DOI: 10.1002/chem.201303964
Communication
& Antibacterial Nanomaterials
Reduced Graphene Oxide Functionalized with a Luminescent Rare-Earth Complex for the Tracking and Photothermal Killing of Drug-Resistant Bacteria Xinjian Yang,[a, b] Zhenhua Li,[a, b] Enguo Ju,[a, b] Jinsong Ren,*[a] and Xiaogang Qu*[a] ering the nonspecific toxicity and less stability of large GO, their clinical usage would be limited. Therefore, it is necessary to explore more advanced system for better treatment of diseases based on bacteria infection with high specificity, excellent temporal/spatial controllability, and ultimately improved efficacy. One promising approach is to use nanomaterial-based photothermal process for selective treatment of bacterial infections.[4] Previous studies have developed light-absorbing photothermal agents to serve as heat carriers for local hyperthermia. These materials can absorb light and convert it into toxic heat upon irradiation.[5] However, to avoid nonspecific heating of healthy cells, photothermal agents must show high absorption and selective aggregation at targeting site over normal tissues. In an effort to improve the safety of thermal therapies, near-infrared (NIR)-absorbing photothermal agents have been explored in combination with laser irradiation to provide sitespecific heating and treatment of diseased regions with a high uptake of the photothermal agents.[5c, 6] Among these NIR-absorbing photothermal agents, reduced graphene oxide (rGO) has been recognized as a suitable architecture for photothermal treatment of cancers owing to its extraordinary electronic, optical, and thermal properties.[7] Nevertheless, little have been done to invest its photothermal killing ability of bacteria.[8] Herein we constructed a graphene-based nanoarchitecture for targeted bacteria imaging and photothermal killing of drug resistant bacteria. To test the design, we attached vancomycin (Van) and a luminescent rare-earth complex to the surface of small rGO covalently. As shown in Scheme 1, Van was introduced to generate multivalent interactions and capture the bacteria.[9] As the 4f orbitals of rare-earth elements are shielded by 5s5p6s orbitals, rare-earth compounds exhibit unique spectroscopic characteristics, such as sharp line-like atomic emission, to overcome autofluorescence and light scattering.[10] Therefore, Van-modified rGO was further functionalized with a europium (Eu3 + ) complex, which would emit strong red luminescence upon excitation of the antenna molecule. Taken together, this novel approach would allow the trace of pathogens as well as bacteria killing upon exposure to a NIR laser beam. GO was synthesized from natural graphite powder using a modified Hummers method. Previous reports have demonstrated that small rGO could act as an efficient photothermal agent for cancer treatment. Therefore we first obtained monodisperse GO with a diameter of less than 150 nm (Figure 1 a, b). Amine-terminated poly(ethylene glycol) (PEG) was then cova-
Abstract: An antibacterial platform based on multifunctional reduced graphene oxide (rGO) that is responsive to near-infrared (NIR) light has been constructed. By introducing a luminescent Eu3 + complex and vancomycin for bacteria tracking into one system, this platform could specifically recognize and light up bacteria. Antibacterial activity of this nanoscale construction under NIR illumination was investigated. Upon illumination with NIR light, this nanoscale architecture generates great heat locally, resulting in the death of drug-resistant bacteria. These results indicate that the ability of this nanoscale platform to kill drug-resistant bacteria has great potential for clinical pathogenic bacteria diagnosis and treatment.
The rapid emergence of antibiotic-resistant bacterial strains[1] threatens public health globally and has sparked great interest in the design of novel approaches to combat the diverse and numerous mechanisms that bacteria employ to provide themselves protection against the action of antibacterial therapeutics. Treatments involving the use of agents that cause physical or chemical damage to the bacteria have drawn increasing attention.[2] For instance, graphene-based nanomaterials have been recently demonstrated to show promising antibacterial activity.[3] The interaction between the nanomaterials and bacteria was found to be nonspecific, and the antibacterial activity of GO sheets was dependenton lateral size.[3c] Larger GO showed stronger antibacterial activity than that of small GO, which is due to their easy coating on the surface of bacteria. However, they were easy to aggregate at physiological environment, which might result in undesired side effects. Consid[a] X. Yang,+ Z. Li,+ E. Ju, Prof. Dr. J. Ren, Prof. Dr. X. Qu Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization Changchun Institute of Applied Chemistry Chinese Academy of Sciences, Changchun, 130022 (China) Fax: (+ 86) 431-85262625 E-mail: [email protected] [email protected] [b] X. Yang,+ Z. Li,+ E. Ju University of Chinese Academy of Sciences Beijing, 100039 (China) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303964. Chem. Eur. J. 2014, 20, 394 – 398
394
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
Scheme 1. a) Illustration of Eu-Van-rGO for targeted bacteria imaging and photothermal killing; b) the structure of the Eu complex; c) the structure of vancomycin.
