A Luminol-Based, Peroxide-Free Fenton Chemiluminescence System Driven by Cu(I)-Polyethylenimine-Lipoic Acid Nanoflowers for Ultrasensitive SARS-CoV-2 Immunoassay

The reliance on unstable hydrogen peroxide (H₂O₂) adversely affects the robustness and simplicity of chemiluminescence (CL)-based immunoassays. We report a novel external H₂O₂-free Fenton CL system integrated into a highly sensitive non-enzymatic immunoassay for the detection of SARS-CoV-2 nucleoprotein, utilizing cuprous-polyethylenimine-lipoic acid nanoflowers (Cu(I)-PEI-LA-Ab NF) as a non-enzymatic tag. The signaling polymer (PEI-LA) was synthesized via EDC/NHS coupling, which conjugated approximately 550 LA units to the PEI backbone. This polymer formed antibody-conjugated NF with various metal ions, and the Cu(I)-based variant was selected for its intense and sustained CL with luminol. The mechanism relies on an in situ Fenton reaction, in which dissolved oxygen is reduced by Cu(I) to H₂O₂, which reacts with oxidized Cu(II), producing hydroxyl radicals that oxidize luminol. Direct calibration of the SARS-CoV-2 nucleoprotein fixed on microplate wells demonstrated excellent linearity in the range of 0.01-3.13 ng/mL (LOD = 3 pg/mL). In a final competitive immunoassay format for samples spiked with the antigen, a decreasing CL signal that correlated with increasing antigen concentration was obtained in the range of 0.1-20.0 ng/mL, achieving excellent recoveries that were favorable compared with those of the sandwich ELISA kit, establishing this H₂O₂-independent platform as a powerful and robust tool for clinical diagnostics.