Fabrication of a Nanobiocomposite Film Containing Heme Proteins and Carbon Nanotubes on a Choline Modified Glassy Carbon Electrode: Direct Electrochemistry and Electrochemical Catalysis

A nanocomposite electrochemical sensing film is assembled on choline (Ch) modified glassy carbon electrode (GCE), which contains multiwalled carbon nanotubes (MWNTs), Nafion cation exchanger, and myoglobin (Mb) or hemoglobin (Hb). The MWNTs provide a 3D porous and conductive network for the enzyme immobilization and Nafion acts as polymeric binder to give cast thin films. Both MWNTs and Nafion provide negative functionalities to bind to the positively charged redox proteins and to attach at the positively charged Ch modified layer, and drive the formation of homogeneous and stable nanocomposite film, the MWNT‐Nafion‐Mb. The nanocomposite film was characterized by field emission scanning electron microscope (FE‐SEM). The Mb in the nanocomposite film showed a pair of well‐defined and nearly reversible cyclic voltammetric peaks at about ?0.32 V vs. SCE at pH 7.0 solution for the heme Fe(III)/Fe(II) redox couple. The immobilized heme proteins can display the features of peroxidase in electrocatalytic reductions of oxygen, hydrogen peroxide, nitric oxide, trichloroacetic acid (TCA), and bromate.     

Fabrication of multilayer-film-modified gold electrode composed of myoglobin,chitosan, and polyelectrolyte-wrapped multi-wall carbon nanotubes by layer-by-layer assembled technique and electrochemical catalysis for hydrogen peroxide and trichloroacetic acid

Electroactive multilayer film of myoglobin (Mb)-, chitosan (CS)-, and poly(dimethyldiallylammonium chloride) (PDDA)-wrapped multi-wall carbon nanotubes (MWNTs) is fabricated on a gold electrode via layer-by-layer (LBL) technique. The assembled multilayer films is characterized by scanning electron microscopy (SEM), UV-vis spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). UV-vis spectroscopy showed that Mb in the films retained its near-native structure. The stable multilayerfilm-modified gold electrodes showed good electroactivity in protein-free buffer solution, which is originated from protein heme Fe(III)/Fe(II) redox couple. The modified electrode exhibited good electrocatalytic property toward reduction of H₂O₂ and trichloroacetic acid, indicating the potential application as amperometric biosensor. Published in Russian in Elektrokhimiya, 2008, Vol. 44, No. 11, pp. 1366–1376. The text was submitted by the authors in English.     

Direct electrochemistry and electrocatalytic characteristic of heme proteins immobilized in a new sol-gel polymer film

Heme proteins were immobilized on glass carbon electrodes by poly (N-isopropylac-yamide-co-3-methacryloxy-propyl-trimethoxysilane) (PNM) and exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about -0.35 V versus a saturated calomel electrode in pH 7.0 buffer solution, corresponding to hemeFe(III)+e-->hemeFe(II). Some electrochemical parameters were calculated by performing nonlinear regression analysis of square wave voltammetry (SWV) experimental data. The formal potential was linearly dependent on pH, indicating the electron transfer of Fe(III)/Fe(II) redox couple accompanied by the transfer of proton. Ultraviolet visible and Fourier transform infrared spectra suggested that the conformation of proteins in the PNM films retained the essential feature of its native secondary structure. Atomic force microscopy images demonstrated the existence of interaction between heme proteins and PNM. N,N-dimethylformamide (DMF) played an important role in immobilizing proteins and enhancing electron transfer between proteins and electrodes. Electrochemical catalytic reductions of hydrogen peroxide and trichloroacetic acid by proteins entrapped in PNM film were also discussed, showing the potential applicability of the film modified electrodes as a biosensor.     

Direct electrochemistry of hemoglobin immobilized in the sodium alginate and SiO2 nanoparticles bionanocomposite film on a carbon ionic liquid electrode

In this paper a carbon ionic liquid electrode (CILE) was fabricated by using a room temperature ionic liquid of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) as binder. By using the CILE as basal electrode, the hemoglobin (Hb) molecule was immobilized on the surface of CILE with a sodium alginate (SA) hydrogel and SiO₂ nanoparticles organic-inorganic composite material. The direct electrochemical behaviors of Hb in the bionanocomposite film were further studied in a pH 7.0 Britton-Robinson (B-R) buffer solution. A pair of well-defined quasi-reversible cyclic voltammetric peaks of Hb was obtained on SA/nano-SiO₂/Hb/CILE with the formal potential (E^0’) at -0.355 V (vs. SCE), which was the characteristic of heme Fe(III)/Fe(II) redox couples. The formal potential of Hb Fe(III)/Fe(II) couple shifted negatively with increasing pH of solution with a slope of -45.2 mV/pH, which indicated that a one electron transfer accompanied with one proton transportation. The immobilized Hb showed good electrocatalytic manner to the reduction of trichloroacetic acid (TCA).     

