Electrospun polymeric patch integrated with an electrochemical biosensor for real-time superoxide monitoring in cell culture models of chronic wounds

Monitoring superoxide anion in chronic wounds is critical for understanding oxidative stress and guiding personalized treatment. We present a novel electrochemical biosensor enabling real-time superoxide detection in cell culture, integrating enzymatic selectivity with a biocompatible electrospun membrane for direct tissue contact. The biosensor employs screen-printed electrodes (SPE) modified with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT), which enhances enzyme immobilization and signal transduction, with superoxide dismutase (SOD) for enhanced selectivity. The analytical performance of SPE/PEDOT/SOD biosensor was evaluated by fixed potential amperometry at +0.3 V with a sensitivity of 130.3 ± 5.6 μA cm^-2·mM^-1, and a 1.5 μM detection limit. A cell-based wound model was developed to simulate chronic wound conditions, using an adhesive membrane modified with electrospun Nylon fibers that provided a biocompatible surface for cell adhesion and proliferation. Cells cultured on the membrane were irradiated with UV light to induce superoxide generation, mimicking oxidative stress in chronic wounds. Although the membrane introduced a diffusion barrier that reduced sensitivity by 40 %, it ensured close contact between cells and the electrode surface, enabling accurate in situ quantification of superoxide. Chronoamperometric detection at +0.3 V revealed sharp photocurrent peaks followed by decay, consistent with ROS generation and enzymatic clearance. The PEDOT matrix exhibited a photo-amplifying effect, nearly tripling the sensitivity under UV irradiation. The developed device is suitable for real-time oxidative stress profiling in wound models with potential for ex vivo and in vivo applications.