Essay: Polyacrylamide/Reduced Graphene Oxide-Ag Nanocomposite as Highly Efficient Antibacterial Transparent Film

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In this study, preparation and characterization of polyacrylamide/reduced graphene oxide-Ag (PAM/rGO-Ag) nanocompositesasas a new nanocomposite film were investigated. Then, the prepared PAM/GO nanocomposite was filled with Ag nanoparticles and thermally annealed in order to achieve high performance nanocomposite film with antimicrobial activities. The prepared nanocomposite was characterized by XRD, FT-IR, SEM, TEM and TGA. The obtainedresults demonstrate that the silver nanoparticles were well decorated and dispersed on the graphene oxide nano sheets. In fact, the GO nanosheets and polyacrylamide chains act as a support and stabilize the Ag nanoparticles. Moreover, antimicrobial activities of the films were also examined and the films containing well-dispersed and stabilized Ag nanoparticles showed high antibacterial activity.
Keywords:Graphene, Graphene oxide, Polyacrylamide, Ag nanoparticles, Antibacterial propert
Since discovery of graphene in 2004, graphene and graphene oxide (GO)have received increasing attention due to their unique structures and outstanding properties and were used in various fields such as nanoscale electronic devices, field emission materials and polymer-enhanced materials, chemical sensors, hydrogen energy storage, fuel cells, Li ion batteries, energy conversion systems and biomedical [1-7].
GO is the oxidation product of graphene and generally involving many oxygen-containing functional groups such as hydroxyl, epoxide, carbonyl and carboxyl groups [8-10]. GO has been widely used for immobilizing numerous metallic nanoparticles [11-13].However, because of their large aspect ratios and strong π-π interactions between the sheets, graphene is inclined to aggregate, resulting in undesirable disperseability in common solvents and matrices, which limits their further applications [14]. Three main strategies have been followed to prepare graphene-polymer nanocomposites using solution blending, melt blending and in situ polymerization [15-18]. Among them, in situ polymerization technique approach is particularly interesting and considered to improve dispersion of graphene.
Design and development of metal nanoparticles (NPs) dispersed in polymeric matrix, have been opened a new research domain, with potential applications in the fields of catalytic process and biological research [19-21]. Commonly, Ag nanoparticles (Ag NPs) use in biological and medical application as an antibacterial due to highly toxic to micro-organisms. In fact, Ag NPs are highly toxic to micro-organisms and is employed as an antibacterial agent [22-24]. In particular, biological activity of silver, especially the antibacterial property, is size dependent. Thus, Ag NPs should be small enough to pass through any bacterial cell membrane. On the other hand, metal NPs tend to agglomerate due to their high active surface area. Therefore, various materials involving polymers, surfactants, graphenenanosheets and its derivatives have been used in order to immobilize or stabilize these NPs [25-27]. Among, GO has been decorated with silver nanoparticles (AgNPs) for use as an antibacterial agent [28-31]. Despite all these efforts, the task of creation of versatile and effective methods for reproducible synthesis of fine dispersion of silver NPs with high aggregative stability is not fulfilled yet.
PAM is a water-soluble polymer with high hydrophilicity, permanent biocompatibility, non-toxicity, inert nature, good stability and resistance to degradation which make it a suitable candidate for applications in medical and pharmacy [32-34]. In related studies, Li et al. synthesized reported PAM/GO nanocomposite hydrogels by in situ polymerization which exhibited superior tensile properties [35]. Moreover, Ye et al. Prepared GO/PAM hydrogels which were crosslinked by N,N-methylene bisacrylamide. The obtained results showed that GO was fully exfoliated into individual sheets and tensile strength of GO/PAM hydrogels wereimproved by increasing of GO content [36].
Design and development of new antibacterial agents with excellent antibacterial activity are very significant in human living environment. Herein, we have focused on the development of new, simple and facile method to produce PAM/rGO-Ag transparent films using in situ polymerization and thermal annealing methods. Antibacterial activities of the prepared nanocomposite films were examined against Gram-negative, pesudom-oas and Gram-positive, Staphylococcus aureus (S. aureus) and Canidida which are the model microorganisms for testing bactericidal properties.
2.Experimental methods:
2.1.Materials and methods:
Natural flake graphite (G), sulfuric acid (98%), nitric acid (65%), hydrogen peroxide (33%), potassium permanganate, potassium peroxodissulfate, acrylamide and sodium borohydride were purchased from Merck.
