ISSN 00201685, Inorganic Materials, 2013, Vol. 49, No. 1, pp. 89–94. © Pleiades Publishing, Ltd., 2013. Original Russian Text © A.I. Orlova, A.E. Kanunov, E.N. Gorshkova, A.N. Shushunov, S.N. Pleskova, E.R. Mikheeva, D.O. Savinykh, E.S. Leonov, 2012, published in Neorga nicheskie Materialy, 2012, Vol. 48, No. 12, pp. 1365–1371.
Synthesis, Luminescence, and Biocompatibility of Calcium and LanthanideContaining NaZr2(PO4)3Type Compounds A. I. Orlovaa , A. E. Kanunova, E. N. Gorshkovab, A. N. Shushunovc, S. N. Pleskovab, E. R. Mikheevab, D. O. Savinykha, and E. S. Leonovc aFaculty
b
of Chemistry, Lobachevsky State University, pr. Gagarina 23, Nizhni Novgorod, 603950 Russia Research and Education Center for the Physics of SolidState Nanostructures, Lobachevsky State University, pr. Gagarina 23, Nizhni Novgorod, 603950 Russia c Research Institute of Physics and Technology, Lobachevsky State University, pr. Gagarina 23/3, Nizhni Novgorod, 603950 Russia email:
[email protected] Received November 9, 2011; in final form, May 12, 2012
Abstract—Ca0.5Zr2(PO4)3:Er/Yb and Ca0.75Zr2(PO4)2.5(SiO4)0.5:Er/Yb compounds have been prepared and their luminescence properties and biocompatibility have been investigated. We found synthesis conditions that ensured phase homogeneity and formation of nanopowders. Their luminescence properties have been studied. Under IR excitation, emission was observed at λ = 0.525 μm. Cells (neutrophil granulocytes) were shown to retain viability in the presence of the compounds studied. DOI: 10.1134/S0020168513010093
INTRODUCTION
Among new materials under development for the optical imaging of cells and tissues, attention should be paid to the family of lanthanidecontaining analogs of NaZr2(PO4)3 (NZP). Isomorphous lanthanide com pounds have been described in a number of reports (see, e.g., reviews by Alamo [6] and Orlova [7]).
One promising direction in the development of materials science is the engineering of biocompatible nanocrystalline materials with good emission charac teristics in the visible spectral region for future use in the in vivo visualization of cells and tissues (bioimaging).
Some of the lanthanidecontaining NZPtype phosphates have been studied as phosphors for white light emitting diodes, e.g., Ca0.5 – xEuxZr2(PO4)3 [8, 9] and Ca0.5 – 1.5xSmxZr2(PO4)3 [9]. Luminescence in these materials is excited by UV radiation (λex = 0.337 μm). The shortwavelength excitation makes processes involving such materials unsafe for biological systems.
Necessary requirements for such materials are bio logical inertness, photoluminescence in the visible range with reasonable intensity, and availability of exci tation sources for their photoluminescence that carry no hazard to biological systems. In connection with this, considerable research effort has focused on nanocrystalline CdSe/ZnS composites (“quantum dots”) [1, 2]; phosphors based on divalent metal silicates, for example, Ca0.2Zn0.9Mg0.5Si2O6, doped with Eu2+, Dy3+, or Mn2+ [3]; and phosphate compounds, including those based on calcium ortho phosphate [4, 5].
The objectives of this work were to synthesize NZPtype phosphates and phosphosilicates containing calcium, Er, and Yb; to examine the influence of the powder preparation procedure and conditions on the formation and microstructure of singlephase powders with the desired structure; to study their luminescence properties under IR excitation (within the hightrans mission therapeutic window); and to assess their toxic ity using biological systems.
As is evident from their elemental composition, the first group of materials are not safe to biological sys tems, even though they possess necessary optical char acteristics (as to the emission wavelength and intensity under excitation with an infrared source). The prepara tion of silicate materials requires high temperatures (⯝1150°C). The possibility of using phosphates, including those with the βCa3(PO4)2 structure, as optical markers has only been mentioned once in the literature [5].
