{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,14]],"date-time":"2026-01-14T19:53:32Z","timestamp":1768420412959,"version":"3.49.0"},"reference-count":40,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2020,5,15]],"date-time":"2020-05-15T00:00:00Z","timestamp":1589500800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>In this paper, a novel electron mediator, 1-methoxy-5-ethyl phenazinium ethyl sulfate (mPES), was introduced as a versatile mediator for disposable enzyme sensor strips, employing representative flavin oxidoreductases, lactate oxidase (LOx), glucose dehydrogenase (GDH), and fructosyl peptide oxidase (FPOx). A disposable lactate enzyme sensor with oxygen insensitive Aerococcus viridans-derived engineered LOx (AvLOx), with A96L mutant as the enzyme, was constructed. The constructed lactate sensor exhibited a high sensitivity (0.73 \u00b1 0.12 \u03bcA\/mM) and wide linear range (0\u201350 mM lactate), showings that mPES functions as an effective mediator for AvLOx. Employing mPES as mediator allowed this amperometric lactate sensor to be operated at a relatively low potential of +0.2 V to 0 V vs. Ag\/AgCl, thus avoiding interference from uric acid and acetaminophen. The lactate sensors were adequately stable for at least 48 days of storage at 25 \u00b0C. These results indicated that mPES can be replaced with 1-methoxy-5-methyl phenazinium methyl sulfate (mPMS), which we previously reported as the best mediator for AvLOx-based lactate sensors. Furthermore, this study revealed that mPES can be used as an effective electron mediator for the enzyme sensors employing representative flavin oxidoreductases, GDH-based glucose sensors, and FPOx-based hemoglobin A1c (HbA1c) sensors.<\/jats:p>","DOI":"10.3390\/s20102825","type":"journal-article","created":{"date-parts":[[2020,5,18]],"date-time":"2020-05-18T02:43:42Z","timestamp":1589769822000},"page":"2825","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Employment of 1-Methoxy-5-Ethyl Phenazinium Ethyl Sulfate as a Stable Electron Mediator in Flavin Oxidoreductases-Based Sensors"],"prefix":"10.3390","volume":"20","author":[{"given":"Maya","family":"Fitriana","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan"},{"name":"Faculty of Biotechnology, Sumbawa University of Technology, Jl. Raya Olat Maras, Batu Alang, Moyo Hulu, Sumbawa Besar 84371, Indonesia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noya","family":"Loew","sequence":"additional","affiliation":[{"name":"Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA"},{"name":"Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arief Budi","family":"Witarto","sequence":"additional","affiliation":[{"name":"Faculty of Biotechnology, Sumbawa University of Technology, Jl. Raya Olat Maras, Batu Alang, Moyo Hulu, Sumbawa Besar 84371, Indonesia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kazunori","family":"Ikebukuro","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Koji","family":"Sode","sequence":"additional","affiliation":[{"name":"Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6299-5267","authenticated-orcid":false,"given":"Wakako","family":"Tsugawa","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,5,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1080\/10408347.2018.1461552","article-title":"Electrochemical Enzyme Biosensors Revisited: Old Solutions for New Problems","volume":"49","author":"Monteiro","year":"2018","journal-title":"Crit. Rev. Anal. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"919","DOI":"10.1016\/0006-291X(89)91546-5","article-title":"Purification and properties of Aerococcus viridans lactate oxidase","volume":"164","author":"Duncan","year":"1989","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1016\/0300-9084(96)88178-8","article-title":"L-lactate oxidase and L-lactate monooxygenase: Mechanistic variations on a common structural theme","volume":"77","author":"Aki","year":"1995","journal-title":"Biochim."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1185","DOI":"10.1107\/S1744309106044678","article-title":"The 2.1 \u00c5 structure of Aerococcus viridans L-lactate oxidase (LOX)","volume":"62","author":"Leiros","year":"2006","journal-title":"Acta Crystallogr. Sect. F Struct. Boil. Cryst. Commun."