Intense near-infrared-II luminescence from NaCeF4:Er/Yb nanoprobes for in vitro bioassay and in vivo bioimaging

We report the controlled synthesis of monodisperse NaCeF4:Er/Yb nanoprobes that exhibit intense NIR-II emission for in vitro bioassay and in vivo bioimaging.

precipitated by addition of 30 mL of ethanol, collected by centrifugation, washed with ethanol several times, and redispersed in cyclohexane.

Synthesis of ligand-free NaCeF 4 :Er/Yb NCs:
Ligand-free NaCeF 4 :Er/Yb NCs were obtained by removing the surface ligands of the OA-capped counterparts through acid treatment. 1 In a typical process, 20 mg of the OA-capped NaCeF 4 :Er/Yb NCs were dispersed in 15 mL of acidic ethanol solution (prepared by adding 112 μL of concentrated hydrochloric acid to 15 mL of absolute ethanol) and stirred for 30 min to remove the surface ligands. After that, the ligand-free NaCeF 4 :Er/Yb NCs were collected by centrifugation, and further purified by adding an acidic ethanol solution (pH 4). The resulting products were washed with ethanol and distilled water several times, and redispersed in distilled water.
Synthesis of Lipo-modified NaCeF 4 :Er/Yb@NaCeF 4 :Er/Yb NCs: Because of the oleate ligands capping on the surface, the as-synthesized NaCeF 4 :Er/Yb@NaCeF 4 :Er/Yb NCs were hydrophobic. To render these NCs hydrophilic and biocompatible, we coated the surface of NCs with a monolayer of functional phospholipids. 2 5 mg of OA-capped NaCeF 4 :Er/Yb@NaCeF 4 :Er/Yb NCs were added into a chloroform solution (3 mL) containing 20 mg of DSPE-PEG phospholipid in a round-bottom flask (5 mL), and the mixture was sonicated for 2 min. Then, the mixture was dried in a rotary evaporator under reduced pressure at 60 °C to form a lipid film on the inside wall of the flask. The lipid film was hydrated with ultrapure water (4 mL), and the NCs became soluble after vigorous sonication for 30 min.
The excess lipids were removed from Lipo-NCs by ultracentrifugation (26600 g, 30 min), washing, and redispersed in distilled water. H 2 O 2 detection based on NaCeF 4 :Er/Yb nanoprobes: Typically, 100 μL of ligand-free NaCeF 4 :Er/Yb NCs (0.5 mg/mL) was mixed with 100 μL of deionized water containing different amounts (0-10 μM) of H 2 O 2 in a 96-well microplate. After incubation at 37 °C for 2 h, the microplate was subjected to photoluminescent (PL) measurement under 980-nm excitation on a fluorescence spectrometer coupled with a multimodal microplate reader.
Uric acid detection based on NaCeF 4 :Er/Yb nanoprobes: 100 μL of UA with different concentration was added to 100 μL of H 3 BO 3 -NaOH buffer solution (10 mM, pH 8.5) containing ligand-free NaCeF 4 :Er/Yb NCs (0.5 mg/mL) and uricase (0.011U/mL) in a 96-well microplate. After incubation at 37 °C for 3 h, the microplate was subjected to PL measurement under 980-nm excitation. For S3 comparison, control experiments were conducted by replacing UA with other analytes (e.g., metal ions, proteins, carbohydrates and amino acids) under otherwise identical conditions. Uric acid detection in serum sample based on NaCeF 4 :Er/Yb nanoprobes: For UA detection in human serum, a calibration curve for UA was first built based on UA depleted human serums. The collected serum samples were pretreated with Sulphur acid to deplete the protein and then NEM was added to eliminate the interference from reducing agents. The treated serum samples were then incubated with H 3 BO 3 -NaOH buffer solution (10 mM, pH 8.5) containing ligand-free NaCeF 4 :Er/Yb NCs (0.5 mg/mL), uricase (0.011 U/mL), and different amounts (0-900 μM) of UA in a 96-well microplate. After incubation at 37 °C for 3 h, the microplate was subjected to spectra measurement upon 980-nm excitation. For real serum sample detection, the samples with different concentration of UA were pretreated with Sulphur acid and diluted by 100 fold with H 3 BO 3 -NaOH buffer solution.
