Biosensors

A biosensor is an analytical device containing an immobilized

biological material (enzyme, antibody, nucleic acid, hormone,
organelle or whole cell) which can specifically interact with an analyte
and produce physical, chemical or electrical signals that can be
measured.

An analyte is a compound (e.g. glucose, urea, drug, pesticide) whose

concentration has to be measured.

1. Biological component—enzyme, cell etc.

2. Physical component—transducer, amplifier etc.
 Desired molecule

Signal
Bioreceptor Transducer Display
Processing

Biosample

Biosensor
Bioreceptor Transducer

Absorption
Antibody Fluorescence
Optical Interference

potentiometric
Enzyme Electrochemical amperometric
conductimetric

Nucleic Acid (DNA) Mass based

Cell Temperature based

Dielectric properties
Electric & Permeability properties
MIP
Magnetic Voltage or Current
Bioreceptor
is designed to interact with the specific analyte of interest to produce an
effect measurable by the transducer. High selectivity and sensitivity for
the analyte among a matrix of other chemical or biological components
is a key requirement of the bioreceptor.
An antibody (Ab), also known as an immunoglobulin
(Ig) is a large, Y-shaped protein produced mainly by
plasma cells that is used by the immune system to
neutralize pathogens such as pathogenic bacteria and
viruses .

In immunology , antigens (Ag) are structures (aka

substances) specifically bound by antibodies (Ab) or a cell
surface version of Ab - B cell antigen receptor (BCR).

An immunosensor utilizes the very specific binding affinity

of antibodies for a specific compound or antigen. The specific
nature of the antibody-antigen interaction is analogous to a lock
and key fit in that the antigen will only bind to the antibody if it
has the correct conformation
Artificial binding proteins

recombinant binding fragments (Fab, Fv or scFv) or domains (VH, VHH) of antibodies have been engineered
In another approach, small protein scaffolds with favorable biophysical properties have been engineered to generate
artificial families of Antigen Binding Proteins (AgBP), capable of specific binding to different target proteins while
retaining the favorable properties of the parent molecule. The elements of the family that specifically bind to a given
target antigen, are often selected in vitro by display techniques: phage display, ribosome display, yeast
display or mRNA display.
The artificial binding proteins are much smaller than antibodies (usually less than 100 amino-acid residues), have a
strong stability, lack disulfide bonds and can be expressed in high yield in reducing cellular environments like the
bacterial cytoplasm, contrary to antibodies and their derivatives. They are thus especially suitable to create biosensors.
Enzymatic interactions
The specific binding capabilities and catalytic activity of enzymes make them popular bioreceptors. Analyte recognition
is enabled through several possible mechanisms:
1) the enzyme converting the analyte into a product that is sensor-detectable,
2) detecting enzyme inhibition or activation by the analyte, or
3) monitoring modification of enzyme properties resulting from interaction with the analyte.

The main reasons for the common use of enzymes in biosensors are:
1) ability to catalyze a large number of reactions;
2) potential to detect a group of analytes (substrates, products, inhibitors, and modulators of the catalytic
activity)
3) suitability with several different transduction methods for detecting the analyte.
4) since enzymes are not consumed in reactions, the biosensor can easily be used continuously. The
catalytic activity of enzymes also allows lower limits of detection compared to common binding
techniques. However, the sensor's lifetime is limited by the stability of the enzyme.
Tissue
Tissues are used for biosensor for the abundance of enzymes existed. Advantages of tissues as biosensors include the
following:
 easier to immobilize compared to cells and organelles
 the higher activity and stability from maintain enzymes in natural environment
 the availability and low price
 the avoidance of tedious work of extraction, centrifuge and purification of enzymes
 necessary cofactors for enzyme to function exists
 the diversity providing a wide range of choice concerning different objectives.
There also exist some disadvantages of tissues, like the lack of specificity due to the interference of other enzymes and
longer response time due to transport barrier.
Cells
Cells are often used in bioreceptors because they are sensitive to surrounding environment and they can respond to all
kinds of stimulants. Cells tend to attach to the surface so they can be easily immobilized. Compared to organelles they
remain active for longer period and the reproducibility makes them reusable. They are commonly used to detect global
parameter like stress condition, toxicity and organic derivatives.
Basic Characteristics of a Biosensor

