催化动力学光度法测定葡萄糖氧化酶活性研究

本文对葡萄糖氧化酶(GO酶)在不同缓冲体系中的活性、酸度、温度的影响进行了研究。表明GO酶在KH_2PO_4-Na_2HPO_4-H_3PO_4体系中活性最大。测定反应基于GO酶催化葡萄糖氧化产生H_2O_2,再用Mo(Ⅵ)催化H_2O_2氧化KI,以淀粉显色,于570nm处测定吸光度,建立了测定生物样品中GO酶的方法。该法在1.6×10~(-3)～8×10~(-2)u/ml酶活力单位浓度范围内有良好的线性关系,相关系数为0.997。并对放置不同时间的峰蜜进行了测定,回收率在92％～110％。结果尚属满意。     

多孔壳聚糖膜固定葡萄糖氧化酶活性的X射线微区分析

甲壳素(chitin)是无脊椎动物,特别是节肢动物,如虾、蟹及昆虫等的外骨骼重要组成部分,其学名为β(1,4)-2-乙酰氨基-2-脱氧-D-葡萄糖。壳聚糖是甲壳素脱乙酰基的衍生物,其结构上具有许多—NH2、—OH等反应基团,对蛋白质具有极高的亲和性,且具有生物相容性好、无毒、可生物降解等     

生物功能电极 III. 葡萄糖氧化酶的电化学固定化研究

利用磷酸盐缓冲溶液中吡咯的电聚合, 将葡萄糖氧化酶(GOD)包埋在聚吡咯(PPy)基质中以构成生物功能电极。讨论了溶液pH和聚合电位对酶固定化的影响, 并用IR和交流阻抗谱对酶膜进行表征。GOD的固定化只有当pH>5.5时才能实现, 由此推测酶是以带负电的粒子嵌入PPy的。交流阻抗谱表明这一电极具有有界多孔电极的特征。探索了酶与电子传递体Fe(CN)_6~(3-)同时固定化的可行性。电化学固定化的GOD保持其生物催化活性, 酶反应表观上遵循Michealis-Menten动力学。     

Amperometric Glucose Determination by Means of Glucose Oxidase Immobilized on a Cellulose Acetate Film: Dependence on the Immobilization Procedures

Glucose microelectrodes were prepared by immobilizing glucose oxidase onto a cellulose acetate film coating a platinum wire. Hexamethylenediamine (HMDA) and Glutaraldehyde (GA) were employed as spacer and coupling agent, respectively. Sensitivities and linear response ranges were studied as a function of the relative amounts of HMDA and GA. The best sensitivity was found when HMDA and GA were 5% and 2.5% in aqueous solutions, respectively. Taking as a reference the functioning of this biosensor, the roles of HMDA and GA percentages appear to be opposed when the extension of the linear response range is considered. Indeed, an increase of one unit in HMDA percentage (from 5 to 6 %) induces an increase in the extension of the linear response range equal to that obtained with a decrease of one unit of GA percentage (from 2.5 to 1.5%).     

Direct Electrochemistry of Glucose Oxidase on Nail‐like Carbon and Its Biosensing for Glucose

Nail‐like carbon (NLC) was synthesized by a simple hydrothermal method. It was the first time that a novel electrochemical biosensing of glucose was explored based on the glucose oxidase (GOx)‐NLC‐chitosan (CHIT) glassy carbon electrode. Morphology and structure of NLC were characterized by scanning electron microscope; meanwhile the chemical composition was determined by X‐ray diffraction and energy dispersive X‐ray spectroscopy. The cyclic voltammetry of immobilized GOx showed a pair of quasireversible redox peaks with the formal potential (E°′) of ?0.458 V and the peak‐to‐peak potential separation was 47 mV at a scan rate of 100 mV s^?1. The present biosensor has a linear range of glucose from 0.02 to 1.84 mM (correlation coefficient of 0.9991) and detection limit of 0.01 mM (S/N=3). Compared with the previous reports based on the carbon material biosensor, it has a high sensitivity of 165.5 μA mM^?1 cm^?2 and low apparent Michaelis–Menten constant of 0.506 mM. Thus, the NLC may have potential applications in the field of bioelectrochemistry, bioelectronics and biofuels.     

