Iron oxide nanoparticles with sizes, shapes and compositions resulting in different magnetization signatures as potential labels for multiparametric detection

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Iron oxide nanoparticles with sizes, shapes and compositions resulting in different magnetization signatures as potential labels for multiparametric detection q

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Université Paris 13, Sorbonne Paris Cité, Laboratoire CSPBAT, CNRS, UMR 7244, F-93017 Bobigny, France Magnisense, F-75008 Paris, France c Université Lyon 1, Laboratoire des Multimatériaux et Interfaces, CNRS, UMR 5615, F-69622 Villeurbanne, France d ICMPE-MCMC, CNRS, UMR 7182, F-94320 Thiais, France e ESPCI ParisTech-UPMC, LPEM, CNRS, UMR 8213, F-75005 Paris, France f APHP, Service de Médecine Nucléaire, Hosp Avicenne, F-93009 Bobigny, France b

a r t i c l e

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Caroline de Montferrand a,b, Ling Hu c, Irena Milosevic a, Vincent Russier d, Dominique Bonnin e, Laurence Motte a, Arnaud Brioude c, Yoann Lalatonne a,f,⇑

i n f o

Article history: Available online xxxx

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Keywords: Nanoparticles Biosensors Superparamagnetism Magnetic properties Ligand exchange

a b s t r a c t Magnetic iron oxide nanoparticles differing in their size, shape (spherical, hexagonal, rods, cubes) and composition have been synthesized and modified using caffeic acid for transfer to aqueous media and stabilization of the particle suspensions at physiological pH. A super quantum interference device and the recently patented magnetic sensor MIAplexÒ, which registered a signal proportional to the second derivative of the magnetization curve, were used to study the magnetization behavior of the nanoparticles. The differences in the magnetic signatures of the nanoparticles (spheres and rods) make them promising candidates for the simultaneous detection of different types of biological molecules. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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1. Introduction

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Among the different chemical composition nanomaterials superparamagnetic iron oxide nanoparticles are certainly the most promising material for medical applications such as cellular labeling, bio-assay, imaging, as drug carriers and for hyperthermia treatment. First, their tunable core sizes, ranging from a few up to tens of nanometers, allow control of the properties appropriate to the intended application. For example, extremely small sized iron oxide nanoparticles (d < 4 nm) can be used as T1 magnetic resonance imaging (MRI) contrast agents [1], whereas 10 nm iron oxide nanoparticles are more efficient T2 MRI contrast agents [2–4]. Second, external magnetic forces induce the movement of nanoparticles, providing various advantages in drug delivery [5–8], cell separation [9,10], transfection [7,11,12] and hyperthermia treatment [13–15]. More recently the use of magnetic particles for magnetic bioassays has grown considerably [16–19], as their physical properties allow faster assay and in some cases improve sensitivity over currently available commercial methods [20]. The binding reaction is mea-

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Presented at the E-MRS Biomaterials Symposium, organized by Prof. Kurosch Rezwan, Dr. Laura Treccani, Prof. Giovanni Marletta and Prof. Miguel Manso Silván. ⇑ Corresponding author at: Université Paris 13, Sorbonne Paris Cité, Laboratoire CSPBAT, CNRS, UMR 7244, F-93017 Bobigny, France. Tel.: +33 148387621. E-mail address: [email protected] (Y. Lalatonne).

sured by the detection of the magnetic signal with sensors based on, for example, the giant magneto-resistance (GMR) effect, the magnetic tunneling effect or the planar Hall effect (PHE) [21–23]. A novel detection and characterization method was developed to take advantage of the nonlinearity of superparamagnetic materials for magnetic immunoassay [24,25]. The MIAplexÒ sensor measures a signal corresponding to the second derivative of magnetization around a zero field. During measurement superparamagnetic nanoparticles are magnetized by an excitation magnetic field formed by the superimposition of a low frequency alternative excitation magnetic field H1 (fH1 = 0.025 Hz, amplitude 37 to 37 kA m1) and a high frequency sinusoidal magnetic field H2 (fH2 = 24.4 kHz, amplitude 0.707 to 0.707 kA m1). Because the magnetization curve for superparamagnetic nanoparticles is nonlinear, the induced MIAplexÒ signal contains multiple order harmonics that can be detected and distinguished from the applied high frequency field (H2) by a simple Fourier transform. Here the second harmonic is exploited for signal detection. Since there is no input to the signal from the biological tissues, the resulting nonlinear measurement has the advantage that it is very sensitive and allows the detection of picograms of particles within biological systems [26]. With this sensor, MIAplexÒ, measurements were performed on microliter sample volumes in a portable device. The simplicity, sensitivity and rapidity of the MIAplexÒ device result in very low purchase

