Gold nanoparticle-enhanced near infrared fluorescent nanocomposites for targeted bio-imaging

Abstract

Low toxicity and near infrared (NIR) fluorescent nanomaterials have many advantages in biological imaging. Herein, based on metal-enhanced fluorescence (MEF), novel NIR fluorescent nanocomposites (Au/SiO₂/Ag₂S core–shell microspheres) are designed and investigated. Characterization with UV, PL, TEM, FTIR and XPS confirms the fact that NIR Au/SiO₂/Ag₂S nanocomposites were prepared. The effects on the MEF including the metal core size (gold nanoparticles), and the distance between the fluorescent molecules (Ag₂S nanoclusters (NCs)) and the metal core are systematically studied. The results show that the developed nanocomposites can effectively enhance the NIR fluorescence signal of the Ag₂S NCs. A maximum enhancement is obtained when the nanocomposites contain a 25 nm gold nanoparticle core and a 34 ± 2 nm silica spacer. SiO₂ as an important scaffold which could prevent NC aggregation and can be easily modified with functional groups (thiol, carboxyl and amine). These characteristics are beneficial for the further conjugation with folic acid (FA). FA-conjugated Au/SiO₂/Ag₂S nanocomposites show specific targeting of HeLa cells compared with 293T cells. With these properties provided, these nanocomposites have potential applications in distinguishing folate receptor-positive cancer cells from normal cells.

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Introduction

Fluorescence imaging is one of the known advanced techniques to achieve non-invasive and non-destructive visualization, and has attracted increasing attention in recent years due to its significant roles in biological and biomedical applications related to prognosis, diagnosis, and therapy of a variety of serious diseases.^1–5 Till now, there is still a challenging subject in targeted bioimaging which is to find an efficient method for distinguishing cells, especially between cancer and normal cells. Folic acid (FA) is a typical cell-targeting agent due to its high affinity for folate receptors (FRs).⁶ The FR is expressed on the surface of a variety of human cancer cells, including those of the mammary gland, lung, kidney and brain.⁷ The level of FRs appears to rise as the stage of the cancer increases, whereas, in normal cells, the FR is only minimally distributed.⁸ These characteristics make FA a promising agent for targeting cancer cells through detecting FRs. At present, various kinds of fluorescent nanomaterials modified with FA have been prepared, such as Au nanoparticles (NPs), organic dyes, quantum dots (QDs) and carbon nanodots.^9–12 Low stability or toxicity of these nanomaterials limit their application. Besides, their fluorescence in the visible region has been known to have low penetrability in vitro, which would make them unsuitable for biological applications.

The inherent near infrared (NIR) fluorescence of nanomaterials has proven to be available as a biological imaging modality, due to its emission in the biological transparency window, low auto-fluorescence and deep tissue penetration.^13,14 Although NIR semiconductor QDs are developing rapidly, the acute and chronic toxic heavy metals (such as Pb, Cd, Hg) limit their application in the biological field.^15–17 To solve this problem, the development of novel NIR nanomaterials with low or no toxicity to living tissues is urgently desired. Ag₂S NPs with a bulk band gap of 1.1 eV exhibited NIR fluorescence and low toxicity to organisms.^18,19 Reported NIR Ag₂S NPs were synthesized through various routes, but the involved preparation at high temperature and dispersion in organic solvents made them unsuitable for biological application.^20–22 Recently, NIR fluorescent Ag₂S nanoclusters (NCs) using the sulfur–hydrazine hydrate complex as the S²⁻ source were easily prepared at room temperature.²³ With these advantages, the NCs may have better potential application in biological labelling. However, this kind of Ag₂S NCs prepared in aqueous phase has low quantum yields (QYs).

