Immobilized Glucose Oxidase in the Potentiometric Detection of Glucose

Previous work has shown that glucose oxidase can be immobilized on platinum to give an electrode that responds potentiometrically to glucose over the clinically useful range of about 10-250 mg glucose/100 mL. The present studies were carried out with electrochemically pretreated platinum and with gold or porous graphite substituted for the platinum support. The presence of the enzyme gave a significantly enhanced potentiometric response over that obtained with the bare support for both the pretreated platinum and the porous graphite, but not with gold. However, with platinum the potentiometric response became more negative with increasing glucose concentration. With porous graphite, the potential changed in the positive direction as the glucose concentration was increased. Hysteresis was demonstrated for the platinum-enzyme electrode. Mass transfer measurements with a rotating ring-disc electrode (RRDE) showed measurable diffusional resistances to the transport of a model electroactive compound (potassium ferrocyanide) through a matrix of immobilized enzyme attached to the disc of the RRDE. These results are part of a larger study to define the source of the potentiometric response by examining the roles of the support and the mass transfer resistances through the immobilized enzyme matrix.     

葡萄糖氧化酶修饰聚苯胺电极的动力学

虽然固定酶有吸附、交联、共价结合和捕集等多种方法,但寻找其新的固定方法仍是感兴趣的研究课题。利用导电高聚物的导电性和掺杂作用,将酶直接固定在导电高聚物上,这种方法简单,固定后的酶仍保持原有的活性。用聚苯胺固定葡萄糖氧化酶已有报道。但电极的活性有效期较短。考虑到固定酶的性质不仅取决于酶本身的性质,而且还受载体性质和固定方法的影响,我们曾用还原后的聚苯胺在酶溶液中氧化而固定酶,本文在文献的基础上,研究了葡萄糖氧化酶修饰聚苯胺电极的动力学特性、pH效应及其使用稳定性。     

Detection of Hydrogen Peroxide and Glucose Based on Immobilized C60‐Catalase Enzyme

The interaction between fullerene C60 and catalase enzyme was studied with a fullerene C60‐coated piezoelectric (PZ) quartz crystal sensor. The partially irreversible response of the C60‐coated PZ crystal sensor for catalase was observed by the desorption study, which implied that C60 could chemically react with catalase. Thus, immobilized fullerene C60‐catalase enzyme was synthesized and applied in determining hydrogen peroxide in aqueous solutions. An oxygen electrode detector with the immobilized C60‐catalase was also employed to detect oxygen, a product of the hydrolysis of hydrogen peroxide which was catalyzed by the C60‐catalase. The oxygen electrode/C60‐catalase detection system exhibited linear responses to the concentration of hydrogen peroxide and amount of immobilized C60‐catalase enzyme that was used. The effects of pH and temperature on the activity of the immobilized C60‐catalase enzyme were also investigated. Optimum pH at 7.0 and optimum temperature at 25 °C for activity of the insoluble immobilized C60‐catalase enzyme were found. The immobilized C60‐catalase enzyme could be reused with good repeatability of the activity. The lifetime of the immobilized C60‐catalase enzyme was long enough with an activity of 93% after 95 days. The immobilized C60‐catalase enzyme was also applied in determining glucose which was oxidized with glucose oxidase resulting in producing hydrogen peroxide, followed by detecting hydrogen peroxide with the oxygen electrode/C60‐catalase detection system.     

纳米聚苯胺修饰石墨电极的葡萄糖双酶传感器

用循环伏安法在石墨电极上制得纳米纤维聚苯胺, 并在其上固定葡萄糖氧化酶(GOD)和辣根过氧化物酶(HRP)制备葡萄糖双酶传感器. 用交流阻抗、SEM等技术对其进行表征; 考察了各种因素对双酶电极响应电流的影响以及双酶电极的稳定性. 该传感器对葡萄糖响应电流的测定在0.05 V(vs SCE)下进行, 有效避免了电活性物质的影响, 线性响应范围为0.05-2.0 mmol·L-1.     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

