Redox protein-polymer films for simultaneous determination of ascorbic Acid and hydrogen peroxide

Layer by layer films of protein and redox polymer were constructed and used to simultaneously analyze ascorbic acid and hydrogen peroxide. The films were made using hemoglobin and poly[4-vinylpyridine Os(bipyridine)(2)Cl]-co-ethylamine (Pos-Ea). The film growth was monitored using cyclic voltammetry, quartz crystal microbalance (QCM) and atomic force microscopy (AFM). Reversible pairs of oxidation-reduction peaks were observed using cyclic voltammetry corresponding to the Os(II)/Os(III) from redox polymer and HbFe(III)/HbFe(II) redox couples at 0.35 and -0.25 V vs. Ag/AgCl, respectively. The two redox centers were independent of each other. This enabled the simultaneous and independent determination of ascorbic acid and hydrogen. Peak currents were linearly related to concentration for both analytes in a mixture. The linear range of ascorbic acid was 0 - 1 mM (R(2) = 0.9996, n = 5) at scan rate of 50 mV s(-1) (sensitivity 3.5 microA/mM) while hydrogen peroxide linear range was 1.0 - 10.0 microM (R(2) = 0.991, n = 6) with sensitivity of 1.85 microA/microM.     

Electropolymerization of iron tetra(<Emphasis Type="Italic">o</Emphasis>-aminophenyl)porphyrin from aqueous solution and the electrocatalytic behavior of modified electrode

Stable electroactive iron tetra(o-aminophenyl)porphyrin (FeTAPP) films are prepared by electropolymerization from aqueous solution by cycling the electrode potential between −0.4 and 1.0 V vs Ag/AgCl at 0.1 V s⁻¹. The cyclic voltammetric response indicates that polymerization takes place after the oxidation of amino groups, and the films could be produced on glassy carbon (GC) and gold electrodes. The film growth of poly(FeTAPP) was monitored by using cyclic voltammetry and electrochemical quartz crystal microbalance. The cyclic voltammetric features of Fe(III)/Fe(II) redox couple in the film resembles that of surface confined redox species. The electrochemical response of the modified electrode was found to be dependent on the pH of the contacting solution with a negative shift of 57 mV/pH. The electrocatalytic behavior of poly(FeTAPP) film-modified electrode was investigated towards reduction of hydrogen peroxide, molecular oxygen, and chloroacetic acids (mono-, di-, and tri-). The reduction of hydrogen peroxide, molecular oxygen, and dichloroacetic acid occurred at less negative potential on poly(FeTAPP) film compared to bare GC electrode. Particularly, the overpotential of hydrogen peroxide was reduced substantially. The O₂ reduction proceeds through direct four-electron reduction mechanism.     

Electron transfer in electroactive poly(glutamic acid)

Electroactive biopolymer was synthesized by incorporation of ferrocene moieties onto poly(glutamic acid) polymer chains. In the presence of the electron acceptor methyl viologen dichloride, the ferrocene-containing poly(glutamic acid) exhibits efficient photoinduced electron transfer. This redox-active polymer’s electrocatalytic activity for the decomposition of hydrogen peroxide and ascorbic acid was investigated by using cyclic voltammetry. As for hydrogen peroxide, the reduction peak current shows proportional response to peroxide concentration in the wide range of 10-100 mM; as for ascorbic acid, the oxidation peak current displays linear dependence on the ascorbic acid concentration under 80 mM, which could lead to the electroactive biopolymer’s applications in catalysis, photosensitizer, sensors, etc. The study could offer a strategy for developing environment-friendly electroactive and photoactive biopolymers.     

超支化聚合物作为放大信号的纳米磁性微球电化学免疫分析法

提出了一种基于超支化聚合物(HBP)固化酶标二抗作为放大信号和纳米磁球相结合的超灵敏的免疫分析新方法。首先羧基纳米磁性微球共价键合乙肝抗体(HBsAb)形成免疫磁性微球,然后与待测乙肝表面抗原(HBsAg)发生特异性结合,加入HBP标记的酶标二抗(HBPS)与结合的抗原结合发生夹心反应。在外加磁场的作用下,抗体抗原免疫复合物易从样品溶液中分离,在含有邻氨基苯酚和H2O2的底液中,快速生成具有电活性的化合物3-氨基吩呃嗪,用示差脉冲伏安法(DPV)测定响应电流,电流强度(I)与乙肝表面抗原浓度(c)在0.05～10.0μg/L范围内呈线性关系,线性回归方程为I(μA)=0.140+16.80 c(μg/L),相关系数r=0.9995,检出限达0.008μg/L,并用于实际样品的测定。     

