Article pubs.acs.org/JPCC
Synthesis of Uniform NaLnF4 (Ln: Sm to Ho) Nanoparticles for Mass Cytometry Lemuel Tong, Elsa Lu, Jothirmayanantham Pichaandi, Guangyao Zhao, and Mitchell A. Winnik* Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada S Supporting Information *
ABSTRACT: Over the past decade, there have been extensive developments in the field of lanthanide-based nanoparticles (NPs). Most studies have focused on the application of upconverting NaYF4-based NPs for deep tissue imaging and paramagnetic NaGdF4 NPs for MRI. Current applications for the remaining members of the lanthanide series are rather limited. Recently, a novel bioanalytical technique known as mass cytometry (MC) has been developed which can benefit from the entire lanthanide series of NPs. MC is a highthroughput multiparametric cell-by-cell analysis technique based on atomic mass spectrometry that uses antibodies labeled with metal isotopes for biomarker detection. NaLnF4 NPs offer the promise of high sensitivity coupled with multiparameter detection, provided that NPs can be synthesized with a narrow size distribution. Here we describe the synthesis of six members of this NP family (NaSmF4, NaEuF4, NaGdF4, NaTbF4, NaDyF4, NaHoF4) with the appropriate size (5−30 nm) and size distribution (CV < 5%) for MC. We employed the coprecipitation method developed by Li and Zhang [Nanotechnology 2008, 19, 345606], and for each member of this series, we examined the heating rate, final reaction temperature, and composition of the reaction mixture in an attempt to optimize the synthesis. For each of the six NaLnF4, in the range of the target sizes, we were able to identify “sweet spots” in the reaction conditions to obtain NPs with a narrow size distribution. In addition, we investigated the oleate surface coverage of the NPs and the effect of longterm storage (2 years) on the colloidal stability of the NPs. Finally, NaTbF4 NPs were rendered hydrophilic via lipid encapsulation and tested for nonspecific binding with KG1a and Ramos cells by mass cytometry.
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INTRODUCTION Lanthanide-based nanoparticles (NPs) are a rapidly growing class of nanomaterials of interest because of their luminescent and magnetic properties.1−4 The lanthanide (Ln) NPs that have received the most attention are NaYF4 NPs doped with different lanthanide ions and NaGdF4 NPs.5 NaYF4 NPs in the hexagonal crystalline phase have been studied extensively as a host lattice for codoping Yb3+ ions with other Ln ions such as Er3+, Tm3+, Eu3+, and Ho3+ ions for upconversion luminescence.6,7 Upconversion is a process in which two or more lowenergy near-infrared photons are converted to one higher energy photon. The upconverting NPs have been shown to have applications for deep-tissue imaging,8 solar cells,9 and photodynamic therapy.10−12 On the other hand, NaGdF413,14 NPs exhibit paramagnetic properties and have been studied for their potential as contrast agents for magnetic resonance imaging (MRI). NPs formed by the other Ln elements have not been studied in great detail, due, in part, to their lack of direct applications as nanomaterials. This situation has recently changed with the development of mass cytometry (MC) for biomarker detection. MC is a high-throughput single-cell analysis technique that uses metal-based single isotope tags attached to antibodies (Abs) to recognize different biomarkers expressed by cells.15 This technique combines single-cell injection with inductively © XXXX American Chemical Society
coupled plasma mass spectrometry (ICP-MS) and time-offlight detection. Lanthanide elements are currently the most commonly used element tags, as they have high mass, low natural abundance in cells, and numerous stable isotopes. Current reagents for mass cytometry are metal-chelating polymers that allow each antibody to carry 150−300 copies of a metal isotope.16−19 While these numbers of atoms/Ab are effective for multiparameter biomarker detection, sensitivity is limited to biomarkers expressed at ca. >104 per cell. Recent reviews of this technique20,21 point out that since the signal detected by ICP-MS increases linearly with the number of metal ions per tag, a 1 to 2 orders of magnitude increase in sensitivity would be possible if the Abs were attached to Ln NPs. In principle, the ideal size of a NP for MC applications would be ca. 10 nm, similar to the size of an Ab. A 10 nm diameter (d) single-element Ln NP such as NaTbF4 would have ca. 8000 159 Tb atoms (Supporting Information, SI, Figure S1). Smaller NPs, e.g., d = 5 nm, may be preferable for intracellular experiments on fixed cells, where the Ab conjugate not only has to enter the cell, but also excess reagent must be washed away Received: January 18, 2016 Revised: February 24, 2016
