Design and characterization of immobilized enzymes in microfluidic systems.
Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.
Mao
Hanbin
H
Department of Chemistry, Texas A&M University, College Station 77842-3012, USA.
Yang
Tinglu
T
Cremer
Paul S
PS
D001426:Bacterial Proteins; D015374:Biosensing Techniques / Q000379:methods*; D004800:Enzymes, Immobilized; D004867:Equipment Design; D005947:Glucose / Q000032:analysis; D005949:Glucose Oxidase / Q000378:metabolism; D006735:Horseradish Peroxidase; D008051:Lipid Bilayers; D008832:Microchemistry
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D008051:Lipid Bilayers; C058865:Strep-avidin conjugated horseradish peroxidase; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D005947:Glucose