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Mohamed S. Selim, Ahmed M. Azzam, [Mohamed A. Shenashen](https://orcid.org/0000-0003-1592-5877), Shimaa A. Higazy, Bayaumy B. Mostafa, [Sherif A. El-Safty](https://orcid.org/0000-0001-5992-9744)

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[Comparative study between three carbonaceous nanoblades and nanodarts for antimicrobial applications](https://mdr.nims.go.jp/datasets/8e6b501b-284f-41df-830c-8ce29a2d813d)

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Preparation and statistical optimization of Losartan Potassium loaded nanoparticles using Box Behnken factorial design: Microreactor precipitationComparative study between three carbonaceous nanoblades and nanodarts for antimicrobial applications M.S. Selim1,2, A.M. Azzam1,3, M.A. Shenashen1,2,* S.A. Higazy2, B.B. Mostafa3, S.A. El-Safty1,4,∗,  1 National Institute for Materials Science (NIMS), 1-2-1 Sengen, Tsukubashi, Ibaraki-ken 305-0047, Japan. 2 Petroleum Application Department, Egyptian Petroleum Research Institute (EPRI), Nasr City 11727, Cairo (Egypt). 3 Department of Environmental Research, Theodor Bilharz Research Institute (TBRI), P.O. Box 30 -12411, Giza, Egypt.  4 Faculty of Engineering and Advanced Manufacturing, University of Sunderland, St Peter's Campus, St Peter's Way, Sunderland SR6 0DD, UK. E-mail: sherif.elsafty@nims.go.jp ; SHENASHEN.Mohameda@nims.go.jp;   Webpage: https://samurai.nims.go.jp/profiles/sherif_elsafty       mailto:sherif.elsafty@nims.go.jpmailto:SHENASHEN.Mohameda@nims.go.jphttps://samurai.nims.go.jp/profiles/sherif_elsafty2  Abstract: This work reports a comparative study between the antimicrobial properties of graphene oxide (GO), reduced graphene oxide (RGO), and single-wall carbon nanotubes (SWCNTs). These nanoblades and nanodarts could have a mechano-bactericidal effect, piercing or slicing bacterial membranes. Nanoblades of GO nanosheets were successfully prepared using a modified Hummer's technique. RGO nanosheets were prepared by a hydrothermal reduction of GO at 180℃. These designed nanomaterials were well-characterized to elucidate the significance of the size, morphology, and elemental composition on the antibacterial activity mechanism.  Gram-negative and gram-positive bacteria and fungi were used to investigate the nanomaterials’ antimicrobial performance. The antibacterial efficacy of GO, RGO, and SWCNTs was assessed using the minimum inhibitory concentration (MIC), standard agar well diffusion, and transmission electron microscopy.  The three nanomaterials were active against all tested microbial strains. These nanomaterials wrapped the bacterial cells completely and disrupted their morphology via microbial cell membrane degradation. They exhibited higher antibacterial efficiency toward gram-positive bacterial strains than gram-negative and fungi microorganisms, because their external cell wall structure and outer membrane proteins can reduce the permeability and protect against toxicants. Results showed that the antimicrobial activity of these nanomaterials was in the order RGO > GO > SWCNTs. RGO had the lowest MIC values (0.062, 0.125, and 0.25 mg/mL against B. subtilis, S. aureus, and E. coli, respectively) as well as minimum fungal concentrations (0.5 mg/mL for both A. fumigatus and C. albicans). Its cell viability values toward different microbial strains reached a complete inhibition after only 12 hr. Microorganisms perching on RGO nanoblades suffered from significant membrane damage, resulting in cell lysis and death. Our work gives an insight into the design of effective graphene-based antimicrobial materials for water treatment and remediation. Keywords: Antimicrobial properties; nanoblades; mechano-bactericidal effect; RGO nanosheets; gram-positive bacteria; cell viability.  3  1. Introduction       Nanomaterial science advancements have led to significant technological advancements in various applications such as medical, manufacturing, energy production, pollution control, and environmental cleanup (Selim et al., 2017). Pollution of water resources, including river pollution and drinking water contamination caused by industrial wastes, animal farms, and agricultural activities. These activities can release unreacted organic dyes and heavy residual materials which are a worldwide environmental threat (Liu et al., 2018; Kumwimba et al., 2018). These organic chemicals in wastewater stimulate the synthesis of hazardous metabolites, increase microbial development in rivers, and cause water-borne disorders because of the high microbial density (Orou et al., 2018). Recent research on "Clean Rivers" to alleviate the problem of water pollution has been prompted by these global threats (Gonzales-Gustavson et al., 2017). Degradation of organic pollutants and microbial growth inhibition by chemical-physical procedures and chemical oxidation processes are examples of modern strategies for minimizing water pollution (Varjani and Upasani et al., 2017). However, in these procedures, the formation of secondary sludge is a big problem (Shabanzade et al., 2018). Furthermore, the capacity of microbial organisms for evolving biological resistance negates the effects of antibacterial materials (Fatthallah et al., 2021). As a result, developing advanced antibacterial and antibiofilm nanomaterials is required for water treatment and sustainable development (Askar et al., 2021).        