Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators.
Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators.
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DOI：
10.1021/ac502758h
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发表时间：
2014-10-21
影响因子：
7.4
通讯作者：
Kennedy, Robert T.
中科院分区：
文献类型：
--
作者：
Guetschow, Erik D.;Steyer, Daniel J.;Kennedy, Robert T.
使用多孔板和荧光板读取器的高通量筛选 (HTS) 是药物发现和评估的强大工具，可在 1 天内完成数以万计的测定。尽管这种方法已经成功，但基于电泳的筛选方法也令人感兴趣，以避免与荧光测定相关的困难，例如工程荧光反应和假阳性的要求。我们开发了一种使用液滴微流体将基于多孔板的检测与微芯片电泳 (MCE) 相结合来筛选酶调节剂的方法。将多孔板中包含的样品重新格式化为样品体积为 8 nL 的塞子，并用不混溶的油进行分段。分段流动样品流与混合聚二甲基硅氧烷-玻璃微流体装置耦合，该装置能够选择性地从液滴流中提取水样，并通过 MCE 和激光诱导荧光检测进行快速分析。该系统通过使用蛋白激酶 A 筛选包含 140 种化合物的测试库进行了演示。对于筛选中的每个样品，都会生成两个液滴，每个样品允许进样大约 6 次 MCE。使用 2000 V/cm 下的 1 秒分离，我们能够在 12 分钟内分析 96 个样品。使用实际样品，内标、底物和产物之间的分离分辨率为 1.2，平均分离效率为 16 000 板/秒。在初步筛选过程中，二十五种化合物被确定为调节剂，并使用剂量反应曲线进行验证。
High-throughput screening (HTS) using multiwell plates and fluorescence plate readers is a powerful tool for drug discovery and evaluation by allowing tens of thousands of assays to be completed in 1 day. Although this method has been successful, electrophoresis-based methods for screening are also of interest to avoid difficulties associated fluorescence assays such as requirements to engineer fluorogenic reactions and false positives. We have developed a method using droplet microfluidics to couple multiwell plate-based assays to microchip electrophoresis (MCE) to screen enzyme modulators. Samples contained in multiwell plates are reformatted in to plugs with a sample volume of 8 nL segmented by an immiscible oil. The segmented flow sample streams are coupled to a hybrid polydimethylsiloxane–glass microfluidic device capable of selectively extracting the aqueous samples from the droplet stream and rapidly analyzing by MCE with laser-induced fluorescence detection. This system was demonstrated by screening a test library of 140 compounds against using protein kinase A. For each sample in the screen, two droplets are generated, allowing approximately 6 MCE injections per sample. Using a 1 s separation at 2000 V/cm, we are able to analyze 96 samples in 12 min. Separation resolution between the internal standard, substrate, and product is 1.2 and average separation efficiency is 16 000 plates/s using real samples. Twenty-five compounds were identified as modulators during primary screening and verified using dose–response curves.
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影响因子：
7.4
作者：
Roman, Gregory T.;Wang, Meng;Shultz, Kristin N.;Jennings, Colin;Kennedy, Robert T.
通讯作者：
Kennedy, Robert T.
影响因子：
7.4
作者：
Sun, Shuwen;Slaney, Thomas R.;Kennedy, Robert T.
通讯作者：
Kennedy, Robert T.
影响因子：
7.4
作者：
Slaney, Thomas R.;Nie, Jing;Hershey, Neil D.;Thwar, Prasanna K.;Linderman, Jennifer;Burns, Mark A.;Kennedy, Robert T.
通讯作者：
Kennedy, Robert T.
影响因子：
7.4
作者：
Pei, Jian;Nie, Jing;Kennedy, Robert T.
通讯作者：
Kennedy, Robert T.
影响因子：
7.4
作者：
Roper, MG;Shackman, JG;Kennedy, RT
通讯作者：
Kennedy, RT