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.     

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.     

Isolation and analysis of tetraheme-bound-cytochrome from photosynthetic reaction centers of Rhodopseudomonas viridis

A tetraheme cytochrome (BCytc) was isolated from the photosynthetic reaction centers (RC) of Rhodopseudomonas viridis while maintaining the redox activity. BCytc was removed from the H-subunit-detached RC by polyacrylamide electrophoresis using an alkyl ether sulfate mixed with sodium dodecyl sulfate. Redox titration of BCytc showed a simple one-step redox titration curve and a lowered midpoint potential than that of one in RC. Direct electron transfer between BCytc and electrode surfaces, such as indium tin oxide, was successfully performed, indicating a potential for molecular electronic material.     

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.

Functionalization of gold electrode surface with heterobifunctional poly(ethylene oxide)s having both mercapto and aldehyde groups

We synthesized heterobifunctional poly(ethylene oxide) (PEO) (α‐formyl‐ω‐mercapto‐PEO; CHO‐PEO₄₀₀‐SH, average molecular weight of PEO part being 400), which had both an aldehyde group as a binding site with amino group of protein and a mercapto group for gold electrode surface. The CHO‐PEO₄₀₀‐SH was adsorbed on a gold electrode surface and cytochrome c (cyt.c) was fixed on this modified electrode. The redox response of covalently immobilized cyt.c was observed on the cyclic voltammetry measurement, showing that CHO‐PEO₄₀₀‐SH can be used as a linker to fix cyt.c on an electrode. Another type of heterobifunctional PEO (α‐formyl‐ω‐(2‐pyridyldithio)‐PEO; CHO‐PEO₃₀₀‐SS‐Py), which had an aldehyde group and a 2‐pyridinethiol (2‐Py) through disulfide bond, was synthesized to form co‐adsorbed monolayer of PEO chain and 2‐Py on an electrode surface. It was expected, due to the spacer with shorter PEO chain and lower surface density, that better redox response of the fixed cyt.c was obtained. However, the redox response of fixed cyt.c was not detected on the CHO‐PEO₃₀₀‐SS‐Py modified gold electrode. Instead, this heterobifunctional PEO was found to function as a good promoter for cyt.c dissolved in phosphate buffer solution. Copyright © 2003 John Wiley & Sons, Ltd.     

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.     

The effects of temperature and electrolyte at acidic and alkaline pH on the electron transfer reactions of cytochrome c at In2O3 electrodes

Spectroelectrochemical and electrochemical methods were used to investigate the characteristics of heterogeneous electron transfer between cytochrome c and indium oxide electrodes. A linear temperature dependence of the formal potential of cytochrome c was observed from 5 to 75 °C in acidic media. This behavior is attributed to a linear variation in the conformation of ferricytochrome c that results in an increase in solvent exposure of the solvent-exposed heme edge. A break in the linear temperature dependence of the formal potential occurred at 40 °C in alkaline media. This reflects a distinct conformational change that accompanies the onset of thermal denaturation of ferricytochrome c. The small change in reaction center entropy, ΔS°_rc, of ca. −54 J K⁻¹ mol⁻¹ in neutral and acidic media (5 to ⩾ 55 °C) and in alkaline media (below 40 °C) is consistent with a small shift to a more stable conformation of cytochrome c that occurs upon reduction.Adsorption of reactant and product was detected. The strength and type of adsorption were found to be temperature- and pH-dependent. The characteristics of electron transfer between cytochrome c and an electrode depend on bulk solvent properties and electrode surface characteristics.     

Design of Maleimide‐Functionalised Electrodes for Covalent Attachment of Proteins through Free Surface Cysteine Groups

Mixed two‐component monolayers on glassy carbon are prepared by electrochemical oxidation of N‐(2‐aminoethyl)acetamide and mono‐N‐Boc‐hexamethylenediamine in mixed solution. Subsequent N‐deprotection, amide coupling and solid‐phase synthetic steps lead to electrode‐surface functionalisation with maleimide, with controlled partial coverage of this cysteine‐binding group at appropriate dilution for covalent immobilisation of a model redox‐active protein, cytochrome c, with high coverage (≈7.5 pmol cm^?2).     

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.

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.     

Electrocatalysis of a Europium-Dependent Bacterial Methanol Dehydrogenase with Its Physiological Electron-Acceptor Cytochrome cGJ

We report the first electrochemical study of a lanthanoid-dependent methanol dehydrogenase (Eu-MDH) from the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV with its own physiological cytochrome c_GJ electron acceptor. Eu-MDH harbours a redox active 2,7,9-tricarboxypyrroloquinoline quinone (PQQ) cofactor which is non-covalently bound but coordinates trivalent lanthanoid elements including Eu³⁺. Eu-MDH and the cytochrome were co-adsorbed with the biopolymer chitosan and cast onto a mercaptoundecanol (MU) monolayer modified Au working electrode. Cyclic voltammetry of cytochrome c_GJ reveals a well-defined quasi-reversible Fe^III/II redox couple at +255 mV vs. NHE at pH 7.5 and this response is pH independent. The reversible one-electron response of the cytochrome c_GJ transforms into a sigmoidal catalytic wave in the presence of Eu-MDH and its substrates (methanol or formaldehyde). The catalytic current was pH-dependent and pH 7.3 was found to be optimal. Kinetic parameters (pH dependence, activation energy) obtained by electrochemistry show the same trends as those obtained from an artificial phenazine ethosulfate/dichlorophenol indophenol assay.     

