DOI: 10.1002/cplu.201402240

Reduced Graphene Oxide Nanosheets Decorated with AuNanoparticles as an Effective Bactericide: Investigation ofBiocompatibility and Leakage of Sugars and ProteinsNajrul Hussain,[a] Animesh Gogoi,[b] Rupak K. Sarma,[b] Ponchami Sharma,[a]

Alexandre Barras,[c] Rabah Boukherroub,*[c] Ratul Saikia,[b] Pinaki Sengupta,[a] andManash R. Das*[a]

Introduction

Graphene, a two-dimensional material, has attracted tremen-dous attention from both experimental and theoretical scientif-ic viewpoints owing to its unique nanostructure and excep-tional properties. The closely packed honeycomb of the sp2-hy-bridized carbon network endows graphene with extraordinaryelectrical, thermal, and mechanical properties.[1, 2] Grapheneand its derivatives have been investigated intensively for po-tential applications in a number of areas such as bio-, electro-chemical, and chemical sensors,[3, 4] batteries,[5] supercapaci-tors,[6] dye-sensitized solar cells,[7] organic solar cells,[8] field

emission devices,[9] catalysts,[10] photocatalysts,[11] nanogenera-tors,[12] and hydrogen storage.[13]

Recently, metal nanoparticle/reduced graphene oxide (rGO)composite materials have received special interest for the de-velopment of advanced materials for utilization in differentfields. Another important aspect of the synthesis of metalnanoparticles on rGO sheets is the prevention of aggregationand restacking of the graphene sheets through p–p interac-tions. The chemical reduction approach is the most efficientand low-cost method for the synthesis of metal nanoparticle/graphene composite materials. Metal nanoparticle/rGO compo-sites are generally synthesized through the simultaneous re-duction of metallic salt and graphene oxide (GO) by using dif-ferent reducing agents such as ethylene glycol, sodium citrate,sodium borohydride (NaBH4), formaldehyde, and hydrazine hy-drate.[14] Sodium borohydride and hydrazine hydrate are thecommonly used reducing agents for the synthesis of metalnanoparticle/rGO composite materials. However, because oftheir toxicity, there is a continuous search for ecofriendly re-ducing agents to replace these reagents. Recently, ascorbicacid has drawn the special attention of researchers as an eco-friendly alternative to toxic reducing agents.[15, 16] In this regard,we have employed ascorbic acid as an ecofriendly reducingagent for the synthesis of gold nanoparticles (Au NPs) on re-duced graphene oxide (rGO) nanosheets through an in situ so-lution chemistry approach under ultrasonication. Au NPs werechosen in this study because of their potential uses in variousareas of chemistry, biology, catalysis, medicine, and nanotech-

[a] N. Hussain, P. Sharma, P. Sengupta, M. R. DasMaterials Science DivisionCSIR-North East Institute of Science and TechnologyJorhat, Assam 785006 (India)Fax: (+ 91) 376-2370011E-mail : mnshrdas@yahoo.com

[b] A. Gogoi, R. K. Sarma, R. SaikiaBiotechnology DivisionCSIR-North East Institute of Science and TechnologyJorhat, Assam 785006 (India)

[c] A. Barras, R. BoukherroubInstitut de Recherche Interdisciplinaire (IRI, USR CNRS 3078)Universit� Lille 1Parc de la Haute Borne50 Avenue de Halley, BP 7047859658 Villeneuve d’Ascq (France)E-mail : rabah.boukherroub@iri.univ-lille1.fr

Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cplu.201402240.&&Please add academic titlesof authors&&

In this article, we report the synthesis of a composite of re-duced graphene oxide nanosheets decorated with gold nano-particles (AuNPs-rGO) through an ecofriendly route, and the in-vestigation of its biocompatibility to HeLa cells and its antibac-terial activity against Gram-positive and Gram-negative bacte-ria. The composite material is synthesized by using an environ-mentally friendly reducing agent, ascorbic acid, underultrasonication. The composite material is characterized by UV/Vis spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), small-angle X-ray scattering (SAXS), high-resolution transmission electron microscopy (HRTEM), and ther-

mogravimetric analysis (TGA). HRTEM analysis reveals the for-mation of spherical Au NPs of 8–45 nm in size on the foldedrGO nanosheets, which is further confirmed by XRD and SAXSanalyses. The AuNPs-rGO shows good biocompatibility withcervix carcinoma, human (HeLa) cells, and possesses high anti-bacterial activity against two Gram-positive bacteria (Staphylo-coccus aureus and Bacillus subtilis) and two Gram-negative bac-teria (Escherichia coli and Pseudomonous aeruginosa). The bac-terial cell death is caused by the leakage of sugars and pro-teins from the cell membrane in contact with the AuNPs-rGOcomposite material.

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nology.[17] The reducing properties of ascorbic acid have beendemonstrated recently for the synthesis of noble metal/gra-phene nanocomposites from metal salts and GO.[18, 19] Iliut et al.used polyvinylpyrrolidone (PVP) to stabilize the AuNPs-rGOcomposite material.[18] In our approach, ascorbic acid plays theroles of both reducing agent and stabilizing agent. Moreover,the advantage of using ultrasonication in our method is the re-duction in the reaction time required.

