An optimised glucose oxidase bioelectrode exhibiting high performance direct electron transfer

A glucose oxidase (GOd) bioelectrode exhibiting high performance, direct electron transfer (DET) has been prepared. Unprecedented redox peak current densities of 1 mA cm(-2) were observed alongside a clear electrochemical response to glucose. This system shows potential as a low cost, high performance enzymatic bioelectrode.     

Biocatalytic anode for glucose oxidation utilizing carbon nanotubes for direct electron transfer with glucose oxidase

Covalently linked layers of glucose oxidase, single-wall carbon nanotubes and poly-l-lysine on pyrolytic graphite resulted in a stable biofuel cell anode featuring direct electron transfer from the enzyme. Catalytic response observed upon addition of glucose was due to electrochemical oxidation of FADH₂ under aerobic conditions. The electrode potential depended on glucose concentration. This system has essential attributes of an anode in a mediator-free biocatalytic fuel cell.     

Engineering of glucose oxidase for direct electron transfer via site-specific gold nanoparticle conjugation

Optimizing the electrical communication between enzymes and electrodes is critical in the development of biosensors, enzymatic biofuel cells, and other bioelectrocatalytic applications. One approach to address this limitation is the attachment of redox mediators or relays to the enzymes. Here we report a simple genetic modification of a glucose oxidase enzyme to display a free thiol group near its active site. This facilitates the site-specific attachment of a maleimide-modified gold nanoparticle to the enzyme, which enables direct electrical communication between the conjugated enzyme and an electrode. Glucose oxidase is of particular interest in biofuel cell and biosensor applications, and the approach of "prewiring" enzyme conjugates in a site-specific manner will be valuable in the continued development of these systems.     

Achievement and assessment of direct electron transfer of glucose oxidase in electrochemical biosensing using carbon nanotubes,graphene, and their nanocomposites

Carbon nanotubes, graphenes, and their hybridized composites with nanoparticles have been attempted to establish a direct electrical communication between the recognition biomolecule and its underlying electrode surface. This review (with 133 refs.) focuses on advances, strategies and technical challenges in the development of reagentless electrochemical biosensors for glucose with enhanced detection sensitivity, selectivity, and simplicity. Specifically, the review commences with a discussion of the relevance of direct electron transfer (DET) in biosensing together with the fundamental of electro-enzymology and kinetics. General aspects of glucose oxidase (GOx), the most popular enzyme with a flavin cofactor, are discussed in view of its historical and important role in the development of electrical biosensors for blood glucose. The next section assesses DET of GOx based on the Marcus theory and the Laviron formalism. The reorganizational energy of the Marcus model and the overpotential play an important role in reaction kinetics and affect the rate of electron transfer significantly. The presence of nanomaterials, particularly for graphene oxide, decreases the electron transfer distance between the enzyme redox center and the underlying electrode surface well beyond 15 Å. The improper Marcus-Hush-Chidsey integral is now simplified to estimate the rate of electron transfer with very good accuracy. Critiques, technical challenges, and future possibilities of glucose electrodes with respect to DET are also presented and discussed.

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Graphical abstract This review (with 133 refs.) focuses on advances, strategies and technical challenges in the development of reagentless electrochemical biosensors for glucose with enhanced detection sensitivity, selectivity, and simplicity.

     

Mediator-free amperometric determination of glucose based on direct electron transfer between glucose oxidase and an oxidized boron-doped diamond electrode

A mediator-free glucose biosensor, termed a “third-generation biosensor,” was fabricated by immobilizing glucose oxidase (GOD) directly onto an oxidized boron-doped diamond (BDD) electrode. The surface of the oxidized BDD electrode possesses carboxyl groups (as shown by Raman spectra) which covalently cross-link with GOD through glutaraldehyde. Glucose was determined in the absence of a mediator used to transfer electrons between the electrode and enzyme. O₂ has no effect on the electron transfer. The effects of experimental variables (applied potential, pH and cross-link time) were investigated in order to optimize the analytical performance of the amperometric detection method. The resulting biosensor exhibited fast amperometric response (less than 5 s) to glucose. The biosensor provided a linear response to glucose over the range 6.67×10⁻⁵ to 2×10⁻³ mol/L, with a detection limit of 2.31×10⁻⁵ mol/L. The lifetime, reproducibility and measurement repeatability were evaluated and satisfactory results were obtained.     

An electrochemically preanodized screen-printed carbon electrode for achieving direct electron transfer to glucose oxidase

Here we report the unique property of a preanodized screen-printed carbon electrode (SPCE1) that can allow direct electron transfer (DET) reaction of glucose oxidase (GOx). The GOx can be immobilized in the composite of oxygen functionalities and edge plane sites generated during preanodization without additional cross-linking agents. The electron transfer rate of GOx is greatly enhanced to 4.38 s⁻¹ as a result of the conformational change of GOx in the microenvironment enabling the accessibility of active site for GOx to the electrode. The analytical versatility is further improved with the aid of Nafion film. As a consequence, the as-prepared electrode can be used as a glucose biosensor and the number of potential foreign species is then restricted by molecular size, permeation and/or (bio)chemical reaction. Most importantly, the disposable nature of the proposed electrode is expected to promote the DET-related researches.     

