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Journal of Inorganic Biochemistry 105 (2011) 1181–1186

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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), UV–visible spectroscopy and fluorescence spectra. The results demonstrate that these silver-nanoprisms assembled on graphene oxide sheets are flexible 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 significant attention from both experimental and theoretical fields recently [5]. Due to its unique electronic properties, graphene sheets provide potential applications in synthesizing nanocomposites [6] and fabricating field-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 mol−1) 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 first 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

and 10 ppm of GO and Ag/GO colloidal dispersions were added to 100 mL liquid culture medium. Subsequently.5. X-ray diffraction (XRD). E. China). Fluorescence spectrometer (EL08013459.03 M) was added in. the silver nanoprisms colloid was formed. 2. 5 ppm. After the serial dilution had been carried out. and epoxy groups on its surface. Antibacterial properties study First of all. respectively in the Erlenmeyer flask shaking in thermostat shaker at rate of 180 rps. Field emission scanning electron microscope (FESEM) images were obtained with a scanning electron microscope system (LEO-1550) operating at 5 kV. Cary eclipse) with a 150 W Xenon lamp as the excitation light source was used at room temperature. hydroxyl (−OH). scanning electron microscopy (SEM). This method did not introduce any environmental toxicity or biological hazards and thus was simple and “green”. remarkable affinity to proteins.1 mL so-made bacteria–GO/Ag mixture or bacteria–GO mixture was diluted with 0. 3. 2. and then cooled to room temperature. One additional plate was poured containing 10 mL of nutrient agar for control purposes. 0. GO was synthesized from natural graphite powder by a modified Hummers method as originally presented by Kovtyukhova et al. the anti-bacterial activity of graphene oxide sheets decorated by silver nanoprisms was also displayed. 2.1182 D. After stirring for 12 h. In this study. used as received without further purification. silver nitrate (0. 2. we make a use of gelatin as a reducing and stabilizing agent to prepare graphene oxide sheets decorated by silver nanoprisms. Reagents Silver nitrate (AgNO3) was obtained from Aldrich.1542 nm).000 rpm for 15 min) and then vacuum- dried at 60 °C for overnight. liquid culture medium and solid culture medium were collocated for Escherichia coli. transmission electron microscopy (TEM). 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. Nanoparticle size was analyzed by TEM using a JEOL 2100 transmission electron microscope operated at an accelerating voltage of 200 kV.9 mL. Graphite was bought from Qingdao Zhongtian Company with a mean particle size of 44 mm. 2.and nitrogen-rich structures in carboxyl and amine groups. the bacteria suspension was diluted 10 5 times. [25]. which was served as a control sample. The results demonstrate that these silver-nanoprisms assembled on graphene oxide sheets are flexible and can form stable suspensions in aqueous solutions. nontoxicity. Briefly. A = the number of bacterial colonies from control sample. Finally. . Preparation of graphene oxide (GO) In this work.2. Zhang et al. Moreover. Then the graphene oxide sheets decorated by silver nanoprisms were formed. The structure of gelatin. the colloid (1) mixed with colloid (2) still kept stirring at the room temperature over 12 h. (1) Rð%Þ = ðA−BÞ = A × 100 ð1Þ where R = the growth inhibition rates.2 mL of each bacteria–GO/Ag mixture or bacteria–GO mixture was added to a Petri dish containing 10 mL warm agar medium. and B = the number of bacterial colonies from GO or GO/Ag mixture. (2) Graphite oxide powder (20 mg) was dispersed in water (30 mL) by sonication for 2 h to form a stable graphene oxide colloid. Results and discussion Gelatin was used as both reducing and stabilizing agent in the synthesis of metal nanoparticles due to its oxygen. 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. / Journal of Inorganic Biochemistry 105 (2011) 1181–1186 and offers distinctive advantages such as good biocompatibility. 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. Scheme 1. The gelatin was from Shanghai Chemical Reagent Co. (Shanghai. The other chemicals were all analytical grade. coli and 1 ppm. Experimental section 2. No GO/Ag or GO colloids were added to one Erlenmeyer flask containing 100 mL liquid culture medium. Characterization UV–vis spectra were recorded on a Shimadzu UV-2500 spectrophotometer in a 1 cm optical path quartz cuvette over a 200–800 nm range at room temperature.3. 0. UV–vis spectroscopy and fluorescence were used to characterize the graphene oxide sheets decorated by silver nanoprisms. and excellent gel-forming ability [24].4. The prepared GO has oxy-functional groups such as carboxyl (−COOH). The final product was centrifuged (10. and the water was deionized. At this time. respectively (Scheme 1). X-ray diffraction patterns were recorded on a Bruker D8 Advanced X-ray diffractometer using Cu Kα radiation (λ = 0.

