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Persistent luminescence strontium aluminate nanoparticles co-doped with Eu2+ and Dy3+ were prepared by urea-assisted combustion synthesis. Different percentages of co-dopants were evaluated in order to optimize luminescence of the nanophosphor. Luminescence measurements showed that excitation of this green-emitting phosphor occurred within a wide range of wavelengths (254 – 460 nm) while the half-life time of persistent luminescence laid within the seconds regime. Presence of Dy3+ as the co-dopant enhanced the green emission in this interval of time, and the entire decay time occurred in minutes. Crystallinity and morphology were evaluated by X-ray diffraction (XRD) and transmission electron microscopy (TEM), respectively. Strontium aluminate co-doped with 1%Eu, and 1%Dy, and 1%Eu, and 3%Dy emitted an intense green signal and long decay time. These crystal nanophosphors displayed sizes of 18 nm and 22 nm, respectively. The cytotoxic effect of nanoparticles was determined by a cell viability test where the tri-methyl-tetrazolium reagent (MTT) was reduced only by metabolically active cells. Different concentrations of bare nanoparticles were tested in a 96-well plate containing 10, 000 cells per well of a human cervix carcinoma cell line (HeLa). Evaluation of cell viability by this cytotoxic assay showed that in most of the cases cell viability was higher than 60% after incubation with bare nanoparticles. Since our bare nanoparticles were not cytotoxic, these results open a broad field of biomedical applications for phosphorescent materials as cell biolabels and imaging research area.
© 2016 Optical Society of America
Corrections
11 April 2016: A correction was made to the author affiliations.
1. Introduction
Persistent luminescence is an optical phenomenon shown by certain materials when they are excited with a certain type of energy (e.g. UV photons or higher energy excitation), as a response, the material emits light for a period of time even when the excitation has stopped. This lapse of emission may last from seconds to hours and usually occurs in the visible light range. Persistent luminescence has also been named afterglow, long-lasting phosphorescence or only phosphorescence [1–3]. Frequently, persistent luminescence is called phosphorescence due to long emission time, however the term phosphorescence must be used in the context of light emission from organic compounds that involves triplet-to-singlet transitions. In contrast the long time in the persistent luminescence occurs by the storage of the excitation energy in traps, and its subsequent release by thermal activation [4].
Green-emitting phosphors such as ZnS : Cu are used in paints [5, 6], which are commonly used for light guides, luminous paint, clock dials and glow-in-the-dark toys. They usually require the addition of radioisotopes such as tritium (H – 3) and promethium (Pm – 147) into the phosphor to maintain the emission, which compromise safety [1, 7]. Indeed, both Zn and its production process damage the environment [7]. To prevent this problem, alternate materials are being investigated such as the alkaline earth aluminates MAl2O4 (M = Ca, Sr, Ba), which also exhibit persistent luminescence [8, 9]. For example, the phosphor SrAl2O4 : Eu2+ emits around 520nm and is bright green to the naked eye. It has long-lasting decay time and is 10 times brighter than ZnS : Cu [1]. Matsuzawa [1], and Takasaki [10] established that both persistent luminescence time and intensity may be enhanced by co-doping, which involves the introduction of two different rare-earth ions or activators into the lattice. For example, SrAl2O4 nanoparticles (NPs) co-doped with Eu2+ and Dy3+ ions show very bright luminescence that lasts for hours at room temperature [1, 10]. Different synthesis methods produce similar persistent luminescence with the doped aluminates [11, 13].
The limited understanding of the persistent luminescence mechanism in SrAl2O4 : Eu, Dy offer several explanations about the role of Eu2+ in the luminescence process. In the Matsuzawa model, the origin of long persistent luminescence of MAl2O4 : Eu2+ was probably firstly due to alkaline earth vacancies [1,14], and was followed by an electronic promotion between Eu2+ and Dy3+ energetic levels. However, the complete mechanism behind the persistent luminescence phenomenon in SrAl2O4 : Eu, Dy is not completely clear, as many research have shown [2, 15–19]. Therefore many models have been developed in recent years predicting the release of electrons by ionization of photo-excited Eu2+ to Eu3+ within SrAl2O4 : Eu, Dy. The freed electrons are supposedly trapped by the rare-earth co-dopants [16], crystal vacancies in the neighborhood [8], or both [17]. Additionally, Holsa [14] found that the enhancement of the persistent luminescence may be due to the increase in number of traps, and for the efficiency of the trapped energy to the vacancies. Also, evidence of partial oxidation of Eu2+ to Eu3+ in SrAl2O4 : Eu, Dy were observed, while the trivalent dysprosium ions remained their valence state [19]. Clearly, these explanations contrast with Matsuzawa model.
