Optical biosensors

There are two main areas of development in 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 (EC 1.11.1.7) and a chromogen (e.g., o-toluidine or 3,3',5,5'-tetramethylbenzidine). The hydrogen peroxide, produced by the aerobic oxidation of glucose (see reaction scheme [1.1]), oxidising the weakly coloured chromogen to a highly coloured dye.

peroxidase
chromogen(2H) + H₂O₂ dye + 2H₂O            [6.24]

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), EC 1.13.12.7) 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)        [6.25]

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.