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. 2018 Jun;34(4):373-381.
doi: 10.1080/02656736.2017.1354403. Epub 2017 Jul 31.

An Optimised Spectrophotometric Assay for Convenient and Accurate Quantitation of Intracellular Iron From Iron Oxide Nanoparticles

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Free PMC article

An Optimised Spectrophotometric Assay for Convenient and Accurate Quantitation of Intracellular Iron From Iron Oxide Nanoparticles

Mohammad Hedayati et al. Int J Hyperthermia. .
Free PMC article

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).

Keywords: Iron oxide nanoparticles; UV/Vis spectrophotometry; intracellular iron; iron quantitation assay; mass spectrometry.

Conflict of interest statement

Disclosure statement

Dr. Cordula Gruettner is an employee of micromod Partikeltechnologie, GmbH, manufacturer of BNF and nanomag®-D-spio particles used in the studies. Dr. Robert Ivkov is an inventor on several issued and pending patents. All patents are assigned to Johns Hopkins University or Aduro Biotech, Inc. All other authors declare no competing interests.

Figures

Figure 1
Figure 1
Comparison of the spectra of the iron standards (0–2 μg Fe+3) in working solution. Ferene-s (A), ferrozine (B), and the corresponding standard curves (0.1–4 μg Fe+3) measured at the peak absorbance for each compound (560 nm for ferrozine and 595 nm for ferene-s), (C).
Figure 2
Figure 2
Time course of absorbance at 595 nm for ferene-s assay in working solution. Iron oxide nanoparticles with various formulations (A) and intracellular BNF-Starch iron oxide nanoparticles (B), DU145 cells with low, medium and high Fe content). Absorbance readings are adjusted to reflect equivalent number of cells.
Figure 3
Figure 3
Digestion with concentrated nitric acid. Intracellular BNF-Starch iron oxide nanoparticles digested at various times at 70 °C prior to addition to the working solution.
Figure 4
Figure 4
Comparison with Riemer’s method. Iron oxide nanoparticles with various formulations (A) and intracellular BNF-Starch iron oxide nanoparticles (B). Identical samples for each formulation of nanoparticles or DU145 cells loaded with BNF-Starch were prepared in duplicates.

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