臺灣博碩士論文加值系統

Guanosine 3,5-cyclic monophosphate由guanosine triphosphate經solube和particulate guanylyl cyclase轉化生成，為細胞內重要之二級訊息傳遞物(secondary messenger)，目前常用Enzyme Immunoassay (EIA)及放射性同位素(radioactive isotope)來進行cGMP的測量，其中Immunoassay的操作程序繁瑣而放射性同位素的使用具危害性，因此本研究的目的利用區域性表面電漿共振之原理開發一種既經濟且安全測量cGMP之方法，將Anti-cGMP之抗體利用共價鍵結固定於金奈米粒子修飾過後的光纖上，用來檢測標準試液及血管平滑肌細胞內cGMP的濃度。實驗結果顯示，製備探針之較佳條件為使用抗體稀釋倍數1:250及浸泡時間2 h。特別的是本研究新開發之探針可偵測cGMP標準液的範圍在2.5×10-3-102 pM之間，其測量cGMP的靈敏度比EIA分析高；在測量完後，探針上之cGMP可使用DPBS (pH 6) 含0.05 % Tween 20清除並且探針可重複檢測cGMP 濃度在20 pM以下的未知樣品。除此之外，將製備好的探針存放在4℃ 1 週後其敏感度並沒有明顯降低的現象。因為乙醯化的cGMP對cGMP 抗體具有高的親合力，利用探針偵測量乙醯化的cGMP 其敏感度較cGMP的測量高於3 個數量級。最後，探針偵測人類動脈平滑肌細胞內之cGMP濃度與常用之cGMP EIA測量數值相當，證實了探針測量的可性度。

Guanosine 3,5-cyclic monophosphate (cGMP) produced from guanosine triphosphate (GTP) via soluble guanylyl cyclase (sGC) and particulate guanylyl cyclase (pGC) is an important secondary messenger in cells. Commonly used methods for measurement of cGMP are the competitive enzyme immunoassay (EIA) and radioimmunoassay. However, the protocol for EIA is tedious and the radioimmunoassay is unsafe for personnel. To overcome the above disadvantages, we developed an optical fiber modified with a self-assembled monolayer of nano-gold particles conjugated with anti-cGMP antibody to determine cGMP concentration based on localized surface plasmon resonance (LSPR). The optimal antibody dilution and incubation time for preparing the probe are 1:250 and 2 h, respectively. Particularly, this senor shows much better sensitivity than EIA with a cGMP detection range of 2.5×10-3-102 pM. After measurement, the cGMP bound to the probe can be washed out using DPBS (pH 6) containing 0.05% Tween 20 and the probe can be reused once for the unknown sample with cGMP concentration less than 20 pM. In addition, the prepared probe can be stored at 4˚C up to 1 week without losing significant sensitivity. Because acetylated cGMP has higher affinity with cGMP antibody than cGMP, the sensitivity of measuring acetylated cGMP by the probe is increased by 3 orders of magnitude as compared to the measurement of cGMP. Finally, the reliability of the probe was confirmed by measuring the cGMP concentration in the cell extract of human aortic smooth muscle cells under various treatments using a commercial cGMP EIA kit.

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中文摘要…………………………….……………………………….....I
英文摘要…………………………….……………………………….....II
目錄……………………………….……………………………….........III
圖目錄…….................…………….……………………………….........IX
表目錄.....................................................................................................XIV
符號……………………………..…..………...…....…….…...…..….…XV

