Complementary Strand-Free Persistent Luminescence Ratiometric Aptasensor for Ultrasensitive and Selective Carbendazim Assay

Carbendazim (CBZ) poses significant health risks, necessitating its selective and sensitive detection. Herein, we report a complementary strand (cDNA)-free ratiometric aptasensor based on persistent luminescence nanoparticles (PLNP) that operates without in-situ excitation for the accurate quantification of trace CBZ in complex matrices. The sensor integrates an aptamer to enhance affinity and specificity, yet eliminates the conventional quencher-labelled cDNA. Methylene blue (MB) is innovatively introduced as a multifunctional substitute: it self-adsorbs onto the aptamer-modified PLNP through π–π stacking, electrostatic attraction, and hydrogen bonding, serving simultaneously as the resonance-energy-transfer acceptor and the second signaling channel. MB quenches the PLNP phosphorescence at 542 nm while generating its own 700 nm emission. Upon CBZ addition, CBZ-aptamer binding releases MB, disrupts the energy-transfer pathway and switches the 542 nm signal “on” while turning the 700 nm signal “off”. This anti-directional dual-channel read-out efficiently eliminates background fluorescence and external interference, enhancing sensitivity. The sensor exhibits a wide linear range of 2.27–500 ng mL −1, a detection limit of 0.682 ng mL −1, and quantitative recovery (90.1–107.4%). The proposed strategy offers superior selectivity, sensitivity, and accuracy. The developed sensor was validated by analyzing a certified reference materials and comparing with an HPLC method and successfully applied to the determination of CBZ in real food samples (cherry tomatoes, oranges, lettuce, green peppers, strawberries, cucumbers, apples, and Chinese chives).