聚乙烯－葡萄糖氧化酶膜的制备和性能研究

聚乙烯－葡萄糖氧化酶膜的制备和性能研究朱如瑾，殷弘浩，刘永盛，黄家湛（成都高分子材料国家重点实验室成都科技大学高分子研究所成都６１００６５）关键词聚乙烯，等离子体，固定化酶，葡萄糖氧化酶固定化酶是６０年代发展起来的生物工程技术Ｉ‘］．酶的固定化方法主...     

甲壳胺-羧甲基纤维素钠高分子复合物固定酶的研究

本文采用天然高分子甲壳胺与羟甲基纤维素钠的高分子复合物作哉体,固定葡萄糖氧化酶和纤维素酶。得到的固定化酶活性较高、热稳定性好。固定化后的葡萄糖氧化酶比固定化前的最适反应温度提高50℃,固定化后的纤维素酶最适反应温度提高20℃。固定酶的贮存稳定性明显提高,并可反复使用。     

核-壳型Fe3O4@Au定向固定葡萄糖氧化酶研究

以核-壳型结构的Fe_3O_4@Au微粒为载体,采用亲和素-生物素系统对葡萄糖氧化酶的固定化进行研究。表征结果表明,固定化酶呈现不规则圆球形,直径为80～180 nm左右。通过单一因素实验,探讨出葡萄糖氧化酶固定化的最优条件:磷酸盐缓冲溶液pH为4、固定化时间为40 min、固定化温度为30℃。游离酶与固定化酶的存贮稳定性表明,固定化葡萄糖氧化酶显示了更好的稳定性,可能与固定酶结构中存在的笼状结构有关。     

高分子对酶,抗体DNA的修饰,固定化及其生物医学应用

为发展适于生物医用的生物功能高分子材料，本实验室近年来研究了可溶性高分子对Ｌ－天冬酰胺酶的修饰，纳米磁性高分子微粒对酶或抗体的固定化，亚微米高分子微球固定化碱性磷酸酶及其在ＤＮＡ检测中的应用，高分子微球固定化酶的合成与性能，酶在导电高分子膜上的固定化及生物传感器制备等，本文对此进行简要总结。     

固定化葡萄糖氧化酶的制备及稳定性研究

选用自制的磁性纳米四氧化三铁为酶固定化的载体,通过交联法来固定葡萄糖氧化酶。通过对酶固定化过程中一系列因素的研究,确定出酶固定化的最佳工艺为:交联剂戊二醛与浓度为0.7 mg/mL酶液的体积比为1:4,固定化时间为0.5 h,固定化温度为室温(≤20 ℃),固定化所用缓冲液pH=7。采用比色法测定其酶活力。结果表明葡萄糖氧化酶经过固定化后,其酶活力较游离酶显著增加。固定化酶的热稳定性、酸碱稳定性、存储稳定性都有很大的提高。     

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...     

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。     

利用壳聚糖的絮凝性质包埋酶的葡萄糖氧化酶电极

本文利用壳聚糖的絮凝性质，将葡萄糖氧化酶包埋在壳聚糖与多聚磷酸盐的絮絮沉淀中，操作简单，固定化效率高。研究了适合了酶电极使用的最佳固定化条件。     

高分子对酶、抗体、DNA的修饰、固定化及其生物医学应用

为发展适于生物医用的生物功能高分子材料,本实验室近年来研究了可溶性高分子对L-天冬酰胺酶的修饰、纳米磁性高分子微粒对酶或抗体的固定化、亚微米高分子微球固定化碱性磷酸酶及其在DNA检测中的应用、高分子微球固定化酶的合成与性能、酶在导电高分子膜上的固定化及生物传感器制备等. 本文对此进行简要总结.     

聚乙烯醇载体制备及其固定化微生物处理污水研究进展

聚乙烯醇在生物活性高分子研究中的作用日趋重要,本文归纳了近年来聚乙烯醇类微生物固定化载体制备与改进方法的研究进展,以及聚乙烯醇载体固定化微生物技术在废水处理中的应用,并展望了聚乙烯醇类固定化微生物载体制备、改性及其在废水处理中应用前景。     

Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane

Gluconic acid was obtained in the permeate side of the bioreactor with glucose oxidase (GOD) immobilized onto anion-exchange membrane (AEM) of low-density polyethylene grafted with 4-vinylpiridine. The electric resistance of the anion-exchange membranes was increased after the enzyme immobilization on the membrane. The gluconic acid productions were relatively low with the GOD immobilized by any method on the AEM. To increase the enzyme reaction efficiency, GOD was immobilized on membrane of AN copolymer (PAN) adjacent to an anion-exchange membrane in bioreactor. Uses of anion-exchange membrane led to selective removal of the gluconic acid from the glucose solution and reduce the gluconic acid inhibition. The amount of gluconic acid obtained in the permeate side of the bioreactor with the GOD immobilized on the PAN membrane adjacent to the AEM under electrodialysis was about 30 times higher than that obtained with enzyme directly bound to the AEM. The optimal substrate concentration in the feed side was found to be about 1 g/l. Further experiments were carried out with the co-immobilized GOD plus Catalase (CAT) on the PAN membrane adjacent to the AEM to improve the efficiency of the immobilize system. The yield of this process was at least 95%. The storage stability of the co-immobilized GOD and CAT was studied (lost 20% of initial activity for 90 d). The results obtained clearly showed the higher potential of the dual membrane bioreactor with GOD plus CAT bound to ultrafiltration polymer membrane adjacent to the AEM. Storage stability of GOD activity in GOD plus CAT immobilized on PAN//AEM membranes and on AEM.     