The antibacterial activity of Au NPs has been explored inrecent years.[20–22] The antibacterial effect of metal NPs hasbeen attributed to their small size and large surface area,which allow them to penetrate bacterial cell walls and mem-branes, and as a result, the antimicrobial effect is directly de-pendent on the size and shape of the NPs.[23, 24] In contrast toAg nanoparticles (Ag NPs), which are used extensively as themost common antimicrobial agent in consumer and medicalproducts, there is only a limited number of reports on the anti-microbial activity of Au NPs. However, there is a high toxico-logical risk associated with the interaction of Ag NPs with bio-logical cells, as suggested by the recent literature focusing onthe risk assessment of Ag NPs.[25–28] The antibacterial activity ofAu NPs with and without polysaccharide stabilizing agents wasinvestigated against Gram-positive and Gram-negative bacteriasuch as Staphylococcus aureus (S. aureus), Streptococcus epider-midis (S. epidermis), Bacillus cereus (B. cereus), Vibrio vulnificus(V. vulnificus), Pseudomonas aeruginosa (P. aeruginosa), E. coli,Flexibacter sp. , and Klebsiella pneumoniae (K. pneumoniae). Itwas shown that Au NPs stabilized with chitosan, which con-tains free positively charged amino groups, showed enhancedantibacterial activity compared with Au NPs without stabilizingagents.[20] Azam et al. examined the antimicrobial activity of AuNPs against Gram-negative bacteria Escherichia coli (E. coli) andfound the zone of inhibition to be about 22 mm. The en-hanced bactericidal effect of the Au NPs was attributed to thelarge surface area of the nanoparticles available for interactionwith the bacteria.[21]

Recent investigations have also demonstrated the antibacte-rial activity and minimal cytotoxicity of GO and rGO nano-sheets.[29–31] Therefore, it is expected that the combination ofAu NPs and GO or rGO will have promising antibacterial activi-ty. Indeed, the antibacterial activity of AuNPs-rGO compositeshas been examined in a few reports.[31–33] He et al. found thatthe antibacterial activity of a TiO2NPs-AuNPs-rGO compositeagainst Gram-positive and Gram-negative bacteria and fungus

was higher than that of rGO, TiO2 NPs, and TiO2NPs-rGO undersolar light irradiation for 2 h. The results also showed that theTiO2NPs-AuNPs-rGO composite was more toxic to Gram-posi-tive bacteria than to Gram-negative bacteria or fungi.[31] Inde-pendently, Wang et al. reported the negligible antibacterial ac-tivity of GO decorated with Au NPs and coated with BSA andBSA-coated GO against Gram-negative E. coli bacteria.[32] Al-though the effect of the BSA coating on the observed reducedantibacterial activity was not established in this study, thisresult is somehow surprising given that the antibacterial activi-ties of both Au NPs and GO have been reported previously inseveral reports. Lin et al. exploited the photothermal propertiesof the AuNPs@Rupby/GO nanocomposite for bacteria killingunder 785 nm irradiation.[33]

The aim of our study is to evaluate the antibacterial activityof the AuNPs-rGO composite material, synthesized by thechemical reduction of Au salt and GO using ascorbic acidunder ultrasonication, against both Gram-positive and Gram-negative bacteria. Two Gram-positive bacteria (S. aureus andB. subtilis) and two Gram-negative bacteria (E. coli and P. aerugi-nosa) were used as models for this study. The leakage of themembrane constituents of the bacterial cell upon interactionwith the AuNPs-rGO composite material was also investigatedin detail.

Results and Discussion

Characterization of the AuNPs-rGO composite material

The AuNPs-rGO composite investigated in this work was syn-thesized through the simultaneous reduction of HAuCl4 andGO with ascorbic acid as a reducing agent under ultrasonica-tion, as depicted in Scheme 1. Figure 1 shows the UV/Vis spec-trum of the starting GO and AuNPs-rGO. GO displays a domi-nant peak with a maximum at 234 nm and a shoulder at302 nm attributed to p–p* and n–p* transitions of aromaticC=C and C=O bonds, respectively. Simultaneous reduction ofboth GO and HAuCl4 with ascorbic acid under ultrasonicationinduced a redshift of the peak from 234 to 265 nm owing tothe p–p* transition, whereas the peak at 302 nm caused bythe n–p* transitions disappeared completely. Moreover, the in-tensity of the absorption tail in the visible region increased sig-nificantly compared with that of GO. These results clearly indi-cate GO reduction and partial restoration of the aromatic net-

Scheme 1. Synthesis process of the AuNPs-rGO composite material.

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work. The formation of Au NPs on the rGO is evidenced fromthe presence of a characteristic surface plasmon resonance(SPR) band at 537 nm in the UV/Vis spectrum.

