Subscriber access provided by Temple University Libraries Biological and Environmental Phenomena at the Interface Antimicrobial Properties of 2D MnO2 and MoS2 Nanomaterials Vertically Aligned on Graphene Materials and Ti3C2 MXene Farbod Alimohammadi, Mohammad Sharifian Gh., Nuwan H. Attanayake, Akila C. Thenuwara, Yury Gogotsi, Babak Anasori, and Daniel R. Strongin Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00262 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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1 2 3 4 Antimicrobial Properties of 2D MnO2 and MoS2 Nanomaterials 5 Vertically Aligned on Graphene Materials and Ti3C2 MXene 6 7 Farbod Alimohammadi1,2, Mohammad Sharifian Gh.1, Nuwan H. Attanayake1,2, Akila C. Thenuwara1,2, 8 Yury Gogotsi3, Babak Anasori3, and Daniel R. Strongin1,2*

9 1 Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, USA. 10 2 Center for Computational Design of Functional Layered Materials (CCDM), Temple University, 1925 North 12th Street, 11 Philadelphia, Pennsylvania 19122, United States 12 3 Department of Materials Science and Engineering, and A. J. Drexel Nanomaterials Institute, Drexel University, Philadelphia, 13 Pennsylvania 19104, USA 14 15 16 ABSTRACT 17 Two dimensional (2D) nanomaterials have attracted considerable attention in biomedical and environmental applications due to their antimicrobial activity. In the interest of investigating the primary 18 antimicrobial mode-of-action of 2D nanomaterials, we studied the antimicrobial properties of MnO and 19 MoS , toward Gram-positive and Gram-negative bacteria. Bacillus subtilis and Escherichia coli bacteria 20 2 were treated individually with 100 µg/mL of randomly oriented and vertically aligned nanomaterials for ∼3 21 2 h in the dark. The vertically aligned 2D MnO2 and MoS2 were grown on 2D sheets of graphene oxide, 22 reduced graphene oxide and Ti3C2 MXene. Measurements to determine the viability of bacteria in the 23 presence of the 2D nanomaterials performed by using two complementary techniques, flow cytometry 24 and fluorescence imaging showed that while MnO and MoS nanosheets show different antibacterial 25 activities, in both cases, Gram-positive bacteria show a higher loss in membrane integrity. Scanning 2 2 26 electron microscopy (SEM) images suggest that the 2D nanomaterials, which have a detrimental effect on 27 bacteria viability, compromise the cell wall, leading to significant morphological changes. We propose 28 that the peptidoglycan mesh (PM) in the bacterial wall is likely the primary target of the 2D nanomaterials. 29 Vertically aligned 2D MnO nanosheets showed the highest antimicrobial activity, suggesting that the 30 edges of the nanosheets were likely compromising the cell walls upon contact. 2 31 32 KEYWORDS 33 2D nanomaterials, antimicrobial, birnessite, mode-of-action, nano-knife, cell wall, peptidoglycan mesh 34 35 INTRODUCTION 36 37 According to the World Health Organization (WHO), antibiotic resistance has become one of the 38 biggest concerns to human health and food security, demanding a continuous development of new 39 40 classes of antimicrobial agents 1-4. In the interest of dealing with the antibiotic resistance, nanomaterials 41 with antibacterial properties (e.g., Ag nanoparticles) have been widely studied for various applications 42 5 6 7 8-9 43 such as water treatment , food packaging , medical devices , and in textile industries . Among the 44 various types of nanomaterials, ones with a two-dimensional (2D) motif and antimicrobial activity have 45 10-12 46 been recently introduced for biomedical and environmental applications . 47 Understanding the primary antimicrobial mode-of-action (MoA) of 2D nanomaterials is crucial in 48 designing new nanomaterials with higher antibacterial activity and lower toxicity toward human cells. 49 50 Among 2D nanomaterials, the graphite family (i.e., graphite, graphene oxide (GO), graphite oxide, and 51 reduced graphene oxide (rGO)) has been well studied for antibacterial applications 10, 13-15. Research has 52 53 shown that antibacterial properties of the graphite family are driven by both chemical and physical factors 54 10, 16-17. For instance, it has been reported that sharp edges of GO nanosheets (i.e., called ‘nano-knives’) 55 10- 56 damage bacterial membranes, which results in the release of cytoplasmic materials and bacteria death 57 58 1 59 60 ACS Paragon Plus Environment Langmuir Page 2 of 21

