An integrated multi-mode detection platform based on CRISPR/Cas 12a and aptamers for ultra-sensitive identification of sulfamethazine and genes associated with sulfonamide resistance.

Abstract

The production and buildup of sulfamethazine (SMZ) and resistance genes for sulfonamide antibiotics (sul1) pose a serious risk to environmental and public health safety. Creating advanced sensing systems that are both highly sensitive and selective for the prolonged observation of SMZ concentrations in the environment, along with the quantification of sul1 gene prevalence, aims to identify trends in resistance, posing a considerable challenge. Here, we devised a platform (SMZ-sul1 multi-mode detection platform) that allows for the fluorescence detection of SMZ in environmental samples. This is achieved through the competition for the aptamer between the complementary base and SMZ, along with the colorimetric, photothermal, and electrochemical tracking of sul1, using a magnetic separation unit (FP@cDNA). MOF-818@PtPd (MPP) nanozymes with high peroxide mimetic enzyme activity were linked to FP@cDNA through Zr-O-P bond and employed as a catalyst for the 3,3',5,5'-tetramethylbenzidine (TMB) oxidation, as well as for electrocatalytic hydrogen peroxide (H₂O₂) reduction. The ability of Cas12a to perform trans cleavage was activated by its precise identification of the sul1, leading to the non-selective cutting of single-stranded DNA (ssDNA). Thereafter, the MPP nanoparticles were released into the supernatant, where they catalyzed the oxidation of TMB. Alternatively, the functioning CRISPR/Cas12a system specifically targeted and cleaved ssDNA present on the electrode, resulting in altered loading of MPP nanozymes and a decrease in the current associated with the catalytic reduction of H₂O₂. The remarkable magnetic separation capabilities of FP@cDNA, combined with the superior target recognition features of CRISPR/Cas12a and aptamer, facilitated the creation of a highly sensitive detection system, achieving detection limits of 0.67 pM for SMZ and 7.6 fM for sul1, and exhibit great potential for monitoring and prediction in the field of public health.