Differential antibacterial response exhibited by graphene nanosheets toward gram‐positive bacterium Staphylococcus aureus

Abstract

Two different morphological forms of graphene nanosheets: improved reduced graphene oxide (IRGO) and modified reduced GO (rGO) (MRGO) have been synthesised by improved and modified methods, respectively. Physical characterisations of these graphene nanosheets were carried out using X‐ray diffraction, Fourier transform infrared spectroscopy, and Raman spectroscopy. Colloidal stability of these nanosheets toward a selected bacterium (e.g. Staphylococcus aureus) was ascertained by zeta potential. In the present study, the authors for the first time made an attempt to study and compare the potentialities of these two different forms of graphene nanosheets as efficient bactericidal agents. Field‐emission scanning electron microscopy and TEM with energy dispersive X‐ray spectroscopy (EDAX) studies of IRGO and MRGO have been carried out to explore their underlying mechanism of antibacterial responses through physical as well as chemical interactions with the selected bacterial species.

1 Introduction

Cytotoxicity studies on carbon‐derived materials fetch a significant attention in the field of antibacterial activity [1]. The antimicrobial potentialities of carbon‐based nanomaterials could be enhanced due to the synergistic influence of both ‘physical’ and ‘chemical’ properties [2, 3]. Intensive physical interactions between nanoparticles and bacterial cells may cause physical damage to the cell membrane resulting in the release of intracellular contents [4, 5]. Chemical dependent cellular oxidative stress may also disrupt the specific microbial process [6]. The net increase in surface area to volume ratio of carbonaceous nanomaterials facilitates a better interaction with the bacterial cells in comparison with conventional microscale systems [2]. Synthetic carbon nanomaterials such as fullerenes and carbon nanotubes (CNTs) have been studied extensively in this regard [7].

CNTs have been built on the fundamental unit of graphene nanosheets besides fullerenes which are cage‐like structures [2] but unable to elicit significant bactericidal activities such as graphene [7]. Graphene and graphene‐based materials have an extraordinary impact due to their fabulous electronic [8] and mechanical properties [9] including high surface area, a higher level of aqueous solubility, and comparatively enhanced stability in physiological solutions such as a human serum. Thus, graphene‐based nanocomposites have become a unique material in the domain of microelectronic devices [10], as well as in the field of biomedicines [11, 12]. Basically, graphene consists of a single atomic layer of sp² carbon atoms. The primary impediment to the achievement of such a single atomic layer of graphene sheet in bulk quantity using mechanical exfoliation technique is to overcome the enormous inter‐layer van der Waals forces in graphite, which is quite difficult in practise [13]. Alternatively, chemical exfoliation technique could be adopted commercially focusing primarily on oxidative intercalation in the intermediate stage as graphene oxide (GO) followed by a reduction in the final stage as reduced GO (rGO). Interestingly, graphene nanosheets have also been reported to exhibit antibacterial properties against pathogenic bacterial strains in comparison with the standard antibiotics [14]. Krishnamoorthy et al. further demonstrated that such antibacterial efficiency was in part due to the free radical generation as a consequence of lipid peroxidation [14]. Similar efforts had been undertaken by other groups to investigate the antibacterial activities of graphene and GOs [15, 16]. The antibacterial activities of graphene and GO nanowalls as studied by Akhavan and Ghaderi [15] and Perreault et al. [16] could only show the antibacterial activity of graphene prepared by a single chemically exfoliated method. The antibacterial activities of GO and rGO have been attributed mainly due to physical and oxidative stresses [3], induced by the rough surface edges of graphene nanosheets on bacterial membrane. Such ruptures brought about significant intracellular contents of the bacterial system besides damaging vital macromolecules such as DNA, protein etc. leading to the eventual death of the bacteria [3, 17].

The present paper adopts the use of two different chemical methods (namely improved method and modified Hummers’ method) for the synthesis of graphene having different surface topographies. To the best of our knowledge, such antibacterial activities of graphene having different surface topographies have not yet been explored extensively in the light of physical as well as chemical interactions with the gram‐positive bacterium, Staphylococcus aureus (S. aureus). It is for the first time we demonstrated and compared the antimicrobial potentialities of two completely different forms of graphene nanosheets, improved rGO (IRGO) and modified rGO (MRGO). Physical characterisations of these chemically synthesised graphene nanosheets have been carried out using X‐ray diffraction (XRD), Fourier transform infrared (FTIR) spectrophotometer, Raman spectroscopy, atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission EM (TEM). EDAX studies have aided in explaining the role of different elements contained in graphene nanosheets, which play crucial roles in eliciting bactericidal responses.

