Nanocomposite material laccase/dispersed carbon carrier for oxygen electrode

The effect of carbon dispersion degree on the effectiveness of oxygen electroreduction by laccase at its immobilization at superdispersed colloid graphite (SCG) or acetylene carbon black (ACB) is studied. A method of synthesizing the highly active composite material based on SCG is suggested providing the optimum orientation of enzyme molecules for the direct bioelectrocatalysis due to the fact that the particles of the carrier (SCG) and laccase molecules are commensurable. The specific current of oxygen reduction per enzyme molecule for the composite of SCG + laccase is five times higher than that at the composite based on ACB. The effect of the specific activity increase is observed only at ultra-thin layers of the composite material at the electrode; for the creation of the gas-diffusion oxygen electrode the active layer structure should be optimized to remove the percolation effects.     

Active Layer of an Oxygen Electrode Based on Nanocomposite Disperse Carbon Carrier + Laccase Material

Effect of the carbon material dispersivity on the efficiency of oxygen electroreduction by laccase immobilized on finely divided colloidal graphite (FCG) and carbon black AD-100 is studied. A highly active composite material based on FCG with laccase immobilized on it is proposed and investigated. This creates optimum conditions for direct bioelectrocatalysis by enzyme molecules. The specific oxygen reduction current calculated per enzyme molecule for nanocomposite FCG + laccase is five times that on an AD-100-based composite. Increasing the active-layer thickness, which is of importance for creating a gas-diffusion oxygen electrode, reduces specific activity of composite and only the activity of ultrathin layers is thickness-independent. This is explained by percolation restrictions on the electron transport, which reduce the number of catalytically active centers in the electrode's active layer that take part in reaction. The FCG particles are presumed to form agglomerates in the active layer. The size of the agglomerates is determined on the basis of computer-aided modeling of percolation processes and experimental data on the dependence of the specific capacitance of the active mass on the active-layer thickness. Hypotheses on the origin of percolation phenomena are put forth. One such hypothesis is that agglomerates of carbon particles are fractal clusters.     

Electroreduction of Oxygen in the Presence of Tyrosinase Adsorbed on Carbon Materials

Electroreduction of oxygen on carbon materials with immobilized tyrosinase is studied, and the effect the carbon support nature and the immobilization technique have on the reaction rate is determined. The tyrosinase activity in a direct bioelectrocatalysis is shown to be lower than that of another copper-containing enzyme, laccase. A maximum activity is obtained when covering the carbon surface with a composite based on tyrosine, tyrosinase, and Nafion.     

Optimizing Weights of Platinum in the Active Layer of the Cathode of a Hydrogen–Oxygen Fuel Cell with a Solid Polymer Electrolyte

The active layer of the cathode of a hydrogen–oxygen fuel cell with a solid polymer electrolyte is computer simulated. The active mass of the electrode consists of substrate grains (agglomerates of carbon particles with Pt particles embedded into them) and grains of a solid polymer electrolyte (Nafion). The substrate grains presumably contain hydrophobic pores, which facilitate the oxygen penetration into the active mass. A calculation of characteristics of such an electrode focuses on the optimization of platinum weights. The principal parameters of the system are concentration and size of grains of substrate and Nafion, Pt concentration in substrate grains, average diameter of hydrophobic pores in substrate grains, and the electrode polarization. The optimum, at a given electrode polarization, electrochemical activity of the active layer, its thickness, and the platinum weight are calculated. A link between these quantities and principal parameters of the active layer is revealed.     

高分子聚合物-多壁碳纳米管复合物固定漆酶及其在玻碳电极上的直接电子迁移

采用不同结构的高分子聚合物与纯化的多壁碳纳米管(MWCNTs)共混的方法,制备得到聚合物非共价功能化多壁碳管复合物,测定了这些载体对漆酶(lac)的担载量、固定漆酶的比活力及稳定性.以固定漆酶的复合物修饰玻碳(GC)电极后,采用循环伏安法研究这些电极在无氧磷酸盐缓冲液(PBS)中的直接电化学行为及催化氧还原活力,粗略地测定了固定漆酶与电极间电子转移的速率常数.实验结果表明,当聚合物中含亲漆酶基团或能与漆酶活性中心发生相互作用的官能团时利于直接电子转移,而且复合物固定漆酶保持了游离漆酶的天然构象.这些电极中,lac/NIPAM-co-BPCP-M WCNTs/GC(NIPAM-co-BPCP:N-烯丙基-1-苯甲酰基-3-苯基-4,5-2H-4-甲酰胺基吡唑-co-N-异丙基丙烯酰胺)在无氧PBS中发生直接电子转移的式电位(605mV)更接近漆酶活性中心的式电位(580mV),具有较快的异相电子转移速率(0.726s-1),较高的漆酶担载量(103.5mg/g)和固定漆酶比活力(1.68U/mg),较高的催化氧还原能力(氧还原起始电位820mV,在650mV时的催化峰电流为85.5μA)以及良好的重复使用性和长期使用性.     

