The synthesis of citrate-modified silver nanoparticles in an aqueous suspension of graphene oxide nanosheets and their antibacterial activity

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Colloids and Surfaces B: Biointerfaces 105 (2013) 128– 136

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Colloids and Surfaces B: Biointerfaces

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The synthesis of citrate-modified silver nanoparticles in an aqueous suspensionof graphene oxide nanosheets and their antibacterial activity

Manash R. Dasa,∗, Rupak K. Sarmab, Sarat Ch. Boraha, Roopa Kumaria, Ratul Saikiab,Ashvini B. Deshmukhc, Manjusha V. Shelkec, Pinaki Senguptaa, Sabine Szuneritsd, Rabah Boukherroubd

a Materials Science Division, CSIR-North East Institute of Science and Technology, Jorhat 785006, Assam, Indiab Biotechnology Division, CSIR-North East Institute of Science and Technology, Jorhat 785006, Assam, Indiac Physical and Materials Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, Indiad Institut de Recherche Interdisciplinaire (IRI, USR-3078), Université Lille1, Parc de la Haute Borne, 50 Avenue de Halley, BP 70478, 59658 Villeneuve d’Ascq, France

a r t i c l e i n f o

Article history:Received 4 May 2012Received in revised form22 December 2012Accepted 27 December 2012Available online xxx

Keywords:Graphene oxideSilver nanoparticlesAntibacterial propertiesStaphylococcus aureusBacillus subtilis

a b s t r a c t

A composite material consisting of silver nanoparticles (Ag NPs) deposited on graphene oxide (GO)nanosheets is prepared by chemical reduction of Ag metal ions by sodium borohydride (NaBH4) in thepresence of trisodium citrate acting as a stabilizing agent to prevent agglomeration of the nanoparticles.The synthesized GO/Ag NPs composite was characterized by UV/vis spectroscopy, X-ray photoelectronspectroscopy (XPS), X-ray diffraction (XRD) and transmission electron microscopy (TEM). TEM analysisconfirmed a high density of Ag NPs on the GO nanosheets with a particle size range of 2–25 nm. The activ-ity of the GO/Ag NPs suspension as an antibacterial agent against Gram positive bacteria Staphylococcusaureus and Bacillus subtilis was investigated. The percentage of the killing bacterial colonies by Ag NPs(without GO) is found to be 96–97% while 100% of killing bacterial colonies is only obtained using GO/AgNPs suspension. Moreover, it was also observed that leakage of sugars and proteins from the cell wallof both S. aureus and B. subtilis in interaction with GO/Ag NPs suspension is higher compared to Ag NPs(without GO) and GO nanosheets.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Graphene has emerged as the most important among con-temporary nanomaterial platforms due to its excellent electrical,mechanical and chemical properties [1,2]. Various strategieshave been developed to produce graphene on a large scale.The most commonly used approach is based on the chemicaloxidation/exfoliation of graphite oxide to graphene oxide (GO)nanosheets, followed by chemical reduction to yield reducedgraphene oxide (rGO) nanosheets. However, van der Waalsinteractions of rGO nanosheets are so strong that aggregationof rGO nanosheets is difficult to prevent. Chemical function-alization of rGO has been considered as a powerful meansto prevent such aggregation. Insertion of molecules such aspolystyrene, acrylonitrile–butadiene–styrene, styrene–butadienerubbers, poly(sodium 4-styrenesulfonate) into graphene sheets isa frequently used strategy [3,4]. The presence of metal nanopar-ticles (NPs) on GO nanosheets has in addition shown to preventrestacking of these nanosheets [5]. Motivated by this property andthe general interest in metallic NPs, enormous efforts have been

∗ Corresponding author. Tel.: +91 9957178399; fax: +91 3762370011.E-mail addresses: [email protected], [email protected] (M.R. Das).

