Aptamer-cDNA-driven nanozyme strategy: A recyclable dual-signal immunosensor for amplified detection of aflatoxin B1 in edible oil.

Aflatoxin B1 (AFB1) poses a serious threat to human health, highlighting theurgent need for sensitive, precise and visual detection methods in daily foods. However,traditional detection methods relying on peroxidase (POD)-mimetic activity are susceptible to external interference, which increases false-positive or false-negative rates and compromise precision and repeatability. To address these challenges, a dual-signal nano-immunosensor was developed using an aptamer-cDNA-assisted colorimetric-fluorescent nanozyme-linked immunosorbent assay for ultrasensitive AFB1 detection in oils. This competitive enzyme-linked immunosorbent assay (ELISA)-type nanosensor incorporates a Cu2O@MnO2 nanozyme that catalyzes the colorimetric reaction to generate the first signal, while the second (fluorescent) signal is produced via rhodamine B isothiocyanate (RBITC) fluorescence quenching through the inner filter effect. This strategy combines the high specificity of aptamers for AFB1 with the exceptional oxidase-mimicking activity of cDNA-decorated Cu2O@MnO2 nanozymes. In addition, the integration of aptamer-conjugated Fe3O4 magnetic nanoparticles (MNPs) enables high specificity for AFB1, as well as magnetic separation and nanozyme recyclability. Compared with traditional ELISA, the composited nanosensor demonstrated remarkable enzymatic capability, achieving 188-fold and 68-fold changes in colorimetric and fluorescent modes, respectively, with detection limits significantly loweredto 0.016 ng/mL for colorimetry and 0.044 ng/mL for fluorescence. Therefore, this immunosensor provides a highly reliable, accurate, sensitive and visible approach for rapid AFB1 detection, offering an advanced strategy for future food safety monitoring .