Journal of Inorganic Biochemistry 105 (2011) 1181–1186

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Journal of Inorganic Biochemistry

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Green synthesis of graphene oxide sheets decorated by silver nanoprisms and theiranti-bacterial properties

Danhui Zhang, Xiaoheng Liu ⁎, Xin Wang ⁎Key Laboratory of Education, Ministry for Soft Chemistry and Functional Materials, Nanjing University of Science and Technology, Nanjing 210094, China

⁎ Corresponding authors. Tel.: +86 25 84315943; faxE-mail addresses: xhliu@mail.njust.edu.cn (X. Liu), w

(X. Wang).

0162-0134/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.jinorgbio.2011.05.014

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 February 2011Received in revised form 19 May 2011Accepted 19 May 2011Available online 27 May 2011

Keywords:Graphene oxide sheetsGelatinSilver nanoprismsGreen synthesis

A widely soluble graphene oxide sheets decorated by silver nanoprisms were prepared through greensynthesis at the room temperature using gelatin as reducing and stabilizing agent. The samples werecharacterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electronmicroscopy (TEM), UV–visible spectroscopy and fluorescence spectra. The results demonstrate that thesesilver-nanoprisms assembled on graphene oxide sheets are flexible and can form stable suspensions inaqueous solutions. Furthermore, the formation mechanism of soluble graphene oxide sheets decorated bysilver nanoprisms was successfully explained. The anti-bacterial properties of graphene oxide sheetsdecorated by silver nanoprisms were tested against Escherichia coli. This work provides a simple and “green”method for the synthesis of graphene oxide sheets decorated by silver nanoprisms in aqueous solution withpromising antibacterial property.

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© 2011 Elsevier Inc. All rights reserved.

1. Introduction

In recent years, the topic of green chemistry has been emphasized inacademic circles since it couldwork out new route for chemical productswith the process reducing or eliminating the use and generation ofhazardous substances [1].With the development of nanotechnology, theprinciples of green chemistry have been applied in synthesis andapplications of nanomaterials. In the green synthetic strategy ofnanoscale materials, two aspects including utilization of nontoxicchemicals and environmental friendly solvents have attracted consider-able attention due to their advantage in reducing the environmental risk.In this sense, biocompatible nanomaterials have received considerableattention for the promising applications in bioimaging, biosensing, anddeveloping of biomedicines [2,3]. Thus, commonly used methods in thepreparation of biocompatible nanoparticles (NPs) should be evaluatedagain in terms of green chemistry viewpoints. As we know that thegreen-synthesis standard is to choose the environmental friendlysolvents used for the synthesis, environmentally benign reducing agents,and nontoxic materials for the stabilization. The synthesis of silvernanoparticles using starch as stabilization and glucose as reducing agentsreported by Wallen et al. [4] is an excellent example of green syntheses.

Graphene, a single-atom-thick sheet of hexagonally arrayed sp2-bonded carbon atoms, has attracted significant attention from bothexperimental and theoretical fields recently [5]. Due to its uniqueelectronic properties, graphene sheets provide potential applications insynthesizing nanocomposites [6] and fabricating field-effect transistors

[7], dye-sensitized solar cells [8], lithium ion batteries [9], electromechan-ical resonators [10], and electrochemical sensors [11]. However, just as forthe other newly discovered allotropes of carbon (fullerenes and carbonnanotubes), material availability and process-ability will be the rate-limiting steps in the evaluation of putative applications of graphene. Forgraphene, that availability is encumbered by having to surmount the highcohesive van der Waals energy (5.9 kJ mol−1) adhering graphitic sheetsto one another [12]. Some methods including an epitaxial growth [13],chemical vapor deposition [14], the solvothermal reduction of grapheneoxide [15], the electrochemical reduction of graphene oxide [16], and thechemical reduction of graphene oxide [17] have been used to prepareindividual graphene sheets and to improve the properties of graphene.More recently, green synthesis of graphene has attracted considerableattention for their bio-application. For example, E.C. Salas et al. [18]showed that graphene oxidewas reduced by the bacterial respiration andP. Laaksonen et al. [19] present a method for the exfoliation andfunctionalization of graphene sheets by an amphiphilic protein.