1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.11.025

Please cite this article in press as: de Montferrand C et al. Iron oxide nanoparticles with sizes, shapes and compositions resulting in different magnetization signatures as potential labels for multiparametric detection. Acta Biomater (2012), http://dx.doi.org/10.1016/j.actbio.2012.11.025

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and running costs when adapted for medical ‘‘point of care’’ testing [27] performed at the site where needed (medical emergency, doctor’s surgery, agricultural production unit, etc.). For such applications a major advance would be multiparametric testing, allowing the detection of several pathogens in one step. Such applications require various magnetic signatures, which can be achieved by exploiting magnetic nanoparticles of different sizes [24,25,28], compositions or shapes. Shape-controlled iron oxide nanoparticles have attracted significant attention in relation to their peculiar magnetic properties due to their shape anisotropy. However, compared with the extensive research on spherical nanoparticles, the use of non-spherical particles with anisotropic configurations has been less well demonstrated, especially for small sized rod structures. This is related to the challenge of the preparation of onedimensional (1-D) nanostructured ferrites. The high symmetry of the spinel structure unfavorably induces 1-D growth, without extra restriction [29]. Nevertheless, recent studies have demonstrated the successful and controllable synthesis of non-spherical iron oxide nanoparticles with cubic [30,31], hexagonal [32], and rod [33–35] morphologies. The nanoparticle surface functionalization itself is a key factor in translating the intrinsic properties of nanoparticles to biomedical applications. First, there is a need for a robust means to stabilize nanoparticles in aqueous media under a variety of processing conditions. Small bifunctional molecules with a robust anchoring surface group such as catechol [36–38] or a member of the 1-hydroxy-1,1-methylene bisphophonic acid family [39–42] are of particular interest, as they can ensure precise control of the number and nature of the terminal functionality presenting on the surface. Furthermore, the molecules of interest, such as antibodies, peptides or polymers, allow specific biological recognition. The formation of amide, ester or carbamate bonds is classically the most widespread chemical methodology used to functionalize nanoparticles with molecules of interest [43–45]. Another promising technique for chemical nanoparticle functionalization is the use of ‘‘click chemistry’’ [42,46]. In this manuscript magnetic iron oxide nanoparticles with spherical, hexagonal, cubic and rod-like shapes were synthesized. For biomedical applications the nanoparticle surface was modified with caffeic acid molecules. Caffeic acid has two major properties as a ligand for water solubilized nanoparticles. First, the catechol functions, as observed for dopamine molecules, are solid anchors on metal oxide surfaces with an irreversible binding affinity [36,47]. Secondly, the large number of COOH functionalities on the magnetic core of the nanoparticle can be used as precursor groups for the covalent coupling of biomolecules, leading to electrostatic interactions between the nanoparticles, dispersing them under physiological conditions [40]. These different properties, allowing good stability over time and control of the interfacial chemistry, are crucial for the use of magnetic nanoparticles in biomedical applications. The magnetic properties of such water dispersed ferrite nanoparticles have been studied at room temperature using a conventional a super quantum interference device (SQUID) and a MIAplexÒ.

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2. Materials and methods

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2.1. Chemicals

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Iron(III) chloride (FeCl3), hydrazine, dopamine, iron(III) acetylacetonate (Fe(acac)3), 1,2-hexadecanediol, oleic acid, dibenzyl ether, and caffeic acid were purchased from Sigma-Aldrich. Oleylamine was provided by Fluka (Sigma-Aldrich).

