隨著醫療技術與微機電系統的日益成熟，細胞晶片的發展越來越受到矚目與重視。如何能利用此成熟的技術，將微機電系統的優點移轉至細胞晶片或細胞感測器上，已成近年來熱門的研究主題。而若要成功發展細胞晶片，如何能夠對蛋白質及細胞進行有效且準確的塗布，佔有相當關鍵的地位。目前已成功發展的塗布技術，如利用壓模轉印圖樣的微壓印技術[1](microcontact printing)、利用微奈米製程的微影技術[2](photolithography)與藉介電泳動效應實現細胞塗布的介電泳動技術[3](dielectrophoresis)等，雖有其各自的優點，但以上幾種方法，皆無法同時達成高解析度、高效率與對細胞無潛在損害的目標。
基於此，本研究中應用電致濕潤現象(eletrowetting)。此現象能依所加電壓而改變表面的親疏水性質，而親疏水間作用力是生物分子間重要的非共價作用力之一。因此經由外加電壓調控，能夠控制表面的性質，即改變表面與生物分子的附著力，並且進而達到生物分子與細胞的精準塗布。於本研究中，證實了此方法的可行性，細胞塗布圖樣可由電極圖樣決定，並且其細胞塗布解析度可達到次微米等級。更重要的，此技術能夠避免不必要的熱能及電解現象產生，亦不會有電場或機械應力而對欲塗布的細胞造成不良影響。由以上結果可證實，本技術未來可望對生物晶片與細胞晶片的塗布技術提供一個新的方向與動力。

As the medical technology and micro-electromechanical systems become mature, the development of cell chips are also more and more important. It has become a hot research topic for decades to try to achieve useful and efficient cell chips by taking advantage of micro-electromechanical technology. Needless to say, to the successful development of cell chips, it is crucial how we effectively and accurately pattern biomolecules and cells. The developed patterning technologies, such as microcontact printing, photolithography, and dielectrophoretic patterning, however, cannot achieve goals of high efficiency, high patterning resolution and no damage to biomolecules.
In order to solve this problem, here we have applied the technique of electrowetting, which can change the surface hydrophobicity/hydrophilicity by applying voltage. The hydrophobicity/hydrophilicity is one of the most important non-covalent forces for biomolecules. Thus, through the regulation of applied voltage, surface properties can be controlled, and therefore the attachment of biomolecules and cells can further be precisely manipulated. In this research, we prove that cell patterns can be confined in the regions of electrode patterns, and patterning resolution can be down to sub-micrometer scale. Besides, this technique can avoid unnecessary generation of electrolysis and heat, and the possible negative influence of the electric field or mechanical stress can also be excluded. Based on previous results, it is certain that this technique can provide a new direction for biomolecular and cell patterning techniques and also benefit the development of biochips and cell chips.

口試委員審定書...............................................#

誌謝.......................................................i

中文摘要 ................................................. ii

ABSTRACT ............................................... iii

目錄......................................................iv

圖目錄 ....................................................vi

表目錄 ................................................. viii

第一章 導論................................................ 1

1.1 前言 ............................................... 1

1.2 動機 ............................................... 2

1.3 論文架構 ............................................ 3

第二章 理論與文獻回顧 ....................................... 4

2.1 細胞塗布技術回顧 ...................................... 5

2.2 電致濕潤現象(Electrowetting) ........................ 14

第三章 原理、製程材料與方法 ................................. 22

3.1 元件設計與操作原理 ................................... 22

3.2 細胞塗布流程 ........................................ 31

第四章 實驗結果與討論 ...................................... 36

4.1 元件穩定性與電致濕潤效果測試 ........................... 37

4.2 細胞塗布流程測試－依序流入蛋白質排斥性分子(Pluronic)與蛋白質 .... 41

4.3 細胞塗布流程測試－同時流入蛋白質排斥性分子(Pluronic)與蛋白質 .... 51

4.4 次微米尺度電極蛋白質塗布螢光測試 .........................56

第五章 結論與展望 .......................................... 61

5.1 總結 .............................................. 61

5.2 展望 .............................................. 62

參考文獻 ................................................. 63

附錄一.....................................................67

附錄二.....................................................68

附錄三.....................................................69

1. Li, H.W., et al., Nanocontact printing: A route to sub-50-nm-scale chemical and biological patterning. Langmuir, 2003. 19(6): p. 1963-1965.

2. Bouaidat, S., et al., Micro patterning of cell and protein non-adhesive plasma polymerized coatings for biochip applications. Lab Chip, 2004. 4(6): p. 632-637.

3. Suzuki, M., et al., Negative dielectrophoretic patterning with different cell types. Biosensors and bioelectronics, 2008. 24(4): p. 1043-1047.

4. Chen, C.S., et al., Geometric control of cell life and death. Science, 1997. 276(5317): p. 1425-1428.

5. Roth, E.A., et al., Inkjet printing for high-throughput cell patterning. Biomaterials, 2004. 25(17): p. 3707-3715.

6. Lee, K.B., et al., Protein nanoarrays generated by dip-pen nanolithography. Science, 2002. 295(5560): p. 1702-1705.

7. Chen, C.S., et al., Cell shape provides global control of focal adhesion assembly. Biochemical and biophysical research communications, 2003. 307(2): p. 355-361.

8. Guilak, F., et al., Control of stem cell fate by physical interactions with the extracellular matrix. Cell Stem Cell, 2009. 5(1): p. 17-26.

