Synthesis of CoâOrganosilaneâAu Nanocomposites via a Controlled

Article pubs.acs.org/cm

Synthesis of Co−Organosilane−Au Nanocomposites via a Controlled Interphasic Reduction Isaac Ojea-Jiménez,*,† Julia Lorenzo,‡ José M. Rebled,§,# Judith Sendra,∥ Jordi Arbiol,§,⊥ and Victor Puntes*,†,⊥ †

Institut Català de Nanotecnologia (ICN), Campus UAB, 08193 Cerdanyola del Vallès, Spain Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i de Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain § Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, 08193 Cerdanyola del Vallès, Spain # LENS-MIND/IN2UB, Dept. d'Electrònica, Universitat de Barcelona, 08028, Barcelona, Spain ∥ Endor Nanotechnologies, S.L., Barcelona Science Park (PCB), 08028 Barcelona, Spain ⊥ Institució Catalana de Recerca i Estudis Avançats (ICREA), 08093 Barcelona, Spain ‡

S Supporting Information *

ABSTRACT: Au-coated magnetic-core nanocomposites with diameters of ∼100 nm were synthesized by the biphasic reduction of Au3+ onto the surface of organosilane-stabilized Co nanoparticles of ∼12 nm in diameter via an iterative gold reduction. The shell consists initially of Au seeds of ∼3 nm in diameter, which can be later grown to form partially fused ∼6 nm Au NPs. A detailed investigation by transmission electron microscopy, scanning electron microscopy, UV−vis, dynamic light scattering, ζ-potential, X-ray diffraction, inductively coupled plasma mass spectrometry, and superconducting quantum interference device magnetometry was performed in order to elucidate the morphology and properties of these nanocomposites. These novel nanoarchitectures, which could be transferred into aqueous phase by means of an amphiphilic polymer, showed low cytotoxicity on human HepG2 cells and presented relaxivity in MRI contrast enhancement experiments. KEYWORDS: cobalt nanoparticles, hybrid nanocomposites, biphasic reduction, gold coating, phase-transfer, contrast enhancement

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INTRODUCTION

Gold represents an excellent model because of its easy reductive preparation, high chemical stability, biocompatibility, and its well established surface chemistry, which allows easy functionalization via amine/thiol terminal groups of organic compounds, such as in the case of bioactive molecules.8 The Au coating additionally provides optical properties to virtually any type of nanoparticle,9 with an aim toward imaging applications and photothermal ablation. Different routes for the synthesis of gold-coated magnetic particles have been reported including laser ablation,10 sonochemical reaction,11 layer-by-layer electrochemical deposition,12 chemical reduction,13−16 galvanic replacement,17,18 and reverse micelle methods.19 However, because of the poor wettability and high immiscibility of Au in the 3D-ferromagnetic metals, the majority of them frequently report a nonuniform coating of gold layers,13,20 which unavoidably results into a significant polydispersity and/or instability of the product. The direct coverage of pure metal magnetic particles with gold is a difficult task since, in addition to the dissimilar nature of the two surfaces, the higher

The interest of magnetic NPs is continuously growing in biotechnology due to their numerous applications in biomagnetic separation,1 magnetic biosensing,2,3 magnetic resonance imaging (MRI),4,5 and hyperthermia treatment,6,7 among others. In fact, MagForce AG and Guerbet Group have already been used in the clinic as therapeutic and diagnostic tools, respectively, based on magnetic iron oxide particles. The extent of biomedical applicability of these particles depends strongly upon their stability and/or dispersibility in solutions of physiological pH and electrolytic concentration as well as the degree to which their surfaces may be chemically functionalized. At room temperature, pure metal magnetic nanoparticles (Fe, Co, Ni, and their alloys) experience a high instability toward oxidation, which increases as the size gets smaller. Besides, magnetic metal oxides, such as ferrites, despite their advanced use, have lower magnetic moments than their metallic parents. As a consequence of this, hybrid nanostructures composed of a magnetic nanoparticle core and a gold shell are greatly being pursued for biological applications due to the combination of the physical nature of the core and the protecting effect of the shell. © 2012 American Chemical Society

Received: March 8, 2012 Revised: September 25, 2012 Published: October 1, 2012 4019

dx.doi.org/10.1021/cm300757j | Chem. Mater. 2012, 24, 4019−4027

Chemistry of Materials

Article

Scheme 1. Schematic Representation of the Synthesis of Co/Au Nanocomposites, Which Involves Functionalization of Co NPs with APTMS Followed by Hydrolysis/Condensation of Organosilane Molecules and Biphasic Reduction of Au+3 Precursor onto the Surface of the Resulting Organosilane

Figure 1. Representative TEM images of first generation (A) and second generation (C) Co/Au nanocomposites, and SEM secondary electron (SE) images of the first generation nanocomposite obtained with an InLens detector (B). UV−vis absorption spectra of as-synthesized Co NPs and Co/ Au nanocomposites (D).

reduction potential of Au3+ results in the core being sacrificed as the reducing agent for the noble metal deposition.18 Recently, some research groups have revealed the possibility of separating both functionalities by the insertion of an organic mediated linker in such a way that interactions between both metals are avoided. This is the case of alkoxysilanes,21−25 polyelectrolytes,26,27 or 3-aminopropylphosphonic acid,20 which were selected because of their strong dual affinity toward both magnetic and Au surfaces. Most of these methods are based on the attractive electrostatic interaction of presynthesized Au seeds with the charged organic linkers making use of water-based protocols under controlled pH conditions. Alternatively, in this article, we describe the remarkable process in which Au seeds were directly formed

in situ on the surface of aminosilane-functionalized Co NPs in an organic phase, which produced 3D spherical assemblies of approximately 100 nm. The seeds were then grown by a second reduction of Au3+ precursor to form a shell consisting of partially fused Au NPs. The morphology and properties of these materials were exhaustively analyzed by a wide range of characterization methods, among them transmission electron microscopy (TEM) techniques such as scanning (S)-TEM in high angular annular dark field (HAADF) mode, electron tomography, electron energy loss spectroscopy (EELS), and energy-dispersive X-ray spectroscopy (EDX). Finally, we demonstrated the biological applicability of these Co/Au nanocomposites first upon transference into aqueous phase employing an amphiphilic polymer and then by evaluating their 4020