undertaken to form GO/metal NPs composite materials [6–25].The large surface area, the presence of reactive surface func-tionalities (hydroxyl, carboxylic, carbonyl, epoxide groups) andthe high water solubility of GO nanosheets are indeed promis-ing for the development of application driven composite materials[26,27]. GO/metallic NPs have shown great interest for chemi-cal sensors, energy and hydrogen storage. However GO/metallicNPs for biological applications is still in its infancy. Only a fewarticles have been published to demonstrate the interaction ofgraphene derivatives with bioorganisms [28–30]. Akhavan andGhaderi investigated the damage of the cell membrane in contactwith graphene and GO nanosheets [31]. The biocompatibility of rGOfilm was investigated by Agarwal et al. [32] and Chen et al. [28]suggesting its suitability for biomedical applications. The interac-tion of GO and rGO nanosheets with bacteria and mammalian cells,demonstrating antibacterial activity of GO and rGO nanosheetswith minimal cytotoxicity was more lately reported by Hu et al.[25]. While GO, rGO [25], and Ag nanoparticle (Ag NPs) [33–36]are studied individually for their antibacterial activity, there areonly a few reports on the antibacterial activity of GO/Ag NPs orrGO/Ag NPs composite materials [37–40]. Mostly these reports arefocused on solution-based methods to reduce silver cations on GOnanosheets [16,38,41–44,12]. Ag NPs anchored on GO nanosheetsby solution chemistry approach agglomerate easily, altering the

0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.colsurfb.2012.12.033


M.R. Das et al. / Colloids and Surfaces B: Biointerfaces 105 (2013) 128– 136 129

antibacterial properties of the resulting composite materials[42].

In this paper we show that the use of citrate capped Ag NPson GO nanosheets inhibits particles agglomeration and results inwell dispersed Ag NPs anchored onto GO nanosheets. The antibac-terial activity of the resulting composite material was investigatedagainst two Gram positive bacteria: Staphylococcus aureus andBacillus subtilis. Indeed, most of the reported investigations usingGO/Ag NPs composite materials concentrated on the antibacterialactivity against Gram negative bacteria.

2. Experimental

2.1. Materials

Graphite powder (<20 �m) was purchased from Sigma–Aldrichand used as-received. Silver nitrate (AgNO3, 99.8%, Qualigen, India),sodium borohydride (NaBH4, >99%, S.D. Fine, India), sulfuric acid(AR grade, Qualigens, India) hydrochloric acid (AR grade, Quali-gens, India), H2O2 (30%, Qualigens, India), potassium permanganate(>99%, NICE-Chemical, India), trisodium citrate (>99%, Qualigens,India), NaOH (99%, Qualigens, India), NH4OH (99%, Qualigens,India), nutrient broth (g/L, peptone 5 g, beef extract 3 g, sodiumchloride 5 g; pH 7; HiMedia, Mumbai, India) were used as-receivedwithout any further purification.

2.2. Preparation of GO and Ag NPs on GO nanosheets

GO nanosheets were prepared as described previously [42]. AgNPs were prepared by reducing AgNO3 with NaBH4 in GO–watersuspension using citrate as stabilizing agent. In a typical pro-cedure, 1 mL of homogeneous suspension of GO (5 mg/mL) wasmixed with the desired amount of aqueous AgNO3 solution(2 × 10−4, 5 × 10−4, 1 × 10−3, 2 × 10−3, 4 × 10−3, 6 × 10−3, 8 × 10−3

and 10 × 10−3 mol dm−3). Then 1 mL of an aqueous trisodiumcitrate solution (0.01 mol dm−3) was added to the reaction mix-ture and stirred for 30 min at room temperature. Freshly preparedaqueous solution of NaBH4 (1 mL, 0.01 mol dm−3) was slowly addedto the reaction mixture of AgNO3–GO suspension under vigorousstirring. Finally water was added to reach a final volume of 10 mL.The color of the reaction mixture turns from dark brown to grayand finally dark green depending on the concentration of AgNO3(see Fig. S1 in supplementary information). The reaction mixturewas stirred for another 5 h at room temperature to achieve com-plete reduction. Similarly Ag NPs (without GO) were also preparedby reducing AgNO3 with NaBH4 in aqueous medium using citrateas stabilizing agent (see supplementary information for synthesisdetails).

2.3. Characterization

Synthesis and anchoring of Ag NPs onto the GO nanosheets wasmonitored by UV/vis spectroscopy. UV/vis spectra were recordedbetween 200 and 800 nm using a Specord 200 (Analytik Zena,Germany) spectrometer. High resolution transmission electronmicroscopy (HRTEM) images were taken by a TECHNAI-T30 modelinstrument operated at an accelerating voltage of 300 kV. X-rayphotoelectron spectroscopy (XPS) measurements were carriedout with VG Micro Tech ESCA 300◦ instrument at a pressure>1 × 10−9 Torr (pass energy: 50 eV, electron take off angle: 60◦

and the overall resolution ∼0.1 eV). X-ray diffraction (XRD) mea-surement was carried out by Rigaku X-ray diffractometer (Model:ULTIMA IV, Rigaku, Japan) with Cu K� X-ray source (� = 1.54056 A)at a generator voltage of 40 kV and a generator current of 40 mAwith the scanning rate of 2◦ min−1.

