Sequential Surface-Modification of Graphene Oxide

The formation and growth of ice crystals is considered to be a critical issue in aerospace and automotive industries as well as in cryopreservation of cells. Pure water undergoes homogenous nucleation of ice at ~ −40 °C. However, the presence of dusts, minerals, birch and conifer pollen and some species of fungus could serve as a nucleator and promotes nucleation of ice much above this temperature. Carbon nanotubes, graphene nano-flakes and carbon soots (from burning of fuels) are promising candidates to promote nucleation of ice crystals.

Base-washing has been shown to be effective in removing oxidative debris from graphene oxide (GO) and enables effective functionalization of the surface of GO with thiols, Au nanoparticles and polymers. Base-washed graphene oxide (bwGO) is a distinct graphene-like material with better qualities than the normal GO. Researchers at Department of Chemistry, Warwick Medical School and Department of Physics, University of Warwick, UK have suggested that surface modification of bwGO would offer a versatile template to evaluate the potential of 2D carbon nanomaterials as ice-nucleating agents as well as to serve as a versatile scaffold to probe the role of surface chemistry.

GO was synthesized by Hummer’s method. About 140 mg of GO was re-dispersed in 250 ml of deionized H₂O by mild sonication followed by addition of 0.140 g of NaOH and heating of the solution to 70 °C for 1 h. The resultant dark brown solution was centrifuged (@12,500 rpm for 30 min). The dark brown solid was washed with water and re-centrifuged. The solid was re-protonated using 0.014 M HCl at 70 °C for 1 h, filtered, thoroughly washed with deionized H₂O and dried under vacuum to yield bwGO (a black solid), which was dispersed in a H₂O/CH₃CN mixture via sonication. Poly(N-isopropylacrylamide), (pNIPAM) with degree of polymerization of 55 and 140 were prepared by polymerization of N-isopropylacrylamide (Fig. 1). pNIPAM hexanethiol, dodecanethiol and octadecanethiol were grafted on the surface of bwGO under Schlenk conditions in N₂ atmosphere (Fig. 2).

Fig. 1 Scheme depicting polymerization of N-isopropylacrylamide

Fig. 2 Scheme depicting polymerization of N-isopropylacrylamide and grafting of polymers and thiols on the surface of base-washed graphene oxide

The ice nucleation activity of unmodified and surface modified GO was quantified by determining the average nucleation temperature to freeze a droplet (1 μL) of water. The droplets were cooled under an atmosphere of dry nitrogen, and the freezing point of each droplet was recorded by visual observation using a microscope. When tested for the nucleation activity, ultra-pure Milli-Q water nucleated at -26 °C, suggesting a heterogeneous nucleation (Fig. 3); Both bwGO and bwGO-Cyst increased the nucleation temperature by over 5 °C, to -20 and -18 °C (Fig. 4).

Fig. 3 Ice nucleation assay: No water droplet is frozen at -20 °C; At -23 °C, two water droplets (marked by red circles) are frozen while all water droplets are frozen at -30 °C.

Fig. 4 Comparison of ice nucleation activity of Milli-Q water, GO and cysteine-functionalized GO

A remarkable nucleation promotion activity is observed for bwGO surface modified with alkane thiols; octadecanethiol modified bwGO increased the nucleation temperature by > 15 °C to –12 °C (Fig. 5(a)). All the alkyl modified GOs are more active than bwGO and the cysteine modified bwGO, which suggests that the increased hydrophobicity plays a dominant role in determining the ice nucleation. The similar activity of pNIPAM-bwGO with that of bwGO (Fig. 5(b)) suggests that modification of the surface of bwGO with polymer molecules exert a very little influence on the ice nucleation temperature.

Fig. 5 Comparison of ice nucleation activity of (a) Milli Q water, GO and GO functionalized with hexanethiol, octadecanethiol and dodecanethiol; and (b) Milli Q water, GO, pNIPAM55 and pNIPAM140.

The surface modified bwGO may find application in cryopreservation and cloud seeding.

