Immunosensors for dual tumor biomarker detection based on ternary electrochemiluminescence using confined CeO2 nanozyme as Co-reactant enhancers for luminol-O2 system.

Sensitive and parallel individual detection of multiple tumor biomarkers is critical for early diagnosis and therapeutic monitoring of pancreatic cancer. Herein, a ternary electrochemiluminescence (ECL) system was developed by employing nanochannel-confined cerium dioxide (CeO2) nanozyme as co-reactant enhancers for the luminol-dissolved oxygen (DO) system, enabling parallel individual immunoassays of carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199). Vertically-ordered mesoporous silica film (VMSF) was rapidly grown on an indium tin oxide (ITO) electrode, forming nanochannel array with thickness of 97 nm and diameters of 2.7 nm. CeO2 nanozyme was synthesized in situ within the VMSF nanochannels via electrochemical deposition. Transmission electron microscopy (TEM), high-angle annular dark-field scanning TEM (HAADF-STEM), and energy-dispersive X-ray spectroscopy (EDX) elemental mapping confirmed CeO2 nanozyme was confined in nanochannels with high dispersion. CeO2 nanozyme exhibited peroxidase-like catalytic activity and enhanced the ECL intensity of the luminol-DO system by 13.9-fold under near-neutral conditions through catalysis of both oxygen reduction and luminol oxidation. After derivatization of the VMSF outer surface with epoxy groups, antibodies specific to CEA or CA199 were covalently immobilized to construct two immunorecognition interfaces. Target binding formed antigen-antibody complexes, increasing interfacial resistance and hindering diffusion of the ECL emitters, enabling a signal-off immunoassay mode. The resulting immunosensors displayed wide detection linear ranges from 0.1 pg mL-1 to 100 ng mL-1 for CEA and 0.1 × 10-3 mU mL-1 to 1.0 × 104 mU mL-1 for CA199, with limit of detection (LOD) of 1.3 fg mL-1 and 0.5 × 10-4 mU mL-1, respectively. Both immunosensors exhibited good selectivity, reproducibility, and storage stability.