2.2. Characterization:
The prepared nanocomposites were characterized with several techniques. UV–vis spectra were recorded by UV–vis instrument (Lambda 850). Raman spectra were performed with Raman spectrometer (SENTERRA 2009). Fourier transform infrared (FT-IR) spectra were performed with Nicloet FTIR spectrophotometer (Avatar 360). Thermo-gravimetric analysis (TGA) was carried out under argon atmosphere at heating rate 10 °C/min using thermo-gravimetric analyser (Q50 V6.3 Bilds189). X-ray diffraction (XRD) was done with an X-ray diffractometer (X-pert Philips, pw 3040/60). The morphology of synthesized GO and film containing nanoparticles were observed with a scanning electron microscopy(SEM) (AIS2100) and field emission scanning electron microscopy (FESEM) (Hitachi Japan S4160). Transmission electron microscopy (TEM) was carried out using Philips instrument (Philips cm30, 300kV).
2.3. Synthesis of GO:
GO was prepared using a modified Hummer’s method [37].Briefly, graphite (1g) and NaNO3 (0.5 g) were added into a 500-ml round-bottom flask followed by adding H2SO4 (60 ml) with stirring in an ice water bath. Next, KMnO4 (3g) was slowly added in 15 min. Cooling was completed in 2 h, and the mixture was allowed to stand for 5 days at room temperature with vigorous stirring. The obtained solution was added to aqueous solution of H2SO4 (100 ml, 5 wt %) in 1 h with stirring, and temperature was kept at 98 °C. the resultant mixture was further stirred for 2 h. Then, 80 ml of hot water (60 °C) and H2O2 (33%, 15 ml) were added to reduce the residual KMnO4 until bubbling is disappeared. Finally, the solid product was separated by centrifugation. After that, the product was filtered and washed with HCl solution (5%) twice to remove the metal ions. Next, the obtained product rinsed with deionized water to remove the acid and get graphite oxide powder after cake was completely dried in air. Finally, a homogenous brown GO solution (2 mg/ml) was made by ultrasonication in deionized water (500 ml) for 20 minutes. The mixture was stirred at room temperature for 3 days. Brown gel was obtained by freezing and drying of the suspension.
2.4. Synthesis of PAM/GO:
The synthesized GO (30 mg) and acrylamide monomer (5 g) were mixed in water (200 ml) for 15 min in an ultrasonic bath and then stirred at room temperature for 30 min. K2S2O8 as the initiator (0.07 g) was added to the mixture and refluxed at 65 °C for 3 h. Finally, the product was precipitated in methanol and dried in vacuum oven at 60 °C for 12 h.
2.5. Preparation of PAM/GO film:
PAM/GO (2 g) was loaded in a 250-ml beaker and water (200 mL) was added and stirred until a homogeneous brown dispersion was obtained. When the homogenization of the mixture was completed, nanocomposite film was obtained by casting the polymeric solution onto a glass plate and drying at 60 °C for 24h.
2.6. Preparation of PAM/rGO-Ag film:
PAM/GO (2 g) was loaded in a 250-ml round bottom flask and water (200 mL) was added in an ice bath and stirred until a homogeneous yellow-brown dispersion is obtained. Then, AgNO3 (0.001 g; 5.8× 10-8mol) was added to the solution of PAM/GO. Next, NaBH4 (as a reduction agent for GO and Ag NPs) (0.0075 g; 1.9 ×10-4mol) was slowly added in 15 min until the yellow solution is appeared. When the homogenization of the mixture was completed, nanocomposite film was obtained by casting the polymeric solution onto a glass plate and drying at 60 °C for 24h.
2.7. Antibacterial properties study:
Antibacterial activity was determined by standard method of NCCLS. Briefly, to carry out the antibacterial activity for these films on Gram negative,pesudomoas and Gram-positive, Staphylococcus aureus (S. aureus) bacteria and Canidida. albicans was investigated according to the agar diffusion method. Initially, nutrient agar medium was prepared by using peptone (5.0 g) and beef extract (3.0 g) in 1000 ml distilled water. pH was adjusted to 7.0 and agar (15.0 g) was added to the solution. The agar medium was sterilized in a conical flask at a pressure of 15 lbs for 30 min. This nutrient agar medium was transferred into sterilized Petri-dishes in a laminar air flan. After solidification of the media, bacteria culture was streaked on the solid surface of the media. To this inoculated Petri-dish, films were cut into sheets with the dimensions of 2 cm × 1 cm (or 20, 40 and 60 µl for solution sample) was added and incubated for 1 day at 37 °C in the incubation chamber. Samples having antimicrobial activity inhibited the growth of the microorganisms and a clear, distinct zone of inhibition was visualized surrounding the sample. The antimicrobial activity of each material was determined by measuring the diameter of inhibition zones in millimetres.
