可選擇性的進行蛋白質與磺胺類藥物檢測之 對環境敏感螢光探針

Environment-sensitive Fluorescent Probes for The Selective Detection of Proteins and Sulfa Drugs

莊鈺德(Yu-De Zhuang)

指導教授 ： 陳貴通

國立清華大學/理學院/化學系/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00384

摘要

有鑒於現代生物與醫學研究發展，可應用於活體細胞內並可專一的檢測蛋白質與特定代謝物非常重要，其濃度在活體細胞內隨代謝活動與生理作用時常變化，研究其變化有助於間接探知活體細胞生理真正的面貌。本實驗皆以對環境敏感分子處於親疏水相中的螢光變化作為訊號開關機理，分別針對蛋白質與代謝物設計一種新型的感測器。
蛋白質檢測方法係以會被水焠熄之對環境敏感螢光分子 SBD 之衍生物結合目標蛋白質專一辨識的嵌合基團做為探針架構，運用蛋白質結構內部普遍較為疏水的特性。當探針結合目標蛋白質，螢光分子將處在疏水環境中，產生強烈螢光訊號，利用此螢光訊號變化量間接得知樣品中蛋白質的含量。本實驗成功以人體碳酸酐酶 (hCA)、胰蛋白酶 (trypsin)、卵白素 (avidin) 與其所對應的嵌合基團對位苯磺胺、對位苄脒 (para-benzamidine) 與生物素 (biotin) 衍生之探針進行此檢測概念的演示，探針合成容易且其螢光增益最高可達 16 倍。
代謝物檢測方法係利用半合成式生物感測器模式，以生物轉殖方法合成重組蛋白 SNAP-hCA，另以有機合成方法合成探針 BG-linker-SBD-SA，以 SNAP-tag 標記技術專一的辨認 BG 基團並將探針標記於 hCA。分子內作用使探針前端的苯磺胺 (SA) 與 hCA 結合並因螢光分子進入 hCA 內部產生螢光。當待測樣品中存在磺胺時，將會競爭性的結合 hCA 並使探針離開活性中心游離至蛋白外，螢光將被焠熄，利用此訊號變化量可反推樣品中的磺胺濃度，最高可達 4.2 倍。
兩種檢測方法均具有普遍性，更換對應蛋白質與探針嵌合基團即可檢測不同蛋白質或代謝物；優化部分亦僅需對有機合成探針進行衍生物合成，極具應用潛力。

關鍵字

對環境敏感螢光分子 ； 蛋白質 ； 磺胺類藥物 ； 螢光探針 ； 生物感測器

並列摘要

Protein, metabolite and drug molecule detection is important in medical diagnosis as well as in biology to investigate cellular processes. Fluorescent probes which can detect specific proteins and small molecules are particularly valuable as they allow for sensitive, simple and specific detection with high signal-to-background ratios. In this thesis, we introduce two new general approaches to generate fluorescent sensor for the selective detection of proteins and small molecules, respectively.
Currently most of the small molecule fluorescent turn-on probes are designed for monitoring enzyme activities, e.g., glycosidases, proteases, lactamases and kinases. Typically, their fluorescence turn-on mechanism is based on the enzymatic reaction with the chemical probes to convert the non-fluorescence substrate into the fluorescence product. On the other hand, the design of fluorescence probes for non-enzymatic proteins remains a challenging task. In the first part of the thesis, we introduce a new type of fluorescent turn-on probes, where a small molecule ligand is conjugated to an environment-sensitive SBD fluorophore, for the selective detection of both enzymes and non-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. Our new fluorescent probe design is modular and versatile as illustrated by the three fluorescent probes synthesized based on this design for the specific detection of hCAII, trypsin and avidin with fluorescent turn-on ratios of up to 17-fold.
In the second part of the thesis, we describe a novel semisynthetic fluorescent sensor for the selective detection of sulfa drugs. The semisynthetic sensor is to mimic open and closed conformation of the periplasmic binding protein upon substrate binding. Covalent labeling of the synthetic sulfonamide inhibitor and environment-sensitive SBD-dye conjugate to the HCAII enzyme was achieved by introducing a self-labeling protein SNAP-tag to HCAII to facilitate quantitative and site-specific labeling via benzylguanine (BG) moiety. We have successfully demonstrated that addition of synthetic molecule to the sensor protein SNAP_HCAII can achieve a fluorescence intensity increase of more than 10-fold. Subsequent addition of sulfonamide drugs to the semisynthetic fluorescent sensor reverses the SBD-dye to its initial non-fluorescent state. This semisynthetic fluorescent sensor has been applied in the detection of several sulfonamide drugs, such as ethoxzolamide and acetazolamide.
The two fluorescent sensors described in this thesis provide a general approach for the selective detection of proteins, metabolites and drug molecules. We believe that these novel fluorescent probe designs will be a very useful approach for a wide range of applications, such as diagnosis and molecular imaging where high fluorescent signal change and simple detection methods are required.

並列關鍵字

environment-sensitive fluorophore ； proteins ； sulfa drugs ； fluorescent probes ； biosensors

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