Flavin Adenine Dinucleotide-dependent Glucose Dehydrogenase Research Articles

Enzymatic Precipitation of Highly Electroactive and Ion-Transporting Prussian Blue for a Sensitive Electrochemical Immunosensor.

Sensitive and/or multiplex electrochemical biosensors often require efficient (bio)catalytic conversion of substrates into insoluble electroactive products. The enzymatic formation and precipitation of coordination polymers under mild conditions offers a promising solution for this purpose. Herein, we report the enzymatic precipitation of Prussian blue (PB), a highly electroactive and ion-transporting coordination polymer, on an immunosensing electrode for application in a sensitive electrochemical immunosensor for detecting thyroid-stimulating hormone (TSH). Five pairs of redox enzymes and their specific reductants were examined to achieve rapid PB precipitation and electrochemical oxidation. Among these pairs, O2-insensitive flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) paired with glucose yielded the highest electrochemical signal-to-background (S/B) ratio. FAD-GDH catalyzed the conversion of Fe(CN)63- to Fe(CN)64-, which coordinated with Fe3+, leading to PB formation and subsequent precipitation through repeated conversions. The resulting PB precipitate, with its close proximity to the electrode, facilitated rapid electrochemical oxidation and generated a strong electrochemical signal. Notably, the precipitation and electrochemical oxidation of PB were more effective than those of its analogues. When applied to a sandwich-type immunosensor for TSH detection, the enzymatic PB precipitation achieved a calculated detection limit of approximately 2 pg/mL in artificial serum, covering the clinically relevant range. These findings indicate the potential widespread utility of PB precipitation and electrochemical oxidation for sensitive multiplex biomarker detection.

Enzymatic Galvanic Redox Potentiometry for In Vivo Biosensing.

Redox potentiometry has emerged as a new platform for in vivo sensing, with improved neuronal compatibility and strong tolerance against sensitivity variation caused by protein fouling. Although enzymes show great possibilities in the fabrication of selective redox potentiometry, the fabrication of an enzyme electrode to output open-circuit voltage (EOC) with fast response remains challenging. Herein, we report a concept of novel enzymatic galvanic redox potentiometry (GRP) with improved time response coupling the merits of the high selectivity of enzyme electrodes with the excellent biocompatibility and reliability of GRP sensors. With a glucose biosensor as an illustration, we use flavin adenine dinucleotide-dependent glucose dehydrogenase as the recognition element and carbon black as the potential relay station to improve the response time. We find that the enzymatic GRP biosensor rapidly responds to glucose with a good linear relationship between EOC and the logarithm of glucose concentration within a range from 100 μM to 2.65 mM. The GRP biosensor shows high selectivity over O2 and coexisting neurochemicals, good reversibility, and sensitivity and can in vivo monitor glucose dynamics in rat brain. We believe that this study will pave a new platform for the in vivo potentiometric biosensing of chemical events with high reliability.

Effect of pore size of MgO-templated porous carbon electrode on immobilized crosslinked enzyme–mediator redox network

Magnesium oxide templated porous carbon (MgOC) is a promising material as an enzyme electrode platform. Herein, a flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and thionine were immobilized using a crosslinker, and the applicability of MgOC with various pore sizes as a platform was investigated. Among the pores of MgOC, the novel designed FAD-GDH/thionine network generated a 1.1 mA cm−2 catalytic current at 100 nm pore size, demonstrating a significantly improved glucose supply and a thin redox composite layer was formed within the nanostructured material. At low loading, the catalytic current is independent of the pore size; however, at high loading, a varied catalytic current is achieved for the different MgOC pore sizes. Further, the stability was improved of porous platform compared to the glassy carbon (GC) electrode. Additionally, a glucose/O2 biofuel cell is constructed with FAD-GDH/thionine on porous MgOC and non-porous GC as an anode, and mediated bilirubin oxidase as a cathode. A remarkably high electricity generation efficiency (0.23 mW cm−2 μg−1 of FAD-GDH) is shown on a porous electrode platform than non-porous and a previously reported immobilized FAD-GDH-based anode. These results demonstrate the potential of porous MgOC as an anode for a novel FAD-GDH/thionine network toward electricity generation.

