Facile Synthesis of Ultrasmall and Hexagonal NaGdF4

Facile Synthesis of Ultrasmall and Hexagonal NaGdF4...
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J. Phys. Chem. C 2010, 114, 21077–21082

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Facile Synthesis of Ultrasmall and Hexagonal NaGdF4: Yb3+, Er3+ Nanoparticles with Magnetic and Upconversion Imaging Properties Jiyoung Ryu,|,† Hye-Young Park,|,† Keumhyun Kim,† Heeyeon Kim,† Jung Ho Yoo,§ Moonsik Kang, Kangbin Im,‡ Regis Grailhe,‡ and Rita Song*,† Nano/Bio Chemistry Laboratory, Institut Pasteur Korea (IP-K), 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea; Applied Microscope Laboratory, Institut Pasteur Korea (IP-K), 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea; and Measurement & Ananlysis Team, National Nanofab Center (NNFC), 355 Gwahakno, Yuseong-Gu, Daejeon-si, South Korea ReceiVed: July 26, 2010; ReVised Manuscript ReceiVed: October 8, 2010

Synthesis of nanomaterials with multi-imaging modality is of great importance in clinical molecular imaging and diagnostics. This work reports novel synthetic strategy to create ultrasmall and hexagonal upconversion nanoparticles (UCNPs), β-NaGdF4: Yb3+, Er3+, and β-NaGdF4: Yb3+, Tm3+, with inherent magnetic and efficient upconversion properties. The use of new combination of lanthanide chloride and sodium TFA as the precursors for UCNPs gave the best results in terms of size (10-40 nm), crystallinity and morphology, and proved to be cost- and time-saving. Water solubilization of both NaGdF4: Yb3+, Er3+, and β-NaGdF4: Yb3+, Tm3+ UCNPs was achieved by homogeneous polymer coating using amphiphilic poly(acrylic acid) derivatives. The strong upconversion and magnetic properties were maintained after extensive polymer coating process. To see the potential of the UCNPs for biological applications, the surface of NaGdF4: Yb3+, Er3+ UCNPs were functionalized with Ni-nitrilotriacetate (NiNTA) moiety. The remarkable specificity of these NiNTAUCNPs for the oligohistidine peptide was clearly shown by both magnetic resonance and optical imaging. Finally, the cellular uptake of these UCNPs was investigated by fluorescence microscope using spectral imaging technique. 1. Introduction Biological applications of nanoparticles have gained considerable interest over the past years with the rapid development of the synthetic strategies for nanomaterials with controlled size, shape, and composition.1 In particular, there has been growing interest to create nanoprobes for non invasive in vivo imaging such as magnetic resonance imaging (MRI) or near-infrared (NIR) optical imaging.2 The use of NIR light for in vivo imaging provides several advantages such as deep penetration and minimal autofluorescence, photobleaching, and phototoxicity. Among NIR emitting fluorophores, new I-III-VI type of NIR quantum dots (QDs), for example, CuInS2 QDs were recently reported.3 The range of the emission of those QDs can be reached to 1000 nm. However, when exited with NIR light source, the efficacy of emission considerably decreases. Alternatively, upconversion nanoparticles (UCNPs), lanthanide-doped rare earth nanomaterials, have been known to convert a NIR light into a visible emission through the upconversion process.4 Recently, UCNPs have been reported for their application for in vivo imaging using their unique optical properties.5 Compared to organic fluorophores and quantum dots, UCNPs exhibit high photochemical stability, sharp emission bandwidths, and large antistokes shifts. In addition, high penetration depth and absence of autofluorescence due to the excitation of NIR source made them ideal materials for biological labeling and in vivo imaging. Considering the great importance of the simplicity and efficiency * To whom correspondence should be addressed. Tel: +82-31-80188230. Fax: +82-31-8018-8014. E-mail: [email protected]. † Nano/Bio Chemistry Laboratory, Institut Pasteur Korea (IP-K). ‡ Applied Microscope Laboratory, Institut Pasteur Korea (IP-K). § Measurement & Ananlysis Team, National Nanofab Center (NNFC). | These authors contributed equally to this study.

