Modifying the magnetic response of magnetotactic bacteria: incorporation of Gd and Tb ions into the magnetosome structure

rsc.li/nanoscale-advances Volume 4 Number 12 21 June 2022 Pages 2525–2764 ISSN 2516-0230 PAPER E. M. Jefremovas, J. Alonso et al. Modifying the magnetic response of magnetotactic bacteria: incorporation of Gd and Tb ions into the magnetosome structure Nanoscale Advances Modifying the magnetic response of magnetotactic bacteria: incorporation of Gd and Tb ions into the magnetosome structure† E. M. Jefremovas, * a L. Gandarias, b L. Marcano, cd A. Gac´ ıa-Prieto, e I. Orue, f A. Muela, bg M. L. Fdez-Gubieda, cg L. Fern´ andez Barqu´ ın a and J. Alonso* a Magnetotactic bacteria Magnetospirillum gryphiswaldense MSR-1 biosynthesise chains of cube–octahedral magnetosomes, which are 40 nm magnetite high quality (Fe 3 O 4 ) nanoparticles. The magnetic properties of these crystalline magnetite nanoparticles, which can be modified by the addition of other elements into the magnetosome structure (doping), are of prime interest in a plethora of applications, those related to cancer therapy being some of the most promising ones. Although previous studies have focused on transition metal elements, rare earth (RE) elements are very interesting as doping agents, both from a fundamental point of view (e.g. significant differences in ionic sizes) and for the potential applications, especially in biomedicine (e.g. magnetic resonance imaging and luminescence). In this work, we have investigated the impact of Gd and Tb on the magnetic properties of magnetosomes by using different complementary techniques. X-ray diffraction, transmission electron microscopy, and X-ray absorption near edge spectroscopy analyses have revealed that a small amount of RE ions, 3–4%, incorporate into the Fe 3 O 4 structure as Gd 3+ and Tb 3+ ions. The experimental magnetic characterisation has shown a clear Verwey transition for the RE-doped bacteria, located at T100 K, which is slightly below the one corresponding to the undoped ones (106 K). However, we report a decrease in the coercivity and remanence of the REdoped bacteria. Simulations based on the Stoner–Wohlfarth model have allowed us to associate these changes in the magnetic response with a reduction of the magnetocrystalline (K C ) and, especially, the uniaxial (K uni ) anisotropies below the Verwey transition. In this way, K uni reaches a value of 23 and 26 kJ m 3 for the Gdand Tb-doped bacteria, respectively, whilst a value of 37 kJ m 3 is obtained for the undoped bacteria. 1 Introduction Magnetosomes are membrane-enclosed single-domain magnetic nanoparticles made of magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) synthesised by magnetotactic bacteria (MTB). These magnetosomes are arranged in one or several chains inside the MTB, which allow the MTB to orient in water by means of the torque exerted by the Earth’s magnetic eld on the chain. 1 The size and shape of magnetosomes strongly depend on the MTB species, their sizes generally being comprised between 35 and 120 nm, and they can be shaped as cube–octahedral, hexagonal prisms, arrows, etc. 2 The potential transfer of magnetosomes towards biomedical applications has boosted the interest of these biosynthesised magnetic nanoparticles in the recent years. Their high purity and crystallinity (referred to as high quality from hereunder), narrow size distribution, good biocompatibility, and relatively easy functionalization have made magnetosomes promising candidates as theranostic agents for magnetic hyperthermia, drug delivery, and magnetic resonance imaging (MRI), among other applications. 3–10 a Dpto. CITIMAC, Universidad de Cantabria, 39005 Santander, Spain. E-mail: [email protected]; javier.alon[email protected] b Dpto. Inmunolog´ ıa, Microbiolog´ ıa y Parasitolog´ ıa, Universidad del Pa´ ıs Vasco (UPV/ EHU), 48940 Leioa, Spain c Helmholtz–Zentrum Berlin f¨ ur Materialien und Energie, Albert-Einstein-Str. 15, 12489 Berlin, Germany d Dpto. Electricidad y Electr´ onica, Universidad del Pa´ ıs Vasco (UPV/EHU), 48940 Leioa, Spain e Dpto. F´ ısica Aplicada, Universidad del Pa´ ıs Vasco (UPV/EHU), 48013 Bilbao, Spain f SGIker Medidas Magn´ eticas, Universidad del Pa´ ıs Vasco (UPV/EHU), 48940 Leioa, Spain g BCMaterials, Basque Center for Materials, Applications and Nanostructures, UPV/ EHU, Spain †Electronic supplementary information (ESI) available: Fig. S1 and S2: Collection of TEM images, where the appearance of R salts attached to the bacterial body and particularities of the magnetosome chains can be inspected. Fig. S3 includes the ZFC/FC M vs. H curves measured at different temperatures for the RE–doped MTB. Fig. S4 offers a comparison between the thermal evolution of DH C and jDM r /M s jof the undoped, Gd [100 : 100], Tb [100 : 100], Mn [480 : 100] and Mn [100 : 100] bacterial samples. See https://doi.org/10.1039/d2na00094f Cite this: Nanoscale Adv., 2022, 4, 2649 Received 9th February 2022 Accepted 1st April 2022 DOI: 10.1039/d2na00094f