lently linked to the epoxide groups to make the GO stable in buffer solutions and ready for further modification. Next, the
GO sheets were reduced by adding sodium borohydride into the solution. During the reduction step, the colour of the solution changed from yellow to black, which indicated the increasing light absorption in the NIR region owing to the recovery of conjugated p network of graphene (Figure 1 c). Centrifugation was then performed to remove any aggregation. Van was firstly modified on the PEG terminal by EDC/NHS chemistry. The Van-rGO was purified using dialysis membrane with a molecular cut-off of 14 000 g mol 1. The modification produces were monitored by FTIR spectra which demonstrated the success of functionalization (Supporting Information, Figure S1). To detect the effects of 808 nm optical excitation of rGO, we carried out a control experiment by irradiating aqueous solutions of Van-rGO at different concentrations (Figure 1 d). Irradiated with 808 nm laser at 1.5 W cm 2 for 300 s, Van-rGO solution (20 mg mL 1) showed a temperature elevation up to 20 8C, and the temperature increase depended on the concentration of the rGO. However, irradiation of the aqueous solution without Van-rGO or with 20 mg mL 1 GO only caused a smaller temperature elevation (Supporting Information, Figure S2). These findings clearly demonstrated the enhanced absorption of the 808 nm light by the rGO and confirmed that the as-prepared Van-rGO had photothermal capability. Thus, the Van-rGO was expected to be employed as photothermal agent for inhibiting the cell growth of target bacteria. Vancomycin is a potent antibiotic that interacts with terminal d-Ala-d-Ala moieties of peptide units located on bacterial
Figure 1. a), b) Images of GO: a) TEM (scale bar 100 nm); b) AFM (scale bar 50 nm; height profile in black determined from white line: 1.12 nm). c) UV/Vis spectra of Van-rGO and d) photothermal heat generated from different concentrations of Van-rGO under NIR irradiation. Chem. Eur. J. 2014, 20, 394 – 398
www.chemeurj.org
395
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication cell walls. Previous studies have demonstrated that Van-modified nanoparticles are capable of recognizing pathogenic bacteria.[9] We then examined the targeting capacity of Van-rGO for several pathogenic bacteria. In our experiment, Escherichia coli (E. coli), kanamyclin drug-resistant E. coli (DR-E. coli), Staphylococcus aureus (S. aureus), and oxacillin drug-resistant S. aureus (DR-S. aureus) were selected as the models to test the feasibility of using Van-rGO as the affinity probes. E. coli and S. aureus bacterial strains were utilized as they are representative of the two major classes of bacteria: Gram-negative and Gram-positive, respectively. Bacteria were allowed to incubate with the Van-rGO for 20 min prior to preparing for SEM analysis. As shown in Figure 2 and the Supporting Information, Figure S3, SEM revealed that all of the four bacteria were indeed covered with Van-rGO. However, in a control experiment, less
Figure 3. a) Fluorescent spectrum of Van-rGO (black) and Eu-Van-rGO (grey); b) photographs Van-rGO and Eu-Van-rGO under daylight and UV light; c), d) fluorescence microscopy images of DR-E. coli and DR-S. aureus incubated with Eu-Van-rGO.
tained DR-E. coli DR-S. aureus after 10 min incubation with EuVan-rGO. As can be seen from the images, both of the bacteria were illuminated, which further indicated that the Van could work as an efficient agent for bacteria targeting and Eu3 + complex could act as an imaging agent for bacteria tracking. Having demonstrated that Van-rGO could strongly absorb NIR radiation and could ultimately transfer this energy into the surrounding environment as heat, we next investigated the photothermal killing of these bacteria by using Van-rGO as photothermal agents under illumination with an NIR laser (808 nm, 1.5 W cm 2) for 3 min. The bacteria were then cultured and recorded with colony counts. As shown in Figure 4, there was a more than 20 % decrease in cell viability upon treatment with Van-rGO at a concentration of 20 mg mL 1. This result demonstrated that Van-rGO showed toxicity toward bacteria which was in accordance with previous reports.[3] However, the antibacterial activity relies on the concentration of VanrGO in the sample. Higher antibacterial activity was observed at higher concentrations (Supporting Information, Figure S6). Interestingly, when the bacteria were treated with Van-rGO upon NIR light irradiation, a high antibacterial activity was uncovered. Control experiments (Supporting Information, Figure S7) demonstrated that there was not obvious change in bacteria survival rate if the bulk solution was just heated to the same temperatures as that obtained by NIR light irradiation. When NIR light was introduced, all of the Gram-negative bacteria were killed at a Van-rGO concentration of 10 mg mL 1, and less than 3 % of Gram-positive bacteria survived at the same condition. This difference in antibacterial ability is because that the cell wall of Gram-positive bacteria is thicker than that of Gram-positive bacteria. As the concentration of Van-rGO increased, the cell growth of bacteria is inhibited completely. This is rational because high concentrate of VanrGO attached on the surface of bacteria can raise the local
Figure 2. SEM images of a) DR-E. coli incubated with Van-rGO, b) DR-E. coli incubated with Van-rGO under NIR irradiation; c) DR-S. aureus incubated with Van-rGO, d) DR-S. aureus incubated with Van-rGO under NIR irradiation. Scale shown is 3.00 mm.
PEG-rGO covered on the surface of bacteria (Supporting Information, Figure S4). These results clearly indicated that the interaction between the nanomaterials and the bacteria cell surface was mediated through specific interactions between vancomycin on the nanomaterials and receptors on the surface of the bacteria, rather than nonspecific interactions. To visualize bacteria by fluorescence microscopy, the van-labelled rGO was further labelled with a fluorescence probe, a Eu3 + complex, through EDC/NHS chemistry (F-Van-rGO). In our experiment, PEG played an important role, which not only made the nanoarchitecture stable, but also protected the Eu3 + complex from quenching. Spectroscopic properties of Eu-VanrGO were then investigated. Both Eu-Van-rGO and Eu3 + -complex have strong absorption at 350 nm and characteristic sharp emission lines in the red light-emitting region that are typical for Eu3 + ions (Supporting Information, Figure S5). Red PL is observable by the naked eye upon UV excitation (Figure 3 b). Therefore, rGO covalently modified by a red PL rareearth complex was designed and synthesized. Figure 3 c, d shows the fluorescent images obtained from samples that conChem. Eur. J. 2014, 20, 394 – 398
www.chemeurj.org
396
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication was introduced or not. When the incubation time increased from 1 to 4 h before NIR illumination, more than 75 % of the cells survived under the set of conditions. A co-culture experiment was then performed to assess the selectivity of the VanrGO when both mammalian and bacterial cells are introduced simultaneously. Most of the HepG 2 cells died and were detached from the cultivation base after 48 h, while the bacteria have proliferated as assessed by visual inspection (Supporting Information, Figure S9 e, h). However, when NIR light was introduced to kill the bacteria, most of the HepG 2 cells survived and no bacteria proliferation was observed (Supporting Information, Figure S9 f, i). Therefore, NIR irradiation to the infected sites will lead to selective photothermal-based killing of individual bacteria with less harmful effects on surrounding tissues. These results demonstrated that Van-rGO exhibited good biocompatibility and could be used as a potential antibacterial nanodrug for clinical application. In summary, we have demonstrated a novel near-infrared light responsive platform for targeted imaging and photothermal killing of drug-resistant bacteria based on multifunctional rGO. This nanoarchitecture was easily constructed by introducing luminescent Eu3 + complex and vancomycin for bacteria tracking. Antibacterial activity of this nanoconstruction under NIR illumination was investigated. Upon illumination of NIR light, this nanoarchitecture generates much heat locally, resulting in the death of bacteria. Our results indicate that drug resistant bacteria killing ability of this nanoplatform is endowed with great potential for clinical pathogenic bacteria diagnosis and treatment.
