Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide, and Reduced Graphene Oxide: Membrane and Oxidative Stress Zongsen Zou Instructor: Dr. Young-Shin Jun Environmental Nanochemistry 2-24-2016

Outline Introduction Objective Results and Discussion Conclusion Further study

1.1 Graphene-based materials 1. Introduction 1.1 Graphene-based materials Graphite(Gt) Graphene Mechanical exfoliation(adhesive tape technique), Graphite(Gt) Graphite oxide(GtO) Graphene oxide(GO) Oxidation (Hummer method) Exfoliation (sonication, stirring) Chemical vapor deposition(CVD) to generate graphene Graphene oxide(GO) Reduced graphene oxide(rGO) Reduction (chemical reduction, thermal annealing)

1.2 Potential applications -Excellent electronic, thermal, and mechanical properties; -Widely potential applications: nanoelectronics, conductive thin films, supercapacitors; 1.3 Antibacterial activities -Before wide applications, cytotoxicity of graphene-based materials need to be thoroughly studied to evaluate its health and environmental impacts firstly; -Previous studies have proved that GO and rGO exhibit strong antibacterial activity[1,2]; -However, there are less studies on the toxicity of graphene-based materials compared with other synthetic carbon nanomaterials, e.g. fullerenes and carbon nanotubes (CNTs); [1] Akhavan, O,, et al. ACS Nano, 2010. [2] Zhang, Y.B., et al. ACS Nano, 2010.

2. Objective The antibacterial activity of four types of graphene-based materials, Graphite(Gt), Graphite oxide(GtO), Graphene oxide(GO) and reduced Graphene oxide(rGO) toward a bacterial model Escherichia coli (E. coli) is studied and compared to help better understand the health and environmental impacts of graphene-based materials. With this knowledge, the physicochemical properties can be better tailored or either reducing their risks or increasing their potential applications.

3. Results and Discussion 3.1 Synthesized Gt, GtO, GO and rGO (a) For Gt, GtO and rGO dispersions, particles will precipitate after standing for 2 h. GO dispersion is stable which is attributed to the large amount of hydrophilic functional groups on GO nanosheets, e.g. carboxyl, hydroxyl and epoxy groups; (b, c) AFM image of GO nanosheet indicates the thickness is around 1 nm; (d, e) SEM images indicate that GO sheets are smooth with small wrinkles at the edges, while rGO sheets are much rougher than GO sheets; Figure1. (a) Photographs of Gt, GtO, GO and rGO dispersions; (b) AFM height image of GO nanosheets; (c) the corresponding height profile of AFM image; (d,e) SEM images of GO(d) and rGO(e) naonsheets

Figure2. Size distributions of Gt(a), GtO(b), rGO© and GO(d) -As shown in Figure 2, GO nanosheet has the smallest size. The size of Gt, GtO, GO, and rGO particles are 6.87±3.12, 6.28±2.50, 0.31±0.20, and 2.75±1.18 µm; -Different aggregation conditions significantly influence the interaction between nanoparticles and bacteria. Stable dispersion and small particles offer opportunities to interact with bacteria.

3.2 Antibacterial activities of Gt, GtO, GO and rGO Figure 3a. Antibacterial activity of Gt(a), GtO(b), rGO© and GO(d); Figure 3b&3c. Time-dependent and concentration –dependent antibacterial activities of GO and rGO -Antibacterial activities: GO(69.3±6.1%)>rGO(45.9±4.8%)>Gt>(26.1±4.8%)>GtO(15.0±3.7%); -GO and rGO have much higher antibacterial activities than Gt and GtO because particle sizes of GO and rGO are much smaller than Gt and GtO; -Antibacterial activities of graphene-based materials are time and concentration dependent;

3.2.1 Membrane stress Figure 4. SEM images of E.coli after incubation in saline solution(a,b), 40 μg/mL GO dispersion(c,d), and 40 μg/mL rGO dispersion(e,f) -One reason why small-sized GO and rGO have higher antibacterial activity than big-sized Gt and GtO is because small particles offer more opportunities to interact with bacteria for bacteria deposition; -After bacteria deposition, membrane stress is induced by sharp edges of graphene nanosheets on bacteria cell, which result in physical damages on cell membranes, leading to the loss of bacterial membrane integrity and the leakage of RNA, finally leading to bacteria death;

3.2.2 Oxidative stress Figure 5a. GSH oxidation by Gt, GtO, GO and rGO; Figure 5b&5c. Time-dependent and concentration-dependent GSH oxidation by GO and rGO -Oxidative stress can disturb or oxidize a vital cellular structure or component, therefore can be another reason causing bacteria death; -Oxidative capacities: rGO>GO>Gt>>GtO. rGO and Gt have higher oxidation capacities than GO and GtO because rGO and Gt have higher electrical conductivity due to their metallic characteristics; -Oxidation of GSH indirectly confirms that graphene-based materials can mediate ROS(reactive oxygen species)-independent oxidative stress toward bacterial cells; -Oxidation capacities of graphene-based materials are time and concentration dependent; Direct measurement of superoxide anion using XTT method doesn’t work, no superoxide anion is tested by XTT method, therefore no ROS mediated oxidative stress. In vitro GSH oxidation method prove that there is ROS-independent oxidative stress. ROS-independent oxidative stress, in which graphene-based materials may disrupt a specific microbial process by disturbing or oxidizing a vital cellular structure or component without ROS production.

3.3 Comparison -GO>GtO: Similar oxidation capacities. GO is much smaller than GtO. Therefore, graphene-based materials with smaller size have higher antibacterial activities than those with bigger size; -Gt>GtO: Similar particle size. Gt has higher oxidation capacity than GtO. Therefore, oxidation capacity also increase the antibacterial activities of graphene-based materials; -GO>rGO: GO is smaller than rGO. rGO has higher oxidation capacity than GO. Therefore, antibacterial activities of graphene-based materials are attributed to the combined effect of dispersibility, size and oxidation capacity.

3.4 Proposed antibacterial mechanism Three-step antibacterial mechanism (1) Bacterial cells firstly deposit on Gt, GtO, GO or rGO during incubation. Stable dispersion and small particles offer more opportunities to interact with bacteria cells for bacteria deposition; (2) After bacteria deposition, by direct contact, graphene nanosheets are able to induce membrane stress by disrupting and damaging cell membranes which can lead to cell death; (3) Thirdly, by direct contact, graphene-based materials are capable of inducing ROS-independent oxidative stress on bacterial stress which can lead to cell death;

4. Conclusion (1) Antibacterial activities of graphene-based materials: GO>rGO>Gt>GtO; (2) Antibacterial activities of graphene based materials are time and concentration dependent; (3) Antibacterial activities of graphene-based materials are attributed to both membrane and oxidative stress applied on bacteria cells. A three-step antibacterial mechanism is proposed to explain this.

5. Further study (1) Antibacterial activities of graphene-oxide materials can be used in system requiring biofouling resistance, such as membrane filtration water treatment system; (2) Study of the antibacterial activities of graphene-based materials may dispersion not be enough. For example, for application in membrane project, we study to study the antibacterial activities of graphene-based materials when they attached on the membrane surface; (3) Besides the two proposed antibacterial mechanisms, membrane stress and oxidative stress, other properties of graphene-based materials, such as photothermal function can also be used to increase the antibacterial activities;

Thank you! Questions?