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

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• 作者：Fabrice Gielen ; Liisa van Vliet ; Bartosz T. Koprowski ; Sean R. A. Devenish ; Martin Fischlechner ; Joshua B. Edel ; Xize Niu ; Andrew J. deMello ; Florian Hollfelder
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：May 7, 2013
• 年：2013
• 卷：85
• 期：9
• 页码：4761-4769
• 全文大小：393K
• 年卷期：v.85,no.9(May 7, 2013)
• ISSN：1520-6882

文摘

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鈥揗enten parameters of 4-nitrophenyl glucopyranoside hydrolysis by sweet almond 尾-glucosidase can be generated based on 24 time-courses taken at different substrate concentrations with a total volume consumption of only 1.4 渭L. 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.