DOI: 10.1002/chem.201304869
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& Carbon Nanodots
Electrochemical Synthesis of Carbon Nanodots Directly from Alcohols Jianhui Deng, Qiujun Lu, Naxiu Mi, Haitao Li, Meiling Liu, Mancai Xu, Liang Tan, Qingji Xie, Youyu Zhang,* and Shouzhuo Yao[a]
Abstract: Carbon nanodots (C-dots) show great potential as an important material for biochemical sensing, energy conversion, photocatalysis, and optoelectronics because of their water solubility, chemical inertness, low toxicity, and photoand electronic properties. Numerous methods have been proposed for the preparation of C-dots. However, complex procedures and strong acid treatments are often required, and the as-prepared C-dots tend to be of low quality, and in particular, have a low efficiency for photoluminescence. Herein, a facile and general strategy involving the electrochemical carbonization of low-molecular-weight alcohols is proposed. As precursors, the alcohols transited into carboncontaining particles after electrochemical carbonization
Introduction Carbon nanodots (C-dots), a new kind of fluorescent carbon nanomaterial consisting of a graphite structure or amorphous carbon core and carbonaceous surfaces with rich oxygen-containing groups, have attracted tremendous interest because of their unique properties, including high surface area, low toxicity, and superior optical properties (tunable, non-blinking, and stable fluorescence emission).[1–4] Since fluorescence C-dots were first prepared in 2004 during the purification of singlewalled carbon nanotubes through preparative electrophoresis,[5] increasing attention has been paid to their synthesis and applications.[6–8] At present, a variety of methods have been developed for the preparation of C-dots, including laser ablation,[8, 9] arc-discharge,[5] pyrolysis,[10–12] hydrothermal synthesis,[13–15] electrochemical etching,[2, 7, 16–19] and ultrasound- and microwave-assisted synthesis.[6, 20, 21] Of these, the electrochemical method is advantageous because of its low cost and easy manipulation.[17, 18]
[a] J. Deng, Q. Lu, N. Mi, Prof. H. Li, M. Liu, Prof. M. Xu, Prof. L. Tan, Prof. Q. Xie, Prof. Y. Zhang, Prof. S. Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education) College of Chemistry and Chemical Engineering Hunan Normal University, Changsha 100084 (P. R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201304869. Chem. Eur. J. 2014, 20, 4993 – 4999
under basic conditions. The resultant C-dots exhibit excellent excitation- and size-dependent fluorescence without the need for complicated purification and passivation procedures. The sizes of the as-prepared C-dots can be adjusted by varying the applied potential. High-quality C-dots are prepared successfully from different small molecular alcohols, suggesting that this research provides a new, highly universal method for the preparation of fluorescent C-dots. In addition, luminescence microscopy of the C-dots is demonstrated in human cancer cells. The results indicate that the as-prepared C-dots have low toxicity and can be used in imaging applications.
The electrochemical approach involves a nonselective “topdown” chemical cutting process of a carbon material such as graphite, carbon nanotube, or carbon fiber electrodes.[7, 16, 17] However, C-dots prepared through the present electrochemical methods have mixed sizes, and require further separation through filtration or chromatography to obtain monodisperse C-dots.[2, 7, 17] In addition, the as-prepared C-dots possess low photoluminescence efficiencies (always lower than 10 %),[2, 7, 17, 22, 23] which limits further study in this field. Furthermore, the size-controllable preparation of high-quality C-dots is difficult and complicated with traditional electrochemical methods.[2, 7] From the point of view of material synthesis, the exploration of new carbon sources for the simple, economical, and controllable synthesis of such C-dots is highly desirable. Early researchers motioned in their reports that carbon-containing particles could be formed from alcohol through a suitable electro-oxidation process.[24, 25] Enlightened by these investigations, we proposed a novel one-pot electrochemical carbonization method for the preparation of C-dots. Uniform Cdots were prepared directly from low-molecular-weight alcohols for the first time. Interestingly, the size of the resultant Cdots could be adjusted by changing the synthesis potential. The as-prepared C-dots exhibited excellent fluorescence properties, with the quantum yield (QY) reaching 15.9 %. Several low-molecular-weight alcohols were used for the successful production of C-dots, indicating the universality of this new strategy. In addition, luminescence microscopy imaging of the C-dots internalized in human cancer cells was performed,
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Scheme 1. Electrochemical synthesis of C-dots. Under basic conditions, two Pt sheets were used as the anode and cathode, respectively, and single and simple organics were used as the carbon source. Under suitable DC control, the organics were transformed into C-dots. The resultant C-dot solutions were obtained through simple purification methods.
the different C-dots reveal that the diffraction contrast is very low with no obvious lattice fringes, indicating that the prepared C-dots have an amorphous structure. For further confirmation of this result, selectedarea electron diffraction (SAED), X-ray diffraction (XRD), and Raman spectroscopy measurements were performed (Figures S1 and S2 in the Supporting Information). The diffuse ring pattern obtained from SAED indicates that the as-prepared Cdots contain no crystal lattice, in
which indicated that the C-dots are promising candidates for use in biolabeled carriers and nanodevices.
Results and Discussion As shown in Scheme 1, it can be imagined that the electrochemical carbonization of an alcohol such as ethanol (EtOH) into ultrasmall particles may offer a straightforward and facile strategy for the preparation of high-quality C-dots. In this work, two Pt sheets were used as the working and auxiliary electrodes and a calomel electrode mounted on a freely adjustable Luggin capillary was used as the reference electrode. The electrolyte was NaOH/EtOH, the synthesis potential was 3.0–9.0 V, and the electrochemical carbonization time was 3– 4 h. The control experiments showed that there was no formation of C-dots in H2SO4/EtOH or Na2SO4/EtOH, whereas highquality C-dots were obtained in alkaline environments (e.g., NaOH/EtOH, Na2HPO4/EtOH, and Na2CO3/EtOH), in which the OH groups play an important role.[24, 25] Figure 1 shows typical transmission electron microscopy (TEM) images and size distributions of C-dots synthesized at 3.0, 4.5, 6.0, and 7.5 V. The images reveal that the as-synthesized C-dots are uniform and monodisperse. The size of the Cdots is strongly dependent on the applied potential, with diameters centered at about 2.1, 2.9, 3.5, and 4.3 nm, respectively, and the size increasing with the applied potential. It has been reported that a large number of hydroxyl free radicals, carbonium ions, and alkoxy radicals can be produced during the electrochemical oxidation of alcohols in alkaline solution.[24–26] Crosslinking and dehydration of these active substances occurs under suitable potentials and in an alkaline environment.[24–28] We speculated that the C-dots are formed from ethanol electrochemical oxidation and dehydration at a suitable potential. The applied potential should thus play a key role in the size control of the C-dots. The higher the applied potential, the greater the number of alcohol molecules oxidized and free radicals and carbonium ions that might be produced to undergo crosslinking and dehydration to form carbon nanoparticles, leading to larger C-dots. In contrast with traditional electrochemical routes,[2, 7, 19, 23] high-resolution TEM (HRTEM) images of Chem. Eur. J. 2014, 20, 4993 – 4999
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Figure 1. a, c, e, g) TEM images and b, d, f, h) corresponding size distributions of C-dots obtained from ethanol at potentials of 3.0, 4.5, 6.0, and 7.5 V, respectively. Insets: corresponding HRTEM images of C-dots; scale bar: 3 nm.
