Multicolor In Vivo Imaging of Upconversion Nanoparticles with

ARTICLE pubs.acs.org/JPCC

Multicolor In Vivo Imaging of Upconversion Nanoparticles with Emissions Tuned by Luminescence Resonance Energy Transfer Liang Cheng,† Kai Yang,† Mingwang Shao,†,* Shuit-Tong Lee,‡ and Zhuang Liu†,* †

Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials, Soochow University, Suzhou, Jiangsu, 215123, China. ‡ Center of Super-Diamond and Advanced Films (COSDAF) and Department of Physics and Materials Science, City University of Hong Kong, Hong Kong SAR, China.

bS Supporting Information ABSTRACT: Upconversion nanoparticles (UCNPs) based on sodium yttrium fluoride (NaYF4) nanocrystals are synthesized, functionalized with an amphiphilic polymer, and loaded with fluorescent and quenching molecules by physical adsorption. The formed supramolecular UCNPdye complexes show tuned visible emission spectra owing to the luminescence resonance energy transfer (LRET) from nanoparticles to the organic dyes under near-infrared (NIR) excitation, and can be well separated in multicolor imaging after spectral decovolution. Our work provides a facile and flexible method to modulate the upconversion luminescence (UCL) spectra of UCNPs for in vivo multicolor UCL imaging in animals.

’ INTRODUCTION Optical imaging has become an essential tool in biological and biomedical research. Traditional optical imaging relying on single-photon fluorescence, although it has been widely used for in vitro and in vivo imaging of biological samples, has various inherent limitations.1-3 Recently, near-infrared (NIR)-to-visible upconversion nanoparticles (UCNPs) have attracted significant interest from different fields including biomedicine.4-10 Compared with down-conversion fluorescent materials such as organic fluorescent dyes and inorganic quantum dots, UCNPs exhibit many advantages including higher photostability, minimum photodamage to living organisms, and deeper light-penetration in tissues because NIR excitation is used.11,12 Moreover, the UCNP based upconversion luminescence (ULC) imaging is free of background autofluorescence, allowing high detection sensitivity.8,11-13 In the past few years, a number of groups have used UCNPs are novel optical nanoprobes for various biosensing and bioimaging applications4,6,14,15 Prasad et al. have reported in vivo whole body imaging of mice injected with UCNPs.16 Using targeting ligand conjugated UCNPs, Li et al. have achieved efficient in vivo tumor targeting and UCL imaging.14,15 The same team further showed that functionalized ultrasmall UCNPs were excreted from mice after intravaneous injection, without causing noticeable toxicity to the treated animals.17 In a recent work by our group, we uncovered that the imaging sensitivity of UCNPs appeared to be significantly better than that of quantum dots (QDs) in a side-by-side comparison experiment.13 By varying the Ln3þ dopants during UCNP synthesis, the UCL emission r 2011 American Chemical Society

spectra of nanoparticles could be well tuned, enabling multicolor UCL imaging in biological systems.13,18,19 Fluorescence resonance energy transfer (FRET) is an optical process in which the energy is transferred from a donor at its excited state to a nearby ground-state acceptor. Efficient FRET process requires the donor and acceptor molecules in very close proximity, and the donor emission spectrum to overlap with the acceptor absorption.20 In recent years, FRET-based analytical methods have gained considerable attention as powerful tools for biological detections.4,21 When UCNPs are used as the energy donor, the upconversion luminescence upon NIR excitation of UCNPs is transferred to donor molecules, a mechanism named as luminescence resonance energy transfer (LRET) similar to FRET. LRET from UCNPs to various types of acceptors including gold nanoparticles,4,22,23 organic dyes,24 and quantum dots24,25 have recently been reported. Xu and co-workers developed an immunoassay of goat antihuman immunoglobulin G antibody based on LRET between UCNPs and gold nanoparticles.22 Zhang and co-workers used silica coated UCNPs encapsulated with organic dyes or QDs for LRET modulation of UCL emission spectra.24 A later work by the same group developed a sensitive UCNP based LRET system to study the releasing behaviors of siRNA molecules in live cells.25 However, to our best knowledge, multicolor in vivo imaging based on LRET of UCNPs has not yet been reported. Received: November 18, 2010 Revised: December 25, 2010 Published: January 20, 2011 2686