On the other hand, the non-radiative property of SrAl2O4 : Eu2+, Dy3+ represents a promising tool in the bio-detection field, for example, in cell detection. Cells contain molecules highly susceptible to becoming fluorescent when receiving UV radiation for an extended time (t ≥ 5 min) [20]. This emission is an intrinsic property of the cells. It is also called auto-fluorescence and is caused by endogenous fluorophores within the mitochondria [21]. Cellular auto-fluorescence can act as background noise for signal detection. It may be avoided with a persistent luminescence material, which requires a single energy pulse.
The advantage of using nanophosphors in bio-detection relies on the stability, efficiency and long decay time of these materials. Additionally, the auto-fluorescence cellular response is minimized because the excitation time that activates the persistent luminescent material is shorter compared with the excitation time for activate the endogenous fluorophores that provide the auto-fluorescence cellular response. These are some of the main reasons for considering nanophosphors as good candidates of biolabel specific cells. However the synthesized nanophosphor requires additional treatment. In a biological environment, the relatively large surface of the nanophosphor gives rise to multiple interactions. To prevent this, it can be isolated by a coating. Besides preserving the nanophosphor, the coating also serves as a platform to add functional groups that can be covalently linked to specific cells, conferring specific targeting and function to the nanophosphor [22,23]. The first step to evaluate a material in contact with living cells is to measure its cytotoxic effects, which may be inferred from the cellular response to the bare nanophosphors. Cytotoxicity assays evaluate various parameters associated with cell death and proliferation; one of these is the reduction of tetrazolium salts by metabolically active cells [24].
This work presents the synthesis of nanophosphors of strontium aluminate co-doped with europium and dysprosium ions. The luminescent properties, quantum yield, decay time, and morphologic characteristics were studied. Additionally, and as novel application, the bare nanophosphors were tested on a cervical adenocarcinoma cell line by a colorimetric cytotoxic assay. Size, morphology, luminescent properties (decay time and quantum yield) and cytotoxicity of this co-doped material open the possibility of using them as cell biolabels for imaging detection.
2. Experimental
2.1. Synthesis of SrAl2O4 : EuDy phosphors
Strontium aluminate doped with europium and dysprosium was prepared by the combustion synthesis method [11, 12]. A stoichiometric precursor solution was prepared using Al(NO3)3 · 9H2O, Sr(NO3)2, Eu(NO3)3 · 9H2O and Dy(NO3)3 · 5H2O [Alfa-Aesar 99.9965]. These precursors were dispersed in 20 mL of de-ionized water with a magnetic stirrer during 30 min to obtain a homogeneous solution. The solution became transparent and urea (CO(NH2)2) was added as fuel in a 2 : 1 molar concentration. The solution was introduced into the pre-heated oven at 500 °C.
Inside the oven, the solution was dehydrated, large amounts of gases (carbon oxides, nitrogen and ammonia) were expelled, and finally the spontaneous combustion occurred and the powders were expanded with a powerful reaction. The whole process occurred in no more than 15 min [11].
The europium-doped powders were annealed in a H2/N2 (5%/95%) atmosphere during 2 hours in a tubular oven (THERMAL Co.) with an alumina tube. The agglomerated nanophosphors required ultrasonication in the VIBRA-Cell, to obtain a homogeneous distribution. For the dissolution, isopropanol was added to the nanophosphors. The parameters of the ultrasonicator were 750 W with an amplitude of 70%, and total time of 60 min. Different percentages of co-dopants (Eu2+, Dy3+) were tested to obtain the best phosphorescent properties regarding intensity, brightness, quantum yield and decay time.
