臺灣博碩士論文加值系統

A microfluidic-driving array is reported in this paper for the count and sortation of micro-scale biological sample based on the Alternating Current Electro Osmosis Flow, which is achieved by the design of the asymmetric electrode pairs array. The features of our proposed design can not only be a micro-flow cytometry but also reduce shear stress on bio-objects. The design, simulation and experimental results of this proposed AC electrokinetic micro-flow cytometry are reported in this paper. The micro-flow cytometry is consisted of titanium electrodes layer. The manipulation electrodes are composed of the asymmetric-pairs electrodes arranged in line and arrow. All of them are embedded in the device chamber, which includes glass bottom and poly-dimethylsiloxane (PDMS) top. The functional electrodes are driven by using a waveform generator that provides the sinusoidal wave excitation of 1 to 10 volts peak to peak. When the ac potentials are applied to these asymmetric electrodes, a uni-direction flow can be generated by the gradient of the electric field. Responding to the frequency of ac potentials applied, the net electroosmosis flow is induced to different direction that results from both capacity charging and faradic charging effects. Then, the bio-particles can be driven with the electroosmosis flow, be focused, separated, and sorted. In this paper, we use HMEC-1 as target objects and special buffer which is low conductivity and the same osmotic pressure on our cells as the experimental medium to demonstrate the manipulation functions. Finally we will discuss cell viability ratio between different situations: “normal cell culture”, “after buffer change”, and “after EOF manipulates cells”, obtaining the excellent results, which are higher than 95%.

一個使用交流電滲流來計數、分選微米級生物樣本之微流體驅動陣列晶片，藉由不對稱電極對的設計來達到驅動的效果。我們所設計的電極陣列不只是一個微型的流式細胞儀,同時它也能降低施加在生物性目標物上的剪應力。這裡所提出來的交流電動力操控晶片設計之模擬與實驗結果也在此篇論文中做討論。微流體驅動之操控陣列是由鈦金屬電極層組成。整個操控陣列是由不對稱電極對以直線與箭形排列而成。且整體被包含在一個由玻璃基板與聚二甲基矽氧烷上蓋組成的微流道腔室當中。驅動電極的交流電訊號源是使用波形產生器所提供，產生一到十伏特波峰對波峰的正弦波。當交流電訊號施加在不對稱電極上時，一個具有相同方向的流體被不對稱的電場所驅動出來。調控所施加的電訊號頻率，由於電容效應以及法拉第效應所造成的結果可使電滲流產生不同方向的淨流量。接著，所要操控的生物性樣本就會被電滲流所驅動，而集中、分散、分選。在這篇論文中，我們使用人類微血管內皮細胞株當作我們的操控目標物，並以細胞相同滲透壓、低導電度的緩衝液當作實驗液體，來呈現整個操控晶片的性能。最後我們討論:正常培養、更換緩衝液後、電滲流操作後的細胞生存率，並且得到高達95%以上的實驗結果。

Chapter 1. Introduction 1
1.1 Background and Motivation 1
1.2 Review of Present Flow Cytometry 2
1.2.1 Flow Cytometry 3
1.2.2 Fluorescence-activated Cell Sorting 4
1.3 Micro-Flow Cytometry 4
1.3.1 Hydrodynamic Focusing 5
1.3.2 Electrokinetical Focusing 7
Chapter 2. Device Development 10
2.1 Fundamental Theory 10
2.1.1 Electroosmosis 10
2.1.2 Electrical Double Layer 11
2.1.3 Alternative Current Electro-osmosis 14
2.2 Design Concept 22
2.3 Simulation Analyses 26
2.3.1 Flow Field Analyses 26
2.3.2 Shear Stress Analyses 31
Chapter 3. Micro Fabrication 36
3.1 Process Flow 36
3.2 Fabrication Results 39
Chapter 4. Experimental Setup and Results 40
4.1 Experimental Setup 40
4.2 Particle Test Results of 1st Design 42
4.3 Particle Test Results of 2nd Design 45
4.4 Particle Test Results of Sorting Zone 48
4.5 Cell Viability Ratio Testing 50
Chapter 5. Conclusion 57

[1] http://www.cyto.purdue.edu
[2] Micromachined Transducers Sourcebook. New York: McGraw-Hill, 1998. G. T.A. Kovacs.