9. Bhatia, S.N., M.L. Yarmush, and M. Toner, Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3T3 fibroblasts. Journal of biomedical materials research, 1997. 34(2): p. 189-199.

10. Albrecht, D.R., et al., Probing the role of multicellular organization in three-dimensional microenvironments. Nature methods, 2006. 3(5): p. 369-375.
64

11. Fukuda, J., et al., Micropatterned cell co-cultures using layer-by-layer deposition of extracellular matrix components. Biomaterials, 2006. 27(8): p. 1479-1486.

12. Camelliti, P., A.D. McCulloch, and P. Kohl, Microstructured cocultures of cardiac myocytes and fibroblasts: a two-dimensional in vitro model of cardiac tissue. Microscopy and Microanalysis, 2005. 11(03): p. 249-259.

13. Zinchenko, Y.S. and R.N. Coger, Engineering micropatterned surfaces for the coculture of hepatocytes and Kupffer cells. Journal of Biomedical Materials Research Part A, 2005. 75(1): p. 242-248.

14. Stenger, D.A., et al., Detection of physiologically active compounds using cell-based biosensors. Trends in Biotechnology, 2001. 19(8): p. 304-309.

15. Pancrazio, J., et al., Development and application of cell-based biosensors. Annals of Biomedical Engineering, 1999. 27(6): p. 697-711.

16. Arnold, M., et al., Cell interactions with hierarchically structured nano-patterned adhesive surfaces. Soft Matter, 2008. 5(1): p. 72-77.

17. Rozkiewicz, D.I., et al., Covalent microcontact printing of proteins for cell patterning. Chemistry-A European Journal, 2006. 12(24): p. 6290-6297.

18. Sanjana, N.E. and S.B. Fuller, A fast flexible ink-jet printing method for patterning dissociated neurons in culture. Journal of neuroscience methods, 2004. 136(2): p. 151-163.

19. Garci-Sanchez, P. and F. Mugele, Fundamentals of Electrowetting and Applications in Microsystems. Electrokinetics and Electrohydrodynamics in Microsystems, 2011: p. 85-125.

20. Fan, S.K., et al., Cross-scale electric manipulations of cells and droplets by frequency-modulated dielectrophoresis and electrowetting. Lab Chip, 2008. 8(8): p. 1325-1331.

21. Prins, M., W. Welters, and J. Weekamp, Fluid control in multichannel structures by electrocapillary pressure. Science, 2001. 291(5502): p. 277-280.

22. Welters, W.J.J. and L.G.J. Fokkink, Fast electrically switchable capillary effects. Langmuir, 1998. 14(7): p. 1535-1538.

23. Kuo, J.S., et al., Electrowetting-induced droplet movement in an immiscible medium. Langmuir, 2003. 19(2): p. 250-255.

24. Satoh, W., M. Loughran, and H. Suzuki, Microfluidic transport based on direct electrowetting. Journal of applied physics, 2004. 96: p. 835.

25. Huh, D., et al., Reversible switching of high-speed air-liquid two-phase flows using electrowetting-assisted flow-pattern change. Journal of the American Chemical Society, 2003. 125(48): p. 14678-14679.

26. Acharya, B.R., et al., Tunable optical fiber devices based on broadband long-period gratings and pumped microfluidics. Applied physics letters, 2003. 83: p. 4912.

27. Krupenkin, T., S. Yang, and P. Mach, Tunable liquid microlens. Applied physics letters, 2003. 82: p. 316.

28. Moon, H., S.K. Cho, and R.L. Garrell, Low voltage electrowetting-on-dielectric. Journal of applied physics, 2002. 92: p. 4080.

29. Hayes, R.A. and B. Feenstra, Video-speed electronic paper based on electrowetting. Nature, 2003. 425(6956): p. 383-385.

30. Heikenfeld, J., et al., Recent progress in arrayed electrowetting optics. Optics and Photonics News, 2009. 20(1): p. 20-26.

31. Cho, S.K., H. Moon, and C.J. Kim, Creating, transporting, cutting, and merging liquid droplets by electrowetting-based actuation for digital microfluidic circuits. Microelectromechanical Systems, Journal of, 2003. 12(1): p. 70-80.

32. Batrakova, E.V. and A.V. Kabanov, Pluronic block copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers. Journal of Controlled Release, 2008. 130(2): p. 98-106.

33. Fan, C.Y., et al., Electrically programmable surfaces for configurable patterning of cells. Advanced Materials, 2008. 20(8): p. 1418-1423.

34. Lhoest, J.B., et al., A new plasma-based method to promote cell adhesion on micrometric tracks on polystyrene substrates. Journal of Biomaterials Science, Polymer Edition, 1996. 7(12): p. 1039-1054.

35. Dewez, J.L., et al., Adhesion of mammalian cells to polymer surfaces: from physical chemistry of surfaces to selective adhesion on defined patterns. Biomaterials, 1998. 19(16): p. 1441-1445.

36. 鍾尚倫, 微奈米生物分子塗布技術之研發, 臺灣大學電子工程學研究所學位論文, 2011(2011年).

37. Kim, L., et al., A practical guide to microfluidic perfusion culture of adherent mammalian cells. Lab Chip, 2007. 7(6): p. 681-694.

38. Fan, C.Y., K. Kurabayashi, and E. Meyhofer, Protein pattern assembly by active control of a triblock copolymer monolayer. Nano letters, 2006. 6(12): p. 2763-2767.