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.     

Structure and Properties of Polymer Biocomposite Materials

Results of studying the structure and properties of biocomposite materials are summarized. The materials in question include an enzyme (laccase, peroxidase), an ion- or electron-conducting polymer (Nafion, polymethylpyrrole), and a carbon substrate (compact, disperse). It is shown that the orientation of a large number of enzyme molecules in an enzyme/Nafion composite material on the substrate surface favors direct bioelectrocatalysis. During co-immobilization of an enzyme and polymethylpyrrole, conditions are realized under which the polymer takes part in the electron transfer between the active center of the enzyme and the surface of the electroconducting substrate. A fresh approach to constructing a biocomposite material is developed. The material is based on an extremely finely divided carbon material (colloidal graphite), which ensures a high specific activity of laccase immobilized on it. The size of colloidal-graphite particles is commensurate with that of the laccase molecule, owing to which the enzyme macromolecule is surrounded by carbon particles. As a result, practically all adsorbed enzyme molecules are electrochemically active and participate in direct bioelectrocatalysis.     

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.     

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).     

Hydrophobized oxygen cathode of a fuel cell with a liquid electrolyte: Calculating overall currents and thicknesses

The mechanism governing operation of hydrophobized cathodes is discussed. A model is proposed for the active-layer structure. The model consists of equidimensional hydrophobic (agglomerates of polytetrafluoroethylene particles) and hydrophilic (agglomerates of carbon black particles with the catalyst on them) grains. The percolation characteristics of the model are calculated: the presence of a gas cluster and an ionic cluster is established, the specific area of contact between these clusters is determined, the magnitude of ionic conductivity is assayed, and so forth. The “model of cylindrical gas pores” is selected for calculating the overall current. Formulas for the bulk current density are determined. The overall characteristics of a cathode with a platinum catalyst on a carbonaceous carrier (on carbon black) in 7 M KOH at a temperature of 60°C are calculated with allowance made for the fact that the Tafel plots for the process of reduction of oxygen on platinum have two segments with different slopes.     

漆酶在纳米金溶胶/多重壁碳纳米管复合载体上固定方法的比较及粒子尺寸效应

以纳米金溶胶（NGS）和多重壁碳纳米管（MWCNTs）的共混物(NGS/MWCNTs)作为固定漆酶的载体,研究了3种固定漆酶方法在酶固定量、比活力上的差异。 研究了不同的固定方法对固定酶热稳定性和重复使用性及纳米金溶胶颗粒粒径对酶固定量和固定酶动力学参数的影响。 实验结果表明,NGS/MWCNTs具有良好的固定漆酶能力和高固酶比活力,NGS/MWCNTs（NGS粒径37 nm）通过简单物理吸附法固定漆酶的量和固酶的比活力最高,分别可达33.80 mg/g和9.433 U/mg。 在NGS-MWCNTs上采用化学键合方法固定的漆酶在70 ℃放置2 h后仍然保持初始活力的75％,重复使用20次后仍保持初始活力的70％。 纳米金溶胶粒子越小（24 nm）,底物和固定漆酶间亲和力越好（KM=0.027 mmol/L）,表观速率常数越大。     

Active layer of fuel cell electrode with polymer electrolyte: Nature of proton and oxygen supply channels

The nature of proton and oxygen supply channels in the active layer of a cathode of fuel cell with polymer electrolyte is discussed. There are three types of electron, proton, and oxygen carriers in the active layer: agglomerates of carbon particles with supported platinum (support grains), agglomerates of Nafion molecules (Nafion grains), and void grains. In computer simulation of the active layer structure, the three types of grains were assumed equal-sized, cube-shaped and arranged into a cubic node lattice (in the terms of the percolation theory). Impossibility of forming on the basis solely the above three grain types of three percolation clusters (??electron??, ??proton??, and ??gas??) that could supply all that is required for the electrochemical process is proved. But in this, the following question arises: how can satisfactory operation of the cathode with polymer electrolyte be provided? The required supply of protons and oxygen can be provided only if the support grains can feature not only electronic conductivity, but can also participate in transport of both protons and oxygen. As a result, the transport of protons and oxygen is carried out via special combined percolation clusters that must include apart from the support grains either Nafion grains (combined ??proton?? cluster) or void grains (combined ??gas?? cluster). The paper describes the technique of calculation of effective specific conductivity of a combined ??proton?? cluster. The effective specific diffusion coefficient of a combined ??gas?? cluster can also be calculated in a similar way.     

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.     

