Antibacterial and antioxidant screening applications of reduced-graphene oxide modified ternary SnO₂-NiO-CuO nanocomposites

2.1 Materials

Graphite, Phosphoric acid (H₃PO₄), 95–97 % conc. Sulfuric acid (H₂SO₄), potassium permanganate (KMnO₄), Hydrogen peroxide (H₂O₂), Hydrochloric acid (HCl), Nickel (II) sulfate hexahydrate (NiSO₄·6H₂O), Copper (II) sulfate pentahydrate (CuSO₄·5H₂O), tin (II) chloride 2-hydrate (SnCl₂·2H₂O), methanol (CH₃OH) and sodium hydroxide (NaOH) were obtained from sigma Aldrich. All the chemicals were of analytical grade and used without further purification. Deionized water (DIW) was utilized throughout the laboratory work. All the glassware was washed with DIW and then rinsed with acetone.

2.2 Preparation of GO

Modified Hummer’s method was used to prepare GO from the graphite flakes. In this method 99 mL of H₂SO₄ was mixed with 11.0 mL of H₃PO₄ with volume ratio 9:1 and placed in an ice bath. The reaction mixture was stirred for 10 min at 10 °C. Then 2 g of graphite was added to the previous mixture under constant stirring followed by the slowly addition of 8 g of KMnO₄. The obtained reaction mixture was stirred for 6 h until its color changed to dark green. Afterward, reaction mixture was stirred for 10 min after the addition of 6.0 mL of H₂O₂ to remove excess KMnO₄. An extensive care was required for this step because it was an exothermic reaction and released high amount of energy. So, this reaction took place in an ice-cold bath to maintain the temperature. The reaction was cooled down at room temperature. Then slowly added 13.0 mL of HCl in 39.0 mL of deionized with the volume ratio 1:3. Then this mixture was centrifuged for 10 min at 4000 rpm. The supernatant was decanted, and the residue was washed with HCl and then with DIW thrice to have its pH around 6 or 7. Finally, the obtained suspension was filtered and then dried in an oven for 24 h at 100 °C. The dried GO powder was black in color.

2.3 Preparation of SNC

For the preparation of nanocomposite of SNC, 3.78 g of SnCl₂·2H₂O, 4.48 g of NiSO₄·6H₂O and 3.93 g of CuSO₄·5H₂O were weighed separately. Then these metallic salts were added in the beakers contain 50.0 mL of DIW and 10 mL of methanol separately. Then these mixtures were stirred for 30 min at 50.0 °C. The pH of the solutions was maintained up to 10 by adding 2 M NaOH solution. All the three mixtures were combined together in a separate beaker and then kept on stirring for 3 h at 50 °C. The mixture was filtered with Whatman No. 1 filter paper. It was dried in an oven at 100 °C and then half of the sample was calcined at 450 °C for 2 h in muffle furnace.

2.4 Preparation of GO-SNC

For the synthesis of GO-SNC, GO was thermally reduced by sonicating 1.0 g of GO into 100 mL of DIW at 100 °C for 2 h. The rGO solution was poured slowly into the solution of previously prepared metallic nanocomposites and was kept on stirring for 4 h. The mixture was washed with DIW and filtered. The residue was dried in an oven at 100 °C. The resulting product in powdered form was GO-SNC. Half of the quantity of sample was calcined at 450 °C for 2 h.

2.5 Characterization

The properties of the synthesized SNC and GO-SNC were studied with the help of XRD, FTIR, and SEM. The FTIR (4000–400 cm⁻¹) was used to check chemical composition of whole sample using Nicolet 6700 (USA) (Dutta 2017). The XRD was performed by Panalytical X-pert Pro operated at 2-theta (20°–80°) to analyze crystalline structure and crystal phases of the sample (Epp 2016). SEM model 5910 made in Japan was used for the determination of surface topography and sample composition (Akhtar et al. 2018).