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DOI: 10.1021/acs.jpcc.6b00570 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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reduced from 300 °C (typically used for NaYF4 synthesis)27 in order to obtain smaller NPs. The most detailed study on the nucleation, growth, and crystal phase evolution for NaLnF4 NPs was carried out by Yan and co-workers.28 They synthesized a series of NaLnF4 NPs (Ln: La to Lu) by a thermal decomposition route using lanthanide trifluoroacetate precursors. The authors categorized the NaLnF4 NPs into three groups (Group 1: La to Nd; Group 2: Sm to Tb; and Group 3: Dy to Lu) based on their growth kinetics and nucleation pattern. They found that lanthanides in Group 1 prefer to nucleate in the LnF3 phase rather than the NaLnF4 phase; those in Group 2 nucleate directly in the hexagonal NaLnF4 phase, which is also the thermodynamically stable phase. Lanthanides in Group 3 nucleate in the cubic NaLnF4 phase, the kinetically favored phase. Subsequent heating of the reaction mixture of Group 3 elements resulted in a transformation to the thermodynamically stable hexagonal phase. Although the Yan group was able to synthesize NPs from the entire lanthanide series, they obtained NPs of different sizes for each of the lanthanides under the same reaction conditions. They found that the size of the NP depends on the lanthanide element’s nucleation pattern and growth kinetics. They also showed that this thermal trifluoroacetate decomposition route to lanthanide NPs suffers from poor reproducibility. It also leads to the release of toxic HF during the reaction. Recently, Haase and co-workers29 studied the transition of Group 1 NaLnF4 NPs (Ln: La to Nd) from the kinetically generated cubic phase to the thermodynamically stable hexagonal phase. They used the coprecipitation process to synthesize and isolate the NPs in the cubic phase and then used this material as the source to transform these precursor NaLnF4 NPs into uniform hexagonal phase NPs. In a second study, they used the same approach to synthesize NaLnF4 NPs (Ln: Sm to Ho) with sizes about 30 nm.30 The important factor to note here is that to prepare the cubic phase NPs as precursor particles they had to use a larger than stoichiometric amount of sodium ions. They discovered that if sodium was not present in large excess, the final size and shape of hexagonal phase NPs was not uniform. In addition, they found that the amount of excess sodium in the reaction also affected the final size of each of the lanthanide NPs. The main conclusion to draw from these studies is that the synthesis of each lanthanide NP in each size regime has its own sweet spot in terms of the reaction conditions needed to obtain NPs with a narrow size distribution. In this paper we report a systematic investigation into factors that affect the size and size distribution of NaLnF4 NPs for group 2 and group 3 elements (Sm to Ho). All experiments employed the one-pot protocol described in ref 16. This is now the most commonly used approach to synthesize NaYF4 and NaGdF4 NPs.13,23,30 Not surprisingly, our attempts to employ the same reaction conditions for all of the Sm to Ho lanthanide elements led to particle sizes that varied in size (from 5 to 35 nm) depending on the lanthanide element and size distributions often outside our target range of CV ≤ 5%. To optimize conditions to obtain uniform NPs, we examined the heating rate, reaction temperature, and reaction time, as well as the OA to ODE ratio in the reaction mixture. For optimized reaction conditions, we evaluated the reproducibility of the NP synthesis and examined the colloidal stability of these NPs after long-term storage (2 years). For all of the elements in the series from Sm to Ho, we were able to find conditions that yielded
prior to MC analysis. For higher sensitivity, NPs such as NaTbF4 with diameters up to 30 nm may be appropriate since they contain about 190 000 159Tb atoms. Large NPs must decompose completely in the plasma torch, and the number of ions generated must not saturate the detector of the MC instrument. More important to our experimental design is that the NPs that we synthesize must have a very narrow size distribution. For quantitative analysis, it is important that each NP−Ab conjugate contains a similar number of metal atoms. As a test of the idea that NPs can provide enhanced sensitivity in MC assays, we carried out a proof-of-concept experiment using a series of aqueous microgel samples as “model cells” with different numbers of streptavidin (SAv) on their surface.22 Using a biotin-containing metal chelating polymer with 50 Tb/polymer, we were able to detect microgels labeled with 104 or more SAvs by MC, but the technique failed to generate a signal with much smaller numbers of this “model biomarker”. In contrast, with 13 nm diameter biotin-labeled NaHoF4 NPs (15 000 Ho/NP), we could monitor the binding of 400, 200, and 100 NPs per microgel, showing that these Ln NPs could enhance the sensitivity of biomarker detection by up to 100-fold. To build on this result, we began an investigation of the synthesis of a series of different lanthanide nanoparticles, particularly NaTbF4, NaSmF4, NaEuF4, NaHoF4, and NaDyF4. We set ourselves an initial target size distribution (CV ≤ 5%) and explored how reaction conditions such as reaction stoichiometry, the reaction medium, heating rate, and final reaction temperature affected the particle diameter and size distribution. We also examined the size evolution of the NPs during the reaction to obtain a deeper understanding of the nucleation and growth kinetics. Although Ln ions in solution have similar chemical and physical properties, each type of lanthanide ion crystallizes under different conditions. Hence, one cannot directly employ the conditions