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2.2. Synthesis of ferrite nanoparticles differing in shape

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Spherical, hexagonal and cubic nanoparticles were prepared, under an inert atmosphere, using Fe(acac)3 as the precursor in a

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high temperature organic solution containing the reducing reagent 1,2-hexadecanediol and the surfactants oleic acid and oleylamine. At the end of synthesis the nanoparticles were precipitated by the addition of 20 ml of ethanol and separated by centrifugation. The resulting product was then dispersed in cyclohexane. Centrifugation was applied once more to remove any undispersed residue. The final product was re-dispersed in cyclohexane.

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2.2.1. Nanospheres Fe(acac)3 (0.3 mmol), 1,2-hexadecanediol (0.3 mmol), oleylamine (6 mmol), oleic acid (6 mmol) and dibenzylether (10 ml) were mixed and magnetically stirred for 30 min. Under reflux the mixture was heated rapidly to 298 °C and kept at this temperature for 30 min, before cooling down to room temperature.

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2.2.2. Nanohexagons Fe(acac)3 (0.3 mmol), 1,2-hexadecanediol (0.3 mmol), oleylamine (6 mmol) and dibenzylether (10 ml) were mixed and magnetically stirred for 30 min. Oleic acid (6 mmol) was then added in this mixture. Under reflux the mixture obtained was heated rapidly to 298 °C and kept at this temperature for 30 min, before cooling down to room temperature.

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2.2.3. Nanocubes Fe(acac)3 (0.25 mmol), 1,2-hexadecanediol (2 mmol), oleylamine (4 mmol), oleic acid (1 mmol), and dibenzylether (5 ml) were mixed and heated at 110 °C for 1 h. The temperature was then raised to 200 °C and kept at this temperature for 30 min. Under reflux the mixture was heated to 298 °C and kept at this temperature for 1 h, before cooling down to room temperature.

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2.2.4. Nanorods Nanorods were prepared in water by a simple and rapid method by microwave (MW)-assisted reduction using akaganeite b-FeOOH nanorods and hydrazine as the precursor and reductor, respectively. Akaganeite b-FeOOH nanorods were obtained by forced hydrolysis (80-90 °C) of aqueous FeCl3/HCl solution (0.025 mol l1/ 0.002 mol l1) in the presence of dopamine as a chemical shapecontrol agent (1.6 mg ml1). The precursor nanorods are then reduced with hydrazine (3.9  102 mol l1), by microwave-assisted reduction. The black product was magnetically collected, washed with deionized water at pH 7 and peptized with HNO3 solution at pH 2 [34].

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2.3. Surface modification for phase transfer into aqueous media

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To stabilize the nanoparticles in aqueous medium the surfactants of hydrophobic (spherical, hexagonal and cubic) nanoparticles oleic acid (OA) and oleylamine were exchanged for caffeic acid (CA), following a method previously described [48]. A 1 ml portion of an aqueous caffeic acid solution (20 mg ml1) at pH 10 was added to 1 ml of the nanoparticle suspension (4 mg ml1) in cyclohexane. The mixture was sonicated for 30 min, then stirred for 2 h. The organic non-polar surfactant was diluted by adding 2 ml of cyclohexane, followed by centrifugation. The supernatant was then discarded. For nanorods, after synthesis, the nanoparticle surface was directly functionalized with caffeic acid. 1 ml of an aqueous caffeic acid solution (5 mg ml1), pH 10, was directly mixed with the bare particles suspension (5 mg ml1) for 2 h. The solution was then centrifugated and the supernatant discarded. In all cases particles in the aqueous phase were washed with acidified water (pH 2) and finally dispersed in 1 ml of pure water (pH 7).

