Journal of Inorganic Biochemistry 105 (2011) 11811186

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Journal of Inorganic Biochemistry

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o

Green synthesis of graphene oxide sheets decorated by silver nanoprisms and their anti-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

a r t i c l e

i n f o

a b s t r a c t
A widely soluble graphene oxide sheets decorated by silver nanoprisms were prepared through green synthesis at the room temperature using gelatin as reducing and stabilizing agent. The samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), UVvisible spectroscopy and uorescence spectra. The results demonstrate that these silver-nanoprisms assembled on graphene oxide sheets are exible and can form stable suspensions in aqueous solutions. Furthermore, the formation mechanism of soluble graphene oxide sheets decorated by silver nanoprisms was successfully explained. The anti-bacterial properties of graphene oxide sheets decorated 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 with promising antibacterial property. 2011 Elsevier Inc. All rights reserved.

Article history: Received 21 February 2011 Received in revised form 19 May 2011 Accepted 19 May 2011 Available online 27 May 2011 Keywords: Graphene oxide sheets Gelatin Silver nanoprisms Green synthesis

1. Introduction In recent years, the topic of green chemistry has been emphasized in academic circles since it could work out new route for chemical products with the process reducing or eliminating the use and generation of hazardous substances [1]. With the development of nanotechnology, the principles of green chemistry have been applied in synthesis and applications of nanomaterials. In the green synthetic strategy of nanoscale materials, two aspects including utilization of nontoxic chemicals and environmental friendly solvents have attracted considerable attention due to their advantage in reducing the environmental risk. In this sense, biocompatible nanomaterials have received considerable attention for the promising applications in bioimaging, biosensing, and developing of biomedicines [2,3]. Thus, commonly used methods in the preparation of biocompatible nanoparticles (NPs) should be evaluated again in terms of green chemistry viewpoints. As we know that the green-synthesis standard is to choose the environmental friendly solvents used for the synthesis, environmentally benign reducing agents, and nontoxic materials for the stabilization. The synthesis of silver nanoparticles using starch as stabilization and glucose as reducing agents reported by Wallen et al. [4] is an excellent example of green syntheses. Graphene, a single-atom-thick sheet of hexagonally arrayed sp2bonded carbon atoms, has attracted signicant attention from both experimental and theoretical elds recently [5]. Due to its unique electronic properties, graphene sheets provide potential applications in synthesizing nanocomposites [6] and fabricating eld-effect transistors
Corresponding authors. Tel.: + 86 25 84315943; fax: + 86 25 84432747. E-mail addresses: xhliu@mail.njust.edu.cn (X. Liu), wxin@public1.ptt.js.cn (X. Wang). 0162-0134/$ see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2011.05.014

[7], dye-sensitized solar cells [8], lithium ion batteries [9], electromechanical resonators [10], and electrochemical sensors [11]. However, just as for the other newly discovered allotropes of carbon (fullerenes and carbon nanotubes), material availability and process-ability will be the ratelimiting steps in the evaluation of putative applications of graphene. For graphene, that availability is encumbered by having to surmount the high cohesive van der Waals energy (5.9 kJ mol1) adhering graphitic sheets to one another [12]. Some methods including an epitaxial growth [13], chemical vapor deposition [14], the solvothermal reduction of graphene oxide [15], the electrochemical reduction of graphene oxide [16], and the chemical reduction of graphene oxide [17] have been used to prepare individual graphene sheets and to improve the properties of graphene. More recently, green synthesis of graphene has attracted considerable attention for their bio-application. For example, E.C. Salas et al. [18] showed that graphene oxide was reduced by the bacterial respiration and P. Laaksonen et al. [19] present a method for the exfoliation and functionalization of graphene sheets by an amphiphilic protein. In the past decade, many efforts have been paid in shaping the metal nanostructures, because the physical and chemical properties are highly dependent on their morphologies [20].The nanoprism, as one of the most important morphologies, is of unique optical properties [21] and has been prepared successfully by two strategies. The rst strategy includes a transformation procedure resulted from the Ostwald ripening process driven by thermal, photochemical or chemical treatment [22]. The other choice is to synthesize nanoprisms through a direct chemical reduction route [23]. In both strategies, the capping agents play an important role. Gelatin is the thermally and hydrolytically denatured product of collagen, which has been extensively applied as the immobilization matrix for the preparation of biosensors. It has a triple-helical structure

