Large-scale synthesis of uniform lanthanide-doped NaREF₄ upconversion/downshifting nanoprobes for bioapplications†

Wenwu You ^ab, Datao Tu *^a, Wei Zheng ^a, Xiaoying Shang ^a, Xiaorong Song ^a, Shanyong Zhou ^a, Yan Liu ^a, Renfu Li ^a and Xueyuan Chen *^ab
aCAS Key Laboratory of Design and Assembly of Functional Nanostructures, and Fujian Key Laboratory of Nanomaterials, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China. E-mail: xchen@fjirsm.ac.cn; dttu@fjirsm.ac.cn; Fax: +86 591 63179421; Tel: +86 591 63179421
^bUniversity of the Chinese Academy of Sciences, Beijing, 100049, China

First published on 17th May 2018

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1. Introduction

Lanthanide (Ln³⁺)-doped inorganic nanocrystals (NCs) with superior physicochemical and optical characteristics have emerged as a new generation of bio-probes in the past few years.^1–15 As a main category of luminescene materials, Ln³⁺-doped NaREF₄ (RE = rare earth) NCs that exhibit high upconversion luminescence (UCL) and downshifting luminescence (DSL) efficiency^16,17 have drawn reviving attention due to their potential applications in bioassays,^18–22 disease therapy,^23–26 and bioimaging.^27–29 The controlled synthesis of monodisperse Ln³⁺-doped NaREF₄ NCs is of fundamental importance for their bioapplications, because the optical properties (e.g., emission intensity and photoluminescence lifetime) of Ln³⁺-doped NCs depend critically on their dimensions and crystalline structures.³⁰ To further promote the clinical applications of these NCs, it is highly desired to reduce the unwanted interbatch variations of the NCs since refining each batch is costly and time-consuming.^31,32

Currently, thermal co-decomposition (TCD) and high temperature co-precipitation (HTCP) methods are the two most frequently used techniques to synthesize monodisperse Ln³⁺-doped NaREF₄ NCs.^33,34 TCD is generally performed in high-boiling-point solvents like oleic acid (OA), oleylamine (OM) and 1-octadecene (ODE), which contain mixed sodium and Ln³⁺ trifluoroacetates as precursors to produce high-quality α- and β-phase NaREF₄ NCs. However, the synthesis experiment must be carefully carried out in well-ventilated chemical hoods, since the pyrolysis of trifluoroacetates may produce toxic fluorinated and oxyfluorinated carbon gases. The requirement of expensive trifluoroacetate precursors along with the generation of highly toxic by-products is unfavorable for the large-scale preparation of Ln³⁺-doped NCs. As compared to the TCD method, HTCP is a user-friendly method for the lab-scale synthesis of Ln³⁺-doped NaREF₄ NCs. This synthetic strategy involves the dissolution of fluoride reagents, and the formation of amorphous NaREF₄ coprecipitates at low temperature, followed by the growth of highly crystalline NCs at elevated temperature. Multiple steps and long reaction time are often required for this method. Thus, the reproducibility of the synthesis is hard to control. So far, it remains challenging to establish a general approach for the large-scale synthesis of Ln³⁺-doped NaREF₄ nanoprobes with cheap reagents, simple procedures and safe operation.

In this regard, we herein develop a facile solid–liquid-thermal-decomposition (SLTD) method for the size- and phase-controlled synthesis of Ln³⁺-doped NaREF₄ NCs, which combines both advantages of simple procedures from TCD and safe operation from HTCP. A kind of cheap reagent of NaHF₂ powder is directly used as the fluoride and sodium precursor (Scheme 1), which decomposes to NaF and HF at an elevated temperature. The produced NaF and HF may react with RE compounds in the liquid phase (OA and ODE) for the nucleation and growth of Ln³⁺-doped NaREF₄ NCs. The crystalline structure, particle size and morphology can be well tuned by adjusting the reaction temperature or the added amounts of NaAc. Moreover, such SLTD strategy can be conveniently applied in the scale-up synthesis of Ln³⁺-doped NaREF₄ core and core/shell NCs (up to 63.38 g, size variation <7%). To verify the practicability of these nanoprobes, we successfully demonstrate their proof-of-concept applications in the sensitive detection of an important tumor marker prostate-specific antigen (PSA) and near-infrared (NIR) in vivo imaging.

