以金纳米粒子为探针可视化监测丝蛋白多肽的自组装研究

发布时间：2018-04-03 02:36

本文选题：丝素蛋白　切入点：多肽　出处：《太原理工大学》2017年硕士论文

【摘要】：家蚕丝素蛋白是一类有着独特的氨基酸组成和结晶结构的动物蛋白,因具有良好的生物相容性、优异的机械性能、无毒和可生物降解等特点而广泛的应用于生物医学、光学和组织工程等领域。以再生丝素蛋白或重组丝蛋白溶液作为原材料,通过仿生的绿色化学方法获取高性能、多功能的丝基材料是实现丝素蛋白应用的基础。丝素蛋白在水溶液中的自组装机理对于深入了解其结构与功能之间的关系具有十分重要的指导意义。目前对丝素蛋白在水溶液中的组装过程和机制没有统一和清晰的认识,采用的研究方法也难以方便直观地监测自组装的过程。金纳米粒子具有与其形状、粒径和聚集程度相关的表面等离子共振效应,金纳米粒子发生聚集时会引发颗粒间的表面等离子耦合,表现出肉眼可见的红色到紫色或蓝色的变化。这种效应的应用逐渐发展成为以金纳米粒子为探针的比色检测方法,并应用于核酸、小分子和蛋白质等物质的比色检测。为此,本文提出了以金纳米粒子为探针研究丝素蛋白水溶液中组装行为的新思路。在家蚕丝素蛋白的整个序列中,高度重复的六肽单元GAGAGS能够形成反平行的β-折叠结构,进而构成丝蛋白的结晶区域,而另外一种六肽单元GAGAGY则有可能构成了其非结晶区。近年来以丝素蛋白序列中某些具有代表性的多肽序列作为简化模型来研究丝素蛋白的组装机理成为一种趋势。本文选用十四肽(GAGSGAGAGSGAGY,GY-14)和八肽(GAGAGAGY,GY-8)分别作为丝素蛋白晶区与非晶区的简化模型,并以多肽末端的酪氨酸原位还原的金纳米粒子作为固定在多肽模型上的探针。通过溶液颜色以及紫外吸收峰的改变,研究外源性因素对多肽溶液组装行为的影响规律,建立以金纳米粒子为探针研究多肽在水溶液中组装行为的方法。本文开展了以下研究:(1)金纳米粒子原位制备条件与性能之间关系的研究:分别以多肽GY-8和GY-14作为还原剂,原位还原氯金酸合成了稳定分散的球形金纳米粒子,并研究了多肽与氯金酸的配比、溶液p H和温度等不同制备条件对金纳米粒子形态、分布以及稳定性的影响。通过紫外-可见吸收光谱和透射电镜表征确定了最佳的制备条件:以多肽GY-8为还原剂时,多肽与氯金酸摩尔比为5:2,p H为9,温度为40℃;以多肽GY-14为还原剂时,多肽与氯金酸摩尔比为1:1,p H为7,温度为35℃。(2)以金纳米粒子为探针对多肽水溶液组装行为的研究。分别研究了多肽GY-14在p H、金属离子浓度以及溶剂组成等外源性因素影响下的自组装行为,并应用紫外光谱、红外光谱、透射电镜和原子力显微镜等方法对组装规律以及组装结构进行了表征。结果表明:多肽GY-14在p H 4的酸性水溶液中构象由无规卷曲转变为β-折叠,溶液由红色变为紫色,紫外吸收峰红移。Ca2+的引入会诱导多肽构象发生转变,当离子浓度为5 m M和10 m M时,多肽自组装形成由粒子连接而成的串珠状的β-折叠结构,溶液分别由红色变为紫色和蓝色,吸收峰红移。异丙醇溶液的加入使得多肽从无规卷曲构象转变为β-折叠构象,同样形成串珠状组装体,溶液中出现紫色絮状物并在短时间内沉淀,紫外吸收峰基本消失。本文以金纳米粒子为探针,将多肽GY-14在水溶液中的组装行为通过溶液颜色的改变以及紫外吸收峰的移动表现出来,为研究多肽的溶液组装行为提供了一种简单直观的新途径,为进一步研究丝素蛋白构象转变机制和过程提供指导。
[Abstract]:Silk fibroin is a kind of a unique amino acid composition and crystal structure of animal protein, with good biocompatibility, excellent mechanical properties, non-toxic and biodegradable characteristics widely used in biomedical, optical and tissue engineering and other fields. The regenerated silk fibroin or recombinant silk protein solution as a raw material, to obtain high performance by the green chemistry method of bionic, multifunctional material filaments is the basis for the application of fibroin protein. The silk fibroin in aqueous solution of the self-assembly mechanism has a very important significance for understanding the relationship between its structure and function. The silk fibroin assembly in water in the solution process and the mechanism is not clear and unified understanding, the research methods are also difficult to conveniently and visually monitoring a process of self-assembly. The gold nanoparticles have its shape, size and The surface plasmon resonance effect is related to the degree of aggregation and aggregation of gold nanoparticles leads to surface plasmon coupling between particles, exhibit visible red to purple or blue. The application of this effect has gradually become the gold nanoparticle colorimetric method for measuring probe, and applied to the nucleic acid, low molecular weight and protein the colorimetric detection is proposed in this paper. Therefore, using gold nanoparticles as a new idea of assembling behavior of silk fibroin solution probe in water. In the entire sequence of silk fibroin, height six peptide unit GAGAGS repeats to form antiparallel beta folding structure, and thus constitute the crystalline regions of silk, and another six GAGAGY peptide unit is likely to constitute the non crystalline region. In the recent years on