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The Journal of Physical Chemistry C Multicolor multiplexed imaging to simultaneously map out different molecular targets is an important trend in the current development of molecular imaging.26-30 In this work, we develop a facile way to generate different colors of UCNPs utilizing LRET for in vivo UCL imaging. Er3þ/Yb3þ-doped NaYF4 nanocrystals are functionalized with a polyethylene glycol (PEG) drafted amphiphilic polymer to afford water solubility and biocompatibility. Several types of organic fluorescent dye and quencher molecules are physically attached on the surface of PEGylated UCNPs via hydrophobic interactions, a supramolecular chemistry approach similar to what we employed to load chemotherapy drugs on UCNPs as reported in our latest work.31 The emssion spectra of UCNPs are thus tuned by the LRET from nanoparticles to the adsorbed dye molecules (part a of Figure 1). As many as five UCNP colors with different UCL emission spectra upon a 980 nm laser excitation are obtained by this method and can be readily distinguished by spectral deconvolution using a Maestro optical imaging system. In vivo multicolor UCL imaging is demonstrated by imaging mice subcutaneously injected with those UCNP-dye complexes.

’ EXPERIMENTAL SECTION Materials. All chemicals involved in this work were analytical grade and used without further purification. Y2O3, Yb2O3, Er2O3, and Tm2O3 were purchased from Shanghai Chemical Industrial Co. All the rare-earth trifluoroacetates were prepared by dissolving the respective rare-earth oxides in trifluoroacetic acid (CF3COOH, Shanghai Chemical Industrial Co.). Oleic acid (OA, 90%), poly (maleic anhydride-alt-1-octadecene) (PMHC18) and 1-octadecene (ODE >90%) were purchased from Sigma-Aldrich. The 5k mPEG-NH2 polymer was purchased from Suzhou PEGBio Inc. Synthesis of NaYF4:Yb, Er (Tm) Nanoparticles. In a typical procedure: 1 mmol of Re (CF3COO)3 (Y: Yb: Er (Tm) = 78%: 20%: 2%), 2 mmol of CF3COONa, and 20 mL solvent (10 mL OA/10 mL ODE) were brought to a 100 mL three-necked flask simultaneously and degassed at 100 °C for 1 h under vacuum. In the presence of nitrogen, the mixture was rapidly heated to 320 °C and kept at this temperature for 30 min under vigorous magnetic stirring. After cooling down to the room temperature, the product was precipitated by addition of ethanol, separated by centrifugation, and washed repeatedly with ethanol and water. The yielded nanoparticles could be redispersed in various nonpolar organic solvents. Surface Modification of NaYF4:Yb, Er (Tm) Nanoparticles. Synthesis of mPEG-PMHC18. mPEG-PMHC18 polymers were synthesized following a previous protocol.32-34 In brief, 71.5 mg of 5k mPEG-NH2 were reacted with 5 mg PMHC18 (PEG/ MHC18 monomer molar ratio = 1:1) in 2 mL dichloromethane for one day in the presence of 10 mg of N-(3-dimethylaminopropyl)-N0 ethylcarbodiimide hydrochloride (EDC, Sigma-Aldrich) and 6 μL triethylamine. The reaction solution was blown-dry by nitrogen, yielding a solid product that was then dissolved in water. The polymer solution was dialyzed against water using a 14 kDa cutoff membrane and then lyophilized. The final product was PEG-PMHC18. 1H NMR (400 MHz, CDCl3) δ: 3.8-3.5 ppm (m, br, CH2 of mPEG), 1.3-1.1 ppm (m, CH2 of C18 chains), 0.88 ppm (m, br, CH3 of PMHC18). The average number of PEG groups bound to PMHC18 chains was determined by 1H NMR in CDCl3, comparing the integrated signal at 3.8-3.5 ppm (broad, CH2 of PEG) with that at 1.1-1.3 ppm

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Figure 1. UCNP-based LRET system. (a) A scheme of our UCNP-LRET system; the hydrophobic layer between the inorganic nanoparticle surface and the external PEG coating allows adsorption of organic dye molecules on UCNPs; (b) a TEM image of PEGylated UCNP1 in water, insert: a high-resolution TEM image of UCNP1; (c) UV-vis spectra of RhB, R6G, and TQ1 in superposition of the UCL spectrum of UCNP1. The green UCL emission band of the UCNP1 donor well overlaps with absorption bands of the three acceptor molecules.

