Total reflection x-ray fluorescence spectroscopy as a tool for evaluation of iron concentration in ferrofluids and yeast samples

Author’s Accepted Manuscript Total reflection x-ray fluorescence spectroscopy as a tool for evaluation of iron concentration in ferrofluids and yeast samples N.A. Kulesh, I.P. Novoselova, A.P. Safronov, I.V. Beketov, O.M. Samatov, G.V. Kurlyandskaya, M. Morozova, T.P. Denisova www.elsevier.com/locate/jmmm

PII: DOI: Reference:

S0304-8853(16)30095-6 http://dx.doi.org/10.1016/j.jmmm.2016.01.095 MAGMA61119

To appear in: Journal of Magnetism and Magnetic Materials Received date: 14 August 2015 Revised date: 30 November 2015 Accepted date: 28 January 2016 Cite this article as: N.A. Kulesh, I.P. Novoselova, A.P. Safronov, I.V. Beketov, O.M. Samatov, G.V. Kurlyandskaya, M. Morozova and T.P. Denisova, Total reflection x-ray fluorescence spectroscopy as a tool for evaluation of iron concentration in ferrofluids and yeast samples, Journal of Magnetism and Magnetic Materials, http://dx.doi.org/10.1016/j.jmmm.2016.01.095 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Total reflection x-ray fluorescence spectroscopy as a tool for evaluation of iron concentration in ferrofluids and yeast samples N.A. Kulesha,*, I.P. Novoselovaa,b, A.P. Safronova,c, I.V. Beketovc, O.M. Samatovc, G.V. Kurlyandskayaa,d, M. Morozovaa, T.P. Denisovae a

Ural Federal University, Mira 19, 620002, Ekaterinburg, Russia Immanuel Kant Baltic Federal University, 236041, Kaliningrad, Russia c Institute of Electrophysics UD RAS, Amundsen 106, 620016, Ekaterinburg, Russia d University of the Basque Country UPV-EHU, 48940, Leioa, Spain e Irkutsk State University, 664003, Karl Marks 1, Irkutsk, Russia b

*[email protected]

Abstract In this study, total reflection x-ray fluorescent (TXRF) spectrometry was applied for the evaluation of iron concentration in ferrofluids and biological samples containing iron oxide magnetic nanoparticles obtained by the laser target evaporation technique. Suspensions of maghemite nanoparticles of different concentrations were used to estimate the limitation of the method for the evaluation of nanoparticle concentration in the range of 1 to 5 000 ppm in absence of organic matrix. Samples of single-cell yeasts grown in the nutrient media containing maghemite nanoparticles were used to study the nanoparticle absorption mechanism. The obtained results were analyzed in terms of applicability of TXRF for quantitative analysis in a wide range of iron oxide nanoparticle concentrations for biological samples and ferrofluids with a simple established protocol of specimen preparation. Keywords Total reflection x-ray fluorescence spectroscopy; Laser target evaporation; Magnetic nanoparticles; Magnetic ferrofluids; Yeast samples * Corresponding author. Tel.: +79089203513; fax: +73432616823. E-mail address: [email protected]

1. Introduction In the last decades, the growing interest in biomedical research has led to the creation of multidisciplinary groups that include not only chemists, physicists and engineers, but also biologists and biochemists. This trend is caused by the high involvement of novel materials and techniques coming from independent branches of science and consolidating into the emerging fields of biophysics and nanotechnology [1,2]. One of the addressed subjects in both basic and applied biomedical research is magnetic nanoparticles (MNPs). Their applications vary from magnetic biosensing, magnetic separation, magnetic resonance imaging to selective cell destruction using 1

