Synthesis of silane ligand-modified graphene oxide

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activity of modified graphene-silver nanocomposite ... Available online 5 May 2017. In this research ... Silane organic compounds as a modifier of carbon based materials ... easily interact with Ag ions through strong and weak covalent binding.

Materials Science and Engineering C 79 (2017) 55–65

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Synthesis of silane ligand-modified graphene oxide and antibacterial activity of modified graphene-silver nanocomposite Soghra Fathalipour ⁎, Mojtaba Mardi Department of Chemistry, Payame Noor University, PO Box: 19395-3697, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 25 July 2016 Received in revised form 26 January 2017 Accepted 4 May 2017 Available online 5 May 2017 Keywords: Silane ligand Ag nanoparticles Modified graphene oxide Antibacterial

a b s t r a c t In this research, a new type of chemically modified graphene oxide (GO) was synthesized based a silane ligand and then used as substrate and stabilizing for the synthesis of monodispersed and small Ag nanoparticles (NPs). First, ligand molecules were successfully grafted onto the surface of GO (LGO) and then, active groups of LGO could effectively interact with Ag ions. The reduction of Ag ions and LGO sheets was carried out by hydrazine under reflux. The resulted nanocomposite was fully characterized by different techniques. Furthermore, the antibacterial behavior of nanocomposite was studied against E. coli and S. aureus. The results showed that nanocomposite exhibits good antibacterial activity against E. coli and S. aureus and also S. aureus showed greater resistance than the E. coli strains against the LG/Ag nanocomposite. © 2017 Published by Elsevier B.V.

1. Introduction Recently, graphene (G) has received increasing attention due to its unique structural, surface properties, extraordinary electronic, thermal and mechanical properties [1–3]. The general method for the preparation of exfoliated graphene sheets is the combination of oxidation and sonication procedures, followed by chemical reducers [4,5]. In solution, obtained graphene sheets are aggregated due to strong Vander Waals interactions and it needs to decrease [6]. Graphene oxide (GO), a graphene derivative, consists of a basal plane with hydroxyl, epoxy, carbonyl and carboxylic groups [7,8]. The surface modification of graphene oxide sheets with classification of the organic chemical functionalization and then inserting of metal nanoparticles (NPs) among two-dimensional (2D) modified GO sheets through the reduction of metal precursors could inhibit the aggregation of graphene sheets [9–11]. On the other hand, the grafting of functional groups to graphene sheets also helps in dispersion a hydrophilic or hydrophobic media and this issue has crucial importance in their applications [12– 16]. Extensive studies have been carried out on the surface modification of graphene sheets and the preparation of graphene-metal nanoparticles composites [17–20]. However, there are limits due to dispersibility, electrical conductivity and the experimental complications. In recent years, silver-graphene nanocomposites have attracted much attention due to their potential applications in catalysis, nanoscale electronics, antibacterial, SERS substrates biomedical field and use in pharmaceuticals [21–26].

⁎ Corresponding author. E-mail address: [email protected] (S. Fathalipour).

http://dx.doi.org/10.1016/j.msec.2017.05.020 0928-4931/© 2017 Published by Elsevier B.V.

