title: Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators.
abstract: High-throughput screening (HTS) using multiwell plates and fluorescence plate readers is a powerful tool for drug discovery and evaluation by allowing tens of thousands of assays to be completed in 1 day. Although this method has been successful, electrophoresis-based methods for screening are also of interest to avoid difficulties associated fluorescence assays such as requirements to engineer fluorogenic reactions and false positives. We have developed a method using droplet microfluidics to couple multiwell plate-based assays to microchip electrophoresis (MCE) to screen enzyme modulators. Samples contained in multiwell plates are reformatted in to plugs with a sample volume of 8 nL segmented by an immiscible oil. The segmented flow sample streams are coupled to a hybrid polydimethylsiloxane-glass microfluidic device capable of selectively extracting the aqueous samples from the droplet stream and rapidly analyzing by MCE with laser-induced fluorescence detection. This system was demonstrated by screening a test library of 140 compounds against using protein kinase A. For each sample in the screen, two droplets are generated, allowing approximately 6 MCE injections per sample. Using a 1 s separation at 2000 V/cm, we are able to analyze 96 samples in 12 min. Separation resolution between the internal standard, substrate, and product is 1.2 and average separation efficiency is 16,000 plates/s using real samples. Twenty-five compounds were identified as modulators during primary screening and verified using dose-response curves.
authors.affiliation: Department of Chemistry, University of Michigan , 930 N. University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Erik D
authors.initials: ED
authors.lastname: Guetschow
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Steyer
authors.forename: Robert T
authors.initials: RT
authors.lastname: Kennedy
mesh_terms: D001681:Biological Assay / Q000379:methods*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D020536:Enzyme Activators / Q000032:analysis; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000032:analysis; D044085:Microfluidics; D008958:Models, Molecular
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases