Microchip Device for Performing Enzyme Assays

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Abstract

An automated enzyme assay was performed within a microfabricated channel network. Precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. Reagent dilution and mixing were controlled by regulating the applied potential at the terminus of each channel, using voltages derived from an equivalent circuit model of the microchip. The enzyme beta-galactosidase (beta-Gal) was assayed using resorufin beta-D-galactopyranoside (RBG), a substrate that is hydrolyzed to resorufin, a fluorescent product. Reaction kinetics were obtained by varying the concentration of substrate on-chip and monitoring the production of resorufin using laser-induced fluorescence. Derived Michaelis--Menten constants compared well between an on-chip and a conventional enzyme assay. Bias in the derived K(m) and kcat was primarily due to the limited solubility of RBG and the associated lack of measurements at substrate concentrations exceeding the K(m). A Ki of 8 microM for the inhibitor phenylethyl beta-D-thiogalactoside (PETG) was determined from plots of initial rate versus substrate concentration obtained at three concentrations of PETG. The relative inhibition of beta-Gal by lactose, p-hydroxymercuribenzoic acid, and PETG was determined by varying the inhibitor concentration with constant enzyme and substrate concentration. An enzyme assay performed on the microchip within a 20-min period required only 120 pg of enzyme and 7.5 ng of substrate, reducing the amount of reagent consumed by 4 orders of magnitude over a conventional assay.

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