1 2 3 12, 16-17. It is proposed that smaller nanosheets might permeate into the microorganism through direct 4 10-12 5 physical penetration or via endocytosis . 6 A goal of the present study is to evaluate the role of ‘nano-knife’ characteristics of 2D 7 8 nanomaterials on their antibacterial properties. While there have been many studies on graphite family 9 nanosheets, there is a little understanding about the antibacterial properties of other common 2D 10 nanomaterials, such as MnO and MoS . Among transition metal chalcogenides 18-20, MoS has been 11 12 widely used for various applications2 that2 have included catalysis 21, energy storage22, drug delivery2 23-26, 13 and environmental chemistry 27. To the best of our knowledge, however, there are only a few studies on 14 15 antibacterial properties of those nanomaterials. Yang et. al. have shown that monolayer MoS with less 16 aggregation has higher antibacterial activity than bulk MoS which is due to their morphology differences,2 17 28 18 including shape and specific surface area . Research has2 reported that production of reactive oxygen 19 species (ROS) is increased in the presence of vertically aligned MoS on a glassy carbon substrate, which 20 29 21 results in higher antibacterial activity of the nanomaterial . Prior 2work has also shown that the basal 22 plane of GO- MoS nanocomposite has antibacterial properties30. MnO nanomaterial has found use in 23 the environmental2 arena as a material capable of destroying aqueous2 pollutants31-32 and showing 24 25 antibacterial activities 31, 33-36 as well. For example, it has been shown that MnO coated onto an 26 ultrafiltration membrane can be used in drinking water treatment. Moreover, antibacterial2 properties of - 27 34 28 MnO are enhanced when decorated onto carbon nanotubes . However, the antibacterial activity αof 29 layer2ed MnO (i.e., birnessite phase) has not been studied yet. 30 31 In the2 current study, we build on prior research and investigate antibacterial properties of MnO 32 toward both Gram-positive (i.e., Gram+) and Gram-negative (i.e., Gram ) bacteria. Our scientific2 33 34 hypothesis to be tested is that the sharp edges of 2D nanosheets play a− key role in damaging the 35 bacterial cell wall, which results in the loss of membrane integrity and bacteria death. To this end, we 36 MnO MoS 37 compare the antibacterial activity of randomly oriented (i.e., dispersed flower-like and ) versus 38 vertically aligned MnO and MoS nanomaterials grown individually on GO , rGO , 2and Ti3C22 MXene 39 MnO /GO2 MoS /rGO2 MoS /MXene 40 substrates (i.e., , , and ). The scientific logic used to test our hypothesis 41 is that if sharp edges2 of the 2D2 nanomaterials 2play a significant role in reducing the bacteria membrane 42 integrity, the antibacterial activity of the nanosheets vertically grown on 2D substrates should be greater 43 44 compared to the randomly oriented sheets of the respective nanomaterial. Escherichia (E.) coli and 45 Bacillus (B.) subtilis bacteria species were chosen as Gram and Gram+ model systems, respectively. To 46 47 reduce the effects of oxidative stress (i.e., mainly ROS−-dependent oxidative stress) in antibacterial 48 properties, we incubated bacteria with the nanomaterials in the dark. Flow cytometry (FC) and 49 50 fluorescence imaging (FI) techniques were used for bacteria viability measurements and scanning 51 electron microscopy (SEM) was used to evaluate interactions of 2D nanomaterials with the bacteria. 52

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1 2 3 RESULTS AND DISCUSSION 4 5 6 Characterization of the 2D nanomaterials 7 GO 8 Atomic force microscopy (AFM) was used to characterize the thickness and lateral size of the 9 and MXene nanomaterials after deposition onto an atomically flat mica surface (see Figure S1 and S2). 10 Visual inspection of the micrographs shows that the GO monolayer has a thickness of ∼1 nm and a lateral 11 12 size varying between 0.3 and 10 µm. The thickness of MXene nanosheets was in the range of ∼1 nm 13 14 (monolayer) to 350 nm, with the latter dimension being associated with the stacking of many nanosheets. 15 Raman spectroscopy of GO (see Figure S3) showed two broad characteristic peaks at 1345 cm 16 −1 (D band) and 1582 cm (G band). The D band is associated with the amount of disorder introduced in to 17 −1 18 the crystalline structure by the presence of defects while the G band reflects the sp2-bonded carbon 19 regions (i.e., tangential radial mode). The presence and intensity of the D band confirms that the 20 21 graphene is oxidized to some extent 37. 22 The morphology of the 2D nanomaterials was also investigated with transmission electron 23 24 microscopy (TEM) and SEM. Figure 1 presents TEM images of MnO , MoS , MXene, MnO /GO, MoS / 25 rGO, and MoS /MXene. Flower-like MnO and MoS had an average particle2 size2 of ∼320 and2 110 nm,2 26 27 respectively (see2 Figures 1, S4, and 2S5). The 2oxide groups on the graphene oxide sheets act as 28 nucleation sites for the growth of MnO and MoS . As depicted in the TEM and SEM images (Figures 1, 29 MnO MoS 2 2 30 S4, S5, and S6), and were uniformly aligned onto the substrate surfaces. TEM confirmed that 31 the few layer MnO and2 MoS were2 vertically aligned on the substrates (Figure 1d, 1e, and 1f). 32 2 2 33 34 Antibacterial activity of and nanomaterials; vertically aligned vs. randomly oriented 35 There are a variety of𝟐𝟐 experimental𝟐𝟐 strategies to determine the viability of bacteria under an 36 𝐌𝐌𝐌𝐌𝐎𝐎 𝐌𝐌𝐌𝐌𝐒𝐒 37 external stress. Among these strategies is the use of fluorescent probes. Propidium iodide (PI), for 38 example, has commonly been used as a stain capable of assessing the viability of bacteria 38-39. PI is 39 40 known to exhibit a fluorescence enhancement of up to 20- to 30-fold upon intercalation into double- 41 stranded regions of DNA 40-41. Given that bacterial DNA is found exclusively within the cytosol, such an 42 43 interaction is possible only if PI can diffuse across the cytoplasmic membrane (CM) of bacteria. Of 44 significance, it has been shown that PI with a concentration of ∼20 µM, does not cross the CM in live 45 46 bacteria, leading to no fluorescence enhancement. Bacteria with damaged membrane, however, exhibit a 47 breakdown-induced permeability enhancement of the CM which results in the uptake of PI into the cytosol, 48 which is followed by fluorescence enhancement. In contrast to PI, the SYTO9 molecule can easily 49 50 transport across the CM of both live and dead bacteria (i.e., with damaged membrane). Using both stains 51 allows us to estimate the ratio of dead/live bacteria (i.e., SYTO9-stained for live; PI-stained for dead). 52 53 Flow cytometry (FC) and fluorescence imaging (FI) of bacteria incubated with concentrations of ∼20 µM 54 PI is a convenient means of evaluating bacterial viability 42-46 and deducing mortality rates of bacteria 55 47 48 56 which are exposed to a stimulus such as antimicrobial treatment and nanoparticles . Here, we 57 58 3 59 60 ACS Paragon Plus Environment Langmuir Page 4 of 21