Figure 2 shows the Raman spectra of rGO and the AuNPs-rGO composite. The spectra display the main features of gra-phene-based materials with a D-band at approximately1359 cm�1 related to disorder in the graphitic material, a G-band at around 1584 cm�1 attributed to the Raman-active E2gphonon (in-plane optical mode), a 2 D-band at approximately2712 cm�1 and a D + D’ band at about 2933 cm�1.[34, 35] The D/Gintensity ratios of rGO and AuNPs-rGO are 1.55 and 1.09, re-spectively. The decrease in the G/D ratio can be attributed toa decrease in edge defects in the AuNPs-rGO composite ascompared to rGO. It is well known that the 2 D Raman band ofgraphene-based materials is highly sensitive to the stacking ofgraphene sheets.[34] From Raman analysis, 2 D/G values of 0.2and 0.09 can be estimated for rGO and AuNPs-rGO, respective-ly, suggesting multilayer (>4 layers) formation.[35]

X-ray photoelectron spectroscopy (XPS) was used to exam-ine the chemical composition of the AuNPs-rGO composite.The XPS survey spectrum of AuNPs-rGO reveals characteristicpeaks at 84, 284, and 532 eV attributed to Au 4f, C 1s, and O 1s,respectively, in accordance with the chemical composition ofthe nanocomposite material(Figure 3). The core-level XPSspectrum of C 1s is displayed inFigure 4 A. This can be deconvo-luted into four components withbinding energies at about 283.8(sp2-hybridized carbon), 284.6(C�H/C�C), 285.8 (C�O), 287.3(C=O), and 289.7 eV (COOH). Thespectrum is dominated by thepeak at 283.8 eV, suggesting thepartial reduction of the initial GOupon reaction with ascorbic acidunder ultrasonication. The Au 4fXPS spectrum of AuNPs-rGO ex-hibits two major peaks at83.4 eV attributed to Au 4f7/2 andat 87.0 eV attributed to Au 4f5/2,

Figure 1. UV/Vis spectra of graphene oxide (GO) and AuNPs-rGO compositematerial synthesized by chemical reduction of GO and HAuCl4 with ascorbicacid under ultrasonication.

Figure 2. Raman spectra of rGO (red) and AuNPs-rGO (black) composite ma-terial.

Figure 3. XPS survey spectrum of the AuNPs-rGO composite.

Figure 4. A) C 1s and B) Au 4f XPS core level spectra of the AuNPs-rGO composite.

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characteristic of metallic Au0, and additional shoulders at 84.6and 88.3 eV attributed to Au+ 4f7/2 and Au+ 4f5/2, respectively(Figure 4 B). The XPS result suggests that Au species are pres-ent in metallic form in the rGO nanosheets. XPS data analysisshows that the loading amount of Au NPs in the compositematerials was 0.14 %.

The XRD pattern of the AuNPs-rGO composite materialshows peaks at 2q values of about 38.208, 44.488, 64.588,77.608, and 81.988, corresponding to d-spacings of 2.35, 2.04,1.44, 1.23, and 1.17 �, which are assigned to the (111), (200),(220), (311), and (222) crystallographic planes of cubic Au NPs,respectively (JCPDS card No 004-0784) (Figure 5). The XRD

characterization further confirms the formation of Au NPs onthe rGO nanosheets. The diffractogram also indicates that thepeak of GO at the 2q value of 11.72, corresponding to the d-spacing value of 8.72 �, decreases considerably upon the for-mation of the AuNPs-rGO composite material, and a newbroad peak appears at a 2q value of approximately 248, corre-sponding to a d-spacing of 3.34 � (002 plane of rGO). The de-crease in the d-spacing value after the formation of the AuNPs-rGO composite material is probably caused by the removal ofmost of the oxygen functional groups upon GO reduction, andthe broadening of the diffraction peak may be attributed tothe formation of small rGO nanosheets. The crystallite size ofthe Au NPs was calculated by the Scherrer equation usingPDXL software, and was found to be 33 nm. The crystalline lat-tice constant value a was determined to be 4.078 �, which issupported by the literature value (JCPDS card No 04-0784).

X-ray scattering (SAXS) characterization was performed toassess the distribution of the Au NPs on the rGO nanosheets.The SAXS intensities of the Au NPs on rGO were plotted asa function of the scattering vector, Q (Figure 6). The scatteringvector Q is defined in terms of scattering angle q and wave-length l of the radiation (Q = 4p sinq/l). The SAXS analysis alsoprovides information on the particle size distribution of the AuNPs in the composite (inset of Figure 6). The average particlesize was determined to be around 38 nm. The average particlesize calculated from SAXS analysis is in good agreement withthe crystallite size (33 nm) estimated from the XRD diffracto-gram and is within the range of the TEM analysis results.

Thermogravimetric analysis (TGA) confirms the conversion ofGO to rGO nanosheets with ascorbic acid under ultrasonicationwith the deposition of Au NPs. Figure 7 shows that the mainweight loss of GO occurs around 200 8C (37.72 %), and is attrib-uted to pyrolysis of the labile oxygen-containing functionalgroups.[36] The weight loss in this region decreases up to10.31 % upon the formation of the AuNPs-rGO composite ma-terial, suggesting the reduction of these oxygen-containingfunctional groups. The TGA curves of GO and AuNPs-rGO bothdisplay weight loss above 500 8C, probably resulting from py-rolysis of the carbon skeleton.[36] Similar results were reportedfor the AuNPs-rGO composite synthesized using ascorbic acidunder microwave irradiation.[37] However, the weight losscaused by pyrolysis of oxygen-containing functional groupsobtained under microwave irradiation was 17.17 %, suggestingthat ultrasonication is more effective for the reduction of GOthan microwave irradiation in the synthesis of AuNPs-rGO com-posites.

Figure 5. XRD pattern of the AuNPs-rGO composite material.

Figure 6. SAXS curve of intensity against scattering vector (Q) of AuNPs-rGOcomposite material. Inset: particle size distribution profile of the Au NPs onrGO nanosheets.

