Amplification-Free Duplex Detection of Bacteria Using Solid-State Nanopores.

Rapid identification of bacterial pathogens remains challenging in clinical settings, particularly when timely decisions are required for acute or polymicrobial infections. Here, we report an amplification-free solid-state nanopore strategy for bacterial DNA detection using 12 nm silicon nitride nanopores and a minimalist Ω-loop probe design. Short DNA-probes hybridize with target sequences to generate defined structural features that encode target identity into single-molecule translocation waveforms. This waveform-level encoding enables bacterial discrimination within a single measurement without enzymatic amplification, long carrier constructs, or multistep chemical signal processing. Using Staphylococcus aureus and Escherichia coli as representative Gram-positive and Gram-negative pathogens, respectively, the platform achieved duplex detection based on distinct translocation substructures. Under optimized conditions, the assay supported reliable detection at low digital PCR-defined input levels and maintained classification performance in heterogeneous clinical matrices. Duplex detection was further validated directly in clinical specimens, showing complete concordance with culture results in the samples examined. These results establish a multiplexable solid-state nanopore framework that combines assay simplicity, waveform-based specificity, and compatibility with clinical samples, advancing nanopore-based bacterial detection toward practical diagnostic applications.