(CH2 of C18 chains). The effective PEGylation degree (on the basis of available carboxyl groups) of 10%-5kPEG-PMHC18 was found to be 89.2%. Functionalization of UCNPs. A 500 μL stock solution of UCNPs nanoparticles was precipitated by centrifuge with the supernatant discarded. The nanoparticles were rinsed twice with ethanol and dispersed in 2 mL chloroform. A second solution of 5 mg polymer (mPEG-PMHC18) in 2 mL chloroform was then added. After blowing-dry chloroform, the residue can be readily dissolved in water. The resultant solution was filtered through a 0.22 μm syringe filter to remove large aggregates. Characterization. The phase and crystallography of the product were characterized by using a Shimadzu XRD-6000 X-ray diffractometer equipped with Cu KR radiation (λ = 0.15406 nm). A scanning rate of 0.05os-1 was applied to record the pattern in the 2θ range of 10-80°. The scanning electron microscopy (SEM) images were taken by using a FEI Quanta 200F scanning electron microscope. Transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) images of the nanocrystals were obtained using a Philips CM300 transmission electron microscope operating at an acceleration voltage of 200 kV. Fourier-transform infrared (FTIR) absorption spectra of the products were recorded by using a ProStar LC240 IR spectrometer at 4 cm-1 resolution, over a wavenumber range of 400-4000 cm-1. TG-DTA measurement of the products was performed in SETARAM-TGA 92 in the temperature range from room temperature to 500 °C at a heating rate of 10 °C min-1. Upconversion fluorescence spectra were obtained on a FluoroMax 4 luminescence spectrometer (HORIBA Jobin Yvon) with an external 980 nm laser diode (1W, continuous wave with 1 m fiber, Beijing Hi-Tech Optoelectronics CO., Ltd.) as the excitation source. UV/vis spectra were obtained with PerkinElmer Lambda 750 UV/vis spectrophotometer. LRET Based on UCNPs. Two milliliters of UCNPs functionalized by PEG-PMHC18 was diluted 8 mL phosphate buffer 2687

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Figure 2. Photos of the UCNP/dye before and after centrifugation. Centrifugation force pulled down UCNPs together with dye molecules, leaving clear colorless supernatant. In marked contrast, no noticeable precipitation was observed after centrifugation of solutions of free dye molecules or PEG-PMHC18 þ dye mixtures. Those photos suggested that three types of dye molecules were adsorbed on the UCNP surface instead of being encapsulated by the amphiphilic PEG-PMHC18 polymer in the UCNP-dye complexes.

(PB). The resulting 10 mL solution was in turn separated into five equal 2 mL aliquots to which the appropriate amount of dye (RhB, R6G, or TQ1) (20, 40, 60, 80, 100 μL) was added. After 30 min incubation with stirring at room temperature in the dark, excessive dye molecules were removed from the solution by centrifugation and water washing (three times). UCL spectra of the final dye-loaded UCNPs were recorded. Animal Experiments and In Vivo Imaging. Athymic nude mice (∼20 g) were obtained from Suzhou Belda Bio-Pharmaceutical Co. and performed under protocols approved by Soochow University Laboratory Animal Center. For subcutaneous injection and UCL imaging, aqueous solutions (15 μL, 1 mg/mL) of UCNP1, UCNP2, UCNP1/RhB, UCNP1/R6G, and UCNP1/TQ1 in 0.9% NaCl were subcutaneously injected into the back area of nude mice (∼20 g). After being anaesthetized by isoflurane inhalation, mice were then imaged by a Maestro in vivo fluorescent imaging system using a 980 nm optical fiber-coupled laser as the excitation source. The laser power density was ∼0.2 W/cm2 during imaging, which was a safe power according to previous in vivo photothermal therapy studies.8 An 850 nm short-pass emission filter was applied to prevent the interference of excitation light to the CCD camera. In vivo spectral imaging from 450 to 850 nm (10 nm step) was carried out with an exposure time of 1000 ms for each image frame. Note that the exposure time required for UCL imaging of UCNPs is generally much longer than that required for fluorescence imaging, due to the rather low quantum yield of UCNPs.35 Multicolor images were obtained by introducing spectra of individual UCNP complexes for spectra unmixing using the software attached to the Maestro in vivo imaging system.