Metal-enhanced fluorescence (MEF) is a unique recent method to improve the detection sensitivity of fluorescence analysis, which is based on interactions of excited-state fluorophores and plasmon resonance of NPs.²⁴ To date, a series of plasmonic nanostructured materials such as gold and silver with enhanced fluorescence has been observed.^25–27 Particularly, based on the MEF method, strongly fluorescent nanoconjugates of metal NPs and fluorescent dyes/QDs have been realized, and these composites can be used as promising fluorescent probes in biomedical detection.^28–30 Herein, we have developed new NIR fluorescent nanocomposites using Au nanoparticle–enhanced NIR fluorescent Ag₂S NCs. These Au/SiO₂/Ag₂S nanocomposites were comprised of a Au NP core, silica-spacer shell of variable thickness, and a Ag₂S NC–dispersed silica shell. Ag₂S NCs were chosen as the like-dye molecule because they show NIR fluorescence at 665 nm. Besides, the core–shell Au/SiO₂/Ag₂S nanocomposites were water-soluble and biocompatible due to the SiO₂ layer on the surface. Moreover, the size of the Au NPs and the spacer distance of SiO₂ between the metal core and the Ag₂S NCs had a large effect on the NIR fluorescence. Finally, the resultant nanocomposites were further conjugated to FA, which allowed targeted bioimaging of HeLa cells.

Experimental

Materials

Reduced glutathione (GSH, molecular weight of 307), folic acid (FA), (3-aminopropyl)trimethoxysilane (KH550), 1-ethyl-3-(3-dimethylaminopropyl)carboxylate (EDC), N-hydroxysuccinimide (NHS) were purchased from Aldrich. Tetrachloroauric(III) acid (HAuCl₄), silver nitrate (AgNO₃, 99%), sulfur sublimed, sodium citrate (Na₃Ct), polyvinylpyrrolidone (PVP-10, average molecular weight of 10 kg mol⁻¹), ammonia solution (NH₄OH, 28.0–30.0%), tetraethylorthosilicate (TEOS, 98%), hydrazine hydrate (N₂H₄·H₂O, 85 wt%), and isopropyl alcohol were of analytical grade. All reagents were used as received without further purification. Deionized water was used in all experiments.

Synthesis of NIR fluorescent Au/SiO₂/Ag₂S nanocomposites

Au nanoparticles (NPs) of various sizes were synthesized according to the Na₃Ct reduction method by changing the molar ratio of Na₃Ct to auric acid.³¹ The silica coating involved two steps: the adsorption of PVP on the surface of the Au NPs and the growth of the silica shell using the Stöber method.³² In the case of coating silica on 25 nm Au NPs, 0.1 g PVP was first mixed with 50 mL of Au NP (0.15 mM) solution under gentle stirring overnight. After removal of the excess PVP using centrifugation, 50.0 mL of 99.0% isopropanol, 20 μL of 98.0% TEOS, and 2.5 mL of NH₄OH were added into the Au NP solution. The solution was continuously stirred for one hour. The second silica layer was formed outside the silica spacer using the Stöber method. Ag₂S NCs were synthesized as mentioned above.²³ During the formation of this silica layer, the Ag₂S NCs were dispersed into the silica shell. In this process, 50 μL of 98.0% TEOS, and 1 mL Ag₂S (5 mM) were added to the previously synthesized silica-coated Au NPs. The solution was stirred in the dark for 12 h and centrifuged at 6000 rpm.

Preparation of FA-conjugated Au/SiO₂/Ag₂S nanocomposites

The as-prepared Au/SiO₂/Ag₂S and 3.5 μL KH550 were mixed in 30 mL isopropyl alcohol solution. Then, the solution was stirred in the dark for 24 h and centrifuged at 5000 rpm. The product could be collected using vacuum freeze-drying. FA could be functionalized on the surface of Au/SiO₂/Ag₂S via conjugation of the amino groups of KH550 to the carboxylates of FA using EDC/NHS. Typically, 15 mg of EDC and 9 mg of NHS were added to 2 mL of PBS buffer (pH 6.86) containing 10 mg of FA. The mixture was treated with sonication in the dark at room temperature for 30 min, and then mixed with 2 mL of an aqueous solution containing 100 mg of the Au/SiO₂/Ag₂S nanocomposites. This solution was allowed to react under magnetic stirring for 8 h. Then, the pH of the reaction mixture was adjusted to ∼9 and the solution was promptly passed through a PD-10 Column (Zeba Desalt Spin Columns provided by Thermo Scientific, India). Successful conjugation of FA to Au/SiO₂/Ag₂S was confirmed using FT-IR and fluorescence spectroscopy.

