Abstract
Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the measurements can be carried out in the absence of a free redox mediator in the solution without the impact of oxygen and at a low enough potential for amperometric measurement to avoid the effect of electrochemically active interferences. However, natural GOx is not capable of DET. Therefore, a simple and rapid strategy to create DET-capable GOx is desired. In this study, we designed engineered GOx, which was made readily available for single-step modification with a redox mediator (phenazine ethosulfate, PES) on its surface via a lysine residue rationally introduced into the enzyme. Thus, PES-modified engineered GOx showed a quasi-DET response upon the addition of glucose. This strategy and the obtained results will contribute to the further development of quasi-DET GOx-based glucose monitoring dedicated to precise and accurate glycemic control for diabetic patient care.
Highlights
Glucose oxidase (β-d-glucose: oxygen 1-oxidoreductases, E.C. 1.1.3.4, GOx) is a flavoprotein that catalyzes the oxidation of β-d-glucose at its hydroxyl group linked to the carbon one using molecular oxygen as the electron acceptor to produce d-glucono-1,5-lactone and hydrogen peroxide (Scheme 1)
AnGOx-WT was modified with PES, and the dye-mediated dehydrogenase activity was determined with an electron mediator, phenazine methosulfate (PMS), and a terminal electron acceptor, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
Considering that both PMS and MTT are negatively charged in their oxidative status, we assumed that the low reactivity of MTT as the primary electron acceptor might be attributed to its bulkiness when compared with PMS
Summary
Glucose oxidase (β-d-glucose: oxygen 1-oxidoreductases, E.C. 1.1.3.4, GOx) is a flavoprotein that catalyzes the oxidation of β-d-glucose at its hydroxyl group linked to the carbon one using molecular oxygen as the electron acceptor to produce d-glucono-1,5-lactone and hydrogen peroxide (Scheme 1). Glucose sensor employing DET-type enzymes has several advantages comparing mediator type glucose sensors, e.g., a smaller number of essential elements for sensing, a smaller number of essential reactions for detection, operation at a lower applying potential than those utilizing hydrogen peroxide, and a major kind of mediator used in second-generation principle such as ferrocene, ferricyanide, and ruthenium complexes [8,9]. This low-applying potential contributes to eliminate interference by electrochemically active ingredients in the samples. The DET-based enzyme sensor principle has been considered ideal for redox enzyme-based electrochemical sensors
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