Silane organic compounds as a modifier of carbon based materials could produce nanomaterial with new chemical and physical properties [27–29]. Yang and coworkers prepared modified graphene via facile covalent functionalization of GO with 3-aminopropyltriethoxysilane and employed as reinforcing components in silica monoliths [30]. The resulting functionalized graphene sheets showed high dispersibility into water, polar solvents such as ethanol, dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO). Chemonne et al. linked chelating groups via a silanization reaction between N-(trimethoxysilylpropyl) ethylenediamine triacetic acid (EDTA-silane) and hydroxyl groups on GO sheets. The modified GO showed high adsorption behavior for pb (II) removal [31]. Yao and coworkers prepared also functionalized graphene by the reaction of N-(3-trimethoxysilylpropyl) diethylenetriamine with the hydroxyl groups of GO and then used as the template for Au nanoparticles (Au NPs). The resultant Au-G nanocomposite showed potential applications in SERS [9]. In this report, a new silane ligand (3,3′-bis-(3-triethoxysilylpropyl)2,2′-dithioxo [5,5′] bithiazolidinylidene-4,4′-dione) has been used for the modification of GO. The silylation modification technique with this ligand on Al2O3 NPs has been previously reported by Hassanpoor et.al. [32]. The resulted Al2O3 nanoparticles were used in ultra-trace determination of arsenic species in environmental waters and food and biological samples. The researchers would like to highlight in fact that this chelating ligand is a superior modifier for GO, and a new type of chemically functionalized graphene oxide sheets with chelating groups via a silanization reaction. On the other hand, it is believed that the silanization of GO with this ligand will be the best method to prepare the monodispersed Ag NPs due to several advantages that includes: (a) chemical reaction between the trialkoxy groups of silane ligand

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and the hydroxyl groups on the surface of graphene oxide that created the higher distance between the layers in the composites than pure GO and prevented from aggregation of GO sheets (b) modification provided a suitable environment for the presence the monodispersed Ag NPs with high density. Several active functional groups, such as carbonyl, thiol, thiocarbonyl, amide and hydroxyl groups in modified GO, could easily interact with Ag ions through strong and weak covalent binding to form the MGO/Ag + complex and then chemical reduction of the complex with hydrazine monohydrate at reflux condition formed the LG/Ag nanocomposite.

2. Experimental section 2.1. Materials Silver nitrate (AgNO3), graphite, carbon disulfide (CS2), dimethyl acetylene dicarboxylate (DMAD), 3-(triethoxysilyl) 1-propanamine

(TEPA) were obtained from Merck and used as received without further purification. Deionized water was used during the samples. 2.2. Equipment UV–Vis spectra were recorded by a PG Instruments T80 UV–Vis spectrophotometer with a scan range of 200–800 nm and NMR spectra were recorded with a Bruker DRX-250 AVANCE (Rheinstetten, Germany) instrument (300.1 MHz) with CDCl3 as solvent. Besides, X-ray diffraction measurements were recorded by a Bruker-D8 ADVANCE 3000 X-Ray diffractometer based on Cu Kα radiation (Kα = 0.1542 nm) at a scanning rate of 10.0ₒ/min, using a voltage of 40 kV and a current of 40 mA. Fourier Transform Infrared (FTIR) spectra were obtained on Shimadzu 8400 spectrometer. The Raman spectra were determined by Dispersive Raman Microscope (Bruker, Germany) in the range of 500– 1800 cm−1 using a laser beam with wavelength of 785 nm. Morphology and particle sizes of composites were noticed using by transmission electron microscopy (TEM, Philips-CM 30). Energy-dispersive X-ray

Scheme 1. Schematic diagram to illustrate the preparation process of LG/Ag nanocomposite.

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Fig. 1. HNMR spectra of the ligand (a) and expand of spectra (b).

spectroscopy (EDX) was performed by an EDX detector on an EMITECHK450X scanning electronic microscope (SEM) with a voltage of 15 kV. Concentrations of silver ions in nanocomposites were quantified by inductively coupled plasma atomic emission spectrometer (ICP-AES).

2.3. Modification of graphene oxide sheets with ligand (LGO) and synthesis of LG/Ag nanocomposite. 2.3.1. Synthesis of ligand (3,3′-bis-(3-triethoxysilylpropyl)-2,2′-dithioxo [5,5′] bithiazolidinylidene-4,4′-dione) The ligand was synthesized according to the previously reported procedure [32]. Drop wised TEPA (0.88 g, 4 mmol) in 5 min was added to the stirred solution of CS2 (0.36 g, 4.8 mmol) and DMAD (0.28 g, 2 mmol). The reaction mixture was allowed to stir for 2 min and then with the addition of EtOH to the reaction mixture, orange crystals (M.p: 330°, decompose) were produced.

