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Recognition elements capable of recognizing multiple PFAS are needed for monitoring PFAS in food. Using Capture-SELEX with PFOA, PFOS, PFNA, and PFHxS as the selection targets, we isolated aptamer S1, which recognized these four compounds as well as PFHpA and PFHpS. Molecular docking and circular dichroism spectroscopy revealed the shared and differential binding sites of S1 with these six PFAS, elucidating the structural basis for their recognition. S1 was immobilized onto streptavidin-coated magnetic beads to construct an Apt-MBs enrichment platform coupled with HPLC-MS/MS. The method exhibited linearity from 0.067 to 50 ng/mL (R2 > 0.997), low detection limits of 0.020–0.050 ng/mL, recoveries of 71.9%–99.3% in spiked meat samples, and relative standard deviations below 6%. This enrichment-LC-MS/MS dual-mode strategy provides a selective, sensitive, and stable tool for monitoring multicomponent PFAS in complex food matrices, with promising applications in nutritional safety assessment and dietary exposure monitoring.
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