Enhanced electrochemical activity of redox-labels in multi-layered protein films on indium tin oxide nanoparticle-based electrode

Facile electrical communication between redox-active labeling molecules and electrode is essential in the electrochemical detection of bio-affinity reactions. In this report, nanometer-sized indium tin oxide (ITO) particles were employed in the fabrication of porous thick film electrodes to enhance the otherwise impeded electrochemical activity of redox labels in multi-layered protein films, and to enable quantitative detection of avidin/biotin binding interaction. To carry out the affinity reaction, avidin immobilized on an ITO electrode was reacted with mouse IgG labeled with both biotin and ruthenium Tris-(2,2′-bipyridine) (Ru-bipy). The binding reaction between avidin and biotin was detected by the catalytic voltammetry of Ru-bipy in an oxalate-containing electrolyte. On sputtered ITO thin film electrode, although a single layer of Ru-bipy labeled avidin exhibited substantial anodic current, attaching the label to the outer IgG layer of the avidin/biotin-IgG binding pair resulted in almost complete loss of the signal. However, electrochemical current was recovered on ITO film electrodes prepared from nanometer-sized particles. The surface of the nanoparticle structured electrode was found by scanning electron microscopy to be very porous, and had twice as much surface binding capacity for avidin as the sputtered electrode. The results were rationalized by the assumption of different packing density of avidin inner layer on the two surfaces, and consequently different electron transfer distance between the electrode and Ru-bipy on the IgG outer layer. A linear relationship between electrochemical current and IgG concentration was obtained in the range of 40-4000 nmol L⁻¹ on the nanoparticle-based electrode. The approach can be employed in the electrochemical detection of immunoassays using non-enzymatic redox labels.     

Redox activity and diffusion of hydrophilic, hydrophobic, and amphiphilic redox active molecules in a bicontinuous cubic phase

The objective was to examine how a bicontinuous cubic phase influences the diffusion and electrochemical activity of dissolved molecules. The cubic phase is a structure with three-dimensional continuous channels of water separated by an apolar membrane. A redox active molecule can dissolve in three different environments. A hydrophobic molecule will prefer the interior of the membrane, a hydrophilic molecule will prefer the water channels, and an amphiphilic molecule will be situated with its headgroup at the surface of the membrane and its tail in the interior. The electrochemical activity was measured with cyclic voltammetry and the transport behavior with chronocoulometry. All the molecules were redox active in the cubic phase; that is, all the molecules could reach the surface of the electrode and react. The cubic phase made the kinetics of the charge transfer slower, showing a quasi-reversible behavior. The reason may be that a layer of the membrane adheres to the hydrophobic electrode surface. The diffusion experiment showed that the diffusion was slower than in solution. The molecules that were restricted to diffuse within the membrane gave particularly low mass transport rates.     

Fabrication and electrochemical characterization of interdigitated nanoelectrode arrays

Interdigitated nanoelectrode arrays with controlled electrode bandwidth and gap geometries ranging from 30 nm to 1 μm were fabricated on glass substrates by a planar process involving high resolution electron beam lithography and lift-off, and their characteristic electrochemical responses to an aqueous ferrocene derivative solution were examined using fundamental electrochemical techniques. Despite the comparatively large electrode area of electrode arrays containing 10 bands to a single band electrode, quasi-steady-state currents with high current density were obtained at a slow potential sweep rate in cyclic voltammograms of ferrocene derivative since the lateral dimension of the nanoelectrode arrays was considerably less than the scale of the diffusion layer of redox species. Additionally, it was demonstrated that the electrode thickness influenced limiting currents of voltammograms in the case of nanoelectrode arrays. In generation-collection mode experiments, furthermore, a collection efficiency as high as ∼99% was attained by 100 nm wide electrode arrays with a gap dimension of 30 nm.     

Surface functionalization with redox active molecule-based imidazolium via click chemistry

In this study, the redox active molecule N-ferrocenylmethyl-N-propargylimidazolium bromide was immobilized onto the surface of an electrode. The surface modification was performed by coupling the electrochemical reduction of the 4-azidophenyldiazonium generated in situ with a copper(I) catalyzed click chemistry reaction. Surface and electrochemical investigations suggest the attachment of a monolayer of redox active molecules containing an ionic liquid framework onto the electrode surface. Furthermore, scanning electrochemical microscopy studies revealed the conductive behavior of the attached ferrocenyl moieties on the ITO surface.     

