Instant synthesis of bimetallic CuCo PBA nanozyme for efficient colorimetric immunoassay of carcinoembryonic antigen

Colorimetric immunoassays are widely used for biomarker detection, offering advantages of simplicity, sensitivity, and cost-effectiveness. Recent advancements focus on improving the catalytic activity of nanozymes for enhancing the sensitivity and accuracy of such assays. Bimetallic CuCo Prussian blue analog (CuCo PBA) has emerged as promising candidates due to their excellent peroxidase-like activity. However, their instant synthesis and integration into immunoassays for the rapid detection of biomarkers like carcinoembryonic antigen (CEA) remain underexplored. This study presents an innovative approach using CuCo PBA nanozymes for colorimetric immunoassays with immediate generation and application. In this study, CuCo PBA nanozymes were synthesized instantly by reacting Cu 2+ with K 3 [Co(CN) 6 ] (<1 min), and their peroxidase-like activity was exploited for a colorimetric immunoassay system targeting CEA. The system demonstrated a clear blue color change upon the reaction of CuCo PBA with H 2 O 2 and 3,3′,5,5′-tetramethylbenzidine (TMB), enabling sensitive detection. The assay was optimized for various parameters, including pH, temperature, and material ratio. A linear response was obtained for CEA detection over a concentration range of 0.05–60 ng/mL with a limit of detection (LOD) of 22 pg/mL. The integration of glucose oxidase (GOx) mediated the generation of H 2 O 2 , triggering the colorimetric reaction. This instantaneous CuCo PBA-based system effectively detected CEA in human serum samples, highlighting its potential for rapid diagnostic applications. This work introduces a novel approach for rapid and sensitive colorimetric immunoassays using CuCo PBA nanozymes that are synthesized on-demand and immediately applied. The system allows for efficient CEA detection with an exceptionally low detection limit, offering great potential for clinical diagnostics. The instant generation and application of CuCo PBA nanozymes in immunoassays represent a significant advancement in point-of-care testing technologies.