DOI: 10.1002/chem.201406151
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& Bioimaging | Very Important Paper |
Oxidized Carbon Nitrides: Water-Dispersible, Atomically Thin Carbon Nitride-Based Nanodots and Their Performances as Bioimaging Probes Junghoon Oh,[a] Ran Ji Yoo,[b] Seung Yeon Kim,[a] Yong Jin Lee,[b] Dong Wook Kim,*[a] and Sungjin Park*[a] Abstract: Three-dimensional (3D) carbon nitride (C3N4)based materials show excellent performance in a wide range of applications because of their suitable band structures. To realize the great promise of two-dimensional (2D) allotropes of various 3D materials, it is highly important to develop routes for the production of 2D C3N4 materials, which are one-atom thick, in order to understand their intrinsic properties and identify their possible applications. In this work, water-dispersible, atomically thin, and small carbon nitride nanodots were produced using the chemical oxidation of
Introduction Two-dimensional (2D) materials, such as graphene,[1–4] MoS2,[5, 6] and hexagonal boron nitride,[7] are attractive for a wide range of applications owing to their intriguing physical, chemical, and morphological properties and high surface areas, which differ from those of the three-dimensional (3D) allotropes. The 3D carbon nitride (C3N4)-based materials, including graphitic C3N4 (g-C3N4) and polymeric g-C3N4, show excellent performances as photocatalysts[8] and electrocatalysts[9] in photovoltaic devices and as luminescent materials in chemical and biosensors[10] because of their suitable band structures. Consequently, the production of new types of 2D carbon nitride materials is a great challenge to understand their intrinsic properties and identify their possible applications. Furthermore, it is highly important to achieve homogeneous colloidal suspensions of individual 2D carbon nitride materials by scalable synthetic methods to facilitate the use of such materials. Herein, we introduce a novel route to produce water-dispersible, atomically thin, [a] J. Oh, S. Y. Kim, Prof. D. W. Kim, Prof. S. Park Department of Chemistry and Chemical Engineering Inha University, 100 Inha-ro, Nam-gu, Incheon 402-751 (Korea) E-mail: [email protected] [email protected] [b] R. J. Yoo, Y. J. Lee Molecular Imaging Research Center Korea Institute of Radiological and Medical Sciences 75 Nowon-ro, Nowon-gu, Seoul 139-706 (Korea) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201406151. Chem. Eur. J. 2015, 21, 6241 – 6246
graphitic C3N4. Various analyses, including X-ray diffraction, X-ray photoelectron, Fourier-transform infrared spectroscopy, and combustion-based elemental analysis, and thermogravimetric analysis, confirmed the production of 3D oxidized C3N4 materials. The 2D C3N4 nanodots were successfully exfoliated as individual single layers; their lateral dimension was several tens of nanometers. They showed strong photoluminescence in the visible region as well as excellent performances as cell-imaging probes in an in vitro study using confocal fluorescence microscopy.
and small carbon nitride-based materials via the chemical oxidation of g-C3N4 and their application in a cell-imaging study as bioimaging probes. Graphitic C3N4 is a stable metal-free layered material. Each layer of g-C3N4 is composed of triazine or tris-s-triazine building units, which contain alternating sp2 hybrid C and N atoms.[11, 12] This layered g-C3N4 material can be a good source material for the generation of single-layer C3N4 materials by exfoliating the individual layers. Although sonication and acid and heat treatments of g-C3N4 produce thin C3N4 materials, the generation of single-layer C3N4 materials that are one atom thick and their homogeneous colloidal suspensions remains a great challenge.[13–17] Chemical oxidation is often a useful approach to produce stable suspensions of carbon-based nanomaterials in polar solvents via the introduction of polar oxygen functional groups onto carbon atoms.[18–20] For example, graphite oxide produced by chemical oxidation of graphite has been widely used to produce homogeneous colloidal suspensions of chemically modified graphene materials.[18] Because the oxygen-containing functional groups of graphite oxide are hydrophilic, singlelayer graphene oxide disperses well in water.[21] Furthermore, the use of graphene-based materials in various applications can be facilitated by further chemical modifications on the reactive oxygen functionalities.[22] To overcome the difficulties of adding other functional groups onto chemically robust g-C3N4,[11, 12] the presence of oxygen functional groups on C3N4-based materials is highly important and will enable additional applications. Although it is thought that gC3N4 networks are stable against acid treatments,[13, 23, 24] we re-
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Full Paper the flat surface structure of g-C3N4 was converted into a porous structure. Colorless, transparent, and homogeneous colloidal suspensions incorporating OCN nanodots were produced by sonication of the OCN powder in water after removal of any undispersed particles by centrifuge. The suspensions showed the Tyndall effect, confirming the presence of particles (Figure 1 d). These aqueous suspensions were stable under acidic, neutral, and basic conditions (see the Supporting Information). TEM and atomic force microscopy (AFM) were used to observe the morphology of the OCN nanodots after drying droplets of the suspension. As shown in an AFM scan and a TEM image (Figure 1 e, f), the dispersed OCN materials primarily comprised 2D thin nanodots. Figure 1. a) Overall Scheme for the production of single-layer carbon nitride-based nanodots; b) SEM images of The lateral dimensions of these the OCN powder samples at different magnifications (inset in the left image: Photo of the OCN powder); c) TEM OCN nanodots were several image of the OCN powder; d) Aqueous colloidal suspension of OCN nanodots showing the Tyndall effect; e) AFM tens of nanometers and some scanning image of OCN nanodots on a mica surface and height profiles of sections marked by the solid (top) and of them were less than 10 nm, dashed (bottom) lines; f) TEM image of the OCN nanodots. which was much smaller than that of the previously reported C3N4-based sheet-like materials.[13, 15, 16, 25] visited the chemical oxidation of g-C3N4 using strong oxidants and acids to generate single-layer C3N4-based materials and The thicknesses of the nanodots, determined via the height profile of the AFM measurements, were in the range of 0.5– their suspensions (Figure 1 a). 1.5 nm. Importantly, the thinnest OCN nanodots were about 0.5 nm thick, which supports the successful exfoliation of OCN nanodots as individual single layers with one-atom thickResults and Discussion ness.[26, 27] Such single-layer nanodots were frequently found in our AFM scans. Since the term “graphitic” suggests 3D stacking A pale yellow powder was obtained by polycondensation of of individual layers, it is not appropriate to use it for singledicyandiamide at 500 8C in a furnace, as was previously reportlayer OCN nanodots. These results were interesting because ed,[12] and the formation of in-plane C3N4 networks and layered structures was confirmed by various characterization methods most of the previously reported C3N4-based sheet-like materials (see the Supporting Information). The as-prepared g-C3N4 was are not single layers with one-atom thickness.[13–17] The demonstrated ability to disperse 2D single-layer C3N4-based materials treated with a strong oxidant (KMnO4) in the presence of in a liquid phase without the use of surfactants or stabilizers H2SO4 ; this method is frequently used to oxidize graphite[21] would be extremely important for their possible application in and afforded oxidized carbon nitride (henceforth, OCN) as various research areas. a white powder (Figure 1 b). Figure 1 b shows scanning elecIt is important to examine the optical properties of the 3D tron microscopy (SEM) images of the OCN powder at two difOCN powder and 2D OCN nanodots to assess their possible ferent magnifications. In the low-magnification SEM image applicability in photocatalytic reactions, optical sensors, and (Figure 1 b, left), it appeared that the surface of the OCN bioimaging applications. First, the UV/Vis adsorption and phopowder was smooth and flat. However, high-magnification toluminescence (PL) properties of 3D materials, such as the asSEM (Figure 1 b, right) revealed a 3D porous structure, which prepared g-C3N4 and 3D OCN powder samples, were comwas much different from the flat morphology of the as-prepared g-C3N4 (see the Supporting Information, Figure S1). pared. The UV/Vis adsorption spectrum of the OCN powder showed a strong adsorption at around 300–400 nm and was Transmission electron microscopy (TEM) confirmed the porous blue-shifted relative to the spectrum of the as-prepared g-C3N4 structure of the OCN materials (Figure 1 c). During oxidation, Chem. Eur. J. 2015, 21, 6241 – 6246