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Fig. 1. UV–vis absorption spectra of GO/Ag NPs suspensions prepared in thepresence of 1 × 10−3 mol dm−3 trisodium citrate and different concentrations ofAgNO3: (a) 2 × 10−4 mol dm−3, (b) 5 × 10−4 mol dm−3, (c) 1 × 10−3 mol dm−3,(d) 2 × 10−3 mol dm−3, (e) 4 × 10−3 mol dm−3, (f) 6 × 10−3 mol dm−3, (g)8 × 10−3 mol dm−3, and (h) 1 × 10−2 mol dm−3. Reducing agent: NaBH4;concentration of GO: 0.5 mg/mL.

2.4. Antibacterial activity

The antibacterial activity of GO/Ag NPs suspensions and Ag NPs(without GO) was studied against Gram positive bacterial strains,S. aureus (MTCC 96) and B. subtilis (MTCC 441) obtained fromMicrobial Type Culture Collection, IMTECH, Chandigarh, India. Thebacterial strains were grown in nutrient broth (NB) at 30 ± 2 ◦C withcontinuous agitation at 200 rpm for 24 h. The bactericidal effect wasstudied in nutrient agar (NA) medium by using 100 �L of GO/AgNPs suspension and Ag NPs (without GO) and 60 �L of bacterialcultures (approx. 108 colony forming unit [CFU]) through agar welldiffusion assay [45]. The zone of inhibition was recorded after 24 hincubation at 35 ◦C for S. aureus and 30 ◦C for B. subtilis.

2.5. Determination of minimum inhibitory concentration andminimum bactericidal concentration

The minimum inhibitory concentration (MIC), the lowest con-centration of the applied material that can inhibit the growth ofan organism, was determined through batch cultures containingdifferent volumes of the GO/Ag NPs suspension (20, 50, 80, 100,120 and 150 �L). GO/Ag NPs suspension of different volumes wasadded to 100 mL of sterile NB taken in Erlenmeyer flask (250 mL)and was properly sonicated to prevent aggregation of the GO/AgNPs composite in sterile conditions [46]. The flasks were theninoculated with 1 mL of 24 h old bacterial suspension (approx.108 CFU) and incubated at 30 ± 2 ◦C with continuous shaking at200 rpm for 18–24 h. After the incubation period, bacterial growthwas observed by measuring optical density (OD) at 600 nm. Bac-terial culture with GO-water suspension (without Ag NPs) wasused as a control experiment. The value of OD for each concen-tration of Ag NPs was subtracted from the value of the controlfor calculation of the killing percentage. The minimum bacteri-cidal concentration (MBC) i.e. the lowest concentration of the NPswhich can kill 100% of tested bacteria was also determined fromthe same batch culture. 60 �L from each flask were inoculated tosterile NA plate and incubated at 35 ◦C for S. aureus and 30 ◦C forB. subtilis. MBC was calculated based on the viable colony countmethod. Culture plate with microbial colony with GO-water sus-pension was used as a control. Each experiment was carried out intriplicate.


130 M.R. Das et al. / Colloids and Surfaces B: Biointerfaces 105 (2013) 128– 136

Fig. 2. TEM images of Ag NPs on GO nanosheets synthesized by NaBH4 reduction process in the presence of trisodium citrate using different AgNO3 concentrations: (a)5 × 10−4 mol dm−3, (b) 1 × 10−3 mol dm−3 (c and d) 2 × 10−3 mol dm−3, (e) 4 × 10−3 mol dm−3 and (f) 8 × 10−3 mol dm−3, GO concentration is 0.5 mg/mL; (d) inset, (g and h)HRTEM images of the Ag NPs on GO nanosheets (1 × 10−3 and 2 × 10−3 mol dm−3 AgNO3); inset (h) HRTEM with fringe spacing (0.236 nm) in enlarged form; (i) SAED imageof the Ag NPs (1 × 10−3 mol dm−3 AgNO3).

