Soft-landed protein voltammetry: a tool for redox protein characterization

Microperoxidase-11 (MP-11) was first soft landed onto the gold surface of a screen-printed electrode. Intact protein deposition was verified by time-of-flight secondary ion mass spectrometry. The coupling of soft landing with electrochemical techniques allowed unique information to be obtained about the deposition features. A full characterization of the direct electron-transfer properties was performed by modeling data obtained from cyclic voltammetry experiments; calculated values of kinetic electron-transfer constant, formal redox potential, and reorganization energy allow us to hypothesize the mechanism involved in soft landing immobilization and demonstrate the different conformation of the enzyme deposited from two different charged species. The strong interaction between MP-11 and the gold surface and long-term stability of the functionalized electrode characterizes the peculiar features of this procedure, which enhance its power with respect to the existing immobilization procedure and ensure its suitability for those practical applications that could benefit from an unmediated bridgeless bioeletrochemical electron transfer (e.g., biosensor transducers or electrode elements in biofuel cells).     

Direct electrochemistry of laccase immobilized on au nanoparticles encapsulated-dendrimer bonded conducting polymer: application for a catechin sensor

The direct electrochemistry of laccase was promoted by Au nanoparticle (AuNP)-encapsulated dendrimers (Den), which was applied for the detection of catechin. To increase the electrical properties, AuNPs were captured in the interiors of the dendrimer (Den-AuNPs) as opposed to attachment at the periphery of dendrimer. To prepare Den-AuNPs, the Au(III) ions were first coordinated in the interior of dendrimer with nitrogen ligands and then reduced to form AuNPs. The size of AuNPs encapsulated within the interior of the dendrimer was determined to be 1.7 +/- 0.4 nm. AuNPs-encapsulated dendrimers were then used to covalently immobilize laccase (PDATT/ Den(AuNPs)/laccase) through the formation of amide bonds between carboxylic acid groups of the dendrimer and the amine groups of laccase. Each layer of the PDATT/Den(AuNPs)/laccase probe was characterized using CV, EIS, QCM, XPS, SEM, and TEM. The PDATT/Den(AuNPs)/laccase probe displayed a well-defined direct electron-transfer (DET) process of laccase. The quasi-reversible redox peak of the Cu redox center of the laccase molecule was observed at -0.03/+0.13 V vs Ag/AgCl, and the electron-transfer rate constant was determined to be 1.28 s (-1). A catechin biosensor based on the electrocatalytic process by direct electrochemistry of laccase was developed. The linear range and the detection limit in the catechin analysis were determined to be 0.1-10 and 0.05 +/- 0.003 microM, respectively. Interference effects from various phenolic and polyphenolic compounds were also studied, and the general applicability of the biosensor was evaluated by selective analysis of real samples of catechin.     

Polypyrrole-entrapped quinohemoprotein alcohol dehydrogenase. Evidence for direct electron transfer via conducting-polymer chains.

It is reported for the first time that direct electron-transfer processes between a polypyrrole (PPY) entrapped quinohemoprotein alcohol dehydrogenase from Gluconobacter sp. 33 (QH-ADH) and a platinum electrode take place via the conducting-polymer network. The cooperative action of the enzyme-integrated prosthetic groups--pyrroloquinoline-quinone and hemes--is assumed to allow this electron-transfer pathway from the enzyme's active site to the conducting-polymer backbone. A hypothetical model of the electron transfer is proposed which is supported by the influence of various parameters, such as, e.g., ionic strength and nature of the buffer salts. This unusual electron-transfer pathway leads to an accentuated increase of the K M app value (102 mM) and hence to a significantly increased linear detection range of an ethanol sensor based on this enzyme.     

