Chpetr 6 Biosensors The use of enzymes in analysis Enzymes make excellent analytical reagents due to their specificity, selectivity and efficiency. They are often used to determine the concentration of their substrates (as analytes) by means of the resultant initial reaction rates. If the reaction conditions and enzyme concentrations are kept constant, these rates of reaction (v) are proportional to the substrate concentrations ([S]) at low substrate concentrations. When [S] < 0.1 Km, equation 1.8 simplifies to give v = (Vmax/Km)[S] (6.1) The rates of reaction are commonly determined from the difference in optical absorbance between the reactants and products. An example of this is the D-galactose dehydrogenase (EC 1.1.1.48) assay for galactose which involves the oxidation of galactose by the redox coenzyme, nicotine-adenine dinucleotide (NAD+). -D-galactose + NAD+ D-galactono-1,4-lactone + NADH + H+ [6.1] A 0.1 mM solution of NADH has an absorbance at 340nm, in a 1 cm pathlength cuvette, of 0.622, whereas the NAD + from which it is derived has effectively zero absorbance at this wavelength. The conversion (NAD + NADH) is, therefore, accompanied by a large increase in absorption of light at this wavelength. For the reaction to be linear with respect to the galactose concentration, the galactose is kept within a concentration range well below the Km of the enzyme for galactose. In contrast, the NAD + concentration is kept within a concentration range well above the K m of the enzyme for NAD+, in order to avoid limiting the reaction rate. Such assays are commonly used in analytical laboratories and are, indeed, excellent where a wide variety of analyses need to be undertaken on a relatively small number of samples. The drawbacks to this type of analysis become apparent when a large number of repetitive assays need to be performed. Then, they are seen to be costly in terms of expensive enzyme and coenzyme usage, time consuming, labour intensive and in need of skilled and reproducible operation within properly equipped analytical laboratories. For routine or on-site operation, these disadvantages must be overcome. This is being achieved by the production of biosensors which exploit biological systems in association with advances in micro-electronic technology. What are biosensors? A biosensor is an analytical device which converts a biological response into an electrical signal (Figure 6.1). 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: 1. 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 greate