Bacterium contact with nanoscale materials in an aqueous media exhibit a significant antibacterial impact (Mokammel et al., 2022; Li et al., 2022). Mechano-bactericidal nanostructures have instant advantages over chemical-based antibacterial therapies, because of these nanostructures’ physical interactions with bacteria and they don’t release antibacterial substances (Wang et al., 2016). Carbonaceous-based antimicrobial surfaces, particularly graphene materials and carbon nanotubes (CNTs) are currently one of the hottest study areas (Samak et al., 2020). Sharp nanostructured materials with high-surface area can swiftly remove adherent microbial strains after only minutes of contact. Graphene oxide (GO), reduced GO (RGO), and single-walled 4  carbon nanotubes (SWCNTs) are smart antimicrobial agents because their morphostructural properties can be controlled to limit their biotoxicity (Lin et al., 2018; Selim et al., 2018). Direct contact with SWCNTs can puncture and destroy the cell wall of E. coli, implying that SWCNTs could be used as antibacterial materials under the mechano-bactericidal mode (Al-Jumaili et al., 2017). Because of the agglomerations of multi-walled CNTs (MWCNTs), they had lower bactericidal efficiency than SWCNTs (Teixeira-Santos et al., 2021; Yuan et al., 2019). SWCNTs were found to have greater antibacterial activity than MWCNTs caused by the high aspect ratio geometry of SWCNTs. Longer nanotubes have better antibacterial action at the same weight concentrations (Safdar et al., 2022). The greater specific surface area allows more interactions with cell membranes and results in more membrane piercing (Yuan et al., 2019). Rigid and thin SWCNTs caused greater penetrating, dart-like damage to gut bacteria membranes than MWCNTs (Chen et al., 2013).        As two-dimensional materials, graphene-derivatives (including GO and RGO nanosheets) have a high aspect ratio and exhibit high antibacterial activity (Xia et al., 2019). The sharp graphene edges cause mechano-bactericidal performance upon physical interaction with bacteria (Linklater et al., 2021). They are biocompatible materials with low toxicity to human and mammalian living cells, making them environmentally friendly. RGO offers various advantages, including superior hydrophobicity, high-surface area, high yield, biocompatibility with human and mammalian live cells, excellent processability, cost savings, and antibacterial performance (Thebo et al., 2018; Naskar et al., 2018). Individually dispersed SWCNTs were more hazardous to bacteria than aggregated SWCNTs (Liu et al., 2009). They acted as many moving "nanodarts" in solution, constantly attacking bacterial cells and causing bacterial cell integrity breakdown, which led to cell death (Parwez, and Budihal, 2019). GO, RGO, and CNTs were heavily regarded as potent antibacterial and antimicrobial active materials. The mechanism happens when bacterial cell integrity is disrupted as a result of direct contact with the selected carbonaceous material, which can 5  kill the cell (Xin et al., 2019; Azizi-Lalabadi et al., 2020). However, the intricate antimicrobial activity mechanisms of GO, RGO, and SWCNTs are still a mystery.         For the first time, this work investigated and compared the antibacterial efficacy between GO and RGO nanoblades and SWCNT nanodarts. These carbonaceous nanomaterials exhibited a diverse range of nanoscale sizes, morphologies, elemental composition, and surface exposure topologies. A modified Hummers' approach was used to create GO sheets. RGO was prepared by a hydrothermal reduction of GO at 180℃. The antibacterial efficiency of GO, RGO, and SWCNT was studied using gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), and fungi (Aspergillus fumigatus and Candida albicans). The cell viability, minimum inhibitor concentration (MIC), and transmission electron microscopy (TEM) were used to investigate the nanomaterials’ bacterial resistance and morphology wrapping and disruption in the cellular membrane. The developed RGO nanoblades exhibited higher antibacterial activity than GO and CNTs against various microbial strains. The antibacterial mechanism of the produced blade-like and dart-like nanomaterials was discussed extensively. This work prompts a modern research gateway for facilitating the next generation of antibacterial carbonaceous nanostructured materials for the sustainable environment.  