3. Results and Discussion:
3.1. Synthesis and characterization of GO and its nanocomposite films:
Herein, we report a simple and practical approach for the first time in order to synthesize graphene oxide reinforced PAM nanocomposite films via in situ polymerization of acrylamide in GO suspension (Scheme1). In the following discussion, we will focus on some of the most important properties of prepared nanocomposite films.
Scheme1. preparation of PAM/rGO-Ag nanocomposite and its film
The successful synthesis of GO and in situ radical polymerization of AM were confirmed by FTIR and TGA analysis. The FT-IR spectra of GO, PAM, PAM/GO, PAM/GO film and PAM/rGO-Ag film were given in Fig. 1. Based on the spectrum of GO, the broad peak at about 3200-3500 cm-1 was attributed to the -OH vibration stretching. It also showed peaks related to C=O carboxylic bond (1650 cm-1), C-O etheric bond (1400 cm-1), C-O epoxy bond (1250 cm-1) and C-O alkoxy (1100 cm-1) situated at the edges of the GO nanosheets. As shown in Fig. 4b, the vibration peaks at 3160 cm-1 result from the stretching vibration of –NH group belonging to the AM. Moreover, the vibration peaks at1683 cm-1, 1457 cm-1 and 1100 cm-1 are associated to C=O, C-O and alkoxy C-O vibration of PAM, respectively [38].
In the spectrum of PAM/GO, PAM/GO and PAM/rGO-Ag film, the appearance of new peak at 2950 cm-1 is associated to the asymmetric stretching vibration of CH2. For PAM/GO (Fig. 2d), the NH band at around 3160 cm-1 has overlapped with the band of OH group located at the surface of GO nanosheets. Moreover, typical stretching absorption bands related to acrylamide unit, C=O and C-O could be found. These results indicate a successful polymerization of PAM in the presence of GO. The -NH bending vibration band appeared at 1603 cm−1 for PAM/GO and PAM/GO film samples, but in the case of the PAM/rGO-Ag film it is shifted to 1628 cm−1. The interaction between the -NH of the polymer support and the metal atoms generally results in a blue shift of the band corresponding to the nitrogen functions in the FT-IR spectra [39]. The observed shifting of the spectral peak for PAM confirms the effective interaction between PAM and Ag nanoparticles. In the spectrum of PAM/rGO-Ag (Fig.1e), a peak could be observed at 3160 cm-1, corresponding to the –NH stretching of the acrylamide unit. Moreover, there is a significant decrease in the intensity of the adsorption bands of the oxygenated functional groups for the PAM/rGO-Ag sample. This can be due probably to the existence of AgNPs on the surface of GO and also to the slight reduction of GO by NaBH4 during the synthesis of the PAM/rGO-Ag nanocomposite.
Fig. 1.FT-IR spectra of GO, PAM, PAM/GO, PAM/GO film and PAM/rGO-Ag film.
The UV−vis spectra of GO, PAM, PAM/GO film and PAM/rGO-Ag film are shown in Fig. 2. GO exhibits a broad band appeared at 230 nm, corresponding to the π → π* transition of C=C of GO. The appearance of a peak at 300 nm is attributed to the n → π* transitions of C=O, suggesting the presence of oxygen functionalities in GO [40,41]. Furthermore, to confirm the formation of Ag NPs in the film, UV–visible absorption studies were also used.
A strong characteristic absorption peak around 440 nm is noted for the Ag NPs in PAM/rGO-Ag due to the surface Plasmon resonance effect (Fig. 2). After reduction byNaBH4, an absorption peak is observed at 260 nm for PAM/rGO-Ag, which is the characteristic absorbance of graphene sheets. The absorption peak of PAM/rGO-Ag is broader than that for PAM/GO film, indicating that the graphene sheets are well dispersed in polyacrylamide matrix. Finally, in all UV–vis spectra related to the films containing AgNPs, there are no peaks identified around 335 and 560 nm. These results show the homogeneous dispersion of Ag nanoparticles and no Ag nanoparticle aggregation or Ag cluster [42,43].
