簡易檢索 / 詳目顯示

研究生: 邱芷欣
Jhih-Hsin Chiu
論文名稱: 微流道晶片表面改質高分子利用雷射檢測子宮內膜癌與循環腫瘤細胞培養之光學研究
Modified microfluidic chip for detection of endometrial cancer by laser system and circulating tumor cell culture optical analysis
指導教授: 陳建光
Jem-Kun Chen
口試委員: 呂建興
Chien-Hsing Lu
陳士勛
Shih-Hsun Chen
黃昭蓮
Jau-Lang Hwang
學位類別: 碩士
Master
系所名稱: 工程學院 - 材料科學與工程系
Department of Materials Science and Engineering
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 189
中文關鍵詞: 聚二甲基矽氧烷明膠循環腫瘤細胞雷射能量分析子宮內膜癌細胞培養與釋放
外文關鍵詞: Polydimethysiloxane, Gelatin, Circulating tumor cell, Laser system, Endometrial cancer, Cell culture and release
相關次數: 點閱:399下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

  • 摘要 I Abstract III 致謝 V 目錄 VIII 圖目錄 XVI 表目錄 XXV 1. 緒論 1 1.1. 研究背景 1 1.2. 研究動機與目的 5 2. 理論與文獻回顧 7 2.1. 循環腫瘤細胞 7 2.1.1. 循環腫瘤細胞介紹 7 2.1.2. 循環腫瘤細胞生物特性標記 9 2.1.3. 循環腫瘤細胞捕獲 11 2.1.4. 循環腫瘤細胞細胞培養 14 2.2. 明膠(Gel) 16 2.3. 聚二甲基矽氧烷 17 2.4. 微影製程及翻模 18 2.5. 光柵效應 21 2.6. 雷射系統偵測 24 2.7. 自組裝單分子層 26 2.8. 共價鍵固定法(EDC/NHS reaction) 29 2.9. 重組蛋白與抗體 31 3. 儀器原理 30 3.1. 高解析度場發射掃描式電子顯微鏡(Field-emission scanning electron microscope,FE-SEM) 30 3.2. 原子力顯微鏡(Atomic Force Microscope, AFM) 32 3.3. X射線光電子能譜儀(X-ray photoelectron spectroscopy,XPS) 36 3.4. 傅立葉轉換紅外線光譜儀 (Fourier-Transform Infrared Spectroscopy, FT-IR) 39 3.5. 接觸角量測儀 (Contact Angle Meter) 44 3.6. 可見光紫外光分光光譜儀 (Ultraviolet-visible spectroscopy,UV-vis) 46 3.7. 雷射掃描式共軛焦顯微鏡 (Laser scanning confocal microscope,LSCM) 49 3.8. 螢光顯微鏡 51 3.9. 全波長多功能微盤分析儀 54 4. 實驗流程與方法 56 4.1. 實驗流程圖 56 4.2. 實驗藥品 57 4.3. 實驗儀器 60 4.4. 實驗步驟 62 4.4.1. 微影製程製備圖案化光阻層 62 4.4.1.1. 表面預處理 62 4.4.1.2. 光阻塗佈 63 4.4.1.3. 軟烤 63 4.4.1.4. 曝光 63 4.4.1.5. 顯影 64 4.4.2. 圖案化轉印於PDMS薄膜 65 4.4.3. PDMS表面改質 66 4.4.3.1. PDMS表面鍍金處理 66 4.4.3.2. 製備自組裝層 66 4.4.3.3. 明膠之接枝 67 4.4.4. PDMS表面改質ProtrinG與Anti-EpCAM 68 4.4.5. Blocking BSA 抗沾黏層於試片表面 69 4.4.6. 微流道晶片製作 70 4.4.7. 紫外光-可見光標準曲線分析TA與Gelatin接枝量 71 4.4.8. 細胞株處理 72 4.4.8.1. HCT-116 72 4.4.8.2. DLD-1 74 4.4.8.3. HeLa 74 4.4.9. 雷射分析儀細胞定量檢測 75 4.4.9.1. 細胞株HCT-116、DLD-1、HeLa接種至微流道晶片 75 4.4.9.2. 細胞試片螢光染色處理 75 4.4.10. 病人檢體之循環腫瘤細胞偵測 76 4.4.11. 細胞培養 76 4.4.11.1. Cell Counting Kit-8 (CCK-8) Assays 判讀細胞活性 76 4.4.11.2. 細胞接種至基材培養 77 4.4.12. 細胞釋放 77 4.4.12.1. 