光誘導電穿孔技術是一種微流體細胞電穿孔的方法。然而現有的光誘導電穿孔晶片，並沒有樣品前處理功能，如溶液置換。其他文獻所提出的微流體細胞電穿孔裝置亦同。因此，本研究提出一連續式溶液置換與光誘導電穿孔之整合式微流體系統。只要將處在原生長溶液的樣品細胞注入此晶片，即可完成無縫溶液置換，自動將樣品細胞置換到特定實驗所需的電穿孔緩衝溶液內。在傳統實驗流程中，溶液置換必須使用實驗室離心機將細胞沉澱，抽取上清液後，注入新的溶液讓細胞重新懸浮，並往復多次，方能達成溶液置換的目的。然而此方法很可能因離心力過大或人為操作失誤導致細胞損失，不利於操作稀有或數量極少的細胞檢體。本研究分別採用微柱狀陣列導引結構（micropost array railing）與決定性側向位移結構（deterministic lateral displacement）作為連續式溶液置換模組，並將之與光誘導電穿孔模組整合。結果證實本整合式系統能成功完成自動式溶液置換，將流程自動化，有效降低上述人工樣品前處理的各種風險。同時，對人類胚胎腎臟細胞（human embryonic kidney 293T）的單一螢光基因（EGFP）轉染，達到現有光誘導電穿孔系統最高之8.3%轉染效率。此外，單一細胞之多重螢光基因（EGFP、ECFP與DsRed）轉染實驗的結果，也證實本系統具備同時進行多種基因轉染之能力，及各種分子生物應用領域之潛能。

Optically-induced electroporation (OIE) is a promising microfluidic-based approach for cell electroporation. However, previously proposed microfluidic cell electroporation devices required tedious sample pre-treatment steps, including media exchange. To facilitate this OIE process for user-friendly cell electroporation, we present herein a new design for continuous OIE on a microfluidic device that is capable of replacing culture media and electroporation buffers in a seamless fashion. The on-chip integration of media exchange with OIE avoided critical issues such as cell loss and damage, both of which are common with traditional, centrifuge-based approaches. Our new system is therefore suitable for handling small or rare cell populations. Medium exchange modules of a micropost array railing structure and a deterministic lateral displacement structure were first explored and integrated with the OIE module respectively. The efficacy of the integrated systems was demonstrated by transfection of a green fluorescent protein (EGFP) plasmid into human embryonic kidney 293T cells with an efficiency of 8.3%. It was the highest efficiency reported so far for the existing OIE systems on microfluidic system. Also, successful co-transfections of three distinct plasmids (EGFP, DsRed, and ECFP) into the cells were demonstrated, suggesting the system is capable of performing multiple gene transfections in to mammalian cells.

Abstract I
摘要 II
致謝 III
Publication list IV
Table of content V
List of figures IX
List of tables XXIII
Abbreviations and nomenclature XXIV
Chapter 1: Introduction 1
1.1 MEMS and Bio-MEMS technology 1
1.2 Electroporation 2
1.3 Microfluidic electroporation 4
1.4 Optically-induced electroporation 8
1.5 Microfluidics for cell separation 10
1.5.1 Active and passive methods for cell separation 10
1.6 Carrier medium exchange 13
1.7 Deterministic Lateral Displacement 17
1.7.1 Design parameters of DLD 20
1.8 Micro-post array railing 24
1.8.1 Design parameters of μPAR 27
1.9 Motivation and objectives 30
Chapter 2: Theory and numeric simulation 33
2.1 Working principle of optically-induced electroporation 33
2.2 Optimization of OIE performance: transmembrane potential 36
2.3 Numerical simulation of OIE performance: transmembrane potential 38
Chapter 3: Materials and methods 48
3.1 Experimental overview and the continuous OIE chip design 48
3.1.1 Chip integration: DLD medium exchange module and OIE module 48
3.1.2 Design of DLD structure 50
3.1.3 Chip integration: µPAR medium exchange module and OIE module 52
3.1.4 Design of µPAR structure 53
