這篇論文研究了幾種基於聚二甲基硅氧烷（PDMS）的微流控芯片，及其對於酶反應和蛋白質結晶的應用。 / 本論文分為兩部份。首先，論文第一部份介紹了兩種微流控芯片。利用到PDMS透氣的性質，第一種微流控芯片是一種基於脫氣PDMS的納升液體進液系統。在第一種微流控芯片的基礎上，我們引進一種可由普通注射器控制的氣閥控制系統，可對反應液體的分配和混合進行更方便和精確的操作。兩種芯片均成功應用於酶反應動力學的測定。在一次實驗中，只需要3~5微升的反應物就可以得到鹼性磷酸酶（alkaline phosphatase）的Michaelis-Menten動力學。 / 論文第二部份研究了“體積效“應對於蛋白質結晶的影響。首先，基於微孔的微流控芯片的蛋白質結晶篩選實驗揭示了在小體積（納升）下，蛋白質的結晶條件比在大體積（微升）下更多。在液滴里的蛋白質結晶實驗結果說明大體積的蛋白質液滴結晶速度更快。最後，蛋白質結晶實驗成功在雙乳液（double emulsion）的內核中進行。 / This thesis describes the design and development of micro dispensing systems for enzyme assay and for protein crystallization. The micro dispensing systems were fabricated by the soft-lithography method with the widely used material poly(dimethylsiloxane) (PDMS), which is gas and water permeable elastomer. / This thesis contains two major parts. In the first part, enzyme assay was performed in two micro dispensing systems, one based on microwells and the other based on pneumatic valves. The complete Michaelis-Menten kinetics measurement of the alkaline phosphatase (AP) with different fluorescein diphosphate (FDP) was achieved in one chip for each system using the fluorescence detection. These micro dispensing systems’ fabrication and operation were simple, and the total sample consumption was about 3~5 μL. / The second part reports the study of the volume effect on protein crystallization. Three micro dispensing systems, the microwell-based, droplet-based and double emulsion-based systems, were developed to perform the protein crystallization. Lysozyme and thaumatin were chosen as the model proteins. First, the protein crystallization screening experiments showed that protein crystallized in more precipitant conditions in the microchip than in conventional microbatch system. Second, the protein crystallization results carried out in droplets showed that protein crystallized faster in larger droplets. Finally, the protein crystallization in double emulsions was demonstrated. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Xiaohu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 87-91). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.vi / List of figures --- p.viii / Chapter Chapter 1 --- Introduction to microfluidics --- p.1 / Chapter 1.1 --- Fabrication of microfluidic devices --- p.2 / Chapter 1.1.1 --- PDMS-based microfluidic device --- p.2 / Chapter 1.1.2 --- PMMA microfluidic device --- p.6 / Chapter 1.1.3 --- Assembly of the microfluidic device --- p.8 / Chapter 1.2 --- Degassed PDMS pumping method --- p.9 / Chapter 1.3 --- Droplet-based microfluidics --- p.13 / Chapter 1.4 --- Thesis organization --- p.16 / Chapter Chapter 2 --- Micro dispensing systems for enzyme assay --- p.17 / Chapter 2.1 --- Introduction to enzyme assay --- p.17 / Chapter 2.1.1 --- Introduction to micro platforms for enzyme assay --- p.17 / Chapter 2.1.2 --- Introduction to enzyme kinetics and Michaelis-Menten kinetics --- p.21 / Chapter 2.2 --- Experimental --- p.26 / Chapter 2.2.1 --- Design of the micro dispensing systems --- p.26 / Chapter 2.2.2 --- Fabrication of the microfluidic devices --- p.30 / Chapter 2.2.3 --- Reagents and operation --- p.32 / Chapter 2.3 --- Results and discussion --- p.32 / Chapter 2.3.1 --- Microwell-based dispensing system --- p.32 / Chapter 2.3.2 --- Micro dispensing system based on pneumatic valves --- p.40 / Chapter 2.4 --- Conclusions --- p.45 / Chapter Chapter 3 --- Micro dispensing systems for protein crystallization --- p.47 / Chapter 3.1 --- Introduction to protein crystallization --- p.47 / Chapter 3.1.1 --- Principle of protein crystallization --- p.49 / Chapter 3.1.2 --- From macrofluidics