UNIT-I

What is a Biosensor?
Biosensor is a powerful and innovative device that is used to convert biological reactions into
measurable signals to detect target analytes. This technological device consists of three
components, the biomolecule, transducer and output system, as depicted in Figure 1.1.
Biomolecules (sensitive bioreceptors) such as enzymes, or bioaffinity groups like antibodies,
tissues and DNA probes are immobilized to support materials. These molecules recognize the
target analytes like enzyme substrates, complementary DNA, antigens, whole cells including
microbial, plant, animal cells, subcellular organelles and lectins. The transducers are used to
convert the signals resulting from the interaction between the biomolecule and target analyte into
observable signals. The signal is then displayed through the output system. The main features of
biosensors are stability, cost, sensitivity, and reproducibility.

Figure 1.1: The main components of biosensors

Main Components of a Biosensor

The block diagram of the biosensor includes three segments namely, sensor, transducer, and
associated electrons. In the first segment, the sensor is a responsive biological part, the second
segment is the detector part that changes the resulting signal from the contact of the analyte and
for the results it displays in an accessible way. The final section comprises of an amplifier which
is known as signal conditioning circuit, a display unit as well as the processor. This technological
device consists of three components, the biomolecule, transducer and output system, as depicted
in Figure 1.1 (and Figure 1.2) . Biomolecules (sensitive bioreceptors) such as enzymes, or
bioaffinity groups like antibodies, tissues and DNA probes are immobilized to support materials.
These molecules recognize the target analytes like enzyme substrates, complementary DNA,
antigens, whole cells including microbial, plant, animal cells, subcellular organelles and lectins.
The transducers are used to convert the signals resulting from the interaction between the
biomolecule and target analyte into observable signals. The signal is then displayed through the
output system. The main features of biosensors are stability, cost, sensitivity, and reproducibility.

Figure 1.2: Main components of a Biosensor

Working Principle of Biosensors

Usually, a specific enzyme or preferred biological material is deactivated by some of the usual
methods, and the deactivated biological material is in near contact to the transducer. The analyte
connects to the biological object to shape a clear analyte which in turn gives the electronic reaction
that can be calculated. In some examples, the analyte is changed to a device which may be
connected to the discharge of gas, heat, electron ions or hydrogen ions. In this, the transducer can
alter the device linked converts into electrical signals which can be changed and calculated.

Classification of Biosensors:
The different types of biosensors are classified based on the sensor device as well as the biological
material that is discussed below.
 1. Electrochemical Biosensor: Generally, the electrochemical biosensor is based on the
reaction of enzymatic catalysis that consumes or generates electrons. Such types of
enzymes are named as Redox Enzymes. The substrate of this biosensor generally includes
three electrodes such as a counter, reference, and working type.

2. Optical Biosensors: Optical Fibers play an important role in Optical Biosensors. The
optical fibers allow detection of the sensing elements based on the different properties of
light like absorption, scattering and fluorescence.

One of the main advantages of using optical biosensors is their non-electrical nature. This
allows them to analyze multiple elements on a single layer just by varying the wavelength
of the light.

3. Piezoelectric Biosensors: are also known as Acoustic Biosensors as they are based on the
principle of sound vibrations i.e. acoustics. When a mechanical force is applied on a
piezoelectric biosensor, they produce an electrical signal.

Biomolecules

The quality of a biosensor is primarily determined by the biochemical specificity of the

biomolecule along with the transducer quality. The biomolecules are mainly chosen depending on
the analyte to be detected. However, the selection also depends on the storage stability in high
temperatures and in acidic, alkaline, hydrophobic and oxidizing environments. Because of their
high specificity, purified enzymes are used extensively as biomolecules in the development of
biosensors.

Immobilization of Biological Materials

Immobilization is a technical process in which enzymes are fixed to or within solid supports,
creating a heterogeneous immobilized enzyme system.

Need for immobilization: The basic requirement of a biosensor is that the biologic material bring
the physicochemical changes in close proximity to a transducer. In this direction, immobilized
enzyme technology has played a major role. Immobilization not only helps in forming the required
close proximity of the biomaterial with the transducer but also in stabilizing it for reuse.

The biologic material has been immobilized directly on the transducer or, in most cases, in
membranes, which can subsequently be mounted on the transducer. Biomaterials can be
immobilized either through adsorption, entrapment, covalent binding, crosslinking, or a
combination of all these techniques. Selection of a technique and support depends on the nature of
enzyme, nature of substrate, and configuration of the transducer used. The choice of support and
technique for the preparation of membranes often has been dictated by the low diffusional
resistance of the membrane. The choice of technique also depends on the biomaterial of interest.
Thus, enzymes and antibodies have normally been immobilized either by covalent binding or
through adsorption followed by crosslinking.

Immobilization methods of biomolecules.

Various methods are available for immobilization of biomolecules (Figure 1.3). The most
commonly used biomaterial immobilization techniques for designing and development of specific
sensors are physical adsorption, entrapment, inter molecular cross-linking and covalent binding:

1. Adsorption: Adsorption is the easiest immobilization method that is based on sticking

biomolecules to the surfaces using hydrogen bonds, Van der Waal’s forces, multiple salt
linkages and through the formation of electron transition complexes. Many
macromolecules like cellulose, collodion, silica gel, glass, hydroxyapatite and collagen
have the ability to adsorb enzymes. This method is commonly used in cell immobilization
as some cells naturally adhere to surfaces.
2. Entrapment: Polymeric gel network prepared in a solution containing biomolecules is
used in this physical immobilization method. The structure is expected to hold the enzyme
inside while allowing the substrate and products to pass through. Usually, the enzyme is
not attached to the support material in this method, differing from covalent binding.
Entrapment is used mainly when cells or cellular organelles are used.
3. Cross-linking: In this method, crosslinkers that have free reactive ends are used to form
covalent bonds that are strong and can anchor biomolecules on the transducer surface.
 Some of the well-known crosslinkers are glutaraldehyde, glyoxal or
hexamethylenediamine. When crosslinking is used, the enzyme should be bound without
significantly affecting its conformation, as even slight changes may have effects on binding
a ligand to a protein. Enzymes and cells can be immobilized on electrodes by immersing
in cross linkable polymers.
4. Covalent binding: It is accomplished through functional group in the enzyme which are
not essential for its catalytic activity. Usually, nucleophilic functional groups present in
amino acid side chains of proteins such as amino, carboxylic, imidazole, thiol, hydroxyl
etc. are used for coupling.

Figure 1.3: Immobilization methods of biomolecules.

Support materials

The term support material usually refers to the solid insoluble matrix where biomolecules can be
attached to. Support materials are widely implemented in an attempt to conserve the tertiary
structure of enzymes used in immobilization, which contributes to the stability and reactivity of
the enzymes. Support materials must display good chemical and mechanical stability, as they can
alter the properties of the immobilized biomolecules. Generally, an ideal support needs to be a
mesoporous material to provide wide surface area and high porosity to increase the amount of the
immobilized biomolecule and to protect it from outer obstructions.
As biomolecules bind to the functional groups, the support materials should also contain groups
that can be easily modified. A variety of organic and inorganic materials that can be used as
supports are shown in Table I and Table II.
In addition to organic and inorganic supports, there are also hybrid support materials produced by
joining inorganic organic, organic-organic and inorganic-inorganic materials to combine their
individual properties.
Table I: The properties of inorganic support materials

Table II: The properties of organic support materials

UNIT-II
2.1 The properties and characteristic of biosensors/Performance influencing
biosensors

As new biosensing techniques are being developed, it is crucial to measure the efficiency of a
device. Different criteria such as selectivity, precision, sensitivity, working range, limit of
detection (LOD), reusability and time factors can be used to quantify the efficiency.
• Selectivity, ability of a biomolecule to detect a certain analyte without reacting with others,
is the most important feature of a biosensor.
• In addition to being selective to a specific analyte, an ideal biosensor should possess good
precision, which is the ability of providing the same results for each measurement, and
 high sensitivity (slope of the calibration curve), which is the minimum quantity of analyte
that can be detected.
• The sensor should also have a wide working range (linear range), the range of analyte
concentration that the sensor can detect and a low LOD, the minimum quantity of an
analyte that is able to induce an identifiable output signal. LOD depends on the affinity
between the recognition element and the analyte, which is expressed via the dissociation
constant “Kd”. A lower Kd value means a higher affinity between the recognition element
and the analyte.
• Reusability represents how many times a sensor can operate before the degradation of the
biomolecule, and this is another desired feature for a biosensor. The reusability of a sensor
also requires high precision in each usage.

Response time, regeneration time and shelf life are key time factors.
• In the context of biosensors, response time is defined as the time needed for the system to
come to equilibrium to make a reliable measurement, and should be short. After an
interaction with the sample, the sensor needs some time to return to its working state. This
is called the regeneration time and should also be quick.
• The shelf life expresses the time range that a biosensor can be used to achieve reliable
results and should be as long as possible. Some of these characteristics are illustrated in
Figure 2.1.
 Figure 2.1: Some characteristics of Biosensor
Notes: (a) LOD, linear range and sensitivity; (b) selectivity (selective to analyte 2); (c)
response time; (d) reusability (number of use) and shelf life (days).

In addition to the characteristics explained above, a successful biosensor must also exhibit the
following features.
• The reaction should not be affected by changes in pH value, temperature and stirring.
This feature shall allow the detection without pretreatment or with minimal treatment.
• The biosensor should be inexpensive, tiny, portable and reusable in addition to being easy
to use.
• If the biosensor is going to be used invasively, the probe should be small to reduce tissue
damage, and should also be biocompatible, possessing no antigenic or toxic effects.
• The sensor should also be sterilizable. The response should be correct, reproducible,
precise and linear over the working range. The response should also be free from noise,
which is often a problem for tools measuring voltage.

2.2 Enzymatic biosensors

Definition: An enzyme biosensor is an analytical device that combines an enzyme with a
transducer to produce a signal proportional to target analyte concentration. This signal can result
from a change in proton concentration, release or uptake of gases, such as ammonia or oxygen,
light emission, absorption or reflectance, heat emission, and so forth, brought about by the reaction
catalyzed by the enzyme. The transducer converts this signal into a measurable response, such as
current, potential, temperature change, or absorption of light through electrochemical, thermal, or
optical means. This signal can be further amplified, processed, or stored for later analysis. Because
of their specificity and catalytic (amplification) properties, enzymes have found widespread use as
sensing elements in biosensors.