3.2 Fabrication of the continuous OIE chip 56
3.2.1 SU-8 lithography 57
3.2.1.1 Substrate pretreatment 57
3.2.1.2 Photoresist coating 57
3.2.1.3 Soft bake 58
3.2.1.4 Exposure, post exposure bake and development 58
3.2.1.5 Hard bake 58
3.2.2 PDMS casting of DLD structure 59
3.2.3 PDMS casting of µPAR structure 62
3.2.4 OIE channel fabrication and chip assembly 64
3.3 Preparation of HEK 293T cells and electroporation buffer 64
3.4 PDMS surface modification 65
3.5 Experimental setup 65
3.6 Experimental parameters 69
Chapter 4: Results and discussion 70
4.1 Simulation of OIE performance 70
4.1.1 Frequency dependence of the transmembrane potential 70
4.1.2 The effect of size and the photoconductivity of the virtual electrode to the transmembrane potential 72
4.1.3 The optimal parameters for maximizing transmembrane potential 75
4.2 Microfluidic medium exchange 77
4.2.1 Exchange efficiency of µPAR structure 77
4.2.2 Cell recovery rate of DLD structure 80
4.2.3 Factors affecting the recovery rates in medium exchange modules 82
4.2.3.1 Shear-stress-induced cell lysis 82
4.2.3.2 Cells being clogged at or failed to be guided by the structure 84
4.3 Electroporation of propidium iodide 87
4.4 Transfection of EGPF plasmid DNA 90
4.5 Co-transfection of three fluorescent Plasmid DNAs 93
4.6 Factors affecting the transfection efficiency of the integrated system 100
Chapter 5: Conclusions and future perspectives 103
5.1 Conclusions 103
5.2 Future perspectives 104
References 105

1. W. J. Jack, "Microelectromechanical systems (MEMS): fabrication, design and applications". Smart Materials and Structures, 2001. 10(6): p. 1115.
2. C.-M. Ho and Y.-C. Tai, "Micro-electro-mechanical-systems (MEMS) and fluid flows". Annual Review of Fluid Mechanics, 1998. 30(1): p. 579-612.
3. J. W. Park, H. J. Kim, M. W. Kang, and N. L. Jeon, "Advances in microfluidics-based experimental methods for neuroscience research". Lab Chip, 2013. 13(4): p. 509-21.
4. E. Nuxoll, "BioMEMS in drug delivery". Adv Drug Deliv Rev, 2013. 65(11-12): p. 1611-25.
5. J.-Y. Yoon and B. Kim, "Lab-on-a-chip pathogen sensors for food safety". Sensors (Basel), 2012. 12(8): p. 10713-41.
6. A. K. Au, H. Lai, B. R. Utela, and A. Folch, "Microvalves and Micropumps for BioMEMS". Micromachines, 2011. 2(4): p. 179-220.
7. A. C. R. Grayson, R. S. Shawgo, A. M. Johnson, N. T. Flynn, Y. Li, M. J. Cima, and R. langer, "A BioMEMS review: MEMS technology for physiologically integrated devices". Proceedings of the Ieee, 2004. 92(1): p. 6-21.
8. E. Neumann, M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider, "Gene transfer into mouse lyoma cells by electroporation in high electric fields". The EMBO journal, 1982. 1(7): p. 841.
9. I. P. Sugar and E. Neumann, "Stochastic model for electric field-induced membrane pores electroporation". Biophysical Chemistry, 1984. 19(3): p. 211-225.
10. S. Movahed and D. Li, "Microfluidics cell electroporation". Microfluidics and Nanofluidics, 2011. 10(4): p. 703-734.
11. J. Kim, I. Hwang, D. Britain, T. D. Chung, Y. Sun, and D.-H. Kim, "Microfluidic approaches for gene delivery and gene therapy". Lab Chip, 2011. 11(23): p. 3941-3948.
12. J. K. Valley, S. Neale, H.-Y. Hsu, A. T. Ohta, A. Jamshidi, and M. C. Wu, "Parallel single-cell light-induced electroporation and dielectrophoretic manipulation". Lab Chip, 2009. 9(12): p. 1714-1720.
13. H.-T. Kuo, Y.-H. Lee, C.-H. Wang, C.-M. Chang, and G.-B. Lee. An optical-induced platform for gene transfection. in Micro TAS. 2012.
14. C.-H. Wang, Y.-H. Lee, H.-T. Kuo, W.-F. Liang, W.-J. Li, and G.-B. Lee, "Dielectrophoretically-assisted electroporation using light-activated virtual microelectrodes for multiple DNA transfection". Lab Chip, 2014. 14(3): p. 592-601.
15. G.-B. Lee, H.-C. Wu, P.-F. Yang, and J. D. Mai, "Optically-induced dielectropheresis sorting with automated medium exchange in an integrated optofluidic device resulting in higher cell viability". Lab Chip, 2014.
16. J. H. Kang and J.-K. Park, "Cell separation technology". Yeast, 2004. 90.
17. N. Pamme, "Continuous flow separations in microfluidic devices". Lab Chip, 2007. 7(12): p. 1644-1659.
18. A. Bhagat, H. Bow, H. Hou, S. Tan, J. Han, and C. Lim, "Microfluidics for cell separation". Medical & Biological Engineering & Computing, 2010. 48(10): p. 999-1014.
19. A. Lenshof and T. Laurell, "Continuous separation of cells and particles in microfluidic systems". Chemical Society Reviews, 2010. 39(3): p. 1203-1217.
20. X. Xuan, J. Zhu, and C. Church, "Particle focusing in microfluidic devices". Microfluidics and Nanofluidics, 2010. 9(1): p. 1-16.
21. H. W. Hou, A. A. S. Bhagat, W. C. Lee, S. Huang, J. Han, and C. T. Lim, "Microfluidic Devices for Blood Fractionation". Micromachines, 2011. 2(3): p. 319-343.
22. J. P. Beech, S. H. Holm, K. Adolfsson, and J. O. Tegenfeldt, "Sorting cells by size, shape and deformability". Lab Chip, 2012. 12(6): p. 1048-51.
23. M. M. Wang, E. Tu, D. E. Raymond, J. M. Yang, H. Zhang, N. Hagen, B. Dees, E. M. Mercer, A. H. Forster, I. Kariv, P. J. Marchand, and W. F. Butler, "Microfluidic sorting of mammalian cells by optical force switching". Nat Biotechnol, 2005. 23(1): p. 83-7.
24. N. Xia, T. P. Hunt, B. T. Mayers, E. Alsberg, G. M. Whitesides, R. M. Westervelt, and D. E. Ingber, "Combined microfluidic-micromagnetic separation of living cells in continuous flow". Biomed Microdevices, 2006. 8(4): p. 299-308.
25. X. Hu, P. H. Bessette, J. Qian, C. D. Meinhart, P. S. Daugherty, and H. T. Soh, "Marker-specific sorting of rare cells using dielectrophoresis". Proc Natl Acad Sci U S A, 2005. 102(44): p. 15757-61.
26. T. Akagi and T. Ichiki, "Cell electrophoresis on a chip: what can we know from the changes in electrophoretic mobility?". Anal Bioanal Chem, 2008. 391(7): p. 2433-41.
27. M. P. MacDonald, G. C. Spalding, and K. Dholakia, "Microfluidic sorting in an optical lattice". Nature, 2003. 426(6965): p. 421-424.
28. F. Petersson, L. Åberg, A.-M. Swärd-Nilsson, and T. Laurell, "Free Flow Acoustophoresis:  Microfluidic-Based Mode of Particle and Cell Separation". Analytical Chemistry, 2007. 79(14): p. 5117-5123.