第一章 緒論………….………………………….………………...…...1
1.1 前言…………………………………………….…………………..1
1.2 文獻回顧………………………………………………………..….2
1.2 表面電漿共振原理……………………………………………..2
1.2.1 漸逝波技術……...………………………………………...….3
1.2.2 表面電漿共振現象………...…………………………………5
1.2.3 光纖簡介.…………………………………………………....10
1.2.4 光纖構造…………………………………………………….11
1.2.5 光纖分類………………..………………………………...…12
1.2.6 光纖傳輸原理…………….............................................……13
1.2.7 探針固定化………………………………………………….15
1.2.8 自我排列單分子層………………………………………….18
1.2.9 金奈米粒子………………………………………………….19
1.2.10 金奈米粒子製備…………………………………………...19
1.2.11 金奈米粒子之光學特性原理………………………...……20
1.2.12 光纖式表面電漿之應用及相關理論公式………………...21
1.2.13 金奈米粒子表面電漿之應用……………………………...22
1.2.14 一氧化氮的生成……................................………………...23
1.2.15 二級訊息傳遞cGMP產生………………………………...23
1.2.16 二級訊息cGMP傳遞造成血管張力的調控……………...25
1.2.17 血管平滑肌細胞……....................………………………...28
1.2.18 放射性同位素分析RIA(radioactive isotope immunoassay)原理…………..............................……………..............…..….29
1.2.19 放射性同位素測定方法………………...…………………29
1.2.20 酵素免疫分析法ELISA原理................................................31
1.2.21 ELISA分析方法....................................................................31
1.2.22 ELISA與RIA分析比較.........................................................32
1.3 研究動機與目的………………………………………………….33

第二章 實驗儀器、器材與藥品……………………….……..….…..34
2.1 實驗儀器……………………………………………………….…34
2.1.1 一般儀器…………………………………………………….34
2.1.2 光學儀器…………………………………………………….35
2.2 實驗器材……………………………………………………….....36
2.2.1 培養細胞材料……………………………………….………36
2.2.2 光纖材料…………………………………………………….36
2.3 實驗藥品………………………………………………….…….....37
2.3.1 細胞培養…………………………………...……...................37
2.3.2 cGMP濃度測定…………………………………...…….......37
2.3.3 蛋白質濃度測定……………………………....………….....38
2.3.4 金奈米光纖測定……………………………………...……..39
2.4 實驗細胞………………………………………………..…...……40

第三章 實驗方法………………………………………..…….……...41
3.1 老鼠胸部大主動脈血管平滑肌細胞的培養.................................41
3.1.1 不同濃度之NO對血管平滑肌細胞產生cGMP的影響.......45
3.1.2 時間的變化NO對血管平滑肌細胞產生cGMP的影響.......46
3.1.3 時間的變化phorone對血管平滑肌細胞產生cGMP的影響……………………………………………...……………..47
3.1.4 Ebselen對血管平滑肌細胞產生cGMP的影響....................48
3.1.5 破碎細胞.................................................................................49
3.1.6 蛋白質濃度測定.....................................................................49
3.2 金奈米粒子之合成、表面修飾化、固定化.................................50
3.2.1 金奈米粒子之合成.................................................................50
3.2.2 光纖表面修飾化.....................................................................51
3.2.3 感測系統偵測蔗糖溶液.........................................................52
3.2.4 光纖表面修飾Anti-cGMP偵測cGMP.................................53
3.2.5 光纖表面Anti-cGMP的活性.................................................57
3.2.6 有乙醯化及無乙醯化cGMP的檢測......................................58
3.2.7 光纖不同表面修飾檢測cGMP...............................................58
3.2.8 光纖表面cGMP的清除..........................................................58
3.2.9 利用ELISA方法偵測cGMP..................................................59