2. Materials & Methods 2.1. Materials       Sigma-Aldrich Company in the United States provided graphite flakes (< 20 μm, synthetic) and sodium nitrate (99%). NaOH (98%) and KMnO4 (99%) were delivered from Aladdin Industrial co., Shanghai, China. H2O2 (35%), H2SO4 (95%), and ethanol (AR) were provided by Acros Company, Belgium. SWCNTs with a diameter distribution of 1.5 nm, length of 500-1000 nm, 99% purity were purchased from Xuzhou Jie-Chuang New Material Technology Co., Ltd. China. The used solvents were of high purity and used exactly without further purification. All the employed materials were 6  analytical reagents used as obtained. All the used microorganisms were obtained from the Environmental Research Department, Theodor Bilharz Research Institute, Egypt (TBRI) and growth media were obtained from Hi-Media, Mumbai, India. 2.2. Synthesis methods 2.2.1. Preparing GO nanosheets The modified Hummer's process generates GO nanosheets by oxidizing graphite powder (Rajapaksha et al., 2019; Liu et al., 2018) as follows: 1.5 g graphite flakes and 1.5 g NaNO3 were dissolved into 72 mL H2SO4 under constant stirring. KMnO4 (9 g) was gently mixed and stirred for 1 hr (35℃) to prevent the suspension temperature from reaching 20℃. After dilution with 120 mL distilled water and slow addition of 15 mL H2O2, the temperature was raised to 90℃. A suspension was created and filtered with deionized H2O until the pH reached 7. The obtained yellow-brown suspension was ultrasonically exfoliated and centrifuged to yield GO nanosheets and eliminate the un-exfoliated precipitation.  2.2.2. Preparing RGO nanosheets       A specific amount of the produced GO sheets was ultrasonically treated in deionized H2O for 30 minutes. This dispersed mixture was put into an autoclave lined with Teflon and subjected to a hydrothermal reduction process for 8 hr at 180℃ (Scheme 1). The reduction product was naturally cooled before being centrifuged to yield RGO sheets. The product was purified by washing it with an ethanol/distilled H2O mixture and then freezing it for 1 day at -45℃ to remove the adsorbed solutions and yield homogeneous RGO sheets (Samak et al., 2022). 2.3. Characterization techniques       FTIR analysis of the developed nanostructures was performed by Nicolet™ iS™10 (Thermo Fisher Scientific, USA) and KBr pellets. X-ray diffraction (XRD) was analyzed by the X'Pert PRO diffraction device (PANalytical, Netherlands) to investigate the products' purity and crystallinity. CuKα radiation was used to get the XRD pattern at 2θ of 5°–80°. A TEM instrument model JEM 7  2100 LaB6 (JEOL, Japan) was used to examine TEM images of the developed nanomaterials at 200 kV. FESEM (JSM530, JEOL,  Japan) was employed at 30 kV to examine the morphology and size of the nanomaterials. Two droplets of the nanomaterials' solutions were put over a glassy sample, dried, and subjected to gold-spatter coating for preventing the electron beam's charging. 2.4. Carbonaceous materials' antimicrobial activity The microbial resistance of GO, RGO, and SWCNTs was investigated by standard agar well diffusion procedure against different bacterial strains, including Staphylococcus aureus and Bacillus subtilis (Gram-positive), Escherichia coli and Pseudomonas aeruginosa (Gram-negative) bacteria and Aspergillus fumigatus and Candida albicans (fungi). These strains represent essential pathogens and are widely spread in water sources (Selim et al., 2020a). They are highly adaptable to different environmental conditions and are extensively applied to assess the biological behavior of nanomaterials (Hao et al., 2020; Selim et al., 2020b).       Fresh pure cultures of these microbes were subcultured in Müller-Hinton and Sabouraud Dextrose broth. Nutritional agar for bacteria and Sabouraud Dextrose agar (SDA) plates were kept standing for 15 min after pouring. The carbonaceous materials' antimicrobial activities were then tested using 6.0 mm holes in plates. 100 µL water suspension (10.0 mg/mL) of the carbonaceous materials was put into each well of all plates using a micropipette. The varying amounts of inhibition zone were evaluated during overnight incubation at 37℃ for bacterial organisms for 2 days, while for fungi the evaluation was performed at 28℃. For every concentration and organism, duplicate plates were utilized (Ranjani et al., 2021). The B. subtilis, S. aureus, P. aeruginosa, and E. coli strains were also cultivated overnight on MHA plates at 37℃ before use. The MIC was evaluated in nutritional broth Hi-Media (Mumbai, India) using successive two-fold dilutions of the selected materials in concentrations ranging from 100-5000 mg/mL, initial bacterial inoculums of 0.5 McFarland turbidity, and incubation for 24 hr at 37℃. MIC represents the lowest antimicrobial concentration which prevents 99% of bacteria from growing. The MIC value for each tested microorganism was determined in triplicate to ensure accuracy (Yayehrad et al., 2022). By plating 8  loopful of A. fumigatus and C. albicans culture onto Sabouraud Dextrose Agar (SDA) plates and incubating under aseptic conditions for 72 hr at 28℃, the MFC of the CNTs, GO, and RGO were determined. The MFC is the lowest concentration of CNTs, GO, and RGO that demonstrated no observable development on solid media (Asl et al., 2021).  