Research Article www.acsami.org
Effect of Lateral Size of Graphene Quantum Dots on Their Properties and Application Fangwei Zhang,† Fei Liu,‡ Chong Wang,‡ Xiaozhen Xin,‡ Jingyuan Liu,‡ Shouwu Guo,*,† and Jingyan Zhang*,‡ †
Department of Electronic Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China ‡ State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, P. R. China S Supporting Information *
ABSTRACT: Well-defined graphene quantum dots (GQDs) are crucial for their biological applications and the construction of nanoscaled optoelectronic and electronic devices. However, as-synthesized GQDs reported in many works assume a very wide lateral size distribution; thus, their apparent properties cannot truthfully reflect intrinsic properties of the well-defined GQDs, and more importantly, the applications of GQDs will be affected and limited as well. In this work, we demonstrated that different sized GQDs with a narrow size distribution could be obtained via gel electrophoresis of the crude GQDs prepared through a photo-Fenton reaction of graphene oxide (GO). It is illustrated that the photoluminesce (PL) emissions of the well-defined GQDs originated mainly from the peripheral carboxylic groups and conjugated carbon backbone planes through fluorescence and UV−vis spectroscopies. More importantly, our findings challenge the notion that the excitation wavelength dependent PL property of the as-synthesized GQDs is the intrinsic property of the size-defined GQDs. Preliminary data at the cellular level indicated that the small sized GQDs exhibit weaker quenching DNA dye ability but higher toxicity to the cells compared to that of the as-synthesized GQDs. This discovery is essential to explore applications of the GQDs in pharmaceutics and to understand the origin of the optoelectronic properties of GQDs. KEYWORDS: graphene quantum dot, gel electrophoresis, lateral size, photoluminescence
1. INTRODUCTION Graphene quantum dots (GQDs) have attracted great research interest and have shown many potential applications in diverse areas.1−5 With a strong quantum confined electronic state and unique edge structure effect, GQDs exhibit an open band gap structure, affording them distinctive optoelectronic properties,6 and thus have been explored in the construction of nanoscaled optoelectronic and electronic devices.7−10 In addition, the intrinsic single atomic layer thickness, nanometer lateral size, and peripheral functional groups provide GQDs with unique application in biomedical research.6 The well-defined GQDs including the size and surface status are critical for their applications. For example, large sized GQDs could alleviate immune-mediated liver damage, but relatively small sized GQDs could not.11 However, the assynthesized GQDs that are prepared with various strategies, in most cases, assume a very wide lateral size distribution.12−16 This will severely affect the understanding of their intrinsic properties and their applications. For instance, the photoluminance (PL) emission wavelength of many GQDs reported in the literature is dependent on the excitation wavelength.17 However, that is different from a quantum dot, whose PL emission peak is independent of the excitation wavelength, but the intensity of the PL emission is dependent on the excitation © 2016 American Chemical Society
wavelength, when its size, morphology, and surface functional groups are defined. Several mechanisms were proposed to illustrate the origin of the excitation wavelength dependence of the PL property of GQDs.18−28 The generally accepted explanation is that quantum size, zigzag edge sites, recombination of localized electron−hole pairs, and defect effects were involved in the PL emission.4,23,28−32 The first three of them were regarded as intrinsic state emission and the last one defined as the defect state emission. However, the dominant factor remains unclear. Zhu et al. prepared three different GQDs with different methods, in which they wished to differentiate the contribution of these factors, but the amination of the GQDs in fact altered the physical property of the starting GQDs; thus, the comparison of the GQDs with the aminated GQDs seems questionable.25 Meanwhile, to obtain well-defined GQDs, especially within a certain size region, several methods have been employed to the separation of as-synthesized GQDs.28,33−35 Fuyuno et al. obtained different sized GQDs by size-exclusion HPLC;34 however, the PL spectra of the different sized GQDs suggested Received: November 4, 2015 Accepted: January 3, 2016 Published: January 3, 2016 2104
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trypsin and plated in 24-well plates at a density of 5 × 104 cells per well to culture sequentially. After 12 h of attaching, the medium containing 50 μg mL−1 of the GQDs was added into the well followed by removing the medium with serum and washing cells with PBS buffer. After 3 h of incubation, the cell morphology was observed and recorded by an inverted microscope. For the fluorescence quenching experiment, after 12 h of attaching, the cell medium was replaced with the medium without serum but containing 0.5 μg mL−1 Hoechst 33258, followed by washing with PBS buffer two times. After 30 min for incubation, the medium was removed, and the cells were washed with PBS buffer. Then, medium containing 20 μg mL−1 GQDs was added, and the cells were incubated for 4 h continuously, while the control cells were incubated in the medium without GQDs. The cells were washed two times with PBS buffer and then fixed with 4% paraformaldehyde (pH 7.4). After 15 min of fixing, the coverslips were placed on slides after washing with PBS buffer. The prepared slides were imaged by a fluorescence microscope.
