本研究主要設計環形雙圓微流道，藉由不同設計之雙圓內接/外切微流道（前者流道寬度較小無圓形阻塊；後者流道寬度較大有圓形阻塊），研究DNA分子於何種微流道受拉伸影響較大。本實驗調配稀薄溶液，使用五種電場強度（1、2.5、5、7.5、10 kV/m）與三種黏滯度（1、10、20cP）之butter solution來拉伸DNA分子，期望產生一均勻延展流及齊次的應變率來觀測DNA分子受水力拉伸情形。本實驗配合光學可視化的量測技術，搭配共軛焦雷射掃瞄顯微鏡（Confocal Laser Scanning Microscopy, CLSM）觀測DNA分子在各參數下受拉伸狀態，統計DNA分子在各區域的數量，並計算出各區域DNA分子之平均長度，探討DNA分子承受之流體阻力及其造成的形態變化和應力鬆弛時間，並且搭配微質點影像測速儀（Micrometer resolution Particle Image Velocimetry, μPIV）計算局部應變率，統計DNA伸長量，希望藉此對DNA分子拉伸形變有更進一步的瞭解。

In this study, we design different inscribed/circumscribed circular torus-type microchannels to investigate the stretching behavior of DNA molecules. Strain rate and relaxation time play an important role in DNA stretching. In order to perform an analysis of the coil-stretch transition of DNA, we develop a method of stretching DNA molecules by using μPIV and CLSM measurements. μPIV is designed to measure the velocity distribution, after which the local strain rate can be estimated. The hydrodynamic stretching of DNA molecules in the elongation flow is observed using a confocal laser scanning microscope (CLSM). The relaxation time of the DNA molecules is then estimated according to the CLSM images analysis. At present, our experiments using the electro-osmotic flow (EOF) driven at various electric fields and viscosities to stretch DNA molecules show how one can investigate the influence of hydrodynamic interactions in the case of stretching of DNA molecules.

目錄 i
表目錄 iii
圖目錄 iv
符號說明 vii
中文摘要 ix
Abstract x
第一章 序論 1
1-1 前言 1
1-2 DNA分子結構與性質 1
1-3 機電系統與生醫晶片 2
1-4 文獻回顧 4
1-5 背景與目的 10
第二章 實驗系統與設備 15
2-1 共軛焦雷射掃瞄顯微鏡量測系統 15
2-2 微質點影像測速儀系統 16
2-3 製程設備 18
2-4 直流電源供應器 19
2-5 其他實驗週邊設備 19
第三章 實驗方法及步驟 28
3-1 微流道設計與製程 28
3-2 工作流體配製 31
3-3 μPIV量測系統建立及原理 31
3-4 CLSM量測系統建立及原理 33
3-5 實驗量測參數 34
第四章 理論分析 43
4-1 DNA分子流變學拉伸理論 43
4-2 無因次參數分析 44
第五章 誤差分析 48
第六章 結果與討論 52
6-1 μPIV之流場量測 52
6-2 DNA分子螢光可視化分析 53
6-3 DNA分子受力分析 56
6-4 DNA分子拉伸模型分析 57
第七章 結論與建議 84
7-1 結論 84
7-2 建議與改進 85
參考文獻 86
附錄 A 92

參考文獻
1. P. G. De Gennes, , 1974, "Coil-Stretch Transition of Dilute Flexible Polymers under Ultrahigh Velocity Gradients," J. Chem. Phys. Vol. 60, pp. 5030-5042.
2. T. T. Perkins, S. Quake, D. Smith, and S. Chu, 1994, "Relaxation of a Single DNA Molecule Observed by Optical Microscopy," Science, Vol. 264, pp. 822-826.
3. T. T. Perkins, D. E. Smith, R. G. Larson, and S. Chu, 1995, "Stretching of a Single Tethered Polymer in a Uniform Flow," Science, Vol. 268, pp. 83-87.
4. S. B. Smith, Y. Cui, and C. Bustamante, 1996, "Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules," Science, Vol. 271, pp.795-799.
5. T. T. Perkins, D. E. Smith, and S. Chu, 1997, "Single Polymer Dynamics in an Elongational Flow," Science, Vol. 276, pp. 2016-2021.
6. D. E. Smith and S. Chu, 1998, "Response of Flexible Polymers to a Sudden Elongational Flow," Science, Vol. 281, pp. 1335-1340.
7. P. S. Doyle and B. Ladoux, 2000, "Stretching Tethered DNA Chains in Shear Flow," Europhys. Lett., Vol. 52, pp. 511-517.
8. H. P. Babcock, R. E. Teixeira, J. S. Hur, E. S. G. Shaqfeh and S. Chu, 2003, "Visualization of Molecular Fluctuations near the Critical Point of the Coil-Stretch Transition in Polymer Elongation," Macromolecules, Vol. 36, pp. 4544-4548.
