本研究主要是透過數位微流體系統的介電濕潤晶片(Electrowetting on dielectric, EWOD)，應用於生殖醫學與微液珠圖案化操作。而介電濕潤晶片的優點在於製程簡單方便且成本低廉、容易操作微流體的生成、傳輸、分離、結合等。
介電濕潤晶片應用於生殖醫學上的應用是將數位微流體晶片作為受精卵胚胎的動態培養平台，在油浴環境中以電濕潤力操控液珠，用以模仿胚胎在母體內之動態移動的環境，並且將實驗分為三組:傳統的微滴培養法作為實驗的對照組、使用晶片培養但不使用電濕潤力驅動的靜態培養組以及晶片培養並使用電濕潤力驅動之動態培養組。晶片靜態胚培養是將受精卵注入平衡後的培養液內並靜置於油浴的介電濕潤晶片中，並每天紀錄觀察生長狀況；動態培養組則是將受精卵注入平衡後的培養液內，並每5分鐘移動培養液一次，持續兩小時，以模擬受精卵由輸卵管中移動至子宮著床之動態過程，並觀察胚胎後續生長狀況，藉此方式來證明數位微流體系統應用於生殖醫學上的可行性。實驗結果顯示傳統組、晶片靜態培養組、晶片動態培養組之發育率分別為77.8%、62.5%、75%，實驗發現傳統的動態培養比起使用傳統培養的還要來的高，但晶片動態培養組與靜態培養組胚胎的發育率有較大的差距，晶片動態培養的胚胎發育率比靜態培養高出12.5%，證實使用介電濕潤晶片驅動後的培養液，更能將培養液內的養分以及胚胎所分泌的激素均勻混合在培養液內，使得動態培養組的聲漲結果較靜態的好。另外實驗發現當胚胎胚齡為E3.5時，動態培養組就已經顯示了相較於靜態培養組有更快的發育速率，動態組發育至早期囊胚期的比例較靜態組高出9.0%，這也顯示了經動態培養的胚胎能改善傳統體外胚胎靜態培養的環境。
介電濕潤晶片應用於微流體圖案化的研究動機希望將數位微流體系統引進印刷式電極油墨的圖案化，因為現有的印刷式技術容易導致模具的刮損造成損壞與壽命簡短，且細小線寬的模具價格昂貴且費時，本研究使用碳酸丙烯酯液體進行的微流體圖案化。實驗成功使用平行板式EWOD系統在120Vpp/20kHz操作液體的定量生成、傳輸、分離、合成。實驗中發現，當液珠體積越大時，要將液珠圖案化所需的電壓也越高，當固定上下板間距為100μm，要使液體體積大小0.563μL附蓋滿5個1.5mm×0.75mm電極需要的電壓為200Vpp/20kHz；要使液體體積大小1.113μL附蓋滿10個1.5mm×0.75mm電極需要的電壓為260Vpp/20kHz。

This study mainly makes use of the electrowetting on dielectric (EWOD) chip to apply in the dynamic culture of mouse embryos and screen printing technologies. The advantage of EWOD chip is simple fabrication, low cost, and easily to operate microfluidic generating, transporting, separating, merging.
In our study, the application of EWOD in reproductive medicine is making use of EWOD chip as a dynamic culture platform for fertilized embryos. We manipulated the HTF droplets with electro-wetting force in an oil bath environment to imitate the dynamic movement of the embryos inside the mother. The experiments contained three groups: the traditional droplet culture method is taken as the experimental control group, the second one is the static culture group on chip without any movement, and the third one is dynamic culture group driven by electric wetting force on the chip. In the static culture group, the fertilized embryos were injected into equilibrated culture medium and placed them in the oil on the EWOD chip. In the dynamic culture group, the fertilized embryos were injected into equilibrated culture medium droplet, and the culture medium droplet was moved every 5 minutes for two hours to imitate the dynamic process of the fertilized embryos moving from the fallopian tube to the uterus. After 3 day cell culture, about 77.8% (N=54) of the embryos eventually developed to the morula and blastocyst stage cultured in the dish(control group); the rate of static group is 62.5% (N=56), and the rate of dynamic group is similar with the control group, 75.0% (N=56) of the embryos developed to the morula and blastocyst stage. The development rate of embryos of dynamic culture was 12.5% higher than the static group at embryo age E4.5. In fact, early blastocysts can be observed in each group at embryo age E3.5. The early blastocyst proportion of dynamic group (23.2%) is 9.0% higher than the static group (14.2%), and similar with the control group (26.0%). The result suggests that the electro-wetting force may not affect the embryos which proves that the biocompatibility of EWOD and the potential of EWOD chip for delivering biological cells and cells culture.


