Characterization of multicomponent monosaccharide solutions using an enzyme-based sensor array

We report the development of a sensor for rapidly and simultaneously measur ing multiple sugars in aqueous samples. In this strategy, enzyme-based assa ys are localized within an array of individually addressable sites on a mic romachined silicon chip. Microspheres derivatized with monosaccharide-speci fic dehydrogenases are distributed to pyramidal cavities anisotropically et ched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping system. Pro duction of fluorescent reporter molecules is monitored under stopped-flow c onditions when localized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to q uantify beta -D-glucose and beta -D-galactose at low micromolar to millimol ar levels, with no detectable cross-talk between assay sites. Analysis is a chieved either through fluorescence detection of an initial dehydrogenase p roduct (NADH, NADPH) or by production of a secondary fluorescent product cr eated by hydride transfer from the reduced nicotinamide cofactor to a fluor ogenic reagent. The array format of this sensor provides capabilities for r edundant analysis of sugars and for monitoring levels of other solution com ponents known to affect the activity of enzymes. The use of this strategy t o normalize raw fluorescence signals is demonstrated by the determination o f glucose and pH on a single chip. Alternatively, uncertainties in the acti vity of an immobilized enzyme can be accounted for using standard additions , an approach used here in the determination of serum glucose. (C) 2001 Aca demic Press.