Direct electron transfer of glucose oxidase on the carbon nanotube electrode

The direct electrochemistry of redox enzymes (or proteins) has received more and more attention[1—9]. These studies developed an electrochemical basis for the investigation of enzyme structure, mechanisms of redox transformations of enzyme molecules and metabolic processes involving redox transformations. From these studies, one can also find potential appli-cations of enzymes in biotechnology. For example, if an enzyme immobilized on electrode surface is ca-pable of the direct electron tra…     

Enhanced direct electron transfer of glucose oxidase based on a protic ionic liquid modified electrode and its biosensing application

A novel glucose oxidase (GOD) biosensor was fabricated with a protic ionic liquid (PIL) N-ethylimidazolium trifluoromethanesulfonate ([EIm][TfO]) as the modifier of a carbon electrode. Due to the excellent conductivity and the conformational changes of the microenvironment around the GOD, the electrochemical and biocatalytic properties of GOD immobilized on the PIL-based electrode were dramatically enhanced. A couple of well-defined redox peaks could be observed, with a formal potential of −0.476 V. The GOD biosensor presented good catalytic activity to the oxidation of glucose in oxygen-saturated phosphate buffer solutions. The cathodic peak currents of GOD decreased along with glucose concentrations. A linear response in the range 0.005–2.8 mM was obtained with a detection limit of 2.5 μM. The sensitivity and the apparent Michaelis–Menten constant (K _m) were estimated to be 14.96 μA mM⁻¹ and 1.53 μM, respectively. In addition, the biosensor remained stable over 30 days, indicating its good chemical and mechanical stability. The glucose content of several serum samples was determined by using the newly developed biosensor, and the results were in good agreement with those obtained by hospital measurements. All results suggested that PILs were a good media for supporting biocatalytic processes on the bioelectrode.     

A molecular wire modified glassy carbon electrode for achieving direct electron transfer to native glucose oxidase

Here we describe a strategy for achieving direct electron transfer to native glucose oxidase (GOx), an enzyme in which the redox active centre is buried deep within the glycoprotein. To achieve this a glassy carbon electrode is modified with a mixed monolayer of 4-carboxyphenyl and a 20 Å long oligo(phenylethynyl) molecular wire (MW), assembled from the respective aryl diazonium salts. Subsequently GOx is adsorbed to the interface, followed by covalent attachment. The redox chemistry of the active centre of glucose oxidase, flavin adenine dinucleotide, was observed at an E_1/2 of –443 mV (vs. Ag|AgCl). The enzyme was shown to retain its activity. Most importantly, in the absence of oxygen the electrode was still able to biocatalytically turn over glucose at −400 mV, thereby demonstrating that the enzyme was being recycled back to its catalytically active oxidized form from its inactive reduced form. The rate of enzyme turnover was 1.1 s⁻¹.     

Fast electron transfer between glucose oxidase and electrodes via phenothiazine mediators with poly(ethylene oxide) spacers attached to the enzyme surface

A series of glucose oxidase hybrids capable of a direct electrical communication with electrodes is synthesized by covalently bonding phenothiazine mediators to surface lysine residues via poly(ethylene oxide) spacers with different lengths. The hybrid with the optimum length of spacer chain exhibits the fast electron transfer between the redox center of enzyme and electrodes. This fast electron transfer in the hybrids realizes a large catalytic current comparable to that for the corresponding freely diffusing mediator systems.     

Ionic liquid/graphene oxide as a nanocomposite for improving the direct electrochemistry and electrocatalytic activity of glucose oxidase

By combination of 1-ethyl-3-methyl immidazolium ethyl sulfate as a typical room temperature ionic liquid (IL) and graphene oxide (GO) nanosheets, a nanocomposite was introduced for improving the direct electrochemistry and electrocatalytic activity of glucose oxidase (GOx). The enzyme on the IL–GO-modified glassy carbon electrode exhibited a quasireversible cyclic voltammogram corresponding to the flavine adenine dinucleotide/FADH₂ redox prosthetic group of GOx. At the scan rate of 100 mV?s^?1, the enzyme showed a peak-to-peak potential separation of 82 mV and the formal potential of ?463 mV (vs Ag/AgCl in 0.1 M phosphate buffer solution, pH?7.0). The kinetic parameters of the charge transfer rate constant, the electron transfer coefficient, and the apparent Michaelis–Menten constant were calculated as 1.36 s^?1 and 0.35 and 2.47 μM, respectively. When the modified electrode was examined as a biosensor for glucose determination, a linear range of 2.5–45 nM with detection limit of 0.175 nM (signal to noise?=?3) was obtained. The biosensor was stable for 2 months.     

