细胞色素C在吡啶、聚吡啶修饰的金电极上的直接电化学

研究了细胞色素C在吡啶修饰的金电极上的电化学反应,结果表明,只具有一个功能团的吡啶分子和它的聚合物对细胞色素C电化学反应也有促进作用,讨论了影响促进剂促进作用的主要因素。     

维生素B1对细胞色素C电化学反应的影响

本文探讨了细胞色素Ｃ在维生素Ｂ１修饰金电极上的电化学反应，结果表明维生素Ｂ１是细胞色素Ｃ电化学反应很好的促进剂而且它的促进作用与金电极表面维生素Ｂ１分子数量相关。     

细胞色素C在单糖修饰金电极上的直接电化学

Hill等发现在4,4′-联吡啶存在时,细胞色素C在金电极上能进行准可逆的电化学反应。在研究细胞色素C的直接电化学过程中,人们又发现一些生物小分子如氨基酸、嘌呤等对细胞色素C的电化学反应有促进作用,但迄今未见有关糖类分子对细胞色素C电化学反应促进作用研究的报道。本文研究了5种单糖对细胞色素C电化学反应的促进作用。     

Reduction of cytochrome c at a lipid bilayer modified electrode

The reduction of horse heart cytochrome c has been investigated at a platinum electrode modified with a lipid bilayer membrane (BLM) which immobilized vinyl ferrocene as an electron mediator. The current—voltage curves show that the direct electrochemistry of cytochrome c at the metal electrode occurs quite efficiently. An adsorption equilibrium constant for cytochrome at the BLM surface, as well as an electron transfer rate constant between the protein and the modified electrode have been estimated from these results. The values of both parameters are much higher than those reported with other types of electrode modifications, indicating that a lipid bilayer-modified platinum electrode system using vinyl ferrocene as a mediator provides substantial improvements in electrochemical activity of cytochrome c at metal electrodes. The potential for modifying and utilizing this new class of “biomembrane-like” electrode surface for metalloprotein electrochemistry is briefly discussed.     

Biosynthesis of gold nanoparticles assisted by Escherichia coli DH5α and its application on direct electrochemistry of hemoglobin

In this communication, biosynthesis of gold nanoparticles assisted by Escherichia coli DH5α and its application on direct electrochemistry of hemoglobin are reported. The gold nanoparticles formed on the bacteria surface are mostly spherical. The direct electrochemistry of hemoglobin can be achieved by incorporated into the bio-nanocomposite films on a glassy carbon electrode.     

Electrochemistry and biosensing activity of cytochrome c immobilized on a mesoporous interface assembled from carbon nanospheres

We report on an amperometric biosensor for hydrogen peroxide. It is obtained via layer-by-layer assembly of ordered mesoporous carbon nanospheres and poly(diallyldimethylammonium) on the surface of an indium tin oxide (ITO) glass electrode and subsequent adsorption of cytochrome c. UV–vis absorption spectroscopy was applied to characterize the process of forming the assembled layers. Cyclic voltammetry revealed a direct and quasi-reversible electron transfer between cytochrome c and the surface of the modified ITO electrode. The surface-controlled electron transfer has an apparent heterogeneous electron-transfer rate constant (k _s ) of 5.9?±?0.2?s^?1 in case of the 5-layer electrode. The biosensor displays good electrocatalytic response to the reduction of H₂O₂, and the amperometric signal increase steadily with the concentration of H₂O₂ in the range from 5?μM to 1.5?mM. The detection limit is 1?μM at pH 7.4. The apparent Michaelis-Menten constant (K _m ) of the sensor is 0.53?mM. We assume that the observation of a direct electron transfer of cytochrome c on mesoporous carbon nanospheres may form the basis for a feasible approach for durable and reliable detection of H₂O₂.

Interfacial electrochemistry of cytochrome c at tin oxide,indium oxide,gold, and platinum electrodes

Cyclic voltammetry has been used to study the heterogeneous electron transfer kinetics of horse heart cytochrome c in pH 7 tris/cacodylate media at several electrode surfaces. Reversible voltammetric responses (formal heterogeneous electron transfer rate constant>10^?2 cm/s) were observed at bare gold electrodes and at tin-doped indium oxide semiconductor electrodes for certain experimental conditions. Quasireversible voltammetric responses were more typically observed at fluorine-doped tin oxide semiconductor electrodes, bare platinum electrodes, and at the indium oxide electrodes. Reaction rates at bare metal electrodes were strongly dependent on pretreatment procedures and experimental protocol. Reaction rates at metal oxide electrodes were strongly dependent on solution conditions, pretreatment procedures, and on the hydration state of the electrode surface. A general mechanistic scheme involving both interfacial electrostatic and chemical interactions is proposed for cytochrome c electrode reactions. The asymmetric distribution of surface charges on cytochrome c appears to play a dominant role in controlling electron transfer rates by its interaction with the electric field at the electrode surface. Electron transfer distances are also considered, and it is concluded that electron transfer between an electrode surface and the exposed heme edge of properly oriented cytochrome c molecules involves maximum distances of ca. 0.6–0.9 nm.     

