Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution

Sensitive detection of small molecules is crucial for many applications, like biomedical diagnosis, food safety, and environmental analysis. Here, we describe a sensitive CRISPR-Cas12a-assisted immunoassay for small molecule detection in homogeneous solution. An active DNA (acDNA) modified with a specific small molecule serves as a competitor for antibody binding and an activator of CRISPR-Cas12a. Large-sized antibody binding with this acDNA probe inactivates the collateral cleavage activity of CRISPR-Cas12a due to a steric effect. When free small molecule target exists, it replaces the small molecule-modified acDNA from antibody, triggering catalytic cleavage of DNA reporters by CRISPR-Cas12a, and strong fluorescence is generated. With this strategy, we achieved detection of three important small molecules as models, biotin, digoxin, and folic acid, at picomolar levels by using streptavidin or antibody as recognition elements. With the progress of DNA-encoded small molecules and antibody, the proposed strategy provides a powerful toolbox for detection of small molecules in wide applications.