Fig. 2.UV-vis Spectrum of PAM, GO, PAM/GO film and PAM/rGO-Ag film
Although GO is thermally unstable and starts to lose mass upon heating even below 100 °C, the major mass loss occurs at 200 °C, presumably due to pyrolysis of the labile oxygen-containing functional groups, yielding CO, CO2 and steam[44,45 ]. The two mass loss peaks at about 200 and 420 °C of the gels are arising from the pyrolysis of the oxygen functional groups and PAM moieties respectively [46]
TGA results indicate that the nanocomposite films have very good thermal stability. According to the TGA results, decomposition temperature of nanocomposite films differs from the GO nanosheets and a considerable increase in the thermal stability of all nanocomposite films in comparison with the GO nanosheets is observed. As illustrated in Fig. 3, PAM, PAM/GO film and PAM/rGO-Ag showed a similar pattern of weight loss, and all have two major mass losses at about 325°C and 420°C due to the decomposition and carbonization of PAM [47, 48]. The overall weight loss of GO is about 34.81% at 600°C, but they were 84.34, 80.44 and 79.21 for PAM, PAM/GO film and PAM/rGO-Ag film, respectively. Degradation of the pure PAM and its film occurred in three steps, the first step was observed at about below 100°C.The second step which is related to thermal decomposition of PAM was considered at 200–325°C and the third step observed at 325-420°C. Final char value of the PAM/rGO-Ag nanocomposites film is somewhat higher than PAM/GO nanocomposite film which is mainly on account of the presence of Ag nanoparticle in this sample.
Fig. 3. TGA curves of GO, PAM, PAM/GO film and PAM/rGO-Ag film
These results indicated that the addition of small amount functionalized graphene improved the thermal stability of the films significantly, which could be reasonably explained by enhanced interfacial interaction between GO nanosheets and PAM matrix.
Raman spectroscopy is a widely used tool for the characterization of carbon products, especially due to this fact that conjugated and double carbon-carbon bonds lead to high Raman intensities. The Raman spectrum of the pristine graphite, as expected, displays a prominent G peak as the only feature at 1581 cm-1, corresponding to the first-order scattering of the E2g mode [49]. Fig. 4 (a,b) illustrates the featured regions of the Raman spectra for graphite oxide, graphene oxide, PAM/GO, and PAM/rGO-Ag film.
Fig.4. Raman spectra of (a) graphite oxide and PAM/rGO-Ag film (b) GO , PAM/GO
Two representative peaks around 1308 and 1584.06 cm-1 are observed for GO, which are generally assigned as D and G band, respectively. G band arises primarily from the presence of a sp2 carbon network, whereas D band is related to the vibrations of sp3 carbon atoms from defects and disorder inherent in the graphite and the edge effect of graphite crystallites. By comparing the relative intensities of D and G band, we can speculate the information about the disordered and ordered crystal structures of carbon. For our samples, the D/G ratios of GO, PAM/GO are 1.26 and 1.31 respectively. This indicates the presence of low localized sp3 defects within the sp2 carbon network.
In addition, the D and G bands in PAM/rGO-Ag exhibit a blue shift compared to GO, the D band shifts from 1308 to1313 cm−1, whereas the G band varies from 1584 to1597 cm−1. The respective blue shift between PAM/rGO-Ag and GO is due to the strong interaction between the three components [50]. Besides, the broader width of D and G bands in case of PAM /rGO-Ag nanocomposite film, compared to GO may be due to deposition of Ag nanoparticles and the formation of covalent link between graphene oxide sheets and polymer chains [51].
The PAM /rGO-Ag nanocomposite film was prepared by in situ polymerization of AM in a GO suspension. After film casting treatment, the size and morphology of the synthesized Ag-NPs on PAM /rGO-Ag nanocomposite film were determined by by SEM, FESM and TEM. The results are given in Fig. 5. The morphology of nanocomposite film before and after addition of Ag NPswere monitored by SEMimages (Fig 5(a b).As shown in Fig. 5(b,c), the Ag NPs dispersed in the PAM /rGOnanocomposite film.The spherical and uniform sized AgNPsare observed on PAM /rGOnanocomposite film.
Moreover, the morphology of GO nanosheets before and after polymerization were also monitored by TEM images. Fig. 5(d,e) shows the TEM images of GO and Ag NPs prepared within the PAM/rGO complex. The TEM images show good uniform distribution of Ag NPs. In addition, based on TEM (Fig. 5e,f), the size of silver particles was estimated between 5-10 nm.The TEM images of PAM/rGO-Ag (fresh) and PAM/rGO-Ag film (one year after production) (Fig. 5 e,f) exhibited the same phenomenon and the uniform AgNPs still anchored on PAM/GO without obvious changes. This implies that AgNPs have excellent stability in PAM/rGO complex during one year, even after exposure to natural light. Therefore, these results indicate that PAM/rGO system has excellent capability to use as Ag NPs carriers and rGO sheets are well dispersed in the case of the PAM/rGO-Ag film.
Fig. 5. (a) Scanning electron microscopy (SEM) image of PAM/GO film, (b) PAM/rGO-Ag film, (c Field emission scanning electron microscopy (FESEM) image of PAM/rGO-Ag film,(d) TEM image of GO, (e) Ag NPs in PAM/rGO film (fresh), and (f) Ag NPs in PAM/rGO film (one year after production).