Figure 7. TGA curves of GO and AuNPs-rGO composite material.

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The decoration of Au NPs on rGO nanosheets was furtherconfirmed by high-resolution transmission electron microscopy(HRTEM). The spherical Au NPs were well dispersed on thefolded rGO nanosheets (Figure 8). The HRTEM images showthat the particle size of the Au NPs is within the range 8–45 nm. The particle size range of the Au NPs on the rGO nano-sheets is found to be larger than that of our previously synthe-sized Au NPs on rGO nanosheets using ascorbic acid as a reduc-ing agent under microwave irradiation.[37] The crystal lattice ofthe Au NPs and fringes of rGO nanosheets are resolved ina few regions&&ok?&&. The measured fringe lattice of AuNPs corresponding to the (111) plane is found to be 0.18 nm(inset of Figure 8). The inset in Figure 8 d shows that the dif-fraction dots are resolved in the selected area of electron dif-fraction (SAED) images, suggesting the crystalline nature of theAu NPs on the rGO nanosheets.

Antibacterial activity of AuNPs-rGO composite material

In this study, we have investigated the antibacterial activity ofthe AuNPs-rGO composite material against two Gram-positivebacteria (S. aureus and B. subtilis) and two Gram-negative bac-teria (E. coli and P. aeruginosa) on NA agar plates with variousvolumes of the AuNPs-rGO suspension. The colony countmethod was adopted to investigate the antibacterial activity ofthe AuNPs-rGO composite. The bacterial colonies were grownon NA plates (�1 � 108 CFU mL�1) as a function of the volumeof AuNPs-rGO suspension (Figure 9 a–d). The killing percentageof the bacterial colonies varies with the volume of the AuNPs-rGO suspension, as depicted in Table 1. Killing of the bacterialcolonies of 100 % was achieved for S. aureus and P. aeruginosaat 250 mL of AuNPs-rGO suspension, and 99.76 % and 97.47 %

were achieved for B. subtilis andE. coli, respectively. In this study,we used 1 � 108 CFU mL�1 as theinitial bacterial concentrationand the MBC of the AuNPs-rGOsuspension calculated forS. aureus, B. Subtilis, E. Coli andP. aeruginosa was 250 mL. Wealso investigated the antibacteri-al activity of the Au NPs alone,that is, without rGO, synthesizedthrough same procedure as forthe AuNPs-rGO composite mate-rials, against two Gram-positivebacteria (S. aureus and B. subtilis)and two Gram-negative bacteria(E. coli and P. aeruginosa) on NAagar plates with different vol-umes of the Au NPs suspension,as shown in Figure S4 (Support-ing Information). The numbersof bacterial colonies grown onthe NA plates as a function ofthe volume of Au NPs alone(without rGO) with approximate-

ly 108 CFU applied to the plates are shown in Figure S4 (a)–(d)and presented in Table S1 (Supporting Information). Killing of

Figure 8. HRTEM images of Au NPs decorated on the rGO nanosheets synthesized using 1 � 10�3 mol dm�3 HAuCl4

solution with 5 mg mL�1 graphene oxide suspension through the ascorbic acid reduction process under ultrasoni-cation: a–e) HRTEM images, f) HRTEM images along with fringe spacing (0.18 nm) in enlarged form; inset ind) SAED image of the Au NPs.

Figure 9. Images of colonies formed by a) Staphylococcus aureus, b) Bacillussubtilis, c) Echerichia coli, and d) Pseaudomonas aeruginosa : 1) control,2) 25 mL, 3) 75 mL, 4) 150 mL and 5) 250 mL of AuNPs-rGO suspension.

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the bacterial colonies of 94.43 % and 93.51 % was achieved forS. aureus and B. subtilis, respectively, with 250 mL of Au NPsalone (without rGO), and values of 45.54 % and 55.43 % wereachieved for E. coli and P. aeruginosa, respectively. This indi-cates that the AuNPs-rGO composite shows more effective an-tibacterial activity than Au NPs alone (without rGO) againstboth Gram-positive and Gram-negative bacteria.

The leakage of reducing sugars in S. aureus, B. subtilis, E. coli,and P. aeruginosa upon interaction with the AuNPs-rGO aque-ous suspension was determined (as shown in Figure 10). Wealso performed a control experiment with both Au NPs alone(without GO) and rGO nanosheets. A gradual increase in theleakage of reducing sugars from all four bacterial strains upon

interaction with rGO, AuNPs, and AuNPs-rGO suspen-sion with increasing incubation time was observed.The experimental results showed that the leakage ofsugars from the bacterial cell membrane was almostnegligible at t = 0 h for rGO nanosheets and Au NPsalone (without GO). However, the leakage of the re-ducing sugars in contact with AuNPs-rGO suspensionat t = 0 h from the cells was 3.59, 3.55, 6.11, and2.51 mg mg�1 for the bacteria S. aureus, B. subtilis,E. coli, and P. aeruginosa, respectively. It reached63.38, 60.48, 62.95, and 60.58 mg mg�1 for the bacteri-

al cells of S. aureus, B. subtilis, E. coli, and P. aeruginosa, respec-tively, after 8 h. On the contrary, the leakage of the reducingsugars was found to be 55.80, 58.15, 48.52, 57.03 mg mg�1 and61.67, 59.98, 60.21, 58.02 mg mg�1 at t = 8 h for the bacterialcells of S. aureus, B. subtilis, E. coli, and P. aeruginosa in contactwith the rGO nanosheets and Au NPs alone (without rGO) sus-pension, respectively. Thus, it can be concluded that the leak-age of the reducing sugars is lower for bacterial cells in contactwith rGO and the Au NPs (without GO) suspension than forthose in contact with the AuNPs-rGO suspension. The highestsugar leakage amount was 63.38 mg mg�1 for S. aureus, and thelowest was 60.48 mg mg�1 for B. subtilis in contact with theAuNPs-rGO suspension after incubation for 8 h.