for synthesis of NaGdF4 or NaYF4 NPs for the synthesis of other Ln NPs. For each element, one needs to tailor the reaction parameters to obtain NPs with a desired size and a narrow size distribution. The most effective syntheses of NaYF4 and NaGdF4 NPs employ a coprecipitation method at high temperatures (230− 300 °C) using a high boiling point solvent (octadecene, ODE) and oleic acid (OA) as a coordinating ligand. Important reaction parameters include the ratio of OA to ODE, the amount and source of sodium and fluoride, the reaction temperature, and reaction time. Li and Zhang23 were the first to report a simple one-pot synthesis to prepare uniform hexagonal phase NaYF4 nanoparticles of different sizes. For reactions on a 1 mmol scale of Ln salt, they found that by changing the amount of OA from 3 to 10 mL with respect to ODE (17 mL) NaYF4:Yb,Er/Tm changes in the size and shape of the NPs obtained from nanoplates (70 nm) to nanospheres (15 nm) to nanoellipses (30 nm). Similarly, Ryu et al.24 employed different ratios of OA to ODE to change the average size of NaGdF4:Yb,Er NPs from 15 to 32 nm. On the other hand, Liu and co-workers25 showed that the size of the NaGdF4 NPs obtained can be increased by reducing the amount of the fluoride source below the required stoichiometric equivalence in the reaction mixture. To further reduce the size of NaGdF4 NPs to less than 10 nm, the van Veggel group13 reduced the ratio of oleic acid to octadecene and obtained sizes between 2.5 and 8 nm. They employed the same process to obtain NaDyF4 NPs with diameters ranging from 3 to 20 nm.26 In both cases they found that the reaction temperature (285 °C) had to be B
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of Ar(g) for 2 h. The thin film was heated to 75 °C under vacuum for 5 min to remove residual CHCl3 and then hydrated with DI H2O. The lipid−NP suspension was heated at 75 °C for 5 min until the thin film was completely hydrated. The NP dispersion was sonicated for 10 min and passed through a 0.2 μm nylon syringe filter to yield a translucent solution. Excess lipids were removed by three sedimentation−redispersion cycles by centrifugation (35 000g, 30 min, 4 °C). The resulting pellet was redispersed in DI H2O (1.5 mL) by vortex and sonication (5−10 min) to yield a clear dispersion. The NP concentration was determined by ICP-MS. Cell Incubation of NPs. Live KG1a cells (2 × 106) were purified from growth media by centrifugation and resuspended in PBS (0.5% BSA). The cells were stained with MaxPar reagents (CD7-Ho165, CD34-Nd148, CD45-Sm154) for 30 min at room temperature. Antibody stained cells were washed and resuspended in PBS (0.5% BSA, 100 μL). In the following washing procedures, cells were pelleted by centrifugation (10 000g, 2 min), and the supernatant was aspirated. Different concentrations of NaTbF4 NPs (107−109, 100 μL) were incubated with cells (2 × 106) for 30 min at 4 °C. Cells were washed three times to remove unbound NPs. The cells were fixed with paraformaldehyde (PFA) (1.6%) overnight at 4 °C and washed. Iridium nucleic acid intercalators were incubated with the cells for 1 h at RT. Cells were washed once more to remove excess intercalators followed by dispersion in PBS (0.5% BSA) in preparation for CyTOF measurement. Mass Cytometry Assay. Mass cytometry experiments were conducted with an Omega mass cytometer (CyTOF) from Fluidigm Canada (Markham, ON). Cells were dispersed in 100 μL of DI H2O, and an aliquot (10−20 μL) of the standard bead stock solution was added to the cells. The data were collected as .fcs file format and processed by FlowJo software. The number of NaTbF4 NPs per cell was calculated using the mass cytometry transmission coefficient for Tb ions of 9.88 × 10−5 and the number of Tb ions/NP (∼7.9 × 103).22 Instrumentation. TEM images of NPs were recorded with a Hitachi H-7000 TEM operating at an accelerating voltage of 100 kV. The crystal lattice planes were imaged with a highresolution TEM (HRTEM) on an FEI Tecnai 20 and a JEOL 2010 instrument at 200 kV. The NP size distribution was measured with the image analysis plugin from ImageJ. The crystal structure of the NPs was determined by powder X-ray diffraction (PXRD) with a Philips PW1830 diffractometer. The scan rate was 0.01° s−1 with a step size of 0.03° from 15° to 60°. A crystalline sample of LaB6 was used to account for the inherent instrument broadening. The Scherrer equation (D = 0.89λ/(β cos θ)) was used to calculate the crystallite size, where λ is the X-ray wavelength (1.54 Å), β the fwhm of the diffraction peak, and θ the corresponding diffraction angle. Thermogravimetric analysis (TGA) was performed with a TA Instruments SDT Q600 to determine the oleate surface coverage on the NPs. Dried NPs (5 mg) were heated in air to 100 °C at 10 °C/min and held isothermally for 30 min to remove moisture and other volatiles and then ramped to 800 °C at 10 °C/min. Dynamic light scattering (DLS) measurements were performed with a Malvern Zetasizer Nano ZS (at a scattering angle of 173°) to determine the hydrodynamic diameter of the NPs in n-hexanes. Samples were measured in triplicate.
NPs of uniform size with some control over particle size, but it still remains a challenge to find that sweet spot in the reaction that corresponds to a targeted size and size distribution.