Figure 4. Viability of bacteria after incubation with Van-rGO (a) and bacteria incubated with Van-rGO under NIR irradiation (b).
temperature more efficiently, thus leading to more effective cell killing. The bacteria were followed with cell membrane disruption (Figure 2 b, d). It has been suggested that cell membrane damage following nanoparticles exposure to NIR radiation could be due to numerous factors, including nanoparticle explosion, shock waves, bubble formation, and thermal disintegration.[4d] Meanwhile, there was no obvious difference of photothermal bacteria killing ability comparing the bacteria with their drug resistant form. Therefore, combining its inherent antibacterial activity with photothermal cell-killing ability, this platform will be an ideal candidate to overcome the drug resistance problem. Based on the American National Standard for the Safe Use of Lasers, the maximum skin exposure for a continuous-wave 808 nm laser is about 0.33 W cm 2.[11] Although the NIR irradiation power used in our system was above the laser safety standards for humans, we could enhance the antibacterial ability of Van-rGO by increasing illumination time at lower irradiation intensity. HepG 2 cells were used as a model to examine whether illumination of these materials under NIR light might affect the growth of human cells. HepG 2 cells were incubated with VanrGO, and additional washing three times with phosphate buffer was carried out to remove remaining Van-rGO in solution before NIR illumination. The MTT results obtained by incubating HepG 2 cells with Van-rGO under different experimental conditions are shown in the Supporting Information, Figure S8. Van-rGO had little impact on the cell growth, whether NIR light Chem. Eur. J. 2014, 20, 394 – 398
www.chemeurj.org
Experimental Section Preparation of Van-PEG-rGO: Small GO was synthesized from natural graphite powder using a modified Hummers method. The PEG-rGO was prepared by vigorously stirring a solution of the graphene oxides (20 mg), amido-PEG (PEG diamine with 35 repeating units; 100 mg), and KOH (100 mg) in H2O (50 mL) at 70 8C for 24 h. Then a NaBH4 solution (10 mL, 1 m) was added, and the reaction was kept on at 70 8C for 2 h. The PEG-rGO was then collected and purified by using a dialysis membrane with a molecular cut-off of 14 000 g mol 1 to remove impurities and the excess of PEG by physical absorption. PEG-rGO (5 mg) in MES (18 mL, 30 mm) buffer was then mixed with vancomycin (2 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; 8 mg), and N-hydroxysuccinimide (NHS; 6 mg) and vigorously stirred for 6 h at room temperature. The resulting vancomycin-modified nanosheets (VanrGO) were purified using a dialysis membrane with a molecular cut-off of 14 000 g mol 1 to ensure all of the free unreacted vancomycin, EDC, and NHS were removed away from Van-rGO. Preparation of Eu-Van-rGO: [Eu(TFAcAcN)3Bpc] was synthesized according to the conventional route as follows: 1-(2-Naphthoyl)3,3,3-trifluoroacetone (TFAcAcN; 160 mg, 0.6 mm) and 2, 20-bipyridine-4,40-dicarboxylic acid (Bpc; 49 mg, 0.2 mm) were dissolved in ethanol (10 mL) under stirring at room temperature. Five drops of NaOH (2 m) were then added to adjust the pH level to 8.0. Thereafter a EuCl3 solution (51 mg, 0.2 mm) in ethanol (3.5 mL) was added dropwise. After complete addition, the solution was stirred for 1 h to ensure a complete precipitation at room temperature. The precipitate was filtered out, washed repeatedly with ethanol
397
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication and water, and then dried overnight in vacuum. [Eu(TFAcAcN)3Bpc] (1 mg), NHS (2 mg), and EDC (4 mg) in DMF/MES were then mixed and stirred for 30 min, and then the mixture was added dropwise into Van-rGO solution (0.2 mg mL 1 10 mL). The solution was then stirred for 12 h at room temperature and dialyzed (dialysis bag MWCO 14 000) for 24 h. The four bacteria strains were grown in LB medium at 37 8C. The resultant bacterial cells were isolated by centrifugation (3500 rpm). The bacterial cells were rinsed with Tris buffer (50 mm, pH 7.4), and designed bacterial concentration was adjusted by measuring the optical density at 600 nm (OD600nm).
[3]
[4]
[5]
To examine the photothermal effect of Van-rGO, the aqueous sample solutions were irradiated using a diode laser (808 nm). The power of the laser was 1.5 W cm 2. The temperature of the resultant solution was measured by a thermocouple. Cell viability test: Two groups of test bacterial suspensions (105– 106 CFU mL 1) were incubated with different concentration of VanrGO for 10 min. One group of the bacteria was then irradiated with NIR light for 3 min. The resultant bacterial suspensions were cultured in Petri dishes containing LB agar medium at 37 8C, overnight. The CFU of the bacteria were then estimated.
[6]
For the co-culture test (HepG 2 cells–(DR-E. coli) and DR-S. aureus), the concentration of cancer cells was fixed at a density of 105 cells per well in 24-well assay plates and infected with DR-E. coli and DR-S. aureus. Van-rGO (20 mg mL 1) was then added with and without NIR light (1.5 W cm 2, 3 min). Co-cultures were incubated at 37 8C and 5 % CO2. Images of HepG 2 cells in the presence of DRE. coli and DR-S. aureus were recorded at 24 and 48 h using an Olympus BX-51 optical system microscope.
