微液滴系統至今已發展超過二十年，此系統可進行眾多程序操作與應用，如混合、萃取、反應等，以兩不互溶液體生成之微液滴可作為獨立之微小反應器，相當適合於分析在兩相界面上進行的生化反應，本研究將先探討微液滴系統之質傳機制，接著研究連續液滴之生成機制，找出最適化之生成條件，最後以連續與靜止兩種微液滴系統建立解脂酶分析平台，進行解脂酶水解反應之快速分析。

微液滴系統中之質傳現象為影響界面反應之重要因素之一，因此在本研究中之第一部分，藉由微液滴系統進行萃取來研究質傳機制。此部分利用螢光染劑於不互溶之連續相與分散相中化學勢之差異作為驅動力，將微液滴中之染劑分子萃取至連續相，並以蔗糖改變染劑溶液之黏滯度，分析質傳的速度隨黏滯度的變化。由於本研究選擇的染劑對連續相親和力相當高，因此染劑於微液滴內部之質傳主導此萃取程序，在黏滯度為0.942及1.699cP時，萃取達平衡的時間分別為3.8與4.7秒，依scale law與Stokes-Einstein equation推論此程序係以微液滴內之循環流動與彎曲流道產生之紊流所主導，而非擴散所主導。

微液滴的表面積為另一控制界面反應速率之重要因素，因此，準確地控制微液滴的生成對於界面反應相當重要。微液滴之生成機制已被廣泛地以實驗或理論模型的方法提出，然而，研究結果只能用於某些條件之下，因此在本研究第二部分，分別以連續和靜止兩種方式生成蛋白質微液滴，並討論連續式生成蛋白質微液滴之生成機制。連續液滴之生成機制與體積和流量比、流道深寬比、黏度比有關，其體積隨流量比增加而減小，當深寬比增加且於低流量比時，微液滴體積隨之增大，當深寬比增加且於高流量比時，微液滴體積幾乎不隨深寬比變化。靜止液滴之體積僅與流道凹槽大小有關，因此改變分散相與連續相溶液仍可簡易生成大小相同之靜止液滴。

了解液滴中質傳機制以及蛋白質液滴之生成機制後，於本研究第三部分，以微液滴系統為基礎，選用來自Burkholderia sp.與Candida Rugosa之解脂酶建立快速酵素分析平台。於連續液滴系統中，僅耗時3秒即可偵測解脂酶水解大豆油之反應速率（以甘油之生成速率測得）分別為4.33 x10-3與3.58 x10-2 nmol/s，然而，連續液滴生成並非相當穩定，原因為其體積由眾多參數所控制，因此對於水解酶反應條件的篩選相當不便，故在本研究最後選擇在反應成分或是條件不同時仍可簡單生成相同大小之靜止液滴進行快速解脂酶分析。於靜止液滴系統中，偵測源自Burkholderia sp.與Candida Rugosa之解脂酶水解之反應速率（甘油之生成速率）分別為1.52 x10-4與1.47 x10-4 nmol/s，此反應速率與對照組相同，推測原因為界面上的解脂酶更新非常緩慢。連續液滴系統反應速率與靜止液滴系統相差243.5倍，但連續式液滴與靜止式液滴比表面積只相差2.9倍，推測原因為連續液滴內具有循環流動與紊流，使得表面解脂酶可自由移動，不斷更新，因此反應速率較快。然而，靜止液滴之體積只與凹槽大小有關，且靜止液滴製造較容易，但其目前此系統所偵測之解脂酶水解反應速率與對照組相同，因此需於此系統中提升解脂酶活性，以解決上述問題。

The first part of this study demonstrates extraction of dye and investigates the rate of mass transfer during the extraction. The equilibrium time for extraction in water and in 20％ sucrose solution were 3.8s and 4.7s, respectively. Form scale law and Stokes-Einstein equation, it is concluded that the mechanism of mass transfer is dominated by recirculating flow and chaotic advection in the micro-droplet, instead of diffusion.

In the second part of this study, protein micro-droplet was generated by dynamic and static methods and the mechanism for dynamic droplet generation is investigated. For generating dynamic droplet, the volume of micro-droplet decreased with increasing volumetric flow rate ratio. When micro-channel aspect ratio increased and flow rate ratio decreased, the droplet volume was constant. For static droplet, the volume only depended on the recess size.

