Efficient synthesis of rice based graphene quantum dots and their fluorescent properties

Abstract

We present a facile green approach to synthesize monodisperse graphene quantum dots (GQDs) of sizes 2–6.5 nm using rice grains as a carbon source. As the size of the GQDs increases from 2–6.5 nm, a red shift (blue to cyan) in the photoluminescence emission spectra is observed due to quantum confinement effect. The colloidal solution of as synthesized GQDs is highly luminescent under 336 nm illumination. The quantum yield (QY) of the as-prepared GQDs in water is size dependent and increases from 16 to 24% with the decrease in size from 6.5 to 2 nm. The potential of these GQDs as biomarkers for cell imaging is explored further. The cytoxicity study with different concentrations of the GQDs confirms the excellent biocompatibility of the GQDs.

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1. Introduction

The graphene quantum dot (GQD) is a recent infant of the carbon nanomaterials family. It shows exciting electrical,^1,2 optical,^3,4 and magnetic^5,6 properties governed by the quantum confinement and edge effects.^7,8 It has received enormous popularity among researchers of various fields due to its potential applications in FET,⁹ optoelectronic,^10–12 bio-labeling,^13–15 and bioimaging devices.^13,16 In fluorescence bioimaging, traditionally fluorescent dyes are used as imaging agents, but their high cost and photoinstability hinders a further development. Semiconductor quantum dots (QDs) are considered a replacement because of their bright and stable fluorescence characteristics.^17–20 However, cytotoxicity is a major issue in most of the conventional QDs (CdS, CdSe, CdTe and PbS).^21–23 GQDs have been found to be less toxic, biocompatible and highly stable against photodegradation and bleaching.^24–27 Therefore, highly fluorescent, low bio-toxic GQDs can be the required alternative.

The photoluminescence (PL) in GQDs is dependent on the size tuning as well as on the synthesis method. The size dependent band gap tuning of graphene is particularly interesting for photovoltaic and emission related applications.^12,28,29 To effectively control the band gap of graphene a promising approach is to alter the 2-D graphene sheets into 0-D GQDs; wherein, the band gap gets tailored from 0 eV to few eV for countable numbers of benzene rings popularly called as GQDs.^30,31 In principle, the band gap of GQDs depends on size,³² shape,³³ and fraction of the sp²–sp³ hybridized domains.³⁴ In present scenario, control of the size of graphene dots (around 1–5 nm) is a great challenge. Thus, to develop a facile, controllable, and scalable technique to prepare GQDs of narrow size distribution is highly desirable.

Till now GQDs are mainly synthesized by two different approaches: top-down and bottom-up. Top down approaches include cutting of carbon precursor such as graphite or graphene oxide,^3,34–36 carbon nanotubes,³⁷ carbon fibres,¹³ and coal,³⁸ via hydrothermal,^34,35 or electrochemical³⁷ processes. However, these methods are limited by special equipment, expensive processing, low chemical stability and poor yield. To overcome these challenges, few bottom-up approaches are also being explored. For example, GQDs are synthesized through controlled thermolysis of glucose,³⁹ carbonization of citric acid,²⁶ and oxidation of polyphenylene dendrites.⁴⁰ Nevertheless, these methods are also tricky and laborious (multiple step processes). Apart from the basic issues, size controlled synthesis has not been achieved in many of the bottom-up synthesis processes. Therefore, a cost effective, quick and environmentally benign method using natural resources to synthesize size controlled luminescent GQDs is the need of the present art.

Herein, we propose a simple, cost effective and green approach to synthesize size controlled monodisperse GQDs (standard deviation, σ ≤ 10%) using rice powder as a carbon resource. The synthesis process does not involve any complex steps, additives like acids, alkali, surfactant etc. As prepared GQDs possess photoluminescence (PL) quantum yield (QY) in the range of 16–24%, which is easily enhanced to 3 times (QY increases from 16% to 54%) after surface functionalization with amine. GQDs show good biocompatibility with the HeLa cells and demonstrate as an excellent probe for bio-imaging.

