本研究目的在測試石墨烯細胞毒性除了靜態方法外，也利用微流道晶片與蠕動幫浦模在血管流動環境下石墨烯的毒性。
為改善石墨烯的疏水特性，本研究在利用電解剝離法製備石墨烯同時接上聚苯乙烯磺酸鈉，依此法製備出的石墨烯擁有良好的分散性，可方便進行毒性試驗。
將混合石墨烯的培養基與人類微血管內皮細胞做靜態培養後，利用MTS試劑測試細胞存活率，結果顯示高濃度(80 ppm)的石墨烯會導致細胞的死亡，電子顯微鏡的觀察顯示石墨烯會沉積在細胞表面而且有被絲狀偽足包覆的情形，可能造成石墨烯被細胞吞噬，進一步造成細胞的死亡。
本研究另使用微流道晶片測試石墨烯流動狀態的毒性，將細胞利用Live/Dead試劑染色且在螢光顯微鏡下觀察，並進一步觀察細胞形狀及計算細胞存活率，在不同石墨烯濃度、剪切力、暴露時間等條件下測試，研究發現無論在哪些條件下均發現培養基中的石墨烯對細胞並未造成影響，推測由於石墨烯在動態流動下，貼附細胞表面機會相對靜態培養為少，所以吞噬作用未發生。本實驗證實石墨烯在動態下不會因片狀結構對細胞產生傷害，可進一步應用於藥物載體上。

第1章 緒論 1
1-1 石墨烯之簡介 1
1-1-1 石墨烯起源與特性 1
1-1-2 石墨烯的製備 2
1-2 細胞簡介 4
1-2-1 細胞定義與分類 4
1-2-2 細胞組成與結構 4
1-2-3 細胞生長需求 5
1-2-4 細胞壞死與凋亡 6
1-3 石墨烯生醫領域的應用 7
1-3-1 癌細胞光熱治療 7
1-3-2 藥物載體 8
1-3-3 石墨烯於加速幹細胞分化之應用 9
1-4 石墨烯毒性的簡介 9
1-4-1 石墨烯的體外毒性簡介 9
1-4-2 石墨烯體內毒性簡介 11
1-5 微流道晶片應用於毒性測試 12
1-6 研究計畫 13
第2章 奈米碳材料的製備 27
2-1 石墨烯的製備 27
2-2 石墨烯形貌與性質分析 28
2-3 螢光標記石墨烯之製備 29
2-4 多壁奈米碳管官能基化接合聚苯乙烯磺酸鈉的製備 31
第3章 石墨烯對於人類微血管內皮細胞的影響 38
3-1 人類微血管內皮細胞培養 38
3-1-1 細胞解凍 38
3-1-2 細胞培養 40
3-2 石墨烯靜態下對內皮細胞的影響 42
3-2-1 石墨烯於靜態下細胞存活率測試 42
3-2-2 石墨烯靜態下與細胞接觸觀察 45
3-2-3 螢光標記石墨烯與細胞接觸情形 48
3-3 石墨烯流動態下對內皮細胞的影響 49
3-3-1 微流道晶片製作 49
3-3-2 微流道內細胞培養 51
3-3-3 石墨烯在微流道於不同剪切力及暴露時間對內皮細胞的影響 52
3-3-4 微流道內細胞與石墨烯接觸觀察 55
3-4 多壁奈米碳管流動態下對內皮細胞的影響 56
第4章 結果與討論 61
4-1 石墨烯溶液製備之結果 61
4-2 石墨烯表面形貌與化學性質分析 61
4-2-1 場發射掃描式電子顯微鏡觀察石墨烯之表面形貌 61
4-2-2 拉曼光譜儀分析之結果 62
4-2-3 原子力顯微鏡之分析結果 63
4-2-4 粒徑分析儀之分析結果 64
4-2-5 表面電位測定儀之分析結果 64
4-2-6 傅立葉轉換紅外線光譜觀察材料之結果 65
4-3 石墨烯靜態下對內皮細胞的影響 66
4-3-1 石墨烯靜態下細胞存活率測試 66
4-3-2 石墨烯靜態下與細胞接觸情形 67
4-3-3 螢光標記石墨烯與細胞接觸情形 68
4-4 石墨烯動態下對內皮細胞的影響 69
4-4-1 石墨烯動態中細胞存活率測試 69
4-4-2 石墨烯動態實驗後微流道內細胞表面形貌 71
4-5 多壁奈米碳管動態下對內皮細胞的影響 72
4-5-1 多壁奈米碳管表面形貌及化學性質分析 72
4-5-2 多壁奈米碳管動態中細胞存活率測試 72
4-5-3 多壁奈米碳管動態實驗後微流道內細胞表面形貌 73
第5章 結論 99
參考文獻 101

[1] H. W. Kroto, J. R. Heath, S. C. O'Brien, R. F. Curl, and R. E. Smalley, “C60: buckminsterfullerene”, Nature, Vol. 318, pp. 162-163, 1985.
