臺灣博碩士論文加值系統

癌症病患在癌細胞轉移發生初期，循環腫瘤細胞會轉移至身體各部位，如果能在發生初期檢測出體內稀少的癌細胞，即可大大降低死亡率，因此進行細胞阻抗量測可以在癌症檢測上提供重要的資訊。在進行量測實驗前先在全血樣本加入紅血球與白血球裂解液，進行紅白血球的細胞裂解，可有效排除樣本中血球細胞對檢測的干擾，實驗發現進行血球細胞裂解後，人類子宮頸癌細胞(HeLa)存活率可達80 %，而紅血球細胞存活率只剩10 %，再利用微流體晶片捕捉癌細胞(HeLa)，並藉由鎖相放大器進行訊號的抓取，最後經由實驗結果的分析，得到細胞的阻抗量測。本研究所使用之晶片，係利用微機電技術製作出微流道與電極，以一交流電壓使電極產生較弱之不均勻電場，產生正介電泳力對細胞進行抓取，最後以微弱的電壓及交流頻率進行量測。實驗結果顯示捕捉細胞只需施予5 到 10 Vpp且頻率為1 MHz之電壓即可對細胞進行捕捉，當進行捕捉與量測切換時，為了確保電極上之細胞不流失，在流速為1 µL/min時，細胞流失率能降低到5 - 8 %，最後施加1 Vpp且頻率為4 kHz進行細胞阻抗量測，本研究設計之量測系統無需複雜流體控制即可達到細胞阻抗量測的效果，對於未來的癌症檢測只需少量的血液樣本結合研製之生物晶片上，觀察其訊號變化即可檢測出血液內是否含有癌細胞。

The detection of rate cells, such as circulating tumor cells (CTCs), circulating fetal cells, is important for medical diagnostics and characterization. In order to detect tumor cells precisely, the red blood cells and white blood cells buffer lysis were added to the whole blood sample before measuring. The RBCs (Red blood cells) and WBCs (white blood cells) were lysed and the experimental result was found that the survival rate of HeLa was about 80 %. However, the survival rate of RBCs was around only 10 %. The main purpose of this study is to trap cancerous cells (HeLa) in the proposed microchip, then measure the impedance of HeLa cells using lock-in amplifier after lysing blood cells. Circular microelectrodes were fabricated by the standard photolithography technique to trap the positive dielectrophoretic cells by applying a AC voltage of 5 to 10 Vpp with a frequency of 1 MHz. After cells were captured, a measuring AC of 1 Vpp with 4 kHz was applied. Meanwhile, the flow rate of 1 μL/min was also applied to reduce the loss rate of captured cells to approximately 5 – 8 %.This microfluidic device is easy to operate and integrate into further biomedical applications and technology. The detection of cancerous cells in the microchip proposed herein is sensitive and the interference of blood cells could be avoided.

摘要 I
致謝 V
目錄 VI
圖目錄 IX
表目錄 XII

第一章 緒論 1
1-1 前言 1
1-2 常見CTCs檢測方法 3
1-2-1 免疫磁分選 4
1-2-2 螢光活化細胞分離 4
1-2-3 密度梯度離心法 6
1-3 細胞操控技術 6
1-3-1 流體力 7
1-3-2 電磁力 8
1-3-3 光學操控技術 10
1-3-4 電場力 11
1-4 研究目的 17

第二章 基本理論 18
2-1 介電泳理論 18
2-2 細胞電導量測之計算公式推導 24
2-3 各式細胞介電泳阻抗量測之文獻探討 27
2-3-1 各式介電泳之種類及應用 27
2-3-2 結合修飾之阻抗量測 30
2-3-3 利用介電泳驅動後進行阻抗量測 31
2-3-4 單細胞定位後之阻抗量測 34
2-4 電場對細胞的傷害及其他影響 36

第三章 研究方法 38
3-1 儀器設備與藥品 38
3-2 細胞培養 41
3-3 細胞樣本配製 42
3-4 晶片設計 44
3-5 晶片製作 46
3-6 阻抗量測之設備架設 50


第四章 結果與討論 52
4-1 紅白血球裂解後之存活率 52
4-2 利用介電泳在環形電極上抓取細胞之結果 54
4-3 捕捉細胞後進行電訊量測之實驗結果 56

