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

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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.

Original language	English
Pages (from-to)	852-861
Number of pages	10
Journal	Lab on a Chip
Volume	20
Issue number	4
DOIs	https://doi.org/10.1039/c9lc01263j
Publication status	Published - 2020 Feb 21

ASJC Scopus subject areas

Bioengineering
Biochemistry
Chemistry(all)
Biomedical Engineering

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Goto, H., Kanai, Y., Yotsui, A., Shimokihara, S., Shitara, S., Oyobiki, R., Fujiwara, K., Watanabe, T., Einaga, Y., Matsumoto, Y., Miki, N., & Doi, N. (2020). Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases. Lab on a Chip, 20(4), 852-861. https://doi.org/10.1039/c9lc01263j

Goto, H, Kanai, Y, Yotsui, A, Shimokihara, S, Shitara, S, Oyobiki, R, Fujiwara, K, Watanabe, T, Einaga, Y , Matsumoto, Y , Miki, N & Doi, N 2020, 'Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases', Lab on a Chip, vol. 20, no. 4, pp. 852-861. https://doi.org/10.1039/c9lc01263j

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

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.",

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

note = "Funding Information: We thank the current and past members of our laboratories, especially Kai Asai, Kunihiko Wakamatsu and Kenichi Horisawa for experimental advice and useful discussion. This work was supported by a Grant-in-Aid for Scientific Research (24656508) from the JSPS of Japan, a Strategic Research Foundation Grant-aided Project for Private Universities (S1411003) from the MEXT of Japan, a Keio Gijuku Academic Development Funds, and the Asahi Glass Foundation. Publisher Copyright: This journal is {\textcopyright} The Royal Society of Chemistry.",

year = "2020",

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doi = "10.1039/c9lc01263j",

language = "English",

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T1 - Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases

AU - Goto, Haruna

AU - Kanai, Yuki

AU - Yotsui, Arisa

AU - Shimokihara, Shota

AU - Shitara, Shunya

AU - Oyobiki, Ryo

AU - Fujiwara, Kei

AU - Watanabe, Takeshi

AU - Einaga, Yasuaki

AU - Matsumoto, Yoshinori

AU - Miki, Norihisa

AU - Doi, Nobuhide

N1 - Funding Information: We thank the current and past members of our laboratories, especially Kai Asai, Kunihiko Wakamatsu and Kenichi Horisawa for experimental advice and useful discussion. This work was supported by a Grant-in-Aid for Scientific Research (24656508) from the JSPS of Japan, a Strategic Research Foundation Grant-aided Project for Private Universities (S1411003) from the MEXT of Japan, a Keio Gijuku Academic Development Funds, and the Asahi Glass Foundation. Publisher Copyright: This journal is © The Royal Society of Chemistry.

PY - 2020/2/21

Y1 - 2020/2/21

N2 - 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.

AB - 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.

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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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