Continuous glucose monitoring (CGM) systems are most important in the current Type I diabetes care and as component for the development of artificial pancreas systems because the amount of insulin ...being supplied is calculated based on the CGM results. Therefore, to stably and accurately control the blood glucose level, CGM should be stable and accurate for a long period. We have been engaged in the biomolecular engineering and application of FAD dependent glucose dehydrogenase complex (FADGDH) which is capable of direct electron transfer. In this study, we report the development of the third-generation type open circuit potential (OCP) principle-based glucose sensor with direct electron transfer FADGDH immobilized on gold electrodes using a self-assembled monolayer (SAM). We developed a novel algorithm for OCP-based glucose sensors. By employing this new algorithm, high reproducibility of measurement and sensor preparation were achieved. In addition, the signal was not affected by the presence of acetaminophen and ascorbic acid in the sample solution. The thus optimized third-generation OCP-based glucose sensor could be operated continuously for more than 9 days without significant change in the signal, sensitivity and dynamic range, indicating its potential application for CGM systems.
•The third-generation type open circuit potential principle-based glucose sensor was developed.•A novel OCP measurement protocol, which discharge charged electron before measurement, was proposed.•This sensor showed high reproducibility in the measurement and electrode preparation.•This sensor signal was not affected by the presence of 340 µM ascorbic acid or 2.6 mM of acetaminophen in the sample solution.•This sensor could measure the glucose concentration stably and continuously during 9 days.
•Interpretation of EIS with a double semicircle and a straight line.•Simultaneous, but different diffusion types depending on position on electrode.•Semi-infinite or finite length diffusion can be ...present in enzyme electrodes.•Explains the relationship between reaction plane location and EIS characteristics.
Herein, a different interpretation of electrochemical impedance spectra with a double semicircle and a straight line obtained for systems involving diffusion processes is proposed. Simulations of electrochemical impedance spectra of mediator-type enzyme electrodes by using the finite element method revealed that under certain conditions, diffusion can be a combination of apparent semi-infinite and finite length, with the diffusion type differing depending on the position on the electrode or the diffusion direction. The Nyquist plot of a system with one charge transfer and the mixed-type diffusion shows a semicircle for the charge transfer, a second semicircle for finite length diffusion (short Warburg impedance), and a straight line for semi-infinite diffusion (Warburg impedance). In mediator-type enzyme electrodes with a free-diffusing enzyme and a mediator, such mixed-type diffusion can be observed at medium substrate concentrations when the reaction plane is close to the electrode at the edges and farther away in the middle of the electrode. These simulation results may help (bio-)electrochemists to more accurately interpret impedance spectra and gain better understanding of the phenomena occurring at mediator-type enzyme electrodes and other cases involving diffusion.
Porous carbon electrodes have considerably improved the performance of biofuel cells and biosensors in recent years. In this paper, we propose a novel in-situ analysis method for porous enzyme ...electrodes. By combining three-dimensional (3D) impedance measurement and a double-channel transmission line model, the stability of porous enzyme electrodes during operation can be evaluated. The proposed method can distinguish between the functional stability of the enzyme and mediator reaction and the general structural stability of the electrode. We demonstrated this method by evaluating bilirubin oxidase-modified carbon cloth (CC) electrodes with and without a magnesium oxide (MgO)-templated carbon coating. In case of the CC electrode, a remarkable increase in the charge transfer resistance within the first 500 s indicated the elution of the enzyme and mediator. When the CC was coated with MgO-templated carbon before enzyme modification, the charge transfer resistance remained constant, indicating an effective suppression of the elution of the enzyme and mediator. The electric double-layer capacitance values of both electrodes indicated that their general electrode structures were stable during the analysis. Thus, the proposed analytical method, based on 3D impedance, can be a powerful tool for simultaneously detecting possible changes in the general electrode structure of enzyme electrodes and in the amount of active enzymes and mediators on the electrode surface.
•Three-dimensional impedance method was proposed for enzyme electrode analysis.•Bilirubin oxidase-modified porous carbon electrodes was analyzed.•A double channel transmission line model was combined with the 3D impedance method.•Elution of enzyme and mediator could be estimated from charge transfer resistance.•Electrode structure could be estimated by electric double layer capacitance.
Most commercially available electrochemical enzyme sensor strips for the measurement of blood glucose use an artificial electron mediator to transfer electrons from the active side of the enzyme to ...the electrode. One mediator recently gaining attention for commercial sensor strips is hexaammineruthenium(III) chloride. In this study, we investigate and compare the preference of enzyme electrodes with two different FAD-dependent glucose dehydrogenases (FADGDHs) for the mediators hexaammineruthenium(III) chloride, potassium ferricyanide (the most common mediator in commercial sensor strips), and methoxy phenazine methosulfate (mPMS). One FADGDH is a monomeric fungal enzyme, and the other a hetero-trimeric bacterial enzyme. With the latter, which contains a heme-subunit facilitating the electron transfer, similar response currents are obtained with hexaammineruthenium(III), ferricyanide, and mPMS (6.8 µA, 7.5 µA, and 6.4 µA, respectively, for 10 mM glucose). With the fungal FADGDH, similar response currents are obtained with the negatively charged ferricyanide and the uncharged mPMS (5.9 µA and 6.7 µA, respectively, for 10 mM glucose), however, no response current is obtained with hexaammineruthenium(III), which has a strong positive charge. These results show that access of even very small mediators with strong charges to a buried active center can be almost completely blocked by the protein.
