本研究利用微機電系統製程技術，設計及製作一新式之整合型細胞晶片用以量測細胞性質。其目的在於藉由晶片培養細胞，再以微幫浦及微型過濾器(Micro-filter)將細胞分離集中，並於偵測區域內量測細胞性質及數目，將可大幅縮短細胞培養時間、亦只需利用極少量細胞，且避免不必要的人為疏失，進而達到快速檢測的效果，以改善傳統微生物檢測耗時及檢測限制的缺點。

　　本研究在整合型晶片製作方面，以玻璃與聚二甲基矽氧烷(Polydimethysiloxane, PDMS)作為基材，成功地藉由三道光罩將細胞培養、樣品傳輸之蠕動式微幫浦(Peristaltic micropump)、樣品分離之微型過濾器與偵測電極元件整合於單一晶片，進而發展出一套簡單快速、低成本並可大量製造的製程。以蠕動式微幫浦來推送微流體，接著利用等向性蝕刻底切特性所製作的過濾結構將細胞集中於偵測區進行量測。另一方面設計與製作量測電路，除了避免不必要的雜訊干擾，更將量測設備微型化，朝實驗室晶片邁進。最後，本研究以聚苯乙烯珠(Polystyrene beads)模擬細胞，針對各元件進行一系列的測試以評估其適用性。藉由此研究成果，相信對未來細胞性質量測之生物晶片研發上，將具有重大貢獻。

　　The present study reports a new microfluidic device fabricated by using MEMS (Micro-electro-mechanical-systems) technology for cell study. The device integrated with four modules, namely a cell culture module, a micro pump, a micro-filter, and an array of micro-electrodes to detect the characteristics and numbers of cells. After cell culturing, the cells are transported by the micro pump, collected by the micro-filter, and finally detected by the micro-electrodes incorporated with a detection circuit. Compared to traditional methods, the integrated microfluidic chip could reduce the time for cell culture and could be used for fast detection by using fewer amounts of samples and reagents.
　　The integrated cell chips are fabricated on soda-lime glass and PDMS (Polydimethysiloxane) substrates. We have successfully integrated these devices including a cell culture module, a peristaltic micro-pump, a micro-filter and micro-electrodes on one chip by a simple three-mask fabrication process. The experimental results demonstrate that polystyrene beads can be successfully concentrated and detected by using the proposed device. The development of the integrated cell chips could be promising for cell study.

[1]. 李國賓, “下一波之生物晶片-微流體生醫晶片之應用及研發,” 《科學發展》月刊, in press, 2004.
[2]. R. Feynman, “There's Plenty of Room at the Bottom,” Journal of Micro
Electro Mechanical Systems, vol. 1, pp. 60-66, 1992.
[3] R. Feynman, “Infinitesimal Machinery,” Journal of Micro Electro Mechanical Systems, vol. 2, pp.4-14, 1993.
[4] M. Madou, Fundamentals of Microfabrication, CRC Press, New York, 1997.
[5] G. H. W. Sander and A. Manz, “Chip-based Microsystem for Genomic
and Proteomic Analysis” Trends in Analytical Chemistry, Vol. 19, pp.364-378, 2000.
[6] J. D. Owens, “Formulation of culture media for conductimetric assays: theoretical consideration,” Journal of General Microbiology, Vol. 131, pp.3055-3076, 1985.
[7] W. J. Spencer, W. T. Corbett, L. R. Dominguez and B. D. Shafer, “An Electronically Controlled Piezoelectric Insulin Pump and Valves,” IEEE Transactions on Sonics and Ultrasonics, Vol. 25, pp. 153-167, 1978.
[8] F. C. M. van de Pol, H. T. G. van Lintel, M. Elwenspoek and J. H. J.
Fluitman, “Thermopneumatic Micropump Based on Micro-engineering
Techniques,” Sensors and Actuators A, Vol. 21, pp. 198-202, 1990.
[9] R. Zengerle, J. Ulrich, S. Kluge, M. Richter and A. Richter, “A
Bidirectional Silicon Micropump,” Sensors and Actuators A, Vol. 50, pp.
81-86, 1995.
[10] W. Zhang and C. H. Ahn, “A Bi-directional Magnetic Micropump on A
Silicon Wafer,” Solid-State Sensor and Actuators, pp. 94-97, 1996.
[11] E. Stemme and G. Stemme, “Valveless Diffuser/Nozzle Based Fluid Pump,” Sensors and Actuators A, Vol. 39, pp. 159-167, 1993.
[12] W. L. Benard, H. Kahn, A. H. Heuer and M. A. Huff, “A Titanium-Nickel
Shape Memory Alloy Actuated Micropump,” Solid-State Sensors and
Actuators, pp. 361-364, 1997.
[13] J. Evans, D. Liepmann and A. P. Pisano, “Planar Laminar Mixer,” IEEE
Micro Electro Mechanical Systems, pp. 96-101, 1997.
