Elucidating the Role of Oxidative Debris in the Antimicrobial Properties

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Elucidating the Role of Oxidative Debris in the Antimicrobial Properties of Graphene Oxide Andreia F. Faria, François Perreault, and Menachem Elimelech ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00332 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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ACS Applied Nano Materials

Elucidating the Role of Oxidative Debris in the Antimicrobial Properties of Graphene Oxide Andreia F. Faria1, François Perreault2, and Menachem Elimelech1*

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Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520-8286, United States

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School of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, Arizona 85287-3005, United States

* Corresponding author: Menachem Elimelech, Email: [email protected], Phone: (203) 432-2789

ACS Paragon Plus Environment

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ABSTRACT

In this paper, we investigate, for the first time, how oxidative debris affect the antimicrobial activity of graphene oxide (GO). Besides the conventional definition of GO structure, our study demonstrates that GO is also composed of one additional component called oxidative debris, small and highly oxidized fragments adsorbed on GO surface. After the removal of oxidative debris using an alkaline washing process, the toxicity of GO sheets to Escherichia coli cells significantly decreased. Raman spectroscopy measurements and acellular oxidation of glutathione indicated that oxidative debris increase the antimicrobial activity of GO sheets by improving their ability to promote bacteria inactivation via cell membrane damage and oxidative stress mechanisms. Given the influence of oxidative debris on the antimicrobial activity of GO, our findings emphasize the need to investigate the presence of oxidative debris before establishing correlations between physicochemical properties and the bio-reactivity of GO sheets.

Keywords: Graphene oxide, oxidative debris, antimicrobial properties, glutathione oxidation, mechanism of toxicity.


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INTRODUCTION The discovery of graphene, a two-dimensional one-atom-thick layer of graphite, has initiated a true revolution in the fields of nanotechnology and materials science.1-2 Its extraordinary electronic properties, combined with its large surface area, prompted the application of graphene as a building block material for the development of sensors, photocatalysts, drug delivery systems, and energy storage devices.3-5 Graphene oxide (GO), originating from the chemical exfoliation of graphite, is currently the most known derivative of graphene.6 GO has drawn attention for its straightforward and inexpensive preparation, good processability in water, and amenability to chemical modification.3, 6 The chemical structure of GO has been widely investigated and discussed.6-8 Aside from some particularities,9 the chemical structure of GO is defined as a single atomic graphitic layer randomly functionalized with oxygen-containing functional groups.6, 10 This conceptual premise has guided the applications for GO over the past years. However, in contrast to this oversimplified view of GO surface chemistry, a number of studies have proposed a much more complex model.11-14 It has been demonstrated that GO is composed of two individual entities that can be separated through a simple base-washing process.13, 15 Specifically, in addition to a single graphene sheet embedded with oxygen atoms, there are small highly oxidized fragments adsorbed on the GO surface. These oxidized fragments, called oxidative debris (or referred to as carboxylated carbonaceous fragments – CCFs), are co-products generated when carbon-based nanomaterials, such as carbon nanotubes (CNTs) and graphene, undergo oxidation via treatment with strong acids.13,

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Oxidative debris are polyaromatic features with a high content of oxygen that can adsorb on the


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GO surface through π-π stacking, hydrogen bonding, and van der Waals interactions. CCFs may act as surfactants, thus playing a role in the colloidal stability of GO sheets.13 Recent studies reported that the quantity of oxidative debris in acid-treated carbon nanotubes15 and GO12 is dependent on the intensity of the oxidation process.16 Whether oxidative debris are categorized as molecules or very small graphenic fragments, their presence modulates the physicochemical properties of GO by offering an additional oxidative component to the nanomaterial.11-12 A number of studies have reported on the toxicity of GO to bacterial cells.17-20 In the vast majority of cases, GO has been shown to destabilize the bacteria membrane cell via physical17-18 or chemical routes21-25. The integrity of cell membranes can be physically damaged by the sharpened edges of GO sheets.17-18 On the other hand, oxidative mechanisms triggered by the generation of reactive oxidative species (ROS) also play a role in the cellular inactivation by GO sheets.19,

21-22, 24

Initially, it was assumed that the overall toxicity of GO may result from a

combination of cell membrane physical damage and oxidative stress.19 However, our recent work demonstrated that the cellular inactivation caused by oxidative stress prevailed over the cell membrane physical damage when E. coli were exposed to GO sheets.24 This finding is supported by evidence that the biological reactivity of GO sheets originates from charge transfer and oxidation reactions.26-28 The toxicity of GO to microorganisms was proven to be affected by its physicochemical characteristics including size of the sheets,24 oxidation level,19 and presence of impurities29. For instance, GO sheets with smaller lateral size have shown higher toxicity against bacteria24, 30. In the context of antimicrobial applications, none of these studies considers the existence of oxidative debris on GO structure or its effect on the antibacterial properties of graphene. Removing debris changes the electrochemical properties11 and biocompatibility31 of GO sheets.


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Realizing that debris have an impact on several physicochemical properties of GO, the question arises: do oxidative debris have an inherent effect on the antimicrobial properties of GO? Recognizing the importance of understanding the numerous factors governing the toxicity of graphene materials, this study investigates the influence of oxidative debris on the antimicrobial properties of GO. As the toxicity of GO is correlated with its physicochemical properties, debris can have a significant effect on the ability of GO to inactivate bacterial cells. To separate the oxidative debris from graphene, the starting GO sample was treated with an alkaline solution, and the isolated fractions were thoroughly characterized. The antimicrobial properties were investigated using aqueous suspensions and graphene-coated surfaces. Finally, based on in vitro oxidative assays, we propose a mechanism of action that provides insights on the participation of oxidative debris in the toxicity of GO sheets to bacteria. MATERIALS AND METHODS Materials and Chemicals. Graphene oxide was purchased from CheapTubes Inc. (Cambridgeport, VT, USA). Sodium hydroxide (NaOH), hydrochloric acid (HCl, 37%), ethanol (anhydrous, 99.5%), paraformaldehyde (95%), sodium bicarbonate (NaHCO3, 99%), Ellman’s reagent (5.5-Dithiobis(2-nitrobenzoic acid), 99%), Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride, 98%), glutathione (GSH, 99%), were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Sodium chloride (NaCl, crystals, ACS reagent) was obtained from J.T. Baker (Phillipsburg, NJ). Trichloro-1, 2, 2-trifluoroethane (Freon, 99%) was purchased from America Refrigerants (Sarasota, FL, USA). Glutaraldehyde solution (50%) was acquired from Amresco (Solon, OH, USA). Sodium cacodylate buffer (pH 7.4) was acquired from Electron Microscopy Sciences (Hatfield, PA, USA). Lysogenic broth (LB) medium and peptone extract for bacteria