細胞至基材釋放 77 4.4.12.2. Live/Dead Cell Imaging Kit 判讀細胞活性 78 4.4.13. 掃描式電子顯微鏡生物試片製備 78 5. 結果與討論 79 5.1. 圖案化表面分析 79 5.1.1. 微影製成光阻圖案 79 5.1.2. PDMS轉印圖案 80 5.2. PDMS圖案化光學性質 82 5.2.1. 可見光繞射 82 5.2.2. 雷射繞射 83 5.3. 硫醇基乙酸與明膠接枝於PDMS之分析 85 5.3.1. XPS能譜圖分析 85 5.3.1.1. PDMS改質後之XPS Wide Scan分析 85 5.3.1.2. S2p能譜分析 87 5.3.1.3. N1s能譜分析 88 5.3.1.4. Si2p能譜分析 88 5.3.1.5. Au4f能譜分析 89 5.3.1.6. C1s能譜分析 90 5.3.1.7. O1s能譜分析 91 5.3.2. FTIR光譜分析 93 5.3.3. 接觸角親疏水測試 94 5.3.4. 硫醇基乙酸接枝於PDMS表面之定量分析 95 5.3.5. 明膠接枝於PDMS表面之定量分析 98 5.3.6. AFM表面型態分析 101 5.3.7. SEM表面型態分析 105 5.3.8. 雷射能量分析 109 5.4. 試片抓取細胞之測試 114 5.4.1. 血液抗沾黏測試 114 5.4.1.1. 螢光顯微鏡分析結果 114 5.4.1.2. 雷射分析結果 115 5.4.2. 不同流速對HCT-116的抓取影響 117 5.4.2.1. 各流速對細胞抓取效率之分析 118 5.4.2.2. 流速30min/1mL、45min/1mL之細胞抓取穩定性之分析 ………………………………..………………………………………………………121 5.4.3. 基材抓取不同顆數細胞對雷射值之影響 124 5.4.4. 基材對不同種類細胞之選擇性 127 5.5. 臨床檢體測試 129 5.6. 細胞培養 135 5.6.1. 細胞生長性測試 135 5.6.2. 螢光顯微鏡與SEM觀察細胞生長 136 5.6.3. 雷射能量分析 139 5.7. 細胞釋放 143 5.7.1. 螢光顯微鏡觀察細胞釋放情形 144 5.7.2. 光柵效應 146 5.7.3. 雷射能量分析 147 5.7.4. Live/Dead細胞活性測試 150 6. 結論 152 參考文獻 154

    [1] Frederic Amant, Philippe Moerman, Patrick Neven, Dirk Timmerman, Erik Van Limbergen, Ignace Vergote, "Endometrial cancer," The Lancet, 2005, 491-505.
    [2] Askin Yildiz, Hakan Yetimalar, Burcu Kasap, Cetin Aydin, Sumeyra Tatar, Ferit Soylu, Fatma Sebnem Yildiz, "Preoperative serum CA 125 level in the prediction of the stage of disease in endometrial carcinoma, " European Journal of Obstetrics & Gynecology and Reproductive Biology, 2012, 191-195.
    [3] Wittekind C, Neid M, "Cancer Invasion and Metastasis," Oncology, 2005, 69(suppl 1):14-16.
    [4] Barradas AM, Terstappen LW, "Towards the Biological Understanding of CTC: Capture Technologies, Definitions and Potential to Create Metastasis," Cancers (Basel), 2013, 1619-42.
    [5] Siyang Zheng, Henry Lin, Jing-Quan Liu, Marija Balic, Ram Datar, Richard J. Cote, Yu-Chong Tai, "Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells," Journal of Chromatography A, 2007, 154-161.
    [6] Seyfried, Thomas N, and Leanne C Huysentruyt, "On the origin of cancer metastasis," Critical reviews in oncogenesis, 2013, 18(1-2):43-73.