agreement with the HRTEM results. At the same time, the XRD pattern shows a broad peak at 2q = 238, revealing an amorphous carbon phase, which agrees well with the HRTEM analysis. In addition, Raman spectra were obtained for the as-prepared C-dots produced through the electrochemical carbonization method and the traditional electrochemical “cutoff”
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Full Paper route.[7] The G-band at 1575 cm 1 and D-band at 1350 cm 1 refer to the graphitic carbon and disordered structure, respectively.[29] The IG/ID ratio for the C-dots obtained from ethanol is clearly lower than that for the C-dots prepared from graphite, indicating that C-dots from alcohols are more disordered and amorphous than those obtained through the traditional electrochemical procedure. In the traditional procedure, C-dots are obtained from a graphitic electrode through an electrochemical “cutoff” mechanism, which leads to their graphitic structural characteristics.[2, 7, 17] However, in this work, C-dots were prepared from low-molecular-weight alcohols through carbonization and dehydration, so the disordered structure is reasonable. These results suggest that our method is totally different from traditional electrochemical routes. Energy-dispersive spectrometry (EDX) and elemental analysis were performed to confirm the components of the as-prepared C-dots. As shown in Figure S3, clear peaks for carbon and copper (holder) are observed, suggesting that the main element contained in the C-dots is carbon. Further elemental analysis was carried out, and as shown in Table S1, the C-dots are composed mainly of C (80.5 %), O (16.9 %), N (0.8 %), and H (1.7 %). As shown in Figure 2, the surface functional groups of the C-dots were detected by Fourier-transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). In the FTIR spectra, the absorption bands of the C-dots prepared at different potentials at 3430, 2930, and 2850 cm 1 (Figure 2 e) are assigned to the stretching vibrations of C OH and C H.[2, 30] The stretching vibrations of C=O, C=C and C O C at about 1700, 1640, and 1250 cm 1, respectively, are observed for all the Cdots,[31–33] and the absorbance peak at 1050 cm 1 is assigned to the epoxide group.[2, 34] XPS analysis was also performed, as shown in Figure 2 a–d. Typically, the peak centered at 284.5 eV is assigned to graphitic sp2 carbon atoms, and those centered at 286.2 and 288.3 eV represent alcoholic (C O) and carbonyl (C=O) carbon atoms, respectively.[2, 34, 35] Further quantitative analysis revealed that the percentage of graphitic carbon increases with increasing potential, whereas the percentage of C O decreases significantly (Table S2, Supporting Information). As summarized in Table S2, the C=O band increases at high potentials, indicating that the C O groups on the C-dots are oxidized to carbonyl C=O groups.[2, 36] Otherwise, the carbon content increases clearly with the applied potential, which is affected by the electrochemical carbonization. These results confirm that C-dots synthesized at higher potentials have a high degree of carbonization, and prove that electrochemical carbonization is the key process for the formation of C-dots. The optical properties of C-dots hold the key for their future applications in optoelectronic devices and biological sensors.[1, 23] UV/Vis absorption and fluorescence (FL) emission spectra of the as-synthesized C-dots were recorded, and are shown in Figure 3. The well-defined UV/Vis absorption at 280 nm is ascribed to the p–p transition of the aromatic sp2 domain within the C-dots.[17, 34] The absorption strength increases clearly, suggesting that the conjugated system increases with the applied potential. The experimental result shows that the potential plays an important role in controlling the structure and size of the as-synthesized C-dots. The fluorescent Chem. Eur. J. 2014, 20, 4993 – 4999
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Figure 2. a–d) XPS of C-dots obtained from ethanol at potentials of 3.0, 4.5, 6.0, and 7.5 V, respectively. e) FTIR spectra of C-dots obtained from ethanol at potentials of 3.0 (black), 4.5 (red), 6.0 (green), and 7.5 V (blue), respectively.
properties of the as-prepared C-dots were explored further. Figure 3 b shows digital photographs of the C-dots obtained at different potentials under 365 nm excitation. A bright blue color was observed for the four C-dots. The emission color of the solutions does not change significantly, but the brightness of the C-dots changes upon changing the applied potential, indicating that the C-dots have different quantum yields under different synthesis potentials (as summarized in Table S2). As shown in Figure 3 c–f, the as-synthesized C-dots exhibit an excitation-dependent FL behavior, similarly to previously reported C-dots.[2, 12, 23] Upon changing the excitation wavelength from 340 to 500 nm, the FL peaks shift correspondingly from 451, 491, 470, and 477 nm to 542, 579, 545, and 565 nm, respectively, for the different applied potentials from 3.0 to 7.5 V.
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Figure 3. a) UV absorbance spectra; b) digital photographs of C-dots obtained at different potentials under 365 nm excitation. c–f) Fluorescence emission spectra of C-dots obtained from ethanol at potentials of 3.0, 4.5, 6.0, and 7.5 V, respectively. The excitations for these emission spectra were 300, 340, 380, 420, 460, and 500 nm, respectively. C-dot concentration: 0.05 mg mL 1 in ultrapure water; pH: 7.0; slit width: 5 nm.
