PT-619

Pharmaceutical Biotechnology (Theory)

Syllabus:
Unit I:
a) Brief introduction to Biotechnology with reference to Pharmaceutical Sciences.
b) Enzyme Biotechnology- Methods of enzyme immobilization and applications.
c) Biosensors – Working and applications of biosensors in Pharmaceutical Industries.
d) Brief introduction to Protein Engineering.
e) Basic principles of genetic engineering.
f) Use of microbes in industry. Production of Enzymes- General consideration - Amylase,
Catalase, Peroxidase, Lipase, Protease, Penicillinase.

Biosensors – Working and Applications of Biosensors In

Pharmaceutical Industries

Introduction
The word "biosensor" was coined by Cammann. The first ‘true’ biosensor was
developed by Leland C. Clark in 1956 for oxygen detection. He is known as the ‘father of
biosensor’ and his invention of the oxygen electrode bears his name ‘Clark electrode’.

According to IUPAC recommendations (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. Essentially it
is an analytical device, which incorporates a biological or biological derived recognition
element to detect a specific bio-analyte integrated with a transducer to convert a biological
signal into an electrical signal.

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.

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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.

Different Components in Biosensor

A typical biosensor consists of the following components.

1. Analyte: A substance of interest that needs detection. For instance, glucose is an

‘analyte’, in a biosensor designed to detect glucose.
2. Bioreceptor: A molecule that specifically recognises the analyte is known as a
bioreceptor. Enzymes, cells, aptamers, deoxyribonucleic acid (DNA) and antibodies are
some examples of bioreceptors. The process of signal generation (in the form of light,
heat, pH, charge or mass change, etc,) upon interaction of the bioreceptor with the
analyte is termed bio-recognition.
3. Transducer: The transducer is an element that converts one form of energy into
another. In a biosensor the role of the transducer is to convert the bio-recognition event
into a measurable signal. This process of energy conversion is known as signalisation.
Most transducers produce either optical or electrical signals that are usually
proportional to the amount of analyte-bioreceptor interactions.
4. Electronics: This is the part of a biosensor that processes the transduced signal and
prepares it for display. It consists of complex electronic circuitry that performs signal
conditioning such as amplification and conversion of signals from analogue into the
digital form. The processed signals are then quantified by the display unit of the
biosensor.
5. Display: The display consists of a user interpretation system such as the liquid crystal
display of a computer or a direct printer that generates numbers or curves
understandable by the user. This part often consists of a combination of hardware and
software that generates results of the biosensor in a user-friendly manner. The output
signal on the display can be numeric, graphic, tabular or an image, depending on the
requirements of the end user.

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Working
Biosensors are operated based on the principle of signal transduction. The components
used in designing of biosensor include a bio-recognition element, a bio-transducer and an
electronic system composed of a display, processor and amplifier. The bio-recognition element,
essentially a bioreceptor (enzyme, DNA, cell, antibody), is immobilized by conventional
methods (physical or membrane entrapment, non-covalent or covalent binding). This
immobilized biological material is in intimate contact with the transducer and allowed to
interact with a specific analyte. The transducer measures heat, gas (O2), electrons or hydrogen

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as the result of interaction and outputs a signal. The intensity of the signal output is proportional
to the concentration of the analyte. The signal is then amplified and processed by the electronic
system.

Advantages
1. Rapid and continuous measurement
2. High specificity
3. Very less usage of reagents required for calibration
4. Fast response time
5. Ability to measure non-polar molecules that cannot be estimated by other conventional
devices.

Types of Biosensors
The types of biosensors can be categorized based on transducers and biorecognition
elements that are utilized for the fabrication of a biosensor.
1. Enzyme Biosensor: The first enzyme-based sensor was reported by Updike and Hicks
(Used the term ‘enzyme electrode’ to describe a device where the enzyme glucose
oxidase was immobilized in a polyacrylamide gel onto a surface of an oxygen electrode
for the rapid and quantitative determination of glucose.) in 1967. Enzyme biosensors
have been devised on immobilization methods, i.e. adsorption of enzymes by van der
Waals forces, ionic bonding or covalent bonding. The commonly used enzymes for this
purpose are oxidoreductases, polyphenol oxidases, peroxidases, and amino-oxidases.

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2. Whole cell biosensor: The first microbe-based or cell-based sensor was designed by
Divies. The tissues for tissue-based sensors arise from plant and animal sources. The
analyte of interest can be an inhibitor or a substrate of these processes. Rechnitz
developed the first tissue-based sensor for the determination of amino acid arginine.
Organelle-based sensors were made using membranes, chloroplasts, mitochondria and
microsomes. However, for this type of biosensor, the stability was high, but the time
was longer, and the specificity was reduced.
Microorganism Analyte Biosensor
Escherichia coli Glutamate Potentiometric (CO2)
Nitrosomonas sp Ammonia Amperometric (O2)
Lactobacillus arsbinosus Nicotinic acid Potentiometric (H+)

