基于邻氟苯基荧光酮荧光探针的荧光光谱法测定牛血清白蛋白

Determination of Bovine Serum Albumin by Fluorescence Spectrometry with o-Fluorophenylfluorone as a Fluorescent Probe

在pH 7.40的磷酸盐缓冲溶液中,牛血清白蛋白(BSA)可使邻氟苯基荧光酮(o-FPF)荧光猝灭,β-环糊精的加入可使荧光猝灭强度(ΔF)增强,据此建立了一种基于o-FPF为荧光探针测定BSA的荧光光谱法。在选定的试验条件下,BSA浓度在6.0×10^(-9)~4.0×10^(-7)mol·L^(-1)范围内与ΔF呈线性关系,方法检出限(3s/k)为7.1×10^(-10)mol·L^(-1)。该方法用于测定牛奶、牛奶粉中的蛋白质含量,结果与考马斯亮蓝法测定结果相吻合,测定值的相对标准偏差(n=5)小于4%。通过测定荧光寿命、探讨温度对猝灭常数的影响以及紫外吸收光谱的变化,确定BSA与o-FPF之间的猝灭过程为静态猝灭。

In PBS with pH 7.40,bovine serum albumin（BSA）could quench the fluorescence intensity of ofluorophenylfluorone（o-FPF）and the fluorescence quenching intensity（ΔF）was enhanced by the addition ofβ-CD.Based on this,a method of fluorescence spectrometry with o-FPF as a fluorescent probe was proposed for the determination of BSA.Under the optimized conditions,a linear relationship was found between ΔF and the concentration of BSA in the range of 6.0×10^（-9）-4.0×10^（-7）mol·L^（-1）,with the detection limit（3s/k）of 7.1×10^（-10）mol·L^（-1）.The method was applied to the determination of BSA in milk and milk powder samples,giving results in consistent with those obtained by coomassie blue staining method and RSDs（n=5）less than 4%.The mechanism of interaction between BSA with o-FPF was studied by measuring the fluorescence lifetime and investigating the temperature effect on the quenching constants and UV absorption spectrometry,and the results showed that the interaction mechanism between BSA and o-FPF was a static quenching process.