高選擇性半胱胺酸與硫化氫螢光化學感測分子之設計與合成及於細胞的應用

Abstract

本論文合成出螢光化學感測分子HPA和AQI，分別用於偵測半胱胺酸與硫化氫。HPA以丙烯醯基團作為與半胱胺酸反應之辨識端，在與半胱胺酸反應前，HPA分子幾乎沒有螢光放射，在加入半胱胺酸之後，HPA分子產生強烈的綠色螢光，並有49倍的螢光增強。HPA在多種胺基酸及硫化氫下，對於半胱胺酸具有高度的選擇性及靈敏度，其偵測極限為0.32 μM (S/N = 3)。在細胞毒性實驗中，當HPA之濃度 達 50 μM時，人體子宮頸癌細胞(HeLa cells)的存活率仍高達約80%。最後，將HPA應用於生物細胞顯影實驗中，利用共軛聚焦顯微鏡，成功觀測到HPA可以在細胞質的位置產生強烈的螢光增強現象。 AQI則是以2-(iodomethyl)benzoate作為硫化氫分子之辨識端，AQI原本僅有微弱螢光放射，在與硫化氫反應後，經由兩次的親核基反應，產生約7倍的黃色螢光增強。AQI在多種陰離子下，對於硫化氫具有高度的選擇性及靈敏度。在細胞毒性實驗中，當AQI之濃度達50 μM時，人體子宮頸癌細胞(HeLa cells)的存活率仍高達約80%。最後，將AQI應用於生物細胞顯影實驗中，成功觀測到AQI主要會存在於細胞質內，僅有少部分會進入細胞核且有螢光增強的現象。

In this thesis, two fluorescent chemosensors HPA and AQI were designed and synthesized for the detection of cysteine and hydrogen sulfide. The acryoyl group in HPA is a cysteine-reactive moiety for cysteine recognization. HPA shows weak fluorescence, whereas 49 folds green fluorescence enhancement is observed after reaction with cysteine. HPA displays good selectivity and sensitivity for cysteine over other amino acids and shows a low detection limit of 21 nM (S/N = 3). In cytotoxicity tests, cell viabilities were about 80 % when the concentration of HPA was 50 μM. In addition, confocal fluorescence microscopy imaging using HeLa cells demonstrates that detection probe HPA could be an efficient fluorescent probe for cysteine in living cells and shows fluorescence enhancement in cytoplasm. In second part, AQI uses 2-(iodomethyl) benzoate moiety as a reconition moiety for hydrogen sulfide. AQI shows weak fluorescence;but 7-folds yellow fluorescence enhancement upon reaction with hydrogen sulfide. AQI displays good selectivity and sensitivity for hydrogen sulfide over other anions. In cytotoxicity tests, cell viabilities were about 80 % when the concentration of AQI were 50 μM. Finally, confocal fluorescence microscopy imaging using HeLa cells demonstrates that the probe AQI could be an efficient fluorescent probe for hydrogen sulfide in living cells; most of AQI localize in cytoplasm and part of nucleus.