Wrinkled Surface-Mediated Antibacterial Activity of Graphene Oxide

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Wrinkled Surface-Mediated Antibacterial Activity of Graphene Oxide Nanosheets Fengming Zou, Hongjian Zhou, Do Young Jeong, Junyoung Kwon, Seong Un Eom, Tae Jung Park, Suck Won Hong, and Jaebeom Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15085 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

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ACS Applied Materials & Interfaces

Wrinkled Surface-Mediated Antibacterial Activity of Graphene Oxide Nanosheets Fengming Zou,†, ‡, ¶ Hongjian Zhou,†, §, ¶ Do Young Jeong,⊥ Junyoung Kwon,† Seong Un Eom,† Tae Jung Park,∥Suck Won Hong*,† and Jaebeom Lee*,† †

Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 46241,

Republic of Korea ‡

High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui 230031, China

§

Key Laboratory of Materials Physics, Centre for Environmental and Energy Nanomaterials,

Anhui Key Laboratory of Nanomaterials and Nanotechnology, Institute of Solid State Physics, Chinese Academy of Sciences, Hefei 230031, China ⊥

ACI Process & Structure Development Group, Samsung Electro-Mechanics, Suwon 16674,

Republic of Korea ∥

Department of Chemistry, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974,

Republic of Korea ¶

These authors contributed equally to this work

*Correspondence should be addressed to S.W.H. (email: [email protected]) and J. L. (email: [email protected])

KEYWORDS: graphene oxide, nanosheet, antibacterial activity, surface roughness, molecular dynamics

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Abstract Surface wrinkles are commonly observed in large-scale of graphene films. As a new feature, wrinkled

surface of graphene films may directly affect bacterial viability by means of various interactions of bacterial cells with graphene sheets. In the present study, we introduce a wrinkled surface geometry of graphene oxide (GO) thin films for antibacterial application. Highly wrinkled GO films were formed by vacuum filtration of a GO suspension through a pre-strained filter. Several types of wrinkled GO surfaces were obtained with different roughness grades determined by root-mean-square values. Antibacterial activity of the fabricated GO films towards three different bacterial species, Escherichia coli, Mycobacterium smegmatis and Staphylococcus aureus, was evaluated in relation to surface roughness. Due to their nanoscopically corrugated nature, the wrinkled GO films exhibited excellent antibacterial properties. Based on our detailed observations, we propose a novel concept of the surrounded contact-based mechanism for antimicrobial activity of wrinkled GO films. It postulates formation of a mechanically robust GO surface “trap” that prompts interaction of bacteria with the diameter-matched GO sink, which results in substantial damages to the bacterial cell membrane. We believe that our approach uncovered a novel use of a promising two-dimensional material for highly effective antibacterial treatment.

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Introduction Antibacterial materials have been actively developed for the treatment of infectious diseases caused by pathogenic bacteria in our daily life. The widespread use of antibiotics has led to increased antimicrobial resistance, resulting in significantly reduced therapeutic efficacy. In this regard, nanomaterials have great potential for advancing the antimicrobial properties in order to extend the capacity of the properties as functional components.1-4 Various nanoscale antimicrobial substances, such as Ag, AgO, TiO2, CuO, ZnO, Au and MgO, exhibits noteworthy broad spectrum anti-pathogenic acitivity.5-13 Carbon-based nanoparticles (e.g., fullerenes and carbon nanotubes, CNTs) characterized by unique chemical, optical and mechanical properties efficiently inhibited bacterial growth when applied in the form of membrane or film substrates.14 The potential of these carbon nanomaterials has demonstrated by several approaches. For example, the protonated amine modified fullerene derivatives were used as chemical disinfectants,15 while the dispersions of highly purified short CNTs showed a strong antimicrobial effect by direct contact-driven interactions.16-19 It has been suggested the antibacterial activity of CNTs could be regulated by adjusting the particle size, surface oxidation degree and functionalization.20 Recently, graphene derivatives (e.g., graphene oxide (GO) and reduced graphene oxide (rGO)), have been paid more attention as potential acute inhibitors of bacterial growth.21,22 In 2010, the research for anti-bacterial activity of GO and rGO nanosheet was first reported, from which the graphene-based nanomaterials were proved to be effective in inhibiting the growth of E. coli.23 Many factors can affect antimicrobial activity of graphene nanomaterials, involved nanomaterial properties (e.g., electronic properties, surface chemical properties, size, etc.) as 3



