Green Strategy to Reduced Nanographene Oxide through Microwave

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A green strategy to reduced nano-graphene oxide through microwave assisted transformation of cellulose Nejla Erdal, Karin H Adolfsson, Torbjörn Pettersson, and Minna Hakkarainen ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03566 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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ACS Sustainable Chemistry & Engineering

A green strategy to reduced nano-graphene oxide through microwave assisted transformation of cellulose

Nejla B. Erdal,1 Karin H. Adolfsson,1 Torbjörn Pettersson1 and Minna Hakkarainen*,1 1

Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, Teknikringen 58, 100 44 Stockholm, Sweden *E-mail: [email protected]

1 ACS Paragon Plus Environment


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KEYWORDS: Carbon nanodots, Biobased, Biomass, Microwave, Hydrothermal, Reducing agent Abstract A green strategy for fabrication of biobased reduced nano-graphene oxide (r-nGO) was developed. Cellulose derived nano-graphene oxide (nGO) type carbon nanodots were reduced by microwave assisted hydrothermal treatment with superheated water alone or in the presence of caffeic acid (CA), a green reducing agent. The carbon nanodots, r-nGO and rnGO-CA, obtained through the two different reaction routes without or with the added reducing agent, were characterized by multiple analytical techniques including FTIR, XPS, Raman, XRD, TGA, TEM, AFM, UV-Vis and DLS to confirm and evaluate the efficiency of the reduction reactions. A significant decrease in oxygen content accompanied by increased number of sp2 hybridized functional groups was confirmed in both cases. The synergistic effect of superheated water and reducing agent resulted in the highest C/O ratio and thermal stability, which also supported a more efficient reduction. Interesting optical properties were detected by fluorescence spectroscopy where nGO, r-nGO and r-nGO-CA all displayed excitation dependent fluorescence behaviour. r-nGO-CA and its precursor nGO were evaluated towards osteoblastic cells MG-63 and exhibited non-toxic behaviour up to 200 µg mL-1, which gives promise for utilization in biomedical applications.


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INTRODUCTION The increasing concerns on the depletion of oil resources and associated environmental problems has directed the interest towards the use of renewable and sustainable resources for chemical and material production. Several studies have demonstrated that bioresources, such as sucrose,1 glucose,2 starch3 and cellulose4 can be used as starting materials for the production of carbon spheres (C-spheres). The C-spheres resulting from hydrothermal carbonization of biomass have gained wide-spread interest due to their sustainable production and potential applications in batteries,5,6 hydrogen storage7 and as adsorbents.8 Valorization of biomass through hydrothermal processing routes involves the use of water at elevated temperature and pressure which carries several benefits since water is green, noncorrosive, nontoxic, nonflammable and inexpensive. Microwave technology is an appealing tool that offers increased efficiency over conventional heating by accelerating chemical processes and give rise to faster transformations.9 A greener carbonization process could, thus be achieved by combining microwave technology and water that readily absorbs microwave irradiation. Recently, it has been shown that microwave-assisted hydrothermal processes offer an effective route to carbonize e.g. cellulose,10 waste paper,11 starch12 and coffee grounds13 to produce C-spheres. An interesting property of the C-spheres is that they can be chemically exfoliated through oxidation to form carbon nanodots such as nano-graphene oxide (nGO).11,14 Unlike the conventional two dimensional (2D) GO, nGO is considered to be zero-dimensional (0D) and has a larger degree of oxygenation.15



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Conventional 2D GO can be reduced to rGO to enhance the hydrophobic or π-interactions by increased degree of sp2-hybridization, while partly maintaining electrostatic-or hydrogen bonding interactions due to some remaining oxygen moieties. Depending on the potential healing of the structural defects of GO the electrical conductivity can be altered or intercalated.16 As an additive in polymer composites the incorporation of rGO can increase the thermal stability of the composites in contrast to GO; which have often been shown to decrease the thermal stability of the composites.17 Chemical reduction of GO has been regarded as one of the most promising methods for large-scale production of rGO at low cost. However, it often involves reducing agents such as hydrazine hydrate and sodium borohydride that are toxic and/or explosive.18 Since the removal of oxygen moieties in GO determines the ultimate properties and to which extent the reduced graphene oxide will resemble pristine graphene a suitable reducing agent for this task is very important. Lately, interest has been directed towards environmentally friendly, cost-effective strategies for producing rGO materials with less harmful reducing agents.19 Up to now a wide variety of natural, non-toxic reducing agents such as vitamin C (ascorbic acid), sugars, certain metals and plant extracts were reported to reduce GO.20 Among them caffeic acid (CA), a naturally abundant antioxidant, has been shown to efficiently reduce GO leading to high C/O ratios.21 Moreover, alternative approaches in the production of graphene derivatives have recently been recognized with studies showing valorisation of e.g. coal tar pitch as industrial carbon waste and starch into patterned graphene films22 and luminescent reduced carbon dots,23 respectively, both interesting for e.g. electrode or photocatalyst applications.

