FAD-dependent Glucose Dehydrogenase Research Articles

Electrosynthesis of Pyrenediones on Carbon Nanotube Electrodes for Efficient Electron Transfer with FAD‐dependent Glucose Dehydrogenase in Biofuel Cell Anodes

AbstractA particularly efficient redox mediator for electron transfer between FAD‐dependent glucose dehydrogenase (FAD‐GDH) and carbon nanotube (CNT) based electrodes can be obtained via electrosynthetic oxidation of pyrene in aqueous buffer solution. 1H‐NMR spectroscopic studies reveal the formation of a 2 : 1 mixture of 1,6‐pyrenedione and 1,8‐pyrenedione at the electrode. The formed pyrenedione exhibits a well‐defined surface‐bound redox system at −0.1 V vs. SCE and provides excellent electron transfer kinetics with this enzyme. Furthermore, the π‐system of pyrenedione allows improved stacking behavior with the CNT walls, leading to enhanced stabilities compared to commonly used mediators like naphthoquinone. The electrosynthesis of pyrenedione for catalytic glucose oxidation is optimal at pH 2 using cyclic voltammetry or chronoamerometry. It is envisioned that the electrosynthetic methodology can be expanded to form different redox mediators for a series of enzymes.

Commentary regarding: "Activity determination of FAD-dependent glucose dehydrogenase immobilized in PEDOT: PSS-PVA composite films for biosensor applications".

In the light of continuous improvement and optimization, recent experiments that the authors conducted give new insights into the applied evaluation method of Riegel etal. [1]: Thorough investigations of the previous results regarding the usage of the Lowry Assay showed discrepancies in the determination of the released amount of protein in the analysis solution. The accurate quantification of this parameter is crucial as it directly influences the calculation of the residual enzymatic activity. In concrete terms, this finding has a major impact on the presented and discussed results in the article "Activity determination of FAD-dependent glucose dehydrogenase immobilized in PEDOT: PSS-PVA composite films for biosensor applications" [1]. Thus, this commentary addresses the new insights concerning the applied evaluation method, explains necessary revisions and discusses new conclusions derived from the adjusted evaluation method.

Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization

FAD-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is an enzyme utilized industrially in glucose sensors. Previously, FAD-GDH isolated from Mucor prainii (MpGDH) was demonstrated to have high substrate specificity for glucose. However, MpGDH displays poor thermostability and is inactivated after incubation at 45 °C for only 15 min, which prevents its use in industrial applications, especially in continuous glucose monitoring (CGM) systems. Therefore, in this study, a chimeric MpGDH (Mr144–297) was engineered from the glucose-specific MpGDH and the highly thermostable FAD-GDH obtained from Mucor sp. RD056860 (MrdGDH). Mr144–297 demonstrated significantly higher heat resistance, with stability at even 55 °C. In addition, Mr144–297 maintained both high affinity and accurate substrate specificity for D-glucose. Furthermore, eight mutation sites that contributed to improved thermal stability and increased productivity in Escherichia coli were identified. Collectively, chimerization of FAD-GDHs can be an effective method for the construction of an FAD-GDH with greater stability, and the chimeric FAD-GDH described herein could be adapted for use in continuous glucose monitoring sensors.

A 3rd generation Biosensor Towards the Continuous Long-Term Glucose Monitoring without Ag/AgCl Reference Electrode