1. LINEARITY Linearity of the sensor should be high

for the detection of high substrate
concentration.
2. SENSITIVITY Value of the electrode response per
substrate concentration.
3. SELECTIVITY Chemicals Interference must be
minimised for obtaining the correct
result.
4.RESPONSE TIME Time necessary for having 95%
of the response.
Types of the Biosensors
1.Calorimetric / Thermal Detection Biosensors
2.Optical Biosensors
3.Resonant Biosensors
4.Piezoelectric Biosensors
5.Ion Sensitive Biosensors
6.Electro Chemical Biosensors
a.Conductimetric Sensor
b.Amperometric Sensor
c.Potentiometric sensors
Typical Sensing Techniques for Biosensors
• Fluorescence
• DNA Microarray
• SPR Surface plasmon resonance
• Impedance spectroscopy
• SPM (Scanning probe microscopy, AFM, STM )
• QCM (Quartz crystal microbalance)
• SERS (Surface Enhanced Raman Spectroscopy)
• Electrochemical
Amperometric Biosensors:

These biosensors are based on the movement of electrons (i.e. determination of electric current) as a
result of enzyme-catalysed redox reactions.

Normally, a constant voltage passes between the electrodes which can be determined. In an enzymatic
reaction that occurs, the substrate or product can transfer an electron with the electrode surface to be
oxidised or reduced

This results in an altered current flow that can be measured. The magnitude of the current is
proportional to the substrate concentration.

Clark oxygen electrode which determines reduction of O , is the simplest form of amperometric
2

biosensor.

Determination of glucose by glucose oxidase is a good example.

Blood-glucose biosensor:

It is a good example of amperometric biosensors

Blood- glucose biosensor looks like a watch pen and has a single use disposable electrode (consisting of a
Ag/AgCI reference electrode and a carbon working electrode) with glucose oxidase and a derivative of
ferrocene (as a mediator). The electrodes are covered with hydrophilic mesh guaze for even spreading of a
blood drop. The disposable test strips, sealed in aluminium foil have a shelf-life of around six months.

An amperometric biosensor for assessing the freshness of fish has been developed. The accumulation of
ionosine and hypoxanthine in relation to the other nucleotides indicates freshness of fish-how long dead and
stored. A biosensor utilizing immobilized nucleoside phosphorylase and xanthine oxidase over an electrode
has been developed for this purpose.
Potentiometric Biosensors:
In these biosensors, changes in ionic concentrations are determined by use of ion- selective electrodes
pH electrode is the most commonly used ion-selective electrode,
many enzymatic reactions involve the release or absorption of hydrogen ions. The other important electrodes
are ammonia-selective and CO selective electrodes.
2

The potential difference obtained between the

potentiometric electrode and the reference electrode can
be measured. It is proportional to the concentration of the
substrate.

The major limitation of potentiometric biosensors is the

sensitivity of enzymes to ionic concentrations such as H +

and NH .+
4

Ion-selective field effect transistors (ISFET) are the low

cost devices that can be used for miniaturization of
potentiometric biosensors. A good example is an ISFET
biosensor used to monitor intra-myocardial pH during
open-heart surgery.
Conduct Metric Biosensors:

There are several reactions in the biological systems that bring about changes in the ionic species. These ionic
species alter the electrical conductivity which can be measured. A good example of conduct metric biosensor is
the urea biosensor utilizing immobilized urease. Urease catalyses the following reaction.

The above reaction is associated with drastic alteration in ionic concentration which can be used for
monitoring urea concentration. In fact, urea biosensors are very successfully used during dialysis and renal
surgery.
Thermometric Biosensors:
Several biological reactions are associated with the production
of heat and this forms the basis of thermometric biosensors.
They are more commonly referred to as thermal biosensors or
calorimetric biosensors. A diagrammatic representation of a
thermal biosensor is depicted in Fig. It consists of a heat
insulated box fitted with heat exchanger (aluminium cylinder).
The reaction takes place in a small enzyme packed bed reactor. As the substrate enters the bed, it gets
converted to a product and heat is generated. The difference in the temperature between the substrate and
product is measured by thermistors. Even a small change in the temperature can be detected by thermal
biosensors.