Immobilization and enzymatic activity of glucose oxidase on polystyrene surface modified with ozone aeration and UV irradiation in distilled water and/or aqueous ammonia solution

Adsorption condition and enzymatic activity of glucose oxidase (GOD) on polystyrene (PS) film surfaces modified with ozone aeration and UV irradiation (O3/UV) treatment were investigated. The total amount of GOD immobilized on the PS film modified with the O3/UV treatment in distilled water (PS-W film) was approximately twice as large as that on the film treated in an aqueous ammonia solution (PS-A film), whereas the specific activity of GOD on the PS-A film was four times higher than that on the PS-W film. In contrast, no enzymatic activity of GOD on the non-treated PS film was observed because of irreversible denaturation of the adsorbed GOD. We therefore conclude that the PS films modified by the O3/UV treatment in the aqueous media are effective in immobilizing GOD.     

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.     

基于室温离子液体的有序多孔金膜葡萄糖传感器

将1-丁基-3-甲基咪唑四氟硼酸盐（[BMIm][BF4]）、N,N-二甲基甲酰胺（DMF）与葡萄糖氧化酶（GOD）的混合物修饰于三维有序大孔（3DOM）金膜电极上,构建了一种新型的葡萄糖传感器．固定的GOD在pH7．0的磷酸缓冲液（PBS）中展现出一对可逆性好的氧化还原峰,这归因于GOD的活性中心黄素腺嘌呤二核苷酸（FAD）的直接电化学行为．研究表明,离子液体（IL）、DMF以及3DOM金膜对GOD的直接电化学都起到了重要的作用．3DOM金膜修饰电极作为基底提高了酶的负载量,加速了GOD与电极表面的电子传递;IL的应用增加了固定GOD的电化学活性;DMF与IL、GOD的协同作用更好地保持了GOD的生物活性．固定在电极表面的GOD对葡萄糖显示出良好的催化性能,其检测线性范围为10～125nmol／L,检测限为3．3nmol／L（S／N=3）,酶催化反应的表观米氏常数Km为0．018mmol／L．     

Facile Fabrication of a Graphene‐based Electrochemical Biosensor for Glucose Detection

A simple and effective glucose biosensor based on immobilization of glucose oxidase (GOD) in graphene (GR)/Nafion film was constructed. The results indicated that the immobilized GOD can maintain its native structure and bioactivity, and the GR/Nafion film provides a favorable microenvironment for GOD immobilization and promotes the direct electron transfer between the electrode substrate and the redox center of GOD. The electrode reaction of the immobilized GOD shows a reversible and surface‐controlled process with the large electron transfer rate constant (k_s) of 3.42±0.08 s^?1. Based on the oxygen consumption during the oxidation process of glucose catalyzed by the immobilized GOD, the as‐prepared GOD/GR/Nafion/GCE electrode exhibits a linear range from 0.5 to 14 mmol·L^?1 with a detection limit of 0.03 mmol·L^?1. Moreover, it displays a good reproducibility and long‐term stability.     

Preparation of the glucose sensor based on three-dimensional ordered macroporous gold film and room temperature ionic liquid

A novel type of glucose sensor was fabricated based on a glucose oxidase (GOD)-N,N-dimethtylformamide (DMF)-[BMIm][BF₄] composites modified three-dimensional ordered macroporous (3DOM) gold film electrode. The immobilized GOD exhibits a pair of well-defined reversible peaks in 50 mM pH 7.0 phosphate buffer solutions (PBS), which could be attributed to the redox of flavin adenine dinucleotide (FAD) in GOD. The research results show that ionic liquid ([BMIm][BF₄]), DMF and 3DOM gold film are crucial for GOD to exhibit a pair of stable and reversible peaks. It is believed that the large active area of 3DOM gold film can increase the amount of immobilized GOD. Simultaneously, the application of IL enhances the stability of GOD and facilitates the electron transfer between GOD and the electrode. The synergetic effect of DMF can help the GOD to maintain its bioactivity better. GOD immobilized on the electrode exhibits the favorable electrocatalytic property to glucose, and the prepared sensor has a linear range from 10 to 125 nM with a detection limit of 3.3 nM at a signal-to-noise ratio of 3σ. The apparent K _m (Michaelis- Menten constant) for the enzymatic reaction is 0.018 mM.     

环糊精聚合物与苯醌的分子包合作用及其在酶电极中的应用

研究了水溶性环糊精预聚合物的存在对苯醌/氢醌体系在铂电极上氧化还原行为的影响, 根据伏安曲线讨论了该预聚合物与苯醌的分子包合作用。环糊精预聚合物与戊二醛缩聚反应而形成的不溶性聚合物膜用于葡萄糖氧化酶的固定化, 以制得新型的第二代葡萄糖电极。由于分子包合作用, 作为电子受体的苯醌在含酶的环糊精聚合物膜中具有较高的浓度, 从而加速了固定化酶的电子传递。测定了酶电极上BQ反应的动力学参数。     

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.     

A Biosensor Composed of Glucose Oxidase‐Containing Liposomes and MnO2‐Based Layered Nanocomposite

Glucose oxidase (GOD) was encapsulated in liposomes, and then the GOD‐containing liposomes were immobilized to a MnO₂‐based multilayered nanocomposite film grown electrochemically. Oxidation of glucose took place on the encapsulated GOD in the manganese oxide film, and the generated H₂O₂ molecules were oxidized catalytically at high valent Mn sites (4+) in the film. Anodic currents due to reoxidation of the reduced Mn ions (3+) were in proportion to the concentration of glucose from 19.6 to 107.1 mM. Such a simple construction of biosensor is applicable to a variety of combinations of liposomal enzymes and substrates.