基于柔性衬底的ZnO葡萄糖酶电极制备及特性

通过水热法在长有ZnO籽晶层的柔性聚酰亚胺(PI)衬底上生长了整齐的ZnO纳米棒,ZnO纳米棒的晶体结构和表面形貌通过X射线衍射(XRD)、扫描电子显微镜(SEM)等进行表征.通过静电吸附方式,将葡萄糖氧化酶(GOx)固定在其表面.分别对GOx及修饰前后的ZnO纳米棒进行了紫外-可见光谱表征,发现修饰后存在ZnO的吸收峰和GOx的特征吸收峰,表明GOx固定在ZnO表面.通过对修饰样品进行傅里叶变换红外(FTIR)光谱测试发现了与GOx相关的吸收峰,这进一步表明GOx仍保持生物活性.最后在循环伏安曲线的测试中,这种在柔性衬底上制备的生物酶电极表现出非常灵敏的电流响应,为制备柔性葡萄糖生物传感器奠定了实验基础.     

Response Characteristics of a Glucose Electrode with a Sensing Membrane Prepared by Plasma Polymerization

A glucose electrode was composed of a dissolved oxygen electrode and an immobilized glucose oxidase membrane prepared by plasma polymerization of propargyl alcohol as a monomer. Fairly good precision of the electrode response to sample solutions was obtained by measurements using the steady-state method or the reaction rate method. Activity of the glucose oxidase immobilized within the membrane and mounted on the electrode lasted for 50 consecutive measurements over 5 days, and, if the membrane was stored in a buffer solution of pH 7.0 at a temperature of 0°C, the activity was preserved for more than 2 months. Such immobilization of the glucose oxidase with the plasma polymer effectively suppressed interference from Cu²⁺ions, which would seriously interrupt oxidation of the glucose in homogeneous solutions, in the sample solutions.     

Bioelectrochemical response of the polyaniline galactose oxidase electrode

Galactose oxidase has been immobilized in a polyaniline film. The response current of the galactose oxidase electrode is a function of the applied potential and increases as the pH increases from 5.61 to 7.25. The optimum pH of the immobilized galactose oxidase is 7.25. The activation energy of the enzyme-catalysed reaction is 41.8 kJ mol⁻¹. The response current of the enzyme electrode shows good reproducibility at temperatures below the optimum temperature of 30.4°C and increases as the galactose concentration increases from 0.2 to 6 mmol dm⁻³. Thus the polyaniline galactose oxidase electrode can be used to determine galactose concentration.     

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.     

纳米铜颗粒-酶-复合功能敏感膜生物传感器

用水合联肼作还原剂研制成亲水纳米铜颗粒,用琥珀酸二异酯磺酸钠／丙三醇／正庚烷反胶束体系合成出憎水纳米铜颗粒,并通过透射电镜和紫外光谱考察了制得的纳米颗粒样品,用憎水纳米铜颗粒及亲水纳米铜颗粒与聚 烯醇缩丁醛构成复合固酶膜基质,用溶胶－凝胶法固定葡萄糖氧化酶,构建葡萄糖生物传感器,实验结果表明,纳米铜颗粒可大幅度提高固定化酶的催化活性,响应电流从相应浓度的几十纳安增强到几千纳安,从理论和实验上证明了     

聚苯胺黄嘌呤氧化酶电极的生物电化学活性

用电化学方法将黄嘌呤氧化酶固定在聚苯胺中以制成聚苯胺黄嘌呤氧化酶电极。该电极呈现典型的酶催化反应动力学特性。且具有快速的生物电化学响应。固定化黄嘌呤氧化酶的表观米氏常数为21×10^-^6mol.dm^-^3, 最适pH为8.4,酶催化反应的活化能为85.1kJ.mol^-^1。酶电极具有较高的稳定性。使用聚苯胺黄嘌呤氧化酶电极能可靠地测定较低的底物浓度, 如2×10^-^6mol.dm^-^3黄嘌呤。     

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。     

中性红掺杂SiO2纳米粒子修饰的酶阵列传感器同时测定葡萄糖、乳酸和L-谷氨酸的研究

本文以中性红为核，二氧化硅为壳，利用反相微乳液技术，通过正硅酸四乙酯的水解制备了掺杂有中性红的二氧化硅纳米粒子，并用TEM技术进行了表征。核中性红能够催化测定葡萄糖，乳酸和L-谷氨酸的反应，而壳二氧化硅不仅克服了电活性物质中性红易流失的缺点，且具有高的生物亲和性。分别与葡萄糖氧化酶、乳酸氧化酶以及L-谷氨酸氧化酶混合后，修饰在碳阵列电极表面。最后在该酶阵列电极表面滴加一层Nafion, 防止电活性物质抗坏血酸、尿酸等的干扰。该酶阵列传感器与流动注射分析技术(FIA)相结合，可应用于同时检测大鼠血样中的葡萄糖，乳酸和L-谷氨酸浓度。该方法无需通过传统的色谱柱的分离，大大简化了实验条件，为这一领域的研究提供了有效的分析方法。     

