Seong Gi Hun, Heo Jinseok, Crooks Richard M
Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77842-3012, USA.
Anal Chem. 2003 Jul 1;75(13):3161-7. doi: 10.1021/ac034155b.
This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume approximately 1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the Lilly-Hornby equation and compared to values obtained from conventional measurements based on the Michaelis-Menten equation. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the beta-galactosidase-catalyzed reaction of nonfluorescent resorufin-beta-D-galactopyranoside to yield D-galactose and fluorescent resorufin. In both cases, the microfluidics-based method yielded the same result obtained from the standard Michaelis-Menten treatment. The continuous-flow method required approximately 10 microL of substrate solution and 10(9) enzyme molecules. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.
本文描述了一种使用连续流动微流控系统测定酶动力学的微分析方法。该分析通过将酶固定在微珠上，将微珠填充到基于芯片的微反应器（体积约为1.0 nL）中，并使底物流过填充床来进行。使用Lilly-Hornby方程分析数据，并与基于米氏方程的传统测量获得的值进行比较。选择研究的两种不同的酶催化反应，使底物为非荧光性而产物为荧光性。第一个反应涉及辣根过氧化物酶催化的过氧化氢与N-乙酰基-3,7-二羟基吩恶嗪（安普列克斯红）之间的反应，生成荧光试卤灵，第二个反应是β-半乳糖苷酶催化的非荧光试卤灵-β-D-吡喃半乳糖苷反应，生成D-半乳糖和荧光试卤灵。在这两种情况下，基于微流控的方法都得到了与标准米氏处理相同的结果。连续流动法需要大约10 μL的底物溶液和10⁹个酶分子。这种方法为在微流控系统中快速测定酶动力学提供了一种新手段，这可能对临床诊断以及药物发现和筛选有用。
