Biofabrication of Reduced Graphene Oxide ...

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Biofabrication of Reduced Graphene Oxide Nanosheets Using Terminalia bellirica Fruit Extract Sireesh B. Maddinedi and Badal K. Mandal* Trace Elements Speciation Research Laboratory, Environmental and Analytical Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632014, India Abstract: A green one step synthesis of polyphenols functionalized reduced graphene oxide (RGO) nanosheets by Terminalia bellirica (T. bellirica) extract was reported herein. The comparative analysis of various characterization results of the formed graphene sheets from graphene oxide disclosed the deoxygenation and subsequent stabilization of nanosheets with plant polyphenols. FTIR spectral data also proved the stabilization of graphene sheets with the oxidized polyphenols of T. bellirica extract.

Keywords: Deoxygenation, graphene oxide, polyphenols, reduced graphene oxide, stabilization, Terminalia bellirica. INTRODUCTION Graphene, a single atom thickness two dimensional material, has been considered as a promising material in nanoscience and nanotechnology due to its extraordinary optical, electronic, catalytic and mechanical properties [1, 2]. Due to these outstanding properties, graphene offers prominent applications in various fields of science. For instance, graphene is being used in sensors [3], fuel cells [4], nanoelectronics [5], supercapacitor [6], and nanocomposites [7] as well as in catalysis [8]. However, great attention is being focussed on the biological applications such as Bioimaging [9], drug delivery [10], cancer therapy [11] and antimicrobial activity [12]. Several synthetic methods have been reported for the preparation of graphene such as epitaxial growth on electrically insulating surfaces [13], micro-mechanical ex-foliation of graphite [14], the exfoliation and reduction of graphite oxide [15-17] and chemical vapour deposition (CVD) [18]. Among all those methods, the low cost production and the ease of bulk synthesis made the chemical routes as prominent for the graphene synthesis. But the chemically reduced GO shows irreversible agglomerations regardless of its distinctive advantages, due to the strong van der Waals interactions between the successive graphene sheets. On the other hand, this may be prevented by surface modification of reduced graphene oxide (RGO) sheets using biomolecules, polymers and surfactants [19-22]. In addition, the negative feature of chemical reduction methods also includes the hazardous nature of reducing agents (hydrazine, sodium borohydride etc) [23-29]. The existence of ultra small amounts of such hazardous materials could have harmful effect, in particular for biological applications. Hence, the introduction *Address correspondence to this author at the Trace Elements Speciation Research Laboratory, Environmental and Analytical Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632014, Tamil Nadu, India; Tel: 91-0416-2202339/9442344774; Fax: 91-0416-2243092; E-mails: [email protected]; [email protected] 1573-4137/15 $58.00+.00

of new green methods for graphene oxide reduction would be an alternate way to overcome the problems of chemical reduction. From the past few years, environmentally friendly methods for RGO synthesis have been reported using various biomolecules [30]. For example, the natural biomolecules like casein [31], bovine serum albumin [32], reducing sugar [33] and L-ascorbic acid [34] have been used as reducing and stabilizing agents for graphene synthesis. The adsorption of biomolecules on the surface of the graphene sheets causes their stabilization by preventing agglomeration and also increases their aqueous dispersibility which makes the green methods prominent towards graphene composites preparations. In recent times, plant extracts have been used as good reducing agents for the synthesis of noble metal nanoparticles (NPs) [35, 36] which could be extended to the preparation of RGO. Although extensive works have been done in this area, but still a lot of attention is gaining to know the reducing potential of plant polyphenols as both bioreductants and stabilizing agents and also the mechanism concerned in the stability and enhanced bioactivity of the prepared nanomaterials. Conversion of the graphene oxide into its reduced form using green plant extracts majorly relies on the abundance levels of the naturally occurring polyphenols in the plant extract. Further identification of the phytochemicals present in the plant extracts promotes scientists to know the plausible mechanism in the deoxygenation of graphene oxide and subsequent stabilization processes. Terminalia bellirica (T. bellirica) is an ayurvedic plant widely used in medicine and other herbal products such as triphala (medicine constituting fruits of T. bellirica, Terminalia chebula and Emblica officinalis) used for curing diarrhoea, skin diseases and cough. T. bellirica belongs to the Combretaceae family which are common in Southeast Asia [37]. This plant fruits are rich in polyphenols and would have high potential as reducing and stabilising agent. © 2015 Bentham Science Publishers

