聚多巴胺薄膜和蛋白质相互作用的原位SPR研究

发布时间：2018-05-08 18:18

本文选题：蛋白质 + 聚多巴胺薄膜　；参考：《西南大学》2017年硕士论文

【摘要】：蛋白质在固体表面上的吸脱附是很多基本生命过程如细胞粘附、信号传导、组织发育等的重要基本步骤,研究蛋白质和表面的相互作用不仅具有重要的基础意义,同时在医用诊断材料、生物芯片、细胞培养、药物输送等方面也有着广泛的应用价值。蛋白质在固体表面的吸附是受各种因素如蛋白质和材料自身的物理化学性质以及周围环境等综合影响的复杂过程,调控固体表面化学组成从而控制蛋白质的吸脱附是当今研究的热点。受贻贝粘附蛋白启发而人工合成的聚多巴胺(PDA)是一种具有良好的生物相容性、亲水性、粘附性和化学活性的聚合物材料,近年来作为多功能涂层已经广泛用于生物传感、药物输送、生物医学成像和组织工程等领域。由于含有邻苯二酚和醌类等官能团,PDA能够通过迈克尔加成或者席夫碱反应共价偶联蛋白质,可以作为通用的蛋白质固定的表面修饰层。蛋白质与PDA表面的相互作用涉及很多相关的生物应用,但其基本规律目前尚不清楚,对于蛋白质在PDA表面的结合密度、反应的动力学特征和固定的稳定性等问题仍然缺乏了解。同时,PDA的形成机理至今未有确切的定论,其表面的精确化学组成仍不清楚,而不同的制备条件也可能会影响PDA膜的厚度、表面粗糙度和化学成分;此外,外界环境的改变也可能会影响PDA膜中活性成分的含量,造成其表面化学成分发生变化,从而影响蛋白质在PDA膜表面上的固定。因此,亟需对蛋白质与PDA表面的相互作用进行实时在线的原位研究;另一方面,尽管PDA作为多功能的表面修饰层拥有独特的优势,但是与其它具有环氧基、羧基衍生的NHS酯或氨基的活性表面相比,PDA的蛋白质固定能力并不存在优势,这在一定程度上限制了它的应用。如果能在认识蛋白质与PDA表面的相互作用规律的基础上,对PDA的表面性质进行调控,进一步提高蛋白质的固定效率,则具有非常重要的意义。本硕士论文针对以上问题展开了系统性的研究,以实时无标记的表面等离子体共振技术(SPR)为主要研究手段,得出蛋白质与PDA表面相互作用的相关规律,并利用这些规律设计了一种增强蛋白质在PDA表面上固定能力的方法。主要为以下两个内容:(1)基于SPR技术探究了PDA膜上蛋白质的吸附行为、蛋白质-PDA相互作用的动力学以及外界因素(生长的介质和pH)对蛋白质吸附的影响。此外,还利用荧光蛋白微阵列技术对蛋白质与PDA相互作用做了定量研究,并与SPR的数据作对比,发现了PDA膜的荧光猝灭现象,随着PDA膜生长条件(生长的介质、pH和时间)的改变,荧光猝灭现象呈现规律性的变化。部分工作帮助解决了关于PDA-蛋白质相互作用长期存在的一些基本问题,将为相关应用提供有益参考。(2)基于电化学与表面等离子共振联用技术(EC-SPR),用电化学的方法调制PDA薄膜,增强了它的蛋白质固定能力。主要过程是通过电化学的方法使PDA中的活性成分对邻苯二酚/醌的平衡向醌基移动,提高PDA中活性醌基团的表面密度,进而改变蛋白质的固定能力。SPR不仅可以实时监测PDA的生长过程,而且还可以定量计算PDA膜上电化学调制前后蛋白质的固定量。研究结果表明,适当的电化学调制能够将PDA的蛋白质固定能力提高27%。此外,光谱分析也证实了电化学调制PDA膜的化学机理。这项工作提供了一种有效可行的方法,用以增强PDA膜上蛋白质的固定能力,为促进其各种生物相关的应用提供了有价值的指导。
[Abstract]:The absorption and desorption of protein on the solid surface is an important basic step in many basic life processes, such as cell adhesion, signal transduction, and tissue development. The study of the interaction of protein and surface is not only of important basic significance, but also widely used in medical diagnostic materials, biochips, cell culture, and drug delivery. The adsorption of proteins on the solid surface is a complex process affected by various factors, such as the physical and chemical properties of protein and material itself, and the surrounding environment. It is a hot spot to regulate the chemical composition of the solid surface and control the desorption of protein. Dopamine (PDA) is a kind of polymer material with good biocompatibility, hydrophilicity, adhesion and chemical activity. In recent years, as a multifunctional coating, it has been widely used in the fields of biosensing, drug delivery, biomedical imaging and tissue engineering. Because of the functional groups such as phthalic and quinones, PDA can be used by Michael. The covalent coupling of protein with Schiff base can be used as a common surface modification layer for protein immobilization. The interaction of protein and PDA surface involves many related biological applications, but its basic rules are not yet clear, for the binding density of the protein on the surface of the PDA, the kinetic characteristics of the reaction and the stability of the immobilization. There is still a lack of understanding. At the same time, the formation mechanism of PDA has not been definitive. The exact chemical composition of the surface is still unclear, and the different preparation conditions may also affect the thickness, surface roughness and chemical composition