that they still have a wide size distribution. The GQDs were also separated by ammonium sulfate through salting out strategy.35 The different fractions obtained by this method had fairly different sizes but exhibited quite similar PL spectra with multiple peaks at one excitation wavelength. Jiang et al. acquired different sized amino-functionalized GQDs with a molecular sieving column,33 and the two fractions obtained from the column assumed different thicknesses, but unexpectedly, their UV−visible and PL spectra were basically identical. Hence, to obtain GQDs with well-defined physical properties, in this work, the as-synthesized GQDs were graded with gel electrophoresis, different sized GQDs with a narrow size distribution were obtained and characterized, and their PL properties were investigated. The results clearly showed that different sized GQDs exhibit different PL emission and UV− visible spectra, and more importantly, the PL emission wavelength of the well-defined GQDs is excitation wavelength independent in general. However, this is not the case when GQDs are in smaller sizes (less than ∼6 nm in this work) because their surface status becomes a dominant factor in their PL properties. In addition, it is found that different sized GQDs exhibit very different behaviors when they interact with cells.
3. RESULTS AND DISCUSSION 3.1. Different Sized GQDs Were Obtained by Gel Electrophoresis of the As-Synthesized GQDs. Gel electrophoresis is widely employed in biology to separate biomacromolecules and their fragments based on size and charge status. By simply varying the ratio of the monomer and cross-linker that composed the gels, gels with different pore size networks could be prepared and are suitable for separating proteins, nucleic acids, or their fragments.37 As we reported previously, the GQDs synthesized from a photo-Fenton reaction of GO sheets assume a defect free carbon basal plane with a large number of peripheral carboxylic groups and a relatively wide lateral size distribution (Figure S1). In principle, each GQD can be considered as a large molecule, and its charge status is determined by its peripheral carboxylic groups. Thereby, in theory, the as-synthesized GQDs could be separated into different sized GQDs through gel electrophoresis. There are possibly some ultrasmall sized GQDs generated during the photon-Fenton reaction of GO;36 electrophoresis was thus first tried with a polyacrylamide gel (PAGE). Unfortunately, most of the GQDs were trapped in the gel, possibly due to the small pore size of the 10% PAGE. An agarose gel with larger matrix pores, often used in nucleic acid molecule separation,37 was then employed. As shown in Figure 1, under a constant voltage, three bands with different colors, blue, green, and orange on the gel were observed under UV light (302 nm) and were labeled as GQDs-1, GQDs-2, and GQDs-3, respectively. In contrast, under white light, only band GQDs-3 is barely visible. On the basis of the general principle of gel electrophoresis, the separation of GQDs in agarose gel is determined both by the lateral dimension of the GQDs and the
2. EXPERIMENTAL SECTION 2.1. Materials and Instruments. GQDs were prepared from the GO by a photon-Fenton reaction as described in our previous work.36 Atomic force microscopy (AFM) images were acquired in tapping mode using a Multimode Nanoscope V scanning probe microscopy system (Bruker, USA), and AN-NSC 10 AFM cantilever tips (SHNIT Co., Russia) with a force constant of ∼37 N m−1 and resonance vibration frequency of ∼300 kHz were used. The specimens for AFM measurement were prepared by solution casting the aqueous suspensions of GQDs on a freshly cleaved mica surface and drying in air. Transmission electron microscope (TEM) images were obtained using a JEM-2100F transmission electron microscope (JEOL, Japan) operated at 200 kV. The samples were prepared by solution casting the aqueous suspensions of GQDs on the copper grids with ultrathin carbon films and drying under ambient conditions in a dryer. The UV−vis measurements were performed on a Cary 50 spectrometer (Varian, USA). The fluorescence spectra were acquired by a Cary Eclipse spectrofluorometer (Varian, USA). Fluorescence images of cells were obtained by fluorescence microscopy (Nikon ECLIPSE, Ti−S). The optical photography was captured by a Panasonic DMX-LX7 camera with a Leica lens (Panasonic, Japan). Agarose gel electrophoresis was carried out with a DYY-6C electrophoresis apparatus (Liuyi Instrumental Co., China). The agarose gels were visualized and digitized with the FR-200A gel image analysis system and analyzed by Smart View software. 