2. Experimental

2.1. Chemicals and materials

NaHF₂, NaAc, poly(acrylic acid sodium salt) (PAA-Na), and cyclohexane were purchased from Sinopharm Chemical Reagent Co., China. Ethanol was purchased from Adamas-beta Ltd (Shanghai, China). Rare earth acetates, OA, ODE, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (China). PSA and PSA antibody were purchased from Sangon Biotech (Shanghai) Co., Ltd. 1,2-Distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG) was purchased from Shanghai Yare Biotech Inc. The 96-well Nunc Immobilizer Amino plates, which have a high affinity for the coupling of peptides and proteins, were purchased from Thermo Fisher Scientific Inc. Deionized (DI) water with a resistivity of 18.2 MΩ cm was used in all the experiments. All chemicals were used as received without further purification.

2.2. Synthesis of 1 mmol of Ln³⁺-doped NaREF₄ NCs

Ln³⁺-Doped NaREF₄ nanocrystals (NCs) were synthesized via a SLTD method as follows. In a typical synthesis of 1 mmol of NaGdF₄:Yb,Er NCs, 0.8 mmol of Gd(CH₃COO)₃, 0.18 mmol of Yb(CH₃COO)₃, and 0.02 mmol of Er(CH₃COO)₃ were mixed with 8 mL of OA and 12 mL of ODE in a 100 mL two-neck round-bottom flask. The resulting mixture was heated to 180 °C under N₂ flow with constant stirring for 20 min to form a clear solution, and then cooled down to room temperature (RT). Thereafter, 2 mmol of NaHF₂ powder was added. The solution was heated to 250 °C under N₂ flow with vigorous stirring for 30 min, and then heated to 310 °C for another 30 min. Subsequently, the mixture was allowed to cool down to RT. The obtained NCs were precipitated by the addition of ethanol, collected by centrifugation, washed with ethanol and cyclohexane several times, and finally re-dispersed in cyclohexane. The phase and size of the NCs can be controlled by adjusting the reaction temperature or adding different amounts (0–1.5 mmol) of NaAc as sodium source in the reaction solution. As control experiments, 1 mmol of NaGdF₄:Yb,Er NCs was also synthesized via the TCD and HTCP methods at 300 °C.^33,34

2.3. Large-scale synthesis of Ln³⁺-doped NaREF₄ NCs

For the synthesis of 5 mmol of β-NaGdF₄:Yb,Er NCs, 4 mmol of Gd(CH₃COO)₃, 0.9 mmol of Yb(CH₃COO)₃, and 0.1 mmol of Er(CH₃COO)₃ were mixed with 10 mL of OA and 5 mL of ODE. The resulting mixture was heated to 180 °C under N₂ flow with constant stirring for 20 min to form a clear solution, and then cooled down to RT. Thereafter, 10 mmol of NaHF₂ powder was added. The solution was slowly heated to 250 °C under N₂ flow and maintained for 30 min, and then heated to 310 °C with vigorous stirring for another 30 min. Finally, the reaction mixture was allowed to cool down to RT. By gradually increasing the quantity of precursors (NaHF₂ powder and rare earth acetates), the synthesis of 20 and 200 mmol of β-NaGdF₄:Yb,Er NCs was carried out in 40 mL ODE/20 mL OA and 400 mL ODE/200 mL OA, respectively.

2.4. Large-scale synthesis of Ln³⁺-doped NaREF₄@NaREF₄ core/shell NCs

For the large-scale synthesis of β-NaGdF₄:Yb,Er@NaYF₄ core/shell NCs, 200 mmol of β-NaGdF₄ core NCs were synthesized based on the abovementioned procedure. After that, 100 mmol of Y(CH₃COO)₃ was added to the reaction solution and heated to 180 °C under N₂ flow for 20 min, and then cooled down to RT. Subsequently, 200 mmol of NaHF₂ powder was added to the solution, which was slowly heated to 250 °C under N₂ flow for 60 min, and cooled down to RT. The obtained NCs were precipitated by the addition of ethanol, collected by centrifugation, washed with ethanol and cyclohexane several times, and finally re-dispersed in cyclohexane.

2.5. Synthesis of PAA-capped NCs

The PAA-capped NCs were synthesized by the following procedures. First, the OA ligands on the surface of as-prepared NCs (50 mg) were removed via a facile acid treatment method.¹⁶ Second, the ligand-free NCs (40 mg mL⁻¹, 500 μL) were mixed with PAA-Na solution (80 mg mL⁻¹, 500 μL) through continuous stirring overnight. Third, PAA-capped NCs were collected by centrifugation, and washed with deionized water several times. Finally, the resulting PAA-capped NCs were dispersed in DI water for further studies.

