Direct electron transfer and bioelectrocatalysis of hemoglobin on nano-structural attapulgite clay-modified glassy carbon electrode

Direct electrochemistry of hemoglobin (Hb) on natural nano-structural attapulgite clay film-modified glassy carbon (GC) electrode was investigated. The interaction between Hb and attapulgite was examined using UV-vis, FTIR spectroscopy, and electrochemical methods. The immobilized Hb displayed a couple of well-defined and quasi-reversible redox peaks with the formal potential (E(0')) of about -0.366 V (versus SCE) in 0.1 M phosphate buffer solution of pH 7.0. The current was linearly dependent on the scan rate, indicating that the direct electrochemistry of Hb in that case was a surface-controlled electrode process. The formal potential changed linearly from pH 5.0 to 9.0 with a slope value of -48.2 mV/pH, which suggested that a proton transfer was accompanied with each electron transfer in the electrochemical reaction. The immobilized Hb exhibited excellent electrocatalytic activity for the reduction of hydrogen peroxide without the aid of an electron mediator. The electrocatalytic response showed a linear dependence on the H(2)O(2) concentration ranging from 5.4 x 10(-6) to 4.0 x 10(-4) M with the detection of 2.4 x 10(-6) M at a signal-to-noise ratio of 3. The apparent Michaelis-Menten constant K(M)(app) for the H(2)O(2) sensor was estimated to be 490 microM, showing a high affinity.     

Electrocatalysis of oxygen at hemoglobin-Au colloid-1,4-benzenedimethanethiol modified electrode

A novel renewable O2 sensor based on the direct electron transfer of hemoglobin (Hb) is proposed. Hb was immobilized on a gold nanoparticles (GNP) associated with a 1,4-benzenedimethanethiol (BDT) monolayer which were modified the electrode. The direct electrochemistry of Hb was investigated by electrochemical methods and cyclic voltammetric showing a pair of redox peaks of Hb. The high efficiency of the Hb/GNP/BDT modified gold electrode towards the catalytic electro-reduction of oxygen has been observed and the potential application of Hb/GNP/BDT modified gold electrode as biosensors to monitor O2 is proposed. The electrocatalytic response showed a linear dependence on the O2 concentration ranging from 2.0 to 40.0 micromol/L.     

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.     

金复合介孔SBA-15吸附血红蛋白在H2O2电催化反应中的应用

以P123嵌段共聚物表面活性剂为模板剂制备介孔氧化硅SBA-15,并用沉积-沉淀(DP)法在SBA-15介孔表面负载纳米Au颗粒制备得到金复合介孔SBA-15材料(Au-SBA-15).再以Au-SBA-15材料制备玻碳修饰电极,将血红蛋白固定于修饰电极上用循环伏安法考察其对不同浓度H₂O₂溶液的电催化反应.在固定了血红蛋白的Hb/Au-SBA-15/GC修饰电极上,H₂O₂在+0.95 V处出现了氧化峰,且随着H₂O₂浓度的增大峰电流不断增加,说明金复合介孔氧化硅材料具有良好的生物兼容性,有利于血红蛋白的固定,其修饰电极对H₂O₂溶液具有一定的电催化作用.     

Hemoglobin on phosphonic acid terminated self-assembled monolayers at a gold electrode: immobilization, direct electrochemistry, and electrocatalysis

Phosphonic acid (--PO(3)H(2)) terminated self-assembled monolayers (SAMs) on a gold surface were used as a functional interface to immobilize hemoglobin (Hb). In situ surface-enhanced infrared absorption spectroscopy (SEIRAS) measurements show that Hb immobilization is a sluggish process due to formation of multilayer Hb structures on the PO(3)H(2)-terminated SAMs, as revealed by ellipsometry, atomic force microscopy (AFM), and cyclic voltammetry (CV). In the multilayered Hb film, the innermost Hb molecules can directly exchange electrons with the electrode, whereas Hb beyond this layer communicates electronically with the electrode via protein-protein electron exchange. In addition, electrochemical measurements indicate that immobilization of Hb on the PO(3)H(2)-terminated SAMs is not driven by the electrostatic interaction, but likely by hydrogen-bonding interaction. The immobilized Hb molecules show excellent bioelectrocatalytic activity towards hydrogen peroxide, that is, the PO(3)H(2)-terminated SAMs are promising for construction of third-generation biosensors.     

