現今臨床醫學中的應用上，即時檢測扮演極重要的角色。以一般疾病檢測來說，細胞培養是非常需要的，然而早期疾病顯現的細胞數量非常少，因此需要大量培養細胞以達到偵測的數量。常用來偵測細胞的方法，有酶聯免疫斑點法(Enzyme- linked Immunospot Assay, ELISPOT)，和流式細胞儀（Flow Cytometry，）。ELISPOT雖可得到較好的偵測靈敏度，卻需要數天的反應時間；而FCM雖偵測快速，但靈敏度大約僅有0.01%，且價錢昂貴。因此本研究將以高靈敏度的檢測概念結合於生醫晶片中，進行癌細胞的偵測。生醫晶片透過大量製造的方式，不僅可以減少成本，晶片一次性的拋棄使用，也可避免生物感染及藥品交叉汙染。本實驗，將以免疫磁珠與螢光結合於微流道系統，利用磁珠分離特定細胞，再利用螢光訊號偵測癌細胞。流道層包含兩個反應區，其中第一混合反應區，是以圓形結構作為設計，配合平行錯位的微管道引入，以及蠕動幫浦驅動達到藥品均勻混和的效果；而第二反應區為偵測區，使用外接488 nm波長雷射激發偵測區樣品，並利用光纖接收樣品的螢光訊號。在本實驗中，癌細胞將與螢光藥物先行反應，再與免疫磁珠進行反應，並於第二反應區底部，外加磁鐵進行吸引，最後進行螢光訊號量測。本實驗結果僅需約5秒的偵測時間即可達到即時檢測目的，並達到0.001%的靈敏度；此外，本實驗系統可精準的傳輸於不同的反應區域，效率可達95%以上；最後，相較於傳統臨床流式細胞儀的檢測極限0.01%，本系統不但可以達成，甚至可達0.001%的靈敏度。

It is an important issue to have real-time and highly sensitive detection of the disease. For a tumor in the early stage, the concentration of disseminated cells circulatinge in the blood is extremely low, making the detection of low concentration cancer cells difficult. Conventional methods for detecting small amount of cells include culture-based techniques and enzyme-linked immunospots (ELISPOT). However, it is labor-intensive and time-consuming to increas the number of cells by culture or ELISPOT to a number that suffices for measurement. Another approach, flow cytometry (FCM) has been established for detecting low concentration of cancer cells. However, the detection sensitivity of FCM is approximately 0.01%.
In this thesis, combining the magnetic bead and fluorescence in the microfluid system was applied to obtain rapid and sensitive detection of cancer cells. Microfluidic system has a number of advantages, including rapid detection of small amounts of samples, low-cost, and disposable.
The detection of cancer cells is made by measuring the fluorescence which is conjugated with the first marker of the specific cells in the micro-reaction chamber. Additionally, the magnetic bead is adopted to conjugated with the second marker of the specific cells. The magnet is applied in the second reaction zone of the microfluid system to isolate the specific cells. The results show the detection sensitivity of the cancer cells is around 0.001% within 5 seconds period of the exposure time. And the percentage of biological cells can be transported into another reaction zone of microfluid system for above 95%. As a result, the immune magnetic bead-based microfluidic system successfully integrates rapid and high sensitivity on cancer cell detection .

[1] R. Feynman, "There is plenty of room at the bottom " Microelectromechanical Systems, vol. 1, pp. 60-66, 1992.
[2] R. Feynman, "Infinitesimal machinery," Microelectromechanical Systems, vol. 2, pp. 4-14, 1993.
[3] N. Maluf, An introduction to microelectromechanical systems engineering. Boston: Artech House, 2000.
[4] K. Seiler, D. J. Harrison, and A. Manz, "Planar chips technology for miniaturization and integration of separation techniques into monitoring systems," Chromatography, vol. 593, pp. 253-258, 1992.
[5] Y. Hathout, "Approaches to the study of the cell secretome," Expert Rev Proteomics, vol. 4, pp. 239-48, 2007.
[6] J. M. Jacobs, J. N. Adkins, W. J. Qian, T. Liu, Y. Shen, D. G. Camp, and R. D. Smith, "Utilizing human blood plasma for proteomic biomarker discovery," Proteome research, vol. 4, pp. 1073-1085, 2005.
[7] J. E. Aledort, A. Ronald, S. M. L. Blancq, R. Ridzon, A. Landay, M. E. Rafael, M. V. Shea, J. Safrit, R. W. Peeling, N. Hellmann, P. Mwaba, K. Holmes, and C. Wilfert, "Reducing the burden of HIV/AIDS in infants: the contribution of improved diagnostics," Nature, vol. 444, pp. 19–28, 2006.
