This report describes a new "lab-on-a-chip" protocol integrating on-line precolumn biocatalytic reactions of multiple (oxidase and dehydrogenase) enzymes and substrates with effective capillary electrophoresis microseparations and amperometric detection. The operation of the new oxidase/dehydrogenase reaction/separation microchip is illustrated for the simultaneous measurement of glucose and ethanol in connection to the corresponding glucose oxidase and alcohol dehydrogenase reactions, respectively. The enzymatic reactions generate hydrogen peroxide and NADH species that are separated (on the basis of their different charges) and detected amperometrically at the end-column thick-film detector. A driving voltage of 2000 V results in peroxide and NADH migration times of 74 and 230 s, respectively. Operating the gold-coated carbon detector at +1.0 V allows simultaneous anodic detection of both reaction products. Factors influencing the reaction, separation, and detection processes are examined and optimized. The applicability of the new multienzyme assay to wine samples is illustrated.
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