2 Experimental details

2.1 Synthesis of graphene nanosheets as rGO

rGO was synthesised from graphite flakes (Sigma Aldrich) using Hummer's different methods (i.e. improved and modified method). For the improved method, the required amount of potassium permanganate (KMnO₄) was added slowly to the solution containing graphite flakes and a cold mixture of concentrated sulphuric acid and phosphoric acid (9:1). The temperature of the mixture was maintained below 35°C with constant stirring for 24 h 30% hydrogen peroxide was added to the mixture after proper dilution with ice‐cold distilled water. The brown colour GO (GO) was then refluxed at 100°C with hydrazine hydrate for the slow reduction of GO to rGO. Finally, the product was centrifuged, washed at least four times with distilled water to avoid any contamination and dried under vacuum at 80°C overnight. In the Hummers’ modified method, an equivalent amount of sodium nitrate (NaNO₃) had been used as an oxidising agent in addition to KMnO₄.

2.2 Characterisation of graphene nanosheets

The crystalline structure and lattice planes were determined by powdered X‐ray diffractometer (Bruker Advance D8) with copper (Cu) Kα (λ  = 1.5406 Å) radiation; where the as‐prepared powdered samples were subjected to characterisation. Raman spectra of the two forms of rGO nanosheets were obtained using a Lab Ram HR800 micro‐Raman spectroscope (Horiba Jobin‐Yvon, France) with an excitation wavelength of 514 nm Ar⁺ ion laser. FTIR spectra at 4500–500 cm⁻¹ were recorded from a Perkin‐Elmer spectrum RX‐1 IR spectrophotometer. Zeta potential measurements were carried out on a Zeta sizer (Zeta sizer NanoZSP) using a laser Doppler microelectrophoresis technique with a 633 nm helium–neon laser as a light source. Surface topography was investigated with field‐emission SEM (FE‐SEM) (EVO‐60, Zeiss company), TEM [FEI‐TECNAI G² 20S‐TWIN, Netherlands (Philips) and AFM (Bruker AXS Pte Ltd., Innova)].

For the sample preparation of as‐prepared IRGO and MRGO nanosheets for high‐resolution transmission electron microscope (HR‐TEM) characterisation, the samples were deposited onto carbon‐coated Cu grids (300 mesh, regular grip, spi 2030C) and allowed to dry for analysis under HR‐TEM (FEI‐TECNAI G2 20S‐TWIN, Netherland) at a frequency of 80 Hz.

To study the topographical morphology of the synthesised nanosheets, powdered samples were deposited onto a cover slip and dried overnight for visualisation under FE‐SEM (EVO‐60, Zeiss Company).

For AFM characterisation, powdered samples were drop cast onto a silicon wafer and allowed to dry for examination under AFM (Bruker AXS Pte Ltd., Innova).

2.3 Antibacterial activity of graphene nanosheets

2.3.1 Disc diffusion test

Bacterial strain, S. aureus American Type Culture Collection 25923 was exploited for implementing the antibacterial activity. It is a gram‐positive bacterium; the cells of which bear grape‐like morphology and do not possess an outer membrane but a thicker peptidoglycan layer of ∼30 nm. Colonies of the bacterial strain, S. aureus, were primarily inoculated in Mueller–Hinton (MH) (0.015% soluble starch, 0.2% beef extract, 1.75% casamino acids and Mueller Hinton Agar (MHA) plates and later stored in a cold room at 4°C after achieving desirable growth of bacterial cells on the plates.