In the past decade, many efforts have been paid in shaping themetal nanostructures, because the physical and chemical propertiesare highly dependent on their morphologies [20].The nanoprism, asone of the most important morphologies, is of unique opticalproperties [21] and has been prepared successfully by two strategies.The first strategy includes a transformation procedure resulted fromthe Ostwald ripening process driven by thermal, photochemical orchemical treatment [22]. The other choice is to synthesize nanoprismsthrough a direct chemical reduction route [23]. In both strategies, thecapping agents play an important role.

Gelatin is the thermally and hydrolytically denatured product ofcollagen, which has been extensively applied as the immobilizationmatrix for the preparation of biosensors. It has a triple-helical structure

1182 D. Zhang et al. / Journal of Inorganic Biochemistry 105 (2011) 1181–1186

and offers distinctive advantages such as good biocompatibility,nontoxicity, remarkable affinity to proteins, and excellent gel-formingability [24].

In this study, we make a use of gelatin as a reducing and stabilizingagent to prepare graphene oxide sheets decorated by silver nanoprisms.The major advantage for gelatin as a stabilizing agent is that it can beused to tailor the nanocomposite properties and also to provide long-term stability of the nanoparticles bypreventing particle agglomeration.This method did not introduce any environmental toxicity or biologicalhazards and thus was simple and “green”. X-ray diffraction (XRD),scanning electronmicroscopy (SEM), transmission electronmicroscopy(TEM), UV–vis spectroscopy andfluorescencewere used to characterizethe graphene oxide sheets decorated by silver nanoprisms. The resultsdemonstrate that these silver-nanoprisms assembled on grapheneoxide sheets are flexible and can form stable suspensions in aqueoussolutions. Moreover, the anti-bacterial activity of graphene oxide sheetsdecorated by silver nanoprisms was also displayed.

2. Experimental section

2.1. Reagents

Silver nitrate (AgNO3) was obtained from Aldrich. Graphite wasbought from Qingdao Zhongtian Company with a mean particle size of44 mm. The gelatinwas fromShanghai Chemical ReagentCo. (Shanghai,China). The other chemicals were all analytical grade, used as receivedwithout further purification, and the water was deionized.

2.2. Preparation of graphene oxide (GO)

In this work, GOwas synthesized from natural graphite powder by amodified Hummers method as originally presented by Kovtyukhovaet al. [25]. The prepared GO has oxy-functional groups such as carboxyl(−COOH), hydroxyl (−OH), and epoxy groups on its surface.

2.3. Synthesis of graphene oxide sheets decorated by silver nanoprisms

The typical procedure for the preparation of silver nanoprisms ongraphene oxide sheets is shown below: (1) A certain amount of gelatinwas completely dissolvedH2O (30 mL) undermagnetic stirring at about60 °C for about 30 min, and then cooled to room temperature. At thistime, silver nitrate (0.03 M) was added in. After stirring for 12 h, thesilver nanoprisms colloid was formed. (2) Graphite oxide powder(20 mg) was dispersed in water (30 mL) by sonication for 2 h to form astable graphene oxide colloid. Finally, the colloid (1)mixedwith colloid(2) still kept stirring at the room temperature over 12 h. The finalproduct was centrifuged (10,000 rpm for 15 min) and then vacuum-

Scheme 1. The struc

dried at 60 °C for overnight. Then the graphene oxide sheets decoratedby silver nanoprisms were formed.