[8] D. Mabey, R. Peeling, A. Ustianowski, and M. Perkins, "Diagnostics for the developing world.," Nature Reviews Microbiology, vol. 3, 2004.
[9] P. Yager, T. Edwards, E. Fu, K. Helton, K. Nelson, M. Tam, and B. Weigl, "Microfluidic diagnostic technologies for global public health," Nature, vol. 442, pp. 412-418, 2006.
[10] WHO, "In Global Summary of the AIDS Epidemic," 2007.
[11] R. A. Fisher, J. M. Bertonis, W. Meier, V. A. Johnson, D. S. Costopoulos, T. Liu, R. Tizard, B. D. Walker, M. S. Hirsch, R. T. Schooley, and R. A. Flavell, "HIV infection is blocked in vitro by recombinant soluble CD4," Nature, vol. 331, pp. 76-78, 1988.
[12] WHO, "In Patient Monitoring Guidelines For HIV Care and ART," 2006.
[13] WHO, "In 3 By 5 Progress Report," 2004.
[14] E. Engvall and P. Perlmann, "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G," Immuno chemistry, vol. 8, pp. 871-874, 1971.
[15] B. Pahar, J. Li, T. Rourke, C. J. Miller, and M. B. McChesney, "Detection of antigen-specific T cell interferon gamma expression by ELISPOT and cytokine flow cytometry assays in rhesus macaques," Immunological Methods, vol. 282, pp. 103-115, 2003.
[16] O. Fornas, J. Garcia, and J. Petriz, "Flow cytometry counting of CD34+ cells in whole blood," Nature Medicine, vol. 7, 2000.
[17] S. Bajaj, J. Welsh, R. Leif, and J. Price, "Ultra-rare-event detection performance of a custom scanning cytometer on a model preparation of fetal nRBCs," Cytometry, vol. 39, pp. 285-294, 2000.
[18] Y. H. Hsieh, S. J. Liu, H. W. Chen, Y. K. Lin, K. S. Liang, and L. J. Lai, "Highly sensitive rare cell detection based on quantum dot probe fluorescence analysis," Analytical and bioanalytical chemistry, vol. 396, pp. 1135-1141, 2010.
[19] Y. H. Hsieh, L. J. Lai, S. J. Liu, and K. S. Liang, "Rapid and sensitive detection of cancer cells by coupling with quantum dots and immunomagnetic separation at low concentrations," Biosensors and Bioelectronics, vol. 26, pp. 4249-4252, 2011.
[20] P. Balakrishnan, M. Dunne, N. Kumarasamy, S. Crowe, G. Subbulakshmi, A. K. Ganesh, A. J. Cecelia, P. Roth, K. H. Mayer, S. P. Thyagarajan, and S. Solomon, "An Inexpensive, Simple, and Manual Method of CD4 T-Cell Quantitation in HIV-Infected Individuals for Use in Developing Countries," Acquired Immune Deficiency Syndromes, vol. 36, pp. 1006-1010, 2004.
[21] S. Thorslund, J. Sanchez, R. Larsson, F. Nikolajeff, and J. Bergquist, "Bioactive heparin immobilized onto microfluidic channels in poly(dimethylsiloxane) results in hydrophilic surface properties," Colloids and Surfaces B: Biointerfaces, vol. 46, pp. 240-247, 2005.
[22] S. Thorslund, R. Larsson, F. Nikolajeff, J. Bergquist, and J. Sanchez, "Bioactivated PDMS microchannel evaluated as sensor for human CD4+ cells—The concept of a point-of-care method for HIV monitoring," Sensors and Actuators B: Chemical, vol. 123, pp. 847-855, 2007.
[23] S. Thorslund, R. Larsson, J. Bergquist, F. Nikolajeff, and J. Sanchez, "A PDMS-based disposable microfluidic sensor for CD4+ lymphocyte counting," Biomed Microdevices, vol. 10, pp. 851-857, 2008.
[24] Z. Wang, S. Y. Chin, C. D. Chin, J. Sarik, M. Harper, J. Justman, and S. K. Sia, "Microfluidic CD4+ T-Cell Counting Device Using Chemiluminescence-Based Detection," Analytical Chemistry, vol. 82, pp. 36-40, 2010.
[25] H. S. Kim, O. T. Son, K. H. Kim, S. H. Kim, S. Maeng, and H. I. Jung, "Separation of apoptotic cells using a microfluidic device," Biotechnol Lett, vol. 29, pp. 1659-63, 2007.
[26] S. H. Song, H. L. Lee, Y. H. Min, and H. I. Jung, "Electromagnetic microfluidic cell labeling device using on-chip microelectromagnet and multi-layered channels," Sensors and Actuators B: Chemical, vol. 141, pp. 210-216, 2009.
