Simplified ratiometric electrochemiluminescence aptasensor for carcinoembryonic antigen detection based on unmodified aptamer using single luminophore and coreactant.

Ratiometric electrochemiluminescence (ECL) aptasensors have garnered significant interest in bioanalysis by simultaneously measuring two distinct emission signals and utilizing their intensity ratio for quantitative output. However, conventional designs face critical limitations. Mandatory aptamer functionalization elevates fabrication complexity and costs. Additionally, dual-luminophore/coreactant systems often suffer from inherent signal mutual interference. To address these issues, we developed a novel ratiometric ECL aptasensing platform employing a single luminophore (luminol) and coreactant (HO) for carcinoembryonic antigen (CEA) detection. This simplified system utilized an unmodified aptamer-G-quadruplex (G4)-hemin complex, which played two roles: (1) suppressing cathodic ECL (E) through hydroxyl radical consumption; (2) enhancing anodic ECL (E) via catalytic acceleration of luminol and HO oxidation. Target-induced CEA-aptamer binding triggered G4-hemin dissociation, generating inversely correlated E enhancement and E reduction. This dynamic enabled quantitative CEA detection through the E/E ratio, achieving an ultrabroad linear range (10 fg mL-10 ng mL) with a detection limit of 0.85 fg mL. Notably, the ratiometric signal is independent of HO concentration. This effectively eliminates interference from HO self-decomposition, significantly improving detection reliability. Successful application in human serum analysis validated clinical utility. This work establishes a new strategy for developing robust ratiometric ECL biosensors, potentially expanding their applications in clinical diagnostics and point-of-care testing.