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and offers distinctive advantages such as good biocompatibility, nontoxicity, remarkable afnity to proteins, and excellent gel-forming ability [24]. In this study, we make a use of gelatin as a reducing and stabilizing agent to prepare graphene oxide sheets decorated by silver nanoprisms. The major advantage for gelatin as a stabilizing agent is that it can be used to tailor the nanocomposite properties and also to provide longterm stability of the nanoparticles by preventing particle agglomeration. This method did not introduce any environmental toxicity or biological hazards and thus was simple and green. X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), UVvis spectroscopy and uorescence were used to characterize the graphene oxide sheets decorated by silver nanoprisms. The results demonstrate that these silver-nanoprisms assembled on graphene oxide sheets are exible and can form stable suspensions in aqueous solutions. Moreover, the anti-bacterial activity of graphene oxide sheets decorated by silver nanoprisms was also displayed. 2. Experimental section 2.1. Reagents Silver nitrate (AgNO3) was obtained from Aldrich. Graphite was bought from Qingdao Zhongtian Company with a mean particle size of 44 mm. The gelatin was from Shanghai Chemical Reagent Co. (Shanghai, China). The other chemicals were all analytical grade, used as received without further purication, and the water was deionized. 2.2. Preparation of graphene oxide (GO) In this work, GO was synthesized from natural graphite powder by a modied Hummers method as originally presented by Kovtyukhova et 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 on graphene oxide sheets is shown below: (1) A certain amount of gelatin was completely dissolved H2O (30 mL) under magnetic stirring at about 60 C for about 30 min, and then cooled to room temperature. At this time, silver nitrate (0.03 M) was added in. After stirring for 12 h, the silver nanoprisms colloid was formed. (2) Graphite oxide powder (20 mg) was dispersed in water (30 mL) by sonication for 2 h to form a stable graphene oxide colloid. Finally, the colloid (1) mixed with colloid (2) still kept stirring at the room temperature over 12 h. The nal product was centrifuged (10,000 rpm for 15 min) and then vacuum-

dried at 60 C for overnight. Then the graphene oxide sheets decorated by silver nanoprisms were formed. 2.4. Antibacterial properties study First of all, liquid culture medium and solid culture medium were collocated for Escherichia coli. Briey, E. coli and 1 ppm, 5 ppm, and 10 ppm of GO and Ag/GO colloidal dispersions were added to 100 mL liquid culture medium, respectively in the Erlenmeyer ask shaking in thermostat shaker at rate of 180 rps. 0.1 mL so-made bacteriaGO/Ag mixture or bacteriaGO mixture was diluted with 0.9 mL. No GO/Ag or GO colloids were added to one Erlenmeyer ask containing 100 mL liquid culture medium, which was served as a control sample. Subsequently, the bacteria suspension was diluted 10 5 times. After the serial dilution had been carried out, 0.2 mL of each bacteriaGO/Ag mixture or bacteriaGO mixture was added to a Petri dish containing 10 mL warm agar medium. One additional plate was poured containing 10 mL of nutrient agar for control purposes. The plates were incubated for 24 h at 37 C and then analyzed for the number of bacterial colonies to determine the growth inhibition rates of GO/Ag or GO in accordance with the Eq. (1) R% = AB = A 100 1

where R = the growth inhibition rates, A = the number of bacterial colonies from control sample, and B = the number of bacterial colonies from GO or GO/Ag mixture. 2.5. Characterization UVvis spectra were recorded on a Shimadzu UV-2500 spectrophotometer in a 1 cm optical path quartz cuvette over a 200800 nm range at room temperature. X-ray diffraction patterns were recorded on a Bruker D8 Advanced X-ray diffractometer using Cu K radiation ( = 0.1542 nm). Nanoparticle size was analyzed by TEM using a JEOL 2100 transmission electron microscope operated at an accelerating voltage of 200 kV. Field emission scanning electron microscope (FESEM) images were obtained with a scanning electron microscope system (LEO-1550) operating at 5 kV. Fluorescence spectrometer (EL08013459, Cary eclipse) with a 150 W Xenon lamp as the excitation light source was used at room temperature. 3. Results and discussion Gelatin was used as both reducing and stabilizing agent in the synthesis of metal nanoparticles due to its oxygen- and nitrogen-rich structures in carboxyl and amine groups, respectively (Scheme 1),

Scheme 1. The structure of gelatin.

