silver nanocomposite for the detection of melamine

Sensors and Actuators B 181 (2013) 885–893

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Preparation of highly water dispersible functional graphene/silver nanocomposite for the detection of melamine S. Vijay Kumar a,∗ , N.M. Huang a,∗∗ , H.N. Lim b,c , M. Zainy a , I. Harrison d , C.H. Chia e a

Low Dimensional Materials Research Centre, Physics Department, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Functional Device Laboratory, Institute of Advanced Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia d School of Chemical and Environmental Engineering, Faculty of Engineering, The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia e School of Applied Physics, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bandar Baru Bangi, Selangor, Malaysia b c

a r t i c l e

i n f o

Article history: Received 28 September 2012 Received in revised form 4 February 2013 Accepted 11 February 2013 Available online xxx Keywords: Nano composites Nano particles Raman spectroscopy Graphene

a b s t r a c t A stable aqueous suspension of a functional graphene/silver (FG/Ag) nanocomposite was prepared by an environmentally friendly hydrothermal method. The precursor, functional graphene oxide (FGO), was prepared by covalent functionalisation of graphene oxide (GO) with a hydrophilic organosilane, N-(trimethoxysilylpropyl) ethylenediaminetriacetic acid trisodium salt (TETA). The attachment of functional groups on the GO surface maintained the aqueous stability of the FG/Ag nanocomposite even after the hydrothermal reduction. Field emission scanning electron microscopy (FESEM) images illustrated a uniform distribution of Ag nanoparticles on the FG surface. The surface enhanced Raman spectroscopy (SERS) activity of the nanocomposite was investigated using p-aminothiophenol (p-ATP) and melamine which can be detected as low as 2 × 10−8 and 2 × 10−7 M, respectively. The impressive water stability and the high SERS sensitivity of the FG/Ag nanocomposite make it a suitable substrate for trace analysis of a variety of drugs, additives or organic contaminants in water. The nanocomposite also showed a positive inhibition effect against the growth of Escherichia coli bacteria, eliminating the possibility of bacterial contamination of the sensor, thus prolonging the shelf-life of the sensing device. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Insertion of nanoparticles (NPs) into a graphene-based matrix is an important study for the exploration of their properties and applications. So far, graphene has been developed as a support to disperse and stabilise metal, metal oxide and semiconductor nanomaterials such as Ag, Au, Pt, Pd, ZnO, TiO2 , Co3 O4 , Fe3 O4 , CdS and ZnS [1–3]. Among these, colloidal metal Ag-NPs have attracted much attention because of their wide applications and unique properties in catalysis [4], biosensing [5], chemical sensing [6], photonics [7], electronics [8] and pharmaceuticals [9]. Their excellent antibacterial activity and low cytotoxicity to human cells favour the use of Ag-NPs in the biomedical and pharmaceutical fields [9–11]. Ag-NPs also strongly absorb and scatter light due to the excitation of localised surface plasmon resonance (SPR), which is dependent on the size, shape and surrounding dielectric environment of the Ag-NPs [12].

∗ Corresponding author. Tel.: +60 102348410. ∗∗ Corresponding author. Tel.: +60 122091008; fax: +60 379674146. E-mail addresses: [email protected] (S.V. Kumar), [email protected] (N.M. Huang). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.02.045

In recent years, Ag-NPs have been widely employed in surface enhanced Raman scattering (SERS) because of their controllable particle size and shape [13]. Generally, Ag-NPs are prepared by a variety of chemical and physical methods, which are not environmentally friendly and suffer from problems which include reproducibility and poor stability of NPs due to colloidal aggregation [14]. To overcome such drawbacks, considerable efforts have been made to prepare Ag-NPs on graphene nanosheets [15–17]. The large surface area and strong van der Waals force between graphene and Ag-NPs significantly reduce NPs aggregation, and the high interfacial interactions ensure the stability and reproducibility of the Ag-NPs [18]. These reduced graphene/silver (RG/Ag) nanocomposites are usually obtained from in situ reduction of silver salts on preformed reduced graphene oxide (RG) or decoration of RG with presynthesised Ag-NPs [15,16,19,20]. The scalable quantity of nanocomposites can be easily obtained by the reduction of graphene oxide (GO) via a chemical oxidation approach [21,22]. However, the combination of Ag-NPs and graphene is usually complicated and associated with some disadvantages, such as uneven sizes, bad dispersion of Ag-NPs and the stability of the composite in aqueous media. The chemical method also suffers from the use of hazardous or toxic reducing agents (NaBH4 , hydrazine and formaldehyde) to reduce both GO