In addition, the maximum fluorescence emissions appear at 485, 490, 500, and 505 nm under the maximum excitations of 390, 395, 400, and 410 nm, respectively, as shown in Figure S4 a of the Supporting Information. According to previous studies, the shift in emission peak positions with different excitation wavelengths arises not only from the different sizes of C-dots, but also from different emissive sites on the Cdots.[1, 2, 8, 12] At certain excitation wavelengths, some corresponding emissive sites would be excited and fluoresce, resulting in the excitation-dependent behaviors of the emission spectra.[2, 8] The fluorescence spectra of the C-dots could reflect the distribution of emissive sites on the C-dots, and not only the size effect.[12] The photoluminescence mechanism of the Cdots could involve quantum-confinement effects,[8, 37] emissive traps,[17, 37] electronic conjugate structures,[38] and free zigzag sites.[12] Although the exact mechanisms responsible for the FL from C-dots remain to be elucidated, the bright and colorful FL may be attributed to the chemical nature of the C-dots. As summarized in Table S3, the quantum yield of C-dots produced from ethanol with an applied potential of 6.0 V at 400 nm excitation is calculated to be about 15 % (by calibrating against quinine sulfate),[2, 17, 39] which is much higher than that at 3, 4.5, and 7.5 V. The full-width at half-maximum (FWHM) values of the emission spectra of C-dots obtained from ethanol at potentials of 3.0, 4.5, 6.0, and 7.5 V excited at 400 nm are only 54, 89, 89, and 104 nm (Figure S4 b), which are much smaller than Chem. Eur. J. 2014, 20, 4993 – 4999
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those reported previously,[2, 4, 17, 34, 39] suggesting a narrow size distribution of the product particles.[40, 41] These results are in agreement with the TEM images and size distributions of Cdots obtained from ethanol at different potentials. Similarly to that of particle size, the increase in the FWHM with applied potential may also occur because of different degrees of crosslinking of free radicals and carbonium ions from the electro-oxidation of ethanol. Different crosslinking degrees occur and result in an increase in the size distribution of C-dots under a high applied potential. For a deeper understanding of the fluorescence properties, size separation of C-dots synthesized at 4.5 and 6.0 V was performed by using three molecularweight cutoff membranes to obtain products equivalent to < 1.0, 1.0–3.5, 3.5–8.0, and > 8.0 kDa. The < 1 kDa fraction exhibits no obvious fluorescence, and there is a small amount of product in the > 8.0 kDa fraction with weak fluorescence, but the 1.0–3.5 and 3.5–8.0 kDa fractions show different blue fluorescence (Figure S6). As shown in Figure S6, for the C-dots obtained at both 4.5 V (Figure S6 a,b) and 6.0 V (Figure S6 c,d), the 1.0–3.5 kDa and 3.5–8.0 kDa fractions all show excitation-dependent emission spectra upon excitation from 300 to 400 nm and 340 to 440 nm. The maximum fluoresce emission appears at 468 and 470 nm under the maximum excitation of 360 nm for the 1.0–3.5 kDa fractions obtained at 4.5 and 6.0 V, respectively, and at 501 and 505 nm under the maximum excitation of 400 nm for the 3.5–8.0 kDa fractions obtained at 4.5 and 6.0 V, respectively. The difference between the FL of the two kinds of C-dots should be attributed to the different sizes of the products. TEM images of the fractions (Figure S5) reveal that both kinds of C-dots are monodisperse and have a uniform spherical shape. Narrow size distributions were obtained: 1.6( 0.3) and 2.9( 0.3) nm in diameter for the 1.0–3.5 and 3.5–8.0 kDa fractions, respectively, of C-dots prepared at 4.5 V, and 1.7( 0.4) and 3.1( 0.4) nm for the 1.0–3.5 and 3.5– 8.0 kDa fractions, respectively, of those prepared at 6.0 V. The FL emission peak is redshifted with an increase in diameter, indicating that the FL properties of the as-prepared C-dots are typically size-dependent; similarly to the results found in a previous study,[2, 17] the slight differences between the two kinds of C-dots are also caused by the size effect. These differences suggest that the applied potential affects the size of the final products. We also measured the quantum yields (QY) of different fractions (1.0–3.5 and 3.5–8.0 kDa) obtained at 4.5 and 6.0 V, and the results are summarized in Table S4. It is found that the different fractions of C-dots obtained from both applied potentials have different QY values, and that the same fraction has a similar QY whether it is obtained at 4.5 or 6.0 V. C-dots (1 g) were separated with cutoff membranes in ultrapure water: 1.0–3.5 and 3.5–8 kDa fractions were obtained (400 mL solutions), and their fluorescence intensities were measured. For the C-dots obtained at 4.5 V, the FL intensity of the 1.0–3.5 kDa fraction is higher than that of the 3.5–8.0 kDa fraction, and the mass ratio of Wt1.0–3.5 kDa/Wt3.5–8.0 kDa is 1.7:1. For the C-dots obtained at 6.0 V, the FL intensity of the 1.0–3.5 kDa fraction is much lower than that of the 3.5–8.0 kDa fraction, and Wt1.0–3.5 kDa/Wt3.5–8.0 kDa is 1:4. These results indicate that the C-dots obtained at 6.0 V are larger (centered at 3.5–8.0 kDa)
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Full Paper than those obtained at 4.5 V (centered at 1.0–3.5 kDa), in agreement with the FL measurement of different fractions Cdots, and confirm the effect of the applied potential on the products. All the above experimental results suggest that the fluorescence properties are affected mainly by the size of the C-dots. However, the exact mechanism of the production of Cdots through this new method is still under investigation. For further exploration of the optical properties of the assynthesized C-dots, a detailed FL study was undertaken in water with different pH values, containing NaCl of various ionic strengths (Figure S7 a,b). The fluorescence intensity of the C-dots is independent of pH under acidic conditions, but decreases under basic conditions. The fluorescence intensity does not change even in an aqueous solution at a high ionic strength of 2.5 m NaCl, indicating that the C-dot aqueous solution is stable. The fluorescence intensity of the C-dots is stable for more than a month without any distinct reduction (Figure S7 c) upon dispersion in water. As mentioned above, abundant oxygen-containing functional groups are present on the surface of the C-dots, which improves their hydrophilicity and stability in aqueous systems. Additionally, the C-dots show excellent photostability: the fluorescence intensity does not change even after continuous excitation with a 150 W Xe lamp (Figure S7 d). For confirmation of the universality of the one-pot electrochemical oxidation method, alcohols such as ethanol, propanol, butanol, ethylene glycol, and glycerine were used as carbon sources for the preparation of C-dots. The TEM results show that the synthesized C-dots are 3–6 nm in diameter (Figure 4 a–d), and all the C-dots exhibit an amorphous structure. Figure 4 e–h shows the UV/Vis absorption and maximum FL excitation and emission spectra of the as-synthesized C-dots. The C-dots prepared from propanol (PC-dots), butanol (BC-dots), ethylene glycol (EGC-dots), and glycerine (GC-dots) display similar UV/Vis absorption properties. Under their maximum excitation, the four solutions all display strong blue FL (QYs summarized in Table S3). The C-dots obtained from different alcohols also exhibit excitation-wavelength-dependent FL properties (Figure S8). Besides alcohols, liquid ketone or amine can also be used as the carbon source for the preparation of C-dots. The experimental results indicate that this new electrochemical carbonization method has wide applicability. Next, we demonstrated the application of the as-prepared C-dots in fluorescence imaging. We first investigated the cytotoxicity of the as-prepared C-dots with a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The cell viability of HeLa cells, a human hepatocellular liver carcinoma line, was tested after exposure to different concentrations of the as-prepared C-dots (0– 250 mg mL 1; see Supporting Information, Figure S9). No significant difference (with no statistical significance < 5 %) between the C-dot-treated groups and the control samples was observed for C-dot concentrations up to 160 mg mL 1 after incubation for 48 h. For the test concentrations, the C-dots displayed no cytotoxicity toward HeLa cells, with the cell viability declining by < 10 %. The bright-field optical images also confirmed that there was no morphological difference between Chem. Eur. J. 2014, 20, 4993 – 4999
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Figure 4. TEM images of a) PC-dots, b) BC-dots, c) EGC-dots, and d) GC-dots. Absorbance (black), excitation (red), and emission (blue) of e) PC-dots, f) BCdots, g) EGC-dots, and h) GC-dots. C-dot concentration: 0.05 mg mL 1; pH: 7.0; slit width: 5 nm. C-dots prepared from propanol, butanol, ethylene glycol, and glycerine are denoted PC-dots, BC-dots, EGC-dots, and GC-dots, respectively.
