Nonlinear Hybridization Chain Reaction Integrated with Fluorescent Immunosensor for Ultrasensitive Detection of Alpha Fetoprotein

As sensitive biomarker detection techniques are desirable for both disease diagnosis and postoperative examination, nonlinear hybridization chain reactions (NHCR) have been widely developed for biosensor platforms as signal amplification techniques to increase signal output. In this study, we developed a flow cytometry immunoassay for the detection of alpha-fetoprotein (AFP) using hairpin-free NHCR. First, the target AFP proteins are captured on the magnetic beads (MBs) modified with capture antibodies. Then, the prepared biotin-streptavidin-biotin (B-S-B) system, which links biotinylated detection antibodies and biotinylated trigger DNAs together through the high affinity between biotin-streptavidin interaction, is added to mark the target AFP protein and form a sandwich complex with three trigger DNA chains. Next, each trigger DNA chain grows a dendritic DNA nanostructure following a nonlinear hybridization chain reaction. As the substrate flue chains have been labeled with fluorophores, the self-assembly process of dendritic DNA is accompanied by the continuous release of fluorophores, and dendrites with strong fluorescence are formed on the surface of MBs afterwards. Finally, the MBs are analyzed using flow cytometry to detect the fluorescence for the analysis of the target AFP. As a result, this developed immunoassay has a high selectivity along with an isothermal, enzyme-free, and exponential amplification efficiency, and exhibited a detection limit of 1.74 pg/ml. This new biosensor has also been successfully used to quantitatively detect AFP in serum samples. Notably, this biosensor has the potential for medical research and disease diagnosis by simultaneously detecting multiple tumor markers by changing the size of MBs and antibody-antigen pairs.