Fluorescent small Au nanodots prepared from large Ag nanoparticles for targeting and imaging cancer cells

Abstract

Developing an approach for targeting and detecting cancer cells has long been a challenge. Herein, the folic acid (FA) conjugated gold nanodots (Au NDs) are designed for specifically targeting and imaging folate receptor (FR) positive cancerous cells. Fluorescent small Au NDs were synthesized using stable Ag NPs with size above 50 nm and GSH as capping agent. This method solves the problems of low stability and difficulty in purification of small Ag NDs as templates. The resulting Au NDs display pink fluorescence with an emission peak at 582 nm. As-prepared Au NDs provide tunable fluorescence with a significant red shift (∼60 nm) with increase in the amount of GSH. In addition, GSH acts as a protecting layer, which could provide original functional groups (thiol, carboxyl and amine) and make Au NDs exhibit outstanding properties such as dispersibility in water, high stability, good biocompatibility and surface bioactivity. These characteristics make them suitable for further conjugation with FA. FA-conjugated Au NDs show the specific target HeLa cancer cells compared with 293T normal cells. These properties provide Au NDs with potential applications in distinguishing FR-positive cancer cells from normal cells.

---

1 Introduction

An exciting new trend in bio/nanotechnology is the development of novel fluorescent nanomaterials that are more suitable for biological and targeted imaging.¹ Fluorescent metal nanoclusters (NCs), as a new kind of fluorescent nanomaterial, such as gold, copper, platinum and silver, have attracted much attention.^2–5 These metal NCs with small size and discrete energy levels bridged the “missing link” between atomic and nanoparticles (NPs) behavior and exhibited molecule-like electronic transitions within the conduction band, which brought about interesting optical properties, especially strong fluorescent emission.^6,7 Similar to quantum dots (QDs), fluorescent metal NCs exhibited tunable fluorescence from the visible to near-infrared regions due to molecule-like electronic transitions within the conduction band or charge-transfer transition from a ligand to metal NCs.^8–10 Besides, these NCs exhibited high photoluminescence quantum yields (QYs) and stability, and could be easily prepared.¹⁰ In addition, fluorescent gold nanodots (Au NDs) have attracted much attention due to their unique physical and chemical properties, including photoluminescence, magnetic properties and low toxicity, and practical applications in various areas such as sensors, bioimaging and catalysis.^11–13

Various synthesis routes to fluorescent Au NDs or NCs have been developed in the last few years, including chemical reduction, ligand etching from large Au nanoparticles, electrochemical techniques and galvanic replacement reactions.^14–17 Among these approaches, galvanic replacement reactions have been demonstrated to be a general and effective method for preparing metallic nanostructures.^18,19 Due to the high efficiency and simplicity, this method has been used in large-scale synthesis of various shaped nanostructures of noble metals such as Au, Pt, and Pd.²⁰ In addition, this method was also suitable for preparing fluorescent Au NDs and Ag/Au alloy NCs.^21,22 In order to prepare strongly fluorescent and highly stable Au NDs by a galvanic replacement reaction, small-sized (below 2 nm) Ag NDs had to be used as templates, whose stability was low.²¹ Moreover, Au NDs were difficult to separate from unreacted small Ag NDs. If stable Ag NPs with large size were used to prepare small fluorescent Au NDs,²³ the synthesis and purification process will be easy and efficient.