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Full Paper Optical/fluorescence imaging of living cells with imaging modalities, such as confocal microscopy, has gained popularity in a variety of biomedical areas.[28] Visualization of cells using these tools requires fluorescent probes that have high biocompatibilities, suitable sizes and morphologies, high PL quantum yields, high stabilities in aqueous media, and low cytotoxicities.[29–31] In recent decades, a range of nanoscale semiconducting materials containing heavy metals, particularly quantum dots, have been applied to a broad range of biological imaging systems.[32–34] However, the high cytotoxicity of these nanomaterials restricted their application in in vivo as well as in vitro studies.[35] Accordingly, it is important to develop “heavymetal free” optical probes with excellent optical properties, high serum stability, and good biocompatibility. To enable biological applications of 2D OCN nanodots, their biocompatibility was evaluated by measuring the amount of viable cells after 48 h of incubation in varying concentrations (0–100 mg mL¢1) of the OCN nanodots using a trypan blue exclusion assay with RAW 264.7 cells (Figure 3 a). The OCN nano-
Figure 2. a) UV/Vis diffuse reflectance absorption spectra of the solid as-prepared g-C3N4 and 3D OCN samples and UV/Vis adsorption spectrum of an aqueous suspension of OCN nanodots; b) PL spectra of the solid as-prepared g-C3N4 and 3D OCN samples and an aqueous suspension of the OCN nanodots.
powder (Figure 2 a). PL emissions in the visible (350–600 nm) and near-IR regions (800–900 nm) were found in a PL spectrum of the OCN powder (Figure 2 b); these peaks were significantly blue-shifted relative to those of the as-prepared g-C3N4, suggesting that the band gap was enlarged by chemical oxidation. This shifting trend was similar to previously reported results using chemically modified g-C3N4-based materials.[13–15] In addition to the optical properties of the 3D solid samples, those of the 2D OCN nanodots in colloidal suspensions were also measured. The suspension containing OCN nanodots visually emitted blue light under exposure to UV light using a UV lamp (see the Supporting Information, Figure S8). A UV/Vis absorption spectrum of the suspensions containing OCN nanodots showed strong adsorption peaks at 227 and 267 nm, which were blue-shifted relative to those of the 3D OCN powder samples (Figure 2 a). The suspensions of the OCN nanodots also showed a strong emission in the visible region and the emitting wavelength was slightly shifted (from 417 to 407 nm) relative to that of the 3D OCN powder (Figure 2 b); this blue-shift could be due to localized structures in the small OCN nanodots.[14] The small size, fluorescent properties, and water dispersibility of the single-layer OCN nanodots are highly beneficial for applications as optical sensors and bioimaging probes. Chem. Eur. J. 2015, 21, 6241 – 6246
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Figure 3. a) In vitro cell viability of RAW 264.7 cells after 48 h of incubation with different concentrations of the OCN nanodots by a trypan blue exclusion assay; b) Confocal fluorescence microscopic image of RAW 264.7 cells after incubation for 24 h at 37 8C with single-layer OCN nanodots.
dots exhibited little cytotoxicity to the RAW 264.7 cells at relatively high concentrations. In particular, the viabilities of the RAW 264.7 cells were 87.2 5.6 %, even at the highest concentration of OCN nanodots (100 mg mL¢1), indicating that the OCN nanodots exhibited high biocompatibility and low cytotoxicity. To investigate the possibility of cell-labeling using these biocompatible single-layer OCN nanodots, we attempted to conduct a bioimaging study using confocal fluorescence microscopy on the RAW 264.7 cells. As can be seen from Figure 3 b and S9 (a merged image of the bright field and confocal images, see the Supporting Information), the OCN nanodots could be used to successfully visualize the RAW cells without any aggregation in the cytoplasm. This confocal image showed that the green-emitting OCN nanodots were internalized into the RAW cells. An interesting phenomenon is that the OCN nanodots did not penetrate into nucleus but instead surrounded it. These results suggested that OCN nanodots could be promising fluorescent probes for biomedical imaging applications. The chemical structure of the 3D OCN powder was determined using X-ray diffraction (XRD), combustion-based elemental analysis, Fourier-transform infrared (FT-IR) and X-ray
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Full Paper After oxidation, the intensities of the peaks corresponding to the N¢H bonds in the FT-IR and XPS N1 s spectra of the 3D OCN powder were enhanced relative to that of the as-prepared gC3N4 (Figure 4). As shown in the FT-IR spectra, the intensities of the peak at 3350 cm¢1, which corresponded to O¢H bonds, and the peak at 1704 cm¢1, which corresponded to C=O bonds, in the spectrum of the OCN powder were stronger than those in the spectrum of g-C3N4.[36, 37] The deconvoluted XPS C1s spectra of the OCN powder showed enhanced intensity of the peak that corresponded to the C=O moieties relative to that of the as-prepared g-C3N4 (Figure 4 d).[1, 22, 38] Elemental analysis of the OCN powder (C/O ratio of 1.46:1) and as-prepared g-C3N4 (C/O ratio of 7.88:1) also supported the introduction of oxygen atoms during the oxidation process. The thermal stabilities of the g-C3N4 and 3D OCN materials were measured by thermogravimetric analysis (TGA). As shown in the TGA curves (Figure 4 e), the as-prepared g-C3N4 showed good thermal stability up to 600 8C, then was completely removed by heat treatment at around 700 8C, which corresponded to decomposition of Figure 4. Chemical characterizations of the as-prepared g-C3N4 and 3D OCN powder samples: a) XRD patterns; the tris-s-triazine moieties of gb) FT-IR spectra; c) XPS N1 s spectra including deconvolution; d) XPS C1 s spectra including deconvolution; e) TGA C3N4.[7, 39] In contrast, the TGA curves. curve of the OCN powder showed stepwise mass losses as the temperature increased. The loss below 300 8C was attributphotoelectron spectroscopy (XPS), and thermogravimetric analed to decomposition of the labile functional groups in the oxiysis (TGA). An XRD pattern of the as-prepared g-C3N4 powder dized carbon nitride networks, which were introduced by the exhibited a peak at 27.38, which corresponded to the interlayer oxidation process. A gradual weight loss from around 60 to distance between the 2D C3N4 networks in g-C3N4, and a peak 0 wt % was observed during heat treatment from 350 to at 13.18, which corresponded to the in-plane hole-to-hole dis700 8C, corresponding to thermal decomposition of the tris-stance.[8, 12] After oxidation, the interlayer distance slightly detriazine moieties. creased and the peak for the in-plane motif almost disapThe combined chemical characterizations suggested the forpeared in the XRD pattern of the 3D OCN powder (Figure 4 a). mation of NH2- and C=O-containing moieties, such as carboxyl This result suggested that the OCN powder featured a graphitic stacking structure of each oxidized C3N4 layer and the in-plane and ketone groups, which could have been generated by decomposition of the N¢C=N groups at the edges of the C3N4 structure was significantly damaged by oxidation. This damage could be one reason for the generation of the porous structure networks during oxidation (see the Supporting Information, of the 3D OCN powder. Figure S10). The presence of these hydrophilic groups at the Chem. Eur. J. 2015, 21, 6241 – 6246
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Full Paper edges enabled the generation of homogeneous suspensions of the single-layer OCN nanodots in water. In addition, the coexistence of basic amine and acidic carboxyl groups could be the reason for the production of stable aqueous suspensions under both acidic and basic conditions. Moreover, the chemically reactive amine and carboxyl groups could be chemical modified to introduce various functional groups onto the C3N4 networks.
