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。     

Application of the Ugi reaction for the preparation of enzyme electrodes

Glucose oxidase and catalase were immobilized via the Ugi reaction by means of cyclohexyl isocyanide and glutaraldehyde on a nylon net partially hydrolysed by hydrochloric acid. A specific enzyme sensor for D-glucose was made by fixing the nylon net with immobilized enzymes on the tip of a Clark-type oxygen sensor. For comparison purposes glucose oxidase and catalase were also co-immobilized in the absence of cyclohexyl isocyanide or only glucose oxidase was immobilized with and without cyclohexyl isocyanide. The prepared biosensors were characterized by the specific activity of glucose oxidase and its dependence on Ph and temperature and by the apparent Michaelis constant. The linear range of the biosensor response to the substrate concentration and the stability of the biosensor were determined. The long-term stabilities of the enzyme electrodes were compared and the advangtage of the developed method was demonstrated.     

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...     

开管柱固定化葡萄糖氧化酶反应器的制备及其在流动注射分析中的应用

本文将葡萄糖氧化酶化学键合到粗糙化的玻璃毛细管内壁上,用开管柱固定化酶反应器-安培检测器进行葡萄糖的流动注射分析。讨论了流速对流动注射分析体系峰电流和分散度的影响,提出了测量开管柱固定化酶表观活力的方法。     

Micromachined Enzyme Reactor for FIA System

An enzyme reactor consisting of a 2.6-m-long silicon capillary with glucose oxidase immobilized on the inner surface was fabricated using micromachining techniques. A V-shaped groove of 100 μm width, formed by anisotropic etching, was anodically bonded to a glass plate to create the capillary. Glucose oxidase was covalently immobilized with 3-aminopropyltriethoxysilane and glutaraldehyde. The reactor was evaluated by connecting it to a Flow injection analysis system for glucose detection. Glucose concentrations were in the range of 10⁻³ to 5 × 10⁻²M with a volume of 0.2 μl of glucose solution.     

Preparation of immobilized glucose oxidase membrane by the plasma polymerization technique

Glucose oxidase was immobilized on a Millipore (MP) filter by coating with plasma-polymerized propargyl alcohol. The resulting immobilized enzyme membrane was used as a glucose sensor. The properties as a glucose electrode system were evaluated by amperometric response with either the steady-state method or the reaction rate method. The response was proportional to concentrations of the glucose solution up to 2 mM and the sensitivity was dependent on the amount of GOD impregnated into the MP filter.     

Technical and economic analysis of lignin conversion to methyl aryl ethers

Glucose oxidase (GOD) and Horseradish peroxidase (HRP) were covalently coupled to alkylamine controlled pore glass by means of glutaraldehyde. About 700-800 U/g of immobilized GOD and 300-400 U/g of immobilized HRP were obtained. Some factors of affecting enzyme immobilization were discussed. The immobilized enzymes were packed into a plastic tube and used in flow-injection analysis (FIA) for glucose in serum. A good linearity range was observed for this immobilized enzyme system at 20 mg/mL to 1000 mg/dL D-glucose, the recovery was 95.4-103.5%, the within-batch imprecision was 0.8-2.2%, and the between-batch imprecision was 2.2-4.2%. More than 100 samples were measured within an hour. One enzyme column with five units of immobilized GOD and HRP, applied for 50 assays/d, has been used for more than 2 mo.     