we can see that the (001) diffraction peak of graphite oxide appears around 10°. 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 fluctuations and keep a meta-stable state [28]. no obvious diffraction peaks of graphite oxide (Fig. 1b. We notice that the content of graphite oxide is relatively lower (about 20% in weight). It means that the nanoparticles are not sphere but orientation along (111) facet to form the structure of nanoprism [26]. (b) Ag/GO composites. As shown in Fig. the surface plasmon resonance (SPR) band of silver nanoprisms is shown. 2. which led to a very tight bond with metal clusters and nanoparticles via electrostatic interactions. In Fig. 4c.3. This is corresponded with the TEM image (Fig. this is duo to the preferred orientation growth of silver nanoparticles. 4 shows the UV–vis absorption spectra of GO dispersion. Moreover. 3c. The spectrum obtained for the GO dispersion shown in Fig. The TEM of GO. respectively (JCPDS card. It is suggested that the formed silver nanoparticles are not sphere. In order to make sure the micrograph of the silver nanoparticles. From the Fig. 2. But the exact coating sites are the wrinkle of graphene oxide sheets. The XRD patterns Fig. but irregular shape. Two reasons are shown below: (1) the ratio of Ag and GO in a composite may affect the XRD patterns. 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. 3. 3. Fig. According to the Mie theory. which is corresponded with the discussion of XRD.1. 3a. From the Fig.D. we can see that there are only two peaks of silver are 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. In Fig. SEM and TEM The micrographs of GO and graphene oxide decorated by silver nanoprisms are shown in Fig. which indicates that the silver nanoparticles are composed of pure crystalline silver. Zhang et al. Fig. Fig. which suggests that the peak actually consists of two or more absorption peaks [29].2. and it is possible that the diffraction signals of silver may cover up those of the carbon sheets. 4a exhibits a maximum at 231 nm (attributed to π–π* transitions of aromatic C–C bonds) and a shoulder at about 300 nm (ascribed to n–π* transitions of C–O bonds). 1a) are observed in the as-synthesized composites. Furthermore. In Fig. GO sheets decorated by silver nanoprisms and silver nanoprisms are shown in Fig. 4b. silver nanoprisms and graphene oxide sheets decorated by silver nanoprisms colloids. This result can be also clearly observed by the TEM which is shown above. it can be seen that the micrograph of GO is sheet with big size. when the XRD peaks of nanoparticles are much narrower. after reaction. According to the Fig. (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]. . No. It shows that the silver nanoparticles are prism with big size. However. we can see that silver nanoprisms coat on the graphene oxide sheets. The SEM of GO (a) and GO sheets decorated by silver nanoprisms (b). From Fig. When the system is polydispersed. it can clearly be observed that large amounts of silver nanoprisms assembled on the surface of GO. 1a. 1. 1 shows the XRD patterns of graphite oxide and Ag/GO composites. we get the detailed views of silver nanoparticles in Fig. 2b. XRD patterns of composites. / Journal of Inorganic Biochemistry 105 (2011) 1181–1186 1183 3. the surface plasmon resonance band of silver nanoprisms disappear. 2a. 04–0783). the peak shape is asymmetric. according to the Scherrer equation D = k · λ / β · cos α. it is suggested that the size of nanoparticles is big. The UV–visible (UV–vis) spectra characterization 3. a band at 231 nm (attributed to π–π* transitions of aromatic C–C bonds) always keep the same but the shoulder at about 300 nm disappear. 3b. the peaks at 38° and 44°can be assigned to the (111) and (200) planes of silver with the face-centered cubic (fcc) structure (space group: Fm3m). 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. 3c). As we known. (a) Graphite oxide. the TEM image of GO clearly illustrates that the transparent sheets are flake-like with wrinkles.