A disposable enzymatic biofuel cell for glucose sensing via short-circuit current

It is essential to construct a biofuel cell-based sensor and develop an effective strategy to detect glucose without any potentiostat circuitry in order to create a simple and miniaturized device. In this report, an enzymatic biofuel cell (EBFC) is fabricated by the facile design of an anode and cathode on a screen-printed carbon electrode (SPCE). To construct the anode, thionine and flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) are covalently immobilized via a crosslinker to make a cross-linked redox network. As a cathode, the Pt-free oxygen reduction carbon catalyst is employed alternative to the commonly used bilirubin oxidase. We proposed the importance of EBFC-based sensors through the connection of anode and cathode; they can identify a short-circuit current by means of applied zero external voltage, thereby capable of glucose detection without under the operation of the potentiostat. The result shows that the EBFC-based sensor could be able to detect based on a short-circuit current with a wide range of glucose concentrations from 0.28 to 30 mM. Further, an EBFC is employed as a one-compartment model energy harvester with a maximum power density of (36 ± 3) μW cm− 2 in sample volume 5 μL. In addition, the constructed EBFC-based sensor demonstrates that the physiological range of ascorbic acid and uric acid shows no significant effect on the short-circuit current generation. Moreover, this EBFC can be used as a sensor in artificial plasma without losing its performance and thereby used as a disposable test strip in real blood sample analysis.

Glucose Sensor Strip Using Flavin Adenine Dinucleotide-dependent Glucose Dehydrogenase with Quinones as Redox Mediators

Glucose Sensor Strip Using Flavin Adenine Dinucleotide-dependent Glucose Dehydrogenase with Quinones as Redox Mediators

Comparison of the stability of Mucor-derived flavin adenine dinucleotide-dependent glucose dehydrogenase and glucose oxidase

Long-term stability at near-body temperature is important for continuous glucose monitoring (CGM) sensors. However, the stability of enzymes used in CGM sensors has often been evaluated by measuring their melting temperature (Tm) values and by short heat treatment but not at around 37°C. Glucose oxidase (GOD) is used in current CGM sensors. In this study, we evaluated the stability of modified Mucor-derived flavin adenine dinucleotide-dependent glucose dehydrogenase (designated Mr144-297) with improved thermal stability at medium to high temperatures and compared it with that of GOD. The Tm value of Mr144-297 was 61.6±0.3°C and was similar to that of GOD (61.4±1.2°C). However, Mr144-297 was clearly more stable than GOD at 40°C and 55°C. At 37°C, the stability of a carbon electrode with immobilized Mr144-297 was higher than that of an electrode with GOD. Our data indicate that Mr144-297 is a more suitable enzyme for CGM sensors than is GOD.

A New Digital Point-of-Care Tool With Advanced Blood Glucose Measuring Technology.

cobas® pulse is a point-of-care blood glucose (BG) measuring system for multiple-patient use in professional healthcare settings. The system provides advances in connectivity and BG measuring technology, and has multiple fail-safes to improve accuracy and reduce the risk of user error. Flavin adenine dinucleotide-dependent glucose dehydrogenase on the working electrode catalyzes oxidation of β-D-glucose in the blood sample. A redox mediator/electron acceptor, on both the working and the counter electrode, facilitates diffusion of electrons in proportion to the glucose concentration and compensates for the effects of potential interfering agents. During development, >1 million test strip measurements were performed using >8000 test scenarios to refine the algorithm model. No clinically relevant interference was identified with extreme variations in blood properties and drugs in whole blood samples.

In Vitro Evaluation of Miniaturized Amperometric Enzyme Sensor Based on the Direct Electron Transfer Principle for Continuous Glucose Monitoring.

