A simplified Hummers’ method was used to produce large-area graphene oxide nanosheets. The large lateral dimension graphite flakes, which were used to produce the large-area graphene oxide, are shown in , and the resulting graphene oxide is shown in . The large graphene oxide has an average area of 7000 μm2 and a lateral dimension of up to 150 μm. After being sonicated for 1 hour, the average area is significantly reduced and the lateral dimension is reduced to <5 μm, as shown in .
Field emission scanning electron microscopy images of (A) graphite flakes, (B) large area graphene oxide sheets, and (C) sonicated graphene oxide sheets.
To prepare graphene hydrogel, graphene oxide underwent hydrothermal treatment at 180°C for 24 hours. The physical appearance of the prepared graphene hydrogel is shown in , with graphene hydrogel prepared at a concentration of 2 mg/mL of graphene oxide, illustrating the largest cylindrical shape with an approximate diameter of 15 mm and height of 30 mm. The surface area of graphene HG-2 was 202.4 ± 2.9 m2
/g compared with the surface area of graphite of approximately 10 m2
As expected, the size of graphene hydrogel was smaller when the concentration of the graphene oxide was reduced. Graphene hydrogel was produced at a concentration as low as 0.5 mg/mL. Previously reported results demonstrated precipitation at these low concentration levels.15
This can be explained by the large-area graphene oxide used in this experiment. It was found that graphene oxide concentration was not the only factor affecting the size of graphene hydrogel; it was also affected by the dimension of the graphene oxide used. The graphene hydrogel formed using graphene oxide that had been sonicated for 1 hour (with a lateral dimension of <5 μm) has a smaller size compared with the nonsonicated graphene oxide, even though the initial concentration of graphene oxide in both the syntheses was 2 mg/mL, as shown in .
Photographs of (A) graphene HG-0.1 (i), graphene HG-0.5 (ii), graphene HG-1 (iii) and graphene HG-2 (iv), and (B) comparison of graphene HGS-2 (i) and graphene HG-2 (ii).
Field emission scanning electron microscopy images of the samples have well defined and interlinked three-dimensional graphene sheets forming a porous network that resembles a loose sponge-like structure, as shown in . The pore walls are made up of thin layers of graphene sheets. The supercritical condition resulted in the graphene oxide sheets converging, overlapping, and coalescing to form cross-links, which give rise to the framework of the graphene hydrogel. The mobility of the large graphene oxide sheets in solution is strongly limited, causing these graphene oxide sheets to orientate randomly in a hydrogel. Moreover, the large conjugated basal planes make the graphene oxide sheets stiff and able to form a stable network.32
The pore size of the graphene hydrogels produced from large area graphene oxide is relatively independent of the concentration of graphene oxide over the measured concentration range (0.5–2 mg/mL). In contrast, graphene HG-2, which was produced from the small-area graphene oxide, has a much smaller pore size than that of graphene HG-2. A plausible explanation for the well-defined large pore size leading to the well-formed cylindrical structure of graphene HG-2 is that the high concentration of large graphene oxide restricts the expansion and flexibility of graphene oxide sheets within the geometry of the autoclave, which is crucial in constructing the three-dimensional microstructure.15
On the other hand, prolonged sonication for 1 hour shattered the graphene oxide sheets (see ) which resulted in a much smaller pore size and much finer pore walls in the network of graphene HGS-2, resulting in the malformed cylindrical shape. Therefore, the pore size of the network is dependent on the lateral size of the graphene oxide nanosheets.
Field emission scanning electron microscopy images of (A) graphene HG-2 and (B) graphene HGS-2.
X-ray diffraction patterns for graphene hydrogel are shown in . The diffraction peaks are 26.5° for pristine graphite, 9.1° for graphene oxide, 13.5° for graphene HG-0.5, 13.7° for graphene HG-2, and 13.3° for graphene HGS-2, corresponding to a layer-to-layer distance of 3.36
, and 6.64
, respectively. The interlayer distance for graphene oxide is significantly larger than for pristine graphite due to the intercalating oxide functional groups.33
The interlayer distance for graphene hydrogel is lower compared with graphene oxide, indicating removal of the oxide functional groups from the graphene sheets after the hydrothermal process. Meanwhile, the appearance of a characteristic peak of graphite for nonsonicated graphene hydrogel, which shifted slightly to the left, suggests the existence of π–π stacking between graphene sheets due to the recovery of a π-conjugated system from the graphene oxide nanosheets upon hydrothermal reduction. Their interlayer spacing was slightly higher than for graphite, which is 3.63
, respectively, for graphene HG-0.5 and graphene HG-2. The diffraction peak and interlayer distance of graphene HG-2 is the closest to that of graphite because of its relatively high concentration. The characteristic graphite diffraction peak is missing from the graphene HGS-2 sample. This can be explained by the small lateral size of the graphene nanosheets, as shown by a small pore size and cotton-like appearance in field emission scanning electron microscopy (). Although some layering is likely to be present due to self-assembly of the graphene oxide nanosheets through van der Waals forces and hydrogen bond interactions, the graphene is disordered enough not to produce the signature π–π stacking diffraction peak.21
The broad peaks of all the graphene hydrogel are indicative of poor ordering of graphene sheets along their stacking direction, which reflects that the three-dimensional structure is made up of a low degree of interlayer separation that mimics graphite.
