Wednesday, 14 June 2023
Chapter : 3 BIOSENSORS
INTRODUCTION
BIOSENSORS: A
biosensor is an analytical device consisting of an immobilized layer of
biological component (e.g. nucleic acid, hormone, antibody, enzyme, whole cell,
tissue) in the intimate contact with the transducer i.e. sensor.
Biosensors are
analytical devices that convert a biological response into an electrical
signal.
The word
"biosensor" was coined by Cammann. According to IUPAC commendations (1999), a biosensor is an
independently integrated receptor transducer device, which is capable of
providing selective quantitative or semi-quantitative analytical information
using a biological recognition element.
The first true
biosensor was developed by Leland C. Clark in 1956 for oxygen
detection He is known as the father of biosensors' and his invention of the
oxygen electrode bear his name "Clark electrode
CHARACTERISTICS OF
BIOSENSORS
1. Linearity: It is defined as the maximum sensor output signal
that can be detected by a sensor. It should be high for a biosensor to detect
substrate concentration
2. Sensitivity.: It is the magnitude of electrode response per unit
substrate concentration
3. Selectivity: The electrode response in the presence of other
interfering chemicals or foreign materials. It is the minimal chemical
interference with the target analyte. It should be minimum for a biosensor
4. Stability: It is the maximum electrode response over a period
of time.
5. Limit-of-detection: The lowest quantity of a substance that can be
determined from the absence of that substance blank signal. It can be
calculated from the mean of the blank and its standard deviation
Parts of
biosensor: Biosensors
are functionally composed of three main components.
1.
BIOLOGICAL ELEMENT: Biological
element is responsible for detecting the analyte (drug, glucose, urea,
pesticide etc.) and generating a response signal. The biological recognition
components are enzymes, microbial cells, plant or animal cells, tissues, immune
binding or receptor proteins.
2. TRANSDUCER: A
transducer is an element which convert one form of energy into another form. A
transducer is a physical component which analyses the biological signals and
converts into an electrical signal which is measurable.
The signal generated by the
biological element is then transformed into a detectable response by the second
component called as transducer. Transducer is the main part of biosensor which
makes use of physical change accompanying the reaction.
3. DETECTOR The
detector which amplifies and processes the signals which are displayed an
electronic display system. The detection systems used in bio sensing are
electrochemical detection, photometric detection, thermal and mass detection,
fluorescence signal processing and instrumentation based detection system.
The various steps in signal
processing of a biosensor is shown in Fig. 3.1.
It is important and beneficial to
use immobilized biocatalyst as it provides reusability, long half-life and
ensures same catalytic activity in a series of analyses. The desired biological
material (enzyme) is immobilized by conventional methods such as adsorption,
entrapment, microencapsulation, covalent or cross binding. This immobilized
biological component is in intimate contact with the transducer.
The system is responsible for the
detection of the analyte and subsequent response in same measurable parameters.
The analyte binds to the biological component to form bound analyte which in
turn produces the electronic response that can be measured. The analyte is
converted to a product which may be associated with the release of heat,
oxygen, electrons, hydrogen ions or the product of ammonium ions. The product
passes through another membrane to the transducer. The transducer converts
product into an electric signal which is amplified. Processor is used to
process the signal by subtracting the base line signal which is taken by an
electrode without a biocatalyst and converts the resultant signal to digital
output.
.
TYPES OF BIOSENSORS
A) Biosensors
are classified as enzyme-based, tissue-based, immunosensors, DNA biosensors and
thermal and piezoelectric biosensors.
1. Enzyme biosensors are prepared
by immobilization methods such as ionic bonding, covalent bonding or van der
Waals forces. The commonly used enzymes are peroxidases, aminoxidases,
polyphenoloxidases, oxidoreductases etc. Bacterial biosensors directly utilize
a chain of bacterial enzyme reactions to achieve detection of one or more
species.
2. Organelle-based sensors are
prepared by using membranes, chloroplasts, mitochondria, microsomes etc.