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EXPERIMENTAL SECTION Materials. The following chemicals were purchased from Sigma-Aldrich and used without further purification: ammonium fluoride (>99.99%), methanol (ACS grade, >99.8%), 1octadecene (ODE, technical grade, 90%), oleic acid (OA, technical grade, 90%), and lanthanide(III) chloride hexahydrate (trace metals basis, 99.9%, Ln: Sm, Eu, Gd, Tb, Dy, and Ho). Sodium hydroxide was purchased from Caledon Laboratory Chemicals. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000] (mPEG2K-DSPE) were purchased from Avanti Polar Lipids Inc. MaxPar reagents for cell staining were obtained from Fluidigm Canada (Markham ON). Synthesis of Sodium Lanthanide Fluoride Nanoparticles. Uniform NaLnF4 NPs were synthesized using the general protocol developed by Zhang and co-workers23,31 for preparing NaYF4-based upconversion NPs. For each Ln, reaction conditions were optimized as described in the Results and Discussion section. In a typical synthesis of NaTbF4 NPs, we first prepared terbium oleate by dissolving TbCl3·6H2O (1 mmol, 0.373 g) in a mixture of OA (16 mL) and ODE (16 mL). The reaction setup is shown in Figure S2. The mixture was heated to 125 °C under vacuum (100 kPa) at a heating rate of 4 °C min−1. After 1 h at 125 °C, the clear mixture was cooled to 50 °C, and the vacuum was released. A methanol solution (10 mL) containing NaOH (2.5 mmol, 0.100 g) and NH4F (4 mmol, 0.148 g) was prepared by sonication for 1 h to dissolve the salts, and then the solution was injected dropwise into the mixture. The mixture, which became turbid, was stirred for 30 min. Methanol was removed under a vacuum by heating slowly (1.7 °C/min) to 100 °C. Then the vacuum was purged with a flow of nitrogen, and the reaction was heated to 300 °C at heating rates of 13 or 19 °C min−1 and held at the final reaction temperature for 2 h. The NPs were purified from excess OA and ODE by sedimentation in ethanol. In a typical washing procedure for TEM analysis, ethanol (1.5 mL) was added to an aliquot (0.5 mL) of the reaction mixture in an Eppendorf tube (2 mL). The NPs were sedimented by centrifugation (11 000g, 10 min). The supernatant was discarded, and the sedimented NPs were redispersed in THF (0.5 mL). This sedimentation−redispersion cycle was repeated two more times before finally redispersing the NPs in cyclohexane. The NP solution was diluted further and drop cast onto a copper grid. The yield of a typical synthesis was approximately 75% based on the mass of washed NPs to 1 mmol of NaTbF4. Lipid Encapsulation of Oleate-Capped NaTbF4 Nanoparticles. Oleate-capped NPs were encapsulated with a monolayer of lipids via a thin-film hydration method. The amount of lipids for encapsulation was based on three molar equivalents of the OA density on the NPs (4 OA/nm2) based on TGA results. The encapsulation of NaLnF4 NPs was modified from the lipid encapsulation of NaGdF4:Yb,Er UCNPs by Yao et al.32 In brief, oleate-capped NaTbF4 NPs (d = 10.4 ± 0.7 nm, 10 mg) (Figure S17), DOPC (11 mg, 70 mol %), and mPEG2K-DSPE (16.9 mg, 30 mol %) were dissolved in anhydrous CHCl3 (5 mL) (Figure S17). The lipid NP mixture in a 2 dram vial was dried into a thin film by a flow C
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RESULTS AND DISCUSSION
Our goal in this project was the synthesis of uniform NaLnF4 NPs for applications in mass cytometry, where narrow size distribution is essential. We examined the series Ln = Sm to Ho, with sizes in the range from 5 to 30 nm, with particle uniformity defined by CV ≤ 5%. These syntheses were based on the one-pot method developed by Li and Zhang.23 In the current state of the art, we lack the fundamental understanding to dial in a particular set of reaction conditions for this series of elements to obtain particles of a desired size. Our goal was more modest, to find specific sets of reaction conditions for each Ln element that yields particles of an appropriate size within the stringent requirements of a narrow size distribution. All reactions were run on a scale of 1 mmol Ln chloride. In brief, each lanthanide chloride salt was dissolved in a mixture of OA and ODE to form its lanthanide oleate precursor. Subsequently, sodium hydroxide and ammonium fluoride were added, and the reaction mixture was heated to 300 or 310 °C. The reaction mixture was held at this temperature for different periods of time ranging from 30 min to 2 h. We began with Group 2 (Sm to Tb), as defined by Yan, and examined the synthesis of NaTbF4 in detail. Major variables