A Hydrogel-Based 3D Microfluidic Platform with Optimized Flow and Mass Transfer for Rapid Antimicrobial Susceptibility Testing

Antibiotic resistance (AMR) is a global health crisis responsible for over five million deaths annually. Rapid antimicrobial susceptibility testing (RAST) is critical for timely clinical decision-making. This study develops a hydrogel-based 3D culture microfluidic platform enclosed within a PMMA box, enabling safe and rapid testing of highly pathogenic bacteria. The microfluidic chip employs a Christmas tree concentration gradient generator, capable of simultaneously delivering four distinct drug concentrations. Theoretical, finite-element method, and experimental analyses demonstrated precise gradient control by tuning inlet flow-rate ratios (<i>Q</i>₁/<i>Q</i>₀), concentration ratios (<i>C</i>₀/<i>C</i>₁), and absolute concentrations (<i>C</i>₀). Optimizing hydrogel porosity (90%) and chamber height (200 μm) enhanced mass transfer, improving bacterial growth and drug delivery. Using <i>Escherichia coli</i> ATCC 25922 as a model, the system determined the minimum inhibitory concentration (MIC, 2 μg/mL) of gentamicin within 2 h─8 to 10-fold faster than standard methods, while matching conventional AST accuracy. From a fluid dynamics perspective, this work optimized the flow and mass transfer processes in AST, thereby enhancing the contact between nutrients, drugs, and bacteria. This hydrogel-based 3D microfluidic system provides a safe, efficient, and scalable RAST platform with strong potential for clinical applications against highly pathogenic and drug-resistant bacteria.