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cultivation were purchased from Becton, Dickinson and Company (Sparks, MD, USA). Deionized (DI) water was supplied by a Millipore System (Millipore Co., Billerica, MA, USA). Debris Removal from Graphene Oxide. Debris removal was performed through a base washing process as previously reported.12-13, 32 Graphene oxide (GO) powder (200 mg) was first mixed with 400 mL DI water and bath-sonicated (FS60 Ultrasonic Cleaner) for 30 minutes to obtain a stable solution. Then, GO dispersion was transferred to a round-flask that was placed on a reflux system at 80 °C. 1.6 g of solid NaOH were added into the flask containing the GO suspension and the mixture kept in reflux at 80 °C for one and a half hours. After base-washing, it was possible to visualize a separation of two phases: 1) a light-brown supernatant that corresponds to the oxidative debris, and 2) a black precipitate, which is the so-called debris-free GO (df-GO) sample. The resulting mixture was left to cool down at room temperature and the supernatant separated from the pellet by vacuum filtration using a 0.22 µm PTFE membrane (Millipore). The debris solution was collected and dialyzed excessively to remove traces of base (3,500 Da membranes, Spectrum Laboratories, CA, USA). The filtered df-GO was then placed into a 50 mL of HCl solution (1 mM) and refluxed at 80 °C for an additional hour to reprotonate their oxygen-containing functional groups. After reprotonation, df-GO was washed by centrifugation with copious amounts of water to remove any remaining residues of salt (NaCl) and acid. Prior to characterization, debris and df-GO samples were frozen in liquid nitrogen and dried by lyophilization. Characterization of GO, df-GO, and Debris. The ultraviolet-visible (UV-Vis) absorption spectra of GO, df-GO, and debris dispersions were acquired in a Hewlett Packard 8453 UV-Vis spectrophotometer. Raman spectra (Horiba Jobin Yvon HR-800) were recorded at an excitation laser of 532 nm, and D and G bands intensities were used as measures for the presence of


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structural defects. X-ray photoelectron (XPS) spectra were recorded on a VG Thermo alpha 110 hemispherical analyzer, operating with an Al-kα X-ray source. Atomic Force Microscopy (AFM) tapping mode images were taken on a Shimadzu SPM-9600 equipped with MESP (Bruker) CoCr coated cantilevers (2.8 N/m force constant, 75 KHz nominal resonance frequency). The morphological characteristics of GO, df-GO, and debris were investigated by Transmission Electron Microscopy (TEM) (FEI Tecnai Osiris), operating with an acceleration voltage of 200 kV. Samples were prepared by dropping a 50 µg mL-1 suspension of the nanomaterials on the surface of a lacey carbon grid (300 meshes). GO, df-GO, and debris-coated surfaces were imaged by Scanning Electron Microscopy (SEM) using an XL-Philips microscope. A Cressington (208 carbon) sputtering machine was applied to coat the sample with a thin layer (10-20 nm) of carbon. Images were taken at an acceleration voltage of 10 kV. Antimicrobial Activity of GO, df-GO, and Debris in Suspension. Escherichia coli was grown overnight in Lysogeny broth (LB) at 37 °C. The E. coli was acquired from the Coli Genetic Stock Center, strain code #7740. The bacterial cells were then diluted (1:50) in fresh LB medium and cultivated for ~2 hours to reach an optical density of 1.0 at 600 nm (OD 600 nm); an OD ~1.0 for E. coli growing in LB at 37 °C corresponds to mid-exponential phase. The bacterial suspension was then washed twice with saline solution (NaCl, 0.9% w/v), and the resulting suspension was diluted to 108 CFU mL-1 in sterile peptone water medium (0.1% w/v). A stock solution of GO (1000 µg mL-1) was prepared and diluted into a peptone water for final concentrations of 500, 250, 125, 50, and 25 µg mL-1. E. coli cells were exposed to the GO dispersions for three hours at room temperature under constant stirring. Right after exposure, the vials were bath-sonicated for 10 minutes to break any possible GO agglomerates, and the suspension was immediately diluted and plated on LB agar plates. The same protocol has been


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used in our previous studies to evaluate the antimicrobial properties of graphene and carbon nanotubes.20, 24, 33-35 The antimicrobial activity of df-GO and debris were evaluated using the IC50% (concentration in which 50% of the bacterial cells are inhibited) of GO as a standard value. Therefore, the antimicrobial assays for df-GO and debris were performed at a fixed of 50 µg mL-1. A 200 µg mL-1 stock solution of each material (GO, df-GO, and debris) was prepared by dispersing their respective powders in DI water followed by a bath-sonication for 30 minutes and a probesonicator for 40 minutes (6.5 kW L-1, Misonix 3000, Misonix Inc., Farmingdale, NY). An ice bath was used to prevent the temperature from rising during sonication. The stock solutions were diluted in sterile peptone water to achieve a concentration of 50 µg mL-1 for each nanomaterial. The scintillation vials were stirred for three hours at room temperature. Before sampling, the vials were bath-sonicated for 10 minutes to break any agglomerates of graphene in the suspension. After exposure, aliquots from the suspension were collected, sequentially diluted, and plated on LB agar plates. The inhibition percentage was calculated by dividing the number of cells in each sample by the number of cells on the control (with no nanomaterial addition). Antimicrobial Properties of GO, df-GO, and Debris-Coated Surfaces. E. coli cells were cultivated in LB media overnight at 37 °C. The bacterial cells were then diluted (1:50) in fresh LB medium and harvested at the exponential growth phase, which corresponds to a concentration of ~109 CFU mL-1. The bacterial suspension was then washed twice with saline solution (0.9%) by centrifugation for two minutes at 10,000 rpm to remove the excess growth medium constituents. The resulting suspension was diluted to 108 CFU mL-1 in sterile peptone water (0.1% w/v).


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To prepare the coated-surfaces, 2-3 mL of a 250 µg mL-1 stock solution of GO, df-GO, and debris were vacuum filtered through 0.22 µm cellulose ester filters (Millipore, Billerica, MA, USA) to form a graphene-coated surface on the membrane. The film was left to dry for 24 hours at room temperature. Next, 2 mL of the bacterial suspension prepared earlier was carefully poured on the top of the GO, df-GO, and debris-coated surfaces. The surface was in contact with bacterial suspension for three hours at room temperature. After the incubation, the bacterial suspension was removed and the surfaces were rinsed with sterile 0.9% saline solution to remove the non-adhered bacteria. The graphene-coated surfaces were transferred to 50 mL falcon tubes containing 10 mL 0.1% peptone solution. Subsequently, the falcon tubes were bath-sonicated (26 W L-1, FS60 Ultrasonic Cleaner) for 15 minutes to detach the bacterial cells from the membrane surface. Aliquots were collected, sequentially diluted in peptone water, and spread on LB agar plates. Plates were incubated overnight at 37 °C for CFU determination. A pristine membrane was used as a control. The percentage of inactivation was determined from the ratio of the number of viable cells on the control divided by the number of viable cells on the graphene’s surfaces. To investigate the influence of oxidative stress mechanisms on the toxicity of GO, df-GO, and debris, the bacterial cells were subjected to a pre-treatment with α-tocopherol (10 mM). Before being exposed to the graphene-coated surfaces, the bacterial suspension was incubated with αtocopherol at 37 °C for 40 minutes. Then, the tocopherol-exposed-bacterial suspension was added on the graphene-coated surfaces and the number of viable cells enumerated as mentioned earlier in this section. The morphology of the E. coli cells after exposure to the nanomaterials was analyzed as described previously.33 The bacteria cells attached to the membrane coupons were fixed using


