Facile and simultaneous synthesis of graphene ...

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Facile and simultaneous synthesis of graphene quantum dots and reduced graphene oxide for bio-imaging and supercapacitor applications† A. Muthurasu,ab P. Dhandapanibc and V. Ganesh*ab A simple and facile method for the simultaneous preparation of water soluble, fluorescent graphene quantum dots (GQDs) and reduced graphene oxide (RGO) is proposed and demonstrated in the present work. This particular method involves constant current electrolysis of a graphite rod either in an aqueous solution or in absolute ethanol medium consisting of sodium methoxide as an electrolyte. The process of electrolysis is carried out at a fixed current density for varied durations. Further chemical reduction of the resultant electrochemically exfoliated graphitic solution produces a clear supernatant solution and a black precipitate. Depending upon the solvent medium, duration of electrolysis and nature of the reducing agents, the size and in turn the emission colour of GQDs could be altered. In this work specifically GQDs that emit blue, green and yellow colours have been prepared. Structural, morphological and chemical characteristics of these GQDs are analyzed by using microscopic [scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM)], spectroscopic (UV-Vis spectroscopy, Fourier transform infrared (FTIR), X-ray diffraction (XRD), Raman) and electrochemical techniques. Microscopic images obtained using HRTEM analysis clearly reveal the formation of lattice fringe patterns for these multi-coloured GQDs along with uniform particle size distribution. Further these fluorescent GQDs are utilized for bio-imaging applications and cytotoxicity studies using Staphylococcus aureus and Escherichia

Received (in Montpellier, France) 22nd February 2016, Accepted 18th August 2016

coli bacteria. Similarly, the resultant black precipitate is identified as a few layered reduced graphene oxide

DOI: 10.1039/c6nj00586a

spectroscopic studies. Further electrochemical investigation indicates the possible application of such

and the structural and morphological characterization is carried out using SEM, FTIR, XRD and Raman layered RGO as a supercapacitor electrode material possessing a fast response time in the order of

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seconds for delivering the stored power.

1. Introduction Graphene has attracted considerable research attention recently due to its unique optical, electrical, physical and chemical properties. The basic building block of graphene materials is essentially derived from graphitic materials consisting of two dimensional (2D) arrangement of carbon atoms leading to the formation of a honeycomb lattice structure. Owing to its extraordinary and intrinsic mechanical, thermal, physical, chemical and optical properties, graphene finds applications in a variety a

Electrodics and Electrocatalysis (EEC) Division, CSIR–Central Electrochemical Research Institute (CSIR–CECRI), Karaikudi – 630003, Tamilnadu, India. E-mail: [email protected], [email protected]; Fax: +91-4565-227779; Tel: +91-4565-241242 b Academy of Scientific and Innovative Research (AcSIR), New Delhi – 110025, India c Microbial Corrosion Division, CSIR–Central Electrochemical Research Institute (CSIR–CECRI), Karaikudi – 630003, Tamilnadu, India † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c6nj00586a

of fields including sensors, actuators, energy storage devices, imaging and electronics applications.1–4 Fluorescent carbon materials like graphene quantum dots (GQDs) and carbon dots (CDs) are usually derived from various precursors like graphene oxide, graphite and other carbonaceous materials. The fluorescence properties of zero dimensional GQDs arise mainly from the quantum confinement, edge effects, surface passivation and also depend on the method employed for their preparation. In general it is possible to tune the optical properties and band gap of these materials by using chemical reduction methods.5,6 These synthetic methods offer additional advantages in terms of obtaining GQDs possessing large optical absorptivity, chemical stability, adequate biocompatibility as well as low toxicity.7–10 Thus GQDs distinguish themselves from other traditional fluorescent materials due to these intrinsic superior properties, making them an ideal candidate for a wide variety of applications including bio-imaging,11 medical diagnosis,12 catalysis,13 photovoltaic devices,14 etc. In general chemically prepared graphene oxide (GO) shows poor emissive properties

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owing to the presence of hydroxyl, carboxylic and epoxy functional groups on the surface resulting in sp2 hybridized clusters leading to localized non-irradiative electron–hole (e–h) pairs.15,16 However, GO could be cleaved and disintegrated into smaller nano-sized particles that exhibit photoluminescence (PL) properties, due to the quantum confinement and edge effects. Conventionally the most widely used method for the preparation of GO and GQDs involves the usage of conc. H2SO4 and conc. HNO3 as strong oxidizing agents by employing intercalating acceptor type of graphite as a source material.17 In this particular method both graphite and GO are soaked for a longer duration in the mixture of strong acids and it requires heating. Moreover, the conventional methods also need some special kind of equipment, a high pressure environment and various chemical reagents like organic solvents and strong acids (HNO3/H2SO4) which are often toxic in nature and harmful to health. In order to avoid such complications, synthesis of GO, GQDs and carbon dots is carried out by using electrochemical methods. It is probably one of the easiest ways to prepare these materials because electrochemical methods offer several advantages in terms of low cost, easy handling, less laborious, environmental friendly approaches, retention of electronic conductivity and high output. Recently, GQDs are prepared by using two different electrolytes namely an aqueous solution of NaOH and K2HPO4 along with ionic liquids by employing a graphite rod as an anode, and Pt foil and Ag/AgCl as counter and reference electrodes respectively. Upon applying a suitable potential, the electrolyte could be electrochemically oxidized to produce -oxy and hydroxyl radicals (O2, OH ) that act as electrochemical scissors and etch the graphite surface to form GQDs.18,19 Similarly, an improved way of exfoliation and intercalation of graphite is reported by using an aqueous solution of alkali metals in ethanol solution to form alkali ethoxide which effectively intercalates into the graphitic network to form the corresponding alkali metal doped GO.20,21 Further Pan et al. have explored the scissoring of GO via a hydrothermal route leading to the formation of GQDs possessing an average size of 3 nm and observed green emission on exposure to UV light.22 Li et al. presented an electrochemical approach for direct preparation of functional GQDs with a uniform size of 3–5 nm and demonstrated them as potential electron acceptors for photovoltaic applications.23 Similarly, Shen et al. proposed hydrazine hydrate reduction of graphene oxide to form GQDs with up-conversion emission.24 All these methods resulted in the formation of GQDs which exhibit an excitation wavelength dependent PL behaviour. Enormous reports are available in the literature on the preparation of GO and GQDs separately and most of these methods involve chemical reduction and cleavage using harsh chemicals and reagents. Usually all these methods also require laborious work-up procedures to separate GQDs. Further chemical functionalization is needed to obtain water soluble GQDs in most of the cases. Keeping this in mind, in this work, we report a facile and simple method for the simultaneous preparation of GQDs and reduced graphene oxide (RGO) (Fig. 1). This method involves two steps. In the first step electrolysis of a graphite rod is performed in a suitable electrolyte consisting of sodium methoxide dissolved either in an aqueous solution or in ethanol by

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Fig. 1 Pictorial representation of the proposed strategy for the simultaneous preparation of GQDs and RGO.

applying a constant current density of 100 mA cm2. During this process, sodium methoxide ions (Na+ OCH3) intercalate into the graphitic network producing -oxy and methoxy radicals (OH and OCH3). Basically, these radicals act as electrochemical scissors and etch the surface to form a black coloured solution. In the second step, a reducing agent (either glucose or hydrazine hydrate) is added to the resulting black coloured solution and this mixture is heated up to 70 1C along with constant stirring for about 8 hours. Afterwards the solution is centrifuged which results in the formation of a clear supernatant solution at the top and a black precipitate settled at the bottom. The supernatant solution is basically GQDs and the precipitate is identified as RGO. Fig. 1 depicts the schematic representation of the proposed strategy adopted in this work for the simultaneous preparation of GQDs and RGO. It is possible to tune the emission colour of these GQDs by controlling the duration of electrolysis and the nature of reducing agents. Particularly here, GQDs that emit blue, green and yellow colours on exposure to UV light have been prepared. These GQDs exhibit excellent photoluminescence (PL) behaviour and good water dispersibility. Several spectroscopic (UV-Vis, FTIR, NMR, Raman and XRD) and microscopic (FESEM, HRTEM) techniques are employed for their characterization and analysis. Further these GQDs are used for bio-imaging application and are found to be biocompatible and low cytotoxic in nature. Similarly, the precipitate RGO is demonstrated to be a potential electrode material for supercapacitors. Electrochemical techniques namely cyclic voltammetry (CV) and electrochemical impedance spectroscopy are used for the studies. Further characteristic parameters of the supercapacitor material are evaluated and analyzed using complex plane and complex capacitance analysis.

