超大孔微球作为蛋白质、核酸等生物大分子的分离纯化介质及固定化酶载体具有很多独特的优势，这些优势使得超大孔微球受到越来越多的重视。在超大孔微球的制备方法中，本课题组发展了一种新颖的反胶团溶胀法，能够制得孔径达500 nm、粒径在30-150 μm可控的大孔微球。与其他方法相比，具有制备简便、重现性好等优点。但是制备得到的产物为弱聚集的微球，虽然用很小的研磨力就能够简单地将微球分散，但当规模制备时会严重影响成本与效率。针对反胶团溶胀法制备超大孔微球发生弱聚集的问题，本文提出了一种新的解决思路。通 过对微球形成弱聚集的机理分析发现，在大孔形成过程中内外水相发生连通、在大孔形成部位原有的油水界面缺失是导致弱聚集发生的原因。如果能在大孔形成的位 置重构界面，应该可以达到减弱聚集的目的。因此，本研究提出双水相的构建策略，一相为油滴内的内水相，一相为外水相，利用双水相之间的界面在大孔位置重构 界面，使稳定剂可以在这个界面处吸附，达到减弱聚集的目的。 论文的工作分为四个部分。 第一部分为构成双水相材料的选择和相图绘制。考察了PEG与葡聚糖、PVA、硫酸铵等构建双水相的材料，以及外界温度对于分相情况的影响。根据分相时溶液的粘度、微球制备的水相情况等的要求，优选出双水相的构建材料为PEG 20000和PVA 217，相图的绘制温度为85°C。通过分相法得到了含有系线的相图。绘制的相图用以指导后续实验利用双水相进行微球的制备过程。 第二部分主要是根据反胶团溶胀法制备超大孔微球的机理，在油滴内外水相连通部位，利用双水相重新构建界面并进行聚（苯乙烯-二乙烯基苯）（P(ST-DVB)） 超大孔微球制备工艺的探索。根据第一部分绘制的相图，对于分相区内的任意一点，由相图可以得到分相之后上下两相的浓度情况和体积之比。据此，设计了先溶胀 后聚合的两步法，使得溶胀吸收的水相为双水相的其中一相，而聚合时的外水相为另一相，由此可构建出界面在大孔表面的双水相体系。利用这种方法，制得的微球 团聚程度有了一定的减弱，但得到的微球粒径与孔径都不是十分理想，需要进一步的优化。 第三部分在双水相的基础上，考察了乳化方法、表面活性剂浓度和溶胀时间等对于微球粒径和孔径的影响。通过对各因素的考察和优化，最终确定了乳化方法为筛网乳化法、表面活性剂质量/单体-交联剂质量为55%，溶胀时间为5min，由此得到的微球其粒径为30 μm左右，孔径为 150 nm左右，均一程度较好且几乎没有发生弱聚集。 第四部分以第三部分PST-DVB超大孔微球的制备为基础，使用甲基丙烯酸缩水甘油酯作为单体进行聚（甲基丙烯酸缩水甘油酯-二乙烯基苯）（P(GMA-DVB)）超大孔微球的制备。观察结果发现，以双水相为基础制备的微球聚集程度有所减弱，但同时粒径大幅度降低，需要通过后续的优化以达到更好的制备结果。 根据弱聚集现象发生机理的分析，通过双水相的构建并对反胶团溶胀法的过程进行优化，能够得到弱聚集程度有效减低的P(ST-DVB)超大孔微球，将该方法应用到规模制备上将有望减少成本、提高微球制备的效率。

Gigaporous microspheres have unique advantages in enzymes immobilization and purification of biomacromolecule such as protein and nucleic acid. There are different methods to make gigaporous microspheres, one of them, named surfactant reverse micelles swelling method had a controllable pore size of 500 nm and particle size of 30-100 μm. This method is convenient for product and has good reproducibility, but there is a problem in production that weak-aggregation of microspheres happened in the preparation process. Though the aggregates could be easily re-dispersed by gentle grinding, it will be a big difficulty for scale up. In order to solve this problem, a new method was presented. The mechanism of the weak-aggregation was that the water inside and outside the pore connected when the gigapore was formed, makes the interface of the oil phase and the water phase lose. If a new interface at the place are rebuilt, the aggregation might be prevented. Thus, an aqueous two phase system (ATPS) was chosen. If the water phase inside and outside the pore can be constructed into ATPS, the interface of the pore can be rebuilt to let the stabilizer absorb on, and the aggregation can be reduced. The work of this paper divided into four parts. The first part was the construction of the ATPS and the phase diagram. The materials such as PEG/Dextran, PEG/PVA, PEG/(NH4)2SO4 were tested to build an ATPS, and the influence of temperature was studied. By considering the viscosity of water and the requirement in microspheres preparation, PEG 20000 and PVA 217 were chosen to build the ATPS, and the temperature was 85°C. By extracting the two phases respectively and measuring the concentration of each phase, the phase diagram with tie-line was determined. This phase diagram could use to guide the producing of microspheres. The second part was according to the mechanism of the reverse micelles swelling method to make the gigaporous microspheres. the ATPS was used to rebuild a new interface between the inner and outer pore, then the poly(styrene-divinyl benzene) [P(ST-DVB)] gigaporous microspheres were prepared. From the phase diagram, the concentrations and volume ratio of the top phase and the bottom phase of each point in the phase separation area can be ensured. According to these properties, a two-step method was put forward to rebuild the interface. The water swelling in the pores was one phase of ATPS, and the outer water phase when polymerization was the other one. The aggregation was reduced by this method, but on the other hand, the particle size and pore size was reduced at the same time, an optimization was needed. Based on the ATPS, the third part was the optimization of the particle size and pore size of the microspheres by changing the emulsification method, the concentration of surfactant and the swelling time. After a series of optimization, the emulsification method was determined as the screen method, the amount of Span 80 was 55% based on the total amount of styrene and divinyl benzene, the swelling time was 5 minutes. The microspheres had uniform particle size about 30 μm, pore size about 150 nm, and no aggregation appeared. The forth part was the preparation of poly(glycidyl methacrylate-divinyl benzene) [P(GMA-DVB)] gigaporous microspheres on the basis of P(ST-DVB) microspheres. The weak aggregation was decreased in this system, and a decrease of particle size happened. A subsequent optimization was needed. By using ATPS to optimization the reverse micelles swelling method, the gigapourous microspheres with less aggregation was got. The cost was reduced and the efficiency was raised.