Thermometric biosensors are in use for the estimation of serum cholesterol. When cholesterol gets oxidized by
the enzyme cholesterol oxidase, heat is generated which can be measured. Likewise, estimations of glucose
(enzyme-glucose oxidase), urea (enzyme-urease), uric acid (enzyme-uricase) and penicillin G (enzyme-P
lactamase) can be done by these biosensors. In general, their utility is however, limited. Thermometric
biosensors can be used as a part of enzyme-linked immunoassay (ELISA) and the new technique is referred to
as thermometric ELISA (TELISA).
Optical Biosensors:

Optical biosensors are the devices that utilize the principle of optical measurements (absorbance, fluorescence,
chemiluminescence etc.). They employ the use of fibre optics and optoelectronic transducers. The word
optrode, representing a condensation of the words optical and electrode is commonly used. Optical biosensors
primarily involve enzymes and antibodies as the transducing elements.

Optical biosensors allow a safe non-electrical remote sensing of materials. Another advantage is that these
biosensors usually do not require reference sensors, as the comparative signal can be generated using the same
source of light as the sampling sensor. Some of the important optical biosensors are briefly described
hereunder.
 Fibre optic lactate biosensor:

Fig. represents the fibre optic lactate biosensor. Its working is based
on the measurement of changes in molecular O concentration by
2

determining the quenching effect of O on a fluorescent dye. The

2

following reaction is catalysed by the enzyme lactate mono-

oxygenase.

The amount of fluorescence generated by the dyed film is dependent on the O . This is because O has a quenching
2 2

(reducing) effect on the fluorescence. As the concentration of lactate in the reaction mixture increases, O is utilized,
2

and consequently there is a proportionate decrease in the quenching effect. The result is that there is an increase in
the fluorescent output which can be measured.
Optical Biosensors for Blood Glucose:
Estimation of blood glucose is very important for monitoring of diabetes. A simple technique involving paper
strips impregnated with reagents is used for this purpose. The strips contain glucose oxidase, horse radish
peroxidase and a chromogen (e.g. toluidine). The following reactions occur

The intensity of the colour of the dye can be measured by using a portable reflectance meter. Glucose strip
production is a very big industry worldwide.

Colorimetric test strips of cellulose coated with appropriate enzymes and reagents are in use for the estimation
of several blood and urine parameters.
Luminescent biosensors to detect urinary infections:

The microorganisms in the urine, causing urinary tract infections, can be detected by employing luminescent
biosensors. For this purpose, the immobilized (or even free) enzyme namely luciferase is used. The
microorganisms, on lysis release ATP which can be detected by the following reaction. The quantity of light
output can be measured by electronic devices.
Piezoelectric Biosensors:
Piezoelectric biosensors are based on the principle of acoustics (sound vibrations), hence they are also called as
acoustic biosensors. Piezoelectric crystals form the basis of these biosensors. The crystals with positive and
negative charges vibrate with characteristic frequencies. Adsorption of certain molecules on the crystal surface
alters the resonance frequencies which can be measured by electronic devices. Enzymes with gaseous substrates
or inhibitors can also be attached to these crystals.

A piezoelectric biosensor for organophosphorus insecticide has been developed incorporating acetylcholine
esterase. Likewise, a biosensor for formaldehyde has been developed by incorporating formaldehyde
dehydrogenase. A biosensor for cocaine in gas phase has been created by attaching cocaine antibodies to the
surface of piezoelectric crystal.

Limitations of Piezoelectric Biosensors:

It is very difficult to use these biosensors to determine substances in solution. This is because the crystals may
cease to oscillate completely in viscous liquids.
 Whole Cell Biosensors:
Whole cell biosensors are particularly useful for multi-step or cofactor requiring reactions. These biosensors may
employ live or dead microbial cells. A selected list of some organisms along with the analytes and the types of
biosensors used is given in Table 21.8

Advantages of microbial cell biosensors:

The microbial cells are cheaper with longer half-lives. Further, they are less sensitive to variations in pH and
temperature compared to isolated enzymes.