The Er3+ and Yb3+ lanthanide cations are known to emit in the visible spectral region, in particular through the luminescence upconversion mechanism, when excited at a wavelength of ~0.98 μm [10]. This feature of the Er3+/Yb3+ pair in NZPtype compounds is addressed in this study. 89
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Table 1. Compositions and designations of the Ca0.5 – 1.5(x + y) ErxYbyZr2(PO4)3 phosphates (system I) and Ca0.75 – 1.5(x + y) ErxYbyZr2(SiO4)0.5(PO4)2.5 phosphosili cates (system II) studied x:y
y
I
II
1:1
0.01 0.05 0.10 0.20 0.01 0.02 0.03 0.02 0.04 0.06
CaLnP (1 : 1) 0.01 CaLnP (1 : 1) 0.05 CaLnP (1 : 1) 0.10 – CaLnP (1 : 4) 0.01 CaLnP (1 : 4) 0.02 CaLnP (1 : 4) 0.03 CaLnP (1 : 10) 0.02 CaLnP (1 : 10) 0.04 CaLnP (1 : 10) 0.06
CaLnPSi (1 : 1) 0.01 CaLnPSi (1 : 1) 0.05 CaLnPSi (1 : 1) 0.10 CaLnPSi (1 : 1) 0.20 – – – – – –
1:4
1 : 10
EXPERIMENTAL Materials. We studied Ca, Zr, Er, and Yb phos phates and phosphosilicates with various concen trations and ratios of Er and Yb in the sys tems Ca0.5 – 1.5(x + y)ErxYbyZr2(PO4)3 (I) and Ca0.75 – 1.5(x + y)ErxYbyZr2(SiO4)0.5(PO4)2.5 (II). The x : y ratios are listed in Table 1. The total Er + Yb content was varied in the ranges 0.01 + 0.0025 ≤ x + y ≤ 0.10 + 0.10 (I) and 0.01 + 0.01 ≤ x + y ≤ 0.20 + 0.20 (II). Synthesis. The compounds for this study were pre pared using various sol–gel processes. To a solution of a stoichiometric mixture of metal salts were added an NH4H2PO4 solution and ethanol. The reaction system was heattreated first at t = 90°C (until the forming gel was completely dry) and then at 600 and 800°C for 24 h at each temperature. Gelation was carried out at t = 0, 20, and 80°C, under sonication in a number of experiments. During heating from 90 to 800°C, the powders were reground several times for 20 min in an agate mortar. The above procedures were employed to prepare the Ca0.5 – 1.5(x + y)ErxYbyZr2(PO4)3 phosphates with x + y = 0.01 + 0.01, 0.05 + 0.05, and 0.10 + 0.10 and x : y = 1 : 1 (cut in system I), which were used as an example to optimize conditions for the preparation of the other phosphates. Characterization techniques. The powders were characterized and investigated by differential scanning calorimetry (DSC), infrared (IR) spectroscopy, Xray diffraction (XRD), atomic force microscopy (AFM), and photoluminescence spectroscopy. The instruments used included a SETARAM LabSys thermal analyzer, Shimadzu Prestige21 IR spectrophotometer, and Shi madzu LabX XRD6000 Xray diffractometer (filtered CuKα radiation, λ = 1.5406 Å). Microstructures were examined by AFM on a Solver Pro scanning probe
microscope (NTMDT, Russia) equipped with a SmenaA scanning head and optical viewing system. Luminescence measurements were made using a setup built around a KSVU23 computerinterfaced multipurpose spectrometer system. The photolumines cence was measured at room temperature in the spectral regions 0.45–0.70 and 1.4–1.65 μm. Excitation was provided by a pulsed semiconductor laser operating at λ = 0.977 μm. The luminescence was detected by an InGaP photodiode (in the nearIR spectral region) or Hamamatsu R406 photomultiplier (in the visible spec tral region) placed behind the exit slit of an MDR23 monochromator. The phosphates were checked for toxicity using a culture of neutrophil granulocytes (cells with high phagocytic activity) isolated from the venous blood of healthy human donors. At the beginning of the experi ment, the fraction of live cells was at least 99% as deter mined by the trypan blue exclusion test. The cell viabil ity in a control sample and after exposure to a phosphate suspension (10–4 M) was assessed from propidium iodide (Sigma, USA) staining results. The cells were fixed on a substrate using ethanol (70%) for 10 min, washed with distilled water, and then stained with a 0.025% propidium iodide solution. After washing five times, the percentage of stained (dead) cells was deter mined. RESULTS AND DISCUSSION Phase formation. From DSC data for a precursor of the Ca0.35Er0.05Yb0.05Zr2(PO4)3 phosphate prepared in the colloid formation step at 20°C and held at 200°C for 24 h, we inferred that there was a heat effect between 570 and 750°C, attributable to the formation of the final product. This allowed us to select a temperature pro