Measurement of enzyme kinetics using a continuous-flow microfluidic system
Measurement of enzyme kinetics using a continuous-flow microfluidic system
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DOI：
10.1021/ac034155b
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发表时间：
2003-07-01
影响因子：
7.4
通讯作者：
Crooks, RM
中科院分区：
文献类型：
--
作者：
Seong, GH;Heo, J;Crooks, RM
本文介绍了一种利用连续流动微流控系统测定酶动力学的微量分析方法。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（体积类似于1.0 nL）中，并使底物流过填充床来进行分析。使用Lilly-Hornby方程分析数据，并与基于Michaelis-Menten方程的常规测量值进行比较。选择研究的两种不同的酶催化反应，以便底物是非荧光的，而产物是荧光的。第一个反应涉及辣根过氧化物酶催化的过氧化氢和N-乙酰基-3，7-二羟基吩恶嗪（amplex红）之间的反应，以产生荧光试卤灵，第二个反应涉及非荧光试卤灵-β-D-吡喃半乳糖苷的β-半乳糖苷酶催化的反应，以产生D-半乳糖和荧光试卤灵。两种情况都是。基于微流体的方法产生与标准Michaelis-Menten处理相同的结果。连续流动法需要类似的10穆尔底物溶液和10（9）个酶分子。该方法为微流控系统中酶动力学的快速测定提供了一种新的手段，可能对临床诊断、药物发现和筛选有用。
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 similar to 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 similar to10 muL 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.