Although bioimaging has been developed rapidly, there is a challenging subject in targeted imaging, namely 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 receptor (FR).²⁴ FR is expressed on the surface of a variety of human cancer cells, including cancers of the mammary gland, lung, kidney and brain.²⁵ The level of FR appears to rise as the stage of the cancer increases, whereas in normal cells FR is only sparsely distributed.²⁶ These characteristics make FA a promising agent for targeting cancer cells by detecting FR. Hitherto, a number of nanomaterials with surface modification by FA have been applied in targeted bioimaging, including Cu nanomaterials, quantum dots, SnO₂ NPs and upconversion NPs.^27–30 However, the large diameters or potentially toxic effects of these functionalized nanomaterials limit their applications. Hence, the smaller-sized and low-toxicity fluorescent FA-Au NDs will become promising materials for targeting cancer cells.³¹

Here, fluorescent Au NDs were synthesized and purified from large-sized Ag NPs by a galvanic replacement method. The resulting Au NDs protected by glutathione (GSH) exhibited red fluorescence with an emission peak at 582 nm and exhibited good bioactivity and dispersion in aqueous solution. After being modified with folic acid (FA), the resulting fluorescent FA-conjugated Au NDs could be used for targeted imaging of HeLa cells, which demonstrates their potential for biomedical applications.

2 Experimental

Materials

Reduced glutathione (GSH, molecular weight of 307), folic acid (FA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were purchased from Aldrich. Tetrachloroauric (III) acid (HAuCl₄), silver nitrate (AgNO₃, 99%), dimethyl sulfoxide (DMSO) and citrate were of analytical grade. All reagents were used as received without further purification. Deionized water was used in all experiments.

Preparing the template of silver nanoparticles (Ag NPs)

The preparation of Ag NPs was similar to a previous citrate reducing method with a slight improvement.³² Firstly, 220 mg AgNO₃ was dissolved in 100 mL aqueous solution and the solution was boiled. Then 2 mL aqueous solution containing 10 mg citrate was added dropwise. After reacting for 1 h, the colorless, transparent solution changed to gray and large-sized Ag NPs were formed. The collected complexes were centrifuged at 7000 rpm. Purification was carried out via centrifuging at 8500 rpm and then redispersing in water three times at room temperature. Ag NPs were ultimately dissolved in 50 mL water (Ag concentration 50 mM) for the next study.

Synthesis of Au NDs by galvanic replacement reaction method

2.5 mL Ag NPs solution mentioned above was mixed with HAuCl₄ (0.5 mL, 50 mM) and GSH (100 mg). The addition of deionized water made the volume of the mixture up to 5 mL. Then 230 μL NaOH was added dropwise to the solution. The reaction was carried out at 80 °C for 2 h. Fluorescent Au NDs were synthesized and stabilized with GSH. In the reaction, some Ag NPs were oxidized to Ag⁺ ions, which would form AgCl precipitate. Purification of the resulting Au NDs was carried out by centrifuging at 12 000 rpm to remove unreacted Ag NPs and AgCl precipitate. This purification process was repeated twice at room temperature. Then the resulting Au NDs were precipitated by adding isopropanol to the solution; the complexes collected via centrifuging at 8000 rpm were then redispersed in water. This purification process was repeated three times at room temperature.

Preparation of FA-conjugated Au NDs

FA (5 mg) was dissolved in DMSO (20 mL) and activated by EDC (2.1 mg) and NHS (6 mg) under a vacuum line. Au NDs in 0.01 M phosphate buffered saline (PBS, 10 mL) were then added to the activated FA solution with continuous stirring in the dark at room temperature for 6 h. Finally, the mixture was loaded into a dialysis bag (MWCO 3500) and dialyzed against ultrapure water for three days. FA-conjugated Au NDs were then stored at 4 °C for further experiments.

Cellular imaging

HeLa cells and 293T cells 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 FA-conjugated Au NDs from the stock solution were prepared with Dulbecco's phosphate buffered saline (DPBS). After sonication for 10 min to ensure complete dispersion, an aliquot (typically 0.01 mL) of the suspension was added to a well of a chamber slide that contained the cells cultured for 24 h. The chamber slide was then incubated at 37 °C in a CO₂ incubator for 24 h for uptake of Au NDs (only 10 μg FA-conjugated Au NDs was added to 150 μL culture medium (10⁵ cells)). Prior to fixation of the cells on the slide for inspection with a confocal fluorescence microscope, excess Au NDs were removed by washing 3 times with warm DPBS.

