臺灣博碩士論文加值系統

近年來，微流體晶片在生物物理領域與生醫檢測方面扮演的角色越來越重要，在微小尺度下，如何將兩種或多種流體在極短時間內混合，是個非常重要的議題。在本論文中，以流體聚焦(Hydrodynamic Focusing)的機制為基礎，比較四組尺寸與結構各異的微流體混合器，並且使用共焦顯微術與螢光相關光譜分析對此四組微流體混合器作定性與定量的分析，得到不同設計的擠壓效果與速度，速度對蛋白質折疊是重要的參數，其影響時間解析度能否解析蛋白質折疊過程；此研究幫助我們了解微流體的行為，並且得到微流體混合器之體積流率比的參數，並且發展了一個最佳的微流體混合器，以便將來對蛋白質折疊作更深入的研究。

In protein folding, early folding events occur on a microsecond to second time scale. In order to study protein kinetics, folding reactions must be triggered in a short time. In this thesis, we design four different micro-mixers based on hydrodynamic focusing. We use con-focal microscopy and fluorescence correlation spectroscopy (FCS) to synthesize qualitatively and quantitatively. Velocity is the most important parameter since it deeply affects the determination of mixing dead time and time resolution of our micro-mixer. Fluorescence correlation spectroscopy is a powerful technique to determine the velocity precisely. We made an overall comparison of four different micro-mixers by FCS and con-focal microscopy.

致謝 ............................................................................................................................... I
摘要 .............................................................................................................................. II
Abstract...........................................................................................................................III
目錄 ............................................................................................................................ IV
圖目錄 ........................................................................................................................... VII
表目錄 ............................................................................................................................ XI
第一章 蛋白質的介紹與折疊 ...............................................................................1
1.1 緒論 .................................................................................................................. 1
1.2 蛋白質的介紹 .................................................................................................. 5
1.2.1 胺基酸 .......................................................................................................... 5
1.2.2 胜肽鍵的形成 .............................................................................................. 6
1.2.3 蛋白質的構形 .............................................................................................. 7
1.3 蛋白質的變性(denaturation)與復性(renaturation) ....................................... 12
1.4 蛋白質的折疊問題與折疊模型 .................................................................... 15
1.4.1 蛋白質折疊之漏斗型位能通道 (The folding funnel) ............................. 15
1.4.2 框架模型 (Framework model) .................................................................. 16
1.4.3 疏水塌縮模型 (Hydrophobic Collapse Model) ........................................ 17
1.4.4 成核-凝聚-生長模型 (Nuclear-Condensation-Growth Model) ................ 17
V
1.5 蛋白質折疊機制的高動力解析方式 ............................................................ 18
1.5.1 超快速混合(Ultrafast mixing) ................................................................... 19
1.5.2 Temperature-jump Method ......................................................................... 22
第二章 流體動力聚焦的原理 .............................................................................25
2.1 低雷諾數之流體系統 .................................................................................... 25
2.2 在圓管中的流動 ............................................................................................ 27
2.3 在方管中的流動 ............................................................................................ 29
2.4 流體動力聚焦方法之原理 ............................................................................ 32
第三章 實驗架設與微流體混合器之設計與製作 .............................................36
3.1 微流體混合器設計 .............................................................................................. 36
3.2 微流體晶片的製作 ............................................................................................. 40
3.2.1 晶片準備與清潔 ............................................................................................ 40
3.2.2 塗底(Priming) ................................................................................................ 42
3.2.3 上光阻 ........................................................................................................... 43
3.2.4 軟烤(Soft bake) ............................................................................................. 45
3.2.5 對準與曝光 ................................................................................................... 45
3.2.6 顯影(Development) ....................................................................................... 46
3.2.7 硬烤(Hard Bake) ........................................................................................... 47
3.2.8 蝕刻(Etching) ................................................................................................ 47
3.2.9 微流道之製程步驟總整理 ........................................................................... 48
3.2.10 鑽孔(Drilling) ............................................................................................. 49
VI
3.2.11 晶片接合(Bonding) .................................................................................... 51
3.2.12 晶片接管(Tubing) ....................................................................................... 52
3.3 共焦顯微鏡系統 (Con-focal microscopy system) ........................................ 54
3.4 螢光相關光譜 (Fluorescence Correlation Spectroscopy, FCS) ................... 56
3.5 實驗架設 ........................................................................................................ 63
第四章 結果與討論 .............................................................................................65
4.1 基本特性描述 ................................................................................................ 65
4.1.1 流體聚焦(Focusing) ................................................................................... 65
4.1.2 噴嘴(Nozzle)的功用 .................................................................................. 66
4.1.3 共焦體積 (focal volume) .......................................................................... 67
4.2 微流體混合器定性與定量結果 .................................................................... 68
4.2.1 微流體混合器I 號 ..................................................................................... 68
4.2.2 微流體混合器II 號 ................................................................................... 74
4.2.3 微流體混合器III 號 .................................................................................. 80
4.2.4 微流體混合器IV 號 .................................................................................. 85
第五章 結論與未來展望 .....................................................................................91
5.1 結論 ..................................................................................................................... 91
5.2 未來展望 ............................................................................................................. 96
參考文獻 .........................................................................................................................97

