以金纳米粒子为探针可视化监测丝素蛋白水溶液中组装行为的研究

In recent years, much attention has been paid to artificially preparation of high-performance and multi-functional materials from reconstituted silk proteins by biomimetic green process in materials research field. In order to reach the goal, it is essential to understand the self-assembling process of silk protein in aqueous solution. However, the underlying assembly mechanisms of silk proteins remains incompletely understood. A variety of methods have been developed to investigate the assembling process, however, most of them are difficult to monitor the assembly behavior directly and characterize the concentrated solution of silk fibroin. Gold nanoparticles(Au NPs) has been widely used for developing various chromatic sensors based on the reversible red-blue color changes between dispersion-aggregation states. Therefore, a new approach for sensing assembly behavior of silk fibroin in aqueous solution is expected to construct with in-situ Au NPs as sensitive indicator. Peptide composed of repetitive glycine-alanine, which obtained from the hydrolysate of Bombxy mori silkworm silk fibroin, is planned to use as simplified model of silk fibroin. Tyrosine residue is able to reduce Au(III) ions to Au NPs in situ and further stabilize them without any external reducing or capping reagents. The conformational change during the assembly process of peptide can be expressed by the color change or the UV-vis absorption spectra change of Au NPs. The influence of physiological factors including pH, metallic ions and concentrations of peptide on self-assembly behavior of peptide in aqueous solution will be studied. The methods for tracking the assembly behavior of peptide will be built based on Au NPs colorimetric assay. Furthermore, the assembly rule of concentrated solution of silk fibroin will be explored by mimicking the microenvironment of silk glands of Bombxy mori. It will be an attractive method used to investigate the assembly of silk fibroin for its simplicity, easy observation and no requirement of sophisticated instruments. This research will not only benefit us to deeply understand the self-assembling rule of silk fibroin, but also provides theoretical guidance for developing new materials from silk.

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近年来材料领域关注的热点，而对丝素蛋白在水溶液中组装过程的深入理解是实现这一目标的关键因素。目前对丝素蛋白组装机理的了解还不够完全，采用的研究方法难以直观监测组装过程以及表征高浓度丝素蛋白溶液。为此，本项目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。 本项目拟选用含甘氨酸与丙氨酸的重复序列多肽作为丝素蛋白的简化模型，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；进而通过模拟家蚕丝腺内的溶液微环境，探索高浓度丝素蛋白在水溶液中的组装规律。本项目将为研究丝素蛋白的溶液组装行为提供了一种简单直观的新方法，有利于组装规律的深入理解，并为人工制备新型丝基材料提供理论指导。

对丝素蛋白在水溶液中组装过程的深入理解是获取高性能、多功能丝基材料的关键因素。目前的研究方法难以直观监测丝素蛋白构象转变的组装行为，金纳米粒子分散与聚集状态发生的颜色改变广泛用于构建比色传感器，本项目采用金纳米粒子为探针来研究丝素蛋白的溶液组装行为。. 本项目主要开展了如下研究：. （1）金纳米粒子原位制备条件与性能之间关系的研究。采用β-折叠结晶区域的重复序列多肽作为丝素蛋白的简化模型，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上的探针。研究了不同制备条件包括多肽与氯金酸配比、溶液pH以及温度对金纳米粒子形态、分布以及稳定性的影响。确定最佳制备条件为多肽与氯金酸摩尔比为5：2，pH为10，温度为30℃。. （2）金纳米粒子为探针对多肽水溶液组装行为的研究。实验结果表明：当溶液pH由中性调节为酸性、加入Ca2+以及异丙醇条件下，体系溶液颜色发生由红色到紫色的转变，伴随紫外吸收峰的红移。透射电镜结果显示金纳米粒子沿多肽组装体分布，跟随多肽同步组装。原子力显微镜结果显示多肽自身在同样条件下也会发生组装，金纳米粒子对组装过程影响不大。建立了以金纳米粒子为探针直观监测多肽水溶液组装行为的方法。. （3）金纳米粒子为探针对丝素蛋白水溶液组装行为的研究探索。以丝素蛋白直接复合金纳米粒子为对照，直接复合体系在广泛pH范围以及高盐离子浓度下都保持稳定，而丝素蛋白原位复合金纳米粒子体系在酸性条件下会发生颜色的改变以及紫外吸收峰的红移。透射电镜结果表明：紫外吸收峰的红移是由于纳米粒子的长大而非粒子间距的改变，探针本身并不稳定。此外金纳米粒子对丝素蛋白的组装过程有明显影响，因而不适合作为监测丝素蛋白组装行为的探针使用。. 本项目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，可以作为其他测试手段的样品筛选方法，也适用于研究其他具有构象转变行为的多肽链段或结构简单的蛋白如β-淀粉样蛋白。