Quantification of Plasma-Generated Hydroxyl Radicals and Amino Acid Oxidation in Aqueous Environments Using Helium Gas in Inert Atmospheric conditions

Cold atmospheric pressure plasma (CAP) is being increasingly explored for biomedical applications, yet fundamental aqueous-phase chemistry underlying plasma–biomolecule interactions remains poorly understood. We present a combined method of chemical dosimetry and mass spectrometric investigation into hydroxyl radical (•OH) generation and free amino acid oxidation during helium CAP treatment of aqueous solutions under inert atmospheric conditions. Terephthalic acid (TA) is used as a selective chemical probe to quantify •OH production by monitoring the fluorescent formation of 2-hydroxyterephthalic acid (HTA) across varying treatment times in phosphate buffer at neutral pH. HTA fluorescence enabled calculation of •OH generation rate to be in the order of 10^-7 M/s. In tandem, liquid chromatography–mass spectrometry (LC-MS) was employed to characterize chemical modifications of amino acids treated under identical plasma conditions with and without TA present. Among nine amino acids treated, tryptophan, methionine, and cysteine exhibited significant transformations, including hydroxylation, and proposed disulfide or thioether-linked multimer formation. These observations provide insight into early oxidative pathways of protein degradation and peptide linkage. This study presents progress toward developeing a mechanistic framework for understanding plasma–biomolecule interactions in aqueous systems and contributes to ongoing research in small biomolecules plasma effects for applications in plasma medicine, protein modification, and radical chemistry