Culture- and PCR-Free Femtomolar Detection of Antibiotic Resistance Genes in Clinical Urine via Cascaded Molecular Machines and Electrokinetic Enrichment

The rapid detection of antibiotic resistance genes (ARGs) is crucial for guiding precise antibiotic therapy and preventing the spread of resistant pathogens. Here, we report a triple-signal amplified biosensor that integrates cascaded molecular machines with on-chip electrokinetic accumulation (CMMEA-Chip) for direct femtomolar-level detection of carbapenemase genes in clinical urine. In this system, the target ARGs first activate a DNAzyme via strand displacement, leading to the release of a key strand (KS). The released KS subsequently triggers a DNA walker (DW) on gold nanoparticles, which autonomously cleaves the FAM-labeled substrate strands to generate fluorescent signals. Liberated fluorophores are further electrokinetically trapped in a three-channel microfluidic chip, completing a triple-amplification process. This method achieves detection limits of 0.89, 0.82, and 0.90 fM for Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), and oxacillinase-48 (OXA-48) genes, respectively, and exhibits excellent concordance with quantitative PCR (qPCR), with an area under the curve (AUC) of 1.000 (95% CI: 1.000–1.000), sensitivity, and specificity of 100%. The platform’s clinical utility is further highlighted by its rapid 55 min analysis time and low cost of $0.43 per test. In summary, the CMMEA-Chip offers a culture- and polymerase chain reaction (PCR)-free, highly sensitive, and cost-effective strategy for rapid ARG detection with strong potential for application in primary healthcare settings.