One-step facile synthesis of fluorescent gold nanoclusters for rapid bio-imaging of cancer cells and small animals - RSC Advances (RSC Publishing) DOI:10.1039/C5RA11321K View PDF VersionPrevious ArticleNext Article  DOI: 10.1039/C5RA11321K(Paper)RSC Adv., 2015, 5, 63821-63826One-step facile synthesis of fluorescent gold nanoclusters for rapid bio-imaging of cancer cells and small animals†

Wanjun Zhang, Jing Ye, Yuanyuan Zhang, Qiwei Li, Xiawei Dong, Hui Jiang and Xuemei Wang*State Key Laboratory of Bioelectronics (Chien-Shiung Wu Lab), School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, PR China. E-mail: xuewang@seu.edu.cn; Tel: +86-25-83792177

Received 14th June 2015, Accepted 20th July 2015

First published on 20th July 2015

Fluorescent bio-imaging has become a major topic of the modern biomedical research field. Fluorescent metal nanoclusters have been proposed as sensitive optical imaging probes aiming for early cancer diagnosis. We have developed a new strategy for the facile synthesis of Au-BSA nanoclusters (NCs) which have stable and bright fluorescence and could be used as a fluorescent probe for bioimaging rapidly and effectively. In this contribution, we have synthesized Au-BSA NCs at 80 °C for 10 minutes with the pH value of 11.5. At the concentration range of 0.1–10 mg mL−1, Au-BSA NCs have no obvious cell cytotoxicity effect on MCF-7, HeLa, L02, U87, and A549 cells. Then the as-prepared Au-BSA NCs were characterized by using TEM and XPS and applied for rapid tumor imaging. The biocompatible BSA stabilized fluorescent gold nanoclusters (NCs) synthesized through one-step hydrothermal reaction possess strong and bright fluorescence that can be readily utilized as a highly sensitive fluorescence probe for tumor-targeted bio-imaging in vitro and in vivo.

IntroductionThe burden of cancer is increasing in economically developing countries as a result of population aging and growth as well as, increasingly, an adoption of cancer-associated lifestyle choices including smoking, physical inactivity, and unhealthy diets.1 Cancer is still a very common disease and seriously threatens human life worldwide and how to realize the early diagnosis of cancer rapidly and effectively has become a major concern in the modern biomedical research field. The early diagnosis of cancer has been explored extensively through the expression of tumor markers at the cellular level.2 Fluorescence imaging has been widely used in the biomedical research field because of its unique advantages, such as high sensitivity, simple operation and intuitive results and so on. In recent decades, series of fluorescent nanomaterials have been proposed as sensitive optical imaging probes aiming for early cancer diagnosis, such as noble metal nanoparticles,3–5 quantum dots (QDs),6–9 fluorescent dyes,10,11 up-conversion nanoparticles,12–14 as well as polymer nanomaterials.15,16

Specific features of noble metal nanoparticles include excellent photostability, water-solubility, size-dependent colors, the lack of swelling, sharp contrast, the ease of characterization by means such as TEM or SEM, and an established surface chemistry (often thiol-based) which is useful if targeted imaging or biosensing is desired. It was relatively late when it was discovered that metal clusters made from metallic gold, silver, copper, for example, display strong intrinsic fluorescence and the surface of these clusters (gold and silver in particular) can be protected with alkanethiolate monolayers.17 Especially, various kinds of gold nanoclusters have been synthesized by coating biological ligands such as albumin from bovine serum (BSA), polyvinyl pyrrolidone (PVP), glutathione (GSH), DL-alpha-lipoic acid (DHLA) and polyethylene glycol (PEG), etc., and accordingly, different kinds of biomedical function have been developed to meet the practical and clinical needs.18 In view these observations, a one-step strategy for synthesis of fluorescent gold nanoclusters (NCs) rapidly and facilely has been explored in this study. Through hydrothermal synthesis of these albumin from bovine serum (BSA) stabilized gold nanoclusters (NCs), it is possible to provide an excellent opportunity for high-sensitive in vitro fluorescent bio-imaging of cancer cells by laser confocal scanning microscopy and in vivo fluorescent bio-imaging through small animal imaging system.

