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系統識別號 U0026-0608201309515100
論文名稱(中文) 應用電性鑑別偵測法於三明治免疫電極檢測晶片之研究
論文名稱(英文) Study of Sandwich-Immunoassay Detection Chip Based on Impedance Measurement
校院名稱 成功大學
系所名稱(中) 工程科學系碩博士班
系所名稱(英) Department of Engineering Science
學年度 101
學期 2
出版年 102
研究生(中文) 蘇冠逢
研究生(英文) Kuan-Feng Su
學號 n96004418
學位類別 碩士
語文別 中文
論文頁數 167頁
口試委員 指導教授-林裕城
口試委員-林弘萍
口試委員-羅錦興
中文關鍵字 電性鑑別  癌胚抗原(CEA)  三明治免疫反應  金奈米粒子  表面化學修飾  微流道 
英文關鍵字 electro-microchip  carcinoembryonic  sandwich-immunoassay  gold nanoparticles  surface chemical modification  microchannel 
學科別分類
中文摘要 本研究將電性鑑別法應用於癌胚抗原(Carcinoembryonic, CEA)三明治免疫反應之檢測,結合微阻抗晶片與結合金奈米粒子抗體標定技術,以助於定性偵側待測檢體之免疫反應並定量阻抗量測分析結果鑑別。實驗中利用電感、電容、電阻量測儀(LCR meter)偵測阻抗晶片之電性訊號,藉由阻抗變化來判斷免疫反應的情況,並建立三明治免疫分析系統。本實驗分為微電極晶片及微流道電極晶片;微電極晶片實驗中成功利用表面化學修飾方法增強抗體與晶片之接合力,並將免疫檢測之檢體使用量降至25 μL及三明治免疫反應偵測時間減少至10分鐘,檢測極限值為1 ng/mL;微流道電極晶片嘗試改量微電極晶片,不經由化學修飾方法直接將微流道與晶片做結合,其檢體量可降至10 μL,反應時間降至8分鐘,檢測極限降至0.1 ng/mL;另外微電極晶片及微流道電極晶片皆在低頻率時(100 Hz)檢測到較高鑑別度之阻抗電性訊號。本研究利用表面修飾方法、阻抗分析晶片、微流道的結合與金奈米粒子免疫鑑別技術,達到快速偵測、低檢體使用量、低成本的目的,提供了醫療與免疫分析一個全新的方向與思維。
英文摘要 This study, a novel micro-electrode biochip for immunoassay was applied to detect sandwich-immunoassay of carcinoembryonic, a gold nanoparticles (AuNPs) as a label of antibody to enhance immunoassay impedance quantitative analysis detection. Compared with traditional enzyme-linked immunosorbent assay (ELISA), we utilized the LCR meter instrument to detect the electric signal on immune-impedance chip. By detecting the impedance changes because of sandwich-immunoassay reaction, a sandwich-immunoassay detection system was established. In this study, the experiment was divided into micro-electrode chip and microchannel electrode chip. Firstly, in micro-electrode chip, we used surface chemical modification method to modify the surface of immune-impedance chip. Then using the contact angle measurement instrument and the fluorescent labeled antibody method verify the results of chip modification. Only used 25 μL of samples that immune detection can be work in the immune detection system, the total time of the immunoassay was about 10 minutes, and the minimum signal detection was 1 ng/mL of carcinoembryonic. Secondly, we combined micro-electrode chip with microchannel into microchannel electrode chip and not by using surface chemical modification. It only used 10 μL of samples in the immune detection system. The total detection time of the immunoassay was about 8 minutes, and the minimum signal detection of microchannel electrode chip was 0.1 ng/mL of carcinoembryonic. Additionally, micro-electrode chip and microchannel electrode chip have the best distinction under 100 Hz in this detection system, according to the relationship between the impedance and carcinoembryonic concentration. In this study, combined impedance changing detection and nanogold labeling technique in sandwich-immunoassay was a novel notion. Impedance measurement was performed for quantitative and qualitative analysis in the aim of decreasing detection time and lowering assay cost. It provided a new immunoassay method for drug-detection and medical analysis sciene.
