Measurement of Enzyme Kinetics Using a Continuous-Flow Microfluidic System

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Abstract
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 ∼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 β-galactosidase-catalyzed reaction of nonfluorescent resorufin-β-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 ∼10 μL of substrate solution and 109 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.

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