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Herein, we report a CRISPR/Cas12a-assisted chemiluminescence sensor for aflatoxin B 1 (AFB 1 ) detection based on functional nucleic-acid-mediated target recognition and in-pipet rolling circle amplification-mediated signal amplification. In this sensor, we performed rolling circle amplification on the inside of the pipet to enrich horseradish peroxidase (pipet-poly-HRP). When AFB 1 is present, it interacts with functional nucleic acids and results in the release of the activator. The activator is designed to activate the CRISPR/Cas12a system, which cleaves the pipet-poly-HRP to liberate HRP. The freed HRP can then be measured by chemiluminescence to quantify AFB 1 . This CRISPR/Cas12a-assisted chemiluminescence sensor enables facile, highly sensitive, and specific detection of AFB 1 , with a linear range from 50 pg/mL to 100 ng/mL and a detection limit of 5.2 pg/mL. Furthermore, it exhibits satisfactory recovery and has successfully challenged AFB 1 detection in cereal samples. The proposed sensor offers a novel rapid screening approach that holds great promise for food security monitoring.

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