2.2.1 Different types of Transducer used in biosensors

Different transducers have been used in enzyme-biosensor construction.
1. Electrochemical Transducers
Three types of electrochemical transducers are used for construction of enzyme electrodes:
potentiometric, amperometric, and conductometric.
a. Potentiometric
Conventional potentiometric enzyme biosensors consist of an ion-selective electrode (ISE)-pH,
ammonium, fluoride, and so forth-or a gas-sensing electrode CO2 and NH3-coated with an
immobilized enzyme layer. The enzymatic reaction with the analyte generates a change in potential
resulting from ion accumulation or depletion. Potentiometric transducers measure the difference
in potential that is generated across an ion selective membrane separating two solutions at virtually
zero current flow.

b. Amperometric

In contrast to potentiometric sensors, where the potential generated across a membrane is used to
convey information on the analyte concentration, amperometric biosensors operate at a fixed
potential with respect to a reference electrode and the current generated by the oxidation or
reduction of species at the surface of the working electrode is measured. Amperometric biosensors
are based on redox enzymes; thus, their appeal is caused by the availability of a large number of
oxido-reductase enzymes that can act on fatty acids, sugars, amino acids, aldehydes, and phenols.
These enzymes use molecular oxygen as an electron acceptor and produce hydrogen peroxide m
the reaction with then substrates.

2. Thermal
Thermal enzyme sensors are based on the principle that the heat evolved in an enzymatic reaction
can be utilized to calorimetrically determine the amount of substrate reacted. Thus, thermometric
indicators or transducers require only a single reaction step producing sufficient or measurable
heat. In thermal enzyme sensors, the enzyme is attached directly to the temperature transducer, a
thermistor, either by crosslinking or by entrapping the enzyme in a membrane enclosing the
thermistor. Alternatively, the enzyme is placed
in a temperature-controlled column and the heat of reaction is measured by
recording the increase in temperature between the inlet and outlet as the sample
flows through the column.

3. Optical
In biosensors based on optical methods, the change in optical properties, such as UV/vis
absorption, bio- and chemiluminescence, reflectance and fluorescence brought by the interaction
of the biocatalyst with the target analyte, is monitored optically.

Potential Applications
The main application areas of enzyme biosensors are m human and animal health care, food and
fermentation industries, environmental monitoring, agriculture and defense.
The second area in which enzyme-biosensors are expected to play a major role is in environmental
monitoring. There is an increasing demand for measuring several classes of substances, including
toxic chemicals, such as polychlorinated biphenyls, polyaromatic hydrocarbons, phenols, dioxins,
organic peroxides, and so forth, pesticides, and heavy metals in water, soil, and air. Enzyme
biosensors will provide rapid, easy to use, and cost-effective on-site testing.

2.2.2 Enzyme Biosensors Based on pH Electrode

A schematic representation of an enzyme biosensor based on pH electrode is given in Fig. 2.2. The
sensitive surface of the pH electrode is in contact with an enzymatic layer and is immersed in a
solution containing the substrate. The substrate migrates toward the interior of the layer and 1s
converted into reaction products, with proton H+ production or consumption, when it reacts with
the immobilized enzyme.
The different steps during the operation of an enzyme sensor based on pH electrode are:
1. Transport of the substrate from the bulk of the solution toward the enzymatic layer,
2. Diffusion of the substrate within this layer, accompanied by the enzymatic transformation
of the substrate in to reaction product with H+ production (or consumption),
3. Migration of H+ toward the pH electrode,
4. Detection of H+ by the pH electrode.
The performances of enzyme pH electrodes will largely depend on the immobilization of the
enzyme onto the electrode to ensure maximal contact and response. Enzymes can be immobilized
by physical entrapment or by crosslinking, which ensures a greater long-term enzymatic stability.
Enzyme pH electrodes can be constructed by dipping the electrode in a solution containing the
enzyme with a crosslinking agent (immersion method) or immobilizing the enzyme directly on the
tip of the pH electrode (direct method).

Figure 2.2: Schematic representation of the diffusion of the substrate S and the products P and H+
in the enzymatic layer on a pH electrode.

2.2.3 Enzyme Biosensors Based on Gas Electrodes (features)

Gas probes exploited for assembly of biosensors have been mainly CO2 and NH, electrodes, and
the range of concentration of most metabolites is 10!" − 10!# M. Enzymes are usually
immobilized on the gas membrane in order to obtain the desired selectivity for specific metabolites.
The gas probes (often called potentiometric gas sensors) are constructed using an ion-selective
electrode (generally a pH glass electrode) and a reference covered by a gas-permeable membrane
(Fig. 2.3). A very thin film of a suitable electrolyte is present between the gas-permeable
membrane and the surface of the ion-selective electrode. When the gaseous form diffuses from the
sample solution through the gas-permeable membrane, it hydrolyzes in the thin film of internal
solution, varying the concentration of some ions (H+ generally) detected by the ion-selective
electrode. The variation of the potential is directly related to the concentration of gas existing in
the sample.
We can consider, for example, the NH, electrode, which is often used in the construction of several
biosensors. In this case the internal solution is 0.1 M NH4Cl, and we will use a pH electrode as
internal electrode.

Figure 2.3: Scheme of a gas probe.

2.2.4 Enzyme Biosensors Based on ISFETs

The ion-sensitive field-effect transistor (ISFET) can be regarded as a successful combination of
two well developed techniques: solid-state integrated circuits and ion-sensitive electrodes.
Bergeveld introduced the ISFET as a new device that combines the chemical-sensitive properties
of glass-membrane electrodes with the impedance-converting characteristics of the metal oxide
semiconductor field-effect-transistor (MOSFET).

2.2.4.1 Operation Principle of ISFETs

As shown from Fig. 2.4, an ISFET consists of an ion-sensitive membrane and an FET-structure in
which the metal electrode (gate) is removed and its function is taken over by the solution under
investigation. Transistors for ISFETs are manufactured according to standard planar silicon
technology, and frequently Si3N4/Si02 /Si structures are used.
The operation principle of an ISFET is the formation of a potential difference at the
membrane/solution interface influencing the width of the source-drain channel of the FET. Since
the value of the interfacial potential difference depends on the concentration of corresponding ions
in solution, the change of the FET characteristics (e.g., drain-source-current ID) will be a measure
of the analyte concentration.

Figure 2.4: Diagram of a pH-ISFET.

2.2.5. Enzyme Biosensors Based on the Hydrogen Peroxide Electrode

The earliest biosensors used pH and oxygen sensing as the method of signal transduction. They
were, however, largely based on oxidase enzymes, most typically glucose oxidase.
Glucose + O2 ↔H2O2 + gluconic acid
In the reaction sequence shown above, it depletes one reactant (oxygen) while producing two
measurable products (hydrogen peroxide and gluconic acid). The measurement of gluconic acid
by pH change is rather insensitive and prone to severe interference from the medium m which the
measurement is made. Oxygen is easily measured using a Clark electrode but again suffers from
signal-to-noise ratio problems. Early researchers found that reversing the polarity on a Clark
electrode allowed them to measure hydrogen peroxide destruction at the working electrode.
Hydrogen peroxide has become by far the most widely used method of signal transduction in
enzyme biosensors. Some 65 % of all biosensors use hydrogen peroxide detection as the method
of signal transduction.
The method of construction of peroxide sensors usually involves a platinum anode and a
silver/silver chloride cathode. A typical electrode configuration is shown in Fig. 2.5. The anode is
poised at +0.6-0.7 V. The method of construction of the hydrogen peroxide- producing biosensor
varies, but, in general, the oxidase enzyme employed is immobilized on the surface of the sensor
by crosslinking with a polymer-forming chemical such as glularaldehyde. Alternatively, the
enzyme may be entrapped either in a polymer such as urethane or by electro-polymerization of a
compound such as a pyrrole.
Figure 2.5: Diagram of a platinum, silver/silver chloride electrode, and an exploded view of an
enzyme membrane.

2.2.6. Enzyme Biosensors Based on Mediator-Modified Carbon Paste Electrode

The mediated mechanism of electron exchange has shown well-defined electrochemistry

associated with the designing of enzyme-based biosensor and has been studied extensively
involving a variety of mediators. The components of the organic metal
(tetracyanequinodimethane) [TCNQ] or tetrathiafulvalene [TTF]) have shown good
electrochemistry and are more hydrophobic as compared with ferrocene derivative. These are
efficient mediators for the regeneration of several enzymatic systems with better practical
applications in designing enzyme-based biosensors. Additionally, these mediators have shown low
background current even when the ratio of mediator/ enzyme is increased to several hundred-fold
within the immobilized phase, which also reduces the problems associated with the kinetic
limitations.

2.2.7. Enzyme Biosensors Based on Electron Transfer Between Electrode and Immobilized
Peroxidases

Peroxidases are widely spread in nature and are classified as oxidoreductases E.C. l. 11.1.X, where
X is determined by a biologic reducer. Heme-containing peroxidases are divided into two
superfamilies, viz., plant and mammalian (see Table 1). The latter includes myeloperoxidase,
lactoperoxidase, thyroid peroxidase, and prostaglandin H synthetase. The superfamily of plant
peroxidases consists of yeast cytochrome c peroxidase (CCP), plant ascorbate peroxidases, fungal
peroxidases, and classic plant peroxidases. Plant enzymes in general are more stable than others,
and among them horseradish peroxidase (HRP) is the most commonly used in practical analytical
applications.
Peroxidases oxidize a wide range of electron donors, such as amines and phenols. One-electron
and proton transfer results in formation of substrate radicals, which could interact with each other
forming polymeric products or attack the enzyme itself causing its inactivation. Rapid inactivation
of peroxidases in the reaction course is a well-known phenomenon and this is one of the main
limitations of sensitivity m peroxidase-based assays.
When reaction la proceeds on an electrode surface Compound I is reduced into ferriperoxidase by
a heterogeneous electron transfer (ET) directly from the electrode material as well as by means of
electron mediators. Both approaches result in a reduction current related to the concentration of
hydrogen peroxide in the contacting solution.