    [7] Javier Mariscal, Patricia Fernandez-Puente, Valentina Calamia, Alicia Abalo, Maria Santacana, Xavier Matias-Guiu,et al, "Proteomic Characterization of Epithelial-Like Extracellular Vesicles in Advanced Endometrial Cancer," Journal of Proteome Research, 2019, 1043-1053.
    [8] Hofman V, Ilie M, Long E, Guibert N, Selva E, Washetine K, et al, "Detection of circulating tumor cells from lung cancer patients in the era of targeted therapy: promises, drawbacks and pitfalls," Curr Mol Med, 2014, 14(4):440-56.
    [9] Jason J. Christiansen, Ayyappan K. Rajasekaran, "Reassessing Epithelial to Mesenchymal Transition as a Prerequisite for Carcinoma Invasion and Metastasis," American Association for Cancer Research, 2006, 8319-8326.
    [10] Tayoun T, Faugeroux V, Oulhen M, Aberlenc A, Pawlikowska P, Farace F, "CTC-Derived Models: A Window into the Seeding Capacity of Circulating Tumor Cells (CTCs)," Cells, 2019, 8(10):1145.
    [11] Chaffer CL, Weinberg RA, "A perspective on cancer cell metastasis, Science, 2011, 331(6024):1,559–64.
    [12] Paterlini-Brechot P, Benali NL, "Circulating tumor cells (CTC) detection: clinical impact and future directions," Cancer Lett, 2007, 253(2):180-204.
    [13] Umer M, Vaidyanathan R, Nguyen NT, Shiddiky MJA, "Circulating tumor microemboli: Progress in molecular understanding and enrichment technologies," Biotechnol Adv, 2018, 36(4):1367-1389.
    [14] Baeuerle PA, Gires O, "EpCAM (CD326) finding its role in cancer," Br J Cancer, 2007, 96(3):417-23.
    [15] M Herlyn, Z Steplewski, D Herlyn, H Koprowski, "Colorectal carcinoma-specific antigen: detection by means of monoclonal antibodies," Proceedings of the National Academy of Sciences, 1979, 76(3):1438-1442.
    [16] Philip T.H Went, Alessandro Lugli, Sandra Meier, Marcel Bundi, Martina Mirlacher, Guido Sauter, et al, "Frequent EpCam protein expression in human carcinomas," Human Pathology, 2004, 122-128.
    [17] De Wit S, van Dalum G, Lenferink AT, Tibbe AG, Hiltermann TJ, Groen HJ, et al, "The detection of EpCAM(+) and EpCAM(-) circulating tumor cells," Sci Rep, 2015, 5:12270.
    [18] Mohid S. Khan, Theodora Tsigani, Mohammed Rashid, Jeremy S. Rabouhans, Dominic Yu, Tu Vinh Luong, Martyn Caplin, Tim Meyer, "Circulating Tumor Cells and EpCAM Expression in Neuroendocrine Tumors," Clin Cancer Res, 2011, 337-345.
    [19] Carlo Patriarca, Roberto Maria Macchi, Anja K. Marschner, Hakan Mellstedt, "Epithelial cell adhesion molecule expression (CD326) in cancer: A short review," Cancer Treatment Reviews, 2012, 68-75.
    [20] M. Yu, S. Stott, M. Toner, S. Maheswaran, and D. A. Haber, "Circulating tumor cells: approaches to isolation and characterization," The Journal of cell biology, 2011, vol. 192, no. 3, pp. 373-382.
    [21] Hyeun Joong Yoon, Molly Kozminsky, Sunitha Nagrath, "Emerging Role of Nanomaterials in Circulating Tumor Cell Isolation and Analysis," ACS Nano, 2014, 8(3):1995-2017.
    [22] Sun W, Jia C, Huang T, Sheng W, Li G, Zhang H, et al. High-Performance Size-Based Microdevice for the Detection Of Circulating Tumor Cells from Peripheral Blood in Rectal Cancer Patients. PloS one 2013;8:e75865.
    [23] Lianidou ES, Markou A. Circulating tumor cells in breast cancer: detection systems, molecular characterization, and future challenges. Clinical chemistry 2011;57:1242-55.
    [24] Chang ZM, Zhou H, Yang C, Zhang R, You Q, Yan R, et al, "Biomimetic immunomagnetic gold hybrid nanoparticles coupled with inductively coupled plasma mass spectrometry for the detection of circulating tumor cells," J Mater Chem B, 2020, 8(23):5019-5025.
