Synthesis and Characterization of Superparamagnetic Nanoparticles

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Synthesis and characterization of superparamagnetic nanoparticles coated with fluorescent gold nanoclusters Xavier Le Guével, Eva-Marie Prinz, Robert Müller, Rolf Hempelmann, Marc Schneider

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Xavier Le Guével, Eva-Marie Prinz, Robert Müller, Rolf Hempelmann, Marc Schneider. Synthesis and characterization of superparamagnetic nanoparticles coated with fluorescent gold nanoclusters. Journal of Nanoparticle Research, Springer Verlag, 2012, 14 (4), pp.727. hal-02337892

HAL Id: hal-02337892 https://hal.archives-ouvertes.fr/hal-02337892 Submitted on 6 Nov 2019

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. J Nanopart Res (2012) 14:727 DOI 10.1007/s11051-012-0727-6

RESEARCH PAPER

Synthesis and characterization of superparamagnetic nanoparticles coated with ﬂuorescent gold nanoclusters

Xavier Le Gue´vel • Eva-Marie Prinz • Robert Mu¨ller • Rolf Hempelmann • Marc Schneider

Received: 26 June 2011 / Accepted: 5 January 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Multifunctional nanoparticles (NPs) com- kept the original fluorescence property of the metal bining the superparamagnetism of Mn-Zn ferrite and clusters with 211-fold increase of the red emission the fluorescence property of gold nanoclusters (NCs) (k = 690 nm) compared to the uncoated NPs. These have been prepared by wet chemistry. Magnetic NPs NPs can be moved with a permanent magnet despite a synthesized by co-precipitation method were coated 72-wt% increase of the non-magnetic fraction due to several times with oppositely charged polyelectrolytes the PE coating and the protein adsorption. (PEs) using the layer-by-layer technique. Common techniques (Fourier transform infrared spectroscopy, Keywords Nanoparticles Á Gold Á Nanoclusters Á electron microscopy, zeta potential, etc.) indicated the Fluorescence Á Superparamagnetism Á Biomedicine monodispersity and the stability of the coated NPs providing a positive charged surface. Fluorescent gold NCs bound to a standard protein bovine serum Introduction albumin were adsorbed on the surface of the magnetic NPs. Structural investigations proved the presence of In recent years, multifunctional nanoscaled particu- small gold clusters (*2 nm) in a shell surrounding the lates in a single entity have attracted great attention for magnetic nanomaterial. The stable nanocomposite their diagnostic and biomedical application (Alexiou et al. 2006; Jain et al. 2008; Liong et al. 2008; Sukhorukov and Mohwald 2007; Zhang et al. 2007). Electronic supplementary material The online version of Especially, the combination of magnetic and optical this article (doi:10.1007/s11051-012-0727-6) contains imaging into a nanostructured system would greatly supplementary material, which is available to authorized users. inspire and support in vitro diagnosis as well as the in X. Le Gue´vel (&) Á M. Schneider vivo monitoring of living cells and then open up the Pharmaceutical Nanotechnology, Saarland University, unique possibility of controlled target-directed appli- Saarbru¨cken, Germany cations (Kim et al. 2009; Koole et al. 2009). These e-mail: [email protected] nanosystems offer a promising approach to serve as all E.-M. Prinz Á R. Hempelmann in one diagnostic and nano-sized drug delivery Department of Physical Chemistry, Saarland University, carriers. For example, in the last few years, several Saarbru¨cken, Germany researchers demonstrated the potential to combine MRI contrast agent and fluorescent organic dyes that R. Mu¨ller Institute of Photonic Technology, P.O. Box 100239, Jena, can allow the detection of cancer through non-invasive Germany MRI and the optical guide during surgery (Veiseh 123 Page 2 of 10 J Nanopart Res (2012) 14:727 et al. 2005; Bridot et al. 2007; Lee et al. 2010). Such nanomaterial for biological applications is the bio- bifunctional nanocomposites have been prepared by compatibility of Mn-Zn ferrite (Beji et al. 2010) and