Effect of the nature of carbonaceous supports on the bioelectrocatalytic activity of peroxidase, immobilized on the supports, in the hydrogen peroxide reduction reaction

Electrochemical properties of such disperse carbonaceous materials as acetylene black AD-100, finely divided colloidal graphite (FCG), ultradisperse diamond (UDD), and carbon nanotubes (CNT) are examined. Effect of the nature of disperse carbonaceous supports on bioelectrocatalytic activity of adsorbed peroxidase (POD) in the hydrogen peroxide reduction reaction is investigated. It is shown that the hydrogen peroxide reduction on the biocatalysts studied proceeds in conditions of direct bioelectrocatalysis independently of the disperse-support type. It is also demonstrated that the biocatalysts’ activity depends on the structure and properties of the surface of the supports defining the magnitude of the POD adsorption in an orientation favorable for direct bioelectrocatalysis. Maximum activity is inherent in the catalysts manufactured on the basis of materials with moderate hydrophobic and hydrophilic properties. By the magnitude of the activity in the hydrogen peroxide reduction reaction, depending on the nature of the carbonaceous support, the fabricated catalysts (carbonaceous material with adsorbed POD) form the series AD-100, CNT > FCG > UDD.     

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.     

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.     

大尺寸SiO2大孔材料固定化漆酶

以表面固定Cu2+的改性大尺寸SiO2大孔材料作为载体,考察了时间、pH和给酶量对漆酶固定化效果的影响,并对固定化漆酶的活性和稳定性进行了研究。结果表明:5 h时吸附达到平衡,pH为4.5、漆酶与载体比例为5 mg·g-1时固定化效果最好,酶活回收率可达到100.4%;固定化漆酶的最适pH和最适温度较游离漆酶的均有升高且范围变宽,固定化后,漆酶的pH稳定性和热稳定性都得到显著提高;固定化漆酶的K m值略高于游离漆酶的;固定化漆酶具有良好的操作稳定性,与底物反应反复操作10批次后剩余酶活为72.7%。     

云芝漆酶在N,N'-亚甲基双丙烯酰胺（BIS）交联聚甲基丙烯酸基元上的固定及其修饰玻碳电极电化学行为

以N,N′-亚甲基双丙烯酰胺（BIS）交联聚甲基丙烯酸作为固定漆酶的载体,以共价偶联法固定云芝漆酶并测定了固定基元的酶固定量和固定漆酶的比活力。 还研究了固定漆酶热稳定性、重复使用性以及固定漆酶催化2,6-二甲氧基苯酚（DMP）氧化的酶动力学参数。 实验结果表明,这种交联聚合物基元通过共价偶联法固定漆酶的量和固定漆酶的比活力分别可达26.37 mg/g和1.202 U/mg;在交联聚合物基元上固定的漆酶在50 ℃下放置2 h后仍然保持初始活力的83％,重复使用10次后仍保持初始活力的80％以上;交联聚合物固定漆酶催化DMP氧化的表观速率常数k_cat可达1090 min^-1,以固定漆酶的BIS交联聚甲基丙烯酸功能化碳纳米管修饰的玻碳电极在pH=4.4磷酸盐缓冲液中氧还原发生在+724 mV(vs.SCE)。     

Porous Electrodes with Immobilized Enzymes: The Problem of Development of Nanocomposites with High Concentrations of Molecules of Active Enzymes

The currents that are generated in a porous electrode with an immobilized enzyme increase with increasing concentration of molecules of an electrochemically active enzyme. However, a finely divided composite, which is manufactured from colloidal particles of a support that have nanodimensions and molecules of the enzyme with the aid of methods of colloid chemistry, has a peculiar structure: it consists of a set of fractal clusters, which are capable of adsorbing only a limited number of enzyme molecules. The paper is devoted to computer simulation of all the stages of immobilization of the enzyme, specifically, producing random fractal clusters of required dimensions and deploying molecules of the enzyme on them. An analysis of the link of the concentration of molecules of an active enzyme with the structure and characteristics of a porous composite makes it possible to give an interpretation to experimental facts obtained by other authors for an oxygen electrode consisting of finely divided colloidal graphite and laccase.     

Porous Electrodes with Immobilized Enzymes: The Fractal-Percolation Properties of Supports Manufactured from Particles of Finely Divided Colloidal Graphite

The development of a porous active layer with an immobilized enzyme of a sufficiently large thickness is one of the problems that unavoidably emerge when constructing biofuel cells with high characteristics. Mounting up the thickness can be obstructed not only by the ohmic and diffusion limitations, which have been studied well enough. One more possibility of limitations (supports manufactured from finely divided colloidal graphite, FDCG), namely a “ fractal-percolation effect,” which has recently been discovered experimentally, is discussed in the paper. The essence of the effect consists of that the particles that are constituting a porous support may gather in random fractal clusters, which are connected with one another (the percolation part of the problem) with a probability that is other than unity. As a result, the electrons that are required for performing bioelectrocatalysis are capable of penetrating into a porous support only to a limited depth. Computer simulation of the fractal and percolation processes is performed in this work. As a result, quantitative relationship of the bulk concentration of FDCG in solution with the size of random fractal clusters, with the probability of their contact with one another, and with the degree of providedness of the material of the support by electrons is established. It may happen that all this information can become useful for the development of porous electrodes with an immobilized enzyme of high activity.__________Translated from Elektrokhimiya, Vol. 41, No. 8, 2005, pp. 943–953.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.     