Viable microbial counts (VMCs) assay was evaluated for each microbial species at different times after being treated with 10 mg/mL of RGO nanosheets, then the count of surviving microbial cells was determined by plate count technique. Using Eq.1, the reduction percentage of total VMCs was calculated after treating cells with RGO: VBCs Reduction % = Viable count at time 0 – Viable count at time xViable count at time 0    X 100               (1) where time 0 is the time before adding RGO nanosheets and time x is the contact time between microbial cells and RGO nanosheets. Additionally, Elbasuney et al. (Elbasuney et al., 2021) reported a qualitative examination of the suppression of biofilm formation. In the absence and presence of carbonaceous nanoparticles, the inhibition of biofilm investigation was conducted using a tube wall method (Elbasuney et al., 2022). The antibiofilm activity of these nanomaterials (at 10.0 mg/mL) was tested against the selected microorganisms (S. aureus, E. coli, and C. albicans) and compared to the control sample. In brief, nutrient broth medium (5 mL) was utilized in the whole tubes, and the examined microbes were inoculated and adjusted to 1–3.5×108 CFU/mL using 0.5 McFarland. The cells were then cultured for 24 hr at 37℃. The media from the control sample and treated tubes were taken out, and then Phosphate Buffered Saline (PBS) with PH=7.0 was mixed and stored. Then, the adhering microbial cells were treated for 20 min with sodium acetate (5 mL, 3.5%). The deionized water was used to clean them. Then, 20 mL Crystal Violet (CV) with 0.15% concentration was employed to dye biofilms followed by rinsing with DW for removing any excess CV. For separating the stained microbial films for the semiquantitative antibacterial evaluation, 5 mL of absolute ethanol was applied. The O.D. (for stained biofilms of bacteria and fungi) was measured at 570 nm with a UV-9  Vis spectrophotometer (El-Batal et al., 2019). Using Eq.2, the microbial biofilm inhibition's proportion was evaluated (Ansari et al., 2014): The percentage of biofilm inhibition = ((O.D. control - O.D. treated)/O.D.control) x 100                    (2) 2.5. The treated bacteria and fungi strains' morphological investigation       A TEM EM 208S instrument (Philips, Netherlands) instrument was used to examine bacterial and fungal cells in both treated and untreated samples. After being fixed in 1% glutaraldehyde and washed in 0.1 M buffer, the specimens were subjected to post-fixing into Osmium tetraoxide (1 wt.%) and washed in 0.1 M buffer.  The specimens were infiltrated and implanted in epoxy resin after being dehydrated in acetone. Lastly, the TEM meshes were dried and captured via the JEOL 1010 instrument (Japan) to investigate morphological changes caused by the selected carbonaceous materials' biocidal effect (Karmakar et al., 2022).      3. Results & Discussion 3.1. GO, RGO, and CNTs morphologies and sizes  The OH stretch modes on the surface of the GO sheets produced FTIR peaks at 3740 cm-1 (Fig. 1A (a)). The symmetric and non-symmetric stretching modes of CH2 units were observed at 2890 and 2956 cm-1. Also, the peaks observed at 1739 and 1339 cm-1 were those of the stretching C=O carboxylic as well as C-OH alcoholic vibrations. The non-oxidized graphene C=C peak was detected at 1628 cm-1. The peak at 665 cm-1 was owed to epoxide units on the GO sheet's edges. The absence and reduction in the hydroxyl and carbonyl groups in the spectrum (Fig. 1A (b)) indicated that GO was reduced to RGO using the hydrothermal technique. This assured the removal or decrease of the surface C-OH and C=O throughout the reduction procedure. The C=O binding changes were also recorded in the region at 1717 cm-1 in the FTIR peak of SWCNTs (Fig. 1A(c)). Furthermore, C-C binding vibrations in CNTs were recorded at 1538 cm-1. Fig. 2 depicted the XRD results of the used materials. XRD analysis of the prepared GO sheets (Fig. 1B(a)) revealed that the main peak observed at 2θ = 10.4° was owed to (001) orientation which exhibited a d-spacing of 10  0.95 nm. This was greater than the 0.335 nm d-spacing of exfoliated natural graphite; which could increase the interlayer distance between the functionalized oxygen on the surface. XRD pattern of RGO was reflected in Fig. 1B(b) at 2θ = 23.28° which owed to (002) crystalline orientation. Those results assured the preparation of nano-RGO sheets in dispersed form without aggregations. Furthermore, CNTs' XRD pattern (Fig. 1B(c)) shows two typical peaks at 2θ = 25.3° as well as 42.8°. These peaks correspond to SWCNTs' (002) and (100) crystal orientations (JCPDS No. 01-075-1621). FETEM device captured the products' morphology (Fig. 2). Fig. 2 (A and B) of the nano-GO showed wrinkled nanosheets including folded regions at the edges. In addition, the nanosheet structure