several routes notably by a combination of colloidal gold NCs labeled to bovine serum albumin (BSA) iron oxide nanocrystals with different classes of (Retnakumari et al. 2010) for possible in vivo fluorophores such as organic dyes and inorganic experiments. semiconductor nanocrystals (so-called quantum dots) Therefore, combining the strong magnetic proper- and give promising hope for advanced therapies (Corr ties of ferrite, the ability of PEs to load model drugs et al. 2008; Kim et al. 2006; Landmark et al. 2008; and protect the NPs with proteins containing small and Liong et al. 2008; Quarta et al. 2008; Tamanaha et al. photostable fluorescent markers offer an interesting 2008; Yang et al. 2008). Moreover, one very potent approach to design a new kind of ‘‘smart’’ delivery approach is the layer-by-layer (LBL) assembly of systems. oppositely charged polyelectrolytes (PEs) from aqueous solution on a host surface, such as charged flat substrates (Lvov et al. 1993; Yoo et al. 1998)or Experimental nanoparticles (NPs) (Shenoy et al. 2003; Sun et al. 2009; Wang et al. 2008; Schneider and Decher 2008), All chemicals were purchased from Sigma-Aldrich to fabricate ultrathin films and multilayer structures of (Germany) and were used without any further purifi- tunable properties for coatings (Schneider and Decher cation. Ultrapure water (18.2 MX) was used in all 2008), electrical materials (Sun et al. 2001), sensors experiments. (Whitney et al. 2005), and drug delivery (Reum et al. A scheme of the synthesis protocol of the multi- 2010). However, some issues have to be tackled functional nanocomposite is depicted in Fig. 1. regarding (i) the fluorescence sensitivity hindered by First, mNPs with the composition Mn0.8Zn0.2Fe2O4 the strong quenching of the fluorophore by the called mNPs were synthesized by the chemical co- magnetic core, (ii) the drug loading efficiency of the precipitation method (Auzans et al. 1999; Alexiou nanomaterials (Labouta and Schneider 2010), and (iii) et al. 2006). Briefly, 40 mL of 0.04 mol FeCl3Á6H2O the protection of the payload which should not be solution was heated up to 80 °C and then mixed with a degraded too fast by catalytic-enzymatic processes. solution of 0.016 mol of MnCl2Á4H2O and 0.004 mol We describe in this article a simple route to design ZnCl2 dissolved in 8.8 mL water. The mixed solution magnetic fluorescent NPs using ferrofluid suspensions was added under vigorous stirring to 200 mL boiling of Mn-Zn ferrites coated with PE multilayers. solution of NaOH (0.25 M). The black suspension was Fluorescent property is brought by adsorbing gold stirred for 1 h at 90 °C. The precipitate was separated nanoclusters (NCs) covalently labeled to a standard with a permanent magnet and washed twice with protein on the surface of these magnetic nanoparticles water. To achieve a ferrofluid which is stable in water, (mNPs). Fluorescent noble metal (Ag, Au) NCs have it is necessary to provide a sufficient stabilization, e.g., emerged as a new family of fluorescent markers, and electrostatic repulsion between the particles. This several studies demonstrated the potential of those implies two steps: an acid treatment of the particle clusters capped in different templates [polymers surface followed by the stabilization with nitrate ions. (Duan and Nie 2007; Zhang et al. 2005; Lin et al. 2009), dendrimers (Zheng et al. 2003, 2007), or proteins (Le Guevel et al. 2011a; Retnakumari et al. 2010; Xie et al. 2009)] for biolabeling, imaging, and sensing. Those clusters have shown remarkable photo- physical properties such as intense fluorescence emission, strong photostability, and high Stokes-shift. Additionally, these clusters are smaller than quantum dots (\2 nm) with a fluorescence tunable in the all visible range due to size-dependent fluorescence (Wu and Jin 2010; Zheng et al. 2007; Zhu et al. 2008). Fig. 1 Scheme of the synthesis of magnetic NPs coated with Another key point regarding the application of this fluorescent gold NCs stabilized in BSA 123 J Nanopart Res (2012) 14:727 Page 3 of 10

