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Anal Chem. 2013 May 7;85(9):4761-9. doi: 10.1021/ac400480z. Epub 2013 Apr 24.

A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition.

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1
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

Abstract

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 Ki values were determined. In both cases, the kinetic parameters obtained in droplets were identical within error to values obtained in titer plates, despite a >10(4)-fold volume reduction, from micro- to nanoliters.

PMID:
23614771
PMCID:
PMC3715888
DOI:
10.1021/ac400480z
[Indexed for MEDLINE]
Free PMC Article
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