2.6 Antibacterial activity

The antibacterial activity of the samples was observed against a GPB (S. aureus) and a gram GNB (E. coli) via agar well diffusion method. These bacterial strains were collected from the department of Biotechnology, University of AJ&K, Muzaffarabad. The apparatus used was sterilized in autoclave. For comparative study, suspensions of various concentrations i.e., 10, 20, 30, 40, 50, 60 mg/3mL were prepared in DIW. The laminar flow was used to perform the activity which was sanitized with the 70 % ethanol solution. Nutrient agar fluid was spread into the petri dishes and allowed to solidify. Afterwards, 50.0 µL of prepared bacterial media was spread on it with micropipette and evenly distributed by swabbing. Sterile microtips were used to bore wells of 5 mm in it and sterile needles were used to eliminate agar plugs. Then, 30.0 µL of each prepared suspension, positive and negative controls, were filled in the separate wells. The petri dishes were placed into an incubator at 37 °C for 16 h. Thus, the zones of inhibitions of calcined and uncalcined samples of SNC and GO-SNC, rGO, graphite and DIW were measured. Finally, in a separate petri dish, same procedure was followed for the calcined samples of SNC and GO-SNC, graphite and water along with an antibiotic. As, they showed highest antibacterial activity at highest concentration, that is why 30.0 µL of each 60 mg/3mL of suspensions were used and then zones of inhibition were measured. Similar procedure was followed for uncalcined samples of SNC and GO-SNC. Likewise, the zones of inhibitions of calcined and uncalcined samples of SNC and GO-SNC were measured against selected bacteria. The solvent (water) as used as negative control whereas the ciprofloxacin was used as positive control.

2.7 Antioxidant activity

Antioxidant activity was performed to examine the potential of calcined and uncalcined samples of SNC and GO-SNC to stabilize ABTS and DPPH free radicals. Various concentrations (5, 10, 15 and 20 µg) of calcined and uncalcined samples were dissolved in 5 mL of DIW and sonicate for 30 min to form suspension. Aqueous solutions of 5.0 mM of potassium persulphate and 14 mM ABTS were prepared individually. Afterwards, these freshly prepared suspensions were mixed in 1:1 V/V and kept in dark for 16 h at room temperature to get blue green solution of ABTS free radical (ABTS^•+). While DPPH^• stock solution was made by dissolving 4.0 mg of DPPH in 50 mL methanol. The resultant deep purple solution was kept for half an hour in dark to get DPPH free radical (DPPH^•).

3.1 XRD analysis

The XRD technique was used to examine the crystalline structure and the chemical composition of the synthesized GO, SNC and GO-SNC. The XRD spectrum given in Fig. 1, exhibits a diffraction peak at 10.78°, which corresponds to the interlayer d-spacing of 8.17 Å, which is almost similar to those reported earlier and this diffraction x-ray correspond to (1 1 1) plane as given in reference card number 01–082-2261. The determined density was found to be 2.12 g/cm³ where the volume of cell was found to be 2834.35 × 10⁶ pm³. It has been evident from the literature, that the peaks for graphite flakes are not appeared in the XRD pattern of GO which suggest that all the graphite was successfully converted into GO.

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Fig. 1

XRD pattern of synthesized GO.

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The XRD pattern of the un-calcined and calcined SNC and GO-SNC are given in the Fig. 2. The pattern for un-calcined nanocomposite beside a sharp signal at 36.55° ascribed to (5 5 1) plane is much noisy suggesting that most the sample is amorphous. However, upon heating, several reflections have been appeared in the diffractogram at 2 θ angle of 23.26, 33.06, 36.55, 42.46, 47.41 and 61.76 due to the miller planes (4 2 2), (4 4 4), (5 5 1), (6 6 0), (6 6 4) and (1200) respectively. All these diffraction bands are matched with reference card no 00–030-0496, confirm the synthesis of Cu, Ni and Sn nanocomposite with chemical formula of Cu₉NiSn₃ with cubic geometrical shape with space group of F-43 m. The length of all three coordinates of the cubic are of 1.801 nm with 90° of alpha, beta and gamma. The average crystallite size calculated by Debye-Scherrer equation is 14 nm, where 0.75 % imperfection was found in the crystal.

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Fig. 2

XRD pattern of both uncalcined and calcined SNC.