Characterization methods

UV-vis absorption spectra were obtained using a Lambda 800 UV-vis spectrophotometer. Photoluminescence (PL) experiments were performed with a Shimadzu RF-5301 PC spectrofluorometer. X-ray photoelectron spectroscopy (XPS) using Mg Kα excitation (1253.6 eV) was carried out with a VG ESCALAB MKII spectrometer. Binding energy calibration was based on C 1s at 284.6 eV. Fourier transform infrared spectroscopy (FT-IR) was performed 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 resulting Au NDs were characterized by a JEM-2100F field emission electron microscope operating at 200 kV. Confocal microscopy images were taken by an Olympus FluoView FV1000. All measurements were performed at room temperature under ambient conditions.

3 Results and discussion

Large-sized Ag NPs that were stabilized with citrate formed a gray, translucent solution and no photoluminescence (PL) under UV light (365 nm) irradiation was observed (Scheme 1a and b). Furthermore, when glutathione (GSH) and HAuCl₄ were reacted with Ag NPs, small-sized Au NDs were obtained. The galvanic replacement reaction occurred due to the standard reduction potential of the AuCl₄⁻/Au pair (0.99 V vs. SHE) being higher than that of the Ag⁺/Ag pair (0.80 V vs. SHE).³³ By simply centrifuging to remove unreacted Ag NPs and AgCl precipitate, the aqueous solution of Au NDs appeared to be transparent and golden-yellow (Scheme 1c). Compared to Ag NPs, which showed no emissions, the aqueous solution of Au NDs exhibited strong pink fluorescence under UV light irradiation (photograph in Scheme 1d), which could be observed by the naked eye; this indicated that highly luminescent Au NDs were formed.

Scheme 1 Schematic of the synthesis of fluorescent Au NDs from large-sized Ag NPs.

The morphology of as-prepared fluorescent Au NDs was confirmed by transmission electron micrography (TEM). As shown in Fig. 1, Au NDs were spherical and their average diameter was less than 2.8 nm. The inset high-resolution TEM image shows lattice planes separated by about 0.235 nm, which corresponds to the (111) lattice spacing of face-centered cubic Au.¹⁴ Moreover, there was no formation of large-sized Au nanoparticles or aggregation due to the unique protective effect of GSH, which could be used for the preparation of small-sized NCs.³⁴ GSH, which is a small tripeptide possessing some functional groups including a free thiol, could be used as a capping agent and thus form an effective protective layer to prevent NCs from growing up to become large nanoparticles.³⁵

Fig. 1 Typical TEM image of as-prepared fluorescent Au NDs. The inset shows a close-up of the crystalline structure of an individual Au ND.

The chemical composition and chemical status of as-prepared Au NDs were determined by X-ray photoelectron spectroscopy (XPS). The binding energies of S 2p, C 1s, N 1s, and O 1s, which give evidence of GSH on the surface, appear (Fig. 2a). Besides, by comparing the FT-IR spectra of as-prepared Au NDs and pure GSH, the former only lacks the characteristic band at 2526 cm⁻¹ of free thiol due to the formation of metal–S complexes, which also indicates that GSH acted as a stabilizing agent on the surface of clusters (Fig. S3†).³⁶ The binding energy of Ag 3d (Fig. 2b) is absent, whereas the appearance of the binding energy of Au 4f, which is located at 88.1 eV and 84.3 eV (Fig. 2c), could explicitly demonstrate that the oxidation state of Au in the luminescent clusters was a combination of Au (0) and Au(I).³⁷ These results indicate that the galvanic replacement reaction occurred and AuCl₄⁻ was replaced by Au NDs completely and also confirmed that as-prepared luminescent clusters were pure Au NDs. Au NDs of such size (3 nm > d > 2 nm) displayed fluorescent properties, as a suitable S–Au(I) complex was bound to the surface of gold nanodots, which resulted in charge-transfer transition from the ligand to metal nanoparticles and could bring about high fluorescence.^10,21,38

Fig. 2 (a) XPS spectrum of resulting fluorescent Au NDs; the inset spectra show the binding energy of Ag 3d (b) and Au 4f (c).