Continuous glucose monitoring (CGM) is highly important, especially, for the diagnostics and control of Type I diabetes. [1] In recent years, automatic insulin pumps (artificial pancreas) are being applied and CGM is necessary for their operation. The insulin pump should deliver a correct dose of insulin based on real-time data about glucose levels in blood. Therefore, CGM sensors should be very selective and accurate, as well as reliable and stable. For this reason, CGM sensors are developed using selective enzymes – glucose oxidase or and FAD-dependent glucose dehydrogenase. [2] They usually operate by measuring either glucose oxidation current (amperometric biosensors) or potential (potentiometric biosensors). Historically, those biosensors are divided into three generations according to the electron acceptor: O2 dependent (1st), mediator depended (2nd) and direct electron transfer (3rd). The 3rd generation biosensors are the most attractive since they are most accurate, sensitive, operate at low potential and their response does not depend on the concentration of external mediator (either O2 or synthetic). However, to this date there are multiple unresolved problems which still hold the development of 3rd generation glucose biosensors for the CGM. Some of those problems include low stability (typically from a few hours to a few days) and miniaturization difficulties due to the usage of bulk reference electrodes for the control of the potential (Ag/AgCl, Hg/Hg2Cl2, etc.). Moreover, typical reference electrodes are poorly suited for long-time operation because they leak electrolyte and ions to the media. [3] A prolonged leakage of electrolyte solution changes the potential of reference electrode and may cause health risk if CGM biosensor in directly implanted in the patient. In this work, we present an open circuit potential (OCP) type 3rd generation glucose biosensor, which addresses the mentioned issues and operates in an electrolyte solution (pH 7.4) which mimics the composition of human serum. Our biosensor is composed of two electrodes: gold nanoparticle-glucose dehydrogenase (AuNPs-GDH) based bioanode and gold nanoparticles-laccase (AuNPs-LAC) based biocathode (Figure A). We have used AuNPs-LAC biocathode, which we have reported earlier [4], as a counter/reference electrode since its potential depends on oxygen reduction reaction. In the majority of solutions, O2 concentration is constant, thus it has a very stable OCP which is independent on glucose concentration and holds a constant value of 0.48 V vs.Ag/AgCl (Sat.) in an electrolyte solution. We have used AuNPs-GDH electrode as a working electrode since its OCP is dependent on glucose concentration raging from 10-6 M to 0.05 M. The OCP difference between two electrodes was -0.374 V and decreased depending on the concentration of glucose (10-6 M – 0.05 M) according to Nernst model (Figure B and C). The biosensor was calibrated in glucose range of 0.001 M to 0.01 M (typical glucose concentrations present in human blood) and a high correlation calibration curve was obtained (Figure D). Moreover, biosensor had showed an excellent stability and was able to operate at least for two weeks without a significant change in OCP. In conclusion, we have developed a promising biosensor for a constant long-term glucose monitoring which operates in an electrolyte solution, mimicking human serum conditions. A future challenge is to apply and optimize our biosensor to operate in serum and blood.

(Invited) Current Understanding and Future Aspects of Direct Electron Transfer Mechanism of Fad Dependent Glucose Dehydrogenase Complex

Direct electron transfer (DET) between enzyme molecule and electrode provides the ideal principle for the enzyme based sensing system. In DET principle the electrons are transferred from enzyme active site to electrode directly in the absence of any additional electron acceptors, or mediators. This intriguing reaction is useful for various biodevices such as biosensors and biofuel-cells, especially by focusing anodic reactions, such as the oxidation of glucose, fructose, and ethanol. The absence of redox mediators in the system enables the operation of the sensor in redox potential closer to the co-factor in the enzyme, reducing the effect of interfering reactions by electroactive compounds in the sample. However, only a limited number of redox enzymes are capable of DET-reaction with electrodes. Among these, several dehydrogenases harboring a redox center in the catalytic subunit/domain and haem which is not involved in catalysis but act as a built-in mediator for electron transfer between enzyme and electrode are reported. One of the representative groups of DET enzymes is the oligomeric dehydrogenases composed of catalytic subunit harboring FAD as a co-factor, and an electron transfer subunit with haem c, the FAD dependent dehydrogenase complexes. Our research group have been engaged in the isolation, characterization, application and engineering of one of the representative FAD-dependent dehydrogenase complexes. FAD glucose dehydrogenase complex (FADGDH). FADGDH is comprised the three distinct subunits: the catalytic subunit (α subunit) that has an FAD cofactor in its redox center, shows catalytic activity, and oxidizes the first hydroxyl group of glucose; the small subunit (γ subunit), a hitch-hiker protein of the bacterial TAT secretion system, which is necessary for the proper folding and secretion of the α subunit; and the three heme-cytochrome c subunit (β subunit) that is in responsible for the transfer of electrons between the active-site cofactor and external electron acceptors. Thanks to the presence of β subunit, FADGDH is capable to transfer electrons directly to an electrode, making FADGDH as the ideal molecule for the 3rd Gen glucose sensors.This paper focuses on biomolecular aspects on DET mechanism of FAD dependent dehydrogenase complexes, considering current increasing number of applicational researches, and recent remarkable progress in understanding their DET mechanism, especially about the recent progress in understanding inter- and intra-molecular electron transfer of FADGDH are introduced. These studies included protein engineering approaches of these molecules, thereby led to construct engineered GDH. Finally, the future prospect of the FAD dependent dehydrogenase complexes will be addressed.

