【作者】 李彤；

【导师】 姚子华；

【作者基本信息】 河北大学 ， 分析化学， 2004， 博士

【摘要】 安培型酶传感器是生物传感器领域中最重要，研究最多，也是最灵敏的一种类型，是最具应用潜力的生物传感器。本工作在研究PB膜修饰电极对H₂O₂高灵敏高选择的电催化还原的基础上，与二氧化硅溶胶凝胶固定酶技术结合制备新型安培型氧化酶传感器。制备了具有“三明治”结构的葡萄糖氧化酶电极、半乳糖氧化酶电极。对它们的制备条件，操作条件进行了优化。对它们的响应性能进行了考察。并通过限制扩散等手段，扩大了葡萄糖传感器的响应范围。研究结果表明：由于PB在低电位下对H₂O₂的高选择及高灵敏电催化还原，使得制备的酶电极具有高灵敏性和好的抗干扰性。溶胶凝胶固定化酶对酶活的增强效应以及电极上修饰层的“三明治”结构，使得制备的酶电极具有快的响应时间和长期稳定性。应用外层膜以限制底物的扩散，制备的酶电极响应的线性范围扩大了2～3倍。利用酶组织化学中酶的定位原理，结合X-射线能谱分析手段，建立了对酶电极上酶活的微区分析方法。以Ce（NO₃）₃为捕捉剂，与底物经氧化酶催化作用的产物H₂O₂反应，在酶活的原位生成铈的沉淀，利用X-射线能谱仪进行定位分析，得到了电极上酶活的分布情况。从微观的角度说明酶电极的性能与表面酶活的关系，并对不同方法制备的酶电极进行X-射线微区分析，结果表明，载体膜的形成及对酶的固定方式对表面酶活大小及分布是有影响的。运用这种表征手段可对制备性能优良的酶电极具有指导意义。利用制备的葡萄糖氧化酶电极，对临床糖尿病人血清的葡萄糖含量进行测定，结果与血糖试剂盒（酶偶联比色法）测定结果一致。回收率实验结果为95.5％～109.8％。制备的葡萄糖氧化酶电极对果汁饮品中的葡萄糖测定研究，用以牛血清白蛋白代替葡萄糖氧化酶制得的电极做对照实验，研究样品基质及可能干扰物对酶电极的测定的干扰影响。实验结果表明，样品基质等对测定没有明显的干扰。测定结果重现性 摘要....曰..........较好，回收率在1似.1%一104.2%.更多还原

【Abstract】 Amperometric enzyme biosensor is the most important and sensitive biosensor in the biosensor area. It has also been paid more attention and has more useful potential. A new approach is described that prepare the amperometric oxidase biosensors based on high sensitive and selective electrocatalysis of Prussian Blue(PB)-modified electrode for H2O2 combined with Si02 sol-gel immobilization in a ’sandwich’ configuration. The glucose oxidase(GOD) electrode and the galactose oxidase(GAO) electrode have been developed. The optimum preparing conditions, experimental conditions as well as the response characteristics of the enzyme electrodes have been investigated. Attempts have been made to extend the response linear range of more conventional glucose electrode by the use of external membranes to restrict diffusion. The results show that the developing sensors exhibit high sensitivity and selectivity due to high sensitive and selective electroreduction of PB for H2O2 at low potential and fast response and higher stability of the sensors are attributed to biocompatible microenviroment provided by the silica sol-gel and the ’sandwich’ construction maintaining the enzyme activity. The linear range of the improved GOD electrode for glucose is extended to 2-3 times as wide as that of the enzyme electrode without the outer membrane. Based on localization of enzyme in the histochemistry and cytochemistry combined with X-ray microanalysis, The microanalysis method of enzyme activity on the enzyme electrode has been established. Ce(NO3)3 serves as capturer. Substrate is catalysed by immobilized oxidase on the electrode to produceH2O2 and the H2O2 is captured by Ce(NO3)3 to form precipitate. The precipitation deposited the active site of immobilized oxidase. Then X-ray microanalysis is employed to locate active site of immobilized enzyme on the electrode. The X-ray microanalysis exhibites the properties of the enzyme electrode depend on the distribution of enzyme activity on its surface in microscopic view. The distribution of activated enzyme on the enzyme electrodes prepared by different methods has been obtained. The results show that the form of carrier membrane and the method of immobilized enzyme effect on enzyme activity and its distribution on the enzyme electrode surface. This analysis method is helpful for preparation of the excellent enzyme biosensor. The novel GOD electrode has been applied for clinical assay of diabetic blood glucose. The recovery was found to be in the 95.5%-109.8%. The measuring results are in satisfactory agreement with those of the spectrophotometric enzyme-coupling method. The developing GOD electrode has also been used to determinate the glucose content in juices and beverages. In this experiment, the BSA electrode made similarly taking BSA instead of GOD, which serves as the reference electrode, has been employed to study the interference of matrix in real sample. The testing results show that matrix and interfering species do not produce any observable interference for the assay. The reproducibility is satisfactory and the recovery is found to be in the 102.1 %-104.2%.更多还原