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Karnovsky’s solution (4% paraformaldehyde and 5% glutaraldehyde diluted in 0.2 M Cacodylate buffer pH 7.4) for three hours. The cells were consecutively dehydrated by immersing the membrane coupons in water-ethanol (50:50, 30:70, 20:80, 10:90, and 100% ethanol) and ethanol-freon solutions (50:50, 25:75, and 100% freon) for 10 minutes. After the sequential dehydration steps, the fiber coupons were dried overnight in a desiccator at room temperature. The samples were then sputter-coated with 10 nm carbon (Cressington coater, 208 carbon), and the bacteria cells were imaged by SEM (XL series-Philips) operating at an acceleration voltage of 10 kV. Glutathione Oxidation by Graphene Oxide. To assess the ability of GO, df-GO, and debris to oxidize biomolecules, we evaluated the oxidation of glutathione (GSH) in acellular conditions.34 The oxidation of glutathione was conducted in a 50 mM bicarbonate buffer (pH 8.6), whereby 0.4 mM of reduced glutathione was exposed to 200 µg mL-1 of GO, df-GO, or debris. The samples were covered with aluminum foil to avoid any photodegradation reaction, and they were kept stirring at room temperature for three hours. The thiol concentration was quantified via a colorimetric method using Ellman’s reagent (5.5'-dithiobis-(2-nitrobenzoic acid)-DTNB). DTNB reacts with the thiols groups on non-oxidized GSH to yield a yellow compound (3-thio-6-nitrobenzoate -TNB) that absorbs light at a wavelength of 412 nm. After exposure, the suspension was filtered through a 0.45 µm polyethersulfone filter (Millipore) to remove any backgrounds provided by the presence of the nanomaterials. An aliquot (900 µL) of this filtered suspension was mixed with 1.57 mL a Tris-HCl buffer (pH 8.3) and 30 µL of 100 DTNB. The yellow-colored suspension resulting from the reaction between the remaining GSH and DTNB was analyzed by a UV-Vis spectrophotometer (Hewlett Packard 8453) at 412 nm using a path length of 1 cm and a molar extinction coefficient of 14150 M−1 cm−1. The oxidation


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of GSH by H2O2 was used as a positive control. The amount of oxidized GSH is calculated and expressed as a percentage loss of GSH.

RESULTS AND DISCUSSION Characteristics of Graphene Oxide (GO), Debris-free GO (df-GO), and Debris. Oxidative debris were separated from GO sheets by washing the original GO sample (Figure 1A) with a NaOH solution (0.1 M) under a reflux system. The washing treatment resulted in a precipitate that corresponds to df-GO (debris-free GO) and a light golden brown suspension containing the debris, as illustrated in Figure 1B. Debris-free GO and oxidative debris were recovered by centrifugation, reprotonated with HCl, and purified.12-13 Figure S1 illustrates the process of debris removal from GO. UV-Vis spectrum usually displays two well-defined peaks for GO samples (blue line). One peak at 230 nm is assigned to π-π* electronic transitions of aromatic C-C bonds, and another small shoulder at 310 nm corresponds to n-π transitions of C=O bonds36 (Figure 1C). The same two peaks are also visualized for debris (black line), but the disappearance of the shoulder at 310 nm in the df-GO spectrum (red line) indicates a lower degree of oxidation after the removal of debris. Another noticeable phenomenon in the UV-Vis spectrum of df-GO is the redshifting of ππ* transitions (~230 nm). Previous studies have reported that changes in the distribution (type and number) of oxygenated functional groups on the GO surface could cause π-π* transition peaks to shift towards longer wavelength regions in the energy spectrum.11, 37-39


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Raman spectra of the graphenic samples show the presence of the typical D and G bands, as depicted in Figure 1D. The G band relates to the graphitic component of GO sheets, while the D band is associated with structural defects or disorder in the graphene layer.40 These structural defects involve the presence of sp3 carbon domains and oxygen-containing functional groups on GO sheets. The ratio between the relative intensities of D and G bands (ID/IG) can be used as an indicator of the prevalence of defects on the graphene sheets.12, 38, 40 Due to a drastic decrease in the D band intensity, a significant decrease in the ID/IG ratio was observed for df-GO, which suggests a reduction in the number of structural defects after the removal of oxidative debris from GO sheets.

Figure 1. Photographs of graphene oxide (GO) suspension (A) before and (B) after the washing process with NaOH solution. C) UV-Vis spectra of GO (blue), debris free-GO (df-GO) (red), and debris (black) showing the absence of n-π electronic transitions on GO sample after the removal


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of debris. D) Raman spectra of GO, df-GO, and debris. Peaks at 1300 cm-1 and 1590 cm-1 are characteristic of the D and G bands of graphene, respectively. E) FTIR spectra of GO, df-GO, and debris showing the distribution of functional groups on each sample. Fourier transform infrared (FTIR) analyses also provided information about the chemical functionalities in GO, df-GO, and debris (Figure 1E). The intensity of a broad band at around 3000-3800 cm-1, assigned to O-H stretching mode (υ(O-H)) of hydroxyl groups, is decreased in the spectrum of df-GO. This observation suggests less dominance of hydroxyl groups on df-GO in comparison to GO. Moreover, the ratio between the vibrational modes of C=O (1720 cm-1) and C=C from aromatic rings (1630 and 1570 cm-1) decreases after debris are washed out from the GO surface, indicating that df-GO possesses a lesser number of oxygen-containing functional groups than pristine GO.11-12 We also observed a clear split of the stretching modes of C=C aromatic bonds (1630 and 1570 cm-1) into two very defined bands in the spectrum of df-GO and debris. As this phenomenon is not revealed in the spectrum of pristine GO (unwashed GO), it is believed the interaction between oxidative debris and the honey carbon structure of graphene affects the vibrational energy of aromatic C=C bonds on GO.12 The intensities of stretching and bending vibrations of carbon-oxygen bonds (C-O) of ketones (1100-1300 cm-1), C-O asymmetric and symmetric stretching for ethers (1040-1280 cm-1), and C-O stretching modes for alcohols and phenols (1000-1250 cm-1) were dramatically decreased with the removal of oxidative debris from GO (Figure 1E). This indicates that df-GO presented a significant decrease in its oxidation level compared to pristine GO, thus confirming that oxygen functionalities were eliminated from GO along with debris. These findings are in accordance with previous studies regarding the surface chemistry of debris-free GO.11-13