[7]
Acknowledgements
[8] [9]
Financial support was provided by the National Basic Research Program of China (Grant 2012CB720602, 2011CB936004) and the National Natural Science Foundation of China (Grants 21072182, 21210002, 91213302). Keywords: imaging · luminescent complexes · rare-earth complexes · photothermal killing · reduced graphene oxide
[10]
[1] a) A. J. Alanis, Arch. Med. Res. 2005, 36, 697 – 705; b) F. C. Tenover, Am. J. Infect. Control. 2006, 34, S3 – S10; c) H. Yoneyama, R. Katsumata, Biosci. Biotechnol. Biochem. 2006, 70, 1060 – 1075. [2] a) I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 2004, 275, 177 – 182; b) C. A. Strassert, M. Otter, R. Q. Albuquerque, A. Hçne, Y. Vida, B. Maier, L. De Cola, Angew. Chem. 2009, 121, 8070 – 8073; Angew. Chem. Int. Ed. 2009, 48, 7928 – 7931; c) W. S. Kuo, C. N. Chang, Y. T. Chang, C. S. Yeh,
[11]
Chem. Eur. J. 2014, 20, 394 – 398
www.chemeurj.org
Chem. Commun. 2009, 4853 – 4855; d) P. C. Ray, S. A. Khan, A. K. Singh, D. Senapati, Z. Fan, Chem. Soc. Rev. 2012, 41, 3193 – 3209. a) S. Liu, T. H. Zeng, M. Hofmann, E. Burcombe, J. Wei, R. Jiang, J. Kong, Y. Chen, ACS Nano 2011, 5, 6971 – 6980; b) W. Hu, C. Peng, W. Luo, M. Lv, X. Li, D. Li, Q. Huang, C. Fan, ACS Nano 2010, 4, 4317 – 4323; c) S. Liu, M. Hu, T. H. Zeng, R. Wu, R. Jiang, J. Wei, L. Wang, J. Kong, Y. Chen, Langmuir 2012, 28, 12364 – 12372; d) O. Akhavan, E. Ghaderi, ACS Nano 2010, 4, 5731 – 5736. a) W. C. Huang, P. J. Tsai, Y. C. Chen, Small 2009, 5, 51 – 56; b) T. J. Yu, P. H. Li, T. W. Tseng, Y. C. Chen, Nanomedicine 2011, 6, 1353 – 1363; c) C. Wang, J. Irudayaraj, Small 2010, 6, 283 – 289; d) S. Wang, A. K. Singh, D. Senapati, A. Neely, H. Yu, P. C. Ray, Chem. Eur. J. 2010, 16, 5600 – 5606. a) M. Chu, X. Pan, D. Zhang, Q. Wu, J. Peng, W. Hai, Biomaterials 2012, 33, 7071 – 7083; b) Y. Luo, Y. S. Shiao, Y. F. Huang, ACS Nano 2011, 5, 7796 – 7804; c) X. Yang, X. Liu, Z. Liu, F. Pu, J. Ren, X. Qu, Adv. Mater. 2012, 24, 2890 – 2895; d) P. K. Jain, X. Huang, I. H. El-Sayed, M. A. ElSayed, Acc. Chem. Res. 2008, 41, 1578 – 1586; e) K. Yang, L. Feng, X. Shi, Z. Liu, Chem. Soc. Rev. 2013, 42, 530 – 547. a) R. Huschka, O. Neumann, A. Barhoumi, N. J. Halas, Nano Lett. 2010, 10, 4117 – 4122; b) M. S. Yavuz, Y. Cheng, J. Chen, C. M. Cobley, Q. Zhang, M. Rycenga, J. Xie, C. Kim, K. H. Song, A. G. Schwartz, L. V. Wang, Y. Xia, Nat. Mater. 2009, 8, 935 – 939; c) W. S. Kuo, C. N. Chang, Y.-T. Chang, M. H. Yang, Y. H. Chien, S. J. Chen, C.-S. Yeh, Angew. Chem. 2010, 122, 2771 – 2775; Angew. Chem. Int. Ed. 2010, 49, 2711 – 2715; d) X. Huang, S. Neretina, M. A. El-Sayed, Adv. Mater. 2009, 21, 4880 – 4910; e) H. Kang, A. C. Trondoli, G. Zhu, Y. Chen, Y. J. Chang, H. Liu, Y. F. Huang, X. Zhang, W. Tan, ACS Nano 2011, 5, 5094 – 5099; f) N. W. Shi Kam, M. O’Connell, J. A. Wisdom, H. J. Dai, Proc. Natl. Acad. Sci. USA 2005, 102, 11600 – 11605; g) K. Yang, S. Zhang, G. Zhang, X. Sun, S. T. Lee, Z. Liu, Nano Lett. 2010, 10, 3318 – 3323; h) M. Li, X. Yang, J. Ren, K. Qu, X. Qu, Adv. Mater. 2012, 24, 1722 – 1728. a) J. T. Robinson, S. M. Tabakman, Y. Liang, H. Wang, H. S. Casalongue, D. Vinh, H. Dai, J. Am. Chem. Soc. 2011, 133, 6825 – 6831; b) W. Zhang, Z. Guo, D. Huang, Z. Liu, X. Guo, H. Zhong, Biomaterials 2011, 32, 8555 – 8561. M. C. Wu, A. D. Deokar, J. H. Liao, P. Y. Shih, Y. C. Ling, ACS Nano 2013, 7, 1281 – 1290. a) A. J. Kell, G. Stewart, S. Ryan, R. Peytavi, M. Boissinot, A. Huletsky, M. G. Bergeron, B. Simard, ACS Nano 2008, 2, 1777 – 1788; b) H. Gu, P. L. Ho, K. W. Tsang, L. Wang, B. Xu, J. Am. Chem. Soc. 2003, 125, 15702 – 15703; c) S. K. Choi, A. Myc, J. E. Silpe, M. Sumit, P. T. Wong, K. McCarthy, A. M. Desai, T. P. Thomas, A. Kotlyar, M. M. Holl, B. K. Orr, J. R. Baker, Jr., ACS Nano 2013, 7, 214 – 228; d) J. H. Gao, L. Li, P. L. Ho, G. C. Mak, H. W. Gu, B. Xu, Adv. Mater. 2006, 18, 3145 – 3148. a) P. Xi, K. Cheng, X. Sun, Z. Zeng, S. Sun, Chem. Commun. 2012, 48, 2952 – 2954; b) A. Fuchsbauer, O. A. Troshina, P. A. Troshin, R. Koeppe, R. N. Lyubovskaya, N. S. Sariciftci, Adv. Funct. Mater. 2008, 18, 2808 – 2814. J. T. Robinson, K. Welsher, S. M. Tabakman, S. P. Sherlock, H. Wang, R. Luong, H. Dai, Nano Res. 2010, 3, 779 – 793.