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/j.bbrc.2006.09.025","article-title":"The crystal structure of L-lactate oxidase from Aerococcus viridans at 2.1 \u00c5 resolution reveals the mechanism of strict substrate recognition","volume":"350","author":"Umena","year":"2006","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1002","DOI":"10.1016\/j.bbrc.2007.05.021","article-title":"Crystallographic study on the interaction of L-lactate oxidase with pyruvate at 1.9 \u00c5 resolution","volume":"358","author":"Li","year":"2007","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1016\/S0003-2670(01)93626-8","article-title":"Microbioassay of phenylalanine in blood sera with a lactate electrode","volume":"119","author":"Karube","year":"1980","journal-title":"Anal. Chim. Acta"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1007\/BF00500746","article-title":"Determination of cell numbers of lactic acid producing bacteria by lactate sensor","volume":"16","author":"Matsunaga","year":"1982","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1097\/00003246-198405000-00011","article-title":"Rapid micromeasurement of lactate on whole blood","volume":"12","author":"Clark","year":"1984","journal-title":"Crit. Care. Med."},{"key":"ref_10","unstructured":"(2020, May 09). Lactate Pro 2. Available online: https:\/\/www.pmda.go.jp\/PmdaSearch\/ivdDetail\/ResultDataSetPDF\/100639_25A2X00001000013_A_01_03."},{"key":"ref_11","unstructured":"(2020, May 09). Nova StatStrip\u00ae Lactate Test Strips-For Use Only with the Nove StatStrip Lactate Hospital Meter. Available online: https:\/\/www.woodleyequipment.com\/docs\/product_insert_lactate_meter_test_strip.pdf."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1002\/prac.199733901109","article-title":"Reagentless Hydrogen Peroxide and L-Lactate Sensors Based on Carbon Paste Electrodes modified with different peroxidases and lactate oxidases","volume":"339","author":"Spohn","year":"1997","journal-title":"J. Prakt. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1016\/S0956-5663(01)00298-6","article-title":"Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine","volume":"17","author":"Moser","year":"2002","journal-title":"Biosens. Bioelectron."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.snb.2011.12.049","article-title":"A contact lens with an integrated lactate sensor","volume":"162","author":"Thomas","year":"2012","journal-title":"Sens. Actuators B Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.electacta.2013.01.080","article-title":"Comparative study of three lactate oxidases from Aerococcus viridans for biosensing applications","volume":"93","author":"Taurino","year":"2013","journal-title":"Electrochim. Acta"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"398","DOI":"10.3390\/bios5030398","article-title":"Characterization of Lactate Sensors Based on Lactate Oxidase and Palladium Benzoporphyrin Immobilized in Hydrogels","volume":"5","author":"Andrus","year":"2015","journal-title":"Biosensors"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1016\/j.bios.2018.08.010","article-title":"Microneedle-based biosensor for minimally-invasive lactate detection","volume":"123","author":"Bollella","year":"2019","journal-title":"Biosens. Bioelectron."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1016\/j.bios.2017.12.018","article-title":"Minimizing the effects of oxygen interference on L-lactate sensors by a single amino acid mutation in Aerococcus viridans L-lactate oxidase","volume":"103","author":"Hiraka","year":"2018","journal-title":"Biosens. Bioelectron."},{"key":"ref_19","first-page":"1703","article-title":"Characterization of electron mediator preference of Aerococcus viridans-derived lactate oxidase for use in disposable enzyme sensor strips","volume":"29","author":"Loew","year":"2017","journal-title":"Sens. Mater."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.bioelechem.2018.04.007","article-title":"Engineered fungus derived FAD-dependent glucose dehydrogenase with acquired ability to utilize hexaammineruthenium(III) as an electron acceptor","volume":"123","author":"Okurita","year":"2018","journal-title":"Bioelectrochemistry"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.jelechem.2012.11.023","article-title":"Electrostatic and steric interaction between redox polymers and some flavoenzymes in mediated bioelectrocatalysis","volume":"689","author":"Nieh","year":"2013","journal-title":"J. Electroanal. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Loew, N., Tsugawa, W., Nagae, D., Kojima, K., and Sode, K. (2017). Mediator Preference of Two Different FAD-Dependent Glucose Dehydrogenases Employed in Disposable Enzyme Glucose Sensors. Sensors, 17.","DOI":"10.3390\/s17112636"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Tsuruoka, N., Sadakane, T., Hayashi, R., and Tsujimura, S. (2017). Bimolecular Rate Constants for FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus and Organic Electron Acceptors. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18030604"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1016\/S0956-5663(01)00313-X","article-title":"Mediated biosensors","volume":"17","author":"Chaubey","year":"2002","journal-title":"Biosens. Bioelectron."