The characteristic plasmon absorption band of Ag NPs appeared at 420 nm, as displayed in the absorption spectra of Fig. 3a, which was in accordance with previous reports.³⁹ As is well known, it is obvious that such large Ag NPs would not display fluorescence, as shown in Fig. 3b. When Ag NPs were reacted to prepare small Au NDs, UV-vis absorption spectra changed markedly. The plasmon feature at 420 nm from large Ag NPs disappeared, which implies that direct rather than gradual core oxidation of Ag happened. The formed Au NDs were very small and did not display optical absorption features (Fig. 3a). Moreover, the absence of absorption at 520 nm, which corresponds to the surface plasmon resonance absorption of large Au nanoparticles,⁴⁰ also demonstrates that small-sized Au NDs were obtained. In contrast to the absence of emission of large Ag NPs in PL spectra, the transparent aqueous solution of Au NDs exhibited excellent fluorescence properties such as strong pink fluorescence with an emission peak at 582 nm and a full width at half maximum around 80 nm (Fig. 3b). Unlike semiconductor quantum dots, these newly developed nanodots provided a clear excitation band with an excitation peak at 370 nm (Fig. S4†). The fluorescence quantum yields (QYs) reached 4% using rhodamine 6G (QYs 0.95 in ethanol) as the standard. Au NDs powder displayed an orange color under visible light (Fig. 3c) and pink luminescence under UV irradiation (Fig. 3d).

Fig. 3 (a) UV-vis absorption and (b) fluorescence spectra of aqueous solution of (1) Ag NPs and (2) Au NDs; images of Au NDs powder under (c) visible and (d) UV light.

Some experimental conditions were critical during the galvanic replacement reaction and controlled the physical properties of Au NDs. These experimental conditions, including the molar ratio of Ag to Au, reaction temperature and reaction time, played essential roles.²¹ Fig. S5† shows that the fluorescence intensity of Au NDs was enhanced with an increase in reaction temperature from 40 °C to 80 °C. With a further rise in the reaction temperature, the fluorescence intensity of Au NDs was reduced gradually. Therefore, 80 °C was identified as the optimal reaction temperature. The reaction time also had an impact on the fluorescence intensity of Au NDs (Fig. S6†). When the reaction time was prolonged from 0.5 to 2 h at 80 °C, the fluorescence intensity of Au NDs was enhanced significantly, whereas the emission wavelength did not change. By further increasing the reaction time, the fluorescence intensity of Au NDs decreased and the emission peak underwent a red shift; therefore, 2 hours was chosen as the optimal reaction time. As the standard reduction potential of the AuCl₄⁻/Au pair (0.99 V vs. standard hydrogen electrode, SHE) is higher than that of the Ag⁺/Ag pair (0.80 V vs. SHE), a galvanic replacement reaction occurred between AuCl₄⁻ and Ag (0).³³ In theory, the reaction stoichiometry is 3 : 1 (Ag/AuCl₄⁻).⁴¹ In the experimental procedure, because large-sized Ag NPs were used as a template, we tried to react all AuCl₄⁻; if a molar ratio of Ag to AuCl₄⁻ of 5 : 1 (which is more than the reaction stoichiometry of 3 : 1) were selected, AuCl₄⁻ would be completely changed to Au (0) to obtain pure Au NDs. Therefore, the fluorescence intensity of Au nanodots is higher than that of the 3 : 1 product (Fig. S7†). Moreover, unreacted large-sized Ag NPs could be removed by simply centrifuging, which made the purification of Au NDs easier. Therefore, the optimal molar ratio of Ag to Au was set at 5 : 1.

