my 2nd sem. presttn biosensor & enzymes as immunosensors

7/31/2019 MY 2nd Sem. Presttn Biosensor & Enzymes as Immunosensors

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BIOSENSOR

& ENZYMES AS IMMUNOSENSORS

PREPARED BY

SAMHITA KALITA

MSc. BIOTECHNOLOGY

( 2nd

SEM. )CENTRE FOR STUDIES INBIOTECHNOLOGY (CSBT)

DIBRUGARH UNIVERSITY


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CONTENTS

Introduction

Biosensor development

Basic components of Biosensor

Working of Biosensor

Types of Biosensor

Enzymes as Immunosensors

Applications of Biosensor

Conclusion

References


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INTRODUCTION

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

It detects, records, and transmits information regarding aphysiological change or process.

It determines the presence and concentration of a

specific substance in any test solution.


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Biosensor Development

1916 First report on the immobilization of proteins: adsorption ofinvertase 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 ofSciences 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 Instrumentsglucose 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]


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BASIC COMPONENTS

Bio-element

Transducer


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BIO-ELEMENT

It is a typically complex chemical systemusually extracted or derived directly from a

biological organism.

Types :

Enzymes Antibodies

Oxidase Tissue

Polysaccharide Nucleic Acid


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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 specificallywith target compound (specificity) is the basis forbiosensor.


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TRANSDUCER

Function :

To convert biological response into an

electrical signal.

Types :

Electrochemical,

Optical,

Piezoelectric


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WORKING OF BIOSENSOR

a- Bio-element

b- Transducer

Figure. Schematic Diagram of Biosensor c- Amplifier

d- Processor

e- Display


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RESPONSE FROM BIO-ELEMENT

Heat absorbed (or liberated ) during theinteraction.

Movement of electrons produced in a redox

reaction.

Light absorbed (or liberated ) during theinteraction.

Effect due to mass of reactants or products.


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TYPES OF BIOSENSOR

Electrochemical biosensor

Calorimetric biosensor

Piezo- electric biosensor

Optical biosensor

Immunosensor


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ELECTROCHEMICAL BIOSENSOR

Principle

Many chemical reactions produce or consume

ions or electrons which in turn cause somechange in the electrical properties of the solutionwhich can be sensed out and used as measuringparameter.

Classification

(1) Amperometric biosensor

(2) Potentiometric biosensor


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AMPEROMETRIC BIOSENSOR

Measuring parameter : Electric current

Based on oxidase enzymes that generate H2O2and consume oxygen.

Formation of H2O2 can be detected by the help ofPt-electrode.


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GLUCOSE BIOSENSOR

Glucose reacts with glucoseoxidase(GOD) to form gluconicacid. Two electrons & twoprotons are also produced.

Glucose mediator reacts withsurrounding oxygen to formH2O2 and GOD.

Now this GOD can reacts with

more glucose. Higher the glucose content,

higher the oxygen consumption.

Glucose content can bedetected by Pt-electrode.


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POTENTIOMETRIC BIOSENSOR

Potentiometric biosensors make use of ion-selectiveelectrodes in order to transduce the biologicalreaction into an electrical signal.

consists of an immobilised enzyme membranesurrounding the probe from a pH-meter, where thecatalysed reaction generates or absorbs hydrogenions.


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Fig.1. A potentiometricbiosensor.

A semi-permeable membrane(a) surrounds the biocatalyst(b) entrapped next to theactive glass membrane (c) ofa pH probe (d). The electricalpotential (e) is generatedbetween the internal Ag/AgClelectrode (f) bathed in dilute

HCl (g) and an external


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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 smallpacked bed columns containing immobilised enzymeswithin a constant temperature environment.


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Fig.2. a calorimetric biosensor.

The sample stream (a)passes through the outerinsulated box (b) to the heat

exchanger (c) within analuminium block (d). Fromthere, it flows past thereference thermistor (e) andinto the packed bed

bioreactor(f), containing thebiocatalyst, where the reactionoccurs. The change intemperature is determined bythe thermistor (g) and thesolution passed to waste (h).

External electronics (l)determines the difference in


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PIEZO- ELECTRIC BIOSENSORS

measures quartz vibrations under the influence of an electricfield.

The frequency of this oscillation (f) depends on their thicknessand 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 toa quartz crystal and sensitive to gaseous formaldehyde.

They are inexpensive, small and robust, and capable of giving arapid response.


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Changes in absorbance between thereactants and products of a reaction

Blood monitoringfor glucose (diabetes)

Cellulose pads with glucose oxidase,

horseradish peroxidase and a chromogen(e.g. o-toluidine)

The hydrogen peroxide, produced by the aerobicoxidation of glucose oxidises the weakly coloured

chromogen to a highly coloured dye.

OPTICAL BIOSENSOR


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Glucoseoxidase

D-glucose D-glucono-1,5-lactone

chromogen(2H) + H2O2 dye + 2H2O

Horseradishperoxidase

Highly coloured

Can be measuredSpectrophotometricaly

at a specific wavelength


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IMMUNOSENSOR


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ImmunosensorsImmunological

Response (ELISA)

ELISA is used to detect and amplify an antigen-antibodyreaction; the amount of enzyme-linked antigen bound

tothe immobilised antibody is determined by the relativeconcentration of the free and conjugated antigen andquantified by the rate of enzymatic reaction.


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Antibodies labelled

with microperoxidaseMP11 for generationof the electrochemicalsignal via

electrocatalyticreduction of H2O2

S AS


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ENZYMES AS

IMMUNOSENSORS

Enzyme was used to demonstrate theselectivity of analytes.

The transduced signal varies with the rate of

reaction of analyte with certain enzyme.


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Advantages:

more specific than cell based sensors

faster responds due to shorter diffusion paths

(no cell walls)

Disadvantages:

more expensive to produce due to theadditional problem of isolating the enzyme

enzymes are often unstable when isolated

many enzymes need cofactors for the detection

of substances


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E1 : Urease

E2 : Penicillinase

An enzyme sensor used to detect theconcentrationof urea and penicillin-V


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APPLICATIONS OF BIOSENSOR

In food industry, biosensors are used to monitor thefreshness of food.

Drug discovery and evaluation of biological activity ofnew compounds.

Potentiometric biosensors are intended primarily formonitoring levels of carbon dioxide, ammonia, and othergases dissolved in blood and other liquids.

Environmental applications e.g. the detection of pesticidesand river water contaminants.


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Determination of drug residues in food, such asantibiotics and growth promoters.

Glucose monitoring in diabetes patients.

Analytical measurement of folic acid, biotin, vitamin B12and pantothenic acid.

Enzyme-based biosensors are used for continuousmonitoring of compounds such as methanol, acetonitrile,

phenolics in process streams, effluents and groundwater.


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CONCLUSION

From all these studies, I conclude that biosensors

are cheap, small, and portable devices.

They are capable of being used by semi-skilledoperators.


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REFERENCES

International Union of Pure and Applied Chemistry."Biosensor". Compendium of Chemical TerminologyInternet 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


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my 2nd sem. presttn biosensor & enzymes as immunosensors

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