Employment of 1-Methoxy-5-Ethyl Phenazinium Ethyl Sulfate as a Stable Electron Mediator in Flavin Oxidoreductases-Based Sensors.

Koji Sode,Wakako Tsugawa,Noya Loew,Arief Budi Witarto,Maya Fitriana,Kazunori Ikebukuro

doi:10.3390/s20102825

Abstract

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 ± 0.12 μA/mM) and wide linear range (0–50 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 °C. 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.

Highlights

Electrochemical enzyme sensors are the most widely studied form of biosensors due to their simple construction and achievable adequate performance
We reported the investigation of electron mediators for enzyme sensors employing Aerococcus viridans-derived engineered LOx (AvLOx), where we concluded that methyl phenazinium methyl sulfate (mPMS) was the best [19]
We have been investigating an alternate mediator for lactate oxidase (LOx)-based enzyme sensors, and we came across the use of methoxy-5-ethyl phenazinium ethyl sulfate (mPES), which is the derivative of mPMS, but is more chemically stable than mPMS

Summary

Introduction

Electrochemical enzyme sensors are the most widely studied form of biosensors due to their simple construction and achievable adequate performance. In electrochemical enzyme sensors, the substrate (or analyte) is oxidized by a redox enzyme, which results in a product and a reduced cofactor of the enzyme in the reductive half reaction; in other words, electrons are transferred from the substrate to the cofactor of the enzyme [1]. Sensors 2020, 20, 2825 the flavoenzyme lactate oxidase (LOx) harboring flavin mononucleotide (FMN) as a cofactor [2,3,4,5,6]. This results in pyruvate and reduced flavin. In second-generation electrochemical biosensors, this problem is solved by utilizing an artificial electron mediator to transfer electrons from the reduced cofactor of the enzyme to the electrode. Especially in disposable strip-type enzyme sensors, because they allow a lower application potential than what is necessary to oxidize H2 O2 , which leads to fewer errors due to redox interference

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Discussion

Conclusion