College of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiaotong University, Shanghai, PR China.
Talanta. 2010 Jun 15;81(4-5):1560-7. doi: 10.1016/j.talanta.2010.03.002. Epub 2010 Mar 7.
In this paper, we developed a highly sensitive homogeneous immunoassay by combining fluorescence correlation spectroscopy (FCS) with silver nanoparticles (SNPs)-antibody conjugates as probes. We first synthesized 14nm SNPs in aqueous solution and then modified SNPs with 11-mercaptoundecanoicacid (MUA) via SNP-S bond. Resonance light scattering correlation spectroscopy (RLSCS) was utilized to characterize SNPs and MUA-functionalized SNPs (MUA-SNPs). The immune reaction of alpha fetal protein (AFP) antigen and its antibody was used as a reaction model and AFP labeled with Alexa Fluor 647 was used as the tracer antigen in homogeneous competitive immunoassay. We observed that the antigen-antibody complexes showed the significant increase in the diffusion times and fluorescence intensity compared to free dye-labeled antigen. On the advantages of the effects of SNPs on fluorescence enhancement and diffusion time, the homogeneous competitive immunoassay was performed by the two-component model analysis of FCS. Under the optimal condition, the detection limit was 1.5pM and the linear range was from 6pM to 60pM (R>0.99). This assay was successfully applied for the determination of the AFP level in human serum samples, the relative standard deviation was about 5%, and the recoveries were over 90%.
本文结合荧光相关光谱（FCS）和银纳米粒子（SNPs）-抗体偶联物作为探针，开发了一种高灵敏度的均相免疫分析方法。我们首先在水溶液中合成了 14nm 的 SNPs，然后通过 SNP-S 键将 SNPs 与 11-巯基十一酸（MUA）修饰。共振光散射相关光谱（RLSCS）用于表征 SNPs 和 MUA 功能化的 SNPs（MUA-SNPs）。将 alpha 胎儿蛋白（AFP）抗原及其抗体的免疫反应用作反应模型，并将 Alexa Fluor 647 标记的 AFP 用作均相竞争免疫分析中的示踪抗原。我们观察到，与游离染料标记的抗原相比，抗原-抗体复合物显示出扩散时间和荧光强度的显著增加。利用 SNPs 对荧光增强和扩散时间的影响，通过 FCS 的两分量模型分析进行了均相竞争免疫分析。在最佳条件下，检测限为 1.5pM，线性范围为 6pM 至 60pM（R>0.99）。该方法成功应用于人血清样品中 AFP 水平的测定，相对标准偏差约为 5%，回收率超过 90%。
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