Cellular imaging

HeLa cells (210 cells per mL) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (DMEM) using a 96-well plate. Suspensions (10 μg mL⁻¹) of the FA-conjugated nanocomposites from the stock solution were prepared with Dulbecco’s phosphate buffer saline (DPBS). After sonication for 10 min to ensure complete dispersion, an aliquot (typically 0.01 mL) of the suspension was added to the well of a chamber slide containing the cells cultured for 4 h. The chamber slide was then incubated at 37 °C in a CO₂ incubator for 4 h for the Au/SiO₂/Ag₂S nanocomposite uptake (only 10 μg of FA-conjugated Au/SiO₂/Ag₂S nanocomposites to 150 μL of culture medium (105 cells) was added). Prior to the fixation of the cells on the slide for inspection with a confocal fluorescence microscope, the excess Au/SiO₂/Ag₂S nanocomposites were removed by washing 3 times with warm DPBS.

Characterization methods

UV-Vis absorption spectra were obtained using a TU-1901 UV-Vis spectrophotometer. Photoluminescence (PL) experiments were performed with a Shimadzu RF-5301 PC spectrofluorimeter. X-ray photoelectron spectroscopy (XPS) using Mg Kα excitation (1253.6 eV) was performed with a VG ESCALAB MKII spectrometer. Binding energy calibration was based on C1s at 284.6 eV. The Fourier transform infrared spectroscopy (FT-IR) was measured at wavenumbers ranging from 500 cm⁻¹ to 4000 cm⁻¹ using a Nicolet Avatar 360 FT-IR spectrophotometer. The morphology and mean diameter of the resultant nanocomposites were characterized using a Hitachi S-4800 scanning electron microscope (SEM) operating at 3 kV and JEM-2100 transmission electron microscope (TEM) operating at 200 kV. Confocal microscopy images were taken with an Olympus Fluoview FV1000. All measurements were performed at room temperature under ambient conditions.

Results and discussion

The preparation of the biocompatible and NIR fluorescent Au/SiO₂/Ag₂S nanocomposites is shown in Scheme 1. In the first step, the Au nanoparticles (NPs) were synthesized using the Na₃Ct reduction method and further protected by PVP. Then, a thin silica layer serving as the spacer was coated on the surface of the Au NPs using the Stöber method. The Ag₂S nanoclusters (NCs) were dispersed into the silica layer of the nanocomposites. This core–shell nanostructure can prevent the aggregation of Ag₂S NCs. Then, the surface of the nanocomposites was modified with amino groups by adding KH550 into the Au/SiO₂/Ag₂S solution. At last a mixture of EDC and NHS was used to cross-link NH₂–Au/SiO₂/Ag₂S with FA.

Scheme 1 Schematic illustration of the synthesis of the metal-enhanced fluorescent nanocomposites.

The structure, composition and properties of the nanocomposites can be studied using transmission electron microscopy (TEM), and X-ray photoelectron (XPS), Ultraviolet-Vis (UV-Vis) and photoluminescence (PL) spectroscopy. As shown in Fig. 1a, the nanocomposites with a core–shell structure showed good dispersion and spherical morphology with an average diameter of nearly 100 nm. The structure of the Au NPs is stable due to PVP as the protective layer on the surface. In the local magnified image of one particle (inset of Fig. 1a, a large view can be found in Fig. S1†), the Ag₂S NCs could be precisely identified. These Ag₂S NCs remained highly dispersed with a diameter of ∼1.9 ± 0.6 nm (Fig. S2†). Fig. 1b displays the UV-Vis absorption of the resultant NIR Au/SiO₂/Ag₂S nanocomposites. The aqueous solution of Au/SiO₂/Ag₂S nanocomposites showed an absorption peak at 527.3 nm, which was consistent with the previous report on Au NPs synthesized using the standard Na₃Ct reduction method.^31,33 Moreover, the elemental composition of the nanocomposites was confirmed using XPS. The binding energies of Au4f located at 88.1 eV and 84.3 eV (Fig. 1c) could clearly demonstrate the electronic structure of the Au NPs.³⁴ The binding energy of Ag3d appeared at 367.8 eV (Fig. 1d), which matched with previous reports on Ag₂S NCs.^23,35,36