2.2. Gel Electrophoresis of GQDs and Recovery of the GQDs from Gel. Agarose gel experiments were carried out using 1.5% agarose gel. Typically, to run the gel electrophoresis, the aqueous suspension of 400 μL (∼2 mg mL−1) of the as-synthesized GQD sample was mixed with 40 μL of glycerin and then was injected into the sample well. A voltage of 50 V was first applied for 6 min, then the voltage was set at 120 V for around 25 min until the brown band reached the middle of the gel. The different colored bands were carefully cut out under UV light after the electrophoresis process. The incised gel bands were then soaked in purified water for 24 h to recover the corresponding GQDs. The resulting GQD solutions were then centrifuged (4000 rpm, 15 min) and dialyzed (dialysis tube with a cutoff of 1000 Da for GQDs-1 and 8000−14 000 Da for the others) to remove the electrolytes. 2.3. Cellular Experiments. Human breast cancer MCF-7 cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in RPMI 1640 medium with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin) at 37 °C under 5% CO2. For the cell morphology observation, MCF-7 cells were detached by
Figure 1. Agarose gel (0.75%) of the as-prepared GQDs aqueous solution. The images were taken under white and UV light (302 nm). The running buffer was Tris-HAc (40 mM, pH 8.35). The voltages were set at 50 V for 6 min and then 120 V for 23 min. 2105
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ACS Applied Materials & Interfaces number of their peripheral carboxylic groups. The number of carboxylic groups of each GQD is proportional to its lateral size, the larger the size is, the more carboxylic groups it contains. However, the surface area of the GQDs grows square proportionally with its lateral dimension. Hence, the density of carboxylic groups in larger lateral sized GQDs is lower than that in smaller sized GQDs. Accordingly, the running speed of the smaller GQDs is faster than that of the larger ones in the gel. As a result, the as-synthesized GQDs were graded according to their lateral sizes in agarose gel, and their sizes should be in the following order: the blue luminescent GQDs-1 < green GQDs2 < orange GQDs-3. The result agrees well with the literature that GQDs with different sizes emitted different PLs.28,38,39 To identify accurately the size of GQDs in each gel band, the bands were recovered from the agarose gel by cutting out the gel stripes according to their color under UV light (302 nm) and incubating them in Milli-Q H2O for 24 h. The resulting aqueous solutions were centrifuged, and the supernatants were dialyzed. As expected, the extracted solutions exhibit the same color as the corresponding gel bands under UV-light, revealing that the GQDs were recovered successfully from the agarose gel. The recovery efficiency was further confirmed by comparing the intensity of the GQDs gel bands under UV light before and after soaking with water (Figure S2). Gel electrophoresis of GQDs not only can provide a partition method of obtaining the GQDs with different sizes, it can also be employed in monitoring the preparation of the GQDs via monitoring the intensity of the color bands under UV-light of the samples (Figure S3). This method makes it possible to track the reaction process within an hour. However, gel electrophoresis is not a ubiquitous method to obtain different sized GQDs with all as-synthesized GQDs. Gel electrophoresis experiments with the GQDs made from glucose (GQDs-glucose)21 showed that GQDs-glucose were trapped inside the gel, suggesting that either their net charge is zero or they contain an extremely high concentration of zigzag sites that are easily trapped in the gel matrix. Similarly, an experiment with GO/DMF (GQDs-DMF)10 displayed that some of them moved forward in the gel but that some of them moved backward (Figure S4), indicating that some GQDsDMF are negatively charged, and some are positively charged. Apparently, these two kinds of GQDs cannot be efficiently separated by gel electrophoresis. 