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Maddinedi and Mandal

This work describes the use of T. bellirica fruit (pericarp) aqueous extracts as green reducing and stabilizing agent for graphene synthesis from graphene oxide. Further, the prepared graphene was characterized by using different spectroscopic and microscopic techniques. Finally, the manuscript describes the possible mechanism for the stabilization of graphene by the plant polyphenols.

ble distilled water was used as blank for spectral measurements. T. bellirica mediated synthesized RGO (TBG) sample was prepared by dispersing the purified dried product in water by ultrasonication. Simultaneously, a diffuse reflectance spectroscopic (DRS) measurement was carried out for the dried TBG powder to know the band gap.

EXPERIMENTAL SECTION

XRD analysis was done by using a Bruker D8 Advance diffractometer with Cu Kα radiation (λ = 1.5406Å) over the angle range of 5o–80o with a step size of 0.02° and at a scanning rate of 1.2º/min. Purified dried TBG powder was used for all analysis.

Materials Graphite powder (100 mesh, 99.9995%), potassium permanganate (KMnO4), hydrogen peroxide (H2O2, 30%), sulphuric acid (98%), sodium nitrate (NaNO3), NH4OH (28% extra pure, Specific gravity = 0.89 g/mL) were purchased from Sigma-Aldrich, Bangalore. Preparation of GO Chemical synthesis of graphene oxide was done by modifying the reported Hummers method [38]. Briefly, 0.5 g of each graphite flakes and sodium nitrate were mixed in 23 mL of H2SO4 (12.1 M) and stirred for 15 min under ice cold conditions (0–5 °C). Then, 4 g of KMnO4 was gradually added to the reaction mixture while the temperature was maintained below 20 °C. The resulting solution was left to stir for 90 min at 40 °C in water bath followed by the addition of 50 mL of distilled water. The formed dark brown mixture was treated slowly with 6 mL of 30% H2O2 solution and diluted with 50 mL distilled water. The resulting GO solution was washed with distilled water to eliminate the excess of manganese salt until neutral pH and then centrifuged. After centrifugation GO was dispersed in water (1 mg/mL) by exfoliation for 3 h using ultrasonication and stored for uses in further experiments. Preparation of T. bellirica Fruit Pericap Extract T. bellirica plant fruits were purchased from local market in vellore, Tamilnadu. Fruit pericap was separated, dried under sunlight and then grinded into a powder. About, 3.5 g of T. bellirica fruit pericap powder was added to 200 mL of distilled water and heated for 1 h at 90 °C, and the resulting solution was filtered using 0.2 µm cellulose nitrate membrane paper before use. Synthesis of Reduced Graphene Oxide (RGO) About 200 mL of T. bellirica extract was added to the 200 mL of GO dispersion (1mg/mL) and the resulting mixture was maintained at pH 12 using NH4OH (5 mM). Then the suspension was heated at 90 °C for about 24 h on a water bath. The successful formation of RGO was confirmed by the color change from brown to black and the formed RGO precipitated out due to the loss of oxygen moieties present on the graphene oxide surface.