第四章 實驗結果與討論……………………………………..…....…66
4.1 製備金奈米粒子的吸收光譜.........................................................66
4.2 反射式光纖感測方法.....................................................................71
4.2.1 分別對不同濃度的蔗糖溶液.................................................71
4.3 計算偵測極限(limit of detection：LOD).........................................74
4.4 光纖上鍵結抗體偵測cGMP...........................................................75
4.4.1 光纖上鍵結抗體浸泡不同時間偵測cGMP...........................75
4.4.2 光纖上鍵結抗體不同濃度偵測cGMP...................................76
4.5 利用不同的pH值Washing buffer將光纖上cGMP清洗..............76
4.6 抗體存放不同時間下活性探討.....................................................77
4.7 cGMP標準液未乙醯化及乙醯化之檢測......................................78
4.8 光纖不同表面修飾檢測cGMP.......................................................84
4.9 測量NO釋放劑對血管平滑肌細胞產生之cGMP........................86
4.9.1 不同濃度之NO釋放劑對血管平滑肌細胞產生cGMP之影響.............................................................................................86
4.9.2 NO釋放劑在不同時間對血管平滑肌細胞產生cGMP之影響.............................................................................................86
4.10 phorone對細胞產生cGMP的影響..............................................89
4.11 Ebselen對細胞產生cGMP的影響..............................................90
4.12 用ELISA方法偵測cGMP............................................................91
第五章 結論未來展望………………………………….………….....98
5.1 結論…………………….………………………….….………......98
5.2 未來展望…………………………....……………..….…………..99

附錄………………….……………………………………….……......100
附錄I 浸泡抗體混合液不同時間檢測cGMP實驗值………….......100
附錄II 不同稀釋倍數之抗體檢測cGMP實驗值..............................103
附錄III 光纖最佳狀態檢測cGMP實驗值........................................106
附錄IV 抗體活性檢測實驗值...........................................................107
附錄V 有乙醯化及無乙醯化cGMP檢測實驗值...........................109
附錄VI 不同鍵結物修飾光纖檢測實驗值.......................................110
附錄VII 一氧化氮對血管平滑肌細胞產生cGMP影響之檢測實驗值………………………………………………………....112
附錄VIII phorone 對血管平滑肌細胞產生cGMP影響之檢測實驗值........................................................................................113
附錄IX Ebselen 對血管平滑肌細胞產生cGMP影響之檢測實驗值........................................................................................114

參考文獻………………….………………………………………...…115












圖目錄

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圖1.1 Snell's Law……............................................…………….….......3
圖1.2 光波與表面電漿波之關係圖…………........…………....……...6
圖1.3 表面電漿波感應圖…...................................................................7
圖1.4 利用Kretschmann組態激發表面電漿波示意圖…............…....9
圖1.5 利用Otto組態激發表面電漿波示意圖….................................10
圖1.6 光纖基本結構圖.............................……………………….…...11
圖1.7 光纖折射率分佈情形………………...................……………..12
圖1.8 光耦合入光纖角度….......................................................….….14
圖1.9 三種不同金表面固定化.............................................................16
圖1.10 金奈米表面固定化聚葡素(dextran)分子的示意圖..................17
圖1.11 內皮細胞NO釋放圖..................................................................24
圖1.12 活化PKG造成血管舒張及縮收圖............................................26
圖1.13 一氧化氮進入血管平滑肌細胞後，產生傳遞訊號路徑圖.......27
圖1.14 sGC及pGC之活化圖..............................................................28
圖1.15 RIA分析原理............................................................................30
圖1.16 ELISA 分析原理......................................................................32
圖3.1 加入IBMX 防止cGMP水解圖.................................................46
圖3.2 合成金奈米溶液流程圖............................................................51
圖3.3 光纖修飾及金奈米的鍵結圖.....................................................55
圖3.4 抗體混合液的鍵結圖................................................................55
圖3.5 光纖鍵結抗體圖........................................................................56
圖3.6 反射式感應系統裝置圖............................................................57
圖3.7 ELISA酵素反應圖.....................................................................62
圖3.8 ELISA酵素反應及cGMP鍵結圖...............................................63
圖3.9 化合物結構圖............................................................................64
圖3.10 實驗總流程圖..........................................................................65
圖4.1 金奈米溶液於氯仿溶液之吸收光譜........................................69
圖4.2 光纖檢測不同濃度之蔗糖水溶液實驗值.............................…72
圖4.3 光纖檢測不同折射率之蔗糖水溶液….................................…73
圖4.4 光纖修飾不同濃度抗體檢測cGMP半對數圖…………........ 79
圖4.5 光纖浸泡抗體不同時間檢測cGMP半對數圖……...........…..80
圖4.6 光纖上cGMP之清洗.................................................................81
圖4.7 抗體於置放不同時間下之活性.................................................82
圖4.8 乙醯化及無乙醯化cGMP標準液檢測.....................................83
圖4.9 光纖表面不同修飾物檢測cGMP標準液.................................85
圖4.10 不同濃度之NO釋放劑對血管平滑肌細胞產生cGMP的影響..............................................................................................87
圖4.11 NO釋放劑在不同時間對血管平滑肌細胞產生cGMP的影響...............................................................................................88
圖4.12 不同時間phorone對血管平滑肌細胞產生cGMP的影響.....93
圖4.13 Ebselen對血管平滑肌細胞產生cGMP的影響......................94
圖4.14 利用ELISA方法偵測的cGMP標準液.....................................95
圖4.15 用ELISA分析比較反射式感應偵測﹕加入不同濃度NO釋放劑...............................................................................................96
圖4.16 用ELISA分析比較反射式感應偵測﹕固定NO釋放劑濃度置放不同時間...............................................................................96
圖4.17 用ELISA分析比較反射式感應偵測﹕用phorone處理細胞置放培養箱30 min.......................................................................97
圖4.18 用ELISA分析比較反射式感應偵測﹕用Ebselen處理細胞置放培養箱30 min.......................................................................97

