Journal of Colloid and Interface Science 440 (2015) 39–45
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Microwave-assisted solvothermal synthesis and upconversion luminescence of CaF2:Yb3+/Er3+ nanocrystals Jing Zhao, Ying-Jie Zhu ⇑, Jin Wu, Feng Chen State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China
a r t i c l e
i n f o
Article history: Received 4 September 2014 Accepted 21 October 2014 Available online 29 October 2014 Keywords: Nanostructures Fluorides Microwave Upconversion Rare earths
a b s t r a c t Water-dispersible CaF2 and Yb3+/Er3+ codoped CaF2 (CaF2:Yb3+/Er3+) nanocrystals with different sizes and different Yb3+ and Er3+ dopant concentrations were synthesized using ionic liquid 1-n-butyl-3-methyl imidazolium tetrafluoroborate as a fluorine source by the rapid microwave-assisted solvothermal method. It was found that the morphology, size and crystallinity of CaF2:Yb3+/Er3+ nanocrystals could be adjusted by using adenosine 50 -triphosphate disodium salt (ATP). Yb3+ and Er3+ ions were doped into CaF2 nanocrystals to enable upconversion luminescence emission, and the as-prepared CaF2:Yb3+/Er3+ samples exhibited upconversion luminescence upon excitation at 980 nm. Confocal laser scanning microscopy images showed that the CaF2:Yb3+/Er3+ nanocrystals could be used for efficient labeling of human gastric carcinoma cells. Moreover, in vitro cytotoxicity experiments indicated that the as-prepared CaF2:Yb3+/Er3+ nanocrystals had essentially little cytotoxicity. These results indicate that the as-prepared CaF2:Yb3+/Er3+ nanocrystals are promising for the application as a luminescent label material in biological imaging. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction It is well known that upconversion fluorescent materials have the ability to convert lower-energy photons into higher-energy photons. Compared with the conventional fluorescent materials, upconversion fluorescent materials have many advantages including greater tissue penetration, minimized autofluorescence, high signal-to-noise ratio and high photochemical stability [1–4]. As a new generation of luminescent materials, lanthanide-doped upconversion nanocrystals (UCNCs) have been widely investigated in the recent years. Efforts have been devoted to developing lanthanide-doped upconversion nanoparticles for applications in biological luminescent labeling, sensing and imaging. For instance, Liu et al. [5] prepared core/shell NaYF4:Tm,Yb nanoparticles and investigated their application in near-infrared-triggered anticancer drug delivery. Multifunctional upconversion luminescent materials, such as upconversion luminescence–magnetic hybrid nanoparticles and multifunctional upconversion nanoprobes, have also been investigated [6–9]. Upconversion luminescent materials generally comprise an inorganic host and lanthanide dopant ions embedded in the host. According to the selection of the host material, the UC luminescent ⇑ Corresponding author. Fax: +86 21 52413122. E-mail address: [email protected] (Y.-J. Zhu). http://dx.doi.org/10.1016/j.jcis.2014.10.031 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
materials can be classified into two kinds. One kind is based on fluorides and the other is based on metal oxide materials. Compared with metal oxide materials, metal fluoride materials such as CaF2, NaYF4, YF3, LaF3 and BiF3 are more appropriate to be used as the host materials because of their lower phonon energies [10,11]. CaF2 crystal is a frequently used phosphor host because of its high solubility and transparence in the range from the near ultraviolet to the middle of infrared [12,13]. Lanthanide-doped CaF2 materials have been extensively studied as one kind of the most efficient upconversion luminescent materials [14]. In general, the as-prepared nanoparticles are difficult to disperse in water due to the presence of hydrophobic organic ligands on the surface of UCNCs. An additional surface functionalization step is necessary to prepare water-dispersible lanthanide-doped UCNCs [15]. For example, Ma et al. [16] employed 6-phosphate-6-deoxy-b-cyclodextrin (bPCD) as the novel surface ligand to fabricate a versatile upconversion luminescent nanoplatform. Using bPCD as the surface ligand not only enhanced the stability and biocompatibility of the UCNCs under physiological conditions but also enabled simple conjugation with various functional molecules. The conjugated upconversion nanoprobe exhibited excellent capability in labeling the cancer cells and tumor tissue slices for luminescent imaging. Guo and Li [10] synthesized rare earth doped lanthanum fluoride nanocrystals (LaF3:5% Yb, 2% Er) via a solvothermal method in octadecylene. Under the irradiation of 980-nm diode laser, these
40
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
nanocrystals emitted strong green upconversion luminescence. These hydrophobic nanocrystals were functionalized with poly(amino acid) to enable them water-dispersible and biocompatible. Sarkar et al. [11] reported the synthesis of water-dispersible BiF3 nanoparticles with sizes of about 6 nm in a poly(vinyl pyrrolidone) matrix by the hydrothermal method. Through suitable Ln3+ doping, BiF3 exhibited strong emissions in the visible region upon both UV and near infrared excitations. Although some progress has been made, it remains a challenge to synthesize UCNCs with a good aqueous dispersibility and high UC luminescence intensity. A number of techniques, such as the thermal decomposition method, hydrothermal/solvothermal method and sol–gel process have been developed to synthesize lanthanide-doped UCNCs. However, some synthetic techniques generally require high reaction temperatures and prolonged reaction times, which lead to the aggregation of the nanoparticles [17–20]. In this study, the rapid microwave-assisted solvothermal method has been developed to synthesize water-dispersible CaF2 and CaF2:Yb3+/Er3+ nanocrystals using ionic liquid 1-n-butyl-3methyl imidazolium tetrafluoroborate as a fluorine source. The morphology, size and crystallinity of CaF2:Yb3+/Er3+ nanocrystals can be adjusted by adenosine 50 -triphosphate disodium salt (ATP). This method combines the advantages of microwave rapid heating and pressurized solvothermal process, thus can achieve high reaction rate and short reaction time. The preparation time of the microwave-assisted heating method can often be reduced by orders of magnitude compared with the conventional heating methods, leading to very high efficiency and energy saving [21– 25]. For instance, Xu et al. [26]. reported the microwave-assisted ionic liquid solvothermal rapid synthesis of hollow microspheres of alkaline earth metal fluorides (MF2, M = Mg, Ca, Sr) using [BMIM]BF4 as a fluorine source. To meet the needs for biological labeling, the UCNCs need to have high water-dispersibility, good biocompatibility, nanometer size and high luminescence intensity [27]. Based on these considerations, we have prepared water-dispersible CaF2:Yb3+/Er3+ nanocrystals by doping Yb3+ and Er3+ ions into CaF2 nanocrystals to enable upconversion (UC) luminescence emission, and the asprepared CaF2:Yb3+/Er3+ samples exhibit UC luminescence upon excitation at 980 nm. The as-prepared CaF2:Yb3+/Er3+ crystals have little cytotoxicity, and can efficiently label human gastric carcinoma cells in vitro. These results indicate that the as-prepared CaF2:Yb3+/Er3+ nanocrystals are promising for the application as a luminescent label material in biological imaging.