Electron-transfer reactivity and enzymatic activity of hemoglobin in a SP Sephadex membrane

Hemoglobin can exhibit a direct electron-transfer reaction after being entrapped in a SP Sephadex membrane. A pair of stable and well-defined redox waves are obtained at a hemoglobin-SP sephadex modified pyrolytic graphite electrode. The anodic and cathodic peak potentials are located at -0.244 and -0.336 V (vs SCE), respectively. On the other hand, the peroxidase activity of the protein in the membrane is also greatly enhanced. The apparent Michaelis-Menten constant is calculated to be 1.9 mM, which shows a large catalytic activity of hemoglobin in the SP Sephadex membrane toward hydrogen peroxide (H2O2). According to the direct electron-transfer property and enhanced peroxidase activity of Hb in the membrane, a Hb/SP Sephadex membrane-based H2O2 biosensor is prepared, with a linear range approximately 5.0 x 10(-6) to 1.6 x 10(-4) mol/L.     

Electrochemistry and electrocatalytic properties of hemoglobin in layer-by-layer films of SiO2 with vapor-surface sol-gel deposition

In this paper, layer-by-layer {Hb/SiO2}n films assembled by alternate adsorption of positively charged hemoglobin (Hb) and vapor-surface sol-gel deposition of silica at 50 degrees C onto a glassy carbon electrode were reported. The result films were characterized with cyclic voltametery, electrochemical impedance spectroscopy, UV-vis spectroscopy, and SEM, and the direct electrochemical and electrocatalytic properties of Hb in these layer-by-layer films were investigated. A pair of well-defined quasi-reversible cyclic voltammetric peaks were observed, and the formal potential of the heme FeIII/FeII redox couple was found to be -0.330 V(vs SCE). The electron-transfer behavior of Hb in {Hb/SiO2}n films was dependent on the vapor temperature, the number of layers, and the pH of the Hb solution, based on which a set of optimized conditions for film fabrication was inferred. The hemoglobin in{Hb/SiO2}n films displayed good electrocatalytic activity to the reduction of hydrogen peroxide, and H2O2 had linear current response from 1.0 x 10(-6) to 2.0 x 10(-4) M with a detection limit of 5.0 x 10(-7) M (S/N = 3). The apparent heterogeneous electron-transfer rate constant (ks) was 1.02 +/- 0.03 s(-1), and the apparent Michaeli-Menten constant (Kmapp) was 0.155 mM, indicating a potential application in the third-generation biosensor.     

Equilibrium and kinetic behavior of Fe(CN)6(3-/4-) and cytochrome c in direct electrochemistry using a film electrode thin-layer transmission cell

We report on the design and performance of a thin-layer electrochemical cell optimized for use with optically transparent film electrodes in combination with UV/vis and IR transmission spectroscopic measurements. The cell allows for measurements under both aerobic and anaerobic conditions. The direct, unmediated electron transfer, as assessed by the current transient, and the corresponding optical response observed for the Fe(CN)(6)(3-/4-) couple were in good agreement with theoretical predictions for voltammetry and optical absorption by an analyte confined in a thin layer. Chronoamperometric and spectroscopic measurements of Fe(CN)(6)(3-/4-) on gold mesh electrode revealed fast kinetics strongly influenced by the electrolyte concentration. Maximal apparent rates exceeding 2 s(-1) in 1 M KCl were observed optically. The direct kinetic and thermodynamic behavior of cytochrome c was compared with several electrode materials using the cell. The results showed heme ligand-dependent changes in the protein-electrode interactions. Mid-UV/visible spectral changes upon redox transitions in native cytochrome c and its cyanide derivative, as well as dissociation of the ferrous cytochrome c-CN complex, are reported.     

Preservation of NADH voltammetry for enzyme-modified electrodes based on dehydrogenase.

Minimizing overpotential and generating high faradaic currents are critical issues for fast-scan voltammetry of beta-nicotinamide adenine dinucleotide (NADH) for the sensitivity of enzyme-modified electrodes based on dehydrogenases. Although NADH voltammetry exhibits high overpotential and poor voltammetric peak shape at solid electrode surfaces, modification of the electrode surface can improve the electrochemical response at carbon fibers. However, these improvements are severely degraded upon the covalent attachment of enzyme. The creation of improved electron-transfer properties and the retention of these properties throughout the enzyme attachment process is the focus of this study. A novel polishing and electrochemical pretreatment method was developed which generated a decreased overpotential and a high faradaic current at carbon-fiber electrodes for NADH. Factors that lead to a degradation of voltammetric response during the enzyme fabrication were investigated, and both the aging and the covalent modification of the pretreated surface contributed to this degradation. Attachment processes that minimized the preparation time, in turn, maximized the retention of the facile electron-transfer properties. These attachment processes included varying the surface attachment reactions for the enzyme. Preparation time reduction techniques included modeling existing techniques and then improving kinetic and mass transport issues where possible. Alternate covalent attachment methods included a direct electrochemical amine reaction and an electrochemically reductive hydrazide reaction. The surface attachment and retention of electron-transfer properties of these probes were confirmed by fluorescence and electrochemical studies.     

