Burke Brian J, Regnier Fred E
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Anal Chem. 2003 Apr 15;75(8):1786-91. doi: 10.1021/ac026173j.
This paper describes a microfabricated enzyme assay system including a micromixer that can be used to perform stopped-flow reactions. Samples and reagents were transported into the system by electroosmotic flow (EOF). Streams of reagents were merged and passed through the 100-pL micromixer in < 1 s. The objective of the work was to perform kinetically based enzyme assays in the stopped-flow mode using a system of roughly 6 nL volume. Beta-galactosidase (beta-Gal) was chosen as a model enzyme for these studies and was used to convert the substrate fluorescein mono-beta-D-galactopyranoside (FMG) into fluorescein. Results obtained with microfabricated systems using the micromixer compared well to those obtained with an external T mixing device. In contrast, assays performed in a microfabricated device by merging two streams and allowing mixing to occur by lateral diffusion did not compare well. Using the microfabricated mixer, Km and kcat values of 75 +/- 13 microM and 44 +/- 3 s(-1) were determined. These values compare well to those obtained with the conventional stopped-flow apparatus for which Km was determined to be 60 +/- 6 microM and kcat was 47 +/- 4 s(-1). Enzyme inhibition assays with phenylethyl-beta-D-thiogalactoside (PETG) were also comparable. It was concluded that kinetically based, stopped-flow enzyme assays can be performed in 60 s or less with a miniaturized system of roughly 6 nL liquid volume when mixing is assisted with the described device.
本文描述了一种微制造酶分析系统，该系统包括一个可用于进行停流反应的微混合器。样品和试剂通过电渗流（EOF）输送到系统中。试剂流在不到1秒的时间内合并并通过100皮升的微混合器。这项工作的目标是使用一个体积约为6纳升的系统，以停流模式进行基于动力学的酶分析。选择β-半乳糖苷酶（β-Gal）作为这些研究的模型酶，并用于将底物荧光素单-β-D-吡喃半乳糖苷（FMG）转化为荧光素。使用微混合器的微制造系统获得的结果与使用外部T形混合装置获得的结果相当。相比之下，在微制造装置中通过合并两股流并通过横向扩散进行混合所进行的分析结果则不太理想。使用微制造混合器，测定的Km和kcat值分别为75±13微摩尔和44±3秒-1。这些值与使用传统停流装置获得的值相当，传统停流装置测定的Km为60±6微摩尔，kcat为47±4秒-1。用苯乙基-β-D-硫代半乳糖苷（PETG）进行的酶抑制分析结果也具有可比性。得出的结论是，当使用所述装置辅助混合时，基于动力学的停流酶分析可以在60秒或更短时间内用一个约6纳升液体体积的小型化系统进行。