Electrodeposition of Pt ultramicroparticles in Nafion films on glassy carbon electrodes

The electrodeposition of platinum particles into Nafion films on a glassy carbon electrode is described. The particles are dispersed three-dimensionally throughout the polymer layer. The active platinum surface area is determined by the charge required for the adsorption of hydrogen. The mass specific surface area of the particles grown in the Nafion film is surprisingly large, suggesting that the platinum particles are highly dispersed in the Nafion film. The average crystal size of the platinum clusters is in the range 10–20 nm. The exchange current density of the hydrogen evolution reaction has been determined in an acidic solution.     

Electrocatalytic Reduction of Hydrogen Peroxide on Polymethylpyrrole–Peroxidase Composite Film Plated on Gold Electrode

Horse radish peroxidase (POD), which is codeposited with polymethylpyrrole on a gold electrode, catalyzes hydrogen peroxide electroreduction. In the case of thin films deposition (Q < 4 mC cm^–2), less than one monolayer of enzyme located on the film's surface is involved in the electrochemical reaction. An increase in the limiting current points to an increase in the number of molecules accessible for the reduction of hydrogen peroxide on the surface of the composite system. The calculated values of layer's thickness suggest, for thin films, the formation of an ensemble of nanosize polymethylpyrrole-peroxidase particles. At a larger thickness of the composite coating (Q > 4 mC cm^–2), the cathodic current increases only at low polarizations. In this case, probably, the POD molecules located on the surface and in the bulk film are involved in the reaction. From these results we conclude that, in the composite system studied, polymethylpyrrole provides the electron transfer to enzyme molecules, and the reaction's mechanism is likely to be the same in the cases of both carbon and gold substrates.     

Bioelectrocatalytic Oxygen Reduction by Laccase Immobilized on Various Carbon Carriers

Laccase is an enzyme that is used for fabricating cathodes of biofuel cells. Many studies have been aimed at searching the ways for enhancing specific electrochemical characteristics of cathode with the laccase- based catalyst. The electroreduction of oxygen on the electrode with immobilized laccase proceeds under the conditions of direct electron transfer between the electrode and active enzyme center. In this work, the effect of oxygen partial pressure on the electrocatalytic activity of laccase is studied. It is shown that, at the concentrations of oxygen dissolved in the electrolyte higher than 0.28 mM, the process is controlled by the kinetics of the formation of laccase–oxygen complex, whereas at lower concentrations and a polarization higher than 0.3 V, the process is limited by the oxygen diffusion. A wide range of carbon materials are studied as the carriers for laccase immobilization: carbon black and nanotubes with various BET specific surface areas. The conditions, which provide the highest surface coverage of carbon material with enzyme in the course of spontaneous adsorptive immobilization and the highest specific characteristics when using a “floating” electrode simulating a gas-diffusion electrode, are determined: 0.2 M phosphate-acetate buffer solution; oxygen atmosphere; the carrier material (nanotubes with a BET surface area of 210 m²/g and a mesopore volume of 3.8 cm³/g); and the composition of active mass on the electrode (50 wt % of carbon material + 50 wt % of hydrophobized carbon black).     

Recent advances in surface chemistry of electrodes to promote direct enzymatic bioelectrocatalysis

Redox enzymes catalyze major reactions in microorganisms to supply energy for life. Their use in electrochemical biodevices requires their integration on electrodes, while maintaining their activity and optimizing their stability. In return, such applicative development puts forward the knowledge on involved catalytic mechanisms, providing a direct electrode connection of the enzyme is fulfilled. Enzymes being large molecules with active site embedded in an insulating moiety, direct bioelectrocatalysis supposes strategies for specific orientation of the enzyme to be developed. In this review, we summarize recent advances during the past 3 years in the chemical modification of electrodes favoring direct electrocatalysis. We present the different methodologies used according to the electrode materials, including metals, carbon-based electrodes, or porous structures and discuss the gained insights into bioelectrocatalysis. We especially focus on enzyme engineering, which appears as an emerging strategy for enzyme anchoring. Remaining challenges will be discussed with regard to these later findings.     

