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题名	尺寸均一可控的琼脂糖凝胶微球的制备及应用研究
作者	周青竹
学位类别	博士
答辩日期	2008-06-05
授予单位	中国科学院过程工程研究所
授予地点	过程工程研究所
导师	马光辉
关键词	微孔膜乳化 琼脂糖凝胶微球 粒径均一 细胞载体 分离介质
其他题名	Preparation and Application of Uniform-sized Controllable Agarose Gel Beads
学位专业	生物化工
中文摘要	随着人们对生物材料研究的日益深入，天然多糖类材料－琼脂糖凝胶的用途越来越广泛。本论文针对传统方法难以制备粒径均一、可控的琼脂糖凝胶微球的问题，提出用微孔膜乳化法制备琼脂糖微球。使用常规膜乳化法制备了粒径均一的微球，之后针对传统制备方法及其常规膜乳化法难以制备小粒径、高琼脂糖含量微球的问题，使用快速膜乳化法制备粒径在10微米以下，琼脂糖含量高达14wt%的琼脂糖微球， 并对膜乳化法制备琼脂糖凝胶微球过程中影响粒径和均一性的因素进行了讨论。最后将膜乳化法制备的琼脂糖微球分别用于细胞载体和分离介质，并对琼脂糖凝胶微球的交联过程进行了研究。 论文工作分为五个部分。第一部分提出用常规膜乳化法制备琼脂糖凝胶微球，系统研究了制备的工艺条件。通过对油相、乳化剂种类及其用量、膜乳化温度、膜孔径及其孔径分布和膜孔形状对琼脂糖凝胶微球粒径及其均一性的影响进行了考察，并从油水相之间界面张力的角度对形成乳液的均一性进行了解释，优化了制备条件，得到平均粒径在15-60微米之间、粒径分布系数小于15%的琼脂糖凝胶微球。第二部分针对常规膜乳化法难以制备平均粒径在10微米以下的琼脂糖凝胶微球，以及当琼脂糖含量较高时所制备的琼脂糖凝胶微球粒径均一性不好的问题，使用快速膜乳化法制备得到小粒径、高浓度的琼脂糖微球，并对制备过程中影响粒径及其均一性的各影响因素进行了系统的研究。发现在快速膜乳化法制备琼脂糖凝胶微球时，操作过程的参数、乳液的配方、和膜有关的参数都可能会对快速膜乳化法所制备的微球的粒径和均一性产生影响。其中，跨膜压力、跨膜次数、油相配方、油相中乳化剂浓度、水相中琼脂糖浓度以及水相和膜表面的接触角均会影响粒径和均一性。研究还发现：快速膜乳化法制备琼脂糖微球时，膜孔径和微球的数平均粒径之间也存在线性关系。第三部分将常规膜乳化法用于制备用作细胞载体的琼脂糖凝胶微球。将取自废水处理厂的活性污泥稀释后的细菌使用常规膜乳化法用琼脂糖微球包埋起来装填入柱体进行培养。当微球内的细菌生长后，再通过流式细胞仪将长菌的微球分选后进行进一步的培养和鉴定，用以研究污泥中细菌种群的微生物多样性。使用常规膜乳化法制备条件温和，被包裹的细菌能够保持其生物活性，并且所制备的微囊粒径均一，所制备的琼脂糖微球填入柱状容器经过14天的培养后其中所包埋的细菌量有所增加，通过流氏细胞仪能成功地将包裹不同细菌的微囊分开。第四部分将膜乳化法制备得到的琼脂糖凝胶微球进行化学交联，分别采用程序升温交联法、反复交联法和两步交联法对膜乳化法制备得到的琼脂糖微球进行交联研究，比较了不同的交联方法制备得到的微球机械强度的差异，讨论不同的交联方法对交联效果的影响，发现在优化交联条件的情况下，采用两步交联法可以得到与杭州争光和GE生产的6FF琼脂糖微球相近的机械强度。第五部分将第四部分交联后的琼脂糖微球进行烯丙基活化，发现可以通过优化得到具有不同烯丙基密度的活化琼脂糖凝胶，在活化后的琼脂糖凝胶上接上离子交换基团可以制备离子交换介质，对所制备的强阳离子交换介质进行蛋白吸附量的考察发现所制备的强阳离子交换介质具有和商品介质相当的离子交换容量和蛋白吸附量。 研究表明，使用不同的膜乳化法可以制备得到粒径均一、可控、且具有各种不同平均粒径和琼脂糖含量的琼脂糖凝胶微球。所制备的琼脂糖凝胶微球可用做包埋细菌的细胞载体，也可以通过化学交联、活化、偶联接入配基用作蛋白质分离纯化的介质。
英文摘要	A natural polysaccharide material-agarose gel has been widely used in biochemistry and biochemical engineering. However, the size distribution of agarose gel beads prepared by conventional methods is broad, and the size of beads is difficult to control. The objective of this dissertation are to develop a novel preparation method-micro-porous membrane emulsification to prepare uniform-sized and size-controllable agarose gel beads, suitable for applications as cells carriers and chromatography media. The dissertation is divided into five parts. The first part focused on the preparation of agarose gel beads with uniform and controllable size by general membrane emulsification. The effect of oil phase, emulsifier and temperature on the uniformity of the beads were investigated and interpreted from interfacial tension between the water phase and the oil phase. The uniform-sized agarose gel beads with different diameters from 15 to 60 μm were successfully prepared and the coefficient of variation (CV) value was less than 15%. However, agarose beads with smaller size and high agarose content can not be prepared by the general membrane emulsification. The second part