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Stö ber synthesis also makes use of APTMS in basic environments, such as ammonia in ethanol and water, to form silica NPs.35 In our control experiments, no reaction occurred for solutions containing only APTMS-Co NPs and NaBH4 nor reduction of the HAuCl4 was achieved in the presence of APTMS-Co NPs without added NaBH4. These control reactions suggested that the polymerization of silanol groups occurred at the interphase, catalyzed by the presence of gold, which simultaneously got reduced, and favored by the basic pH conditions of the aqueous borohydride phase. Additionally, in the absence of Co NPs, the reaction did not lead to nanoparticle formation, which confirms the need of at least one Co nanocrystal as a platform to create these nanocomposites. Moreover, the presence of either single or multiple magnetic cores due to the aggregation of Co NPs prior to or during the coating process is also possible.27 From inductively coupled plasma mass spectrometry (ICP-MS), TEM, and image analysis, it could be estimated that each Co/Au nanocomposite contained an average of 3× Co NPs.36 The particles could be purified by centrifugation/resuspension cycles and were fully stable for years. The stability of the Co/ Au nanocomposites was investigated by monitoring the Cocontent in DCB solvent by ICP-MS. After 1 year, no significant increase of Co could be observed in solution, which corroborates the stability of the particles. On the basis of dynamic light scattering (DLS), the measured hydrodynamic diameter in volume size of the APTMS-coated Co NPs in DCB was 43.5 nm (0.17 Pdl), what could be explained by either the presence of multilayers of organosilane molecules29 or dimers of Co NPs bridged by these ligands. The mean volume size of the resulting Co/Au nanocomposites measured by DLS in DCB solution was 62.8 nm (0.08 Pdl), which contrasts with the higher size measured by TEM (101 ± 17 nm) in the dry state. Such a small hydrodynamic radius measured appears as limitation of the DLS technique itself when measuring hybrid materials of different chemical compositions with very disparate refractive indexes. In DLS, the speed at which the particles are diffusing due to Brownian motion is measured by the rate at which the intensity of the scattered light fluctuates. The diameter that is obtained by this technique is the diameter of a compact sphere of gold that has the same translational diffusion coefficient as the particle. As gold is not homogeneously distributed around the surface of the Co/Au nanocomposites, the intensity of the scattered light will be lower as expected for a compact sphere gold of ∼100 nm. SEM images further revealed the surface morphology of the Co/Au nanocomposites and an average particle diameter similar to TEM analysis (Figure 1B). The presence of Co, Si, and Au was confirmed by EDX, even though their relative intensities could only be taken as estimative values due to the limited resolution of the instrument (Supporting Information, Figure S2). The formation of a solid Au-shell onto the magnetic silanized spheres would offer the possibility of engineering them with optical properties to provide imaging and photothermal ablation applicability. To this aim, the growth of initial Au seeds on the first generation Co/Au nanocomposites was carried out by the addition of additional reducing aliquots of Au+3-precursor. This method comprising an initial nanoparticulate Au-shell has the advantage of simplifying the process of completing the formation of the solid shell because gold only has to fill the voids between the NPs in the second step.27 The reaction was performed in a similar way than in the

cytotoxicity on human hepatocellular carcinoma cell line HepG2 and their potential use as imaging contrast agents. Although poor relaxivity values were obtained, these findings offer new opportunities for imaging as in tumor early staging of the liver and lymph nodes: large biocompatible particles are rapidly recognized and phagocytized by monocytes without producing inflammation, what can be used for imaging atheromas and the lymphatic system.

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RESULTS AND DISCUSSION Synthesis of Co/Au Nanocomposites. Highly crystalline Co NPs were prepared by thermal decomposition of Co2(CO)8 in the presence of oleic acid and TOPO as surfactants and using DCB as solvent, which resulted in monodispersed particles of approximately 12 nm in diameter (Supporting Information, Figure S1A).28 The surface of as-synthesized Co NPs was then functionalized with an organosilane by first stirring the oleic acid-protected Co NPs with APTMS for 12 h, followed by cleaning the particles by centrifugation/redispersion. TEM analysis revealed that most of the Co NPs were surrounded by a thick layer of organic material (Supporting Information, Figure S1B), which suggested a replacement of the original oleic acid coating. Several cases of organosilane coating of ferrite magnetic nanoparticles have been described in the literature.23−25,29,30 Among them, some authors have demonstrated the successful replacement of oleic acid by trialkoxysilane ligands employing organic solvents, such as in the case of Fe3O4 NPs21 and CoFe2O4 NPs.29 The high affinity of silanol groups to Co/CoO surfaces leaves the terminal-NH2 groups of APTMS molecules available for Au deposition in the organic phase (Scheme 1). The formation of Au seeds on top of the APTMS-coated Co NPs was achieved via modification of the two-phase liquid−liquid system developed by Brust and Schiffrin,31 in which AuCl4− was transferred from the aqueous phase into a toluene solution employing a phase-transfer agent, TOAB, and then reduced at the interphase with an aqueous NaBH4 solution. The competition between nucleating new Au NPs and growing of the primary Au seeds was controlled by a combination of solution temperature, concentration of reagents, and type of capping agent. During the reaction, we assumed that those Co NPs that were not completely coated by APTMS became rapidly oxidized and disintegrated32 since metallic Co can also act as a sacrificial reducing agent in the presence of Au3+precursor in a galvanic replacement reaction.17,18 By TEM, the resulting Co/Au nanocomposites appear as fairly spherical and relatively monodispersed NPs of 101 ± 17 nm in diameter (SD ≈ 17%), which were composed of small clusters of Au NPs (Figure 1A). The large sizes obtained contrast with what would be expected for a structure composed of a Co-core (∼12 nm), a monolayer of APTMS molecules (0.5−2 nm),33 and a coating of Au NPs (3−4 nm). This experimental evidence suggested that the structure of the resulting Co/Au nanocomposites was indeed composed of a multilayer of APTMS molecules, which hydrolyzed and condensed on top of the Co NP surfaces (Scheme 1). Through a hydrolysis reaction, the methoxy groups (−OMe) of APTMS can be replaced by hydroxyl groups (−OH) to form reactive silanol moieties, which condense with other silanol groups to produce siloxane bonds (Si−O−Si). A few examples can be found in the literature,32,34 where silanization of Co NPs was performed employing trialkoxysilanes, among them APTMS, in the presence of NaBH4. Besides, the elaborated recipe of the 4021



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Figure 2. HRTEM images of first generation (A,B) and second generation (C,D) Co/Au nanocomposites, showing the fringes of crystal planes for Au domains with the corresponding lattice-resolved image of a section.