Electorchemical Studies of Cytochrome c on Electodes Modified by Single—Wall Carbon Naotubes

Single-wall carbon nanotubes(SWNTs) modified gold electrodes were prepared by using two different methods.The electrochemical behavior of cytochrome c on the modified gold electrodes was investigated.The first kind of SWNT-modified electrode (noted as SWNT/Au electrode)was prepared by the adsorption of carboxylterminated SWNTs from DMF dispersion on the gold electrode.The oxidatively processed SWNT tips were covalently modified by coupling with amines (AET) to form amide linkage.Via Au-S chemical bonding,the self-assembled monolayer of thiol-unctionalized nanotubes on gold surface was fabricated so as to prepare the others SWNT-modified electrode (noted as SWNT/AET/Au electrode).It was shown from cyclic voltammetry cxperiments that cytochrome c exhibited direct electrochemical responses on the both electrodes, but only the current of controlled diffusion existed on the SWNT/Au electrode while both the currents of controlled diffusion and adsorption of cytochrome c occurred on the SWNT/AET/Au electrode.Photoelastic Modulation Infared Reflection Absorpthion Spectroscopy (PEM-IRRAS) and Quartz Crystal Microbalance (QCM) were employed to verify the adsorption of SWNTs on the gold electrodes.The results proved that SWNTs could enhance the direct electron transfer proecss between the electrodes and redox proteins.     

Direct electrochemistry of cytochrome c on a phosphonic acid terminated self-assembled monolayers

The direct electrochemistry of cytochrome c (cyt c) on a gold electrode modified with 3-mercaptopropylphosphonic acid [HS-(CH₂)₃-PO₃H₂, MPPA] self-assembled monolayers (SAMs) was for the first time investigated. Electrochemical measurements and surface-enhanced infrared absorption spectroscopic reveal that the adsorption kinetics of cyt c on the MPPA-SAMs is very fast (saturation adsorption is completed within 5 s) and the immobilized cyt c molecules retain their native secondary protein structure. The nature of interaction between cyt c and -PO₃H₂ groups is mainly the electrostatic interaction. The direct electrochemistry of the immobilized cyt c on the -PO₃H₂ terminated SAMs with short chain is nearly reversible. Its formal potential (E⁰′ = 18 ± 3 mV vs. SCE) is very close to that of cyt c in an aqueous solution (E⁰′ = 18-22 mV vs. SCE). In addition, the electron transfer rate of cyt c immobilized on -PO₃H₂ terminated SAMs is relatively slow as compared to -SO₃H and -COOH terminated SAMs, indicating excess negative charge density on the SAMs surface will decrease the electron transfer rate of cyt c.     

Direct Electrochemistry of Cytochrome c at Gold Electrode Modified with Fumed Silica

Direct electrochemistry of horse heart cytochrome c (cytc) has been obtained at a gold electrode constructed by self‐assembling fumed silica particles (FSPs) onto a silane monolayer. A pair of well‐defined and nearly symmetrical redox peaks of cytc is obtained at the FSPs film modified gold electrode. Cyclic voltammetry (CV) and tapping‐mode atomic force microscopy (AFM) are used to characterize the FSPs film modified electrode, showing that the FSPs can provide a favorable microenvironment for cytc and facilitate the direct electron transfer between the cytc and the gold electrode, which may propose an approach to realize the direct electrochemistry of other proteins.     

A Simple Fabrication Method for Three‐Dimensional Gold Nanoparticle Electrodes and Their Application to the Study of the Direct Electrochemistry of Cytochrome c

A simple method for constructing gold nanoparticle‐modified electrodes with three‐dimensional nanostructures is demonstrated. The electrodes were prepared by casting citrate‐reduced AuNPs onto polycrystalline gold electrodes. The resultant electrodes had a large surface area‐to‐volume ratio, adequate for high protein loading and conferring high stability. The gold nanoparticle electrodes were covered with a self‐assembled monolayer of 11‐mercaptoundecanoic acid for electrostatic immobilization of cytochrome c (cyt c). At the electrode, direct, reversible electron transfer from cyt c was observed with remarkable stability. Moreover, an extremely high surface coverage of electrochemically active cyt c, 167 fully packed monolayers, was obtained through use of the electrode.     