[2] S. Iijima, “Helical microtubules of graphitic carbon”, Nature, Vol. 354, pp. 56-58, 1991.
[3] S. Iijima and T. Ichihashi, “Single-shell carbon nanotubes of 1-nm diameter”, Nature, Vol. 363, pp. 603-605, 1993.
[4] A. K. Geim and K. S. Novoselov, “The rise of graphene”, Nature Materials, Vol. 6, pp. 183-191, 2007.
[5] K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva, and A. A. Firsov, “Electric field effect in atomically thin carbon films”, Science, Vol. 306, pp. 666-669, 2004.
[6] C. Lee, X. Wei, J. W. Kysar, and J. Hone, “Measurement of the elastic properties and intrinsic strength of monolayer graphene”, Science, Vol. 321, pp. 385-388, 2008.
[7] A. A. Balandin, S. Ghosh, W. Bao, I. Calizo, D. Teweldebrhan, F. Miao, and C. N. Lau, “Superior thermal conductivity of single-layer graphene”, Nano Letters, Vol. 8, pp. 902-907, 2008.
[8] J. H. Chen, C. Jang, S. Xiao, M. Ishigami, and M. S. Fuhrer, “Intrinsic and extrinsic performance limits of graphene devices on SiO2”, Nature Nanotechnology, Vol. 3, pp. 206-209, 2008.
[9] Y. Zhu, S. Murali, W. Cai, X. Li, J. W. Suk, J. R. Potts, and R. S. Ruoff, “Graphene and graphene oxide: synthesis, properties, and applications”, Advanced Materials, Vol. 35, pp. 3906-3924, 2010.
[10] V. Singh, D. Joung, L. Zhai, S. Das, S. I. Khondaker, and S. Seal, “Graphene based materials: past, present and future”, Progress in Materials Science, Vol. 56, pp. 1178-1271, 2011.
[11] A. Varykhalov and O. Rader, “Graphene grown on Co (0001) films and islands: electronic structure and it precise magnetization dependence”, Physical Review B, Vol. 80, pp. 035437 (6), 2009.
[12] A. Varykhalov, J. Sánchez-Barriga, A. M. Shikin, C. Biswas, E. Vescovo, A. Rybkin, D. Marchenko, and O. Rader, “Electronic and magnetic properties of quasifreestanding graphene on Ni”, Physical Review B, Vol. 101, pp. 157601 (4), 2008.
[13] X. Li, W. Cai, J. An, S. Kim, J. Nah, D. Yang, R. Piner, A. Velamakanni, I. Jung, E. Tutuc, S. K. Banerjee, L. Colombo, and R. S. Ruoff, “Large-area synthesis of high-quality and uniform graphene films on copper foils”, Science, Vol. 324, pp. 1312-1314, 2009.
[14] M. Losurdo, M. M. Giangregorio, P. Capezzuto, and G. Bruno, “Graphene CVD growth on copper and nickel: role of hydrogen in kinetics and structure”, Physical Chemistry Chemical Physics, Vol. 13, pp. 20836-20843, 2011.
[15] X. Li, Y. Zhu, W. Cai, M. Borysiak, B. Han, D. Chen, R. D. Piner, L. Colombo, and R. S. Ruoff, “Transfer of large-area graphene films for high-performance transparent conductive electrodes”, Nano Letter, Vol. 9, pp. 4359-4363, 2009.
[16] B. C. Brodie, “On the atomic weight of graphite”, Philosophical Transactions of the Royal Society of London, Vol. 149, pp. 249-259, 1859.
[17] W. S. Hummers and R. E. Offeman, “Preparation of graphitic oxide”, Journal of the American Chemical Society, Vol. 80, p. 1339, 1958.
[18] D. Li, M. B. Müller, S. Gilje, R. B. Kaner, and G. G. Wallace, “Processable aqueous dispersions of graphene nanosheets”, Nature Nanotechnology, Vol. 3, pp. 101-105, 2008.
