Electrochemical studies of cytochrome c on gold electrodes with promotor of humic acid and 4-aminothiophenol

p-Aminothiophenol (PATP) and humic acids (HA or HAs) were applied jointly as the electron transfer accelerants of redox reactions of cytochrome c (Cyt c) on gold electrodes. The electrochemical properties of the modified electrodes were studied by field emission scanning electron microscope, ultraviolet-visible spectroscopy, electrochemical impedance spectroscopy, Raman spectroscopy and cyclic voltammetry. The immobilized Cyt c displayed a couple of stable and well-defined redox peaks with a formal potential of −0.101 V (vs. SCE) in pH 7.0 phosphate buffer solution. Cyt c adsorption is in the form of a monolayer with average surface coverage of 5.28 pmol cm⁻². The electron transfer rate constant was calculated to be 2.14 s⁻¹. It indicate that the HA film acted as a good adsorption matrix for Cyt c and an excellent accelerant for the redox of Cyt c. The Cyt c-HA modified gold electrode showed a new couple of well-marked redox peaks when 2,4-dichlorophenol was added to the test solution.     

Carboxyphenyl Covalent Immobilization of Heme Proteins and its Favorable Biocompatible Electrochemical and Electrocatalytic Characteristics

A novel carboxyphenyl covalent immobilization technique has been successfully developed to realize the effective attachment of two typical heme proteins, hemoglobin (Hb) and cytochrome c (Cyt‐c), onto underlying glassy carbon electrode (GCE). Primarily, the GCE surface is functionalized with aromatic 4‐carboxyphenyl (4‐CP) group by the electrochemical reduction of diazonium cations, producing covalently linked carboxyl‐terminated active GCE surface to work as a ‘bridge’. Then, Hb and Cyt‐c are readily attached to GCE through the ‘bridge’ by functional covalently combination between ? NH₂ terminal groups of proteins and ? COOH terminal groups of 4‐CP, obtaining Hb/4‐CP/GCE and Cyt‐c/4‐CP/GCE. On both electrodes, well‐defined peaks attributing to the Fe^III/Fe^II couple of heme group of Hb and Cyt‐c are clearly observed with the electron transfer rate constant (k_s) evaluated to be 2.48±0.05 s^?1 and 2.73±0.05 s^?1, respectively. It is attractive that the formal potential (E°') of the immobilized Hb and Cyt‐c are estimated to be 50 and 100 mV (vs. SCE), respectively, which are closer to the standard redox potential of native Hb and Cyt‐c in solution, owing to the good biocompatibility of 4‐CP groups. The electrodes also exhibit fast response, high sensitivity and well stability for the amperometric detection of H₂O₂ at a fairly mild potential of 0 V without any mediators, obtaining rather small apparent Michaelis‐Menten constant (K_M^app) values of 113 μM for Hb/4‐CP/GCE and 101 μM for Cyt‐c/4‐CP/GCE. All the experimental results indicated that the covalent graft 4‐carboxyphenyl group plays an important role in constructing a “biocompatible bridge” to help the direct electron transfer of Hb and Cyt‐c with favorable biocompatibility and good bio‐ electrocatalytic affinity in virtue of the substituted aryl group only consisting of C, H and O elements, which is similar with the constitutes of organics. It makes the system of functionalized covalent immobilization of proteins onto carbon electrode a promising platform for fabricating the third‐generation biosensors. A new approach for realizing direct electrochemistry of proteins, as well as design of novel bioelectronic devices has been accordingly provided.     

Regulating Effect of Cytochrome b5 Overexpression on Human Breast Cancer Cells

Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b₅ (Cyt b₅) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b₅ content. Based on the low content of Cyt b₅ in MDA-MB-231 cells, the overexpression of Cyt b₅ was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b₅ MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H₂O₂, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b₅ most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b₅ has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b₅ and provides clues for clinical treatment.     

