Synthesis, Structure, and Luminescent Properties of Europium-Doped Hydroxyapatite Nanocrystalline Powders

The luminescent europium-doped hydroxyapatite (Eu:HAp, Ca10−xEux(PO4)6(OH)2) with 0 ≤ x ≤ 0.2 nanocrystalline powders was synthesized by coprecipitation. The structural, morphological, and textural properties were well characterized by Xray diffraction, scanning electron microscopy, and transmission electron microscopy. The vibrational studies were performed by Fourier transform infrared, Raman, and photoluminescence spectroscopies. The X-ray diffraction analysis revealed that hydroxyapatite is the unique crystalline constituent of all the samples, indicating that Eu has been successfully inserted into the HAp lattice. Eu doping inhibits HAp crystallization, leading to a decrease of the average crystallite size from around 20 nm in the undoped sample to around 7 nm in the sample with the highest Eu concentration. Furthermore, the samples show the characteristic D0 → F0 transition observed at 578 nm related to Eu3+ ions distributed on Ca2+ sites of the apatitic structure.


Introduction
Hydroxyapatite (HAp, Ca 10 (PO 4 ) 6 (OH) 2 ) belongs to the apatite family with general formula Ca 10 (PO 4 ) 6 X 2 , X being a fluorine, chlorine ion, or a hydroxyl group.HAp being the major inorganic component in natural bones crystallizes in hexagonal system.Synthetic HAp has the same chemical composition as biological HAp and thus mimics many properties of natural bone [1][2][3].On the other hand, synthetic HAp is limited in use due to high in vivo solubility and poor mechanical properties, such as low-impact resistance [4,5].The physical, chemical, and biological properties of HAp are controlled by its crystal structure and composition.Substitution of Ca 2+ with other metal ions into the HAp structure is one of the effective ways to improve the properties of HAp.Metal ions such as Ag + , Cu 2+ , and Zn 2+ in the HAp structure can affect its crystallinity, morphology, and lattice parameters.The advantages of doping HAp with foreign elements pertinent to orthopedic applications were reported by others.For example, Y-doped calcium oxyhydroxyapatite [6] has been studied for humidity sensor applications due to its high affinity for water molecules [7].The doping of HAp with 2 mol% of Zn 2+ significantly increased osteoblast adhesion [8].Webster et al. [9] reported that the Bi 3+ would be the best choice of dopant to enhance properties of HAp pertinent for bone implant applications.
Being an important emitter in the red region of the visible spectrum, Eu 3+ ions have been utilized extensively in color television and high-efficiency fluorescent lamps [10,11].Therefore, Eu 3+ ions have been widely used as a local structure probe [12][13][14][15][16]. Labeling using organic fluorescent molecules is popular in clinical use for years.In recent years, a lot of inorganic components even nanoparticles were suggested to be such candidates [15].However, the toxicity of particles used is an important issue in practical application due to composition and nanosize nature.A luminescent agent with great biocompatibility is ideal for implantation and clinical application [16].Particularly, in biomedicine field, nanoparticles can be used as drug-delivery vehicles that can target tissues or cells [17] and can be functionalized with special characteristics (such as magnetization, fluorescence, and near-infrared absorption) for qualitative or quantitative detection of tumor cells [18].As we know, the nanoscale fluorescent materials have attracted much interest due to the increasing demand for efficient photosensitive materials not only for sophisticated optoelectronic and photonic devices but also for a broad range of biomedical application [19].In biomedical areas, the luminescent materials, mainly including fluorescent organic molecules [20,21] and semiconductor nanoparticles [22,23], have been widely investigated in biological staining and diagnostics.However, some serious problems of photobleaching and quenching of fluorescent organic molecules and the toxicity of semiconductor quantum dots are critically pronounced, which have seriously limited their applications in biomedical areas [23,24].Furthermore, high performance in function-specific biological applications requires that the composites possess some unique characteristics, such as uniform morphology, large surface areas, good dispersion, and so forth [24].Recently, a class of the stable, efficient, and self-activated luminescent materials, whose emission is induced by the defects or impurities in the host lattices, has been prepared by various synthesis routes [25][26][27][28].These novel self-activated inorganic materials may be a promising fluorescent material for biodetection due to their good optical properties and nontoxicity.
In this study, the objective towards this goal was to synthesize nanocrystalline HAp doped with Eu ions at low temperature and characterize their resulting properties.In this paper we investigate the doping of hydroxyapatite by europium ions and the influence of the atomic ratio Eu/(Eu + Ca) on the structure, morphology and optical properties.Europium-doped hydroxyapatite Ca 10−x Eu x (PO 4 ) 6 (OH) 2 with 0 ≤ x ≤ 0.2 was synthesized by coprecipitation method at 100 • C mixing Eu(NO 3 ) 3 •6H 2 O, Ca(NO 3 ) 2 •4H 2 O and (NH 4 ) 2 HPO 4 in deionized water.The structure, morphology, and vibrational and optical properties of the obtained samples were systematically characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR), and FT-Raman spectroscopies.The powders were also characterized by steady-state photoluminescence.HPO 4 was dissolved in deionised water to make 300 mL P-containing solution.The [Ca + Eu]-containing solution was put into a Berzelius and stirred at 100 • C during 30 minutes.Meanwhile the pH of P-containing solution was adjusted to 10 with NH 3 and stirred continuously for 30 minutes.The P-containing solution was added drop by drop into the [Ca + Eu]-containing solution and stirred for 2 h and the pH was constantly adjusted and kept at 10 during the reaction.After the reaction the deposited mixtures were washed several times with deionised water.The resulting material was dried at 100 • C for 72 h.