Single-molecule mapping of long-range electron transport for a cytochrome b(562) variant

Cytochrome b(562) was engineered to introduce a cysteine residue at a surface-exposed position to facilitate direct self-assembly on a Au(111) surface. The confined protein exhibited reversible and fast electron exchange with a gold substrate over a distance of 20 ? between the heme redox center and the gold surface, a clear indication that a long-range electron-transfer pathway is established. Electrochemical scanning tunneling microscopy was used to map electron transport features of the protein at the single-molecule level. Tunneling resonance was directly imaged and apparent molecular conductance was measured, which both show strong redox-gated effects. This study has addressed the first case of heme proteins and offered new perspectives in single-molecule bioelectronics.     

Peptide nanotube-modified electrodes for enzyme-biosensor applications

The fabrication and notably improved performance of composite electrodes based on modified self-assembled diphenylalanine peptide nanotubes is described. Peptide nanotubes were attached to gold electrodes, and we studied the resulting electrochemical behavior using cyclic voltammetry and chronoamperometry. The peptide nanotube-based electrodes demonstrated a direct and unmediated response to hydrogen peroxide and NADH at a potential of +0.4 V (vs SCE). This biosensor enables a sensitive determination of glucose by monitoring the hydrogen peroxide produced by an enzymatic reaction between the glucose oxidase attached to the peptide nanotubes and glucose. In addition, the marked electrocatalytic activity toward NADH enabled a sensitive detection of ethanol using ethanol dehydrogenase and NAD+. The peptide nanotube-based amperometric biosensor provides a potential new tool for sensitive biosensors and biomolecular diagnostics.     

Assembly of Ni(OH)2 nanoparticle films on aqueous surfaces induced by small amounts of toluene, and the study of their unmediated electrocatalytic oxidation toward some small biomolecules

A simple strategy for the preparation of a Ni(OH)2 nanoparticle film is described. Ni(OH)2 nanoparticles were synthesized in an aqueous solution of Ni2+ and tert-butylamine in the presence of small amounts of toluene, which induced the nanoparticles to assemble a thin film on the aqueous surface. The obtained Ni(OH)2 nanoparticle film was easily transferred onto the electrode surfaces and exhibited stable electrochemical performance. The electrochemical behavior of various small biomolecules, including cysteine, homocysteine, glutathione, histidine, glycine, cystine, methionine, lysine, aspartic acid, glutamic acid, phenylalanine, ascorbic acid, uric acid and dopamine, were studied at the Ni(OH)2 nanoparticle-film-modified electrode. The Ni(OH)2 nanoparticle film exhibits excellent direct, unmediated electrocatalysis toward the oxidation of cysteine, homocysteine and ascorbic acid in a pH 7.4 buffer solution with a low onset potential and a high oxidation signal. This behavior differs from many reports in which small organic molecules are electrocatalyzed indirectly by the Ni(OH)2/NiOOH redox couple in a strongly alkaline solution.     

All-solid-state Z-scheme in CdS-Au-TiO2 three-component nanojunction system

Natural photosynthesis, which achieves efficient solar energy conversion through the combined actions of many types of molecules ingeniously arranged in a nanospace, highlights the importance of a technique for site-selective coupling of different materials to realize artificial high-efficiency devices. In view of increasingly serious energy and environmental problems, semiconductor-based artificial photosynthetic systems consisting of isolated photochemical system 1 (PS1), PS2 and the electron-transfer system have recently been developed. However, the direct coupling of the components is crucial for retarding back reactions to increase the reaction efficiency. Here, we report a simple technique for forming an anisotropic CdS-Au-TiO2 nanojunction, in which PS1(CdS), PS2(TiO2) and the electron-transfer system (Au) are spatially fixed. This three-component system exhibits a high photocatalytic activity, far exceeding those of the single- and two-component systems, as a result of vectorial electron transfer driven by the two-step excitation of TiO2 and CdS.     

