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BCH410 Note
BCH410 Note
COURSE OUTLINE:
Enzyme Immobilization
Biosensors
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ENZYME IMMOBILIZATION
ADSORPTION METHOD
Using adsorption as immobilization method is the easiest technique and includes
reversible surface interaction between carrier and enzyme. The forces formed are weak forces,
mostly electrostatic, for example Van der Waals forces, ionic bond and hydrogen bonding
interactions, although hydrophobic bonding can be significant, but although these forces are
very weak, but sufficiently large in number to enable reasonable binding. This method is done
by mixing the enzyme(s) and a support material with each other in adsorption properties, at
optimum pH, ionic strength, etc., for a time. After that, immobilized enzyme are collected and
washed to remove unbound enzymes.
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The most significant disadvantage is the separation of enzyme from the support
material, desorption may be happen under changes in pH, temperature, and also ionic
strength. Desorption may be happen as a result of physical factors, for example, flow rate,
agitation, particle-particle Collisions. Nonspecific binding may be lead to diffusion limitation
and reaction kinetic problem, with consequent alteration in parameter Vmax and Km8.
Further, binding of protons to the support with consequent shift in pH optimum (1-2 pH),
which may be important enzymes with precise pH optimum requirement. Unless carefully
controlled, overloading the support can lead to low catalytic activity, and the presence of a
suitable spacer between the enzyme molecule and the support can produce problems related
to steric hindrance.
ENTRAPMENT METHOD
One of the easiest techniques of immobilization is entrapment. In recent years, calcium
alginate has attraction as an immobilization support material. It has been utilized for
immobilization of variety of cell types, sub-cellular organelles, multi-component systems, and
enzymes. The physicochemical characteristics of this matrix in gel form have an important
effect on the reactions of entrapped biologically active material in the gel. Critical parameter in
selecting a matrix is pore size. The difference between entrapment technique and adsorption
and covalent binding is that however the enzyme is restricted in movement by the structure of
a gel lattice but it is free in solution. The pore size of a gel lattice is controlled to ensure that
the structure become tight enough to prevent loose of enzyme or cells, it also allow free
movement of the substrate and product. The support acts as a barrier to mass transfer, and
although this have serious reaction kinetics implications, but it can prevent interaction
between harmful cell, proteins, and enzymes and immobilized biocatalyst.
There are several major methods of entrapment:
1. Ionotropic gelation of macromolecules with multivalent cations (e.g. alginate).
2. Temperature-induced gelation (e.g. agarose, gelatin).
3. Organic polymerization reaction by chemical/photochemical (e.g. Polyacrylamide).
4. Precipitation from an immiscible solvent (e.g. polystyrene).
Entrapment can be accomplished by cross linking the polyionic polymer material with
multivalent cations in an ion-exchange reaction after mixing with enzyme to form a structure
that traps the enzymes/cells (ionotropic gelation). Change in temperature is a simple way of
gelation by phase transition utilizing 1-4% solutions of gelation. It is possible to mix the enzyme
with material that is then polymerized to frame a cross-linked polymeric system, trapping the
enzyme in the internal spaces of the lattice. The last method is more widely utilized, and
various acrylic materials are available for the formation of hydrophilic copolymers. For
example, acrylamide monomer is polymerized to form polyacrylamide and methylacrylate is
polymerized to form polymethacrylate. In addition to the monomer, a cross-linking agent is
added during polymerization to form cross linkage between the polymer chains and help to
create a three-dimensional network lattice. The formed polymer may be broken up into
particles of a desired size, or polymerization can be arranged to form beads of defined size.
Precipitation occurs by phase separation rather than by chemical reaction, but does bring the
enzymes/cells into contact with a water-miscible organic solvent, and most enzymes/cells are
not tolerant of such solvents. Thus, this method is limited to highly stable/previously stabilized
enzymes or nonliving cells.
This method is depending on localization of an enzyme inside polymer network or
membrane lattice. Entrapment has been advanced and broadly utilized for the immobilization
of cells more than for enzymes. It is limited for enzymes immobilization as it may be lost during
repeatedly using because of the small molecular size of enzyme compared to the cells.
