A label-free impedance cytometry platform for multi-functional and high-precision bacterial characterization

Rapid bacterial detection remains a critical need in clinical diagnostics, environmental monitoring, and food safety, yet conventional approaches are often limited by the small size and physiological variability of bacteria. To address this, we have developed a label-free impedance cytometry platform that combines a planar double-differential electrode configuration with an upper sheath fluid-assisted vertical compression. This design actively positions bacteria toward the bottom of the microchannel, where the electric field is strongest, thereby substantially improving signal stability and detection sensitivity. Through systematic optimization, a sheath-to-sample flow rate ratio of 2:1 was established as the optimal operating condition, providing an approximately 37% enhancement in impedance amplitude for bacteria while sustaining chip stability. The platform enables comprehensive evaluation of bacterial species, thermal viability, and antimicrobial susceptibility through real-time, dual-frequency (1.5 and 9 MHz) electrical profiling at the single-cell level. It effectively discriminates Escherichia coli (E. coli) from Bacillus subtilis (B. subtilis) based on their distinct amplitude and phase opacity profiles, assesses thermal viability in E. coli by detecting an approximately 11% increase in diameter after heat treatment, and performs rapid antimicrobial susceptibility testing-capturing an approximately 15% increase in average diameter (from 1.326 μm to 1.532 μm) within 20 min of polymyxin B (PMB) exposure. These results confirm the platform's ability to deliver multi-functional and high-precision bacterial characterization, offering a versatile tool for rapid microbiological analysis in clinical diagnostics and research.