3.2. Characterization of the Isolated GQDs. TEM images of the GQDs-1, GQDs-2, and GQDs-3 were acquired, and the results were shown in Figure 2a−c. Clearly, the average sizes of the GQDs are in the order GQDs-1 < GQDs-2 < GQDs-3, which is highly consistent with the aforementioned gel electrophoresis results. Figure S5 shows the high resolution TEM (HRTEM) image of the GQDs-2. The basal plane of GQDs-2 maintains the pristine graphene structure with a lattice parameter of 0.23 nm (for (100) facet of graphite) and 0.34 nm (for (002) facet of graphite),36,40 indicating that no damage to the GQDs is occurred during the electrophoresis. The isolated GQDs were further characterized by taping mode atomic AFM (Figure 2d−f). The average sizes of the GQDs estimated from their AFM images are 5.5, 12.5, and 16 nm in diameter for GQDs-1, GQDs-2, and GQDs-3, respectively (Figure S6). Notably, the diameter data were measured directly from AFM images of the GQDs and calibrated with the AFM tip deconvolution parameters obtained using a simplified model shown in Figure S7.41 Obviously, the estimated lateral sizes of GQDs based on their AFM images are given along with the
Figure 2. TEM images of the GQDs-1 (a), GQDs-2 (b), and GQDs-3 (c). Scale bar equals 20 nm. The inset in panel a is the zoomed-in image of the GQDs-1, and the scale bar equals 10 nm. (d−f) Taping mode AFM phase images of the GQDs-1, GQDs-2, and GQDs-3, respectively. The scale bar equals 100 nm.
electrophoresis and TEM results. The thickness of most GQDs detected in taping mode AFM images is around 0.8 nm, showing a single layer motif (Figures S6 and S8).36,42 These results together demonstrate that the GQDs isolated by gel electrophoresis show different sizes with a narrow size distribution. 3.3. PL Properties of the GQDs-1, GQDs-2, and GQDs3. Although GQDs exhibit unique optoelectronic properties owing to the quantum confinement effect, the apparent properties of the GQDs are usually collected on the mixture of GQDs with different sizes, and it thus cannot reflect the intrinsic property of certain sized GQDs. The PL properties of the as-synthesized GQDs, GQDs-1, GQDs-2, and GQDs-3 thus were investigated, and their spectra are displayed in Figure 3a. Overall, under the same excitation wavelength, the PL spectrum of the as-synthesized GQDs (Figure 3a, black line) is broad suggesting that it is a combination of the PL spectra of different sized GQDs. While the PL spectra of GQDs-1, GQDs-2, and GQDs-3 are much narrow, especially for GQDs-1, the full width at half maximum (FWHM) is about 90 nm, which is much narrower than that reported in several works.39 It has been demonstrated that the π* → n transition of carbonyl or carboxylic groups connected to carbon nanotubes and other fullerene carbon nanostructured materials produce PL emission around 430 nm with excitation wavelength at around 320 nm.43 Therefore, the emission at 450 nm of the GQDs-1 is partially contributed by the π* → n transition of carbonyl or carboxylic of GQDs. The other contribution to PL of the GQDs-1 might be the conjugated basal carbon skeleton. Notably, the main PL emission peak of the GQDs-1 shifted gradually to the longer wavelength with the increase of the excitation wavelength (Figure 3b). However, the size distribution of the GQDs-1 is narrow based on AFM statistics results (Figure S6), implying that the emission wavelength shifts with the excitation wavelength of GQDs-1 are possibly caused by other reasons (see the Discussion later). Figure 3c showed the PL spectra of the GQDs-2. Overall, it can be deconvoluted into two peaks centered at ∼425 and 510 nm. Similar to the GQDs-1, the emission at 425 nm originated from the π* → n transition of the carboxylic groups, and the emission at 510 nm is from the conjugated carbon backbone of the GQDs.17 In contrast to the GQDs-1, the emission wavelength of the GQDs-2 is independent of the excitation wavelength. Similarly, as illustrated in Figure 3d, the PL 2106
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Figure 3. PL properties of the GQDs separated by gel electrophoresis. (a) The comparison of the PL spectra of as-synthesized GQDs with GQDs-1, GQDs-2, and GQDs-3 that excited at 340 nm (concentrations were not calibrated). (b, c, and d) PL spectra of the GQDs-1, GQDs-2, and GQDs-3 that were excited from 320 to 400 nm, respectively. The insets in each figure are the pictures of each sample in cuvettes taken under UV−vis light (302 nm).
