A facile synthesis of NaYF₄:Yb³⁺/Er³⁺ nanoparticles with tunable multicolor upconversion luminescence properties for cell imaging

Abstract

The ability to manipulate the upconversion luminescence of lanthanide-ion doped fluoride upconversion nanoparticles (UCNPs) is critical and highly desired due to their wide applications in color displays, multiplexing bioassays and multicolor imaging. Here, we developed a facile strategy for simultaneously tuning color output and enhancing upconversion emission of Yb³⁺/Er³⁺ doped fluoride UCNPs, based on adjusting the crystalline phases and particle size. With the transformation of crystalline phases from α, a transition state of α mixed β, and finally to β phase, the upconversion emission color output changed from red, yellow, and finally turned green. That is, the UCNPs' emission is sensitive to the crystallite size and phase. Moreover, the fluorescence intensity of the obtained UCNPs became stronger with the crystalline phase changing from a to β. Three kinds of UCNPs including α-UCNPs, α mixed β-UCNPs, and β-UCNPs were used for cell imaging, while the transition state phase α mixed β-UCNPs was the better candidate.

---

Introduction

During the past decade, luminescent nanomaterial-based fluorescence imaging has caught much attention due to its high sensitivity and excellent selectivity in monitoring molecular localization, cellular processes, and gene expression even in early cancer identified in live cells. General bioimaging nanomaterials such as organic dyes¹ and semiconductor quantum dots (QDs)^2,3 have intrinsic limitations. Organic dyes exhibit rapid photobleaching, and QDs are inherently toxic and chemically unstable. Compared to the above luminescent materials, lanthanide-doped UCNPs can convert continuous-wave near-infrared (NIR) excitation to visible emission via a two-photon or multiphoton mechanism with a large anti-stokes shift.^4–12 Francois Auzel¹³ and his coworkers were the primordial and seminal groups who discovered it and have produced the wholesome of basic results regarding lanthanide-doped UCNPs' luminescence and basic properties. More importantly, UCNPs have been proven to exhibit conceivable advantages including greater tissue penetration,^14,15 minimized auto fluorescence, low cytotoxicity,^16–18 high signal-to-noise ratio,^19,20 and no photobleaching in bioimaging.^21,22 Although most of their bulk counterparts have been well studied previously, the ability to tune the multicolor emission of UCNPs is also particularly important for their myriad applications in vitro and in vivo bio-imaging.^23,24

Several attempts have been made to tune multicolor upconversion emission, and the typical approach is doping multiactivators and adjusting their concentration in upconversion nanomaterials.^24–30 Multicolor output of UCNPs can be obtained by adjusting the intensities of the different emission peaks with various activators through controlling their doping level in nanoparticles, due to the characteristic emission bands of lanthanide ions.²⁶ However, the presence of high concentrations of dopant and multi-activators can result in the reduction of upconversion intensity.^28,31 There was also report demonstrated that high excitation irradiance could alleviate concentration quenching in upconversion luminescence when combined with higher activator concentration.³⁰ Alternatively, multicolor tuning from a single lanthanide-ion activator can also greatly enrich the colors of UCNPs by controlling the relative intensities of different emission peaks,^28,29,31,32 but the upconversion emission intensity was still sacrificed, which may limit applications of fluoride UCNPs on the cell imaging due to the reduced emission intensity. Researchers have attempted to increase the emission intensity recently. For example, core–shell structured upconversion fluorescent materials,^33–40 NIR-to-NIR upconversion (UC) Tm³⁺ and Yb³⁺ doped fluoride nanophosphors,^41,42 polymer-coated NaYF₄:Yb³⁺/Er³⁺ upconversion nanoparticles⁴³ have been utilized in the biological imaging to improve the intensity of emission peaks,^14,37,38,44 and the oxygen doping method has also been used to tune color output and enhance upconversion emission of Yb/Er doped fluoride UCNPs.²⁴ However, it is still a substantial challenge for developing a facile method to both tune color output and enhance upconversion emission.