X-ray Diffraction (XRD)

Fourier Transform Infrared Spectroscopy (FTIR) JASCO FTIR 4100 instrument was used for Fourier transform infrared (FTIR) analysis over the wave number range of 400–4000 cm-1 in the diffuse reflectance mode at a resolution of 4 cm-1. Pellet was prepared by mixing TBG powder with KBr powder. The dried fruit extract sample was prepared by drying the extracts in hot air oven to get dried powder for attenuated total reflectance (ATR)-FTIR analysis. ATR-FTIR analysis of the residue of T. bellirica extract was carried out before and after completion of reduction of GO. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX) Carl Zeiss SEM instrument attached to EVO MA 15 was used for SEM and EDX analysis. Images were taken at diverse magnifications for the solid TBG powder spread over the surface of carbon tape attached on a metallic disk. A simultaneous measurement of EDX spectrum was obtained at certain areas of the TBG solid surface to acquire atomic distribution on TBG surface. Transmission Electron Microscopy (TEM) JEOL-2100 F electron microscope was used to obtain high resolution transmission electron microscopic (HRTEM) images at an operating voltage of 200 kV. TEM samples were prepared by ultrasonication of the dried TBG powder in water (1 mg/mL) and placing a drop of it on a Lacey carbon coated copper grid followed by drying. Dynamic Light Scattering (DLS) Study Malvern Instruments were used for dynamic light scattering (DLS) analysis of TBG. Sample was prepared by the dispersing the TBG powder in water under ultrasonication conditions to get a fine dispersion suitable for TEM analysis. Thermogravimetric Analysis (TGA)

CHARACTERIZATION

A thermal analyser, TG50 (Simadzu) was used for the TGA analysis. Purified TBG powder was used for the analysis and the instrument operation was carried out at a heating rate of 10 °C /min with a nitrogen flow rate of 30 mL/min.

Ultraviolet–Visible (UV–Vis) Spectroscopy

RESULTS AND DISCUSSION

Jasco V-670 UV-Vis double beam spectrophotometer was used for UV–Visible spectroscopic analysis where dou-

In this work water extract of T. bellirica fruits pericarp was used for the synthesis of RGO as illustrated in (Fig. 1),

Biofabrication of RGO

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Fig. (1). Schematic illustration of green reduction of GO using Terminalia bellirica extract.

Briefly, T. bellirica extract was added to graphene oxide aqueous solution and the mixture was refluxed on water bath for 24 h. The colour of reaction mixture was changed from brown to black during the completion of the reduction process which was the sign of indication for reduction process. Another reaction was conducted as blank test under similar conditions without the addition of T. bellirica extract and no color change was observed even after three days (data not shown). UV- VISIBLE SPECTRA ANALYSIS The preliminary confirmation of graphene oxide reduction process was done by taking the UV–Visible absorption spectra of as-synthesized TBG as a function of reaction time. UV absorption peak for GO showed a maximum absorption peak at 232 nm due to the π–π* transitions of the aromatic C–C bonds and a weak shoulder at 300 nm ascribed to the n– π* transitions of C=O bonds. Successful completion of reduction process was indicated by the appearance of a new peak obtained due to the red shift of typical GO peak at 232

Fig. (2). UV–visible absorption spectra of GO (blue) and TBG (green).

nm to 275 nm (Fig. 2), signifying the restoration of electronic conjugation of the formed RGO sheets. However, the maximum red shift value observed for the RGO represents the efficiency of the reducing agent used for reduction of GO [39]. For example, RGO prepared by using T. bellirica extract exhibits an absorption band at 275 nm, which is higher than that of phenyl hydrazine [40] and hydrazine [41] reduced graphene (270 nm), representing the reducing efficiency of polyphenols present in T. bellirica extracts towards the reduction of GO. A characteristic broad shoulder peak at 367 nm may be the indication of surface capping of graphene sheets with polyphenols of T. bellirica [42, 43]. FTIR STUDY Fourier transform infrared (FTIR) analysis of TBG and GO were carried out to confirm the reduction of GO to TBG. Fig. (3) shows a broad band at 3100-3400 cm-1 characteristic of O–H stretching vibration. Additionally, the presence of absorption bands around 1722 cm-1 and 1618 cm-1 are corresponding to C=O stretch of carbonyl group and the O-H



Fig. (3). FTIR spectra of GO (black) and dried TBG (red).