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and Ag+ , posing environmental and health risks [23]. Moreover, the resulting composites have poor stability and reproducibility and are mostly in the form of aggregates or multilayers due to ␲–␲ stacking interactions between graphene nanosheets, in which the obtained hybrid materials have a relatively low surface area [24]. It is already well-established that the properties (chemical or physical) and the SERS enhancement factors decrease with an increasing number of graphene layers [25,26]. To maintain aqueous stability and to control the morphology of Ag-NPs on graphene, a surfactant is usually added, which passivates some SERS-active sites but also limits the pharmaceutical applications of the nanocomposite [27]. Thus, the preparation of stable, environmentally friendly, narrow dispersed Ag-NPs on monolayer graphene is desirable. In this paper, we demonstrate the preparation of a highly water dispersible functional graphene/silver (FG/Ag) nanocomposite using an environmentally friendly, one-pot hydrothermal preparation method. The precursor, functional graphene oxide (FGO), was prepared by covalent functionalisation using the water soluble organosilane N-(trimethoxysilylpropyl) ethylenediaminetriacetic acid trisodium salt (TETA). The formation of FG/Ag was accomplished by hydrothermally heating a mixture of FGO and an aqueous solution of AgNO3 at 140 ◦ C for 6 h without the introduction of other reducing agents or a surface modifier. In this approach, the FGO served as a substrate, and consequently the functional carboxyl groups attached to the surface of FGO acted as a stabiliser for the Ag-NPs. The functional groups also helped to retain the high water stability of the FG and FG/Ag nanocomposite. We further demonstrated the successful application of the nanocomposite as an SERS substrate for the detection of p-aminothiophenol (p-ATP) and melamine. The antibacterial property of the nanocomposite was tested against Escherichia coli bacteria.

2.3. Synthesis of functionalised graphene oxide

2. Experimental

2.5. SERS experiment

2.1. Materials

The SERS samples were prepared by drop casting and drying the aqueous solutions of FG/Ag and the analyte (melamine and p-ATP) on a clean glass slide followed by analysis through Raman spectra. Briefly, 50 ␮L of the FG/Ag nanocomposite (1 mg/mL) was mixed with the 200 ␮L of the analyte solution in a vial. After the mixture was shaken, it was allowed to stand for 3 h to reach the adsorption equilibrium. Then, 50 ␮L of the mixture was withdrawn using a micropipette and drop cast onto a clean glass plate and allowed to dry at 40 ◦ C for 1 h. The SERS spectra were recorded on five different spots and the highest intensity spectrum was selected for analysis.

Graphite flakes (3061, Asbury Graphite Mill Inc.), silver nitrate (AgNO3 ) (98%), potassium permanganate (98%), sodium hydroxide and p-aminothiophenol (p-ATP) (97%) were purchased from Merck Chemicals, (NJ, USA). Melamine (99%) was purchased from Acros Organics (NJ, USA). N-(trimethoxysilylpropyl) ethylenediaminetriacetic acid sodium salt (TETA) in aqueous solution (45%) was purchased from Gelest Inc. (Morrisville, PA, USA). Ammonia solution (25%), sulphuric acid (98%), phosphoric acid (98%), hydrochloric acid (HCl) and hydrogen peroxide (H2 O2 , 30%) were purchased from Systerm (Kuala Lumpur, Malaysia). All chemicals were used as received without further purification.

