Yingcan
Zhao
and
Chad T.
Jafvert
*
Lyles School of Civil Engineering and Division of Environmental and Ecological Engineering, Purdue University, West Lafayette, Indiana 47907, USA. E-mail: jafvert@purdue.edu; Tel: +1 (765)494 2196
First published on 2nd March 2015
Graphene oxide (GO) is a carbonaceous nanomaterial that is a precursor material in the preparation of graphene, and because of its unique properties, it will likely be used in a number of industrial and consumer products in the future. Despite its name, it contains many epoxy, hydroxyl, and carboxyl functional groups on its edges and surface, making it easy to suspend in water. However, how it is transformed or mineralized in natural aquatic environments and its effects on natural processes within these environments remain largely unknown. Therefore, in this study, we report on the photochemical reactivity of single layered GO dispersed in water and irradiated with light within the solar spectrum that reaches the water bodies at the earth's surface (λ ≥ 300 nm). Upon irradiation, the visible color of a 5 mg L−1 GO suspension shifted from pale to dark brown, possibly indicating the repair of some of the π-bond structures; however, Raman spectroscopy indicated an increase in nonaromatic defects. To further examine how oxidation or reduction of the GO surface may occur upon solar light irradiation, we probed the production of various reactive oxygen species (ROS). By monitoring ROS production with selective and highly reactive chemical probes, formation of superoxide anions (O2˙−), but not single oxygen (1O2) or hydroxyl radicals (·OH), was detected, indicating electron transfer from GO to dissolved molecular oxygen (O2). However, further electron transfer through reduction of O2˙− did occur, as hydrogen peroxide (H2O2) was found to accumulate, forming 3 μM H2O2 in a suspension of 5 mg L−1 GO after 4 hours of irradiation.
Nano impactIn addition to fullerenes and carbon nanotubes, graphene and graphene oxide are major types of carbon-based nanomaterials. As a precursor material in the preparation of graphene, and because of its unique properties, graphene oxide (GO) will likely be used in a number of industrial and consumer products in the future. The types of products in which it will find application will partially depend on whether inclusion within these products contributes to human and environmental exposure, and the degrees of risk associated with these exposures. Because it contains many hydrophilic functional groups, it is easy to suspend in water. Hence, if it is released to the environment, exposure is likely to occur in aquatic environments. Yet, very limited research has been conducted regarding the environmental fate of GO, its transport mechanisms in the environment, or its toxicity to aquatic species. As a result, this study investigates the photochemical reaction of GO as it is one of the more likely fate processes acting on GO in aquatic environments. This study provides evidence that GO is chemically altered upon irradiation with light within the solar spectrum and that GO serves as an electron donor, transferring electrons to molecular oxygen to form reactive oxygen species (including superoxide anions and hydrogen peroxide). This type of information is critical for assessing potential impacts of graphene oxide on aquatic environments. |
Because it is easy to disperse in water, GO has been studied both as a potential aquatic pollutant and as a potential material to selectively kill cancer cells due to its toxicity to human cells.6,10 For example, Liao et al. observed that sonicated (smaller) GO exhibited greater hemolytic activity to human cells compared to larger GO materials. Viability assay experiments revealed that both graphene and GO have toxic effects on human skin fibroblasts.11 Another research group reported that single-layer GO had dose-dependent toxicity to human lung epithelial cells and fibroblasts and caused obvious toxicity when doses were above 50 mg L−1, indicating risk to human health.12 Hu et al. found that GO could disrupt cell membranes by direct interactions occurring between cell membranes and GO nanosheets.13 In contrast, another research group used different sized GO and found that all the different sized materials showed no toxicity to A549 cells, which are typical human lung cells.14 The variability among the results might be attributed to the various methods by which GO and graphene were produced or suspended in water. However, there is little doubt that GO might cause some toxicological effects on humans, motivating further studies on the fate and effects of GO in the natural environment. Environmental effects may include toxicity to micro-organisms and other organisms further up the food chain. Indeed, Akhavan et al. showed that hydrazine-reduced GO was more toxic to bacteria than the parent GO, and suggested that this resulted from the “sharper” nanowalls on the reduced GO (RGO).15