論文目次 目錄

中文摘要 I
Abstract II
誌謝 IV
目錄 V
表目錄 X
圖目錄 XII
第一章緒論 1
1-1 研究背景 1
1-2 癌症的簡介 3
1-2-1 癌症的發展史 3
1-2-2 腫瘤標記物 6
1-2-3 腫瘤指數與用途 6
1-2-4 癌胚抗原 8
1-3 免疫分析法 9
1-3-1 免疫分析基本理論 10
1-3-2 抗體與抗原定義 10
1-3-3 抗原與抗體的結合力 12
1-3-4 免疫分析檢測種類 13
1-3-5 免疫分析偵測方法 14
1-3-6 傳統酵素聯結免疫吸附分析法-ELISA 17
1-4 文獻回顧 20
1-4-1 免疫分析法之發展 20
1-5 自組裝單層薄膜 30
1-6 研究動機與目的 33
1-7 研究架構 34
第二章阻抗式微電極晶片及微流道整合電極晶片設計與製作 35
2-1 電極晶片之等效電路模型 35
2-2 電極晶片和微流道光罩設計 36
2-3 晶片設計結構 38
2-3-1 阻抗式免疫檢測於微電極晶片結構 38
2-3-2 阻抗式免疫檢測於微流道電極晶片結構 40
2-4 電極晶片製程 43
2-4-1 玻璃基材清洗 44
2-4-2 微影(Lithography) 45
2-4-3 電極蝕刻 50
2-5 免疫電極晶片反應區製作 53
2-6 免疫微流道電極晶片反應區製作 57
2-6-1 母模製作 57
2-6-2 PDMS灌注成形技術及翻製流程 64
2-7 晶片接合與組裝 66
第三章實驗與研究方法 69
3-1 實驗儀器與設備 69
3-1-1 接觸角量測儀 69
3-1-2 螢光光學顯微鏡偵測系統 70
3-1-3 真空抽氣系統 72
3-1-4 阻抗免疫偵測系統之建立 72
3-2 實驗藥品 75
3-3 實驗方法 77
3-3-1 免疫微電極晶片表面修飾 78
3-3-2 三明治型免疫偵測實驗 82
3-3-2-1 免疫分析檢測模型 83
3-3-2-2 抗體與免疫晶片接合 85
3-3-2-3 三明治免疫阻抗分析實驗-CEA-Ab濃度測試 86
3-3-2-4 三明治免疫阻抗分析實驗-檢體量測試於微電極晶片 87
3-3-2-5 體積流量測試於微流道電極晶片 87
3-3-2-6 三明治免疫阻抗分析實驗-反應時間 88
3-3-2-7 三明治免疫阻抗分析實驗-頻率測試 89
3-3-2-8 三明治免疫阻抗分析-CEA檢測 90
3-3-2-9 三明治免疫金膜之阻抗分析-CEA檢測 93

第四章結果與討論 95
4-1 微電極晶片實驗 95
4-1-1 晶片表面改質之接觸角驗測量測 95
4-1-2 晶片表面改質之螢光驗測量測 97
4-1-3 三明治免疫反應之CEA-Ab濃度測試 99
4-1-4 三明治免疫反應之反應時間測試 102
4-1-5 三明治免疫反應之檢體檢測量測試 104
4-1-6 三明治免疫反應之頻率測試 106
4-1-7 CEA癌胚抗原之阻抗分析 108
4-1-7-1 CEA-GC稀釋倍率影響免疫反應測試 108
4-1-7-2 稀釋10倍CEA-GC之CEA癌胚抗原免疫反應 111
4-1-7-3 稀釋30倍CEA-GC之CEA癌胚抗原免疫反應 113
4-1-7-4 稀釋70倍CEA-GC之CEA癌胚抗原免疫反應 115
4-1-8 CEA-GC稀釋倍數與CEA癌胚抗原偵測結果比較 117