emission peak of GQDs-3 centered at 513 nm is mainly from the conjugated carbon backbone. The slight red shift of the emission wavelength of the GQDs-3 with the increase of the excitation wavelength is likely due to the wider size distribution compared to that of GQDs-1 and GQDs-2, as evidenced by their broad PL peak. Notably, the PL intensity of the larger sized GQDs-3 is much weaker because the contribution of the quantum effect apparently is less prominent. To further understand the phenomenon of the emission wavelength dependent on the excitation wavelength that we observed in the GQDs-1, but not in GQDs-2, two adjacent blue gel bands were cut out from the gel and recovered, the bottom band was named as GQDs-1a, and the top band was named as GQDs-1b as illustrated in Figure 4. GQDs-1a are marginally smaller than GQDs-1b because GQDs-1a run slightly faster than GQDs-1b in the gel. The PL spectra of these two samples were compared. When they were excited at 340 nm, their emission spectra are almost identical, but when they were excited at 380 nm, the emission wavelengths of both samples shifted to longer wavelengths, and it was 23 nm shift for the GQDs-1b, which was 6 nm more than that for the GQDs-1a. Since the GQDs-1a and GQDs-1b are very similar in terms of size distribution and surface status, the difference in emission wavelength shift apparently is caused by their small difference in size. As mentioned earlier, PL spectra of the GQDs were contributed by the conjugated carbon skeleton and surface status. These two contributions are both excitation wavelength independent in principle; however, when the excitation wavelength shifts to a longer wavelength, the relative ratio of these two contributions will change, the overall apparent emission spectrum thus will be excitation wavelength dependent. In the case of GQDs-1, the contribution of surface status is
Figure 4. Recovery of GQDs-1a and GQDs-1b from the gel bands. Their pictures were taken under UV−vis light in cuvettes (302 nm). The PL spectra of two samples were acquired at excitation wavelengths of 340 and 380 nm.
dominant because of their smaller sizes; thus, the main peak of PL centers at ∼450 nm, and the contribution from the carbon skeleton shows up only when the GQDs-1 are excited at a longer wavelength. While for the GQDs-2, the larger carbon 2107
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Figure 5. (a) Bright field images of MCF-7 cells incubated with 50 μg mL−1 of the as-synthesized GQDs and GQDs-1 for 3 h. (b) MCF-7 cells were preincubated with 0.5 μg mL−1 of Hoechst for 30 min and then incubated with 20 μg mL−1 of the GQDs for 4 h. Excitation wavelength, 340 nm. Scale bar: 50 μm. (c) The PL of Hoechst was quenched by the various concentrations of the as-synthesized GQDs and GQDs-1 in vitro.