Limitations of microbial cell biosensors:

The whole cells, in general, require longer periods for catalysis. In addition, the specificity and sensitivity of whole
cell biosensors may be lower compared to that of enzymes.
Immuno-Biosensors:
Immuno-biosensors or immunochemical biosensors
work on the principle of immunological specificity,
coupled with measurement (mostly) based on
amperometric or potentiometric biosensors. There
are several possible configurations for immuno-
biosensors and some of them are depicted in Fig.
21.18, and briefly described hereunder

1. An immobilized antibody to which antigen can directly

bind (Fig . A).

2. An immobilized antigen that binds to antibody which in

turn can bind to a free second antigen (Fig.B).

3. An antibody bound to immobilized antigen which can be

partially released by competing with free antigen (Fig. C).

4. An immobilized antibody binding free antigen and

enzyme labeled antigen in competition (Fig.D).
Transducers- Optical methods
Concept: Capture analyte and detect binding by optical tag or binding-sensitive
optical phenomenon

Absorption

I1/I0 = e−αlc
l is the pass length
C is the concentration of absorbing material
α is the absorption coefficient
Transducers- Optical methods- Absorption

A device to determine a patient’s blood oxygen content: “the oximeter”

The absorption spectra (α) of haemoglobin (Hb) and oxyhaemoglobin (HbO2) differ,
which makes it possible to measure the ratio of both concentrations in blood by
measuring the absorption of light of two different wavelengths, e.g. 660 nm and 805 nm.
 Transducers-Optical methods- Florescence

Fluorescence is a molecular absorption of light at one

wavelength and its instantaneous emission of at longer
wavelengths. Some molecules fluoresce naturally and others
such as DNA can be modified for fluorescence detection by
attachment of special fluorescent dyes

An optical system for

Florescence measurement
 Transducers-Optical methods- Florescence

Grating

Antigens modified
by florophor dye

Evanescent-field.
Planer
waveguide

Excitation light Florescence

detector

A device for florescence measurement

Transducers-Optical methods- Florescence

Taq-Man probe in its

preliminary condition for
real time PCR devive

T  55 c


T  72 c


Hybridisation of primer and Taq-Man Releasing fluorophore dye

probe at annealing temperature during extension
 Transducers-Optical methods- Florescence

Real time Chamber PCR device with integrated

fluorescence detection
Photodiode pattern to
reduce direct illumination

Photodiode implanted at the

Optical fibre used to Pyrex bottom of the chamber and
introduce excitation energy Si CdS film covers the
photodiode

Pyrex

Si

Temperature sensor
and heaters
Transducers-Optical methods-refractive index

Mach-Zehnder interferometer
Transducers-Optical methods-refractive index

The reflected wavelength (λB), called the Bragg

wavelength, is defined by
 Transducers-Optical methods-refractive index
Optical waveguide based biosensor
Transducers-Electrochemical methods
The underlying principle for this class of biosensors is that many chemical reactions produce
or consume ions or electrons which in turn cause some change in the electrical
properties of the solution which can be sensed out and used as measuring parameter
Transducers-Electrochemical methods-amperometric

amperometric glucose biosensors

Transducers-Electrochemical methods-amperometric

amperometric glucose biosensors

Transducers-Electrochemical methods-potentiometric

Schematic diagram of an integrated enzyme-based flow-through glucose sensor.

Transducers-Electrochemical methods-potentiometric
Transducers-Electric methods
Concept: Capture analyte and detect changes in electrical parameters of sample

Immunosensing at micro-sized Au electrodes based on the change of conductivity

between the Au strips upon binding of Au nanoparticles
Transducers-Electric methods

Capacitive sensor by
MIP dielectric

Variation of
Transducers-Electric methods

F= 20KHz, AC amplitude of 40 mV peak to

peak
Transducers-Mass based methods

The principle is to change in the frequency of vibrating element. when the mass
increases due to binding of chemicals, the oscillation frequency of the device changes
and the resulting change can be measured electrically and be used to determine the
a d d i t i o n a l m a s s .
Transducers-Mass based methods

Detection by cantilevers
Transducers-Mass based methods

Detection by cantilevers

Change in the resonance frequency: AcV1 antibody

(green) and baculovirus particles (red).
Transducers-Mass based methods

Detection by cantilevers
(a) A mechanical cantilever resonator
containing an embedded microfluidic channel.

