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論文中文名稱:以SAM修飾之金奈米粒子應用於免疫分析之研究 [以論文名稱查詢館藏系統]
論文英文名稱:Application of SAM modified gold nanoparticles for the enhancement of immunoassay efficiency [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
出版年度:97
中文姓名:黃俊彥
英文姓名:Chun-Yen Huang
研究生學號:95688003
學位類別:碩士
語文別:中文
口試日期:2008-07-15
論文頁數:84
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:王勝仕;黃聲東;黃榮堂
口試委員英文名:Sheng-Shih Wang;Sheng-Tung Huang;Jung-Tang Huang
中文關鍵詞:自我集結單分子層金奈米粒子抗體
英文關鍵詞:Self-assembled monolayersgold nanoparticlesantibody
論文中文摘要:SAMs是自我集結單分子層,可以自發性的在不同的金屬基材上形成,其分子藉由化學吸附於基材上,而分子之間靠著凡得瓦爾力集結排列成高度次序、取向性、緊密的單分子層。一般以金奈米粒子為標記物的免疫分析方法中,大部分抗體是以物理性吸附於金奈米粒子上,我們認為有不穩定的問題存在,因此,本篇論文主要是發展一種以SAMs為基礎經由共價結合抗體於金奈米粒子上的技術應用於免疫分析上。首先金奈米粒子在界面活性劑Tween 20的保護下,用16-MHDA和1-UDT以1:100的比例修飾,隨後加入EDC和NHS活化16-MHDA分子末端的羧基,最後半乳糖苷酶抗體以胺基與之鍵結,如此便完成了構築。與物理性吸附抗體的方法做比較,分別實驗於dot-blot、免疫層析試紙以及耐受性的測試,實驗結果發現以SAMs修飾過的金奈米粒子不但較穩定,不會有非專一性蛋白質吸附的問題,而且所使用抗體的量也較少,即可達到相同的偵測極限,減少了分析的成本。
論文英文摘要:Self-assembled monolayers (SAMs), formed by spontaneous chemisorption of head group of selected organic molecule on suitable materials, represent one major trend in surface chemistry. Van der Waals forces between long alkyl chains orient and stabilize the monolayers.
In this paper, we fabricated the covalently-linked bioconjugates of antibody/gold nanoparticles (AuNPs) for immunoassay. The AuNPs were first modified by 16-mercaptohexadecanoic acid (16-MHDA)/1-Undecanethiol (1-UDT) mixture of molar ratio 1:100 under protection of Tween 20. Subsequently, the peripheral carboxylic groups were activated by EDC/NHS as coupling agents. The as-formed NHS-terminated AuNPs covalently combined with the amino groups of anti-β galactosidase antibody and the target bioconjugates were constructed.
A comparison of antibody adsorption between chemisorption and physisorption has been done for sensitivity and durability in applying dot-blot and immunochromatography methods.
Although covalent immobilization involves more steps technically, antibody covalently bonded on AuNPs are much more stable than antibody immobilized by physisorption. Moreover, SAMs-based method allowed for the more economical use of valuable antibodies.
論文目次:目 錄

中文摘要 i
英文摘要 ii
誌 謝 iv
目 錄 v
表目錄 vii
圖目錄 viii
第一章 緒論 1
1.1 前言 1
1.2 免疫分析簡介 1
1.3 金奈米粒子標記抗體技術 2
1.4 自我集結單分子層(SAMs)介紹 3
1.5 研究動機及目的 5
第二章 研究背景與原理介紹 6
2.1 免疫分析方法及原理 6
2.1.1 放射免疫分析法( radioimmunoassay ) 6
2.1.2 酵素免疫分析法( enzyme immunoassay ) 7
2.1.3 螢光免疫分析法( fluorescence immunoassay ) 10
2.1.4 化學冷光免疫分析法( chemiluminescence immunoassay ) 11
2.2 金奈米粒子標記免疫分析 12
2.2.1 金奈米粒子標記抗體 12
2.2.2 點漬免疫分析(dot-blot immunoassay) 13
2.2.3 免疫層析試驗(immunochromatographic test) 14
2.3 金奈米粒子之表面修飾與自我集結單分子層(self-assembled monolayers , SAMs) 17
2.4 不同SAM官能基修飾金奈米粒子的方法 20
2.4.1 表面置換反應(place-exchange reaction) 20
2.4.2 SN2親核取代反應 21
2.4.3 偶合反應(coupling reaction) 22
2.5 SAM修飾金奈米粒子於生技上的應用 24
第三章 實驗方法與步驟 33
3.1 實驗架構 33
3.2 修飾不同比例的SAM分子結合抗體於金奈米粒子上 36
3.3 修飾SAM 1:100比例結合不同濃度抗體於金奈米粒子上 37
3.4 不同濃度抗體吸附於金奈米粒子上 38
3.5 點漬免疫分析實驗(dot-blot immunoassay) 38
3.6 定量分析 39
3.7 耐受性測試(durability test) 40
3.8 免疫層析試驗(immunochromatographic test) 40
3.9 凝膠電泳(agarose gel electrophoresis)分析 41
3.10 動態光散射(dynamic light scattering)粒徑分析 42
3.11 紫外光/可見光光譜(UV/VIS)分析 42
3.12 螢光光譜(fluorescence spectrometer)分析 42
3.13 實驗藥品與設備 44
3.13.1 實驗藥品 44
3.13.2 實驗設備 46
第四章 結果與討論 48
4.1 修飾不同比例的SAM分子結合抗體於金奈米粒 48
4.2 修飾SAM 1:100比例結合不同濃度抗體於金奈 51
4.3 不同濃度抗體吸附於金奈米粒子上 53
4.4 耐受性測試(durability test) 55
4.5 免疫層析試驗(immunochromatographic test) 60
4.6 SAM及吸附方式結合抗體之製備及特性 61
4.6.1 凝膠電泳(agarose gel electrophoresis)分析 62
4.6.2 動態光散射(dynamic light scattering)粒徑分析 63
4.6.3 紫外光/可見光光譜(UV/VIS)分析 63
4.6.4 螢光光譜(fluorescence spectrometer)分析 65
4.6.5 蛋白質非專一性吸附測試 69
第五章 結論 70
參考文獻 71
附錄 78

