Hemoglobin adsorption into TiO2 phytate multi-layer films: particle size and conductivity effects

Hemoglobin (molecular weight 64.5 kDa, isoelectric point 7.4) in 0.1 M phosphate buffer solution at pH 5.5 readily adsorbs onto mesoporous TiO₂ phytate films, which have been formed in a layer-by-layer deposition process from TiO₂ nanoparticles (ca. 6–10 nm diameter) and phytic acid at tin-doped indium oxide (ITO) electrodes. Quartz crystal microbalance data, voltammetry, and SEM evidence are consistent with hemoglobin adsorption only into the outer TiO₂ phytate surface layer. The size of the tetrametric hemoglobin protein (ca. 6 nm diameter) appears to be too big for a homogeneous film to form.The modified ITO electrode immersed in 0.1 M phosphate buffer solution at pH 5.5 allows reversible electron transfer for hemoglobin to be observed with a midpoint potential of 0.01 vs. SCE. Characteristic TiO₂ phytate film thickness and pH effects are observed with both thicker films and lower proton activity causing ‘decoupling’ of the protein redox chemistry due to a reduced electrical conductivity of the TiO₂ phytate film connecting hemoglobin with the electrode. This is the first example of a bi-layer nanofilm structure where the underlying TiO₂ phytate film controls the electrochemical properties of the hemoglobin modified top-layer.     

Protein adsorption on nanoporous TiO2 films: a novel approach to studying photoinduced protein/electrode transfer reactions

We have investigated the use of nanoporous TiO2 films as substrates for protein immobilisation. Such films are of interest due to their high surface area, optical transparency, electrochemical activity and ease of fabrication. These films moreover allow detailed spectroscopic study of protein/electrode electron transfer processes. We find that protein immobilisation on such films may be readily achieved from aqueous solutions at 4 degrees C with a high binding stability and no detectable protein denaturation. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of up to 850 for an 8 microns thick film). We demonstrate that the redox state of proteins such as immobilised cytochrome-c (Cyt-c) and haemoglobin (Hb) may be modulated by the application of an electrical bias potential to the TiO2 film, without the addition of electron transfer mediators. The binding of Cyt-c on the TiO2 films is investigated as a function of film thickness, protein concentration, protein surface charge and ionic strength. We demonstrate the potential use of immobilised Hb on such TiO2 films for the detection of dissolved CO in aqueous solutions. We further show that protein/electrode electron transfer may be initiated by UV bandgap excitation of the TiO2 electrode. Both photooxidation and photoreduction of the immobilised proteins can be achieved. By employing pulsed UV laser excitation, the interfacial electron transfer kinetics can be monitored by transient optical spectroscopy, providing a novel probe of protein/electrode electron transfer kinetics. We conclude that nanoporous TiO2 films may be useful both for basic studies of protein/electrode interactions and for the development of novel bioanalytical devices such as biosensors.     

Direct Electrochemistry of Cytochrome c on a Silica Sol‐Gel Film Modified Electrode

Dilute silica sol‐gel was simply dropped on the surface of a basal plane graphite electrode (BPGE) to form a silica sol‐gel film modified electrode. Direct electrochemical response of cytochrome c (Cyt c) on the modified electrode was observed by cyclic voltammetry (CV). The results suggested that Cyt c could be tightly adsorbed on the surface of the silica sol‐gel film modified electrode. A couple of well‐defined and nearly reversible redox peaks can be observed in a phosphate buffer solution (pH 7.0), which anodic and cathodic peak potentials were at ?0.243 and ?0.306 V (vs. Ag/AgCl), respectively. Cyt c adsorbed on the surface of silica sol‐gel film shows a remarkable electrocatalytic activity for the reduction of oxygen. Based on these, a third‐generation biosensor could be constructed to detect the concentration of oxygen in aqueous solution.     

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

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

The fabrication of a colloidal gold-carbon nanotubes composite film on a gold electrode and its application for the determination of cytochrome c

Colloid Au (Au_nano) with a diameter of about 20 nm was prepared and used in combination with the multi-wall carbon nanotubes (MWNTs) to modify a gold electrode. Dihexadecylphosphate (DHP) dispersed in Au_nano aqueous solution was used to solubilize MWNTs. Deposition of Au_nano on MWNTs was realized as illustrated by TEM micrographs. The DHP formed a network that connected Au_nano and MWNTs to the gold electrode surface. The Au_nano–MWNTs–DHP composite film on the gold electrode surface was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammmetry (CV). The composite film modified gold electrode was used to detect cytochrome c and a pair of well-defined redox waves was obtained. It was found that the composite film promoted the redox of horse heart cytochrome c and its effect was developed for the determination of cytochrome c.     