the C-dot-treated groups and the control groups. All these results indicate that the C-dots are of low toxicity to cell lines. Fluorescence microscope images of HeLa live cells loaded with C-dots for 2 h at 37 8C show intracellular blue fluorescence, as shown in Figure 5. A large number of HeLa cells with good morphology exhibiting blue fluorescence were observed upon incubation with C-dots, suggesting the excellent cytocompatibility of the obtained C-dots. In control experiments on cells without C-dots, no blue fluorescence was observed under the same exposure conditions (Figure S10). These results not only indicate that the C-dots can be utilized to take images of live cells, but also further confirm that the as-prepared C-dots can potentially be used as fluorescence probes in biochemical applications. In fact, there are few reports on C-dots prepared through the electrochemical etching method for cell imaging. This may be because of the lower fluorescence quantum yield and poor biocompatibility of C-dots produced through electrochemical etching. Therefore, the C-dots prepared here through the electrochemical carbonization method have more advantages for further applications.
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Full Paper In vitro cytotoxicity study
Figure 5. Fluorescence microscopy images of HeLa cells (b) incubated with C-dots (0.05 mg mL 1) for 12 h. a) Bright-field images of (b). Images acquired with 360 nm excitation. C-dots prepared from ethanol at a potential of 6.0 V.
The cell viability was evaluated on HeLa cells by using a standard MTT assay. Briefly, cells were seeded in 96-well culture plates at a density of 5 103 cells per well in culture medium and allowed to grow over 12 h (the cells reached 70–80 % confluence). The medium was replaced with fresh Dulbecco’s modified Eagle medium (DMEM; 90 mL), and C-dots (obtained from ethanol at 6.0 V, 10 mL) were added to each well to keep the final concentration in the range 0–250 mg mL 1 for incubation for 24 or 48 h. MTT solution (20 mL, 0.1 mg) was added to each well and the cells were incubated for another 4 h. N,N’-Dimethyl sulfoxide (150 mL) was used to liberate the formazan crystals completely. The absorbance at 490 nm was measured for calculation of the cell survival rate. The cells treated with phosphate buffer solution (PBS) that did not contain C-dots were taken as the control group, and six parallel samples were tested in each group. Three independent experiments were performed under identical conditions.
Conclusion In summary, we have demonstrated that low-molecular-weight alcohols can be used as the single carbon source for the controllable synthesis of fluorescent C-dots through a one-pot electrochemical carbonization method. Compared to traditional electrochemical routes involving etching from carbon material, the present method is more efficient, and several low-molecular-weight alcohols can be applied for C-dot preparation, suggesting the high universality of our new strategy. The resultant C-dots exhibit high FL quantum yields, and their sizes and maximum emission wavelengths can be controlled by varying the applied potential. The high-quality fluorescent C-dots can also be used for the fluorescence imaging of live cells. Because of its excellent universality and outstanding products, our new method opens up a new route for the synthesis of C-dots and extends the application of electrochemistry in the preparation of nanomaterials.
Experimental Section Preparation of C-dots A CHI 660 A electrochemical workstation (CHI Instrument Inc., USA) was used in this work. C-dots were synthesized conveniently through one-pot electrochemical carbonizations of low-molecularweight alcohols. Scheme 1 shows an overview of the preparation of C-dots. A traditional three-electrode system was used: two Pt sheets (4 4 cm2) were employed as the working and counter electrodes, and a calomel electrode mounted on a freely adjustable Luggin capillary acted as the reference electrode; these were fixed with a rubber plug, and the distance between the two Pt sheets was about 3 cm. The alcohols (140 mL) were mixed with water (10 mL), and then, NaOH (1.2–1.5 g) was added under stirring. The reaction proceeded for about 4 h at a suitable potential until the transparent solution turned dark brown. The current density was 15–100 mA cm 2. Ethanol (150 mL) was added to salt out the NaOH, and the mixture was then kept still, overnight. The mixture was evaporated at 80 8C for 24 h, and a yellow powder was obtained. The product solution was dialyzed against ultrapure water through a dialysis membrane (with 1000 Da MWCO) to obtain the C-dots. Chem. Eur. J. 2014, 20, 4993 – 4999
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Cell imaging using C-dots The potential of the C-dots for bioimaging was tested by using HeLa cells. Cells were incubated on 96-well plate and cultured using the DMEM growth medium with 10 % fetal bovine plasma at 37 8C with 5 % CO2 for 24 h. In this step, a spot of polyethyleneimine (PEI, about 5 mg mL 1) was used. The mixture was washed with PBS, and the mixture of modified C-dots (0.05 mg mL 1, synthesized from ethanol at 6.0 V) and DMEM growth medium was added to each well. After incubation for 12 h in 5 % CO2 at 37 8C, the cells were washed with PBS to remove extracellular C-dots. Fluorescence images were recorded on an inverted fluorescence microscope (NIKON Eclipse Ti-S, Japan) with a 40 objective lens.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21275051, 21375037), the Scientific Research Fund of Hunan Provincial Education Department (12 A084) and Science and Technology Department (13 JJ2020), and the Doctoral Fund of the Ministry of Education of China (20134306110006). Keywords: carbon nanodots · cell imaging · electrochemical carbonization · fluorescence · low-molecular-weight alcohols · nanostructures
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[28] W. T. Dixon, R. O. C. Norman, A. L. Buley, J. Chem. Soc. 1964, 3625. [29] X.-H. Li, S. Kurasch, U. Kaiser, M. Antonietti, Angew. Chem. 2012, 124, 9827 – 9830; Angew. Chem. Int. Ed. 2012, 51, 9689. [30] Y. Q. Dong, R. X. Wang, G. L. Li, C. Q. Chen, Y. W. Chi, G. N. Chen, Anal. Chem. 2012, 84, 6220. [31] S. J. Zhu, Q. N. Meng, L. Wang, J. H. Zhang, Y. B. Song, H. Jin, K. Zhang, H. C. Sun, H. Y. Wang, B. Yang, Angew. Chem. 2013, 125, 4045 – 4049; Angew. Chem. Int. Ed. 2013, 52, 3953. [32] P. G. Luo, S. Sahu, S. T. Yang, S. K. Sonkar, J. P. Wang, H. F. Wang, G. E. LeCroy, L. Cao, Y. P. Sun, J. Mater. Chem. B 2013, 1, 2116. [33] Y. H. Deng, D. X. Zhao, X. Chen, F. Wang, H. Song, D. Z. Shen, Chem. Commun. 2013, 49, 5751. [34] Y. Xu, M. Wu, Y. Liu, X. Z. Feng, X. B. Yin, X. W. He, Y. K. Zhang, Chem. Eur. J. 2013, 19, 2276. [35] H. P. Liu, T. Ye, C. D. Mao, Angew. Chem. 2007, 119, 6593; Angew. Chem. Int. Ed. 2007, 46, 6473. [36] X. N. Dou, Z. Lin, H. Chen, Y. Z. Zheng, C. Lu, J. M. Lin, Chem. Commun. 2013, 49, 5871. [37] Y. P. Sun, B. Zhou, Y. Lin, W. Wang, K. A. S. Fernando, P. Pathak, M. J. Meziani, B. A. Harruff, X. Wang, H. F. Wang, P. G. Luo, H. Yang, M. E. Kose, B. L. Chen, L. M. Veca, S. Y. Xie, J. Am. Chem. Soc. 2006, 128, 7756. [38] J. H. Shen, Y. H. Zhu, X. L. Yang, C. Z. Li, Chem. Commun. 2012, 48, 3686. [39] Y. Q. Dong, R. X. Wang, H. Li, J. W. Shao, Y. W. Chi, X. M. Lin, G. N. Chen, Carbon 2012, 50, 2810. [40] X. H. Zhong, Y. Y. Feng, W. Knoll, M. Y. Han, J. Am. Chem. Soc. 2003, 125, 13559. [41] P. Yu, X. M. Wen, Y. R. Toh, J. Tang, J. Phys. Chem. C 2012, 116, 25552.
