聚邻苯二胺膜电极中辣根过氧化物酶的电子传递

利用酸的电化学固定法制备含辣根过氧化物酶的聚邻苯二胺膜电极，研究其伏安行为及对Ｈ２Ｏ２还原的生物电催化作用，结果表明，在所述生物电催化反应中酶与聚合物基质 直接电子传递，但对新制的酶电极而言，电聚合时生成并包埋在酶膜中的寡聚体可作为电子传递体加速氧化态酶的再生，根据酶电极电流响应实验曲线的拟合，发现经态酶的再生速度随是极电位的变化表观上符合Ｔａｆｅｌ关系式，提出了酶反应学参数的测定方法。     

固定化辣根过氧化物酶在有机/水混合溶液中的电化学

用魔芋多糖(KGM)将辣根过氧化物酶(HRP)固定在玻碳电极(GCE)表面, 制备了HRP-KGM膜修饰电极. 在乙醇等亲水性有机溶剂与水的混合溶液中, 包埋在KGM中的HRP 可以与电极发生直接电子传递, 且能催化还原过氧化氢、氢过氧化异丙基苯、氢过氧化叔丁基、过氧化丁酮等过氧化物. HRP-KGM膜修饰电极具有较好的稳定性和重现性, 可用于这些物质的定量检测.     

Mesoporous Materials Promoting Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase

The direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) immobilized on hexagonal mesoporous silicas (HMS) matrix was studied. The interaction between HRP and HMS was examined by using Fourier transform infrared spectroscopy, nitrogen adsorption isotherms and electrochemical methods. The immobilized HRP at a modified glassy carbon electrode showed a good direct electrochemical behavior, which depended on the specific properties of the HMS. Two couples of redox peaks corresponding to Fe(III) to Fe(II) conversion of the HRP intercalated in the mesopores and adsorbed on the external surface of the HMS were observed with the formal potentials of ?0.315 and ?0.161 V in 0.1 M pH 7.0 PBS, respectively. The amount of HRP intercalated in the mesopores of HMS proved to be related to the pore size. The HRP intercalated in the mesopores showed a surface controlled electrode process with a single proton transfer. The immobilized HRP displayed an excellent electrocatalytic response to the reduction of hydrogen peroxide (H₂O₂) without the aid of an electron mediator. The HMS provided a novel matrix for protein immobilization and direct electron transfer study of the immobilized protein.     

Direct Electrochemistry and Electrocatalysis of Myoglobin Immobilized on Graphene‐CTAB‐Ionic Liquid Nanocomposite Film

This paper describes the direct electrochemistry and electrocatalysis of myoglobin immobilized on graphene‐cetylramethylammonium bromide (CTAB)‐ionic liquid nanocomposite film on a glassy carbon electrode. The nanocomposite was characterized by transmission electron microscopy, scanning electron microscopy, X‐ray photoelectron spectroscopy, and electrochemistry. It was found that the high surface area of graphene was helpful for immobilizing more proteins and the nanocomposite film could provide a favorable microenvironment for MB to retain its native structure and activity and to achieve reversible direct electron transfer reaction at an electrode. The ionic liquid may play dual roles here: it keeps the protein's activity and improves stability of the nanocomposite film; it also serves as a binder between protein and electrode, therefore, enhancing the electron transfer between the protein and the electrode. The nanocomposite films also exhibit good stability and catalytic activities for the electrocatalytic reduction of H₂O₂.     

辣根过氧化物酶在表面活性剂膜中的直接电化学

利用3种表面活性剂分别将辣根过氧化氢酶固定在裂解石墨棱面（edge-plane pyrolytic graphite,EPG)电极表面,研究了辣根过氧化物酶（HRP）中Fe(Ⅲ）／Fe（Ⅱ）电对与电极之间的直接电子传递过程以及酶催化双氧化还原过程。实验结果表明：（1）表面活性剂是一种固定酶的理想材料;（2）这种体系可能构造第三代生物传感器,对解释生物体代谢过程具有理论意义,对制备第三代生物传感器具有应用价值。     

Direct Electrochemistry of Horseradish Peroxidase Immobilized in Calcium Carbonate Microsphere Doped with Phospholipids

Protein electrochemistry affords a direct method to study the biological electron transfer processes. However, supplying a biocompatible environment to maintain the native state of protein is all‐important and challengeable. Here, we chose vaterite, one of the crystalline polymorphs of calcium carbonate, with highly porous nature and large specific surface area, which was doped with phospholipids, as the matrix to immobilize horseradish peroxidase (HRP). The integrity of HRP was kept during the simple immobilization procedure. By virtue of this organic/inorganic complex matrix, the direct electrochemistry of HRP was realized, and the activity of HRP for catalyzing reduction of O₂ and H₂O₂ was preserved.     

