【作者】 丁家旺；

【导师】 杜丹；

【作者基本信息】 华中师范大学 ， 分析化学， 2008， 硕士

【摘要】 酶生物传感器自发现以来,因具有高度的选择性、结构简单、自动、价廉而备受研究者的关注。特别是微电子技术、纳米材料制备技术、生物技术的发展为扩展生物传感器的应用范围、批量生产、集成化、微型化打下了坚实的基础,极大地促进了酶生物传感器的研究与应用。基于酶的生物传感器已经广泛用于农药的检测,尤其是以乙酰胆碱酯酶（AChE）的催化活性为基础的抑制型酶电极已大量应用于氨基甲酸酯和有机磷类农药的检测。酶的固定技术是制备生物传感器的关键步骤,这将直接影响传感器的稳定性、灵敏度、检出限、响应时间和酶的活性、存活时间等。酶的固定技术主要包括酶的固定方法和固定酶的载体材料的选择,当前国内外对生物传感器中酶的固定技术进行了广泛的研究,并取得了许多重要进展。电沉积是一种电化学过程,也是一种氧化还原过程。电化学诱导的聚合物沉积已经用于生物分子的固定。壳聚糖是一种具有良好生物相容性的线型聚合物,分子中的—NH₂能通过离子吸附的方式有效地应用于固定酶技术,从而作为生物传感器的材料。本论文一方面基于电沉积构建了三种生物界面,（1）壳聚糖（2）金纳米-壳聚糖复合膜（3）金纳米-自组装膜。我们在这些生物兼容膜上固定乙酰胆碱酯酶,用于农药的检测,检出限可达ng/mL级。另一方面,我们利用乙酰胆碱酯酶催化底物氯化乙酰硫代胆碱,产生的硫代胆碱的特性进行农药检测。（1）硫代胆碱具有还原性,硫代胆碱可以还原溶液中的四氯化金在界面生成金纳米,生成的金纳米可以调控界面的电子传导,基于铁氰化钾探针电流的变化建立了农药测定的电化学传感器。（2）用硫代胆碱在电极表面的化学吸附和脱附过程,构建了乙酰胆碱酯酶生物传感器,用于高灵敏农药检测。我们构建的生物传感器,一方面为农药残留速测问题的解决提供了一种新的方法。另一方面据不同种类农药的生物活性不同以及它们与靶标酶作用的机理和结合能力不同,可以用于农药和药物的高通量筛选,具有快速、灵敏、简便和不受筛选试材生长状况影响等优点。更多还原

【Abstract】 Enzymes are the most powerful molecular recognition elements in biosensing, since the sensing signal can be amplified millions of times through enzymatic reactions. This signal amplification enables the designing of highly sensitive biosensing systems. Amperometric enzymatic biosensors have been considered to be the most suitable for biochemical analysis due to their good selectivity, sensitivity, rapid response, miniature size, and reproducible results. A combination of enzymatic reactions with electrochemical method allowed to develop different enzyme based electrochemical biosensors for sensitive and rapid determination of environmental analysis. Among these, amperometric acetylcholinesterase (AChE) biosensors based on the inhibition of AChE have shown satisfactory results for pesticides analysis, in which the enzymatic activity is employed as an indicator of quantitative measurement of insecticides.A significant challenge to development of sensitive and stable sensors comes from effective immobilization of enzyme to solid electrode surface. Electrochemical deposition has been reported recently as an effective immobilization method for enzyme due to the formation of sol-gel films.In our experiment, on the one hand, three different kinds of interfaces were constructed based on the electrodeposition of chitosan or gold nanoparticles. These interfaces displayed excellent biocompatibility and good stability for immobilization of AChE. Under the optimal electrodeposition, amperometric sensors for the fast determination of pesticide were developed. On the other hand, the immobilized AChE mediated hydrolysis of ATC1 and yielded a reducing agent thiocholine. A simple strategy for the design of an electrochemical sensor based on enzyme-induced growth of gold nanoparticles (AuNPs) without adding gold nano-seeds was proposed.Chemisorption/desorption process seems to be useful for determination of thiol compounds (and thiol-producing catalysts) with high sensitivity. We also applied the chemisorption/desorption process of thiocholine on a gold nanoparticles modified electrode to determine pesticide.To sum up, these proposed AChE biosensors could be readily extended toward the determination of pesticide and the investigation of medicine or pesticide sensitivity.更多还原