in clinical diagnosis, the development of the potential dualmodal contrast agent is of particular interest. In this context, imparting magnetic properties to the upconversion nanoparticles is highly valuable approach for the molecular imaging application. However, the synthesis of those nanoparticles needed complex or inconvenient synthetic procedures.6 Recently, Muhammad et al. reported dual functional core/shell NaYF4: Yb3+, Er3+/NaGdF4 UCNPs for in vivo imaging.7 Li et al. also demonstrated hydrothermal synthetic approach for preparing NaGdF4: Yb3+, Er3+, for which water solublilization was performed by oxidation of oleic acid ligand to azelaic acid.5a The crystal structure of these particles was hexagonal phase with broad size distribution from 25-50 nm. NaGdF4: Yb3+, Er3+ UCNPs with a cubic structure have been known to exhibit low photoluminescence (PL) efficiency and necessitate additional shell formation in order to increase the PL efficiency.7,8 Alternatively, hexagonal NaYF4 UCNPs have been known to exhibit about an order of magnitude enhancement of upconversion efficiency compared to cubic ones.9 Therefore, the progress on the synthetic method to obtain high quality of hexagonal UCNPs with the biology-relevant small size should be of great importance. Here, we report a new approach for the facile synthesis of ultrasmall and hexagonal phase NaGdF4: Yb3+, Er3+, and NaGdF4: Yb3+, Tm3+ UCNPs obtained from the commercial lanthanide chlorides and sodium trifluoroacetate (TFA) precursors. The effects of surfactant concentration and reaction time on the formation of hexagonal NaGdF4: Yb3+, Er3+, and the dual optical/magnetic properties were also presented. Moreover, the feasibility as a biological labeling agent was demonstrated in in vitro cell experiment after water solubilization by amphiphilic polymer coating.

10.1021/jp107725r  2010 American Chemical Society Published on Web 11/17/2010

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2. Experimental section 2.1. Chemicals and Materials. LnCl3 (Ln ) Gd, Yb, Er, and Tm, 99.99%), sodium trifluoroacetate (98%), 1-octadecene (ODE, 90%), oleic acid (OA, 90%), Poly(acrylic acid) (PAA, MW. 1,800), octylamine (99%), tetramethyl-ammonium hydroxide (TMAH), diamino PEG (DAPEG, MW. 987) N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), dimethylsulfoxide (DMSO) were purchased from Aldrich. All chemicals were of analytical grade and used without further purification. N-hydroxysuccinimide (NHS) and N,Nbis(carboxymethyl)-L-lysine (NTA) from Fluka, 6xhistidine peptide (6His) and 5xGly-His-2xGly peptide (5GH2G) were purchased from Peptron Inc. (Daejeon, Korea). Glyoxal-agarose beads were purchased from ABT (Tampa, FL). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT, Sigma), Fetal bovine serum (FBS), penicillin-streptomycin, bovine serum albumin (BSA), phosphate buffered saline (PBS), and tris/borate/ EDTA (TBE) buffers were purchased from Invitrogen. 2.2. Synthesis of Hexagonal NaGdF4: Yb3+, Er3+ Nanocrystals. In a typical procedure, 0.8 mmol of GdCl3, 0.18 mmol of YbCl3, and 0.02 mmol of ErCl3 were mixed in a 100 mL flask. After adding 6 mL of oleic acid and 15 mL of 1-octadecene, the solution was heated to 120 °C under vacuum for 1 h and cooled to room temperature, to which 2.5 mmol sodium trifluoroacetate was then added. The temperature of the solution was increased to 300 °C under the nitrogen atmosphere and maintained for 2 h. After cooling to room temperature, the nanocrystals were precipitated with ethanol, collected after centrifugation, and redispersed in toluene. To study the effect of OA/ODE ratio on the size and morphology, the syntheses were carried out varying OA/ODE ratios (6/15, 8/12, 10/10, 14/6). Kinetic studies were also carried out by collecting aliquots of the reaction solution at different time course. The synthesis of hexagonal NaGdF4: Yb3+, Tm3+ nanocrystals was carried out by the same method above- mentioned replacing ErCl3 to TmCl3. 2.3. Polymer Coating (PC) and PEG Modification. Water solubilization of UCNPs was performed by coating the particles with amphiphilic polymer, octylamine modified PAA.10 Briefly, octylamine modified PAA (20 mg) was dissolved in 2 mL of chloroform. TMAH was added to the polymer solution to raise the pH up to 10. With continued stirring, NaGdF4: Yb3+, Er3+ and NaGdF4: Yb3+, Tm3+ (10 mg/mL) solution was added dropwise to the polymer solution in chloroform. After the reaction, chloroform was evaporated in vacuo at 80 °C to yield a thin film of the polymer coated UCNPs on the wall of the flask. Five ml of distilled water was added to the flask and centrifuged (14 000 ×g, 15 min) to remove aggregates. The excess amount of polymer was removed using ultracentrifugation (500 000 ×g, 60 min). Polymer coated UCNPs were then reacted with diamino PEG molecules via EDC coupling reaction.1 Briefly, to a solution of NaGdF4: Yb3+, Er3+ and NaGdF4: Yb3+, Tm3+ (16 mg/mL), EDC (90 µmol) and diaminoPEG (45 µmol) were added and the mixture was stirred for 2 h and then purified using the membrane filter (MWCO 100K). Finally, the pegylated- and polymer-coated NaGdF4: Yb3+, Er3+ and NaGdF4: Yb3+, Tm3+ UCNPs were dispersed in deionized water. 2.4. NiNTA Conjugation Reaction. The synthesis of NTA ligand was carried out as previously reported in the literature.11 NTA conjugation to the UCNP was achieved using a bifunctional linker, sulfo-SMCC. To the solution of DAPEG-PCNaGdF4: Yb3+, Er3+ (1.5 nmol), an excess amount of sulfoSMCC (200 equiv.) was added and reacted for 1 h followed by the addition of NTA (200 equiv.) in DMSO. After removing