3. Immunosensors are prepared on the
fact that antibodies have high affinity towards their respective antigens, i.e.
antigenntibody specific reaction properties. Labelled antigens or antibodies
may be used in biosensors based on enzyme linked immunoassary (ELISA)
principles.
4. The DNA biosensors are based
on the property that single-strand nucleic acid molecule is able to recognize
and bind to its complementary strand in a sample.
B)
Biosensors can be classified according to the mode of physicochemical
transduction or the type of bio recognition element. Based on the transducer,
biosensors can be classified as
1. Electrochemical biosensor
2. Optical biosensor (measure the
absorbed or emitted light)
3. Thermal biosensor
4. Piezoelectric (detect stress)
biosensors.
1. Electrochemical biosensors
can be further classified as
a) Amperometric biosensors
(measure the electrical current),
b) Potentiometric biosensors
(measure electrical voltage)
c) Conductometric biosensors
(measure electrical conductance).
a) Amperometric Biosensor
These
biosensors are based on the movement of electrons as a result of
enzyme-catalysed redox reactions. The amperometric biosensors contain either
enzyme-electrode or chemically modified electrodes. A redox reaction catalysed by
enzymes is directly coupled to an electrode where enzyme is presented with the
oxidizable substrate. The electrons are transferred from the substrate to the
electrode via enzyme and redox mediator (Fig. 3.2). The magnitude of the
current is proportional to the substrate concentration. The most common
amperometric biosensor is glucose biosensor which is based on a Clark oxygen
electrode. In this case, the platinum cathode is held next to an oxygen
permeable membrane on which glucose oxidase enzyme is immobilized. This
membrane is also permeable to substrate glucose
b) Potentiometric Biosensor
In
potentiometric biosensor, changes in ionic concentrations are measured by use
of ion-selective electrodes (pH electrode). These biosensors consist of a
membrane containing immobilized enzyme and surrounding the probe from a pH
meter. The potential difference obtained between the potentiometric electrode
(Fig. 3.3) and the reference electrode can be measured. It is proportional to
the concentration of the substrate. The major limitation of these biosensors
are the sensitivity of enzymes to pH, ammonia, carbon dioxide or other
analytes. Ion-selective field effect transistors (ISFET) can be used for
miniaturization of potentiometric biosensors. The intramyocardial pH is
monitored by an ISFET biosensor during human open-heart surgery.
c) Conductimetric Biosensors
The
changes in the ionic species is transferred or altered into electrical
conductivity in conductimetric biosensors. Conductivity measurement is based on
the biocatalytic reaction of the sample on an electrode. The conductimetric
transducer consists of reference electrode and working electrode. Both
electrodes are coated with a nata de coco membrane and enzyme is immobilized
only on working electrode. The reaction produces ions which results in the
change of conductivity. Urea biosensor utilizing immobilized urease is a
example of conductimetric biosensors.
2. Thermometric or Calorimetric
Biosensors
Many enzyme catalyzed reactions
are exothermic (production of heat), which may be used as a basis for measuring
the rate of reaction. These changes are determined by thermal biosensors or
calorimetric biosensors. In this method, substrate enters into the enzyme
packed bed reactor and then it converted to a product by generating heat. The
difference in the temperature between the substrate and product is measured by
thermistors (thermal biosensors) calorimetric microsensors have been used for
detection of cholesterol in blood serum based on production of heat by
enzymatic reaction.
3. Optical Biosensors :
Optical
biosensors are the devices that utilize the principle of optical measurements
i.e. fluorescence, absorbance, chemiluminescence, internal reflection
spectroscopy; etc. These involve determining changes in light absorption
between the reactants and products of a reaction or measuring the light output.
Optical biosensors primarily involve enzyme and antibodies as the transducing
elements. It allow a safe non-electrical remote sensing of materials and these
biosensors usually do not require reference sensors. The most common
application of this biosensor is estimation of blood glucose for monitoring of
diabetes.
