Highly sensitive homogenous immunoassay of cancer biomarker using silver nanoparticles enhanced fluorescence correlation spectroscopy.

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%.