rsc.li/nanoscale-advances © 2022 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2022, 4,2649–2659 | 2649 Nanoscale Advances PAPER Open Access Article. Published on 26 April 2022. Downloaded on 10/3/2022 2:40:56 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue Furthermore, magnetosomes have also been considered as reliable models to investigate the relationship between the structural and magnetic properties of magnetite at the nanoscale, 11–14 a matter of debate that has attracted great interest for many years in the scientic community. To this respect, magnetosomes can be employed to investigate different relevant issues, including the survival of the Verwey transition of magnetite at the nanoscale or the role of shape anisotropy in faceted nanoparticles. 15–17 Nevertheless, despite these very promising features, magnetosomes present some drawbacks, especially when compared with their chemically synthesised counterparts. These include, for example, the restricted tunability of their shape, size and chemical composition, as these features are strictly genetically determined. 18,19 These restrictions constitute a nuisance when trying to modify the magnetic response of magnetosomes for different applications. 20–23 However, alternative routes have been devised to overcome some of these limitations. MTB exhibit a high affinity and specicity towards iron, which they extract from the medium in order to synthesise magnetosomes. In the same way, it has been demonstrated that MTB can also synthesise magnetosomes doped with some transition metals such as manganese, titanium, copper, or cobalt, 23–28 by adding limited amounts of these metals to the growth medium. There are however very few studies describing the incorporation of other elements, 29 which underlines the inherent complexity associated with the doping process. Among all the possible doping candidates, the incorporation of Rare Earth (RE) ions into magnetosomes would be considerably appealing. RE doping opens the door to modifying the internal structure and the magnetic properties of the nanoparticles, both being accomplished at the same time. Moreover, RE elements are currently used in several top-notch elds, such as biomedicine, catalysis, and/or solar cells. 30,31 The fascination towards RE does not stop at their potential biomedical and technological transfer, yet there is also room for the emergence of new magnetic phenomena. In this way, from a fundamental point of view, the large unquenched orbital angular momentum and high spin–orbit coupling of the 4f electrons in some RE ions can give rise to more pronounced magnetic features in comparison to transition metal ions. 32,33 Therefore, there is also a great potential for investigation on RE-doped magnetosomes, apart from the ones doped with transition metals. To the best of our knowledge, the only work that has been published in this area is the one by Shimoshige et al. 34 Specically, they doped Magnetospirillum magneticum RSS-1 with Sm ions, obtaining core/shell magnetosomes made of magnetite in the core and samarium oxide in the shell. In our work, we have been able to incorporate, for the rst time, Gd 3+ and Tb 3+ ions into magnetosomes from the Magnetospirillum gryphiswaldense strain MSR-1. Gd 3+ is a S-state ion (L¼0) with seven unpaired electrons, which has been investigated, among other things, to develop gadolinium-doped iron oxide nanoparticles exhibiting a T 1 –T 2 dual-model MRI contrast. 35,36 On the other hand, Tb 3+ is an ion with six unpaired electrons, which has attracted attention for the possibility of providing magnetite nanoparticles with luminescence properties, which can be useful for monitoring the nanoparticles within the context of several biomedical applications. 36,37 Furthermore, the incorporation of Gd and Tb ions into the magnetite structure has also attracted attention due to the modulation of the magnetic properties of magnetite when the larger Gd 3+ and Tb 3+ ions are incorporated into its inverse spinel structure. 38 Bearing all these considerations in mind, we present here a combination of experimental and theoretical results to investigate the role of Gd 3+ and Tb 3+ cations in the magnetic response of magnetosomes. The morphological and structural properties of these RE-doped magnetosomes have been studied by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The incorporation of the RE ions into the magnetosome structure has been investigated by X-ray absorption near edge spectroscopy (XANES) experiments, carried out in large scale Synchrotron facilities. In addition, the magnetic response of these doped magnetosomes has been thoroughly analysed, and compared with undoped magnetosomes, by using different experimental magnetic measurements, including zero-eld cooling/eld-cooling (ZFC/FC) curves and hysteresis loops (M vs. H). Finally, a modied Stoner–Wohlfarth model has been employed to simulate the experimental M vs. H loops. This has allowed us to pinpoint the specic magnetic changes taking place, and to relate these changes to the intrinsic modication of the effective anisotropies of these Gdand Tb-doped magnetosomes. 