【作者】 彭运平；

【导师】 于淑娟；

【作者基本信息】 华南理工大学 ， 制糖工程， 2013， 博士

【摘要】 纳米微球胶体金和胶乳颗粒作为功能性载体材料已广泛的应用于细胞生物学、生命科学、临床医学、免疫诊断等领域，其与靶标物质免疫技术、侧向层析微流控技术联合形成的免疫层析诊断技术及产品，具有反应迅速、操作简单、灵敏度高及稳定性好等优点，被广泛应用于靶向研究及临床、家庭诊断产品中。但基于纳米金和纳米胶乳微球的免疫层析技术也存在灵敏度不高（假阴）及特异性（假阳）问题，以及灵敏度和特异性如何平衡及根本原因不清楚等问题。所以，研究纳米免疫微球制备及应用过程中发生的各种变化及其影响因素，探索假阴和假阳现象的本质原因，对于促进纳米微球及免疫层析技术的进一步应用显得至关重要。本研究选取两类纳米微球材料胶体金和胶乳颗粒，与目标抗体蛋白通过吸附或共价偶联制备对应的免疫微球，通过透射电镜、Zeta电位及动态光散射等分析方法对免疫微球的稳定性进行比较分析，结果表明：胶体金免疫微球（最适pH值为5.0、最佳蛋白浓度为20μg/mL）及胶乳免疫微球（以EDC/NHS为活化剂、活化时间15min、采用pH6.0MES缓冲反应体系、偶联时间2h和抗体蛋白浓度为800μg/mL）在上述条件下，电镜观察显示免疫胶体金和免疫胶乳复合物形成，Zeta电位变化结果表明两类微球与抗体蛋白作用后都处于较稳定的状态，动态光散射结果显示免疫胶体金微球的水合粒径随着pH值的增加而逐渐减小，而免疫胶乳微球的水合粒径随着抗体蛋白浓度的增加而逐渐增加。采用傅立叶变换红外光谱技术对不同结合条件下制备的免疫胶体金和免疫胶乳上抗体蛋白二级结构的变化进行表征，利用计算机辅助分析技术发现，pH值的升高，微球浓度的增加，抗体蛋白浓度的增加，都可以使抗体蛋白的有序结构含量增加。利用荧光光谱法对两类免疫微球中微球与抗体之间的相互作用机理进行了探讨，结果表明：两类免疫微球对抗体蛋白的内源荧光有显著的猝灭作用，猝灭机制均为静态猝灭；胶体金与抗体蛋白的结合是一个自发的物理吸附过程，主要通过疏水作用力和氢键结合，胶乳颗粒-抗体蛋白复合物则主要是静电相互作用。这些相互作用力使抗体蛋白的三级结构发生变化，其抗体分子上的氨基酸处于更为疏水的环境中，从而导致抗体蛋白表面疏水性也发生相应的变化。利用纳米金和纳米胶乳颗粒两种微球分别研制免疫层析检测方法及产品，并对两类免疫微球上抗体蛋白的分子结构与灵敏度、特异性的关系进行了研究，结果表明：免疫胶体金和免疫胶乳上抗体蛋白的分子结构与灵敏度和特异性间有显著的关系，一定条件下有序结构含量的增加及无序结构含量的减少会使抗体蛋白的灵敏度和特异性都升高。更多还原

【Abstract】 In recent years, nanospheres colloidal gold and latex as the functional carrier materialare widely used in cell biology, life sciences, clinical medicine, immune diagnosis and otherfields. The immunochromatographic diagnostic technology and related products came out forthe combination of nanospheres, target substance immunoassay and lateral flow microfluidictechnology. It has the advantage of quick response, simple operation, high sensitivity andgood stability, and has widely used in targeted research and clinical, home-version diagnostickits. However, the deficiencies such as low sensitivity(false negative), specific problems(falsepositive), the problems of how to balance the relationship between sensitivity and specificity,and what was the root cause of the appearance of false negative and false positivephenomenon, which showed adverse impact in the application of immunochromatographicassay. Therefore, in order to promote the further application of nanospheres andimmunochromatographic assay, it is very important to research the preparation ofnano-immunomicrosphere, to study various changes in the process of application and itsinfluential factors, to explore the nature of false negative and false positive phenomenon.In this paper, two kinds of nanospheres material, colloidal gold and latex, were selectedto react with target protein. Immune colloidal gold and immunolatex were prepared by themethods of physical adsorption and covalent coupling, respectively. The stability ofimmunomicrospheres were also studied by transmission electron microscope, Zeta potentialand dynamic light scattering. Results showed that immune colloidal gold (the optimum pHvalue5.0and the optimal protein concentration20μg/mL) and immunolatex (activatorEDC/NHS, active time15min, the reaction buffer pH value6.0MES, coupling time2.5h,and the antibody concentration800μg/mL) complex could be clearly observed by electronmicroscopy. Zeta potential results showed that the colloidal gold and latex microspheres werein a relatively stable state after they were coupled with antibody protein. Dynamic lightscattering showed that the hydrated particle size of immune colloidal gold graduallydecreased as the pH increased, and the hydrated particle size of immunolatex increased as theantibody protein concentration gradually increased.The secondary structure of antibody protein on nano-immunomicrospheres, prepared in different conditions, was detailed using FTIR combining with computer aided analysistechnology. Results showed that the content of ordered structure of antibody protein increasedalong with the increase of pH value, microspheres concentration and antibody proteinconcentration.The interaction mechanism between nanospheres and antibody protein was revealed byfluorescence spectrum technology. Results showed that colloidal gold and latex nanosphereshad a significant quenching effect on the intrinsic fluorescence of antibody protein. Thequenching mechanisms were all static quenching. However, the interaction force between thetwo kinds of nanospheres and antibody protein were different. The binding process ofcolloidal gold and antibody protein was a physical spontaneous adsorption process, in whichhydrophobic force and hydrogen bonds played a major role. The main interaction betweenlatex nanospheres and antibody protein was electrostatic interaction. These interaction forceschanged the tertiary structure of antibody protein to some extent, the hydrophobicity of themicroenvironment around the amino acids residues increased, and made a significant changein the hydrophobicity of antibody protein.The colloidal gold and latex immunochromatographic assay strips were prepared by thetwo kinds of nanospheres, and the relationship between the structure of antibody protein andsensitivity and specificity were detailed. Results showed that the structure of antibody proteincombined with nanospheres had a significant relationship with the sensitivity and specificityof the test strips, which were enhanced when the content of ordered structure of antibodyprotein increased.更多还原