本研究提出一種新的連續粒子分類方法，藉由粒子在邊鞘流中流
動，其將可產生擠壓躍遷現象(Squeeze-Jumping Effect)，即在微流體聚
焦作動下，粒子無法穩定流動在寬度小於該粒子直徑之流層當中，粒
子將被擠壓，而從寬度較小的流層躍遷至寬度較大的流層中。藉由此
一粒子擠壓躍遷現象，其可應用於不同大小粒子之分類。而藉著微機
電系統(Micro-electro-mechanical Systems, MEMS)製程技術的發展，研
究亦成功的設計並製作兩種粒子/細胞分類器，一為多階邊鞘流連續粒
子/細胞分類器，另一改良後的串接式連續粒子/細胞分類器。研究中除
了理論數學推導及數值模擬之分析外，並利用實驗予以佐證。藉由實
驗與模擬的結果，本研究所發展的微型粒子分類器，可得到獲得率(%
Recovery)與收集率(% Yield)分別為87.7%與94.1%之高分類效能，其可
應用於微全分析系統之領域。
此外，有感於在微全分析系統之發展下，流體試劑均需精密地配
製，並作微量的傳輸，因此本研究亦以簡單的邊鞘流理論為基礎，發
展一晶片式比例調劑裝置，可以用來作試劑的合成、樣本的稀釋、藥
物的調配等生化反應之前置處理步驟，其簡單之架構將易於整合在各
類功能性晶片中。而為驗證上述比例調劑裝置之可行性，研究也提出
一比例調劑混合反應晶片，其中包含：比例調劑裝置、T 型微混合器、蛇型反應區域。實驗亦以細胞裂解反應之應用作為說明，利用比例調
劑裝置配製細胞裂解液，並與細胞於微混合器中進行混合，最後在蛇
型反應區域使之有足夠時間進行裂解反應。由實驗結果之觀察，此一
比例調劑反應晶片，確實可用以進行細胞裂解反應。
本研究藉由微機電製程成功的製作出：微型細胞分類器、比例調
劑裝置、比例調劑混合反應晶片。且利用簡單的流體力學原理，推導
微管道邊鞘流行為之數學模式，並以電腦數值模擬方法，分析所設計
微流體晶片之流場、電場、壓力場、速度場之分佈，不僅如此，也由
實驗之結果作為對照與驗證。盼望藉由研究所得之成果，能對發展整
合型之微小生化分析系統有所貢獻，以實現微全分析系統之構想。

This study proposes a navel method for continuously particle sorting utilizing cascade squeeze jumping effect under microfluidic configuration. Microparticles with different sizes can be successfully separated at different stages of squeezing sheath flow. The method is based on that particles can not flow stably within a flow stream with the smaller stream width than their sizes. Big particles will jump from their original flow stream into the wider neighboring sheath flow. In this study, we have successfully designed and fabricated two kinds of particles/cells sorters using MEMS (Micro-electro-mechanical Systems) technology. The proposed microchip device includes a multi-stage sheath flow particles/cells sorter and an improved design of a cascade squeezed flow scheme. In the study, theoretical formulations, computer simulations and experimental operations are used to analyze the flow field in the microchip and evaluate the sorting performance of the devices. Results show the good sorting performance with cell recovery rate of 87.7% and yield rate of 94.1% can be obtained using the proposed micro particles/cells sorter.
Furthermore, it is also important to continiously prepare reagents for in-column bio-chemical reactions. Therefore, this study presents a sheath-flow based microfluidic device for concentration fraction delivery of liquid samples. The simple and novel structure proposed in this study is able to prepare reagent with different concentration and is also easy to be integrated with other multifunctional microfluidic device. In order to demonstrate the feasibility and performance of the proposed concentration fraction delivery device, this study designs an integrated microchip device for in-line preparation of lysin reagent for cell lysis and an integrated T-form microfluidic mixer for demonstration of RBC lysis in the same microchip. Reagents for cell lysis are firstly prepared by the concentration faction delivery part of the chip. The prepared reagent is mixed with RBC sample downstream in the reaction channel using the T-form mixer. Results show a high RBC lysing rate of upto 100% in 10 mm downstream the T-junction can be achieved utilizing the proposed chip.