mersed in a water bath set at 35 8C and stirred with a magnetic stirring bar for 2 h. Then, H2O (200 mL) was slowly added to the flask in an ice bath. Excess hydrogen peroxide (30 % in water, Sigma–Aldrich) was then added to the flask until no gas bubbling was observed, followed by the addition of excess water. The resultant mixture was filtered through an Anodisc membrane filter (47 mm of diameter, 0.1 mm of pore sizes; Whatman). The filtrate was further washed several times with H2O then dried under vacuum at room temperature for 12 h.
Conclusion
Production of aqueous suspensions of the 2D OCN nanodots
We synthesized novel OCN materials using a liquid process via the treatment of g-C3N4 with KMnO4 in the presence of sulfuric acid. During the reaction, polar functional groups, such as amine, carboxyl, and ketone groups, were introduced into the C3N4 network, as confirmed by XPS, FT-IR, TGA, and elemental analysis. XRD patterns showed the presence of a graphitic layered structure in the 3D OCN powder, and the SEM and TEM images showed the generation of a porous structure. The presence of functional groups on the C3N4 network will be highly useful for further chemical modifications to introduce specific functional groups onto the C3N4-based materials. Homogeneous colloidal suspensions of 2D OCN nanodots were produced by sonication of the 3D OCN powder in water and showed strong photoluminescence in the visible region. The lateral size of OCN nanodots was several tens of nanometers and their thicknesses were in the range of 0.5–1.5 nm, which indicated that the OCN nanodots were successfully exfoliated as very thin and small 2D materials; some portion of the generated nanodots were determined to comprise a single layer with one-atom thickness (ca. 0.5 nm). This approach for dispersing single-layer C3N4-based materials in a liquid phase without the use of surfactants or stabilizers should be extremely useful for the production of a wide range of colloidal suspensions that can be used to study the fundamental properties of 2D C3N4-based materials; this will also facilitate the discovery of their potential applications in catalysis, bio-sensing, biomedical imaging, fabrication of composites, and thin-film technologies. In particular, the water-dispersible OCN nanodots showed good performance in a cell-imaging study using confocal fluorescence microscopy. Given their favorable properties, such as high biocompatibility, low cytotoxicity, good dispersibility, and stability in aqueous media, as well as suitable optical characteristics, these “heavy metal free” OCN nanodots can be regarded as physiologically friendly alternatives to conventional nanoscale metal-based optical probes for biomedical imaging.
Experimental Section Preparation of OCN The as-prepared g-C3N4 powder (1 g, see the Supporting Information for synthetic procedure) and sulfuric acid (50 mL, 98 %, Dae Jung Chemicals, Korea) were loaded into an Erlenmeyer flask. Potassium permanganate (KMnO4, 3.5 g, Dae Jung Chemicals, Korea) was slowly added to the flask in an ice bath. The flask was imChem. Eur. J. 2015, 21, 6241 – 6246
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OCN powder (10 mg) was added into a vial filled with H2O (10 mL). The vial was then sonicated using an ultrasonic cleaner (Bransonic 8510, Danbury, CT, USA) for 200 min, affording colorless colloidal suspensions of the 2D OCN nanodots. Undispersed particles were separated by centrifugation (Supra 22 K centrifuge; Hanil Science Industrial, Incheon, Korea) at 2000 rpm for 30 min.
Cell culture Mouse macrophage cell lines (RAW 264.7) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; WelGENE Inc., Korea) containing 10 % fetal bovine serum (FBS) and 1 % antibiotics and grown in a humidified incubator at 37 8C under 5 % CO2.
In vitro viability test Solutions of each concentration of OCN nanodots (0, 50, and 100 mg mL¢1) in phosphate buffered saline (PBS) were added to a suspension of 1 Õ 106 RAW 264.7 cells in 1 mL of cell-culture media, and the mixture was incubated at 37 8C for 2 h. After centrifugation (250 Õ g, 5 min), the supernatant was removed and the cells were washed twice with 1 mL of PBS. The OCN-labeled RAW 264.7 cells were plated in a 6-well culture plate and incubated with DMEM containing 10 % FBS at 37 8C under humidified 5 % CO2 for 48 h. The cell viability of OCN-labeled RAW 264.7 cells was determined via the trypan blue exclusion test. All data are expressed as mean standard deviation.
In vitro confocal fluorescence microscopy The labeled RAW 264.7 cells (2 Õ 105) with OCN nanodots were plated in 8-well chamber slides and incubated with DMEM containing 10 % FBS at 37 8C under humidified 5 % CO2 for 4 h. After incubation, the supernatant was removed and the cells were washed twice with PBS. Phenol red-free DMEM containing 2 % FBS was refreshed after cell washing. Fluorescence images were then captured using a laser-scanning confocal microscope (LSM710, Carl Zeiss MicroImaging, Jena, Germany).
Instruments XPS study was performed using an angle-resolved X-ray photoelectron MXR1 Gun, 400 mm, 15 kV spectrometer (Theta probe, Thermo Fisher Scientific, UK); binding energies were determined versus the C1 s peak at 284.6 eV. Elemental analysis for C, N, H, and O was conducted using a FLASH EA1112 instrument (Thermo Electron, Italy). XRD patterns were obtained using a Multiplex spectrometer (Rigaku, Tokyo, Japan). PL spectra were recorded with a HR800 UV spectrometer (HORIBA JovinYvon, 325 nm excitation, He Cd laser) for the as-prepared g-C3N4 powder and PI-MAX3 spectrometer (Princeton Instruments, 325 nm excitation, He Cd laser) for the
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Full Paper OCN powder and suspensions. FT-IR spectra of the samples in KBr pellets were obtained using an FT-IR vacuum spectrometer (VERTEX 80 V, Bruker, Germany) and included 64 scans. SEM images of Pt-coated samples were collected using a field emission scanning electron microscope (S-4300, Hitachi, Tokyo) at an accelerating voltage of 15 kV. TEM images were obtained using a JEOL JEM2100F instrument (JEOL Co. Ltd., Japan) at 200 kV. TEM samples were prepared by drying a droplet of a mixture including 3D OCN powder in water or diluted suspension incorporating 2D OCN nanodots on a mesh copper grid coated with carbon film (CF200Cu, Electron Microscopy Sciences). TGA measurements (SDT Q600, TA Instruments) were performed at a heating rate of 5 8C min¢1 from 30 to 800 8C in a N2 gas environment. AFM images were obtained using a XE-100 microscope (PSIA, Korea) in non-contact mode.
