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系統識別號 U0002-2707201521043800
中文論文名稱 用於可攜式液晶型免疫分析系統的表面修飾策略研究
英文論文名稱 The Surface Modification Strategy of Portable Liquid Crystal-based Immunoassay
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 103
學期 2
出版年 104
研究生中文姓名 莊惠喻
研究生英文姓名 Hui-Yu Chuang
學號 602180084
學位類別 碩士
語文別 中文
口試日期 2015-06-25
論文頁數 92頁
口試委員 指導教授-陳志欣
委員-李偉
委員-陳巧貞
中文關鍵字 液晶  免疫分析  表面修飾策略  三聚氰胺 
英文關鍵字 liquid crystal  immunoassay  modification strategy  melamine 
學科別分類 學科別自然科學化學
中文摘要 近年來,利用液晶分子作為訊號輸出來源的液晶型免疫分析系統已經發展成為新型的檢測平台,我們使用不同的表面修飾策略以研究對於液晶型免疫分析系統靈敏度的影響。由於頻傳的食安事件,促使我們開發食品中非法添加的危害物的感測器,以三聚氰胺為檢測物,設計了三種免疫分析方法來進行檢測,並利用不同的表面修飾方法來將抗體或抗原修飾於表面上。將氧化三聚氰胺一級抗體以共價鍵方式修飾於表面的策略,在直接型免疫分析方法中雖然無法測得三聚氰胺的存在,但能於間接型免疫分析方法中輔以卵白素結合二級抗體得到2.5 ng/mL檢測極限值。競爭型免疫分析方法使用生物素共軛三聚氰胺抗體結合於卵白素修飾表面的策略,能檢測含有50 ng/mL三聚氰胺的溶液。
英文摘要 Recently, the liquid crystal (LC)-based immunoassay which applies liquid crystal molecules as a signal output has been a new detecting platform for immunoassay. In this study, we tried to study the sensitivity impact to LC-based immunoassay by using different surface modification strategy. Due to frequent food safety issues, we decided to develop a sensor for detecting the illegal additives in food. We designed three types of immunoassay to detect melamine, an illegal adulteration in infant formula and pet food, and tried different surface modification strategy to modify antibody or antigen on surface. In direct immunoassay by using the strategy of modified oxidized 1st anti-melamine on surface with covalent bond, it can’t detect the presence of melamine. Within streptavidin combine to 2nd anti-melamine in indirect assay, we can get a detection limit of 2.5 ng/mL melamine. Furthermore, we used the strategy of decorating biotin conjugated anti-melamine on streptavidin modified surface and it could detect the solution containing 50 ng/mL of melamine.
論文目次 目錄
第一章 緒論...1
1-1 液晶型分析系統...1
