Microfluidic gradient generator based on serial injection mode and EIS sensing for rapid biofilm antibiotic resistance analysis

Biofilms are highly organized microbial communities formed under specific environmental conditions, and their strong antibiotic resistance poses major challenges in clinical treatment and environmental management. Traditional resistance testing methods suffer from cumbersome procedures, high sample consumption, and lack of real-time monitoring. Here, a microfluidic platform integrating a serial injection mode, a multi-stage concentration gradient generator, and electrochemical impedance sensing was developed for rapid, parallel analysis of biofilm resistance. The chip was fabricated by soft lithography and oxygen plasma bonding, integrating five interdigitated-electrode culture chambers; two syringe pumps delivered LB medium and gentamicin through the serial injection and gradient generator, and EIS signals were recorded using a five-channel multiplexer connected to an electrochemical workstation. Its novelty lies in three aspects: (i) a serial injection mode that forms a three-layer fluid structure, doubling the interfacial contact area and enhancing diffusive mixing without external energy input or complex chaotic structures; (ii) a connecting-channel width offset (Δw = 125 μm) that overcomes pressure-gradient dilution and enables a stable linear gradient of 24.3-91.8% (5-18 μg/L gentamicin); and (iii) an integrated interdigitated-electrode EIS system for real-time, label-free monitoring of biofilm formation and drug response across five concentrations. The mixing index increased from 46.6% at the initial section to 91.4% at the outlet. The normalized impedance of a 72-h mature biofilm was 0.311, decreasing to 0.007 after 24 h treatment with 18 μg/L gentamicin, indicating near-complete biofilm removal, whereas the 5 μg/L group remained at 0.174. Confocal fluorescence imaging agreed with the EIS results, validating the platform. This platform enables parallel resistance analysis of five drug concentrations with low sample consumption, offering a simple, real-time, and parallel-analysis solution for biofilm resistance assessment and antimicrobial screening.