NanoTiO2-CNT复合膜电极在DMF溶液中对糠醛的异相电催化还原

通过在乙醇中电化学溶解Ti金属阳极合成前驱体Ti(OEt)4和溶胶-凝胶法在Ti表面修饰一层纳米TiO2-碳纳米管(nanoTiO2-CNT)复合膜, 采用循环伏安和电解合成法研究了nanoTiO2-CNT复合膜电极在N, N-二甲基甲酰胺(DMF)中的氧化还原行为以及对糠醛(furfural)还原的电催化活性. 结果发现, nanoTiO2-CNT电极在阴极扫描时有两对氧化还原峰, 可逆半波电位E r1/2 分别为－1.27 V和－2.44 V(vs SCE, 扫描速度100 mV•s-1), 分别对应于TiO2/Ti2O3氧化还原电对的可逆电极过程和TiO2/Ti(OH)3电对的准可逆电极过程；在DMF电解液中nanoTiO2-CNT复合膜中的Ti(IV)/Ti(III)氧化还原电对作为媒质间接电还原糠醛为糠醇, 反应机理为电化学偶联随后化学催化反应(EC′)机理.     

纳米尺度TiO2聚苯胺多孔膜电极光电化学研究

用光电流作用谱，光电流－电势图等光电化学方法研究了ＴｉＯ２／聚本胺复合多孔膜电极在不含氧化还原和含有没氧化还原对体系中的光电转换过程。结果说明，ＴｉＯ２／聚苯胺复合多孔膜电极为双层ｍ－型半导体结构，ＴｉＯ２多孔膜的禁带宽度为３．２ｅＶ，外层聚苯胺膜的禁带宽度为２．８８ｅＶ。     

A conductive crosslinked graphene/cytochrome c networks for the electrochemical and biosensing study

The direct electrochemistry of catalytically active cytochrome C (Cyt c) adsorbed together with a 3-dimensional network of chemically synthesized graphene on glassy carbon electrode has been readily obtained in aqueous phosphate buffer. Direct electrical communication between the redox center of Cyt c and the modified graphene-based electrode was established. The modified electrode was employed as a high-performance hydrogen peroxide (H₂O₂) biosensor. The Cyt c present in modified electrode exhibited a pair of quasi-reversible redox peaks with a midpoint potential of ?0.380 and ?0.2 V, cathodic and anodic, respectively. Investigations into the electrocatalytic activity of the modified electrode upon hydrogen peroxide exposure revealed a rapid amperometric response (5 s). Under optimized conditions, the linear range of response to H₂O₂ concentration ranged from 5 × 10^?7 to 2 × 10^?4 M with a detection limit of 2 × 10^?7 M at a signal-to-noise ratio of 3. The stability, reproducibility, and selectivity of the proposed biosensor are discussed in relation to the morphology and composition of the modified electrode.     

Triplet state photosensitization of nanocrystalline metal oxide electrodes by zinc-substituted cytochrome c: application to hydrogen evolution

The interfacing of nanostructured semiconductor photoelectrodes with redox proteins is an innovative approach to the development of artificial photosynthetic systems. In this paper, we have investigated the photoinduced electron-transfer reactions of zinc-substituted cytochrome c, ZnCyt-c, immobilized on mesoporous, nanocrystalline metal oxide electrodes. Efficient electron injection from the triplet state of ZnCyt-c is observed into TiO(2) electrodes (t(50%) approximately 100 micros) resulting in a long-lived charge-separated state (lifetime of up to 0.4 s). Further studies were undertaken as a function of electrolyte pH and metal oxide employed. Optimum yield of a long-lived charge-separated state was observed employing TiO(2) electrodes at pH 5, consistent with our previous studies of analogous dye-sensitized metal oxide electrodes. The addition of EDTA as a sacrificial electron donor to the electrolyte resulted in efficient photogeneration of molecular hydrogen, with a quantum yield per one absorbed photon of 10 +/- 5%.     

Amperometric and voltammetric detection of hydrazine using glassy carbon electrodes modified with carbon nanotubes and catechol derivatives