Lipase from Burkholderia sp. and Candida Rugosa are chosen to demonstrate the rapid enzyme analysis platform based on micro-droplet. For dynamic droplet, the analysis of hydrolyzing soybean oil only took 3s and the reaction rate of Burkholderia sp. and Candida Rugosa are 4.33 x10-3 and 3.58 x10-2 nmol glycerol/s, respectively. For static droplet, the generated rate of glycerol by Burkholderia sp. and Candida Rugosa are as same as control due to slow lipase renewal at the interphase. Therefore, the activity of lipase needs to be enhanced in static droplet system to resolve the aforementioned issue. The rate of reaction in dynamic droplet system is 243.5 times faster than static droplet system. However, the specific surface area of dynamic droplet is only 2.9 times larger than static droplet. The circular flow and chaotic advection inside micro-droplet resulted in rapid lipase renewal at the interphase. Therefore, the dynamic droplet resulted in much higher reaction rate. Therefore, the activity of lipase needs to be enhanced in static droplet system to resolve the aforementioned issue.

1. Le, H.P., Progress and Trends in Ink-Jet Printing Technology. Journal of Imaging Science and Technology, 1998. 42(1): p. 49-62.
2. Terry, S.C., J.H. Jerman, and J.B. Angell, A Gas Chromatographic Air Analyzer Fabricated on a Silicon Wafer. Electron Devices, IEEE Transactions on, 1979. 26(12): p. 1880-1886.
3. Shoji, S., M. Esashi, and T. Matsuo, Prototype Miniature Blood Gas Analyser Fabricated on a Silicon Wafer. Sensors and Actuators, 1988. 14(2): p. 101-107.
4. Van Lintel, H., F. Van de Pol, and S. Bouwstra, A Piezoelectric Micropump Based on Micromachining of Silicon. Sensors and actuators, 1988. 15(2): p. 153-167.
5. Gass, V., et al., Integrated Flow-Regulated Silicon Micropump. Sensors and Actuators A: Physical, 1994. 43(1): p. 335-338.
6. Chen, Y.Y., Z.M. Chen, and H.Y. Wang, Enhanced Fluorescence Detection Using Liquid-Liquid Extraction in a Microfluidic Droplet System. Lab on a Chip, 2012. 12(21): p. 4569-75.
7. Song, H., D.L. Chen, and R.F. Ismagilov, Reactions in Droplets in Microfluidic Channels. Angewandte chemie international edition, 2006. 45(44): p. 7336-56.
8. Teh, S.Y., et al., Droplet Microfluidics. Lab on a Chip, 2008. 8(2): p. 198-220.
9. Seemann, R., et al., Droplet Based Microfluidics. Reports on progress in physics, 2012. 75(1): p. 016601.
10. Hwang, D.K., D. Dendukuri, and P.S. Doyle, Microfluidic-Based Synthesis of Non-Spherical Magnetic Hydrogel Microparticles. Lab on a Chip, 2008. 8(10): p. 1640-7.
11. Dewan, A., et al., Growth Kinetics of Microalgae in Microfluidic Static Droplet Arrays. Biotechnology and bioengineering, 2012. 109(12): p. 2987-2996.
12. Mary, P., V. Studer, and P. Tabeling, Microfluidic Droplet-Based Liquid-Liquid Extraction. Analytical chemistry, 2008. 80(8): p. 2680-2687.
13. Kintses, B., et al., Microfluidic Droplets: New Integrated Workflows for Biological Experiments. Current opinion in chemical biology, 2010. 14(5): p. 548-555.
14. Thorsen, T., et al., Dynamic Pattern Formation in a Vesicle-Generating Microfluidic Device. Physical Review Letters, 2001. 86(18): p. 4163-4166.
15. Anna, S.L., N. Bontoux, and H.A. Stone, Formation of Dispersions Using “Flow Focusing” in Microchannels. Applied physics letters, 2003. 82(3): p. 364-366.
16. Zagnoni, M., J. Anderson, and J.M. Cooper, Hysteresis in Multiphase Microfluidics at a T-Junction. Langmuir, 2010. 26(12): p. 9416-9422.