2 Materials and methods 2.1 Magnetotactic bacteria: culture and magnetosome isolation Magnetospirillum gryphiswaldense MSR-1 (DMSZ 6631) was grown without shaking at 28 C in Flask Standard Medium (FSM) (Heyen and Sch¨ uler 39 ) containing (per litre of deionized water) 0.1 g KH 2 PO 4 , 0.15 g MgSO 4 $7H 2 O, 2.38 g HEPES, 0.34 g NaNO 3 , 0.1 g yeast extract, 3 g soybean peptone, 0.3% (wt/vol) of sodium pyruvate as the carbon source and 100 mM of Fe(III)- citrate. For gadolinium and terbium doping of bacteria, 100 mM of Gd(III)-quinate and Tb(III)-quinate were added, respectively. Bacteria were grown in 100 mL bottles lled with 80 mL of the culture media to obtain the desired oxygen concentration conditions. The inoculation in FSM containing 100 mM of Gd/ Tb-quinate is made from a 48 hour culture grown in FSM in a 1/10 dilution. In order to ensure that the bacteria are in contact with the dopant long enough, two subcultures of 48 hours are made in FSM containing 100 mM of Gd/Tb-quinate. The reproducibility was assured with more than 10 replicates of the RE doped bacteria obtained at different times since the beginning of the studies. Two different samples were employed in the subsequent experimental measurements: whole cells and isolated magnetosomes from the bacteria. First, the whole cell samples were harvested by centrifugation, xed in 2% glutaraldehyde, and washed three times in Milli Q water. Second, the isolation of magnetosomes was performed following the protocol described by Gr¨ unberg et al. 30 with minor modications. The cells, 2650 |Nanoscale Adv., 2022, 4,2649–2659 © 2022 The Author(s). Published by the Royal Society of Chemistry Nanoscale Advances Paper Open Access Article. Published on 26 April 2022. Downloaded on 10/3/2022 2:40:56 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online suspended in 20 mM HEPES–4 mM EDTA (pH ¼7.4), were disrupted using a French press (P¼1.4 kbar). To promote the separation of the magnetosomes, the lysated cells were sonicated and centrifuged at 600 g for 5 min to remove cell debris. Then, magnetic separation was employed to collect the magnetosomes from the supernatant, and aerwards, they were rinsed 10 times with 10 mM Hepes–200 mM NaCl (pH ¼7.4). 2.2 Transmission electron microscopy Transmission electron microscopy (TEM) was carried out on both unstained whole bacteria (i.e., whole cells) and the isolated magnetosomes extracted from the bacteria. In both cases, samples were adsorbed onto 300 mesh carbon-coated copper grids. The images were obtained with a JEOL JEM-14000 Plus electron microscope at an accelerating voltage of 120 kV. The particle size distribution was analysed by using ImageJ so- ware. 40 More than 130 magnetosomes from different cells were measured one-by-one in order to ensure good statistics. 2.3 X-ray diffraction X-Ray diffraction (XRD) measurements were performed on Gdand Tb-doped whole bacteria (whole cells) using a Bruker D8 Advance diffractometer working in Bragg–Bentano geometry with Cu-K a (l¼1.5418 ˚ A) radiation. The selected range for the 2qBragg angle was 18to 95, with an angular step of 0.02at a counting rate of 1 second/step. The obtained XRD patterns were analysed using Rietveld renements. All the measurements were carried out in the whole cells in order to minimise possible oxidation of the magnetosomes aer extraction. 2.4 X-ray absorption near edge spectroscopy X-ray absorption near edge spectroscopy (XANES) was performed on Gdand Tb-doped magnetosomes extracted from the bacteria at both the Fe–K and RE-L 3 edges (7112 eV for Fe–K, 7514 eV for Tb-L 3 , and 7243 eV for Gd-L 3 ). Measurements were carried out at the CLAESS beamline of the ALBA synchrotron at room temperature. Fe K-edge and Tb L 3 -edge measurements were carried out in transmission mode, and Gd L 3 -edge measurements were carried out in uorescence mode. In all cases, the measurements were performed using a double Si crystal monochromator oriented in the [111] direction. 2.5 Magnetic measurements The magnetic characterisation was carried out on the whole bacteria (whole cells). The samples were freeze-dried and encapsulated in gelatin capsules. Magnetic measurements were performed in a superconducting quantum interference device magnetometer (Quantum Design MPMS-5). Magnetisation vs. temperature (M vs. T) curves were measured following the usual zero-eld-cooling/eld-cooling (ZFC/FC) protocol, with an applied magnetic eld of 5 mT. Magnetisation vs. magnetic eld (M vs. H) loops were measured at different temperatures, 10–300 K, applying magnetic elds up to 1 T. 3 Results and discussion 3.1 Structural characterisation Transmission electron microscopy (TEM) was employed to study the size, shape, and arrangement of the RE doped magnetosomes. Fig. 1(a)–(c) show representative images of the magnetosomes extracted from the bacteria corresponding to the undoped, Gd-, and Tb-doped bacteria, respectively. We have included in Fig. S1 and S2 of the ESI†additional TEM images of the MTB and their magnetosome chains. In both Gdand Tbdoped bacteria, the magnetosomes clearly exhibit the faceted cube-octahedral morphology typical of M. gryphiswaldense (see Fig. 1(a)). However, some of these RE doped magnetosomes seem to present a less faceted morphology compared to their undoped counterparts (see Fig. S1 in the ESI†). Along these lines, it has been reported that the presence of doping salts in the culture medium and the incorporation of the doping elements into the magnetosome structure can impose stress in the biomineralisation process. 