Synthesis and functionalization of biocompatible Tb:CePO4 nanophosphors with spindle-like shape

1 Synthesis and functionalization of biocompatible Tb:CePO4 nanophosphors with spindle-like shape Sonia Rodriguez-Liviano,† Francisco J. Aparicio,† Ana I. Becerro,† Jorge García-Sevillano,‡ Eugenio Cantelar,‡ Sara Rivera,# Yulán Hernández,# Jesus M. de la Fuente, # Manuel Ocaña†* †Instituto de Ciencia de Materiales de Sevilla (CSIC-US), Americo Vespucio 49, Isla de La Cartuja, 41092 Sevilla, Spain. #Instituto de Nanociencia de Aragon, Universidad de Zaragoza, Mariano Esquillor s/n, Zaragoza, 50018, Zaragoza, Spain ‡ Dpto. Física de Materiales, C-04. Universidad Autónoma de Madrid, 28049 Madrid, Spain. *Corresponding Author: Manuel Ocaña. E-mail: [email protected] Abstract Monoclinic Tb:CePO4 nanophosphors with a spindle-like morphology and tailored size have been prepared though a very simple procedure, which consists of aging, at low temperature (120ºC), ethylene glycol solutions containing only cerium and terbium acetylacetonates and phosphoric acid, not requiring the addition of surfactants or capping agents. The influence of the heating mode (conventional convection oven or Manuscript Click here to download Manuscript: Manuscript JNR.doc Click here to view linked References 2 microwave oven) and the Tb doping level on the luminescent, structural and morphological features of the precipitated nanoparticles have been also analyzed. This study showed that microwave-assisted heating resulted in an important beneficial effect on the luminescent properties of these nanophosphors. Finally, a procedure for the functionalization of the Tb:CePO4 nanoparticles with aspartic-dextran is also reported. The functionalized nanospindles presented negligible toxicity for Verocells and a high colloidal stability when dispersed in water (pH=6.7), which along with theirs excellent luminescent properties, make them suitable for biomedical applications. Key words: Nanoparticles ∙ Cerium phosphate ∙ Terbium ∙ Luminescence ∙ Functionalization∙ Aspartic-dextran 3 Introduction Cerium(III) phosphate doped with Tb3+ cations (Tb:CePO4) is a well known green emitting phosphor with low toxicity, high thermal and chemical stability, high luminescence efficiency and sharp emission bands, which finds important applications in chemical and biological sensing. For example, this phosphor has been recently proposed as an efficient oxygen sensor making use of the luminescence quenching produced when it is exposed to an oxidizing ambient, as a consequence of the partial oxidation of Ce3+ to Ce4+ (Di et al. 2010a). The application of this material for detection of molecules of biological interest, such as vitamin C, has been also suggested taking advantage of the recovering of luminescence occurred when previously oxidized Tb:CePO4 nanophosphors are put into contact with such molecules (Di et al. 2010b). Finally, Tb:CePO4 nanowires have been also used for bioimaging purposes (Zhang and Wong 2010). For most of these uses, the control of particle morphology has been shown to be essential to optimize the nanophosphors performance (Chen et al. 2011). In particular, for biomedical applications, it has been well established that the particle geometry plays an important role in cell-material interactions, affecting cellular uptake and cell functioning. For example, Huang et al. (2010) have shown recently that anisometric nanoparticles are taken up by cells in larger amounts and have faster internalization rates than those with small aspect ratios. They also found that particles with larger aspect ratios have a greater impact on different aspects of cellular functions including cell proliferation, apoptosis, cytoskeleton formation, adhesion and migration. Particle size is another important issue for bioapplications. Thus, for biolabeling, especially for the sensitive analysis of molecules such as DNA, RNA or proteins, nanosized 4 phosphors are required, since their reduced size greatly increases surface area and, therefore, the