神经肽分子印迹聚合物阵列芯片的制备及荧光免疫分析研究

发布时间：2018-05-04 04:08

本文选题：缩胆囊素 + 脑啡肽　；参考：《武汉大学》2017年硕士论文

【摘要】：缩胆囊素神经肽和脑啡肽是广泛分布于中枢神经系统中的两类多功能性神经肽,对多种生理过程如神经传递,癫痫,抑郁,痛觉等有重要的调控作用。目前报道的脑脊液及血液中缩胆囊素神经肽和脑啡肽的定量主要基于放射免疫法和酶联免疫法,这些免疫分析方法虽然灵敏度高,但需使用昂贵的抗体,且操作繁琐,存在辐射污染等问题。因此,研发经济、高特异性的人工抗体来代替生物抗体用于多肽及蛋白质的分析检测具有重大意义。分子印迹聚合物生物芯片是将分子印迹技术和生物芯片相结合,对目标分子具有专一性识别能力的新型生物芯片,兼具分子印迹聚合物高特异性,耐酸碱,抗高温和生物芯片高通量,快速,微量消耗的特点。本研究将分子印迹聚合物的高选择性识别能力、微阵列芯片和微孔板芯片的高通量以及荧光检测的高灵敏度三大优势相结合,研究制备脑啡肽分子印迹聚合物阵列芯片、缩胆囊素分子印迹聚合物微孔板芯片和缩胆囊素分子印迹膜,基于此阵列芯片和微孔板芯片建立了荧光免疫分析方法对人脑脊液中低丰度的缩胆囊素神经肽和脑啡肽进行定量分析。本文主要进行了以下研究:1.研究制备脑啡肽分子印迹聚合物微阵列芯片。将光刻技术与抗原决定簇法相结合,通过光刻掩膜法在硅烷化的玻片表面原位聚合制备对脑啡肽具有特异性识别能力的分子印迹聚合物微阵列芯片。研究并优化印迹聚合物阵列芯片合成制备条件。借助分子模拟,计算模板-单体复合物之间的结合能;用扫描电镜和红外光谱等对印迹材料进行表征。以平衡吸附实验和MALDI-TOF-MS分析研究评估印迹材料特异性及专一性。最后基于该印迹聚合物芯片,研究建立了固相竞争荧光免疫分析方法,利用倒置荧光显微镜—CCD图像分析系统对人脑脊液中低丰度脑啡肽进行定量分析。2.缩胆囊素神经肽分子印迹聚合物微孔板芯片的制备及荧光免疫分析应用。采用抗原决定簇法沉淀聚合制备对缩胆囊素神经肽具有特异性识别能力的分子印迹聚合物微球,以此MIP微球代替昂贵的缩胆囊素神经肽抗体,用聚乙烯醇(PVA)固定于96孔板中,制备缩胆囊素神经肽分子印迹聚合物微孔板芯片。优化印迹聚合物微孔板芯片的合成制备条件,用扫描电镜对印迹微球进行表征。以平衡吸附实验探索印迹材料特异性及选择性。最后基于分子印迹聚合物微孔板芯片,研究建立固相竞争荧光免疫分析方法,利用倒置荧光显微镜—CCD图像分析系统对人脑脊液中缩胆囊素神经肽进行定量分析。3.缩胆囊素神经肽分子印迹聚合膜的制备及其荧光免疫分析应用。采用抗原决定簇法,在硅烷化的玻片表面原位合成制备对缩胆囊素神经肽具有特异性识别能力的分子印迹膜。优化印迹膜合成制备条件,并用扫描电镜和红外光谱对其进行表征,通过吸附平衡实验对印迹膜的吸附容量及选择性进行评价。最后基于该分子印迹膜,研究建立固相竞争荧光免疫分析方法,利用倒置荧光显微镜—CCD图像分析系统对人脑脊液中缩胆囊素神经肽进行定量分析。
[Abstract]:Cholecystokinin and enkephalin are two kinds of multifunctional neuropeptides widely distributed in the central nervous system. They have important regulatory effects on many physiological processes, such as neurotransmission, epilepsy, depression, and pain. The current reported quantitative of cholecystokinin and enkephalin in cerebrospinal fluid and blood is mainly based on radioimmunoassay and enzyme. Although these immunoassays are sensitive, they need to use expensive antibodies and have complicated operation and radiation pollution. Therefore, it is of great significance to develop economic, highly specific artificial antibodies instead of biological antibodies for the analysis and detection of peptides and proteins. Molecular imprinted polymer biochips are the molecules of the molecular imprinted polymer. A new biochip with the ability to identify the target molecules with the specificity of the molecularly imprinted polymer, with the characteristics of high specificity of molecularly imprinted polymers, acid resistance and alkali resistance, high temperature resistance and high throughput, rapid and micro consumption of biochips. The high selectivity recognition ability of molecularly imprinted polymers, microarray chips and micropores in this study The high throughput of the chip and the high sensitivity of the fluorescence detection are combined to study the preparation of the enkephalin molecularly imprinted polymer array chip, the molecular imprinted polymer microchip and the cholecystokinin molecularly imprinted membrane. Based on this array chip and microplate chip, the fluorescence immunoassay method is established for the human cerebrospinal fluid (CSF). The quantitative analysis of the moderate and low abundance of cholecystokinin neuropeptides and enkephalin was