Cas12a Autocatalytic Amplification Coupled with a DNA-Regulated COF Nanozyme Activation for the Colorimetric Detection of Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a major foodborne and clinical pathogen, and its rapid and accurate detection are essential for food safety monitoring and clinical diagnostics. Here, we developed a colorimetric platform for quantifying S. aureus that integrates circular DNA (Cir-DNA)-mediated Cas12a autocatalytic amplification with DNA regulation of a covalent organic framework (COFTph-BT) nanozyme. Recognition of the 16S rDNA target by the Cas12a/crRNA complex activates trans-cleavage activity, which linearizes Cir-DNA and initiates an autocatalytic amplification circuit that drives cascade cleavage of probe S1. Signal transduction exploits the differential binding affinities of COFTph-BT for single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and its peroxidase activities could be reversely regulated by the surface modification of ssDNA. Cleavage of S1 inhibits its hybridization with COFTph-BT surface-bound S2, maintaining S2 predominantly in its single-stranded form and preserving the catalytic activity of COFTph-BT-S2. The nanozyme subsequently catalyzes the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB), generating a colorimetric readout. The assay can be completed within 90 min and achieves a limit of detection (LOD) of 7 CFU mL−1. Integration with smartphone-based image analysis further enables portable colorimetric detection with an LOD of 87 CFU mL−1. This strategy provides an approach for the sensitive colorimetric detection of foodborne pathogens, with potential applications in food safety monitoring and clinical diagnostics.