to microfluidics --- p.51 / Chapter 3.2 --- Experimental --- p.56 / Chapter 3.2.1 --- Design of the micro dispensing systems --- p.56 / Chapter 3.2.2 --- Fabrication of the microfluidic devices --- p.58 / Chapter 3.2.3 --- Reagents and operation --- p.60 / Chapter 3.2.4 --- Layer-by-layer modification --- p.61 / Chapter 3.3 --- Results and discussion --- p.65 / Chapter 3.3.1 --- Demonstration of the micro dispensing systems --- p.65 / Chapter 3.3.2 --- Protein crystallization screening results --- p.69 / Chapter 3.3.3 --- Protein crystallization in droplets --- p.73 / Chapter 3.3.4 --- Protein crystallization in w/o/w double emulsions --- p.77 / Chapter 3.4 --- Conclusions --- p.80 / Appendix --- p.82 / References --- p.87

Zhou, Xuechang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 51-53). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.ii / Acknowledgement --- p.iii / Table of contents --- p.iv / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Protein crystallization --- p.1 / Chapter 1.1.1 --- The principle of protein crystallization --- p.3 / Chapter 1.1.2 --- Protein crystallization approaches --- p.4 / Chapter 1.1.3 --- Screening strategies and approaches --- p.6 / Chapter 1.2 --- Microfluidic systems for protein crystallization --- p.7 / Chapter 1.2.1 --- Integrated valve-controlled microfluidic system --- p.8 / Chapter 1.2.2 --- Droplet-based microfluidic system --- p.9 / Chapter 1.2.3 --- Objective of the research --- p.10 / Chapter 2 --- Nanoliter Liquid Dispensing Method --- p.12 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.2 --- Experimental --- p.14 / Chapter 2.2.1 --- The fabrication of SU-8 master --- p.14 / Chapter 2.2.2 --- The fabrication of PDMS microfluidic device --- p.16 / Chapter 2.2.3 --- The fabrication of glass and PMMA microwells --- p.17 / Chapter 2.2.4 --- The liquid dispensing into microwells --- p.17 / Chapter 2.3 --- Results and discussions --- p.20 / Chapter 2.3.1 --- The internal vacuum pumping source --- p.20 / Chapter 2.3.2 --- The efficiency of the pumping --- p.23 / Chapter 2.3.3 --- The removal of PDMS channel patch --- p.26 / Chapter 2.4 --- Conclusion --- p.29 / Chapter 3 --- The Screening of Protein Crystallization Conditions --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Experimental --- p.31 / Chapter 3.2.1 --- The design and fabrication of the screening chip --- p.31 / Chapter 3.2.2 --- The screening of protein crystallization conditions --- p.32 / Chapter 3.2.3 --- Crystallization and X-ray diffraction of an unknown protein --- p.33 / Chapter 3.3 --- Results and discussions --- p.33 / Chapter 3.3.1 --- Sparse matrix screening strategy in microwell arrays --- p.33 / Chapter 3.3.2 --- The results of the sparse matrix screening --- p.39 / Chapter 3.3.3 --- Crystal extraction and X-ray diffraction --- p.40 / Chapter 3.4 --- Conclusion --- p.41 / Chapter 4 --- Conclusion --- p.43 / Chapter 4.1 --- Summary --- p.43 / Chapter 4.2 --- Discussions and future directions --- p.44 / Appendix Information --- p.47 / References --- p.51

This thesis describes a novel microfluidic platform to control the macromolecular concentrations and their applications in constructing the phase diagram of polymer aqueous solutions and in the high-throughput screening of protein crystallization conditions at the nanoliter scale. The microfluidic platform was fabricated using the soft-lithography method and based on poly(dimethylsiloxane) (PDMS) material, which is widely used in microfluidic device. PDMS is gas and water permeable elastomer. By exploiting the permeability of the gas and water