TY - JOUR
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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U2 - 10.1039/c9lc01263j
DO - 10.1039/c9lc01263j
M3 - Article
C2 - 31984406
AN - SCOPUS:85079745682
SN - 1473-0197
VL - 20
SP - 852
EP - 861
JO - Lab on a Chip - Miniaturisation for Chemistry and Biology
JF - Lab on a Chip - Miniaturisation for Chemistry and Biology
IS - 4
ER -