具有可逆物理交联结构的表面蛋白质印迹纳米粒子的可控合成及其表面功能化

Protein-imprinted polymer (PIP) nanoparticles have been considered to be promising substitutes for the costly antibodies now widely used in a variety of areas. Addressing the problems such as relatively low imprinting performance and difficulty in template removal and surface functionalization which hinder their practical applications, we put forward a design idea for constructing PIPs based on combining utilization of both reversible physical cross-links and chemical cross-links concurrently formed in the PIP matrix. According to this conception, we formulate a novel synthesis strategy based on aqueous surface-initiated RAFT precipitation polymerization. It primarily involves adoption of di(ethylene glycol) methyl ether methacrylate (MEO2MA), a thermo-responsive monomer with only proton receptors but without donor. Combined with other functional and cross-linking monomers in the presence of a protein template, MEO2MA is used to construct thermo-responsive PIP nanoshells containing the template via controlled aqueous precipitation polymerization over the nanoparticles, whose surface is modified with both template capturing groups and the RAFT agent. Besides the chemical cross-links resulting from the cross-linking monomer participating in the polymerization, reversible physical cross-links are also produced inside the imprinted shells based on the hydrophobic association among the formed polyMEO2MA chains. These physical cross-links together with the introduced template capturing groups are advantageous to the formation of high-quality imprinting sites inside the PIP shells. Also, dissociation of the reversible cross-links by lowering the temperature can facilitate the removal of the embedded template. Furthermore, surface functionalization of the PIP shells can be readily realized by utilization of the active RAFT ends attached on the shell surface and the well-known highly efficient thiol–ene click reactions, without significant impairing of the imprint sites formed beneath. The composition of the pre-polymerization system and modification of the support nanoparticles’ surface will be tailored according to the features of the typical proteins to be employed as templates, followed by tuning of the PIP shells’ structures and functionalization of their outer surface. As a result, the intrinsic dependence of the imprinting performance and surface-related characteristics of the core–shell protein-imprinted nanoparticles will be clarified, which is expected to the provide foundation for their practical applications in the future.

蛋白质印迹聚合物（PIP）纳米粒子有望在多个领域替代昂贵的抗体，但要付诸应用仍面临印迹性能较低、不易去除模板和表面功能化等问题。为此，提出同时利用化学交联和可逆物理交联构筑PIP的构想，设计了基于表面引发RAFT水相沉淀聚合的合成方法。采用仅含质子受体的温敏聚合物单体（MEO），联合其它功能单体和交联剂，在纳米粒子的表面修饰RAFT试剂和模板结合基团，再通过可控沉淀聚合接枝含有模板蛋白质的温敏PIP壳层。除化学交联外，壳层中还形成了基于聚MEO疏水缔合的可逆物理交联，加之模板结合基团，都有利于产生高效印迹位点。通过降温将物理交联解离，则有利于去除壳层中的模板。利用壳层表面的活性RAFT端基和巯基-烯烃点击易于进行表面功能化。将根据模板的性质，设计纳米载体的表面修饰和预聚合体系的组成，研究壳层的结构调控及其表面功能化，揭示影响印迹粒子的印迹性能及其表面相关特性的内在规律，为实际应用提供依据。

蛋白质印迹聚合物纳米粒子是具有良好应用前景的人工抗体，但目前的合成方法仍面临印迹性能较低、不易去除模板和表面功能化等问题。为此，我们提出基于仅含质子受体的温敏聚合物单体2-(2-甲氧基乙氧基)甲基丙烯酸乙酯（MEO2MA），采用表面引发RAFT水相沉淀聚合（SI-RAFT-APP）或自由基引发水相沉淀聚合（RI-APP）的方法，在纳米粒子表面接枝同时具有化学交联和可逆物理交联结构的温敏壳层。（1）利用铜催化叠氮-炔环化点击化学，合成了同时修饰RAFT试剂和模板结合基团的亲水性SiO2纳米粒子载体，其中RAFT试剂的接枝密度高达1.5个/nm2。（2）通过SI-RAFT-APP合成了核-壳表面蛋白质印迹纳米粒子。通过聚合时间可调控壳层的厚度，从而优化印迹性能。利用印迹壳层表面的RAFT端基，通过二次SI-RAFT-APP在其表面接枝PMEO2MA链，成功实现表面聚乙二醇（PEG）化，可大幅度提高印迹粒子的识别选择性，印迹因子可从2.1提高到9.1。（3）将MEO2MA的RI-APP与金属配位作用相结合，合成了高选择性和高灵敏度的荧光响应表面蛋白质印迹纳米粒子，印迹因子达5.8，检测限20 nmol。因大多数蛋白质表面具有可与过渡金属离子配位的组氨酸残基，故该方法具有通用性。（4）利用多巴胺自聚合的生物启发表面蛋白质印迹法已有大量的研究，但这种由聚多巴胺（PDA）构成的印迹层有两个重要缺陷：即较高非特异结合及其在酸性介质中欠稳定，对此一直没有好的解决办法。我们利用金属配位作用在PDA层表面引入可聚合双键，再利用RI-APP接枝交联PMEO2MA层，实现表面PEG化，从而简单有效地解决了这些问题，蛋白质印迹纳米粒子的印迹因子从2.6提高到6.4。该法也可应用于以其它生物大分子或小分子化合物为模板的PDA印迹层的功能化。以上研究结果为解决蛋白质印迹研究中存在的问题提供新的解决思路。