1 2 3 evaluate antibacterial properties of the 2D nanomaterials (i.e., MnO and MoS ) against the two common 4 5 Gram and Gram+ bacterial species (i.e., E. coli and B. subtilis,2 respectively)2 by performing viability 6 measurements− of the samples using the two complementary methods, FC and FI techniques. 7 8 Figure 2 exhibits FC and FI results of E. coli and B. subtilis bacteria species, untreated and 9 treated with 100 µg/mL of MnO and MnO /GO for 3 h. For the FI and FC experimental methods and data 10 11 analysis, the reader is referred 2to the SI document.2 As shown in Figure 2a, the population ratio of the PI- 12 stained to the SYTO9-stained bacteria (i.e., red/green ratio) is higher for the MnO /GO treated bacterial 13 population (Figure 2a right panels), which is consistent with MnO /GO having a 2stronger antimicrobial 14 15 activity than MnO (Figure 2a left panels). Of significance is that 2the antibacterial activity of MnO /GO 16 against the Gram+2 bacteria (i.e., B. subtilis) is noticeably higher than that for the Gram species (i.e.,2 E. 17 18 coli). While the FI results show stronger antibacterial activity of the vertically aligned Mn− O , it does not 19 provide statistical information over a large population of bacteria. However, FI results allow us2 to visualize 20 21 the bacterial populations. Figure 2b depicts the complementary results obtained by the FC technique, 22 where the percentage of viable populations for untreated bacteria is shown for comparison. The strong 23 24 antibacterial activity of randomly oriented MnO is reflected in the significant drop of the viable B. subtilis 25 bacteria from 97 to 38%. We stress that all bacteria2 l samples were exposed to the 2D nanomaterials in 26 27 the dark, and therefore, we expect that oxidative stress due to ROS is not an important consideration. 28 Data presented in Figure 2 show that the activity of MnO is significantly increased for the vertically 29 aligned motif (i.e. MnO /GO) where the population of the viable2 bacteria decreases to 10%. A similar trend 30 31 is observed for the Gram2 species where the percentages of viable bacteria were 87 and 82% for 32 MnO /GO and MnO treated samples, respectively. Similar to previous reports on other 2D nanomaterials 33 − 34 49-51, 2the antibacterial2 activities of MnO against Gram+ bacteria are higher than against Gram bacteria. 35 While the mass of aligned nanosheets 2in solution are lower in comparison to the randomly oriented MnO 36 − 37 sheets, the higher antibacterial activity has been observed for the aligned nanosheets. It is important to2 38 mention that the entire substrate was covered by vertically aligned MnO and we do not expect any 39 40 physical interaction of the microbes with the substrate. By vertically aligning2 the MnO2 sheets the density 41 of edges has been increased. The results confirm that the increasing the density of edges improve 42 43 antibacterial activity. We believe that the edges play a pivotal role in the antibacterial activity of the 44 nanomaterials, likely via a nano-knife mode-of-action. 45 Figure 3 exhibits results for viability measurements of the bacterial samples that were treated 46 47 with 100 µg/mL MoS for 3 h. As depicted, the antibacterial activity of MoS toward both types of bacteria 48 is lower than MnO . However,2 the number of viable bacteria reduced from 95%2 (i.e., for untreated bacteria) 49 50 to 60% and 75% 2for MoS /rGO treated and MoS /MXene treated B. subtilis, respectively. Although, we 51 observe a slightly different2 activity for MoS on the2 two substrates (i.e., which can be attributed to the 52 53 different physical properties of substrates 2including dimension and flexibility), we observe a stronger 54 antibacterial activity for the vertically aligned samples for both bacterial species. Similar to MnO , the 55 56 antibacterial activity of MoS (i.e., and aligned samples) is stronger against Gram+ than Gram bacteria.2 57 2 − 58 4 59 60 ACS Paragon Plus Environment Page 5 of 21 Langmuir