In this study, we have successfully demonstrated three kinds of microfluidic device including a micro particles/cells sorter, a concentration fraction delivery device and a cell lysis reactor. Numerical analysis and experimental investigation confirm the proposed concepts and performance of the microfluidic devices. The contributions of the study are highly potential for developing a low-cost bioreactor system in the

目 錄 ……………………………………………………………i
圖 目 錄 ……………………………………………………………v
表 目 錄 …………………………………………………………ix
摘 要 …………………………………………………x
Abstract …………………………………………………………xii
致 謝 …………………………………………………………xiv
第一章 緒論 …………………………………………………………1
1-1 前言……………………………………………………………1
1-2 微流體系統 …………………………………………………2
1-3 文獻回顧 ……………………………………………………5
1-3.1 微型細胞分類器 …………………………………5
1-3.2 微型調劑傳遞裝置 …………………………………11
1-3.3 整合型晶片實驗室 …………………………………16
1-4 研究動機與目的 ……………………………………………22
1-4.1 電驅動式邊鞘流粒子/細胞分類器…………………22
1-4.2 樣本比例調劑並傳輸裝置……………………………24
1-4.3 細胞裂解反應處理系統………………………………26
1-5 論文架構 ……………………………………………………27

第二章 理論分析與數學推導………………………………30
2-1 電雙層形成機制……………………………………………30
2-2 電滲透流理論 ………………………………………………31
2-3 流場分析數值模擬 …………………………………………33
2-4 微流體聚焦壓縮現象 ………………………………………34
2-5 粒子/細胞於壓縮流體之擠壓彈跳效應……………………38

第三章 晶片設計與實驗方法………………………………………40
3-1 晶片設計 ……………………………………………………40
3-1.1 多階邊鞘流薄層微型粒子/細胞分類器 ……………40
3-1.2 串接式連續粒子/細胞分類器 ………………………41
3-1.2 比例調劑混合反應系統……………………………42
3-2 晶片製作 ……………………………………………………43
3-2.1光罩製作 ……………………………………………44
3-2.2 晶片清洗.………..…………………………………...…45
3-2.3 微影…………………………………………………45
3-2.4 蝕刻……………………………………………………46
3-2.5 鑽孔……………………………………………………47
3-2.6 晶片接合………………………………………………47
3-3 材料與生物樣本之準備………………………………………48
3-4 實驗架設………………………………………………………50
3-4.1連續粒子/細胞分類裝置……………………50
3-4.2 整合比例調劑混合裝置之生化反應系統……………51

第四章 結果與討論…………………………………………………54
4-1 多階邊鞘流薄層連續粒子/細胞分類裝置.…………………54
4-1.1 多階邊鞘流聚焦壓縮情形……………………………54
4-1.2 粒子分離結果…………………………………………57
4-2 新型串接式連續粒子/細胞分類裝置………………………59
4-2.1 樣本流聚焦壓縮情形…………………………………60
4-2.2 粒子分類結果…………………………………………65
4-2.3 效率評估………………………………………………68
4-3 整合比例調劑混合裝置於細胞裂解反應之應用……………73
4-3.1 試劑濃度比例配製……………………………………73
4-3.2 細胞裂解反應之結果…………………………………75

第五章 結論與未來展望.……………………………………………76
5-1 結論……………………………………………………………76
5-2 未來展望………………………………………………………78
參考文獻…………………………………………………………………80
自 述…………………………………………………………………85



































