title: Measurement of enzyme kinetics using a continuous-flow microfluidic system.
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 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.
authors.affiliation: Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77842-3012, USA.
authors.forename: Gi Hun
authors.initials: GH
authors.lastname: Seong
authors.forename: Jinseok
authors.initials: J
authors.lastname: Heo
authors.forename: Richard M
authors.initials: RM
authors.lastname: Crooks
mesh_terms: 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
chemical_list: D004800:Enzymes, Immobilized; C105363:N-acetyl-3,7-dihydroxyphenoxazine; D010078:Oxazines; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase