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Chan Wang,a,c Chuanxi Wang,*a Lin Xu,b Hao Cheng,a Quan Lin*b and Chi Zhang*a 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x The development of functional copper nanoclusters (Cu NCs) is becoming increasingly widespread in consumer technologies due to their applications in cellular imaging and catalyst. Herein, we reported a simple protein-directed synthesis of stable, water–soluble and fluorescent Cu NCs, using BSA as the stabling agent. Meanwhile, in this study, hydrazine hydrate (N2H4.2H2O) was used as the reducing agent. N2H4.2H2O was a mild reducing agent and suggested all process to be operated at room temperature. Asprepared Cu NCs showed red fluorescence with peaking center at 620 nm (quantum yield 4.1%). The fluorescence of as-prepared BSA-Cu NCs was responsive to pH that the intensity of fluorescence increased rapidly with decreasing the pH from 12 to 6. Besides, with an arresting set of features including water-dispersible, red fluorescent, good biocompatible, surface-bioactive and small size, the resultant BSA-Cu NCs could be used as a probe for cellular imaging and catalyst. In this study, CAL-27 cells and the reaction of oxidation of styrene are used as models to achieve the fluorescence imaging and to elucidate the catalytic activity of as-prepared BSA-Cu NCs.
1. Introduction The development of functional nanomaterials is becoming increasingly widespread in consumer technologies.1-3 Metal nanoclusters (NCs) defined as isolated particles less than 2 nm in size with several to a hundred atoms, which bridge the gap between traditional organometallic compounds and crystalline metal nanoparticles, considered novel potential practical nanomaterials.4,5 Metal NCs with discreted energy levels showed molecule–like electronic transitions within the conduction band, and exhibited unique physical and chemical properties, as well as had practical applications in various areas, such as in cell labeling, ion sensor, and catalysts.6-9 Compared with traditional quantum dots, some superior properties such as low toxicity, good biocompatibility and multifunctional surface chemistry made metal NCs receive much attention in biological field.10-13 For example, Yeh reported fluorescent gold NCs as a biocompatible marker for in vitro and in vivo tracking of endothelial cells;12 DNA-encapsulated fluorescent silver NCs could be used as biolabels.13 On the other hand, small colloidal metal NCs exhibited superior catalytic activity due to their high surface to volume ratio and high surface energy that made surface atoms fairly active.14 Recent reports also suggested size-dependent catalytic activity of ultrasmall gold NCs, including Au25, Au55, Au38, and Au144.15 However, to date extensive research progress for NCs focused on luminescent and small Au and Ag nanomaterials.16,17 Studies for the synthesis, properties and applications of copper (Cu) NCs are still scarce primarily because This journal is © The Royal Society of Chemistry [year]
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of the difficulty in preparing highly stable and extremely tiny particles.18,19 Recently, biomimetic synthesis had become a hot topic and various biological molecules including amino acids, peptides, proteins, DNA and RNA, had been widely used for synthesizing various nanomaterials due to their advantages in controlling nucleation and growth of nanoparticles.20-24 The biomoleculeconjugated nanomaterials were more suitable for bio-applications owing to the bioactive surface.25 For example, proteins would play an important role in directing the synthesis of functional nanomaterials under mild conditions due to the amine, carboxyl, and thiol groups in proteins which can serve as effective stabilizing agents for nanoparticles.26 Bovine serum albumin (BSA), a commercially available protein, has been frequently adopted to synthesize various inorganic nanocrystals due to the presence of 35 thiol groups (from the 35 cysteine (Cys) residues) in a BSA monomer.27,28 Inspired by the protein-directed inorganic nanomaterials synthesis, fluorescent Au and Ag NCs stabilized by BSA had been studied extensively.29,30 Besides, water soluble Cu NCs capped with BSA were successfully prepared,31 however, the blue emitting fluorescence limited their practical application in biological field. Therefore, it would be of great interest to develop protein-directed synthesis of stable and biocompatible Cu NCs with red emission. Herein, we reported a simple protein-directed synthesis of stable, water–soluble and fluorescent Cu NCs, using BSA as [journal], [year], [vol], 00–00 | 1
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Protein-directed synthesis of pH-responsive red fluorescent copper nanoclusters and applications in cellular imaging and catalysts
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2. Experimental Section
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Materials: Bovine serum albumin (BSA, molecular weight of 66000) was purchased from Sigma. Copper sulfate (CuSO4), copper nitrate (Cu(NO3)2), copper chloride (CuCl2), copper acetate (Cu(AcO)2), hydrazine hydrate (N2H4.2H2O, 85 wt%) and isopropanol were analytical grade. All reagents were used as received without further purification. De–ionized water was used in all experiments. Preparation of BSA stabilized Cu nanoclusters: In a typical experiment, BSA (100 mg) was added to CuSO4 solution (5 mL, 5 mM). The solution was stirred at room temperature for 10 min, and the hydrogel was formed due to the coordination between Cu ions and the various functional groups of BSA such as –NH, –OH, and –SH. Adjusting the pH to 12 by 1 M NaOH solution, a purple transparent solution was received. Slow injection of N2H4.2H2O (1 ml) to the reaction mixture and stirring at room temperature for 4 h, the solution changed to light yellow which suggested Cu NCs were prepared. And the solution after synthesis are dialyzed against doubly distilled water for more than 24 h with a water change every 4 h, to remove all small molecular impurity. The resultant BSA-Cu NCs were freeze-drying for further application. Cellular imaging: CAL-27 cells (210 cells/ml) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicil-lin/streptomycin (DMEM) using 96-well plate. Suspensions (10 µg/mL) of BSA-Cu NCs 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 24 h. The chamber slide was then incubated at 37 0C in a CO2 incubator for 24 h for BSA-Cu NCs uptake (only 10 µg of BSA-Cu NCs to 150 µL of culture medium (105 cells) was added). Prior to fixation of the cells on the slide for inspection with a confocal fluorescence microscope, the excess BSA-Cu NCs were removed by washing 3 times with warm DPBS. Catalyst experiment: Styrene oxidation catalyzed by supported copper: 1.6 mmol of styrene, 20 mL of acetonitrile, and 18 mg of supported catalyst reacted for 16 h. When the temperature of the oil bath at was 75 °C, t-butyl hydroperoxide (TBHP, 5 mol%) was added. The products were styrene epoxide (1), benzaldehyde (2), acetophenone (3), and 2-phenylacetaldehyde (4). A blank experiment was performed in identical conditions, but without the catalyst. The products were analyzed with GCMS. Characterization Methods: 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 collected in a VG ES2 | Journal Name, [year], [vol], 00–00
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CALAB MKII spectrometer. Binding energy calibration was based on C 1s 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 spectropho-tometer. The morphology and mean diameter of resultant BSA-Cu NCs were characterized by TECNAI F20 transmission electron microscope (TEM) operating at 200 kV. The CD spectrum was measured in a Jasco 815 spectropolarimeter with a Peltier setup for the temperaturedependent measurements. CD studies were done with 10 mm path length cell. The confocal microscopy images were taken at Olympus Fluoview FV1000. All measurements were performed at room temperature under ambient conditions.
3. Results and Discussion
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The process of protein-directed synthesis of stable, water–soluble and fluorescent copper nanoclusters (Cu NCs) was shown in scheme 1. Cu NCs conjugated to BSA were synthesized in aqueous solution at room temperature using N2H4.2H2O as the reducing agent. N2H4.2H2O was a mild reducing agent and had been extensively used to synthesis nanomaterials.32 Herein BSA was chosen as the model protein to stabilize the clusters and provide steric protection due to the 17 disulfide bonds and 1 free cysteine.33 It had been widely used in the synthesis of fluorescent Ag and Au NCs.29,30 Slow injection of N2H4.2H2O to the reaction mixture of BSA and Cu ions, the solution changed to light yellow with stirring at room temperature for 4 h, which suggested Cu NCs were prepared. This preparation procedure was simple and robust. Photographs of resultant BSA-Cu NCs aqueous solution and powder are shown in Fig 1. As we can see, as-prepared BSACu NCs had good dispersion in aqueous solution and showed a color of light yellow (Fig 1a). Moreover, there was no noticeable precipitation, which was attributed to capping layer of BSA to prevent NCs from agglomeration. Under UV irradiatation (365 nm), BSA-Cu NCs showed red fluorescence (Fig 1b). The solution upon precipitation and washing gave an orange powder (Fig 1c) which showed red luminescence in the solid state (Fig 1d). The photoluminescence (PL) emission and excitation spectra were shown in Fig 1E. In aqueous solution, the BSA-Cu NCs exhibited excitation at 524 nm and emission at 625 nm with the full width at half maximum around 85 nm. The quantum yield of the as-prepared BSA-Cu NCs in aqueous solution at room temperature was found to be 4.1% using rodamine6G (QYs, 0.95 in ethanol) as the standard. As well known, this is the first report for red fluorescent Cu NCs with such high QYs.
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Scheme 1. The facile formation process of protein-directed synthesis of stable and water–soluble red fluorescent Cu NCs.
The size distribution of resultant red fluorescent BSA-Cu NCs was characterized by high-resolution transmission electron microscope (HRTEM). HRTEM images of resultant fluorescent BSA-Cu NCs was given in Fig 2a. The average size of as– prepared BSA-Cu NCs was 2.7 ± 0.4 nm. The crystal lattice This journal is © The Royal Society of Chemistry [year]
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stabling agent. Meanwhile, in this study, a mild reducing agent of hydrazine hydrate (N2H4.2H2O) was used which suggested all process could be operated at room temperature. As-prepared Cu NCs showed red fluorescence with peaking center at 620 nm and the fluorescence was responsive to pH that with adjusting the pH from 12 to 6, the fluorescent intensity increased rapidly. Additionally, resultant Cu NCs capping with BSA showed good biocompatibility and could be used as probe for cells. On the other hand, the small size of as-prepared Cu NCs suggested the NCs exhibited superior catalytic activity.
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intensity due to forming large Cu NCs (Fig S4). In addition to CuSO4, other Cu source including Cu(AcO)2, CuCl2, and Cu(NO3)2, could be used to prepare red fluorescent BSA-Cu NCs (Fig S5). Raising reaction temperature, short time would complete reaction (40 0C for 30 min, 60 0C for 5 min), however, poor fluorescence property suggested the room temperature would be the best choice (Fig S6).
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Fig. 1. Photographs of BSA-Cu NCs aqueous solution (a, b) and powder (c, d) under (a, c) visible and (b, d) UV light. (E) The excitation (EX) and emission (EM) spectra of resultant fluorescent BSA–Cu NCs in aqueous solution. 90
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Fig. 2. a, the typical TEM image of resultant red fluorescent BSA-Cu NCs. The average size of as–prepared BSA-Cu NCs was 2.7 ± 0.4 nm; b, XPS spectrum show the binding energy of Cu 2p in Cu nanoclusters, which demonstrated that the Cu was in the zero-valent state
Synthesis conditions including the pH value, the molar ratio of BSA and Cu ions, the amount of N2H4.2H2O, the reaction temperature, and various kinds of Cu source were studied. An optimal composition of 20.0 mg/mL BSA, 5.0 mM CuSO4 1 ml N2H4.2H2O and pH =12 produces the highest fluorescence intensity of the BSA-Cu NCs bioconjugates. The pH of reaction solution is an important consideration in the synthesis of fluorescent BSA-Cu NCs as shown in Fig S1. Experiments conducted in neutral (pH =6) and acidic pH (pH =3) did not result in the clusters (Fig S1), only the blue emission of BSA (seeing the PL spectrum of BSA without copper source in Fig S2). At high pH, the fluorescence of Cu NCs appeared (pH =9) and formed optimal fluorescence intensity of the BSA-Cu NCs at pH =12 due to the phenolic moieties of tyrosine in BSA (Fig S1). Fig S3 demonstrated that an increase in the molar ratio of BSA and Cu ions would increase the yield of BSA-Cu NCs bioconjugates and further increasing the molar ratio will decrease the fluorescence intensity. N2H4.2H2O as the reducing agent, an increase of the amount would produce high fluorescence intensity; however, further increasing would quench the fluorescence This journal is © The Royal Society of Chemistry [year]
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Fig. 3 The relative fluorescence intensity was determined by calculating the ratio of the fluorescence intensities of BSA-Cu NCs in the presence and absence of common anions (0.10 M).
The stability of luminescent nanomaterials was an important factor to assess their applications.36 The as-prepared fluorescent Cu NCs was conjugated to BSA which was confirmed by FITR and XPS spectra, which provided resultant Cu NCs with good stability. Fig S7 depicts the FITR spectra of the free BSA and BSA-Cu NCs bioconjugates. There were not any differences in their spectra, inferring that the Cu NCs embedded in BSA would not affect the surface-structure of BSA.37 Besides, the XPS spectra of Fig S8 gave the binding energy of S 2p, C 1s, N 1s, and O 1s which was another evidence of existing BSA in as-prepared Cu NCs.38 In summary, metallic Cu NCs are successfully synthesized and embedded in BSA. The stability was investigated on the interference of some common anions (0.10 M) on the fluorescence of BSA-Cu NCs. The relative fluorescence intensity was determined by calculating the ratio of the fluorescence intensities of BSA-Cu NCs in the presence and absence of the interference ions as shown Fig 3. Most of these ions showed either no or slight interferences. The fluorescence spectra of BSA-Cu NCs in various common anions in detail were given in Fig S9. It is well known that BSA has different conformations at different pH values which would affect the physical properties of nanomaterials.39 Interestingly strong optical responses to pH changing were observed for as-prepared BSA-Cu NCs. To study pH responses property, a series of universal buffer solutions with pH ranging systematically from 6 to 12 were prepared and the fluorescence intensities of BSA-Cu NCs in buffers of different pH were monitored. Significant changes in fluorescence intensity were observed as the pH was varied, as depicted in Fig 4a. When the pH changed from 12 to 6, the fluorescence intensity of BSAJournal Name, [year], [vol], 00–00 | 3
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fringes are 2.05 Å apart which indicates the (111) planes of the metallic Cu (Fig 2c). Moreover, there was no formation of larger size metal nanoparticles or aggregation due to BSA as protective layer. XPS analysis is carried out to determine the oxidation state of copper in the BSA–Cu NCs. In the present study, the binding energy of Cu 2p3/2 was observed at 932.3 eV, which is assigned to the binding energies of the of Cu(0),34 and a weak peak can be observed at 953eV, suggesting very minimal presence of Cu(II) in the system.35 While, the existed oxidation state on the surface of NCs would ensure stability and enhance the florescence.36
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Cu NCs increased nearly 2-fold. Within the neutral pH range 6-7, the highest fluorescence intensity were found, which indicated resultant BSA-Cu NCs was more suitable for cellular imaging. We also verified that these changes were reversible between pH 6 and 12, as shown in Fig 4b. Besides, there were no discernible shifts in the peak emission at different pH values. Hence the reason for responses to pH was neither shell degradation and hole trapping from thiolate formation nor aggregation through irreversible ligand loss.40,41
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Fig. 5 The circular dichroism (CD) spectroscopy show the secondary structures of BSA in the reaction system at different pH values.
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Fig. 4 a, fluorescence intensity of BSA-Cu NCs responses to pH ranging systematically from 6 to 12, inset showing when the pH changed from 12 to 6, the fluorescence intensity of BSA-Cu NCs increased nearly 2-fold; b, reversible between pH 6 and 12 for four times was observed
The changes in the PL at different pH may broadly be attributed to corresponding structural changes of BSA.42 The secondary structures of BSA in the reaction system were investigated by circular dichroism (CD) spectroscopy at different pH values (Fig 5), which is a valuable spectroscopic technique for studying protein and its complex. With the decrease in pH of the solution, more and more α helix were stretched and transformed into β sheets, which could contribute to the impairment or break of hydrogen bonds. In previous report, the β sheet secondary structure could form a suitable conformation for the oriented growth of NPs which result in NPs uniformly coated with BSA.43 With the decrease of pH, β sheet as well as random coil structural elements became more predominant which made more functional groups, like –OH, –NH, –COOH, accessible to interact with the surface of NCs, hence, the luminescence intensity of the as-prepared materials is high at low pH owing to improved surface passivation of NCs. Therefore, these dramatic changes of the secondary structure may thus play a significant role in the observed trends in pH responsiveness of BSA-Cu NCs. Copper, one of the transition metals, is an essential trace element for life. Although excessive amounts of copper can result in severe diseases, low amounts of copper ions play a pivotal role in many fundamental physiological processes in organisms, such as
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Fig. 6. Viability of Cal-27 cells after 24 h incubation with different concentrations of BSA-Cu NCs in the cell medium as determined by a MTT assay. 90
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With an arresting set of features, including water-dispersible, red fluorescent, good biocompatible and surface-bioactive, asprepared BSA-Cu NCs indicated great potential in cellular marking. The cell internalization and intracellular distribution of resultant BSA-Cu NCs were evaluated by confocal laser fluorescence microscopy. Fig 7a–c respectively revealed the bright field, confocal fluorescent and overlay images of CAL-27 cells incubating with as-prepared BSA-Cu NCs for 24 h. From the bright field image of CAL-27 cells, we could see the cells incubating with resultant BSA-Cu NCs are still maintain their normal morphology, thus indicating the good biocompatibility of as-prepared BSA-Cu NCs in this specific dose and time point. The fluorescent image irradiated by 515 nm, showed bright red fluorescence within the cells that indicated the uptake behaviors of CAL-27. It could be seen that the fluorescent signal was distributed not only in cytoplasm but also most in cellular nucleus. This result demonstrated BSA-Cu NCs could be used as a probe for real-time cellular imaging, especially labeling the oligonucleotide as a DNA probe for specific detection of nucleic acids. This result was consistent with previous reports about fluorescent NCs with similar small size.45
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Fig. 7 (a) bright field, (b) confocal fluorescent and (c) overlay images of Cal-27 cells incubating with BSA-Cu NCs for 24 h.
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bone formation, connective tissue development and cellular respiration.44 Besides, as-prepared BSA-Cu NCs was protected by a layer of BSA. These conditions suggested as-prepared BSACu NCs displayed the low or no toxic to cells at suitable concentration. MTT assay and apoptosis assay were used to evaluate the cytotoxicity of as-prepared BSA-Cu NCs and the viability of cells. From the results of MTT assay (Fig 6), cell viability still remained above 70% after incubating with asprepared BSA-Cu NCs even at the concentration of 80 µg/mL for 24 h. Thus the result showed that GNDs had low acute toxicity to cells.
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Notes and references a
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Fig. 8 Styrene oxidation catalyzed by as-prepared BSA-Cu NCs; see Table 1 for details Table 1. Styrene oxidation catalyzed by supported various Cu NCs. The products were styrene epoxide (1), benzaldehyde (2), acetophenone (3), and 2phenylacetaldehyde (4); see Figure 9 for product details
Entry
Cu(NO3)2 CuCl2 CuSO4 Cu(OAc)2 30
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Cu cluster loading (%)
Styrene conversion [mol %]
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Product selectivity [mol %] 85
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2 72 69 70 66
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4. Conclusion In summary, a simple protein-directed synthesis method was applied to prepare stable, water–soluble and fluorescent copper nanoclusters (Cu NCs). The surface of as-prepared BSA-Cu NCs was protected by a layer of BSA and showed stability in various common anions. With the help of mild reducing agent of N2H4.2H2O, the BSA-Cu NCs could be easily prepared at room temperature. As-prepared BSA-Cu NCs showed red fluorescence with peaking center at 620 nm (quantum yield 4.1%). The fluorescence of as-prepared BSA-Cu NCs was responsive to pH. The intensity of fluorescence increased rapidly with decreasing the pH from 12 to 6 and the responsive property was reversible. It is also verified that the resultant BSA-Cu NCs could be used a probe for cells and as the catalyst due to an arresting set of features, including water-dispersible, red fluorescent, good biocompatible, surface-bioactive and small size. In this study, the resultant BSA-Cu NCs was used as a probe for labeling CAL-27 cells. Moreover, BSA-Cu NCs was used as catalyst in the reaction of styrene oxidation and displayed high (70%) selectivity for benzaldehyde as the major product and with 70% conversion of the styrene
Acknowledgements This journal is © The Royal Society of Chemistry [year]
This work was supported by the National Natural Science Foundation of China (Grants No.21174048 and No. 51373061) and Programme of Introducing Talents of Discipline to Universities (111 Project B06009).
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China-Australia Joint Research Centre for Functional Molecular Materials, School of Chemical & Material Engineering, Jiangnan University, Wuxi 214122, P. R. China E–mail: [email protected]. b State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, 130012, P. R. China E–mail: linquan@ jlu.edu.cn c State Key Laboratory of Materials-oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009, P. R. China † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/ 1. C. X. Wang, D. Zhang, L. Xu, Y. N. Jiang, F. X. Dong, B. Yang, K. Yu and Q. Lin, Angew. Chem. Int. Ed. 2011, 50, 7587 –7591. 2. Y. Z. Lu and W. Chen, Chem. Soc. Rev. 2012, 41, 3594–3623. 3. T. Udayabhaskararao, Y. Sun, N. Goswami, K. P. Samir, K. Balasubramanian and T. Pradeep, Angew. Chem. Int. Ed. 2012, 51, 2155 –2159. 4. H. F. Qian, M. Z. Zhu, Z. K. Wu and R. C. Jin, Accounts Of Chemical Research. 2012, 45, 1470–1479. 5. H. X. Xu and S. S. Kenneth, Adv. Mater. 2010, 22, 1078–1082. 6. B. S. González, M. J. Rodríguez, C. Blanco, J. M. A. LópezQuintela and J. M. G. Martinho, Nano Lett. 2010, 10, 4217–4221. 7. S. I. Tanaka, J. Miyazaki, D. K. Tiwari, T. Jin and Y. Inouye, Angew. Chem. Int. Ed. 2011, 50, 431 –435. 8. C. C. Huang, Z. S. Yang, K. H. Lee and H. T. Chang, Angew. Chem. Int. Ed. 2007, 46, 6824 –6828. 9. M. Hyotanishi, Y. Isomura, H. Yamamoto, H. Kawasaki and Y. Obora, Chem. Commun. 2011, 47, 5750–5752. 10. Y. Y. Cui, Y. L. Wang, R. Liu, Z. P. Sun, Y. T. Wei, Y. L. Zhao and X. Y. Gao, ACS Nano. 2011, 5, 8684–8689. 11. S. Kumar, M. D. Bolan and T. P. Bigioni, J. Am. Chem. Soc. 2010, 132, 13141–13143. 12. H. H. Wang, C. J. Lin, C. H. Lee, Y. C. Lin, Y. M. Tseng, C. L. Hsieh, C. H. Chen, C. H. Tsai, C. T. Hsieh, J. L. Shen, W. H. Chan, W. H. Chang and H. I. Yeh, ACS Nano. 2011, 5, 4337–4344. 13. J. H. Yu, S. M. Choi and R. M. Dickson, Angew. Chem. Int. Ed. 2009, 48, 318 –320. 14. S. Biswas, J. T. Miller, Y. H. Li, K. Nandakumar and C. S. S. R. Kumar, Small. 2012, 8, 688-698. 15. H. J. Zhang, T. Watanabe, M. Okumura, M. Haruta and N. Toshima, Nat. Mater. 2012, 11, 49. 16. Z. K. Wu, M. Wang, J. Yang, X. H. Zheng, W. P. Cai, G. W. Meng, H. F. Qian, H. M. Wang and R. C. Jin, Small. 2012, 8, 2028–2035. 17. W. J. Cho, Y. Kim and J. K. Kim, ACS Nano. 2012, 6, 249–255. 18. W. T. Wei, Y. Z. Lu, W. Chen and S. W. Chen, J. Am. Chem. Soc. 2011, 133, 2060–2063. 19. X. F. Jia, J. Li, L. Han, J. T. Ren, X. Yang and E. K. Wang, ACS Nano. 2012, 6, 3311–3317. 20. L. Turyanska, T. D. Bradshaw, J. Sharpe, M. Li, S. Mann, N. R. Thomas and A. Patane, Small. 2009, 5, 1738–1741. 21. M. X. Yu, C. Zhou, J. B. Liu, J. D. Hankins and J. Zheng, J. Am. Chem. Soc. 2011, 133, 11014–11017. 22. M. Naito, K. Iwahori, A. Miura, M. Yamane and I. Yamashita, Angew. Chem. Int. Ed. 2010, 49, 7006 –7009. 23. 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, 5038–5039. 24. Y. H. Lin, M. L. Yin, F. Pu, J. S. Ren, X. G. Qu, Small. 2011, 7, 1557–1561. 25. T. Li, L. B. Zhang, J. Ai, S. J. Dong and E. K. Wang, ACS Nano. 2011, 5, 6334–6338.
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On the other hand, NCs can be applied in catalysts owing to their ultrasmall size and high surface energy which had been extensively studied.9,46 Copper nanoparticles have been previously shown to be active catalysts for a number of organic reactions especially for cross-coupling reactions.47 However, catalysis of Cu NCs had scarcely been investigated.14,18 Herein, the reaction of oxidation of styrene was used as a model reaction to elucidate the catalytic activity of as-prepared BSA-Cu NCs. Thermal gravimetric analysis (TGA) shows that the organic weight loss of as-prepared BSA-Cu NCs is 65% and therefore, the Cu content of BSA-Cu NCs is calculated as 35% (Fig S10). The styrene oxidation resulted in several reaction products (Table 1; Fig 8) with a high (70%) selectivity for benzaldehyde as the major product and with 70% conversion of the styrene. The oxidation reaction without catalyst occurs very slowly with a final conversion of only 7%.
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26. H. Kawasaki, K. Hamaguchi, I. Osaka and R. Arakawa, Adv. Funct. Mater. 2011, 21, 3508–3515. 27. Y. L Wang, J. J. Chen and J. Irudayaraj, ACS Nano. 2011, 5, 9718– 9725. 28. H. W. Li, K. L. Ai and Y. Q. Wu, Chem. Commun. 2011, 47, 9852– 9854. 29. J. P. Xie, Y. G. Zheng and J. Y. Ying, J. Am. Chem. Soc. 2009, 131, 888–889. 30. X. L. Guevel, B. Hotzer, G. Jung, K. Hollemeyer, V. Trouillet and M. Schneider, J. Phys. Chem. C. 2011, 115, 10955–10963. 31. N. Goswami, A. Giri, M. S. Bootharaju, P. L. Xavier, T. Pradeep and S. K. Pal, Anal. Chem. 2011, 83, 9676–9680. 32. 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 – 1656. 33. A. Mathew, P. R. Sajanlal and T. Pradeep, J. Mater. Chem. 2011, 21, 11205. 34. X. Yuan, Z. T. Luo, Q. B. Zhang, X. H. Zhang, Y. G. Zheng, J. Y. Lee and J. P. Xie, ACS Nano. 2011, 5, 8800–8808. 35. C. Vazquez-Vazquez, M. Banobre-Lopez, A. Mitra, M. A. LopezQuintela and J. Rivas, Langmuir. 2009, 25, 8208–8216. 36. 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 37. A. Retnakumari, S. Setua, D. Menon, P. Ravindran, H. Muhammed, T. Pradeep, S. Nair and M. Koyakutty, Nanotechnology. 2010, 21, 055103 38. L. Yan, Y. Q. Cai, B. Z. Zheng, H. Y. Yuan, Y. Guo, D. Xiao and M. M. F. Choi, J. Mater. Chem. 2012, 22, 1000. 39. D. Ghosh, S. Mondal, S. Ghosh and A. Saha, J. Mater. Chem. 2012, 22, 699. 40. W. H. Liu, H. S. Choi, J. P. Zimmer, E. Tanaka, J. V. Frangioni and M. Bawendi, J. Am. Chem. Soc. 2007, 129, 14530-14531. 41. S. Jeong, M. Achermann, J. Nanda, S. Ivanov, V. I. Klimov and J. A. Hollingsworth, J. Am. Chem. Soc. 2005, 127, 10126-10127. 42. Y. Z. Wu, S. Chakrabortty, R. A. Gropeanu, J. Wilhelmi, Y. Xu, S. K. Er, S. L. Kuan, K. Koynov, Y. Chan and T. Wei, J. Am. Chem. Soc. 2010, 132, 5012–5014. 43. N. E. Kadi, N. Taulier, J. Y. L. Huérou, M. Gindre, W. Urbach, I. Nwigwe, P. C. Kahn and M. Waks, Biophysical Journal. 2006, 91, 3397–3404. 44. X. J. Liu, C. H. Zong and L. H. Lu, Analyst. 2012, 137, 2406. 45. 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, 3137–3142. 46. X. T. Nie, H. F. Qian, Q. J. Ge, H. Y. Xu and R. C. Jin, ACS Nano. 2012, 6, 6014–6022. 47. L. D. Pacho, J. H. VanMaarseveen and G. Rothenberg, Adv. Synth. Catal. 2005, 347, 811 –815.
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TOC A simple and green protein-directed synthesis of stable, water–soluble and fluorescent Cu NCs. Asprepared Cu NCs could be applied in cellular imaging and catalysts fields.
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Chan Wang,a,c Chuanxi Wang,*a Lin Xu,b Hao Cheng,a Quan Lin*b and Chi Zhang*a 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x The development of functional copper nanoclusters (Cu NCs) is becoming increasingly widespread in consumer technologies due to their applications in cellular imaging and catalyst. Herein, we reported a simple protein-directed synthesis of stable, water–soluble and fluorescent Cu NCs, using BSA as the stabling agent. Meanwhile, in this study, hydrazine hydrate (N2H4.2H2O) was used as the reducing agent. N2H4.2H2O was a mild reducing agent and suggested all process to be operated at room temperature. Asprepared Cu NCs showed red fluorescence with peaking center at 620 nm (quantum yield 4.1%). The fluorescence of as-prepared BSA-Cu NCs was responsive to pH that the intensity of fluorescence increased rapidly with decreasing the pH from 12 to 6. Besides, with an arresting set of features including water-dispersible, red fluorescent, good biocompatible, surface-bioactive and small size, the resultant BSA-Cu NCs could be used as a probe for cellular imaging and catalyst. In this study, CAL-27 cells and the reaction of oxidation of styrene are used as models to achieve the fluorescence imaging and to elucidate the catalytic activity of as-prepared BSA-Cu NCs.
1. Introduction The development of functional nanomaterials is becoming increasingly widespread in consumer technologies.1-3 Metal nanoclusters (NCs) defined as isolated particles less than 2 nm in size with several to a hundred atoms, which bridge the gap between traditional organometallic compounds and crystalline metal nanoparticles, considered novel potential practical nanomaterials.4,5 Metal NCs with discreted energy levels showed molecule–like electronic transitions within the conduction band, and exhibited unique physical and chemical properties, as well as had practical applications in various areas, such as in cell labeling, ion sensor, and catalysts.6-9 Compared with traditional quantum dots, some superior properties such as low toxicity, good biocompatibility and multifunctional surface chemistry made metal NCs receive much attention in biological field.10-13 For example, Yeh reported fluorescent gold NCs as a biocompatible marker for in vitro and in vivo tracking of endothelial cells;12 DNA-encapsulated fluorescent silver NCs could be used as biolabels.13 On the other hand, small colloidal metal NCs exhibited superior catalytic activity due to their high surface to volume ratio and high surface energy that made surface atoms fairly active.14 Recent reports also suggested size-dependent catalytic activity of ultrasmall gold NCs, including Au25, Au55, Au38, and Au144.15 However, to date extensive research progress for NCs focused on luminescent and small Au and Ag nanomaterials.16,17 Studies for the synthesis, properties and applications of copper (Cu) NCs are still scarce primarily because This journal is © The Royal Society of Chemistry [year]
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of the difficulty in preparing highly stable and extremely tiny particles.18,19 Recently, biomimetic synthesis had become a hot topic and various biological molecules including amino acids, peptides, proteins, DNA and RNA, had been widely used for synthesizing various nanomaterials due to their advantages in controlling nucleation and growth of nanoparticles.20-24 The biomoleculeconjugated nanomaterials were more suitable for bio-applications owing to the bioactive surface.25 For example, proteins would play an important role in directing the synthesis of functional nanomaterials under mild conditions due to the amine, carboxyl, and thiol groups in proteins which can serve as effective stabilizing agents for nanoparticles.26 Bovine serum albumin (BSA), a commercially available protein, has been frequently adopted to synthesize various inorganic nanocrystals due to the presence of 35 thiol groups (from the 35 cysteine (Cys) residues) in a BSA monomer.27,28 Inspired by the protein-directed inorganic nanomaterials synthesis, fluorescent Au and Ag NCs stabilized by BSA had been studied extensively.29,30 Besides, water soluble Cu NCs capped with BSA were successfully prepared,31 however, the blue emitting fluorescence limited their practical application in biological field. Therefore, it would be of great interest to develop protein-directed synthesis of stable and biocompatible Cu NCs with red emission. Herein, we reported a simple protein-directed synthesis of stable, water–soluble and fluorescent Cu NCs, using BSA as [journal], [year], [vol], 00–00 | 1
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Protein-directed synthesis of pH-responsive red fluorescent copper nanoclusters and applications in cellular imaging and catalysts
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2. Experimental Section
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Materials: Bovine serum albumin (BSA, molecular weight of 66000) was purchased from Sigma. Copper sulfate (CuSO4), copper nitrate (Cu(NO3)2), copper chloride (CuCl2), copper acetate (Cu(AcO)2), hydrazine hydrate (N2H4.2H2O, 85 wt%) and isopropanol were analytical grade. All reagents were used as received without further purification. De–ionized water was used in all experiments. Preparation of BSA stabilized Cu nanoclusters: In a typical experiment, BSA (100 mg) was added to CuSO4 solution (5 mL, 5 mM). The solution was stirred at room temperature for 10 min, and the hydrogel was formed due to the coordination between Cu ions and the various functional groups of BSA such as –NH, –OH, and –SH. Adjusting the pH to 12 by 1 M NaOH solution, a purple transparent solution was received. Slow injection of N2H4.2H2O (1 ml) to the reaction mixture and stirring at room temperature for 4 h, the solution changed to light yellow which suggested Cu NCs were prepared. And the solution after synthesis are dialyzed against doubly distilled water for more than 24 h with a water change every 4 h, to remove all small molecular impurity. The resultant BSA-Cu NCs were freeze-drying for further application. Cellular imaging: CAL-27 cells (210 cells/ml) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicil-lin/streptomycin (DMEM) using 96-well plate. Suspensions (10 µg/mL) of BSA-Cu NCs 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 24 h. The chamber slide was then incubated at 37 0C in a CO2 incubator for 24 h for BSA-Cu NCs uptake (only 10 µg of BSA-Cu NCs to 150 µL of culture medium (105 cells) was added). Prior to fixation of the cells on the slide for inspection with a confocal fluorescence microscope, the excess BSA-Cu NCs were removed by washing 3 times with warm DPBS. Catalyst experiment: Styrene oxidation catalyzed by supported copper: 1.6 mmol of styrene, 20 mL of acetonitrile, and 18 mg of supported catalyst reacted for 16 h. When the temperature of the oil bath at was 75 °C, t-butyl hydroperoxide (TBHP, 5 mol%) was added. The products were styrene epoxide (1), benzaldehyde (2), acetophenone (3), and 2-phenylacetaldehyde (4). A blank experiment was performed in identical conditions, but without the catalyst. The products were analyzed with GCMS. Characterization Methods: 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 collected in a VG ES2 | Journal Name, [year], [vol], 00–00
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CALAB MKII spectrometer. Binding energy calibration was based on C 1s 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 spectropho-tometer. The morphology and mean diameter of resultant BSA-Cu NCs were characterized by TECNAI F20 transmission electron microscope (TEM) operating at 200 kV. The CD spectrum was measured in a Jasco 815 spectropolarimeter with a Peltier setup for the temperaturedependent measurements. CD studies were done with 10 mm path length cell. The confocal microscopy images were taken at Olympus Fluoview FV1000. All measurements were performed at room temperature under ambient conditions.
3. Results and Discussion
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The process of protein-directed synthesis of stable, water–soluble and fluorescent copper nanoclusters (Cu NCs) was shown in scheme 1. Cu NCs conjugated to BSA were synthesized in aqueous solution at room temperature using N2H4.2H2O as the reducing agent. N2H4.2H2O was a mild reducing agent and had been extensively used to synthesis nanomaterials.32 Herein BSA was chosen as the model protein to stabilize the clusters and provide steric protection due to the 17 disulfide bonds and 1 free cysteine.33 It had been widely used in the synthesis of fluorescent Ag and Au NCs.29,30 Slow injection of N2H4.2H2O to the reaction mixture of BSA and Cu ions, the solution changed to light yellow with stirring at room temperature for 4 h, which suggested Cu NCs were prepared. This preparation procedure was simple and robust. Photographs of resultant BSA-Cu NCs aqueous solution and powder are shown in Fig 1. As we can see, as-prepared BSACu NCs had good dispersion in aqueous solution and showed a color of light yellow (Fig 1a). Moreover, there was no noticeable precipitation, which was attributed to capping layer of BSA to prevent NCs from agglomeration. Under UV irradiatation (365 nm), BSA-Cu NCs showed red fluorescence (Fig 1b). The solution upon precipitation and washing gave an orange powder (Fig 1c) which showed red luminescence in the solid state (Fig 1d). The photoluminescence (PL) emission and excitation spectra were shown in Fig 1E. In aqueous solution, the BSA-Cu NCs exhibited excitation at 524 nm and emission at 625 nm with the full width at half maximum around 85 nm. The quantum yield of the as-prepared BSA-Cu NCs in aqueous solution at room temperature was found to be 4.1% using rodamine6G (QYs, 0.95 in ethanol) as the standard. As well known, this is the first report for red fluorescent Cu NCs with such high QYs.
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Scheme 1. The facile formation process of protein-directed synthesis of stable and water–soluble red fluorescent Cu NCs.
The size distribution of resultant red fluorescent BSA-Cu NCs was characterized by high-resolution transmission electron microscope (HRTEM). HRTEM images of resultant fluorescent BSA-Cu NCs was given in Fig 2a. The average size of as– prepared BSA-Cu NCs was 2.7 ± 0.4 nm. The crystal lattice This journal is © The Royal Society of Chemistry [year]
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stabling agent. Meanwhile, in this study, a mild reducing agent of hydrazine hydrate (N2H4.2H2O) was used which suggested all process could be operated at room temperature. As-prepared Cu NCs showed red fluorescence with peaking center at 620 nm and the fluorescence was responsive to pH that with adjusting the pH from 12 to 6, the fluorescent intensity increased rapidly. Additionally, resultant Cu NCs capping with BSA showed good biocompatibility and could be used as probe for cells. On the other hand, the small size of as-prepared Cu NCs suggested the NCs exhibited superior catalytic activity.
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intensity due to forming large Cu NCs (Fig S4). In addition to CuSO4, other Cu source including Cu(AcO)2, CuCl2, and Cu(NO3)2, could be used to prepare red fluorescent BSA-Cu NCs (Fig S5). Raising reaction temperature, short time would complete reaction (40 0C for 30 min, 60 0C for 5 min), however, poor fluorescence property suggested the room temperature would be the best choice (Fig S6).
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Fig. 1. Photographs of BSA-Cu NCs aqueous solution (a, b) and powder (c, d) under (a, c) visible and (b, d) UV light. (E) The excitation (EX) and emission (EM) spectra of resultant fluorescent BSA–Cu NCs in aqueous solution. 90
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Fig. 2. a, the typical TEM image of resultant red fluorescent BSA-Cu NCs. The average size of as–prepared BSA-Cu NCs was 2.7 ± 0.4 nm; b, XPS spectrum show the binding energy of Cu 2p in Cu nanoclusters, which demonstrated that the Cu was in the zero-valent state
Synthesis conditions including the pH value, the molar ratio of BSA and Cu ions, the amount of N2H4.2H2O, the reaction temperature, and various kinds of Cu source were studied. An optimal composition of 20.0 mg/mL BSA, 5.0 mM CuSO4 1 ml N2H4.2H2O and pH =12 produces the highest fluorescence intensity of the BSA-Cu NCs bioconjugates. The pH of reaction solution is an important consideration in the synthesis of fluorescent BSA-Cu NCs as shown in Fig S1. Experiments conducted in neutral (pH =6) and acidic pH (pH =3) did not result in the clusters (Fig S1), only the blue emission of BSA (seeing the PL spectrum of BSA without copper source in Fig S2). At high pH, the fluorescence of Cu NCs appeared (pH =9) and formed optimal fluorescence intensity of the BSA-Cu NCs at pH =12 due to the phenolic moieties of tyrosine in BSA (Fig S1). Fig S3 demonstrated that an increase in the molar ratio of BSA and Cu ions would increase the yield of BSA-Cu NCs bioconjugates and further increasing the molar ratio will decrease the fluorescence intensity. N2H4.2H2O as the reducing agent, an increase of the amount would produce high fluorescence intensity; however, further increasing would quench the fluorescence This journal is © The Royal Society of Chemistry [year]
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Fig. 3 The relative fluorescence intensity was determined by calculating the ratio of the fluorescence intensities of BSA-Cu NCs in the presence and absence of common anions (0.10 M).
The stability of luminescent nanomaterials was an important factor to assess their applications.36 The as-prepared fluorescent Cu NCs was conjugated to BSA which was confirmed by FITR and XPS spectra, which provided resultant Cu NCs with good stability. Fig S7 depicts the FITR spectra of the free BSA and BSA-Cu NCs bioconjugates. There were not any differences in their spectra, inferring that the Cu NCs embedded in BSA would not affect the surface-structure of BSA.37 Besides, the XPS spectra of Fig S8 gave the binding energy of S 2p, C 1s, N 1s, and O 1s which was another evidence of existing BSA in as-prepared Cu NCs.38 In summary, metallic Cu NCs are successfully synthesized and embedded in BSA. The stability was investigated on the interference of some common anions (0.10 M) on the fluorescence of BSA-Cu NCs. The relative fluorescence intensity was determined by calculating the ratio of the fluorescence intensities of BSA-Cu NCs in the presence and absence of the interference ions as shown Fig 3. Most of these ions showed either no or slight interferences. The fluorescence spectra of BSA-Cu NCs in various common anions in detail were given in Fig S9. It is well known that BSA has different conformations at different pH values which would affect the physical properties of nanomaterials.39 Interestingly strong optical responses to pH changing were observed for as-prepared BSA-Cu NCs. To study pH responses property, a series of universal buffer solutions with pH ranging systematically from 6 to 12 were prepared and the fluorescence intensities of BSA-Cu NCs in buffers of different pH were monitored. Significant changes in fluorescence intensity were observed as the pH was varied, as depicted in Fig 4a. When the pH changed from 12 to 6, the fluorescence intensity of BSAJournal Name, [year], [vol], 00–00 | 3
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fringes are 2.05 Å apart which indicates the (111) planes of the metallic Cu (Fig 2c). Moreover, there was no formation of larger size metal nanoparticles or aggregation due to BSA as protective layer. XPS analysis is carried out to determine the oxidation state of copper in the BSA–Cu NCs. In the present study, the binding energy of Cu 2p3/2 was observed at 932.3 eV, which is assigned to the binding energies of the of Cu(0),34 and a weak peak can be observed at 953eV, suggesting very minimal presence of Cu(II) in the system.35 While, the existed oxidation state on the surface of NCs would ensure stability and enhance the florescence.36
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Cu NCs increased nearly 2-fold. Within the neutral pH range 6-7, the highest fluorescence intensity were found, which indicated resultant BSA-Cu NCs was more suitable for cellular imaging. We also verified that these changes were reversible between pH 6 and 12, as shown in Fig 4b. Besides, there were no discernible shifts in the peak emission at different pH values. Hence the reason for responses to pH was neither shell degradation and hole trapping from thiolate formation nor aggregation through irreversible ligand loss.40,41
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Fig. 5 The circular dichroism (CD) spectroscopy show the secondary structures of BSA in the reaction system at different pH values.
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Fig. 4 a, fluorescence intensity of BSA-Cu NCs responses to pH ranging systematically from 6 to 12, inset showing when the pH changed from 12 to 6, the fluorescence intensity of BSA-Cu NCs increased nearly 2-fold; b, reversible between pH 6 and 12 for four times was observed
The changes in the PL at different pH may broadly be attributed to corresponding structural changes of BSA.42 The secondary structures of BSA in the reaction system were investigated by circular dichroism (CD) spectroscopy at different pH values (Fig 5), which is a valuable spectroscopic technique for studying protein and its complex. With the decrease in pH of the solution, more and more α helix were stretched and transformed into β sheets, which could contribute to the impairment or break of hydrogen bonds. In previous report, the β sheet secondary structure could form a suitable conformation for the oriented growth of NPs which result in NPs uniformly coated with BSA.43 With the decrease of pH, β sheet as well as random coil structural elements became more predominant which made more functional groups, like –OH, –NH, –COOH, accessible to interact with the surface of NCs, hence, the luminescence intensity of the as-prepared materials is high at low pH owing to improved surface passivation of NCs. Therefore, these dramatic changes of the secondary structure may thus play a significant role in the observed trends in pH responsiveness of BSA-Cu NCs. Copper, one of the transition metals, is an essential trace element for life. Although excessive amounts of copper can result in severe diseases, low amounts of copper ions play a pivotal role in many fundamental physiological processes in organisms, such as
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Fig. 6. Viability of Cal-27 cells after 24 h incubation with different concentrations of BSA-Cu NCs in the cell medium as determined by a MTT assay. 90
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With an arresting set of features, including water-dispersible, red fluorescent, good biocompatible and surface-bioactive, asprepared BSA-Cu NCs indicated great potential in cellular marking. The cell internalization and intracellular distribution of resultant BSA-Cu NCs were evaluated by confocal laser fluorescence microscopy. Fig 7a–c respectively revealed the bright field, confocal fluorescent and overlay images of CAL-27 cells incubating with as-prepared BSA-Cu NCs for 24 h. From the bright field image of CAL-27 cells, we could see the cells incubating with resultant BSA-Cu NCs are still maintain their normal morphology, thus indicating the good biocompatibility of as-prepared BSA-Cu NCs in this specific dose and time point. The fluorescent image irradiated by 515 nm, showed bright red fluorescence within the cells that indicated the uptake behaviors of CAL-27. It could be seen that the fluorescent signal was distributed not only in cytoplasm but also most in cellular nucleus. This result demonstrated BSA-Cu NCs could be used as a probe for real-time cellular imaging, especially labeling the oligonucleotide as a DNA probe for specific detection of nucleic acids. This result was consistent with previous reports about fluorescent NCs with similar small size.45
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Fig. 7 (a) bright field, (b) confocal fluorescent and (c) overlay images of Cal-27 cells incubating with BSA-Cu NCs for 24 h.
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bone formation, connective tissue development and cellular respiration.44 Besides, as-prepared BSA-Cu NCs was protected by a layer of BSA. These conditions suggested as-prepared BSACu NCs displayed the low or no toxic to cells at suitable concentration. MTT assay and apoptosis assay were used to evaluate the cytotoxicity of as-prepared BSA-Cu NCs and the viability of cells. From the results of MTT assay (Fig 6), cell viability still remained above 70% after incubating with asprepared BSA-Cu NCs even at the concentration of 80 µg/mL for 24 h. Thus the result showed that GNDs had low acute toxicity to cells.
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Notes and references a
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Fig. 8 Styrene oxidation catalyzed by as-prepared BSA-Cu NCs; see Table 1 for details Table 1. Styrene oxidation catalyzed by supported various Cu NCs. The products were styrene epoxide (1), benzaldehyde (2), acetophenone (3), and 2phenylacetaldehyde (4); see Figure 9 for product details
Entry
Cu(NO3)2 CuCl2 CuSO4 Cu(OAc)2 30
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Cu cluster loading (%)
Styrene conversion [mol %]
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Product selectivity [mol %] 85
1 25 27 25.5 28
2 72 69 70 66
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4 1 1 1.5 1
4. Conclusion In summary, a simple protein-directed synthesis method was applied to prepare stable, water–soluble and fluorescent copper nanoclusters (Cu NCs). The surface of as-prepared BSA-Cu NCs was protected by a layer of BSA and showed stability in various common anions. With the help of mild reducing agent of N2H4.2H2O, the BSA-Cu NCs could be easily prepared at room temperature. As-prepared BSA-Cu NCs showed red fluorescence with peaking center at 620 nm (quantum yield 4.1%). The fluorescence of as-prepared BSA-Cu NCs was responsive to pH. The intensity of fluorescence increased rapidly with decreasing the pH from 12 to 6 and the responsive property was reversible. It is also verified that the resultant BSA-Cu NCs could be used a probe for cells and as the catalyst due to an arresting set of features, including water-dispersible, red fluorescent, good biocompatible, surface-bioactive and small size. In this study, the resultant BSA-Cu NCs was used as a probe for labeling CAL-27 cells. Moreover, BSA-Cu NCs was used as catalyst in the reaction of styrene oxidation and displayed high (70%) selectivity for benzaldehyde as the major product and with 70% conversion of the styrene
Acknowledgements This journal is © The Royal Society of Chemistry [year]
This work was supported by the National Natural Science Foundation of China (Grants No.21174048 and No. 51373061) and Programme of Introducing Talents of Discipline to Universities (111 Project B06009).