2.4. Antibacterial properties study

First of all, liquid culture medium and solid culture medium werecollocated for Escherichia coli. Briefly, E. coli and 1 ppm, 5 ppm, and10 ppm of GO and Ag/GO colloidal dispersions were added to 100 mLliquid culture medium, respectively in the Erlenmeyer flask shaking inthermostat shaker at rate of 180 rps. 0.1 mL so-made bacteria–GO/Agmixture or bacteria–GO mixture was diluted with 0.9 mL. No GO/Ag orGO colloids were added to one Erlenmeyer flask containing 100 mLliquid culture medium, which was served as a control sample.Subsequently, the bacteria suspension was diluted 105 times. Afterthe serial dilution had been carried out, 0.2 mL of each bacteria–GO/Agmixture or bacteria–GO mixture was added to a Petri dish containing10 mLwarm agarmedium. One additional plate was poured containing10 mL of nutrient agar for control purposes. The plates were incubatedfor 24 h at 37 °C and then analyzed for the number of bacterial coloniesto determine the growth inhibition rates of GO/Ag or GO in accordancewith the Eq. (1)

R %ð Þ = A−Bð Þ= A × 100 ð1Þ

where R = the growth inhibition rates, A = the number of bacterialcolonies from control sample, and B = the number of bacterialcolonies from GO or GO/Ag mixture.

2.5. Characterization

UV–vis spectra were recorded on a Shimadzu UV-2500 spectro-photometer in a 1 cm optical path quartz cuvette over a 200–800 nmrange at room temperature. X-ray diffraction patterns were recordedon a Bruker D8 Advanced X-ray diffractometer using Cu Kα radiation(λ=0.1542 nm). Nanoparticle size was analyzed by TEM using a JEOL2100 transmission electron microscope operated at an acceleratingvoltage of 200 kV. Field emission scanning electron microscope(FESEM) images were obtained with a scanning electron microscopesystem (LEO-1550) operating at 5 kV. Fluorescence spectrometer(EL08013459, Cary eclipse) with a 150 W Xenon lamp as theexcitation light source was used at room temperature.

3. Results and discussion

Gelatin was used as both reducing and stabilizing agent in thesynthesis of metal nanoparticles due to its oxygen- and nitrogen-richstructures in carboxyl and amine groups, respectively (Scheme 1),

ture of gelatin.

Fig. 1. XRD patterns of composites. (a) Graphite oxide. (b) Ag/GO composites.

1183D. Zhang et al. / Journal of Inorganic Biochemistry 105 (2011) 1181–1186

which led to a very tight bond with metal clusters and nanoparticlesvia electrostatic interactions.

3.1. The XRD patterns

Fig. 1 shows the XRD patterns of graphite oxide and Ag/GOcomposites. From the Fig. 1a, we can see that the (001) diffractionpeak of graphite oxide appears around 10°. In Fig. 1b, the peaks at 38°and 44°can be assigned to the (111) and (200) planes of silver withthe face-centered cubic (fcc) structure (space group: Fm3m),respectively (JCPDS card, No. 04–0783), which indicates that thesilver nanoparticles are composed of pure crystalline silver. As weknown, according to the Scherrer equation D=k·λ /β·cos α, whenthe XRD peaks of nanoparticles are much narrower, it is suggestedthat the size of nanoparticles is big. Furthermore, we can see thatthere are only two peaks of silver are shown, this is duo to thepreferred orientation growth of silver nanoparticles. It means that thenanoparticles are not sphere but orientation along (111) facet to formthe structure of nanoprism [26]. However, no obvious diffractionpeaks of graphite oxide (Fig. 1a) are observed in the as-synthesizedcomposites. Two reasons are shown below: (1) the ratio of Ag and GOin a composite may affect the XRD patterns. We notice that thecontent of graphite oxide is relatively lower (about 20% in weight),and it is possible that the diffraction signals of silver may cover upthose of the carbon sheets. (2) Another reason of the disappearance ofgraphite oxide signals may be the exfoliation of graphite oxidethrough sonication to form the graphene oxide [27].