Ryu et al. the unreacted NTA, NiCl2 · 6H2O (200 equiv.) was added. After stirring for 1 h at room temperature, the final NiNTA-modified UCNP were purified by membrane filtration (MWCO 100 K). 2.5. Specific Binding Study of NiNTA-Modified UCNP with 6His-Modified Bead. Two different agarose beads (AB) functionalized with different peptides were prepared with the method that we previously reported.12 This experiment allowed us to examine the potential of NiNTA-UCNPs as a site-specific fluorescent probes for the 6His-tagged proteins. The beads were modified with 2G6H or 5GH2G by reductive amination of the glyoxal group. The functionalized bead, AB-2G6H, was used as a model for cells expressing 6His on their membranes. AB5GH2G, which represents a random amino acid sequence, was used as a negative control. Both ABs were incubated with NiNTA-UCNPs for 15 min at room temperature and the unbound UCNPs were removed by washing with 10 mM PBS. The specificity of the binding was investigated by MR and fluorescence imaging. 2.6. Characterization. The analyses on the morphology and size distribution of NaGdF4: Yb3+, Er3+ were carried out by dynamic light scattering (DLS) and scanning transmission electron microscope (STEM, Philips CM-30). Absorption and emission spectra of NaGdF4: Yb3+, Er3+ and NaGdF4: Yb3+, Tm3+ were obtained by UV-vis spectrometer (CARY 5000, Varian) and fluoro-spectrophotometer (Fluorolog, Horiba Jovin Yvon) using NIR laser operated at 50-100 mW (980 nm, Daeduk optical instruments, Korea), respectively. X-ray Diffractometery (XRD) analysis was carried out using Philips XPERT XPD equipped with PW3020 goniometer (Theta/2theta), 3 kW generator (Cu anode), Nickel filter and operated using K-R1 ()1.54 Å). Ni2+ elemental analysis was performed using ELAN DRC (Perkin-Elmer) inductively coupled plasma mass spectrometer (ICP-MS, KIST, Korea) equipped with quadrupole mass analyzer. All MR imaging experiments were performed with a 4.7 T animal MRI instrument (Bruker, Germany) with a 72-mm volume coil (KBSI, Osan, Korea). Proton relaxation time was measured by the Carr-Purcell-Meiboom-Gill (CPMG) sequence at room temperature: TR 7 s, 100 echoes with 8.7 ms even echo space, number of acquisition 1, point resolution of 417-469 µm and section thickness of 2 mm. Serial dilution of PEG-PC-UCNPs (10 mg/mL) in DI water were placed in the tubes for T1-weighted MR imaging. The resulting T1 (spin-lattice relaxation time) values were averaged and plotted as 1/T1 vs Gd3+ molar concentration. The slopes of the graph provided the molar relaxavity r1. For fluorescence imaging, a fluorescence microscope equipped with near-infrared CCD camera (KP-F2A, Hitachi) and NIR (980 nm) laser operated at 50-100 mW (Daeduk Optical Instruments, Korea) was built in the laboratory. 2.7. Cell Experiment. HeLa cells were cultured at 37 °C in a humidified atmosphere and 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 IU/mL streptomycin (Invitrogen). Media were changed 2 to 3 times per week. Cell viability was measured by the MTT assay. Spectrophotometric data were obtained by measuring the absorbance using Victor 3 microplate reader (Perkin-Elmer). For cellular imaging, the HeLa cells were incubated with the UCNPs (0.5 mg/mL) for 1 h. 3. Results and Discussion 3.1. Synthesis and Characterization. A variety of synthetic strategies for NaGdF4: Yb3+, Er3+ have been recently reported reflecting the great interest for noninvasive dual imaging.7,8,13 The precursors used in the thermolysis reaction were sodium TFA and lanthanide TFA salts that were obtained from the reaction of lanthanide oxide and TFA. These reactions usually

