糖基化金纳米粒子的制备及其与伴刀豆球蛋白A相互作用的研究

发布时间：2018-01-30 10:36

本文关键词： 糖基化金纳米粒子特异性识别表面等离子体共振　出处：《东北师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：糖基化纳米粒子是一种重要的具有生物活性的粒子,它能够通过多价交互的方式增强对蛋白质的特异性识别能力,因此在糖科学和生物医学等领域具有重要的应用价值。金纳米粒子由于具有尺寸小、比表面积大等优点,使其具有独特的光学、热学、催化等特性。糖类是一种有机碳水化合物,不仅是人体生命活动的主要能量来源,并且在细胞的生命活动中具有重要的功能,包括细胞增殖、细胞识别、病原体感染与信号传递等。研究细胞表面的糖与蛋白质的特异性识别作用(CPIs)有助于了解肿瘤的产生与转移过程。为了研究糖与蛋白质的相互作用,本文制备了三种糖基化金纳米粒子纳米复合材料,利用金纳米粒子的表面等离子体共振(SPR)特性,定性分析糖基化纳米粒子与伴刀豆球蛋白A(ConA)特异性绑定结合力的强弱。利用浊度法计算糖基化金纳米粒子与ConA的表面解离常数,比较不同种类的糖与蛋白质的绑定结合力大小,最后利用金纳米棒的光热效应,通过生物实验研究糖基化金纳米粒子靶向细胞的能力。本论文的具体研究内容包括:(1)首先利用晶种生长法制备了粒径均一、分散性良好的两种形状(棒形、球形)的金纳米粒子,通过改变加入晶种的量,调控金纳米粒子的尺寸,研究了加入反应参数的量如AgNO3、AA和CTAB的量对金纳米棒SPR吸收峰位的影响。随后又引入FeCl3刻蚀法在室温下动态调控金纳米棒的形貌,这种金属离子刻蚀反应优先发生在金纳米棒的尖端,通过研究反应机理得到氧化产物,并研究了刻蚀剂浓度、刻蚀时间、CTAB浓度以及反应温度对反应速率的影响。相比于晶种生长法,刻蚀法更加快速方便的获得不同形貌的金纳米粒子。(2)以金纳米粒子为核,通过共价耦合以及金纳米粒子催化协同作用,制备了三种糖包覆的金纳米粒子(Glc-、Lac-、Gal-)。通过对其进行Zeta电位和FTIR表征,得到糖分子成功作用在金纳米粒子表面,研究实验反应温度与加入糖的浓度对粒子SPR吸收峰位的影响。此外,还研究了糖基化金纳米粒子在不同盐溶液和pH值溶液中的稳定性,结果发现粒子在复杂的环境下不发生聚集,保持良好的稳定性,糖基化提高了金纳米粒子的稳定性。通过MTT法对其进行体外细胞毒性测试,实验结果表明糖基化金纳米粒子对细胞没有毒性,对生物体安全,糖基化提高了金纳米粒子的生物相容性。(3)选用ConA作为模型蛋白,评价前面准备的三种糖基化金纳米粒子的生物活性。通过对其进行UV-vis、TEM、DLS等表征,定性比较三种糖与蛋白质绑定结合力的强弱关系。比较发现,Glc-AuNPs与ConA相互作用力最强。以葡萄糖糖基化的金纳米粒子为例,讨论了这种绑定结合力的强弱与糖包覆密度、加入蛋白质浓度的关系。此外还研究了糖基化金纳米粒子对蛋白的特异识别能力,定量计算了糖基化金纳米粒子与ConA的表面解离常数(Kd),计算结果与定性分析结果一致。最后利用金纳米粒子的光热效应,采用MTT法检测其体外细胞毒性,证明了糖基化金纳米粒子对细胞具有靶向识别功能,未来在疾病治疗方面具有广阔的应用前景。
[Abstract]:Glycosylation of nanoparticles is an important bioactive particle, it can enhance the ability to identify specific proteins by multivalent interaction, so it has important application value in the fields of biomedical science and sugar. Gold nanoparticles because of its small size, large surface area etc., make its unique the optical, thermal and catalytic properties. Sugar is a carbohydrate, is not only the main energy source of human life, and has important function in cellular activity, including cell proliferation, cell recognition, pathogen infection and signal transfer. Specific recognition of cell surface carbohydrate and protein (CPIs) contributes to the understanding of tumor formation and metastasis process. In order to study the interaction of sugar and protein, we prepared three kinds of glycosylation of gold nanoparticles nano composite The use of materials, the surface plasmon resonance of gold nanoparticles (SPR) characteristics, qualitative analysis of glycosylation of nanoparticles and concanavalin A (ConA) specific binding force. The surface dissociation constants of gold nanoparticles and ConA calculation of sugar based by the turbidity method, comparison of different sugars and protein binding finally the use of force, the photothermal effects of gold nanorods, gold nanoparticles targeting ability of cells through biological experimental study of carbohydrate. The specific research contents of this thesis include: (1) the seeding growth was prepared by the method of uniform size and dispersion of two kinds of good shape (cylindrical, spherical) gold nanoparticles, by changing the seeding quantity control of gold nanoparticles size, to study the reaction parameters such as AgNO3, AA and CTAB absorption peak of the influence of the gold nanorods with SPR. After the introduction of FeCl3 etching method In the dynamic regulation of gold nanorod morphology at room temperature, the metal ion etching reaction occurs preferentially in the gold nanorod tip, obtained by studying the reaction mechanism of oxidation products, and study the etchant concentration, etching time, CTAB concentration and reaction temperature on the reaction rate. Compared to the seed growth method, etching method is more rapid and convenient the different morphologies of gold nanoparticles. (2) using gold nanoparticles as the core, the synergistic effect of gold nanoparticles through covalent coupling and catalysis, three kinds of sugar coated gold nanoparticles were prepared (Glc-, Lac-, Gal-). The Zeta potential and FTIR characterization of the sugar molecules successful role in gold nanoparticles surface study on the experiment, the reaction temperature and adding sugar concentration effect of absorption peak of SPR particles. In addition, also study the glycosylation of gold nanoparticles in aqueous solution is stable in different salt solution and pH And found that the particles do not occur together in a complex environment, good stability, glycosylation improves the stability of gold nanoparticles. The in vitro cytotoxicity test by the MTT method, the experimental results show that glycosylation of gold nanoparticles had no cytotoxicity to the organism, safety, improving the compatibility of glycosylation gold nanoparticles. (3) ConA was chosen as the model protein, three kinds of sugar based evaluation of gold nanoparticles prepared in front of the biological activity. Through UV-vis, the TEM, DLS characterization, qualitative comparison of three kinds of sugar and protein binding force. In comparison, Glc-AuNPs and ConA the strongest force to glucose. The glycosylation of gold nanoparticles as an example, discusses the strength and the binding force of the sugar coating density, the relationship between the addition of protein concentration. Furthermore glycosylation of gold nanoparticles Specific recognition ability of protein surface, quantitative calculation of the dissociation constants of glycosylation of gold nanoparticles and ConA (Kd), the calculation results and the qualitative analysis results. Finally the photothermal effects of gold nanoparticles, to detect the cytotoxicity by MTT method, proved that the glycosylation of gold nanoparticles on the cell targeted identification function and has a wide application prospect in the treatment of disease in future.

【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.1;O614.123

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