Nonlinear hybridization chain reaction-based flow cytometric immunoassay for the detection of prostate specific antigen

Sensitive detection of biomarkers is highly desirable for disease diagnosis and postoperative examination. As a signal amplification technique, nonlinear hybridization chain reactions (NHCR) based on DNA self-assembly has been widely adopted to versatile biosensor platforms for signal output and sensitivity enhancement. Herein, we proposed a novel hairpin-free NHCR based flow cytometric immunoassay for prostate specific antigen (PSA) detection. In this study, Ab1-Ag-Ab2-streptavidin-trigger DNA complexes were formed on the magnetic beads (MBs), and each trigger DNA initiated a round of NHCR amplification to form dendritic DNA nanostructures with many fluorescent signal molecules. The robust flow cytometric fluorescent analysis was finally employed for the quantitation of target protein on the MBs. As far as we know, this is the first time to combine the hairpin-free NHCR strategy with fluorescent immunoassay on MBs to detect protein biomarkers. In addition to the high selectivity of immunoassay itself, the characteristics of isothermal, enzyme-free, and exponential amplification efficiency of hairpin-free NHCR endow this developed immunoassay with a detection limit that exceeds 100-folds that of commercially available PSA kits. Moreover, this MBs-based platform of this immunoassay is also amenable to target enrichment and removal of spontaneous NHCR signal through magnetic separation, greatly eliminating the background signal interference. With our efforts, this newly developed biosensor exhibits a detection limit of 1.92 pg/mL and shows high selectivity. It has also been successfully applied to the quantitative detection of PSA in serum samples. With these merits, this convenient biosensor platform has the potential for medical research and disease diagnosis. [Display omitted] •A novel sandwich immune-NHCR assay was developed to achieve highly sensitive detection of prostate-specific antigen.•High selectivity were ensured via NHCR signal amplification with magnetic enrichment.•Flow cytometry enables direct analysis of NHCR products on each magnetic bead.•This method was proven to be robust for the detection of human serum.