2. Experimental section 2.1.

Chemicals

Graphite rods were purchased from Sigma Aldrich, Bangalore, India, and used as such without any pretreatment. Hydrazine monohydrate, sodium methoxide and glucose were also obtained


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from Sigma Aldrich, Bangalore, India. Sodium chloride which was used as a supporting electrolyte in electrochemical studies was procured from Merck. Millipore water having a resistivity of 18.2 MO cm was used for the preparation of aqueous solutions used for the electrochemical investigation. All other chemicals were used as such without any further purification. 2.2.

Preparation of graphene quantum dots

A simple two-step method was employed for the preparation of GQDs. The first step involved electrolysis using a graphite rod as an anode and platinum (Pt) foil as a cathode with 0.1 M sodium methoxide in water as an electrolyte. The electrodes were placed parallel to each other with a fixed distance of separation between them. A constant current density of 100 mA cm2 was applied for a period of 3 hours. During electrolysis, the solution changed from colourless to a homogeneous black coloured solution. The resultant solution was further separated into two parts. About 1 ml of hydrazine monohydrate was added to one part and 0.1 M glucose was added to the other part. Here both hydrazine monohydrate and glucose act as reducing agents. After the addition, the corresponding solutions were heated at 70 1C along with continuous stirring for about 8 hours. Later on they were cooled down to room temperature and after 30 minutes it was observed that a black precipitate was settled at the bottom and a clear solution was separated out at the top as a supernatant. Then the supernatant solution was carefully decanted to separate the black precipitate and the clear solution. Afterwards the clear solution was centrifuged at 12 000 rpm for about 30 minutes to remove the remaining black precipitate. This procedure was repeated for 2–3 times until we obtain no residue at the bottom after centrifugation. Using this procedure, we collected the black precipitate and the clear solution separately that were used for the subsequent analysis and characterization. In addition, the resultant slight yellow coloured solution was further purified using dialysis with millipore water by employing a dialysis bag possessing a molecular weight of 4 kDa. Dialysis was carried out for about 14–15 hours. From the analysis, we found that the clear supernatant solution exhibits fluorescence and it was found to emit yellow colour in the case of hydrazine and green colour in the case of glucose being used as reducing agents upon exposure to UV light. The black precipitate was identified as RGO. In a separate experiment, the electrolyte was changed to 0.1 M sodium methoxide dissolved in ethanol and a similar procedure as described above was followed for performing electrolysis. After 8 hours of continuous stirring with heating, the solution was allowed to cool down to room temperature. Then a small amount of MgSO4 was added and the solution was stirred for 30 minutes. Later it was just left alone for about 24 hours in order to remove the excess salt and water. Afterwards the solution was decanted carefully and a clear solution was separated that was centrifuged at 12 000 rpm for 30 minutes in order to remove the black precipitate settled at the bottom. The top supernatant solution was further purified using silica gel column chromatography by employing a mixture of petroleum ether and diethyl ether as solvents. The clear supernatant

solution was observed to be fluorescent and it emitted a dark blue colour under illumination with UV light (365 nm) and the black precipitate was analyzed to be RGO. The fluorescent solutions were further analyzed using UV-Vis spectroscopy, PL measurements, NMR, etc., and the size and morphology of the resultant GQDs were evaluated using HRTEM analysis. Furthermore, FTIR, XRD, Raman spectroscopy and FESEM studies were carried out to evaluate and characterize the black precipitate settled at the bottom, namely RGO, in order to understand the structure and chemical properties. 2.3.

Bio-imaging analysis

In the present work, we have demonstrated the utilization of blue, green and yellow colour emitting GQDs for bio-imaging applications using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. The bacterium under study was procured from IMTECH (Institute of Microbial Technology, Sector 39-A), Chandigarh – 160036, India. Cell culture of S. aureus (MTCC 3160) was carried out in a nutrient broth of the following composition (g L1): peptone 5.0, yeast extract 2.0, beef extract 1.0 and NaCl 5.0. The bio-imaging experiments were carried out using overnight bacterial cultures. The cells were placed in neutral phosphate buffer (pH = 7.0) medium and treated in an aqueous suspension with varying concentrations of GQDs such as 0.1, 0.3 and 0.5 mg ml1. Initially dose–survival curves were recorded for all these GQDs and analyzed for the optimum amount of GQDs and time to be employed for bio-imaging and cytotoxic studies. Basically the GQDs obtained after the preparation were in solution form. In order to obtain these GQDs in powder form, these solutions were evaporated using a rotary evaporator. The resultant powders were removed carefully and appropriate amounts of these GQDs were employed for bio-imaging studies. Further toxicity level analysis was carried out after incubation at various time intervals such as 3, 6, 12 and 24 hours using MTT assay.25 After an incubation time of 3 hours, the medium was stripped by centrifugation at 10 000 rpm for 10 minutes, and the uptake of these individual GQDs by the bacterial cells was observed by using a Nikon Epi-fluorescence microscope (model E200 COOLPIX 5400 with imaging software NIS-Elements, Nikon, Tokyo, Japan). Bio-imaging applications using these GQDs of varying size and in turn of different emission colours namely blue, green and yellow were investigated and the results were analyzed. 2.4.

Supercapacitor studies using electrochemical techniques

Electrochemical techniques namely cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed for exploring RGO as a potential electrode material for supercapacitor applications. These studies were carried out using a three-electrode cell assembly with the help of potentiostat– galvanostat equipment. A Pt foil having a large surface area was used as a counter electrode along with a saturated calomel electrode (SCE) as a reference electrode and RGO modified glassy carbon (GC) electrode as a working electrode. The working


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electrode was prepared by mixing the active material namely RGO (B1 mg) with a few drops of diluted Nafion solution and made into a paste, which was coated over the GC electrode. Then the modified GC electrode was dried at B100 1C for about an hour and used as a working electrode. CV experiments were performed within the potential window of 0 V to +0.8 V vs. SCE using an aqueous solution of 0.1 M NaCl as an electrolyte. The effect of scan rate on the capacitive behaviour was also investigated by performing CV studies over a wide range of scan rates varying from 2 mV s1 to 500 mV s1. Similarly impedance measurements were carried out using the same NaCl aqueous solution as the electrolyte by applying a sinusoidal signal of 10 mV peak-to-peak amplitude in the frequency range of 100 kHz to 100 mHz. The impedance data were analyzed in terms of complex capacitance and complex power in order to determine the relaxation time constant (t0), which defines the speed at which the material can deliver the stored power, a characteristic parameter of supercapacitor electrode materials. 2.5.

