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A rapid enzyme assay for β-galactosidase using optically gated sample introduction on a microfabricated chip

Author:
Xu, Hongwei, Ewing, Andrew G.
Source:
Analytical and bioanalytical chemistry 2004 v.378 no.7 pp. 1710-1715
ISSN:
1618-2642
Subject:
Escherichia coli, beta-galactosidase, cattle, drugs, enzyme substrates, fluorescein, fluorescence, hydrolysis, lactose, liver, screening
Abstract:
The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction. The hydrolysis of fluorescein mono-β-D-galactopyranoside (FMG) by β-D-galactosidase (β-Gal) is continuously monitored using a microchip for 5 to 10 min. The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein. Kinetic information about β-Gal has been successfully obtained by varying the concentration of FMG. β-Gal enzymes from two different sources including bovine liver and E. coli., have been examined and compared to each other and to results obtained using traditional assay methods. In addition, the competitive inhibition of β-Gal by phenylethyl β-D-thiogalactoside (PETG) and β-lactose has been studied using this technique. PETG is found to have higher inhibition than lactose in the hydrolysis. This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method. Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method. By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays. This technique should therefore be valuable for high-throughput screening in the drug discovery industry.
Agid:
4380757