Enzyme Electrode: Enzyme based biosensor

Dr. Denoj Sebastian Department of Life Sciences University of Calicut

11/18/2012

Dr.Denoj Sebastian

What is a biosensor?
a biosensor is a chemical sensing device in which a biologically derived recognition entity is coupled to a transducer, to allow the quantitative development of some complex biohemical parameter A biosensor is an analytical device incorporating a deliberate and intimate combination of a specific biological element (that creates a recognition event) and a physical element (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 the presence as well as concentration of an analyte. The bioelement may be an enzyme, antibody, living cells, tissue, etc and a sensor-element- an asembly that actually converts the biochemical signal into electrical signal which may be amplified and read on a digital panel or recorded on a suitable recording device.

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 of detector or biosensor that have been exclusively designed for the potentiometric or amperometric assay of substrates, for instance : alcohol, amino acids, glucose, and lactic acid. The enzyme electrode is a combination of any electrochemical probe (amperometric, potentiometric or conductimetric ) with a thin layer (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 particular substrate molecule

The anylate is sensed by the immobilized enzyme.

Following this, the progress of the enzyme reaction is 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 or conductivity

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Design of Enzyme electrodes

Composed of a electrochemical sensor which in close contact with a thin-permeable enzyme membrane. 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 solely on the enzymatic reactions, that are rapidly detected by the particular sensor. The magnitude of the response gives the precise estimations of the prevailing concentration of the substrate.

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Design of Enzyme electrodes

The biological component
An Enzyme A Multi-enzyme System An Antibody Organelle Microbial Cell Whole Slices Of Tissue.

The out put of device would be

Current (Amperometric) Voltage (Potentiometric) Conductivity (Conductimetric)

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Choice of Method of Analysis

The main considerations are;
Does the enzyme contain redox active groups Are the products of the reaction electroactive Is one of the substrates/cofactors electroactive Required speed of response Final application of the sensor
First three criteria will depend largely on the system under investigation

Latter three depends on the requirements and application of the sensor

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History of Enzyme electrodes

The history of biosensors started in the year 1962 with the development of amperometric enzyme electrode for glucose by the scientist Leland C. Clark.

The year 1969 marks first potentiometric biosensor: urease immobilized on an ammonia electrode to detect urea.

During the year 197275, first commercial glucose biosensor was developed by yellow spring instruments.

Since then, several biosensors including enzyme electrodes were developed

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Systems based on oxygen or peroxide electrochemistry

The most commonly used enzymes in the design of enzyme electrodes contain redox groups which change redox state during the biochemical reaction. Enzymes: Enzyme electrode with redox group of this type are the oxidases and the pyrroloquinoline quinone (PQQ) dependent dehydrogenases.

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Systems based on oxygen or peroxide electrochemistry

In nature, oxidase enzymes such as glucose lactate and cholesterol oxidase act by oxidising their substrates, accepting electrons in the process and thereby changing to an inactivated reduced state.

These enzymes are normally returned to their active oxidized state by transferring these electrons to molecular oxygen, resulting in the production of hydrogen peroxide (H2O2).

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Systems based on oxygen or peroxide electrochemistry

Both oxygen and hydrogen peroxide are electrochemically active

The reaction progress can be followed by

reducing the oxygen (co-substrate) oxidising the hydrogen peroxide (product).

The method based upon oxygen reduction at an O2 electrode or measurements based upon hydrogen peroxide oxidation, is the most popular approach
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Glucose Biosensors
The first historic experiment that served as the origin of glucose biosensors was carried out by Leland C. Clark. He used platinum (Pt) electrodes to detect oxygen.

The most commercially successful biosensors are amperometric glucose biosensors. These biosensors have been made available in the market in various shapes 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 form hydrogen 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 glucose content results in more hydrogen peroxide. Hence, either the consumption of oxygen or the production of hydrogen peroxide can be detected by the help of platinum electrodes and this can serve as a measure for glucose concentration.

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Mediated systems
A major limitation of the peroxide system is the high operating voltage required to oxidise the hydrogen peroxide resulting in the possibility of interference. Mediators (molecules which can shuttle electrons between the redox centre of the enzyme and the electrode) can minimise this problem as they can be regenerated at potentials where interference from species such as ascorbate, urate and paracetamol.

Mediators based on ferrocene and its derivatives and metal complexes are common

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Mediated systems

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Bi-enzyme systems
Based on direct electrical communication between an enzyme and the electrode.

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 the organic reductant is obviated since the electrode itself provides the reducing equivalents

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Multienzymes Systems

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