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.     

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 modifiers for the promotion of direct electrochemistry of cytochrome c

Fifty-four bifunctional organic compounds were studied to assess their ability to promote the direct electrochemistry of horse heart cytochrome c at a modified gold electrode. From the results of the survey it was possible to identify those features important for successful promotion of the electrochemical activity. It is suggested that it is necessary to provide groups on the electrode surface which can hydrogen bond or form salt bridges to the positively charged lysine side chain groups around the heme crevice on cytochrome c. The functional groups, Y, may be anionic or weakly basic and can be attached to the electrode using a bifunctional compound X ～ Y. The group, X, adsorbs or binds to the gold surface through nitrogen, phosphorus, or sulphur. A “pre-activation” step for the adsorption of some surface modifiers has been discovered. The molecular structure of a compound which promotes cytochrome c electrochemistry can be either conformationally rigid or flexible, aromatic or aliphatic, but it should direct Y out from the electrode. The length of the molecule does not appear to affect the rate of electron transfer. The presence of a hydrophobic zone in the structure is neither necessary nor sufficient for successful promotion of cytochrome c electrochemistry.     

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.     

Remarkable Electrochemical Responses of Ferrocene/NaY Zeolite Composite modified Electrode Based on Hydrophobic Ionic Liquid

The ferrocene/NaY zeolite composites (Fc/NaY) are introduced on the surface of a glassy carbon electrode together with the hydrophobic ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF₆). The modified electrode thus constructed exhibits a pair of reversible redox peaks corresponding to ferrocene. Additionally the peak separation remains almost constant (58–75 mV) and the value of the ratio i_pa/i_pc is close to 1 for scan rates in the range from 10 to 1000 mV s^?1. The effects of the scan rate, aqueous supporting electrolytes, hydrophobic ionic liquid and the contents of ferrocene encapsulated by electrochemistry are investigated. The extrazeolite electron transfer process is discussed. Furthermore, the Fc/NaY/IL‐modified electrode shows good mediation towards oxidation of ascorbic acid, dopamine, hydroquinone, and catechol.     

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.

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.

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.     

Voltammetric investigation of cytochrome c on gold coated with a self-assembled glutathione monolayer

The direct, reversible electrochemistry of horse-heart cytochrome c (cyt. c) was realized on a self-assembled glutathione (GSH) monolayer modified Au electrode. The voltammetric responses of cyt. c on GSH/Au electrode were found to be affected by pH during the electrode modification, metal ions and surfactants. Using potassium ferricyanide [K4Fe(CN)6] as a probe, these effects on the voltammetric responses of cyt. c were characterized by electrochemical methods. It was found that the pH during the electrode modification, metallic ions and surfactants changed GSH monolayer's charge state and the conformation on the electrode surface, and resulted in the influence on the voltammetric responses of cyt. c. The experimental results provided us information to understand the mechanism of the interfacial electron transfer of electrode-protein, as well as the electron transfer of cyt. c in life system.     

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.     

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.     

Evaluation of a glassy carbon electrode modified by a bilayer lipid membrane with incorporated DNA

The objective of the present work was the evaluation and characterization of a glassy carbon (GC) electrode modified by a bilayer lipid membrane (BLM) with incorporated single-stranded deoxyribonucleic acid fss DNA). Various procedures were developed and tested for the incorporation of ss DNA at the electrode modified by the lipidic membrane: Differential pulse voltammetry (i.e. oxidation of guanine and adenine residues) was used to monitor the incorporation of ss DNA at the GC electrode modified by the BLM. The results have shown that the lipid membrane enhances the stability of ss DNA during a "medium-exchange" of the electrode and prohibits its diffusion from the electrode surface. The third scheme was proven to be the most appropriate as both electrode modification by the BLM and DNA adsorption occur in one stage and much faster (as no BLM thinning process is required) as compared to the former two techniques; furthermore, maximized loading of DNA in BLMs is achieved which reduces by ca. 10-fold the DNA amounts that can be detected electrochemically. Conventional planar "free-suspended" and self-assembled metal supported BLMs were used to monitor in situ the incorporation of ss DNA in these membranes. The results have shown that the adsorption of ss DNA at lipid membranes (as a medium for DNA incorporation on an electrode surface) can occur much faster, using milder conditions and smaller amounts of DNA than by previously described techniques.     

Ferrocene clicked poly(3,4-ethylenedioxythiophene) conducting polymer: Characterization, electrochemical and electrochromic properties

The “click” chemistry, Cu(I)-catalyzed azide–alkyne cycloaddition reaction, was applied to covalently functionalize the poly(3,4-ethylenedioxythiophene) (PEDOT) conducting polymer film with an excellent electron transfer mediator (ferrocene). Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy were used to characterize the ferrocene-grafted PEDOT conducting polymer film, and it was proved that the grafting procedure via click reaction had a high efficiency. The ferrocene groups covalently grafted in the polymer films turned out to own a relatively fast electron transfer rate and show multi-color states via adjusting applied potential.     

