Non-covalent functionalization of graphene oxide using self-assembly of silver-triphenylphosphine for bactericidal formulations

Journal Pre-proof Non-covalent functionalization of graphene oxide using self-assembly of silvertriphenylphosphine for bactericidal formulations Samir Bouchareb, Rachida Doufnoune, Farid Riahi, Hafsa Cherif-Silini, Belbahri Lassaad PII:

S0254-0584(19)31408-7

DOI:

https://doi.org/10.1016/j.matchemphys.2019.122598

Reference:

MAC 122598

To appear in:

Materials Chemistry and Physics

Received Date: 16 November 2019 Revised Date:

19 December 2019

Accepted Date: 30 December 2019

Please cite this article as: S. Bouchareb, R. Doufnoune, F. Riahi, H. Cherif-Silini, B. Lassaad, Noncovalent functionalization of graphene oxide using self-assembly of silver-triphenylphosphine for bactericidal formulations, Materials Chemistry and Physics (2020), doi: https://doi.org/10.1016/ j.matchemphys.2019.122598. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Graphical Abstract GO-Ag nanohybrid

Living bacteria

Bacteria death

Adhesion, Physical perforation, Stress Oxidative

Non-covalent functionalization of graphene oxide using self-assembly of silver-triphenylphosphine for bactericidal formulations Samir Bouchareb1,2, Rachida Doufnoune1,2*, Farid Riahi3 and Hafsa CherifSilini4 and Lassaad Belbahri5, * 1

Unité de Recherche des Matériaux Emergents –Sétif–URMES, Equipe de Valorisation des

Polymères, Université Ferhat ABBAS Sétif-1, Algérie. 2

Département de Génie des Procédés, Faculté de Technologie, Université Ferhat ABBAS Sétif-1,

Algérie. 3

Laboratoire des Matériaux Polymériques Multiphasiques (LMPMP), Faculté de Technologie,

Université Ferhat ABBAS Sétif-1, Algérie. 4

Laboratoire de Microbiologie Appliquée, Département de Microbiologie, Faculté des Sciences de la

Nature et de la Vie, Université Ferhat ABBAS Sétif-1, Algérie. 5

Laboratory of Soil Biology, University of Neuchatel, Neuchatel, Switzerland.

*Corresponding author: [email protected] Phone: +213 (0) 658 487 133; Fax: +213 (0) 036 61 12 37 E-mail: [email protected]

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ABSTRACT GO-Ag nanohybrids have attracted tremendous attention thanks to their several potential properties. In this research work, a new approach was adopted, where Triphenylphosphine (PPh3) was used as a linkage to decorate the surface of Graphene oxide (GO) nanosheets by Silver nanoparticles (AgNPs) via a simple method. The

Fourier transform infrared

spectroscopy (FTIR), Ultraviolet-visible spectroscopy (UV-Vis), X-ray diffraction analysis (XRD), Raman spectroscopy, Thermo-gravimetric analysis (TGA), X-ray fluorescence (XRF), Zeta potential analysis, Scanning electron microscopy (SEM), and Atomic force microscopy (AFM) techniques were used to reveal that AgNPs have covered the surface of GO sheets through non covalent and permanent bonding, altering new structural and electronic properties, leading to the appearance of the oxidative stress phenomenon, which considered as a key step of the antibacterial mechanism of this kind of hybrids, causing death of both Gram-positive (Bacillus subtilis, Enterococcus faecalis, Methicillin-resistant Staphylococcus aureus - MRSA, and Staphylococcus aureus) and Gram-negative (Escherichia coli, Serratia marcescens, Shigella sp, Salmonella sp, Serratia microorganisms liquefaciens, Proteus sp, Enterobacter cloacae and Pseudomonas aeruginosa). Keywords: Graphene oxide · Nanohybrid · Oxidative stress · Antibacterial activity· Silver nanoparticles. Self-assembly.

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1. INTRODUCTION The microbial world despite being invisible to the naked eye is a truly vast world even more than we imagine it to be, because of its great diversity and its enormous capacity for replication. The bacterial community represents an important class of this world. Bacteria are around us and exist everywhere; in our food, water, clothing, our skin, in the air we breathe and in all the equipment that we use habitually and even in our digestive system [1,2]. Fortunately, many bacteria are inoffensive and are rather useful purpose [3]. Nevertheless, another type of bacteria and microorganisms represent a threat to our health by its great pathological capacity. It is exactly the kind that we try to fight by the different preventive means like hygiene or other biological means such as drugs, antibiotics and chemicals such as disinfectants. However, with the proven effectiveness of these ways, their side effects cannot be negated. It is therefore necessary to invent and develop other products which more safe for health and the environment. Furthermore, not only the pathological capacity or the exacerbation of resistance to antibiotics of these microorganisms that pose a problem but also their ability to adhere on surfaces and build-up biofilms which will have significant sanitary and economic disadvantages [4,5]. Over the last few decades, nanomaterials found applications in many products used in our everyday life. In parallel, the engineering of hybrid nanostructures has attracted tremendous attention due to its diverse functionalities integrated in a single object. Graphene based materials (GBMs) which include few-layer graphene, graphene nanosheets, graphene oxide and reduced graphene oxide are one of the basic constituents of these hybrid materials [6]. Graphene, a monolayer of sp2-hybridized carbon atoms bonded in the hexagonal lattice, has a unique structure which provides this material with exceptional properties such as a large specific surface area, an excellent electrical conductivity, a good mechanical flexibility and a

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strong thermal/chemical stability [7]. It has therefore been considered as an ideal base for nanohybrids syntheses [8]. Recently, it was reported that graphene, like the other carbon nanomaterials, such as fullerenes and carbon nanotubes exhibits strong antibacterial or cytotoxicity activity with few environmental or health risks that allows its incorporation into products which may come into contact with the biosphere [9,10]. In spite of the strong push for understanding the interaction of graphene with cells and bacteria, the cytotoxicology mechanism of graphene remains ambiguous. Akhavan et al. [11] confirmed that the edges of GO lead to the destruction of the cell wall and then kill both Gram negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The profound study was carried out by Liu et al. [12] for the investigation of the antibacterial activity and antimicrobial mechanism of four kinds of graphene. The study concluded that the antimicrobial mechanism includes three stages: an initial cell deposition on graphene-based materials, the membrane stress caused by the direct contact with the sharp nanosheets and, the ensuing superoxide anion-independent oxidation. On the other hand, silver nanoparticles are known to exhibit the highest bactericidal activity and biocompatibility amongst all the antibacterial nanomaterials and have been used as biocide agents in health, food and textile applications [2,13]. So, we can imagine the synergistic antibacterial effect obtained by the hybridization of GO with AgNPs. Many studies [14,15] demonstrated a time-dependent and concentration-dependent antibacterial activity for GO–Ag nanohybrids against Gram-negative strain such as E. coli. According to these investigations, bacterial viability has been shown to depend on the materials, concentration, time of exposure and the physico-chemical properties. Moreover, Andreia et al. [16] and Shen et al. [17] reported that GO–Ag nanohybrids presented a high biocide property with a minimum inhibitory concentration. 4

But, the majority of the chemical processes to synthetize GO-Ag nanohybrids are based on the reduction of GO sheets using a wide variety of reducing toxic agents such as hydrazine [18], sodium borohydride [19], hydroquinone [20] and aqueous alkaline solution [21]. Furthermore, the reduction process led to a bad dispersity and less stability of GO–Ag nanohybrids [22], which weakened the contact with the bacterial cells and decreased the antibacterial effectiveness [23,24]. Therefore, the objective of our study is not only to provide support and investigate the antibacterial activity of the GO–Ag nanohybrids but also to develop a simple method for the synthesis with a few and effective chemical products to ensure a simple process on one hand, and a good dispersion and high stability of GO–Ag nanohybrids on the other hand. Herein and to reach that, triphenylphosphine (PPh3) was employed as a linkage to create a strong contact and permanent deposition of the AgNPs on GO sheets, in order to ensure unlimited use of this hybrid. 2. EXPERIMENTAL 2.1. Materials Graphite powder with particle size < 20 µm and triphenylphosphine (PPh3,) were purchased from Sigma-Aldrich. All chemical reagents necessary for the preparation of GO such as: sulfuric acid (H2SO4, 98 %) hydrogen peroxide (H2O2, 30 %), hydrochloric acid (HCl, 36 %) and sodium nitrate (NaNO3, 99 %) were analytical grades procured from Sigma-Aldrich and used without any further purification. Potassium permanganate (KMnO4, 99.5 %) was purchased from Fisher and used as received. 2.2. Synthesis of graphene oxide (GO) GO was synthesized from graphite powder using Hummers approach with a slight modification [25]. Typically, 4 g of graphite and 2.5 g of NaNO3 were mixed together 5

followed by the addition of 100 ml of concentrated H2SO4. The mixture was then ultrasonicated for 30 min and stirred for a period of 4 h, keeping the temperature at approximately 5 °C using an ice bath. Then 12 g of KMnO4 were added gradually keeping the temperature of the mixture below 15 °C under constant stirring. The mixture was stirred for 24 h at 35 °C and the resulting solution was diluted by adding 200 ml of water. In order to reduce the residual KMnO4 and MnO2, the resulting slurry was further treated with 20 ml of 30 % H2O2 solution [26]. The obtained mixture was then washed successively with HCl and with distilled water many times until the pH of the solution became neutral. The bulk of the water was removed by oven drying at 65 °C for 36 h. 2.3. Synthesis of GO–Ag nanohybrid The GO-Ag nanohybrid was prepared by a simple two-step strategy (Scheme 1). Briefly, 20 mg of GO were dispersed in 60 ml of PPh3 followed by sonication for 1 h. After that, the mixture was refluxed under vigorous stirring for 24 h at 200 °C. Another solution was prepared by dissolving 1.4 mg of AgNO3 in 20 ml of PPh3, and then was added to the GO– PPh3 slurry. After ultrasonication for 30 min, the mixture was maintained for 24 h at 80 °C under continuous stirring in a refluxing system until the colour of the slurry turned blackgreen indicating the formation of Ag particles. The final product was then centrifuged at 4000 rpm for 1 h and washed with ethanol ten times. Finally, GO–Ag nanohybrid was dried under vacuum at 80 °C for 24 h. 2.4. Characterization The optical absorption of the different samples analyzed was measured in the 200–800 nm range using a Shimadzu UV-1800 apparatus. FTIR characterization was carried out using a JASCO-IR spectrometer by signal averaging 16 scans at a resolution of 4 cm−1 with a spectral range of 4000–400 cm−1. A small amount of samples (1 wt%) was mixed with an excess of KBr powder (99 wt%) and ground down again to form a uniform consistency. Aliquots of the 6

mixture were compressed using hydraulic press to form thin pellets. To get detailed information about the chemical structure and molecular interactions, the Raman spectroscopy was performed at room temperature in the 1000-2000 cm-1 range using HORIBA LabRAMHR Evolution apparatus. The crystallographic structural analysis was performed using a Phillips System XPERT-PRO diffractometer operating with CuKα X-ray source (λ = 1.54 Å) at a generator voltage of 40 kV. The diffractograms were scanned in the 2θ range from 5 to 60° at a rate of 2°/min. TGA of the samples was conducted using a TA-SDT Q 600 thermal analyzer under nitrogen atmosphere at a flow rate of 20 ml/min from 25 to 800 °C at a heating rate of 10 °C/min. To determine the composition and the amount of the elements existing in the different samples the X-ray fluorescence analysis (XRF) was performed. Pellets of each sample were characterized using a ZSX Primus IV instrument under a vacuum atmosphere at 38 °C with a power supply of 4.0 W 30 kV in the semi-quantitative mode. Both particle size distribution and zeta potential (the magnitude of the particle surface charge) measurements were performed by dynamic light scattering at room temperature using HORIBA Nano Partica SZ-100 apparatus. The morphology of the studied samples was analyzed using a NEOSCOPE scanning electron microscope at an acceleration voltage of 10 kV. AFM measurements were performed on a BRUKER dimension 3100V atomic force microscope, using the tapping mode. The tips used have a resonance of 350 kHz. Samples (5 µl, 0.47 mg/ml) after sonication were dropped on cut mica, then air dried prior to analysis. 2.5. Antimicrobial activity The antimicrobial activity of both GO and GO–Ag nanohybrid was evaluated against twelve different species of Gram-negative (Escherichia coli ATCC 25922 , Serratia marcescens, Shigella sp, Salmonella sp, Serratia liquefaciens, Proteus sp, Enterobacter cloacae, Pseudomonas aeruginosa ATCC 27853) and Gram-positive bacteria (Bacillus subtilis 168, Enterococcus faecalis, Methicillin-resistant Staphylococcus aureus -MRSA-, Staphylococcus 7

