Locking-DNA network regulated CRISPR-Cas12a fluorescent aptasensor based on hollow flower-like magnetic MoS2 microspheres for sensitive detection of sulfadimethoxine

Sulfadimethoxine (SDM) poses a potential threat to the food safety and human health due to its overuse in aquatic or animal husbandry. Therefore, a rapid and sensitive identification method is urgently needed to monitor SDM in food and the environment. Herein, a turn-on fluorescence aptasensor based on entropy-driven catalysis (EDC) and CRISPR-Cas12a was proposed for the sensitive determination of SDM. Particularly, to overcome the barriers of coupling the EDC circuit to CRISPR-Cas12a, a novel EDC circuit with a Y-shaped substrate scaffold was designed to lock the activation sequence of CRISPR-Cas12a. Multifunctional hollow flower-like microsphere H-Fe3O4@MoS2 was synthesized as a fluorescence resonance energy transfer (FRET) energy acceptor for ssDNA-FAM to quenching its fluorescence. In the presence of the SDM, activated Cas12acrRNA-A rapidly cleaved the ssDNA-FAM adsorbed on the MoS2 nanosheet to restore the fluorescence signal in the reporter system, which could be utilized to determine the concentration of SDM. Due to the prominent quenching properties and rapid magnetic separation of H-Fe3O4@MoS2 along with the double signal amplification of locking-EDC-CRISPR-Cas12a, the developed aptasensor exhibited the appealing analytical performance with a log-linear range of 0.005-100 ng mL-1 and a limit of detection of 2.86 pg mL-1. Furthermore, the practicability and credibility of the locking-EDC-Cas12a sensoring strategy were validated by spiked recovery experiments of SDM in real samples. Hence, the current strategy provides a promising candidate analytical tool for food and environmental safety.

Automated summary

A turn-on fluorescence aptasensor based on entropy-driven catalysis (EDC) and CRISPR-Cas12a was developed for the sensitive determination of sulfadimethoxine (SDM). A novel EDC circuit was designed to lock the activation sequence of CRISPR-Cas12a, and H-Fe3O4@MoS2 microsphere was used as a fluorescence resonance energy transfer (FRET) acceptor for quenching the fluorescence of ssDNA-FAM. The developed aptasensor showed excellent analytical performance with a wide dynamic range and low detection limit.