2.6. Bioconjugation of the PAA-capped NCs with antibody

Antibodies were attached onto the PAA-capped NC surface using the EDC/NHS coupling reaction as previously described.³⁵ EDC (20 mg) and NHS (10 mg) in MES buffer (500 μL, pH 5.4) were added to DI water (500 μL) containing 10 mg of PAA-capped NCs, which were incubated for 20 min. The resulting NCs were collected by centrifugation and washed with DI water several times. Thereafter, these NCs were mixed with 30 μg of PSA antibody in HEPES buffer (500 μL, pH 7.4) and incubated for 30 min. The PSA antibody-conjugated NCs were collected by centrifugation and purified by washing with DI water three times. Finally, the PSA antibody-conjugated NCs were dispersed in 2% BSA solution (500 μL) and stored at 4 °C.

2.7. UCL heterogeneous assay based on PSA antibody conjugated NCs

Typically, 100 μL of PSA antibody (10 μg mL⁻¹) coating buffer (100 mM carbonate buffer, pH 9.6) were added to the wells of a 96-well Nunc Immobilizer Amino plate. The plate was incubated at 37 °C for 1 h. Then 300 μL of blocking solution (100 mM carbonate buffer containing 0.2% of 2-aminoethanol, pH 9.6) was added in each well and incubated at 37 °C for 1 h to block the unbinding sites on the plate. Thereafter, different amounts of PSA dissolved in 100 μL of phosphate-buffered saline (PBS) (pH 7.4) were added and incubated at 37 °C for 1 h followed by washing with PBST three times. Afterwards, 100 μL of antibody-conjugated NC solution (100 μg mL⁻¹) was added and incubated at 37 °C for 30 min. After washing with PBS four times, the plate was subjected to UCL detection for PSA on a custom-built microplate reader. The control experiment was conducted by using BSA as an analyte under otherwise identical conditions.

2.8. Synthesis of DSPE-PEG-capped NCs

The DSPE-PEG-capped NCs were synthesized by the following procedures. First, 40 mg of DSPE-PEG and 20 mg of OA-capped NCs were dispersed in 2 mL of chloroform solution, and the mixture solution was sonicated for 5 min. Second, the chloroform solution was removed by using a rotary evaporator at RT. Third, DSPE-PEG-capped NCs were washed with DI water and collected by centrifugation. Finally, the resulting DSPE-PEG-capped NCs were dispersed in DI water for further studies.

2.9. Cytotoxicity assay

Cell viability was measured by using a CCK-8 assay on the HELF cells. In a typical experiment, HELF cells were seeded in 96-well plates and then incubated with 100 μL of varying concentrations of DSPE-PEG-capped β-NaGdF₄:Yb,Er@NaYF₄ NCs for 24 h at 37 °C under a humidified 5% CO₂ atmosphere. The optical density at 450 nm (OD450) of each well was measured on a multimodal microplate reader (Synergy 4, BioTek). The inhibition rate of cell growth was calculated by the following formula: cell viability (%) = (mean of absorbance value of treatment group/mean of absorbance value of control) × 100%.

2.10. DSL NIR imaging in vivo

Small animal imaging was performed following the tail vein injection of the DSPE-PEG-capped β-NaGdF₄:Yb,Er@NaYF₄ NCs (5 mg mL⁻¹, 200 μL) into mice. The in vivo DSL NIR imaging was recorded by using a custom-built imaging system equipped with an InGaAs camera. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Fujian Medical University, and the protocol was approved by the Animal Ethics Committee of Fujian Medical University.

2.11. Characterization

Powder X-ray diffraction (XRD) patterns of the samples were collected with an X-ray diffractometer (MiniFlex 600, Rigaku) with Cu Kα1 radiation (λ = 0.154187 nm). Both the low- and high-resolution transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) were performed by using a TECNAI G² F20 TEM equipped with an energy dispersive X-ray (EDX) spectrometer. Scanning electron microscopy (SEM) was performed by using a JSM 6700F SEM. Thermogravimetric analysis (TGA) experiments were conducted on a Netzsch STA449C thermal analysis system under a N₂ atmosphere at a rate of 10 °C min⁻¹. The zeta potential and hydrodynamic diameter distribution of the NCs were determined by means of dynamic light scattering (DLS) measurement (Nano ZS ZEN3600, Malvern). Fourier-transform infrared (FTIR) spectra were recorded in KBr discs on a Magna 750 FTIR spectrometer. UCL and DSL emission spectra were recorded by using a fluorescence spectrometer (FLS980, Edinburgh Instrument) with a continuous-wave 980 nm semiconductor NIR laser diode. UCL and DSL decays were measured with a customized ultraviolet (UV) to mid-infrared steady-state and phosphorescence lifetime spectrometer (FSP920-C, Edinburgh) equipped with a digital oscilloscope (TDS3052B, Tektronix) and a tunable mid-band Optical Parametric Oscillator (OPO) pulse laser as the excitation source (410–2400 nm, 10 Hz, pulse width 5 ns, Vibrant 355II, OPOTEK). The UCL heterogeneous assays were carried out on a custom-built microplate reader. The in vivo NIR imaging was performed with a custom-built imaging system. External 0–2 W adjustable CW 980 nm semiconductor laser was used as the excitation light source. A near-infrared camera which employs a 640 × 512 pixel InGaAs sensor (C12741-11, Hamamatsu Photonics K.K.) was used as the signal collector.

