A microfluidic platform for producing nanoliter-scale double emulsion and its application in protein crystallization

• Other Authors: Zhu, Deyong (author.)
• Format: Others
• Language: English; Chinese
• Published: 2015
• Subjects: Microreactors; Emulsions; TP159.M53 Z58 2015eb
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1291466

This thesis describes the design and construction of a microfluidic platform for producing stable double emulsions and its application in protein crystallization. Emulsion-based systems has been widely utilized to encapsulate various substances in both industrial applications and fundamental researches. Microfluidics is a promising technique to handle small volumes of liquid and produce such kind of emulsion-based microreactors. === Two different types of microfluidic devices were constructed to generate double emulsions: one was the planar PDMS-based device, and the other was the 3D Glass-Teflon hybrid device. Both devices were demonstrated with the ability to produce a high level of monodispersity in the size distribution of double emulsions with volumes ranging from dozens to hundreds of nanoliter. Compared to the PDMS-based microfluidic devices which are commonly used in the microfluidic field but require sophisticated soft-lithography procedures and selective surface modifications, the Glass-Teflon hybrid microfluidic devices are more competitive in the aspects of low cost, ease of fabrication, reliable performance, and reusability. Double emulsions stored in confined environments were proved to be more stable than that in open environments. With the prolonged lifespan of double emulsions, long-term reactions could be conducted inside the microreactors. In addition, the permeability of the oil shell in double emulsions provided means to precisely control the bio/chemical environments of the core droplets by adjusting the composition of the oil phase or the osmotic pressure differences between the inner and outer phases. The microfluidic platform was established to be a simple and reliable tool for producing stable double emulsion-based microreactors. === We employed the microfluidic platform to perform protein crystallization in the inner aqueous phase of double emulsions.Double emulsions can provide ideal conditions to explore protein crystallization for the advantages of convection-free environment and homogeneous interface. Both vapor diffusion method and microbatch method were implemented in the double emulsion-based approaches using four model proteins to grow crystals successfully. The property of water-oil interface was demonstrated to be a critical factor for nucleation and appropriate surfactants should be chosen to prevent protein adsorption at interface. The results from the volume effect study showed a trend of fewer crystals formed and longer incubation time needed with a reduction in the protein solution volume, suggesting that the nucleation in protein crystallization process can be controlled by changing the volume of protein solutions.Finally, sparse matrix screening was achieved using the double emulsion-based microbatch method. The double emulsion-based approaches for protein crystallization were established to be a promising tool for reducing heterogeneous nucleation and gaining the chance to obtain one crystal in one reactor. === 本論文發展了一種用於產生和存儲穩定雙乳液（double emulsion）的微流控實驗平台，對其製備方法以及把雙乳液用作微型反應容器并在其內核進行蛋白質結晶做了系列的研究和探討。 === 首先，我們製作了兩種製備雙乳液的微流控裝置：第一種是基於聚二甲基硅氧烷（PDMS）的平面型微流控芯片；另一種是由Teflon管、硅膠管和玻璃毛細管組裝而成的三維結構微流控裝置。兩套裝置均可以產生不同納升體積的、具有高單一分散性的雙乳液。製作第一種微流控芯片需要軟光刻及選擇性表面修飾等相關的儀器和步驟；相比之下，第二種微流控裝置在製作消耗的時間和材料，製作簡易性、性能可靠性和可重複使用等方面證明有明顯優勢。通過把雙乳液以低密度存儲在受限容器中，雙乳液的壽命被顯著延長，為將其作為微型反應器提供了可能。此外，雙乳液的中間油層可以起到半透膜功能，可以用調節油相組成及內外水相滲透壓的方式精確控制雙乳液內核溶液的濃度環境。 === 雙乳液的內核是一個沒有對流影響的環境及具有高度均一的界面，可以為蛋白質結晶提供理想空間。我們以製備的雙乳液為微型反應容器，在其內核進行了一系列蛋白質結晶實驗。四種蛋白質分別以類氣相擴散方法（vapor diffusion method）和類微液滴方法（microbatch method）在納升量級進行了結晶實驗，并得到了良好的蛋白質晶體。雙乳液界面會對結晶過程產生明顯影響，在實驗中我們選用了適當的表面活性劑來阻止蛋白質分子的界面吸附。在研究體積效應對蛋白質結晶的影響中，我們發現隨著體積減小，在單一容器內會得到更少數目的晶體，但結晶時間會增加。這為通過控制蛋白質溶液體積來研究成核過程提供可能的途徑。最後，我們把這種基於雙乳液的微型反應容器應用到大數量的結晶條件篩選，通過偏光顯微鏡觀察對比，得到了與普通微液滴方法一致的結晶條件。這種基於雙乳液的微型反應容器為研究蛋白質結晶提供了一種可有效阻止異相成核的新方法。 === Zhu, Deyong. === Thesis Ph.D. Chinese University of Hong Kong 2015. === Includes bibliographical references (leaves 80-90). === Abstracts also in Chinese. === Title from PDF title page (viewed on 05, October, 2016). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.