the silk fibroin peptide sequence of protein sequences in some representative as a simplified model to The assembly mechanism of silk fibroin has become a trend. This paper selected fourteen peptides (GAGSGAGAGSGAGY, GY-14) and eight (GAGAGAGY, GY-8) peptides were used as the silk fibroin crystalline region and amorphous region of the simplified model, and the in situ reduction of tyrosine peptide gold nanoparticles as probes immobilized on the polypeptide model. The color of the solution. And the ultraviolet absorption peak changes, the influence of exogenous factors on the peptide solution assembly behavior, build with gold nanoparticle assembly of peptide probe method in aqueous solution. In this paper, we conducted the following research: (1) study on the relationship between gold nanoparticles in situ preparation conditions and performance respectively with polypeptides GY-8 and GY-14 as the reducing agent, in situ reduction of chloroauric acid stable dispersion of spherical gold nanoparticles were synthesized, and studied the ratio of peptides with chloroauric acid solution, P H and different temperature etc. The morphology of gold nanoparticles preparation conditions, distribution and stability. The influence by UV Vis absorption spectroscopy and transmission electron microscopy characterization of the best preparation condition was determined: the peptide GY-8 as reducing agent, polypeptide and chloroauric acid molar ratio of 5:2, P and H was 9, the temperature of 40 DEG GY-14 for reduction in polypeptide; agent, polypeptide and chloroauric acid molar ratio of 1:1, P and H was 7, the temperature of 35 DEG. (2) research on probe polypeptide assembly behavior in aqueous solution by using gold nanoparticles as GY-14 in P H. The peptide self-assembly behavior were studied, the effect of metal ions concentration and solvent composition under the exogenous factors then, using ultraviolet spectrum, infrared spectroscopy, transmission electron microscopy and atomic force microscopy were used to characterize the assembly rules and assembly structure. The results show that the conformation of peptide GY-14 in P H 4 acid solution by random coil transition folding beta, the solution from red to purple The introduction of color, UV absorption peak red shift.Ca2+ will induce peptide conformational change when the ion concentration of 5 m M and 10 m M, peptide self-assembly formed by particles formed by connecting the beaded beta folding structure, solution was changed from red to purple and blue absorption peak red shift. Isopropanol the solution of adding polypeptides from random coil conformation into p-sheet conformation, the same form of beaded assembly, solution purple floc and precipitation in a short period of time, the ultraviolet absorption peak disappeared. In this paper, gold nanoparticles as probe, the assembly of peptide GY-14 in aqueous solution by the color of the solution change and UV absorption peak of mobile performance, provides a new way for the solution of a simple polypeptide assembly, to provide guidance for further research on the conformation transition of silk fibroin mechanism and process.

【学位授予单位】：太原理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.1;O629.7

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