[3] S. Shoji and M. Esashi, “ Micro Flow Devices and Systems,” Journal of Micromechanics and Microengineering, 1994, Vol. 4, pp. 157-171.
[4] P. Gravesen, J. Branebjerg and O. S. Jensen, “ Microfluidics – A Review,” Journal of Micromechanics and Microengineering, 1993, Vol. 3, pp. 168-182.
[5] Gwo-Bin Lee, Chen-I Hung, Bin-Jo Ke, Guan-Ruey Huang, Bao-Herng Hwei, “Hydrodynamic Focusing for a Micromachined Flow Cytometer”, Journal of Fluids Engineering (Transactions of the ASME), 2001, Vol. 123, no. 3, pp. 672-679.
[6] Nam-Trung Nguyen, Xiaoyang Huang, ” Mixing in Microchannels Based on Hydrodynamic Focusing and Time-Interleaved Segmentation: Modelling and Experiment”, Proc. of SPIE, Vol. 6036, pp. 1-10.
[7] T. Vestad, D.W.M. Marr, J. Oakey, “Flow Control for Capillary-Pumped Microfluidic Systems”, Journal of Micromechanics and Microengineering, Vol. 14, 2004, pp. 1503-1506.
[8] Andreas Jahn, Wyatt N. Vreeland, Michael Gaitan, Laurie E. Locascio, “Controlled Vesicle Self-Assembly in Microfluidic Channels with Hydrodynamic Focusing”, J. Am. Chem. Soc., 2004, Vol. 126, no. 9, pp. 2674–2675.
[9] James B. Knight, Ashvin Vishwanath, James P. Brody, and Robert H. Austin, “Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds”, Phys. Rev. Lett., Vol. 80, pp. 3863–3866.
[10] Seiji Takayama, Hiroko Shimosato, Hiroshi Shiba, Miyuki Funato, Fang-Sik Che, Masao Watanabe, Megumi Iwano2 and Akira Isogai, ”Direct Ligand–Receptor Complex Interaction Controls Brassica Self-Incompatibility”, Nature, 2001, Vol. 413, pp. 534-538.
[11] Ruey-Jen Yang, Chih-Chang Chang, Sung-Bin Huang and Gwo-Bin Lee, “A New Focusing Model and Switching Approach for Electrokinetic Flow Inside Microchannels”, Journal of Micromechanics and Microengineering, 2005, Vol. 15, pp. 2141-2148.
[12] Gwo-Bin Lee, Che-Hsin Lin, Lung-Ming Fu, Rey-Jen Yang, Bao-Herng Hwei, “Fabrication and Application of Micro Flow Cytometers”, Journal of Instrument Today, 2003, Vol. 132, pp. 15-24,
[13] M. P. Hughes, “AC Electrokinetics: Applications for Nanotechnology,” Nanotechnology, 2000, Vol. 11, pp. 124–132.
[14] Lisen Wang, Lisa A. Flanagan, Noo Li Jeon, Edwin Monuki and Abraham P. Lee, ” Dielectrophoresis Switching with Vertical Sidewall Electrodes for Microfluidic Flow Cytometry”, Lab Chip, 2007, Vol. 7, pp. 1114–1120.
[15] Hsin-Yu Wu and Cheng-Hsien Liu, “An Electrokinetic Micromixer for Micro-Fluidic Bio-Chip Application”, Master Thesis, Department of PME, NTHU, 2003.
