A novel chemiluminescence immunoassay for highly sensitive and specific detection of protein using rolling circle amplification and the multiplex binding system

A simple, sensitive and specific chemiluminescence immunoassay (CLIA) strategy has been developed for protein detection by integrating rolling circle amplification (RCA), multiplex binding of the biotin-streptavidin (B-SA) system and enzymatic amplification. The cascade signal amplification methodology is initiated by specifically recognition of target protein based on sandwich immunoassay, which can combine with circular DNA for triggering RCA via biotin-streptavidin. Upon RCA, thousands of repeated sequences are generated for hybridizing with biotinylated detection probes. Then the streptavidin-horseradish peroxidases (ST-HRPs) are bound to biotinylated detection probes, which subsequently catalyze the oxidation of luminol by H2O2 and yield an enhanced chemiluminescence (CL) signal. Taking human prolactin (PRL) as a model, under optimal conditions, the CL intensity was proportional to the logarithm value over six orders from 10 fg mL−1 to 10 ng mL−1 with a detection limit of 0.16 fg mL−1. The established approach was successfully applied for the detection of human PRL in serum samples. Thus, it might be a potential tool for protein detection in clinic biomedical application.