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研究生:吳信輝
研究生(外文):Hsin-Hui Wu
論文名稱:開放式循環腫瘤細胞濃縮收集晶片之研製
論文名稱(外文):Study On the Open-Top Microreservoir for CTCs Isolation
指導教授:任春平
指導教授(外文):Chun-Ping Jen
口試委員:林派臣江佩如楊奕玲
口試委員(外文):Pai-Chen LinPei-Ju Chiang
口試日期:2013-06-25
學位類別:碩士
校院名稱:國立中正大學
系所名稱:機械工程學系暨研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:76
中文關鍵詞:微流體晶片細胞分離循環腫瘤細胞介電泳稀少細胞
外文關鍵詞:microfluidic chipcellular concentrationcirculating tumor cellsdielectrophoresisrare cells
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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重要器官,如能早期檢測體內血液之稀少癌細胞,可降低死亡率。而進行循環腫瘤細胞檢測,須先將癌細胞從血液中分離或濃縮,而過去濃縮細胞方式需要使用大型儀器,且在大樣本量下進行,不利於個人醫療檢測。本研究目的主要是在不破壞細胞之前提下且無需外加微幫浦及電源訊號產生器,製作手持式裝置針對全血樣本進行分離癌細胞與紅血球細胞,利用稀少細胞與紅血球不同的臨界電壓的驅動特性,進行濃縮與分離。因此設計及製作一大面積圓形平面電極之細胞濃縮晶片,以步進式電場切換的動作,利用兩相鄰電極上施加的電壓使兩電極間距產生強電場區域,正介電泳會使細胞朝向位於兩電極間強電場的方向移動,以達到同時驅動及濃縮細胞之目的。於研究中也將使用商用計算流體力學軟體CFD-ACE+來模擬電場強度之變化趨勢,於實驗中先以子宮頸癌細胞進行實驗測試,分別施加15 V向外及10 V向內之步進電場且頻率為1 MHz,成功將紅血球細胞分離於外圈電極,而子宮頸癌細胞則濃縮至內圈電極部分。此外,本研究亦加入紅血球裂解液,輔助進行乳癌循環腫瘤細胞之濃縮,只需要8 V向內之步進電場即可達到濃縮乳癌循環腫瘤細胞之目的。
The detection of rare cells, such as circulating tumor cells (CTCs), circulating fetal cells, and stem cells, is important for medical diagnostics and characterization. The present study develops a handheld electric module which provides stepping electric fields for dielectrophoresis (DEP) to selectively concentrate CTCs from red blood cells, making it low-cost and automated. The circular microelectrodes were designed to generate a stepping electric field by a handheld electric module with a voltage-frequency converter and an operational amplifier. The transparent electrodes were fabricated by patterning the indium tin oxide (ITO) coated glass to observe the experimental results apparently. The large volume open-top cistern for 100 μL was employed to replace conventional microchannel. Positive dielectrophoretic cells were guided toward the center of the microchamber due to the movement of the high-electric-field region. The magnitude of the DEP force acting on HeLa cells is about seven-fold that acting on red blood cells under a given electric field distribution, making it possible to separate HeLa cells from normal blood cells without any hydrodynamic force. The concentrations of HeLa cells for 500 cells/mL were selectively concentrated. The voltages for outward and inward stepping electric field were 15 Vpp and 10 Vpp respectively at a frequency of 1 MHz. Besides, the RBC lysis buffer was employed to concentrate the CTCs of MCF-7 in this study. Experiment results demonstrate the MCF-7 cells also can be concentrated by the open-top concentrate chip.
目錄
摘要 I
Abstract II
致謝 IV
目錄 V
圖目錄 VIII
表目錄 XI
第一章 緒論 1
1-1 前言 1
1-2 既有之稀少細胞濃縮技術 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 分子生物學 5
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 9
1-3-4 電場力 10
1-4 研究目的 15
第二章 基本理論 17
2-1 介電泳理論 17
2-2 流場與電位能理論 24
2-3 各式介電泳力操控生物粒子之文獻探討 25
2-3-1 正介電泳力 25
2-3-2 負介電泳力 27
2-3-3 絕緣式介電泳 28
2-3-4 旅波式介電泳 30
2-4 操控介電泳力附加影響因子 31
2-4-1 交流電滲流 31
2-4-2 電泳力的影響 32
2-4-3 電場對細胞的傷害及其他影響 32
第三章 研究方法 34
3-1 儀器設備與藥品 34
3-2 細胞培養 37
3-3 細胞樣本配製 38_Toc358977250
3-4 晶片設計 40
3-5 晶片製作 43
3-6 交流電源供應及控制電路 46
第四章 結果與討論 49
4-1 圓型電極濃縮稀少細胞實驗結果 49
4-2 改良式電路操控進行稀少癌細胞與紅血球細胞分離/濃縮之結果 53
4-3 利用紅血球裂解液輔助稀少細胞濃縮實驗結果 56
第五章 結論 64
第六章 未來發展與建議 67
參考文獻 68


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