Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices

Ryo Oyobiki, Taisuke Kato, Michinobu Katayama, Ai Sugitani, Takeshi Watanabe, Yasuaki Einaga, Yoshinori Matsumoto, Kenichi Horisawa, Nobuhide Doi

研究成果: Article

17 引用 (Scopus)

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Although oxidoreductases are widely used in many applications, such as biosensors and biofuel cells, improvements in the function of existing oxidoreductases or the discovery of novel oxidoreductases with greater activities is desired. To increase the activity of oxidoreductases by directed evolution, a powerful screening technique for oxidoreductases is required. In this study, we demonstrate the utility of boron-doped diamond (BDD) microelectrodes for quantitative and potentially high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. We first confirmed that BDD microelectrodes can quantify the activity of low concentrations (10-100 pM) of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase with a measuring time of 1 ms per sample. In addition, we found that poisoning of BDD microelectrodes can be repressed by optimizing the pH and by adding l-arginine to the enzyme solution as an antiaggregation agent. Finally, we fabricated a microfluidic device containing a BDD electrode for the first time and observed the elevation of the oxidation current of NADH with increasing flow rate. These results imply that the combination of a BDD microelectrode and microfluidics can be used for high-throughput screening of an oxidoreductase library containing a large number (>106) of samples, each with a small (nanoliter) sample volume.

元の言語English
ページ(範囲)9570-9575
ページ数6
ジャーナルAnalytical chemistry
86
発行部数19
DOI
出版物ステータスPublished - 2014 10 7

ASJC Scopus subject areas

  • Analytical Chemistry

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