血红蛋白在海藻酸钠膜中的电化学和电催化行为

用海藻酸钠(SodiumAlginate,SA)将血红蛋白(Hb)固定在热裂解石墨电极表面,制备了Hb SA膜修饰电极。包埋在海藻酸钠膜中的血红蛋白与电极直接传递电子。在pH7.0的磷酸盐缓冲溶液中可得到一对可逆的血红蛋白辅基血红素Fe(Ⅲ) Fe(Ⅱ)电对氧化还原峰,式电势为-0.364V(vs.SCE)。其式电势随溶液pH值增加而负移且成线性关系,直线斜率为-36.0mV pH,说明血红蛋白的电子传递过程伴随有质子的转移。并研究了Hb SA膜修饰电极对O2、H2O2和NO的电催化性质。     

Direct electrochemistry and electrocatalysis of hemoglobin entrapped in composite matrix based on chitosan and CaCO3 nanoparticles

The direct electron transfer between hemoglobin (Hb) and the underlying glassy carbon electrode (GCE) can be readily achieved via a high biocompatible composite system based on biopolymer chitosan (CHT) and inorganic CaCO₃ nanoparticles (nano-CaCO₃). Cyclic voltammetry of Hb-CHT/nano-CaCO₃/GCE showed a pair of stable and quasi-reversible peaks for HbFe(III)/Fe(II) redox couple in pH 7.0 buffer. The electrochemical reaction of Hb immobilized in CHT/nano-CaCO₃ composite matrix exhibited a surface-controlled process accompanied by electron and proton transfer. The electron transfer rate constant was estimated to be 1.8 s⁻¹. This modified electrode showed a high thermal stability up to 60 °C. The apparent Michaelis–Menten constant was calculated to be 7.5 × 10⁻⁴ M, indicating a high catalytic activity of the immobilized Hb toward H₂O₂. The interaction between Hb and this nano-hybrid material was also investigated using FT-IR and UV–vis spectroscopy, indicating that Hb retained its native structure in this hybrid matrix.     

肌红蛋白在海藻酸钠水凝胶中的电化学和电催化特性

海藻酸钠(Sodium Alginate,SA)是由L-葡萄糖醛酸与D-甘露糖醛酸组成的高分子线性糖醛酸,常作为固定化酶包埋材料。本文研究了海藻酸钠水凝胶膜中的肌红蛋白在磷酸盐缓冲溶液中的直接电化学和酶催化性质,探讨了测定H2O2和NO2^-的可能性。     

Direct electrochemistry and electrocatalysis of heme proteins entrapped in agarose hydrogel films in room-temperature ionic liquids

The electrochemistry and electrocatalysis of a number of heme proteins entrapped in agarose hydrogel films in the room-temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) have been investigated. UV-vis and FTIR spectroscopy show that the heme proteins retain their native structure in agarose film. The uniform distribution of hemoglobin in agarose-dimethylformamide film was demonstrated by atomic force microscopy. Cyclic voltammetry shows that direct electron transfer between the heme proteins and glassy carbon electrode is quasi-reversible in [bmim][PF(6)]. The redox potentials for hemoglobin, myoglobin, horseradish peroxidase, cytochrome c, and catalase were found to be more negative than those in aqueous solution. The charge-transfer coefficient and the apparent electron-transfer rate constant for these heme proteins in [bmim][PF(6)] were calculated from the peak-to-peak separation as a function of scan rate. The heme proteins catalyze the electroreduction of trichloroacetic acid and tert-butyl hydroperoxide in [bmim][PF(6)]. The kinetic parameter I(max) (maximum current at saturation concentration of substrate) and the apparent K(m) (Michaelis-Menten constant) for the electrocatalytic reactions were evaluated.     

琼脂糖水凝胶固定化血红素蛋白质的电化学研究

利用琼脂糖（agarose）水凝胶将肌红蛋白（Mb）、血红蛋白（Hb）、辣根过氧化物酶（HRP）和过氧化氢酶（Cat）4种血红素蛋白质固定在裂解石墨电极表面,形成稳定的血红素蛋白质-agarose膜修饰电极。在agarose膜中,Mb、Hb、HRP和Cat直接与电极传递电子。4种血红素蛋白质的式电势都随溶液pH的增加而负移且呈线性关系,表明电子传递过程伴随着质子转移。     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized by methacrylic acid