gram of the synthesis. Figure 1 shows the IR spectra of some of the synthe sized compounds. The absorption bands in the range 950–1200 cm–1 are assignable to the P–O asymmetric stretching mode ν3 in the phosphorus tetrahedron. The features in the range 900–1000 cm–1 are due to the symmetric stretching mode ν1. The asymmetric bend ing mode ν4 is represented by three bands in the range 520–630 cm–1. The absorption band at 430–450 cm–1 corresponds to the symmetric bending mode ν2. Analysis of the XRD patterns of the phases formed during heat treatments at 90, 600, and 800°C (Fig. 2) indicated that the target phase formed at t ⯝ 800°C, in agreement with the above DSC data. The final heat treatment temperature for singlephase material forma tion was not influenced by the variation in the gelation temperature from 0 to 80°C or sonication (t = 20°C). The only effect of sonication was broadening and a INORGANIC MATERIALS
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2
Transmission
3
ν2
520
630 555
450
ν1
ν4
1050
slight decrease in the intensity of diffraction peaks, due to the reduction in average crystallite size. Based on the above data, the gelation temperature in all subsequent syntheses was 20°C and no sonication was performed in order to simplify the process. After heat treatment at 800°C, the XRD patterns of the phosphates and phosphosilicates studied were simi lar in the position and relative intensity of diffraction peaks. As an illustration, Fig. 3 presents XRD data for the samples with the highest lanthanide content, that is, with partial silicon substitution for phosphorus. The XRD patterns were indexed using the Ca0.5Zr2(PO4)3 phosphate as an analog [11]. The calculated unitcell parameters of these phosphates lie in the ranges a = 8.761–8.785 Å, c = 22.658–22.850 Å, and V = 1507.547–1519.072 Å3. The particle size of the phosphate samples had a nearly normal distribution, which depended on syn thesis conditions (Table 2, Fig. 4). Raising the gela tion temperature from 0 to 80°C led to an increase in average crystallite size, whereas regrinding between heattreatment steps and sonication reduced the crystallite size. The smallest average particle size was 40 ± 10 nm (tgelation = 20°C, regrinding and sonica tion) and the largest one was 110 ± 50 nm (tgelation = 80°C, no regrinding), with a difference by more than a factor of 2.5 (Table 2). Photoluminescence. We studied the luminescence properties of compounds containing various concen trations and ratios of Er3+ and Yb3+ in the systems Ca0.5 – 1.5(x + y)ErxYbyZr2(PO4)3 (x + y = 0.01 + 0.01, 0.05 + 0.05, and 0.1 + 0.1 at x : y = 1 : 1; 0.0025 + 0.01, 0.005 + 0.02, and 0.0075 + 0.03 at x : y = 1 : 4; and 0.002 + 0.02, 0.004 + 0.04, and 0.006 + 0.06 at x : y = 1 : 10) and Ca0.75 – 1.5(x + y)ErxYbyZr2(SiO4)0.5(PO4)2.5 (x + y = 0.01 + 0.01, 0.05 + 0.05, 0.1 + 0.1, and 0.2 + 0.2 at x : y = 1 : 1). Their spectra have two characteristic emission ranges: in the visible and nearIR spectral regions. The luminescence spectra in the visible range are identical. As an example, Fig. 5 shows the spectra of the Ca0.5 – 1.5(x + y)ErxYbyZr2(PO4)3 phosphates with x : y = 1 : 4 and all values of x + y. The spectra contain two bands, at 0.525 and 0.625 μm. The former band is brighter. Note that the bands differ in position and shape from those known for Er3+ luminescence upcon version. The emission intensity is essentially indepen dent of lanthanide content (x + y). Its highest values are presented in Fig. 5. The nearIR photoluminescence spectra in Fig. 6 contain a band near 1.55 μm, characteristic of Er3+, 4I which corresponds to the 4I13/2 15/2 transition, and a number of narrow bands due to the Stark splitting of
91
ν3 Wavenumber, сm–1 Fig. 1. IR spectra of Ca0.5 – 1.5(x + y)ErxYbyZr2(PO4)3 phos phates with x = y = (1) 0.01, (2) 0.05, and (3) 0.10.
the 4I15/2 level of this transition. These bands arise from the crystalline environment of the erbium ion: the material is crystalline. The lanthanide content, x + y, influences the luminescence intensity, but there is no
Table 2. AFM data for Ca0.35Er0.05Yb0.05Zr2(PO4)3 tgelation, °C
Average particle size, nm without regrinding
with regrinding
0
90 ± 30
60 ± 20
20
100 ± 60
50 ± 20
20*
90 ± 10
40 ± 10
80
110 ± 50
90 ± 20
* With sonication.
