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

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)9570-9575
Number of pages6
JournalAnalytical chemistry
Volume86
Issue number19
DOIs
Publication statusPublished - Oct 7 2014

Fingerprint

Diamond
Boron
Microelectrodes
Microfluidics
NAD
Screening
Oxidoreductases
Throughput
Biological fuel cells
Alcohol Dehydrogenase
Glucosephosphate Dehydrogenase
Biosensors
Arginine
Flow rate
Oxidation
Electrodes
Enzymes

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices. / Oyobiki, Ryo; Kato, Taisuke; Katayama, Michinobu; Sugitani, Ai; Watanabe, Takeshi; Einaga, Yasuaki; Matsumoto, Yoshinori; Horisawa, Kenichi; Doi, Nobuhide.

In: Analytical chemistry, Vol. 86, No. 19, 07.10.2014, p. 9570-9575.

Research output: Contribution to journalArticle

Oyobiki, R, Kato, T, Katayama, M, Sugitani, A, Watanabe, T, Einaga, Y, Matsumoto, Y, Horisawa, K & Doi, N 2014, 'Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices', Analytical chemistry, vol. 86, no. 19, pp. 9570-9575. https://doi.org/10.1021/ac501907x
Oyobiki, Ryo ; Kato, Taisuke ; Katayama, Michinobu ; Sugitani, Ai ; Watanabe, Takeshi ; Einaga, Yasuaki ; Matsumoto, Yoshinori ; Horisawa, Kenichi ; Doi, Nobuhide. / Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices. In: Analytical chemistry. 2014 ; Vol. 86, No. 19. pp. 9570-9575.
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