BIOSENSOR

Prof. Anant Achary

Dept. of Biotechnology
Kamaraj College of Engineering and Technology
• A biosensor is defined as an analytical device
consisting of a biological component (e.g.,
enzyme, antibody, entire cell, DNA) and a
physical transducer (e.g.,electrode, optical device).
• Biosensors are mostly designed for routine
analysis, such as clinical diagnosis, quality control
of food, in-process control of fermentations, and
in environmental analysis.
• Different strategies for the immobilization of biomaterials a)
adsorption on the solid surface. b) Integration on the gel
material. C) covalent coupling
• Amperometric sensors have also been used for screening natural products.
A biosensor specific for polyphenols was developed by Romani et al.
(2000).
• Polyphenols are commonly found e.g., in food, in vegetables, and medicinal
plants. They play an important role in the human diet and health, but they
also influence the sensorial properties of many foods and act as natural
antioxidants.
• Tyrosinase is a relatively unspecific enzyme oxidizing a wide variety of polyphenolic
compounds.
• Different compounds such as phenolic acids, flavones, flavonols, catechins, tannins, and
oleuropein were tested with this system.
• Calibration was performed in a range between 20 and 80 µM of catechin.
• This method can be used for a rapid and sensitive screening of plant extracts for
polyphenols.
• Thust et al. (1996) and Poghossian et al. (2000) introduced a system
using a pH-sensitive electrolyte-insulator-semiconductor (EIS) as
physical transducer and immobilized the enzyme penicillinase on its
active surface.
• The obtained biosensor was sensitive towards penicillin G, ampicillin,
and amoxicillin and could be operated over 250 days.
• The sensor's detection limit was at about 0.1 mM penicillin G and
could be lowered by introduction of a diffusion barrier (down to 0.5
µg penicillin per analyzed sample).
• It can probably be assumed that this sensor will be able to detect other
penicillin derivatives and may be therefore useful for the screening of
antibiotics.
• Immobilization can be achieved by a variety of techniques and is
one of the key steps in the development of biosensors.
• There are stringent demands to this process:
(1) immobilization should give a stable layer of biomolecules;
(2) the bio-component should not be destroyed by the procedure;
(3) activity of enzymes and binding capacity of antibodies
should not be reduced significantly;
(4) substrate specificity of the bio-component must not change.
 AMPEROMETRIC BIOSENSOR