aureus ATCC 25923) . The referenced strains were supplied from The Laboratory of Applied Microbiology of University Sétif -1- while the unreferenced ones were acquired from the microbiology laboratory of Sétif hospital. Using the agar diffusion method, the bacterial species were grown in Luria-Bertani (LB) medium at 37 °C for 24 h. Bacterial cell suspensions were diluted with physiological water (0.1 % w/v) until a concentration of 106 CFU/mL was reached. With a sterile swab, the bacterial suspension was dipped into the inoculums and streaked over the surface of the MH agar plates. Then, aliquots of 25 µL of GO and GO-Ag solutions (50 µg /ml) were placed on the surface of each inoculated plate using sterile forceps and the plates were incubated at 37 °C for 24 h. The formation of transparent halos on the medium surface (inhibition zone) indicates the antimicrobial effect of the samples to be tested. 2.6.Time-dependent and concentration-dependent antibacterial activity The spread plate method was used to investigate the effect of contact time and concentration on the antimicrobial activity of GO–Ag nanohybrid against Gram-positive (Bacillus subtilis 168, Staphylococcus aureus ATCC 25923) and Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853). 100 µl of bacterium cells (106 to 107 CFU/mL) were incubated with 10 ml of GO-Ag dispersions in physiological water in a shaker incubator at 200 rpm for 2 h. Series of 10-fold cell dilutions (100 µL each) were spread onto agar plates, and incubated at 37 °C for 24 h . The reading was performed after this period by counting the colonies and compared with those of the control plates to estimate changes in the bacterium growth inhibition. To study the effect of contact time and concentration of GO–Ag nanohybrid on the microbial activity, the same experimental protocol was carried out with GO–Ag nanohybrid except that the incubation time was 4 h instead of 2 h to study the effect of contact time. Various concentrations of GO-AgNPs (5, 10, and 15 µg/mL) were prepared and incubated with 8

bacterium cells (106-107 CFU/mL) for 2 h at 37 °C under a shaking speed of 200 rpm in order to study the effect of concentration on the antimicrobial activity. The loss of bacteria viability (%) was determined by the counts of colonies according to the following formula:

% =

−

∗ 100

where: LV: is the loss of bacteria viability Nc: is the number of colonies in the control petri dishes Nt: is the number of colonies in the treated petri dishes The number of colonies in both control and treated petri dishes was counted in the same dilution ratio. To observe E. coli morphology, cell suspensions were passed through a membrane of low porosity (0.22 µm). The particles were retained on the surface of the filter and fixed with glutaraldehyde and osmium tetroxide, followed by air-drying. Finally, the E. coli cells morphology was examined by the NEOSCOPE Scanning Electron Microscope. 3. RESULTS AND DISCUSSION 3.1. UV-Visible UV-Vis absorption spectra of the PPh3, GO and GO–Ag nanohybrid was conducted to confirm the adsorption of PPh3 molecules as well as the formation of AgNPs on the surface of GO sheets, and the results are delineated in Figure 1. As shown in Figure 1a the GO spectrum displays two absorption peaks. The first one at λ = 230 nm deriving from the π-π* transition and is due to the C=C bond in an aromatic ring and the second one at λ = 300 nm is attributed to the n-π* transition of C=O bond resulting from the oxidation process of graphite [26-28].

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The UV–Vis absorption spectrum of PPh3 (Figure 1c) shows an intensive peak at λ = 221 nm which is attributed to the n-σ* transition of PPh3 and another peak at λ = 275 nm related to the π-π* transition of the C=C bond in the aromatic ring of the PPh3. Figure 1b displays the UV-Vis spectrum of GO–Ag nanohybrid. The presence of a surface plasmon band at λ = 400 nm in the GO–Ag sample indicates the formation of silver nanoparticles resulting from the reduction of Ag+ ions by the PPh3 already adsorbed onto the flat plane of GO by π-π stacking interaction. These results are confirmed by the shifting of 4 nm marked for the peak at 279 nm corresponding to the electronic π-π* transition of the aromatic ring compared to the peak at 275 nm for PPh3 spectra [29,30]. 3.2. FT-IR analysis In order to confirm the oxidation of graphite, the adsorption of PPh3 and the deposition of AgNPs onto the graphene carbon network, ATR-FTIR analysis was carried out. As shown in Figure 2, the difference between the spectrum of graphite and GO is very clear; the absorption band at 1640 cm-1 (Figure 2a) is due to the graphitic structure of pristine graphite [31]. The GO spectrum (Figure 2b) exhibits several characteristic absorption bands of oxygencontaining groups. The broad band in the range of 3770 – 3240 cm−1 refers to the stretching vibration of the OH and COOH groups. The band at 1729 cm−1 is attributed to the C=O stretching of COOH group and the bands located at 1221 cm−1 and 1051 cm−1 are characteristic of the stretching vibration of the C–O–C bonds [32]. Moreover, the most characteristic feature in the PPh3 spectrum (Figure 2c) is the adsorption band corresponding to the C=C stretching vibration at 1400 ~ 1600 cm-1. The spectrum of GO-Ag (Figure 2d) also possessed the obvious adsorption band in addition to other bands with a moderate shifting even though they did not appear in the GO spectrum. This is what confirms the π-π stacking interaction between PPh3 molecules and the conjugated graphene carbon network. These results support those obtained in the UV-Vis analysis. 10

3.3. Raman spectroscopy As it is shown in Figure 3, the Raman spectra of pristine graphite (Figure 3a) exhibit a dominant G band at approximately 1568 cm-1 which refers to the bond stretching of the sp2 carbon network and, a second smaller D band, which is spotted at 1326 cm-1 and is due to a breathing mode of the hexagonal carbon rings. This band can reflect the disorder along the edges of the graphite pattern [33]. After the oxidation process (Figure 3b), the intensity of the D band increases significantly, indicating an increased disorder and a symmetry breaking in the graphene layers. The D band appears wider with a moderate shifting at 1360 cm-1, accompanied by a significant decrease of the G band’s intensity that also undergoes a moderate shifting at 1595 cm-1. In the case of GO–Ag nanohybrid (Figure 3c), the intensity of both D and G bands increased in a very remarkable way under the dual effect of PPh3 adsorbed molecules and AgNPs deposition on the GO sheets. In this case, the ratio of the intensity of the D band to the G band ID/IG of GO–Ag nanohybrid was equal to 0.98 which was the highest value compared to 0.1 and 0.8 values of the ID/IG ratios of both pristine graphite and GO samples respectively. This important increase resulting from the asymmetric breathing modes of the six-atom rings that occur due to non-covalent interaction between the GO and the PPh3 moieties [34] on one hand and, the increase in the disorder degree of the GO matrix on the other hand, due to a chemical bond between the carbon matrix and AgNPs [35]. 3.4. XRD analysis The effect of the oxidation as well as the efficiency is very clear through a small comparison between pristine graphite and GO XRD spectra (Figure 4). For the graphite pattern (Figure 4a), there is a sharp diffraction peak (002) at 2θ = 26.5° with a very low interlamellar distance 11