NaYF₄ is known to exist in two modifications, namely cubic (α) and hexagonal (β) phases.⁴⁵ Most groups were focused on only single phase (α or β-UCNPs) for cell imaging,^38–40 but few concerned about the contrast study of different crystalline phases for cell imaging. Here, we developed a facile solvothermal route to synthesize water-soluble NaYF₄:Yb³⁺/Er³⁺ UCNPs using direct modification of the hydrophobic surface ligands-polyethylenimine (PEI) molecules, which provided a platform for direct surface functionalization of biomolecules by bioconjugate chemistry. The choice of solvent was important in tuning color output and enhancing upconversion emission. The reaction parameters, different crystalline phases and particle size of samples synthesized by this simple method were discussed and characterized in detail. Then practical issues for cell imaging were also reported, showing their excellent biocompatibility and great potential as fluorescent labels for in vitro or in vivo bioapplications.

Experimental section

Materials

All chemicals involved in this work were analytical grade and used without further purification. Ultrapure water (18.2 MΩ cm) obtained from a Milli-Q A10 water purification system (MILLIPORE, USA) was used throughout. Y₂O₃ (99.99%), Yb₂O₃ (99.99%) and Er₂O₃ (99.99%) were purchased from Shanghai Chemical Industrial Co., Ltd. NaCl, NaF, NH₄F, KCl, NaOH, HCl, KH₂PO₄, Na₂HPO₄, ethanol, ethylene glycol (EG, 99%), and diethyleneglycol (DEG, 99%) were supplied from Tianjin Kewei Co., Ltd. All the rare-earth nitrates Re(NO₃)₃ (RE = Y_0.80 Yb_0.18 Er_0.02) were prepared by dissolving the respective rare-earth oxides in concentrated nitric acid (HNO₃). Polyethyleneimine (PEI, branched, M_W 10 000, 99%, liquid) was purchased from Alfa Aesar. The phosphate buffer solution (PBS) (0.01 M) was made up of 8.0 g of NaCl, 0.2 g of KCl, 3.63 g of Na₂HPO₄·12H₂O, and 0.24 g of KH₂PO₄ in 1 L ultrapure water and adjusted to a desired pH = 7.4 for direct use.

Materials characterization

Powder X-ray diffraction (XRD) measurements were performed on a Bruker D8 diffractometer at a scanning rate of 1° min⁻¹ in the 2θ range from 5 to 80°. The dynamic light scattering (DLS) and the zeta potential were measured by Malvern (British) Zetasizer Nano ZS (red badge) with 633 nm He–Ne laser. The size and morphology of UCNPs, the energy-dispersive X-ray spectroscopy (EDX) were determined at 200 kV with a Tecnai G2 F20 (FEI, USA). These as-prepared samples were dispersed in water and dropped on the surface of a copper grid for low to high resolution transmission electron microscope (TEM, HRTEM) test, Fourier transform infrared (FTIR) spectra were performed using an NICOLET 6700 FT-IR spectroscope with KBr pellets. Three samples' composition quantitative analysis was analyzed on the X7 Series Inductively coupled plasma mass spectrometry (ICP-MS) (Thermo Electron Corporation, USA). Steady-state fluorescent experiment at room temperature was measured on an Cary Eclipse Fluorescence spectrophotometer, but using an external 0–2 W adjustable 980 nm semiconductor laser (Beijing Hi-Tech Optoelectronic Co., Ltd. China). The upconversion luminescence emission spectra also was recorded on a laser scanning confocal microscope (Olympus FluoView FV1000 IX system) attached with a external 980 nm laser (input power: 500 mW, Connet Fiber Optics Company, Shanghai, China). The photos of upconversion luminescence were obtained digitally on a Nikon multiple CCD Camera.

Synthesis of UCNPs nanoparticles

In a typical synthesis of α-UCNPs,²⁵ 1 mmol of Re(NO₃)₃ (RE = Y_0.80 Yb_0.18 Er_0.02), 0.20 g of PEI were dissolved in 9 mL of EG or 1 mL of water and 8 mL of EG, then mixed thoroughly to form a transparent solution A, the solution A was then added into 6 mL of EG containing 10 mmol of NaF (solution B) under magnetic stirring. The resulting mixture was agitated for 15 min, transferred to a 25 mL of Teflon-lined autoclave, rapidly heated to 200 °C and kept at this temperature for 10 h. After cooling down to the room temperature, the products were precipitated by addition of ethanol, separated by centrifugation, and washed repeatedly by ethanol and water. The as-prepared nanoparticles could be redispersed in water.

The synthesis procedure of β-UCNPs was similar to that of α-NaYF₄:Yb³⁺/Er³⁺, and we modified the synthesis procedure by varying the value of x (x is the volume of water, 6 ≤ x < 15, and the EG is 15 − x mL). We also only modified the synthesis procedure of α mixed β-UCNPs by varying the value of x (x is the volume of water, 1 < x < 6, and the EG is 15 − x mL) in the reaction system.

