Synthesis, characterization and antibacterial activity of Ag incorporated ZnO–graphene nanocomposites

Abstract

The present work reports on the successful one-pot surfactant-free in situ synthesis of silver incorporated ZnO–chemically converted graphene (CCG) nanocomposites (AZG) by adopting a low temperature solution technique from zinc acetate dihydrate, silver nitrate and graphene oxide, and the varying silver content (up to 20% Ag with respect to Zn) in the precursors. X-ray diffraction and transmission electron microscopy studies confirmed the presence of Ag and ZnO nanoparticles (NPs), distributed uniformly with CCG. FTIR, Raman, UV-visible and X-ray photoelectron spectroscopic analyses confirmed the existence of interaction between CCG with the inorganic moieties (ZnO/Zn²⁺ and Ag NPs) of the AZG samples. In vitro cytotoxicity and quantitative cell viability of the human ovarian teratocarcinoma cell line (PA1) was studied up to a maximum sample concentration of 200 μg ml⁻¹. Antibacterial activity was also measured on E. coli and S. aureus to confirm the efficiency of the nanocomposite, especially for killing bacterial cells without any major effect on the surrounding cells. Among the nanocomposites, the 10% Ag incorporated sample at a 6.25 μg ml⁻¹ dose showed excellent antibacterial activity with negligible cytotoxicity. This simple strategy could be applied in the synthesis of Ag incorporated different metal oxide–CCG nanohybrids for antibacterial applications.

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1. Introduction

In the medical and food storage sectors, the rapid rise of multidrug resistant pathogens from the use of broad spectrum antibacterial drugs is a serious global problem.¹ These pathogens are (e.g. bacteria, fungi) capable of surviving antimicrobials or antibiotics.² According to the report of the World Health Organization, Escherichia coli (such as cephalosporins, fluoroquinolones), Staphylococcus aureus (methicillin), Hepatitis B virus (lamivudine) and Cryptococcus neoformans (fluconazole) are the most important examples of some kinds of microorganisms that have shown tremendous resistivity against their respective drugs.³ Moreover, repetitive use of antimicrobial drugs is also a worry due to different side effects in human beings.⁴ Most of the textile and fibre industries are also facing acute problems of microbial attack because of the use of antimicrobial fabrics in hospitals, hotels and restaurants.^5,6 Hence, there is a great challenge towards providing an improved antimicrobial agent which could destroy the multidrug resistant pathogens safely and cost effectively.

Nanocomposite materials could be used as nano weapons to tackle effectively the problem of multidrug resistant pathogens. Metal oxide nanoparticles have already been used in drug delivery,⁷ bio-imaging,⁸ antimicrobial activity⁹ etc. This is because the materials in nano dimension could easily enter into the living cells through the pores of plasma membrane proteins¹⁰ owning to high surface to volume ratio as well as physiochemical properties of nanomaterials. These could prove excellent protector against multidrug pathogens.¹¹ The basic difference of antibiotics with nanocomposite materials consisting metal oxide is that the later prefer multiple target approach rather than mechanical approach of antibiotics¹² and this could be a reason, why microbes fail to acquire resistance against nanocomposite materials. For this purpose, a wide variety of inorganic nanomaterials including copper oxide,¹³ zinc oxide,¹⁴ titanium oxide,¹⁵ gold¹⁶ and silver nanoparticles (NPs),¹⁶ graphene¹⁷ etc. had already been investigated explicitly. However, silver based nanocomposites had shown the most effective inhibitory properties against microorganisms including 16 major species of bacteria.¹⁸ In fact, Ag NPs are also known as an environmental friendly non-toxic material for biomedical applications.¹⁹

Among several metal oxide nanoparticles, zinc oxide (ZnO) is believed to be nontoxic, biocompatible and safe material [(21CFR182.8991) (FDA, 2011)]. Moreover, ZnO possesses a special ability to change/tailor a specific property of living cells in the biological system.²⁰ Reduced graphene oxide (rGO)/chemically converted graphene (CCG) has also received strong attention due to unique physiochemical properties such as large surface area,⁸ easy to functionalize with other materials including metal oxides²¹ and high biocompatibility. The CCG also helps to perform antibacterial activity by disrupting bacterial cell membrane with the help of oxidative and membrane stresses generated by sharp edges of graphene nanosheets to kill bacteria.^22,23

An improvement in antibacterial activity of the materials are possible by using cellulose nanocrystal,^24–27 nanoparticles (NPs) of Ag and ZnO as well as graphene.^9,12,28,29 The main disadvantage of cellulose nanocrystal is that it has no antimicrobial activity²⁴ but it could show an improved antibacterial activity after coupling with ZnO^24,25 and Ag NPs.^26,27 Ag–ZnO nanocomposite also showed excellent antibacterial activity at 550 μg ml⁻¹ and 60 μg ml⁻¹ of nanocomposite for green fluorescent protein expressing antibiotic resistant E. coli and S. aureus, respectively.¹² For poly(acrylonitrile/maleic acid)–silver nanocomposites, it was 25 μg ml⁻¹ for Gram-negative (S. typhimurium, E. coli) and 50 μg ml⁻¹ for Gram-positive (S. aureus, B. cereus) bacteria.²⁸ The concentration required for complete bacterial inhibition growth is found to be 12.5 μg ml⁻¹ of Ag–rGO nanocomposite for E. coli bacteria.²⁹ A relatively high antibacterial activity with 10 μg ml⁻¹ of ZnO–GO composite had also been reported for E. coli.⁹

The main problem with Ag NPs is it's stability.³⁰ For the synthesis of stable Ag NPs, several techniques like sol–gel,³¹ laser ablation,³² sonochemical,³³ electrochemical processes³⁴ etc. could be adopted. Although, surfactants such as sodium borohydride,³⁵ sodium citrate,³⁶ hydrazine hydrate³⁷ are mainly used in these processes to enhance the stability of silver NPs but it simultaneously weaken the antibacterial efficiency.²⁹ Another problem of Ag NPs is its aggregation problem particularly in the nano size regime¹² similar to ZnO²¹ which could be mitigated by coupling with CCG/rGO for the use in living cell system.^38,39 Therefore, the challenge now stand is to synthesise stable Ag NPs–ZnO–rGO/CCG nanocomposite from surfactant free precursor solution technique, which could act effectively against bacteria without harming surrounding living cells.