圖目錄
圖 1.1利用多孔隙微結構分類粒子之微流體晶片……………………6
圖 1.2利用重力結合聚焦薄層分流之連續粒子分類晶片……………7
圖 1.3整合界電泳力之連續細胞分類器……………………………8
圖 1.4整合磁性微奈米尖端之磁珠/細胞分類器……………………9
圖 1.5整合微加工聲波換能器於微管道之流體切換開關…………10
圖 1.6利用光鉗操縱生物細胞之微流體細胞分類裝置……………10
圖 1.7利用擠壓流體結合末端多非對稱分支管道之粒子分類器…11
圖 1.8利用液壓控制樣本之M x N型微流體晶片…………………13
圖 1.9利用流體體積法控制樣本切換之微流體裝置…...……………14
圖 1.10雙十字形聚焦微量進樣晶片…………………15
圖 1.11利用管道長度控制混合濃度之微流體裝置……………15
圖 1.12可產生濃度梯度混合溶液之樹狀分之微流體裝置………16
圖 1.13整合梯度液相層析法所需流體元件之微晶片裝置…………18
圖 1.14整合電滲幫浦、微混合器、溫度控制系統之微流體裝置……19
圖 1.15一種可微量平行處理多種樣本之核酸純化處理晶片……20
圖 1.16整合PCR、電動注射分離、光纖偵測等裝置之微流體晶片…21
圖 1.17安捷倫公司所推出之(a)高效能液相層析晶片及(b)微流體晶片分析平台……………………………………………………………22
圖 1.18微流體粒子/細胞分類器之工作原理示意圖………………24
圖 1.19比例調劑裝置示意圖………………………………………26
圖 1.20整合比例調劑裝置及微混合器之細胞裂解反應處理系統…27
圖 1.21整合型生化分析系統架構圖..……………………………28
圖 2.1電荷分佈與電雙層形成示意圖………………………………31
圖 2.2電滲透流原理示意圖…………………………………………32
圖 2.3微流體聚焦壓縮現象示意圖…………………………………36
圖 2.4(a)樣本流經邊鞘流擠壓，將壓縮後之樣本流流體層視為可滑動之庫頁流(Coutte Flow)，(b) 粒子受力示意圖，粒子於管道中流動受流體推力及黏滯力所影響，使之無法穩定進入寬度較小之流層中，將會被擠壓而跳出並流到另一側寬度較大之流體層……………………39
圖 3.1多階邊鞘流薄層微型粒子/細胞分類器之尺寸大小示意圖…41
圖 3.2串接式連續粒子/細胞分類器之尺寸大小示意圖……………42
圖 3.3比例調劑混合反應裝置之尺寸大小示意圖…………………43
圖 3.4晶片製程示意圖………………………………………………44
圖 3.5微流體細胞分類晶片實體圖………………………………48
圖 3.6比例調劑混合反應裝置之晶片實體圖……………………48
圖 3.7粒子/細胞分類器系統架設圖………………………………51
圖 3.8比例調劑混合裝置之系統架設圖…………………………53
圖 4.1流道電滲透流聚焦實驗與模擬比較(a)多流道聚焦，拍攝於主管與三側管交界處，聚焦寬度分別約為7、13、25 μm，(b)實驗局部放大圖，(c)、(d)為利用CFDRC®軟體進行多層流體聚焦的模擬圖，該計算之流層壓縮寬度與實驗結果吻合……………………………………56
圖 4.2聚焦寬度與主/側管施加電壓關係圖………………………57
圖 4.3直徑為20 μm的粒子被壓縮之流體擠出之連續影像………58
圖 4.4粒子分離圖。圖中綠色箭頭所指的為直徑5 μm的綠色螢光粒子，白色箭頭所指的為直徑20 μm的無螢光粒子…………………59
圖 4.5流道電滲透流聚焦實驗與模擬比較(a) 流體聚焦影像，(b) 實驗局部放大圖，(c)、(d)、(e)為利用商用軟體CFDRC®進行多層流體聚焦的模擬圖……………………………………………………………61
圖 4.6利用模擬與數學推導之分析結果，(a)流場分佈，(b)速度場分佈，(c)加速度場分佈，(d)受力分佈……………………………………62
圖 4.7在主管與側管間有無輔助聚焦微結構之模擬比較圖………63
圖 4.8粒子第一次分離之連續實驗影像……………………………64
圖 4.9粒子第一次收集之連續實驗影像……………………………65
圖 4.10粒子第二次分離之連續實驗影像…………………………67
圖 4.11粒子第二次收集之連續實驗影像…………………………68
圖 4.12酵母菌於顯微鏡底下之影像圖形…………………………69
圖 4.13酵母菌與微粒子之連續實驗分類影響……………………70
圖 4.14酵母菌與塑膠小球之效率評估圖……………………………72
圖 4.15試劑調劑比例與主管管道長度關係圖。結果顯示，在不同管道長度下可以快速調配不同濃度之比例調劑…………………………74
圖 4.16紅血球裂解影像……………………………………………75



























表目錄
表 3.1紅血球之裂解液成分參數…………………………………50
表 4.1粒子實驗收集記錄表………………………………………72