3. Results and discussion

In the SLTD method, the crystalline phase and particle size of Ln³⁺-doped NaREF₄ NCs can be readily tuned by varying the reaction temperature (Fig. 1). According to the TGA, NaHF₂ powder began to lose weight at 160 °C and decomposed to NaF and HF at 235 °C (Fig. S1†). In a typical synthesis of Ln³⁺-doped NaGdF₄ NCs, the reaction temperature was set at 250 °C in the first stage to ensure the complete decomposition of NaHF₂ powder (Fig. 1a). After 30 min, α-NaGdF₄:Yb,Er NCs (3.5 ± 0.3 nm) were obtained (Fig. 1b, c and S2†). It was reported previously that small α-NaREF₄ NCs may redissolve at high temperature to grow the larger β-NaREF₄ NCs through Ostwald ripening.^36,37 As such, we then raised the reaction temperature in the second stage (Fig. 1a). As the reaction temperature reached 290 °C, mixed-phase NCs (α- and β-NaGdF₄:Yb,Er) with 7.7 ± 1.2 nm were yielded (Fig. 1b and d). However, uniform β-NaGdF₄:Yb,Er NCs with sizes of 25.0 ± 1.9 nm and 41.2 ± 1.7 nm were produced at 300 °C and 310 °C, respectively (Fig. 1b, e and f). The XRD and TEM results indicate that raising the reaction temperature may not only overcome the dynamic energy barrier of α to β phase transition, but also modulate the size and morphology of the NCs. It should be noted that the NCs synthesized at 300 °C via SLTD are slightly larger than those synthesized via the TCD and HTCP methods (Fig. S3†). The small-sized NCs are of particular importance for bioimaging, since body clearance for small-sized NCs is reported to be more efficient, allowing the use of a higher dose of imaging agents.^38,39 In the SLTD synthesis, the addition of NaAc as the sodium source was found to decrease the size of the final NaGdF₄:Yb,Er NCs from 41.2 to 12.8 nm (Fig. S4†). The size decrease of the NCs can be attributed to the high concentration of Na⁺, which increases the number of seeds for the formation of β-NaREF₄ NCs.⁴⁰ Similarly, we extended the SLTD method for the synthesis of other isomorphic NaREF₄ NCs, such as NaSmF₄ (11.7 ± 1.0 nm), NaEuF₄ (16.2 ± 0.8 nm), NaGdF₄ (15.1 ± 1.0 nm), NaTbF₄ (27.7 ± 1.5 nm) and NaYF₄ (55.8 ± 2.2 nm) (Fig. 1g–k and S5†), which indicates the universality of our proposed strategy.

By virtue of its mild reaction conditions and facile operation, the SLTD strategy was exploited for the large-scale production of Ln³⁺-doped NaREF₄ NCs. For the synthesis of 1 mmol of NaREF₄ NCs, 20 mL of reaction solvents (8 mL of OA and 12 mL of ODE) were used. In an effort to make the scale-up synthesis more economical, the ratio of solvent (mL) to rare earth acetates (mmol) was reduced from 20 to 3 for the synthesis of 5, 20 and 200 mmol of Ln³⁺-doped NaREF₄ NCs (Table S1†). As shown in Fig. 2a–c, the particle sizes of the produced 5, 20 and 200 mmol of β-NaGdF₄:Yb,Er NCs were measured as 27.8 ± 1.5 nm, 24.7 ± 1.3 nm and 23.5 ± 1.5 nm, respectively. All the as-prepared β-NaGdF₄:Yb,Er NCs showed a narrow size variation (<7%). In addition to their high crystallinity and monodispersity (Fig. S6†), their optical performance was also preserved during scaling up. Upon excitation at 980 nm, the UCL intensity for the 200 mmol β-NaGdF₄:Yb,Er NCs was observed to be comparable to that of the synthesized 1 mmol counterparts (25.0 ± 1.9 nm) (Fig. 2d).