X-ray diffraction patterns of (A) graphite flakes, (B) graphene oxide nanosheets, (C) graphene HG-0.5, (D) graphene HG-2, and (E) graphene HGS-2.
To verify the formation of graphene oxide and graphene hydrogel, the infrared spectra of the samples were measured and are compared in with the spectrum taken from graphite. In the infrared spectrum for graphite (), a peak occurs at approximately 860 cm−1
and is attributed to the aromatic C–H bonds.34
The trough-like absorption peaks located between 1240 and 1590 cm−1
are associated with the stretching vibrations of both double and single C–C bonds and the stretching of C–H bonds,24
whilst the peak centered at 1600 cm−1
is assigned to skeletal vibrations of unoxidized graphitic domains.35
For graphene oxide, as shown in , the characteristic vibrations include the appearance of the broad peak from 900 to 1200 cm−1
attributed to C–O stretching, a C–O–C peak at 1246 cm−1
, and a C–OH peak at 1400 cm−1
The disappearance of the peaks between 1440 cm−1
and 1590 cm−1
shows that the C=C bonds have been oxidized during the chemical oxidation process. Graphene oxide exhibits a similar peak to that of graphite at 1620 cm−1
, signifying nonexfoliated graphite. On the other hand, graphene hydrogel regained most of the graphitic features as illustrated in , indicating the reduction of graphene oxide during the hydrothermal process.
Fourier transform infrared spectra of (A) graphite flakes, (B) graphene oxide, and (C) graphene hydrogel.
Cell proliferation on the surface of graphene hydrogel was studied using an MTT assay analysis. illustrates cell proliferation on graphene HG-2 after a culture period of 1, 3, 5, and 7 days and by measuring the optical density at a wave-length of 570 nm. On the first day of culture, cell proliferation was observed on the graphene hydrogel. The biocompatible graphene hydrogel could potentially stimulate interaction between cells and the material, which may result in an ideal condition for cell adhesion and regeneration. By increasing the culture time to 3 days, the cells continued to flourish on the hydrogel, although there was a drop in the proliferation rate. This could be due to the formation of confluent cellular layers on the surface that lowered the growth rate of cells.36
However, there was a tremendous decrease in cell proliferation on the fifth day, although there was an increase in cell proliferation rate on the seventh day. Similarly, a decrease in cell viability was also observed by Robinson’s group after 2 days of treatment with nanoreduced graphene oxide28
and Chang’s work after 1 day of graphene oxide exposure, as a result of oxidative stress.27
Optical density measurement of cell proliferation on graphene HG-2 and control after 1, 3, 5, and 7 days of culture time (P < 0.001, n = 3).
The MTT results are in good agreement with the observation of cell anchorage on the surface of graphene hydrogel. shows the field emission scanning electron microscopy results for graphene HG-2, and when compared with the original field emission scanning electron microscopy image of the original surface, the cell adherence, spreading, and growth on the surface of the hydrogel after 7 days of culture time is apparent. The images display extensive flaky layers of adhered cells with rough cellular surfaces even from the early stage of day 1, when the hydrogel was entirely covered with cells. The confluent layer appeared to be thicker after 3 days. At higher magnifications, graphene hydrogel demonstrated pronounced cell attachment where cells with flattened morphology were seen to adhere to each other, with cellular microextensions on the third and seventh days of culture as illustrated in , which is similar to the observation of Bodhak et al.36
Guided filopodia protrusions, as indicated by the arrows in , were probably caused by the enhanced cell adhesion of MG63 on graphene hydrogel.37
Additionally, the morphology of the spreading MG63 cells suggests their high adaptation to the as-prepared graphene hydrogel substrate. Park et al demonstrated similar cell morphologies in their in vitro study, which involved the culture of MC3T3-El on a highly organized ZnO nanoflower array.38
Field emission scanning electron microscopy images of the cells grown on graphene HG-2 after culturing for 1, 3, 5, and 7 days. All the images share the same scale bar.
Guided filopodia protrusions of MG63 on graphene HG-2 after 3 and 7 days of culture time observed at higher magnifications.