Fig. 2. UV–Vis absorption of the LGO and LG/Ag nanocomposite. In the inset, the UV–Vis absorption of GO is shown.

2.3.2. Modification of graphene oxide with silane ligand (LGO) GO was prepared by oxidizing natural graphite powder based on an improved Hummers' method as originally presented by Marcano et al. [33]. The resultant GO was modified with silane ligand according to the following method: GO (10 mg) was dispersed in ethanol (50 ml) and after ultrasonication for 1.0 h, ethanol solution of synthesized ligand (1 ml of a 1.0 wt%) and triethylamine (0.2 ml) were added and stirred for 18 h through reflux. The precipitate was collected by filtration and then washed with ethanol, water and acetone, respectively. The final product was vacuum-dried and could be defined as silane ligandfunctionalized graphene oxide: LGO.

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2.3.3. Preparation of LG/Ag nanocomposites Dried LGO powder (25 mg) was added to ethanol (50 ml) and a homogeneous dispersion was obtained by ultrasonication. To this dispersion, freshly prepared silver nitrate solution (0.001 mM, 10 ml) was added drop wise and mixed under ultrasonication at room temperature for 1 h. The resulted mixture was heated with hydrazine (0.01 ml) at 95 °C for 1 h and then was filtered and repetitively washed with DI water and ethanol to remove excess hydrazine and ligand. Finally, resultant precipitate was dried with vacuum at room temperature to result in LG/Ag nanocomposite. For comparison, the G-Ag nanocomposite and

LG were also synthesized according to the same procedure with LG/Ag nanocomposite. 2.4. Antimicrobial activity The antibacterial properties of the nanocomposites were tested against S-arouses (ATCC 29213) and E. coli bacteria (ATCC 25922) by the Agar well diffusion and Broth-dilution methods. 2.4.1. Agar well diffusion method S. aureus and E. coli cells were cultured in nutrient agar medium and after centrifution, bacteria suspensions were diluted with sterile physiological solution to 1.5 × 106 CFU/ml. For the agar well diffusion method, bacterial suspension was spread on the surface of the agar plate [34]. Then, a hole with a diameter of 3 mm is punched aseptically with a sterile corn borer and 40 μl of GO, LGO, ligand/Ag, G/Ag and LG/Ag with concentration of 1 mg/ml is introduced in to the well. Agar plates were incubated at 37 °C for 24 h. The antimicrobial agent diffused in the agar medium and inhibited the growth of the microbial strain tested. Finally, the diameters of inhibition growth zones were measured. 2.4.2. 2.4.2 Broth-dilution method The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by Broth-dilution method. 10 μl of bacterial suspension was transferred to tubes containing different concentration of nanocomposites (40, 20, 10, 5, 2.5, 1.25, 0.6, 0.3, 0.15, 0.075 mg/ml) and incubated for 24 h in an incubator shaker at 80 rpm. The MIC is the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in tubes as detected by the unaided eye [34]. The MBC is defined as the lowest concentration of antimicrobial agent needed to kill 99.9% of the final inoculum after incubation for 24 h [34].

Fig. 3. XRD patterns of the LGO, LG, ligand and LG/Ag nanocomposite. In the inset, the XRD pattern of GO is shown.

Fig. 4. FT-IR spectra of the GO, LGO and LG/Ag nanocomposite.

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2.5. Time kill curves of GO and LG/Ag nanocomposite 100 μl of bacterial suspensions (1.5 × 10 6 CFU/ml) was added to LG/ Ag dispersions (0, 0.3, 0.6 and 1.25 mg/ml) and tubes were incubated in an incubator shaker at different times of incubation at 37 °C for 6 h. 10 μl of each bacteria-LG/Ag mixture was diluted with physiological solution 104 times and then 10 μl of the mixtures was dripped over the nutrient agar plates and all plates were incubated at 37 °C for another 24 h. Also a negative control was carried out in a tube without GO and LG/Ag. The number of colonies was counted and expressed in CFU/ml and the antibacterial ratio (R) was determined according to the following equation. R % = (A− B)/A × 100Where A and B are the numbers of viable bacterial colonies incubated with the control and GO or LG/Ag, respectively. 3. Results and discussion The synthesis procedure of silane ligand, LGO and LG/Ag nanocomposite was schematically illustrated in Scheme 1. Synthesize of silane ligand was confirmed by HNMR spectroscopy and the results were summarized in Fig. 1. 1H NMR (CDCl3): δ ppm 0.66 (t, 2H, J = 7.8,