Table 1. Killing percentage with respect to different volumes of GO/Au NPs suspen-sion.

OrganismsBlank (0 mL) 25 mL 75 mL 150 mL 250 mL

No. of Visible Colonies Killing % Killing % Killing % Killing %

B. subtilis 1663 34.88 58.99 96.87 99.76S. aureus 2063 15.12 44.98 69.22 100.00E. coli 1896 29.06 55.64 90.35 97.47P. aeruginosa 2275 21.19 58.64 94.37 100.00

Figure 10. Leakage of sugars from Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa in contact with rGO, Au NPs (withoutrGO), and AuNPs-rGO (error bar : standard deviation, n = 3, n = number of experimental results).

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The leakage of proteins from the cell membrane of the bac-terial cells of S. aureus, B. subtilis, E. coli, and P. aeruginosa incontact with AuNPs-rGO was also investigated in this study(Figure 11). The release of proteins from the bacterial cellmembrane of S. aureus, B. subtilis, E. coli, and P. aeruginosa wasdetermined to be 3.05, 3.00, 6.87, and 2.67 mg mg�1, respective-ly, for cells in contact with the AuNPs-rGO suspension at the in-itial stage (t = 0 h). These values increased to 13.95, 17.65,16.48, and 11.48 mg mg�1 for S. aureus, B. subtilis, E. coli, andP. aeruginosa, respectively, after 8 h in contact with AuNPs-rGO.The highest protein leakage (17.65 mg mg�1) was observed forB. subtilis, and the lowest (11.48 mg mg�1) was found for P. aeru-ginosa. The leakage of proteins from S. aureus, B. subtilis, E. coli,and P. aeruginosa in contact with rGO nanosheets and Au NPs(without rGO) was found to be 2.01, 2.28, 2.87, and2.02 mg mg�1 and 2.68, 3, 5.82, and 2.67 mg mg�1, respectively,at t = 0 h, and reached 11.85, 12.19, 11.09, and 10.99 mg mg�1

and 13.55, 14.75, 14.82, and 11 mg mg�1, respectively, after 8 h.These results suggest that the AuNPs-rGO composite is moreeffective than both rGO nanosheets and Au NPs (without rGO)for sugar and protein leakage. Indeed, after 8 h contact, theAuNPs-rGO composite materials improved the leakage of re-ducing sugars from S. aureus, B. subtilis, E. coli, and P. Aerugino-

sa by 13.58 %, 4.0 %, 29.74 %, and 6.22 % and 2.77 %, 0.83 %,4.55, and 4.4 %, respectively, compared with rGO nanosheetsand Au NPs (without GO), respectively. Similarly, after 8 h con-tact, AuNPs-rGO improved the leakage of proteins from theS. aureus, B. subtilis, E. coli and P. Aeruginosa by 17.72 %,44.79 %, 48.60 %, and 4.45 % and 2.95 %, 19.66 %, 11.20 %, and4.36 % of, respectively, compared with rGO nanosheets and AuNPs (without GO), respectively. Notably, the leakage of reduc-ing sugars was higher than the leakage of proteins in contactwith the AuNPs-rGO suspension under similar experimentalconditions. The leakage of reducing sugars from S. aureus,B. subtilis, E. coli, and P. aeruginosa was 49.43, 42.83, 46.47, and49.10 mg mg�1 greater than that of proteins from the same bac-terial cells in contact with AuNPs-rGO for 8 h. In our previousstudy, it was clearly demonstrated that the presence of oxygenfunctionalities cause the rGO nanosheets to wrap the bacterialcell membrane owing to the formation of hydrogen bonds be-tween them.[38] This phenomenon prevents the bacterial cellfrom taking nutrients or proliferating.[29, 39, 40] It is well describedin the literature that both GO and rGO have good antibacterialactivity owing to their sharp edges.[29, 30, 39, 41] The presence ofoxygen functionalities and the 2 D sheet structure mean thatboth GO and rGO serve as coating materials for bacterial cells.

Figure 11. Leakage of protein from Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa in contact with rGO, Au NPs (withoutrGO), AuNPs-rGO (error bar : standard deviation, n = 3, n = number of experimental results).