Received: October 10, 2013 Published online on December 10, 2013
398
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
& Antibacterial Nanomaterials
Reduced Graphene Oxide Functionalized with a Luminescent Rare-Earth Complex for the Tracking and Photothermal Killing of Drug-Resistant Bacteria Xinjian Yang,[a, b] Zhenhua Li,[a, b] Enguo Ju,[a, b] Jinsong Ren,*[a] and Xiaogang Qu*[a] ering the nonspecific toxicity and less stability of large GO, their clinical usage would be limited. Therefore, it is necessary to explore more advanced system for better treatment of diseases based on bacteria infection with high specificity, excellent temporal/spatial controllability, and ultimately improved efficacy. One promising approach is to use nanomaterial-based photothermal process for selective treatment of bacterial infections.[4] Previous studies have developed light-absorbing photothermal agents to serve as heat carriers for local hyperthermia. These materials can absorb light and convert it into toxic heat upon irradiation.[5] However, to avoid nonspecific heating of healthy cells, photothermal agents must show high absorption and selective aggregation at targeting site over normal tissues. In an effort to improve the safety of thermal therapies, near-infrared (NIR)-absorbing photothermal agents have been explored in combination with laser irradiation to provide sitespecific heating and treatment of diseased regions with a high uptake of the photothermal agents.[5c, 6] Among these NIR-absorbing photothermal agents, reduced graphene oxide (rGO) has been recognized as a suitable architecture for photothermal treatment of cancers owing to its extraordinary electronic, optical, and thermal properties.[7] Nevertheless, little have been done to invest its photothermal killing ability of bacteria.[8] Herein we constructed a graphene-based nanoarchitecture for targeted bacteria imaging and photothermal killing of drug resistant bacteria. To test the design, we attached vancomycin (Van) and a luminescent rare-earth complex to the surface of small rGO covalently. As shown in Scheme 1, Van was introduced to generate multivalent interactions and capture the bacteria.[9] As the 4f orbitals of rare-earth elements are shielded by 5s5p6s orbitals, rare-earth compounds exhibit unique spectroscopic characteristics, such as sharp line-like atomic emission, to overcome autofluorescence and light scattering.[10] Therefore, Van-modified rGO was further functionalized with a europium (Eu3 + ) complex, which would emit strong red luminescence upon excitation of the antenna molecule. Taken together, this novel approach would allow the trace of pathogens as well as bacteria killing upon exposure to a NIR laser beam. GO was synthesized from natural graphite powder using a modified Hummers method. Previous reports have demonstrated that small rGO could act as an efficient photothermal agent for cancer treatment. Therefore we first obtained monodisperse GO with a diameter of less than 150 nm (Figure 1 a, b). Amine-terminated poly(ethylene glycol) (PEG) was then cova-
Abstract: An antibacterial platform based on multifunctional reduced graphene oxide (rGO) that is responsive to near-infrared (NIR) light has been constructed. By introducing a luminescent Eu3 + complex and vancomycin for bacteria tracking into one system, this platform could specifically recognize and light up bacteria. Antibacterial activity of this nanoscale construction under NIR illumination was investigated. Upon illumination with NIR light, this nanoscale architecture generates great heat locally, resulting in the death of drug-resistant bacteria. These results indicate that the ability of this nanoscale platform to kill drug-resistant bacteria has great potential for clinical pathogenic bacteria diagnosis and treatment.
The rapid emergence of antibiotic-resistant bacterial strains[1] threatens public health globally and has sparked great interest in the design of novel approaches to combat the diverse and numerous mechanisms that bacteria employ to provide themselves protection against the action of antibacterial therapeutics. Treatments involving the use of agents that cause physical or chemical damage to the bacteria have drawn increasing attention.[2] For instance, graphene-based nanomaterials have been recently demonstrated to show promising antibacterial activity.[3] The interaction between the nanomaterials and bacteria was found to be nonspecific, and the antibacterial activity of GO sheets was dependenton lateral size.[3c] Larger GO showed stronger antibacterial activity than that of small GO, which is due to their easy coating on the surface of bacteria. However, they were easy to aggregate at physiological environment, which might result in undesired side effects. Consid[a] X. Yang,+ Z. Li,+ E. Ju, Prof. Dr. J. Ren, Prof. Dr. X. Qu Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization Changchun Institute of Applied Chemistry Chinese Academy of Sciences, Changchun, 130022 (China) Fax: (+ 86) 431-85262625 E-mail: [email protected] [email protected] [b] X. Yang,+ Z. Li,+ E. Ju University of Chinese Academy of Sciences Beijing, 100039 (China) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201303964. Chem. Eur. J. 2014, 20, 394 – 398
394
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
Scheme 1. a) Illustration of Eu-Van-rGO for targeted bacteria imaging and photothermal killing; b) the structure of the Eu complex; c) the structure of vancomycin.