Cell culture and cytotoxicity

Prostate cancer cells (PC cells) were provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). The cells were seeded into a 24-well cell culture plate at 1 × 10⁵ per well, under 100% humidity, and were cultured in RPMI 1640 medium at 37 °C and 5% CO₂ for 24 h; different concentrations of PEI–UCNPs (50 μL; 0, 0.625, 1.25, 2.5, 5.0, 10.0 mg mL⁻¹, diluted in PBS solution) were then added to the wells for subsequent incubation for 12 h. Thereafter, MTT (10 μL; 5 mg mL⁻¹) was added to each well and the plate was incubated for an additional 4 h at 37 °C under 5% CO₂. After the addition of 120 μL DMSO, the assay plate was gently shaken for 10 min. The optical density OD570 value (Abs.) of each well, with background subtraction at 570 nm, was measured by means of a Tecan Infinite M200 monochromator-based multifunction microplate reader. The following formula was used to calculate the inhibition of cell growth: cell viability (%) = (mean of Abs. value of treatment group/mean Abs. value of control) × 100%.

Upconversion luminescence (UCL) bioimaging of living cells

PC cells were plated into 24-well plates (1 × 10⁵ per well) and cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. All cultures were maintained at 37 °C in a 5% CO₂ atmosphere. 50 μL of α-UCNPs, α mixed β-UCNPs, β-UCNPs (1.0 mg mL⁻¹) were added to each well, followed by incubation for 3 h at 37 °C. The treated cells were washed three times with PBS (0.01 M, pH 7.4) to remove unbounded UCNPs. The internalization of α-UCNPs, α mixed β-UCNPs, β-UCNPs was observed using a laser scanning confocal microscope (10× objective, Olympus FluoView FV1000 IX system) attached with a external 980 nm laser (input power: 500 mW, Connet Fiber Optics Company, Shanghai, China). Upconversion luminescence signals were detected in the green channel (520–560 nm) and red channel (630–670 nm).

Results and discussion

Preparation and structure characterization of NaYF₄:Yb³⁺/Er³⁺ UCNPs

The FTIR spectra of the resultant PEI–UCNPs, UCNPs and pure PEI samples were shown in Fig. S1.† The presence of six characteristic absorption bands of PEI (3420 cm⁻¹ for N–H stretch, 2930 and 2870 cm⁻¹ for C–H stretch, 640 cm⁻¹ for N–H bend, 1460 cm⁻¹ for C–H bend, and 1110 cm⁻¹ for C–N stretch),⁴⁶ confirming the capping of PEI on the surface of UCNPs. In addition, both shape changes and wavelength shift in peaks of N–H stretch and N–H bend indicated a strong interaction between amino groups of PEI and Y, Yb or Er. The zeta-potentials (Fig. S1,† inset) also demonstrated the successful modification of positively charged PEI onto their corresponding UCNPs. The lanthanide doped concentrations in NaYF₄:Yb/Er (Y : Yb : Er = 80 : 18 : 2 mol%) nanoparticles were confirmed by a X7 Series inductively coupled plasma mass spectrometry (ICP-MS). Three samples' measured molar ratios of Y : Yb : Er were in close proximity to 80% : 18% : 2% (Table S1†). Compositional analysis of an individual particle by energy-dispersive X-ray spectroscopy (EDX) revealed the presence of the doped elemental Y, Yb and Er, and the strong signals for Cu (Fig. S2†) coming from the copper TEM grid.

