Fluorescence measurements of peroxynitrite/peroxynitrous acid in cold air plasma treated aqueous solutions

Abstract

Qualitative detection of peroxynitrite/peroxynitrous acid (ONOO⁻/ONOOH) as one of the key bactericidal agents produced in cold air plasma activated aqueous solutions is presented. We examined the use of the 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) fluorescent dye to detect ONOO⁻/ONOOH in plasma activated non-buffered water (PAW) or buffered solution (PAPB) generated by DC-driven self-pulsed transient spark discharge at atmospheric pressure in ambient air. The diagnostic selectivity of H₂DCFDA to reactive oxygen and nitrogen species (RONS) typical of plasma activated aqueous solutions was examined by using various scavengers of RONS. This cross-reactivity study showed the highest sensitivity of the H₂DCFDA dye to ONOO⁻/ONOOH. However, besides ONOO⁻/ONOOH, H₂DCFDA also exhibited sensitivity to hypochlorite anions/hypochlorous acid (OCl⁻/HOCl), showing that for a selective study it is important to have an idea about the possible constituents in the studied solutions. The sensitivity of H₂DCFDA to other RONS even in much higher concentrations was negligible. The presence of nitrites (NO₂⁻) and hydrogen peroxide (H₂O₂) in PAW led predominantly to the production of peroxynitrous acid with a strong fluorescence response of H₂DCFDA in PAW. Plasma treatment of buffered solutions led to the weak response of H₂DCFDA. The fluorescence induced in PAW decreased after scavenging individual reactants, namely NO₂⁻ and H₂O₂, as well as by scavenging the product of the peroxynitrite forming reaction, proving that the fluorescence response of H₂DCFDA is primarily due to the formation of ONOO⁻/ONOOH. A chemical kinetics analysis of post-discharge processes and the pseudo-second order reaction between H₂O₂ and NO₂⁻ confirms formation of peroxynitrous acid in PAW with a rate in the order of tens of nM per second. The post-discharge evolution of the ONOOH formation rate was clearly correlated with the parallel detection of ONOO⁻/ONOOH by fluorescence spectroscopy using the H₂DCFDA dye.