1 2 3 Prior research has reported that the density of edges on graphene nanosheets play an important 4 12 5 role in antibacterial activity . Vertically oriented nanosheets were shown to have maximum antibacterial 6 activity. Our results show that an increase in the density of MnO edges improves the antibacterial activity 7 8 of the nanomaterial. We should note that, in both studies, the2 substrates are fully covered by the 2D 9 nanomaterials. Therefore, the antibacterial activities of the materials are expected to be dominated by the 10 properties of the MnO and MoS 2D nanomaterials rather than the substrates (i.e., GO and MXene). 11 12 2 2 13 Proposed inhibition mechanism of 2D nanomaterials 14 15 The viability measurements of the bacterial species treated with the 2D nanomaterials (data in 16 Figures 2 and 3) suggest that the Gram bacteria are more resistant to the nanomaterials. While MnO 17 18 and MoS show different antibacterial activities− against the bacterial species, in both cases, Gram+2 19 bacteria show2 a higher loss in membrane integrity when exposed to the materials. These results are in a 20 MXene 21 good agreement with previous reports which were carried out for and graphene family 22 nanomaterials 49-51. Here, with the purpose of investigating interactions of the nanosheets with the 23 bacterial surfaces (i.e., to better understand the antibacterial MoA of our 2D nanomaterials), we used 24 25 SEM 52 to visualize the bacterial morphology changes due to those physical interactions. Specifically, we 26 investigated the interaction of randomly oriented and vertically aligned MnO nanosheets with B. subtilis. 27 28 Figure 4 presents the SEM images of the Gram+ bacteria, untreated2 B. subtilis, and after being 29 exposed individually to GO, MnO and MnO /GO nanomaterials at a loading of 100 µg/mL for 3 h. 30 31 Untreated bacteria (Figure 4a) have2 a rod-shape2 morphology with a smooth surface and we interpret this 32 image as being representative of intact bacterial membranes. The physical interaction of the graphene 33 34 oxide before aligning sheets on the surface is presented in figure 4b. Inspection of the micrograph 35 suggests that graphene oxide sheets wrap around the bacteria. Wrapping or trapping (in addition to the 36 37 nano-knife mechanism) of the cell by graphene is another proposed antimicrobial mechanism of the 38 graphene10-11, 53-54. Thin layers of graphene oxides are flexible allowing them to wrap around the bacteria, 39 isolating the cell from the environment. The lack of the physiochemical conditions and nutrients 40 41 presumably would ultimately lead to a loss bacterial viability. However, the bacteria morphology does not 42 change, and it has been shown after removing graphene oxide they can proliferate53. Inspection of Figure 43 44 4c, shows that the physical interaction of the MnO nanomaterial with the bacterial surface alters the 45 morphology of bacteria to a significant extent. Regional2 shrinkages of the cell are observed all around the 46 47 bacteria where the nanomaterial is attached on to the bacterial wall. This observation confirms that direct 48 interactions of the nanomaterial with the bacteria can cause morphological changes and presumably 49 bacterial death. presents the SEM image of B. subtilis exposed to MnO /GO nanomaterial. As 50 Figure 4d 51 expected, vertically aligned MnO causes more drastic change to the bacteria2 l morphology and 52 membrane integrity. The wrapping 2mechanism which was observed for graphene oxide (substrate, figure 53 54 4b) is not observed here which shows that the substrate likely does not play a key role in the antibacterial 55 activity. However, graphene oxide is a very thin layer and flexible and it can probably facilitate the 56 57 58 5 59 60 ACS Paragon Plus Environment Langmuir Page 6 of 21

