Iron 2 sulphate heptahydrate

Melanin nanoparticles were prepared at stock solutions of 2 mM and 10 mM. Iron (II) sulphate heptahydrate (Sigma, Poole) stock solutions were prepared at double the final concentration required. Equal volumes of melanin nanoparticle and iron sulphate solution were mixed to give final concentrations of 1 mM and 5 mM melanin, in varying concentrations of iron. The solutions were incubated at 37 °C for either 24 or 48 h. After incubation the solutions were centrifuged at 15,000 rpm for 3 min and the supernatant containing unbound iron was collected.
To the collected supernatant (810 μl) add 1/10 volume of sodium acetate buffer (equal volumes of 6 M acetic acid and 5 M NaOH) and incubate at room temperature for 10 min. Next, add 10 μl of hydroxylamine solution (10 wt% in water), and incubate for a further 10 min. Phenathroline solution (prepared by adding 0.1 wt% phenathroline to heated water (60 °C) was then added, to give a final volume of 1 mL per sample. Sulphuric acid (8 μl) was added to each sample to ensure that the pH was in the range 4–7. The solutions were mixed and incubated for 20 min to allow the colour reaction to take place. The absorbance of the solutions was measured at 450 nm.
A calibration curve was obtained using a series of iron sulphate solutions (0, 0.32, 0.48, 0.8, 0.96, 1.62, 2.43, 3.25, 4.0, 6.8, 8.0 mM). A linear relationship was found (R² = 0.9934).

Analytical, HPLC and LCMS-grade chemicals were used in this study. Gallic acid, Folin–Ciocalteu reagent, vanillin, L-ascorbic acid, iron(III) chloride hexahydrate (Fe[III]Cl₃•6H₂O), sodium phosphate dibasic hepta-hydrate (Na₂HPO₄•7H₂O), hexahydrate aluminium chloride sodium phosphate, sodium phosphate monobasic monohydrate (Na₂HPO₄•H₂O), trichloroacetic acid (TCA), hydrochloric acid (HCl), ferrozine, hydrated sodium acetate, ethylene diamine tetra acetic acid (EDTA), potassium ferrocyanide (III), iron (II) and iron (III) chloride, 3-hydrobenzoic acid (C₇H₆O₃), ammonium molybdate, iron (II) sulphate heptahydrate, catechin, quercetin, DPPH, 2,4,6-tripyridyl-s-triazine (TPTZ) and ABTS were purchased from the Sigma Aldrich (Castle Hill, Australia) for the estimation of polyphenols and antioxidant potential. Hydrogen peroxide (30%) and Sodium carbonate anhydrous were purchased from Chem-Supply Pty Ltd. (Adelaide, Australia) and 98% sulfuric acid was purchased from RCI Labscan (Rongmuang, Thailand). HPLC and LC-MS grade reagents include ethanol, methanol, acetonitrile, formic acid, glacial acetic acid and iron (III) chloride anhydrous were purchased from Thermo Fisher Scientific Inc (Scoresby, Australia). HPLC standards were also purchased from Sigma Aldrich.

Carbon felt was obtained from Alfa Aesar (A Johnson Matthey Company, 67300 Schiltigheim, France). The pretreated carbon felt was denoted as raw CF. AO7 (Orange II sodium salt), sodium sulfate (anhydrous, ≥99%), concentrated nitric acid, terephthalic acid (98%), iron (II) sulphate hepta-hydrate (99%), and DMF (99.8%) were purchased from Sigma Aldrich (38070 Saint-Quentin-Fallavier, France) and used without any further purification.

Analytical grade iron (II) sulphate hepta-hydrate (FeSO₄·7H₂O) and Alconox were purchased from Sigma-Aldrich. The glassware used in all experiments was washed with Alconox and rinsed thrice with DI H₂O. All aqueous solutions were prepared with double distilled deionized (DI) H₂O.

Graphite Powder (≥99.99%, Daejung Korea), Sodium Nitrate (≥99.0%, Sigma-Aldrich), Concentrated Sulphuric acid (clear, assay: 99.99%, Sigma-Aldrich), Hydrogen peroxide (30 %, Sigma-Aldrich, Germany), Iron (III) Chloride hexahydrate (99.0%, Daejung Korea) Iron (II) Sulphate heptahydrate (≥98.0%, Sigma-Aldrich), Cobalt chloride hexahydrate (99.0%, Daejung Korea), and Liquid ammonia (Sigma-Aldrich) were used in this study. All chemicals were analytical grade reagents and were used as received without further purification. Distilled water was used throughout the study.

All the starting materials were analytical grade and used as received (i.e., without further purification). Rice starch (RS) of 99.9% purity (with ~30% of amylose and ~70% of amylopectin content) was purchased from Euramco (M) Ptd Ltd., Johor, Malaysia. Methylene blue (MB), ethanol, and hydrochloric acid (HCl) were purchased from MERCK (M) Sdn Bhd, Bandar Sunway, Selangor, Malaysia, and acetone was purchased from HmbG Chemical, Ludolfstr., Hamburg, Germany. Iron(II) sulphate heptahydrate (FeSO₄·7H₂O, 99.9%) was purchased from Sigma-Aldrich (M) Sdn Bhd, Subang Jaya, Selangor, Malaysia. In a typical RS-assisted synthesis, the HNPs were synthesized according to a calculated molar composition of H₂O/RS/FeSO₄·7H₂O at 1:1:4. Firstly, an appropriate amount of FeSO₄·7H₂O and RS were added to double distilled water (w/w) for 1 h at room temperature before the samples were finally dried and collected as a reddish-brown powder, which is referred to as Sp-HNP.