2.2. Graphene oxide synthesis The preparation of GO was carried out using simplified Hummers’ method [28], Briefly, H2 SO4 :H3 PO4 (320:80 mL), graphite flakes and KMnO4 (18 g) were mixed using a magnetic stirrer. After adding all the materials slowly, the one-pot mixture was left for stirring for 3 days to allow the oxidation of graphite. The colour of the mixture changed from dark purplish green to dark brown. Later, an H2 O2 solution was added to stop the oxidation process, and the colour of the mixture changed to bright yellow, indicating a high oxidation level of graphite. The graphite oxide formed was washed three times with a 1 M aqueous solution of HCl and with deionised water until a pH of 5–6 was achieved. The obtained thick solution of GO was dried at 60 ◦ C for 2 days in a hot air oven to obtain dry GO.

FGO was prepared according to the literature [29] with slight modifications as follows: 150 mg of dry GO was placed in a 500 mL three-neck round bottomed flask containing 300 mL of ethanol and dispersed by ultrasonication for 1 h. To this, 15 mL of a freshly prepared 3% aqueous solution of TETA was added dropwise under vigorous stirring. The reaction flask was placed in an oil bath and maintained at 60 ◦ C for another 16 h. After the completion of the reaction, the flask was cooled to room temperature and diluted with an excess of methanol and washed three times with a methanol and water mixture (60:40). Finally, the product was washed with acetone and dried at 80 ◦ C for 12 h to obtain a dark brown powder of FGO. 2.4. Preparation of functionalised graphene/silver nanocomposite The functionalised graphene/silver nanocomposite (FG/Ag) was prepared by the hydrothermal reduction method. Briefly, 25 mg of FGO powder was added to a 100 mL conical flask containing 40 mL of deionised water and sonicated for half an hour to obtain a homogeneous dispersion. To this solution, 10 mL of aqueous silver nitrate solution (0.001 mM) was added dropwise under stirring and stirred for 2 h at room temperature to form the FGO–Ag ion complex. The solution was transferred to a Teflon-lined autoclave and heated at 140 ◦ C for 6 h, and then allowed to cool to room temperature. The obtained black product was washed several times with deionised water and collected by high speed centrifugation. Finally, the FG/Ag nanocomposite was dried in an oven at 80 ◦ C. The general method for the synthesis of FGO and the FG/Ag nanocomposite is shown in Scheme 1. GO and FGO were also treated following the similar procedure in the absence of silver nitrate, and the reduced products are designated as RG and FG, respectively.

2.6. Antibacterial study The antibacterial activity of the samples was tested against Escherichia coli (E. coli) (Gram-negative). The bacteria (105 CFU) were inoculated in a nutrient broth and incubated with AgNPs and nanocomposite samples at five different concentrations (6.25–100 ␮g/mL) at a volume ratio of 1:1 for 4 h at 37◦ C. After the incubation, 0.1 mL of the mixture for each sample was spread on a nutrient agar plate, followed by incubation at 37◦ C for another 24 h. Control sample (sterilised distilled water) and 100 ␮g/mL nanocomposites were prepared and spread on agar plates for standard comparison. All the agar plates were visually inspected for the presence of bacterial growth and the results were recorded as digital images. 2.7. Characterisation The prepared samples were confirmed by an ultraviolet–visible (UV–vis) spectroscopy. The spectra were recorded using diluted

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Scheme 1. Preparation of the FG/Ag nanocomposite by hydrothermal reduction.

