1. Introduction

In the past few decades, tomographic imaging techniques have played an important role in the investigation of novel biomedical imaging methods. It is due to their superiority in terms of unlimited penetration depth [1]. However, because of long image acquisition times they can’t visualize real-time dynamics and have limited spatial resolution [2]. In comparison, photoluminescence (PL)-based biomedical imaging has the advantages of fast feedback, high sensitivity, and high resolution [3]. But it has a inevitable drawback, low tissue penetration depth. Optical probes that emit in the visible range (400-750nm) can only penetrate approximately 1 mm into the tissue because of the absorption and scattering of the photons [3,4]; for those emitting in the “biological transparency window”, that is the first near-infrared (NIR-I) window (750-900 nm), the penetration depth only reaches several millimeters [5,6]. Thus, a new imaging method with high spatial resolution, fast feedback, and deep tissue penetration is desired to optimize the accuracy and sensitivity of biomedical imaging.

First of all, the most basic work is to realize high spatial resolution. Near-infrared contrast agents have generated particular interest in recent years due to their emission at longer wavelengths where scattering is reduced, and auto-fluorescence is weak [7–9]. These advantageous features enable imaging with increased signal-to-background ratio to greater depths within tissue and realize high spatial resolution imaging. Extending this principle, materials emitting even further into the infrared, in the so-called NIR II region (1100-1600 nm), such as quantum dots [10,11], carbon nanotubes [12], and rare-earth ions have recently gained attention due to their ability to provide signal from even deeper within scattering tissues [13, 14].

Compared with the previously reported handful of NIR II organic dyes and QDs, lanthanide-doped nanoparticles possess outstanding chemical and optical properties, including low cytotoxicity, high efficiency, low photo-bleaching, long luminescence and photochemical degradation [15, 16]. More importantly, they can emit UV and visible light after excitation by near-IR (NIR) light, through a process known as photon upconversion [17]. At the same time, because of the fast feedback and deep tissue penetration features, some efforts have been focused on the study of the temperature-dependent UC luminescence due to significant potential applications in temperature sensors, thermal imaging and cancer treatment as well as an optical heater [18–22]. These unique properties render lanthanide-doped nanoparticle particularly useful in bio-sensing and bio-imaging applications.

Lanthanide-doped nanoparticles may provide a much needed solution for the above-mentioned problems. Herein, we selected co-precipitation in a binary solvent mixture of oleic acid and 1-octadecene to synthesize the hexagonal-phase NaYF₄:Yb³⁺, Er³⁺ NPs which have been widely used as the most efficient NIR-to-visible upconversion (UC) luminescent and probe in bioscience [23, 24]. For the medical applications of this material, we exploited not only the function of dual-mode emission of β-NaYF₄: Yb³⁺, Er³⁺ NPs in the near infrared (NIR) and visible regions with single wavelength excitation at 980 nm, but also the function of physiological temperature sensing with the luminescence of Er³⁺ in the visible region. The obtained nanoparticle can be realized water-dispersible through surface coating with silica. Also, the strategy to design dual mode sensing and imaging composites may be extended to prepare multi-functional platforms for targeted multi-mode imaging of various biological systems.

2. Experimental

2.1 Materials

Y(CH₃CO₂)₃·xH₂O, (99.9%), Yb(CH₃CO₂)₃·4H₂O, (99.9%), Er(CH₃CO₂)₃·xH₂O, (99.9%), 1-octadecene (90%), oleic acid (90%), H₅NO, were all purchased from Sigma-Aldrich. NH₄F (98%) and Polyvinylpyrrolidone (PVP-40) were obtained from Aladdin. NaOH (AR), methanol (AR), ethanol (AR), hydrochloric acid (AR), were supplied by Sinopharm Chemical Reagent Company. Ethyl silicate (AR) was purchased from Shanghai Chemical Reagent Purchase And Supply Wulian Chemical Factory.