2.2. Characterization
The luminescent properties of the nanophosphors were analyzed using a fluorescence spectrophotometer (PL, Hitachi F-7000), with excitation at λ = 254 nm, and the emission was evaluated on the visible range (400 – 700 nm). Nanophosphor X-ray diffraction (XRD) was obtained by the XPert-MPD (Philips) with CuKα radiation at λ = 0.15406 nm, operated at 40 kV and 30 mA and measurements in a 2θ = 10° – 80° range taken with a step size of 0.1° per point. Subsequently the spectra were compared with the PCPDFWIN database. Transmission electron microscopy (TEM) was used for morphological studies including defects in the lattice and nanoparticle size distribution. TEM images were obtained with a JEOL JEM-2010 operated at 200 kV accelerating voltage. The temporal limit of the spectrophotometer is as short as a fluorescent material kept emitting (t ≤ 10−8 s); by contrast, decay time for persistent luminescence materials must be in seconds, minutes or higher. The decay time was measured in an optical arrangement shown in Fig. 1. The UV-light (254 nm) excites the powder sample (PS), the light is emitted and collected by a lens that conducted it into the monochromator. Inside, the light is separated by a diffraction grating according of the wavelength peak in the emission spectra, in this case 525 nm. Finally, the photodetector (Det) receive all the emission information using an algorithm in MatLab 8.0 and GPIB interface. The monochromator (ORIEL-260i), power detector (NEWPORT RS-3828), focal lens (f = 10 cm), mirror and UV -source (UVS – 28) were pre-tested separately. The properties of luminescence, quantum yield and long decay time of strontium aluminate co-doped with different levels of Eu– and Dy– co-dopants were compared.
Fig. 1 Optical arrangement for decay time measurements: The UV-source (λ = 254 nm) excites the powder sample (PS). The emission generated by the PS is collected by a lens and conducted into the monochromator. Within the monochromator the light is guided by a set of lenses. Light is selected in the emission range (λ = 254 nm), and finally detected by a power detector (Det).
2.3. Cell viability assay
Cytotoxicity of bare nanophosphors was tested on cervical adenocarcinoma HeLa cells (ATCC CCL-2). The HeLa cell line was cultured at 37°C with 5% CO2 in RPMI-1640 media supplemented with 10% fetal bovine serum (Gibco), 1% antibiotic/antimicotic (Sigma-Aldrich) and 1% of L-gluthamine (Gibco). A 96-well plate was used to seed 10, 000 HeLa cells per well in supplemented RPMI-1640 media. Cells were then incubated at 37°C with 5% CO2 for 24 h prior the treatment with nanoparticles. Before the incubation with HeLa cells, nanophosphors were dispersed by ultrasonication for 15 min and different dilutions of nanoparticles were prepared in supplemented RPMI-1640 media. HeLa cells were exposed to 0.001; 0.01; 0.1 and 1 μg/μL of nanophosphors for 24 h at 37°C with 5% CO2. Cell viability was analyzed by a colorimetric assay based on the reduction of MTT reagent (methy-134-thiazolyltetrazolium, Sigma-Aldrich). The cytotoxic effect of different concentrations of bare nanophosphors was compared with a negative control of cell viability exposed to DMSO (dimethyl sulfoxide), which induces cell death. Whereas, incubation for 24 h of HeLa cells with RPMI-1640 media without nanophosphors acted as positive control, simulating cell behavior under ideal conditions. Absorbance measurements at 570 and 690 nm were taken with a plate reader (Thermo Scientific), and used to compare cell viability controls with the cytotoxicity induced by different nanophosphor concentrations to the HeLa cells.
3. Results
Figures 2 and 3 show the excitation and emission spectra of some synthesized co-doped strontium aluminates, respectively. Co-dopant percentages are indicated by two consecutive numbers (ij) after the lattice denomination (SAO). The first number is for the percentage of europium and the second for dysprosium. Similar results were obtained for other synthesized nanophosphors.
Fig. 3 Emission spectrum of SrAl2O4: Eu2+, Dy3+ phosphors (SAOij). The first number (i) is for the percentage of europium and the second (j) for dysprosium. Excitation and emission spectra are shown in arbitrary units (A.U.).