in PDMS, we developed the degassed-PDMS nanoliter liquid dispensing system, the controlled microevaporation method in constructing the phase diagram of polymer aqueous solution, and the screening platform of protein crystallization conditions. / This thesis describes two types of degassed-PDMS nanoliter liquid dispensing system. One is dispensing without the microvalve, in which various liquids are dispensed through the degassed PDMS microchannel. It involves two steps: in the first step, the PDMS microchannel patch (or the entire microchip) is placed in a vacuum chamber for a certain time; in the second step, the target liquid is deposited at the inlet of the PDMS channel and dispensed into the PDMS microchamber. The other method is dispensing with the aid of PDMS microvalve. This method combines the valve control and degassed PDMS pumping source, which provides more control over on the liquid dispensing, such as isolating, mixing, etc. / Zhou, Xuechang. / Adviser: Bo Zheng. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 53-56). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Li, Yuefang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 46-48). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.ii / Acknowledgement --- p.iii / Table of contents --- p.iv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Introduction to protein crystallization --- p.1 / Chapter 1.1.1 --- Principles of protein crystallization --- p.2 / Chapter 1.1.2 --- Classical methods to crystallize protein --- p.4 / Chapter 1.2 --- Crystal growth in unique environments: the pursuit of better crystals --- p.6 / Chapter 1.2.1 --- Protein crystallization in space --- p.6 / Chapter 1.2.2 --- Crystallization in gel and capillary --- p.7 / Chapter 1.3 --- Microfluidic methods for protein crystallization: high through-put screenings --- p.9 / Chapter 1.3.1 --- Valve-controlled methods --- p.10 / Chapter 1.3.2 --- Droplet-based methods --- p.11 / Chapter 1.3.3 --- Microwell-based methods --- p.11 / Chapter 1.4 --- Objective of the project --- p.13 / Chapter Chapter 2 --- Rehydratable hydrogel in nanoliter microwells --- p.15 / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Experimental --- p.17 / Chapter 2.2.1 --- Fabrication of SU-8 mould --- p.17 / Chapter 2.2.2 --- Fabrication of the PDMS device --- p.19 / Chapter 2.2.3 --- Liquid dispensing in PDMS device --- p.20 / Chapter 2.2.4 --- Polymerization of PA gel --- p.21 / Chapter 2.2.5 --- Drying and Rehydration of PA gel --- p.22 / Chapter 2.3 --- Results and discussions --- p.23 / Chapter 2.3.1 --- Preparation of PA gel in PDMS device --- p.23 / Chapter 2.3.2 --- Immobilization of PA gel in microwells --- p.25 / Chapter 2.3.3 --- Dehydration and Rehydration of PA gel --- p.25 / Chapter 2.3.4 --- Liquid dispensing in the gel-preloaded microwells --- p.29 / Chapter 2.4 --- Conclusion --- p.31 / Chapter Chapter 3 --- Protein crystallization by gel-based FID --- p.32 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.2 --- Experimental --- p.34 / Chapter 3.2.1 --- Conditions used for crystallize proteins --- p.34 / Chapter 3.2.2 --- Protein crystallization by microbatch method --- p.34 / Chapter 3.2.3 --- Protein crystallization in microchip --- p.35 / Chapter 3.3 --- Results and discussions --- p.35 / Chapter 3.3.1 --- Crystallization in microplate --- p.36 / Chapter 3.3.2 --- Crystallization in microwells --- p.38 / Chapter 3.4 --- Conclusion --- p.41 / Chapter Chapter 4 --- Conclusions --- p.43 / Chapter 4.1 --- Summary of the work --- p.43 / Chapter 4.2 --- Future perspectives --- p.44 / Reference --- p.46