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CH2-Si), 1.23 (t, 9H, J = 6.9, 3CH3), 1.71–1.81 (m, 2H, CH2), 3.83 (q, 6H, J = 6.9, OCH2), 4.12(t, 2H, J = 7.5 Hz, NCH2). Fig. 2 displays the UV–Vis spectra of GO, LGO and LG/Ag dispersed in ethanol. As shown in Fig. 2, GO exhibits a strong absorption peak at 230 nm and a shoulder at around 300 nm (Fig. 2, inset). This maximum absorption peak is attributed to the π/π* transitions of aromatic C\\C bonds while the shoulder occurred due to the n/π* transition of C_O bonds (carbonyl groups) [35]. The maximum absorption peak of LGO shifted to 248 nm which can be attributed to the linking of silane molecules to the GO sheets. The modification of GO was also accompanied with the presence of a new peak at 403 nm and a color change from light brown to gray which are attributed to partially deoxygenation of GO sheets upon the attaching of ligand (Fig. 2 and inset in Scheme 1). For LG/Ag, the 248 nm and 403 nm peaks were further red-shifted to 264 nm and 430 nm which these changes could be attributed to the reduction of LGO sheets and the excitation of surface plasmons of Ag NPs, respectively [10,36]. Meanwhile, in comparison with UV–Vis of LGO, the intensity of peak at 430 nm increased confirming the formation of Ag NPs in composite. When LGO and AgNO3were treated under hydrothermal reduction, oxygen groups especially hydroxyl and epoxy group could be removed from LGO surfaces and Ag NPs loaded on the surface of LG sheets, thus reflect on the color changes from dark brown to black (Scheme 1, inset). XRD measurement was used to analyze the changes in crystalline arrangements in LGO and LG/Ag nanocomposite. The XRD patterns of GO, LGO, LG, ligand and LG/Ag nanocomposite are shown in Fig. 3. The comparison of XRD patterns of GO and LGO showed that the characteristic peak of GO at 2θ ≈ 11(001), was absent in LGO, while there were new peaks at 2θ:24.7 and 43.1 for LGO. For comparison, the XRD patterns of LG and ligand were shown in Fig. 3. In the XRD pattern of ligand, characteristic peak was appeared at 2θ: 24.8 but the XRD pattern of LG was almost similar to that of LGO, except the high of peak increase after the reduction. These results revealed that during the silylation process, the lattice structure of GO changed because of the attached functional groups of the surface and suggested that the GO sheets were completely exfoliated. In the spectrum of LG/Ag, the major diffraction peaks at 38°, 44°, 66° and 77° were assigned to the (111), (200), (220) and (311) planes of silver with a face-centered cubic (Fcc) structure, respectively [10]. The broad diffraction peaks of Ag indicated a relatively small crystal size, which demonstrate the exfoliated and disordered structure formed during sonication and chemical reaction process. On the other hand, in XRD pattern of LG/Ag nanocomposite, the increase of the intensity of peak at 2θ: 24.8 compared to LGO confirmed the successful reduction of LGO to graphitic structures [37]. Grafting of the silane ligand to the graphene oxide surface and the synthesis of LG/Ag nanocomposite was further confirmed through FTIR spectra. As shown in Fig. 4, the pristine GO displayed four peaks at 3428, 1725, 1231 and 1061 cm− 1 characteristic of the hydroxyl broad absorption, carbonyl, the stretching vibration of the –CO– groups, and epoxy groups, respectively. Compared with the spectrum of GO, there are new peaks appeared in the spectrum of LGO at 1355, 1243, 1158 and 604 cm−1 which could be attributed to C_S and C\\N, C\\S and Si\\O\\C vibrations and also characteristic peaks of CH2 groups at 2895 and 2976 cm−1 proving the presence of firmly bonded of ligand on the surface of GO [32,38]. Moreover, the increasing of the intensity of the absorption peaks around 1727and 1061 cm−1 could be related to the presence of C_O vibration of ligand and the formation Si-O-Si