Direct cytochrome c electrochemistry at boron-doped diamond electrodes

Highly boron-doped diamond electrodes are characterized voltammetrically employing Ru(NH₃)₆^3+/2+, Fe(CN)₆^3−/4−, benzoquinone/hydroquinone, and cytochrome c redox systems. The diamond electrodes, which are polished to nanometer finish, are initially `activated' electrochemically and then pretreated by oxidation, reduction, or polishing. All electrodes give reversible cyclic voltammetric responses for the reduction of Ru(NH₃)₆³⁺ in aqueous solution.Redox systems other than Ru(NH₃)₆^3+/2+ show characteristic electrochemical behavior as a function of diamond surface pretreatment. In particular, the horse heart cytochrome c redox system is shown to give reversible voltammetric responses at Al₂O₃ polished boron-doped diamond electrodes. No voltammetric response for cytochrome c is detected at anodically pretreated diamond electrodes. The observations are attributed to preferential interaction of the polished diamond surface with the reactive region of the cytochrome c molecule and low interference due to a lack of protein electrode fouling.     

Microscale Crystals of Cytochrome c and Calixarene on Electrodes: Interprotein Electron Transfer between Defined Sites

The assembly of redox proteins on electrodes is an important step in biosensor development. Recently, p‐sulfonato‐calix[4]arene was shown to act as “molecular glue” for the assembly and crystallization of cytochrome c (cyt c). Electrochemical data are presented for microscale cyt c–calixarene crystals grown on self‐assembled monolayers (SAM)‐modified Au electrodes. The crystals were characterized by cyclic voltammetry and exceptionally high concentrations of electroactive cyt c were obtained. The peak currents were found to increase linearly with the square root of the scan rate, thus allowing an evaluation of the rate constant for electron self‐exchange. This study revealed high electroactivity accompanied by fast interprotein electron transfer in crystals, which may have implications for the construction of novel bioelectronic devices.     

Interfacing cytochrome c to electrodes with a DNA – carbon nanotube composite film

Multi-walled carbon nanotube (MWCNT) is successfully immobilized on the surface of platinum electrode by mixing with DNA. The DNA/MWCNT modified electrodes are characterized by electrochemical impedance spectroscopy and cyclic voltammetry. Further research indicates that cytochrome c can strongly adsorbed on the surface of the modified electrode, and forms an approximate monolayer. The immobilized MWCNT can promote the redox of horse heart cytochrome c which gives reversible redox peaks with a formal potential of 81 mV vs SCE.     

Direct Electrochemistry of Cytochrome c Based on Poly(diallyldimethylammonium Chloride)‐ Graphene Nanosheets/Gold Nanoparticles Hybrid Nanocomposites and Its Biosensing

A novel biosensor was developed by entrapping cytochrome c (Cyt c) in thin films of the room temperature ionic liquid (RTIL) containing nanocomposites of poly(diallyldimethylammonium chloride)‐graphene nanosheets‐gold nanoparticles (PDDA‐Gp‐AuNPs) at a 11‐mercaptoundecanoic acid‐6‐mercapto‐1‐hexanol modified gold electrode. The synthesized PDDA‐Gp‐AuNPs hybrid nanocomposites were characterized by UV‐vis spectroscopy, Raman spectroscopy, scanning electron microscopy and atomic force microscopy. The PDDA‐Gp‐AuNPs nanocomposites could increase the effective surface of the electrode, enhance the fixed amount of Cyt c on the electrode surface, promote the electron transfer and facilitate the catalytic activity of Cyt c. The RTIL could provide a biocompatible microenvironment to keep Cyt c biological activities, act as an effective mediator to immobilize a large number of Cyt c on the electrode and have good conductivity to improve electron transfer. Therefore, the resultant electrode exhibited good electrochemical performance and electrocatalytic activity. It could be used for electrochemical detection of H₂O₂ with rapid response, high sensitivity, wide linear range and low detection limit, as well as good stability, repeatability and selectivity. The sensor might be promising for practical application.     

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.     

