Multifunctional BiF3:Ln3+ (Ln = Ho, Er, Tm)/Yb3+ nanoparticles: an investigation on the emission color tuning, thermosensitivity, and bioimaging

Pure cubic phase and uniform BiF3:Ln3+ (Ln = Ho, Er, Tm)/Yb3+ nanoparticles (NPs) were prepared by coprecipitation. The growth mechanism of BiF3:2%Er3+/20%Yb3+ NPs was proposed based on evolution analysis of the time-dependent morphology, in which BiF3:2%Er3+/20%Yb3+ was formed through the growth process of “nucleation to crystallization and Ostwald ripening”. The upconversion luminescence (UCL) properties and mechanism of BiF3:Ln3+ (Ln = Ho, Er, Tm)/Yb3+ under dual-wavelength excitation were also systematically investigated. The emission intensity of BiF3:2%Er3+/20%Yb3+ by dual-wavelength excitation (λ = 980 nm + 1550 nm) was 1.49 times more than that excited by 1550 nm or 980 nm individually. Furthermore, the properties of the bright white and multicolor UCL showed that yellow, purple, green, or pinkish light could be observed by controlling the doping concentration of Ln3+ (Ln = Yb, Er, Tm, and Ho), indicating that they had potential applications in backlight sources of color displays and security labeling. The temperature sensitivity of BiF3:2%Er3+/20%Yb3+ exhibited a downward tendency and its max value was about 0.0036 K−1 at 273 K. Cell toxicity tests showed that the UCNPs in phospholipid aqueous solution presented low cytotoxicity. Also, in vivo imaging and X-ray imaging revealed that the BiF3:2%Er3+/20%Yb3+ NPs had deep penetration and high contrast, which meant it could be used as a potential probe and contrast agent in in vivo optical bioimaging.


Introduction
][10][11][12][13][14][15][16] Therefore, they have great potential in different elds. 17It is well known that UC emission can be accurately controlled through adjusting the concentrations of different activator ions (Er 3+ , Tm 3+ , Ho 3+ ).Also, white and multicolor UCLNPs have attracted much attention due to their potential applications in backlight sources in color displays and security labeling. 18,194][25][26][27] Zhang et al. reported the excellent matrix NaBiF 4 without a rare earth ion fabricated by a super facile synthetic method, in which the reaction only took 1 min at room temperature. 28Yu et al. used the same method to synthesize NaBiF 4 :Yb 3+ /Er 3+ NPs and BiF 3 :Yb 3+ /Er 3+ NPs and investigated their UCL properties and temperature sensitivity. 29,30However, investigations on multicolor UCL, UCL excited by dual-wavelength, and the bioimaging of BiF 3 :Yb 3+ /Er 3+ have not been performed yet, but are very important for their application. 5,18,31Herein, this motivated us to further study these excellent matrix BiF 3 :Yb 3+ / Ln 3+ (Ln ¼ Ho, Er, Tm) NPs in a deeper investigation.
In this work, the cubic phase of BiF 3 :Ln 3+ (Ln ¼ Ho, Er, Tm)/ Yb 3+ UCNPs was designed and synthesized by the same facile strategy.Their UCL properties were investigated by excitation at 1550 nm and 980 nm, simultaneously.The growth mechanism and the effect of time on their morphology was also explored.The function of different doped concentrations of Er 3+ , Ho 3+ , and Tm 3+ ions for the multicolor and white emission were also investigated.In vivo and X-ray imaging of the BiF 3 :2%Er 3+ /20% Yb 3+ NPs were studied.

Preparation
All reagents were purchased from Aladdin Chemical Reagent Factory (China) in this research and used without further treatment.The synthesis of the cubic BiF 3 :Ln 3+ /Yb 3+ UCNPs proceeded as reported in ref. 30 (see ESI †).