of 0.345 nm which corresponds to the typical graphitic structure that completely changes under the effect of oxidation [36]. During this process, the insertion of water molecules and the implantation of voluminous oxygen groups on the basal plane and on the edges of GO sheets weaken the maintenance energy of the typical graphitic network and increase the intersheet distance [37,38]. This is what explains the disappearance of the acute peak previously observed for the case of pristine graphite, and the appearance of a new less sharp peak at 2θ = 12.5° (Figure 4b) accompanied by an increase in the interplanar distance by 0.21 nm. The Xray diffraction analysis was also used to confirm that AgNPs anchored onto the GO sheets. The XRD pattern of GO–Ag (Figure 4c) shows four peaks at 37.9°, 44.8°, 64.2° and 77.1°, which respectively refer to (111), (100), (220) and (311) crystalline planes of the face-centred cubic of silver nanoparticles with the complete disappearance of the characteristic peak of GO [16,39]. These results do not leave any doubt about the formation of GO-Ag hybrid. 3.5. TGA analysis TGA was used to evaluate the thermal decomposition of the samples in order to obtain additional information about their composition (Figure 5). It is to be noted that pristine graphite has a high thermal stability as no weight loss was marked below 630 °C (Figure 5a). The GO curve (Figure 5b) shows three distinctive weight loss regions, which are directly linked to oxidation process. The first region observed around 130 °C is due to the evaporation of water molecules inserted between the GO sheets [40]. The second one around 250 °C refers to the decomposition of unstable oxygen containing functionalities releasing a number of gases as carbon dioxide that can contribute to the early thermal decomposition of the carbonic structure as it is noted through the last region approximately at 480 °C [40,41]. The TGA curve of GO-Ag (Figure 5c) exhibits two–step weight losses at 190 °C and at 400 °C with a significant reduction of the decomposition temperatures. This reflects the catalytic effect of the silver particles [42]. The first weight loss may be attributed to the decomposition of 12

oxygen-based functional groups fixed on the GO sheets surface and to the degradation of PPh3 molecules that play the role of an intermediate agent between the π-conjugated system of the graphene sheets and the AgNPs. The last stage weight loss is attributed to the decomposition of the GO carbon skeleton. Moreover, the amount of residues remained above 700 °C is associated with the mass of the AgNPs on the nanohybrid which is within the limits of 20 % (w/w) (Figure 5c). 3.6. X-ray fluorescence The results of the XRF analysis (Table 1 and Figure 6) show a significant difference between the three characterized samples. The presence, with an abundant level of oxygen element (43.1 %) in the GO specimen (Figure 6a) compared to the pristine graphite, which contains only the carbon element, mainly refer to the strong effect of the oxidation process which lead to the appearance of new elements. The results of the XRF analysis of the GO–Ag nanohybrid (Figure 6b) were very different in terms of quantity and quality. First, there is an oxygen content of 15.2 % which represents a decrease of 27.9 % compared to the GO specimen. In addition, two new components were added (Figure 6b): the P (phosphorus) element with 5.8 % which refers to the adsorbed PPh3 molecules, and the Ag element with 24.3 % which refers to the reduction of Ag+ ions. It is noticeable that the amount of the silver element deposited corresponds to that already found by the TGA analysis. 3.7. Zeta potential analysis The zeta potential of different colloid solutions was shown in Figure 7. The GO sheets (Figure 7a) show a moderate negative surface charge (-11 mV) resulting from electronegative functional groups formed at the graphite skeleton during the oxidation process [43]. These oxygenated groups, especially carboxylic acid groups (COOH) can dissociate by giving COO-

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and H+ ions at the surface, resulting in an increase of the zeta potential and leading to more or less stable GO colloidal aqueous suspension [44] . The zeta potential of GO–Ag nanohybrid (Figure 7b) exhibits a significant negative charge at approximately – 62.9 mV which refers to the loss of Ag+ ions their charges during the reduction reaction with PPh3 molecules prevent the formation of aggregates, thanks to the repulsive forces leading to a change of the electrical properties of the interfacial layers in the dispersion and an improvement of the stability of GO–Ag colloidal suspensions [45]. According to Figure 8 which illustrates the particle size distribution of AgNPs in GO–Ag nanohybrid, an average nanoparticle size of 5 ± 1.4 nm was found. This result confirms the role played by GO sheets in the nucleation and stabilization processes by allowing the formation of smaller nanoparticles [36]. 3.8. SEM and AFM analysis The morphological analysis of the different samples studied, as illustrated by the SEM micrographs (Figure 9), gives a strong argument to prove the effectiveness of oxidation, the adsorption of the PPh3 and the anchoring of AgNPs on the GO sheets. The attachment of oxygenated bulky groups during the oxidation process on both edges and surfaces of carbon layers led to a severe expansion; that is to say a significant widening of the interlayer space which led in turn to the decrease of the size of the GO layers. On the other hand, self-assembled sheets form, thanks to the hydrogen bridges that were created between the oxygenated functions mentioned above [46,47]. When the GO undergoes these changes, it takes bulky agglomerates form with a smooth surface (Figure 9b) unlike the pristine graphite where the sheets appear wide and flat (Figure 9a) . The phenomenon of adsorption of PPh3 on the GO network gives another morphological aspect different from that appearing previously. The aggregation of GO in the GO-PPh3 nanohybrid seems to be slighter (Figure 9c), implying 14

that the adsorption of PPh3 on the GO sheets plays an important role in precluding the restacking of graphene sheets. The SEM image of GO-Ag (Figure 9d) has a similar morphological aspect compared to the GO–PPh3 image with a uniform deposition of AgNPs on the GO surface. This is what is illustrated in the AFM image (Figure 10), where numerous AgNPs are observed with a small thickness since their surface root mean square (RMS) value is equal to 4.43 nm. 3.9. Antibacterial activity of GO and GO-Ag nanohybrid After three repetitions of the tests under the same conditions, the results obtained were very promising; a transparent circle which indicates that there was an inhibition was observed in most of the species tested for both Gram-positive and Gram-negative bacteria representing seven types (E. coli, S. marcescens, Salmonella sp, S. liquefaciens, Proteus sp, E. cloacae, B. subtilis) which were sensitive to all the total twelve species tested. This reflects the great antibiotic effect of GO-Ag nanohybrid especially against Gram-negative bacteria as it shown in Figure 11, although it was used at a low concentration. These results are contrary to those obtained by Andreia Fonseca et al. [42,47] who found that only one type of Gram-negative bacteria, P. aeruginosa, among thirteen Gram-positive and negative which exhibited a strong resistance to GO-AgNPs. These results also showed that, no inhibitory effect of GO was observed (Figure 11) against the same species previously tested. This resistance could be attributed to the low amount that did not reach the minimum inhibitory concentration (MIC). Despite the fact that many studies have confirmed the high bacterial toxicity of GO against several bacterial strains [6,11,12,48]. To evaluate the effect of time and concentration on the bioactivity of the GO–Ag nanohybrid, the choice of strains tested was based on the results obtained from the previous experiment. Four elements were selected; two of them were sensitive to GO–Ag (Escherichia coli ATCC