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 and superficial characterization of DNA-cytochrome c-MUA films on chemically modified gold surface

Cytochrome c (Cyt. c) was immobilized on the 11-mercaptohendecanoic acid (MUA)-modified gold electrode. The electrode was stable and sensitive to Cyt. c. Later, DNA was also immobilized on the two-layer modified electrode. Cyclic voltammetry studies show that Cyt. c can interact with dsDNA and ssDNA. The binding site sizes were determined to be 15 base pairs per Cyt. c molecule with dsDNA and 30 nucleotides binding 1 Cyt. c molecule with ssDNA. The modified electrodes were characterized by quartz crystal microbalance (QCM), impedance spectroscopy and atomic force microscope (AFM). The modified electrode can be used for determining DNA.     

Direct electrochemistry of bacterial cytochrome c551 at surface-modified gold electrodes

The direct electrochemistry of the single heme cytochrome c₅₅₁ from the bacterium Pseudomonas aeruginosa has been investigated at gold electrodes surface-modified through chemisorption of polyfunctional organic molecules. The results have been compared and contrasted with those obtained under the same conditions for the eukaryotic cytochrome c from horse heart. Both cytochromes give a quasi-reversible electrode reaction at pH 6.0 at a modified interface presenting only 4-pyridyl groups to the solution suggesting the occurrence, in both cases, of a hydrogen bonding interaction from lysine side-chains on the protein to pyridyl-nitrogens on the electrode surface. However, in contrast, gold electrodes modified by Pyridine-n-AldehydeThioSemicarbazones (n = 2, 3, 4) give electrochemistry which is strongly isomer-dependent in the case of horse heart cytochrome c but completely isomer-independent in the case of cytochrome c₅₅₁. It is suggested that interaction of the eukaryotic protein with surfaces is dominated by its lysine residues only, but that interaction of the bacterial cytochrome is through hydrogen bonding from the surface to both lysines and carboxylate groups of aspartate residues. This is supported by observation of the loss of cytochrome c₅₅₁ electrochemistry at 4-pyridyl-only modified gold at pH 9.0 compared with the good, quasi-reversible electrochemistry maintained under the same conditions at PATS-4 modified gold. It is concluded that, while the two cytochromes show many similarities with respect to their structures and functions, they have quite different interfacial electron transfer reactions, particularly at PATS-modified electrodes. This may correlate with the known large differences between the two proteins in net electrostatic charge and surface charge distribution.     

纳米ZnO修饰电极的制备及其对血红蛋白, 细胞色素c的直接电化学研究

A novel electrochemical method as a sensitive and convenient technique for the determination of heme proteins based on their interaction with ZnO nanorods was developed. A ZnO nanorod modified glassy carbon electrode (ZnO/GCE) was prepared and the electrochemical behaviors of heme proteins, such as hemoglobin (HB) and cytochrome c (Cyt-c), on this modified electrode have been studied. The results showed that both HB and Cyt-c could be oxidized on the modified electrode and the oxidation currents were linear to the concentrations of the analytes in aqueous solutions. In addition, the results of flow injection analysis (FIA) further suggested the high stability and reproducibility of the ZnO nanorod modified electrode. So this method can be applied to the determination of HB and Cyt-c in biological systems.     

Bioelectrochemical detection of L-lactate respiration using genetically modified Hansenula polymorpha yeast cells overexpressing flavocytochrome b2

In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, also known as flavocytochrome b₂, FC b₂) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b₂ the O₂ reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b₂, cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN⁻, lactate respiration could be proven for causing the decrease in the O₂ reduction.     

The adsorption of cytochromes on a modified surface of gold electrodes

The adsorption of cytochromes b ₅ and c on the surface of gold electrodes, including the surface modified with cysteine, was studied. The quartz crystal microbalance method with parallel dissipation energy measurements, microcontact printing, and atomic-force microscopy were used to show that the special features of the structure and morphology of two-component cytochrome b ₅ and c films were determined by the nature of the proteins themselves and the influence of the modifying “sublayer.” The largest changes in the weight of films and dissipation energy were observed in the adsorption of cytochrome b ₅ on a cytochrome c film deposited on a cysteine sublayer. Atomic-force microscopy measurements showed that strong interaction between cytochrome c and b ₅ molecules on the surface of gold modified with cysteine could be related to the formation of the corresponding protein complex.     

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).     

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.     

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.     

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.     

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.     

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 ]