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研究生:林家鴻
研究生(外文):Chia-Hung Lin
論文名稱:非標定式阻抗分析免疫感測器於Enrofloxacin藥物的檢測
論文名稱(外文):Label-Free Impedimetric Immuno-Sensor for the Detection of Enrofloxacin
指導教授:吳靖宙
指導教授(外文):Ching-Chou Wu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物產業機電工程學系所
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:2008
畢業學年度:96
語文別:中文
論文頁數:65
中文關鍵詞:enrofloxacin免疫感測器電化學阻抗分析法非標定式
外文關鍵詞:enrofloxacinimmunosensorelectrochemical impedance spectroscopylabel-free
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Enrofloxacin是fluoroquinolones家族中最常被使用的抗菌劑之一,用來預防與治療家禽與畜類的疾病。研究中以11-mercapto-undecanoic acid (MUA)與金電極表面進行自組性單層膜之共架性鍵結,然後用EDC/NHS活化MUA尾端COOH官能基,使抗enrofloxacin抗體能被固定在MUA上以進行enrofloxacin檢測,電極修飾特性與免疫反應則在含有等量Fe(CN)6 3-/4-的磷酸緩衝溶液(PBS)中,以電化學阻抗分析法(electrochemical impedance spectroscopy, EIS)和循環伏安法(cyclic voltammetry, CV)進行分析。研究結果顯示最佳修飾條件為:(1)在37℃、15% RH的條件時MUA修飾最穩定;(2)在24小時內大部分的MUA可被EDC/NHS活化;(3)抗體在37℃、15%RH固定2小時最穩定;(4) bovine serum albumin (BSA)的阻隔效應在1小時內可達穩定;(5)利用Tween 20清洗10分鐘即可清除非特異性鍵結。在等效電路模擬上,一個電阻串聯兩個並聯電阻與電容(2R//C)之等效電路,可分辨電極與修飾膜間和修飾膜與溶液間的特性,而一個電阻串聯一個並聯電阻與電容(1R//C)之等效電路則具有分析簡單與低變異性等特點。研究結果也顯示,修飾條件的最佳化可使enrofloxacin檢測極限從10 ng/ml降低到1 pg/ml,此非標定式免疫感測器可以大幅簡化免疫檢測的步驟,並能提供足夠靈敏之檢測極限,將來可用於食品安全或臨床醫學低濃度生物分子的檢測。
Enrofloxacin, that is most extensive approval antibiotic in the fluoroquinolone family, is frequently used to treat and prevent the disease of food-producing animals. To immobilize the specific antibodt for enrofloxacin on an electrode can develop a lable-free immunosensor. The immunosensors were prepared by covalently binding anti-enrofloxacin antibodies onto a 11-mercapto-undecanoic acid (MUA) monolayer with the pretreatment of EDC/NHS activation on a gold electrode. Each modification process for the Au electrodes was investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) in phosphate buffer saline(PBS) solution with Fe(CN)6 3-/4-. The optimal experiment conditions are : (1) MUA modification at 37℃ in 15% RH is more stable, (2) EDC/NHS can activate the most part of MUA after 24 h, (3)antibody immobilized at 37℃ in 15% RH presents stable after 2 h, (4) after 1 h, bovine serum albumin(BSA) blocking can finish, and(5) the nonspecific adsorption can be rinsed out for 10 min in Tween 20. In the equivalent circuit models, one resistor in series with two parallel RC circuits (2R//C) can clearly distinguish the impedance properties of the electrode-membrane and membrane-solution interface, and one resistor in series with one parallel circuit of resistor and capacitor (1R//C) possesses the advantage of simplification and stability of measurement. The optimal experiment conditions promote the limit of detection from 10 ng/ml to 1 pg/ml. The label-free impedance immunosensor supplies a sensitive and simple method for the enrofloxacin detection. In the future, the sensor can be applied to detect low concentration bio-molecule in food safety and clinical medical field.
摘要 I
Abstract II
表目錄 V
圖目錄 VI
第一章 緒論 1
1-1 前言 1
1-2 Quinolone 類藥物簡介 2
1-2-1 Quinolone 類藥物之作用機制 2
1-2-2 檢測enrofloxacin之技術文獻探討 5
1-2-3 酵素免疫分析法 6
1-3 免疫感測電極簡介 8
1-3-1 抗原 8
1-3-2 抗體 9
1-3-3 抗體與抗原的交互作用 10
1-3-4 抗體修飾技術簡介 11
1-4 電化學檢測 13
1-4-1 循環伏安法原理 13
1-4-2 電化學阻抗分析頻譜原理 15
1-4-3 電化學阻抗分析法之應用 22
1-5 研究動機與目的 26
1-6 實驗架構圖 27
第二章 實驗原理與方法 28
2-1 實驗設備與試劑 28
2-1-1 實驗試劑 28
2-1-2 實驗設備 29
2-1-3 抗體製備 30
2-2 實驗步驟與方法 33
2-2-1 電極表面修飾 33
2-2-2 循環伏安法檢測流程 35
2-2-3 電阻抗分析檢測流程 35
2-2-4 電路模型之模擬 36
第三章 結果與討論 39
3-1 最短時間製程之免疫感測器特性探討 39
3-1-1 在不同修飾步驟之CV行為 39
3-1-2 EIS的量測 40
3-1-3 電路模型的選用 41
3-1-4 Enrofloxacin 的檢量線 43
3-2 修飾條件最佳化的探討 44
3-2-1 Dropping法於MUA修飾最佳化 44
3-2-2 EDC/NHS活化的最佳條件 45
3-2-3 抗體固定時間的最佳化 48
3-2-4 BSA界面阻隔時間最佳化 50
3-2-5 Tween 20清洗時間最佳化 51
3-2-6 修飾最佳化下的檢測範圍 53
第四章 結論 57
參考文獻 58
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