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.

Fungal FAD-dependent glucose dehydrogenases concerning high activity, affinity, and thermostability for maltose-insensitive blood glucose sensor

Six FAD-dependent glucose dehydrogenase (FAD-GDH) genes, for blood glucose sensors, from the genomic DNAs of Aspergillus species were identified by degenerated PCR screening. The enzymatic properties of the gene products expressed by Pichia pastoris were characterized. Investigation of substrate selectivity to fourteen saccharides revealed that these enzymes were maltose-insensitive FAD-GDHs. A.bisporus, A.terreus var. aureus, and A. phoenicis FAD-GDHs showed high substrate selectivity; they exhibited reactivity to only one or two saccharides. A. phoenicis FAD-GDH specifically exhibited insensitivity to d-xylose. Furthermore, six FAD-GDHs exhibited superior parameter values, such as maximum velocity (Vmax), Michaelis-Menten constant (Km), and apparent transition midpoint temperature (Tm). A. phoenicis and A. terreus var. aureus FAD-GDHs exhibited high activity, with Vmax values of (8.66 ± 0.17) × 104 U/μmol and (1.14 ± 0.03) × 105 U/μmol, respectively. A. brunneo-uniseriatus, A.carneus, and A. malignus FAD-GDHs exhibited very high affinity for glucose, with Km values between 0.41 and 0.96 mM. A. bisporus FAD-GDH exhibited high thermostability, with a Tm value of 72.5 °C. Therefore, we propose that the FAD-GDHs will be practical in blood glucose sensors and in other bioelectrochemical devices exhibiting high substrate selectivity, high current output, high sensitivity, and/or high stability to storage and use.

(Invited) Enzymatic Reduction of Oxygen for the Development of Biofuel Cells and Hybrid Fuel Cells

