Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases

Haruna Goto; Yuki Kanai; Arisa Yotsui; Shota Shimokihara; Shunya Shitara; Ryo Oyobiki; Kei Fujiwara; Takeshi Watanabe; Yasuaki Einaga; Yoshinori Matsumoto; Norihisa Miki; Nobuhide Doi

doi:10.1039/C9LC01263J

Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases†

Haruna Goto,^a Yuki Kanai,^a Arisa Yotsui,^a Shota Shimokihara,^a Shunya Shitara,^a Ryo Oyobiki,^a Kei Fujiwara,

^a Takeshi Watanabe,^b Yasuaki Einaga,

^c Yoshinori Matsumoto,^d Norihisa Miki^e and Nobuhide Doi*^a

Author affiliations

* Corresponding authors

^a Department of Biosciences and Informatics, Keio University, Yokohama 223-8522, Japan
E-mail: doi@bio.keio.ac.jp

^b Department of Electrical Engineering and Electronics, Aoyama Gakuin University, Sagamihara 252-5258, Japan

^c Department of Chemistry, Keio University, Yokohama 223-8522, Japan

^d Department of Applied Physics and Physico-Informatics, Keio University, Yokohama 223-8522, Japan

^e Department of Mechanical Engineering, Keio University, Yokohama 223-8522, Japan

Abstract

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.

Lab on a Chip

Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases†

Abstract

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Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases

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