The direct electrochemistry of hemoglobin (Hb) incorporated in methacrylic acid (MAA) film on a paraffin-impregnated graphite electrode (PIGE) was described. A pair of well-defined and quasi-reversible cyclic voltametric peaks are obtained. The formal potentials (E ^0′) linearly depend on the pH of solution, indicating that the electron transfer was proton-coupled. Ultraviolet-visible (UV-Vis) spectra showed that the secondary structure of Hb in the MAA film was similar to individual Hb. The immobilized Hb retained its biological activity well and exhibited a nice response to the reduction of both NO₂⁻, and H₂O₂, on the basis of which a new biosensor has been developed. Published in Russian in Elektrokhimiya, 2008, Vol. 44, No. 9, pp. 1079–1086. The text was submitted by the authors in English.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized in bimodal mesoporous silica and chitosan inorganic–organic hybrid film

Hemoglobin modified electrode was successfully fabricated to realize direct electrochemistry by immobilizing of Hemoglobin (Hb) in bimodal mesoporous silica (BMS) and chitosan (CS) inorganic–organic hybrid film. Here, BMS acted as a support to immobilize Hb due to its large pores and CS acted as a binder to increase film adherence and stabilizer to prevent the leakage of Hb. The resulting electrode (Hb/BMS/CS) gave a well-defined, reversible redox couple for HbFe(III)/Fe(II) with a formal potential of about −0.32 V (vs. Ag/AgCl) in pH 7.0 phosphate buffer solution. Hb/BMS/CS electrode showed a better electrocatalytial performance to H₂O₂ with wider linear detection range, lower detection limit, and higher sensitivity than that at electrode without BMS. The improved electrocatalytic performance for Hb/BMS/CS electrode was possibly contributed to BMS bimodal structure, whose large pores with 10–40 nm provide favorable conditions for protein immobilization and small pores with 2–3 nm avoid the mass-transfer limitations. In addition, UV–Vis and FTIR spectra indicated that Hb well maintained its native structure in the hybrid film.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized in TiO2 nanotube films

Titanium oxide nanotubes (TiO₂-NTs) synthesized by the hydrothermal method had been prepared as the co-immobilization matrix to incorporate hemoglobin (Hb) successfully. The nanostructures of TiO₂-NTs were investigated by X-ray diffraction and high-resolution electron microscopy. The Hb immobilized in TiO₂-NTs had a similar structure to the native of Hb and retained its near-native conformations as characterized by the UV–vis and FT-IR spectroscopy. A couple of quasi-reversible redox peaks with a formal potential of −0.34 V (vs. SCE) in 0.10 M pH 7.0 phosphate buffered saline (PBS) were observed. The amperometric response of the immobilized Hb linearly to H₂O₂ concentration ranged from 4 μM to 64 μM with a detection limit of 4.637 × 10⁻⁶ M and the high stability of the immobilized Hb in TiO₂-NTs constituted a promising platform for the development of biosensors.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized in a magnetic nanoparticles-chitosan film

Magnetic nanoparticles (Fe₃O₄) were synthesized by a chemical coprecipitation method. X-ray diffraction (XRD) and transmission electron microscope (TEM) were used to confirm the crystallite structure and the particle's radius. The Fe₃O₄ nanoparticles and chitosan (CS) were mixed to form a matrix in which haemoglobin (Hb) can be immobilized for the fabrication of H₂O₂ biosensor. The Fe₃O₄-CS-Hb film exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks due to the redox of Hb-heme Fe (III)/Fe (II) 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 4.0-10.0 with a slope of 46.5 mV pH⁻¹, indicating that electron transfer was accompanied with single proton transportation in the electrochemical reaction. The surface coverage of Hb immobilized on Fe₃O₄-CS film glassy carbon electrode was about 1.13 × 10⁻¹⁰ mol cm⁻². The heterogeneous electron transfer rate constant (k_s) was 1.04 s⁻¹, indicating great facilitation of the electron transfer between Hb and magnetic nanoparticles-chitosan modified electrode. The modified electrode showed excellent electrocatalytic activity toward oxygen and hydrogen peroxide reduction. The apparent Michaelis-Menten constant for H₂O₂ was estimated to be 38.1 μmol L⁻¹.     

Direct electrochemistry and electrocatalysis of nitrite based on nano-alumina-modified electrode

Simple and sensitive electrochemical method for the determination of nitrite, based on a nano-alumina-modified glassy carbon electrode (GCE), is described. Nitrite yields a well-defined oxidation peak whose potential is 0.74 V at the nano-alumina-coated GCE in 0.1 mol L⁻¹ phosphate buffer (pH 5.0). Compared with bare GCE, the nano-alumina-modified GCE has evident catalytic effect towards the oxidation of nitrite, and its peak current can be significantly enhanced. Some of the experimental parameters were optimized for the determination of nitrite. The oxidation peak current was proportional to nitrite concentration in the range of 5.0 × 10⁻⁸–1.1 × 10⁻³ mol L⁻¹, and a detection limit of 1.0 × 10⁻⁸ mol L⁻¹ was obtained. This method has been successfully used to the determination of nitrite in sausage sample. Furthermore, results obtained by the method have been compared with spectrophotometric method.     

