Sandra Vergara

Dylan A Lukes

Michael W Martynowycz

Ulises Santiago

Germán Plascencia-Villa

Simon C Weiss

M Jason De

La Cruz

David M Black

Marcos M Alvarez

Xóchitl López-Lozano

German Plascencia-Villa

M Jason De La Cruz

Xochitl Lopez-Lozano

Christopher O Barnes

Guowu Lin

Hans- Christian Weissker

Robert L Whetten
email: robert.whetten@utsa.edu

Tamir Gonen
email: tgonen@ucla.edu

Miguel Jose-Yacaman
email: miguel.yacaman@utsa.edu

Guillermo Calero

MicroED Structure of Au 146 (p-MBA) 57 at Subatomic Resolution Reveals a Twinned FCC Cluster

Keywords: Gold cluster, electron diffraction, single crystal X-ray, structure determination, stacking fault

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Within the field of nanotechnology, metal clusters and nanoparticles are of central interest for their optical, electronic, and chemical properties. However, advances in this field have been limited by a lack of fidelity in the growth of nanoparticles. One strategy to achieve precise control over the shape and size of nanoparticles is the use of ligands as surfaceprotecting agents. This approach has enabled the synthesis of numerous stable clusters with atomically well-defined composition. Spectroscopic studies reveal that smaller metallic clusters have molecule-like electronic structures while larger ones support collective plasmon excitation similar to bulk-like nanoparticles [START_REF] Negishi | A Critical Size for Emergence of Nonbulk Electronic and Geometric Structures in Dodecanethiolate-Protected Au Clusters[END_REF][START_REF] Zhou | Evolution from the Plasmon to Exciton State in Ligand-Protected Atomically Precise Gold Nanoparticles[END_REF][START_REF] Zeng | Gold Quantum Boxes: On the Periodicities and the Quantum Confinement in the Au(2)(8), Au(3)(6), Au(4)(4) , and Au(5) (2) Magic Series[END_REF] . Solving the atomic structure of protected metal clusters is fundamental to understanding their size-dependent properties. Single crystal X-ray diffraction (XRD) has previously revealed the structure of several thiolate-protected gold clusters [START_REF] Zeng | Gold Quantum Boxes: On the Periodicities and the Quantum Confinement in the Au(2)(8), Au(3)(6), Au(4)(4) , and Au(5) (2) Magic Series[END_REF][START_REF] Zeng | Chiral Structure of Thiolate-Protected 28-Gold-Atom Nanocluster Determined by X-Ray Crystallography[END_REF][START_REF] Zeng | Total Structure and Electronic Properties of the Gold Nanocrystal Au36(Sr)24[END_REF][START_REF] Crasto | Single Crystal Xrd Structure and Theoretical Analysis of the Chiral Au30s(S-T-Bu)18 Cluster[END_REF][START_REF] Qian | Total Structure Determination of Thiolate-Protected Au38 Nanoparticles[END_REF][START_REF] Yang | A New Crystal Structure of Au36 with a Au14 Kernel Cocapped by Thiolate and Chloride[END_REF][START_REF] Heaven | Crystal Structure of the Gold Nanoparticle [N(C8h17)4[END_REF][START_REF] Zeng | Atomic Structure of Self-Assembled Monolayer of Thiolates on a Tetragonal Au92 Nanocrystal[END_REF][START_REF] Jadzinsky | Structure of a Thiol Monolayer-Protected Gold Nanoparticle at 1.1 a Resolution[END_REF][START_REF] Chen | Crystal Structure of Barrel-Shaped Chiral Au130(P-Mbt)50 Nanocluster[END_REF][START_REF] Zeng | Structural Patterns at All Scales in a Nonmetallic Chiral Au(133)(Sr)(52) Nanoparticle[END_REF][START_REF] Zeng | Emergence of Hierarchical Structural Complexities in Nanoparticles and Their Assembly[END_REF] . Although some small clusters display FCC-like kernels [START_REF] Zeng | Gold Quantum Boxes: On the Periodicities and the Quantum Confinement in the Au(2)(8), Au(3)(6), Au(4)(4) , and Au(5) (2) Magic Series[END_REF][START_REF] Zeng | Chiral Structure of Thiolate-Protected 28-Gold-Atom Nanocluster Determined by X-Ray Crystallography[END_REF][START_REF] Zeng | Total Structure and Electronic Properties of the Gold Nanocrystal Au36(Sr)24[END_REF] , for several clusters -including the larger ones with {102, 130, 133, & 246} Au atoms-non-crystalline icosahedra and truncated decahedra (known as multiply twinned particles, or MTPs) have been found [START_REF] Jadzinsky | Structure of a Thiol Monolayer-Protected Gold Nanoparticle at 1.1 a Resolution[END_REF][START_REF] Chen | Crystal Structure of Barrel-Shaped Chiral Au130(P-Mbt)50 Nanocluster[END_REF][START_REF] Zeng | Structural Patterns at All Scales in a Nonmetallic Chiral Au(133)(Sr)(52) Nanoparticle[END_REF][START_REF] Zeng | Emergence of Hierarchical Structural Complexities in Nanoparticles and Their Assembly[END_REF] . However, for sufficiently large clusters, a transition to bulk-like FCC packing should occur. Although some studies suggest a geometric and electronic transition occurring in the range of Au 144 to Au 329, the critical size for this transition is not clear [START_REF] Negishi | A Critical Size for Emergence of Nonbulk Electronic and Geometric Structures in Dodecanethiolate-Protected Au Clusters[END_REF][START_REF] Zhou | Evolution from the Plasmon to Exciton State in Ligand-Protected Atomically Precise Gold Nanoparticles[END_REF][START_REF] Philip | Evolution of Nonlinear Optical Properties: From Gold Atomic Clusters to Plasmonic Nanocrystals[END_REF][START_REF] Yi | Nanometals: Identifying the Onset of Metallic Relaxation Dynamics in Monolayer-Protected Gold Clusters Using Femtosecond Spectroscopy[END_REF][START_REF] Cleveland | Structural Evolution of Smaller Gold Nanocrystals: The Truncated Decahedral Motif[END_REF] . Therefore, the case of ubiquitous clusters of ~29kDa core mass (1.7-nm core diameter), commonly identified as Au 144 (SR) 60 , is of particular interest. After two decades since this class of compounds was first reported [START_REF] Whetten | Nanocrystal Gold Molecules[END_REF] , the precise composition and structure of the ~29kDa cluster remains elusive. The current models predict that Au 144 (SR) 60 should have the icosahedral symmetry [START_REF] Lopez-Acevedo | Structure and Bonding in the Ubiquitous Icosahedral Metallic Gold Cluster Au 144(Sr) 60[END_REF][START_REF] Bahena | Stem Electron Diffraction and High-Resolution Images Used in the Determination of the Crystal Structure of the Au144(Sr)60 Cluster[END_REF] whereas aqueous-phase samples may present polymorphism with competing icosahedral and truncated decahedral cores [START_REF] Jensen | Polymorphism in Magic-Sized Au144(Sr)60 Clusters[END_REF] . Unfortunately, structural efforts to reveal definitively its structure have thus far been unproductive. Herein we present a novel gold cluster in the ~29kDa size regime, Au 146 (SR) 57 , whose twinned-FCC structure departs from previous icosahedral and decahedral models proposed for Au 144 (SR) 60 .

Synthesis of the gold nanoclusters was achieved by a modified two-phase method [START_REF] Plascencia-Villa | Analytical Characterization of Size-Dependent Properties of Larger Aqueous Gold Nanoclusters[END_REF] , where clusters were precipitated by incubation in cold methanol or in cold methanol with 100mM ammonium acetate. The product was separated by native-PAGE (Figure S1). In both cases, analysis via liquid chromatography / high-resolution electrospray ionization mass spectroscopy (HPLC / ESI-MS) methods is consistent with the Au 146 (p-MBA) 57 molecular formula (Figure S2). Thus, Au 146 (p-MBA) 57 does not constitute an artifact of crystallization as it existed in solution prior to crystallization. Samples without ammonium acetate formed poorly-diffracting hexagonal plate crystals similar to those previously reported [START_REF] Jensen | Polymorphism in Magic-Sized Au144(Sr)60 Clusters[END_REF][START_REF] Mustalahti | Ultrafast Electronic Relaxation and Vibrational Cooling Dynamics of Au144(Sc2h4ph)60 Nanocluster Probed by Transient Mid-Ir Spectroscopy[END_REF][START_REF] Wong | Ligand Symmetry-Equivalence on Thiolate Protected Gold Nanoclusters Determined by Nmr Spectroscopy[END_REF] , whereas samples co-precipitated with ammonium acetate crystallized as needles and plates in the presence of 25% polyethylene glycol 8000 and 50mM sodium potassium phosphate (Table S1). Large crystals as well as fragmented nanocrystals (Figure 1a) were selected for X-ray, free electron laser (FEL) or MicroED experiments [START_REF] Shi | Three-Dimensional Electron Crystallography of Protein Microcrystals[END_REF][START_REF] Nannenga | High-Resolution Structure Determination by Continuous-Rotation Data Collection in Microed[END_REF] , respectively. X-ray analysis provided resolution of 1.3 Å, while hardware limitations restricted FEL data to 1.7 Å only. Remarkably, subatomic resolution was only achieved with electron diffraction by MicroED where crystals were analyzed in a frozen hydrated state by electron diffraction (Figures 1b andS3). A total of 146 gold atoms and 57 p-MBA ligands were identified in the electron density maps correlating with ESI-MS (Figure 1c). The surface representation of the metal cluster shows a globular and well-ordered structure that is effectively coated by p-MBA, preventing further growth (Figure 1d and Movie S1). The overall architecture of the cluster is illustrated stereographically by Figure 1e. The root-mean-square deviation between MicroED and X-ray atomic positions was 0.05 Å for the cluster kernel (innermost 13 Au atoms) and 0.1 Å for the entire gold structure, confirming the equivalence of the two methods. This MicroED structure represents the highest resolution structure for such aqueous phase gold clusters and the highest resolution reported to date by any cryoEM method.