were the heating rate and the final reaction temperature. The optimum set of conditions, with some tweaking, was also appropriate for NaGdF4 and NaEuF4, where we monitored the evolution of particle size as a function of reaction time. More drastic changes were needed to obtain NaSmF4 NPs within our size and size distribution range. Here the key variable was the ratio of OA to ODE. Finally, we turned our attention to NaDyF4 and NaHoF4 NPs, in Yan’s Group 3. Most reactions led to broad or bimodal size distributions, but by varying the OA content of the reaction as well as the final reaction time and temperature, we found conditions that gave uniform elliptical particles. In the sections below, we first summarize the results for the most effective syntheses of each type of NP. Then we provide details of the synthesis optimization, beginning with Group 2 elements (NaTbF4, NaGdF4, and NaEuF4, then NaSmF4), followed by the more complicated case of NaDyF4 and NaHoF4 NPs in Group 3. We demonstrate the robustness of these syntheses by testing their reproducibility and long-term storage. Finally we look at a single application to mass cytometry, where we examine the ability of lipid-coated NaTbF4 NPs to resist nonspecific interaction with live and fixed cells. Properties of the Nanoparticles Obtained under Optimized Synthesis Conditions. Before turning our attention to the factors that were varied in the synthesis of each type of NP, we summarize our results for the most effective syntheses of each type of NP. The TEM images (Figure 1) and corresponding size histograms in Figure S3 show that the NaLnF4 NPs obtained from the syntheses are uniform and have a narrow size distribution. In each case, the yield of NPs from each reaction exceeded 75% based on Ln salt. The high-resolution TEM (HRTEM) images (insets in Figure 1) and XRD measurements (Figure 2) show that these NPs are highly crystalline. The peaks in the XRD spectra reveal that all NaLnF4 NPs belong to the P63/m space group, corresponding to the hexagonal crystal phase, which is also the thermodynamically stable phase for the NaLnF4 NPs. The lattice fringe spacing of 2.6 and 3.0 Å observed for (200) and (110) planes in the HRTEM images further corroborates the fact that the NaLnF4 NPs are in the hexagonal phase. The size of the
Figure 1. TEM and HRTEM images of NaLnF4 NPs: (A) NaSmF4, (B) NaEuF4, (C) NaGdF4, (D) NaTbF4, (E) NaDyF4, and (F) NaHoF4 synthesized according to the conditions listed in Table 1. Scale bar is 100 nm. HRTEM shows the (110) and (200) planes highlighted. Inset scale bar is 5 nm.
Figure 2. PXRD patterns of NaLnF 4 NPs (Ln: Sm−Ho) corresponding to the pure hexagonal crystal phase with the P63/m space group according to the reference spectra: JCPDS 00-027-0779 (β-NaSmF4), 00-049-1897 (β-NaEuF4), 00-027-0699 (β-NaGdF4), 00027-0809 (β-NaTbF4), 00-027-0687 (β-NaDyF4), and 00-049-1896 (β-NaHoF4).
NaLnF4 NPs calculated from the TEM images (Figure 1) and the reaction conditions to obtain uniform NaLnF4 NPs are summarized in Table 1. The crystallite sizes calculated from the XRD data using the Scherrer equation also agree with the NP diameter determined by TEM image analysis for NaLnF4 (Ln: D
DOI: 10.1021/acs.jpcc.6b00570 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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Table 1. Optimized Reaction Conditions for the Synthesis of NaLnF4 NPs (Ln = Sm to Ho) on a 1 mmol Scale with a Size Distribution Below 5% CV NP
oleic acid (mL)
octadecene (mL)
temperature (°C)
heating time (min)a
diameter/dimensions (nm)
NaSmF4 NaEuF4 NaGdF4 NaTbF4 NaDyF4 NaHoF4
14 16 16 16 12 10
16 16 16 16 16 16
300 300 300 300 300 310
120 60 90 60 90 120
16.9 ± 0.8 12.8 ± 0.4 12.7 ± 0.5 13.9 ± 0.4 21 × 17 30 × 24
XRD crystallite size (nm)b 16.0 12.6 11.0 12.4 16.0 18.9
± ± ± ± ± ±
0.4 0.9 0.7 0.3 0.6 0.8
a Reactions were heated to the final reaction temperature at 13 °C/min. bCrystallite sizes were calculated with the Scherrer equation from the diffraction peaks corresponding to the (100) and (111) crystal lattice planes.
Figure 3. TEM images and size distributions of NaTbF4 nanoparticles synthesized (A, C) at 300 °C and (B, D) at 310 °C for 1 h with a heating rate of 19 °C/min in a reaction mixture consisting of 1 mmol Tb precursor and 16 mL each of OA and ODE. (E) PXRD patterns of NaTbF4 nanoparticles synthesized at 300 and 310 °C with a heating rate of 19 °C/min. The reference spectra of cubic FCC-Na5Tb9F32 (JCPDS 27-0808) (blue) and hexagonal β-NaTbF4 (JCPDS 27-0809) (red) are represented by vertical bars.
