Measurement of enzyme kinetics using a continuous-flow microfluidic system.
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.
Seong
Gi Hun
GH
Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77842-3012, USA.
Heo
Jinseok
J
Crooks
Richard M
RM
D002384:Catalysis; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence; D008863:Microspheres; D010078:Oxazines / Q000378:metabolism
D004800:Enzymes, Immobilized; C105363:N-acetyl-3,7-dihydroxyphenoxazine; D010078:Oxazines; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase