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There are two main
areas of development in chemical-based optical biosensors. These involve determining changes in
light absorption between the reactants and products of a reaction, or measuring
the light output by a luminescent process. The former usually involve the widely
established, if rather low technology, use of colorimetric test strips. These
are disposable single-use cellulose pads impregnated with enzyme and reagents.
The most common use of this technology is for whole-blood monitoring in diabetes
control. In this case, the strips include glucose oxidase, horseradish
peroxidase and a chromogen (e.g. o-toluidine or 3,3',5,5'-tetramethylbenzidine).
The hydrogen peroxide, produced by the aerobic oxidation of glucose, oxidising
the weakly coloured chromogen to a highly coloured dye.
peroxidase
chromogen(2H) + H2O2
dye + 2H2O
The evaluation of
the dyed strips is best achieved by the use of portable reflectance meters,
although direct visual comparison with a coloured chart is often used. A wide
variety of test strips involving other enzymes are commercially available at the
present time.A most promising biosensor involving luminescence uses firefly
luciferase (Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) to detect the
presence of bacteria in food or clinical samples. Bacteria are specifically
lysed and the ATP released (roughly proportional to the number of bacteria
present) reacted with D-luciferin and oxygen in a reaction which produces yellow
light in high quantum yield.
luciferase
ATP + D-luciferin + O2
oxyluciferin + AMP + pyrophosphate + CO2 + light (562 nm)
The light produced
may be detected photometrically by use of high-voltage, and expensive,
photomultiplier tubes or low-voltage cheap photodiode systems. The sensitivity
of the photomultiplier-containing systems is, at present, somewhat greater (< 104
cells ml-1, < 10-12 M ATP) than the simpler photon
detectors which use photodiodes. Firefly luciferase is a very expensive enzyme,
only obtainable from the tails of wild fireflies. Use of immobilised luciferase
greatly reduces the cost of these analyses. |