Acknowledgements This work was supported by Inha University and grants from the Center for Advanced Soft Electronics funded by the Ministry of Science, ICT & Future Planning as a Global Frontier Project (CASE-2014M3A6A5060938)and from the Basic Science Research Program (Grant no. NRF-2012R1A1A2044945) of the Ministry of Science, ICT & Future Planning, South Korea. S.P. thanks the Busan Center at the Korea Basic Science Institute (KBSI) for the XPS analysis. Keywords: bioimaging · carbon oxidation · photoluminescence
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[1] D. R. Dreyer, S. Park, C. W. Bielawski, R. S. Ruoff, Chem. Soc. Rev. 2010, 39, 228 – 240. [2] W. J. Lee, U. N. Maiti, J. M. Lee, J. Lim, T. H. Han, S. O. Kim, Chem. Commun. 2014, 50, 6818 – 6830. [3] K. E. Lee, J. E. Kim, U. N. Maiti, J. Lim, J. O. Hwang, J. Shim, J. J. Oh, T. Yun, S. O. Kim, ACS Nano 2014, 8, 9073 – 9080. [4] J. Shim, J. M. Yun, T. Yun, P. Kim, K. E. Lee, W. J. Lee, R. Ryoo, D. J. Pine, G. Yi, S. O. Kim, Nano Lett. 2014, 14, 1388 – 1393. [5] B. Radisavljevic, A. Radenovic, J. Brivio, V. Giacometti, A. Kis, Nat. Nanotechnol. 2011, 6, 147 – 150. [6] K. Chang, Z. Mei, T. Wang, Q. Kang, S. Ouyang, J. Ye, ACS Nano 2014, 8, 7078 – 7087. [7] C. Zhi, Y. Bando, C. Tang, H. Kuwahara, D. Golberg, Adv. Mater. 2009, 21, 2889 – 2893. [8] X. Wang, K. Maeda, A. Thomas, K. Takanabe, G. Xin, J. M. Carlsson, K. Domen, M. Antonietti, Nat. Mater. 2009, 8, 76 – 80.
Chem. Eur. J. 2015, 21, 6241 – 6246
www.chemeurj.org
[9] Y. Zheng, Y. Jiao, J. Chen, J. Liu, J. Am. Chem. Soc. 2011, 133, 20116 – 20119. [10] Y. Tang, H. Song, Y. Su, Y. Lv, Anal. Chem. 2013, 85, 11876 – 11884. [11] Y. Wang, X. Wang, M. Antonietti, Angew. Chem. Int. Ed. 2012, 51, 68 – 89; Angew. Chem. 2012, 124, 70 – 92. [12] A. Thomas, A. Fischer, F. Goettmann, M. Antonietti, J. Mìller, R. Schlçgl, J. M. Carlsson, J. Mater. Chem. 2008, 18, 4893 – 4908. [13] L. Chen, D. Huang, S. Ren, T. Dong, Y. Chi, G. Chen, Nanoscale 2013, 5, 225 – 230. [14] X. She, H. Xu, Y. Xu, J. Yan, J. Xia, L. Xu, Y. Song, Y. Jiang, Q. Zhang, H. Li, J. Mater. Chem. A 2014, 2, 2563 – 2570. [15] J. Tian, Q. Liu, A. M. Asiri, A. O. Al-Youbi, X. Sun, Anal. Chem. 2013, 85, 5595 – 5599. [16] S. Yang, Y. Gong, J. Zhang, L. Zhan, L. Ma, Z. Fang, R. Vajtai, X. Wang, P. M. Ajayan, Adv. Mater. 2013, 25, 2452 – 2456. [17] P. Niu, L. Zhang, G. Liu, H. Cheng, Adv. Funct. Mater. 2012, 22, 4763 – 4770. [18] S. Park, R. S. Ruoff, Nat. Nanotechnol. 2009, 4, 217 – 224. [19] V. Datsyuk, M. Kalyva, K. Papagelis, J. Parthenios, D. Tasis, a. Siokou, I. Kallitsis, C. Galiotis, Carbon 2008, 46, 833 – 840. [20] J. Figueiredo, M. Pereira, M. Freitas, J. Orfao, Carbon 1999, 37, 1379 – 1389. [21] S. Park, J. An, R. Piner, I. Jung, Chem. Mater. 2008, 20, 6592 – 6594. [22] J. Han, L. L. Zhang, S. Lee, J. Oh, K. Lee, J. R. Potts, J. Ji, X. Zhao, R. S. Ruoff, S. Park, ACS Nano 2013, 7, 19 – 26. [23] T. Y. Ma, Y. Tang, S. Dai, S. Z. Qiao, Small 2014, 10, 2382 – 2389. [24] Y. Zhang, A. Thomas, M. Antonietti, X. Wang, J. Am. Chem. Soc. 2009, 131, 50 – 51. [25] Q. Wang, W. Wang, J. Lei, N. Xu, F. Gao, H. Ju, Anal. Chem. 2013, 85, 12182 – 12188. [26] J. Xu, L. Zhang, R. Shi, Y. Zhu, J. Mater. Chem. A 2013, 1, 14766 – 14772. [27] F. Zhao, H. Cheng, Y. Hu, L. Song, Z. Zhang, L. Jiang, L. Qu, Sci. Rep. 2014, 4, 5882. [28] J. Frangioni, Curr. Opin. Chem. Biol. 2003, 7, 626 – 634. [29] S. Achilefu, Angew. Chem. 2010, 122, 10010 – 10012. [30] S. Achilefu, Angew. Chem. Int. Ed. 2010, 49, 9816 – 9818; Angew. Chem. 2010, 122, 10010 – 10012. [31] K. P. Loh, Q. Bao, G. Eda, M. Chhowalla, Nat. Chem. 2010, 2, 1015 – 1024. [32] G. M. Whitesides, Nat. Biotechnol. 2003, 21, 1161 – 1165. [33] P. Zrazhevskiy, M. Sena, X. Gao, Chem. Soc. Rev. 2010, 39, 4326 – 4354. [34] L. Y. T. Chou, K. Ming, W. C. W. Chan, Chem. Soc. Rev. 2011, 40, 233 – 245. [35] A. Gnach, A. Bednarkiewicz, Nano Today 2012, 7, 532 – 563. [36] T. Yeh, J. Syu, C. Cheng, T. Chang, H. Teng, Adv. Funct. Mater. 2010, 20, 2255 – 2262. [37] S. Park, K. Lee, G. Bozoklu, W. Cai, S. T. Nguyen, R. S. Ruoff, ACS Nano 2008, 2, 572 – 578. [38] D. Marton, K. Boyd, A. Al-Bayati, S. Todorov, J. Rabalais, Phys. Rev. Lett. 1994, 73, 118. [39] Z. Wu, P. a. Webley, D. Zhao, J. Mater. Chem. 2012, 22, 11379 – 11389. Received: November 19, 2014 Published online on March 11, 2015
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& Bioimaging | Very Important Paper |
Oxidized Carbon Nitrides: Water-Dispersible, Atomically Thin Carbon Nitride-Based Nanodots and Their Performances as Bioimaging Probes Junghoon Oh,[a] Ran Ji Yoo,[b] Seung Yeon Kim,[a] Yong Jin Lee,[b] Dong Wook Kim,*[a] and Sungjin Park*[a] Abstract: Three-dimensional (3D) carbon nitride (C3N4)based materials show excellent performance in a wide range of applications because of their suitable band structures. To realize the great promise of two-dimensional (2D) allotropes of various 3D materials, it is highly important to develop routes for the production of 2D C3N4 materials, which are one-atom thick, in order to understand their intrinsic properties and identify their possible applications. In this work, water-dispersible, atomically thin, and small carbon nitride nanodots were produced using the chemical oxidation of
Introduction Two-dimensional (2D) materials, such as graphene,[1–4] MoS2,[5, 6] and hexagonal boron nitride,[7] are attractive for a wide range of applications owing to their intriguing physical, chemical, and morphological properties and high surface areas, which differ from those of the three-dimensional (3D) allotropes. The 3D carbon nitride (C3N4)-based materials, including graphitic C3N4 (g-C3N4) and polymeric g-C3N4, show excellent performances as photocatalysts[8] and electrocatalysts[9] in photovoltaic devices and as luminescent materials in chemical and biosensors[10] because of their suitable band structures. Consequently, the production of new types of 2D carbon nitride materials is a great challenge to understand their intrinsic properties and identify their possible applications. Furthermore, it is highly important to achieve homogeneous colloidal suspensions of individual 2D carbon nitride materials by scalable synthetic methods to facilitate the use of such materials. Herein, we introduce a novel route to produce water-dispersible, atomically thin, [a] J. Oh, S. Y. Kim, Prof. D. W. Kim, Prof. S. Park Department of Chemistry and Chemical Engineering Inha University, 100 Inha-ro, Nam-gu, Incheon 402-751 (Korea) E-mail: [email protected] [email protected] [b] R. J. Yoo, Y. J. Lee Molecular Imaging Research Center Korea Institute of Radiological and Medical Sciences 75 Nowon-ro, Nowon-gu, Seoul 139-706 (Korea) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201406151. Chem. Eur. J. 2015, 21, 6241 – 6246
graphitic C3N4. Various analyses, including X-ray diffraction, X-ray photoelectron, Fourier-transform infrared spectroscopy, and combustion-based elemental analysis, and thermogravimetric analysis, confirmed the production of 3D oxidized C3N4 materials. The 2D C3N4 nanodots were successfully exfoliated as individual single layers; their lateral dimension was several tens of nanometers. They showed strong photoluminescence in the visible region as well as excellent performances as cell-imaging probes in an in vitro study using confocal fluorescence microscopy.
