隨著科學及醫療領域的持續發展，目前已有許多有別於傳統的檢測方法被提出並受到重視。其中，側向層析分析法（Lateral flow assay, LFA）是一種廣泛使用的即時檢測技術，在多個研究領域得到應用，例如：醫學診斷、環境監測、食品安全、農業獸醫應用及藥物檢測等。相較於其他現有的檢測方式，側向層析分析法具有簡易使用、快速檢測、低成本及具有可攜帶性等優點。然而此方法雖具有迅速且專一的特性，目前三明治型側向層析分析法的大多使用於大分子，例如抗原、蛋白質及核酸等，偵測小分子則較常使用競爭型側向層析分析法進行檢測，如此易導致結果判讀複雜性。於這篇論文中我們提出一種透過調控親和力的方式，並且基於側向層析分析平台的半合成蛋白開關，以增強訊號開啟方式來偵測有機小分子。此原理為透過目標小分子與探針上的配體競爭蛋白之結合位點，進而調控探針上之生物素與測試線上鏈黴親和素的結合能力，並透過特殊探針結構修飾使訊號明顯增強，達到提高靈敏度的效果。
在不含有目標小分子的樣品中，探針上生物素衍生物因周圍會產生較大的立體障礙，使生物素無法與測試線上之鏈黴親和素結合，呈現訊號關閉的狀態。當樣品中具有目標小分子，生物素周圍的空間立障會被消除，於流經測試線時會與鏈黴親和素結合，並因為探針上特殊結構修飾而使訊號放大，呈現訊號明顯開啟的狀態。基於這項研究設計，我們利用此訊號表達及增強機制、模塊化的探針特性及側向層析分析法的優點，成功實現偵測小分子的目的，並希望於未來擴大此技術的應用範疇以達到更多重要生物分子的即時檢測。

With the continuous development of science and medical treatment, many novel detection methods have been proposed and attracted much attention. Among them, Lateral flow assay (LFA) has been widely applied in many research fields, including medical diagnosis, environmental monitoring, food safety, agricultural and veterinary applications and drug detection. Compared with other detection methods, lateral flow assay has the advantages of simple use, rapid detection, low cost and portability. However, most of the sandwich-type LFA methods are used for macromolecules, such as antigens, proteins, and nucleic acids. Small molecules are more commonly detected by competitive type LFA, it is easy to lead to the result interpretation complexity. To detect small organic molecules by signal-on format, we propose an affinity-tunable semisynthetic protein switch based on lateral flow assay. The principle is regulating the binding affinity by utilizing the competition between the target small molecule and the ligands, and amplifying the signal through specific structural modification.
In the absence of target molecule, the biotin cannot combine with the streptavidin on the test line due to the large steric hindrance, so the signal is turned off. When the sample contains the target small molecule, the steric hindrance will be eliminated, and it will be captured by streptavidin when it flows through the test line. As a result, the signal will be turned on. Besides, the signal will be amplified due to the special structural modification so we can achieve lower detection limit. Based on this research design, we have successfully realized the purpose of detecting small molecules by taking advantage of semisynthetic protein switch and lateral flow assay, and believe that the application of this technology will be expanded to achieve more point-of-care detection of important biomolecules.

