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研究生:曾柏儒
研究生(外文):Bo-Ru Tzeng
論文名稱:利用微流道電極晶片裝置捕捉支氣管上皮細胞及阻抗量測之實驗研究
論文名稱(外文):Capture of bronchial epithelial cell for impedance measurements in microelectrode device
指導教授:陳志敏陳志敏引用關係吳嘉哲
指導教授(外文):Jerry M. ChenChia-Che Wu
口試委員:郭正雄陳明志廖恩慈
口試委員(外文):Cheng-Hsiung KuoMing-Jyh CHERNEn-Chih Liao
口試日期:2016-07-21
學位類別:碩士
校院名稱:國立中興大學
系所名稱:機械工程學系所
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:2016
畢業學年度:104
語文別:中文
論文頁數:67
中文關鍵詞:微流道阻抗人類支氣管上皮細胞液動力過濾尼古丁
外文關鍵詞:microfluidicsimpedancehuman bronchial epithelial cellhydrodynamic filtrationNNK
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本研究利用液動力分離原理於微流道裝置捕捉單一細胞,並於捕捉槽內設置微電極以對細胞進行阻抗量測與分析。本實驗利用黃光微影技術製作微流道母模,再翻模於聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)流道結構,並以氧氣電漿將流道與量測阻抗之微電極基板對準接合。我們對人類支氣管上皮細胞(BEAS-2B)投入菸草致癌物(4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK),將其培養24小時後進行阻抗量測,頻率為0.1~100 kHz,電壓0.5 V,分析細胞投入NNK致癌物前後之阻抗變化。量測後發現投致癌物後細胞之阻抗值大小會高於未投致癌物細胞之阻抗大小值,對以投致癌物細胞而言其阻抗值大小會隨著添加致癌物濃度增加而升高。

In this study, microchannels composed of a main channel and several branch channels were employed to capture single human bronchial epithelial cells (BEAS-2B) cells using the hydrodynamic filtration method for investigation of tobacco-specific carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) effects. The effects of tobaccoc carcinogen NNK on the cell were investigated by impedance measurements through the microelectrodes fabricated on each of the cell trapping chambers positioned between the main and branch channels. The lithography techniques were used to manufacture the microchannel mold and the microelectrodes on a glass substrate. Then the oxygen plasma was utilized to bond the microchannel structure, which was made of Polydimethylsiloxane (PDMS) from the replica molding, to the substrate with microelectrodes. The BEAS-2B cells were treated for 24 hours with the tobacco carcinogen NNK at concentrations of 7 and 500 µM before the impedance measurements. Single BEAS-2B cells were successfully captured in the present microfluidic device for impedance a frequency range of 100 Hz to 100 kHz at a supply voltage of 0.5V. The impedance measured for the BEAS-2B cell treated with NNK is higher than that of the BEAS-2B cell without NNK treatment by one order of magnitude. It is found that the magnitude of impedance of BEAS-2B cell increases with the higher NNK concentration. The phase of the impedance also shows significant difference in distributions for the BEAS-2B cell treated with or without NNK.

目錄
摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vii
第一章 前言 1
1.1研究動機 1
1.2 文獻回顧 2
1.2.1細胞分離與捕捉方法 3
1.2.2微電極設計與量測 6
1.3研究目的 8
1.4論文架構 9
第二章 理論分析與結構設計 10
2.1流道運作原理 10
2.1.1捕捉原理 10
2.2設計流程 12
2.3流道參數設計 12
2.3.1分支流量堆導 12
2.3.2分支流阻推導 13
2.3.3流道分支長度 14
2.3.4微流道結構尺寸 16
2.4電極設計 17
第三章 微流道電極晶片製作與相關知識 19
3.1微流道製作 19
3.1.1微流道母模微影製程與參數 19
3.1.2軟微影技術 21
3.2微電極晶片製作 23
3.2.1微電極母模微影製程及參數 23
3.2.2微電極金屬蒸鍍與Lift-off技術 24
3.3微流道與微電極晶片對準接合 28
3.4細胞培養條件與環境 30
3.4.1影響細胞生長之因素 30
3.4.2細胞介紹 30
3.4.3無菌操作台使用流程 31
3.4.4培養液(RPMI 1640)配製 31
3.4.5細胞解凍 32
3.4.6細胞培養與繼代 32
3.4.7細胞凍存 33
3.4.8細胞計數 34
3.4.9 NNK溶液配置與細胞投入致癌藥培養 35
3.5實驗步驟 37
第四章 實驗結果與討論 39
4.1 BEAS-2B細胞捕捉 39
4.2細胞於不同頻率下之響應 42
4.3阻抗量測儀器校準與補償 43
4.4 BEAS-2B細胞與細胞培養液之量測比較 44
4.5等效電路模型建立與擬合 46
純培養液之等效電路 46
含單一細胞培養液之等效電路 48
4.6 BEAS-2B細胞投入NNK致癌藥劑之量測比較 51
4.6.1不同濃度NNK致癌藥劑溶液阻抗量測 51
4.6.2 BEAS-2B細胞投入500µM與7µM NNK致癌藥劑之量測比較 52
4.6.3 BEAS-2B細胞與投入NNK致癌藥劑BEAS-2B細胞之量測比較 55
4.6.4 實驗誤差範圍 57
第五章 結論與建議 59
5.1結論 59
5.2建議 59
參考文獻 60
附錄(A) 62
人類肺腺癌(A549)之量測阻抗結果 62
附錄(B) 63
實驗設備與材料 63