bending, epoxide ring vibrations respectively. The absorption band at 1050 cm-1 is due to the C-O stretching vibrational peak of carboxylic acid located on the graphene oxide surface. The presence of tertiary alcohol (C–OH) group shows the absorption band at around 1400 cm-1. However, the decreased intensities of absorption bands of O–H, C=O, C-O and tertiary C–OH bands in the FTIR spectra of TBG after reduction indicates the deoxygenation of GO and the obtained results are similar to the graphene prepared by using extracts of Citrus sinensis, Mesuaferrea Linn and Colocasiaesculenta [44]. XRD STUDY A comparative XRD analysis of TBG and GO approves the T. bellirica extract mediated reduction of GO to reduced graphene oxide. Fig. (4) shows a single diffraction peak at 11.2o (002) for GO with consequent interlayer d-spacing of 0.77nm, indicating the existence of oxygen containing moie-

Fig. (4). XRD patterns of GO and TBG.

ties between the graphite layers upon oxidation. On the other hand, TBG obtained by the reduction of GO with T. bellirica extract exhibits a broad diffraction peak at 2θ of 25.0o (111) with related d-spacing of 0.34nm. The complete vanishing of GO peak at 2θ of 11.2o and a comparatively decreased dspacing value of RGO synthesized further confirms the inclusive removal of oxygen moieties in GO after reduction. SEM AND EDX ANALYSIS The surface morphological investigation of GO and TBG were done by SEM analysis. From Fig. (5A), it is clear that GO exists as stacked layers containing rough surface which may be because of the oxidation of sheets. In contrast, the TBG shows the thin layered structures with sharp edges (Fig. 5B). Further, the EDS spectrum of the produced GO and TBG reveals the percentage of oxygen as 43.2, 24.8 atom% subsequently (Fig. 5C, D).



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Fig. (5). SEM images at different magnifications (A) 2 µm (B) 1 µm and EDS spectrum of GO (C) and TBG (D).

TEM ANALYSIS Fig. (6A and B) show the HRTEM microscopic images of the formed TBG. It shows that TBG exists as thin, transparent, silk like graphene sheets after reduction. The edges of the graphene film allowed the cross-sectional view which also concluded the existence of three to four layers of RGO sheets. MECHANISM OF STABILIZATION OF TBG From our previous work, it is known that the aqueous extract of T. bellirica fruit pericap contains five different polyphenols namely, ascorbic acid, resorcinol, gallic acid, pyrogallol and methyl gallate [45]. However, the gallic acid exists as a major constituent in T. bellirica extracts indicating its major role in the reduction. In addition, to know the surface capping and the reduction mechanism, the FTIR spectra for the dried T. bellirica extract before and after the reduction reaction were taken. Fig. (7) represents the existence of infra red bands at 3196 and 1332, 1203 cm-1 in the

T. bellirica extract which relates to the O-H and C-O stretching of polyphenolic groups signifying the rich polyphenolic substance in T. bellirica aqueous extract. Notably, the disappearance of C-O stretching vibrations at 1322 and 1203 cm-1 associated with the T. bellirica polyphenolic groups after reduction suggests the participation of phenolic OH groups in the reduction of GO. Additionally, the emergence of new vibrational band at 1629 cm-1 (corresponding to ketone group) indicating the transformation of polyphenolic groups into their quinone forms during the reaction and also confirms the existence of conjugation as well as intramolecular hydrogen bonding in the polyphenolic structures after reduction. This strongly suggests that the polyphenols of T. bellirica extract acted as both reductant and stabilizer by adsorbing on the formed TBG surface. Hence, the resultant oxidized polyphenols may interact with TBG nanosheets via π-π stacking interactions, creating the electrostatic repulsions between the reduced graphene oxide layers causing their stabilization. Fig. (8) represents the π-π interactions between the oxidized polyphenols and the graphene sheets which pro-

Fig. (6). HR-TEM images of TBG at different magnifications (A) 200 nm and (B) 50 nm.



Fig. (7). FTIR spectrum of T. bellirica fruit pericap extract before (black) and after reduction (red).