X-Ray Diffraction (XRD).
The samples were characterized by X-ray diffraction using a Bruker D8-Advance Xray diffractometer, in powder setting, equipped with copper target X-ray tube, Ni filter, and a high-efficiency linear detector of Lynx Eye type, operated in integration mode.The patterns were scanned in the 2θ range 15-140 • , with a step size of 0.02 • and 36 s measuring time per step.

Scanning Electron Microscopy (SEM).
The structure and morphology of the samples were studied using a HITACHI S2600N-type scanning electron microscope (SEM), operating at 25 kV in vacuum.The SEM studies were performed on powder samples.For the elemental analysis the electron microscope was equipped with an energy dispersive X-ray attachment (EDAX/2001 device).

Transmission Electron Microscopy (TEM).
Studies were carried out using a JEOL 200 CX.The specimen for TEM imaging was prepared from the particles suspension in deionised water.A drop of well-dispersed supernatant was placed on a carbon-coated 200 mesh copper grid, followed by drying the sample at ambient conditions before it is attached to the sample holder on the microscope.

FT-IR Spectroscopy.
The functional groups present in the prepared powder and in the powders calcinated at different temperatures were identified by FTIR (Spectrum BX Spectrometer).For this 1% of the powder was mixed and ground with 99% KBr.Tablets of 10 mm diameter for FTIR measurements were prepared by pressing the powder mixture at a load of 5 tons for 2 min, and the spectrum was taken in the range of 400 to 4000 cm −1 with resolution 4 and 128 times scanning.

Raman Spectroscopy.
Raman studies were performed in a backscattering geometry, under laser excitation wavelengths 1064 nm, using a RFS 100 FT-Raman Bruker spectrophotometer.

Steady-State Photoluminescence (PL).
Steady-state photoluminescence spectra are collected from the front-face geometry of the samples with a Jobin-Yvon Fluorolog spectrometer using a xenon lamp (500 W) as an excitation source.