Diffusion limitations are also disadvantages for this method. This method may be classified
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into five categories: lattice, microcapsule, liposome, membrane, and reverse micelle. The most
widely one is the lattice method, in this type enzyme is entrapped in the lattice of the different
natural or synthetic polymers. Alginate which is naturally occurring polysaccharide that has the
ability to form gels by ionotropic gelation, is the most popular one. Another type,
microcapsule, involves entrapment to porous polymer. The preparation of micro capsules
containing enzyme requires highly controlled conditions. The entrapment in liposome is
increasingly recognized as a technique of protecting biocatalysts from inactivation by
proteolytic enzymes. Also, this enzyme, liposome, offers a noticeable increment in thermal
protection. In the third type, reversed micelle, entrapping within the reversed micelles, it is
formed by adding a surfactant with an organic solvent, such as aerosol OT/isooctane reverse
micelles and used to entrap β-galactosidase. While in the membrane type, the enzyme is
isolated from the reaction solution microfiltration membrane or a hollow fiber.
ENCAPSULATION METHOD
Encapsulation of enzymes as well as cells can be accomplished by wrapping the
biological components inside different forms of semi permeable membranes. It is as
entrapment in that the enzymes/cells are free in movements, however limited in space. Vast
proteins or enzymes cannot out or inter capsule, however small substrates and products can
go freely across the semi permeable membrane. Numerous materials have been utilized to
form microcapsules are in range of 10-100 μm in diameter; such as, nylon and cellulose
nitrate. Rupture of the membrane is a problem associated with diffusion may be result if
products from a reaction accumulate rapidly.
Biological cells also may be used as capsules, and a famous example of this is the use of
erythrocytes (red blood cells). The membrane of the erythrocytes is normally just permeable
to small molecules. However, when erythrocytes are placed in hypotonic solution, they swell,
expanding the cell membrane and substantially expanding the penetrability. In this condition,
erythrocytes proteins go out of the cell and enzymes can inter into the cell. Returning these
erythrocytes, swollen, to the isotonic solution enables the cell membrane to return to the
normal state, and the enzymes inside the cell can't leak out. A distinct advantage of this
technique is co immobilization. Cells and/or enzymes may be immobilized in any type of
combination to be suitable for particular application.
Liposomal encapsulation involves the use of liposomes, which are small spherical
vesicles with at least one bilayer. They are amphipathic in nature. They can be used as a
vehicle for the administration of nutrients and pharmaceutical drugs. Cells consist of
phospholipids in their membrane bilayer structures. On contact with water, the geometric
structure properties vary depending on the class of phospholipids involved. The enzyme to be
encapsulated is mixed with the phospholipids and subjected to ultrasonic treatments
(sonication). Examples of such phospholipids include phosphatidylcholine, phosphatidylserine,
and phosphatidic acid.
In microbial encapsulation, the microbial cells hosting the target enzymes are
entrapped within insoluble matrices. This mode is found useful in the study of intracellular
enzymes. For example, sodium alginate has been used to immobilize thermophilic bacterium,
bacillus strain, in order to generate β-lactamase and proteases of industrial importance. Some
organisms like E. coli are used in whole cell membrane entrapment by hosting aspartic acid.
Ammonium lyase can be trapped on polyacrylamide for production of L-aspartic acid.
Pseudomonas, which hosts arginine deaminase, when immobilized in polyacrylamide results in
bulk production of citrulline (used in deamination). E. coli also penicillin amidase when
attached to polyacrylamide or cellulose carriers in the production of 6-aminopenicillanic acid.
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CROSS-LINKING METHOD
This method of immobilization depends only on enzyme and it is support-free as it
done by joining the enzyme (or the cells) to each other to prepare a large, three-dimensional
complex structure, and it can be done chemically or physically. Chemical type of cross-linking
normally includes formation of covalent linkage between the cells by means of a bi- or
multifunctional reagent, for example glutaraldehyde and toluene diisocyanate. However,
limiting factors can be used in this method for living cells and many enzymes because of
harmful materials. To minimize the close problems that can be found because of cross-linking
of single enzyme, both albumin and gelatin have been used.
This technique uses a bi- or multifunctional compounds, which serve as the reagent for
intermolecular cross-linking of the biocatalyst. Covalent binding or cross-linking methods are
done under relatively severe conditions in comparison with those of physical adsorption or
encapsulation. Hence, in the previous cases, conformational change of the enzyme structure
and partial destruction of the active site may occur. Accordingly, unless covalent binding
method is done under well controlled conditions, immobilized enzyme having high activity
cannot be obtained. Also, the enzyme cannot easily be lost from carriers because of the strong
binding forces between the enzyme and carrier.