The need for clean methods of producing electricity for medical applications has stimulated the emergence of biofuel cells as a new generation of fuel cells. This subcategory of fuel cells, mainly rely on redox enzymes, which are very efficient and selective biocatalysts that can advantageously replace rare and expensive platinum-based catalysts in classic fuel cell devices. Enzymes provide exceptional specificities towards their substrates, thus enabling the assembly of both the anode and cathode electrodes of a biofuel cell without the need for membranes [1,2]. Recent advances in the design of biocathodes based on electrically wired enzymes onto carbon nanotube coatings for the reduction of oxygen will be reported. In particular, different strategies for achieving a controlled orientation of laccase or bilirubin oxidase on carbon nanotube-based electrodes will be presented [3,4]. In particular, a new generation of flexible buckypaper electrodes was produced by using linear polynorbornene polymers containing multiple pyrene groups as crosslinker. Robust buckypapers using copolymers containing both pyrene and activated ester groups for cross-linking and tethering, respectively, will be applied to the covalent binding of redox groups or enzymes [5]. Moreover, buckypapers based on bilirubin oxidase and FAD-dependent glucose dehydrogenase, were developed for the direct electron transfer and the mediated electron transfer, respectively. The resulting biofuel cell based on the O2/glucose system, provides the highest volumetric power reported until now, namely 24.07 mW cm-3 [6]. The design of hybrid biofuel cells will be also reported. In particular, cubic Pd nanoparticles were synthesized and evaluated for the catalytic oxygen reduction. These nanoparticles were employed for the development of an air-breathing cathode modified by multiwalled carbon nanotubes. The latter was combined with a phenanthrolinequinone/glucose dehydrogenase-based anode to form a complete glucose/O2 hybrid biofuel cell delivering a maximal power output of 184 ± 21 µW cm-2 [7]. Finally, an innovative approach based on the electrical wiring of enzymes in solution by redox glyconanoparticles resulting from the self-assembly of bio-sourced block copolymers will be presented [8]. We demonstrate the self-assembly, characterization and bioelectrocatalysis of redox-active cyclodextrin-coated nanoparticles. The nanoparticles with host-guest functionality are easy to assemble and permit entrapment of hydrophobic redox molecules in aqueous solution. Bis-pyrene-ABTS encapsulated nanoparticles (diameter: 195 nm) were used as electron shuttles between electrode and bilirubin oxidase. Enhanced current densities for enzymatic O2 reduction are observed with the redox nanoparticle system compared to equivalent bioelectrode cells with dissolved mediator [9]. Acknowledgements This work is partly supported by the French National Research Agency in the framework of the "Investissements d’avenir” program Glyco@Alps (ANR-15-IDEX-02), the LabEx ARCANE (ANR-11-LABX-0003-01), and Institut Carnot PolyNat (ANR-16-CARN-0025-01). References Cosnier, A. Le Goff, M. Holzinger, Electrochem. Commun. 38 19-23 (2014)Cosnier A. J. Gross, A. Le Goff, M. Holzinger, J. Power Sources 325 252-263 (2016)Lalaoui, R. David, H. Jamet, M. Holzinger, A. Le Goff, S. Cosnier. ACS Catal., 6 4259-4264 (2016)Lalaoui, M. Holzinger, A. Le Goff, S. Cosnier. Chem. Eur. J., 22 10494-10500 (2016)J. Gross, M. P. Robin, Y. Nedellec, R. K. O’Reilly, D. Shan, S. Cosnier, Carbon, 107 542-547 (2016)Gross, X. Chen, F. Giroud, C. Abreu, A. Le Goff, M. Holzinger, S. Cosnier. ACS Catalysis, 7 (2017) 4408-4416.Faggion Junior, R. Haddad, F. Giroud, M. Holzinger, C. Eduardo Maduro de Campos, J. J. S. Acuña, J. B. Domingos, S. Cosnier. Nanoscale, 8 10433-10440 (2016)J. Gross, R. Haddad, C. Travelet, E. Reynaud, P. Audebert, R. Borsali, S. Cosnier. Langmuir, 32 11939-11945 (2016). J. Gross, X. Chen, F. Giroud, C. Abreu, A. Le Goff, M. Holzinger, S. Cosnier, J. Am. Chem. Soc. accepted.

Engineered fungus derived FAD-dependent glucose dehydrogenase with acquired ability to utilize hexaammineruthenium(III) as an electron acceptor