The bacterial derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (FADGDH) is the most promising enzyme for the third-generation principle-based enzyme sensor for continuous glucose monitoring (CGM). Due to the ability of the enzyme to transfer electrons directly to the electrode, recognized as direct electron transfer (DET)-type FADGDH, although no investigation has been reported about DET-type FADGDH employed on a miniaturized integrated electrode. The miniaturized integrated electrode was formed by sputtering gold (Au) onto a flexible film with 0.1 mm in thickness and divided into 3 parts. After an insulation layer was laminated, 3 openings for a working electrode, a counter electrode and a reference electrode were formed by dry etching. A reagent mix containing 1.2 × 10-4 Unit of DET-type FADGDH and carbon particles was deposited. The long-term stability of sensor was evaluated by continuous operation, and its performance was also evaluated in the presence of acetaminophen and the change in oxygen partial pressure (pO2) level. The amperometric response of the sensor showed a linear response to glucose concentration up to 500 mg/dL without significant change of the response over an 11-day continuous measurement. Moreover, the effect of acetaminophen and pO2 on the response were negligible. These results indicate the superb potential of the DET-type FADGDH-based sensor with the combination of a miniaturized integrated electrode. Thus, the described miniaturized DET-type glucose sensor for CGM will be a promising tool for effective glycemic control. This will be further investigated using an in vivo study.

Disposable electrochemical glucose sensor based on water-soluble quinone-based mediators with flavin adenine dinucleotide-dependent glucose dehydrogenase

Glucose level measurement is essential for the point-of-care diagnosis, primarily for persons with diabetes. A disposable electrochemical glucose sensor is constructed using flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and redox mediator for electron transfer from the enzyme to the electrode surface. Ideally, a suitable mediator should have high water solubility, high kinetic constant, high stability, and redox potential between −0.2 and 0.1 V vs. Ag|AgCl|sat. KCl. We designed and synthesized two new quinone-based water-soluble mediators: quinoline-5,8-dione (QD) and isoquinoline-5,8-dione (IQD). The formal potentials for both QD and IQD at pH 7.0 were −0.07 V vs. Ag|AgCl|sat. KCl. The logarithms of the electron exchange rate constants (k2/(M−1 s−1)) between QD/IQD and FAD-GDH were 7.7 ± 0.1 and 7.4 ± 0.1 for QD and IQD, respectively, which are the highest value among the water-soluble mediators for FAD-GDH reported to date. Disposable amperometric glucose sensors were fabricated by dropping FAD-GDH and QD or IQD onto a test strip. The sensor achieved a linear response up to glucose concentrations of 55.5 mM. The linear response was obtained even when the mediator loading was low (0.5 nmol/strip); loading was only 0.2 mol% of glucose. The results proved that the response current was primarily controlled by glucose diffusion. In addition, the sensor using QD exhibited high stability over 3 months at room temperature.

Improved glucose oxidation catalytic current generation by an FAD-dependent glucose dehydrogenase-modified hydrogel electrode, in accordance with the Hofmeister effect

Herein, we describe the effect of varying anions in an electrolyte solution on current generation by a redox hydrogel electrode. The electrode surface is coated with a thin film of hydrogel matrix, consisting of an osmium (Os) redox polymer including tethered Os complexes, polymer backbone, and a redox enzyme. In this case, the enzymes employed are flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH), which catalyzes glucose oxidation, and the result was compared with that reported earlier for glucose oxidase (GOx). The hydrogel matrix facilitates efficient electron transfer from glucose to the electrode via collision of the Os complexes and thus acts as a mediator. The degree of impact of anions on current generation is characteristic of the Hofmeister series. Chaotropic anions, such as nitrate and chloride, increase and decrease the catalytic current produced by FAD-GDH and GOx hydrogel electrodes, respectively. Such anions can adsorb onto the cationic region of the FAD-GDH surface and induce a negative charge, which enhances electrostatic interactions between the enzyme and the positively charged Os polymer. Kosmotropic anions, such as sulphate and phosphate increase the catalytic current due to hydrogel shrinkage, which increases the relative concentrations of both enzyme and mediator within the hydrogel architecture due to an increase in density. High-performance electrode design depends on understanding the impact of ion identity on catalytic current responses of redox hydrogel electrodes.

