An optimised spectrophotometric assay for convenient and accurate quantitation of intracellular iron from iron oxide nanoparticles

Abstract

We report the development and optimisation of an assay for quantitating iron from iron oxide nanoparticles in biological matrices by using ferene-s, a chromogenic compound. The method is accurate, reliable and can be performed with basic equipment common to many laboratories making it convenient and inexpensive. The assay we have developed is suited for quantitation of iron in cell culture studies with iron oxide nanoparticles, which tend to manifest low levels of iron. The assay was validated with standard reference materials and with inductively coupled plasma-mass spectrometry (ICP-MS) to accurately measure iron concentrations ~1 × 10−6 g in about 1 × 106 cells (~1 × 10−12 g Fe per cell). The assay requires preparation and use of a working solution to which samples can be directly added without further processing. After overnight incubation, the absorbance can be measured with a standard UV/Vis spectrophotometer to provide iron concentration. Alternatively, for expedited processing, samples can be digested with concentrated nitric acid before addition to the working solution. Optimization studies demonstrated significant deviations accompany variable digestion times, highlighting the importance to ensure complete iron ion liberation from the nanoparticle or sample matrix to avoid underestimating iron concentration. When performed correctly, this method yields reliable iron ion concentration measurements to ~2 × 10−6 M (1 × 10−7 g/ml sample).

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“…The detailed protocol for conducting the modified ferene-s measurement of iron associated with cells after exposure to BNF nanoparticles has been previously described (24). Briefly, cells were trypsinized and washed with PBS thoroughly and were incubated at 37°C with BP (0.5 mg/ml), BH, or trastuzumab (Her 2 g/ml) alone for 3 hours in growth media (DMEM + 10% FBS) with occasional shaking/tapping of tubes to maximize distribution and prevent settling of cells.…”

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“…S6, and evidencing that no iron leakage occurs. Other analytical chemical methods such as spectrophotometric assays (45) would also lead to the measurement of total iron content in the cellular sample, but not iron within a nanoparticle superparamagnetic structure, which can only be assessed by magnetic measures. Finally, we performed additional magnetic measurements using a third method that allows quantification at shows the distribution of the magnetic moments over the cell population.…”

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“…Samples were loaded into Kel-F liquid capsules (LakeShore Cryogenics), and sealed with epoxy to preserve water during measurement under vacuum. Data are normalized to either total iron content, determined by ferene-s method previously described 60 , or by total solid content.…”