The 146 gold atoms in the Au 146 (p-MBA) 57 may be decomposed into two constituent sets: 119 core atoms and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure (Figure 2a), whereas the surface gold atoms follow a C 2 rotational symmetry about an axis bisecting the twinning plane (Figure 2b). While FCC structures may be described morphologically as nested cuboctahedra, twinned FCC structures may be described as nested anti-cuboctahedra (Figure S4). Anti-cuboctahedra are also known as J27 under the Johnson solid classification system. The eight triangular faces of a J27 correspond to {111} planes, and the six square faces to {100} planes. The core of Au 146 (p-MBA) 57 is comprised of three nested J27 shells: a first shell (J27-1) formed by the 12 immediate neighbors of the central Au-atom site, a second shell (J27-2) of 42 sites, and an incomplete third shell (J27-3) comprising 60 sites (115 in total). Two pairs of additional gold atoms located on {100} facets complete the 119-atom core. The observed truncation of 32 sites, with respect to an ideal J27-3 shell, rounds the morphology (Figure S5a-b), as these truncations occur at sites of low coordination-number: 12 at vertices, and 20 along edges (Figure S5c-h). In particular, sites on the boundary between mirror {111} planes are not occupied. In addition, in four of the {111} facets, we observe a lattice distortion that resembles the HCP packing. The occupied positions as well as the distorted positions in the third shell are symmetric across mirror planes. However, non-mirror {111} planes are distinct throughout the third shell. The view along the [111] direction (Figure 2c) shows that atoms belonging to J27-1 and J27-2 as well as the majority of atoms in J27-3 lie on the expected positions for an FCC, while most of the peripheral atoms deviate from ideal positions (Figures 2b andS6). The view along the [1-10] direction shows the twin in the Au 146 structure.

Alternatively, one notes the presence of a 79-site twinned truncated octahedron (t-TO+) as a substructure of Au 146 (Figure 2d). The superior stability of the Marks decahedron and t-TO+ was previously predicted for gold clusters as compared to the icosahedron or the untwinned TO+ [START_REF] Cleveland | Structural Evolution of Smaller Gold Nanocrystals: The Truncated Decahedral Motif[END_REF][START_REF] Doye | Structural Consequences of the Range of the Interatomic Potential -a Menagerie of Clusters[END_REF] . The 79-atom t-TO+ substructure may be recovered by capping each of the six {100} facets of J27-2 with four gold atoms. This t-TO+ also appears as a distinguishable group in the radial distance histogram from the central atom (Figure S7).

The protective layer of Au 146 (p-MBA) 57 comprises 7 bridging motifs (-S-), 19 monomeric staple motifs (-S-Au-S-), and 4 dimeric staple motifs (-S-Au-S-Au-S-) arranged around the C 2 symmetry axis bisecting the twinning plane (Figure 3a-b). Four of the bridging motifs lie on {100} facets, two connect mirror {111} facets and one is located on the screw axis. This bridging motif has been observed primarily on {100} facets [START_REF] Qian | Total Structure Determination of Thiolate-Protected Au38 Nanoparticles[END_REF][START_REF] Zeng | Atomic Structure of Self-Assembled Monolayer of Thiolates on a Tetragonal Au92 Nanocrystal[END_REF] , but has recently been identified linking {111} facets in the structure of Au 246 (p-MBT) 80 [START_REF] Zeng | Emergence of Hierarchical Structural Complexities in Nanoparticles and Their Assembly[END_REF] (Figure S8). The monomeric staples connect adjacent facets. The four dimeric staples are anchored to {111} facets, two of them directly linked to atoms in the J27-2. Dimeric staples have been observed forming a "V" shape with the sulfur atoms in almost coplanar positions [START_REF] Heaven | Crystal Structure of the Gold Nanoparticle [N(C8h17)4[END_REF][START_REF] Jadzinsky | Structure of a Thiol Monolayer-Protected Gold Nanoparticle at 1.1 a Resolution[END_REF] . In Au 146 (p-MBA) 57 , the three sulfur atoms (-S-Au-S-Au-S-) are not coplanar resulting in a bending of the staple. Interestingly, both characteristics of dimeric staples, the exclusive binding on {111} facets and the bending, are also present in the nonaqueous system Au 246 (p-MBT) 80

14

(Figure S8). The overall distribution of the staples is presented in Figure 3c.

Starting from the experimental coordinates we have analyzed the structure by density functional theory (DFT) methods. The optimized (relaxed) structure thus obtained retains all connectivity of the original, while removing much of its irregularities in interatomic distances, thereby enhancing the C 2 -symmetry feature (Figure S9). In particular, Au-S bond lengths show a bimodal distribution, in agreement with experiment, where this double-peak structure is rather broad. The first peak is at 2.297 Å and corresponds to peripheral Au-S bonds, while the second is at 2.360 Å and corresponds to core Au-S bonds. We also observe a strong correlation between peaks of the radial distribution function and those of an FCC structure, with a peak gold-gold distance of 2.88 Å and most atoms in the range 2.73Å-3.1Å.

Gold clusters may manifest both crystalline FCC and non-crystalline structures. FCC cores have been observed in some small clusters [START_REF] Zeng | Gold Quantum Boxes: On the Periodicities and the Quantum Confinement in the Au(2)(8), Au(3)(6), Au(4)(4) , and Au(5) (2) Magic Series[END_REF][START_REF] Zeng | Chiral Structure of Thiolate-Protected 28-Gold-Atom Nanocluster Determined by X-Ray Crystallography[END_REF][START_REF] Zeng | Total Structure and Electronic Properties of the Gold Nanocrystal Au36(Sr)24[END_REF][START_REF] Crasto | Single Crystal Xrd Structure and Theoretical Analysis of the Chiral Au30s(S-T-Bu)18 Cluster[END_REF][START_REF] Yang | A New Crystal Structure of Au36 with a Au14 Kernel Cocapped by Thiolate and Chloride[END_REF] and more recently, in the tetragonal-shaped Au 92 [START_REF] Zeng | Atomic Structure of Self-Assembled Monolayer of Thiolates on a Tetragonal Au92 Nanocrystal[END_REF] . Other clusters, including the larger ones (Au 102 , Au 130 , Au 133 , and Au 246 ), display cores with five-fold symmetry (MTPs) [START_REF] Qian | Total Structure Determination of Thiolate-Protected Au38 Nanoparticles[END_REF][START_REF] Heaven | Crystal Structure of the Gold Nanoparticle [N(C8h17)4[END_REF][START_REF] Jadzinsky | Structure of a Thiol Monolayer-Protected Gold Nanoparticle at 1.1 a Resolution[END_REF][START_REF] Chen | Crystal Structure of Barrel-Shaped Chiral Au130(P-Mbt)50 Nanocluster[END_REF][START_REF] Zeng | Structural Patterns at All Scales in a Nonmetallic Chiral Au(133)(Sr)(52) Nanoparticle[END_REF][START_REF] Zeng | Emergence of Hierarchical Structural Complexities in Nanoparticles and Their Assembly[END_REF] . Figure 4 shows the six largest gold clusters solved to date. Au 92 10 presents an FCC core; Au 102 [START_REF] Jadzinsky | Structure of a Thiol Monolayer-Protected Gold Nanoparticle at 1.1 a Resolution[END_REF] , Au 130 [START_REF] Chen | Crystal Structure of Barrel-Shaped Chiral Au130(P-Mbt)50 Nanocluster[END_REF] , and Au 246 14 display decahedra cores; and Au 133 [START_REF] Zeng | Structural Patterns at All Scales in a Nonmetallic Chiral Au(133)(Sr)(52) Nanoparticle[END_REF] has a Mackay icosahedral core. Au 146 is the largest solved cluster with a FCC core. In addition, it is the smallest gold nanoparticle observed containing a stacking fault. These planar defects are very common in much larger particles (such as colloidal gold) but have never been observed at this low number of atoms.

Although icosahedral and decahedral structures display energetically-favorable close-packed outer facets, as the size of the particle increases, a preference for bulk-like FCC structures is expected. While nucleation mechanisms remain unclear, most likely FCC nanoparticles should grow starting from FCC seeds. The favorability of a cluster as a seed for an FCC particle is linked to the ability to grow quickly and leave the subcritical size region before disintegration [START_REF] Vandewaal | An Atomic-Scale Model of Fcc Crystal-Growth[END_REF] . The twinned-FCC anti-cuboctahedral kernel of Au 146 may therefore be a more suitable seed than a cuboctahedral kernel, as the saturation of {100} facets in J27 generates extra sites with 4-coordination on the twin plane than are absent in the untwinned structure (Figure S10). This creation of 4-coordination sites is similar to the well-established effect of twins in the growth of nanoparticles [START_REF] Lofton | Mechanisms Controlling Crystal Habits of Gold and Silver Colloids[END_REF] . In the case of nanoparticles, the introduction of twins favors a kinetic growth on the twin that leads to thermodynamicallyunfavorable shapes like triangular plates or rods in a similar fashion that screw dislocations favor the growth of large crystals [START_REF] Burton | The Growth of Crystals and the Equilibrium Structure of Their Surfaces[END_REF] .