Hybrid bioelectrocatalyst for hydrogen peroxide reduction: immobilization of enzyme within organic-inorganic film of structured Prussian Blue and PEDOT

Fabrication of structured film (on glassy carbon substrate) composed of compact Prussian Blue (that has been prepared by alternate immersions and through assembling within ultra-thin layers of 4(pyrrole-1-yl)-benzoic acid, PPyBA) and poly(3,4-ethylendioxythiophene), PEDOT, is described. This functionalized film has been characterized by fast charge propagation, and it has served as a redox conducting template for permanent attachment of a model enzyme, horseradish peroxidase, HRP. The resulting organic-inorganic system acts as an effective hybrid bioelectrocatalyst for electroreduction of hydrogen peroxide, a model reactant for biosensors and biofuel cells. Among important issues are rigidity, permanence of enzyme attachment, morphology, hydrophilicity, and attractive mediating capabilities of the PEDOT-stabilized Prussian Blue based structured film.     

NiCo2O4纳米线对 H2O2电还原的催化性能

以无模板生长法制备了泡沫镍载NiCo2O4纳米线正极材料, XRD和SEM表征结果表明, 所得材料为NiCo2O4纳米线, 以循环伏安法和计时电流法研究了泡沫镍载NiCo2O4纳米线对H2O2电还原的催化性能. 结果显示, 在0.4 mol/L H2O2 和 3.0 mol/L NaOH 溶液中, 当电压为-0.4 V(vs. Ag/AgCl)时, 循环伏安的电流密度达到125 mA/cm2; 当电压为-0.2, -0.3和 -0.4 V 时, 在30 min 的测试时间内, 计时电流密度几乎均为一常数, 表明以泡沫镍载NiCo2O4纳米线为催化剂电还原H2O2具有很高的活性和很好的稳定性.     

Electrocatalytic Oxidation of Hydrogen Peroxide on Poly(m‐toluidine)‐Nickel Modified Carbon Paste Electrode in Alkaline Medium

The poly(m‐toluidine) film was prepared by using the repeated potential cycling technique in an acidic solution at the surface of carbon paste electrode. Then transition metal ions of Ni(II) were incorporated to the polymer by immersion of the modified electrode in a 0.2 M NiSO₄, also the electrochemical characterization of this modified electrode exhibits stable redox behavior of the Ni(III)/Ni(II) couple. The electrocatalytic ability of Ni(II)/poly(m‐toluidine)/modified carbon paste electrode (Ni/PMT/MCPE) was demonstrated by electrocatalytic oxidation of hydrogen peroxide with cyclic voltammetry and chronoamperometry methods in the alkaline solution. The effects of scan rate and hydrogen peroxide concentration on the anodic peak height of hydrogen peroxide oxidation were also investigated. The catalytic oxidation peak current showed two linear ranges with different slopes dependent on the hydrogen peroxide concentration and the lower detection limit was 6.5 μM (S/N=3). The catalytic reaction rate constant, (k_h), was calculated 5.5×10² M^?1 s^?1 by the data of chronoamperometry. This modified electrode has many advantages such as simple preparation procedure, good reproducibility and high catalytic activity toward the hydrogen peroxide oxidation. This method was also applied as a simple method for routine control and can be employed directly without any pretreatment or separation for analysis cosmetics products.     

Visualisation of the local bio-electrocatalytic activity in biofuel cell cathodes by means of redox competition scanning electrochemical microscopy (RC-SECM)

Optimisation of biocatalytic systems for the electroreduction of molecular O₂ in biofuel cell cathodes implies screening of the catalytic activity of enzyme/redoxpolymer assemblies. Os-complex modified electrodeposition polymers are suggested for linking bilirubin oxidase catalysed O₂ reduction via an electron hopping sequence along the redox polymer to the electrode. They can be non-manually precipitated on electrode surfaces by electrochemically induced pH modulation. Cyclic voltammetry provides a good estimation of the electrocatalytic activity of a redox polymer/enzyme modified electrode surface. In addition, scanning electrochemical microscopy operating in redox competition mode (RC-SECM) supplies images of the spatial distribution of the biocatalytic activity.     