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X-ray photoelectron spectroscopy (XPS) was performed to track relative changes in the chemical composition of GO, df-GO, and oxidative debris (Figure 2). C1s deconvoluted curves presented four typical peaks that correspond to C-C bonding (sp2 and sp3, ~285 eV), epoxy and hydroxyl (C-O, ~286 eV), carbonyl (C=O, ~288 eV), and carboxyl groups (O-C=O, ~290 eV) of graphene-based materials.12, 24 The intensity of the peak at 286 eV (C-O) in the spectrum of dfGO is decreased, whereas the intensity of the peak at 285 eV (C-C) for df-GO increases compared to that of pristine GO (Figures 2A and B). In addition to a decreased content of hydroxyl and epoxy groups, the removal of debris also led to a slightly decreased number of carbonyl groups (Figure 2B). The concentration of oxygen-containing functional groups in GO was significantly lowered with the removal of debris. The ratio intensity between carbon (C-C) and oxygen (C-O) bonds for GO, df-GO, and debris were 2.0, 3.1, and 1.4, respectively, confirming that the debris had an impact on the chemical composition of pristine GO. Similar findings have been reported for GO before and after removing oxidative debris.11-13, 41


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Figure 2. X-ray photoelectron microscopy (XPS) spectra of (A) GO, (B) df-GO, and (C) debris. The deconvoluted curves are associated with different carbon functional groups on the graphenerelated materials. Red and purple lines represent the survey and baseline curves, respectively. Representative TEM micrographs of (D) GO, (E) df-GO, and (F) debris. Images were taken at an acceleration voltage of 20 kV. Our characterization demonstrated that oxidative debris contribute to the overall oxidation degree and content of structural defects on GO surface. It appears that the total functionality of pristine GO results from a combination of its own functional groups and the oxygen-containing functional groups present in the debris. Even though oxygen-containing functionalities are less dominant in df-GO, it is clear that df-GO still possesses the same oxygen functionalities as pristine GO. This observation rules out arguments suggesting that our washing process could be


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leading to the chemical reduction of GO. Additionally, XPS spectra have shown the same peak profile for GO, df-GO, and debris (Figures 2A, B, and C), which would not likely occur if the base wash were promoting the reduction of GO sheets. We note that deoxygenation of GO under strong alkaline conditions (NaOH, 8 mol L-1) has been reported.42 However, our protocol applies a much more diluted NaOH solution (0.1 mol L-1). We neither believe that oxidation-reduction reactions are taking place during the removal of debris. Redox reaction are unlikely since sodium and hydrogen in NaOH at their highest oxidation state, and, therefore, cannot work as proton or electron donors.13 The base treatment seems to be only separating debris from GO rather than inducing the reduction of oxygen functional groups on the GO surface. Results obtained elsewhere show that the removal of debris is nothing more than a cleaning process that reveals the true monolayer structure of GO.14 As aforementioned, GO became darker (dark brown to black) after treatment with NaOH solution (Figures 1A and B). The literature has reported that changes in color for GO samples are usually associated with differences in water solubility.43 Similar changes in the water solubility for df-GO have been described elsewhere.12-13 The df-GO sample presented a loss in solubility, although it was still able to form stable dispersions in water for hours. These differences in solubility between GO and df-GO are reflected in their TEM and AFM images (Figures 2 and 3). The TEM image of pristine GO portrays flat, well dispersed, and electrontransparent sheets (Figure 2D). In contrast, df-GO sheets displayed a darker color and a clear tendency to agglomeration (Figure 2E).


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Figure 3. Atomic force microscopy (AFM) images of (A) GO, (B) df-GO, and (C) debris. Graphics on the down panel represent the thickness distribution of (D) GO, (E) df-GO sheets, and (F) oxidative debris. AFM images depicted GO as single-layered sheets (~ 1 nm thick), while agglomerates and overlapping layers with thickness varying from 2 to 8 nm were evidenced for df-GO (Figures 3A, B, D, and E). This result confirms the information given by TEM imaging. Also, AFM demonstrates that oxidative debris consist mostly of very small fragments with lateral size and thickness ranging the scale of nanometers (Figure 3C). According to the height distribution histograms, debris featured an average thickness of about 1.73 ± 0.28 nm (Figure 3F). The


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average thickness of GO and df-GO were 1.08 ± 0.27 and 3.09 ± 1.93 nm, respectively (Figures 3D and E). This discrepancy in thickness between GO and df-GO indicates that debris play a role in the stabilization of GO sheets. It is most likely that oxidative debris adsorbs on the backbone structure of pristine GO sheets and work as surfactants to solubilize GO in water.12-13 Recent studies have questioned whether debris have a molecular component or if these fragments are only very tiny graphene-like flakes that adhere to the graphitic layer of GO sheets. For example, mass spectrometry analyses have been used to determine the approximate molecular weight of debris. Mass spectra have shown peak patterns from 100 m/z to 6000 m/z for debris41, which reassemble chemical structures similar to highly oxidized polyaromatic molecules (fulvic and humic acids)13, 41. While the chemical nature and origin of oxidative debris is still a question open to discussion, several reports consistently demonstrated the influence of debris on the adsorption capacity,32, 44 electrochemical activity,11, 44 and voltammetric response45 of as-prepared GO. Previous studies have demonstrated that the content of debris can vary from 14%12 to approximately 30%13 of the total GO weight. Concentration-dependent Antimicrobial Activity of GO, df-GO, and Debris towards E. coli in Suspension. The IC50% (inhibitory concentration where 50% of the cells are inactivated) was determined for pristine GO using Escherichia coli as a model bacteria. The antimicrobial assay was performed using different concentrations of pristine GO (25, 50, 100, 250, and 500 µg mL-1) dispersed in peptone water (0.1% w/v). Before diluting and plating, the scintillation vials containing both nanomaterials and E. coli cells (107 cell mL-1) were gently sonicated in a bath sonicator. Sonication was applied to disrupt very small GO agglomerates and release bacterial cells that may have been trapped by these agglomerates. Therefore, each GO sample was sonicated and immediately plated to avoid interference from agglomeration.


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The loss of viability as a function of GO concentration is plotted in Figure 4A. A greater loss of viability is observed as the concentration of GO increases in the aqueous suspension, thereby indicating a concentration-dependent toxicity for GO. E. coli cells were almost completely inhibited at GO concentrations higher than 100 µg mL-1, while GO at the concentration of 50 µg mL-1 was able to inactivate 50% of the cells. This finding suggests that the IC50% of pristine GO is approximately 50 µg mL-1. Our IC50% is consistent with other studies reporting the antimicrobial properties of GO.18-19 21 The percent loss of viability demonstrated by pristine GO is consistent with a previous study that shows inactivation rates of 81.5% for GO samples after contact with E. coli for two hours.19 No aggregates were visualized within the three-hour experiment, which indicates that the three graphene samples were well suspended in the assay media. The antimicrobial activity displayed by GO sheets can be attributable to their intrinsic properties and not to the presence of contaminants such as heavy metals, as previously suggested for CNTs and catalysts residual.46 According to the supplier (Cheap Tubes), GO is repeatedly rinsed until water is pH neutral. This cleaning procedure is sufficient to remove traces of chemicals (nitrogen, sulfur, potassium, phosphorous, and silicon) or lower their concentration to levels that are not harmful to bacteria cells. Moreover, unless the precursor graphite is contaminated, the presence of heavy metals is unlikely to occur. The elemental analysis reported by Cheap Tubes shows the presence of C (35%), O (45-55%), and H (3-5%) in their GO sample, and there is no mention of the presence of heavy metals or any other toxic contaminants. Reported XPS analysis of Cheap Tubes’ GO material support the supplier’s analysis.12, 47-49 Next, we examined the antimicrobial properties of GO, df-GO, and debris at the same concentration of 50 µg mL-1. The assay tubes were also sonicated before plating, as mentioned