Received: December 13, 2013 Revised: January 27, 2014 Published online on March 12, 2014
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& Carbon Nanodots
Electrochemical Synthesis of Carbon Nanodots Directly from Alcohols Jianhui Deng, Qiujun Lu, Naxiu Mi, Haitao Li, Meiling Liu, Mancai Xu, Liang Tan, Qingji Xie, Youyu Zhang,* and Shouzhuo Yao[a]
Abstract: Carbon nanodots (C-dots) show great potential as an important material for biochemical sensing, energy conversion, photocatalysis, and optoelectronics because of their water solubility, chemical inertness, low toxicity, and photoand electronic properties. Numerous methods have been proposed for the preparation of C-dots. However, complex procedures and strong acid treatments are often required, and the as-prepared C-dots tend to be of low quality, and in particular, have a low efficiency for photoluminescence. Herein, a facile and general strategy involving the electrochemical carbonization of low-molecular-weight alcohols is proposed. As precursors, the alcohols transited into carboncontaining particles after electrochemical carbonization
Introduction Carbon nanodots (C-dots), a new kind of fluorescent carbon nanomaterial consisting of a graphite structure or amorphous carbon core and carbonaceous surfaces with rich oxygen-containing groups, have attracted tremendous interest because of their unique properties, including high surface area, low toxicity, and superior optical properties (tunable, non-blinking, and stable fluorescence emission).[1–4] Since fluorescence C-dots were first prepared in 2004 during the purification of singlewalled carbon nanotubes through preparative electrophoresis,[5] increasing attention has been paid to their synthesis and applications.[6–8] At present, a variety of methods have been developed for the preparation of C-dots, including laser ablation,[8, 9] arc-discharge,[5] pyrolysis,[10–12] hydrothermal synthesis,[13–15] electrochemical etching,[2, 7, 16–19] and ultrasound- and microwave-assisted synthesis.[6, 20, 21] Of these, the electrochemical method is advantageous because of its low cost and easy manipulation.[17, 18]
[a] J. Deng, Q. Lu, N. Mi, Prof. H. Li, M. Liu, Prof. M. Xu, Prof. L. Tan, Prof. Q. Xie, Prof. Y. Zhang, Prof. S. Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education) College of Chemistry and Chemical Engineering Hunan Normal University, Changsha 100084 (P. R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201304869. Chem. Eur. J. 2014, 20, 4993 – 4999
under basic conditions. The resultant C-dots exhibit excellent excitation- and size-dependent fluorescence without the need for complicated purification and passivation procedures. The sizes of the as-prepared C-dots can be adjusted by varying the applied potential. High-quality C-dots are prepared successfully from different small molecular alcohols, suggesting that this research provides a new, highly universal method for the preparation of fluorescent C-dots. In addition, luminescence microscopy of the C-dots is demonstrated in human cancer cells. The results indicate that the as-prepared C-dots have low toxicity and can be used in imaging applications.
The electrochemical approach involves a nonselective “topdown” chemical cutting process of a carbon material such as graphite, carbon nanotube, or carbon fiber electrodes.[7, 16, 17] However, C-dots prepared through the present electrochemical methods have mixed sizes, and require further separation through filtration or chromatography to obtain monodisperse C-dots.[2, 7, 17] In addition, the as-prepared C-dots possess low photoluminescence efficiencies (always lower than 10 %),[2, 7, 17, 22, 23] which limits further study in this field. Furthermore, the size-controllable preparation of high-quality C-dots is difficult and complicated with traditional electrochemical methods.[2, 7] From the point of view of material synthesis, the exploration of new carbon sources for the simple, economical, and controllable synthesis of such C-dots is highly desirable. Early researchers motioned in their reports that carbon-containing particles could be formed from alcohol through a suitable electro-oxidation process.[24, 25] Enlightened by these investigations, we proposed a novel one-pot electrochemical carbonization method for the preparation of C-dots. Uniform Cdots were prepared directly from low-molecular-weight alcohols for the first time. Interestingly, the size of the resultant Cdots could be adjusted by changing the synthesis potential. The as-prepared C-dots exhibited excellent fluorescence properties, with the quantum yield (QY) reaching 15.9 %. Several low-molecular-weight alcohols were used for the successful production of C-dots, indicating the universality of this new strategy. In addition, luminescence microscopy imaging of the C-dots internalized in human cancer cells was performed,
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Scheme 1. Electrochemical synthesis of C-dots. Under basic conditions, two Pt sheets were used as the anode and cathode, respectively, and single and simple organics were used as the carbon source. Under suitable DC control, the organics were transformed into C-dots. The resultant C-dot solutions were obtained through simple purification methods.
the different C-dots reveal that the diffraction contrast is very low with no obvious lattice fringes, indicating that the prepared C-dots have an amorphous structure. For further confirmation of this result, selectedarea electron diffraction (SAED), X-ray diffraction (XRD), and Raman spectroscopy measurements were performed (Figures S1 and S2 in the Supporting Information). The diffuse ring pattern obtained from SAED indicates that the as-prepared Cdots contain no crystal lattice, in
which indicated that the C-dots are promising candidates for use in biolabeled carriers and nanodevices.