Strategies for the reversible immobilization of enzymes by use of biotin-bound anti-enzyme antibodies

Glucose oxidase (E.C. 1.1.3.4) is reversibly immobilized in a reactor coupled to a flow-injection analysis system using an immunological reaction. The antibody used is irreversibly immobilized on the reactor support by an avidin-biotin linkage. The bond between avidin and biotin is nearly irreversible under normal elution conditions for antibody-antigen reactions. The reactor is packed with a support on which avidin is covalently attached and a biotin-bound second antibody is passed over the reactor packing, which immobilizes this antibody. An immune complex of the enzyme, or first anti-enzyme antibody followed separately by enzyme, is introduced into the flow system, resulting in enzyme immobilization. The reactor produced can be used in the determination of 1 x 10(-11) -1 x 10(-6) mole of glucose with a sample size of 20 mul and a sample throughput of 20-30/hr. These results are comparable to or better than those obtained with glucose oxidase directly immobilized on the same support. The enzyme can be removed by elution with low-pH buffers, and the reactor regenerated by injection of the anti-enzyme antibody and the enzyme.     

Characterization and Potential Applications of Immobilized Glucose Oxidase and Polyphenol Oxidase

Pyrrole functionalized polystyrene (PStPy) was copolymerized with pyrrole to obtain a conducting copolymer, P(PStPy‐co‐Py) which is used as the immobilization matrix. Glucose oxidase and polyphenol oxidase enzymes were immobilized via the entrapment method by electrochemical polymerization. Enzyme electrodes were prepared by electrolysis at a constant potential using sodium dodecyl sulfate (SDS) as the supporting electrolyte during the copolymerization of PStPy with pyrrole. Maximum reaction rates (V_max) and enzyme affinities (Michaelis‐Menten constants, K_m) were determined for the enzyme entrapped both in polypyrrole (PPy) and P(PStPy‐co‐Py) matrices. Optimizations of enzyme electrodes were done by examining the effects of temperature and pH on enzymes' activities along with the shelf life and operational stability investigations. Glucose oxidase enzyme electrodes were used for human serum analysis and glucose determination in two brands of orange juices. Polyphenol oxidase enzyme electrodes were used for the determination of phenolics in red wines of Turkey.     

The Immobilization of Glucose Oxidase and its Improved Method

The best conditions of immobilizing glucose oxidase using different supports and different methods were studied and compared. An improved method of glucose oxidase immobilization by glutar aldehyde was studied. One end of a glutaraldehyde molecule was first protected by die thanolamine and the other end was linked to the carrier. Then the protected end was hydrolysed and glucose oxidase was linked to it. Glucose oxidase immobilized according to the above method has the highest activity. The enzyme activity was two fold higher than the polyacrylamide and 10% higher than the diazo method.     

氨基化树枝状介孔二氧化硅固定葡萄糖氧化酶用于检测葡萄糖

以三乙胺为碱源合成了树枝状介孔二氧化硅纳米粒子（DMSNs）,并用3-氨基丙基三乙氧基硅烷（APTES）进行氨基修饰合成了氨基化树枝状介孔二氧化硅纳米粒子（DMSNs-NH₂）,将其用于葡萄糖氧化酶（GOD）的固定化研究.采用扫描电子显微镜、透射电子显微镜、红外光谱仪、X射线衍射仪、氮气吸附仪及热重分析仪对固定化GOD（DMSNs-NH₂-GOD）进行了表征,测定了其活性及蛋白载量.结果表明,固定化GOD的直径约为200 nm,形状均一,呈分散的球形微粒;在最佳固定条件下,蛋白载量达225 mg/g,酶活性达215 U/mg;固定化GOD检测葡萄糖的最低检测限为0.0014 mg/mL.利用固定化GOD检测了血清和饮料中的葡萄糖,重复使用36次以上其相对酶活性仍剩余80%.该方法操作方便、准确度高,提高了酶的pH稳定性、热稳定性及重复使用性,降低了检测成本.     

Development of a Temperature-pulse Enhanced Electrochemical Glucose Biosensor and Characterization of its Stability via Scanning Electrochemical Microscopy

Glucose oxidase (GOx) is an enzyme frequently used in glucose biosensors. As increased temperatures can enhance the performance of electrochemical sensors, we investigated the impact of temperature pulses on GOx that was drop-coated on flattened Pt microwires. The wires were heated by an alternating current. The sensitivity towards glucose and the temperature stability of GOx was investigated by amperometry. An up to 22-fold increase of sensitivity was observed. Spatially resolved enzyme activity changes were investigated via scanning electrochemical microscopy. The application of short (<100 ms) heat pulses was associated with less thermal inactivation of the immobilized GOx than long-term heating.     

