Sie sind auf Seite 1von 6

Draft by Dr. V.K.

Mishra, DPMC, Dehradun

Enzyme Electrode: Enzyme based biosensorT

What is a biosensor
Various definitions and terminologies are used depending on the field of
application. Biosensors are known as: immunosensors, optrodes,
chemical canaries, resonant mirrors,glucometers, biochips, biocomputers, and so on.

A commonly cited definition is: “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 biochemical 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)”.

The name “biosensor” signifies that the


device is a combination of two parts:
(i) a bio-element, and
ii) a sensor-element.
The basic concepts of a biosensor’s operation can be illustrated with the following
figure1.

Elements of Biosensors : A biosensor


essentially comprise of the following two
major parts, namely :

(a) Biological component –i.e., for


sensing the presence as well as
concentration of an analyte. The
bioelement may be an enzyme,
antibody, living cells, tissue, etc., and

(b) Transducer device — i.e., an


asembly that actually converts the
biochemical signal into the
corresponding electrical signal which
may be adequately amplified and read
finally either on a digital panel or
recorded on a suitable recording
device .
Draft by Dr. V.K. Mishra, DPMC, Dehradun

The schematic outline of a biosensor exhibiting the various integral components


associated with it is given in fig.2

Electrochemical
Electrode or Enzyme
Electrode

Electrochemical electrodes (or Enzyme eletrodes) are 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.

History
Enzyme electrodes are a type of biosensor that have enzyme as a biological
component. 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 1972-75, first commercial glucose biosensor
Draft by Dr. V.K. Mishra, DPMC, Dehradun

was developed by yellow spring instruments. Since then, several biosensors including
enzyme electrodes were developed.

Design
The electrochemical electrode(enzyme electrode) is 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 ever 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.

The biological component present in a biosensor may invariably be an enzyme or a


multi-enzyme system, that could also be an antibody or organelle or microbial cell or
even whole slices of tissue.

In these devices, enzyme is combined with electrochemical probe .The out put of
device would be in terms of current (amperometric), voltage (potentiometric) or
conductivity (conductimetric).)

Types of Enzyme Electodes

1. Amperometric Enzyme Electrode


2. Potentiometric Enzyme Electrode
3. Conductimetric Enzyme Electrode
Draft by Dr. V.K. Mishra, DPMC, Dehradun

• 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.

I. 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 pyrroloqui
noline quinone (PQQ) dependent
dehydrogenases.

• 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).

 Because both oxygen and hydrogen peroxide are both electrochemically


active, the progress of the biochemical reaction can be followed by either
reducing the oxygen (co-substrate) or oxidising the hydrogen peroxide
(product). The method based upon oxygen reduction at an O2 electrode or
measurements based upon hydrogen peroxide
oxidation, is indeed represents by far the most
popular approach.

Glucose Biosensors

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.

The first historic experiment that served as theFig. The Clark experiment
origin of glucose biosensors was carried out by Leland
Draft by Dr. V.K. Mishra, DPMC, Dehradun

C. Clark. He used platinum (Pt) electrodes to detect oxygen. 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.
Fig. 7 shows the reaction catalyzed by GOD. 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.

Mediated systems

A major limitation of the peroxide


system described above, is the high
operating voltage (circa 0.8 volts vs
the Ag/AgCl reference lectrode)
required to oxidise the hydrogen
peroxide resulting in the possibility of
interference. The use of mediators
(molecules which can shuttle electrons
between the redox centre of the
enzyme and the electrode) can
minimise this problem as they can, depending on the compound used, be regenerated
at potentials where interference from species such as ascorbate, urate and
paracetamol. A vast number of compounds are capable of acting as enzyme mediators
and the groups which are most frequently used in the construction of enzyme
electrodes are detailed below. Of these, mediators based on metal complex

Bi-enzyme systems

Recently, work has focused on the


direct electrical communication
between an enzyme and the
electrode. Although 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.
Draft by Dr. V.K. Mishra, DPMC, Dehradun

When the enzyme is linked electrically to an electrode however, the need for the
organic reductant is obviated since the electrode itself provides the reducing
equivalents.

Das könnte Ihnen auch gefallen