As a preliminary screening process to assess the antimicrobial potential of graphene nanosheets (IRGO and MRGO), Disc diffusion technique was the method of choice that was implemented based on standard protocols [18]. In this regard, MH broth culture (100 µl) of each test bacteria was pipetted and allowed to seed onto the MHA plates. Thereafter, discs (sterilised Whatman filter paper) of ∼5 mm in diameter impregnated with an equal volume of selected concentrations of graphene nanosheets (MRGO and IRGO) ranging from a concentration of 125 to 2000 µg/ml were placed on the surface of the solid medium. Each and everyone of the plates were incubated at 37°C for a span of 24 h. As soon as optimum growth was observed, the plates were retrieved from the incubator and clear zones were measured with an antibiotic scale (Himedia) and corresponding results were tabulated. Afterwards, with the help of broth dilution test, the study was conducted by taking the positive result. Every set of experiment was carried out thrice. The mean ± standard deviation (SD) of the zone of inhibitions (ZOIs) were used to calculate the antimicrobial activity of both forms of graphene nanosheets.

2.3.2 Microbroth dilution test

For further confirmation, the antimicrobial activity of graphene nanosheets (IRGO and MRGO) was assessed through microbroth dilution method. Sterile 96 flat‐bottom well micro‐dilution plates were used in preparing two‐fold serial dilutions of graphene nanosheets dispersed in sterile Millipore water. To derive a standardised inoculum, the test organism was subjected to grow overnight in MH broth and later on re‐inoculated in a fresh MH broth for obtaining a typical turbidity with a value of optical density (OD) = 0.003 at 600 nm wavelength. All wells of the micro‐dilution plates were fed with 190 µl of the inoculum along with the 10 µl of graphene nanosheet solution. To prepare the control wells, 190 µl of MH broth and 10 µl of graphene nanosheets (IRGO and MRGO) were mixed. Thereafter post‐effective mixing, the plates were properly sealed with parafilms. The readings were then calculated using a microplate reader (Bio Rad) at 590 nm wavelength after subjecting the micro‐dilution trays in a shaker‐incubator for 37°C for 24 h. The proportion of growth of bacteria in the wells was monitored in comparison with controls. The complete experiment was repeated twice. The expression for the relative inhibition percentage (%) of the test sample is given in equation below:

$$\mathrm{relative}\mathrm{inhibition}(\text{\%})=\frac{\mathrm{O}{\mathrm{D}}_{\left(\mathrm{graphene}+\mathrm{bacteria}\right)}-\mathrm{O}{\mathrm{D}}_{\left(\mathrm{graphene}+\mathrm{uninoculated}\mathrm{media}\right)}}{\mathrm{O}{\mathrm{D}}_{\left(\mathrm{bacterial}\mathrm{inoculated}\mathrm{media}\right)}}\times 100$$ (1)

Moreover, the minimum inhibitory concentration (MIC) of the graphene nanosheets was expressed in terms of IC₅₀ value.

For understanding the inner morphology of bacteria after being treated with the graphene nanosheets, equal parts of graphene sample and bacterial cells were made to interact in the form of a solution and thereafter used for biological sample preparation with the help of 2.5% glutaraldehyde and 1% osmium tetroxide, and finally subjected to HR‐TEM (FEI‐TECNAI G² 20S‐TWIN, Netherlands) characterisation at 80 Hz.

3 Results and discussion

3.1 Sample characterisation

XRD has been employed for studying the diffraction angle at which characteristic crystalline peaks are formed for IRGO and MRGO nanosheets. Figs. 1 a and b describe the XRD patterns of IRGO and MRGO, respectively. Shifting of broad diffraction peak (002) of rGO at 24.30 Ǻ compared with the characteristic peak (002) of graphite at 26.58 Ǻ indicters the increase of inter‐layer distance between graphene sheets due to the introduction of oxygen‐containing functional groups such as hydroxyl, epoxy, carboxyl etc. [19]. The further appearance of a small diffraction peak at 42.84 Ǻ in rGO reveals the short‐range order in stacked graphene layers [20].

Fig. 1.

XRD & Zeta‐potential of graphene nanosheets

(a) X‐ray diffraction of IRGO (blue), (b) X‐ray diffraction of MRGO (red), (c) Zeta potential of MRGO nanosheets, (d) Zeta potential of IRGO nanosheets

FTIR studies have been carried out to ascertain the major functional groups present in the nanosheets as shown in Figs. 2 a and b. The characteristic absorption peak at 1600 cm⁻¹ is ascribed to the aromatic skeleton structure (–C = C–) in the graphene nanosheets.