表目錄
表2.1 微流道結構尺寸 16
表3.1微流道母模製程參數表 22
表3.2微電極製程參數表 27
表4.1等效電路元件擬合參數表 50
表4.2為Medium (RPMI 1640)、BEAS-2B細胞、BEAS-2B w/500 µM NNK細胞與BEAS-2B w/7 µM NNK細胞各段頻率阻值與誤差 58


圖目錄
圖1.1 Yamada and Seki [12] 梳狀流道示意圖 3
圖1.2 Park et al. [13]所製作的流道結構圖與細胞捕捉情形 4
圖1.3 Noguchi et al. [14] 流道與單一細胞抓取示意圖 5
圖1.4 細胞(a)相位;(b)電阻量測結果(Park et al. [17]) 6
圖1.5 (a)電極結構示意圖;(b)細胞貼附量測電極示意圖(Kang et al.[18]) 7
圖1.6 (a)平行式電極與橢圓式電極;(b)細胞位於平行式電極不同位置量測圖;(c)細胞位於橢圓式電極不同位置量測圖(Mondal et al. [20]) 8
圖2.1微流道結構示意圖 10
圖2.2細胞捕捉原理示意圖 11
圖2.3 流道內各部位流量示意圖 13
圖2.4流道分支設計示意圖 15
圖2.5 流道尺寸示意圖 16
圖2.6 電極轉角處示意圖[25] 18
圖3.1微影製程流程圖 21
圖3.2 軟微影製程示意圖 22
圖3.3流道結構影像 22
圖3.4電極母模製作流程 23
圖3.5電極Lift-off製作流程示意圖 25
圖3.6 Lift-off完成後之微電極影像 25
圖3.7正立式顯微鏡下之電極母模,過曝和過顯所造成之結構圖 26
圖3.8 金批覆層不均勻產生之剝離現象,微電極之Lift-off之結果圖26
圖3.9 流道與電極接合示意圖 28
圖3.10流道(PDMS)、電極晶片與對準平台 29
圖3.11 (a)微流道電極晶片(尺寸:5×2.5 cm)與(b)於虛線區為晶片之細部影像 29
圖3.12 BEAS-2B細胞培養(a)物鏡10X貼付狀況 (b)物鏡20X貼付狀況 31
圖3.13添加Trypan-Blue後BEAS-2B細胞於計數盤上之影像 35
圖3.14 (a)NNK致癌物(b)NNK分子化學式(Toronto Research Chemicals) 37
圖3.15細胞捕捉與阻抗量測實驗架構圖 38
圖4.1(a)於物鏡10X第一與第二分支道捕捉到單一BEAS-2B細胞(b)捕捉單一細胞之影像 40
圖4.2 BEAS-2B細胞於第一與第二分支流道順序捕捉影像,當細胞進入捕捉槽後,流阻上升,使流體流往後方流道,因此細胞可依序進入後方捕捉槽 41
圖4.3細胞被擠壓於流道細分支與重複捕捉單一細胞之影像流道重複捕捉單一細胞影像 42
圖4.4 ((a)低頻時,電力線於細胞外部分布圖 (b)高頻時,電力線於細 胞內分布圖(Mernier et al. [26]) 43
圖4.5 校準結果圖(a)開路校準結果 (b)閉路校準結果後 43
圖4.6單一BEAS-2B細胞與培養液量測結果(a)阻抗大小(b)相位大小 45
圖4.7細胞之類比電路模型 46
圖4.8 不含細胞之純培養液之等效電路模型 47
圖4.9培養液實驗數據與等效電路模型擬合結果 48
圖4.10含單一細胞之純培養液之等效電路模型 48
圖4.11含單一細胞實驗數據與等效電路模型擬合結果 49
圖4.12培養液RPMI含(W/)500 µM 、7 µM NNK與培養液RPMI1不含(W/O) NNK量測結果(a)阻抗大小 (b)相位大小 51
圖4.13於捕捉槽捕之單一細胞之影像(a)BEAS-2B (b) BEAS-2B W/500 µM NNK (c) BEAS-2B W/7 µM NNK 53
圖4.14 BEAS-2B細胞與投入500 µM與7 µM NNK藥劑之細胞量測結果(a)阻抗大小(b)相位大小 54
圖4.15 BEAS-2B細胞投入500 µM NNK藥劑之相位斜率變化,於頻率20200 Hz時斜率由正轉負 54
圖4.16 BEAS-2B細胞與投NNK藥劑BEAS-2B細胞之阻抗量測結果(a)阻抗大小(b)相位大小 56
圖A為類肺腺癌細胞(A549)阻抗量測結果(a)阻值大小(b)相位大小 62





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