The spherical morphology of the α-NaYF₄:Yb³⁺/Er³⁺ particles with an average diameter of 30 nm were obtained (Fig. 1A1–A3). High resolution TEM (Fig. 1A4) showed lattice fringes with an observed d-spacing of 0.32 nm, which was in good agreement with the lattice spacing in the (111) planes of NaYF₄ (0.316 nm, JCPDS file number 77-2042). The TEM image of α mixed β-NaYF₄:Yb³⁺/Er³⁺ in Fig. 1B1–B3 showed that these nanoparticles were spherical and hexagonal with an average diameter of 60 nm, high resolution transmission electron microscopy (HRTEM) (Fig. 1B4) showed lattice fringes and the capping reagents layer. A 2 nm thick capping reagent layer around the crystallized nanocrystals was visible in the Fig. 1B4, verifying the attachment of PEI on UCNPs. The TEM images of as-prepared β-NaYF₄:Yb³⁺/Er³⁺ were shown in Fig. 1C1–C3. The particles remained a little shape change or aggregation, and an average diameter of 150 nm. HRTEM analysis (Fig. 1C4) showed lattice fringes (0.52 nm), which was indicative of the high crystallinity and the hexagonal NaYF₄ structure. The mean size of the nanoparticles increased from 30 nm, 60 nm to 150 nm as the amount of water increased from 0, 3 mL, to 6 mL, respectively. This was because the added water promoted the growth of NaYF₄:Yb³⁺/Er³⁺ UCNPs, and resulted in the increased nanoparticle size. It was in agreement with the DLS that the size of UCNPs became bigger with the increase of the amount of water (Fig. S3†).

Fig. 1 TEM images of (A1–A3) α-NaYF₄:Yb³⁺/Er³⁺, (B1–B3) α mixed β-NaYF₄:Yb³⁺/Er³⁺, and (C1–C3) β-NaYF₄:Yb³⁺/Er³⁺; HRTEM images of (A4) α-NaYF₄:Yb³⁺/Er³⁺, (B4) α mixed β-NaYF₄:Yb³⁺/Er³⁺, and (C4) β-NaYF₄:Yb³⁺/Er³⁺.

UCNPs with different crystalline phases were prepared by adjusting the volume ratio of water in the mixture solvents, and the XRD patterns were shown in Fig. 2. XRD of samples exhibited a single α-phase (spherical) when the amount of water was 0–1 mL. With increasing the volume of water, the α-phase was drastically transformed to the transition state α (spherical) mixed β-phase (hexagonal) when the volume of water was 2–6 mL. The coexistence of the both phases was totally transformed to the β-phase, as the volume of water increased more than 6 mL. All positions and intensities of the diffraction peaks of UCNPs were in agreement with data for the reference spherical and hexagonal phase (JCPDS no.77-2042 and JCPDS no. 28-1192).

Fig. 2 XRD patterns of α-UCNPs, α mixed β-UCNPs, β-UCNPs (0, 1, 3, 4, 6, 9 were x, x was the volume of water). The position of the α-UCNPs and β-UCNPs peaks were identified at the bottom of the figure.

Multicolor tunability and enhancement of upconversion emission

The as-prepared UCNPs were dispersed in PBS, and the solutions were largely transparent to visible light as evidenced by the optical transmission spectrum shown in Fig. 3A, which were similar to what had been reported previously for these materials.²⁵

Fig. 3 (A) The images of α-UCNPs, α mixed β-UCNPs, β-UCNPs. (B and D) The corresponding fluorescence emission spectra and (C) chromatic graph. UCNPs were dispersed at the concentration of 500 μg mL⁻¹ in PBS under excitation at an external 0–2 W adjustable 980 nm semiconductor laser (0.6 A).

Fig. 3 and 4 showed the upconversion luminescence spectra of UCNPs with a dispersion concentration of 500 μg mL⁻¹ in water under 980 nm excitation wavelength. The upconversion emission color and the intensity ratios of the green and red emissions of as-prepared UCNPs could be tuned by adding water to the absolute EG solvent. The solvent was controlled at 15 mL, x was the volume of water, and 15 − x represented the volume of EG. When 0 ≤ x ≤ 1, the crystallite phase of UCNPs was simple α, and the red intensity was much larger than the green intensity. When x was 1 < x < 6, the phase of UCNPs were α mixed β transition state, and the red intensity was as much as the green intensity. When x was 6 ≤ x ≤ 15, the phase of UCNPs were simple β, and the green intensity was much bigger than the green intensity. Together with their size and crystalline phases from α to α mixed β, then to β (Fig. S4† and 4), the color was transformed from red to yellow and finally to green. This luminescent evolution was closely connected with the phase ratio (R_p = I_α/(I_α + I_β)), where I_α and I_β were the XRD intensity of α-phase (111) at 29.89° and β-phase (100) at 17.13°, respectively. Fig. 4D showed the relationship between R_p and the emission intensity ratio of red to green (R_e = I_R/(I_G + I_R)), where I_G and I_R represented the emission intensity at 540 and 655 nm, respectively. This figure apparently showed the relationship between R_e and R_p, revealing that the variation of R_e was just proportional to that of R_p. From this result it was concluded that the UCNPs emission was sensitive to the crystallite phase. This was according with Lee et al., the α-phase was dominantly contributed to the red emission, while the green emission was mainly ascribed to the β-phase.⁴⁷