biosensor sensitivity (Li et al. 2008). Therefore, the development of methods suitable for the fabrication of monoclinic Tb:CePO4 nanophosphors with uniform morphologies and narrow size distribution in the nanometer range is of high interest. Recently, several procedures for the synthesis of uniform Tb:CePO4 based nanophophors with anysometric particle shape have been reported. Most of these methods conducted to long (in the micron range) nanowires (Bao et al. 2010, Bu et al. 2005, Di et al. 2010, Yu et al. 2009, Zhang et al. 2009, Zhang et al. 2010), nanorods (Komban et al. 2011) or nanotubes (Chen et al. 2008) with hexagonal structure. However, much fewer reports can be found regarding to CePO4 nanoparticles with monoclinic structure, which is preferred since the presence of water in the hexagonal phase partially quenches the luminescence of this kind of phosphors (Niraj Luwang et al. 2011). Actually, to the best of our knowledge, only two precipitation methods have been developed which yield one-dimensional CePO4 nanoparticles (<200 nm) with monoclinic structure, involving high synthesis temperatures (200ºC) and the use of toxic surfactants (CTAB) (Buchold and Feldman 2008) or complexing agents (EDTA) (Ma et al. 2008). In this paper, we describe a much simpler (it does not require the use of toxic additives) low temperature (120ºC) route for the synthesis of Tb:CePO4 nanophosphors with spindle-like morphology and narrow size distribution in the nanometer size range. The procedure is based on a homogeneous precipitation process in biocompatible ethylene glycol (EG) solutions containing only phosphoric acid (H3PO4) and Ln acethylacetonates (Ln(acac)3) as phosphate and Ln precursors, respectively. We also have investigated the influence of the heating source (conventional convection oven or 5 microwave oven) on the morphological, structural and luminescent features of the precipitated nanoparticles, finding an important beneficial effect of microwave-assisted heating on the luminescent properties of these nanophosphors. Finally, it is also well known that surface modification of the nanoparticles is important for most bioapplications since the surface properties determine the colloidal stability of the nanoparticles and the ability to provide anchors for adding functional ligands such as antibodies and drugs (Chatterjee et al. 2010). Although several biomedical applications have been suggested for Tb-CePO4 nanophosphors, very few works dealing with their functionalization have been reported. In fact, we have only found in literature a recent work on the functionalization of Tb-CePO4 nanorods with “Quat-primer” polymers (Komban et al. 2011). Herein, we also describe for the first time a procedure for the functionalization of the Tb-doped CePO4 nanospindles with aspartic-dextran polymers. The so functionalized nanophosphors showed negligible toxicity and a high colloidal stability in aqueous dispersions, which make them suitable for biomedical applications. Experimental section Reagents Cerium(III) acetylacetonate hydrate (Ce(C5H7O2)3·xH2O, Aldrich, 99.9%), terbium acetylacetonate hydrate (Tb(C5H7O2)3·xH2O, Aldrich, 99.9%), orthophosphoric acid (H3PO4, Aldrich, 85%) and ethylene glycol (EG, Fluka, < 99.5%) were used as received for the synthesis of nanoparticles. Albumin from bovine serum (BSA, ≥ 98% Sigma- Aldrich); tetraethylenepentamine (TEPA, C8H23N5, Aldrich), sodium citrate tribasic 6 dehydrate (C6H5Na2O7·2H2O, ≥ 99.5 Sigma) and aspartic-dextran polymers were used for functionalization. Dextran from Leuconostoc mesenteroides (average mol wt 15,000-30,000, Sigma), sodium (meta)periodate (≥99.0%, Sigma-Aldrich), sodium borohydride (≥98,0% Sigma-Aldrich), L-aspartic acid (≥98%, Sigma), borane trimethylamine complex ( >95%, Fluka), hydrochloric acid (37%, Prolabo), sodium hydroxide (≥98%, Sigma-Aldrich) and sodium acetate anhydrous (>99.5%, Fluka) were used for the synthesis of aspartic-dextran. Synthesis of nanospindles Weighted amounts of Ce(acac)3 and Tb(acac)3 were dissolved