NO sensors, nitric oxide biomarkes, inflammation, 2D culture monitoring, osteoarthrtis, poly-o-phenylenediamine

Nitric oxide (NO), a key metabolite released during inflammation, can be a reliable biomarker for conditions such as trauma, infection or during inflammation. Standard NO detection methods, including electron paramagnetic resonance (EPR) and griess assay, are limited in their ability to provide real-time monitoring, a critical feature for studying drug responses and enabling clinical applications. Electrochemical sensors can be used for continuous real-time NO measurement but require careful design to ensure selectivity in complex biological environments. In this study, platinum wire electrodes were used, either unmodified or coated with poly-o-phenylenediamine (PPD), to improve selectivity. Sensors were calibrated using S-nitroso-N acetylpenicillamine (SNAP),an NO-donor, and tested against common biological interferents, as ascorbic acid, fetal bovine serum, antibiotics, and several electrolytes. For real time NO detection, a tissue culture plate was customized to allow sensor connection guaranteeing a closed and sterile environment for cell culture. Amperometry was performed over 48 hours in 2D chondrocyte inflammation model cultures with and without IL-1β stimulation. Results demonstrated that the PPD coating was homogeneously distributed on the sensor surface and significantly reduced background noise from ascorbic acid. Furthermore, PPD-modified sensors showed enhanced selectivity and reproducibility during real-time NO monitoring, particularly in distinguishing NO levels in inflamed versus non-inflamed chondrocyte cultures. The findings highlight the importance of surface modifications to mitigate interference from non-relevant analytes in complex biological environments. These results suggest that real-time NO monitoring with electrochemical sensors could offer a valuable tool for assessing inflammation, with potential future applications in both in vitro and in vivo settings.