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.     

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).     

Pt/十六烷基三甲基溴化铵纳米聚集体修饰玻碳电极促进析氢(英文)

A novel, cost‐effective, and simple electrocatalyst based on a Pt‐modified glassy carbon electrode (GCE), using cetyltrimethylammonium bromide (CTAB) as a cationic surfactant, is reported. Am‐phiphilic CTAB molecules were adsorbed on GCE by immersion in a CTAB solution. The positively charged hydrophilic layer, which consisted of small aggregates of average size less than 100 nm, was used for accumulation and complexation of [PtCl6]2? anions by immersing the electrode in K2PtCl6 solution. The modified electrode was characterized using scanning electron microscopy, energy‐dispersive X‐ray spectroscopy, impedance spectroscopy, and electrochemical methods. The electrocatalytic activity of the Pt particles in the hydrogen evolution reaction (HER) was investigat‐ed. The results show that the CTAB surfactant enhances the electrocatalytic activity of the Pt parti‐cles in the HER in acidic solution.     

基于聚亚甲基蓝-碳纳米管复合膜固载血红蛋白的H2O2生物传感器

采用吸附和电化学聚合修饰方法,制得了聚亚甲基蓝-碳纳米管聚合膜玻碳电极(PMB-MWNTs/GCE),再将血红蛋白(Hb)固定在PMB-MwNTs/GCE表面,制备了稳定的Hb/PMB-MwNTs//GCE的H2O2生物传感器,并用循环伏安法对修饰电极的生物电催化行为进行了表征.研究结果表明,固定在PMB-MWNTs/...     

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.     

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.     

Effects of a Surfactant on the Electrocatalytic Activity of Cobalt Hexacyanoferrate Modified Glassy Carbon Electrode Towards the Oxidation of Dopamine

The cobalt hexacyanoferrate film (CoHCF) was deposited on the surface of a glassy carbon (GC) electrode with a potential cycling procedure in the presence and absence of the cationic surfactant, cetyl trimethylammonium bromide (CTAB), to form CoHCF modified GC (CoHCF/GC) electrode. It was found that CTAB would affect the growth of the CoHCF film, the electrochemical behavior of the CoHCF film and the electrocatalytic activity of the CoHCF/GC electrode towards the electrochemical oxidation of dopamine (DA). The reasons of the electrochemical behavior of CoHCF/GC electrode influenced by CTAB were investigated using FTIR and scanning electron microscope (SEM) techniques. The apparent rate constant of electrocatalytic oxidation of DA catalyzed by CoHCF was determined using the rotating disk electrode measurements.     

Single-chain surfactant monolayer on carbon paste electrode and its application for the studies on the direct electron transfer of hemoglobin

A variety of single-chain surfactants with different charge properties and tail lengths can spontaneously adsorb on the hydrophobic surface of carbon paste electrode and form stable monolayers on the electrode surface. Hemoglobin (Hb) was successfully immobilized on these surfactant monolayers to form stable protein-surfactant composite films regardless of the charge and the tail length of surfactants. The resulting surface-confined Hb exhibited well-defined direct electron-transfer behaviors in all positively, neutrally and negatively charged surfactant films, suggesting the important role of hydrophobic interactions in the adsorption of Hb on surfactant films. When the density of surfactant monolayers was controlled to be the same, Hb was found to possess a better direct electron-transfer behavior on monolayers of cationic surfactants with a longer tail length. This, in combination with the tunneling effect in the direct electron transfer of Hb on surfactant films, demonstrated that the adsorption of Hb on surfactant monolayers may be mainly achieved by the partial intercalation of Hb in the loose structures of surfactant films through hydrophobic interactions between the alkane chains of surfactants and the hydrophobic regions of Hb. The native conformation of Hb adsorbed on these surfactant films was proved to be unchanged, reflected by the unaltered ultraviolet-visible (UV-vis) and reflection-absorption infrared (RAIR) spectra, and by the catalytic activity toward hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) in comparison with the free Hb molecules.     