in EG under magnetic stirring while heating at low temperature (~75ºC) to favor dissolution. Then, these solutions were cooled to room temperature and proper amounts of phosphoric acid were admixed so that the final H3PO4 concentration was 0.15 mol dm-3. The final Ce+Tb concentration was kept constant at 0.004 mol dm-3, whereas the Tb/Ce+Tb ratio was varied in the 0-15% molar range. After homogenization, the final solutions (total volume = 10 cm3) placed in tightly closed reactors were aged at 120ºC in a convection oven preheated at such temperature (hereafter it will be referred as conventional heating, CH) or in a microwave oven (Sineo MDS-8), which was heated at the target temperature using a heating rate of 14ºC min-1 (hereafter it will be referred as MW heating). The dispersions obtained after aging were cooled down to room temperature, centrifuged to remove the supernatants and washed twice with ethanol and once with double distilled water. Finally, the precipitates were dispersed in water. For some specific analyses, the powders were dried at room temperature. 7 For functionalization, solutions with adjusted pH of sodium citrate (20 mmol dm-3, pH=4-13), BSA (10 g cm-3, pH=5), TEPA (20 mmol dm-3, pH=5-8) and asparticdextran (20% oxidized) (0.18 mmol dm-3, pH=5-8) were first prepared. Then, a weighted amount of Tb:CePO4 nanoparticles was added to such solutions so that their final concentration was 0.06 mg cm-3. The resulting dispersions were sonicated in cold water during 1 h. After this treatment, the so functionalized nanoparticles were washed several times with MilliQ water by centrifugation and finally redispersed in MilliQ water. The aspartic-dextran polymers solutions were prepared following a procedure previously reported by Fuentes et al. (2004) (details are provided in supporting information). Characterization Particle shape was examined by transmission (TEM, Philips 200CM) electron microscopy. Particle size distributions were obtained by counting several hundreds of particles from the TEM micrographs. EDX spectroscopy couplet to the TEM microscope was used to obtain information on the particles composition. The quantitative composition of the samples was analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Horiba Jobin Yvon, Ultima 2). The crystalline structure of the prepared samples was assessed by X-ray diffraction (XRD, Panalytical, X´Pert Pro with an X-Celerator detector). Unit cell parameters were determined from the XRD data (collected at intervals of 0.03° (2) for an accumulation time for interval of 3000 s) by using the Rietveld method with the JANAsoftware (Petricek et al. 2006). The starting parameters were taken from (Ni et al. 1995). To gain 8 additional information on the structural features of the synthesized nanoparticles, they were also characterized by high resolution transmission electron microscopy (HRTEM, Philips 200CM). The digital diffraction patterns (DDP) were obtained from the HRTEM images using Digital Micrograph software. The infrared spectra (FTIR) of the nanophosphors diluted in KBr pellets were recorded in a Jasco FT/IR-6200 Fourier transform spectrometer. Thermogravimetric analyses (TGA) were performed in air at a heating rate of 10°C min-1, using a Q600 TA Instrument. The excitation and emission spectra of the nanophosphors dispersed in water (0.5 mg cm-3) were recorded in a Horiba Jobin-Yvon Fluorolog3 spectrofluorometer operating in the front face mode. Lifetime measurements have been performed under pulsed excitation (10 ns) by using a Nd:YAG laser (Spectra Physics model DCR 2/2A 3378) linked to a harmonic generator. The fourth harmonics at 266 nm has been selected for Ce3+ excitation. Tb3+ luminescence at = 544 nm (5D4 → 7F5 transition) was detected by using a photomultiplier tube and averaged by a digital oscilloscope. The photographs showing the luminescence of the nanophosphors in water dispersions were taken under illumination with ultraviolet radiation (= 254 nm), filtered from a Hg discharge a lamp. The X-ray