CRISPR/Cas12a-mediated fluorescent aptasensor based on DNA walker amplification for oxytetracycline detection.

Nowadays, although most oxytetracycline (OTC) are used only in animals, their leaching into the environment and residues in food lead to noticeable health problems. Fortunately, by studying the molecular dynamics of OTC aptamers and the mutations of key bases, aptamer's base fragments involved in identifying OTC was confirmed for the first time, which provided theoretical guidance for the ingenious design of aptamer detection. Thereafter, benefiting from the amplification impact by DNA walker, the disadvantage of low efficiency caused by CRISPR/Cas12a was effectively avoided. In the established delicate aptasensing platform, small quantity of OTC promoted DNA walker to produce a massive of activators, which stimulated the trans-cleavage activity of CRISPR/Cas12a. By implying nanoclusters (NCs) with covalent organic framework (COF), the sensitive fluorescence "turn-on" method for OTC detection was realized. Under the optimum conditions, the linear range of aptasensor was 0.05-50 μM, and the detection limit was 0.041 μM. In addition, the aptasensing method can be successfully used for the quantitative detection of OTC in milk and honey samples with a recovery rate of 95.67%-104.10% and 95.33%-109.70%, implying excellent analytical performance and practical application prospect.