2. Experimental

2.1. Synthesis process

Rice grains are used as the carbon source for the synthesis of GQDs. At first rice grains were cleaned with de-ionized (DI) water and grinded to get rice powder. The cleaned rice powder was fried in a cooking pan at around 200 °C. Different batches of rice powder were taken and heated for different time. The size of GQDs was controlled by varying the heating time as 3 min (2 nm), 5 min (4 nm), 7 min (5.2 nm) and 10 min (6.5 nm). The schematic of the experimental set-up is shown in Fig. S1 (see ESI†). To collect GQDs, the fried rice powder samples were dispersed in DI water using ultrasonication. When the particle size is smaller, the color of the solution is light brownish and the solution appears dark brownish for large particle size. The unreacted rice grains were separated out through the millipore porous membrane (0.22 μm pore size) using vacuum filtration technique and clean/transparent brown color filtrate was collected. The filtrate was then washed and dialyses at pH ∼ 4 for 2–3 days to remove any unwanted carbohydrates and then the purified samples were used for further characterizations. A plausible mechanism of the synthesis process is depicted in Fig. 1. Rice powder contains a large amount of starch in the form of amylose (unbranched D-glucose chain) and amylopectin (glucose units are linked in linear fashion with α(1 → 4) glycosidic bonds and branching takes place with α(1 → 6) bonds occurring every 24 to 30 glucose units).⁴¹ While heating the rice powder, thermal breakdown of starch leads to glucose oligomers (Fig. 1a).⁴² When the oligomers of glucose are heated at high temperature (200 °C) nucleation takes place (Fig. 1b) and pyrolization of the glucose molecule leads to the growth of the particle (Fig. 1c).⁴³ The size of the GQDs is controlled between 2–6.5 nm by sheer increase of the heating time from 3 min to 10 min. The heating in hot plate is homogenous and fast which leads to the uniform size distribution of GQDs. In a typical large scale synthesis (1.4 g), 2.5 g cleaned rice is taken and powder is made out of it and then fried it on a hot plate for 10 min. The percentage yield of GQDs from rice grain is ∼56%. Further, GQDs are functionalized with amine to modify the surface of the GQDs. A mixture of 200 mg of GQDs powder (of size 6.5 nm) and 5 ml of oleylamine was first heated to 120 °C for 30 min then to 180 °C for 4 h.

Fig. 1 A plausible mechanism for the synthesis of GQDs from rice powder [in the chemical structure of GQD: red color dots – carboxyl groups, blue color dots – epoxy groups, yellow color dots – hydroxyl groups].

2.2. Cell viability and cellular imaging

Human cervical cancer cells (HeLa) were cultured in the Dulbecco's modified Eagle's medium (DMEM, HiMedia), supplemented with 10% of fetal bovine serum (FBS, HiMedia) and 1% of antibiotic antimycotic solution (penicillin/streptomycin, HiMedia) in a humidifier incubator at 37 °C, and 5% CO₂ environment. Cytotoxicity was examined by sulforhodamine B (SRB, Sigma-Aldrich) assay. HeLa cells were seeded into 96 well plates at ∼1 × 10⁴ cells per well and incubated overnight. After incubation, cells were treated with fresh media containing different concentrations of GQDs. Next, the treated cells were fixed with 10% trichloroacetic acid (TCA) for 1 h at 4 °C and washed thrice with fresh autoclaved water. Thereafter, cells were incubated with 0.06% SRB solution for 20 min at room temperature. Following which, cells were washed with 1% acetic acid to dissolve the unbounded SRB. Then, plate was processed for the measurement of cell viability which was calculated using the following formula:

For imaging purposes, ∼4 × 10⁵ cells were seeded on cover slips which were previously placed in a 24 well plate and transferred to the humidifier incubator. After overnight incubation, cells were treated with fresh media containing 0.5 mg ml⁻¹ of GQDs samples at 37 °C. Over a period of 24 h, treated cells were washed twice with fresh sterile phosphate buffered saline (PBS) to remove the extra GQDs in the media. Then, the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. After that, the cells were washed with PBS twice and cover slips were placed on clean glass slides with mounting agent.

2.3. Characterizations

The high-resolution transmission electron microscopy (HR-TEM) images and selected area diffraction patterns (SAED) were obtained with a JEOL JEM 2100F field emission gun transmission electron microscope (FEG-TEM). Atomic force microscope (AFM) measurements were performed using digital instruments Nanoscope IV Multimode scanning probe microscopy. Images were taken in tapping mode using etched silicon probes. Raman spectra were recorded at room temperature using confocal micro-Raman spectrometer equipped with Ar⁺ laser (514.5 nm). A Thermo VG Scientific Multilab 2000 photoelectron spectrometer was used for X-ray photoelectron spectroscopy (XPS) measurements. FTIR spectra were taken on a Bruker, Vertex-80 spectrometer using KBr pellets. UV-visible spectra were carried using Lambda 950 (Perkin-Elmer) in the wavelength range 200–600 nm using quartz cell. All the photoluminescence (PL) investigations were carried out on Varian Cary Eclipse Fluorescence spectrophotometer at room temperature. Confocal microscopic images were taken using a Confocal Laser Raman Spectroscopy (CLSM, Zeiss LSM 780) system.

