A biosensor is an analytical device which converts a biological response into an electrical signal. The term ‘biosensor’ is often used to cover sensor devices used in order to determine the concentration of substances and other parameters of biological interest even where they do not utilise a biological system directly. This very broad definition is used by some scientific journals (e.g. Biosensors, Elsevier Applied Science) but will not be applied to the coverage here. The emphasis of this Chapter concerns enzymes as the biologically responsive material, but it should be recognised that other biological systems may be utilised by biosensors, for example, whole cell metabolism, ligand binding and the antibody-antigen reaction. Biosensors represent a rapidly expanding field, at the present time, with an estimated 60% annual growth rate; the major impetus coming from the health-care industry (e.g. 6% of the western world are diabetic and would benefit from the availability of a rapid, accurate and simple biosensor for glucose) but with some pressure from other areas, such as food quality appraisal and environmental monitoring. The estimated world analytical market is about ?12,000,000,000 year-1 of which 30% is in the health care area. There is clearly a vast market expansion potential as less than 0.1% of this market is currently using biosensors. Research and development in this field is wide and multidisciplinary, spanning biochemistry, bioreactor science, physical chemistry, electrochemistry, electronics and software engineering. Most of this current endeavour concerns potentiometric and amperometric biosensors and colorimetric paper enzyme strips. However, all the main transducer types are likely to be thoroughly examined, for use in biosensors, over the next few years.
A successful biosensor must possess at least some of the following beneficial features:
The biocatalyst must be highly specific for the purpose of the analyses, be stable under normal storage conditions and, except in the case of colorimetric enzyme strips and dipsticks (see later), show good stability over a large number of assays (i.e. much greater than 100).
The reaction should be as independent of such physical parameters as stirring, pH and temperature as is manageable. This would allow the analysis of samples with minimal pre-treatment. If the reaction involves cofactors or coenzymes these should, preferably, also be co-immobilised with the enzyme .
The response should be accurate, precise, reproducible and linear over the useful analytical range, without dilution or concentration. It should also be free from electrical noise.
If the biosensor is to be used for invasive monitoring in clinical situations, the probe must be tiny and biocompatible, having no toxic or antigenic effects. If it is to be used in fermenters it should be sterilisable. This is preferably performed by autoclaving but no biosensor enzymes can presently withstand such drastic wet-heat treatment. In either case, the biosensor should not be prone to fouling or proteolysis.
The complete biosensor should be cheap, small, portable and capable of being used by semi-skilled operators.
There should be a market for the biosensor. There is clearly little purpose developing a biosensor if other factors (e.g. government subsidies, the continued employment of skilled analysts, or poor customer perception) encourage the use of traditional methods and discourage the decentralisation of laboratory testing.
The biological response of the biosensor is determined by the biocatalytic membrane which accomplishes the conversion of reactant to product. Immobilised enzymes possess a number of advantageous features which makes them particularly applicable for use in such systems. They may be re-used, which ensures that the same catalytic activity is present for a series of analyses. This is an important factor in securing reproducible results and avoids the pitfalls associated with the replicate pipetting of free enzyme otherwise necessary in analytical protocols. Many enzymes are intrinsically stabilised by the immobilisation process , but even where this does not occur there is usually considerable apparent stabilisation. It is normal to use an excess of the enzyme within the immobilised sensor system. This gives a catalytic redundancy (i.e. h << 1) which is sufficient to ensure an increase in the apparent stabilisation of the immobilised enzyme (see, for example, Figures 3.11, 3.19 and 5.8). Even where there is some inactivation of the immobilised enzyme over a period of time, this inactivation is usually steady and predictable. Any activity decay is easily incorporated into an analytical scheme by regularly interpolating standards between the analyses of unknown samples. For these reasons, many such immobilised enzyme systems are re-usable up to 10,000 times over a period of several months. Clearly, this results in a considerable saving in terms of the enzymes’ cost relative to the analytical usage of free soluble enzymes.