1-1-1 液晶...1
1-1-2 液晶型分析系統...4
1-1-3 液晶型分析系統介紹...6
1-1-3-1 液晶-固體介面(LC-solid interfaces)...7
1-1-3-2 液晶-水溶液介面(LC-aqueous interfaces)...9
1-1-3-3 液晶液滴(LC droplets)...11
1-1-4 液晶型分析系統優點...12
1-2 液晶型免疫分析系統...13
1-2-1 免疫分析方法...13
1-2-2 液晶型免疫分析方法...14
1-2-2-1 小分子分析...17
1-2-2-1-1 三聚氰胺的檢驗...17
1-3 免疫分析方法的表面修飾策略...19
1-3-1 修飾抗體於表面的策略...20
1-3-1-1 非共價性修飾策略...22
1-3-1-2 共價性修飾策略...22
1-3-2 液晶-固體介面的表面修飾策略...24
1-4研究動機...25
第二章 實驗方法...27
2-1 實驗藥品...27
2-2 實驗材料...29
2-3 實驗儀器...29
2-4 實驗方法...30
2-4-1 製備溶液...30
2-4-1-1 製備緩衝溶液...30
2-4-1-2 製備牛免疫球蛋白溶液...30
2-4-1-3 製備氧化牛免疫球蛋白溶液...31
2-4-1-4 製備三聚氰胺抗體溶液...31
2-4-1-5 製備生物素共軛三聚氰胺抗體溶液...31
2-4-1-6 製備氧化三聚氰胺抗體溶液...31
2-1-4-7 製備卵白素溶液...32
2-4-1-8 製備牛血清蛋白溶液...32
2-4-1-9 製備三聚氰胺溶液...32
2-4-1-10 製備三聚氰酸溶液...32
2-4-1-11 製備醋酸鉀溶液...33
2-4-1-12 製備過碘酸鈉溶液...33
2-4-1-13 製備甘油溶液...33
2-4-1-14 製備氰基硼氫化鈉溶液...33
2-4-2 製備官能基修飾載玻片...33
2-4-2-1 清潔載玻片...33
2-4-2-2 DMOAP修飾載玻片...34
2-4-2-3 醛基修飾載玻片...34
2-4-2-4 胺基修飾載玻片...35
2-4-2-5 氧化牛免疫球蛋白抗體修飾之不同官能基修飾載玻片...35
2-4-2-6 卵白素修飾載玻片...36
2-4-2-7 牛血清蛋白修飾載玻片...36
2-4-2-8 三聚氰胺修飾載玻片...36
2-4-2-9 三聚氰胺-牛血清蛋白修飾載玻片...37
2-4-3 製備三聚氰胺檢測晶片...37
2-4-3-1 氧化三聚氰胺一級抗體檢測晶片...37
2-4-3-2 生物素共軛三聚氰胺抗體檢測晶片...37
2-4-4 三聚氰胺檢測方法...38
2-4-4-1 直接型免疫分析方法...38
2-4-4-2 間接型免疫分析方法...38
2-4-4-3 競爭型免疫分析方法...38
2-4-4-3-1 方法一...38
2-4-4-3-2 方法二...39
2-4-4-3-3生物素共軛三聚氰胺抗體檢測晶片檢測三聚氰胺...39
2-4-5 生物素共軛三聚氰胺...39
2-4-6 製備液晶元件...40
2-4-7 液晶元件成像...40
2-4-8 接觸角測量儀...40
第三章 結果與討論...41
3-1 官能基修飾載玻片於抗體修飾的影響...41
3-1-1 官能基修飾載玻片最佳製備條件...41
3-1-1-1 DMOAP修飾載玻片...41
3-1-1-2 醛基修飾載玻片...43
3-1-1-3 胺基修飾載玻片...45
3-2 官能基修飾載玻片水珠接觸角比較...47
3-3 官能基修飾載玻片於抗體修飾及其免疫結合力影響...48
3-3-1 修飾抗體於官能基修飾載玻片對液晶光學訊號影響...48
3-3-2 修飾抗體於官能基修飾載玻片對免疫結合力影響...51
3-4 不同修飾策略於三聚氰胺檢驗的影響...54
3-4-1 直接型免疫分析方法...54
3-4-1-1 氧化三聚氰胺一級抗體於胺基修飾載玻片的修飾...55
3-4-1-2 氧化三聚氰胺一級抗體於胺基修飾載玻片最佳條件...56
3-4-1-3 直接型免疫分析方法檢測三聚氰胺...57
3-4-2 間接型免疫分析方法...59
3-4-2-1 界面活性劑於液晶光學訊號影響...59
3-4-2-2 間接型免疫分析方法檢測三聚氰胺...60
3-4-2-3 氧化三聚氰胺一級抗體檢測晶片特異性探討...63
