單一細胞的電特性可以對其病理狀態提供重要的資訊，在醫療應用上已引起臨床醫師極大的興趣。本篇論文提出微流道捕捉單顆人類子宮頸癌細胞，並藉由阻抗分析儀量測其阻抗，達成單一細胞的電性分析。在微流道組成部分是由帶有金屬電極的玻璃基板與有微柱結構的聚二甲基矽氧烷之流道所組成。而為分析微流道中的流體，我們利用了流體力學模擬軟體分析流道中之流體運動情形，根據模擬結果，再利用三柱結構來達成10%細胞捕捉機率。此外，為進行單細胞電特性分析，我們對整個系統建立了一個等效電路並使用有限元素法進行模擬分析與驗證，並建立一個單細胞之等效電路模型，其等效電路在1k到100k赫茲操作頻率與0.1到1伏特電壓下，單顆人類子宮頸癌細胞的導電係數、介電係數、及阻抗變化。從模擬與驗證之結果來看，等效電路模型與實驗量測結果是極為符合的。在0.2伏特的操作電壓下，實驗與模擬結果對於單顆人類子宮頸癌細胞的振幅與相位，最大誤差分別為9.5%與4.2%。而因為單顆人類子宮頸癌細胞有電容性的表現，可看出在所有操作電壓下振幅都會隨頻率增加而變小。除此之外，因為一個強的電場可能會促進細胞質與等張溶液中的離子交換，所以增加操作電壓會使的單顆人類子宮頸癌細胞的振幅變小。從等效電路來看，在操作電壓在0.9伏特以下時，系統阻抗的特性在30k赫茲以下可以用並聯電路表示，而在30k到100k赫茲的範圍可以串聯電路表示。而當在操作電壓超過0.8伏特時，由於細胞阻抗特性較明顯，單顆人類子宮頸癌細胞阻抗的相位特性可以串聯電路表示。而根據單顆人類子宮頸癌細胞的電特性分析，單顆人類子宮頸癌細胞的導電系數與介電係數都會隨操作電壓增加而增加。在操作電壓下0.1到1伏特，單顆人類子宮頸癌細胞的導電係數會隨著頻率增加而增加，但是單顆人類子宮頸癌細胞介電係數在操作電壓0.6到1伏特，卻會隨著頻率增加會減少。此外在模擬與實驗結果的基礎上，我們建立了經驗公式來預測單顆人類子宮頸癌細胞在特定操作頻率及電壓下的導電係數及介電係數。在操作頻率為0.2伏特下，導電系數與介電係數在經驗公式預測結果與模擬結果間的最大差異值分別只有0.5%和4.5%。
此外，為了促進單細胞阻抗量測分析方法於醫療上的疾病診斷及治療之廣泛應用，本論文亦使用之前之單一細胞量測方法發展了一可攜式阻抗分析儀。可攜式阻抗分析儀是由一個電源供應晶片一個阻抗量測晶片以及一個電腦微控制器晶片所組成。這些應用晶片提供了可攜帶、低成本、操作簡單以及能自動收集數據的能力。此外，為驗證本可攜式阻抗分析儀之特性，本論文使用了一台精準的商用阻抗分析儀來確定這個可攜式阻抗分析儀的準確性及可靠度。根據可攜式阻抗分析儀量測結果，一個乳膠小珠的準確性及可靠度大約是97.10%與 96.34%。最後，使用這個可攜式阻抗分析儀量測單顆人類子宮頸癌細胞以及乳線腫瘤細胞的強度，結果證明了所提出的阻抗量測平台能成功地在11到101 kHz這範圍分辨出單顆人類子宮頸癌細胞以及乳線腫瘤細胞。

The electrical properties of single cells provide fundamental insights into their pathological condition and are therefore of immense interest to medical practitioners. Accordingly, this thesis captures single HeLa cells using a microfluidic device and then measures their impedance properties using a commercial impedance spectroscopy system. The microfluidic device includes a glass substrate with metal electrodes and a PDMS (Poly-dimethylsiloxane) channel with micro pillars. The computation fluid dynamics software is used to study the flow of the microstructures in the channel. According to simulation results, the probability of cell capture by three micro pillars is about 10%. The experimental system is modeled by an equivalent electrical circuit and FEM (Finite element method) simulations are then performed to establish the conductivity, permittivity and impedance of single HeLa cells under various operational frequencies ranging from 1 to 100 kHz and voltages between 0.1 and 1.0V. The equivalent circuit model of the device is established and fits closely to the experimental results. At an operational voltage of 0.2 V, the maximum deviation between the experimental and simulation results for the magnitude and phase of the HeLa cell impedance is found to be 9.5% and 4.2%, respectively. The magnitude of the HeLa cell impedance declines at all operation voltages with frequency because the HeLa cell is capacitive. Additionally, increasing the operation voltage reduces the magnitude of the HeLa cell because a strong electric field may promote the exchange of ions between the cytoplasm and the isotonic solution. Below an operating voltage of 0.9 V, the system impedance response is characteristic of a parallel circuit at under 30 kHz and of a series circuit at between 30 and 100 kHz. The phase of the HeLa cell impedance is characteristic of a series circuit when the operation voltage exceeds 0.8 V because the cell impedance becomes significant. In general, both sets of results show that the conductivity and permittivity of single HeLa cells increase with an increasing operational voltage. Moreover, an increasing frequency is found to increase the conductivity of HeLa cells at all values of the operational voltage, but to reduce the permittivity for operational voltages in the range 0.6–1.0 V. Based upon the simulation and experimental results, empirical equations are constructed to predict the conductivity and permittivity of single HeLa cells under specified values of the operational voltage and frequency, respectively. The maximum discrepancy between the predicted results and the simulation results for the permittivity and conductivity of the HeLa cells at an operational voltage of 0.2V is found to be just 0.5% and 4.5%, respectively.