For comparison, we carried out the control experiments for the synthesis of 20 mmol β-NaGdF₄:Yb,Er NCs based on the TCD and HTCP approaches.^33,34 For the TCD method, when the temperature was raised to 260 °C, lots of gas bubbles were produced immediately and spurted from the vessel owing to the drastic decomposition of trifluoroacetates (Fig. 2e, left), which is dangerous and should be handled with care. For the HTCP method, 50 mmol NaOH and 80 mmol NH₄F dissolved in 200 mL methanol were added into the flask to provide sodium and fluorine sources. As a result, a long reaction time was required to completely evaporate methanol. When the mixture was heated at 50 °C for as long as 3 h under N₂ flow, large amounts of methanol remained in the flask to form a turbid suspension (Fig. 2e, middle). As such, these results show unambiguously that neither the TCD method nor the HTCP method is suitable for scale-up syntheses. In contrast, the SLTD method has several advantages in the large-scale synthesis of β-NaREF₄ NCs. First, the synthetic process is user-friendly and economical, since cheap NaHF₂ powder was employed directly as the fluoride and sodium precursor (Fig. 2e, right). In particular, the produced HF due to the decomposition of NaHF₂ was essentially consumed in the nucleation and growth of Ln³⁺-doped NaREF₄ NCs (Fig. S7†). Second, SLTD is based on the one-pot reaction route, which is easy to perform and time-saving (within 2 h). Third, the proposed strategy is universal for fabricating not only Ln³⁺-doped NaREF₄ core NCs, but also their core/shell NCs.

Core/shell structure is considered to be an effective strategy to improve the optical performance of the Ln³⁺-doped β-NaREF₄ NCs.⁴¹ For traditional methods (e.g., TCD and HTCP), complicated separation and washing steps are often needed, which are labor-intensive and time-consuming.^42,43 Moreover, it is difficult to grow a controllable and uniform shell on the core NCs, especially for the large-scale NCs. Nevertheless, monodisperse large-scale NaREF₄:Ln³⁺@NaREF₄ core/shell NCs with a uniform shell can be produced without sacrificing the narrow size distribution via SLTD (Fig. 3a). After the synthesis of core NCs, the shell precursor was added directly into the solution containing core NCs. It is worth noting that the presence of core NCs may reduce the energy barrier for the formation of a β-NaREF₄ shell. At 250 °C, uniform β-NaGdF₄:Yb,Er@NaYF₄ NCs were acquired with a yield of 63.38 g in a one-pot reaction (Fig. 3b–d), which is more than 30 times greater than the highest yield reported previously.⁴⁴ Note that the SLTD method is also applicable for other core/shell NCs, for instance, 58.58 g of β-NaYF₄:Yb,Er@NaYF₄ core/shell NCs (Fig. S8†). To the best of our knowledge, such large-scale synthesis of monodisperse Ln³⁺-doped NaREF₄ core/shell NCs had never been achieved before.

The successful coating of a uniform NaYF₄ shell on the NaGdF₄:Yb,Er core NCs can be verified by the TEM image of the as-prepared β-NaGdF₄:Yb,Er@NaYF₄ NCs (Fig. 3b and S9†). After coating a NaYF₄ layer, the particle size increased from 23.5 ± 1.5 nm to 27.8 ± 1.7 nm, indicative of the formation of a 2.2 nm layer coating on the inner core (Fig. 3e). The electron energy-loss spectroscopy (EELS) analysis also revealed that Gd³⁺ ions were distributed in the inside region of the NCs while Y³⁺ ions were located in the outside layer (Fig. S9†). The synthesized β-NaGdF₄:Yb,Er@NaYF₄ core/shell NCs can be well dispersed in cyclohexane, which displayed intense green emission upon irradiation with a 980 nm laser (Fig. 3c). Compared with the core NCs, the UCL and DSL emissions of core/shell NCs were significantly enhanced by 17 and 11 times, respectively (Fig. 3f). The enhanced UCL and DSL are attributed to the protection of an inert shell layer, which greatly hinders the energy migration to the surface defects or other quenchers.^45,46 The protection effect of the shell layer can be further verified by the prolonged fluorescence lifetime of Er³⁺ (Fig. S10†).

