Chaos to order: an eco-friendly way to synthesize graphene quantum dots

Abstract

We developed a rapid, simple and pollution-free method to synthesize highly ordered graphene quantum dots (GQDs), which adopts cheap and readily available activated carbon and environmentally friendly hydrogen peroxide as raw materials through simple microwave and hydrothermal treatment, and the fine products are obtained as uniformly sized particles. The proposed strategy enables the difficult transformation from amorphous carbon to highly ordered GQDs for the first time while completely avoiding the use of concentrated sulphuric acid, concentrated nitric acid and other caustic reagents, and the purification procedure is relatively simple. Furthermore, the as-prepared products possess low toxicity, high biological compatibility and good fluorescence properties, which are excellent properties for bio-labelling applications.

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1. Introduction

Graphene, a two-dimensional (2D) monolayer graphite consisting of sp²-hybridized carbon atoms, was first obtained by K. S. Novoselov and A. K. Geim via mechanical exfoliation in 2004.¹ Its extremely high mechanical strength, chemical stability, good electrical conductivity at room temperature than any other known materials and long-range π-conjugation, which yields a half-integer quantum Hall effect and an ambipolar electric field effect, make graphene a superstar in the materials realm.²

In comparison with conventional two-dimensional (2D) graphene, zero-dimensional (0D) graphene quantum dots (GQDs) are clusters stacked from several layers of graphene with nanometer diameter, which usually contain oxygen-rich functional groups such as hydroxy, carboxyl, or epoxy groups on their edges.³ Furthermore, GQDs with high quantum yield also contain some nitrogen-containing functional groups after passivating treatment.^4,5 As a newly coming carbonaceous nanomaterial derived from graphene, GQDs not only possess a quantum Hall effect at room temperature, but also show characteristic quantum confinement effects and strong photoluminescence (PL) character. Compared with the traditional semiconductor quantum dots, which are synthesized from toxic metal compounds such as CdS,⁶ the outstanding biological compatibility, low toxicity and good dispersibility of GQDs in many kinds of solvents make them a promising material in the bioimaging, photovoltaic and light emitting fields.³

Up to now, the reported methods to synthesize GQDs mainly include nanolithography techniques,⁷ electrochemical scissoring,⁸ ultrasonic shearing,⁹ hydrothermal and solvothermal cutting of graphene sheets (GSs),^10,11 nanotomy assisted exfoliation,¹² precursor pyrolysis¹³ and stepwise organic synthesis.¹⁴ Almost all the methods require the use of sophisticated, expensive equipment and/or require harsh conditions. Moreover, the separation and purification processes are extremely cumbersome and the purity of product is not satisfactory. It should also be noted that the precursors used in most of these methods are highly ordered graphite or its derivatives. Not only are these raw materials relatively expensive, but also the modified Hummers method must be employed to obtain graphite oxide before the preparation of GQDs.^15–17 As is well known, during the preparation of graphite oxide via Hummers method, harsh and environmental unfriendly chemicals such as fuming sulfuric acid, concentrated nitric acid, and potassium permanganate are unavoidably used. Moreover, washing the product requires a large volume of water and the proceedings inevitably introduce secondary pollution to the product. All these deficiencies have become bottlenecks in the commercial production of GQDs.

In order to solve the problems mentioned above, we innovatively exploited a green method for synthesizing GQDs, which adopts cheap and readily available activated carbon and environmentally friendly hydrogen peroxide instead of other negligibly degradable reagents as raw materials through microwave assisted preliminary ordering and purification and simple hydrothermal reaction, and the fine products (GQDs-SH) were obtained as uniformly sized particles after dialysis. The overall synthetic procedure is illustrated in Scheme 1. GQDs-SH exhibit good stability and dispersity and excellent solubility (stored for 6 months without aggregation). The particle size distribution is uniform with 85.9% located in 4.5–10 nm intervals, and the quantum yield is as high as 9.8% (without using any passivant). The as-prepared GQDs-SH exhibit good chemical stability, low toxicity, excellent solubility and biocompatibility.