product. Control sample 1 was synthesized using 0.147 g CaCl22H2O, 0.110 g adenosine 50 -monophosphate disodium salt hexahydrate (AMP) and 3 mL [BMIM]BF4 under the same conditions. Control sample 2 was synthesized using 0.147 g CaCl22H2O, 0.110 g NaH2PO4 and 3 mL [BMIM]BF4 under the same conditions. Control sample 3 was prepared using 0.147 g CaCl22H2O and 3 mL [BMIM]BF4 in the absence of any additive under the same conditions. 2.2. Preparation of CaF2:Yb3+/Er3+ nanocrystals Water-dispersible CaF2:Yb3+/Er3+ nanocrystals with varying Yb and Er3+ doping concentrations and varying sizes were prepared. For the synthesis of CaF2:Yb3+/Er3+ (20/2 mol% Yb3+/Er3+) UCNCs, 0.0076 g Er2O3 and 0.0788 g Yb2O3 were dissolved in HNO3 under vigorous stirring. After the evaporation of excessive HNO3, the residual Er(NO3)3 and Yb(NO3)3 powders were dissolved in 30 mL deionized water. Then, 0.147 g CaCl22H2O, 0.110 g ATP, 10 mL ethylene glycol and 3 mL ionic liquid [BMIM]BF4 were added into the above solution under magnetic stirring. The resulting solution was transferred into a 60 mL autoclave, sealed and microwave heated in a microwave oven at 150 °C for 1 h. After cooling to room temperature, the product was separated by centrifugation, washed with deionized water and ethanol, and dried at 60 °C. The as-prepared CaF2:Yb3+/Er3+ (20/2 mol% Yb3+/Er3+) UCNCs were named as sample A. CaF2:Yb3+/Er3+ (10/1 mol% Yb3+/Er3+) UCNCs (sample B) were synthesized by the same procedure except that the amounts of Er2O3 and Yb2O3 were 0.0039 g and 0.0398 g, respectively. The synthesis of the ATP-free samples (samples C and D) follows a similar procedure. The reactant amounts of the four samples are shown in Table 1. 3+
2.3. In vitro cytotoxicity study In vitro cytotoxicity tests were carried out by the standard MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay [28]. Human gastric carcinoma cells (SGC-7901) cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C for 24 h were used for cell viability tests. Cells were seeded in a 96-well flat-bottomed microassay plate at a concentration of 1 104 cells mL1 and cultured for 24 h. The sterilized samples (samples A, B, C and D) were added to the wells at concentrations ranging from 0.1 to 100 mg L1 and cocultured with the cells for 24 h. Cell viability was quantified by the MTT assay. Each data point was represented by the mean of three parallel tests.
2. Materials and methods 2.1. Preparation of CaF2 nanocrystals The water-dispersible CaF2 nanocrystals were synthesized as follows: 0.147 g CaCl22H2O and 0.110 g adenosine 50 -triphosphate disodium salt (ATP) were dissolved in 30 mL deionized water, and then 10 mL ethylene glycol and 3 mL ionic liquid 1-n-butyl-3methylimidazolium tetrafluoroborate ([BMIM]BF4) were added into the solution under magnetic stirring. The resulting solution was transferred into a 60 mL autoclave, sealed and microwave heated at 150 °C in a microwave oven (MDS-6, Sineo, China). The reaction time was set as 30 min, 1 h and 2 h, respectively. After cooling to room temperature, the products were separated by centrifugation, washed with deionized water and ethanol, and then dried at 60 °C. The influence of pH value was also investigated. The initial pH value of the solution was adjusted to 3.0, 5.0 and 7.0 by using dilute NaOH solution, respectively, and then microwave heated under the same conditions. In this study, other additives were also investigated to adjust the morphology of the CaF2
2.4. Confocal laser scanning microscopy observation of CaF2:Yb3+/Er3+ UCNCs For confocal laser scanning microscope observations, human gastric carcinoma cells (SGC-7901, 1 104 cells mL1) were seeded in glass bottom dishes, and co-cultured with CaF2:Yb3+/Er3+ UCNCs (100 mg L1) for 12 h at 37 °C. Then for every dish, the culture medium was removed and 0.5 mL 4,6-diamidino-2-phenylindole
Table 1 The amounts of Er2O3, Yb2O3 and ATP used to synthesize CaF2:Yb3+/Er3+ crystals with different sizes. The reaction conditions were the same for all the samples.
Er2O3 (g) Yb2O3 (g) ATP (g) Yb3+/Er3+ (mol%) Mean size (nm)
Sample A
Sample B
Sample C
Sample D
0.0076 0.0788 0.110 20/2 26
0.0039 0.0398 0.110 10/1 23
0.0076 0.0788 0 20/2 793
0.0039 0.0398 0 10/1 645
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
(DAPI) solution in phosphate buffered saline (PBS) (10 %) was added. The cells were incubated for 15 min to stain the nuclei and fix the cells. After the incubation, the cells were washed and observed under a confocal laser scanning microscope (FluoView FV 1000, Olympus). 2.5. Characterization Transmission electron microscopy (TEM) micrographs and selected-area electron diffraction (SAED) patterns were obtained on a transmission electron microscope (HITACHI, H-800, Japan). The scanning electron microscopy (SEM) micrographs and energy dispersive spectra (EDS) of the samples were performed using a field-emission scanning electron microscope (S-4800, Japan). The crystal phases of the samples were determined by X-ray powder diffraction (XRD) using an X-ray diffractometer with a graphite monochromator (Cu Ka radiation, k = 1.54178 Å, Rigaku D/max 2550V, Japan). Thermogravimetric (TG) analysis was carried out on a simultaneous thermal analyzer (Netzsch, STA 409PC, Germany) at a heating rate of 10 °C min1 in flowing air. Upconversion luminescence spectra of the samples were recorded at room temperature by a spectrofluorometer (Fluorolog-3, Jobin Yvon). A 980 nm continuous wave diode laser was used as the excitation source. 3. Results and discussion In this study, we synthesized water-dispersible CaF2 nanocrystals using CaCl22H2O, an ionic liquid [BMIM]BF4 and ATP in mixed solvents of deionized water and ethylene glycol by the microwaveassisted solvothermal method. In this method, [BMIM]BF4 acts as a fluorine source. In order to investigate how the synthetic conditions influence the size and morphology of the CaF2 product, the
41
reaction time and pH value were varied. Fig. 1a–c shows the TEM micrographs of the products prepared using CaCl22H2O, an ionic liquid [BMIM]BF4 and ATP in mixed solvents of deionized water and ethylene glycol (pH 7.0) by the microwave-assisted solvothermal method at 150 °C for different microwave solvothermal times. CaF2 nanocrystals with sizes of tens of nanometers were obtained at 150 °C for 30 min by microwave heating (Fig. 1a). Fig. 1b shows nearly monodisperse CaF2 nanocrystals with relatively uniform sizes of about 100 nm prepared by microwave heating at 150 °C for 1 h. Fig. 1c shows that the sizes of CaF2 crystals increased to about 150 nm when the microwave heating time was prolonged to 2 h, indicating that the crystal size of CaF2 increased with increasing reaction time. In the subsequent studies, the microwave heating time of 1 h was chosen considering that the corresponding product had relatively uniform morphology, size and good monodispersity. The influence of pH value on the product was also investigated. When the pH value was 3.0, the product consisted of dispersed nanoparticles and nanoparticle-assembled microspheres with sizes from hundred of nanometers to about 1 lm (Fig. 1d). When the pH values were 5.0 and 7.0, the products were dispersed nanocrystals (Fig. 1e and b). The above-discussed samples were prepared using ATP as an additive. We also explored the effect of other additives on the morphology of the product. The product was composed of CaF2 polyhedra with sizes in the range of 50–200 nm when using adenosine 50 -monophosphate disodium salt hexahydrate instead of ATP (Fig. 1f). When NaH2PO4 instead of ATP was used, CaF2 hollow microspheres constructed by the self-assembly of nanoparticles were obtained (Fig. 1g). Fig. 1h shows that the product synthesized in the absence of any additive consisted of CaF2 polyhedra with sizes of about 1 lm. Fig. 2a shows the XRD pattern of the CaF2 sample prepared using 0.147 g CaCl22H2O, 3 mL [BMIM]BF4 and 0.110 g ATP by
Fig. 1. TEM micrographs of CaF2 samples synthesized using 0.147 g CaCl22H2O, 3 mL [BMIM]BF4 and 0.110 g ATP in mixed solvents of deionized water and ethylene glycol by the microwave-assisted solvothermal approach at 150 °C under different synthetic conditions: (a) pH 7.0, 30 min; (b) pH 7.0, 1 h; (c) pH 7.0, 2 h; (d) pH 3.0, 1 h; (e) pH 5.0, 1 h; (f) 0.110 g adenosine 50 -monophosphate disodium salt hexahydrate instead of ATP, pH 7.0, 1 h; (g) 0.110 g NaH2PO4 instead of ATP, pH 7.0, 1 h; and (h) in the absence of ATP and any additive, pH 7.0, 1 h.
