本文選題：乙酰膽堿酯酶 + 有機(jī)磷農(nóng)藥　； 參考：《湖南大學(xué)》2012年碩士論文

【摘要】：乙酰膽堿酯酶(Acetylcholinesterase, AChE)是動(dòng)物中樞神經(jīng)系統(tǒng)中的一種催化酶，參與細(xì)胞的發(fā)育和成熟，能夠促進(jìn)神經(jīng)元的發(fā)育和神經(jīng)的再生。乙酰膽堿是乙酰膽堿酯酶的水解底物，是中樞膽堿能系統(tǒng)中重要的神經(jīng)遞質(zhì)之一，其主要功能是維持意識(shí)的清醒，在學(xué)習(xí)和記憶中起重要作用。人的腦組織中有大量的乙酰膽堿，但乙酰膽堿的含量會(huì)隨著年齡的增加而出現(xiàn)下降。有機(jī)磷與AChE結(jié)合會(huì)形成磷�；疌hE。磷�；疌hE很穩(wěn)定，使酶失去催化水解乙酰膽堿的能力并逐漸老化，引起乙酰膽堿積聚，最終導(dǎo)致膽堿能神經(jīng)先興奮后抑制。阿爾茨海默病AD即老年癡呆癥，是一種神經(jīng)衰退癥，嚴(yán)重影響人的認(rèn)知、記憶、情感等功能。世界較為認(rèn)可的AD病理為大腦內(nèi)乙酰膽堿的缺失。目前，全世界的患病者已達(dá)到一千七百到二千五百萬(wàn)，給家庭和社會(huì)造成嚴(yán)重的負(fù)擔(dān)。所以實(shí)現(xiàn)對(duì)影響乙酰膽堿酯酶活性以及與其相關(guān)物質(zhì)的準(zhǔn)確、快速、簡(jiǎn)單的檢測(cè)，對(duì)于AD和很多神經(jīng)疾病的發(fā)病機(jī)理研究及早期診斷治療非常重要。因此，開(kāi)發(fā)一些易于操作、價(jià)格低廉且又精確有效的乙酰膽堿酯酶?jìng)鞲屑夹g(shù)意義重大。 酶?jìng)鞲衅骷夹g(shù)因具有選擇性好、結(jié)構(gòu)簡(jiǎn)單、靈敏度高等優(yōu)點(diǎn)已經(jīng)被廣泛應(yīng)用。納米金作為研究和應(yīng)用最廣泛的納米材料之一，具有獨(dú)特的光學(xué)、電學(xué)和催化性質(zhì)。本文中，我們將酶?jìng)鞲衅骱图{米金信號(hào)放大技術(shù)有機(jī)地結(jié)合起來(lái)，構(gòu)建了電化學(xué)和液晶酶?jìng)鞲衅�，�?shí)現(xiàn)對(duì)乙酰膽堿酯酶抑制劑、乙酰膽堿酯酶底物和乙酰膽堿酯酶抗體的靈敏檢測(cè)。主要內(nèi)容如下： (1)設(shè)計(jì)了利用乙酰膽堿酯酶的催化活性，水解底物氯化硫代乙酰膽堿生成硫代膽堿。硫代膽堿具有還原性，能夠還原氯金酸生成納米金顆粒。再將納米金粒子作為銅沉積的催化劑和種子。銅納米粒子在納米金顆粒上長(zhǎng)大后，利用溶出伏安法將沉積了的銅溶出。然后通過(guò)線性伏安掃描法定量檢測(cè)沉積的銅得到的電化學(xué)溶出峰，來(lái)檢測(cè)乙酰膽堿酯酶抑制劑有機(jī)磷，其濃度和減弱的溶出峰信號(hào)成正比。有機(jī)磷的檢測(cè)下限達(dá)到0.02ng/mL。(第二章) (2)利用納米金沉積信號(hào)放大的乙酰膽堿酯酶液晶傳感器，實(shí)現(xiàn)對(duì)乙酰膽堿酯酶底物乙酰膽堿和乙酰膽堿酯酶抑制劑有機(jī)磷農(nóng)藥的低濃度檢測(cè)。實(shí)驗(yàn)中利用氯化硫代乙酰膽堿與乙酰膽堿競(jìng)爭(zhēng)與酶的結(jié)合，調(diào)控硫代膽堿還原生成的納米金顆粒，來(lái)擾亂液晶分子的有序性排列，引起液晶的偏光效應(yīng)。在偏光顯微鏡下觀察，獲得的偏光圖像實(shí)現(xiàn)對(duì)乙酰膽堿的簡(jiǎn)單、靈敏、有效的檢測(cè)，檢測(cè)下限達(dá)到15nM/mL。乙酰膽堿酯酶的水解催化活性受有機(jī)磷農(nóng)藥的特異性抑制，，可以將其作為監(jiān)控有機(jī)磷農(nóng)藥的靶標(biāo)物質(zhì)。有機(jī)磷農(nóng)藥抑制酶的水解活性，減弱酶對(duì)底物的水解，還原劑硫代膽堿就會(huì)相應(yīng)減少，從而導(dǎo)致納米金顆粒數(shù)量的減少，對(duì)液晶有序性的擾亂度也減弱，偏光效應(yīng)也相應(yīng)的減弱，最終實(shí)現(xiàn)對(duì)有機(jī)磷農(nóng)藥的檢測(cè),檢測(cè)下限達(dá)到0.1ng/mL。此方法是一種簡(jiǎn)單、直觀、快速且較靈敏地適用于乙酰膽堿和有機(jī)磷農(nóng)藥檢測(cè)的技術(shù)，具有較強(qiáng)的通用性。(第三章) (3)建立了一種基于乙酰膽堿抗體催化活性的液晶傳感器檢測(cè)方法。該法考察了乙酰膽堿抗體酶具有和酶相似催化水解活性的特點(diǎn)，水解硫代乙酰膽堿，生成具有還原性的硫代膽堿，硫代膽堿還原氯金酸生成納米金顆粒，構(gòu)建納米金信號(hào)放大的液晶生物傳感器。通過(guò)在偏光顯微鏡下觀察獲得偏光圖像，實(shí)現(xiàn)對(duì)乙酰膽堿酯酶抗體超低濃度的檢測(cè)，檢測(cè)下限達(dá)到0.01pg/mL。該方法比傳統(tǒng)的方法更靈敏地檢測(cè)了乙酰膽堿酯酶抗體，是一種價(jià)格低廉、選擇性好、靈敏度高的適用于蛋白目標(biāo)物分析檢測(cè)的技術(shù)。(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)
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 丁強(qiáng),左謙,丁銘臣,董秀敏,沈正萱;癡呆患者腦脊液膽堿酯酶活性初步研究[J];北京醫(yī)學(xué);1992年05期