The motivation of patterning of microfluidic is hoped that the digital microfluidic system can be used in printing technologies. Because current printing technologies easily cause the mother's mold damage and short life, and molds of small line width are expensive and time-consuming, this paper used EWOD chip to realize microfluidic patterning of propylene carbonate fliud. The experiment generated, transported, separated and merged droplets by using the parallel plate EWOD system at 120Vpp / 20kHz. The experiment found that when the volume of the droplet is larger, the voltage required to pattern the droplet is higher. When the gap of the top plate and bottom plate is fixed to 100μm, the 0.563μL droplet cab be covered with 5 electrodes (one electrode size:1.5mm × 0.75mm) by applying 200 Vpp / 20 kHz, and the 1.113μL droplet can be covered with 10 electrodes by applying 260 Vpp / 20 kHz.

中文摘要 2
ABSTRACT 4
目錄 6
圖目錄 9
表目錄 14
第一章 緒論 15
1.1 前言 15
1.2 研究目標 16
1.3 本文架構 16
第二章 文獻回顧 17
2.1 介電濕潤現象與微液珠操控 17
2.2 介電層材料之探討 24
2.3 體外胚胎培養裝置與動態培養相關文獻 28
2.4 介電濕潤系統應用於生醫檢測及細胞培養 32
2.5 微流體圖案化與印刷電極相關文獻 39
第三章 研究方法及系統架設 45
3.1 介電濕潤原理 45
3.2 共平面式介電濕潤系統 47
3.3 介電濕潤晶片製程 48
3.4 介電濕潤晶片之NI-USB 6509系統架設及建立 58
3.5 老鼠胚胎樣品來源 61
3.6 液體圖案化實驗製備與架構 63
第四章 鼠胚動態培養實驗結果 65
4.1 EWOD晶片之微液滴操控 65
4.2 共平面式晶片之DI 水操控測試 65
4.3 EWOD晶片應用於小鼠受精卵培養應用 67
4.3.1 晶片之生物相容性驗證 68
4.3.2介電濕潤系統之胚胎動態培養情形 71
4.3.3培養液液珠含少數卵子之動態培養比較 74
第五章 微流體圖案化實驗結果 78
5.1 平板式EWOD晶片應用於微流體圖案化 78
5.2 平行板式陣列電極晶片之液珠定位 78
5.3 交流電壓對於微流體圖案化之影響 79
5.4 碳酸丙烯酯液體之液珠圖案化 80
第六章 結論 83
第七章 未來工作 85
6.1受精卵母體植回 85
6.2 判斷是否為孤雌生殖 85
6.3共平面式陣列電極晶片之液珠圖案化 86
6.4 L-DEP與EWOD電極的結合 88
文獻參考 92

[1] R. P. Feynman, "There's plenty of room at the bottom," Journal of Microelectromechanical Systems, vol. 1, pp. 60-66, 1992.
[2] R. Feynman, "Infinitesimal machinery," Journal of Microelectromechanical Systems, vol. 2, pp. 4-14, 1993.
[3] S. K. Cho, H. Moon, J. Fowler, and C.-J. Kim, Splitting a liquid droplet for electrowetting-based microfluidics vol. 2, 2001.
[4] M. G. Pollack, A. D. Shenderov, and R. B. Fair, "Electrowetting-based actuation of droplets for integrated microfluidics," Lab on a Chip, vol. 2, pp. 96-101, 2002.
[5] C. Sung Kwon, M. Hyejin, and K. Chang-Jin, "Creating, transporting, cutting, and merging liquid droplets by electrowetting-based actuation for digital microfluidic circuits," Journal of Microelectromechanical Systems, vol. 12, pp. 70-80, 2003.
[6] A. Papathanasiou, A. T. Papaioannou, and A. Boudouvis, "Illuminating the connection between contact angle saturation and dielectric breakdown in electrowetting through leakage current measurements" ,vol. 103, 2008.
[7] Y. Li, W. Parkes, L. I. Haworth, A. W. S. Ross, J. T. M. Stevenson, and A. J. Walton, "Room-Temperature Fabrication of Anodic Tantalum Pentoxide for Low-Voltage Electrowetting on Dielectric (EWOD)," Journal of Microelectromechanical Systems, vol. 17, pp. 1481-1488, 2008.
[8] Y. S. Heo, L. M. Cabrera, C. L. Bormann, C. T. Shah, S. Takayama, and G. D. Smith, "Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates," Human Reproduction, vol. 25, pp. 613-622, 2010.
