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研究生:陳京瑤
論文名稱:自組裝單層膜技術在蛋白質生物晶片的應用:利用烷基硫醇化合物將山葵過氧化酵素固定在晶片的金表層
論文名稱(外文):Self-assembled monolayers technology in protein biochip:immobilization of horseradish peroxidase on gold-coated chip modified by alkanethiols
指導教授:吳東昆
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
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
中文關鍵詞:自組裝單層膜
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本研究是在生物晶片的製程中,利用自組裝單層膜技術在晶片表面上模擬天然的生物膜並進行生物分子之固定化,藉此研究蛋白質分子與晶片介面間的反應性,進而發展生物晶片之生物感測介面;也希望以此技術在生物技術和積體光電元件間達到橋樑作用,進而加速生物晶片的發展。
由於晶片的表面特性會被不同長度或是不同末端官能基的自組裝單層膜所控制,並且影響生物分子在晶片上的穩定度、活性及專一性,因此實驗中選用3-mercaptopropionic acid ( 3-MPA )、11-mercaptoundecanoic acid ( 11-MUA )、16-mercaptohexadecanoic acid ( 16-MHA ) 等不同長度的烷基硫醇化合物分子在晶片的金表層上形成自組裝單層膜,以此控制蛋白質的活性中心與晶片之間的距離,並利用山葵過氧化酵素做為蛋白質固定化的模擬系統,再以螢光技術、化學發光法、呈色反應法所得到參數值來判斷固定在晶片上的山葵過氧化酵素之活性,找出最佳的自組裝單層膜晶片製程。
由實驗結果中可發現 11-MUA 有最佳的山葵過氧化酵素固定化效率;若以 11-MUA 之自組裝單層膜晶片進行測試,則晶片上的活性酵素量約為 0.424 ~ 0.426 nmol/mm2 ,而過氧化氫偵測範圍約為 0 ~ 4.425 mol/mm2 ;此外,晶片在重複使用 9 次之後,酵素的固定化效率仍然高於 80 ﹪。在未來希望可利用建立出的自組裝單層膜製程進行多種蛋白質生物晶片之研究及發展。
中文摘要……………………………………...……………………….……I
英文摘要………………...………………………………...………………II
謝誌………………………………………………...……….………….....III
目錄……………………………………………………………..………...IV
圖目錄………………………………………………...…………….…..VIII
表目錄……………………………………………………...………..…...ⅩI
第一章 緒論
1-1 生物晶片簡介……...………………………………...………………..1
1-1-1 源起……………………………………………………………1
1-1-2 定義……………………………………………………………2
1-1-3 應用領域………………………………………………………2
1-2 蛋白質晶片概述……………………………………………..…….…5
1-2-1 發展背景…………………………………………………..…..5
1-2-2 分類及應用………………………………………………..…..6
1-2-3 發展難題……………………………………………...…..…...8
1-2-4 技術及應用展望…………………………………...……..….10
1-3 蛋白質晶片固定化技術………………………………….………....12
1-3-1 生物晶片技術………………………………………......……12
1-3-2 固定化技術…………………………………………….…….14
1-3-3 自組裝單層膜(Self-Assembled Monolayers;SAMs)在 蛋白質晶片上之應用…………………………….…………..20
1-4 山葵過氧化酵素晶片………………………..……………..………..24
1-4-1 山葵過氧化酵素特性………………..………….…………...24
1-4-2 山葵過氧化酵素晶片之過氧化氫偵測系統……………..…28
1-5 研究目的……………………………………………….………….....29
第二章 實驗材料與方法
2-1 實驗材料與藥品………………………………………...……...........30
2-2實驗儀器與設備……………….……………………………...……...31
2-3實驗溶液……………………….……………………………...……...31
2-4 鍍金矽晶片製造……………….……………………………...……..35
2-5自組裝單層膜 ( Self-assembled monolayers; SAMs ) 形成與山葵 過氧化酵素 ( Horseradish Peroxidase; HRP ) 之固定化…...……..36
2-5-1 晶片前處理….……………………………...…......................37
2-5-2 烷基硫醇化合物之自組裝單層膜製造….………..….……..37
2-5-3 自組裝單層膜之活化….……………..…………….....……..37
2-5-4 山葵過氧化酵素固定化……………………………......…....37
2-6 山葵過氧化酵素 ( Horseradish Peroxidase; HRP ) 偵測系統立.....39
2-6-1 螢光酵素法….………………………………...…..……...….39
2-6-2 螢光抗體法.…………………………………...……….....…..40
2-6-3 化學發光法.…………………………………...……….....…..41
2-6-4 呈色法.………………………………………...……….....…..43
2-6-5 循環伏特安培法…………………………...………………....45
2-6-6 晶片重複使用效率…………………………..…...……..…...46
2-6-7 過氧化氫偵測範圍大小………………………..…....……....46
第三章 結果與討論.
3-1 鍍金矽晶片製造………….…...………...……………...……...….....47
3-2 自組裝單層膜 ( Self-Assembled Monolayers ; SAMs ) 製造……..48
3-2-1 烷基硫醇化合物自組裝單層膜形成條件..............................47
3-2-3 EDC/NHS進行SAMs末端官能基活化..................................51
3-2-3山葵過氧化酵素 ( HRP ) 固定化...........................................51
3-3 山葵過氧化酵素 ( HRP ) 固定化效率偵測…….…………..……..54
3-3-1 螢光酵素法……………………………………..…................54
3-3-1-1 FITC-HRP固定化之反應濕度比較..….........................55
3-3-1-2 FITC-HRP最佳固定化濕度之螢光強度分析…………60
3-3-1-3 FITC-HRP最佳固定化效率.…………...…………..….....62
3-3-2 螢光抗體法…………………….…………………….…..…..…63
3-3-2-1 HRP和antiFITC-HRP間最佳反應濃度………..…..…...63
3-3-2-2 FITC-antiHRP螢光強度分析………………….…….…...65
3-3-2-3 最佳HRP固定化與FITC-antiHRP反應效率………….68
3-3-3 化學發光法….……………………………..…..………….……69
3-3-3-1化學發光法最佳之HRP反應濃度.….……….….…….…69
3-3-3-2固定化HRP化學發光強度之定量分析…….……………71
3-3-4呈色法….……….………...….………………………………..…74
3-3-4-1呈色法最佳之HRP反應濃度………..…………….…..…74
3-3-4-2 固定化HRP呈色反應之定量分析…..…………….…….76
3-3-5循環伏特安培法…..…………………..…………………..……..79
3-3-5-1 SAMs工作電極之品質測試……...…………………........80
3-3-6山葵過氧化酵素固定化結果之探討…...……………………….82
3-3-7 晶片的重複使用效率...…………..…….………………………83
3-3-8過氧化氫偵測範圍大小……………………………………...….85
第四章 結論與未來展望…………………………………………..…...87
第五章 參考文獻………………………………………….…….……...89
附錄
附錄1……………………………………….…….……………………....97
附錄2……………………………………….…….……………………....98
附錄3……………………………………….…….……………………....98
附錄4……………………………………….…….……………………....98
附錄5……………………………………….…….……………………....99
附錄6……………………………………….…….……………………....99
附錄7……………………………………….…….……………………..100
附錄8……………………………………….…….……………………..101
附錄9……………………………………….…….……………………..102
附錄10………………..…………………….…….……………………..103
附錄11………………..…………………….…….……………………..104
附錄12………………..…………………….…….……………………..105
附錄13………………..…………………….…….……………………..106
附錄14………………..…………………….…….……………………..107
附錄15………………..…………………….…….……………………..108
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