Electron transfer between ferrocene-modified Au/octadecanethiol/lipid BLM electrode and redox couples in solution

Bilayers incorporated with ferrocene consisting of self-assembled octadecanethiol and lipid monolayer on gold substrates were fabricated. Its electrochemical behaviors in solutions containing different redox couples were investigated by cyclic voltammetry and ac impedance. The transmembrane electron transfer reaction across octadecanethiol self-assembled film and an adsorbed phospholipid layer mediated by ferrocene have been observed in the solution of Fe(CN)6(3-/4-). The formal potential difference between mediator in bilayer lipid membrane (BLM) and redox couple in solution has a great impact on the transmembrane electron transfer behavior. The ferrocene-modified BLM electrodes might be useful for constructing a bilayer-based electrochemical current rectifying device.     

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.     

Glucose and lactate biosensors prepared by a layer-by-layer deposition of concanavalin A and mannose-labeled enzymes: electrochemical response in the presence of electron mediators.

An electrochemical response of glucose and lactate biosensors which were prepared by coating a platinum electrode with a thin film composed of concanavalin A and mannose-labeled glucose oxidase (GOx) or lactate oxidase (LOx) was evaluated in the presence of ferrocene derivatives as electron mediator. Both glucose and lactate biosensors showed catalytic current to glucose and lactate, respectively, in cyclic voltammetry, suggesting that the ferrocene derivatives can mediate electron transport smoothly from the reduced forms of GOx and LOx in the thin films to the electrode. Among the three kinds of ferrocene derivatives used, ferrocenylmethanol was found to be the most suitable electron mediator because of its low oxidation potential. The glucose and lactate sensors gave useful calibration graphs, in which higher detection limits were reached as compared with those observed when the sensors were operated in the absence of electron mediator.     

Immobilization and electrochemistry of cytochrome c on amino-functionalized mesoporous silica thin films

The immobilization and electrochemistry of cytochrome c (cyt c) on amino-functionalized mesoporous silica thin films are described. The functionalized silica films with an Im3m cubic phase structure were deposited on conducting ITO substrate by co-condensation of tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) in the presence of Pluronic F127 under acidic conditions. The high specific surface area, large pore size and functional inner surface of mesoporous silica thin films result in a high cyt c loading, and the cyt c immobilization on this silicate framework is stable. After adsorption of cyt c, the ordered cubic structure of mesoporous silica and the redox activity of immobilized cyt c are retained as demonstrated by X-ray diffraction (XRD), Transmission electron microscope (TEM) and cyclic voltammetry. The redox behavior of the cyt c/silica film-modified ITO electrode is a surface-controlled quasi-reversible process for the experimental conditions used in this work and the electron transfer rate constant is calculated is 1.33 s⁻¹. The ITO electrode modified by cyt c/silica film possesses a high stability; even cyt c retains its redox activity following immobilization for several months. Furthermore, the electrocatalytic activities of the modified ITO electrode to hydrogen peroxide and ascorbic acid have been studied. Since these behaviors are quite pronounced, the modified electrode can be used for detection of hydrogen peroxide and ascorbic acid.     

Activation of electrode surfaces: semiquantitative characterization of electrode surfaces modified with heteropolyanions

Several Techniques have been used successively or in combination to approach a better understanding of surfaces modified by silicotungstic heteropolyanion. Due to the strikingly high activity of these electrodes for the hydrogen evolution reaction, in particular, there exists a need to identify the chemical composition of the catalyst, to evaluate the amount of catalyst on the surface, and to study the electrochemical behaviour of the “catalyst” itself. Plasma emission spectrometry shows semi-quantitatively that the catalyst resembles the starting material closely, although it is not possible at present to give the exact chemical formula. Electron microprobe analysis (EMA) confirms the presence of tungsten, and especially silicon, on the surface, even though the concentration of this last element is very low. The absence of platinum in the starting HPA, as well as on the electrode surface, is also clearly demonstrated by EMA, which is of prime importance in showing that the “HPA catalyst” is active by itself. Combined coulometry and UV-visible spectroscopy have been used to evaluate the amount of HPA consumed during electrolysis. Cyclic voltammetry shows a surface redox couple corresponding to the catalyst, and well separated from the first redox couple of SiW₁₂O^4-₄ in 0.5 M H₂SO₄ solution. A better understanding of the activation-deactivation processes of the catalytic electrode surface ensues.     

Direct and Cytochrome c Mediated Electrochemistry of Bilirubin Oxidase on Gold

Direct electron transfer (DET) of bilirubin oxidase from Myrothecium verrucaria (BOD) was established on promoter‐modified gold electrodes. The electrochemical behavior of the enzyme in solution was studied by means of cyclic voltammetry evaluating the biocatalytic reduction of dioxygen. The reaction of BOD at Au electrodes was shown to be efficient only at low pH. In addition, a novel interaction between BOD and cytochrome c (cyt.c) was found. It was shown that BOD efficiently accepts cyt.c as an electron donor in both cases when cyt.c is in solution and electrostatically adsorbed. The results suggest that cyt.c can play the role of a mediator facilitating electron transfer in a pH range where no DET could be observed between the enzyme and the electrode. For the interaction between cyt.c and BOD in solution the reaction kinetics has been studied electrochemically and spectrophotometrically.     

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.