’ RESULTS AND DISCUSSION Er3þ/Yb3þ- and Er3þ/Tm3þ-doped NaYF4 UCNPs emitting green (UCNP1) and NIR (UCNP2) light upon the 980 nm excitation respectively were synthesized following a literature procedure with slight modifications. Transmission electron microscopy (TEM) images of UCNP1 (part b of Figure 1) revealed that those nanoparticles had an average diameter of ∼34 nm (Figure S3 of the Supporting Information). High-resolution TEM showed the lattice fringes with an observed d spacing of 0.52 nm (insert of part b of Figure 1), which was in good agreement with the lattice spacing in the (100) planes of hexagonal NaYF4 (Figure S1 of the Supporting Information). Similar electron microscopy results were also observed for UCNP2 sample (Figures S2 and S4 of the Supporting Information). Assynthesized UCNPs coated by oleic acid were not soluble in water. A PEG modified poly (maleic anhydride-alt-1-octadecene) (PEG-PMHC18) was synthesized based on a literature method and used to transfer the hydrophobic UCNPs into the aqueous phase. The coating of PEG-PMHC18 on UCNPs was formed by hydrophobic interactions between hydrocarbon chains of the PMHC18 and the oleic acid layer on the nanoparticle surface. After polymer coating, these nanocrystals were well dispersible in water with excellent stability (Figure S5 of the Supporting Information). The successfully PEGylation of UCNPs was also evidenced by IR spectra and thermogravimetric analysis (Figures S6 and S7 of the Supporting Information). The hydrodynamic diameters of UCNP samples were measured to be ∼55 nm by dynamic light scattering (DLS), suggesting that PEGylated UCNPs were mostly monodispersed in water (Figure S8 of the Supporting Information). 2688

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Figure 3. Efficiencies of LRET in relationship with UCNP:dye ratios. (a, c, e) The absorption qualities of RhB (a), R6G (b), and TQ1 (c) on PEGylated UCNP1 at different dye concentrations. (b, d, f) UCL spectra of three UCNP/dye complexes with different loadings of RhB (b), R6G (d), and TQ1 (f). Dye molecule concentrations: black, 0 μM; red, 5 μM; green, 10 μM; blue, 20 μM; cyan, 25 μM. Insert of (b) and (d): The corresponding magnification of the UCL spectra between 540 to 640 nm.

The UCL emission spectra of two types of UCNPs in aqueous solutions were recorded by a fluorometer using a 980 nm laser as the excitation light (Figure S9 of the Supporting Information). UCNP1 exhibited green and red emission bands at ∼520-545 nm and ∼660 nm, respectively; whereas UCNP2 showed a dominate NIR emission peak at ∼805 nm. An absolute absence of signal was noted in the region of 550-650 nm where many organic fluorescent dyes emit. UCNP1 was selected as the LRET donor with its green emission energy transferred to dye molecules (part a of Figure 1). Two fluorescent molecules, Rhodamine B (RhB) and Rhodamine 6G (R6G) with different fluorescence emission peaks, and one quencher, Tide Quencher 1 (TQ1), all with optical absorption in the range of 510-560 nm, were thus chosen as acceptors in our UCNP-LRET system (part c of Figure 1). We found that dye molecules (RhB, R6G, TQ1) could be noncovalently adsorbed to the surface of PEGPMHC18 functionalized UCNPs upon simple mixing (Figure 2), via hydrophobic interactions with the oleic acid layer on the inorganic nanoparticle surface (part a of Figure 1). Control experiments excluded the possibility of polymer micelle encapsulation of dyes in our UCNP-dye systems (Figure 2). The close proximity between dye molecules and nanoparticles facilitates energy transfer between each other. The LRET donor, UCNPs, can be excited using a NIR laser at 980 nm where the