Fibre optic lactate biosensor is
one of important example of optical biosensors. The principle of this biosensor
is based on the measurement of changes in molecular oxygen concentration. This
reaction is catalysed by the enzyme lactate monooxygenase,
Lactate
monooxygenase
Oxygen has quenching (reducing)
effect on the fluorescence and hence, the amount of fluorescence generated by
the dyed film is dependent on the oxygen. Lactate present in the reaction
mixture utilizes oxygen and proportionate decrease in the reducing effect of
oxygen. This results, increase in the fluorescent output which can be measured.
Optical fibre sensing devices are used for measuring pH, PCO, and po, in
critical care and surgical monitoring.
4. Piezoelectric or Acoustic
Biosensor
Piezoelectric
biosensors are based on the principle of sound vibrations (acoustics).
Piezoelectric crystals with positive and negative changes vibrate with
characteristics frequencies. The optimal resonant frequency for acoustic wave
transmission is dependent on the physical dimensions and properties of the
piezoelectric crystals. Adsorption of molecules on the crystal surface alters
the resonance frequencies which can be measured by electronic devices. A
piezoelectric biosensor has been developed for organophosphorus insecticide by
incorporating acetylcholine esterase, for formaldehyde by incorporating
formaldehyde dehydrogenase and for cocaine in gas phase by attaching cocaine
antibodies to the surface of piezoelectric crystal.
5. Whole Cell Biosensor
In
the whole cell biosensor or microbial biosensor, immobilized whole cell of
microorganisms or their organelles are used. These biosensors may employ live
or dead microbial cells. The microbial cells are easily available with less
cost and they are less sensitive to variations in pH and temperature compared
to isolated enzymes. The specificity and sensitivity of whole cell biosensors
may be less compared to that of enzymes.
6.Blood glucose Biosensors are
used widely throughout the world for diabetic patients. It has a single-use
disposable electrode with glucose oxide and derivatives of a mediator (Ferrocene)
and the shape of the blood glucose Biosensor looks like a watch pen. With the
help of hydrophilic mesh, electrodes are converted. Blood glucose Biosensor is
a good example of an Amperometric Biosensor.
7.Potenciometric biosensor: In this type
of Biosensors changes the concentration of ionic is determined by the
ion-selective electrodes in this pH electrodes are used most commonly. Hence a
large amount of enzymatic reactions is involved in the release of
hydrogen ions. Ammonia-selective
and Carbondioxide selective electrodes are
some other important electrodes.
The Potentiometric electrode and the
reference electrode can be measured with the help of potential difference and
it is directly proportional to the substrate concentration. The Potentiometric
Biosensors is the sensitivity of enzymes to ionic concentration like H+
and NH4+ The
ion-selective field-effect transistors are
lower-price devices. It can be
used in the miniaturization of Potentiometric Biosensors. An example of the ISFET Biosensor is to monitor
intra-myocardial for open-heart surgery.
8. The immune Biosensors: It
work on the principle of immunological specificity
and mostly coupled with
measurement on the Potentiometric Biosensors.
9.Fiber Optic Lactate Biosensor: The working of the fiber optic lactate Biosensor
is based on the measurement of change in oxygen concentration, molecular by
identifying the effects of oxygen in the fluorescent dye.
APPLICATIONS OF BIOSENSORS
Biosensors
are widely applied in many fields such as pharmaceutical industries, medicine,
food industry, pollution control, military etc. They are very popular in many
areas due to the small size, easy handling, low cost, and better stability and
sensitivity. Pharmaceutical and biomedical applications:
1.
Biosensors are mainly used for the quantitative estimation of biologically
important substances in body fluids e.g.
glucose, cholesterol, urea etc.
2. Glucose
biosensors are used for diagnosis of diabetes mellitus.
3.
Optical biosensors can be used for measurement of patient's blood glucose level
by combining bacterial magnitude with
a chromophoric system.
4.