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2.4. Analytical and characterization methods

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Transmission electron microscopy (TEM) was performed using a Topcon EM 002B microscope operating at 200 keV. Samples were prepared by air drying drops of the nanoparticles suspension on carbon-coated copper grids. Iron weight was determined using elemental analyses carried out using an ICP-AES Activa Jobin Yvon. TGA experiments were performed to determine the extent of the nanoparticle coating layer using a Steram evo Lab system. Consequently, the dry weight of the iron oxide had been determined by subtracting the coating layer from the total mass sample. Nanoparticle surface charge was characterized using the Zetasizer Nano ZS MalvernFTIR spectra recorded in a ThermoScientific Nicolet 380. The magnetic properties of nanoparticles were recorded at room temperature in powder form using a superconducting quantum interference device. The MIAplexÒ magnetic sensor had been developed by Magnisense SE. The instrument measures a signal which is proportional to the second derivative of the magnetization curve [49]. The corresponding magnetic signatures are recorded at room temperature in liquid suspensions 0.5 wt.% ().

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3. Results and discussion

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3.1. Shape control

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The spherical, hexagonal and cubic iron oxide nanoparticles were prepared by the reduction of Fe(acac)3 in a high boiling point inert organic solvent, in the presence of oleic acid and oleylamine. Fig. 1A–C (left, black) show typical TEM images of iron oxide nanoparticles with different shapes prepared using the present method. Magnetic nanospheres (diameter 12 nm) and nanohexagons (width 12 nm) were obtained by direct heating of the reaction mixture from room temperature to reflux (298 °C). This rapid rise in temperature promoted decoupling of the nucleation and growth stages. The cubic nanoparticles (side length 26 nm) were synthesized according to the procedure developed by Yang et al. [31]. Compared with the protocols to prepare spheres and hexagons, the molar ratio between reagents and the reaction time were changed in this synthesis procedure. Such modifications are based on the selective binding ability of the two stabilizers used (oleic acid and oleylamine) onto different energy crystal facets [50]. Magnetic nanorods were successfully prepared in water by reduction of akaganeite b-FeOOH nanorods with hydrazine. Elongated paramagnetic akaganeite precursors were synthesized using dopamine as a green chemical shape control agent. After 2 min microwave reduction leads to iron oxide nanorods with an aspect ratio of 3.2. XRD and Raman spectroscopy indicated a heterogeneous composition corresponding to 30% akaganeite/70% maghemite [34]. A typical TEM image of a nanorod sample is shown in the Fig. 1D (left, black). The average length and width are 38 and 12 nm, respectively.

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3.2. Phase transfer and colloidal stability

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Nanoparticle dispersivity in water is a key point for biological applications,. By ligand exchange a catechol ligand (CA) was used to replace the original surfactant OA on the surface of nanoparticles (nanospheres, nanohexagons, and nanocubes). After synthesis the nanorods were uncoated and dispersed in acidic medium. CA was added to the bare nanoparticle solution in order to disperse the nanoparticles at physiological pH. TEM images (Fig. 1 A–D, right, red) highlight morphology conservation during phase transfer. Catechol derivative anchor groups possess a high binding affinity for iron oxide [51]. Hence we chose CA to replace the OA coating