42
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
Fig. 2. XRD patterns: (a) the CaF2 sample prepared using 0.147 g CaCl22H2O, 3 mL [BMIM]BF4 and 0.110 g ATP in mixed solvents of deionized water and ethylene glycol (pH 7.0) by the microwave-assisted solvothermal approach at 150 °C for 1 h and (b) the product prepared in the absence of ATP under the same conditions.
the microwave-assisted solvothermal method at 150 °C for 1 h. Fig. 2b shows the XRD pattern of the product prepared in the absence of ATP under the same conditions. One can see that the diffraction peaks of both samples can be indexed to single-phase face-centered cubic CaF2 (JCPDS No. 35-0816, space group: Fm3m). The lattice parameter of both CaF2 products prepared with and without ATP was calculated to be 5.45 Å based on the XRD data, which is in good agreement with that reported in the literature [29]. In this work, Er3+ was selected as the activator and Yb3+ was codoped as a sensitizer to enhance upconversion efficiency, and the ionic liquid [BMIM]BF4 was used as the fluorine source. The synthesis of CaF2:Yb3+/Er3+ samples was carried out under the same conditions as those for the synthesis of single-phase CaF2 at 150 °C for 1 h. The morphologies of the products were observed by SEM and TEM. Fig. 3 shows the TEM and SEM micrographs and size distributions of the as-prepared CaF2:Yb3+/Er3+ samples. Samples A and B were CaF2:Yb3+/Er3+ (20/2 mol%) nanocrystals and CaF2:Yb3+/Er3+ (10/1 mol%) nanocrystals prepared in the presence of ATP as an additive, respectively. The average diameters of as-prepared CaF2:Yb3+/Er3+ nanocrystals were obtained by the
Fig. 3. SEM micrographs (a, d, g, j), TEM micrographs (b, e, h, k) and size distributions (c, f, i, l) of the CaF2:Yb3+/Er3+ samples prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a–c) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) nanocrystals synthesized in the presence of ATP; (d–f) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) nanocrystals synthesized in the presence of ATP; (g–i) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; (j–l) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
Fig. 4. EDS patterns of the as-prepared CaF2:Yb3+/Er3+ samples prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
Fig. 5. TG curves of the CaF2:Yb3+/Er3+ samples prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
analysis of SEM micrographs. One can see that the morphology of CaF2:Yb3+/Er3+ samples (Fig. 3a, b, d, and e) is different from that of undoped CaF2 crystals obtained by the same method (Fig. 1b). Fig. 3a, b, d, and e shows that the as-prepared CaF2:Yb3+/Er3+ nanocrystals were spherical in shape, and the average diameter was measured to be 26.5 ± 3.7 nm and 23.3 ± 4.1 nm, respectively, smaller than that of undoped CaF2 particles prepared under the same conditions (about 100 nm). Insets of Fig. 3b and e show the selected-area electron diffraction (SAED) patterns of samples A and B. Diffraction rings and some bright diffraction spots in the SAED patterns indicate that the as-prepared CaF2:Yb3+/Er3+ nanocrystals had high crystallinity. CaF2:Yb3+/Er3+ crystals of samples C and D were prepared in the absence of ATP. TEM and SEM micrographs (Fig. 3g, h, j, and k) show highly crystalline CaF2:Yb3+/Er3+ polyhedral crystals with an average size of 793.2 ± 103.9 nm and 645.6 ± 107.6 nm, respectively. The SAED patterns (insets of Fig. 3h and k) of these two samples exhibit single-crystalline diffraction spots. These results indicate that ATP has a significant effect on the morphology, size and crystallinity of the product. The possible reason is that the
43
Fig. 6. XRD patterns of the CaF2:Yb3+/Er3+ samples prepared by the microwaveassisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/ 2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
Fig. 7. Upconversion luminescence spectra of the as-prepared CaF2:Yb3+/Er3+ samples in aqueous suspensions (1 mg mL1) prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
Fig. 8. In vitro viabilities of human gastric carcinoma cells after the 24 h incubation with the CaF2:Yb3+/Er3+ samples prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
44
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
Fig. 9. Confocal laser scanning microscopy images of the human gastric carcinoma (SGC-7901) cells incubated with the CaF2:Yb3+/Er3+ samples prepared by the microwaveassisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/ 1 mol%) polyhedral crystals obtained in the absence of ATP. Insets of b–d are magnified images of cell nuclei.
ATP chains can adsorb on the crystal surfaces of CaF2 and affect the growth of CaF2 crystals. The doping of lanthanide ions into CaF2 crystals was also confirmed by the EDS spectra (Fig. 4). For all CaF2:Er3+/Yb3+ samples, peaks attributed to Yb and Er ions were detected. The Yb and Er peak intensities of samples A and C (CaF2:Yb3+/Er3+ (20/2 mol%)) were obviously higher than those of samples B and D (CaF2:Yb3+/ Er3+ (10/1 mol%)). TG analysis was employed to determine the contents of water and organics in the CaF2:Yb3+/Er3+ samples (Fig. 5). The total mass losses of the four samples were less than 4 %, indicating that the adsorption amounts of [BMIM]BF4 and ATP on CaF2:Yb3+/Er3+ crystals were very small. The XRD patterns of the as-prepared CaF2:Yb3+/Er3+ samples are shown in Fig. 6, where the diffraction peaks are in good agreement with the standard data of face-centered cubic CaF2 (JCPDS No. 35-0816). Compared with the XRD patterns of undoped CaF2 samples (Fig. 2), the appearance of (200) peak in the XRD patterns of CaF2:Yb3+/Er3+ samples indicates the incorporation of rare earth ions in CaF2 host [30]. According to the XRD analysis, the lattice constants of rare earth doped samples A, B, C and D were 5.48 Å, 5.49 Å, 5.46 Å and 5.48 Å, respectively, larger than that of the undoped CaF2 sample (5.45 Å). That is because CaF2 has a fluorite structure, in which Ca2+ ions lie at the nodes in a face-centered lattice, while F ions lie at the centers of octants. When Yb3+ or
Er3+ ions substitute for Ca2+, F ions would occupy the interstitial fluoride cubic sites for the charge balance. Thus, the electronic repulsion between fluoride ions occurs not only at the normal fluoride ion sites but also at interstitial fluoride ion sites, and makes the lattice parameters increase [31]. The increase in the cell volume also indicates that Yb3+ and Er3+ ions have been doped into the CaF2 crystal lattice. The upconversion luminescence spectra of the CaF2:Yb3+/Er3+ samples in aqueous suspensions (1 mg mL1, inset of Fig. 7) were measured under a 980 nm laser excitation at room temperature. Fig. 7 shows the measured upconversion emission spectra at the wavelengths ranging from 450 to 700 nm. The four samples had similar emission spectra with the same wavelength positions except for the emission intensities. The upconversion emission peaks in the green1 (520 and 540 nm) and red (658 nm) regions are attributed to the transitions from the excited states. The emission peaks observed at 520 and 540 nm are assigned to the (2H11/2, 4 S3/2)–4I15/2 transitions of Er3+ ions, while the observed red emission at about 658 nm is attributed to the Er3+ 4F9/2–4I15/2 transition. Many factors may influence the luminescence of UCNCs, such as crystal size, shape, crystallinity and lanthanide dopant amount [32]. In 1 For interpretation of color in Figs. 7 and 9, the reader is referred to the web version of this article.