29. J. A. Davis, D. W. Inglis, K. J. Morton, D. A. Lawrence, L. R. Huang, S. Y. Chou, J. C. Sturm, and R. H. Austin, "Deterministic hydrodynamics: Taking blood apart". Proceedings of the National Academy of Sciences, 2006. 103(40): p. 14779-14784.
30. P. Sethu, A. Sin, and M. Toner, "Microfluidic diffusive filter for apheresis (leukapheresis)". Lab Chip, 2006. 6(1): p. 83-9.
31. J. Takagi, M. Yamada, M. Yasuda, and M. Seki, "Continuous particle separation in a microchannel having asymmetrically arranged multiple branches". Lab Chip, 2005. 5(7): p. 778-784.
32. M. Yamada and M. Seki, "Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics". Lab Chip, 2005. 5(11): p. 1233-9.
33. M. Yamada, K. Kano, Y. Tsuda, J. Kobayashi, M. Yamato, M. Seki, and T. Okano, "Microfluidic devices for size-dependent separation of liver cells". Biomed Microdevices, 2007. 9(5): p. 637-45.
34. S. S. Kuntaegowdanahalli, A. A. S. Bhagat, G. Kumar, and I. Papautsky, "Inertial microfluidics for continuous particle separation in spiral microchannels". Lab Chip, 2009. 9(20): p. 2973-80.
35. S. Nagrath, L. V. Sequist, S. Maheswaran, D. W. Bell, D. Irimia, L. Ulkus, M. R. Smith, E. L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U. J. Balis, R. G. Tompkins, D. A. Haber, and M. Toner, "Isolation of rare circulating tumour cells in cancer patients by microchip technology". Nature, 2007. 450(7173): p. 1235-9.
36. S. Yang, A. Undar, and J. D. Zahn, "A microfluidic device for continuous, real time blood plasma separation". Lab Chip, 2006. 6(7): p. 871-80.
37. S. Choi, S. Song, C. Choi, and J.-K. Park, "Continuous blood cell separation by hydrophoretic filtration". Lab Chip, 2007. 7(11): p. 1532-8.
38. F. Petersson, A. Nilsson, H. Jönsson, and T. Laurell, "Carrier Medium Exchange through Ultrasonic Particle Switching in Microfluidic Channels". Analytical Chemistry, 2005. 77(5): p. 1216-1221.
39. M. Yamada, J. Kobayashi, M. Yamato, M. Seki, and T. Okano, "Millisecond treatment of cells using microfluidic devices via two-step carrier-medium exchange". Lab Chip, 2008. 8(5): p. 772-778.
40. L. R. Huang, E. C. Cox, R. H. Austin, and J. C. Sturm, "Continuous Particle Separation Through Deterministic Lateral Displacement". Science, 2004. 304(5673): p. 987-990.
41. D. W. Inglis, J. A. Davis, R. H. Austin, and J. C. Sturm, "Critical particle size for fractionation by deterministic lateral displacement". Lab Chip, 2006. 6(5): p. 655-658.
42. R. Quek, D. V. Le, and K.-H. Chiam, "Separation of deformable particles in deterministic lateral displacement devices". Physical Review E, 2011. 83(5): p. 056301.
43. N. M. Karabacak, P. S. Spuhler, F. Fachin, E. J. Lim, V. Pai, E. Ozkumur, J. M. Martel, N. Kojic, K. Smith, P.-i. Chen, J. Yang, H. Hwang, B. Morgan, J. Trautwein, T. A. Barber, S. L. Stott, S. Maheswaran, R. Kapur, D. A. Haber, and M. Toner, "Microfluidic, marker-free isolation of circulating tumor cells from blood samples". Nat Protoc, 2014. 9(3): p. 694-710.
44. Z. Liu, F. Huang, J. Du, W. Shu, H. Feng, X. Xu, and Y. Chen, "Rapid isolation of cancer cells using microfluidic deterministic lateral displacement structure". Biomicrofluidics, 2013. 7(1): p. 011801.
