HIGHLY SENSITIVE DETECTION OF L-LACTATE AND PYRUVATE BY AMPEROMETRIC FLOW-INJECTION ANALYSIS BASED ON ENZYMATIC SUBSTRATE RECYCLING AND SELECTIVE DETECTION OF HYDROGEN-PEROXIDE

The poly(1,2-diaminobenzene)film-coated platinum electrode was used as an amperometric detector in a flow-injection system with the lactate dehydrogenase/lactate oxidase coimmobilized reactor involving amplific ation by substrate recycling. Both L-lactate and pyruvate are recycled enzymatically during their passage through the enzyme reactor in the presence of reduced nicotinamide adenine dinuceotide (NADH) and oxygen in the carrier stream. As a result, a large amount of hydrogen peroxi de is generated in the enzyme reactor. The poly(1,2-diaminobenzene)-co ated electrode could only selectively detect the hydrogen peroxide gen erated in the enzyme reactor, without an increase in the base-line cur rent owing to the direct oxidation of NADH, because the poly(1,2-diami nobenzene film effectively prevented NADH in the carrier stream from r eaching the electrode surface and allowed only the hydrogen peroxide t o penetrate into the polymerized film. In the present flow-injection a nalysis (FIA) system, both L-lactate and pyruvate were determined with a 400-fold increase in sensitivity compared with the unamplified resp onses. The detection limit was 2 x 10(-9) M (20 fmol) for a 10 mu L sa mple injection.