hyperthermia, drug delivery and many others [3-6]. The problems related to toxicity and accumulation of MNPs in specific parts of the body become very important. The last trends have led to special requests: big size of the batch for thorough characterization of MNPs, due to the fact that their properties can vary from batch to batch even with well controlled fabrication techniques [7] and a precise control of the MNPs concentration in tissues and biofluids. Magnetite (Fe3O4) is one of the most versatile biocompatible magnetic materials with a high saturation magnetization and relatively weak magneto-crystalline anisotropy [8]. On the other hand, the long-term instability of the properties of magnetite MNPs is encouraging researchers to study maghemite (Fe2O3) for particular applications. One of the most productive techniques for obtaining big batches of spherical maghemite MNPs is laser target evaporation (LTE) technique which doesn’t suffer from the well known disadvantages of chemical synthesis, such as low production rates, limited purity, deviations from the sphericity, wide size distributions and a high environmental cost of the final product [9]. It is important to characterize each batch by different techniques, i.e. to measure the same quantity with multiple methods caused a new tendency to develop protocols for characterization of the properties of biological samples by techniques initially developed for different kind of the samples. One of such techniques is the total reflection x-ray fluorescent spectrometry (TXRF) [10]. TXRF is a relatively new method of elemental analysis that can be applied to samples in the form of thin near-surface layer including dried drops of homogenized solutions, suspensions of fine particles, or even a thin layer of whole cells. High sensitivity and reliability of the analysis results, in case of proper sample preparation, combined with the availability of highly effective desktop spectrometers lead to wider environmental and biomedical research involving TXRF spectrometry [10]. Although the TXRF method without a pressure controlled chamber proved to be limited for low Z (such as carbon, nitrogen, and oxygen) elements’ quantification [11], it can be successfully applied for the determination of elements with higher-energy characteristic lines. The study of biological samples (cells or tissues) has certain testing limitations when physical methods are used. These limitations are caused by the wide variety of complex processes that exist in the living matter and the wide variety of morphologies of each particular sample. To make biophysical research more effective, one can select model systems, which reproduce the main features and properties of more complex living system. Single-cell yeasts are good model subjects [4] that can be used not only for the study of the MNPs’ accumulation mechanism and analysis of MNPs’ toxicity, but also as a system potentially suitable for graduating a magnetic biosensor [12]. We applied TXRF spectrometry to evaluate the concentration of iron oxide MNPs in simple biological samples having a light matrix as well as ferrofluids of different concentrations. The aim of the present work is to estimate the applicability and limitations of TXRF spectrometry for iron quantification in ferrofluids and simple single-cell organisms elaborating a simple established protocol of specimen preparation in a wide range of MNP concentrations. 2. Samples and techniques Iron oxide MNPs were synthesized by LTE method based on the evaporation of the solid pellet by the laser beam followed by condensation of vapors in the gas phase [4, 9]. These conditions provided the formation of spherical particles with controlled dispersion parameters. For synthesis of individual MNPs with similar size the evaporation was performed in a constant gas flow, which prevented particles from agglomerating. The LTE laboratory setup provided continuous synthesis of iron oxide MNPs with a production rate of 50 g/h. Evaporation was performed in pulsed regime with a 5 kHz frequency and 60 μs pulse duration favoring the formation of uniform fine MNPs and narrow particle size distribution.

