Cover Picture

The cover picture shows schematically the elements of the rapidly developing research fields of bioelectronics and optobioelectronics. Immobilization of redox enzymes as monolayers or thin films on conductive supports yields functionalized biocatalytic electrodes. The formation of electrical contact between the redox center of the enzyme and the electrode surface, which is possible in various ways, provides a means of assembling integrated enzyme electrodes for bioelectronic applications. Photoswitchable redox proteins assembled on electrode surfaces form the basis of optobioelectronic devices. A further element of bioelectronic and optobioelectronic systems is the possibility for the microscale patterning of biomaterials onto the solid supports-through photolithography, micromachining, and stamping. More on the different aspects of bioelectronics and optobioelectronics can be found in the review by I. Willner and E. Katz on page 1180 ff.[ Magnified Cover Picture ]     

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.     

Integration of Layered Redox Proteins and Conductive Supports for Bioelectronic Applications

Integration of redox enzymes with an electrode support and formation of an electrical contact between the biocatalysts and the electrode is the fundamental subject of bioelectronics and optobioelectronics. This review addresses the recent advances and the scientific progress in electrically contacted, layered enzyme electrodes, and discusses the future applications of the systems in various bioelectronic devices, for example, amperometric biosensors, sensoric arrays, logic gates, and optical memories. This review presents the methods for the immobilization of redox enzymes on electrodes and discusses the covalent linkage of proteins, the use of supramolecular affinity complexes, and the reconstitution of apo-redox enzymes for the nanoengineering of electrodes with protein monolayers of electrodes with protein monolayers and multilayers. Electrical contact in the layered enzyme electrode is achieved by the application of diffusional electron mediators, such as ferrocene derivatives, ferricyanide, quinones, and bipyridinium salts. Covalent tethering of electron relay units to layered enzyme electrodes, the cross-linking of affinity complexes formed between redox proteins and electrodes functionalized with relay-cofactor units, or surface reconstitution of apo-enzymes on relay-cofactor-functionalized electrodes yield bioelectrocatalytic electrodes. The application of the functionalized electrodes as biosensor devices is addressed and further application of electrically "wired" enzymes as catalytic interfaces in biofuel cells is discussed. The organization of sensor arrays, self-calibrated biosensors, or gated bioelectronic devices requires the microstructuring of biomaterials on solid supports in the form of ordered micro-patterns. For example, light-sensitive layers composed of azides, benzophenone, or diazine derivatives associated with solid supports can be irradiated through masks to enable the patterned covalent linkage of biomaterials to surfaces. Alternatively, patterning of biomaterials can be accomplished by noncovalent interactions (such as in affinity complexes between avidin and a photolabeled biotin, or between an antibody and a photoisomerizable antigen layer) to provide a means of organizing protein microstructures on surfaces. The organization of patterned hydrophilic/hydrophobic domains on surfaces, by using photolithography, stamping, or micromachining methods, allows the selective patterning of surfaces by hydrophobic, noncovalent interactions. Photoactivated layered enzyme electrodes act as light-switchable optobioelectronic systems for the amperometric transduction of recorded photonic information. These systems can act as optical memories, biomolecular amplifiers, or logic gates. The photoswitchable enzyme electrodes are generated by the tethering of photoisomerizable groups to the protein, the reconstitution of apo-enzymes with semisynthetic photoisomerizable cofactor units, or the coupling of photoisomerizable electron relay units.     

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.     

壳聚糖-二茂铁复合物的制备及其在电化学传感器上的应用

壳聚糖-二茂铁复合物(CHIT-Fc)由二茂铁羧酸的羧基和脱乙酰壳多糖的羟基缩合合成,并通过红外光谱检测.合成得到的壳聚糖-二茂铁复合物通过物理吸附作为固定胆固醇氧化酶(CHOx)的基体.同时,使用Nafion稀释液可以消除诸如抗坏血酸和尿酸的影响.最佳测试条件下,采用示差脉冲(DPV)研究胆固醇生物传感器的响应,在4.0×10-6mol/L～1.0×10-4 mol/L范围内,氧化峰电流与胆固醇浓度呈现良好的线性关系,线性方程为Ipa=0.0223c-0.0875(Ipa:μA,c:μmol/L,R=0.9982),检测限为5.0×10-7mol/L(S/N=3).     