Towards immunoassay in whole blood: separationless sandwich-type electrochemical immunoassay based on in-situ generation of the substrate of the labeling enzyme

An 18 minute separationless amperometric ELISA-type sandwich immunoassay, utilizing only stable reagents and having no washing steps is described. The platform for the assay was an electron conducting redox hydrogel on a vitreous carbon electrode. Avidin and choline oxidase were co-immobilized on the redox hydrogel and the biotinylated antibody to the antigen to be assayed (the biotin-labeled F(ab′)₂ fragment of goat anti-rabbit IgG) was bound to the gel. When the antigen (goat anti-rabbit IgG) was present in the analyzed solution, then its binding to the immobilized antibody made the electrode receptive to the complementary peroxidase-labeled antibody (horseradish peroxidase-labeled F(ab′)₂ fragment of goat anti-rabbit IgG). Its binding resulted in electrical contact (“wiring”) of the horseradish peroxidase label to the redox hydrogel, and converted the non-catalytic hydrogel into an electrocatalyst for the reduction of hydrogen peroxide to water at –0.07 V (SCE) and resulted in the flow of a cathodic current. The electroreduced hydrogen peroxide was not added to the solution and was therefore not significantly accessible to hydrogen peroxide decomposing agents such as catalase. Instead, it was generated within the coating of the electrode through reacting dissolved choline with oxygen. This reaction was catalyzed by the immobilized choline oxidase. The reaction centers of choline oxidase, unlike those of horseradish peroxidase, are not connected to the electrode by the redox hydrogel.     

Hydrogen peroxide electroreduction on composite PEDOT films with included gold nanoparticles

Composite PEDOT/Au films were obtained by chemical deposition of dispersed gold nanoparticles into PEDOT (poly-3,4-ethylenedioxythiophene) conducting polymer matrix. Morphology of the obtained gold-containing films was studied by SEM and TEM methods. To study the kinetics of the hydrogen peroxide electroreduction that proceeds on glassy carbon electrodes modified with such films, we used phosphate buffer solutions containing addenda of hydrogen peroxide species. It was observed that the electroreduction process takes place on both the gold clusters?? surface and the film surface free of metal inclusions. The rate of the process is higher in the first case and rises with increasing the gold content in modifying films, but in the limit of large gold contents it is limited only by diffusion of hydrogen peroxide species in the bathing solution. A simple theory of such parallel electroreduction is proposed, which appears to allow for quantitative treatment of the obtained results.     

Reagentless enzyme electrode based on phenothiazine mediation of horseradish peroxidase for subnanomolar hydrogen peroxide determination

The development and characterization of a highly sensitive enzyme immobilized carbon based electrode for the determination of subnanomolar concentrations of hydrogen peroxide in aqueous samples is described. The biosensor consists of horseradish peroxidase (HRP) immobilized in solid carbon paste along with a suitable redox mediator. The latter allows the acceleration of the electroreduction of HRP in the presence of hydrogen peroxide. Several phenothiazines as mediators are investigated in a comparative manner and with respect to dimethylferrocene using cyclic voltammetry and amperometry. Insolubilization of the HRP in the solid carbon paste is achieved by cross-linking the enzyme with glutaraldehyde and bovine serum albumin. Several experimental parameters such as pH, mediator and enzyme content are considered. The hydrogen peroxide determination is better carried out in 0.1 M acetate buffer, pH 4.5, by amperometry at an applied potential of 0.0 V versus Ag/AgCl, 3 M NaCl concentration and by using the phenothiazine base as redox mediator. The biosensor response is linear over the concentration range 2 nM-10 microM with a detection limit of 1 nM. The linear range of the hydrogen peroxide response without a mediator in the biosensor is found between 2 and 40 microM. The biosensor can be used for more than 180 measurements. Additional modification of the electrode by incorporation of Nafion SAC-13 microparticles in the solid carbon paste allows detection of concentrations of hydrogen peroxide as low as 0.1 nM.     

