過氧亞硝酸鹽螢光探針Ds-DAB之特性研究

Abstract

過氧亞硝酸鹽是生物體中重要的生物分子之一，牽涉著許多生理與病理作用。有效偵測過氧亞硝酸鹽可以使我們進一步了解它在生物體扮演的角色。本研究發展了一個過氧亞硝酸鹽的螢光探針，是以dansyl為基本架構的Ds-DAB。Ds-DAB (Φ = 0.004)是利用過氧亞硝酸鹽與二級胺基進行亞硝基化反應，進而形成產物 benzotriazol 及 dansyl acid (Φ = 0.148)，藉螢光訊號的改變作為偵測的方法。此探針不但反應速率快(20秒)且在一系列的活性氧化物及活性氮化物中具有極佳選擇性；對於過氧亞硝酸鹽具有良好線性關係的及偵測極限(52.4 nM)，說明Ds-DAB具有定量分析的潛力。此外，Ds-DAB呈現低毒性，並且能成功地進行細胞內過氧亞硝酸鹽的光學造影。且Ds-DAB於pH值5.8至11.9具有穩定性，不會受所處生理環境酸鹼值的改變而影響螢光性質。

Peroxynitrite (ONOO－) is an important signaling molecule in organism. It is involved in diverse physiological and pathological pathways. Visualization of peroxynitrite using bioimaging techniques provide us to advance the understanding of roles played by peroxynitrite in organism. The fluorescent ONOO－probe, Ds-DAB, was developed in this study. The design strategy of Ds-DAB is based on the peroxynitrite anion might react with secondary amine to form nitrosamine by direct nucleophilic nitrosation. Then, the benzotriazol was released and formed dansyl acid with high fluorescence intensity. Ds-DAB has not only fast reaction rate (20 sec.) but high specificity for peroxynitrite over other reactive nitrogen species and oxygen species. Besides, an excellent linearity between the fluorescence intensity and peroxynitrite concentration could provide the quantitative measurement through fluorometric method. The detection limit was 52.4 nM. In addition, Ds-DAB can be applied to monitor the endogenous peroxynitrite with low cytotoxicity. Ds-DAB had hardly any effect on the fluorescence intensity over the pH range from 5.8 to 11.9. Therefore, the probe is expected to work well under physiological condition.