新的光散射体系测定蛋白质的研究

以1,6-己二胺二吖啶为荧光探针，建立了一种新的蛋白质共振散射检测体系。实验考察了该吖啶荧光探针的共振散射特征光谱、散射反应稳定性，研究了缓冲介质pH、探针浓度等参数对测定蛋白质的影响。结果表明，当pH 8.9时，1,6-己二胺二吖啶染料的散射波长为470 nm;加入BSA，反应约9 min后，体系光散射强度达到最大并维持稳定，2 h内无明显变化。当1,6-己二胺二吖啶探针(浓度1.00×10-4mol·L-1)用量为3.00 mL时，蛋白质与1,6-己二胺二吖啶的光散射增强作用显著，共振散射强度随着BSA的增加而明显增强，光散射强度与蛋白质浓度成良好线性关系，线性浓度范围为1.00～5.00 μg·mL-1,检测限(3σ/K)为0.085 μg·mL-1。测定了血清样品，浓度为2.00～4.00 μg·mL-1,回收率为98.0%～101.7%，测定偏差小于1.7％。

Studies on the Determination of Protein by a New Resonance Rayleigh Scattering System

Using 1,6-hexanediaminediacridine as a spectrum probe,a new method of quantitative determination of protein by resonance Rayleigh scattering (RRS) has been developed. Characteristics of the resonance scattering reaction of the acridine probe with bovine serum albumin (BSA) were investigated. Effects of the concentration of acridine probe and pH value of the buffer solution,as well as the stability of the resonance scattering interaction,were also studied,and then the optimum condition was obtained. In the Tris-HCl buffer solution,the intensity of resonance Rayleigh scattering spectrum remained stable without any notable change in the pH range of 6.0-7.5. While pH ranged from 8.0 to 8.9,the interaction of BSA and acridine was accelerated. The resonance scattering was increasing and the peak value was gained at pH 8.9. However,with the augmentation of pH from 10.0 to 11.0 in Na2B4O7-NaOH or Na2HPO4-NaOH buffer medium，some negative effects on the molecular structure and nature of BSA might occur and resulted in a too high background scattering noise,which caused a rapid decrease in the resonance scattering intensity. A good calibration curve of the protein was obtained in the range of 1.00-5.00 μg·mL-1 with a detection limit (3σ/K) of 0.085 μg·mL-1. Applied to the quantitative analysis of BSA simulant samples,the result was satisfied and the recoveries were 98.0%-101.7% at the concentrations ranging from 2.00 to 4.00 μg·mL-1. The relative standard deviation was less than 1.7%.