Direct electrochemical behavior of cytochrome c on DNA-modified glassy carbon electrode and its application to cytochrome c biosensor.

DNA was immobilized on glassy carbon electrodes to fabricate DNA-modified electrodes. The direct electron transfer of horse heart cytochrome c on DNA-modified glassy carbon electrode was achieved. A pair of well-defined redox peaks of cytochrome c appeared at Epc = -0.017 V and Epa = 0.009 V (vs. Ag/AgCl) in 10 mM phosphate buffer solution (pH 7.0) at a scan rate of 50 mV/s. The electron transfer coefficient (alpha) and the standard rate constant of the surface reaction (Ks) of cytochrome c on DNA-modified electrodes could be estimated to be 0.87 and 34.52 s(-1), respectively. The DNA-modified glassy carbon electrode could be applied to detect cytochrome c by means of differential pulse voltammetry (DPV). The cathodic peak current was proportional to the quantity of cytochrome c in the range of 4.0 x 10(-6) M to 1.2 x 10(-5) M. The correlation coefficient is 0.996, and with the detection limit was 1.0 x 10(-6) M (three times the ratio of signal to noise, S/N = 3).     

Conductive wiring of immobilized photosynthetic reaction center to electrode by cytochrome C

The photosynthetic reaction center (RC) found in photosynthetic bacteria is one of the most advanced photoelectronic devices developed by nature. However, after immobilization on the electrode surface, the efficiency of electron transfer (ET) between the RC and the electrode is relatively low. This inefficiency has limited the possibility of using the RC for technological applications. Here we show that photoinduced electron transfer between the immobilized RC and a gold electrode can be increased by several tens-fold by incorporation of cytochrome c into the RC-self-assembled monolayer (SAM)-electrode complex. The effect does not depend on the initial redox state of the cytochrome and seems to be the result of the formation of a complex between the RC and the cytochrome c serving as an ET wire. This observation opens the possibility for electrochemical analysis of the special pair in the RC protein that is deeply buried inside the protein globe and is barely electrically addressable from the electrode surface.     

Direct electrochemical behavior of cytochrome c, and its determination on phytic acid modified electrode

Phytic acid (PA) with its unique structure was attached to a glassy carbon electrode (GCE) to form PA/GCE modified electrode which was characterized by electrochemical impedance. The electrochemical behavior of cytochrome c (Cyt c) on the PA/GCE modified electrode was explored by cyclic voltammetry and differential pulse voltammetry. The Cyt c displayed a quasi-reversible redox process on PA modified electrode pH 7.0 phosphate buffer solution with a formal potential (E ^0′) of 57 mV (versus Ag/AgCl). The peak currents were linearly related to the square root of the scan rate in the range of 20–120 mV·s^?1. The electron transfer rate constant was determined to be 12.5 s^?1. The PA/GCE modified electrode was applied to the determination of Cyt c, in the range of 5?×?10^?6 to 3?×?10^?4 M, the currents increase linearly to the Cyt c concentration with a correlation coefficient 0.9981. The detection limit was 1?×?10^?6 M (signal/noise?=?3).     

Recognition and modulation of cytochrome c's redox properties using an amphiphilic homopolymer

An amphiphilic homopolymer scaffold has been used to bind to the protein, cytochrome c. This interaction is analyzed using cyclic voltammetry, native gel electrophoresis, UV-visible absorption, and circular dichroism spectroscopy. The polymer binds to cytochrome c with micromolar affinity and the association of polymer with cytochrome c leads to a structural change of the protein. This conformational change exposes the heme unit of the protein, which affords an opportunity to reversibly modulate its electron-transfer properties. We have also shown that the electrostatic binding of polymer to cytochrome c can be used to disrupt its interaction with its natural partner, cytochrome c peroxidase.     

Application of silver electrode to the electrochemical studies of hemoglobin

Several aspects of the application of silver electrode to the electrochemical studies of hemoglobin have been discussed in this paper . The silver electrode could not only be used directly as the electrode for the electrochemical studies of hemoglobin, but also react with phenothiazine and benzimidazole to give stable and useful mediator-coated electrodes. In addition, the silver electrode could help sodium dodecyl sulfate to give full play to its promoting effect on the protein.     

