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研究生:林勃伸
研究生(外文):Po-Shen Lin
論文名稱:藉由新穎之表面電漿共振儀表面改質方式於人類血清白蛋白的偵測之研究
論文名稱(外文):A novel method for detecting microalbuminuria by surface plasmon resonance
指導教授:陳文逸陳文逸引用關係
指導教授(外文):Wen-Yin Chen
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程研究所
學門:工程學門
學類:化學工程學類
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:150
中文關鍵詞:人類血清白蛋白染料分子表面電漿共振儀
外文關鍵詞:HSASPRCibacron Blue
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人類血清白蛋白為人體血漿中含量最豐富的蛋白質。在健康的腎臟中,腎小球會將其過濾使其不會隨著尿酸跟廢棄物經由尿液排出;但是一個不健康的腎臟,因腎小球被破壞造成無法過濾的情形,白蛋白則會隨著尿液排出。依程度不同分成微蛋白尿、蛋白尿等,所以尿液中的白蛋白含量在臨床診斷則為一個重要的生理訊息。
本研究以仿生染料(Cibacron Blue F3GA)做為觸手,將其和具有抗非特異性吸附特性的PEG結合,以自我組裝技術固定在生物晶片上。結果發現,此種生物晶片在表面電漿共振儀之系統中,當檢測環境為緩衝溶液pH值4、外加鹽離子濃度為0.05M時對人類血清白蛋白具有高親和性以及高吸附量的特性及可重覆性,此外,對照於CD圖譜人類血清白蛋白峰值偏移的結果,亦發現pH值的改變會對人類血清白蛋白的結構產生變化,此顯示在同一緩衝溶液的鹽濃度環境下,pH值能影響人類血清白蛋白結構,而此結構改變可能也是造成親和性常數升高的重要原因之ㄧ。在酵素鏈結免疫分析確認了,CB對人類血清白蛋白之高特異性。定量方面,找出了適宜之檢測濃度範圍為0.01 mg/ml ~0.1mg/ml,可用來偵測人類血清白蛋白,增加了應用的價值。
最後,配合熱力學數據的探討,計算△H、△S、△Cp,說明CB與人類血清白蛋白之間的交互作用之機制,實驗證實人類血清白蛋白的結構會隨著溫度而改變,且與CB之吸附反應為自發性行為,為一種entropy driven與enthalpy cost的程序。ΔH之所以為正值,推測吸附反應可能包含了人類血清白蛋白的劇烈結構變化、疏水作用力、靜電作用力與氫鍵的破壞等。
Human serum albumin (HSA) is the most abundant protein in human blood plasma. It is produced in the liver. Albumin comprises about half of the blood serum protein. It is soluble and monomeric. In the healthy kidney, albumin''s size and negative electric charge exclude it from excretion in the glomerulus. But in some diseases including diabetic nephropathy, it can cross the glomerulus and stockpile in the bladder. Depending on the amount of albumin in the urine, a patient may have normal renal function, microalbuminuria, or albuminuria.So urine HSA is an important bio-marker to diagnose nephropathy. Cibacron Blue F3G-A is an anthraquinone dyes which has special affinity for HSA and it`s structure is similar to bilirubin. In this study, Cibacron Blue F3G-A was covalently attached onto PEG monolayer provided with low-fouling property which was established by self-assembled monolayer. The goals of this study used surface plasmon resonance (SPR) to detect HSA and to establish the optimum condition. The results exhibited the chip had high affinity, high capacity and could regeneration in the condition of pH 4. The results also show the pH value can change the HSA’s structure and the structure change is an important factor to raise the affinity constant. The same result also proved by ELISA. In addition, linear response appeared in concentration of HSA in the range of 0.01~0.1mg/ml. The detection limit was 4μg/ml . In other words, SPR biosensor is used as a sensitive technique for direction of diagnosis is powerful.
目錄
中文摘要..................................................I
Abstract................................................III
誌謝....................................................IV
目錄.....................................................V
圖目錄...................................................IX
表目錄..................................................XV
第一章 序論............................................1
第二章 文獻回顧........................................3
2.1 生物晶片之簡介.......................................3
2.2 對於人類血清白蛋白之研究與介紹.......................7
2.2.1 微量尿白蛋白定義及量測.............................7
2.2.2 人類血清白蛋白結構、特性之描述.....................9
2.2.3白蛋白與觸手(receptor)之關係.......................11
2.2.4現行使用之檢測系統.................................16
2.2.5 動機..............................................18
2.3 其他實驗室所發展之分離、檢測方式....................20
2.4 表面改質與分子固定化................................28
2.4.1 自我組裝單層膜....................................30
2.4.1.1 硫醇............................................32
2.4.1.2 矽烷或矽氧類....................................33
2.4.1.3 混合性自我組裝膜................................35
2.4.2 表面分子之固定化..................................39
2.4.2.1仿生染料之固定化.................................39
2.4.2.2去氧核醣核酸固定化...............................42
2.4.2.3蛋白質和抗體固定化...............................42
2.5 偵測生物分子間交互作用力之儀器......................44
2.5.1表面電漿共振儀.....................................44
2.5.2石英晶體微天平.....................................49
第三章 實驗藥品與儀器設備...............................51
3.1實驗藥品.............................................51
3.2儀器設備.............................................53
3.3實驗步驟.............................................54
3.3.1 衝溶液的配置......................................54
3.3.2生物晶片的改質.....................................54
3.3.3 表面電漿共振儀偵測實驗............................55
3.3.3.1 儀器設定及操作..................................55
3.3.3.2實驗設計.........................................56
3.3.3.2.1 最佳吸附條件的找尋............................56
3.3.3.2.2檢量線的製作...................................57
3.3.3.2.3 恆溫吸附曲線的製作............................57
3.3.4 圓二色光譜儀測量實驗..............................58
3.3.5 原子力顯微鏡測量實驗..............................58
3.3.6接觸角測量器.......................................59
3.3.7 BCA Protein Assay Kit.............................60
3.3.8 酵素連結免疫吸附分析(ELISA) ......................60
3.3.9 SPR數據之處理與軟體分析...........................61
第四章 實驗結果.........................................63
4.1 表面鑑定............................................63
4.2 檢測條件最佳化......................................72
4.3 特異性實驗之實驗結果................................77
4.3.1親和性測試.........................................77
4.3.2特異性比較實驗.....................................78
4.3.3競爭反應...........................................79
4.4酵素連結免疫吸附分析之實驗結果.......................85
4.5人類血清白蛋白之結構變化.............................89
4.6熱力學性質之演算.....................................93
4.7偵測人類血清白蛋白之檢量線的製備....................101
第五章 討論............................................107
5.1 緩衝溶液pH值的影響.................................107
5.2鹽離子濃度的影響....................................110
5.3 人類血清白蛋白與CB之間的交互作用過程...............112
第六章 結論............................................113
第七章 參考文獻........................................116
附件--投稿中之文章初稿.................................124
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