胺氧化酶修饰聚苯胺电极的生物电化学响应特性

采用聚合物掺杂方法将胺氧化酶固定在聚苯胺膜中制成聚苯胺/胺氧化酶电极。该电极对组胺有快速的生物电化学响应,电极反应受酶动力学控制。固定化胺氧化酶的表观米氏常数为0.21mmol/L.最适pH值为7.3～7.6,酶催化反应的表观活化能为76kJ·mol^-1.酶电极具有较好的稳定性,可用来测定组胺。此外,酶电极的电化学性质通过循环伏安和交流阻抗进行了表征。     

基于层-层自反应的葡萄糖氧化酶有序多层膜电极

以胱胺修饰的金电极为基础电极, 利用席夫碱反应使经高碘酸根氧化的葡萄糖氧化酶在该电极表面进行自身的层-层有序组装. 用电化学交流阻抗法对多层酶膜形成过程的跟踪结果表明, 该多层酶膜的生长是一个逐步形成的均匀过程. 用循环伏安法和I-t曲线法研究了该酶电极对葡萄糖的电催化氧化. 实验结果表明, 当采用羟基二茂铁作为人工电子转移媒介体时, 该酶电极对葡萄糖具有很好的电催化氧化功能. 该传感器制作简便, 响应迅速, 性能稳定, 催化电流与葡萄糖浓度在一定范围内成正比, 并且可以通过控制葡萄糖氧化酶的组装层数来调节该生物传感器的灵敏度与检测限.     

用聚苯胺膜从牛乳中直接分离黄嘌呤氧化酶

酶电极;新鲜牛乳;固定分离;用聚苯胺膜从牛乳中直接分离黄嘌呤氧化酶     

Glucose oxidase/colloidal gold nanoparticles immobilized in Nafion film on glassy carbon electrode: Direct electron transfer and electrocatalysis

The direct electron transfer of glucose oxidase (GOD) was achieved based on the immobilization of GOD/colloidal gold nanoparticles on a glassy carbon electrode by a Nafion film. The immobilized GOD displayed a pair of well-defined and nearly reversible redox peaks with a formal potential (Eo ') of -0.434 V in 0.1 M pH 7.0 phosphate buffer solution and the response showed a surface-controlled electrode process. The dependence of Eo ' on solution pH indicated that the direct electron transfer reaction of GOD was a two-electron-transfer coupled with a two-proton-transfer reaction process. The experimental results also demonstrated that the immobilized GOD retained its electrocatalytic activity for the oxidation of glucose. So the resulting modified electrode can be used as a biosensor for detecting glucose.     

基于钯纳米颗粒修饰直立碳纳米管电极的电化学葡萄糖生物传感器

将电化学氧化生成的Pd(Ⅳ)离子配合到直立碳纳米管(ACNTs)上, 使其还原为纳米颗粒(Pb nps), 从而制得Pd nps-ACNTs纳米复合物电极, 经过葡萄糖氧化酶(GOD)进一步修饰后, 制成GOD/Pds nps/ACNTs酶电极, 通过测量GOD和葡萄糖酶促反应中产生的H₂O₂含量, 进而监测葡萄糖浓度. 实验结果表明, 电极表面大量Pd纳米颗粒的存在显著提高了传感器的检测灵敏度, 使酶电极具有响应时间短(<5 s)及检测电位低(<0.4 V)等优点.     

Glucose enzyme electrode with extended linearity: Application to undiluted blood measurements

An enzyme electrodes is described for glucose determination in unstirred, undiluted whole blood. The system comprises an H₂O₂-detecting electrode upon which is placed a membrane laminate incorporating glucose oxidase. The external membrane was pretreated with methyltrichlorosilane. The electrode response was linearly dependent on glucose concentration up to 50 mmol l^?1 glucose, it had a decreased dependence on dissolved oxygen concentrations and gave response times of 30–90 s. Whole blood glucose measurements correlated well with a routine spectrophotometric method.     

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.