The objective of the present work is to synthesize an effective nanocomposite having Ag, ZnO and CCG components for enhancing the antibacterial activity of the material due to the synergistic effect of the individual components. Herein, we report, a facile one pot surfactant free low temperature (95 °C) solution process for the synthesis of Ag–ZnO–CCG (AZG) nanocomposite using optimized contents of zinc acetate dihydrate, graphene oxide and silver nitrate as precursor materials. Gram-negative – Escherichia coli and Gram-positive – Staphylococcus aureus has been used to test the antibacterial activity and the human ovarian teratocarcinoma cell line (PA1) has been taken for the cytotoxicity study of AZG nanocomposite materials.

2. Experimental

2.1 Synthesis of Ag–ZnO–CCG (AZG) nanocomposites

For the preparation of graphene oxide (GO), modified Hummer's method was used.⁴⁰ A facile low temperature (95 °C) solution process was adopted for the synthesis of Ag nanoparticle–ZnO–chemically converted graphene (AZG) nanocomposites. In this respect, the samples were prepared separately and in each preparation, a fixed amount (40 mg) of as-prepared GO was uniformly dispersed in dimethyl formamide (DMF, Merck) solvent (40 ml) by ultrasonication for about 2 h duration. In addition, a fixed quantity (1 g) of zinc acetate dihydrate [Zn(CH₃COO)₂·2H₂O, Sigma-Aldrich, ≥98%] and silver nitrate (AgNO₃, Qualigens, ≥99.9%) of different contents (0, 2, 5, 10 and 20 atomic percent, at%; with respect to Zn) was uniformly dispersed in 200 ml of DMF during continuous stirring. The products were designated as ZG, AZG2, AZG5, AZG10 and AZG20 where the content (at%) of Ag used in the precursors was 0, 2, 5, 10, and 20, respectively.

Further, the GO dispersed DMF was mixed with zinc acetate and silver nitrate solution while stirring continuously for 2 h to form precursors of the nanocomposites. Subsequently, the precursors were kept in an air oven at 95 °C for 9 h.⁴¹ In this condition, the formation of Ag–ZnO–CCG (AZG) nanocomposites was observed from the change of colour (black to greyish white, ZG or yellowish white, AZG2, AZG5, AZG10 and AZG20) of the suspensions. The solid materials were then separated out by centrifugation using double distilled water and ethanol as washing solvents. Finally, the samples were dried in an air oven at ∼60 °C for 3 h. The same procedure was also followed for the synthesis of pristine ZnO nanoparticles (ZO) where no GO was used. It is worthy to mention that reduced graphene (rGO) could generally be formed due to reduction of GO via thermal annealing (>1000 °C),⁴² photochemical reduction⁴³ and electrochemical reduction processes⁴⁴ but in the present work, none of these processes was adopted and therefore, the presence of GO in the nanocomposites could be termed as chemically converted graphene (CCG).⁴⁵

2.2 Characterizations

2.2.1 Materials properties. For X-ray diffraction (XRD) study of the samples, X-ray diffractometer (Bruker D8 Advance with DAVINCI design X-ray diffraction unit) with nickel filtered CuK_α radiation source (λ = 1.5418 Å) was used in the 2θ range, 5–80°. A field emission scanning electron microscope (FESEM and FESEM-EDS, ZEISS, SUPRA™ 35VP) was used to analyze the morphology, microstructure and elemental mapping of AZG10 sample. The transmission electron microscopy (TEM)/high resolution TEM (HRTEM) along with TEM-EDS studies were done by FEI Company make (Tecnai G2 30S-Twin, Netherlands) machine at the accelerating voltage of 300 kV. Carbon coated 300 mesh Cu grids were used for the placement of samples. Initially, the samples were dispersed in methanol by ultrasonication and the dispersed nanocomposites were placed on the Cu-grid carefully. Moreover, to characterize the chemically converted graphene (CCG) in AZG10 nanocomposite, the TEM measurement was also performed by using a JEOL JEM-2100F (FEG) high-resolution electron microscope operated at an accelerating voltage of 200 kV. X-ray photoelectron spectra (XPS) in the binding energy range of 200–1200 eV of a representative sample, AZG10 along with CCG precursor GO was carried out to determine the chemical state of elements and chemical interaction/complexation of CCG with inorganic moiety present in the sample by employing PHI Versaprobe II Scanning XPS microprobe surface analysis system using Al-K_α X-rays (hν, 1486.6 eV; ΔE, 0.7 eV at room temperature). In the XPS analysis chamber, the pressure was better than 5 × 10⁻¹⁰ mbar. The energy scale of the spectrometer was calibrated with pure Ag sample. The position of C 1s peak was taken as standard (binding energy, 284.5 eV). Moreover, a Netzsch STA 409 C/CD thermoanalyzer was used for thermogravimetric analysis (TG-DTA) of the composite using Al₂O₃ as a reference material maintaining the heating rate of 10 K min⁻¹ in air atmosphere. For the TG-DTA run, a maximum temperature was chosen up to 700 °C. FTIR spectral study was carried out by Thermo Electron Corporation, USA make FTIR spectrometer (Nicolet 5700). In each experiment, the number of scans was fixed at 100 (wavenumber resolution, 4 cm⁻¹). For measurement of UV-vis spectra of the samples, diffused reflectance method was adopted using an UV-VIS-NIR spectrophotometer (UV3600, Shimadzu, Japan) with ISR 3600 attachment. Raman spectra were also recorded using micro-Raman (Renishaw inVia Raman microscope) with an argon ion laser of incident wavelength of 514 nm as an excitation source.

