一、使用分子信籤感測器通過非酵素的方法來偵測肝素裡的過硫酸化硫酸軟骨素
肝素(Heparin)是一種具有多電荷的葡萄糖胺多聚醣(Glycosaminoglycan)，在醫療上常用來當抗凝血劑使用，在2008 年，報導指出有數十人因施打肝素導致休克而死亡，上百人出現不同程度的過敏反應，美國食品暨藥物管理局(FDA)進而對肝素進行研究，發現裡面含有結構相似的汙染物-過硫酸化硫酸軟骨素(OSCS)，此汙染物無法使用一般的偵測方法檢驗出所以本實驗設計一種高選擇性、靈敏度且快速簡單的分子信籤(Molecular Beacon)來偵測肝素裡的汙染物，使用5’與3’端帶有螢光團(FAM)與消光團(DABCYL)的分子信籤，其loop 端含有22個鹼基，stem端各含有8個腺嘌呤(Adenine，A)鹼基，加入coralyne 會在stem 端產生A2-coralyne-A2 的錯合物，使分子信籤產生髮夾彎的結構，致使螢光團與消光團碰撞產生消光現象，藉由加入Ca2+離子，OSCS會比Heparin帶有更多的負電荷位置，所以跟polyA8競爭coralyne的程度不同，進而將不同數量的corlayne置換出來，利用螢光恢復程度的差異即可分辨肝素裡的汙染物OSCS。本實驗在實際注射液可偵測到0.01%的OSCS，代表此偵測方法步驟簡單，具高靈敏度、選擇性且不須使用肝素酵素(Heparinase)即可快速地去偵測肝素裡的汙染物。

二、利用分子信籤感測器偵測第二信使c-di-AMP與訊號分子Ap5A
c-di-AMP是細菌裡重要的第二信使，可以調控多種生理功能如鉀離子的輸送、脂肪酸的形成、細胞壁的平衡、DNA損傷的偵測等功能；其結構為兩端各含一個腺嘌呤中間接兩個磷酸根形成環狀分子，此應用將第二信使c-di-AMP作為目標物，利用Poly A20分子信籤作為探針，5'端及3'端帶有螢光團(FAM)與消光團(BHQ1)，加入coralyne會與Poly A20形成A2-coralyne-A2二聚體，使螢光團與消光團發生螢光碰撞消光反應，接著加入目標物c-di-AMP反應，因其兩端含有腺嘌呤，可與A20·coralyne作競爭將coralyne從分子信籤中置換出來，使螢光產生回復現象，成功地偵測到c-di-AMP且LOD為0.1μM。利用同樣機制也能偵測兩端含有腺苷的類似物Ap5A，其同樣為調控數種生理機制的訊息分子，結構兩端也含有腺嘌呤，中間由五個磷酸根連接，利用相同的感測機制成功於真實眼淚樣品中偵測到Ap5A，LOD為3μM。

(a) Molecular Beacon-Based Fluorescent Assay for Specific Detection of Oversulfated Chondroitin Sulfate Contaminants in Heparin without Enzyme Treatment
Heparin belongs to a family of naturally occurring negatively charged glycosaminoglycans (GAGs) which are complex linear polysaccharides comprised of repeating disaccharide units. A sudden increase of severely adverse responses to heparin treatment of patients (including 94 deaths) was reported in U.S. during 2007-2008 period. The U.S. Food and Drug Administration’s (FDA) investigation of this problem found that the origin of these fatal side effects was oversulfated chondroitin sulfate (OSCS) that was present in some heparin products. Here we presents a highly selective, sensitive, enzyme free, simple and fast method to detect OSCS in heparin. The MB (A8–MB–A8) contains a 22-mer loop, a pair of 8-mer adenosine (A) bases at stem, a fluorophore unit at the 5’-end, and a quencher at the 3’-end. Hairpin-shaped MB with A2-coralyne-A2 complex at stem has formed after addition of coralyne. This study found that with present of Ca2+ ions can efficiently suppress the negative charges of heparin, they do not neutralize the negative charge of OSCS, the result of OSCS can remove coralyne from the MB stem more than heparin. This system can detect 0.01% w/w OSCS in heparin without enzyme treatment, also determine 0.01% w/w OSCS in the pharmaceutical heparin under 5 min.
 