and small carbon nitride-based materials via the chemical oxidation of g-C3N4 and their application in a cell-imaging study as bioimaging probes. Graphitic C3N4 is a stable metal-free layered material. Each layer of g-C3N4 is composed of triazine or tris-s-triazine building units, which contain alternating sp2 hybrid C and N atoms.[11, 12] This layered g-C3N4 material can be a good source material for the generation of single-layer C3N4 materials by exfoliating the individual layers. Although sonication and acid and heat treatments of g-C3N4 produce thin C3N4 materials, the generation of single-layer C3N4 materials that are one atom thick and their homogeneous colloidal suspensions remains a great challenge.[13–17] Chemical oxidation is often a useful approach to produce stable suspensions of carbon-based nanomaterials in polar solvents via the introduction of polar oxygen functional groups onto carbon atoms.[18–20] For example, graphite oxide produced by chemical oxidation of graphite has been widely used to produce homogeneous colloidal suspensions of chemically modified graphene materials.[18] Because the oxygen-containing functional groups of graphite oxide are hydrophilic, singlelayer graphene oxide disperses well in water.[21] Furthermore, the use of graphene-based materials in various applications can be facilitated by further chemical modifications on the reactive oxygen functionalities.[22] To overcome the difficulties of adding other functional groups onto chemically robust g-C3N4,[11, 12] the presence of oxygen functional groups on C3N4-based materials is highly important and will enable additional applications. Although it is thought that gC3N4 networks are stable against acid treatments,[13, 23, 24] we re-
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Full Paper the flat surface structure of g-C3N4 was converted into a porous structure. Colorless, transparent, and homogeneous colloidal suspensions incorporating OCN nanodots were produced by sonication of the OCN powder in water after removal of any undispersed particles by centrifuge. The suspensions showed the Tyndall effect, confirming the presence of particles (Figure 1 d). These aqueous suspensions were stable under acidic, neutral, and basic conditions (see the Supporting Information). TEM and atomic force microscopy (AFM) were used to observe the morphology of the OCN nanodots after drying droplets of the suspension. As shown in an AFM scan and a TEM image (Figure 1 e, f), the dispersed OCN materials primarily comprised 2D thin nanodots. Figure 1. a) Overall Scheme for the production of single-layer carbon nitride-based nanodots; b) SEM images of The lateral dimensions of these the OCN powder samples at different magnifications (inset in the left image: Photo of the OCN powder); c) TEM OCN nanodots were several image of the OCN powder; d) Aqueous colloidal suspension of OCN nanodots showing the Tyndall effect; e) AFM tens of nanometers and some scanning image of OCN nanodots on a mica surface and height profiles of sections marked by the solid (top) and of them were less than 10 nm, dashed (bottom) lines; f) TEM image of the OCN nanodots. which was much smaller than that of the previously reported C3N4-based sheet-like materials.[13, 15, 16, 25] visited the chemical oxidation of g-C3N4 using strong oxidants and acids to generate single-layer C3N4-based materials and The thicknesses of the nanodots, determined via the height profile of the AFM measurements, were in the range of 0.5– their suspensions (Figure 1 a). 1.5 nm. Importantly, the thinnest OCN nanodots were about 0.5 nm thick, which supports the successful exfoliation of OCN nanodots as individual single layers with one-atom thickResults and Discussion ness.[26, 27] Such single-layer nanodots were frequently found in our AFM scans. Since the term “graphitic” suggests 3D stacking A pale yellow powder was obtained by polycondensation of of individual layers, it is not appropriate to use it for singledicyandiamide at 500 8C in a furnace, as was previously reportlayer OCN nanodots. These results were interesting because ed,[12] and the formation of in-plane C3N4 networks and layered structures was confirmed by various characterization methods most of the previously reported C3N4-based sheet-like materials (see the Supporting Information). The as-prepared g-C3N4 was are not single layers with one-atom thickness.[13–17] The demonstrated ability to disperse 2D single-layer C3N4-based materials treated with a strong oxidant (KMnO4) in the presence of in a liquid phase without the use of surfactants or stabilizers H2SO4 ; this method is frequently used to oxidize graphite[21] would be extremely important for their possible application in and afforded oxidized carbon nitride (henceforth, OCN) as various research areas. a white powder (Figure 1 b). Figure 1 b shows scanning elecIt is important to examine the optical properties of the 3D tron microscopy (SEM) images of the OCN powder at two difOCN powder and 2D OCN nanodots to assess their possible ferent magnifications. In the low-magnification SEM image applicability in photocatalytic reactions, optical sensors, and (Figure 1 b, left), it appeared that the surface of the OCN bioimaging applications. First, the UV/Vis adsorption and phopowder was smooth and flat. However, high-magnification toluminescence (PL) properties of 3D materials, such as the asSEM (Figure 1 b, right) revealed a 3D porous structure, which prepared g-C3N4 and 3D OCN powder samples, were comwas much different from the flat morphology of the as-prepared g-C3N4 (see the Supporting Information, Figure S1). pared. The UV/Vis adsorption spectrum of the OCN powder showed a strong adsorption at around 300–400 nm and was Transmission electron microscopy (TEM) confirmed the porous blue-shifted relative to the spectrum of the as-prepared g-C3N4 structure of the OCN materials (Figure 1 c). During oxidation, Chem. Eur. J. 2015, 21, 6241 – 6246
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Full Paper Optical/fluorescence imaging of living cells with imaging modalities, such as confocal microscopy, has gained popularity in a variety of biomedical areas.[28] Visualization of cells using these tools requires fluorescent probes that have high biocompatibilities, suitable sizes and morphologies, high PL quantum yields, high stabilities in aqueous media, and low cytotoxicities.[29–31] In recent decades, a range of nanoscale semiconducting materials containing heavy metals, particularly quantum dots, have been applied to a broad range of biological imaging systems.[32–34] However, the high cytotoxicity of these nanomaterials restricted their application in in vivo as well as in vitro studies.[35] Accordingly, it is important to develop “heavymetal free” optical probes with excellent optical properties, high serum stability, and good biocompatibility. To enable biological applications of 2D OCN nanodots, their biocompatibility was evaluated by measuring the amount of viable cells after 48 h of incubation in varying concentrations (0–100 mg mL¢1) of the OCN nanodots using a trypan blue exclusion assay with RAW 264.7 cells (Figure 3 a). The OCN nano-