2.3 Cell culture for cytotoxicity study

Human ovarian teratocarcinoma cell line, PA1, procured from ATCC, USA was used for the cytotoxicity study of ZO, ZG, AZG2, AZG5, AZG10 and AZG20 samples with their varying concentrations. In this work, Dulbecco's modified eagles medium (DMEM, Gibco, USA), fetal bovine serum (FBS, Gibco, USA), PenStrep (Gibco, USA) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, Sigma, USA) were also used as required chemicals and reagents for the cytotoxicity study. At first, PA1 cells were cultured in DMEM (Gibco) supplemented with 10% FBS and antibiotic (1% penicillin/streptomycin) and incubated at 37 °C under a humidified atmosphere with 5% CO₂. For the study, the cells from exponentially growing cultures were used.

2.3.1 Measurement of in vitro cellular cytotoxicity, MTT assay. The MTT assay was performed to evaluate the cytotoxicity of Ag–ZnO–CCG nanocomposites. In a brief procedure, PA1 (10⁴ cells per well) were seeded onto a flat bottom 96-well plate and incubated at 37 °C in 5% CO₂. Then, the Ag–ZnO–CCG nanocomposites were introduced separately to the cells with varying concentrations ranging from 0 to 200 μg ml⁻¹ in a culture medium for 24 h. It is noted that the cells were also treated without any nanocomposite, considered as the control. Finally, it was subjected to MTT assay. Then, 20 μl of MTT (5 mg ml⁻¹) was added to each well. After 4 h of incubation, the media was removed and cells were dispersed in 100 μl of dimethyl sulphoxide (DMSO) solvent. Absorbance spectra of the samples were measured at 595 nm wavelength by Qualigens plate reader-96 well. With respect to control values, the cell viability values were expressed in percentages.

2.4 Antibacterial property

Antibacterial experiment was carried out with Gram-positive bacteria – Staphylococcus aureus (S. aureus ATCC 25923) and Gram-negative bacteria – Escherichia coli (E. coli MTCC 2939) as model microorganisms. Muller–Hindon-Broth (MHB) medium was used for the growth of bacteria. An approximately 10⁷ cfu ml⁻¹ E. coli/S. aureus bacteria cells were grown in 15 ml liquid MHB medium supplemented with different concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg ml⁻¹) of samples (ZO, ZG, AZG2, AZG5, AZG10, AZG20) and incubated at 37 °C. After that the bacterial growth rate was investigated by optical density analysis where optical density (OD) of the treated and untreated bacteria (control) were measured at 600 nm using UV-visible spectrophotometer (SpectraMax M5) up to 10 h with 2 h intervals. Then, the results were further analysed to check the antibacterial activity or their bacterial growth inhibition efficiency.

3. Results and discussion

3.1 Materials properties

3.1.1 Phase structure. The crystallinity and crystal phases of as-synthesized GO, pristine ZnO (ZO), ZnO–CCG (ZG) and Ag incorporated ZnO–CCG (AZG) nanocomposites were analysed by X-ray diffraction study (Fig. 1). In GO, a strong diffraction peak (2θ) was found at ∼10.1° along with a relatively weak intensity peak observed at ∼42.4°, could correspond to the crystal planes, (002) and (100), respectively.²¹ However, in ZG, AZG2, AZG5, AZG10, and AZG20 nanocomposites, these peaks were found to be disappeared completely after 9 h of reaction time.⁴¹ This could be an indication of the transformation of GO into chemically converted graphene (CCG)⁸ in the sample. This transformation was further confirmed by FTIR (Fig. 4) and Raman spectral (Fig. 5) analyses. All the XRD peaks of ZO and ZG, except the XRD peaks appeared at 38.1°, 44.3°, 64.5°, 77.4° of AZG2, AZG5, AZG10 and AZG20 samples fully matched with hexagonal ZnO (h-ZnO) [JCPDS 36-1451] as observed in our previous work.⁴⁵ The XRD peaks observed at 38.1°, 44.3°, 64.5°, 77.4° of Ag incorporated AZG2, AZG5, AZG10 and AZG20 samples were the characteristics of cubic Ag nanoparticles (Ag NPs) (JCPDS 04-0783).⁴⁶ It is also very significant to mention that the peak intensity of cubic Ag was found to be improved with increasing silver nitrate concentration in the precursors, indicating an enhancement in Ag content in the nanocomposites. It is further noted that there was a small shift in 2θ values (∼0.15°) towards lower diffraction angles in Ag incorporated nanocomposites, implying that some lattice site of zinc would be replaced by silver. In this respect, a limited substitution of Ag⁺ ions in Zn²⁺ lattice and interstitial sites of hexagonal ZnO lattice would be a reason.¹² The average crystallite size (D) of hexagonal ZnO and cubic Ag crystallites was⁴⁷ along (101) and (111) planes, respectively was measured by using Debye–Scherrer's equation (eqn (1)).

D = kλ/β cos θ, (1)

where, k is the proportionality constant (k = 0.89), λ is the wavelength of X-ray (1.5406 Å), β is the FWHM (full width at half maximum) of the peak of maximum intensity in radians, θ is the diffraction angle and D is the crystallite size.

Fig. 1 XRD patterns of ZO, ZG, AZG2, AZG5, AZG10 and AZG20 samples along with as-synthesized graphene oxide (GO).

Fig. 2 (a) FESEM image of as-prepared AZG10 nanocomposite and its corresponding elemental mapping images for (b) oxygen (c) zinc and (d) silver elements.

Fig. 3 TEM images of ZG (a, b) and AZG10 (d, e) nanocomposites. HRTEM images of ZG, and AZG10 nanocomposites are shown in (c) and (f), respectively. (e1) and (e2) show the HRTEM images of the particles of Ag and ZnO, respectively. Histograms for the particle size distributions of ZG and AZG10 nanocomposites are given in the inset of (b) and (e), respectively. Insets of (c) and (f) show the TEM-EDS of ZG and AZG10, respectively.

Fig. 4 FTIR spectra of ZO, ZG, AZG2, AZG5, AZG10 and AZG20 along with as-synthesized graphene oxide (GO).

Fig. 5 Raman spectra of GO, ZG and AZG10 samples. Respective I_D/I_G values are embedded in the figure.