Spatially Resolved Confocal Resonant Raman Microscopic Analysis of Anode‐Grown Geobacter sulfurreducens Biofilms

When grown on the surface of an anode electrode, Geobacter sulfurreducens forms a multi‐cell thick biofilm in which all cells appear to couple the oxidation of acetate with electron transport to the anode, which serves as the terminal metabolic electron acceptor. Just how electrons are transported through such a biofilm from cells to the underlying anode surface over distances that can exceed 20 microns remains unresolved. Current evidence suggests it may occur by electron hopping through a proposed network of redox cofactors composed of immobile outer membrane and/or extracellular multi‐heme c‐type cytochromes. In the present work, we perform a spatially resolved confocal resonant Raman (CRR) microscopic analysis to investigate anode‐grown Geobacter biofilms. The results confirm the presence of an intra‐biofilm redox gradient whereby the probability that a heme is in the reduced state increases with increasing distance from the anode surface. Such a gradient is required to drive electron transport toward the anode surface by electron hopping via cytochromes. The results also indicate that at open circuit, when electrons are expected to accumulate in redox cofactors involved in electron transport due to the inability of the anode to accept electrons, nearly all c‐type cytochrome hemes detected in the biofilm are oxidized. The same outcome occurs when a comparable potential to that measured at open circuit (?0.30 V vs. SHE) is applied to the anode, whereas nearly all hemes are reduced when an exceedingly negative potential (?0.50 V vs. SHE) is applied to the anode. These results suggest that nearly all c‐type cytochrome hemes detected in the biofilm can be electrochemically accessed by the electrode, but most have oxidation potentials too negative to transport electrons originating from acetate metabolism. The results also reveal a lateral heterogeneity (x–y dimensions) in the type of c‐type cytochromes within the biofilm that may affect electron transport to the electrode.     

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.     

The Effect of Ionic Liquid Covalent Bonding to Sol‐Gel Processed Film on Ion Accumulation and Transfer

Accumulation of electroactive anions into a silicate film with covalently bonded room temperature ionic liquid film deposited on an indium tin oxide electrode was studied and compared with an electrode modified with an unconfined room temperature ionic liquid. A thin film containing imidazolium cationic groups was obtained by sol‐gel processing of the ionic liquid precursor 1‐methyl‐3‐(3‐trimethoxysilylpropyl)imidazolium bis(trifluoromethylsulfonyl)imide together with tetramethylorthosilicate on the electrode surface. Profilometry shows that the obtained film is not smooth and its approximate thickness is above 1 μm. It is to some extent permeable for a neutral redox probe – 1,1′‐ferrocene dimethanol. However, it acts as a sponge for electroactive ions like Fe(CN)₆^3?, Fe(CN)₆^4? and IrCl₆^3?. This effect can be traced by cyclic voltammetry down to a concentration equal to 10^?7 mol dm^?3. Some accumulation of the redox active ions also occurs at the electrode modified with the ionic liquid precursor, but the voltammetric signal is significantly smaller compare with the bare electrode. The electrochemical oxidation of the redox liquid t‐butyloferrocene deposited on silicate confined ionic liquid film is followed by the expulsion of the electrogenerated cation into an aqueous solution. On the other hand, the voltammetry obtained with the electrode modified with t‐butyloferrocene solution in the ionic liquid precursor exhibits anion sensitive voltammetry. This is explained by anion insertion into the unconfined ionic liquid deposit following t‐butylferricinium cation formation.     

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.     

Electrocatalysis of Water Oxidation by H2O‐Capped Iridium‐Oxide Nanoparticles Electrodeposited on Spectroscopic Graphite

Electrocatalysis of water oxidation by 1.54 nm IrO_x nanoparticles (NPs) immobilized on spectroscopic graphite electrodes was demonstrated to proceed with a higher efficiency than on all other, hitherto reported, electrode supports. IrO_x NPs were electrodeposited on the graphite surface, and their electrocatalytic activity for water oxidation was correlated with the surface concentrations of different redox states of IrO_x as a function of the deposition time and potential. Under optimal conditions, the overpotential of the reaction was reduced to 0.21 V and the electrocatalytic current density was 43 mA cm^?2 at 1 V versus Ag/AgCl (3 M KCl) and pH 7. These results beneficially compete with previously reported electrocatalytic oxidations of water by IrO_x NPs electrodeposited onto glassy carbon and indium tin oxide electrodes and provide the basis for the further development of efficient IrO_x NP‐based electrocatalysts immobilized on high‐surface‐area carbon electrode materials.     