2

For MNPs structural characterization, X-ray diffraction (XRD) studies were performed by DISCOVER D8 (Bruker) diffractometer using Cu-Kα radiation (λ = 1.5418 Å). Bruker software TOPAS-3 with Rietveld full-profile refinement was employed for a quantitative analysis. The average size of coherent diffraction domains was estimated using the Scherrer approach [13]. Transmission electron microscopy (TEM) was performed by JEOL JEM2100 microscope operating at 200 kV. The specific surface area (Ssp) of MNPs was measured by the low-temperature sorption of nitrogen Brunauer-Emmett-Teller physical adsorption technique (BET) [14] using the Micromeritics TriStar3000 analyzer. Suspensions of iron oxide MNPs were prepared in distilled water with the addition of sodium citrate (electrostatic stabilizer) in 5 mM concentration. Coarse suspensions in an initial concentration of 5 g/L were treated by a Cole-Parmer CPX-750 ultrasound processor at 300 W average power output level. De-aggregation of the suspension was performed in the same way as described in our previous reports [9]. During the ultrasound treatment the diminishing of an average hydrodynamic diameter of aggregates in suspension was monitored by dynamic light scattering. When a constant value of the hydrodynamic diameter was achieved, the suspensions were centrifuged using a Hermle Z383 centrifuge. The supernatant was separated with a syringe and used for further studies. The aggregation state of MNPs in water suspension (both with and without living cells) was evaluated by dynamic light scattering using the Brookhaven Zeta Plus analyzer. The electrokinetic zeta potential of the suspensions was measured by electrophoretic light scattering (ELS) using the same analyzer. For the study of maghemite MNP’s accumulation process two nonpathogenic strains of yeasts were used: Exophiala nigrum (black yeasts) and a mutant strain of red yeasts which were isolated from Lake Baikal and the Schumak River in Russia respectively. A liquid and gelatinous agar nutrient medium was used for cultivation. Maghemite MNPs in form of a stabilized aqueous suspension were introduced in a liquid and gelatinous nutrient medium directly with a final MNPs concentration of 0.5 g/l and 0.3 g/l consequently. The samples for TXRF measurements were prepared by diluting the yeasts colony of known mass (2 to 4 mg for yeasts grown on gel) in 0.5 ml of deionized water. The volume of the specimen placed onto the polyimide-coated substrate was 40 μl, including 20 μl of the yeasts suspension and 20 μl of the vanadium internal standard (200 ppm). The presence of a small quantity of iron in the nutrient media and, consequently, in the yeasts samples was taken into account by subtracting the iron concentration obtained on the reference yeast colony grown in similar condition but without adding MNPs to the nutrients. TEM images of cell cultures were obtained with a Philips EM208S electron microscope at an accelerating voltage of 70 kV. Cell morphology was also studied by optical and scanning (Hitachi S-4800 electron microscope, SEM) microscopy. The study of the magnetic properties of yeasts and ferrofluidic samples was carried out by vibrating sample magnetometer (VSM). All TXRF measurements were carried out by a Nanohunter spectrometer. For every experiment the same parameters were used: exposure time of 500 s, angle of 0.05°, x-ray tube with Cu anode as a primary beam source. Samples were dried at 50 °C in a Rigaku Ultradry chamber at normal pressure. For all calculations of iron concentration, we neglected the matrix effects assuming thin film sample geometry. 3. Results and discussion Spherically shaped nanoparticles were obtained by LTE using a target made of pressed coarse maghemite powder [9]. (Fig.1a). The graphical analysis of the TEM images gives a lognormal  particle size distribution (PSD) function: Pn (d )  A exp  1  ln d  ln(d 0 )  d     2

2

  , where A, σ, d (nm) 0   

parameters of lognormal PSD for as-prepared MNPs. Number averaged (dn) and weight averaged 3