Notice that the photoluminescence spectra of all SAOij are similar on the shape and only the relative intensity varies according with the co-dopant concentration. A specific excitation occurs between 245 nm and 265 nm. The SAOij at different concentrations is also excited with the blue light of the visible spectrum (400–460 nm). The emission spectrum (Fig. 3) shows that co-dopants greater than 2%, such as SAO22 and SAO33 include not only green emissions, but yellow (593 nm), and red emissions (613 nm, 661 nm), and these are due to presence of Eu3+ where the main emission lines in the range of 595 to 615 nm take place [19]. On the other hand, co-dopants with minimal europium co-dopant [SAO(1/2)1: 0.5% Eu2+, 1% Dy3+] exhibit low intensity, and are comparable in relative intensity with the lattice without co-dopants (SAO).
Strontium aluminate co-doped with 1% Eu2+ and 1% Dy3+ (SAO11), and with 1% Eu2+ and 3% Dy3+ (SAO13) have very good persistent luminescence properties, i.e. long decay time and high quantum yield (Fig. 4). Decay time of SAOij was obtained with a high level of confidence using the optical arrangement shown in the previous section (Fig. 1). The longest decay time was registered for SAO13, followed by SAO11, and it was detected for longer than one minute with the power detector, and for more than three minutes with the naked eye. A quantum yield of 26.67% for SAO11, and 39.14% for SAO13 corroborated the optimal properties of these persistent luminescence nanomaterials. Other co-doped phosphors showed faster decay time and lower quantum yield, such as SAO11, SAO33, SAO21, and SAO22, with less than 23% of quantum yield. Emission and excitation spectra obtained in Fig. 2 and 3 were similar to the obtained in the literature [1–3,5]. However, a comparison between the quantum yield and decay time values was not founded for similar nanoparticles of SrAl2O4. Several factors are different such as: synthesis method, fuel, annealing temperature and nanoparticle size, and a possible comparison is not possible at this time.
Fig. 4 Central graph: Decay time of SrAl2O4:Eu, Dy phosphors with different percentage of co-dopant, additionally quantum yield (QY) percentage for the SAO11 and SAO13 samples. Right upper inset: decay time curve, where the quantum yield is less than 20% for other do-doped nanophosphors.
In Fig. 5 the curves for SAO11 and SAO13 fit an exponential decay model described by the equation [25]:
where I1 and I2 are the luminescence intensities at time 0 and at a given time t, the resulting I will be a linear combination of both. Persistent luminescence half-life time is represented by τ. Decay time curves can be fitted very well by Eq. 1, and is implicit that there are two different emissions (Fig. 5). The major half-life time value for the fitted curve of SAO11 occurs at τ1 = 5.67 s, and the second at τ2 = 0.64 s. In the case of fitted SAO13, τ1 = 13.22 s and τ2 = 1.12 s. Both values represent the emission half- life time for a persistent luminescence material, this decay time is in the order of seconds, and both are associated to different time of de-excitation that occurs for Eu2+ and Dy3+ into the lattice [1, 25].Decay time for SAO13 is longer than for SAO11 (Fig. 5); these decay time values are supported by the high quantum yield of the materials. The detected persistent luminescence intensity is greater for SAO13 (Fig. 3).
Figure 6 indicates that the SAOij phosphors have a monoclinic structure, according with the database (PCPDFWIN No. 740794). No difference in the monoclinic structure was observed with different co-dopant percentages (Fig. 6).
Fig. 6 X-Ray Diffraction of Strontium Aluminum Oxides (SAOij). According to PCPDFWIN No. 740794 the obtained structure was monoclinic for all nanophosphors.
Figures 7(a) and 7(b) display transmission electron microscope (TEM) images before and after the ultrasonication process, respectively. The difference between the agglomerates and ultrasonicated nanophosphors is clear.
Fig. 7 TEM images of SAO11: (a) without the ultrasonication process. The phosphors showed a predominant spherical morphology. (b) After ultrasonication process. The phosphors are more separated after this process.
The synthesis of nanophosphors (SAOij) generated different nanoparticle size (average size: 18 – 80 nm), but we found no correlation between size and co-dopant percentage. Figure 8 displays the nanoparticle size distribution of SAO11, and SAO13; the averaged diameter of these phosphors is 18 nm, and 22 nm, respectively. Other co-doped nanophosphors were larger than SAO11 and SAO13, for example, for SAO12 the average size was 63 nm, and for SAO31, 184 nm.