Figure 3. HAADF STEM (Z-contrast) general view of the 1st generation of Co/Au nanocomposite (A). Ortho slice of the Z-axis obtained from the 3D reconstruction model (B). EELS spectra obtained at red and green points highlighted in A. Au signal is found everywhere on the nanocomposite (red) whereas Co signal is found at its center (green) (C). A 3D movie showing the reconstructed tomography can be found elsewhere.38

previous step, but employing 4-fold lesser amounts of Au3+precursor. TEM analysis confirmed that the process led to increased size of the Au NPs from approximately 3−4 to 6−7

nm, while maintaining a similar size distribution of the spheres (Figure 1C) but left unclear whether a dense surface coverage was far from being obtained. In addition, free Au NPs could 4022



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weight, depending upon the region from which the spectrum was obtained (Supporting Information, Figure S4). Crystal Structure and Atomic Composition. In the powder X-ray diffraction (XRD) spectrum (Figure 4A), the as-

also be observed in solution, which could be well-separated by centrifugation. The resulting roughness of these second generation Co/Au nanoparticles could be an attractive property to investigate further applications as surface enhanced raman spectroscopy (SERS) surfaces. These second generation of Co/ Au nanocomposites are stable in toluene solution, but at this point, attempts to continuing the increase of the shell thickness just by adding more Au3+-precursor resulted in lower colloidal stability and aggregation of the particles. Measurements of the surface plasmon (SP) resonance band provided further evidence of the formation of Co/Au nanocomposites. In contrast to the largely silent feature in the visible region for as-synthesized Co NPs, the Co/Au nanocomposites show a SP band characteristic of the unique optical property of gold nanostructure (Figure 1D). After the first reduction step, when the small domains of Au are formed, the SP band is very weak, which was explained by the low concentration of Au NPs in the composite material as well as their small size.37 As the Au seeds grow, there is an increase in absorbance, and the coalescence between neighboring Au NPs induces a broadening and red-shift of the SP peak (531 nm) due to a collective intraparticle interaction of the electrons of the interconnected grains at the NP surface. Structural Characterization by HRTEM and STEM. Analysis by HRTEM of the atomic planes of Au in the first generation of Co/Au nanocomposites revealed that the monocrystalline Au seeds have a different orientation from particle to particle and exhibit lattice fringes with a spacing of 2.35 Å, corresponding to the {111} planes of the fcc phase from pure Au NPs (Figure 2A,B). By contrast, in the second generation, the Au NPs have multiple lattice fringe distortions and dislocations typical for polycrystalline structures (Figure 2C,D), which agrees with the fact that the newly formed Au domains and the Au seeds have different growing histories. As can be seen in Figure 3A, the STEM image taken under HAADF mode confirms the distribution of the Au nanoparticles, corresponding to the brightest spots. However, it was difficult to ascertain whether the particles were just in the surface or in the interior of the composites too. A series of Bright Field-STEM vs HAADF-STEM mode micrographs were taken at different stages for the growth of the Au domains (Supporting Information, Figure S3). In order to obtain the 3D spatial distribution of the Au particles within the first generation of Co/Au nanocomposites, we acquired TEM and HAADF STEM tilt series to reconstruct tomography models.38 Although the Au seeds were mainly distributed around the surface of the sphere, the formation of Au seeds inside the organosilane matrix was also possible, as shown in the orthoslice cut obtained from the middle of the reconstructed model (Figure 3B). Unfortunately, it was found difficult to localize the Co NPs due to the reduced contrast of Co in comparison to the Au atoms. Further analysis of the EELS spectra obtained on different points over the first generation Co/Au nanocomposite revealed the presence of Co traces in the inner points of the sphere, together with signals corresponding to the rest of expected elements such as Au, O, and Si (Figure 3A,C). Additionally, the composition of the particles was investigated by microanalysis using the EDX detector of the TEM microscope, which also confirmed the presence of both Co and Au in the particles. The relative intensities of both elements indicated that the particles were largely constituted of Au and that Co was only present in approximately 5−10% of atomic

Figure 4. (A) XRD pattern of as-synthesized Co NPs and first and second generation Co/Au nanocomposites. The triangles represent reference values of ε-cobalt, and the cubic Au (Fm3m) values are indicated with circles. (B) Magnetization measurements of the first generation Co/Au nanocomposites as a function of applied field. Inset shows a detail of the hysteresis loop. Plots are normalized to the mass of Co.

synthesized Co NPs showed no distinct peak corresponding to CoO, indicating absence of oxidation in the starting singlephase ε-cobalt.39 All the observed peaks in the samples corresponding to the Co/Au nanocomposites can be indexed as fcc Au with cell parameters a = b = c = 4.0699 Å and space group Fm3m (225) (JCPDS card no. 98-000-0230). The enhanced scattering and heavy atom effect of Au made difficult the determination of the Co peaks and thus the chemical composition of the Co-containing core by XRD. This observation is consistent with a dense coverage of the Co core in the Co/Au nanocomposite structures and has been previously reported in the literature.10,15,20 Some of these studies have determined that Au coatings above ∼2.5 nm thickness are sufficient to completely dominate the XRD pattern.16 Further analyses by ICP-MS technique of the metal composition (Co and Au) of the nanocomposites provided additional important findings. Although both Co and Au were detected in the two types of particles, the first generation Co/ Au nanocomposites presented a higher percentage of Co (Co/ Au ratio 34:66). This observed ratio was of the same order as the EDX analyses performed with SEM (Co/Au ratio 40:60) and TEM techniques (Co/Au ratio 37:63) (Supporting Information, Figures S2 and S4). By contrast, the second generation nanocomposite presented a Co/Au ratio of 3:97, which is consistent with the growth of the Au domains in the second step. Magnetic Measurements. Magnetic properties of the first generation Co/Au nanocomposites have been studied by hysteresis loops at different temperatures and zero-field cooled and field cooled (ZFC/FC) curves. All the magnetization measurements were performed with noninteracting particles embedded in a paraffin matrix and were systematically normalized with respect to the calculated mass of Co, estimated by extrapolation from the Co mass fraction measured by ICPMS (∼230 μg). At low temperatures, the width of the hysteresis loop gives information of the magnetic anisotropy and the barrier for magnetization reversal (Figure 4B). The small coercive field (Hc) of 32 Oe at 5 K is symmetric with respect to the x and y axes and has a superparamagnetic component. The symmetry of the hysteresis loop was preserved after field cooling indicating the absence of exchange bias.40 At 50 kOe, 4023