Here we aimed to develop a microwave induced hydrothermal reduction process for nGO derived from cellulose, as a green and sustainable strategy for the production of nano-sized rGO (r-nGO). Water is easily heated above its boiling point in a microwave reactor due high


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pressure. Such water is called superheated water and has a higher H3O+ ion concentration compared to normal liquid water and the ability to acid-catalyse organic reactions.24 It was thus, hypothesized that superheated water could, alone or in combination with a green reducing agent, promote the reduction process of nGO. The aim was to transform cellulose into value added r-nGO carbon nanodots through two different, simple and reproducible routes with or without separately adding the green, nontoxic reducing agent, CA. The reduction processes were evaluated and the properties of the resulting carbon nanodots were assessed with several techniques. Graphene derivatives have shown tremendous potential in the field of biomedical materials in applications such as: drug delivery,25 biosensing,26 bioimaging,27 bacteria inhibiton28 and bioactive scaffolds.29,30 On the basis of the potential of the synthesized r-nGO in biomedical applications its’ in vitro cytotoxicity towards osteoblastic cells was also determined.

EXPERIMENTAL SECTION Materials. Sulphuric acid (H2SO4) (95–98%), nitric acid (HNO3) (70%), α-cellulose and caffeic acid (CA) (≥ 98%) were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany and St. Louis, USA). Standard RC Membrane Spectra/Por® 7 Pre-treated dialysis tubing (MWCO 1 kDa) was obtained from Spectrum Labs. Dulbecco’s modified Eagle’s medium, phosphate-buffered saline (10×), and trypsin−EDTA solutions were purchased from Sigma-Aldrich. Fetal bovine serum, penicillin−streptomycin, and alamarBlue cell viability reagent were purchased from Fisher. All chemicals were used as received. nGO synthesis. nGO were synthesized from cellulose via a microwave-assisted process according to our previously reported procedure.10,31 Briefly, 2 g of α-cellulose was treated in 20 ml aqueous 0.01 g mL−1 H2SO4 solution in Milestone UltraWAVE (Milestone Inc) microwave device in dynamic mode with a supreme power of 950 ± 50 W. The temperature



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was first set to increase up to 180 °C under a ramp-time of 20 minutes and then kept at isothermal conditions by input irradiation for 2 hours and a pressure of 40 bar assisted with nitrogen. The resulting black solid, carbon spheres (C-spheres) were filtered from the solution and washed with 500 mL of deionized H2O. The C-spheres were then dried in a vacuum oven overnight at 25 °C. To synthesize nGO, a 15 ml solution of C-spheres in 70 % HNO3 (1 : 100, w/w) was kept in a 100 ml one-neck round-bottom flask, bathsonicated 0.5 h at 45 °C and then heated 0.5 h at 90 °C with magnetic stirring. After the oxidation, the solution was poured into 50 ml of cold deionized H2O (15 °C) to dilute the acidic medium. An orange/red solid was received after removal of the acidic water by rotary evaporation. The product was freeze dried and then kept in vacuum oven at room temperature for one week to remove any residues of water and acid. r-nGO and r-nGO-CA synthesis. For the production of r-nGO typically 1 mg nGO was dispersed in deionized water for further treatment in Milestone UltraWAVE (Milestone Inc) microwave as above. The temperature was increased to 180 °C under a ramp-time of 20 minutes and maintained at 180 °C for 2 hours starting with an external pressure of 40 bar nitrogen. The resulting black solid product r-nGO was freeze dried. To synthesize r-nGO-CA, nGO was dispersed in deionized water and CA powder (1:3 w:w nGO:CA) was added to the aqueous solution containing nGO. After mixing, the solution was treated in the microwave as above. The resulting black solid product was poured into dialysis membrane (MWCO1 kDa) and put in 500 mL deionized water for 24-48 hours with stirring to separate r-nGO-CA from excess CA. The dialysis solution was changed 2-3 times. The solution was freeze dried to obtain r-nGO-CA. Both r-nGO and r-nGO-CA were stored for 5 days in vacuum oven at room temperature to remove excess water. Fourier Transform Infrared Spectroscopy. Fourier transform infrared spectra (FTIR) of nGO, r-nGO and r-nGO-CA were obtained by PerkinElmer Spectrum 2000 FTIR


