蛋白質在細胞中的位置、轉移與交互作用等，往往提供了生物學研究重要訊息。而利用螢光方式對蛋白質進行標記較為靈敏且方便觀測，是最容易應用於生物體內的方法之一。1990年代螢光蛋白質的發展對蛋白質標記技術造就卓越的貢獻，利用本身具有螢光的蛋白質以基因轉殖方式可以精確地標記在細胞表面或胞器上，但其光穩定性、螢光強度、分子量大小等問題，限制了其發展。以螢光分子標記蛋白質的方式不僅選擇多，更可以有效控制標記的時間與位置，是最具有發展潛力的標記方式。然而，過高的背景訊號是一直以來的問題，常需透過清洗步驟洗去未結合之螢光物。清洗步驟會影響我們對樣品觀測的真實性與即時性。因此，發展免清洗的螢光分子標記探針是蛋白質標記所亟需。
在本研究中，我們以SBD（4-Sulfamonyl-7-aminobenzoxa-diazole）環境敏感螢光分子發展SNAP-tag蛋白之螢光增益型探針，並成功標記於融合SNAP-tag蛋白的細胞表面與細胞內部。由於我們的螢光探針只有在與目標蛋白反應後，螢光團被引入SNAP-tag蛋白質疏水區域時才會產生螢光，因此可以免除標記後的清洗步驟，螢光增益效果甚至可達280倍，可有效降低背景訊號的影響，並具有標記速率快、易於合成、可穿透細胞膜、高選擇性等優點。由於探針顯著的螢光增益性，我們以類似的結構，更換為生物素與抗生物素蛋白系統（Biotin-avidin system, BAS），發展螢光增益蛋白質辨識。目前我們已成功開發螢光增益抗生物素蛋白探針，並預期未來能改善蛋白質辨識訊號放大等問題。

To study protein functions and dynamics such as motion and localization in living cells, protein labeling by synthetic fluorescent probes is a powerful technique. We developed novel SNAP-tag labeling fluorescent probes using environment-sensitive fluorophore to achieve large fluorescence enhancement, high cellular permeability, quick reaction rate, and no-wash labeling. In our strategy, we attached environment-sensitive fluorophore, 4-sulfamonyl-7-aminobenzoxadiazole (SBD), to O6-benzylguanine (BG), substrate of SNAP-tag protein, to construct fluorogenic probes. The environment-sensitive fluorophore exhibits very weak fluorescence in polar environment while increases fluorescence dramatically as it is introduced to the protein hydrophobic domain. Our method was successfully applied in living cells to image SNAP-tag expressed membrane, cytosol, and nucleus with no additional washout procedure and also feasible for flow cytometric analysis. We believe this method will be an attractive tool for tracking dynamic protein behaviors and various biological studies. Furthermore, we applied the similar probe design to biotin-avidin system (BAS), a high affinity ligand-protein interaction used in versatile immunoassays and tumor cell diagnoses, by changing the BG moiety to biotin. Our aim is to develop a method for signal turn-on protein recognition. So far, we have successfully created several biotinylated probes to achieve good fluorescence enhancement upon binding to avidin. We expect that this method will contribute to a widespread bioanalytical applications.

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