Fig. 4 (A) The chromatic graph of α-UCNPs, α mixed β-UCNPs and β-UCNPs. (500 μg mL⁻¹, 0.6 A. 0, 1, 3, 4, 6, 9 were x, x was the volume of water) (B and C) The fluorescence emission spectra (500 μg mL⁻¹, 0.6 A. 0, 3, 4, 6 were x, x was the volume of water) (D) Upconversion emissions spectra (1) and the relationship between R_p and R_e (2) of NaYF₄:Yb³⁺, Er³⁺ powders (0, 3, 4, 6 were x, x was the volume of water).

The fluorescence emission spectra and chromatic graph were shown in Fig. 3B and C and 4A–C. It was observed that the upconversion emission intensity increased with the change of solvent proportion at the same dispersion concentration. First, under the 980 nm NIR light, an electron of the Yb³⁺ ion was excited from the ²F_7/2 to ²F_5/2 level due to strong absorption at 980 nm of the Yb³⁺ ion. Then the electron transferred back to the ground state (²F_7/2), while simultaneously the energy was nonradiatively transferred to Er³⁺, resulting in a population of Er³⁺ from ⁴I_15/2 to ⁴I_11/2. A second 980 nm photon transferred by the excited Yb³⁺ ion could then populate a higher ⁴F_7/2 energetic state of the Er³⁺ ion, whose energy lay in the visible region. The Er³⁺ ion could then relax nonradiatively by a fast multiphonon decay process to the ²H_11/2 and ⁴S_3/2 levels. Finally, radiant transitions from these levels yield emissions at 523 nm (²H_11/2 → ⁴I_15/2), 541 nm (⁴S_3/2 → ⁴I_15/2), and 653 nm (⁴F_9/2 → ⁴I_15/2) (Fig. S5†).^25,36 The subtle variations in the solvent (water and EG) led to prominent changes in the green (²H_11/2, ⁴S_3/2 → ⁴I_15/2) and red (⁴F_9/2 → ⁴I_15/2) spectral region. When the temperature was elevated, precursor decomposed slowly, rare-earth ions were released from the precursor, and reacted with sodium and fluorine ions at the solvent to form tiny particles.⁴⁸ Since the boiling point of EG was 197 °C, the growth temperature was set at 200 °C. The boiling point of the solvent was dropped when the volume of water was added, which was equivalent to improve the collision times and severity rate of cores. Then the particle size became bigger and at the same time the crystalline phases changed, which ultimately resulted in the upconversion emission color output changed from red, yellow, and finally turned into green.

Application of UCNPs as a luminescent probe for living cells imaging

Due to the modification of water-soluble and biocompatible polymers, UCNPs showed excellent stability without any noticeable agglomeration in ultrapure water (Fig. S6†). Their long-term water stability and nanoscale properties made them suitable for bioimaging applications. In order to apply the UCNPs to cell imaging, the particle size and fluorescence property must be carefully controlled to meet certain standards, so it is crucial important to optimize the parameters in the synthesis of UCNPs. The reaction parameters, including reaction time, the type of fluorine source, the amount of NaF, the amount of PEI, and the solvent as well as reaction temperature,⁴⁴ were studied in detail (Fig. 5).

Fig. 5 Evolution of the fluorescent spectra of PEI–UCNPs synthesized under different reaction parameters: (A) time (0.5–24 h) (B) F source (NaF and NH₄F) (C) NaF amounts (4–10 mmol) (D) PEI amounts (0.15–0.30 g) (E) The solvent (EG/DEG and water) and (F) temperature (160–200 °C).