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spectrometer (Norwalk, CT). The instrument was equipped with attenuated total reflectance (ATR) accessory (golden gate) from Graseby Specac (Kent, United Kingdom). X-ray photoelectron spectroscopy. The X-Ray Photoelectron Spectroscopy (XPS) spectra of nGO, r-nGO and r-nGO-CA were collected by a Kratos Axis Ultra DLD electron spectrometer using monochromated Al Kα source operated at 150 W. Pass energy of 160 eV were applied for wide spectra analyzer and pass energy of 20 eV for individual photoelectron lines. Spectrometer charge neutralization system was employed to stabilize the surface potential. The binding energy (BE) scale was referenced to the C1s line of aliphatic carbon, set at 285.0 eV for nGO and 284.7 eV for r-nGO and r-nGO-CA. Kratos software was used for data processing. Raman Spectroscopy. The Raman spectra of the nGO, r-nGO and r-nGO-CA samples were obtained using a home-built spectrometer.32 The radiation laser source used had a wavelength of 532 nm CW laser (Laser Quantum, U.K.). X-ray Diffraction. X-ray diffraction (XRD) spectra were recorded for nGO, r-nGO and rnGO-CA using the X-ray source CuKR radiation (λ = 0.1541 nm). The diffraction was measured by PANalytical X'Pert PRO diffractometer at 25 °C with a silicium mono-crystal sample holder. The intensity was determined in a 2θ angular range between 5–40° with a step size of 0.017° for all analyses. Thermal Gravimetric Analysis. Mettler-Toledo TGA/SDTA 851e was utilized for the thermogravimetric analysis (TGA) of nGO, r-nGO and r-nGO-CA. 3-4 mg of each sample was placed into 70 µl alumina cup. The samples were then heated at a rate of 10 °C min−1 from 25 °C to 600 °C with an N2 flow rate of 80 mL min−1. Transmission Electron Microscopy. Transmission electron microscopy (TEM) images were taken by HITACHI HT7700 instrument with the software version 02/05. Dispersions of nGO, r-nGO and r-nGO-CA were prepared in acetone at a concentration of 0.1 mg mL-1 and drop-



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casted on the TEM grid ultrathin carbon coated copper grid (Ted Pella, Inc.). The samples were left to dry prior to analyses in high-contrast mode TEM. Atomic Force Microscopy. Height images of nGO, r-nGO and r-nGO-CA were acquired using a Nanoscope MultiMode 8 atomic force microscopy (AFM) (Bruker, Santa Barbara, CA) in QNM PeakForce tapping mode under ambient conditions (20–25 °C). Freshly cleaved mica (grade V-1, Electron Microscopy Sciences) was used as a surface for all the samples. Dispersions of the carbon nanodots in acetone 0.1 mg mL-1 were prepared and drop-casted on the mica surface. The samples were left to dry prior to analyses. Ultraviolet−Visible Spectroscopy. Ultraviolet-visible (UV/Vis) absorption of nGO, r-nGO and r-nGO-CA was measured by SHIMADZU UV-2550 UV/Vis. Solutions were prepared in deionized water at a concentration of 0.1 mg mL-1. Fluorescence Spectroscopy. Fluorescence spectroscopy measurements were performed by Cary Eclipse spectrophotometer from Varian at medium voltage. Solutions of nGO, r-nGO and r-nGO-CA were prepared in deionized water and in acetone at a concentration of 0.1 mg mL-1. Excitation wavelengths from 254 to 510 nm were used. The instrument used xenon lamp technology. In vitro cell viability test. MTT assays were performed to measure the cytotoxicity of nGO and r-nGO-CA. Osteoblastic cells MG63 were cultured in complete growth medium (CGM; Dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units mL-1 of penicillin, and 100 µg mL-1 of streptomycin) at 37 °C in 5% CO2 and 95% relative humidity. The cells were seeded on the bottom of the wells in a 96-well tissue-culture polystyrene plate at a concentration of 8000 cells/well. Five different concentrations of nGO and r-nGO-CA: 20, 50, 100, 200, 1000 and 10 000 µg mL-1 in CGM were prepared and poured into the wells of the 96-wellplate and incubated during 24 hours. Fresh CGM without any carbon nanodots was used as a control group. After 24 hours the