photoelectron spectra (XPS) of the samples were measured with a VG Escalab apparatus (Model 220) using the AlK excitation source. Calibration of the spectra was done at the P2p peak corresponding to phosphate group taken at 132.4 eV (Wagner et al. 1973). Z potential measurements were carried out in a Malvern Zetasizer Nano-ZS90 apparatus. The isoelectric point (i.e.p.) of the nanophosphors was determined by measuring the values of Z potential as a function of pH. For this, the nanophosphors were dispersed in 0.01 mol dm-3 NaCl solutions to keep the ionic strength constant, and 9 the pH was varied by adding HCl or NaOH as needed. After adjusting pH, the suspensions were stabilized overnight. The colloidal stability of the nanophosphors suspensions having a nanoparticle content of 0.5 mg cm-3 was monitored by analyzing the evolution of their UV-visible absorbance spectrum with aging time. Cell culture Vero (monkey kidney epithelial cells) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 5% glutamine and 5% penicillin/streptomycin. Cell cultures were incubated at 37ºC and equilibrated in 4% CO2 and air. Viability Testing Cell viability and proliferation were analyzed by the MTT colorimetric assay. For the cytotoxicity assay 5000 cells were seeded in each well of 96-well plates and grown for 24 h. After 24 h, the medium was replaced with fresh medium containing the different types of nanoparticles in varying concentrations. After cultivation again for 24 h, 20 µL of MTT dye solution (5 mg/mL in PBS) was added to each well. After 4 h of incubation at 37 °C and 5% CO2, the medium was removed, the cells were washed with fresh medium, and formazan crystals were dissolved in 100 µL of DMSO. The absorbance of each well was read on a microplate reader (Biotek ELX800) at 570 nm. The spectrophotometer was calibrated to zero absorbance using culture medium without cells. The relative cell viability (%) related to control wells containing cell culture 16 shoulder at 255 nm, which are due to 4f→5d electronic transitions of Ce3+ (Riwotzki et al. 2000). Also as expected, the emission spectrum, which was recorded using an excitation wavelength of 255 nm for a better observation of all the emission features of the Ce3+ ions, displayed a broad band centred at 352 nm corresponding to the transition from the excited 5d1 configuration to the 2F5/2 and 2F7/2 levels of Ce3+ (Guan et al. 2007). As observed in Fig. 6 (top), the excitation spectrum monitored at the main Tb emission line at 542 nm (Riwotzki et al. 2000) for sample Tb0.02Ce0.98PO4 prepared by conventional heating was quite similar to that of undoped CePO4 (Fig. 5) indicating that the Tb emission is produced after an energy transfer from excited Ce3+ to Tb3+ cations, which confirmed the success of the doping procedure. The emission spectra obtained for all Tb doped samples prepared by conventional heating (Fig. 6, bottom) clearly showed the characteristics emissions of Tb3+ (from 450 to 650 nm) arising from the 5D4-7Fj (J = 6, 5, 4, 3) transitions (Guan 2007), the most intense appearing at 542 nm, which is the main responsible for the green luminescence of this nanophosphor (inset in Fig. 6, bottom). The broad band associated to the Ce ions was still detected in these spectra, which indicated that the energy transfer from the latter to the Tb cations was not complete. Fig. 6 (bottom) also shows that the increase of the Tb content from 2 to 15% resulted in an increase of the emissions intensity, in apparent contrast with a previous report on Tb:CePO4 nanowires, which has showed a decrease of luminescence intensity above a 5% Tb content due to the well known concentration quenching effect (Bao et al. 2010). In order to get information on the luminescence efficiency of our samples, the values of lifetime for the 5D4 Tb3+ manifold were measured after pulsed excitation using the fourth harmonics of a Nd:YAG laser (exc = 266 nm). It was verified that the emission spectra under this excitation are coincident with those obtained after 255 nm excitation