Effects of surfactants on the electroreduction of dioxygen at an acetylene black electrode.

An acetylene black electrode modified by an adsorbed cationic surfactant, cetyltrimethylammonium bromide (CTAB), was developed. The influences of various types of surfactants on the electroreduction of O2 were investigated. It was demonstrated that a cationic surfactant, CTAB, on the surface of the electrode could significantly decrease the overpotential of dioxygen reduction, and the reduction peak current of O2 was also remarkably increased. The reduction of O2 at a CTAB modified acetylene black electrode was studied using cyclic voltammetry, chronocoulometry and controlled potential coulometry. The total number of electrons, the exchange current density (j(o)) and the transfer coefficient (alpha) for reduction of O2 at the surface of this modified electrode were determined.     

Direct probing of the folding/unfolding event of bovine hemoglobin at montmorillonite clay modified electrode by adsorptive-transfer voltammetry

An efficient way was proposed for probing the folding/unfolding event of bovine hemoglobin (Hb) through adsorptive-transfer voltammetry. Hb molecules in native and pre-unfolded in different urea conditions for 23 h were adsorbed onto the montmorillonite clay modified glassy carbon electrode (Hb/clay/GCE and uHb/clay/GCE, respectively). Cyclic voltammograms of Hb/clay/GCE and uHb/clay/GCE showed that the unfolding of Hb caused great change in the direct electron transfer between the heme irons within Hb and electrode surface, which was facilitated on clay film. From the amount of the electroactive Hb (W_Hbe) and the adsorbed Hb (Γ) on clay per unit mass, the minimal electroactive portion (MEP) of the adsorbed Hb was calculated to assess the unfolding state of Hb. With the increase of urea concentration, MEP showed a sigmoid curve. Thermodynamic parameters related to the unfolding event of Hb were also obtained based on the linear free energy model (LEM), including the free energy of folding in water (ΔG_U^water), the slope of the Santoro-Bolen equation (m), and the urea concentration required in for achieving half of the total change (S_m) in the unfolding curves. This work gave the first try for investigating protein unfolding at nano-materials modified electrode using adsorptive-transfer voltammetry, which improved the sensitivity of analysis and avoided the disadvantages involved in the existing electrochemical methods for protein unfolding. The proposed method will benefit the electrochemical studies of protein.     

ZnO-MWCNTs/Nafion Inorganic-organic composite film： Preparation and application in bioelectrochemistry of hemoglobin

Multi-walled carbon nanotubes(MWCNTs) were coated with ZnO by a hydrothermal method.The resulting nanocomposites were mixed with the Nafion solution to form a composite matrix for the fabrication of hemoglobin(Hb) biosensor.To prevent the leak of Hb molecules of the biosensor,silica sol-gel film was coated on the surface of the Hb/ZnO-MWCNTs/Nafion electrode.The silica sol-gel/Hb/ZnO-MWCNTs/Nafion film exhibited a pair of well-defined,quasi-reversible redox peaks.This biosensor showed excellent electroca...     

Diazonium-functionalized horseradish peroxidase immobilized via addressable electrodeposition: direct electron transfer and electrochemical detection

A simple one-step procedure is introduced for the preparation of diazonium-enzyme adducts. The direct electrically addressable deposition of diazonium-modified enzymes is examined for electrochemical sensor applications. The deposition of diazonium-horseradish peroxidase leads to the direct electron transfer between the enzyme and electrode exhibiting a heterogeneous rate constant, ks, of 10.3 +/- 0.7 s-1 and a DeltaEp of 8 mV (v = 150 mV/s). The large ks and low DeltaEp are attributed to the intimate contact between enzyme and electrode attached by one to three phenyl molecules. Such an electrode shows high nonmediated catalytic activity toward H2O2 reduction. Future generations of arrayed electrochemical sensors and studies of direct electron transfer of enzymes can benefit from protein electrodes prepared by this method.     