Fig. 1 (a) TEM images of silica-coated AuNPs doped with Ag₂S during the formation of the second silica layer. (b) UV-Vis spectrum of the nanocomposites. XPS spectra of resultant nanocomposites: (c) Au4f and (d) Ag3d.

The PL spectra of the pure Ag₂S NCs and resultant NIR Au/SiO₂/Ag₂S nanocomposites are displayed in Fig. 2. The aqueous phase of previously synthesised Ag₂S NCs showed very weak fluorescence at 665 nm. More interestingly, the nanocomposites showed a nearly ∼28-fold enhancement because of the formation of the Au/SiO₂/Ag₂S core–shell nanostructure. According to metal-enhanced fluorescence (MEF), such nanostructures could improve the quantum yields (QYs) of Ag₂S NCs due to interactions of the excited-state Ag₂S NCs and plasmon resonance of the Au NPs. The PL QYs of the resultant nanocomposites reached up to 7.8% using Rhodamine 6G (QY: 0.95 in ethanol) as the standard. Under UV light (365 nm) irradiation, strong and red fluorescence of the nanocomposites in aqueous solution is clearly observed (inset photograph in Fig. 2). In contrast, the aqueous solution of as-prepared Ag₂S NCs gave off red fluorescence barely visible by the naked eye. According to the results of UV, PL, TEM and XPS, the NIR Au/SiO₂/Ag₂S nanocomposites were successfully synthesized.

Fig. 2 Demonstration of the fluorescence enhancement by measuring the fluorescence intensity of the 1 mM nanocomposites with (top curve) and without the metal core (bottom curves). Inset: photographs of Au/SiO₂/Ag₂S, Ag₂S and SiO₂/Ag₂S in aqueous solution under UV light. The excitation wavelength is 550 nm.

To optimize the experimental conditions for better MEF, the size of the Au NPs was varied. In this work, Au NPs were prepared using the citrate reduction method, which was the most popular and controllable method for synthesizing Au NPs. By adjusting the ratio of Na₃Ct to HAuCl₄, Au NPs with various sizes were prepared.³¹ In this article, Au NPs with the following sizes were prepared: 17 ± 2.1, 25 ± 1.8, and 28 ± 2.7 nm (Fig. S3†). With increasing the size of the Au NPs, their absorption peak slightly red-shifted, as shown in Fig. S4.† Using the Stöber method, three kinds of Au/SiO₂/Ag₂S nanocomposites with variously sized Au NPs were synthesized. Their core–shell structures were characterized using TEM. In Fig. 3a–c, the cores of the Au NPs were 17 ± 2.1, 25 ± 1.8, and 28 ± 2.7 nm, and the SiO₂ layer on the surface was nearly 32 ± 2.5 nm. These nanocomposites were very stable and well-dispersed in aqueous solution for a long time without aggregation due to the SiO₂ shell as a protective layer. Pure Au NPs are good quenchers for metal NCs due to the resonant energy transfer from the metal NCs to the Au NPs. In our experiments, the PL spectra of the three kinds of nanocomposites were measured (Fig. 3d). When the nanocomposites had a 25 ± 1.8 nm Au NP core coated with a 32 ± 2.5 nm SiO₂ shell, they showed strong fluorescence. With a similar spacer thickness, the fluorescence reduced with decreasing or increasing the Au core, as shown for 17 ± 2.1 nm or 28 ± 2.7 nm. The differences in fluorescence may be due to differences in their effective surface area. Although the Au NPs with a diameter of 17 ± 2.1 nm have a larger surface area than those of the 25 ± 1.8 and 28 ± 2.7 nm Au NPs, the effective surface area in contact with the Ag₂S NCs may not be that large, leading to a relatively low quenching efficiency. Also, the photographs show that the nanocomposites with a 25 ± 1.8 nm Au NP core had bright red fluorescence (inset of Fig. 3d).