Fungal FAD-dependent glucose dehydrogenases (FADGDHs) are considered to be superior enzymes for glucose sensor strips because of their insensitivity to oxygen and maltose. One highly desirable mediator for enzyme sensor strips is hexaammineruthenium(III) chloride because of its low redox potential and high storage stability. However, in contrast to glucose oxidase (GOx), fungal FADGDH cannot utilize hexaammineruthenium(III) as electron acceptor. Based on strategic structure comparison between FADGDH and GOx, we constructed a mutant of Aspergillus flavus-derived FADGDH, capable of utilizing hexaammineruthenium(III) as electron acceptor: AfGDH-H403D. In AfGDH-H403D, a negative charge introduced at the pathway-entrance leading to the FAD attracts the positively charged hexaammineruthenium(III) and guides it into the pathway. The corresponding amino acid in wild-type GOx is negatively charged, which explains the ability of GOx to utilize hexaammineruthenium(III) as electron acceptor. Electrochemical measurements showed a response current of 46.0 μA for 10 mM glucose with AfGDH-H403D and hexaammineruthenium(III), similar to that with wild-type AfGDH and ferricyanide (47.8 μA). Therefore, AfGDH-H403D is suitable for constructing enzyme electrode strips with hexaammineruthenium(III) chloride as sole mediator. Utilization of this new, improved fungal FADGDH should lead to the development of sensor strips for blood glucose monitoring with increased accuracy and less stringent packing requirements.

Mutagenesis Study of the Cytochrome c Subunit Responsible for the Direct Electron Transfer-Type Catalytic Activity of FAD-Dependent Glucose Dehydrogenase.

The FAD-dependent glucose dehydrogenase from Burkholderia cepacia (FADGDH) is a hetero-oligomeric enzyme that is capable of direct electron transfer (DET) with an electrode. The cytochrome c (cyt c) subunit, which possesses three hemes (heme 1, heme 2, and heme 3, from the N-terminal sequence), is known to enable DET; however, details of the electron transfer pathway remain unknown. A mutagenesis investigation of the heme axial ligands was carried out to elucidate the electron transfer pathway to the electron mediators and/or the electrode. The sixth axial ligand for each of the three heme irons, Met109, Met263, and Met386 were substituted with His. The catalytic activities of the wild-type (WT) and mutant enzymes were compared by investigating their dye-mediated dehydrogenase activities and their DET abilities toward the electrode. The results suggested that (1) heme 1 with Met109 as an axial ligand is mainly responsible for the electron transfer with electron acceptors in the solution, but not for the DET with the electrode; (2) heme 2 with Met263 is responsible for the DET-type reaction with the electrode; and (3) heme 3 with Met386 seemed to be the electron acceptor from the catalytic subunit. From these results, two electron transfer pathways were proposed depending on the electron acceptors. Electrons are transferred from the catalytic subunit to heme 3, then to heme 2, to heme 1 and, finally, to electron acceptors in solution. However, if the enzyme complex is immobilized on the electrode and is used as electron acceptors, electrons are passed to the electrode from heme 2.

Dawson-type polyoxometalate nanoclusters confined in a carbon nanotube matrix as efficient redox mediators for enzymatic glucose biofuel cell anodes and glucose biosensors

Two new inorganic-organic hybrid materials based on heteropolyoxometalates (POMs): (C4H10N)6[P2Mo18O62]·4H2O (P2Mo18) and (C6H8NO)4[H2P2W18O62]·6H2O (P2W18) are reported as mediators for electron transfer between FAD-dependent glucose dehydrogenase (FAD-GDH) and a multiwalled carbon nanotube (MWCNT) matrix for glucose biofuel cell and biosensor applications. These polyoxometalates were chosen due to their promising redox behavior in a potential range for mediated electron transfer with the glucose oxidizing enzyme, FAD-GDH. P2Mo18 and P2W18 were immobilized on 1-pyrenemethylamine (PMA) functionalized MWCNT deposits. After immobilization of FAD-GDH, the P2W18-modified MWCNT electrode demonstrated mediated electron transfer and provided a catalytic current density of 0.34 mA cm−2 at 0.2 V vs SCE with an open circuit potential (OCP) of −0.08 V vs SCE. A 10-fold increase in catalytic current to 4.7 mA cm−2 at 0.2 V vs SCE and a slightly lower OCP of −0.10 V vs SCE was observed for an equivalent electrode modified with P2Mo18.The apparent superiority of P2Mo18 is related, at least in part, to its improved incorporation in the MWCNT matrix compared to P2W18. Both POM-modified bioanodes showed exceptional stabilities with 45% of their initial performances remaining after 15 days. The mediated electron transfer capacities of the POMs were also evaluated in a glucose sensor setup and showed very satisfying performances for glucose detection, including a sensitivity of 0.198 mA mol L−1 cm−2, a satisfying linear range between 1 mmol L−1 and 20 mmol L−1, and good reproducibility for the P2Mo18 electrode.