Electrochemistry of individual molecules in zeptoliter volumes

Electrochemical experiments were carried out in a nanometer-sized cylindrical thin layer cell (TLC) formed by etching the surface of a disk-type platinum nanoelectrode (5- to 150-nm radius). Using high frequency ac voltage, the surface of such an electrode was etched to remove a very thin (> or = 1-nm-thick) layer of Pt. The resulting zeptoliter-scale cavity inside the glass sheath was filled with aqueous solution containing redox species, and the etched electrode was immersed in a dry (no external solution) pool of mercury to produce a TLC. Several approaches based on steady-state voltammetry and scanning electrochemical microscopy (SECM) were developed to independently evaluate the electrode radius and the etched volume. The number of redox molecules in the TLC could be varied between one and a few hundred by changing its volume and solution concentration. In this way, the transition between a random and deterministic number of trapped molecules was observed. High quality steady-state voltammograms of > or = 1 molecules were obtained for different neutral and charged redox species and different concentrations of supporting electrolyte. The analysis of such voltammograms yields information about mass transfer, adsorption, electron transfer kinetics, and double-layer effects on the nanoscale.     

Motion of redox molecules in solution monitored by the highly-sensitive EQCM technique

The behavior of redox molecules in solution that was not detected by electrochemical techniques was measured by a highly-sensitive electrochemical quartz crystal microbalance (EQCM) technique that has been improved in this study to obtain a high sensitivity of EQCM measurement in solution. The improved EQCM technique allowed to monitor the motion of a redox molecule, that is an access of the molecule to an electrode surface and repulsion from the surface during redox. An EQCM technique currently in use has measured adsorption of redox molecules on an electrode surface or polymerization on the surface caused by a chemical reaction following redox, which exhibits an enough large mass change response to detect with an EQCM measurement. However, access and repulsion of redox molecule, which is a slight motion of the molecule near on electrode surface, has not been detected and investigated by an EQCM technique, because the mass change response seems to be very small. In this study, the redox behavior of methyl viologen on a bare gold surface, pyridinethiol surface and methylpyridinethiol surface was investigated. Although the three electrodes give the same cyclic voltammogram of methyl viologen, the three are different in QCM response recorded at the same time as the voltammetry. Access/repulsion of methyl viologen within an electrical double layer was monitored by the highly-sensitive EQCM technique.     

Motion of redox molecules in solution monitored by the highly-sensitive EQCM technique

The behavior of redox molecules in solution that was not detected by electrochemical techniques was measured by a highly-sensitive electrochemical quartz crystal microbalance (EQCM) technique that has been improved in this study to obtain a high sensitivity of EQCM measurement in solution. The improved EQCM technique allowed to monitor the motion of a redox molecule, that is an access of the molecule to an electrode surface and repulsion from the surface during redox. An EQCM technique currently in use has measured adsorption of redox molecules on an electrode surface or polymerization on the surface caused by a chemical reaction following redox, which exhibits an enough large mass change response to detect with an EQCM measurement. However, access and repulsion of redox molecule, which is a slight motion of the molecule near on electrode surface, has not been detected and investigated by an EQCM technique, because the mass change response seems to be very small. In this study, the redox behavior of methyl viologen on a bare gold surface, pyridinethiol surface and methylpyridinethiol surface was investigated. Although the three electrodes give the same cyclic voltammogram of methyl viologen, the three are different in QCM response recorded at the same time as the voltammetry. Access/repulsion of methyl viologen within an electrical double layer was monitored by the highly-sensitive EQCM technique.     

Polymer-based bilayer interfaces for electrochemical rectification

In this paper, the electrochemical current rectification phenomenon exhibited at an electrochemical interface constituted by a glassy carbon electrode covered with a bilayer of polymer films is discussed. The authors have shown that Methylene Blue (MB) redox species can be confined to a very thin insulating polymer film formed from orthophenylene diamine. The poly(opd) film exhibited excellent blocking properties to redox molecules in solution. On the other hand, the insulating poly(opd) film trapped with MB could mediate electron transfer between the redox molecules in solution and the electrode. Further, a second polymeric layer (Nafion film) trapped with ferrocene redox species was formed as the outer layer over the inner poly (opd) film containing MB. This bilayer-modified electrode, due to the significant difference in the redox potentials of the MB and ferrocene species immobilized in the inner and outer layers, respectively, exhibits unidirectional current flow and the results of the voltammetric investigations on the modified electrodes are described in this communication.     