Fig. 3 (a–c) TEM images of the nanocomposites with differently sized Au NPs; (d) fluorescence emission spectra of nanocomposites with differently sized Au NP cores ((1), 25 ± 1.8 nm; (2), 17 ± 2.1 nm; (3), 28 ± 2.7 nm). Inset: photographs of 1 mM nanocomposite solutions under UV light.

On the other hand, MEF strongly depends on the distance between the fluorescent dyes and metal NPs.²⁴ If the distance is too short, the fluorescence will be quenched. While a far distance does not meet the criteria for fluorescence enhancement. To achieve fluorescence enhancement, a suitable distance between the Au NPs and Ag₂S NCs is crucial. Therefore, the length of the spacer needs to be investigated. PVP-protected Au NPs with a size of 25 ± 1.8 nm were added to the TEOS solution. By adjusting the amount (5 μL, 10 μL, 20 μL, 50 μL, 100 μL) of TEOS before adding the Ag₂S NCs, NIR Au/SiO₂/Ag₂S nanocomposites of various diameters were prepared. Fig. 4 shows that when a 22 ± 0.8 nm spacer was employed, the nanocomposites had the weakest fluorescence. When the size of the spacer increased to 25 ± 1.7 nm, the fluorescence intensity increased. When the spacer size further increased to 32 ± 2.5 nm, the fluorescence was enhanced to its maximum. When the size of the spacer further increased, the fluorescence intensity decreased obviously. When the distance between the Ag₂S NCs and the Au NPs is too close (<35 nm), energy transfer could occur and thus quench the fluorescence signal of the NCs. Meanwhile, a far distance (>35 nm) cannot enhance fluorescence efficiently. The PL shown in Fig. S5† also displays the emission of the nanocomposites. Thereby, the fluorescence intensity depended not only on the metal core size but also on the distance between the metal core and the NCs. Therefore, these unique nanocomposites with enhanced fluorescence, based on the investigated effects on MEF, will have a better application as fluorescent labels in clinical diagnostics.

Fig. 4 Distance-dependent fluorescence enhancement of the Ag₂S NCs using the 25 ± 1.8 nm Au NP core. Inset: photographs of the nanocomposites with different SiO₂ thicknesses under UV light. The concentration of the nanocomposites is 1 mM. The excitation wavelength is 550 nm.

The SiO₂ shell of the nanocomposites has many advantages, such as low toxicity, well-established surface chemistry and biocompatibility.^37,38 The silica surface can be modified with suitable functional groups, such as thiol, carboxyl and amine groups.^39,40 Fig. 5 displays these modified nanocomposites with good dispersion and a diameter of ∼100 nm in aqueous solution after adding KH550 (Fig. 5a). Fig. 5b shows that the nanocomposites modified with FA have a similar size. The structure and PL of the modified nanocomposites have been characterized using TEM and PL spectra (Fig. 5c and d). They show that these modified nanocomposites have a core–shell structure, good dispersion and an average diameter of ∼100 nm. After modification, the NIR fluorescence properties did not change and the fluorescence signal still appeared at 665 nm. Under UV light, the deep red fluorescence could clearly be seen in the aqueous solution (inset of Fig. 5d).

Fig. 5 SEM images of the amino (a) and FA modified (b) nanocomposites. As-prepared nanocomposites after FA modification: (c) TEM image and (d) PL spectrum. The excitation wavelength is 550 nm. Inset: photograph under UV light of the 1 mM modified nanocomposites.

Comparing the FT-IR spectra of the surface-modified nanocomposites and pure FA, all the characteristic vibrational modes associated with FA can be observed (Fig. 6). The N–H stretch appeared at 3330 cm⁻¹, and the aromatic ring stretch of the pyridine and p-aminobenzoic acid moieties were observed in the range of 1476–1695 cm⁻¹.^12,41 The most characteristic bands of folic acid at 1690 cm⁻¹ (amide I) and 1572 cm⁻¹ (amide II) have become more prominent and intense in the Au/SiO₂/Ag₂S conjugates. The peaks at 1312 cm⁻¹ and 918 cm⁻¹ show the presence of aromatic C–H in-plane and out-of-plane bending, respectively.⁴² This may provide evidence for the formation of an extra amide bond during the attachment of folic acid.

