乙酰胆碱酯酶相关物质的生物传感器检测

发布时间：2018-06-07 05:01

本文选题：乙酰胆碱酯酶 + 有机磷农药　；参考：《湖南大学》2012年硕士论文

【摘要】：乙酰胆碱酯酶(Acetylcholinesterase, AChE)是动物中枢神经系统中的一种催化酶，参与细胞的发育和成熟，能够促进神经元的发育和神经的再生。乙酰胆碱是乙酰胆碱酯酶的水解底物，是中枢胆碱能系统中重要的神经递质之一，其主要功能是维持意识的清醒，在学习和记忆中起重要作用。人的脑组织中有大量的乙酰胆碱，但乙酰胆碱的含量会随着年龄的增加而出现下降。有机磷与AChE结合会形成磷酰化ChE。磷酰化ChE很稳定，使酶失去催化水解乙酰胆碱的能力并逐渐老化，引起乙酰胆碱积聚，最终导致胆碱能神经先兴奋后抑制。阿尔茨海默病AD即老年痴呆症，是一种神经衰退症，严重影响人的认知、记忆、情感等功能。世界较为认可的AD病理为大脑内乙酰胆碱的缺失。目前，全世界的患病者已达到一千七百到二千五百万，给家庭和社会造成严重的负担。所以实现对影响乙酰胆碱酯酶活性以及与其相关物质的准确、快速、简单的检测，对于AD和很多神经疾病的发病机理研究及早期诊断治疗非常重要。因此，开发一些易于操作、价格低廉且又精确有效的乙酰胆碱酯酶传感技术意义重大。酶传感器技术因具有选择性好、结构简单、灵敏度高等优点已经被广泛应用。纳米金作为研究和应用最广泛的纳米材料之一，具有独特的光学、电学和催化性质。本文中，我们将酶传感器和纳米金信号放大技术有机地结合起来，构建了电化学和液晶酶传感器，实现对乙酰胆碱酯酶抑制剂、乙酰胆碱酯酶底物和乙酰胆碱酯酶抗体的灵敏检测。主要内容如下： (1)设计了利用乙酰胆碱酯酶的催化活性，水解底物氯化硫代乙酰胆碱生成硫代胆碱。硫代胆碱具有还原性，能够还原氯金酸生成纳米金颗粒。再将纳米金粒子作为铜沉积的催化剂和种子。铜纳米粒子在纳米金颗粒上长大后，利用溶出伏安法将沉积了的铜溶出。然后通过线性伏安扫描法定量检测沉积的铜得到的电化学溶出峰，来检测乙酰胆碱酯酶抑制剂有机磷，其浓度和减弱的溶出峰信号成正比。有机磷的检测下限达到0.02ng/mL。(第二章) (2)利用纳米金沉积信号放大的乙酰胆碱酯酶液晶传感器，实现对乙酰胆碱酯酶底物乙酰胆碱和乙酰胆碱酯酶抑制剂有机磷农药的低浓度检测。实验中利用氯化硫代乙酰胆碱与乙酰胆碱竞争与酶的结合，调控硫代胆碱还原生成的纳米金颗粒，来扰乱液晶分子的有序性排列，引起液晶的偏光效应。在偏光显微镜下观察，获得的偏光图像实现对乙酰胆碱的简单、灵敏、有效的检测，检测下限达到15nM/mL。乙酰胆碱酯酶的水解催化活性受有机磷农药的特异性抑制，，可以将其作为监控有机磷农药的靶标物质。有机磷农药抑制酶的水解活性，减弱酶对底物的水解，还原剂硫代胆碱就会相应减少，从而导致纳米金颗粒数量的减少，对液晶有序性的扰乱度也减弱，偏光效应也相应的减弱，最终实现对有机磷农药的检测,检测下限达到0.1ng/mL。此方法是一种简单、直观、快速且较灵敏地适用于乙酰胆碱和有机磷农药检测的技术，具有较强的通用性。(第三章) (3)建立了一种基于乙酰胆碱抗体催化活性的液晶传感器检测方法。该法考察了乙酰胆碱抗体酶具有和酶相似催化水解活性的特点，水解硫代乙酰胆碱，生成具有还原性的硫代胆碱，硫代胆碱还原氯金酸生成纳米金颗粒，构建纳米金信号放大的液晶生物传感器。通过在偏光显微镜下观察获得偏光图像，实现对乙酰胆碱酯酶抗体超低浓度的检测，检测下限达到0.01pg/mL。该方法比传统的方法更灵敏地检测了乙酰胆碱酯酶抗体，是一种价格低廉、选择性好、灵敏度高的适用于蛋白目标物分析检测的技术。(第四章)
[Abstract]:Acetylcholinesterase (AChE) is a catalytic enzyme in the central nervous system of animals. It participates in the development and maturation of cells and can promote the development of neurons and the regeneration of nerves. Acetylcholine is the substrate for the hydrolysis of acetylcholinesterase. It is one of the important neurotransmitters in the central cholinergic system. Its main function is There is a lot of acetylcholine in human brain tissue, but the content of acetylcholine will decrease with the increase of age. The combination of organophosphorus and AChE will form phosphorylated ChE. phosphorylated ChE, which causes the enzyme to lose the ability to catalyze the hydrolysis of acetylcholine and gradually aging. The accumulation of acetylcholine eventually causes the cholinergic nerve to first excited and suppressed. Alzheimer's disease AD, Alzheimer's disease, is a neurodegenerative disorder that seriously affects human cognition, memory, emotion and other functions. The world's more recognized AD pathology is the absence of acetylcholine in the brain. The disease of the world has reached one thousand and seven hundred to two thousand before the eyes. Five million, causing serious burdens to the family and society, so it is very important to realize the accurate, rapid and simple detection of acetylcholinesterase activity and its related substances, which is very important for the study of the pathogenesis of AD and many neural diseases and early diagnosis and treatment. Therefore, it is easy to operate, cheap and accurate and effective. The sensing technique of acetylcholinesterase is of great significance.
Enzyme sensor technology has been widely used because of its good selectivity, simple structure and high sensitivity. As one of the most widely used nanomaterials, nano gold has unique optical, electrical and catalytic properties. In this paper, we organically combine enzyme sensor and nano gold signal amplification technology to build electrochemistry. The sensor and the liquid crystal enzyme sensor are sensitive to acetylcholinesterase inhibitors, acetylcholinesterase substrates and acetylcholinesterase antibodies.