Rapid sucrose monitoring in green coffee samples using multienzymatic biosensor

Amperometric biosensor utilizing FAD-dependent glucose dehydrogenase (FAD-GDH) for a specific sucrose monitoring in green coffee is described. FAD-GDH was co-immobilized with invertase and mutarotase on a thin-layer gold planar electrode using chitosan. The biosensor showed a wide linearity (from 10 to 1200 μM), low detection limit (8.4 μM), fast response time (50 s), and appeared to be O2 independent. In addition the biosensors exhibited a good operational (3 days) and storage (1 year) stability. Finally, the results achieved from the biosensor measurements of sucrose in 17 samples of green coffee (Coffea arabica, C. canephora and C. liberica) were compared with those obtained by the standard HPLC method. The good correlation among results of real samples, satisfactory analytical performance and simple use of the presented biosensor make it suitable for application in coffee industry.

The electrochemical behavior of a FAD dependent glucose dehydrogenase with direct electron transfer subunit by immobilization on self-assembled monolayers

Continuous glucose monitoring (CGM) is a vital technology for diabetes patients by providing tight glycemic control. Currently, many commercially available CGM sensors use glucose oxidase (GOD) as sensor element, but this enzyme is not able to transfer electrons directly to the electrode without oxygen or an electronic mediator. We previously reported a mutated FAD dependent glucose dehydrogenase complex (FADGDH) capable of direct electron transfer (DET) via an electron transfer subunit without involving oxygen or a mediator. In this study, we investigated the electrochemical response of DET by controlling the immobilization of DET-FADGDH using 3 types of self-assembled monolayers (SAMs) with varying lengths. With the employment of DET-FADGDH and SAM, high current densities were achieved without being affected by interfering substances such as acetaminophen and ascorbic acid. Additionally, the current generated from DET-FADGDH electrodes decreased with increasing length of SAM, suggesting that the DET ability can be affected by the distance between the enzyme and the electrode. These results indicate the feasibility of controlling the immobilization state of the enzymes on the electrode surface.

Mediator Preference of Two Different FAD-Dependent Glucose Dehydrogenases Employed in Disposable Enzyme Glucose Sensors.

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.

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.

A High Power Buckypaper Biofuel Cell: Exploiting 1,10-Phenanthroline-5,6-dione with FAD-Dependent Dehydrogenase for Catalytically-Powerful Glucose Oxidation

Enzymatic biofuel cells generate electrical energy from renewable sources with high selectivity and environmental benefits compared to lithium batteries and traditional fuel cells. For enzymatic fuel cells to become competitive, major improvements in electrode design are required to enhance power density, voltage output, and stability. Here we have developed a freestanding paper biofuel cell comprising redox molecule embedded multiwalled carbon nanotube papers for electrical wiring of enzymes. The drop-coat and one-pot fabrication methods provide flexibility and permit easy scalability of functionalized bioelectrodes via commercially available materials. Buckypaper functionalized with 1,10-phenanthroline-5,6-dione (PLQ) as an efficient electron mediator for fungal-derived FAD-dependent glucose dehydrogenase (FADGDH) shows very high steady-state current densities for glucose oxidation of Imax = 5.38 ± 0.54 mA cm–2 at 0.15 V vs SCE at neutral pH. When coupled with a bioinspired protoporphyrin IX buckypaper ...