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well as interaction conditions, such as the incubation time, concentration, medium, or other external parameters.24-28 It has been reported that several primary mechanisms determine the potential antimicrobial activity of graphene-based materials. For example, many research has demonstrated that physical interactions between bacterial cells and GO nanosheets may cause membrane perturbations due to piercing with sharp edges of atomically thin GO nanosheets.29 Such local perturbations affect the membrane potential of the lipid bilayer of bacterial cells, disturb cellular metabolism and eventually damage the cell membrane resulting in bacterial cell lysis and death.30,31 Oxidative stress caused by GO nanosheets may be involved in irreversible damage to the bacterial cells leading to the disruption of cell integrity.32,33 The damaging effect of graphene on the cell membrane can also be mediated by the charge transfer interaction due to the direct contact of the microbial membrane with graphene, which acts as an electron acceptor under the direct contact with bacteria.42. In addition, bacterial cell wrapping in graphene-based surface coatings was observed, which could also contribute to inactivation of bacteria by biologically isolating them from the growth medium in graphene nanosheet suspensions.34 Thin graphene sheets intrinsically exhibit surface corrugations such as wrinkles or ripples with a smooth undulation.35,36 The wrinkles of graphene films with periodic orders of magnitude could be important because new features of unique properties can be expected, and thereby lead to remarkable changes in the performance of graphene-based nanomaterials (e.g. the charge transport behavior and local chemical reactivity). Crumpling of graphene sheets with ridges or folds can be produced by the localized mechanical instabilities controlled by the prestrains and subsequent surface relaxations of a stretchable substrate in a particular order.37-41 Such structures, considered as unique architectures with features on multi-length scale, can be used for supercapacitor electrodes or flexible electronics.42 However, little attention has been 4


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paid to biological interactions on wrinkled graphene surfaces. Because the enlarged surface area provides a more sensitive environment and allows tighter interactions with biological systems compared to planar bio-interface,39 we postulate that finely tuned wrinkled graphene surfaces may directly affect bacterial viability. In the present study, we explored critical interactions of graphene sheets with three species of bacteria (Escherichia coli, Mycobacterium smegmatis and Staphylococcus aureus), and, for the first time, demonstrated antibacterial activity of highly wrinkled GO films. The antimicrobial properties were evaluated in relation to the parameters of defined surface roughness of the wrinkled GO films, which were prepared by vacuum filtration of GO suspension through a pre-strained filter paper. After vacuum filtration and drying, highly wrinkled GO films spontaneously formed following the release from pre-straining. Antimicrobial activity of GO nanostructures was investigated in terms of geometrical functionality of graphenebased nanomaterials. The morphological features of the GO films were characterized using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Antibacterial activity of the nanostructured surfaces of GO was determined by the colony count method and fluorescent staining. In addition, molecular dynamics (MD) simulations was also employed for the further study the antibacterial mechanisms of GO nanosheets.

Experimental section Synthesis of graphene oxide The expanded graphite was able to produce graphene oxide (GO) from its acid-based exfoliation synthetics according to modified Hummers method.43 As original material, some amount of expandable graphite (Asbury Carbon, Grade 1721, NJ, USA) in 500 mL beaker was heated for 5



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~10 s in a microwave oven, which has triggered the graphite expansion over ~100 times from its original volume. After that, a 250 mL flask was filled with 50 mL of concentrated sulfuric acid with magnetic stirring and then placed into an ice bath to maintain the temperature at 0 °C for the acid treatment. Next, the suspension was made in a falsk by slowly pouring the expanded graphites (~1.5 g), then potassium permanganate (4 g) was added into the flask slowly. The suspension was conducted under the tempreture of 35 °C with 2 hours of constant gentle stirring. Afterwards, the mixture was cooled down by placing flask in an ice bath, followed by slowly adding deionized water, while the temperature was maintained below 70 °C. To remove the unreacted potassium permanganate, H2O2 was slowly added into the flask, which has trigerred vigorous bubbles and apparent color change of the suspension from dark brown to yellow. After filtered for several times, the suspension was removed the acid completely by being diluted with deionized water. The pH level of the dispersion in this process should be monitored until pH 6. Finally, the suspension was under suction drying over 12 h for the final completion of GO platelets.