25,27,28 In fact, similar shape irregularities have also been reported, for example, in Mndoped magnetosomes. 27 Indeed, high-resolution transmission electron microscopy (HRTEM) or similar high resolution imaging techniques would be needed for quantitative analyses. We have also observed that the chains of magnetosomes inside the RE-doped MTB occasionally present minor irregularities and deformations, as depicted in Fig. S1 and S2 in the ESI.† Moreover, the RE doped bacteria tend to form larger chains (27 magnetosomes/chain) compared to the undoped bacteria (20) (see Table 1). Histograms accounting for the sizedistribution of the magnetosomes are shown in Fig. 1(d)–(f), together with the corresponding Gaussian ts. For the undoped magnetosomes, two size distributions can be observed, one centered around 47(8) nm and the other one centered at 22(8) nm. This double size distribution is typical of these M. gryphiswaldense bacteria, and accounts for the difference in size between the magnetosomes located at the ends of the chain (smaller) and those located at the inner positions (larger). Just in the same way, two size distributions are also observed for the Tb-doped magnetosomes, centered at 42(6) nm and 29(2) nm. However, only a single size distribution centered at 33(9) nm is obtained for the Gd-doped magnetosomes. What is clear according to these TEM analyses, is that the Gdand Tb-doped bacteria tend to synthesise longer chains with smaller magnetosomes. A similar size reduction was also found for M. gryphiswaldense bacteria doped with other elements, such as Mn and Co. 26–28 A possible explanation for the presence of longer chains in RE doped bacteria could be that an increase in the magnetosomes/chain ratio would compensate for the reduction of the magnetic moment per magnetosome, given the smaller average size of the RE doped magnetosomes compared to the undoped ones. As a result, the net magnetic moment per chain would remain similar in both cases. However, further work will be needed to conrm this. X-ray diffraction (XRD) analyses were performed to detect the possible presence of internal structural changes in the REdoped magnetosomes. Fig. 1(g)–(i) show the XRD patterns for © 2022 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2022, 4,2649–2659 | 2651 Paper Nanoscale Advances Open Access Article. Published on 26 April 2022. Downloaded on 10/3/2022 2:40:56 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online the undoped, Gdand Tb-doped bacterial samples, respectively, together with the corresponding Rietveld renements 41 (background was effectively subtracted during the analysis). The obtained Bragg errors R B are always below 4% for Gd-doped, and 15% for Tb-doped samples, ensuring the reliability of the performed analysis. The peak identication of the XRD patterns, as shown by the vertical green bars below them, has conrmed the presence of magnetite (Fe 3 O 4 ) in both RE-doped bacteria (25.2(3)% content for Gd-, and 12.31(1)% for Tb-doped bacteria). Apart from the Fe 3 O 4 phase, the XRD patterns also present some reections corresponding to NaCl (66.0(2)% for Gd-, and 68.66(1)% Tb-doped bacteria) and KCl salts (8.5(1)% for Tb-doped bacteria). These contributions come from the PBS medium employed for washing the harvested bacteria. Besides, a poorly crystallised contribution related to the GdCl 3 salt (8.8%) has also been shown in Fig. 1(h). We must clarify that the XRD contributions of these additional salts are well differentiated from the one corresponding to the magnetosomes, and therefore they do not affect the analysis of the Fe 3 O 4 phase. Rietveld renements shown in Fig. 1(g)–(i) (black colour) corroborate the presence of well-formed crystalline magnetosomes in the undoped and RE doped bacteria. The obtained lattice parameters for each ensemble are a¼8.3598(3) ˚ A for Gddoped, and a¼8.3815(1.1) ˚ A for Tb-doped samples. These values are slightly reduced (<0.4%) with respect to the one typically reported for bulk Fe 3 O 4 (a¼8.397 ˚ A) 42 and undoped magnetosomes (a¼8.3985(2) ˚ A). 9 This slight contraction of the unit cell parameter could in principle seem counterintuitive, since the ionic radius of Gd 3+ (1.08 ˚ A) and Tb 3+ (1.06 ˚ A) is larger than that of Fe 3+ (0.63–0.78 ˚ A) or Fe 2+ (0.92 ˚ A). 