In order to obtain high-quality fluorescent Au NDs, some factors were investigated which played dominant roles in the fluorescence properties of as-prepared Au NDs. Firstly, as-prepared Au NDs displayed tunable fluorescence by varying the amount of GSH. As shown in Fig. 4, tunable PL emission was observed at room temperature and the emission peak exhibited a significant red shift (∼60 nm) from 555 nm to 615 nm with an increase in the concentration of GSH from 0.027 M to 0.133 M. To our knowledge, metal NCs were similar to quantum dots and exhibited size-dependent emission from visible red to NIR due to the quantum size effect.^42–44 Fluorescent Au NDs with different concentrations of GSH were tested by TEM. The average diameter of as-prepared Au NDs was 2.1 nm when the concentration of GSH was 0.040 M (Fig. S8a†). When the concentration of GSH increased to 0.100 M, the average diameter increased to ∼2.8 nm (Fig. 1). With a further increase in the concentration of GSH to 0.133 M, the average diameter of Au NDs was 3.1 nm (Fig. S8b†). Here, we suggest that the tunable emission wavelength that occurs with an increase in the GSH concentration results from the change in size of Au NDs.

Fig. 4 Tunable PL emission of resulting fluorescent Au NDs with different concentrations of GSH.

Secondly, the pH value of the reaction system was investigated, which had an effect on the fluorescence of Au NDs. When the pH value of the reaction system was 4.2, the galvanic replacement reaction did not occur and no fluorescent substance appeared (Fig. 5a). With an increase in the pH value from 5.4 to 9.1, the fluorescence intensity of Au NDs was enhanced markedly, which indicates that neutral and alkaline conditions are suitable for the preparation of highly fluorescent Au NDs. Moreover, the emission peak of Au NDs did not shift with an increase in the pH value of the reaction system. A further increase in pH value resulted in a rapid decrease in the emission intensity of Au NDs (Fig. S9†). Finally, the size of Ag NPs was taken into consideration. To determine its influence on fluorescence properties, three Ag NPs of various sizes were synthesized by adjusting the molar ratio of AgNO₃ to citrate (Fig. S10†). The fluorescence of as-prepared Au NDs was red-shifted from 558 nm to 595 nm and the PL intensity decreased with an increase in the size of Ag NPs from 50 nm to 78 nm (Fig. 5b). With a further increase in the size of Ag NPs to 90 nm, the fluorescence of Au NDs was blue-shifted to 575 nm (Fig. 5b). Therefore, we chose 78 nm Ag NPs as the optimal size to synthesize Au NDs based on the position of the emission peak and the fluorescence intensity. The concentration of GSH, pH value and size of Ag NPs were the three factors that controlled the fluorescence of as-prepared Au NDs. Increasing the pH value or size of Ag NPs could decrease the stability of Ag NPs, which would favor the galvanic replacement reaction to form highly fluorescent Au NDs. Besides, large Ag NPs were easily separated from small Au NDs. However, an excessively high pH value (>10.0) of the reaction system or too large a size (90 nm) of Ag NPs would reduce the quality of Au NDs.⁴⁵

Fig. 5 (a) Fluorescence spectra of resulting Au NDs prepared at different pH values; (b) fluorescence spectra of resulting Au NDs prepared from different sizes of Ag NPs: (1) 50 nm; (2) 78 nm; and (3) 90 nm.

Because GSH and the S–Au(I) complex were both attached to the surface of as-prepared fluorescent Au NDs, they played important roles in the stability of Au NDs. From our previous report, it has been proved that Au NDs with this structure display outstanding stability over a wide pH span, a long time and various metal ions.²¹ Especially, Au NDs could provide excellent fluorescence properties, which were suitable for applications of biological detection under physiological conditions. As-prepared Au NDs have many advantages including being water-dispersible, highly fluorescent, highly biocompatible and surface-bioactive, which make them possess great potential in cellular marking. Au NDs that were incubated with HeLa cells were evaluated by confocal laser fluorescence microscopy. As shown in Fig. S11,† fluorescence signals in cells could not be observed when Au NDs were incubated with HeLa cells for 4 h, but with an increase in incubation time to 12 h, there were strong fluorescence emissions, which demonstrated that Au NDs could be used as a probe for optical cellular imaging.