Additionally, in order to facilitate the development of single cell analysis for disease diagnosis and detection in the medical, portable impedance analyzers is also presented in this thesis. The minimized impedance spectroscopy consists of a power supply chip (ADR423), an impedance measurement chip (AD5934) and a microcontroller chip (C8051F320). The functional chip is developed specially to provide a portable, low cost, easy to use, and it is performed to automatic collection of data. Moreover, the measurement accuracy and reliable obtained by the proposed system and a conventional precision impedance analyzer are verified. The measurement accuracy and reliability of the minimized impedance spectroscopy using single latex beads are about 97.10% and 96.34%, respectively. Finally, the proposed system is used to measure the magnitude of HeLa cells and MCF-7 cells. The results demonstrate that the proposed impedance sensing platform as well as the equivalent circuit model with frequency range successfully distinguishes the HeLa cells and MCF-7 cells at between 11 and 101 kHz.

[1]. Y.H Cho, T. Yamamoto, Y. Sakai, T. Fujii and B. Kim, “Development of microfluidic device for electrical/physical characterization of single cell”, Journal of Microelectromechanical Systems, Vol. 15, No. 2, pp. 287-295, 2006.
[2]. S. Pecorelli, G. Favalli, L. Zigliani and F. Odicino, “Cancer in women”, International Journal of Gynecology & Obstetrics, Vol. 82, No. 3, pp. 369-379, 2003.
[3]. R. Carlson, C. Gabel, S. Chan and R. Austin, “Self-sorting of white blood cells in a lattice”, Physical Review Letters, Vol. 79, pp. 2149–2152. 1997.
[4]. O. Bakajin, R. Carlons, C. Chou, S. Chan, C. Gabel, J. Knight, T. Cox and R. Austin, “Sizing, Fractionation and Mixing of Biological Objects via Microfabricated Devices”, MicroTAS, pp. 193–198, 1998
[5]. H. Andersson, W.V.D. Wijngaart, P. Enoksson and G. Stemme, “Micromachined flow-through filter-chamber for chemical reactions on beads”, Sensors and Actuators, Vol. 67, pp. 203–208, 2000.
[6]. C. Rusu, R.V. Oever, M.D. Boer, H. Jansen, J. Berenschot, J. Bennink, J. Kanger, B.D. Grooth, M. Elwenspoek, J. Grever, J. Brugger and A.V.D. Berg, “Direct integration of micromachined pipettes in a flow channel for single DNA molecule study by optical tweezers”, Journal of Microelectronic Systems, Vol. 10, pp. 238–247. 2001.
[7]. H. Liang, W.H. Wright, C.L. Rieder, E.D. Salmon, G. Profeta, J. Andrews, Y. Liu, G.J. Sonek and M.W. Berns, “Directed movement of chromosome arms and fragments in mitotic newt lung cells using optical scissors and optical tweezers”, Experimental Cell Research, Vol. 213, pp. 308-312, 1994.
[8]. J. Xu, L. Wu, M. Huang, W. Yang, J. Cheng and X. Wang, “Dielectrophoresis Separation and Transportation of Cells and Bioparticles on Microfabricated Chips”, MicroTAS, pp. 565–566. 2001.
[9]. S. Fiedler, S. Shirley, T. Schnelle and G. Fuhr, “Dielectrophoretic sorting of particles and cells in a microsystem”, Analytical Chemistry, Vol. 70, pp. 1909–1915, 1998.
[10]. Y. Huang, K. Ewalt, M. Tirado, R. Haigis, A. Forster, D. Ackley, M. Heller, J. O’Connell and M. Krihak, “Electric manipulation of bioparticles and macromolecules on microfabricated electrodes”, Analytical Chemistry, Vol. 73, pp. 1549–1559, 2001.