17. Liu, H. and Y. Zhang, Droplet Formation in a T-Shaped Microfluidic Junction. Journal of Applied Physics, 2009. 106(3): p. 034906.
18. Gupta, A., S.M.S. Murshed, and R. Kumar, Droplet Formation and Stability of Flows in a Microfluidic T-Junction. Applied Physics Letters, 2009. 94(16): p. 164107.
19. Gupta, A. and R. Kumar, Effect of Geometry on Droplet Formation in the Squeezing Regime in a Microfluidic T-Junction. Microfluidics and Nanofluidics, 2009. 8(6): p. 799-812.
20. Xu, J.H., et al., Shear Force Induced Monodisperse Droplet Formation in a Microfluidic Device by Controlling Wetting Properties. Lab on a Chip, 2006. 6(1): p. 131-6.
21. Christopher, G.F. and S.L. Anna, Microfluidic Methods for Generating Continuous Droplet Streams. Journal of Physics D: Applied Physics, 2007. 40(19): p. R319-R336.
22. Gupta, A. and R. Kumar, Flow Regime Transition at High Capillary Numbers in a Microfluidic T-Junction: Viscosity Contrast and Geometry Effect. Physics of Fluids, 2010. 22(12): p. 122001.
23. Abate, A., et al., Impact of Inlet Channel Geometry on Microfluidic Drop Formation. Physical Review E, 2009. 80(2).
24. Huebner, A., et al., Static Microdroplet Arrays: A Microfluidic Device for Droplet Trapping, Incubation and Release for Enzymatic and Cell-Based Assays. Lab on a Chip, 2009. 9(5): p. 692-8.
25. Sun, M., S.S. Bithi, and S.A. Vanapalli, Microfluidic Static Droplet Arrays with Tuneable Gradients in Material Composition. Lab on a Chip, 2011. 11(23): p. 3949-52.
26. Wang, W., C. Yang, and C.M. Li, On-Demand Microfluidic Droplet Trapping and Fusion for on-Chip Static Droplet Assays. Lab on a Chip, 2009. 9(11): p. 1504-6.
27. Chen, H., et al., Microfluidic Chip-Based Liquid-Liquid Extraction and Preconcentration Using a Subnanoliter-Droplet Trapping Technique. Lab on a Chip, 2005. 5(7): p. 719-25.
28. Song, H., et al., Experimental Test of Scaling of Mixing by Chaotic Advection in Droplets Moving through Microfluidic Channels. Applied Physics Letters, 2003. 83(12): p. 4664-4666.
29. DeMello, A.J., Control and Detection of Chemical Reactions in Microfluidic Systems. Nature, 2006. 442(7101): p. 394-402.
30. Liau, A., et al., Mixing Crowded Biological Solutions in Milliseconds. Analytical chemistry, 2005. 77(23): p. 7618-7625.
31. Roach, L.S., H. Song, and R.F. Ismagilov, Controlling Nonspecific Protein Adsorption in a Plug-Based Microfluidic System by Controlling Interfacial Chemistry Using Fluorous-Phase Surfactants. Analytical chemistry, 2005. 77(3): p. 785-796.
32. Song, H. and R.F. Ismagilov, Millisecond Kinetics on a Microfluidic Chip Using Nanoliters of Reagents. Journal of the American Chemical Society, 2003. 125(47): p. 14613-14619.
33. Jaeger, K.-E., et al., Bacterial Lipases. FEMS microbiology reviews, 1994. 15(1): p. 29-63.
34. Jaeger, K., B. Dijkstra, and M. Reetz, Bacterial Biocatalysts: Molecular Biology, Three-Dimensional Structures, and Biotechnological Applications of Lipases. Annual Reviews in Microbiology, 1999. 53(1): p. 315-351.
35. Pandey, A., et al., The Realm of Microbial Lipases in Biotechnology. Biotechnology and applied biochemistry, 1999. 29(2): p. 119-131.
36. Gupta, R., N. Gupta, and P. Rathi, Bacterial Lipases: An Overview of Production, Purification and Biochemical Properties. Applied Microbiology and Biotechnology, 2004. 64(6): p. 763-81.
37. 劉建宏, 脂肪分解酵素菌株之篩選及其應用. 2008.