Results and Discussions
The XRD patterns of the Eu:Hap, Ca 10−x Eu x (PO 4 ) 6 (OH) 2 nanoparticles prepared with different x values (0 ≤ x ≤ 0.2) are shown in Figure 1.It is obvious that all the XRD diffractions of each sample can be well indexed as a pure hexagonal phase (P6 3 /m space group), agreeing well with the values of the standard data (JCPDS no.9-0432).In the case of the samples with x / = 0, the characteristic diffractions of hexagonal HAp (x = 0) are still obvious, and no other phases related with doped component can be detected.
The samples with x = 0.01 (HAp E1) and x = 0.02 (HAp E2) are almost identical from XRD point of view with the undoped HAp (x = 0), showing very small differences concerning the relative intensities of the peaks.Furthermore, for x = 0.1 and x = 0.2, it can be found that the relative intensities of the diffractions decrease with the increasing of the Eu concentration, suggesting that the doping inhibits the HAp crystal growth.Besides, the fluorescence of Eu excited by the absorption of Cu Kα radiation on the Eu L shell may contribute substantially to this increased background intensity.(The cross section of the photoelectric effect is quite large (412.5 cm 2 /g) because the L absorption edges of Eu, E EuL2 = 7.617 KeV and E EuL3 = 6.977KeV, approaches the Cu Kα radiation energy used for analysis (E CuKα = 8.049 KeV) [29]).The deviations of the relative intensities of the diffraction peaks from the reference are more visible in the pattern of the sample with the highest Eu concentration.Insightful analyses of the doped HAp structures, carried out by Rietveld whole-powder pattern fitting using the MAUD code [30], showed that Eu enters the HAp lattice by substituting Ca, with comparable probabilities for the two crystallographic sites of calcium in the HAp unit cell.The lattice parameters did not modify significantly after substitution.The XRD analysis using the anisotropic microstructure analysis implemented in MAUD as "Popa rules" [31] shows that the calculated lattice constants for Hap:Eu are in good agreement with the standard data of a = b = 0.9418 nm, c = 0.6884 nm.The unit-cell parameters of the corresponding samples are summarized in Table 1; the standard data of the JCPDS no.9-0432 for hydroxyapatite are presented for comparison.
Figure 2 displays the TEM images of pure HAP (x = 0) and Eu:HAp (0.01 ≤ x ≤ 0.2) with low resolution and selected areas electron diffraction (SAED).Figures 2(a It can be seen from the HRTEM image of Eu:HAp with atomic ratio Eu/[Eu + Ca] = 20% (Figure 3) that the crystalline phase of hydroxyapatite with well-resolved lattice fringes can be observed.The distances (2.82 Å and 2.72 Å) between the adjacent lattice fringes agree well with the d 211 and d 004 spacing of the literature values (0.2814 nm and 0.2778 nm; CJPDS no.09-0432).
The HRTEM images of the sample further confirm that the synthesized samples are well-crystallized single crystals.
The SEM images and EDS analysis of Eu:HAp samples prepared at the different atomic ratio Eu/[Eu + Ca] are displayed in Figure 4.
SEM images provide the direct information about the size and typical shapes of the prepared samples.It is found that all the samples Eu:HAp consists of relatively uniform rod-like particles.The result suggests that the doping Eu 3+ has little influence on the morphology.These materials present quite different morphology, which reveals a homogeneous aspect of the synthesized particles for all samples.The samples prepared at the atomic ratio Eu/[Eu + Ca] 2% exhibit smaller particle size.When the atomic Figure 5 shows the FT-IR results obtained from Eu:HAp, when the atomic ratio Eu/(Ca + Eu) increases from 1 to 20%.For all the samples the presence of strong OH vibration peak (632 cm −1 ) could be noticed.The broad bands in the regions 1600-1700 cm −1 and 3200-3600 cm −1 correspond to H-O-H bands of lattice water [32][33][34].
The strong absorption bands at 3429 cm −1 can be assigned to OH and H 2 O for all the samples.Bands characteristics of the phosphate and hydrogen phosphate groups in apatitic environment were observed: 565 cm −1 , 632 cm −1 , 603 cm −1 , 962 cm −1 , and 1000-1100 cm −1 for the PO 4 3− groups [35,36] and at 875 cm −1 for the HPO 4 2− ions.The bands at 1093 and 1033 cm −1 can be attributed to the triply degenerated ν 3 antisymmetric stretching of P-O band.The 962 cm −1 band can be due to the ν 1 non-degenerated symmetric stretching of P-O band.The bands at 603 cm −1 and 565 cm −1 are associated with the triply degenerated band was present in all the spectra, even for high values of Eu/(Ca + Eu) atomic ratio.The CO 3 2− band was presented in the spectra with atomic ratio Eu/(Ca + Eu) higher than 10% at 1400 cm −1 and 1508 cm −1 [3], respectively.The intensity of vibration peak decreases with the increasing of the Eu concentration.A broadening of peak vibration with the decreasing of the Eu concentration is also observed.These resultants suggest that the amounts of specific surface area of Eu:Hap increased with the Eu concentration.This compartment was observed by Owada et al. [6] in sintered Y-doped hydroxyapatite.