[9] K. Matsuura, N. Hayashi, Y. Kuroda, C. Takiue, R. Hirata, M. Takenami, Y. Aoi, N. Yoshioka, T. Habara, T. Mukaida, and K. Naruse, "Improved development of mouse and human embryos using a tilting embryo culture system," Reproductive BioMedicine Online, vol. 20, pp. 358-364, 2010.
[10] Y.-H. Chang, G.-B. Lee, F.-C. Huang, Y.-Y. Chen, and J.-L. Lin, "Integrated polymerase chain reaction chips utilizing digital microfluidics," Biomedical Microdevices, vol. 8, pp. 215-225, 2006.
[11] V. N. Luk, G. C. H. Mo, and A. R. Wheeler, "Pluronic Additives: A Solution to Sticky Problems in Digital Microfluidics," Langmuir, vol. 24, pp. 6382-6389, 2008.
[12] S. H. Au, P. Kumar, and A. R. Wheeler, "A New Angle on Pluronic Additives: Advancing Droplets and Understanding in Digital Microfluidics," Langmuir, vol. 27, pp. 8586-8594, 2011.
[13] D. Witters, N. Vergauwe, S. Vermeir, F. Ceyssens, S. Liekens, R. Puers, and J. Lammertyn, "Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications," Lab on a Chip, vol. 11, pp. 2790-2794, 2011.
[14] S. Srigunapalan, I. A. Eydelnant, C. A. Simmons, and A. R. Wheeler, "A digital microfluidic platform for primary cell culture and analysis," Lab on a Chip, vol. 12, pp. 369-375, 2012.
[15] I. Barbulovic-Nad, S. H. Au, and A. R. Wheeler, "A microfluidic platform for complete mammalian cell culture," Lab on a Chip, vol. 10, pp. 1536-1542, 2010.
[16] D. G. Pyne, J. Liu, M. Abdelgawad, and Y. Sun, "Digital Microfluidic Processing of Mammalian Embryos for Vitrification," PLOS ONE, vol. 9, p. e108128, 2014.
[17] N. Tsukada, K.-i. Kudoh, M. Budiman, A. Yamamoto, T. Higuchi, M. Kobayashi, K. Sato, K. Oishi, and K. Iida, "Development of Automated Nuclear Transplantation System," Journal of Mammalian Ova Research, vol. 18, pp. 106-109, 2001.
[18] H. Y. Tseng, D. J. Yao, C. H. Tien, C. J. Li, and H. Y. Huang, "Using control microfluidic system to enhance the sperm motility sorting efficiency," in 2012 IEEE 6th International Conference on Nano/Molecular Medicine and Engineering (NANOMED),pp. 47-50, 2012.
[19] H.-Y. Huang, T.-L. Wu, H.-R. Huang, C.-J. Li, H.-T. Fu, Y.-K. Soong, M.-Y. Lee, and D.-J. Yao, "Isolation of Motile Spermatozoa with a Microfluidic Chip Having a Surface-Modified Microchannel," Journal of Laboratory Automation, vol. 19, pp. 91-99, 2013.
[20] J. E. Swain and G. D. Smith, "Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment," Human Reproduction Update, vol. 17, pp. 541-557, 2011.
[21] 曾新堯, "利用控制微流體系統以提升精蟲篩選之效能," 清華大學奈米工程與微系統研究所學位論文, 清華大學, 2013.
[22] H. C. Zeringue, I. K. Glasgow, D. J. Beebe, J. T. Lyman, and M. B. Wheeler, "Micro fluidic single embryo culture systems in PDMS," in Proceedings of the First Joint BMES/EMBS Conference. 1999 IEEE Engineering in Medicine and Biology 21st Annual Conference and the 1999 Annual Fall Meeting of the Biomedical Engineering Society (Cat. N, p. 851 vol.2, 1999.
[23] S.-K. Fan, P.-W. Huang, T.-T. Wang, and Y.-H. Peng, "Cross-scale electric manipulations of cells and droplets by frequency-modulated dielectrophoresis and electrowetting," Lab on a Chip, vol. 8, pp. 1325-1331, 2008.
[24] H. Geng, J. Feng, L. M. Stabryla, and S. K. Cho, "Dielectrowetting manipulation for digital microfluidics: creating, transporting, splitting, and merging of droplets," Lab on a Chip, vol. 17, pp. 1060-1068, 2017.
[25] H. Geng, J. Feng, L. M. Stabryla, and S. K. Cho, "Droplet manipulations by dielectrowetting: Creating, transporting, splitting, and merging," in 2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS), pp. 113-116, 2017.
[26] Y. C. Lin, K. C. Chuang, T. T. Wang, C. P. Chiu, and S. K. Fan, "Integrated Digital and Analog Microfluidics by EWOD and LDEP," in 2006 1st IEEE International Conference on Nano/Micro Engineered and Molecular Systems, pp. 1414-1417, 2006.
