同步荧光光谱比率法检测过氧亚硝酸根及其清除剂的研究

利用同步荧光光谱建立了一种新的荧光比率法检测过氧亚硝酸根(ONOO－)及评价其清除剂的方法。酪氨酸自身有荧光，在pH 8.5的磷酸缓冲溶液中，酪氨酸与CO₂和ONOO－反应生成强 荧光的酪氨酸二聚体，利用同步荧光光谱技术, 在荧光发射光谱中能同时获得酪氨酸单体(λ_em=364 nm)与二聚体的荧光峰(λ_em=406 nm)，且两峰的荧光强度的比值与ONOO－的浓度呈计量相 关。结果表明荧光强度的比值不受实验参数改变的影响，与传统的荧光法相比，有利于扩大检测的线性范围，提高灵敏度。荧光强度的比值与ONOO－的浓度在1.60×10－7 mol·L-1～6.00× 10－6 mol·L-1范围内呈线性关系，线性相关系数为0.999，检测限为1.84×10－8 mol·L-1。对浓度为1.00×10－6 mol·L-1 ONOO－ 平行测定8次，其相对标准偏差为2.4％。利用该法测得抗癌药米托蒽醌的IC₅₀为0.065 μg·mL-1。方法简便易行，试剂廉价易得，体系稳定，可作为一种检测ONOO-及筛选其清除剂的方法。

Study on Ratiometric Determination of Peroxynitrite and Its Scavenger Based on Synchronous Fluorescence Spectroscopy

A ratiometric fluorimetry is proposed for the determination of peroxynitrite (ONOO-) and the evaluation of its scavenger based on synchronous fluorescence spectroscopy.L-tyrosine, an intrinsic fluorescent aminoacid, reacts with peroxynitrite and carbon dioxide, yielding a highly fluorescent dimer of tyrosine in pH 8.5 PBS buffer solution.By synchronous fluorescence scanning, both monomer and dimer emission bands of L-tyrosine appeared at 364 and 406 nm，respectively.The ratio of F₄₀₆/F₃₆₄ is quantitatively related to the concentration of peroxynitrite, where F₄₀₆ represents the intensity of synchronous emission band of dimer and F₃₆₄ represents that of monomer.The method has been showing merit of being insensitive to the changes in experimental parameters and offers a higher sensitivity and a broader responding linear range of the analyte concentration, compared to fluorimetric method, giving a LOD of 1.84×10－8 mol·L-1 and a linear range of 1.60×10－7 mol·L-1- 6.00×10－6 mol·L-1 for ONOO-, respectively, with a deviation of 2.4％ for 1.00×10－6 mol·L-1 ONOO-(n=8).A IC₅₀ of 0.065 μg·mL-1 for mitoxantrone，an antioxidant and anticancer drug，was also obtained.This method has proved to be simple and speedy.It would be easily used in the determination of ONOO- and its scavenger.