Fig. 8 Nanoparticle size distribution. The average size was: a) 18 nm for SAO11, and b) 22 nm for SAO13.
To evaluate if the synthetized nanophosphors are suitable for biomedical applications, the cytotoxic effect was tested. Figure 9(a) and 9(b) show the cytotoxicity results of bare nanophosphors SAO11 and SAO13, respectively tested on HeLa cells. A comparison between the absorbance measurements of the positive control (cells incubated with RPMI-1640 media) and different concentrations of nanophosphors was done. As shown in Fig. 9(a), exposition of HeLa cells to SAO11 nanophosphor resulted to induce the survival of approximately 60% of the cells for all the tested concentrations. Moreover, Fig. 9(b) depicted the cytotoxic effect of SAO13 nanophosphor when exposed to HeLa cells; it is evident that for concentrations from 0.001 to 0.1 μg/μL cell survival was 60 to 70%. However, when a dose of 1 μg/μL of SAO13 was used, the survival of the cells increased up to 80%. This effect was caused by the agglomeration and precipitation of SAO13 bare nanoparticles in the bottom of the well rather to the cytotoxic effect. We have observed that SAO13 nanoparticles, tend to aggregate at concentrations higher than 0.1 μg/μL. To overcome the smooth cytotoxic effect of both SAO11 and SAO13, it is possible to cover the nanophosphors with a biocompatible polymer such as polyethyleneglycol (PEG) or oxide silica.
Fig. 9 Cytotoxic effects of SAO11 and SAO13 nanophosphors on HeLa cells. Cell viability percentage is showing for cells incubated with SAO11 (a) and SAO13 (b). Positive control (C+) represents the viability of cells incubated with RPMI-1640 media under normal conditions. Bars represent the mean and standard deviation of a threefold independent experiments.
4. Discussion
Homogeneous strontium aluminate nanophosphors co-doped with Eu2+, and Dy3+ were prepared by combustion synthesis. These nanophosphors were excited with a single energy pulse from a UV-light, or more energetic such as X-ray. Even the blue light in the visible spectrum could excite them (Fig. 2). Different concentrations of co-dopants were tested; however the spectra of the photoluminescence properties (excitation and emission) remained constant for all concentrations: the nanophosphors always exhibited their characteristic green emission and maintained the broad emission spectra. This means that the excitation energy (UV-light) over the Eu2+ constantly promoted the same energetic holes in the f orbitals due to the excited states (4 f65d1), and independently of the co-dopant percentage.
From the combustion synthesis we obtained nanophosphors of different sizes and morphologies. In Fig. 7(a) and 7(b) the predominant nanophosphors are quasi-spherical, but we also found irregular grains. We corroborated, with the size distribution the existence of large phosphors including micro-sized phosphors. In this work, the most intense co-doped aluminates occurred for 1% Eu2+ and 1% Dy3+ (labelled SAO11), fabricated by combustion synthesis and, in agreement with Matsuzawa [1], calcining the appropriate precursors.
We obtained another intensely emitting nanophosphor; it was doped with 1% Eu2+ and 3% Dy3+ (SAO13). It showed similar quantum yield and decay response as SAO11 (Fig. 4). The use of urea as fuel may be the difference in the size of nanophosphors and the annealing conditions to maintain crystallinity. To the best of our knowledge, this co-doped nanophosphor has not been previously reported.
As reported elsewhere [13, 15], the strontium aluminate phase and the crystallinity were altered by modifications in the time and temperature of the annealing treatment due to the complexity of the phase equilibrium of the SrO – Al2O3 system. Aitasalo et al [17] studied these parameters and found that the most stable phase occurs at 1200°C with post-annealing treatment in a reductive H2/N2 atmosphere. The same procedure was followed in the present study to obtain the most stable phase of strontium aluminate.
The decay time curves are well fitted by Eq. 1 and this implies that two emissions are present: Eu2+ and Dy3+ emitting centers, in accordance with Lin [25].