2.6. Effect of GO/Ag NPs and Ag NPs (without GO) on leakage ofreducing sugars and proteins from the membrane in S. aureus andB. subtilis

150 �L of GO/Ag NPs suspension or Ag NPs (without GO) areadded to 100 mL of NB containing 108 CFU/mL of bacterial cultureand incubated at 30 ± 2 ◦C with continuous shaking at 200 rpm.1 mL of suspension treated bacterial culture was collected at every2 h interval up to 8 h. Samples were centrifuged at 12,000 rpm for5 min and the supernatants were immediately frozen at −20 ◦C.The concentrations of reducing sugars and proteins from each sam-ple were determined immediately by standard reported method[47,48]. A bacterial culture with GO suspension (without Ag NPs)was considered as control.

2.7. Bacterial growth kinetics

The bacterial growth kinetics was studied using a colorimet-ric method [49]. 60 �L of bacterial suspension was inoculated

individually in 10 mL of NB supplemented with 5 �L of differentdoses of GO/Ag NPs suspension. Then it was incubated at 30 ± 2 ◦Cwith continuous agitation at 200 rpm. Growth kinetics was deter-mined by measuring OD at 600 nm at every 1 h interval from thetime of inoculation with 3 replications.

3. Results and discussion

3.1. Characterization of GO/Ag NPs suspension

GO nanosheets were synthesized adopting the Hummersand Offeman method and characterized as reported previously[42,50,51]. The resulting GO nanosheets were decorated with AgNPs by the reduction of AgNO3 with NaBH4 in the presence oftrisodium citrate as capping agent. The ratio between AgNO3 andtrisodium citrate/NaBH4 was varied while keeping the concentra-tion of trisodium citrate and NaBH4 constant (1 × 10−3 mol dm−3).

The reduction of Ag cations by NaBH4 in the presence of citratewas monitored by UV/vis spectroscopy as shown in Fig. 1. The



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Fig. 3. C 1s XPS spectra of GO nanosheets before (a) and after deposition of Ag NPs (b); Ag 3d core level spectrum of GO after deposition of Ag NPs (c). Concentration of GO0.5 mg/mL, AgNO3 2 × 10−3 mol dm−3, reducing agent: NaBH4 in the presence of 1 × 10−3 mol dm−3 trisodium citrate.

appearance of a characteristic sharp surface plasmon band at� = 398 nm implies the formation of Ag NPs. Such a value is in theusual range of those of spherical Ag NPs for which the reported max-imum falls between 400 and 500 nm [52,53]. Addition of trisodiumcitrate as a stabilizing agent thus helped efficiently in preventingthe agglomeration of the NPs. The sharp localized surface plas-mon resonance (LSPR) peak at � = 398 nm did not shift to longerwavelengths with increasing the AgNO3 concentration as shownpreviously [42]. However, the AgNO3 concentration has a signifi-cant influence on the LSPR signal of the GO/Ag NPs composite asseen in Fig. 1 [52]. The morphology and structure of the GO/AgNPs suspensions formed from different concentrations of AgNO3were investigated by transmission electron microscopy (TEM).Fig. 2(a)–(d) shows that a high density of Ag NPs is deposited ontoGO nanosheets with their size ranges from 2 to 25 nm. The wrin-kles of the GO nanosheets (shown with arrows) are also observed,suggesting that the GO nanosheets are thin. The spherical Ag NPsgradually evolve into elongated forms for AgNO3 concentrations of4 × 10−3 and 8 × 10−3 mol dm−3 (Fig. 2e and f). The HRTEM imagesof Ag NPs embedded on the GO nanosheets are shown in Fig. 2(inserts d and h). The crystal lattice of the Ag NPs and fringes of GOnanosheets are resolved at few regions. The measured fringe latticeof Ag NPs is found to be 0.236 nm (inset of Fig. 2h). Fig. 2(i) shows

that the diffraction dots are resolved in the selected area of elec-tron diffraction (SAED) images, implying the crystalline nature ofthe Ag NPs on the GO nanosheets [8,37,38]. The results are compa-rable to those reported for the synthesis of Ag NPs on GO nanosheetsusing glucose or poly(N-vinyl-2-pyrrolidone) (PVP) as reducing andstabilizing agent [38,54].

