High-Throughput Steady-State Enzyme Kinetics Measured in a Parallel Droplet Generation and Absorbance Detection Platform.
High-Throughput Steady-State Enzyme Kinetics Measured in a Parallel Droplet Generation and Absorbance Detection Platform.
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DOI：
10.1021/acs.analchem.2c03164
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发表时间：
2022-12-06
影响因子：
7.4
通讯作者：
Gielen, Fabrice
中科院分区：
文献类型：
--
作者：
Neun, Stefanie;van Vliet, Liisa;Hollfelder, Florian;Gielen, Fabrice
微流体油包水乳液液滴正在成为实验生物学的支柱，它们取代了经典的试管。在大多数应用中，例如超高通量定向进化，液滴含量对于所有区室化测定反应是相同的。然而，当乳液液滴用于动力学或其他功能测定时，需要定义Michaelis-Menten参数的初始速率的浓度依赖性。按需液滴系统满足了这一需求，但提取大量数据具有挑战性。在这里，我们介绍了一个多路复用液滴吸光度检测器，它耦合到半自动液滴生成，形成一个基于管道的液滴按需系统，能够生成和提取定量数据集，从定义的浓度梯度在多个时间点的多个系列的液滴。通过在每次检测后反转流动方向，在多个可调时间点阅读液滴组的吸光度来检测产品的出现，使得液滴多次通过线扫描相机。检测多路复用允许测量12个不同位置的吸光度值，并记录无标记浓度梯度的酶动力学，该浓度梯度由每个约60个液滴组成，覆盖尽可能多的浓度。每小时约8640个数据点的吞吐量，与先前报道的单点检测方法相比，实现了10倍的改进。在单个实验中，确定了12个高分辨率和高准确度Michaelis-Menten动力学的完整数据集，以证明糖苷酶底物的酶表征潜力，其酶促水解范围为7个数量级（kcat/KM）。简单的设置，高通量，优良的数据质量，和宽的动态范围，允许覆盖不同的活动表明，该系统可以作为一个小型化的分光光度计的详细分析出现的克隆从大规模的组合实验。
Microfluidic water-in-oil emulsion droplets are becoming a mainstay of experimental biology, where they replace the classical test tube. In most applications, such as ultrahigh-throughput directed evolution, the droplet content is identical for all compartmentalized assay reactions. When emulsion droplets are used for kinetics or other functional assays, though, concentration dependencies of initial rates that define Michaelis–Menten parameters are required. Droplet-on-demand systems satisfy this need, but extracting large amounts of data is challenging. Here, we introduce a multiplexed droplet absorbance detector, which—coupled to semi-automated droplet generation—forms a tubing-based droplet-on-demand system able to generate and extract quantitative datasets from defined concentration gradients across multiple series of droplets for multiple time points. The emergence of a product is detected by reading the absorbance of the droplet sets at multiple, adjustable time points by reversing the flow direction after each detection, so that the droplets pass a line scan camera multiple times. Detection multiplexing allows absorbance values at 12 distinct positions to be measured, and enzyme kinetics are recorded for label-free concentration gradients that are composed of about 60 droplets each, covering as many concentrations. With a throughput of around 8640 data points per hour, a 10-fold improvement compared to the previously reported single point detection method is achieved. In a single experiment, 12 full datasets of high-resolution and high-accuracy Michaelis–Menten kinetics were determined to demonstrate the potential for enzyme characterization for glycosidase substrates covering a range in enzymatic hydrolysis of 7 orders of magnitude in kcat/KM. The straightforward setup, high throughput, excellent data quality, and wide dynamic range that allows coverage of diverse activities suggest that this system may serve as a miniaturized spectrophotometer for detailed analysis of clones emerging from large-scale combinatorial experiments.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
16.6
作者：
Colin PY;Kintses B;Gielen F;Miton CM;Fischer G;Mohamed MF;Hyvönen M;Morgavi DP;Janssen DB;Hollfelder F
通讯作者：
Hollfelder F
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：
3.8
作者：
Doerr, Mark;Fibinger, Michael P. C.;Bornscheuer, Uwe T.
通讯作者：
Bornscheuer, Uwe T.
影响因子：
6.2
作者：
Horvath, Daniel G.;Braza, Samuel;Abbyad, Paul
通讯作者：
Abbyad, Paul