Fig. 2. The SEM of GO (a) and GO sheets

3.2. SEM and TEM

The micrographs of GO and graphene oxide decorated by silvernanoprisms are shown in Fig. 2. From Fig. 2a, it can be seen that themicrograph of GO is sheet with big size. As shown in Fig. 2b, it canclearly be observed that large amounts of silver nanoprismsassembled on the surface of GO.

The TEM of GO, GO sheets decorated by silver nanoprisms andsilver nanoprisms are shown in Fig. 3. In Fig. 3a, the TEM image of GOclearly illustrates that the transparent sheets are flake-like withwrinkles, which may be the key point leading to a gain in elasticenergy for the quasi-two dimension crystallite to avoid dislocationscaused by thermal fluctuations and keep a meta-stable state [28].From the Fig. 3b, we can see that silver nanoprisms coat on thegraphene oxide sheets. But the exact coating sites are the wrinkle ofgraphene oxide sheets. In order to make sure the micrograph of thesilver nanoparticles, we get the detailed views of silver nanoparticlesin Fig. 3c. It shows that the silver nanoparticles are prismwith big size,which is corresponded with the discussion of XRD.

3.3. The UV–visible (UV–vis) spectra characterization

Fig. 4 shows the UV–vis absorption spectra of GO dispersion, silvernanoprisms and graphene oxide sheets decorated by silver nanopr-isms colloids. The spectrum obtained for the GO dispersion shown inFig. 4a exhibits a maximum at 231 nm (attributed to π–π* transitionsof aromatic C–C bonds) and a shoulder at about 300 nm (ascribed ton–π* transitions of C–O bonds). In Fig. 4b, the surface plasmonresonance (SPR) band of silver nanoprisms is shown, it can be seenthat the band at about 475 nm and the value of the full width at half-maximum (fwhm) are very large. According to the Mie theory, whenthe solution system is monodispersed (narrow size distribution) thepeak shape is symmetric and the value of the full width at half-maximum (fwhm) is small. When the system is polydispersed, thepeak shape is asymmetric, which suggests that the peak actuallyconsists of two or more absorption peaks [29]. It is suggested that theformed silver nanoparticles are not sphere, but irregular shape. This iscorresponded with the TEM image (Fig. 3c). According to the Fig. 4c,after reaction, a band at 231 nm (attributed to π–π* transitions ofaromatic C–C bonds) always keep the same but the shoulder at about300 nm disappear. Moreover, the surface plasmon resonance band ofsilver nanoprisms disappear, this is mean that all the formed silvernanoprisms load on the surface of graphene oxide sheets and no freesilver nanoprisms exist in the solution. This result can be also clearlyobserved by the TEM which is shown above.

decorated by silver nanoprisms (b).

Fig. 3. The TEM of GO (a), GO sheets decorated by silver nanoprisms (b) andsilver nanoprisms (c).

Fig. 4. UV–vis absorption spectra of GO (a), silver nanoprisms (b) and GO sheetsdecorated by silver nanoprisms (c).

Fig. 5. Fluorescence spectra of gelatin (a), GO (b), gelatin-conjugated silver nanoprisms(c) and GO sheets decorated by silver nanoprisms (d).

1184 D. Zhang et al. / Journal of Inorganic Biochemistry 105 (2011) 1181–1186

3.4. Fluorescence spectrum

Fluorescence spectra excited at 320 nm of Gelatin, GO, gelatin-conjugated silver nanoprisms and GO decorated by silver nanoprismsare shown in Fig. 5. The emission peak of gelatin shown in Fig. 5a

includes only one peak at 425 nm, this belongs to the electron transferof π–π* [30]. Fig. 5b displays the emission peak of GO, it can be seenthat there is also only one peak at 525 nm [31,32]. From the emissionspectra of gelatin-conjugated Ag nanoprisms in Fig. 5c, it is clear thatthe fluorescence intensity was drastically quenched in presence of Agnanoprisms, which implies that efficiency energy transfer takes placebetween gelatin and Ag nanoprisms. Furthermore, from the emissionspectra of GO decorated by silver nanoprisms in Fig. 5d, it is alsoclearly shown that the fluorescence intensity was drasticallyquenched. It is suggested that efficiency energy transfer occursamong gelatin, GO and Ag nanoprisms.

