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系統識別號 U0026-0812200914002903
論文名稱(中文) 結合奈米金-免疫抗體作用於SAM 修飾電極上之電化學阻抗特性變化進行微量Protein A 的檢測
論文名稱(英文) Combining Gold Nanoparticles with Antibody on SAM Modified Electrode for Trace Protein A Detection Based on Electrochemical Impedance Spectroscopy
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 95
學期 2
出版年 96
研究生(中文) 黃建豪
研究生(英文) Chien-hao Huang
電子信箱 P8694115@ccmail.ncku.edu.tw
學號 P8694115
學位類別 碩士
語文別 中文
論文頁數 80頁
口試委員 口試委員-葉晨聖
口試委員-丁信智
口試委員-鄭國順
指導教授-張憲彰
口試委員-吳靖宙
中文關鍵字 蛋白質A  免疫分析  電化學阻抗分析  奈米金粒子  自組性單層薄膜 
英文關鍵字 Protein A  Au nanoparticles  Self assembled monolayer  Electrochemical impedance  Immunoassay 
學科別分類
中文摘要 某些存在於血液中與疾病相關的特定蛋白質,其濃度變化或是結構上的改變,被視為疾病發展的重要指標,而目前在臨床上,檢驗這些物質之方法,主要採用免疫分析法;其檢測方式係利用抗原與抗體間高度專一性的結合反應,可用以定量抗原或抗體的濃度,是目前最常用的檢驗技術,由於其具有相當高的特異性及靈敏度(約1-10 ng/ml),因此成為分子生物領域的一項重要分析技術。但現行免疫分析儀器缺點為價格昂貴、測試項目過於窄化、操作繁瑣耗時等。基於此,本研究將電化學阻抗分析應用於免疫分析技術上,以期發展一高靈敏、低檢測極限之免疫感測器。實驗中以金電極為基材,修飾上一自組性單層薄膜(self assembled monolayer,SAM;本研究使用1,6-hexanedithiol)以固定奈米金粒子,利用奈米金粒子提高抗體固定量,檢測不同濃度之protein A;而本實驗主要是利用特殊功能化之奈米金粒子(Functionalized Au nanoparticles, FAuNPs)進行二次標定,使免疫反應訊號放大,以期降低檢測極限。從電化學阻抗分析結果顯示,利用FAuNPs標定之方式相當成功,且可以得知Ret對protein A的檢測濃度在5-1000 pg/ml間有良好之線性範圍,其檢測極限可達1 pg/ml。於傳統酵素免疫分析法比較可發現,其偵測極限高出2-3級數。探討實驗結果可知,電化學阻抗分析於anti-protein A/protein A親和性之檢測,已有不錯之結果,因此未來可利用實驗建立之技術平台,將電化學阻抗技術應用於其他親和性物質的檢測。
英文摘要 The variation in concentration or the change of the structure of some special proteins related with diseases, which existing in the blood had passed for an important index of the development of diseases. Immunoassay, the measurement of antibody or antigen concentrations based on biospecific recognition interactions, has been considered as a major analytical method in the fields of clinic diagnostics and environmental analysis. It has been commonly used for detection of a variety of antigens and antibodies. Basically, it is sensitive (around 1-10 ng/ml) and specific. But the shortcoming of current immune analytical instrument is high price, narrow test project, convoluted operation. We try to develop an electrochemical impedance immunosensor to shorten the detection time and improve the detection limits of the target antigens. We used colloidal gold to enhance the amount of antibody immobilized on a gold electrode and ultimately monitored the interaction of antigen-antibody by impedance measurement. We used a novel amplification procedure with the application of an Au-colloid labeled antibody and special molecule as the amplifying probe. As a result, it showed that the detection of limit could reach to 5-1000 pg/ml of antigen concentration and higher than the traditional method in sensitivity. In summary, EIS provides good linear range to detection of antigen-antibody complex. To develop biosensing technique, impedance analysis and experimental parameter could be applied to detection of other affinity biosample.
論文目次 摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VI
表目錄 VIII

第一章 緒論 1
1.1 前言 1
1.2 生物感測器 2
1.2.1生物感測器的簡介 2
1.2.2生物感測器的分類 3
1.3 免疫修飾電極簡介 6
1.3.1 免疫分析法的基本理論 6
1.3.2 抗原與抗體的定義 6
1.3.3 抗原與抗體的結合力 6
1.3.4 酵素免疫分析法 7
1.3.5 免疫感測器 8
1.4 金黃色葡萄球菌之簡介 11
1.5 電化學阻抗分析法 14
1.5.1 電極的測量方式 17
1.5.2 電路參數的測量 18
1.6 阻抗圖譜在檢測上之意義 22
1.6.1 阻抗圖譜與電路元件之關聯性 22
1.6.2 電化學阻抗分析之文獻回顧 24
1.7 研究動機與目的 26
1.8 研究架構圖 26

第二章 文獻回顧及實驗原理與方法 27
2.1 文獻回顧 27
2.1.1 自主性單層薄膜之特性簡介 27
2.1.2 奈米材料之簡介 29
2.2 電化學檢測之原理 31
2.2.1 循環伏安法 32
2.2.2 電化學阻抗分析 34
2.3 實驗設備與試劑 41
2.3.1 實驗設備 41
2.3.2 實驗試劑 41
2.4 實驗方法 43
2.4.1 電極表面修飾 43
2.4.2 特殊功能化的奈米金粒子 46
2.4.3特殊功能化奈米金粒子之製作 47
2.4.4分析檢測流程 48

第三章 結果與討論 50
3.1 模擬電路之建立 50
3.2 SAM不同修飾時間的緻密度變化 54
3.3 奈米金不同修飾時間對阻抗值的影響 58
3.4 抗體及BSA之固定 59
3.4.1 抗體固定之濃度 61
3.4.2 抗體及BSA固定後其Ret值之變化 62
3.5 電化學阻抗分析對抗原之檢測 63
3.5.1 抗體與抗原反應訊號 63
3.5.2 經由FAuNPs放大後之免疫反應訊號 64
3.5.3 FAuNPs放大免疫反應訊號之驗證 65
3.5.4 Protein A之電化學阻抗檢測 67

第四章 結論與展望 71
4.1 電化學阻抗分析於免疫感測之探討 71
4.2未來的發展與應用 72

參考文獻 73
附錄 77
自述 79
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