organic dyes have no absorption, thereby completely avoiding direct excitation of the LRET acceptor by the external light. We next studied the LRET effect in our UCNP-organic dye system by measuring UCL emission spectra of UCNP1 loaded with RhB, R6G, and TQ1 at varying UCNP/dye ratios. UCNP1 solutions at 0.2 mg/mL were incubated with different concentrations (0, 5, 10, 20, and 50 μM) of RhB, R6G, and TQ1 at room temperature for 5 min. After washing to remove excess dye molecules by repeated centrifugation, the amounts of dyes adsorbed on UCNPs were determined by their characteristic UV-vis absorption peaks (parts a, c, and e of Figure 3). While the dye load increases, the intensity of green UCL emission peak at 545 nm decreased gradually, whereas the RhB and R6G emissions at 585 and 565 nm respectively occurred via the resonance energy transfer from UCNP1 to those LRET acceptor molecules (parts b and d of Figure 3). As a commonly used fluorescence quencher, TQ1 loaded on UCNP1 caused a significant quenching of green emission without giving a new emission peak (part f of Figure 3). The red emission of UCNP1 at 660 nm was not affected after loading of different molecules since those organ dyes had no absorption at this wavelength. We further tested the stability of our UCNP-FRET systems in the biological environment by incubating three UCNP-dye complexes in serum over 24 h. On the basis of the UCL spectra, it was found that the dye loading on 2689

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Figure 4. Multicolor UCL imaging of UCNP-dye complexes. (a-c) Multicolor UCL images of three UCNP-dye complexes and a mixture of the three. The images were obtained by the Maestro in vivo imaging system after spectral unmixing. (d) A merged UCL image. Solution 1, UCNP1/RhB; solution 2, UCNP1/R6G; solution 3, UCNP/TQ1; solution 4, the mixture. Note that the solution 4 was an equal mixture of solutions 1-3. Therefore the concentration of each UCNP/dye complex in the solution 4 was diluted, resulting in its observed weaker individual colors. (e) UCL emission spectra of UCNP1/RhB, UCNP1/R6G, UCNP/TQ1, and the mixture, recorded by the Maestro in vivo imaging system.

Figure 5. Multicolor in vivo UCL imaging of LRET-tuned UCNPs in mice. (a) UCL emission spectra of solutions of UCNP1, UCNP2, UCNP1/RhB, UCNP1/R6G and UCNP1/TQ1 recorded by the Maestro in vivo imaging system under the 980 nm NIR laser excitation. Insert: down-conversion fluorescence spectra of RhB and R6G under green light excitation. (b-g) In vivo multicolor UCL images of a nude mouse subcutaneously injected with five colors of UCNPs solutions after spectral unmixing. Different colors were individually separated in (b-f) and merged in (g). (h) A white light image of the imaged mouse. (h-j) In vivo multicolor down-conversion fluorescence images of the mouse under green light excitation. Fluorescence of RhB and R6G from UCNP1/RhB and UCNP1/R6G complexes were detected and differentiated also by spectral unmixing.

UCNPs, although by simple physical adsorption without covalent bonding, appeared to be fairly stable in serum with only small spectral changes, likely correlated to the partial detachment of dye molecules from nanoparticles or possible photobleaching of those organic dyes (Figure S10 of the Supporting Information). The good stability of our UCNP-dye complexes allow us to use them as spectrally modified contrast agents for multicolor imaging, at least when long-term imaging and tracking is not required. Although tuned by LRET, the different UCNP-dye complexes still showed certain overlap in their emission spectra (e.g., the red emission). To verify the capability of spectral unmixing to

well differentiate various UCNP-dye colors, we imaged aqueous solutions of UCNP1/RhB, UCNP1/R6G, UCNP1/TQ1, and a mixture of these three UCNP-dye complexes by a modified Maestro in vivo spectral imaging system. A 980 nm optical fibercoupled laser was used as the excitation source and an 850 nm short-pass filter was placed in front of the CCD camera to cut the excitation light (Figure S11 of the Supporting Information). A spectral image was taken by scanning the emission wavelength from 450 to 850 nm with 10 nm for each step. The emission spectra of three individual UCNP-dye complexes were then introduced to deconvolute the acquired spectral image by linear fitting, a classical method widely used in multicolor spectral 2690

