Nanoliter Quantitative High-Throughput Screening with Large-Scale Tunable Gradients Based on a Microfluidic Droplet Robot under Unilateral Dispersion Mode.

Performing quantitative high throughput screening (qHTS) is in urgent need in current chemical, biological, and medical research. In this work, we developed an automated microfluidic dilution and large-scale screening system in the nanoliter range, by combining the droplet-based microfluidic robot technique with a novel unilateral Taylor-Aris dispersion-based dilution approach. The unilateral dispersion approach utilizes multiphase microfluidic design to generate a concentration gradient with fast gradient generation time, low sample/reagent consumption, and high operation efficiency over the widely used bilateral Taylor-Aris dispersion approach adopted in previous dilution systems. The present system is capable of automatically generating a large and tunable range of concentration gradients covering ca. 6 orders of magnitude in droplet arrays and achieving qHTS of a large number of different samples. We applied the microfluidic droplet system in miniaturized enzyme kinetic assay in 8-nL droplets and high-throughput quantitative screening of enzyme inhibitors with a library of 102 compounds. Only 9.8 μL of enzyme solution was consumed in 2448 droplet assays containing 102 compounds and 24 concentrations, representing an approximate 1600-fold reduction compared with multiwell plate-based assays. In the screening, dose-response curves of each tested compound were established and 4 hits (CP-471474, ilomastat, batimastat, and marimastat) were screened to have inhibitory activity to matrix metallopeptidase-9 (MMP-9), which demonstrated that the present system has the potential to provide a miniaturized qHTS platform for drug discovery.