1,4-二羟基-9-10-蒽醌-2-醛缩-4-氟苯基氨基硫脲探针测定生物样品中蛋白质含量的光谱法

Microdetermination of Proteins in the Biological Samples by the Synchronous Fluorescence Method with EDMHT

在模拟人体生理条件下,基于1,4-二羟基-9-10-蒽醌-2-醛缩-4-氟苯基氨基硫脲(EDMHT)与人血清白蛋白(HSA)相互作用生成复合物,导致血清白蛋白的内源荧光产生特异性变化,且体系的同步荧光强度与溶液中HSA的浓度呈良好的线性关系,从而建立了以EDMHT为分子探针,用固定波长同步荧光光谱分析测定蛋白质含量的新方法.体系荧光光谱特征及强度受Δλ值、pH、离子强度及试剂加入顺序等因素的影响.在20℃,pH=7.40Tris-HCl缓冲溶液及离子强度为0.1mol/L的NaCl溶液中,体系的Δλ值为50nm的同步荧光强度(ISF)与人血清白蛋白在5.52～276mg/L的浓度范围内呈现良好的线性关系.本实验对人血清、尿样、唾液样品进行了测定,回收率在95.18%～98.32%之间,对11份空白样品进行平行测定,求得相对标准偏差为2.87%,方法的检测限可达0.348mg/L.实验结果表明,本方法具有简单、快速、灵敏度较高,线性范围宽,精密度和回收率较好等优点,可直接用于生物样品中蛋白质总量的测定,结果令人满意,有望用于生化分析和临床分析.

EDMHT can react with human serum albumin to form a complex compound-protein. It can make intrinsic fluorescence of HSA produce specific change and the synchronous fluorescence intensity of the system was in good proportion to the concentration of protein. Based on this, a new method for the determination of proteins by synchronous fluorescence spec-trometry under simulated physiological conditions was established using EDMHT as a molecular probe. I he spectrat characterization and intensity of synchronous fluorescence were related to the value of AX, pH, ionic strength and the order of the reagents. The synchronous fluorescence intensity of the system was in proportion to the concentration of protein in the range of 5.52-276 mg/I. for HSA when T=20℃, △λ= 50 nm, Tris-HCl buffer （pH 7.40） and ionic strength was 0.1 mol/L of NaCI solution. The serum, urine and saliva were determined in the experiment and its recovery was in the range of 95.18- 98.32% with RSD of 2.87% The detection limit was 0. 348mg/L（n=11） for HSA. The results suggested that the method possessed characteristics of eonvinience, rapidity, high sensitivity, broad linear range, better precision and recovery. Above all it had been applied to the determination of proteins in biological samples with satisfactory results. Key words： （E）-2-[（1,4- dihydroxy- 9,10- 2 yl） methylene -N （4 - fluorophenyl） hydrazinecarbothioamide（EDM- HT）; Human Serum Albumin; Synchronous Fluorescence Method; Molecular Probe