ExperimentalChemicals, materials and cellsAuric chloride acid (HAuCl4·6H2O) was purchased from Sinopharm Chemical Reagent Co., Ltd NaOH was obtained from Nanjing Chemical Reagent Co., Ltd. A 0.01 M phosphate buffer solution (PBS, pH 7.4) comprising Na2HPO4 and NaH2PO4 was used as the supporting electrolyte. DMEM (high glucose) medium, RMPI-1640 medium and trypsin were purchased from Hyclone. Albumin from bovine serum (BSA), thiazolyl blue tetrazolium bromide (MTT), streptomycin, penicillin and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (St.Louis, USA), and stored in the dark. Dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co. (China). All reagents were of analytical grade purity level, there is no need purification before use. All the solutions were prepared by Milli-Q purified water (18.2 MΩ cm).

Cells like MCF-7, HeLa and L02 cells (purchased from Cell Bank of Chinese Academy of Sciences, Shanghai) were applied in our study. MCF-7 and L02 cells were cultured in RMPI-1640 medium supplemented with 1% streptomycin/penicillin and 10% fetal bovine serum, while, HeLa cells were cultured in DMEM medium supplemented with 1% streptomycin/penicillin and 10% fetal bovine serum. All cells were placed at 37 °C in a carbon dioxide cell incubator with 5% CO2 and 95% relative humidity.