Immobilization and direct electrochemistry of cytochrome <Emphasis Type="BoldItalic">c</Emphasis> at a single-walled carbon nanotube-modified electrode

A single-walled carbon nanotube (SWNT)-modified electrode was fabricated and characterized by SEM and ac impedance techniques. The direct electrochemistry of cytochrome c (Cyt c), which was adsorbed on the surface of the SWNT, was studied by cyclic voltammetry. The results from cyclic voltammetry and infrared spectroscopy indicated that Cyt c remained in its original structure and did not undergo structural change after its immobilization on the SWNT. Further results demonstrated that the SWNT had promotional effects on the direct electron transfer of Cyt c and also indicated that the immobilized Cyt c retained its electrocatalytic activity to the reduction of H₂O₂. This modified electrode might be used in development of new biosensors and the biofuel cells.     

Analysis of a high redox potential heme in tetraheme cytochrome c3 by direct electrochemistry

Cytochrome c₃ from Desulfovibrio vulgaris (Miyazaki F), a redox protein, contains four bis-histidine-coordinated hemes and has lower redox potential than other heme proteins. Direct electrochemical measurements of cytochrome c₃ were carried out using a pyrolytic graphite edge (PGE) electrode. A low redox potential, already measured by redox titration, and a high redox potential (− 245 mV vs. Ag/AgCl) were observed at room temperature. The high redox potential of cytochrome c₃ was similar to that observed for the loss of an axial ligand at heme. To investigate the loss of the histidine ligand, we explored the electrochemistry of four cytochrome c₃ mutants, in which the sixth coordinated histidine was replaced by methionine. The electrochemistry of the cytochrome c₃ mutants indicated that only Heme III undergoes loss of its axial histidine ligand.     

455 — Effect of pH on the electroactivity of horse heart cytochrome c

The effect of pH on the electroactivity of horse heart cytochrome c has been studied by differential pulse polarography, differential pulse voltammetry at the gold electrode, cyclic voltammetry and spectro-photometry; the gold electrode has been activated by 4.4′-bipyridine. The experimental results have been interpreted on the basis of the existence of a pH-dependent equilibrium between two electroactive cytochrome c forms; in agreement with the Lambeth et al.'s scheme the existence of transient species can be postulated.From spectrophotometric and differential pulse voltammetric experiments the pK_a value of 8.1±0.1 has been calculated for the equilibrium between the neutral and alkaline cytochrome c forms: state III ? state IV+H⁺.     

Direct electron transfer of cytochrome c and its biosensor based on gold nanoparticles/room temperature ionic liquid/carbon nanotubes composite film

A robust and effective composite film based on gold nanoparticles (GNPs)/room temperature ionic liquid (RTIL)/multi-wall carbon nanotubes (MWNTs) modified glassy carbon (GC) electrode was prepared by a layer-by-layer self-assembly technique. Cytochrome c (Cyt c) was successfully immobilized on the RTIL-nanohybrid film modified GC electrode by electrostatic adsorption. Direct electrochemistry and electrocatalysis of Cyt c were investigated. The results suggested that Cyt c could be tightly adsorbed on the modified electrode. A pair of well-defined quasi-reversible redox peaks of Cyt c was obtained in 0.10 M, pH 7.0 phosphate buffer solution (PBS). RTIL-nanohybrid film showed an obvious promotion for the direct electron transfer between Cyt c and the underlying electrode. The immobilized Cyt c exhibited an excellent electrocatalytic activity towards the reduction of H₂O₂. The catalysis currents increased linearly to the H₂O₂ concentration in a wide range of 5.0 × 10⁻⁵– 1.15 × 10⁻³ M. Based on the multilayer film, the third-generation biosensor could be constructed for the determination of H₂O₂.     

UV-ozone dry-cleaning process for indium oxide electrodes for protein electrochemistry

The present study reports, for the first time, on electrochemical responses of cytochrome c at a UV-ozone treated indium oxide electrode. Results from surface tension measurements indicate that UV-ozone treatment is an efficient cleaning procedure to remove organic species contamination on surfaces. Well-defined redox responses for cytochrome c were observed at a UV-ozone treated fully hydrophilic indium oxide electrode. Electrochemical parameters, including the diffusion coefficient, the heterogeneous electron transfer rate constant and the redox potential, were in good agreement with those previously reported. However, decrease in peak current for cytochrome c and [Fe(CN)₆]⁴⁻ were observed at a UV-ozone treated electrode. From XPS results, this behavior would be understood to indicate a decrease in homogeneous active electrode surface area by a decrease in conductivity of the indium oxide surface by UV-ozone treatment. Simple and effective UV-ozone treatment methods are useful for surface contamination sensitive electrochemistry.     