Characterization
Details on the instruments used are presented in the ESI.†

In vitro cytotoxicity evaluation, and in vivo and X-ray imaging
The relevant evaluation 32 of cytotoxicity in vitro and in vivo and X-ray imaging were presented in ESI.†

Characterization of the phase and morphology
The crystal phases of BiF 3 :Ln 3+ /Yb 3+ NPs were determined by XRD as shown in Fig. 1, in which they presented similar crystal cubic phases (JCPDS: 51-0944).The SEM image of BiF 3 :2%Er 3+ / 20%Yb 3+ NPs, as an example, exhibited regular NPs (Fig. 2a), which was conrmed by the TEM image (Fig. 2b).Fig. 2c shows that the d-spacing was 0.13 nm, which matched with the crystal plane (331) of the cubic phase BiF 3 NPs.According to the results of element mapping (Fig. 2d-g), it was clear that the elements of Bi, Yb, Er, and F were uniform on the NPs.These elements were also conrmed by the EDX spectrum (Fig. 2h).BiF 3 :20% Yb 3+ ,0.5%Tm 3+ /2% Ho 3+ are presented in Fig. S1 † and their SEM images are shown in Fig. S2, † which further illustrates the formation of BiF 3 :Ln 3+ (Ln ¼ Ho, Er, Tm)/Yb 3+ UCNPs.
The stability of the BiF 3 :2%Er 3+ /20%Yb 3+ NPs was investigated.The TG-DSC curve (see Fig. S4 †) showed that the NPs had a mild loss of weight below 600 C. The loss of water or crystalline water could be calculated at about 100 C. When heated at 200 C, the glycol on the surface of the sample gradually disappeared, and the surface defects of the sample gradually decreased.When the temperature reached up to 600 C, the sample itself began to decompose and then the mass declined linearly.
To verify whether the reaction time affected the phase and morphology, XRD patterns and SEM/TEM images of BiF 3 :2% Er 3+ /20%Yb 3+ NPs prepared at different reaction times were investigated (Fig. 3 and 4).The results showed that the diffraction peaks matched with BiF 3 (JCPDS: 51-0944), indicating that the prepared compounds were single phase and had better crystallinity (Fig. 3).It can be seen from Fig. 3b that the diffraction peaks at 26.34 of Yb 3+ , Er 3+ -doped BiF 3 had a slight shi compared to BiF 3 (JCPDS: 51-0944).The reason should be that the ionic radii of both Er 3+ (1.004 Å) and Yb 3+ (0.985 Å) are   smaller than that of Bi 3+ (1.17 Å).In the BiF 3 host lattice, the ions (Yb 3+ , Er 3+ ) could enter the BiF 3 crystal site through substituting for the Bi 3+ ions or in the interstitial sites or could coexist in these two ways.Different doping methods could make the XRD peaks of the samples prepared at different time shi by different degrees.This indicated that Yb 3+ and Er 3+ had been doped into the BiF 3 matrix. 33The XRD of BiF 3 :20%Yb 3+ ,0.5% Tm 3+ /2%Ho 3+ showed similar results, as shown in Fig. S5.† The SEM and TEM images of BiF 3 :2%Er 3+ /20%Yb 3+ obtained at different reaction times are presented in Fig. 4. It can be clearly observed that there are a large number of well-dispersed NPs with similar morphologies, indicating that the morphologies were almost unchanged even as the time was prolonged, which was conrmed by the TEM images (insets of Fig. 4).In addition, BiF 3 :20%Yb 3+ ,2%Ho 3+ /0.5%Tm 3+ in Fig. S6 and S7 † showed similar results.
In order to better understand the formation process of BiF 3 :2%Er 3+ /20%Yb 3+ within 1 min, the growth mechanism was carefully studied by XRD and TEM at different reaction times (5, 30, 50, and 60 s), as shown in Fig. S8 † and 5. Some impurity peaks emerged compared with the standard peaks in 5 s (Fig. S8 †) and some small particles appeared in the TEM (Fig. 5a), indicating that the crystal nucleus was completed quickly.As the reaction time was further increased, the impurity peaks gradually disappeared (Fig. S8 †) and the basic BiF 3 spheroids gradually further grew or aggregated (see Fig. 5b and  c).These could be deemed as indicating the ripening process of BiF 3 NPs, meaning that larger spherical NPs were gradually formed due to the thermodynamic minimization of the surface energies of smaller particles, which is commonly known as Ostwald ripening. 34BiF 3 :2%Er 3+ /20%Yb 3+ with regular morphologies were formed and the whole system achieved equilibrium when the time was 1 min (Fig. 5d).Meanwhile, XRD patterns were well matched with the pure cubic phase of BiF 3 (Fig. S8 †).Based on the morphology evolution, the possible growth mechanism is proposed as follows (Scheme 1).First, the precursor was converted to BiF 3 :2%Er 3+ /20%Yb 3+ nuclei in the nucleation stage.Subsequently, the nuclei grew to rudimental NPs and then underwent the Ostwald ripening process until well-crystallized BiF 3 NPs were formed, therefore the process could be described as "Nucleation / Crystallization / Ostwald ripening" (Scheme 1).
