BIOSENSOR & ENZYMES AS IMMUNOSENSORS

PREPARED BY SAMHITA KALITA MSc. BIOTECHNOLOGY ( 2nd SEM. ) CENTRE FOR STUDIES IN BIOTECHNOLOGY (CSBT) DIBRUGARH UNIVERSITY

CONTENTS

Introduction Biosensor development Basic components of Biosensor Working of Biosensor Types of Biosensor Enzymes as Immunosensors Applications of Biosensor Conclusion References

INTRODUCTION

It is an analytical device which converts a biological response into an electrical signal.

It detects, records, and transmits information regarding a physiological change or process. It determines the presence and concentration of a specific substance in any test solution.

Biosensor Development

1916 First report on the immobilization of proteins: adsorption of invertase on activated charcoal. 1956 Invention of the first oxygen electrode [Leland Clark] 1962 First description of a biosensor: an amperometric enzyme electrode for glucose. [Leland Clark, New York Academy of Sciences Symposium] 1969 First potentiometric biosensor: urease immobilized on an ammonia electrode to detect urea. [Guilbault and Montalvo] 1970 Invention of the Ion-Selective Field-Effect Transistor (ISFET). 1972/5 First commercial biosensor: Yellow Springs Instruments glucose biosensor. 1976 First bedside artificial pancreas [Clemens et al.] 1980 First fiber optic pH sensor for in vivo blood gases. 1982 First fiber optic-based biosensor for glucose 1983 First surface plasmon resonance (SPR) immunosensor. 1987 Launch of the blood glucose biosensor[ MediSense]

BASIC COMPONENTS
Bio-element

Transducer

BIO-ELEMENT

It is a typically complex chemical system usually extracted or derived directly from a biological organism. Types : Enzymes Oxidase Polysaccharide

Antibodies Tissue Nucleic Acid

BIO-ELEMENT

Function To interact specifically with a target compound i.e. the compound to be detected. It must be capable of detecting the presence of a target compound in the test solution. The ability of a bio-element to interact specifically with target compound (specificity) is the basis for biosensor.

TRANSDUCER

Function : To convert biological response into an electrical signal.

Types :
Electrochemical, Optical, Piezoelectric

WORKING OF BIOSENSOR

Figure. Schematic Diagram of Biosensor

a- Bio-element b- Transducer c- Amplifier d- Processor e- Display

RESPONSE FROM BIO-ELEMENT

Heat absorbed (or liberated ) during the interaction. Movement of electrons produced in a redox reaction. Light absorbed (or liberated ) during the interaction. Effect due to mass of reactants or products.

TYPES OF BIOSENSOR
Electrochemical biosensor Calorimetric biosensor Piezo- electric biosensor Optical biosensor Immunosensor

ELECTROCHEMICAL BIOSENSOR

Principle Many chemical reactions produce or consume ions or electrons which in turn cause some change in the electrical properties of the solution which can be sensed out and used as measuring parameter.

Classification (1) Amperometric biosensor (2) Potentiometric biosensor

AMPEROMETRIC BIOSENSOR

Measuring parameter : Electric current

Based on oxidase enzymes that generate H2O2 and consume oxygen. Formation of H2O2 can be detected by the help of Pt-electrode.

GLUCOSE BIOSENSOR

Glucose reacts with glucose oxidase(GOD) to form gluconic acid. Two electrons & two protons are also produced. Glucose mediator reacts with surrounding oxygen to form H2O2 and GOD. Now this GOD can reacts with more glucose. Higher the glucose content, higher the oxygen consumption. Glucose content can be detected by Pt-electrode.

POTENTIOMETRIC BIOSENSOR

Potentiometric biosensors make use of ion-selective electrodes in order to transduce the biological reaction into an electrical signal.

consists of an immobilised enzyme membrane surrounding the probe from a pH-meter, where the catalysed reaction generates or absorbs hydrogen ions.

Fig.1. A potentiometric biosensor.