shown in Fig. 6. The normalized temporal decays of the 5D4 level, measured 17 atem = 544 nm (5D4 → 7F5 transition), for different Tb concentrations are shown in Fig. 7. The observed Tb luminescence decays are multi-exponential, as it has been reported for other nano-crystalline hosts (K. Riwotzky 2000, Pankratov 2010). In order to quantify the decay we have used the effective lifetime, , which is defined as (Inokuti 1965):   00 )()( dttIdttIt  It can be observed that the emission lifetime varies from  = 1.05 ms to  = 0.54 ms when terbium concentration is increased from 2% to 15%, as it is depicted in the inset of Fig. 7. It has been also checked that the same values are obtained when the decays are measured in the other luminescence bands associated to the radiative decay from the 5D4 Tb level. Consequently, it can be concluded that concentration quenching is also present in our samples for doping levels above 2%, in agreement with previous reports (Bao et al 2010). The increase of luminescence observed as increasing the Tb content must be then mainly attributed to the increase of luminescence centers, although the increase of crystallite size as increasing the Tb content (see Table S1 in the supplementary information), should also have a positive effect on the increase of the Tb luminescence since crystal imperfections may act as effective luminescence quenchers (Zhu 2010). As expected, the features of the excitation and emission spectra of the Tb0.02Ce0.98PO4 nanophosphor prepared by MW-assisted heating (Fig. 8) were found to be similar to those of the samples prepared by conventional heating. However, as observed in Fig. 8 (bottom), the intensity of the emissions of the former (Tb doping level = 2%) was even higher than that associated to the sample prepared by conventional heating having the strongest emission (Tb0.15Ce0.85PO4), which contained a much higher Tb content (15%). This finding is in agreement with lifetime measurements (Fig. 7) since the lifetime 18 value obtained for the sample synthesized by MW heating was much ( = 1.86 ms) higher than those corresponding to all samples prepared by conventional heating (Fig. 7, inset), which ranged from 0.54 (15% Tb) to 1.04 ms (2%Tb). To explain the higher luminescence efficiency in the sample prepared by MW-assisted heating, we must take into account that crystal imperfections and the presence of impurities are among the main known factors that may quench the luminescence of lanthanide ions. An additional cause of luminescence quenching in Tb3+/Ce3+ codoped systems is the partial oxidation of Ce3+ to Ce4+ (Di et al, 2010a). In our case, the only impurity contained in the nanospindles prepared according to the two heating methods was a certain amount of hydroxyl anions and/or water molecules (see Fig. S8 in supplementary information), as also observed for the undoped samples (Fig. 3). TGA analyses (Fig. S9 in supplementary information) revealed that the amount of these impurities contained in both kinds of samples was very similar (~6%), which suggest that the quenching by impurities does not seem to be the main cause of their different luminescence intensity. In order to identify the Ce oxidation state in these samples, their XPS spectra were also recorded. As observed in Fig. 9, almost identical spectroscopic features were detected for the two main Ce3d peaks measured for both samples, which appeared at the binding energy (903.5 and 885.3 eV) expected for Ce3+ species (Di et al. 2010a). The presence of Ce4+ in both samples can be disregarded in view of the absence of a peak at a binding energy of 917 eV, which is characteristic of tetravalent cerium (Di et al. 2010a). Therefore, we conclude that the much stronger luminescence of the sample prepared by microwave-assisted heating is due to its much higher crystallinity, as detected by XRD and HRTEM measurements. It should be noted that the lifetime obtained for our optimum nanophosphor (Tb0.02Ce0.98PO4 synthesized by MW-assisted heating) ( = 1.86 ms) was among the 19 