3. Results and discussion

Fig. 2a–d show the representative transmission electron micrographs of the GQDs prepared by heating rice powder for different times. A statistical analysis has been done for each sample by counting almost 50–60 GQDs using the lognormal distribution (see inset, Fig. 2). The average size of GQDs increases from 2 to 6.5 nm (σ ≤ 10%) with increase of the calcination time from 3–10 min. Controlling the size of the GQDs ≤ 5 nm with highly monodispersed (std deviation ≤ 15%) nature is a great challenge.^3,35,36 So far, there are only very few reports on size controlled synthesis of highly monodisperse GQDs.^37,39,43 High resolution TEM micrograph (Fig. 3a) shows the crystallinity of the as-synthesized GQDs with an inter-planar spacing of 0.33 nm corresponding to (002) plane of graphite.¹³ Fig. 3b shows a topographical atomic force microscopy (AFM) image of 4 nm sized GQDs along with the sectional analysis. The height profile of as synthesized quantum dots is found to be around 1–2 nm which corresponds to one to three layers of graphene.^4,44 GQDs usually represents up to ten of layers of graphene with size less than 30 nm.⁴³

Fig. 2 TEM images of GQDs of different sizes (a) 2 nm, (b) 4 nm, (c) 5.2 nm and (d) 6.5 nm. Inset shows the corresponding size distribution histogram with a count of 50–60 particles.

Fig. 3 (a) High resolution TEM image of the GQDs of size 4 nm shows the crystallinity and the fringe pattern with d-spacing of 0.33 nm corresponding to (002) lattice planes. (b) AFM image of 4 nm sized GQDs and (c) the cross-sectional analysis showing the average height of the GQDs to be 1–2 nm.

Fig. 4a shows the Raman spectra of different sized GQDs with G band at 1590 cm⁻¹ which is assigned to the E_2g phonon of sp² carbon atoms and D band at 1350 cm⁻¹ which corresponds to the extent of defects and originates from a breathing κ-point phonon with A_1g symmetry.⁴⁵ A 2D band (overtone of D-band) is also observed at 2700 cm⁻¹, which is due to double resonance transition resulting in production of two phonons with opposite momentum.⁴⁶ The high value of I_D/I_G ratio (0.83) confirms the presence of defects in the prepared GQDs. I_D/I_G ratio can be related to the particle size according to Tuinstra and Koenig (TK) relation: I_D/I_G = C(λ)/L_a, where L_a is the cluster size, C(λ) is an empirical constant that depends on the excitation laser energy and C(λ = 514.5 nm) = 4.4 nm.^45,47 The average size of the GQDs is L_a = 5.3 nm, and the result falls within the 2–6.5 nm range as obtained by TEM analysis described above. The high resolution TEM study shows the (002) facet of graphite,¹³ along-with the sectional analysis by AFM which shows the height as 1–3 layers of the graphene,^4,44 and the explanation of G-band and D-band by Raman spectroscopy confirms that the synthesized dots are graphene quantum dots and not merely carbon nanoparticles or carbon dots.

Fig. 4 (a) Raman spectra of as-prepared GQDs (b) XPS survey spectrum of 4 nm GQDs and (c) slow scanned deconvoluted C1s spectrum of 4 nm GQDs. (d) FTIR spectra of the GQD samples of size 4 nm.

