開關式環境敏感螢光探針對非酶蛋白質之檢測

Environment-sensitive Fluorescent Turn-On Probes for Non-Enzymatic Proteins

李秉原(Bing-Yuan Lee)

指導教授 ： 陳貴通

國立清華大學/理學院/化學系/碩士(2015年)

摘要

摘要
蛋白質是一種具生物性的有機大分子，其在生物體內的濃度異常往往是許多疾病的變因，因此偵測蛋白質是一個很重要的課題。傳統上最典型的例子為酵素連結免疫吸附分析法(ELISA)，ELISA已經成功用於許多分析物的檢測，但由於其複雜的前處理過程及步驟具有侷限性，因此我們設計出一個簡單、方便、快速及免清洗的偵測方法。
在本論文中，我以環境敏感之螢光分子與蛋白質之親和性配體結合，設計了一個對目標蛋白質具有專一性偵測的螢光探針。並且運用蛋白質結構內部普遍較為疏水的特性，當探針與目標蛋白質結合時，螢光分子將處在疏水的環境中，產生強烈螢光訊號，利用此螢光訊號變化量可以得知樣品中蛋白質的含量。我們以SBD(4-sulfamonyl-7-aminobenzoxa-diazole)環境敏感螢光分子成功應用於二氫葉酸合成酶(DHPS)之專一性檢測，同時我們也可將此螢光探針運用於多種磺胺類藥物的檢測。此外我們也以此概念，將SBD環境敏感螢光分子應用於生物素與抗生素蛋白系統(Biotin-Avidin System, BAS)，我們以SBD分子結合生物素(Biotin)，利用生物素與抗生素蛋白(Avidin)可產生專一性鍵結，可以用來檢測抗生素蛋白，其探針合成容易、具高度選擇性且螢光增益可達數十倍，其在醫學上具有相當的應用潛力，如免疫沉澱、生物辨識等等。

關鍵字

螢光探針 ； 蛋白質 ； 開關式

並列摘要

Proteins detection is very important in biology and medicine because abnormal protein concentrations in cells are indicative of many diseases. Traditional protein detection methods such as western blot and enzyme-linked immunosorbent assay (ELISA) which uses a covalently labeled enzyme-antibody conjugate to carry out specific analyte detection and signal amplification, are laborious and time consuming. Currently, development of a rapid, selective and sensitive protein detection method remains a challenging task.
Herein, I introduce a new type of protein-specific fluorescent turn-on probes, where a small molecule ligand is conjugated to an environment-sensitive fluorophore, for the selective detection of both non-enzymatic proteins and enzymatic proteins. The fluorescent turn-on mechanism is based on the binding of the ligand to a hydrophobic ligand binding domain of the target protein whereby the close proximity to the hydrophobic environment can influence the environment-sensitive fluorophore to exhibit stronger fluorescence. With this design, we have successfully constructed an environment-sensitive fluorescent probe for specific detection of dihydropteroate synthase (DHPS) as well as sulfonamide detection. We also applied the similar probe design to Biotin-Avidin System (BAS) in which biotin was used as the ligand for the interaction with avidin. We believe that this design of fluorescence probes can have many future applications, such as immuno-precipitation and biometric.

並列關鍵字

probe ； protein

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