highest values so far reported for this system (1.4-1.97 ms) (Yu et al 2009, Li et al 2010). This sample was then selected for functionalization, colloidal stability measurements, and citotoxicity analyses. Functionalization and cell viability For the functionalization of the nanophosphors different charged molecules were used at different pH to be linked to the nanoparticles by ionic adsorption, such as BSA, citrate, TEPA or aspartic-dextran polysaccharide synthesized as described in Supporting Information. Among them, the only successful functionalization methodology was that using aspartic-dextran polysaccharide working at pH = 5, which was substantiated by FTIR spectroscopy. Thus, the spectrum of the functionalized nanoparticles (Fig. 11) displayed bands in the 1400-1600 cm-1 region, similar to those of aspartic-dextran polymer indicating the presence of these species on the nanoparticles surface. It should be noted that at pH = 5, aspartic-dextran is negatively charged, while the surface of the nanoparticles, whose i.e.p. was 6.7 (Fig. 10), is positively charged, guaranteeing ionic interactions between nanoparticles and polymer. The establishment of multipunctual interactions between the carboxilated dextran and the nanoparticles reinforce the ionic interactions making the dextran-nanoparticles more stable. The success of the functionalization procedure was further confirmed by the surface charge reversal observed for the functionalized nanoparticles (Z potential = -19 mV, at pH = 5.1) when compared with the untreated ones (Z potential = 7.3 mV, at pH =5.1). The amount of aspartic-dextran species attached to the nanoparticles surface was quantified by TGA analysis. As observed in Fig. 12, the TGA curve obtained before functionalization showed a weight loss of 4% in the 25-230ºC range mainly due to the 20 release of water, whereas that of the functionalized sample presented and additional weight loss of 4.5% in the 230-800ºC range associated to the decomposition of the adsorbed organic species. It is important to mention that the Tb luminescence intensity remained almost unaltered after functionalization (Fig. S10 in supplementary information) indicating the absence of significant quenching associated to the adsorbed aspartic-dextran ploymer. The UV-visible absorbance spectra recorded for suspensions in water (pH=6.7) of the Tb:CePO4 nanophosphor after different aging times up to 24 h (Fig. 13) showed the expected two bands at 260 and 275 nm due to the absorption of phosphate groups (Zhang et al. 2010). The lack of variation of the intensity of these bands on aging discarded the presence of sedimentation thus manifesting the high colloidal stability of such suspenions in aqueous media. Biocompatibility studies of the nanophosphors were undertaken by evaluating the cell viability of Vero cells by the MTT assay [Mosmann 1993]. This assay relies on the mitochondrial activity of fibroblasts activity of fibroblasts and represents a parameter for their metabolic activity. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide) assay is a simple non-radioactive colorimetric assay to measure cell cytotoxicity, proliferation, or viability. MTT is a yellow, water-soluble tetrazolium salt. Metabolically active cells are able to convert this dye into a water-insoluble dark-blue formazan by reductive cleavage of the tetrazolium ring. Formazan crystals, then, can be dissolved in an organic solvent such as dimethylsulphoxide (DMSO) and quantified by measuring the absorbance of the solution at 550 nm, and the resultant value is related to the number of living cells. The metabolic activity and proliferation of fibroblasts was thus measured after 24 hours’ culture, and showed negligible toxicity effects (Fig. 14) with viability percentages as high as 80-90% for concentrations up to 0,5 mg/mL. 21 Conclusions We have shown that the aging of ethylene glycol solutions containing cerium and terbium acetylacetonates and phosphoric acid at low temperature (120ºC) in a conventional oven yields, under