Fluorescence and Scattering Light Cross Correlation Spectroscopy and Its Applications in Homogeneous Immunoassay

In this work, we propose fluorescence and scattering light cross-correlation spectroscopy (FSCCS) based on laser confocal configuration using silver nanoparticle (SNPs) and Alexa Fluor 488 (Alexa) as probe pairs. FSCCS is a single molecule (particle) method, and its principle is similar to that of fluorescence cross-correlation spectroscopy (FCCS). We established the setup of FSCCS using single wavelength laser and developed an immunoassay model of FSCCS. The reliability and adaptability of FSCCS method were evaluated by homogeneous sandwich immunoassay mode. In the study, liver cancer biomarker alpha-fetoprotein (AFP) was used as an assay model, two different antibodies were labeled with SNPs and fluorophore Alexa Fluor 488, respectively. In the optimal conditions, the linear range of AFP covers 5 pM to 580 pM and the detection limit is 3.1 pM. This method was successfully applied for direct determination of AFP levels in human serum samples, and the obtained results were in good agreement with data obtained via ELISAs. The advantage of this method lies in its simplicity, attractive SNPs probes, high sensitivity and selectivity and high efficiency. We believe that FSCCS method exhibits promising potential applications in homogeneous bioassays and study on the molecular interaction and nanoparticle-molecule interaction.