2.2.8. Enzyme Biosensors Based on Redox Polymers

Enzymes can be immobilized near electrode surfaces by trapping them in a crosslinked polymer.
Gregg and Heller (I) introduced an interesting extension of this technique by incorporating the
polymer into the sensor transduction mechanism. The strategy was to attach redox complexes to
the polymer backbone and use the resultant redox polymer to mediate electron transfer between
the immobilized enzymes and the substrate electrode.
The principles behind the operation of these enzyme electrodes are essentially identical to those
behind sensors that use soluble mediators. The main difference IS that the redox functionalities
pendant to the crosslinked polymer cannot participate in molecular diffusion, Rather, the electron
transport process occurs by a series of self-exchange reactions between neighboring redox centers,
a process referred to as electron hopping (2).
The polymeric mediator offers several important features. Perhaps the most important is the
opportunity to construct very small self-contained sensors. Tightly binding the mediator and the
enzymes to the electrode eliminates the need for a dialysis membrane to trap the enzymes and
eliminates the need to add soluble mediator to the sample. These features have been invaluable to
our work on microsensors for measurements m the brain tissue of living animals (3), in which rt
is vital to make the sensors as small as possible and it is not feasible to add reagents to the
sample.
Several factors impinge on the choice of redox complex: it must be possible to attach the complex
to a crosslinkable polymer; the complex must readily participate in electron self-exchange as well
as crossexchange with enzymes and electrodes; the complex must be chemically stable in two
oxidation states; and it must possess an appropriate half-wave potential.

2.2.9. Enzyme Biosensors Based on Metallized Carbon Electrodes

Early work on metal-dispersed carbon biosensors focused on the utility of platinized carbon for
enhancing the sensitivity of oxidase- or dehydrogenase-based devices. Palladium-dispersed or
sputtered particles displayed similar improvements. The dramatically improved sensitivity towards
the liberated peroxide or NADH species is attributed to the tine dispersion of the metal
microparticles m the carbon matrix. Such dispersion results in highly catalytic surfaces, as long as
the particle size is comparable to that of the electrical double layer. Unfortunately, the dispersed
platinum or palladium particles are also highly electrocatalytic toward interfering substances.
Investigation has led to the identification of dispersed rhodium, ruthenium and iridium particles as
selective promoters of the peroxide (and to a lesser extent NADH) reactions.

2.2.10. Enzyme Biosensors Based on Conducting Polymers

A major concern in the development of amperometric biosensors is the tight and reproducible
immobilization of enzymes with high activity on the electrode surface. Additionally, aiming on
the development of miniaturized amperometric enzyme electrodes, it should be possible to
predefine the site for the immobilization of the enzyme without using manual deposition
techniques,
In this respect, conducting polymers like polypyrrole, polythiophene, polyaniline, and polyindole
show distinct advantages over nonconducting polymers because they can be electrochemically
grown exclusively on the surface of an electrode.
The formation of conducting polymers is usually attained by means of an electrochemically
induced polymerization of the monomer in O2-free solution. The first step is the oxidation of monomers
at an appropriate electrode potential, leading to radical cations in the vicinity of the electrode, which
can react in a second step with another monomeric or oligomeric radical cation, or a neutral
monomer under formation of a dimeric radical cation or a dimeric diradical dication. The diradical
dication may lose two protons, leading to the dimer (or to higher oligomers). Since the thus
obtained dimers and oligomers have lower oxidation potentials than the monomer itself, the
polymer formation process leads predominaantly to a chain propagation, and after a critical chain
length for precipitation is attained, the deposition of the polycationic polymer occurs on the surface
of the anode. The intermediate radical cations may undergo reactions with nucleophilic species in the
solution, preventing chain propagation. Thus, exclusion of oxygen and the proper choice of the
polymerization solution is indispensable for reproducible deposition of conducting polymer films.
Local changes of pH as a result of the liberation of protons during the polymerrzatron reaction
have to be taken mto account. The resulting polymer has a net positive charge that is neutralized
by incorporation of anions from the electrolyte.

2.2.11. Enzyme Sensors Based on Conductimetric Measurement

Enzyme sensors based on conductimetric measurement exploit the fact that the changes in
substrate and product concentrations resulting from the catalytic action of some enzymes may be
accompanied by a net change in solution electrical conductivity.
There are a number of mechanisms by which an enzymatic reaction can lead to a conductivity
change. The magnitude of the conductivity change, and to some extent the suitability of a particular
enzyme for the construction of a conductimetric biosensor, depends on the reaction mechanism,
the background conductivity, and the nature of the buffer used.
Electrical conductivity measurement is one of the oldest methods of the analytical chemist.
Conductimetric biosensors are based on the measurement of enzymatically catalyzed changes in
conductivity, but the devices differ in important ways from the analytical chemist’s conductivity
cell.

2.2.12. Enzyme Biosensors Based on Thermal Transducer/Thermistor

The thermistor-based calorimeters popularly known as “enzyme thermistors” (ET) were designed
to cover a broader range of applications (6), which included determination of metabolites,
monitoring of enzyme reactions, bioprocess monitoring, characterization of immobilized
biocatalysts, bio-separations, reactions in nonaqueous medium, environmental control, and more
recently investigation of ionic interactions.
The metabolites determined include alcohols, glucose, and lactate, using alcohol oxidase, glucose
oxidase, and lactate oxidase, respectively. The detection limit for these compounds is in the
submicromolar range m pure solutions. The normal procedure is to coimmobilize the oxidases
with catalase to increase total heat production, reduce oxygen consumption, and eliminate
hydrogen peroxide.

2.2.13. Enzyme Biosensors Based on Fluorometric Detection

Measurement of specific analytes employing the combination of fluorimetry and the enzymatic
reaction offers a number of advantages including high specificity, ease of measurement, and low
cost. The use of fluorescence-based enzyme techniques can provide the basis for a variety of
bioanalytical and biosensor assays. Fluorescence-based enzyme biosensors typically employ
dehydrogenases and the oxidases to target specific analytes by measuring the changes in the
fluorescence emission behavior of a product or an indicator fluorophore. Depending on the type
of enzyme employed, several options are available to achieve the desired transduction. These may
include measuring the direct fluorescence of an enzyme product or cofactor, or (2) changes in the
fluorescence of an indicator fluorophore resulting from an interaction with a product of the
enzymatic reaction.
Enzyme reactions are coupled to fluorescence detection through either the disappearance of a
substrate or the appearance of products. The substrates or products typically linked to fluorescence
measurement include H+, H2O2, O2, or enzyme cofactors. The interaction of these products with
an indicator fluorophore can allow the enzymatic reaction to be monitored in addition to providing
a means of measuring substrate concentrations.
Modulation of fluorescence as a result of the changes m the pH within a bioreactor has been used
to develop a variety of biosensors, including those for glucose, penicillin, ammonia, and urea.
Kiefer et al. described a lactose sensor based on a lipid bilayer containing the protein lactose
permease. Lactose permease 1s a cotransporter for lactose and H+. When lactose is present on one
side of the membrane, cotransport produces an increase in the concentration of H+ on the opposite
side of the membrane. Changes in pH are typically measured using a fluorescent dye indicator.

Methods of Enzyme Immobilization:

Based on support or matrix and the type of bonds involved, there are five different methods of
immobilization of enzyme.
1. Adsorption
2. Covalent bonding
3. Entrapment
4. Copolymerization
5. Encapsulation
2.3 Immune Sensors:
Immunosensors are solid-state devices in which the immunochemical reaction is coupled to a
transducer. They form one of the most important classes of affinity biosensors based on the specific
recognition of antigens by antibodies to form a stable complex, in a similar way to immunoassay.
Depending on the type of transducer there are four types of immunosensor: electrochemical,
optical, microgravimetric and thermometric. The most commonly used bioelements for the
development of electrochemical immunosensors are antibodies (Ab), followed by aptamers (Apt)
and, in the last five years, microRNA (miRNA).

2.3.1 Basic Principles of an Immunosensor

The immune system is an attractive subject in much scientific research because of its ability to
process information. The primary function of an immune system is to recognize and identify all
cells and molecules in the system and sort these biological entities as either harmful or not harmful,
as part of the system's defense mechanism. In the presence of foreign substances (i.e. antigens),
specialized immune system cells produce immunoglobulins (i.e. antibodies) that specifically bind
these antigens. This phenomenon has a number of functions including the development of sensors.
A sensor that is based on the concept of immunology (also known as an immunosensor) uses an
antibody (as a bioreceptor) for specific molecular recognition of antigens and subsequently forms
a stable immunocomplex. The immunocomplex is determined and measured by coupling this
reaction to the surface of a transducer (e.g. thermistor). The transducer detects and converts the
reaction to an electrical signal where it can be processed, recorded and viewed. Transducers can
be classified into different categories based on the signal output (e.g. electrical) or alteration in
properties (e.g. mass change) upon the formation of antigen–antibody complexes. Ideally, an
immunosensor should be designed with the following specifications: (i) ability to identify target
antigens quickly; (ii) ability to generate immunocomplexes without the need to add supplementary
reagents; (iii) ability to give results with high reproducibility; and (iv) ability to easily detect the
target in real samples. In immunosensors, detection of the target analyte may be direct by observing
the formation of immunocomplexes, or indirect by utilizing a label, for example, enzymes or gold
nanoparticles to observe a binding event.

Immunosensors can be categorized according to the transducers employed: electrochemical,

optical and piezoelectric.

2.3.2 Electrochemical Immunosensor

An electrochemical immunosensor measures an electrical signal displaying either an increase or

decrease in the electrical signal when an antigen–antibody complex forms.9 The use of
electrochemistry in immunosensors for analyte detection has several advantages as this technique
is economical, easy to operate, portable and simple to construct. Since electrochemistry is a
surface-based method, reaction volume is not important where minute samples are only required
for detection purposes. With the use of modern and evolving technology, this analytical technique
is sensitive, selective and able to produce instantaneous results thus making an electrochemical
immunosensor an attractive candidate for wide sensing applications.
2.3.3 Optical Immunosensors

Optical immunosensors exploit other methods for the detection of analytes that are based on
chemiluminescence, light absorbance, fluorescence, phosphorescence, light polarization and
rotation, with surface plasmon resonance being the most widely employed. Optical-based
immunosensors observe a variation in phase, polarization speed or frequency of input light that
corresponds to the formation of an antigen–antibody complex. The concept involved in optical
sensors for the identification of analytes is based on the higher dielectric permittivity acquired by
all proteins, cell and DNA, than air and water causing these biomolecules to reduce the propagation
speed of the electromagnetic fields flowing through them. Because all molecules contain atomic
nuclei and electrons in varying orbital states, these molecules are able to interact with the
electromagnetic fields that pass through them. By placing these molecules in oscillating
electromagnetic fields analogous to the propagation of light, electrons within the molecules will
vibrate due to the force subjected to them. Free electrons will then polarize in the presence of
light's magnetic field generating polarization current, resulting from the movement of electrons
where it moves much slower though a biomolecule than in free space. Typically, optical
immunosensors employ light either coming from a laser, diode or white-hot light bulb and enable
observations of any alterations in the attributes of the light reflected from or passed through the
sensor. Measurements can be concurrently or successively carried out by casting light upon the
sensor at various angles on a single sensor plane. An advantage of this type of sensor is the fact
that changes in the light's characteristic is measured and all the light is sourced externally, thus
making it energy efficient; only especially low illumination power is required to generate signals.
Several kinds of transducing components can be used to generate optical change: a grating
couplers resonant mirror, surface plasmon resonance (SPR) interferometry, reflectrometric
interference spectroscopy, ellipsometry, and total internal reflection fluorescence (TIRF).