Direct Electrochemistry of Hemoglobin in Layer‐by‐Layer Films Assembled with DNA and Room Temperature Ionic Liquid

Based on electrostatic interaction and electrodeposition, poly‐anionic deoxyribonucleic acid (DNA), room temperature ionic liquid 1‐butyl‐3‐methyl‐imidazolium tetrafluoroborate (BMIMBF₄), hemoglobin (Hb) and Poly(diallyldimethylammonium chloride) (PDDA) were successfully assembled into Hb/IL/DNA/PDDA layer‐by‐layer complex films on the surface of ITO electrode. FTIR spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the composite film. The obtained results demonstrated that the Hb molecule in the film kept its native structure and showed its good electrochemical behavior. A pair of well‐defined redox peaks of Hb with the formal potentials (E°′) of ?0.180 V (vs. SCE) was appeared in phosphate buffer solution (PBS, pH 7.0). The Hb/IL/DNA/PDDA/ITO modified electrode also showed an excellent electrocatalytic behavior to the reduction of hydrogen peroxide (H₂O₂). Therefore, the IL/DNA/PDDA complex film as a novel matrix open up a possibility for further study on the direct electrochemistry of other proteins and the fabrication of the third‐generation electrochemical biosensors.     

辣根过氧化物酶修饰电极在离子液体[EMIM]BF4中的直接电化学

将壳聚糖(Chi)-辣根过氧化物酶(HRP)-多壁碳纳米管(MWCNTs)的复合物修饰在玻碳电极(GCE)表面, 制备了HRP修饰电极(Chi-HRP-MWCNTs/GCE), 并将其用于在亲水性离子液体1-乙基-3-甲基咪唑四氟硼酸([EMIM]BF4)中HRP的直接电化学研究. 紫外可见光谱和红外光谱表明, HRP在复合物内保持了其原始构象. 电化学研究表明, 该修饰电极在[EMIM]BF4中的循环伏安图上出现了一对峰形良好、几乎对称的氧化还原峰, 式量电位为－0.247 V (vs. Ag/AgCl), 说明包埋在Chi-MWCNTs中的HRP与电极之间发生了直接电子传递; HRP在电极表面直接电子转移的速率常数ks为3.12 s－1; 在65 ℃的[EMIM]BF4中HRP仍然保持其活性; HRP修饰电极对过氧化氢的还原具有电催化作用, 其表观米氏常数Km为5.6×10－5 mol•L－1, 催化电流与过氧化氢浓度在5.0×10－7～5.0×10－5 mol•L－1范围内呈线性关系, 检出限为2.0×10－7 mol•L－1. 该研究为非水相生物传感器的构制提供了一种新途径.     

反相胶束中辣根过氧化物酶的停流光谱研究

用停流光谱法研究了HRP在AOT、CTAB和SDS反相胶束中的吸收光谱和反应动力学，实验结果显示在AOT反相胶束中，HRP的吸收峰位置与水相中相同；而另外两种反相胶束对HRP的分子结构产生了较大影响，快速反应动力学研究显示在反相胶束中HRP形成化合物Ⅰ的速率常数远远高于化合物Ⅰ形成HRP—Ⅱ的反应速率常数，推测这是反相胶束的特殊性质造成的结果。     

Preparation of Magnetic Ordered Mesoporous Carbon Composite and Its Application in Direct Electrochemistry of Horseradish Peroxidase

A novel magnetic ordered mesoporous carbon composite was prepared. Electrochemical measurements showed that the ordered mesoporous carbon composite provided an excellent matrix for the co‐adsorption of horseradish peroxidase (HRP). HRP could be separated and collected by the application of a magnetic field and its direct electron‐transfer could be achieved in the solution, not on the electrode thereby preventing the degradation of the enzyme. The cyclic voltammetric experimental results of HRP indicated that HRP displayed a pair of stable peaks with a formal potential of ?0.306 V in PBS. The resulting biosensor exhibited fast amperometric response to hydrogen peroxide.     