Synthesis of Au-BSA NCsAu-BSA NCs were fabricated according to a previously reported method with a slight modification.19 The HAuCl4 aqueous solution (10 mL, 10 mM) was added to BSA aqueous solution (10 mL, 50 mg mL−1) under vigorous stirring. Then, NaOH solution (1 mL, 1 M) was introduced to adjust the pH to about 11.5, and the mixture was incubated at 80 °C for 10 minutes under vigorous stirring. The relevant color of the solution changed from light yellow to light brown, and then deep brown. After that, to remove all small molecular impurity, the solution were dialyzed with a dialysis bag (molecular weight cut-off 8–14 kDa) extensively against doubly distilled water for more than 24 h with a water change every 4 h. The obtained gold nanoclusters were stored at 4 °C in dark before use.Characterization and apparatusA Shimadzu RF-5301 PC instrument was used for the fluorescence spectral measurements. The UV-Vis absorption spectroscopy measurements were carried out on Thermo BioMate 3S UV-Vis spectrophotometer. The transmission electron microscopy (TEM) images of Au-BSA NCs were collected on a JEM-2100 microscope with an accelerating voltage of 200 kV to characterize the size and size distribution. X-ray photoelectron spectroscopy (XPS) measurements were measured on a PHI-Quantera SXM system equipped with a monochromatic Al Kα X-ray under UHK (6.7 × 108 Pa). All XPS spectra were referenced to the C1s peak at 284.8 eV from the adventitious hydrocarbon contamination. Fluorescence lifetimes were obtained by using an FLS920 fluorescence spectrometer (Edinburgh Instruments Ltd, United Kingdom). Cells fluorescence imaging was collected by confocal fluorescence microscopy (Leica TCS SP2). The fluorescence images of the mice were obtained with Perkin-Elmer animal imaging system (USA, IVIS Lumina XRMS Series, with excitation wavelength of 520 nm and emission wavelength of 710 nm). Fluorescence images were recorded by using the CCD camera with constant exposure time.Toxicity evaluation in cellsMTT assay was utilized for in vitro cytotoxicity studies of Au-BSA NCs on MCF-7, HeLa and L02 cells. MCF-7, HeLa and L02 cells were trypsinized and seeded respectively in 96-well plates at a density of 6 × 103 cells per well in 0.1 mL of medium and incubated overnight under 37 °C in a 5% CO2 humidified environment. Then different concentrations (i.e., 0.1, 0.5, 1, 5, 10 mg mL−1) of Au-BSA NCs were added into the wells and cultured for 24 hours. A set of samples without the addition of fluorescent gold nanoclusters was treated as the control group. Then, 20 μL MTT solution (5 mg mL−1) was added per well and all cells were incubated for another 4 h. Next, the supernatant was removed and 150 μL DMSO was added to each well. Finally, all samples were shaken for 10 min. The cell viabilities were measured by the standard MTT assay. The optical density (OD) was measured at 490 nm with a microplate reader (MK3, Thermo Fisher), and the viability of cells could be calculated on the basis of the OD values. There are also two other kinds of cancer cells were used to demonstrate that Au-BSA NCs have good bio-compatibility (Fig. S5, ESI†).Confocal fluorescence microscopic studies in cellsFirst of all, MCF-7, HeLa and L02 cells were incubated with 1 mg mL−1 Au-BSA NCs in a 6-well culture plate for 24 h. For control experiments, DMEM (high glucose) medium or RMPI-1640 medium was used instead of Au-BSA NCs. The cells were washed three times with PBS, then added 4% p-formaldehyde for 15 min to fix the cells. Afterwards, cells were mounted on microscope slides for fluorescence imaging. In order to get the bio-imaging of the above-mentioned three cells treated with Au-BSA NCs, confocal fluorescence microscopy which is called Leica TCS SP2 was fully utilized at an excitation wavelength of 488 nm.Small animal imagingThe athymic nude mice harboring MCF-7 cells and HeLa cells (female, weighed 18–20 g, aged 4–5 weeks) were initially prepared. All animal studies were conducted according to protocols approved by the Animal Ethics Committee of Southeast University. Now, we expect to examine the feasibility of in vivo fluorescence imaging of tumors based on intratumoral injection with Au-BSA NCs. For this purpose, we relied on xenografted tumor nude mice of none injection as self-control, almost no fluorescence was observed in the control group. Then, the injection of Au-BSA NCs (10 mg mL−1, 100 μL) was performed around xenograft tumors. Nude mice were anesthetized with isoflurane through the gas anesthesia system. The resulting images were processed and analyzed by subtracting the background tissue auto-fluorescence from the fluorescence of the probe under the assistance of Perkin-Elmer Image software of the small animal imaging system. We obtained the in vivo fluorescent images at different time points after the relevant fluorescent probes were injected via intratumoral injection, with the excitation and emission wavelengths of 520 nm and 710 nm, respectively. The ex vivo fluorescence images were obtained immediately after the major organs/tumors were excised from the tested nude mice (Fig. 1). Fig. 1 Illustration of the synthesis of fluorescent Au-BSA NCs. Mixed with a certain concentration of BSA and HAuCl4, then the mixture was placed in a water bath with 80 °C for 10 minutes. The purified Au-BSA NCs were applied in bioimaging and measured by fluorescence spectrometer and transmission electron microscope. One-step rapid synthesis and characterization of Au-BSA NCsMany literatures reported that BSA, lysozyme, trypsin, and ovalbumin were separately used as templates for the preparation of protein-Au NCs. During the synthesis of Au-BSA NCs, BSA acted as both a stabilizer and a reductant. BSA also played an important role in restricting the size of the AuNCs during the growth and nucleation processes.20,21 NaOH was used to increase the pH value of the reaction solution (pH ≥ 10) to improve the reducing power of BSA and thus accelerate the reduction of Au(III) into Au(0).22 Additionally, reaction time, temperature, and pH values were also significant for obtaining the nanoclusters with bright and stable fluorescence. As shown in Fig. 2A, the optimal reaction temperature was 80 °C. Upon heating, BSA tends to undergo partial denaturation to expose amino residues such as tyrosine, histidine, and cysteine to interact with Au(I)/Au(III) more firmly. However, when even higher thermal energy was provided at 90 °C, denaturation of BSA occurred to a greater extent and formed some insoluble gel, leading to its weaker interactions with Au(III) ions and the as-prepared Au NCs.23 Red-shifts in the fluorescence, while the fluorescence intensity decreased greatly can be observed upon increasing the reaction time. Fig. 2B revealed that the most suitable reaction time is 10 minutes when the reaction temperature is 80 °C. Fig. 2B displays that it took 10 min to obtain fluorescent Au-BSA NCs that emission peak located at 640 nm. Since pH value affects the structure of proteins, its role in the formation of Au-BSA NCs should not be under-estimated.19 From Fig. 2C we can draw a conclusion that the pH values of the whole reaction solution play important role during the synthesis of Au-BSA NCs. When the pH value of the whole reaction solution is 11.5, the relevant fluorescence intensity reached maximum. When the pH value of the whole reaction solution is increasing from 11.5 to 12, there appear about 10 nm red-shifts in the fluorescence, but there is a sharp decline in fluorescence intensity unfortunately. Based on the above observations, the optimum reaction condition for preparing Au-BSA NCs is at 80 °C for 10 minutes and the pH value of the whole reaction solution is 11.5. Upon heating, BSA tends to undergo partial denaturation to expose amino residues such as cysteine, tyrosine, and histidine to interact with Au(I)/Au(III) more firmly. Fig. 2 Fluorescence spectra of Au-BSA NCs prepared in different conditions. (A) Fluorescence spectra of Au-BSA NCs prepared in solutions (pH* 11.5) at (a) 50, (b) 60, (c) 70, (d) 80 and (e) 90 °C for 10 min. The fluorescence intensities (IF) are plotted in arbitrary units (a.u.). (B) Fluorescence spectra of Au-BSA NCs prepared at 80 °C (pH* 11.5) for different periods of time (a) 5, (b) 10, (c) 15, and (d) 20 min. (C) Fluorescence spectra of Au-BSA NCs obtained at different pH* values: (a) 8.0, (b) 10.0, (c) 11.0, (d) 11.5, and (e) 12, adjusted with addition of NaOH.