The bare graphitic particles are hydrophobic in nature. The hydrophilicity (water solubility in our case) results from the surface functional groups which might be attached to the interface of the GQDs. We have performed X-ray photoelectron spectroscopy (XPS) in order to confirm the surface functional groups at the GQDs surface. XPS survey spectra (Fig. 4b) for GQDs shows a predominant graphite C1s peak at 284.8 eV and an O1s peak at 532 eV. Fig. 4c shows the deconvoluted C1s XPS spectrum of as prepared GQDs. The peak at 284.4 eV and 285.5 eV corresponds to sp²C carbon (graphitic) and sp³C carbon, while the component at 289.6 eV is attributed to the π–π* shake up satellite of the 284.4 eV.⁴⁸ The peaks at 286.5 eV, 287.4 eV, and 288.4 eV respectively, are assigned to oxygen functionalities, namely C–O, C=O, and COOH.⁴⁹ The O/C ratio calculated from the C1s peak for GQDs is 0.44 (Table S1 in the ESI†). To further confirm the surface functional groups Fourier transform infrared (FTIR) studies are carried out. The surface components of the GQDs as determined by the XPS data are in good agreement with FTIR results (Fig. 4d). The stretching vibrations of O–H at 3450 cm⁻¹, C–H at 2923 cm⁻¹ and 2850 cm⁻¹, vibrational absorption band of C=O at 1640 cm⁻¹ and an epoxide band (C–O–C) at 1020 cm⁻¹ confirms that the prepared GQDs are functionalized by oxygen functional groups. XPS and FTIR confirm the presence of C–H, C–O, C=O, and C–O–C on the surface of the GQDs. As prepared GQDs form a stable colloidal suspension as the surface functional groups might be preventing the GQDs from aggregation and hence remain stable without additives. The GQDs solution is in stable condition for almost one year under the ambient condition. The functional groups at the surface of the GQDs act as a “passivation” layer for the GQDs. This self passivated layer facilitates the solubility of GQDs in water and it also plays an important role for efficient photoluminescence properties which will be discussed later.

To study the optical properties of the as-prepared GQDs, UV-Vis absorption and photoluminescence (PL) measurements of the GQDs suspension are carried out. In UV-Vis spectra of GQDs, (Fig. 5a) the π → π* transition peak centred at a wavelength between 200 nm and 270 nm and the n → π* transition peak is observed at wavelength longer than 260 nm.⁵⁰ We have observed that both absorption peaks at 220 nm and 270 nm become stronger with the increase in size, which is similar to earlier reports.^39,43 Sometimes the former peak is not distinguishable as seen in the GQDs sample of smaller size, this might be because of the screening of the strong background absorption.^4,33 As the size of the GQDs increases from 2 to 6.5 nm, their absorption peak exhibits a red shift. This red shift with GQDs size confirms the quantum confinement effect and is in agreement with the literature value reported for GQDs.⁵¹

Fig. 5 (a) UV-Vis spectra of different sized GQDs (b) size dependent PL spectra measured at a fixed excitation wavelength of 360 nm (c) excitation dependent PL spectra of 4 nm sized GQDs. (d) The optical image of the GQDs along with the change in luminescence from blue to cyan under the UV light illumination (λ = 336 nm) with the increase in size.

The photoluminescence (PL) property can be tailored either by synthesizing different sizes of GQDs or by functionalizing the GQDs. Fig. 5b shows the size dependent PL spectra of as prepared GQDs at λ_ex of 360 nm. With increase of the GQDs size from 2–6.5 nm, a red shift (blue to cyan) in the PL emission spectra is observed. The change in size of GQDs results in a change in HOMO–LUMO gap and thus fluorescence color changes from blue to cyan, as shown in Fig. S2.† Fig. 5c shows the excitation dependent PL spectra of 4 nm GQDs. When the excitation wavelength is varied from 340 to 500 nm, the PL peaks are linearly shifted from 440 (blue) to 556 nm (green). The linear trend of λ_peak of the PL emission spectra with λ_ex is shown in Fig. S3(b).† The excitation-dependent PL is extensively studied in fluorescent nano-sized carbon materials including GQDs and is considered to be coming from the optical selection of quantum sizes and defects in the GQDs.^4,13,52 This excitation dependent PL behavior may be induced by surface chemistry distribution,⁵³ as well as excited electron relax to different energy level.⁵⁴ A similar trend also has been observed in 2 nm, 5.2 nm and 6.5 nm sized GQDs as shown in Fig. S4.†

The quantum yield (QY) of 4 nm sized GQDs is found to be ca. 23%. Table S2 (see ESI†) shows the QY of the GQD samples as a function of GQD size and the obtained QY varies between 16 to 24%. The QY observed for the pristine GQDs is higher than the earlier reported GQDs synthesized via different approaches.^{3,35,37,39,55,56} The QY is inversely proportional to the diameter of the GQDs as already been observed in earlier reports.³⁹ The decreasing trend of QY with the increase in size of the GQDs may be due to the domination of the non-radiative processes as observed in GQDs,³⁹ and silicon nanocrystals.⁵⁷ Surface defects and traps, vibration relaxation and the existence of a transition from nanocrystalline to nanoamorphous forms can be the reasons for non-radiative pathways.⁵⁷ In general, sp² carbon domains increase upon amination and this might enhance the luminescence of GQDs. Furthermore, electron-donating amine groups have a better ability to donate electrons than hydroxyl groups and can further enhance the luminescence of GQDs.⁵⁸ So, to enhance the QY, the as-produced GQDs are subjected to surface functionalization with oleylamine (σ donor ligand).⁵⁸ The QY of the amine functionalized GQDs of size 6.5 nm is found to increase to 54%, showing that QY of the amine-GQDs is increased 3.4 fold (Table S2 in the ESI†). Surface functionalization based methods have been shown to produce GQDs with high quantum yield.^55,59–61 The increase in QY with functionalization of amino groups is consistent with previously reported methods.^60,62–64 These results show that GQDs can be an excellent candidate for the use in bioimaging and other biomedical applications.