XPS analysis of GO nanosheets before and after Ag NPs depo-sition was performed to follow the chemical changes occurringon the GO nanosheets. XPS survey spectrum of as prepared GOnanosheets shows bands at 285 and 530 eV due to C 1s and O 1s,respectively (data not shown). The C 1s XPS core level spectrum ofGO nanosheets is displayed in Fig. 3(a). It can be deconvoluted intothe three components with binding energy at about the 283.9 eVis due to C C, 285.90 is due to C C/C H and 287.81 eV is due toC O [55]. The C 1s and Ag 3d XPS core level spectra of GO afterdeposition of Ag NPs (synthesized using AgNO3 concentration of2 × 10−3 mol dm−3) are shown in Fig. 3(b) and (c). The C 1s corelevel XPS spectrum can be deconvoluted into three componentswith binding energies at 285.1, 286.7 and 288.1 eV assigned to C C,C O and C O, respectively (Fig. 3b) [56]. The intensity of the bandat 286.7 (C O) decreased which indicates the partial reduction ofGO nanosheets to rGO nanosheets. The presence of signals at 368.2and 374.1 eV due to Ag 3d3/2 and Ag 3d5/2, respectively suggests



the formation of metallic Ag on the GO nanosheets (Fig. 3c)[38].

The formation of the Ag NPs on GO nanosheets is further con-firmed by XRD analysis as shown in Fig. 4. The prominent peakat 2� values of about 38.1◦, 44.4◦, 64.8◦ and 77.3◦, correspond-ing d-spacing values of the Ag NPs are 2.36, 2.04, 1.44 and 1.23 A,respectively are assigned to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1)crystallographic planes of face-centered cubic (fcc) Ag NPs, [JCPDScard no. 04-0783]. The high intense diffraction peak observed at38.1◦, corresponding to crystalline Ag, confirms that the NPs arecomposed of pure crystalline Ag. The crystallite size of the Ag NPsis calculated by Sherrer equation using PDXL software and foundto be ∼11–20 nm which supports the TEM analysis results.

3.2. Antibacterial activity of GO/Ag NPs and Ag NPs (without GO)

The bactericidal effect of GO/Ag NPs composites using differ-ent concentrations of AgNO3 [5 × 10−4 (AgNP1), 1 × 10−3 (AgNP2),2 × 10−3 (AgNP3), 4 × 10−3 (AgNP4), and 8 × 10−3 mol dm−3

(AgNP5)] were investigated and zones of inhibition are recordedfrom 9 to 21 mm. The zones of inhibition of the S. aureus and B. sub-tilis produced by GO/Ag NPs suspensions are presented in Fig. S3.The varying diameter of zone of inhibition reflects the degree ofsusceptibility of the bacteria tested. More susceptible organismto the material exhibits a larger zone, while a smaller zone rep-resents comparatively less susceptible organism. As-synthesizedGO/Ag NPs suspensions were found to be more effective against S.aureus than B. subtilis. In S. aureus, AgNP5 showed the highest zoneof inhibition of 21 ± 1.53 mm (n = 3, n = number of experimentalresults) followed by AgNP4 (17 ± 1.15 mm), AgNP3 (14 ± 0.47 mm),AgNP2 (12 ± 1.53 mm) and AgNP1 (10 ± 0.58 mm). GO/Ag NPs com-posites synthesized using lower AgNO3 concentrations (i.e. AgNP1and AgNP2) could not inhibit the growth of B. subtilis. The B. sub-tilis exhibits maximum growth inhibition zone of (16 ± 1.53 mm)for AgNP5 followed by AgNP4 (6 ± 1.15 mm) and then AgNP3(5 ± 1.15 mm).