[16] T. M. Squires and S. R. Quake, “Microfluidics: Fluid physics at the nanoliter scale”, Rev. Mod. Phys., 2005, Vol. 77, pp. 977.
[17] A. Ramos, H. Morgan, N. G. Green, and A. Castellanos, “AC Electric-Field-Induced Fluid Flow in Microelectrodes,” Journal of Colloid Interface Sci., 1999, Vol. 217, pp. 420-422.
[18] A. Ajdari, ” Pumping Liquids Using Asymmetric Electrode Arrays”, Phys. Rev. E, 2000, Vol. 61, pp. 45-48.
[19] A. B. D. Brown, C. G. Smith, and A. R. Rennie, “Pumping of Water with AC Electric Fields Applied to Asymmetric Pairs of Microelectrodes,” Phys. Rev. E, 2001, Vol. 63, pp. 1-8.
[20] N. G. Green, A. Ramos, A. Gonzalez, H. Morgan, and A. Castellanos, Phys. Rev. E, 2002, Vol. 66.
[21] M. Mpholo, C.G. Smith, A.B.D. Brown, “Low Voltage Plug Flow Pumping Using Anisotropic Electrode Arrays” Sensors and Actuators B, 2003, Vol. 92, pp. 262–268.
[22] V. Studer, A. Pepin, Y. Chen, and A. Ajdari, “An Integrated AC Electrokinetic Pump in a Microfluidic Loop for Fast and Tunable Flow Control”, Analyst, 2004, Vol. 129, pp. 944-949.
[23] A. Ramos, A. Gonzalez, A. Castellanos, N. G. Green, and H. Morgan,Phys, “Pumping of Liquids with AC Voltages Applied to Asymmetric Pairs of Microelectrodes”. Rev. E, 2003, Vol. 67.
[24] L. H. Olesen, H. Bruus, and A. Ajdari, Phys. Rev. E, 2006, Vol. 73.
[25] John Paul Urbanski, Todd Thorsena, Jeremy A. Levitan and Martin Z. Bazant, “Fast AC Electro-Osmotic Micropumps with Nonplanar Electrodes”, Applied Physics Letters, 2006, Vol. 89.
[26] Dmitri Lastochkin, Ronghui Zhou, Ping Wang, Yuxing Ben, and Hsueh-Chia Chang, “Electrokinetic Micropump and Micromixer Design Based on AC Faradaic Polarization”, Journal of Applied Physics, 2004, Vol. 96.
[27] Sarah Stolberg, Kara E. McCloskey, “Can Shear Stress Direct Stem Cell Fate?”, Chemical Engineers Biotechnol, 2009, Vol. 25, pp. 10-19.
[28] M. Kathryn Thompson and John M. Thompson, “Multi-Physics Analysis of Microfluidic Devices with Hydrodynamic Focusing and Dielectrophoresis”, Proceedings of 2004 International ANSYS Users Conference & Exhibition, Pittsburgh, PA. 14 pages.
[29] Sigma; cat no. E 7637 http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/e7637pis.Par.0001.File.tmp/e7637pis.pdf
[30] H. Yang, J. Acker, A. Chen and L. McGann, “In situ assessment of cell viability”, Cell Transplantation, 1998, Vol. 7, pp. 443–451.
[31] George C. Engelmayr Jr, Virna L. Sales, John E. Mayer Jr., Michael S. Sacks, "Cyclic flexure and laminar flow synergistically accelerate mesenchymal stem cell-mediated engineered tissue formation: Implications for engineered heart valve tissues", Biomaterials, 2006, Vol. 27, pp. 6083-6095.
[32] Duane L. Garner, “Flow cytometric sexing of mammalian sperm”, Theriogenology, 2006, Vol. 65, pp. 943–957.
[33] T.K. Suh and J.L. Schenk, “Pressure during flow sorting of bull sperm affects post-thaw motility characteristics”, Theriogenology, 2003, Vol. 59, pp. 516.