ROS generation by reduced graphene oxide (rGO) induced by visible light showing antibacterial activity: comparison with graphene oxide (GO)

Abstract

Reduced graphene oxide (rGO) generates reactive oxygen species (ROS) under visible light in air via a singlet oxygen–superoxide anion radical pathway which readily kills Enterobacter sp. The rGO⁺ intermediate reacts with a hydroxyl ion to produce graphene oxide (GO) as a coating on the surface of rGO resulting in enhanced fluorescence and a slow down in photo-induced ROS formation. GO is not toxic but on ageing it gets a surface coating of rGO and shows toxicity.

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The synthesis of graphene oxide from graphite is dominated by the classic method introduced by Hummers et al.¹ This GO is soluble in water as it contains several functional groups like carboxylic and hydroxyl groups.² This solubility feature attracted several studies to explore its utility in diverse fields. GO is used in catalytic oxidation,^3–5 biotechnology^6–9 and as a surfactant.¹⁰ However, a straightforward readily available conversion of GO to graphene has not been observed as it is difficult to get rid of all those attached hydrophilic oxo groups in GO. Graphene oxide under reduction converts into a reduced GO form which improves the electrical conductivity.^11,12 The reactivity of GO under varied chemical environments has been investigated. One important aspect of such a reaction is the reduction of GO. It was observed that GO is changed to reduced graphene oxide (rGO) under exposure to reducing chemicals or bio-molecules including bacteria or even under bright light.¹³ This reduction of GO to rGO diminished its dispersibility in water and fluorescence properties as well.¹⁴ The physicochemical changes of GO on reduction to rGO led to the development of newer optoelectronic properties which are being used in physical and biochemical applications.¹³ It is now known that graphene and GO are generally non-toxic to humans.¹⁵ However, rGO, the intermediate species, perhaps, with no fixed stoichiometry with respect to C : O (representing the attached oxo functional groups) in between GO and graphene, has not been checked for its benign role towards human health. Therefore, we undertook the work and herein show that rGO generates ROS from aerial oxygen even under visible light and such reaction though harmful to humans may be used to combat hospital pathogens.

The GO for this work was prepared by the well-known Hummers method¹ and was characterized. This GO was reduced by using four conventional reducing agents like hydrazine hydrate, sodium borohydride, hypophosphorous acid and sodium dithionite to get rGOs of different shades (see S1†). All of these rGOs are now subjected to sunlight (or indoor tungsten lamp 60 W light irradiation) using a water filter and glass tube to cut off thermal and UV irradiation. In a typical experiment rGO, (hydrazine hydrate reduced) (0.5 mg mL⁻¹) was just dispersed mechanically in water containing 0.6 μmol nitro blue tetrazolium chloride (NBT). The pale yellow solution of the mixture slowly changed to blue under light exposure within 30 minutes (Fig. 1).

Fig. 1 (a) Blue diformazan formation on adding rGO (5 mg) in sunlight into a 0.6 μmol NBT solution; (b) the increase in diformazan formation with the increase in the light exposure time.

The change in yellow to blue colour of the NBT solution is characteristic to the formation of the diformazan dye.¹⁶ On extending the time of light exposure, the intensity of the blue colour steadily increased (a part of diformazan is precipitated out when formed in excess) to a point and on further irradiation ceased to develop further. Similar reactions in the presence of either sodium azide or dimethylsulfoxide of fresh rGO with NBT does not produce any blue diformazan. As the azide ion and DMSO are well known quenchers of singlet oxygen (¹O₂) and hydroxyl radical respectively¹⁷ it is apparent that the light induced reaction of rGO with oxygen proceeded via the formation of such species.

Thus the reaction proves that light induced excited rGO* transfers its energy to triplet oxygen (³O₂) to form singlet oxygen (¹O₂) at the initial stage and then other reactive oxygen species including hydroxyl radicals are finally formed. The next step reaction could be the excited rGO* reducing ¹O₂ to a superoxide ion, O₂˙⁻ which is known to react with NBT involving the abstraction of protons from water (Scheme 1).

Scheme 1 Reaction scheme for photo-induced superoxide generation by rGO, the self generation of other ROS including OH radicals from superoxide radical anions has not been shown.

Therefore diformazan formation increases the pH of the reaction medium with progress in time (Fig. 2a) and this generated OH⁻ can react with rGO⁺ to produce GO (Scheme 1). The alternate direct hydroxylations by the hydroxyl radicals of rGO to produce GO is possible. The formation of hydroxyl radicals is anticipated as observed by the quenching ability of DMSO in this reaction.¹⁷

Fig. 2 (a) The increase in pH of the NBT–rGO solution on exposure to sunlight λ ≥ 300 nm with time intervals of 15 min. (b) The slowdown of diformazan formation (O₂˙⁻ generation by rGO on its reuse in several cycles (photo-exposure time per cycle was an hour)), (c) electronic spectrum of the formed GO in ethanol (i) where rGO is virtually insoluble (ii) (broken line).