Antibacterial tests were also performed against the Gram posi-tive bacterium S. aureus and B. subtilis on NA agar plates at variablevolume of the GO/Ag NPs suspensions (AgNP3). The number of bac-terial colonies grown on NA plates as a function of the volume ofGO/Ag NPs suspension (AgNP3) when approximately 108 CFU wereapplied to the plates is shown in Figs. 5 and 6. The percentage of thekilling bacterial colonies with the variable volume of the GO/Ag NPssuspension (AgNP3) is presented in Table S1 (supplementary infor-mation). It is observed that 100% of the bacterial colonies werekilled at 150 �L of GO/Ag NPs suspension (AgNP3). The antibacterialactivity of Ag NPs alone i.e. without GO (prepared by NaBH4 reduc-tion of 2 × 10−3 mol dm−3 AgNO3, see supplementary informationfor synthesis and characterization) was also investigated againstGram positive bacterium S. aureus and B. subtilis on NA agar platesat variable volume of the Ag NPs (without GO). The numbers of bac-terial colonies grown on NA plates as a function of the volume ofAg NPs (without GO) when approximately 108 CFU were applied tothe plates is shown in Figs. S7 and S8 (supplementary information).The percentage of the killing bacterial colonies by Ag NPs (withoutGO) at 150 �L is found to be 96–97% (shown in Table S2 of sup-plementary information). 100% of killing bacterial colonies is onlyobtained using GO/Ag NPs suspension (AgNP3).

We have in addition investigated the MIC and MBC using GO/AgNPs (AgNP3) sample where more densely packed Ag NPs areanchored on the GO nanosheets with a size range of 2–25 nm. Val-ues are 100 �L for S. aureus and 120 �L for B. subtilis. Increasingthe amount of GO/Ag NPs beyond the MIC limit, no bacteria growthwas recorded. However, it is known that the bactericidal effect ofAg NPs is highly dependent on the initial bacterial concentration[49]. In this study, we have used 108 CFU/mL as initial bacterial

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Fig. 4. XRD of the synthesized Ag NPs on GO nanosheets using differ-ent concentrations of AgNO3 (a) 1 × 10−3 mol dm−3, (b) 2 × 10−3 mol dm−3, (c)4 × 10−3 mol dm−3, (d) 6 × 10−3 mol dm−3, and (e) 8 × 10−3 mol dm−3 in the presenceof 1 × 10−3 mol dm−3 citrate and NaBH4 as reducing agent.

concentration and recorded MBC at 150 �L for each bacteria. GO/AgNPs suspension (AgNP3) was found to be most effective in mem-brane leakage of reducing sugars in both strains of S. aureus andB. subtilis. The leakage of sugars and proteins in both S. aureusand B. subtilis in interaction with GO/Ag NPs suspension (AgNP3)compared to Ag NPs (without GO, 2 × 10−3 mol dm−3 AgNO3) andGO nanosheets taken as controls is depicted in Figs. 7 and 8. Itwas observed that with increasing the incubation time, there wasa gradual increase of sugar and protein leakage, as compared toAg NPs (without GO) and GO nanosheets alone. In both bacteria,initially, leakage of reducing sugars from cells was absent in thecontrol experiment (GO), while 9.67 and 4.39 �g/mg were obtainedfor S. aureus and B. subtilis, respectively using 150 �L of Ag NPs(without GO) and 21.33 and 11.67 �g/mg were obtained in case ofusing GO/Ag NPs suspension (AgNP3). After a regular interval of2 h, up to 8 h the leakage amount of reducing sugars was found toincrease to 21.67, 51, 61.67 and 82 �g/mg for S. aureus and 12.77,22.55, 42.03 and 55.84 �g/mg for B. subtilis using Ag NPs (withoutGO) as compared to 43.66, 69, 80 and 109 �g/mg for S. aureus and19.33, 42.66, 62 and 67.66 �g/mg for B. subtilis using GO/Ag NPssuspension (AgNP3) (Figs. 7 and 8). From this observation, it can beconcluded that the action of GO/Ag NPs suspension (AgNP3) on thecell wall of S. aureus is more effective than for B. subtilis. In case of S.aureus, 41 �g/mg extra sugar leakage was observed as compared toB. subtilis after 8 h under the same experimental conditions. More-over it was also observed that leakage of sugar from the cell wall ofboth S. aureus and B. subtilis in interaction with GO/Ag NPs (AgNP3)is higher compared to Ag NPs (without GO).

