Enzyme Electrodes

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Enzyme Electrode:

Enzyme based biosensor

Dr. Denoj Sebastian

Department of Life Sciences

University of Calicut

11/18/2012 Dr.Denoj Sebastian 1


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What is a biosensor?

a biosensor is a chemical sensing device in which abiologically derived recognition entity is coupled to atransducer, to allow the quantitative development of somecomplex biohemical parameter

A biosensor is an analytical device incorporating adeliberate and intimate combination of a specific biologicalelement (that creates a recognition event) and a physicalelement (that transduces the recognition event)

Biosensors are known as: immunosensors, optrodes,chemical canaries, resonant mirrors, glucometers, biochips,biocomputers

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What is a biosensor?

a bio-element -sensing thepresence as well as concentrationof an analyte. The bioelementmay be an enzyme, antibody,living cells, tissue, etc and

a sensor-element- an asembly

that actually converts thebiochemical signal into electricalsignal which may be amplifiedand read on a digital panel orrecorded on a suitable recordingdevice.

Combination

of two parts:



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Elements of Biosensor



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Schematic outline of a biosensor



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The Enzyme Electrode

Enzyme Electrode is a new type ofdetectororbiosensor that have been exclusively designedfor the potentiometric oramperometric assayof substrates, for instance : alcohol, amino

acids, glucose, and lactic acid.

The enzyme electrode is a combination of anyelectrochemical probe (amperometric,

potentiometric or conductimetric ) with a thinlayer (10 - 200mm) of immobilised enzyme.



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Design of Enzyme electrodes

Function of the enzyme is to provide selectivity by

virtue of its biological affinity for a particularsubstrate molecule

The anylate is sensed by the immobilized enzyme.

Following this, the progress of the enzyme reactionis monitored by the rate of formation of product or

the disappearance of a reactant.

If either the product or reactant are electroactive,then the progress of the reaction can be monitored as

out put in form of current or potential orconductivity



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Composed of a electrochemical sensor which inclose contact with a thin-permeable enzymemembrane.

The embedded enzymes located in the

membrane produce products, such as H+ ions,oxygen (O2), NH4+ ions, carbon dioxide (CO2)or even other small molecules depending solelyon the enzymatic reactions, that are rapidlydetected by the particular sensor.

The magnitude of the response gives the preciseestimations of the prevailing concentration ofthe substrate.



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The biological component

An Enzyme

A Multi-enzyme System

AnAntibody

Organelle

Microbial Cell

Whole Slices Of Tissue.

The out put of device would be

Current (Amperometric)

Voltage (Potentiometric)

Conductivity (Conductimetric)



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History of Enzyme electrodes

The history ofbiosensors started inthe year 1962 withthe development of

amperometricenzyme electrode for

glucose by thescientist Leland C.

Clark.

The year 1969 marksfirst potentiometricbiosensor: ureaseimmobilized on an

ammonia electrode todetect urea.

During the year 1972-75, first commercial

glucose biosensor wasdeveloped by yellowspring instruments.

Since then, severalbiosensors includingenzyme electrodes

were developed



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Systems based on oxygen or peroxide electrochemistry

The most commonly used enzymesin the design of enzyme electrodescontain redox groups which changeredox state during the biochemical

reaction.

Enzymes: Enzyme electrode withredox group of this type are theoxidases and the pyrroloquinolinequinone (PQQ) dependentdehydrogenases.



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In nature, oxidase enzymes such asglucose lactate and cholesterol oxidaseact by oxidising their substrates,accepting electrons in the process andthereby changing to an inactivated

reduced state.

These enzymes are normally returned totheir active oxidized state by transferring

these electrons to molecular oxygen,resulting in the production of hydrogenperoxide (H2O2).



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Both oxygen and hydrogen peroxide areelectrochemically active

The reaction progress can be followed by

reducing the oxygen (co-substrate)

oxidising the hydrogen peroxide (product).

The method based upon oxygen reduction atan O2 electrode or measurements basedupon hydrogen peroxide oxidation, is themost popular approach



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Glucose Biosensors

The first historic experiment that served asthe origin of glucose biosensors wascarried out by Leland C. Clark. He usedplatinum (Pt) electrodes to detect oxygen.

The most commercially successfulbiosensors are amperometric glucosebiosensors. These biosensors have beenmade available in the market in variousshapes and forms such as glucose pens,glucose displays, etc.



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Glucose Biosensors

The enzyme glucose oxidase (GOD) was placed very close to the surface of platinum by physically

trapping it against the electrodes with a piece of dialysis membrane.

The enzyme activity changes depending on the surrounding oxygen concentration.

Glucose reacts with glucose oxidase (GOD) to form gluconic acid while producing two electrons

and two protons, thus reducing GOD.

The reduced GOD, surrounding oxygen, electrons and protons (produced above) react to formhydrogen peroxide and oxidized GOD (the original form).

This GOD can again react with more glucose.

The higher the glucose content, more oxygen is consumed. On the other hand, lower glucosecontent results in more hydrogen peroxide. Hence, either the consumption of oxygen or theproduction of hydrogen peroxide can be detected by the help of platinum electrodes and this canserve as a measure for glucose concentration.



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Mediated systemsA major limitation of the peroxide system is the highoperating voltage required to oxidise the hydrogenperoxide resulting in the possibility of interference.

Mediators (molecules which can shuttle electrons

between the redox centre of the enzyme and theelectrode) can minimise this problem as they can beregenerated at potentials where interference from speciessuch as ascorbate, urate and paracetamol.

Mediators based on ferrocene and its derivatives andmetal complexes are common



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Mediated systems



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Bi-enzyme systemsBased on direct electrical communication between an enzyme and theelectrode.

Success in this field has been limited

One enzyme which has achieved this goal is horseradish peroxidase (HRP).

HRP catalyses the reduction of hydrogen peroxide at the expense of a number

of organic reducing compounds.

When the enzyme is linked electrically to an electrode, the need for theorganic reductant is obviated since the electrode itself provides the reducingequivalents



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Multienzymes Systems


Enzyme Electrodes

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