Results and Discussion As shown in Scheme 1, it can be imagined that the electrochemical carbonization of an alcohol such as ethanol (EtOH) into ultrasmall particles may offer a straightforward and facile strategy for the preparation of high-quality C-dots. In this work, two Pt sheets were used as the working and auxiliary electrodes and a calomel electrode mounted on a freely adjustable Luggin capillary was used as the reference electrode. The electrolyte was NaOH/EtOH, the synthesis potential was 3.0–9.0 V, and the electrochemical carbonization time was 3– 4 h. The control experiments showed that there was no formation of C-dots in H2SO4/EtOH or Na2SO4/EtOH, whereas highquality C-dots were obtained in alkaline environments (e.g., NaOH/EtOH, Na2HPO4/EtOH, and Na2CO3/EtOH), in which the OH groups play an important role.[24, 25] Figure 1 shows typical transmission electron microscopy (TEM) images and size distributions of C-dots synthesized at 3.0, 4.5, 6.0, and 7.5 V. The images reveal that the as-synthesized C-dots are uniform and monodisperse. The size of the Cdots is strongly dependent on the applied potential, with diameters centered at about 2.1, 2.9, 3.5, and 4.3 nm, respectively, and the size increasing with the applied potential. It has been reported that a large number of hydroxyl free radicals, carbonium ions, and alkoxy radicals can be produced during the electrochemical oxidation of alcohols in alkaline solution.[24–26] Crosslinking and dehydration of these active substances occurs under suitable potentials and in an alkaline environment.[24–28] We speculated that the C-dots are formed from ethanol electrochemical oxidation and dehydration at a suitable potential. The applied potential should thus play a key role in the size control of the C-dots. The higher the applied potential, the greater the number of alcohol molecules oxidized and free radicals and carbonium ions that might be produced to undergo crosslinking and dehydration to form carbon nanoparticles, leading to larger C-dots. In contrast with traditional electrochemical routes,[2, 7, 19, 23] high-resolution TEM (HRTEM) images of Chem. Eur. J. 2014, 20, 4993 – 4999
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Figure 1. a, c, e, g) TEM images and b, d, f, h) corresponding size distributions of C-dots obtained from ethanol at potentials of 3.0, 4.5, 6.0, and 7.5 V, respectively. Insets: corresponding HRTEM images of C-dots; scale bar: 3 nm.
agreement with the HRTEM results. At the same time, the XRD pattern shows a broad peak at 2q = 238, revealing an amorphous carbon phase, which agrees well with the HRTEM analysis. In addition, Raman spectra were obtained for the as-prepared C-dots produced through the electrochemical carbonization method and the traditional electrochemical “cutoff”
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Full Paper route.[7] The G-band at 1575 cm 1 and D-band at 1350 cm 1 refer to the graphitic carbon and disordered structure, respectively.[29] The IG/ID ratio for the C-dots obtained from ethanol is clearly lower than that for the C-dots prepared from graphite, indicating that C-dots from alcohols are more disordered and amorphous than those obtained through the traditional electrochemical procedure. In the traditional procedure, C-dots are obtained from a graphitic electrode through an electrochemical “cutoff” mechanism, which leads to their graphitic structural characteristics.[2, 7, 17] However, in this work, C-dots were prepared from low-molecular-weight alcohols through carbonization and dehydration, so the disordered structure is reasonable. These results suggest that our method is totally different from traditional electrochemical routes. Energy-dispersive spectrometry (EDX) and elemental analysis were performed to confirm the components of the as-prepared C-dots. As shown in Figure S3, clear peaks for carbon and copper (holder) are observed, suggesting that the main element contained in the C-dots is carbon. Further elemental analysis was carried out, and as shown in Table S1, the C-dots are composed mainly of C (80.5 %), O (16.9 %), N (0.8 %), and H (1.7 %). As shown in Figure 2, the surface functional groups of the C-dots were detected by Fourier-transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). In the FTIR spectra, the absorption bands of the C-dots prepared at different potentials at 3430, 2930, and 2850 cm 1 (Figure 2 e) are assigned to the stretching vibrations of C OH and C H.[2, 30] The stretching vibrations of C=O, C=C and C O C at about 1700, 1640, and 1250 cm 1, respectively, are observed for all the Cdots,[31–33] and the absorbance peak at 1050 cm 1 is assigned to the epoxide group.[2, 34] XPS analysis was also performed, as shown in Figure 2 a–d. Typically, the peak centered at 284.5 eV is assigned to graphitic sp2 carbon atoms, and those centered at 286.2 and 288.3 eV represent alcoholic (C O) and carbonyl (C=O) carbon atoms, respectively.[2, 34, 35] Further quantitative analysis revealed that the percentage of graphitic carbon increases with increasing potential, whereas the percentage of C O decreases significantly (Table S2, Supporting Information). As summarized in Table S2, the C=O band increases at high potentials, indicating that the C O groups on the C-dots are oxidized to carbonyl C=O groups.[2, 36] Otherwise, the carbon content increases clearly with the applied potential, which is affected by the electrochemical carbonization. These results confirm that C-dots synthesized at higher potentials have a high degree of carbonization, and prove that electrochemical carbonization is the key process for the formation of C-dots. The optical properties of C-dots hold the key for their future applications in optoelectronic devices and biological sensors.[1, 23] UV/Vis absorption and fluorescence (FL) emission spectra of the as-synthesized C-dots were recorded, and are shown in Figure 3. The well-defined UV/Vis absorption at 280 nm is ascribed to the p–p transition of the aromatic sp2 domain within the C-dots.[17, 34] The absorption strength increases clearly, suggesting that the conjugated system increases with the applied potential. The experimental result shows that the potential plays an important role in controlling the structure and size of the as-synthesized C-dots. The fluorescent Chem. Eur. J. 2014, 20, 4993 – 4999
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Figure 2. a–d) XPS of C-dots obtained from ethanol at potentials of 3.0, 4.5, 6.0, and 7.5 V, respectively. e) FTIR spectra of C-dots obtained from ethanol at potentials of 3.0 (black), 4.5 (red), 6.0 (green), and 7.5 V (blue), respectively.
properties of the as-prepared C-dots were explored further. Figure 3 b shows digital photographs of the C-dots obtained at different potentials under 365 nm excitation. A bright blue color was observed for the four C-dots. The emission color of the solutions does not change significantly, but the brightness of the C-dots changes upon changing the applied potential, indicating that the C-dots have different quantum yields under different synthesis potentials (as summarized in Table S2). As shown in Figure 3 c–f, the as-synthesized C-dots exhibit an excitation-dependent FL behavior, similarly to previously reported C-dots.[2, 12, 23] Upon changing the excitation wavelength from 340 to 500 nm, the FL peaks shift correspondingly from 451, 491, 470, and 477 nm to 542, 579, 545, and 565 nm, respectively, for the different applied potentials from 3.0 to 7.5 V.
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Figure 3. a) UV absorbance spectra; b) digital photographs of C-dots obtained at different potentials under 365 nm excitation. c–f) Fluorescence emission spectra of C-dots obtained from ethanol at potentials of 3.0, 4.5, 6.0, and 7.5 V, respectively. The excitations for these emission spectra were 300, 340, 380, 420, 460, and 500 nm, respectively. C-dot concentration: 0.05 mg mL 1 in ultrapure water; pH: 7.0; slit width: 5 nm.