• In amperometric sensors, an enzyme is immobilized at the surface

of an amperometric electrode.
• The enzyme reacts with the substrate (e.g., sugar or phenolic
compound), a current is produced that depends on the concen. of
the analyte.
• In the simplest biosensoric configuration, an
oxygen-consuming enzyme (e.g., glucose oxidase,
phenolase) is immobilized on a platinum electrode
and the reduction of oxygen at the electrode
results in a current that is inversely proportional to
the analyte concentration.
• A disadvantage of this type of sensor is its sensitivity
to endogenous components within a sample. Interfering
substances can be either oxidized or reduced under the
selected working conditions.
• Alternative amperometric detection principles such as
those based on the electrochemical determination of
hydrogen peroxide.
• This method is more robust and suitable for enzyme
systems, which either consume or produce hydrogen
peroxide.
 POTENTIOMETRIC BIOSENSOR
• The potentiometric biosensors,an enzyme is immobilized
on the surface of a pH sensitive device.
• This device may be a pH glass electrode or a gas electrode
based on a pH electrode (e.g., ammonia gas electrode) or
an ion-selective field effect transistor (ISFET).
• ISFET allows miniaturization of the sensor, an advantage
that has led to hundreds of variations of this sensor type
developed in recent years.
• Measurements were usually done in “zero current” mode
and, therefore, no electrochemical reactions with further
substances in the sample can be expected.
• A major limitation of the potentiometric approach is the fact
that sensors are sensitive to pH-active components of the
sample and have to be operated at low buffer concentrations.
• The latter demand is often in conflict with the stability of the
enzymes used.
• Liu et al.(1997) developed penicillin biosensor based on a pH
sensitive field effect transistor and penicillin acylase was the
enzyme.
• Operated in phosphate buffer at pH 7, the linear range of the
calibration curve was between 0.5 and 8 mM penicillin G.
• The sensor was used over a 6-month period, in which time more
than 1,000 single analyses were performed without a significant
decrease in the output value.
• The biosensoric detection of cysteine sulfoxides has also
been studied intensively (Krest et al. 1997; Keusgen 1999;
Milka et al. 1999). Cysteine sulfoxides are amino acid
derivatives typically found in garlic (Allium sativum),
onions (A. cepa) and leek (A. porrum). These compounds
are reportedly responsible for the health benefits of these
plants.
• A cholesterol-lowering effect, an anti-atherosclerotic
activity and a cancer-protective activity have been shown
for cysteine sulfoxides and further sulfur-containing
substances derived from these plants.
• The cysteine–sulfoxide biosensor is based on an ammonia-gas electrode
and the enzyme alliinase. The ammonia-gas electrode is a modified pH-
sensitive glass electrode. The enzyme alliinase specifically cleaves
cysteine sulfoxides into allylsulfenic acid, pyruvic acid and ammonia.
• Ammonia is specifically detected by the electrode.
• The sensor had a detection limit of 0.5 µM alliin, which is a typical
cysteine sulfoxide.
• used over 400 h for real-sample measurements. The enzyme was found
to be stable over more than 250 days.
• The sensor may be also realized on the basis of an EIS structure as
physical transducer, which is under investigation.
• Currently, this biosensor is used routinely for screening of Allium
species available worldwide for the presence of cysteine sulfoxides.
• A sensor for the screening on cyanogenic compounds, like cyanogenic
glycosides, has also been developed on the basis of an ammonia electrode
(Keusgen et al.2001c). This electrode was combined with the enzyme
cyanidase, which hydrolyses cyanide to formic acid and ammonia.
• Cyanogenic glycosides are widespread in nature and occur in more than
2,500 known plants, many of which are used as food or medicinally.
• If plant material containing these compounds is eaten, cyanide is liberated
by the action of the enzyme glycosidase.
• The recently developed sensor could thus be used for screening purposes
to check on the levels of cyanogenic glycosides of plant material.
• The detection limit of this sensor was around 0.1 mM cyanide.
Interestingly, the enzyme cyanidase seems to be stable over several years.
• Aldicarb is a synthetic carbamate pesticide
used on a variety of corps.
• Organophosphates and carbamates are the
widely used pesticide.
• They have replaced organochlorines like
DDT, aldrine, lindane etc.
• These pesticide inhibits the action of enzyme of nervous
system of insects and mammals.
• AChE transforms acetylcholine to choline.
• Accumulation of actylcholine prevents smooth
transmission of nerve impulses across the junction
between nerves.
• Results in loss of muscular coordination, convulsions and
death.
• Carbamate and organophosphate inhibits
AChE.
• Inhibition is monitored by oxygen probe.
• Oxygen probe detects the oxygen
consumption for the oxidation of choline.
• AChE and CHO is immobilized together.
• Organophosphorus hydrolase (OPH), hydrolyze a range of organophosphate
esters, pesticides such as parathion, coumaphos and acephate, and chemical
warfare agents such as soman, sarin, VX, and tabun.

• The catalytic hydrolysis of each molecule of these compounds releases two

protons, the measurement and correlation of which to the OP concentration
forms the basis of a potentiometric enzyme electrode.

• OPH-catalyzed hydrolysis products of OPs offers the advantages of simpler,

more direct, and quicker measurements of only organophosphorus–type nerve
agents over that based on the inhibition of acetylcholinesterase activity,
• Potentiometric biosensors for the direct determination of OP were reported.
These sensors consisted of OPH-expressing recombinant Escherichia coli cells
cryoimmobilized by entrapment in Poly(vinyl)alcohol gel.

• The immobilized cells were either suspended in a batch reactor with a pH

electrode or packed in a column and placed upstream of a flow cell with a pH
electrode.

• The long response time, attributable to the various mass transfer resistances
present in the system, and the need of special equipment for
cryoimmobilization of the cells are the limitations of the reported biosensing
systems.

• OPH immobilized via cross linking on the surface of a pH electrode that can be
potentially used for on-line monitoring of detoxification processes.
• Phenolic compounds are wide spread in environment that
has deleterious effect.
• Biosensors are attractive alternative technique.
• Polyphenoloxidase / tyrosinase is a bifunctional Cu
containing oxidase having catecholase and cresolase
activity.
• First the phenolic compounds are oxidised to catechol and
in next step catechol to o-quinone
• o-quinone can be reduced back to catechol in an electrode
at an appropriate potential.