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medium in the wells was suctioned and the wells were washed thoroughly with PBS to remove all free carbon nanodots. Then, 100 µL of 1× resazurin (alamarBlue cell viability reagent) solution in PBS was added to each assay well and incubated for approximately 1 hour. The fluorescence of each well was measured by a Tecan Infinite 200 PRO multifunctional microplate reader (excitation wavelength = 560 nm; emission wavelength = 590 nm). The cell viability (%) was calculated from 100 × ([Isample] − [Iblanc])/([Inegative][Iblanc]), where [Isample] represent the fluorescence intensity values of the wells with the carbon nanodots, [Iblanc] without cells, and [Inegative] without carbon nanodots. The fluorescence intensity was the average value measured from three wells in parallel for each sample. An optical microscopy at ×400 was used to evaluate the morphology of the cells after 24 hours incubation. Dynamic Light Scattering. Dynamic light scattering (DLS) measurements of nGO and rnGO-CA solutions in deionized water (D.I.) and complete growth medium (CGM) (25 µg mL-1) at 20°C were carried out on Zetasizer Nano ZS from Malvern Instruments (Malvern, UK), to measure hydrodynamic diameter and ζ-potential. The solutions were sonicated for 10 minutes in an ultrasonication bath before analyses and graphene oxide (refractive index,

n=1.33) was used as standard for all calculations.

RESULTS AND DISCUSSION Previously we synthesized highly oxidized nano graphene oxide (nGO) from α-cellulose with carbon spheres (C-spheres) as an intermediate product according to Figure 1.11 Here, the reduction of the cellulose derived nGO was evaluated through two different microwaveassisted routes. The first route was reduction in superheated water (r-nGO) alone and the second route also included CA as a reducing agent (r-nGO-CA) in superheated water. nGO formed a bright-yellow suspension in water. This suspension turned black after the microwave assisted reductions, see Figure 1, providing a clear visual indication of reductions. 9 ACS Paragon Plus Environment


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The obtained black products were characterized by multiple techniques to evaluate the reduction process and properties of the products.

Figure 1. Illustration of the reduction process through two different routes.

Chemical characterization of nGO, r-nGO and r-nGO-CA. Figure 2 compares the FTIR spectra of nGO and the reductions products, r-nGO and r-nGO-CA. nGO shows the characteristic vibrational modes of alcohols (C-OH stretching 3560 cm-1), carboxylic acids (C=O stretching at 1710 cm-1, broad C-OH stretch at 2500-3350 cm-1 and C-O stretching at 1100 cm-1), epoxides (C-O-C stretching 1288 cm-1) and aromatic sp2 hybridized carbons (C=C stretching at 1618 cm-1). As illustrated by the spectra, there is a significant decrease in the intensities of the absorption bands representing oxygen-containing functionalities for rnGO-CA indicating successful reduction process, whereas the change is smaller for r-nGO. Still the spectrum of r-nGO shows a decrease in carbonyl peak intensity and a narrowing of the broad carboxylic acid band and the hydroxyl band suggesting that some reduction has taken place leading to loss or conversion of some of the original oxygen functionalities.


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Figure 2. FTIR transmittance spectra of nGO, r-nGO and r-nGO-CA. The spectra are not normalized against each other. The relevant chemical features are marked. The reduction of the oxygen-containing groups decorating nGO was further evaluated and confirmed with XPS measurements. Figure 3a shows the XPS survey spectra of nGO, r-nGO and r-nGO-CA. There is an increasing reduction level going from nGO to r-nGO and then further to r-nGO-CA. This is illustrated by the decreased intensity of O1 peaks and the increased C/O ratios changing from 1.65 for nGO to 2.30 for r-nGO and 4.41 for r-nGO-CA. As can be seen in Figure 3d the carbon nanodots contained C-C, C=C, C-O, C=O, O=C-O components in different intensities and percentages. The decreasing intensities for the different oxygen functionalities in r-nGO and r-nGO-CA were accompanied by the appearance of π-π* transitions in the XPS spectra of r-nGO and r-nGO-CA, see Figure 3b, implying formation of sp2 hybridization and recovery of conjugated structures, which were possibly disturbed by the rich oxygen functionalities in nGO. The XPS analysis, thus, further confirmed a substantially decreased concentration of oxygen-containing groups. Accordingly, SEM-EDX (See SI, Figure S1, S2 and S3) analyses illustrated increasing C/O ratios changing from 1.0 for nGO to 2.2 for r-nGO and 3.25 for r-nGO-CA. This shows a similar trend with the values determined by XPS, although the C/O ratios determined by SEM-EDX should be considered more indicative than absolute. 11 ACS Paragon Plus Environment