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The XRD pattern of the GO/ Cu₉NiSn₃ nanocomposite (both un-calcined and calcined) are shown in the Fig. 3, which displays the diffraction peak at 2 θ position as that were observed in the XRD spectrum of Cu₉NiSn₃ nanocomposite as shown in Fig. 3. No shift was seen in the position of diffraction bands is the evidence that the Cu₉NiSn₃ was only incorporated on graphene oxide sheet and chemical linkage was formed. However, the d-spacing value for the plane (5 5 1) is 2.523 Å in case of pure Cu₉NiSn₃ nanocomposite whereas this value decreases to 2.458 Å which confirm the incorporation of graphene oxide on the Cu₉NiSn₃ nanocomposite. This shifting of d-spacing to lower value is because of the reorganization of the lattice position after the mixing of GO and Cu₉NiSn₃ nanocomposite. As the ionic radii of the Cu²⁺, Ni²⁺ and Sn⁴⁺ is larger and the space between is larger and after the incorporation of GO, the carbon with small ionic radius adjusted between the metal atoms led the reduction of d-spacing. The average crystallite size increase from 14 nm to 57.45 nm and the lattice strain reduced to 0.59 %, proposed that after the incorporation of GO, the nanocomposite become more stable.

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Fig. 3

XRD pattern of both uncalcined and calcined GO-SNC.

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3.2 SEM analysis

The SEM image shown in Fig. 4(a) described the morphological features of the synthesized GO, where a layer type structure were detected. The multilayers structure of the GO is observed that has high surface roughness. The cavities/cracks are seen in the GO sheets which might be due to the grinding of the sample after dehydration. The morphological study of the as-synthesized and calcined Cu₉NiSn₃ nanocomposite was conducted through SEM analysis and the micrographs are shown in Fig. 4(b and c). The snowball-like structures (grains) that are observed in the micrograph (4b) can result from the aggregation of smaller particles, which are formed during the synthesis process.These snowball-like structures are irregular dispersion are clump together, forming larger structures led to the size vibration of the particles. The background of the sample shows high degree of agglomeration, which may be due to high surface energy of the particles which held the particles together. The calcination of sample at high temperature can cause the individual snowball-like structures (grain) in the sample to grow by diffusion-driven processes, such as grain boundary migration and recrystallization. The growth of the individual grains can result in an increase in the average grain size of the sample. The cavities of distinct size are formed due to the irregular arrangement of these grains. The grain size for as-synthesized estimated from the SEM images is ranging from 40.75 to 98.93 nm with average size of 84.55 nm, whereas those for calcined sample ranging from 68 to 123 nm with average size of 101 nm. The SEM analysis was also carried out after the incorporation of metals (Cu, Ni and Sn) in the GO matric to examine the effect on the morphology of the resultant composite. The obtained SEM images are shown in Fig. 4(d and e), reveals that the morphology of the sample is completely changed after mixing of metals and GO. The metal nanocomposite deeply incorporated in GO layer, which have changed the morphology of the sample. The as-synthesized sample shows that the grains are embedded in the GO layer, where majority of the cavities that seen in the Cu₉NiSn₃ NC (Fig. 4(b)) have been filled due to the deposition of GO. Upon calcination, the GO layer became more prominent and converted to a semi porous sheet and boundaries between copper nickel tin nanocomposite grains are almost disappeared.

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Fig. 4

SEM images of GO (a), as-synthesized (b) and calcined (c) of SNC; as-synthesized (d) and calcined (e) of GO-SNC.

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3.3 FTIR analysis

In order to describe the bond structure of rGO, calcined and uncalcined SNC and GO-SNC transmission spectra of KBr sample pellets were acquired using Fourier transforms infrared spectroscopy (FTIR). The Figs. 5, 6 and 7 shows the FTIR spectrum of the GO, SNC (both uncalcined and calcined) and GO-SNC (both uncalcined and calcined) in the range of 3800 to 550 cm⁻¹. In FTIR plot of GO (Fig. 5), two peaks were observed at 3404.10 cm⁻¹ correspond to the stretching vibration of O–H (N. Baali, A. Khecha 2019). The peaks at 2929.68 cm⁻¹ and 2834.47 cm⁻¹ indicated the symmetric and asymmetric stretching of the C–H group respectively (Parthipan et al. 2021). The peak at 2308.34 cm⁻¹ referred to the C = O group (O = C = O) the node at 1717.37 cm⁻¹ attributed to C = C vibrations (Ebnalwaled et al. 2019). The peaks located at 1607.38 cm^{−1 is} related to the C = C of aromatic ring whereas the peaks at 1381.66 cm⁻¹ and 1118.19 cm⁻¹ are assigned to C = C and C-O moieties, respectively (Alam et al. 2017). The peak at 600.27 cm⁻¹ is might be due to the out plane bending vibration of C–H moiety (Shahriary Leila and Athawale Anjali A. 2014; Yoo and Park 2019).