Table 1 Positions of D, G bands, ID/IG and La of samples obtained in Raman spectra.

Fig. 5. Raman spectra of the GO, LGO, LG and LG/Ag nanocomposite.

Sample

ID/IG

La (nm)

D (cm−1)

G(cm−1)

GO LGO LG LG/Ag

1.14 1.21 1.31 1.33

3.86 3.64 3.36 3.31

1332 1327 1310 1311

1598 1597 1597 1595

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band, respectively [39]. In FT-IR of LG/Ag nanocomposite, the peak at 1727 cm− 1 was almost disappeared and the intensities of all FTIR peaks that correlated to the oxygenous groups decreased and shifted to the low energy. These changes could be related to removal of oxygenated groups or the presence of Ag NPs [40]. Raman spectroscopy is a powerful tool for investigating the structural changes that occur in graphene materials. Fig. 5 shows the Raman spectra of GO, LGO, LG and LG-Ag nanocomposite. The Raman spectra of GO displayed two characteristic peaks of the D band and the G band

at 1332 cm−1 and 1598 cm−1 which the D band is attributed to defects in the edges and arises from the vibration of sp3 while the G band related to the first of the E2g mode of sp2 bonded carbon atoms [41]. In the Raman spectra of LGO, D band shifted to lower frequencies by ∼5 cm−1 and for the LG and LG/Ag shifted by ∼20 cm−1 in relation to that of GO which could be related to increasing of defects in the edges indicating that the covalent grafting of ligand and Ag NPs to the surface of GO and also the reduction of LGO sheets causes further damage to the modified graphene surface. Table 1 shows the order of the intensity ratio of D

Fig. 6. SEM images of the LGO (a and b), LG/Ag nanocomposite (c and d) and LG sheets (e and f).