density of carboxylic groups in the GQDs-1 and GQDs-2 is higher as shown in gel electrophoresis. In contrast, the peak at 300 nm of the GQDs-3 sample is almost invisible, revealing that the surface density of carboxylic groups is relatively low in the GQDs-3, and their size distribution is wider. The results agree with the observation that GQDs-1 and GQDs-2 move faster than GQDs-3 in the agarose gel (Figure 1). The red shift of the onsets of the absorption around 300 nm in the UV−vis spectra of the GQDs-1, GQDs-2, and GQDs-3 also indicates that the lateral dimension increases from GQDs-1 to GQDs-3. 3.5. GQDs Obtained by Gel Electrophoresis Exhibit a Unique Effect on the Cells. Interestingly, with these isolated GQDs, it is possible to understand some of their biological effect because they have well-defined properties. Figure 5 compared the effects of the as-synthesized GQDs and the GQDs-1 to the MCF-7 cells. The bright field images of the MCF-7 cells that were incubated with the as-synthesized GQDs and GQDs-1 for 3 h clearly show the difference. With the GQDs-1, the cells were round and unhealthy, but with the assynthesized GQDs, cells are in normal healthy state. Furthermore, when MCF-7 cells were prestained with the nuclei dye Hoechst and then incubated with the as-synthesized GQDs and GQDs-1, the fluorescence of the nuclei in the former was quenched rapidly, while it was not in the latter, which agrees well with the in vitro molecular level experiment as shown in Figure 5c. The fluorescence of the Hoechst was quenched upon the addition of as-synthesized GQDs, but it cannot be quenched by the GQDs-1 under the same condition. The slight increase of the fluorescence intensity of the Hoechst with GQDs-1 was caused by the contribution of the fluorescence of the GQDs-1 themselves (Figure 3a). The result suggests that smaller sized GQDs-1 have a weaker fluorescence quench capability because of their poor electron transport ability compared to the larger sized GQDs. Though more work in depth is necessary to understand these preliminary findings, it indicates unambiguously that the welldefined GQDs exhibit different properties compared to those of the as-synthesized GQDs with a wide size distribution. More importantly, it is necessary to have well-defined GQDs for the research of GQDs in biomedical applications, such as in drug delivery.
skeleton is a major contributor to the PL, and peripheral carboxylic groups have a minor effect; thus, the change of the excitation wavelength would not cause a larger shift to the emission wavelength. The same is true for the GQDs-3. These results together showed that the excitation wavelength dependence of the emission wavelength of the GQDs is not their intrinsic property when they are larger than 5 nm; it is caused by the wide size distribution of the GQDs. Our result is different from the literature report that the GQDs with sizes from 1.5 to 5.5 nm, prepared by the hydrothermal method, exhibited emission wavelength independent PL properties.44 The authors proposed that the surface status of the GQDs prepared by the hydrothermal method dominated the PL property, and thus, a small size variation would not cause the shift of emission wavelength.44 This possibly is true; the GQDs generated from a mild photon-Fenton reaction of GO would be unlikely to have many zigzag sites (this is in along with their low quantum yield discussed later), and thus only when they are in small sizes is the contribution of surface state dominant. Nevertheless, through the gel electrophoresis method, the information on the size and PL property of the GQDs could be obtained concomitantly. The PL quantum yields of GQDs-1, GQDs-2, and GQDs-3 are higher than the