A FullyUnsupervised Compartment-on-Demand Platformfor Precise Nanoliter Assays of Time-Dependent Steady-State EnzymeKinetics and Inhibition

摘要

The ability to miniaturize biochemical assays in water-in-oil emulsion droplets allows a massive scale-down of reaction volumes, so that high-throughput experimentation can be performed more economically and more efficiently. Generating such droplets in compartment-on-demand (COD) platforms is the basis for rapid, automated screening of chemical and biological libraries with minimal volume consumption. Herein, we describe the implementation of such a COD platform to perform high precision nanoliter assays. The coupling of a COD platform to a droplet absorbance detection set-up results in a fully automated analytical system. Michaelis–Menten parameters of 4-nitrophenyl glucopyranoside hydrolysis by sweet almond β-glucosidase can be generated based on 24 time-courses taken at different substrate concentrations with a total volume consumption of only 1.4 μL. Importantly, kinetic parameters can be derived in a fully unsupervised manner within 20 min: droplet production (5 min), initial reading of the droplet sequence (5 min), and droplet fusion to initiate the reaction and read-outover time (10 min). Similarly, the inhibition of the enzymatic reactionby conduritol B epoxide and 1-deoxynojirimycin was measured, and Ki values were determined. In both cases, thekinetic parameters obtained in droplets were identical within errorto values obtained in titer plates, despite a >10⁴-foldvolume reduction, from micro- to nanoliters.