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to G band (ID/IG) of samples increased as LG/Ag N LG N LGO N GO which can be related to decreasing of mean crystallite size (La) or the variation of sp3 and sp2 bands upon the reduction of LGO to LG sheets [40,42]. The La of the samples was calculated by employing the ratio, La (nm) = 4.4 (IG/ID) whose order is consistent with the order of ID/IG ratio of samples (Table 1) [43,44]. Fig. 6 displays FESEM images of LGO, LG and LG-Ag nanocomposite. From Fig. 6, it could be understood that with the modification of GO, the reduction of LGO and the synthesis of nanocomposite, different morphologies were appeared. The SEM images of LGO show crumpled structure which is related to strict functionalization (Fig. 6a and b) [45]. In the modification process, GO sheets were attached to the surface of ligand via covalent bond to form a shell and the GO shell collapsed forming a crumpled shell around ligand molecules. Fig. 6 (c and d) shows the presence of nanoparticles on the surface of layered structure. The presence of these nanoparticles was confirmed by TEM images. With loading of Ag NPs on the surface of LG sheets via reduction process, the crumpled structure of LGO was decreased and layered structure of G was obtained which could be attributed to the removal of oxygenated groups. For comparison, the SEM images of LG sheets are displayed (Fig. 6 (e and f)). As it shows, the SEM images of LG are similar to LG/ Ag with this different that SEM images of LG doesn't have Ag nanoparticles and this result confirmed the obtaining of layered structure upon the reduction process not the existence of nanoparticles. EDX can also be used to track the modification of GO and synthesize of Ag NP on the surface of modified graphene via the analysis of the surface element contents. As shown in Fig. 7, the presence of S, C, O, and Si at LGO, LG and LG/Ag surface was approved by the signal of above elements. The presence of Si element signal confirmed the presence of silane ligand on to LGO, LG and LG/Ag nanocomposite. In EDX of LG/Ag, the signal of silver element with weight ratio (S:Ag, 1:111) confirmed the successful loading of Ag NPs on the surface of modified graphene sheets. Fig. 8 displays TEM images of G/Ag and LG/Ag nanocomposite with different magnifications. The comparison of TEM images of nanocomposites showed that in LG/Ag nanocomposite, monodispersed spherical nanoparticles (8 nm) with high particle number density have been dispersed on surface of LG layered sheets while in G/Ag nanocomposite, Ag NPs (70 nm) have been dispersed with low density on the surface of G sheets. The density of Ag NPs on the surfaces of LG and G sheets was confirmed with AES-ICP technique. The results (Table 2) showed the high density of loaded Ag NPs on LG sheets (146,704.80 ppm and also the presence of S element with concentration ratio (S:Ag: 1:100) and these confirmed the data of EDX. The interaction between Ag NPs and LGO layers is through strong covalent interaction thiol groups or through weak covalent Ag\\N or Ag\\O bonds [9] which is the main reason for the higher coverage of Ag NPs on the surface of LG. In the G/Ag nanocomposite, Ag NPs were loaded on the surface of GO sheets via week covalent interaction (Ag\\O), therefore Ag NPs were aggregated compared to that of LG/Ag nanocomposite. The SEM and TEM figures show that modification of GO with silane ligand via the formation of covalent bond, plays an important role in the preparation of monodispersed Ag-graphene nanocomposite. With the silanization of GO, from the aggregation of graphene layers and Ag NPs is prevented and also many nanoparticles on graphene layers are dispersed. The insets of Fig. 8 (b and f) show average particle size of Ag NPs dispersed on the surfaces of LG/Ag and G/Ag nanocomposites, respectively. The size of Ag NPs in G/Ag nanocomposite is between 2 and 12 nm with the maximum close to 8 nm and for G/Ag size is between 10 and 110 nm with the maximum close to 70 nm. To evaluate the antibacterial behavior of the resultant LG/Ag nanocomposite, Agar well diffusion and Broth-dilution methods were carried out. In order to compare GO, LGO, ligand/Ag and G/Ag were also tested. Fig. 9 shows optical images of bacterial colonies against E. coli (A) and S. aureus (B) corresponding to the GO, LGO, ligand/Ag, G/Ag and LG/Ag using agar well diffusion. Table 3 summarized the results of Agar well

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Fig. 7. EDX spectrum of the LGO, LG and LG/Ag nanocomposite.

diffusion for GO, LGO, ligand/Ag, G/Ag nanocomposite and LG/Ag nanocomposite. Regarding Table 3, GO and LGO showed no inhibition bacterial growth which the results of GO is similar to some previous studies but some of studies have showed strong antibacterial activities of GO against a wide of spectrum of microorganisms because of the oxidant nature and the sharp edges of GO [46]. On the other hand, no having of antibacterial activity of LGO could be related to its low dispersity due to the presence of hydrophobic groups. In contrast, ligand/Ag and both nanocomposites showed antimicrobial activities against bacteria. The comparison of all samples shows that in this research, the

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Fig. 8. TEM images of the LG/Ag nanocomposite (a–d) and G/Ag nanocomposite (e–h) with different magnifications. In the inset, the size distribution of Ag NPs in LG/Ag and G/Ag nanocomposite is shown, respectively.

antibacterial activity is related to the presence of Ag NPs. The mechanism of bactericidal effect of Ag NPs can be described that Ag NPs may attach to the surface of the cell membrane and interact with sulfur and phosphorous containing compound such as DNA because of high affinity to such compounds and then inactive the bacteria [47,48]. Since Agar well diffusion cannot distinguish bactericidal and bacteriostatic effects, so MIC and MBC of LG/Ag and G/Ag nanocomposite were