In particular, the reaction time and the fluoride source are two important reaction parameters, which could not only influence the particle size and phase structure, but also regulate the fluorescence intensity. It was noticed that the emission intensity of the UCPNs increased as the reaction time prolonged from 0.5 to 24 h (Fig. 5A), and the NH₄F used as fluoride source could weaken the red emission and improve the green emission relative to NaF (Fig. 5B), this is consistent with the other literature reports.^44,49 To obtain the UCNPs with suitable size and multicolor for cell imaging, the growth time was finally set at 10 h and the fluoride sources was chosen as NaF. Fluoride ions increased from 4 to 10 mmol were used to stabilize Ln³⁺ ions, subsequently controlled the growth of UCNPs and increased the emission intensity (Fig. 5C).²⁵ The PEI was chosen as the capping reagent in order to acquire positively charged UCNPs, which could significantly enhance cellular uptake in several human cell lines.^25,43 Moreover, PEI can not only improve the solubility of UCNP nanoparticles in PBS, but also strengthen the upconversion intensity, however excess amount of PEI can also lead to evident aggregation between nanoparticles (Fig. 5D). Water and EG or DEG were used as mixed solvents to control the formation and growth of UCNPs by adjusting molar ratio of different solvents, where the mixed solvents of water and EG made the emission stronger (Fig. 5E). In order to obtain suitable UCNPs with shape-regulation and strong upconversion emission, the optimal molar ratio of Y, Yb, and Er was 80 : 18 : 2 (Fig. S7†). Other synthetic parameters, such as the growth temperature was optimized at 200 °C (Fig. 5F).

Since the as-prepared PEI–UCNPs could be expected to apply for bioprobes and contrast agents in biomedical application, the cytotoxicity and cell permeability characteristics of these nanoparticles is necessary to evaluate. Cell viability test based on the standard thiazolyl blue tetrazolium bromide (MTT) assay was carried out on PC cells, revealing no obvious cytotoxicity to cells even at high nanoparticle concentrations up to 500 μg mL⁻¹ (Fig. S8†). Good water-solubility and low cytotoxicity implied that PEI–UCNPs could serve as a potential probe for luminescence imaging.

The interaction of living cells with PEI–UCNPs was also investigated by laser scanning upconversion luminescence microscopy (LSUCLM). After incubation with 1 mg mL⁻¹ serum-free medium containing α-UCNPs, α mixed β-UCNPs, β-UCNPs for 3 h at 37 °C, an intense UCL signal was observed from the PC cells (Fig. 6A1, B1 and C1) images collected at green (520–560 nm) channels and Fig. 6A2, B2 and C2 images collected at red (630–670 nm) channels. Fig. 6A3, B3 and C3 merged images of (Fig. 6A1 and A2, B1 and B2, C1 and C2).

Fig. 6 (A) Fluorescent images collected at green (520–560 nm) channels, (B) fluorescent images collected at red (630–670 nm) channels, and (C) merged images of A and B.

Together with crystalline phases from α, α mixed β and finally to β, the upconversion emission images became brighter, which were according with their upconversion emission spectra. Moreover, it should be pointed out that no autofluorescence signal could be found in the UCL images of PC cells. The red emission of α-UCNPs (Fig. 6A2) was much larger than the green intensity (Fig. 6A1), but the merged image Fig. 6A3 was much weaker. The red emission of β-UCNPs (Fig. 6C2) was much lower than the green intensity (Fig. 6C1), the merged image Fig. 6C3 was brightest, but the background was so strong. It was apparent to see that the green emission (Fig. 6B1) was as much as the red emission (Fig. 6B2), and the merged image (Fig. 6B3) was the best. These results indicate that α mixed β-UCNPs could be expected to use as a luminescent probe for living PC cell imaging.

Conclusion

In conclusion, we have successfully demonstrated a strategy to simultaneously manipulate multicolor output and enhance upconversion emission of Yb³⁺/Er³⁺ doped NaYF₄ upconversion nanoparticles based on adjusting the crystalline phases and particle size by adding water amount from absolute EG solvent. The upconversion emission color output could be changed from red to yellow, and finally turned into green, since the UCNPs emission was sensitive to the size and the structure of particles. The FTIR analysis confirmed the successful linking between UCNP surface and PEI. The HRTEM and XRD further indicated the changes in the crystalline phases from α to the transition state of α mixed β and finally to β phase, together with the increase of the as-prepared particle size. In addition, the mechanism for the increase of upconversion fluorescence with the growth of nanocrystals was also investigated in detail. Finally, the cytotoxicity of UCNPs was evaluated, three kinds of UCNPs including α-UCNPs, α mixed β-UCNPs and β-UCNPs were used for cell imaging, but only the α mixed β-UCNPs could be chosen as better candidate for cell imaging.