Instrumentation

Electrochemical studies were carried out using AUTOLAB equipment procured from the Netherlands. The corresponding experiments and their analyses were carried out using General Purpose Electrochemical Software (GPES) provided by AUTOLAB. Absorbance and fluorescence spectra were recorded using UV-Vis Perkin Elmer Lambda 650 and Infinite M200MPC. TEM images were analyzed using the TECNAI G2 20 FEI model operated at 200 kW. TEM samples were prepared by the drop casting method on a copper grid. The FTIR spectrum was recorded using the Bruker Optics GmbH TENSOR 27 model operated with the 162 software Opus version 6.5 m. SEM studies were performed using Hitachi model S3000-H. XRD analysis was carried out using an XPERT-PRO multipurpose X-ray diffractometer procured from the Netherlands, using Cu Ka radiation with a wavelength of 1.540 Å along with 2y values ranging from 10 to 90 degrees. LASER Raman measurements were performed by using the Renishaw InVia Raman Microscope model obtained from UK, by employing a He–Ne laser possessing a wavelength of 633 nm and a power of 18 mW. NMR spectra were recorded using Avance 400 MHz, BRUKER, procured from Switzerland. Bio-imaging studies were carried out using a Nikon epifluorescence microscope (model E200 COOLPI 5400 with imaging software NIS-Elements, Nikon, Tokyo, Japan). Zeta potential values were measured by using the Beckman Coulter Delsat Nano C model purchased from USA.

that by using hydrazine monohydrate yellow colour emitting GQDs namely y-GQDs and in the case of glucose being used as a reducing agent green colour emitting GQDs denoted as g-GQDs and finally for the case of ethanol medium blue colour emitting GQDs represented as b-GQDs are obtained. Subsequently the chemical nature, structural aspects and morphological characteristics are analyzed using spectroscopic and microscopic techniques. 3.1. Optical property analysis of GQDs using UV-visible and photoluminescence spectroscopic studies It is important to understand the chemical nature and surface states of the resultant GQDs in order to analyze their absorbance and fluorescence properties. UV-visible and photoluminescence (PL) spectroscopic studies provide information about the optical properties of these GQDs. Fig. 2 shows the UV-visible spectra (A) of different GQDs prepared using hydrazine hydrate and glucose as a reducing agent in aqueous and ethanol medium respectively. In addition, the photographic images of the resultant supernatant solutions obtained in each case under visible (B) and upon UV light (C) illumination are also displayed. It is clearly evident from these photographs that upon exposure to UV light (365 nm), these GQDs display different emission colours namely blue (i) in the case of ethanol medium, yellow (ii) for hydrazine hydrate and green (iii) for glucose being used as reducing agents in the aqueous medium. Interestingly all these solutions under visible light show slightly yellow colour after the hydrothermal treatment. Consecutively these GQDs are termed as b-GQDs (blue), y-GQDs (yellow) and g-GQDs (green) respectively. The optical properties of these GQDs are analyzed by using UV-visible and PL spectroscopic studies. Fig. 2A shows the corresponding UV-Vis absorbance spectra of (a) b-GQDs, (b) y-GQDs and (c) g-GQDs. A typical absorption peak below 300 nm is observed in all the cases and is assigned to p–p* transition of aromatic

3. Results and discussion Basically, in this work, a simple and facile method involving electrochemical exfoliation of graphite sheets followed by chemical reduction is proposed for the simultaneous preparation of GQDs and RGO. It has been demonstrated that by changing the nature of the reducing agent and by changing the solvent used for electrolysis it is possible to tune the emission colour of these GQDs. Particularly here, we have shown

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Fig. 2 (A) UV-visible absorption spectra of different colour emitting GQDs namely b-GQDs (a), y-GQDs (b) and g-GQDs (c) respectively. Photographic images of these GQDs’ dispersion under normal visible light (B) and upon UV light illumination (C) at a fixed wavelength of 365 nm showing their respective emission colours, viz., blue obtained using ethanol medium (i), yellow produced using hydrazine monohydrate as a reducing agent (ii) and finally green obtained by employing glucose as a reducing agent (iii) respectively.


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sp2 domains and another shoulder peak observed at 335 nm indicates n–p* transition especially in the case of b-GQDs. Further the fluorescence properties of these GQDs are investigated using PL studies and the respective PL spectra are displayed in Fig. 3. The fluorescence spectra of the corresponding y-GQDs (a), g-GQDs (b) and b-GQDs (c) excited at a fixed wavelength of 360 nm are shown in Fig. 3A. It can be seen from the spectra that the maximum intensity for emission occurs at 550 nm for y-GQDs, 535 nm for g-GQDs and 525 nm for b-GQDs upon excitation at 360 nm. To further explore the optical properties of these GQDs, a detailed PL study had been carried out by recording the emission of all these GQDs at various excitation wavelengths ranging from 360 nm to 450 nm. Fig. 3B displays the various PL spectra recorded for y-GQDs obtained by using hydrazine hydrate as a reducing agent in aqueous medium, by exciting at different wavelengths ranging from 360 nm to 420 nm. It can be seen that y-GQDs show the PL emission maximum at 550 nm and it is independent of excitation wavelengths suggesting the homogeneous and uniform particle size distribution of GQDs. In this case, the maximum fluorescence emission intensity is obtained at an excitation wavelength of 360 nm. Moreover, hydrazine monohydrate is a strong reducing agent that is capable of reducing all the functional groups present on the surface leading to surface passivation with amine functionalities. Such

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passivated amine groups create a new surface state and result in a single transition mode energy level.26 Hence, the PL property of y-GQDs exhibits excitation wavelength independent emission behaviour. Similarly Fig. 3C and D display the respective PL emission spectra of g-GQDs and b-GQDs obtained at different excitation wavelengths ranging from 360 nm to 450 nm. Interestingly both g-GQDs and b-GQDs exhibit excitation wavelength dependent emission behaviour unlike many other fluorescent QDs. But this behaviour is similar to that of carbon dots which exhibit excitation dependent PL emission and this is one of the most regarded features of photoluminescent carbon dots. To be precise, the emission wavelength is red shifted in the case of g-GQDs and it is blue shifted in the case of b-GQDs. Particularly the PL emission peak shifted from 535 nm to 555 nm in the case of g-GQDs (Fig. 3C) when the excitation wavelength is varied from 360 nm to 450 nm. Similarly for b-GQDs (Fig. 3D), the PL emission peak is shifted from 525 nm to 470 nm for a similar excitation wavelength range. The maximum fluorescence emission is observed at lem = 550 nm at an excitation wavelength of lex = 450 nm for g-GQDs. Similarly for the case of b-GQDs the maximum fluorescence emission is observed at lem = 475 nm at an excitation wavelength of lex = 420 nm. This behaviour is quite uncommon for fluorescent materials and is mainly attributed to the different energy levels associated with

Fig. 3 (A) PL emission spectra recorded for (a) y-GQDs, (b) g-GQDs and (c) b-GQDs respectively obtained by using hydrazine monohydrate and glucose as reducing agents in aqueous medium and in ethanol medium at a fixed excitation wavelength of 360 nm. (B), (C) and (D) display various PL emission spectra obtained for y-GQDs, g-GQDs and b-GQDs respectively at different excitation wavelengths ranging from 360 nm to 450 nm.


