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.