3. Immuno-sensors: Immunosensors were established on the fact that antibodies have

high affinity towards their respective antigens, i.e. the antibodies specifically bind to
pathogens or toxins, or interact with components of the host’s immune system.
4. DNA biosensor: The DNA biosensors were devised on the property that single-strand
nucleic acid molecule is able to recognize and bind to its complementary strand in a
sample. The interaction is due to the formation of stable hydrogen bonds between the
two nucleic acid strands.
5. Magnetic biosensors: Miniaturized biosensors detecting magnetic micro and
nanoparticles in microfluidic channels using the magnetoresistance effect have great
potential in terms of sensitivity and size.
6. Thermometric or thermal biosensors or calorimetric biosensors are developed by
assimilating biosensor materials as into a physical transducer. Several biological
reactions involve production of heat and form the basis of thermometric biosensors. A
thermal biosensor consists of a heat insulated box fitted with a heat exchanger.
7. Piezoelectric biosensors are of two types: the quartz crystal microbalance and the
surface acoustic wave device. They are based on the measurement of changes in
resonance frequency of a piezoelectric crystal due to mass changes on the crystal
structure.
8. Optical biosensors consist of a light source, as well as numerous optical components
to generate a light beam with specific characteristics and a modified sensing head along
with a photodetector. Optical biosensors are the devices that utilize the principle of
optical measurements (absorbance, fluorescence, luminescence, etc.). They employ the
use of fibre optics and optoelectronic transducers. 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.
9. Amperometric biosensor: Amperometric biosensors measure either the current or
potential resulting from a chemical reaction of electroactive materials on transducer
surface while a constant potential or current, respectively, is applied. The change in
current is related to the concentration of the target species. The working electrode (WE)
of the amperometric biosensor is usually a noble metal (gold, titanium, nickel, etc.),

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indium tin oxide (ITO), or carbon covered by the bioreceptor elements. In an

amperometric biosensor, on application of potential, the current (typically nA to mA
range) due to catalytic conversion or the absorption of proteins that occurs at the
electrode surface is measured. Glucose biosensor is a good example of amperometric
biosensor.
10. Potentiometric biosensor: Potentiometric biosensors can detect potential from a
chemical reaction of electroactive materials when constant current is applied.
Potentiometric biosensors can measure species such as pH, H+, NH4+ and other ions, as
well as biomolecules including glucose, urea, penicillin, etc. 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.
11. 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.

Applications
Biosensors applied in many fields namely food industry, medical field, marine sector
etc., and they provide better stability and sensitivity as compared with the traditional methods.
• In food processing, monitoring, food authenticity, quality and safety:
• Detection of ageing of beer using enzymatic biosensors, based on cobalt
phthalocyanine.
• Detection of pathogens in food. Presence of Escherichia coli in vegetables is a
bioindicator of faecal contamination in food. E. coli has been measured by detecting
variation in pH caused by ammonia (produced by urease - E. coli antibody
conjugate) using potentiometric alternating biosensing systems.
• Enzymatic biosensors are also employed in the dairy industry. A biosensor, based
on a screen-printed carbon electrode, was integrated into a flow cell. Enzymes were
Immobilized on electrodes by engulfment in a photo cross-linkable polymer. The
automated flow-based biosensor could quantify the three organophosphate
pesticides in milk.
• Biosensors are used in determining the quality of the food.
• Biosensors are used in monitoring key analytes like glucose.
• Biosensors are used for detection of biogenic amines or in other words food
freshness marker determining parameters in industry.
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• In fermentation processes:
• Biosensors can be utilized to monitor the presence of products, biomass, enzyme,
antibody or by-products of the process to indirectly measure the process conditions.
• Glutaminase-based microfluidic biosensor chip with a flow-injection analysis for
electrochemical detection has been used for detection in frequentation process.
• Environmental field:
• Biosensors are used in detecting environmental pollutants.
• Biosensors are used for measuring concentration of various metal ions by specific
protein concentration or by using genetically modified organisms.
• Biosensors are used in the BOD measurement during waste water treatment.
• Biosensors are used in the detection of polyaromatic hydrocarbons present in water.
• Medical field:
• Biosensors are used in both clinical and laboratory use in medical care.
• Tumour cells are used as biosensors to monitor the susceptibility of
chemotherapeutic drugs.
• Biosensors are also used to monitor the manufacturing of pharmaceutical
compounds.
• Biosensors are used in organ replacement procedures such as an artificial pancreas
replacement in patient with diabetes.
• Biosensors are being used pervasively in the medical field to diagnose infectious
disease.
• Fluorescent biosensors are effectively employed for early detection of biomarkers
in molecular and clinical diagnostics, for monitoring disease progression and
response to treatment/ therapeutics, for intravital imaging and image guided
surgery.
• Biosensors are also used in medical research.
• Biodefense biosensing applications:
Biosensors can be used for military purposes at times of biological attacks. The
main motive of such biosensors is be to sensitively and selectively identify
organisms posing threat in virtually real time called biowarfare agents (BWAs)
namely, bacteria (vegetative and spores), toxins and viruses.

Reference:
Satyalakshmi S. Biosensors. In: Current Trends in Biotechnology. 2019 ed. Pee Vee.
2019:59-67.

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