Modular solid-state unit for electrochemical studies

A relatively inexpensive unit based upon solid-state operational amplifiers is described; its modular design makes it an extremely versatile instrument for many electrochemical techniques, e.g., normal direct current polarography and linear sweep voltammetry, cyclic voltammetry, alternating current polarography, and coulometry and electrolysis at controlled electrode potential. It can be readily adapted to many other functions.     

Direct electrochemical response of cytochrome c on a room temperature ionic liquid,N-butylpyridinium tetrafluoroborate,modified electrode

Cytochrome c (Cyt c) and a room temperature ionic liquid (RTIL), 2-methyl-N-butylpyridinium tetrafluoroborate ([2-MBPy][BF₄]), were immobilized on the surface of a basal plane graphite (BPG) electrode. Direct electron transfer from Cyt c to BPG electrode was clearly observed. A pair of well-defined and reversible redox peaks could be obtained in a 0.13 M [2-MBPy][BF₄] aqueous solution. The anodic and cathodic peak potentials of Cyt c were at −0.064 and −0.020 V (vs. Ag/AgCl), respectively. The results showed that [2-MBPy][BF₄] promoted the direct electron transfer between Cyt c and the BPG electrode. Cyt c immobilized on BPGE can catalyze the reduction of hydrogen peroxide. Based on this, a biosensor can be constructed to detect quantitatively hydrogen peroxide.     

细胞色素c在无机复合纳米材料修饰电极上的直接电化学研究

采用超声辅助湿法合成的方法制备了羟基磷灰石与碳纳米管(HAp/MWNT)无机复合纳米材料,并将其修饰到玻碳电极表面上。研究了细胞色素c(Cyt c)在该复合纳米材料修饰电极表面上的电化学行为。实验结果表明,复合修饰材料对细胞色素c有很好的催化作用,其氧化还原峰峰差(ΔEp)为78 mV,式电位为15mV。在6.0×10-7-5.0×10-5mol/L浓度范围内,Cyt c的峰电流与其浓度呈良好的线性关系,其检测下限为3.0×10-7mol/L。关键词:细胞色素c;羟基磷灰石;碳纳米管;HAp/MWNT复合材料     

Direct electrochemistry of heme proteins: effect of electrode surface modification by neutral surfactants

Direct electrochemical studies on horse heart myoglobin and horseradish peroxidase (HRP) have been carried out using tin-doped indium oxide (ITO) and surfactant modified glassy carbon working electrodes. These proteins show very slow electron transfer kinetics at metal or untreated electrodes. Moreover, small amounts of surface-active impurity were drastically affects the electrode reaction of these proteins. The results showed that modification of the electrode surface with neutral surfactants significantly improves the electrochemical response of myoglobin as well as of HRP. The electrode response was found to depend on the structure of the surfactants. The amount of surfactant required per unit area of the electrode surface to promote the maximum electron transfer rate constants was found to be constant. This indicated that the surfactant molecules interacted with the electrode surface in a specific manner and anchored the protein molecules to align in the suitable orientation. The hydrophobicity of the surfactants rather than their charge was found to be crucial in promoting the electrode response of these proteins at the glassy carbon electrode.     

Myoglobin/arylhydroxylamine film modified electrode: Direct electrochemistry and electrochemical catalysis

The adsorption processes and electrochemical behavior of 4-nitroaniline (4-NA) adsorbed onto glassy carbon electrodes (GCE) have been investigated in aqueous 0.1 M nitric acid (HNO₃) electrolyte solutions using cyclic voltammetry (CV). 4-NA adsorbs onto GCE surfaces, and upon potential cycling past −0.2 V, is transformed into the arylhydroxylamine (ArHA) derivative which exhibits a well-behaved pH dependent redox couple centered at 0.32 V at pH 1.5. It is noted as arylhydroxylamine modified glassy carbon electrodes (HAGCE). This modified electrode can be readily used as an immobilization matrix to entrap proteins and enzymes. In our studies, myoglobin (Mb) was used as a model protein for investigation. A pair of well-defined reversible redox peaks of Mb (Fe(III)-Fe(II)) was obtained at the Mb/arylhydroxylamine modified glassy carbon electrode (Mb/HAGC) by direct electron transfer between the protein and the GCE. The formal potential (E^0′), the apparent coverage (Γ^*) and the electron-transfer rate constant (k_s) were calculated as −0.317 V, 8.26 × 10⁻¹² mol/cm² and 51 ± 5 s⁻¹, respectively. Dramatically enhanced biocatalytic activity was exemplified at the Mb/HAGC electrode by the reduction of hydrogen peroxide (H₂O₂), trichloroacetic acid (TCA) and oxygen (O₂). The Mb/arylhydroxylamine film was also characterized by UV-visible spectroscopy (UV-vis), scanning electron microscope (SEM) indicating excellent stability and good biocompatibility of the protein in the arylhydroxylamine modified electrode. This new Mb/HAGC electrode exhibited rapid electrochemical response (2 s) for H₂O₂ and had good stability in physiological condition, showing the potential applicability of the films in the preparation of third generation biosensors or bioreactors based on direct electrochemistry of the proteins.     