The calculated ‘D’ values of ZnO crystallites in ZO, ZG, AZG2, AZG5, AZG10, and AZG20 nanocomposites were ∼15 nm, ∼11 nm, ∼10.0 nm, ∼9.0 nm, ∼8.5 nm and ∼8 nm, respectively while the size of Ag crystallites was ∼30 nm for all the silver incorporated nanocomposites. Therefore, the crystallite size of ZnO was found to be decreased with increasing Ag loading content which is in well agreement with the reported literature.⁴⁶ The small crystallite size could result in increased surface area which might contribute to enhance the antibacterial activity of AZG nanocomposite.

3.1.2 Morphology and microstructure. FESEM surface morphology and elemental mapping particularly for O, Zn and Ag elements present in a representative nanocomposite (AZG10) are shown Fig. 2. The FESEM image (Fig. 2a) confirmed the presence of spherical shaped nanocomposite particles (within 1–2 μm) whereas the elemental mapping (Fig. 2b to d) revealed the uniformly distributed O, Zn and Ag elements in the sample.

The TEM study of two representative samples, namely ZG and AZG10 were also performed. The TEM microstructure of ZnO–CCG (ZG) sample is shown in Fig. 3a where the hollow ZnO–CCG microsphere is clearly visible. It is worthy to note that we have already reported this observation in our previous report.⁴¹ The formation of microspheres could be happen by oriented attachment of ZnO nanoparticles through self-assembly.²¹ On the other hand, the Fig. 3b shows the ZnO NPs are well anchored in CCG layers. The corresponding HRTEM image of the sample as given in Fig. 3c shows the distinct lattice fringes with an interplanar distance of 0.28 nm, corresponded to (100) plane of hexagonal ZnO. This result is fully supported to XRD data (Fig. 1) of the samples. The particle size distribution of ZG is shown in the histogram (inset, Fig. 3b). From the histogram, the measured average particle size of ∼12.0 nm is obtained which corroborated with the crystallite size measured from XRD patterns (Fig. 1). The microstructure of AZG10 nanocomposite is displayed in Fig. 3d where Ag and ZnO NPs co-exist within the microsphere. However, the particle size of Ag is found to be much higher than that of ZnO nanoparticles. This observation is well supported the XRD result (Fig. 1). The nanoparticles of Ag and ZnO in AZG10 sample are clearly visible (Fig. 3e) and the lattice fringes (Fig. 3e1, e2 and f) with interplanar distances of 0.23 nm and 0.28 nm are observed from the HRTEM image of AZG10 nanocomposite. These could be indexed to the (100) plane of hexagonal ZnO and (111) plane of cubic Ag, respectively. Moreover, a wavy like lattice fringes with an average interplanar distance of 0.34 nm could confirm the existence of CCG layer as observed from the HRTEM image of ZG and AZG10 nanocomposites (Fig. 3c and f). It is also important to note that the particle size of ZnO was found to be decreased (∼9.0 nm) in AZG10 compare to ZG as evidenced from the histogram (Fig. 3e) for particle size distribution and it is in agreement with crystallite size as measured from XRD patterns (Fig. 1) of the sample. In this respect, the presence of Ag⁺ ions (Fig. 6) would inhibit⁴⁶ the growth of hexagonal ZnO crystal during the formation of nanocomposite. However, the particles in the nano regime of Ag were much larger than ZnO. The TEM-EDX spectra of ZG and AZG10 are shown in the individual insets of Fig. 3c and f, respectively. As revealed from the EDX spectra for both the samples, the presence of C and Cu could be originated from the carbon coated Cu grid that was used for the TEM study. Another source of carbon could be the CCG that was the organic part of organic–inorganic (ZnO/Zn²⁺ with Ag) hybrid AZG nanocomposite. However, the existence of ZnO and CCG could show the signals for Zn and O elements (Fig. 3c and f) in ZG and AZG10 samples whereas the EDX spectrum of AZG10 (Fig. 3f) evident the existence of Ag in the sample. The observation is fully supported the XRD result (Fig. 1). A semi quantitative analysis by TEM-EDX of AZG10 nanocomposite showed the presence in atomic percentage of 86.59 ± 3.83% and 13.40 ± 3.03% Zn and Ag, respectively.

Fig. 6 XPS data of AZG10 nanocomposite: typical XPS data for the binding energy curves of C 1s along with their Gaussian-fitted components for (a) GO and (b) CCG. Binding energy curves of (c) Zn 2p and (d) Ag 3d core levels.

3.1.3 FTIR spectra. FTIR spectra of ZO, ZG, AZG2, AZG5, AZG10 and AZG20 samples are displayed in Fig. 4. The FTIR peaks appeared at 1732, 1622, 1208 and 1045 cm⁻¹ in GO, attributed to COOH stretching vibration in carboxylic acid groups, skeletal vibration of unoxidized graphitic domains as well as for the vibrations of C–O stretching, and C–OH stretching, respectively.⁸ A prominent vibration observed at ∼470 cm⁻¹ for pristine ZnO and ZG samples assigned to Zn–O stretching vibration²¹ of h-ZnO which strongly supported by the XRD result (Fig. 1). However, in Ag incorporated sample (AZG2, AZG5, AZG10 and AZG20), the vibration due to Zn–O was found to be shifted gradually towards lower wavenumber region (AZG2 ∼ 459 cm⁻¹, AZG5 ∼ 450 cm⁻¹, AZG10 ∼ 442 cm⁻¹, and AZG20 ∼ 435 cm⁻¹, respectively). The peak shifting could suggest an introduction of Ag ions into the h-ZnO crystal lattice of the nanocomposites.⁴⁸ In this regard, the FTIR result also supported the XRD data (Fig. 1). The presence of hydroxyl groups is also characterized by the presence of a broad vibration appeared at ∼3435 cm⁻¹ in all the samples. Moreover, a new peak observed at ∼1565 cm⁻¹, in GO loaded samples (ZG, AZG2, AZG5, AZG10, and AZG20) indicated the existence of CCG in the nanocomposites.²¹ It is also noted that the ∼1565 cm⁻¹ vibration was absent in pristine graphene oxide and all the FTIR vibrations responsible for oxygen functional groups of GO became very weak or nearly disappeared in the ZG, AZG2, AZG5, AZG10, and AZG20 samples, implying the transformation of GO to CCG took place during the synthesis process.