As shown in Fig. S1 (ESI†), the size of the as-prepared Au-BSA NCs is evaluated by TEM. This typical TEM image of the resulting Au-BSA NCs evidenced their relatively uniform sizes of 1–2 nm and high mono-dispersion, while the high resolution image (HRTEM) of Au-BSA NCs showed clear crystal of metallic structure. HRTEM (inset in Fig. S1, ESI†) illustrated that the gold nanoclusters kept their interplanar Au–Au spacing at ca. 0.2 nm.

XPS was used to investigate the valence of gold after the formation of Au-BSA NCs. From Fig. 3A we can observe that two peaks located at the binding energy of 84.25 and 87.875 eV (7440-57-5), respectively, which were consistent with the emission of 4f7/2 and 4f5/2 photoelectrons from Au (0).

Fig. 3 Elemental analysis of Au-BSA NCs. (A) XPS spectrum showing the binding energy of Au 4f. (B) XPS spectrum showing the binding energy of all elements of the product. Fluorescence of Au-BSA NCsFrom Fig. S2(f),† we can find out that the as-prepared Au-BSA NCs exhibited bright red fluorescence by the naked eyes under a UV lamp (λex = 365 nm) and were highly dispersed in aqueous solution. The as-prepared Au-BSA NCs solution shows an excitation and emission maxima wavelength at 490 and 640 nm, respectively. The optical and fluorescence photographs of Au NCs products stabilized and reduced with BSA for 10 minutes incubation at 80 °C were illustrated in Fig. S2.† As shown in Fig. S3 (ESI†), the max relaxation time for the fluorescence decay of the relevant Au-BSA NCs was 463 ns.

Moreover, we have measured the fluorescence quantum yields of the as-prepared Au-BSA NCs. The fluorescence quantum yield is measured to be around 9.4% by using the comparative method with rhodamine B (QY = 95% in ethanol, see the ESI†).

Application of Au-BSA NCs for cancer cell imagingThe MTT assays revealed that the as-prepared Au-BSA NCs have good bio-compatibility. From Fig. 4A–C, we can find out that the viabilities of MCF-7, HeLa and L02 cells maintained above 85% when they had been incubated with Au-BSA NCs in the concentration range of 0.1–10 mg mL−1 for 24 h, respectively. Fig. 4 The viabilities of cells after incubation 24 h with Au-BSA NCs. Three kinds of cells were tested for studying the toxicity of the as-obtained materials against the cells: (A) MCF-7 cell, (B) HeLa cell, and (C) L02 cell.

It is noteworthy that, the as-prepared Au-BSA NCs were well distributed in both MCF-7 and HeLa cells so that the relevant edges and morphologies of the above two cancer cells were neatly delineated. Interestingly, as early as 2 h incubation, large numbers of red-emitting Au-BSA NCs were found specifically attaching to the cell membrane of the above two cancer cells (Fig. 5B and 6B). Moreover, the fluorescence intensity increased with the increase of incubation time (As shown in Fig. 5C and 6C). On the other hand, no obvious staining of cell membrane or cellular uptake could be seen in control groups involving L02 cells, which indicated that there was no significant fluorescence for normal cells subjected to the same incubation time and concentration of Au-BSA NCs(Fig. 6D–F). Importantly, the above results demonstrate the capability of these fluorescent gold nanoclusters for target cancer cells imaging rapidly and sensitively.