[11]. L.S. Jang, P.H. Huang and K.C. Lan, “Single-cell trapping utilizing negative dielectrophoretic quadrupole and microwell electrodes”, Biosensors and Bioelectronics, Vol. 24, pp. 3637-3644, 2009.
[12]. K.H. Gilchrist, L. Giovangrandi and G..T.A. Kovacs, “Analysis of Microelectrode-Recorded Signals from a Cardiac Cell Line as a Tool for Pharmaceutical Screening”, The 11th International Conference on Solid-State Sensors and Actuators, pp. 10-14, 2001.
[13]. R. Schmukler, G. Johnson, J.Z. Bao and C.C. Davis, “Electrical Impedance Of Living Cells : A Modified Four Electrode Approach”, IEEE Engineering in Medicine & Biology Society, pp. 899-901, 1988.
[14]. J.Z. Bao, C.C. David and R.E. Schmukler, ”Impedance Spectroscopy of Human Erythrocytes:System Calibration and Nonlinear Modeling”, IEEE Transactions on Biomedical Engineering, Vol. 40, No. 4, pp. 364-378, 1993.
[15]. C. Ren, H. Wang, Y. An, H. Sha and G. Lin, “Development Of Electrical Bioimpedance Technology In The Future”, The 20th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Vol. 20, No. 2, pp. 1052-1054, 1998.
[16]. C. Ren, “EIT-An attractive technology of medical imaging”, Electronic science & technology review, 1996.
[17]. C. Ren and X. Chi et al, “Electrical bioimpedance tomography”, Chinese journal of medical physics, 1997.
[18]. C. Liao, G. Lee, H. Liu, T. Hsieh and C. Luo, “Micro Reverse-Transcription Polymerase Chain Reaction System For Clinical Diagnosis of RNA-based Viruses”, Nucleic Acids Research, Vol. 33, pp. 156-162, 2005.
[19]. H.E. Ayliffe, A.B. Frazier and R.D. Rabbitt, “Electric impedance spectroscopy using microchannels with integrated metal electrodes”, Journal of Microelectromechanical Systems, Vol. 8, Issue 1, pp. 50–57. 1999.
[20]. Y.H. Cho, N. Takama, T. Yamamoto, T. Fujii and B.J. Kim, “MEMS-based Bio-chip for the Characterization of Single Red Blood Cell”, Microtechnologies in Medicine and Biology, pp. 60-63, 2005.
[21]. S. Li and L. Lin, “A single cell electrophysiological analysis device with embedded electrode”, Sensors and Actuators, Vol. 134, pp. 20–26, 2006.
[22]. B. Matthews and J.W. Judy, “Design and Fabrication of a Micromachined Planar Patch-Clamp Substrate with Integrated Microfluidics for Single-cell Measurements”, Journal of Microelectromechanical Systems, Vol. 15, pp. 214-222, 2006.
[23]. S. Pecorelli, G. Favalli, L. Zigliani and F. Odicino, “Cancer in women”, International Journal of Gynecology & Obstetrics, Vol. 82, No. 3, pp. 369-379, 2003.
[24]. T. Sharrer, “"HeLa" Herself”, Scientist, Vol. 20, Issue 7, pp. 22, 2006.
[25]. A.S. Levenson and V.C. Jordan, ”MCF-7: the first hormone-responsive breast cancer cell line”, Cancer Research, Vol. 57, pp. 3071 – 3078, 1997.
[26]. J.C. Weaver and Y.A. Chizmadzhev, “Theory of electroporation: A review” Bioelectrochemistry and Bioenergetics, Vol. 41, pp. 135-160, 1996.
[27]. C. Yao, C. Sun, Y. Mi, S. Wang and L. Xiong, “Study on electroporation of cell membrane and tumor treatment”, Gaodianya Jishu/High Voltage Engineering, Vol. 30, pp. 45-47, 2004.
[28]. S. Grimnes and O.G. Martinsen, “Bioimpedance and bioelectricity basics”, New York, ACADEMIC PRESS, 2000.
[29]. K.R. Foster and H.P. Schwan, “Dielectric properties of tissues and biological materials: a critical review“, Critical Reviews™ in Biomedical Engineering, Vol. 17, pp. 25-104, 1989.
[30]. H.P. Schwan, “Electrical properties of tissue and cell suspensions”, Advances in Biological and Medical Physics, Vol. 5, pp. 147–209, 1957.
[31]. K.C. Lan and L.S. Jang, “Single-cell trapping and impedance measurement utilizing electrothermal effect quadrupole structure and negative dielectrophoretic microwell electrodes”, Submit.
[32]. M.B. Somasekhar and J. Gorski, “Two elements of the rat prolactin 5′ flanking region are required for its regulation by estrogen and glucocorticoids”, Gene, Vol. 69, pp. 13–21, 1988.