(b) Decrease in resonant frequency as the

density inside the embedded channel
increases.

(c) Frequency modulation by single particle

movment
Transducers-Mass based methods

Detection by cantilevers

Microcantilever technology: a) with immobilised

protein for a specific bacterium b) bending after
adsorption of bacteria to the protein
Transducers-Mass based methods
Transducers-Temperature methods
This type of biosensor is exploiting one of the fundamental properties of biological
reactions, namely absorption or production of heat, which in turn changes the
temperature of the medium in which the reaction takes place. They are constructed by
combining immobilized enzyme molecules with temperature sensors. When the analyte
comes in contact with the enzyme, the heat reaction of the enzyme is measured and is
calibrated against the analyte concentration. The total heat produced or absorbed is
proportional to the molar enthalpy and the total number of molecules in the reaction
Transducers-Temperature methods

A three-dimensional schematic representation of the proposed microcalorimeter

with integrated microfluidic channels.
 Potential Applications

• Clinical diagnostics
• Food and agricultural processes
• Environmental (air, soil, and water) monitoring
• Detection of warfare agents.
Father of the Biosensor

Professor Leland C Clark Jnr

1918–2005
 History of Biosensors

• 1916 First report on immobilization of proteins : adsorption of invertase on activated

charcoal

• 1922 First glass pH electrode

• 1956 Clark published his definitive paper on the oxygen

electrode.

• 1962 First description of a biosensor: an amperometric

enzyme electrodre for glucose (Clark)

• 1969 Guilbault and Montalvo – First potentiometric

biosensor:urease immobilized on an ammonia
electrode to detect urea

• 1970 Bergveld – ion selective Field Effect Transistor (ISFET)

• 1975 Lubbers and Opitz described a fibre-optic sensor with immobilised indicator to
measure carbon dioxide or oxygen.
 History of Biosensors

• 1975 First commercial biosensor ( Yellow springs

Instruments glucose biosensor)

• 1975 First microbe based biosensor, First immunosensor

• 1976 First bedside artificial pancreas (Miles)

• 1980 First fibre optic pH sensor for in vivo blood gases (Peterson)

• 1982 First fibre optic-based biosensor for glucose

• 1983 First surface plasmon resonance (SPR) immunosensor

• 1984 First mediated amperometric biosensor: ferrocene used with

glucose oxidase for glucose detection
 History of Biosensors

• 1987 Blood-glucose biosensor launched by

MediSense ExacTech

• 1990 SPR based biosensor by Pharmacia BIACore

• 1992 Hand held blood biosensor by i-STAT

• 1996 Launching of Glucocard

• 1998 Blood glucose biosensor launch by LifeScan

FastTake

• 1998 Roche Diagnostics by Merger of Roche and

Boehringer mannheim

• CurrentQuantom dots, nanoparicles, nanowire, nanotube, etc

 Wearable Biosensors

Ring Sensor

Smart Shirt
 Biosensors on the Nanoscale
 Molecular sheaths around the nanotube are developed that
respond to a particular chemical and modulate the
nanotube's optical properties.

 A layer of olfactory proteins on a nanoelectrode react with

low-concentration odorants (SPOT-NOSED Project).
Doctors can use to diagnose diseases at earlier stages.

 Nanosphere lithography (NSL) derived triangular Ag

nanoparticles are used to detect streptavidin down to
one picomolar concentrations.

The School of Biomedical Engineering has developed an anti-

body based piezoelectric nanobiosensor to be used for
anthrax,HIV hepatitis detection.
 Potential Applications

• Clinical diagnostics
• Food and agricultural processes
• Environmental (air, soil, and water) monitoring
• Detection of warfare agents.
 Application of Biosensor

 Food Analysis
 Study of biomolecules and their interaction
 Drug Development
 Crime detection
 Medical diagnosis (both clinical and laboratory use)
 Environmental field monitoring
 Quality control
 Industrial Process Control
 Detection systems for biological warfare agents
 Manufacturing of pharmaceuticals and replacement
organs
 • Biosensors play a part in the field of
environmental quality, medicine and industry
mainly by identifying material and the degree
of concentration present