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The Journal of Physical Chemistry C imaging previously.13,26,27,29,30 It was found that the three colors from UCNP1/RhB, UCNP/R6G, and UCNP1/TQ1 were clearly separated with minimal cross-talk (Figure 4) after spectra unmixing, allowing us to further use them for multicolor in vivo imaging. Next, we used LERT tuned UCNPs for in vivo imaging. UCNP1 without dye loading and the NIR emitting UCNP2 were also introduced for additional two more colors (Figures S4 and S8 of the Supporting Information). UCL emission spectra from a total of five solutions were recorded by spectral imaging (part a of Figure 5) using the Maestro system, showing distinctive spectral patterns that allow convenient spectral unmixing. To demonstrate the feasibility of in vivo UCL multicolor imaging based on LRET, we subcutaneously injected five different UCNPs solutions (UCNP1, UCNP1/RhB, UCNP1/R6G, UCNP1/TQ1, and UCNP2) into the back of nude mice, which were imaged afterward using the Maestro system with the 980 nm laser as the excitation light (power density = ∼0.2 W/cm2). UCL emission spectra of five UCNP solutions were introduced to unmix the obtained spectral images, showing clearly distinguished colors at their corresponding injection sites without noticeable interference between different colors (parts b-h of Figure 5). Green, blue, magenta, yellow, and red were artificially assigned to UCNP1, UCNP1/RhB, UCNP1/R6G, UCNP1/TQ1, and UCNP2, respectively. Down-conversion fluorescence from RhB and R6G in UCNP/RhB and UCNP/R6G were also imaged by green light excitation (523 nm) and differentiated by spectral unmixing (insert of part a of Figure 5, parts i-k of Figure 5). These results suggest the promise of our UCNP-LRET system for future UCL multiplexed molecular imaging of different targets simultaneously.

’ CONCLUSIONS In summary, a UCNP-LRET system is developed in this work by adsorption of fluorescence molecules and quenchers on the nanoparticle surface. Modulated UCL emission spectra were obtained via the efficient resonance transfer of energy from UCNPs to organic dyes under the NIR light excitation. In vivo five-color UCL imaging using UCNPs and dye-loaded UCNPs were achieved in a proof-of-concept animal experiment by spectral deconvolution. To our best knowledge, this is the first success of in vivo multicolor UCL imaging of UCNPs based on the LRET mechanism. Many other different down-conversion organic fluorescent dyes and inorganic quantum dots could also be coupled with UCNPs for LRET-based multiplexing UCL imaging to obtain even more imaging colors. Compared with varying Ln3þ doping compositions to tune the UCNP spectra, which requires much synthetic effort and is limited by the number of available Ln3þ doping elements,13 the UCNP-LRET system is easy to fabricate by simple mixing and has much more spectral flexibilities due to a wide range selections of LRET acceptors. ’ ASSOCIATED CONTENT

bS

Supporting Information. Experimental details and UCNP characterization data, and a photo of the imaging setup. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (Z.L.), [email protected] (M.S.).