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.ica.2014.03.013","article-title":"Electrochemical and chemical aspects of ruthenium(II) and (III) ammines in basic solution: The role of the ruthenium(IV) species","volume":"416","author":"Metzker","year":"2014","journal-title":"Inorg. Chim. Acta"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1016\/0005-2728(73)90001-7","article-title":"Oxidation-reduction potential of the ferro-ferricyanide system in buffer solutions","volume":"292","year":"1973","journal-title":"Biochim. Biophys. Acta (BBA) Bioenerg."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"271","DOI":"10.2116\/analsci.1.271","article-title":"Electrochemical oxidation of nicotinamide-adenine dinucleotide (NADH) by modified pyrolytic graphite electrode","volume":"1","author":"Kimura","year":"1985","journal-title":"Anal. Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1007\/s00775-020-01752-9","article-title":"Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster\u2019s Blue in methanol dehydrogenase assays","volume":"25","author":"Jahn","year":"2020","journal-title":"J. Boil. Inorg. Chem."},{"key":"ref_29","unstructured":"(2020, May 09). Methoxy PES. Available online: https:\/\/www.dojindo.co.jp\/products\/M470\/."},{"key":"ref_30","first-page":"293","article-title":"Synthesis and characterization of 1-substituted 5-alkylphenazine derivatives carrying functional groups","volume":"179","author":"Yomo","year":"1989","journal-title":"JBIC J. Boil. Inorg. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.1007\/s10529-015-1774-8","article-title":"Stabilization of fungi-derived recombinant FAD-dependent glucose dehydrogenase by introducing a disulfide bond","volume":"37","author":"Sakai","year":"2015","journal-title":"Biotechnol. Lett."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1007\/s10529-011-0787-1","article-title":"Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions","volume":"34","author":"Kim","year":"2011","journal-title":"Biotechnol. Lett."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1139\/cjc-2014-0024","article-title":"Electrochemical studies of ascorbic acid, dopamine, and uric acid at a dl-norvaline-deposited glassy carbon electrode","volume":"92","author":"Kamel","year":"2014","journal-title":"Can. J. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1016\/S0731-7085(01)00695-1","article-title":"Electrochemical analysis of acetaminophen using a boron-doped diamond thin film electrode applied to flow injection system","volume":"28","author":"Wangfuengkanagul","year":"2002","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1839","DOI":"10.1016\/0039-9140(95)01638-4","article-title":"Catalytic oxidation of ascorbic acid by some ferrocene derivative mediators at the glassy carbon electrode. Application to the voltammetric resolution of ascorbic acid and dopamine in the same sample","volume":"42","author":"Ojani","year":"1995","journal-title":"Talanta"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"951","DOI":"10.1016\/S0039-9140(97)00201-4","article-title":"Benzoquinone modified electrode for sensing NADH and ascorbic acid","volume":"45","author":"Murthy","year":"1998","journal-title":"Talanta"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1007\/s100080050172","article-title":"Attempt to incorporate ferrocenecarboxylic acid into polypyrrole during the electropolymerization of pyrrole in chloroform: Its application to the electrocatalytic oxidation of ascorbic acid","volume":"3","author":"Ojani","year":"1999","journal-title":"J. Solid State Electrochem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/s100080050004","article-title":"Electrochemistry and electrocatalytic activity of polypyrrole\/ferrocyanide films on a glassy carbon electrode","volume":"4","author":"Ojani","year":"2000","journal-title":"J. Solid State Electrochem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1193","DOI":"10.1002\/elan.200503516","article-title":"Electrocatalytic Determination of Ascorbic Acid at the Surface of 2,7-Bis(ferrocenyl ethyl)fluoren-9-one Modified Carbon Paste Electrode","volume":"18","author":"Raoof","year":"2006","journal-title":"Electroanalysis"},{"key":"ref_40","unstructured":"(2020, May 09). VC Sensor for Pocket Chem VC. Available online: http:\/\/www.arkray-vc.com\/spec\/#."}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/20\/10\/2825\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:29:21Z","timestamp":1760174961000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/20\/10\/2825"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,5,15]]},"references-count":40,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2020,5]]}},"alternative-id":["s20102825"],"URL":"https:\/\/doi.org\/10.3390\/s20102825","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,5,15]]}}}