To validate that the synthesized NCs can be used as feasible bioprobes, we employed them for in vitro PSA assay and in vivo NIR imaging. To begin with, we removed the surface oleate ligands of the as-prepared NCs via an acid-washing treatment to render them hydrophilic (Fig. S11†). Then, the ligand-free NCs were functionalized with PAA, which were confirmed by FTIR spectra (Fig. S12†), TGA (Fig. S13†), DLS and zeta potential (Fig. S14†). Subsequently, antibodies were conjugated onto the PAA-capped NCs through an EDC/NHS coupling reaction (Fig. S15†). Finally, the UCL bioassay of PSA was performed in a typical heterogeneous sandwich-type bioassay (Fig. 4a). The concentration of PSA was quantified by measuring the UCL signal of the PSA antigen-conjugated β-NaGdF₄:Yb,Er@NaYF₄ nanoprobes. As shown in Fig. 4b and c, the UCL signal gradually increased with the concentration of PSA. Specifically, the calibration curve for the concentration of PSA showed a logarithmic dependence in the range of 2.1–500 ng mL⁻¹. The limit of detection, defined as the concentration that corresponds to three times the standard deviation above the signal measured in the control experiment, was thus determined as 1.8 ng mL⁻¹. It was reported that the PSA level in the serum of normal humans is below 4 ng mL⁻¹, and above 10 ng mL⁻¹ in prostate cancer patients.⁴⁷ As such, the large-scale synthesized UC nanoprobes show promising applications for monitoring PSA as well as other disease markers. Meanwhile, both the excitation (980 nm) and emission (1450–1650 nm) of β-NaGdF₄:Yb,Er@NaYF₄ nanoprobes are located in the NIR window, which can effectively suppress the scattering of photons and avoid autofluorescence.¹² Besides, the potential cellular toxicity of DSPE-PEG-capped β-NaGdF₄:Yb,Er@NaYF₄ NCs was also assessed for the HELF cells. The viability percentages of HELF cells were higher than 90% even when the concentration of the DSPE-PEG-capped β-NaGdF₄:Yb,Er@NaYF₄ NCs reached 500 μg mL⁻¹ (Fig. S16†), which reveals the remarkably low cytotoxicity of the DSPE-PEG-capped β-NaGdF₄:Yb,Er@NaYF₄ NCs. Therefore, we performed in vivo mouse imaging (Fig. 4d–f) based on the DSPE-PEG-capped β-NaGdF₄:Yb,Er@NaYF₄ nanoprobes via tail vein injection. After 10 s of blood circulation, the blood vessels of the mouse can be clearly distinguished with a high signal-to-noise ratio of 12 (Fig. 4e and f), indicating that the large-scale synthesized NaGdF₄:Yb,Er@NaYF₄ nanoprobes exhibited deep tissue penetration and high spatial resolution for in vivo imaging.

4. Conclusions

In summary, we have presented a general SLTD strategy for the controlled synthesis of a series of Ln³⁺-doped NaREF₄ NCs, and afforded monodisperse β-phase NaSmF₄, NaEuF₄, NaGdF₄, NaTbF₄ and NaYF₄ NCs. The novel strategy, proved to circumvent the limitations of traditional approaches, can be readily applied in the scale-up synthesis of Ln³⁺-doped NaREF₄ core and core/shell NCs. Specifically, a record-high yield of 63.38 g of uniform β-NaGdF₄:Yb,Er@NaYF₄ core/shell NCs with narrow size variation (<7%) has been successfully achieved. These core/shell NCs, exhibiting intense UCL and DSL emissions upon 980 nm excitation, have been revealed as excellent nanoprobes for sensitive in vitro PSA assay and in vivo NIR imaging. The developed SLTD method can be further extended to the large-scale preparation of other inorganic NCs, and thereby may pave the way for the exploration of lanthanide nanoprobes for versatile clinical applications in commercial bioassay kits or fluorescent bioimaging.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB20000000 and XDA09030307), the 973 program of the MOST (No. 2014CB845605), the NSFC (No. 11774345 and 21771185), the CAS/SAFEA International Partnership Program for Creative Research Teams, the Fujian Provincial Natural Science Foundation (No. 2017I0018, 2017J01105, and 2018J01089) and the Youth Innovation Promotion Association of CAS (No. 2014264).

