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研究生:詹子欣
研究生(外文):Tsu-Shin Chan
論文名稱:光纖生物感知器在心臟血管疾病致病因子C-reactiveprotein之快速檢驗
論文名稱(外文):Rapid Identification of C-reactive Protein by Use of Fiber-optic Biosensor
指導教授:周晟周晟引用關係
指導教授(外文):Chien Chou
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:放射醫學科學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:82
中文關鍵詞:光纖生物感知器心臟病動力學螢光免疫分析法C反應蛋白
外文關鍵詞:fiber-optic biosensorcoronary heart diseasekineticsC-reactive proteinFITCPMMA
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心臟病是台灣居民死亡的第三病因,僅次於惡性腫瘤及腦血管疾病;如果把第十大死因高血壓性疾病也考慮在內的話,則心血管方面的疾病將會與腦血管疾病的死亡率不相上下。在美國及歐洲的幾個國家,因心臟病發作而死亡的人口也是居第二位,僅次於惡性腫瘤。而隨著年齡的老化,患有心血管方面疾病的機率也會隨之上升。
我們利用自行研發之光纖生物感知器,以三明治螢光免疫分析法之原理,將蛋白質抗體吸附在塑膠光纖表面,之後與其專一性抗原以及螢光標誌抗體做特異性結合形成一三明治複合體,再將波長為488 nm之雷射光束透過一N.A.值與光纖N.A.值相吻合之顯微物鏡使雷射光束進入光纖進行全反射傳導。雷射光束在光纖去纖衣部分進行全反射時會產生瞬逝波,作用範圍大約是100 nm,激發波長為520 nm之FITC螢光,並由架設於反應腔側端之光電倍增管接收螢光訊號進行分析。
本實驗成功地對IgG — anti IgG完成了定性定量以及動力學參數之量測,偵測靈敏度可到達15 ng/ml。另外也針對心血管方面疾病所發現之新危險因子C-reactive protein做了一系列的實驗,包括CRP — anti CRP之定性定量以及動力學分析,mCRP之量測,患有急性心肌梗塞病人之血清液量測等等。而偵測靈敏度是100 ng/ml,與現今檢測方法之靈敏度不相上下。證明本系統的確可應用在蛋白質交互作用之快速偵測以及行為分析,而且具有簡單、快速、高靈敏度與特異性佳的優點。
A novel fiber-optic fluorescence biosensor on monitoring biomolecular interactions in real time is setup. A higher detection sensitivity on the measurement of IgG-anti IgG protein interaction is measured. An Ar+ (488 nm) laser beam propagates in a multiple mode plastic fiber where FITC-labeled anti IgG is excited by the evanescent wave adjacent to the fiber surface at the unclad region of the fiber. The sandwich structure of <anti IgG — IgG - FITC labeled anti IgG> is immobilized on the surface of unclad plastic fiber where the fluorescence signal is measured by a photomultiplier tube at side. Then the absorption and dissociation coefficients of IgG-anti IgG interaction are calculated simultaneously. In addition, the kinetics of C-reactive protein (CRP) induced by coronary heart disease (CHD) is studied. The experimental result indicates larger association coefficient in CRP-anti CRP interaction, which agrees with the acute inflammation response in CHD.
致謝………………………………………………………………… Ⅰ
中文摘要…………………………………………………………… Ⅱ
英文摘要…………………………………………………………… Ⅳ
目錄………………………………………………………………… Ⅴ
圖目錄……………………………………………………………… Ⅸ
表目錄…………………………………………………………..… ⅩⅡ
縮寫……………………………………………………………..… ⅩⅢ
第一章 序論…………………………………………………… 1
1.1 研究動機……………………………………………………. 1
1.1.1 傳統的心臟病危險因子……………………………….. 3
1.1.2 新發現的心臟病危險因子…………………………….. 4
1.1.3 傳統檢測方法………………………………………….. 6
1.2 研究方法……………………………………………………. 7
第二章 基礎原理……………………………………………… 11
2.1 C-reactive protein與coronary heart disease之間的關係 11
2.1.1 什麼是C-reactive protein…………………………… 11
2.1.2 發炎反應……………………………………………….. 15
2.1.3 CRP在發炎反應中所扮演的角色…………………… 16
2.2 免疫分析…………………………………………………… 19
2.2.1 競爭型免疫分析法…………………………………… 21
2.2.2 三明治型免疫分析法………………………………… 22
2.3 光纖………………………………………………………… 24
2.3.1 光纖之結構與種類…………………………………… 24
2.3.2 光纖之傳導模式……………………………………… 26
2.4 數值孔徑…………………………………………………… 28
2.4.1 光纖之數值孔徑……………………………………… 28
2.4.2 顯微物鏡之數值孔徑………………………………… 29
2.4.3 數值孔徑與光纖半徑和傳導介質之關係…………… 29
2.5 瞬逝波……………………………………………………… 30
2.5.1 產生原理……………………………………………… 31
2.6 表面處理…………………………………………………… 32
2.6.1 生物分子之固定法…………………………………… 33
2.6.2 表面活化技術………………………………………… 34
2.7 螢光物質…………………………………………………… 38
2.7.1 螢光物質應具備之條件……………………………… 38
2.7.2 FITC(fluorescein isothiocyanate)之結構與特性 38
2.8 動力學……………………………………………………… 42
第三章 實驗材料與方法……………………………………… 45
3.1 方法………………………………………………………… 45
3.1.1 實驗架構……………………………………………… 45
3.1.2 系統校正……………………………………………… 47
3.1.3 螢光物質標誌抗體之品質檢測……………………… 47
3.1.4 光纖之前處理………………………………………… 48
3.1.5 光纖表面之三明治結構吸附處理…………………… 49
3.1.6 表面活化……………………………………………… 50
3.1.6.1 放射線照射……………………………………… 50
3.1.6.2 連接層之運用…………………………………… 51
3.1.7 IgG動力學參數之量測………………………………. 51
3.1.8 CRP之量測…………………………………………… 53
3.1.9 全血之CRP量測……………………………………… 53
3.1.10 CRP專一性實驗…………………………………….. 53
3.2 材料…………………………………………………………. 55
3.2.1 藥品及試劑…………………………………………… 55
3.2.2 儀器及組件…………………………………………… 56
第四章 實驗結果與討論……………………………………… 57
4.1 系統穩定度………………………………………………… 57
4.2 IgG動力學參數之量測…………………………………….. 57
4.3 系統靈敏度………………………………………………… 62
4.4 利用放射線照射活化光纖表面之結果…………………… 63
4.5 利用protein G當作連結層之結果…………………….…. 65
4.6 Native CRP之量測………………………………………… 66
4.7 CRP靈敏度測試…………………………………………… 69
4.8 mCRP之量測………………………………………….…… 71
4.9 人類血清液之CRP量測…………………………….…….. 72
4.10 CRP專一性實驗結果…………………………….……….. 75
第五章 結論與未來研究方向………………………………… 76
參考文獻………………………………………………………….. 79
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