In this report we have shown that even a FCC structure with such few atoms (Au146, ~1.7 nm) contains a well-defined twinning plane. Considering that decahedral structures have 5 twin-planes and icosahedra have 30 twin-planes, twinning appears to be a critical component of nanoparticle structure from a very early stage of growth. Moreover, to our knowledge, the subatomic resolution structure of Au 146 (p-MBA) 57 presented here is the first such structure of a metal cluster obtained by electron diffraction of frozen hydrated samples, establishing MicroED as a new and important tool for the characterization of nanomaterials.

EXPERIMENTAL METHODS

Synthesis and Purification of Gold Nanoclusters

The gold nanoclusters were produced by the two-phase method with some minor modifications 23, 31-33 . First, stock solution of para-mercaptobenzoic acid (p-MBA) at 100 mM was prepared in 300 mM NaOH by vigorous stirring for at least 3 h or overnight and pulsed sonication. Nanoclusters were typically produced in 25 ml batches in 50 % v/v methanol in a 50 ml round flask by adding aqueous HAuCl 4 to a final concentration of 3 mM and p-MBA to 9 mM. The color of this solution was light yellow. The mixture was stirred overnight until solution was almost clear/colorless. Then freshly prepared ice-cold aqueous NaBH 4 was added to achieve 1.5 mM final concentration, and the reaction proceeded for 2 h. The entire content was transferred to a 50 ml conical tube. Volume was completed to 50 ml with cold methanol. Two distinct approaches were used to precipitate the as-prepared cluster product: cold methanol or cold methanol with 100 mM ammonium acetate. The content was mixed gently and incubated at 4°C. Nanoclusters were concentrated with centrifugation at 2000-3000 rpm for15 min, supernatant removed, and the pellet resuspended in 25 ml methanol. Nanoclusters were again concentrated by centrifugation at 2000-3000 rpm for 15 min, supernatant removed, and the pellet air dried. Finally, the pellet was dissolved in a small volume with ultrapure ddH 2 O (0.5-1 ml). Product quality was assessed by native 10-12 % PAGE in TBE buffer at 100 V. Major bands corresponding to gold nanoclusters were cut from the gel to separate the fractions of interest and then reconcentrated by precipitation with methanol or rotary evaporator. Aliquots in ultrapure ddH 2 O were stored at 4°C for further analysis. Stability of gold nanoclusters in both samples, with and without ammonium acetate coprecipitation, was monitored by routine 10-12 % PAGE, UV-Vis and Transmission Electron Microscopy (TEM), confirming no perceivable changes over 6 months.

Gold cluster crystallization

The two cluster samples, with and without ammonium acetate coprecipitation, were submitted to crystallization screenings. The sample without ammonium acetate formed poorly-diffracting hexagonal plates, whereas the sample coprecipitated with ammonium acetate crystallized in the presence of 25% polyethylene glycol 8000 (PEG8K) and 50 mM sodium potassium phosphate. Crystals were transferred to a stabilizing solution for cryoprotection containing 25% methyl pentanediol (MPD), 5% PEG 8K and 50mM sodium potassium phosphate.

Electron Micro-Diffraction (MicroED)

Crystals were too big for MicroED thus a fragmentation protocol was designed [START_REF] De La Cruz | Atomic-Resolution Structures from Fragmented Protein Crystals with the Cryoem Method Microed[END_REF] . Crystals in stabilizing solution (see above) were harvested in a 0.5 ml Eppendorf tube and 30 mgs of stainless steel beads (1 mm diameter) were placed for fragmentation [START_REF] Stevenson | Transmission Electron Microscopy for the Evaluation and Optimization of Crystal Growth[END_REF][START_REF] Barnes | Assessment of Microcrystal Quality by Transmission Electron Microscopy for Efficient Serial Femtosecond Crystallography[END_REF] . Sample was vortexed several times and the presence of homogeneously sized fragments was verified using bright field microscopy. Stainless steel beads were removed using a magnet and samples were centrifuged at 1000 rpms to pellet the fragments. Excess of mother liquour was removed to obtain a concentrated slurry and stabilizing cryo-protecting solution was added and mixed carefully with the slurry. This procedure was repeated three times to ensure full buffer exchange. Holey 300 mesh carbon Quantifoil® R2/2 grids were negatively glow discharged at 25 mA and 2*10^-1 mbar for 30s from both sides. Then 2 µl of the concentrated and cryoprotected nanocrystal solution were pipetted onto the grid and incubated for 1 minute, before the excess liquid was removed by careful blotting from the backside using Fisherbrand P2 filter paper. Afterwards the grid was flash frozen by plunging it into liquid nitrogen. The grids were stored in liquid nitrogen until they were used for data collection.

MicroED Data were collected using an FEI Titan Krios TEM equipped with a field emission gun operating at 300kV, corresponding to an electron wavelength of 0.0197Å [START_REF] Shi | Three-Dimensional Electron Crystallography of Protein Microcrystals[END_REF][START_REF] Nannenga | High-Resolution Structure Determination by Continuous-Rotation Data Collection in Microed[END_REF] . Data was recorded with a TVIPS TemCam-F416 4K × 4K CMOS camera with sensor pixel dimensions of 15.6 µm × 15.6 µm. Images were taken in rolling-shutter mode with 2X-pixel binning, resulting in final images of 2048 px × 2048 px. Frames were acquired under a continuous rotation of 0.1°/s every 5 s, or a 0.5° wedge of reciprocal space per frame. Data was converted using open-source software and corrected for pixel truncation as previously described [START_REF] Hattne | Modeling Truncated Pixel Values of Faint Reflections in Microed Images[END_REF][START_REF] Hattne | Microed Data Collection and Processing[END_REF] . MicroED experiments were performed at liquid nitrogen temperatures, ~77K. Nano crystals diffracted to 0.7Å resolution by MicroED.

Structure Refinement

X-ray and MicroED data were processed in space group P1 using the software package XDS 39 . Reflections were indexed using the program XPREP in P2(1)/n or P2(1)/c. The Xray structure was solved using the program SHELXT [START_REF] Sheldrick | Shelxt -Integrated Space-Group and Crystal-Structure Determination[END_REF] yielding 146 gold and 57 sulfur atoms. The Fo-Fc difference map using X-ray data set 2 (Table S1) showed positive density for forty pMBA molecules. The full model was completed using the difference map (Fo-Fc) from X-ray data set 1 (Table S1) which revealed all fifty-seven pMBA molecules (Figure S3c-d). The MicroED structure was solved using a partial solution obtained with the program Shake and Bake [START_REF] Miller | Snb -Crystal-Structure Determination Via Shakeand-Bake[END_REF] where 79 Au atoms were clearly identified in the Patterson map. The remaining Au atoms and all 57 sulfurs were easily identified as positive density in the Fo-Fc map. Only a few p-MBA molecules were observed in the MicroED data.

The MicroED and X-ray structures were refined using the programs SHELXL [START_REF] Sheldrick | A Short History of Shelx[END_REF] and Phenix [START_REF] Adams | Phenix: A Comprehensive Python-Based System for Macromolecular Structure Solution[END_REF] . Several rounds of manual building and refinement were employed to place the pMBA molecules.

Mass Spectrometry

Samples were analyzed by HPLC-ESI-ToF-MS. The LC-MS method resolves (separates) multi-component samples (chromatographically) prior to the identification of each component (mass-spectrometrically), reducing interference from adducts [START_REF] Black | Capillary Liquid Chromatography Mass Spectrometry Analysis of Intact Monolayer-Protected Gold Clusters in Complex Mixtures[END_REF][START_REF] Black | Triethylamine Solution for the Intractability of Aqueous Gold-Thiolate Cluster Anions: How Ion Pairing Enhances Esi-Ms and Hplc of Aq-Aun(Pmba)P[END_REF] . Au 102 (pMBA) 44 clusters were used for validation of the quality of the technique. The Au 146 clusters were detected intact. The major peak could be cleanly attributed to the intact (146,57) species, in the charge-states = [4-] thru [7-], with [5-] dominant. Minor satellite peaks could be attributed to water adduction.

Computational DFT methods

The ab initio DFT structural optimization started from the experimental structure.

Subsequently, the structure was relaxed using the VASP code [START_REF] Kresse | Efficiency of Ab-Initio Total Energy Calculations for Metals and Semiconductors Using a Plane-Wave Basis Set[END_REF][START_REF] Kresse | From Ultrasoft Pseudopotentials to the Projector Augmented-Wave Method[END_REF] with PAWs until all forces were smaller than 0.02 eV/ Å. An energy cutoff of 280 eV was employed. A charge state of 3-(three additional electrons) was used to comply with the expected electronic shell closing at 2008 electrons according to the superatom model. Among our various optimizations with distinct exchange-correlation functionals, ligand models (SR: Cl-, R:CH 3 , or SR:pMBA), and charge states {3+, 1+, 1-, 3-}, the LDA optimization chg.= 3turns out to best represent the Au-Au bonds, unlike the calculations done using a GGA functional (PBE; not shown), to which the missing hydrogen atoms were added.

