Efficient Separation-Free and Wash-Free Immunoassay for Sensitive Detection of Biomarkers Based on the Distance-Driven Chemiluminescent Technique

The wash-free method represents a promising strategy for enhancing the detection efficiency of automated chemiluminescent (CL) immunoassay analyzers. Herein, a novel separation-free and wash-free immunoassay was developed for the first time based on the distance-driven CL technique. In the CL immunoassay, the well-designed Au-Co metal nanoclusters (NCs) exhibited excellent peroxidase-like activity and good stability, allowing for efficient catalysis of luminol or its analogue (ABEI)-H₂O₂ system even at low concentrations. Furthermore, the large specific surface area of Au-Co NCs facilitated the accommodation of a greater number of antibodies, thereby enhancing the capture of antigens and achieving dual amplification of the CL signal. The distance-driven CL technique relied on the formation of sandwich-type immunocomplexes. Upon the generation of hydroxyl radicals and superoxide anion radicals through the catalytic decomposition of H₂O₂ by Au-Co NCs, the ABEI within sandwich-type immunocomplexes could efficiently react with these radicals, leading to a significant enhancement in CL signals. Furthermore, C-reactive protein (CRP) was chosen as the model analyte to evaluate the practicability of the proposed immunoassay. Notably, the proposed immunoassay presented high sensitivity, selectivity, reproducibility, and stability, successfully determining CRP in serum samples with recoveries of 96.55-106.29%. Accordingly, the proposed strategy with the advantage of separation-free, wash-free, and reliable characteristics could drastically simplify the detection operation steps and enhance the detection efficiency, which would make significant advances in the revolution of traditional CL immunoassay.