1 2

3 interaction of the aligned edges of the MnO2 with the bacteria. The increased amount of morphological 4 5 change is likely the result of the higher density of edge sites associated with the vertically aligned 6 nanosheets, which play the role of nano-knives. The shrinkage of the bacteria is likely due to the loss of 7 8 cytoplasmic material. SEM images show that there is no observable interaction of the microbes with the 9 substrate (GO), suggesting the entire antimicrobial activity is due to the vertically aligned nanosheets. 10 To investigate the probable MoA of the 2D nanomaterials, we need to review the membrane 11 12 ultrastructure of bacteria in more details. As depicted in Figure 5a, Gram bacteria are composed of a 13 pair of distinct lipoprotein membranes: a lipopolysaccharide (LPS) coated outer membrane (OM) and an 14 − 15 inner cytoplasmic membrane (CM), which are separated by a peptidoglycan mesh (PM) 55-56. Conversely, 16 Gram+ bacteria (Figure 5a, right panel) are comparatively simpler and possess only a single lipid bilayer 17 18 membrane (i.e., CM), though their PM is typically ca. 10−20+ times thicker than that found in Gram 19 55 bacteria . Figure 5b is a schematic representation depicting possible direct physical interactions of the− 20 bacterial surface with sharp edges of vertically aligned nanosheets. 21 22 We note that previous studies have not explicitly appreciated the presence of PM in the bacterial 23 wall and the role it might play in antibacterial MoA of the 2D nanomaterials. Of significance, in both 24 25 bacterial classes, PM (i.e., the bacterial cell wall scaffold) has an important role in maintaining the 26 morphological integrity of the bacteria. Inhibition or degradation of the PM results in bacterial shrinkage 27 57 28 and eventually cell lysis . If the integrity of the PM is compromised by an external stimulus (e.g., 29 interaction with the nanomaterials), a turgor pressure of ∼4 atm from the cytoplasmic space toward the 30 57-58 31 exoplasmic region is created which results in loss of the bacterial membrane integrity . Furthermore, 32 for any external molecule or nanomaterial to reach the lipid bilayer of the bacteria CM, PM plays the role 33 34 of a diffusion barrier. In Gram+ species, the PM function is much more pronounced due to its thickness. A 35 majority of previous research (i.e. especially, molecular dynamics (MD) simulations) has proposed that 36 graphene nanosheets pierce into the lipid bilayer of the bacterial membrane 59 resulting in the extraction 37 38 of the lipid molecules from the bilayer. This mechanism, however, does not consider the presence of the 39 PM in the cell wall of the bacteria. We believe that this particular mechanism does not fully explain the cell 40 41 shrinkage that has been noted in prior research and in our research presented here. 42 Here, we propose that PM is likely the primary target of the 2D nanomaterials. We note that 43 58, 60 44 although Gram bacteria has a thinner layer of PM , it possesses an external protective OM which 45 protects the PM− from external stimulus (i.e., PM in Gram+ bacteria which has no OM is more susceptible 46 47 to the interaction of the sharp nano-knives of the 2D nanosheets). It has also been reported that Gram 48 bacteria exhibit more resistance to a direct contact interaction caused by an AFM tip than Gram+ due to− 49 the presence of the OM in Gram bacterial species 61. It is also worthwhile to note that the isoelectric 50 51 point of the surface of bacteria is− in the range of 1.75 - 4.15 for Gram+ and 2.07 - 3.65 for Gram 52 bacteria 49, 62-64. Therefore, at the pH used in our research (i.e., pH 7.3), both bacterial species have a 53 − 54 negatively charged surface which are expected to be repelled by the negatively charged 2D 55 nanomaterials (i.e., the zeta potential of the 2D nanomaterials are measured and shown in Table S1). 56 57 58 6 59 60 ACS Paragon Plus Environment Page 7 of 21 Langmuir

1 2 3 Therefore, the bacterial surface charge is likely not the driving force for the interactions of the 2D 4 5 nanomaterials with bacterial species. However, the slightly higher negative charge on the Gram 6 bacterial surface may be another reason for higher resistance of Gram species (e.g., E. coli) to 2D− 7 8 nanomaterials. − 9 10 CONCLUSIONS 11 12 We have investigated the antimicrobial mode-of-action of 2D nanomaterials. The viability 13 measurements of the bacteria species (i.e., B. subtilis as a Gram+ and E. coli as a Gram classes) 14 15 treated with the vertically aligned and randomly oriented MnO and MoS nanosheets confirm− that the 16 sharp edges of the nanosheets play a significant role in damaging2 the bacteria2 l cell wall and reducing 17 18 membrane integrity. Although MnO and MoS nanosheets show different antibacterial activities against 19 the bacteria species, in both cases,2 Gram+ bacteria2 show higher loss in membrane integrity. We propose 20 21 that the PM in bacteria cell wall is likely the primary target of the 2D nanosheets which is supported by the 22 experimental observation that show that 2D nanomaterials show a higher antibacterial activity toward 23 Gram+ species. Finally, though we propose PM as an important target of the 2D nanomaterials, more 24 25 studies are needed to understand the molecular-level nature of the interaction. Our study shows that 26 vertically aligned 2D nanosheet motif show a higher antibacterial activity against both bacteria classes 27 28 than 2D nanomaterials that have been previously investigated. Toxicity and environmental impacts of the 29 vertically aligned layered material, however, needs to be studied to evaluate them for further 30 31 environmental applications or industrial use. 32 33 34 MATERIALS AND METHODS 35 All reagents were analytical grade and were used without further purification. 36

37 38 Synthesis of and 39 The GO nanosheets were synthesized via the modified Hummers’ method 65. The MXene (i.e., Ti C Tx) 2D 40 𝐆𝐆𝐆𝐆 𝐌𝐌𝐌𝐌𝐌𝐌𝐌𝐌𝐌𝐌 41 sheets were synthesized by hydrochloric acid-lithium fluoride etching and delamination method3 2as has 42 been reported in detail previously 66. 43 44 45 Synthesis of and / 46 MnO 𝟐𝟐 𝟐𝟐 67 GO 47 nanomaterial𝐌𝐌𝐌𝐌𝐎𝐎 was synthesized𝐌𝐌𝐌𝐌𝐎𝐎 𝐆𝐆𝐆𝐆 via the Redox reaction as reported in ref. . About 80 mg of 48 nanomaterial2 was added to deionized water (DI-water) and was sonicated for 1 h. Then, 170 mg of 49 Mn(NO ) and 60 mg copolymer (i.e., poly(ethylene glycol), poly(propylene glycol), poly(ethylene glycol) 50 51 triblock3 copolymer)2 were added to the GO solution and stirred for 30 min. The solution was heated to 45°C 52 and then 10 ml of 0.1 M KMnO was added into the solution dropwise. The solution was centrifuged and 53 54 the collected pellet was rinsed five4 times to eliminate byproducts. Pristine MnO was synthesized through 55 the same procedure, but without addition of the GO nanomaterial. 2 56 57 58 7 59 60 ACS Paragon Plus Environment Langmuir Page 8 of 21

