Microbiosensors for acetylcholine and glucose.

Microbiosensors based on carbon and and platinum fibers are described. Carbon fibers were used to construct microelectrodes of 7 microm diameter. Electrochemical operations for pre-electrolysis and measuring were examined for the highly sensitive determination of hydrogen peroxide. A triangular potential (-2 to +2V vs Ag/AgCl) was applied before measuring each pair of double pulses (first pulse: 750 mV; second pulse: 1100 mV). The determination limit was 0.1 microM of hydrogen peroxide. The reproducible determination of hydrogen peroxide is possible even in samples containing albumin protein. The separation of hydrogen peroxide from ascorbic acid is also possible because the oxidation potential of ascorbic acid is different from that of hydrogen peroxide. An acetylcholine microsensor was fabricated by immobilizing acetylcholine esterase and choline oxidase on the carbon fiber by entrapment with poly(vinyl alcohol)-quarternized stilbazole (PVA-SbQ). This sensor gave a linear calibration plot for the range 0.1-1.0 mM with a linear correlation coefficient of 0.9842. Glucose oxidase (GOD) and glucose dehydrogenase (GDH) immobilized cylindrical platinum microelectrodes were fabricated, and their characteristics were evaluated, respectively, by using 1,4-benzoquinone (BQ) and ferricyanide as electron mediators. Each enzyme was immobilized by using PVA-SbQ on a cylindrical microelectrode of 2 microm diameter. A linear range in the calibration curve of the GOD-based glucose microsensor was observed to be wider than that obtained using a disk electrode of 1 mm diameter. The mediated response of the 2 microm glucose sensor was compared with the response resulting from hydrogen peroxide detection. This result showed that a higher response and a wider linear range were observed with highly concentrated mediator. A much higher response of the GDH immobilized 2 microm microelectrode was obtained when not only ferricyanide but also diaphorase was employed to reoxidize the NADH produced by the enzyme reaction of GDH. The GHD-based glucose microsensor was found to be unaffected by the concentration of dissolved oxygen.