(dw) particle diameters can be calculated on PSD data basis [9]. The specific surface area Ssp determined by the low-temperature adsorption of nitrogen was used for the calculation of surface 6 average diameter (ds) of spherical MNPs according to the equation [16]: d s  , where  is the S sp density of iron oxide MNPs (4.6 g/cm3). The calculated value of ds was in good agreement with TEM results dw ≈ ds ≈ 14 ± 2 nm. Fig. 1 shows XRD plots of the as-prepared LTE MNPs. Although the experimental points are fitted well by the magnetite XRD database, it is well known that the Fe3O4 structure cannot be distinguished from maghemite (γ-Fe2O3) based only on XRD data evaluation. Therefore, the chemical composition of MNPs was determined by the comparative analysis of Red-Ox titration data and XRD analysis of the spinel lattice period. The chemical composition was Fe2.68O4, i.e. very close to the stoichiometric γ-Fe2O3 (Fe2.67O4) composition, but contained slightly fewer cation vacancies per cell (inverse spinel structure with Fd-3 m space group). In order to determine the MNPs concentration range in which TXRF can be used for direct quantification, 13 aqueous ferrofluidic samples with MNPs concentration varying from 10 to 0.001 g/l were prepared. Two series of five specimens for each concentration were prepared. In the first series, suspensions were homogenized by MNPs dissolution in 5% hydrochloric acid, while the second one was not treated except for the addition of the vanadium internal standard. For all samples, a mixture of 10 μl of the ferrofluid and 10 μl of the internal standard was taken, placed onto the polyimide-coated substrate and dried at 50 ˚C. When selecting the substrate for sample preparation we considered 25 mm × 75 mm reflectors made of different materials: quartz, soda-lime glass, polyimide-coated soda-lime glass, vinyl polychloride, acrylic, and polystyrene slides. The main criteria for an optimal reflector were low iron concentration, smooth surface (which means less intense scattering of the primary x-ray beam and consequently lower background on the spectrum) and optimal surface tension. In the initial experiment, angle dependencies of the background in the iron Kα line region were obtained and compared in order to estimate potential income from the substrate due to fluorescent yield or primary x-ray beam’s scattering on the sample. The lowest Fe contamination was observed for vinyl polychloride, quartz, polyimide, and acrylic substrates (from lower to higher Fe concentration). The estimation of spectrum background in the region of 4 to 7 keV performed on the control bacteria specimen prepared for each reflector, revealed similar values except for vinyl polychloride substrates, which had two times higher background on average. Taking into account the easiness of polyimide-coated glass substrate preparation

Intensity (arb. un.)

(311)

(400) (200)

(222) (400)

(111)

15

(511)

(422) (311)

25

35

45

55

(511) (533) (620)(622) (444)

65

75

2θ (o)

4

Fig. 1. TEM image of iron oxide MNPs. Inset: particle size distribution (2040 particles) (a). XRD plots of asprepared MNPs with bright peaks Miller indexes. Points – experimental diffraction values, line – fitting by the database magnetite structure (b).

(disposable adhesive polyimide tape attached to the surface of ordinary lime-soda glass slide), good dried drop shape, single use and low cost, we chose this type of reflector for the TXRF measurements. The measurements of iron concentration in dissolved ferrofluids demonstrated a rather good linearity of the dependency of nominal vs calculated concentrations for the entire range of MNP concentrations (Fig. 2a). The total mass of iron oxide deposited onto the carrier was estimated to be from 12 to 5 ×105 ng, giving an average surface density between 0.16 and 640 ng/µm2. The results obtained on the series of untreated ferrofluids also showed linear behavior, but suffered from relatively large standard deviation (SD) (Fig. 2b). Possible reasons are nonuniformity of the dried drop, which can cause partial shading of the specimen, and nonsymmetrical residue distribution on the reflector. Another important factor is the nonlinear dependency between x-ray fluorescence intensity and mass of iron oxide deposited onto the carrier, which can be observed starting from the critical value [17]. Thus, for highly concentrated samples, the uniformity of the internal standard distribution in the residue becomes critical and a slight inhomogeneity can lead to a high deviation of the obtained iron concentration from the nominal one and consequently to increased SD. Auxiliary investigation of the dried drops’ profile made by contact profilometer confirmed a nonuniform distribution of the residue with strong peaks on the boundaries and in the central part (Fig.2c-e). The effect is especially strong for MNPs concentrations exceeding 1000 ppm. The nonuniformity of particle distribution in the highly concentrated suspension leads to inadequately calculated concentrations for suspensions diluted with deionized water. The experiment on maghemite MNPs suspensions demonstrated potential applicability of TXRF spectrometry for MNPs quantification in the entire concentration range considered. However, the nonhomogeneous residue distribution and large mass of the sample to be analyzed can lead to low TXRF measurement reproducibility, especially in case of suspensions without electrostatic stabilizer.