1 2 3 4 5 Synthesis of , / , and / 6 The procedure𝐌𝐌𝐌𝐌 for𝐒𝐒𝟐𝟐 𝐌𝐌𝐌𝐌synthesis𝐒𝐒𝟐𝟐 𝐆𝐆𝐆𝐆 of Mo𝐌𝐌𝐌𝐌S𝐒𝐒𝟐𝟐 and𝐌𝐌𝐌𝐌𝐌𝐌𝐌𝐌𝐌𝐌 MoS /GO nanomaterials have been reported in details 7 68 GO 2 2 8 previously . Briefly, 8 mg nanosheets were added in to 15 ml DMF solution and then sonicated for 30 9 min. Subsequently, 30 mg (NH ) MoS was added in to the solution and stirred for 30 min. The solution 10 was transferred to a 25 ml Teflon4 2-Lined4 autoclave and heated to 200°C for 10 h (i.e., without ramping or 11 12 cooling rate control). The product in the form of a precipitate was recovered by centrifugation (6,000 rpm) 13 for 15 min. The precipitate was rinsed in DI-water to remove the solvent and byproducts. This procedure 14 15 was repeated using ethanol and acetone to completely remove residual solvent and byproducts. Pristine 16 MoS was synthesized through the same procedure, but without the addition of GO nanosheets. To 17 18 synthesize2 MoS on the MXene substrate, we first sonicated MXene in dimethylformamide (DMF) for 1 h to 19 exfoliate the material.2 Thereafter, the same procedure was applied to synthesize MoS nanosheets on to 20 21 the MXene as that on GO. 2 22 23 24 Characterization of the nanomaterials 25 SEM images of the nanomaterials were obtained by using a FEI Quanta 450 FEG instrument. Samples 26 were prepared by drop-drying from a water suspension on to Si wafers. TEM images were obtained by a 27 28 JEOL JEM-1400 instrument. Samples were prepared by drop-drying a diluted suspension onto copper 29 grids covered with lacy carbon films. X-ray diffraction (XRD) was performed on a Bruker d8 instrument. 30 31 Zeta potential measurements were performed by using a Zetasizer Nano (Malvern Instruments). AFM 32 measurements were performed with an Agilent 5100 (Agilent technologies, AZ, USA) operating in tapping 33 34 mode in air. Conical tips with aluminum reflex coating were obtained from MikroMasch (radius < 8 nm, 35 spring constant = 5 N/m, resonant frequency = 160 kHz). Samples were prepared by drop-drying a diluted 36 suspension onto mica substrate. The WSXM software was used for the image analysis 69. 37 38 39 Bacterial strains 40 41 The antibacterial properties of the nanomaterials were evaluated using the Gram E. coli (mc4100 strain, 42 ATCC 35695) and the Gram+ B. subtilis bacterial strains (Ehrenberg Cohn 168 strain, ATCC 23857). The 43 − 44 bacterial strains were cultivated on Lauria Broth agar medium plates (LB Broth with agar Lennox, Cat. No.: 45 L2897, Sigma-Aldrich) at 37°C for 24 h and then stored at 4°C for future use. 46 47 48 Bacteria preparation 49 A discrete colony of each bacterial strain was grown aerobically at 37°C in 50 mL Terrific Broth solution in 50 51 a shaking flask at 150 rpm for 8 hours (i.e., at middle-to-late exponential phase). The harvested bacteria 52 were centrifuged (i.e., 1500xg, 2 min, room temperature) and then washed twice with phosphate buffer 53 54 saline (i.e., 1xPBS; pH = 7.3) to remove waste and residual Terrific Broth. For each washing step, we 55 used a Rotamix (10101-RKVSD, ATR Inc.) at 20 rpm to suspend the pellet cells in 1xPBS with no 56 57 58 8 59 60 ACS Paragon Plus Environment Page 9 of 21 Langmuir

1 2 3 biomechanical forces applied to the bacteria during the resuspensions. After twice washing with enough 4 5 1xPBS, the supernatant was removed and the pellets were collected for preparing the E. coli and B.