Fig. 6 FT-IR spectra of (a) pure FA and (b) as-prepared Au/Ag₂S/SiO₂–FA.

FA is widely applied in cell targeting, because the FA receptor (FR) can be expressed on the surface of a variety of cancer cells while its expression is highly restricted in normal tissue.^43,44 Thus, the FA-modified fluorescent nanomaterials are highly effective in targeted bio-imaging. What is more, the future clinical application of fluorescent nanomaterials in bio-imaging eventually depends on their cytotoxicity. Cytotoxicity evaluation demonstrated that the as-prepared nanocomposites have good biocompatibility and low cytotoxicity.^45,46 In this research, a methylthiazo-lyldi-phenyltetrazolium (MTT) assay and apoptosis assay were used to evaluate the cytotoxicity of the FA-conjugated nanocomposites and the cell viability. As shown in Fig. 7a, the viability of HeLa cells maintained above 80% after incubation with the FA-nanocomposites for 24 h in the concentration range of 0–100 μg mL⁻¹, which is much higher than the standard incubating concentration and time for cellular imaging.

Fig. 7 (a) Viability of HeLa cells after 24 h of incubation with different concentrations of nanocomposites in the cell medium as determined by an MTT assay; overlay images of (b) 293T cells and (c) HeLa cells incubated with nanocomposites for 4 h. The luminescence was in the NIR region with an emission wavelength λ_max at 665 nm.

The functionalized nanocomposites have inherent advantages: NIR fluorescence, water-solubility, and low toxicity. With these advantages, the FA-modified nanocomposites can be used as a target imaging agent. The FA-conjugated nanocomposites were incubated under physiological conditions with 293T cells and HeLa cells. Both cell lines are known to express different levels of FR on the cell surface. After 4 hours of incubation, the cells were washed and imaged using a confocal microscope, as shown in Fig. 7. Fig. 7b and c show the overlay images of the 293T cells and HeLa cells incubated with FA-nanocomposites, respectively. The 293T cells display weak fluorescence due to low nonspecific binding of the FA-conjugated nanocomposites to the normal cells (Fig. 7b). In contrast, the HeLa cells show bright fluorescence (Fig. 7c), which suggests a highly specific interaction between FA on the FA-conjugated nanocomposites and FRs on the cells. These results indicate that the FA-conjugated nanocomposites could be used for specific targeting and imaging of FR-positive cancer cells.

Conclusions

In summary, metal–silica core–shell fluorescent nanocomposites with Ag₂S NCs dispersed in the outer silica shell were synthesized. The as-prepared Au/SiO₂/Ag₂S nanocomposites with a diameter of ∼100 nm displayed near-infrared fluorescence centered at 665 nm with a quantum yield of 7.8%. By controlling the experimental conditions, the maximum fluorescence enhancement was observed by applying 25 ± 1.8 nm Au NPs as the metal core with a 32 ± 2.5 nm silica spacer. Besides, the core–shell Au/SiO₂/Ag₂S nanocomposites show good water-solubility, low toxicity and good biocompatibility due to the SiO₂ shell. In addition, the surface of SiO₂ was modified with FA. The results of the MTT assay confirmed that the modified nanocomposites had no acute toxicity to cells. These advantages suggest that the nanocomposites have potential applications in biological labeling. Based on this study, the modified nanocomposites could be used as probes for labeling HeLa cells.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 50925207 and no. 51432006), the Ministry of Science and Technology of China for the International Science Linkages Program (no. 2011DFG52970), the Natural Science Foundation of Jiangsu Province, China (no. BK20140157), the Programme of Introducing Talents of Discipline to Universities (111 Project B13025), and the Fundamental Research Funds for the Central Universities (JUSRP11418).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra12066c

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