A ROS-based droplet microfluidic platform for rapid antimicrobial susceptibility testing and single-cell heterogeneity analysis

Rapid and accurate antimicrobial susceptibility testing (AST) is crucial for combating the global health threat of antimicrobial resistance (AMR), yet current clinical methods require multi-step incubations. Here, we present a reactive oxygen species (ROS)-based droplet microfluidic platform for single-cell AST. This microfluidic device enables the real-time detection of antibiotic-induced ROS generation, allowing for the determination of minimum inhibitory concentrations (MICs) within 90 min. In our microfluidic device, bacteria, ROS-sensitive dye, and antibiotics are co-encapsulated into droplets to create a controlled concentration gradient. By quantifying the fold change of normalized ROS, we determined MIC values that achieved high concordance (R² > 0.95) with the gold-standard broth microdilution (BMD) across eight antibiotics tested against Escherichia coli and Staphylococcus aureus . Application to 20 clinical isolates, including MRSA, multidrug-resistant S. aureus , colistin-resistant, and susceptible E. coli , also demonstrated accurate MIC calls within ±1 two-fold dilution of BMD results. This high concordance suggests the robustness of our platform and its potential for reliable use in real-world clinical settings. Furthermore, this approach revealed subpopulations with reduced ROS responses, uncovering phenotypic heterogeneity and potential persister-like cells that are often overlooked by bulk assays. Our approach not only reduces AST time but also provides a versatile platform for both high-throughput, clinically relevant framework for point-of-care diagnostics and advanced mechanistic insight of antibiotic action and resistance at the single-cell level. This capability promises to bridge the critical gap between the diagnosis and effective treatment, thereby paving the way for improved patient outcomes.