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In addition to the physical damage of the cells and generationof Reactive Oxygen Species (ROS), wrapping of the bacteria byaggregating graphene sheets is also proposed as one of themechanisms involved in the bacterial inactivation.[42]

Au NPs alone do not show this coating property, but theydo show antibacterial activity.[20–22] Annamalai et al. investigat-ed the antibacterial activity of Au NPs biosynthesized usingthe leaf extract of Euphorbia hirta L. against E. coli, P. aerugino-sa, and K. pneumoniae, and found that very low concentrationsof Au NPs (1.25–200 mg mL�1) inhibit the growth of the bacteri-al strains.[22] Prema and Thangapandiyan also showed their ef-fective antibacterial activity against Gram-positive and Gram-negative bacteria such as S. aureus, S. epidermidis, B. cereus,V. vulnificus, E. coli, P. aeruginosa, K. pneumoniae, and Flexibactersp.[20] Thus, the AuNPs-rGO composite acts as a good coatingmaterial, but also shows antibacterial activity against the bac-terial cells of both Gram-positive (S. aureus, B. subtilis) andGram-negative (E. coli, P. aeruginosae) bacteria. AuNPs-rGOshows enhanced antibacterial activity because the rGO nano-sheets trap the bacterial cell membrane and the Au NPs dis-solve the outer envelope of the bacterial cell walls, and theconsequent leakage of the reducing sugars and proteins re-sults in cell death.

Cytotoxicity studies

The cytotoxic effects of graphene-based materials have beendescribed in several reports.[28, 36–40] For example, recent studieshave demonstrated the size- and concentration-dependentcyto- and even genotoxicity of graphene-based materials onhuman mesenchymal stem cells and spermatozoa.[43–45] Al-though there have been several investigations on the cytotox-icity of graphene-based materials on various cell lines,[30, 46–50]

there are only few reports on the biocompatibility of AuNPs-rGO composites.[51, 53] [ref. &&52?&&] For the assessment ofthe biocompatibility of AuNPs-rGO, its cytotoxicity to HeLacells was evaluated in vitro. We employed the water-solubletetrazolium salt (CCK-8), instead of the commonly used MTTassay, to evaluate the cell viability. The choice of CCK-8 wasmade on the basis of previous reports highlighting the inter-ference of carbon-based materials with cytotoxicity dyes in-cluding MTT.[53–55] For example, single-walled carbon nanotubes(SWCNTs) appeared to interact with some tetrazolium saltssuch as MTT, but not with WST-1, INT, and XTT. The interferenceof SWCNTs was not associated with the enzymatic activity butattributed to the insoluble nature of MTT-formazan.[53] Similarly,MTT assay also failed to predict the cytotoxicity of GO and rGObecause of the spontaneous reduction of MTT by these materi-als.[47] Figure 12 depicts the viability of HeLa cells determinedfrom CCK-8 assay data after 24 h exposure to different concen-trations of rGO and AuNPs-rGO. The data suggest good bio-compatibility of these materials up to 50 mg mL�1 with the rela-tive cell viability above 90 %, indicating the good biocompati-bility of rGO nanosheets and the AuNPs-rGO composite withHeLa cells under our experimental conditions. At the highestconcentration of 100 mg mL�1, the AuNPs-rGO compositeshowed a slight decrease in cell viability (�80 %). These results

are comparable to the cell viability reported for HeLa cellsusing Au NPs encapsulated in GO sheets.[51]

Conclusion

The AuNPs-rGO composite material has been synthesized suc-cessfully by using an ecofriendly and nontoxic reducing agentunder ultrasonication. Au NPs in the size range 8–45 nm weredeposited successfully on the rGO nanosheets. The compositematerial was utilized for the investigation of its antibacterialactivity against Gram-positive (S. aureus, B. subtilis) and Gram-negative (E. coli, P. aeruginosa) bacteria. The AuNPs-rGO com-posite showed an enhanced antibacterial activity against allfour bacteria compared with GO nanosheets. Total (100 %) kill-ing of the bacterial cell was observed with the AuNPs-rGOcomposite. The leakage of reducing sugars and proteins wasenhanced significantly by using the AuNPs-rGO compositecompared with Au NPs alone (without rGO) and rGO nano-sheets. Finally, it can be concluded that the rGO nanosheetstrap the bacterial cell membrane and the Au NPs break thepermeability of the outer membrane of the bacterial cell andenhance the leakage of the reducing sugars and proteins, re-sulting in cell death. The effective antibacterial activity of theAuNPs-rGO composite material combined with its good bio-compatibility hold promise for its potential application as aneffective graphene-based biomaterial. Moreover, the high sur-face area of the nanocomposite combined with the ease ofrGO surface functionalization can be exploited to design effec-tive antimicrobial formulations against resistant bacterialstrains. Indeed, the AuNPs-rGO surface can be loaded with dif-ferent bactericides and/or antibiotics to form more efficient an-tibacterial platforms.

Experimental Section

Materials

Graphite powder (<20 mm) was purchased from Sigma–Aldrichand used as received. HAuCl4 (Arrora-Matthy, India), hydrochloricacid (HCl, AR grade, Qualigens, India), sulfuric acid (H2SO4, ARgrade, Qualigens, India), ascorbic acid (>99 %, Fisher Scientific,India), hydrogen peroxide (H2O2, 30 %, Qualigens, India), potassium

Figure 12. Viability of HeLa cells determined from CCK-8 assay data after24 h exposure to different concentrations of rGO and AuNPs-rGO.

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permanganate, KMnO4 (>99 %, E-Merck, India), nutrient broth(g L�1, peptone 5 g, beef extract 3 g, sodium chloride 5 g; pH 7; Hi-Media, Mumbai, India) were used as received without any furtherpurification.

Preparation of graphene oxide nanosheets

GO nanosheets were produced from graphite powder through animproved Hummers and Offeman method. The detailed experi-mental conditions are reported in a recently published work.[56] Ahomogeneous yellow–brown suspension of GO nanosheets inwater was achieved by ultrasonication for 3 h.

