有機小分子（例如代謝物、神經遞質和藥物）之感測是最基本的生物過程之一。其中，細胞表面為一個不斷變化的小分子微環境，因為許多細胞代謝過程是通過改變小分子的濃度來調節的。迄今為止，由螢光團標記之變構及半合成蛋白是僅有的兩種能夠在細胞表面對小分子進行細胞顯影之方法。然而，這些方法受到螢光基團無法隨意更換的限制，僅適用於基因工程細胞。在本論文中，我們開發出一種新型小分子半合成生物素蛋白，其可以反應小分子結合目標，與鍊黴親和素藉由可控之親和力結合。這種方法可用於快速且準確地檢測細胞表面的各種磺胺類藥物和甲氨蝶呤。我們相信用於變構控制鍊黴親和素-生物素結合親和力的半合成蛋白質設計可以成為快速、專一性和高靈敏地檢測許多有機小分子的泛用工具。

Small organic molecule (e.g. metabolites, neurotransmitters and drugs) sensing is one of the most fundamental biological processes. Notably, the extracellular cell surface represents an extremely dynamic microenvironment for small molecules sensing as many cellular processes are regulated by changing the concentration of small molecules. To date, allosteric and semisynthetic proteins based on Förster resonance energy transfer (FRET) were the only two methods enabling rapid imaging of small molecules on the cell surface. However, these approaches are restricted by their limited selection of fluorophores and are applicable only in genetic engineered cells. In this thesis, we report a new type of biotinylated semisynthetic protein which can undergo affinity-switchable binding with streptavidin in response to a small molecule binding target. This general approach can be applied to rapidly and accurately detect various sulfonamide drugs and methotrexate on the cell surface. We believe that our biotinylated protein design to allosterically control streptavidin-biotin binding affinity can be a versatile tool for rapid, specific and sensitive detection of many small organic molecules.

p.I 摘要
p.II Abstract
p.III 謝誌
p.VI 目錄
p.1 第一章、 緒論
p.1 1-1 小分子之介紹
p.2 1-1-1 菸鹼醯胺腺嘌呤二核苷酸 ( Nicotinamide adenine dinucleotide, NAD )
p.3 1-1-2 三磷酸腺苷 (Adenosine triphosphate, ATP)
p.4 1-2 小分子偵測方法
p.5 1-2-1 液相層析質譜分析法 (Liquid Chromatography-Mass Spectrometry LC-MS)
p.8 1-2-2 酵素免疫分析法 (Enzyme-Linked Immunosorbent Assay, ELISA)
p.11 第二章、 文獻回顧
p.12 2-1 變構蛋白
p.13 2-1-1 G蛋白偶聯受體（G Protein-Coupled Receptors, GPCRs）
p.15 2-1-2 變構蛋白於小分子偵測之應用
p.17 2-2 半合成蛋白
p.20 第三章、 探針之設計與探討
p.20 3-1 設計構想
p.22 3-2 配體選擇
p.24 3-3 探針之設計
p.26 第四章、實驗結果及討論
p.26 4-1 探針1
p.26 4-1-1 探針1之合成
p.28 4-1-2 探針1之MCF7細胞顯影實驗
p.31 4-2 探針2
p.31 4-2-1 探針2之合成
p.33 4-2-2 探針2之MCF7細胞顯影實驗
p.36 4-2-3 探針2之A549細胞顯影實驗
p.37 4-2-4 探針2之磺胺類藥物滴定實驗
p.41 4-2-5 探針2之穩定性
p.42 4-3探針3
p.42 4-3-1 探針3之合成
p.44 4-3-2 探針3之HeLa細胞顯影實驗
p.45 4-3-3 探針3之穩定性
p.46 4-4 探針4之探討
p.46 4-4-1 探針4之結構
p.47 4-4-2 探針4之MCF7細胞影像實驗
p.48 第五章、實驗結論
p.49 第六章、實驗部分
p.49 6-1 實驗器材與藥品
p.51 6-2 有機合成及光譜資料
p.65 第七章、參考資料
p.73 附錄

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