6 subtilis stock samples in 1xPBS with the cell density of OD = 0.15 ± 0.05. We point out that we applied 7 8 experimental conditions that were deemed least detrimental600 to the viability of the bacteria (e.g., osmotic 9 shock, mechanical force) to make sure that all antibacterial activity in our experiments stemmed only from 10 exposure to the nanomaterials. 11 12 13 Fluorescence imaging of bacteria 14 15 In each fluorescence imaging experiment, a 20 µL aliquot of the untreated and treated bacterial 16 suspensions were placed on microscope glass slides for performing the viability staining. For treated 17 18 samples, bacterial stock samples were exposed to 100 µg/mL of each 2D nanomaterial for 3 hours in 19 complete darkness (i.e., nanomaterial stock suspensions of 200 µg/mL were sonicated at 37 kHz for 1 h 20 21 before use). Then, the samples were incubated with 20 µM propidium iodide, PI (Sigma-Aldrich) and 5 µM 22 SYTO9 (Molecular ProbesTM) for 15 min at room temperature. To avoid osmotic stress on bacterial 23 24 samples, the PI/SYTO9 solution was prepared in 1xPBS. The samples were enclosed by glass coverslip 25 slides and were mounted on the microscope stage. Epi-fluorescence images of at least 15 filed-of-view 26 27 (FOV) were recorded for each glass slide (i.e., each sample) and more than 2,000 cells were counted for 28 three separate experiments. The SYTO9-stained bacteria (i.e., green) correspond to the live bacteria and 29 the PI-stained bacteria (i.e., red) correspond to the dead bacteria. The percentage of viable bacteria was 30 31 calculated by using these two values. 32

33 34 Fluorescence microscope setup and image analysis 35 A Nikon ECLIPSE TE200 microscope with a 40x/0.60 PlanFlour (Nikon) objective lens coupled to a digital 36 37 image capture system (Hamamatsu C11440) was used to record images by the NIS Elements (ver. 4.20) 38 software. Images were saved using the tagged image file format (TIFF). For the fluorescence imaging, we 39 40 used a epi-fluorescence configuration. A EXFO X-cite 120 Fluorescence Illuminator system was used as 41 the light source to excite the PI and SYTO9 molecules and the red and green fluorescence emission were 42 recorded in the backward direction through appropriate filter cubes. Filter cubes had an excitation and 43 44 detection wavelengths respectively centered at 560 and 630 nm for PI (Prod. No.: 49008, CHROMA) and 45 480 and 535 nm for SYTO9 molecules (Prod. No.: 49011, CHROMA). Image analysis was performed by 46 47 using ImageJ software (National Institutes of Health, 1.43u). In a typical image analysis, the images 48 recorded by the two filter cubes were stacked to show the PI-stained and SYTO9-stained bacteria in a 49 50 single FOV. 51 52 53 Flow cytometry of bacteria 54 Bacterial stock samples were exposed to 100 µg/mL of each 2D nanomaterial for 3 h in the dark (i.e., 2D 55 nanomaterial stock suspensions of 200 µg/mL were sonicated at 37 kHz for 1 h before use). 100 µL of 56 57 58 9 59 60 ACS Paragon Plus Environment Langmuir Page 10 of 21

1 2 3 each treated and untreated bacterial sample was added in to a 96 flat-bottom well and incubated with 20 4 5 µM PI (Sigma-Aldrich) and 5 µM SYTO9 (Molecular ProbesTM) in 1xPBS (i.e., 1xPBS was used to avoid 6 osmotic shock) for 15 min at room temperature. Samples were then placed on to the flow cytometer, FC 7 8 (BD Accuri® C6 Flow Cytometer) stage for analysis. The FC analysis was carried out with a medium fluid 9 rate and a limit of 100,000 events. The PI and SYTO9 were illuminated with a 15 mW argon ion laser 10 (488 nm) and their fluorescence signals were collected through the FL2 and FL1 channels with the 11 12 detection wavelengths of 585 ± 20 nm and 533 ± 15 nm, respectively. To obtain statistical results, three 13 FC trials were done on each sample and for three separate bacterial suspensions. The fluorescence 14 15 signals and the forward angle scattering signal were amplified with the logarithmic mode. The FC data 16 were collected and analyzed by the BD Accuri® C6 Software. For the details of FC data analysis, see SI. 17 18 19 SEM images of bacteria 20 21 Visual inspection of the morphological changes of bacteria resulting from their interaction with 2D 22 nanomaterials was accomplished by using SEM (FEI Quanta 450 FEG instrument). For treated bacteria, 23 bacterial stock samples were exposed to 100 µg/mL of each 2D nanomaterial for 3 h (i.e., 2D 24 25 nanomaterial stock suspensions of 200 µg/mL were sonicated at 37 kHz for 1 h before use). The freeze- 26 µ 27 dry method was used to prepare the bacterial samples for SEM imaging experiments. About 50 L aliquot 28 of the untreated and nanomaterial-treated bacterial suspensions were dropped on to a silicon wafer (i.e., 29 silicon wafer was first cleaned and then attached onto a SEM stub before adding bacteria). The silica was 30 31 then inserted into a mixture of dry ice and ethanol (ca. – 60°C) to quickly freeze the samples 70. The 32 frozen samples were then quickly placed in to a manifold freeze-dryer for at least two days. 33 34 35 ASSOCIATED CONTENT 36 37 Supporting Information 38 The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/. 39 40 Experimental methods, including nanomaterials characterization and FC data analysis. 41 42 AUTHOR INFORMATION 43 44 Corresponding Author 45 *E-mail: [email protected] 46 47 ORCHID IDs 48 Farbod Alimohammadi: https://orcid.org/0000-0002-5143-2933 49 50 Mohammad Sharifian Gh: https://orcid.org/0000-0003-3867-1611 51 Nuwan H. Attanayake: https://orcid.org/0000-0001-7622-2337 52 Akila C. Thenuwara: https://orcid.org/0000-0002-6146-9238 53 54 Yury Gogotsi: https://orcid.org/0000-0001-9423-4032 55 Babak Anasori: https://orcid.org/0000-0002-1955-253X 56 57 58 10 59 60 ACS Paragon Plus Environment Page 11 of 21 Langmuir