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
general, high crystallinity and large size lead to strong upconversion emissions. In this work, samples A and B exhibit relatively lower upconversion intensities than those of samples C and D. This phenomenon may be explained by the reason that the presence of ATP molecules produced CaF2:Yb3+/Er3+ nanocrystals of samples A and B with smaller crystal size and lower crystallinity. The upconversion emission process has different mechanisms, which have been discussed and summarized in several review articles [33,34]. Excited state absorption (ESA), photon avalanche (PA) and energy transfer upconversion (ETU) are the most accepted basic mechanisms. We propose that the luminescence mechanism of CaF2:Yb3+/Er3+ crystals is an energy transfer upconversion process, which is the most efficient upconversion process [35,36]. Due to the promising applications of upconversion luminescence crystals in various biological fields, it becomes an indispensable demand to evaluate the cytotoxicity of the as-prepared CaF2:Yb3+/Er3+ nanocrystals. The MTT assay was employed to assess the cytotoxicity of CaF2:Yb3+/Er3+ samples with human gastric carcinoma (SGC-7901) cells, and the results are shown in Fig. 8. One can see that as the concentration of CaF2:Yb3+/Er3+ samples increased from 0.1 to 100 mg L1, no obvious toxicity to the cells was observed after 24 h co-incubation for the four CaF2:Yb3+/Er3+ samples at low concentrations. Even at a high concentration of 100 mg L1, the cell viability still remained above 80%. The results show that the as-prepared CaF2:Yb3+/Er3+ samples had low cytotoxicity and the cell growth was not obviously affected. In order to evaluate the cell luminescence labeling ability of the as-prepared CaF2:Yb3+/Er3+ samples, human gastric carcinoma (SGC-7901) cells were incubated with the CaF2:Yb3+/Er3+ samples at 37 °C for 12 h. After the incubation, the cells were washed and stained blue with DAPI. Fig. 9 shows the human gastric carcinoma cells under observation with a confocal laser scanning microscope at the 980 nm radiation. The first column shows the cell nuclei stained with DAPI (blue), whereas the corresponding fluorescence images are displayed in the second (green) and third (red) columns. The fourth column is the superimposed images. From Fig. 9, one can see that the upconversion luminescence was observed in samples B, C and D. The yellow fluorescence represents that CaF2:Yb3+/Er3+ crystals were located around the cell nucleus regions. This result demonstrates that a certain amount of samples B, C and D crystals had been taken up by the cells. However, no fluorescence could be detected from sample A because of the low upconversion intensity. 4. Conclusions In this work, water-dispersible CaF2 and CaF2:Yb3+/Er3+ crystals with different sizes and different Yb3+ and Er3+ dopant amounts have been successfully prepared using an ionic liquid 1-n-butyl3-methyl imidazolium tetrafluoroborate ([BMIM]BF4) as the fluorine source by the one-step, fast and environmentally friendly microwave-assisted solvothermal technique. It has been found that the microwave solvothermal time, pH value and ATP have effects on the morphology, size and crystallinity of the product. The as-prepared CaF2 and CaF2:Yb3+/Er3+ crystals are hydrophilic and
45
easily dispersed in water. The as-prepared CaF2:Yb3+/Er3+ samples show strong upconversion emission peaks in the green (520 and 540 nm) and red (658 nm) regions under 980 nm excitation and can efficiently label human gastric carcinoma cells in vitro. The excellent cell labeling ability, high biocompatibility and good water-dispersibility indicate the as-prepared CaF2:Yb3+/Er3+ samples are potential fluorescence labels for biological applications. Acknowledgment Financial support from the National Natural Science Foundation of China (51172260) is gratefully acknowledged. References [1] J. Chen, J.X. Zhao, Sensors 12 (2012) 2414. [2] J.C. Boyer, F. Vetrone, L.A. Cuccia, J.A. Capobianco, J. Am. Chem. Soc. 128 (2006) 7444. [3] D.K. Chatteriee, A.J. Rufalhah, Y. Zhang, Biomaterials 29 (2008) 937. [4] F. Wang, X.G. Liu, Chem. Soc. Rev. 38 (2009) 976. [5] J.N. Liu, W.B. Bu, L.M. Pan, J.L. Shi, Angew. Chem. Int. Ed. 52 (2013) 4375. [6] X.M. Li, D.Y. Zhao, F. Zhang, Theranostics 3 (2013) 292. [7] J.N. Liu, W.B. Bu, L.M. Pan, S.J. Zhang, F. Chen, L.P. Zhou, K.L. Zhao, W.J. Peng, J.L. Shi, Biomaterials 33 (2012) 7282. [8] F. Chen, P. Huang, Y.J. Zhu, J. Wu, C.L. Zhang, D.X. Cui, Biomaterials 32 (2011) 9031. [9] F. Chen, P. Huang, Y.J. Zhu, J. Wu, D.X. Cui, Biomaterials 33 (2012) 6447. [10] C. Guo, M.G. Li, Acta Chim. Sin. 72 (2014) 215. [11] S. Sarkar, A. Dash, V. Mahalingam, Chem. Asian J. 9 (2014) 447. [12] J.S. Wang, Z.W. Wang, X. Li, S. Wang, H.D. Mao, Z.J. Li, Appl. Surf. Sci. 257 (2011) 7145. [13] G.F. Wang, Q. Peng, Y.D. Li, J. Am. Chem. Soc. 131 (2009) 14200. [14] L. Zhao, A. Kutikov, J. Shen, C.Y. Duan, J. Song, G. Han, Theranostics 3 (2013) 249. [15] L.M. Song, J.H. Gao, R.J. Song, J. Lumin. 130 (2010) 1179. [16] C. Ma, T. Bian, S. Yang, C.H. Liu, T.R. Zhang, J.F. Yang, Y.H. Li, J.S. Li, R.H. Yang, W.H. Tan, Anal. Chem. 86 (2014) 6508. [17] F. Wang, D. Banerjee, Y. Liu, X.Y. Chen, X. Liu, Analyst 135 (2010) 1839. [18] C.Y. Cao, W.P. Qin, J.S. Zhang, Y. Wang, G.F. Wang, G.D. Wei, P.F. Zhu, L.L. Wang, L.Z. Jin, Opt. Commun. 281 (2008) 1716. [19] Z.G. Xia, P. Du, J. Mater. Res. 25 (2010) 2035. [20] M. Zahedifar, E. Sadeghi, Z. Mohebbi, Nucl. Instrum. Methods Phys. Res., Sect. B 274 (2012) 162. [21] Y.J. Zhu, F. Chen, Chem. Rev. 114 (2014) 6462. [22] Y.J. Zhu, W.W. Wang, R.J. Qi, X.L. Hu, Angew. Chem., Int. Ed. 43 (2004) 1410. [23] M. Baghbanzadeh, L. Carbone, P.D. Cozzoli, C.O. Kappe, Angew. Chem., Int. Ed. 50 (2011) 11312. [24] C. Qi, Y.J. Zhu, B.Q. Lu, X.Y. Zhao, J. Zhao, F. Chen, J. Wu, Chem. Eur. J. 19 (2013) 5332. [25] J. Zhao, Y.J. Zhu, J.Q. Zheng, F. Chen, J. Wu, Microporous Mesoporous Mater. 180 (2013) 79. [26] J.S. Xu, Y.J. Zhu, CrystEngComm 14 (2012) 2630. [27] T.Y. Cao, Y. Yang, Y. Gao, J. Zhou, Z.Q. Li, F.Y. Li, Biomaterials 32 (2011) 2959. [28] B.R. Schroeder, M.I. Ghare, C. Bhattacharya, R. Paul, Z.Q. Yu, P.A. Zaleski, T.C. Bozeman, M.J. Rishel, S.M. Hecht, J. Am. Chem. Soc. 136 (2014) 13641. [29] A. Bensalah, M. Mortier, G. Patriarche, P. Gredin, D. Vivien, J. Solid State Chem. 179 (2006) 2636. [30] P. Aubry, A. Bensalah, P. Gredin, G. Patriarche, D. Vivien, M. Mortier, Opt. Mater. 31 (2009) 750. [31] M. Pedroni, F. Piccinelli, T. Passuello, M. Giarola, G. Mariotto, S. Polizzi, M. Bettinelli, A. Speghini, Nanoscale 3 (2011) 1456. [32] H.X. Mai, Y.W. Zhang, L.D. Sun, C.H. Yan, J. Phys. Chem. C 111 (2007) 13721. [33] H. Dong, L.D. Sun, C.H. Yan, Nanoscale 5 (2013) 5703. [34] M. Wang, G. Abbineni, A. Clevenger, C. Mao, S. Xu, Nanomed.-Nanotechnol. Biol. Med. 7 (2011) 710. [35] Y. Kishi, S. Tanabe, S. Tochino, G. Pezzotti, J. Am. Ceram. Soc. 88 (2005) 3423. [36] D. Chen, Y. Wang, E. Ma, Y. Yu, F. Liu, Opt. Mater. 29 (2007) 1693.
