開發SNAP-tag及抗生物素蛋白結合的螢光探針以提升螢光訊號及抑制非專一性訊號

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研究生：	高若晴 Kao, Ruo Ching
論文名稱：	開發SNAP-tag及抗生物素蛋白結合的螢光探針以提升螢光訊號及抑制非專一性訊號 Nonspecific Signal Suppression and Fluorescence Amplification of Fluorescence Probes by SNAP-tag and Avidin
指導教授：	陳貴通 Chen, Kui Thong
口試委員:	黃郁棻 Huang, Yu Fen 王聖凱 Wang, Sheng Kai
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：	2016
畢業學年度：	104
語文別：	中文
論文頁數：	90
中文關鍵詞：	螢光、探針、專一性、血漿、血清
外文關鍵詞：	fluorescence, probe, specificity, plasma, serum
相關次數：	點閱：2 下載：0
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儘管螢光探針在偵測酵素和活性小分子的應用上不斷的突破，兩個基本的問題還是有待解決：(一)許多染料在水溶液中的螢光低弱；(二)在高濃度蛋白質的樣品內受到的非專一性訊號。在本篇論文當中，我們將介紹一個新穎的概念：“包裹在蛋白質空腔內的螢光探針”- fluorescent probe encapsulated in protein cavity (FPEPC)。我們在環境敏感性分子修飾上BG (benzylguanine)基團或者生物素(biotin)基團，BG會和SNAP-tag蛋白質以共價鍵的方式鍵結，而生物素和抗生物素蛋白(avidin)是目前所知最強的非共價鍵親和力。透過將探針和SNAP-tag蛋白質或抗生物素蛋白結合，我們成功的在血漿以及血清樣品內偵測硝基還原酶和硫化氫。當沒有SNAP-tag或抗生物素蛋白與環境敏感性探針結合時，會因為非專一性訊號的干擾而高估待測物濃度。另外一方面，透過與SNAP-tag的結合也大幅度的提升探針螢光訊號，因此提高了偵測靈敏度。相較於傳統利用聚合物或奈米粒子包覆螢光探針的方法，本論文中提出的概念，簡單並克服了許多問題，像是染料滲漏、長時間且複雜的準備步驟以及只能偵測小分子的限制。

Despite the promising improvements made recently in fluorescence probes for enzymes and reactive small molecules detection, two fundamental problems remain: weaker fluorescence of many dyes in aqueous buffers and strong nonspecific signals in samples containing high protein levels. In this paper, we introduce a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAP-tag and avidin protein. The probes were constructed by following the current probe design for enzymes and reactive small molecules, but with an additional benzylguanine moiety for selective SNAP-tag conjugation or a short biotin linker for the conjugation to avidin protein. The SNAP-tag conjugated probes or avidin conjugated probes achieved quantitative nitroreductase and hydrogen sulfide detection in blood plasma and serum, whereas analyte concentrations were overestimated when bare fluorescent probes were employed for detection. Furthermore, detection sensitivity was increased dramatically as our probes displayed fluorescence enhancement upon SNAP-tag conjugation. In contrast to conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple and general approach successfully overcome many key issues such as dye leakage, long preparation steps and limited application to detect only small metabolites.

目錄
摘要    i
Abstract    ii
謝誌    iii
著作列表    v
縮寫對照表    vi
目錄    x
第一章 緒論    1
1-1生物分子(Biomolecules)    1
1-2小分子螢光增益探針(Small-Molecule Fluorogenic Probes)    3
1-2-1鎖住基團的螢光分子(Block group fluorophore)    4
1-2-2螢光共振能量轉移(Förster resonance energy transfer, FRET)    5
1-2-3自組裝/解離(Self-Assembly / Disassembly)    7
1-2-4聚集誘導放光 (Aggregation-Induced Emission, AIE)    8
1-2-5染料摻雜型奈米探針(Dye-doped nanoparticles)    9
1-2-5.1染料包覆型聚合物奈米探針(polymer-based dye-doped nanoparticles)    10
1-2.5.2染料包覆型矽奈米探針(dye-doped silica nanoparticles)    12
1-2-6環境敏感性分子(Environment-sensitive fluorophore)    13
1-2-6.1黏度敏感螢光分子(Viscosity-sensitive fluorophore)    13
1-2-6.2極性敏感螢光分子(Polarity-sensitive fluorophore)    17
1-3 實驗動機及目的    20
第二章 文獻回顧    23
2-1硝基還原酶之螢光偵測    23
2-2硫化氫之螢光偵測    25
2-2-1. 親核反應型探針(nucleophilic reaction)    26
2-2-2. 硫化物沉澱(sulfide precipitation)    28
2-2-3. 疊氮基還原反應的探針(Azide reduction-based probes)    28
2-2過氧化氫之螢光偵測    30
第三章 SNAP-tag結合式螢光探針    33
3-1探針設計    33
3-1-1 探針構想    33
3-1-2 探針設計    36
3-1-3 SNAP-tag蛋白質純化    37
3-2結果與討論    38
3-2-1螢光探針性質測試及討論    38
3-2-2血漿和血清的螢光測試    48
第四章 抗生物素蛋白結合式螢光探針    59
4-1探針設計    59
4-1-1探針構想    59
4-1-2 探針設計    61
4-2結果與討論    62
4-2-1螢光探針性質測試及討論    62
4-2-2 血清的螢光測試    67
第五章 結論    72
第六章 實驗部分    73
6-1實驗藥品與儀器    73
6-2 蛋白質純化與表現    74
6-2.1 配製儲備液 (Stock solution) 及緩衝溶液 (緩衝溶液)    74
6-2.2 蛋白質表現 (Protein Expression)    76
6-2.3 蛋白質純化 (Protein Purification)    77
6-2.4 SDS-膠體電泳 (SDS-Page)    78
6-3 螢光偵測實驗流程    79
6-3.1硝基還原酶偵測程序    79
6-3.2硫化氫偵測程序    80
6-3.2過氧化氫以及葡萄糖偵測程序    80
6-4 HPLC追蹤實驗    81
6-5膠體電泳螢光(in-gel fluorescence scanning)    81
6-6 血清及血漿實驗    82
6-6.1血漿    82
6-6.2血清    82
6-6.3回收率測試    82
文獻參考    84


                                

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