1 2 3 Daniel R. Strongin: https://orcid.org/0000-0002-1776-5574 4 5 Author Contributions 6 F.A., M.S.G., and D.R.S. conceived and designed the experiments; F.A. and M.S.G. performed the 7 experiments; F.A. and M.S.G. analyzed the data; F.A., M.S.G., and D.R.S. interpreted the results; and 8 F.A., M.S.G., and D.R.S. contributed to writing the manuscript. N.H.A., Y.G., B.A., and A.C.T. contributed 9 in the material synthesis and preparations. 10 11 Notes 12 13 The authors declare no competing financial interest. 14 15 16 ACKNOWLEDGEMENTS 17 This work was supported by the Center for the Computational Design of Functional Layered Materials, an 18 19 Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, and Basic 20 Energy Sciences under Award No. DESC0012575. M.S.G. (see author contributions) was supported by 21 the National Science Foundation under Grant CHE-1465096. We are grateful to Joel B. Sheffield 22 23 (Temple University) for helpful guidance with bacterial sample preparations for the SEM technique. 24 25 26 ABBREVIATIONS 27 FC, flow cytometry; FI, fluorescence imaging; SEM, scanning electron microscopy; TEM, transmission 28 29 electron microscopy; AFM, atomic force microscopy; XRD, X-ray diffraction; OM, outer membrane; CM, 30 cytoplasmic membrane; PM, peptidoglycan mesh; PI, propidium iodide; E. coli, Escherichia coli; B. subtilis, 31 Bacillus subtilis; MoA, mode-of-action; 2D, two-dimensional; GO, graphene oxide; rGO, reduced 32 33 graphene oxide; MXene, Ti C T ; Gram+, Gram-positive; Gram , Gram-negative; ROS, reactive oxygen 34 species. 3 2 X 35 − 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 11 59 60 ACS Paragon Plus Environment Langmuir Page 12 of 21

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1 2 3 FIGURES 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 f ) 19 20 21 22 23 24 25 26 27 28 29 30 31 MnO MoS MXene MnO /GO MoS /rGO 32 Figure 1. TEM images of (a), (b), Ti3C2 (substrate) (c), (d), (e), MoS /MXene 33 and (f). The TEM2 images confirm2 the individual nanosheets aligned2 on the substrates2 . The 34 scale bar2 is 100 nm. 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 16 59 60 ACS Paragon Plus Environment Page 17 of 21 Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Figure 2. Antibacterial activity of MnO and MnO /GO nanomaterials against E. coli and B. subtilis 26 bacteria. Fluorescence imaging (a) and flow cytometry (b) results of bacteria treated with 100 µg/mL of 27 2 2 the nanomaterials for ca. 3 hours in the dark. In fluorescence images, the live bacteria are green (SYTO9- 28 stained) and the dead bacteria are red (PI-stained). In flow cytometry results, the percentage of viable 29 populations for untreated bacteria is shown for comparison. The errors are obtained from three separate 30 experiments on each bacterial strain. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 17 59 60 ACS Paragon Plus Environment Langmuir Page 18 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Figure 3. Antibacterial activity of MoS , MoS /rGO, and MoS /MXene nanomaterials against E. coli and B. subtilis bacteria. Fluorescence imaging (a) and flow cytometry (b) results of bacteria treated with 100 24 2 2 2 25 µg/mL of the nanomaterials for ca. 3 hours in the dark. In fluorescence images, the live bacteria are green 26 (SYTO9-stained) and the dead bacteria are red (PI-stained). In flow cytometry results, the percentage of 27 viable populations for untreated bacteria are shown for comparison. The errors are obtained from three separate experiments on each bacterial strain. 28

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1 2 3 4 5 a) c) 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 b) d) 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37 38 39 40 Figure 4. SEM images of the B. subtilis bacteria untreated (a), treated with 100 µg/mL of graphene oxide 41 (substrate) (b), and treated with 100 µg/mL of MnO (c), and MnO /GO (d) nanomaterials for 3 hours in 42 dark. The scale bar is 1 µm. 2 2 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 19 59 60 ACS Paragon Plus Environment Langmuir Page 20 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Figure 5. (a.) General membrane ultrastructure of Gram and Gram+ bacterial species. Gram bacteria 27 contain a pair of lipoprotein membranes (i.e., OM and CM) separated by a rigid PM. Gram+ bacteria have 28 a significantly thicker PM and a single lipoprotein membrane− (i.e., CM). (b.) Schematic representation− of 29 direct physical interaction of the bacterial surface with sharp edges of vertically aligned nanosheets onto a 30 substrate. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 20 59 60 ACS Paragon Plus Environment Page 21 of 21 Langmuir

1 2 3 TABLE OF CONTENTS GRAPHIC 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

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