Table 2 ICP-AES data of LG/Ag and G/Ag nanocomposites. Tube

Sample labels

Element Ag

1 2 3

Blank (ppm) G/Ag (ppm) LG/Ag (ppm)

0.00 15,817.60 146,704.80

Wavelength 338.289

determined. The results show that MIC and MBC of both nanocomposites against E. coli are the same (0.6 mg/ml and 1.25 mg/ml, respectively) but different against S-aureus. The MIC and MBC values of LG/Ag are 0.6 and 1.25 mg/ml and for G/Ag are 2.5 and 5 mg/ml against S-aureus,

Table 3 Inhibition zones evaluated using the agar well diffusion method for: GO, LGO, ligand-Ag, G/Ag and LG/Ag nanocomposites at concentration of 1 g/ml. Tested sample

Element S 0.00 – 1464.62

Wavelength 181.972

GO LGO Ligand/Ag G/Ag LG/Ag

Inhibition Zone (mm) E. coli

S. aureus

No effect No effect 8 18 20

No effect No effect 11 16 23

Fig. 9. Optical images of bacterial colonies against E. coli (A) and S. aureus (B) corresponding to the GO (a), LGO (b), ligand/Ag (c), G/Ag (d) and LG/Ag (e) using agar well diffusion.

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respectively. Fig. 10 shows the bacteria growth on agar plates at different concentrations of LG/Ag (upper) and G/Ag (bottom) against E. coli (A) and S. aureus (B)·The studies show that size, shape and medium

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of Ag NPs have significant effect on their antibacterial activity [47]. In this work, smaller (8 nm) and more (146,704.8 ppm) Ag NPs were loaded on LG rather than loaded Ag NPs on G (70 nm, 15,817.6 ppm).

Fig. 10. The bacteria growth on agar plates at different concentrations of LG/Ag (upper) and G/Ag (bottom), Time-Kill curves (C and D) and Antibacterial ratios (E and F) of LG-Ag at different concentrations, E. coli (A, C and E) and S. aureus (B, D and F).

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Smaller Ag NPs with having of large surface area showed more bactericidal effect than the larger Ag NPs [47,48]. Because of the structural differences in cell walls of bacteria, small Ag NPs not only interact with surface of membrane but can also penetrate inside the S-aureus and shows higher antibacterial activity. Meanwhile, the dispersity of LG/Ag nanocomposite is lower than G/Ag and all of these reasons effect on antibacterial activity of resulted nanocomposites. So, for the synthesis of dispersive and small Ag nanoparticles with antibacterial activity it is need the presence of GO and ligand simultaneously. Further experiments were carried out to characterize the bacteria-killing performance and antibacterial kinetics of LG-Ag against E. coli and S. aureus. Fig. 10 shows time-kill curves (C and D) and antibacterial ratios (E and F) of LG-Ag nanocomposite at different concentrations. Fig. 10 (C-F) shows the significant antibacterial activity against E. coli and S. aureus at concentrations of 0.6 and 1.2 mg/ml of LG-Ag. Meanwhile, antibacterial ratio against S. aureus is lower than that against E. coli, probably due to the difference in cell walls between bacteria.

4. Conclusion In summary, a new silane ligand was used as modifier of GO and the modified GO (LGO) acted as substrate and stabilizer for Ag NPs. Ag NPs with monodispersed size (8 nm) were well dispersed on the surface of LG nanosheets. The results showed that ligand on the surface of LG sheets prevented from the agglomeration of both Ag NPs and graphene nanosheets. The resulted nanocomposite exhibited effective antibacterial activities against E. coli and S, aureus and also S, aureus showed greater resistance rather than the E. coli strains against the nanocomposite. This new modified GO can be used for stabilizing of other metal nanocomposite and a different antibacterial application.

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