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The COOH functionalities decreased significantly during both reduction processes. nGO and r-nGO both had about 50% of the oxygen functionalities in C=O and –OH/C-O-C form, respectively. However, the concentrations of these groups greatly decreased when nGO was reduced to r-nGO. An even larger decrease was seen in the case of r-nGO-CA which had 85.6% of the oxygen functionalities in the form of –OH/C-O-C groups and the relative amount of C=O had decreased to only 14.4%, again in agreement with the FTIR results. In addition to the changes in the type of oxygen functionalities, the XPS results also manifested that the total amount of oxygen functionalities had been greatly reduced in comparison to nGO. Low intensity peaks indicating presence of nitrogen and sulphur were also detected in the XPS spectra of all three samples. The trace amounts probably originated from the synthesis step of C-spheres and nGO, which involved H2SO4 and HNO3 respectively that further decreased during the reductions (See SI file, Figure S4).


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Figure 3. (a) XPS survey spectra for C1s and O1s, respectively (b) High resolution XPS spectra of C1s (c) High resolution XPS spectra of O1s and (d) Table of C1s and O1s positions and intensities obtained by deconvolution of the original “fitted spectra” the for nGO, r-nGO and r-nGO-CA.

Structural disorder, crystallinity and thermal properties. The reduction of the particles was additionally investigated with Raman spectroscopy (Figure 4a). The two main bands referred to as D band and G band and are marked in the spectra. The D band is assigned to structural imperfections caused by hydroxyl and epoxide groups33 and the G band reflects the graphite lattice.34. A broad D band noticed for GO is possibly due to hydrogen bonding since the hydroxyl groups generally yield a series of bands around 1300 cm-1.35 The intensity ratio of these two bands gives information of the extent of structural disorder of graphene/graphitic materials. The intensity ratio of D to G peak (ID/IG) was 0.88 for nGO, 1 for r-nGO, and 0.85 for r-nGO-CA. As depicted there is an increase in the ratio for r-nGO suggesting that there are certain defects such as grain boundaries, vacancies and amorphous carbon species present in r-nGO.34,36,37 Interestingly, for r-nGO-CA the trend is the opposite and a small decrease in ID/IG ratio is noticed. A decrease would indicate recovery of conjugated graphitic framework 13 ACS Paragon Plus Environment


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upon defunctionalisation of epoxide, hydroxyl and other oxygen moieties38 during the microwave treatment suggesting an enhanced reduction.39,40 However, it should be noted that the difference between nGO and n-rGO-CA is only minor.

XRD spectra were recorded to further understand the reduction process, see Figure 4b. After the microwave treatment of semicrystalline cellulose, the obtained C-spheres displayed an amorphous character with a broad peak at approximately 2θ= 22° that corresponds to the (002) of the graphitic plane with an interlaying distance of 0.40 nm as previously reported by our group.11,41 After oxidation of C-spheres to form nGO the graphitic peak remained, suggesting that the graphitic plane is maintained with a peak located at around 22°, that has been reported for GO in the literature.42 A different diffraction pattern is obtained probably due to salt originating from the preparation steps involving H2SO4 and HNO3 as indicated also by the trace amounts of sulphur and nitrogen found in the XPS survey spectra (See SI file, Figure S4). Another possibility is that a polydisperse crystal structure is created by the assembly of multilayer graphene oxide structures to graphite oxide in solid state.43 The peaks around (2θ = 15°, 19°, 26°) may in addition be attributed to functional oxygen groups that cause nGO to stack more loosely than GO.14 As for the r-nGO and r-nGO-CA a broad peak centring around 2θ=22° could be observed corresponding to the (002) of the graphitic plane.44 A transformation from merely crystalline (r-nGO) to fully amorphous character (r-nGO-CA) is revealed when the reduction reaction is aided with a reducing agent.