Site-specifically wired and oriented glucose dehydrogenase fused to a minimal cytochrome with high glucose sensing sensitivity

Direct electron transfer based enzymatic biosensors are highly efficient systems where electrons are transferred directly from the enzyme's electroactive site to the electrode. One way of achieving it is by ‘wiring’ the enzyme to the electrode surface. The wiring of enzymes to electrode surfaces can be reached in many different ways but controlling its orientation towards the electrode surface is still a challenge. In this study we have designed a Flavin-adenine dinucleotide dependent glucose dehydrogenase that is fused to a minimal cytochrome with a site-specifically incorporated unnatural amino acid to control its orientation towards the electrode. Several site-specifically wired mutant enzymes were compared to each other and to a non-specifically wired enzyme using atomic force microscopy and electrochemical techniques. The surface and activity analyses suggest that the site-specific wiring through different sites maintains the correct folding of the enzyme and have a positive effect on the apparent electrochemical electron transfer rate constant kETapp. Electrochemical analysis revealed an efficient electron transfer rate with more than 15 times higher imax and 10-fold higher sensitivity of the site-specifically wired enzyme variants compared to the non-specifically wired ones. This approach can be utilized to control the orientation of other redox enzymes on electrodes to allow a significant improvement of their electron transfer communication with electrodes.

Sensitive electrochemical immunosensor using a bienzymatic system consisting of β-galactosidase and glucose dehydrogenase.

Bienzymatic systems are often used with electrochemical affinity biosensors to achieve high signal levels and/or low background levels. It is important to select two enzymes whose reactions do not exhibit mutual interference but have similar optimal conditions. Here, we report a sensitive electrochemical immunosensor based on a bienzymatic system consisting of β-galactosidase (Gal, a hydrolase enzyme) and flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH, a redox enzyme). Both enzymes showed high activities at neutral pH, the reactions catalyzed by them did not exhibit mutual interference, and the electrochemical-enzymatic redox cycling based on FAD-GDH coupled with enzymatic amplification by Gal enabled high signal amplification. Among the three amino-hydroxy-naphthalenes and 4-aminophenol (potential Gal products), 4-amino-1-naphthol showed the highest signal amplification. Glucose, as an electro-inactive, stable reducing agent for redox cycling, helped in achieving low background levels. Our bienzymatic system could detect parathyroid hormone at a detection limit of ∼0.2 pg mL-1, implying that it can be used for highly sensitive electrochemical detection of parathyroid hormone and other biomarkers in human serum.

(Invited) Glucose/Oxygen Wearable Biofuel Cells Based on Poly(glycidyl methacrylate)-Grafted MgO-Templated Porous Carbon

There are a lot of recent researches on high power enzymatic glucose/O2 biofuel cells (BFCs). We have reported that the use of MgO-templated porous carbon (MgOC) at the electrodes of biofuel cells dramatically improves the output power [1-4]. Using MgOC can improve the effective electrode surface area, leading to increased current density and output density. Supporting enzymes on these types of porous materials is expected to lead to increased output density. In the past researches [1-4], the enzymes physically adsorbed inside the porous carbon gradually leach from the carbon surface after long-term operation or during the replacement of the fuel solution.In this study, to prevent enzyme leakage, the enzyme or mediator were immobilized on the MgOC surface via multipoint covalent bonding; the grafting of polymers bearing pendant glycidyl groups was achieved by the polymerization of glycidyl methacrylate (GMA), initiated by electron beam irradiation on the carbon surface. Poly(glycidyl methacrylate) (poly(GMA)) bearing pendant glycidyl groups, grafted on the surface of MgO-templated carbon, is useful for forming strong multipoint covalent bonds with amino functional groups on the surface of flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH), Azure A and aminoferrocene.For example, the immobilized FAD-GDH can generate glucose-oxidation catalytic current using 1,2-naphthoquinone (12NQ) as the redox mediator, which is also captured on the electrode surface. The catalytic current is more stable than that obtained using the FAD-GDH-MgOC electrode without poly(GMA) because the leaching of FAD-GDH and 12NQ is suppressed by the enzyme capping effect.The BFC performance using the anode measured in 1.0 M phosphate buffer (PBS) containing 100 mM glucose exhibited the open circuit voltage of 0.8 V, maximum power density of 1.4 mW/cm2, and maximum current density of 4.25 mA/cm2.In the present paper, we totally discuss three types glucose/oxygen biofuel cells based on the poly(glycidyl methacrylate)-grafted MgO-templated porous carbon.