Cell viability was tested using the MTT assay, which evaluated cell survival of HeLa cells incubated with different concentrations of bare nanophosphors. The concentrations used in the present study (0.001–1 μg/μL) allowed the cells to survive approximately 60% when exposed to SAO11, see Fig. 9(a). However, at 1 μg/μL of SAO13, this nanoparticle tend to agglomerate and interfere with the absorbance measurements resulted in a false positive of cell viability of 80% at this concentration. After analyzing the remaining viability in all the concentrations tested, it is possible to conclude that cell viability for SAO13 is in between 60% and 70%. Strictly, a compound is considered cytotoxic when cell viability is as low as 20% [26]. Therefore, none of the present preparations showed this effect. To reduce the smooth cytotoxic effect of SAO11 and SAO13, nanophosphors could be covered by a polymer or an inert surface such as polyethyleneglycol or silica. This methodologies have been successfully used to reduce the reactivity of nanoparticles and to diminish their cytotoxic effect in cell cultures [27]. Additionally, cell responses to nanoparticles would depend not only to the chemical composition of the nanoparticles, but also to the size and morphology. It has been reported that cell responses like nanoparticle internalization inside the cell, may vary according to the size of the nanoparticle tested [28]. In this case SAO11 nanophosphors is co-doped with 1% Eu2+ and 1% Dy3+ while SAO13 is co-doped with 1% Eu2+ and 3% Dy3+. Indeed both nanoparticles have different size distribution as observed in Fig. 8. Where SAO11 resulted to be smaller (18 nm) than SAO13 (22 nm). This may explain also the tendency of SAO13 to precipitate and the cell response given when exposure to SAO13 was achieved.
Therefore this result opens the possibility to evaluate the usage of the SAO11 and SAO13 nanophosphors as further biolabels to detect cancer cells.
5. Conclusions
SrAl2O4 co-doped with Eu2+ and Dy3+ at nanoscale was successfully prepared via combustion synthesis. Synthesized nanophosphors displayed sizes of 18 nm and 22 nm for strontium alumi-nate co-doped with 1% Eu2+, and 1% Dy3+ (SAO11), and 1% Eu2+, and 3% Dy3+ (SAO13), respectively. We tested different concentrations of co-dopants to obtain the material with the best photoluminescence properties. We observed differences in intensity counts, but in each co-doped material, the highest emission or emission peak was observed around 525 nm independently of the concentration of the co-dopants. SAO11 and SAO13 revealed the highest intensity in the green emission. This was corroborated with the high quantum yield that both nanophosphors showed when were exposed to UV-light (λ = 254 nm); however, blue light (λ = 400 – 460 nm) in the visible spectrum excited them too.
As confirmed by XRD, the SrAl2O4 nanophosphors maintained a monoclinic structure independently of the co-dopants percentage. In this structure, the Dy3+ substituting the Al3+ places certainly enhanced the persistent luminescence and yielded a long decay time [29].
Decay time for SrAl2O4 nanophosphors occurs in seconds and even in minutes. To measure this time, the spectrophotometer was limited in the time scale to measure fluorescent materials only (t ≤ 10−8 s), but using the optical arrangement proposed we were able to detected the longer decay time with a high confidence level and reproducibility. The half-life time values were of the order of seconds.
The SrAl2O4 nanophosphors have potential applications due of its green emission, which is considered the most visible o evident color to the human eyes. This emission was activated by a wide energy spectrum (λ = 254 – 460 nm) with a single pulse of energy, restraining the use of high energies and consequently hindering unnecessary damage.
Cell viability determined by a cytotoxic assay showed that the cell percentage damaged by the bare nanoparticles was less than 40%. These nanoparticles are therefore considered noncytotoxic in the used concentrations.
Size, morphology, luminescent properties (emission, decay time, and quantum yield), and low cytotoxicity of these co-doped materials offer the possibility to be used as cell biolabels for detection of cancer cells.
Further studies in order to evaluate cell viability are still under progress and should include the polymeric coating and functionalization of the nanophosphors for a complementary and detailed evaluation of cytotoxicity.
