2,pH etc.
 Implantable subcutaneous glucose sensors are also
being used to adjust the dose of insulin
 Intravascular sensors that release nitric oxide have
been developed to decrease the possibility of
thrombosis
 Detection of pathogens
 Determining levels of toxic substances before and
after bioremediation
 Detection and determining of organophosphate
 Routine analytical measurement of folic
acid, biotin, vitamin B12 and pantothenic acid as an
alternative to microbiological assay
 Determination of drug residues in food, such
as antibiotics and growth promoters, particularly
meat and honey.
 Drug discovery and evaluation of biological activity of
new compounds.
 Protein engineering in biosensors
 Detection of toxic metabolites such as mycotoxins