A competitive aptamer binding-based CRISPR-cas biosensor for sensitive detection of tetracycline residues in biological samples.

Tetracycline (TC) is widely used in veterinary medicine and animal feed; however, TC residues in food pose a risk to human health. Thus, the sensitive and selective detection of TC is needed to ensure food safety. Herein, we developed a CRISPR-Cas12a biosensor with competitive aptamer binding to detect TC residues. The aptasensor, formed by hybridizing activator DNA with TC-specific aptamers on streptavidin-modified magnetic beads, releases activator DNA in a TC concentration-dependent manner. This activated the Cas12a-crRNA complex, which cleaved single-strand DNA reporters to generate a detectable fluorescence signal. The TC signal was amplified through a two-step incubation reaction, with a detection limit as low as 9.45 × 10 μg L. The assay showed high selectivity and good recovery rates in various biological samples (e.g., honey, milk, fish), demonstrating the applicability of the biosensors in pollutant detection.