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Figure 1. (a)TEM Bright field image, (b) HRTEM image of NaGdF4:Yb3+, Er3+, and digital diffractogram by Fourier transformed operation (inset), (c) SAD pattern, and (d) Powder XRD pattern of NaGdF4:Yb3+, Er3+ reacted in 10 mL OA/10 mL ODE for 2 h at 300 °C.

resulted in polydispersed UCNPs crystallized in cubic phase.2a,13a,14 Another variation of the synthesis is to use lanthanide chlorides and methanolic solution of NaOH and NH4F.7,9 This reaction was proven to produce relatively small UCNPs (∼20 nm) in hexagonal phase. In the present work, the new combination of lanthanide chloride and sodium TFA as the precursors for UCNPs gave the best results in terms of size and morphology, and proved to be cost and time saving. This synthetic approach demonstrated several advantages such as short reaction time, high monodispersity, high crystallinity with a hexagonal structure, ultrasmall size (∼10 nm) and the use of cheap and commercial precursors. The crystal structures of the NaGdF4: Yb3+, Er3+ obtained in the solvent mixture of OA/ODE (1:1) at 300 °C were analyzed by TEM, SAD (Selective Area Diffraction) pattern and XRD analysis. Figure 1(a) shows typical TEM bright field image of the NaGdF4: Yb3+, Er3+ UCNPs which are uniform spherical shape with a diameter of 15 ( 2 nm. High resolution TEM (HRTEM) image presented in Figure 1(b) showed the lattice distance of 0.532 nm, corresponding to d spacing for the {100} lattice plane in the hexagonal NaGdF4 structure. Moreover, from the diffractogram obtained from HRTEM image (inset of Figure 1(b)), it was clearly shown that the nanoparticles were synthesized in single crystal with hexagonal phase. NaGdF4 UCNPs has been known to be crystallized in cubic (R-phase) or hexagonal (β-phase) phases depending on the kind of precursor and reaction conditions. The UCNPs with hexagonal structure has been reported to show much higher fluorescent efficiency than cubic ones.15 In addition, the SAD pattern in Figure 1(c) shows the clear diffraction rings corresponding to the specific (100), (110), (111), and (201) plans of the hexagonal lattice, which indicates high crystallinity of this UCNP. The crystal structure of the NCs was further