proper reagents concentrations, polycrystalline Tb:CePO4 nanophosphors with a spindle-like morphology and monoclinic structure. The size of these nanoparticles was found to increase as the Tb content increased, whereas their axial ratio decreased. We have also demonstrated that the use of microwave assisted heating instead of the conventional heating mode resulted in smaller nanospindles with much higher crystallinity (single crystals), which has an important beneficial effect on the on the luminescent properties of these nanophosphors, which could be further functionalized with aspartic-dextran polymers providing anchors for adding functional ligands such as antibodies and drugs. These features along with the negligible toxicity of these functionalized nanophosphors and their stability when dispersed in aqueous media make them suitable for biomedical applications. Acknowledgements. This work has been supported by Junta de Andalucía (grant FQM6090), the Spanish CICYT (Grant MAT2011-23593) and ERC-Sarting Grant NANOPUZZLE. S. R. Liviano wants to thank the Spanish Ministerio de Ciencia y Tecnología for an FPI fellowship. J.M.F. wants to thank ARAID for financial support. Authors thank V. Grazu for fruitful discussion and I. Echaniz for technical support. 22 References Bao J, Yu R, Zhang J, Wang D, Deng J, Chen J, Xing X (2010) Scripta Mater 62:133- 136 Blasse G, Brill A (1967) J Chem Phys 47:1920-1926 Bu W, Hua Z, Chen H, Shi J (2005) J Phys Chem B 109:14461-14464 Buchold BHM, Feldman C (2008) Adv Funct Mater 18:1002-1011 Chatterjee DK, Gnanasammandhan MK, Zhang Y (2010) Small 6:2781-2795 Chen G, Sun S, Zhao W, Xu S, You T (2008) J Phys Chem C 112:20217-20221 Chen Y, Chen H, Zhang S, Chen F, Zhang L, Zhang J, Zhu M, Wu H, Guo L, Feng J, Shi J (2011) Adv Funct Mater 21:270-278 Di W, Wang X, Ren X (2010) Nanotech 21:075709 (a) Di W, Shirahata N, Zheng A, Sakka Y (2010) Nanotech 21:365501 (b) Feldmann C (2003) Adv Funct Mater 13:101-107 Fuentes M, Mateo C, García L, Tercero JC, Guisán JM, Fernández-Lafuente R, (2004) Biomacromolecules 5:883-888 Gamarnik MY (1992) phys stat sol 170:27-37 Guan M, Sun, J, Han M, Xu Z, Tao F, Yin G, Wei X, Zhu J, Jiang X (2007) Nanotech 18:415602 Huang XL, Teng X, Chen D, Tang FQ (2010) Biomaterials 31:438-448 Imteyaz Ahmad MD, Bhattacharya SS (2009) Appl Phys Letters 95:191906 Inokuti M, Hirayama F (1965) J Chem Phys 43, 1978-1989 Komban R, Beckmann R, Rode S, Ichilmann S, Kühnle, Beginn W, Haase M (2011) Langmuir 27:10174-10183 Lemine OM, Sajieddine M, Bououdina M, Msalam R, Mufti S, Alyamani A (2010) J Alloys Comp 502:279-282 23 Lewis DA, Summers JD, Ward TC, McGrath JEJ (1992) J Polym Sci 30A:1647-1653 Li H, Zhu G, Ren H, Li Y, Hewitt IJ, Qiu S (2008) Eur J Inorg Chem 2033-2037 Li Y, Sun Z, Lun M, Zhang X, Yao M, Joly AG, Liu Z, Chen W (2010) Nanotech. 21:125604 (7 pp) Ma L, Che WX, Zheng YF, Xu ZD (2008) Mater Res Bull 43:2840-2849 Matijević E (1993) Chem Mater 5:412-426 Mosmann, TJ (1993) J Immunol Methods 95:55-63 Mullica DF, Milligan WO, Grossie DA, Beall GW, Boatner LA (1984) 95:231-236 Nakamoto K (1986) Infrared and Raman Spectra of Inorganic and Coordination Compounds, John Wiley & Sons, New York. Ni YX, Hughes JM, Mariano AN (1995) Amer Miner 80:21-26 Niraj Luwang M, Ningthoujam Jagannath RS, Srivastava SK, Vatsa RK (2010) J Am Chem Soc 132:2759-2768 Nuñez NO, Ocaña M (2007) Nanotech 18:455606 Nyutu EK, Chen CH, Dutta PK, Suib SL (2008) J Phys Chem C 112:9659-9667 Pankratov V, Popov AI, Chernov SA, Zharkouskaya A, Feldmann C (2010) Phys Status Solidi B 247:2252-2257 Petricek V, Dusek M, Palatinus L (2006) Institute of Physics, Academy of Sciences of the Czech Republic, Praha. http://www-xray.fzu.cz/jana/jana.html Poul L, Ammar S, Jouini N, Fievet F (2003) J Sol-Gel Sci Tech 26:261-265 Riwotzki K, Meyssamy H, Kornowski A, Haase M (2000) J Phys Chem B 104:2824- 2828 Shannon RD (1976) Acta Cryst A32:751-767 Wagner D, Riggs WH, Davis LE, Moulder JF, Muilinberg GE (1973) Handbook of X- Ray Photoelectron Spectroscopy, Perkin-Elmer Corporation, Minnesota 