Cell viability study and cellular imaging of GQDs is performed to evaluate the biocompatibility and bioimaging capability of GQDs, respectively. Cytotoxic effect of GQDs is observed at different dosage (0.015–1 mg ml⁻¹ of GQDs) on the HeLa cells as shown in Fig. 6. It is observed that the GQDs shows cell viability above 90% after 24 h incubation from lower to higher concentration of 1 mg ml⁻¹. GQDs have shown very low toxicity and good biocompatibility which is in tandem with the previous results of the literature.^4,13,50,60 This result indicates that GQDs show good cell compatibility in HeLa cells.

Fig. 6 Relative cytotoxic effect of GQDs observed at different concentration 0.015–1 mg ml⁻¹ in the HeLa cells with 24 h incubation time.

To evaluate the bioimaging capability, GQDs treated HeLa cells are imaged using a 375 nm, 488 nm and 561 nm laser. Fig. 7a shows the bright field image of untreated control where Fig. 7b–d images are in different fluorescence phases and Fig. 7e is a merged image of a, b, c and d. In control experiments, cells do not show any intense fluorescence with blue, green and red laser. Fig. 7f shows the bright field image of GQDs treated cells. In case of GQDs treatment, strong blue and green emission is observed due to internalized quantity of GQDs in the cells where red emission is negligible in Fig. 7g–i. The blue and green fluorescence demonstrate that the GQDs are distributed as agglomerates in the cytoplasm and nucleus site of the cells in Fig. 7j (merged image of f, g, h and i). In GQDs treatments, cells do not undergo obvious morphological changes in the presence of GQDs, suggesting good biocompatibility. The luminescence of the normal cells is recorded as compared to GQDs treated HeLa cells. The bright field images of the cells are much weaker than the bright field images of the GQDs incubated HeLa cells and it is consistent at all the wavelengths of 375 nm, 488 nm and 561 nm. The cytotoxicity on human cells of our GQDs is significantly lower (approx. 10-fold) than cadmium-based semiconductor quantum dots such as CdSe and CdTe.⁶⁵ This result suggests that GQDs synthesized by this green approach can be useful for bioimaging application.

Fig. 7 Cellular imaging of GQDs using HeLa cells. (a–j) Both untreated control and GQD treated row show confocal fluorescence image: (a & f) bright field images of untreated control and GQD treated cells, (b & g) blue (pseudocolour) at 375 nm excitation, (c & h) green (pseudocolour) at 488 nm, (d & i) red (pseudocolour) at 561 nm and (e & j) merged images. (Scale bar = 50 μm in all cases).

4. Conclusions

In conclusion, we have successfully synthesized monodisperse GQDs of average diameter 2–6.5 nm by a green aqueous based method. The synthesized GQDs are of high crystallinity and the thickness is 1–3 layers of graphene. GQD samples show excellent fluorescence property with an emission range of blue to cyan with an increase in their size. The QY of the as synthesized water based GQDs is found to be 16–24%. The QY increases by 125% with amine surface functionalization due to better electron donating ability of the amine groups. The GQDs are used as a nano probe for fluorescence imaging of the HeLa cells. The colloidal solution of GQDs is easily uptaken by the HeLa cells. The GQDs incubated HeLa cells show lower cytotoxicity with the cell viability more than 90% upto a concentration of 1 mg ml⁻¹. Low cytotoxicity, ease of labeling make GQDs an eco-friendly material and excellent candidate for application in biological imaging and biolabelling. This work could be a stepping stone for promoting the natural resources based synthesis process of GQDs. This study opens new avenues to extend the application of such fascinating nanoparticles in various fields especially in biochemical and biomedical studies.

Acknowledgements

The authors would like to thank IRCC, IIT Bombay for Scanning Probe Microscope (SPM) facility, Electron Spectrometer for Chemical Analysis (ESCA) facility and the Centre for Research in Nanotechnology and Science (CRNTS), IIT Bombay.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25706a

‡ These authors contributed equally to this work.

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