Ultrasensitive Dual-Mode Visual/Photoelectrochemical Bioassay for Antibiotic Resistance Genes through Incorporating Rolling Circle Amplicons into a Tailored Nanoassembly

As emerging contaminants in the environment, antibiotic resistance genes (ARGs) have aroused a global health crisis and posed a serious threat to ecological safety and human health. Thus, efficient and accurate onsite detection of ARGs is crucial for environmental surveillance. Here, we presented a colorimetric-photoelectrochemical (PEC) dual-mode bioassay for simultaneous detection of multiple ARGs by smartly incorporating rolling circle amplification (RCA) into a stimuli-responsive DNA nanoassembly, using the tetracycline resistance genes tetA and tetC as models. The tailored DNA nanoassembly containing RCA amplicons hybridized with specific signal probes: CuO nanoflowers-anchored signal DNA1 and HgO nanoparticles-anchored signal DNA2, respectively. Upon exposure to an acidic stimulus, numerous Cu²⁺ and Hg²⁺ were released, serving as the reporting agent of colorimetric/PEC dual-mode assay. The released Cu²⁺ and Hg²⁺ induced localized surface plasmon resonance shifts in Au nanorods and triangular Ag nanoplates through an etching process, respectively, enabling visual analysis of ARGs with distinguishing color changes. Meanwhile, numerous Cu²⁺ and Hg²⁺ triggered the amplified PEC variations via reacting with the photoactive layers of CuS/CdS and ZnS, respectively. Thus, a rapid and ultrasensitive colorimetric/PEC dual-mode detection of multiple ARGs was achieved with the detection limit down to 17.2 aM. Furthermore, such dual-mode bioassay could discriminate single-base mismatch and successfully determine ARGs in E. coli plasmids and sludge samples, holding great promise for point-of-care genetic diagnostics.