Preparation of graphene oxide films Wrinkled graphene oxide (GO) films were fabricated by vacuum filtration method, employing a disc-type membrane filter (Whatman: PTFE and Millipore: nylon, pore size = 0.45 µm) between the funnel (Pyrex, 250 ml) and filtration flask (Pyrex, 1000 ml). Due to the reduced pressure (~74 kPa), only water passed through a slightly pre-stretched membrane filter (biaxial mode, 2-3% stretching from the original size) and GO sheets were uniformly stacked up on top of the membrane filter (the fabrication procedure is illustrated in Figure 1a); For complete drying out of the GO sheets, the actual pressure was under monitoring by pump-gauge (Welch, 2030B-01). GO 6


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films has four defined roughness grades, and to accomplish which, the membrane filter was required appropriate adjustment and strain with a clamp (Figure 1a) should be employed well. As for the filters, PTFE filters by stretching 2% and 2.5% were employed, while for nylon filters from 2.5% to 3% with lower stiffness were for contrast. Consequently, the surface wrinkle formation combined with the surface relief, which effected on two different types of polymeric materials (i.e., PTFT and nylon) with the production of four different nanostructured surfaces (Figure 1g). Final thickness of these GO films (~2 µm) was controlled by adjusting the volume of the GO suspension (c = 1 mg/mL). The prepared GO films were dried at 50 °C under vacuum overnight. Afterwards, freestanding wrinkled GO films could be slowly peeled off from the membrane filters.

Characterization of graphene oxide Morphology and size of GO samples were studied with atomic force microscopy (AFM, XE7, Park System, South Korea) and field-emission SEM (FE-SEM, JSM-6700F, JEOL, Japan). The surface roughness of GO film was measured at five random sites of GO film by AFM. The X-ray photoelectron spectroscopy (XPS) spectra was acquired with the help of an ESCALAB 250 Xray photoelectron spectrometer (Thermo, America) equipped with Al Kα1,2 monochromatized radiation at 1486.6 eV X-ray source. Raman spectroscopy measurements (LabRAM HR800, Horiba Jobin Yvon, France) were characterized with a 532 nm excitation laser (2.36 mW at the sample's surface) and the holographic notch filter was used to remove Rayleigh-scattered (RS) light. Bacterial culture In this experiment, three representative bacterial species were selected, including the gram7



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negative bacterium E. coli and two gram-positive bacteria, M. smegmatis and S. aureus. M. smegmatis was obtained from the Prof. Taejung Park’s lab at the Chung-Ang University (Seoul, South Korea). S. aureus (ATCC®27217TM) was purchased from ATCC (Manassas, VA, USA). E. coli was obtained from Life Technologies, South Korea. E. coli and S. aureus were cultured in the Luria-Bertani (LB) broth medium in a humidified incubator under stable temperature of 37 °C as well as constant agitation for 8 h. M. smegmatis was cultured in the Sauton’s liquid medium in a CO2 incubator at 37 °C without shaking. The bacterial cultures in the midexponential growth phase were collected by centrifugation at 6000 rpm for 5 min. Afterwards, discarding the supernatant, pellets were washed three times with PBS buffer to remove the medium. And then the cells were re-suspended in PBS buffer, and gradually diluted to a desired concentration of 107–108 colony forming units (CFU) per mL.

In vitro antimicrobial assays The antimicrobial activity of GO nanosheets was evaluated by the drop test method.37,38 E. coli and S. aureus cells were first grown on a nutrient agar plate (Difco LB agar, BD biosciences, USA) at 37 °C for 24 h. A single colony from the plate was picked and inoculated into 5 mL of the LB broth (BD). The bacterial culture was then incubated at 37 °C and 200 rpm until its optical density (OD) at 600 nm reached 1.0. Then, 1 mL of the culture was centrifuged at 13,000 rpm for 2 min to obtain bacterial pellets. The pellets were first resuspended in 1 mL of PBS buffer and then diluted 100-fold with PBS. The number of live M. smegmatis cells in the PBS buffer was calculated by counting colonies on LB agar plates following serial 10-fold dilutions after incubating cells at 37 °C for 24 h. Next, 1 µL of PBS containing M. smegmatis cells (approximately 2.95×107 cells/mL) was dropped on a free-standing film (40-mm diameter) in a 8


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petri dish (60-mm diameter). On each freestanding film, random sites were chosen for four different drops according to the drop test method.44 The GO film was exposed in the dark for 15 min at room temperature before its surface was washed with 5 mL of PBS, and live cells were counted using the above-mentioned colony counting method.

Fluorescence microscopy imaging Briefly, a 1mL bacterial suspension (107–108 CFU per mL) was collected into a centrifuge tube after treatment with GO nanosheets. After centrifugation at 6000 rpm and resuspension in 1 mL of PBS buffer, the cells were stained with 10 µL of propidium iodide (PI) for 15 min, subsequently, using 10 µL of 4'-6-diamidino-2-phenylindole (DAPI) to counter-stain for another 5 min under the dark condition. Finally, the samples were measured by an inverted fluorescence microscope (IX71, Olympus, Japan).