Development of a novel enzyme reactor and application as a chemiluminescence flow-through biosensor

A novel enzyme reactor was prepared using calcium alginate fiber (CAF) and amine-modified nanosized mesoporous silica (AMNMS) as a support. Combination of the adsorption of the enzyme on AMNMS with the cage effect of the polymer greatly increases the catalytic activity and the stability of the immobilized enzyme. It was shown that the lifetime, stability, and catalytic activity of the enzyme reactor were greatly improved by incorporating AMNMS into CAF to efficiently encapsulate the enzyme. Glucose oxidase was chosen as a model enzyme to explore the possibility of using CAF–AMNMS as a matrix for enzyme immobilization in the design of a chemiluminescence (CL) flow-through biosensor. The sensitivity of the flow-through biosensor combined with a novel luminol-diperiodatonickelate CL system was higher than for other reported CL biosensors. The proposed biosensor exhibits short response time, easy operation, long lifetime, high catalytic activity, high sensitivity, and simple assembly.     

Activation of Polymer-Metal Complexes on Enzyme

In this paper a new type of activation of polymer-metal complexes on an enzyme immobilized in them will be described. The polymer is partially quaternized poly(dimethylaminomethylstyrene) (PQPD). The metals (Me) include the fourth-period transition elements trivalent chromium and divalent iron, cobalt, nickel, copper, zinc, and manganese. Glucose oxidase (GO) is the enzyme. These complexed metallic ions had some activation effects on GO for most PQPD-Me-GO complexes. Specifically, the activity of the GO was increased up to 1.8-fold by chromium ion in the complex compared to immobilized GO without metal ion. The extent of activation of the polymer-metal complexes on GO was found to be related to the ionic radius and could be expressed quantitatively by a regression equation a = (4.5 – 42.3r) × 100, where a represents the relative activity of the immobilized enzyme and r represents the metal ion radius in the polymer-metal-enzyme complex.     

纳米增强型毛细管酶柱用于葡萄糖液滴生物传感器的研究

葡萄糖的检测在临床医学以及食品工业等领域中十分重要.以往的检测方法主要包括化学发光法_{[1]、吸光光度法[2]、电化学法[3]和荧光法[4]等.固定化酶柱的制作是发展葡萄糖传感器的关键技术之一.传统的固定化方法主要是将具有生物活性的酶通过物理吸附、共价键合和交联的方法固定于载体基质上或包埋于有机聚合物的基质中.近期研究[5,6]表明,采用溶胶凝胶(Sol-gel)法将蛋白质和酶等生物活性物质包埋于无机陶瓷或玻璃材料内,保持生物组分的活性,且SiO2作为基质材料具有较好的坚固性、抗磨性、化学惰性以及高的光稳定性和透过性,但目前该法多用于电化学型生物传感器[7,8].本文利用纳米颗粒的比表面积大和吸附能力强等特点,将酶吸附在SiO2纳米颗粒表面,用易成膜的聚乙烯醇缩丁醛(PVB)作辅助基质在毛细管上固定酶,并采用分立式酶柱,克服了以往混合型酶柱普遍存在的酶促效率不高和使用寿命较短的局限性.所制得的酶柱具有表面反应活性高、表面活性中心多和催化效率高等特点.结合自行设计的液滴光化学传感装置[9,10],建立了一种高效、快速、微量的葡萄糖实时检测方法.}     

