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.