The inuence of the temperature on the phase and morphology of BiF 3 :2%Er 3+ /20%Yb 3+ were also investigated by XRD, SEM, and TEM (Fig. S9 † and 6).It could be seen that the XRD spectra of NPs obtained at different temperatures (À25, 0, 30, 50, and 70 C) matched well with the standard cubic phase (Fig. S9 †).No impurities peaks were found in these patterns, indicating these samples had higher purity.However, a few new diffraction peaks appeared at 100 C, indicating that the pure cubic phase of BiF 3 :2%Er 3+ /20%Yb 3+ NPs tended to be formed at a lower temperature.Meanwhile, SEM images of BiF 3 :2%Er 3+ / 20%Yb 3+ NPs synthesized at different temperatures, sizes, and morphologies also showed no obvious changes, except that they became irregular at 100 C (Fig. S9 † and 6f).The corresponding TEM images are shown in Fig. 6g-l, in which the results were in good agreement with the XRD results.From the above analysis, it could be concluded that the regular BiF 3 :2%Er 3+ /20%Yb 3+ NPs could be formed at lower temperature under coprecipitation conditions.annealed at 50 C was the same as that without calcination (Fig. S10b †).However, the UCL intensity did not change with increasing irradiation time when it was annealed at 100 C, 200 C, or 400 C (Fig. S10c-e †).The reason for this might be that there was a large amount of hydroxyl groups (OH) on the sample's surface before calcination or certain temperature, which could increase the non-radiative relaxation rate and decrease the luminescence efficiency. 35The results could be further conrmed by FT-IR and XRD studies of BiF 3 :2%Er 3+ / 20%Yb 3+ before and aer calcination.The XRD patterns and FT-IR spectra of BiF 3 :2%Er 3+ /20%Yb 3+ NPs at different calcination temperatures were recorded, as shown in Fig. S11 † and 7.The diffraction peaks were matched with a cubic phase of BiF 3 , except when annealed at 400 C in Fig. S11.† The reason might be that the temperature reached its phase inversion temperature and then the crystalline phase began to change.The morphologies of samples calcined at different temperatures were also analyzed, as shown in Fig. S12.† From the gure, we can clearly see that the morphology of calcined samples became irregular when the calcination temperature was 400 C.This result was consistent with the XRD results.According to the FT-IR spectra measured for the calcinated NPs (Fig. 7), the intensity of the characteristic peaks changed.The band at 552 cm À1 arose from the vibration mode of Bi-F, which showed almost no change, but then changed at 400 C to form another phase.The band approximately at 3388 cm À1 and 1650 cm À1 ascribed to O-H and some organic molecular decreased with the rising calcination temperature.The enhancement of UCL intensity might be caused by the reduction in the number of OH groups 35 and in the organic component.This was in agreement with the result of TG-DSC.BiF 3 :2%Er 3+ /20%Yb 3+ treated at 200 C was used for the following investigation on UCL.
3.2.2Investigation on UCL mechanism.The UC emission spectra of BiF 3 :Ln 3+ (Ln ¼ Tm, Er, Ho)/Yb 3+ NPs irradiated at 980 nm were measured and the corresponding results are given in Fig. 8a.It could be seen that there were three different colors.To better understand the mechanism of every UCL, the variation of UCL intensities with pump power density was measured (Fig. 8b-d).It is well known that the relationship between the pump power density (P) and luminescence intensity (I) is I ¼ P n , by which information of the n photons in the UCL process can be provided. 36As presented in Fig. 8b, the slopes for the 526, 547, and 656 nm bands were 1.45, 1.57, and 1.49, respectively, showing a two-photon absorption process for the BiF 3 :2%Er 3+ / 20%Yb 3+ NPs.As presented in Fig. 8c, the slopes were 1.87 and 1.61 for Ho 3+ ion, meaning a two-photon process for BiF 3 :2% Ho 3+ /20%Yb 3+ NPs.The slopes for BiF 3 :0.5%Tm3+ /20%Yb 3+ NPs were 1.97, 2.50, and 2.60, corresponding to two-photon (red NIR) and three photon (red and blue) energy-transfer processes in Fig. 8d.
The possible UCL mechanism of Yb 3+ , Er 3+ , Ho 3+ , and Tm 3+ ions are shown in Fig. S13.† For BiF 3 :2%Er 3+ /20%Yb 3+ NPs, the Yb 3+ ion absorbs a photon and is excited to 2 F 5/2 , then drops back to the ground state and transfers the energy to a nearby Er 3+ ion to populate the 4 I 11/2 .The Yb 3+ can then transfer the second photon to populate the 4 F 7/2 and relax to 2 H 11/2 or 4 S 3/2 levels non-radiatively.Through non-radiative relaxation, the 4 I 11/2 level can also relax to the 4 I 13/2 level and aer that a transferred photon from Yb 3+ ions can populate the 4 F 9/2 .Red   emission is transmitted through 4 F 9/2 / 4 I 15/2 of Er 3+ , as shown in Fig. S13a.† The red and blue emissions of BiF 3 :20%Yb 3+ /0.5% Tm 3+ NPs could be conrmed as a three-photon process, as shown in Fig. S13b.† The 1 G 4 level could be populated by a three-step energy transfer of Yb 3+ through 3 H 4 and 3 F 4 .From the transitions of 1 G 4 / 3 F 4 , 3 H 4 / 3 H 6 and 1 G 4 / 3 H 6 , red, NIR, and blue emissions of Tm 3+ could be obtained, respectively.As for BiF 3 :20%Yb 3+ /2%Ho 3+ NPs, red emission through the bridge of the 5 I 7 level was pumped to the 5 F 5 level and the green emission was then a two-photon process (Fig. S13b †). 