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different surface states arising from the presence of various functional groups on the surface of these GQDs that are responsible for excitation wavelength dependent emission properties. Specifically the oxygen containing functional groups such as –COOH, –OH and C–O–C moieties are present on the surface of these GQDs depending upon the preparation method used. These functional groups have created various surface states and hence the fluorescence emission could take place from these altered energy levels leading to excitation dependent fluorescence emission behavior for these GQDs. In contrast, in the case of y-GQDs even if the excitation wavelength is changed from lower to higher, the corresponding PL spectra do not show any shift in the emission wavelength. This could be explained by the high uniformity in both size and surface states of sp2 clusters associated with GQDs. Furthermore b-GQDs are prepared by using ethanol as an electrolytic medium where the solvent also plays a major role in affecting PL behaviour.26,27 Furthermore the quantum yield values of all these GQDs are measured by recording the emission spectra over a range of excitation wavelength using a standard fluorescent probe. In the case of y-GQDs and g-GQDs where hydrazine monohydrate and glucose are being used as the reducing agent in aqueous medium quinine sulfate is employed as the standard probe, whereas for b-GQDs that are prepared in ethanol medium Rhodamine B and fluorescein are being used as the standard probe for PL quantum yield measurements. The experiments are repeated thrice and the average quantum yield values are reported here. These values are found to be 36 1% for y-GQDs, 2 0.4% for g-GQDs and 8.5 0.9% for b-GQDs (ESI,† S1). Similarly, the

surface charge on these GQDs is also analyzed using zeta potential measurements and the values are estimated to be 3.74 mV for y-GQDs, 12.72 mV for g-GQDs and 12.88 mV for b-GQDs respectively, indicating the presence of negative charges on the surface of all these GQDs. Further nuclear magnetic resonance (NMR) spectroscopic studies are performed to investigate the nature of the carbon environment within these GQDs (ESI,† S2). NMR results clearly show the formation of a single carbon peak at 170 ppm indicating the presence of –CQO groups in the case of y-GQDs prepared using hydrazine monohydrate as a reducing agent in aqueous medium. Similarly the NMR spectrum of g-GQDs obtained by employing glucose as a reducing agent in water displays several peaks at 20, 23, 68, 161, 181 and 183 ppm suggesting the presence of various functional groups such as secondary carbons, –C–OH, –COOMe, –CQC– and –CQO groups on the surface of g-GQDs. These results also agree very well with the zeta potential measurements indicating that the negative charges arise predominantly from these functional groups present on the surface of GQDs. 3.2. Structural and morphological characterization using microscopic studies Structural morphology and particle size of the resultant GQDs and the precipitate are further analyzed using scanning electron microscopic (SEM) and high-resolution transmission electron microscopic (HRTEM) studies. Fig. 4 shows both TEM and HRTEM images of y-GQDs (A), g-GQDS (B) and b-GQDs (C) respectively. It can be clearly seen from these images that all these GQDs are spherical in nature and their respective HRTEM

Fig. 4 TEM (A) and HRTEM (B and C) images of y-GQDs (A) and g-GQDs (B) obtained using hydrazine monohydrate and glucose as reducing agents in aqueous medium along with b-GQDs (C) prepared in ethanol medium respectively. These images clearly indicate the formation of lattice fringe structures (insets) and their corresponding particle size distribution graphs are shown in (D), (E) and (F) respectively.

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images clearly reveal the formation of lattice fringe patterns, as shown in the corresponding insets indicating the crystalline and ordered nature of these GQDs. Lattice parameters in terms of in-plane lattice spacing of all these GQDs are determined to be 0.20–0.21 nm from HRTEM images which reveal clearly the higher crystalline nature of GQDs arising from the (100) diffraction planes of sp2 graphitic carbon and consistent with the reports of graphene.28–31 Among these y-GQDs show the formation of more or less homogeneous uniform particles and in contrast b-GQDs display the formation of particles of largely deviating sizes. The corresponding particle size distribution graphs are shown in Fig. 4D–F. The average particle size is estimated to be 3.5 0.5 nm, 5.8 0.7 nm and 7.3 0.4 nm in the case of y-GQDs, g-GQDs and b-GQDs respectively. Further the structural morphology of the black precipitate obtained during the preparation of these GQDs is analyzed using SEM and TEM analyses and the corresponding images are shown in Fig. 5. It can be clearly seen from these images that the black precipitate obtained in all the cases shows the formation of a layered structure with not a perfectly flat shape rather a shape with crumbled sheets, randomly aggregated, and wrapped. Moreover these sheets are closely packed with each other and display clear wrinkles and a wavy structure suggesting the typical characteristics of RGO that is confirmed later with spectroscopic characterization. Fig. 5D depicts the TEM image of such a sheet obtained in the case of y-GQDs where hydrazine monohydrate is being used as a reducing agent in aqueous medium which shows the formation of a very thin and transparent layer structure. These studies reveal that RGO

Fig. 5 SEM images of the black precipitates identified as RGO, obtained during the preparation of y-GQDs (A), g-GQDs (B) and b-GQDs (C) respectively showing the formation of ultra-thin layers with twisted paper like structures. (D) Shows the TEM image of RGO obtained for the case of y-GQDs prepared by using hydrazine monohydrate as a reducing agent in aqueous medium displaying a very thin and transparent layer like structure.

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obtained in all the cases possesses a few layered sheets with flake-like morphology.32–34 3.3. Investigation of structural characteristics of RGO using spectroscopic and diffraction studies The black precipitate was furthermore characterized and analyzed using Raman spectroscopy and X-ray diffraction (XRD) studies. Raman spectroscopy is a powerful tool to investigate the structure of carbon based materials in order to identify the order and disorder nature of graphitic structure especially for graphene and its derivatives. Fig. 6A shows the Raman spectra of the black precipitate obtained during the preparation of y-GQDs (b) and g-GQDs (c) using hydrazine monohydrate and glucose as reducing agents in aqueous medium respectively. For comparison the Raman spectrum of pristine graphite (a) is also shown in the figure. As expected, the pristine graphite displays a prominent G peak at 1581 cm1 corresponding to the in-plane vibrations of the sp2 bonded carbon atom indicating the predominant graphitic nature of the material. In contrast the black precipitate obtained in both the cases clearly displays two distinguishable peaks ascribed to the famous D and G bands of the carbon materials. The D band is formed due to the defects present within the graphitic structure arising out of sp3 bonds caused by oxidation. Moreover this particular band can also result from grain boundaries, edge defects, vacancies and amorphous carbon species in the disordered structure of carbon. Similarly the G band originates from the phonon scattering associated with the E2g vibration mode of the sp2 bonded carbons (in-plane CQC) in the graphitic structure.35–39 Formation of these two bands is indicative of typical characteristics of RGO. In the case of y-GQDs, D and G bands appeared at 1350 cm1 and 1578 cm1 and for the case of g-GQDs these bands are formed at 1351 cm1 and 1583 cm1 respectively. These wavenumber values are slightly different from that of pristine graphene (ESI,† Fig. S3) due to the presence of various functional groups resulting in more structural defects. A large change in the intensity and the large band width (B150 cm1 for the D band and B90 cm1 for the G band) observed for these materials are indicative of significant structural disorder. Further, it is well assessed using the ID/IG ratio which is a measure of quantity of defects within the graphene plane. In our case, ID/IG ratio values of 1.19 and 0.95 were determined for RGO obtained during the preparation of y-GQDs and g-GQDs. The increase in the ratio is attributed to a larger amount of new graphitic domains created during the reduction process arising out of sp3 hybridized carbon atoms at the edge of the exfoliation and it results in a low degree of defects within the graphene plane.35–39 Nevertheless these results obtained from Raman spectroscopic studies vividly prove that RGO possesses a significantly disordered structure. Interestingly, the Raman spectrum of graphene based materials also shows a 2D band described by the double resonance model that is very sensitive and a trade mark of stacking of graphene sheets.40,41 For single-layer graphene sheets, this particular band is expected to appear generally centered at 2679 cm1 whereas for multilayer graphene sheets including a few layered graphene