Direct electrochemistry and electrocatalysis of hemoglobin in gelatine film modified glassy carbon electrode

Direct electrochemical and electrocatalytic behavior of hemoglobin (Hb) immobilized on glass carbon electrode (GCE) containing gelatine (Gel) films was investigated. The characteristics of Hb/Gel film modified GC electrode were performed by using SEM microscopy, UV-vis spectroscopy and electrochemical methods. The immobilized Hb showed a couple of quasi-reversible redox peak with a formal potential of −0.38 V (versus SCE) in 0.1 M pH 7.0 PBS. The formal potential changed linearly from pH 4.03 to 8.41 with a slope value of −52.0 mV pH⁻¹, which suggested that a proton transfer was accompanied with each electron transfer (ET) in the electrochemical reaction. The Hb/gelatine/GCE displayed a rapid amperometric response to the reduction of H₂O₂ and nitrite.     

Direct electrochemistry and electrocatalysis of hemoglobin on gold nanoparticle decorated carbon ionic liquid electrode

In this paper a carbon ionic liquid electrode (CILE) was fabricated by using ionic liquid 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM]EtOSO₃) as modifier and further gold nanoparticles were in situ electrodeposited on the surface of CILE. The fabricated Au/CILE was used as a new platform for the immobilization of hemoglobin (Hb) with the help of a Nafion film. Electrochemical experimental results indicated that direct electron transfer of Hb was realized on the surface of Au/CILE with a pair of well-defined quasi-reversible redox peaks appeared. The formal peak potential (E⁰′) was obtained as −0.210 V (vs. SCE) in pH 7.0 phosphate buffer solution (PBS), which was the characteristic of Hb heme Fe(III)/Fe(II) redox couple. The fabricated Nafion/Hb/Au/CILE showed excellent electrocatalytic activity to the reduction of trichloroacetic acid (TCA) and the reduction peak current was in proportional to TCA concentration in the range from 0.2 to 18.0 mmol/L with the detection limit as 0.16 mmol/L (S/N = 3). The proposed electrode showed good stability and reproducibility, and it had the potential application as a new third-generation electrochemical biosensor.     

Direct electrochemistry and electrocatalysis of hemoglobin on undoped nanocrystalline diamond modified glassy carbon electrode

Direct electrochemistry of hemoglobin (Hb) was observed at glassy carbon electrode (GCE) modified with undoped nanocrystalline diamond (UND) and Hb multilayer films via layer-by-layer assembly. UV-VIS absorbance spectroscopy, electrochemical impedance spectroscopy and cyclic voltammograms were employed to characterize the film. The results showed that the UND had the effect of enhancing the electron transfer between Hb and the electrode surface. Hb in the multilayer films maintained its bioactivity and structure. It also exhibited a good catalytic activity towards the reduction of H(2)O(2). The reciprocal of catalytic current showed a linear dependence on the reciprocal of H(2)O(2) concentration ranging from 0.5 microM to 0.25 mM with a detection limit of 0.4 microM. The apparent Michaelis-Menten constant was estimated to be 0.019 mM.     

Direct electrochemistry of cytochrome c entrapped in agarose hydrogel in room temperature ionic liquids

Direct electrochemistry of cytochrome c (cyt-c) entrapped in agarose hydrogel on gold electrode (Au), edge plane pyrolytic graphite electrode (EPPGE) and glassy carbon electrode (GC) in two room temperature ionic liquids was investigated. The effects of the addition of N,N-dimethylformamide (DMF) in the agarose-cyt-c film, water concentration in ionic liquids and exterior metal ions on the electrochemical behavior of cyt-c were monitored, and electrocatalytic properties of cyt-c were also done. Results showed that a good quasi-reversible redox behavior of cyt-c could be found after adding DMF in agarose-cyt-c film, and peak shape would not change after continuously scanning for 50 cycles. In addition, a certain amount of water in hydrophilic ionic liquids is necessary to maintain electrochemical activities of cyt-c, electrochemical performance of cyt-c is the best when the water content is 5.2% and 5.8% for 1-butyl-3-methylimidazolium bromide ([Bmim][Br]) and 1-butyl-3-methylimidazolium tetrafluoroborate([Bmim][BF(4)]) respectively. However, electrochemical activities of cyt-c are inhibited by exterior metal ions. Interestingly, cyt-c entrapped in agarose hydrogel on EPPGE and GC could catalyze the electroreduction of trichloroacetic acid (TCA) and tert-butyl hydroperoxide (t-BuOOH) in [Bmim][BF(4)], but could not in [Bmim][Br]. Reasons for above-mentioned differences of electrochemical properties of cyt-c in different ionic liquids were preliminarily discussed.     