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and thus to optimally disperse ferrite nanoparticles at physiological pH. The aqueous suspension of CA-coated nanoparticles remained stable after several months storage at room temperature. This high stability could be explained by electrostatic repulsion between particles, which was quantified by a high negative zeta potential (40 mV). Fig. 2 displays infrared spectra of spherical nanoparticles coated with OA (black line) and spherical, hexagonal, cubic and rod-shaped nanoparticles coated with CA (red, green, blue, and dark yellow lines, respectively). The infrared spectra of pure OA (black dotted line) and CA (red dotted line) are given for reference. The most relevant peak assignment of OA is the carbonyl absorbance at 1711 cm1 [52]. CA is characterized by different vibration bands: aromatic ring C@C stretching vibration bands at 1639, 1602, 1522 and 1376 cm1, the C–O vibration band of carboxylic acid at 1293 cm1 and the C–O stretching vibration band at 1280 cm1.[53] In the spectrum of nanoparticles coated with OA (Fig. 2, black line) two new peaks at 1558 and 1405 cm1 are assigned to bidendate (–COO–Fe) mode OA binding [51]. Absence of the peak at 1711 cm1 corresponding to the carboxylic function of free OA (Fig. 2, black dotted line) indicates that no unbound OA remained within the nanoparticles coated with OA and strong interaction between carboxylate and the nanoparticles occurred [54]. Comparing nanoparticles coated with OA and CA (Fig 2, red, green, blue, and dark yellow lines), a number of changes are observed in the related infrared spectra confirming ligand exchange of CA for OA. The most notable is the appearance of a strong band characteristic of a conjugated aromatic ketone (1632 cm1) [55], confirming bidentate bonding of catechol to the surface of the nanoparticles. It should be noted that for nanocubes the infrared spectrum indicated the presence of an excess of CA. Concerning the Fe–O stretching region, some differences are observed depending on the nanoparticle shape. Nanospheres, nanohexagons and nanocubes (Fig 2, red, green, and blue lines) present a strong peak at 588 cm1, corresponding to a Fe–O vibration band within the Fe3O4 magnetite structure, and a shoulder at 617 cm1, due to the cFe2O3 maghemite phase [56]. Thus these nanoparticles presented a mixed composition of magnetite and maghemite, confirmed by the electronic diffraction patterns (Supplementary material). The nanorods (Fig. 2, dark yellow line) present a strong peak at 699 cm1 and a shoulder at 617 cm1, due to the presence of both b-FeOOH akaganeite [57–60] and cFe2O3 maghemite phases in the nanocrystals, as previously reported from XRD and Raman spectra [34].

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3.3. High field magnetic properties at room temperature

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At room temperature (300 K) – the magnetization curves between 3000 and 3000 kA m1 of nanospheres, nanohexagons and nanocubes coated with OA are characteristic of superparamagnetic behavior (black curves in Fig. 3A–C, respectively). This behavior is preserved after transfer to water with CA (red curves in Fig. 3A–C, respectively). Taking into account the measurement error in normalized saturation magnetization (8%) one can consider that saturation magnetization is not or only slightly changed by ligand exchange. Usually, for a given crystalline composition a change in saturation magnetization is correlated with the crystal volume. Hence, considering different nanoparticle shapes with the same composition and same unit size (d = 12 nm, corresponding to the diameter for nanospheres, the width for nanorods and nanohexagons (cuboctahedrons in three dimensions), and the side length for nanocubes), Vnanocube > Vnanorod > Vnanosphere > Vcuboctahedron. Consequently the same order of saturation magnetization of these nanocrystals is expected [61]. The nanosphere and nanohexagon magnetization curves have comparable saturation magnetizations

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Fig. 1. TEM images after synthesis (left, black) and after CA coating (right, red) for (A) spherical, (B) hexagonal, (C) cubic and (D) rod-shaped ferrite nanoparticles. Scale bar 50 nm.

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of about 80 ± 7 emu g1 (Fig. 3A and B). This value is close to that of magnetite bulk (90 emu g1). Comparing the volume of a sphere (0.5236 d3) and the volume of a cuboctahedron (0.4536 d3) the deviation of 14% is of the same order of magnitude as that of the TEM size distribution (17%) of both nanospheres and nanohexa-

gons (Fig. 1A and B). Moreover, both samples present a mixed composition of magnetite and maghemite phases, as observed in the infrared spectra (Fig. 2, red and green lines). Hence, this volume difference remains weak and can explain the similarity between the saturation magnetization.

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Fig. 2. Infrared spectra of free oleic acid OA (black dotted line), nanospheres coated with OA (black line), nanospheres, nanohexagons, nanocubes, nanorods coated with CA (red, green, blue, dark yellow lines, respectively) and free CA (red dotted line).

Fig. 3. Room temperature magnetization curves (300 K) of (A) spherical, (B) hexagonal, cubic (C) and (D) rod-shaped ferrite nanoparticles in powder form functionalized with oleic acid (black curves) and caffeic acid (red curves).

