Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system
Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system
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DOI：
10.1021/ac800553g
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发表时间：
2008-07-01
影响因子：
7.4
通讯作者：
Kennedy, Robert T.
中科院分区：
文献类型：
--
作者：
Pei, Jian;Dishinger, John F.;Kennedy, Robert T.
研制了一种用于快速电泳酶检测的平行通道微流控芯片。径向排列的通道和一个常见的废物通道允许芯片与16和36个电泳单元制造在7.62 x 7.62厘米(2)玻璃基板上。利用氙气弧光灯和商用电荷耦合器件（CCD）相机实现荧光检测，对各个通道中的迁移分析物区域进行成像。以BODIPY-GTP为酶底物，对芯片上G蛋白GTPase活性进行电泳测定，评价芯片性能。16通道设计被证明是有用的提取动力学信息，允许串行电泳分析从16种不同的酶反应混合物在20秒的间隔平行。该系统用于快速测定酶浓度、最佳酶反应条件和Michaelis-Menten常数。采用36通道芯片筛选G蛋白- rgs蛋白相互作用的调节因子，通过分析含有测试化合物的酶反应混合物中产物的形成量。在30秒内进行了36次电泳分析，表明在适当的样品处理程序下，潜在通量可达4320次/小时。两种设计均表现出良好的峰迁移时间和峰面积重现性。在16通道和36通道设计中，酶产物BODIPY-GDP归一化峰面积的相对标准偏差分别为5%和11%。
A microfluidic chip consisting of parallel channels de-signed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.