3.1.4 Raman spectra. Micro-Raman spectral study (Fig. 5) was performed on GO, ZG and AZG10 samples to understand the change in structural defects/layer by layer exfoliation in graphene moiety of the nanocomposites with respect to the precursor GO used for the synthesis. Two distinct Raman peaks were observed at ∼1350 cm⁻¹ and at ∼1595 cm⁻¹, assigned to D band (defect) and G (graphene) band, respectively. The D band could be attributed to the exfoliation/breaking of graphene layers whereas the G band (graphene) could be originated due to the presence of E_2g phonon in sp² carbon atoms of graphene moiety.²¹ The intensity ratio of the D (I_D) and G (I_G) bands could also give a significant information about the chemical interaction that happened between CCG and the inorganic moiety.⁸ From Fig. 5, it is clear that the I_D/I_G value is much higher in AZG10 compare of ZG. The relative increase in I_D/I_G (Fig. 5) could be accounted for the decrease of in-plane sp² domain size of graphene. This could happen by layer by layer exfoliation/breaking of graphene layers due to chemical interaction/complexation with the inorganic moiety.²¹ Moreover, removal of oxygen functional groups during the reduction process could also be the reason for the I_D/I_G increament.²¹ In this respect, the Ag nanoparticles would assist for the exfoliation or further structural change in CCG moiety in Ag incorporated nanocomposite.⁴⁹ In addition, a prominent shift of G band towards lower wavenumber region was found in AZG10 compared to GO and ZG samples, confirmed the further interaction of CCG with Ag–ZnO inorganic moiety.⁴⁹ This interaction would result in enhancement of antibacterial activity.

3.1.5 XPS spectra. XPS measurement is generally performed to understand the surface chemical bonding/chemical interaction of materials.⁵⁰ The XPS data of a representative sample AZG10 is shown in Fig. 6. The figure shows the binding energy signals of Zn 2p, Ag 3d, and C 1s. The asymmetric C 1s signals in GO and AZG10 samples are given in Fig. 6a and b. This C 1s signal could be resolved into three Gaussian fitted peaks (components). The XPS curve of GO (Fig. 6a) shows three distinct Gaussian fitted peaks found at 284.5 eV, 286.5 eV and 288.2 eV, could be assigned to C–C, C–O (epoxy and hydroxyl) and C=O (aromatic),⁵¹ respectively. It is noted that the C 1s peaks with similar binding energies were also found in AZG10 (Fig. 6b). It is further noted that the intensity of the peaks for oxygen containing functional groups in AZG10 was much lower compare to precursor GO. This result implies the transformation of GO to CCG happened in the AZG10 nanocomposite.⁴⁰ The strong binding energy signals of Zn 2p (Fig. 6c) was observed at 1021.9 eV and 1045 eV, could be assigned to the binding energy of Zn 2p_3/2 and Zn 2p_1/2, respectively.⁸ An energy difference of ∼23.1 eV between Zn 2p_3/2 and Zn 2p_1/2 binding energy levels could also confirm the existence of Zn²⁺ in the nanocomposite.⁸ On the other hand, the formation of Ag nanoparticles was also assessed by the XPS curve of AZG10 and the peaks found at 367.8 and 373.8 eV (Fig. 6d) could be assigned to Ag 3d_5/2 and Ag 3d_3/2, respectively.⁵² Moreover, the existence of Ag⁺ ions was also evidenced by the presence of low intensity XPS peaks at ∼371 eV and ∼378 eV.⁵² It is worthy to note that the presence of Ag⁺ ions also supported the XRD 2θ peak shifting towards lower angles in hexagonal ZnO present in AZG nanocomposites (Fig. 1). We have also calculated the Ag and Zn contents from the corresponding peak area of XPS signal for silver (Ag⁰ and Ag⁺) and zinc (Zn²⁺) and the contents of silver and Zn were ∼7% and ∼93%, respectively. Therefore, the experimentally measured Ag content was slightly less compare to the content that was taken (10%) from silver nitrate as the source of silver for the synthesis of AZG nanocomposite. This lower value of silver content in the nanocomposite could indicate that some silver ions removed during the purification process using ethanol and deionized water.

3.1.6 UV-vis spectra. Fig. 7 displays UV-vis absorption spectra (measured by diffused reflectance method) of ZO, ZG, AZG2, AZG5, AZG10 and AZG20 nanocomposites. The Kubelka–Munk algorithm was used to obtain the absorption spectra from the reflectance spectra of the samples. A typical UV absorption peak appeared at 360 nm could be assigned to HOMO–LUMO transition of ZnO, due to electron transition from the valence band to the conduction band, related the intrinsic band-gap of ZnO semiconductor.⁴⁶ A distinct and broad shoulder appeared at 415 nm (inset i, Fig. 7) could also be seen in AZG2, AZG5, AZG10 and AZG20 nanocomposites, implied the formation of Ag nanoparticles⁴⁶ and fully supported the XRD (Fig. 1) and TEM (Fig. 3) analyses. It is also noted that, with increasing Ag loading level (content) in the nanocomposites, the absorption edge was found to be shifted towards higher wavelength region⁴⁶ indicating a decrease in particle size of ZnO. The higher absorbance in ZG sample compared to pristine ZnO could be due to incorporation of CCG that modified the optical properties of ZnO.⁴¹ This UV peak shifting towards higher wavelength region due to increased Ag loading level could favour the antibacterial activity of the AZG nanocomposite.

Fig. 7 Absorption spectra of ZO, ZG, AZG2, AZG5, AZG10 and AZG20 measured by diffused reflectance method. Inset (i) shows the enlarged spectrum (as marked) of AZG2, AZG5, AZG10 and AZG20 nanocomposites.

Based on XRD (Fig. 1), FESEM (Fig. 2), TEM (Fig. 3), FTIR (Fig. 4), Raman (Fig. 5), UV-vis (Fig. 7), TG-DTA (Fig. S1†) and XPS (Fig. 6) analyses, a probable formation mechanism and structure of AZG nanocomposite is displayed in Scheme 1.