Direct electrochemistry and electrocatalysis with hemoglobin in water-soluble quantum dots film on glassy carbon electrode

The direct electrochemistry of hemoglobin can be performed by immobilizing hemoglobin in a water-soluble quantum dots (CdSe-ZnS) film on glassy carbon electrode.     

Direct electrochemistry and electrocatalysis of hemoglobin on a gold ion implantation-modified indium tin oxide electrode

In this paper, a gold nanoparticle-modified indium tin oxide electrode (Au/ITO) was prepared without the use of any cross-linker or stabilizer reagent. The prepared Au/ITO was used as a new platform to achieve the direct electron transfer between Hb and the modified electrode. The proposed electrode exhibited a pair of well-defined redox peaks with a formal potential of ?0.073 V (vs. Ag/AgCl). The immobilized Hb showed excellent electrocatalytic activity toward H₂O₂ and the electrocatalytic current values were linear with the increasing concentration of H₂O₂ ranging from 1.0?×?10^?6?M to 7.0?×?10^?4?M. The detection limit was 2.0?×?10^?7?M (S/N?=?3) and the Michaelis–Menten constant was calculated to be 0.2 mM. The proposed electrode also showed high selectivity, long-term stability, and good reproducibility.     

Direct electrochemistry and enhanced electrocatalytic activity of hemoglobin entrapped in graphene and ZnO nanosphere composite film

A biocomposite film for sensing hydrogen peroxide (HP) is described that is based on nanospheres made from hemoglobin (Hb), graphene, and zinc oxide. The composition, morphology and size of the film were studied by transmission electron microscopy. UV-vis spectroscopy revealed that the Hb entrapped in the graphene and ZnO nanosphere retains its native structure. A pair of stable and well-defined quasi-reversible redox peaks of Hb was obtained, with a formal potential of ?30 mV at pH 6.5. Hb exhibits excellent long-term bioelectrocatalytic activity towards HP. The apparent heterogeneous electron transfer rate constant is 1.0 s^?1, indicating that the presence of graphene in the composite film facilitates the electron transfer between matrix and the electroactive center of Hb. The sensor responds linearly to HP in the range from 1.8 μM to 2.3 mM, with a detection limit of 0.6 μM (at S/N?=?3). The apparent Michaelis-Menten constant is 1.46 mM. The biosensor displays high sensitivity, good reproducibility, and long-term stability.

Direct electrochemistry and electrocatalysis of heme-protein based on N,N-dimethylformamide film electrode

The heme-protein including myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP) were immobilized on normal graphite electrode by using N,N-dimethylformamide (DMF). The proteins undergo direct electron-transfer reactions. The current is linearly dependent on the scan rate, indicating that the direct electrochemistry of heme-protein in that case is a surface-controlled electrode process. The E°s are linearly dependent on solution pH (redox-Bohr effect), indicating that the electron transfer was proton-coupled. Ultraviolet-visible (UV-vis) and reflection-absorption infrared (RAIR) spectra suggest that the conformation of proteins in the presence of DMF are little different from that proteins alone the conformation changes reversibly in the range of pH 3.0-10.0. The catalytic activity of proteins were examined by hydrogen peroxide and nitrite.     

Direct electrochemistry and electrocatalysis of hemoglobin on an indium tin oxide electrode modified with implanted carboxy ions

A flow injection on-line preconcentration system was developed for the determination of lead by hydride generation atomic fluorescence spectrometry (HG-AFS). It is based on a simple micro-column filled with multiwalled carbon nanotubes (MWCNTs). The preconcentration of lead on the MWCNTs was carried out based on the adsorptive retention of analyte via on-line introducing the sample into the micro-column system. A 0.3 mol L^?1 HNO₃ was introduced to elute the retained analyte and merged with KBH₄ solution for HG-AFS detection. Under the optimal experimental conditions, an enhancement factor of 26 was obtained with a sample consumption of 14.4 mL. The limit of detection was 2.8 ng L^?1 and the precision (RSD) of 11 replicate measurements of 0.2?μg L^?1 Pb was 4.4%. The method was validated by analyzing three certified reference materials, and was successfully applied to the determination of trace lead in natural water samples.