臺灣博碩士論文加值系統

在在現在的細胞治療及基因轉殖的技術上，主要的關鍵在於如何能夠突破細胞膜的限制，把基因或藥物送入，藉此得知單一種分子對細胞的影響。在傳統電穿孔儀器上，所需要的電壓、樣品量及產生的廢熱都會比較高，且對象只能針對大量細胞，分子對細胞的影響只能藉由表現的平均值的方式去推算，無法針對單一細胞進行了解。
因此本研究致力於發展微流道裝置進行單一細胞之電穿孔，實驗以轉染腺病毒E1A基因的人腎上皮細胞系(HEK 293T cell)為例，利用流體集中(hydrodynamic focusing)的方法配合橫向流(cross flow)的壓力將目標細胞推送至抓取L型結構陣列中。接著對結構的兩側的ITO電極施加不同的脈衝電壓，造成細胞膜破洞，執行電穿孔，同時將螢光染劑及pEGPF-c1螢光蛋白送入細胞，以利觀察，驗證電穿孔的結果。本研究所設計的微流道裝置成功的達到針對單一目標細胞電穿孔的效果。

One of the key techniques in gene delivery and cell therapy is to break through the limit of the cell membrane and to send gene or drug into the cell. Electroporation is one of such methods. However, it conventionally suffers the drawbacks in high voltage, large sample amount and high heat generation. The gene/drug delivery results can be only estimated for the average gene expression of the group of the cells, instead of a single cell. To realize a particular gene expression onto a single cell, our thesis thus is devoted to perform electroporation on a single cell while to improve the limitation of the traditional electroporation techniques.
A microfluidic device for single cell electroporation was presented. The HEK 293T cells were tested. The cells were first confined at the L-shape trapping structure with cross-flow cell filters enhanced by hydrodynamic focusing forces. Once the cells were trapped, the different voltage were applied for electroporation. Fluorescence dye and pEGFP-c1 plasmid were successfully delivered into the cells to validate the devices.

誌謝 i
中文摘要 ii
Abstract iii
List of Figures vii
List of Tables xiv
Chapter 1 Introduction 1
1.1 Gene Delivery 1
1.2 Single Cell Analysis 4
1.3 Overall Structure of Thesis 5
Chapter 2 Literature Review 7
2.1 Single Cell Technique 7
2.1.1 Single Cell Trap 7
2.1.2 Cross Flow Filters with Hydrodynamic Focusing 12
2.2 Electroporation Principle 13
2.3 Commercial Electroporation 19
2.3.1 Traditional Electroporation 19
2.3.2 Neon Transfection System 20
2.4 Microfluidic Electroporation 22
2.4.1 Flow Type of Electroporation 24
2.4.2 Membrane Type of Electroporation 27
2.4.3 Trapping Type of Electroporation 29
2.5 Localized Electroporation 31
Chapter 3 Material and Method 34
3.1 Chip Design 34
3.2 Trap Design 36
3.3 Numerical Simulations 38
3.4 Device Fabrication 38
3.4.1 Soft Lithography 39
3.4.2 Patterning ITO Glass 43
3.4.3 Microchannel Bonding 43
3.5 Experiment Preparation 44
3.5.1 Bead Separation 44
3.5.2 Cell Preparation 44
3.5.3 Experiment Setup 47
Chapter 4 Result and Discussion 49
4.1 Electric Field Simulation 49
4.2 Trapping 53
4.3 Electroporation 57
Chapter 5 Conclusion 66
5.1 Conclusion 66
5.2 Future Perspectives 66
Reference 70

Bordignon, C., L. D. Notarangelo, N. Nobili, G. Ferrari, G. Casorati, P. Panina, E. Mazzolari, D. Maggioni, C. Rossi, P. Servida, A. G. Ugazio and F. Mavilio (1995). "Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients." Science 270(5235): 470-475.
Brennecke, P., S. Anders, J. K. Kim, A. A. Kolodziejczyk, X. Zhang, V. Proserpio, B. Baying, V. Benes, S. A. Teichmann, J. C. Marioni and M. G. Heisler (2013). "Accounting for technical noise in single-cell RNA-seq experiments." Nature Methods 10(11): 1093-1095.
Chang, S., S. Takashima and T. Asakura (1985). "Volume and Shape Changes of Human-Erythrocytes Induced by Electrical Fields." Journal of Bioelectricity 4(2): 301-316.
Chen, S. C., T. S. Santra, C. J. Chang, T. J. Chen, P. C. Wang and F. G. Tseng (2012). "Delivery of molecules into cells using localized single cell electroporation on ITO micro-electrode based transparent chip." Biomed Microdevices 14(5): 811-817.
Chiu, Y. Y., C. K. Huang and Y. W. Lu (2016). "Enhancement of microfluidic particle separation using cross-flow filters with hydrodynamic focusing." Biomicrofluidics 10(1).