Scheme 1 Proposed mechanism for the formation of silver incorporated ZnO–CCG (AZG) nanocomposite.

3.2 Biomedical properties

3.2.1 Antibacterial activity. The antibacterial properties of Ag–ZnO–CCG nanocomposites were evaluated using Gram-negative bacteria – Escherichia coli (E. coli) and Gram-positive bacteria – Staphylococcus aureus (S. aureus). The bacterial inhibition growth curve (Fig. 8) was used to correlate the antibacterial efficacy of the nanocomposites with the observed bacterial density at 600 nm and examined the antibacterial properties of the nanocomposites. Confocal laser scanning microscope (CLSM) was also used to investigate the growth inhibition effect of AZG10 nanocomposite on S. aureus (Fig. S2†). Fig. 8 shows the effect of samples (6.25 μg ml⁻¹ concentration of each sample) on the growth of E. coli and S. aureus, respectively. Although, different concentration (200 μg ml⁻¹, 100 μg ml⁻¹, 50 μg ml⁻¹, 25 μg ml⁻¹, 12.5 μg ml⁻¹, 6.25 μg ml⁻¹, 3.125 μg ml⁻¹ and 1.5625 μg ml⁻¹) were tested for each sample, the bacterial inhibition growth curve was considered only at the concentration of 6.25 μg ml⁻¹. This is because the minimum concentration was required for both AZG10 and AZG20 samples for antibacterial activity. In this respect the recorded absorbance (OD) values of AZG10 nanocomposite from low to high concentration (1.5625–200 μg ml⁻¹) was also given in Fig. 9 to prove that the 6.25 μg ml⁻¹ was sufficient for obtaining maximum antibacterial activity of the sample. This required concentration was also less than the reported concentrations in the literatures (Table S1, ESI†). It was also seen that the bacterial inhibition rate was more for higher concentration of Ag based nanocomposites but we found the higher concentration of sample could kill the surrounding cells (Fig. 10). Hence, AZG10 nanocomposite was selected for killing the bacterial cells. In this respect, the MTT graph (Fig. 10) showed that at 6.25 μg ml⁻¹ concentration, the cell cytotoxicity was found to be minimum (∼7% only) for the human ovarian teratocarcinoma cell line, PA1. Therefore, the bacterial inhibition growth curve (Fig. 8) clearly reveals that the AZG10 nanocomposite was most effective than the others and the change of the effectiveness was found in the order AZG20 ∼ AZG10 > AZG5 > AZG2 > ZG > ZO. In this respect, pristine ZnO (ZO) sample showed the antibacterial activity compared to the control. It is worthy to note that ZG nanocomposite showed greater antibacterial activity than ZO, as CCG was hybridized with pristine ZnO. Therefore, the presence of CCG could help to enhance the antibacterial activity but it showed very low activity compare to AZG nanocomposites (AZG2, AZG5, AZG10 and AZG20). It was seen that the antibacterial activity increased with increasing Ag loading level (AZG20 ∼ AZG10 > AZG5 > AZG2). However, in case of AZG10 sample at a certain concentration, it remained same. The reason behind the similar antibacterial activity as noticed from AZG10 and AZG20 nanocomposites could be attributed to the lack of bacterial cells in the cell medium i.e. the concentration of AZG10 could be sufficient to kill all the bacterial cells at 10 h time frame. These results also clearly demonstrated the combined effect of silver nanoparticles (Ag) and chemically converted graphene (CCG) for the antibacterial activity of Ag–ZnO–CCG in which the main protagonist could be the Ag nanoparticles. However, the mechanism of the antibacterial activity of silver nanoparticles in the nanocomposite is not clearly known. However, several suggested mechanisms are available in the literature.^18,53 In one of these, Ag nanoparticles could penetrate into the bacterial cell wall and make structural changes in cell membrane causing the bacterial cell death.¹⁸ Formation of free radicals which disrupt the cell membrane would also be considered for the bacterial cell death.¹⁸ Moreover, Ag NPs could terminate the bacteria by disrupting the bacterial DNA replication.¹⁸ In another mechanism involving the interaction between silver ion and proteins could also be considered for the cell death.¹⁸ However, all the above reasons could depend upon the formation of Ag NPs and Ag ions. It is noteworthy to mention that the existence of the Ag species was already proved by XPS spectra (Fig. 6). Further, ZnO (ZO) also has the ability to interact with the membrane lipid of bacteria which is in turn disrupting the membrane of the bacteria and eventually the death of it.⁵³ ZnO at nano regime could also produce toxic oxygen radicals by entering the bacterial cells and could kill the bacteria.⁵³ The presence of CCG could also helped in enhancing antibacterial activity by disrupting the cell wall of bacteria by oxidative and membrane stresses with the help of the sharp edges of graphene sheets.^22,23 It also assists ZnO and Ag nanoparticles to distribute homogeneously in the Ag–ZnO–CCG nanocomposites.¹⁹ Hence, the interaction of ZnO and Ag NPs with CCG could be the pivotal factor for superior antibacterial activity of AZG10 sample. The interaction of CCG with the inorganic moiety has already been proved by Raman spectra (Fig. 5) where I_D/I_G value was found to be increased in AZG10 nanocomposite. Therefore, the superior antibacterial activity of Ag incorporated AZG10 sample could be due to the synergistic effect of ZnO and Ag nanoparticles with chemically converted graphene (CCG) of the nanocomposite.

Fig. 8 Bacterial inhibition growth curves of (a) E. coli, (b) S. aureus after treatment with ZO, ZG, AZG2, AZG5, AZG10 and AZG20 at 6.25 μg ml⁻¹ sample concentration. Insets of (a) and (b) show the larger views of the growth curves for AZG10 and AZG20 samples.

Fig. 9 Bacterial inhibition growth curves of (a) E. coli, (b) S. aureus after treatment with AZG10 at different (1.5625–200 μg ml⁻¹) sample concentrations. Insets of (a) and (b) show the larger views of the growth curves for 6.25–200 μg ml⁻¹ sample concentrations.