反相胶束对辣根过氧化物酶催化反应的影响

胶束体系是酶学研究比较理想的体系,因为它所具有的诸如热力学稳定、光学透明及能增溶亲水分子、亲油分子或两性分子等性质,使许多酶在胶束体系中的反应速率远远高于在水相中,即人们发现的所谓“超活性”［‘j．辣根过氧化物酶（HRP）是一种比较稳定的酶,且价廉易得,具备一般过氧化物酶的典型反应．在研究中人们发现,HRP在反相胶束体系中同样具有“超活性”,由于HRP能够催化大量底物进行反应,因此“超活性”对HRP的催化反应具有重要意义．已有研究者［’、’j对CTAB反相胶束体系中HRP的性质进行了探讨,但反相胶束对HRP的…     

Direct Electrochemistry of Horseradish Peroxidase Immobilized in a Low Molecular Weight Gel

The ternary system of dodecylpyridinium bromide (DDPB)/acetone/H₂O with appropriate composition can form a gel spontaneously and the gel is stable in hydrophobic ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([Bmim]PF₆). Based on the gelation phenomenon we observed, the low molecular weight gelator (LMWG) was first tried to immobilize horseradish peroxidase (HRP) on glassy carbon electrode (GCE). The scanning electron microscope (SEM) images, the UV‐Vis spectra and the bioactivity measurement indicate that the gel is suitable for the immobilization of HRP. The direct electrochemistry of the HRP‐gel modified GCE (HRP‐gel/GCE) in [Bmim]PF₆ shows a pair of well‐defined and quasi‐reversible redox peaks with the heterogeneous electron transfer rate constant (k_s) being 14.4 s^?1, indicating that the direct electron transfer between HRP and GCE is fast. The HRP‐gel/GCE is stable and reproducible. Also the electrode exhibits good electrocatalytic effect on the reduction of trichloroacetic acid (TCA), showing good promise in bioelectrocatalysis.     

Mediatorless Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized on 4‐Carboxyphenyl Film Electrografted on Gold Electrode

A novel method to fabricate a third‐generation hydrogen peroxide biosensor was reported. The electrode was first derivatized by electrochemical reduction of in situ generated 4‐carboxyphenyl diazonium salt (4‐CPDS) in acidic aqueous solution yielded stable 4‐carboxyphenyl (4‐CP) layer. The horseradish peroxidase (HRP) enzyme was then covalently immobilized by amidation between NH₂ terminus of enzyme and COOH terminus of 4‐CP film making use of the carbodiimide chemistry. Electrodeposition conditions used to control electrode functionalization density and film electron transfer kinetics were assessed by chronoamperometry and electrochemical impedance spectroscopy. The immobilized HRP displayed excellent electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediators. The effect of various operational parameters was explored for optimum analytical performance. The reported biosensor exhibited fast amperometric response (within 5 s) to H₂O₂. The detection limit of the biosensor was 5 μM, and linear range was from 20 μM to 20 mM. Furthermore, the biosensor exhibited high sensitivity, good reproducibility, and long‐term stability.     

Direct Electron Transfer and Electrocatalysis of Myoglobin Based on its Direct Immobilization on Carbon Ionic Liquid Electrode

Direct electron transfer of myoglobin (Mb) was achieved by its direct immobilization on carbon ionic liquid electrode (CILE) with a conductive hydrophobic ionic liquid, 1‐butyl pyridinium hexaflourophosphate ([BuPy][PF₆]) as binder for the first time. A pair of well‐defined, quasi‐reversible redox peaks was observed for Mb/CILE resulting from Mb redox of heme Fe(III)/Fe(II) redox couple in 0.1 M phosphate buffer solution (pH 7.0) with oxidation potential of ?0.277 V, reduction potential of ?0.388 V, the formal potential E°′ (E°′=(E_pa+E_pc)/2) at ?0.332 V and the peak‐to‐peak potential separation of 0.111 V at 0.5 V/s. The average surface coverage of the electroactive Mb immobilized on the electrode surface was calculated as 1.06±0.03×10^?9 mol cm^?2. Mb retained its bioactivity on modified electrode and showed excellent electrocatalytic activity towards the reduction of H₂O₂. The cathodic peak current of Mb was linear to H₂O₂ concentration in the range from 6.0 μM to 160 μM with a detection limit of 2.0 μM (S/N=3). The apparent Michaelis–Menten constant (K and the electron transfer rate constant (k_s) were estimated to be 140±1 μM and 2.8±0.1 s^?1, respectively. The biosensor achieved the direct electrochemistry of Mb on CILE without the help of any supporting film or any electron mediator.     

酚类聚合物在水相胶束中的酶促合成

本文报道辣根过氧化物酶在水相胶束中催化聚合取代酚类的研究。水相胶束中酶催化活力高，产物分子量均一；通过调节表面活笥剂和单体的浓度，可以有效地控制产物的分子量，从而建立了不同聚合度有机纳米材料酶促合成的新方法。产物具有较强的蓝紫色荧光及较好的耐热性能，是一种很有应用前景的蓝光材料。     

离子液体在电化学中的应用

离子液体作为绿色替代溶剂在电化学中的应用涉及电解、电镀、电催化、电池和电容技术等 ,在全球环境问题日益严峻的今天 ,电化学及其技术将显示重要作用。本文就离子液体这一新型的反应介质及其在电化学中的应用和研究进展加以阐述     

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized in Carbon Nanotubes/ SBA‐15 Film

A new third‐generation biosensor for H₂O₂ assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes (CNTs)‐SBA‐15 modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV‐vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H₂O₂. The effects of the experimental variables such as solution pH and working potential were investigated using steady‐state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range from 1 µM to 7 mM and a detection limit of 0.5 µM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.     