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Fig. 5

FTIR spectrum of the synthesized GO.

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Fig. 6

FTIR spectrum of the uncalcined and calcined SNC.

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Fig. 7

FTIR spectrum of the uncalcined and calcined GO-SNC.

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FTIR spectroscopic analysis of SNC (uncalcined and calcined) shows (Fig. 6) the peak in the range of 3524.18 cm⁻¹ shows the vibration of non-bonding water molecules adsorbed on the surface of sample (Shah et al. 2023). The band at 3473 cm⁻¹ corresponds to the stretching vibration of OH group due to the fact that calcined powder tends to physically absorb water. The intensity of these peak decrease after the calcination, suggest the evaporation of water molecules from the same. The peak at 1385.83 cm⁻¹ correspond to the C = O stretching vibration (El-Kemary et al. 2013). The peak attributed at 1044.60 cm⁻¹ correspond the Cu–O-Cu vibration (Sorbiun et al. 2018). The peak at 571.72 cm⁻¹ correspond to Ni-O stretching vibration (Haq et al. 2021c). The peak at 887.58 cm⁻¹ ascribed C–C vibration (Al-Hada et al. 2018).

The FTIR analysis of GO-SNC (uncalcined and calcined) shows a wide wave band centered at 3398.46 cm⁻¹ shows that O–H stretching vibration (Fig. 7) and the decrease intensity of this peak decrease after the calcination, suggest the evaporation of water molecules from the same. The peak at 1624.89 cm⁻¹ correspond to the C = O vibration (Haghshenas et al. 2019). The peak at 1490.30 cm⁻¹ ascribed C-O-C vibration (Haq et al. 2018b). The peak at 1362.47 cm⁻¹ due to the vibration of O-C = O group (Paquin et al. 2015). The peak at 1028.36 cm⁻¹ show the Cu–O-Cu vibration (Hamid et al. 2021). The peak at 544.63 cm⁻¹ attributed to the Ni-O vibration (Haq et al. 2021c). The peak at 799.27 cm⁻¹ show the Sn-O stretching vibration (Shoukat et al. 2019).

3.4 Antibacterial activity

The uncalcined and calcined of SNC and GO-SNC were tested against Gram-positive and Gram-negative bacteria. Agar well diffusion method was used to evaluate the antibacterial activity of samples under study. E. coli and S. aureus were selected as GNB and GPB respectively. The inhibition zones showing (Fig. 8) bactericidal activity were measured in millimeter (mm) show in Table 1. The microbial activity of samples was analyzed at different concentrations of 10, 20, 30, 40, 50, and 60 µg/mL. It was examined that bacterial activity was directly related to the concentration of samples. As in aqueous solution, metallic nanocomposites form metal cations, hydroxyl radical, superoxide radical anions. The cations are reactive towards the thiol group of fundamental bacterial enzyme. This interaction caused the inactivation of cell activities which lead to the cell death. However, radicals thus formed interact with the cell surface which is negatively charged. Therefore, cell wall ruptured and caused loss of cell content which inhibited cell growth (Haq et al. 2021a). Likewise, aqueous suspensions of tested samples generated reactive species to more extent by increasing concentration per mL. This resulted in increase in the inhibition capacity of the sample against bacteria.

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Fig. 8

Antibacterial activity of uncalcined and calcined SNC and GO-SNC; H = -ve control (solvent), AB=+ve control (Antibiotic), GO = graphene oxide, G = GO-SNC, WG = SNC, GC = calcined GO-SNC, and WGC = calcined SNC.

