Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors

We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzymeinhibitor systems in a single experiment, for the first time utilizing droplet picoinjectors for bioanalysis. This device enables the scanning of multiple inhibitors, inhibitor concentrations and substrate conditions in a single, highly time resolved experiment yielding the Michaelis constant (Km), the turnover number (Kcat) the mode of inhibition and the inhibitor enzyme binding constants (Ki, Ki). Using this device we determine Km and Kcat for β-galactosidase and the fluorogenic substrate Resorufin β-D-galactopyranoside (RBG) to 252 μM and 477 s-1, respectively. Furthermore, we examine the inhibitory effects of Phenylethyl β-D-thiogalactopyranoside (PETG) on this system.