Glucose Oxidase/Cellulose–Carbon Nanotube Composite Paper as a Biocompatible Bioelectrode for Biofuel Cells

Biofuel cells are devices for generating electrical energy directly from chemical energy of renewable biomass using biocatalysts such as enzymes. Efficient electrical communication between redox enzymes and electrodes is essential for enzymatic biofuel cells. Carbon nanotubes (CNTs) have been recognized as ideal electrode materials because of their high electrical conductivity, large surface area, and inertness. Electrodes consisting entirely of CNTs, which are known as CNT paper, have high surface areas but are typically weak in mechanical strength. In this study, cellulose (CL)–CNT composite paper was fabricated as electrodes for enzymatic biofuel cells. This composite electrode was prepared by vacuum filtration of CNTs followed by reconstitution of cellulose dissolved in ionic liquid, 1-ethyl-3-methylimidazolium acetate. Glucose oxidase (GOx), which is a redox enzyme capable of oxidizing glucose as a renewable fuel using oxygen, was immobilized on the CL–CNT composite paper. Cyclic voltammograms revealed that the GOx/CL–CNT paper electrode showed a pair of well-defined peaks, which agreed well with that of FAD/FADH_2, the redox center of GOx. This result clearly shows that the direct electron transfer (DET) between the GOx and the composite electrode was achieved. However, this DET was dependent on the type of CNTs. It was also found that the GOx immobilized on the composite electrode retained catalytic activity for the oxidation of glucose.     

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.     

Tetrathiafulvalene-based molecular nanowires

A new molecular wire suitably functionalized with sulfur atoms at terminal positions and endowed with a central redox active TTF unit has been synthesized and inserted within two atomic-sized Au electrodes; electrical transport measurements have been performed in STM and MCBJ set-ups in a liquid environment and reveal conductance values around 10(-2) G0 for a single molecule.     

Protein electrochemistry using aligned carbon nanotube arrays

The remarkable electrocatalytic properties and small size of carbon nanotubes make them ideal for achieving direct electron transfer to proteins, important in understanding their redox properties and in the development of biosensors. Here, we report shortened SWNTs can be aligned normal to an electrode by self-assembly and act as molecular wires to allow electrical communication between the underlying electrode and redox proteins covalently attached to the ends of the SWNTs, in this case, microperoxidase MP-11. The efficiency of the electron transfer through the SWNTs is demonstrated by electrodes modified with tubes cut to different lengths having the same electron-transfer rate constant.     

Layer-by-layer electrostatic deposition of biomolecules on surfaces for molecular recognition, redox mediation and signal generation

Layer-by-layer supramolecular structures composed of alternate layers of negatively charged enzymes and cationic redox polyelectrolyte have been assembled. Glucose oxidase (GOx), lactate oxidase (LOx) and soybean peroxidase (SBP) have been electrically wired to the underlying electrode by means of poly(allylamine) with [Os(bpy)2ClPyCOH]+ covalently attached (PAA-Os) in organized structures with high spatial resolution. Biotinylated glucose oxidase has also been used to assemble step-by-step on antibiotin goat immunoglobulin (IgG) layers and the enzyme was electrically wired by PAA-Os. These spatially organized multilayers with mono- and bienzymatic schemes can work efficiently in molecular recognition, redox mediation and generation of an electrical signal. The concentration of redox mediator integrated into the multilayers, obtained from the voltammetric charge and an estimation of the layer thickness, exceeds by 100-fold the amount of deposited enzyme assessed by quartz crystal microbalance. Differences in GOx electrical wiring efficiency have been detected with the different assembling strategies. The surface concentration of electrically wired enzyme represents a small proportion of all the enzyme molecules present in the multilayers which can be oxidized by the soluble mediator [Os(bpy)2Cl PyCOOH]Cl. This proportion, as well as the rate of FADH2 oxidation by PAA-Os, increases with the number of electrically wired enzyme layers and with the spatial accessibility of the Os moiety to the enzyme active center.     

