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研究生:張瑋廷
研究生(外文):Wei-Ting Chang
論文名稱:即時檢測沙門氏桿菌及其抗藥性之適體傳感器
論文名稱(外文):Aptamer-based sensors using carbon nanotubesto detect antibiotics resistance of Salmonella rapidly
指導教授:黃榮堂黃榮堂引用關係曲立全曲立全引用關係
口試委員:方旭彬侯劭毅
口試日期:2013-07-22
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:機電整合研究所
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:66
中文關鍵詞:微機電製程技術單壁奈米碳管核酸適體抗藥性檢測
外文關鍵詞:MENSCarbon NanotubeAptamerThe drug resistance of bacteria
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在亞洲地區當中,沙門氏菌是幼兒腸道炎常見的病菌,而現行檢測沙門氏菌抗藥性的塗盤方式需耗費三至五天的時間,若能縮短檢測抗藥性的時間,不但能夠幫助醫師選擇適當的抗生素,也可以避免抗生素濫用的情形。
本研究的目的是發展一種可即時檢測菌體存在,並在數小時內完成細菌抗生素篩選的適體傳感器。採用新式奈米材料-單壁奈米碳管,使其鍵結與沙門氏桿菌具有專一性之核酸適體,並利用微機電製程技術,製作以鉻金屬為材料之平行梳狀電極,再使用微量吸管吸取沙門氏桿菌液滴定於載具容器的電極上,讓其與核酸適體接合產生電荷傳遞,並可直接於電極上做細菌的培養。當電極上的細菌隨著時間的增生,電訊號也會因此改變,最後加入抗生素於培養載具中,使用微電流計與阻抗分析儀擷取電訊號,紀錄其訊號變化差異性。本研究得到馬偕醫院檢驗科的協助,提供實驗用的標準菌,包括大腸桿菌、沙門氏桿菌、檸檬酸桿菌、以及變形桿菌等不同的腸內菌種來測試晶片的專一性,並藉由沙門氏菌增生而改變的電流變化率,來判斷菌種是否具有抗藥性,這種簡易的判讀方式有助於臨床實驗的操作,未來修飾更多細菌種類的核酸適體晶片,即可為一種細菌檢驗的使用平台。


The development of an aptamer-based sensor for typing of bacteria is presented. Highly specific DNA aptamers to Salmonella enteritidis were selected via Cell-SELEX technique. This study developed a method of detecting bioparticles such as Salmonella by a biochip using single-walled carbon nanotubes modified by aptamer. A label-free impedance sensor for rapid detection of Salmonella was developed by aptamer onto an chromium interdigitated array (IDA) microelectrode. The cross-test between Salmonella and E. coli on the biochip exhibits high specificity for Salmonella. Each comprises a positive selection step against Salmonella enteritidis and a negative selection step against a mixture of related pathogens, including Salmonella, Escherichia coli, Citrobacter freundii, and Proteus to ensure the species-specificity of the selected aptamers. The electron-transfer resistance was correlated with the concentration of Salmonella. A variety of different antibiotics were dropped in the wells with volume of 0.5 ml on the biochip to detect antibiotics resistance of Salmonella.

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 vi
圖目錄 vii
第一章 緒論 …1
1.1 前言 1
1.2 生物感測器起源與介紹 1
1.3 微機電系統 3
1.4 生物微粒檢測技術 4
1.5 沙門氏桿菌抗藥性及抗生素探討 7
1.6 研究目的 7
第二章 文獻探討與原理 9
2.1 阻抗檢測原理 9
2.1.1 平行梳狀電極 10
2.1.2 法拉第反應與非法拉第反應 10
2.2 細菌的抗藥性機制 12
2.3 核酸適體簡介 12
2.4 奈米碳管改質原理 14
2.4.1 奈米碳管溶液 14
2.4.2 SAM 自組裝分子溶液 14
2.4.3 建構碳管表面官能基 15
2.4.4 適體接合 17
2.5 適體與目標物結合原理 19
2.6 酵素連結免疫吸附法 19
第三章 檢測晶片的設計與製作 22
3.1 實驗設計 22
3.2 感測原件製作流程 23
3.3 SAM 表面修飾溶液製備 25
3.4 奈米碳管溶液製備與表面改質 25
3.5 奈米碳管表面改質與接合核酸適體 26
3.6 光罩設計與製作 26
3.7 晶片製作 27
3.7.1 玻璃晶片清洗 28
3.7.2 黃光製程 29
3.7.3 金屬濺鍍 32
3.8 檢測晶片的微影製程 33
第四章 實驗設備與材料 35
4.1 實驗架構 35
4.2 實驗設備 35
4.3 實驗材料 38
4.3.1 腸內菌的濃度調配 38
4.3.2 沙門氏菌的尺寸量測 39
第五章 實驗結果與討論 40
5.1 檢驗晶片上的奈米碳管 40
5.2 金屬電極的附著度測試 42
5.3 碳管與適體的接合分佈 45
5.4 適體與菌體的專一性測試 47
5.5 晶片的菌體殘留率測試 50
5.6 檢測濃度的標準曲線 52
5.7 沙門氏菌直接於晶片培養測試 53
5.8 沙門氏菌的抗藥性測試 54
5.9 阻抗式頻譜分析法應用於定量分析 57
5.10 單一頻率的阻抗分析應用於細菌生長 58
第六章 結論與未來展望 60
6.1 結論 60
6.2 未來工作與展望 61
6.2.1 多種類病菌的抗藥性測試 61
6.2.2 檢測晶片的保存性 61
6.2.3 奈米碳管的均勻度與方向性 61
6.2.4 未來展望 61
參考文獻 62


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