Application of power spectra patterns in Fourier transform square wave voltammetry to evaluate electrode kinetics of surface-confined proteins

This paper describes an application of Fourier transform (FT) voltammetry that provides a quantitative evaluation of the electron-transfer kinetics of protein molecules attached to electrode surfaces. The potential waveform applied in these experiments consists of a large-amplitude square wave of frequency f superimposed onto the traditional triangular voltage used in dc cyclic voltammetry. The resultant current-time response, when Fourier transformed into the frequency domain, provides patterns of data at the even harmonic frequencies that arise from nonlinearity in the Faradaic response. These even harmonic contributions are ideally suited for kinetic evaluation of electron-transfer processes because they are highly selective to quasi-reversible behavior (insensitive to reversible or irreversible processes) and almost devoid of background charging current. Inverse FT methods can then be used to provide the wave shapes of the dc as well as the ac voltammetric components and other characteristics employed to detect the level of nonideality present relative to theoretical models based upon noninteracting surface-confined molecules. The new form of data evaluation has been applied to the electron-transfer properties of a typical biological electron carrier, the blue copper protein azurin, immobilized on polycrystalline gold electrodes modified with self-assembled monolayers of different length alkanethiols. Details of the electrode kinetics (rates of electron transfer, dispersion, and charge-transfer coefficients) as a function of alkanethiol, apparent surface coverage, and capacitance are all deduced from the square wave (FT-inverse FT) protocol, and the implications of these findings are considered.     

Detailed analysis of the electron-transfer properties of azurin adsorbed on graphite electrodes using DC and large-amplitude Fourier transformed AC voltammetry

The analysis of dc cyclic voltammograms of surface-confined metalloproteins is complicated by large background currents, significant ohmic iRu drop, and frequency dispersion related to protein and electrode surface inhomogeneity. The use of large-amplitude Fourier transform ac voltammetry for the quantification of the electron-transfer properties of a thin film of redox-active protein azurin adsorbed onto edge-plane, basal-plane, and highly oriented pyrolytic graphite electrode surfaces has been evaluated and compared to results obtained by dc cyclic voltammetry. In principle, it has been established that fourth and higher harmonic sine-wave data are ideally suited for analysis of electron-transfer processes as they are almost completely devoid of background capacitance current contributions. However, uncompensated resistance has a higher impact on these components, as is the case with fast scan rate dc techniques, so strategies to include this term in the simulations have been investigated. Application of recommended strategies for the evaluation of the electron-transfer properties of azurin adsorbed onto three forms of graphite, each having different background or uncompensated resistance values, is described and compared to results obtained by traditionally used forms of cyclic voltammetry. The electron-transfer rate constant, k0', of azurin at a highly oriented pyrolytic graphite electrode surface was approximately 250 s(-1), compared with > or =1000 s(-1) at edge-plane and basal-plane graphite electrodes. The significantly lower k0' value found at the highly oriented pyrolytic graphite electrode was related to the relatively low level of edge-plane defect sites present at the surface of this electrode. However, analysis of high ac harmonics suggests that frequency dispersion is substantial at all electrode surfaces. Such effects in these diffusionless situations are significantly enhanced relative to solution-phase voltammetry, where overlay of diffusion layers minimizes the impact of heterogeneity.     

