Detection of antibiotic-resistance genes in bacterial pathogens using a Cas12a/3D DNAzyme colorimetric paper sensor

The rapid detection of antibiotic-resistant genes in bacterial pathogens is critical in combating global health crises. Herein, we report a CRISPR/Cas12a-based colorimetric paper sensor, where the trans-cleavage activity of Cas12a was post-amplified by rolling circle replication, resulting in the generation of a 3D DNAzyme. The 3D DNAzyme adhered strongly to the paper surface, creating a highly bioactive paper sensor containing high densities of functional DNAzymes. This assay was effective for the rapid detection of the antibiotic-resistant gene, NDM-1, with high sensitivity. In the absence of the NDM-1 gene, the 3D DNAzyme catalyzed a colorimetric reaction, resulting in a blue-colored signal while in the presence of NDM-1, collateral cleavage activity of Cas12a was activated, leading to cleavage of the circle template, thus preventing the generation of the 3D DNAzyme and producing no colorimetric signal. This paper sensor provides rapid and low-cost detection of antibiotic-resistant genes carried by various pathogenic microorganisms with femtomolar-level sensitivity and results that are visible to the naked eye. The entire analysis requires less than 90 minutes of assay time. Due to the highly programmable design of the CRISPR probe, the platform has significant potential for quick responses to new global epidemics.