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Figure 5. Cytotoxicity of aqueous first generation Co/Au nanocomposites in human hepatocellular carcinoma (HepG2) cells after 24 h as determined by (A) XTT-cell viability test. Data represent the average of four replicate wells with standard deviation. (B) Trypan blue test analyzed by comparative optical microscope images. Blue color determines dead cells.

approximately 2, 10, and 20 nM of particles. Further toxicity assessment was also performed by a standard Trypan blue exclusion assay after 24 h of incubation with Co/Au nanocomposites at the same concentration of particles (Figure 5B). Comparative optical microscope images show that cells were alive, as indicated by the absence of blue color inside the cells. Overall, there was no indication of any effects the particles might have on cell metabolism or proliferation in the hepatocellular carcinoma cells tested. However, we are well aware that the cell type can have quite a significant role in the definition of suitable pathways for detoxification of NPs, which has deep implications for biomedical applications.49,50 The potential of these novel Co/Au nanocomposites as contrast agents in MRI was measured in a 7 T magnetic field. As qualitatively shown in Figure 6, these particles exhibited a

magnetizations (M) of 150.1 emu/g and 133.1 emu/g could be observed corresponding to 5 and 10 K measurements, respectively. These values remain distant from the ones expected for bulk Co at room temperature (Ms = 162 emu/ g),41 which involves either partial oxidation of the surface of Co NPs or surface spin canting induced by the organic/polymeric coating, thus significantly decreasing both the Ms and relaxivity in small magnetic NPs.42,43 Phase-Transfer into Aqueous Solution, Cytotoxicity, and Investigation of MR Contrast Enhancement. The surface features of nanomaterials are generally considered very important for biomedical applications since these dominate the interactions with the blood components and target both cells and organs.44,45 In our case, water-solubility and biocompatibility was obtained by modifying the surface of the nanocomposites with an amphiphilic polymer possessing both (i) hydrophobic alkyl side chains for intercalation with the hydrophobic surfactant layer (TOAB) on the nanoparticle surface and (ii) a hydrophilic backbone that provided water solubility through electrostatic repulsion between charged COO− groups (Supporting Information, Figure S5).46 Such a coating was easily achieved by mixing the nanoparticle solution with an excess of polymer (100-fold excess) in chloroform, which after solvent evaporation and redispersion of the pellet in aqueous NaOH (0.1 M) did not leave apparent aggregation even after a few months, as observed by DLS and TEM (Supporting Information, Figure S6). These hydrophilic nanoparticles possessed an increased effective hydrodynamic diameter by volume size of 115.6 nm (0.131 Pdl) as a result of the polymeric coating and a surface charge of −64.3 mV (±10.3 mV) at pH 7.5 due to the carboxylate groups. Noninvasive detection of tumors at an early stage by MRI needs highly sensitive contrast agents, the size and surface properties of which are the main factors that control their characteristics, such as blood half-life, biodistribution, and extravasation ability. Since large nanoparticles (∼100 nm) have short plasma-circulation times due to their fast clearance rate by the mononuclear phagocytic system and the rapid uptake by the liver and spleen, it is envisaged that these Co/Au nanocomposites could be particularly useful for tumor staging of the liver and lymph nodes.47,48 Therefore, cell viability was evaluated using the well-established XTT assay on human hepatocellular carcinoma (HepG2) cells (we choose these cell line as NPs normally accumulate in the tumor), which measures the respiratory activity of the cells (Figure 5A). Importantly, after 24 h of incubation with Co/Au nanocomposites, no significant toxicity could be observed at concentrations of

Figure 6. T1-weighted (A) and T2-weighted (B) MR images of Co/Au nanocomposites at 14, 7, 3.5, and 1.75 μg/mL of Co in agarose gel (1.3% in water).

MRI signal attenuation effect even though there is a relatively small concentration of Co present in the samples. The longitudinal (r1) and transverse (r2) relaxitivity values, a measure of the efficacy of the nanoparticles to behave as MRI contrast agents, were calculated and compared to other MRI contrast agents (Tables 1 and S1, Supporting Information).51−55 Preliminary data indicated that our particles exhibit modest transverse relaxivity, r2 = 13.2 mMCo−1 s−1, and poor longitudinal relaxivity, r1 = 0.52 mMCo−1 s−1, compared to conventional Gd and clinical iron oxide-based nanoparticles. Given the fact that millimolar concentrations of Fe are usually needed with current clinical agents, the poor relaxivities observed for the Co/Au nanocomposites would then at least require concentration in the tens of millimolar range of Co, which is somehow inadequate for biomedical applications. 4024



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Table 1. Relaxivities (r1 and r2) of the 1st Generation Co/Au Nanocomposites and Comparison with Other Commercial Contrast Agents; X Refers to the Metal Atom in Each Case (Co and/or Fe) sample Co/Au nanocomposite Sinerem (Guerbet) Abdoscan VSOP-C184 (Ferropharm) Resovist SHU-555C (ref 595) (Schering) Magnevist (Schering) FeCo (4 nm)/graphitic carbon-PEG FeCo (7 nm)/graphitic carbon-PEG CoFe2O4/2,3dimercaptosuccinic acid

r1 (mMX−1 −1 s )

r2 (mMX−1 −1 s )

