Abstract
Cold atmospheric plasmas (CAP) are a versatile tool in medical applications like wound healing. Its therapeutic benefits are partially attributed to the generation of biologically active reactive oxygen and nitrogen species (RONS). Characterization of RONS, however, typically only occurs after treatment. Here we report the first real-time in situ detection of CAP-generated nitric oxide (NO), and the simultaneous detection of cellular calcium ions (Ca²⁺) release using electrochemical sensors during CAP treatment of murine wounds. In vivo, NO rose rapidly within the first minute of CAP treatment but accumulated less overall than in PBS, reflecting reactions with wound-bed targets. In situ measurements revealed nearly double the concentrations of static endpoint assays, underscoring the importance of real-time detection. Ca²⁺ signals displayed transient, burst-like increases, likely due to CAP-induced membrane permeability and as response to oxidative stress. We also investigated the sensitivity, selectivity, and stability of the graphene oxide coated NO sensors and ion-selective Ca²⁺ sensors. Interference studies showed that the NO sensor also responds to H(2)O(2) and NO(2) (-) yet remains most sensitive to NO. Raman microscopy revealed progressive degradation of the graphene oxide layer after only one hour of CAP exposure, drastically reducing sensor currents. Improvements in NO sensor design will enable more accurate measurements for feedback control for plasma-based wound therapies. Ca²⁺ sensors are more robust and retained full functionality after three hours and repeated use providing a reliable diagnostic for immediate biological response. The results establish real-time electrochemical sensing as a powerful approach to monitor CAP-tissue interactions.