3. 5c. As we know. the chemically exfoliated graphene oxides include a variety of functional groups such as Fig. GO and Ag nanoprisms. 5b displays the emission peak of GO.32]. Furthermore. The emission peak of gelatin shown in Fig. GO sheets decorated by silver nanoprisms (b) and silver nanoprisms (c). includes only one peak at 425 nm.5. From the emission spectra of gelatin-conjugated Ag nanoprisms in Fig. gelatinconjugated silver nanoprisms and GO decorated by silver nanoprisms are shown in Fig.4. GO. 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 2.1184 D. 5a Fig. 3. so the formed silver nanoprisms were capped by the gelatin at first. 4. it can be seen that there is also only one peak at 525 nm [31. The TEM of GO (a). Zhang et al. Furthermore. . Fig. 3. It is suggested that efficiency energy transfer occurs among gelatin. this belongs to the electron transfer of π–π* [30]. In this work. it is also clearly shown that the fluorescence intensity was drastically quenched. 5. GO (b). gelatin-conjugated silver nanoprisms (c) and GO sheets decorated by silver nanoprisms (d). / Journal of Inorganic Biochemistry 105 (2011) 1181–1186 Fig. UV–vis absorption spectra of GO (a). 5d. silver nanoprisms (b) and GO sheets decorated by silver nanoprisms (c). we know that the silver nanoprisms are attached with the carboxy− directly. 5. Fluorescence spectrum Fluorescence spectra excited at 320 nm of Gelatin. Fluorescence spectra of gelatin (a). which implies that efficiency energy transfer takes place between gelatin and Ag nanoprisms. it is clear that the fluorescence intensity was drastically quenched in presence of Ag nanoprisms. from the emission spectra of GO decorated by silver nanoprisms in Fig. make a comparison about the capping ability between the carboxy− and amino− of gelatin. the silver nanoprisms were first synthesized through green method using gelatin as reducing and stabilizing agent.

Abbreviations XRD X-ray diffraction TEM Transmission electron microscopy UV–vis Ultraviolet–visible GO Graphene oxide SPR Surface plasmon resonance FESEM Field emission scanning electron microscope fwhm Full width at half-maximum 3.34]. the silver nanoprisms are on the wrinkle of the graphene oxide. The anti-bacterial activity study suggested that GO/Ag composites exhibits satisfactory antibacterial property. Evidently. Based on growth inhibition rates shown in Table 1. Zhang et al.6. The formation mechanism of graphene oxide sheets decorated by silver nanoprisms. 4. Thus. GO/Ag has strong anti-bacterial effect.9 38 . coli 99. concentration of GO was 10 ppm. hydroxyl (C–OH) and epoxide (C–O–C) groups in addition to carbonyl (C=O) and carboxyl (−COOH) groups usually present at the defects and edges of the sheets. The results of UV–vis spectra and transmission electron microscopy revealed that Ag nanoprisms are well-dispersed with different sizes on the surface of graphene oxide.9% as the concentration of GO/Ag was 10 ppm. when the graphene oxide was added in the above solution. coli was 99.D. coli. Conclusions The biomolecule of gelatin used to synthesis graphene oxide decorated silver nanoprisms is ideal according to the criteria of green synthesis. it is clear that the growth inhibition rates against E. and then the silver nanoprisms are carried in the range of graphene oxide.3 5 ppm 85 32 10 ppm 99. making them close enough to each other for the fluorescence resonance energy transfer. 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. while the growth inhibition rates against E. Table 1 summarizes the results obtained for growth inhibition rates of the GO/Ag and GO particles against E. but it weakened significantly with decrease of GO/Ag concentrations. coli. So. but changes of inhibition rates are not very obvious with the decrease of concentration of GO [33. coli was only 38% as the Table 1 Growth inhibition rates of GO/Ag and GO against Escherichia coli. the formation of graphene oxide decorated silver nanoprisms synthesized using gelatin as reducing and protecting agent is “green” method.9% as the concentration of GO/Ag was 10 ppm.1 25. Growth inhibition rates (%) Concentrations GO/Ag GO 1 ppm 73. Anti-bacterial activity of particles The antibacterial activity study of GO/Ag and GO were carried out against E. This observation of fluorescence quenching also suggests that silver nanoprisms was coated on the surfaces of graphene oxide. the carboxy− on the edge of graphene oxide can interact with the amino− of gelatin. Accordingly. the growth inhibition rates against E. / Journal of Inorganic Biochemistry 105 (2011) 1181–1186 1185 Scheme 2.

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