Label-free electrochemical sensing platform for sensitive detection of ampicillin by combining nucleic acid isothermal enzyme-free amplification circuits with CRISPR/Cas12a.

The residue of ampicillin (AMP) in food and ecological environment poses a potential harm to human health. Therefore, a reliable system for detecting AMP is in great demand. Herein, a label-free and sensitive electrochemical sensor utilizing NH-Co-MOF as an electrocatalytic active material for methylene blue (MB) was developed for rapid and facile AMP detection by combining hybridization chain reaction (HCR), catalytic hairpin assembly (CHA) with CRISPR/Cas12a. The surface of glassy carbon electrode modified with NH-Co-MOF was able to undergo HCR independent of the AMP, forming long dsDNA complexes to load MB, resulting in strong original electrochemical signal. The presence of AMP could trigger upstream CHA circuit to activate the CRISPR/Cas12a system, thereby achieving rapid non-specific cleavage of the trigger ssDNA of HCR on the electrode surface, hindering the occurrence of HCR and reducing the load of MB. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the AMP with a detection limit as low as 1.60 pM (S/N = 3). The proposed sensor exhibited good stability, selectivity, and stability, and achieved reliable detection of AMP in milk and livestock wastewater samples, demonstrating its promising application prospects in food safety and environmental monitoring.