solutions of the samples in the range between 200 and 800 nm on a Specord 200 apparatus (AnalytikZena, Germany). Fourier transform infrared spectroscopy (FTIR) spectra were recorded on a Thermo Nicolet iS10 spectrophotometer (Washington, USA) using the KBr method. X-ray diffraction measurements were carried out on a Rigaku X-ray diffractometer (ULTIMA IV, Rigaku, ˚ at a generator voltJapan) with a CuK␣ X-ray source ( = 1.5405 A) age of 40 kV and a generator current of 40 mA with a scanning rate of 2◦ min−1 . Transmission electron microscopy images were taken by a Philip model instrument operated at an accelerating voltage of 120 kV. Samples for transmission electron microscopy (TEM) imaging were prepared by placing a drop of the solution sample in deionised water onto a carbon-coated Cu grid (3 nm thick, deposited on a commercial copper grid), dried in air and loaded into the electron microscope’s chamber. A JEOL JSM-6700F field emission scanning electron microscope (FESEM) was used to capture images of the samples. The thermal stability of the samples was studied on a TA Instruments TGA-Q-500 series at a heating rate of 20 ◦ C/min under a nitrogen atmosphere. The Raman spectra were recorded on a Renishaw inVia Raman microscope system. The excitation source was a 514 nm laser with power below 0.5 mW to avoid laser-induced heating of the samples.

3. Results and discussion Fig. 1 shows the digital images of sonicated aqueous suspensions of GO, RG, FGO and FG solution at a concentration of 0.5 mg/mL. The top row images were taken one hour after sonication and

Fig. 1. Aqueous solutions of GO, RG, FGO and FG at a concentration of 0.5 mg/mL.

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Fig. 2. (a) UV–vis and (b) XRD spectra of the samples.

the bottom row images of the same samples were taken after three months. The GO solution separated into two phases within a week of the stability test. The RG solution aggregated upon the removal of the oxygenated groups from GO through the hydrothermal treatment. Even after strong sonication, the RG sheets sedimented quickly and formed a thin black layer at the bottom of the bottle. On the contrary, both the FGO and FG solutions were perfectly stable even after three months. This impressive stability was attributed to the electrostatic stabilisation of large numbers of negatively charged functional carboxylate ions attached to the surfaces of the FGO and FG sheets. In the previous studies, polymers with carboxyl groups have been used as nucleation sites for the deposition of metal nanoparticles. The polymers serve as template substrates for controlling the nucleation and growth of silver nanoparticles as well as supporting matrices to stabilise the incipient metal colloids that are thermodynamically metastable [30,31]. Recently, Li et al. demonstrated that poly(styrene-co-acrylic acid) nanospheres containing carboxyl groups facilitated the attachment of silver ions onto the surfaces by ion-pairing upon reduction, which allowed the formation of uniform sites of silver nuclei that served as seeds for further growth to form Ag-NP shells on the surface of the nanospheres [32]. In this study, FGO was used as a matrix for the synthesis of FG/Ag via the hydrothermal method in the absence of stabilisers. The mechanism for the synthesis of FG/Ag is shown in Scheme 1. FGO was obtained by functionalising GO with hydrophilic silane TETA molecules. The golden brown colour of GO solution turned to dark brown for FGO due to partial deoxygenation during functionalisation. FGO contains a large number of carboxylic groups, which interact easily with Ag ions through electrostatic binding to form the FGO/Ag+ complex. The colour change of FGO from brown to greenish grey indicates the formation of the complex. The hydrothermal treatment of the complex results in simultaneous reduction of both FGO and Ag ions to form the FG/Ag nanocomposite. The reduction of FGO and Ag ions was confirmed by the UV–vis spectra, as shown in Fig. 2a. The appearance of a sharp peak for GO at 227 nm was contributed by the ␲ → ␲* transition of aromatic C C bonds. The evident red-shift to 246 nm for FGO was due to the partial restoration of electronic conjugation in the aromatic carbon structure. Functionalisation of GO with TETA increased the solution pH to alkali; under the alkaline condition, FGO underwent partial deoxygenation which resulted in the red-shift of the absorption peak [33]. For FG/Ag, the 246 nm peak was further red-shifted to 264 nm, along with the appearance of a new peak at 421 nm due to the excitation of surface plasmons of Ag-NPs [34], confirming