TGA analysis additionally support the above described spectral analysis. Figure 4c shows the TGA curves for nGO, r-nGO and r-nGO-CA. The thermal stability of both r-nGO and r-nGOCA was clearly enhanced as compared to nGO, as the reduced particles show significantly higher thermal stability around 100 °C in contrasts to nGO. At 600 °C the total weight loss


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was only 48.5% and 38.0% for r-nGO and r-nGO-CA, respectively, as compared with 76.7% for nGO. The differences in weight losses can be attributed to the different amounts of oxygen functional groups11 before and after the microwave reduction and this further supports the more efficient reduction and further lowering of the oxygen content in the case of r-nGO-CA as compared to r-nGO.

Figure 4. (a) Raman spectra, (b) XRD spectra and (c) TGA thermograms for nGO, r-nGO and r-nGO-CA. In a) and b) the spectra are shifted in height in offset.

Morphology and size of the particles. The size and shape of the different carbon nanodots were monitored by TEM (Figure 5). The dispersions were sonicated prior to the analysis to 15 ACS Paragon Plus Environment


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prevent the formation of large agglomerates. TEM images showed particles with circular features and narrow size distribution in the case of r-nGO and r-nGO-CA (casted from acetone 0.1 mg mL-1). The diameters were calculated to be 22 ± 19 nm for nGO, 70 ± 14 nm for r-nGO and 12 ± 3 nm for r-nGO-CA (the diameters were measured using software ImageJ). The diameters of the basal planes of the carbon nanodots were, thus, less than 100 nm revealing a zero dimensional (0D) characteristics of the carbon nanodots.45,46 The images illustrate the clear distinction in dimensionality between the carbon nanodots and the conventional GO structures that typically display a 2D character with expanded sheets.18

Figure 5. TEM images of nGO, r-nGO and r-nGO-CA (left) and the diameter size distributions based on image analysis of TEM images (right).

AFM images (Figure 6) of the nGO, r-nGO and r-nGO-CA surfaces were in agreement with the TEM images in Figure 5 although the measured sizes were around twice as large. According to AFM, nGO particles had a diameter around 50 nm and consisted of a few nGO 16 ACS Paragon Plus Environment

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sheets or layers stacked together, as revealed by the average height, which was up to 3 nm (Figure 6a,b,c). A single sheet 2D GO is characterized by a thickness of around 1 nm suggesting that 0D nGO imaged here consists of nanosized GO with a few stacking layers.47 The small deviation in size as compared to TEM measurements can be due to the association of the dots and lateral interaction between functional groups at the edges during the drying process, as well as an effect of tip convolution. The size of r-nGO and r-nGO-CA as measured by AFM and marked in the figures was 160 nm and 25 nm respectively (Figure 6f,i). r-nGO seem to expand and diplay flake like appearance (Figure 6d) while r-nGO-CA tend to shrink into dot-like structures (Figure 6g). The lateral size of r-nGO appears to be significantly larger than the lateral size of nGO and r-nGO-CA. One likely reason could be self-assembly of smaller objects into larger objects due to edge interactions as well as a tendency of r-nGO to extend more than r-nGO-CA since it has a thickness of around 1 nm while r-nGO-CA has a thickness of 5 nm (Figure 6e,h). However, the height profile of rnGO appears to fluctuate a lot which could possibly indicate thinner dots than 1 nm that are stacked together with different number of layers and smaller lateral dimensions resulting in an uneven height profile (Figure 6f). This also explains the size difference of the reduced carbon nanodots vizualized in the TEM images (Figure 5). The thickness of GO has been reported to reduce after the reduction process due to loss of oxygen groups on the surface and values around 0.8-1 nm have been reported.21,48 Altough the size of the carbon nanodots deviates due to self-assembly one obvious difference is that r-nGO seem to elucidate few layered structure while r-nGO-CA seem to stack to a higher extent possibly due to stronger intersheet attractions.