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above. As seen in Figure 4B, df-GO presented an average inactivation rate of 55%, while debris showed a slightly weaker inhibition percentage of 44%. Pristine GO, therefore, revealed the strongest toxicity compared to both df-GO and debris, with an inhibition percentage of 75%. This suggests that the antimicrobial properties of these three samples are correlated with the presence of oxidative debris on the GO sample. Although debris showed the lowest toxicity compared to GO and df-GO, their role in the toxicity of GO cannot be dismissed. According to our findings, df-GO was able to preserve a portion of the original toxicity displayed by pristine GO. This indicates that oxidative debris enhance the antimicrobial activity of GO but are not entirely responsible for it. In this way, the toxicity of GO seems to result from a synergistic effect between debris and the df-GO matrix, and not from an additive effect from debris. The combination of debris and df-GO favors the antimicrobial activity of GO, and it is clear that both systems contribute to its total toxicity. Therefore, in order to discern the effect of debris, it is necessary to look at the whole system and understand how these two parts (debris and df-GO matrix) relate to the final toxicity of GO.


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Figure 4. (A) Concentration-dependent toxicity of pristine GO against E. coli cells after a threehour exposure. The antimicrobial assay was performed in suspension and the scintillation vials were bath sonicated for ~ 10 minutes before plating to disrupt aggregates. (B) Inhibition of E. coli cells in suspension after exposure to 50 µg mL-1 of GO, df-GO, and debris for three hours. The loss of cellular viability was expressed as a percentage relative to the control containing no nanomaterials. Asterisks indicate statistical difference (p < 0.05) from pristine GO sample.


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Another aspect that should be taken into consideration is the chemical structure and composition of debris. Our AFM and TEM images (Figures 2 and 3) show the presence of small and monolayer graphene-like fragments. However, mass spectrometry has previously demonstrated the presence of oxygen-containing polyaromatic molecules with molecular weight varying from 200 to 700 m/z13 or from 100 to 6000 m/z41. So far, there is no consensus regarding the chemical and structural characteristics of debris, even though previous findings have been able to offer valuable insights.11, 13, 41, 44-45 Because of this complex chemical composition, it is difficult to establish the relationship between structure/composition and the toxicity of debris. Further studies are necessary not only to reveal the true chemical nature of debris but also to better address their toxicity to microorganisms. Antimicrobial Activity of GO, df-GO, and Debris-coated Surfaces. To obtain more insights on the toxicity of GO-based materials, we conducted antimicrobial tests on graphene-coated surfaces. Samples freshly sonicated in water were deposited on filter surfaces by vacuum filtration. SEM images of the graphene coated-surfaces are shown in Figure S2. The cell viability was determined by plate counting after E. coli cells were in contact with the graphene coated-surfaces for three hours. As shown in Figure 5, the losses of viability of E. coli cells attached to graphene-coated filters were of 92%, 73%, and 57% for GO, df-GO, and debris, respectively. Particularly, GO exhibited greater antibacterial activity compared to either df-GO or debris. In fact, removal of debris yielded a reduction of ~20% in the original inactivation rate of pristine GO. Our findings show for the first time that the presence of debris has an effect on the toxicity of GO-based materials.


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Figure 5. Antimicrobial activity of GO, df-GO, and debris-coated surfaces before and after the pre-treatment of E. coli cells with α-tocopherol. For both antimicrobial assays, bacterial cells were kept in contact with coated-surfaces for three hours at room temperature. Before contacting the graphene coated surfaces, the E. coli cells in suspension were exposed to α-tocopherol (10 mM) for 40 minutes at 37 °C. The number of cells was quantified by plate counting method and the result expressed as percentage of inhibition compared to the control. The single asterisk indicates statistical difference from GO at an interval of 95%. The double asterisk indicates statistical difference from experiments conducted in absence of α-tocopherol. Both suspended and coated-surface assays presented a similar trend, with GO showing the strongest toxicity to E. coli cells. Furthermore, all three samples revealed a systematic improvement in their antimicrobial activities in the contact-based assay compared to the experiments performed in suspension (Figures 4 and 5). The explanation lies in the fact that graphene-coated surfaces are likely to provide a larger surface area for contact with bacterial cells, which results in enhanced antimicrobial properties. Similar antimicrobial studies have reported on the importance of allowing cells to enter into direct contact with nanomaterials-


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coated surfaces.17-18, 24, 26, 50 Contact-based antimicrobial assays can be easily applied to assess the toxicity of nanomaterials without any concern regarding their stability in water. Up to now, our results suggest that GO is able to inactivate E. coli cells, and the toxicity seems to be associated with its oxidative nature and larger number of structural defects, which are attributable to the occurrence of debris on GO. It has been shown that GO sheets can induce perturbations on the cellular membrane through the generation of oxidative stress,21-22,

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although physical damage caused by the contact between cells and the sharp edges of graphene is also an accepted mechanism17, 50. In this process, GO sheets may trigger the production of ROS and oxidation of membrane components such as lipids and proteins.21 For instance, lipid peroxidation has been found to be one the most important mechanisms of toxicity for GO-based materials.51 To investigate the participation of oxidative stress mechanisms on the toxicity of GO, df-GO, and debris, E. coli cells underwent a pre-treatment with α-tocopherol before contacting the graphene-coated surfaces. α-Tocopherol, also known as vitamin E, is a powerful antioxidant that is naturally present in the cell membrane of microorganisms.52-53 Tocopherol can suppress lipid peroxidation by donating hydrogen atoms to ROS and lipid radicals.52 After pre-incubating E. coli cells with α-tocopherol, these antioxidant molecules are expected to be absorbed in the cellular membrane where oxidative stress is more likely to occur during exposure to GO sheets.24 This absorption is facilitated by the interaction between the hydrophobic side chain of tocopherol and the fatty acids in the cellular membrane.53 Cells pre-incubated with α-tocopherol were exposed to the graphene-coated surfaces for three hours, and viable cells were quantified by CFU plate count. It should be noted that treated E. coli cells were less susceptible to the toxic effects of GO sheets (Figure 5). Compared to non-treated


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cells, the cellular inactivation for all three graphene-based surfaces suffered a reduction of roughly 50% after E. coli cells were pre-exposed to α-tocopherol (Figure 5). As an example, the cellular inactivation percentage of pristine GO was decreased from 92% to ~45% after preincubation of E. coli with the antioxidant. This is an indication that oxidative stress has a significant role in the antimicrobial activity of GO, df-GO, and debris. However, α-tocopherol was unable to defeat the oxidative activity of GO (the most oxidative sample), and the cellular viability was not completely recovered. One possible explanation is that it is impossible to load enough α-tocopherol to totally suppress oxidative stress. This result also suggests that the toxicity induced by GO is not only mediated by oxidative stress mechanisms but may be caused by other phenomena including physical disruption of the cellular membrane. To characterize the cellular morphology after the contact with GO, df-GO, and debris, the attached cells were analyzed by SEM. After exposure to the GO-based surfaces (Figure 6), the bacteria cells revealed an apparent loss in morphological integrity with the presence of darkened patches and flattened and shrunken morphology compared to the cells deposited on the control (polymeric filter without graphene) (Figure 6, left panel). For comparison, α-tocopherol treated E. coli cells attached to the graphene surfaces were also analyzed, as seen in Figure 6 (right panel). The majority of cells were still intact and maintained their morphology integrity, showing a much less compromised shape in comparison to the non-treated cells in Figure 6 (left panel). These results are consistent with the CFU plate count shown in Figure 5. Therefore, the protection provided by α-tocopherol confirms the participation of membrane-mediated oxidative stress in the toxicity mechanism of GO-related materials.


