Direct electron transfer in the system gold electrode–recombinant horseradish peroxidases

The kinetics of the bioelectrocatalytic reduction of hydrogen peroxide has been studied at gold electrodes modified with different forms of horseradish peroxidase (HRP). Native HRP, wild type recombinant HRP (rec-HRP) and its two mutant forms containing a six-histidine tag at the C- or N-terminus, C_Hisrec-HRP and N_Hisrec-HRP, respectively, have been used for an adsorptive modification of the gold electrodes. The histidine sequences, i.e. histidine tags, were introduced into the peroxidase structure by genetic engineering of non-glycosylated rec-HRP using an Escherichia coli expression system. Experiments with a gold rotating disc electrode demonstrated that electrodes with the adsorbed rec-HRP forms exhibited high and stable current response to H₂O₂ due to its bioelectrocatalytic reduction based on direct (mediatorless) ET between gold and the active site of HRP. The heterogeneous ET rate constants were evaluated to be in the order of 20 or 33 s^?1 between rec-HRP or its histidine mutants and gold, respectively, in 0.01 M phosphate buffer (pH 7.4) containing 0.15 M NaCl. The increase in the heterogeneous ET rate found for C_Hisrec-HRP and N_Hisrec-HRP is probably due to the interaction of the histidine tag with the electrode surface. The kinetic data demonstrate that new possibilities for enhancing the catalytic activity of the enzyme at the electrode ∣ solution interface can be achieved by genetic engineering design of the enzyme molecules.     

Electrochemical reactions of benzoic acid on platinum and palladium studied by DEMS. Comparison with benzyl alcohol

The electrochemical reactivity of benzoic acid on porous platinum and palladium electrodes has been studied by cyclic voltammetry and differential electrochemical mass spectrometry (DEMS) in aqueous 0.1 M HClO₄. The objective of this work is to investigate the adsorption processes and the reactivity of this compound on different noble metals, in order to compare these results with those obtained for related aromatic compounds. On-line mass spectroscopy analysis of volatile products reveals that the adsorption of benzoic acid is irreversible at platinum while it is mainly reversible on palladium. Accordingly, different catalytic activity of platinum and palladium is demonstrated during the electrooxidation process. The anodic stripping of the adsorbates formed from benzoic acid yields CO₂ as the sole oxidation product of platinum. Cathodic stripping of the residues results in the partial desorption of the organic adlayer from the electrode. At palladium, only small amounts of irreversibly adsorbed species, which also oxidise to CO₂ are detected. The effect of the nature of the electrode on the electrochemical behaviour of these substances is also analysed.     

In situ monitoring of the mercury electrode surface in HClO4 solutions by means of infrared reflection absorption spectroscopy

In situ IRAS at a Hg–Au electrode in HClO₄ solutions shows that the adsorption state of water molecules on mercury changes depending on the applied potential as in the case on gold rather than on platinum. At the negatively charged electrode surface, it is expected that the dipole moment of the adsorbed water molecules is oriented toward the surface to some extent. The mercury–water interaction is considered to be stronger than the gold–water interaction. As the sample potential changes in the positive direction, the adsorbed water molecules are expelled and/or reorient and perchlorate ions adsorb on the Hg–Au electrode surface.     

On the adsorption and reduction of NO3− ions at Au and Pt electrodes studied by in situ FTIR spectroscopy

The adsorption of nitrate ions on gold and platinum electrodes in acid solution has been studied in acidic solution using in situ FURS. It is found that nitrate is adsorbed probably with a two-fold coordination on gold electrodes as suggested by the adsórbate potential-dependent spectral feature centered between 1440 and 1460 cm^?1. In addition, partial reduction of the nitrate ions at more negative potentials (ca. 0.05 V) generates nitrite ions in solution. These ions are co-adsorbed with nitrate ions in the double layer region of potentials. The nitrite ions seem to be adsorbed O-down with a one-fold or two-fold coordination. Platinum electrodes are found to be much more active catalytically to nitrate reduction than gold electrodes. The reduction of nitrate ions at potentials below 0.8 V generates an adsorbed product presenting a potential-dependent band at 1540 to 1580 cm^?1 which has been identified as adsorbed NO.     

