A Fully Unsupervised Compartment-on-Demand Platform for Precise Nanoliter Assays of Time-Dependent Steady-State Enzyme Kinetics and Inhibition

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 b...

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Bibliographic Details
Published in:	Analytical chemistry (Washington) Vol. 85; no. 9; pp. 4761 - 4769
Main Authors:	Gielen, Fabrice, van Vliet, Liisa, Koprowski, Bartosz T, Devenish, Sean R. A, Fischlechner, Martin, Edel, Joshua B, Niu, Xize, deMello, Andrew J, Hollfelder, Florian
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 07-05-2013
Subjects:	Analytical chemistry beta-Glucosidase - antagonists & inhibitors beta-Glucosidase - metabolism Enzyme kinetics Kinetics Nanotechnology - instrumentation Particle Size Prunus - enzymology Substrates Time Factors
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Summary:	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-out over time (10 min). Similarly, the inhibition of the enzymatic reaction by conduritol B epoxide and 1-deoxynojirimycin was measured, and K i values were determined. In both cases, the kinetic parameters obtained in droplets were identical within error to values obtained in titer plates, despite a >104-fold volume reduction, from micro- to nanoliters.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0003-2700 1520-6882
DOI:	10.1021/ac400480z