[1] J. B. Lee, J. English, C. H. Ahn, and M. G. Allen, "Planarization techniques for vertically integrated metallic MEMS on silicon foundry circuits," Journal of Micromechanics and Microengineering, vol. 7, pp. 44-54, 1997.
[2] B. Ziaie, A. Baldi, M. Lei, Y. D. Gu, and R. A. Siegel, "Hard and soft micromachining for BioMEMS: review of techniques and examples of applications in microfluidics and drug delivery," Advanced Drug Delivery Reviews, vol. 56, pp. 145-172, 2004.
[3] M. Yano, F. Yamagishi, and T. Tsuda, "Optical MEMS for photonic switching-compact and stable optical crossconnect switches for simple, fast, and flexible wavelength applications in recent photonic networks," IEEE Journal of Selected Topics in Quantum Electronics, vol. 11, pp. 383-394, 2005.
[4] C. L. Goldsmith, Z. M. Yao, S. Eshelman, and D. Denniston, "Performance of low-loss RF MEMS capacitive switches," IEEE Microwave and Guided Wave Letters, vol. 8, pp. 269-271, 1998.
[5] A. C. R. Grayson, R. S. Shawgo, A. M. Johnson, N. T. Flynn, Y. W. Li, M. J. Cima, and R. Langer, "A BioMEMS review: MEMS technology for physiologically integrated devices," Proceedings of the IEEE, vol. 92, pp. 6-21, 2004.
[6] A. Manz, N. Graber, and H. M. Widmer, "Miniaturized total chemical analysis systems: A novel concept for chemical sensing," Sensors and Actuators B, vol. 1, pp. 244-248, 1990.
[7] E. Verpoorte and N. F. De Rooij, "Microfluidics meets MEMS," Proceedings of the IEEE vol. 91, pp. 930-953, 2003.
[8] J. Khandurina and A. Guttman, "Bioanalysis in microfluidic devices," Journal of Chromatography A, vol. 943, pp. 159-183, 2002.
[9] D. Erickson and D. Q. Li, "Integrated microfluidic devices," Analytica Chimica Acta, vol. 507, pp. 11-26, 2004.
[10] M. N. Sabry, "Scale effects on fluid flow and heat transfer in microchannels," IEEE Transactions on Components and Packaging Technologies, vol. 23, pp. 562-567, 2000.
[11] A. Nag, B. R. Panda, and A. Chattopadhyay, "Performing chemical reactions in virtual capillary of surface tension-confined microfluidic devices," Pramana-Journal of Physics, vol. 65, pp. 621-630, 2005.
[12] D. M. Hobbs and F. J. Muzzio, "Reynolds number effects on laminar mixing in the Kenics static mixer," Chemical Engineering Journal, vol. 70, pp. 93-104, 1998.