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024
104
116
110
113
92
128 134 0210 226
208 119 306
214 300 018
015
012 003 101
Intensity
3
2
1 10
20
30 2θ, deg
40
50
Fig. 2. XRD patterns of the Ca0.35Er0.05Yb0.05Zr2(PO4)3 phosphate after heat treatment at t = (1) 90, (2) 600, and (3) 800°C.
4
Intensity
3
10
20
30 2θ, deg
119 306 128 134 226
018 214 300 208
116
024
113
003 101 012
104
110
2
40
1
50
Fig. 3. XRD patterns of the phosphates (1) CaLnP(1 : 1) with y = 0.1, (2) CaLnP(1 : 4) with y = 0.03, (3) CaLnP(1 : 10) with y = 0.06, and (4) CaLnPSi(1 : 1) with y = 0.2. INORGANIC MATERIALS
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Count
Count
8
8 6
6 4
4
2
2 0 40
(b)
10
10
93
60
80 100 120 140 160 180 Particle size, nm
0
20
40
60 80 Particle size, nm
100
120
Fig. 4. Particle size distributions of Ca0.35Er0.05Yb0.05Zr2(PO4)3 (a) without and (b) with regrinding between heattreatment steps (tgelation = 0°C).
7 Intensity, arb. units
6 Intensity, arb. units
5
1 2 3
5 4 3 2
4
1 2 3
(a)
3 2 1 0
1 0.6 Wavelength, μm
0.7
Fig. 5. Visible photoluminescence spectra of the CaLnP(1 : 4) phosphates with y = (1) 0.01, (2) 0.02, and (3) 0.03 (same designations as in Table 1).
monotonic dependence (Fig. 6). The highest intensity was obtained in the Ca0.5 – 1.5(x + y)ErxYbyZr2(PO4)3 phosphates with x : y = 1 : 10 and all values of x + y (Fig. 6c). The luminescence decay time was ⯝3 ms (at λ = 1.55 μm) and decreased with increasing Er3+ + Yb3+ concentration in all of the samples. There was no corre lation between the luminescence intensity and decay time as functions of x + y. This can be interpreted as evi dence that concentration quenching plays an insignifi cant role in the luminescence decay in the materials studied. The luminescence intensity as a function of Er3+ + 3+ Yb concentration in the visible range does not corre Fig. 6. NearIR photoluminescence spectra of the phos phates and phosphosilicates: (a) CaLnP(1 : 1), y = (1) 0.01, (2) 0.05, (3) 0.10; (b) CaLnP(1 : 4), y = (4) 0.01, (5) 0.02, (6) 0.03; (c) CaLnP(1 : 10), y = (7) 0.02, (8) 0.04, (9) 0.06; (d) CaLnPSi(1 : 1), y = (10) 0.01, (11) 0.05, (12) 0.10, (13) 0.20 (same designations as in Table 1). INORGANIC MATERIALS
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(b)
0.5
0 Intensity, arb. units
0.5
Intensity, arb. units
0
Intensity, arb. units
1.0
7 6 5 4 3 2 1 0
2
(c)
7 8 9
(d)
10 11 12 13
1
0 1.40
1.45
1.50 1.55 Wavelength, μm
1.60
1.65
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late with that in the nearIR spectral region. Like the above characteristics of the nearIR spectra, this is inconsistent with the expected upconversion mecha nism. Additional work is necessary to clarify the lumi nescence mechanism. On the whole, it follows from the present lumines cence data for the phosphates and phosphosilicates that these materials offer visible luminescence bright enough to be detected by the naked eye under IR excitation (within the therapeutic window). Therefore, their opti cal properties satisfy the requirements for bioimaging materials. Biocompatibility. The percentage of dead cells (neu trophil granulocytes) determined as described above indicates that, after 30 min of incubation with the phos phates, cell viability was 95–98%. For comparison, note that, in a previous study [12], cell viability in the presence of fluorophores with an active Er/Yb center was estimated at 27.0 ± 6.6%. CONCLUSIONS Using the compositions and procedures described above, we obtained nanostructured crystalline and Ca0.5Zr2(PO4)3:Er/Yb Ca0.75Zr2(PO4)2.5(SiO4)0.5:Er/Yb materials uniform in phase composition, with a rhombohedral unit cell
ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research, project no. 110497036 r_Povolzh'e_a. REFERENCES 1. Jaiswal, J.K., Mattoussi, H., Mauro. J.M., and Simon, S.M., LongTerm Multiple Color Imaging of Live Cells Using Quantum Dot Bioconjugates, Nat. Biotechnol., 2003, vol. 21, pp. 47–51. 2. Manzoor, K., Johny, S., Thomas, D., et al., Bioconju gated Luminescent Quantum Dots of Doped ZnS: A CytoFriendly System for Targeted Cancer Imaging, Nanotechnology, 2009, vol. 20, pp. 1–13. 3. Le Masne, Q., Scherman D., Bessodes, M., et al., Nan oprobes with NearInfrared Persistent Luminescence for In Vivo Imaging, Proc. Natl. Acad. Sci. U. S. A., 2007, vol. 104, pp. 9266–9271. 4. Le Masne, Q., Scherman, D., Bessodes, M., et al., Nanoparticules a luminescence persistante pour leur utilisation en tant qu’agent de diagnostique destine a l’imagerie optique In Vivo, CNRS Patent, Internat. Ext., WOEP06067950, WO2007048856, 2006. 5. Benhamou, R.A., Bessiere, A., Walles, G., et al., New Insight in the Structure–Luminescence Relationship of Ca9Eu(PO4)7, J. Solid State Chem., 2009, vol. 182, pp. 2319–2325.
(sp. gr. R 3 ).
6. Alamo, J., Chemistry and Properties of Solids with the [NZP] Skeleton, Solid State Ionics, 1993, vols. 63–65, pp. 547–561.
Our results are the first to demonstrate that the pres ence of Er and Yb in NZPtype compounds ensures emission in the visible spectral region at λ = 0.525 μm with reasonable intensity under IR excitation.
7. Orlova, A.I., Isomorphism of Crystalline NaZr2(PO4)3 Type Phosphates and Radiochemical Problems, Radiokhimiya, 2002, vol. 44, no. 5, pp. 385–403.
We determined the emission ranges in the visible spectral region with the highest intensity at an Er to Yb ratio x : y = 1 : 10, independent of the total lanthanide content x + y. The present results lead us to assume that the visible luminescence is unrelated to upconversion. We found excellent cell viability characteristics in the presence of the nanocrystallites studied. On the whole, the present results open up new pos sibilities for the engineering of crystalline materials based on NZPtype compounds containing tetrahe drally coordinated oxyanions (PO4 and SiO4) for bio logical applications. The most basic prerequisite for this is the isomorphism of (iso and heterovalent) cations and anions in the structure of such compounds, which would ensure the formation of various compounds with controlled luminescence characteristics.
8. Hirayama M., Sonoyama, N., Yamada, A., and Kan no, R., Structural Investigation of Eu2+ Emission from Alkaline Earth Zirconium Phosphate, J. Solid State Chem., 2009, vol. 182, pp. 730–735. 9. Glorieux, B., Orlova, A., Garcia, A., et al., New Phos phors for White LEDs, the Case of Phosphate Doped with Divalent Europium and Other Luminescent Ions, 5th Forum on New Materials, CIMTEC2010, Tuscany, 2010, p. 100. 10. Kaminskii, A.A. and Antipenko, B.M., Mno gourovnevye funktsional’nye skhemy kristallicheskikh lazerov (Multilevel Functional Diagrams of Crystalline Lasers), Moscow: Nauka, 1989, p. 270. 11. Alamo, J. and Rodrigo, J.L., High Temperature Neu tron Diffraction Study of CaZr4(P04)6, Solid State Ion ics, 1993, vols. 63–65, pp. 678–683. 12. Pleskova, S.N., Gorshkova, E.N., Mikheeva, E.R., and Shushunov, A.N., Study of Biocompatibility of Nano particles with Er/Yb Fluorescent Centers in System with Neutrophil Granulocytes, Cell Tissue Biol., 2011, vol. 5, no. 4, pp. 332–338. INORGANIC MATERIALS
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