of nano-RGO with 2 nm sheet thickness was confirmed (Fig. 2 (C and D)). Also, FETEM analysis could study the SWCNTs' shape and size. The observed SWCNTs observed was homogeneous and had a diameter of nearly 1 nm, as seen in the HRTEM image (Fig. 2 (E and F)). The results also showed that the tested material was pure, i.e. free of metallic impurities and amorphous carbon deposits. Fig. 3 depicted the morphology and nanostructures of the materials produced using FESEM images. FESEM captures in Fig. 3 (A and B) depicted the designed GO shape and structure. The FESEM of RGO revealed layered nanosheets with wrinkled edge structures (Fig. 3 (C and D)). Meanwhile, FESEM images of SWCNTs showed nanotube lengths ranging from 100 nm to 500 nm with uniform diameters of 1.5 nm, Fig. 3 (E and F)). The images of the SWCNT showed a smooth and uniform surface.  3.2. Antimicrobial activity of carbonaceous nanomaterials       The different inhibition zones of three tested carbonaceous nanomaterials (10.0 mg/mL) and positive control antibiotics against different microbes were illustrated in Fig. 4. The highest recorded inhibition zone was presented by RGO. Also, the selected carbonaceous materials exhibited high antibacterial performance toward gram-positive bacteria than gram-negative strains and fungi. Moreover, B. subtilis and S. aureus (G-positive bacteria) exhibited the highest inhibition zones, 20.0±2.0 and 19.0±1.5 mm with RGO, respectively. However, E. coli, A. fumigatus, C. 11  albicans, and P. aeruginosa were recorded at 17.5±1.0, 14.0±1.0, 12.7±1.0, and 12.5±1.0 with RGO, respectively. These results are in agreement with those reported by Naseer et al. (Naseer et al., 2020). They stated that Fe2O3/RGO nanocomposite could significantly inhibit the growth of S. aureus, vancomycin-resistant S. aureus, and ciprofloxacin-resistant S. aureus bacteria in the range of 10–5000 μg/mL  Fig. 5 showed the MIC of CNTs, GO, and RGO that were detected for different bacterial and fungal species. The recorded MIC values of RGO were the lowest ones recorded at 0.062, 0.125, 0.25, 0.5, 0.5, and 1.0 mg/mL against B. subtilis, S. aureus, E. coli, A. fumigatus, C. albicans, and P. aeruginosa, respectively. However, the CNTs and GO recorded higher concentrations. Thus, RGO is a promising antimicrobial agent that can be developed and utilized in wastewater purification for the inactivation of waterborne microbial species that causes harmful diseases for human and contaminates our hydrosphere. Kellici and his coworkers (Kellici et al., 2014) recorded MIC 125 μg mL−1 for RGO against the gram-negative bacterium E. coli. As shown in Fig. 6, the VMCs reduction percent of B. subtilis and S. aureus bacteria that treated with RGO at 9 and 12 hr of contact time reached 100% inhibition, respectively, whereas, E. coli and A. fumigatus was treated for 18 hr. However, complete inactivation occurred for P. aeruginosa and C. albicans after 24 hr of contact time.  3.3. Antibiofilm performance assessments Exo-polysaccharide synthesis promotes the biofilm formation by pathogenic bacteria and fungi (Singh et al., 2021). The antibiofilm behaviour of carbonaceous materials against unicellular microbes was defined using a tube design. On the other hand, a substantial negative influence was detected in the tubes containing microbial cells and the three carbonaceous nanomaterials, as the cells didn't create biofilm layers and the ring building was stopped. Furthermore, the adhering cells' color was only a faint blue tint formed once ethyl alcohol was added (Kasim et al., 2016). Fig. 7 shows the percentage of biofilm inhibition for S. aureus, E. coli, and C. albicans after the addition 12  of 10.0 mg/mL of three carbonaceous nanomaterials. RGO demonstrated the highest percentage inhibition of 55%, 45%, and 40% against S. aureus, E. coli, and C. albicans, respectively. GO was also slightly more active than CNTs. Notably, RGO inactivated the formation and cohesion strength of the biofilm and provided high antibacterial performance (Zhang et al., 2020). The difference in the hindrance % could be attributable to the antimicrobial agent's high capacity to connect to the surface due to RGO's structure. The attack style of these groups also has an impact on their interactions and affiliation with biofilm-producing bacteria (El-Batal et al., 2020). The antimicrobial efficiency of graphene materials is usually dependent on the physicochemical interactions between the sheets and pathogenic microbes (Omran et al., 2022). RGO nanosheets can pierce the bacterial cellular membrane with their sharp edges, resulting in pore formation, changes in bacterial osmotic pressure, and cell death (Pham et al., 2015). Choudhary et al. (2017) reported that the presence of carboxyl, hydroxyl, amine, and phosphate groups of the intracellular protein provided enormous binding sites for conjugation with RGO through covalent, hydrophobic, and π–π stacking interactions with sp2 bonded carbon atom. The RGO thus interacts with cell membrane proteins through hydrogen bonding, π−π stacking, and electrostatic interactions by making contact with the microorganism. Membrane stress is initiated when RGO pierces the bacterial cell membrane with blade-like edges, causing leakage of intracellular constituents, and cell death.  