TY - JOUR
T1 - A rapid enzyme assay for β-galactosidase using optically gated sample introduction on a microfabricated chip
AU - Xu, Hongwei
AU - Ewing, Andrew G.
N1 - Funding Information:
Acknowledgments Funding for this research is provided by the National Institute of Heath and National Science Foundation. The authors thank Marc Maxson, at the Huck Institute, Pennsylvania State University, for helpful suggestions, the EMPRL Nanofabri-cation Facility, Pennsylvania State University, for assistance with microchip fabrication, and Dr Anne M. Andrews, Department of Chemistry, for helpful suggestions with the manuscript.
PY - 2004/4
Y1 - 2004/4
N2 - 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.
AB - 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.
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U2 - 10.1007/s00216-003-2317-z
DO - 10.1007/s00216-003-2317-z
M3 - Article
C2 - 14634707
AN - SCOPUS:7644240186
SN - 1618-2642
VL - 378
SP - 1710
EP - 1715
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 7
ER -