固酶氮掺杂碳纳米复合物基燃料电池性能

利用掺杂氮介孔材料(NDMPC)和羧甲基壳聚糖(CMCH)机械共混的纳米复合物作为固酶载体,以滴涂-干燥法分别制备了固定漆酶(Lac)阴极和固定葡萄糖氧化酶阳极,组装了有Nafion离子交换膜的葡萄糖/O₂酶燃料电池.固定漆酶电极作为燃料电池阴极和氧电化学传感器的性能以结合旋转圆盘电极技术的循环伏安法、线性扫描伏安(LSV)法以及计时电流法进行表征,同时使用紫外-可见分光光度法和石墨炉原子吸收光谱法研究酶分子在电极表面的构型和估算电极表面载体对酶的担载量.测试结果表明:固酶阴极在无电子中介体时可以实现漆酶活性中心T₁与导电基体之间的直接电子迁移(表观电子迁移速率为0.013 s^-1),而且具有较小的氧还原超电势(150 mV).通过进一步定量比较分子内电子传递速率(1000 s^-1)、底物转化速率(0.023 s^-1)以及前述酶-导电基体间电子迁移速率,可以发现此电极催化氧还原循环受制于酶-电极之间的电子迁移过程;这种电极对氧的传感性能良好:低检测限(0.04 μmol·dm^-3)、高灵敏度(12.1 μA·μmol^-1·dm³)和良好的对氧亲和力(K_M = 8.2 μmol·dm^-3),这种固酶阴极还具有良好的重现性、长期使用性、热稳定性和pH耐受性.组装的生物燃料电池的开路电压为0.38 V,最大能量输出密度为19.2 μW·cm^-2,最佳工作条件下使用3周后输出功率密度仍可保持初始值的60%以上.     

Bioelectrocatalytic Reduction of Peroxide Compounds on the Peroxidase–Nafion Composite

Electrochemical reactions of peroxide compounds (hydrogen peroxide and peracetic and perbenzoic acids) on an electrode of pyrocarbon with immobilized horseradish peroxidase (HRP) are studied. The immobilization of HRP is performed in the composition of a composite with Nafion whose structure is studied by a method of scanning tunneling microscopy. The proposed composite material provides for a high catalytic activity and stability of enzyme in the reaction of reduction of peroxide compounds. It is shown that the electrocatalytic reduction of the studied compounds on the electrode with the peroxidase–Nafion composite proceeds in conditions of direct bioelectrocatalysis. The effect of the solution pH and the concentration of substrates on the electrocatalytic activity of HRP in the composition of the composite is studied. On the basis of the obtained results a possible mechanism of the electrocatalytic reduction of peroxide compounds in the presence of HRP is suggested. The rate of a bioelectrocatalytic process is defined by the nature and concentration of the substrate as well as by the electrode potential and the solution pH.     

Direct and electrically wired bioelectrocatalysis by hydrogenase from Thiocapsa roseopersicina.

Hydrogen enzyme electrodes based on direct and mediated bioelectrocatalysis were developed. Direct bioelectrocatalysis of hydrogen oxidation/evolution was observed for hydrogenase adsorbed on carbon filament material. The equilibrium hydrogen potential was achieved on mediatorless hydrogen enzyme electrodes in hydrogen atmosphere. The electrocatalytic activity of hydrogenase in direct bioelectrocatalysis of hydrogen oxidation was two orders of magnitude higher compared to platinum. The reported electrode remained 50% activity after 6 months of storage with periodical testing. Wired bioelectrocatalysis was achieved by adsorption of hydrogenase onto electropolymerized redox mediator N-methyl-N'-(12-pyrrol-1-yl-dodecyl)-4,4'-bipyridinium ditetrafluoroborate.     

Calculating Electrochemical Activity of Regular-Structure Porous Electrodes with an Immobilized Enzyme

A porous electrode of regular structure with an immobilized enzyme is studied. The electrode carcass, which consists of substrate particles, is a system of two sets of mutually perpendicular planes crossing one another (cellular structure). A monomolecular layer of enzyme molecules is deployed on the inner surface of such a porous substrate. In the center of each cell of the substrate gas pores, which are cylinders of porous grains of a hydrophobizing agent one grain thick, are situated. The rest of the cell space is filled by a solid polymer electrolyte. The ultimate goal of calculations is to estimate the electrochemical activity of such an electrode. The estimation is done for an oxygen electrode with an enzyme whose characteristics are close to those of laccase. The calculation assumes that active centers of enzyme molecules undergo a direct, i.e. without participation of mediators, reduction. It is shown that at an overvoltage of 30 mV, it would be possible to obtain a current density of 0.44 A cm^–2 in an electrode 16 m thick.     