Fig. 2.

FT‐IR & Raman spectroscopy of graphene nanosheets

(a) FT‐IR spectra of IRGO (blue), (b) FT‐IR spectra of MRGO (red), (c) Raman spectra of IRGO (blue), (d) Raman spectra of MRGO (red) nanosheets

The spectrum also reveals a dip around 3400 cm⁻¹ in case of IRGO characterisation, which indicates the occurrence of O–H stretching vibrations [21]. The peak tends to grow sharper in case of MRGO that might be due to a variation in oxygen content in MRGO. Again, a very small absorption peak of ∼1714 cm⁻¹ is observed for both IRGO and MRGO cases, which could be an evidence for the presence of functional groups such as quinones and/or single ketones (–C = O) [22] in the analysed materials [23]. Dipping is also noted around ∼1020 cm⁻¹ wavenumber, which informs that alkoxy (C–O) group [22] is also found in the materials (IRGO and MRGO). Absorption peaks for carboxyl and epoxy groups are absent in the FTIR spectra. Raman spectroscopy has been carried out to study the crystallinity, disorder, and defect levels in graphene nanosheets [24]. In general, the Raman spectrum of graphite exhibits peaks at a ‘G‐band’ (1570 cm⁻¹) and at a ‘D‐band’ (1350 cm⁻¹). The observation of the ‘G‐band’ is due to the first‐order scattering of the E_2g mode as shown in Figs. 2 c and d, while the ‘D‐band’ belongs to the defects in the graphitic lattice [25].

The G‐peak in IRGO occurs at a frequency of 1530 cm⁻¹, whereas the G‐peak in MRGO is shifted to 1540 cm⁻¹ [23]. This blue shift in the G peaks could be attributed due to the re‐graphitisation of carbon network resulting into the creation of a large number of sp² carbon species in graphene network [22, 25]. The presence of less number of defects in MRGO is further supported by the narrow D‐band in the Raman spectra [26]. Using the relative intensity ratio, i.e. I _D /I _G, the strength of defects present in the crystal lattice of a material can be evaluated. The I _D /I _G for IRGO is 1.35 while the I _D /I _G for MRGO is 1.15. This shows that the concentration of defects present in IRGO is higher than the defect concentration present in MRGO. The increasing number of defects found in IRGO can be attributed to the formation of many internal benzene‐derived sp² domains in a typical graphene structure [22, 27], which have resulted on account of the rigorous reduction process that employed harsher reagents. On the contrary, the use of NaNO₃ (as an oxidising agent) with KMnO₄ has helped to bring down the total number of defects found in MRGO in comparison with IRGO.

The colloidal stability of the nanomaterials in the dispersion state is one of the vital criteria in the field of biomedical applications [28].

The zeta potential of graphene nanosheets has been studied in an aqueous medium to investigate the colloidal stability of nanomaterials in the dispersion state. The zeta potential value of MRGO is −26 mV as shown in Fig. 1 c, which is well above the acceptable limit of colloidal stability (± 25 mV) in dispersion state [29]. In contrast, the IRGO nanosheet shows less colloidal stability (−20 mV) in the dispersion state as shown in Fig. 1 d.

The surface topologies of IRGO and MRGO have been investigated by AFM as shown in Figs. 3 a and b.

Fig. 3.

AFM & FE‐SEM of graphene nanosheets

(a) AFM image of IRGO nanosheets, (b) AFM image of MRGO nanosheets, (c) FE‐SEM image of IRGO nanosheets, (d) FE‐SEM image of MRGO nanosheets

The graphene nanosheets of both IRGO and MRGO were deposited on a glass substrate. The surface topography of the graphene nanosheets prepared by modified method (i.e. MRGO) is more uneven and irregular as compared with graphene nanosheets synthesised by the improved method (i.e. IRGO). This surface crumpling can be explained based on the deposition of single and/or multilayers of GO sheets in random orientation and with a higher density [26], thus once again validating its significant role in the enhancement of the bactericidal effect. To determine the thickness of the graphene nanosheets [25], AFM had been performed. The average thickness of the exfoliated graphene nanosheets is of ∼2.5 nm. The wavy features in surface topographies of IRGO and MRGO are also reflected in the images of FE‐SEM as shown in Figs. 3 c and d, wherein few nanosheets appear almost perpendicular to the surface of the substrate.