Whether rGO is catalytic or not has now been tested by re-using the used rGO with fresh NBT solution in several subsequent cycles. We observe that the production of diformazan decreases gradually in subsequent cycles (Fig. 2b). This shows that after every cycle the ability of rGO to generate superoxide radicals reduces. Interestingly, washing the used rGO with 10% sodium hydroxide followed by its acid wash workup with 6 N HCl and drying under anaerobic conditions in the dark showed a weight loss of around 30% but the residual alkali–acid treated rGO regains its ability to produce superoxide anions (diformazan production) from air under exposure to light. The NaOH leached part on neutralization with dilute HCl acid and after evaporation has been checked. This residue on extraction with ethanol when subjected to electronic absorption displayed the spectrum of GO as shown in Fig. 2c. Interestingly rGO is not soluble in ethanol (Fig. 2c). Therefore such an apparent solubility difference between GO and rGO can readily be made to identify both the species.

Furthermore the alkali washes were found to be fluorescent (not shown) and this part contains GO as shown in Fig. 2c. The leaching of GO in alkaline medium showing enhanced fluorescence has been reported.¹⁸ These results led to the conclusion that the excited rGO donates an electron to singlet oxygen to create a superoxide ion. The NBT–superoxide–water reaction generates diformazan dye liberating HO⁻ ions. This hydroxyl ion reacts with rGO⁺ to produce GO which may coat the surface of the insoluble bulk rGO to passivate it against subsequent reactions occurring. The superoxide anions generate other reactive oxygen species (ROS) like hydroxyl radicals that may directly react with rGO to produce GO. This establishes that the activity of rGO to produce superoxide is hindered by the deposition of GO on its surface produced by light induced ROS. The general belief that GO generates superoxide radicals from aerial oxygen and thus is toxic because of the production of ROS is wrong as freshly prepared GO has been checked and it has been found that it does not have the capability to generate superoxide radicals in air under light exposure (see S2†). These experiments when carried out in phosphate buffer saline (PBS) (0.01 M) in the pH range between 6.8 and 7.4 under indoor tungsten light (60 W) showed a similar result indicating that rGO is capable of producing ROS to damage cells under physiological pH. The starting rGO and light exposed rGO at pH 6.8 have been subjected to fluorescence microscopy to show that rGO does not show any observable fluorescence but after tungsten light irradiation fluorescence at all three visible wavelengths was observed (Fig. 3) which is characteristic to GO.

Fig. 3 (i) Fluorescence of fresh rGO, (ii) fluorescence of rGO after light exposure in air for two hours in PBS buffer, pH 6.8. The excitation wavelengths are (a) 385, (b) 488, (c) 561 nm.

This ability of rGO to generate ROS under indoor light in air made us research its antibacterial property. This is more important to combat strains like New Delhi metallo-beta-lactamase-1 (NDM-1) producing Enterobacteriaceae.¹⁹ Enterobacter sp. was chosen to find the effect of rGO under light and air. 20 μL of Enterobacter sp. suspension was inoculated in two sets of nutrient broth (Hi-Media Laboratories, India) separately. To one of these sets 0.5 mg rGO was added in the culture media inoculated with Enterobacter sp. and another set of Enterobacter sp. culture was used as a control. Both of the sets were allowed to stand under 60 W glowing tungsten bulb light for 2 hours at 37 °C. Then these bacterial cultures were incubated for 24 hours at 37 °C. After 24 hours, 10 μL of cell suspension from both the rGO-treated and control were taken and smeared onto two clean glass slides separately. The smears were observed under a Nikon inverted microscope (Fig. 4). A similar operation when carried out under argon (in the presence of the necessary amount of CO₂ in all cases) treated incubation of the bacterial culture showed a distinct difference in the growth rate. The reduced growth of the bacterial cells in the presence of rGO when compared with the control clearly established the influence of rGO in generating superoxide. The traces of air present under argon medium resulted in residual proliferation of the cells.

Fig. 4 Activity of rGO on the growth of Enterobacter sp. treated under visible light: (a) without rGO, (b) with rGO, (a and b) in air, (c) without rGO and (d) with rGO, (c and d) in argon.

This clearly suggests that microbial contamination can be readily avoided by using rGO to combat the growth of hospital pathogens. The gradual loss of the activity of rGO may be replenished by the reduction of the formed GO coating using hydrazine vapour or by washing off the GO coating with an alkali.

Conclusions

In summary this work demonstrates that handling of rGO under ambient conditions in air and under visible light should be carried out with caution as it readily generates harmful ROS. To combat hospital pathogens, light and air should be used carefully and it should be stored in the dark when it is not needed. This work reveals a caveat that one should use GO with the utmost care as it gets slowly reduced even in the open environment on ageing with the formation of a surface coating of non-stoichiometric rGO. Such toxicity is related to ROS generation and this is due to the rGO contamination and not GO.

Acknowledgements

RS thank Dr D. S. Kothari Post-doctoral fellowship, BP acknowledges the support of CSIR, New Delhi for a SRF and SS thanks SERB, DST India for a Ramanna Fellowship (SR/S1/RFIC-01/2011).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra14061g

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