2.3.4 Immunosensor based on Piezoelectric crystal device

Recently, techniques exploiting the use of piezoelectric crystal instruments have been designed
and developed for immunosensor utilization. The piezoelectric immunosensor makes use of the
mass sensitivity of piezoelectric quartz crystal in response to the specific antibody–antigen
interaction. This type of immunosensor is becoming popular because of the simplicity, high
sensitivity, specificity, speed, label-free, stability and safety of this technique and is being used in
a number of sectors such as in clinical diagnosis and environmental pollution supervision. Another
advantage of the piezoelectric immunosensor is its ability to detect analytes in real time, which
obeys the pseudo-first order kinetics. The basic principle involved in the mass sensitive
immunosensor is based on the change in mass during the attachment of the analyte to the binding
sites of the antibody. This mass change can be examined by quartz crystal, a main constituent of
the piezoelectric sensor. The piezoelectric crystal oscillates at a specific frequency in conjunction
with the use of an electrical signal at a certain frequency. By applying electrical voltage to the
quartz crystal via two electrodes, the orientation of the crystal is altered and this causes a distortion
in the crystal lattice that causes a mechanical oscillation at a characteristic vibrational
frequency i.e. the crystal's natural resonant frequency. In the event of the formation of
immunocomplexes, the surface of the crystal is loaded with an extra mass, which changes the
frequency of oscillation of the crystal and the mass change can be determined electrically.

2.3.5 Thermometric Immunosensor

Biological reactions are often accompanied by liberation or absorption of heat. This concept is
exploited in the thermal immunosensor to measure temperature change from evolved or absorbed
heat as a consequence of a specific analyte–antibody/antigen reaction as the detection mode.
Variations in temperature can straightforwardly be converted to an electrical signal, which makes
the electrical method the most efficient procedure to determine temperature when constructing a
thermometric immunosensor. The entire heat evolved or absorbed can be measured by evaluating
the molar enthalpy and sum of products produced in the biochemical reaction. The transducer
commonly chosen for the thermometric sensor is a thermistor acquiring an extremely high negative
temperature coefficient of resistance.
The temperature-based detection mode is often coupled with an enzyme thermistor since virtually
all reactions involving enzymes are accompanied with enthalpy changes. It is a common practice
to combine thermal detection of enzymatic reactions with the flow-injection assay (FIA) method
that leads to the development of the thermometric immunosensor. An advantage of this principle
has paved the way for the use of theoretically any type of enzyme conjugated with detection
antibodies.
2.3.6 Fiberoptic lmmunosensors with Continuous Analyte Response
Fiberoptic fluorescence signal transmission has several advantages for immunosensor design:
physical flexibility for remote sensing, no risk of electrical interference, high signal-to-noise ratio
with little attenuation over distance, and the capacity to both measure several analytes with
fluorescence from a single fiber and bundle fibers without significant crosstalk. For many
immunosensors these possibilities have offered little advantage because they have been designed
for single use, or they have required regeneration that usually cannot be accomplished in situ.
Basis of the lmmunosensor
The indicator system is membrane-encapsulated at the end of an optical fiber and depends on
changes in fluorescence energy transfer between the donor, B-phycoerythrin (BPE), and the
acceptor, Texas Red (TR). The intensity of fluorescence from BPE labeled with phenytoin (BPE-
PHT) or other analyte is decreased when the antibody, labeled with TR, binds to phenytoin. Figure
1A shows the condition when antibody-hapten binding places the BPE and TR in close proximate
geometry for efficient energy transfer and thus quench of BPE fluorescence. In the absence of
analyte phenytoin, antibody molecules are fully packed around BPE and fluorescence quenching
is maximal. When free phenytoin is present it competes with the BPE-PHT for TR- antibody. A
portion of the TR-antibody binds to analyte phenytoin, making it unavailable to quench BPE,
increasing the fluorescence signal (Fig. 1B). At equllibrium, the concentration of free phenytoin
in the test solution produces a characteristic fraction of TR-antibody bound to BPE-PHT, with a
unique and reproducible fluorescence intensity. In this design the main determinants of response
time are the effective kdls, the reagent solution viscosity, and membrane interactions. Simulations
indicate that the association rate constant (kass) has little impact on the time to reach equilibrium
after a change in analyte concentration, whereas a large kdrs is essential for a rapid response.
Changing kass by three orders of magnitude has little effect on the simulated response time.
However, if the kass is held constant at 4 x 106 L/mol/s, increasing the kdls, one order of magnitude,
for example from 4 x 10-4 to 4 x 110-3 s-1, results in a 10-fold reduction in response time (162 to
16 min). Because kdls rates may vary by as many as eight orders of magnitude, antibodies differ in
suitability for continuous measurement systems and should be chosen Judiciously to achieve an
appropriate sensor response time. The phenytoin sensor described here uses antibodies with a kdls
of 4 x 10-3 s-l, and intact sensors have response times of 15 m in aqueous solution. Reagent viscosity
can impact response time because the free analyte must diffuse into and throughout the reagent
chamber to affect a change m fluorescence. For example, when dextran 70 kDa is included in the
sensor for plasma or blood to balance oncotic pressure in the specimen, the response time is 1 h.
Because of viscosity effects, and other unpredictable interactions between reagents and the
encapsulation membrane, sensor performance may differ substantially from theoretical. The best
approach for sensor design may be to select antibodies with a relatively large kdls and satisfactory
specificity, then test intact sensors to determine if the response time is appropriate.
Materials Used

Instrumentation
1. The optical components of the immunosensor are shown in Fig. 2. 1. Excitation source: a
100-mW air-cooled argon Ion laser (model 5500A from Ion Laser Technology, Salt Lake
City, UT) tuned to 5 14.5 nm and attenuated to 0.1% with a neutral-density filter.
2. Reference channel beam splitter (conventional glass microscope slide at 45” to the incident
beam) and photometer
3. Off axis parabolic mirror (#02POAOl5, Melles Gnat, Irvine, CA) of 66-mm focal length
with a 2-mm hole drilled on the optical axis.
4. Focusing lens, fused silica with a 25-mm focal length (#OlLQB028, Melles Griot).
5. Fiber positioner (#FP-2, Newport, Fountain Valley, CA).
6. Emission monochrometer set at 577 nm.
7. Optical fiber 100/l 10 l.un core/cladding fiber (Superguide G #B2-0007-20, Fiberguide
Industries, Stirling, NJ)
8. The remaining optical components (breadboard, photomultiplier tubes, photometers, lens
mounts, shutter, and filters) are standard.
Affinity Biosensing Based on Surface Plasmon Resonance Detection

This is an optical method capable of monitoring biological interaction phenomena at surfaces in

real time, i.e., under continuous flow conditions, in which one of the molecules in the so-called
interaction pair, the ligand, is covalently attached to the surface. The optical-detection method is
based on total internal reflection (TIR) and surface plasmon resonance (SPR), a collective
oscillation of the electrons with respect to the nuclei in the near surface region of certain metals
like gold, silver, and aluminum. The surface plasmon oscillation can be regarded as an optical
wave that is driven by an external light source. It is a strongly localized wave that propagates along
the interface between the metal and the ambient medium (e.g., a buffer or a biofluid). This wave
is extremely sensitive to changes in refractive index near the metal surface, for example, caused
by adsorption or binding of biomolecules to the surface. The sensing signal m an SPR experiment
is defined as the change in angle of incidence ⊚$% of an incident light beam, and is denoted as
∆ ⊚$% . It is, for small angular shifts, proportional to the changes in refractive index and
consequently to the mass concentration of the biomolecules at the surface of the metal. The
outcome of an SPR experiment is strongly dependent on the accessibility and presentation of the
active region’s “epitopes” of the immobilized ligand.

Isolated Receptor based Biosensors

The idea that receptors could be incorporated with potentiometric electrodes to produce biosensors
was proposed as early as 1975. Neurotransmitter receptor proteins are excellent candidates as
sensing elements in biosensors because of their high affinity and selectivity for specific ligands.
They can recognize families of chemicals of physiological, pharmacological and toxicological
significance, ranging from amino acids and peptides to therapeutics, drugs of abuse and toxicants.
However, the highly complex structures of neuroreceptors, their generally labile nature at room
temperature, and the difficulty in obtaining sufficient quantities of receptor protein for biosensor
studies have restricted attempted biosensor applications of these proteins until recently.
 UNIT III

DNA based Biosensor

Deoxyribonucleic acids (DNA) are arguably the most important of all biomolecules. The unique
complementary structure of DNA between the base pairs adenine/thymine and cytosine/guanine
has been the basis for genetic analysis over the last few decades. The ability of a single stranded
DNA (ssDNA) molecule to ‘seek out’, or hybridize to, it’s complementary strand in a sample is
the foundation of DNA-based detection systems. There is a great potential market for simple,
cheap, rapid, and quantitative detection of specific genes. Areas of application include clinical,
veterinary, medico-legal, environmental, and the food industry.
The DNA-based biosensor is a device that incorporates immobilized DNA, as molecular
recognition element in the biological active layer on the surface, and measures specific binding
processes with DNA mainly using electrochemical, optical and piezoelectric transducers. The fact
that the DNA sequences are unique to each organism means that any self-replicating biological
organism can be discriminated. The DNA-based biosensor is also a complementary tool for the
study of biomolecular interaction mechanisms of compounds with double-stranded DNA (dsDNA)
enabling the screening and evaluation of the effect caused to dsDNA by health hazardous
compounds and oxidizing substances.