SEM observation of cell morphology To observe morphological changes in the E. coli, S. aureus and M. smegmatis cells upon their interactions with the GO nanosheets, 1 µL of the bacterial suspension (1.0×107 cells/mL) was dropped on the film. After 15 min at 25 °C, the E. coli, S. aureus and M. smegmatis cells on the films were treated with 2.5% glutaraldehyde (GLA) for 30 min, washed with PBS and fixed with 1% osmium tetraoxide for 30 min. After fixation, the E. coli, S. aureus, and M. smegmatis cells were gradually dehydrated with progressive washes with 30%, 50%, 70%, 80%, 90%, and 100% ethanol. Every washing treatment was performed for 15 min. The dried free-standing films were sputter coated with platinum for FE-SEM measurements as described previously.

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Simulation methods and models The structure of 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), commonly found in the outer membrane of E. coli, S. aureus, and M. smegmatis cells, was used in our simulations. Two types of graphene nanomaterials, graphene and GO, were simulated in our system. The simulation of the interaction of a GO film with bacteria was performed by constructing a layer of slab with a periodic boundary and by applying the whole system to MD operated at 298 K. The MD simulation was performed by the Material Studio (MS) v. 4.2 package (Accelrys, San Diego, CA, USA) with condensed-phase optimized molecule potentials for atomistic simulation studies (COMPASS) force fields.

Results and discussion Spontaneous wrinkles with periodic amplitudes in specific dimensions commonly form on various natural surfaces across a range of scales, from meters to nanometers. When forced to extend or shrink in its plane, the sheet or layer will be wrinkled or buckled along a certain direction in a limited planar space under some geometric boundary conditions.38 In this study, we examined antibacterial properties of free-standing GO films that exhibited instability-driven, hierarchical, micro/nanostructured wrinkles. Intriguingly, vacuum-drying mediated deposition of a GO suspension through the slightly stretched (i.e., pre-strained) porous filter membrane causes the emergence of wrinkled hierarchical structures composed of GO nanosheets with multiscale lengths. There are two competing parameters to achieve the amplitude of the wrinkles, that is, the initial thickness of GO films and the mechanical properties of the membrane filters, which directly influence the final geometries of the wrinkled surface of GO nanosheets.39 Figure 1a 10


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shows the schematics of the fabrication process and the insets illustrate in detail how the filter paper was placed in between the funnel and the flask. This manipulation induced constrained shrinking along the elongated direction (Figure1a, upper schematic drawing inset). The combination of a mild strain applied to the filter membrane and subsequent vacuum filtration of the GO solution produced periodically ordered GO structures with wrinkles (i.e., strain-induced surface relief structures) over the entire area in a highly efficient manner (Figure 1a, lower schematic drawing). The resulted surface of GO film was measured by an optical microscope and the formation of the uniaxial mode wrinkles was confirmed (Figure 1b). Morphological details of GO films with wrinkled surfaces were explored by SEM. A representative SEM image of a highly regular wrinkled GO surface formed on the filter paper is appeared in Figure 1c. The combination of surface wrinkle formation with the surface relief effect generated nanostructured surfaces with certain wavelengths. The average periodicity of the intertwined wavelength, λ was measured to be ~9.2 µm and the width of each wall of wrinkles was ~1.1 µm. Abundant wrinkles with a highly oriented configuration generated by the directional strain relief clearly indicated that large compressive stress affected the GO thin film after the removal of strain. Highly magnified SEM images show that markedly sharp edges of the GO wrinkles popped up and crumpled at different length scales in a certain thickness criteria (~2 µm) of the GO films (Figure 1d–f). AFM was used for a more extensive examination of morphology of the wrinkled GO film structures (Figure 1g). We found that the fabricated GO films exhibited wavy wrinkles and nanoscale grooves with characteristic surface roughness values (Rq, Root-mean-square) in the range of 465–1179 nm. It is important to note that surface roughness (i.e., RMS values) can be a general parameter that quantitatively characterizes wrinkled nanostructures composed by GO nanosheets. According to our measurements, four 11



