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研究生： 陳相榮
Chen, Hsiang-Jung.
論文名稱： 可轉換式近紅外光之部分花青染料 ─ 蛋白質標記特定胞器與細胞顯影
Near-Infrared Fluorescence Switchable Merocyanine Dye for Organelle-Specific Protein Labeling and Imaging in Living Cells
指導教授： 陳貴通
Tan, Kui-Thong
口試委員: 王聖凱
Wang, Sheng-Kai
吳淑褓
Wu, Shu-Pao
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2017
畢業學年度： 105
語文別： 中文
論文頁數： 132
中文關鍵詞： 螢光探針 、部分花青染料 、細胞顯影 、不須清洗
外文關鍵詞： fluorescent probe, merocyanine, cell image, no wash
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    • 近來螢光分子探針蓬勃發展， 而多數近紅外光染料都是由花青染
    料及羅丹名所衍生。 但因為結構上有著永久性的正電荷，花青染料和羅丹
    名傾向於堆積在粒線體上，使它們不利於在特定胞器的細胞顯影上。 在本
    論文中， 我們發展一個新穎可轉換式無電荷的近紅外光部分花青染料， 與
    典型的部分花青染料做比較，他對於黏稠的環境更為敏感而且吸收及放射
    波長紅移至紅外光區。 從不同 溶劑中測量的吸收和發射光譜，我們觀察到
    相同的電子帶。因此，可以得出結論，涉及電荷轉移狀態的光化學在該系
    統中 影響是較不大的。 藉由修飾上不同的蛋白質配體（ SNAP-tag and hCAII
    proteins） ，當這些探針偵測到目標蛋白質後展現出顯著的螢光增益（ 最高
    可達到 300 倍的螢光提升） 。 巨大的螢光增益可以使我們進行細胞影像時
    免去洗滌的步驟， 並使在不同 的細胞胞器 的 SNAP-tag 蛋白質影像中達到
    最低的螢光背景。 此蛋白質探針也成功地應於觀察細胞中變異之核纖層蛋
    白。

    Most near-infrared dyes are based on the derivatives of cyanine and
    rhodamine. However, due to their permanent cationic charge, cyanine and
    rhodamine tend to accumulate preferentially in the mitochondria which make
    them not suitable for organelle-specific imaging. In this paper, we present a
    novel charge-free fluorescence switchable near-IR dye based on merocyanine.
    As compared to the classical merocyanines, the new dye is highly sensitive to
    crowded surroundings and exhibits substantial red-shifted absorption and
    emission within near-IR region. Computational study and emission spectra
    measurement in different solvents showed that photochemistry involving the
    charge transfer state is insignificant in this new dye. By incorporating a protein
    specific ligand to the dye, the probes （ for SNAP-tag and hCAII proteins）
    exhibit dramatic fluorescence increase （ up to 300-fold） upon binding with its
    target protein. The large fluorescence enhancement enabled no-wash labeling
    and imaging of the SNAP-tag protein in different subcellular compartments
    with minimum background fluorescence. This protein probe was successfully
    applied to visualize incomplete posttranslational processing of nuclear envelope
    protein, Lamin A, in living cells.

    摘要 I Abstract II 謝誌 III 著作列表 V 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 生物感測器 4 § 1-3蛋白質偵測方法 5 第二章、文獻回顧 7 § 2-1螢光探針種類 7 2-1.1 超分子自組裝螢光探針 7 2-1.2 共振能量轉移探針（Förster resonance energy transfer, FRET） 10 2-1.3 環境敏感型探針 12 2-1.4 聚集誘導螢光放射型探針 19 2-1.5 金奈米粒子探針 22 § 2-2螢光探針限制 24 第三章、蛋白質探針之設計構想 26 § 3-1 設計構想 26 § 3-2 部分花青染料的優化 26 第四章、實驗結果及討論 28 § 4-1 探針之測試與討論 28 4-1.1 探針P-Mero4與Mero4的合成 28 4-1.2 探針P-Mero4的光譜性質 29 4-1.3 理論計算 35 § 4-2 偵測hCAII的探針P-Mero4SA 38 4-1.1 hCAII 蛋白質 38 4-1.2 偵測hCAII的探針 39 4-1.3 螢光探針P-Mero4SA的選擇性 40 4-1.4 螢光探針P-Mero4SA的偵測極限 41 § 4-3 偵測SNAP-tag蛋白質 42 4-2.1 SNAP-tag 蛋白質 42 4-2.2 偵測SNAP-tag的探針P-Mero4BG 44 4-2.3 螢光探針P-Mero4BG的選擇性 47 4-2.4 螢光探針P-Mero4BG的偵測極限 48 4-2.5 In-gel 螢光分析 49 4-2.6 苯環取代對探針P-Mero4BG的影響 49 § 4-4 模型之延伸 51 4-4.1 苯環取代基的改變 51 4-4.2 親水性官能基的加入 54 § 4-5 細胞實驗 55 4-5.1 利用可標記SNAP-tag的P-Mero4BG螢光探針，可免去洗涤步驟並且針對不同細胞器的標記細胞影像 56 4-5.2 變異Lamin A的特異性標記 60 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 第七章、參考文獻 84

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