the formation of FG/Ag. The functional carboxylic groups acted as a platform for the attachment of Ag ions, which later nucleated and grew into Ag-NPs under the hydrothermal condition. The formed Ag-NPs were stabilised by the carboxylic groups on the FG. The electrons for the reduction of Ag ions were supplied by the surface oxygenated groups of FGO [3]. The profound stability of the FG/Ag solution was attributed to the presence of free carboxylate ions, and that of the Ag-NPs on the surface of FG, hindering the restacking of FG sheets. We observed that the FG/Ag nanocomposite powder could be easily redispersed in water by mild sonication. The strong XRD diffraction peak at 27◦ in Fig. 2b was attributed to the (0 0 2) reflection of the hexagonal graphite structure; its intensity reflects the degree of graphitisation of the carbon material. For GO, the most striking difference was the intensity and shifting of the peak to a lower angle at 2 ≈ 10◦ , indicating an increase in the ‘d’ spacing in comparison to pristine graphite. Diffraction peaks were almost entirely absent for FGO, suggesting a higher degree of disorder and complete exfoliation of graphene sheets. The negatively charged bulky functional groups on both sides of the FGO surface decreased the interaction between the sheets and thus increased the ‘d’ spacing, leading to complete exfoliation. A similar XRD pattern was also observed for the FG powder, suggesting that the sheets were completely exfoliated. In the spectrum of FG/Ag, the major diffraction peaks at 38◦ , 44◦ , 61◦ and 77◦ were assigned to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of silver with a face-centred cubic (fcc) structure, respectively. The broad diffraction peaks of Ag indicate a relatively small crystal size [35]. In Fig. 3a, the FTIR spectrum of GO shows the characteristic peaks, including O H stretching at 3400 cm−1 and C OH deformation at 1400 cm−1 . The peak around 1020 cm−1 was attributed to C O (C OH and C O C merged). Functionalisation of GO with TETA resulted in new chemical bonds, which in turn gave new absorption peaks in the FGO spectrum. The appearance of two new bands at 2925 and 2858 cm−1 were assigned to the stretching vibration of the methylene groups, whereas the band at 1218 cm−1 was associated with the C N vibration of TETA-silane molecules. The peak intensity of the C OH and C O C (epoxide) groups at 1400 and 1020 cm−1 , respectively, decreased in FGO, suggesting the attachment of TETA to GO. Meanwhile, the appearance of a peak at 694 cm−1 indicates the formation of new Si O C bond. A low intensity peak at 923 cm−1 was observed because of the presence of unreacted Si OH groups, which completely disappeared after the hydrothermal reduction that resulted in FG, forming a new Si O Si peak at 1025 cm−1 which suggests self-condensation of Si OH groups [36,37]. The typical peaks of carbonyl and ionised carboxyl groups belonging to TETA of FGO and FG appeared to have the same

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Fig. 3. (a) FTIR spectra and (b) TGA thermogram of GO, FGO, FG and the FG/Ag nanocomposite.

strength at 1628 cm−1 , respectively. This indicates that only the oxygenated groups directly attached to the surface of GO had been reduced in FG during the hydrothermal treatment. In contrast, the peak intensity of OH and carboxyl ions around 3000–3700 cm−1 was negligible in the FG/Ag nanocomposite, which was attributed to the interaction between Ag-NPs and carboxyl groups [38]. It was also observed that the interaction between the Ag-NPs and FG was

so strong that they remained attached to the surface even after washing and strong sonication. The weight loss pattern of the prepared samples was studied by TGA and is shown in Fig. 3b. The initial weight loss for all the samples below 140 ◦ C was ascribed to the elimination of adsorbed water. Apart from this, GO showed four distinctive degradation steps. The first and the second major weight losses (29%)

Fig. 4. (a) TEM image of FGO, FESEM images of (b) FG after reduction, (c) Ag-NPs decorated uniformly on FG at a low magnification and (d) FG/Ag at a high magnification.