4-2 微流道電極晶片實驗 119
4-2-1 三明治免疫反應之CEA-Ab濃度測試 119
4-2-2 三明治免疫之反應時間測試 123
4-2-3 微流道之體積流量實驗 125
4-2-4 三明治免疫反應之頻率測試 126
4-2-5 CEA癌胚抗原之阻抗分析 128
4-2-5-1 CEA-GC稀釋倍率影響免疫反應測試 128
4-2-5-2 稀釋10~30倍之CEA癌胚抗原之免疫反應 131
4-2-5-3 稀釋40~50倍之CEA癌胚抗原之免疫反應 134
4-2-5-4 稀釋70倍之CEA癌胚抗原之免疫反應 137
4-2-6 驗證微流道電極晶片之稀釋倍數實驗 139
4-2-7 CEA-GC稀釋倍數與CEA癌胚抗原偵測結果比較 143
4-2-8 CEA-GC稀釋之金膜與CEA癌胚抗原偵測結果比較 146
第五章結論與未來展望 149
5-1 結論 149
5-2 未來展望 154
參考文獻 155
自述 167

表目錄

表2-1光源(汞燈)強度與曝光時間參數表 49
表3-1 E4980A LCR meter 規格表 74
表3-2微電極晶片檢測步驟 91
表3-3微流道電極晶片檢測步驟 92
表4-1 CEA-Ab濃度測試實驗之檢體濃度參數表 101
表4-2微電極晶片反應時間測試實驗之參數表 103
表4-3微電極晶片檢體量測試實驗之檢體濃度參數表 105
表4-4微電極晶片頻率測試實驗之檢體濃度參數表 107
表4-5 CEA-GC稀釋倍率免疫反應測試之檢體濃度參數表 109
表4-6不同稀釋倍率之CEA-GC之阻抗電性訊號比較 110
表4-7 CEA-GC稀釋10倍之CEA之檢體濃度參數表 112
表4-8 CEA-GC稀釋30倍之CEA之檢體濃度參數表 114
表4-9 CEA-GC稀釋70倍之CEA之檢體濃度參數表 116
表4-10不同稀釋倍數之CEA-GC偵測機制比較 118
表4-11 CEA-Ab濃度測試實驗之檢體濃度參數表 121
表4-12微流道電極晶片反應時間測試實驗之參數表 124
表4-13微流道電極晶片頻率測試實驗之檢體濃度參數表 127
表4-14 CEA-GC稀釋倍率免疫反應測試之檢體濃度參數表 129
表4-15不同稀釋倍率之CEA-GC之阻抗電性訊號比較 130
表4-16 CEA-GC稀釋10~30倍之CEA之檢體濃度參數表 132
表4-17 CEA-GC稀釋40~50倍之CEA之檢體濃度參數表 135
表4-18 CEA-GC稀釋70倍之CEA之檢體濃度參數表 138
表4-19 CEA-GC稀釋10~50倍之CEA之檢體濃度參數表 140
表4-20未用PBS清洗之不同稀釋倍數之CEA-GC偵測機制比較 145
表4-21用PBS清洗之不同稀釋倍數之CEA-GC偵測機制比較 145
表4-22 CEA-GC稀釋10~40倍金膜之CEA之檢體濃度參數表 147


圖目錄

圖1-1生物感測分析原理 9
圖1-2免疫蛋白(IgG)的結構 12
圖1-3免疫分析檢測之四種方式:(a)直接型,(b)三明治型,(c)競爭型,(c)間接型 13
圖1-4放射免疫分析所採用的競爭性免疫結合反應示意圖 15
圖1-5酵素連結免疫吸附測定法的原理 16
圖1-6傳統ELISA檢測流程示意圖:三明治型式,利用酵素為標記物搭配特定基質,來使酵素催化基質以進行螢光反應分析 18