All chemicals were used without further purification. Doubly distilled water was used in all experiments.

2.2 General procedure for the synthesis of core nanoparticles

Hexagonal-phase NaYF₄: Yb³⁺, Er³⁺ nanoparticles were prepared using a modified version of the synthesis previously published by Liu et al [25–27]. All reactions were carried out in a 50 mL three-neck roundbottom flask. Briefly, 2mL water solution of Ln (CH₃CO₂)₃ (0.2 M, Ln = Y, Yb, and Er), oleic acid (3 mL) and 1-octadecene (7 mL) was added to a 50-mL flask. The mixture was heated at 150 °C for 30 min to remove the water content from the solution before cooling down to 50 °C. Shortly thereafter, 5 mL of methanol solution containing NH₄F (1.60 mmol) and NaOH (1 mmol) was added and the resultant solution was stirred for 30 min. After the methanol was evaporated, the solution was heated to 290 °C under argon for 1.5 h and then cooled down to room temperature. The resulting nanoparticals were precipitated by addition of ethanol, collected by centrifugation at 6000 rpm for 5 min, washed with ethanol several times, and re-dispersed in 4 mL of cyclohexane or dried at 60 °C for 12 h.

2.3 General procedure for the synthesis of water-dispersible NaYF₄: Yb³⁺, Er³⁺ @ SiO₂ nanocomposite

The 1 mL as-prepared oleic acid-capped nanoparticles were dispersed in a 2-mL HCl solution (0.1 M) and ultra sonicated for 10 min to remove the surface ligands. After the reaction, the nanoparticles were collected via centrifugation at 12000 rpm for 20 min, and further purified by adding an acidic ethanol solution (pH 4; prepared by adding 0.1 M HCl aqueous solution to absolute ethanol). The nanoparticles were washed with ethanol several times until transparent, and re-dispersed in 2 mL of ethanol. Typically, 0.2 g PVP was dissolved in 4 mL deionized water with magnetic stirring at room temperature for 0.5 h. Then 1 mL ligand-free nanoparticles was added to the mixture solution, and added 1 mL ammonium hydroxide solution after stirring for 4 h. Finally, 1 mL TEOS was added dropwise to the solution with stirring at room temperature for 12 h to obtain a homogeneous water-dispersible NaYF₄: Yb³⁺, Er³⁺ @ SiO₂ nanocomposite [28–30].

2.4 Characterization of NPs

The X-ray diffraction (XRD) pattern of Er³⁺-Yb³⁺ codoped NaYF₄ was recorded using Bruker D8 Advance X-ray diffractometer with Cu-Kα (1.5405 Å) radiation within 10-80︒ range of 2θ. Scanning electron microscopy (SEM) was performed on a SU8010 FE-SEM scanning electron microscope (HITACHI, Japan) operated at 1.0 kV and the SEM image of the water-dispersible β-NaYF₄: 3 mol.% Er³⁺, 20 mol.% Yb³⁺ @ SiO₂was operated at 10.0 kV. The UC and NIR II spectra were obtained at room temperature by using a PL3-211-P spectrometer (HORIBA JOBIN YVON, America). For UC and NIR II steady-state spectra investigation, a 980 nm continuous diode laser was used to pump the samples. The experimental setup for temperature sensing was a TAP-02 analyzer of high temperature fluorescence (ORIENT KOJI, China).