1. Crick, F.H.C., On Protein Synthesis. in Symp. Soc. Exp. Biol. XII, 139-163,1958.
2. Anfinsen C. B., Principles that govern the folding of protein chains, Science, 181,
223-230, 1973.
3. P. Berg, Dissections and Reconstructions of Genes and Chromosomes, Science, 213,
296-303, 1981.
4. Roder H., Stepwise helix formation and chain compaction during protein folding,
Proc. Natl. Acad. Sci. U.S.A., 101, 1793-1794, 2004.
5. Jones, C. M., Henry, E. R., Hu, Y., Chan, C.-K., Luck, S. D., Bhuyan, A., Roder, H.,
Hofrichter, J., Eaton, W. A, Fast events in protein folding initiated by nanosecond
laser photolysis, Proc. Natl. Acad. Sci. U.S.A., 90, 11860-11864, 1993.
6. Jacob, M., Holtermann, G., Perl, D., Reinstein, J., Schindler, T., Geeves, M. A.,
Schmid, F. X., Microsecond Folding of the Cold Shock Protein Measured by a
Pressure-Jump Technique, Biochemistry, 38, 2882-2891, 1999.
7. Hagen, S. J., Eaton, W. A., Two-state Expansion and Collapse of a Polypeptide, J.
Mol. Biol., 301, 1037-1045, 2000.
8. Pollack, L., Tate, M. W., Darnton, N. C., Knight, J. B., Gruner, S. M., Eaton, W. A.,
Austin, R. H., Compactness of the denatured state of a fast-folding protein meas98
ured by submillisecond small-angle x-ray scattering, Proc. Natl. Acad. Sci. U.S.A.,
96, 10115-10117, 1999.
9. Fujita H., A decade of MEMS and its future, Proceedings of IEEE MEMS Symposium
(invited talk), 1-8,1997.
10. Walter Lang, Reflexions on the future of microsystem, Sensor and Actuators, Sensor
and Actuators, 72, 15, 1999.
11. 馬漢茂, Hammer, Andreas Pigulla Die Anfaenge der historisch orientierten China-
wissenschaften im deutschsprachigen Raum, Chinawissenschaften-
Deutschsprachige Entwicklungen, Geschichte, Personen, Perspektiven, Hamburg,
117-145, 1999.
12. 生物化學基礎講義,台灣大學生化科技系莊榮輝教授,2008.
13. Armstrong, F. B. Biochemistry. Oxford, 1983.
14. Nelson D. L., Cox M. M., Lehninger Principles of Biochemistry, 3E, Worth Publishers,
New York, 2000.
15. C. Dobson, A. Sali, M. Karplus, Angew., Protein Folding: A perspective from
theory and experiment, Chem. Int. Ed., 37, 7, 868-893, 1998.
16. Dyson, H. J., P. E. Wright., Peptide conformation and protein folding, Curr. Opin.
Struct. Biol., 3, 60-65, 1993.
17. DILL K. A., BROMBERG S., YUE K., FIEBIG K. M., YEE D. P., THOMAS P. D.,
99
CHAN H. S., Principles of protein folding: A perspective from simple exact models,
J. Protein Sci.,4, 561-602, 1995.
18. Abkevich V., Gutin A., Shakhnovich E., Specific nucleus as the transition state for
protein folding: evidence from the lattice model., Biochemistry, 33, 0026–10036,
1994.
19. Nolting B., Andert K., Mechanism of Protein Folding, Proteins, 41, 288-298, 2000.
20. Nicole N.-W. Kuo, Joseph J.-T. Huang, Jaroslava Miksovska, Rita P.-Y. Chen,
Randy W. Larsen, Sunney I. Chan, Effects of Turn Stability on the Kinetics of Refolding
of a Hairpin in a â-sheet, J. Am. Chem. SOC., 127, 16945-16954, 2005.
21. Nolting B., Protein Folding Kinetics: Biophysical Methods, Springer, Berlin Heidelberg
New York Tokyo (presents methods and application on protein folding),
2005.
22. Knight J. B., Vishwanath A., Brody J. P., Austin R. H., Hydrodynamic Focusing on a
Silicon Chip: Mixing Nanoliters in Microseconds, Phys. Rev. Lett. 80, 3863 – 3866,
1998.
23. Brody J. P., Yager P., Goldstein R. E., Austin R. H., Biotechnology at low Reynolds
numbers., Biophys. J., 71, 3430–3441,1996.
24. Chen C. F., Kung C. F., Chen H. C., Chu C. C., Chang C. C., Tseng F. G., A microfluidic
nanoliter mixer with optimized grooved structures driven by capillary pump100
ing, J. Micromech. Microeng., 16, 1358–1365, 2006.
25. Cha J., Kim J., Ryu S. K., Park J., Jeong Y., Park S., Park S., Kim H. C., Chun K., A
highly efficient 3D micromixer using soft PDMS bonding, J. Micromech. Microeng.,
16, 1778–1782, 2006.
26. 楊禮綾, 超快混合微流體元件的製作, 國立台灣大學碩士論文, 2003.
27. Park H. Y., Qiu X., Elizabeth R., Jonas K., Lisa W. K., Warren R. Z., Watt W. W.,
Lois P., Achieving Uniform Mixing in a Microfluidic Device: Hydrodynamic Focusing
Prior to Mixing, Anal. Chem., 78, 4465-4473, 2006.
28. Hertzog D. E., Michalet Xa., Jäger M., Kong X., Santiago J. G., Weiss S., Bakajin
O., Femtomole Mixer for Microsecond Kinetic Studies of Protein Folding, Anal.
Chem., 76, 7169-7178, 2004.
29. Kim S.A., Schwille P., Intracellular applications of fluorescence correlation spectroscopy:
prospects for neuroscience, Current Opinion in Neurobiology, 13, 583-590, 2003.
30. P. Schwille and E. Haustein, Online textbook article：Fluorescence Correlation
Spectroscopy (FCS),2002.