Principles of the DNA Biosensor

The DNA techniques, including hybridization, amplification, and recombination, are all based on
the double helix structure of the DNA. Nucleic acid hybridization is the underlying principle of
DNA biosensors in 1953, Watson and Crick described the structure of DNA and the role of the
DNA molecule in holding the information for cell reproduction and function. DNA is composed
of four repeating nucleotides (sometimes called nucleotide bases or simply bases): adenine,
guanine, cytosine, and thymine. DNA is coiled to form a double helix (double-stranded DNA, or
dsDNA) composed of two strands held together by hydrogen bonds that can be broken by heat or
high pH. The single stranded DNA (ssDNA) is relatively stable, but on removal of the heat source
or pH extreme, the DNA molecule will re-form (reanneal) into the double stranded configuration.
Reannealing between the ssDNAs from different sources is called hybridization. The reannealing
of the dsDNA is possible because nucleotide bases will re-form hydrogen bonds only with specific
complementary bases: adenine pairs with thymine, and cytosine pairs with guanine. In RNA, the
nucleotide base uracil replaces thymine and pairs with adenine. The stability of the hybridization
depends on the nucleotide sequences of both strands. A perfect match in the sequence of
nucleotides produces very stable dsDNA, whereas one or more base mismatches impart increasing
instability that can lead to weak hybridization of strands. The same basic principles apply to RNAs,
the transcriptional products of cell growth and reproduction. RNA-RNA and RNA-DNA
hybridizations can occur. Like other biosensors, DNA sensors are usually in the form of electrodes,
chips, and crystals; hence, hybridization on a sensory surface is a solid-phase reaction. Solution
hybridization is more rapid than hybridization on solid-supports, but unless the assay is a
homogeneous assay, a separation step is required before final detection. Solid phase or filter
hybridization is the longest established and the most often used.

The DNA-based sensors should have a rapid response time and should be quantitative, sensitive,
suitable for automation, cost effective, disposable and solve analytical problems in a wide range
of industrial contexts in order to be commercially viable.

DNA Biosensors based on Optical Transduction

Several kinds of optical transducers can form the basis of a DNA biosensor. Such transducers
include fluorescence, surface plasmon resonance and Raman spectroscope.

Fiber Optics
The fluorescent DNA stain ethidium bromide (EB) is a commonly used dye for the detection of
DNA. The fluorescent ethidium cation strongly associates with dsDNA by intercalation into the
base stacking region and, in some cases, the major groove of the double helical structure. EB has
an absorption maximum of 510 nm; detection of dsDNA is achieved by exposing it to an EB
solution, washing off the unbound EB, and measuring the fluorescence intensity by UV visible
spectrometry. The fluorescence intensity is directly proportional to the amount of EB-intercalated
DNA. Biosensors can be designed to use this principle.

Resonant Mirror
Optical methods, such as those using surface plasmon resonance (SPR) and monomode dielectric
wave guides, have been extensively developed in recent years. The resonant mirror is an
evanescent wave sensor which has been designed to combine the simple construction of SPR
devices with the enhanced sensitivity of wave guiding devices. Detection of enzyme substrate,
antibody-antigen, and protein-cell interactions by the resonant mirror device has been reported.
This technology has recently been applied to direct and rapid detection of DNA-DNA
hybridization. Biotinylated oligonucleotide probes were immobilized on the sensor’s surface, via
streptavidin, and hybridization of a complementary target oligonucleotide (40-mer) was monitored
in real time. The interaction at the sensory surface was shown to be sequence specific under
conditions of low stringency.

Raman Spectroscopy
Raman spectroscopy is a useful tool for chemical analysis because of its excellent capability for
chemical group identification. One limitation of conventional Raman spectroscopy is its low
sensitivity; hence, effective measurements often require powerful and costly laser sources for
excitation. However, this method has attracted renewed attention because of the recent observation
that Raman scattering efficiency can be enhanced by factors of up to 10& when a compound is
adsorbed on or near special metal surfaces. This modified technique is known as surface-enhanced
Raman scattering (SERS) spectroscopy. In 1994, Vo-Dinh et al. reported the development of a
new type of DNA probe based on surface-enhanced Raman scattering detection. These surface-
enhanced Raman gene (SERG) probes do not require the use of radioactive labels, while having a
great potential for sensitivity and selectivity.

Biomolecular Interaction Analysis Developments in instrumentation for biomolecular interaction

analysis (BIA) using biosensor technology have made real-time monitoring biological events
possible. The biosensor often used, BIAcore (Pharmacia Biosensor), is based on surface plasmon
resonance (SPR) for detection of changes in refractive index over time at the sensory surface.
DNA Biosensors Based on Electrochemical Transduction
Millan et al. attempted development of a DNA sequence-selective biosensor employing
electrochemical transduction. A DNA probe sequence was covalently bound to the surface of an
amperometric electrode. Hybridization of the immobilized probe sequence with its dissolved
complement produced an immobilized double strand that could be detected using redox-active
metal/polypyridine complexes that associate selectively and reversibly with double-stranded
DNA. The presence of the immobilized double strand caused preconcentration of the metal
complex in the DNA layer near the surface of the electrode, and it resulted in much larger
voltammetric peak currents than are observed for immobilized single-stranded DNA.

Apparently electrochemical transduction-based DNA sensors are easy to construct, needing only
two electrodes and a voltammeter; however, the sensitivity and specificity of such constructs is
poor.

DNA Biosensors Based on Piezoelectric Transducers

Piezoelectric materials have been used in a variety of configurations as microgravimetric detectors,
and their general theory and use has been well reviewed. They offer an attractive, near-universal
mode of transducing the biorecognition event, but only if the changes in detector mass that
accompany analyte binding are sufficiently large.
Piezoelectric transducers also offer the advantages of a solid-state construction, chemical inertness,
durability, and ultimately the possibility of low-cost mass production. Attention to date has been
mainly on AT-cut quartz crystals as the piezoelectric material that can function in a ‘microbalance’
mode. In order to carry out a measurement, an external voltage is used to deform the quartz crystal
plate so that there is relative motion between the two parallel crystal surfaces (thickness shear),
crystal relaxation and oscillation at the resonant frequency then being maintained by means of an
appropriate external circuit. The change in frequency (∆' ) resulting from any added mass (∆( ) to
the device can be described by the Sauerbrey equation. Thus, at least to a first approximation, the
change in mass per unit area of the crystal is directly proportional to the change in frequency. A
number of basic assumptions underlie this relation, however, and various modifying theories have
been proposed to account for deviations in both gas- and liquid-phase operation.
Biosensors Based on DNA Intercalation Using Light Polarization
The intercalation of polyaromatic compounds by DNA can serve as a basis for a simple and
sensitive method for detection and quantification of carcinogens. The experimental technique is
based on monitoring the decrease of polarization, caused by the displacement of an intercalated
fluorescent dye molecule by the analyte molecule (carcinogen). The magnitude of the polarization
decrease is proportional to the concentration of the analyte. Intercalation is a reversible insertion
of a guest species into a lamellar host structure. Study of the reactions between guest molecules
and the host molecule (double-stranded DNA) has been ongoing since 1947, when Michaelis
observed and correlated dramatic changes m the visible absorption spectra of basic dyes when
binding to DNA. Quantitative binding studies have been made by using equilibrium dialysis,
thermodynamic models, such as Scatchard plots, viscosity, NMR, and fluorescence spectroscopy.
The intercalative interactions of dyes with DNA have been intensively studied and characterized
by using many different methods. In addition to dyes, other compounds, such as aminoquinolines,
fused aromatics, such as diamino-phenyl indoles, a large number of polycyclic aromatic
hydrocarbons, and benzopyrenediol epoxide also intercalate into the DNA. The assay architecture
is analogous to a protocol generally used for competitive immunoassay, whereby an intercalating
dye competes with an analyte for a binding site on the DNA or is displaced by the analyte. The
initial guide for this study was a US patent by Richardson and Schulman, who used the classic
intercalators acridine orange, ethidium bromide, and proflavin and calf thymus DNA to measure
small quantities of the drug actinomycin D. The competitive binding assay described here used
DNA-acridine orange as a competitive agent to the intercalating test compound. The fluorescence
from unbound acridine orange is not polarized because of the random orientation and free rotation
of the dye molecules. For a freely rotating molecule the polarization of the fluorescence will be
completely random, even if the excitation light is polarized. After the acridine orange Intercalates
into the DNA, its orientation is fixed and it is unable to freely rotate. The fluorescence emitted
from the bound acridine orange will then have the same or similar polarization as the excitation
light. The displacement of intercalated acridine orange by a carcinogen is monitored by a reduction
m the polarized fluorescence intensity. One advantage of this method is that any test molecule,
fluorescent or not, that binds to DNA is detected by the displacement of the fluorescent
intercalator. The action is monitored by using excitation and emission wavelengths specific for the
fluorescent intercalator. In most cases, any fluorescence of test compounds will not interfere if
they do not strongly overlap with the chosen detector dye; also, the quantum efficiency of the dyes
will be orders of magnitude greater, reducing interferences. Advantages of using fluorescence
polarization and DNA intercalation are its rapid analysis time, simple experimental apparatus, and
good sensitivity as a result of the signal amplification of the multiple binding sites in the DNA.
The ability to measure many varieties of carcinogens allows for use m surveys and m screening
environmental sites for carcinogen contamination.
Materials Used