Interdigitated Array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia coli O157:H7

A label-free electrochemical impedance immunosensor for rapid detection of Escherichia coli O157:H7 was developed by immobilizing anti-E. coli antibodies onto an indium-tin oxide interdigitated array (IDA) microelectrode. Based on the general electronic equivalent model of an electrochemical cell and the behavior of the IDA microelectrode, an equivalent circuit, consisting of an ohmic resistor of the electrolyte between two electrodes and a double layer capacitor, an electron-transfer resistor, and a Warburg impedance around each electrode, was introduced for interpretation of the impedance components of the IDA microelectrode system. The results showed that the immobilization of antibodies and the binding of E. coli cells to the IDA microelectrode surface increased the electron-transfer resistance, which was directly measured with electrochemical impedance spectroscopy in the presence of [Fe(CN)(6)](3-/4-) as a redox probe. The electron-transfer resistance was correlated with the concentration of E. coli cells in a range from 4.36 x 10(5) to 4.36 x 10(8) cfu/mL with the detection limit of 10(6) cfu/mL.     

Superoxide dismutase-based third-generation biosensor for superoxide anion

A third-generation biosensor for superoxide anion (O2-) was developed by immobilizing superoxide dismutase (SOD) on a self-assembled monolayer of cysteine on gold electrode; i.e., a SOD/cysteine-modified gold electrode (SOD/Cys/Au) was fabricated. A rapid and direct electron transfer of SOD was realized at the gold electrode by using the cysteine molecule as an electron-transfer promoter. The promoted direct electron transfer of SOD and biomolecular recognition by the exploitation of specific and significant enzyme-substrate reactivity of SOD toward O2- combined with the low operating potential enabled a sensitive measurement of O2-. At SOD/Cys/Au, O2- could be specifically oxidized and reduced to O2 and hydrogen peroxide, respectively, through the inherent catalytic reaction of SOD. This allowed us to measure O2- by polarizing the electrode both anodically and cathodically. We could successfully measure O2- by suitably polarizing the electrode, typically at 300 and -200 mV versus Ag/AgCl without the virtual interference from physiological levels of H2O2, ascorbic acid, uric acid, and metabolites of neurotransmitters. The response mechanism of SOD/Cys/Au to O2- and its sensor characteristics are also presented and discussed.     

Electroanalytical performance of carbon films with near-atomic flatness

Physicochemical and electrochemical characterization of carbon films obtained by pyrolyzing a commercially available photoresist has been performed. Photoresist spin-coated on to a silicon wafer was pyrolyzed at 1,000 degrees C in a reducing atmosphere (95% nitrogen and 5% hydrogen) to produce conducting carbon films. The pyrolyzed photoresist films (PPF) show unusual surface properties compared to other carbon electrodes. The surfaces are nearly atomically smooth with a root-mean-square roughness of <0.5 nm. PPF have a very low background current and oxygen/carbon atomic ratio compared to conventional glassy carbon and show relatively weak adsorption of methylene blue and anthraquinone-2,6-disulfonate. The low oxygen/carbon ratio and the relative stability of PPF indicate that surfaces may be partially hydrogen terminated. The pyrolyzed films were compared to glassy carbon (GC) heat treated under the same conditions as pyrolysis to evaluate the electroanalytical utility of PPF. Heterogeneous electron-transfer kinetics of various redox systems were evaluated. For Ru(NH3)6(3+/2+), Fe(CN)6(3-/4-), and chlorpromazine, fresh PPF surfaces show electron-transfer rates similar to those on GC, but for redox systems such as Fe3+/2+, ascorbic acid, dopamine, and oxygen, the kinetics on PPF are slower. Very weak interactions between the PPF surface and these redox systems lead to their slow electron-transfer kinetics. Electrochemical anodization results in a simultaneous increase in background current, adsorption, and electron-transfer kinetics. The PPF surfaces can be chemically modified via diazonium ion reduction to yield a covalently attached monolayer. Such a modification could help in the preparation of low-cost, high-volume analyte-specific electrodes for diverse electroanalytical applications. Overall, pyrolysis of the photoresist yields an electrode surface with properties similar to a very smooth version of glassy carbon, with some important differences in surface chemistry.     