0.5

25.4

14.0

13.2 53.1 33.4

10.7

r2/r1

magnetic field (T)

ref

2.4

7 1.5 1.5

56 53

38.0

3.6

1.5

53

4.6 31.0

4.5 185.0

1.0 6.0

1.5 1.5

52 52

70.0

644.0

9.2

1.5

52

1.5

54,55

172.0

kV. Digital images were analyzed with the ImageJ software and a custom macro performing smoothing (3 × 3 or 5 × 5 median filter), manual global threshold, and automatic particle analysis provided by the ImageJ. The macro can be downloaded from http://code.google. com/p/psa-macro. High-resolution (HR)-TEM and EELS were performed on a JEOL2010F field emission gun microscope operated at 200 kV. HAADF, STEM, and electron tomography were obtained on a JEOL2100 operated at 200 kV equipped with an EDX detector. HAADF tilt series for tomography were obtained by using the GATAN 3D Tomography Acquisition Software and the reconstructions by using the 3D Reconstruction and 3D Visualization GATAN PlugIns.57 This technique has been demonstrated to be powerful in order to obtain the 3D morphology of a wide range of nanostructures.58,59 The samples (10 μL) for TEM and HRTEM were drop-casted either onto ultrathin Formvar-coated 200-mesh copper grids (Tedpella, Inc.) or special holey carbon filmed grids (SPI Supplies, Inc.), respectively, and left to dry under Ar. Previous to HRTEM characterization, samples were subjected to oxygen plasma cleaning (2 min) for the removal of organic contaminants. SEM images and EDX microanalysis were performed in a MERLIN FESEM (Carl Zeiss) operating at an acceleration voltage of 3 and 20 kV, respectively, at the Servei de Microscopia (UAB). Samples were prepared by evaporating a drop of the reacted solution on top of an aluminum support. DLS measurements and ζ-potential were performed on a Malvern Zetasizer ZS-Nano instrument. Data was analyzed with the Malvern DTS software using the general purpose algorithm for DLS and the Smochulowski model for obtaining ζpotential values. DLS values are given as volume distributions. UV−vis absorption spectra were recorded with a Shimadzu UV-2401PC spectrophotometer at room temperature. Organic samples were measured in DCB solution using 1 mm quartz cuvettes (Hellma). XRD data were collected on a PANalytical X'Pert diffractometer using a Co Kα radiation source (λ = 1.78901 Å). In a typical experiment, the 2θ diffraction (Bragg) angles were measured by scanning the goniometer from 30° to 90° at a speed of 0.0014 s−1. Samples were prepared by precipitating the particles by centrifugation (7000 rcf, 15 min) and smeared onto (510) off-axis silicon wafers (Silicon Materials). Atomic composition of nanocomposites was analyzed by ICP-MS Agilent instrument (Model: 7500cx) with a detection limit of 0.02386 ppb. Dry amounts of the samples were dissolved in aqua regia at 100 °C before ICP-MS analysis. Ga was used as the internal standard and the integration time/point and time/mass were 0.1 and 0.3 s, respectively, with a 3× times repetition. Magnetization measurements were performed on a superconducting quantum interference device (SQUID) magnetometer (MPMS Quantum Design). The hysteresis cycles were obtained at different temperatures in a magnetic field varying from +50 to −50 kOe. The sample preparation was performed by dispersing the particles in paraffin to have a solid dispersion of noninteracting NPs, which was introduced then into gelatin capsules under ambient conditions. MRI experiments were performed in a 7 T Bruker BioSpec 70/30 USR spectrometer with a maximum gradient of 400 mT/m (Bruker BioSpin GmbH, Karlsruhe, Germany) equipped with a circular polarized 1H RF coil with 7.2 cm inner diameter. The T2-weigthed (T2w) image was acquired using a fast spin−echo sequence with echo train length of 8, effective echo time (TE) of 60 ms, repetition time (TR) of 9 s, acquisition matrix of 256 × 256, field of view (FOV) = 6 × 6 cm2, and slice thickness 1.8 mm. For T1-weigthed imaging (T1w), a spin−echo sequence was used with TE 8 ms, TR 350 ms, and with the same geometry parameters used for the T2w image. MRI data were processed on a Linux computer using ParaVision 4.0 software (Bruker BioSpin GmbH, Karlsruhe, Germany). Sample preparation was performed by mixing different concentrations of the first generation Co/Au nanocomposites (1.75, 3.5, 7, and 14 μg/mL of Co) with agarose gel (1.3% in water) (Supporting Information, Figures S7 and S8). Synthesis of Co NPs. Monodispersed oleic acid-stabilized Co NPs (approximately 2 × 1015 NPs/mL) of 11.6 ± 1.4 nm in diameter were prepared following a previous procedure.28 Briefly, TOPO (0.15 g) and oleic acid (0.1 mL) were degassed in Ar in a flask for 20 min.

Although the relaxivities presented by these nanocomposites in MRI could afford effective magnetic relaxations of the proton spins around the particles, the distance separating the magnetic core from the solvent water molecules was high as a result of the relatively thick silane coatings, which significantly reduced both the transverse and longitudinal relaxivities.53 Experiments in order to decrease the thickness of the condensed silanized layers on top of the Co-cores and thus reduce the size of the Co/Au nanocomposites are subject to an ongoing study.

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CONCLUSIONS We have shown a novel route toward the synthesis of Co/Au nanocomposites through the biphasic reduction of Au3+ onto the surface of organosilane-stabilized Co NPs. The first generation of Co/Au nanocomposites exhibit an abnormal size of ∼100 nm in diameter, which was attributed to the formation of polymerized silane multilayers over the Co NPs. We also demonstrated that the initial Au seeds could be grown by further reduction of additional Au3+ precursor. These Co/Au nanocomposites offer not only a system with magnetic and plasmonic properties but also a surface with high chemical stability, biocompatibility, and easy functionalization. We also analyze in detail the morphological transformations of these hybrid systems at the nanoscale as well as its optical and magnetic properties. Preliminary measurements of the relaxivity in MRI render these composites as attractive agents for advanced negative-contrast cell imaging. Continuing efforts in the synthesis of these Co/Au nanocomposites should improve the control of the size, size uniformity, and the stability of the Co core.

