Electrochemical biosensors

Electrochemical biosensors are the most common especially when the biological component is an enzyme. Many enzyme reactions involve electroactive species being either consumed or generated and can be monitored by ampero-metric, potentiometric or conductimetric techniques, although the latter are the least developed and will not be discussed further.

Amperometric biosensors

These devices measure the current that is produced when a constant potential difference is applied between the transducing and reference electrodes. A current is produced when the species of interest is either oxidized at the anode or reduced at the cathode. The amount of current flowing is a function of the concentration of that species at the electrode surface. This type of

> Monoclonal antibodies - see Section transducer is applicable to substances that are easily electrochemically oxidized or reduced at an electrode.

Biosensors for the measurement of glucose which are based on glucose oxidase are examples of devices which use amperometric detection. The overall reaction can be monitored in several ways.

glucose + 02 = gluconic acid + H202

Originally, a Clark oxygen electrode was used to measure a reduction in current due to the consumption of oxygen. Anodic detection of the hydrogen peroxide by oxidation at a platinum or carbon electrode was then introduced but, owing to the high electrode potential required, suffered from interference from other electroactive compounds in the sample.

Attempts to reduce interference and minimize the effect of variations in oxygen tension have resulted in the development of biosensors with improved linear ranges which operate at lower electrode potentials. They incorporate artificial electron acceptors, called mediators, to transfer electrons from the flavoenzyme (e.g. glucose oxidase) to the electrode and thus are not dependent on oxygen. Ferrocene (bisir^-cyclopentadienyljiron) and its derivatives are examples of redox mediators for flavoenzymes. The reaction now becomes glucose + 2Fe+ = gluconic acid + 2Fe + 2H+

(ferricinium) (ferrocene)

The ferrocene is detected at the anode, which also regenerates the ferricinium ion for further use.

Construction of enzyme-based amperometric biosensors usually involves coating the electrode surface with both the enzymes and the mediator. The method adopted and concentrations used are important factors in the performance of the device. For extended use, covalent immobilization is the most satisfactory method.

Potentiometric biosensors

These measure the potential difference between the transducing electrode and a reference electrode under conditions of zero current. Three types of potentiometric detectors are commonly employed: ion-selective electrodes (ISE), gas-sensing electrodes and field effect transistors (FET).

Gas-sensing electrodes are modified ISEs and consist of either a hydrophobic gas-permeable membrane or an air gap between the sample solution and a pH electrode. Only gases that can permeate through the membrane and produce a change in pH, e.g. carbon dioxide, ammonia, will be detected by the electrode. Gas-sensing electrodes have been used in devices incorporating entrapped microorganisms as the biological component which metabolize the analyte of interest ami produce a detectable gas.

Field effect transistors are miniature, solid-state, potentiometric transducers (Figure 4.22) which can be readily mass produced. This makes them ideal for use as components in inexpensive, disposable biosensors and various types are being developed. The function of these semiconductor devices is based on the fact that when an ion is absorbed at the surface of the gate insulator (oxide) a corresponding charge will add at the semiconductor surface and the current flowing between the source and drain electrodes will change. The resulting voltage shift is a measure of the absorbed ionic species.

Variation of the nature of the gate electrode results in the different types of FET. For example, in the metal oxide semiconductor FET (MOS-FET) palladium/palladium oxide is used as the gate electrode. This catalyti-cally decomposes gases such as hydrogen sulphide or ammonia with the production of hydrogen ions, which pass into the semiconductor layer. An enzyme may be coated on the palladium, e.g. urease, which catalyses the production of ammonia from urea and thus provides a device for the measurement of this substrate.

In ion-selective FETs (ISFETs), an ion-selective membrane replaces the gate electrode. When an enzyme-loaded gel is combined with the membrane, the device can be used to measure substrates which enzymically generate charged species.

Gate electrode

Gate electrode

Figure 4.22 Schematic diagram of a Held effect transistor. The silicon-silicon dioxide system exhibits good semiconductor characteristics for use in FETs. The free charge carrier concentration, and hence the conductivity, of silicon can be increased by doping with impurities such as boron. This results in p-type silicon, the 'p' describing the presence of excess positive mobile charges present. Silicon can also be doped with other impurities to form n-type silicon with an excess of negative mobile charges.

Figure 4.22 Schematic diagram of a Held effect transistor. The silicon-silicon dioxide system exhibits good semiconductor characteristics for use in FETs. The free charge carrier concentration, and hence the conductivity, of silicon can be increased by doping with impurities such as boron. This results in p-type silicon, the 'p' describing the presence of excess positive mobile charges present. Silicon can also be doped with other impurities to form n-type silicon with an excess of negative mobile charges.

Self test questions

Sections 4.2-5

1. Which of the following can be used for both qualitative and quantitative analysis?

(a) Conductimetry.

(b) Coulometry.

(c) Amperometry.

(d) Polarography.

Self test questions

2. What is the technique in which a reactive ion is generated at an electrode for analytical purposes?

(a) Conductimetry.

(b) Coulometry.

(c) Amperometry.

(d) Polarography.

3. Enzymes are unsuitable for use in biosensor systems BECAUSE

biosensors require both a component to bind and convert the analyte to a product and a transducer to detect the product.

4. The increase in conductivity of a solution is directly proportional to an increase in ionic concentration

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