A DNAzyme amplifier-based immunoassay for small molecule detection

Detection of small molecules is of urgent demand for many applications, such as drug discovery, disease diagnosis, food safety, and environmental monitoring. Developing simple, selective, and sensitive methods to detect small molecules has attracted wide attention from researchers. In this work, we report a DNAzyme amplifier-based immunoassay for sensitive detection of small molecules. This strategy combines the benefits of the specific binding between antibodies and antigens with the superior signal amplification capability of DNAzymes. Antigen-labeled 10-23 DNAzyme binds with immobilized antibodies on a microplate, catalyzing the cleavage of numerous fluorescently labeled substrate molecules in the presence of Mg²⁺, thus generating amplified fluorescence signals. In the presence of small molecule targets, the targets compete with the antigen-labeled DNAzyme for binding with the immobilized antibodies on the microplate, resulting in a decrease in the fluorescence signal. We successfully detected small molecules, digoxin and folic acid, at picomolar levels through the proposed immunoassay. The immunoassay can specifically identify small molecules and enable their detection in complex sample matrices. This strategy provides a simple and sensitive approach to selectively detect small molecules, inspiring researchers to develop DNAzyme-based immunoassays with multiple functions.