Fig. (8). Proposed stabilization mechanism of TBG using the T. bellirica polyphenols

tects agglomeration of RGO sheets. On the other hand, the RGO synthesized by using chemical reagents such as hydrazine, phenylhydrazine and sodiumborohydride is not stable due to lack of stabilizing agents on its surface to prevent the aggregation of graphene sheets and inturn causes serious obstruct to their production, storage and transfer. But, the RGO prepared by using T. bellirica extract is well stabilized by the oxidised polyphenols present in the extract (which are confirmed from FTIR and UV data). These biomolecules present on the surface of RGO increase its stability by preventing the agglomeration, and hence extending its scope of applications in various fields of science, as most of the properties of graphene are related to the individual sheet. Additionally, the surface polyphenols also increase its aqueous dispersibility without adding any external surfactants and hence increasing the development of new composite materials synthesis [46]. Most of the plant extract reduced nanomaterials are biocompatible due to the non-toxic nature of poly-

phenols present in the extract. However, the biocompatibility of plant extract mediated RGO is already reported [47]. In contrast, the GO reduced by chemical reagents may lead to new toxicity issues due to the adsorption of these toxic chemicals onto the surface of graphene sheets making it less promising towards the environmental and biological related applications. All these properties explain the important advantages of TBG in biological applications such as drug delivery and cancer therapy. DLS ANALYSIS Additionally, to know the size distribution of TBG particles in water dispersion, DLS analysis was carried out using a standard spherical model (Fig. 9A). The graphene sheets were broken down into the particles under prolonged sonication conditions and the average particle size was found between 600 to 700 nm.



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Fig. (9). (A) DLS of TBG aqueous dispersion (B) TGA thermogram of TBG (C) Optical diffuse reflectance spectra of T. bellirica reduced graphene oxide.

THERMO GRAVIMETRIC ANALYSIS (TGA) Further, the thermal stability of TBG was studied by TGA after knowing the presence of oxygen containing functional groups. Fig. (9B) represents the thermogram of asprepared TBG which showed a 17% weight loss at below 200 °C because of the removal of oxygen functionalities, phytochemicals and absorbed water present on the graphene surface. Consequently, it also showed a huge weight loss of 86.5% at below 500 °C, due to the pyrolysis of the residual oxygen constituents and the burning of the carbon ring. However, the high weight loss below 200 °C, compared to the other biosynthesized graphene is clearly representing the biomolecular accumulation on the formed graphene sheets after reduction [48, 46]. BAND GAP STUDIES The optical band gap of TBG was determined by UV– Visible diffuse reflectance spectrophotometer using the following equation as α = c (hν- Ebulk) 1/2/ hν, where α is absorption coefficient, Ebulk is bulk ‘band gap’, c is a constant and hν is the photon energy. A plot between hν verses (αhν)2 for the TBG is shown in Fig. (9C). The band gap of TBG was found to be 3.75 eV from the plot (Fig. 9C). The calculated band gap in the present study is in close agreement with the reported values of graphene synthesized by using other aqueous extracts of Mesuaferrea Linn, Colocasiaesculenta and Citrus sinensis [44]. CONCLUSION We showed a facile, low-cost ecofriendly synthetic method for the production of reduced graphene oxide by

using polyphenols from T. bellirica fruit extracts. FTIR analysis concluded that the naturally occurring polyphenols present in T. bellirica extract were responsible for the reduction of GO to TBG. It is also found that polyphenols in T. bellirica stabilized the formed reduced graphene oxide nanosheets by preventing their aggregation. This paper further shows an ecofriendly method to produce RGO in bulk scale by using low cost polyphenols rich plant extracts. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS Mr. SBM deeply acknowledges the help of VIT University, Vellore-632014, India for the platform and the financial help given to do this research. He also acknowledges the help in characterising nanomaterials from Department of physics, Sri Venkateswara University, Tirupati-517502, India and NIIST, CSIR, Thiruvananthapuram-695 019, India. REFERENCES [1] [2] [3] [4]

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Received: January 08, 2015

Revised: January 27, 2015

Accepted: February 16, 2015

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