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.

Ämnesord

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

Nyckelord

Droplet microfluidics

Enzyme inhibitors

Enzyme kinetics

High throughput

Drops

Experiments

Kinetics

Microfluidics

Reaction kinetics

Fluorogenic substrate

Michaelis constants

Multiplex analysis

Substrate conditions

Time resolved experiments

Enzyme inhibition

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