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different surface roughness grades could be distinguished in wrinkled GO nanostructures (Figure S1). The GO film with 465-nm surface roughness (denoted as GO-465) exhibited wrinkles of 7.75 ± 0.5 µm in width and 0.74 ± 0.12 µm in height. GO-505 wrinkles were 9.2 ± 1.1 µm in width and 1.1 ± 0.28 µm in height, while the GO-845 film comprised wrinkles of 12.7 ± 0.9 µm and 2.09 ± 0.32 µm in width and height, respectively. As for GO-1179, its wrinkles were 15.2 ± 1.4 µm and 4.26 ± 0.27 in width and height, respectively. It is worth to note that fabrication of these different types of nanostructured GO films with highly wrinkled configuration was highly reproducible and robust, while the procedure of applying the strain to the membrane filter before filtration of the GO solution was very simple. The analysis of the chemical composition of the wrinkled GO films was carried out by Raman and X-Ray photoelectron spectroscopy (XPS). As shown in Figure S2, two typical bands were presented in the Raman spectra. The G band was broad and shifted to 1595 cm-1, whereas the D band at 1350 cm-1 due to extensive oxidation. The ID/IG ratio of this fabricated GO film was 0.96. The G band represents sp2-bonded carbon, whereas the D band demonstrates disorder level introduced to the crystalline structure with defects, indicating the well going process of graphitic oxidation.39 The XPS spectra survey reveals the O1s signal (oxygen) is higher than the C1s (carbon) signal characteristics (Figure 3Sa). The ratio of atomic percentage concentration of C-C/C-H functional groups to all carbon-oxygen functional groups is 1.09, which denotes the oxidation degree of GO. A considerable degree of oxidation of the fabricated GO film was also confirmed by the presence of different functional oxygen-containing groups in the GO structure (e.g. carbonyl, epoxy and hydroxyl groups) revealed by C1s XPS spectra (Figure 3Sb).40 Such abundant oxygen-containing groups in GO nanosheets were considered significant for the antimicrobial activity of GO. We hypothesized that such finely tuned GO wrinkled structures could endow GO coated 12


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surfaces with antimicrobial properties. To evaluate the antibacterial activity of the wrinkled GO nanosheet surfaces, gram-negative E. coli, gram-positive M. smegmatis and gram-positive S. aureus were selected as test microorganisms. Gram-negative bacteria have a thin (2–3 nm) layer of peptidoglycan between the inner and outer cell membranes, whereas gram-positive bacteria have a thicker peptidoglycan layer (20–80 nm) in their single layer cell wall. These structural differences underlie distinct sensitivity of gram-negative and gram-positive bacteria to various antimicrobial treatments. Thus, separate strategies are usually used to inhibit the growth of gramnegative and gram-positive bacteria, because their morphological features affect their sensitivity to particular antimicrobials. Initially, we characterized the morphology of the three bacterial species cultured on a flat surface by SEM (Figure 2). Magnified SEM images of each individual bacterium were used to determine their characteristic properties summarized in Figure S4. Next, we performed conceptual experiments to evaluate the relationship between GO nanosheet surface roughness and bacterial viability. Antibacterial activity was investigated by measuring the viability of the selected bacterial pathogens after their exposure to wrinkled GO nanostructures. For this experiment, the antibacterial drop test was used (see the Experimental section for details). The survival rate of the bacterial cells after exposure to GO nanosheets with different grades of surface roughness is presented in Figure 3. We observed that in the presence of nanostructured surfaces, bacterial growth was significantly inhibited, whereas in control experiments, in the absence of the wrinkled structures, the growth of bacterial cells was unaffected. Notably, GO samples with different roughness grades showed distinct patterns of antibacterial activity. GO-505 with surface roughness value in the range of ~500 nm exhibited the strongest antibacterial effect against E. coli and S. aureus, whose viability decreased to approximately 20% of control values. However, in the case of M. smegmatis, GO-845 with the 13



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surface roughness value of ~850 nm showed the most potent antibacterial effect with a decrease in bacterial viability to approximately 30% of control values. Overall, the results obtained clearly indicate that GO nanostructures can exhibit substantial antimicrobial activity toward different types of bacterial pathogens within a relatively short period (i.e., 15 min). This was confirmed by the consecutively lower number of colonies grown on the LB plates as a result of the bactericidal effect. In our experimental approach, the time-dependent changes in the bacterial cell survival rate were not taken into consideration. The control period of incubation with the wrinkled GO surfaces usually lasted 15 min, because, after that time, we observed a nearly complete loss of cell viability (~99%). In addition, the observed differences in the bactericidal effects of GO films on E. coli and M. smegmatis may be mainly attributed to the differences in their native cell wall characteristics.24 The zeta potential of GO nanosheet and three kinds of bacterials were measured and the results of which of GO nanosheet, E. coli, M. smegmatis, and S. aureus is -37.3 eV, -57.7 eV, -58.1 eV and -48.2 eV, are demonstrated respectively in Figure S6. From the above measurements, negative charges have been observed on the GO sheets edges along with carboxylate groups, and the three kinds of bacterial cell wall in aqueous suspensions with neutral pH. The ionization of the acidic/basic functional groups in their cell wall has empowered some bacterial species the quality of adhesion. The negative charge of the bacterial cells functions as a communicator with the sheet edges, electron acceptors as well as the reason of bacterial cell membrane damage. In the case of M. smegmatis, the peptidoglycan layer is protected from chemical

attacks

under

the

cover

of

an

additional

outer

membrane

containing

lipopolysaccharides. Furthermore, graphene-mediated toxicity toward bacterial species not only relies on intrinsic graphene properties but also depends on several factors, such as the cell membrane composition, diameter, and morphology of treated bacteria.45 14