[14] A. Richter, A. Plettner, K. A. Hofmann and H. Sandmaier, “A
Micromachined Electrohydrodynamic Pump,” Sensors and Actuators A,
Vol. 29, pp. 159-168, 1991.
[15] C. Vieider, O. Ohman and Elderstig, “A Pneumatically Actuated Micro
Valve with a Silicone Rubber Membrane for Integration with
Fluid-Handling Systems,” Solid-State Sensors and Actuators, Vol. 2, pp.
284-286, 1995.
[16] H. Jerman, “Electrically-Activated ,Normally-Closed Diaphragm Valve,”
Solid-State Sensors and Actuators, pp. 1045-1048, 1991.
[17] M. A. Huff, J. Gilbert and M. A. Schmidt, “Flow Characteristics of a Pressure-Balanced Microvalve,” Solid-State Sensors and Actuators, pp.
98-101, 1993.
[18] K. Yanagisawa, H. Kuwano and A. Tago, “An Electromagnetically Driven Microvalve,” Solid-State Sensors and Actuators, pp. 102-105, 1993.
[19] S. Quake and A. Scherer, “From micro- to nanofabrication with soft materials,” Science ,Vol. 290, pp.1536-1540, 2000.
[20] J. G. Spencer, “Piezoelectric Micropump with Three Valves Working Peristaltically,” Sensor and Actuators A, Vol. 21-23, pp. 203-206, 1990.
[21] M. A. Unger, H. P. Chou and T. Thorsen, A. Scherer and S. R. Quake,“Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography,” Science, Vol. 288, pp. 113-116, 2000.
[22] J. J. Hawkes, M. S. Limaye and W. T. Coakley, “An Ultrasonic Method
for Filtering and Separating Cell Suspensions” IEEE Ultrasonic Symposium, pp.1069-1072 , 1995.
[23] K. V. I. S. Kaler, A. Docoslis, N. Kalogerakis and L. A. Behie, “A
Micromachined DEP Cell Filtration Device” IEEE IAS Ann.Meeting,
vol. 4, pp. 1932-1939, 1996.
[24] T. M. Liakopoulos, J. W. Choi and C. H. Ahn, “A bio-magnetic bead separator on glass chips using semi-encapsulated spiral electromagnets,” Solid State Sensors and Actuators, Vol. 1, pp. 485-488, 1997.
[25] C. J. M. van Rijn and M. C. Elwenspoek, “Micro Filtration Membrane
Sieve with Silicon Micro Machining for Industrial and Biomedical
Applications” IEEE Micro Electro Mechanical Systems, pp.485-488, 1995.
[26] X. Yang , J. M. Yang , X. Q. Wang , E. Meng , Y. C. Tai and C. M. Ho,
“Micromachined membrane particle filters,” IEEE Micro Electro Mechanical Systems, pp137-142, 1998.
[27] Y. K. Yoon, J. H. Park, F. Cros, and M. G. Allen, “Integrated vertical screen microfilter system using inclined SU-8 structures, ” IEEE Micro
Electro Mechanical Systems, pp227-230, 2003.
[28] R. D. Oleschuk, L. L. Shultz-Lockyear, Y. Ning, and D. J. Harrison,
“Trapping of Bead-Based Reagents within Microfluidic Systems: On-Chip Solid-Phase Extration and Electrochromatography, ” Analytical Chemistry, Vol. 72, pp. 585-590, 2000.
[29] 劉恆惠,“微型生化反應槽之設計製作及測試,”國立成功大學工程科學研究所碩士論文(2003).
[30] N. S. Hobson and P. F. Turner, “Review article: Microbial detection,” Biosensors & Bioelectronic, Vol. 11, pp.455-477, 1996.
[31] D. Ivnitski, I. Abdel-Hamid, P. Atanasov and E. Wilkins, “Biosensors for detection of pathogenic bacteria,” Biosensors & Bioelectronics, Vol.14, pp. 599-624, 1999.
[32] C. M. Haris, R. W. Todd, S. J. Bungard, R. W. Lovitt, J. Gareth and D. B.
Kell, “Dielectric permittivity of microbial suspensions at ratio frequencies : a novel method for the real-time estimation of microbial biomass,” Enzyme and Microbial Technology, Vol. 9, pp. 235-243, 1899.
[33] M. Ciureanu, W. Levadoux and S. Goldstein, “Electrical impedance studies on a culture of a newly discovered strain of Streptomyces,” Enzyme and Microbial Technology, Vol. 21, pp. 41-449, 1997.
[34] P. A. Nobel, M. Dziuba, D. J. Harrison and W. L. Albritton, “Factors influencing capacitance-based monitoring of microbial growth,” Journal of Microbiological Methods, Vol. 37, pp. 51-64 , 1999.
[35] J. J. Wu, A. H. Huang, J. H. Dai, T. C. Chang, “Rapid detection of oxacillin-resistant Staphylococcus aureus in blood culture by an impedance method,” Journal of Clinical Microbiology, Vol. 35, pp. 1460-1464, 1997.
