隨著醫療資源的發展，大腦的疾病仍然沒有明確的診斷方案或可量化的化學生物標記物質，因此在治療上增加了一定的難易度。許多研究指出，神經傳導物質在這方面影響甚深。常見的神經傳導物質，如興奮性的Glutamate和抑制性的GABA，亦或作為調節劑的Acetylcholine，這些分子的濃度和分佈都會影響訊號的變化，因此瞭解它們與大腦之間的時空關係，有助於生物醫學的發展。目前已有許多基於小分子和基因編碼的螢光感測器用於檢測各式的神經活動，其中，合成蛋白質開關是一項具有前瞻性的方法，因此我們欲延伸其技術來擴大對這領域的應用，在此研究中，我們提出一系列可調控生物素化蛋白開關來偵測細胞膜表面之小分子，其原理為藉由改變生物素周圍的空間障礙作為螢光訊號的開關。在未存在小分子的狀態下，生物素會因被包覆在結合蛋白與目標蛋白之間，呈現封閉構型，使其無法與Streptavidin結合；當加入小分子後，其會與探針之配體端進行競爭，使生物素呈現開放構型，同時活化了與Streptavidin的作用力，並透過修飾在Streptavidin上的染料Cy5來達標記的效果。我們預期能將此設計應用在不同的神經傳導物質偵測上，以及最後能順利引入到生物體內進行相關實驗。

With the development of medical resources, there are still no clear diagnostic schemes or quantifiable chemical biomarkers for brain disorders, which increase the difficulty of treatment. Researches have pointed out that most of them are affected by neurotransmitters. There are several excitatory, inhibitory, and modulatory neurotransmitters, such as Glu, GABA and ACh, respectively, will impact the change of signal by their concentration and distribution. Thus, determining the spatiotemporal relationship between these chemical signals and the brain is beneficial for biomedical development. Nowadays, numerous small-molecule based and genetically encoded sensors have been developed to track neural activity. Synthetic protein switch is one of the prospective methods in this field. Hence, we refer to these techniques to expand the applications. In this thesis, we introduce a series of tunable biotin-derived protein switch to detect small molecules on cell surface. The principle of this design is switching the fluorescent signal on and off by altering the steric hindrance of biotin. In the absence of analyte, the protein switch forms a closed conformation, and the biotin, meanwhile, will be trapped between the binding proteins causing it is unable to interact with streptavidin. While adding the analyte will let the analyte compete with the ligand of the probe, and lead the protein switch to form an open conformation activating the interaction between biotin and streptavidin. Consequently, the exposing biotin binds to streptavidin-Cy5 and generates the fluorescent turn-on signal. Based on the design, we expect these kinds of protein switch can be applied to detect different kinds of neurotransmitters, and be also expressed in vivo.

摘要 i
Abstract ii
誌謝辭 iii
目錄 iv
第一章、緒論 1
1-1 神經傳導物質 (Neurotransmitter) 1
1-1-1 合成與釋放 (Synthesis and Release) 1
1-1-2 生理作用 (Physiological Action) 2
1-2 神經傳導物質檢測方法 (Neurotransmitter Detection) 3
1-2-1 電化學 (Electrochemical Method) 3
1-2-2 微透析 (Microdialysis) 3
1-2-3 光學成像 (Fluorescence Imaging) 4
第二章、文獻回顧 5
2-1 基因編碼感測器 (Genetically Encoded Sensor) 5
2-2-1 Circular Permutation 5
2-2-2 GPCR-Based Sensor 6
2-2-3 PBP-Based Sensor 7
2-2 混合感測器 (Hybrid Sensor) 9
2-1-1 Glutamate Optical Sensor (EOS) 9
2-1-2 Fluorescin Arsenical Hairpin Binder (FlAsH) 9
2-1-3 Snifit 10
2-1-4 Target Probe 12
第三章、研究動機 13
3-1 探針設計與策略 (Probe Design and Strategy) 13
3-2 設計原理 (Design Principle) 16
3-3 反應端與配體端之蛋白選擇 (Protein Selection) 17
3-3-1 結合蛋白 (Binding Protein) 17
3-3-2 目標蛋白 (Target Protein) 18
3-4 螢光標記之選擇 (Fluorescent Labeling Selection) 20
第四章、實驗結果與討論 21
4-1 探針合成路徑 (Synthetic Routes) 21
4-1-1 Synthesis of HaloTag Reaction Site 21
4-1-2 Synthesis of LC-Glu 23
4-1-3 Synthesis of LC-HCA 24
4-1-4 Synthesis of LC-GABA 25
4-1-5 Synthesis of LC-DHFR 26
4-1-6 Synthesis of LC-SNAP 27
4-2 實驗結果 28
4-2-1 HaloTag Control Test 28
4-2-2 LC-HCA 29
4-2-3 LC-GABA 30
4-2-4 LC-DHFR 31
4-2-5 LC-SNAP 33
第五章、結論 36
第六章、實驗步驟 37
6-1 實驗器材與藥品 (Instruments and Chemicals) 37
6-2 細胞影像實驗 (Experiment for Cell Imaging) 39
6-3 有機合成與光譜資料 (Synthesis and Spectrum) 40
第七章、參考資料 61
附錄 68

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