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3.2. SEM and TEM The micrographs of GO and graphene oxide decorated by silver nanoprisms are shown in Fig. 2. From Fig. 2a, it can be seen that the micrograph of GO is sheet with big size. As shown in Fig. 2b, it can clearly be observed that large amounts of silver nanoprisms assembled on the surface of GO. The TEM of GO, GO sheets decorated by silver nanoprisms and silver nanoprisms are shown in Fig. 3. In Fig. 3a, the TEM image of GO clearly illustrates that the transparent sheets are ake-like with wrinkles, which may be the key point leading to a gain in elastic energy for the quasi-two dimension crystallite to avoid dislocations caused by thermal uctuations and keep a meta-stable state [28]. From the Fig. 3b, we can see that silver nanoprisms coat on the graphene oxide sheets. But the exact coating sites are the wrinkle of graphene oxide sheets. In order to make sure the micrograph of the silver nanoparticles, we get the detailed views of silver nanoparticles in Fig. 3c. It shows that the silver nanoparticles are prism with big size, which is corresponded with the discussion of XRD.

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

which led to a very tight bond with metal clusters and nanoparticles via electrostatic interactions. 3.3. The UVvisible (UVvis) spectra characterization 3.1. The XRD patterns Fig. 1 shows the XRD patterns of graphite oxide and Ag/GO composites. From the Fig. 1a, we can see that the (001) diffraction peak of graphite oxide appears around 10. In Fig. 1b, the peaks at 38 and 44can be assigned to the (111) and (200) planes of silver with the face-centered cubic (fcc) structure (space group: Fm3m), respectively (JCPDS card, No. 040783), which indicates that the silver nanoparticles are composed of pure crystalline silver. As we known, according to the Scherrer equation D = k / cos , when the XRD peaks of nanoparticles are much narrower, it is suggested that the size of nanoparticles is big. Furthermore, we can see that there are only two peaks of silver are shown, this is duo to the preferred orientation growth of silver nanoparticles. It means that the nanoparticles are not sphere but orientation along (111) facet to form the structure of nanoprism [26]. However, no obvious diffraction peaks of graphite oxide (Fig. 1a) are observed in the as-synthesized composites. Two reasons are shown below: (1) the ratio of Ag and GO in a composite may affect the XRD patterns. We notice that the content of graphite oxide is relatively lower (about 20% in weight), and it is possible that the diffraction signals of silver may cover up those of the carbon sheets. (2) Another reason of the disappearance of graphite oxide signals may be the exfoliation of graphite oxide through sonication to form the graphene oxide [27]. Fig. 4 shows the UVvis absorption spectra of GO dispersion, silver nanoprisms and graphene oxide sheets decorated by silver nanoprisms colloids. The spectrum obtained for the GO dispersion shown in Fig. 4a exhibits a maximum at 231 nm (attributed to * transitions of aromatic CC bonds) and a shoulder at about 300 nm (ascribed to n* transitions of CO bonds). In Fig. 4b, the surface plasmon resonance (SPR) band of silver nanoprisms is shown, it can be seen that the band at about 475 nm and the value of the full width at halfmaximum (fwhm) are very large. According to the Mie theory, when the solution system is monodispersed (narrow size distribution) the peak shape is symmetric and the value of the full width at halfmaximum (fwhm) is small. When the system is polydispersed, the peak shape is asymmetric, which suggests that the peak actually consists of two or more absorption peaks [29]. It is suggested that the formed silver nanoparticles are not sphere, but irregular shape. This is corresponded with the TEM image (Fig. 3c). According to the Fig. 4c, after reaction, a band at 231 nm (attributed to * transitions of aromatic CC bonds) always keep the same but the shoulder at about 300 nm disappear. Moreover, the surface plasmon resonance band of silver nanoprisms disappear, this is mean that all the formed silver nanoprisms load on the surface of graphene oxide sheets and no free silver nanoprisms exist in the solution. This result can be also clearly observed by the TEM which is shown above.