38. Sidhu, P., et al., Production of Extracellular Alkaline Lipase by a New Thermophilicbacillus Sp. Folia microbiologica, 1998. 43(1): p. 51-54.
39. Koritala, S., et al., Biochemical Modification of Fats by Microorganisms: A Preliminary Survey. Journal of the American Oil Chemists' Society, 1987. 64(4): p. 509-513.
40. Arpigny, J. and K. Jaeger, Bacterial Lipolytic Enzymes: Classification and Properties. Biochemical Society, 1999. 343: p. 177-183.
41. Jaeger, K.-E. and M.T. Reetz, Microbial Lipases Form Versatile Tools for Biotechnology. Trends in biotechnology, 1998. 16(9): p. 396-403.
42. Benjamin, S. and A. Pandey, Candida Rugosa Lipases: Molecular Biology and Versatility in Biotechnology. Yeast, 1998. 14(12): p. 1069-1087.
43. Sugihara, A., T. Tani, and Y. Tominaga, Purification and Characterization of a Novel Thermostable Lipase from Bacillus Sp. Journal of biochemistry, 1991. 109(2): p. 211-216.
44. Lesuisse, E., K. Schanck, and C. Colson, Purification and Preliminary Characterization of the Extracellular Lipase of Bacillus Subtilis 168, an Extremely Basic Ph‐Tolerant Enzyme. European Journal of Biochemistry, 1993. 216(1): p. 155-160.
45. Dunhaupt, A., S. Lang, and F. Wagner, Properties and Partial Purification of a Pseudomonas Cepacia Lipase. GBF monographs, 1991. 16: p. 389-392.
46. Kojima, Y., M. Yokoe, and T. Mase, Purification and Characterization of an Alkaline Lipase from Pseudomonas Fluorescens Ak102. Bioscience, biotechnology, and biochemistry, 1994. 58(9): p. 1564-1568.
47. Rathi, P., et al., A Hyper-Thermostable, Alkaline Lipase from Pseudomonas Sp. With the Property of Thermal Activation. Biotechnology letters, 2000. 22(6): p. 495-498.
48. Rathi, P., R. Saxena, and R. Gupta, A Novel Alkaline Lipase from Burkholderia Cepacia for Detergent Formulation. Process Biochemistry, 2001. 37(2): p. 187-192.
49. Bradoo, S., et al., Microwave-Assisted Rapid Characterization of Lipase Selectivities. Journal of biochemical and biophysical methods, 2002. 51(2): p. 115-120.
50. Ting, W.-J., et al., Application of Binary Immobilized Candida Rugosa Lipase for Hydrolysis of Soybean Oil. Journal of Molecular Catalysis B: Enzymatic, 2006. 42(1): p. 32-38.
51. Benzonana, G. and S. Esposito, On the Positional and Chain Specificities of Candida Cylindracea Lipase. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism, 1971. 231(1): p. 15-22.
52. Liu, Y.-Y., J.-H. Xu, and Y. Hu, Enhancing Effect of Tween-80 on Lipase Performance in Enantioselective Hydrolysis of Ketoprofen Ester. Journal of Molecular Catalysis B: Enzymatic, 2000. 10(5): p. 523-529.
53. Cheirsilp, B., W. Kaewthong, and A. H-Kittikun, Kinetic Study of Glycerolysis of Palm Olein for Monoacylglycerol Production by Immobilized Lipase. Biochemical Engineering Journal, 2007. 35(1): p. 71-80.
54. Macrae, A., Lipase-Catalyzed Interesterification of Oils and Fats. Journal of the American Oil Chemists’ Society, 1983. 60(2): p. 291-294.
55. Meher, L., D. Vidya Sagar, and S. Naik, Technical Aspects of Biodiesel Production by Transesterification—a Review. Renewable and sustainable energy reviews, 2006. 10(3): p. 248-268.
56. Schmidt, M. and U.T. Bornscheuer, High-Throughput Assays for Lipases and Esterases. Biomolecular engineering, 2005. 22(1): p. 51-56.
57. Moore, B.D., et al., Rapid and Ultra-Sensitive Determination of Enzyme Activities Using Surface-Enhanced Resonance Raman Scattering. Nature biotechnology, 2004. 22(9): p. 1133-1138.