18.2.3Multicolor tuning and bright white emission.In order to expand those UCNPs potential applications, the bright white and multicolor UCL has been studied.To get multicolor emission, such as bright white emission, the concentrations of Er 3+ , Ho 3+ , and Tm 3+ ions could be properly adjusted, in which different samples were obtained (Table S1 †).The bright yellow green, pinkish, bright white, and bluish white UCL emissive spectra and photographs are shown in Fig. 9, which agreed well with the results of the color coordinate in Fig. S14.† Those results indicated that BiF 3 nanomaterials have potential applications as backlight sources in color displays and security labeling.
3.2.4Performance of UCL under dual-wavelength excitation (1550 nm and 980 nm).8][39] According to Fig. 10, the ratio of green/red emission intensities was similar.Under dual-excitation at 1550 nm and 980 nm, the intensity of UCL was greater than the sum of the intensities at 1550 nm and 980 nm, respectively.It was 1.49 times the sum of the separate single wavelength excitations, indicating there was a dual-wavelength synergistic effect in this system. 40,412.5 Thermometric property of BiF 3 :2%Er 3+ /20%Yb 3+ NPs.It was well known that the change of the transitions of 2 H 11/2 / 4 I 15/2 (524 nm) and 4 S 3/2 / 4 I 15/2 (542 nm) at different temperatures owing to 2 H 11/2 and 4 S 3/2 levels were related to the thermal coupling of Er 3+ ion.The ratio (R) of emission intensities at 524 nm and 542 nm followed the formula based on the Boltzmann distribution theory: [42][43][44] where A is the proportionality constant, DE is the energy gap between 2 H 11/2 and 4 S 3/2 levels, T is the absolute temperature, and k is the Boltzmann's constant.Eqn (1) could also be expressed as: Fig. 11a presents the UC emission spectra of BiF 3 :2%Er 3+ / 20%Yb 3+ excited at 980 nm with various temperatures.For green (524 nm and 542 nm) emissions, the UC emission intensities changed with increasing temperature.Fig. 11b presents a plot of 1/T versus ln(R), in which the slope (ÀDE/k) is equal to À501 and the intercept is 1.17.It could be also seen from Fig. 11c that R increased with the increasing temperature and presented a certain rule.In addition, the sensor sensitivity is another very important factor.The sensor sensitivity (S) can be calculated by using the following formula: [45][46][47][48] It was noteworthy that the sensitivity exhibited a downward tendency with the increasing temperature from 273 K to 453 K and its max value was about 0.0036 K À1 at 273 K (Fig. 11d), suggesting that the BiF 3 :2%Er 3+ /20%Yb 3+ NPs had a certain Fig. 9 UCL emissive spectra and corresponding luminescence photographs of: (a) BiF 3 :20%Yb 3+ /1%Er 3+ /0.15%Ho 3+ , (b) BiF 3 :20% Yb 3+ /1%Er 3+ /0.5%Tm 3+ , (c) BiF 3 :20%Yb 3+ /0.5%Tm 3+ /0.15%Ho 3+ , (d) BiF 3 :20%Yb 3+ /1%Er 3+ /0.5%Tm 3+ /0.15%Ho 3+ NPs excited at 980 nm.thermometric property, which might be employed as a temperature sensor.
3.2.6 Cell toxicity test.In order to evaluate the potential biosafety, cytotoxicity tests must be investigated. 49,50As illustrated in Fig. 12, the cytotoxicity of a NPs phospholipid aqueous solution with different concentrations was assessed by CCK-8 assay under 5% CO 2 at 37 C for 24 h.The viability of the B16-F10 cells decreased to 90% at a density of 1 mg mL À1 .When the concentration varied from 50 mg mL À1 to 100 mg mL À1 , the cellular viability of B16-F10 cells decreased to 82%, indicating that the UCNPs phospholipid aqueous solution presented lower cytotoxicity.
3.2.7In vivo imaging.100 mL BiF 3 :2%Er 3+ /20%Yb 3+ UCNPs phospholipid aqueous solution (100 mg mL À1 ) was injected into a Kunming mouse subcutaneously or intraperitoneally for deeper tissue imaging.In a comparative experiment, the luminous point on a Kunming mouse without treatment with NPs by exciting at 980 nm could not be observed with UCL (Fig. 13a and  b).On the other hand, aer intraperitoneal and subcutaneous injection in the corresponding area (100 mL, 100 mg mL À1 ), yellow UCL could be observed in dark upon the excitation at 980 nm laser (Fig. 13c and d).These indicated that UCNPs had a high contrast and deep penetration for in vivo optical bioimaging, which could be used as probe. 5.2.8In vivo X-ray imaging.A certain amount of BiF 3 :2% Er 3+ /20%Yb 3+ (200 mL, 100 mg mL À1 ) UCNPs phospholipid aqueous solution was injected subcutaneously in a Kunming mouse for X-ray imaging.Obvious X-ray signals marked by a white arrow could be observed for the Kunming mouse injected with NPs (Fig. 14b) compared to non-injection (Fig. 14a).It has been proven that they can absorb X-rays.The results showed that they could be utilized as X-ray imaging contrast agents.