Preparation of reduced graphene oxide nanosheets

The rGO nanosheets were prepared by the reduction of GO withascorbic acid under ultrasonication. The detailed experimental con-ditions are reported in our previous publication.[57]

Preparation of AuNPs-rGO composite

The AuNPs-rGO composite material was synthesized by using anecofriendly reducing agent, ascorbic acid, under ultrasonication.Briefly, water (5 mL) was added to GO water suspension (5 mL,100 g L

�1) under magnetic stirring for 10 min, and then an aqueousHAuCl4 solution (2 mL, 0.01 m) was added. The reaction mixturewas stirred for a further 30 min. Finally, 2 mL of ascorbic acid aque-ous solution (0.1 m) was added along with a balance amount ofwater to give a final suspension volume of 20 mL. The reactionmixture was then subjected to ultrasonication for 2 h. The productwas treated with 30 % H2O2 solution to remove excess ascorbicacid, and separated by centrifugation. Then, the product wasrinsed with ethanol to remove excess H2O2. Finally, it was washedwith a copious amount of deionized water and separated by cen-trifugation. The product was dried in an oven at 110 8C, and subse-quently, was used for cytotoxicity studies and antibacterial investi-gations against both Gram-positive and Gram-negative bacteria.

Preparation of Au NPs (without reduced graphene oxide)

HAuCl4 (2 mL, 0.02 m) solution was put in a 25 mL round-bottomedflask&&ok?&& and ascorbic acid (2 mL, 0.1 m) was added. Final-ly, a balance amount of water was added to give a final suspensionvolume of 20 mL. The resulting mixture was subjected to ultrasoni-cation for 2 h. The product was treated with 30 % H2O2 solution toremove excess ascorbic acid, and treated with ethanol to removeexcess H2O2.

Sample characterization

UV/Vis spectrophotometry

UV/Vis spectra were recorded in quartz cells of path length 1 cmbetween 200 and 800 nm using a UV 1800 spectrometer (Model:TCC 240A, Shimadzu Corporation, Japan).

Raman spectroscopy

Micro-Raman spectroscopy measurements were performed witha Horiba Jobin Yvon LabRam HR Micro-Raman system combinedwith a 473 nm laser diode as the excitation source. Visible lightwas focused by a 100 � objective. The scattered light was collected

by the same objective in backscattering configuration, dispersedby a monochromator (1800 mm focal length) and detected bya CCD. The sample for Raman analysis was prepared by castinga concentrated aqueous solution of rGO or AuNPs-rGO onto a sili-con substrate and drying at 100 8C for 1 h.

X-ray photoelectron spectroscopy (XPS)

XPS measurements were performed with an ESCALAB 220 XL spec-trometer from Vacuum Generators featuring a monochromatic AlKa

X-ray source (1486.6 eV) and a spherical energy analyzer operatedin constant analyzer energy (CAE) mode (CAE = 100 eV for surveyspectra and CAE = 40 eV for high-resolution spectra), using theelectromagnetic lens mode. The detection angle of the photoelec-trons was 308, as referenced to the sample surface. The Au 4f at84 eV was used to calibrate the other elements. After subtractionof the Shirley-type background, the core-level spectra were decom-posed into their components with mixed Gaussian–Lorentzian(30:70) shape lines using CasaXPS software. Quantification calcula-tions were performed using sensitivity factors supplied by PHI.

Transmission electron microscopy (TEM)

High-resolution transmission electron microscopy (HRTEM) wasperformed with a TECHNAI T30 model instrument operated at anaccelerating voltage of 200 kV.

X-ray diffraction (XRD)

XRD measurements were made with a Rigaku X-ray diffractometer(Model: ULTIMA IV, Rigaku, Japan) with a CuKa X-ray source (l=1.54056 �) at a scanning rate of 38min�1. Small-angle X-ray scatter-ing (SAXS) was also performed with the Rigaku X-ray diffractome-ter operated at 40 kV and 40 mA at a scanning rate of 18min�1.The scattered X-ray intensities were measured in the 2q range0.18–88 and processed in NANOsolver version 3.5 software.

Thermogravimetric analysis (TGA)

Thermogravimetric analysis of the samples was performed witha TA-SDT thermogravimetric analyzer (Model: Q600DT, TA Instru-ments, USA) at a heating rate of 5 8C min�1 in an Ar atmosphere.

Antibacterial activity of the AuNPs-rGO composite material

The antibacterial activities of the rGO (without Au NPs) suspensionas a control, Au NPs (without rGO), and AuNPs-rGO composite ma-terial were studied against two Gram-positive bacterial strains,S. aureus (MTCC 96) and B. subtilis (MTCC 441), and two Gram-neg-ative bacteria, E. coli (MTCC 739) and P. aeruginosa (MTCC 2453).Bacterial cultures were obtained from the Microbial Type CultureCollection, CSIR-IMTECH, Chandigarh, India. The procedure adoptedfor these experiments was the same as in our previous study.[38, 56]

Briefly, the bacterial strains were grown in nutrient broth (NB) at30�2 8C with continuous agitation at 180 rpm for 24 h. The mini-mum bactericidal concentration (MBC) was measured by applyingrGO (without Au NPs) and the AuNPs-rGO suspension against100 mL of bacterial cultures (�1 � 108 colony forming unit [CFU])on nutrient agar (NA) plates by the viable colony count method.[58]