    [25] Yoon HJ, Shanker A, Wang Y, Kozminsky M, Jin Q, Palanisamy N, et al, "Tunable Thermal-Sensitive Polymer-Graphene Oxide Composite for Efficient Capture and Release of Viable Circulating Tumor Cells," Adv Mater, 2016, 28(24):4891-7.
    [26] Hongmei Chen1, Zhichao Zhang, Hailian Liu1, Changming Lin, Bin Wang, "Hybrid magnetic and deformability based isolation of circulating tumor cells using microfluidics," AIP Advances 9, 2019, 025023.
    [27] Wang S., Wang H., Jiao J., Chen K.-J., Owens G., Kamei K.-i., et al, "Three-Dimensional Nanostructured Substrates toward Efficient Capture of Circulating Tumor Cells," Angewandte Chemie International Edition, 2009, 48: 8970-8973.
    [28] Kayla Duval, Hannah Grover, Li-Hsin Han, Yongchao Mou, Adrian F.Pegoraro, Jeffery Fredberg, Zi Chen, "Modeling Physiological Events in 2D vs. 3D Cell Culture," Physiology, 2016, Vol. 32, No. 4.
    [29] Roskelley CD, Bissell MJ, "Dynamic reciprocity revisited: a continuous, bidirectional flow of information between cells and the extracellular matrix regulates mammary epithelial cell function," Biochem Cell Biol, 1995, 73(7–8):391–7.
    [30] Cukierman E, Pankov R, Stevens DR, Yamada KM, "Taking cell–matrix adhesions to the third dimension," Science, 2001, 294(5547):1708–12.
    [31] Wan Y., Mahmood M. A. I., Li N., Allen P. B., Kim Y.-t., Bachoo R., et al, "Nanotextured Substrates with Immobilized Aptamers for Cancer Cell Isolation and Cytology," Cancer, 2012, 118, 1145–1154.
    [32] Shunqiang Wang, Yuan Wan, Yaling Liu, "Effects of nanopillar array diameter and spacing on cancer cell capture and cell behaviors," Nanoscale, 2014, 6:12482–12489 .
    [33] I.J. Haug, K.I. Draget, "5 - Gelatin," Handbook of Food Proteins, 2011, 92-115.
    [34] Zhang Y., Ouyang H., Lim C.T., Ramakrishna S., Huang, Z.-M., "Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds," J. Biomed. Mater. Res., 2005, 72B: 156-165.
    [35] Su K., Wang C., "Recent advances in the use of gelatin in biomedical research," Biotechnol Lett, 2015, 37:2139–2145.
    [36] A. J. Kuijpers, P. B. van Wachem, M. J. A. van Luyn, J. A. Plantinga, G. H. M. Engbers, J. Krijgsveld, et al, "In vivo compatibility and degradation of crosslinked gelatin gels incorporated in knitted Dacron," J. Biomed. Mater, 2000, 51:136-145.
    [37] Zhou J., Ellis A.V., Voelcker N.H, "Recent developments in PDMS surface modification for microfluidic devices," ELECTROPHORESIS, 2010, 31:2-16.
    [38] H. Xiao, "半導體製程技術導論,羅正忠和張鼎張譯," ed:二版,臺灣培生教育出版,臺北市,民國九十三年, 2007.
    [39] Yu Hongbin, Zhou Guangya, Chau Fook Siong, Wang Shouhua and Lee Feiwen, "Novel polydimethylsiloxane (PDMS) based microchannel fabrication method for lab-on-a-chip application," Sensors and Actuators B: Chemical, 2009, 754-761
    [40] Chen-Chieh Yu, Hsuen-Li Chen, "Nanoimprint technology for patterning functional materials and its applications," Microelectronic Engineering,2015,98-119.
    [41] Lee J., Lee M, "Improved light harvest in diffraction grating-embedded TiO2 nanoparticle film," Appl. Phys. A, 2017, 123:737.
    [42] Chang C.-L., Acharya,G., Savran C. A, "In situ assembled diffraction grating for biomolecular detection," Appl. Phys. Lett, 2007, 90:233901.
