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).     

Amperometric glucose-responding property of enzyme electrodes fabricated by covalent immobilization of glucose oxidase on conducting polymer films with macroporous structure

Macroporous conducting polymer films were prepared by the electrochemical copolymerization of 3-methylthiophene and thiophene-3-acetic acid on the ITO-coated glass plates bearing different sizes of polystyrene template particles, and enzyme electrodes were fabricated by covalent immobilization of glucose oxidase on the macroporous copolymer films. It was found that the doping level and conductivity of the copolymer films was significantly affected by the treatment with solvent to remove the polystyrene particles, which was considered to result in deterioration in amperometric glucose-responding property of the enzyme electrodes fabricated with the copolymer films. Three-dimensionally ordered macroporous structure on the copolymer films led to enhancement of amperometric response of the enzyme electrodes, and this effect was attributed to the geometry of the interconnected channel structure formed by the linkage of macropores. It was suggested that the amperometric response of the enzyme electrodes was determined by whether the interconnected channel structure on the copolymer films had long distance regularity and a proper size to allow the enzyme and electron-mediator molecules to penetrate into the interior pores of the copolymer film. In particular, the interconnected channel structure seemed to play an important role in the electron-transfer reaction between the mediator molecules and the surface of electrodes.     

Kinetics of redox polymer-mediated enzyme electrodes

Oxygen-reducing enzyme electrodes are prepared from laccase of Trametes versicolor and a series of osmium-based redox polymer mediators covering a range of redox potentials from 0.11 to 0.85 V. Experimentally obtained current density generated by the film electrodes is analyzed using a one-dimensional numerical model to obtain kinetic parameters. The bimolecular rate constant for mediation is found to vary with mediator redox potential from 250 s(-1) M(-1) when mediator and enzyme are close in redox potential to 9.4 x 10(4) s(-1) M(-1) when the redox potential difference is large. The value of the bimolecular rate constant for the simultaneously occurring laccase-oxygen reaction is found to be 2.4 x 10(5) s(-1) M(-1). The relationship between mediator-enzyme overpotential and bimolecular rate constant is used to determine the optimum mediator redox potential for maximum power output of a hypothetical biofuel cell with a planar cathode and a reversible hydrogen anode. For laccase of T. versicolor (E(e)(0) = 0.82), the optimum mediator potential is 0.66 V (SHE), and a molecular structure is presented to achieve this result.     

AFM study of conducting polymer polypyrrole nanoparticles formed by redox enzyme - glucose oxidase - initiated polymerisation

Redox enzyme – glucose oxidase E.C. 1.1.3.4 from Penecillum vitale (GOx) – initiated polypyrrole (Ppy) synthesis was applied for the formation of polypyrrole based nanoparticles. The increase in optical absorbance at λ = 460 nm was exploited for the monitoring of polypyrrole polymerisation process. The shape and size of the formed Ppy nanoparticles was also monitored by means of contact mode AFM. The highest increase in the diameter of the formed Ppy nanoparticles was detected during 15-day period. AFM imaging was performed in contact mode to investigate the shape and flexibility of particles deposited on the SiO₂ and Pt surfaces. Contact mode AFM investigations allowed us to conclude that after drying at 50 °C the formed Ppy particles are more flexibly deposited on the Pt electrode if compared to those deposited on the SiO₂ substrate. The application of well-shaped Ppy nanoparticles in biomedicine, chromatography and bioanalysis may be predicted.     

Low-potential cyclometalated osmium(II) mediators of glucose oxidase

The osma(II)cycles [Os(phpy)(LL)(2)]PF(6) (LL = 1,10-phen (3a) and 4,4'-Me(2)-2,2'-bpy (3b)) are made from [(eta(6)-C(6)H(6))Os(micro-Cl)Cl](2) (1) either via transmetalation using the [Hg(phpy)(2)] organomercurial in MeOH or via the sp(2)-C-H bond cleavage of 2-phenylpyridine (phpyH) in MeCN to afford [(eta(6)-C(6)H(6))Os(phpy)Cl] or [(eta(6)-C(6)H(6))Os(phpy)(MeCN)]PF(6), respectively. The latter two react cleanly with LL to give 3a and 3b, the M(II/III) redox potentials of which equal 30 and -100 mV (vs Ag/AgCl), respectively. The electrochemically made Os(III) species oxidize rapidly reduced glucose oxidase. The second-order rate constant equals 1.1 x 10(7) M(-)(1) s(-)(1) for 3a at 25 degrees C, pH 7.     

A glucose/oxygen enzymatic fuel cell based on redox polymer and enzyme immobilisation at highly-ordered macroporous gold electrodes

Highly ordered macroporous electrodes are prepared by electro-deposition of gold through a polystyrene sphere template. Drop-coating redox polymer and either glucose oxidase, for the anode, or Melanocarpus albomyces laccase, for the cathode on the macroporous gold provides film-coated electrodes for assembly of membrane-less glucose/oxygen enzymatic fuel cells (EFC) in pH 7.4 buffer containing 10 mM glucose and 0.15 M NaCl. Under these conditions the maximum power density of 17 μW cm(-2) for EFCs using films adsorbed to planar gold electrodes increased to 38 μW cm(-2) for films adsorbed to 2? sphere gold macroporous electrodes.     