[27] K. Y. Lien, L. Y. Hung, T. B. Huang, Y. C. Tsai, H. Y. Lei, and G. B. Lee, "Rapid detection of influenza A virus infection utilizing an immunomagnetic bead-based microfluidic system," Biosensors Bioelectronics, vol. 26, pp. 3900-3907, 2011.
[28] K. Y. Lien, Y. H. Chuang, L. Y. Hung, K. F. Hsu, W. W. Lai, C. L. Ho, C. Y. Chou, and G. B. Lee, "Rapid isolation and detection of cancer cells by utilizing integrated microfluidic systems," Lab on a Chip, vol. 10, pp. 2875-2886, 2010.
[29] Y. K. Cho, J. G. Lee, J. M. Park, B. S. Lee, Y. Lee, and C. Ko, "One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device," Lab on a Chip, vol. 7, pp. 565-573, 2007.
[30] M. A. Unger, H. P. Chou, T. Thorsen, A. Scherer, and S. R. Quake, "Monolithic microfabricated valves and pumps by multilayer soft lithography," Science, vol. 288, pp. 113-116, 2000.
[31] J. H. Kim, K. H. Na, C. J. Kang, D. Jeon, and Y. S. Kim, "A disposable thermopneumatic-actuated microvalve stacked with PDMS layers and ITO-coated glass," Microelectronic Engineering, vol. 73-74, pp. 864-869, 2004.
[32] A. Baldi, G. Yuandong, P. E. Loftness, R. A. Siegel, and B. Ziaie, "A hydrogel-actuated environmentally sensitive microvalve for active flow control," Microelectromechanical Systems, vol. 12, pp. 613-621, 2003.
[33] C. R. Neagu, J. G. E. Gardeniers, M. Elwenspoek, and J. J. Kelly, "An electrochemical active valve," Electrochim Acta, vol. 42, pp. 20-22, 1997.
[34] B. K. Paul and T. Terhaar, "Comparison of two passive microvalve designs for microlamination architectures," Micromechanics and Microengineering, vol. 10, pp. 15-20, 2000.
[35] B. Bae, N. Kim, H. Kee, S. H. Kim, Y. Lee, S. Lee, and K. Park, "Feasibility test of an electromagnetically driven valve actuator for glaucoma treatment," Microelectromechanical Systems, vol. 11, pp. 344-542, 2002.
[36] B. T. Chia, X. Y. Yang, M. Y. Cheng, C. W. Lin, and Y. J. Yang, "An electromagnetically-driven microfluidic platform with indirect-heating thermo-pneumatic valves," BioChip Journal, vol. 5, pp. 97-105, 2011.
[37] V. Studer, R. Jameson, E. Pellereau, A. Pepin, and Y. Chen, "A microfluidic mammalian cell sorter based on fluorescence detection," Microelectronic Engineering, vol. 73-74, pp. 852-857, 2004.
[38] N. T. Nguyen and Z. Wu, "Micromixers—a review," Micromechanics and Microengineering, vol. 15, pp. R1-R16, 2005.
[39] H. Mirzadeh, F. Shokrolashi, and M. Daliri, "Effect of silicon rubber crosslink density in fibroblast cell behavior in vitro," Biomedical Materials Research, vol. 67A, pp. 727-732, 2003.
[40] J. N. Lee, X. Jiang, D. Ryan, and G. M. Whitesides, "Compatibility of mammalian cells on surfaces of polydimethylsiloxane," Langmuir, vol. 20, pp. 11684-11691, 2004.
[41] S. G. Charati and S. A. Sterm, "Diffusion of gases in silicone polymers: molecular dynamic simulations," Macromolecules, vol. 31, pp. 5529-5535, 1998.
[42] J. R. Anderson, D. T. Chiu, J. C. McDonald, R. J. Jackman, O. Cherniavskaya, H. Wu, S. Whitesides, and G. M. Whitesides, "Fabrication of topologically complex three-dimensional microfluidic systems in PDMS by rapid prototyping," Analytical Chemistry, vol. 72, pp. 3158-3164, 2000.
[43] M. H. Wu, J. P. G. Urban, Z. Cui, and Z. F. Cui, "Development of PDMS microbioreactor with well-defined and homogenous culture environment for chondrocyte 3-D culture," Biomedical Microdevices, vol. 8, pp. 331-340, 2006.
[44] "Data sheet for NANOTM SU8 negative tone photoresists, formulations 50 & 100, released by MICRO-CHEM. corp."
[45] "Data sheet for NANOTM SU8 negative tone photoresists, formulations 2-25, released by MICRO-CHEM. corp."
[46] 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," Chromatography A, vol. 948, pp. 225–233, 2002.