Paper-Based Lactate Biofuel Cell Using Poly(glycidyl methacrylate)-Grafted MgO-Templated Carbon

Enzymatic lactate/O2 biofuel cells (BFCs) are energy conversion systems in which enzymes oxidize lactate at a bioanode and reduce oxygen at a biocathode. These enzymes are highly safe toward humans, and highly selective for substrates. In addition, lactate is contained abundant in a human sweat. Thus, the lactate/O2 BFCs have been attracting attention as a next-generation wearable power source1).We have produced a paper-based lactate biofuel cell in which lactate oxidase (LOx) and a mediator are physically absorbed on MgO-templated carbon (MgOC) having mesopores suitable for enzyme2).By using MgOC for electrode, long-term stability may be improved because the elution of the enzyme is suppressed by entrapping the enzyme in the mesopore3). On the other hand, covalent immobilization of mediators and enzymes seems to be effective for further improvement of stability of the electrodes. We recently reported that poly(glycidyl methacrylate) (poly(GMA)) bearing pendant glycidyl groups, grafted on the surface of MgO-templated carbon (GMgOC), was useful for forming strong multipoint covalent bonds with amino functional groups on the surface of flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) molecules4). The stability of FAD-GDH-immobilized GMgOC electrode showed good stability with a simple immobilization scheme by suppressing elution of FAD-GDH from the electrode surface.In this study, we newly fabricated a paper-based lactate biofuel cell using a GMgOC electrode formed by screen printing. A lactate biofuel cell anode, in which Azure A, as a mediator, was covalently immobilized on the GMgOC. LOx was also immobilized in the GMgOC. We evaluated the output current and the stability of the electrode. The synthesis reaction of the GMgOC is shown in Fig. 1(a). MgOC was irradiated with an electron beem to generate radicals, and then glycidyl methacrylate was introduced to form GMgOC on the electrode surface. Polyvinylidene difluoride(PVdF) as a binder and N-methyl-2-pyrrolidinone(NMP) as a solvent were added to GMgOC as a carbon ink.The paper-based carbon electrodes were fabricated by screen printing. A carbon leads and MgOC ink was printed on a water-repellent paper substrate, successively. The AzureA as a mediator and LOx as an enzyme were dropped on the porous carbon electrode surface. Chronoamperometry was performed using the prepared bioanode. The measurement was performed by the three-electrode method using a platinum wire as the counter electrode and a saturated KCl/Ag/AgCl electrode as the reference electrode, respectively. The chlonoamperometry was performed in 1 mol dm-3 phosphate buffer containing 0.1 mol dm-3 lactate. The potential was set at 0.1 V vs. Ag/AgCl.Figure 1(b) shows the cyclic voltammetry of the GMgOC electode. A catalytic oxidation current was observed at a nobler potential than -0.1 V. The peak current value of the present enzyme electrode was larger than that of an enzyme electrode in which MgOC was used as the electrode material. The results indicated that the use of GMgOC will obtain a larger peak value because of immobilizing mediator and enzymes makes electrons to transport more efficiently to the electrode.A chronoamperograms using GMgOC and MgOC electrodes were measured at 0.1 V. Figure 1(c) shows that the current value of MgOC was 0.09 mA cm-2 after 30000 s, and that of GMgOC was 1.7 mA cm-2. It was found to exhibit that the current density was about twice as high as MgOC. It’s because, mediators and enzymes are immobilized by physical adsorption on the carbon surface when using MgOC, while elution of LOx could be suppressed by using GMgOC because the amino group of the enzyme shell and the epoxy group on the carbon surface are bonded successfully.Reference1) E. Katz et al., J. Electroanal. Chem., 479 (1999) 64.2) I. Shitanda et al., J. Electrochem. Soc, 166(12) (2019) B1063-B1068.3) S. Tsujimura et al., Electrochemistry 83 (2015) 372.4) I. Shitanda, T. Kato et al., Bull. Chem. Soc. Jpn., 93 (2020) 32.AcknowledgmentsThis work was partially supported by JST-ASTEP Grant Number JPMJTS1513, JSPS Grant Number 17H02162 and Private University Research Branding Project (2017-2019) from Ministry of Education, Culture, Sports, Science and Technology, and Tokyo University of Science Grant for President's Research Promotion． Figure 1

