Enzymatic Charge Switching in Asymmetric Dual-Barrel Nanopipettes for Multiplex and Label-Free Bacterial Detection

Bacterial detection remains a global public health challenge, demanding rapid and precise detection strategies. Conventional nucleic acid or antigen-based assays face challenges of high costs and contamination risks. Conversely, bacterial enzymes represent attractive targets due to their strain-specific expression and inherent catalytic amplification capacity. Herein, we developed an enzyme-responsive dual-barrel nanopipette with heterogeneously modified nanointerfaces for sensitive, label-free, multiplex bacterial detection. Two independently functionalized channels integrate enzyme-triggered surface charge switching with nanofluidic ionic current rectification (ICR) characterization. Exposure to β-galactosidase (β-Gal) or β-glucuronidase (β-Glu) selectively alters channel surface charges, thereby modulating interchannel charge asymmetry. Leveraging bacterial enzymatic fingerprints, the nanopipette generates species-specific ICR signatures for label-free quantification within 30 min, achieving detection limits of 12 CFU/mL for E. coli and 126 CFU/mL for K. pneumoniae. The practical applicability was validated across diverse complex matrices: recovery rates of 97.49–106.94% in milk and drinking water, and 101.37–113.91% in serum (5000–50,000 CFU/mL), demonstrating its potential for diagnosing moderate bacterial infections. The innovation lies in the label-free operation, rapid response, and multiplexing capability of this sensor, which addresses critical limitations in current bacterial monitoring systems and offers a cost-effective on-site pathogen screening platform for applications spanning food safety and clinical diagnostics.