Electrocatalysis of the Oxygen Reaction by Pyropolymers of N4 Complexes

Results of research into structural and electrocatalytic properties of metalloporphyrins and metallophthalocyanines pyrolyzed on carbon supports of various dispersion degree in the oxygen electroreduction reaction (OER) are analyzed. The pyrolysis products (pyropolymers) that form at T 800° in inert atmosphere contain centers Co(Fe)–N surrounded by carbon particles. The oxygen electroreduction reaction on pyropolymers in acid and alkali solutions is studied on a model gas-diffusion electrode and a rotating ring–disk electrode. The slopes of Tafel plots in an acid solution are 60 and 120 mV. On a disk electrode covered with a pyropolymer, the intermediate product of OER, hydrogen peroxide, is fixed on the ring electrode throughout the entire range of OER potentials. The activity of pyropolymers in the hydrogen peroxide electroreduction reaction in an acid solution is insignificant. In an acid environment, OER occurs via a parallel–successive mechanism with a slow stage of the attachment of the first electron. In alkali media, slopes of Tafel plots equal 40 and 120 mV at low and high polarizations, respectively. The amount of hydrogen peroxide fixed on the ring electrode corresponds to 2–5% of the disk electrode current. A pyropolymer is active in the hydrogen peroxide reduction. The slow stage in OER in an alkali environment is the attachment of the second electron at a low polarization and the attachment of the first electron, at a high polarization. In acid and alkali solutions a pyropolymer is methanol-tolerant.     

Hydrogen peroxide electroreduction on composite PEDOT films with included gold nanoparticles

Composite PEDOT/Au films were obtained by chemical deposition of dispersed gold nanoparticles into PEDOT (poly-3,4-ethylenedioxythiophene) conducting polymer matrix. Morphology of the obtained gold-containing films was studied by SEM and TEM methods. To study the kinetics of the hydrogen peroxide electroreduction that proceeds on glassy carbon electrodes modified with such films, we used phosphate buffer solutions containing addenda of hydrogen peroxide species. It was observed that the electroreduction process takes place on both the gold clusters’ surface and the film surface free of metal inclusions. The rate of the process is higher in the first case and rises with increasing the gold content in modifying films, but in the limit of large gold contents it is limited only by diffusion of hydrogen peroxide species in the bathing solution. A simple theory of such parallel electroreduction is proposed, which appears to allow for quantitative treatment of the obtained results.

     

Biochemical synthesis of water soluble polyanilines: Poly(p-aminobenzoic acid)

This report describes the synthesis of a water soluble polyaniline through a biochemical synthetic route. The oxidative free radical coupling mechanism for the synthesis of poly(p-aminobenzoic acid) is catalyzed by horseradish peroxidase in the presence of hydrogen peroxide. The resulting polymer is electrochemically active and undergoes reversible redox reactions. The polymer as synthesized is self doped and undergoes undoping in alkaline or ammonia solutions.     

A laccase-wiring redox hydrogel for efficient catalysis of O2 electroreduction

Laccase was earlier wired to yield an O2 electroreduction catalyst greatly outperforming platinum and its alloys. Here we describe the design, synthesis optimization of the composition, and characterization of the +0.55 V (AgAgCl) laccase-wiring redox hydrogel, with an apparent electron diffusion coefficient (D(app)) of 7.6 x 10(-7) cm2 s(-1). The high D(app) results in the tethering of redox centers to the polymer backbone through eight-atom-long spacer arms, which facilitate collisional electron transfer between proximal redox centers. The O2 flux-limited, true-area-based current density was increased from the earlier reported 560 to 860 microA cm(-2). When the O2 diffusion to the 7-microm-diameter carbon fiber cathode was cylindrical, half of the O2 flux-limited current was reached already at 0.62 V and 90% at 0.56 V vs Ag/AgCl, merely -0.08 and -0.14 V versus the 0.7 V (Ag/AgCl) reversible O2/H2O half-cell potential at pH 5.     

Copper redox cycling in the prion protein depends critically on binding mode

The prion protein (PrP) takes up 4-6 equiv of copper in its extended N-terminal domain, composed of the octarepeat (OR) segment (human sequence residues 60-91) and two mononuclear binding sites (at His96 and His111; also referred to as the non-OR region). The OR segment responds to specific copper concentrations by transitioning from a multi-His mode at low copper levels to a single-His, amide nitrogen mode at high levels (Chattopadhyay et al. J. Am. Chem. Soc. 2005, 127, 12647-12656). The specific function of PrP in healthy tissue is unclear, but numerous reports link copper uptake to a neuroprotective role that regulates cellular stress (Stevens, et al. PLoS Pathog.2009, 5 (4), e1000390). A current working hypothesis is that the high occupancy binding mode quenches copper's inherent redox cycling, thus, protecting against the production of reactive oxygen species from unregulated Fenton type reactions. Here, we directly test this hypothesis by performing detailed pH-dependent electrochemical measurements on both low and high occupancy copper binding modes. In contrast to the current belief, we find that the low occupancy mode completely quenches redox cycling, but high occupancy leads to the gentle production of hydrogen peroxide through a catalytic reduction of oxygen facilitated by the complex. These electrochemical findings are supported by independent kinetic measurements that probe for ascorbate usage and also peroxide production. Hydrogen peroxide production is also observed from a segment corresponding to the non-OR region. Collectively, these results overturn the current working hypothesis and suggest, instead, that the redox cycling of copper bound to PrP in the high occupancy mode is not quenched, but is regulated. The observed production of hydrogen peroxide suggests a mechanism that could explain PrP's putative role in cellular signaling.     

