快速测定生物样品中蛋白含量的同步荧光法研究

最佳实验条件下,基于脱氧尿苷与人血清白蛋白(HSA)相互作用,导致血清白蛋白的同步荧光光谱发生特异性变化,且体系的同步荧光强度和溶液中白蛋白的浓度呈良好的线性关系,建立了以脱氧尿苷为探针,运用固定波长同步荧光光谱快速测定生物样品中蛋白质总含量的新方法.深入考察了△λ值、反应介质、试剂用量、离子强度、加入顺序、反应时间等因素对测定的影响.在最佳实验条件下,体系的同步荧光强度与血清白蛋白在2.76～524.4μg/mL范围内的线性相关系数为0.9991,方法的检出限可达0.11μg/mL.运用本方法对人血清、唾液和尿液等生物样品进行测定并进行加标回收实验,回收率在98.0%～102.5%之间.对11份空白溶液进行平行测定,相对标准差为1.2%.用经典的考马斯亮蓝G-250(CBB)法做了对照实验,两种方法测定结果基本一致.还考察了一些常见离子和有机物存在时对蛋白质测定的影响.本方法已用于血清、唾液和尿样等生物样品中蛋白质总量的快速测定.

Study on determination of the total protein in biological samples by synchronous fluorescence technique

Under the optimum experimental conditions, 2'-Deoxyuridine could interact with human serum albumin (HSA), resulting in the specific changes of the intrinsic fluorescence of serum albumin. Synchronous fluorescence in-tensity of the system had a good linear relationship with the serum albumin concentration in the range of 2.76～524.4 μg/mL, and the detection limit of this method was 0.11 μg/mL. A novel method for the determination of the proteins with 2'-Deoxyufidine as a molecular probe was developed. The effect factors for spectral characterization and intensity of synchronous fluorescence such as the value of △λ, reaction medium, reagent concentration, ionic strength, addition sequence, reaction time were also investigated. The recoveries were 98.0%～102.5% and 11 blank solut-ions to the parallel experimental data obtained the relative standard deviation of 1.12%. The CBB G-250 method was done as the control experiment and the results were in good accordance with those by synchronous fluorescence method. The results indicated that this method was simple, rapid, and had high sensitivity, wide linear range, good stability and high selectivity. It was successfully applied to the determination of the total proteins in human body fluids such as human serum, saliva and urine samples, and the results were satisfactory.