Figure 2. a) UV/Vis diffuse reflectance absorption spectra of the solid as-prepared g-C3N4 and 3D OCN samples and UV/Vis adsorption spectrum of an aqueous suspension of OCN nanodots; b) PL spectra of the solid as-prepared g-C3N4 and 3D OCN samples and an aqueous suspension of the OCN nanodots.
powder (Figure 2 a). PL emissions in the visible (350–600 nm) and near-IR regions (800–900 nm) were found in a PL spectrum of the OCN powder (Figure 2 b); these peaks were significantly blue-shifted relative to those of the as-prepared g-C3N4, suggesting that the band gap was enlarged by chemical oxidation. This shifting trend was similar to previously reported results using chemically modified g-C3N4-based materials.[13–15] In addition to the optical properties of the 3D solid samples, those of the 2D OCN nanodots in colloidal suspensions were also measured. The suspension containing OCN nanodots visually emitted blue light under exposure to UV light using a UV lamp (see the Supporting Information, Figure S8). A UV/Vis absorption spectrum of the suspensions containing OCN nanodots showed strong adsorption peaks at 227 and 267 nm, which were blue-shifted relative to those of the 3D OCN powder samples (Figure 2 a). The suspensions of the OCN nanodots also showed a strong emission in the visible region and the emitting wavelength was slightly shifted (from 417 to 407 nm) relative to that of the 3D OCN powder (Figure 2 b); this blue-shift could be due to localized structures in the small OCN nanodots.[14] The small size, fluorescent properties, and water dispersibility of the single-layer OCN nanodots are highly beneficial for applications as optical sensors and bioimaging probes. Chem. Eur. J. 2015, 21, 6241 – 6246
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Figure 3. a) In vitro cell viability of RAW 264.7 cells after 48 h of incubation with different concentrations of the OCN nanodots by a trypan blue exclusion assay; b) Confocal fluorescence microscopic image of RAW 264.7 cells after incubation for 24 h at 37 8C with single-layer OCN nanodots.
dots exhibited little cytotoxicity to the RAW 264.7 cells at relatively high concentrations. In particular, the viabilities of the RAW 264.7 cells were 87.2 5.6 %, even at the highest concentration of OCN nanodots (100 mg mL¢1), indicating that the OCN nanodots exhibited high biocompatibility and low cytotoxicity. To investigate the possibility of cell-labeling using these biocompatible single-layer OCN nanodots, we attempted to conduct a bioimaging study using confocal fluorescence microscopy on the RAW 264.7 cells. As can be seen from Figure 3 b and S9 (a merged image of the bright field and confocal images, see the Supporting Information), the OCN nanodots could be used to successfully visualize the RAW cells without any aggregation in the cytoplasm. This confocal image showed that the green-emitting OCN nanodots were internalized into the RAW cells. An interesting phenomenon is that the OCN nanodots did not penetrate into nucleus but instead surrounded it. These results suggested that OCN nanodots could be promising fluorescent probes for biomedical imaging applications. The chemical structure of the 3D OCN powder was determined using X-ray diffraction (XRD), combustion-based elemental analysis, Fourier-transform infrared (FT-IR) and X-ray
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Full Paper After oxidation, the intensities of the peaks corresponding to the N¢H bonds in the FT-IR and XPS N1 s spectra of the 3D OCN powder were enhanced relative to that of the as-prepared gC3N4 (Figure 4). As shown in the FT-IR spectra, the intensities of the peak at 3350 cm¢1, which corresponded to O¢H bonds, and the peak at 1704 cm¢1, which corresponded to C=O bonds, in the spectrum of the OCN powder were stronger than those in the spectrum of g-C3N4.[36, 37] The deconvoluted XPS C1s spectra of the OCN powder showed enhanced intensity of the peak that corresponded to the C=O moieties relative to that of the as-prepared g-C3N4 (Figure 4 d).[1, 22, 38] Elemental analysis of the OCN powder (C/O ratio of 1.46:1) and as-prepared g-C3N4 (C/O ratio of 7.88:1) also supported the introduction of oxygen atoms during the oxidation process. The thermal stabilities of the g-C3N4 and 3D OCN materials were measured by thermogravimetric analysis (TGA). As shown in the TGA curves (Figure 4 e), the as-prepared g-C3N4 showed good thermal stability up to 600 8C, then was completely removed by heat treatment at around 700 8C, which corresponded to decomposition of Figure 4. Chemical characterizations of the as-prepared g-C3N4 and 3D OCN powder samples: a) XRD patterns; the tris-s-triazine moieties of gb) FT-IR spectra; c) XPS N1 s spectra including deconvolution; d) XPS C1 s spectra including deconvolution; e) TGA C3N4.[7, 39] In contrast, the TGA curves. curve of the OCN powder showed stepwise mass losses as the temperature increased. The loss below 300 8C was attributphotoelectron spectroscopy (XPS), and thermogravimetric analed to decomposition of the labile functional groups in the oxiysis (TGA). An XRD pattern of the as-prepared g-C3N4 powder dized carbon nitride networks, which were introduced by the exhibited a peak at 27.38, which corresponded to the interlayer oxidation process. A gradual weight loss from around 60 to distance between the 2D C3N4 networks in g-C3N4, and a peak 0 wt % was observed during heat treatment from 350 to at 13.18, which corresponded to the in-plane hole-to-hole dis700 8C, corresponding to thermal decomposition of the tris-stance.[8, 12] After oxidation, the interlayer distance slightly detriazine moieties. creased and the peak for the in-plane motif almost disapThe combined chemical characterizations suggested the forpeared in the XRD pattern of the 3D OCN powder (Figure 4 a). mation of NH2- and C=O-containing moieties, such as carboxyl This result suggested that the OCN powder featured a graphitic stacking structure of each oxidized C3N4 layer and the in-plane and ketone groups, which could have been generated by decomposition of the N¢C=N groups at the edges of the C3N4 structure was significantly damaged by oxidation. This damage could be one reason for the generation of the porous structure networks during oxidation (see the Supporting Information, of the 3D OCN powder. Figure S10). The presence of these hydrophilic groups at the Chem. Eur. J. 2015, 21, 6241 – 6246
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Full Paper edges enabled the generation of homogeneous suspensions of the single-layer OCN nanodots in water. In addition, the coexistence of basic amine and acidic carboxyl groups could be the reason for the production of stable aqueous suspensions under both acidic and basic conditions. Moreover, the chemically reactive amine and carboxyl groups could be chemical modified to introduce various functional groups onto the C3N4 networks.