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To directly observe morphological changes of the microbial cells after their exposure to wrinkled GO nanostructures, SEM measurements were performed in 15 min after the drop test (Figure 4). The SEM images show that the bacterial cells came in close contact with the intertwined walls of wrinkled GO nanostructures. In particular, in addition to the direct contact with the wall edges, GO “blankets” that slightly peeled off from the bulky GO substrate trapped and fully covered individual and aggregated bacterial cells. In accordance to previously reported observations,41 bacterial cells of each of the three species tested appeared to be completely enveloped in continuous thin GO layers regardless of their original shape (i.e., rod or spherical). Such isolation of cells from the medium by complete or partial wrapping may prevent nutrient absorption and, thereby, block the active sites on the bacterial cell surfaces.46 Furthermore, as can be seen from images of GO nanosheet-treated cells, their shape and size dramatically changed. The length and diameter of E. coli shrunk to 1.3 ± 0.2 µm and 0.6 ± 0.15 µm, respectively, which was considerably smaller than dimensions of untreated cells (Figure 2). Similar changes in size were also noted for M. smegmatis. The diameter of spherical S. aureus cells treated with GO nanosheets decreased to 0.65 ± 0.1 µm that indicates a ~30% reduction in volume compared to the measurements in untreated cells (Figure 2). Eventually, the cell membranes became deformed and collapsed, so a significant amount of bacterial debris could be observed on the surface of the GO nanosheets. Figure S5 demonstrates the SEM image of E. coli on the wrinkled GO surfaces, which it was the clear evidence for the leakage of intracellular substances for substantial degradation of cell membranes. In some cases, the cell membranes were completely decomposed (Figure S6). These results suggest that nanoscale geometry of GO nanosheets significantly affects their antimicrobial activity. When the surface roughness of GO nanosheets was consistent with the diameter of bacteria, the larger direct contact area was introduced between the GO 15



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nanosheets and the model bacteria surfaces. The physical structure of the GO surface, both high surface area and deep terrains, is a prominent factor affecting interaction with bacterial cells and improving bacterial cell adhesion. The surface wrinkles were composed of peaks and valleys on the GO surface, and the sharp edge of the former can inhibit attachment of bacteria cells and their abundance of sharp edges with better charge transfer in the GO nanosheets causing destruction of the bacterial cell membrane (Figure 4 a, c and e). The deep terrains in the roughened GO nanostructures was proved to be able to trap bacteria of matching diameter, enhance the strong interaction with the bacterial cells and promote direct oxidation of cellular components. In addition, improved physical disruption of the bacterial cellular membrane may result from the interaction between the GO nanosheet interface and the cell surface leading to the destructive extraction of the membrane lipids29 and subsequent release of the intracellular contents.24,43 The antimicrobial activity of the GO nanosheets was also confirmed by fluorescence imaging (Figure 5). In these experiments, we used PI and DAPI fluorescent dyes to assess cell viability. DAPI can pass through live cells with an intact membrane during which process the dye was witnessed to strongly bind to DNA and emit blue fluorescence. Membrane-impermeable PI is widely used in staining cells with damaged or compromised membranes (i.e., dead cells), and it emits red fluorescence. Thus, fluorescent staining of live and dead cells that allowed assessing the integrity of the cell membrane was conducted. E. coli, M. smegmatis and S. aureus grew vigorously in control conditions on a cellulose membrane surface (Figure 5). In contrast, when exposed to the wrinkled GO nanostructures, the bacterial cells clumped together and became significantly degraded (Figure 5 d, f, h), as uptake of PI by treated cells was considerably higher than that before treatment (Figure 5b). 16


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Based on the above results, the relationship between antibacterial activity and surface roughness of the wrinkled GO nanostructures can be explained as follows. In addition to the well-established mode of antimicrobial activity mediated by direct contact, our approach reveals a potent antimicrobial effect of the geometrically tuned GO surfaces that do not release bactericidal substances. Only a thin peptidoglycan layer could be observed from the cell wall of gram-negative bacteria, so when bacteria come into contact with the highly wrinkled GO surfaces, their outer membranes may undergo piercing or laceration across the lipid bilayer because its three dimensional sharp edges become surrounded by nanoscale wrinkles (Figure 1b, c). This physical effect changes membrane integrity or directly damages the membrane,32 which represents the initial impact of nanostructures on the bacterial cell.47 Subsequently, when bacteria become trapped or caught in the GO nanogrooves during the three-dimensional contact interaction with the wrinkled GO surfaces, the developing oxidative stress effectively complements the initial antibacterial activity.48-51 Indeed, the high defect density in GO nanostructures induces substantial oxidative stress in bacterial cells for adsorption of O2 on the defect sites in the basal plane and edges of GO nanosheets.48,52 Subsequently, there occurs oxidation of glutathione that serves as a redox state mediator in bacteria.53 High oxidation capacity of the GO sheets stimulates generation of reactive oxygen species that may cause a significant structural damage to the cell membranes by disrupting energy transduction, respiration,

