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論文中文名稱:以奈米金粒子為標記物之半抗原分子檢測 [以論文名稱查詢館藏系統]
論文英文名稱:Development of a high detection sensitivity method of hapten using a gold nanoparticle immunoassay [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
中文姓名:陳錫圭
英文姓名:Hsi-kuei Chen
研究生學號:92688005
學位類別:碩士
語文別:中文
口試日期:2006-06-28
論文頁數:90
指導教授中文名:侯劭毅
指導教授英文名:Shao-yi Hou
口試委員中文名:郭文正;阮雪芬
中文關鍵詞:奈米金粒子半抗原免疫檢測生物素
英文關鍵詞:gold nanoparticlehaptenimmunoassayBiotin
論文中文摘要:半抗原分子(hapten)是一種分子量小於5kD的小分子,單獨存在時並不會引發免疫反應,但是當半抗原分子(例如生物素,Biotin)與載體(carrier)以共價鍵結結合並注射到動物體內即會引發免疫抗體反應,所產生的抗體對半抗原分子有專一性的結合,即使半抗原分子不再與載體結合。有一些環境污染物,特別是農業上常用的除草劑如digoxin、甲基巴拉松或是戴奧辛等等都是半抗原分子。常見的半抗原分子檢測方法為酵素免疫分析法(ELISA),這種方法的缺點是需要較長的分析時間和昂貴的分析儀器。因此,我們希望能開發出一種簡單和敏感度極高的方法來作為現場即時監測系統。在我們的研究中,我們是以具有紅色色澤的奈米金粒子作為標記物並將其接在抗體上。當分析樣品中存在著我們要偵測的半抗原分子時,帶有奈米金粒子的抗體即會去認此一半抗原分子,試紙因奈米金粒子的聚集作用而呈現出紅色標記。由研究結果中我們發現,試紙上所呈現的紅色色澤強度與Biotin的濃度成一正比例關係,肉眼可觀察到紅色色澤變化的極限值約為10 femtomole。而為了提高此種檢測方法的靈敏度,我們還以銀還原方法來放大奈米金的呈色效果。經過銀還原反應後,其靈敏度可以大幅提昇到100 zeptomole,約60,000個Biotin分子。由於此種奈米金粒子免疫檢測方法靈敏度相當高,加以不需要任何貴重或是笨重的儀器,因此我們預測這種檢測方法未來在現場即時檢測上將扮演著相當重要地角色。
論文英文摘要:The objectives of the present work are to develop an easy and inexpensive gold nanoparticle immunoassay to detect hapten in field. To immobilize samples, biotin, our model molecule for hapten, was covalent linkage to a peptide, SGQSWRPQGRFG, and the biotin-peptide bound on nitrocellulose membrane. The binding is due to that the strong dipole of the nitrate ester interacts with the strong dipole of the peptide bond. Then antibody-coated gold nanoparticles that served as a detection reagent were added to the membrane. By human eyes, red dots were observed for 10 femtomole of biotin and no dot was observed for negative control. The assay sensitivity can be increased by silver enhancement to 100 zeptomole. The assay will allow future studies for fast detection of environmental pollutants, such as DTT, dioxin and so on.
論文目次:目 錄

中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 前言 1
1.2 研究動機及目的 4
第二章 研究背景與原理介紹 6
2.1 半抗原特性 6
2.2 現行半抗原檢測方法及原理 7
2.2.1氣相質譜儀(GC/MA) 7
2.2.2 核磁共振(nuclear magnetic resonance) 7
2.2.3 螢光免疫分析法( fluorescence immunoassay ) 8
2.2.4 電化學式生物感測器(Electrochemical biosensor) 9
2.2.4.1電化學式感測器傳導機制 10
2.2.4.2酵素固定化電化學感測器(Electrochemical enzyme-immobilized biosensor) 11
2.2.4.3電化學式免疫感測器(Electrochemical immunoassay) 13
2.2.4.4電化學式核酸感測器(Electrochemical sensors of nucleic acids) 14
2.2.5 生物感測器(Biosensor) 15
2.2.6 酵素免疫分析法( enzyme-linked immunosorbent assay, ELISA) 16
2.3 奈米金免疫檢測技術介紹 18
2.3.1 膠體金結構 18
2.3.2 膠體金特性 19
2.3.3 膠體金粒子吸收光譜 19
2.3.4 膠體金製備方法 19
2.3.5 膠體金粒子於DNA檢測上之原理及應用 19
2.3.6 膠體金粒子於免疫檢測上之原理及應用 24
2.4 文獻回顧 28
第三章 實驗方法與步驟 32
3.1 胜肽(peptide)固相合成 32
3.1.1 Fmoc固相胜肽合成法的基本原理 33
3.1.2 Fmoc合成法中的固相載體 33
3.1.3 Fmoc合成法中的樹脂接臂 33
3.1.4 Fmoc-氨基酸的製備和側鏈保護 34
3.1.4.1 Asp和Glu常用側鏈保護基 35
3.1.4.2 Ser、Thr和Tyr常用側鏈保護基 35
3.1.4.3 Asn和Gln常用側鏈保護基 35
3.1.4.4 His常用側鏈保護基 36
3.1.4.5 Cys常用側鏈保護基 36
3.1.4.6 Arg常用側鏈保護基 36
3.1.4.7 Lys常用側鏈保護基 36
3.1.5 Fmoc基團的側鏈脫除 37
3.2 Biotin與胜肽結合反應 37
3.2.1 實驗材料 37
3.2.2 Biotin與胜肽結合反應操作步驟 38
3.3 奈米金粒子與抗Biotin抗體結合反應 38
3.3.1 實驗材料 38
3.3.2 奈米金粒子與抗Biotin抗體結合反應操作步驟 39
3.4 Biotin-peptide固定與奈米金粒子免疫檢測實驗 39
3.4.1 Biotin-peptide固定於硝化纖維膜上實驗 39
3.4.2 奈米金粒子免疫檢測實驗 40
3.5 銀還原反應 40
3.5.1 實驗材料 40
3.5.2 銀還原反應操作步驟 40
3.6 定量分析 40
3.7 奈米金粒子免疫檢測實驗流程 41
第四章 結果與討論 42
4.1 胜肽(peptide)固相合成 42
4.2 Biotin與胜肽結合反應 42
4.3 奈米金粒子與抗Biotin抗體結合反應 43
4.4 奈米金粒子免疫檢測實驗 44
4.4.1 操作範圍實驗 45
4.4.2 重覆實驗 47
4.4.3 溫度變數的影響 50
4.4.4 溶劑pH值的影響 54
4.4.5 載體變數的影響 59
4.4.6 溶劑變數的影響 62
4.4.7 Matrix變數的影響 66
4.4.8 Matrix中真實Biotin量檢測 70
第五章 結論 74
參考文獻 76
論文參考文獻:參考文獻

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