Voltammetry of immobilized cytochrome c on novel binary self-assembled monolayers of thioctic acid and thioctic amide modified gold electrodes

Horse heart cytochrome c (cyt c) was adsorbed on the binary self-assembled monolayers (SAMs) composed of thioctic acid (T-COOH) and thioctic amide (T-NH₂) at gold electrodes via electrostatic interaction. The cyt c adsorbed on the modified gold electrode exhibited well-defined reversible electrochemical behavior in 10 mM phosphate buffer solution (PBS, pH 7.0). The surface concentration (Γ) of electroactive species, cyt c, on the binary SAMs was higher than that in single-component SAMs of T-COOH, and reached a maximum value of 9.2 × 10⁻¹² mol cm⁻² when the ratio of T-COOH to T-NH₂ in adsorption solution was of 3:2, and the formal potential (E^0′=(E_pa+E_pc)/2) of cyt c was −0.032 V (vs. Ag|AgCl (3 M NaCl)) in a 10 mM PBS. The interaction between cyt c and the binary SAMs made the E^0′ shift negatively when compared with that of cyt c in solution (+0.258 V vs. NHE, i.e., +0.058 V vs. Ag|AgCl (3 M NaCl)). The fractional coverage of bound cyt c was a 0.64 theoretical monolayer. The standard electron transfer rate constant of cyt c immobilized on the binary SAMs was also higher than that on single-component SAMs of T-COOH, and the maximum value of 15.8 ± 0.6 s⁻¹ was obtained when the ratio of T-COOH to T-NH₂ in adsorption solution was at 3:2. The results suggest that the electrode modified with the binary SAMs functions better than the electrode modified with single-component SAMs of T-COOH.     

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.     

Disposable electrochemical immunosensor for myeloperoxidase based on the indium tin oxide electrode modified with an ionic liquid composite film containing gold nanoparticles, poly(o-phenylenediamine) and carbon nanotubes

A disposable electrochemical myeloperoxidase (MPO) immunosensor was fabricated based on the indium tin oxide electrode modified with a film composed of gold nanoparticles (AuNPs), poly(o-phenylenediamine), multi-walled carbon nanotubes and an ionic liquid. The composite film on the surface of the electrode was prepared by in situ electropolymerization using the ionic liquid as a supporting electrolyte. Negatively charged AuNPs were then adsorbed on the modified electrode via amine-gold affinity and to immobilize MPO antibody. Finally, bovine serum albumin was employed to block possible remaining active sites on the AuNPs. The modification of the electrode was studied by cyclic voltammetry and scanning electron microscopy. The factors affecting the performance of the immunosensor were investigated in detail using the hexacyanoferrate redox system. The sensor exhibited good response to MPO over two linear ranges (from 0.2 to 23.4 and from 23.4 to 300 ng.mL^?1), with a detection limit of 0.05 ng.mL^?1 (at an S/N of 3).

Electroanalytical properties of cytochrome c by direct electrochemistry on multi-walled carbon nanotubes incorporated with DNA biocomposite film

A novel conductive biocomposite film (MWCNTs–DNA–cyt c) which contains multi-walled carbon nanotubes (MWCNTs) along with the incorporation of DNA and cytochrome c (cyt c) has been synthesized on glassy carbon electrode (GCE), gold (Au), indium tin oxide (ITO) and screen printed carbon electrode (SPCE) by potentiostatic methods. The presence of both MWCNTs and DNA in the biocomposite film enhances the surface coverage concentration (Γ), increases the electron transfer rate constant (K_s) up to 21% and decreases the degradation of cyt c during the cycling. The biocomposite film also exhibits a promising enhanced electrocatalytic activity towards the reduction of halogen oxyanions and oxidation of biochemical compounds such as ascorbic acid and l-cysteine. The cyclic voltammetry has been used for the measurement of electroanalytical properties of analytes by means of biocomposite film modified GCEs. The sensitivity of MWCNTs–DNA–cyt c modified GCE possess higher values than the values obtained for DNA–cyt c film modified GCE. Further, the reduction potentials of halogen oxyanions E_pc, clearly shows that the activity of the biocomposite is dependent on the electronegativity of halogen oxyanions. Electrochemical quartz crystal microbalance studies revealed the enhancements in the functional properties of MWCNTs, DNA and cyt c. We have studied the surface morphology of the biocomposite films using scanning electron microscopy and atomic force microscopy, which revealed that DNA and cyt c have been incorporated on MWCNTs. Finally, the flow injection analysis has been used for the amperometric detection of analytes at MWCNTs–DNA–cyt c film modified SPCE.