圖1-7利用SPR技術進而分析微量相位變化與反射行為於病原菌的檢測模型 21
圖1-8利用AFM偵測平台結合蛋白質自組裝(Self-assembly)方法分析表面型態於抗體與抗原之接合狀況生物感測分析原理 22
圖1-9利用QCM偵測平台,根據其上載體特性轉換成頻率特性,所量測的頻率變化反應了接觸介面的特性,因此可運用於免疫感測器(Immunosensors)之檢測上 22
圖1-10利用標定物技術搭配表面增強拉曼散射方法,對已具有標記不同物質之膠體金來進行拉曼偵測,並可同時檢測分析不同物質 23
圖1-11 酵素標定物之免疫分析法:(a)三明治免疫分析之標準流程圖,(b)微流道應用於九十六孔盤之圖 24
圖1-12利用螢光物質奈米量子點(Quantum dot)為標定物,並運用螢光亮度再搭配TEM量測系統,來探討免疫分析後之結果 25
圖1-13利用電化學發光法檢測以標記之蛋白質或DNA 25
圖1-14增強免疫偵測之研究流程 27
圖1-15電免疫感測微小晶片之示意圖:(a)免疫電極檢測晶片分解示意圖,(b)電免疫檢測模型示意圖 29
圖1-16定量橫向流動式免疫傳感器之檢測模型示意圖 30
圖1-17自組裝單層模示意圖 31
圖1-18自組裝單層模(GPMS)鍵結步驟 32
圖1-19實驗研究架構 34
圖2-1濕式阻抗量測之等效電路模型 36
圖2-2免疫電極阻抗量測晶片光罩設計尺寸圖:(a)8組免疫電極阻抗量測晶片於ITO玻片擺放設計圖,(b)檢測區電極尺寸示意圖 37
圖2-3微流道光罩設計圖:左圖為全區域設計圖,右圖為放大圖 38
圖2-4阻抗式免疫檢測微電極晶片示意圖:(a)左:結構分解圖,右:結構組裝圖, (b)結構上視圖 40
圖2-5阻抗式免疫檢測微流道電極晶片示意圖:(a)上:結構分解圖,下:結構組裝圖, (b)結構上視圖 42
圖2-6電極晶片製作流程圖 43
圖2-7底切效應示意圖 50
圖2-8電極檢測晶片實體圖 52
圖2-9 PDMS主劑、固化劑與混合固化後之分子結構式 54
圖2-10 PDMS固化溫度與固化時間關係圖 55
圖2-11微流體晶片製作流程 57
圖2-12 THB-151N微結構製程示意圖,製程各步驟依序為清洗基材、光阻塗佈、曝光、顯影,完成後便可將該凸模結構應用於PDMS翻模製作上 58
圖2-13 THB-151N光阻塗佈轉速與厚度的關係圖 60
圖2-14 THB-151N微結構母模 63
圖2-15 PDMS翻模流程示意圖,清洗THB-151N母模後,再將配製好的PDMS溶液澆注到母模中,經烘烤後即可製得PDMS凹模晶片 64
圖2-16氧電漿接合PDMS原理:(a)常溫下PDMS表面帶CH3官能基,(b)經氧電漿活化後表面可接上OH官能基,(c)給予外力擠壓後,PDMS間將以共價鍵結力緊密接合 66
圖2-17氧電漿接合PDMS晶片:(a) 氧電漿轟擊機操作平台,(b) 氧電漿處理平台,將欲活化接合之晶片面朝上擺放,即可進行氧電漿處理過程 67
圖2-18組裝完成之電極晶片:(a)微電極晶片實體圖,(b)微流道電極晶片實體圖 68
圖3-1影像式接觸角量測儀 70
圖3-2螢光光學顯微鏡與影像擷取系統,利用數位即時影像來作為拍攝實驗結果的依據,並利用軟體將CCD所擷取出來的影像做存檔紀錄 71
圖3-3真空抽氣系統 72
圖3-4阻抗免疫分析偵測平台架構 73
圖3-5微電極晶片免疫阻抗分析示意圖 77