1. The fluorescence polarization measurements are made using an SLM 8000C scanning
spectrofluorometer, manufactured by SLM Aminco Instruments, Inc * (Urbana, IL). Other
spectrofluorometers with similar specifications and components can also be used This
instrument uses a 450-W xenon arc lamp as the excitation source and a double-grating
monochromator for the selection of the excitation wavelength. A single-grating
monochromator monitored the fluorescence. The excitation and emission paths contained
adjustable Glan-Thompson polarizers and the normal 90’ fluorescence geometry was used.
Photomultipliers monitored two channels; a reference channel monitoring the xenon lamp,
consisting of a concentrated solution of rhodamine B, which served as a quantum counter,
and the fluorescence signal channel. The fluorescence signal was measured and normalized
by the reference signal to minimize the effects of lamp fluctuations.
2. A UV-VIS spectrophotometer, used for measurement of the DNA absorption at 260 nm for
concentration determinations, is available from Perkin-Elmer Corp. (Norwalk, CT), Milton
Roy (Rochester, NY), and many other instrument companies.
3. Standard buffer: 8 mM Tris-HCl, 50 mM NaCl, and 1 mM EDTA in nanopure distilled
water; adjust pH to 7.0 with 3 M HCl. Autoclave the buffer for 20 min at 20 psi.
4. Calf thymus DNA (sodium salt): Take one vial (2 mg) and dissolve m 50 mL of buffer by
placing the DNA and buffer m a screw-capped vial and agitate for at least 12 h. Determine
the concentration of DNA in solution by measuring the absorption at 260 nm in a 1 -cm
path length quartz cuvet (1.00 absorption units of duplex DNA is assumed equal to 50
𝜇g/mL of DNA in solution). The typical DNA concentration range is between 32 and 33
𝜇g/mL.
5. Dilute suspension of glycogen.
6. Acridine orange: 2 x 10-5 M in buffer
Cell-Based Biosensor
Cell-based biosensor can detect biochemical effects directly via living cells and convert these
effects into digital electrical signals by sensors or transducers. Hence, it serves as the bridge
between biology and electronics. Cell-based biosensors combine living cells and sensors or
transducers for cellular physiological parameter detection, pharmaceutical effect analysis,
environmental toxicity test, etc. In contrast to molecule-based approaches, cell-based biosensors
have a broad spectrum of detection capabilities. Moreover, in addition to analyte sensing and
detecting, cell-based biosensors can provide the advantages of rapid and sensitive analysis for in
situ monitoring with cells. Cells naturally encapsulate molecular sensor arrays. Enzymes,
receptors, and ion channels, all with a stable status, could respond to their corresponding analytes
via a native cellular mechanism. Compared with molecular biosensors, cell-based biosensors are
expected to respond optimally to bioactive analytes. Therefore, cell-based biosensors provide a
useful tool to study the physiological effects of analytes.
Because of the obvious advantages of cell-based biosensors, e.g., long-term recording in a
noninvasive way, fast response time, and label-free experimentation, they have been widely
utilized in many fields such as cellular physiological analysis, pharmaceutical evaluation,
environmental monitoring, and medical diagnosis. In these applications, cell lines and primary
cultured cells are mainly selected as the cell sources. Cell lines divide actively in vitro, which
offers the convenience of preparation and culture, if the desired cell type is available.
The conventional cell-based biosensors usually use cell population recording to study the behavior
of cell populations on the basis of sensor platforms. The recorded data are often the collective
responses of many cells detected by the sensor electrodes, which can be applied for cell−cell
interaction study, such as cell invasion and barrier function assessment.
Cell-based biosensors enable analysis and monitoring of drug-ligand interactions, effect of
bioactive agents, environmental toxicity studies, etc. in a more physiologically relevant way. With
the use of suitable substrates, it is possible now to use cell-based biosensors for in situ (ex vivo in
some cases) monitoring with living cells in their native environment. Different cell types such as
bacteria, yeast, fungi, algae, and other higher eukaryotes including fish, rat and human cells have
been utilized for biosensor fabrication (Figure 3.1). Microbial cells, including bacteria, fungi,
yeast and algae, have largely been utilized in biosensors for water quality monitoring and toxicity
assessment.
Figure 3.1: Schematic showing the different cell types (left) and functional strategies (right)
utilized in cell-based biosensors.

Types of immobilization
Passive immobilization
Several types of immobilization strategies have been explored for attachment of living cells onto
transducer/detecting platforms (Figure 3.2). In general, the factors that help in determining a
particular immobilization strategy for a biosensor include: 1) the cell types used as bioreceptors,
2) the type of material/substrate onto which cells are to be immobilized, 3) toxicity of the substrate,
4) cell viability and growth requirements, and 5) the application of the biosensor. For example,
applications such as monitoring of biochemical oxygen demand (BOD) and cell migration rely on
the viability and metabolism of the cells used, hence it is imperative that immobilization should
not hinder the cell growth or viability.
The functional strategy of a biosensor is also an important determinant of immobilization method.
For example, bio-luminescence mediated detection of target analytes (e.g. heavy metals)
necessitates the microbial cells to be metabolically active and expressing the bioluminescent gene
in a stable manner. Thus, such biosensors often involve immobilization strategies that do not affect
cell viability, such as physical adsorption, hydrogel/sol gel entrapment/encapsulation and biofilm
formation. Since such techniques rely on the cells’ natural ability to adhere to surfaces, they are
collectively referred to as passive immobilization techniques.
Entrapment in sol gel/hydrogel is also a prominent cell immobilization technique. Some of the
most commonly used gel matrices are calcium alginate (or, alginate-starch), agarose and chitosan
Nanomaterials have emerged as promising candidates for cell immobilization. Magnetic Fe3O4
nanoparticles have been employed for labeling Bacillus subtilis cells electrostatically and
subsequently immobilizing them on ultramicroelectrode array (UMEA) using external magnetic
field. This modified UMEA was subsequently utilized as a BOD microsensor and shown to be
regenerable and reproducible with better sensitivity than dissolved oxygen (DO)-based and
mediated BOD sensors. Another study has been reported on the immobilization of E. coli cells on
nanoporous gold for selective detection of sulfide. Silicon-based nanostructures have been utilized
for both immobilization of breast cancer cells and improving sensitivity of the biosensor in terms
of monitoring drug effects at very low concentrations (2 nM). Thus, nanomaterials have shown
potential as robust substrates that serve the dual purpose of cell immobilization and signal
enhancement.

Active immobilization
Active immobilization techniques are an efficient alternative to passive immobilization since these
techniques involve the utilization of chemical linkers or other agents for immobilization of living
cells onto a suitable substrate, e.g. glutaraldehyde crosslinking. These methods allow tighter
binding of the cells to transducer surface leading to enhanced sensitivity. A recent study highlights
the significance of covalent (or active) immobilization of cells for improving the stability and
sensitivity of an optical biosensor against methyl parathion. Biomolecules that interact directly
with receptors and other moieties present on the cell surface have been used to immobilize bacterial
cells on various substrates. Leonard et al. developed an antimicrobial susceptibility testing (AST)
platform by immobilizing E. coli cells on Si micropillar arrays via wheat germ agglutinin (WGA).
Bacteria-specific antibodies have also been utilized for efficient immobilization on plastic and
glass surfaces.
Some immobilization strategies are unique for higher eukaryotic cells, such as utilization of the
extracellular matrix (ECM, a semi-solid network of various proteins and fluids to which cells are
bound in a multicellular organism) and its components for immobilization. It has been reported
that surface modification by ECM components enables uniform immobilization of cells while
maintaining their viability, which is a crucial factor in many biosensing applications. Collagen,
fibronectin and laminin together with peptides such as poly-L-lysine have been predominantly
used for the immobilization of various cell types including mouse germ cells, taste receptor cells,
myocytes and MCF-7 breast cancer cells.

Microbial biosensors
Many different types of microorganisms have been utilized for biosensor fabrication over the
years, including bacteria, photosynthetic algae and yeast cells with prominent applications in
environmental monitoring and antimicrobial susceptibility testing. Some of these biosensors
exploit the natural ability of a particular microorganism to sense a specific analyte while several
others are dependent on the introduction of specifically designed genetic constructs in a host
microorganism for detection of bioavailable analytes. Different types of transducing/detecting
systems have been utilized in microbial biosensors, electrochemical and optical being the most
prominent. Electrochemical readouts exploit the activation/production of redox active species by
the microbes for biosensing. In some cases, redox-active mediators may be externally
supplemented to improve electron transfer across the electrode. Thus, genetic modification is not
always required in electrochemical sensing. However, electrochemical systems are often
vulnerable to high noise and background due to interfering species. Optical readouts, on the other
hand, rely heavily on genetic modification for the microbes to produce a visual/fluorescent
response against a specific stimulus. Most genetic modifications involve the introduction of an
analyte-specific promoter upstream to a ‘bioreporter’ gene in the host microbial cells. Upon
exposure to the target, the promoter gets activated and the bioreporter gene expresses fluorescent
proteins. Thus, optical biosensors are generally more specific than electrochemical ones, however
they require a number of additional steps involving gene modification and hence, increase the cost
of the device considerably.

Animal cell-based biosensors

Animal cells (higher eukaryotes) differ from microbial cells in terms of their nutritional
requirements, growth rate, and many morphological and structural characteristics. Thus,
biosensors based on animal cells face altogether different types of design and fabrication
challenges. These biosensors have important applications in the fields of food safety, disease
modeling, and drug toxicity assessment. Electrical cell-substrate impedance sensing (ECIS) and
light-addressable potentiometric sensor (LAPS) are the two most prominently used transduction
techniques for animal cell-based biosensors. ECIS, developed by Giaever and Keese (1993),
utilizes gold interdigitated electrodes (IDEs) for monitoring of impedance (or, capacitance) with
changes in cell density.

Applications of Cell-based biosensors:

1. Water toxicity/quality monitoring
Different microbial sensors have been developed for the detection of various environmental
contaminants and physicochemical parameters including biochemical oxygen demand
(BOD), heavy metals, plasticizers, toxic gases, pharmaceuticals, and endocrine-disrupting
agents.
2. Clinical applications
Antimicrobial susceptibility testing (AST) has emerged as one of the most significant
clinical applications of microbial biosensors in the recent years owing to the rise of
multidrug resistant ‘superbugs.
3. Cancer research
Cancer cells-based biosensors have enabled non-destructive analysis of various cellular
parameters including growth, migration, cell surface interactions, cell-cell interactions,
cell-drug interactions, etc. by monitoring the electrical properties of cancer cells. In
addition, these biosensors have allowed the study of cellular parameters and ligand
interactions at single cell resolution, thus providing new insights in cancer pathology,
biology, and drug development.
4. Clinical and health care applications
In addition to cancer pathogenesis, cell-based biosensors have interesting applications in
monitoring the response of normal living cells toward changes in their microenvironment,
including presence of drugs and other bioactive molecules.
 5. Food safety and technology
Taste and odor perception and analysis are important parameters in food quality and safety
assessment. Several attempts have been made to fabricate efficient ‘bioelectronic tongue
and nose’ that mimic human olfaction and gustation for food quality and safety evaluation.
6. Pharmaceutical and pharmacological research
Drug screening and toxicity evaluation are important part of pharmaceutical research.
Recently, induced pluripotent stem cell (iPSC)- derived CM-based biosensors have
emerged as a powerful tool in assessing drug toxicity. Several MEA-based CM biosensors
have been reported for assessing the potential of drugs to initiate arrhythmia. Efforts are
being made to validate and standardize MEA-based CM sensors for drug proarrythmic
potential evaluation. Recently, a computer simulation technique was developed to analyze
the relationship between field potentials measured by MEA and cardiac AP.
7. Environmental applications
Cell culture-based assays, particularly based on fish and mammalian cell lines, can prove
fruitful for water quality assessment in terms of determination of the effects of different
toxicants on cell metabolism and growth. A portable ECIS-based sensor utilizing fish
RTgill-W1 gill epithelial cells has been developed for use in the US Army. Human cell
lines have been utilized toward development of devices for water toxicity monitoring.