36,43 Nevertheless, similar reductions in the lattice parameter have been reported in other RE-doped Fe 3 O 4 nanoparticles, and understood in terms of the RE-mediated strain 44 and/or surface stress. 45 Rietveld renements also provide information on the mean diameter and microstrain. The obtained values of the mean diameter hDiof the magnetosomes are 34.8(2) nm for the GdTable 1 Average (TEM) diameter, hDi, number of magnetosomes per chain, N, and lattice parameter, a, for the undoped, Gdand Tb-doped samples. The error in the average diameter corresponds to the standard deviation, s Sample Undoped Gd-doped Tb-doped hDi(nm) 47(8), 22(8) 33(9) 42(6), 29(2) N20 27 27 a(˚ A) 8.3985(2) 8.3598(3) 8.3815(1.1) Fig. 1 Representative TEM images (a)–(c) of the magnetosomes (extracted from the bacteria), size-distribution histograms (d)–(f) and XRD patterns (g)–(i), together with Rietveld refinements, corresponding to the undoped, Gdand Tb-doped bacteria, respectively. The size-distributions are fitted with Gaussian distribution. In (g)–(i), the position of the hkl reflections are marked below the XRD patterns in green lines. In all cases, the Fe 3 O 4 phase gives rise to the most intense peaks. XRD refinements for the undoped bacteria are reproduced from ref. 9 with permission. Insets in XRD show a representative TEM image of M. gryphiswaldense bacteria. 2652 |Nanoscale Adv., 2022, 4,2649–2659 © 2022 The Author(s). Published by the Royal Society of Chemistry Nanoscale Advances Paper Open Access Article. Published on 26 April 2022. Downloaded on 10/3/2022 2:40:56 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online doped, and 32.7(3) nm for Tb-doped samples, values that are in good agreement with the results obtained by TEM (see above), conrming the single crystalline nature of the magnetosomes. On the other hand, microstrain values of h¼1.92(9)% (Gd) and h¼3.9(1)% (Tb) have been obtained. These strain values indicate that the presence of both doping ions distorts the crystalline structure of the Fe 3 O 4 magnetosomes. Similar results have been reported for other doped magnetosomes. 22,25,26 At this point, TEM and XRD results have revealed that the crystalline structure of the magnetosome is mostly maintained despite the presence of Gd and Tb ions inside the Fe 3 O 4 lattice. This structural characterisation has been completed by investigating the incorporation of the Gd and Tb ions into the magnetosomes using XANES. XANES is a very powerful elementsensitive synchrotron technique that has provided us accurate information on the oxidation state and site occupancy of the Gd and Tb ions in the spinel structure of magnetite. 26,46 XANES experiments were carried out on Gdand Tb-doped magnetosomes, extracted from the bacteria, both at the Fe–K and RE-L 3 edges. Since the XANES signal of the RE salts attached to the bacterial body is so large that it masks any signal due to the RE doped magnetosomes, this time we have worked with isolated magnetosomes instead of the whole bacteria in order to avoid this effect. Fig. 2(a) and (b) show the XANES spectra for the Gdand Tb-doped magnetosomes at the Gd-L 3 (7243 eV) and Tb-L 3 (7514 eV) edges, respectively. The presence of a clear absorption edge for both samples is an indicator of the incorporation of both Tb and Gd into the magnetosome structure. Nevertheless, we cannot completely discard the possibility of the presence of some Gd/Tb salts attached to the membrane of the magnetosomes, despite the multiple washings to remove any remaining salts aer extraction. It should also be noted that, regardless the low Tb-content would, in principle, have led to measuring the Tb-L 3 edge in uorescence mode, the emission lines of Tb-L 3 overlap with the Fe–K ones, imposing the use of transmission measuring mode. Hence, the normalised transmission spectrum shown in Fig. 2(b) corresponds to an extremely low absorption jump. It is also noticeable that the high absorption white line for the RE-doped magnetosomes, associated with the number of holes in the 5d band (valence) and the location of the 5d states. The overall shapes of the XANES spectra resemble those of the reference compounds shown in Fig. 2, i.e., GdCl 3 and Tb(NO 3 ) 3 . The overlapping edge position is a clear-cut indicator of the oxidation state of the absorbing atom, 47 indicating that the oxidation state of the RE ions inside magnetosomes is that of RE 3+ . The incorporation of the RE 3+ ions into the Fe 3 O 4 structure of the magnetosomes is further conrmed by absorption measurements on the Fe K-edge. Fig. 2(c) and (d) show the Fe Kedge XANES spectra of the Gdand Tb-doped magnetosomes, Fig. 2 (a) and (b) normalised Gdand Tb-L 3 -edge XANES spectra for Gdand Tb-doped magnetosomes, respectively. The corresponding XANES spectrum for the GdCl 3 and Tb(NO 3 ) 3 reference samples have been included for comparison. (c) and (d) normalised Fe–K edge XANES spectra of magnetosomes from Gdand Tb-doped magnetosomes, respectively. The control spectrum (i.e., undoped magnetosomes) has been included as the reference. The