A semi-permeable membrane (a) surrounds the biocatalyst (b) entrapped next to the active glass membrane (c) of a pH probe (d). The electrical potential (e) is generated between the internal Ag/AgCl electrode (f) bathed in dilute HCl (g) and an external

CALORIMETRIC BIOSENSOR

Changes in heat used to determine concentrations.

The temperature changes are usually determined by means of thermistors at the entrance and exit of small packed bed columns containing immobilised enzymes within a constant temperature environment.

Fig.2. a calorimetric biosensor. The sample stream (a) passes through the outer insulated box (b) to the heat exchanger (c) within an aluminium block (d). From there, it flows past the reference thermistor (e) and into the packed bed bioreactor(f), containing the biocatalyst, where the reaction occurs. The change in temperature is determined by the thermistor (g) and the solution passed to waste (h). External electronics (l) determines the difference in

PIEZO- ELECTRIC BIOSENSORS

measures quartz vibrations under the influence of an electric field. The frequency of this oscillation (f) depends on their thickness and cut, each crystal having a characteristic resonant frequency. A simple use of such a transducer is a formaldehyde biosensor, utilising a formaldehyde dehydrogenase coating immobilised to a quartz crystal and sensitive to gaseous formaldehyde. They are inexpensive, small and robust, and capable of giving a rapid response.

OPTICAL BIOSENSOR
Changes in absorbance between the reactants and products of a reaction Blood monitoring for glucose (diabetes) Cellulose pads with glucose oxidase, horseradish peroxidase and a chromogen (e.g. o-toluidine) The hydrogen peroxide, produced by the aerobic oxidation of glucose oxidises the weakly coloured chromogen to a highly coloured dye.

Glucose oxidase

D-glucose

D-glucono-1,5-lactone
Horseradish peroxidase

chromogen(2H) + H2O2

dye + 2H2O

Highly coloured
Can be measured Spectrophotometricaly at a specific wavelength

IMMUNOSENSOR

Immunosensors

Immunological Response (ELISA)

ELISA is used to detect and amplify an antigenantibody reaction; the amount of enzyme-linked antigen bound to the immobilised antibody is determined by the relative concentration of the free and conjugated antigen and quantified by the rate of enzymatic reaction.

Antibodies labelled with microperoxidase MP11 for generation of the electrochemical signal via electrocatalytic reduction of H2O2

ENZYMES AS IMMUNOSENSORS

Enzyme was used to demonstrate the selectivity of analytes. The transduced signal varies with the rate of reaction of analyte with certain enzyme.

Advantages: more specific than cell based sensors faster responds due to shorter diffusion paths (no cell walls) Disadvantages: more expensive to produce due to the additional problem of isolating the enzyme enzymes are often unstable when isolated many enzymes need cofactors for the detection of substances

E1 : Urease E2 : Penicillinase An enzyme sensor used to detect the concentration of urea and penicillin-V

APPLICATIONS OF BIOSENSOR

In food industry, biosensors are used to monitor the freshness of food. Drug discovery and evaluation of biological activity of new compounds. Potentiometric biosensors are intended primarily for monitoring levels of carbon dioxide, ammonia, and other gases dissolved in blood and other liquids. Environmental applications e.g. the detection of pesticides and river water contaminants.

Determination of drug residues in food, such as antibiotics and growth promoters. Glucose monitoring in diabetes patients. Analytical measurement of folic acid, biotin, vitamin B12 and pantothenic acid. Enzyme-based biosensors are used for continuous monitoring of compounds such as methanol, acetonitrile, phenolics in process streams, effluents and groundwater.

CONCLUSION

From all these studies, I conclude that biosensors are cheap, small, and portable devices. They are capable of being used by semi-skilled operators.

REFERENCES

International Union of Pure and Applied Chemistry. "Biosensor". Compendium of Chemical Terminology Internet edition. www.lsbu.ac.uk/biology/enztech/optical.html. www.lsbu.ac.uk/biology/enztech/biosensors.html www.ornl.gov/ornlreview/rev293/text/biosens.html www.wikipedia.org/wiki/Biosensor Encyclopedia of chemical technology, by Kirth and Othmer (vol.4) page no.208-220