Electrochemical Biosensor
The electrochemical biosensor is one of the typical sensing devices based on transducing the
biochemical events to electrical signals. In this type of sensor, an electrode is a key component
that is employed as a solid support for immobilization of biomolecules and electron movement.
Thanks to numerous nanomaterials that possess the large surface area, synergic effects are enabled
by improving loading capacity and the mass transport of reactants for achieving high performance
in terms of analytical sensitivity.
The electrochemical biosensor is the analytical devices that transduce biochemical events such as
enzyme-substrate reaction and antigen-antibody interaction to electrical signals (e.g., current,
voltage, impedance, etc.). Since Clark developed the 1st version of electrochemical biosensor for
blood glucose, various types of biosensor have consecutively been introduced and commercialized
for diverse applications. In this electrochemical biosensor, an electrode is a key component, which
is employed as a solid support for immobilization of biomolecules (enzyme, antibody and nucleic
acid) and electron movement. Various chemical modification methods are applied for this purpose
via amine- and carboxyl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide: EDC), aldehyde-
(hydrazide) and thiol (maleimide), depending on the chemical groups on the electrode in the
presence of or absence of supporting materials. Since inappropriate immobilization may cause loss
of activity, less specificity, and low biocompatibility, it is crucial not only to maintain orientation
and biological activity of the biomolecules upon immobilization. In addition, employing proper
functional material for the electrode is a key process for the high performance of biosensors.

Transduction in electrochemical biosensors

Conductometric, potentiometric, amperometric, voltammetric transistors together with
impedimetric transistors have already been used in electrochemical biosensors. To apply these
techniques, suitable electrodes are needed. Usually three types of electrodes, namely working,
reference, and auxiliary electrodes, have been utilized for conducting these electrochemical
techniques.
The working electrode is the electrode at which the concerning reactions take place. Considering
electrochemical biosensors, these electrodes can be solid electrodes like carbon, gold, platinum
(Pt), diamond, etc. The other types of electrodes, composite electrodes, are also frequently used as
working electrodes. Carbon-based composite electrodes like the carbon paste electrode (CPE) and
the glassy carbon paste electrode (GCPE) are two important examples of these electrodes.
Recently, nanomaterials have been introduced in these electrodes to enhance their electrochemical
performance. The reference electrode provides a constant against the changeable working
electrode’s potential. Silver/silver chloride and saturated calomel electrodes are the most
convenient reference electrode types. The auxiliary electrode is generally utilized for voltammetric
and impedimetric measurements and is known as current-carrying electrode. An inert solid
electrode like Pt or graphite can serve as an auxiliary electrode.
Going back to the electrochemical methods, conductometry can be defined as a method that
measures the conductivity difference of a medium. To process the method, charged species, ions,
must be present in the medium. In conductometric biosensors, charged species are either produced
or consumed which causes a definite conductivity change in the sample solution.
On the other hand, in potentiometry, the potential difference between reference and working
electrode is monitored under zero or very small current. Usually, ion-selective electrodes (ISE)
have been used to observe conversion of the biorecognition process into a measurable
potentiometric signal. For example, an enzyme can be immobilized on a pH-meter electrode. The
bioelectrochemical reaction produces or absorbs H+. The change in the H+ concentration
definitely causes the change in pH which can be easily monitored with the pH meter. Glass pH
electrodes coated with a selective gas-permeable membrane for compounds like CO2, NH3, or H2S,
and AgS together with AgX-selective thin-membrane electrodes are most commonly used ISEs as
potentiometric biosensor transducers.
The other type of electrochemical biosensors, amperometric biosensors, measure the current
under the influence of a constant potential that is applied between the reference and working
electrodes. Many types of electrodes have been used in amperometric biosensors.
Impedimetry, also called electrochemical impedance spectroscopy (EIS), has usually been used
in affinity biosensors for monitoring the changes that happen on the electrode surface. Upon
binding of the new layer, the capacitance or charge-transfer resistance of the electrode has changed
and this can be easily followed using EIS. EIS provides the monitoring of a wide variety of
molecules. In addition, it provides label-free detection which makes it practical compared to other
techniques like the fluorescent-based or magnetic-based biosensors.

Types of electrochemical biosensors based on biological recognition elements

Enzymatic electrochemical biosensors
Enzymes can be described as biological catalysts that respond specifically to particular substrates.
To obtain enzymatic electrochemical biosensors, a suitable enzyme has to be immobilized onto
the electrode surface. Then the oxidation or reduction of a specific analyte is monitored via current
change. One of the oldest and the most remarkable enzymatic biosensor is glucose biosensor.

Bioaffinity-based electrochemical biosensors

Compared to electrochemical enzymatic detection, it can be said that affinity-based interactions
help us monitor assays that are more complex. Electrochemical techniques are very proper
techniques to follow the biorecognition events in affinity-based sensors. Although amperometric
detection is more practical relative to EIS, especially for monitoring the changes at the electrode
surface, EIS is preferred. Genosensors [deoxyribonucleic acid (DNA)-based biosensors],
immunosensors, cytosensors, and aptamer-based biosensors (aptasensors) are types of affinity-
based biosensors.
In electrochemical genosensors, usually a single-stranded (ss) oligonucleotide probe is
immobilized onto the electrode surface. The transduction is completed with hybridization of this
probe with its complementary DNA sequence via base pairing. In terms of electrochemical
detection, the hybridization of two DNA sequences is monitored via the increased-current signal
of an indicator that binds to the DNA duplex or from other hybridization-based electrochemical
changes like the current decrease of guanine base found in the DNA sequence.
On the other hand, immunosensors rely on monitoring antibody–antigen interplay after the
hybridization occurs onto the electrode surface. In some assays, antigen or antibody has been
labeled with a substrate, usually with an enzyme that produces an electrochemically measurable
signal. Besides enzymes, metal markers and redox tags have also been used for electrochemical
monitoring of antigen–antibody interplay.
The third type of affinity-based biosensor is the cytosensor. Cytosensors can be defined as a kind
of biosensor that evaluates the cells. In the fabrication process, the electrode surface is modified
to be selective to significant cells. In the case of electrochemical cytosensors, the change in the
current, impedance, and capacitance have been measured to obtain information about the cells.
Detection of cancer cells has widely been conducted with cytosensors by capturing proper cell
lines that are found in the sample solution.
The last type of affinity-based biosensors that will be discussed here is aptasensors. Aptamers are
short and stable single-stranded oligonucleic acid molecules, in another words single-stranded
DNA or RNA molecules with high specificity to various ligands like amino acids, drugs, and
proteins. They are the cores of aptasensors. It is easy to label aptamers with proper tags like
fluorescence probes, quenchers, electrochemical indicators, nanoparticles, or enzymes. The ease
in modification procedures makes it possible to immobilize aptamers onto different supports with
high stability.

Nanomaterials in electrochemical biosensors

Nowadays, to increase sensitivity and selectivity of electrochemical applications, besides new
techniques, chemical modification and functionalization of electrodes have also been conducted.
In recent years, new electrode materials like GCPE and bismuth film electrode (BiFE) have been
developed and applied to electrochemical biosensor systems. As mentioned earlier, nanomaterials
like carbon-based nanomaterials, metallic nanoparticles, and nanoballs have been introduced into
electrode structure. On the other hand, carbon-based nanomaterials like carbon nanotubes (CNTs)
and graphene have been widely used because they increase the electron-transfer rate and provide
higher a surface area for the immobilization of biological molecules. In addition, decoration of
these two carbon-based nanomaterials with metal nanoparticles is easy and produces robust
material in terms of electrochemical catalysis. Besides decoration, sometimes nanohybrid or
hybrid nanomaterials have been produced and used in electrochemical biosensors.

Optical Biosensors
An optical biosensor is a compact analytical device containing a biorecognition sensing element
integrated with an optical transducer system (Figure 3.2). The basic objective of an optical
biosensor is to produce a signal which is proportionate to the concentration of a measured
substance (analyte). The optical biosensor can use various biological materials, including enzymes,
antibodies, antigens, receptors, nucleic acids, whole cells and tissues as biorecognition elements.
Surface plasmon resonance (SPR), evanescent wave fluorescence and optical waveguide
interferometry utilize the evanescent field in close proximity to the biosensor surface to detect the
interaction of the biorecognition element with the analyte. Optical detection in these sensors is
performed by exploiting the interaction of the optical field with a biorecognition element.

Figure 3.2: Optical Biosensors

Types of Optical Biosensors

1. Surface plasmon resonance-based biosensors
SPR is currently the most widespread and well-known technology, which detects the shift in
refractive index caused by molecular interaction at a metal surface via surface plasmon wave.
Surface plasmon occurs when photons from incident wave interact with electrons in the metal
surface. The plasmon propagates parallel along the metal surface thereby forming a surface
plasmon wave. The SP wave propagates on the boundary between metal and dielectric and
attenuates exponentially in the dielectric. Surface plasmon can be inspired optically at a metal
dielectric interface, only when the wave vector of the optical wave matching that of the SP mode.
In most cases, SP mode cannot be excited directly by a light due to the wave vector of the SP mode
usually larger than that of the incident wave. The increasement of the light wavenumber can be
achieved by the attenuated total reflection (ATR) or diffraction which can be performed in a
coupling device. The most common used coupling structures are prism couplers, waveguide
couplers, grating couplers, and fiber couplers.
For prism couplers, Kretschmann configuration of the ATR method take advantages over Otto
structure, due to its easily accessible geometry with no space between a glass prism and a gold
film. Incident light passes through a glass prism and is totally reflected at the base of the glass. As
a result, an evanescent field is generated and penetrated into a gold layer. By tuning incident angle,
evanescent wave along the prism-gold interface can be coupled to the SP wave on the surface of
the gold film. SP wave can be also inspired by grating couplers.
Grating couplers without suffering from a metal thickness are easy-to-make using standard
micro- and nano- fabrication. Fiber couplers with the ability of remote controlling and particular
the single mode fiber immune to the disturbance of multimode make them extensively used as the
coupling scheme. The bandwidth and moderate sensitivity, relative to a prism coupler, and the
requirement of theoretical design pose the biggest challenges which need to be overcome.
Waveguide coupler resembles the principle of prism couplers, i.e. optical mode propagates in a
waveguide core and its evanescent tail penetrates in the ambient medium with low refractive index.
When the light goes into the waveguide region with metal film covered, a SP mode can be excited
at the outer face of the metal film. Only when the propagation constants of the guided mode and
the SP mode are equal, will the coupling condition be fulfilled. Waveguide couplers which are
compatible with integration technology have become the most promising couplers.