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25922 and Bacillus subtilis 168) and the two others exhibited a resistant behaviour (Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923). It is still observed through the results obtained, that there is a proportional correlation (Figure 12 and Figure 13) between the duration of the contact time and the mortality rate of the bacteria, reflecting the increase in the antibacterial efficacy of GO–Ag nanohybrid against the four species tested, especially E. coli and B. subtilis where the mortality rate reached almost 100 % after 4 h of contact. Knowing that it was about 97 % after 2 h of contact. Staphylococcus aureus and P. aeruginosa cells also recorded a significant ratio of nearly 98 and 95 %, respectively after 4 h of contact since they scored 96 and 84 %, respectively for a contact time of 2 h. It should be mentioned, that these two types recorded no response to GOAg nanohybrid in the first experiment where the agar diffusion method was adopted, which confirms the limitations of this technique. Concerning the effect of the concentration of GO-Ag nanohybrid on the antimicrobial activity, it also showed a proportionality (Figure 14 and Figure 15). In this context, the biological response reached its maximum with the highest concentration used, 15 µg. For example, the mortality rate recorded for P. aeruginosa was 53, 65 and 84 % with concentrations of 5, 10 and 15 µg, respectively where the effect was very pronounced. Escherichia coli cells also recorded a mortality rate of 91, 96 and 98 % with the corresponding concentrations of 5, 10 and 15µg respectively. This is conforming with the results of many reported studies [16,49,51]. The theory proposed by Chad et al. [52] which clarifies the cytotoxicity effect of carbon nanotubes against bacterial cells remains the most probable and valid to explain the results obtained in our investigation. According to this theory, the mechanism of action occur in three steps: adhesion of bacterial cells on graphene nanosheets, physical perforation of the cell

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membrane in other words membrane stress and thirdly the appearance of oxidative stress. The effect of the second step is dependent on the type of species (Gram-positive or Gramnegative) while the third step is strongly related to the electronic structure of the material, the more the material is a good current driver, the higher its effectiveness antimicrobial is strong, this is appropriate for the situation studied ,where the oxidation process of graphite leads to a decrease in the level of electricity transfer of GO produced [53], which reduces his biological effectiveness, especially if it is used with low concentration, that what explains its biological inertia and its inability to stop the proliferation of bacteria during the first experiment. Unlike the GO which despite the fact that it was used at the same concentration as that of GOAg nanohybrid. This latter exhibited a great bacterial cytotoxicity with the majority of the species tested. This difference could be attributed to the appearance of a new electronic structure resulting from the combination of the AgNPs and GO sheets. GO–Ag nanohybrid is nano-sized so it is able to be placed on the insulating lipid bilayers of the cytoplasmic membrane of the bacterial cell by acting as a transmission bridge for the resulting electrons during metabolic processes from the intracellular medium to the extracellular medium [52,54] as it is shown in Scheme 2. This transmission may generate an excess of reactive oxygen species (ROS) which are capable of inducing Deoxyribonucleic acid (DNA) mutations, destruction of cellular proteins, lipid peroxidation and membrane dysfunction [55]. Furthermore, it is known that Ag+ ions can deactivate cell enzymes and bacterial DNA by coordinating with electron donating groups such as thiols, carboxylates, amides, imidazoles, indoles and hydroxyls. They would then inhibit the bacteria's ability to breathe and replicate [33]. In addition, the shaking process ensures the good and the permanent dispersion of GO–Ag nanohybrid as it was demonstrated by the zeta potential analysis, leading to an increase in the specific area of nanoparticles which ensures the active contact between these latter and the 17

bacteria cells. Consequently, this contact causes an increase in oxidative stress effect as well as the mechanical perforation of the cell wall by the acute edges of GO, especially for Gramnegative species whose cell wall is formed of a single thin layer of peptidoglycan [12,54,56]. However Gram-positive species whose wall is formed by a thick layer of peptidoglycan, they resist relatively the mechanical stress, these effects are confirmed by SEM images obtained (Figure 16), where the difference in the bacterial cells shape is evident. All E. coli cells appear rod-shaped and uniform (Figure 16a) which is the typical shape of this type of bacteria, indicating the safety of the cell wall [56]. However as shown in Figure 16b the E. coli cells take an unusual and irregular shape confirming the perforation and rupture of the cell wall which is its most important function that ensures the bacteria integrity and maintain its shape [43]. This reflects also the results found in the first experiments where the majority of the species that exhibit a sensitivity against GO–Ag nanohybrid are those of Gram-negative bacteria. These results confirm the proportionality between the increase of contact time as well as concentration with the antibacterial effect. 4. CONCLUSIONS GO–Ag nanohybrid was successfully synthesized by a convenient two-step strategy based on π-π stacking interactions between GO–PPh3 molecules and reduction of Ag+ ions. Many of analysis were released to confirm the decorated GO sheets with AgNPs. The anti-bacterial activity of GO–Ag nanohybrid against the two kinds of bacteria strains, Gram-positive and Gram-negative was investigated and promising results were obtained. However, the contradictory effects observed with respect to the sensitivity of the same Grambacteria type against GO–Ag nanohybrid prompts us to intensify research in this vast and complex area in order to achieve a clear and a precise understanding of the antibacterial mechanism of this nanohybrid. Furthermore, another question remains whether this property 18

is reserved when incorporate this nanohybrid in different matrices, that what exactly will be investigated in future studies. REFERENCES [1] R. Diego, K Roberto, Will biofilm disassembly agents make it to market?, Trends Microbiol. 19 (2011) 304-306. http://dx.doi.org/10.1016/j.tim.2011.03.003. [2] What are bacteria and what do they do? www.medicalnewstoday.com/articles/157973.php /Medical News Today /Yvette Brazier/, 2019 (accessed 12 February 2019). [3] M.E. Davey, G.A. O'toole, Microbial biofilms: from ecology to molecular genetics, Microbiol Mol Biol Rev. 64 (2000) 847-867.http://dx.doi.org/10.1128/MMBR.64.4.847867.2000 [4] M. Simo˜es, L.C. Simo˜es , M.J. Vieira, A review of current and emergent biofilm control strategies LWT Food Sci. Technol. 43 (2010) 573- 583. http://dx.doi.org/10.1016/j.lwt.2009. 12.008. [5] R.F. Al-Thani, N.K. Patan, M.A. AlMa'adeed, Graphene oxide as antimicrobial against two gram-positive and two gram-negative bacteria in addition to one fungus, J. Biol. Sci. 14 (2014) 230-239. http://dx.doi.org/10.3844/ojbssp.2014.230.239. [6] X.A.T. Simmons, R. Shah, C. Wolfe, K.M. Lewis, M. Washington, S.K. Nayak, S. Talapatra, S. Kar, Stable aqueous dispersions of noncovalently functionalized graphene from graphite and their multifunctional high-performance applications, Nano Lett. 10 (2010) 42954301. https://doi.org/10.1021/nl903557p. [7] D. Galpaya, M. Wang, M. Liu, N. Motta, E. Waclawik, C. Yan, Recent advances in fabrication and characterization of graphene-polymer nanocomposites, Graphene. 1 (2012) 30-49. http://dx.doi.org/10.4236/graphene.2012.12005.