第五章 結論 60

參考文獻 62

[1]Department of health, Executive Yu, R.O.C.,"主要死因統計"Available at: http://bhp.dog.gov.tw
[2]Cheng-Hsin Chuang, C. H. Wei and Y. M Shu, "Impedance Sensing of Bladder Cancer Cells based on a Single cell based DEP Microchip," IEEE sensors, 2009.
[3]H. A. Pohl, "Some Effects of Nonuniform Fields on Dielectrics," Journal of Applied Physics, pp. 1182-1188, 1958.
[4]J. L. Hong, K. C. Lan and L. S. Jang, "Electrical characteristics analysis of various cancer cells using a microfluidic device based on single cell impedance measurement," Sensors and actuators B : Chemical pp. 927-934, 2012
[5]Junya Suehiro, R. Yatsunami, Ryo Hamada and Masanori Hara, "Quantitative estimation of biological cell concentration suspended in aqueous medium by using dielectrophoretic impedance measurement method," J. Phys. D: Appl. Phys. 32 2814, pp. 2814–2820, 1999.
[6]林君彥, "細胞蛋白感測晶片之研製-使用氧化銦錫電極之連續阻抗量測," 碩士論文, 私立台北醫學大學口腔科學研究所, 2007.
[7]D. D. Carlo, N. Aghdam and L. P. Lee, "Single-Cell Enzyme Concentrations, Kinetics, and Inhibition Analysis Using High-Density Hydrodynamic Cell Isolation Arrays," Analytical Chemistry, pp. 4925-4930, 2006.
[8]D. R. Gossett , H. T. Tse, S. A. Lee, Y. Ying, A. G. Lindgren, O. O. Yang, J. Rao, A. T. Clark and D. D. Carlo, "Hydrodynamic Stretching of Single Cells for Large Population Mechanical Phenotyping,," Proceeding of the National Academy of Science, pp. 7630-7635, 2012.
[9]Y. Haik, V. Pai and C. J. Chen, "Development of magnetic device for cell separation," Journal of Magnetism and Magnetic Materials, pp. 254-261, 1999.
[10]M. Zborowski and J. Chalmers, "Rare Cell Separation and Analysis by Magnetic Sorting," Analytical Chemistry, pp. 8050-8056, 2011.
[11]M. Dao, C. T. Lim and S. Suresh, "Mechanics of the human red blood cell deformed by optical tweezer," Journal of the Mechanics and Physics of Solid, pp. 2259-2280, 2003.
[12]D. C. N. Silva, C. N. Jovino and C. A. L. Silva, H. P. Fernandes, M. M. Filho, S. C. Lucena, A. M. D. N. Costa, C. L. Cesar, M. L. Barjas-Casrto, B. S. Santos and A. Fontes, "Optical Tweezers as a New Biomedical Tool to Measure Zeta Potential of Stored Red Blood Cells," Plos One, p. e31778, 2012.
[13]J. R. Du, Y. J. Juang, J. T. Wu and H. H. Wei, "Long-Range and Superfast trapping of DNA molecules in an ac electromagnetic funnel," Biomicrofluidics, p. 044103, 2008.
[14]M. Koklu, S. Park, S. D. Pillai and A. Beskok,, "Negative dielectrophoretic capture of bacterial spores in food matrices," Biomicrofluidics, p. 034107, 2010.
[15]F. Yang, X. Yang, H. Jiang, P. Bulkhaults, P. Wood, W. Hrushesky and G. Wang, "Dielectrophoretic separation of colorectal cancer cells," Biomicrofluidics, p. 013204, 2010.
[16]M. Sancho, G. Martinez, S. Muñoz, J. L. Sebastián and R. Pethig, "Interaction between cell in dielectrophoresis and electrorotation experiment," Biomicrofluidics, p. 022802, 2010.
[17]R. Pethig, "Preface to special topic: Dielectrophoresis," Biomicrofluidics, p. 022701, 2010.
[18]M. D. Pysher and M. A. Hayes, "Electrophoretic and Dielectrophoretic Field Gradient Technique for Separating Bioparticles," Analytical Chemistry, pp. 4552-4557, 2007.
[19]A. Alazzam, I. Stiharu, R. Bhat and A. N. Meguerditchian, "Interdigitated comb-like electrodes for continuous separation of malignant cells from blood using dielectrophoresis," Electrophoresis, pp. 1327-1336, 2011.
[20]F. F. Becker, X. B. Wang, Y. Huang, R. Pethig, J. Vykoukal and P. R. C. Gascoyne, "Separation of human breast cancer cells from blood by differential dielectric affinity," Proceeding of the National Academy of Science, pp. 860-864, 1995.
[21]L. Wu, L.-Y. L. Yang and K.-M. Lim, "Dielectrophoretic capture voltage spectrum for measurement of dielectric properties and separation of cancer cells," Biomicrofluidics, p. 014113, 2012.
[22]K. Kwon, H. S. Moon, S. I. Kim, H. Han, J. Sohn, S. Lee and H. I. Jung, "Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP)," Lab on a Chip, pp. 1118-1125, 2011.
[23]J Cheng, E. L. Sheldon, L. Wu, M. J. Heller and J. P. O’Connell, "Isolation of Cultured Cervical Carcinoma Cells Mixed with Peripheral Blood Cells on a Bioelectronic Chip," Analytical Chemistry, pp. 2321-2326, 1998.
[24]J. Noshari, P. R. C. Gascoyne, T. J. Anderson and F. F. Becker, "Isolation of rare cells from cell mixtures by dielectrophoresis," Electrophoresis, pp. 1388-1398, 2009.