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’ ACKNOWLEDGMENT This work was partially supported by the National Natural Science Foundation of China (51002100, 51072126), a National “973” Program of China (2011CB911002), and Research Grants Council of Hong Kong SAR - CRF Grant (CityU5/CRF/08). Liang Cheng was supported by the Innovation Program of Graduate Students in Jiangsu Province (CX10B_036Z). ’ REFERENCES (1) Wagnieres, G. A.; Star, W. M.; Wilson, B. C. Photochem. Photobiol. 1998, 68, 603–632. (2) Aubin, J. E. J. Histochem. Cytochem. 1979, 27, 36–43. (3) Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7, 626–634. (4) Wang, L.; Yan, R.; Huo, Z.; Wang, L.; Zeng, J.; Bao, J.; Wang, X.; Peng, Q.; Li, Y. Angew. Chem., Int. Ed. 2005, 44, 6054–6057. (5) Downing, E.; Hesselink, L.; Ralston, J.; Macfarlane, R. Science 1996, 273, 1185–1189. (6) Kumar, R.; Nyk, M.; Ohulchanskyy, T. Y.; Flask, C. A.; Pras, P. N. Adv. Funct. Mater. 2009, 19, 853–859. (7) Wang, F.; Han, Y.; Lim, C. S.; Lu, Y.; Wang, J.; Xu, J.; Chen, H.; Zhang, C.; Hong, M.; Liu, X. Nature 2010, 463. (8) Yi, G. S.; Chow, G. M. Adv. Funct. Mater. 2006, 16, 2324–2329. (9) Liu, C.; Wang, H.; Li, X.; Chen, D. J. Mater. Chem. 2009, 19, 3546–3553. (10) Mai, H.; Zhang, Y.; Si, R.; Yan, Z.; Sun, L.; You, L.; Yan, C. J. Am. Chem. Soc. 2006, 128, 6426–6436. (11) Cao, T.; Yang, T.; Gao, Y.; Yang, Y.; Hu, H.; Li, F. Inorg. Chem. Commun. 2010, 13, 392–394. (12) Wang, F.; Banerjee, D.; Liu, Y. S.; Chen, X. Y.; Liu, X. G. Analyst 2010, 135, 1839–1854. (13) Cheng, L.; Yang, K.; Zhang, S.; Shao, M.; Lee, S. Nano Res. 2010, 3, 722–732. (14) Xiong, L.; Chen, Z.; Tian, Q.; Cao, T.; Xu, C.; Li, F. Anal. Chem. 2009, 81, 8687–8694. (15) Xiong, L. Q.; Chen, Z. G.; Yu, M. X.; Li, F. Y.; Liu, C.; Huang, C. H. Biomaterials 2009, 30, 5592–5600. (16) Nyk, M.; Kumar, R.; Ohulchanskyy, T. Y.; Bergey, E. J.; Prasad, P. N. Nano Lett. 2008, 8, 3834–3838. (17) Xiong, L. Q.; Yang, T. S.; Yang, Y.; Xu, C. J.; Li, F. Y. Biomaterials 2010, 31, 7078–7085. (18) Kobayashi, H.; Kosaka, N.; Ogawa, M.; Morgan, N. Y.; Smith, P. D.; Murray, C. B.; Ye, X.; Collins, J.; Kumar, G. A.; Bell, H.; Choyke, P. L. J. Mater. Chem. 2009, 19, 6481–6484. (19) Wang, F.; Liu, X. J. Am. Chem. Soc. 2008, 130, 5642–5643. (20) Gill, R.; Zayats, M.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 7602–7625. (21) Clapp, A. R.; Pons, T.; Medintz, I. L.; Delehanty, J. B.; Melinger, J. S.; Tiefenbrunn, T.; Dawson, P. E.; Fisher, B. R.; O’Rourke, B.; Mattoussi, H. Adv. Mater. 2007, 19, 1921–1926. (22) Wang, M.; Hou, W.; Mi, C.-C.; Wang, W.-X.; Xu, Z.-R.; Teng, H.-H.; Mao, C.-B.; Xu, S.-K. Anal. Chem. 2009, 81, 8783–8789. (23) Zhang, S.-Z.; Sun, L.-D.; He Tian, a. Y. L.; Wang, J.-F.; Yan, C.H. Chem. Commun. 2009, 2547–2549. (24) Li, Z.; Zhang, Y.; Jiang, S. Adv. Mater. 2008, 20, 4765–4769. (25) Jiang, S.; Zhang, Y. Langmuir 2010, 26, 6689–6694. (26) Kobayashi, H.; Koyama, Y.; Barrett, T.; Hama, Y.; Regino, C. A. S.; Shin, I. S.; Jang, B.-S.; Le, N.; Paik, C. H.; Choyke, P. L.; Urano, Y. ACS Nano 2007, 1, 258–264. (27) Longmire, M.; Kosaka, N.; Ogawa, M.; Choyke, P. L.; Kobayashi, H. Cancer Sci. 2009, 100, 1099–1104. (28) Kobayashi, H.; Longmire, M. R.; Ogawa, M.; Choyke, P. L.; Kawamoto, S. Lancet Oncol. 2010, 11, 589–595. (29) Chan, W. C. W.; Maxwell, D. J.; Gao, X. H.; Bailey, R. E.; Han, M. Y.; Nie, S. M. Curr. Opin. Biotechnol. 2002, 13, 40–46. (30) Liu, Z.; Tabakman, S.; Sherlock, S.; Li, X. L.; Chen, Z.; Jiang, K. L.; Fan, S. S.; Dai, H. J. Nano Res. 2010, 3, 222–233. 2691

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