螢光增益探針由於能快速且靈敏地偵測目標分析物，使此類型探針在生物與醫學研究上逐漸受到重視。近年來，許多螢光分子被用來偵測酶蛋白，透過酶的催化反應使探針中的非螢光基團受質結構改變以產生螢光訊號。然而，因非酶蛋白不具催化能力，發展快速偵測非酶蛋白的螢光增益探針仍有諸多限制。本論文發展出新型的蛋白質偵測策略，藉由在探針中引入二氟苯酚作為反應基團以增加探針反應性。當加入目標蛋白後，蛋白質表面的胺基酸殘基和探針中的反應基團進行親核取代反應後斷鍵，再經由自解離機制釋放出螢光基團，依螢光訊號改變量來達到偵測目的。本論文成功以人體碳酸酐酶 (hCA) 及抗生物素蛋白 (avidin) 作為此概念的展示。此種檢測方式具有普遍性，只需更換蛋白質所對應的配體即可檢測不同蛋白質，另外，此類探針具有高選擇性與高靈敏度的優點，且能夠快速檢測，極具有應用潛力。

Currently, most of the fluorogenic probes are designed for the detection of enzymes which work by converting the non-fluorescence substrate into the fluorescence product via an enzymatic reaction. On the other hand, the design of fluorogenic probes for non-enzymatic proteins remains a great challenge. Herein, we report a general strategy to create protein-activated fluorogenic probes based on difluorophenyl pivalate chemistry. In this approach, difluorophenyl pivalate will act as a reactive moiety in which the binding of the target protein would attack the reactive group to trigger fluorescence enhancement. This difluorophenyl pivalate chemistry allows us to study different proteins by changing specific ligand such as biotin and benzenesulfonamide. We expect that this novel protein detection method will be useful for rapid fundamental research and disease diagnosis.

摘要 I
Abstract II
謝誌 III
第一章、緒論 1
1-1 蛋白質 1
1-1.1 酶與非酶蛋白 1
1-2 蛋白質偵測方法 2
1-2.1 質譜分析法 3
1-2.2 免疫分析方法 4
第二章、文獻回顧 7
2-1 酶激活螢光探針 7
2-1.1 反應型籠閉螢光探針 7
2-1.2 共振能量轉移螢光探針 10
2-2 非酶蛋白螢光探針 12
2-2.1 自組裝螢光探針 12
2-2.2 環境敏感型螢光探針 16
2-3 螢光探針限制 22
第三章、蛋白質探針之設計構想 23
3-1 探針設計構想 23
3-2 探針合成設計與策略 25
3-3 人類碳酸酐酶 27
第四章、實驗結果及討論 28
4-1 hCA探針之測試及討論 28
4-1.1 探針11、16及22之合成 28
4-1.2 探針11、16及22之螢光測試比較 31
4-1.3 探針11、22測試及比較 34
4-1.4 探針11之測試與討論 38
4-1.5 探針11細胞測試 40
4-2 模型之延伸 43
4-2.1 連接基團長度對偵測效果的影響 45
4-2.2 對抗生物素蛋白探針之設計 51
第五章、實驗結論 56
第六章、實驗部分 57
6-1 實驗藥品及器材 57
6-2 有機合成與光譜資料 59
第七章、參考文獻 91
附錄 96

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