transport

of

biologically

active

substances

and

energetic

balance

of

phospholipids.46,49 One theory was proved by coarse-grained molecular dynamics (CGMD) simulations that large amounts of phospholipids can be extracted directly from cell membranes by graphene sheets as which conducts dispersed interaction with hydrophobic lipid molecules.46 In that study, graphene sheets were positioned in horizontal or vertical way inside of the 17



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phospholipid

bilayer

formed

by

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1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

phospholipids (POPC) and possible interactions between graphene and lipid bilayers were visualized. It was found that graphene micelles were found successively releasing the monolayer of lipid tails and hybrid sandwiched graphene-membrane superstructures were also formed after being merged with the membrane. Because the phospholipid bilayer, as the basal plane of the cellular membrane, serves as a protective barrier that keeps ions, proteins and other molecules at necessary concentrations inside the cell, its destruction by graphene micelles justified a detailed study of their potential antimicrobial activity.49 GO nanosheets containing large π-conjugated structures with strong hydrophobicity can interact with phospholipid molecules in cellular membranes through hydrophobic interactions and, thereby, affect viability of microorganisms.50 When the lipid tails are extracted from cell membrane, nanoscale dewetting of water is accelerated by hydrophobic interactions due to phospholipids were all over the entire graphene sheet surface to maximize contact.22 Therefore, the MD simulation based on the force field of atoms is supportive to identify the molecular mechanism by finding out which GO sheets may alter cell membrane integrity.22 In this context, to obtain insight into the mechanisms of antibacterial effects of graphene-based nanomaterials, we designed two models with a spatial length ranging from several angstroms to nanometers, where graphene and GO were modelled to interact with POPE, a common constituent of the outer membrane of bacterial cells (Figure S8).32,33,46 We refer to them as the POPE-graphene (PG) and the POPE-GO (PGO) models. MD simulations were conducted under the canonical ensemble NVT (amount of substance (N), volume (V) and temperature (T)) with a time step of 1 fs for 300 ps until reaching the state of system equilibrium. MD simulations were performed additional time step of 1 fs for 100 ps for further analysis. As 18


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a consequence, snapshots before and after MD simulations of the PG and PGO models were produced (Figure 6). Notably, MD simulations indicated significant transformation of POPE on the graphene nanomaterial layer, which supports our experimental results. Adhesion between POPE and the graphene nanomaterial layer can be evaluated through interaction energy. Table S1 lists values of the energy of interaction between POPE and graphene nanomaterials, which were calculated as following:

= − ( + )

(1)

Here, EGraphene is the energy of the graphene nanolayer, EPOPE represent the energy of POPE and ETotal is the total energy of POPE with graphene nanomaterials. It is noted that high interaction energy value demonstrates strong adhesion between POPE and graphene nanomaterials, while negative and positive values of interaction energy represents attractive and repulsive forces between POPE and graphene nanomaterials, respectively. After evaluating the simulation results, the negative values of interaction energy in all systems clearly proved that POPE adhered to graphene nanomaterials. The simulation results were consistent with the snapshots of every MD simulation model. The value of the interaction energy in the PGO model was much higher than that of the PG model. Our findings indicate that GO nanosheets strongly interact with bacterial cell membranes. Furthermore, the values of non-binding energy between POPE and graphene nanomaterials were calculated in COMPASS force field (Table S2). POPE and graphene nanomaterials clearly interacted via electrostatic and van der Waals forces, and the electrostatic force was the dominant one. Based on this experiment, we conclusively presumed that direct adsorption of bacteria by GO nanosheets potently disrupted bacterial cell wall and cytoplasmic membrane resulting in a substantial loss of bacterial viability. 19