Cathode of a hydrogen-oxygen fuel cell with a solid polymer electrolyte: The effect of the flooding of pores by water on the characteristics of the active layer

A computer model of the active layer of the cathode of a hydrogen-oxygen fuel cell with a solid polymer electrolyte is studied. The active mass of the electrode consists of equidimensional grains of the substrate (agglomerates of carbon particles with platinum particles embedded in them) and a solid polymer electrolyte (Nafion). The flooding by water can be experienced by both the pores in the substrate grains, which facilitate the oxygen penetration into the active layer of the electrode, and the voids between the grains. All possible versions of the flooding of these pores by water are considered. A calculation of the optimum, at a given polarization of the electrode, value of electrochemical activity, the thickness of the active layer, and the weight of platinum is performed. The major parameters of the system are the concentrations of grains of the substrate and solid polymer electrolyte, the size of these grains, the platinum concentration in the substrate grains, the average diameter of pores in the substrate grains, and the polarization of electrodes. The ultimate aim of the work is to estimate how the flooding of pores of the active layer of the cathode by water affects the magnitude of the optimum current, the effective thickness of the active layer, and the weight of platinum.Translated from Elektrokhimiya, Vol. 41, No. 1, 2005, pp. 35–47.Original Russian Text Copyright © 2005 by Chirkov, Rostokin.     

Fuel cell oxygen cathode with Nafion and platinum: the effect of active layer heating on overall cathode characteristics

A fuel cell with Nafion and platinum is considered. The effect of heating of the oxygen cathode active layer on the cathode overall characteristics (current and power density) is taken into account for the first time. Attention is focused on calculations of Tafel plots of oxygen cathodes and also on how the active layer temperature changes with the potential. Calculation parameters are as follows: fuel cell initial temperature, cathode active layer thickness, gas-diffusion layer effective heat conductivity and thickness. The following conditions of cathode operation are studied: (1) heat formed in the cathode active layer is almost completely removed, no active layer heating is observed, the active-layer temperature remains equal to that of fuel cell operation; (2) heat removal is impeded, the heat conductivity of the gas-diffusion layer is insufficiently high to remove heat. In the latter case, the active layer temperature may increase by several tens of degrees. A fundamental difference of Tafel plots for the catalytic layers studied in model experiments and the cathodic active layers is demonstrated. In the latter case, the first Tafel plot segment may extend further up to potentials of ∼0.6 V.     

聚苯并咪唑-漆酶复合物修饰电极无电子传递媒介体催化氧还原性能

采用循环伏安法将聚苯并咪唑和漆酶的复合物共沉积在玻碳电极表面。 制备的漆酶基电极在O2气饱和的磷酸盐缓冲液中可以观察到明显的催化还原电流,实现了无媒介体的酶-电极间直接电子迁移,电极静止时氧还原起始电位为645 mV,近于漆酶活性位T1的式电位580 mV,而极限扩散催化电流密度可达318.5×10-6 A/cm2。 但由于O2气在致密的固酶导电聚合物修饰层中扩散不够快(扩散系数只有在溶液中的1.25％),导致电极以较高速度旋转时极限扩散催化电流密度仅仅增加到1×10-3 A/cm2。 根据静态时极限催化电流密度求算得到的固定漆酶催化氧还原平均转化率为21.7/s。 这种漆酶基电极具有良好的重现性和长期使用性(储存10 d后催化活力仍然保持了初始值的80％以上),在人体生理温度和弱酸性条件下具有最佳催化活力。 这种漆酶基电极作为氧传感器具有良好的传感性能：检测限低(0.5 μmol/L),灵敏度高(71.1 μA·L/mmol),且对O2具有良好的亲和力(KM=89.9 μmol/L)。     

Carbon ceramic nanoparticulate film electrode prepared from oppositely charged particles by layer-by-layer approach

Carbon ceramic nanoparticulate film electrode was prepared from negatively charged sulfonated carbon nanoparticles and positively charged silicate submicrometre particles with appended imidazolium groups. They were immobilized on an indium tin oxide surface by layer-by-layer method: alternative immersion into suspension of positive and negative particles. The film formation is confirmed by scanning electron microscopy – after larger number of immersion and withdrawal steps more material is deposited on the electrode substrate. The nanoparticulate film is stable and the obtained electrode exhibits a significant increase of capacitive current and faradaic current corresponding to hydrogen peroxide electroreduction. The effect of the number of immersion and withdrawal steps is strikingly visible on dioxygen bioelectrocatalytic reduction current after adsorption of the enzyme laccase.