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Figure 6. E. coli cells were exposed to the graphene-coated surfaces for three hours and the number of viable cells quantified by agar CFU agar plate counting. Left panel: SEM micrographs revealing morphological damages to E. coli cells after contact with GO, df-GO, and debriscoated surfaces. Right panel: SEM micrographs showing a much less compromised cellular integrity of E. coli cells compared to those without a pre-incubation with the antioxidant. Antimicrobial Activity of Graphene Oxide is Likely Driven by Oxidative Stress and Defects-Mediated Mechanisms. Experiments conducted with tocopherol provided important insights regarding the contribution of oxidative stress mechanisms to the antimicrobial properties


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of GO (Figure 5). To further evaluate the involvement of oxidative stress in the toxicity of graphene-based samples, we used acellular oxidation of glutathione to probe the oxidative properties of GO, df-GO, and debris (Figure 7). Glutathione (GSH) is a natural antioxidant that promotes degradation of ROS and protects the cell from oxidative stress.54-55 The consumption of glutathione has been considered a biomarker for oxidative stress mechanisms induced by nanomaterials.19, 24, 30, 34

Figure 7. Oxidation of glutathione by GO, df-GO, and debris (black line). Glutathione (0.4 mM) was in contact with 200 µg mL-1 of each individual nanomaterial for three hours at room temperature in an acellular condition. H2O2 was used as a positive control. The intensity ID/IG ratios obtained from Raman spectra of GO, df-GO, and debris (blue line). Asterisks show that the loss of glutathione for df-GO and debris are significantly different from that of GO. In an in vitro assay, free thiol groups on GSH are quantified via reaction with Ellman’s reactant (DTNB). When oxidized, thiol groups (-SH) on GSH are converted to disulfide (S-S),


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which impedes the ability of GSH to react with DTNB. Therefore, Ellman’s assay is used to verify the concentration of unoxidized thiol groups and evaluate the oxidation of GSH.34,

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Results suggest that GO, df-GO, and debris were able to mediate the oxidation of GSH, but at different intensities (Figure 7). As debris was removed from pristine GO, the loss of GSH decreased, confirming that debris are partially involved in the oxidation of GSH by the GO sheets. After a three-hour exposure to pristine GO, nearly all glutathione was oxidized, and this greater depletion of GSH is in agreement with the increased toxicity shown by GO in the antimicrobial assays (Figures 4 and 5). Similar to the antimicrobial results (Figures 4 and 5), debris presented the lowest GSH oxidation compared to GO or df-GO (Figure 7). Although small GO sheets were previously shown to have high GSH oxidation potential24, debris cannot be considered as simply small GO fragments as their structure is more complex and heterogeneous12-13, 41. Therefore, debris cannot be expected to be similar to GO sheets in terms of GSH oxidation behavior. As seen in Figure 7, df-GO was still able to oxidize GSH, indicating that most of the oxidative properties of GO remains in df-GO after debris removal. Rather than an increment from debris, it seems that the combination of debris and df-GO affects the ability of GO to generate oxidative stress. The fact that df-GO retained part of GO’s ability to generate oxidation prompts us to believe that the final oxidative properties of GO benefit from a synergistic effect between the df-GO matrix and debris fragments. The results provided by Ellman’s assay indicate that oxidative stress has a major influence on the cytotoxicity of GO and that oxidative debris have an important role in the cellular inactivation via oxidative stress mechanisms. Glutathione oxidation also suggests that the


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cellular damage demonstrated by SEM images in Figure 6 may be the result of not only physical perturbation but also the degradation of the cellular membrane components via oxidative stress. The extensive glutathione depletion is probably associated with the generation of ROS by the nanomaterials. According to a previous study, the presence of structural defects plays a crucial role in the production of ROS and subsequent oxidation of GSH.57 In a first step, molecular oxygen (O2) reacts with defects and edges on the graphene sheets, which leads to surface-bound oxygen intermediate species such as superoxides and hydroperoxyl. In contact with these oxygen species, GSH is oxidized to GSSH, and carbon bonds on the graphene surface are simultaneously restored in a catalytic cycle that depletes the concentration of the antioxidant. The restoration of carbon bonds on the graphene surface, upon reduction by GSH, can lead to the delivery of oxidative species (by-products) in solution, which also increase the consumption of GSH. The defective surface of graphene works as a catalyst by providing active sites for the adsorption of dissolved O2, ROS generation, and consequent degradation of GSH.57 Assuming that the ROS production and loss of glutathione are related to the presence of defects in the graphene structure, we used Raman spectroscopy to estimate the number of structural defects on GO, df-GO, and debris, as reported in our previous study.24 The intensity of the ID/IG ratio serves as an indicator of the number of defects (edges and surface defects) on graphene sheets. The ID/IG ratio for GO and df-GO are 0.88 ± 0.08 and 0.75 ± 0.07, respectively, thus providing evidence that GO undergoes a reduction in the number of structural defects after the removal of oxidative debris (Figure 7). In fact, it appears that some of the defects present in pristine GO are attributed to oxidative debris, which showed a high ID/IG ratio of 0.9 ± 0.05. However, in the case of debris, the increase in ID/IG ratio is also attributable to the smaller size of the fragments, which increase the intensity of the D band.58 The increased ID/IG ratio found for


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pristine GO agrees with the loss of glutathione, indicating that an increase in the number of structural defects can lead to better oxidative capacity and improved cytotoxicity. This observation also corroborates our hypothesis that oxidative debris have a strong influence on both physicochemical and biological properties of GO. These highly oxidative fragments can change GO surface properties by adding oxygen-containing groups and structural defects, which improves the ability of GO to induce oxidative stress. For debris, the ID/IG ratio does not follow the same trend as the GO and df-GO materials; however, as previously mentioned, the ID/IG ratio here may also reflect a contribution from the much smaller dimensions of the debris compared to GO and df-GO. Two different molecular mechanisms seem to explain the toxicity of GO to E. coli cells. Primarily, chemical interactions play an important role in the antimicrobial property of GO sheets. Several studies have demonstrated that oxidative mechanisms are involved in the cytotoxicity of graphene-based materials.21, 24-25 For instance, GO and rGO were found to induce the production of a significant amount of superoxide radical anion during inactivation of Pseudomonas aeruginosa.21 The oxidation of cell membrane components has been considered the primary cause of cell inactivation by oxidative stress.19, 24-25 By reacting with polyunsaturated lipids on the cell membrane, ROS can initiate lipid peroxidation, promote the collapse of membrane nutrient transport, and interrupt the membrane energy-transducing systems, which ultimately culminate in cell death.25 In addition to enabling oxidative mechanisms, we assume GO sheets are also capable of generating physical damage to the cell membrane via a contact-mediated mechanism. In fact, intimate contact between cells and the material has been described as one of the most critical steps in cellular inactivation by carbon nanomaterials.17, 24, 34, 50 For instance, a simulation and