Therefore, a 2-M HNO3 solution was added to the 3 mL AuBSA (15 mg/mL) under stirring for 30 min. washed particles and stirred at room temperature for The pH of the AuBSA was adjusted to 8 in order to 6 min. In parallel, 60 mL of an aqueous solution promote the electrostatic interaction between the containing 0.02 mol Fe(NO3)3Á9H2O and 8.5 mL of a magnetic NPs—positively charged—and the gold 0.008 mol MnCl2Á4H2O and 0.002 mol ZnCl2 was NCs-labeled BSA. Then, the mixture was centrifuged prepared at 80 °C. The two solutions were mixed and two times at high speed (15,000 rpm) and resuspended subsequently added to the particle suspension. The in water. The new solution mNPs-PE-AuBSA (5 mg/ resulting solution was stirred for 30 min at 80 °C. The mL) has a dark brown color and was kept refrigerated stabilized particles called mNPs were separated with a before characterization. permanent magnet and taken up in 200 mL of water. X-ray powder diffraction (XRD) patterns were In order to obtain stable ferroﬂuids at pH 7, the nitrate obtained with a Siemens D 500 diffractometer, using ions have been exchanged with citrate ions using a a Cu-Ka radiation (k = 1.54 A˚ ) at room temperature. dialysis against 0.01 M citrate solution for 7 days. The They were recorded in the range 20°-70° (2H) with a solution was then dialyzed against distilled water for typical step size of 0.02°. The XRD spectra were 7 days to remove the excess of citrate. compared to the Joint Committee on Powder Diffrac- A standard LBL technique was used to coat mNPs: tion Standards database (JCPDS) database. The esti- 9 mL of mNPs (23 mg/mL) was added dropwise to mation of the particle size is made via Scherrer 9 mL solution containing 50 mg poly(allylamine formula. Matrix-assisted laser desorption/ionization hydrochloride) (PAH, Mw = 1.800) under sonication (MALDI-TOF MS) (Applied Biosystems 4800) mea- for 20 min and then kept under stirring in the dark and surements of AuBSA were performed using a-cyano- at room temperature for 12 h. The solution was then 4-hydroxycinnamic acid (CHCA) as matrix. The puriﬁed by dialysis (membrane from Medicall Int.; spectrum was collected in the positive mode. The

Mwco 12–14,000 Da) against water for 2 days. A particle morphology was observed by transmission negative layer was coated by the same method as the electron microscopy (TEM) using JOEL Model JEM positive layer using in this case is a polyacrylic acid 2010 instrument (JOEL GmbH, Eching, Germany)

(PAA, Mw = 15,000) solution (50 mg in 9 mL of operated at an accelerating voltage of 120 kV. The water). A third PAH layer was adsorbed using the presence of the gold NCs was detected by energy- same protocol as previously and resulted in a positive dispersive X-ray spectroscopy (EDX). For each exper- f-potential of the NPs at neutral pH. The new sample iment, a drop of the colloidal solution of particles in mNPs-PE was kept refrigerated in the dark before water was deposited on the copper grid ([ = 3 mm). further experiments. The hydrodynamic diameters and the surface charge of Fluorescent gold NCs stabilized in BSA have been the particles were analyzed after each functionaliza- synthesized using the protocol reported by Xie et al. tion step using a Zetasizer Nano ZS from Malvern.