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.     

Comparison of amperometric measuring principles for determinations of glucose with electrodes based on glucose oxidase

The three measuring principles for glucose determination (H₂O₂ formation, O₂ consumption, or enzymatically reduced acceptor) were studied with an apparatus that was the same for all measurements, with virtually identical electrode design. The methods were compared with regard to measurement range, sensitivity, time behaviour, operational lifetime, precision and accuracy. Essential differences were found with respect to measurement range as well as to time. Measurements under anaerobic conditions have advantages over those done aerobically.     

A glucose biosensor using methyl viologen redox mediator on carbon film electrodes

A new methyl viologen-mediated amperometric enzyme electrode sensitive to glucose has been developed using carbon film electrode substrates. Carbon film electrodes from resistors fabricated by pyrolytic deposition of carbon were modified by immobilization of glucose oxidase through cross-linking with glutaraldehyde in the presence of bovine serum albumin. The mediator, methyl viologen, was directly immobilised with the enzyme together with Nafion cation-exchange polymer. The electrochemistry of the glucose oxidase/methyl viologen modified electrode was investigated by cyclic voltammetry and by electrochemical impedance spectroscopy. The biosensor response to glucose was evaluated amperometrically; the detection limit was 20 μM, the linear range extended to 1.2 mM and the reproducibility of around 3%. When stored in phosphate buffer at 4 °C and used every day, the sensor showed good stability over more several weeks.     

Characterization of mediated and non-mediated oxidase enzyme based glassy carbon electrodes

The performance and analytical characteristics of a glassy carbon glutaraldehyde immobilized glucose oxidase electrode have been established with regard to the direct detection of hydrogen peroxide produced from the reaction of glucose with oxygen. Measurements were performed at + 1.1 V vs. SCE, and selectivity was obtained by casting the surface with a cellulose acetate membrane. Results compared favorably with the classical platinum-enzyme probe. The mechanism of ascorbic acid interference in hydrogen peroxide detection is reported. Mediated detection was also investigated for oxidase enzymes (glucose oxidase and xanthine oxidase) immobilized on the bare glassy carbon electrode. The probes were characterized using a specific enzyme mediator in solution (phenazine methosulfate or dichlorophenol-indophenol) plus hexacyanoferrate(III) as an electrochemical mediator. The electrode was poised at + 0.36 V vs. SCE for the detection of hexacyanoferrate(II). The advantages of this dual mediator configuration include high stability and sensitivity of the electrochemical signal and the ability to use less positive potentials for increased selectivity. Application to other enzymes, such as hydrogenases, using such a binary redox configuration is suggested.     

Amperometric enzyme sensor for glucose based on graphite paste-modified electrodes

Amperometric enzyme electrode for glucose is described based on the incorporation of glucose oxidase (GOD) into graphite paste modified with tetracyanoquinodimethane (TCNQ). The incorporated enzyme exhibits high activity and long-term stability over the earlier TCNQ-based glucose sensor (1). The sensor provides a linear response to glucose over a wide concentration range. The response time of the sensor is 15-50 sec, and the detection limit is 0.5 mM. Stable response to the substrate was obtained during a period of 35 d. Application of the sensor in the plasma analysis is reported.     

Amperometric enzyme electrodes: Part II. Conducting salts as electrode materials for the oxidation of glucose oxidase

Results are reported for the direct oxidation of the enzyme glucose oxidase on 7 different one-dimensional conducting donor acceptor salts. Experiments conducted with the enzyme in bulk solution are shown to be in good agreement with theory. Three salts, made of the cations tetrathiafulvalinium (TTF⁺), N-methylphenazinium or quinolinium with the anion tetracyanoquinodimethanide (TCNQ^?) had the lowest background currents and were used to make membrane sensors for glucose. Analysis of the variation of current with glucose concentration identified the rate limiting processes as transport of gluycose through the membrane and electrochemical kinetics under unsaturated and saturated conditions respectively. The electrochemical rate constants for these three materials were all greater than 10^?2 cm s^?1. TTF⁺TCNQ^? is the material of choice and linear calibration plots were obtained for glucose concentrations between 50 μmol dm^?3 and 10 mmol dm^?3.     

Organic oxidations by osmium and ruthenium oxo complexes

TMC Literature Highlight-21,Transition Met. Chem.,14, 79 (1989).     

Improved stability of redox enzyme layers on glassy carbon electrodes via covalent grafting

One of the challenges in the field of enzymatic biofuel cells is to significantly improve their current limited lifetime. In the present work, we report the covalent immobilization of enzyme layers on glassy carbon electrodes, functionalized via electrochemical reduction of in situ generated aryldiazonium salts bearing carboxylic acid groups. We present the performance and the stability over time of the modified electrodes. For glucose oxidase – modified electrodes, stable catalytic activity is observed for a minimum of 6 weeks.     