Photochemical Generation of Ferrocene‐Based Redox‐Polymer Networks

A photochemical approach toward the generation of enzyme‐containing redox polymer networks, which are the key material in enzymatic sensors and biofuel cells, is described. The approach is based on the incorporation of photo‐reactive benzophenone groups into the redox polymers. The obtained polymers are then deposited on the surface of glassy carbon electrodes and cross‐linked by illumination with UV light at 365 nm. If this step is done in the presence of the enzyme glucose oxidase, functional electrodes are obtained that yield electrical power upon addition of glucose. This work specifically addresses the question of electrode stability in buffer and demonstrates how slight variations in the chemistry of the redox polymer have a dramatic effect on the electrochemical performance of the electrodes. Different ferrocene‐containing redox polymer networks are synthesized and their properties in physiological buffer are studied.

     

Electric double layer effect on observable characteristics of the tunnel current through a bridged electrochemical contact

Scanning tunneling microscopy and electrical conductivity of redox molecules in conducting media (aqueous or other media) acquire increasing importance both as novel single-molecule science and with a view on molecular scale functional elements. Such configurations require full and independent electrochemical potential control of both electrodes involved. We provide here a general formalism for the electric current through a redox group in an electrochemical tunnel contact. The formalism applies broadly in the limits of both weak and strong coupling of the redox group with the enclosing metal electrodes. Simple approximate expressions better suited for experimental data analysis are also derived. Particular attention is given to the effects of the Debye screening of the electric potential in the narrow tunneling gap based on the limit of the linearized Poisson-Boltzmann equation. The current/overpotential relation shows a maximum at a position which depends on the ionic strength. It is shown, in particular, that the dependence of the maximum position on the bias voltage may be nonmonotonous. Approximate expressions for the limiting value of the slope of the current/overpotential dependence and the width of the maximum on the bias voltage are also given and found to depend strongly on both the Debye screening and the position of the redox group in the tunnel gap, with diagnostic value in experimental data analysis.     

Oriented immobilization of a membrane-bound hydrogenase onto an electrode for direct electron transfer

The interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble enzymes than with membrane enzymes because of the higher structural complexity and instability of the latter proteins. In this work, we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H(2)-oxidation reversibly and is associated with the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, allowing only mediated electron transfer between the enzyme and electrode. However, a one-step immobilization procedure favors the formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted into a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.     

Integrated electrochemical transistor as a fast recoverable gas sensor

A new design of conductometric chemical sensors based on conducting polymers as chemosensitive elements was suggested. The sensor includes six electrodes. Four inner electrodes coated by chemosensitive polymer are used for simultaneous two- and four-point resistance measurements thus providing information on the bulk polymer resistance and on the resistance of the polymer/electrode contacts. Two outer electrodes wired to inner electrodes by polymeric electrolyte are used for electrical control of redox state of the chemosensitive polymer. The outer electrodes are connected to potentiostat as reference and counter electrodes. It allows us to control redox state of the inner (working) electrodes. This new measurement configuration, resembling chemosensitive electrochemical transistors, provides an internal test of the sensor integrity and an electrically driven sensor regeneration. It was tested as a sensor for the detection of nitrogen dioxide. Polythiophene or polyaniline was used as receptors. Cyclic voltammograms of these polymers on the sensor surface measured in air atmosphere were very similar to that measured in aqueous electrolyte. A control of conductivity of these chemosensitive polymers by electrical potential applied vs. incorporated reference electrode was demonstrated. This effect was used for the regeneration of the chemosensitive material after exposure to nitrogen dioxide: in comparison to usual chemiresistors displaying an irreversible behavior in such test even in the time scale of hours, a completely reversible sensor regeneration within few minutes was observed.     