Figure 1 .

 1 Figure 1. MicroED and X-ray determination of the crystal structure of Au 146 (p-MBA) 57 . a, Transmission electron micrograph of Au 146 (p-MBA) 57 crystals, and b, typical MicroED data extending well beyond 1Å. c, MicroED density map (2Fo-Fc, contoured at 2 σ) shown as red mesh, identifies atomic positions of Au (white spheres) and S (yellow spheres) atoms. Ligands (p-MBA) are shown as blue framework. d, Surface representation of the cluster with oxygen in red, carbon in white, sulfur in cyan, and gold in green. The full set of p-MBA ligands was determined using X-ray data. e, Stereoscopic representation of the cluster including all gold atoms and p-MBA ligands. Gold atoms colored by shells for visualization purposes (see Figure 2).

Figure 2 .

 2 Figure 2. Configuration of gold atoms in Au 146 (p-MBA) 57 . a, Core structure (119-atoms) of the Au 146 cluster. The central atom, purple, and the first shell J27-1, red, comprise the kernel of the cluster; second shell J27-2, brown; third shell J27-3, light green; extra atoms in the core, dark yellow. Twin plane parallel to xz-plane. b, Location of peripheral gold atoms (27atoms) in dark green, with core displayed in wires. c, View of crystallographic planes. Plane directions are absolute to an FCC unit cell. d, Selection of 79-atom corresponding to a twinned truncated octahedron, t-TO+. Remaining atoms from core displayed on wires.

Figure 3 .

 3 Figure 3. Configuration of ligands (p-MBA) on Au146(p-MBA)57. a, Distribution of the three types of staples on the core surface of Au146(p-MBA)57. Bridging motifs (left) in pink, monomeric staples (center) in light pink, and dimeric staples (right) in purple. b, Rotational symmetric distribution of staples. Screw axis parallel to z-axis bisecting the twin plane (xzplane). c, Stereoscopic view of Au146(p-MBA)57 displaying all different types of staples, J27-2 atoms are displayed as solid surface.

Figure 4 .

 4 Figure 4. Atomic structures of the largest gold clusters solved to date. a, Kernel structures of Au 92 (conjoined cuboctahedra with no unique central atom); Au 102 and Au 246 (decahedra); Au 130 (Ino decahedron); Au 133 (icosahedron); and Au 146 (anti-cuboctahedron). b, The view along the [111] axis for Au 92 and Au 146 shows the atomic positions following the close-packing of FCC structures, whereas the projection along [110] axis for Au 102 , Au 130 , Au 133 , and Au 246 shows five-fold symmetry.

J Phys Chem Lett. Author manuscript; available in PMC 2018 January 16.

Acknowledgments

We would like to acknowledge May Elizabeth Sharp and Meitan Wang at the Swiss Light Source (SLS). We would also like to acknowledge Michael Becker and Craig Ogata at GM/CA (Argonne National Laboratory). We would also like to acknowledge John Chrzas and Rod Salazar at the South East Regional Collaborative Access Team (SERCAT, Argonne National Laboratory). The authors acknowledge Aina Cohen and Jinhu Song, from the Macromolecular Femtosecond Crystallography (MFX) at the Linac Coherent Light Source (LCLS) for their support during data collection. We thank D. Lee for home-source X-ray technical support at the University of Pittsburgh. Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities (NIMHD) of the National Institutes of Health (#G12-MD007591) and by The Welch Foundation grants #AX-1615 and #AX-1857. GC acknowledges University of Pittsburgh startup funds and support from NIH grants P50-GM082251, R01-GM112686 and BioXFEL-STC1231306. GM/CA@aps has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The Gonen laboratory is supported by the Howard Hughes Medical Institute and this work was also supported by the Janelia Research Campus visitor program. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

data summary, Mass Spectroscopy and DFT results. The following files are available free of charge. Au146-PMB57.cif This file corresponds to X-ray Dataset 1 (SLS: XO6SA-PXI). Au146-S17-PMB40.cif This file corresponds to X-ray Dataset 2 (APS: SERCAT-22ID) Au146-S32-PMB25.cif

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Niéli Daffé

Fadi Choueikani

Sophie Neveu

Marie-Anne Arrio

Amélie Juhin

Philippe Ohresser

Vincent Dupuis

Philippe Sainctavit
email: philippe.sainctavit@upmc.fr

Magnetic anisotropies and cationic distribution in CoFe 2 O 4 nanoparticles prepared by co-precipitation route: Influence of particle size and stoichiometry

Keywords:

Magnetic anisotropies and crystallographic structures of cobalt-iron nanospinels obtained with the co-precipitation synthesis process are revealed using X-ray Absorption Spectroscopy and X-ray Magnetic Circular Dichroism. The chemical process allows to obtain nanoparticles of various sizes and chemical composition, but it is the site symmetry environment of Co 2+ that is found to be the crucial parameter that governs the magnetic anisotropies of the nanospinels. The distribution of Co 2+ among the crystallographic sites of the structure is directly linked to the temperature of the synthesis process. In parallel, the results also revealed that a superficial rich shell of iron is formed for the iron-cobalt nanospinels stable in acidic medium leading to chemically inhomogeneous nanoparticles.

Introduction

Ferro-/ferrimagnetic materials present attractive properties for modern applications due to the magnetic anisotropies arising from the system. Even more enhanced magnetic properties, not shown in the bulk, appear in the nanoscale of ferro-/ferrimagnetic materials. Such enhanced magnetic properties associated to the nanoscale range make them good candidates for applications in the biomedical field or for magnetic devices [START_REF] Torres-Díaz | Recent progress in ferrofluids research: novel applications of magnetically controllable and tunable fluids[END_REF]. Approaches to use magnetic nanoparticles in biomedical applications rely on the large magnetization at saturation and on the large remanent magnetization of ferromagnetic materials that are mandatory for Magnetic Resonnance Imaging (MRI) [START_REF] Shokrollahi | Magnetic resonance imaging by using nano-magnetic particles[END_REF] while advances in anticancer treatment take the advantage on the specific loss power of magnetic nanoparticles at radiofrequencies [START_REF] Périgo | Fundamentals and advances in magnetic hyperthermia[END_REF]. In the field of magneto-optical devices, integrated optics [START_REF] Amata | Hybrid magneto-optical mode converter made with a magnetic nanoparticles-doped SiO 2 /ZrO 2 layer coated on an ion-exchanged glass waveguide[END_REF], core optical fibers [START_REF] Zou | Magnetic-fluid core optical fiber[END_REF] or tunable beam splitter [START_REF] Horng | Tunable optical switch using magnetic fluids[END_REF] incorporate magnetic nanoparticles for their tunable magneto-optical properties. For such applications, ferrites with a spinel structure MFe 2 O 4 (with M = Fe, Co, Mn, Ni, Zn…) present appealing properties [START_REF] Mathew | An overview of the structure and magnetism of spinel ferrite nanoparticles and their synthesis in microemulsions[END_REF]. Among them cobalt ferrite (CoFe 2 O 4 ) is a promising magnetic material for high-storage devices [START_REF] Frey | Magnetic nanoparticles: synthesis, functionalization, and applications in bioimaging and magnetic energy storage[END_REF] taking advantage of its large magneto-cristalline anisotropy energy [START_REF] Rondinone | Characterizing the magnetic anisotropy constant of spinel cobalt ferrite nanoparticles[END_REF], associated with a large coercive field H c , and a moderate magnetization at saturation M s . However the magnetic properties of CoFe 2 O 4 nanoparticles can be strongly influenced by the morphology, the size, the chemical composition and the crystallographic structure. Ultimately, processes used to synthesize nanoparticles can be adjusted to the requirement of the application. In particular, chemical processes offer to tune a quasi-infinite number of parameters (chemical agents, pH, temperature, atmosphere and so on). During the last decades, various preparation modes of magnetic nanoparticles in fluids have been adopted [START_REF] Lefebure | Monodisperse magnetic nanoparticles: preparation and dispersion in water and oils[END_REF][START_REF] Caruntu | Synthesis of variable-sized nanocrystals of Fe 3 O 4 with high surface reactivity[END_REF][START_REF] Abou-Hassan | Synthesis of cobalt ferrite nanoparticles in continuous-flow microreactors[END_REF][START_REF] Hyeon | Synthesis of highly crystalline and monodisperse maghemite nanocrystallites without a size-selection process[END_REF][START_REF] Pérez-Mirabet | One-pot synthesis of stable colloidal solutions of MFe 2 O 4 nanoparticles using oleylamine as solvent and stabilizer[END_REF]. Among them, the co-precipitation route [START_REF] Massart | Preparation of aqueous magnetic liquids in alkaline and acidic media[END_REF] developed by R. Massart is a highly reliable, very convenient and cost-effective process to obtain well-crystallized and long term stable spinel nanoparticles dispersed in aqueous media. It is already known that the acidity of the medium or Abbreviations: XMCD, .