Photoelectrochemically-assisted biofuel cell constructed by redox complex and g-C3N4 coated MWCNT bioanode

Herein, we report a membraneless glucose and air photoelectrochemical biofuel cell (PBFC) with a visible light assisted photobioanode. Flavin adenine dinucleotide dependent glucose dehydrogenase (FADGDH) was immobilized on the combined photobioanode for the visible light assisted glucose oxidation (GCE|MWCNT|g-C3N4|Ru-complex|FADGDH) with a quinone mediated electron transfer. Bilirubine oxidase (BOx) immobilized on MWCNT coated GCE (GCE|BOx) was used as the cathode with direct electron transfer (DET). An improvement of biocatalytic oxidation current was observed by 6.2% due in part to the light-driven electron-transfer. The large oxidation currents are probably owing to the good contacting of the immobilized enzymes with the electrode material and the utilization of light assisted process. Under the visible light, the photobioanode shows an anodic photocurrent of 1.95 μA cm2 at attractively low potentials viz. −0.4 vs Ag/AgCl. The lower-lying conduction band of g-C3N4 as compared to Ru-complexes decreases the rate of hole and electron recombination and enhances the charge transportation. The bioanode shows maximum current density for glucose oxidation up to 6.78 μA cm−2 at 0.2 V vs Ag/AgCl at pH:7. The performance of three promising Ru-complexes differing in chemical and redox properties were compared as electron mediators for FADGDH. Upon illumination, the PBFC delivered a maximum power density of 28.5 ± 0.10 μW cm−2 at a cell voltage of +0.4 V with an open circuit voltage of 0.64 V.

Wearable glucose/oxygen biofuel cell fabricated using modified aminoferrocene and flavin adenine dinucleotide-dependent glucose dehydrogenase on poly(glycidyl methacrylate)-grafted MgO-templated carbon

We have developed a new electrode material for biofuel cells (BFCs) in which a mediator was covalently immobilized on MgO-templated porous carbon (MgOC). Grafted MgOC (GMgOC) was prepared by modifying poly (glycidyl methacrylate) on the MgOC surface by electron beam graft polymerization. Furthermore, aminoferrocene (AmFc) and flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) were immobilized by covalent bonding between the amino group and pendant glycidyl group. The current density estimated by cyclic voltammetry was approximately 21.2 mA cm−2 at 0.49 V in a 1 mol L−1 phosphate buffer containing 100 mmol L−1 glucose. The current value was stable because of the suppression of AmFc elution after 10 h of continuous measurement. A glucose/O2 BFC was fabricated by combining an anode modified with FAD-GDH/poly(GMA)-modified AmFc and a BOD/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-modified water-repellent cathode and applying 1 mol L−1 PBS containing 300 mmol L−1 glucose (pH 7.0). The BFC showed a high power density of 3.6 mW cm−2. We fabricated a wearable BFC based on hydrogels with ortho hydroquinone groups that can maintain adhesiveness on human skin. The power density of the wearable cell was 0.42 mW cm−2 at 0.38 V in the air, which is sufficient output to power a small electronic device.

