ROS study using 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFH-DA) probe

In order to further support the enhancement of the catalytic activity of Fer in the presence of SnF₂, we used photoluminescence (PL) method using DCFH-DA (Sigma-Aldrich, ≥97% purity) as a ROS probing agent³⁶. First, stock solutions of Fer (0.5 mg of Fe/ml) with or without SnF₂ (0.5 mg/ml) were incubated in 0.1 M sodium acetate buffer (pH 4.5) for 1 h at room temperature. Afterward, the working solution (final volume 2 mL) containing DCFH-DA (30 μM) and Fer (20 µg of Fe/ml) with or without SnF₂ (20 µg/ml) was prepared in 0.1 M sodium acetate buffer (pH 4.5). Subsequently, PL intensity was recorded at 520 nm with an excitation wavelength of 505 nm. H₂O₂ (1%, v/v) was then mixed to the reaction mixture to initiate the reaction, and the PL intensity was recorded at 520 nm at different incubation times with the excitation wavelength of 505 nm. For the control, vehicle was used.