DNA-modified electrodes. Part 2. Electrochemical characterization of gold electrodes modified with DNA

Gold electrodes were modified with DNA by adsorption. The DNA-modified electrodes were electrochemically characterized with Co(bpy)₃³⁺, a electroactive DNA-binding complex, as an indicator. It is interesting that the pair of redox peaks of Co(bpy)₃³⁺ split into two pairs at dsDNA-modified gold electrodes. One pair of peaks shifts negative, and the peak current increases notably; another pair of peaks shifts positive. These suggest that dsDNA has been immobilized onto gold electrode surfaces and the layer of dsDNA on the surfaces can bind Co(bpy)₃³⁺ in two different ways. Gold electrodes can be modified also with ssDNA by adsorption but only one pair of peaks of Co(bpy)₃³⁺ appears at ssDNA-modified gold electrodes. The amount of Co(bpy)₃³⁺ enriched by the layers of dsDNA or ssDNA adsorbed at gold electrodes was estimated from the peak charge of Co(bpy)₃³⁺ reduction at the electrodes obtained by CV. The stability of the DNA-modified electrodes was investigated. The DNA modification layer on gold surfaces is unstable to alkali and to heat, but stable to acid solutions and very stable in long stock in a dry state. A comparison of modifications of gold, platinum and glassy carbon with DNA was carried out.     

Electrosynthesis of a new enzyme-modified electrode for the amperometric detection of glucose

The electrooxidation of a new aminonaphthalene compound modified with a carboxylic acid function, 1-(5-aminonaphthyl)ethanoic acid (ANEA) was studied, with a view to binding glucose oxidase (GOD) covalently. Its electrooxidation leads to thin films (PANEA) on bare glassy carbon (GC) platinum (Pt) electrodes and also on prepolymerized aminonaphthoquinone (PANQ) films. Polymer-modified bilayer electrodes Pt/PANQ/PANEA were then constructed. GOD was covalently bound onto these electrodes, leading to a Pt/PANQ/PANEA/GOD glucose sensor. The amperometric response to glucose was monitored in aerated and deaerated conditions. The results show very efficient covalent binding of GOD and good enzyme activity on the electrode surface.     

Improvement of alternariol monomethyl ether detection at gold electrodes modified with a dodecanethiol self-assembled monolayer

The electro-oxidation of alternariol monomethyl ether (AME), one of the main metabolites of the Alternaria genus mycotoxins, is studied at 1-dodecanethiol (DDT)-modified gold electrodes, in acetonitrile (ACN) – aqueous phosphate buffer solutions of different pH values, by using cyclic (CV) and square-wave (SWV) voltammetries. The AME voltammetric response at the bare electrode suffers from two drawbacks: it appears at potentials close to the onset of gold oxide formation, and it is hampered by a fouling of the electrode surface due to the accumulation of oxidized products. These shortcomings are circumvented by the use of DDT-coated electrodes, since the intervening monolayer inhibits gold oxide formation and surface passivation by the electrochemical products, without affecting the oxidation kinetics of AME significantly. Diagnostic criteria based on the voltammetric peak parameters show that the electrochemical behavior of AME at the modified electrode is mainly controlled by reactant diffusion from solution, with a weak adsorption of both the mycotoxin and its oxidation products at monolayer defects. Calibration curves were constructed from the AME square-wave voltammetric response and a detection limit of 9.1 × 10^?8 mol dm^?3 was determined, which is about three times smaller than a previous estimate at platinum and glassy carbon electrodes, and about fifty times smaller than the limit derived from measurements carried out at a polyphenol oxidase-modified carbon paste electrode.     

Electrochemical characterisation of gold on Pt{h k l} for ethanol electrocatalysis

The cyclic voltammetric (CV) behaviour of gold adsorbed on Pt{h k l} electrodes is reported. It is found that stepped surfaces vicinal to Pt{1 1 1} exhibit preferential adsorption of gold at step sites. However, for stepped surfaces containing Pt{1 0 0} terraces, adsorption of gold at step and terrace site occurs with equal probability, although for Pt{n 1 1} electrodes with n < 7 (i.e. electrodes containing short Pt{1 0 0} terraces), slight preference for step site adsorption is still observed. This behaviour is similar to that reported previously for Bi adsorption on Pt{h k l} and is interpreted in terms of slower rates of gold atom surface diffusion across Pt{1 0 0} vs. Pt{1 1 1} terraces. By comparing the electrochemical response of gold adlayers on Pt{h k l} with previously published bulk Au{h k l} voltammetry, it is deduced that the gold overlayers on Pt{h k l} are pseudomorphic in nature. This finding is in agreement with previous UHV and electrochemical results for gold deposition on platinum. Using such gold modified Pt{h k l} electrodes as model surfaces, we also report, for the first time, the electrooxidation of ethanol on Au/Pt{h k l} in alkaline aqueous solution. The ethanol oxidation reaction (EOR) is shown to be structure sensitive for submonolayer gold coverages. In fact, a maximum in the rate of EOR at platinum sites adjacent to gold islands is observed between approximately 0.4–0.6 monolayers of gold on Pt{1 0 0}, Pt{1 1 1} and Pt{1 1 0} in 0.1 M NaOH(aq). A second electrooxidation state at more positive potentials for 0 < θ_Au < 1 ascribed to EOR at pure gold sites is also observed, at least for Pt{1 1 1} and Pt{1 0 0}. On Pt{1 1 1}, the potential range and activity of EOR in this second process is similar to bulk gold whereas for submonolayer gold films on Pt{1 0 0}, the peak is shifted to more negative potentials.     

Kinetics of oxygen reduction on gold nanoparticle/multi-walled carbon nanotube hybrid electrodes in acid media

In this paper, the electrochemical reduction of oxygen has been studied on gold nanoparticle/multi-walled carbon nanotube (AuNP/MWCNT) modified glassy carbon (GC) electrodes in 0.5 M H₂SO₄ using the rotating disk electrode (RDE) method. The AuNP/MWCNT catalysts were prepared by chemical deposition of AuNPs onto MWCNTs spontaneously grafted with 4-nitrophenyl groups. The composite electrode was characterised by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and cyclic voltammetry (CV). The oxygen reduction behaviour of these electrodes was compared with that of a bulk gold electrode. The AuNP/MWCNT catalyst showed a pronounced electrocatalytic activity towards O₂ reduction in acid media. The half-wave potential of O₂ reduction on the AuNP/MWCNT catalyst shifted ca 80 mV to more positive potentials as compared to that of a polished Au electrode. The kinetic parameters of oxygen reduction were determined and the specific activity of the hybrid electrode was slightly higher than that of the bulk Au electrode.     

The influence of surfactants on the electron-transfer reaction at self-assembled thiol monolayers modifying a gold electrode

Monolayer films of thiols with different terminal groups (CH₃, COOH and NH₂) were prepared on gold electrodes using a self-assembling procedure. Cyclic voltammetry was used to investigate the electron-transfer reaction of potassium ferricyanide at the thiol modified gold electrode in the presence of surfactants. It was found that surfactants show some effects on the electron-transfer reaction at the thiol modified electrode. Cationic surfactants can improve the reversibility of a redox reaction at the thiol modified gold electrodes, while anionic surfactants cause a little inhibition of electron transfer of the redox at both methyl and carboxyl terminal thiol modified gold electrodes. Surfactants can interact with thiol monolayers in different ways, changing the structure and properties of the monolayer film, and can further affect electron transfer at the modified gold electrode.     

Fabrication of a mediated glucose oxidase recessed microelectrode for the amperometric determination of glucose

Miniaturized electrochemical biosensors for glucose were constructed, using recessed gold microelectrodes (diameter 350 to 425 μm) and ultramicroelectrodes (diameter less than 25 μm) as the base sensors. The gold was a substrate for chemically attached thiolated glucose oxidase containing adsorbed hydroquinonesulfonate, which served as a mediator of electron transfer between the electrode surface and the enzyme. The chemically modified electrodes were coated with Nafion® to reduce the effects of anionic interferences, such as ascorbic acid, and were tested in sodium phosphate buffered glucose solutions (pH 7.4) at 25°C by cyclic voltammetry and steady-state amperometry at positive potentials suitable for glucose detection.Cyclic voltammetry confirmed that the glucose oxidase and hydroquinonesulfonate were both securely immobilized at the surface of the electrodes. The amperometric t₉₀ response times for the ultramicroelectrodes and microelectrodes were under 20 and 100 s respectively. The current response of both electrode types was linear up to glucose concentrations of 50 mM, and the response to ascorbic acid at physiological concentrations was reduced by Nafion® by 53.3%. Key advantages of this electrode are that it is miniaturized for optimum in vivo analysis, it is unresponsive to variable oxygen tension, and its recess allows for increased response stability and faster response times.     

Mechanism of the catalysis by Alcaligenes eutrophus H16 hydrogenase of direct electrochemical reduction of NAD+

The mechanism of direct electrochemical reduction of NAD⁺ into NADH catalysed by Alcaligenes eutrophus H16 hydrogenase was analysed in thin layer electrochemical cells with platinum and carbon electrodes. Two phases can be distinguished in the catalytic reaction occurring on platinum electrodes. In the potential range from approximately ?0.620 to ?0.675 V (SCE) direct electron transfer occurred via the diaphorase-like dimer of the hydrogenase. Below ?0.69 V versus SCE the hydrogenase used hydrogen species adsorbed onto the platinum electrode, but no molecular hydrogen was required for this second catalytic phase. The mechanism was quite similar to those which had been previously determined for Rhodococcus opacus hydrogenase. This confirmed the very great similarity of the two enzymes, even if the maximum NAD⁺ reduction rate of 0.36 mM min^?1 obtained here remains lower than those reached with R. opacus hydrogenase in a previous study. Careful analysis of the experimental data obtained on a carbon electrode led to the conclusion that no direct electron transfer was observed on this material under the operating conditions used. On the other hand, the voltammetric experiments performed on a carbon electrode in a thin layer cell showed clearly the occurrence of a catalytic current due to the hydrogenase-catalysed reduction of NAD⁺ by molecular hydrogen. This may be a useful tool for further analysis of the hydrogenase kinetics.     

Oxidation of methylamine and ethylamine on Pt single crystal electrodes in acid medium

Cyclic voltammetry (CV) and Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) have been combined to analyse the oxidation of methylamine and ethylamine on platinum single crystal electrodes in an acidic medium. The oxidation of both amines on Pt(hkl) electrodes gives rise to the formation of adsorbed cyanide ad-layers, which have been detected by in situ infrared spectroscopy. In the case of Pt(111) the resulting adsorbed cyanide is rather stable but, on the contrary, the adsorbed CN-like species is highly reactive on Pt(100). It yields either adsorbed NO and CO₂ when the electrode is polarised above 0.7 V or adsorbed CO below 0.4 V. The detection of adsorbed cyanide is difficult to achieve at the Pt(110) surface. In the case of ethylamine, these adsorbed species are present in lower coverage than for methylamine. The substitution of one hydrogen atom by a methyl group in the methylamine makes the amine molecule more stable. So, the behaviour of ethylamine provides evidence for its lower electrochemical reactivity when compared with that of the methylamine molecule.     

Electrochemical oxidation of o-aminophenol in aqueous acidic medium: formation of film and soluble products

The electrochemical oxidation of o-aminophenol (OAP) has been studied by cyclic voltammetry on platinum (Pt) and glassy carbon (GC) electrodes. Films formed on Pt exhibited a voltammetric response that is dependent on the final potential applied during the electropolymerization. Although a less electroactive film has been obtained on Pt electrodes cycled at higher potentials, modified GC electrodes showed a well-defined redox response independently of the potential range used. This result is attributed to an activation process of the GC electrode rather than to an electroactive film deposited on the electrode. The deposition process of thin films was also investigated with quartz crystal microbalance experiments. Poly-o-aminophenol (POAP) films were insoluble with an electrochromic response from colorless to brown, and a ladder structure confirmed by infrared spectra. Exhaustive electrolysis of OAP produced a reasonable amount of soluble product, which after solvent extraction, was characterized as 2-aminophenoxazin-3-one (APZ), a product also prepared by chemical oxidation of OAP. A route of electrooxidation of OAP has been proposed based on electrochemical and spectroscopic results.     

Self-assembled monolayers of long-chain xanthic acids on gold studied by voltammetry

A new method for the formation of xanthic acids H(CH₂)_nCS₂H (n=8, 10, 12) in a non-polar solvent from the corresponding potassium salts was developed. It was found that the xanthic acids spontaneously adsorb onto gold electrodes, via a mechanism that is proposed to be similar to the adsorption of thiols on gold surfaces. The xanthate modified gold electrodes were studied by cyclic voltammetry. It was found that the xanthate layers efficiently block the current from a ferro/ferri redox couple in aqueous solution. Studies of the electrochemical desorption of the xanthate layer supported the fact that xanthic acids spontaneously form a self-assembled monolayer on the electrode.     

Effect of pH on the catalytic electrooxidation of NADH using different two-electron mediators immobilised on zirconium phosphate

A detailed study is reported on the electrocatalytic oxidation of β-nicotinamide adenine dinucleotide at three different carbon paste electrodes modified with redox mediators commonly used in bioelectrochemistry, viz. Meldola blue, methylene green and riboflavin, adsorbed on zirconium phosphate. Cyclic voltammetric investigations of these chemically modified electrodes performed in aqueous tris buffer solutions at different pHs allowed us to conclude that the immobilised redox species presented very stable redox properties, with midpoint potentials (E_m, ?125, ?40, and ?140 mV versus SCE for Meldola blue, methylene green and riboflavin, respectively) virtually independent of the pH of the contacting solution. The second-order rate constant for electrocatalytic oxidation of NADH as well as its pH dependence (from 6 to 8) were evaluated from rotating disk electrode (RDE) experiments, using the Koutecky–Levich approach.     

Adsorption of human serum albumin on electrochemical titanium dioxide electrodes: Protein–oxide surface interaction effects studied by electrochemical techniques

The adsorption of Human Serum Albumin (HSA) on a semiconductor TiO₂ electrochemical oxide was investigated using Cyclic Voltammetry (CV), Capacity–Potential curves (C–E) and time-resolved techniques as a function of electrode potential and protein concentration. The presence of HSA adsorbed on the electrode surface modifies the voltamperometric behavior of the hydrogen evolution reaction (her) and also produces a major modification in the diffusional layer thickness of the H⁺ ions. The adsorbed amounts of HSA were analyzed throughout different adsorption isotherms. The experimental data were modeled with a modified Langmuir type isotherm, considering a weak chemisorption on a surface with heterogeneity in site-energy distribution with some degree of attractive lateral interactions between the adsorbed protein molecules. The effect of the adsorption potential (E_ads) was investigated polarizing the electrode at −0.70, −0.50 and −0.08 V vs. SCE. The capacity response obtained from the different impedance experiments was determined by the space charge region in the semiconductor. It was possible to correlate processes produced by the protein adsorption on the surface (occurring in the electrolyte side of the interface) with changes in the semiconductor properties of the TiO₂ (in the oxide side of the interface). The adsorption of HSA produces an increase in donor concentration (N_D) of the semiconductor and a shift of the E_fb to more negative values. These effects are more pronounced with an increase in the protein concentration. The relative change in N_D is lower and the change in E_fb is higher when the adsorption occurs at less negative applied potentials. Adsorption kinetics and thermodynamic parameters were calculated for a wide protein concentration range.     

Preparation of a platinum layer-modified aluminum electrode by electrochemical and electroless cementations and its use for the electrooxidation of methanol

The deposition of platinum on the aluminum electrode by two methods involving electroplating by single step potential from an aqueous buffered H₂PtCl₆ solution (pH 7) and electroless cementation from an aqueous solution of H₂PtCl₆ saturated with NaF is described. The electrochemical characteristic of the platinum layer-modified aluminum electrode (Pt/Al) in aqueous solution with various pHs was investigated. The Pt/Al layers were characterized by imaging with scanning electron microscopy and optimum conditions for the preparation of the Pt/Al modified electrode was chosen. The modified electrode was used as electrocatalyst for the electrooxidation of methanol in 0.1 M H₂SO₄ solution. After characterization of some features of the electrochemical system on the stationary and rotating electrode, an oxidation pathway is suggested. The current density of the methanol oxidation at Pt/Al electrode measured in optimized conditions and compared with that obtained on the smooth Pt, Pt/Pt, and Pt/PoPD/Pt electrocatalysts. The stability of the electrode was followed over the course of the time and its stability and reproducibility was confirmed.     

Characterization of protein monolayers by surface plasmon resonance combined with cyclic voltammetry ‘in situ’

Surface plasmon resonance (SPR) and cyclic voltammetry (CV) have been used to characterize monolayers of cytochrome-c and cytochrome-c-oxidase adsorbed on gold surfaces modified with several alkanethiol self-assembled monolayers. A direct comparison between protein surface coverages calculated from SPR and cyclic voltammetric measurements carried out with the same sample allows one to make some conclusions about the amount and orientation of cytochrome-c adsorbed on the gold surface. Time differential surface plasmon resonance measurements combined with cyclic voltammetry ‘in situ’ has been used to investigate the redox processes of adsorbed cytochrome-c and cytochrome-c-oxidase. The potential-dependent time differential SPR response observed for cytochrome-c adsorbed on Au ∣ MPA (3-mercapto-1-propionic acid) electrodes is explained as arising from a potential-dependent adsorption/desorption process. By contrast, similar experiments carried out with cytochrome-c oxidase adsorbed on Au ∣ MPA electrodes can be explained as the result from a potential-dependent conformational change between the ‘resting’ and the ‘pulsed’ states of the adsorbed oxidase.     

Preparation and methanol oxidation catalysis of Pt-CeO2 electrode

An electrochemical methodology to prepare platinum/cerium oxide electrodes by using glassy carbon substrates has been developed. The method involves an “occlusion deposition” of CeO₂ particles at glassy carbon electrodes from a platinum bath in acid solution at an applied constant potential. The depositions were made by applying a controlled potential of ?200 mV vs. Ag/AgCl in 0.5 M H₂SO₄ containing K₂PtCl₆ and CeO₂ suspensions. The deposition was done at different concentrations of K₂PtCl₆ and constant supersaturation concentrations of CeO₂. The final composite electrodes were characterized by using X-ray fluorescence/energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscopy. The catalytic properties of the composite electrodes were tested for the electrooxidation of methanol in acid solution by chronoamperometry and were compared to Pt only deposits on glassy carbon.