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Microwave-assisted solvothermal synthesis and upconversion luminescence of CaF2:Yb3+/Er3+ nanocrystals Jing Zhao, Ying-Jie Zhu ⇑, Jin Wu, Feng Chen State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, PR China
a r t i c l e
i n f o
Article history: Received 4 September 2014 Accepted 21 October 2014 Available online 29 October 2014 Keywords: Nanostructures Fluorides Microwave Upconversion Rare earths
a b s t r a c t Water-dispersible CaF2 and Yb3+/Er3+ codoped CaF2 (CaF2:Yb3+/Er3+) nanocrystals with different sizes and different Yb3+ and Er3+ dopant concentrations were synthesized using ionic liquid 1-n-butyl-3-methyl imidazolium tetrafluoroborate as a fluorine source by the rapid microwave-assisted solvothermal method. It was found that the morphology, size and crystallinity of CaF2:Yb3+/Er3+ nanocrystals could be adjusted by using adenosine 50 -triphosphate disodium salt (ATP). Yb3+ and Er3+ ions were doped into CaF2 nanocrystals to enable upconversion luminescence emission, and the as-prepared CaF2:Yb3+/Er3+ samples exhibited upconversion luminescence upon excitation at 980 nm. Confocal laser scanning microscopy images showed that the CaF2:Yb3+/Er3+ nanocrystals could be used for efficient labeling of human gastric carcinoma cells. Moreover, in vitro cytotoxicity experiments indicated that the as-prepared CaF2:Yb3+/Er3+ nanocrystals had essentially little cytotoxicity. These results indicate that the as-prepared CaF2:Yb3+/Er3+ nanocrystals are promising for the application as a luminescent label material in biological imaging. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction It is well known that upconversion fluorescent materials have the ability to convert lower-energy photons into higher-energy photons. Compared with the conventional fluorescent materials, upconversion fluorescent materials have many advantages including greater tissue penetration, minimized autofluorescence, high signal-to-noise ratio and high photochemical stability [1–4]. As a new generation of luminescent materials, lanthanide-doped upconversion nanocrystals (UCNCs) have been widely investigated in the recent years. Efforts have been devoted to developing lanthanide-doped upconversion nanoparticles for applications in biological luminescent labeling, sensing and imaging. For instance, Liu et al. [5] prepared core/shell NaYF4:Tm,Yb nanoparticles and investigated their application in near-infrared-triggered anticancer drug delivery. Multifunctional upconversion luminescent materials, such as upconversion luminescence–magnetic hybrid nanoparticles and multifunctional upconversion nanoprobes, have also been investigated [6–9]. Upconversion luminescent materials generally comprise an inorganic host and lanthanide dopant ions embedded in the host. According to the selection of the host material, the UC luminescent ⇑ Corresponding author. Fax: +86 21 52413122. E-mail address: [email protected] (Y.-J. Zhu). http://dx.doi.org/10.1016/j.jcis.2014.10.031 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
materials can be classified into two kinds. One kind is based on fluorides and the other is based on metal oxide materials. Compared with metal oxide materials, metal fluoride materials such as CaF2, NaYF4, YF3, LaF3 and BiF3 are more appropriate to be used as the host materials because of their lower phonon energies [10,11]. CaF2 crystal is a frequently used phosphor host because of its high solubility and transparence in the range from the near ultraviolet to the middle of infrared [12,13]. Lanthanide-doped CaF2 materials have been extensively studied as one kind of the most efficient upconversion luminescent materials [14]. In general, the as-prepared nanoparticles are difficult to disperse in water due to the presence of hydrophobic organic ligands on the surface of UCNCs. An additional surface functionalization step is necessary to prepare water-dispersible lanthanide-doped UCNCs [15]. For example, Ma et al. [16] employed 6-phosphate-6-deoxy-b-cyclodextrin (bPCD) as the novel surface ligand to fabricate a versatile upconversion luminescent nanoplatform. Using bPCD as the surface ligand not only enhanced the stability and biocompatibility of the UCNCs under physiological conditions but also enabled simple conjugation with various functional molecules. The conjugated upconversion nanoprobe exhibited excellent capability in labeling the cancer cells and tumor tissue slices for luminescent imaging. Guo and Li [10] synthesized rare earth doped lanthanum fluoride nanocrystals (LaF3:5% Yb, 2% Er) via a solvothermal method in octadecylene. Under the irradiation of 980-nm diode laser, these
40
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
nanocrystals emitted strong green upconversion luminescence. These hydrophobic nanocrystals were functionalized with poly(amino acid) to enable them water-dispersible and biocompatible. Sarkar et al. [11] reported the synthesis of water-dispersible BiF3 nanoparticles with sizes of about 6 nm in a poly(vinyl pyrrolidone) matrix by the hydrothermal method. Through suitable Ln3+ doping, BiF3 exhibited strong emissions in the visible region upon both UV and near infrared excitations. Although some progress has been made, it remains a challenge to synthesize UCNCs with a good aqueous dispersibility and high UC luminescence intensity. A number of techniques, such as the thermal decomposition method, hydrothermal/solvothermal method and sol–gel process have been developed to synthesize lanthanide-doped UCNCs. However, some synthetic techniques generally require high reaction temperatures and prolonged reaction times, which lead to the aggregation of the nanoparticles [17–20]. In this study, the rapid microwave-assisted solvothermal method has been developed to synthesize water-dispersible CaF2 and CaF2:Yb3+/Er3+ nanocrystals using ionic liquid 1-n-butyl-3methyl imidazolium tetrafluoroborate as a fluorine source. The morphology, size and crystallinity of CaF2:Yb3+/Er3+ nanocrystals can be adjusted by adenosine 50 -triphosphate disodium salt (ATP). This method combines the advantages of microwave rapid heating and pressurized solvothermal process, thus can achieve high reaction rate and short reaction time. The preparation time of the microwave-assisted heating method can often be reduced by orders of magnitude compared with the conventional heating methods, leading to very high efficiency and energy saving [21– 25]. For instance, Xu et al. [26]. reported the microwave-assisted ionic liquid solvothermal rapid synthesis of hollow microspheres of alkaline earth metal fluorides (MF2, M = Mg, Ca, Sr) using [BMIM]BF4 as a fluorine source. To meet the needs for biological labeling, the UCNCs need to have high water-dispersibility, good biocompatibility, nanometer size and high luminescence intensity [27]. Based on these considerations, we have prepared water-dispersible CaF2:Yb3+/Er3+ nanocrystals by doping Yb3+ and Er3+ ions into CaF2 nanocrystals to enable upconversion (UC) luminescence emission, and the asprepared CaF2:Yb3+/Er3+ samples exhibit UC luminescence upon excitation at 980 nm. The as-prepared CaF2:Yb3+/Er3+ crystals have little cytotoxicity, and can efficiently label human gastric carcinoma cells in vitro. These results indicate that the as-prepared CaF2:Yb3+/Er3+ nanocrystals are promising for the application as a luminescent label material in biological imaging.
product. Control sample 1 was synthesized using 0.147 g CaCl22H2O, 0.110 g adenosine 50 -monophosphate disodium salt hexahydrate (AMP) and 3 mL [BMIM]BF4 under the same conditions. Control sample 2 was synthesized using 0.147 g CaCl22H2O, 0.110 g NaH2PO4 and 3 mL [BMIM]BF4 under the same conditions. Control sample 3 was prepared using 0.147 g CaCl22H2O and 3 mL [BMIM]BF4 in the absence of any additive under the same conditions. 2.2. Preparation of CaF2:Yb3+/Er3+ nanocrystals Water-dispersible CaF2:Yb3+/Er3+ nanocrystals with varying Yb and Er3+ doping concentrations and varying sizes were prepared. For the synthesis of CaF2:Yb3+/Er3+ (20/2 mol% Yb3+/Er3+) UCNCs, 0.0076 g Er2O3 and 0.0788 g Yb2O3 were dissolved in HNO3 under vigorous stirring. After the evaporation of excessive HNO3, the residual Er(NO3)3 and Yb(NO3)3 powders were dissolved in 30 mL deionized water. Then, 0.147 g CaCl22H2O, 0.110 g ATP, 10 mL ethylene glycol and 3 mL ionic liquid [BMIM]BF4 were added into the above solution under magnetic stirring. The resulting solution was transferred into a 60 mL autoclave, sealed and microwave heated in a microwave oven at 150 °C for 1 h. After cooling to room temperature, the product was separated by centrifugation, washed with deionized water and ethanol, and dried at 60 °C. The as-prepared CaF2:Yb3+/Er3+ (20/2 mol% Yb3+/Er3+) UCNCs were named as sample A. CaF2:Yb3+/Er3+ (10/1 mol% Yb3+/Er3+) UCNCs (sample B) were synthesized by the same procedure except that the amounts of Er2O3 and Yb2O3 were 0.0039 g and 0.0398 g, respectively. The synthesis of the ATP-free samples (samples C and D) follows a similar procedure. The reactant amounts of the four samples are shown in Table 1. 3+
2.3. In vitro cytotoxicity study In vitro cytotoxicity tests were carried out by the standard MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay [28]. Human gastric carcinoma cells (SGC-7901) cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C for 24 h were used for cell viability tests. Cells were seeded in a 96-well flat-bottomed microassay plate at a concentration of 1 104 cells mL1 and cultured for 24 h. The sterilized samples (samples A, B, C and D) were added to the wells at concentrations ranging from 0.1 to 100 mg L1 and cocultured with the cells for 24 h. Cell viability was quantified by the MTT assay. Each data point was represented by the mean of three parallel tests.