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Fig. 6 (A) Raman spectra of pristine graphite (a) and RGO obtained in the case of preparation of y-GQDs (b) and g-GQDs (c) respectively. (B) XRD pattern of RGO formed during the preparation of y-GQDs (a) and g-GQDs (b). Here y-GQDs and g-GQDs are prepared in aqueous medium by using hydrazine monohydrate and glucose as reducing agents. For comparison the XRD spectrum of pristine graphite is also shown in the inset.

sheets this band is shifted to higher wavenumbers and displays a wider peak width as observed for pristine graphene (ESI,† Fig. S3).42 In our case, the 2D band appeared at 2706 cm1 and 2712 cm1 for RGO obtained for y-GQDs and g-GQDs (Fig. 6A) respectively along with a low intensity shoulder at higher wavenumbers. The appearance of 2D bands at higher wavenumbers in our case suggested that RGO obtained here possesses multilayered graphene sheets where the sheets were aggregated and closely packed with each other. These results correlate very well with our microscopic observations (Fig. 5). Furthermore, the crystalline structure and morphology of the resultant RGO were analyzed using XRD studies and typical XRD patterns recorded are shown in Fig. 6B. For comparison the XRD spectrum of pristine graphite is also shown as an inset and this exhibits a sharp peak centered at a 2y value of 261 corresponding to the (002) plane and the interlayer spacing is determined to be 0.34 nm.43,44 However, RGO obtained for the cases of y-GQDs (a) and g-GQDs (b) displays a sharp and weakly broad peak with less intensity at a 2y value of around 26.51 showing the formation of similar (002) planes and the interlayer spacing is estimated to be 0.23 nm–0.25 nm. The reduction in the d-spacing of RGO when compared to that of pristine graphite occurs due to the reduction of various functional groups such as carboxyl at the periphery and epoxide and hydroxide groups between the planes during the exfoliation followed by the chemical reduction process.45 Further FTIR spectroscopic studies confirm the presence of such functional groups in the case of RGO obtained during the preparation of y-GQDs and g-GQDs by using hydrazine monohydrate and glucose as reducing agents respectively (ESI,† Fig. S4). Similarly the absence of a characteristic peak at a 2y value of 11.41 and formation of the (002) diffraction line indicate the complete conversion of graphite, the starting material, into RGO. It is also important to point out here that this particular peak appeared at the same position for RGO obtained using both the reducing agents indicating that a complete and comparable degree of reduction is achieved using these reducing agents.

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3.4. Electrochemical investigation of RGO as an electrode material for supercapacitor applications Electrochemical techniques namely cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are used to investigate the electrochemical properties and characteristics of the resultant RGO. It is worth mentioning here that although RGO obtained in all three cases exhibits a similar kind of behaviour in electrochemical studies, here we present results obtained for RGO prepared during the synthesis of y-GQDs by using hydrazine monohydrate as a reducing agent in aqueous medium alone as an example. Formation of a perfect rectangular shaped voltammogram with large current separation between the forward and reverse scans along with symmetry in both anodic and cathodic directions is an indicator of an ideal capacitor. Fig. 7A shows the typical comparison of cyclic voltammograms obtained for a bare glassy carbon (GC) electrode (a) and RGO modified electrode (b) at a fixed scan rate of 50 mV s1 in 0.1 M NaCl aqueous solution with the potential scanned in the double layer region of 0 V to +0.8 V vs. SCE. It can be noticed that these CVs do not show any visible peak formation suggesting a pure capacitive behaviour and the RGO modified electrode displays a significantly higher current density (at least by 400 times) when compared to the bare GC electrode indicating the enhanced capacitive property. Further the effect of scan rate on the capacitive behaviour of RGO is investigated by recording CVs over a wide range of scan rates varying from 10 mV s1 to 100 mV s1 in 0.1 M NaCl aqueous solution with the potential scanned in the double layer region of 0 V to +0.8 V vs. SCE and the corresponding CVs are shown in Fig. 7B. It can be seen that these CVs show no visible peak formation but show a large current separation between forward and reverse scans. In addition these CVs also possess almost a rectangular shape especially at lower scan rates and display symmetry across both the anodic and cathodic directions indicating a pure electrochemical double layer capacitive behaviour. The capacitance values are calculated by measuring the ratio of the magnitude of current separation and the scan rate. The fact that all the CVs display


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Fig. 7 (A) Cyclic voltammograms obtained at a fixed scan rate of 50 mV s1 for (a) bare glassy carbon (GC) and (b) RGO modified electrodes. (B) Comparative CVs recorded at various scan rates ranging from (a) 10 mV s1, (b) 20 mV s1, (c) 30 mV s1, (d) 40 mV s1, (e) 50 mV s1, (f) 60 mV s1, (g) 70 mV s1, (h) 80 mV s1 and (i) 100 mV s1 respectively using the RGO modified GC electrode. (C) Nyquist plots recorded at different potentials starting from 0 V to 0.8 V with a 200 mV interval using electrochemically exfoliated RGO as a supercapacitor electrode material. Inset: Nyquist plot measured at 0.4 V for a bare GC electrode is also shown for comparison. Electrolyte used: aqueous solution of 0.1 M NaCl.

good rectangular features even at higher scan rates with higher current density values indicates a good electrochemical activity arising out of the higher electrochemical active surface area and high power density values for RGO employed as an electrode material. Table 1 shows the double layer capacitance and specific capacitance values determined for RGO at different potential scan rates. It can be noted that the capacitance values vary with the scan rate. A maximum capacitance value of 419.45 mF cm2 is obtained at a scan rate of 2 mV s1 which corresponds to a specific capacitance of 75.29 F g1. It is interesting to note that specific capacitance varies with the scan rate and it exponentially increases with decreasing scan rate (ESI,† Fig. S5). Furthermore the capacitance value determined in the present work is comparable with the specific capacitance value obtained for carbon materials. Moreover, for comparison the effect of scan rate on the capacitive behaviour of bare GC is also investigated and there is no significant change in the capacitance value with respect to increasing scan rate (ESI,† Fig. S6). Further EIS is used to investigate the electrochemical characteristics of the RGO coated electrode–electrolyte interface and analyze the capacitive behaviour. The corresponding Nyquist plots are shown in Fig. 7C and the dc bias potential is varied

Table 1 Double layer capacitance and specific capacitance values obtained from CV studies at different scan rates using RGO as an electrode material

Scan rate (mV s1)

Double layer capacitance (mF cm2)

Specific capacitance (F g1)

2 5 10 20 30 40 50 60 70 80 90 100 150 200 300 400

419.45 399.02 408.13 92.64 93.26 108.29 91.21 76.01 75.32 73.3 70.14 68.41 59.77 51.18 37.34 27.2

75.29 70.83 72.44 16.44 16.45 19.22 16.19 13.49 13.28 13.03 12.45 12.15 10.61 9.08 6.63 4.83

between 0 V and +0.8 V. For comparison a similar plot obtained using a bare GC electrode at 0.4 V is also shown in the inset. It can be seen from these plots that the impedance response is almost parallel to the imaginary axis at all the potentials used


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for the study indicating a typical capacitive nature of the RGO electrode material. In the high frequency region the RGO electrode material displays a low resistance value and in the low frequency regime RGO shows a complete capacitive behaviour suggesting the potential applicability as a supercapacitor electrode material. The impedance data are best fitted to an appropriate equivalent circuit model proposed by Conway for supercapacitor electrode materials namely R(CR) where C dominates almost over the entire range of frequency used for the study. The double layer capacitance value is determined to be 417 mF cm2. Moreover, a phase angle value of 861891 is determined from the Bode plot suggesting that the RGO material is suitable for the fabrication of low-leakage capacitors. Among the various potentials investigated, the impedance response obtained at +0.4 V shows a perfect capacitive behaviour whereas the impedance response obtained for other potentials displays a capacitive behaviour with minor additional contribution arising from the resistor component. 3.5. Determination of the relaxation time constant using complex capacitance and complex power analysis The utility of RGO as a supercapacitor electrode material is further validated by analyzing the impedance data using the complex capacitance and complex power method.46,47 Basically several methods and models have been proposed to describe the frequency dependence of supercapacitor materials and their behaviour by employing the transmission line model (TLM) and models based on the size and shape of the pores namely the pore size distribution model, etc. In this case, a simple method of analysis using complex capacitance C(o) and complex power S(o) is used (ESI,† S4). The complex capacitance consists of a real part, C 0 (o), and an imaginary part, C00 (o). Similarly the complex power, S(o), contains the real part active power, P(o), and the imaginary part reactive power, Q(o). These parameters are expressed by the following equations: C(o) = C 0 (o) jC00 (o)

