PCR-free and visual detection of antibiotic resistance gene in feces genome of aquatic products by coupling CuO nanoparticles concatemers-Au@Fe3O4 and 3D DNA walker based on a portable blue light gel imager

Abstract

Antibiotic resistance genes (ARGs) present a significant health issue, and the sensitive detection of ARGs is essential for preventing antibiotic resistance. Herein, a novel highly sensitive and visual biosensor was developed by combining gold nanoparticle (AuNP)-coated Fe3O4 (Au@Fe3O4), CuO nanoparticles (CuONPs) concatemers, Cu2+-triggered 3D DNA walker (DW, which included DNAzyme-capped AuNPs (DNAzyme@AuNPs) and AuNPs capped by substrate DNA modified with fluorescein (FAM-Sub@AuNPs), namely CuONPs-Au@Fe3O4-DW method. When the target ARG was present in the sample, it acted as a bridge between the complementary strand DNA (cDNA) on the surface of Au@Fe3O4 and H1-CuONP which paired with repeatedly H2-CuONP by a hybridization chain reaction, resulting in an ARG coupling multiple CuONPs (CuONPs concatemers) effects. After magnetic separation, hydrochloric acid was added to dissolve the CuONPs concatemers to release plenty of Cu2+ ions, which activated the DNAzyme@AuNPs of the DW. Subsequently, the FAM-Sub@AuNP of the DW was sheared by its DNAzyme, resulting that the FAM molecules kept away from AuNPs, and generated high fluorescence signals. The fluorescence signals can be measured by a 96-well microplate reader with a limit of detection (LOD) of 0.89 pM and visualized using a portable blue light gel imager with a LOD of 8.1 pM. Remarkably, the results of the qPCR method are high consistent with those of the microplate reader (r = −0.935) and the blue light gel imager (r = −0.994) in detecting tetA of aquatic faecal samples. Furthermore, our PCR free, high sensitivity and visual method is also suitable for the identification of other ARGs by replacing the corresponding gene sequence, and enables on-site detection of the ARGs in aquatic products by integrating the blue light gel imager.