荧光素-曙红Y能量转移荧光猝灭法测定微量白蛋白研究

Study on Energy Transfer in Fluorescein-Eosin Y System and the Fluorescence Quenching Method for Determination of Trace Protein

在λex/λem=405/547 nm,于缓冲溶液和表面活性剂存在的情况下,荧光素和曙红Y能够发生有效能量转移,而牛血清白蛋白(BSA)的加入使得曙红Y荧光猝灭,该体系可用于微量蛋白质的测定。系统探讨了荧光素-曙红Y能量转移体系发生荧光猝灭的条件,最佳条件为:2.0 mL pH=3.8的B-R缓冲溶液,0.4 mL 0.05%曲拉通X-100,1.5 mL 1.0×10-4mol/L的荧光素水溶液,2.0 mL 1.0×10-4mol/L的曙红Y水溶液,最佳实验时间为溶液配制完成静置15 min后60 min内,最佳加入顺序为pH=3.8缓冲溶液+荧光素+曙红Y+曲拉通+蛋白质标准溶液或样品。在优化的实验条件下,蛋白质含量在0～2.0μg/mL范围内与荧光猝灭强度呈良好的线性关系。检出限为6.6 ng/mL;测定样品的相对标准偏差(RSD)在±5%以内;样品加标回收率为90.4%～95.3%。该法可用于人血清、牛奶中蛋白质含量的测定。

In the condition of λex/λem=405/547 nm and with the existence of buffer solution and surfactant,an effective energy transfer could take place between fluorescein and eosin Y,and the fluorescence intensity of eosin Y is quenched with the addition of BSA.The energy transfer fluorescence quenching was used to determine trace BSA.The best conditions of fluorescence quenching of fluorescein-eosin Y system were as follow：2.0 mL B-R buffer solution with pH=3.8,0.4 mL 0.05% triton X-100,1.5 mL 1.0×10-4 mol/L fluorescein solution and 2.0 mL 1.0×10-4mol/L Eosin Y solution.The reaction time and the order of reagent addition were studied systematically in this paper.The influences of coexistent substances were also explored.Under the optimized experimental conditions,the fluorescence quenching value was linearly proportional to the amount of BSA in the range of 0~2.0 μg/mL.The detection limit was 6.6 ng/mL.RSD in the determination of samples was within ±5% and the recovery was 90.4%~95.3%.This method was applied to the determination of protein content of human serum and milk samples with satisfying results.