Scheme 1 Graphical representation of the synthesis of GQDs.

2. Results and discussion

2.1. XRD and PL characterization

The structure of the GQDs-SH was studied using X-ray diffraction (XRD) as shown in Fig. 1. The diffraction peak at 2θ = 26.56° is attributable to the limited ordering (only a few layers) of the graphite structure in microwave treated activated carbon.¹⁸ Compared with the original activated carbon, the peak at 2θ = 26.56° sharpens after treatment in a microwave oven, which indicates that microwave irradiation may lead to a pre-ordering process of the activated carbon as well as the removal of organic impurities in the raw materials. Microwave irradiation has been accepted as a useful method for syntheses of organic and inorganic compounds due to its distinct advantages such as uniform, rapid and volumetric heating and energy efficiency,^19,20 which result in a quick increase of temperature in the system and more efficient removal of some small molecules and functional groups in the activated carbon. After irradiation, the activated carbon acquires a more compact and orderly structure, which is verified by XRD spectra. As shown in Fig. 1, the (001) and (002) diffraction peaks of GQDs-SH correspond to graphite oxide and the characteristic peaks of graphite,²¹ respectively. The (002) peak indicates that the GQDs-SH contain graphite-like structures²² and the graphite oxide (001) peak denotes that the product contains functional groups such as –OH, –COOH, and epoxy/ether, originating from the oxidization of H₂O₂ and O₂, which is rooted in the decomposition of H₂O₂. Because of these groups the interlayer spacing of the GQDs-SH becomes larger than graphite, the 2θ angle is smaller than 26.56°. These functional groups are also confirmed by Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS).

Fig. 1 XRD spectra of GQDs-SH (line 4), activated carbon after microwave treatment for six minutes (line 3), activated carbon after microwave treatment for three minutes (line 2), and raw activated carbon (line 1).

A detailed PL study was carried out using different excitation wavelengths to explore the optical properties of the GQDs-SH. The PL spectra with excitation wavelengths from 280 to 440 nm are shown in Fig. 2. From the figure we can see that the emission intensity increases initially with the increase of excitation wavelength, and the emission intensity reaches a climax when the excitation wavelength is 320 nm, and then the emission intensity decreases with increase in excitation wavelength. Generally, the PL spectra of most luminescent carbon nanoparticles are dependent on excitation wavelength. However, the GQDs-SH show an excitation-independent PL feature. When the excitation wavelength is changed from 280 to 360 nm, the PL spectra are almost identical and show a strong peak at ca. 420 nm (see Fig. S1, ESI†), which indicates that both the size and the surface state of the sp² clusters are uniform.²³ The UV-vis absorption spectrum of the GQDs-SH (see Fig. S2, ESI†) shows an absorption band at ca. 286 nm, which reveals a blue shift of ca. 36 nm with respect to that of the reported GQDs.²⁴ It has been reported that due to the localization of electron–hole pairs, the isolated sp² hybridized clusters with size of ca. 3 nm within the carbon oxygen matrix could yield band gaps consistent with blue emission,²⁵ which, along with the previous observation of PL from nanosized graphene oxide,²⁶ indicates that the oxygen-rich groups make a significant contribution to the observation of blue shift in the PL emission, in addition to the size and surface effects.²⁷

Fig. 2 PL emission spectra of GQDs-SH.

2.2. XPS analysis

Fig. 3(a) and (b) show the XPS analysis of C1s and O1s of GQDs-SH, respectively. The XPS C1s spectra of GQDs-SH show three characteristic peaks at 283.2, 284.9, and 286.7 eV. The binding energy (BE) of 283.2 eV is in close agreement with the values obtained by conventional XPS.²⁸ The 284.9 eV peak reveals that the products exhibit graphite-like sp² hybrid carbons, which is in good accordance with the XRD results. Furthermore, the 286.7 eV –C–O BE provides further proof of the C–OH stretching vibration observed by FTIR. The O1s peak at 531.8 eV in Fig. 3(b) is assigned to contributions from C=O and O=C–OH groups, referring to the peaks of C1s, and the peak at 533.2 eV is assigned to C–OH groups. In addition, the area of the peak at 533.2 eV is larger in Fig. 3(b), which indicates that the hydroxyl content is greater than that of carboxyl.

