Electrochemical and biosensing properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens.

We investigated the bioelectrochemical properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens (TvGDH) and its electrochemical behaviour when immobilized on a graphite electrode. TvGDH was recently shown to have an unusual substrate spectrum and to prefer maltose over glucose as substrate, and hence could be of interest as recognition element in a maltose sensor. In this study, we determined the redox potential of TvGDH, which is -0.268 ± 0.007 V vs. SHE, and advantageously low to be used with many redox mediators or redox polymers. The enzyme was entrapped in, and wired by an osmium redox polymer (poly(1-vinylimidazole-co-allylamine)-{[Os(2,2'-bipyridine)2Cl]Cl}) with formal redox potential of +0.275 V vs. Ag|AgCl via poly(ethylene glycol) diglycidyl ether crosslinking onto a graphite electrode. When the TvGDH-based biosensor was tested with maltose it showed a sensitivity of 1.7 µA mM-1cm-2, a linear range of 0.5-15 mM, and a detection limit of 0.45 mM. Furthermore, it gave the lowest apparent Michaelis-Menten constant (KM app) of 19.2 ± 1.5 mM towards maltose when compared to other sugars. The biosensor is also able to detect other saccharides including glucose, maltotriose and galactose, these however also interfere with maltose sensing.

Versatile potentiometric metabolite sensing without dioxygen interference.

The field of electrochemical biosensors has been dominated by amperometric and voltammetric sensors; however, these are limited greatly in their signal dependence on electrode size. Open circuit potentiometric sensors are emerging as an alternative due to their signal insensitivity to electrode size. Here, we present a second-generation biosensor that uses a modified chitosan hydrogel to entrap a dehydrogenase or other oxidoreductase enzyme of interest. The chitosan is modified with a desired electron mediator such that in the presence of the analyte, the enzyme will oxidize or reduce the mediator, thus altering the measured interfacial potential. Using the above design, we demonstrate a swift screening method for appropriate enzyme-mediator pairs based on open circuit potentiometry, as well as the efficacy of the biosensor design using two dehydrogenase enzymes (FADGDH and ADH) and peroxidase. Using 1,2-naphthoquinone as the mediator for FADGDH, dynamic ranges from 0.1 to 50 mM glucose are achieved. We additionally demonstrate the ease of fabrication and modification, a lifetime of ≥28 days, insensitivity to interferents, miniaturization to the microscale, and sensor efficacy in the presence of the enzyme's natural cofactor. These results forge a foundation for the generalized use of potentiometric biosensors for a wide variety of analytes within biologically-relevant systems where oxygen can be an interferent.

Creation of a novel DET type FAD glucose dehydrogenase harboring Escherichia coli derived cytochrome b562 as an electron transfer domain.

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b562 (cyt b562) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b562 at either the N- or C-terminus of fungal FADGDH, (cyt b562-GDH or GDH-cyt b562), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b562 specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b562. Consequently, both cyt b562-GDH and GDH-cyt b562 showed DET abilities toward electrode. Interestingly, cyt b562-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b562. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.

Direct electron transfer-type bioelectrocatalysis of FAD-dependent glucose dehydrogenase using porous gold electrodes and enzymatically implanted platinum nanoclusters.

The direct electron transfer (DET)-type bioelectrocatalysis of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) from Aspergillus terreus (AtGDH) was carried out using porous gold (Au) electrodes and enzymatically implanted platinum nanoclusters (PtNCs). The porous Au electrodes were prepared by anodization of planar Au electrodes in a phosphate buffer containing glucose as a reductant. Moreover, PtNCs were generated into AtGDH by an enzymatic reduction of hexachloroplatinate (IV) ion. The modification was confirmed by native polyacrylamide gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses. The AtGDH-adsorbed porous Au electrode showed a DET-type bioelectrocatalytic wave both in the presence and absence of PtNCs; however, the current density with PtNCs (~1 mA cm-2 at 0 V vs. Ag|AgCl|sat. KCl) was considerably higher than that without PtNCs. The kinetic and thermodynamic analysis of the steady-state catalytic wave indicated that inner PtNCs shortened the distance between the catalytic center of AtGDH (=FAD) and the conductive material, and improved the heterogeneous electron transfer kinetics between them.

Third generation impedimetric sensor employing direct electron transfer type glucose dehydrogenase.