mersed in a water bath set at 35 8C and stirred with a magnetic stirring bar for 2 h. Then, H2O (200 mL) was slowly added to the flask in an ice bath. Excess hydrogen peroxide (30 % in water, Sigma–Aldrich) was then added to the flask until no gas bubbling was observed, followed by the addition of excess water. The resultant mixture was filtered through an Anodisc membrane filter (47 mm of diameter, 0.1 mm of pore sizes; Whatman). The filtrate was further washed several times with H2O then dried under vacuum at room temperature for 12 h.
Conclusion
Production of aqueous suspensions of the 2D OCN nanodots
We synthesized novel OCN materials using a liquid process via the treatment of g-C3N4 with KMnO4 in the presence of sulfuric acid. During the reaction, polar functional groups, such as amine, carboxyl, and ketone groups, were introduced into the C3N4 network, as confirmed by XPS, FT-IR, TGA, and elemental analysis. XRD patterns showed the presence of a graphitic layered structure in the 3D OCN powder, and the SEM and TEM images showed the generation of a porous structure. The presence of functional groups on the C3N4 network will be highly useful for further chemical modifications to introduce specific functional groups onto the C3N4-based materials. Homogeneous colloidal suspensions of 2D OCN nanodots were produced by sonication of the 3D OCN powder in water and showed strong photoluminescence in the visible region. The lateral size of OCN nanodots was several tens of nanometers and their thicknesses were in the range of 0.5–1.5 nm, which indicated that the OCN nanodots were successfully exfoliated as very thin and small 2D materials; some portion of the generated nanodots were determined to comprise a single layer with one-atom thickness (ca. 0.5 nm). This approach for dispersing single-layer C3N4-based materials in a liquid phase without the use of surfactants or stabilizers should be extremely useful for the production of a wide range of colloidal suspensions that can be used to study the fundamental properties of 2D C3N4-based materials; this will also facilitate the discovery of their potential applications in catalysis, bio-sensing, biomedical imaging, fabrication of composites, and thin-film technologies. In particular, the water-dispersible OCN nanodots showed good performance in a cell-imaging study using confocal fluorescence microscopy. Given their favorable properties, such as high biocompatibility, low cytotoxicity, good dispersibility, and stability in aqueous media, as well as suitable optical characteristics, these “heavy metal free” OCN nanodots can be regarded as physiologically friendly alternatives to conventional nanoscale metal-based optical probes for biomedical imaging.
Experimental Section Preparation of OCN The as-prepared g-C3N4 powder (1 g, see the Supporting Information for synthetic procedure) and sulfuric acid (50 mL, 98 %, Dae Jung Chemicals, Korea) were loaded into an Erlenmeyer flask. Potassium permanganate (KMnO4, 3.5 g, Dae Jung Chemicals, Korea) was slowly added to the flask in an ice bath. The flask was imChem. Eur. J. 2015, 21, 6241 – 6246
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OCN powder (10 mg) was added into a vial filled with H2O (10 mL). The vial was then sonicated using an ultrasonic cleaner (Bransonic 8510, Danbury, CT, USA) for 200 min, affording colorless colloidal suspensions of the 2D OCN nanodots. Undispersed particles were separated by centrifugation (Supra 22 K centrifuge; Hanil Science Industrial, Incheon, Korea) at 2000 rpm for 30 min.
Cell culture Mouse macrophage cell lines (RAW 264.7) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; WelGENE Inc., Korea) containing 10 % fetal bovine serum (FBS) and 1 % antibiotics and grown in a humidified incubator at 37 8C under 5 % CO2.
In vitro viability test Solutions of each concentration of OCN nanodots (0, 50, and 100 mg mL¢1) in phosphate buffered saline (PBS) were added to a suspension of 1 Õ 106 RAW 264.7 cells in 1 mL of cell-culture media, and the mixture was incubated at 37 8C for 2 h. After centrifugation (250 Õ g, 5 min), the supernatant was removed and the cells were washed twice with 1 mL of PBS. The OCN-labeled RAW 264.7 cells were plated in a 6-well culture plate and incubated with DMEM containing 10 % FBS at 37 8C under humidified 5 % CO2 for 48 h. The cell viability of OCN-labeled RAW 264.7 cells was determined via the trypan blue exclusion test. All data are expressed as mean standard deviation.
In vitro confocal fluorescence microscopy The labeled RAW 264.7 cells (2 Õ 105) with OCN nanodots were plated in 8-well chamber slides and incubated with DMEM containing 10 % FBS at 37 8C under humidified 5 % CO2 for 4 h. After incubation, the supernatant was removed and the cells were washed twice with PBS. Phenol red-free DMEM containing 2 % FBS was refreshed after cell washing. Fluorescence images were then captured using a laser-scanning confocal microscope (LSM710, Carl Zeiss MicroImaging, Jena, Germany).
Instruments XPS study was performed using an angle-resolved X-ray photoelectron MXR1 Gun, 400 mm, 15 kV spectrometer (Theta probe, Thermo Fisher Scientific, UK); binding energies were determined versus the C1 s peak at 284.6 eV. Elemental analysis for C, N, H, and O was conducted using a FLASH EA1112 instrument (Thermo Electron, Italy). XRD patterns were obtained using a Multiplex spectrometer (Rigaku, Tokyo, Japan). PL spectra were recorded with a HR800 UV spectrometer (HORIBA JovinYvon, 325 nm excitation, He Cd laser) for the as-prepared g-C3N4 powder and PI-MAX3 spectrometer (Princeton Instruments, 325 nm excitation, He Cd laser) for the
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Full Paper OCN powder and suspensions. FT-IR spectra of the samples in KBr pellets were obtained using an FT-IR vacuum spectrometer (VERTEX 80 V, Bruker, Germany) and included 64 scans. SEM images of Pt-coated samples were collected using a field emission scanning electron microscope (S-4300, Hitachi, Tokyo) at an accelerating voltage of 15 kV. TEM images were obtained using a JEOL JEM2100F instrument (JEOL Co. Ltd., Japan) at 200 kV. TEM samples were prepared by drying a droplet of a mixture including 3D OCN powder in water or diluted suspension incorporating 2D OCN nanodots on a mesh copper grid coated with carbon film (CF200Cu, Electron Microscopy Sciences). TGA measurements (SDT Q600, TA Instruments) were performed at a heating rate of 5 8C min¢1 from 30 to 800 8C in a N2 gas environment. AFM images were obtained using a XE-100 microscope (PSIA, Korea) in non-contact mode.