Amperometric glucose enzyme electrode by immobilizing glucose oxidase in multilayers on self-assembled monolayers surface

A novel and robust amperometric enzyme electrode for the determination of glucose was constructed by immobilizing glucose oxidase (GOD) and Os(bpy)(2)Cl-poly(4-vinyl)pyridine (Os-PVP) complex multilayers on thiol self-assembled monolayers surface. The apparent Michaelis-Menton constant K(m)' increased with increasing the number of Os-PVP/GOD multilayers. The concentration range of linear response and detection limit were 0.1-10 and 0.05 mM, the interference of ascorbic acid and uric acid were eliminated by the presence of SAMs and the enzyme electrodes were stable over 3 weeks. The preparation technique may be useful for controlling the performance of multilayer enzyme electrodes by changing the enzyme content.     

Bioelectrocatalytic current based on direct heterogeneous electron transfer reaction of glucose oxidase adsorbed onto multi-walled carbon nanotubes synthesized on platinum electrode surfaces

Multi-walled carbon nanotubes (MWCNTs) were synthesized on platinum plate electrodes by the chemical vapor deposition (CVD) method. The MWCNTs synthesized on the Pt plate (MWCNTs/Pt) electrode were immediately immersed into solutions of glucose oxidase (GOX) to immobilize these enzymes onto the MWCNTs/Pt electrode surfaces. After the GOX was immobilized onto the MWCNTs/Pt electrode, a well-defined catalytic oxidation current was increased from ca. −0.45 V (vs. Ag/AgCl/saturated KCl), which was close to the redox potential of flavin adenine dinucleotide (FAD) as a prosthetic group of GOX under physiological pH values.     

Direct electron transfer of glucose oxidase and dual hydrogen peroxide and glucose detection based on water-dispersible carbon nanotubes derivative

A water-dispersible multi-walled carbon nanotubes (MWCNTs) derivative, MWCNTs-1-one-dihydroxypyridine (MWCNTs-Py) was synthesis via Friedel–Crafts chemical acylation. Raman spectra demonstrated the conjugated level of MWCNTs-Py was retained after this chemical modification. MWCNTs-Py showed dual hydrogen peroxide (H₂O₂) and glucose detections without mutual interference by adjusting pH value. It was sensitive to H₂O₂ in acidic solution and displayed the high performances of sensitivity, linear range, response time and stability; meanwhile it did not respond to H₂O₂ in neutral solution. In addition, this positively charged MWCNTs-Py could adsorb glucose oxidase (GOD) by electrostatic attraction. MWCNTs-Py-GOD/GC electrode showed the direct electron transfer (DET) of GOD with a pair of well-defined redox peaks, attesting the bioactivity of GOD was retained due to the non-destroyed immobilization. The high surface coverage of active GOD (3.5 × 10⁻⁹ mol cm⁻²) resulted in exhibiting a good electrocatalytic activity toward glucose. This glucose sensor showed high sensitivity (68.1 μA mM⁻¹ cm⁻²) in a linear range from 3 μM to 7 mM in neutral buffer solution. The proposed sensor could distinguish H₂O₂ and glucose, thus owning high selectivity and reliability.     

Difference in enzyme activity and conformation of glucose oxidase before and after purification

Enzyme activity of commercial glucose oxidase was enhanced after purification through a strong anionic exchange resin. In order to get a better insight into this phenomenon, surface pressure–area (π–A) isotherms and surface pressure–time (π–t) isotherms was used to study the interaction and the absorption at different pH values of the subphases between octadecylamine and glucose oxidase purified by a styrene system quaternary ammonium type strongly basic anionic exchange resin. Circular dichroism (CD), electrophoresis and enzyme activity measurements were conducted to study these phenomena. A preliminary hypothesis has been suggested to explain why the enzyme activity of purified glucose oxidase was higher than that of the commercial one.     

Study on the direct electron transfer process of superoxide dismutase

The electron transfer process of biological important species, superoxide dismutase (SOD), was studied using cyclic voltammetric method at different conditions. The rate of electron transfer process of SOD at conventional bare gold electrode was very low. Different methods were used to enhance the rate of this process. Gold microdisk electrode and gold minigrid electrode were used to replace the used conventional size gold electrode and the electron transfer rate was enhanced obviously. Different promoters, such as bis(4-pyridyl)disulfide, histidine and arginine, were used to promote the electron transfer process also. Promising results were observed with the help of those promoters. The rate enhancement through the participation of amino acids might be more interesting for the exploration of the real process of SOD-related reaction inside the human body.     

Evaluation of a biosensor based on reduced graphene oxide and glucose oxidase enzyme on the monitoring of second-generation ethanol production

Journal of Solid State Electrochemistry - An easily prepared biosensor based on reduced graphene oxide (rGO) and glucose oxidase (GOx) enzyme was developed to monitor the enzymatic hydrolysis...     

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer-modified nanoporous gold electrodes

Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (±0.4), 16.0 (±0.1) and 2.0 (±0.1) μM were obtained for CtCDH-modified lactose and glucose biosensors and GOx-modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells composed of GOx- and CtCDH-modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase as cathodes, in biofuel cells. A maximum power density of 41 μW/cm² was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O₂-saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).