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EXPERIMENTAL SECTION

Chemicals. Dicobalt octa-carbonyl (5−10% in hexane), (3aminopropyl)trimethoxysilane (APTMS, 97%), tetraoctylammonium bromide (TOAB, 98%), gold(III) chloride trihydrate (>99.9%), trioctylphosphine oxide (TOPO, 99%), oleic acid (99%), sodium borohydride (99.9%), 1,2-dichlorobenzene anhydrous (DCB, 99%), and toluene anhydrous (99.8%) were purchased from Sigma-Aldrich and used as received without further purification. Unless otherwise stated, all syntheses were carried out under Ar-atmosphere conditions, using the standard Schlenk line setup. Characterization Methods. Low-magnification TEM analysis was performed on a JEOL1010 microscope at an accelerating voltage of 80 4025



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Then, 15 mL of DCB was introduced into the flask under an Ar atmosphere. The solution was heated to the reflux temperature of DCB (182 °C), and 0.54 g of Co2(CO)8 diluted in 3 mL of DCB was quickly injected into the mixture. The reaction continued for another 10 min, and then, the black colloidal solution was extracted using an airtight syringe and stored in a glass vial under Ar. Particle size was verified by TEM by measuring the size of 200 particles to obtain the average and the size distribution. The absence of oxidation was confirmed by XRD.39 Functionalization of Co NPs. For the synthesis of Co/Au nanocomposite material, we used colloidal solutions of Co NPs in DCB that exhibit stability for several months when kept under Ar conditions. APTMS (0.33 mL, 2.0 mmol) was added to a stirred solution of Co NPs (1 mL, 0.02 mM Co, 1.2 mg Co/mL) in 9 mL of DCB and the resulting mixture was stirred for a further 12 h at room temperature. The particles were separated by centrifugation (10 000 rcf, 15 min) and redispersed in DCB (10 mL). Synthesis of First Generation Co/Au Nanocomposites. An aqueous solution of HAuCl4·3H2O (0.04 g, 0.1 mmol) in 3.33 mL of distilled H2O was mixed with a solution of TOAB (0.07 g, 0.12 mmol) in 5 mL of dry DCB. The two-phase mixture was vigorously stirred until all AuCl4− was transferred into the organic layer (30 min), which was then separated and added to the DCB solution of APTMSfunctionalized Co NPs. A freshly prepared aqueous solution of sodium borohydride (2.75 mL, 0.4 M) was slowly added (approximately 5 min) to the organic mixture under vigorous stirring. The resulting particles were separated by centrifugation (5000 rcf, 15 min) and redispersed in DCB, toluene, or chloroform (10 mL). Synthesis of Second Generation Co/Au Nanocomposites. A solution of Co/Au nanocomposites in toluene (1 mL) was dissolved in 4 mL of toluene. An aqueous solution of HAuCl4·3H2O (0.01 g, 0.1 mmol) in 3.33 mL of distilled H2O was mixed with a solution of TOAB (0.03 g, 0.25 mmol) in 5 mL of dry toluene. The two-phase mixture was vigorously stirred until all AuCl4− was transferred into the organic layer (30 min), which was then separated and added to the solution containing the Co/Au nanocomposites. A freshly prepared aqueous solution of sodium borohydride (2.75 mL, 0.4 M) was slowly added to the organic mixture under vigorous stirring. The particles were separated by centrifugation (5000 rcf, 15 min) and redispersed in either DCB or toluene (1 mL). Transfer of First Generation Co/Au Nanocomposites from Organic into Aqueous Phase. The phase-transfer reaction was performed by employing a previously designed amphiphilic polymer for nanoparticle coating,46 resulting from the reaction of polymer(isobutylene-alt-maleic anhydride) (Sigma-Aldrich, MW 6000 corresponding to roughly 39 monomer units per polymer chain) and dodecylamine. A solution of the first generation Co/Au nanocomposites (150 μL) was dissolved in toluene (0.5 mL), centrifuged (7000 rcf, 15 min), and the resulting precipitate dissolved in CHCl3 (0.5 mL). The amphiphilic polymer (50 μL, 0.55 M of monomer units) in CHCl3 was added to the nanoparticle solution, vortexed for 15 min, and the solvent evaporated. The resulting dry nanoparticles were redissolved in aqueous (0.5 mL) NaOH (0.1 M) and then brought to pH 7.5 with diluted HCl (0.1 M). Excess polymer was cleaned by repeated (minimum of 3 times) centrifugation (7000 rcf, 15 min) and resuspension of the particles in fresh Milli-Q water. Cytotoxicity Assays. Human hepatocellular carcinoma cell line HepG2 (American Type Culture Collection, ATCC) was maintained in Minimum Essential Medium (MEM alpha, Invitrogen) supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, Invitrogen). Cells were grown in a humidified incubator (Heraus, Germany) with 5% CO2 at 37 °C. The growth inhibitory effect of the compound on cells was measured by the XTT assay.60 Cells were seeded in 96-well plates (6 × 103 cells per well) and incubated overnight to allow for cell attachment. After overnight incubation, cells were washed with PBS, and fresh culture medium (200 μL) was added containing the nanocomposites at the desired concentration and incubated at 37 °C for 24 h. All conditions were performed in quadruplicate. The concurrent cell viability assay was performed using the XTT assay (Biomedica) in 96-well plates according to the

manufacturer’s instructions. In brief, XTT reagent (2,3-bis-(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) was added (20 μL) to each well and incubated for 3 h. The color formed was quantified with a spectrophotometric plate reader at 490 nm (Victor 3, PerkinElmer) (reference wavelength 620 nm). The percentage of cell viability was calculated by dividing the average absorbance of the cells treated with the complex by that of the control. Cell viability was additionally determined by a Trypan blue dye exclusion assay. After 24 h of exposure to Co/Au nanocomposites, cells were rinsed with fresh medium, and 0.4% Trypan blue solution was added and left for 3 min. The cells were then rinsed thoroughly with fresh medium and inspected under a phase contrast microscope.