Acknowledgments
We would like to acknowledge DGAPA-UNAM (Grant IN-109913) and CONACYT (scholarship No. 237502, grant No. 232608). Technical assistance by E. Aparicio, I. Gradilla, J. Mendoza and F. Ruiz is also gratefully appreciated. Special thanks to I. Pérez-Montfort for reviewing the manuscript.
References and links
1. T. Matsuzawa, Y. Aoki, N. Takeuchi, and G. Murayama, “A new long phosphorescent phosphor with high brightness, SrAl2O4 : Eu2+, Dy3+,” J. Electrochem. Soc. 13(8), 2670–2673 (1996). [CrossRef]
2. K. Van den Eeckhout, P.F. Smith, and D. Poelman, “Persistent luminescence in Eu2+ doped compounds: a review,” Materials 3(4), 2536–2566 (2010). [CrossRef]
3. E. Shafia, A. Aghaei, M. Bodaghi, and M. Tahiri, “Combustion synthesis, structural and photo-physical characteristics of Eu2+ and Dy3+ co-doped SrAl2O4 phosphors nanopowders,” J. Mater. Sci. -Mater. Electron. 22(8), 1136–1142 (2011). [CrossRef]
4. T. Maldiney, D. Scherman, and C. Richard, “Persistent luminescence nanoparticles for diagnostics and imaging,” ACS: ACS Symp. Ser.Washington (2012).
5. V. Chernov, T. M. Piters, R. Melndrez, W. M. Yen, E. Cruz-Zaragoza, and M. Barbosa-Flores, “Photoluminescence, afterglow and thermoluminescence in SrAl2O4 : Eu2+, Dy3+ irradiated with blue and UV light,” Radiat. Meas. 42(4), 668–671 (2007). [CrossRef]
6. M. Nazarov, “Persistent phosphors for painting, medical and biological applications,” MJPS 12 (N1-2), 102–118 (2013).
7. X. Zhang, L. Yang, Y. Li, H. Li, W. Wang, and B. Ye, “Impacts of lead/zinc mining and smelting on the environment and human health in China,” Environ. Monit. Assess. 184(4), 2261–2273 (2012). [CrossRef]
8. F. Clabau, X. Rocquefelte, S. Jobic, P. Deniard, M. H. Whangbo, A. Garcia, and T. Le Mercier, “Mechanism of phosphorescence appropriate for the long-lasting phosphors Eu2+ doped with codopants Dy3+ and B3+,” Chem. Mater. 17(15), 3904–3912 (2005). [CrossRef]
9. D. Haranath, V. Shanker, H. Chander, and P. Sharma, “Tuning of emission colours in strontium aluminate long persisting phosphor,” J. Phys. D: Appl. Phys. 36(18), 2244–2248 (2003). [CrossRef]
10. H. Takasaki, S. Tanabe, and T. Hanada, “Long-lasting afterglow characteristics of Eu, Dy codoped SrOAl2O3 phosphor,” J. Ceram. Soc. Jpn. 104(4), 322–326 (1966). [CrossRef]
11. N. M. Son, L. T. Thao, L. V. Khoa, and N. N. Trac, “Synthesis of SrAl2O4 : Eu2+, Dy3+ phosphorescence nanosized powder by combustion method and its optical properties,” J. Phys. Conf. Ser.187(1), (2009). [CrossRef]
12. T. Peng, H. Yang, X. Pu, B. Hu, Z. Jiang, and C. Yan, “Combustion synthesis and photoluminescence of SrAl2O4 : Eu, Dy phosphor nanoparticles,” Mater. Lett. 58(3), 352–356 (2004). [CrossRef]
13. P. D. Sarkisov, N. V. Popovich, and A. G. Zhelnin, “Luminophores based on strontium aluminates produced by the sol-gel method,” Glass Ceram. 60(9–10), 309–312 (2003). [CrossRef]
14. J. Hölsä, H. Jungnerc, M. Lastusaaria, and J. Niittykoskia, “Persistent luminescence of Eu2+ doped alkaline earth aluminates, MAl2O3 : Eu2+,” J. Alloys Compd. 323, 326–330 (2001). [CrossRef]
15. V. Abbruscato, “Optical and electrical properties of SrAl2O4 : Eu2+,” J. Electrochem. Soc. -Solid State Science 118(6), 930–933 (1971).