Blood gas monitoring for pH, PCO, and pO2 is carried out by optical biosensors during critical care and surgical
monitoring of patients.
5. Mitomycin,
an aflatoxin, causes cancer in inborne infants. Mutagenicity of such chemicals can be detected by using
biosensors.
6.
Several toxic compounds produced in the body can also be detected by
biosensors.
7.
Biosensors are being used in the medical field to diagnose infectious disease
mainly urinary tract infections.
8.
A novel biosensor (hafnium oxide, HfO;) has been used for early stage detection
of human interleukin-10.
Interaction between recombinant human IL-10 with corresponding monoclonal antibody is studied for early
detection of cytokine.
9.
Fluroescent biosensors can probe ions, metabolites, and protein biomarkers with
great sensitivity. They are used
in protein localization, probing gene expression and conformation in fields such as signal transduction,
transcription and apoptosis.
10. Fluorescent
biosensors can be used for detection of cancer, inflammatory diseases,
cardiovascular and neuro-degenerative and viral infections.
11. Fluorescent biosensors are
also used in drug discovery for the identification of the high thorough put and high content screening
approaches. These are considered tools for preclinical evaluation and clinical
validation of active drug molecules.
12.
The biosensors are effectively used for early detection of biomarkers in
molecular and clinical diagnostics, for monitoring disease status and response
to treatments.
13.Potentiometric
biosensors are developed with lon-Selective Field Effect Transisto (ISFETS) for
different applications such as chiral amine salt detection, enzyme inhibitors
acetyl choline esterase, detection of pharmaceutical preparations such as
procaine, tetracaine and lidocaine, and detection of anionic surfactants like
sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide.
14.Biosensors
can be used in fermentation industry for monitoring fermentation products,
biomass, enzymes and estimation of various ions Biosensors are commonly used to
control the fermentation process and produce reproducible results.
15.
In fermentation process, glucose biosensors are used to monitor the process to
saccharification to produce glucose.
16.
Glutamate biosensors are used to conduct experimentation on ion exchange
retrieval of an isoelectric liquor supernatant of glutamate.
Non-medical applications:
Biosensors are used in many
non-medical fields such as food industry, environmental monitoring, defense,
agriculture and related industries.
Food industry:
1.
Biosensors are commonly used in food industry to detect the odour and freshness
of food. Freshness of stored
fish can be detected by ATPase. ATP is not found in spoiled fish and this can be detected by using ATPase.
2.
Biosensors are used for the detection of pathogens in food. E. coli
contaminated in food or
vegetables can be measured by detecting variation in pH caused by ammonia using potentiometric
alternating bio sensing systems,
3. Enzymatic
biosensors are used in the dairy industry.
4. The
automated flow-based biosensor is used to quantify the three organophosphate pesticides in milk.
5. Biosensors
are also used to defect artificial sweeteners in food additives.
6. Food
safety is one of most important parameters and quality of food refers to the appearance, taste, smell, freshness,
flavour, nutritional value and chemicals. Nanotechnology
and electromechanical systems are striding in to make sensing technology imminent for use in
ensuring food quality and safety.
7.
Biosensorsare used to detect pesticides organophosphates and carbamic insecticide
8.
Biosensors also detect pesticides in wine and orange juice.
Environmental Monitoring:
Biosensors
are very helpful in environmental monitoring and pollution control. They are
useful for monitoring pollutants, chemical residues, pesticides, toxins or
microbes in marine water, rivers and reservoirs. The concentration of
pesticides and the biological oxygen demand (BOD) can be measured by
biosensors. Biosensors coupled with oxygen electrode and immobilized Trichosporon
cutaneum is used for measuring BOD. The whole cell biosensor in conjugation
with oxygen electrode and immobilized Salmonella typhimurium and Bacillus
subtilis can be used to measure mutagenicity or carcinogenecity of several
chemical compounds.
Defense:
Biosensors are used by the
infantry to detect and identify the toxic gases and other chemical agents used
during war.
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