[19] H. J. Shin, K. K. Kim, A. Benayad, S. M. Yoon, H. K. Park, I. S. Jung, M. H. Jin, H. K. Jeong, J. M. Kim, J. Y. Choi, and Y. H. Lee, “Efficient reduction of graphite oxide by sodium borohydride and its effect on electrical conductance”, Advanced Functional Materials, Vol. 19, pp. 1987-1992, 2009.
[20] C. Zhu, S. Guo, Y. Fang, and S. Dong, “Reducing sugar: new functional molecules for the green synthesis of graphene nanosheets”, ACS Nano, Vol. 4, pp. 2429-2437, 2010.
[21] T. Kuila, S. Bose, P. Khanra, A. K. Mishra, N. H. Kim, and J. H. Lee, “A green approach for the reduction of graphene oxide by wild carrot root”, Carbon, Vol. 50, pp. 914-921, 2012.
[22] Y. Wang, Z. Shi, and J. Yin, “Facile synthesis of soluble graphene via a green reduction of graphene oxide in tea solution and its biocomposites”, ACS Applied Materials & Interfaces, Vol. 3, pp. 1127-1133, 2011.
[23] N. Liu, F. Luo, H. Wu, Y. Liu, C. Zhang, and J. Chen, “One-step ionic-liquid-assisted electrochemical synthesis of ionic-liquid-
functionalized graphene sheets directly from graphite”, Advanced Functional Materials, Vol. 18, pp. 1518-1525, 2008.
[24] G. Wang, B. Wang, J. Park, Y. Wang, B. Sun, and J. Yao, “Highly efficient and large-scale synthesis of graphene by electrolytic exfoliation”, Carbon, Vol. 47, pp. 3242-3246, 2009.
[25] C. Y. Su, A. Y. Lu, Y. Xu, F. R. Chen, A. N. Khlobystov, and L. J. Li, “High-quality thin graphene films from fast electrochemical exfoliation”, ACS Nano, Vol. 5, pp. 2332-2339, 2011.
[26] 林良平等譯, 細胞分子生物學, 茂昌圖書有限公司, 1984.
[27] L. Hayflick and P. S. Moorhead, “The serial cultivation of human diploid cell strains”, Experiment Cell Research, Vol. 25, pp. 585-621, 1961.
[28] J. F. R. Kerr, A. H. Wyline, and A. R. Currie, “Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics”, British Journal of Cancer, Vol. 26, pp. 239-257, 1972.
[29] N. K. Kuan and E. Passaro, “Apoptosis: programmed cell death”, Archives of Surgery, Vol. 133, pp. 773-775, 1998.
[30] S. M. Moghimi, A. C. Hunter, and J. C. Murray, “Nanomedicine: current status and future prospects”, The FASEB Journal, Vol. 19, pp. 311-330, 2005.
[31] M. Nikfarjam, V. Muralidharan, and C. Christophi, “Mechanisms of focal heat destruction of liver tumors”, Journal of Surgical Research, Vol. 127, pp. 208-223, 2005.
[32] Z. M. Markovic, L. M. Harhaji-Trajkovic, B. M. Todorovic-
Markovic, D. P. Kepic, K. M. Arsikin, S. P. Jovanovic, A. C. Pantovic, M. D. Dramicanin, and V. S. Trajkovic, “ In vitro comparison of the photothermal anticancer activity of graphene nanoparticles and carbon nanotubes”, Biomaterials, Vol. 32, pp. 1121-1129, 2011.
[33] K. Yang, S. Zhang, G. Zhang, X. Sun, S. T. Lee, and Z. Liu, “Graphene in mice: ultrahigh in vivo tumor uptake and efficient photothermal therapy”, Nano Letters, Vol. 10, pp. 3318-3323, 2010.
[34] Z. Liu, J. T. Robinson, X. Sun, and H. Dai, “PEGylated nanographene oxide for delivery of water-insoluble cancer drugs”, Journal of the American Chemical Society, Vol. 130, pp. 10876-10877, 2008.
[35] X. Sun, Z. Liu, K. Welsher, J. T. Robinson, A. Goodwin, S. Zaric, and H. Dai, “Nano-graphene oxide for cellular imaging and drug delivery”, Nano Research, Vol. 1, pp. 203-212, 2008.