Because the disrupted π-bond structure in GO absorbs a significant amount of light within the solar spectrum, environmental fate processes acting on GO are expected to include photochemical processes. Indeed, it is well known that photo-irradiation is a good method for “reducing” GO, at least with lamp light that occurs in UV regions which may or may not occur solely within the solar spectrum.16 For example, in a study reporting on photo-reduction of GO conducted by Matsumoto et al.,16 experiments were performed using a xenon lamp of unknown spectral output, although this type of lamp generally emits light down to 280 nm, approximately 20 nm below the spectral limit of solar light measured at the earth's surface. Matsumoto et al., however, did measure CO2 and H2 generation from aqueous GO suspensions and noted a drastic decrease in H2 production when xenon lamp light was filtered through a 390 nm cutoff filter. While not reported, it seems likely that a similar decrease in CO2 production would occur under a 390 nm cutoff filter. It is somewhat interesting that while the overall reaction can be termed photo-reduction of GO, it is not clear if any of the remaining carbon in GO has actually been reduced, as the simple loss of CO2 from the carboxyl groups of GO is the result of a rearrangement reaction, where the carbon–carboxylic acid bond is broken and replaced with a carbon–hydrogen bond. Hence, although the average oxidation state of carbon in GO is lower, it may result from the loss of carbon that was already highly oxidized, as previously reported to occur during photolysis of carboxylated multi-walled carbon nanotubes,17 and not from any oxidation reaction of specific carbon atoms in GO. Similar to the lack of information in the literature regarding photochemical carbonaceous products, there is a general lack of information on the generation of reactive oxygen species (ROS) by GO under solar light. Hou et al.18 irradiated an aqueous dispersion of single layer GO with light not strictly within the solar spectrum (at energies in the range 3.94–4.43 eV; i.e., at λ = 280–315 nm) and, similar to Matsumoto et al.,16 noted that GO became visibly darker over the irradiation period, but suggested through XPS analysis that this occurred due to the loss of hydroxyl groups through hemolytic removal of ·OH groups and formation of more conjugated π-bonds, rather than through decarboxylation alone. Further, through electron paramagnetic resonance spectroscopy, dose-dependent exponential growth in radical production on the GO surface was shown to occur, presumably after the loss of ·OH; however, it was reported that ·OH was not observed, and the methodology of ·OH detection was not reported.
For other carbon-based nanomaterials, several reactive oxygen species have been shown to be generated under sunlight conditions (λ > 300 nm). For example, singlet oxygen (1O2) was generated in aqueous suspensions of C60 clusters (i.e., nano-precipitates), oxidizing C60 to more polar water-soluble products.19–21 Aqueous dispersions of carboxylated and PEG-functionalized single walled carbon nanotubes (SWCNTs) produced a significant amount of ROS, including singlet oxygen (1O2), superoxide anions (O2˙−), and hydroxyl radicals (·OH).22,23 As a result, in this study, the ability of aqueous dispersions of single-layered GO to generate reactive oxygen species (ROS) upon exposure to light within the solar spectrum (λ = 300–410 nm) was investigated. Based on the experimental results of this study and the previous work cited above, a mechanism for ROS generation by photosensitized GO in water is proposed. Information on ROS generation during solar irradiation is significant not only to evaluate the ecological risks associated with GO, but also to better understand the transformation pathways of carbon within GO.
Raman spectroscopy is often used to probe the differences in the electronic properties of carbon nanomaterials.30Fig. 2 shows the Raman spectral changes in GO as a function of irradiation time. The spectra show D, G, and 2D bands, which are three characteristic peaks of GO. The G band occurs at 1580 cm−1 which results from the vibration of sp2-bonded carbon, and is an indication of the relative extent of aromaticity. The D band at 1350 cm−1 is assigned to the vibration of sp3 carbon atoms (i.e., nonaromatic carbon). The relative intensity, or the ratio of the D-to-G band intensities (ID/IG), is often used as a qualitative measure for the degree of disorder caused by nonaromatic sp3 carbon defects that often occur at the edges or as ripples or holes within the GO structure.31Fig. 2 shows that after 24 h of irradiation, the ID/IG ratio increased from 0.45 (0 h) to 0.68 (24 h). This increase suggests an increase in the number of defects (e.g., functionalized carbon) on the already functionalized graphene oxide sheets. These defects may be sites for ROS production, which is discussed subsequently.