Multilayered construction of glucose oxidase on gold electrodes based on layer-by-layer covalent attachment

A feasible approach to construct multilayered enzyme film on the gold electrode surface for use as biosensing interface is described. The film was fabricated by alternate layer-by-layer deposition of periodate-oxidized glucose oxidase (GOx) and poly(allylamine) (PAA). The covalent attachment process was followed and confirmed by electrochemical impedance spectroscopy (EIS). X-ray diffraction (XRD) experiments revealed that the film was homogeneous and formed in an ordered manner with a thickness of 2.6 ± 0.1 nm per bilayer. The gold electrodes modified with the GOx/PAA multilayers showed excellent electrocatalytical response to the oxidation of glucose when ferrocenemethanol was used as an artificial redox mediator, which was studied by cyclic voltammetry (CV). From the analysis of voltammetric signals, the coverage of active enzyme on the electrode surface was estimated, which had a linear relationship with the number of GOx/PAA bilayers. This suggests that the analytical performance such as sensitivity, detection limit, and so on, is tunable by controlling the number of attached bilayers. The six GOx/PAA bilayer electrode exhibited a sensitivity of 15.1 μA mM⁻¹ cm⁻² with a detection limit of 3.8 × 10⁻⁶ M. In addition, the sensor exhibited good reproducibility and stability.     

Metal-alloy-dispersed carbon-paste enzyme electrodes for amperometric biosensing of glucose

The preferential electrocatalytic detection of hydrogen peroxide and the selective biosensing of glucose at carbon-paste enzyme electrodes dispersed with bimetallic (Ru–Pt) alloy particles are described. Unlike the marked acceleration of the redox reaction of the peroxide product (versus single metal catalysts), the signals of common interfering compounds shift to higher potentials. Such use of carbon-supported alloy particles thus results in a greatly enhanced sensitivity compared to the dispersion of pure metals, without compromising the remarkable selectivity inherent to metallized carbon biosensors.     

A glucose biosensor based on electrodeposited biocomposites of gold nanoparticles and glucose oxidase enzyme.

Electrodeposition was used for the codeposition of glucose oxidase enzyme and a gold nanoparticle-silicate network onto an indium tin oxide (ITO) glass electrode. This co-entrapment of glucose oxidase enzyme in a gold nanoparticle-silicate network imparts biocatalytic activity to the film. The gold nanoparticles in the network catalyse the oxidation and reduction of H2O2, the by-product of the enzymatic reaction. The low operating potential of the sensor eliminates the interference from common interferents, such as acetaminophen, ascorbic acid, dopamine, etc.     

Enzyme electrode studies of glucose oxidase modified with a redox mediator

Glucose oxidase modified by the covalent attachment of ferrocenecarboxylic acid or ferrocene-acetic acid groups undergoes direct oxidation at metal electrodes. Studies of the comparative stability of the two modified enzymes on storage and on electrochemical cycling show that the material modified with ferroceneacetic acid is the more stable. Amperometric studies of enzyme electrodes based on these modified forms of glucose oxidase show that their application in practical biosensors is severely limited by the poor stability of the oxidized form of the covalently attached ferrocene mediator. A comparison of the results obtained with the native enzyme and with that modified with ferroceneacetic acid, for the oxidation of glucose, d-mannose, 2-deoxy-d-glucose, d-xylose and d-galactose, suggests that the modification procedure has little effect on the selectivity of the enzyme.     

Enzyme sequence and competition electrodes based on immobilized glucose oxidase,peroxidase and catalase

The toxicologically important peroxidase substrates bilirubin and aminopyrine can be determined by combination of immobilized glucose oxidase, horseradish peroxidase and catalase, forming so-called enzyme sequence and enzyme competition electrodes. Bilirubin and aminopyrine are determined in the concentration range 5–50 μM.     

Development and evaluation of electrochemical glucose enzyme biosensors based on carbon film electrodes

Electrochemical glucose enzyme biosensors have been prepared on carbon film electrodes made from carbon film electrical resistors. Evaluation and characterisation of these electrodes in phosphate buffer saline solution has been carried out with and without pretreatment by cycling in perchloric acid or at fixed applied potential. Both pretreatments led to a reduction in the carbon surface oxidation peak and enabled better detection of hydrogen peroxide in the pH range of 5-7. Glucose oxidase enzyme was immobilised on the carbon surface by mixing with glutaraldehyde, bovine serum albumin and with and without Nafion. The performance of these two types of electrode was similar, that containing Nafion being more physically robust. Linear ranges were up to around 1.5 mM, with detection limits 60 μM, and pretreatment of the carbon film electrode at a fixed potential of +0.9 V versus SCE for 5 min was found to be the most beneficial. Michaelis-Menten constants between 5 mM and 10 mM were found under the different experimental conditions. Coating the immobilised enzyme layer with a thin layer of Nafion was found to give similar results in the determination of glucose to mixing it but with benefits against interferences for the analysis of complex matrices, such as wine. Potentialities, for a short-term-use or disposable sensors, are indicated.