To start, we synthesized a series of NPs with a heating rate of 19 °C/min to reach 300 or 310 °C. Heating the reaction mixture to a final reaction temperature of 300 °C resulted in bimodal NPs for varying amounts of oleic acid (10, 14, and 16 mL) as shown in Figure S5 and Figures 3A,C. [In discussing mean dimensions calculated from TEM images, we will use d to indicate size (diameter) for quasi-spherical NPs, whereas for elliptical or platelet NPs, we will use l to indicate the longest dimension.] XRD analysis of the NPs synthesized with 16 mL of OA and 16 mL of ODE at 300 °C revealed that the bimodal mixture consists of NPs in both the hexagonal and cubic phases. By correlating the diffraction peaks to the crystallite sizes using the Scherrer equation, we discovered that the cubic phase corresponds to the smaller NPs (d ∼ 8.3 ± 1.9 nm) in the mixture, while the hexagonal phase corresponds to the larger NPs (d ∼ 23.2 ± 2.0 nm). When the final reaction temperature was 310 °C, we obtained uniform NPs in the hexagonal phase with d ∼ 26.7 ± 2.7 nm. An example is shown in Figure 3B, D, and E. As a comparison, we carried out similar syntheses of NaTbF4 NPs at a slower heating rate (13 °C/min). The final reaction temperatures were 290, 300, or 310 °C, and the corresponding TEM images and size distributions of the NPs are presented in Figure 4. When the final reaction temperature was 290 °C, the resulting NPs were small and polydisperse (d = 6.4 ± 1.7 nm). Increasing the final temperature to 300 °C resulted in uniform NPs (d = 10.9 ± 0.4 nm), which XRD analysis showed to be in
Sm to Tb) NPs. In the case of elliptical NaDyF4 and NaHoF4 NPs, the crystallite sizes calculated from the Scherrer equation correlate with the short axis. These uniform NPs (CV < 5%) were obtained after optimizing the reaction parameters (ratio of oleic acid to octadecene, heating rate, the final reaction time, and reaction temperature) for each type of NaLnF4. A description of the optimization of the reaction conditions for each type of NP and the corresponding NP sizes are presented in the following sections. NaTbF4, NaGdF4, and NaEuF4 NPs: Effect of Heating Rate and Temperature. We begin with an examination of the synthesis of three NaLnF4 NPs (Ln: Tb, Gd, Eu) from Group 2. On the basis of the work of Yan and co-workers,28 we anticipate similarities in the reaction conditions. To optimize conditions for the synthesis of NaTbF4 NPs, we examined different heating rates and final reaction temperatures to observe their influence on the final size and shape of the NPs. These reactions were run on a 1 mmol scale of TbCl3, and the ratio of the OA to ODE in the reaction mixture was kept constant at 1:1 (v/v, 16 mL each). We examined the effect of two heating rates (13 and 19 °C/min) to reach a final reaction temperature of 290, 300, or 310 °C. Once the reaction mixture reached the final temperature, we could maintain this temperature within 1−2 °C. A representative temperature profile of the heating conditions is presented in Figure S4, and the reaction conditions are shown in Table S2. E
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hexagonal crystalline phase. There is no evidence that these smaller NPs nucleated initially in the cubic phase. We next turned our attention to the synthesis of NaGdF4 and NaEuF4 NPs. Using a similar heating profile and the same reaction conditions as for the NaTbF4 NP synthesis (13 °C/ min to 300 °C), we obtained NPs for both NaGdF4 and NaEuF4. The specific conditions employed are presented in Table S3. Both the NaGdF4 and NaEuF4 NPs are hexagonal in shape as shown in Figure 5, with an average size of 11.0 ± 0.6
Figure 4. TEM images and size distributions of NaTbF4 nanoparticles heated at 13 °C/min to a final temperature of 290 °C (A, D), 300 °C (B, E), and 310 °C (C, F) with a heating rate of 13 °C/min with OA (16 mL) and ODE (16 mL) for 1 h.
Figure 5. TEM images of (A, B) NaGdF4 and (C, D) NaEuF4 NPs synthesized at a heating rate of 13 °C/min to 300 or 310 °C and then held at that temperature for 1 h. Reactions involved 1 mmol of LnCl3 in a mixture of 16 mL each of OA and ODE. NaGdF4 NPs synthesized at (A) 300 °C (d = 11.0 ± 0.6 nm) and (B) 310 °C (d = 16.0 ± 0.7 nm). NaEuF4 NPs synthesized at (A) 300 °C (d = 10.7 ± 0.7 nm) and (B) 310 °C (d = 27.3 ± 1.3 nm).
