A signal-on fluorescent probe integrating Au@Ag NPs-based fluorescence resonance energy transfer quenching and enzyme-free catalytic hairpin assembly for c arcinoembryonic antigen detection

Carcinoembryonic antigen (CEA) is a pivotal broad-spectrum tumor marker, yet its accurate quantification in early-stage cancer remains challenging due to the limited sensitivity and operational complexity of conventional immunoassays. We report a signal-on fluorescent probe that synergistically integrates the fluorescence resonance energy transfer (FRET) quenching capability of Au@Ag NPs with enzyme-free catalytic hairpin assembly (CHA) amplification for ultrasensitive detection of CEA. In this design, a Cy3-labeled hairpin DNA (H1) is anchored onto Au@Ag NPs to construct Au@Ag-H1 NPs, where the fluorophore is efficiently quenched. In the presence of CEA, the target specifically displaces a complementary DNA (cDNA) trigger from its aptamer duplex, initiating a CHA cascade reaction that repeatedly opens H1, spatially separates Cy3 from the nanoparticle surface, and restores the fluorescence emission. This strategy endows the probe with both an ultralow background signal and exponential signal amplification. Under optimized conditions, the proposed probe exhibited a wide linear range from 0.1 to 50 ng·mL − 1 with a limit of detection as low as 67.2 pg·mL − 1 , alongside excellent selectivity, reproducibility, and stability. Its practical utility was further validated through successful application in spiked human serum and clinical samples from cancer patients. Collectively, this approach provides a simple, rapid, and highly sensitive platform for CEA quantification, holding considerable promise for early cancer screening and diagnostics. Graphical abstract