Acknowledgements This work was supported by Inha University and grants from the Center for Advanced Soft Electronics funded by the Ministry of Science, ICT & Future Planning as a Global Frontier Project (CASE-2014M3A6A5060938)and from the Basic Science Research Program (Grant no. NRF-2012R1A1A2044945) of the Ministry of Science, ICT & Future Planning, South Korea. S.P. thanks the Busan Center at the Korea Basic Science Institute (KBSI) for the XPS analysis. Keywords: bioimaging · carbon oxidation · photoluminescence
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[1] D. R. Dreyer, S. Park, C. W. Bielawski, R. S. Ruoff, Chem. Soc. Rev. 2010, 39, 228 – 240. [2] W. J. Lee, U. N. Maiti, J. M. Lee, J. Lim, T. H. Han, S. O. Kim, Chem. Commun. 2014, 50, 6818 – 6830. [3] K. E. Lee, J. E. Kim, U. N. Maiti, J. Lim, J. O. Hwang, J. Shim, J. J. Oh, T. Yun, S. O. Kim, ACS Nano 2014, 8, 9073 – 9080. [4] J. Shim, J. M. Yun, T. Yun, P. Kim, K. E. Lee, W. J. Lee, R. Ryoo, D. J. Pine, G. Yi, S. O. Kim, Nano Lett. 2014, 14, 1388 – 1393. [5] B. Radisavljevic, A. Radenovic, J. Brivio, V. Giacometti, A. Kis, Nat. Nanotechnol. 2011, 6, 147 – 150. [6] K. Chang, Z. Mei, T. Wang, Q. Kang, S. Ouyang, J. Ye, ACS Nano 2014, 8, 7078 – 7087. [7] C. Zhi, Y. Bando, C. Tang, H. Kuwahara, D. Golberg, Adv. Mater. 2009, 21, 2889 – 2893. [8] X. Wang, K. Maeda, A. Thomas, K. Takanabe, G. Xin, J. M. Carlsson, K. Domen, M. Antonietti, Nat. Mater. 2009, 8, 76 – 80.
Chem. Eur. J. 2015, 21, 6241 – 6246
www.chemeurj.org
[9] Y. Zheng, Y. Jiao, J. Chen, J. Liu, J. Am. Chem. Soc. 2011, 133, 20116 – 20119. [10] Y. Tang, H. Song, Y. Su, Y. Lv, Anal. Chem. 2013, 85, 11876 – 11884. [11] Y. Wang, X. Wang, M. Antonietti, Angew. Chem. Int. Ed. 2012, 51, 68 – 89; Angew. Chem. 2012, 124, 70 – 92. [12] A. Thomas, A. Fischer, F. Goettmann, M. Antonietti, J. Mìller, R. Schlçgl, J. M. Carlsson, J. Mater. Chem. 2008, 18, 4893 – 4908. [13] L. Chen, D. Huang, S. Ren, T. Dong, Y. Chi, G. Chen, Nanoscale 2013, 5, 225 – 230. [14] X. She, H. Xu, Y. Xu, J. Yan, J. Xia, L. Xu, Y. Song, Y. Jiang, Q. Zhang, H. Li, J. Mater. Chem. A 2014, 2, 2563 – 2570. [15] J. Tian, Q. Liu, A. M. Asiri, A. O. Al-Youbi, X. Sun, Anal. Chem. 2013, 85, 5595 – 5599. [16] S. Yang, Y. Gong, J. Zhang, L. Zhan, L. Ma, Z. Fang, R. Vajtai, X. Wang, P. M. Ajayan, Adv. Mater. 2013, 25, 2452 – 2456. [17] P. Niu, L. Zhang, G. Liu, H. Cheng, Adv. Funct. Mater. 2012, 22, 4763 – 4770. [18] S. Park, R. S. Ruoff, Nat. Nanotechnol. 2009, 4, 217 – 224. [19] V. Datsyuk, M. Kalyva, K. Papagelis, J. Parthenios, D. Tasis, a. Siokou, I. Kallitsis, C. Galiotis, Carbon 2008, 46, 833 – 840. [20] J. Figueiredo, M. Pereira, M. Freitas, J. Orfao, Carbon 1999, 37, 1379 – 1389. [21] S. Park, J. An, R. Piner, I. Jung, Chem. Mater. 2008, 20, 6592 – 6594. [22] J. Han, L. L. Zhang, S. Lee, J. Oh, K. Lee, J. R. Potts, J. Ji, X. Zhao, R. S. Ruoff, S. Park, ACS Nano 2013, 7, 19 – 26. [23] T. Y. Ma, Y. Tang, S. Dai, S. Z. Qiao, Small 2014, 10, 2382 – 2389. [24] Y. Zhang, A. Thomas, M. Antonietti, X. Wang, J. Am. Chem. Soc. 2009, 131, 50 – 51. [25] Q. Wang, W. Wang, J. Lei, N. Xu, F. Gao, H. Ju, Anal. Chem. 2013, 85, 12182 – 12188. [26] J. Xu, L. Zhang, R. Shi, Y. Zhu, J. Mater. Chem. A 2013, 1, 14766 – 14772. [27] F. Zhao, H. Cheng, Y. Hu, L. Song, Z. Zhang, L. Jiang, L. Qu, Sci. Rep. 2014, 4, 5882. [28] J. Frangioni, Curr. Opin. Chem. Biol. 2003, 7, 626 – 634. [29] S. Achilefu, Angew. Chem. 2010, 122, 10010 – 10012. [30] S. Achilefu, Angew. Chem. Int. Ed. 2010, 49, 9816 – 9818; Angew. Chem. 2010, 122, 10010 – 10012. [31] K. P. Loh, Q. Bao, G. Eda, M. Chhowalla, Nat. Chem. 2010, 2, 1015 – 1024. [32] G. M. Whitesides, Nat. Biotechnol. 2003, 21, 1161 – 1165. [33] P. Zrazhevskiy, M. Sena, X. Gao, Chem. Soc. Rev. 2010, 39, 4326 – 4354. [34] L. Y. T. Chou, K. Ming, W. C. W. Chan, Chem. Soc. Rev. 2011, 40, 233 – 245. [35] A. Gnach, A. Bednarkiewicz, Nano Today 2012, 7, 532 – 563. [36] T. Yeh, J. Syu, C. Cheng, T. Chang, H. Teng, Adv. Funct. Mater. 2010, 20, 2255 – 2262. [37] S. Park, K. Lee, G. Bozoklu, W. Cai, S. T. Nguyen, R. S. Ruoff, ACS Nano 2008, 2, 572 – 578. [38] D. Marton, K. Boyd, A. Al-Bayati, S. Todorov, J. Rabalais, Phys. Rev. Lett. 1994, 73, 118. [39] Z. Wu, P. a. Webley, D. Zhao, J. Mater. Chem. 2012, 22, 11379 – 11389. Received: November 19, 2014 Published online on March 11, 2015
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