Fig. 2 The Raman spectra of GO before and after irradiating an aqueous GO suspension (100 mg L−1) for 24 hours, where the spectra have been normalized to the intensity of the G band. |
By monitoring ROS production with selective and highly reactive chemical probes, formation of O2˙−, but not 1O2 or ·OH, was detected. Irradiated samples containing furfuryl alcohol as a scavenging probe for 1O2 showed no decay in furfuryl alcohol after 24 h of irradiation (Fig. S3†). However, in GO suspensions containing XTT, a significant increase in light absorbance at 470 nm occurred upon irradiation and after filtering out GO after irradiation over time. This increase in absorbance occurs when the reaction product between XTT and O2˙− has an absorbance maximum22 (Fig. 3). Fig. 3a also indicates that addition of SOD almost completely inhibited XTT reduction, further suggesting that XTT product formation was caused directly by the reaction of XTT with O2˙− as SOD rapidly converts O2˙− to H2O2 through a disproportionation reaction, reducing the amount of the XTT product formed. In the absence of XTT, the irradiated and dark control GO samples showed little change in absorbance at 470 nm over the same time period. Note that because the light absorption spectral changes shown in Fig. 3b (and reported at 470 nm in Fig. 3a) are on samples filtered through 0.2 μm membrane filters (i.e., after removal of GO), the increase in absorbance at 470 nm is due only to XTT product formation and not to the increase in light absorbance caused by GO on unfiltered samples, as shown in Fig. 1. An image showing the course of the reaction from 0 to 3 hours for a 5 mg L−1 GO suspension containing 0.1 mM XTT, prior to filtration, is provided in the ESI† (Fig. S4). Even with the increase in the overall absorbance caused by GO, the pink product of the reaction between O2˙− and XTT is evident.
With significant O2˙− formation, there is a high probability that hydrogen peroxide will form also, and potentially accumulate in solution. As noted above, H2O2 can be formed by disproportionation of O2˙− with enzymes such as SOD, accelerating the rate of the reaction considerably. As the disproportionation reaction suggests, conversion can occur also through transfer of another electron to the protonated form of the superoxide anion, HO2· (hydroperoxyl radical), which upon electron transfer extracts a proton from solution to form H2O2. Whether it occurs by disproportionation or another electron transfer process, both electrons must originate from GO in the absence of an additional electron donor. Hence, accumulation of H2O2 was measured by using the DPD–horseradish peroxidase (HRP) assay, with DPD/HRP added to the aqueous samples after irradiating the GO dispersions, and then after removing GO by filtration. Although H2O2 is somewhat reactive in this system, it has a much longer half-life than the other ROS, even under solar light irradiation. Fig. 4a clearly shows that the H2O2 concentration in the GO dispersions increased over an irradiation period of 4 h, whereas no increase occurred in the dark control samples or in the absence of GO. The H2O2 concentrations shown in Fig. 4a are calculated from the sample absorbance values measured at 551 nm after filtration, using the H2O2 standard curve shown in Fig. 4b. Hence, the initial values at time zero of 0.2–0.5 μM are due to absorbance reading at or below 0.005, and likely due to trace contamination resulting in a small positive interference. Despite this, the results indicate that H2O2 was indeed produced and accumulated during irradiation of 5 mg L−1 GO with light within the solar spectrum, accumulating to over 3 μM after an irradiation period of 4 h. Assuming a carbon content of 80% in the original GO, the 5 mg L−1 GO concentration translates to a molar concentration of 3 mM carbon. Hence, over the 4 h irradiation period, approximately 1 molecule of H2O2 was produced for every 1000 carbon atoms in GO.
Although it is known that sunlight can cleave the oxygen–oxygen bond in H2O2 to form ·OH, the reaction is slow.23,32 Alternatively, ·OH may be formed more rapidly if transfer of an additional electron from GO to H2O2 occurs, as was found to be the case for carboxylated single walled carbon nanotubes under solar irradiation.23 In order to determine whether there are hydroxyl radicals produced by GO, pCBA was added to some samples, as it rapidly scavenges ·OH resulting in the loss of pCBA. However, no pCBA decay was observed for both irradiated and dark control samples over the 24 hour time period of the experiments (see the ESI,† Fig. S5), suggesting that a negligible amount of ·OH was produced or that ·OH scavenging by GO was rapid and significant, reducing its pseudo-steady-state concentration. Although scavenging of ·OH by GO is likely to occur, as the initial electron transfer that results in its formation would occur at the GO surface such that the site of its generation would be in close proximity to GO π-bonds at which it could be consumed, it is also likely that not much is produced, otherwise the concentration of its precursor, H2O2, would not accumulate to such a degree, and the chromophore content of GO would not be enhanced as irradiation proceeds.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4en00209a |
This journal is © The Royal Society of Chemistry 2015 |