圖3-6微流道電極晶片免疫阻抗分析示意圖 78
圖3-7晶片表面修飾接合抗體技術示意圖 79
圖3-8微電極晶片改質處理示意圖 81
圖3-9三明治免疫偵測機制示意圖 84
圖3-10微流道電極晶片結合金膜示意圖:(a)整體架構圖, (b)上視圖 94
圖4-1微電極晶片改質處理後接觸角量測圖:(a)清洗並未改質,(b)經氧電漿處理,(c)浸泡過Toluene,(d)浸泡過GPTS-Toluene 96
圖4-2螢光標記抗體接合微電極晶片之螢光反應偵測:(a)未改質微電極晶片之螢光免疫反應,(b)已經由化學改質修飾的微電極晶片之螢光免疫反應 98
圖4-3不同濃度CEA-Ab對應三種不同CEA濃度之免疫偵測阻抗分析圖 101
圖4-4濃度為200 ng/mL之CEA抗原對應Negative Control三明治免疫反應時間阻抗分析圖 103
圖4-5濃度200 ng/mL之CEA檢測檢體量測試實驗阻抗分析結果圖 105
圖4-6 CEA癌胚抗原之三明治免疫反應與阻抗分析之頻率比較圖 107
圖4-7 Negative Control與不同稀釋倍率之CEA-GC與CEA 200 ng/mL在固定頻率100 Hz下進行三明治免疫反應偵測之阻抗電性分析圖 110
圖4-8在頻率100 Hz下,利用稀釋10倍之CEA-GC偵測不同濃度的CEA阻抗分析圖 112
圖4-9在頻率100 Hz下,利用稀釋30倍之CEA-GC偵測不同濃度的CEA阻抗分析圖 114
圖4-10在頻率100 Hz下,利用稀釋70倍之CEA-GC偵測不同濃度的CEA阻抗分析圖 116
圖4-11稀釋10X, 30X and 70X之CEA-GC偵測不同CEA之對數座標線性趨勢圖 118
圖4-12不同濃度CEA-Ab對應三種不同CEA濃度之免疫偵測阻抗分析圖 122
圖4-13濃度為50 ng/mL之CEA抗原對應Negative Control三明治免疫反應時間阻抗分析圖 124
圖4-14微流道之不同體積對應時間之分析圖 125
圖4-15 CEA癌胚抗原之三明治免疫反應與阻抗分析之頻率比較圖 127
圖4-16 Negative Control與不同稀釋倍率之CEA-GC與CEA 50 ng/mL在固定頻率100 Hz下進行三明治免疫反應偵測之阻抗電性分析圖 130
圖4-17稀釋10~30倍之CEA-GC偵測不同濃度的CEA阻抗分析:(a)10X, (b)20X, (c)30X之CEA-GC 133
圖4-18稀釋40~50倍之CEA-GC偵測不同濃度的CEA阻抗分析:(a)40X, (b)50X之CEA-GC 136
圖4-19稀釋70倍之CEA-GC偵測不同濃度的CEA阻抗分析 138
圖4-20 PBS清洗之稀釋10~50倍之CEA-GC偵測不同濃度的CEA阻抗分析:(a)10X, (b)20X, (c)30X, (d)40X, (e)50X之CEA-GC 142
圖4-21稀釋倍數之CEA-GC偵測不同CEA濃度:(a)未用PBS清洗,(b)用PBS清洗之對數座標線性趨勢圖 144
圖4-22稀釋10~40倍之CEA-GC金膜偵測不同濃度的CEA阻抗分析:(a)10X, (b)20X, (c)30X, (d)40X之CEA-GC金膜 149

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