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China-Australia Joint Research Centre for Functional Molecular Materials, School of Chemical & Material Engineering, Jiangnan University, Wuxi 214122, P. R. China E–mail: [email protected]. b State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, 130012, P. R. China E–mail: linquan@ jlu.edu.cn c State Key Laboratory of Materials-oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009, P. R. China † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/ 1. C. X. Wang, D. Zhang, L. Xu, Y. N. Jiang, F. X. Dong, B. Yang, K. Yu and Q. Lin, Angew. Chem. Int. Ed. 2011, 50, 7587 –7591. 2. Y. Z. Lu and W. Chen, Chem. Soc. Rev. 2012, 41, 3594–3623. 3. T. Udayabhaskararao, Y. Sun, N. Goswami, K. P. Samir, K. Balasubramanian and T. Pradeep, Angew. Chem. Int. Ed. 2012, 51, 2155 –2159. 4. H. F. Qian, M. Z. Zhu, Z. K. Wu and R. C. Jin, Accounts Of Chemical Research. 2012, 45, 1470–1479. 5. H. X. Xu and S. S. Kenneth, Adv. Mater. 2010, 22, 1078–1082. 6. B. S. González, M. J. Rodríguez, C. Blanco, J. M. A. LópezQuintela and J. M. G. Martinho, Nano Lett. 2010, 10, 4217–4221. 7. S. I. Tanaka, J. Miyazaki, D. K. Tiwari, T. Jin and Y. Inouye, Angew. Chem. Int. Ed. 2011, 50, 431 –435. 8. C. C. Huang, Z. S. Yang, K. H. Lee and H. T. Chang, Angew. Chem. Int. Ed. 2007, 46, 6824 –6828. 9. M. Hyotanishi, Y. Isomura, H. Yamamoto, H. Kawasaki and Y. Obora, Chem. Commun. 2011, 47, 5750–5752. 10. Y. Y. Cui, Y. L. Wang, R. Liu, Z. P. Sun, Y. T. Wei, Y. L. Zhao and X. Y. Gao, ACS Nano. 2011, 5, 8684–8689. 11. S. Kumar, M. D. Bolan and T. P. Bigioni, J. Am. Chem. Soc. 2010, 132, 13141–13143. 12. H. H. Wang, C. J. Lin, C. H. Lee, Y. C. Lin, Y. M. Tseng, C. L. Hsieh, C. H. Chen, C. H. Tsai, C. T. Hsieh, J. L. Shen, W. H. Chan, W. H. Chang and H. I. Yeh, ACS Nano. 2011, 5, 4337–4344. 13. J. H. Yu, S. M. Choi and R. M. Dickson, Angew. Chem. Int. Ed. 2009, 48, 318 –320. 14. S. Biswas, J. T. Miller, Y. H. Li, K. Nandakumar and C. S. S. R. Kumar, Small. 2012, 8, 688-698. 15. H. J. Zhang, T. Watanabe, M. Okumura, M. Haruta and N. Toshima, Nat. Mater. 2012, 11, 49. 16. Z. K. Wu, M. Wang, J. Yang, X. H. Zheng, W. P. Cai, G. W. Meng, H. F. Qian, H. M. Wang and R. C. Jin, Small. 2012, 8, 2028–2035. 17. W. J. Cho, Y. Kim and J. K. Kim, ACS Nano. 2012, 6, 249–255. 18. W. T. Wei, Y. Z. Lu, W. Chen and S. W. Chen, J. Am. Chem. Soc. 2011, 133, 2060–2063. 19. X. F. Jia, J. Li, L. Han, J. T. Ren, X. Yang and E. K. Wang, ACS Nano. 2012, 6, 3311–3317. 20. L. Turyanska, T. D. Bradshaw, J. Sharpe, M. Li, S. Mann, N. R. Thomas and A. Patane, Small. 2009, 5, 1738–1741. 21. M. X. Yu, C. Zhou, J. B. Liu, J. D. Hankins and J. Zheng, J. Am. Chem. Soc. 2011, 133, 11014–11017. 22. M. Naito, K. Iwahori, A. Miura, M. Yamane and I. Yamashita, Angew. Chem. Int. Ed. 2010, 49, 7006 –7009. 23. 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, 5038–5039. 24. Y. H. Lin, M. L. Yin, F. Pu, J. S. Ren, X. G. Qu, Small. 2011, 7, 1557–1561. 25. T. Li, L. B. Zhang, J. Ai, S. J. Dong and E. K. Wang, ACS Nano. 2011, 5, 6334–6338.
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On the other hand, NCs can be applied in catalysts owing to their ultrasmall size and high surface energy which had been extensively studied.9,46 Copper nanoparticles have been previously shown to be active catalysts for a number of organic reactions especially for cross-coupling reactions.47 However, catalysis of Cu NCs had scarcely been investigated.14,18 Herein, the reaction of oxidation of styrene was used as a model reaction to elucidate the catalytic activity of as-prepared BSA-Cu NCs. Thermal gravimetric analysis (TGA) shows that the organic weight loss of as-prepared BSA-Cu NCs is 65% and therefore, the Cu content of BSA-Cu NCs is calculated as 35% (Fig S10). The styrene oxidation resulted in several reaction products (Table 1; Fig 8) with a high (70%) selectivity for benzaldehyde as the major product and with 70% conversion of the styrene. The oxidation reaction without catalyst occurs very slowly with a final conversion of only 7%.
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26. H. Kawasaki, K. Hamaguchi, I. Osaka and R. Arakawa, Adv. Funct. Mater. 2011, 21, 3508–3515. 27. Y. L Wang, J. J. Chen and J. Irudayaraj, ACS Nano. 2011, 5, 9718– 9725. 28. H. W. Li, K. L. Ai and Y. Q. Wu, Chem. Commun. 2011, 47, 9852– 9854. 29. J. P. Xie, Y. G. Zheng and J. Y. Ying, J. Am. Chem. Soc. 2009, 131, 888–889. 30. X. L. Guevel, B. Hotzer, G. Jung, K. Hollemeyer, V. Trouillet and M. Schneider, J. Phys. Chem. C. 2011, 115, 10955–10963. 31. N. Goswami, A. Giri, M. S. Bootharaju, P. L. Xavier, T. Pradeep and S. K. Pal, Anal. Chem. 2011, 83, 9676–9680. 32. 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 – 1656. 33. A. Mathew, P. R. Sajanlal and T. Pradeep, J. Mater. Chem. 2011, 21, 11205. 34. X. Yuan, Z. T. Luo, Q. B. Zhang, X. H. Zhang, Y. G. Zheng, J. Y. Lee and J. P. Xie, ACS Nano. 2011, 5, 8800–8808. 35. C. Vazquez-Vazquez, M. Banobre-Lopez, A. Mitra, M. A. LopezQuintela and J. Rivas, Langmuir. 2009, 25, 8208–8216. 36. 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 37. A. Retnakumari, S. Setua, D. Menon, P. Ravindran, H. Muhammed, T. Pradeep, S. Nair and M. Koyakutty, Nanotechnology. 2010, 21, 055103 38. L. Yan, Y. Q. Cai, B. Z. Zheng, H. Y. Yuan, Y. Guo, D. Xiao and M. M. F. Choi, J. Mater. Chem. 2012, 22, 1000. 39. D. Ghosh, S. Mondal, S. Ghosh and A. Saha, J. Mater. Chem. 2012, 22, 699. 40. W. H. Liu, H. S. Choi, J. P. Zimmer, E. Tanaka, J. V. Frangioni and M. Bawendi, J. Am. Chem. Soc. 2007, 129, 14530-14531. 41. S. Jeong, M. Achermann, J. Nanda, S. Ivanov, V. I. Klimov and J. A. Hollingsworth, J. Am. Chem. Soc. 2005, 127, 10126-10127. 42. Y. Z. Wu, S. Chakrabortty, R. A. Gropeanu, J. Wilhelmi, Y. Xu, S. K. Er, S. L. Kuan, K. Koynov, Y. Chan and T. Wei, J. Am. Chem. Soc. 2010, 132, 5012–5014. 43. N. E. Kadi, N. Taulier, J. Y. L. Huérou, M. Gindre, W. Urbach, I. Nwigwe, P. C. Kahn and M. Waks, Biophysical Journal. 2006, 91, 3397–3404. 44. X. J. Liu, C. H. Zong and L. H. Lu, Analyst. 2012, 137, 2406. 45. 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, 3137–3142. 46. X. T. Nie, H. F. Qian, Q. J. Ge, H. Y. Xu and R. C. Jin, ACS Nano. 2012, 6, 6014–6022. 47. L. D. Pacho, J. H. VanMaarseveen and G. Rothenberg, Adv. Synth. Catal. 2005, 347, 811 –815.
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DOI: 10.1039/C3NR04835G
TOC A simple and green protein-directed synthesis of stable, water–soluble and fluorescent Cu NCs. Asprepared Cu NCs could be applied in cellular imaging and catalysts fields.
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