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.
Abstract
In this study, the performance of a paper-based, screen-printed biofuel cell with mesoporous MgO-templated carbon (MgOC) electrodes was improved in two steps. First, a small amount of ...carboxymethyl cellulose (CMC) was added to the MgOC ink. Next, the cathode was modified with bilirubin prior to immobilizing the bilirubin oxidase (BOD). The CMC increased the accessibility of the mesopores of the MgOC, and subsequently, the performance of both the bioanode and biocathode. CMC also likely increased the stability of the electrodes. The pre-modification with bilirubin improved the orientation of the BOD, which facilitated direct electron transfer. With these two steps, an open circuit potential of 0.65 V, a maximal current density of 1.94 mA cm
−2
, and a maximal power density of 465 μW cm
−2
was achieved with lactate oxidase as bioanode enzyme and lactate as fuel. This is one of the highest reported performances for a biofuel cell.
Faradaic electrochemical impedance spectroscopy (faradaic EIS) is an attractive measurement principle for biosensors. However, there have been no reports on sensors employing direct electron transfer ...(DET)-type redox enzymes based on faradaic EIS principle. In this study, we have attempted to construct the 3rd-generation faradaic enzyme EIS sensor, which used DET-type flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase (GDH) complex, to elucidate its characteristic properties as well as to investigate its potential application as the future immunosensor platform. The gold disk electrodes (GDEs) with DET-type FADGDH prepared using self-assembled monolayer (SAM) showed the glucose concentration dependent impedance change, which was confirmed by the change in the charge transfer resistance (Rct). The Δ(1/Rct) values were also affected by DC bias potential and the length of SAM. Based on the Nyquist plot and Bode plot simulations, glucose sensing by imaginary impedance monitoring under fixed frequency (5 mHz) was carried out, revealing the higher sensitivity at low glucose concentration with wider linear range (0.02–0.2 mM). Considering this high sensitivity toward glucose, the 3rd-generation faradaic enzyme EIS sensor would provide alternative platform for future impedimetric immunosensing system, which does not use redox probe.
•First impedimetric sensor with direct electron transfer type glucose dehydrogenase.•First impedimetric enzyme sensor employing direct electron transfer principle.•Glucose-dependent impedance change with direct electron transfer principle.•Successful measurement of faradaic impedance without redox probe.•Suitable for measurement of very low concentrations of glucose.
Simulations enable fundamental electrochemistry to be more deeply understood and are useful tools for the further development of electrochemical processes. However, important parameter restrictions ...have thus far limited the applicability of electrochemical simulations. In this study, we developed a realistic model of mediator-type enzyme electrodes to accurately simulate cyclic voltammetry and electrochemical impedance spectroscopy by considering the current arising from electrochemical double-layer charging. Implementation of the finite element method in these calculations resulted in a model with very few boundary conditions. The model was successfully tested for various enzyme and electrode reaction kinetics and subsequently adapted to describe immobilized enzymes and mediators. The insight gained through this study has implications for the further development of biosensor and biofuel cell technologies.
Glycated albumin (GA) is an important glycemic control marker for diabetes mellitus. This study aimed to develop a highly sensitive disposable enzyme sensor strip for GA measurement by using an ...interdigitated electrode (IDE) as an electrode platform. The superior characteristics of IDE were demonstrated using one microelectrode of the IDE pair as the working electrode (WE) and the other as the counter electrode, and by measuring ferrocyanide/ferricyanide redox couple. The oxidation current was immediately reached at the steady state when the oxidation potential was applied to the WE. Then, an IDE enzyme sensor strip for GA measurement was prepared. The measurement of fructosyl lysine, the protease digestion product of GA, exhibited a high, steady current immediately after potential application, revealing the highly reproducible measurement. The sensitivity (2.8 nA µM
) and the limit of detection (1.2 µM) obtained with IDE enzyme sensor strip were superior compared with our previously reported sensor using screen printed electrode. Two GA samples, 15 or 30% GA, corresponding to healthy and diabetic levels, respectively, were measured after protease digestion with high resolution. This study demonstrated that the application of an IDE will realize the development of highly sensitive disposable-type amperometric enzyme sensors with high reproducibility.
In this study, magnesium oxide (MgO)-templated mesoporous carbon (MgOC) and chitosan cross-linked with genipin (chitosan-genipin) were considered bio-composite inks for screen-printed bioanodes. The ...fabrication processes were optimized using rheological and structural data, and a bioanode ink containing glucose oxidase (GOx) and 1,2-naphthoquinone (1,2-NQ) was successfully developed. The optimal bioanode-ink contained MgOC pre-treated by washing to achieve a hydrophilic and neutral surface, which helped maintain enzyme activity and resulted in a highly porous electrode structure, which is essential for the accessibility of glucose to GOx. A bioanode fabricated using this ink showed a linear response current dependency up to 8 mM glucose with a sensitivity of 25.83 μA cm
−2
mM
−1
. Combined with a conventional biocathode, an electromotive force of 0.54 V and a maximal power density of 96 μW cm
−2
were achieved. These results show that this bio-composite ink can be used to replace the multi-step process of printing with conventional ink followed by drop-casting enzyme and mediator with a one-step printing process.
Bio-composite inks based on magnesium oxide (MgO)-templated mesoporous carbon (MgOC) and chitosan cross-linked with genipin for one-step screen-printing process.
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