Glucose Biosensor Using Glucose Oxidase and Electrospun Mn2O3‐Ag Nanofibers

The highly porous Mn₂O₃‐Ag nanofibers were fabricated by a facile two‐step procedure (electrospinning and calcination). The structure and composition of the Mn₂O₃‐Ag nanofibers were characterized by SEM, TEM, XRD, EDX and SAED. The as‐prepared Mn₂O₃‐Ag nanofibers were then employed as the immobilization matrix for glucose oxidase (GOD) to construct an amperometric glucose biosensor. The biosensor shows fast response to glucose, high sensitivity (40.60 µA mM^?1 cm^?2), low detection limit (1.73 µM at S/N=3), low K_m,app value and excellent selectivity. These results indicate that the novel Mn₂O₃‐Ag nanfibers‐GOD composite has great potential application in oxygen‐reduction based glucose biosensing.     

A Stable Enzyme Biosensor Using Concanavalin a Complex of Glucose Oxidase for Glucose Determination

Abstract

The thermal stability of an insoluble concanavalin A (ConA) complex of glucose oxidase (GOD) was researched. The thermal deactivation rate constants of the complexes were obtained. It was found that the GOD-ConA complexes were less sensitive to thermal inactivation than the native enzyme GOD. By using the complexes, ferrocene-mediated enzyme electrodes were constructed. The results suggested that the GOD-Con A complex electrodes had good thermal stability at room temperature.     

Construction of a Novel Glucose Biosensor Based on Covalent Immobilization of Glucose Oxidase on Poly(glycidyl methacrylate‐co‐vinylferrocene)

A novel glucose biosensor was constructed via direct covalent attachment of glucose oxidase onto epoxy group containing polymeric electron transfer mediator, Poly(glycidyl methacrylate‐co‐vinylferrocene). A copolymer of glycidyl methacrylate (GMA) and vinylferrocene (VFc) with different molar ratios has been prepared by free radical copolymerization. These copolymers have been utilized as polymeric mediators for amperometric glucose sensing. The catalytic electrochemistry of the enzyme electrode with the copolymer was investigated. Copolymer acts as an electron transfer mediator between the redox center of Glucose oxidase (GOx) and the electrode. The stability, reusability, pH and temperature response of the biosensor as well as its kinetic parameter have also been studied.     

再生丝素固定葡萄糖氧化酶及其传感器应用

再生丝素固定葡萄糖氧化酶及其传感器应用钱江红，刘永成，刘海鹰，于同隐，邓家祺（复旦大学化学系高分子科学系，上海，２００４３３）关键词再生丝素，葡萄糖氧化酶，传感器，酶电极酶电报的各项性能在很大程度上取决于酶的固定比方法，葡萄糖氧化酶的固定化方法很多１...     

An Amperometric Glucose Electrode Based on Adsorbed Glucose Oxidase on Palladium/Gold Modified Graphite

Abstract

A glucose electrode was constructed by adsorbing glucose oxidase (GOD) on a modified electrode for H₂ O ₂ oxidation, consisting of Pd/Au sputtered on graphite. Maximally, 0.8 U cm^?2 of GOD could be adsorbed. The electrode was used in a f.i.a. manifold for determination of glucose. Linear calibration curves were obtained in the concentration range 3. 10^?6 4. 10^?3 mol L^?1 glucose. The applied potentials for glucose determination were + 300 mV vs. Ag/AgCl at pH 8.0, + 350 mV at pH 7.0, + 400 mV at pH 6.0 and + 500 mV at pH 5.0. The activity vs. pH profile of adsorbed GOD was broad having an optimum between pH 5 and 6. The apparent kinetic parameters for adsorbed GOD, K_M ^app and i_max, were found to be 50 mM and 160 uA at optimal pH.     

Reagentless Glucose Biosensor Based on the Direct Electrochemistry of Glucose Oxidase on Carbon Nanotube‐Modified Electrodes

The direct electrochemistry of glucose oxidase (GOD) was revealed at a carbon nanotube (CNT)‐modified glassy carbon electrode, where the enzyme was immobilized with a chitosan film containing gold nanoparticles. The immobilized GOD displays a pair of redox peaks in pH 7.4 phosphate buffer solutions (PBS) with the formal potential of about ?455 mV (vs. Ag/AgCl) and shows a surface‐controlled electrode process. Bioactivity remains good, along with effective catalysis of the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, which could be used for reagentless detection of glucose with a linear range from 0.04 to 1.0 mM. The proposed glucose biosensor exhibited high sensitivity, good stability and reproducibility, and was also insensitive to common interferences such as ascorbic and uric acid. The excellent performance of the reagentless biosensor is attributed to the effective enhancement of electron transfer between enzyme and electrode surface by CNTs, and the biocompatible environment that the chitosan film containing gold nanoparticles provides for immobilized GOD.     