insets show the pre-edge and edge regions in more detail. © 2022 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2022, 4,2649–2659 | 2653 Paper Nanoscale Advances Open Access Article. Published on 26 April 2022. Downloaded on 10/3/2022 2:40:56 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online together with undoped magnetosomes. As shown, the Fe K-edge spectra for both Gdand Tb-doped magnetosomes are very alike, and they qualitatively reproduce the shape of the spectrum recorded for the control magnetosomes. However, for both the Gdand Tbdoped magnetosomes, there is a minor damping of the oscillations of the XANES spectra, that can be traced for instance, in the white line (7131 eV) and the valley (7160 eV) amplitudes. A similar damping has been observed in XANES of magnetite nanoparticles when the purity and/or crystallinity of magnetite is slightly reduced. 47,48 On top of that, a displacement towards lower energies of the absorption edge can be observed, while for the undoped magnetosomes, the absorption edge is located at 7123.1 eV, being its position shied towards 7122.5 eV for both RE-doped magnetosomes. Therefore, this negative shiof 0.6 eV could indicate a reduction of the average valence state of Fe in the RE-doped magnetosomes. Considering that the difference between the edge position for Fe 2+ and Fe 3+ is 7 eV, we can estimate the valence of the RE-doped magnetosomes to be z2.55–2.57, while in the undoped MTB, the valence is 2.66. This estimation has been made taking into account the fact that the edge position depends linearly on the valence, therefore, a substitution of approximately 3–4% of Fe 3+ ions by Gd 3+ or Tb 3+ can account for the aforementioned valence reduction. In order to obtain a more accurate estimate, additional techniques such as X-ray magnetic circular dichroism may prove useful. At the same time, an increase in the pre-edge peak amplitude can also be observed for the RE-doped magnetosomes. This modication reects a change of the symmetry around the Fe atoms, towards a more non-centrosymmetric site. Therefore, these results suggest a reduction in the number of the centrosymmetric octahedral sites occupied by Fe 3+ ions, as a consequence of their substitution by Gd 3+ and Tb 3+ ions. 3.2 Magnetic characterisation The magnetic response of the doped-MTB has been analysed by tracing the Mvs.Tand Mvs.Hdependence. All the measurements were performed in whole bacteria in order to minimise the effect of the interchain interactions, but also to allow a better comparison with the results obtained for undoped bacteria. 26,49 Fig. 3(a) shows the ZFC–FC curves of the RE-doped and undoped bacteria, shied in the Y-axis for clarity purposes. Starting with the undoped sample, the Mvs.Tcurves present a strong irreversibility in the whole temperature range studied, and a sharp transition in the ZFC curve around T V 105 K, which is also accompanied by a smaller peak in the FC curve. This transition corresponds to the well-known Verwey transition, constituting a ngerprint of the presence of stoichiometric magnetite. 13,50 Concerning the Gdand Tb-doped bacteria, their overall Mvs.Tevolution is very similar to the one of the undoped bacteria. The ZFC–FC curves evidence clear irreversibility, and the presence of the Verwey transition is also evident, although it seems to be now slightly displaced towards lower Tvalues, 95 K for the Gd-doped and 99 K for Tb-doped bacteria. This displacement becomes more evident by comparing the derivatives of the ZFC curves for the three samples, as shown in Fig. 3(b). There, we can observe that the T V , marked by the point at which the derivative becomes null, is slightly shied towards lower values for both the Gdand Tb-doped bacteria. In addition, the peak of the derivative, which marks the onset of the transition, is broader, less intense, and also displaced towards lower temperatures for the RE-doped bacteria. It must be noted that the survival of the Verwey transition in magnetite nanoparticles is strongly dependent on the crystallinity and stoichiometry. Small changes in the magnetite structure, for example by doping with other elements or by creating defects/vacancies, 12,26,51 can quickly lead to the displacement and disappearance of this transition. Finally, on the low-temperature side, a strong paramagnetic contribution appears in both RE doped bacteria below T25 K. This is caused by the presence of the Gd and Tb salts attached to the bacterial body, as shown by the TEM images (see Fig. S1 and S2 in the ESI†). To further explore the magnetic behaviour of the Gdand Tbdoped magnetosomes, we have also analysed their magnetic Fig. 3 (a) M–Tcurves measured following the ZFC–FC protocol for the undoped (cyan), Gd- (red) and Tb-doped (blue) MTB. Note that the curves are displaced in the Y-axis for clarity purposes. (b) Derivatives of the ZFC magnetisation curves (dMdT 1 ) of these three samples. In both (a) and (b), the measurements for undoped MTB are included for comparison purposes, and the position of the Verwey transition corresponding to the undoped bacteria is marked with a gray line. 2654 |Nanoscale Adv., 2022, 4,2649–2659 © 2022 The Author(s). Published by the Royal Society of Chemistry Nanoscale Advances Paper Open Access Article. Published on 26 April 2022. Downloaded on 10/3/2022 2:40:56 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online response as a function of the applied magnetic eld. Hysteresis loops, M vs. H, have been measured at different temperatures, from 5 to 300 K, aer a cooling process with either no applied magnetic eld (zero-eld-cooling, ZFC) or with an applied eld of 1 T (eld-cooling, FC). We have included several of these M vs. Hloops in Fig. S3 of the ESI.