目錄
摘要 I
Abstract II
謝誌 III
目錄 V
第一章、緒論 (Introduction) 1
1-1小分子介紹 (Introduction of small molecules) 1
1-1-1常見之生物小分子 (Common biomolecules) 2
1-1-2神經傳導物質 (Neurotransmitters) 4
1-1-3藥物小分子 (Small drug molecule) 5
1-2傳統小分子檢測方式 (Traditional small molecule detection methods) 9
1-2-1液相層析質譜分析法 (Liquid chromatography mass spectrometry) 9
1-2-2酵素結合免疫分析法 (Enzyme-linked immunosorbent assay) 11
第二章、文獻回顧 (Literature review) 14
2-1 側向流體分析法 (Lateral flow assay, LFA) 15
2-1-1三明治型及競爭型側向流體分析法 (Sandwich and competitive lateral flow analysis) 15
2-1-2檢測小分子之訊號開啟型側向流體分析法 (Signal turn-on lateral flow assay for the detection of small molecules) 20
2-2變構蛋白開關 (Allosteric protein switch) 27
2-2-1合成蛋白質開關 (Synthetic protein switch) 28
2-2-2半合成蛋白開關 (Semi synthetic protein switch) 29
2-3 LFA訊號增益常見方法 (Common methods of LFA signal enhancement) 31
2-3-1 透過雷射激發放大訊號 (Signal amplified by laser excitation) 31
2-3-2 增加螢光基團增益LFA訊號 (Adding fluorophores to improve LFA signal) 33
2-3-3透過配對標記識別增強信號強度 (Enhanced signal strength through paired marker recognition) 35
2-3-4透過聚集奈米金增益LFA訊號 (Amplification of LFA signal by aggregating AuNPs) 37
第三章、實驗設計 (Experimental design) 39
3-1生物素與抗生物素 (Biotin and anti-biotin molecule) 39
3-2半合成蛋白開關應用 (Semisynthetic protein switch application) 41
3-3探針結構及實驗設計 (Probe structure and experimental design) 43
3-3-1探針結構 (Probe structure) 43
3-2-2標記端、載體蛋白及配體端選擇 (Label site, carrier protein and ligand selection) 45
第四章、實驗結果與討論 (Experimental results and discussion) 46
4-1可調控親和力生物素探針於LFA檢測磺胺類藥物 (EZA) 探討 (Discussion on the detection of sulfonamides (EZA) with tunable affinity biotin probes in LFA) 46
4-2可調控親和力生物素探針訊號改良 (Signal improvement of affinity-switchable biotin probes) 50
4-2-1增強LFA訊號方法 (Enhance LFA signal method) 50
4-2-2 炔丙基修飾之可調控親和力探針 (Propargyl-modified affinity-switchable probe) 52
4-3 具炔丙基修飾之可調控親和力生物素探針偵測磺胺類藥物結果 (Results of affinity-switchable biotin probe with propargyl modification for detection of sulfonamide drugs) 54
4-3-1定性測試 (Activity test) 55
4-3-2 選擇性測試 (Selectivity test) 58
4-3.3 靈敏度測試 (Sensitivity test) 60
4-3-4 再現性測試 (Reproducibility test) 61
4-3-5 動力學測試 (Kinetic test) 62
4-3-6 穩定性測試 (Stability test) 63
第五章、實驗結論 (Experimental results) 64
第六章、實驗部分 (Experimental part) 65
6-1實驗藥品與儀器 (Experimental drugs and instruments) 65
6-2蛋白質製作 (Protein production) 65
6-2-1 實驗菌種及藥品 (Experimental strains and drugs) 65
6-2-2蛋白質表達 (Protein expression) 66
6-2-3 蛋白質純化 (Protein purification) 67
6-3化學品配置及試紙製備方法 (Chemical and test paper preparation method) 67
6-3-1奈米金粒子 (AuNPs) 製備 (Gold nanoparticles preparation) 67
6-3-2奈米金粒子結合載體蛋白 (AuNPs-HaloTag) 製備 (Preparation AuNPs-HaloTag) 68
6-3-2探針 (AuNPs-HaloTag-ASB probe) 製備 (AuNPs-HaloTag-ASB probe preparation) 68
6-3-3奈米金粒子結合抗體 (AuNPs-Mouse IgG) 製備 (AuNPs-Mouse IgG preparation) 69
6-3-4側向層析試紙前處理 (Pretreatment of lateral flow test paper) 69
6-3-5測試磺胺類藥物小分子之條件 (Conditions for testing small molecules of sulfonamide drugs) 69
第七章、參考資料 (References) 71
附錄 77

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