固定化葡萄糖氧化酶活性的X射线微区分析

利用X射线微区分析方法,对固定化活性葡萄糖氧化酶进行了定位分析;葡萄糖作为底物,FeSO4和KI作为捕捉剂,底物经固定化葡萄糖氧化酶催化产生H2O2,后者和捕捉剂反应生成沉淀,可以确定固定化葡萄糖氧化酶的催化活性部位。结果表明：颗粒越小,酶活越高,活性葡萄糖氧化酶在凝胶内分布均匀,且绝大多数葡萄糖氧化酶固定在凝胶的内部。作者还研究了固定化活性葡萄糖氧化酶定位的最佳条件。     

Direct Electrochemistry of Glucose Oxidase Immobilized on Titanium Carbide‐Au Nanoparticles‐Fullerene C60 Composite Film and Its Biosensing for Glucose

The direct electrochemistry of glucose oxidase (GOD) immobilized on the designed titanium carbide‐Au nanoparticles‐fullerene C₆₀ composite film modified glassy carbon electrode (TiC‐AuNPs‐C₆₀/GCE) and its biosensing for glucose were investigated. UV‐visible and Fourier‐transform infrared spectra of the resulting GOD/TiC‐AuNPs‐C₆₀ composite film suggested that the immobilized GOD retained its original structure. The direct electron transfer behaviors of immobilized GOD at the GOD/TiC‐AuNPs‐C₆₀/GCE were investigated by cyclic voltammetry in which a pair of well‐defined, quasi‐reversible redox peaks with the formal potential (E^0′) of ‐0.484 V (vs. SCE) in phosphate buffer solution (0.05 M, pH 7.0) at the scan rate of 100 mV·s^?1 were obtained. The proposed GOD modified electrode exhibited an excellent electrocatalytic activity to the reduction of glucose, and the currents of glucose reduction peak were linearly related to glucose concentration in a wider linearity range from 5.0 × 10^?6 to 1.6 × 10^?4 M with a correlation coefficient of 0.9965 and a detection limit of 2.0 × 10^?6 M (S/N = 3). The sensitivity and the apparent Michaelis‐Menten constant (K_M^app) were determined to be 149.3 μA·mM^?1·cm^?2 and 6.2 × 10^?5 M, respectively. Thus, the protocol will have potential application in studying the electron transfer of enzyme and the design of novel electrochemical biosensors.     

Electrochemical Activity Studies of Glucose Oxidase (GOx)‐Based and Pyranose Oxidase (POx)‐Based Electrodes in Mesoporous Carbon: Toward Biosensor and Biofuel Cell Applications

A simple study using a fixed amount of mesoporous carbon (MSU‐F‐C) was performed for the comparison of pyranose oxidase (POx) and glucose oxidase (GOx) in their electrochemical performance under biosensor and biofuel cell operating modes. Even though the ratio of POx to GOx in the glucose oxidation activity per unit weight of MSU‐F‐C was 0.35, the ratios of POx to GOx in sensitivity and power density were reversed to be 6.2 and 1.4, respectively. POx with broad substrate specificity and an option of large scale production using recombinant E. coli has a great potential for various electrochemical applications, including biofuel cells.     

葡萄糖氧化酶共价交联于蛋膜上的葡萄糖传感器

以牛血清白蛋白－戊二醛为交联剂，将葡萄糖氧化酶固定地鸡蛋膜上，氧电极作电化学敏感元件，制成葡萄糖氧化酶电极。传感器的响应范围为４．０×１０＾－６－２．４×１０＾－３ｍｏｌ／Ｌ；检测限为１．２１０＾－６ｍｏｌ／Ｌ。该传感器具有线性范围宽，灵敏度高，使用寿命长等优点。     

蜘蛛丝素和聚乙烯醇固定葡萄糖氧化酶 制备葡萄糖传感器

用蜘蛛丝素和聚乙烯醇的混合材料把葡萄糖氧化酶固定在氧电极表面,制成葡萄糖氧化酶电极。传感器对葡萄糖有灵敏的响应,平均响应时间为20s,电位变化值与葡萄糖浓度在3.0×10     

聚苯胺葡萄糖氧化酶电极的催化过程

用电化学方法固定在直径为0.5mm铂丝上的聚苯胺(PANI)葡萄糖(GOD)电极对葡萄糖有催化氧化作用.在0～-0.6V(vs.SCE)的电极范围内,在电极的循环伏安曲线上观察到与葡萄糖浓度有关的氧的还原峰和GOD还原态的氧化峰,用此GOD还原态的氧化峰电流可定量检测葡萄糖的浓度。本文提出在PANI电极上存在着酶反应氧化还原电荷直接传递的可能性。     