2. Materials and methods 2.1. Preparation of CaF2 nanocrystals The water-dispersible CaF2 nanocrystals were synthesized as follows: 0.147 g CaCl22H2O and 0.110 g adenosine 50 -triphosphate disodium salt (ATP) were dissolved in 30 mL deionized water, and then 10 mL ethylene glycol and 3 mL ionic liquid 1-n-butyl-3methylimidazolium tetrafluoroborate ([BMIM]BF4) were added into the solution under magnetic stirring. The resulting solution was transferred into a 60 mL autoclave, sealed and microwave heated at 150 °C in a microwave oven (MDS-6, Sineo, China). The reaction time was set as 30 min, 1 h and 2 h, respectively. After cooling to room temperature, the products were separated by centrifugation, washed with deionized water and ethanol, and then dried at 60 °C. The influence of pH value was also investigated. The initial pH value of the solution was adjusted to 3.0, 5.0 and 7.0 by using dilute NaOH solution, respectively, and then microwave heated under the same conditions. In this study, other additives were also investigated to adjust the morphology of the CaF2
2.4. Confocal laser scanning microscopy observation of CaF2:Yb3+/Er3+ UCNCs For confocal laser scanning microscope observations, human gastric carcinoma cells (SGC-7901, 1 104 cells mL1) were seeded in glass bottom dishes, and co-cultured with CaF2:Yb3+/Er3+ UCNCs (100 mg L1) for 12 h at 37 °C. Then for every dish, the culture medium was removed and 0.5 mL 4,6-diamidino-2-phenylindole
Table 1 The amounts of Er2O3, Yb2O3 and ATP used to synthesize CaF2:Yb3+/Er3+ crystals with different sizes. The reaction conditions were the same for all the samples.
Er2O3 (g) Yb2O3 (g) ATP (g) Yb3+/Er3+ (mol%) Mean size (nm)
Sample A
Sample B
Sample C
Sample D
0.0076 0.0788 0.110 20/2 26
0.0039 0.0398 0.110 10/1 23
0.0076 0.0788 0 20/2 793
0.0039 0.0398 0 10/1 645
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
(DAPI) solution in phosphate buffered saline (PBS) (10 %) was added. The cells were incubated for 15 min to stain the nuclei and fix the cells. After the incubation, the cells were washed and observed under a confocal laser scanning microscope (FluoView FV 1000, Olympus). 2.5. Characterization Transmission electron microscopy (TEM) micrographs and selected-area electron diffraction (SAED) patterns were obtained on a transmission electron microscope (HITACHI, H-800, Japan). The scanning electron microscopy (SEM) micrographs and energy dispersive spectra (EDS) of the samples were performed using a field-emission scanning electron microscope (S-4800, Japan). The crystal phases of the samples were determined by X-ray powder diffraction (XRD) using an X-ray diffractometer with a graphite monochromator (Cu Ka radiation, k = 1.54178 Å, Rigaku D/max 2550V, Japan). Thermogravimetric (TG) analysis was carried out on a simultaneous thermal analyzer (Netzsch, STA 409PC, Germany) at a heating rate of 10 °C min1 in flowing air. Upconversion luminescence spectra of the samples were recorded at room temperature by a spectrofluorometer (Fluorolog-3, Jobin Yvon). A 980 nm continuous wave diode laser was used as the excitation source. 3. Results and discussion In this study, we synthesized water-dispersible CaF2 nanocrystals using CaCl22H2O, an ionic liquid [BMIM]BF4 and ATP in mixed solvents of deionized water and ethylene glycol by the microwaveassisted solvothermal method. In this method, [BMIM]BF4 acts as a fluorine source. In order to investigate how the synthetic conditions influence the size and morphology of the CaF2 product, the
41
reaction time and pH value were varied. Fig. 1a–c shows the TEM micrographs of the products prepared using CaCl22H2O, an ionic liquid [BMIM]BF4 and ATP in mixed solvents of deionized water and ethylene glycol (pH 7.0) by the microwave-assisted solvothermal method at 150 °C for different microwave solvothermal times. CaF2 nanocrystals with sizes of tens of nanometers were obtained at 150 °C for 30 min by microwave heating (Fig. 1a). Fig. 1b shows nearly monodisperse CaF2 nanocrystals with relatively uniform sizes of about 100 nm prepared by microwave heating at 150 °C for 1 h. Fig. 1c shows that the sizes of CaF2 crystals increased to about 150 nm when the microwave heating time was prolonged to 2 h, indicating that the crystal size of CaF2 increased with increasing reaction time. In the subsequent studies, the microwave heating time of 1 h was chosen considering that the corresponding product had relatively uniform morphology, size and good monodispersity. The influence of pH value on the product was also investigated. When the pH value was 3.0, the product consisted of dispersed nanoparticles and nanoparticle-assembled microspheres with sizes from hundred of nanometers to about 1 lm (Fig. 1d). When the pH values were 5.0 and 7.0, the products were dispersed nanocrystals (Fig. 1e and b). The above-discussed samples were prepared using ATP as an additive. We also explored the effect of other additives on the morphology of the product. The product was composed of CaF2 polyhedra with sizes in the range of 50–200 nm when using adenosine 50 -monophosphate disodium salt hexahydrate instead of ATP (Fig. 1f). When NaH2PO4 instead of ATP was used, CaF2 hollow microspheres constructed by the self-assembly of nanoparticles were obtained (Fig. 1g). Fig. 1h shows that the product synthesized in the absence of any additive consisted of CaF2 polyhedra with sizes of about 1 lm. Fig. 2a shows the XRD pattern of the CaF2 sample prepared using 0.147 g CaCl22H2O, 3 mL [BMIM]BF4 and 0.110 g ATP by
Fig. 1. TEM micrographs of CaF2 samples synthesized using 0.147 g CaCl22H2O, 3 mL [BMIM]BF4 and 0.110 g ATP in mixed solvents of deionized water and ethylene glycol by the microwave-assisted solvothermal approach at 150 °C under different synthetic conditions: (a) pH 7.0, 30 min; (b) pH 7.0, 1 h; (c) pH 7.0, 2 h; (d) pH 3.0, 1 h; (e) pH 5.0, 1 h; (f) 0.110 g adenosine 50 -monophosphate disodium salt hexahydrate instead of ATP, pH 7.0, 1 h; (g) 0.110 g NaH2PO4 instead of ATP, pH 7.0, 1 h; and (h) in the absence of ATP and any additive, pH 7.0, 1 h.
