Cu/SiO₂ nanozyme-amplified colorimetric immunosensor for high-fidelity pancreatic cancer detection with...

Pancreatic cancer (PC) remains one of the most lethal malignancies due to the lack of reliable tools for early-stage diagnosis. Therefore, developing highly sensitive and robust detection strategies that accurately quantify CA19-9 in complex biological samples is crucial for improving early diagnostic outcomes. A Cu/SiO₂ nanozyme, which exhibits intrinsic peroxidase-mimetic activity, has been utilized for the first time in an immunodetection assay for the sensitive and selective analysis of PC-biomarker CA19-9. Cu/SiO₂ nanozyme oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) with hydrogen peroxide (H₂O₂), producing blue-colored oxTMB with absorbance at 650 nm. An indirect ELISA was performed using a mouse anti-CA19-9 monoclonal antibody (MAC IgG) and a goat anti-mouse IgG secondary antibody (GAM IgG) conjugated to a Cu/SiO₂ nanozyme. Cu/SiO₂ nanozyme kinetics were performed using Michaelis-Menten and Lineweaver-Burk plots. The obtained parameters were 7.43 × 10^-3 M, 8.65 × 10^-7 M s^-1, and 3.67 × 10^-4 s^-1 for K_M, V_max, and K_cat, respectively. The developed colorimetric immunoassay demonstrated high sensitivity with a limit of detection (LOD) of 0.000771 U mL^-1. The applicability of the developed method was evaluated using human serum samples, achieving 100.18-103.15% recovery. Software-assisted analysis was utilized for rapid detection, achieving an outstanding LOD of 0.00139 U mL^-1, respectively. Overall, this developed colorimetric immunoassay demonstrates its applicability for sensitive and clinically relevant detection of the PC biomarker CA19-9.