Conclusions
In summary, multi-functional BiF 3 UCNPs were successfully synthesized by a facile co-precipitation method, both below and above room temperature (À25 C, 100 C).The growth mechanism indicated that the obtained BiF 3 NPs were formed through the growth process of "Nucleation / Crystallization / Ostwald ripening".Fluorescence intensity ratio technology was used to investigate the temperature sensitivity of BiF 3 :2%Er 3+ / 20%Yb 3+ and the max sensitivity was found to be 0.0036 K À1 at 273 K.The integrated emission of BiF 3 :2%Er 3+ /20%Yb 3+ NPs excited by dual-wavelength simultaneously was 1.49 times more than that excited by two single wavelengths (1550 nm and 980 nm), respectively.Multicolor and bright white light could be obtained upon excitation at 980 nm by modulating the content of Ho 3+ , Er 3+ , and Tm 3+ , by which they might be used as security labeling and backlight sources for color displays.The low cytotoxicity of BiF 3 :2%Er 3+ /20%Yb 3+ was conrmed by cell toxicity tests with phospholipid aqueous solution.BiF 3 :2%Er 3+ / 20%Yb 3+ NPs presented better properties of penetration and contrast in the in vivo imaging and X-ray imaging tests, indicating that they could be used as potential probe and contrast agents in in vivo optical bioimaging.

Live subject statement
All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Zhengzhou University and experiments were approved by the Animal Ethics Committee of Henan Laboratory Animal Center.

Fig. 8
Fig. 8 (a) UC emission spectra and corresponding luminescence photographs, (b-d) pump power dependence of blue, green, red, and NIR UCL emission intensities in BiF 3 :Ln 3+ NPs excited at 980 nm.

Fig. 13
Fig. 13 In vivo UC imaging of a Kunming mouse: (a and b) without injection of UCNPs, (c) subcutaneous injection, and (d) intraperitoneal injection of UCNPs [100 mL (100 mg mL À1 ) for c and d].