    [43] Miquel Avella-Oliver, Vicente Ferrando, Juan A. Monsoriu, Rosa Puchades, Angel Maquieira, "A label-free diffraction-based sensing displacement immunosensor to quantify low molecular weight organic compounds," Analytica Chimica Acta, 2018, 173-179.
    [44] Ghanashyam Acharya, Chun-Li Chang, and Cagri Savran, "An Optical Biosensor for Rapid and Label-Free Detection of Cells," Journal of the American Chemical Society, 2006, 128 (12).
    [45] Ghanashyam Acharya, Chun-Li Chang, Derek D. Doorneweerd, Erina Vlashi, Walter A. Henne, Lynn C Hartmann, et al, "Immunomagnetic Diffractometry for Detection of Diagnostic Serum Markers," Journal of the American Chemical Society, 2007, 129 (51).
    [46] W.C. Bigelow, D.L. Pickett, W.A. Zisman, "Film adsorbed from solotion in non-polar liquids," Journal of Colloid Science, 1946, vol. 1, pp. 513-538.
    [47] R. G. Nuzzo and D. L. Allara, "Adsorption of bifunctional organic disulfides on gold surfaces," Journal of the American Chemical Society, 1983, vol. 105, pp. 4481-4483.
    [48] Colin D. Bain, E. Barry Troughton, Yu Tai Tao, Joseph Evall, George M. Whitesides, Ralph G. Nuzzo, "Formation of monolayer films by the spontaneous assembly of organic thiols from solution onto gold," Journal of the American Chemical Society, 1989, 111(1):321-335.
    [49] Jacques Cousty, Alexandr Marchenko, "Substrate-induced freezing of alkane monolayers adsorbed on Au(111) dependant on the alkane/gold misfit," Surface Science, 2002, 128-136.
    [50] Kien Cuong Nguyen, "Quantitative analysis of COOH-terminated alkanethiol SAMs on gold nanoparticle surfaces," Adv. Nat. Sci: Nanosci. Nanotechnol, 2012, 3:045008.
    [51] John A. Howarter, Jeffrey P. Youngblood, "Optimization of Silica Silanization by 3-Aminopropyltriethoxysilane," Langmuir, 2006, vol. 22, pp. 11142-11147.
    [52] A Simon, T Cohen-Bouhacina, M C Porté, J P Aimé, C Baquey, "Study of two grafting methods for obtaining a 3-aminopropyltriethoxysilane monolayer on silica surface," Journal of colloid and interface science, 2002, 251.2: 278-283.
    [53] Knani D, Foox M, Zilberman M., "Simulation of the bioadhesive gelatin-alginate conjugate loaded with antibiotic drugs," Polym Adv Technol, 2019, 30:519–528.
    [54] Hamid Goodarzi, Khosrow Jadidi, Samiramis Pourmotabed, Esmaeel Sharifi, Hossein Aghamollaei, "Preparation and in vitro characterization of cross-linked collagen–gelatin hydrogel using EDC/NHS for corneal tissue engineering applications," International Journal of Biological Macromolecules, 2019, 620-632.
    [55] Cuie Wang, Qin Yan, Hong-Bo Liu, Xiao-Hui Zhou, Shou-Jun Xiao, "Different EDC/NHS Activation Mechanisms between PAA and PMAA Brushes and the Following Amidation Reactions," Langmuir, 2011, 12058–12068.
    [56] Akerström B, Brodin T, Reis K, Björck L, "Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies," J Immunol, 1985, 135(4):2589-92.
    [57] Asta Makaraviciute, Almira Ramanaviciene, "Site-directed antibody immobilization techniques for immunosensors," Biosensors and Bioelectronics, 2013, 460-471.
    [58] Welch NG, Scoble JA, Muir BW, Pigram PJ, "Orientation and characterization of immobilized antibodies for improved immunoassays (Review)," Biointerphases, 2017, 12(2):02D301.
    [59] Han-Yu Peng, Wei Wang, Fuhua Gao, Shuo Lin, Lu-Yue Liu, Xing-Qun Pu, et al, "Ultrasensitive diffraction gratings based on smart hydrogels for highly selective and rapid detection of trace heavy metal ions",J. Mater. Chem. C, 2018, 11356-1136.

    無法下載圖示 全文公開日期 2031/08/18 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)
    全文公開日期 本全文未授權公開 (國家圖書館:臺灣博碩士論文系統)
    QR CODE