Quantification of hydrogen peroxide (H2O2) in PBS(Ca2+/Mg2+) using sodium orthovanadate (Na3VO4) or titanium(IV) oxysulfate (TiOSO4)

The concentration of H₂O₂ in untreated and plasma-treated PBS(Ca²⁺/Mg²⁺) was determined using two methods. In the first method, H₂O₂ reacts with sodium orthovanadate to produce pervanadate, which is colourless³². In the second method, H₂O₂ reacts with titanium oxysulfate to produce pertitanic acid, which is yellow^33,34. The formation of each product is detected spectrophotometrically. For the establishment of H₂O₂ standard curves by Na₃VO₄-based assay (method 1), serial dilutions of H₂O₂ were prepared in 500 μl of PBS(Ca²⁺/Mg²⁺), and Na₃VO₄ was added to a final concentration of 1 mM. For the establishment of H₂O₂ standard curves by TiOSO₄-based assay (method 2), serial dilutions of H₂O₂ were prepared in 400 μl of PBS(Ca²⁺/Mg²⁺), 15 μl of 200 mM NaN₃ were added and then 200 μl of 2% TiOSO₄ diluted in 3 M H₂SO₄. NaN₃ is used to scavenge nitrites and other ROS that can interfere with TiOSO4. For the determination of H₂O₂ concentration in plasma-treated PBS(Ca²⁺/Mg²⁺) by the method 1, 500 μl of PBS(Ca²⁺/Mg²⁺) containing (direct treatment) or not (indirect treatment) 1 mM Na₃VO₄ were exposed to He plasma for various times. For indirect treatment, Na₃VO₄ was added post treatment to plasma-treated PBS(Ca²⁺/Mg²⁺) from a stock solution at 200 mM. For the determination of H₂O₂ concentration in plasma-treated PBS(Ca²⁺/Mg²⁺) by the method 2, 500 μl of PBS(Ca²⁺/Mg²⁺) were exposed to He plasma for various times. Thereafter, 400 μl of plasma-treated PBS were mixed to 15 μl of 200 mM NaN₃ and 200 μl of 2% TiOSO₄ diluted in 3 M H₂SO₄. The samples were incubated protected from light for 30 min at room temperature to allow the reaction to occur, and the absorbance was measured at 260 and 270 nm (method 1) or at 407 nm (method 2). All reagents (Na₃VO₄, TiOSO₄, H₂O₂ and NaN₃) were from Sigma-Aldrich.