the process temperature can influence the crystallographic order, the size, the shape and the stoichiometry of the nanocrystals, as well as the valence states of the cations [START_REF] Zhang | Composition and magnetic properties of cobalt ferrite nano-particles prepared by the co-precipitation method[END_REF][START_REF] Zhang | Magnetic behavior of nanocrystalline CoFe 2 O 4[END_REF].However, so far we know, it does not exist any systematic study linking the synthesis conditions of co-precipitation to the different magnetic properties of the nanospinels obtained. To obtain a clear picture of the relations existing between the co-precipitation synthesis conditions of iron-cobalt nanospinels and their magnetic properties, we have investigated six different sets of iron-cobalt nanospinels with different size and stoichiometry. The present study focuses on Co x Fe y O 4 nanoparticles with sizes ranging from 7

to 21 nm and with molar ratios X M equal to either 25% or to 33%. The cobalt molar ratio X M is defined as follow:

( Intermediate situations between direct and inverse spinels are characterized by a degree of inversion γ which is the fraction of divalent cations in octahedral sites. Hence, the cationic order of iron-cobalt spinels is described by Eq. ( 2) as:

(

The inversion degree illustrates the crystallographic disorder. In the case of cubic spinels structure, the crystallographic order is deeply related to the magnetic anisotropies arising from the system. The crystallographic order of the nanoparticles have been investigated in various studies where the different cations distribution among T d and O h sites can be associated to different synthetic processes. Depending on the preparation mode of CoFe 2 O 4 nanoparticles, inversion parameters from 0.20 to 0.96 have been obtained [START_REF] Peddis | Spin-glass-like freezing and enhanced magnetization in ultra-small CoFe 2 O 4 nanoparticles[END_REF][START_REF] Peddis | Cationic distribution and spin canting in CoFe 2 O 4 nanoparticles[END_REF][START_REF] Sawatzky | Cation Distributions in Octahedral and Tetrahedral Sites of the Ferrimagnetic Spinel CoFe 2 O 4[END_REF][START_REF] Sawatzky | Mössbauer Study of Several Ferrimagnetic Spinels[END_REF][START_REF] Murray | Linnett (the late), Mössbauer studies in the spinel system Co x Fe 3 -x O 4[END_REF]. In parallel, Peddis and co-workers also established the major role played by surface spin-canting in the magnetic properties of small nanoparticles [START_REF] Peddis | Spin-glass-like freezing and enhanced magnetization in ultra-small CoFe 2 O 4 nanoparticles[END_REF][START_REF] Peddis | Beyond the effect of particle size: influence of CoFe 2 O 4 nanoparticle arrangements on magnetic properties[END_REF]. Among the technics used to characterized the cationic distributions in the spinels structure, if Mssbauer provides good results, more recently the composition and structure of Co-doped maghemite γ-Fe 2 O 3 nanoparticles have been studied by Extended X-ray Absorption Fine Structure at the Fe and Co K-edge [START_REF] Carta | HRTEM/STEM, and X-ray absorption spectroscopy studies of co-substituted maghemite nanoparticles[END_REF] and X-ray Absorption Near-Edges Spectra was used to study nanoparticles with different sizes prepared in a polyol medium [START_REF] Artus | Size-dependent magnetic properties of CoFe 2 O 4 nanoparticles prepared in polyol[END_REF]. In the present study, we investigate iron-cobalt nanospinels with X-ray Absorption Spectroscopy (XAS) and X-ray Magnetic Circular Dichroism (XMCD). These element selective spectroscopies have already been applied to investigate magnetic and cationic structure of such system [START_REF] Li | Cobalt-ferrite nanoparticles: structure, cation distributions, and magnetic properties[END_REF][START_REF] Hochepied | X-ray absorption spectra and X-ray magnetic circular dichroism studies at Fe and Co L 2,3 edges of mixed cobalt-zinc ferrite nanoparticles: cationic repartition, magnetic structure and hysteresis cycles[END_REF][START_REF] Kita | X-ray magnetic circular dichroism in cobalt-iron spinels and electronic states of Co ions[END_REF][START_REF] Carta | X-ray absorption investigation of the formation of cobalt ferrite nanoparticles in an aerogel silica matrix[END_REF][START_REF] Henderson | Cation occupancies in Mg Co, Ni, Zn, Al ferrite spinels: a multi-element EXAFS study[END_REF][START_REF] Staniland | Controlled cobalt doping of magnetosomes in vivo[END_REF][START_REF] Coker | Probing the site occupancies of Co-, Ni-, and Mn-substituted biogenic magnetite using XAS and XMCD[END_REF][START_REF] Coker | Harnessing the extracellular bacterial production of nanoscale cobalt ferrite with exploitable magnetic properties[END_REF][START_REF] Subías | Investigation of pressure-induced magnetic transitions in Co x Fe 3 -xO 4 spinels[END_REF][START_REF] Kim | Nanomedicine for targeted drug delivery[END_REF][START_REF] Gyergyek | Influence of synthesis method on structural and magnetic properties of cobalt ferrite nanoparticles[END_REF][START_REF] Aquilanti | Cation distribution in Zn doped cobalt nanoferrites determined by X-ray absorption spectroscopy[END_REF][START_REF] Matzen | Restoration of bulk magnetic properties by strain engineering in epitaxial CoFe 2 O 4 (0 0 1) ultrathin films[END_REF][START_REF] Byrne | Controlled cobalt doping in biogenic magnetite nanoparticles[END_REF][START_REF] Schmitz-Antoniak | Electric in-plane polarization in multiferroic CoFe2O4/BaTiO3 nanocomposite tuned by magnetic fields[END_REF][START_REF] Abdulwahab | A one-pot synthesis of monodispersed iron cobalt oxide and iron manganese oxide nanoparticles from bimetallic pivalate clusters[END_REF][START_REF] Pearce | Direct determination of cation site occupancies in natural ferrite spinels by L2,3 X-ray absorption spectroscopy and X-ray magnetic circular dichroism[END_REF][START_REF] Erdem | CoFe 2 O 4 and CoFe 2 O 4 -SiO 2 nanoparticle thin films with perpendicular magnetic anisotropy for magnetic and magneto-optical applications[END_REF]. As local probes of the electronic and magnetic properties, XAS and XMCD measurements allow to solve the crystallographic structure and disentangle the magnetic signature of Co 2 + and Fe 3 + in CoFe 2 O 4 nanoparticles. In the following, we first describe the various coprecipitation synthesis routes that allowed to obtain the nanoparticles with variable sizes and X M ratios, and then the characterization methods are detailed. Magnetometry measurements and XAS and XMCD results are presented and show that the magnetic properties of the nanospinels are not simply given by their composition and their size but are also governed by the organization on the cationic sublattices.

Experimental methods

Synthesis

The Co-precipitation synthesis yields nano-clusters dispersed in aqueous media with sizes ranging between 5 and 25 nm and different stoichiometries. We investigate here synthesis routes benefiting from significant improvements and new added treatments. In the following, the samples are named CoFeX_Y where X is the value of the molar ratio X M and Y is the diameter in nm of the iron cobalt nanospinels. The list of the six nanospinels under study is reported in Table 1.

Co-precipitation route

The synthesis described in this paragraph leads to nanoparticles with a mean size of 9 nm and a molar ratio X M = 25%, named CoFe25_9. The process is detailed in 5 steps. Initially, precipitation of hydroxides were made at 100°C as described by Tourinho et al. [START_REF] Tourinho | Aqueous ferrofluids based on manganese and cobalt ferrites[END_REF]. We modified this one-step procedure into two steps further described (Step 1 and Step 2).

Step 1 -Hydroxides precipitation. The precipitation of Co(II) and Fe(III) hydroxides is carried out at room temperature. Sodium hydroxyde (4 mmol) solution is added to a mixture of Co(NO 3 ) 2 •6H 2 O (25 mmol) and Fe(NO 3 ) 3 •6H

2 O (3 mmol) dissolved in distilled water (80 mL) under stirring. The mixture was kept under vigorous stirring for 30 min and results in nonmagnetic amorphous suspension of hydroxides species.

Step 2 -Ferrite formation. The reaction medium is stirred and heated at 100°C for 2 h. During this step hydroxides transform into cobalt ferrites with sizes ranging between 8 nm and 13 nm [START_REF] Kim | Synthesis and characterization of CoFe 2 O 4 magnetic nanoparticles prepared by temperature-controlled coprecipitation method[END_REF].

Step 3 -Dispersion in acidic medium. To eliminate spurious non magnetic and amorphous hydroxydes, nitric acid (0.2 mmol, pH = 2) is added to the suspension. This step favor the dispersions of cobalt ferrites, as (-O -) anions initially at the surface after Step 1 are protonated and give ( ) species. Successive precipitations with acetone were used to remove the excess of acid. As cobalt ferrites are not necessary stable in acidic media, further treatments are used to prevent their degradation [START_REF] Soler | Aging investigation of cobalt ferrite nanoparticles in low ph magnetic fluid[END_REF].

Step 4-Stabilization. Following the Massart process, ferric nitrate is added and the solution is kept boiling during 30 min. The nitrate treatment allows the stable dispersion of cobalt ferrites nanoparticles in acidic medium but leads to the molar ratio X M = 25% [START_REF] Tourinho | Synthesis and magnetic properties of managanese and cobalt ferrite ferrofluids[END_REF].

Step 5 -Dispersion in aqueous solvent. The ferrites obtained are washed several times with acetone and ether and dispersed in the aqueous solvent, which yields a colloidal solutions of cobalt ferrites nanoparticles.