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theoretical study has demonstrated that GO sheets can penetrate into the cellular membrane and extract large portions of phospholipids from the lipid bilayer.51 The process of piercing and membrane internalization has been found to vary according to the size and oxidation level of the graphene sheets. Prior studies have shown that the oxidation level influences how the graphene nanosheets interact with the lipid bilayer.59-60 While low oxidized sheets tend to be trapped within the hydrophobic interior of the lipid bilayer (parallel configuration)59, 61, highly oxidized sheets are more likely to concentrate across the membrane (orthogonal orientation) where the contact between lipid heads and the oxygen-containing groups of GO is maximized and freeenergy reduced59-60. Therefore, a higher oxidation state would facilitate the entry of graphene sheets into cells and generate a larger extent of irregular perturbations to the cellular membrane.59 Since oxidative debris imparts a higher level of oxidation to GO sheets, we can assume that debris-containing GO is able to cause more damage to the cellular membrane than df-GO if membrane perturbation is part of the mechanism of toxicity of GO. Penetration and internalization would increase with the number of structural defects on GO sheets provided by the adsorbed debris. Given the acellular degradation of GSH, we surmise that GO sheets are capable of oxidizing cellular components when in contact with bacteria cells. Furthermore, the presence of structural defects, confirmed by Raman analyses, indicates that GO sheets are potentially able to inactivate bacteria via membrane stress. Our results provide evidence that the oxidation capacity of GO and its ability to cause membrane perturbation are likely associated with the coexistence of oxidative debris with GO. Oxidative debris increase the biological oxidation potential of GO, which in turn leads to improved toxicity compared to df-GO. Similar observations were described in our previous study, where smaller GO sheets enabled higher toxicity by increasing the number of


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structural defects on the graphitic layer.24 Through Raman measurements, GSH oxidation, and experiments with α-tocopherol, we tried to offer insights into the role of debris in the toxicity of GO. We recognize, however, that the mechanism of toxicity may be more complex than that proposed herein, especially regarding the molecular interactions between debris and the bacteria cells. Further investigation is needed to evaluate the possible involvement of additional modes of action in the antimicrobial activity of GO, df-GO, and debris. The antimicrobial properties of GO have been found to be dependent on its content of oxygenfunctional groups,19 size,24, 30 and presence of impurities29. However, ours is the first study that shows the influence of oxidative debris on the antibacterial activity of GO. In fact, the presence of debris and its implication for the physicochemical properties of GO has been omitted in the vast majority of publications. Along with other problems involved in the manufacturing of GObased nanomaterials, such as the source of graphite, oxidation method, and purification, the lack of knowledge about the presence of oxidative debris complicates efforts to compare the antimicrobial properties among the different samples of GO. Considering the impact of the physicochemical properties on the antimicrobial activity of GO, we believe that it is necessary to recognize whether debris are present and understand how this reflects on the toxicity of GO sheets. In order to establish useful correlations between the existence of oxidative debris and cytotoxicity of GO, a more careful and detailed characterization of the material will be required before performing the standard antimicrobial assays. CONCLUSION This study provides a mechanistic insight into the role of oxidative debris in the antimicrobial properties of GO. The physicochemical characterization of the graphene-based samples shows that debris are major contributors of oxygen-containing functional groups and structural defects


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on GO. The adsorption of oxidative debris on GO surface can modify or even modulate properties that have been attributed initially to GO itself. Our results provide evidence that debris have a significant role in the toxicity of GO. The removal of debris led to a decrease in the antimicrobial activity of GO. By contributing to a large number of defects on the GO sheets and high content of oxygen-containing functional groups, oxidative debris increased GO toxicity via membrane damage and oxidative stress mechanisms. This correlation between the presence of debris and cytotoxicity of GO emphasizes the need for a more elaborate discussion on how the intrinsic characteristics of GO can affect its biological properties. This subject is of growing importance as commercial applications of graphene-based materials have attracted considerable attention in past years. SUPPORTING INFORMATION AVAILABLE: A scheme illustrating the removal of debris from graphene oxide (Figure S1) and scanning electron microscopy (SEM) images of graphene-coated surfaces (Figure S2) are present in the supporting information file. AUTHOR INFORMATION Corresponding Author *Menachem Elimelech ([email protected]). Department of Chemical and Environmental Engineering, Yale University, New Haven, CT 06520-8286, USA. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. GRANTS AND AWARDS Author Andreia Fonseca de Faria received financial support from the National Council for Technological and Scientific Development (CNPq-Brazil), grant 246407/2012-3. François


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Perreault received funding from the Natural Sciences and Engineering Research Council of Canada, grant PDF-420633-2012. This work was supported by the NSF Nanosystems Engineering Research Center for Nanotechnology-Enabled Water Treatment (ERC-1449500.

ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Council for Technological and Scientific Development (CNPq-Brazil) through the “Science without Borders” Program. F.P. thanks the Natural Sciences and Engineering Research Council of Canada for financial support through a postdoctoral fellowship. The authors are grateful to Dr. Douglas Soares da Silva from University of Campinas in Brazil for his contributions to the AFM analysis. The authors thank the Brazilian Nanotechnology National Laboratory (LNNano), Dr. Cristiane Silva, and Dr. Ana Mazarin Moraes for their help with the XPS measurements. We also would like to thank Dr. Kanani Lee for granting access to the Raman spectrometer and Dr. Zhenting Jiang and Dr. Jennifer Girard for their support on the SEM and Raman analyses. Additionally, the authors acknowledge the Yale Institute of Nanoscale and Quantum Engineering (YINQE) for their support on the TEM analyses.