(1)

S(o) = P(o) + jQ(o)

(2)

Interestingly, the relaxation time constant [to = 1/(2pfo)], which is also known as dielectric relaxation time of the supercapacitor, is the figure of merit of the supercapacitor electrode material. This can be determined from this analysis using the imaginary complex capacitance C00 (o) vs. frequency plot. This relaxation time constant is essentially determined at the resonant frequency corresponding to the phase angle of 451 and it represents the transition for the electrochemical capacitor between a pure resistive (for f 4 1/to) and a pure capacitive (for f o 1/to) behaviour.48–52 Fig. 8A shows the plots of the imaginary component of the complex capacitance C00 (o) vs. frequency (in logarithmic scale) at two different potentials namely 0 V and 0.4 V. Similar plots determined for other potential values are shown in the ESI† (Fig. S7). This plot clearly shows a characteristic peak formation at the frequency, fo, measured to be 0.1 Hz for the RGO electrode. This fo value does not change with respect to change in potential used for recording the impedance data, suggesting that the frequency determined is characteristic and real representation of RGO employed as an electrode material for supercapacitors. Similarly Fig. 8B shows the plots of the normalized real part |P|/|S| and the imaginary part |Q|/|S| of the complex power vs. frequency (in logarithmic scale) obtained for the RGO electrode at two different potentials namely 0 V and 0.4 V. The power dissipated into the system can be analyzed from this plot using the normalized active power and the reactive power. Generally, at high frequency all the power is dissipated into the system (P = 100%) where the supercapacitor acts as a pure resistor whereas no power is dissipated in the low frequency regime in which it acts as a pure capacitor. In fact, this can be noted from Fig. 8B wherein the values of the normalized active power, |P|/|S|, and the reactive power, |Q|/|S|, show an opposite trend with frequency. The value of |P|/|S| decreases with decreasing frequency while the value of |Q|/|S| increases with decreasing frequency. The maximum value of |Q|/|S| occurs at low frequency where the supercapacitor acts like a pure capacitor.48–52 Ideally the crossing of two plots occurs at a frequency known as the resonant frequency, fo, from which the relaxation time constant, to,

Fig. 8 Plots of the imaginary part of the complex capacitance vs. frequency (in logarithmic scale) [A] and the plots of normalized active power and reactive power vs. frequency (in logarithmic scale) [B] obtained at two different potentials namely 0 V and 0.4 V using complex power analysis for RGO being used as an electrode material for supercapacitor applications.

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can be determined explicitly as mentioned above. From Fig. 8B, the to value of 1.034 s is determined for the RGO electrode which implies faster power dissipation into the system. This value is much better than the previous values reported for carbon based materials and others whose to values are in the range of 10 to 100 s.48–52 A low to value is preferred for devices having a fast charging–discharging process and the value obtained in the present case where the RGO electrode is used for supercapacitors suggests that this material is able to deliver its stored energy at a much faster time scale with high power. 3.6. Exploration of fluorescent GQDs for bio-imaging application In order to explore the applications of versatile fluorescence associated with different GQDs prepared in the present work, bio-imaging application is demonstrated here. Owing to their stable photoluminescence, low cytotoxicity, very small size, multi-colour emission property and excellent biocompatibility, generally fluorescent nanoparticles and QDs are employed for bio-imaging applications, which are critical in the field of biomedics.53–56 Particularly in this work GQDs possessing different emission colours such as y-GQDs and g-GQDs obtained by using hydrazine monohydrate and glucose as reducing agents in aqueous medium and b-GQDs in ethanol medium respectively are prepared and further explored for bio-imaging and cytotoxic studies. In this case two different bacteria namely S. aureus and E. coli are used for the demonstration of bio-imaging using all three GQDs. These bacteria are incubated with bright green, blue and yellow coloured emission GQDs (1 mg/10 ml) for 3 hours and the experiments are repeated thrice to check the reproducibility and validity of our observations. The representative epi-fluorescence microscopic images recorded at an excitation wavelength of 365 nm are shown in Fig. 9A. These images clearly reveal the successful translocation of all the GQDs through cell membranes and hence all three kinds of GQDs are able to penetrate into the cells. Moreover they can also be adsorbed onto the cell wall of the outer membrane. Previously it has been reported that GQDs could possibly penetrate into the human cell line cultures through electrostatic interaction force leading to clear visualization of cell morphology.53–56 In our work, comparatively S. aureus cell culture shows a higher fluorescence intensity with clear bio-images of the cells for all the three GQDs when compared to E. coli cells. This is mainly attributed to the effective electrostatic interaction between the GQDs and the bacterial cell wall membrane. Similarly among the different GQDs studied in this work, y-GQDs exhibit high intense fluorescence bio-images in comparison to the other two due to the higher quantum yield value and the uniform size of the dots observed for y-GQDs. The order of increasing fluorescence is identified as y-GQDs 4 b-GQDs 4 g-GQDs. Besides, these GQDs (blue, green and yellow) retained their respective fluorescence properties while being internalized within the cells. In addition, these GQDs are hydrophilic in nature with active surface functional groups like hydroxyl and carboxylic groups as confirmed from FTIR spectroscopic studies discussed earlier and these functional groups aid the effective internalization into bacteria.

Fig. 9 (A) Epifluorescence microscopic images of S. aureus and E. coli bacterial cell cultures obtained by incubation for 3 hours with g-GQDs, b-GQDs and y-GQDs respectively at an excitation wavelength of 365 nm. (B) Plots obtained for cytotoxic studies using g-GQDs, b-GQDs and y-GQDs respectively by investigating bacterial viability as a function of added GQD concentrations by employing S. aureus bacterial culture for a time duration of 3 hours along with error bars obtained for three repeated independent measurements.

Moreover these treated cells become more visible with intense and appreciable fluorescence with increasing time and retain this fluorescence even after exposure for several hours. These observations lucidly indicate the high photostability of all these GQDs. Furthermore SEM images obtained for both the control (without incubation of GQDs) and GQDs incubated S. aureus bacteria clearly indicate that GQDs are distributed uniformly and homogeneously over the entire surface of the bacteria (ESI,† Fig. S8). Finally cytotoxic studies are also carried out in order to validate the potential utility of these GQDs for bio-imaging applications by repeating the experiments thrice and the average values are calculated. The corresponding results obtained using S. aureus bacteria are shown in Fig. 9B. Bacterial viability or toxicity studies are performed using methyl thiazolyl tetrazolium (MTT) assay by following its reduction.57,58 The viability of bacteria in the case of untreated GQDs is assumed to be 100% and displayed as a control in Fig. 9B. Further the amount of GQDs incubated with S. aureus bacteria is varied as 1 mg/10 ml,