2. Localized surface plasmon (LSP) based biosensor

When a surface plasmon is confined in a nanoparticle (NP) with dimension comparable to an
incident wavelength and the particle’s free electrons engaged in the collective oscillation, and it is
named a localized surface plasmon. The LSP which is nonpropagating surface plasmon is
distributed in a small region near the particle surface and the LSP resonance can be adjusted by
the particle properties, i.e. size, shape, and composition. The electric fields near the particle surface
are strongly enhanced and the enhancement decaying rapidly with distance. The optical extinction
of the particle reaches the maximum value at the frequency of plasmon resonance, which always
appears at visible wavelengths for noble metal particle. The extinction peak which is determined
by the refractive index of the ambient medium is the foundation for biosensing. Different types of
nanoparticles have been reported to enhance the SPR signal in the case of detecting interested and
hard–to-find targets, i.e. gold or silver NPs, magnetic NPs, carbon-based NPs, latex NPs, and
liposome NPs.

Figure 3.3: Schematic illustration of LSPR transmission (left) and reflection (right) modes.

3. Evanescent wave fluorescence biosensors

Figure 3.4: Schematic diagram of an evanescent wave planar optical waveguide chip

In these biosensors, the biological recognition and the consequent binding event occur within the
confines of an evanescent wave. The evanescent wave arises from the manner in which light
behaves when confined in an optical waveguide or fibres. Guided light is totally internally reflected
when it meets the interface of the waveguide/fibre and a surrounding medium with a lower index
of refraction, as a result an electromagnetic field called an evanescent wave extends out from the
interface into the lower index medium. The evanescent wave decays exponentially with distance
from the surface, generally over the distance of 100 nm to approximately a wavelength. Since the
evanescent wave is such a near-surface phenomena, detection employing evanescent wave
excitation to generate the fluorescent signal is surface-sensitive, meaning that only fluorescent
molecules near the surface are excited (Figure 3.4). This geometric limitation can help to minimize
unwanted background signal from a bulk sample while only enhancing the signal from
fluorophores captured on the surface. A profuse variety of biosensors was developed based on this
principle with a wide array of applications ranging from clinical diagnostics to biodefence to food
testing.

4. Bioluminescent optical fibre biosensors

This technique uses recombinant bioluminescent cells and the bioluminescent signal is transferred
from the analyte by an optical fibre. An Escherichia coli strain, genetically modified to emit a
luminescent signal in the presence of genotoxic agents, was immobilized on to a fibre optic and
the optrode response to the genotoxin atrazine achieved a detection limit of 10 𝑝𝑔. 1!) . A live cell
array bioluminescent biosensor fabricated by immobilizing bacterial cells on the optical fibres
arranged in a high-density array of microwells was developed. Each microwell accommodated a
single genetically engineered bacterium responding to a specific analyte and the array biosensor
enabled the multidetection of genotoxins.

5. Optical waveguide interferometric biosensors

An integrated planar optical waveguide interferometric biosensor is a combination of evanescent
field sensing and optical phase difference measurement methods. By probing the near-surface
region of a grating sensor area with the evanescent field, any change of the refractive index of the
probed volume induces a phase shift of the guided mode compared with a reference field, typically
of a mode propagating through the reference arm of the same waveguide structure. The interfering
fields of these modes produce an interference signal detected at the sensor’s output, whose
alteration is proportional to the refractive index change and the signal is related to the concentration
of the analyte. This technique, also called resonant waveguide grating (RWG), is suitable for
detecting the redistribution of cellular contents, studying cellular responses and cellular processes,
and was also applied to the detection of the avian influenza virus.
 6. Ellipsometric biosensors
An ellipsometric biosensor measures changes in the polarization of light when it is reflected from
a surface. This platform was applied in detecting the binding of influenza A virus strains with a
panel of glycans of diverse structures. The apparent equilibrium dissociation constants (avidity
constants, 10–100 pM) were used as characterizing parameters of viral receptor profiles.
Microarray biosensors based on total internal reflection imaging ellipsometry for the detection of
the serum tumour biomarker CA19-9 had an estimated detection limit of CA19-9 of
18.2 𝑢𝑛𝑖𝑡𝑠 . 𝑚𝑙 !) , which is lower than the cut-off value for a normal level [29].

7. Reflectometric interference spectroscopy biosensors

Reflectometric interference spectroscopy (RIfS) is a label-free and time-resolved method where
the simple optical setup is based on white light interference at thin layers. Changes in the phase
and amplitude of polarized light provides information about the thickness and refractive index of
the adsorbed protein layer. This method was used for the detection and quantification of diclofenac
in bovine milk and the obtained limit of detection was 0.112 𝜇𝑔 . 1!) in the complex milk matrix.
An RIfS biosensor for the detection of circulating tumour cells was capable of the selective
detection of cancer cells within a concentration range of 1000 − 100000 𝑐𝑒𝑙𝑙𝑠. 𝑚𝑙 !) .

8. Surface-enhanced Raman scattering biosensors

Surface-enhanced Raman scattering (SERS) is a biosensing technique which enhances the
intensity of the vibration spectra of a molecule by several orders of magnitude when it is in close
proximity to nano-roughened metallic surfaces or nanoparticles made of gold or silver. A SERS-
active surface fabricated on the tip of the optical fibres was applied to the sensitive detection of
cancer proteins (∼100 pg) in a low sample volume (∼10 nl). A SERS biosensor for the fast and
sensitive detection of a protein biomarker of endocrine-disrupting compounds in an aquatic
environment, with a limit of detection 5 𝑛𝑔. 1!) has been reported.
Microbial Biosensors Based on Respiratory Inhibition

Biosensors generally provide a rapid and convenient alternative to conventional methods for
monitoring chemical substances in fields as diverse as medicine, environmental monitoring, and
food processing. A biosensor is a device that combines a biologic-sensing element with an
electronic signal-transducing element. As biologic-sensing elements, enzymes, antibodies,
receptors, organelles, and microorganisms, as well as animal and plant cells, can be used. Among
these biologic-sensing elements, microorganisms have several advantages, as listed below (I): 1.
Wide range of suitable pH and temperature. 2 Long lifetime. 3. Low cost (because no isolation of
the active enzyme is needed) Purified biomolecules, such as enzymes, are used as the sensing
element in most biosensors. Although enzymes show high specificity for their substrates, they are
generally expensive and have poor stability. Therefore, microorganisms have been employed as
the sensing element of biosensors to solve these problems. They are suitable for on-line
environmental monitoring that requires long-term stability. Microbial biosensors have been
developed for assaying biochemical oxygen demand (BOD), a value related to the total content of
organic material in waste water. Microbial sensors, of course, have some disadvantages, as listed
below: 1. Long response time. 2. Long time to return to baseline. 3. Lower selectivity (because a
microorganism contains many enzymes)
Therefore, it is necessary to optimize measurement and storage conditions to shorten the
measurement time of microbial sensors. The low selectivity of microbial sensors can be
complemented by use of membranes, such as a gas-permeable membrane. More recently
developed microbial sensors are reviewed by Karube et al. The principle of microbial sensors is
shown in Fig. 1. A typical microbial sensor consists of an oxygen electrode to which a membrane
containing immobilized aerobic microorganisms is attached. The respiratory and metabolic
functions of the microorganisms are used to detect particular substances. Immobilization of the
microorganisms is a key step in the fabrication of microbial sensors. Immobilization should not
harm the microorganism, and rt should, ideally, improve their stability for use in continuous
monitoring. When analytes are degraded by immobilized microorganisms, respiratory activity
increases and dissolved oxygen is consumed. The oxygen concentration is monitored using the
oxygen electrode. A large decrease m current coming from the oxygen electrode indicates high
metabolic activity of the immobilized microorganisms, which in turn indicates a high
concentration of analyte. Some sensors use microorganisms whose respiration is inhibited by the
target analyte, as in the case of sensors for cyanide or pesticides.

Immobilization of Microorganisms
Immobilization methods must not harm the immobilized microorganisms and yet provide a sensor
with a good stability under the prevailing conditions of temperature, ionic strength, pH, redox
potential, and chemical composition. Major methods of immobilization are listed as follows: 1.
Physical adsorption method. 2. Entrapment method. 3 Crosslinking method. 4 Covalent binding
method.
Methods of microorganism immobilization are usually modified versions of methods used for
immobilizing enzymes. Immobilization of microorganisms is also carried out in reactors used in
the food industry (e.g., beer and wine production) and in waste water treatment reactors. The most
widely used methods of immobilization are physical adsorption or entrapment methods. Since
chemical methods often damage the cell membrane and decrease biological activity, those methods
have seldom been successfully applied.
Oxygen Electrodes
One of the most suitable transducers for microbial sensors is an electrode, especially the Clark-
type oxygen electrode. The oxygen electrode measures the change in dissolved oxygen
concentration resulting from the respiration of the immobilized microbes. Clark-type electrodes
are either polarographic or galvanic in nature. Polarographic electrodes consist of a platinum
cathode and a silver anode, both of which are immersed in an electrolyte solution of saturated
potassium chloride. A suitable polarization voltage between the anode and the cathode is -0.6 or -
0.7 V, and oxygen is selectively reduced at the cathode at this voltage.

Microbial Sensors for Cyanide Determination

Yeast can be used as a microorganism for such sensors, and thus cyanide can be measured by
respiratory inhibition using an oxygen electrode. Cyanide is extremely toxic because it inhibits the
respiration of life forms. The respiratory chain in the inner mitochondrial membrane in yeast
contains the enzyme complexes through which electrons pass from NADH to O2. Compounds such
as potassium cyanide and sodium cyanide bind to heme a in cytochrome a and cytochrome a3 in
the cytochrome oxidase complex. The resultant inactivation of heme a causes a decrease in the
respiration. The cyanide induced decrease in respiratory activity causes a decrease in oxygen
consumption. The cyanide concentration can be measured from the change in the oxygen
consumption of the immobilized microorganisms with an oxygen electrode. Cyanide is mainly
discharged from industries, such as electroplating plants, coke plants, chemical industries, and
petroleum refineries, and there have been several accidents of cyanide contamination of rivers.
Cyanide may enter the environment from many sources, in the form of water or air pollution.
Therefore, a cyanide-detection system should be urgently established for river water analysis.
Cyanide is commonly determined using spectrometric methods. A spectrofluorimetric method was
also reported but these methods require laborious and time-consuming procedures. Flow-Injection
analysis (FIA) was applied to these methods for rapid analysis, but these methods require many
chemical reagents and expensive instruments. A cyanide electrode has also been reported, but it
suffers from interference problems. Therefore, conventional analysis methods are not suitable for
monitoring cyanide in the environment. A microbial sensor for cyanide detection is one of the
prospective methods for monitoring cyanide (Fig. 2).