纳米溶胶-凝胶膜修饰电极及电化学催化性能

报道了以纳米硅溶胶-凝胶（sol-gel）膜为载体的化学修饰电极。用sol-gel法在金电极上固定亚甲蓝及硫堇，发现固定于纳米硅溶胶-凝胶膜内的亚甲蓝和硫堇均有良好的电化学活性，并对同时固定于膜内的NADH、血红蛋白等生物分子产生显著的催化氧化还原作用。     

Monolayers of photosystem II on gold electrodes with enhanced sensor response—effect of porosity and protein layer arrangement

Mass transport of the bulk of the analyte to the electrode and through the bioactive layer can be significantly improved by use of the nanoelectrode array and defined arrangement of protein film. This phenomenon has been studied by (i) atomic-force microscopy, (ii) electrochemical measurements of PSII activity, and (iii) digital simulations for an oriented monolayer of histidine-tagged photosystem II (PSII) immobilized on nitrilotriacetic acid (NTA)-modified gold electrodes. The output signal of the electrochemical biosensor is controlled by (i) mass transport from the bioactive layer to electrode and (ii) mass transport between the bulk of the analyte and the electrode. Mass transport through the bioactive layer was electrochemically studied for PSII self-assembled on gold screen-printed electrodes. A densely packed monolayer of PSII has a significant shielding effect toward the diffusion of redox mediator duroquinone (DQ). Mass transport to the planar electrode surface was improved by co-immobilization of bovine-serum albumin (BSA) as spacer biomolecule in the monolayer of PSII. Correlation between the electrochemical properties and surface arrangement of the resulting protein films was clearly observable and confirmed the improved mass-transport properties of structured enzyme monolayers. On the basis of this observation, the application of a bottom-up approach for improvement of electrode performance was proposed and digitally simulated for an infinite array of electrodes ranging in diameter from 50 nm to 5 m. The nanoelectrode array, with the optimum time window selected for measurements, enables enhancement of mass transport between the bulk of the analyte and the macroelectrode by a factor of up to 50 in comparison with classical planar electrodes. Use of a time window enables minimization of crosstalk between individual electrodes in the array. The measurements require methods which suppress the double-layer capacity.     

Electrochemical catalysis with redox polymer and polyion-protein films

Supramolecular redox-active assemblies on electrodes are of fundamental interest and can be used to create functioning devices such as sensors, biosensors, and bioreactors. The ability of redox-active films to mediate electron transfer reactions in 3-D dramatically increases the sensitivity with which target molecules can be determined. Metallopolyion hydrogel films immobilized on electrode surfaces exhibit many properties that are reminiscent of those shown by redox-active proteins. This review discusses the electrochemical properties and applications of such films, including mediating electron transfer between electrodes and oxidase enzymes. In addition, polyion-protein films grown layer by layer have certain advantages in device fabrication, including facilitating direct electron transfer for many proteins, mechanical stability, use of tiny amounts of protein, and control of film architecture. This review presents examples of iron heme proteins in films grown layer by layer by alternate electrostatic adsorption for catalytic reduction of hydrogen peroxide and trichloroacetic acid and for oxidation of styrene.     

Strategies for "wiring" redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology

In this tutorial review the basic approaches to establish electrochemical communication between redox-active proteins and electrodes are elucidated and examples for applications in electrochemical biosensors, biofuel cells and nanotechnology are presented. The early stage of protein electrochemistry is described giving a short overview over electron transfer (ET) between electrodes and proteins, followed by a brief introduction into experimental procedures for studying proteins at electrodes and possible applications arising thereof. The article starts with discussing the electrochemistry of cytochrome c, the first redox-active protein, for which direct reversible ET was obtained, under diffusion controlled conditions and after adsorption to electrodes. Next, examples for the electrochemical study of redox enzymes adsorbed on electrodes and modes of immobilization are discussed. Shortly the experimental approach for investigating redox-active proteins adsorbed on electrodes is outlined. Possible applications of redox enzymes in electrochemical biosensors and biofuel cells working by direct ET (DET) and mediated ET (MET) are presented. Furthermore, the reconstitution of redox active proteins at electrodes using molecular wire-like units in order to "wire" the proteins to the electrode surface and possible applications in nanotechnology are discussed.     