References

1. Y. Liu, Y. Lu, X. Yang, X. Zheng, S. Wen, F. Wang, X. Vidal, J. Zhao, D. Liu, Z. Zhou, C. Ma, J. Zhou, J. A. Piper, P. Xi and D. Jin, Nature, 2017, 543, 229–233 CrossRef PubMed .
2. F. Wang, S. Wen, H. He, B. Wang, Z. Zhou, O. Shimoni and D. Jin, Light: Sci. Appl., 2018, 7, 18007 CrossRef .
3. L. Sun, R. Wei, J. Feng and H. Zhang, Coord. Chem. Rev., 2018, 364, 10–32 CrossRef .
4. S. Chen, A. Z. Weitemier, X. Zeng, L. He, X. Wang, Y. Tao, A. J. Y. Huang, Y. Hashimotodani, M. Kano, H. Iwasaki, L. K. Parajuli, S. Okabe, D. B. L. Teh, A. H. All, I. Tsutsui-Kimura, K. F. Tanaka, X. Liu and T. J. McHugh, Science, 2018, 359, 679–684 CrossRef PubMed .
5. R. Gao, C. Hao, L. Xu, C. Xu and H. Kuang, Anal. Chem., 2018, 90, 5414–5421 CrossRef PubMed .
6. Q. Li, X. Li, L. Zhang, J. Zuo, Y. Zhang, X. Liu, T. Langping, B. Xue, Y. Chang and X. Kong, Nanoscale, 2018 10.1039/C8NR00446C .
7. M.-H. Chan, Y.-T. Pan, Y.-C. Chan, M. Hsiao, C.-H. Chen, L. Sun and R.-S. Liu, Chem. Sci., 2018, 9, 3141–3151 RSC .
8. B. Shen, S. Cheng, Y. Gu, D. Ni, Y. Gao, Q. Su, W. Feng and F. Li, Nanoscale, 2017, 9, 1964–1971 RSC .
9. A. Qu, X. Wu, L. Xu, L. Liu, W. Ma, H. Kuang and C. Xu, Nanoscale, 2017, 9, 3865–3872 RSC .
10. Q. S. Qin, P. Z. Zhang, L. D. Sun, S. Shi, N. X. Chen, H. Dong, X. Y. Zheng, L. M. Li and C. H. Yan, Nanoscale, 2017, 9, 4660–4664 RSC .
11. Q. Zhan, H. Liu, B. Wang, Q. Wu, R. Pu, C. Zhou, B. Huang, X. Peng, H. Agren and S. He, Nat. Commun., 2017, 8, 1058 CrossRef PubMed .
12. Y. Zhong, Z. Ma, S. Zhu, J. Yue, M. Zhang, A. L. Antaris, J. Yuan, R. Cui, H. Wan, Y. Zhou, W. Wang, N. F. Huang, J. Luo, Z. Hu and H. Dai, Nat. Commun., 2017, 8, 737 CrossRef PubMed .
13. X. Zhu, W. Feng, J. Chang, Y. Tan, J. Li, M. Chen, Y. Sun and F. Li, Nat. Commun., 2016, 7, 10437 CrossRef PubMed .
14. R. Wang, X. Li, L. Zhou and F. Zhang, Angew. Chem., Int. Ed., 2014, 126, 12282–12286 CrossRef .
15. M. Haase and H. Schafer, Angew. Chem., Int. Ed., 2011, 50, 5808–5829 CrossRef PubMed .
16. D. Peng, Q. Ju, X. Chen, R. Ma, B. Chen, G. Bai, J. Hao, X. Qiao, X. Fan and F. Wang, Chem. Mater., 2015, 27, 3115–3120 CrossRef .
17. J.-C. Boyer and F. C. J. M. van Veggel, Nanoscale, 2010, 2, 1417–1419 RSC .
18. J. Peng, W. Xu, C. L. Teoh, S. Han, B. Kim, A. Samanta, J. C. Er, L. Wang, L. Yuan, X. Liu and Y. T. Chang, J. Am. Chem. Soc., 2015, 137, 2336–2342 CrossRef PubMed .
19. X. Hu, Y. Wang, H. Liu, J. Wang, Y. Tan, F. Wang, Q. Yuan and W. Tan, Chem. Sci., 2017, 8, 466–472 RSC .
20. C. Zhang, Y. Yuan, S. Zhang, Y. Wang and Z. Liu, Angew. Chem., Int. Ed., 2011, 50, 6851–6854 CrossRef PubMed .
21. M. K. Tsang, W. Ye, G. Wang, J. Li, M. Yang and J. Hao, ACS Nano, 2016, 10, 598–605 CrossRef PubMed .
22. P. Huang, D. Tu, W. Zheng, S. Zhou, Z. Chen and X. Chen, Sci. China Mater., 2015, 58, 156–177 CrossRef .
23. X. Lin, X. Chen, W. Zhang, T. Sun, P. Fang, Q. Liao, X. Chen, J. He, M. Liu, F. Wang and P. Shi, Nano Lett., 2018, 18, 948–956 CrossRef PubMed .
