O2-·荧光探针法检测及其应用

Development and Application of Fluorescence Probe Method for Superoxide Anion Radical Detection

以2-氨基吡啶与吡啶-2-甲醛为原料,经亲核取代反应合成了一种新型荧光探针——2-(2’-吡啶亚胺甲基)吡啶(2-APC),所得目标产物经熔点测定、元素分析、红外光谱与核磁共振波谱等方法表征确认。利用2-APC与超氧阴离子自由基(O2-.)发生荧光淬灭反应的原理,建立了一种测定邻苯三酚自氧化体系产生O2-.的方法,该方法反应体系最佳条件为反应pH8.2、反应温度(T)40℃、反应时间(t)40min,测定条件为:λex=295nm、λem=365nm,狭缝宽度5nm。邻苯三酚在0.4×10-6～8.0×10-6mol/L浓度范围内与相对荧光强度呈良好线性关系,线性回归方程为y=37.567x+55.581,R2=0.9834。应用该方法对L-抗坏血酸清除O2-.能力进行评价,结果表明L-抗坏血酸清除O2-.的IC50值为0.182mmol/L。

A novel fluorescence probe 2-APC was synthesized based on nucleophilic substitution reaction of 2-aminopyridine and 2-pyridineearbaldehyde in this study. The reaction product was characterization by melting test, elemental analysis, infrared spectrum and 1HNMR. A fluorescence probe method for superoxide anion radical detection was established using the probe. The optimum reaction conditions were pH 8.2, 40℃ and 40 min. The conditions of fluorimetric determination were established as follows： λex -- 295 nm, λex = 365 nm and slit width = 5 nm. In the range from 0.4 × 10^6 to 8.0× 10^-6 mol/L, relative fluorescence intensity （y） and pyrogallol concentration （x） showed a good linear relationship： y =37.567x ＋ 55.581, R2 = 0.9834. Superoxide anion radical （O2-） scavenging activity of L-ascorbic acid was evaluated using the method and an IC50 of 0.182 mmol/L was obtained.