Au-Fe₃O₄ nanozyme coupled with CRISPR-Cas12a for sensitive and visual antibiotic resistance diagnosing

The accumulation and spread of antibiotic resistance bacteria (ARB) in the environment may accelerate the formation of superbugs and seriously threaten the health of all living beings. The timeliness and accurate diagnosing of antibiotic resistance is essential to controlling the propagation of superbugs in the environment and formulating effective public health management programs. Herein, we developed a speedy, sensitive, accurate, and user-friendly colorimetric assay for antibiotic resistance, via a synergistic combination of the peroxidase-like property of the Au-Fe₃O₄ nanozyme and the specific gene identification capability of the CRISPR-Cas12a. Once the CRISPR-Cas12a system recognizes a target resistance gene, it activates its trans-cleavage activity and subsequently releases the Au-Fe₃O₄ nanozymes, which oxidizes the 3,3,5,5-tetramethylbenzidine (TMB) with color change from transparent to blue. The diagnosing signals could be captured and analyzed by a smartphone. This method detected kanamycin-resistance genes, ampicillin-resistance genes, and chloramphenicol-resistance genes by simple operation steps with high sensitivity (<0.1 CFU μL^-1) and speediness (<1 h). This approach may prove easy for the accurate and sensitive diagnosis of the ARGs or ARB in the field, thus surveilling and controlling the microbial water quality flexibly and efficiently.