介孔分子筛上的蛋白质直接电化学

将介孔分子筛用于不同含血红素蛋白质的直接电子传递研究,分别研究了辣根过氧化物酶、血红蛋白和肌红蛋白在六方介孔硅(HMS)上的直接电化学,探讨了介孔分子筛与这些蛋白质间的相互作用,构建了过氧化氢和亚硝酸根的新型的生物传感器. 这些工作扩展了HMS在蛋白质固定、直接电子传递研究和无试剂生物传感器制备方面的应用.     

A hydrogen peroxide biosensor based on the direct electron transfer of hemoglobin in the nanosheets of exfoliated HNb3O8

In the present work, hemoglobin (Hb) was entrapped into the nanosheets of a pre-exfoliated layered material HNb₃O₈. UV–vis spectra analysis displayed that no significant denaturation occurred to the entrapped protein. Electrochemical results showed that the entrapment of Hb into layered HNb₃O₈ enhanced the direct electron transfer ability between protein molecules and electrode. A pair of well-defined redox peaks was observed at ?0.39 and ?0.34 V on the glassy carbon electrode modified with the Hb/HNb₃O₈ composite. The electrode reactions showed a surface-controlled process with a single electron transfer at the scan rate of 50–400 mV/s, and the electron transfer rate was very fast. The entrapped Hb retained its biological activity well and the sensor constructed by the Hb/HNb₃O₈-composite-modified electrode displayed excellent response to the reduction of hydrogen peroxide (H₂O₂) with wide linear range, low detection limit, and good stability.     

Carboxyphenyl Covalent Immobilization of Heme Proteins and its Favorable Biocompatible Electrochemical and Electrocatalytic Characteristics

A novel carboxyphenyl covalent immobilization technique has been successfully developed to realize the effective attachment of two typical heme proteins, hemoglobin (Hb) and cytochrome c (Cyt‐c), onto underlying glassy carbon electrode (GCE). Primarily, the GCE surface is functionalized with aromatic 4‐carboxyphenyl (4‐CP) group by the electrochemical reduction of diazonium cations, producing covalently linked carboxyl‐terminated active GCE surface to work as a ‘bridge’. Then, Hb and Cyt‐c are readily attached to GCE through the ‘bridge’ by functional covalently combination between ? NH₂ terminal groups of proteins and ? COOH terminal groups of 4‐CP, obtaining Hb/4‐CP/GCE and Cyt‐c/4‐CP/GCE. On both electrodes, well‐defined peaks attributing to the Fe^III/Fe^II couple of heme group of Hb and Cyt‐c are clearly observed with the electron transfer rate constant (k_s) evaluated to be 2.48±0.05 s^?1 and 2.73±0.05 s^?1, respectively. It is attractive that the formal potential (E°') of the immobilized Hb and Cyt‐c are estimated to be 50 and 100 mV (vs. SCE), respectively, which are closer to the standard redox potential of native Hb and Cyt‐c in solution, owing to the good biocompatibility of 4‐CP groups. The electrodes also exhibit fast response, high sensitivity and well stability for the amperometric detection of H₂O₂ at a fairly mild potential of 0 V without any mediators, obtaining rather small apparent Michaelis‐Menten constant (K_M^app) values of 113 μM for Hb/4‐CP/GCE and 101 μM for Cyt‐c/4‐CP/GCE. All the experimental results indicated that the covalent graft 4‐carboxyphenyl group plays an important role in constructing a “biocompatible bridge” to help the direct electron transfer of Hb and Cyt‐c with favorable biocompatibility and good bio‐ electrocatalytic affinity in virtue of the substituted aryl group only consisting of C, H and O elements, which is similar with the constitutes of organics. It makes the system of functionalized covalent immobilization of proteins onto carbon electrode a promising platform for fabricating the third‐generation biosensors. A new approach for realizing direct electrochemistry of proteins, as well as design of novel bioelectronic devices has been accordingly provided.