Cooperative Split DNA Activation of CRISPR-Cas12a for Sensitive and Selective Detection of Flunixin

Flunixin, a nonsteroidal anti-inflammatory drug widely used in veterinary therapy, presents a risk of residues entering both the food supply and the environment. Therefore, developing sensitive methods for the treatment of flunixin is of great importance. Herein, we report a highly sensitive and selective CRISPR-Cas12a based fluorescent assay for flunixin measurement. By employing functionalized magnetic beads which carry double-stranded DNA (dsDNA) formed by a flunixin binding aptamer and its short partially complementary single-stranded DNA (ssDNA) hybridization probe and taking advantage of two split ssDNA strands to synergistically trigger the <i>trans</i>-cleavage activity of Cas12a, flunixin can be detected with a limit of detection (LOD) reaching as low as 0.35 nM. Furthermore, our sensor was highly selective: other commonly used veterinary drugs such as ciprofloxacin, neomycin, sulfanilamide, sulfamethoxazole, ketoprofen, meloxicam, and mefenamic acid would not interfere with the detection of flunixin. In addition, the simulated water samples were successfully analyzed. The cooperative split DNA activation of the CRISPR-Cas12a sensing strategy developed in this work should find useful application in the development of CRISPR sensors for various non-nucleic acid species, including large biomolecules such as proteins and small molecules and ionic species like metals.