| Autor: |
Shota Shimokihara, Shunya Shitara, Kei Fujiwara, Ryo Oyobiki, Yoshinori Matsumoto, Nobuhide Doi, Norihisa Miki, Yuki Kanai, Haruna Goto, Yasuaki Einaga, Takeshi Watanabe, Arisa Yotsui |
| Rok vydání: |
2020 |
| Předmět: |
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| Zdroj: |
Lab on a chip. 20(4) |
| ISSN: |
1473-0189 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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