CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules

CRISPR/Cas systems are powerful tools for sensitive nucleic acid molecular diagnosis due to their specific nucleic acid recognition and high trans-cleavage activity and have also allowed for quantification of non-nucleic acid targets, relying on a strategy to convert the target detection to analysis of nucleic acids. Here, we describe a CRISPR/Cas12a-powered immunosorbent assay for sensitive small-molecule detection by using the antibody coated on the microplate to recognize the target and the small molecule-labeled active DNA (acDNA) to trigger the activity of CRISPR/Cas12a. In the absence of small-molecule targets, acDNA probes are captured by the antibody on the microplate and then activate Cas12a in catalytic trans-cleavage of fluorescent DNA reporters, generating strong fluorescence. The presence of small-molecule targets displaces the acDNA probes from the antibody, causing a decrease of acDNA probes on the microplate and reduction of activated Cas12a, so the fluorescence signal decreases, and small molecules can be detected by monitoring the fluorescence change. After systematically optimizing experimental conditions (e.g., Cas12a reaction), the proposed method achieved the detection of three model small molecules, biotin, digoxin, and folic acid, with low detection limits, and a flexible detection concentration range was obtained by simply changing the amount of acDNA probes and immobilized antibodies. The assay showed high selectivity and good applicability in complex media. The integration of the CRISPR/Cas12a system improves the analytical performance of immunoassay, broadening and facilitating its applications in rapid, simple, and sensitive small molecule analysis.