In addition, the maximum fluorescence emissions appear at 485, 490, 500, and 505 nm under the maximum excitations of 390, 395, 400, and 410 nm, respectively, as shown in Figure S4 a of the Supporting Information. According to previous studies, the shift in emission peak positions with different excitation wavelengths arises not only from the different sizes of C-dots, but also from different emissive sites on the Cdots.[1, 2, 8, 12] At certain excitation wavelengths, some corresponding emissive sites would be excited and fluoresce, resulting in the excitation-dependent behaviors of the emission spectra.[2, 8] The fluorescence spectra of the C-dots could reflect the distribution of emissive sites on the C-dots, and not only the size effect.[12] The photoluminescence mechanism of the Cdots could involve quantum-confinement effects,[8, 37] emissive traps,[17, 37] electronic conjugate structures,[38] and free zigzag sites.[12] Although the exact mechanisms responsible for the FL from C-dots remain to be elucidated, the bright and colorful FL may be attributed to the chemical nature of the C-dots. As summarized in Table S3, the quantum yield of C-dots produced from ethanol with an applied potential of 6.0 V at 400 nm excitation is calculated to be about 15 % (by calibrating against quinine sulfate),[2, 17, 39] which is much higher than that at 3, 4.5, and 7.5 V. The full-width at half-maximum (FWHM) values of the emission spectra of C-dots obtained from ethanol at potentials of 3.0, 4.5, 6.0, and 7.5 V excited at 400 nm are only 54, 89, 89, and 104 nm (Figure S4 b), which are much smaller than Chem. Eur. J. 2014, 20, 4993 – 4999
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those reported previously,[2, 4, 17, 34, 39] suggesting a narrow size distribution of the product particles.[40, 41] These results are in agreement with the TEM images and size distributions of Cdots obtained from ethanol at different potentials. Similarly to that of particle size, the increase in the FWHM with applied potential may also occur because of different degrees of crosslinking of free radicals and carbonium ions from the electro-oxidation of ethanol. Different crosslinking degrees occur and result in an increase in the size distribution of C-dots under a high applied potential. For a deeper understanding of the fluorescence properties, size separation of C-dots synthesized at 4.5 and 6.0 V was performed by using three molecularweight cutoff membranes to obtain products equivalent to < 1.0, 1.0–3.5, 3.5–8.0, and > 8.0 kDa. The < 1 kDa fraction exhibits no obvious fluorescence, and there is a small amount of product in the > 8.0 kDa fraction with weak fluorescence, but the 1.0–3.5 and 3.5–8.0 kDa fractions show different blue fluorescence (Figure S6). As shown in Figure S6, for the C-dots obtained at both 4.5 V (Figure S6 a,b) and 6.0 V (Figure S6 c,d), the 1.0–3.5 kDa and 3.5–8.0 kDa fractions all show excitation-dependent emission spectra upon excitation from 300 to 400 nm and 340 to 440 nm. The maximum fluoresce emission appears at 468 and 470 nm under the maximum excitation of 360 nm for the 1.0–3.5 kDa fractions obtained at 4.5 and 6.0 V, respectively, and at 501 and 505 nm under the maximum excitation of 400 nm for the 3.5–8.0 kDa fractions obtained at 4.5 and 6.0 V, respectively. The difference between the FL of the two kinds of C-dots should be attributed to the different sizes of the products. TEM images of the fractions (Figure S5) reveal that both kinds of C-dots are monodisperse and have a uniform spherical shape. Narrow size distributions were obtained: 1.6( 0.3) and 2.9( 0.3) nm in diameter for the 1.0–3.5 and 3.5–8.0 kDa fractions, respectively, of C-dots prepared at 4.5 V, and 1.7( 0.4) and 3.1( 0.4) nm for the 1.0–3.5 and 3.5– 8.0 kDa fractions, respectively, of those prepared at 6.0 V. The FL emission peak is redshifted with an increase in diameter, indicating that the FL properties of the as-prepared C-dots are typically size-dependent; similarly to the results found in a previous study,[2, 17] the slight differences between the two kinds of C-dots are also caused by the size effect. These differences suggest that the applied potential affects the size of the final products. We also measured the quantum yields (QY) of different fractions (1.0–3.5 and 3.5–8.0 kDa) obtained at 4.5 and 6.0 V, and the results are summarized in Table S4. It is found that the different fractions of C-dots obtained from both applied potentials have different QY values, and that the same fraction has a similar QY whether it is obtained at 4.5 or 6.0 V. C-dots (1 g) were separated with cutoff membranes in ultrapure water: 1.0–3.5 and 3.5–8 kDa fractions were obtained (400 mL solutions), and their fluorescence intensities were measured. For the C-dots obtained at 4.5 V, the FL intensity of the 1.0–3.5 kDa fraction is higher than that of the 3.5–8.0 kDa fraction, and the mass ratio of Wt1.0–3.5 kDa/Wt3.5–8.0 kDa is 1.7:1. For the C-dots obtained at 6.0 V, the FL intensity of the 1.0–3.5 kDa fraction is much lower than that of the 3.5–8.0 kDa fraction, and Wt1.0–3.5 kDa/Wt3.5–8.0 kDa is 1:4. These results indicate that the C-dots obtained at 6.0 V are larger (centered at 3.5–8.0 kDa)
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Full Paper than those obtained at 4.5 V (centered at 1.0–3.5 kDa), in agreement with the FL measurement of different fractions Cdots, and confirm the effect of the applied potential on the products. All the above experimental results suggest that the fluorescence properties are affected mainly by the size of the C-dots. However, the exact mechanism of the production of Cdots through this new method is still under investigation. For further exploration of the optical properties of the assynthesized C-dots, a detailed FL study was undertaken in water with different pH values, containing NaCl of various ionic strengths (Figure S7 a,b). The fluorescence intensity of the C-dots is independent of pH under acidic conditions, but decreases under basic conditions. The fluorescence intensity does not change even in an aqueous solution at a high ionic strength of 2.5 m NaCl, indicating that the C-dot aqueous solution is stable. The fluorescence intensity of the C-dots is stable for more than a month without any distinct reduction (Figure S7 c) upon dispersion in water. As mentioned above, abundant oxygen-containing functional groups are present on the surface of the C-dots, which improves their hydrophilicity and stability in aqueous systems. Additionally, the C-dots show excellent photostability: the fluorescence intensity does not change even after continuous excitation with a 150 W Xe lamp (Figure S7 d). For confirmation of the universality of the one-pot electrochemical oxidation method, alcohols such as ethanol, propanol, butanol, ethylene glycol, and glycerine were used as carbon sources for the preparation of C-dots. The TEM results show that the synthesized C-dots are 3–6 nm in diameter (Figure 4 a–d), and all the C-dots exhibit an amorphous structure. Figure 4 e–h shows the UV/Vis absorption and maximum FL excitation and emission spectra of the as-synthesized C-dots. The C-dots prepared from propanol (PC-dots), butanol (BC-dots), ethylene glycol (EGC-dots), and glycerine (GC-dots) display similar UV/Vis absorption properties. Under their maximum excitation, the four solutions all display strong blue FL (QYs summarized in Table S3). The C-dots obtained from different alcohols also exhibit excitation-wavelength-dependent FL properties (Figure S8). Besides alcohols, liquid ketone or amine can also be used as the carbon source for the preparation of C-dots. The experimental results indicate that this new electrochemical carbonization method has wide applicability. Next, we demonstrated the application of the as-prepared C-dots in fluorescence imaging. We first investigated the cytotoxicity of the as-prepared C-dots with a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The cell viability of HeLa cells, a human hepatocellular liver carcinoma line, was tested after exposure to different concentrations of the as-prepared C-dots (0– 250 mg mL 1; see Supporting Information, Figure S9). No significant difference (with no statistical significance < 5 %) between the C-dot-treated groups and the control samples was observed for C-dot concentrations up to 160 mg mL 1 after incubation for 48 h. For the test concentrations, the C-dots displayed no cytotoxicity toward HeLa cells, with the cell viability declining by < 10 %. The bright-field optical images also confirmed that there was no morphological difference between Chem. Eur. J. 2014, 20, 4993 – 4999
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Figure 4. TEM images of a) PC-dots, b) BC-dots, c) EGC-dots, and d) GC-dots. Absorbance (black), excitation (red), and emission (blue) of e) PC-dots, f) BCdots, g) EGC-dots, and h) GC-dots. C-dot concentration: 0.05 mg mL 1; pH: 7.0; slit width: 5 nm. C-dots prepared from propanol, butanol, ethylene glycol, and glycerine are denoted PC-dots, BC-dots, EGC-dots, and GC-dots, respectively.