Thermally Expanded Graphite as Functional Material in the Technology of Electrode Material with Mixed Conductivity

Methods of differential thermal analysis and cyclic voltammetry were used to examine the functional properties of thermally expanded graphite in an electrode material for electrochemical systems. This material contains platinum, carbon black, and proton-conducting polymer Nafion. It was shown that addition of thermally expanded graphite to the electrode material makes higher the thermal stability of Nafion. Under an electrochemical treatment, thermally expanded graphite compares well in stability against this treatment with the commonly used carbon black of the Vulcan XC-72 type. A mechanism is suggested for stabilizing the proton-conducting polymer Nafion in the presence of thermally expanded graphite. It was shown that thermally expanded graphite is promising for being used in the technology of electrode materials with mixed conductivity, which contain a proton-conducting polymer of the Nafion type, as a functional additive serving to improve the thermal stability.     

An Amperometric Biosensor Based on Laccase Immobilized in Polymer Matrices for Determining Phenolic Compounds

An amperometric enzyme electrode based on laccase for determining phenolic compounds is proposed. The following three types of polymer materials were used for enzyme immobilization on the surface of a glassy-carbon electrode: positively charged cetyl ethyl poly(ethyleneimine) (CEPEI) and negatively charged commercial Nafion and Eastman AQ 29D polymers. The advantages and disadvantages of each of the above polymers for enzyme immobilization are discussed. The detection limits of the model phenolic compounds hydroquinone and pyrocatechol in a buffer solution on laccase immobilization in a Nafion membrane were 3.5 × 10⁻⁸ and 5.0 × 10⁻⁸ M, respectively, at a signal-to-noise ratio of 3. Electrodes with laccase immobilized in Nafion and Eastman AQ 29D membranes exhibited the shortest response time. The operating stability and the stability in storage can be significantly improved by the additional incorporation of gelatin in the polymer matrices. Gelatin prevents enzyme inactivation as a result of enzyme modification by the free-radical oxidation products of phenolic compounds.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 6, 2005, pp. 624–628.Original Russian Text Copyright © 2005 by Yaropolov, Shleev, Morozova, Zaitseva, Marko-Varga, Emneus, Gorton.Presented at the VI All-Russia Conference (with international participation) on Electrochemical Methods of Analysis (EMA-2004, Ufa, May 23–27, 2004).     

Fructose/dioxygen biofuel cell based on direct electron transfer-type bioelectrocatalysis

One-compartment biofuel cells without separators have been constructed, in which d-fructose dehydrogenase (FDH) from Gluconobacter sp. and laccase from Trametes sp. (TsLAC) work as catalysts of direct electron transfer (DET)-type bioelectrocatalysis in the two-electron oxidation of d-fructose and four-electron reduction of dioxygen as fuels, respectively. FDH adsorbs strongly and stably on Ketjen black (KB) particles that have been modified on carbon papers (CP) and produces the catalytic current with the maximum density of about 4 mA cm(-2) without mediators at pH 5. The catalytic wave of the d-fructose oxidation is controlled by the enzyme kinetics. The location and the shape of the catalytic waves suggest strongly that the electron is directly transferred to the KB particles from the heme c site in FDH, of which the formal potential has been determined to be 39 mV vs. Ag|AgCl|sat. KCl. Electrochemistry of three kinds of multi-copper oxidases has also been investigated and TsLAC has been selected as the best one of the DET-type bioelectrocatalyst for the four-electron reduction of dioxygen in view of the thermodynamics and kinetics at pH 5. In the DET-type bioelectrocatalysis, the electron from electrodes seems to be transferred to the type I copper site of multi-copper oxidases. TsLAC adsorbed on carbon aerogel (CG) particles with an average pore size of 22 nm, that have been modified on CP electrodes, produces the catalytic reduction current of dioxygen with a density of about 4 mA cm(-2), which is governed by the mass transfer of the dissolved dioxygen. The FDH-adsorbed KB-modified CP electrodes and the TsLAC-adsorbed CG-modified CP electrodes have been combined to construct one-compartment biofuel cells without separators. The open-circuit voltage was 790 mV. The maximum current density of 2.8 mA cm(-2) and the maximum power density of 850 microW cm(-2) have been achieved at 410 mV of the cell voltage under stirring.     

壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮-多壁碳纳米管复合物固定漆酶基化学传感器的性能

通过壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮(CTS-g-N-CSIDZ)非共价功能化多壁碳纳米管(MWCNTs)的方式制备固定漆酶载体,该复合物载体主要通过物理吸附和漆酶活性中心与载体上配体之间的配位作用来固定漆酶,较大程度地保持了游离漆酶活性位原始构象.将固定了漆酶的复合物附着在裸玻碳电极上便构筑了复合物固定漆酶修饰玻碳电极.在以分光光度法测定了这种复合物载体对漆酶的担载量、固定漆酶比活力、稳定性、重复使用性及其催化2,6-二甲氧基苯酚(DMP)氧化动力学参数的基础上,还对基于此种复合物固定漆酶修饰玻碳电极作为化学传感器(以DMP作为底物)的性能进行了研究.结果表明,该复合物具有较高的固酶担载量(81.7 mg/g)和固定漆酶比活力(1.33 U/mg);而作为电化学传感器的复合物固定漆酶修饰玻碳电极对底物DMP具有较高的亲和力(对DMP的米氏常数KM是0.0918 mmol/L),较高的灵敏度( 3680 mA· L/mol),较低的检测限(3.3×10-4 mmol/L),较高的响应选择性,良好的重现性、重复使用性和长期稳定性.这种漆酶基电极有望用作电流型特定结构的酚类传感器.     

Xanthine and hypoxanthine sensors based on xanthine oxidase immobilized in poly(mercapto-p-benzoquinone) film

The construction and performance of an enzyme electrode as an amperometric sensor of xanthine and hypoxanthine is described. Xanthine oxidase has been immobilized in a conductive redox polymer, poly(mercapto-p-benzoquinone), by means of an electropolymerization of mercaptohydroquinone in the presence of the enzyme. An Au-electroplated glassy carbon electrode coated with the resulting polymer film functioned well as a direct response type of sensor, where the polymer chain served as a conductive molecular chain between the active sites in the enzyme and the substrate electrode. Response characteristics as well as kinetic parameters have been evaluated.     

Computer Simulation of the Structure of Porous Electrodes with an Immobilized Enzyme and Nanosized Support Particles

The efficiency of the operation of a porous electrode with an immobilized enzyme is defined, in particular, by a lucky structure of its active layer, which can contain nanosized particles of the support. The composites of such a kind are prepared with the aid of methods of colloidal chemistry. The aim of this particular investigation is to perform a computer simulation of processes of coagulation of particles of the support and their possible heterocoagulation with molecules of the enzyme. Algorithms of the formation of nanocomposite structures in solution are suggested. Calculations show that the concentration of the enzyme molecules in the nanocomposite structures cannot exceed a certain critical value. On the other hand, at a fixed value of the concentration of the enzyme molecules, the concentration of the support particles must not fall below a certain threshold quantity, which provides for the passing of current through the active layer. In order for all the enzyme molecules, rather than for a fraction of these, in the composite to take part in the process of bioelectrocatalysis, the concentration of support particles must be increased even higher, to an optimum value.__________Translated from Elektrokhimiya, Vol. 41, No. 6, 2005, pp. 738–747.Original Russian Text Copyright © 2005 by Chirkov, Rostokin.     

Bioelectrocatalytic and Enzymic Activity of Laccase in Water–Ethanol Solutions

The effect the composition of a water–ethanol mixture has on the enzymic (in the pyrocatechol oxidation reaction) and bioelectrocatalytic in a broad potential range (in the oxygen reduction reaction) activity of laccase (L) is studied. On the basis of obtained results conclusions are made about the influence exerted by ethanol in the composition of the water–ethanol mixture on the activity of laccase solubilized and immobilized in the composition of a composite (laccase–Nafion). The decrease in the activity in both the enzymic and the bioelectrocatalytic reactions is probably caused by the denaturation of laccase, which is due to the replacement of the hydration shell of a protein globule by a solvation shell. Besides, there take place a retardation of the kinetic stage of the formation of a laccase–substrate complex (LHO₂OH) because of the slowness of the diffusion of water into an active center and an inhibiting effect of ethanol, which is capable of binding itself to an enzyme globule in the vicinity of the active center of laccase.