Biosensing and electrochemical properties of flavin adenine dinucleotide (FAD)-Dependent glucose dehydrogenase (GDH) fused to a gold binding peptide

In the present work, direct electron transfer (DET) based biosensing system for the determination of glucose has been fabricated by utilizing gold binding peptide (GBP) fused flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Burkholderia cepacia. The GBP fused FAD-GDH was immobilized on the working electrode surface of screen-printed electrode (SPE) which consists of gold working electrode, a silver pseudo-reference electrode and a platinum counter electrode, to develop the biosensing system with compact design and favorable sensing ability. The bioelectrochemical and mechanical properties of GBP fused FAD-GDH (GDH-GBP) immobilized SPE (GDH-GBP/Au) were investigated. Here, the binding affinity of GDH-GBP on Au surface, was highly increased after fusion of gold binding peptide and its uniform monolayer was formed on Au surface. In the cyclic voltammetry (CV), GDH-GBP/Au displayed significantly high oxidative peak currents corresponding to glucose oxidation which is almost c.a. 10-fold enhanced value compared with that from native GDH immobilized SPE (GDH/Au). As well, GDH-GBP/Au has shown 92.37% of current retention after successive potential scans. In the chronoamperometry, its steady-state catalytic current was monitored in various conditions. The dynamic range of GDH-GBP/Au was shown to be 3-30 mM at 30 °C and exhibits high selectivity toward glucose in whole human blood. Additionally, temperature dependency of GDH-GBP/Au on DET capability was also investigated at 30-70 °C. Considering this efficient and stable glucose sensing with simple and easy sensor fabrication, GDH-GBP based sensing platform can provide new insight for future biosensor in research fields that rely on DET.

Mediated electrochemical oxidation of glucose via poly(methylene green) grafted on the carbon surface catalyzed by flavin adenine dinucleotide-dependent glucose dehydrogenase

Electrochemically polymerized phenothiazines (thionine, methylene green, methylene blue, and toluidine blue) on carbon electrodes were investigated as electron transfer mediators of glucose oxidation by flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) for biosensor and biofuel cell applications. Among the tested polyphenothiazines grafted on a glassy carbon electrode, clear redox-mediating activity was observed for poly(methylene green), and the catalytic oxidation current depended on the concentrations of glucose and enzymes and the amount of polymer deposited on the electrode surface. The poly(methylene green)-grafted porous carbon electrodes showed 3 mA cm−2 of glucose oxidation current catalyzed by FAD-GDH.

Toward an ideal platform structure based on MgO-templated carbon for flavin adenine dinucleotide-dependent glucose dehydrogenase-Os polymer-hydrogel electrodes

For the efficient electrochemical glucose oxidation on the electrode modified with redox hydrogel involving Os-tethered polymer and flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH), the effect of pore size of MgO-templated carbon (MgOC) as an electrode material was studied. The MgOC was modified on glassy carbon electrode by ink-drop-casting technique. The MgOC pore size clearly affected on the current generation efficiency for glucose oxidation. As the pore size increased above 100 nm, a glucose oxidation current density of more than 100 mA cm−2 was achieved with 1000 μg cm−2 of hydrogel loading. Both high specific surface area and macrostructure of MgOC, which does not impede mass transport even if hydrogel loading is increased, are important factors in designing the porous structure of the MgOC layer via the ink-drop-casting process during electrode fabrication.

Improved operational stability of mediated glucose enzyme electrodes for operation in human physiological solutions

Stability of glucose-oxidising enzyme electrodes is affected by substances in physiological solutions, hampering deployment as long-term implantable biosensors or fuel cells. The performance of Nafion over-coated enzyme electrodes, consisting of multiwalled carbon nanotubes and flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) or glucose oxidase (GOx) crosslinked with osmium-complex based redox polymer, was compared to uncoated electrodes in presence of uric acid and artificial plasma. Nafion over-coating resulted in lower glucose oxidation current densities compared to no over-coating. The highest initial current density for Nafion over-coated electrodes in artificial plasma in 100 mM glucose was 8.0 ± 2.0 mA cm−2 for GOx electrodes with 0.5% w/v Nafion coating. These electrodes retained 83% of initial current after 12 h continuous operation in artificial plasma while similarly prepared FADGDH electrodes retained 58% signal. This is compared to retention of only 73% or 31% observed for GOx or FADGDH electrodes in artificial plasma with no Nafion membrane. Enzyme electrodes over-coated with Nafion maintain improved signal stability when tested continuously in the presence of uric acid, identified as being the main contributing substance to FADGDH enzyme electrode instability, showing promise for application to continuous use glucose-oxidising enzyme electrodes.