The MBC of the AuNPs-rGO suspensions was determined followingthe guidelines of the Clinical and Laboratory Standards Institute

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considering the rGO-water (without Au NPs) suspension as a con-trol.[59] The MBC is defined as the lowest concentration of AuNPs-rGO composite material that results in a �99.9 % reduction ingrowth compared with the starting test inoculums.[60] Stock solu-tions of 20 mg mL�1 rGO, Au NPs (without rGO), and AuNPs-rGO inwater were prepared. The bacterial batch cultures were preparedin four different volumes (25, 75, 150, and 250 mL) of both AuNPs-rGO and Au NPs (without rGO) suspensions and of the rGO-water(without Au NPs) suspension as a control. The different volumes ofthe AuNPs-rGO suspension were added to 100 mL of sterile NBtaken in a 250 mL Erlenmeyer flask and then sonicated properly toprevent aggregation of the AuNPs-rGO composite material.[61] Theflasks were then inoculated with 1 mL of 24-hour-old bacterial sus-pension (�1 � 108 CFU) and incubated at 30�2 8C with continuousagitation at 200 rpm for 18–24 h. A volume of 70 mL from eachflask was inoculated to a sterile NA plate and incubated at 35 8C.Each experiment was carried out in triplicate.

The leakage of reducing sugars and proteins from the bacterial cellmembrane in contact with different volumes of the AuNPs-rGOand Au NPs (without rGO) suspensions was also investigated,adopting the same procedure as published previously by ourgroup.[38] The rGO-water (without Au NPs) was taken as a control.The concentrations of reducing sugars and proteins from eachsample were determined by standard reported methods.[62, 63]

Flasks with bacterial cultures in NB medium (108 CFU mL�1) were in-oculated separately with 150 mL of AuNPs-rGO, Au NPs (withoutrGO), and rGO-water suspensions, and incubated at 30�2 8C withcontinuous shaking at 200 rpm. Samples (1 mL) of the suspension-treated bacterial cultures were collected at 2 h intervals up to 8 h.The collected samples were centrifuged at 12,000 rpm for 5 min,and the supernatants were frozen immediately at �20 8C.

Cytotoxicity of the AuNPs-rGO composite material

Cell culture

HeLa (cervix carcinoma, human) cells were maintained in DMEMcontaining 4.5 g L�1

d-glucose and supplemented with 10 % (v/v)fetal calf serum (FCS) and penicillin/streptomycin in a 5 % CO2 at-mosphere at 37 8C. Cells were seeded into 96-well plates (1 �104 cells/100 mL media/well) 48 h before experiments.

Cytotoxicity (CCK-8) assay

Cells were seeded into 96-well plates (1 � 104 cells/100 mL media/well) 24 h before experiments. Prior to incubation, the cells werewashed with PBS. The medium was replaced with 100 mL of freshmedium containing rGO and AuNPs-rGO at different concentra-tions (2.5 to 100 mg mL�1). After 24 h incubation, the medium wasremoved and the cell viability was measured colorimetrically byperforming CCK-8 assays. The Cell Counting Kit-8 (CCK-8) allowsconvenient assays using WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodiumsalt), which produces a water-soluble formazan dye upon biore-duction in the presence of an electron carrier, 1-methoxy PMS. Thecells were incubated for 2 h with 100 mL of serum-free mediumcontaining CCK-8 solution (10 mL per 100 mL). After incubation, theabsorbance intensity of orange formazan was measured directly bya microplate reader (PHERAstar FS, BMG LABTECH GmbH, Germa-ny) at 450 nm with background subtraction at 650 nm. The experi-ment for each set of conditions was replicated three times. Thecell viability was determined as the percentage of live cells pertotal non-treated cells.

Acknowledgements

The authors thank the Department of Science and Technology,New Delhi for financial support (SR/FT/CS-136/2011 and CSIR-NEIST project No. GPP 269) and the Director, CSIR-North East In-stitute of Science and Technology, Jorhat, India for the interest inthis work and facilities. P.S. is thankful to CSIR, New Delhi. A.B.and R.B. gratefully acknowledge financial support from TheCentre National de Recherche Scientifique (CNRS), the Universit�Lille 1, and the Nord Pas de Calais region.

Keywords: antibacterial activity · cytotoxicity · gold ·nanoparticles · nanocomposites · reduced graphene oxide

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Received: July 25, 2014Published online on && &&, 0000

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FULL PAPERS

N. Hussain, A. Gogoi, R. K. Sarma,P. Sharma, A. Barras, R. Boukherroub,*R. Saikia, P. Sengupta, M. R. Das*

&& –&&

Reduced Graphene Oxide NanosheetsDecorated with Au Nanoparticles asan Effective Bactericide: Investigationof Biocompatibility and Leakage ofSugars and Proteins

Antibacterial activity: Gold nanoparti-cle/reduced graphene oxide compositesare synthesized through an ecofriendlysynthesis route and their biocompatibil-ity to cervix carcinoma, human (HeLa)cells, and antibacterial activity against

Gram-positive (Staphylococcus aureusand Bacillus subtilis) and Gram-negativebacteria (Escherichia coli and Pseudomo-nous aeruginosa) are investigated (seefigure).

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