Dev, S. B., D. P. Rabussay, G. Widera and G. A. Hofmann (2000). "Medical applications of electroporation." Ieee Transactions on Plasma Science 28(1): 206-223.
Di Carlo, D., N. Aghdam and L. P. Lee (2006). "Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays." Analytical Chemistry 78(14): 4925-4930.
Di Carlo, D., H. T. Tse and D. R. Gossett (2012). "Introduction: why analyze single cells?" Methods in Molecular Biology 853: 1-10.
Edd, J. F., D. Di Carlo, K. J. Humphry, S. Koster, D. Irimia, D. A. Weitz and M. Toner (2008). "Controlled encapsulation of single-cells into monodisperse picolitre drops." Lab on a Chip 8(8): 1262-1264.
Efroni, I. and K. D. Birnbaum (2016). "The potential of single-cell profiling in plants." Genome Biology 17: 65.
Fei, Z., S. Wang, Y. Xie, B. E. Henslee, C. G. Koh and L. J. Lee (2007). "Gene transfection of mammalian cells using membrane sandwich electroporation." Analytical Chemistry 79(15): 5719-5722.
Felgner, P. L., T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold and M. Danielsen (1987). "Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure." Proceedings of the National Academy of Science of the U.S.A. 84(21): 7413-7417.
Fox, M. B., D. C. Esveld, A. Valero, R. Luttge, H. C. Mastwijk, P. V. Bartels, A. van den Berg and R. M. Boom (2006). "Electroporation of cells in microfluidic devices: a review." Analytical and Bioanalytical Chemistry 385(3): 474-485.
Fukuda, J., T. Kaneko, M. Egashira and K. Oshimi (1998). "Direct measurement of CD34+ blood stem cell absolute counts by flow cytometry." Stem Cells 16(4): 294-300.
Geng, T. and C. Lu (2013). "Microfluidic electroporation for cellular analysis and delivery." Lab on a Chip 13(19): 3803-3821.
Ji, H. M., V. Samper, Y. Chen, C. K. Heng, T. M. Lim and L. Yobas (2008). "Silicon-based microfilters for whole blood cell separation." Biomedical Microdevices 10(2): 251-257.
Kandušer, M. and D. Miklavčič (2008). Electroporation in Biological Cell and Tissue: An Overview. Electrotechnologies for Extraction from Food Plants and Biomaterials. New York, NY, Springer New York: 1-37.
Kang, W., J. P. Giraldo-Vela, S. S. Nathamgari, T. McGuire, R. L. McNaughton, J. A. Kessler and H. D. Espinosa (2014). "Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons." Lab on a Chip 14(23): 4486-4495.
Karra, D. and R. Dahm (2010). "Transfection techniques for neuronal cells." Journal of Neuroscience 30(18): 6171-6177.
Khine, M., A. Lau, C. Ionescu-Zanetti, J. Seo and L. P. Lee (2005). "A single cell electroporation chip." Lab on a Chip 5(1): 38-43.
Kim, J., I. Hwang, D. Britain, T. D. Chung, Y. Sun and D. H. Kim (2011). "Microfluidic approaches for gene delivery and gene therapy." Lab on a Chip 11(23): 3941-3948.
Kim, J. A., K. C. Cho, M. S. Shin, W. G. Lee, N. C. Jung, C. I. Chung and J. K. Chang (2008). "A novel electroporation method using a capillary and wire-type electrode." Biosensors and Bioelectronics 23(9): 1353-1360.
Lashford, L. S. (1997). "Possibilities of gene therapies for cancer." Annals of Medicine 29(1): 1-4.
Lee, P. J., P. J. Hung, R. Shaw, L. Jan and L. P. Lee (2005). "Microfluidic application-specific integrated device for monitoring direct cell-cell communication via gap junctions between individual cell pairs." Applied Physics Letters 86(22).
Lin, Y. C., C. M. Jen, M. Y. Huang, C. Y. Wu and X. Z. Lin (2001). "Electroporation microchips for continuous gene transfection." Sensors and Actuators B-Chemical 79(2-3): 137-143.
Lo, S. J. and D. J. Yao (2015). "Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis." International Journal of Molecular Sciences 16(8): 16763-16777.
Lowe, S. W. and A. W. Lin (2000). "Apoptosis in cancer." Carcinogenesis 21(3): 485-495.
Lu, H., M. A. Schmidt and K. F. Jensen (2005). "A microfluidic electroporation device for cell lysis." Lab on a Chip 5(1): 23-29.
Luft, C. and R. Ketteler (2015). "Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues." Journal of Biomolecular Screening 20(8): 932-942.
M.A.Barber (1911). "A Technic for the Inoculation of Bacteria and Other Substances into Living Cells." The Journal of Infectious Diseases 8(3): 348-360.