identified by XRD as shown in Figure 1(d). The position of well-defined diffraction peaks of UCNPs was consistent with that of reference crystals of NaGdF4 in hexagonal structure (JCPDS 27-0699). Figure 2(a) and 2(b) show the emission and excitation spectra of the NaGdF4: Yb3+, Er3+ and NaGdF4: Yb3+, Tm3+, respectively. Both UCNPs showed characteristic emission profiles as reported previously. Especially, NaGdF4: Yb3+, Tm3+ exhibited a strong NIR emission peak at 800 nm. The photostability of as-synthesized UCNPs was investigated under the continuous laser excitation at 980 nm with power density of 500 mW/cm3 (Figure 2(c)). The intensity of the photoluminescence of the UCNPs solution did not change over two hours of continuous irradiation. This remarkable photostability can be a great beneficial property for long-term bioimaging application. In order to adapt further UCNPs to biological application, water solubilization of nanoparticles synthesized in organic solvent was performed using polymer coating method. These UCNPs were coated by amphiphilic poly(acrylic acid) derivatives and DLS analysis showed high monodispersity (See Supporting InformationS1). As seen in Figure 2(d), the UCNPs were clearly attracted by the magnet and as well showed high luminescence properties. For in vivo imaging application, a variety of near IR emitting organic dyes or quantum dots have been recently explored to overcome the phototoxicity, low penetration, and high background in the biological specimens. The shortcoming of small animal in vivo imaging using near IR emitting organic dyes or quantum dots is the usage of visible range of excitation source that is absorbed by the tissue. Our NaGdF4: Yb3+, Tm3+ UCNPs, however, were selectively excited using NIR excitation source, at 980 nm and detected at 800 nm. Such spectral characteristics in both excitation and emission

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Figure 2. (a) Emission spectra of NaGdF4:Yb3+, Tm3+ (blue line) and NaGdF4:Yb3+, Er3+ (green line). (b) Absorption spectra of NaGdF4:Yb3+, Tm3+. (c) The chloroform solution of NaGdF4:Yb3+, Er3+ showed remarkable photostability under the continuous irradiation with 980 nm laser. (d) As-prepared NaGdF4:Yb3+, Tm3+ UCNPs aggregates induced in ethanol were attracted to permanent magnet (top left). The luminescence of NaGdF4: Yb3+, Tm3+ solution (chloroform, top middle), NaGdF4:Yb3+, Er3+ solution (chloroform, top right), and polymer coated NaGdF4:Yb3+, Er3+ solution (borate buffer(pH 8.5), bottom) under the excitation of 980 nm laser.

Figure 3. TEM images of NaGdF4:Yb3+, Er3+ synthesized in the solvent mixture of OA/ODE (a) 6 mL/15 mL, (b) 8 mL/12 mL, (c) 10 mL/10 mL, and (d) 14 mL/6 mL at different reaction times. The scale bar is 20 nm.

in NIR range are particularly well adapted to thick tissues and small animal in vivo imaging. 3.2. Effects of OA/ODE Ratio on the Size and Morphology of UCNPs. It has been reported that the presence of oleic acid in the solvent plays an important role for tuning the size and morphology of UCNPs. To understand the reaction process and the effect of OA/ODE ratio in our synthetic condition, the shape evolution of NaGdF4: Yb3+, Er3+ has been investigated varying the ratios of OA/ODE at different reaction time. Figure 3 shows the TEM image of NaGdF4: Yb3+, Er3+ synthesized in various OA/ODE ratios, i.e, 6/15, 8/12, 10/10, and 14/6, at different times. XRD analysis of all NaGdF4: Yb3+, RE3+ UCNPs synthesized at 300 °C showed hexagonal crystalline structures (See the Supporting InformationS2). This result

suggests that the reaction time and the composition of the solvent have little effect in the formation of crystalline structure. On the other hand, the final size and shape of the UCNPs were strongly affected by the solution compositions (Figure 3). The size of final UCNPs at the different ratio of OA/ODE, 6/15, 8/12, 10/10, and 14/6, were 32 ( 8 nm, 22 ( 2 nm, 15 ( 2 nm, and 22 ( 3 nm, respectively. The size decreased with increasing ratio of OA until the amount of OA is reached to the same amount of ODE. When the ratio was 14/6, the UCNPs became polydispersed and the size increased greatly with the average size of 20 nm. These results clearly show that the optimum amount of OA is critical factor for producing ultasmall and monodispersed NaGdF4: Yb3+, Er3+, and NaGdF4: Yb3+, Tm3+. Moreover, OA was found to be an important capping

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Figure 4. (a) T1 weighted magnetic resonance images of UCNPs at various [Gd3+] concentrations in water. (b) Agarose bead test; (a) NiNTA-UCNPs reacted with agarose bead-2G6H (Left) and agarose bead-5GH2G (right).