as-synthesized GQDs (0.3%, 0.07%, and 0.02% vs 0.006% using quinine sulfate as a reference) but are incomparable to those of organic PL dyes. The low quantum yield of the GQDs possibly is caused by the way they were prepared, where the precursor GO was not treated as harshly as that in the hydrothermal method that generated a high concentration of the zigzag site.12 3.4. UV−Vis Absorption of the GQDs-1, GQDs-2, and GQDs-3. Differently sized GQDs also exhibit dissimilar UV− visible spectra. The as-synthesized GQDs display a wide absorption at UV and visible regions (black line), but GQDs-1 adsorb primarily UV light (blue line), and GQDs-2 and GQDs3 are in between (Figure S9). All of the GQD samples show absorption at 225 nm, which could be assigned to the π → π* transition of aromatic sp2 domains;45 but the intensity of the peak at 225 nm of GQDs-1 is much higher than that of GQDs2 and GQDs-3, indicating that smaller sized GQDs-1 assume better defect free aromatic sp2 domains. The shoulder at 300 nm appeared in the spectra of GQDs-1, GQDs-2, and GQDs-3 (enlarged spectra are in the inset of Figure S9), is assigned to the n → π* transition of CO bonds.46 Apparently, the 2108
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(4) Qu, D.; Sun, Z.; Zheng, M.; Li, J.; Zhang, Y.; Zhang, G.; Zhao, H.; Liu, X.; Xie, Z. Three Colors Emission from S,N Co-Doped Graphene Quantum Dots for Visible Light H2 Production and Bioimaging. Adv. Opt. Mater. 2015, 3, 360−367. (5) Benítez-Martínez, S.; Valcárcel, M. Graphene Quantum Dots in Analytical Science. TrAC, Trends Anal. Chem. 2015, 72, 93−113. (6) Li, L.; Wu, G.; Yang, G.; Peng, J.; Zhao, J.; Zhu, J. J. Focusing on Luminescent Graphene Quantum Dots: Current Status and Future Perspectives. Nanoscale 2013, 5, 4015−4039. (7) Guo, C. X.; Yang, H. B.; Sheng, Z. M.; Lu, Z. S.; Song, Q. L.; Li, C. M. Layered Graphene/Quantum Dots for Photovoltaic Devices. Angew. Chem., Int. Ed. 2010, 49, 3014−3017. (8) Gupta, V.; Chaudhary, N.; Srivastava, R.; Sharma, G. D.; Bhardwaj, R.; Chand, S. Luminscent Graphene Quantum Dots for Organic Photovoltaic Devices. J. Am. Chem. Soc. 2011, 133, 9960− 9963. (9) Li, L.; Liu, K.; Yang, G.; Wang, C.; Zhang, J.; Zhu, J. Fabrication of Graphene-Quantum Dots Composites for Sensitive Electrogenerated Chemiluminescence Immunosensing. Adv. Funct. Mater. 2011, 21, 869−878. (10) Zhu, S.; Zhang, J.; Qiao, C.; Tang, S.; Li, Y.; Yuan, W.; Li, B.; Tian, L.; Liu, F.; Hu, R.; Gao, H.; Wei, H.; Zhang, H.; Sun, H.; Yang, B. Strongly Green-Photoluminescent Graphene Quantum Dots for Bioimaging Applications. Chem. Commun. 2011, 47, 6858−6860. (11) Volarevic, V.; Paunovic, V.; Markovic, Z.; Markovic, B. S.; Misirkic-Marjanovic, M.; Todorovic-Markovic, B.; Bojic, S.; Vucicevic, L.; Jovanovic, S.; Arsenijevic, N.; Holclajtner-Antunovic, I.; Milosavljevic, M.; Dramicanin, M.; Kravic-Stevovic, T.; Ciric, D.; Lukic, M. L.; Trajkovic, V. Large Graphene Quantum Dots Alleviate Immune-Mediated Liver Damage. ACS Nano 2014, 8, 12098−12109. (12) Pan, D.; Zhang, J.; Li, Z.; Wu, M. Hydrothermal Route for Cutting Graphene Sheets into Blue-Luminescent Graphene Quantum Dots. Adv. Mater. 2010, 22, 734−738. (13) Liu, F.; Jang, M. H.; Ha, H. D.; Kim, J. H.; Cho, Y. H.; Seo, T. S. Facile Synthetic Method for Pristine Graphene Quantum Dots and Graphene Oxide Quantum Dots: Origin of Blue and Green Luminescence. Adv. Mater. 2013, 25, 3657−3662. (14) Kotchey, G. P.; Allen, B. L.; Vedala, H.; Yanamala, N.; Kapralov, A. a.; Tyurina, Y. Y.; Klein-Seetharaman, J.; Kagan, V. E.; Star, A. The Enzymatic Oxidation of Graphene Oxide. ACS Nano 2011, 5, 2098− 2108. (15) Han, T. H.; Huang, Y. K.; Tan, A. T.; Dravid, V. P.; Huang, J. Steam Etched Porous Graphene Oxide Network for Chemical Sensing. J. Am. Chem. Soc. 2011, 133, 15264−15267. (16) Lu, J.; Yeo, P. S.; Gan, C. K.; Wu, P.; Loh, K. P. Transforming C60 Molecules into Graphene Quantum Dots. Nat. Nanotechnol. 