Catalysis‐Electrochemical Detection of Horseradish Peroxidase at Zeptomole Level in Capillary Zone Electrophoresis

Capillary zone electrophoresis with catalysis‐electrochemical detection has been developed and applied to determining horseradish peroxidase (HRP) at zeptomole levels. In this method, an on‐line enzyme catalysis reactor with a reaction capillary was designed. Isoenzymes of HRP were separated by capillary zone electrophoresis, and then they catalyzed the enzyme substrate 3,3′,5,5′‐tetramethylbenzide (TMB(Red)) and H₂O₂ in the reaction capillary. The reaction product, TMB(Ox), could be determined using amperometric detection on a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Because of enzyme amplification, a significant amount of TMB(Ox) could be produced for detection. Therefore, the limit of detection (LOD) of HRP is very low. The optimum conditions of the method are 1.5×10^?2 mol/L borate (pH 7.4) for the run buffer, 2×10^?3 mol/L for the concentration of H₂O₂, 2×10^?4 mol/L TMB(Red)+2.0×10^?2 mol/L citrate‐phosphate (pH 5.0) for the substrate solution, 40 cm for the liquid pressure height, 20 kV for the separation voltage, 100 mV for the detection potential. HRP could be measured with a detection limit of 4.8×10^?12 mol/L or 47.5 zmol (S/N=3). The linear range is from 2.40×10^?11 to 2.40×10^?8 mol/L. Using this method, commercial HRP was measured at zeptomole within ten minutes.     

Novel Composite of Multiwalled Carbon Nanotubes and Gold Nanoparticles Stabilized by Chitosan and Hydrophilic Ionic Liquid for Direct Electron Transfer of Glucose Oxidase

A novel composite was fabricated through dispersing multiwalled carbon nanotubes (MWNTs) in gold nanoparticle (GPs) colloid stabilized by chitosan and ionic liquid (i.e., 1‐butyl‐3‐methylimidazolium tetrafluoroborate, BMIMBF₄). Transmission electron microscopy (TEM) experiment showed that the GPs highly dispersed on the MWNTs probably due to the electrostatic interaction among GPs, MWNTs and the imidazolium cation of BMIMBF₄. X‐ray photoelectron spectroscopy (XPS) indicated that thus‐formed gold nanostructure was mediated by BMIMBF₄. When glucose oxidase (GOD) was immobilized on the composite (MWNTs‐GPs) its ultraviolet‐visible absorption spectrum kept almost unchanged. The immobilized GOD coated glassy carbon electrode (GOD/MWNTs‐GPs/GC) exhibited a pair of well‐defined peaks in 0.10 M pH 7.0 phosphate buffer solution (PBS), with a formal potential of ?0.463 V (vs. SCE). The electrochemical process involved two‐electron transfer. The electron transfer coefficient was ca.0.56 and the electron transfer rate constant was 9.36 s^?1. Furthermore, the immobilized GOD presented good catalytic activity to the oxidation of glucose in air‐saturated PBS. The K_m and I_m values were estimated to be 13.7 μM and 0.619 μA. The GOD/MWNTs‐GPs/GC electrode displayed good stability and reproducibility.     

Determination of Fluoride,Cyanide, and Thiocyanate Using Horseradish Peroxidase Immobilized on Modified Silica Gel

Abstract

Silica gels modified with different functional groups (amino, epoxy, cycloepoxy, isocyanate, and thiocyanate) were used for the covalent immobilization of horseradish peroxidase (HRP). The catalytic activity and stability of the obtained enzyme preparations were studied using the reaction of o‐dianisidine oxidation with hydrogen peroxide as an indicator. The covalent immobilization of horseradish peroxidase using silica gel modified with thiocyanate groups provided not only the improvement of the enzyme stability, but also the development of the sensitive, rapid, and simple procedures for the determination of fluoride, cyanide, and thiocyanate. Enzymatic determination of inorganic anions is based on their inhibitory effect on the enzyme as the ligands capable to form stable complexes with Fe(III)‐HRP cofactor. The proposed procedures were applied for the determination of F^? in mineral and drinking waters; CN^? and SCN^?—in biological fluids (blood and saliva).