lently linked to the epoxide groups to make the GO stable in buffer solutions and ready for further modification. Next, the
GO sheets were reduced by adding sodium borohydride into the solution. During the reduction step, the colour of the solution changed from yellow to black, which indicated the increasing light absorption in the NIR region owing to the recovery of conjugated p network of graphene (Figure 1 c). Centrifugation was then performed to remove any aggregation. Van was firstly modified on the PEG terminal by EDC/NHS chemistry. The Van-rGO was purified using dialysis membrane with a molecular cut-off of 14 000 g mol 1. The modification produces were monitored by FTIR spectra which demonstrated the success of functionalization (Supporting Information, Figure S1). To detect the effects of 808 nm optical excitation of rGO, we carried out a control experiment by irradiating aqueous solutions of Van-rGO at different concentrations (Figure 1 d). Irradiated with 808 nm laser at 1.5 W cm 2 for 300 s, Van-rGO solution (20 mg mL 1) showed a temperature elevation up to 20 8C, and the temperature increase depended on the concentration of the rGO. However, irradiation of the aqueous solution without Van-rGO or with 20 mg mL 1 GO only caused a smaller temperature elevation (Supporting Information, Figure S2). These findings clearly demonstrated the enhanced absorption of the 808 nm light by the rGO and confirmed that the as-prepared Van-rGO had photothermal capability. Thus, the Van-rGO was expected to be employed as photothermal agent for inhibiting the cell growth of target bacteria. Vancomycin is a potent antibiotic that interacts with terminal d-Ala-d-Ala moieties of peptide units located on bacterial
Figure 1. a), b) Images of GO: a) TEM (scale bar 100 nm); b) AFM (scale bar 50 nm; height profile in black determined from white line: 1.12 nm). c) UV/Vis spectra of Van-rGO and d) photothermal heat generated from different concentrations of Van-rGO under NIR irradiation. Chem. Eur. J. 2014, 20, 394 – 398
www.chemeurj.org
395
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication cell walls. Previous studies have demonstrated that Van-modified nanoparticles are capable of recognizing pathogenic bacteria.[9] We then examined the targeting capacity of Van-rGO for several pathogenic bacteria. In our experiment, Escherichia coli (E. coli), kanamyclin drug-resistant E. coli (DR-E. coli), Staphylococcus aureus (S. aureus), and oxacillin drug-resistant S. aureus (DR-S. aureus) were selected as the models to test the feasibility of using Van-rGO as the affinity probes. E. coli and S. aureus bacterial strains were utilized as they are representative of the two major classes of bacteria: Gram-negative and Gram-positive, respectively. Bacteria were allowed to incubate with the Van-rGO for 20 min prior to preparing for SEM analysis. As shown in Figure 2 and the Supporting Information, Figure S3, SEM revealed that all of the four bacteria were indeed covered with Van-rGO. However, in a control experiment, less
Figure 3. a) Fluorescent spectrum of Van-rGO (black) and Eu-Van-rGO (grey); b) photographs Van-rGO and Eu-Van-rGO under daylight and UV light; c), d) fluorescence microscopy images of DR-E. coli and DR-S. aureus incubated with Eu-Van-rGO.
tained DR-E. coli DR-S. aureus after 10 min incubation with EuVan-rGO. As can be seen from the images, both of the bacteria were illuminated, which further indicated that the Van could work as an efficient agent for bacteria targeting and Eu3 + complex could act as an imaging agent for bacteria tracking. Having demonstrated that Van-rGO could strongly absorb NIR radiation and could ultimately transfer this energy into the surrounding environment as heat, we next investigated the photothermal killing of these bacteria by using Van-rGO as photothermal agents under illumination with an NIR laser (808 nm, 1.5 W cm 2) for 3 min. The bacteria were then cultured and recorded with colony counts. As shown in Figure 4, there was a more than 20 % decrease in cell viability upon treatment with Van-rGO at a concentration of 20 mg mL 1. This result demonstrated that Van-rGO showed toxicity toward bacteria which was in accordance with previous reports.[3] However, the antibacterial activity relies on the concentration of VanrGO in the sample. Higher antibacterial activity was observed at higher concentrations (Supporting Information, Figure S6). Interestingly, when the bacteria were treated with Van-rGO upon NIR light irradiation, a high antibacterial activity was uncovered. Control experiments (Supporting Information, Figure S7) demonstrated that there was not obvious change in bacteria survival rate if the bulk solution was just heated to the same temperatures as that obtained by NIR light irradiation. When NIR light was introduced, all of the Gram-negative bacteria were killed at a Van-rGO concentration of 10 mg mL 1, and less than 3 % of Gram-positive bacteria survived at the same condition. This difference in antibacterial ability is because that the cell wall of Gram-positive bacteria is thicker than that of Gram-positive bacteria. As the concentration of Van-rGO increased, the cell growth of bacteria is inhibited completely. This is rational because high concentrate of VanrGO attached on the surface of bacteria can raise the local
Figure 2. SEM images of a) DR-E. coli incubated with Van-rGO, b) DR-E. coli incubated with Van-rGO under NIR irradiation; c) DR-S. aureus incubated with Van-rGO, d) DR-S. aureus incubated with Van-rGO under NIR irradiation. Scale shown is 3.00 mm.
PEG-rGO covered on the surface of bacteria (Supporting Information, Figure S4). These results clearly indicated that the interaction between the nanomaterials and the bacteria cell surface was mediated through specific interactions between vancomycin on the nanomaterials and receptors on the surface of the bacteria, rather than nonspecific interactions. To visualize bacteria by fluorescence microscopy, the van-labelled rGO was further labelled with a fluorescence probe, a Eu3 + complex, through EDC/NHS chemistry (F-Van-rGO). In our experiment, PEG played an important role, which not only made the nanoarchitecture stable, but also protected the Eu3 + complex from quenching. Spectroscopic properties of Eu-VanrGO were then investigated. Both Eu-Van-rGO and Eu3 + -complex have strong absorption at 350 nm and characteristic sharp emission lines in the red light-emitting region that are typical for Eu3 + ions (Supporting Information, Figure S5). Red PL is observable by the naked eye upon UV excitation (Figure 3 b). Therefore, rGO covalently modified by a red PL rareearth complex was designed and synthesized. Figure 3 c, d shows the fluorescent images obtained from samples that conChem. Eur. J. 2014, 20, 394 – 398
www.chemeurj.org
396
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication was introduced or not. When the incubation time increased from 1 to 4 h before NIR illumination, more than 75 % of the cells survived under the set of conditions. A co-culture experiment was then performed to assess the selectivity of the VanrGO when both mammalian and bacterial cells are introduced simultaneously. Most of the HepG 2 cells died and were detached from the cultivation base after 48 h, while the bacteria have proliferated as assessed by visual inspection (Supporting Information, Figure S9 e, h). However, when NIR light was introduced to kill the bacteria, most of the HepG 2 cells survived and no bacteria proliferation was observed (Supporting Information, Figure S9 f, i). Therefore, NIR irradiation to the infected sites will lead to selective photothermal-based killing of individual bacteria with less harmful effects on surrounding tissues. These results demonstrated that Van-rGO exhibited good biocompatibility and could be used as a potential antibacterial nanodrug for clinical application. In summary, we have demonstrated a novel near-infrared light responsive platform for targeted imaging and photothermal killing of drug-resistant bacteria based on multifunctional rGO. This nanoarchitecture was easily constructed by introducing luminescent Eu3 + complex and vancomycin for bacteria tracking. Antibacterial activity of this nanoconstruction under NIR illumination was investigated. Upon illumination of NIR light, this nanoarchitecture generates much heat locally, resulting in the death of bacteria. Our results indicate that drug resistant bacteria killing ability of this nanoplatform is endowed with great potential for clinical pathogenic bacteria diagnosis and treatment.