T.S.N. Sankara Narayanan

For more information, the reader may kindly refer: Caroline I. Biggs et al., Impact of sequential surface-modification of graphene oxide on ice nucleation, Phys. Chem. Chem. Phys., 2017,19, 21929-21932

Achieving Enhanced Antibacterial Activity by Suitably Aligning Graphene Oxide Nanosheets

Graphene-based nanomaterials (GBNs) due to their exceptional mechanical, electronic, and thermal properties assumed significance in a variety of applications. The cytotoxic properties of GBNs are also important for their biomedical applications. GBNs have been shown to be cytotoxic toward a variety of cell types. However, the impact of alignment of nanosheets on the antibacterial activity has not been established. Researchers at Department of Chemical and Environmental Engineering, Yale University, USA, have investigated orientation-dependent interaction of graphene oxide (GO) nanosheets aligned in different orientations using a magnetic filed with Escherichia coli (E. coli). The GO nanosheets with vertical orientation exhibit an enhanced antibacterial activity when compared to those with random and horizontal orientations and the mechanism responsible is also suggested.

The schematic illustration of alignment of GO nanosheets with different orientations using magnetic field and alignment quality of GO nanosheets suspended in the monomer solution at different field strengths evaluated by 2D small-angle X-ray scattering (SAXS) are shown in Fig. 1.

Fig. 1 (a) Schematic illustration of alignment of GO nanosheets with different orientations using magnetic field; and (b) alignment quality of GO nanosheets

The various stages involved in the fabrication of GO composite films is shown in Fig. 2. Suspensions of GO nanosheets (with a thickness of ∼0.8 nm) in 2-hydroxyethyl methacrylate (HEMA), doped with cross-linker and photo initiator, were sealed between two glass substrates with a 300-μm spacer and aligned in a magnetic field of 6 T. Samples were subsequently cross-linked under UV irradiation to form polymer films, which preserved the orientation of the aligned GO nanosheets. The composite films were then detached from the glass substrates and irradiated using UV/O₃ to etch away the outer polymer and expose GO nanosheets on the surface. The resultant films are tough, mechanically coherent and resistant to water swelling, which are critical in preserving the GO orientation in aqueous environments.

Fig. 2 Various stages involved in the fabrication of GO composite film

The GO composite films were contacted with E. coli in suspension for 3 h. The bacteria attached on the surface were stained using SYTO 9 dye and propidium iodide and evaluated for live and dead cells. The vertical-GO film showed a lower cell viability (56.0 ± 8.7%) when compared to those with random (75.3 ± 3.5%) and planar (81.8 ± 5.1%) orientation. Morphological features indicate that E. coli on No-GO film showed an intact cell morphology, indicating no cytotoxicity of the pure polymer. E. coli on planar- and random-GO films largely retained their morphological integrity whereas cells on vertical-GO films became flattened and wrinkled, suggesting loss of viability and possible damage to the cell membrane (Fig. 3).Fig. 3 SEM micrographs of E. coli cells on etched GO composite films. The scale bar is 1 μm.

The mechanism for the enhanced antibacterial activity of vertically aligned GO nanosheets is explained based on (i) physical disruption; and (ii) chemical oxidation using lipid vesicles and oxidation of glutathione, respectively. GO nanosheets with a vertical orientation induced physical disruption of the lipid bilayer structure, resulting in loss of membrane integrity of the GO/lipid vesicle system. GO nanosheets with a vertical orientation also increased the extent of oxidation of glutathione (27.6%) with limited generation of reactive oxygen species, suggesting that the oxidation occurs through a direct electron transfer mechanism. Thus, both mechanisms contribute to the enhanced antibacterial activity of the vertical-GO film. Nevertheless, both of them require direct, edge-mediated contact with cells. The exposed edges of GO nanosheets with a vertical orientation could induce enhanced physical penetration and promote greater levels of electron transfer. Hence, the enhanced antibacterial activity of the film with vertically aligned GO nanosheets can be attributed to the increased density of edges with a preferential orientation for membrane disruption. The orientation-dependent cytotoxicity of GO nanosheets has direct implications on the design of engineering surfaces using graphene based nanomaterials.

T.S.N. Sankara Narayanan

For more information, the reader may kindly refer: X. Lu et al., Enhanced antibacterial activity through the controlled alignment of graphene oxide nanosheets, PNAS 2017 114: E9793-E9801