Issue 9, 2013

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

Abstract

Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine and industrial bioprocess technology. We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzyme substrate inhibitor systems in a single experiment utilizing an integrated droplet picoinjector for bioanalysis. This device enables the scanning of multiple fluorescently-barcoded inhibitor concentrations and substrate conditions in a single, highly time-resolved experiment yielding the Michaelis constant (Km), the turnover number (kcat) and the enzyme inhibitor dissociation constants (ki, ki′). Using this device we determine Km and kcat for β-galactosidase and the fluorogenic substrate Resorufin β-D-galactopyranoside (RBG) to be 442 μM and 1070 s−1, respectively. Furthermore, we examine the inhibitory effects of isopropyl-β-D-thiogalactopyranoside (IPTG) on β-galactosidase. This system has a number of potential applications, for example it could be used to screen inhibitors to pharmaceutically relevant enzymes and to characterize engineered enzyme variants for biofuels production, in both cases acquiring detailed information about the enzyme catalysis and enzyme inhibitor interaction at high throughput and low cost.

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

Supplementary files

Article information

Article type
Paper
Submitted
20 Dec 2012
Accepted
12 Feb 2013
First published
13 Feb 2013

Lab Chip, 2013,13, 1754-1761

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

S. L. Sjostrom, H. N. Joensson and H. A. Svahn, Lab Chip, 2013, 13, 1754 DOI: 10.1039/C3LC41398E

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