Fig. 3 XPS spectra of GQDs-SH: (a) C1s peaks and (b) O1s peaks.

2.3. TEM images and FT-IR spectra

The transmission electron microscope (TEM) images of GQDs-SH are shown in Fig. 4. From the TEM images, we can reach a conclusion that GQDs-SH synthesized by this green strategy possess good dispersibility and stability, which is further demonstrated by TEM images acquired six months later, in which there are no obvious agglomerations even after six months of storage (see Fig. S3, ESI†). The size distribution histogram (see Fig. 4(c)) indicates that the particle sizes of GQDs-SH are mostly (85.9%, calculated by statistics) concentrated in the 4.5–10 nm range, suggesting that the GQDs-SH are uniform in dimension, and the average diameter is 7 nm (calculated), similar to a previously reported value.²⁹

Fig. 4 TEM images, scale bar: (a) 50 nm, (b) 100 nm. (c) Size distribution of GQDs-SH. (d) FT-IR spectra. Inset: photographs of aqueous solutions of the GQDs-SH (left) under visible light and (right) under UV light (365 nm).

Since the reaction is conducted under hydrothermal conditions at 180 °C in a hydrothermal tank and H₂O₂ readily decomposes into H₂O and O₂ under high temperature and pressure conditions, the as-synthesized GQDs-SH consequently contain some oxygen-containing functional groups, similar to the traditional method. As can be seen from the FT-IR spectra in Fig. 4(d), the GQDs-SH display a stretching vibration of O–H at 3024 cm⁻¹ and a characteristic peak of C=O at 1718 cm⁻¹, while the peak intensity is very weak, indicating low carbonyl content. The peaks at 1587 cm⁻¹, 1490 cm⁻¹ and 1407 cm⁻¹ demonstrate that the product possesses a benzene ring structure, which is in accordance with the XRD results. Moreover, a strong C–OH stretching vibration peak appeared at 1089 cm⁻¹, indicating that the GQDs-SH are rich in hydroxyl groups, which could be further explored for applications in fields such as biolabeling and fluorescence detection.

2.4. AFM characterization and cytotoxicity

The AFM image and the height profile of the GQDs-SH are shown in Fig. 5. The topographic heights of the products are mainly between 2.5–5 nm according to the image. Consequently, it can be concluded that most of the GQD-SH particles consist of fewer than 5 layers of graphene, although some heights in the profile are greater than 10 nm, which is probably due to the preparation process of the AFM testing samples in which the GQD-SH solution is repeatedly dripped onto a mica plate and the overlap of GQDs-SH could be inevitable.

Fig. 5 (a) AFM image of the GQDs-SH on Si substrate, and (b) the height profile of the GQDs-SH.

As the method proposed in this article completely avoids the use of toxic raw materials and reagents and the quantum yield is satisfactory in the absence of a passivating agent, we investigated the biocompatibility and bio-labelling applications of the GQDs-SH using HeLa cells. After 24 h incubation, the survival rate was higher than 80% even if the concentration of GQDs-SH was increased up to 400 μg mL⁻¹ (see Fig. 6(a)), which indicates the fairly low toxicity of the GQDs-SH. The excellent biocompatibility demonstrated by the as-prepared GQDs-SH could be attributed to the completely green preparation procedure, which adapts non-toxic H₂O₂ and active carbon as raw materials. Next, cell imaging was performed on an inverted fluorescence microscope after incubating HeLa cells with GQDs-SH (100 μg mL⁻¹) for 2 h. The bright-field image in Fig. 6(b) shows that the treated cells retain their original fusiform morphology, confirming the low toxicity of the GQDs-SH. The fluorescence image in Fig. 6(c) and the overlay image in Fig. 6(d) both show that HeLa cells labeled with GQDs-SH have a strong blue fluorescence under UV irradiation, which further indicates that our materials could serve as promising agents for optical imaging.