Enzymes Confinement in CNT Doped Carbon Microstructures Electrodes (CNT-CME) for Miniaturized Enzyme Biofuel Cell Application

Fast development of biomedical technology has led us to the invention and use of implantable medical devices to be utilized for direct monitoring of human health. In general, batteries is used as the main micropower supply of these implanted devices, however it has some drawbacks as it has to be replenished in due time. One of the promising continuous energy supplies to replace the use of batteries is enzyme biofuel cell (EBFC) which harvests electrical energy from the oxidation-reduction reactions as it generates electricity by glucose oxidation and oxygen reduction by the anode and cathode enzymes respectively; in which two of its main substrate are abundant and essential components in human physiological fluid. EBFC is expected to be minimal in size but still concerning to have maximized performance during operation including; high electrode potential range, current and power densities which dependent to the stability of the enzyme used and enzyme environment construction. In order to achieve long operation of EBFC, it is necessary to be assured that enzyme will not be denatured due to external potent factor such as shear stress, especially if EBFC is employed as micro-power supply of implanted devices in human in which the blood velocity will depend on the diameter of blood vessel as implantation site. One of the modest methods to protect immobilized enzyme from the shear stress is by using polymer such as Nafion to form physical entrapment layer. However, there is high possibility that Nafion as polymer will be degraded under shear stress leading to leaching of enzyme during long operation. Previous studies showed that encapsulated protein in porous materials, or immobilized inside layer by layer material showed more stability due to less exposure of the external stress. The use of mesoporous material such as sol-gels and entrapment in mesoporous silica have proven to increase the enzyme stability, however this materials are lack of conductivity which is not suitable for bioelectronics application. Due to the necessity of electrical communication, another study was employing conducting material such as Si wafer with micro culverts as enzyme protection platform which enhance the electron transfer between enzyme-electrodes material . The success of enzyme immobilization also depends on its carrier used for particular application. The platform for enzyme immobilization should have high capacity as enzyme anchoring sites, chemically stable, and having the structural ability to prolong enzyme stability. The combination of nanostructures with enzyme immobilization will be a promising direction since it is possible to engineer enzyme environment to limit the effect of external forces as called enzyme-in-cage. Carbon as the most popular and widely studied as electrode materials due to its physical and electrochemical stability properties as inert material, low cost and easy to be functionalized and decorated. Carbon can be used for miniaturized electrode fabricated through nanolithography by pyrolizing photoresist, decompose co-composite, leaving only carbon in microstructured pattern. The use of photoresist to form carbon after pyrolysis step has the advantage due to its ability to be patterned by photolithography techniques which is reproducible. The properties of pyrolized electrodes are comparable than glassy carbon electrode (GCE) which has similar electrochemical properties such as low background current as the effect of low capacitance Further advancement for this system is the integration of CNT which has high surface area, excellent conductivity due to its sp2carbon hybridization along its wall. In order to prevent any proximity effect of CNT-pyrolyzed carbon electrode, CNT was grown directly in carbon post in which the catalyst of CNT growth was deposited through electrostatic spray deposition (ESD). Grown CNT on pyrolyzed carbon is expected to keep high structural stability. In this study we elaborated the fabrication and application of CNT doped carbon microstructures (CNT-CME) as platform for enzyme confinement and immobilization. Thus eventually this method then applied to immobilize the FAD-dependent glucose dehydrogenase (FADGDH) α-subunit with menadione as mediator (FADGDH/menadione) system which has proven to have better performance as anode for biofuel cell application as its native character of low Km value. The parameter such as K mof free and immobilized enzyme, current and power densities of the EBFC were also tested. Figure 1

Enzymes Confinement in Gold Nanospikes Electrodes Fabricated By Metal-Assisted Chemical Etching for Enhanced Electron Transfer