16. P. Dorenbos, “Mechanism of persistent luminescence in Eu2+ and Dy3+ codoped aluminate and silicate compounds,” J. Electrochem. Soc. 152(7), H107–H110 (2005). [CrossRef]
17. T. Aitasalo, P. Deren, J. Hölsä, H. Jungner, J. C. Krupa, M. Lastusaari, J. Legendziewicz, J. Niittykoski, and W. Strek, “Persistent luminescence phenomena in materials doped with rare earth ions,” J. Solid State Chem. 171(1), 114–122, (2003). [CrossRef]
18. F. Clabau, X. Rocquefelte, S. Jobic, P. Deniard, M. H. Whangbo, A. Garcia, and T. Le Mercier, “On the phosphorescent mechanism in SrAl2O4 : Eu2+ and its codoped derivatives,” Solid State Sciences 9(7), 608–612 (2007). [CrossRef]
19. K. Korthout, K. Van den Eeckhout, J. Botterman, S. Nikitenko, D. Poelman, and P. F. Smet, “Luminescence and x-ray absorption measurements of persistent SrAl2O4 : Eu, Dy powders: Evidence for valence state changes,” Phys. Rev. B 84(8), 085140 (2011). [CrossRef]
20. R. C. Benson, R. A. Mayer, M. E. Zaruba, and G. M. McKhann, “Cellular autofluorescence Is it due to flavins,” J. Histochem. Cytochem. 27(1), 44–48 (1979). [CrossRef] [PubMed]
21. M. Melici, “Cell and tissue autofluorescence research and diagnostic applications,” Biotechnol. Annu. Rev. 11, 227–256 (2005). [CrossRef]
22. K. D. Wegner, P. T. Lanh, T. Jennings, E. Oh, V. Jain, S. M. Fairclough, J. M. Smith, E. Giovanelli, N. Lequeux, T. Pons, and N. Hildebrandt, “Influence of luminescence quantum yield, surface coating, and functionalization of quantum dots on the sensitive of time-resolved free bioassays,” ACS Appl. Mater. Interfaces 5(8), 2881–2892 (2013). [CrossRef] [PubMed]
23. C. J. Lin, R. A. Sperling, J. K. Li, T. Yang, P. Li, M. Zanella, W. H. Chang, and W. J. Parak, “Design of an amphiphilic polymer for nanoparticle coating and functionalization,” Small 4(3), 334–341 (2008). [CrossRef] [PubMed]
24. J. Weyermann, D. Lochmann, and A. Zimmer, “A practical note on the use of cytotoxicity assays,” Int. J. Pharm. 288(2), 369–376 (2005). [CrossRef]
25. Y. Lin, Z. Zhang, Z. Tang, J. Zhang, Z. Zheng, and X. Lu, “The characterization and mechanism of long afterglow in alkaline earth aluminates phosphors co-doped by Eu2O3 and Dy2O3,” Mater. Chem. Phys. 70(2), 156–159 (2001). [CrossRef]
26. D. Sgouras and R. Duncan, “Methods for the evaluation of biocompatibility of soluble synthetic polymers which have potential for biomedical use: 1-use of the tetrazolium-based colorimetric assay (MTT) as a preliminary screen for evaluation of in vitro cytotoxicity,” J. Mater. Sci. -Mater. Med. 1(2), 61–68 (1990). [CrossRef]
27. H. Otsuka, Y. Nagasaki, and K. Kataoka, “PEGylated nanoparticles for biological and pharmaceutical applications,” Adv. Drug Deliv. Rev. 64, 246–255 (2012). [CrossRef]
28. W. Jiang, B. Y. S. Kim, J. T. Rutka, and W. C. W. Chan, “Nanoparticle mediated cellular response is size-dependent,” Nat. Nanotechnol. 3(3), 145–150 (2008). [CrossRef] [PubMed]
29. T. Katsumata, T. Nabae, K. Sasajima, S. Komuro, and T. Morikawa, “Effects of composition on the long phosphorescent SrAl2O4 : Eu2+, Dy3+ phosphor crytals,” J. Electrochem. Soc. 144(9), L243–L245 (1997). [CrossRef]
