Characterization of Multicomponent Monosaccharide Solutions Using an Enzyme-Based Sensor Array Theodore E. Curey,* Adrian Goodey,* Andrew Tsao,* John Lavigne,* Youngsoo Sohn,² John T. McDevitt,* Eric V. Anslyn,* Dean Neikirk,² and Jason B. Shear* , ‡ ,1 *Department of Chemistry & Biochemistry, ²Department of Computer & Electrical Engineering, and ‡Institute for Cellular & Molecular Biology, University of Texas, Austin, Texas 78712 Received December 1, 2000; published online May 15, 2001 We report the development of a sensor for rapidly and simultaneously measuring multiple sugars in aqueous samples. In this strategy, enzyme-based as- says are localized within an array of individually ad- dressable sites on a micromachined silicon chip. Mi- crospheres derivatized with monosaccharide-specific dehydrogenases are distributed to pyramidal cavities anisotropically etched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping sys- tem. Production of fluorescent reporter molecules is monitored under stopped-flow conditions when local- ized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to quantify b-D-glucose and b-D- galactose at low micromolar to millimolar levels, with no detectable cross-talk between assay sites. Analysis is achieved either through fluorescence detection of an initial dehydrogenase product (NADH, NADPH) or by production of a secondary fluorescent product cre- ated by hydride transfer from the reduced nicotin- amide cofactor to a fluorogenic reagent. The array format of this sensor provides capabilities for redun- dant analysis of sugars and for monitoring levels of other solution components known to affect the activ- ity of enzymes. The use of this strategy to normalize raw fluorescence signals is demonstrated by the deter- mination of glucose and pH on a single chip. Alterna- tively, uncertainties in the activity of an immobilized enzyme can be accounted for using standard addi- tions, an approach used here in the determination of serum glucose. © 2001 Academic Press Routine analysis of complex chemical samples gen- erally requires the use of chromatography or electro- phoresis to fractionate components before detection, procedures that are relatively time consuming and in- strumentally complex. Recently, compact sensors have been developed that rapidly characterize gas-phase mixtures (1–7). In some cases, sensing by these “elec- tronic nose” devices is accomplished through differen- tial adsorption of analytes onto a series of electrically conductive polymers and polymer composite systems, a process that causes specific changes in polymer resis- tivity indicative of volatile components within a mix- ture. Because many analytes can be detected in paral- lel, electronic noses typically can report on environmental conditions within seconds or less. The speed and promise for compact size of these sensors make them useful in applications that require real- time chemical profiling of temporally and spatially het- erogeneous environments. Complex solution-phase samples also can be ana- lyzed using parallel analyte-receptor assays (8 –15), although several challenges exist that are not encoun- tered in gas-phase sensing. Analytes in biological sam- ples—such as viral antigens and mRNA transcripts— commonly are present at low concentrations within matrices containing many chemically similar species. Biological samples also may contain highly diverse components of interest; characterization of blood se- rum, for example, may require determinations of small inorganic ions, hydrophobic triglycerides, and amphi- pathic proteins. Moreover, the efficiency of analyte- receptor binding depends on interactions of both spe- cies with the solvent, a fact that must be considered in selecting receptors and solution parameters (e.g., di- electric constant, pH, ionic strength, solubility). We describe the development of a versatile strategy for rapidly characterizing sugars and small ions in 1 To whom correspondence should be addressed. E-mail: jshear@mail.utexas.edu. 178 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 293, 178 –184 (2001) doi:10.1006/abio.2001.5114, available online at http://www.idealibrary.com on