42
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
Fig. 2. XRD patterns: (a) the CaF2 sample prepared using 0.147 g CaCl22H2O, 3 mL [BMIM]BF4 and 0.110 g ATP in mixed solvents of deionized water and ethylene glycol (pH 7.0) by the microwave-assisted solvothermal approach at 150 °C for 1 h and (b) the product prepared in the absence of ATP under the same conditions.
the microwave-assisted solvothermal method at 150 °C for 1 h. Fig. 2b shows the XRD pattern of the product prepared in the absence of ATP under the same conditions. One can see that the diffraction peaks of both samples can be indexed to single-phase face-centered cubic CaF2 (JCPDS No. 35-0816, space group: Fm3m). The lattice parameter of both CaF2 products prepared with and without ATP was calculated to be 5.45 Å based on the XRD data, which is in good agreement with that reported in the literature [29]. In this work, Er3+ was selected as the activator and Yb3+ was codoped as a sensitizer to enhance upconversion efficiency, and the ionic liquid [BMIM]BF4 was used as the fluorine source. The synthesis of CaF2:Yb3+/Er3+ samples was carried out under the same conditions as those for the synthesis of single-phase CaF2 at 150 °C for 1 h. The morphologies of the products were observed by SEM and TEM. Fig. 3 shows the TEM and SEM micrographs and size distributions of the as-prepared CaF2:Yb3+/Er3+ samples. Samples A and B were CaF2:Yb3+/Er3+ (20/2 mol%) nanocrystals and CaF2:Yb3+/Er3+ (10/1 mol%) nanocrystals prepared in the presence of ATP as an additive, respectively. The average diameters of as-prepared CaF2:Yb3+/Er3+ nanocrystals were obtained by the
Fig. 3. SEM micrographs (a, d, g, j), TEM micrographs (b, e, h, k) and size distributions (c, f, i, l) of the CaF2:Yb3+/Er3+ samples prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a–c) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) nanocrystals synthesized in the presence of ATP; (d–f) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) nanocrystals synthesized in the presence of ATP; (g–i) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; (j–l) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
Fig. 4. EDS patterns of the as-prepared CaF2:Yb3+/Er3+ samples prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
Fig. 5. TG curves of the CaF2:Yb3+/Er3+ samples prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
analysis of SEM micrographs. One can see that the morphology of CaF2:Yb3+/Er3+ samples (Fig. 3a, b, d, and e) is different from that of undoped CaF2 crystals obtained by the same method (Fig. 1b). Fig. 3a, b, d, and e shows that the as-prepared CaF2:Yb3+/Er3+ nanocrystals were spherical in shape, and the average diameter was measured to be 26.5 ± 3.7 nm and 23.3 ± 4.1 nm, respectively, smaller than that of undoped CaF2 particles prepared under the same conditions (about 100 nm). Insets of Fig. 3b and e show the selected-area electron diffraction (SAED) patterns of samples A and B. Diffraction rings and some bright diffraction spots in the SAED patterns indicate that the as-prepared CaF2:Yb3+/Er3+ nanocrystals had high crystallinity. CaF2:Yb3+/Er3+ crystals of samples C and D were prepared in the absence of ATP. TEM and SEM micrographs (Fig. 3g, h, j, and k) show highly crystalline CaF2:Yb3+/Er3+ polyhedral crystals with an average size of 793.2 ± 103.9 nm and 645.6 ± 107.6 nm, respectively. The SAED patterns (insets of Fig. 3h and k) of these two samples exhibit single-crystalline diffraction spots. These results indicate that ATP has a significant effect on the morphology, size and crystallinity of the product. The possible reason is that the
43
Fig. 6. XRD patterns of the CaF2:Yb3+/Er3+ samples prepared by the microwaveassisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/ 2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
Fig. 7. Upconversion luminescence spectra of the as-prepared CaF2:Yb3+/Er3+ samples in aqueous suspensions (1 mg mL1) prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
Fig. 8. In vitro viabilities of human gastric carcinoma cells after the 24 h incubation with the CaF2:Yb3+/Er3+ samples prepared by the microwave-assisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/1 mol%) polyhedral crystals obtained in the absence of ATP.
44
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
Fig. 9. Confocal laser scanning microscopy images of the human gastric carcinoma (SGC-7901) cells incubated with the CaF2:Yb3+/Er3+ samples prepared by the microwaveassisted solvothermal approach at 150 °C for 1 h: (a) sample A, CaF2:Yb3+/Er3+ (20/2 mol%) UCNCs synthesized in the presence of ATP; (b) sample B, CaF2:Yb3+/Er3+ (10/1 mol%) UCNCs synthesized in the presence of ATP; (c) sample C, CaF2:Yb3+/Er3+ (20/2 mol%) polyhedral crystals obtained in the absence of ATP; and (d) sample D, CaF2:Yb3+/Er3+ (10/ 1 mol%) polyhedral crystals obtained in the absence of ATP. Insets of b–d are magnified images of cell nuclei.
ATP chains can adsorb on the crystal surfaces of CaF2 and affect the growth of CaF2 crystals. The doping of lanthanide ions into CaF2 crystals was also confirmed by the EDS spectra (Fig. 4). For all CaF2:Er3+/Yb3+ samples, peaks attributed to Yb and Er ions were detected. The Yb and Er peak intensities of samples A and C (CaF2:Yb3+/Er3+ (20/2 mol%)) were obviously higher than those of samples B and D (CaF2:Yb3+/ Er3+ (10/1 mol%)). TG analysis was employed to determine the contents of water and organics in the CaF2:Yb3+/Er3+ samples (Fig. 5). The total mass losses of the four samples were less than 4 %, indicating that the adsorption amounts of [BMIM]BF4 and ATP on CaF2:Yb3+/Er3+ crystals were very small. The XRD patterns of the as-prepared CaF2:Yb3+/Er3+ samples are shown in Fig. 6, where the diffraction peaks are in good agreement with the standard data of face-centered cubic CaF2 (JCPDS No. 35-0816). Compared with the XRD patterns of undoped CaF2 samples (Fig. 2), the appearance of (200) peak in the XRD patterns of CaF2:Yb3+/Er3+ samples indicates the incorporation of rare earth ions in CaF2 host [30]. According to the XRD analysis, the lattice constants of rare earth doped samples A, B, C and D were 5.48 Å, 5.49 Å, 5.46 Å and 5.48 Å, respectively, larger than that of the undoped CaF2 sample (5.45 Å). That is because CaF2 has a fluorite structure, in which Ca2+ ions lie at the nodes in a face-centered lattice, while F ions lie at the centers of octants. When Yb3+ or
Er3+ ions substitute for Ca2+, F ions would occupy the interstitial fluoride cubic sites for the charge balance. Thus, the electronic repulsion between fluoride ions occurs not only at the normal fluoride ion sites but also at interstitial fluoride ion sites, and makes the lattice parameters increase [31]. The increase in the cell volume also indicates that Yb3+ and Er3+ ions have been doped into the CaF2 crystal lattice. The upconversion luminescence spectra of the CaF2:Yb3+/Er3+ samples in aqueous suspensions (1 mg mL1, inset of Fig. 7) were measured under a 980 nm laser excitation at room temperature. Fig. 7 shows the measured upconversion emission spectra at the wavelengths ranging from 450 to 700 nm. The four samples had similar emission spectra with the same wavelength positions except for the emission intensities. The upconversion emission peaks in the green1 (520 and 540 nm) and red (658 nm) regions are attributed to the transitions from the excited states. The emission peaks observed at 520 and 540 nm are assigned to the (2H11/2, 4 S3/2)–4I15/2 transitions of Er3+ ions, while the observed red emission at about 658 nm is attributed to the Er3+ 4F9/2–4I15/2 transition. Many factors may influence the luminescence of UCNCs, such as crystal size, shape, crystallinity and lanthanide dopant amount [32]. In 1 For interpretation of color in Figs. 7 and 9, the reader is referred to the web version of this article.