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Figure 6. AFM results from the different carbon nanodots; on each row left and middle image are AFM height (in top view and 3D-view, respectively) and left shows a short height section as indicated in the left image. Row (a) to (c) shows nGO, row (d) to (f) shows r-nGO, and row (g) to (i) shows r-nGO-CA. Optical properties. Figure 7 presents the UV-Vis absorbance spectrum of nGO, r-nGO, rnGO-CA and CA. nGO and r-nGO show comparable absorbance’s at 200 nm and 245 nm. rnGO-CA exhibit a red-shift from 245 nm to 283 nm indicating that the reduction of nGO to rnGO-CA partly restored the π-conjugation network.24 As the π -conjugation increases, less energy is required for the transition, which corresponds to the observed shift of the absorption to the longer wavelength region.39 This transition is not detected for r-nGO suggesting, in accordance with the results reported above, a less efficient reduction process. However, CA that underwent the same hydrothermal microwave treatment as nGO showed an absorbance at around 276 nm. This shift connected to microwave treated CA could also contribute to the


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observed shift in the case of r-nGO-CA, although r-nGO-CA shifted to even higher wavelength than CA.

Figure 7. UV-Vis spectra of nGO, r-nGO, r-nGO-CA and CA.

Another interesting optical property of the synthesized carbon nanodots was the fluorescence behaviour, which was clearly visible under UV-light in deionized water solutions with a concentration of 0.1 mg mL-1. Figure 8 shows the fluorescence emission spectra for the excitation wavelengths (λexc) ranging from 254 nm to 510 nm. Broad emission bands appeared between 400 – 600 nm for all the carbon nanodots, but r-nGO displayed the highest intensities. It has been reported that the fluorescence intensity for thin films of GO reaches a maximum when GO is only slightly reduced compared to highly reduced GO.49 This could explain the higher fluorescence intensities exhibited by r-nGO in contrast to nGO and r-nGOCA. However, there is a general uncertainty regarding the origin of fluorescence for carbon nanodots and other parameters that prevail the fluorescence behaviour involve size, shape and relative fraction of sp2 hybridized domains. Differences in these mentioned properties might consequently yield different fluorescence intensities.50 Two emission bands were observed for all the carbon nanodots below λex =370 nm, although the second peak was weak for nGO.



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Only one emission band was noticed at 370 nm < λex where the emission bands started to shift to higher wavelengths. The carbon nanodots were thus found to exhibit an excitationdependent photoluminescence behaviour. This striking behaviour is common for most luminescent carbon nanoparticles51–53 and may reflect not only the effects from particles of different sizes, but also a distribution of different emissive sites on the particles.54 For r-nGO and r-nGO-CA a third peak appeared at λex =254 nm. The images showing the fluorescing solutions of nGO, r-nGO and r-nGO-CA were irradiated with a UV-lamp at λex =254 nm. The intensity of the fluorescence emission intensities was higher in acetone although it followed the same trend as in water (See SI file, Figure S5).

Figure 8. Fluorescence spectra of nGO, r-nGO and r-nGO-CA in deionized water. 20 ACS Paragon Plus Environment

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In vitro cytotoxicity. Based on the above characterization of the reduced carbon nanodots, rnGO-CA was found to be more efficiently reduced as shown by the C/O ratio and thermal stability. Additionally, the yield was higher for r-nGO-CA (75 wt%) than in the case of rnGO (45 wt%). r-nGO-CA was therefore selected for further exploration as a potential material for use in biomedical applications. The cytotoxicity of r-nGO-CA and nGO in complete growth medium (CGM) at different concentrations was evaluated by MTT assay using osteoblast MG63 cell lines. Figure 9 shows that both nGO and r-nGO displays no obvious cell toxicity for MG63 cells, in fact the cells seemed to thrive in the company of the carbon nanodots as demonstrated by the relative cell viability that is maintained at a range of 102 % - 109 % up to a concentration of at least 200 µg mL-1. r-nGO-CA demonstrated good cell viability (90 ± 5 %) even up to a concentration of 1000 µg mL-1 while the cell viability of nGO was low at 1000 µg mL-1. At higher concentrations from 1000 to 10 000 µg mL-1 a dramatic cell viability loss is observed for both nGO and r-nGO-CA. However, the cell viability of the carbon nanodots was very good at the concentration range that is usually tested and reported, i.e., 10-200 µg mL-1.55,56 One possible reason to the observed loss of cell viability for nGO at 10 000 µg mL-1 is the acidic environment that caused a decrease in pH due to high concentration of nGO that is rich in carboxylic acid groups (see Figure 10). Additionally, nGO was not dialysed in contrast to r-nGO-CA and carried traces of nitrogen and sulphur as demonstrated by the XPS results. Previous studies have shown that size57 and surface charge58 of the carbon nanodots play important role for the in vitro cytotoxicity, since the cell membranes themselves are negatively charged.