Manjila, S. B., J. N. Baby, E. N. Bijin, I. Constantine, K. Pramod and J. Valsalakumari (2013). "Novel gene delivery systems." International Journal of Pharmaceutical Investigation 3(1): 1-7.
Meir, A. and B. Rubinsky (2014). "Alternating electric field capacitively coupled micro-electroporation." RSC Advances 4(97): 54603-54613.
Movahed, S. and D. Q. Li (2011). "Microfluidics cell electroporation." Microfluidics and Nanofluidics 10(4): 703-734.
Muller, K. J., V. L. Sukhorukov and U. Zimmermann (2001). "Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration." Journal of Membrane Biology 184(2): 161-170.
Nawarathna, D., K. Unal and H. K. Wickramasinghe (2008). "Localized electroporation and molecular delivery into single living cells by atomic force microscopy." Applied Physics Letters 93(15).
Neumann, E., M. Schaefer-Ridder, Y. Wang and P. H. Hofschneider (1982). "Gene transfer into mouse lyoma cells by electroporation in high electric fields." EMBO J 1(7): 841-845.
Neumann, E., A. E. Sowers and C. A. Jordan (1989). Electroporation and electrofusion in cell biology. New York, Plenum Press.
Nickoloff, J. A. (1995). Animal cell electroporation and electrofusion protocols. Totowa, N.J., Humana Press.
O''Brien, J. and S. C. Lummis (2002). "An improved method of preparing microcarriers for biolistic transfection." Brain Research Protocols 10(1): 12-15.
Ohashi, T., S. Boggs, P. Robbins, A. Bahnson, K. Patrene, F. S. Wei, J. F. Wei, J. Li, L. Lucht, Y. Fei and et al. (1992). "Efficient transfer and sustained high expression of the human glucocerebrosidase gene in mice and their functional macrophages following transplantation of bone marrow transduced by a retroviral vector." Proceedings of the National Academy of Science of the U.S.A. 89(23): 11332-11336.
Olofsson, J., K. Nolkrantz, F. Ryttsen, B. A. Lambie, S. G. Weber and O. Orwar (2003). "Single-cell electroporation." Current Opinion in Biotechnology 14(1): 29-34.
Sako, Y., S. Minoghchi and T. Yanagida (2000). "Single-molecule imaging of EGFR signalling on the surface of living cells." Nature Cell Biology 2(3): 168-172.
Santra, T. S., C. W. Chen, H. Y. Chang and F. G. Tseng (2016). "Dielectric passivation layer as a substratum on localized single-cell electroporation." Rsc Advances 6(13): 10979-10986.
Santra, T. S. and F. G. Tseng (2013). "Recent Trends on Micro/Nanofluidic Single Cell Electroporation." Micromachines 4(3): 333-356.
Schwan, H. P. (1957). "Electrical properties of tissue and cell suspensions." Advances in Biological and Medical Physics 5: 147-209.
Shah, P., X. Zhu, C. Chen, Y. Hu and C. Z. Li (2014). "Lab-on-chip device for single cell trapping and analysis." Biomed Microdevices 16(1): 35-41.
Valero, A., J. N. Post, J. W. van Nieuwkasteele, P. M. Ter Braak, W. Kruijer and A. van den Berg (2008). "Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device." Lab on a Chip 8(1): 62-67.
Valley, J. K., S. Neale, H. Y. Hsu, A. T. Ohta, A. Jamshidi and M. C. Wu (2009). "Parallel single-cell light-induced electroporation and dielectrophoretic manipulation." Lab on a Chip 9(12): 1714-1720.
Varga, C. M., K. Hong and D. A. Lauffenburger (2001). "Quantitative analysis of synthetic gene delivery vector design properties." Molecular Therapy 4(5): 438-446.
Weiss, S. (2000). "Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy." Nature Structural Biology 7(9): 724-729.
Wenzel, G., O. Schieder, T. Przewozny, S. K. Sopory and G. Melchers (1979). "Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs." Theoretical and Applied Genetics 55(2): 49-55.
Yun, H. and S. C. Hur (2013). "Sequential multi-molecule delivery using vortex-assisted electroporation." Lab on a Chip 13(14): 2764-2772.
Zhan, Y., J. Wang, N. Bao and C. Lu (2009). "Electroporation of cells in microfluidic droplets." Analytical Chemistry 81(5): 2027-2031.
Zhu, T., C. Luo, J. Huang, C. Xiong, Q. Ouyang and J. Fang (2010). "Electroporation based on hydrodynamic focusing of microfluidics with low dc voltage." Biomedical Microdevices 12(1): 35-40.
Zimmermann, U. (1986). "Electrical Breakdown, Electropermeabilization and Electrofusion." Reviews of Physiology Biochemistry and Pharmacology 105: 175-256.