Effects of nonionic surfactants on electrochemical behavior of ubiquinone and menaquinone incorporated in a carbon paste electrode

A carbon paste electrode, in which the carbon particles were coated with a thin layer of a nonionic surfactant (NIS), was constructed with a pasting liquid containing ubiquinone (UQ) or menaquinone (MQ). It has revealed that the layer acts not only as a diffusion barrier but also as a matrix for the redox reaction of quinones at electrode surface, and its effects on the electrochemical behavior of quinones depend on both the physico-chemical structure of a surfactant and the kind of quinones. Further, such a modification was applied to the preparation of an enzyme electrode in which the quinone molecule act as a redox mediator and the influences on the sensitivity of the glucose biosensor was demonstrated.     

Transport of redox probes through single pores measured by scanning electrochemical-scanning ion conductance microscopy (SECM-SICM)

We report scanning electrochemical microscopy-scanning ion conductance microscopy (SECM-SICM) experiments that describe transport of redox active molecules which emanate from single pores of a track-etch membrane. Experiments are performed with electrodes which consist of a thin gold layer deposited on one side of a nanopipet. Subsequent insulation of the electrode with parylene results in a hybrid electrode for SECM-SICM measurements. Electrode fabrication is straightforward and highly parallel. For image collection, ionic current measured at the nanopipet both controls the position of the electrode with respect to the membrane surface and reports the local conductance in the vicinity of the nanopipet, while faradaic current measured at the Au electrode reports the presence of redox-active molecules. Application of a transmembrane potential difference affords additional control over migration of charged species across the membrane.     

Combining aryltriazenes and electrogenerated acids to create well-defined aryl-tethered films and patterns on surfaces

Immobilization of submonolayers to 4-5 multilayers of organic molecules on carbon surfaces can be performed by in situ generation of aryl radicals from aryltriazenes. The central idea consists of oxidatively forming an electrogenerated acid of N,N'-diphenylhydrazine to convert the aryltriazene to the corresponding diazonium salt in the diffusion layer of the electrode. In a second step, the diazonium salt is reduced at the same electrode to give a surface of covalently attached aryl groups. In this manner, various moieties tethered to the aryl groups can be immobilized on the surface. Here a ferrocenyl group was introduced as redox marker, the electrochemical signal of which is extraordinarily well-defined. This behavior is independent of film thickness, the latter being easily controlled by the number of repetitive cycles performed. It is also demonstrated that the new approach is suitable for patterning of surfaces using scanning electrochemical microscopy.     

Electrochemistry in nanometer-wide electrochemical cells

The electrochemical properties of an electrochemical cell defined by two concentric spherical electrodes, separated by a 1 to 20-nm-wide gap, and a freely diffusing electrochemically active molecule (e.g., ferrocene) have been investigated by coupling of Brownian dynamics simulations with long-range electron-transfer probability values. The simulation creates a trajectory of a single molecule and calculates the likelihood that the molecule undergoes a redox reaction during each time interval based on a probability-distance function derived from literature first-order kinetic data for a surface-bound ferrocene. Steady-state voltammograms for the single-molecule concentric spherical electrochemical cell are computed and are used to extract a heterogeneous electron-transfer rate for the freely diffusing molecule redox reaction. The Brownian dynamics simulations also indicate that long-range electron transfer, between the redox molecule and electrode, leads to nonsigmoidal-shaped i-E characteristics when the distance between electrodes approaches the characteristic redox tunneling decay length. The long-range electron transfer generates a "tunneling depletion layer" that results in a potential-dependent diffusion-limited current.     

