Quantifying the concentration and penetration depth of long-lived RONS in plasma-activated water by UV absorption spectroscopy

Reactive oxygen and reactive nitrogen species (RONS) are believed to play a key role in biomedical applications, which means that RONS must reach the target tissue to produce a therapeutic effect. Existing methods (electron spin spectrometry and microplate reading) to determine the RONS concentration are not suitable for experimental real-time measurements because they require adding an indicating reagent to the plasma-treated medium, which may alter the chemical composition of the medium. In this paper, we propose a method to measure the long-lived RONS concentration in plasma-activated water (PAW) by using UV absorption spectroscopy. Based on an analysis and fit of the absorption spectra of standard solutions (H2O2, NaNO2, and NaNO3), we propose a detailed fitting procedure that allows us to calculate the concentrations of simplex H2O2, NO2−, and NO3−. The results show that the pH and the cross reactivity between RONS in PAW correlate strongly with the absorption spectra. To confirm the accuracy of the calculations, we also use a microplate reader and add chemical reagents to measure the concentrations of H2O2, NO2−, and NO3−. The results show that the concentrations calculated by the proposed fitting method are relatively accurate and that the error range is acceptable. Additionally, the time-dependent diffusion of RONS in PAW is measured and analyzed at different depths in the PAW. This fitting approach constitutes a nonintrusive approach to measure RONS at different depths in PAW.