the hexagonal phase (Figure S6). Further increase of the final reaction temperature to 310 °C resulted in bimodal NPs (d = 10.9 ± 0.8 and 22.7 ± 1.2 nm) as shown in the TEM images and size histograms of the NPs in Figure 4. Since the smaller NPs synthesized at 310 °C are the same size as those obtained in the synthesis at 300 °C, we infer that the larger NPs are formed as a result of Ostwald ripening. We find two interesting differences between the types of NPs formed at the more rapid heating rate (19 °C/min) compared to the slower heating rate (13 °C/min). The first difference is that larger NaTbF4 NPs are formed at the faster heating rate. The implication of this result is that fewer nuclei are formed leading to a smaller number of larger particles. At the slower heating rate, which takes 20 min to reach the final reaction temperature, nucleation must be more prominent. As a result, more nuclei are formed, leading to a larger number of smaller particles. The second difference has to do with the types of NPs that were formed. Heating at 19 °C/min favored the formation of the NaTbF4 NPs in the cubic (kinetic) phase. Subsequent heating at 300 °C led to a slow transformation of the NPs to the hexagonal (thermodynamic) phase. After 60 min, we found a mixture consisting of polydisperse NPs in the cubic crystalline phase accompanied by a narrow distribution of larger NPs in the hexagonal phase. After 60 min at a final reaction temperature of 310 °C, all of the particles in the sample were the larger NPs in the hexagonal phase. In contrast, at the lower heating rate and after 60 min at final reaction temperatures of 300 and 310 °C, all of the NPs formed were found to be in the
nm for the NaGdF4 NPs and 10.7 ± 0.7 nm for the NaEuF4 NPs. Increasing the final reaction temperature from 300 to 310 °C resulted in uniform NPs for both NaGdF4 and NaEuF4 NPs. Under these conditions, the average size of NaGdF4 NPs increased to 16.0 ± 0.7 nm, and that of NaEuF4 NPs increased to 27.3 ± 1.3 nm. Remarkably, this 10 °C temperature difference led to an average size increase of 5 nm for NaGdF4 NPs and 17 nm for NaEuF4 NPs. The most likely explanation for the NP size difference between the two elements at 310 °C is that at this temperature the growth rate of NaEuF4 NPs is much higher than that of NaGdF4 NPs. Even though Eu and Gd are neighboring elements, their respective NaLnF4 NPs nucleate and grow differently in solution under similar reaction conditions. To obtain additional information about the growth of these NPs, we monitored the size evolution of NaEuF4, NaGdF4, and NaTbF4 NPs based on the optimized reaction conditions described above (13 °C/min, 300 °C) in Table 1. Aliquots were taken from each reaction mixture at half hour increments after the reaction temperature reached 300 °C. These samples were characterized by TEM as shown in Figure S7. The sizes determined from these TEM images are plotted in Figure 6, where the error bars refer to the standard deviation of the size distributions. The key observation is that both NaTbF4 and F
DOI: 10.1021/acs.jpcc.6b00570 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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Figure 6. Evolution of the NP size as determined by analysis of TEM images of (A) NaSmF4, (B) NaEuF4, (C) NaGdF4, (D) NaTbF4, (E) NaDyF4, and (F) NaHoF4 NPs. Each reaction was carried out on a 1 mmol scale with a heating rate of 13 °C/min, employing the reaction conditions (OA/ ODE ratio and final reaction temperature) listed in Table 1. Aliquots were taken at the times indicated. The size distributions are based upon analysis of several hundred particles in a single TEM image. Portions of each image are presented in Figure S7. The error bars on the plots refer to the standard deviations of the size distribution. Distributions that were visually bimodal are indicated by “b” on these plots, whereas polydisperse distributions are denoted “p”. The TEM images corresponding to A and D at 90 min reaction time show uniform NPs. The broad distributions associated with these samples are due to the presence of tiny particles in these two samples. In contrast, sample B at 90 min is polydisperse.
For example, when we decreased the OA content of the reaction mixture to 14 mL (OA/ODE = 0.88 v/v), we found a bimodal size distribution consisting of small (d = 5.5 ± 0.9 nm) and large (d = 10.1 ± 0.4 nm) NPs at 30 min, which became uniform quasi-spherical NPs (d = 17.5 ± 0.7 nm) after 60 min. In a reaction run for 90 min, we also observed some small polydisperse NPs (d = 5.3 ± 2.3 nm), which disappeared after further heating for 120 min. Here we obtained uniform NPs with d = 16.9 ± 0.8 nm. For 12 mL of OA (OA/ODE = 0.75 v/ v), we observed a bimodal mixture of small (d = 5.0 ± 0.9) and larger (d = 9.9 ± 0.6 nm) NPs at 30 min. These NPs converged to an average size of d = 19 nm after 60 min and maintained their size for reactions run up to 120 min. Ostwald ripening appears to play an important role in the growth of NaSmF4 NPs. Under each of the reaction conditions described above, a bimodal distribution formed initially (30 min) consisting of a mixture of NPs with d ∼ 5 and 10 nm. Subsequent heating at 300 °C led to a depletion of the smaller NPs and a growth in size of the larger NPs, and at OA/ODE < 1, the NPs formed were uniform in size. NaDyF4 and NaHoF4 NPs: Effect of Oleic Acid Content. In the Yan classification,28 Dy and Ho are Group 3 elements. In these experiments, we were not successful in our attempts to synthesize small uniform nearly spherical NaDyF4 and NaHoF4 NPs. We examined the effect of final reaction temperature while keeping other factors constant, but all of these reactions yielded bimodal NP distributions. Further details on the synthesis of NaDyF4 and NaHoF4 NPs are reported in SI (Figures S9 and S10 and Table S5). To obtain uniform NPs, we varied the OA to ODE ratio and monitored the NP growth at 30 min time intervals during the heating process. For the synthesis of NaDyF4 NPs, we reduced the OA to 12 mL and obtained uniform elliptical NPs (l = 20.2 ± 0.6 nm). As for NaHoF4 NPs, decreasing the OA to 12 mL yielded uniform hexagonal NPs (d = 26.5 ± 1.0), and reducing the OA to 10 mL resulted in uniform elliptical NPs (l = 29 ± 1 nm).