19

[8] V.C. Sanchez, A. Jachak, R.H. Hurt, A.B. Kane, Biological interactions of graphenefamily nanomaterials: an interdisciplinary Review, Chem. Res. Toxicol. 25 (2012) 115-34. https://doi.org/10.1021/tx200339h. [9] M. Shannon, N.R.J. Crawford, P.E. Ivanova, Bacterial interaction with graphene particles and surfaces, in: M. Aliofkhazraei (Eds.), Advances in Graphene Science., Hawthorn VIC, (2003) 100-118. [10] O. Akhavan, E. Ghaderi, Toxicity of graphene and graphene oxide nanowalls against bacteria, ACS Nano. 4 (2010) 5731-5736. https://doi.org/10.1021/nn101390x. [11] S. Liu, T.H. Zeng, M. Hofmann, E. Burcombe, J. Wei, R. Jiang, J. Kong , Y. Chen, Antibacterial activity of graphite, graphite oxide, graphene oxide, and reduced graphene oxide:

membrane

and

oxidative

stress,

ACS

Nano.

5

(2011)

6971–6980.

http://dx.doi.org/10.1021/nn202451x. [12] K. M.M. Abou El-Noura, A.E. Abdulrhman, A. Reda , A.A. Ammarb, Synthesis and applications

of

silver

nanoparticles,

Arabian

J.

Chem.

3

(2010)

135-140.

https://doi.org/10.1016/j.arabjc.2010.04.008. [13] M. Das, R. Sarma, R .Saikia, V. Kale, M .Shelke, P. Sengupta, Synthesis of silver nanoparticles in an aqueous suspension of graphene oxide sheets and its antimicrobial activity, Colloids Surf B. 83 (2011) 16-22. http://dx.doi.org/10.1016/j.colsurfb.2010.10.033. [14] L. Liu, J. Liu, Y. Wang, X. Yana , D.D. Sun, Facile synthesis of monodispersed silver nanoparticles on graphene oxide sheets with enhanced antibacterial activity, New J. Chem. 35 (2011) 1418-1423. http://dx.doi.org/10.1039/C1NJ20076C. [15] A.F. Faria, A.C.M. Moraes, P.D Marcato, Eco-friendly decoration of graphene oxide with biogenic silver nanoparticles: antibacterial and antibiofilm activity, J Nanopart Res. 16 (2014) 2110-2117. https://doi.org/10.1007/s11051-013-2110-7.

20

[16] J. Shen, M. Shi, N. Li, J. Shen, M. S. Li, B.Y. Hongwei, M.Y. Hu, M.Ye, Facile synthesis and application of Ag-chemically converted graphene nanocomposite, Nano Res. 3 (2010) 339-349. https://doi.org/10.1007/s12274-010-1037-x. [17] Y. Zhou, J. Yang, T. He, H. Shi, X. Cheng, Y. Lu, Highly stable and dispersive silver nanoparticle–graphene composites by a simple and low energy consuming approach and their antimicrobial activity, Graphene Compos. 9 (2013) 3445-3454. https://doi.org/10.1002/smll.201202455. [18] J. Ma, J. Zhang, Z. Xiong, Y. Yonga, X. S. Zhao, Preparation, characterization and antibacterial properties of silver-modified graphene oxide, J. Mater. Chem. 21 (2011) 33503352. http://dx.doi.org/10.1039/C0JM02806A. [19] R. Moosavia, S. Ramanathana, Y.Y. Leea, K.C.S. Linga, A. Afkhamib, G. Archunand , P. Padmanabhanc, B. Gulyásc, M. Kakran, S.T. Selvan, Simple green synthesis of metal nanoparticles and their composites for antibacterial applications, RSC Adv. 5 (2015) 7644276450. http://dx.doi.org/10.1039/C5RA15578A. [20] S. William, J. Hummers, E.R. Offeman, Preparation of graphitic oxide, J. Am. Chem. Soc. 80 (1958) 1339. https://doi.org/10.1021/ja01539a017. [21] U. Kanta, V. Thongpool, W. Sangkhun, N. Wongyao, Preparations, characterizations, and a comparative study on photovoltaic performance of two different types of graphene/TiO2 nanocomposites photoelectrodes, J. Nanomater. 1 (2017) 1-13. http://dx.doi.org/10.1155/2017/2758294. [22] Y. Dong, J. Shao, C. Chen, H. Li, R. Wang, Y. Chi, X. Lin, G. Chen, Blue luminescent graphene quantum dots and graphene oxide prepared by tuning the carbonization degree of citric acid, Carbon. 50 (2012) 4738-4743. https://doi.org/10.1016/j.carbon.2012.06.002.

21

[23] H. Fuan, F.Jintu, M. Dong, Z. Liming, L. Chiwah, C.H. Laiwa, The attachment of Fe3O4 nanoparticles to graphene oxide by covalent bonding, Carbon. 48(2010)3139-3144. http://dx.doi.org/10.1016/j.carbon.2010.04.052. [24] T.F. Emiru, D.W. Ayele, Controlled synthesis, characterization and reduction of graphene oxide: A convenient method for large, scale production Egypt. J. Bas. Appl. Sci. 4 (2017) 74-79. http://dx.doi.org/10.1016/j.ejbas.2016.11.002. [25] C. Xu, X. Wang, Fabrication of flexible metal-nanoparticle films using graphene oxide sheets as substrates. Small. 5 (2009) 2212-2217. http://dx.doi.org/10.1002/smll.200900548. [26] R. Manash, K.R. Dasa, S.R.S. Vinayak. V.K. Manjusha, S.P. Sengupta, Synthesis of silver nanoparticles in an aqueous suspension of graphene oxide sheets and its antimicrobial activity, Colloids Surf., B. 83 (2011) 16-22. https://doi.org/10.1016/j.colsurfb.2010.10.033. [27] M. Saravanana, A. Nandab, Extracellular synthesis of silver bionanoparticles from Aspergillus clavatus and its antimicrobial activity against MRSA and MRSE, Colloids Surf ., B. 77 ( 2010) 214-218. https://doi.org/10.1016/j.colsurfb.2010.01.026. [28] R.A. Friedel, F.L. Carlson, Infrared spectra of ground graphite, J. Phys. Chem. 75 (1971) -1149-1151. https://doi.org/10.1021/j100678a021. [29] Z. Huanga, Q. Sunb, K. Lva, Z. Zhanga, M. Lia, B. Lic, Effect of contact interface between TiO2 and g-C3N4 on the photoreactivity of g-C3N4/TiO2 photocatalyst: (001) vs (101) facets of TiO2, Appl. Catal., B.164 (2015) 420-427. https://doi.org/10.1016/j.apcatb.2014. 09.043. [30] G. Zhao, L .Jiang, Y. He, J. Li, H. Dong, X. Wang, W. Hu, Sulfonated graphene for persistent aromatic pollutant management, Adv Mater. 23 (2011) 3959-3963. http://dx.doi.org/10.1002/adma.201101007. [31] B. Karimi, B. Ramezanzadeh, A comparative study on the effects of ultrathin luminescent graphene oxide quantum dot (GOQD) and graphene oxide (GO) nanosheets on