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Fig. 5. Raman spectra of (a) GO, FGO and FG/Ag, (b) detection of p-ATP at different concentrations adsorbed on FG/Ag, (c) comparison of intensities of solid melamine, FGO, melamine adsorbed on FG and melamine adsorbed on FG/Ag and (d) detection of melamine at different concentrations adsorbed on FG/Ag.

between 145 and 210 ◦ C were caused by the decomposition of labile oxygen groups (epoxide, carboxyl, anhydride, or lactone groups), whereas the lower weight loss (11.6%), measured in the third step from 210 to 500 ◦ C, was attributed to the removal of more stable

Fig. 6. The plots of SERS intensity of p-ATP (black line) at 1077 cm−1 and melamine (red line) at 671 cm−1 vs. solution concentration. (For interpretation of the references to colour in figure legend, the reader is referred to the web version of the article.)

oxygen groups (e.g. phenol, carbonyl and quinone), and the last step above 600 ◦ C was due to the degradation of the carbon skeleton (data not shown). Compared to the curve of GO, the weight loss of FGO was different and showed higher thermal stability than GO. The first degradation step of FGO appeared almost in the similar range to that of GO (145–173 ◦ C), whereas the second degradation step appeared at a much higher temperature of 365–415 ◦ C. During the functionalisation of GO with TETA, the labile oxygen groups underwent chemical reaction to form a strong covalent bond with the silane groups of TETA, which considerably decreased the amount of labile groups in FGO. Thus, very low weight loss was observed at around 145–173 ◦ C in FGO. This result is also supported by the decrease in the FTIR peak intensity of C O C at 1020 cm−1 (see Fig. 3a). The weight-loss region from 365 ◦ C to 450 ◦ C is caused by the pyrolysis of the silane moieties of FGO [36]. At 500 ◦ C, GO and FGO showed 50% and 40% weight loss, respectively, which supports the partial reduction of FGO during heating. The weight loss of hydrothermally reduced FG was very negligible in the range of 145–210 ◦ C, indicating the complete reduction of labile oxygenated functional groups. However, there was still a significant mass loss between 220 and 365 ◦ C, suggesting that the reduction process could not remove highly stable functional groups. A gradual single-step weight loss was observed from 365 to 500 ◦ C due to the degradation of functional TETA. Compared to both GO and FGO, FG showed higher stability and lower weight loss (20%) at

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Fig. 7. Digital photographs of colonies of E. coli grown on agar plates obtained from incubated suspensions with (a) water (control), (b) GO, (c) FGO and (d) FG/Ag.

500 ◦ C. In the case of the FG/Ag nanocomposite, the weight loss of the sample was greatly restricted. The reason is that the FG imposed a restriction on the mobilisation of silver nanoparticles, resulting in homogeneous heating and the avoidance of heat concentration. This also supports a strong interaction between FG and Ag-NPs. The TEM image of FGO shows a smooth and transparent surface in Fig. 4a. The morphology remained the same after the hydrothermal treatment which produced FG, as shown in Fig. 4b. The FESEM images of FG/Ag nanocomposites in Fig. 4c and d illustrate uniformly distributed Ag-NPs on the graphene sheets. The average size of the nanoparticles measured from the FESEM image is around 30–50 nm. Raman spectroscopy has been accepted as a very versatile and purely optical technique for the characterisation of graphitic materials. In Fig. 5a, the Raman spectra of GO, FGO and FG/Ag show two characteristic peaks, namely the D band and the G band. The D band is ascribed to defects in the edges due to the vibration of sp3 bonded carbon atoms and disordered carbon, whereas the G band arises from the zone centre E2 g mode, corresponding to ordered sp2 -bonded carbon atoms. In the spectrum, the D band appeared at 1350 cm−1 and the G band at 1597 cm−1 , and the intensity ratio (ID /IG ) of D band to G band for GO is about 0.96, which is much higher than that of 0.10 in graphite (see Fig. S2 in the supplementary information). This is an indication of disorder, originating from defects associated with vacancies, edge defects and amorphous carbon species [39]. Functionalisation of GO (FGO) with TETA further increased the intensity of the D band and the ID /IG ratio to 1.15. The prominent D band in FGO was attributed to increased sp3 bonds due to the presence of the TETA ethylene group. The ID /IG ratio of FG/Ag decreased to 1.02, which was lower than that of FGO, indicating increased