RGO nanosheets have a relatively high surface area due to their sharp edges, making them attractive as direct-contact bactericidal agents (Mann et al., 2021). The cellular membranes trapped the graphene tail end as a result of van der Waals attraction forces and hydrophobic interactions. The nanosheet can pierce the lipid bilayer membrane. RGO destabilizes cell membrane by inducing mechanical stress and also by triggering reactive oxygen species (ROS) generation.  Sengupta et al. (2019) reported the antibacterial activity of GO and RGO on gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa bacteria. They concluded that GO destructed bacteria by cell membrane damage through chemical reaction whereas, RGO sheets induced mechanical stress and pierced the cell membrane.  Gusev and his coworkers (Gusev et al., 2019)  performed 13  microscopic and zeta potential studies of RGO-bacteria interactions. They indicated the electrostatic nature of the interaction of RGO and bacteria, which could be mediated via hydrogen bonding between the cell walls of E. coli and RGO functionalities.  Because of the presence of these oxygen functional groups in both RGO and cell  membranes, the aggregation of RGO sheets and bacteria mediated via hydrogen bonding was expected.  Their optical photographs studies showed that the bacteria were localized on the RGO but not on a bare substrate, suggesting strong interaction between bacteria and RGO, which was likely mediated by hydrogen bonding.  3.4. Bacterial morphological studies The putative antibacterial mechanism against S. aureus and E. coli was investigated using TEM imaging analysis. In the absence of RGO nanomaterial, TEM inspection of the control bacteria revealed that they were growing normally with typical budding surfaces and solid cell membranes, as shown in Fig. 8 (A and C). Both bacterial species showed significant morphological abnormalities after RGO treatment (Fig. 8 (B and D)), including entire cell membrane lysis, as demonstrated by bacterial cell deformation. Furthermore, the RGO produced full lysis of the bacterial cells as well as cell deformation, resulting in a decrease in the total number of viable cells. Effective mechanisms, including the dispersion of ROS, may be created by the active groups of the RGO surface (El-Batal et al., 2018). The RGO nanomaterial is thought to start its activity by wrapping and attaching to the microbial cells' outer surface, inducing membrane breakdown and altering the transport potential (Maksoud et al. 2020). All internal structures, including plasmid, DNA, and other important organelles, are divided when microbial cells' interior components are reacted with RGO. Finally, cellular toxicity occurs as a result of oxidative stress induced by the ROS's production which causes cellular and genotoxicity as a result of interactions among negatively charged essential organelles (Ashour t al., 2018).  - The RGO action mechanism combines the synergetic effects of the nanosheets' blade-like edges.  - The RGO nanosheets' edges function as nanoblades, putting bacterium-mechanical stress over microbial cell membranes and inflicting physical damage. The disintegration of the cell wall causes 14  RNA spills. Carbon-free radicals develop on the microbial surface, causing cell structural damage and creating oxidative stress. Thus, the cell membrane broke and shattered, triggering lysis, which forced the cellular components out of the cell.  - The the antibacterial characteristics of SWCNTs are caused by the high the aspect ratio and small diameter. Graphene materials with larger degrees of sharp edge-asperities follow the same pattern. The antibacterial activity of graphene materials in both colloids and nanotopography is increased by enhancing the aspect ratio by developing sharper or thinner nanosheets. The presence of many wrinkles on the RGO surface can reduce surface energy and improve the stability of the aqueous medium (Boukherroub, 2016). The high RGO edges' oxygen content can minimize the repulsion between hydrophilic edge atoms and hydrophobic lipid tails, resulting in a trans-membrane nanostructure. RGO alters the osmotic pressure of microbial cells and causes cells to enlarge and die by acting as pores in the cell membrane (Pham et al., 2015). - RGO nanosheets' lateral edge may increase the permeability of cell membrane, leading to the tearing cell wall (Linklater et al., 2018). The cell wall break disrupts cellular signaling and electron transport through the cell membrane. The high oxidative stress on microbial cells leads to more disturbance in the bioactivities of cells through inhibition of growth and lysis of internal cellular components or death.  