[36] T. R. Nayak, H. Andersen, V. S. Makam, C. Khaw, S. Bae, X. Xu, P. L. R. Ee, J. H. Ahn, B. H. Hong, G. Pastorin, and B. Özyilmaz, “Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells”, ACS Nano, Vol. 5, pp. 4670-4678, 2011.
[37] Y. Zhang, S. F. Ali, E. Dervishi, Y. Xu, Z. Li, D. Casciano, and A. S. Biris, “Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells”, ACS Nano, Vol. 4, pp. 3181-3186, 2010.
[38] A. Sasidharan, L. S. Panchakarla, A. R. Sadanandan, A. Ashokan, P. Chandran, C. M. Girish, D. Menon, S. V. Nair, C. N. R. Rao, and M. Koyakutty, “Hemocompatibility and macrophage response of pristine and functionalized graphene”, Small, Vol. 8, pp. 1251-1263, 2012.
[39] O. Akhavan, E. Ghaderi, and A. Akhavan, “Size-dependent genotoxicity of graphene nanoplatelets in human stem cells”, Biomaterials, Vol. 33, pp. 8017-8025, 2012.
[40] K. Wang, J. Ruan, H. Song, J. Zhang, Y. Wo, S. Guo, and D. Cui, “Biocompatibility of graphene oxide”, Nanoscale Research Letters, Vol. 6, pp. 8 (8), 2011.
[41] S. Zhang, P. Xiong, X. Yang, and X. Wang, “Novel PEG functionalized graphene nanosheets: enhancement of dispersibility and thermal stability”, Nanoscale, Vol. 3, pp. 2169-2174, 2011.
[42] K. Yang, H. Gong, X. Shi, J. Wan, Y. Zhang, and Z. Liu, “In vivo biodistribution and toxicology of functionalized nano-graphene oxide in mice after oral and intraperitoneal administration”, Biomaterials, Vol. 34, pp. 2787-2795, 2013.
[43] D. Kim, Y. S. Lin, and C. L. Haynes, “On-chip evaluation of shear stress effect on cytotoxicity of mesoporous silica nanoparticles”, Analytical Chemistry, Vol. 83, pp. 8377-8382, 2011.
[44] S. Mao, D. Gao, W. Liu, H. Wei, and J. M. Lin, “Imitation of drug metabolism in human liver and cytotoxicity assay using a microfluidic device coupled to mass spectrometric detection”, Lab on a Chip, Vol. 12, pp. 219-226, 2012.
[45] T. G. Papaioannou and C. Stefanadis, “Vascular wall shear stress basic principles and methods”, Hellenic Journal of Cardiology, Vol. 46, pp. 9-15, 2005.
[46] A. H. Cory, T. C. Owen, J. A. Barltrop, and J. G. Cory, “Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture”, Cancer Communications, Vol. 3, pp. 207-212, 1991.
[47] 陳家全、李家維、楊瑞森, ”生物電子顯微鏡學”, 國科會精儀中心, pp. 25-35, 1991.
[48] N. J. Kent, L. Basabe-Desmonts, G. Meade, B. D. MacCraith, B. G. Corcoran, D. Kenny, and A. J. Ricco, “Microfluidic device to study arterial shear-mediated platelet-surface interactions in whole blood: reduced sample volumes and well-characterised protein surfaces”, Biomedical Microdevices, Vol. 12, pp. 987-1000, 2010.
[49] F. Tuinstra and J. L. Koenig, “Raman spectrum of graphite”, Journal of Chemical Physics, Vol. 53, pp. 1126-1130, 1970.
[50] A. C. Ferrari, J. C. Meyer, V. Scardaci, C. Casiraghi, M. Lazzeri, S. Piscanec, D. Jiang, K. S. Novoselov, S. Roth, and A. K. Geim, “Raman spectrum of graphene and graphene layers”, Physical Review Letters, Vol. 97, pp. 187401 (4), 2006.
[51] C. Bussy, H. Ali-Boucetta, and K. Kostarelos, “Safety considerations for graphene lessons learnt from carbon nanotubes”, Accounts of Chemical Research, Vol. 46, pp. 692-701, 2013.
[52] H. Yue, W. Wei, Z. Yue, B. Wang, N. Luo, Y. Gao, D. Ma, G. Ma, and Z. Su, “The role of the lateral dimension of graphene oxide in the regulation of cellular responses”, Biomaterials, Vol. 33, pp. 4013-4021, 2012.