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

Aflatoxin B 1 (AFB 1 ) 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 AFB 1 detection in oils. This competitive enzyme-linked immunosorbent assay (ELISA)-type nanosensor incorporates a Cu 2 O@MnO 2 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 AFB 1 with the exceptional oxidase-mimicking activity of cDNA-decorated Cu 2 O@MnO 2 nanozymes. In addition, the integration of aptamer-conjugated Fe 3 O 4 magnetic nanoparticles (MNPs) enables high specificity for AFB 1 , 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 AFB 1 detection, offering an advanced strategy for future food safety monitoring .