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BIOSENSORS
The Schematic diagram shown below for the biosensor is mainly divided into three
sections:
(i) Sensor: a sensitive biological element (biological material (e.g. tissue, microorganisms,
organelles, cell receptors, enzymes, antibodies, nucleic acids, etc),
(ii) Transducer: it is the detector element (works in a physicochemical way; optical,
piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of
the analyte with the biological responsible for the display of the results in a user-friendly way
(iii) The third section is the associated electronics, which comprises of signal conditioning
circuit (amplifier), processor and a display unit.
There are initially two following types of transducer technologies. In the first one, the
estimates were made by measurement of electric current (amperometric) and in the second
case for urea measurements, the estimates were based on the measurement of charge on the
electrode (potentiometric). The biosensors based on the use of enzymes involving catalytic
action are described as catalytic biosensors as against bioaffinity biosensors developed later,
which do not make use of enzymes, but instead make use of antibodies, receptor molecules,
etc., which have high affinity with the analyte. In 1980s, the first bioaffinity biosensors were
developed, in which radiolabelled receptors were immobilized on to a transducer surface.
Biosensors based on ELISA have also been developed using labelled antibody or labelled
antigen coupled with a suitable transducer.
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Major components of a biosensor
The electrical signal from the transducer is often low and superimposed upon a
relatively high and noisy (i.e. containing a high frequency signal component of an apparently
random nature, due to electrical interference or generated within the electronic components
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of the transducer) baseline. The signal processing normally involves subtracting a 'reference'
baseline signal, derived from a similar transducer without any biocatalyst membrane, from the
sample signal, amplifying the resultant signal difference and electronically filtering (smoothing)
out the unwanted signal noise. The relatively slow nature of the biosensor response
considerably eases the problem of electrical noise filtration. The analogue signal produced at
this stage may be output directly but is usually converted to a digital signal and passed to a
microprocessor stage where the data is processed, manipulated to desired units and output to
a display device or data store.
The technology used for transducer can be any one of the four types listed below and
depend upon the biological sensor used. In biosensors, suitable transducers are designed,
keeping in view the following:
(i) Specific desired interaction between the analyte and the biological elements
(ii) The intended use of the biosensors
(iii) Manufacturing cost of the device.
Biosensing Method
The critical aspect of the biosensor is matching the appropriate biological and
electronic components to produce a relevant signal during analysis. Isolation of the biological
component is very much essential to ensure that only the molecule of interest is bound or
immobilized on the electronic component or the transducer. Attachment of the biological
component to the electronic component is vital for the success of these devices. The stability
of the biological component is also quite critical, since it is being used outside its normal
biological environment.
TYPES OF BIOSENSORS
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Biosensors can be grouped according to their biological element or their transduction
element. Biological elements include enzymes, antibodies, micro-organisms, biological tissue,
and organelles. The method of transduction depends on the type of physicochemical change
resulting from the sensing event. Primarily biosensors based on transducer element are mass
based (piezoelectric, etc), electrochemical biosensors (potentiometric, amperometric, etc),
and optical types of biosensors (fiber optics, etc).
B. Electrochemical Sensors: Electrochemical biosensors have been the subject of basic as well
as applied research for nearly fifty years. Leland C. Clark introduced the principle of the first
enzyme electrode with immobilized glucose oxidase at the New York Academy of Sciences
Symposium in 1962. In this configuration, sensing molecules are either coated onto or
covalently bonded to a probe surface. A membrane holds the sensing molecules in place,
excluding interfering species from the analyte solution. The sensing molecules react
specifically with compounds to be detected, sparking an electrical signal proportional to the
concentration of the analyte. Based on their operating principle, the electrochemical
biosensors can employ potentiometric, amperometric and impedimetric transducers
converting the chemical information into a measurable amperometric signal.
C. Optical Sensors: In optical biosensors, the optical fibers allow detection of analytes on the
basis of absorption, fluorescence or light scattering. Here both catalytic and affinity reactions
can be measured. The reaction causes a change in fluorescence or absorbance resulting due to
change in the refractive index of the surface between two media which differ in density. For
instance, if antibodies bind on a metal layer, the refractive index of the medium in contact
with this layer will change. Since they are non-electrical, optical biosensors have the
advantages of lending themselves to in vivo applications and allowing multiple analytes to be
detected by using different monitoring wavelengths. The versatility of fiber optics probes is
due to their capacity to transmit signals that reports on changes in wavelength, wave
propagation, time, intensity, distribution of the spectrum, or polarity of the light.