Fig. 10 Cell viability from in vitro cellular cytotoxicity, MTT assay of human ovarian teratocarcinoma cell line, PA1 for ZO, ZG, AZG2, AZG5, AZG10 and AZG20 samples with varying concentrations. The error bars represent ±SD (P < 0.05).

3.3 In vitro cytotoxicity

The cytotoxicity percentage (Fig. 10) of ZO, ZG, AZG2, AZG5, AZG10 and AZG20 samples on human ovarian teratocarcinoma cell line, PA1 were evaluated by varying the concentration of samples. Each bar graph is the representation of an average of three different measurements in which composite dose-related induced cell viability loss could be found. From the result, it is clearly seen that the cell viability percentage (CVP) decreased in the order, ZG > AZG2 > AZG5 > AZG10 > AZG20 where the activity of ZO could be compared with AZG2 nanocomposite. The toxicity of ZO could attribute to the dissolution of ZnO nanoparticles and cellular uptake of the dissolute ions.²¹ In this work, one important observation was the improved cell viability of ZG nanocomposite. This could be due to the biocompatibility of CCG²¹ coupled with ZnO. The prime objective of cell viability test of ZO, ZG, AZG2, AZG5, AZG10 and AZG20 nanocomposites was to examine the action of nanocomposites on the cancer cells at 6.25 μg ml⁻¹ sample concentration and finally, to confirm the selective antibacterial nature of AZG10 nanocomposite. Therefore, from the cell viability result, we could see that the cytotoxicity of AZG10 nanocomposite for the cancer cells was very low (∼7% only) at 10 μg ml⁻¹. However, at 6.25 μg ml⁻¹ sample concentration the antibacterial activity of AZG10 was found to be maximum, the cytotoxicity effect could be low. Moreover, we have seen that AZG10 nanocomposite not only showed a superior antibacterial activity but also it exhibited minimum cytotoxicity, fulfilling the objective of this work towards selective killing of bacterial cells by using Ag incorporated ZnO–CCG nanocomposite.

4. Conclusion

In summary, we developed a new strategy by synthesizing highly efficient antibacterial Ag incorporated ZnO–CCG nanocomposite by low temperature solution technique without using any surfactant in the precursors. A series of Ag–ZnO–CCG (AZG) nanocomposites had been prepared by changing silver content. Without use of additional reducing agent/surfactant, silver nitrate and graphene oxide precursors were found to be reduced in situ in the reaction medium. The AZG nanocomposite synthesized from 10% Ag content in precursor showed an excellent antibacterial activity towards Gram negative and Gram positive bacteria with a very low cytotoxicity towards human ovarian teratocarcinoma cell line, PA1 at 6.25 μg ml⁻¹ sample concentration. Therefore, Ag–ZnO–CCG nanocomposite was found to be an able antimicrobial agent that selectively kills bacteria without harming surrounding cells. This synthesis strategy could open an avenue in Ag incorporated different metal oxide–CCG nanocomposites for biomedical applications.

Acknowledgements

Authors are grateful to the Director, CSIR-CGCRI, Kolkata for his kind support to publish this work. The authors, AN, SB, PS, and RB thankfully acknowledge UGC-RGNF and CSIR, Govt. of India for providing their Ph.D. research fellowships. The authors also acknowledge the help rendered by Nanostructured Materials Division, Bioceramic and Coating Division and Electron Microscopy Section for several characterizations. The work has been done as an associated research work of 12^th Five Year Plan project of CSIR (No. ESC0202).