the C-dot-treated groups and the control groups. All these results indicate that the C-dots are of low toxicity to cell lines. Fluorescence microscope images of HeLa live cells loaded with C-dots for 2 h at 37 8C show intracellular blue fluorescence, as shown in Figure 5. A large number of HeLa cells with good morphology exhibiting blue fluorescence were observed upon incubation with C-dots, suggesting the excellent cytocompatibility of the obtained C-dots. In control experiments on cells without C-dots, no blue fluorescence was observed under the same exposure conditions (Figure S10). These results not only indicate that the C-dots can be utilized to take images of live cells, but also further confirm that the as-prepared C-dots can potentially be used as fluorescence probes in biochemical applications. In fact, there are few reports on C-dots prepared through the electrochemical etching method for cell imaging. This may be because of the lower fluorescence quantum yield and poor biocompatibility of C-dots produced through electrochemical etching. Therefore, the C-dots prepared here through the electrochemical carbonization method have more advantages for further applications.
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Figure 5. Fluorescence microscopy images of HeLa cells (b) incubated with C-dots (0.05 mg mL 1) for 12 h. a) Bright-field images of (b). Images acquired with 360 nm excitation. C-dots prepared from ethanol at a potential of 6.0 V.
The cell viability was evaluated on HeLa cells by using a standard MTT assay. Briefly, cells were seeded in 96-well culture plates at a density of 5 103 cells per well in culture medium and allowed to grow over 12 h (the cells reached 70–80 % confluence). The medium was replaced with fresh Dulbecco’s modified Eagle medium (DMEM; 90 mL), and C-dots (obtained from ethanol at 6.0 V, 10 mL) were added to each well to keep the final concentration in the range 0–250 mg mL 1 for incubation for 24 or 48 h. MTT solution (20 mL, 0.1 mg) was added to each well and the cells were incubated for another 4 h. N,N’-Dimethyl sulfoxide (150 mL) was used to liberate the formazan crystals completely. The absorbance at 490 nm was measured for calculation of the cell survival rate. The cells treated with phosphate buffer solution (PBS) that did not contain C-dots were taken as the control group, and six parallel samples were tested in each group. Three independent experiments were performed under identical conditions.
Conclusion In summary, we have demonstrated that low-molecular-weight alcohols can be used as the single carbon source for the controllable synthesis of fluorescent C-dots through a one-pot electrochemical carbonization method. Compared to traditional electrochemical routes involving etching from carbon material, the present method is more efficient, and several low-molecular-weight alcohols can be applied for C-dot preparation, suggesting the high universality of our new strategy. The resultant C-dots exhibit high FL quantum yields, and their sizes and maximum emission wavelengths can be controlled by varying the applied potential. The high-quality fluorescent C-dots can also be used for the fluorescence imaging of live cells. Because of its excellent universality and outstanding products, our new method opens up a new route for the synthesis of C-dots and extends the application of electrochemistry in the preparation of nanomaterials.
Experimental Section Preparation of C-dots A CHI 660 A electrochemical workstation (CHI Instrument Inc., USA) was used in this work. C-dots were synthesized conveniently through one-pot electrochemical carbonizations of low-molecularweight alcohols. Scheme 1 shows an overview of the preparation of C-dots. A traditional three-electrode system was used: two Pt sheets (4 4 cm2) were employed as the working and counter electrodes, and a calomel electrode mounted on a freely adjustable Luggin capillary acted as the reference electrode; these were fixed with a rubber plug, and the distance between the two Pt sheets was about 3 cm. The alcohols (140 mL) were mixed with water (10 mL), and then, NaOH (1.2–1.5 g) was added under stirring. The reaction proceeded for about 4 h at a suitable potential until the transparent solution turned dark brown. The current density was 15–100 mA cm 2. Ethanol (150 mL) was added to salt out the NaOH, and the mixture was then kept still, overnight. The mixture was evaporated at 80 8C for 24 h, and a yellow powder was obtained. The product solution was dialyzed against ultrapure water through a dialysis membrane (with 1000 Da MWCO) to obtain the C-dots. Chem. Eur. J. 2014, 20, 4993 – 4999
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Cell imaging using C-dots The potential of the C-dots for bioimaging was tested by using HeLa cells. Cells were incubated on 96-well plate and cultured using the DMEM growth medium with 10 % fetal bovine plasma at 37 8C with 5 % CO2 for 24 h. In this step, a spot of polyethyleneimine (PEI, about 5 mg mL 1) was used. The mixture was washed with PBS, and the mixture of modified C-dots (0.05 mg mL 1, synthesized from ethanol at 6.0 V) and DMEM growth medium was added to each well. After incubation for 12 h in 5 % CO2 at 37 8C, the cells were washed with PBS to remove extracellular C-dots. Fluorescence images were recorded on an inverted fluorescence microscope (NIKON Eclipse Ti-S, Japan) with a 40 objective lens.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21275051, 21375037), the Scientific Research Fund of Hunan Provincial Education Department (12 A084) and Science and Technology Department (13 JJ2020), and the Doctoral Fund of the Ministry of Education of China (20134306110006). Keywords: carbon nanodots · cell imaging · electrochemical carbonization · fluorescence · low-molecular-weight alcohols · nanostructures
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Received: December 13, 2013 Revised: January 27, 2014 Published online on March 12, 2014
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