(b) A Simple Molecular Beacon-based Fluorescent Sensor for Sensing Second Messenger c-di-AMP and Signalling Molecular Ap5A
c-di-AMP is an important bacterial second messenger found in Gram-positive and mycobacterial. Second messenger will enhancement, differentiation, integration and passed to the receptors to regulate physiological processes, in response to a changing environment. c-di-AMP is produced from two molecules of ATP by diadenylyl cyclase (DAC) enzyme, made up of two adenosine moieties joined in double 3’-5’ linkage by two phosphodiester. Recently, we develop a simple molecular beacon-based senor for detection of c-di-AMP. The MB contains 20-mer adenosine (A) bases, a fluorophore unit (FAM) at the 5’-end, and a quencher (BHQ1) at the 3’-end. The presence of coralyne form a hairpin-shaped MB with A2-coralyne-A2 complex. After addition of c-di-AMP, it will compete with PolyA20 and displace coralyne out, and turn on fluorescence. Under optimal conditions (10nM MB, 0.5μM coralyne), dtection limit (LOD) of c-di-AMP is 0.1μM. Same mechanism is used in signaling molecular-Ap5A. Studies had demonstrated a ubiquitous occurrence of Ap5A in the whole spectrum of organisms from bacteria to higher eukaryotes, it is made up of two adenosine moieties joined in 5’-5’ linkage by a chain of five phosphodiester linkages. The MB sensor can also detect Ap5A in tears sample, with detection limit of 3μM.

論文審定書 i
論文公開授權書 ii
摘要 iii
目錄 vii
圖次 x
表次 xi
縮寫表 xii
第 1 章 、使用分子信籤感測器通過非酵素的方法來偵測肝素裡的過硫酸化硫酸軟骨素 1
一、前言 1
1-1 藥物Heparin的介紹 1
1-2 偵測藥物肝素汙染物-OSCS的方法 1
1-3 研究動機 4
二、實驗部分 5
2-1 實驗藥品 5
2-2 化學結構 7
2-3 儀器裝置 8
2-4 樣品配置方法 9
2-5 實驗過程 10
三、結果與討論 11
3-1 感測機制的建立 11
3-2 探討鈣離子對OSCS與Heparin螢光值的影響 13
3-3 探討反應時間對螢光值的影響 15
3-4 以圓二色光譜法來分辨Heparin與OSCS 15
3-5 探討coralyne與Heparin、OSCS之鍵結常數 18
3-5 探討分子信籤尾端長度對分辨Heparin與OSCS之影響 23
3-6 探討不同金屬離子對分子信籤探針分辨葡萄糖胺多聚醣之選擇性 25
3-7 探討Ca2+離子濃度對分子信籤探針分辨葡萄糖胺多聚醣之影響 25
3-8 探討不同陰離子對分子信籤探針分辨葡萄糖胺多聚醣之影響 28
3-9 標準Heparin溶液裡定量分析OSCS 28
3-10 海派注射液裡定量分析OSCS 32
四、結論 35
五、參考文獻 37
第 2 章 、利用分子信籤感測器偵測第二信使c-di-AMP與訊號分子Ap5A 42
一、前言 42
1-1 目標物c-di-AMP與Ap5A的介紹 42
1-2 偵測c-di-AMP與Ap5A的方法 43
1-3 研究動機 45
二、實驗部分 47
2-1 實驗藥品 47
2-2 化學結構 49
2-3 儀器裝置 50
2-4 樣品配置方法 51
2-5 實驗過程 52
三、結果與討論 54
3-1 感測機制的建立 54
3-2利用Poly A20分子信籤作為螢光探針偵測c-di-AMP之螢光光譜圖 57
3-3 探討緩衝溶液最佳化條件與反應時間 59
3-4 coralyne與c-di-AMP形成二聚體之螢光光譜圖與吸收光譜圖 62
3-5 利用極化螢光鑑定coralyne．c-di-AMP二聚體之生成 66
3-6 探討coralyne與c-di-AMP之鍵結常數 68
3-7 探討不同核苷酸對實驗之選擇性影響 70
3-8 c-di-AMP的定量分析 72
3-9 相同實驗方法偵測腺苷類似物Diadenosine polyphosphates之感測機制 74
3-10 探討ApnAs中間連接之磷酸根數量影響 77
3-11利用Poly A20分子信籤作為螢光探針偵測Ap5A之螢光光譜圖 77
3-12 探討緩衝溶液最佳化條件與反應時間 80
3-13 coralyne與Ap5A形成二聚體之螢光光譜圖與吸收光譜圖 83
3-14 利用極化螢光證實coralyne．Ap5A二聚體之生成 86
3-15 標準品Ap5A的定量分析 86
3-16 在模擬淚液下對Ap5A定量分析 89
3-17 人體真實淚液裡定量分析Ap5A 90
四、結論 93
五、參考文獻 94

第1章
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第2章
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