5

(a)

1000

100

10

Without MNPs dissolution Dissolved MNPs o 45 line

1

Standard deviation (%)

Measured MNPs concentration (ppm)

10000

30

10

100

1000

20 15 10 5

10000

Nominal MNPs concentration (ppm)

1 ppm

(c)

1

10

1000 ppm

1 2 Lengh (mm)

3

(d)

10000

10 000 ppm

(e)

20

2 1 0 0

1000

Heigth (m)

Height (m)

Height (m) 0.0 0

100

Measured MNPs concentration (ppm)

3

0.2

Without MNPs dissolution Dissolved MNPs

25

0 1

(b)

1

2 3 4 Lengh (mm)

5

0 0

1

2 3 4 Lengh (m)

5

Fig. 2. Dependencies of MNP’s concentrations obtained by TXRF on nominal concentrations for untreated ferrofluids and for ferrofluids homogenized by addition of 5% HCl (a). (b) Dependencies of standard deviation on MNPs concentration obtained by TXRF for corresponding ferrofluid samples. Microphotographs (c), (d), (e) taken with optical microscope show overall view of the residue of dried drops of different concentrations on the reflector. Corresponding profile obtained with mechanical profilometer is shown under each photograph.

Fig. 3 shows morphological features of yeast samples which are very similar to morphologies of control groups, i.e. groups grown in the same conditions but without MNPs (data for them are not shown here). The average size of the unicellular yeasts was approximately 5 μm × 2 μm for black and 3.5 μm × 3.5 μm for red yeasts. In both cases, rather uniform individual morphologies were obtained. TXRF measurements performed on yeast samples revealed much more effective MNP absorption from the liquid nutrient medium than from the gelatinous one. According to TXRF data, for gelatinous medium Fe concentrations were in the 100 to 300 ppm range, whereas for liquid medium from 500 to 1500 ppm depending on the yeast type and stabilizer used for suspensions (Fig.4). One can clearly see significantly enhanced iron concentration for the MNPs grown in the presence of LTE MNPs in nutrient (Fig.4a) compared to yeast grown without MNPs in the nutrient (Fig.4b). 6

Fig. 3. Exophiala nigrum (black yeast): optical microscopy (a); SEM (b). Mutant strain (red yeasts) optical microscopy (c); SEM (d). Both colonies were grown on gelatinous agar nutrient with of LTE MNPs in Petri dish.

For several reference samples, results obtained by TXRF spectrometry were verified by magnetic measurements of dried ferrofluids and dried yeast samples. As the MNPs concentration was calculated from saturation magnetization (Ms) of the corresponding dried yeast sample, the final values can be related to iron contribution corresponding to iron oxide in a magnetic state (Table 1). Iron concentrations obtained by TXRF and magnetic measurements showed usually the following tendency: TXRF values for the concentration were higher. This fact is not surprising because the mechanisms of iron adsorption can be different including particle dissolution and change of the iron oxidation state. One can understand the difference in the concentrations obtained by TXRF and magnetic measurements better if we take into account that for γ-Fe2O3 MNPs of 14 nm the surface/volume contribution is approximately 70%. Recalculation of the real amount of iron oxide gives much higher values for iron concentrations comparing with data for direct magnetic measurements.

7

(b)

(a)

(c) Black yeast/MNPs -Fe2O3 MNPs

0 0.01

M (emu/g)

M (emu/g)

20

0.00

-0.01

-20

-6

-60

-30

0

30

-3

0

3H (kOe) 6

60 H (kOe)

Fig. 4. TXRF spectra obtained for Exophiala nigrum (black yeast) sample grown without LTE MNPs in nutrient (a) and with LTE MNPs in nutrient (b). Green lines indicate parts of spectra related to iron. Hysteresis loops of LTE MNPs and Exophiala nigrum dried sample for yeast grown in the presence of LTE MNPs in nutrient, measurements at room temperature (c). TEM image for red yeast sample for study of cell morphology and determination of MNPs localization/ iron accumulation (d).