2011, 6, 247−252. (17) Zhu, S.; Song, Y.; Zhao, X.; Shao, J.; Zhang, J.; Yang, B. The Photoluminescence Mechanism in Carbon Dots (Graphene Quantum Dots, Carbon Nanodots, and Polymer Dots): Current State and Future Perspective. Nano Res. 2015, 8, 355−381. (18) Wunsch, B.; Stauber, T.; Guinea, F. Electron-Electron Interactions and Charging Effects in Graphene Quantum Dots. Phys. Rev. B 2008, 77, 035316. (19) Hu, Y.; He, D. W.; Wang, Y. S.; Duan, J. H.; Wang, S. F.; Fu, M.; Wang, W. S. An Approach to Controlling the Fluorescence of Graphene Quantum Dots: From Surface Oxidation to Fluorescent Mechanism. Chin. Phys. B 2014, 23, 128103. (20) Liang, F. X.; Jiang, Z. T.; Lv, Z. T.; Zhang, H. Y.; Li, S. Energy Levels of Double Triangular Graphene Quantum Dots. J. Appl. Phys. 2014, 116, 123706. (21) Yang, P.; Zhou, L.; Zhang, S.; Wan, N.; Pan, W.; Shen, W. Facile Synthesis and Photoluminescence Mechanism of Graphene Quantum Dots. J. Appl. Phys. 2014, 116, 244306. (22) Zhu, X.; Xiao, X.; Zuo, X.; Liang, Y.; Nan, J. Hydrothermal Preparation of Photoluminescent Graphene Quantum Dots Characterized Excitation-Independent Emission and Its Application as a Bioimaging Reagent. Part. Part. Syst. Charact. 2014, 31, 801−809.
4. CONCLUSIONS In summary, we employed a convenient gel electrophoresis strategy to obtain GQDs in average sizes of 5.5, 12.5, and 16 nm with a narrow size distribution. With these well-defined GQDs, we demonstrated that the PL emission of the welldefined GQDs originated mainly from the peripheral carboxylic groups and conjugated carbon skeleton. More importantly, it is revealed that the contribution of surface status to PL properties of the GQDs is dominant when GQDs are in small sizes (less than 6 nm in this work) and that the contribution of the carbon skeleton only shows up when they are excited at a longer wavelength. While for the large sized GQDs (∼12 and 16 nm), the larger carbon skeleton is a major contributor to their PL, and peripheral carboxylic groups have a minor effect. Their PL emission spectra are independent of the excitation wavelength. We thus can infer that the dependence of emission wavelength on the excitation wavelength of the GQDs that reported in most literature was caused by their wide size distributions. Preliminary experimental data also showed that the well-defined small sized GQDs (∼5.5 nm) obtained from gel electrophoresis exhibit unique behavior to cells and to small molecules. The results should be beneficial to gaining a deep understanding of the origin of the optoelectronic property of GQDs and their biological applications as drug, gene delivery systems.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b10602. UV−vis spectra of the different sized GQDs, AFM images of the as-synthesized GQDs, high resolution TEM of the different sized GQDs, and other information (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*(S.G.) Tel/Fax: 0086-21-34206915. E-mail:
[email protected]. cn. *(J.Z.) Tel/Fax: 0086-21-64253846. E-mail: jyzhang@ecust. edu.cn. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was carried out with financial support from the national “973 Program” of China (Nos. 2014CB260411 and 2015CB931801), National Science Foundation of China (Nos. 11374205, 21376148), the State Key Laboratory of Bioreactor Engineering (No. 2060204), 111 Project (No. B07023), the Shanghai Committee of Science and Technology (No. 11DZ2260600), and national “863” Program of China (No. 2012AA022603).
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