Layer-by-layer electrodeposition of redox polymers and enzymes on screen-printed carbon electrodes for the preparation of reagentless biosensors

Layer-by-layer electrodeposition of redox polymer/enzyme composition films on screen-printed carbon electrodes for fabrication of reagentless enzyme biosensors has been proposed and the resulting films were found to be very stable and rigid.     

Electroanalytical measurements without electrolytes: Conducting polymers as probes for redox titration in non-conductive organic media

Electroanalytical methods have been applied only in conducting media. An application of conducting polymers allows to overcome this limitation. If such material is in electrochemical equilibrium with dissolved redox active species, its electrical conductivity depends on the redox potential of these species. Therefore, conductometric measurements with conducting polymers can provide about the same information as classical redox electrodes. The approach was applied for redox titration. Equivalent points obtained by this titration in aqueous and organic electrolytes were identical. Then the approach was applied for determination of bromine number by redox titration in non-conducting organic phase.     

Electrochemical studies on reconstituted horseradish peroxidase modified carbon paste electrodes

Horseradish peroxidase (HRP) is a heme protein that acts specifically on H(2)O(2) as the electron acceptor. Hemin (Ferriprotoporhyrin-IX) is the prosthetic group of the enzyme. A direct molecular wire to the redox center of the enzyme is expected to enhance the electrochemical response of the enzyme. Native HRP was immobilized onto the surface of glassy carbon (GC) matrix using a 16-atom spacer arm. We have also immobilized the redox center of the enzyme (hemin) through one of the propionate groups onto the surface of glassy carbon matrix using an 11-atom spacer arm with amino terminus. Apoperoxidase was isolated according to the Teale's method and was allowed to reconstitute with the hemin-bound matrix for enzyme reconstitution. The HRP paste and reconstituted-HRP (rec-HRP) paste electrodes were used to study the electrochemical response to substrate H(2)O(2) using electrochemical techniques like cyclic voltammetry (CV) and flow injection (FI) studies. Flow injection studies using HRP paste electrode showed a linearity from 25 to 200 microM H(2)O(2). The rec-HRP paste showed approximately 100 times increase in the electron transfer rates compared to native HRP paste, and substrate linearity from 25 to 100 microM was observed.     

Conducting polymer based amperometric enzyme electrodes

The construction and the properties of conducting-polymer based amperometric enzyme electrodes are reviewed. The main aim is to focus on the properties of conducting polymer films which are important for the construction of amperometric enzyme electrodes. Additionally, the review is focused on electron-transfer pathways between conducting-polymer integrated immobilized enzyme molecules and the modified electrode using free-diffusing redox mediators as well as direct electron transfer via the conducting-polymer wires. Possible future applications using microstructured conducting-polymer films will be discussed.     

Direct Heterogeneous Electron Transfer Reactions of Bacillus halodurans Bacterial Blue Multicopper Oxidase

Direct electron transfer reactions of Bacillus halodurans bacterial multicopper oxidase on bare spectrographic graphite, as well as bare and thiol‐modified gold electrodes were studied using cyclic voltammetry, potentiometry, amperometry, and spectroelectrochemistry. The redox potential of the T1 site of the enzyme was measured using mediatorless redox titration and found to be 325 mV±10 mV vs. NHE. From measurements with a mercaptopropionic acid‐modified gold electrode under aerobic conditions a midpoint potential of 360 mV vs. NHE for the T2/T3 copper cluster is deduced. Differing from most other characterized laccases of fungal and plant origins this bacterial enzyme exhibits bioelectrocatalytic activity at neutral pH and tolerates high chloride concentrations (200 mM), conditions that usually strongly inhibit catalysis. Moreover, it has the very high affinity towards molecular oxygen both in solution and in the adsorbed state (K_M≤50 μM).