The sharp surface edge of such nanosheets is capable of effectively rupturing the membrane of the bacteria, thereby inducing bactericidal activity.

FE‐SEM characterisation was mainly performed for EDAX analyses. The EDAX plots highlighting the distribution of the major elemental players present in IRGO and MRGO are given in Figs. 4 a and b.

Fig. 4.

FE‐SEM EDAX & HR‐TEM EDAX of graphene nanosheets

(a) FE‐SEM EDAX of IRGO nanosheets, (b) FE‐SEM EDAX of MRGO nanosheets, (c) HR‐TEM EDAX of IRGO nanosheets, (d) HR‐TEM EDAX of MRGO nanosheets

Integrated HR‐TEM images for the pristine nanomaterials and the interaction taking place between S. aureus and the two types of rGO nanosheets are represented through Fig. 5.

Fig. 5.

HR‐TEM studies of Graphene nanosheets and its antibacterial activities

(a) HR‐TEM image of IRGO nanosheets, (b) HR‐TEM image of MRGO nanosheets, (c) HR‐TEM image highlighting the differential effects of IRGO and MRGO nanosheets on control S. aureus, causing changes in structural morphology of control S. aureus to IRGO‐treated S. aureus and MRGO‐treated S. aureus, respectively. Minor damaged region is shown by an arrow in case of IRGO‐treated S. aureus and heavily damaged region is shown by another arrow in case of MRGO‐treated S. aureus

Figs. 5 a and b depict the typical TEM images of the graphene nanosheets of IRGO and MRGO, respectively.

The morphology of graphene is characterised by the possession of high optical transparency. It also indicates the presence of wrinkles and folded regions on the surface of the synthesised graphene nanosheets [21]. EDAX plots under HR‐TEM analysis in Figs. 4 c and d show the presence of oxygen in the graphene nanosheets. Elemental profiles are in at.% as tabulated in Table 1.

Table 1.

Elemental distribution profile in at.% of carbon and oxygen for IRGO and MRGO nanosheets

Element	IRGO nanosheet, at.%	MRGO nanosheet, at.%
carbon	92.67	90.01
oxygen	7.33	9.99

The concentration of the oxygen content in MRGO (9.99 at.%) is more than that of IRGO (7.33 at.%).

This difference in the oxygen concentration could also be linked to the differential antibacterial responses of IRGO and MRGO graphene nanosheets treated with bacteria.

3.2 Antibacterial studies

The antibacterial activities against S. aureus were performed using IRGO and MRGO graphene nanosheets, respectively, by means of disc diffusion method followed by microbroth dilution method. The antibacterial results of IRGO and MRGO nanosheets by disc diffusion method were depicted in Table 2 and as insets in Figs. 6 b and c.

Table 2.

Antibacterial activity of graphene nanosheets by an agar‐well diffusion method

Test samples	Mean ZOI ± SD, mm
	2000.0 µg/ml	1000.00 µg/ml	500 µg/ml	250 µg/ml	125 µg/ml	Control (0.0 µg/ml)
MRGO nanosheets	18.90 ± 1.0	17.58 ± 1.0	16.80 ± 1.0	15.90 ± 1.0	15.50 ± 1.0	0.00
IRGO nanosheets	18.10 ± 1.0	17.10 ± 1.0	16.30 ± 1.0	15.45 ± 1.0	15.20 ± 1.0	0.00

Fig. 6.

HR‐TEM EDAX of bacterial interaction with graphene nanosheets

(a) HR‐TEM EDAX profile of control bacteria (S. aureus), (b) HR‐TEM EDAX profile of IRGO‐treated S. aureus (Disk Diffusion test in the inset), (c) HR‐TEM EDAX profile of MRGO‐treated S. aureus (Disk Diffusion test in the inset), (d) Elemental Distribution profile of bacteria treated with IRGO and MRGO nanosheets respectively

The further confirmatory antibacterial activities of IRGO and MRGO nanosheets were done by microbroth dilution method with estimation of MIC (IC₅₀) and the results were presented in Table 3.