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ASSOCIATED CONTENT

S Supporting Information *

TEM images of as-synthesized Co NPs and Co NPs after treatment with APTMS; EDX analysis of the first generation Co/Au nanocomposites by SEM and STEM modes; STEM mode micrographs taken at different stages of the growth of Au domains; schematic representation of the amphiphilic polymer used for the phase-transfer; TEM images of Co/Au nanocomposites after transfer into aqueous phase; images of the phantom used for MRI and longitudinal (T1) and transverse (T2) relaxation times for the different concentrations of Co/Au nanocomposites. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (I.O.-J.); [email protected] (V.P.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mr. Bernat Bozzo-Closas from ICMAB for SQUID magnetometer measurements, Mr. Marcos Rosado from Servei de Microscopia (UAB) for SEM measurements, Dr. Silvia Lope-Piedrafita from Servei de Ressonància Magnètica Nuclear (UAB) for MR contrast enhancement measurements, and Dr. Belén Ballesteros from Catalan Institute of Nanotechnology (ICN) for help with EDX/TEM measurements. We also gratefully acknowledge Dr. Ralph Sperling for the kindly donation of the amphiphilic polymer, Dr. Stephanie Lim for fruitful discussions about the manuscript, and Technological Centers (CCiT-UB) which allowed the use of their facilities. The work was supported in part by Ministerio de Ciencia e Innovación under grant number PTQ-06-2-0839 (for I.O.-J.) and Consolider Nanobiomed Ingenio 2010. J.M.R thanks the CSIC for a JAE predoc Grant.

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REFERENCES

(1) Safarik, I.; Safarikova, M. BioMag. Res. Technol. 2004, 2 (1), 7. (2) Graham, D. L.; Ferreira, H. A.; Freitas, P. P. Trends Biotechnol. 2004, 22 (9), 455. (3) Megens, M.; Prins, M. J. Magn. Magn. Mater. 2005, 293 (1), 702. (4) Berry, C. C.; Curtis, A. S. G. J. Phys. D: Appl. Phys. 2003, 36 (13), R198. (5) Neuberger, T.; Schöpf, B.; Hofmann, H.; Hofmann, M.; von Rechenberg, B. J. Magn. Magn. Mater. 2005, 293 (1), 483. (6) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26 (18), 3995. (7) Mornet, S.; Vasseur, S.; Grasset, F.; Duguet, E. J. Mater. Chem. 2004, 14 (14), 2161.

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particle was calculated using the particle diameter measured by TEM (3 nm for Au NPs and 12 nm for Co NPs), the density of bulk metals, and their molecular weights; the values were 546 Au atoms per Au NP and 8.2 × 104 Co atoms per Co NP. By means of image analysis with ImageJ software, it could be estimated around 250−350 Au NPs per each Co/Au nanocomposite. (37) Hostetler, M. J.; Wingate, J. E.; Zhong, C.-J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir 1998, 14 (1), 17. (38) Complete tilt series and 3D reconstruction models can be found as a live motion movie at http://www.icmab.cat/gaen. (39) Dinega, D. P.; Bawendi, M. G. Angew. Chem., Int. Ed. 1999, 38 (12), 1788. (40) Tracy, J. B.; Weiss, D. N.; Dinega, D. P.; Bawendi, M. G. Phys. Rev. B 2005, 72 (6), 064404. (41) O’Handley, R. C. Modern Magnetic Materials: Principles and Applications; John Wiley & Sons: New York, 2000. (42) Smolensky, E. D.; Park, H.-Y. E.; Berquó, T. S.; Pierre, V. C. Contrast Media Mol. Imaging 2011, 6 (4), 189. (43) Herea, D. D.; Chiriac, H. Optoelectron. Adv. Mater. 2008, 2, 549. (44) Hirsch, L. R.; Jackson, J. B.; Lee, A.; Halas, N. J.; West, J. L. Anal. Chem. 2003, 75 (10), 2377. (45) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Nat. Mater. 2009, 8 (7), 543. (46) Lin, C.-A. J.; Sperling, R. A.; Li, J. K.; Yang, T.-Y.; Li, P.-Y.; Zanella, M.; Chang, W. H.; Parak, W. J. Small 2008, 4 (3), 334. (47) Stark, D. D.; Weissleder, R.; Elizondo, G.; Hahn, P. F.; Saini, S.; Todd, L. E.; Wittenberg, J.; Ferrucci, J. T. Radiology 1988, 168 (2), 297. (48) Weissleder, R. Radiology 1994, 193 (3), 593. (49) Laurent, S.; Burtea, C.; Thirifays, C.; Häfeli, U. O.; Mahmoudi, M. PLoS one 2012, 7 (1), e29997. (50) Mahmoudi, M.; Laurent, S.; Shokrgozar, M. A.; Hosseinkhani, M. ACS Nano 2011, 5 (9), 7263. (51) Gao, J.; Liang, G.; Cheung, J. S.; Pan, Y.; Kuang, Y.; Zhao, F.; Zhang, B.; Zhang, X.; Wu, E. X.; Xu, B. J. Am. Chem. Soc. 2008, 130 (35), 11828. (52) Seo, W. S.; Lee, J. H.; Sun, X.; Suzuki, Y.; Mann, D.; Liu, Z.; Terashima, M.; Yang, P. C.; McConnell, M. V.; Nishimura, D. G.; Dai, H. Nat. Mater. 2006, 5 (12), 971. (53) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R. N. Chem. Rev. 2008, 108 (6), 2064. (54) Jun, Y.-W.; Seo, J.-W.; Cheon, J. Acc. Chem. Res. 2008, 41 (2), 179. (55) Lee, J.-H.; Huh, Y.-M.; Jun, Y.-W.; Seo, J.-W.; Jang, J.-T.; Song, H.-T.; Kim, S.; Cho, E.-J.; Yoon, H.-G.; Suh, J.-S.; Cheon, J. Nat. Med. 2007, 13 (1), 95. (56) Rohrer, M.; Bauer, H.; Mintorovitch, J.; Requardt, M.; Weinmann, H.-J. Invest. Radiol. 2005, 40 (11), 715. (57) Gatan 3D Tomography-Acquisition Software. http://www. gatan.com/products/software/tomography.php. (58) Bouhelier, A.; Beversluis, M. R.; Novotny, L. Ultramicroscopy 2004, 100 (3−4), 413. (59) Heigoldt, M.; Arbiol, J.; Spirkoska, D.; Rebled, J. M.; ConesaBoj, S.; Abstreiter, G.; Peiro, F.; Morante, J. R.; Morral, A. F. I. J. Mater. Chem. 2009, 19 (7), 840. (60) Cory, A. H.; Owen, T. C.; Barltrop, J. A.; Cory, J. G. Cancer Commun. 1991, 3 (7), 207.

