Long-Lived, Nonenzymatic Signal Generation Chemiluminescence Immunoassay for Pretreatment-Free Detection of...

To address the technical limitations of the short chemiluminescence (CL) lifetime and poor stability of chemiluminescence emission in conventional CL systems, a novel CL with prolonged lifetime and high intensity with nonenzymatic signal generation was innovatively developed. Deep red carbon dots (DRCDs) served as efficient energy acceptors, while bis(2,4,6-trichlorophenyl) oxalate (TCPO)-H2O2 acted as energy donors. Compared with traditional systems, the luminescence lifetime was extended by 2 orders of magnitude, accompanied by an excellent CL quantum yield of 6.99 × 10-3 einsteins/mol. Based on this, a TCPO-H2O2-DRCDs-based CL immunoassay was first developed for lincomycin detection in milk. The method achieved a detection limit as low as 0.025 ng/mL, demonstrating a 39.2-fold improvement in sensitivity over reported methods. Benefiting from the nonenzymatic signal emission mechanism and long-lived glow-type signal output, the assay exhibited significantly enhanced stability and allowed quantitative analysis without relying on expensive, large-scale transient signal detection equipment. Moreover, samples could be analyzed without any pretreatment, greatly improving analytical throughput and operational convenience. Results from 10 real samples showed high consistency with confirmatory LC-MS/MS data (R2 > 0.99). The constructed stable signal-output, long-lived CL signaling platform lays a solid theoretical and technical foundation for the development of next-generation highly sensitive and robust immunoassays suitable for point-of-care testing and resource-limited settings.