[13] S. V. Ermakov, S. C. Jacobson, and J. M. Ramsey, "Computer simulations of electrokinetic injection techniques in microfluidic devices," Analytical Chemistry, vol. 72, pp. 3512-3517, 2000.
[14] T. Stiles, R. Fallon, T. Vestad, J. Oakey, D. W. M. Marr, J. Squier, and R. Jimenez, "Hydrodynamic focusing for vacuum-pumped microfluidics," Microfluidics and Nanofluidics, vol. 1, pp. 280-283, 2005.
[15] H. Andersson and A. van den Berg, "Microfluidic devices for cellomics: a review," Sensors and Actuators B, vol. 92, pp. 315-325, 2003.
[16] S. Metz, C. Trautmann, A. Bertsch, and P. Renaud, "Flexible microchannels with integrated nanoporous membrances for filtration and separation of moleculles and particles," Proc. IEEE 17th International MEMS Conference (IEEE MEMS 2004), pp. 81-84, 2002.
[17] J. Moorthy and D. J. Beebe, "In situ fabricated porous filter-characterization and biologicalapplications," Proc. IEEE 15th International MEMS Conference (IEEE MEMS 2002), pp. 514-517, 2002.
[18] J. Moorthy and D. J. Beebe, "In situ fabricated porous filters for microsystems," Lab on a Chip, vol. 3, pp. 62-66, 2003.
[19] M. H. Moon, S. G. Yang, J. Y. Lee, and S. Lee, "Combination of gravitational SPLITT fractionation and field-flow fractionation for size-sorting and characterization of sea sediment," Analytical and Bioanalytical Chemistry, vol. 381, pp. 1299-1304, 2005.
[20] I. Doh and Y. H. Cho, "A continuous cell separation chip using hydrodynamic dielectrophoresis (DEP) process," Sensors and Actuators A, vol. 121, pp. 59-65, 2005.
[21] F. Arai, A. Ichikawa, M. Ogawa, T. Fukuda, K. Horio, and K. Itoigawa, "High-speed separation system of randomly suspended single living cells by laser trap and dielectrophoresis," Electrophoresis, vol. 22, pp. 283-288, 2001.
[22] X. B. Wang, J. Vykoukal, F. F. Becker, and P. R. C. Gascoyne, "Separation of polystyrene microbeads using dielectrophoretic/gravitational field-flow-fractionation," Biophysical Journal, vol. 74, pp. 2689-2701, 1998.
[23] R. Rong, J. W. Choi, and C. H. Ahn, "A functional magnetic bead biocell sorter using fully integrated magnetic micro nano tips.," Proc. IEEE 16th International MEMS Conference (IEEE MEMS 2003), pp. 530-533, 2003.
[24] C. B. Fuh, H. Y. Tsai, and J. Z. Lai, "Development of magnetic split-flow thin fractionation for continuous particle separation," Analytica Chimica Acta, vol. 497, pp. 115-122, 2003.
[25] H. Jagannathan, G. G. Yaralioglu, A. S. Ergun, and B. T. Khuri-Yakub, "An implementation of a microfluidic mixer and switch using micromachined acoustic transducers.," Proc. IEEE 16th International MEMS Conference (IEEE MEMS 2003), pp. 104-107, 2003.
[26] M. Ozkan, M. Wang, C. Ozkan, R. Flynn, A. Birkbeck, and S. Esener, "Optical manipulation of objects and biological cells in microfluidic devices," Biomedical Microdevices, vol. 5, pp. 61-67, 2003.
[27] M. Yamada, M. Nakashima, and M. Seki, "Pinched flow fractionation: Continuous size separation of particles utilizing a laminar flow profile in a pinched microchannel," Analytical Chemistry, vol. 76, pp. 5465-5471, 2004.
[28] J. Takagi, M. Yamada, M. Yasuda, and M. Seki, "Continuous particle separation in a microchannel having asymmetrically arranged multiple branches," Lab on a Chip, vol. 5, pp. 778-784, 2005.