(2009). Briefly, 5 mL aqueous solution of HAuCl4 Fourier transform infrared spectroscopy (FTIR) spec- (10 mM) was reacted to 5 mL of BSA (96%; 20 mg/ tra of coated and uncoated ferrofluids were measured mL) under vigorous stirring at 37 °C. Then, 50 lLof on a ZnSe slide by a Perkin Elmer spectrometer ascorbic acid (0.35 mg/mL) was added dropwise. Ascor- between 750 and 4,000 cm-1. The characteristic bic acid allowed to trigger the formation of gold NCs vibrations of the organic groups present in the nano- without using a large amount of BSA. After 5 min, composite have been collected in the supporting 0.5 mL of NaOH solution (1 M) was introduced, and the information. UV–visible absorption spectra of the reaction proceeded for 5 h at 37 °C. The solution labeled protein AuBSA were measured using a changed the color from pale yellow to deep brown after Lambda 35 scan UV–visible spectrophotometer (Per- 3 h. Two more hours are necessary to complete the kin Elmer) in transmission mode. Fluorescence scans reduction of BSA-encapsulated Au precursor. The excess of the samples were performed on a Tecan infinite of the unreacted gold salt was removed by dialysis for M200 instrument using microplates. Static magnetic 15 h. The new solution AuBSA (*15 mg/mL) was measurements were performed on a Vibrating Sample TM stable for over 5 months when kept refrigerated. Magnetometer MicroMag 3900 (Princeton Measure- AuBSA was adsorbed on the surface of mNPs-PE ments Corp., USA). The magnetic cores before and by adding dropwise 1 mL mNPs-PE (5 mg/mL) to after PE coating and fluorescent protein adsorption 123 Page 4 of 10 J Nanopart Res (2012) 14:727 were characterized both in fluid state and in immobi- peaks on the XRD pattern suggests the formation of a lized dry state. The immobilized samples were single phase of Mn-Zn ferrite during the synthesis prepared by drying at 35 °C (uncoated particles: without any impurity phases. The weak broad band at 40 °C) over night and pressing the powder in a quartz 2h = 22° is attributed to the substrate used for the tube. The magnetization loop M(H) of a dry sample measurement. Hydrodynamic diameter of mNPs was measured within 10 min, the fluid samples within determined by dynamic light scattering (DLS) 30 min in order to reduce the noise. All measurements (Fig. 3a) indicates a larger diameter d = 28 ± 4nm were normalized to the sample mass. The specific than by TEM. This behavior could be related to the saturation magnetization values, taken from the mag- water shell on the surface of the magnetic NPs or to a netization loops, allowed to estimate the amount of possible aggregation despite a polydispersity index bound non-magnetic material for all samples in dry below 0.2 suggesting narrow distributed particles in condition (coating) and in solution (mNP concentra- the sample. Infrared spectroscopy of mNP sample tion). The magnetization data of fluid samples were shows a broad band located at 1,600 cm-1 corre- corrected by the diamagnetic effect of the sample sponding to the vibration of the carboxylate groups holder and container. and hence confirms the presence of citrate groups on Because of the superposition of the diamagnetic the surface of the magnetic NPs. effect (linear with negative slope) of the carrier fluid The LBL technique has been optimized to coat the and the ferrimagnetic effect of the particles, the magnetic NPs with PE layers (three layers: PAH- specific saturation magnetization cannot be taken from PAA-PAH) using the positively charged PAH and the the magnetization curve at high-field strength (in the negatively charged PAA. The outer layer was PAH. case of very low-mNPs concentrations). The maxi- Successful modification of the surface charge after mum magnetization appeared at lower fields because each coating step and the presence of PAH and PAA the diamagnetic effect is low. In the present case, M of on the NPs has been confirmed by zeta potential the particles considerably depends on H, and a field- measurement and infrared spectroscopy (Supplemen- dependent correction factor M(H)/MS (MS: saturation tary Fig. S2). TEM micrograph of mNPs-PE (Fig. 2b) magnetization) was introduced, which was taken from indicates no change of the morphology and no a magnetization loop of non-coated dry ferrite parti- significant aggregation of the magnetic NPs after the cles. According to Msample-corrected(H) = Msample(H)/ PE coating. The PE coating led to an increase of the (Mferrite(H)/MSferrite), the maximum of the corrected NPs diameter from 28 to 35 nm (Fig. 3a) accompa- curve was used. nied by a rise of the surface charge value from f = -49.4 mV to ?50 mV (Fig. 3b) due to the presence of the protonated amino groups of PAH at pH 7. The real Results and discussion thickness of the PE shell is expected to be *3nm (Nooney et al. 2010). However, due to the strongly Structural investigation charged surface of mNPs-PE NPs, the hydrodynamic diameter gives a higher value. The rise of the thickness