surfactant affecting not only the size distribution but the morphology. When the synthesis was carried in pure ODE, nanoparticles were found to form big aggregates and the size distribution was far from the monodispersity (data not shown). On the basis of the characterization results described above, OA/ODE ratio was proven to play an important role in the formation of high quality of NaGdF4: Yb3+, Er3+, and NaGdF4: Yb3+, Tm3+ nanoparticles in our experimental condition. The insufficient amount of OA gave the UCNPs mixed with various shapes and sizes. The size and morphological evolution was also investigated at 10, 30, 60, 90, and 120 min at 300 °C. Initial nucleation of the UCNPs occurred within 10 min of the reaction time. The prolongation of the reaction time up to 2 h resulted in the monodispersed and highly luminescent UCNPs. (See the Supporting Information S3). 3.3. Magnetic and Optical Imaging Properties of UCNPs. To further evaluate the magnetic properties of NaGdF4: Yb3+, Er3+, the aqueous solution of UCNPs at various concentrations was measured for their T1 relaxation time. MR images in Figure 4 show the Gd3+ concentration dependent contrast effect of the UCNPs. UCNPs with smaller size of 10 nm exhibited higher T1 contrast enhancing effect. Linearity of the concentration dependent contrast effect is obtained by plotting 1/T1 as a function of [Gd3+] (See Supporting InformationS4). The specific relaxivities (r1) for 10 and 40 nm UCNPs were determined to be 0.99 and 0.47 mM-1s-1, respectively. The result showing the size dependent relaxivity was also reported in the literature. Park et al. claimed that the decrease in relaxivity in the larger particles is due to the reduced surface area.7(a) Specific labeling ability of UCNPs after functionalization with biorecognition molecules was further studied using agarose bead model system. The UCNPs were modified with NiNTA because NiNTA is a well-known binding molecule for oligohistidine. The binding specificity of NiNTA-UCNPs was tested employing ABs modified with 6His peptide on their surface. ABs modified with 5GH2G were used as a negative control. Figure 4(b) illustrates the representative fluorescence and MR contrast images of 6Hisand 5GH2G-modified ABs after the reaction with NiNTAUCNPs. The specific binding of NiNTA-UCNPs with 6His modified ABs was clearly visualized upon excitation with NIR laser while no visible fluorescence was observed for 5GH2Gmodified ABs. The MR contrast images also confirmed the specific binding of NiNTA-UCNPs on 6His-ABs showing the pronounced contrast effect in the T1 weighted image. These

Figure 5. Fluorescence imaging of HeLa cells incubated in the presence of UCNPs at different concentrations. (a) 0.1 mg/mL, (b) 0.5 mg/mL, (c) 1.0 mg/mL, and (d) 2.0 mg/mL. Incubation time was 1 h. The scale bar represents 20 µm.

results provide the compelling evidence that ultrasmall and hexagonal UCNPs can be used as an efficient and specific biological probe exhibiting both optical and MR contrast effect. In addition, the NaGdF4: Yb3+, Er3+ UCNPs were evaluated in vitro to examine their biolabeling capacities. First, the MTT cell proliferation assay showed the EC50 values of 402 and 225 µg/mL in 1 and 3 days of incubation, respectively (see Supporting InformationS5). In order to determine, if UCNPs could be used for cellular microscopy, we next looked at UCNPs passive staining of Human stable cells lines. The in vitro fluorescence microscopic images of UCNPs were obtained using fluorescence microscope coupled with a Ti-Sapphire laser. The excitation wavelength was set to 980 nm. Figure 5 shows increase of the cell surface cellular labeling depending on the UCNPs concentration. We found that HeLa cells became spherical at concentration above 1 mg/mL, indicative for cytotoxicity in this concentration range as investigated in MTT assay. 4. Conclusions New synthetic method for rare earth ion-dopped NaGdF4: Yb3+, Er3+ and NaGdF4: Yb3+, Tm3+ UCNPs was developed by using new combination of lanthanide chloride and sodium TFA as the precursors. This synthetic approach was proven to give ultrasmall UCNPs crystallized in hexagonal structure with high crystallinity. Both hydrophobic and water-soluble UCNPs exhibited efficient upconverting and magnetic resonance properties. These UCNPs were successfully functionalized with NiNTA moiety and showed high potential as a site-specific nanoprobes. In addition, the labeling capacity and internalization of these UCNPs was visualized in HeLa cells using spectral imaging microscope. Further specific labeling ability of these UCNPs in living cells is under investigation. Acknowledgment. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MEST), Gyeonggi-do (K204EA000001-09E0100-00110) through

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