Surface functions of 2-mercaptopyridine, 2-mercaptopyrazine and 2-mercaptoquinoxaline modified Au(1 1 1) electrodes for direct rapid electron transfer of cytochrome c

Surface functions of modified electrodes for the rapid electron transfer of cytochrome c have been examined by using new surface modifiers. 2-Mercaptopyrazine (2-PyZSH) and 2-mercaptoquinoxaline (or 2-mercaptobenzopyrazine, 2-MQ) modified Au(1 1 1) electrodes gave well-defined cyclic voltammmograms of cytochrome c, while a 2-mercaptopyridine (2-PySH) modified electrode gave no response. The STM images of 2-PySH and 2-PyZSH modified surfaces were similar to each other, suggesting 2-PySH and 2-PyZSH adsorbed at both thiolate S and pyridine (or pyrazine) N atoms with pyridine (or pyrazine) ring being perpendicular to the electrode surface. The 2-PyZSH modified surface has another pyrazine N atom faced to the solution, through which cytochrome c can interact, and the double layer capacitance data of the electrode gave more hydrophilic nature than the 2-PySH modified surface. On the other hand, although 2-MQ was suggested to adsorb on the electrode in a similar manner to 2-PySH and 2-PyZSH to give N atom at the solution side, the 2-MQ modified surface showed less hydrophilicity than the 2-PySH modified surface due to the quinoxaline ring. These results clearly reveal that the pyridine and pyrazine N atoms faced to the solution (rather than the hydrophilicity of the electrode surface) are important for the rapid electron transfer of cytochrome c on these modified electrodes.     

Oxygen-reducing electrodes based on layer-by-layer assemblies of cytochrome c and laccasse

Electroactive multilayer assemblies combining the redox protein cytochrome c and the enzyme laccase were fabricated by the layer-by-layer adsorption technique on gold electrodes and were shown to be capable of direct oxygen reduction. Laccase from trametes versicolor was electrostatically immobilized on multilayer films consisting of cytochrome c and the polyelectrolyte polyanilinesulfonic acid. The layer formation was monitored by quartz crystal microbalance. The electrochemical behavior of the electrodes was investigated by cyclic voltammetry. The resulting assembly exhibited a catalytic oxygen reduction current. This indicates a multi-step electron transport chain from the electrode via the cytochrome c layers towards laccase, and finally, to molecular oxygen. The catalytic efficiency of the electrodes was examined in the pH range from 4.5 to 7.0, showing highest enzymatic oxygen reduction at pH 4.5. Furthermore, the catalytic current was found to correlate linearly with the oxygen content of the solution. This suggests that the overall current is limited by the catalytic reduction of oxygen by the laccase rather than by the preceding electron transfer steps.     

Electrochemistry of Mitochondria: A New Way to Understand Their Structure and Function

In this article, electrochemistry of mitochondria is achieved. Cyclic voltammograms of freshly prepared mitochondria were obtained by immobilizing mitochondria together with glutaraldehyde and bovine serum albumin on the surface of a pyrolytic graphite electrode. Two pairs of redox peaks could be observed which were ascribed to the electron transfer reactions of cytochrome c and FAD/FADH₂. Study of submitochondrial particles was also conducted, which could confirm the results of the study of the entire mitochondria. The redox wave of NADH could be obtained due to the destruction of the membrane of mitochondria. We have also checked the function of succinate in mitochondria by employing the electrochemical method. This work is not only the first to be able to obtain the direct electrochemistry of mitochondria, but is also beneficial to the further understanding of the structure and function of mitochondria in vitro.     

Electrochemistry and biosensing activity of cytochrome c immobilized in macroporous materials

An amperometric biosensor for hydrogen peroxide (H₂O₂) has been constructed by immobilizing cytochrome c on an indium/tin oxide (ITO) electrode modified with a macroporous material. Cyclic voltammetry showed that the direct and quasi-reversible electron transfer of cytochrome c proceeds without the need for an electron mediator. A surface-controlled electron transfer process can be observed with an apparent heterogeneous electron-transfer rate constant (k_s) of 29.2?s^?1. The biosensor displays excellent electrocatalytic responses to the reduction of H₂O₂ to give amperometric responses that increase steadily with the concentration of H₂O₂ in the range from 5???M to 2?mM. The detection limit is 0.61???M at pH?7.4. The apparent Michaelis-Menten constant (K_m) of the biosensor is 1.06?mM. This investigation not only provided a method for the direct electron transfer of cytochrome c on macroporous materials, but also established a feasible approach for durable and reliable detection of H₂O₂.