Ferroﬂuids made from stable mNPs Mn0.8Zn0.2Fe2O4 layer could be also attributed to the permeability and have been prepared following a protocol described by the swelling properties of PE layers in aqueous Massart et al. (Auzans et al. 1999). Electron micros- environment. Indeed, numerous surveys demonstrate copy image in Fig. 2a shows a relative monodisperse the crucial role of pH, temperature, and nature of the distribution of the mNPs cores (mNPs) with a size PE to the water adsorption into the PE layers around 15 nm. The estimation of the crystallite size (Schonhoff 2003; Harris and Bruening 2000; Gao was made via the Scherrer formula using the half- et al. 2003). FTIR spectrum of mNPs-PE in Fig. 3c maximum width of the (440) X-ray diffraction line. indicates the presence of PAA with the characteristic The crystallite size was determined to be 13 nm what stretching vibration peaks of C=O and the deproto- is in agreement with the TEM pictures of uncoated nated carboxyl groups at 1,710 and 1,545 cm-1, particles. The XRD spectra (Supplementary Fig. S1) respectively. N–H, CH2–CO, and C–N at 1450, -1 indicate the characteristic peaks of Mn0.8Zn0.2Fe2O4 1385, and 1032 cm are also clearly visible on compared to the JCPDS database. The absence of extra mNPs-PE spectrum. The standard vibration peaks of 123 J Nanopart Res (2012) 14:727 Page 5 of 10

Fig. 2 TEM images of a mNPs, b mNPs-PE, c mNPs-PE- protein AuBSA. Picture of the protein shell at higher AuBSA, and d zoom of the coating surrounding the magnetic magnification (d) indicates the presence of monodisperse gold NPs revealing AuBSA. Analysis of the micrographs showed a NCs with sizes smaller than 3 nm inside the protein BSA distribution of 15 nm magnetic NPs embedded in the labeled the PEs are gathered in the supporting information shell surrounding the dark magnetic cores (high (Supplementary Table S1). electron density). Higher magnification of the shell Gold NCs labeled to the protein BSA, called here between the magnetic cores (Fig. 2d) indicates the AuBSA, have been already the subject of previous presence of objects smaller than 3 nm called NCs. studies (Le Guevel et al. 2011b, c; Simms et al. 2009). Elemental analysis confirmed the gold nature of those Structural and chemical investigations followed by NCs (Supplementary Fig. S6). Size of the particles is MALDI-TOF and XPS indicated a polydisperse increasing from 35 to 100 nm but shows a relatively distribution of 5-33 gold atoms—partially oxidized narrow distribution (PDI = 0.210). The zeta potential with Au0 (90%) and AuI (10%)—covalently bound to measurement of mNPs-PE-AuBSA indicated the BSA via the thiol groups of the cysteine residues adsorption of the protein—negatively charged at pH (Supplementary Figs. S3, S4). The labeling involves a 8—on the PE-coated magnetic NPs with a drop of the modification of the secondary structure of the protein surface charge from ?50 to -17 mV. Infrared spec- confirmed by infrared measurements. AuBSA is stable trum of mNPs-PE-AuBSA plotted in Fig. 3c showed in a wide range of pH from 4 to 11 and in PBS buffer the presence of the characteristic vibration bands of (Supplementary Fig. S5) without significant aggrega- AuBSA located at 1,650 and 1,545 cm-1 correspond- tion. TEM image (Fig. 2c) shows the coating of ing to the amide I (mainly CO stretch) and the amide II AuBSA on the NPs with the presence of a translucent bands (C–N stretch coupled with N–H bending mode), 123 Page 6 of 10 J Nanopart Res (2012) 14:727