9. P. K. Wong, Y. K. Lee, and C. M. Ho, 2003, "Deformation of DNA Molecules by Hydrodynamic Focusing," J. Fluid Mech., Vol. 497, pp. 55-65.
10. G. C. Randall and P. S. Doyle, 2004, "Electrophoretic Collision of a DNA Molecule with an Insulating Post," Phys. Rev. Lett., Vol. 93, pp. 58102 (4pp).
11. Y.-J. Juang, S. Wang, X. Hu, and L. J. Lee, 2004, "Dynamics of single polymers in a stagnation flow induced by electrokinetics," Physical Review Letters, Vol. 93, pp. 268105 (4pp).
12. J. B. Heng, A. Aksimentiev, C. Ho, P. Marks, Y. V. Grinkova, S. Sligar, K. Schulten, and G. Timp, 2005, "Stretching DNA Using the Electric Field in a Synthetic Nanopore," Nano Lett., Vol. 5, pp. 1883-1888.
13. S. Jeong, S. K. Park, J. K. Chang, and S. H. Kang, 2005, "Ultra-Sensitive Real-Time Single-DNA Molecules Detection at a Fused-Silica/Water Interface Using TIRFM Technique," Bull. Korean Chem. Soc., Vol. 26, pp. 979-982.
14. G. C. Randall and P. S. Doyle, 2005," DNA Deformation in Electric Fields: DNA Driven Past a Cylindrical," Macromolecules, Vol. 38, pp. 2410-2418.
15. J. W. Larson, G. R. Yantz, Q. Zhong, R. Charnas, C. M. D’Antoni, M. V. Gallo, K. A. Gillis, L. A. Neely, K. M. Phillips, G. G. Wong, S. R. Gullans, and R. Gilmanshin, 2006, "Single DNA Molecule Stretching in Sudden Mixed Shear and Elongational Microflows," Lab Chip, Vol. 6, pp.1187-1199.
16. T. F. Chan, C. Ha, A. Phong, D. Cai, E. Wan, L. Leung, P. Y. Kwok, and M. Xiao, 2006, "A Simple DNA Stretching Method for Fluorescence Imaging of Single DNA Molecules," Nucleic Acids Res., Vol. 34, pp.e113.
17. B. Yang, V. R. Dukkipati, D. Li, B. L. Cardozo, and S. W. Pang, 2007, "Stretching and Selective Immobilization of DNA in SU-8 Micro- and Nanochannels," J. Vac. Sci. Technol., Vol. 25, pp. 2352-2356.
18. J. Tang and P. S. Doyle, 2007, "Electrophoretic Stretching of DNA Molecules Using Microscale T Junctions," Applied Physics Letters, Vol. 90, pp. 224103 (3pp).
19. J. H. Kim, W. X. Shi, and R. G. Larson, 2007, "Methods of Stretching DNA Molecules Using Flow Fields," Langmuir, Vol. 23, pp. 755-764.
20. L. H. Thamdrup, A. Klukowska and A. Kristensena, 2008, "Stretching DNA in polymer nanochannels fabricated by thermal imprint in PMMA," Nanotechnology, Vol.19, pp. 125301(6pp).
21. K. Yamashita , M. Miyazaki, Y. Yamaguchi, M. P. Briones , H. Nakamura , and H. Maeda, 2008, "Direct Circular Dichroism Spectra Measurement of Stretching Long-strand DNA in a Tapering Microchannel," Chem. Eng. J., Vol.135, pp. S288-S291.
22. S.S. Hsieh, J. H. Liou, 2009, "DNA molecules dynamics in converging-diverging microchannels," Biotechnol. Appl. Biochem, Vol. 52, pp. 29-40.
23. H. T. Hoang, I. M. Segers-Nolten, J.W. Berenschot, M. J. de Boer, N. R. Tas, J. Haneveld and M. C. Elwenspoek, 2009, "Fabrication and Interfacing of Nanochannel Devices for Single-molecule Studies," J. Micromech. Microeng., Vol. 19, pp. 065017 (10pp).
24. F. Natali, I. M. Gregory, P. T. Christopher, B. Christopher, I. W. Mark, T. P. Y. Joseph, S. D. Mark, R. W. Martin and E. M. Justin, 2010, "Visualizing Helicases Unwinding DNA at the Single Molecule Level", Nucleic Acids Res., pp. 1-10.
25. A. Asanov, A. Zepeda and L.Vaca, 2010, "A Novel form of Total Internal Reflection Fluorescence Microscopy (LG-TIRFM) Reveals Different and Independent Lipid Raft Domains in Living Cells," Biochimica et Biophysica Acta, Vol. 1801, pp. 147-155.