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3 mg/10 ml and 5 mg/10 ml respectively for all three GQDs and the cytotoxic effect is noted down. Initially dose–survival curves are recorded for all these GQDs (ESI,† Fig. S9). These curves describe the relationship between the amount (dose) of GQDs and the fraction of cells that survive that dose. It is mainly used to assess the biological effectiveness of the dosage. In general these curves display two distinct features namely the initial shoulder shape and the terminal linear straight line portion. Interestingly the initial shoulder regime is used for bio-imaging applications where the chances of survival are usually higher and the terminal portion follows an exponential relationship indicating the same dose increment resulting in equal reduction of surviving numbers of cells. If the percentage of survival of cells is higher even after the higher loading and for longer exposure time, then those materials could have potential anti-bacterial activity. In our case, both y-GQDs and b-GQDs show the initial shoulder region and a linear relationship at higher dose and in contrast g-GQDs display a linear relationship throughout (ESI,† Fig. S9). It is interesting to note that upon increasing the amount of GQDs more bacterial cell death occurs for all three GQDs.57,58 The order of bacterial survival is as follows: 1 mg 4 3 mg 4 5 mg. Upon the addition of all the GQDs up to 1 mg in 10 ml for a period of 3 hours, 97%, 96% and 98% bacterial survival is observed for the case of b-GQDs, g-GQDs and y-GQDs respectively. These results clearly indicate the biocompatibility and significant cytotoxic effects of these GQDs at lower concentrations. Further on increasing the concentrations of these GQDs to 3 mg and 5 mg in 10 ml, even more bacterial cell death is observed. Specifically the chance of survival becomes 85% for 3 mg/10 ml and below 70% for 5 mg/10 ml. On average 83% bacterial cells are still alive and 17% bacterial growth inhibition is observed. Among the three GQDs, g-GQDs show a poor cytotoxicity and the order of increasing bacterial viability is g-GQDs o b-GQDs o y-GQDs as observed from the dose–survival curves. Similar results and trends are also observed upon increasing the exposure time from 6 to 12 and 24 hours. Among the various GQDs analyzed in this work, y-GQDs show more than 82% of cell survival even after exposure for 24 hours, suggesting an impressive biocompatibility of these GQDs and g-GQDs show a poor survival of 63% after 24 hours of exposure. It is important to analyze the toxicity levels and the optimum concentration of GQDs to be used for bio-imaging applications. Our cytotoxic studies and dose–survival curve analyses clearly show that higher the amount of loading, higher the bacterial cell death, and longer the incubation time, lesser the chance of bacterial survival for all the three GQDs. It can also be seen from Fig. 9B that after 3 hours of incubation in 1 mg/10 ml of GQDs, there is almost 98% chance of bacterial survival. Hence, 1 mg/10 ml and 3 hours of incubation time are identified as optimum for bioimaging applications, which is also supported by dose–survival curve analysis. In summary though all the GQDs namely g-GQDs, b-GQDs and y-GQDs possess excellent biocompatibility along with low cytotoxic effects, only y-GQDs show significant and better results when compared to other two GQDs suggesting the possible use of this in bio-imaging applications.

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4. Conclusions In this work, a simple, facile and simultaneous preparation of graphene quantum dots (GQDs) along with reduced graphene oxide (RGO) is demonstrated. By varying the nature of reducing agents from hydrazine monohydrate to glucose in aqueous medium the emission colours of these graphene quantum dots are tuned between yellow and green. Further when the medium of electrolysis is changed from water to ethanol, an intense blue colour emission is observed for the resultant graphene quantum dots. Interestingly, the resultant black precipitate is identified as reduced graphene oxide. Spectroscopic, microscopic and electrochemical techniques are employed for structural, morphological and functional characterization. High resolution transmission electron microscopic images clearly reveal that these GQDs exhibit beautiful lattice fringe patterns along with homogeneous and uniform particle size distribution. Further, these fluorescent GQDs are explored for bio-imaging applications where all the GQDs investigated in this work display bright, intense fluorescence emission along with good bio-compatibility as well as low cytotoxicity. Among them y-GQDs show a significantly higher rate of cell survival when compared to other two GQDs indicating the potential utility for bio-imaging applications and toxicity studies. Electrochemical studies prove that reduced graphene oxide could potentially be used as an electrode material for supercapacitor applications. In this case, the specific capacitance value of this material is determined to be 75.29 F g1. Moreover, complex capacitance and complex power analysis is employed for the determination of relaxation time constant (to) and it is calculated to be 1.034 s. These observations suggest that reduced graphene oxide is an ideal candidate for supercapacitors that could deliver its stored energy at a much faster time scale with high power.

Acknowledgements The authors would like to thank Central Instrumentation Facility (CIF) of CSIR – CECRI, Karaikudi, for providing the necessary equipment for characterization and analysis. AM and PD acknowledge the Council of Scientific and Industrial Research (CSIR), India, for providing Senior Research Fellowship (SRF) for pursuing their PhD degree program.

References 1 K. S. Novoselov, K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva and A. A. Firsov, Science, 2004, 306, 666–669. 2 K. Zhang, L. L. Zhang, X. S. Zhao and J. Wu, Chem. Mater., 2010, 22, 1392–1401. 3 C. N. R. Rao, A. K. Sood, K. S. Subrahmanyam and A. Govindaraj, Angew. Chem., Int. Ed., 2009, 48, 7752–7777. 4 Y. Xu, H. Bai, G. Lu, C. Li and G. Shi, J. Am. Chem. Soc., 2008, 130, 5856–5857. 5 (a) X. Li, X. Wang, L. Zhang, S. Lee and H. Dai, Science, 2008, 319, 1229–1232; (b) S. Fujii and T. Enoki, J. Am. Chem. Soc., 2010, 132, 10034–10041.