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Conclusions In summary, we investigated antibacterial activity of wrinkled GO nanostructures fabricated by simple vacuum filtration of GO suspensions through a pre-strained filter paper. Roughness of the wrinkled GO films was characterized by the Rq values, and three different bacterial species, E. coli, M. smegmatis and S. aureus were treated with the wrinkled GO films in a drop test to evaluate antibacterial activity of the nanostructures. We found that the extent of the antibacterial effect of GO nanostructures depended on the relative correspondence of the surface roughness grade to the bacterial size. For E. coli and S. aureus, the GO films with ~500 nm roughness grade had the strongest antibacterial effect due to the match of the wrinkle size to the bacterial diameter. For M. smegmatis, the GO film with the ~845 nm roughness grade showed the best antibacterial activity. The main advantage of using GO nanosheets with wrinkled geometry as antibacterial films is that preparation of wrinkled GO substrate does not require any complicated procedures or expensive materials. Adhesion of bacterial cells onto GO surfaces is promoted by characteristic wrinkled geometry of GO nanosheets that trap shape-matched bacteria within a larger direct contact area. The tight contact of the GO substrate with the cell wall or cytoplasmic membrane probably causes significant membrane stress and disruption of the cell membranes, leading to the leakage of intracellular substances and, eventually, cell death. We believe that our approach validates a promising 2-D material as an efficient platform with antibacterial properties that can be fabricated with high yields. ASSOCIATED CONTENT Supporting Information

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The supporting information is available free of charge via the internet at http://pubs.acs.org. AFM image and representative height profiles of GO film; XPS of GO; size distributions of three kinds of bacteria; SEM images of E. coli and M. smegmatis on the wrinkled GO surface; models of POPE and graphene nanomaterials; values of energy interaction and non-bonding energy between POPE and graphene nanomaterials. AUTHOR INFORMATION

Corresponding Author * Email: [email protected] (S. W. Hong) * Email: [email protected] (J. Lee)

Author contributions ¶

These authors contributed equally to this work

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C0862), National Research Foundation of Korea Grant funded by the Korean Government (NRF-2014R1A1A2058350), and the National Natural Science Foundation of China (51502296).

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Figure 1. Overview of the fabrication process and the characteristic features of wrinkled GO film surface. (a) Left: schematic illustration of the vacuum filtration process to fabricate wrinkled GO film, Right: the mounted filter paper membrane between the funnel and the flask (i.e., prestraining and shrinkage). Inset: a schematic drawing of the wrinkled GO surface. (b) A typical optical micrograph and a digital image of the wrinkled GO film (inset). (c–e) Representative SEM images of the wrinkled GO film surface with difference length scale; the sharp wall of edges are shown with micron- and nanoscale. (f) The thickness of a freestanding GO film measured by SEM. (g) Representative AFM images of the wrinkled GO film surfaces. Rq (rootmean-squire) values determine the surface roughness grades. From left to right, corresponding Rq values are 465 nm, 505 nm, 845 nm, and 1179 nm, respectively. 26


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Figure 2. SEM images of aggregated and individual bacteria. (a and b) Rod-shaped E. coli (length: 2.16 ± 0.4 µm; diameter: 0.76 ± 0.096 µm). (c and d) Rod-shaped M. smegmatis (length: 2.94 ± 0.46 µm; diameter: 0.62 ± 0.1 µm). (e and f) Spherically shaped S. aureus with a diameter of 0.90 ± 0.07 µm.

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Figure 3. Antibacterial activity of wrinkled GO nanostructures under dark conditions. The survival rates are displayed as a function of surface roughness for E. coli (a), M. smegmatis (b) and S. aureus (c) cultures. Inset images show digital images of agar plate colonies. 28


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Figure 4. SEM images of E. coli (a and b), M. smegmatis (c and d) and S. aureus (e and f) after the drop test on the wrinkled GO surfaces. Bacterial cells of each species exposed to the wrinkled GO films were completely enveloped with thin GO nanosheets. The arrows on the images point to local disruptions of the cell membrane caused by exposure to GO nanosheets.

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Figure 5. Cell viability measurements using staining of live and dead cells. (a–h) Fluorescence micrographs of E. coli, M. smegmatis and S. aureus after staining with PI and DAPI on the wrinkled GO nanostructures (Rq = 505, 845 and 505 nm for E. coli, M. smegmatis and S. aureus, respectively). This fluorescence assay shows increased antimicrobial activity as a higher percentage of bacteria that stained with PI (red color), i.e., lost their viability. 30


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Figure 6. MD simulation snapshots of (a) POPE-graphene and (b) POPE-GO models. Color codes: carbon atom (gray), hydrogen atom (white), oxygen atom (red), phosphorus atom (pink), and nitrogen atom (blue). The frame in the structure designate a resonance state, which is simulated by the amorphous cell module.

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Table of Contents (TOC) Graphics

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Wrinkled Surface-Mediated Antibacterial Activity of Graphene Oxide

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