†Here it can be seen that at 300 K, the M vs. H loops of the 3 samples (undoped, Gd-doped, and Tbdoped) are very similar, whereas clear differences emerge when decreasing the temperature, especially, below the Verwey temperature (100 K). Fig. 4 shows the thermal evolution of the most relevant hysteresis parameters, i.e., the coercive eld, m 0 H C (lepanels), and the magnetisation remanence, normalised by the saturation magnetisation, M r /M s (right panels). These have been measured under ZFC (top), and FC (middle) protocols, to nally compare them by plotting the difference (in absolute value) between the FC and ZFC values (bottom). There, in all cases (ZFC, FC and difference), it can be seen that either the coercive eld or the remanent magnetisation corresponding to the doped and undoped bacteria no longer overlap below T V , getting more and more differentiated with decreasing temperature, all the way down to 5 K. The same happens for the M vs. H loops shown in Fig. S3 of the ESI†. We will now analyse the coercive eld and remanence magnetisation in greater detail. Concerning the coercive eld, both the ZFC and FC coercive eld curves for the doped bacteria [Fig. 4 (a) and (c)] remain nearly constant down to 100 K, with a lower m 0 H C value (0.017 T) than the undoped bacteria (0.023 T). Then, the m 0 H C slowly increases up to 0.024 T at 50 K, and nally rises more steeply reaching a value of 0.045 T at 5 K, again smaller than the one obtained for the undoped bacteria. The differences in coercivity between RE-doped and undoped magnetosomes can be seen more clearly if we focus on Fig. 4(e), where the difference between ZFC and FC values, jDm 0 H C jcurves, is shown. The Verwey transition, delimited by the non-zero value of jDm 0 H C j, is clearly dened around 107 K for the undoped bacteria, while in the case of the Gdand Tbdoped samples, this transition is less abrupt and smoother. This result agrees well with the magnetic behaviour observed in the ZFC/FC M vs. T curves, indicating that the RE ions inside the magnetosomes, on one hand, reduce the effective anisotropy, Fig. 4 Evolution with Tof the coercive field, m 0 H C , ((a), (c) and (e)), and the normalised remanence, M r /M s ((b), (d) and (f)) for the undoped, Gd-, and Tb-doped bacteria. Samples were cooled under no field (ZFC, (a) and (b)), and under a field of 1 T (FC, (c) and (d)). (e) and (f) depict the difference between FC and ZFC measurements, in absolute value, of the m 0 H C and the M r /M s values. © 2022 The Author(s). Published by the Royal Society of Chemistry Nanoscale Adv., 2022, 4,2649–2659 | 2655 Paper Nanoscale Advances Open Access Article. Published on 26 April 2022. Downloaded on 10/3/2022 2:40:56 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online and, on the other hand, slightly modify the Verwey transition due to minor structural changes. These results are further supported by the M r /M s curves shown in Fig. 4(b), (d), and (f). There, it can be seen how the shoulders found at T107 and 50 K for the undoped bacteria become smooth and broadened in the case of the RE-doped bacteria. Although some small differences can be observed between the values of the M r /M s curves of the Gdand Tb-doped samples, when plotting the change of remanence jDM r /M s jin Fig. 4(f), both curves overlap, as for jDm 0 H C j. Following a similar evolution to that of the jDm 0 H C jcurves shown in (e), jDM r /M s jfor the RE-doped magnetosomes remains very small (<0.005) down to 90 K. Then, it slowly starts increasing up to 0.01 at 50 K, and below that temperature, the increase becomes more abrupt, although the maximum values reached (0.18) are again smaller than those obtained for the undoped magnetosomes (0.24). A comparison between the thermal evolution of jDm 0 - H C jand jDM r /M s jof the undoped, Gd-doped, Tb-doped, and Mn-doped bacterial samples is presented in Fig. S4 of the ESI†. In order to shed light on the specic changes that are taking place in the intrinsic magnetic properties of the Gdand Tbdoped samples, we have carried out magnetic simulations of the M vs. H loops measured at different temperatures. For this, we have employed a modied Stoner–Wohlfarth approach, which has been extensively described in our previous studies. 