A simple procedure was developed to prepare a glassy carbon (GC) electrode modified with carbon nanotubes (CNTs) and catechol compounds. First, 25 microL of DMSO-CNTs solutions (0.4 mg/mL) was cast on the surface of GC electrode and dried in air to form a CNTs film. Then the GC/CNTs modified electrode immersed into a chlorogenic acid, catechine hydrate and caffeic acid solution (electroless deposition) for a short period of time (2-80s). The cyclic voltammogram of the modified electrode in aqueous solution shows a pair of well-defined, stable and nearly reversible redox couple (quinone/hydroquinone) with surface confined characteristics. The combination of unique electronic and electrocatalytic properties of CNTs and catechol compounds results in a remarkable synergistic augmentation on the response. The electrochemical reversibility and stability of modified electrode prepared with incorporation of catechol compound into CNTs film was evaluated and compared with usual methods for attachment of catechols to electrode surfaces. The transfer coefficient (alpha), heterogeneous electron transfer rate constants (k(s)) and surface concentrations (Gamma) for GC/CNTs/catechol compound modified electrodes were calculated through the cyclic voltammetry technique. The modified electrodes showed excellent catalytic activity, fast response time and high sensitivity toward oxidation of hydrazine in phosphate buffer solutions at pH range 4-8. The modified electrode retains its initial response for at least 2 months if stored in dry ambient condition. The properties of modified electrodes as an amperometric sensor for micromolar or lower concentration detection of hydrazine have been characterized.     

Redox processes in mesoporous oxide membranes: layered TiO2 phytate and TiO2 flavin adenine dinucleotide films

Thin films of TiO2 (anatase) nanoparticles are assembled at an electrode surface via a layer-by-layer deposition process employing phytic acid, pyromellitic acid, or flavin adenine dinucleotide (FAD) as molecular binders. With all three types of binders, layers of typically 30 nm thickness are formed each deposition cycle. FAD as an electrochemically active component immobilized at the surface of the TiO2 particles is reduced to FADH2 and reoxidized in a chemically reversible two electron-two proton redox process. Two distinct voltammetric signals are observed for the immobilized FAD redox system associated with (i) hopping of electrons at the TiO2 surface (reversible) and (ii) conduction of electrons through the TiO2 assembly (irreversible). The conduction of electrons through the TiO2 assembly is possible by diffusion over considerable distances as well as through a "spacer" layer of TiO2 phytate. An order of magnitude (upper limit) estimate for the diffusion coefficient of electrons through TiO2 phytate, D(electron) approximately 10(-6) m(2) s(-1), is obtained from voltammetric data. Finally, it is demonstrated that the calcination of TiO2 assemblies causes dramatic changes in the electron transfer kinetics for the immobilized FAD/FADH2 redox system.     

间苯二酚在TiO2-石墨烯修饰玻碳电极上的电化学行为及测定

制备了TiO2-石墨烯修饰玻碳电极。用循环伏安法(CV)和差分脉冲伏安法(DPV)对间苯二酚在该修饰电极的电化学行为进行了研究。实验结果表明,在pH值为6.0的磷酸盐缓冲液(PBS)中,该修饰电极对间苯二酚具有良好的电催化作用。对TiO2-石墨烯用量、支持电解质、pH和扫描速度等实验条件进行了优化。在优化条件下,利用DPV测定,间苯二酚的氧化峰电流与其浓度在1.0×10-6～1.0×10-4mol/L范围内呈良好的线性关系,线性相关系数为0.995。检出限为2×10-7mol/L。将该方法应用于模拟水样中间苯二酚的测定,回收率为96.5～104.2%。     

Electrochemical characterisation and anodic stripping voltammetry at mesoporous platinum rotating disc electrodes

Using the technique of liquid crystal templating a rotating disc electrode (RDE) was modified with a high surface area mesoporous platinum film. The surface area of the electrode was characterised by acid voltammetry, and found to be very high (ca. 86 cm(2)). Acid characterisation of the electrode produced distorted voltammograms was interpreted as being due to the extremely large surface area which produced a combination of effects such as localised pH change within the pore environment and also ohmic drop effects. Acid voltammetry in the presence of two different types of surfactant, namely Tween 20 and Triton X-100, suggested antifouling properties associated with the mesoporous deposit. Further analysis of the modified electrode using a redox couple in solution showed typical RDE behaviour although extra capacitive currents were observed due to the large surface area of the electrode. The phenomenon of underpotential deposition was exploited for the purpose of anodic stripping voltammetry and results were compared with data collected for microelectrodes. Underpotential deposition of metal ions at the mesoporous RDE was found to be similar to that at conventional platinum electrodes and mesoporous microelectrodes although the rate of surface coverage was found to be slower at a mesoporous RDE. It was found that a mesoporous RDE forms a suitable system for quantification of silver ions in solution.     

Redox reactions of cytochrome c facilitated by silver-imidazole complex

An imidazole modified silver electrode is prepared by immersing the substrate silver electrode in a 2% imidazole solution of ethanol at 50℃ for 10 min. The modified electrode is then swept in a cytochrome c solution and the modified layer takes off because the modified electrode is very unstable. Although the amount of the silver-imidazole complex is very small compared with the amount of cytochrome c in the protein solution, it greatly facilitates redox reactions involving the biomacromolecules.     