3-4-2-4 以卵白素及生物素共軛三聚氰胺二級抗體檢測三聚氰胺...64
3-4-3 競爭型免疫分析方法...66
3-4-3-1 修飾三聚氰胺於載玻片表面方法...68
3-4-3-1-1 方法一...68
3-4-3-1-2 方法二...70
3-4-3-3 生物素共軛三聚氰胺抗體修飾載玻片檢測三聚氰胺...73
第四章 結論...82
第五章 參考資料...85

圖目錄
圖1 液晶相圖...2
圖2 液晶特殊光學訊號...2
圖3 液晶分子結構...3
圖4 熱致型液晶...4
圖5 液晶型分析系統檢測機制 ...5
圖6 交叉偏光板顯微鏡成像原理示意圖...6
圖7 4-cyano-4'-pentylbiphenyl...8
圖8 DMOAP...8
圖9 液晶-固體介面方法示意圖及偏光顯微鏡光學訊號圖...9
圖10 液晶-水溶液介面方法示意圖及偏光顯微鏡光學訊號圖...10
圖11 液晶液滴方法示意圖及偏光/自然光顯微鏡光學訊號圖...12
圖12 免疫分析方法示意圖...14
圖13 液晶光學訊號...15
圖14 液晶檢測元件裝置示意圖...16
圖15 三聚氰胺及其衍生物...19
圖16 免疫球蛋白G示意圖...21
圖17 抗體隨機修飾於表面可能發生情形示意圖...21
圖18 DMOAP修飾載玻片製備示意圖...34
圖19 醛基修飾載玻片製備示意圖...35
圖20 胺基修飾載玻片製備示意圖...35
圖21 卵白素修飾載玻片製備示意圖...36
圖22 不同修飾載玻片組合的液晶元件光學訊號圖及其裝置示意圖...42
圖23 不同DMOAP塗佈時間的液晶元件光學訊號圖...43
圖24 紫外光照射時間於光學訊號影響結果圖及表面修飾示意圖...45
圖25 聚乙烯亞胺分子結構及其常見片段...46
圖26 不同聚乙烯亞胺溶液濃度的液晶元件光學訊號圖...47
圖27 牛免疫球蛋白抗體於不同官能基修飾載玻片實驗結果圖...49
圖28 界面活性劑對牛免疫球蛋白抗體於官能基修飾載玻片影響結果圖...50
圖29 界面活性劑於液晶光學訊號影響實驗結果圖...52
圖30 牛免疫球蛋白對牛免疫球蛋白抗體修飾之不同官能基修飾載玻片的免疫結合力影響實驗結果圖...53
圖31 直接型免疫分析方法檢測三聚氰胺示意圖...55
圖32 氧化三聚氰胺一級抗體檢測晶片光學訊號圖及螢光結果圖...56
圖33 不同濃度氧化三聚氰胺抗體修飾於胺基載玻片實驗結果圖...57
圖34 直接型免疫分析方法檢測三聚氰胺實驗結果圖...58
圖35 三聚氰胺於胺基修飾載玻片實驗結果圖...58
圖36 間接型免疫分析方法檢測三聚氰胺示意圖...59
圖37 Tween20最佳添加濃度實驗結果圖...60
圖38 間接型免疫分析方法檢測三聚氰胺實驗結果圖...61
圖39 不同液晶元件於三聚氰胺檢驗影響實驗結果圖...62
圖40 特異性免疫結合實驗結果圖...64
圖41 提升靈敏度間接型免疫分析示意圖...65
圖42 卵白素提升間接型免疫分析方法靈敏度實驗結果圖...66
圖43 競爭型免疫分析方法檢測三聚氰胺示意圖...67
圖44 修飾三聚氰胺於醛基修飾載玻片實驗示意圖...69
圖45 修飾三聚氰胺於醛基修飾載玻片實驗結果圖...69
圖46 三聚氰胺抗體於三聚氰胺修飾表面實驗結果圖...70
圖47 利用牛血清蛋白及戊二醛修飾三聚氰胺實驗示意圖...71
圖48 牛血清蛋白修飾載玻片實驗結果圖...72
圖49 三聚氰胺抗體於三聚氰胺-牛血清蛋白修飾載玻片實驗結果圖...73
圖50 生物素共軛三聚氰胺結合於卵白素修飾載玻片實驗示意圖...74
圖51 生物素共軛三聚氰胺合成途徑示意圖...75
圖52 卵白素修飾載玻片實驗結果圖...76
圖53 T20濃度於生物素共軛三聚氰胺抗體檢測晶片影響結果圖...77
圖54 競爭型免疫分析方法檢測三聚氰胺實驗結果圖...78
圖55競爭型免疫分析方法莫爾數調整後檢測三聚氰胺實驗結果圖...80

表目錄
表 1 實驗藥品清單...27
表 2 實驗蛋白質清單...28
表 3 實驗材料...29
表 4 實驗儀器...29
表 5 官能基修飾載玻片水珠接觸角...48
表 6 三聚氰胺、生物素共軛三聚氰胺及卵白素莫爾數比較表...79
表 7 調整後三聚氰胺、生物素共軛三聚氰胺及卵白素莫爾數表...80
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