High-throughput screening of enzyme inhibition using an inhibitor gradient generated in a microchannel.
A new rapid microfluidic method for measuring enzyme inhibition is presented. The assay relies upon the creation of a uniform concentration of substrate and a microfluidically generated concentration gradient of inhibitor using a single microchannel and a single initial inhibitor concentration. The IC(50) values of two enzyme inhibitors were determined using the new technique and validated using a conventional microtiter plate assay. Using both experimental and computational simulation techniques, the assay was shown to be sensitive to inhibitor potency and the distribution of inhibitor in the system. The method has the potential to be more accurate than conventional methods because of the comparatively large amount of data that may be collected. Recommendations for use of the assay are provided, including its use for high-throughput screening in drug discovery and general use in measurement of enzyme inhibition.
Garcia
Elena
E
Department of Bioengineering, University of Washington, Box 355061, Foege N530J, 1705 NE Pacific Street, Seattle, WA 98195-2255, USA.
Hasenbank
Melissa S
MS
Finlayson
Bruce
B
Yager
Paul
P
D002626:Chemistry, Pharmaceutical / Q000379:methods; D003198:Computer Simulation; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008832:Microchemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D013678:Technology, Pharmaceutical / Q000379:methods
D004791:Enzyme Inhibitors