Standalone self-compartmentalizing microfluidic chip for digital single-cell antimicrobial susceptibility testing

The growing threat of antibiotic-resistant bacteria calls for antimicrobial susceptibility testing (AST) methods that are both rapid and accessible. In this study, we demonstrate a novel approach to digital cell-based AST that leverages a passive microfluidic picochamber array to quantify bacterial growth and determine drug susceptibility at single-cell resolution. By combining stochastic confinement of individual E. coli cells with resazurin-based fluorescence detection, we establish a streamlined workflow for rapid phenotypic AST without the need for specialized equipment. The device uses capillary burst valves to autonomously guide fluid into 12,800 picoliter-sized chambers, enabling sample loading and partitioning using only a pipette and syringe. This method enables quantification of viable bacterial concentrations and determination of gentamicin minimum inhibitory concentration (MIC) breakpoints within 4 h. To evaluate its clinical potential, we tested ten clinical isolates and achieved 100 % categorical agreement with standard laboratory AST results. This work highlights a user-friendly and scalable strategy for digital AST, paving the way for broader applications in clinical diagnostics and resource-limited settings.