References

1. Z. Ma, D. Kim, A. T. Adesogan, S. Ko, K. Galvao and K. C. Jeong, ACS Appl. Mater. Interfaces, 2016, 8, 10700–10709.
2. S. Huijben, A. S. Bell, D. G. Sim, D. Tomasello, N. Mideo, T. Day and A. F. Read, PLoS Pathog., 2013, 9(9), 1003578.
3. J. Tanwar, S. Das, Z. Fatima and S. Hameed, Interdiscip. Perspect. Infect. Dis., 2014, 2014, 541340.
4. F. A. Minhas, H. Rehaman, A. Yasin, Z. I. Awan and N. Ahmed, Afr. J. Biotechnol., 2013, 12(48), 6754–6760.
5. B. Mahltig, H. Haufe and H. Böttcher, J. Mater. Chem., 2005, 15, 4385–4398.
6. A. Yıldız and M. Değirmencioğlu, Bioinorg. Chem. Appl., 2015, 2015, 215354.
7. Q. Quan, J. Xie, H. Gao, M. Yang, F. Zhang, G. Liu, X. Lin, A. Wang, H. S. Eden, S. Lee, G. Zhang and X. Chen, Mol. Pharmaceutics, 2011, 8, 1669–1676.
8. S. Bera, M. Ghosh, M. Pal, N. Das, S. Saha, S. K. Dutta and S. Jana, RSC Adv., 2014, 4, 37479–37490.
9. Y. W. Wang, A. Cao, Y. Jiang, X. Zhang, J. H. Liu, Y. Liu and H. Wang, ACS Appl. Mater. Interfaces, 2014, 6, 2791–2798.
10. A. Verma and F. Stellacci, Small, 2010, 6(1), 12–21.
11. K. Elumalai and S. Velmurugan, Appl. Surf. Sci., 2015, 345, 329–336.
12. I. Matai, A. Sachdev, P. Dubey, S. U. Kumar, B. Bhushan and P. Gopinath, Colloids Surf., B, 2014, 115, 359–367.
13. M. Hans, A. Erbe, S. Mathews, Y. Chen, M. Solioz and F. Mücklich, Langmuir, 2013, 29, 16160–16166.
14. K. R. Raghupathi, R. T. Koodali and A. C. Manna, Langmuir, 2011, 27, 4020–4028.
15. H. A. Foster, I. B. Ditta, S. Varghese and A. Steele, Appl. Microbiol. Biotechnol., 2011, 90, 1847–1868.
16. Y. Zhou, Y. Kong, S. Kundu, J. D. Cirillo and H. Liang, J. Nanobiotechnol., 2012, 10, 19.
17. F. Perreault, A. F. Faria, S. Nejati and M. Elimelech, ACS Nano, 2015, 9, 7226–7236.
18. S. Prabhu and E. K. Poulose, Int. Nano Lett., 2012, 2, 32.
19. B. Pant, P. Pokharel, A. P. Tiwari, P. S. Saud, M. Park, Z. K. Ghouri, S. Choi, S. J. Park and H. Y. Kim, Ceram. Int., 2015, 41(4), 5656–5662.
20. J. W. Rasmussen, E. Martinez, P. Louka and D. G. Wingett, Expert Opin. Drug Delivery, 2010, 7(9), 1063–1077.
21. A. Naskar, S. Bera, R. Bhattacharya, S. S. Roy and S. Jana, Polym. Adv. Technol., 2016, 27, 436–443.
22. S. Liu, T. H. Zeng, M. Hofmann, E. Burcombe, J. Wei, R. Jiang, J. Kong and Y. Chen, ACS Nano, 2011, 5(9), 6971–6980.
23. O. Akhavan and E. Ghaderi, ACS Nano, 2010, 4, 5731–5736.
24. H.-Y. Yu, G.-Y. Chen, Y.-B. Wang and J.-M. Yao, Cellulose, 2015, 22, 261–273.
25. S. Azizi, M. Ahmad, M. Mahdavi and S. Abdolmohammadi, BioResources, 2013, 8, 1841–1851.
26. Z. Shi, J. Tang, L. Chen, C. Yan, S. Tanvir, W. A. Anderson, R. M. Berry and K. C. Tam, J. Mater. Chem. B, 2015, 3, 603–611.
27. N. Drogat, R. Granet, V. Sol, A. Memmi, N. Saad, C. K. Koerkamp, P. Bressollier and P. Krausz, J. Nanopart. Res., 2011, 13, 1557–1562.
28. A. R. Allafchian and S. A. H. Jalali, J. Taiwan Inst. Chem. Eng., 2015, 57, 154–159.
29. W. P. Xu, L. C. Zhang, J. P. Li, Y. Lu, H. H. Li, Y. N. Ma, W. D. Wang and S. H. Yu, J. Mater. Chem., 2011, 21, 4593–4597.
30. B. F. Leo, S. Chen, Y. Kyo, K. L. Herpoldt, N. J. Terrill, I. E. Dunlop, D. S. McPhail, M. S. Shaffer, S. Schwander, A. Gow, J. Zhang, K. F. Chung, T. D. Tetley, A. E. Porter and M. P. Ryan, Environ. Sci. Technol., 2013, 47, 11232–11240.
31. R. C. Gamez, E. T. Castellana and D. H. Russell, Langmuir, 2013, 29, 6502–6507.
32. D. O. O. Galindo, R. M. Mejia, N. Bogdanchikova and J. D. M. Morales, J. Colloid Interface Sci., 2016, 12, 1–4.
33. M. Darroudi, A. K. Zak, M. R. Muhamad, N. M. Huang and M. Hakimi, Mater. Lett., 2012, 66, 117–120.
34. B. Yin, H. Ma, S. Wang and S. Chen, J. Phys. Chem. B, 2003, 107, 8898–8904.
35. T. Simon, C. S. Wu, J. C. Liang, C. Cheng and F. H. Ko, New J. Chem., 2016, 40, 2036–2043.
36. Z. S. Pillai and P. V. Kamat, J. Phys. Chem. B, 2004, 108, 945–951.
37. P. Fageria, S. Gangopadhyay and S. Pande, RSC Adv., 2014, 4, 24962–24972.
38. I. Ocsoy, M. L. Paret, M. A. Ocsoy, S. Kunwar, T. Chen, M. You and W. Tan, ACS Nano, 2013, 7(10), 8972–8980.
39. A. S. M. I. Uddin and G. S. Chung, Sens. Actuators, B, 2014, 205, 338–344.
40. S. Bera, A. Naskar, M. Pal and S. Jana, RSC Adv., 2016, 6, 36058–36068.
41. S. Bera, M. Pal, A. Naskar and S. Jana, J. Alloys Compd., 2016, 669, 177–186.
42. S. Pei and H. M. Cheng, Carbon, 2012, 50, 3210–3228.
43. W. C. Hou, I. Chowdhury, D. G. Goodwin Jr, W. M. Henderson, D. H. Fairbrother, D. Bouchard and R. G. Zepp, Environ. Sci. Technol., 2015, 49, 3435–3443.
44. M. A. Raj and S. A. John, J. Phys. Chem. C, 2013, 117, 4326–4335.
45. S. Bera, A. Naskar, M. Pal and S. Jana, RSC Adv., 2016, 6, 40854–40857.
46. M. Ahmad, E. Ahmed, Z. L. Hong, N. R. Khalid, W. Ahmed and A. Elhissi, J. Alloys Compd., 2013, 577, 717–727.
47. A. Naskar, S. Bera, A. K. Mallik and S. Jana, Adv. Nanopart., 2016, 5, 9–17.
48. G. Murtaza, R. Ahmad, M. S. Rashid, M. Hassan, A. Hussnain, M. A. Khan, M. E. U. Haq, M. A. Shafique and S. Riaz, Curr. Appl. Phys., 2014, 14, 176–181.
49. X. Lv and J. Weng, Sci. Rep., 2013, 3, 3285.
50. S. Bera, H. Khan, I. Biswas and S. Jana, Appl. Surf. Sci., 2016, 383, 165–176.
51. J. Wang, S. Liang, L. Ma, S. Ding, X. Yu, L. Zhou and Q. Wang, CrystEngComm, 2014, 16, 399–405.
52. L. Xu, D. Zhang, L. Ming, Y. Jiao and F. Chen, Phys. Chem. Chem. Phys., 2014, 16, 19358–19364.
53. W. Salem, D. R. Leitner, F. G. Zingl, G. Schratter, R. Prassl, W. Goessler, J. Reidl and S. Schild, Int. J. Med. Microbiol., 2015, 305, 85–95.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra14808e

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