SYNTHESIS OF SOLUBLE POLY（3,4-DIHYDROXY-o-TOLYLENE）： REDOX POLYMER

Conventional chloromethylation, paraformaldehyde/hydrogen chloride in acetic acid medium, was applied to 1,2- dimethoxybenzene. Chloroform-soluble poly（3,4-dimethoxy-o-tolylene） was obtained with an intrinsic viscosity of 0.034 dL g^-1. The polymer was evaluated as a condensation redox polymer precursor formed by a Friedel-Crafts reaction. Cleavage of the methoxy groups present in this polymer resulted in poly（3,4-dihydroxy-o-tolylene） which manifested a great air-oxidation resistance. The redox property of the latter polymer was found to be 1017 mV by potentiometric titration with 0.05 N ceric ammonium nitrate at 25℃. This midpotential was compared to that of catechol, a monomeric analogue, under the same titration conditions.     

Structure and electrochemical properties of composite films based on poly-3,4-ethylenedioxythiophene with metallic palladium inclusions

The electrochemical behavior of PEDOT/Pd composite films obtained by the chemical deposition of ultradisperse Pd particles in the poly-3,4-ethylenedioxythiophene (PEDOT) polymer matrix was studied. The structure of the films was determined by electron microscopy and energy-dispersion X-ray fluorescence analysis. The electrochemical properties of PEDOT/Pd composite films in solutions containing hydrogen peroxide was also studied. Special attention was paid to the effect of the time of the chemical deposition of palladium in the polymer structure on the electroreduction of hydrogen peroxide in phosphate buffer solutions.     

Oxygen reduction reaction on polycrystalline gold. Pathways of hydrogen peroxide transformation in the acidic medium

The mechanism of oxygen electroreduction on polycrystalline gold is studied in the acidic medium. Hydrogen peroxide is the main reaction product. However, two potential regions can be singled out in which the oxygen electroreduction reaction proceeds by different pathways. The first region is the potential interval close to the steady-state potential. Here, the oxygen electroreduction virtually completely produces peroxide. The second interval is the potential range of considerable cathodic polarization values. In this case, peroxide can be reduced to water. The low energy of hydrogen peroxide adsorption on gold determines the considerable overpotential of peroxide reduction. It is shown that on the gold electrode surface, the catalytic decomposition of peroxide occurs. The use of the method of electrochemical impedance spectroscopy allows the peculiarities of the oxygen reaction associated with hydrogen peroxide transformations to be revealed. In the acidic medium, the reactions of consecutive reduction of oxygen through the intermediate formation of hydrogen peroxide and the catalytic decomposition of the intermediate product are shown to proceed simultaneously. The ratio of rate constants of electrochemical stages depends on the potential. The chemical decomposition is observed both near the steady-state potential and in the cathodic region where considerable electrochemical reduction of peroxide occurs.     

Amperometric Response of Hydrogen Peroxide at Carbon Nanotubes Paste Electrodes Modified with an Electrogenerated Poly(Fe(III)‐5‐amino‐phenantroline). Analytical Applications for Glucose Biosensing

This work reports the catalytic activity of a polymer electrogenerated from Fe(III)‐5‐amino‐1,10‐phenantroline solution at a carbon nanotubes paste electrode (CNTPE) towards the oxidation and mainly the reduction of hydrogen peroxide. The important role of carbon nanotubes on the generation of poly(Fe(III)‐5‐amino‐1,10‐phenantroline) is demonstrated through the comparison with the behavior of graphite paste electrode (CPE). The polymer electrogenerated at CNTPE largely improves the amperometric detection of hydrogen peroxide at ?0.100 V. The analytical application of the resulting electrode is demonstrated in connection with the design of a glucose biosensor based on the deposition of GOx and diluted Nafion on the top of the polymer‐modified CNTPE. The quantification of glucose in human serum samples showed a good correlation with the values obtained by the spectrophotometric technique.