[27] H.-H. Shen, L.-Y. Chung, and D.-J. Yao, "Improving the dielectric properties of an electrowetting-on-dielectric microfluidic device with a low-pressure chemical vapor deposited Si3N4 dielectric layer," Biomicrofluidics, vol. 9, 2015.
[28] H.-Y. Huang, H.-H. Shen, C.-H. Tien, C.-J. Li, S.-K. Fan, C.-H. Liu, W.-S. Hsu, and D.-J. Yao, "Digital Microfluidic Dynamic Culture of Mammalian Embryos on an Electrowetting on Dielectric (EWOD) Chip," PLOS ONE, vol. 10, 2015.
[29] H. Y. Huang, H. H. Shen, L. Y. Chung, Y. H. Chung, C. C. Chen, C. H. Hsu, S. K. Fan, and D. J. Yao*, "Fertilization of Mouse Gametes in Vitro Using a Digital Microfluidic System," IEEE Transactions on NanoBioscience, vol. 14, pp. 857-863, 2015.
[30] L. Y. Chung, H. H. Shen, Y. H. Chung, C. C. Chen, C. H. Hsu, H. Y. Huang, and D. J. Yao, "In vitro dynamic fertilization by using EWOD device," in 2015 28th IEEE International Conference on Micro Electro Mechanical Systems (MEMS), pp. 519-522, 2015.
[31] A. Karimpor Malekshah and A. Esmailnejad Moghaddam, "The effect of culture medium volume on in vitro development of mouse embryos," International Journal of Reproductive BioMedicine; Vol 3, 2005.
[32] M. Lane and D. K. Gardner, "Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro," Hum Reprod, vol. 7, pp. 558-62, 1992.
[33] Y. Fukui, E. S. Lee, and N. Araki, "Effect of medium renewal during culture in two different culture systems on development to blastocysts from in vitro produced early bovine embryos," J Anim Sci, vol. 74, pp. 2752-8, 1996.
[34] B. Heindryckx, A. Rybouchkin, J. Van Der Elst, and M. Dhont, "Effect of culture media on in vitro development of cloned mouse embryos," Cloning, vol. 3, pp. 41-50, 2001.
[35] T. Otoi, L. Willingham, T. Shin, D. C. Kraemer, and M. Westhusin, "Effects of oocyte culture density on meiotic competence of canine oocytes," Reproduction, vol. 124, pp. 775-8, 2002.
[36] C. C. Li, Y. W. Hsu, H. Y. Huang, and S. K. Fan, "Dynamic embryo culture on a digital microfluidic chip," in 2012 IEEE 6th International Conference on Nano/Molecular Medicine and Engineering (NANOMED), pp. 74-77, 2012.
[37] B. Zhao, J. S. Moore, and D. J. Beebe, "Surface-Directed Liquid Flow Inside Microchannels," Science, vol. 291, p. 1023, 2001.
[38] C. P. Chiu, W. J. Chen, and S. K. Fan, "Enhanced Droplet Mixer by LDEP on Spiral Microelectrodes," in 2007 2nd IEEE International Conference on Nano/Micro Engineered and Molecular Systems, pp. 951-954, 2007.
[39] J. Noh, D. Yeom, C. Lim, H. Cha, J. Han, J. Kim, Y. Park, V. Subramanian, and G. Cho, Scalability of Roll-to-Roll Gravure-Printed Electrodes on Plastic Foils vol. 33, 2010.
[40] T. B. Jones, M. Gunji, M. Washizu, and M. J. Feldman, Dielectrophoretic liquid actuation and nanodroplet formation vol. 89, 2001.
[41] T. B. Jones, K. L. Wang, and D. J. Yao, "Frequency-Dependent Electromechanics of Aqueous Liquids:  Electrowetting and Dielectrophoresis," Langmuir, vol. 20, pp. 2813-2818, 2004.
[42] K. Wang and T. B. Jones, "Saturation effects in dynamic electrowetting", vol. 86, 2005.
[43] T. B. Jones, "More about the electromechanics of electrowetting," Mechanics Research Communications, vol. 36, pp. 2-9, 2009.
[44] S. Bansal and P. Sen, "Frequency response of a liquid interface segment actuated using AC EWOD," in 2016 3rd International Conference on Emerging Electronics (ICEE), pp. 1-3, 2016.
[45] W. Wang, J. Chen, and J. Zhou, "Droplet generating with accurate volume for EWOD digital microfluidics," in 2015 IEEE 11th International Conference on ASIC (ASICON), pp. 1-4, 2015.