3. Results and discussion

3.1 Structure and morphology

Figures 1(a-e) shows SEM images of Er³⁺-Yb³⁺ codoped NaYF₄. The concentration of Er³⁺ ions in the codoped NaYF₄ is varied keeping Yb³⁺ ions concentration fixed at 20 mol%. It can be seen that the nanoparticles displayed uniform spherical morphologies and narrow size distributions. The particle size distribution confirms that with the increase of erbium ion concentration, the concentrations of erbium ion in the codoped NaYF₄ is 0.5 mol.%, 1 mol.%, 2 mol.%, 3 mol.%, 10 mol.%, the average diameters are 35.5 nm, 30.0 nm, 29.0 nm, 27.0 nm, 25.5 nm, respectively. The diffraction peaks of all the nanoparticles [Fig. 1(f)] can be indexed as a pure hexagonal NaYF₄:Yb³⁺, Er³⁺ phase (PDF No.16-0334), which is contributing to the upconversion luminescence properties. With further increase in Er³⁺ ion concentration, no extra diffraction peaks were observed. The well defined peaks indicate the high crystallinity of the synthesized nanoparticles. As shown in Fig. 1(f), the (110) and (101) peak shifts towards higher diffraction angles with the increase of erbium ion concentration. As a result of shrinkage in unit-cell volume owing to the substitution of Y³⁺ ions by smaller Yb³⁺ ions and Er³⁺ in the host lattice [i.e. (r (Yb³⁺ 0.868 Å) < r (Er³⁺ 0.890 Å) < r (Y³⁺ 0.900 Å)] [21, 32], implying that doping-introduced lattice shrinkage in Er-doped NaYF₄.. Importantly, we also observed that the diffraction peaks broaden with increasing Er³⁺ dopant content, also indicating a reduction of the average crystallite size which is consistent with SEM images.

 

Fig. 1 (a-e) SEM images of β-NaYF₄: Yb³⁺, Er³⁺, the concentration of Er³⁺ ion in the codoped NaYF₄ is 0.5 mol.%, 1 mol.%, 2 mol.%, 3 mol.%, 10 mol.%, respectively. The upper right inset is the corresponding particle size distribution. (f) The X-ray diffraction patterns of β-NaYF₄: Yb³⁺, Er³⁺; inset is the partially enlarged XRD pattern showing the shift of peaks.

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3.2 Upconversion and Down-shifting study under 980 nm excitation

Figure 2(a) shows the corresponding up-conversion luminescence spectra of NaYF₄ with different Er³⁺/Yb³⁺ ratio. The concentration of Er³⁺ ions in the codoped NaYF₄ nanoparticles is varied keeping Yb³⁺ ions concentration fixed at 20 mol.%. Under 980 nm laser excitation, green and red up-conversion emissions centered at around 521 nm, 541 nm, and 654 nm are observed, corresponding to the ²H_11/2→⁴I_15/2, ⁴S_3/2→⁴I_15/2, ⁴F_9/2→⁴I_15/2 transitions of Er³⁺ ions, respectively. Among these bands the intensity of green bands decreases more prominently while the intensity of red band decreases not significantly. It is due to the cross-relaxation processes (²I_11/2/ ⁴S_3/2 + ⁴I_11/2→ 2⁴F_9/2) as well as the non-radiative processes within the Er³⁺ ions [33]. The intensity of all the UC emission peaks decreases with increasing the concentration of erbium ions up to 10 mol. %, the UC emission lifetime decreases with increasing the concentration of erbium ion, which is due to the concentration quenching. In order to understand the number of pump photons involved in the UC mechanisms the monolog plot of upconversion emission intensities, the excitation power-dependent UC emissions of 521, 541 and 654 nm were calculated. It is well known that the relation between the UCL intensity (I_UC) and the infrared excitation power (P_pump) can be expressed as [34, 35]

 

Fig. 2 (a) The UC emissions in β-NaYF₄: Yb³⁺-Er³⁺ crystals with different concentration of Er³⁺ ions; (b)Log–log plot of UC emission intensity in β-NaYF₄: 3 mol.% Er³⁺-20 mol.% Yb³⁺ versus pump power ; (c) Calculated peak area ratio of the red region (⁴F_9/2 → ⁴I_15/2) to the green region (²H_11/2, ⁴S_3/2 → ⁴I_15/2); (d)Schematic energy level diagram for Er³⁺-Yb³⁺ energy transfer system under 980 nm excitation; (e) The SWIR emissions in β-NaYF₄: Yb³⁺, Er³⁺ crystals with different concentration of Er³⁺ ions; (f) Log–log plot of DC emission intensity in β-NaYF₄: 3 mol.% Er³⁺-20 mol.% Yb³⁺ versus pump power.