[36] A. H. Huang, J. J. Wu, Y. M. Weng, H. C. Ding and T. C. Chang, “Direct antimicrobial susceptibility testing of gram-negative bacilli in blood cultures by an electrochemical method,” Journal of Clinical Microbiology, Vol. 36, pp. 2882-2886, 1998.
[37] H. C. Chang and A. H. Huang, “Rapid differentiation of fermentative from nonfermentative gram-negative bacilli in positive blood cultures by an impedance method,” Journal of Clinical Microbiology, pp. 3589-3594, 2000.
[38] H. C. Chang, J. J. Chang, A. H. Huang and T. C. Chang, “Evaluation of a capacitance method for direct antifungal susceptibility testing of yeasts in positive blood cultures,” Journal of Clinical Microbiology, pp. 971-976, 2000.
[39] J. J. Wu, Y. C. Lee, S. N. Leaw, M. C. Lin and T. C. Chang, “Evaluation of an impedance method for subtyping of Pseudomonas aeruginosa,” Diagnostic Microbiology & Infectious Disease, Vol. 48, pp. 181-189, 2004.
[40] 郭盈成,“新型微幫浦與微閥門之設計與製作,”國立成功大學工程科學研究所碩士論文(2003).
[41] S. W. Park, K.S. Kim, and J. B. Lee, “Polydimethylsiloxane(PDMS) Elastomer for Polymer and Metallic High Aspect Ratio Microstructure
, ” University of Taxas, 2001.
[42] B. E. Slentz, N. A. Penner and F. E. Regnier, “Capillary electrochromatography of peptides on microfabricated poly (dimethylsiloxane) chips modified by cerium(IV)-catalyzed polymerization ,” Journal of Chromatography A, vol. 948, pp. 225–233, 2002.
[43] Y. Xia and G. M. Whitesides, “Soft Lithography,” Angewandte Chemie, pp. 550-575, 1998.
[44] 李正中, 薄膜光學與鍍膜技術, 藝軒圖書出版社, 2001.
[45] E. W. Becker, W. Ehreld, P. Hagmann, A. Maner, D. Munchmeyer, “Fabrication of microstructures with high aspect ratios and greatstructural heights by synchrotron radiation lithography, galvanoforming and plastic molding(LIGA process),” Microelectronic Engineering 4, pp. 35-36, 1986.
[46] C. Birnai, J. Mphr, P. Bley, “Fabrication of capactive acceleration sensors
by the LIGA technique,” Sensors and Actuators A , pp. 44-48,1991.
[47] B. Choi, E.G. Lovell, H. Guckel, T. R. Christenson, K.J. Skrobis, J. W. Kang, “Development of pressure transducers utilizing deep X-ray lithography,” International Conference on Solid-State Sensors and Actuators, 1991 , pp. 393-396.
[48] C. H. Chiou, Z. F. Tseng , G. B. Lee, “A Novel Magnetic Tweezers for Manipulation of a Single DNA Molecule,” IEEE 17th International Micro Electro Mechanical Systems Conference, 2004.
[49] 張瑞斌,“微電鍍技術及其在生物晶片之應用,” 國立成功大學工程科學研究所碩士論文(2002).
[50] A. T. Woolley and R. A. Mathies, “Ultra-High-Speed DNA fragment separations using microfabrication capillary array electrophoresis chips,” Proceedings of the National Academy of Sciences of the United States of America , pp.11348-52,
[51] M. Stjernström and J. Roeraade, “Method for fabrication of microfluidic
system in glass,” J. Micromech. Microeng., Vol. 8, pp. 33-38.
[52] F. Delahaye, L. Montagne, G. Palavit, J. C. Touray and P. Baillif, “Acid dissolution of sodium-calcium metaphosphate glasses,” Journal of Non-Crystalline Solids, Vol. 242, pp. 25-32.
[53] 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.
[54] 林哲信, 李國賓,“ Polymer-MEMS及其微流體生醫晶片之應用,”電子月刊, Vol. 90, pp. 144-156, 2003.
[55] D. Armani, C. Liu and N. Aluru, “Re-Configurable Fluid Circuits by PDMS Elastomer Micromachining,” IEEE Micro Electro Mechanical Systems, pp. 222-227, 1999.
[56] C. H. Wang, G. B. Lee, M. C. Li and Y. C. Wang,“Pneumatic micro-valves and micro-pumps for applications on multiple bio-sampling chips,” the 27th conference on theoretical and applied mechanics, pp. 760-768, 2003.
[57] 微機電系統技術與應用, 行政院國家科學委員會精密儀器發展中心, 台灣, pp.703-718, 2003.
[58] 江昭皚, 感測電路, 高立出版, 1997.
[59] 劉國雄, 工程材料科學, 全華出版, 1991.
[60] S. Timosheko, Theory of Plate and Shells, pp. 4-27, McGraw-Hill, New
York, 1970.
[61] 蓋永鋒,“微型壓阻式壓力感測器製作之研究,”國立成功大學工程科學研究所碩士論文(2000).