␲-conjugation in aromatic carbons after the hydrothermal reduction. Generally, SERS can be explained in terms of electromagnetic (high enhancement factors up to 1012 ) [40] and chemical (high enhancement factors up to 102 ) enhancements [35]. However, both the D and G intensities of FG/Ag synchronously increased by more than 500% as compared with those of GO under similar test conditions. The mechanism for this SERS enhancement is thought to be a short-range chemical effect arising mainly from the charge-transfer interaction between GO and Ag-NPs [41–43]. To evaluate the SERS activity of the FG/Ag nanocomposite, we first used p-aminothiophenol (p–ATP) as a model Raman probe because it has been well-characterised by SERS, and most of the prominent Raman bands have been identified [44]. Fig. 5b displays the SERS spectra for different concentrations of p-ATP adsorbed on FG/Ag. In the normal Raman spectrum of solid p-ATP (see Fig. S3 in the supplementary information), strong and medium-strong bands appear at 464, 1089, and 1595 cm−1 , whereas the p-ATP adsorbed on FG/Ag shows noticeable difference in the SERS spectrum, and the five strong bands are observed at 1074, 1141, 1390, 1435, and 1575 cm−1 (Fig. 5b). These significantly different values arose because of the formation of a p-ATP chemical bond with AgNPs and the charge transfer from Ag-NPs to p-ATP [45]. The SERS intensity of p–ATP on the FG/Ag nanocomposite at a higher concentration (2 × 10−5 ) is pronounced, but the intensity decreased with a decrease in the concentration of p-ATP. However, p–ATP peaks could be detected at 2 × 10−8 M using the FG/Ag nanocomposite. This result confirms the SERS activity of the FG/Ag nanocomposite. Melamine (2,4,6-triamino-1,3,5-triazine) is a nitrogen-rich chemical commonly used to produce melamine resin, a synthetic heat-tolerant polymer. Although melamine is not inherently a carcinogenic and toxic chemical, illegal large-dose adulteration in