The hysteresis loops of ferrofluids and yeast samples (see Fig.4c as an example) show that, despite the low total magnetic moment for biocomposites, the shape of the M(H) loops in all the cases was similar to the shape of the MNPs loop, confirming not simply the existence of the iron accumulation process, but also the fact that it is probably accumulated in the shape of iron oxide MNPs with Ms value typical for iron oxide superparamagnetic MNPs of about 14 nm in diameter. Taking into account the higher iron concentration observed for red yeasts, which have a round shape, smaller cell size and, therefore, larger surface area, membrane absorption can be proposed as a possible mechanism. At the same time, at present this question is still open as we were not able to localize confidently iron residues inside the cell of neither black nor red yeasts (Fig. 4 d). Zeta potential is the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle. Its evaluation for suspensions (both with and without living cells) was studied here. The examples of the values of zeta potential of suspensions are given in Table 1. The threshold for the colloidal stability of an aqueous suspension is usually achieved for the absolute value of zeta potential above 20 mV. Therefore, the observed values of -50.6 mV for black yeasts zeta potential were substantially higher (by 20 mV), providing the effective 8

electrostatic stabilization (good stability). The observed values of -20.7 mV were very close to the threshold for red yeasts (incipient instability). Table 1. Selected examples of physical properties for ferrofluid and Red yeast composites and C(Fe)m – iron concentration from magnetic measurements; C(Fe) – iron concentration from NanoHunter TXRF measurements; Red yeast - data for samples grown in liquid. Red yeast* - data for samples grown on gelatinous medium.

Sample Ferrofluid MNPs Red yeast Red yeast*

Zeta-potential (mV) 20.7 20.7

Ms (emu/g) 21.3 0.008 0.002

C(Fe)m (ppm)

C(Fe) (ppm)

C(Fe)m/ C(Fe)

260 65

338 74

0.77 0.88

4. Conclusion MNPs of γ-Fe2O3 were fabricated by LTE technique and water based ferrofluids were prepared on their basis. The TXRF spectrometry was applied for compositional characterization of ferrofluids and Exophiala nigrum (black yeast) and mutant strain of red yeast samples. It was shown for ferrofluids of different concentrations that iron content values obtained with internal standard method are in good agreement with the nominal ones obtained by the standard dissolution method. The enhancement of standard deviation and evolution of dried drop profile for high MNPs concentration demonstrate an increasing influence of shading and thick layer effects on the concentrations measured on non-dissolved samples. According to the dependence of SD on MNPs concentration measured for suspension with dissolved MNPs (with more homogeneous residue distribution) critical concentration for the significant SD increase is about 1000 ppm. Data obtained on yeast samples by TXRF spectrometry and magnetic measurements confirmed an effective iron uptake from nutrient medium, especially for the yeasts grown in liquid medium. A comparison between concentrations measured by these techniques revealed that most part of the iron is absorbed in form of MNPs rather than iron in nonmagnetic complexes. Additional studies are required to clarify mechanisms of MNPs accumulation. 5. Acknowledgments Part of this work was performed with the financial support of the Ministry of Education and Science of the Russian Federation, project № 1362, within the state job No. 0389-2014-0002, RFBR mol-nr 15-32-50369 grant, and Grant MAT2014-55049-C2-1-R, Ministerio de Economia y Competitividad of Spain. Selected measurements were performed at SGIker service of UPV-EHU. We thank A.M. Murzakayev, A.I. Medvedev, R. Andrade, G.S. Kuprianova, for special support. References [1] J.L. West, N.J. Halas, Engineered nanomaterials for biophotonics applications: Improving sensing, imaging, and therapeutics, Annual Review of Biomedical Engineering. 5 (2003) 285-292. [2] C.R. Martin, P. Kohli, The emerging field of nanotube biotechnology, Nature Reviews Drug Discovery. 2(1) (2003) 29-37. [3] X. Chen, H.J. Schluesener, Nanosilver: A nanoproduct in medical application, Toxicology Letters. 176(1) (2008) 1-12. [4] J. P. Novoselova, A.P. Safronov, O. M. Samatov, I. V. Beketov, H. Khurshid, Z. Nemati, H. Srikanth, T. P. Denisova, R. Andrade, G. V. Kurlyandskaya, Laser target evaporation Fe2O3 9