The produced ROS can injure cells while also functioning as an antibacterial agent. - RGO nanosheets exhibited ultra-higher conductivity than nano-GO, which is electrically insulating (Devi and Kumar, 2018). Thus, RGO with higher conductivity can exhibit better glutathione oxidation capabilities than the lower conductive GO sheets. RGO was found to have better antibacterial action than GO against both bacterial types in a fascinating investigation (Mohamed et al., 2020). Also, RGO increases the conductivity through cell membrane lipid layers, interfering with electron transport from the bacterial inner milieu to the exterior environment. RGO antibacterial action is based on electron transfer rather than ROS (Li et al., 2015). Another study found that RGO suppressed E. coli multiplication while showing no cytotoxicity 15  (Zheng et al., 2017; Lu et al., 2016). RGO nanosheets exhibited high surface area and strength. As a result, RGO generations partially restore graphene properties. In a fascinating investigation, RGO outperformed GO in bactericidal effect against S. aureus and E. coli. Another study found that RGO suppressed E. coli multiplication while GO had no cytotoxicity. In addition, the wrinkled edges of RGO nanosheets interact with aqueous media, stabilizing the RGO combination's structure and resulting in long-lasting antibacterial action. The RGO exhibited a large dispersion capability in an aqueous medium due to their potent microbicidal effect and the wide surface that interact with microbes.       Herein, the TEM results showed cellular alteration and damage appeared in the form of cellular fragmentation, wrinkled abnormalities, viability, and integrity loss. These alterations are the consequences of oxidative stress caused upon contact of blade-like edges of RGO and the bacterial cell walls. Generally, oxidative stress is the major symptom of RGO toxicity. The produced ROS by RGO nanosheets and their sharp edges lead to cellular lysis upon contact with bacterial cell walls to end with microbial cytotoxicity and death (Shulga and Shulga, 2019). TEM, MIC, and cell viability are the major tools employed to express the RGO antibacterial mode of action. Selim and coworkers (Selim et al., 2020a) demonstrated the antimicrobial efficiency of the blade-like GO/Cu2O which was elucidated by electron microscopy. They concluded that the complete microbial cell is subjected to oxidative stress of high levels of ROS leading to DNA damage and cellular degradation ending in microbial growth inhibition, cytotoxicity, or death. Samak et al. (2022) used SEM analysis to study the antibacterial mechanism of RGO/metal oxide nanocomposite and concluded that the lateral edges of the RGO nanosheets and electrostatic interactions of metal oxide nanoparticles may increase the permeability of the cellular membrane. The microbial cell is then exposed to oxidative stress, which causes cellular disintegration leading to microbial growth suppression, cytotoxicity, or death (Pieper et al., 2016). Mohamed et al. (2020) reported that graphene materials hold a high affinity to the membrane proteoglycans where they accumulate leading to membrane damage. After internalization, they can interact with bacteria interrupting the replicative stage. RGO interacts with bacteria through hydrogen 16  bonding, π-π stacking, and electrostatic adsorption due to the presence of oxygen- and nitrogen-containing groups in addition to the π-conjugated structure (He et al., 2010). Graphene materials' permeation into the microorganism alters the DNA structures and properties leading to the inactivation or death of the microorganism (Hadidi and Mohebbi, 2022).  4. Conclusions:        To combat the negative effects of microbial strains, the current study introduces three carbonaceous materials with high antibacterial properties for water resource control and remediation. Mechano-bactericidal nanostructures, as antibacterial surfaces that do not emit chemicals, provide opportunities for the development of long-lasting antimicrobial materials. For the first time, this study compared the antibacterial and antibiofilm properties of three different carbon-based materials (GO, RGO, and CNTs). A modified Hummer's method was used to synthesize ~2 nm sheet thickness of nano-GO. RGO sheet morphology was produced using a hydrothermal method. Carbonaceous-mediated toxicity is determined by physiological properties such as size, concentration, and surface features. Discussing the carbon-based antibacterial mechanism against various bacterial organisms was unavoidable. RGO nanosheets exhibited a uniform distribution and outperformed GO and SWCNTs in antibacterial performance. RGO demonstrated the highest antibacterial activity toward various microbes with 55 %, 45 %, and 40 % antibiofilm inhibition percentages for the tested unicellular microbes S. aureus (gram-positive bacterial strains), E. coli (gram-negative organisms), and C. albicans (fungi), respectively. The RGO sheets' antimicrobial performance was discussed through microbial physical wrapping as well as covalent and π-π interactions with the microbial cellular membranes. The RGO nanobaldes with high surface area induce oxidative stress, cellular destruction and lysis, physical wrapping, mechanical stress, and cytotoxicity. These fascinating antimicrobial carbon-based materials can introduce an advanced and controlled model for water treatment and antibiotic resistance. The current study encourages the development of futuristic RGO-derived nanocomposites for 17  environmental sustainability. Understanding bacterial interactions with mechano-bactericidal nanostructures will aid in defining optimal geometry and dimensions in the future. It presents a simple model that may inspire further experimental and theoretical research on carbonaceous-based materials as antibacterial active agents.  Conflicts of interest The authors declare no conflicts of interest.  Acknowledgement This work was supported by the Center for Functional Materials, National Institute for Materials Science (NIMS in Japan), Egyptian Petroleum Research Institute (EPRI in Egypt), and Nano-Environmental Uint (NEU), Theodor Bilharz Research Institute (TBRI in Egypt).  References: Al-Jumaili, A., Alancherry, S., Bazaka, K., Jacob, M.V., 2017. Review on the antimicrobial properties of carbon nanostructures. Materials 10, 1066. 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Graphene-engineered cementitious composites: small makes a big impact. Nanomater. Nanotechnol. 7, 1-18.   https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorRaw=Xin%2C+Qihttps://onlinelibrary.wiley.com/action/doSearch?ContribAuthorRaw=Shah%2C+Hameedhttps://onlinelibrary.wiley.com/action/doSearch?ContribAuthorRaw=Nawaz%2C+Asmathttps://onlinelibrary.wiley.com/action/doSearch?ContribAuthorRaw=Xie%2C+Wenjinghttps://onlinelibrary.wiley.com/action/doSearch?ContribAuthorRaw=Akram%2C+Muhammad+Zainhttps://onlinelibrary.wiley.com/action/doSearch?ContribAuthorRaw=Batool%2C+Aisha25  List of Figures: Scheme 1. Schematic illustration of the GO preparation through a modified Hummers' method and its reduction by a hydrothermal procedure to yield RGO nanosheets. The Scheme also illustrates the antibacterial influences for GO and RGO nanoblades as well as the SWCNT nanodarts. The major effects underpinning the antibacterial capability of RGO nanosheets are depicted in this diagram: (i) The direct friction between the carbonaceous nanomaterials and cell membrane leads to the tearing of the cell wall; (ii) ROS released from RGO nanosheets interacting with the microbial cell contents; (iii) RGO nanosheets cause cell membrane damage, alter transport activity, and block ion transport from and to the microbial cells. RGO creates and increases ROS, resulting in cell damage through interactions with extracellular and intracellular contents (cytoplasm, DNA, and ribosomes). Fig. 1. FETEM captures for (A,B) GO nanosheets, (C,D) RGO nanosheets, and (E,F) SWCNTs at different magnifications. Inside (F) a high magnification TEM image of SWCNTs. Fig. 2. FESEM captures for (A,B) GO nanosheets, (C,D) RGO nanosheets, and (E,F) SWCNTs  at different magnifications. Fig. 3. (A) FTIR and (B) XRD analyses for (a) GO, (b) RGO nanosheets, and (c) SWCNTs. Fig. 4. Inhibition zones of treated microorganisms with GO nanosheets, RGO nanosheets, SWCNTs, and antibiotics including (Ampicillin, Gentamicin, and Amphotericn B). (Error bars represent ±0.2 standard deviations based on three determinations) Fig. 5. MIC evaluations for GO nanosheets, RGO nanosheets, and SWCNTs against various microbes. Fig. 6. Viable microbial counts reduction% of tested microorganisms at different contact times with RGO nanosheets. Fig. 7. Antibiofilm inhibition percent of GO, RGO, and SWCNTs nanomaterials against different unicellular microbes. Fig. 8. TEM images for S. aureus as well as E. coli untreated (A, C) and after interacting with RGO nanosheets (B, D), respectively.   26  Scheme 1.  27   Fig. 1.  28  Fig. 2. 29   Fig. 3.   30  Fig. 4.     Fig. 5.  31   Fig. 6.    Fig. 7.   32   Fig. 8.            For the first time, this work investigated and compared the antibacterial efficacy between GO and RGO nanoblades and SWCNT nanodarts. These carbonaceous nanomaterials exhibited a diverse range of nanoscale sizes, morphologies, elemental composit... Liu, S., Wei, L., Hao, L., Fang, N., Chang, M.W., Xu, R., et al., 2009. Sharper and faster “Nano Darts” kill more bacteria: A study of antibacterial activity of individually dispersed pristine single-walled carbon nanotube. ACS Nano 3(12), 3891-3902. Parwez, K., Budihal, S.V., 2019. Quality control and risk management of carbon nanomaterials, in Perspective of Carbon Nanotubes. London, United Kingdom: IntechOpen. Safdar, M., Kim, W., Park, S., Gwon, Y., Kim, Y‑O., Kim, J. 2022. Engineering plants with carbon nanotubes: a sustainable agriculture approach. J. Nanobiotechnol. 20, 275.