Fig. 6 Cellular toxicity and cellular imaging of GQDs. (a) Effect of GQDs-SH on HeLa cell viability. (b) Bright field, (c) fluorescent field, (d) overlay images of labelled HeLa cells.

3. Conclusions

In summary, GQDs-SH with strong blue fluorescence have been prepared via simple microwave treatment and hydrothermal reaction from activated carbon, and the difficult transformation from amorphous carbon to highly ordered GQDs was achieved for the first time by such a method. This approach is energy, material, and labour efficient; moreover, it satisfies several principles of green chemistry. Fluorescence quantum yield of the product is as high as 9.8% without the usage of any passivating agent. Furthermore, the resulting GQDs-SH disperse well and has an excitation-independent PL feature. This novel nanomaterial possesses the ability to easily penetrate live cells, remarkable PL properties, and nontoxicity to cells and holds great potential for biomedical applications.

4 Experimental section

4.1 Microwave irradiation procedure

0.6 g of accurately weighted activated carbon (AC) was placed in a smaller quartz crucible, which was then placed into a larger crucible, and the interspaces between the two crucibles were filled with graphite powder. Then, the crucibles were placed in a modified domestic microwave oven (adjusted to 800 W) and heated for six minutes under atmospheric conditions. After the heating procedure, the samples were cooled to room temperature in air.

4.2 Hydrothermal procedures

The microwave purified AC was naturally cooled to room temperature, then the AC was mixed with 15 mL H₂O₂ (30%) and transferred to a 25 mL Teflon®-lined stainless steel autoclave, which was then sealed and heated in an oven equipped with a thermostat or an automatic temperature control unit. The reaction temperature was raised from room temperature to 180 °C in 90 minutes and maintained at that temperature for 15 h. After cooling, the obtained colloidal solution was dialyzed in a dialysis bag (retained molecular weight 8000–14 000 Da) overnight, and then diluted with distilled water to 25 mL.

4.3 Dialysis treatment

A dialysis bag (6–8 cm) was boiled for 5 minutes, and then submerged in 50% ethanol for half an hour. After that, the bag was washed with EDTA and distilled water. The raw product was then sealed in the dialysis bag and dialyzed in water for 24 h to obtain uniformly sized particles.

4.4 Quantum yield (QY) calculation

Quinine sulfate in 0.10 M H₂SO₄ solution was used as a standard. The QYs of GQDs (in water) were computed according to the following formula:
where Φ is the quantum yield, I is the measured integrated emission intensity, η is the refractive index of the solvent, and A is the optical density. The subscript R refers to the standard index of quinine sulfate (data are listed in Table S1†).

4.5 Cell cultivation and cytotoxicity test

HeLa cells were cultured with Dulbecco's modified Eagle's medium (DMEM, high glucose) supplemented with 10% fetal bovine serum seeded in a flask under a humidified environment at 37 °C with 5% CO₂ concentration. In vitro cytotoxicity was tested by performing a CCK-8 assay on the cells. HeLa cells were incubated in 96-well culture plates at a density of 2 × 10³ cells per cell in 100 μL fresh culture medium and cultivated for 24 h. Then, the cells were washed with PBS, and the medium was replaced with fresh medium containing a predetermined amount of testing materials. After 24 h, the cells were washed with PBS and cultivated in DMEM containing 10% CCK-8 for 2 h. The absorbance of each well was measured at 450 nm with a plate reader.

Acknowledgements

The financial supports from the National Natural Science Foundation of China (Grants 21171122, 21271132, 91126016, J1210004, J1103315 and J1310008), the Chinese Academy of Sciences (Grant KJCX2-YW-N50-3) and the International Collaboration Project of Science and Technology Program of Sichuan Province, China (Grant 2010HH0008) are gratefully acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra06757f

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