View Article Online


NJC

6 (a) L. A. Ponomarenko, F. Schedin, M. I. Katsnelson, R. Yang, E. W. Hill, K. S. Novoselov and A. K. Geim, Science, 2008, 320, 356–358; (b) Y. W. Son, M. L. Cohen and S. G. Louie, Nature, 2006, 444, 347–349. 7 D. Pan, L. Guo, J. Zhang, C. Xi, Q. Xue, H. Huang, J. Li, Z. Zhang, W. Yu, Z. Chen, Z. Li and M. Wu, J. Mater. Chem., 2012, 22, 3314–3318. 8 S. Zhu, J. Zhang, X. Liu, B. Li, X. Wang, S. Tang, Q. Meng, Y. Li, C. Shi, R. Hu and B. Yang, RSC Adv., 2012, 2, 2717–2720. 9 K.-J. Jeon, Z. Lee, E. Pollak, L. Moreschini, A. Bostwick, C.-M. Park, R. Mendelsberg, V. Radmilovic, R. Kostecki, T. J. Richardson and E. Rotenberg, ACS Nano, 2011, 5, 1042–1046. 10 J. Shen, Y. Zhu, C. Chen, X. Yang and C. Li, Chem. Commun., 2011, 47, 2580–2582. 11 S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, B. Li, L. Tian, F. Liu, R. Hu, H. Gao, H. Wei, H. Zhang, H. Sun and B. Yang, Chem. Commun., 2011, 47, 6858–6860. 12 Y. Xu, Z. Liu, X. Zhang, Y. Wang, J. Tian, Y. Huang, Y. Ma, X. Zhang and Y. Chen, Adv. Mater., 2009, 21, 1275–1279. 13 C.-H. Lu, H.-H. Yang, C.-L. Zhu, X. Chen and G.-N. Chen, Angew. Chem., Int. Ed., 2009, 48, 4785–4787. 14 V. Gupta, N. Chaudhary, R. Srivastava, G. D. Sharma, R. Bhardwaj and S. Chand, J. Am. Chem. Soc., 2011, 133, 9960–9963. 15 B. Seger and P. V. Kamat, J. Phys. Chem. C, 2009, 113, 7990–7995. 16 D. Yanga, A. Velamakannia, G. Bozoklub, S. Parka, M. Stollera, D. Richard, S. Stankovichc, I. Junga, A. Daniel, A. Ventrice and R. S. Rouff, Carbon, 2009, 47, 145–152. 17 L. M. Viculis, J. J. Mack, O. M. Mayer, H. T. Hahn and R. B. Kaner, J. Mater. Chem., 2005, 15, 974–978. 18 M. Zhang, L. Bai, W. Shang, W. Xie, H. Ma, Y. Fu, D. Fang, H. Sun, L. Fan, M. Han, C. Liu and S. Yang, J. Mater. Chem., 2012, 22, 7461–7467. 19 Y. Li, Y. Hu, Y. Zhao, G. Shi, L. Deng, Y. Hou and L. Qu, Adv. Mater., 2011, 23, 776–780. 20 K. Parvez, Z.-S. Wu, R. Li, X. Liu, R. Graf, X. Feng and ¨llen, J. Am. Chem. Soc., 2014, 136, 6083–6091. K. Mu 21 S. Stankovich, D. Dikin, R. D. Piner, K. Kohlhaas, A. Kleinhammes, Y. Jia, Y. Wu, S. T. Nguyen and R. S. Ruoff, Carbon, 2007, 45, 1558–1565. 22 D. Pan, J. Zhang, Z. Li and M. Wu, Adv. Mater., 2010, 22, 734–738. 23 Y. Li, Y. Hu, Y. Zhao, G. Shi, L. Deng, Y. Hou and L. Qu, Adv. Mater., 2011, 23, 776–780. 24 J. H. Shen, Y. H. Zhu, C. Chen, X. L. Yang and C. Z. Li, Chem. Commun., 2011, 47, 2580–2582. 25 C. Sun, G. Pratx, C. M. Carpenter, H. Liu, Z. Cheng, S. S. Gambhir and L. Xing, Adv. Mater., 2011, 23, H195–H199. 26 X. Li, S. Zhang, S. a. Kulinich, Y. Liu and H. Zeng, Sci. Rep., 2014, 4, 4976. 27 S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, B. Li, L. Tian, F. Liu, R. Hu, H. Gao, H. Wei, H. Zhang, H. Sun and B. Yang, Chem. Commun., 2011, 47, 6858–6860.

Paper

28 Q. Li, S. Zhang, L. Dai and L. Li, J. Am. Chem. Soc., 2012, 134, 18932–18935. 29 S. H. Jin, D. H. Kim, G. H. Jun, S. H. Hong and S. Jeon, ACS Nano, 2013, 7, 1239–1245. 30 S. Kim, D. Hee Shin, C. Oh Kim, S. Seok Kang, S. Sin Joo and S. H. Choi, Appl. Phys. Lett., 2013, 102, 053108. 31 Y. Li, Y. Zhao, H. Cheng, Y. Hu, G. Shi, L. Dai and L. Qu, J. Am. Chem. Soc., 2012, 134, 15–18. 32 A. J. Cooper, N. R. Wilson, I. Kinloch and R. W. Dryfe, Carbon, 2014, 66, 340–350. 33 V. Loryuenyong, K. Totepvimarn, P. Eimburanapravat, W. Boonchompoo and A. Buasri, Adv. Mater. Sci. Eng., 2013, 1–5. ´lvarez, C. Blanco, M. D. Gutie ´rrez, P. Ares, 34 C. Botas, P. A ńdez, R. Zamani, J. Arbiol, J. R. Morante and R. Mene RSC Adv., 2012, 2, 9643–9650. 35 P. Cui, J. Lee, E. Hwang and H. Lee, Chem. Commun., 2011, 47, 12370–12372. 36 K. N. Kudin, B. Ozbas, H. C. Schniepp, R. K. Prud’Homme, I. Aksay and R. Car, Nano Lett., 2008, 8, 36–41. 37 J. Peng, W. Gao, B. K. Gupta, Z. Liu, R. Romero-Aburto, L. Ge, L. Song, L. B. Alemany, X. Zhan, G. Gao, S. A. Vithayathil, B. A. Kaipparettu, A. Marti, T. Hayashi, J.-J. Zhu and P. M. Ajayan, Nano Lett., 2012, 12, 844–849. 38 K. Parvez, R. Li, S. R. Puniredd, Y. Hernandez, F. Hinkel, ¨llen, ACS Nano, 2013, 7, S. Wang, X. Feng and K. Mu 3598–35606. 39 M. Zhou, J. Tang, Q. Cheng, G. Xu, P. Cui and L.-C. Qin, Chem. Phys. Lett., 2013, 572, 61–65. 40 A. C. Ferrari, J. C. Meyer, V. Scardaci, C. Casiraghi, M. Lazzeri, F. Mauri, S. Piscanec, D. Jiang, K. S. Novoselov, S. Roth and A. K. Geim, Phys. Rev. Lett., 2006, 97, 187401. 41 C. Thomsen and S. Reich, Phys. Rev. Lett., 2000, 85, 5214–5217. 42 D. Graf, F. Molitor, K. Ensslin, C. Stampfer, A. Jungen, C. Hierold and L. Wirtz, Nano Lett., 2007, 7, 238–242. 43 K. Chen and D. Xue, J. Colloid Interface Sci., 2014, 436, 41–46. 44 K. S. Rao, J. Senthilnathan, Y.-F. Liu and M. Yoshimura, Sci. Rep., 2014, 4, 4237–4242. 45 S. Stankovich, D. Dikin, R. Piner, K. Kohlhaas, A. Kleinhammes, Y. Jia, Y. Wu, S. Nguyen and R. Ruoff, Carbon, 2007, 45, 1558–1565. 46 P. L. Taberna, P. Simon and J. F. Fauvarque, J. Electrochem. Soc., 2003, 150, A292–A300. 47 E. Lust, A. Janes and M. Arulepp, J. Electroanal. Chem., 2004, 562, 33–42. 48 P. Khanra, T. Kuila, S. H. Bae, N. H. Kim and J. H. Lee, J. Mater. Chem., 2012, 22, 24403–24410. 49 V. Ganesh, S. Pitchumani and V. Lakshminarayanan, J. Power Sources, 2006, 158, 1523–1532. 50 K. S. Cole and R. H. Cole, J. Chem. Phys., 1941, 9, 341–352. 51 B. E. Conway, J. Electrochem. Soc., 1991, 138, 1539–1548. 52 V. Ganesh, S. Pitchumani and V. Lakshminarayanan, Electrochem. Solid-State Lett., 2005, 8, A308–A312.


New J. Chem., 2016, 40, 9111--9124 | 9123

View Article Online


Paper

NJC

53 L. Li, G. Wu, G. Yang and J. Peng, Nanoscale, 2013, 5, 4015–4039. 54 Q. Xue, H. Huang, L. Wang, Z. Chen and M. Wu, Nanoscale, 2013, 5, 12098–12103. 55 Z. Zhang, L. Xing, Y. He, N. Zhang, Y. Lu, Z. Zhang and J. Zhang, J. Nanosci. Nanotechnol., 2012, 12, 2924–2928.

9124 | New J. Chem., 2016, 40, 9111--9124

56 S. Chen, X. Hai, C. Xia, X.-W. Chen and J.-H. Wang, Chem. – Eur. J., 2013, 19, 15918–15923. 57 H. Sun, L. Wu, N. Gao, J. Ren and X. Qu, ACS Appl. Mater. Interfaces, 2013, 5, 1174–1179. 58 S. X. Sun, C. Pratx, G. Carpenter, C. M. Liu, H. Cheng and Z. Gambhir, Adv. Mater., 2011, 23, H195–H199.