The PL properties of the sample were further characterized by the PL excitation and emission spectra, as shown in Figures 7 and 8.The excitation spectrum was obtained by the Eu 3+ 5 D 0 → 7 F 2 transition at 614 nm (Figure 7).In these excitation spectra, some sharp peaks originating from the f-f transitions of Eu 3+ can be observed at 300 nm [37].The highest excitation peaks are observed at 250 nm and 398 nm, in the UV region.Besides, excitation in the visible region is possible at 454 nm.The broad peak at 250 nm was attributed to a charge transfer transition between Eu 3+ and O 2− [38,39].In the longer wavelength region, the weak peaks may arise from the direct excitation of the Eu 3+ ground state into higher levels in the 4f 6 configuration, which can be assigned to 7 F 0 → 5 H 6 (320 nm), 7 F 0 → 5 D 4 (365 nm), 7 F 0 → 5 G 2 (382 nm), 7 F 0 → 5 L 6 (398 nm), 7 F 0 → 5 D 3 (421 nm), 7 F 0 → 5 D 2 (454 nm), respectively [38].
Upon excitation at 394 nm, the characteristic transition lines from the excited 5 D 0 level of Eu 3+ ions can be detected in the emission spectra [40], as shown in Figure 8, and the locations of the emission lines with their assignments are labeled as well.Spectral features are observed in three different ranges: 570-582, 582-603, and 603-640 nm.They were ascribed to the 5 D 0 → 7 F 0 , 5 D 0 → 7 F 1 , and 5 D 0 → 7 F 2 transitions, respectively.The 5 D 0 → 7 F 0 transition observed at 578 nm is related to Eu 3+ ions distributed on Ca 2+ sites of the apatitic structure [1].The two main characteristic peaks from 5 D 0 → 7 F 1 (591 nm) and 5 D 0 → 7 F 2 (614 nm) are dominant.The more efficient emission was observed for the hypersensitive 5 D 0 -7 F 2 transitions whose maximum intensity was obtained at 614 nm.PL intensity increased with increasing Eu doping concentration, and no significant modification of emission spectra was observed.It is worth noting that the characteristic emission lines are obvious in the emission spectra for all the spectra, showing the potential application to be tracked or monitored by the luminescence.The selective observations were made for excitation at the respective 5 D 0 → 7 F 0 transition.For the 5 D 1 level, three bands were observed in agreement with data reported for Eu 3+ :Sr 5 (PO 4 ) 3 F [41].The assignment of the 7 F 1 and 7 F 2 components corresponds to those given by Vooronko et al. [42] in their study on Eu 3+ :Ca 5 (PO 4 ) 3 F.A coupled substitution Ca 2+ , PO 4 3− ↔ Ln 3+ , SiO 4 4− has been proposed by other authors to explain the replacement of calcium by a rare earth Ln 3+ in apatites [43,44].Such processes could not occur in our experimental conditions (absence of Na+ or SiO 4 4− ions).Lin et al. [45] in their studies on CeHAp with x Ce from 0 to 0.2 have shown that cerium can partially substitute for calcium and enters the structure of HAp.In this case, after substitution, the crystallinity and the IR wavenumbers of bonds in CeHAp nanoparticles decreased gradually with the increased of x, and the morphology of the nanoparticles changed from the rod-shaped Hap to the needle-shaped CeHAp.
To the best of our knowledge, this is the first paper that demonstrates Eu-doped nanocrystalline hydroxyapatite by coprecipitation method at low temperature.Our studies show that the morphology of the nanoparticles does not change when the ratio of Eu/(Ca + Eu) increases.To determine the influence of Eu concentration on cell functions, drug loading, and release properties, future studies on this topic would be of significance for the development of materials and the design of bioceramics.

Conclusions
The aim of this work was to study the impact of the atomic ratio of Eu/(Ca + Eu) on the structural, morphological, and vibrational properties of hydroxyapatite nanocrystalline powders.In summary, a simple coprecipitation method has been developed to synthesize luminescent Eu 3+ doped hydroxyapatite nanoparticles.We have synthesized europium-doped hydroxyapatite nanoparticles by coprecipitation method at low temperature with the atomic ratio (a)   could be interesting for academic and industry researchers in biomaterials, medical materials, and drug carriers.
), 2(c), 2(e), and 2(g) presents TE micrograph of the Eu:HAp with low resolution for different values of x.Figures 2(b), 2(d), 2(f), and 2(h) shows selected areas electron diffraction (SAED) pattern of the synthesized powder, which confirms its crystallinity.As shown in Figures2(a), 2(c), 2(e), and 2(g), all the samples exhibit a rod-like morphology which is consistent with the SEM results.