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35. Rodrigues, D. F.; Elimelech, M., Toxic Effects of Single-Walled Carbon Nanotubes in the Development of E. coli Biofilm. Environmental Science & Technology 2010, 44, 4583-4589. 36. de Faria, A. F.; Martinez, D. S. T.; Meira, S. M. M.; de Moraes, A. C. M.; Brandelli, A.; Filho, A. G. S.; Alves, O. L., Anti-Adhesion and Antibacterial Activity of Silver Nanoparticles supported on Graphene Oxide Sheets. Colloids and Surfaces B: Biointerfaces 2014, 113, 115124. 37. Zhang, J.; Yang, H.; Shen, G.; Cheng, P.; Zhang, J.; Guo, S., Reduction of Graphene Oxide Vial-Ascorbic Acid. Chemical Communications 2010, 46, 1112-1114. 38. Li, D.; Muller, M. B.; Gilje, S.; Kaner, R. B.; Wallace, G. G., Processable Aqueous Dispersions of Graphene Nanosheets. Nat Nano 2008, 3, 101-105. 39. López-Díaz, D.; Mercedes Velázquez, M.; Blanco de La Torre, S.; Pérez-Pisonero, A.; Trujillano, R.; Fierro, J. L. G.; Claramunt, S.; Cirera, A., The Role of Oxidative Debris on Graphene Oxide Films. ChemPhysChem 2013, 14, 4002-4009. 40. Malard, L. M.; Pimenta, M. A.; Dresselhaus, G.; Dresselhaus, M. S., Raman Spectroscopy in Graphene. Physics Reports 2009, 473, 51-87. 41. Chen, X.; Chen, B., Direct Observation, Molecular Structure, and Location of Oxidation Debris on Graphene Oxide Nanosheets. Environmental Science & Technology 2016, 50, 85688577. 42. Fan, X.; Peng, W.; Li, Y.; Li, X.; Wang, S.; Zhang, G.; Zhang, F., Deoxygenation of Exfoliated Graphite Oxide under Alkaline Conditions: A Green Route to Graphene Preparation. Advanced Materials 2008, 20, 4490-4493. 43. Park, S.; An, J.; Jung, I.; Piner, R. D.; An, S. J.; Li, X.; Velamakanni, A.; Ruoff, R. S., Colloidal Suspensions of Highly Reduced Graphene Oxide in a Wide Variety of Organic Solvents. Nano Letters 2009, 9, 1593-1597. 44. Ma, D.; Dong, L.; Zhou, M.; Zhu, L., The Influence of Oxidation Debris Containing in Graphene Oxide on the Adsorption and Electrochemical Properties of 1,10-phenanthroline-5,6dione. Analyst 2016, 141, 2761-2766. 45. Jia, L.; Dong, L.; Zhu, L., Stripping Voltammetry at Graphene Oxide: The Negative Effect of Carbonaceous Debris. Applied Materials Today 2017, 8, 26-30. 46. Pulskamp, K.; Diabaté, S.; Krug, H. F., Carbon Nanotubes show no Sign of Acute Toxicity but Induce Intracellular Reactive Oxygen Species in Dependence on Contaminants. Toxicology Letters 2007, 168, 58-74. 47. Alzate-Carvajal, N.; Basiuk, E. V.; Meza-Laguna, V.; Puente-Lee, I.; Farias, M. H.; Bogdanchikova, N.; Basiuk, V. A., Solvent-Free One-Step Covalent Functionalization of Graphene Oxide and Nanodiamond with Amines. RSC Advances 2016, 6, 113596-113610. 48. Prabakar, S. J. R.; Park, C.; Ikhe, A. B.; Sohn, K.-S.; Pyo, M., Simultaneous Suppression of Metal Corrosion and Electrolyte Decomposition by Graphene Oxide Protective Coating in Magnesium-Ion Batteries: Toward a 4-V-Wide Potential Window. ACS Applied Materials & Interfaces 2017, 9, 43767-43773. 49. Du, T.; Adeleye, A. S.; Keller, A. A.; Wu, Z.; Han, W.; Wang, Y.; Zhang, C.; Li, Y., Photochlorination-Induced Transformation of Graphene Oxide: Mechanism and Environmental Fate. Water Research 2017, 124, 372-380. 50. Hui, L.; Piao, J.-G.; Auletta, J.; Hu, K.; Zhu, Y.; Meyer, T.; Liu, H.; Yang, L., Availability of the Basal Planes of Graphene Oxide Determines Whether It Is Antibacterial. ACS Applied Materials & Interfaces 2014, 6, 13183-13190.


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Figure 1. Photographs of graphene oxide (GO) suspension (A) before and (B) after the washing process with NaOH solution. C) UV-Vis spectra of GO (blue), debris free-GO (df-GO) (red), and debris (black) showing the absence of n-π electronic transitions on GO sample after the removal of debris. D) Raman spectra of GO, dfGO, and debris. Peaks at 1300 cm-1 and 1590 cm-1 are characteristic of the D and G bands of graphene, respectively. E) FTIR spectra of GO, df-GO, and debris showing the distribution of functional groups on each sample. 308x190mm (96 x 96 DPI)



Figure 2. X-ray photoelectron microscopy (XPS) spectra of (A) GO, (B) df-GO, and (C) debris. The deconvoluted curves are associated with different carbon functional groups on the graphene-related materials. Red and purple lines represent the survey and baseline curves, respectively. Representative TEM micrographs of (D) GO, (E) df-GO, and (F) debris. Images were taken at an acceleration voltage of 20 kV. 254x167mm (96 x 96 DPI)


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Figure 3. Atomic force microscopy (AFM) images of (A) GO, (B) df-GO, and (C) debris. Graphics on the down panel represent the thickness distribution of (D) GO, (E) df-GO sheets, and (F) oxidative debris. 254x190mm (96 x 96 DPI)



Figure 4. (A) Concentration-dependent toxicity of pristine GO against E. coli cells after a three-hour exposure. The antimicrobial assay was performed in suspension and the scintillation vials were bath sonicated for ~ 10 minutes before plating to disrupt aggregates. (B) Inhibition of E. coli cells in suspension after exposure to 50 µg mL-1 of GO, df-GO, and debris for three hours. The loss of cellular viability was expressed as a percentage relative to the control containing no nanomaterials. Asterisks indicate statistical difference (p < 0.05) from pristine GO sample. 150x261mm (300 x 300 DPI)


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Figure 5. Antimicrobial activity of GO, df-GO, and debris-coated surfaces before and after the pre-treatment of E. coli cells with α-tocopherol. For both antimicrobial assays, bacterial cells were kept in contact with coated-surfaces for three hours at room temperature. Before contacting the graphene coated surfaces, the E. coli cells in suspension were exposed to α-tocopherol (10 mM) for 40 minutes at 37 °C. The number of cells was quantified by plate counting method and the result expressed as percentage of inhibition compared to the control. The single asterisk indicates statistical difference from GO at an interval of 95%. The double asterisk indicates statistical difference from experiments conducted in absence of α-tocopherol. 146x115mm (300 x 300 DPI)



Figure 6. E. coli cells were exposed to the graphene-coated surfaces for three hours and the number of viable cells quantified by agar CFU agar plate counting. Left panel: SEM micrographs revealing morphological damages to E. coli cells after contact with GO, df-GO, and debris-coated surfaces. Right panel: SEM micrographs showing a much less compromised cellular integrity of E. coli cells compared to those without a pre-incubation with the antioxidant.


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Figure 7. Oxidation of glutathione by GO, df-GO, and debris (black line). Glutathione (0.4 mM) was in contact with 200 µg mL-1 of each individual nanomaterial for three hours at room temperature in an acellular condition. H2O2 was used as a positive control. The intensity ID/IG ratios obtained from Raman spectra of GO, df-GO, and debris (blue line). Asterisks show that the loss of glutathione for df-GO and debris are significantly different from that of GO. 130x97mm (300 x 300 DPI)


Elucidating the Role of Oxidative Debris in the Antimicrobial Properties

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