Figure 4. Viability of bacteria after incubation with Van-rGO (a) and bacteria incubated with Van-rGO under NIR irradiation (b).
temperature more efficiently, thus leading to more effective cell killing. The bacteria were followed with cell membrane disruption (Figure 2 b, d). It has been suggested that cell membrane damage following nanoparticles exposure to NIR radiation could be due to numerous factors, including nanoparticle explosion, shock waves, bubble formation, and thermal disintegration.[4d] Meanwhile, there was no obvious difference of photothermal bacteria killing ability comparing the bacteria with their drug resistant form. Therefore, combining its inherent antibacterial activity with photothermal cell-killing ability, this platform will be an ideal candidate to overcome the drug resistance problem. Based on the American National Standard for the Safe Use of Lasers, the maximum skin exposure for a continuous-wave 808 nm laser is about 0.33 W cm 2.[11] Although the NIR irradiation power used in our system was above the laser safety standards for humans, we could enhance the antibacterial ability of Van-rGO by increasing illumination time at lower irradiation intensity. HepG 2 cells were used as a model to examine whether illumination of these materials under NIR light might affect the growth of human cells. HepG 2 cells were incubated with VanrGO, and additional washing three times with phosphate buffer was carried out to remove remaining Van-rGO in solution before NIR illumination. The MTT results obtained by incubating HepG 2 cells with Van-rGO under different experimental conditions are shown in the Supporting Information, Figure S8. Van-rGO had little impact on the cell growth, whether NIR light Chem. Eur. J. 2014, 20, 394 – 398
www.chemeurj.org
Experimental Section Preparation of Van-PEG-rGO: Small GO was synthesized from natural graphite powder using a modified Hummers method. The PEG-rGO was prepared by vigorously stirring a solution of the graphene oxides (20 mg), amido-PEG (PEG diamine with 35 repeating units; 100 mg), and KOH (100 mg) in H2O (50 mL) at 70 8C for 24 h. Then a NaBH4 solution (10 mL, 1 m) was added, and the reaction was kept on at 70 8C for 2 h. The PEG-rGO was then collected and purified by using a dialysis membrane with a molecular cut-off of 14 000 g mol 1 to remove impurities and the excess of PEG by physical absorption. PEG-rGO (5 mg) in MES (18 mL, 30 mm) buffer was then mixed with vancomycin (2 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; 8 mg), and N-hydroxysuccinimide (NHS; 6 mg) and vigorously stirred for 6 h at room temperature. The resulting vancomycin-modified nanosheets (VanrGO) were purified using a dialysis membrane with a molecular cut-off of 14 000 g mol 1 to ensure all of the free unreacted vancomycin, EDC, and NHS were removed away from Van-rGO. Preparation of Eu-Van-rGO: [Eu(TFAcAcN)3Bpc] was synthesized according to the conventional route as follows: 1-(2-Naphthoyl)3,3,3-trifluoroacetone (TFAcAcN; 160 mg, 0.6 mm) and 2, 20-bipyridine-4,40-dicarboxylic acid (Bpc; 49 mg, 0.2 mm) were dissolved in ethanol (10 mL) under stirring at room temperature. Five drops of NaOH (2 m) were then added to adjust the pH level to 8.0. Thereafter a EuCl3 solution (51 mg, 0.2 mm) in ethanol (3.5 mL) was added dropwise. After complete addition, the solution was stirred for 1 h to ensure a complete precipitation at room temperature. The precipitate was filtered out, washed repeatedly with ethanol
397
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication and water, and then dried overnight in vacuum. [Eu(TFAcAcN)3Bpc] (1 mg), NHS (2 mg), and EDC (4 mg) in DMF/MES were then mixed and stirred for 30 min, and then the mixture was added dropwise into Van-rGO solution (0.2 mg mL 1 10 mL). The solution was then stirred for 12 h at room temperature and dialyzed (dialysis bag MWCO 14 000) for 24 h. The four bacteria strains were grown in LB medium at 37 8C. The resultant bacterial cells were isolated by centrifugation (3500 rpm). The bacterial cells were rinsed with Tris buffer (50 mm, pH 7.4), and designed bacterial concentration was adjusted by measuring the optical density at 600 nm (OD600nm).
[3]
[4]
[5]
To examine the photothermal effect of Van-rGO, the aqueous sample solutions were irradiated using a diode laser (808 nm). The power of the laser was 1.5 W cm 2. The temperature of the resultant solution was measured by a thermocouple. Cell viability test: Two groups of test bacterial suspensions (105– 106 CFU mL 1) were incubated with different concentration of VanrGO for 10 min. One group of the bacteria was then irradiated with NIR light for 3 min. The resultant bacterial suspensions were cultured in Petri dishes containing LB agar medium at 37 8C, overnight. The CFU of the bacteria were then estimated.
[6]
For the co-culture test (HepG 2 cells–(DR-E. coli) and DR-S. aureus), the concentration of cancer cells was fixed at a density of 105 cells per well in 24-well assay plates and infected with DR-E. coli and DR-S. aureus. Van-rGO (20 mg mL 1) was then added with and without NIR light (1.5 W cm 2, 3 min). Co-cultures were incubated at 37 8C and 5 % CO2. Images of HepG 2 cells in the presence of DRE. coli and DR-S. aureus were recorded at 24 and 48 h using an Olympus BX-51 optical system microscope.