The crystal structure and particle size distribution of materials were characterized by XRD. The XRD determines the changes of interlayer distance in graphite and GO nanosheets as shown in Fig. 6a. The peak appears at 2ϴ= 26.1 for pristine graphite, whereas the same peak is shifted to 2ϴ =11.1 after the oxidation of the layers in graphite oxide, corresponding to the layer-to-layer distance of near 0.78 nm. It is significantly larger than that of pristine graphite (near 0.34 nm), due to the intercalating oxide functional groups [52].
Fig. 6 X-ray diffraction pattern of (a) graphite and GO (b)PAM and PAM/rGO-Ag
According to Fig. 6b, for pure PAM, only weak amorphous diffraction peaks can be found, which indicates that it is completely amorphous. In addition, for PAM/rGO-Ag film, there is no peak at 11.6°, which confirms the reduction of GO Sheets. In addition, the XRD patterns of the obtained films demonstrate the existence of silver in PAM/rGO-Ag. The major diffraction lines can be indexed to the Ag face centered cubic (fcc) phase. The broad diffraction peaks of Ag indicate relatively small crystal size. The average particle size of the deposited Ag nanoparticles is calculated to be ca. 6 nm from the (111) peak in terms of the Scherrer’s equation.
3.2. Antibacterial activity:
Recent studies suggested that GO and also Ag NPs-functionalized graphene-based materials possesses high antibacterial activity [53-56].graphene and its derivatives,such as GO and rGO, can inhibit the growth of bacterial cells bydamaging bacterial cell membranes with their extremely sharpedges. graphene has been combined with nanomaterials to improvetheir antibacterial activity. graphene was also conjugated with organicantibiotics to increase their antibacterial activity. Hence, it is expected that the prepared films nanocomposite containing GO and Ag NPs should demonstrate great antibacterial properties. Therefore, the task of the current study includes testing the developed PAM/rGO-Ag and PAM/GO nanocomposite films for the purpose of antibacterial activity. Fig. 7 and Table1 illustrates the antibacterial effect of GO, PAM/GO, PAM/rGO-Agon pseudomonas, Staphylococcus aureus (S. aureus) and Canidida. albicans. The reaction conditions were optimized, and the results are presented in Table 1. It was found that the best result was obtained by the fresh PAM/rGO-Ag film (Table 1, entry 3) whereas the pure PAM did not show any effect psudomoasGram- negative, Staphylococcusaureus Gram-positive (S. aureus) and Canidida.albicans. It was noticed that higher inhibition zone sizes against bacteria were observed with PAM/rGO-Ag film. According to the obtained results, Antibacterial test was repeated for the entry 3-5(fresh, six month and one year after production). The results showed that the antibacterial properties of the films have not changed. Hence, it can also be noted that because of the size, excellent stability and good uniform distribution of Ag NPs, strong inhibition effect with a minimum sample amount is obtained. By combining the obtained results, we conclude that the PAM/rGO-Ag nanocomposite films can used as excellent antibacterial material for biological applications.
Fig. 7 The antibacterial effect of (a) GO (0.3mg/ml ), (b) PAM/GO(10mg/ ml ), ( c) 10mg/ ml PAM/rGO-Ag(Pd;25×10-8mmol; fresh ) and (d) 10mg/ ml PAM/rGO-Ag(Pd; 25×10-8mmol; one year after production) on S.aureus.(1:20 µl, 2: 40µl and 3:60 µl).
Table 1: Anti-bacterial activity of GO, PAM/GO and PAM/rGO-Ag nanocomposite films a
Conditions:a PAM/GO (1mg/ ml; 60 µl ), GO (0.3mg/ml; 60 µl), PAM/rGO-Ag (10mg/ ml; 25×10-8mmol, 60µl ).b PAM/rGO-Ag (fresh), c PAM/rGO-Ag (six monthafter production) and d PAM/rGO-Ag( one year after production)
4. Conclusion:
We report for the first time a simple and practical approach to synthesize graphene oxide-reinforced PAM nanocomposite films via in situ polymerization. Interfacial interaction was dramatically enhanced due to chemical linkage and hydrogen bonding between GO and PAM backbone, then AgNO3 was added to the solution of PAM/GO and Ag NPs was prepared by NaBH4. The combination of PAM, GO and Ag NPs are selected because of their more relevance for biomedical applications. On the other hand, because of the size, excellent stability and good uniform distribution of Ag NPs, the highest growth inhibition zone with a minimum sample amount is obtained.
We are thankful to the Research Council of the University of Tehran.
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