NaGdF4 NPs were initially polydisperse at 30 min. As the reaction proceeded for 2 h, the size distribution converged into a uniform size suggesting that the uniformity was achieved via self-focusing.33 NaEuF4 NPs also evolved from a polydisperse distribution to a uniform size at 60 min (d ∼ 7.0 ± 0.5 nm). This size evolution and narrowing of the size distribution indicate that self-focusing took place in the reaction. However, heating this reaction for longer periods of time (90 min, 120 min) led to a broadening of the size distribution (d ∼ 5−22 nm). We note that one can see (Figure S7) smaller particles as well as larger particles, which suggests a competition between secondary nucleation and Ostwald ripening. The main point that we wish to emphasize, however, is that in the synthesis of NaTbF4 NPs and NPs for neighboring lanthanides each element exhibits a different nucleation and growth behavior. NaSmF4: Effect of Oleic Acid. According to the Yan classification, Sm is in Group 2 (Sm to Tb), and NaSmF4 NPs are predicted to nucleate directly in the thermodynamically favored hexagonal phase. We found, however, that the reaction conditions needed to obtain uniform NaSmF4 NPs were different from those used to obtain uniform NaEuF4, NaGdF4, and NaTbF4 NPs. In Table S4, we present the results of time evolution studies of the synthesis of these particles with a heating rate of 13 °C/min and a final reaction temperature of 300 °C. TEM images are presented in Figure S8. On a 1 mmol scale and with 16 mL each of OA and ODE in the reaction, the size evolution was rather complicated. Small NPs (d = 4.5 ± 0.8 nm) found at 30 min evolved into a bimodal mixture of small (d = 5.5 ± 1.3 nm) and larger (d = 10.7 ± 0.4 nm) NPs at 60 min. At 90 min, we found a mixture of disk-shaped (d ∼ 14 nm) and large hexagonal (d ∼ 40 nm) NPs in addition to small (d ∼ 5 nm) NPs. All attempts to obtain uniform particles of the proper size failed when we varied only the reaction time and temperature but maintained the reaction recipe. As a consequence, we varied the relative amounts of OA and ODE in the reaction. G
DOI: 10.1021/acs.jpcc.6b00570 J. Phys. Chem. C XXXX, XXX, XXX−XXX
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Figure 7. Different batches of NaGdF4 NPs synthesized under the same reaction conditions: 1 mmol scale, OA:ODE = 16:16 mL, 13 °C/min, and 1 h at 300 °C. Batch, (d ± std.dev.): 1 (9.8 ± 0.5 nm), 2 (7.4 ± 0.5 nm), 3 (11.6 ± 0.6 nm), 4 (9.7 ± 0.5 nm), 5 (12.0 ± 0.6 nm), 6 (11.0 ± 0.6 nm). Scale bar is 50 nm.
Figure 8. DLS CONTIN plots for NaLnF4 (Ln: Sm to Tb) NPs in hexanes for particle samples stored 2 years in their original reaction mixture and then purified. (A) NaSmF4, (B) NaEuF4, (C), NaGdF4, (D) NaTbF4. For NaSmF4 (dh = 44.7 ± 0.1, PDI = 0.224 ± 0.005) and NaEuF4 (dh = 36.2 ± 0.5, PDI = 0.266 ± 0.005), values of dh were estimated from the peak position of the CONTIN plots. For NaGdF4 (dh = 13.7 ± 0.1 nm, PDI = 0.067 ± 0.007) and NaTbF4 (dh = 17.4 ± 0.1 nm, PDI = 0.113 ± 0.011) dh values were calculated by a cumulant analysis of the autocorrelation decay. Standard deviations were calculated from three independent measurements.
However, smaller NP sizes could not be obtained by varying the OA to ODE ratio. Reproducibility of the Synthesis. Reproducing the synthesis of NPs to obtain a particular size can be challenging especially when the reaction is subjected to subtle variations in the laboratory environment. Evidence is largely anecdotal,34 but reproducibility difficulties point to small changes in reaction conditions that one normally thinks of as “identical”. Recently, Suter et al.,35 in the context of detailed studies of the formation of NaYF4:Yb,Er UCNPs, examined the reproducibility of various stages of the reaction. Their experiments took advantage of in situ real-time monitoring of the upconversion PL of the reaction mixture over the entire course of the reaction, from the initial heat-up to the 300 °C final reaction temperature. They noted a relatively large synthesis-tosynthesis variation in the time required for the cubic-tohexagonal phase conversion to begin. Nevertheless, at the end of the reaction, they found very reasonable reproducibility in
particle size, with mean sizes for six different batches ranging from 37 to 41 nm. To test the reproducibility of our reaction conditions, we synthesized six batches of NaGdF4 NPs and five batches of NaTbF4 NPs, each under the optimized reaction conditions listed in Table 1. Figure 7 shows TEM images and a histogram of mean size and size distribution for the six batches of NaGdF4 NPs. Corresponding data for the five batches of NaTbF4 NPs are presented in Figure S12. These images show that the NPs formed in each batch were uniform in size, with a standard deviation in the particle diameter