22

the interfacial interactions and mechanical properties of an epoxy composite, J Colloid Interface Sci. 1(2017) 62-76. http://dx.doi.org/10.1016/j.jcis.2017.01.013. [32] R. Doufnoune, T. Baouz, S. Bouchareb, Influence of functionalized reduced graphene oxide and compatibilizer on mechanical, thermal and morphological properties of polypropylene/polybutene-1 (PP/PB-1) blends, J. Adhes. Sci. Technol. 33 (2019) 1729-1757. https://doi.org/10.1080/01694243.2019.1611367. [33] M.R. Dasa, R.K. Sarmab, S.C. Boraha, R. Kumaria, R. Saikiab, A. B. Deshmukhc, M.V. Shelkec, P. Sengupta, S. Szuneritsd, R. Boukherroubd, The synthesis of citrate-modified silver nanoparticles in an aqueous suspension of graphene oxide nanosheets and their antibacterial activity, Colloids Surf ., B.105 (2013) 128-136. http://dx.doi.org/10.1016/j.colsurfb.2012.12.033 [34] M.H. Alonso, A. Abdala, M. McAllister, I. Aksay, R. Prud'homme, Intercalation and stitching of graphite oxide with diaminoalkanes, Langmuir. 23 (2007) 10644-10649. http://dx.doi.org/10.1021/la0633839. [35] J. Rourke, P. Pandey, J. Moore, M. Bates, I. Kinloch, R. Young, N. Wilson, The real graphene oxide revealed: stripping the oxidative debris from the graphene-like sheets, Angew Chem Int Ed Engl. 50 (2011) 3173-3177. http://dx.doi.org/10.1002/anie.201007520. [36] A.F. Faria, D.S.T. Martinez, S.M.M. Meira, A.C.M. Moraes, A. Brandelli, A.G.S. Filho, O.L. Alvesa, Anti-adhesion and antibacterial activity of silver nanoparticles supported on graphene oxide sheets, Colloids Surf ., B. 113 (2014) 115-124. https://doi.org/10.1016/j.colsurfb.2013.08.006. [37] S. Rattanaae, N. Chaiyakunae, N. Witit-anunae, P. Nuntawongb, S. Chindaudomb, C. Oaewc, P.K. Limsuwande, Preparation and characterization of graphene oxide nanosheets, Proc.Eng. 32 (2012) 759-764. https://doi.org/10.1016/j.proeng.2012.02.009.

23

[38] D. Li, M. Müller, S. Gilje, R. Kaner, G.Wallace, Processable aqueous dispersions of graphene nanosheets, Nat Nanotechnol. 3 (2008) 101-105. http://dx.doi.org/10.1038/nnano.2007.451. [39] B.A. Bourlinos, D. Gournis, D. Petridis, T. Szabó, A. Szeri, I. Dékány, Graphite oxide chemical reduction to graphite and surface modification with primary aliphatic amines and amino acids, Langmuir. 19 (2003) 6050-6055. https://doi.org/10.1021/la026525h. [40] G. Wang, J. Yang, J. Park, X. Gou, B. Wang, H. Liu, J. Yao, Facile synthesis and characterization of graphene nanosheets, J. Phys. Chem.

22 (2008) 8192-8195.

https://doi.org/10.1021/jp710931h. [41] S. Park, J. An, R.D. Piner, I. Jung, D. Yang, A. Velamakanni, S.B.T. Nguyen, R.S. Ruoff, Aqueous suspension and characterization of chemically modified graphene sheets, Chem. Mater. 20 (2008) 6592-6594. http://dx.doi.org/10.1021/cm801932u. [42] S. Rattanaae, N. Chaiyakunae, N. Witit-anunae, P. Nuntawongb, S. Chindaudomb, C. Oaewc, P. Kedkeawd, Limsuwande, Preparation and characterization of graphene oxide nanosheets, Proc.Eng. 32 (2012) 759-764. https://doi.org/10.1016/j.proeng.2012.02.009. [43] I. Barbolina, C.R. Woods, N. Lozano, K. Kostarelos , K.S. Novoselov, I.S Roberts, Purity of graphene oxide determines its antibacterial activity, 2D Mater. 3 (2016) 025025. http://dx.doi.org/10.1088/2053-1583/3/2/025025. [44] L. Liu, J. Liu, Y. Wang, X. Yana , D.D. Sun, Facile synthesis of monodispersed silver nanoparticles on graphene oxide sheets with enhanced antibacterial activity, New J. Chem. 35 (2011) 1418-1423. http://dx.doi.org/10.1039/C1NJ20076C. [45] W. Xu, L. Zhang, J. Li, Y. Lu, H. Li, Y. Ma, W. Wang, S. Yu, Facile synthesis of silver@graphene oxide nanocomposites and their enhanced antibacterial properties, J. Mater. Chem. 21 (2011) 4593-4597. http://dx.doi.org/10.1039/C0JM03376F.

24

[46] D. Chad, R. Vecitis, K. Zodrow, S. Kang, M. Elimelech, Electronic-structure-dependent bacterial cytotoxicity of single-walled carbon nanotubes, ACS Nano. 4 (2010) 5471-5479. http://dx.doi.org/10.1021/nn101558x. [47] R. Wang, D. Zhuo, Z. Weng, L. Wu, X. Cheng, Y. Zhou, J. Wanga , B. Xuanac, A novel nanosilica/graphene oxide hybrid and its flame retarding epoxy resin with simultaneously improved mechanical, thermal conductivity, and dielectric properties, J Mater Chem A. 3 (2015) 9826-9836. https://doi.org/10.1039/C5TA00722D . [48] C.V. Gomez, E. Robalino, D. Haro, T. Tene, P. Escudero, A. Haro, J. Orbeb, Structural and electronic properties of graphene oxide for different degree of oxidation, Mater. Today: Proc. 3 (2016) 796-802. https://doi.org/10.1016/j.matpr.2016.02.011. [49] G.W. Thorp, C.S. Fong, N. Alic, V.J. Higgins, I.W. Dawes, Cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative-stressresponse genes, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 6564-6569. http://dx.doi.org/10.1073/pnas.0305888101. [50] The Structure of Bacterial Cell Wall, glycopedia. https://glycopedia.eu/e-chapters/thestructure-of-bacterial-cell/article/the-bacterial-cell-wall,/N. Jean, C. Bougault, J.P. Simorre/ 2019 (accessed 21 April 2019). [51] M.A. Pedro, F. Cava, Structural constraints and dynamics of bacterial cell wall architecture, Front. Microbiol. 8 (2015) 446-449. https://doi.org/10.3389/fmicb.2015.00449. [52] I. Jung, D. Dikin, R. Piner, R. Ruoff, Tunable electrical conductivity of individual graphene oxide sheets reduced at "low" temperatures, Nano Lett. 12 (2008) 4283-4287. http://dx.doi.org/10.1021/nl8019938. [53] K. Krishnamoorthy, M. Veerapandian, K. Yun, S. Kim, The chemical and structural analysis of graphene oxide with different degrees of oxidation, Carbon. 53 (2013) 38-49. http://dx.doi.org/10.1016/j.carbon.2012.10.013.