carried out. The following studies were carried out in this paper: 1. the preparation of enkephalin molecularly imprinted polymer microarray was studied. The photolithography technique was combined with the antigen determinant method to prepare enkephalin on the surface of silanized glass by photolithography. Molecularly imprinted polymer microarray for sexual recognition. Study and optimize the synthesis conditions of imprinted polymer array chips. By means of molecular simulation, the binding energy between template and monomers is calculated. The imprinted materials are characterized by scanning electron microscopy and infrared spectroscopy. The evaluation of the adsorption and MALDI-TOF-MS analysis is evaluated by the scanning electron microscopy and infrared spectroscopy. The specificity and specificity of the imprinted material. Finally, based on the imprinted polymer chip, the solid phase competitive fluorescence immunoassay was established, and the quantitative analysis of the low abundance enkephalin in the human cerebrospinal fluid by the inverted fluorescence microscope CCD image analysis system was used to prepare the.2. cholecystokinin neuropeptide molecularly imprinted polymer microplate chip The molecularly imprinted polymer microspheres with specific recognition ability for cholecystokinin neuropeptides were prepared by precipitation polymerization with antigenic determinant method. The MIP microspheres were replaced by the expensive cholecystokinin neuropeptide antibody and polyvinyl alcohol (PVA) was immobilized in 96 pore plates to prepare the cholecystokinin neuropeptide molecularly imprinted polymer. Microporous plate chip. Optimization of synthesis conditions of imprinted polymer microplate chips, characterization of imprinted microspheres by scanning electron microscopy. The specificity and selectivity of imprinted materials are explored by equilibrium adsorption experiments. Finally, based on molecularly imprinted polymer microplate chips, solid phase competitive fluorescence immunoassay method was established and inverted fluorescence was used. Quantitative analysis of cholecystokinin neuropeptide in human cerebrospinal fluid by micromirror CCD image analysis system the preparation of.3. cholecystokinin neuropeptide molecularly imprinted polymer membrane and its application in fluorescence immunoassay. The preparation conditions of the imprinted membrane were optimized, and the preparation conditions were synthesized and characterized by scanning electron microscope and infrared spectrum. The adsorption capacity and selectivity of the imprinted membrane were evaluated by adsorption equilibrium experiment. Finally, based on the molecularly imprinted membrane, the solid phase competitive fluorescence immunoassay method was established, and the inverted fluorescence microscope CCD image was used. A systematic analysis of cholecystokinin neuropeptides in human cerebrospinal fluid was carried out.

【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O631.3;O657.3

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