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With the help of schematic energy level diagram [(Fig. 2(d)] the UC emissions in the green and red regions are due to the following mechanisms [36, 37]

It is worth mentioning that the slope of 654 nm (1.74) emission is larger than 541 nm (1.63), therefore, the UC emission in the red region is proposed mainly due to the process 2). In addition, the five samples not only exhibit obvious change of the relative intensities but also have different relative intensity ratios of red (600–700 nm) to emission green (500–600 nm) (R_R/G). G is the integrated intensities corresponding to green bands and R is the integrated intensities corresponding to red band. As can be seen from Fig. 2(c), the red/green ratio increases linearly with Er³⁺ concentration. It should be noted that the ratio of the intensities of the red band and the green one is turned by the Er³⁺ ions concentration; an increase in the content of Er³⁺ ions induces a increase in R_R/G.

Figure 2(e) shows the corresponding NIR II PL of NaYF₄ with different concentration of Er³⁺ ions. In the down shifting emission spectrum bands around 1525 nm are observed [38]. The intensity of the short-wavelength infrared light increases with increasing the concentration of erbium ions up to 3 mol. %, but beyond this concentration of erbium ions it decreases due to concentration quenching. As shown in Fig. 2(f), the slope of the linear fit of 1525 nm emission in the sample is below 1, indicating that only a single-photon process is involved to produce the infrared emission.

Interestingly in the present case it is clearly observed that the intensities of upconversion emission peaks decrease when increasing the doping level of Er³⁺ from 0.5 to 3 mol.%, however, the intensities of down shifting emission peaks increase substantially. It is plausible that the cross-relaxation process (⁴S_3/2 + ⁴I_13/2 → ⁴F_9/2 + ⁴I_11/2) plays an important role in changing the R_R/G [39]. An increase in the Er³⁺ ion concentration enforces the Er³⁺-Er³⁺ interaction due to the shorter ion-ion distance leading to higher cross relaxation rates. The increased cross relaxation rates will cause a increase in the R_R/G with the increase of Er³⁺ ions which is in good agreement with the observation shown in the inset of Fig. 2(c) [40]. Figure 2(d) shows the simplified Er³⁺-Yb³⁺ energy level diagram of the 980 nm down shifting luminescence of the samples.

3.3 Study of temperature sensing behavior about β-NaYF₄: 20 mol. %Yb³⁺, 3 mol. %Er³⁺

It is known that intensity-based measurements may suffer from variation of the sensor concentration and drifts of the optoelectronic system. Fluorescence ratio, however, is more reliable due to the freedom from these drawbacks. Different researchers have investigated the temperature sensing behavior for different rare earth doped materials by using the fluorescence intensity ratio (FIR) technique [31–43]. Er³⁺ doped NPs can display a highly temperature-dependent emission, large Stokes’ shifts and long lifetimes, which make such materials strong candidates for fluorescent sensing of the temperature [44–46]. In this work, depending on the temperature dependent physic-chemical property of the synthesized material the temperature sensing property has been studied, especially in the physiological range (25-55 ︒C). Figure 3(a) represents the upconversion spectra of the optimal doping concentration 3 mol.% Er³⁺ - 20 mol.% Yb³⁺ codoped NaYF₄ under 980 nm excitation in the physiological range from 25︒C to 55 ︒C. The excitation intensity was well below 0.5 W/cm².

 

Fig. 3 (a) Upconversion emission spectra obtained at six different cuvette temperatures (λ_exc = 980 nm); (b) The monolog plot of the FIR (I₅₂₁/I₅₄₁) as a function of inverse absolute temperature; (c) The sensitivity as a function of the absolute temperature; (d) SEM image of the water-dispersible β-NaYF₄: 3 mol.% Er³⁺, 20 mol.% Yb³⁺ @ SiO₂.