Electrochemical characterization of binderless, recompressed exfoliated graphite electrodes: electron-transfer kinetics and diffusion characteristics

Exfoliated graphite (EG) is prepared by the thermal exfoliation of graphite intercalation compounds at different temperatures. Surface and bulk physicochemical properties of EG are followed by spectroscopic and analytical methods and are observed to be a function of exfoliation temperature. EG particles can be recompressed without any binder and used as surface-renewable electrodes. Surface preparation is accomplished by either polishing or roughening the electrode surface using emery sheets. Effects of exfoliation temperature and the surface preparation on the electron-transfer kinetics and on the diffusion characteristics have been followed by electrochemical methods using several benchmark redox systems. It is found that the electron-transfer kinetics and the diffusion of K(4)[Fe(CN)(6)] are affected by the nature of the EG surface while that of iron(II)(1,10-phenanthroline)(3) and cobalt(II)(1,10-phenanthroline)(3) are not affected by the surface preparation. The redox systems are classified into different groups according to their kinetic sensitivity. Diffusion of electroactive species toward the EG electrodes is found to nonlinear. Current-time plots suggest that the recompressed EG electrodes can be modeled as fractals.     

Influence of microstructure on the electrochemical performance of tin-doped indium oxide film electrodes

The effect of the microstructure of tin-doped indium oxide (ITO) films on their electrochemical performance was studied using three redox probes, tris(2,2'-bipyridyl ruthenium(II) chloride (Ru(bpy)3(2+/3+)), ferrocyanide (Fe(CN)6(4-/3-)), and ferrocenemethanol (FcCH2H(0/+)). ITO films were deposited using dc magnetron sputtering under a variety of conditions that resulted in films having different degrees of crystallinity, crystallographic texture, sheet resistance, surface roughness, and percent tin. It was found that the electron transfer for all three redox probes used in this study was more efficient at polycrystalline films than at amorphous ITO films. This effect is more pronounced at faster scan rates. The crystallographic texture of the ITO films, surface roughness, and a change in sheet resistance from 7.9 to 13.7 ohms/square did not have an effect on electron-transfer kinetics. ITO films deposited using a 1 wt % SnO2 target and having sheet resistance comparable to films deposited using a 10 wt % SnO2 target had dramatically different microstructure from the films with higher weight percent Sn and were shown to perform poorly when used as electrode materials. We believe that the dramatic differences in electron-transfer kinetics observed at the various ITO films can be attributed to either the different density of defect sites along the grain boundaries or defect sites caused by substitutional Sn in the film.     

Bridge-mediated hopping or superexchange electron-transfer processes in bis(triarylamine) systems

Hopping and superexchange are generally considered to be alternative electron-transfer mechanisms in molecular systems. In this work we used mixed-valence radical cations as model systems for the investigation of electron-transfer pathways. We show that substituents attached to a conjugated bridge connecting two triarylamine redox centres have a marked influence on the near-infrared absorption spectra of the corresponding cations. Spectral analysis, followed by evaluation of the electron-transfer parameters using the Generalized Mulliken-Hush theory and simulation of the potential energy surfaces, indicate that hopping and superexchange are not alternatives, but are both present in the radical cation with a dimethoxybenzene bridge. We found that the type of electron-transfer mechanism depends on the bridge-reorganization energy as well as on the bridge-state energy. Because superexchange and hopping follow different distance laws, our findings have implications for the design of new molecular and polymeric electron-transfer materials.     

Studies of electron-transfer and charge-transfer coupling processes at a liquid/liquid interface by double-barrel micropipet technique

Investigation of a heterogeneous electron-transfer (ET) reaction at the water/1,2-dichloroethane interface employing a double-barrel micropipet technique is reported. The chosen system was the reaction between Fe(CN)63- in the aqueous phase (W) and ferrocene in 1,2-dichloroethane (DCE). According to the generation and the collection currents as well as collection efficiency, the ET-ion-transfer (IT) coupling process at such an interface and competing reactions with the organic supporting electrolyte in the organic phase can be studied. In addition, this technique has been found to be an efficient method to distinguish and measure the charge-transfer coupling reaction between two ions (IT-IT) processes occurring simultaneously at a liquid/liquid interface. On this basis, the formal Gibbs energies of transfer of some ions across the W/DCE interface, such as NO3-, NO2-, Cl-, COO-, TBA+, TPAs+, Cs+, Rb+, K+, Na+, and Li+, for which their direct transfers are usually difficult to obtain because of the IT-IT coupling processes, were quantitatively evaluated.