Fig. 5 Laser confocal fluorescence micrographs of MCF-7 cancer cells. (A) MCF-7 cells without Au-BSA NCs treatment. (B) MCF-7 cells treated with 1 mg mL−1 Au-BSA NCs solutions for 2 h. (C) MCF-7 cells treated with 1 mg mL−1 Au-BSA NCs solutions for 4 h. (D) Relative fluorescence intensity variations along cross-sections a (in A), b (in B), or c (in C) (the color gradient coding illustrates the direction of the sampling). Fluorescence micrographs were collected by using a 488 nm fluorescence excitation wavelength. Fig. 6 Laser confocal fluorescence micrographs of HeLa cells and L02 cells. (A) HeLa cells without Au-BSA NCs treatment. (B) HeLa cells treated with 1 mg mL−1 Au-BSA NCs solutions for 2 h. (C) HeLa cells treated with 1 mg mL−1 Au-BSA NCs solutions for 4 h. (D) L02 cells without Au-BSA NCs treatment. (E) L02 cells treated with 1 mg mL−1 Au-BSA NCs solutions for 2 h. (F) L02 cells treated with 1 mg mL−1 Au-BSA NCs solutions for 4 h. Fluorescence micrographs were collected by using a 488 nm fluorescence excitation wavelength. Application of Au-BSA NCs for small animal imagingTo indicate the feasibility of the fluorescent gold nanoclusters for small animal imaging, the fluorescent gold nanoclusters was intratumorally injected into the two kinds of mice modeled with MCF-7 and HeLa tumors, as shown in Fig. 7 and 8 respectively. The two kinds of tumor-bearing mice were real-time imaged after injection. Upon injecting the fluorescent gold nanoclusters into the tumor-bearing mice, the substantially enhanced NIR fluorescence signal was detected after 60 min, and the NIR fluorescence signal in tumors was becoming weaker gradually. After 24 h, the fluorescence signal was almost cleared. No obvious toxic effects were observed during the whole experimental trail, suggesting that Au-BSA NCs can be administered for rapid and high-sensitive in vivo fluorescence cancer imaging. Fig. 7 In vivo fluorescence imaging of the nude mice modeled with MCF-7 tumors after intratumoral injection of 10 mg mL−1 Au-BSA NCs solution for different periods of time (A) 0, (B) 30, (C) 60, and (D) 120 min. Fig. 8 In vivo fluorescence imaging of the nude mice modeled with HeLa tumors after intratumoral injection of 10 mg mL−1 Au-BSA NCs solution for different periods of time (A) 0, (B) 30, (C) 60, and (D) 120 min.

The ex vivo fluorescence images of various organs/tissues were also obtained via thoracotomy, which were obtained 12 h post-injection of the Au-BSA NCs. Representative organs/tissues including heart, liver, spleen, lung, kidney and tumor were excised, washed with PBS buffer, and then used for fluorescence imaging. From Fig. 9, we can find that the ex vivo HeLa tumors showed much stronger fluorescence, while almost no fluorescence was observed in other organs.

