银纳米颗粒—蛋白质量圈吸附性质及光谱特性的研究

发布时间：2018-02-05 20:10

本文关键词： 银纳米颗粒牛血清蛋白离散型共聚物荧光淬灭构象变化　出处：《内蒙古农业大学》2016年博士论文　论文类型：学位论文

【摘要】：有机分子特别是生物大分子与无机纳米颗粒形成的纳米结构具有优良特性,在生物医药研究及应用领域引起的关注日益增多,尤其是在血液中的检测、呈递、诊疗等方面得到了迅速的发展。血浆中含量最丰富的成分之一是血清白蛋白,无论是内源性物质还是外源性物质,包括纳米颗粒都容易与其发生相互作用。纳米颗粒作为药物载体进入血液后会不可避免地附着血清白蛋白,其功能不可避免会受到影响。因此,纳米颗粒与蛋白质相互作用研究是设计和构建基于纳米颗粒的生物传感器的先决条件,对于探索纳米材料在生命科学领域的应用具有重要意义。为了实现纳米颗粒在血液环境下的应用,本文对银纳米颗粒一一牛血清白蛋白晕圈这种复合物的特性及形成机制进行了系统的研究。利用双亲性聚合物(Amphiphilic polymer, AP)进行表面改性可以使疏水银纳米颗粒具有水溶性,在此过程中颗粒粒径变化较大,并引起此外最大吸收峰的轻微蓝移,然而外部形貌特征仍然比较规则且分散均匀。在水相环境中与血液相关的不同蛋白质混合,常温下反应,牛血清白蛋白(Bovine serum albuinin, BSA)表现出与双亲性聚合物包裹的银纳米颗粒具有突出的吸附特性,通过调节混合比例,能够得到吸附BSA数量可控的复合物。结合这种通用的表面修饰策略和物理吸附形成的纳米颗粒一蛋白复合物,双亲性聚合物的外壳不仅可以为纳米颗粒表面提供亲水基团,同时也形成了一个较厚的隔离层,蛋白分子并不直接接触金属纳米颗粒,弱化了金属本身与蛋白之间的相互作用,对于维持纳米颗粒一蛋白复合物稳定性至关重要。与化学耦合方法相比,物理吸附介导蛋白——纳米颗粒复合物拥有容易合成,结合稳定的以及可逆的偶联一解离等诸多的优势,在纳米生物医药领域具有更加广泛应用前景。纳米颗粒与离散蛋白质仅可以通过一个简单的混合过程,随后进行琼脂糖凝胶电泳分离,由此产生基于物理吸附形成稳定的复合物足以抵抗常见的凝胶电泳等分离技术,而不会造成纳米颗粒表面BSA的脱离。这种强烈相互作用,源于双亲性聚合物外壳赋予银颗粒的水溶性与颗粒表面同BSA分子形成物理吸附,两个因素之间相互平衡、共同作用。结合Stern-Volmer和Hill等方程并采用荧光猝灭的方法,系统地研究了两亲性聚合物包覆的纳米银颗粒与牛血清白蛋白之间的相互作用。这种结合力为1.30x107M-1并且其相互作用从热力学的观点来说是自发的通过物理吸附实现的。双亲性聚合物修饰的银纳米颗粒表面会形成蛋白晕圈,当精确控制摩尔比混合时这样的结构可以形成离散的纳米颗粒一蛋白共聚物。同时使用荧光光谱的方法研究结合纳米颗粒引起的蛋白构象的变化,结果显示纳米颗粒引起BSA的强烈物理吸附,蛋白的空间构象存在轻微的改变。
[Abstract]:Organic molecules, especially biomolecules and inorganic nanoparticles, have excellent properties, and have attracted increasing attention in biomedical research and applications, especially in the detection and presentation of blood. One of the most abundant components in plasma is serum albumin, whether endogenous or exogenous. Nanoparticles are easy to interact with them. As drug carriers, nanoparticles will inevitably adhere to serum albumin, and its function will inevitably be affected. The study of the interaction between nanoparticles and proteins is a prerequisite for the design and construction of biosensors based on nanoparticles. In order to realize the application of nanoparticles in blood environment, it is of great significance to explore the application of nanomaterials in the field of life science. In this paper, the characteristics and formation mechanism of silver nanoparticles, bovine serum albumin halo ring, were systematically studied. The amphiphilic polymer Amphiphilic polymer was used. The surface modification of APs can make the hydrophobic silver nanoparticles water-soluble, and the particle size changes greatly during the process, which also leads to the slight blue shift of the maximum absorption peak. However, the external morphologies are still regular and evenly dispersed. In aqueous environment, different proteins associated with blood are mixed and reacted at room temperature. Bovine serum albumin (BSA) exhibited outstanding adsorption properties with silver nanoparticles coated with amphiphilic polymers. By adjusting the mixing ratio, the adsorbed BSA complex with controllable number can be obtained, which combines the general surface modification strategy with the physical adsorption to form a nano-particle protein complex. The shell of amphiphilic polymer can not only provide hydrophilic groups on the surface of nanoparticles, but also form a thick isolation layer. Protein molecules do not directly contact metal nanoparticles. It weakens the interaction between metal and protein, which is very important to maintain the stability of nanoparticles and protein complexes, compared with the chemical coupling method. Physical adsorption mediated protein-nanoparticle complexes have many advantages, such as easy synthesis, stable and reversible coupling dissociation and so on. Nanoparticles and discrete proteins can only be separated by a simple mixing process and then separated by agarose gel electrophoresis. The formation of stable complexes based on physical adsorption is sufficient to resist common separation techniques such as gel electrophoresis without causing the separation of BSA on the surface of nanoparticles. The water solubility of silver particles is attributed to the amphiphilic polymer shell, and the surface of the particles is physically adsorbed with BSA molecules, and the two factors balance each other. Combined with Stern-Volmer and Hill equations and fluorescence quenching method. The interaction between amphiphilic polymer-coated silver nanoparticles and bovine serum albumin (BSA) has been systematically studied. The binding force is 1.30x107M-1 and the interaction is from a thermodynamic point of view. Amphiphilic polymer modified silver nanoparticles will form protein halos on the surface. When the molar ratio is accurately controlled, this structure can form a discrete nano-particle protein copolymer. Meanwhile, the conformational changes of proteins induced by the combination of nanoparticles are studied by fluorescence spectroscopy. The results showed that BSA was strongly physically adsorbed by nanoparticles and the spatial conformation of protein changed slightly.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TB383.1;Q51

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