(1) the catalytic activity of acetylcholinesterase was designed, and the substrate was hydrolyzed to produce thiocholine by chlorination of thioacetylcholine. Thiocholine was reductive and able to reduce the formation of gold nanoparticles by chloroauric acid. Then the nano gold particles were used as the catalyst and seed of copper deposition. After the copper nanoparticles were grown on gold nanoparticles, the nanoparticles were dissolved and dissolved. An electrochemical dissolution peak of the deposited copper was detected by the method of linear voltammetry to detect the organic phosphorus of the acetylcholinesterase inhibitor, which was proportional to the signal of the dissolution peak. The lower limit of the detection of organophosphorus was 0.02ng/mL. (second chapters).
(2) a low concentration detection of acetylcholine acetylcholine and acetylcholinesterase inhibitor organophosphorus pesticides by using the acetylcholinesterase liquid crystal sensor amplified by gold nanoparticle deposition signal. In the experiment, the combination of thioacetylcholine chloride and acetylcholine is used to regulate the reduction of thiocholine by the binding of acetylcholine and acetylcholine. Gold particles, which disturb the ordered arrangement of liquid crystal molecules, cause the polarizing effect of liquid crystals. The polarizing images obtained under polarizing microscope can realize simple, sensitive and effective detection of acetylcholine, and detect the hydrolysis catalytic activity of 15nM/mL. acetylcholinesterase to be inhibited by the specific inhibition of organophosphorus pesticides. In order to monitor the target substance of organophosphorus pesticides, the hydrolytic activity of the organophosphorus pesticide inhibitor, the hydrolysis of the enzyme to the substrate and the reduction of the thiocholine of the reductant will be reduced, which leads to the decrease of the number of nano gold particles, the disturbance of the order of liquid crystal and the weakening of the polarization effect, and finally the detection of organophosphorus pesticides. It is a simple, intuitive, fast and sensitive technique for the detection of acetylcholine and organophosphorus pesticides, which has strong generality. (third chapter).
(3) a method for the detection of liquid crystal sensors based on the catalytic activity of acetylcholine antibody was established. The method investigated the characteristics of the acetylcholine antibody enzyme with enzyme similar catalytic hydrolysis activity, hydrolyzed thioacetylcholine, generated reductive thiocholine, thiocholine and prochloroauric acid produced nano gold particles, and constructed nanoscale gold signal. An amplified liquid crystal biosensor is used to detect the ultra low concentration of acetylcholinesterase antibody by observing the polarizing image under the polarizing microscope, and detecting the lower limit of 0.01pg/mL., which is more sensitive than the traditional method to detect the acetylcholinesterase antibody. It is a kind of low price, good selectivity and high sensitivity. Techniques for the analysis and detection of protein targets (Chapter fourth)
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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