Steps 1, 2 and 4 are modified in order to produce, the five other nanospinels than CoFe25_9. A scheme that detailed the steps followed to obtain the six samples is given in Supporting Informations (Fig. S1).

Step 1′. In order to obtain smaller cobalt ferrites nanoparticles, hydroxides precipitation is performed in the presence of complexing species [START_REF] Bee | Synthesis of very fine maghemite particles[END_REF]. Step 1 is modified by adding tartrate ions to the mixture of Co(NO 3 ) 2 •6H 2 O and Fe(NO 3 ) 3 •6H 2 O before the addition of sodium hydroxide.

Step 1′ is used instead of Step 1 to obtain CoFe25_7 ferrofluid.

Step 2′. From the litterature, larger size of nanoparticles can be obtained with hydrothermal treatment [START_REF] Daou | Hydrothermal synthesis of monodisperse magnetite nanoparticles[END_REF]. After Step 1, the mixture is transferred into an autoclave reactor set at 200 °C for 1 h. Step 2′ is used instead of Step 2 to obtain CoFe33_13 ferrofluid.

Step 2″. The duration of the hydrothermal treatment influenced the size of the nanoparticles [START_REF] Cabuil | Ionic magnetic fluid based on cobalt ferrite nanoparticles: influence of hydrothermal treatment on the nanoparticle size[END_REF]. In Step 2″, the hydrothermal treatment at 200 °C is performed during 24 h in order to obtain larger particles. Step 2″ is used instead of Step 2 to obtain CoFe33_21 ferrofluid.

Step 4′. Chemical composition of X M = 33% can be obtain for nanoparticles further dispersed in acidic medium with a cobalt nitrate treatment [START_REF] Gomes | Synthesis of core-shell ferrite nanoparticles for ferrofluids: chemical and magnetic analysis[END_REF]. Step 4 is modified by adding a solution of Co(II) nitrate (3 mmol) and the mixture is boiled for 30 min. Step 4′ is used in a fraction of the synthesis batch of CoFe25_9 after Step 3 to obtain CoFe33_9.

Step 4″. To obtain a stable ferrofluid at neutral pH, hydroxyl groups at the surface of the particles are exchanged with ligands that ensure long-term stability of ferrofluids combining steric and electrostatic repulsions [START_REF] Bee | Synthesis of very fine maghemite particles[END_REF].

Characterization

Transmission electron microscopy

The morphology of the nanospinels for the six samples has been measured on a JEOL 100 CX2 Transmission Electron Microscope (TEM) (Electronic microscopy platform of the Fédération des Matériaux -UPMC). Fig. 1 shows TEM micrographs and size histograms. The TEM images reveal irregular spheroidal shape nanoparticles for the six samples. The analysis of the diameter of more than 2000 particles for each sample leads to size histograms fitted with a log-normal distribution. Results are reported in Table 1.

X-ray diffraction

The coherent domain size of the nanoparticles were determined with a PANALYTICAL X'pert Pro MPD diffractometer with the CoKα radiation (Kα = 1.79 Å) (See Supporting Information Fig. S2). All diffraction peaks can be indexed with the CoFe 2 O 4 spinel structure (reference code pattern 96-5941-0064). From the XRD profile of the (311) peak, one can compute the nanospinel size (d XRD ) with the Debye-Sherrer expression [START_REF] Scherrer | Bestimmung der Gröe und der inneren Struktur von Kolloidteilchen mittels Röntgenstrahlen[END_REF][START_REF] Holzwarth | The Scherrer equation versus the 'Debye-Scherrer equation[END_REF]. The mean d XRD sizes are reported in Table 1.

Atomic absorption spectrometry

The cationic concentrations of Cobalt and Iron are determined by Atomic Absorption Spectrometry after degradation of the nanoparticles in concentrated hydrochloric acid. We used a Perkin Elmer Analyst 100 spectrometer with an air-acetylene flame at a mean temperature of 2300°C. Experiments were repeated at least three times on each sample. The molar ratio X M is determined from the Fe and Co concentrations (Table 1).

SQUID magnetometry

Magnetometry measurements were recorded using either a Superconducting QUantum Interference Device (SQUID) Magnetometer (Quantum Design MPMS-XL7) or a Vibrating Sample Magnetometer (QuantumDesign PPMS) (Plate-forme de mesures physiques basse température -UPMC). Measurements are performed on dried powders of nanoparticles at 300 K and 4 K, and on liquid ferrofluids frozen at 4 K. The 4 K magnetization curves for frozen ferrofluids and for dried nanoparticles almost superimpose. The small discrepancies observed between the dried nanoparticles and the ferrofluids measurements show that inter-particles interactions can be neglected in nanoparticles solid phase compare to ferrofluids where colloids are far from each others. Table 1 reports the values of the magnetization at saturation (M S ) for powders nanoparticles at 300 K. Magnetization curves for frozen ferrofluids are plotted in Fig. 4.

X-ray magnetic circular dichroism

XMCD at L 2,3 edges is a powerful method to probe the 3d magnetic orbitals of transition metal ions. XMCD possesses the chemical and orbital selectivities of XAS and is sensitive to the site symmetry and the valence of the absorbing atom. XMCD is a magnetic technique that can mostly be explained in the electric dipole approximation so that it chiefly yields information on the orbital magnetic moment of the absorbing atom. When spin-orbit is large enough as it is the case for spin-orbit coupling acting on the 2p core-hole, one can also gather information on the spin magnetic moment. As a consequence, it is a unique tool to separate contributions from the different magnetic ions in a ferrimagnetic system such as Iron-Cobalt nanospinels. The silicon plates are screwed on a non-magnetic sample holder which is entered into the air-lock chamber and transfered into the DEIMOS cryomagnet [START_REF] Ohresser | DEIMOS: a beamline dedi cated to dichroism measurements in the 350-2500 eV energy range[END_REF]. Measurements are performed on dried magnetic nanoparticles. For XAS the isotropic absorption cross-section is taken as . This assumption is fully satisfied for powders of nanoparticles with a cubic crystallographic structure.

By following the variation XMCD intensity as a function of the external magnetic field, one can measure element-specific magnetization curves. One can select energies that correspond to specific spinel sites such as 707. 98 sites. Magnetization curves are measured by using the EMPHU-65 undulator which allows fast switching (5 Hz) of the X-ray circular helicity.

Results and discussion

XAS and XMCD spectra

In Fig. 2 we present the XAS and XMCD spectra of Fe and Co L 2,3 edges for sample CoFe33_9. The different features of the spectra are labelled to simplify the discussion and the labels are reported in Fig. 2.

As expected from the stoichiometry, we notice the absence of Fe for the discussion have labels beginning with "I" for the features of the isotropic spectrum edges and with "X" for the features of the XMCD signal.

ions [START_REF] Brice-Profeta | Magnetic order innanoparticles: a XMCD study[END_REF]. Iron ions thus exist only as fully oxidized Fe that have been performed following procedures detailed in previous papers [START_REF] Hochepied | X-ray absorption spectra and X-ray magnetic circular dichroism studies at Fe and Co L 2,3 edges of mixed cobalt-zinc ferrite nanoparticles: cationic repartition, magnetic structure and hysteresis cycles[END_REF][START_REF] Brice-Profeta | Magnetic order innanoparticles: a XMCD study[END_REF]. The calculated XAS and XMCD signals can be found in Supporting Informations (Figs. S8 andS9). Samples are grouped into three sets for the presentation and the discussion of the results. The four samples of the first set (CoFe33_9, CoFe33_11, CoFe33_13 and CoFe33_21) present a stoichiometric composition with a molar ratio X M = 33%. The second set (CoFe25_7, and CoFe25_9) groups samples with X M = 25% where vacancies are present on the O h sublattice. For the two first sets of samples, the cationic distribution is discussed as a function of size and synthesis route. The third set (CoFe33_9, CoFe25_9) groups the two samples with 9 nm size nanospinels and for which the cationic distribution is examined as a function of two different X M and two different amounts of vacancies in the O h sub-lattice.

First set -Influence of particle size for X M = 33%

The isotropic and XMCD spectra measured at the Fe L 2,3 edges for samples CoFe33_9, CoFe33_11, CoFe33_13 and CoFe33_21 are plotted in Fig. 3 ((A) and (B) panels). One observes that the ratio I1/I2 for the isotropic spectra and X1/X2 for the XMCD signals both increase when the particles size increases. These two variations can be straightforwardly interpreted as an increase of the occupation of the T d sites with respect to the occupation of the O h sites when the size of the nanospinels increases. Hence the larger the particles, the higher the fraction of Fe 3 + on the T d sites.

