Additional Molecular Biological Amplification Strategy for Enhanced Sensitivity of Monitoring Low-Abundance Protein with Dual Nanotags

A new signal-on immunoassay protocol for sensitive electronic detection of alpha-fetoprotein (AFP) was developed by coupling with metal sulfide nanolabels and a silver nanocluster (AgNC)-based rolling circle amplification (RCA) strategy. Initially, a sandwiched immunocomplex was formed on a primary antibody-coated microplate using a PbS nanoparticle-labeled polyclonal anti-AFP antibody (PbS-pAb2) as the detection antibody, and then the carried PbS-pAb2 was dissolved by acid to release a large number of lead ions, which could induce the cleavage of lead-specific DNAzyme immobilized on the electrode. The residual single-stranded DNA on the electrode could be used as the primer to produce numerous repeated oligonucleotide sequences via the RCA reaction for the hybridization with many AgNC-labeled detection probes, resulting in the amplification of the electronic signal due to the unique properties of silver nanoclusters. Under optimal conditions, the developed immunoassay exhibited high sensitivity for the detection of AFP with a dynamic range of 0.001-200 ng mL(-1) and a detection limit (LOD) of 0.8 pg mL(-1). Intra-assay and interassay coefficients of variation were below 8.0% and 10%, respectively. Importantly, the methodology was evaluated by analyzing 12 clinical serum specimens, and no significant differences were encountered in comparison with the conventional enzyme-linked immunosorbent assay (ELISA) method.