Chitosan-Functionalized Graphene Nanocomposite Films: Interfacial Interplay and Biological Activity

Graphene oxide (GO) has recently captured tremendous attention, but only few functionalized graphene derivatives were used as fillers, and insightful studies dealing with the thermal, mechanical, and biological effects of graphene surface functionalization are currently missing in the literature. Herein, reduced graphene oxide (rGO), phosphorylated graphene oxide (PGO), and trimethylsilylated graphene oxide (SiMe3GO) were prepared by the post-modification of GO. The electrostatic interactions of these fillers with chitosan afforded colloidal solutions that provide, after water evaporation, transparent and flexible chitosan-modified graphene films. All reinforced chitosan–graphene films displayed improved mechanical, thermal, and antibacterial (S. aureus, E. coli) properties compared to native chitosan films. Hemolysis, intracellular catalase activity, and hemoglobin oxidation were also observed for these materials. This study shows that graphene functionalization provides a handle for tuning the properties of graphene-reinforced nanocomposite films and customizing their functionalities.

Graphene oxide (GO) was obtained from graphite flakes using the Hummers method [19]. In a typical procedure, graphite flakes (5 g) and NaNO3 (2.5 g) are mixed in 150 mL of H2SO4 (98%) in a 1000 ml volumetric flask kept under at ice bath (0 °C) with continuous stirring. The mixture was stirred for 4 h at this temperature, and potassium permanganate (15 g) was added to the suspension very slowly. The mixture is diluted with the very slow addition of 200 ml water and kept under stirring for 2 h. Then, the ice bath was removed, and the mixture was stirred at 35 °C for 2 h. The above mixture is kept in a reflux system at 98 °C for 10-15 min. After cooling, the mixture kept under stirring for 2 h at 25 °C. The solution is finally treated with 40 ml of H2O2 and then 200 mL of water. Then, it is kept without stirring for 3-4 h, where the particles settle at the bottom and the remaining water is poured through a filter. The resulting mixture is washed repeatedly by centrifugation with 10% HCl and then with deionized (DI) water several times until it forms a gel-like substance (pHneutral). After centrifugation, the gel-like substance is vacuum dried at 60 °C for more than 6 h to GO powder.
Phosphorylated GO (PGO) was obtained through the phosphorylation of graphene oxide using POCl3 as the phosphorus source. The details can be found in the supporting information. In a typical procedure using POCl3 as the phosphorylating agent, 80 mg of lyophilized graphene oxide is dispersed in 250 mL of THF. The mixture is sonicated until the formation of a homogeneous suspension. Then, 42.9 mmol of K2CO3 is added to the suspension, and the mixture is kept under stirring for 3 h. Subsequently, 42.9 mmol of POCl3 is dropped into the suspension, and the mixture is kept under stirring for 4 days. The phosphorylated product is dispersed in deionized water and kept under stirring for 18 h to hydrolyze the residual Cl that did not react with GO. Finally, the product is collected through filtration and is thoroughly washed/rinsed with DI water and ethanol.  Solid-state 13 C SSNMR of graphene oxide reveals the presence of epoxide groups evidenced by the signal at 60 ppm and the presence of alcohols (signal at 68 ppm), and the signal at 131 ppm is attributed to aromatic carbons of graphitic domains.
Solid-state 13 C SSNMR of phosphorylated graphene oxide (PGO) reveals the presence of epoxide groups evidenced by the signal around 63 ppm; the formation of C-O-P bonds might be evidenced by the signal at 69 ppm, and the signal at 127 ppm is attributed to aromatic carbons of graphitic domains. In GO's Raman spectrum, two bands corresponding to the D band and the G band appear at 1354 cm −1 and 1601 cm −1 , respectively. The ID/IG found for GO is 0.87.
In the case of PGO, these two bands are shifted to lower values (1348 cm −1 for the D band and 1586 cm −1 for the G band). An increase of the ID/IG value is observed (0.91). This increase and the shift of the two bands are a consequence of the creation of more defective sites on the sheets or a decrease of the graphitic domains due to sonication.
In the case of rGO, these two bands are shifted to lower values (1349 cm −1 for the D band and 1594 cm −1 for the G band). An increase of the ID/IG value is observed (1.26). The integral C1s spectrum of GO can be deconvoluted into three components at 284.5 eV, 286.5 eV, and 288.5 eV corresponding to carbon atoms of graphitic domains, carbon atoms of epoxides and tertiary alcohols, and carboxyl and ester groups carbon O-C=O respectively.
The O1s spectrum of GO evidences the presence of oxygenated functional groups. The C1s spectrum of PGO is different from the one found for GO. The integral C1s spectrum of PGO can be deconvoluted into four components at 284.5 eV, at 286.3 eV, 287.2 and 288.7 eV, corresponding to carbon atoms of graphitic domains, carbon atoms of tertiary alcohols, carbon atoms of epoxides, and carboxyl and ester groups carbon O-C=O respectively.
The O1s spectrum of GO evidences the presence of oxygenated functional groups. The P2p spectrum is deconvoluted to one component mainly due to the presence of orthophosphate groups, since the binding energy observed indicates that phosphorus is linked to oxygen atoms.
The integral C1s spectrum of PGO can be deconvoluted into five components at 284.7 eV, 286.2 eV, 286.9, 288.3 eV, and 289.2 eV corresponding to carbon atoms of graphitic domains, carbon atoms of tertiary alcohols, and the carbon atoms of epoxides, carboxyl, and ester groups carbons O-C=O and C-Si, respectively. The 289.2 eV might be due to the subsistence of residual Si-CH3 groups upon the functionalization of GO with HMDS.
The Si2p spectrum of SiMe3GO can be deconvoluted to one component, and this is mainly due to the presence of Si-O bonds.
The N1s peak at 401.3 indicates the formation of C-NH and the peak around 399.8 eV suggests the formation of C-N + bonds.