Similarly, GO/Ag NPs (AgNP3) also accelerated the leakage ofproteins through the membrane of both S. aureus and B. subtilisorganisms. Initially, protein leakage from cells in control experi-ment (only GO) was found to be 3.6 and 1.67 �g/mg for S. aureusand B. subtilis, respectively, while leakage of proteins from bacte-ria treated with Ag NPs (without GO, 2 × 10−3 mol dm−3 AgNO3)was found to be 8.37 and 2.96 �g/mg for S. aureus and B. subtilis,respectively and 11.33 and 7.07 �g/mg for S. aureus and B. subtilis,respectively using GO/Ag NPs suspension (AgNP3). The leakage of



Fig. 5. Photographs of bacterial colonies formed by Staphylococcus aureus: (1) GO (control), (2) 50 �L, (3) 80 �L, (4) 100 �L, (5) 120 �L and (6) 150 �L GO/Ag NPs suspension;sample: AgNP3.

proteins treated with Ag NPs (without GO) increased up to 16.67and 15.01 �g/mg in S. aureus and B. subtilis, respectively after 8 has compared to 21.33 and 18.41 �g/mg in S. aureus and B. subtilis,respectively after 8 h using GO/Ag NPs suspension (AgNP3). Theleakage of proteins is more important (∼3 �g/mg higher) in case of

S. aureus than B. subtilis. The leakage of proteins upon interactionwith GO/Ag NPs suspension (AgNP3) is ∼12 and ∼9 �g/mg morethan that of GO nanosheets and ∼5 and ∼3 �g/mg more than thatof Ag NPs (without GO) in case of S. aureus and B. subtilis organisms,respectively after 8 h.

Fig. 6. Photographs of bacterial colonies formed by Bacillus subtilis: (1) GO (control), (2) 50 �L, (3) 80 �L, (4) 100 �L, (5) 120 �L and (6) 150 �L GO/Ag NPs suspension; sample:AgNP3.



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Fig. 7. Leakage of sugars and proteins from Staphylococcus aureus organism (error bar: standard deviation, n = 3, n = number of experimental results). Concentration of GO:0.5 mg/mL, Ag NPs (without GO): prepared at the concentration of 2 × 10−3 mol dm−3 AgNO3 solution, GO/Ag NPs sample: AgNP3.

The GO/Ag NPs suspension (AgNP3) has considerable effect onmembrane leakage of reducing sugars and proteins compared toAg NPs (without GO) and GO nanosheets. GO/Ag NPs suspension(AgNP3) enhanced the sugar leakage up to 65.15% in S. aureus and32.69% in B. subtilis after 8 h of incubation while protein leakageenhancement was 137% and 106.85% in S. aureus and B. subtilis,respectively compared to GO nanosheets. In comparison to Ag NPs(without GO, 2 × 10−3 mol dm−3 AgNO3), GO/Ag NPs suspension(AgNP3) enhanced the sugar leakage up to 32.93% in S. aureus and21.18% in B. subtilis after 8 h of incubation while protein leakageenhancement was 27.95% in S. aureus and 22.65% in B. subtilis,respectively. Thus, the percentage of sugar and protein leakage inboth bacteria was maximal upon interaction with GO/Ag NPs sus-pension in comparison to Ag NPs (without GO) and GO nanosheetsalone. GO/Ag NPs suspension enhances more sugar and proteinleakage in S. aureus than in B. subtilis. In spite of the presence of thickpeptidiglycan, leakage of protein in both bacteria is good enough,indicating good antibacterial activity of the synthesized Ag NPs onGO nanosheets.

Both GO nanosheets and Ag NPs (without GO) show weakerantimicrobial activity compared to GO/Ag NPs toward Gram pos-itive bacteria, S. aureus and B. subtilis. 100% of killing bacterialcolonies is only obtained using GO/Ag NPs suspension due to syn-ergistic effect of Ag NPs (without GO) and GO nanosheets. Both

bacterial cell (due to the presence of phosphate groups in teichoicacids of lipid bilayer) and GO nanosheets are negatively chargedand they repel each other. GO nanosheets contains oxygen contain-ing surface functionalities such as hydroxyl, carboxylic, carbonyland epoxide groups. The hydrogen bonding between the oxygenfunctional groups of the GO nanosheets and lipid bilayer of cellmembranes facilitated to wrap GO nanosheets to bacteria cellmembrane and isolating from growth medium inhibiting themfrom taking nutrients or proliferate [31,38,57]. GO and graphenenanosheets also show good antimicrobial activity due to their sharpedge [31]. Akhavan and Ghaderi reported that graphene and GOnanowalls could damage the cell membrane by direct contact withthe bacteria [31]. Recently, it has been reported that GO and rGOnanosheets have also antibacterial activity and could inhibit thegrowth of E. coli [25]. It has been suggested that after 30 min contactwith GO/Ag NPs, both the outer and inner membranes of bacterialcells were damaged [38]. GO not only serves as a functional materialbut also can act as coating material for living cell due to its sheetlike structure that usually lacks in Ag NPs alone. GO nanosheetshave hydrophobic sp2 carbon, found to be a prominent bactericidalagent by construction of a strong interaction with the lipid bilayerof cell membranes [57]. Ma et al. also reported that the significantdecrease of the negative charges of GO/Ag NPs compared to GOnanosheets facilitated strong contact between the cell membrane

