We report the development of a micro total analysis system (μTAS) based on electrochemical measurements and dielectrophoretic sorting for screening of NAD(P)-dependent oxidoreductases. In this system, the activity of enzymes immobilized on microbeads, together with their encoding DNA, can be measured with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. The 30 nL droplets containing microbead-displayed genes of enzymes with higher activity can then be recovered by dielectrophoretic sorting. Previously, we developed the NAD(P)H-measuring device containing the BDD electrode for high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. In this study, we fabricated an encapsulating device and a droplet-sorting device for nanoliter-size droplets, for the first time, and then combined these three devices to construct a μTAS for directed evolution of NAD(P)-dependent oxidoreductases. We confirmed that this system works by proof-of-principle experiments and successfully applied this system for screening of randomized libraries of NAD-dependent dehydrogenases.

中文翻译：

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基于硼掺杂金刚石电极和介电泳分选的NAD（P）依赖的氧化还原酶的定向进化的微流筛选系统。

我们报告的发展基于电化学测量和介电电泳筛选NAD（P）依赖的氧化还原酶的微量总分析系统（μTAS）。在该系统中，固定在微珠上的酶的活性及其编码DNA可以用微流体系统每个隔室（〜30 nL）中的掺硼金刚石（BDD）电极测量。然后可以通过介电泳分选回收包含微珠展示的具有更高活性的酶基因的30 nL液滴。以前，我们开发了包含BDD电极的NAD（P）H测量设备，用于高通量测量NAD（P）依赖性氧化还原酶的活性。在这项研究中，我们首次制造了用于纳升级液滴的封装设备和液滴分选设备，然后结合这三个装置构建一个μTAS，用于NAD（P）依赖性氧化还原酶的定向进化。我们证实该系统通过原理验证实验工作，并成功地将该系统用于筛选NAD依赖性脱氢酶的随机文库。