24 Xia Y, Yang P, Sun Y, Wu Y, Mayers B, Gates B, Yin Y, Kim F, Yan H (2003) Adv Mater 15:353-389 Yu C, Yu M, Li C, Liu X, Yang J, Yang P, Lin J (2009) J Solid State Chem 182:339- 347 Zhang F, Chan SW, Spanier JE, Apak E, Jin Q, Robinson RD, Herman IP (2002) Appl Phys Letters 80:127129 Zhang YW, Yang ZG, You LP, Si R, Yan CH (2003) Eur J Inorg Chem 4099-4104 Zhang C, Chen J, Zhu X, Zhou Y, Li D (2009) Chem Mater 21:3570-3575 Zhang F, Wong SS (2010) ACS Nano 4:99-112 Zhu X, Zhang Q, Li Y, Wang H (2010) J Mater Chem 20:1766-1771 25 Table 1: Size and axial ratio of the Tb:CePO4 particles synthesized by using conventional or microwave heating. Sample Tb/Tb+Ce (%molar) Heating mode Length (nm) Width (nm) Axial ratio L/W CePO4 0 Conventional 244±40 57±11 4.3 Tb0.02Ce0.98PO4 2 Conventional 318±48 81±16 3.9 Tb0.05Ce0.95PO4 5 Conventional 409±70 128±21 3.2 Tb0.10Ce0.90PO4 10 Conventional 650±108 277±60 2.4 Tb0.15Ce0.85PO4 15 Conventional 821±95 410±47 2.0 CePO4 0 Microwave 171±47 48±8 3.6 Tb0.02Ce0.98PO4 2 Microwave 216±42 58±8 3.7 Tb0.05Ce0.95PO4 5 Microwave 256±70 74±13 3.4 Tb0.10Ce0.90PO4 10 Microwave 170±62 68±18 2.5 Tb0.15Ce0.85PO4 15 Microwave 103±29 41±10 2.7 Table 2. Unit cell parameters of the monoclinic Tb:CePO4 samples synthesized by using conventional or microwave heating. Errors are included in parenthesis. Sample Tb/Tb+Ce (% molar) Heating mode a (Å) b (Å) c (Å) β (º) CePO4 0 Conventional 6.8143(34) 7.0303(30) 6.4807(30) 103.897(40) Tb0.02Ce0.98PO4 2 Conventional 6.8107(25) 7.0326(24) 6.4835(23) 103.973(28) Tb0.05Ce0.95PO4 5 Conventional 6.8001(24) 7.0203(23) 6.4800(22) 103.995 (25) Tb0.10Ce0.90PO4 10 Conventional 6.7884(24) 7.0007(21) 6.4738(21) 104.011(23) Tb0.15Ce0.85PO4 15 Conventional 6.7799(26) 6.9967(23) 6.4634(23) 104.046(27) CePO4 0 Microwave 6.7960(9) 7.0184(8) 6.4707(8) 103.817(9) Tb0.02Ce0.98PO4 2 Microwave 6.7887(5) 7.0090(4) 6.4653 (4) 103.840(5) Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Figure 7 Click here to download high resolution image Figure 8 Click here to download high resolution image Figure 9 Click here to download high resolution image Figure 10 Click here to download high resolution image Figure 11 Click here to download high resolution image Figure 12 Click here to download high resolution image Figure 13 Click here to download high resolution image Figure 14 Click here to download high resolution image 0246810 12 14 Intensity (a.u.) Energy (KeV) Cu Ce Tb P O Fig. S4. EDX spectrum obtained for the Tb0.15Ce0.85PO4 sample prepared by conventional heating Fig. S5. X-ray diffraction patterns recorded for the Tb doped CePO4 nanoparticles prepared by conventional heating. The JCPDS file for monoclinic CePO4 is also included. 10 20 30 40 50 60 70 80 2θ (degree) JCPDS: 00-032-0199 Tb 0,02 Ce 0,98 PO 4 Tb 0,05 Ce 0,95 PO 4 Tb 0,10 Ce 0,90 PO 4 Tb 0,15 Ce 0,85 PO 4 CePO 4 Fig. S6. TEM images of the Tb doped CePO4 nanoparticles prepared with different Tb content (% Tb/Ce+Tb mol ratio) by microwave-assisted heating. 20 30 40 50 60 70 2θ (degree) JCPDS: 00-032-0199 Tb0,02Ce0,98PO4 CePO4 Fig. S7. X-ray diffraction patterns recorded for the CePO4 and Tb0.02Ce0.98PO4 nanoparticles prepared by microwave heating. The JCPDS file for monoclinic CePO4 is also included. 4000 3500 3000 2500 2000 1500 1000 500 Microwave Conventional Absorbance (a.u.) Wavenumber (cm-1) Fig. S8. FTIR spectra recorded for the Tb0.02Ce0.98PO4 sample obtained by microwave heating and for the Tb015Ce0.85PO4 sample synthesized by conventional heating. Fig. S9. TGA curves obtained for the Tb0.15Ce0.85PO4 sample obtained by conventional heating and for Tb0.02Ce0.98PO4 sample obtained by microwave heating 0200 400 600 800 85 90 95 100 Weight loss (%) Temperature (ºC) CePO 4 Conventional CePO 4 Microwave 300 350 400 450 550 600 650 700 750 Intensity (a.u) Wavelength (nm) TbCePO 4 functionalized TbCePO 4 as prepared Fig. S10. Emission spectrum (λex= 255 nm) recorder for the Tb0.02Ce0.98PO4 sample obtained by microwave heating before and after functionalization with aspartic-dextran.