碳糊电极上无机膜固载血红蛋白的直接电化学

报道了用硅溶胶-凝胶(Sol-gel)膜将血红蛋白(Hb)固载于碳糊电极上的直接电化学行为.研究结果表明,Hb-Sol-gel修饰的碳糊电极在pH=7.0的缓冲溶液中于-0.275V(vs.Ag/AgCl)处有一对可逆的循环伏安氧化-还原峰,为Hb血红素辅基Fe(Ⅲ)/Fe(Ⅱ)电对的特征峰.HbFe(Ⅲ)/Fe(Ⅱ)电对的式量电位在pH5.0～11.0范围内与溶液pH值呈线性关系,表明Hb的电化学还原很可能是一个质子伴随着一个电子的电极过程.FTIR光谱证实,Sol-gel膜对Hb的固载没有破坏其天然结构.Hb-Sol-gel修饰的碳糊电极能够催化还原H₂O₂,可望将其用于制作第三代生物传感器.     

Studies on direct electron transfer and biocatalytic properties of hemoglobin in polyacrylonitrile matrix

The direct electrochemistry of hemoglobin (Hb) immobilized in polyacrylonitrile (PAN) modified glassy carbon electrode was described. The protein-PAN film exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks for Hb Fe(III)/Fe(II) redox couple in a pH 7.0 phosphate buffer. The formal potential of Hb heme Fe(III)/Fe(II) couple varied linearly with the increase of pH in the range of 5.0-9.0 with a slope of 54 mV pH(-1), which implied that a proton transfer was accompanied with each electron transfer in the electrochemical reaction. Position of Soret absorption band of Hb-PAN film suggested that the Hb kept its secondary structure similar to its native state in the PAN matrix. The Hb in PAN matrix acted as a biologic catalyst to catalyze the reduction of hydrogen peroxide. The electrocatalytic response showed a linear dependence on the H(2)O(2) concentration ranging from 8.3 x 10(-6) to 5 x 10(-4) mol L(-1) with a detection limit of 8.3 x 10(-6) mol L(-1) at 3 sigma. The apparent Michaelis-Menten constant K(M)(app) for H(2)O(2) sensor was estimated to be 0.9 mmol L(-1).     

Direct Electrochemistry of Hemoglobin in Chitosan/Multiwalled Carbon Nanotubes/Ionic Liquid–Modified Carbon-Paste Electrode

Abstract

The direct electrochemistry of hemoglobin (Hb) was realized on chitosan and multiwalled carbon nanotubes (MWCNTs)–modified carbon ionic liquid electrode (CILE). The CILE was fabricated first and further modified by MWCNTs to get an electrode as MWCNTs/CILE. The Hb was immobilized on the surface of MWCNTs/CILE with the help of chitosan film. Ultraviolet–visible (UV-vis) and Fourier transform–infrared (FT-IR) spectra indicated that Hb kept its native structure in the modified film. A pair of well-defined quasi-reversible redox peaks of heme Fe(III)/Fe(II) couple appeared with the formal potential (E^0′) as ?0.314 V (vs. SCE) in pH 7.0 phosphate buffer solution (PBS). The modified electrode showed good electrocatalytic ability for the reduction of trichloroacetic acid.     

Electrodeposited nanogold decorated graphene modified carbon ionic liquid electrode for the electrochemical myoglobin biosensor