4,27 Briey, the equilibrium conguration of the magnetic moment of each magnetosome is calculated as the sum of three contributions: (i) the magnetocrystalline anisotropy energy, E C ; (ii) the effective uniaxial anisotropy energy, E uni , arising from the competition between the magnetosome shape anisotropy and the dipolar interactions between magnetosomes inside the chain; and (iii) the Zeeman energy term, E Z . 26,52 In spherical coordinates, considering the h100icrystallographic directions of magnetite as the reference system, the total energy density is given by: E(q,f)¼E C (q,f)+E uni (q,f)+E Z (q,f)(1) being ECðq;fÞ¼KCsin4qsin2fþsin22q 4 Euniðq;fÞ¼Kunih1ð ^ um$ ^ uuniÞ2i EZðq;fÞ¼m0MHð^ um$ ^ uHÞ (2) where qand faccount for the polar and azimuthal angles of the magnetic moment of each magnetosome, respectively. K C and K uni stand for the magnetocrystalline and uniaxial anisotropy constants, respectively. The ˆ u i represents the unitary vector along the magnetic moment (ˆ u m ), the uniaxial anisotropy vector (ˆ u uni ) and the external magnetic eld (ˆ u H ) directions, respectively. As proved in previous studies, by SANS and electron cryotomography imaging, among other techniques, the ˆ u m forms an angle of 20with the chain axis direction, h111i. 15,52 Based on these considerations, the ZFC M vs. H loops at different temperatures have been simulated employing the dynamical approach already described in ref. 52 and 53. The anisotropy terms, K C and K uni , have been adjusted to attain the best match between experimental M vs. H loops and the corresponding simulations. As shown in Fig. 5(a)–(f), the calculated loops closely follow the experimental ones. The thermal evolution of K C and K uni for the undoped, Gdand Tb-doped samples is shown in Fig. 5(g) and (h). At room temperature, the values of K C for the three samples are similar: 11.0 kJ m 3 for the undoped and Gd-doped samples, and 12.0 kJ m 3 for the Tbdoped sample. These values are close to the theoretical K C value for bulk magnetite, 10.8 kJ m 3 . With decreasing temperature, jK C j(in absolute value) slightly increases for the undoped and Gd-doped bacteria, while it instead decreases for the Tb-doped sample, but overall, the change is small, remaining around jK C j z11–12 kJ m 3 . However, below 180 K, jK C jtends to decrease for the undoped sample, becoming null at 110 K, around the Verwey transition. This indicates that the role of the cubic magnetocrystalline anisotropy becomes negligible below T V for the undoped magnetosomes, as reported before. 26,27 A similar behaviour is also observed for the RE doped bacteria, but the drop in jK C jis displaced towards lower temperatures for both the Gdand Tb-doped bacteria, becoming practically null at 90 and 100 K, respectively. This follows the same trend observed in the ZFC–FC curves, which indicates, again, that the incorporation of RE ions into the magnetite structure is modifying the Verwey transition. The particular differences in the K C values and evolution between Gdand Tb-doped bacteria may be associated with differences in the incorporation of the Gd 3+ and Tb 3+ -ions into the magnetosome structure. Concerning the uniaxial anisotropy term, K uni , it remains almost constant for the three samples down to T V :11.5 kJ m 3 for the undoped and Gd-doped, and 10 kJ m 3 for the Tbdoped bacteria. In both cases, the K uni value is smaller than the one obtained for the undoped magnetosomes (12 kJ m 3 ). Being above the Verwey transition, K uni is mainly related to the effect of shape anisotropy and dipolar interactions, and this decrease could be ascribed to differences in the size distribution and/or morphology of the magnetosomes, as already observed in the TEM images. These differences are more appreciable in the case of the Tb-doped bacteria. Below T V ,K uni increases substantially for the undoped bacteria, up to 37 kJ m 3 . However, for the Gdand Tb-doped bacteria, the increase is slower, and the onset is not so well dened (90–100 K). Besides, the change in the slope obtained for K uni of the undoped magnetosomes below 50 K is also present in the Gdand Tb-doped bacteria, but is less obvious, especially in the case of the Gd-doped bacteria. In the end, at 10 K, a maximum K uni value of 23 and 26 kJ m 3 is reached for the Gdand Tb-doped bacteria, respectively. Qualitatively similar results were obtained in the case of Mn-doped bacteria. 27 All these results clearly indicate that there is amodication of both the magnetocrystalline and uniaxial anisotropies in REdoped magnetosomes. The changes above the Verwey transition can be most likely associated to modications in the shape/size of the RE-doped magnetosomes in comparison to the undoped ones. On the other hand, at low temperatures, the observed 2656 |Nanoscale Adv., 2022, 4,2649–2659 © 2022 The Author(s). Published by the Royal Society of Chemistry Nanoscale Advances Paper Open Access Article. Published on 26 April 2022. Downloaded on 10/3/2022 2:40:56 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online