[29] G. B. Lee, B. H. Hwei, and G. R. Huang, "Micromachined pre-focused M x N flow switches for continuous multi-sample injection," Journal of Micromechanics and Microengineering, vol. 11, pp. 654-661, 2001.
[30] R. Y. Chein and S. H. Tsai, "Microfluidic flow switching design using volume of fluid model," Biomedical Microdevices, vol. 6, pp. 81-90, 2004.
[31] L. M. Fu, R. J. Yang, and G. B. Lee, "Electrokinetic focusing injection methods on microfluidic devices," Analytical Chemistry, vol. 75, pp. 1905-1910, 2003.
[32] M. Yamada, T. Hirano, M. Yasuda, and M. Seki, "A microfluidic flow distributor generating stepwise concentrations for high-throughput biochemical processing," Lab on a Chip, vol. 6, pp. 179-184, 2006.
[33] N. L. Jeon, S. K. W. Dertinger, D. T. Chiu, I. S. Choi, A. D. Stroock, and G. M. Whitesides, "Generation of solution and surface gradients using microfluidic systems," Langmuir, vol. 16, pp. 8311-8316, 2000.
[34] J. Xie, Y. N. Miao, J. Shih, Y. C. Tai, and T. D. Lee, "Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures," Analytical Chemistry, vol. 77, pp. 6947-6953, 2005.
[35] C. Y. Lee, G. B. Lee, J. L. Lin, F. C. Huang, and C. S. Liao, "Integrated microfluidic systems for cell lysis, mixing/pumping and DNA amplification," Journal of Micromechanics and Microengineering, vol. 15, pp. 1215-1223, 2005.
[36] J. W. Hong, V. Studer, G. Hang, W. F. Anderson, and S. R. Quake, "A nanoliter-scale nucleic acid processor with parallel architecture," Nature Biotechnology, vol. 22, pp. 435-439, 2004.
[37] G. B. Lee, C. H. Lin, F. C. Huang, C. S. Liao, C. Y. Lee, and S. H. Chen, "Microfluidic chips for DNA amplification, electrophoresis separation and on-line optical detection.," Proc. IEEE 16th International MEMS Conference (IEEE MEMS 2003), pp. 423-426, 2003.
[38] "http://www.agilent.com."
[39] S. C. Jacobson and J. M. Ramsey, "Electrokinetic focusing in microfabricated channel structures," Analytical Chemistry, vol. 69, pp. 3212-3217, 1997.
[40] J. R. Marchesi, T. Sato, A. J. Weightman, T. A. Martin, J. C. Fry, S. J. Hiom, and W. G. Wade, "Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA," Applied and Environmental Microbiology, vol. 64, pp. 795-799, 1998.
[41] W. Janusz, I. Kobal, A. Sworska, and J. Szczypa, "Investigation of the electrical double layer in a metal oxide/monovalent electrolyte solution system," Journal of Colloid and Interface Science, vol. 187, pp. 381-387, 1997.
[42] R. J. Yang, L. M. Fu, and G. B. Lee, "Variable-volume-injection methods using electrokinetic focusing on microfluidic chips," Journal of Separation Science, vol. 25, pp. 996-1010, 2002.
[43] C. H. Lin, L. M. Fu, and Y. S. Chien, "Microfluidic T-form mixer utilizing switching electroosmotic flow," Analytical Chemistry, vol. 76, pp. 5265-5272, 2004.
[44] C. H. Lin, G. B. Lee, Y. H. Lin, and G. L. Chang, "A fast prototyping process for fabrication of microfluidic systems on soda-lime glass," Journal of Micromechanics and Microengineering, vol. 11, pp. 726-732, 2001.
[45] B. C. Ferrari, G. Oregaard, and S. J. Sorensen, "Recovery of GFP-labeled bacteria for culturing and molecular analysis after cell sorting using a benchtop flow cytometer," Microbial Ecology, vol. 48, pp. 239-245, 2004.
[46] "EPICS® ALTRA™ Flow Cytometer," http://www.beckman.com.