3.5. The formation mechanism of graphene oxide sheets decorated bysilver nanoprisms

The formation mechanism of graphene oxide sheets decorated bythe silver nanoprisms is shown in Scheme 2. In this work, the silvernanoprisms were first synthesized through green method usinggelatin as reducing and stabilizing agent, so the formed silvernanoprisms were capped by the gelatin at first. Furthermore, makea comparison about the capping ability between the carboxy− andamino− of gelatin, we know that the silver nanoprisms are attachedwith the carboxy− directly. As we know, the chemically exfoliatedgraphene oxides include a variety of functional groups such as

Scheme 2. The formation mechanism of graphene oxide sheets decorated by silver nanoprisms.

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hydroxyl (C–OH) and epoxide (C–O–C) groups in addition to carbonyl(C=O) and carboxyl (−COOH) groups usually present at the defectsand edges of the sheets. Thus, when the graphene oxide was added inthe above solution, the carboxy− on the edge of graphene oxide caninteract with the amino− of gelatin, and then the silver nanoprismsare carried in the range of graphene oxide. So, the silver nanoprismsare on the wrinkle of the graphene oxide.

3.6. Anti-bacterial activity of particles

The antibacterial activity study of GO/Ag and GO were carried outagainst E. coli. Table 1 summarizes the results obtained for growthinhibition rates of the GO/Ag and GO particles against E. coli. Evidently,GO/Ag has strong anti-bacterial effect. Based on growth inhibitionrates shown in Table 1, it is clear that the growth inhibition ratesagainst E. coli 99.9% as the concentration of GO/Ag was 10 ppm, butit weakened significantly with decrease of GO/Ag concentrations;while the growth inhibition rates against E. coli was only 38% as the

Table 1Growth inhibition rates of GO/Ag and GO against Escherichia coli.

Growth inhibition rates (%)

Concentrations 1 ppm 5 ppm 10 ppm

GO/Ag 73.1 85 99.9GO 25.3 32 38

concentration of GO was 10 ppm, but changes of inhibition rates arenot very obvious with the decrease of concentration of GO [33,34].

4. Conclusions

The biomolecule of gelatin used to synthesis graphene oxidedecorated silver nanoprisms is ideal according to the criteria of greensynthesis. Accordingly, the formation of graphene oxide decorated silvernanoprisms synthesized using gelatin as reducing and protecting agent is“green”method. The results of UV–vis spectra and transmission electronmicroscopy revealed that Ag nanoprisms are well-dispersed withdifferent sizes on the surface of graphene oxide. This observation offluorescence quenching also suggests that silver nanoprisms was coatedon the surfaces of graphene oxide, making them close enough to eachother for the fluorescence resonance energy transfer. The anti-bacterialactivity study suggested that GO/Ag composites exhibits satisfactoryantibacterial property, the growth inhibition rates against E. coliwas99.9% as the concentration of GO/Ag was 10 ppm. The post-reactionGO/Ag particleswere proven to have antibacterial capabilities that renderthempotentially useful as antibacterial agents for a variety of applications.

AbbreviationsXRD X-ray diffractionTEM Transmission electron microscopyUV–vis Ultraviolet–visibleGO Graphene oxideSPR Surface plasmon resonanceFESEM Field emission scanning electron microscopefwhm Full width at half-maximum

1186 D. Zhang et al. / Journal of Inorganic Biochemistry 105 (2011) 1181–1186

Acknowledgement

This work was financially supported by the National NaturalScience Foundation of China (No. 10776014) and the High TechnicalFoundation of Jiangsu Province of China (No. BG2007047).

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