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

• Main Authors: Zhuang, Yu-De, 莊鈺德
• Other Authors: Tan, Kui-Thong
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/17997464546993851799

碩士 === 國立清華大學 === 化學系 === 101 === 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.