Table 3.

Antibacterial activity of graphene nanosheets by Broth dilution method

Test samples	Percentage of inhibition (%) ± S.D.					IC50 value (µg/ml)
	2000 µg/ml	1000 µg/ml	500 µg/ml	250 µg/ml	125 µg/ml
MRGO nanosheets	99.3 ± 0.08a	99.3 ± 0.16a	65.1 ± 0.08b	45.0 ± 0.08c	5.6 ± 2.1d	297.04 ± 0.14
IRGO nanosheets	99.6 ± 0.16a	99.4 ± 0.04a	60.5 ± 0.70b	34.5 ± 0.61c	1.67 ± 0.47d	377.92 ± 0.42

Mean value followed with different superscripts a, b, c and d in Table 3 represents the percentage inhibition range of the bacterial growth (S. aureus) as follows

a = > 90%

b = 90‐50%

c = 50‐10%

d = <10%

As seen from Table 3, the data are expressed as a percentage inhibition of S. aureus and represent the mean ± SD (n  = 3). Antimicrobial activity: exponentially growing S. aureus cells were treated with different concentrations of MRGO and IRGO nanosheets for 24 h and cell growth inhibition was analysed through broth dilution assay. In each row, mean values followed with different superscripts significantly differ from each other according to Duncan's multiple range test (P  < 0.05). IC₅₀ is defined as the concentration, which results in a 50% reduction in cell numbers as compared with that of the control cultures (graphene nanosheets). The values represent the mean ± SD of three individual observations.

From both the standard methods of antibacterial activity, MRGO nanosheets (IC₅₀  = 297.04 ± 0.14 µg/ml) exhibited more antibacterial activity than IRGO nanosheets. Several mechanisms were described by different researchers regarding the bactericidal effect. Oxygen plays a significant role in the generation of the clear ZOI using such method for inducing the cidal activity in living systems [30]. Indeed, oxygen facilitates the production of reactive oxygen species (ROS) by interacting with the living cells, thereby causing cell damage and death [31]. The underlying mechanism resulting into the manifestation of such augmented ROS levels in the cellular interior is the responsible cause of allowing the cells to enter a state of oxidative stress leading to the damage of vital cellular macromolecules such as DNA, lipids, and proteins [32, 33]. Carbon‐based nanomaterials such as CNTs and fullerenes also exhibit such similar oxidative stress mechanism in the biological systems [34]. In the present paper, IRGO exhibited a less precise clear ZOI in comparison with MRGO as shown in Figs. 6 b and c. Experiments were conducted in triplicates and the results were consistent. The antibacterial activity exhibited by IRGO over the gram‐positive bacterium (S. aureus) may be accounted for the presence of less atomic oxygen (∼7.33%) in comparison with MRGO nanosheets containing more oxygen concentration in its structure (9.99 at.%).

To visualise the impacts of IRGO and MRGO nanosheets on S. aureus at the nanometre scale, HR‐TEM studies were carried out. Untreated bacteria (control) displayed a smooth outer envelope as shown in Fig. 5 c. However, major grooves and lesions are further observed in the MRGO‐treated bacteria in comparison with the IRGO‐treated bacteria indicating pronounced antibacterial activity of MRGO over IRGO against the bacteria.

Next, the elemental analyses of the bacterial species (S. aureus) treated with IRGO and MRGO, respectively, through EDAX were performed and determined under standard conditions as shown in Figs. 6 a –c.

Elemental analyses confirm the presence of oxygen, carbon, silicon, phosphorus, potassium, chlorine, sulphur, sodium, and bromine in varied proportions. Besides, a comparative elemental distribution profile of bacteria treated with the two types of nanosheets is depicted in Fig. 6 d. It is evident from the profile that the percentages of oxygen content in case of MRGO‐treated graphene nanosheets are subsequently high.

It has been demonstrated elsewhere that the generation of large number of ROS species such as O⁻, O²⁻, and O₂ ²⁻ are responsible for the creation of oxidative stress in damaging the integrity of the bacterial cell systems [35], and this was further supported by the reduction in the concentration of sulphur in MRGOs as compared with IRGOs as shown in Fig. 6 d.