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The hydrodynamic diameter of both nGO and r-nGO-CA measured in CGM was less than 10 nm, see Figure 9. The smaller sizes displayed by the carbon nanodots in CGM might be due to less aggregation in CGM as in comparison to TEM and AFM. However, DLS size characterization of carbon nanodots is problematic due to their anisotropic morphology and fluorescence properties which results in a diameter that is somewhat over or under estimated.59–61 In addition the AFM and TEM characterizations were made on dispersions in acetone and similar as for polymers and polyelectrolytes, different solvents affect the size as if the molecules are fully extended or coiled-up in different degrees.62 Therefore the selected solvents will also affect the self-assembly and estimated size of the carbon nanodots.

Figure 9. Relative cell viability of nGO and r-nGO-CA as a function of concentration (left), optical microscopy images at highest concentration where the cells are still alive (middle) and hydrodynamic diameter for nGO and r-nGO-CA in CGM at 0.025 mg ml-1.

The zeta (ζ)-potential of nGO and r-nGO-CA were negative in deionized water and CGM at 25°C, see Figure 10. This could contribute to the good cell viability of the carbon nanodots in CGM since previous reports have found that positively charged particles were more toxic to cells than negatively charged particles.58,63 CGM contains series of amino acids, iron and other salts, some of these could interact and adsorb on the surface of the carbon nanodots, which could explain the evident differences in ζ-potential values in CGM as compared to D.I. The ζ-potential of plain CGM is -11.4 mV and is only slightly changed with addition of nGO 22 ACS Paragon Plus Environment

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and r-nGO-CA. Additionally, the pH of the solutions measured at 25 µg mL-1 (see Figure 10) influences the charges on the carbon nanodots and subsequently the measured ζ-potential values.

Figure 10. pH as function of the concentration of nGO and r-nGO-CA in CGM at 37°C (top) and ζ-potential for nGO and r-nGO-CA in deionized water (D.I.) and CGM at a concentration of 25 µg mL-1 at 20°C (bottom).

CONCLUSIONS A green strategy for fabrication of biobased reduced nano-graphene oxide was demonstrated. Cellulose derived nGO was successfully reduced in a microwave process using superheated water without (r-nGO) or with the presence of additional green reducing agent, caffeic acid (CA) (r-nGO-CA). Two environmentally benign and straightforward routes were thus, shown where the addition of CA further enhanced the reduction efficiency. This was proven by the even higher decrease of oxygen functional groups and higher C/O ratios as displayed through FTIR and XPS and further confirmed by XPS. This was also supported by the thermal stability that increased from r-nGO to r-nGO-CA and was in both cases significantly higher than the thermal stability of nGO as determined by TGA. The morphological observations 23 ACS Paragon Plus Environment


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revealed zero dimensional characteristics of few layered structures. In addition, the morphological characterization by different techniques and in different solvents, indicated solvent dependent self-assembly or self-aggregation behaviour, with the measured average sizes varying from under 10 nm to slightly over 100 nm. According to the in vitro cell viability assays r-nGO-CA exhibited non-toxic behaviour towards osteoblastic MG63 cells at up to 1000 µg mL-1 and nGO up to 200 µg mL-1. The cell viability loss for nGO at concentrations above 200 µg mL-1 was probably caused by a decreased pH with increased nGO concentration due to its high concentration of carboxyl groups. The carbon nanodots also showed interesting fluorescence behaviour with excitation dependent photoluminescence. As such, r-nGO and r-nGO-CA may find use as nano-enhancers in composite materials in a variety of fields.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: The supporting information includes SEM-EDX results of the C/O ratios, additional XPS data, fluorescence spectra of nGO, r-nGO and r-nGO-CA in acetone.

AUTHOR INFORMATION Corresponding author *Email: [email protected] Notes The authors declare no competing financial interest. Author contribution


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The manuscript was written through contribution of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The Swedish Research Council (VR) is acknowledged for the financial support (contract grant number 2014-4091).

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For Table of Contents Use Only

Synopsis A green microwave-assisted route turns cellulose to reduced nano-graphene oxide without use of toxic reducing agents.


Green Strategy to Reduced Nanographene Oxide through Microwave

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