表目錄

表2.1 Murry以不同配體進行表面置換反應的結果比較 21
表2.2 MPC與不同試劑進行偶合反應 22
表2.3 SAM修飾金奈米粒子之特性探討 28
表2.4 SAM修飾金奈米粒子於生物技術應用相關文獻 29
表3.1 實驗藥品 44
表3.2 實驗設備 46
表4.1 金奈米粒子不同修飾階段之DLS粒徑分析 63
表4.2 SAM修飾金奈米粒子鍵結抗體重複實驗之螢光強度統計 67
表4.3 抗體吸附金奈米粒子重複實驗之螢光強度統計 68
表4.4 BSA非專一吸附測試重複實驗之螢光強度統計 69

圖目錄

圖1.1 SAMs分子結構圖 4
圖2.1 同質酵素免疫分析法示意圖 8
圖2.2 競爭型酵素免疫分析示意圖 8
圖2.3 三明治型酵素免疫分析示意圖 9
圖2.4 間接型酵素免疫分析示意圖 10
圖2.5 perylene標記之螢光免疫分析法示意圖 11
圖2.6 化學冷光免疫分析法偵測甲狀腺促進素(THS)示意圖 12
圖2.7 抗體結合於金奈米粒子上作用力示意圖 13
圖2.8 點漬免疫試驗示意圖 14
圖2.9 免疫層析試紙示意圖 15
圖2.10 競爭型免疫層析檢測gentamicin示意圖 16
圖2.11 競爭型免疫層析檢測不同濃度及不同環境中的gentamicin 16
圖2.12 Brust-Schiffrin法製備dodecanethiol保護之金奈米粒子示意圖 18
圖2.13 Kunitake法製備MPA-Na保護之金奈米粒子示意圖 18
圖2.14 金奈米粒子經Tween 20保護及未保護下以烷基硫醇分子修飾之示意圖 19
圖2.15 表面置換反應示意圖 20
圖2.16 金奈米粒子上溴基與一級胺反應示意圖 21
圖2.17 合成tripeptides(Phe,Ile,Ala)修飾的金奈米粒子 22
圖2.18 EDC/NHS反應示意圖 23
圖2.19 dextran修飾金奈米粒子及葡萄糖偵測示意圖 25
圖2.20 dextran修飾金奈米粒子及生物分子辨識偵測示意圖 26
圖2.21 MUA修飾金奈米粒子結合glucose oxidase示意圖 27
圖3.1 實驗流程圖 34
圖3.2 SAM修飾之金奈米粒子共價結合抗體示意圖 35
圖3.3 定量分析流程圖 39
圖3.4 免疫層析試紙組裝示意圖 41
圖4.1 SAM不同比例修飾金奈米粒子之實驗結果圖 49
圖4.2 SAM1:100修飾金奈米粒子結合不同濃度抗體之實驗結果圖 52
圖4.3 吸附方式結合不同濃度抗體於金奈米粒子上之實驗結果圖 54
圖4.4 不同構築方式的免疫金探針懸浮於PBS之耐受性測試結果圖 56
圖4.5 不同構築方式的免疫金探針懸浮於PBSB之耐受性測試結果圖 57
圖4.6 不同構築方式的免疫金探針懸浮於PBST之耐受性測試結果圖 59
圖4.7 免疫層析實驗結果圖 61
圖4.8 金奈米粒子不同修飾階段之凝膠電泳分析圖 62
圖4.9 SAM修飾金奈米粒子共價鍵結抗體之紫外光/可見光光譜 64
圖4.10 抗體吸附結合於金奈米粒子上之紫外光/可見光光譜 65
圖4.11 SAM修飾金奈米粒子鍵結抗體之螢光光譜 67
圖4.12 抗體吸附金奈米粒子之螢光光譜 68
圖4.13 BSA非專一吸附測試之螢光光譜 69
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