Inlaid Multi‐Walled Carbon Nanotube Nanoelectrode Arrays for Electroanalysis

The rapid development in nanomaterials and nanotechnologies has provided many new opportunities for electroanalysis. We review our recent results on the fabrication and electroanalytical applications of nanoelectrode arrays based on vertically aligned multi‐walled carbon nanotubes (MWCNTs). A bottom‐up approach is demonstrated, which is compatible with Si microfabrication processes. MWCNTs are encapsulated in SiO₂ matrix leaving only the very end exposed to form inlaid nanoelectrode arrays. The electrical and electrochemical properties have been characterized, showing well‐defined quasireversible nanoelectrode behavior. Ultrasensitive detection of small redox molecules in bulk solutions as well as immobilized at the MWCNT ends is demonstrated. A label‐free affinity‐based DNA sensor has shown extremely high sensitivity approaching that of fluorescence techniques. This platform can be integrated with microelectronics and microfluidics for fully automated microchips.     

Electrically driven motion of an air bubble on hemispherical oil/water interface by three-phase boundary reactions

Our electrochemical cell consisted of a ferrocene-included hemispherical nitrobenzene (NB) droplet on the glassy carbon (GC) electrode which was immersed in the aqueous solution including sodium sulfate and sodium dodecyl sulfate (SDS). When an air bubble was injected near the boundary between the oil and the aqueous phase, it stayed at the top of the hemisphere on the boundary so that the lower half of the bubble was put in oil and the other half was in water. From the force balance of surface tension and buoyancy of the bubble, the bubble took an energetic minimum at the interface. It sank into the oil phase when ferrocene in the oil was electrochemically oxidized through the GC electrode by the three-phase boundary reaction. The electrochemical reduction caused the bubble to move back toward the aqueous phase. The motion of the bubble was synchronized with the redox reaction of ferrocene. The potential step oxidation showed such a rapid response that the motion could not be attributed to diffusion of ferricenium ion from the three-phase boundary to the bubble. Our idea of explaining the rapidity was the translational motion of the SDS layer along the boundary, which was driven by the difference in the surface concentration of SDS caused by the electrochemical generation of the ferricenium ion. The motion of the SDS layer was demonstrated by the shrinkage of the oil layer spread on the water surface when SDS solution was dropped on the oil layer. The spreading velocity was close to the velocity of propagating the oxidation of ferrocene to the bubble.     

Electrochemical immunoassay at a 17beta-estradiol self-assembled monolayer electrode using a redox marker

A simple electrochemical immunoassay was demonstrated using a 17beta-estradiol modified electrode. 17beta-estradiol was immobilized on the gold electrode surface with a self-assembly technique. The specific binding between estradiol antibody and 17beta-estradiol on the electrode surface was evaluated by monitoring the change in the electrode response with three hydrophilic redox markers. The decrease in the electrode response for the redox marker was observed, when the antibody was bound to the estradiol self-assembled monolayer (SAM) electrode surface. The change in the electrode response of the redox marker is attributed to the steric hindrance between the antibody on the electrode surface and the redox marker. The relative standard deviation at 30 microg ml(-1) estradiol antibody was 4.1% (n = 3). The competitive reaction between the antigen in the solution and 17beta-estradiol immobilized on the electrode surface for the limited binding sites on the antibody produced an increase in the electrode response with hydroquinone as the marker. The binding affinity of three antigens including 17beta-estradiol to the estradiol antibody was evaluated. Furthermore, the result obtained from this method was compared with the previously reported enzyme binding assay using the biotinylated estradiol and the biotin-immobilized microtiter plate.     

Colloidal Supercapattery: Redox Ions in Electrode and Electrolyte

Redox chemistry is the cornerstone of various electrochemical energy conversion and storage systems, associated with ion diffusion process. To actualize both high energy and power density in energy storage devices, both multiple electron transfer reaction and fast ion diffusion occurred in one electrode material are prerequisite. The existence forms of redox ions can lead to different electrochemical thermodynamic and kinetic properties. Here, we introduce novel colloid system, which includes multiple varying ion forms, multi‐interaction and abundant redox active sites. Unlike redox cations in solution and crystal materials, colloid system has specific reactivity‐structure relationship. In the colloidal ionic electrode, the occurrence of multiple‐electron redox reactions and fast ion diffusion leaded to ultrahigh specific capacitance and fast charge rate. The colloidal ionic supercapattery coupled with redox electrolyte provides a new potential technique for the comprehensive use of redox ions including cations and anions in electrode and electrolyte and a guiding design for the development of next‐generation high performance energy storage devices.