In this paper, a carbon ionic liquid electrode (CILE) was fabricated using ionic liquid 1-hexylpyridinium hexafluorophosphate as modifier, which was further in situ electrodeposited with graphene (GR) and gold nanoparticles step by step to get an Au/GR nanocomposite modified CILE. Myoglobin (Mb) was further immobilized on the Au/GR/CILE surface with Nafion film to get the modified electrode denoted as Nafion/Mb/Au/GR/CILE. Cyclic voltammetric experiments indicated that a pair of well-defined quasi-reversible redox peaks appeared in pH 3.0 phosphate buffer solution with the formal potential (E ^0′) located at ?0.197 V (vs. saturated calomel electrode), which was the typical characteristics of Mb heme Fe(III)/Fe(II) redox couples. Thus, the direct electron transfer rate between Mb and the modified electrode was promoted due to the high conductivity and increased surface area of Au/GR nanocomposite present on electrode surface. Based on the cyclic voltammetric data, the electrochemical parameters of Mb on the modified electrode were calculated. The Mb-modified electrode showed excellent electrocatalytic activities towards the reduction of trichloroacetic acid and H₂O₂ with wider linear range and lower detection limit. Using GR and Au nanoparticles modified CILE, a new third-generation electrochemical Mb biosensor was constructed with good stability and reproducibility.     

Electrocatalysis of Hemoglobin in ZnO Nanoparticle/Ionic Liquid Composite Film Modified Glassy Carbon Electrode

A nanobiocompatible composite containing hemoglobin (Hb), ZnO nanoparticles (nano‐ZnO) and ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF₆) was fabricated and further modified on the glassy carbon electrode (GCE). The electrochemical behaviours of Hb in the composite film were carefully studied and a pair of quasi‐reversible redox peaks appeared in pH 7.0 phosphate buffer solution, which was attributed to the electrode reaction of Hb heme Fe(III)/Fe(II) redox couple. The presences of nano‐ZnO and BMIMPF₆ in the film can retain the bioactivity of Hb and greatly enhance the direct electron transfer of Hb. The immobilized Hb showed high stability and good electrocatalytic ability to the reduction of hydrogen peroxide and O₂.     

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.     

A hydrogen peroxide biosensor based on Langmuir-Blodgett technique: Direct electron transfer of hemoglobin in octadecylamine layer

The present paper describes the modification of hemoglobin (Hb)-octadecylamine (ODA) Langmuir-Blodgett (LB) film on a gold electrode surface to develop a novel electrochemical biosensor for the detection of hydrogen peroxide. Atomic force microscopy (AFM) image of Hb-ODA LB film indicated Hb molecules existed in ODA layer in a well-ordered and compact form. The immobilized Hb displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of 4.58 ± 0.95 s⁻¹ and a formal potential of −185 mV (versus Ag/AgCl) in phosphate buffer (1.0 mM, pH 5.0) contain 0.1 M KCl at a scan rate of 200 mV s⁻¹, characteristic of Hb heme Fe(III)/Fe(II) redox couple. The formal potential of Hb heme Fe(III)/Fe(II) redox couple in ODA film shifted linearly between pH 5 and 8 with a slope of −23.8 mV pH⁻¹, suggesting that proton took part in electrochemical reaction. The ODA could accelerate the electron transfer between Hb and the electrode. This modified electrode showed an electrochemical activity to the reduction of hydrogen peroxide (H₂O₂) without the aid of any electron mediator.     

Direct electrochemistry and electrocatalysis of myoglobin immobilized on L-cysteine self-assembled gold electrode

Myoglobin (Mb) has been successfully immobilized on a self-assembled monolayer (SAM) of L-cysteine (Cys) on a gold electrode, Au/Cys. The presence of a pair of well-defined and nearly reversible waves centered at ca. 0.086 V vs Ag/AgCl (pH 6.5) suggests that the native character of Mb heme Fe(III/II) redox couple has been obtained. The formal potential of Mb on Cys SAM exhibited pH-dependent variation in the pH range of 5-9 with a slope of 55 mV/pH, indicating that the electron transfer is accompanied by a single proton exchange. Thermodynamic and kinetic aspects of Mb adsorption processes on Au/Cys were studied by using voltammetric and quartz-crystal microbalance methods. The Au/Cys electrode with immobilized Mb exhibited electrocatalytic activity toward ascorbic acid (AA) oxidation with an overpotential decrease of over 400 mV and a linear dependence of current on the AA concentration from 0.5 to 5.0 mmol L(-1).     