Enzymatic Sensors
Inhibitor sensors are intended for determination of substances reducing the activity of
an enzyme. An example is the determination of organophosphorus pesticides inhibiting
acetylcholine hydrolysis catalyzed by acetylcholinesterase. The most common enzymatic
biosensors are glucose and urea biosensors.
Immunosensors
DNA Sensors
The biochemical components of DNA sensors are nucleic acids (DNA). Most frequently,
they are not natural components isolated from a living organism, but their fragments called
DNA probes or DNA primers. They are selected so that they reflect the specificity of the DNA
structure as a whole. DNA probes are synthesized by DNA amplification via a polymerase chain
reaction. They can be additionally modified so as to enhance their stability or facilitate their
introduction into a biosensor. Oligonucleotide sequences having no natural analogue, selected
according to their capability to interact with certain biomolecules, are also used in DNA
sensors. These synthetic nucleic receptors received the name of aptamers. Since present-day
science is unable to predict the aptamer structure required for each particular ligand, one has
to synthesize all possible oligonucleotides for obtaining an aptamer (imposing a reasonable
limit of, e.g., 40–100 nucleotides) and then select those which bind most strongly to the target.
In most common microbial biosensors, the biological component is separated from the
recording device. This is due to the fact that the response of microorganisms to variations in
the chemical composition of the medium is rather sluggish compared to the response of
enzymes or antibodies, because the former is mediated by matter transfer across a
biomembrane. For this reason, it is necessary to create a higher concentration of living cells
than is allowed by the geometry of the transducer. A microbial biosensor may be a columnar
or membrane reactor or a suspension of microorganisms in a solution with an immersed
sensor. The microorganisms employed in these sensors can execute various functions. They
can convert the analyte using enzymes they secrete into the culture medium during their
metabolism or enzymes remaining in their living cells.
These sensors are similar to enzymatic sensors, with the only difference that a group of
enzymes, not necessarily a single one, may be involved in the conversion of the substrate. The
action of microorganisms is based on the fact that they change their respiratory activity as
they assimilate organic substances. These microbial biosensors are called respiratory
biosensors. They are used in the determination of the total amount of oxidizable organics in,
e.g., wastewater. Respiratory microbial biosensors are also usable in the determination of
antimicrobial agents suppressing microbial respiration.
APPLICATIONS OF BIOSENSORS
The advantages of biosensors include low cost, small size, quick and easy use, as well as
a sensitivity and selectivity greater than the current instruments. Biosensors have many uses in
clinical analysis, general health care monitoring. The most popular example is glucose oxidase-
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based sensor used by individuals suffering from diabetes to monitor glucose levels in blood.
Biosensors have found potential applications in the industrial processing and monitoring,
environmental pollution control, also in agricultural and food industries. The introduction of
suitable biosensors would have considerable impact in the following areas:
A. Clinical and Diagnostic Applications: Among wide range of applications of biosensors, the
most important application is in the field of medical diagnostics. The electrochemical variety is
used now in clinical biochemistry laboratories for measuring glucose and lactic acid. One of the
key features of this is the ability for direct measurement on undiluted blood samples.
Consumer self-testing, especially self-monitoring of blood components is another important
area of clinical medicine and healthcare to be impacted by commercial biosensors. Nowadays
reusable sensors also permit calibration and quality control unlike the present disposable
sticks where only one measurement can be carried out. Such testing will improve the efficiency
of patient care, replacing the often slow and labour intensive present tests. It will bring clinical
medicine closer to bedside, facilitating rapid clinical decision-making.
E. Food Industry: Biosensors for the measurement of carbohydrates, alcohols, and acids are
commercially available. These instruments are mostly used in quality assurance laboratories or
at best, on-line coupled to the processing line through a flow injection analysis system. Their
implementation in-line is limited by the need of sterility, frequent calibration, analyte dilution,
etc. Potential applications of enzyme based biosensors to food quality control include
measurement of amino acids, amines, amides, heterocyclic compounds, carbohydrates,
carboxylic acids, gases, cofactors, inorganic ions, alcohols, and phenols. Biosensors can be used
in industries such as wine beer, yogurt, and soft drinks producers. Immunosensors have
important potential in ensuring food safety by detecting pathogenic organisms in fresh meat,
poultry, or fish.
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