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The nanocubes are expected to present the highest magnetization saturation value due to their shape and their average side length of 26 nm. Indeed, the reported saturation magnetization for 26 nm magnetite nanocubes at 5 K is 82 emu g1 [62] and for 14.5 nm maghemite nanocubes at room temperature is 75 emu g1 [63]. Our sample presents a surprisingly weak room temperature saturation magnetization of about 40 ± 3 emu g1 (Fig. 3C). This unexpected saturation magnetization could be related to size poly-

dispersity and polymorphism (the coexistence of smaller cubes and triangular shaped particles, Fig. 1C), to eventual polycrystallinity [64], to surface spin canting [65–67] and to the crystalline composition. The magnetization curve for nanorods presents a weakly ferromagnetic behavior with a coercive field Hc = 4.4 kA m1 (Fig. 3D). The saturation magnetization (18 emu g1, Fig. 3C) is reached at 3979 kA m1 and is smaller than the saturation magnetization

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reported for maghemite nanorods [68]. This behavior was related to the presence of the paramagnetic akaganeite phase and the spin canting effect, as well as shape anisotropy [34]. 3.4. MIAplexÒ magnetic signatures in low fields of water dispersible nanoparticles of different shapes: new nanoprobes for multiparametric detection Fig. 4 shows the magnetic behaviour measured with the MIAplex device for nanoparticles coated with OA and dispersed in cyclohexane (black curves) and dispersed in water after ligand exchange for – (red curves). Comparing these magnetic signatures (Fig. 4) with the room temperature SQUID magnetization curves (Fig. 3) it can be observed that magnetic detection with the MIAplexÒ at very low magnetic fields where the magnetization slope is most important. Hence, slight changes in the MIAplexÒ signal depending on the particles size and shape are expected. The corresponding MIAplexÒ measurements (Fig. 4D) show differences depending on the shape of the particles, in particular considering signal amplitude Sptp, the intensity difference between the peaks, and DHmax, the peak to peak line width (Table 1). Hysteresis loops are also observed and are attributed to the short time measurement of the MIAplex technology (105 s) compared with SQUID time measurement (100 s) [28]. Replacing the OA coating of the particles with CA, since the corresponding molecule chain length decreases, one expects an increase in dipolar interparticle interactions, resulting in a decrease in the relaxation time and, consequently, the onset of hysteresis cycle opening (Fig. 4, black and red curves). Ligand exchange induces an increase in the MIAplexÒ signal intensity Sptp and a decrease in the peak to peak line width DHmax (Fig. 4 and Table 1). This relation dependence between Sptp and DHmax was previously reported for spherical maghemite nanoparticles differing in size and dispersed in water [24]. The change observed between hydrophobic and hydrophilic nanoparticles has to be correlated with solvent viscosity g (gwater = 1.0 mPa s1, ghex1 ) inducing an increased Brownian relaxation time ane = 0.33 mPa s

sB (sB = 3VHg/(kbT), where kb is the Boltzmann constant, T is the temperature, VH is the particle hydrodynamic volume and g is the solvent viscosity) [17,69]. The frequency dependence of the imaginary part of the complex magnetic susceptibility v00 (x) exhibits a maximum also known as a relaxation or absorption peak xmax. The Brownian relaxation time sB varies inversely with xmax. Consequently, xmax decreases after phase transfer, getting closer to the MIAplexÒ characteristic frequency (2.4  104 s1). This explains the increase in the MIAplexÒ intensity Sptp. Comparing the nanoparticles functionalized with CA (Fig. 4, red curves), the spheres, hexagons and rods have similar Sptp values. DHmax decreases from hexagons to spheres and presents a very small value for nanorods. These three particles have a common crystalline diameter of 12 nm and are distinguished by their surface/volume ratio. The nanorods have a higher surface/volume ratio than spheres and hexagons. This indicates that the Sptp and DHmax values roughly correlate with the nanocrystal diameter and the shape-dependent surface/volume ratio, respectively. The signature of the nanocubes shows hysteretic opening and a small intensity Sptp (Fig. 4C, red curve). Their side length (26 nm) is higher than the nanosphere, nanohexagon and nanorod diameters (12 nm). This could explain the decrease in Sptp. Indeed, in a previous work we have shown that an increase in particle size induces a decrease in MIAplexÒ intensity Sptp [24]. The hysteretic opening (Fig. 4C, red curve) can be explained by the larger volume of the nanocubes, inducing dipolar interactions, as well as by a high surface anisotropy of the cubes compared with the spheres [61]. These differences should lead to different MIAplexÒ signatures which can be used in multiparamagnetic immunoassays. Indeed, the nanoparticle has two essential roles: to act as a probe, due to its specific magnetic properties, and to carry on its surface precursor groups for the covalent coupling of biological recognition molecules, such as antibodies and nucleic acids. The peak to peak line width DHmax varies from 1.7 (nanorods) to 7.5 kA m1 (nanospheres). Thus the spherical and rod-shaped nanoparticles appear to be the most interesting candidates for decomposition signal treatment, in the light of the results of the multiparametric tests.