The field of enzyme based biofuel cells (EBFC) which utilize enzymes that can consume complex fuels (i.e. sucrose, fructose, etc.) and fuel mixtures to produce electrical energy has expanded over the last 3 decades, and it is now possible to consider applications for implantable fuel cells, bio-battery and aquatic device power source. The last three decades have seen major advancements in the field to enhance the open circuit potentials, current and power densities, fuel efficiency and biocatalyst stability. These advances have resulted in open circuit potentials increasing from 175 mV to almost 1 V and current densities increasing from μA/cm2 to mA/cm2. These increases come from enhanced electrochemically assessable surface areas with nanomaterials, increasing the oxidation reaction activity of anodes with enzyme cascades, metabolons, and optimal cell design to minimize internal resistance. However, further researches into improving power density over 1~5 mW are still needed to supply the power for stable operation of target devices. In this situation, the stable power output is hampered by a low level of electron transfer rate since electron transfer is known as crucial factor that determines performance of EBFC systems. The low level of electron transfer rate is resulted from several reasons that are related to protein structure and enzyme-layer formation. In detail, the redox center where electron is generated is often buried inside the protein matrix and moreover, enzymes are randomly oriented or forms multi-layers of enzymes during loaded on electrode, all of which attribute to long distance of electron transfer and degrade the performance of enzymatic electrode as well. To alleviate these problems, many groups have studied to develop the methods to enhance the electron transfer in these circumstances. Especially, construction of nanostructured high surface electrodes has been spotlighted because it could facilitate the electron transfer and thus performance of enzymatic electrode. In this research, we attempted to fabricate the nanostructured bioanode, optimizing the electron transfer from enzymes to electrode by control of enzyme layer thickness on the electrode surface. Genetically expressed FAD-dependent glucose dehydrogenase is immobilized on Au electrode modified by metal assisted chemical etching (MaCE). Recently, the fabrication of silicon (Si) subwavelength structure (SWS) by means of metal-assisted chemical etching (MaCE) has received increasing attention for its several merits during the modification of electrode surface that low-cost, fast, and scalable. Through this methodology, we fabricated nanospikes in pyramidal structure on the electrode surface, in which the nanospikes exist periodically with a sub-wavelength pitch all over the electrode surface. The effect of nanofabrication in this study is that immobilized enzyme on electrode surface are facilitated to be evenly distributed on the electrode surface. The periodically inclined structure grants equal opportunity of enzyme loading for unit area of electrode surface. By achieving the uniform distribution of enzymes, the distance between enzyme redox center and electrode surface can be reduced by excluding enzyme multilayers. Here, we experimentally studied the effect of pyramidal structure on enzymatic behaviors. During the experiment, the silicon (Si) wafer was selected with high conductive type for eliminating the possible overpotential that could be occured from the support material. Ag nanoparticles were used to assist the etching process of Si substrate, providing catalytic sites. The fabrication utilizing various diameter of Ag nanoparticles and etching times were conducted for having electrode candidates with different periodicities and pitch sizes. The optimized structure was confirmed by testing with electrochemical method, cyclic voltammetry (CV), and the optical slope which includes periodicity and pitch size was determined. Based on this research, it was concluded that electrode morphology is parameter that determines the performance of EBFC. Therefore, electrode structure for EBFC should be constructed in consideration of bio-electrode compatibility, not just of high surface area. Figure 1

Lowering the potential of electroenzymatic glucose oxidation on redox hydrogel-modified porous carbon electrode

Herein, we demonstrate high current densities per projected surface area for the electroenzymatic oxidation of glucose at low potential using a hierarchically structured electrode based on MgO-templated carbon. The modified electrode was prepared by assembling glucose oxidase (GOx) with an osmium-based redox polymer with a low formal potential (−0.03V vs. Ag|AgCl), in which Os complexes are tethered to the polymer backbone via a 13-atom alkyl chain and a crosslinker. A glucose oxidation current density of 15mAcm−2 was measured at 0V vs. Ag|AgCl at 37°C and pH 7, with a plateau value of 50mAcm−2 at 0.3V. The hydrogel electrodes composed of the same redox polymer and FAD-dependent glucose dehydrogenase delivered only 10% of the current densities obtained with the GOx electrode.