Fig. 3 a Size measurements determined by DLS and b zeta potential of mNPs, mNPs-PE, and mNPs-PE-AuBSA samples. Results conﬁrmed the size enhancement and the change of the surface charge after PE coating and protein adsorption on the magnetic NPs. c Infrared spectra between 850 and 1,750 cm-1 of NPs after each functionalization step. Characteristic vibration peaks of the PE and the protein BSA conﬁrmed the presence of PE and gold NCs-labeled BSA on magnetic NPs for the sample mNPs-PE-AuBSA

respectively. Compared to AuBSA, mNPs-PE-AuBSA presents the aspect of a saturated signal independent presents a broader amide I band due to the overlapping of the NPs concentration in solution, which might be with the broad band of the deprotonated carboxyl related to the scattering of the particles. Another groups of PAA. We also notice the absence of the C=O possible explanation for this trend could be associated peak which proves a complete deprotonation of the to the intrinsic nature of the gold NCs exhibiting photo- carboxyl groups after the protein adsorption. The other physical properties similar as quantum dots (Bawendi characteristic peaks of the PEs were clearly detected et al. 1992). Furthermore, an additional excitation peak on mNPs-PE-AuBSA sample with the presence of the is also detected at 280 nm (Supplementary Fig. S8). carboxylate groups (1,610 cm-1), the aliphatic groups This peak is attributed to the absorption of the aromatic -1 -1 (1,520 cm ), and CH2CO groups (1,395 cm ). This residues of the protein, which fluoresce at lower behavior was confirmed with the vibration of the wavelengths (kem \ 400 nm) and then contribute to aliphatic groups at higher wavenumbers between the energy transfer between the protein and the gold 2,700 and 3,800 cm-1 (Supplementary Fig. S7). NCs. Figure 4a illustrates the presence of an intense emission peak centered at 690 nm. A full excitation/ Fluorescence and magnetic properties emission spectrum of AuBSA indicates as well a weak emission band located at 450 nm (Supplementary Fig. The multifunctional NPs mNPs-PE-AuBSA present S9). These multiple and broad emission bands are multiple excitation peaks: one located at 380 nm explained by the polydispersity in size of the gold partially overlapped by the strong absorption of the clusters in the protein (Simms et al. 2009; Negishi et al. magnetic core and a second one located at 530 nm 2005). Indeed, in the last few years, many authors have (Fig. 4a). The excitation band in the UV region described the size-dependent fluorescence emission of 123 J Nanopart Res (2012) 14:727 Page 7 of 10

Au NCs in several templates with the presence of transition between ligand (cysteine groups of BSA) clusters of different sizes emitting in the blue-green and the gold cluster core and (2) to the plasmonic region (kexc/kem = 380 nm/450 nm) and in the red resonance of few gold NPs present with sizes bigger region (kexc/kem = 530 nm/690 nm) (Bao et al. 2007; than 2 nm trapped in BSA. The absorbance spectrum Le Guevel et al. 2011b). The strong red emission under of the coated magnetic mNPs-PE-AuBSA showed only UV irradiation could be attributed to the energy the strong absorption of the magnetic core in the visible transfer between the small clusters to the bigger ones. range, which overlaps the absorption band of the gold Compared to AuBSA (Supplementary Fig. S9), no shift NCs. of the excitation and emission bands of the mNPs-PE- The quantum yield of AuBSA has been determined AuBSA has been observed conﬁrming the photosta- by comparison with a standard organic dye (Rhoda- bility of the AuBSA on the magnetic cores. Fluorescent mine 6G, 95% in ethanol) with a value between 5 and properties of noble metals (Au, Ag) are strongly 6% in agreement with the literature (Xie et al. 2009). dependent on the oxidation state of the metal and the Fluorescence measurements of mNPs-PE-AuBSA and nature of the ligand (Negishi et al. 2005; Wu and Jin mNPs-PE samples indicate a 211-fold increase of the

2010). Therefore, it is crucial to keep the integrity of relative fluorescence intensity (kexc/kem = 390 nm/ AuBSA using a soft-coating method in order to prevent 690 nm, gain = 100) with the presence of the gold a drop in fluorescence intensity. Absorbance spectra of clusters. The main limitation to obtain brighter NPs in AuBSA and mNPs-PE-AuBSA are plotted in Fig. 4b. our case was related to the formation of a single Strong contribution of scattered light for lower wave- protein monolayer around mNPs-PE and to the strong lengths overlapping with the specific absorption of the absorption of the magnetic core at the lower wave- NCs is clearly visible. Nevertheless, the presence of a length (k \ 450 nm), which reduces the excitation weak broad peak for AuBSA located at 530 nm can be efficiency of the gold NCs. seen. It could be speculated that this shoulder might be The magnetic measurements of the dried mNPs attributed to two different aspects: (1) an electronic sample showed a slightly lower specific saturation