Fig. 4. Room temperature MIAplexÒ magnetic signatures for (A) spherical, (B) hexagonal, (C) cubic and (D) rod-shaped ferrite nanoparticles in solution before (black curves) and after (red curves) surface functionalization with CA.

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Table 1 MIAplexÒ intensity Sptp and peak to peak line width DHmax of the spherical, hexagonal, cubic and rod-shaped nanoparticles in suspension before and after surface functionalization with caffeic acid. Nanospheres

Sptp (u.MIAplexÒ mg1) DHmax (kA m1)

Nanohexagons

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OA

CA

OA

CA

OA

CA

2249 ± 160 9.99 ± 0.70

3080 ± 220 7.5 ± 0.53

1680 ± 120 14.5 ± 1.0

2820 ± 200 9.0 ± 0.63

190 ± 13

1430 ± 100 5.6 ± 0.39

1850 ± 130 1.7 ± 0.12

2990 ± 210 1.7 ± 0.12

In a preliminary multiparametric test these two magnetic nanoparticles were mixed at two different ratios (Fig. 5). Fig. 5 shows the MiaplexÒ signatures for mixtures of the two kinds of particles at proportions of nanospheres/nanorods equal to 0.25/ 0.75 for sample A (red curve) and 0.75/0.25 for sample B (black curve). The two signatures are clearly different. The measured peak to Q5 peak line widths DHmax were 5990 and 2210 A m1 for samples A and B, respectively. This preliminary result on mixed nanoparticles demonstrates that the sensor is able to discriminate between various superparamagnetic nanoparticles differing in their magnetic signatures. This confirms the high sensitivity of measuring d2B(H)/dH2 for characterization and separation purposes. We have shown that this sensor is able to discriminate between the signatures of superparamagnetic nanoparticles differing in their shape anisotropy. These first results are very promising for the use of the MIAplexÒ technology for multiparametric detection.

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4. Conclusions

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In conclusion, we have designed a new magnetic nano-platform for multiparametric detection using magnetic nanoparticles of different sizes, shape and compositions coated with CA for dispersivity and stabilization in aqueous media at physiological pH. It has been demonstrated that the magnetic properties differ dramatically according to the nanocrystal shape and structure. Particles differing in shape display varied MIAplexÒ signatures. Hence, promising candidates could be identified to allow multiparametric testing. These results pave the way for the simultaneous detection of different types of biological molecules. Today these hybrid nanosystems are being evaluated for bioconjugation and use in bioassays.

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Nanorods

CA

Fig. 5. MIAplexÒ signatures of nanospheres and nanorods mixed in the proportions: (A) 75% nanospheres–25% nanorods (black curve); (B) 25% nanospheres–75% nanorods (red curve).

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Nanocubes

OA

Acknowledgements

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We gratefully acknowledge Hicham Jouni (Université Paris 13) for assistance with the ligand exchange process. This work was supported by ANR Biotecs and a grant from Region Île-de-France.

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Appendix A. Figures with essential colour discrimination

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Certain figures in this article, particularly Figs. 1–5, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2012. 11.025

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