近來螢光分子探針，大部分是用在酶蛋白偵測上，如醣苷酶(glycosidases)、蛋白酶(proteases)等。其方法是透過酶的催化使探針上的非螢光基團發生結構變化，使其轉變成具螢光性質的基團。本論文開發以Cy5螢光探針為主的非酶蛋白偵測方法，在聚次甲基的γ位有苯基取代的Cy5螢光分子對蛋白質之親和性配體結合後，可不經由酶的催化，選擇性的偵測蛋白質。進一步的研究發現，此螢光探針具自組裝性質，並會形成J-型聚集使螢光淬熄。當目標分析物存在時，因配體和目標物的結合使得螢光探針的聚集拆卸，並發出強烈的近紅外螢光。基於探針的自組裝/拆卸使得螢光顯著增益的性質，本論文設計了一系列螢光探針，並可成功標記癌細胞表面之標記物：跨膜型碳酸酐酶IX。

Abstract
Currently most of the fluorogenic probes are designed for the detection of enzymes which work by converting the nonfluorescence 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 near-IR fluorogenic probes, where a small molecule ligand is conjugated to a novel γ-phenyl-substituted Cy5 fluorophore, for the selective detection of proteins through a non-enzymatic process. Detail mechanistic studies reveal that the probes self-assemble to form fluorescence-quenched J-type aggregate. In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate. This Cy5 fluorophore is a unique self-assembly/disassembly dye as it gives remarkable fluorescence enhancement. Based on the same design, three different fluorogenic probes were constructed and one of them was applied for the no-wash imaging of tumor cells for the detection of hypoxia-induced cancer-specific biomarker, transmembrane-type carbonic anhydrase IX.

目錄
摘要 I
Abstract III
謝誌 I
第一章、緒論 1
§1-1 蛋白質 (Proteins) 1
1-1.1 酶蛋白與非酶蛋白 1
§1-2 花青染料(Cyanine dyes) 3
1-2.1 花青染料簡介 3
1-2.2 常見之花青染料與其應用 5
§1-3 J-型聚集(J-aggregate) 7
§1-4 實驗動機及目的 9
第二章、文獻回顧 10
§2-1 選擇性蛋白質螢光探針 10
§2-2 髮夾型螢光探針 10
§2-3 自組裝/拆卸型螢光探針 12
2-3.1 超分子化學 12
2-3.2 聚集和拆卸 13
2-3.3 超分子自組裝螢光探針 13
2-3.4 苝拆卸聚集型螢光探針 15
§2-4 聚集誘導螢光放射型探針 17
§2-5 金奈米粒子探針 19
§2-6 環境敏感型探針 21
第三章、蛋白質探針之設計構想 23
§3-1 設計構想 23
§3-2 花青染料的優化 23
第四章、實驗結果及討論 25
§4-1 探針之測試與討論 25
4-1.1 偵測hCAII的探針1 25
4-1.2 螢光探針1的選擇性 29
4-1.3 螢光探針1的環境干擾測試 29
4-1.4 苯環取代對偵測效果的影響 30
4-1.5 探針1自體聚集的螢光性質 32
4-1.6 探針1自體聚集的物理特性 33
4-1.7 探針1的配體數量對偵測效果的影響 36
4-1.8 修飾探針1 37
§4-2 偵測SNAP-tag蛋白質 39
4-2.1 偵測SNAP-tag的Cy5型探針3 39
4-2.2 螢光探針3的選擇性 42
4-2.3 螢光探針3的反應速率 43
4-2.4 探針3的配體數量對偵測效果的影響 44
4-2.5 修飾探針3 45
§4-3 模型之延伸 46
4-3.1 對胰蛋白酶探針之設計 46
4-3.2 對生物素蛋白探針之設計 48
4-3.3 探針疏水性質研究 49
§4-4 拆卸聚集誘導放光式近紅外螢光探針對醣類偵測 51
4-4.1 探針4偵測Fructose 51
§4-5 細胞實驗 55
4-5.1 探針1的細胞顯影 55
4-5.2 探針3的細胞顯影 61
第五章、實驗結論 62
第六章、實驗部分 63
§6-1 一般實驗用品 63
§6-2 有機合成及光譜資料 65
七、參考文獻 84

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