24. D. Yang, P. Ma, Z. Hou, Z. Cheng, C. Li and J. Lin, Chem. Soc. Rev., 2015, 44, 1416–1448 RSC .
25. Q. Xiao, X. Zheng, W. Bu, W. Ge, S. Zhang, F. Chen, H. Xing, Q. Ren, W. Fan, K. Zhao, Y. Hua and J. Shi, J. Am. Chem. Soc., 2013, 135, 13041–13048 CrossRef PubMed .
26. Y. Dai, H. Xiao, J. Liu, Q. Yuan, P. Ma, D. Yang, C. Li, Z. Cheng, Z. Hou, P. Yang and J. Lin, J. Am. Chem. Soc., 2013, 135, 18920–18929 CrossRef PubMed .
27. X. Ding, J. Liu, D. Liu, J. Li, F. Wang, L. Li, Y. Wang, S. Song and H. Zhang, Nano Res., 2017, 10, 3434–3446 CrossRef .
28. H. Dong, L.-D. Sun and C.-H. Yan, Chem. Soc. Rev., 2015, 44, 1608–1634 RSC .
29. J. C. Bünzli, Chem. Rev., 2010, 110, 2729–2755 CrossRef PubMed .
30. 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 RSC .
31. H. J. Kwon, K. Shin, M. Soh, H. Chang, J. Kim, J. Lee, G. Ko, B. H. Kim, D. Kim and T. Hyeon, Adv. Mater., 2018, e1704290 CrossRef PubMed .
32. P. L. Saldanha, V. Lesnyak and L. Manna, Nano Today, 2017, 12, 46–63 CrossRef .
33. H. Mai, Y. Zhang, R. Si, Z. Yan, L. Sun, L. You and C. Yan, J. Am. Chem. Soc., 2006, 128, 6426–6436 CrossRef PubMed .
34. Z. Li and Y. Zhang, Nanotechnology, 2008, 19, 345606 CrossRef PubMed .
35. D. Kim, H. J. Kwon, K. Shin, J. Kim, R. E. Yoo, S. H. Choi, M. Soh, T. Kang, S. I. Han and T. Hyeon, ACS Nano, 2017, 11, 8448–8455 CrossRef PubMed .
36. R. Komban, J. P. Klare, B. Voss, J. Nordmann, H.-J. Steinhoff and M. Haase, Angew. Chem., Int. Ed., 2012, 51, 6506–6510 CrossRef PubMed .
37. H. Mai, Y. Zhang, L. Sun and C. Yan, J. Phys. Chem. C, 2007, 111, 13730–13739 Search PubMed .
38. G. Chen, T. Y. Ohulchanskyy, R. Kumar, H. Agren and P. N. Prasad, ACS Nano, 2010, 4, 3163–3168 CrossRef PubMed .
39. H. Li, L. Xu and G. Chen, Molecules, 2017, 22, 2113 CrossRef PubMed .
40. T. Rinkel, A. N. Raj, S. Duhnen and M. Haase, Angew. Chem., Int. Ed., 2016, 55, 1164–1167 CrossRef PubMed .
41. Y. Liu, D. Tu, H. Zhu and X. Chen, Chem. Soc. Rev., 2013, 42, 6924–6958 RSC .
42. F. Wang, R. Deng and X. Liu, Nat. Protoc., 2014, 9, 1634–1644 CrossRef PubMed .
43. W. Shao, G. Chen, A. Kuzmin, H. L. Kutscher, A. Pliss, T. Y. Ohulchanskyy and P. N. Prasad, J. Am. Chem. Soc., 2016, 138, 16192–16195 CrossRef PubMed .
44. S. Wilhelm, M. Kaiser, C. Wurth, J. Heiland, C. Carrillo-Carrion, V. Muhr, O. S. Wolfbeis, W. J. Parak, U. Resch-Genger and T. Hirsch, Nanoscale, 2015, 7, 1403–1410 RSC .
45. N. J. Johnson, S. He, S. Diao, E. M. Chan, H. Dai and A. Almutairi, J. Am. Chem. Soc., 2017, 139, 3275–3282 CrossRef PubMed .
46. Q. Chen, X. Xie, B. Huang, L. Liang, S. Han, Z. Yi, Y. Wang, Y. Li, D. Fan, L. Huang and X. Liu, Angew. Chem., Int. Ed., 2017, 56, 7605–7609 CrossRef PubMed .
47. J. Xu, S. Zhou, D. Tu, W. Zheng, P. Huang, R. Li, Z. Chen, M. Huang and X. Chen, Chem. Sci., 2016, 7, 2572–2578 RSC .

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Footnote

† Electronic supplementary information (ESI) available: Table S1 and Fig. S1–S16. See DOI: 10.1039/c8nr03252a

This journal is © The Royal Society of Chemistry 2018