2 陳玲;;生物傳感器的研究進(jìn)展綜述[J];傳感器與微系統(tǒng);2006年09期

3 唐建設(shè),項(xiàng)麗;毛細(xì)管電泳在農(nóng)藥殘留檢測(cè)上的應(yīng)用[J];分析測(cè)試技術(shù)與儀器;2005年03期

4 許翠玲,王改萍,胡勝水;有機(jī)磷農(nóng)藥生物電化學(xué)傳感器的研究進(jìn)展[J];分析科學(xué)學(xué)報(bào);2002年06期

5 黨育平,張亮,武彩霞;應(yīng)用定量免疫印跡技術(shù)分析人類自身反應(yīng)抗體庫(kù)[J];國(guó)外醫(yī)學(xué)(免疫學(xué)分冊(cè));2002年04期

6 王文雷,趙明輝,袁偉棟;毛細(xì)管膠束電動(dòng)色譜法檢測(cè)4種有機(jī)磷農(nóng)藥[J];化學(xué)分析計(jì)量;2005年05期

7 李平;張建林;王愛(ài)勤;;殼聚糖及其衍生物在分析化學(xué)中的應(yīng)用[J];化學(xué)進(jìn)展;2006年04期

8 曾艷;宋豐;王麗榮;陸光漢;;甲殼素和殼聚糖在電化學(xué)分析中的應(yīng)用進(jìn)展[J];化學(xué)研究與應(yīng)用;2005年06期

9 袁愛(ài)華;汪萍;沈小平;;一維納米結(jié)構(gòu)材料制備方法的研究進(jìn)展[J];化學(xué)研究與應(yīng)用;2006年02期

10 朱美財(cái),辛顏彬,孫曼霽,方允中;人腦乙酰膽堿酯酶的分離純化及特性[J];中國(guó)科學(xué)(B輯 化學(xué) 生命科學(xué) 地學(xué));1992年08期

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