In order to prepare a target agent, some functional groups in the molecule of GSH, such as free carboxyl and amine groups, enabled Au NDs to exhibit good dispersion in an aqueous phase (Scheme 1c) and easy surface modification. FA was conjugated to Au NDs by carboxyl moieties of FA being coupled to amine groups of GSH, which was capped on the surface of Au NDs, via formation of an amide bond activated by EDC and NHS.⁴⁶ Fig. S12† shows FTIR results for FA-conjugated Au NDs and pure FA, which confirms that the surface of resulting Au NDs was conjugated to FA.⁴⁷

The biocompatibility of luminescent nanomaterials is an important factor. Future clinical applications of Au NDs in diagnosis and treatment of cancers are dependent on their potential cytotoxicity. In this research, a methylthiazolyl-diphenyltetrazolium (MTT) assay and an apoptosis assay were used to evaluate the cytotoxicity of Au NDs conjugated to FA and the viability of cells. From the results of the MTT assay (Fig. 6, Fig. S13†), the viability of both HeLa cells (cancer cells) and 293T cells (normal cells) still remained above 80% after they were incubated with Au NDs even at a high concentration of 50 μg mL⁻¹ for 24 h. These results demonstrated that Au NDs conjugated to FA displayed no obvious acute toxicity to cells.

Fig. 6 Viability of HeLa cells after 24 h incubation with different concentrations of fluorescent FA-conjugated Au NDs in the cell medium as determined by a MTT assay.

For further targeted bioimaging, FA-conjugated Au NDs were incubated with 293T and HeLa cells under physiological conditions. These two cell lines are known to express different levels of folate receptors (FR) on the cell surface. After the same incubation time (4 hours), the cells were washed and imaged using a confocal microscope as shown in Fig. 7. Note that HeLa cells produced bright fluorescence (Fig. 7a), which demonstrates high specific interaction between FA on FA-conjugated Au NDs and FR on cancer cells. In contrast, 293T cells that were incubated with FA-conjugated Au NDs (Fig. 7b) displayed rather weak luminescence, which suggests low non-specific binding of FA-conjugated Au NDs to normal cells. These results indicate that FA-conjugated Au NDs possess potential applications in the short term, specifically targeting and imaging FR-positive cancerous cells.

Fig. 7 Images of immunofluorescent cell imaging captured by laser scanning confocal microscopy: (a) HeLa cells and (b) 293T cells incubated with FA-conjugated Au NDs.

4 Conclusions

In summary, fluorescent small Au NDs were synthesized by a galvanic replacement reaction using large Ag NPs as templates and GSH as capping agent. This method can simplify the procedure of synthesis and purification. As-prepared Au NDs displayed red fluorescence with an emission peak at 582 nm and the full width at half maximum was around 80 nm. Moreover, tunable PL emission from 555 nm to 615 nm of Au NDs was observed by controlling the amount of GSH. In addition, Au NDs have outstanding properties such as water-dispersibility, high stability, good biocompatibility and surface bioactivity. These characteristics provide Au NDs with potential applications in biological labeling. In this study, the resulting Au NDs were modified by FA and FA-conjugated Au NDs could be used as a probe for specifically targeting and imaging FR-positive cancerous cells.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grants 21174048 and 51373061) and Graduate Innovation Fund of Jilin University (2014046); Dr C. X. Wang thanks the Natural Science Foundation of Jiangsu Province, China (no. BK20140157) and the Fundamental Research Funds for the Central Universities (JUSRP11418).