Fig. 2. The SEM of GO (a) and GO sheets decorated by silver nanoprisms (b).

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Fig. 4. UVvis absorption spectra of GO (a), silver nanoprisms (b) and GO sheets decorated by silver nanoprisms (c).

includes only one peak at 425 nm, this belongs to the electron transfer of * [30]. Fig. 5b displays the emission peak of GO, it can be seen that there is also only one peak at 525 nm [31,32]. From the emission spectra of gelatin-conjugated Ag nanoprisms in Fig. 5c, it is clear that the uorescence intensity was drastically quenched in presence of Ag nanoprisms, which implies that efciency energy transfer takes place between gelatin and Ag nanoprisms. Furthermore, from the emission spectra of GO decorated by silver nanoprisms in Fig. 5d, it is also clearly shown that the uorescence intensity was drastically quenched. It is suggested that efciency energy transfer occurs among gelatin, GO and Ag nanoprisms. 3.5. The formation mechanism of graphene oxide sheets decorated by silver nanoprisms The formation mechanism of graphene oxide sheets decorated by the silver nanoprisms is shown in Scheme 2. In this work, the silver nanoprisms were rst synthesized through green method using gelatin as reducing and stabilizing agent, so the formed silver nanoprisms were capped by the gelatin at rst. Furthermore, make a comparison about the capping ability between the carboxy and amino of gelatin, we know that the silver nanoprisms are attached with the carboxy directly. As we know, the chemically exfoliated graphene oxides include a variety of functional groups such as

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

3.4. Fluorescence spectrum Fluorescence spectra excited at 320 nm of Gelatin, GO, gelatinconjugated silver nanoprisms and GO decorated by silver nanoprisms are shown in Fig. 5. The emission peak of gelatin shown in Fig. 5a

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

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

hydroxyl (COH) and epoxide (COC) groups in addition to carbonyl (C=O) and carboxyl (COOH) groups usually present at the defects and edges of the sheets. Thus, when the graphene oxide was added in the above solution, the carboxy on the edge of graphene oxide can interact with the amino of gelatin, and then the silver nanoprisms are carried in the range of graphene oxide. So, the silver nanoprisms are on the wrinkle of the graphene oxide.

concentration of GO was 10 ppm, but changes of inhibition rates are not very obvious with the decrease of concentration of GO [33,34]. 4. Conclusions The biomolecule of gelatin used to synthesis graphene oxide decorated silver nanoprisms is ideal according to the criteria of green synthesis. Accordingly, the formation of graphene oxide decorated silver nanoprisms synthesized using gelatin as reducing and protecting agent is green method. The results of UVvis spectra and transmission electron microscopy revealed that Ag nanoprisms are well-dispersed with different sizes on the surface of graphene oxide. This observation of uorescence quenching also suggests that silver nanoprisms was coated on the surfaces of graphene oxide, making them close enough to each other for the uorescence resonance energy transfer. The anti-bacterial activity study suggested that GO/Ag composites exhibits satisfactory antibacterial property, the growth inhibition rates against E. coli was 99.9% as the concentration of GO/Ag was 10 ppm. The post-reaction GO/Ag particles were proven to have antibacterial capabilities that render them potentially useful as antibacterial agents for a variety of applications. Abbreviations XRD X-ray diffraction TEM Transmission electron microscopy UVvis Ultravioletvisible GO Graphene oxide SPR Surface plasmon resonance FESEM Field emission scanning electron microscope fwhm Full width at half-maximum

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

Table 1 Growth inhibition rates of GO/Ag and GO against Escherichia coli. Growth inhibition rates (%) Concentrations GO/Ag GO 1 ppm 73.1 25.3 5 ppm 85 32 10 ppm 99.9 38

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Acknowledgement This work was nancially supported by the National Natural Science Foundation of China (No. 10776014) and the High Technical Foundation of Jiangsu Province of China (No. BG2007047). References
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