26. T. T. Perkins, D. Smith, and S. Chu, 1994, "Direct Observation of Tube-like Motion of a Single Polymer Chain," Science, Vol. 264, pp. 819-822.
27. P. Cluzel, A. Lebrun, C. Heller, R. Lavery, J. L. Viovy, D. Chatenay, and F. Caron, 1996, "DNA: an Extensible Molecule," Science, Vol. 271, pp. 792-794.
28. T. Strick, J. F. Allemand, D. Bensimon, A. Bensimon, and V. Croquette, 1996, "The Elasticity of a Single Supercoiled DNA Molecule," Science, Vol.271, pp. 1835-1837.
29. C. Gosse, and D. Wirtz, 2000, "Magnetic Tweezers for DNA Micromanipulation," Rev. Sci. Instrum., Vol. 71, pp. 4561-4570.
30. S. B. Smith, L. Finzi, and C. Bustamante, 1992, "Direct Mechanical Measurements of The Elasticity of Single DNA Molecules by Using Magnetic Beads," Science, Vol. 258, pp. 1122-1126.
31. P. G. De Gennes, 1974, "Coil-Stretch Transition of Dilute Flexible Polymers Under Ultrahigh Velocity Gradients," J. Chem. Phys. Vol. 60, pp. 5030-5042.
32. Y. Lin, S. X. Cheng , J. J. Wang, B. Lei, Z. L. Zhang and D. W. Pang, 2007, "Measuring Radial Young’s Modulus of DNA by Tapping Mode AFM," Chin. Sci. Bull., Vol. 52, pp. 3189-3192.
33. R. G. Larson, T. T. Perkins, D. E. Smith, and S. Chu, 1997, "Hydrodynamics of a DNA Molecule in a Flow Field," Phys. Rev. E., Vol. 55, pp. 1794-1797.
34. D. E. Smith, H. P. Babcock, and S. Chu, 1999, "Single Polymer Dynamics in Steady Shear Flow," Science, Vol. 283, pp. 1724-1727.
35. E. C. Lee and S. J. Muller, 1999, "Flow Light Scattering Studies of Polymer Coil Conformation in Solutions in Extensional Flow," Macromolecules, Vol. 32, pp. 3295-3305.
36. P. S. Doyle, B. Ladoux, and J. L. Viovy, 2000, "Dynamics of a Tethered Polymer in Shear Flow," Phys. Rev. Lett., Vol. 84, pp. 4769-4772.
37. J. P. Rothstein, G. H. McKinley, 1999, "Extensional Flow of a Polystyrene Boger Fluid Through a 4:1:4 Axisymmetric Contraction/Expansion," J. Fluid Mech. Vol. 86, pp. 61-88.
38. P. J. Shrewsbury, S. J. Muller, and D. Liepmann, 2001, "Effect of Flow on Complex Biological Macrcmolecules in Microfluidic Devices," Biomedical Microdevices, Vol. 3, pp. 225-238.
39. P. J. Shrewsbury, D. Liepmann, and S., J. Muller, 2002, "Concentration Effects of a Biopolymer in a Microfluidic Device," Biomedical Microdevices, Vol. 4, pp. 17-26.
40. S. S. Hsieh, C. H. Liu, and J. H. Liou, 2007, "DNA Molecules Dynamics in a Cross-Slot Microchannel," Meas. Sci. Technol., Vol. 18, 2907-2915.
41. A. G. Balducci, J. Tang, and P. S. Doyle, 2008, "Electrophoretic Stretching of DNA Molecules in Cross-Slot Nanoslit Channels," Macromolecules, Vol. 41, pp. 9914-9918.
42. S. S. Hsieh, C. Y. Lin, C. F. Huang, and H. H. Tsai, 2004, "Liquid Flow in a Microchannel," J. Micromech. Microeng., Vol. 14, pp. 436-445.
43. S. S. Hsieh, H. C. Lin, and C. Y. Lin, 2006, "Electroosmotic Flow Velocity Measurements in a Square Microchannel," Colloid and Polymer Science, Vol. 284, pp. 1275-1286.
44. S. S. Hsieh, and T. T. Yang, 2007, "Electroosmotic Flow in Rectangular Microchannels with Joule Heating Effects," J. Micromech. Microeng., Vol. 18, 025025 (11pp).
45. R. G. Larson, 2005, "The Rheology of Dilute Solutions of Flexible Polymers: Progress and Problems," Journal of Rheology, Vol. 49, pp. 1-70.
46. J. R. Taylor, 1997, "An Introduction to Error Analysis," University Science Books, Sausalito.
47. 黃彥祥, 2003 "DNA分子在漸縮微渠道中流動的拉伸及動態特性," 國立交通大學機械工程學系碩士論文.
48. 伍秀菁, 汪若文, 林美吟編輯, 2003 "微機電系統技術與應用," 國科會精密儀器發展中心出版.