Three independent experiments were carried out to yield the average value and deviation.
In vivo bioimaging measurements: The in vivo upconversion (UC) imaging experiments were performed on an IVIS Lumina II imaging system with an external 0~2 W adjustable 980-nm continuous-wave (CW) laser as the excitation source. In vivo NIR imaging was performed with a modified NIR vana 640 CCD camera (Princeton Instruments Inc.), with quantum efficiency of ~85% for 1530 nm. A 980-nm CW semiconductor laser was used as the excitation source, in combination with two long-pass optical filters (1000LP from Chroma Corp and 1200LP from thorlabs). A NIR-CCD Xeva-1.7-640 Xenics Co. with a band pass filter of 1538±82 nm (semrock FF01-1538/82-25) was used as the signal collector. NIR luminescence signals were analyzed with Kodak Molecular Imaging Software. For in vivo imaging, 1 mL of water soluble NCs (0.1 mg/mL) was imbued to the tail of a nude mouse by using gastric syringe. All the animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee of Fudan University and performed in accordance with institutional guidelines on animal handling.
Characterization: Powder X-ray diffraction (XRD) patterns of the samples were collected with an Xray diffractometer (MiniFlex2, Rigaku) with Cu Kα1 radiation (λ = 0.154187 nm). Both the low-and high-resolution transmission electron microscopy (TEM) measurements were performed by using a JEOL-2010 TEM equipped with the energy dispersive X-ray (EDX) spectrum. Downshifting (DS) and UC emission spectra were carried out upon 980-nm excitation provided by a CW semiconductor laser.
Thermogravimetric analysis (TGA) experiments were conducted on a Netzsch STA449C thermal analysis system under N 2 atmosphere at a rate of 10 °C/min. Zeta potential and hydrodynamic diameter S4 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. The absolute quantum yield (QY) of NaCeF 4 :Er/Yb NCs was measured at RT by employing a barium sulfate coated integrating sphere (Edinburgh) as the sample chamber that was mounted on the FLS920 spectrometer with the entry and output port of the sphere located in 90 geometry from each other in the plane of the spectrometer. All the spectral data collected were corrected for the spectral response of both the spectrometer and the integrating sphere.
Photographs of the NCs solution were taken by a Canon digital camera without using any filter. The homogeneous assays of H 2 O 2 and UA were carried out on a custom-built microplate reader. Table   Table S1. Comparison of the UA levels in 24 human serum samples determined based on ligand-free NaCeF 4 :Er/Yb nanoprobes and commercial kit (mean ± standard deviation (SD), coefficient of variation (CV), n = 3).  NCs. The ζ-potential for ligand-free NaCeF 4 :Er/Yb NCs dispersed in aqueous solution (pH 6.9) was determined to be 21.9 ± 0.9 mV due to the existence of positively charged Ln 3+ ions (i.e., Er 3+ , Yb 3+ and Ce 3+ ) on the surface of ligand-free NCs. By contrast, the ζ-potential for Lipo-modified NaCeF 4 :Er/Yb@NaCeF 4 NCs was determined to be -11.7 ± 0.8 mV, as a result of the negatively charged DSPE-PEG phospholipid bound on the NC surface. Besides, the ligand-free NaCeF 4 :Er/Yb NCs had a HD of 23.6 ± 0.9 nm, which is similar to the size of OA-capped NaCeF 4 :Er/Yb NCs (25.2 ± 2.7 nm). Meanwhile, the Lipo-modified NaCeF 4 :Er/Yb@NaCeF 4 NCs had a HD of 22.3 ± 1.1 nm, which is a little larger than that of OA-capped NaCeF 4 :Er/Yb@NaCeF 4 NCs (18.1 ± 1.9 nm) due to the coating of DSPE-PEG phospholipid on the NC surface.