Notes and references

1. B. Hötzer, I. L. Medintz and N. Hildebrandt, Small, 2012, 8, 2290.
2. J. Zheng, J. T. Petty and R. M. Dickson, J. Am. Chem. Soc., 2003, 125, 7780.
3. W. T. Wei, Y. Z. Lu, W. Chen and S. W. Chen, J. Am. Chem. Soc., 2011, 133, 2060.
4. S. I. Tanaka, J. Miyazaki, D. K. Tiwari, T. Jin and Y. Inouye, Angew. Chem., Int. Ed., 2011, 123, 451.
5. W. W. Guo, J. P. Yuan, Q. Z. Dong and E. K. Wang, J. Am. Chem. Soc., 2010, 132, 932.
6. Y. Z. Lu and W. Chen, Chem. Soc. Rev., 2012, 41, 3594.
7. I. Díez and R. H. A. Ras, Nanoscale, 2011, 3, 1963.
8. L. Shang, S. J. Dong and G. U. Nienhaus, Nano Today, 2011, 6, 401.
9. S. Choi, R. M. Dickson and J. H. Yu, Chem. Soc. Rev., 2012, 41, 1867.
10. J. Zheng, C. Zhou, M. X. Yu and J. B. Liu, Nanoscale, 2012, 4, 4073.
11. Y. Chen, H. P. Zhou, Y. Wang, W. Y. Li, J. Chen, Q. Lin and C. Yu, Chem. Commun., 2013, 49, 9821.
12. M. X. Yu, C. Zhou, J. B. Liu, J. D. Hankins and J. Zheng, J. Am. Chem. Soc., 2011, 133, 11014.
13. G. Li and R. C. Jin, Acc. Chem. Res., 2013, 46, 1749.
14. L. Shang, N. Azadfar, F. Stockmar, W. Send, V. Trouillet, M. Bruns, D. Gerthsen and G. U. Nienhaus, Small, 2011, 7, 2614.
15. R. J. Zhou, M. M. Shi, X. Q. Chen, M. Wang and H. Z. Chen, Chem.–Eur. J., 2009, 15, 4944.
16. B. S. González, M. J. Rodríguez, C. Blanco, J. Rivas, M. A. López-Quintela and J. M. G. Martinho, Nano Lett., 2010, 10, 4217.
17. C. X. Wang, L. Xu, X. W. Xu, H. Cheng, H. C. Sun, Q. Lin and C. Zhang, J. Colloid Interface Sci., 2014, 416, 274.
18. J. S. Mohanty, P. L. Xavier, K. Chaudhari, M. S. Bootharaju, N. Goswami, S. K. Pal and T. Pradeep, Nanoscale, 2012, 4, 4255.
19. C. X. Wang, H. Cheng, Y. Q. Sun, Z. Z. Xu, H. H. Lin, Q. Lin and C. Zhang, Microchim. Acta, 2015, 182, 695.
20. G. D. Moon, S. W. Ko, Y. H. Min, J. Zeng, Y. N. Xia and U. Jeonga, Nano Today, 2011, 6, 186.
21. C. X. Wang, Y. Wang, L. Xu, X. D. Shi, X. W. Li, X. W. Xu, H. C. Sun, B. Yang and Q. Lin, Small, 2013, 9, 413.
22. T. Udayabhaskararao, Y. Sun, N. Goswami, S. K. Pal, K. Balasubramanian and T. Pradeep, Angew. Chem., Int. Ed., 2012, 51, 2155.
23. L. Dhanalakshmi, T. Udayabhaskararao and T. Pradeep, Chem. Commun., 2012, 48, 859.
24. S. Santra, C. Kaittanis, J. Grimm and J. M. Perez, Small, 2009, 5, 1862.
25. Y. C. Song, W. Shi, W. Chen, X. H. Li and H. M. Ma, J. Mater. Chem., 2012, 22, 12568.
26. D. Feng, Y. C. Song, W. Shi, X. H. Li and H. M. Ma, Anal. Chem., 2013, 85, 6530.