血红蛋白在壳聚糖修饰碳纳米管上的电化学特性及对过氧化氢的电催化分析

用壳聚糖对多壁碳纳米管进行修饰,构建了一种用于固定血红蛋白的新型复合材料,并研究了血红蛋白在该碳纳米管上的电化学性质及其对过氧化氢的电催化活性.扫描电镜结果表明,壳聚糖修饰的多壁碳纳米管呈单一的纳米管状,并能均匀分散在玻碳电极表面.紫外光谱分析表明血红蛋白在该复合膜内能很好地保持其原有的二级结构.将该材料固定在玻碳电极上后,血红蛋白能成功地实现其直接电化学.根据峰电位差随着扫描的变化,计算得到血红蛋白在壳聚糖修饰的碳纳米管膜上的电荷转移系数为0.57,表观电子转移速率常数为7.02 s-1.同时,该电极对过氧化氢显示出良好的催化性能,电流响应信号与H2O2浓度在1.0×10-6 ～1.5×10-3 mol/L间呈线性关系,检出限为5.0×10-7 mol/L.修饰电极显示了良好的稳定性.     

Direct Electrochemistry of Hemoglobin and its Electrocatalysis Based on a Carbon Nanotube Paste Electrode

A new hemoglobin (Hb) and carbon nanotube (CNT) modified carbon paste electrode was fabricated by simply mixing the Hb, CNT with carbon powder and liquid paraffin homogeneously. To prevent the leakage of Hb from the electrode surface, a Nafion film was further applied on the surface of the Hb‐CNT composite paste electrode. The modified electrode was characterized by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Direct electrochemistry of hemoglobin in this paste electrode was easily achieved and a pair of well‐defined quasi‐reversible redox peaks of a heme Fe(III)/Fe(II) couple appeared with a formal potential (E^0′) of ?0.441 V (vs. SCE) in pH 7.0 phosphate buffer solution (PBS). The electrochemical behaviors of Hb in the composite electrode were carefully studied. The fabricated modified bioelectrode showed good electrocatalytic ability for reduction of H₂O₂ and trichloroacetic acid (TCA), which shows potential applications in third generation biosensors.     

Electrochemistry and electrocatalysis of hemoglobin in Nafion/nano-CaCO3 film on a new ionic liquid BPPF6 modified carbon paste electrode

Room temperature ionic liquid N-butylpyridinium hexafluorophosphate (BPPF6) was used as a binder to construct a new carbon ionic liquid electrode (CILE), which exhibited enhanced electrochemical behavior as compared with the traditional carbon paste electrode with paraffin. By using the CILE as the basal electrode, hemoglobin (Hb) was immobilized on the surface of the CILE with nano-CaCO3 and Nafion film step by step. The Hb molecule in the film kept its native structure and showed good electrochemical behavior. In pH 7.0 Britton-Robinson (B-R) buffer solution, a pair of well-defined, quasi-reversible cyclic voltammetric peaks appeared with cathodic and anodic peak potentials located at -0.444 and -0.285 V (vs SCE), respectively, and the formal potential (E degrees') was at -0.365 V, which was the characteristic of Hb Fe(III)/Fe(II) redox couples. The formal potential of Hb shifted linearly to the increase of buffer pH with a slope of -50.6 mV pH-1, indicating that one electron transferred was accompanied with one proton transportation. Ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopy studies showed that Hb immobilized in the Nafion/nano-CaCO3 film still remained its native arrangement. The Hb modified electrode showed an excellent electrocatalytic behavior to the reduction of H2O2, trichloroacetic acid (TCA), and NaNO2.     

Preparation,Characterization, and Electrocatalytic Properties of Cobalt Oxide and Cobalt Hexacyanoferrate Hybrid Films

Polynuclear mixed‐valent films of cobalt oxide and cobalt hexacyanoferrate (CoOCoHCF) have been deposited on electrode surfaces from a solution of Co²⁺ and Fe(CN)₆^3? ions by repetitive potential cycling method. Simultaneous cyclic voltammetry and electrochemical quartz crystal microbalance measurements demonstrate the steady growth of modified film. The effect of type of monovalent cations as well as acidity of the supporting electrolyte on film growth and redox behavior of resulting film was investigated. In pure supporting electrolyte, electrochemical responses of modified electrode resemble with that of a surface immobilized redox couple. The hybrid film electrodes showed electrocatalytic activity toward oxidation of NADH, hydrazine and hydroxylamine. The feasibility of using our modified electrodes for analytical application was also explored.     