0 2 4 6 80

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Fig. 8. Leakage of sugars and proteins from Bacillus subtilis organism (error bar: standard deviation, n = 3, n = number of experimental results). Concentration of GO: 0.5 mg/mL,Ag NPs (without GO): prepared at the concentration of 2 × 10−3 mol dm−3 AgNO3 solution, GO/Ag NPs sample: AgNP3.



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AgNP4

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Fig. 9. Growth kinetics curves of (a) Staphylococcus aureus and (b) Bacillus subtilisin nutrient broth (error bar: standard deviation, n = 3, n = number of experimentalresults).

of the Gram positive bacteria (S. aureus and B. subtilis) and GO/AgNPs [38]. When Ag NPs are supported on GO nanosheets, the antimi-crobial activity increases because the GO nanosheets trapped thebacteria and the Ag NPs dissolve the outer envelope of bacterial cellwall and thereby leakage of cellular constituents, resulting in celldeath. Thus GO/Ag NPs attach on the bacteria cell wall more pro-foundly than the AgNPs (without GO), which thereby acceleratedthe bacterial cell death.

The bacterial growth kinetics was monitored in 10 mL NB sup-plemented with GO/Ag NPs (AgNP1, AgNP2, AgNP3, AgNP4 andAgNP5). It showed that the Ag NPs on GO nanosheets preparedusing different concentrations of AgNO3 caused a growth delayof S. aureus and B. subtilis. The growth kinetics studies (shown inFig. 9) revealed that the slope of the bacterial growth curve con-tinuously decreased. The growth curves included four phases: lagphase, log phase, exponential phase, and stabilization phase. How-ever, decline phases could not be determined, since we only assayedthe total numbers of bacteria, including live and dead ones. TheAg NPs activity on the bacterial growth delay follows the order asAgNP1 < AgNP2 < AgNP3 < AgNP4 < AgNP5 (Fig. 9a and b). The bac-terial growth kinetics was studied based on the value of OD value at600 nm and found that the growth kinetics increased with increas-ing the AgNO3 concentration [58]. Sondi and Salopek observed thatincreasing the concentration of Ag NPs caused a growth delay ofEscherichia coli [58]. Thus, the growth delay of S. aureus and B.subtilis with increasing concentration of the precursor (AgNO3) dur-ing the synthesis of Ag NPs on GO nanosheets indicates that the

concentration of precursor has considerable effect on bacterialgrowth [42].

4. Conclusions

The prevention of agglomeration and control of Ag NPs size syn-thesized on GO nanosheets by using trisodium citrate as stabilizingagent was demonstrated. The GO/Ag NPs suspension holds greatpromise as antimicrobial agent against Gram positive bacteria S.aureus and B. subtilis by inhibiting their growth and multiplica-tion. Using GO/Ag NPs resulted in a total (100%) killing of bacterialcolonies. The leakage of reducing sugars and proteins from cellwall of both Gram positive bacteria S. aureus and B. subtilis uponinteraction with the GO/Ag NPs suspensions has been found tobe significantly higher compared to Ag NPs (without GO) and GOnanosheets taken separately. A concise study on the mode of actionof Ag NPs demonstrated that, it can break the permeability ofbacterial outer membrane, stimulate the leakage of membrane con-stituents, resulting in cell putrefaction and death. In addition, thehigh antibacterial activity of the GO/Ag NPs suspensions suggeststhat the materials may be used as graphene-based biomaterials.

Acknowledgements

The authors thank the Ministry of Mines, Govt. of India, NewDelhi and DST New Delhi for financial support and also the Director,CSIR-North East Institute of Science and Technology, Jorhat, Indiafor the interest in this work and facilities.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2012.12.033.

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The synthesis of citrate-modified silver nanoparticles in an aqueous suspension of graphene oxide nanosheets and their antibacterial activity

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