Direct and mediated electrochemical oxidation of ammonia on boron-doped diamond electrode

Direct (non-mediated) electrochemical oxidation of ammonia on boron-doped diamond (BDD) electrode proceeds mainly at high pH (> 8) via free ammonia (NH₃) oxidation. To enhance ammonia oxidation on BDD at low pH (< 8), where mainly ammonium (NH₄⁺) is present, oxidation of ammonia was mediated by active free chlorine. In this process, electro-generated in situ active chlorine rapidly reacts with ammonia instead of being further electro-oxidized to chlorate at the electrode surface. Thus, active chlorine effectively removes ammonia from an acidic solution, while the formation of by-products such as chlorate and possibly perchlorate is minimized.     

An improved solution-tight electrode holder for variable temperature electrochemical studies

With the help of oscillopolarographic methods, the formation of additional adsorption-desorption peaks or incisions in the curves i=f(E) and dE/dt=f(E) was observed for saturated solutions of higher fatty acids in 1 M H₂SO₄. In virtue of the analysis of the first and second polarization cycle, it was found, that, a previous electrode polarization to the adsorption potentials was necessary, if the additional peaks were to be formed. A supposition was made, that the additional peaks originate in the desorption and adsorption of the dimerized molecules of the acids.     

Temperature-dependent studies of NO recombination to heme and heme proteins

The rebinding kinetics of NO to the heme iron of myoglobin (Mb) is investigated as a function of temperature. Below 200 K, the transition-state enthalpy barrier associated with the fastest (approximately 10 ps) recombination phase is found to be zero and a slower geminate phase (approximately 200 ps) reveals a small enthalpic barrier (approximately 3 +/- 1 kJ/mol). Both of the kinetic rates slow slightly in the myoglobin (Mb) samples above 200 K, suggesting that a small amount of protein relaxation takes place above the solvent glass transition. When the temperature dependence of the NO recombination in Mb is studied under conditions where the distal pocket is mutated (e.g., V68W), the rebinding kinetics lack the slow phase. This is consistent with a mechanism where the slower (approximately 200 ps) kinetic phase involves transitions of the NO ligand into the distal heme pocket from a more distant site (e.g., in or near the Xe4 cavity). Comparison of the temperature-dependent NO rebinding kinetics of native Mb with that of the bare heme (PPIX) in glycerol reveals that the fast (enthalpically barrierless) NO rebinding process observed below 200 K is independent of the presence or absence of the proximal histidine ligand. In contrast, the slowing of the kinetic rates above 200 K in MbNO disappears in the absence of the protein. Generally, the data indicate that, in contrast to CO, the NO ligand binds to the heme iron through a "harpoon" mechanism where the heme iron out-of-plane conformation presents a negligible enthalpic barrier to NO rebinding. These observations strongly support a previous analysis (Srajer et al. J. Am. Chem. Soc. 1988, 110, 6656-6670) that primarily attributes the low-temperature stretched exponential rebinding of MbCO to a quenched distribution of heme geometries. A simple model, consistent with this prior analysis, is presented that explains a variety of MbNO rebinding experiments, including the dependence of the kinetic amplitudes on the pump photon energy.     

