Accelerated CRISPR/Cas12a-based small molecule detection using bivalent aptamer.

CRISPR/Cas holds great promise for biosensing applications, however, restricted to nucleic acid targets. Here, we broaden the sensing target of CRISPR/Cas to small molecules via integrating a bivalent aptamer as a recognition component. Using adenosine 5'-triphosphate (ATP) as a model molecule, we found that a bivalent aptamer we selected could shorten the binding time between the aptamer and ATP from 30 min to 3 min, thus dramatically accelerating the detection of ATP. The accelerated bivalent aptamer binding to ATP was mainly ascribed to the extended conformation of the aptamer, which was stabilized through linking with a 5 T bases connector on specific loops of the monovalent aptamer. To facilitate on-site detection, we integrated lateral flow assay (LFA) with the CRISPR/Cas sensing strategy (termed BA-CASLFA) to serve as a visual readout of the presence of ATP. In addition, in the CASLFA platform, due to the unique characteristics of LFA, the thermal step of Cas12a inactivation can be omitted. The BA-CASLFA could output a colorimetric "TURN ON" signal for ATP within 26 min, which could be easily discriminated by the naked eye and sensitively quantified by the portable reader. Furthermore, we showed the versatility of BA-CASLFA for detecting kanamycin using a kanamycin bivalent aptamer obtained through the same design as the ATP bivalent aptamer. Therefore, this strategy is amenable to serve as a general sensing strategy for small molecular targets. The above work opened a new way in developing CRISPR-based on-site sensors for clinic diagnosis, food safety, and environmental analysis.