附錄圖....................................................................................................100
圖I.1 光纖浸泡抗體混合液0.5 h檢測不同濃度之cGMP實驗值...100
圖I.2 光纖浸泡抗體混合液1 h檢測不同濃度之cGMP實驗值......100
圖I.3 光纖浸泡抗體混合液1.5 h檢測不同濃度之cGMP實驗值...101
圖I.4 光纖浸泡抗體混合液2 h 檢測不同濃度之cGMP實驗值....101
圖I.5 光纖浸泡抗體混合液4 h 檢測不同濃度之cGMP實驗值....102
圖II.1 光纖修飾抗體原液稀釋100倍檢測cGMP實驗值................103
圖II.2 光纖修飾抗體原液稀釋250倍檢測cGMP實驗值................103
圖II.3 光纖修飾抗體原液稀釋500倍檢測cGMP實驗值................104
圖II.4 光纖修飾抗體原液稀釋1000倍檢測cGMP實驗值..............104
圖II.5 光纖修飾抗體原液稀釋2000倍檢測cGMP實驗值..............105
圖III.1 光纖檢測不同濃度之cGMP實驗值......................................106
圖IV.1 光纖修飾抗體incubate 4 ℃ 1 week檢測cGMP ...................107
圖IV.2 光纖修飾抗體incubate 4 ℃ 2 week檢測cGMP...................107
圖IV.3 光纖修飾抗體incubate 4 ℃ 4 week檢測cGMP...................108
圖V.1 反射式感應系統檢測無乙醯化cGMP標準液......................109
圖V.2 反射式感應系統檢測有乙醯化cGMP標準液......................109
圖VI.1 裸光纖檢測不同濃度cGMP……...........................................110
圖VI.2 光纖直接修飾抗體檢測不同濃度cGMP...............................110
圖VI.3 光纖直接修飾金奈米檢測不同濃度cGMP...........................111
圖VI.4 光纖修飾金奈米及抗體檢測不同濃度cGMP.......................111
圖VII.1 不同濃度之一氧化氮釋放劑對血管平滑肌細胞產生cGMP.......................................................................................112
圖VII.2 一氧化氮釋放劑在不同時間對血管平滑肌細胞產生cGMP.......................................................................................112
圖VIII.1 phorone預處理時間為30 min對血管平滑肌細胞產生cGMP………...........................................................................113
圖VIII.2 phorone預處理時間為60 min對血管平滑肌細胞產生cGMP………...........................................................................113
圖IX.1 Ebselen對血管平滑肌細胞產生cGMP實驗值......................114













表目錄
表4.1 可見光部份的波長與其所顯示的各種顏色及其互補色.........70

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