Ionic Liquid‐Hemoglobin‐Carbon Paste Composite Bioelectrode and Its Electrocatalytic Activity

A new carbon ionic liquid paste bioelectrode was fabricated by mixing hemoglobin (Hb) with graphite powder, ionic liquid 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (EMIMBF₄) and liquid paraffin homogeneously. Nafion film was cast on the electrode surface to improve the stability of bioelectrode. Direct electrochemistry of Hb in the bioelectrode was carefully investigated. Cyclic voltammetric results indicated that a pair of well‐defined and quasi‐reversible electrochemical responses appeared in pH 7.0 phosphate buffer solution (PBS), indicating that direct electron transfer of Hb was realized in the modified electrode. The formal potential (E^0′) was calculated as ?0.316 V (vs. SCE), which was the typical characteristic of the electrochemical reaction of heme Fe(III)/Fe(II) redox couple. Based on the cyclic voltammetric results the electrochemical parameters of the electrode reaction were calculated. This bioelectrode showed high electrocatalytic activity towards the reduction of trichloroacetic acid (TCA) with good stability and reproducibility.     

Myoglobin on multi-walled carbon nanotubes modified electrode: direct electrochemistry and electrocatalysis

Electrochemical behavior of myoglobin (Myb) incorporated on multi-walled carbon nanotubes (MWNTs) modified GC electrode is investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The results indicate that Myb can be strongly adsorbed on the surface of the MWNT-modified electrode to form an approximate monolayer film. MWNTs can greatly promote the redox of horse heart Myb, on which a pair of well-defined and nearly reversible CV peaks for Myb Fe(III)/Fe(II) redox couple were obtained in pH 7.0 buffers. The formal potential of Myb on MWNT modified electrode shifts linearly with pH with a slope of −56.4 mV/pH, showing that the electron transfer is accompanied by single proton transportation. The high efficiency of the Myb/MWNT modified electrode towards the catalytic electro-reduction of nitric oxide has been observed. Potential application of Myb/MWNT modified electrode as biosensors to monitor NO is proposed.     

Electrochemistry and Electrocatalysis with Myoglobin in Biomembrane-Like DHP-PDDA Polyelectrolyte-Surfactant Complex Films

The polyelectrolyte-surfactant complex DHP-PDDA was prepared by reacting the anionic surfactant dihexadecylphosphate (DHP) with polycationic poly(diallyldimethylammonium) (PDDA). Thin films made from DHP-PDDA on solid substrates demonstrated an ordered multibilayer structure by XRD and DSC. Incorporated myoglobin (Mb) in DHP-PDDA films on pyrolytic graphite (PG) electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks for the Mb Fe(III)/Fe(II) couple at about -0.3 V vs SCE in pH 7.0 buffers. Electron transfer between Mb and PG electrodes was greatly facilitated in the film microenvironment. The positions of the Soret absorption band suggest that Mb maintains its secondary structure similar to its native state in DHP-PDDA films in the medium pH range. Mb could act as an enzyme-like catalyst in DHP-PDDA films as demonstrated by catalytic reduction of trichloroacetic acid, nitrite, and oxygen with a decrease in the electrode potentials required. Mb-DHP-PDDA films may thus have potential application as biosensors. Copyright 2001 Academic Press.     

Electroactive porous films of myoglobin within calcium alginate

In this work, a novel two-step construction strategy for protein assembly films was proposed. The first step was the preparation of porous calcium alginate (CA) films by spraying calcium chloride (CaCl₂) solution over the mixture surface of sodium alginate and polyethylene glycol on various solid substrates. The second step involved the cast of myoglobin (Mb) onto the porous CA films and then formed the electroactive porous Mb-CA films. The nitrogen adsorption desorption isotherm, scanning electron microscope, alternating current impendence and cyclic voltammetry were used to characterize the porous films. Fully hydrated porous CA films had nearly 90 wt% water contents and UV–vis showed that Mb in the porous films retained its near native conformation at medium pH. The stable films modified on glassy carbon electrode demonstrated good electroactivity in protein-free buffer, which was originated from protein heme Fe(III)/Fe(II) redox couples. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k _s) and formal potential (E ^o′) were estimated by fitting the data of square-wave voltammetry with nonlinear regression analysis. It was observed that the formal potential of the Mb Fe(III)/Fe(II) couple in porous CA films shifted linearly between pH 4.0 and 11.0 with a slope of −52.7 mV/pH, suggesting that one proton transfer was coupled to each electron transfer in the electrochemical reaction. The porous Mb-CA films showed the electrocatalytic activity toward dioxygen, hydrogen peroxide, and nitrite with significant decreases in the electrode potential required, and exhibited good operational and storage stability, reproducibility and fast response time for H₂O₂ detection. It is showing the possible future application of the films for biosensors and biocatalysis.