Fig. 9 Representative ex vivo fluorescence images of excised HeLa tumors and other visceral organs in Au-BSA NCs-injected xenograft tumor mouse after 12 h treatment. (a) spleen (b) liver (c) kidney (d) heart (e) tumor (f) lung. ConclusionsTo sum up, the fluorescent gold nanoclusters were successfully developed with stable and bright fluorescence for tumor imaging and small animal imaging application through a facile synthetic routine. We prepared fluorescent Au-BSA NCs (em at 640 nm) at 80 °C for 10 min, which is considerably quicker in comparison to the reaction being carried out at 37 °C (12 h). Moreover, the prepared fluorescent gold nanoclusters offer various advantages of ultra-small size, good photo-stability, strong fluorescence emission, excellent water solubility, and bio-compatibility. More importantly, the prepared fluorescent gold nanoclusters applied in cancer imaging showed excellent tumor-targeted imaging in vitro and in vivo during a relatively short period of time. Thus, the as-prepared fluorescent Au-BSA NCs holds excellent potential as a nanoscaled platform for rapid tumor diagnosis in future clinic applications.AcknowledgementsThis work is supported by the National Natural Science Foundation of China (81325011), National High Technology Research Development Program of China (2015AA020502, 2012AA022703), and the Major Science Technology Project of Suzhou (ZXY2012028).Notes and referencesA. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward and D. Forman, Ca-Cancer J. Clin., 2011, 61, 69–90 CrossRef PubMed.G. L. Wong, H. L. Chan, Y. K. Tse, H. Y. Chan, C. H. Tse, A. O. Lo and V. W. Wong, Hepatology, 2014, 59, 986–995 CrossRef CAS PubMed.J. M. Liu, J. T. Chen and X. P. Yan, Anal. Chem., 2013, 85, 3238–3245 CrossRef CAS PubMed.L. Shang, S. J. Dong and G. Ulrich Nienhaus, Nano Today, 2011, 6, 401–418 CrossRef CAS PubMed.D. H. Chen, S. P. Gao, W. Ge, Q. W. Li, H. Jiang and X. M. Wang, RSC Adv., 2014, 4, 40141–40145 RSC.H. S. Choi, Y. Kim, J. C. Park, M. H. Oh, D. Y. Jeon and Y. S. Nam, RSC Adv., 2015, 5, 43449–43455 RSC.L. J. Shao, Y. F. Gao and F. Yan, Sensors, 2011, 1, 11736–11751 CrossRef PubMed.Y. He, Y. L. Zhong, Y. Y. Su, Y. M. Lu, Z. Y. Jiang, F. Peng, T. T. Xu, S. Su, Q. Huang, C. H. Fan and S. T. Lee, Angew. Chem., Int. Ed., 2011, 50, 5695–5698 CrossRef CAS PubMed.C. L. Zhang, X. H. Ji, Y. Zhang, G. H. Zhou, X. L. Ke, H. Z. Wang, P. Tinnefeld and Z. K. He, Anal. Chem., 2013, 85, 5843–5849 CrossRef CAS PubMed.S. L. Luo, E. L. Zhang, Y. P. Su, T. M. Cheng and C. M. Shi, Biomaterials, 2011, 32, 7127–7138 CrossRef CAS PubMed.W. B. Edwards, W. J. Akers, Y. P. Ye, P. P. Cheney, S. Bloch, R. Laforest and S. Achilefu, Mol. Imaging, 2009, 8, 101–110 CAS.Q. Liu, T. S. Yang, W. Feng and F. Y. Li, J. Am. Chem. Soc., 2012, 134, 5390–5397 CrossRef CAS PubMed.S. J. Zeng, M. K. Tsang, C. F. Chan, K. L. Wong, B. Fei and J. H. Hao, Nanoscale, 2012, 4, 5118–5124 RSC.Z. W. Wei, L. N. Sun, J. L. Liu, J. Z. Zhang, H. R. Yang, Y. Yang and L. Y. Shi, Biomaterials, 2014, 35, 387–392 CrossRef CAS PubMed.Z. S. Haidar, Polymers, 2010, 2, 323–352 CrossRef CAS PubMed.S. H. Hu and X. H. Gao, J. Am. Chem. Soc., 2010, 132, 7234–7237 CrossRef CAS PubMed.O. S. Wolfbeis, Chem. Soc. Rev., 2015, 44, 4743–4768 RSC.E. C. Dreaden, A. M. Alkilany, X. H. Huang, C. J. Murphy and M. A. El-Sayed, Chem. Soc. Rev., 2012, 41, 2740–2779 RSC.J. P. Xie, Y. G. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888–889 CrossRef CAS PubMed.L. Shang and G. U. Nienhaus, Biophys. Rev., 2012, 4, 313–322 CrossRef CAS.D. M. Chevrier, A. Chatt and P. Zhang, J. Nanophotonics, 2012, 6, 064504 CrossRef PubMed.P. Zhang, X. X. Yang, Y. Wang, N. W. Zhao, Z. H. Xiong and C. Z. Huang, Nanoscale, 2014, 6, 2261–2269 RSC.P. C. Chen, C. K. Chiang and H. T. Chang, J. Nanopart. Res., 2013, 15, 1336 CrossRef.