Droplet Microfluidics Coupled to Microchip Electrophoresis for High-Throughput Enzyme Modulator Screening.

Microfluidics;Electrophoresis;segmented flow;High-throughput screening;Chemistry;Science;Chemistry

Guetschow, Erik D.Ruotolo, Brandon Thomas ;

University of Michigan

关键词: Microfluidics;    Electrophoresis;    segmented flow;    High-throughput screening;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/120766/edguetsc_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

High-throughput screening (HTS) represents a powerful tool for drug discovery by allowing tens of thousands of assays to be completed within a single day. Typically, assays are performed in multiwell plates (MWPs) utilizing fluorogenic substrates for detection. These substrates increase development time and can introduce artifactual results. Therefore, alternate screening strategies based around natural substrates are necessary.We developed a screening platform that couples nanoliter volume samples to microchip electrophoresis for analysis using a novel polydimethylsiloxane (PDMS)-and-glass microfluidic device. The system was demonstrated by screening a small library against protein kinase A (PKA), which regulates metabolism within the cell. It was chosen for its well-characterized kinetic parameters and commercially available peptide substrates. Sample throughput of 0.16 Hz was achieved allowing at least 6 replicate MCE injections from each sample in a high quality assay (Z’-factor = 0.8).To demonstrate the ability to screen larger libraries, we developed a novel assay for sirtuin 5 (SIRT5) using a naturally derived peptide substrate. SIRT5 impacts metabolism and has reported oncogenic functions making inhibitor identification of clinical importance. Compared to the PKA assay previously developed, assay throughput was increased 3-fold and 1406 samples were analyzed within 46 minutes (0.5 Hz). Using a 250 ms separation, each assay sample could be analyzed 8 times by MCE generating over 11,000 electropherograms. Several previously unreported inhibitors of SIRT5 were identified and verified by dose-response analysis. Finally, work toward miniaturization of high-throughput assays was demonstrated by performing sample preparation and analysis completely within nanoliter volume droplets. A simple to use and easy to fabricate PDMS microfluidic device was developed to allow addition of assay reagents to nanoliter volume samples. Reagent use, relative to assays performed in 384 well plates, could be reduced 1,000-fold and sample-to-sample carryover was less than 5 percent under typical experimental conditions. Analysis of samples prepared in droplet format was demonstrated with droplets containing a fluorescent dye and addition of a fluorescent peptide. These samples could be analyzed by MCE at 0.33 samples per second but increased throughput should be possible by using higher flow rates.