[7]
Acknowledgements
[8] [9]
Financial support was provided by the National Basic Research Program of China (Grant 2012CB720602, 2011CB936004) and the National Natural Science Foundation of China (Grants 21072182, 21210002, 91213302). Keywords: imaging · luminescent complexes · rare-earth complexes · photothermal killing · reduced graphene oxide
[10]
[1] a) A. J. Alanis, Arch. Med. Res. 2005, 36, 697 – 705; b) F. C. Tenover, Am. J. Infect. Control. 2006, 34, S3 – S10; c) H. Yoneyama, R. Katsumata, Biosci. Biotechnol. Biochem. 2006, 70, 1060 – 1075. [2] a) I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 2004, 275, 177 – 182; b) C. A. Strassert, M. Otter, R. Q. Albuquerque, A. Hçne, Y. Vida, B. Maier, L. De Cola, Angew. Chem. 2009, 121, 8070 – 8073; Angew. Chem. Int. Ed. 2009, 48, 7928 – 7931; c) W. S. Kuo, C. N. Chang, Y. T. Chang, C. S. Yeh,
[11]
Chem. Eur. J. 2014, 20, 394 – 398
www.chemeurj.org
Chem. Commun. 2009, 4853 – 4855; d) P. C. Ray, S. A. Khan, A. K. Singh, D. Senapati, Z. Fan, Chem. Soc. Rev. 2012, 41, 3193 – 3209. a) S. Liu, T. H. Zeng, M. Hofmann, E. Burcombe, J. Wei, R. Jiang, J. Kong, Y. Chen, ACS Nano 2011, 5, 6971 – 6980; b) W. Hu, C. Peng, W. Luo, M. Lv, X. Li, D. Li, Q. Huang, C. Fan, ACS Nano 2010, 4, 4317 – 4323; c) S. Liu, M. Hu, T. H. Zeng, R. Wu, R. Jiang, J. Wei, L. Wang, J. Kong, Y. Chen, Langmuir 2012, 28, 12364 – 12372; d) O. Akhavan, E. Ghaderi, ACS Nano 2010, 4, 5731 – 5736. a) W. C. Huang, P. J. Tsai, Y. C. Chen, Small 2009, 5, 51 – 56; b) T. J. Yu, P. H. Li, T. W. Tseng, Y. C. Chen, Nanomedicine 2011, 6, 1353 – 1363; c) C. Wang, J. Irudayaraj, Small 2010, 6, 283 – 289; d) S. Wang, A. K. Singh, D. Senapati, A. Neely, H. Yu, P. C. Ray, Chem. Eur. J. 2010, 16, 5600 – 5606. a) M. Chu, X. Pan, D. Zhang, Q. Wu, J. Peng, W. Hai, Biomaterials 2012, 33, 7071 – 7083; b) Y. Luo, Y. S. Shiao, Y. F. Huang, ACS Nano 2011, 5, 7796 – 7804; c) X. Yang, X. Liu, Z. Liu, F. Pu, J. Ren, X. Qu, Adv. Mater. 2012, 24, 2890 – 2895; d) P. K. Jain, X. Huang, I. H. El-Sayed, M. A. ElSayed, Acc. Chem. Res. 2008, 41, 1578 – 1586; e) K. Yang, L. Feng, X. Shi, Z. Liu, Chem. Soc. Rev. 2013, 42, 530 – 547. a) R. Huschka, O. Neumann, A. Barhoumi, N. J. Halas, Nano Lett. 2010, 10, 4117 – 4122; b) M. S. Yavuz, Y. Cheng, J. Chen, C. M. Cobley, Q. Zhang, M. Rycenga, J. Xie, C. Kim, K. H. Song, A. G. Schwartz, L. V. Wang, Y. Xia, Nat. Mater. 2009, 8, 935 – 939; c) W. S. Kuo, C. N. Chang, Y.-T. Chang, M. H. Yang, Y. H. Chien, S. J. Chen, C.-S. Yeh, Angew. Chem. 2010, 122, 2771 – 2775; Angew. Chem. Int. Ed. 2010, 49, 2711 – 2715; d) X. Huang, S. Neretina, M. A. El-Sayed, Adv. Mater. 2009, 21, 4880 – 4910; e) H. Kang, A. C. Trondoli, G. Zhu, Y. Chen, Y. J. Chang, H. Liu, Y. F. Huang, X. Zhang, W. Tan, ACS Nano 2011, 5, 5094 – 5099; f) N. W. Shi Kam, M. O’Connell, J. A. Wisdom, H. J. Dai, Proc. Natl. Acad. Sci. USA 2005, 102, 11600 – 11605; g) K. Yang, S. Zhang, G. Zhang, X. Sun, S. T. Lee, Z. Liu, Nano Lett. 2010, 10, 3318 – 3323; h) M. Li, X. Yang, J. Ren, K. Qu, X. Qu, Adv. Mater. 2012, 24, 1722 – 1728. a) J. T. Robinson, S. M. Tabakman, Y. Liang, H. Wang, H. S. Casalongue, D. Vinh, H. Dai, J. Am. Chem. Soc. 2011, 133, 6825 – 6831; b) W. Zhang, Z. Guo, D. Huang, Z. Liu, X. Guo, H. Zhong, Biomaterials 2011, 32, 8555 – 8561. M. C. Wu, A. D. Deokar, J. H. Liao, P. Y. Shih, Y. C. Ling, ACS Nano 2013, 7, 1281 – 1290. a) A. J. Kell, G. Stewart, S. Ryan, R. Peytavi, M. Boissinot, A. Huletsky, M. G. Bergeron, B. Simard, ACS Nano 2008, 2, 1777 – 1788; b) H. Gu, P. L. Ho, K. W. Tsang, L. Wang, B. Xu, J. Am. Chem. Soc. 2003, 125, 15702 – 15703; c) S. K. Choi, A. Myc, J. E. Silpe, M. Sumit, P. T. Wong, K. McCarthy, A. M. Desai, T. P. Thomas, A. Kotlyar, M. M. Holl, B. K. Orr, J. R. Baker, Jr., ACS Nano 2013, 7, 214 – 228; d) J. H. Gao, L. Li, P. L. Ho, G. C. Mak, H. W. Gu, B. Xu, Adv. Mater. 2006, 18, 3145 – 3148. a) P. Xi, K. Cheng, X. Sun, Z. Zeng, S. Sun, Chem. Commun. 2012, 48, 2952 – 2954; b) A. Fuchsbauer, O. A. Troshina, P. A. Troshin, R. Koeppe, R. N. Lyubovskaya, N. S. Sariciftci, Adv. Funct. Mater. 2008, 18, 2808 – 2814. J. T. Robinson, K. Welsher, S. M. Tabakman, S. P. Sherlock, H. Wang, R. Luong, H. Dai, Nano Res. 2010, 3, 779 – 793.
Received: October 10, 2013 Published online on December 10, 2013
398
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
