title: Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices.
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 (>10(6)) of samples, each with a small (nanoliter) sample volume.
authors.affiliation: Department of Biosciences and Informatics, Keio University , 3-14-1 Hiyoshi, Yokohama 223-8522, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Oyobiki
authors.forename: Taisuke
authors.initials: T
authors.lastname: Kato
authors.forename: Michinobu
authors.initials: M
authors.lastname: Katayama
authors.forename: Ai
authors.initials: A
authors.lastname: Sugitani
authors.forename: Takeshi
authors.initials: T
authors.lastname: Watanabe
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Einaga
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Kenichi
authors.initials: K
authors.lastname: Horisawa
authors.forename: Nobuhide
authors.initials: N
authors.lastname: Doi
mesh_terms: D001895:Boron / Q000737:chemistry*; D018130:Diamond; D057166:High-Throughput Screening Assays; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D008839:Microelectrodes; D044085:Microfluidics / Q000295:instrumentation*; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*
chemical_list: D018130:Diamond; D009247:NADH, NADPH Oxidoreductases; D001895:Boron