Acknowledgements

We are grateful for the financial support from the National Natural Science Foundation of China (21375095, 21403155 and 20975054), the Tianjin Natural Science Foundation (12JCZDJC21700), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD-201023), the open fund state Key Laboratory of Medicinal Chemical Biology (Nankai University, 20140418), the Program for Innovative Research Team in University of Tianjin (TD12-5038) and Program for young backbone talents in Tianjin (2012).

Notes and references

1. H. Ow, D. R. Larson, M. Srivastava, B. A. Baird, W. W. Webb and U. Wiesner, Nano Lett., 2005, 5, 113–117.
2. G. Ruan, A. Agrawal, A. I. Marcus and S. Nie, J. Am. Chem. Soc., 2007, 129, 14759–14766.
3. K. T. Yong, J. Qian, I. Roy, H. H. Lee, E. J. Bergey, K. M. Tramposch, S. He, M. T. Swihart, A. Maitra and P. N. Prasad, Nano Lett., 2007, 7, 761–765.
4. L. Xiong, Z. Chen, M. Yu, F. Li, C. Liu and C. Huang, Biomaterials, 2009, 30, 5592–5600.
5. W. Feng, L. Sun, Y. Zhang and C. Yan, Coord. Chem. Rev., 2010, 254, 1038–1053.
6. H. S. Mader, P. Kele, S. M. Saleh and O. S. Wolfbeis, Curr. Opin. Chem. Biol., 2010, 14, 582–596.
7. J. Wang, F. Wang, C. Wang, Z. Liu and X. Liu, Angew. Chem., Int. Ed., 2011, 50, 10369–10372.
8. Z. Xu, P. Ma, C. Li, Z. Hou, X. Zhai, S. Huang and J. Lin, Biomaterials, 2011, 32, 4161–4173.
9. M. Haase and H. Schäfer, Angew. Chem., Int. Ed., 2011, 50, 5808–5829.
10. J. Zhou, Z. Liu and F. Li, Chem. Soc. Rev., 2012, 41, 1323–1349.
11. B. Yan, J. C. Boyer, D. Habault, N. R. Branda and Y. Zhao, J. Am. Chem. Soc., 2012, 134, 16558–16561.
12. M. K. G. Jayakumar, N. M. Idris and Y. Zhang, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 8483–8488.
13. F. Auzel, Chem. Rev., 2004, 104, 139–173.
14. L. Cheng, K. Yang, Y. Li, J. Chen, C. Wang, M. Shao, S. T. Lee and Z. Liu, Angew. Chem., Int. Ed., 2011, 50, 7385–7390.
15. Z. Hou, C. Li, P. Ma, Z. Cheng, X. Li, X. Zhang, Y. Dai, D. Yang, H. Lian and J. Lin, Adv. Funct. Mater., 2012, 22, 2713–2722.
16. W. Wei, T. He, X. Teng, S. Wu, L. Ma, H. Zhang, J. Ma, Y. Yang, H. Chen, Y. Han, H. Sun and L. Huang, Small, 2012, 8, 2271–2276.
17. J. Zhao, Z. Lu, Y. Yin, C. McRae, J. A. Piper, J. M. Dawes, D. Jin and E. M. Goldys, Nanoscale, 2013, 5, 944–952.
18. Y. Yang, F. Liu, X. Liu and B. Xing, Nanoscale, 2013, 5, 231–238.
19. D. Vennerberg and Z. Lin, Sci. Adv. Mater., 2011, 3, 26–40.
20. B. Dong, B. Cao, Y. He, Z. Liu, Z. Li and Z. Feng, Adv. Mater., 2012, 24, 1987–1993.
21. J. Wu, Q. Tian, H. Hu, Q. Xia, Y. Zou, F. Li, T. Yi and C. Huang, Chem. Commun., 2009, 4100–4102.
22. Y. I. Park, J. H. Kim, K. T. Lee, K. S. Jeon, H. B. Na, J. H. Yu, H. M. Kim, N. Lee, H. ChoiS, S. I. Baik, H. Kim, S. P. Park, B. J. Park, Y. W. Kim, S. H. Lee, S. Y. Yoon, I. C. Song, W. K. Moon, Y. D. Suh and T. Hyeon, Adv. Mater., 2009, 21, 4467–4471.