The Co spectra measured at the Co L 2,3 edges can be found in Fig. 3 (panels (B) and (C)). When the size of the nanospinels increase, one observes that peak I3 and the ratio I4/I5 increase. As a mirror effect of the I4/I5 variation, one observes a similar trend at the Co L 2 edge. From Fig. From SQUID magnetometric measurements reported in Fig. 4, one observes that the larger the nanoparticles, the larger the coercive field. Indeed, the coercive fields are equal to 0.7 Tesla for CoFe33_9 and CoFe33_11 particles, 0.9 Tesla for CoFe33_13 particles and 1.2 Tesla for CoFe33_21 particles. Same variation is obtained from the XMCD detected magnetization curves (See Supporting Information Fig. S5, see also Table 1). In iron cobalt nanospinels, the magnetic anisotropy mainly arises from the Co ions [START_REF] Slonczewski | Origin of magnetic anisotropy in cobalt-substituted magnetite[END_REF]. For the four different samples with X M = 33%, the proportion between iron and cobalt ions is constant so that an increase of coercivity cannot be attributed to an increase of Co ions concentration. The Co anisotropy depends on the sites it occupies. The origin of the coercivity for Co ions comes from a strong interplay between spin-orbit coupling and crystal field in O h symmetry. Indeed the electronic configuration for Co(II) ions is 3d T 1 which is split by first-order spin-orbit coupling and a large magnetic moment is found in the ground state [START_REF] Van Der Laan | Influence of crystal field on anisotropic x-ray magnetic linear dichroism at the Co 2 + L 2,3 edges[END_REF]. Moreover, O h Co(II) undergoes a strong Jahn-Teller distortion. In the spinel structure, the exact symmetry for the 6-fold site is not O h but D 3d so that Jahn-teller distortion is fully compatible with the crystal structure and the interplay between spin-orbit coupling and crystallographic distortion is then at the origin a large orbital magnetic moment and magnetocrystalline anisotropy for Co ions in distorted O h symmetry [START_REF] Tachiki | Origin of the magnetic anisotropy energy of cobalt ferrite[END_REF]". Many studies have reported discrepancies of the magnetocrystalline anisotropy observed between the bulk and the nanoparticles in terms of spin-canting [START_REF] Coey | Noncollinear spin arrangement in ultrafine ferrimagnetic crystallites[END_REF][START_REF] Tronc | Surface effects in noninteracting and interacting γ-Fe 2 O 3 nanoparticles[END_REF][START_REF] Vazquez-Vazquez | Finite size and surface effects on the magnetic properties of cobalt ferrite nanoparticles[END_REF]. Spin-canting arises from the subtle interplay between the chemical disorder, that modifies the superexchange interactions, and the formation of an external layer of non-collinear spin arrangement at the surface of the nanoparticles where the ion coordination symmetry is broken [START_REF] Peddis | Spin-glass-like freezing and enhanced magnetization in ultra-small CoFe 2 O 4 nanoparticles[END_REF][START_REF] Peddis | Cationic distribution and spin canting in CoFe 2 O 4 nanoparticles[END_REF][START_REF] Peddis | Spin-canting and magnetic anisotropy in ultrasmall CoFe 2 O[END_REF]. In the present case, we have measured magnetization curves by setting the monochromator at energies specific for Fe ions on O h and T d sites and for Co ions on O h and T d sites.

The magnetization curves for one specific sample clearly show the expected antiferromagnetic coupling between O h and T d sub-lattices (See Supporting Informations Fig. S4). However, the shape of the curves are exactly the same, so that is no indication for any specific magnetic disorder originating from surface defects. On the other hand, there is a clear indication that the synthesis route that imposes the particle size also induced a different repartition of Co ions between T d and O h sites.

From the isotropic and XMCD spectra, we know that when the particle size increases, the amount of Co ions in O h sites and the amount of Fe ions in T d sites both increase. Thus in the case of the present series of samples with X M = 33%, one can conclude that the increase in magnetic anisotropy is governed by larger concentration of Co ions in O h sites which can be illustrated by a larger inversion parameter γ. Numerical values of γ were calculated from the determination of the cations occupancy in LFM calculations fitted with XMCD experimental data(See the variation of γ in Supporting Informations Table 2). From the co-precipitation synthesis, the variation of γ with the size can be directly related to heat treatments. Heat treatments have already been reported as being at the origin of the cations distribution in CoFe 2 O 4 [START_REF] Sawatzky | Cation Distributions in Octahedral and Tetrahedral Sites of the Ferrimagnetic Spinel CoFe 2 O 4[END_REF][START_REF] Sawatzky | Mössbauer Study of Several Ferrimagnetic Spinels[END_REF][START_REF] Murray | Linnett (the late), Mössbauer studies in the spinel system Co x Fe 3 -x O 4[END_REF]. Kim et al. have also reported that temperature can increase the coercivity [START_REF] Kim | Synthesis and characterization of CoFe 2 O 4 magnetic nanoparticles prepared by temperature-controlled coprecipitation method[END_REF]. Here the largest γ is found for the CoFe33_21 which underwent heat treatment at higher temperature of 200°C for several hours. The hydrothermal treatment was already known to increase the size and the crystallinity of the grains. This step also improves the preferential distribution of Co 2 + ions in O h sites.

Second set -Influence of particle size for X M = 25%

CoFe25_7 and CoFe25_9 have been obtained from two rather different synthesis routes leading both to a partial loss of stoichiometry from pure CoFe 2 O 4 nanospinels. For the two samples CoFe25_9 and CoFe25_7 X M = 25% one has [Fe] = 3x [Co]. From the XAS spectra at the Fe L 2,3 edges one notices that the decrease of divalent cobalt ions is not accompanied by the appearence of divalent iron ions. The presence of vacancies on the O h sites is therefore expected as it is commonly observed in maghemitizated iron bearing spinels. If the nanospinels are homogeneous with a constant composition (X M ) within the whole particle, then the actual chemical formula is which correspond to a small fraction (1/11) of O h vacancies per formula unit.

In order to account for the degree of inversion, the formula for a sample with X M = 25% can be written

. It is to be stressed that if Fe 2 + ions had been present to compensate the small amount of cobalt, then the stoichiometry formula would have been much different.

But in the absence of Fe 2 + , the chemical composition of cobalt iron nanospinels with X M = 25% differs from only 1/11 of vacancies to the ones showing X M = 33%.

At the Fe L 2,3 edges, XAS spectra of CoFe25_7 and CoFe25_9 are almost similar with a slight difference in the ratio of I1/I2 peaks (Fig. 5). XMCD signals for CoFe25_7 and CoFe25_9 particles have also much similar X3/X4 ratios (Fig. 5) indicating that the distribution of iron ions between the T d and O h sites is very similar for both samples. This is slightly different from what has been observed for the samples with X M = 33%

, where a size increase was associated to an increase of Fe ions in the sites. On the other hand, one sees that the larger the proportion of Co ions in O h sites, the larger the coercive field and this conclusion is fully satisfied for both X M = 33% and X M = 25% series (Fig. 4).

T

Influence of the cationic molar ratio X M

From the two previous series X M = 33% and X M = 25%, it is clear that the knowledge of the size of the nanoparticles does not give non ambiguous clues concerning the expected magnetic anisotropy or the coercive field. One important physical parameter that scales with the magnetic anisotropy is the content of Co ions in the O h sites. It then appears interesting to compare the magnetic properties of the two samples with the same size (9 nm) but belonging to the two different series X M = 33% and X M = 25%. Sample CoFe25_9 is obtained from the same synthesis batch as CoFe33_9 except that an excess of Fe(III) nitrate has been added in the case of CoFe25_9 while Co(II) nitrate were used in the synthesis of CoFe33_9 (see Supporting information Fig. S1).

Following the analysis developed for the X M = 33% series, one notices trates would induce the growth of a small shell of γ-Fe 2 O 3 or oxy-hydroxydes in the core of CoFe 2 O 4 that would contribute to lower the X M value [START_REF] Gomes | Synthesis of core-shell ferrite nanoparticles for ferrofluids: chemical and magnetic analysis[END_REF]. The stoichiometry of the core would be then X M = 33% with a distribution of the Co ions in O h sites that is similar to the one observed for CoFe33_9. Another evidence of a core@shell structure for CoFe25_9 nanospinels is brought by calculated sites occupancy performed in the LFM theory (See Table 2 in Supporting Informations). Calculations were performed taking into account the formula for CoFe25 samples while cationic distributions in CoFe33 samples are obtained using the formula O 4 . Cationic distributions calculated are consistent with coercive fields measured in XMCD detected magnetic (see Supporting Informations Table 2) for all samples except for the CoFe25_9 sample. For the CoFe25_9 nanospinels one obtains a smaller amount of Co 2 + in O h sites than for the CoFe33_9 while they show identical coercive fields. One can explain these discrepancies assuming a inhomogeneous chemical composition inside CoFe25_9 nanospinels. In that case, the formula O 4 cannot be applied for CoFe25_9 as for CoFe25_7 nanospinels which exhibit a steady chemical composition X M = 25% in a whole nanospinel.

The comparison of the magnetization curves either measured by SQUID or at the Fe or the Co L 2,3 edges show that the magnetic properties for the two samples are almost identical. Coercive fields and remanent magnetization are the same with similar general shapes. The main conclusion that can be drawn from the comparison between CoFe33_9 and CoFe25_9 is that when the proportion of Co ions in O h sites is constant then the magnetization curves are almost identical. This observation further confirms the hypothesis concerning the core@shell structure of sample CoFe25_9. Indeed if the core for CoFe25_9 is similar to CoFe33_9 with a surrounding γ-Fe 2 O 3 shell, one would indeed expect that both samples have very similar magnetic properties despite different apparent stoichiometry. On the contrary CoFe25_7 is obtained by blocking the growth of the nanoparticles by adjunction of complexing species agent (tartrate ions). For these very small particles, one would observe a partial, non-congruent dissolution of Co and Fe so that the initial X M = 33% stoichiometry is reduced to X M = 25%. The complexation for this very small particles coupled to a preferential dissolution T d Co ions would then lead to a larger concentration of Co ions in O h sites.

