基于小分子识别诱导的蛋白质超分子组装及表面固定研究

Protein immobilization on nonfouling substrate has important application significance. However, conventional methods based on complex chemical/physical process often suffers some serious drawbacks, and could not meet the requirements of high specificity and sensitivity. This project proposes an innovative design that is able to directly immobilize target biomolecules on nonfouling substrates from undiluted mixture solution (e.g. cell lysate) by utilizing a newly discovered small molecular-induced phase transition of lysozymes, therefore solving the core problem in this field.The research will firstly study how thes small molecules i.e. HEPES and TCEP could induce the phase transition of lysozyme under mild quasi-physiological conditions, and it is expected that small molecule-based specific recognition with lysozyme and subsequently resulting conformation change of lysozyme would be responsible to this phenomenon.After that, the research will move on the study of the phase transition product and its adhesion mechanism with nonfouling substrate, and thus describe at molecular level the self-assembling process during phase transition and interaction model with surfaces. On the basis of the above-mentioned studies, the research finally endevoures to establish a set of methodlogy that could directly immobilize target proteins on arbitrary substrates by using biotin-labeled lysozyme, streptavidin and biotin-labeled antigen/antibody.The establishment of this scientific method is expected to completely abandon the tedious activation and passivation steps used in traditional methods for protein immobilization on nonfouling substrates, so as to effectively solve the key issues in the field of surface immobilization of biomolecules and provide a scientific toolbox for directly fishing target proteins from the actual complex system with high sensitivity and specificity.

在零污染背景上固定蛋白质具有重要应用意义，但目前采用的化学/物理固定方法过程复杂，其特异性和灵敏度不能满足要求。本项目创新性地利用小分子（如HEPES和TCEP）对溶菌酶特异性识别及相转变的诱导作用，研究建立溶菌酶分子在零污染背景基材表面的直接固定方法，解决在零污染背景上固定目标蛋白质分子的关键核心难题。通过在分子水平上研究小分子对溶菌酶特异性识别和诱导其相转变的微观进程，以及相转变产物与包括零污染背景在内的各种表面之间的相互作用，阐明直接固定方法的作用机理。在实现溶菌酶相转变产物与零污染背景稳定结合的基础之上，通过生物素标记或溶菌酶与目标蛋白质偶联，研究建立一整套目标生物分子在零污染背景上直接固定的科学方法。该科学方法的建立拟完全摒弃传统方法中对零污染背景进行重复活化和钝化的繁琐步骤，从而有效解决生物分子表面固定中的关键问题，为从实际复杂体系中高灵敏度钩钓靶向蛋白质提供科学依据。

材料表界面改性具有重大的战略价值和社会经济意义。然而目前该领域存在如下三方面的关键难点：在化学结构调控方面，与无机、金属不同，高分子链结构的长程有序性控制极难，很难实现高复杂度和高规整性的高分子材料制备；在物理结构调控方面，微纳米加工所用刻蚀剂环保性差，绿色微纳米刻蚀技术缺乏；在化学和物理结构调控均涉及的方面，缺少对高分子、金属和无机都适合的普适性表面改性方法。我们设计和开发了三种策略以解决这三方面难点，包括：.第一，发现并提出基于蛋白质beta-sheet组装的大分子介晶概念，突破传统意义上的高分子链结构有序性控制；.第二，创制基于类淀粉样蛋白质组装的普适性材料表界面改性方法，实现了在包括零污染背景在内的各类表面上的蛋白质等功能分子和胶体的固定；.第三，基于核酸与贵金属的配位机制，发展材料表界面的绿色微纳米刻蚀及绿色提金技术。.References.Chem. Rev. 2013, 113, 5547; ACS Appl. Mater. Interf. 2014, 6, 3759; Macromol. Biosci. 2012, 12, 1053; Angew. Chem. Int. Ed. 2017, 10.1002/anie.201706843; Angew. Chem. Int. Ed. 2017, 56, 9331 (Hot Paper); Adv. Mater. 2016, 28, 579 (VIP paper); Adv. Mater. 2016, 28, 7414 (Frontispiece); Adv. Funct. Mater. in press