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With the increasing of temperature, the FIR of 521 nm and 541 nm bands change due to the thermal population, while the all green emissions exhibit a little bit of quenching. The FIR varies according to the relation

Where I₅₂₁ and I₅₄₁ are the integrated intensities corresponding to green bands around 521 nm and 541 nm respectively. C is proportionality constant. ΔE is the energy difference between the thermally coupled levels ⁴S_3/2 and ²H_11/2, K = 0.695 K⁻¹ cm⁻¹ is the Boltzmann’s constant. As the energy difference between ²H_11/2 and ⁴S_3/2 level is ~700cm⁻¹, thermal coupling is established between these levels. Figure 3(b) shows the monolog plot of FIR as a function of inverse absolute temperature. In accordance, with expression 1, a linear behavior is obtained; the best fit being Ln(I₅₂₁/I₅₄₁) = 2.00 – 969 (1/T) (T given in K). This clearly demonstrates that the temperature can be accurately measured from the ratio of the 521 and 541 nm fluorescent intensities in the temperature range relevant for most biological systems. The linear fitting of the experimental data gives the slope value of about 969. This slope actually gives the ΔE/K value. From this value the calculated ΔE value comes out to be~674 cm⁻¹. This deviation of ΔE value from the actual value is small.

It is worthwhile to know the sensitivity (S) of the material and it is defined as the rate at which the FIR changes with temperature. Sensitivity can be defined as

As the temperature rises from 25︒C to 55 ︒C, the sensitivity of the present material decreases. The maximum value of sensitivity is observed to be 0.1091 K⁻¹ at 298 K whereas the minimum value of the sensitivity is 0.0895 K⁻¹ at 333 K. Figure 3(c) shows the sensitivity as a function of the absolute temperature, the sensitivity is higher than the reference which sensitivity is observed to be 0.0091K⁻¹ at 303 K temperature [46].

In order to satisfy biocompatibility of these UCNPs, we provide a simple and versatile protocol for hydrophilic modification of the as-synthesized nanoparticles through silica coating. The coating of silica arises from the hydrophobic interactions of the alkyl chains of silica with the octadecyl group of oleic acid on the surface of hydrophobic NPs. After hydrolysis and polycondensation of silanes, the hydrophilic silanol groups of silanes extend outwards, rendering NPs water soluble and allowing attachment of biomolecules. Figure 3(d) shows the SEM image of the water-dispersible NaYF₄: 3 mol. % Er³⁺, 20 mol.% Yb³⁺ @ SiO₂. The SEM images showed that there is a very uniform layer of silica on the NaYF₄: 3 mol.% Er³⁺, 20 mol.% Yb³⁺ nanoparticle; obviously, the average diameters is 92.0nm, the nanoparticles are monodisperse in water. Furthermore, the thickness of the silica shell was about 35 nm and could be adjusted by changing the amount of TEOS. In this way, multi-functional biological luminescent materials can be achieved.

4. Conclusions

In summary, the temperature sensing and dual-mode emission in the near infrared (NIR) and visible regions with single wavelength under 980 nm irradiation were achieved in β-NaYF₄: Yb³⁺, Er³⁺ nanoparticles. The efficient NIR II PL can be used for bioimaging and UC emission can be used for physiological temperature sensing. The FIR of the green bands corresponding to the ²H_11/2→ ⁴I_15/2 and ⁴S_3/2→ ⁴I_15/2 transition has been studied as a function of temperature to examine its temperature sensing behavior. In addition, we have shown that the obtained nanoparticles can be dispersed in water through use of a silica surface coating. This suggests that the strategy to design dual mode sensing and imaging composites may be extended to prepare multi-functional platforms for targeted multi-mode molecular imaging of various biological systems.