Silicon nanotube array/gold electrode for direct electrochemistry of cytochrome c

The highly ordered Si nanotube (SiNT) arrays have been fabricated and demonstrated to be an attractive matrix to accommodate macromolecules, such as proteins, and exhibit facile direct electrochemistry of cytochrome c without any pretreatment. A pair of well-defined redox waves can be observed in the aqueous solution containing cytochrome c on this SiNT electrode using cyclic voltammetry. The peak separation (DeltaEp) between the anodic and the cathodic peaks is 63 mV at the sweep rate of 10 mV/s, and the peak current is proportional to the square root of the sweep rates, which indicates that the redox process is a quasi-reversible process. The experimental results show that the good biocompatibility and nanoscale hollow structure of the SiNT arrays can be applicable to preparing Si-based biosensors and protein characterizations.     

纳米尺度TiO2／聚吡咯多孔膜电极光电化学研究

用光电流作用谱，光电流－电势图和ＵＶ－Ｖｉｓ光说研究了ＴｉＯ２／聚吡咯多孔膜电极在不含氧化还原对和含不同氧化还原体系电解质溶液中的光电转换过程。ＴｉＯ２／聚吡咯多孔膜电极双层ｎ型半导体结构，内层ＴｉＯ２多孔膜的禁带宽度为３．２６ｅＶ，外层聚吡咯膜的禁带宽度为２．２ｅＶ。     

Direct Electrochemistry of Cytochrome c at Gold Electrode Modified with Fumed Silica

Direct electrochemistry of horse heart cytochrome c (cytc) has been obtained at a gold electrode constructed by self‐assembling fumed silica particles (FSPs) onto a silane monolayer. A pair of well‐defined and nearly symmetrical redox peaks of cytc is obtained at the FSPs film modified gold electrode. Cyclic voltammetry (CV) and tapping‐mode atomic force microscopy (AFM) are used to characterize the FSPs film modified electrode, showing that the FSPs can provide a favorable microenvironment for cytc and facilitate the direct electron transfer between the cytc and the gold electrode, which may propose an approach to realize the direct electrochemistry of other proteins.     

Oligonucleotide-modified electrodes for fast electron transfer to cytochrome c

Gold electrodes were modified with short ds-oligonucleotides via thiol binding to form a thin and stable surface layer. The modification was characterised by impedance measurements and used as a promoter for fast electron transfer to cytochrome c. The protein was investigated both immobilised and in solution showing reversible electrochemical behaviour in each case. The modification proved to have a good adsorption capability for the redox protein which was also found to be reversible. In the immobilised state at the electrode cytochrome c reacted with superoxide radicals in solution, exemplified by cyclic voltammetric measurements.     

Quartz crystal microbalance monitoring of density changes in mesoporous TiO2 phytate films during redox and ion exchange processes

Nanofilm deposits of TiO₂ nanoparticle phytates are formed on gold electrode surfaces by ‘directed assembly’ methods. Alternate exposure of a 3-mercapto-propionic acid modified gold surface to (i) a TiO₂ sol and (ii) an aqueous phytic acid solution (pH 3) results in layer-by-layer formation of a mesoporous film. Ru(NH₃)₆³⁺ is shown to strongly adsorb/accumulate into the mesoporous structure whilst remaining electrochemically active. Scanning the electrode potential into a sufficiently negative potential range allows the Ru(NH₃)₆³⁺ complex to be reduced to Ru(NH₃)₆²⁺ which undergoes immediate desorption. When applied to a gold coated quartz crystal microbalance (QCM) sensor, electrochemically driven adsorption and desorption processes in the mesoporous structure become directly detectable as a frequency response, which corresponds directly to a mass or density change in the membrane. The frequency response (at least for thin films) is proportional to the thickness of the mass-responsive film, which suggests good mechanical coupling between electrode and film. Based on this observation, a method for the amplified QCM detection of small mass/density changes is proposed by conducting measurements in rigid mesoporous structures.     

Atomic Force Microscopic and Electrochemical Investigations of an Electrostatically Fabricated Single‐Wall Carbon Nanotubes Modified Electrode

Single‐wall carbon nanotubes (SWNTs) sub‐monolayer film has been prepared by simply electrostatically adsorbing nanotubes onto a 2‐aminoethanethiol self‐assembled monolayer (SAM) on a gold bead electrode. Tapping‐mode atomic force microscopy (TM‐AFM) is used to characterize the SWNT film, which exhibits that the orientation of SWNTs on the SAM is horizontal and the surface coverage is quite low. The SWNTs modified electrode shows nearly ideal electrochemical response to Fe(CN) /Fe(CN) redox probe. The electrode with such a low SWNTs coverage, however, shows good electrocatalytic behavior to cytochrome c.