An aptamer-CRISPR/Cas12a biosensor for rapid and sensitive detection of florfenicol

Abstract

Florfenicol (FF), a broad-spectrum antibacterial agent widely used in livestock and poultry farming, has raised significant food safety concerns due to the accumulation of its residues in animal-derived products (e.g., eggs), posing potential threats to human health. Herein, we developed a novel aptamer-CRISPR/Cas12a biosensor for the rapid and sensitive detection of FF. The biosensor employs streptavidin-modified magnetic beads (SA-MBs) as a solid carrier to achieve efficient enrichment of FF-specific aptamers (APT), while integrating the dual advantages of the APT's high-specificity target recognition and the CRISPR/Cas12a system's powerful signal amplification capability. The detection mechanism is based on a competitive displacement: APT pre-hybridizes with its complementary strand (APT-c) to form stable duplexes. The presence of FF triggers the release of APT-c from APT, and the liberated APT-c then activates the trans-cleavage activity of the CRISPR/Cas12a system. This process converts the small-molecule FF into a CRISPR/Cas12a-detectable nucleic acid signal and enables quantitative FF detection. Under optimized conditions, the biosensor demonstrated a linear detection range of 10 nM to 100 µM for FF (R² = 0.9907) and a limit of detection (LOD) of 1.41 nM. The accuracy and practicality were confirmed through spiked recovery experiments in egg samples, yielding recoveries between 97.1% and 100.8%. Furthermore, the modular design of this platform allows its easy adaptation for detecting other antibiotics simply by replacing the specific APT and its corresponding APT-c, highlighting its considerable potential for broad applications in food safety monitoring.