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.

From fundamentals to applications of bioelectrocatalysis: bioelectrocatalytic reactions of FAD-dependent glucose dehydrogenase and bilirubin oxidase.

In this review, I present the main highlights of my works in the development of bioelectrocatalysis, which can be used in widespread applications, particularly for the design of biosensor and biofuel cells. In particular, I focus on research progress made in two key bioelectrocatalytic reactions: glucose oxidation by flavin adenine dinucleotide-dependent glucose dehydrogenase and oxygen reduction by bilirubin oxidase. I demonstrate the fundamental principles of bioelectrocatalysis and the requirements for enhancing the catalytic performance, including the choice of a mediator of redox reactions, immobilization, and electrode materials. These methods can allow for achieving control of the bioelectrocatalytic reaction, thereby overcoming obstacles toward their industrial applications.

Development of a third-generation glucose sensor based on the open circuit potential for continuous glucose monitoring.

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.

Diffusion-controlled Mediated Electron Transfer-type Bioelectrocatalysis Using Microband Electrodes as Ultimate Amperometric Glucose Sensors.

We performed numerical simulations on an extremely fast, mediated, electron transfer-type bioelectrocatalytic reaction using a microband electrode. The simulations under fast-enzyme-kinetics conditions predicted that the decrement of the current density by increaseing the microband thickness would effectively improve the upper limit of detection. These predictions were accurate for an ultrathin-ring with thickness of 100 nm and gold leaf with thickness of 10 µm electrodes, acting as novel amperometric glucose sensors with FAD-dependent glucose dehydrogenase. The gold leaf electrode provided pseudo-steady-state currents which were proportional to the glucose concentration up to a concentration of 20 times higher than the mediator concentration.

Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi.

FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T m) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.

Novel fungal FAD glucose dehydrogenase derived from Aspergillus niger for glucose enzyme sensor strips.

In this study, a novel fungus FAD dependent glucose dehydrogenase, derived from Aspergillus niger (AnGDH), was characterized. This enzyme's potential for the use as the enzyme for blood glucose monitor enzyme sensor strips was evaluated, especially by investigating the effect of the presence of xylose during glucose measurements. The substrate specificity of AnGDH towards glucose was investigated, and only xylose was found as a competing substrate. The specific catalytic efficiency for xylose compared to glucose was 1.8%. The specific activity of AnGDH for xylose at 5mM concentration compared to glucose was 3.5%. No other sugars were used as substrate by this enzyme. The superior substrate specificity of AnGDH was also demonstrated in the performance of enzyme sensor strips. The impact of spiking xylose in a sample with physiological glucose concentrations on the sensor signals was investigated, and it was found that enzyme sensor strips using AnGDH were not affected at all by 5mM (75mg/dL) xylose. This is the first report of an enzyme sensor strip using a fungus derived FADGDH, which did not show any positive bias at a therapeutic level xylose concentration on the signal for a glucose sample. This clearly indicates the superiority of AnGDH over other conventionally used fungi derived FADGDHs in the application for SMBG sensor strips. The negligible activity of AnGDH towards xylose was also explained on the basis of a 3D structural model, which was compared to the 3D structures of A. flavus derived FADGDH and of two glucose oxidases.

Employing FAD-dependent glucose dehydrogenase within a glucose/oxygen enzymatic fuel cell operating in human serum.

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) is emerging as an oxygen-insensitive alternative to glucose oxidase (GOx) as the biocatalyst for bioelectrodes and bioanodes in glucose sensing and glucose enzymatic fuel cells (EFCs). Glucose EFCs, which utilize oxygen as the oxidant and final electron acceptor, have the added benefit of being able to be implanted within living hosts. These can then produce electrical energy from physiological glucose concentrations and power internal or external devices. EFCs were prepared with FAD-GDH and bilirubin oxidase (BOx) to evaluate the suitability of FAD-GDH within an implantable setting. Maximum current and power densities of 186.6±7.1 µA cm(-2) and 39.5±1.3 µW cm(-2) were observed when operating in human serum at 21 °C, which increased to 285.7±31.3 µA cm(-2) and 57.5±5.4 µW cm(-2) at 37 °C. Although good stability was observed with continual near-optimal operation of the EFCs in human serum at 21 °C for 24 h, device failure was observed between 13-14 h when continually operated at 37 °C.