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Int J Hyperthermia. 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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a Department of Radiation Oncology and Molecular Radiation Sciences , Johns Hopkins University School of Medicine , Baltimore , MD , USA.
b Department of Environmental Health Sciences , Johns Hopkins Bloomberg School of Public Health , Baltimore , MD , USA.
c Micromod Partikeltechnologie GmbH , Rostock , Germany.
d Department of Oncology, Sidney Kimmel Comprehensive Cancer Center , Johns Hopkins University School of Medicine , Baltimore , MD , USA.
e Institute for NanoBioTechnology , Johns Hopkins University , Baltimore , MD , USA.
f Department of Materials Science and Engineering , Whiting School of Engineering, Johns Hopkins University , Baltimore , MD , USA.
g Department of Mechanical Engineering , Whiting School of Engineering, Johns Hopkins University , Baltimore , MD , USA.


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).


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

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