23. R. Liu, D. Wu, S. Liu, K. Koynov, W. Knoll and Q. Li, Angew. Chem., Int. Ed., 2009, 48, 4598–4601.
24. W. Niu, S. Wu, S. Zhang, L. Su and A. Yoong Tok, Nanoscale, 2013, 5, 8164–8171.
25. F. Wang and X. Liu, J. Am. Chem. Soc., 2008, 130, 5642–5643.
26. S. Wu, G. Han, D. J. Milliron, S. Aloni, V. Altoe, D. V. Talapin, B. E. Cohen and P. J. Schuck, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 10917–10921.
27. Y. Liu, D. Tu, H. Zhu, R. Li, W. Luo and X. Chen, Adv. Mater., 2010, 22, 3266–3271.
28. G. Wang, Q. Peng and Y. Li, Acc. Chem. Res., 2011, 44, 322–332.
29. E. M. Chan, G. Han, J. D. Goldberg, D. J. Gargas, A. D. Ostrowski, P. J. Schuck, B. E. Cohen and D. J. Milliron, Nano Lett., 2012, 12, 3839–3845.
30. J. Zhao, D. Jin, E. Schartner, Y. Lu, Y. Liu, A. Zvyagin, L. Zhang, J. Dawes, P. Xi, J. Piper, E. Goldys and T. Monro, Nat. Nanotechnol., 2013, 8, 729–734.
31. G. Tian, Z. Gu, L. Zhou, W. Yin, X. Liu, L. Yan, S. Jin, W. L. Ren, G. M. Xing, S. J. Li and Y. L. Zhao, Adv. Mater., 2012, 24, 1226–1231.
32. W. Niu, S. Wu and S. Zhang, J. Mater. Chem. C, 2011, 21, 10894–10902.
33. W. Niu, S. Wu, S. Zhang, J. Li and L. Li, Dalton Trans., 2011, 40, 3305–3314.
34. S. Huang, Y. Fan, Z. Cheng, D. Kong, P. Yang, Z. Quan, C. Zhang and J. Lin, J. Phys. Chem. C, 2009, 113, 1775–1784.
35. X. Kang, Z. Cheng, C. Li, D. Yang, M. Shang, P. Ma, G. Li, N. Liu and J. Lin, J. Phys. Chem. C, 2011, 115, 15801–15811.
36. Y. Dai, P. Ma, Z. Cheng, X. Kang, X. Zhang, Z. Hou, C. Li, D. Yang, X. Zhai and J. Lin, ACS Nano, 2012, 6, 3327–3338.
37. C. Zhong, P. Yang, X. Li, C. Li, D. Wang, S. Gai and J. Lin, RSC Adv., 2012, 2, 3194–3197.
38. X. Li, Z. Hou, P. Ma, X. Zhang, C. Li, Z. Cheng, Y. Dai, J. Lian and J. Lin, RSC Adv., 2013, 22, 8517–8526.
39. Y. Wu, D. Yang, X. Kang, P. Ma, S. Huang, Y. Zhang, C. Li and J. Lin, Dalton Trans., 2013, 42, 9852–9861.
40. C. Li, Z. Hou, Y. Dai, D. Yang, Z. Cheng, P. Ma and J. Lin, Biomaterials, 2013, 1, 213–223.
41. X. Zhang, P. Yang, C. Li, D. Wang, J. Xu, S. Gai and J. Lin, Chem. Commun., 2011, 47, 12143–12145.
42. N. Niu, P. Yang, F. He, X. Zhang, S. Gai, C. Li and J. Lin, J. Mater. Chem., 2012, 22, 10889–10899.
43. J. Jin, Y. Gu, C. W. Y. Man, J. Cheng, Z. Xu, Y. Zhang, H. Wang, V. H. Y. Lee, S. H. Cheng and W. T. Wong, ACS Nano, 2011, 5, 7838–7847.
44. X. Wang, J. Chen, H. Zhu, X. Chen and X. Yan, Anal. Chem., 2013, 85, 10225–10231.
45. C. Li, Z. Quan, P. Yang, J. Yang, H. Lian and J. Lin, J. Mater. Chem., 2008, 18, 1353–1361.
46. Y. Zhang, J. Liu and X. Yan, Anal. Chem., 2013, 85, 228–234.
47. J. S. Lee and Y. J. Kim, Opt. Mater., 2011, 33, 1111–1115.
48. M. Wang, J. Liu, Y. Zhang, W. Hou, X. Wu and S. Xu, Mater. Lett., 2009, 63, 325–327.
49. Y. Chen, W. He, H. Wang, X. Hao, Y. Jiao, J. Lu and S. Yang, J. Lumin., 2012, 132, 2404–2408.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra05205f

---