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routine dairy products can result in urinary calculi, acute renal failure and even infant death. Thus, developing an accurate and rapid on-site melamine screening method is a very important task [46]. The inset of Fig. 5c shows the Raman spectrum of pure solid melamine powder with the most prominent peak around 674 cm−1 , which is assigned to the ring breathing mode II and in-plane deformation of the triazine ring [47], accompanied by other peaks at 384,582, 780 and 982 cm−1 . These peaks are not prominent with the FGO-melamine and FG-melamine mixtures at a concentration of 2 × 10−5 M. However, when the same concentration of melamine was adsorbed on FG/Ag, the main peaks at 671, 802, and 966 cm−1 appeared. The shift in the peaks possibly occurred because of the interaction of melamine with Ag-NPs. The enhancement of the carbon peaks of the FG/Ag-melamine mixture at 1350 and 1954 cm−1 is also evident for the SERS activity of the nanocomposite. Fig. 5d shows the SERS spectra of FG/Ag-melamine with different concentrations of melamine (2 × 10−5 , 2 × 10−6 and 2 × 10−7 M). The main peak of melamine at 671 cm−1 is distinguishable to 2 × 10−7 . These results suggest that melamine can be detected with the FG/Ag nanocomposite at a concentration as low as 2 × 10−7 M. The graph of SERS intensities vs. concentration of melamine and p-ATP is presented in Fig. 6. The detection sensitivity of p-ATP is higher than the melamine. The formation of chemical bond between p-ATP thio group and Ag-NPs resulted in significant increase in the SERS signals compared to the melamine adsorbed on Ag-NPs. Thus, the strong interaction of p- ATP shows higher sensitivity than the melamine. The antibacterial activity of FG/Ag was carried out against E. coli bacteria. The digital images of the antibacterial test results of GO, FGO and the FG/Ag nanocomposite are displayed in Fig. 7. The water and agar containing GO and FGO, respectively, were hazy, signifying the presence of bacterial colonies. In contrast, the agar containing FG/Ag was completely clear, indicating maximum bacterial inhibition. This is evident that FG/Ag has an excellent antibacterial effect, as opposed to GO and FGO (see Fig. 7). The impressive aqueous stability and positive antibacterial effect of the FG/Ag nanocomposite could be effectively utilised for long-term storage of sensing devices. 4. Conclusion Stable aqueous suspensions of FG/Ag nanocomposite was prepared by an environmentally friendly, one-step hydrothermal method. Improved water stability of the nanocomposite was attributed to the electrostatic repulsion between negatively charged functional groups on the surface. Uniform decoration of Ag-NPs on the FG surface showed surface enhanced Raman scattering, and was able to detect melamine at concentrations as low as 2 × 10−7 M, demonstrating the nanocomposite’s potential application as a substrate for SERS studies. The bactericidal property of the nanocomposite prevented bacterial growth, which could enhance the sensitivity of sensing materials as well as enhance their storage life. Acknowledgements This work was financially supported by a High Impact Research Grant from the University of Malaya (UM.C/625/1/HIR/030), a High Impact Research Grant from the Ministry of Higher Education Malaysia (UM.C/625/1/HIR/MOHE/05) and a Postgraduate Research Grant from the University of Malaya (UM.C/241/9). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2013.02.045

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Biographies S. Vijay Kumar received his B.Sc M.Sc and PhD degree from Kuvempu University, Karnataka, India. In 2009 he joined Yonsei University, South Korea as a visiting scientist for one and half years, later he moved to University Malaya in 2011 as a postdoc fellow and continued his research on graphene and polymer nanocomposites. His research area mainly focused on structural property relationship of polymers, preparation of graphene and polymer nanocomposites and their applications. N.M. Huang received his B.Sc, M.Sc and PhD degrees from Universiti Kebangsaan Malaysia. He joined University of Malaya in 2009 as Senior Lecturer in the Department of Physics, Faculty of Science. He started working on graphene and graphene related materials in the same year and had published more than 20 articles on graphene since then. H.N. Lim received her B.Sc. and M.Sc. degrees from Universiti Kebangsaan Malaysia in 2002 and 2004, respectively. She was awarded a Ph.D. degree in Chemistry from University Putra Malaysia in 2010. She was an Assistant Professor at the Nottingham University Malaysia Campus and a Senior Lecturer at the University of Malaysia before joining Universiti Putra Malaysia as a Senior lecturer. Since 2009, she has been actively involved in graphene related research, encompassing the synthesis of graphene-based nanomaterials and their applications. M. Zainy received a Master degree in Physics from Universiti Putra Malaysia (UPM), Malaysia in 2010. She is currently a PhD candidate at the Low Dimensional Materials Research Centre (LDMRC), University Malaya and working in the research of synthesizing and characterization of graphene and metal nanocomposite field. I. Harrison has worked on electronic materials and devices for over 25 year and has published over 150 journal papers. He was appointed as a lecturer in the Department of Electrical and Electronic Engineering at the University of Nottingham in 1987. In September 2009 he was seconded to the Malaysia Campus where he is currently the Dean. C.H. Chia is currently a Senior Lecturer in the Materials Science Program, School of Applied Physics, Universiti Kebangsaan Malaysia (UKM) (which also known as National University of Malaysia). He is also the Deputy President of Polymer Committee of the Institute of Materials Malaysia. He obtained his PhD from the Materials Science (UKM, Malaysia) in 2007. He had performed research on Magnetic Paper at The Australian Pulp and Paper Institute, Monash University in 2006 and 2007.