45. S. H. Holm, J. P. Beech, M. P. Barrett, and J. O. Tegenfeldt, "Separation of parasites from human blood using deterministic lateral displacement". Lab Chip, 2011. 11(7): p. 1326-1332.
46. K. J. Morton, K. Loutherback, D. W. Inglis, O. K. Tsui, J. C. Sturm, S. Y. Chou, and R. H. Austin, "Hydrodynamic metamaterials: Microfabricated arrays to steer, refract, and focus streams of biomaterials". Proceedings of the National Academy of Sciences, 2008. 105(21): p. 7434-7438.
47. J. V. Green, M. Radisic, and S. K. Murthy, "Deterministic Lateral Displacement as a Means to Enrich Large Cells for Tissue Engineering". Analytical Chemistry, 2009. 81(21): p. 9178-9182.
48. W. I. David, L. Megan, and E. N. Robert, "Scaling deterministic lateral displacement arrays for high throughput and dilution-free enrichment of leukocytes". Journal of Micromechanics and Microengineering, 2011. 21(5): p. 054024.
49. R. D. Sochol, S. Li, L. P. Lee, and L. Lin, "Continuous flow multi-stage microfluidic reactors via hydrodynamic microparticle railing". Lab Chip, 2012. 12(20): p. 4168-4177.
50. R. D. Sochol, D. Corbett, S. Hesse, W. E. R. Krieger, K. T. Wolf, M. Kim, K. Iwai, S. Li, L. P. Lee, and L. Lin, "Dual-mode hydrodynamic railing and arraying of microparticles for multi-stage signal detection in continuous flow biochemical microprocessors". Lab Chip, 2014. 14(8): p. 1405-9.
51. W. Choi, S.-H. Kim, J. Jang, and J.-K. Park, "Lab-on-a-display: a new microparticle manipulation platform using a liquid crystal display (LCD)". Microfluidics and Nanofluidics, 2007. 3(2): p. 217-225.
52. H.-y. Hsu, A. T. Ohta, P.-Y. Chiou, A. Jamshidi, S. L. Neale, and M. C. Wu, "Phototransistor-based optoelectronic tweezers for dynamic cell manipulation in cell culture media". Lab Chip, 2010. 10(2): p. 165-172.
53. Y.-H. Lin and G.-B. Lee, "An optically induced cell lysis device using dielectrophoresis". Applied Physics Letters, 2009. 94(3).
54. P. Marszalek, D.-S. Liu, and T. Y. Tsong, "Schwan equation and transmembrane potential induced by alternating electric-field". Biophys J, 1990. 58(4): p. 1053-1058.
55. C. Kremer, C. Witte, S. L. Neale, J. Reboud, M. P. Barrett, and J. M. Cooper, "Shape-dependent optoelectronic cell lysis". Angew Chem Int Ed Engl, 2014. 53(3): p. 842-6.
56. R. W. Glaser, S. L. Leikin, L. V. Chernomordik, V. F. Pastushenko, and A. I. Sokirko, "Reversible electrical breakdown of lipid bilayers: formation and evolution of pores". Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988. 940(2): p. 275-287.
57. C. G. Malmberg and A. A. Maryott, "Dielectric constants of aqueous solutions of dextrose and sucrose". Journal of Research of the National Bureau of Standards, 1950. 45(4): p. 299-303.
58. M. Pavlin and D. Miklavcic, "The Effective Conductivity and the Induced Transmembrane Potential in Dense Cell System Exposed to DC and AC Electric Fields". Plasma Science, IEEE Transactions on, 2009. 37(1): p. 99-106.
59. C. Chen, J. A. Evans, M. P. Robinson, S. W. Smye, and P. O. Toole, "Measurement of the efficiency of cell membrane electroporation using pulsed ac fields". Phys Med Biol, 2008. 53(17): p. 4747.
60. K. Loutherback, K. Chou, J. Newman, J. Puchalla, R. Austin, and J. Sturm, "Improved performance of deterministic lateral displacement arrays with triangular posts". Microfluidics and Nanofluidics, 2010. 9(6): p. 1143-1149.