[25]M. B. Sano, E. A. Henslee, E. Schmelz and R. V. Davalos, "Contactless dielectrophoretic spectroscopy: Examination of the dielectric properties of cells found in blood," Electrophoresis, pp. 3164-3171, 2011.
[26]A. Salmanzadeh, L. Romero, H. Shafiee, R. C. Gallo-Villanueva, M. A. Stremler, S. D. Cramer and R. V. Davalos, "Isolation of prostate tumor initiating cells (TICs) through their dielectrophoretic signature," Lab on a Chip, pp. 182-189, 2012.
[27]T. B. Jones, "Electromechanics of particles," Cambridge University Press, 1995.
[28]C. P. Jen and T. W. Chen, "Selective trapping of live and dead mammalian cells using insulator- based dielectrophoresis within open- Top Microstructures," Biomedical Microdevices, pp. 507-607, 2009.
[29]K. Khoshmanesh, S. Baratchi, F. J. Tovar-Lopez, S. Nahavandi, D. Wlodkowic, A. Mitchell and K. Kalantar-zadeh, "On-chip separation of Lactobacillus bacteria from yeasts using dielectrophoresis," Microfluid Nanofluid, pp. 597-606, 2012.
[30]R. S. Thomas, H. Morgan and N. G. Green , "Negative DEP traps for single cell immobilisation," Lab on a Chip, pp. 1534-1540, 2009.
[31]S. K. Srivastava, J. L. Baylon.-Cardiel, B. H. Lapizco-Encinas, A. R. Minerick, "A continuous DC-insulator dielectrophoretic sorter of microparticles," Journal of Chromatography A, pp. 1780-1789, 2011.
[32]C. P. Jen, H. H. Chang and C. T. Huang and K.H. Chen, "A microfabricated module for isolating cervical carcinoma cells from perpheral blood utilizing dielectrophoresis in stepping electric fields," Microfluid Nanofluid, pp. 1887-1896, 2012.
[33]Y. K Chung, J. Reboud, K. C. Lee, H. M Lim and P. Y. Lim, "An electrical biosensor for the detection of circulating tumor cells," Biosensor and bioelectronics, pp. 2520-2526, 2011.
[34]Junya Suehiro, Akio Ohtsubo, Tetsuji Hatano and Masanori Hara, "Selective detection of bacteria by a dielectrophoretic impedance measurement method using an antibody-immobilized electrode chip," Sensors and Actuators B pp. 319-326, 2006.
[35]R. Hamada, H. Takayama and Y. Shonishi, "Improvement of dielectrophretic impedance measurement method by bacterial concentration utilizing negative dielectrophoresis," Joural of physics; conference series, p. 012031, 2011.
[36]H. Park, D. Kim and K. S. Yun, "Single cell manipulation on microfluidic chip by dielectrophoretic actuation and impedance detection," Sensors and Actuators B: Chemical, pp. 167-173, 2010.
[37]K. C. Lan and L. S. Jang, "Integration of single cell trapping and impedance measurement utilizing microwell electrodes," Biosensors and Bioelectronics, pp. 2025-2031, 2011.
[38]L. S. Jang, K. C. Lan and J. L. Hong, "Electrical characteristics analysis of various cancer cells using a microfluidic device based on single cells impedance measurement," Sensors and Actuators B: Chemical, pp. 927-937, 2012.
[39]F. Han, Y. Wang, C. E. Sims, M. Bachman, R. Chang, G. P. Li and N. L. Allbrittion, "Fast electrical lysis of cells for capillary electrophresis," Analytical Chemistry, pp. 3688-3696, 2003.
[40]D. Walz, H. Berg and G. Milazzo, "Bioelectrochemistry of cells and tissues," Birkhauser Verlag, 1995.
[41]S. Nishimura, H. Matsumura, K. Kosuge and T. Yamaguchi, "Enhance Pearl-Chain Formation by Electrokinetic Interaction with the bottom Surface of Vessel," Langmuir, pp. 8789-8797, 2007.
[42]H. Arjomandi, S. S. Barceona, S. L. Gallocher and M. Vallejo, "Biofluid dynamics of the human circulatory system," Congress on Biofluid Dynamics of Human Body Systems at Biomedical Engineering - FIU, 2003.
[43]Zuckerman K., "Approach to the anemias. In: Goldman L," Saunders Elsevier, p. 162, 2007.
[44]M. Toner and D. Irimia, "Blood-on-a-chip," Annual Review Biomedical Engineering, pp. 77-103, 2005.
[45]C. Iliescu, D. P. Poenar, M. Carp and C. C. Loe, "A microfluidic device for impedance spectroscopy analysis of biological samples," Sensors and Actuators B, pp. 168-176, 2007.
[46]J. L. Hong, K. C. Lan and L. S. Jang, "Electrical characteristics analysis of various cancer cells using a microfluidic device based on single cells impedance measurement," Sensors and Actuators B: Chemical, pp. 927-937, 2012.
[47]Jung A Lee, S. Hwang, J. Kwak, S. I Park, S. S. Lee. and K. C. Lee "An electrochemical impedance biosensor with aptamer- modified pyrolyzed carbon electrode for label free protein detection," Sensors and Actuators B, pp. 372-379, 2008.