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.

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