Homogenous Immunoassay By Fluorescence Correlation Spectroscopy

Immunoassay is currently one of very widely used analytical technologies in clinical diagnosis, food and environmental analyses and biological and biomedical studies. Compared to heterogeneous assays, homogeneous assay is to directly determine analytes in immune reaction mixture. This method is usually fast and amenable to miniaturization and automation. Recently, homogeneous immunoassay is an attractive alternative to conventional heterogeneous immunoassays due to its fast and amenable operation. However, homogeneous immunoassay is not satisfied for current requirement in clinical diagnosis for its low sensitivity in high-noise bioassay.In this paper, on the basis of novel nanoparticle, we developed a highly sensitive homogeneous immunoassay by combining fluorescence correlation spectroscopy (FCS) with silver nanoparticles (SNPs)-antibody conjugates as probes. The main contributions are as follows:1. We first synthesized 14 nm 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). MUA-SNPs were covalently linked with antibody in different molar ratios of antibody/SNPs (2:1 and 10:1), and low-antibody-SNPs and high-antibody-SNPs conjugates were obtained. 2. The immune reaction of alpha fetal protein (AFP) antigen and its antibody was used as a reaction model in this study. 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 increase of about 60-fold (low-antibody-SNPs) and 120-fold (high-antibody-SNPs) in the diffusion times compared to free dye-labeled antigen. Meanwhile, the antigen-antibody complexes showed more than 18-fold and 49-fold increase in fluorescence intensity and about 10-fold and 20-fold increase in signal-to-noise ratio, respectively. 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 1.5 pM and the linear range was from 6 pM to 60 pM (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%.In summary, the use of SNPs labeled antibody in FCS based immunoassays not only increased the resolution of the antigen and the antigen-antibody complexes, but also significantly enhanced the sensitivity of immunoassays. We believe that SNPs enhanced fluorescence correlation spectroscopy possesses great potential applications in clinical diagnosis, environmental analysis and biological and biomedical studies.