25

[54] H. Raza., Graphene nanoelectronics: Metrology, synthesis, properties and applications, first ed., Springer-Verlag Berlin Heidelberg, 2012. [55] J. Cho, I. Jeon, S.Y. Kim, S. Lim, J.Y. Jho, Improving dispersion and barrier properties of polyketone/graphene nanoplatelet composites via noncovalent functionalization using aminopyrene,

ACS

Appl.

Mater.

Interfaces.

9

(2017)

27984-27994.

https://doi.org/10.1021/acsami.7b10474. [56] Q. Bao, D. Zhang, P. Qi, Synthesis and characterization of silver nanoparticle and graphene oxide nanosheet composites as a bactericidal agent for water disinfection, J Colloid Interface Sci. 2 (2011) 463-470. http://dx.doi.org/10.1016/j.jcis.2011.05.009.

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Table 1. Results of XRF analysis of pristine graphite, GO and GO-Ag nanohybrid Element

Pristine graphite (%)

GO (%)

GO-Ag (%)

C

99.9

56,4

51,5

O

-

43,1

15,2

P

-

-

05,8

Ag

-

-

24,3

Sonication & Reflux (π-π interaction)

+

GO

GO–Ag nanohybrid Scheme 1.

+

Ag+

230

300

Absorbance (a.u.)

270

Absorbance

221

(c) 300 400 500 Wavelength (nm)

200 266

(a) 490

(b)

400

200

300

400

500

600

Wavelength (nm) Figure 1.

700

800

(d)

OH

C=O C=C

1400

Transmittance (a.u.)

(c) (b)

C-O-C

(a)

1735

1221

C=C 3240

3770

4500

Figure 2.

4000

3500

1059

1640

3000 2500 2000 1500 Wavenumber (cm-1)

1000

500

G band

D band

Intensity (a.u.)

(c)

(a)

(b)

1000

Figure 3.

1200

1400 1600 Wavenumber (cm-1)

1800

2000

Intensity (a.u.)

(111)

(200)

(001)

(220) (311)

(c) (b)

(a) 10 Figure 4.

20

30

40 2-Theta (°)

50

60

70

80

100

(a)

Weight (%)

90

80 (c) 70

60 (b) 50

Figure 5.

100

200

300 400 500 Temperature (°C)

600

700

C

(a)

O

20

40

60 80 2-Theta (°)

100

120

140

C

(b)

Ag O P

20 Figure 6.

40

60

100 80 2-Theta (°)

120

140

Total counts

20000 15000 10000

(b) (a)

5000 0 -150

Zeta potential (mV)

0 -10 -20 -30 -40

-50 -60 -70

Figure 7.

-100

-50 0 50 Zeta potential (mV)

(a)

(b)

100

150

35

Number of particles

30 25 20 15 Xc = 5

10 5 0

Figure 8.

0

20

80 40 60 Particle Diameter (nm)

100

(b)

(a)

20µm (c)

(d)

20µm Figure 9.

20µm

20µm

(a)

(c)

Figure 10.

(b)

(d)

Figure 11.

Figure 12.

Loss of Viability (%)

P. aeruginosa

2 4 Incubation time (h)

E. coli B. subtilis S. aureus P. aeruginosa E. coli B. subtilis S. aureus P. aeruginosa

100

S. aureus

80

0

B. subtilis

60

40

20

0 E. coli

Figure 13.

P. aeruginosa

S. aureus

B. subtilis

E. coli Control 2h 4h

Figure 14.

Loss of Viability (%)

E. coli B. subtilis

S. aureus P. aeruginosa

15

E. coli B. subtilis S. aureus P. aeruginosa

100

10 Concentration (µg/ml)

S. aureus P. aeruginosa

80

5

B. subtilis

60

40

20

0

E. coli

P. aeruginosa

S. aureus

B. subtilis

E. coli

Control

Figure 15. 5µg 10µg 15µg

Insulating Bacterial Cytoplasmic Membrane Intracellular space

Extracellular space

Metabolic Processes (Breathing/Fermentation )

GO-Ag Transmission Bridge

Scheme 2.

(a)

(b)

2µm Figure 16.

2µm

Scheme and Figure captions Scheme 1: A scheme illustrating the mechanism of Ag adsorption on GO sheets. Figure 1: UV–Vis absorption spectra of (a) GO, (b) GO–Ag and (c) PPh3. Figure 2: FT–IR spectra of (a) neat graphite, (b) GO, (c) PPh3 and (d) GO–Ag hybrid. Figure 3: Raman spectra of (a) pristine graphite, (b) GO and (c) GO-Ag nanohybrid. Figure 4: XRD patterns of (a) pristine graphite, (b) GO and (c) GO–Ag nanohybrid. Figure 5: TGA curves of (a) pristine graphite, (b) GO and (c) GO–Ag nanohybrid. Figure 6: XRF spectra of (a) GO and (b) GO-Ag nanohybrid. Figure 7: Zeta potential analysis of (a) GO and (b) GO-Ag nanohybrid. Figure 8: Particle size distribution of Ag nanoparticles in GO-Ag nanohybrid. Figure 9: SEM images of (a) pristine graphite, (b) GO, (c) GO–PPh3 and (d) GO-Ag. Figure 10: AFM images of (a,c) 2D GO-Ag and (b,d) 3D GO-Ag nanohybrid. Figure 11: Results of the bacterial response against GO and GO-Ag nanohybrids. Figure 12: Loss of Viability of bacterial cells after incubation with GO-Ag nanohybrid

suspensions at different times. Figure 13: Results of contact time effect on antimicrobial activity (the photos of each strain

were taken for the same dilution). Figure 14: Loss of Viability of bacterial cells after incubation with GO-Ag nanohybrid

suspensions at different concentrations. Figure 15: Results of concentration effect on antimicrobial activity (the photos of each strain

were taken for the same dilution).

Scheme 2: Demonstrative scheme of the role of GO-Ag nanohybrid in the process of

oxidative stress. Figure 16: SEM images of (a) E. coli after incubation for 2 h without GO-Ag nanohybrid and

(b) E. coli after incubation with GO-Ag dispersion (15 µg/mL) for 2 h.

Highlights: •

A nanohybrid material based on GO and AgNPs was effectively synthetized

•

The PPh3 molecules play a dual effect during the synthesis of GO-Ag nanohybrid

•

The GO-Ag showed a high bactericidal effect against both Gram-positive and Gramnegative bacteria strain.

•

The antibacterial mechanism of GO-AgNPs occurs in three main stages

Declarations of interest statement

Authors attest that there's no financial/personal interest or belief that could affect their objectivity.