J. Zhao et al. / Journal of Colloid and Interface Science 440 (2015) 39–45
general, high crystallinity and large size lead to strong upconversion emissions. In this work, samples A and B exhibit relatively lower upconversion intensities than those of samples C and D. This phenomenon may be explained by the reason that the presence of ATP molecules produced CaF2:Yb3+/Er3+ nanocrystals of samples A and B with smaller crystal size and lower crystallinity. The upconversion emission process has different mechanisms, which have been discussed and summarized in several review articles [33,34]. Excited state absorption (ESA), photon avalanche (PA) and energy transfer upconversion (ETU) are the most accepted basic mechanisms. We propose that the luminescence mechanism of CaF2:Yb3+/Er3+ crystals is an energy transfer upconversion process, which is the most efficient upconversion process [35,36]. Due to the promising applications of upconversion luminescence crystals in various biological fields, it becomes an indispensable demand to evaluate the cytotoxicity of the as-prepared CaF2:Yb3+/Er3+ nanocrystals. The MTT assay was employed to assess the cytotoxicity of CaF2:Yb3+/Er3+ samples with human gastric carcinoma (SGC-7901) cells, and the results are shown in Fig. 8. One can see that as the concentration of CaF2:Yb3+/Er3+ samples increased from 0.1 to 100 mg L1, no obvious toxicity to the cells was observed after 24 h co-incubation for the four CaF2:Yb3+/Er3+ samples at low concentrations. Even at a high concentration of 100 mg L1, the cell viability still remained above 80%. The results show that the as-prepared CaF2:Yb3+/Er3+ samples had low cytotoxicity and the cell growth was not obviously affected. In order to evaluate the cell luminescence labeling ability of the as-prepared CaF2:Yb3+/Er3+ samples, human gastric carcinoma (SGC-7901) cells were incubated with the CaF2:Yb3+/Er3+ samples at 37 °C for 12 h. After the incubation, the cells were washed and stained blue with DAPI. Fig. 9 shows the human gastric carcinoma cells under observation with a confocal laser scanning microscope at the 980 nm radiation. The first column shows the cell nuclei stained with DAPI (blue), whereas the corresponding fluorescence images are displayed in the second (green) and third (red) columns. The fourth column is the superimposed images. From Fig. 9, one can see that the upconversion luminescence was observed in samples B, C and D. The yellow fluorescence represents that CaF2:Yb3+/Er3+ crystals were located around the cell nucleus regions. This result demonstrates that a certain amount of samples B, C and D crystals had been taken up by the cells. However, no fluorescence could be detected from sample A because of the low upconversion intensity. 4. Conclusions In this work, water-dispersible CaF2 and CaF2:Yb3+/Er3+ crystals with different sizes and different Yb3+ and Er3+ dopant amounts have been successfully prepared using an ionic liquid 1-n-butyl3-methyl imidazolium tetrafluoroborate ([BMIM]BF4) as the fluorine source by the one-step, fast and environmentally friendly microwave-assisted solvothermal technique. It has been found that the microwave solvothermal time, pH value and ATP have effects on the morphology, size and crystallinity of the product. The as-prepared CaF2 and CaF2:Yb3+/Er3+ crystals are hydrophilic and
45
easily dispersed in water. The as-prepared CaF2:Yb3+/Er3+ samples show strong upconversion emission peaks in the green (520 and 540 nm) and red (658 nm) regions under 980 nm excitation and can efficiently label human gastric carcinoma cells in vitro. The excellent cell labeling ability, high biocompatibility and good water-dispersibility indicate the as-prepared CaF2:Yb3+/Er3+ samples are potential fluorescence labels for biological applications. Acknowledgment Financial support from the National Natural Science Foundation of China (51172260) is gratefully acknowledged. References [1] J. Chen, J.X. Zhao, Sensors 12 (2012) 2414. [2] J.C. Boyer, F. Vetrone, L.A. Cuccia, J.A. Capobianco, J. Am. Chem. Soc. 128 (2006) 7444. [3] D.K. Chatteriee, A.J. Rufalhah, Y. Zhang, Biomaterials 29 (2008) 937. [4] F. Wang, X.G. Liu, Chem. Soc. Rev. 38 (2009) 976. [5] J.N. Liu, W.B. Bu, L.M. Pan, J.L. Shi, Angew. Chem. Int. Ed. 52 (2013) 4375. [6] X.M. Li, D.Y. Zhao, F. Zhang, Theranostics 3 (2013) 292. [7] J.N. Liu, W.B. Bu, L.M. Pan, S.J. Zhang, F. Chen, L.P. Zhou, K.L. Zhao, W.J. Peng, J.L. Shi, Biomaterials 33 (2012) 7282. [8] F. Chen, P. Huang, Y.J. Zhu, J. Wu, C.L. Zhang, D.X. Cui, Biomaterials 32 (2011) 9031. [9] F. Chen, P. Huang, Y.J. Zhu, J. Wu, D.X. Cui, Biomaterials 33 (2012) 6447. [10] C. Guo, M.G. Li, Acta Chim. Sin. 72 (2014) 215. [11] S. Sarkar, A. Dash, V. Mahalingam, Chem. Asian J. 9 (2014) 447. [12] J.S. Wang, Z.W. Wang, X. Li, S. Wang, H.D. Mao, Z.J. Li, Appl. Surf. Sci. 257 (2011) 7145. [13] G.F. Wang, Q. Peng, Y.D. Li, J. Am. Chem. Soc. 131 (2009) 14200. [14] L. Zhao, A. Kutikov, J. Shen, C.Y. Duan, J. Song, G. Han, Theranostics 3 (2013) 249. [15] L.M. Song, J.H. Gao, R.J. Song, J. Lumin. 130 (2010) 1179. [16] C. Ma, T. Bian, S. Yang, C.H. Liu, T.R. Zhang, J.F. Yang, Y.H. Li, J.S. Li, R.H. Yang, W.H. Tan, Anal. Chem. 86 (2014) 6508. [17] F. Wang, D. Banerjee, Y. Liu, X.Y. Chen, X. Liu, Analyst 135 (2010) 1839. [18] C.Y. Cao, W.P. Qin, J.S. Zhang, Y. Wang, G.F. Wang, G.D. Wei, P.F. Zhu, L.L. Wang, L.Z. Jin, Opt. Commun. 281 (2008) 1716. [19] Z.G. Xia, P. Du, J. Mater. Res. 25 (2010) 2035. [20] M. Zahedifar, E. Sadeghi, Z. Mohebbi, Nucl. Instrum. Methods Phys. Res., Sect. B 274 (2012) 162. [21] Y.J. Zhu, F. Chen, Chem. Rev. 114 (2014) 6462. [22] Y.J. Zhu, W.W. Wang, R.J. Qi, X.L. Hu, Angew. Chem., Int. Ed. 43 (2004) 1410. [23] M. Baghbanzadeh, L. Carbone, P.D. Cozzoli, C.O. Kappe, Angew. Chem., Int. Ed. 50 (2011) 11312. [24] C. Qi, Y.J. Zhu, B.Q. Lu, X.Y. Zhao, J. Zhao, F. Chen, J. Wu, Chem. Eur. J. 19 (2013) 5332. [25] J. Zhao, Y.J. Zhu, J.Q. Zheng, F. Chen, J. Wu, Microporous Mesoporous Mater. 180 (2013) 79. [26] J.S. Xu, Y.J. Zhu, CrystEngComm 14 (2012) 2630. [27] T.Y. Cao, Y. Yang, Y. Gao, J. Zhou, Z.Q. Li, F.Y. Li, Biomaterials 32 (2011) 2959. [28] B.R. Schroeder, M.I. Ghare, C. Bhattacharya, R. Paul, Z.Q. Yu, P.A. Zaleski, T.C. Bozeman, M.J. Rishel, S.M. Hecht, J. Am. Chem. Soc. 136 (2014) 13641. [29] A. Bensalah, M. Mortier, G. Patriarche, P. Gredin, D. Vivien, J. Solid State Chem. 179 (2006) 2636. [30] P. Aubry, A. Bensalah, P. Gredin, G. Patriarche, D. Vivien, M. Mortier, Opt. Mater. 31 (2009) 750. [31] M. Pedroni, F. Piccinelli, T. Passuello, M. Giarola, G. Mariotto, S. Polizzi, M. Bettinelli, A. Speghini, Nanoscale 3 (2011) 1456. [32] H.X. Mai, Y.W. Zhang, L.D. Sun, C.H. Yan, J. Phys. Chem. C 111 (2007) 13721. [33] H. Dong, L.D. Sun, C.H. Yan, Nanoscale 5 (2013) 5703. [34] M. Wang, G. Abbineni, A. Clevenger, C. Mao, S. Xu, Nanomed.-Nanotechnol. Biol. Med. 7 (2011) 710. [35] Y. Kishi, S. Tanabe, S. Tochino, G. Pezzotti, J. Am. Ceram. Soc. 88 (2005) 3423. [36] D. Chen, Y. Wang, E. Ma, Y. Yu, F. Liu, Opt. Mater. 29 (2007) 1693.
