過氧亞硝酸鹽螢光探針 4-MB 之特性研究

Abstract

過氧亞硝酸鹽，主要是由一氧化氮與超氧離子快速反應生成的產物。其具有高氧化及硝化的特性，可與許多生物分子產生反應，使之在生物體內扮演重要角色。本研究著重於發展一個可偵測過氧亞硝酸鹽的螢光探針， 4-MB。4-MB是利用過氧亞硝酸鹽與螢光探針進行親核性反應，藉螢光訊號的改變作為偵測判讀的方法。4-MB與過氧亞硝酸鹽反應後，螢光波長會右移，為一種具有比例特性的螢光探針。4-MB與過氧亞硝酸鹽反應速度快，在一分鐘內，螢光訊號強度即有明顯變化，與不同濃度的過氧亞硝酸鹽反應，其螢光強度也隨之增加，偵測極限為29.8 nM，對過氧亞硝酸鹽具有良好的選擇性，於不同酸鹼值(pH 5.9-11.1)環境中具有穩定性。最後，4-MB，也成功地應用於細胞進行外源性與內源性之過氧亞硝酸鹽的光學造影。

Peroxynitrite (ONOO-) is an important signaling molecule in organism. It is produced from nitric oxide and superoxide radical by the diffusion-controlled reaction. Peroxinitrite is a strong oxidant and good neucleophile, it can react with different biomolecules. It has been related to a list of disease such as cardiovascular diseases, circulatory shock, diabetes, neurodegenerative disorders, and cancer. However, the effects of ONOO- in physiology and pathology remain controversial. There has been a strong demand for reliable and real-time analytical methods for ONOO- detection. The aim of this project is to design and synthesis a new fluorescent probe for detecting peroxynitrite. The design strategy of this ONOO- fluorescent probe, 4-MB, is based on the peroxynitrite anion might react with this probe by nucleophilic attack. The response time of 4-MB to react with ONOO- is about 4 minutes. 4-MB also has a good selectivity for ONOO- over other biologically relevant RNS and ROS. The detection limit of 4-MB for ONOO- is 29.8 nM. Moreover, 4-MB is successfully applied to detection of exogenous and endogenous ONOO- in living cells.