investigated the preparation of uniform-sized agarose beads with size smaller than 10 μm and high agarose content by premix membrane emulsification. The effects of preparation conditions and emulsion composition on emulsification results were examined. It was found that the transmembrane pressure, number of passes, composition of oil phase, concentration of PO-500 and concentration of agarose in the water phase, all affected the size and uniformity. The investigation on membrane parameters showed that the pore size distribution and the shape of pore opening did not affect the emulsification results apparently within a wide range of membrane pore size distribution, the contact angle between the water phase and the membrane surface must be large enough in order to obtain uniform-sized agarose beads. The results also showed that there was a linear relationship between the average number diameter of agarose beads and pore size of membranes in premix membrane emulsification. In the third part, bacterial cells from suspensions of sludge after dilution were encapsulated in uniform-sized agarose microcapsules prepared by general membrane emulsification method. These agarose gel beads containing bacterial cells were transferred into a column vessel and were incubated to form micro-colonies. Then, the cultured microcapsules were analyzed by flow cytometry to investigate the biological diversity of cells from sludge. The result showed that mild membrane emulsification process preserved the viability of cells and this method was effective to create niches for various bacterial cells growing in high throughput cultivation vessels. The fourth part dealt with cross-linking of agarose gel beads. Agrose gel beads prepared by micro-porous membrane emulsification were cross-linked by gradually heated cross-linking method, repeated cross-linking method and two-step cross-linking method, respectively. The effect of different cross-linking methods on the mechanical stability was studied. It was found that the agarose gel beads cross-linked by two-step cross-linking showed the best mechanical stability. In the last part, agarose gel beads after cross-linked were allylated, then -SO3- groups were coupled on allylated agarose gel beads, and the strong cation-exchanger, SP-agarose was obtained. It indicated that agarose beads prepared in this study were suitable for matrix of separation medium. In conclusion, micro-porous membrane emulsification is a potential technique to prepare uniform-sized and size-controllable agarose gel beads for application as cells carriers and chromatography media.
语种	中文
公开日期	2013-09-13
页码	150
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1180]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	周青竹. 尺寸均一可控的琼脂糖凝胶微球的制备及应用研究[D]. 过程工程研究所. 中国科学院过程工程研究所. 2008.

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