Direct electrochemistry and electrocatalysis of cytochrome c immobilized on gold nanoparticles-chitosan-carbon nanotubes-modified electrode

A robust and effective nanohybrid film based on gold nanoparticles (GNPs)/chitosan (Chit)/multi-walled carbon nanotubes (MWNTs) was prepared by a layer-by-layer self-assembly technique. Cytochrome c (Cyt c) was successfully immobilized on the nanohybrid film modified glassy carbon (GC) electrode by cyclic voltammetry. The direct electron transfer between Cyt c and the modified electrode was investigated in detail. Cyt c shows a couple of quasi-reversible and well-defined cyclic voltammetry peaks with a formal potential (E^0′) of −0.16 V (versus Ag/AgCl) in pH 7.0 phosphate buffer solution (PBS). The Cyt c/GNPs/Chit/MWNTs modified GC electrode gives an improved electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂). The sensitivity is 92.21 μA mM⁻¹ cm⁻² and the calculated apparent Michaelis-Menten constant () is 0.791 mM, indicating a high-catalytic activity of Cyt c. The catalysis currents increase linearly to the H₂O₂ concentration in a wide range of 1.5 × 10⁻⁶ to 5.1 × 10⁻⁴ M with a correlation coefficient 0.999. The detection limit is 9.0 × 10⁻⁷ M (at the ratio of signal to noise, S/N = 3). Moreover, the modified electrode displays rapid response (5 s) to H₂O₂, and possesses good stability and reproducibility.     

Carbon nanotube wiring for signal amplification of electrochemical magneto immunosensors: application to myeloperoxidase detection

In this work, chronoamperometric myelo-peroxidase (MPO) detection was accomplished using immunofunctionalized magnetic microparticles (MPs), disposable carbon screen-printed electrodes (C-SPEs), and a ready-to-use commercially available tetramethylbenzidine (TMB)-based enzymatic substrate. In order to reach the limit of detection (LOD) needed to study real blood serum samples, assay performance was additionally improved by exploiting CNT wiring, which amplified the signal and decreased the LOD. The optimized assay can be performed in 30 min and yields LODs of 6 and 55 ng mL^?1 in PBS and undiluted human serum, respectively, making it useful for the identification of patients at risk of cardiovascular disease. These results demonstrate that electrode nanostructuring can be accomplished “post-assay,” which favors the development of enhanced magneto immunosensors based on the exploitation of cheap and simple SPE devices.     

Direct electrochemistry behavior of cytochrome c/L-cysteine modified electrode and its electrocatalytic oxidation to nitric oxide

Cytochrome c (Cyt c) was successfully immobilized on L-cysteine modified gold electrode by multicyclic voltammetry method. The electrochemical behavior of Cyt c on the L-cysteine modified electrode was explored. In 0.10 M, pH 7.0 phosphate buffer solution (PBS), Cyt c showed a quasi-reversible electrochemical redox behavior with E(pc)=0.180 V, E(pa)=0.208 V (versus Ag/AgCl). The Cyt c/L-cysteine modified electrode gave an excellent electrocatalytic activity towards the oxidation of nitric oxide, and the catalysis currents were proportional to the nitric oxide concentration in the range of 7.0 x 10(-7) to 1.0 x 10(-5) M, the linear regression equation is I (microA)=-0.124-0.003 C(NO) (microM), with a correlation coefficient 0.996, The detection limit was 3.0 x 10(-7) M (times the ratio of signal to noise, S/N=3).     

细胞色素c在Fe_2O_3修饰电极上的直接电化学和电催化特性

采用全氟磺酸树脂Nafion将金属氧化物Fe2O3颗粒细胞色素c(Cyt c)固定玻碳电极(GCE)表面,制备了Nafion-Cyt c-Fe2O3修饰的玻碳电极,构建了基于直接电子传递的过氧化氢生物传感器。在0.10mol/L pH7.0的磷酸盐缓冲溶液中,修饰电极的循环伏安曲线上显示出一对准可逆的氧化还原峰,式量电位为22mV。Cyt c在修饰电极表面的异相电子转移速率常数为1.21s-1。修饰后的电极对过氧化氢有良好响应,响应时间小于10s,电极的安培响应与过氧化氢浓度在2.0×10-6～3.0×10-3mol/L范围内成线性关系,检出限为1.0×10-6mol/L,米氏常数为1.35mmol/L,显示出较好的亲和力。     

Direct electrochemical interaction between a modified gold electrode and a bacterial membrane extract

A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity.