Direct electron transfer of cytochrome P450 2B4 at electrodes modified with nonionic detergent and colloidal clay nanoparticles

A method for construction of biosensors with membranous cytochrome P450 isoenzymes was developed based on clay/detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated cytochrome P450 2B4 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bioelectrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay-modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between -0.292 and -0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bioelectrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of product formation also indicates the bioelectrocatalysis by CYP2B4.     

Direct electron transfer of heme- and molybdopterin cofactor-containing chicken liver sulfite oxidase on alkanethiol-modified gold electrodes

Direct heterogeneous electron transfer (ET) of sulfite oxidase (SOx), a heme- and molybdopterin cofactor-containing intermembrane enzyme, was studied on alkanethiol-modified Au electrodes both with SOx entrapped between the modified Au electrode and a permselective membrane and with SOx adsorbed at the electrode surface, in the absence of any membrane. SOx in direct electronic communication with the electrode surface gave a quasi-reversible electrochemical signal with a midpoint potential of--120 mV vs Ag/AgCl corresponding to the redox transformations of the heme domain of SOx and with a heterogeneous ET constant in the order of 15 s(-1). The efficiency of the bioelectrocatalytic 2e- oxidation of sulfite catalyzed by SOx in direct ET exchange with the electrode was shown to depend essentially on the nature of the alkanethiol layer. Adsorption and orientation of SOx on an 11-mercapto-1-undecanol (MuD-OH) self-assembled monolayer, i.e., terminally functionalized with OH groups, provided efficient catalytic oxidation of sulfite, contrary to nonfunctionalized alkanethiols, e.g., 1-decanethiol, or alkanethiol layers terminally functionalized with NH2 groups. Comparative studies with short-chain alkanethiols, e.g., cysteamine and 2-mercaptoethanol, revealed an evidently different mode of adsorption of SOx on these layers, onto which SOx was not catalytically active. Coadsorption of MuD-OH and 11-mercapto-1-undecanamine improved the surface properties of the SAM, resulting in a higher surface coverage with bioelectrocatalytically active SOx but not in an increased apparent catalytic rate constant, kcat, ranging in the order of 18-24 s(-1) at pH 7.4. The achieved efficiency of SOx bioelectrocatalysis in direct ET reaction between the modified electrode and the enzyme approached the rates characteristic for the catalysis mediated by cytochrome c, the natural redox partner of SOx, thus implying the retention of the biological function of SOx under the heterogeneous electrode reaction conditions. Results obtained enable the development of a third-generation biosensor for sulfite monitoring.     

Electron transfer reactions of glucose oxidase at Au111 electrodes modified with phenothiazine derivatives

The catalytic reaction of glucose oxidase (GOx) mediated by 3-(10-phenothiazyl)propionic acid (PT-PA) and phenothiazine-labeled poly(ethylene oxide) (PT-PEO1000) that are covalently bonded to Au(111) electrodes has been investigated. The PT-PA and PT-PEO1000 are reacted with 2-aminoethanethiol (AET), followed by the formation of a self-assembled monolayer (SAM) onto the Au surface. The PT group immobilized on the SAM of AET acts as an effective mediator for the electron transfer (ET) between the electrode and the FAD center of freely diffusing GOx in solution. The ET rate constant estimated from the catalytic current using a newly derived equation is larger by 1 order of magnitude for the PT-PA-modified system (1.1 x 10(5) dm(3) mol(-1) s(-1)) than for the PT-PEO1000 system (1.4 x 10(4) dm(3) mol(-1) s(-1)). The order of the magnitude of the ET rate constant clearly contrasts with the GOx hybrid systems that we previously investigated (Anal. Chem. 2003, 75, 910-917), in which the presence of the PEO spacer enhances the ET reaction rate. The reduction in the apparent PT concentration at the electrode interface due to the high mobility of the PEO chain, leading to low efficiency in the formation of an enzyme-mediator complex, is a possible reason for the lower mediation ability of PT-PEO1000 than that of PT-PA for the ET between the FAD group and PT(+) immobilized on the electrode. Inhibition of the penetration of GOx molecules into the monolayer and of the accessibility of some part of PT groups to GOx molecules could also be reasons for the lower mediation ability of PT-PEO1000 thickly modified on the electrode.     

Towards combined electrochemistry and surface-enhanced resonance Raman of heme proteins: Improvement of diffusion electrochemistry of cytochrome c at silver electrodes chemically modified with 4-mercaptopyridine

To date, a successful combination of surface-enhanced resonance Raman spectroscopy (SERRS) and electrochemistry to study heme proteins is inhibited by the problems raised by the prerequisite to use silver as electrode metal. This paper indicates an approach to overcome these problems. It describes a quick and reproducible procedure to prepare silver electrodes chemically modified with 4-mercaptopyridine suitable to perform diffusion electrochemistry of cytochrome c (cyt c). The method involves the employment of a mechanical and a chemical treatment and avoids the use of alumina slurries and any electrochemical pretreatment. Cyclic voltammetry (CV) was used to test the electrochemical response of cyt c, and the CV signals were found identical with those obtained on gold electrodes under the same experimental conditions. Compared to previous literature, a significant improvement of the CV signal of cyt c at silver electrodes was achieved. Preliminary results show that this treatment can be also successfully employed for the preparation of SERRS-active electrodes.     

Efficient direct electron transfer of PQQ-glucose dehydrogenase on carbon cryogel electrodes at neutral pH

We present a comprehensive study of the direct electron transfer reaction of soluble PQQ-GDH from Acinetobacter calcoaceticus. Wild-type PQQ-sGDH nonspecifically adsorbed on carbon cryogel electrodes retained its enzymatic activity for glucose and maltose oxidation at pH 7.2 and 37 °C. The cyclic voltammograms in the absence of enzymatic substrate showed 2 redox peaks that suggest a two-step, one-electron oxidation/reduction of PQQ. Calibration curves showed a linear amperometric response for a wide glucose concentration range, including the values normally found in blood. At saturation, the catalytic current reached 0.93 mA cm(-2). Altogether the experimental results suggest that the amperometric output of the electrodes and the shape of the calibration curves represent a combination of the intrinsic enzyme kinetics, the maximum rate of heterogeneous electron transfer and the substrate accessibility to the enzyme's active center caused by the confinement of the enzyme into the mesoporous structure. A new mutant enzyme, N428C, developed in our group that shows almost twice the maximum catalytic activity in homogeneous experiments in solution, also showed a DET signal on carbon cryogel electrodes for glucose electro-oxidation. The higher activity for the mutant enzyme was also verified on the electrode surface.     

Field-induced electron emission from broad-area YBaCuO high-T c electrodes

A transparent anode facility has been used to compare the field-induced electron emission characteristics of planar YBaCuO high-T _c electrodes at room temperature and in the superconducting state. It was found that virgin electrodes under ambient conditions exhibit an initial current switch-on event at typical field levels of 30 MV m^–1, with the emission coming from a single point site: a large hysteresis effect was also observed when the field was cycled. In contrast, at low temperatures, no switch-on events were observed, and the hysteresis effect was significantly smaller. Also, if an emission current in the nanoamp range was recorded under constant-field conditions as the temperature was lowered through T _c, there was an abrupt (i.e. step-like) fall in both the emission current and its associated noise at the superconducting transition.     

Direct electrochemistry of cytochrome c at a glassy carbon electrode modified with single-wall carbon nanotubes

The electrochemistry of horse heart cytochrome c was studied by cyclic voltammetry at a glassy carbon electrode modified with single-wall carbon nanotubes (SWNTs). A pair of well-defined redox waves was obtained in cytochrome c aqueous solution at an activated SWNT film-modified electrode. The optimal conditions for activating the SWNT film-modified electrode has been determined. The electrode reaction of cytochrome c is a diffusion-controlled process. The peak current increases linearly with the concentration of cytochrome c in the range from 3.0 x 10(-5)-7.0 x 10(-4) M. The detection limit is 1.0 x 10(-5) M. The activated SWNT film was characterized by scanning electron microscopy. Furthermore, interaction of cytochrome c with adenine was characterized by electrochemical and spectral methods.     

Fabrication of silver nanowire transparent electrodes at room temperature

Silver nanowires (AgNWs) surrounded by insulating poly(vinylpyrrolidone) have been synthesized by a polyol process and employed as transparent electrodes. The AgNW transparent electrodes can be fabricated by heat-treatment at about 200 °C which forms connecting junctions between AgNWs. Such a heating process is, however, one of the drawbacks of the fabrication of AgNW electrodes on heat-sensitive substrates. Here it has been demonstrated that the electrical conductivity of AgNW electrodes can be improved by mechanical pressing at 25 MPa for 5 s at room temperature. This simple process results in a low sheet resistance of 8.6 Ω/square and a transparency of 80.0%, equivalent to the properties of the AgNW electrodes heated at 200 °C. This technique makes it possible to fabricate AgNW transparent electrodes on heat-sensitive substrates. The AgNW electrodes on poly(ethylene terephthalate) films exhibited high stability of their electrical conductivities against the repeated bending test. In addition, the surface roughness of the pressed AgNW electrodes is one-third of that of the heat-treated electrode because the AgNW junctions are mechanically compressed. As a result, an organic solar cell fabricated on the pressed AgNW electrodes exhibited a power conversion as much as those fabricated on indium tin oxide electrodes. These findings enable continuous roll-to-roll processing at room temperature, resulting in relatively simple, inexpensive, and scalable processing that is suitable for forthcoming technologies such as organic solar cells, flexible displays, and touch screens.      

Surface-enhanced Raman scattering of dopamine at polymer-coated silver electrodes

Attenuation of dopamine surface-enhanced Raman scattering (SERS) spectra at silver electrodes by protein adsorption is described. Polymer-modified electrodes eliminate protein adsorption effects. Partially hydrolyzed cellulose acetate coatings prevent protein adsorption and allow transport of dopamine to and from the electrode surface. Dopamine SERS spectra from these electrodes are similar to those obtained at uncoated electrodes. Perfluorosulfonate (Nafion) coatings also prevent protein adsorption. However, dopamine transported to the electrode remains trapped inside the coating and cannot be removed.     

Quasi-steady-state voltammetry of rapid electron transfer reactions at the macroscopic substrate of the scanning electrochemical microscope

We report on a novel theory and experiment for scanning electrochemical microscopy (SECM) to enable quasi-steady-state voltammetry of rapid electron transfer (ET) reactions at macroscopic substrates. With this powerful approach, the substrate potential is cycled widely across the formal potential of a redox couple while the reactant or product of a substrate reaction is amperometrically detected at the tip in the feedback or substrate generation/tip collection mode, respectively. The plot of tip current versus substrate potential features the retraceable sigmoidal shape of a quasi-steady-state voltammogram although a transient voltammogram is obtained at the macroscopic substrate. Finite element simulations reveal that a short tip-substrate distance and a reversible substrate reaction (except under the tip) are required for quasi-steady-state voltammetry. Advantageously, a pair of quasi-steady-state voltammograms is obtained by employing both operation modes to reliably determine all transport, thermodynamic, and kinetic parameters as confirmed experimentally for rapid ET reactions of ferrocenemethanol and 7,7,8,8-tetracyanoquinodimethane at a Pt substrate with ～0.5 μm-radius Pt tips positioned at 90 nm-1 μm distances. Standard ET rate constants of ～7 cm/s were obtained for the latter mediator as the largest determined for a substrate reaction by SECM. Various potential applications of quasi-steady-state voltammetry are also proposed.     

Direct electron transfer and biosensing of glucose oxidase immobilized at multiwalled carbon nanotube-alumina-coated silica modified electrode

Investigations are reported regarding the direct electrochemical performance of glucose oxidase (GOD) immobilized on a film of multiwalled carbon nanotube-alumina-coated silica (MWCNT-ACS). The surface morphology of the GOD/MWCNT-ACS nanobiocomposite is characterized by scanning electron microscopy. In cyclic voltammetric response, the immobilized GOD displays a pair of well-defined redox peaks, with a formal potential (E°′) of ? 0.466 V versus Ag/AgCl in a 0.1 M phosphate buffer solution (pH 7.5) at a scan rate of 0.05 V s^? 1; also the electrochemical response indicates a surface-controlled electrode process. The dependence of formal potential on solution pH indicates that the direct electron transfer reaction of GOD is a reversible two-electron coupled with a two-proton electrochemical reaction process. The glucose biosensor based on the GOD/MWCNT-ACS nanobiocomposite shows a sensitivity of 0.127 A M^? 1 cm^? 2 and an apparent Michaelis–Menten constant of 0.5 mM. Furthermore, the prepared biosensor exhibits excellent anti-interference ability to the commonly co-existed uric acid and ascorbic acid.     

Chemical stabilization of gold coated by silver core-shell nanoparticles via electron transfer

Silver nanoparticles are notoriously susceptible to oxidation, yet gold nanoparticles coated in silver exhibit a unique electronic interaction that occurs at the interface of the two metals, leading to enhanced stability properties for the silver shell. In order to probe the phenomenon, the stability of gold nanoparticles coated by silver was studied in the presence of various chloride-containing electrolytes. It was found that a critical silver shell thickness of approximately 1 nm exists that cannot be oxidatively etched from the particle surface: this is in contrast to the observation of complete oxidative etching for monometallic silver nanoparticles. The results are discussed in terms of particle composition, structure and morphology before and after exposing the particles to the electrolytes. Raman analysis of the reporter molecule 3-amino-1,2,4-triazole-5-thiol adsorbed on the particle surface illustrates the feasibility of using gold coated by silver nanoparticle probes in sensing applications that require the presence of high levels of salt. The results provide insight into the manipulation of the electronic and stability properties for gold- and silver-based nanoparticles.     

Direct binding of a redox protein for single-molecule electron transfer measurements

An electron transfer protein is engineered with two thiol groups introduced at different positions in the molecular structure to allow robust binding to two gold electrodes. Atomic force microscopy and scanning tunneling microscopy single-molecule studies show that the engineered proteins: (1) bind to a gold electrode in defined orientation dictated by the thiol-pair utilised, and (2) have a higher conductance than the wild-type proteins indicating a more efficient electron transmission due to the strong gold-thiol contacts.     

Redox reactions at titanium electrodes covered with doped passive films

The rate of electron transfer reactions at titanium electrodes covered with thin platinum-doped passive films was investigated. The doping enhances the current considerably. It is suggested that the platinum atoms in the film serve as intermediate states in an electronic resonance-tunnelling mechanism. The corresponding model calculations show a satisfactory agreement with the experimental data. From these calculations the energy levels of the platinum states in the film are estimated.     

Direct electrochemical oxidation of disulfides at anodically pretreated boron-doped diamond electrodes

Anodically oxidized diamond electrodes have been used to oxidize disulfides, thiols, and methionine in aqueous acidic media and tested for amperometric detection of these compounds after chromatographic separation. Cyclic voltammetric signals for 1 mM glutathione disulfide (GSSG) were observed at 1.39 and 1.84 V vs SCE, the values being less positive than those of its as-deposited counterpart as well as glassy carbon electrode. The voltammetric and chronocoulometric results have indicated the high stability of the electrode with negligible adsorption. A positive shift in the peak potential with increasing pH indicated the attractive electrostatic interaction between the anodically oxidized diamond surface and the positively charged GSSG in acidic media that promoted its analytical performance. The results of the electrolysis experiments of disulfides and thiols showed that the oxidation reaction mechanism of glutathione (GSH) and GSSG involves oxygen transfer. Following separation by liquid chromatography (LC), the determination of both GSH and GSSG in rat whole blood was achieved at a constant potential (1.50 V vs Ag/AgCl), and the limits of detection for GSH and GSSG were found to be 1.4 nM (0.028 pmol) and 1.9 nM (0.037 pmol) with a linear calibration range up to 0.25 mM. These detection limits were much lower than those reported for the amperometry using Bi-PbO2 electrodes and LC-mass spectrometry, and the LC method using diamond electrodes were comparable with enzymatic assay in real sample analysis. The high response stability and reproducibility together with the possibility of regeneration of the electrode surface by on-line anodic treatment at 3 V for 30 min further support the applicability of anodically pretreated diamond for amperometric detection of disulfides.     

Direct DNA hybridization at disposable graphite electrodes modified with carbon nanotubes

The performance of glassy carbon (GCE) and graphite pencil electrodes (PGE) modified with multiwalled carbon nanotubes (CNTs) are compared, based on the direct electrochemical detection of nucleic acids. This is accomplished by monitoring the differential pulse voltammetry changes of the guanine signal. CNT-modified PGE compares favorably to that of the commonly used CNT-modified GCE owing to the intrinsic improved performance of the supporting PGE. The better intrinsic characteristics of the PGE are related to its composite structure and higher level of porosity compared to GCE. The performance characteristics of the direct DNA hybridization on the disposable CNT-modified PGE are studied in terms of optimum analytical conditions such as probe concentration, target concentration, hybridization time, and selectivity. The new DNA biosensor described here has shown some important advantages such being inexpensive, sensitive, selective, and able to generate reproducible results using a simple and direct electrochemical protocol.     

Light-induced ultrafast proton-coupled electron transfer responsible for H2 evolution on silver plasmonics

Light-driven proton-coupled electron transfer (PCET) reactions on nanoplasmonics would bring temporal control of their reactive pathways, in particular, prolong their charge separation state. Using a silver nano-hybrid plasmonic structure, we observed that optical excitation of Ag-localized surface plasmon instigated electron injection into TiO₂ conduction band and oxidation of isopropanol alcoholic functionality. Femtosecond transient infrared absorption studies show that electron transfer from Ag to TiO₂ occurs in ca. 650?fs, while IPA molecules near the Ag surface undergo an ultrafast bidirectional PCET step within 400?fs. Our work demonstrates that ultrafast PCET reaction plays a determinant role in prolonging charge separation state, providing an innovative strategy for visible-light photocatalysis with plasmonic nanostructures.     

Direct electrochemistry and electrocatalytic activity of cytochrome c covalently immobilized on a boron-doped nanocrystalline diamond electrode

Cytochrome c (Cyt c) was covalently immobilized on a boron-doped nanocrystalline diamond (BDND) electrode via surface functionalization with undecylenic acid methyl ester and subsequent removal of the protecting ester groups to produce a carboxyl-terminated surface. Cyt c-modified BDND electrode exhibited a pair of quasi-reversible and well-defined redox peaks with a formal potential (E(0)) of 0.061 V (vs Ag/AgCl) in 0.1 M phosphate buffer solution (pH 7.0) and a surface-controlled process with a high electron transfer constant (ks) of 5.2 +/- 0.6 s(-1). The electrochemical properties of as-deposited and Cyt c-modified boron-doped microcrystalline diamond (BDMD) electrodes were also studied for comparison. Investigation of the electrocatalytic activity of the Cyt c-modified BDND electrode toward hydrogen peroxide (H2O2) revealed a rapid amperometric response (5 s). The linear range of response to H2O2 concentration was from 1 to 450 microM, and the detection limit was 0.7 microM at a signal-to-noise ratio of 3. The stability of the Cyt c-modified BDND electrode, in comparison with that of the BDMD and glassy carbon counterpart electrodes, was also evaluated.