近年來，大部分的螢光增益探針多應用於酵素分析，其機制主要是透過斷鍵或重排反應以達到螢光增益的效果。然而，這種方式無法應用於偵測無催化能力的非酶蛋白。本論文分成兩個部分，在第一部分中，我們開發出一種具有自解離連接器雙氟苯酚酯基 (self-immolative difluorophenyl ester) 的螢光探針以模擬酵素受質解離的過程，快速分析非酶蛋白。雖然已有許多自解離試劑應用於傳感器 (sensor) 、藥物傳遞與材料化學，但是它們主要透過酵素或是反應小分子的觸發而導致自解離的產生。在我們的方法中，探針透過與特異性蛋白-配體之間的相互作用，誘導自解離基團與蛋白質的胺基酸進行化學反應，從而觸發串聯反應 (cascade reaction) ，導致螢光訊號的釋放。通過這類型的探針，可以實現細胞內以及細胞外內源性腫瘤標誌物的活體細胞成像。
在本論文的第二部分，我們開發出一種新穎的葉酸結合蛋白標記探針。葉酸結合蛋白是一種重要的膜蛋白，掌控著生物體對於葉酸的吸收。該蛋白也在許多腫瘤細胞上過度表達，因此在生物醫學上常作為一種重要的生物標記物。然而，在過往偵測蛋白質的策略中，有著耗時、高成本以及無法應用於活體細胞等問題，為了解決這些缺點，我們運用配體導向化學的概念開發出能夠快速標記葉酸結合蛋白的螢光探針，該探針擁有高穩定性與良好的反應性。在細胞影像上，此類探針也成功地標記到細胞膜上的葉酸結合蛋白，並在細胞裂解實驗中證明探針的高度專一性與靈敏性。我們相信這種新穎的探針設計能夠成為生物學研究、藥物開發以及醫學診斷的有力工具。

Currently most fluorogenic probes are developed for the analysis of enzymes, where a bond breaking or rearrangement reaction is required to transform a non-fluorescent enzymatic substrate into a fluorescent product. However, this approach cannot be used for proteins which do not possess enzymatic activities. In the first part of thesis, we show that fluorogenic probes with a self-immolative difluorophenyl ester linker can mimic the bond disassembly processes of fluorogenic enzyme substrates for the rapid analysis of non-enzymatic proteins. Although numerous self-immolative reagents have shown promising applications in sensors, drug delivery systems, and material chemistry, all of them are triggered by either enzymes or small reactive molecules. In our approach, the probe binds to the protein via a specific protein–ligand interaction, inducing a chemical reaction between the self-immolative linker and an amino acid of the protein, thereby triggering a cascade reaction that leads to the release and activation of fluorogenic reporter. With this probe design, live cell imaging of extracellular and intracellular endogenous tumor marker proteins can be achieved with high selectivity and sensitivity.
In the second part, we developed a new strategy for the labeling of folate binding proteins. Folate binding proteins are important membrane proteins, which can regulate the absorption of the folic acid. Folate binding proteins are also expressed in many kinds of malignant tumors. Therefore, it is an important biomarker in biomedical science. However, there are some limitations in the existing approaches to analyze these proteins, such as time consuming, expensive and not suitable for living cells. In order to solve these problems, we developed a new labeling probe design for folate binding protein by using ligand-direct chemistry. The probe shows good stability, high reactivity and can be applied to label and imaging folate binding proteins in living cells. We believe that our novel approach would be a powerful tool in biological researches, drug discovery and medical diagnosis.

摘要.....I
Abstract.....III
謝誌......V
著作列表......VIII
第一章、 緒論......1
§ 1-1 研究動機......1
§ 1-2酶與非酶蛋白......1
§ 1-3蛋白質偵測方法......2
1-3-1 西方墨點法 (Western Blotting)......2
1-3-2 酵素免疫分析法 (Enzyme-Linked Immunosorbent Assay, ELISA)......3
第二章、文獻回顧......5
§ 2-1酶活性螢光探針......5
2-1-1 籠閉型螢光增益探針......6
2-1-2 光誘導電子轉移型螢光探針......8
2-1-3 共振能量轉移型螢光探針......11
§ 2-2非酶蛋白螢光探針......13
2-2-1 可轉換式螢光增益探針......13
2-2-2 聚集誘導螢光放光型探針......18
2-2-3 信標式蛋白質螢光探針......21
第三章、螢光探針之設計與探討......23
§ 3-1 探針構想......23
§ 3-2 自解離探針設計與偵測策略......26
第四章、實驗結果及討論......29
§ 4-1探針1對人類碳酸酐酶之螢光測試.....29
§ 4-2 探針1之選擇性及靈敏度測試......32
§ 4-3 探針1之穩定性與動力學測試......35
§ 4-4 探針1之細胞影像......39
4-4-1 探針1之MCF7細胞影像......39
4-4-2 探針1之A549細胞影像......40
§ 4-5 自解離探針模型之延伸......41
4-5-1 探針3之螢光測試......42
4-5-2 探針3之靈敏度與選擇性測試......44
第五章、文獻回顧與探針設計......46
§ 5-1 葉酸結合蛋白......46
§ 5-2 葉酸結合蛋白之偵測與應用......47
5-2-1 免疫組織化學染色法......48
5-2-2 TMP-tag標記技術......50
5-2-3 活體螢光顯影技術......51
§ 5-3 探針構想......52
§ 5-4配體導向螢光探針設計......55
第六章、實驗結果及討論......57
§ 6-1 探針5~10與DHFR反應活性與反應速率測試......57
6-1-1 探針5~10與DHFR之反應活性測試......57
6-1-2 探針5~9之反應速率測試......61
§ 6-2 探針5之選擇性測試......62
§ 6-3 探針5之穩定度測試......64
§ 6-4 探針5之細胞實驗......67
6-4-1 探針5之Hela細胞影像......67
6-4-2 探針5之MCF7細胞影像......68
6-4-3 探針5之細胞裂解實驗......69
第七章、實驗結論......72
第八章、實驗部分......73
§ 8-1 實驗藥品與器材......73
§ 8-2 螢光測試條件......74
§ 8-3 蛋白質表現及純化......74
8-3-1 蛋白質表現......74
8-3-2 蛋白質純化 (Protein Purification)......75
8-3-3 SDS-PAGE膠體電泳......76
§ 8-4 細胞培養及細胞影像實驗......78
8-4-1 培養基及試劑......78
8-4-2 細胞繼代培養 (Cell Subculture)......79
8-4-3 細胞影像 (Cell Imaging)......79
8-4-4 細胞裂解液 (Cell Lysates)......81
第九章、參考文獻.......82
附錄......86

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