(8) Park, H.-Y.; Schadt, M. J.; Wang, L.; Lim, I. I. S.; Njoki, P. N.; Kim, S. H.; Jang, M.-Y.; Luo, J.; Zhong, C.-J. Langmuir 2007, 23 (17), 9050. (9) Wang, H.; Brandl, D. W.; Nordlander, P.; Halas, N. J. Acc. Chem. Res. 2006, 40 (1), 53. (10) Zhang, J.; Post, M.; Veres, T.; Jakubek, Z. J.; Guan, J.; Wang, D.; Normandin, F.; Deslandes, Y.; Simard, B. J. Phys. Chem. B 2006, 110 (14), 7122. (11) Wei, W.; Quanguo, H.; Hong, C.; Jianxin, T.; Libo, N. Nanotechnology 2007, 18 (14), 145609. (12) Spasova, M.; Salgueirino-Maceira, V.; Schlachter, A.; Hilgendorff, M.; Giersig, M.; Liz-Marzan, L. M.; Farle, M. J. Mater. Chem. 2005, 15 (21), 2095. (13) Lyon, J. L.; Fleming, D. A.; Stone, M. B.; Schiffer, P.; Williams, M. E. Nano Lett. 2004, 4 (4), 719. (14) Wang, L.; Luo, J.; Fan, Q.; Suzuki, M.; Suzuki, I. S.; Engelhard, M. H.; Lin, Y.; Kim, N.; Wang, J. Q.; Zhong, C.-J. J. Phys. Chem. B 2005, 109 (46), 21593. (15) Wang, L.; Luo, J.; Maye, M. M.; Fan, Q.; Rendeng, Q.; Engelhard, M. H.; Wang, C.; Lin, Y.; Zhong, C.-J. J. Mater. Chem. 2005, 15 (18), 1821. (16) Xu, Z.; Hou, Y.; Sun, S. J. Am. Chem. Soc. 2007, 129 (28), 8698. (17) Ban, Z.; Barnakov, Y. A.; Li, F.; Golub, V. O.; O’Connor, C. J. J. Mater. Chem. 2005, 15 (43), 4660. (18) Mandal, S.; Krishnan, K. M. J. Mater. Chem. 2007, 17 (4), 372. (19) Mikhaylova, M.; Kim, D. K.; Bobrysheva, N.; Osmolowsky, M.; Semenov, V.; Tsakalakos, T.; Muhammed, M. Langmuir 2004, 20 (6), 2472. (20) Smolensky, E. D.; Neary, M. C.; Zhou, Y.; Berquo, T. S.; Pierre, V. C. Chem. Commun. 2011, 47 (7), 2149. (21) Caruntu, D.; Cushing, B. L.; Caruntu, G.; O’Connor, C. J. Chem. Mater. 2005, 17 (13), 3398. (22) Fang, C.-L.; Qian, K.; Zhu, J.; Wang, S.; Lv, X.; Yu, S.-H. Nanotechnology 2008, 19 (12), 125601. (23) Levin, C. S.; Hofmann, C.; Ali, T. A.; Kelly, A. T.; Morosan, E.; Nordlander, P.; Whitmire, K. H.; Halas, N. J. ACS Nano 2009, 3 (6), 1379. (24) Pita, M.; Abad, J. M.; Vaz-Dominguez, C.; Briones, C.; MateoMartí, E.; Martín-Gago, J. A.; del Puerto Morales, M.; Fernández, V. M. J. Colloid Interface Sci. 2008, 321 (2), 484. (25) Wang, H.; Brandl, D. W.; Le, F.; Nordlander, P.; Halas, N. J. Nano Lett. 2006, 6 (4), 827. (26) Spuch-Calvar, M.; Pérez-Juste, J.; Liz-Marzán, L. M. J. Colloid Interface Sci. 2007, 310 (1), 297. (27) Salgueirino-Maceira, V.; Correa-Duarte, M. A.; Farle, M.; LopezQuintela, A.; Sieradzki, K.; Diaz, R. Chem. Mater. 2006, 18 (11), 2701. (28) Puntes, V. F.; Krishnan, K. M.; Alivisatos, A. P. Science 2001, 291 (5511), 2115. (29) De Palma, R.; Peeters, S.; Van Bael, M. J.; Van den Rul, H.; Bonroy, K.; Laureyn, W.; Mullens, J.; Borghs, G.; Maes, G. Chem. Mater. 2007, 19 (7), 1821. (30) Yamaura, M.; Camilo, R. L.; Sampaio, L. C.; Macêdo, M. A.; Nakamura, M.; Toma, H. E. J. Magn. Magn. Mater. 2004, 279 (2−3), 210. (31) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 7, 801. (32) Kobayashi, Y.; Horie, M.; Konno, M.; Rodriguez-Gonzalez, B.; Liz-Marzan, L. M. J. Phys. Chem. B 2003, 107 (30), 7420. (33) Wasserman, S. R.; Tao, Y. T.; Whitesides, G. M. Langmuir 2002, 5 (4), 1074. (34) Salgueirino-Maceira, V.; Correa-Duarte, M. A. J. Mater. Chem. 2006, 16 (36), 3593. (35) Stöber, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26 (1), 62. (36) The measured concentration by ICP-MS of Au3+ and Co+2 ions was divided by the number of atoms per particle to obtain the concentration of each type of NP in solution (2 × 1015 Au NP/mL and 2 × 1013 Co NP/mL), which left an estimated ratio of 100 Au NP/1 Co NP. The number of either Au or Co atoms per each type of 4027


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