β沸石固定酶修饰四氰基醌二甲烷电流型葡萄糖传感器的研究

本文介绍了一种新型的酶传感器, 首次以具有一定规整结构的β沸石固定酶, 以7, 7', 8, 8'-四氰基醌二甲烷作为电子传递媒介体,研制成葡萄糖传感器, 运用傅利叶变换红外光谱研究了β沸石与葡萄糖氧化酶之间的相互作用, 通过载体孔径分布和等电点的测定, 研究了载体的孔结构及表面酸性质对酶吸附的影响规律, 并对电极的制备条件和响应性能进行了优化。     

Enzymatic immunoassay of α-fetoprotein,insulin and 17-α-hydroxyprogesterone base on chemiluminescence in a flow-injection system

Highly-sensitive enzymatic immunoassay procedures based on a chemiluminescent reaction are described. Glucose oxidase was used as the labelling enzyme conjugated with anti-α-fetoprotein IgG, insulin or 17-α-hydroxyprogesterone. Free and bound fractions present after the immune reaction were separated by an immobilized antibody or a second antibody. The enzyme activity was measured by the chemiluminescence produced by luminol and hydrogen peroxide, catalyzed by potassium hexacyanoferrate(III) after incubation with glucose. The chemiluminescence was measured in a flow-injection system, with a home-made luminescence detector equipped with a spiral flow cell. The detection limits for each substance were 10^?15–10^?17 mol. Recoveries of added α-fetoprotein from diluted serum were quantitative.     

Determination of glucose in blood by flow injection analysis and an immobilized glucose oxidase column

A flow-injection system for glucose determination is described. Glucose oxidase is immobilized on controlled porosity glass (CPG) and used in a glass column (2.5 mm diameter × 2.5 cm). The hydrogen peroxide produced by the enzymatic reaction (? 1 × 10^?6 M) is detected by the current produced in a flow-through cell, with two platinum electrodes having a potential difference of 0.6 V. Glucose (0–20 mmol l^?1) can be determined in blood plasma either with a dialyser in the system or, better, by incorporating a column of copper(II) diethyldithiocarbamate on CPG before the enzyme column. The results compared well with those obtained by a conventional analyser system. The glucose oxidase column showed little change in activity over a 10-month period.     

Feasibility Study of Introducing Redox Property by Modification of PMBN Polymer for Biofuel Cell Applications

In this study, the feasibility of introducing redox property to an amphiphilic phospholipid polymer (PMBN) was investigated. The active ester group in the side chain of the polymer was used to react with pyrroloquinoline quinine (PQQ). Redox peaks that corresponded to PQQ redox potentials were observed after the modification. Glucose oxidase was immobilized to the modified polymer. Electrochemical oxidation of glucose was carried out with the polymer electrode. The oxidation current increased with elevating glucose concentration indicating electron transfer established between the electrode and enzyme. It suggests that by modification, PMBN is possible to use for enzyme electrode for bioelectronics.     

Determination of Urinary Glucose by a Flow Injection Analysis Amperometric Biosensor and Ion-Exchange Chromatography

A practical biosensor system has been developed for the determination of urinary glucose using a flow-injection analysis (FIA) amperometric detector and ion-exchange chromatography. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immobilized enzyme column. On the basis of its negative charge at pH 5.5, endogenous urate in urine samples was effectively retained by an upstream anion-exchange resin column. The biosensor system possessed a sensitivity of 160 ±2.4 RU μM^-1 (RU or relative unit is defined as 2.86 μV at the detection output) for glucose with a minimum detection level of 10 μM. When applied for the determination of urinary glucose, the result obtained compared very well with that of the widely accepted hexokinase assay. The immobilized glucose oxidase could be reused for more than 1000 repeated analyses without losing its original activity. The reuse of the acetate anion-exchange column before replacement would be about 25–30 analyses. Acetaminophen and ascorbic acid were also effectively adsorbed by the acetate anion exchanger. The introduction of this type of anion exchanger thus greatly improved the selectivity of the FIA biosensor system and fostered its applicability for the determination of glucose in urine samples.