Although a decent amount of sulphur is essential to enhance the overall antibacterial potential of the GO nanosheet as previously demonstrated by Barbolina et al. [36] and Marković et al. [37], the presence of other elemental impurities such as oxygen and oxygen radicals do significantly influence the antibacterial behaviour of a material.

In the present paper, the percentage of sulphur in both MRGO and IRGO is insufficient to cause minimum antibacterial response but a higher composition of oxygen in MRGO is observed to elicit good antibacterial response toward S. aureus when the concentration of MRGO (to be used on bacteria) was raised from 125 to 2000 µg/ml, in comparison with IRGO with low‐oxygen composition. Higher content of oxygen in the GO nanosheets is responsible for giving rise to increased ROS while causing bactericidal effects. Oxygen is seen to dominate the elemental distribution profile of the GO nanosheets and also the underlying mechanism of killing the bacterial cells by damaging the cell wall and inducing DNA mutation and protein denaturation. Other physico‐chemical attributes such as higher zeta potential of ∼26 mV and the presence of a pointed and uneven surface in case of MRGO lattice together with more oxygen impurities make MRGO a potent antibacterial agent.

AFM studies also support the pronounced antibacterial activity of MRGO nanosheets over IRGO nanosheets and are depicted in Fig. 7. It could be seen clearly from Fig. 7 that MRGO‐treated bacterial (S. aureus) cells exhibited more surface damage as compared with IRGO‐treated bacterial cells.

Fig. 7.

AFM of bacterial interaction with graphene nanosheets

(a) AFM image of untreated S. aureus, (b) AFM image of IRGO‐treated S. aureus, (c) AFM image of MRGO‐treated S. aureus [arrows indicate bacterial damage caused by nanosheets]

Furthermore, the antibacterial potentials of MRGO and IRGO nanosheets are studied using FE‐SEM as shown in Fig. 8. It could be clearly seen from this figure that MRGO nanosheets have exhibited more surface damage to S. aureus as compared with IRGO sheets. The surface damages were manifested as intense corrugation (on MRGO treatment) on the bacterial surface.

Fig. 8.

FE‐SEM of bacteria‐graphene interaction

(a) FE‐SEM image of untreated S. aureus, (b) FE‐SEM image of IRGO‐treated S. aureus, (c) FE‐SEM image of MRGO‐treated S. aureus [arrows indicate bacterial damage caused by nanosheets]

The bactericidal effect of graphene nanosheets, other than oxidative stress and mechanical damage, could similarly be attributed due to the wrapping tendency of bacterial cells by graphene nanosheets. It seems likely that on wrapping, there is possibly a biological isolation of the bacterial cells from its surrounding nutrient medium. The isolation causes insufficiency of nutrient transport in and out of the bacterial cells resulting in starvation and eventual death of the same [38]. The hydrophobic surface entices bacteria more than the hydrophilic surfaces [38, 39]. The wrapping tendency of the bacteria toward MRGO is significantly prominent compared with IRGO because of having more negative surface charges (−26 mV) compared with IRGO (−20 mV). Further investigation is required to understand the real underlying mechanism of antibacterial responses involved in the physical as well as the chemical interactions between the two types of graphene nanosheets (namely IRGO and MRGO) with the bacterial culture (e.g. S. aureus).

4 Conclusion

Two different chemical methods have been adopted for the syntheses of IRGO and MRGO graphene nanosheets, respectively, in two different surface topography forms. Structural characterisations of both IRGOs and MRGOs have been carried out by XRD, FTIR, and Raman spectroscopy. Zeta potential demands the colloidal stability of the MRGO (−26 mV) better than IRGO (−20 mV). The bactericidal responses for the graphene nanosheets toward gram‐positive bacteria S. aureus has been assessed by disc diffusion method and transmission electron microscopic analysis. The sharper and wavy surface topography of MRGO compared with IRGO, as revealed from AFM, FE‐SEM, and TEM studies, are responsible for executing bactericidal effect to a greater extent toward the gram‐positive bacteria S. aureus. In conclusion, the present paper establishes MRGO as a potential candidate in the field of biomedical applications as it displays a higher magnitude of ROS generating capacity toward gram‐positive bacteria.