nanoparticles for water-based ferrofluids for biomedical applications, IEEE Trans. Magn. 50(11) (2014) 4600504. [5] P. Tartaj, M. Del Puerto Morales, S. Veintemillas-Verdaguer, T. González-Carreño, C.J. Serna, The preparation of magnetic nanoparticles for applications in biomedicine, Journal of Physics D: Applied Physics. 36(13) (2003) R182-R197. [6] M. Luisa Fdez-Gubieda, Alicia Muela, Javier Alonso, Ana Garcıa-Prieto, Luca Olivi, Rodrigo Fernandez-Pacheco, and Jose Manuel Barandiaran, Magnetite Biomineralization in Magnetospirillum gryphiswaldense: Time-Resolved Magnetic and Structural Studies, ACS Nano. 7(4) (2013) 3297–3305. [7] J. H. Grossman and S. E. McNeil, Nanotechnology in Cancer Medicine, Physics Today, 38 (August, 2012). [8] R. C. O´Handley, Modern Magnetic Materials (John Wiley & Sons, New York, 1972) p. 740. [9] A.P. Safronov, I.V. Beketov, S.V. Komogortsev, G.V. Kurlyandskaya, A. I. Medvedev, D. V. Leiman, A. Larranaga, S. M. Bhagat, Spherical magnetic nanoparticles fabricated by laser target evaporation, AIP Advances. 3 (2013) 052135. [10] N. Szoboszlai, Z. Polgári, V.G. Mihucz, G. Záray, Recent trends in total reflection X-ray fluorescence spectrometry for biological applications, Analytica Chimica Acta. 633 (2009) 1–18. [11] Z. Polgári, N. Szoboszlai, , V.G. Mihucz, G. Záray, Possibilities and limitations of the total reflection X-rayfluorescence spectrometry for the determination of low Z elements in biological samples, Microchemical Journal. 99 (2011) 339–343. [12] G.V. Kurlyandskaya, Giant magnetoimpedance for biosensing: Advantages and shortcomings, J. Magn. Magn. Mater. 321 (2009) 659–662. [13] Rietveld H M, Appl. Crystallogr. 2 (1969) 65-71. [14] S.J. Gregg, K.S.W. Sing Adsorption, Surface Area and Porosity. Academic Press. London. 1982. [15] I.P. Babieva, I.Yu. Chernov, Biology of Yests, Moscow State University (2004) 1-239. [16] P.C. Hiemenz, R. Rajagopalan Principles of Colloid and Surface Chemistry. Marcel Dekker: New York. 1997. [17] R. Fernández-Ruiz, R. Costo, M.P. Morales, O. Bomatí-Miguel, S. Veintemillas-Verdaguer, Total-reflection X-rayfluorescence: An alternative tool for the analysis of magnetic ferrofluids, Spectrochimica Acta Part B. 63 (2008) 1387–1394. [18] I. V. Beketov, A. P. Safronov, A. I. Medvedev, J. Alonso, G. V. Kurlyandskaya, and S.M. Bhagat, Iron oxide nanoparticles fabricated by electric explosion of wire: focus on magnetic nanofluids, AIP Advances 2 (2012) 022154. Highlights Ferrofluids and yeasts samples were analysed by TXRF spectroscopy. Simple protocol for iron quantification by means of TXRF was proposed. Results were combined with magnetic, structural, and morphological characterisation. Preliminary conclusion on nanoparticles uptake mechanism was made.

10