An Amperometric Glucose Biosensor Based on Poly (Pyrrole‐2‐Carboxylic Acid)/Glucose Oxidase Biocomposite

A newly developed amperometric glucose biosensor based on graphite rod (GR) working electrode modified with biocomposite consisting of poly (pyrrole‐2‐carboxylic acid) (PCPy) particles and enzyme glucose oxidase (GOx) was investigated. The PCPy particles were synthesized by chemical oxidative polymerization technique using H₂O₂ as initiator of polymerization reaction and modified covalently with the GOx (PCPy‐GOx) after activation of carboxyl groups located on the particles surface with a mixture of N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). Then the PCPy‐GOx biocomposite was dispersed in a buffer solution containing a certain amount of bovine serum albumin (BSA). The resulting biocomposite suspension was adsorbed the on GR electrode surface with subsequent solvent airing and chemical cross‐linking of the proteins with glutaraldehyde vapour (GR/PCPy‐GOx). It was determined that the current response of the GR/PCPy‐GOx electrodes to glucose measured at +300 mV vs Cl reference electrode was influenced by the duration of the PCPy particles synthesis, pH of the GOx solution used for the PCPy particles modification and the amount of immobilized PCPy‐GOx biocomposite. An optimal pH of buffer solution for operation of the biosensor was found to be 8.0. Detection limit was determined as 0.039 mmol L⁻¹ according signal to noise ratio (S/N: 3). The proposed glucose biosensor was tested in human serum samples.     

Biosensor Based on Self‐Assembling Glucose Oxidase and Dendrimer‐Encapsulated Pt Nanoparticles on Carbon Nanotubes for Glucose Detection

A novel amperometric glucose biosensor based on layer‐by‐layer (LbL) electrostatic adsorption of glucose oxidase (GOx) and dendrimer‐encapsulated Pt nanoparticles (Pt‐DENs) on multiwalled carbon nanotubes (CNTs) was described. Anionic GOx was immobilized on the negatively charged CNTs surface by alternatively assembling a cationic Pt‐DENs layer and an anionic GOx layer. Transmission electron microscopy images and ζ‐potentials proved the formation of layer‐by‐layer nanostructures on carboxyl‐functionalized CNTs. LbL technique provided a favorable microenvironment to keep the bioactivity of GOx and prevent enzyme molecule leakage. The excellent electrocatalytic activity of CNTs and Pt‐DENs toward H₂O₂ and special three‐dimensional structure of the enzyme electrode resulted in good characteristics such as a low detection limit of 2.5 μM, a wide linear range of 5 μM–0.65 mM, a short response time (within 5 s), and high sensitivity (30.64 μA mM^?1 cm^?2) and stability (80% remains after 30 days).     

Unadulterated Glucose Biosensor Based on Direct Electron Transfer of Glucose Oxidase Encapsulated Chitosan Modified Glassy Carbon Electrode

Glucose oxidase (GOD) was encapsulated in chitosan matrix and immobilized on a glassy carbon electrode, achieving direct electron transfer (DET) reaction between GOD and electrode without any nano‐material. On basis of such DET, a novel glucose biosensor was fabricated for direct bioelectrochemical sensing without any electron‐mediator. GOD incorporated in chitosan films gave a pair of stable, well‐defined, and quasireversible cyclic voltammetric peaks at about ?0.284 (E_pa) and ?0.338 V (E_pc) vs. Ag/AgCl electrode in phosphate buffers. And the peak is located at the potentials characteristic of FAD redox couples of the proteins. The electrochemical parameters, such as midpoint potential (E_1/2) and apparent heterogeneous electron‐transfer rate constants (k_s) were estimated to ?0.311 V and 1.79 s^?1 by voltammetry, respectively. Experimental results indicate that the encapsulated GOD retains its catalytic activity for the oxidation of glucose. Such a GOD encapsulated chitosan based biosensor revealed a relatively rapid response time of less than 2 min, and a sufficient linear detection range for glucose concentration, from 0.60 to 2.80 mmol L^?1 with a detection limit of 0.10 mmol L^?1 and electrode sensitivity of 0.233 μA mmol^?1. The relative standard deviation (RSD) is under 3.2% (n=7) for the determination of practical serum samples. The biologic compounds probably existed in the sample, such as ascorbic acid, uric acid, dopamine, and epinephrine, do not affect the determination of glucose. The proposed method is satisfactory to the determination of human serum samples compared with the routine hexokinase method. Both the unique electrical property and biocompatibility of chitosan enable the construction of a good bio‐sensing platform for achieved DET of GOD and developed the third‐generation glucose biosensors.