Ferrozine assay and ICP-OES based Fe quantification

The samples (particles and particle-labelled cells) were dissolved in 1.2 M HCl. Fe standard curves were made from the known concentrations of Fe and from these standard curves the unknown concentrations of Fe in the samples were determined.

For ferrozine assay, the Fe standard (50 μL, 4 μg in 1.2 M HCl) was serially diluted in HCl (1.2 M) followed by addition of 50 μL water so that final Fe concentration in standards was 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.031 μg in HCl (0.6 M; 100 μL). Blank was 100 μL HCl (0.6 M). In the samples with unknown Fe content, 50 μL HCl (1.2 M) was added and samples, Fe standards and blank were placed on a heat block at 65 °C for 2 h. After heat-assisted acid dissolution, the samples and standards were cooled down to room temperature and centrifuged to collect the condensation products along the walls of tubes. HCl concentration was adjusted to 0.6 M by adding water. 30 μL ferrozine reagent was added to 100 μL of each sample including standards and blank, mixed and left at room temperature for 30 min. Their absorption was measured at 590 and 780 nm using a plate reader. For background correction the absorbance at 780 nm was subtracted from the absorbance at 590 nm and blank was subtracted from all values. A standard curve for Fe content was plotted and was used to quantify Fe contents in the samples with unknown Fe concentrations.

(Note: the ferrozine reagent was prepared by dissolving 2.4 g ammonium acetate, 2.2 g ascorbic acid, 0.02 g ferrozine, and 0.02 g neocuproine (dissolved in small volume of ethanol) in 6.25 mL water, mixed, frozen in small aliquots and protected from light).

For ICP-OES Fe quantifications after dissolution with 1.2 M HCl, the samples were diluted in water so that final acid content will not exceed 5%. ICP-OES measurements were performed using 3 readings per wavelength and 11 wavelengths per sample. The concentration determination was performed using calibration curve for Fe consisting of 6 measurement points of freshly prepared Fe concentrations (0–10 μg mL⁻¹) derived from iron standard solutions from Inorganic Ventures (100 mg L⁻¹).