双孔道亲水性双连续聚苯乙烯整体柱的制备、结构调控及分离性能研究

Polymer monoliths are of particular interest as bioseparation supports for their major advantages in good permeability and rapid separation of biomacromolecules. However, there are still some ongoing challenges for polymer monoliths, i.e., the separation performance of polymer monoliths cannot match that of packed columns, the bad biocompatibility of some monoliths will result in denaturation of proteins, and the poor chemical stability of (meth) acrylate-based monoliths cannot promise their long run use. Inspired by bicontinuous microemulsion structure, we will develop a novel preparation method of polymer monoliths based on multifunctional amphiphilic diblock glycopolymers (ADG). Taking ADG as surfactant and aliphatic alcohol as cosurfactant, the hydrophilic bicontinuous polystyrene (PS) monoliths with dual pores were prepared via living polymerization of bicontinuous miniemulsion by coordinated regulation of macro and microphase separation during polymerization process. The monoliths have homogeneous skeletons, analogous polysaccharide surfaces and a balance separation efficiency and capacity. Also, the formation mechanism of bimodal pore and bicontinuous structure of monoliths will be studied in detail. In addition, we will put emphasis on studying the chromatographic performance of PS monolith for various molecular scales of biomacromolecules. In order to establish a relationship between thermodynamic parameters and separation results, Isothermal titration calorimetry (ITC) will be introduced to analyze the interaction between biomacromolecules and ligands, which will provide basic information to elucidate the high efficiency of separation mechanism and build separation model of PS monoliths. These results will lay theoretical foundation for the practical application of this novel PS monoliths.

聚合物整体柱作为生物大分子分离介质具有分离速度快、背压低、传质阻力小的优势，是生物技术下游产业一种高效节能的分离手段；但目前还存在分离效果无法与颗粒填充柱相比、生物相容性较差易使蛋白变性、化学稳定性差使用周期短等问题。本项目从微乳液两相连续体结构获得启发，提出一种基于多功能双亲性两嵌段含糖聚合物（ADG）为主导的新型整体柱制备方法。利用ADG协同脂肪醇，将活性聚合和双连续相微乳液聚合有机结合，通过协同调控聚合过程中的宏观/微观相分离行为，制备一种兼顾分离效率与分离容量、骨架结构均匀、表面性质类似多糖的双孔道亲水性双连续聚苯乙烯整体柱，探明整体柱双孔道双连续结构形成机理，研究介质对不同分子量生物大分子的分离效果，并引入等温滴定微量热法分析蛋白质与配基之间相互作用，实现微观热力学参数和宏观分离行为的关联，阐明其快速高效分离机理，建立分离模型，为新型聚苯乙烯整体柱的应用奠定理论基础。

有机聚合物整体柱制备方法简单、可选单体多元化、容易修饰衍生、机械强度和化学稳定性好，作为生物大分子分离介质具有分离速度快、背压低、传质阻力小的优势，是生物技术下游产业一种高效节能的分离手段，近年来受到持续关注。聚合物整体柱目前还存在分离效果无法与颗粒填充柱相比、生物相容性较差易使蛋白变性、化学稳定性差使用周期短等问题。本项目从微乳液两相连续体结构获得启发，提出一种基于多功能双亲性两嵌段含糖聚合物（ADG）为主导的新型整体柱制备方法。.首先采用两种活性自由基聚合技术-原子转移自由基聚合（ATRP）反应及可逆加成-断裂链转移聚合（RAFT）-合成了分子量和亲油亲水平衡（HLB）值可控的双亲性两嵌段含糖聚合物（ADG）。接着利用ADG和PF127通过油水界面协同自组装作用制备了双连续中内相乳液，采用倒置荧光显微镜和激光共聚焦显微镜系统考查了不同HLB值的ADG和PF127在油水界面的自组装对乳液结构的影响，为后期双连续整体柱的制备奠定了基础。最后以双连续中内相乳液为模板，采用ATRP/RAFT反应制备得到双孔道亲水性双连续聚苯乙烯整体柱。所得整体柱表面具有含糖聚合物，除了可以采用常规方法衍生之外，还可以直接作为亲水色谱模式分离糖蛋白。.本项目取得的创新性成果如下：1）首次通过ADG和PF27在油水界面的氢键自组装作用得到双连续中内相乳液（水滴细小且紧密相连），提出了双连续中内相乳液模型，对于制备双连续超大孔结构聚合物材料具有重要的指导意义；2）双孔道亲水性双连续聚苯乙烯整体柱兼顾分离效率与分离容量、骨架结构均匀、表面性质类似多糖，具有贯穿的超大孔（1.2 μm）和扩散介孔（50 nm）结构，在生物大分子色谱分离、细胞培养以及酶的固定化领域具有广阔的应用前景。后续试验中我们将进一步优化制备条件，争取早日实现分离应用。