A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition.
A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition.
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DOI：
10.1021/ac400480z
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发表时间：
2013-05-07
影响因子：
7.4
通讯作者：
Hollfelder, Florian
中科院分区：
文献类型：
--
作者：
Gielen, Fabrice;van Vliet, Liisa;Koprowski, Bartosz T.;Devenish, Sean R. A.;Fischlechner, Martin;Edel, Joshua B.;Niu, Xize;deMello, Andrew J.;Hollfelder, Florian
在油包水乳液液滴中进行生化测定的能力允许反应体积的大规模缩小，使得可以更经济和更有效地进行高通量实验。在按需隔室（COD）平台中产生这样的液滴是以最小体积消耗快速自动筛选化学和生物文库的基础。在本文中，我们描述了这样的COD平台的实现，以执行高精度纳升测定。COD平台与液滴吸光度检测装置的耦合导致全自动分析系统。甜杏仁β-葡萄糖苷酶水解4-硝基苯基吡喃葡萄糖苷的Michaelis-Menten参数可以基于不同底物浓度下的24个时间过程来获得，总体积消耗仅为1.4 μL。重要的是，可以在20分钟内以完全无人监督的方式导出动力学参数：液滴产生（5分钟），液滴序列的初始阅读（5分钟），以及液滴融合以启动反应并随时间读出（10分钟）。类似地，测量conduritol B环氧化物和1-脱氧野尻霉素对酶反应的抑制，并测定Ki值。在这两种情况下，在液滴中获得的动力学参数在误差范围内与在滴定板中获得的值相同，尽管从微升到纳升体积减少>104倍。
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 >104-fold volume reduction, from micro- to nanoliters.
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影响因子：
6.1
作者：
Churski, Krzysztof;Michalski, Jacek;Garstecki, Piotr
通讯作者：
Garstecki, Piotr
DOI：
10.1007/978-1-62703-354-1_16
发表时间：
2013-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Devenish, Sean R A;Kaltenbach, Miriam;Hollfelder, Florian
通讯作者：
Hollfelder, Florian
影响因子：
16.6
作者：
Ali-Cherif, Anais;Begolo, Stefano;Malaquin, Laurent
通讯作者：
Malaquin, Laurent
影响因子：
7.4
作者：
Bui, Minh-Phuong Ngoc;Li, Cheng Ai;Seong, Gi Hun
通讯作者：
Seong, Gi Hun
影响因子：
--
作者：
Kintses, Balint;Hein, Christopher;Hollfelder, Florian
通讯作者：
Hollfelder, Florian