of the PDA film. In addition, the changes in the external environment may also affect the content of the active components in the PDA membrane, resulting in the surface of the membrane. The chemical composition of the surface changes, thus affecting the immobilization of protein on the surface of the PDA membrane. Therefore, it is urgent to study the interaction of the protein and the surface of the PDA in real time. On the other hand, although PDA has a unique advantage as a multifunctional surface modification layer, it is the NHS ester or ammonia derived from other epoxy groups with carboxyl groups. Compared with the active surface of the base, the protein immobilization ability of PDA does not exist, which limits its application to a certain extent. It is of great significance to regulate the surface properties of PDA on the basis of the understanding of the interaction between protein and PDA surface, and to further improve the immobilization efficiency of protein. In this paper, a systematic study is carried out on the above problems. Using the real time unmarked surface plasmon resonance (SPR) as the main research method, the relevant laws of the interaction between protein and PDA surface are obtained, and a method for enhancing the immobilization ability of protein on the surface of PDA is designed by using these rules. The main two methods are as follows. Content: (1) based on SPR technology, the adsorption behavior of protein on PDA membrane, kinetics of protein -PDA interaction and the influence of external factors (medium and pH) on protein adsorption were investigated. In addition, the interaction between protein and PDA was quantitatively studied by using fluorescent protein microarray technology, and the results were compared with the data of SPR. Fluorescence quenching of PDA film, with the change of PDA film growth conditions (medium, pH and time), the fluorescence quenching phenomenon is changed regularly. Some work helps to solve some basic problems on the long-term existence of PDA- protein interaction, and will provide useful reference for the related applications. (2) based on electrochemistry and surface isolation. The PDA film is modulated by electrochemical method (EC-SPR) to enhance its protein immobilization ability. The main process is to move the equilibrium of the active component in PDA to quinone by electrochemistry, to increase the surface density of the active quinone in the PDA, and to change the immobilization capacity of the protein,.SPR The growth process of PDA can only be monitored in real time, and the fixed amount of protein before and after the electrochemical modulation on the PDA membrane can be calculated. The results show that the appropriate electrochemical modulation can increase the protein immobilization ability of PDA in addition to 27%.. The spectral analysis also confirms the chemical mechanism of the electrochemically modulated PDA membrane. An effective and feasible method is used to enhance the immobilization capacity of proteins on PDA membrane and provide valuable guidance for the promotion of various biological applications.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O647.31

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