Fig. 4 a Excitation and emission spectra of mNPs- PE-AuBSA sample between 300 and 850 nm. b Extinction spectra of AuBSA (dashed line) and mNPs-PE-AuBSA (solid line). c Pictures of mNP-PE- AuBSA solution—1 mg/mL in water—under UV irradiation (k = 366 nm) with the presence of a permanent magnet at t = 0 (on the left) and t = 30 min (on the right). Images clearly show the impact of the magnetic ﬁeld and the distribution of the red emission of the multifunctional NPs. (Color ﬁgure online)

123 Page 8 of 10 J Nanopart Res (2012) 14:727

Fig. 5 Magnetic measurements of mNPs, mNPs-PEs, and mNPs-PE- AuBSA in dry state (a) and in ﬂuid state (b) showing the superparamagnetic behavior after each synthesis step

magnetization (Fig. 5a) than usual maghemite NPs mNPs, mNPs-PE, and mNPs-PE-AuBSA, respec- (54.8 and 65 Am2/kg, respectively, at room temper- tively. Despite the decrease of the magnetic strength ature). The coercivity of the sample was determined at after the PE coating and the adsorption of the labeled 0.24 kA/m, which is smaller than for iron oxide (0.5- protein, these multifunctional NPs still can be moved 1 kA/m assuming a diameter of NPs around 13 nm), with the presence of permanent magnet (Fig. 4c). but for a final result a size distribution indicating a non-superparamagnetic fraction had to be determined. The remanence ratio (remanent magnetization/saturation magnetization) is 0.013, i.e., the particles are Conclusions mostly superparamagnetic considering remanence ratio of superparamagnetic and ferrimagnetic com- We report in this article a simple route to synthesize pounds with statistical orientation at 0 and 0.5, bifunctional NPs. mNPs coated with PEs multilayers respectively. The PE-coating and AuBSA binding are highly monodisperse and possibly allow the lead to an increase of the non-magnetic fraction in the incorporation of macromolecules (drugs, proteins, dry samples at 67 and 72 wt%, respectively. Addi- peptides, etc.) for use as drug delivery carriers. tionally, the diamagnetic effect of the pure AuBSA- Furthermore, the fluorescence is brought by gold cluster is depicted in Supplementary Fig. S10. NCs labeled to the standard protein BSA. They are From magnetization data of the uncoated cores, the adsorbed on those NPs by electrostatic interaction and mean diameter d and the deviation r of a lognormal emit in the red region with a high-fluorescence signal size distribution can be calculated according to (*200-fold compared to the uncoated NPs) without Chantrell et al. (1978) assuming a superparamagnetic modification of the photo-physical properties of the behavior (Langevin-magnetization curve). We calcu- metal clusters. The multifunctional NPs are stable in lated the diameter of the particles d = 11.2 nm and aqueous solution at different pH and can be moved r = 0.34 taking the measured specific saturation easily with a permanent magnet. Therefore, the study magnetization into account (see above) and a density shows the potential of using this new family of of 5 g/cm3 for the dried sample. These results biolabels—metal NCs—to design NPs with fluores- correlated well with the structural investigations. cence and magnetic properties for several biomedical The concentration of the mNPs in solution applications such as imaging and drug delivery decreased by the necessary dilution during the coating systems. However, before performing any in vivo step procedures (Fig. 5b). Using the measured mag- application, critical issues (cytotoxicity, degradation, netization of the cores and taking into account the uptake mechanism, etc.) still need to be addressed field-dependent correction factor mentioned in the to demonstrate the applicability of these dual mag- experimental section, we determined these concentra- netic fluorescent nanocomposites in biological tion values to be around 1.8, 0.96, and 0.014 wt% for environment. 123 J Nanopart Res (2012) 14:727 Page 9 of 10

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