From a simple geometric estimation, one determines that a surrounding γ-Fe 2 O 3 shell of ≈0.5 nm accounts for the change of stoichiometry.

Conclusion

Magnetic solutions of CoFe

2 O 4 nanoparticles with different sizes are obtained from the versatile coprecipitation process. Playing with the temperature treatments or with the adjunction of tartrate ions, Fe nitrates or Co nitrates, nanoparticle sizes are tuned between 7 nm and 21 nm. For most syntheses, the expected stoichiometry of 1 Co for 2 Fe ions are obtained, and in two cases the stoichiometry is partially lowered to 1 Co ion for 2 Fe ions. The main outcome of the present study is that neither the size nor the stoichiometry can determine the magnetic properties such as the coercive field nor the remanent magnetization. XMCD allowed the determination of the proportion of Co ions in O h sites and that this is the parameter governing magnetic coercivity.

In addition, the comparison of the data for both Fe and Co L 2,3 edges showed that some of the particles were very likely built from a core with high Co concentration surrounded by an iron rich shell (probably of γ-Fe 2 O 3 . Such study could be extend to investigate the intimate relations existing between the magnetic properties and crystallographic structures of magnetic nanoparticles obtain with others synthesis processes. Inferences to be drawn would helped chemists for the development of rationalized synthesis.

  Step 4 is modified by adding citrate trisodium salt (3 mmol) to the sol. During citratation the hydroxyl groups at the surface of the particles are replaced by citrate anions. The particles ob tained by Step 4″ were dispersed in distilled water in Step 5. Step 4″ is used instead of Step 4 to obtain CoFe25_7, CoFe33_11, CoFe33_13 and CoFe33_21.

Fig. 1 .

 1 Fig. 1. TEM images and size distribution histograms of (A) 7 nm non-stoichiometric nanoparticles CoFe25_7, (B) 9 nm non-stoichiometric nanoparticles CoFe25_9, (C) 9 nm stoichiometric nanoparticles CoFe33_9, (D) 11 nm stoichiometric nanoparticles CoFe33_11, (E) 13 nm stoichiometric nanoparticles CoFe33_13, (F) 21 nm stoichiometric nanoparticles CoFe33_21.

  XAS and XMCD signals for both Fe and Co L 2,3 edges were recorded on the DEIMOS beamline at the French synchrotron, SOLEIL [55]. All spectra were measured in Total Electron Yield (TEY) at 4.2 K and in UHV conditions (10 -10 mbar). In the presence of an external magnetic field H + (respectively H -) parallel (respectively antiparallel) to the X-ray propagation vector, XMCD signals are obtained by taking the difference σ XMCD = σ --σ + where and , and σ L (respectively σ R ) is the absorption cross-section measured with left (respectively right) circularly polarized X-rays. The XMCD signals were recorded by flipping both the circular polarization (either left or right helicity) and the external magnetic field (either +6.6 Tesla or -6.6 Tesla). The circularly polarized X-rays are provided by an Apple-II HU-52 helical undulator for both XAS and XMCD measurements. Ferrofluids are drop-casted on silicon plates and dried at room temperature.

2 +Fig. 2 .

 22 Fig. 2. XAS and XMCD spectra at the Fe (left side) and Co (right side) L 2,3 edges for the CoFe33_9 sample measured at 4 K with an applied field of ±6.6 Tesla. The different peaks relevant

3 ,

 3 one notices that the intensity of the XMCD feature X3 increases while X4 decreases when the size of nanospinels increases. All these variations indicate that the proportion of Co ions in O h site increases for the larger particle sizes. Hence, the increase of cobalt ions occupancy in O h sites coincides with the increase of the Fe 3 + proportion in T d sites when the size of nanospinels increases.

Fig. 3 .

 3 Fig. 3. Isotropic XAS and XMCD spectra at the Fe L 2,3 edges ((A) and (B)) and Co L 2,3 edges ((C) and (D)) for CoFe33_9, CoFe33_11, CoFe33_13, CoFe33_21 samples at 4 K. XMCD spectra are plotted in percent of the maximum of the XAS spectra. Inset: zoom in on the L 3 edges of the XMCD spectra.

Fig. 4 .

 4 Fig. 4. Magnetization curve versus field measurement of ferrofluids measured at 4 K of: CoFe33_9, CoFe33_11, CoFe33_13 and CoFe33_21 (A), CoFe25_9 and CoFe25_7 (B), CoFe25_9 and CoFe33_9 (C).

  d sites. A smaller dichroic amplitude for CoFe25_9 sample compared to CoFe25_7 sample indicates a slightly smaller magnetization. Despite very similar XMCD signals at the Fe L 2,3 edges, a major difference in the XMCD detected magnetization curve is observed (Fig. 5). The smaller CoFe25_7 particles present a slightly larger coercive field and a much smaller remanent magnetization accompanied by a slanted shape for high fields. The Co L 2,3 edges spectra of the CoFe25_7 and CoFe25_9 samples are presented in Fig. 5 panels (C) and (D). At the difference of what has been observed at the Fe L 2,3 edges, the two spectra present a series of differences that can be readily analyzed as it has been done for the X M = 33% series. A sharper I3 peak, a larger ratio of I4/I5 and the shape of the L 2 edge (see Fig. S8 in Supporting Informations) indicate that the CoFe25_7 sample contains a larger amount of Co ions in the O h sites than the CoFe25_9 sample. This trend is fully supported by the XMCD signals at the Co L 2,3 edges which present a smaller X3/X4 ratio for CoFe25_7 sample compare to CoFe25_9 sample (see Fig. 5). Contrary to what has been observed for the X M = 33% series, one obtains that the larger the particles the smaller the proportion of Co ions on the O h

Fig. 5 .

 5 Fig. 5. Isotropic XAS and XMCD spectra at the Fe L 2,3 edges ((A) and (B)) and Co L 2,3 edges ((C) and (D)) for CoFe25_7 and CoFe25_9 samples at 4 K.

Fig. 6 .

 6 Fig. 6. Isotropic XAS and XMCD spectra at the Fe L 2,3 edges ((A) and (B)) and Co L 2,3 edges ((C) and (D)) for CoFe33_9 and CoFe25_9 samples at 4 K.

Table 1

 1 Physicochemical characteristics of the magnetic iron-cobalt nanospinels. d 0 and σ are the mean diameter and the polydispersity deduced from TEM micrographs,d DRX the particle diameter measured from patterns, X M the molar ratio and M S the saturation magnetization measured at 300 K.

	Sample	d 0 (nm)	σ	d XRD (nm)	X M (%)	2 • M S (A•m kg -1 )
	CoFe25_7 CoFe25_9 CoFe33_9 CoFe33_11 CoFe33_13 CoFe33_21	6.8 8.9 8.6 10.7 13.3 20.9	0.4 0.5 0.5 0.4 0.4 0.5	5.4 10.6 10.5 12.8 13.0 18.0	26.9 26.4 32.7 35.3 35.5 34.2	65.8 72.8 67.8 72.5 76.2 76.1

  eV for Fe ions in T d sites, 708.72 eV for Fe ions in O h sites, 776.56 eV for Co ions in T d sites and 777.55 eV for Co ions in O h

  3 + ions distributed between T d and the O h sites. It is well known from literature that the X1 peak at 707.98 eV is the signature of Fe 3 + in T d sites whereas the X2 peak at 708.72 eV is the signature of Fe 3 + in O h sites [57]. The corresponding signals have opposite signs which reveal the antiferromagnetic coupling between the T d sub-lattice and the O h one. The All XAS spectra are normalized to 1 at the maximum of the L 3 edge and the XMCD spectra are plotted with respect to the maximum of the isotropic XAS signal. The assignment of the spectral features for Fe and Co L 2,3 edges is confirmed by Ligand Field Multiplet calculations

	Co L 2,3 edges spectra are typical for an iron cobalt spinel containing Co 2 + ions in both T d and O h sites [34]. In the Co 2 + XMCD spectrum the X3 peak at 776.56 eV corresponds mainly to the T d sites (where a small fraction of this positive contribution arises from the signal of the O h sites -See Fig. S8 in Supporting Informations) while the X4 peak at 777.54 eV corresponds to the contribution of Co 2 + in O h symme-try.

  When the Co(II) ion is in a T d site, its configuration is in the ground state where the e orbitals are at lower energy than the t 2 ones.

	The corresponding spectroscopic term is

7

.

[START_REF] Amata | Hybrid magneto-optical mode converter made with a magnetic nanoparticles-doped SiO 2 /ZrO 2 layer coated on an ion-exchanged glass waveguide[END_REF]

A 2 with no orbital degeneracy thus no orbital moment is expected. On the opposite, when the Co(II) ion is in a O h site, its configuration is in the ground state where the t 2g orbitals are at lower energy than the e g ones. The corre sponding spectroscopic term is 4

nanoparticles, J. Phys. Chem. B 112 (2008) 8507-8513.

Acknowledgments

This work was supported by French state funds management by the ANR within the Investissements d'Avenir programm under reference ANR-11-IDEX-004-02, and more specifically within the framework of the Cluster of Excellence MATISSE. The authors are very grateful to SOLEIL and the DEIMOS beamline for the good measurement conditions. We express our gratitude to Aude Michel for Atomic Absorption Spectroscopy characterizations and Ludovic Delbes for its technical support on the X-ray Diffractometer.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, athttps://doi.org/10.1016/j.jmmm.2018.03.041.