可控結合生物素探針於超氧自由基的偵測及成像

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研究生：	簡婉菁 Chien, Wan-Ching
論文名稱：	可控結合生物素探針於超氧自由基的偵測及成像 Superoxide Anion Detection and Imaging by Streptavidin-Biotin Controlled Binding Probes
指導教授：	陳貴通 Tan, Kui-Thong
口試委員:	黃郁棻 Huang, Yu-Fen 許馨云 Hsu, Hsin-Yun
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：	2018
畢業學年度：	106
語文別：	中文
論文頁數：	108
中文關鍵詞：	可控結合、生物素探針、超氧自由基
外文關鍵詞：	Superoxide, Controlled binding probes, Streptavidin-Biotin
相關次數：	點閱：2 下載：0
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小分子螢光探針的分析方法具備簡單、快速、靈敏等優點，已被廣泛應用於眾多領域，如生物和醫事檢驗上。
在本研究中，我們發展生物素探針，其螢光增益機制是源自鏈黴親和素蛋白與生物素的可控結合。此探針克服了傳統螢光化學探針之限制，如：過高的背景訊號、一個目標物對應一個螢光訊號模式及非專一性訊號的干擾等問題。由於其不同的螢光增益機制，可控結合生物素探針(controlled binding probe)被視為一種新型的化學探針。其優點展現了更低的背景值，更優越的螢光增益性，且不受複雜環境中其他大分子的干擾。
當分析物不存在時，鍵結上辨識基團的生物素和鏈黴親和素蛋白之間的微弱親和力，可以簡單的的清洗步驟，達到去除背景螢光訊號的目的。當目標分析物存在的狀況下，分析物會解除籠閉生物素的籠閉狀態，使得生物素與帶有螢光染料的鏈黴親和素結合，產生螢光增益。由於超氧自由基與免疫方面有緊密之關係，藉由此種策略，我們研發可偵測此種活性小分子的可控結合生物素探針，以偵測細胞表面的超氧自由基。
我們相信此種可控結合生物素策略可以克服傳統探針所遇到的問題，成為一種可準確地進行生物小分子檢測及定量分析的方法。

Recently, the developments and applications of small molecule-based fluorescent probes for analyte detection have gained much attention, such as basic biological research and medical diagnosis.
In this thesis, we report a novel chemical probe with caged-biotin strategy to detect superoxide radical from the cells. There are three limitations of traditional fluorescent probes. The first limitation is that a fluorescent probe reacts with one target molecule to produce a fluorescence signal that has limited the sensitivity of the fluorescent probes. The second limitation of caged-fluorophores is caused by the intrinsic background fluorescence when the fluorescent probe is at the "off" state. Furthermore, fluorescent probes easily produce nonspecific signals in biological samples, like blood or plasma. Hence, we use the controlled binding of biotin with streptavidin to overcome the limitations of the fluorescent probes. As compared to the conventional caged-probes, streptavidin-biotin controlled binding probes exhibit extremely low background with significant signal amplification and simple procedure to remove nonspecific signals.
In the absence of the target analyte, the caged-biotin probe on the cell surface is not able to bind with fluorophore conjugated streptavidin due to the low binding affinity of caged biotin with streptavidin. After simple washing, the fluorophore conjugated streptavidin can then be washed away to give no background. When target analyte is present, it will uncage the probe. After adding fluorophore conjugated streptavidin, it will bind to the biotin probe to achieve signal amplified detection. With this new streptavidin-biotin controlled binding approach, we can image secreted superoxide radical along the extracellular plasma membrane.
We believe that the streptavidin-biotin controlled binding strategy might provide an opportunity to overcome the problems and challenges encountered for the chemical probe design and have an impact on biomolecules detection and quantification.

摘要    I
Abstract    II
謝誌    IV
目錄    V
縮寫對照表    VIII
第一章 緒論    1
1-1 研究動機    1
1-2 活性氧化物(reactive oxygen species, ROS)    2
1-2-1超氧自由基( superoxide radical, O2•−)    3
1-2-2氫氧自由基(hydroxyl radical, OH•)    3
1-2-3過氧化氫(hydrogen peroxide, H2O2)    4
1-3 NADPH氧化酶(NADPH Oxidase)    4
第二章 文獻回顧    6
2-1 光活化型籠閉螢光探針(photoactivatable caged-fluorescence probe)    7
2-1-1 籠閉螢光團(caged-fluorophore)    7
2-1-2 籠閉螢光素(caged-luciferin)    13
2-1-3 籠閉生物素(caged-biotin)    15
2-2 反應型籠閉螢光探針(reactive caged-fluorescence probe)    18
2-2-1 籠閉螢光團(caged-fluorophore)    18
2-2-2 籠閉螢光素(caged-luciferin)    20
2-3偵測活性小分子之螢光探針    22
2-3-1 活性氧化物之螢光探針    22
2-3-2 活性氮化物之螢光探針    28
第三章 探針構想與設計    32
3-1 籠閉生物素探針設計構想(caged-biotin probe, CBP)    32
3-2籠閉生物素探針設計    34
3-3 探針8螢光機制    35
3-4 探針8結構設計    36
第四章 結果與討論    38
4-1 探針8螢光與選擇性測試    38
4-2 探針8細胞測試    45
第五章 結論    51
第六章 實驗部分    52
6-1 實驗藥品及器材    52
6-2 有機合成與光譜資料    54
6-3 螢光測試條件    67
6-4 選擇性測試中不同ROS與RNS溶液配製    67
6-5 細胞培養及細胞影像實驗    68
6-5-1 培養基及試劑    68
6-5-2 細胞繼代培養    68
6-5-3 RAW264.7細胞影像    69
參考文獻    70
附錄    74




                                

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