27. C. X. Wang, H. Cheng, Y. Q. Sun, Q. Lin and C. Zhang, Chem. Nanostruct. Mater., 2015, 1, 27.
28. M. X. Zhao, H. F. Huang, Q. Xia, L. N. Ji and Z. W. Mao, J. Mater. Chem., 2011, 21, 10290.
29. H. F. Dong, J. P. Lei, H. X. Ju, F. Zhi, H. Wang, W. J. Guo, Z. Zhu and F. Yan, Angew. Chem., Int. Ed., 2012, 51, 4607.
30. F. F. Li, C. G. Li, X. M. Liu, Y. Chen, T. Y. Bai, L. Wang, Z. Shi and S. H. Feng, Chem.–Eur. J., 2012, 18, 11641.
31. C. L. Zhang, Z. J. Zhou, Q. R. Qian, G. Gao, C. Li, L. L. Feng, Q. Wang and D. X. Cui, J. Mater. Chem. B, 2013, 1, 5045.
32. J. Turkevich, P. C. Stevenson and J. Hiller, Discuss. Faraday Soc., 1951, 11, 55.
33. Y. G. Sun and Y. N. Xia, J. Am. Chem. Soc., 2004, 126, 3892.
34. C. X. Wang, L. Xu, Y. Wang, D. Zhang, X. D. Shi, F. X. Dong, K. Yu, Q. Lin and B. Yang, Chem.–Asian J., 2012, 7, 1652.
35. C. X. Wang, Y. Wang, L. Xu, D. Zhang, M. X. Liu, X. W. Li, H. C. Sun, Q. Lin and B. Yang, Small, 2012, 8, 3137.
36. I. Odriozola, I. Loinaz, J. A. Pomposo and H. J. Grande, J. Mater. Chem., 2007, 17, 4843.
37. Y. Chen, Y. Wang, C. X. Wang, W. Y. Li, H. P. Zhou, H. P. Jiao, Q. Lin and C. Yu, J. Colloid Interface Sci., 2013, 396, 63.
38. Y. M. Guo, Z. Wang, H. W. Shao and X. Y. Jiang, Analyst, 2012, 137, 301.
39. D. G. Shchukin, I. L. Radtchenko and G. B. Sukhorukov, ChemPhysChem, 2003, 4, 1101.
40. J. Li, J. Wu, X. Zhang, Y. Liu, D. Zhou, H. Z. Sun, H. Zhang and B. Yang, J. Phys. Chem. C, 2011, 115, 3630.
41. Y. G. Sun and Y. N. Xia, Nano Lett., 2003, 3, 1569.
42. C. I. Richards, S. M. Choi, J. C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y. L. Tzeng and R. M. Dickson, J. Am. Chem. Soc., 2008, 130, 5038.
43. C. C. Huang, Z. Yang, K. H. Lee and H. T. Chang, Angew. Chem., Int. Ed., 2007, 46, 6824.
44. H. Xu and K. S. Suslick, Adv. Mater., 2010, 22, 1078.
45. I. Díez, M. Pusa, S. Kulmala, H. Jiang, A. Walther, A. S. Goldmann, A. H. E. Müller, O. Ikkala and R. H. A. Ras, Angew. Chem., Int. Ed., 2009, 48, 2122.
46. J. Y. Chang, G. Q. Wang, C. Y. Cheng, W. X. Lin and J. C. Hsu, J. Mater. Chem., 2012, 22, 10609.
47. H. Cheng, C. X. Wang, Z. Xu, H. Lin and C. Zhang, RSC Adv., 2015, 5, 20.

---

Footnote

† Electronic supplementary information (ESI) available: SEM measurement and XRD pattern of Ag NPs and FT-IR spectra, PL spectra and TEM images of Au NDs were given. See DOI: 10.1039/c5ra06946g

---