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It was observed that GO-SNC showed higher antibacterial activity then SNC against GNB and GPB. GO supported metallic nanocomposites show higher antibacterial activity as compared to bare metallic nanocomposites (Ahmad et al. 2016). The oxygen vacancy present at the GO surface which can act as sorption center of water molecules will react with water molecules thus resulting in the formation of the hydroxyl radicals. The direct contact of graphene sheets edges and metal ions ruptures the bacterial cell to form pores. In this way there occur losses of cytoplasmic material which inhibit the growth of bacteria. Furthermore, bacteria’s can be entrapped into graphene sheets which causes inactivation of bacterial cells. Moreover, GO sheets trap the bacterial cell and isolate it from its environment. In this way, bacterial growth is retarded due to inactivation of cell system (Ji et al. 2016).

The zones of inhibition of calcined samples of SNC and GO-SNC were reduced as compared to uncalcined ones. The potential explanation of this behavior could be the particle size. Upon calcination, particle size increased and surface area to volume ratio decreased. As a result, reactive sites of the particles reduced which gave rise to the less interaction of NCs with the bacterial surfaces. While in uncalcined samples, surface area of the NCs was large because of the smaller size of nanoparticles. Consequently, NCs strongly interacted with the bacterial cell wall. In this way, bacterial cell death became easier and maximum zones of inhibition were observed (Hamid et al. 2021).

The prepared NCs showed greater zones of inhibition against GNB as compared to GPB. It is attributed to the variation in chemical composition of each bacterial cell wall. GPB have thick layer of peptidoglycan in cell wall which strongly resist the penetration of foreign particles into the cell. Whereas the outer layer of GNB contains thin layer of peptidoglycan and its extent of resistance is low which led to the penetration of particles through the cell wall. Another possible reason for enhanced activity of NCs against GNB was due to the strong interaction of metal cations with the highly negative surface of bacteria. The teichoic acid in the cell wall of GPB makes its surface to be partially negative, thus have weaken interaction between cations and bacteria. The less interaction is responsible for the increased resistance of GPB against the tested samples (Haq et al. 2021b).

The obtained antibacterial activity results against both bacteria were compared with available the literature. The GO based trimetallic oxide nanocomposites are not reported yet. However, antibacterial activity of GO based monometallic and bimetallic nanocomposites against both E. coli and S. aureus are reported and the zones of inhibition are listed in Table 2. The listed results shows that the GO based trimetallic nanocomposite reported in this study is more active than the composites reported in literature.

3.5 Antioxidant activity

The manifestation of free radical scavenging potential of molecules against sustained free radical reduction is the key principle of antioxidant action. The antioxidant potential of prepared NCs was analyzed against DPPH and ABTS free radicals at different concentrations as shown in Table 3 and Table 4. Both of these unstable free radicals were reduced by the target sample. Therefore, reduction leads to the stabilization of the unstable free radicals. With increasing the concentration of the sample, the absorbance was found to be decreasing. Decrease in absorbance revealed the free radical scavenging activity of the sample. Accordingly, percentage radical scavenging activity of the sample kept on increasing. It was observed that antioxidant activity of the sample increased with increasing the sample dose. This increased antioxidant activity is attributable to a greater concentration of sample that can oppose more reactive oxygen species (DPPH and ABTS) (Haq et al. 2020).

It was also observed that GO-SNC nanocomposite show more antiradical efficacy as compared to SNC. This might be explained by the production of a radical adduct at sp² carbon sites, which then results in radical annihilation. The production of a second adduct occurs as a result of spin delocalization across the conjugated backbone of graphene. Electron transport and hydrogen donation from hydroxyl functional groups connected to graphene are also significant. Despite the fact that the basal plane of graphene acts as the primary scavenging location, hydrogen donation from oxygen-containing functional groups is still a significant factor in radical scavenging instance (Hastak et al. 2018). GO-SNC had higher antioxidant activity than SNC, which might be attributed to synergetic effects between SNC and GO. Because of the characteristics of SNC and GO, GO-SNC might form reactive oxygen species. These species donate electrons to the free radicals thus making them stable (Shadmehri et al. 2019).

The antioxidant results were obtained against both DPPH and ABTS free radicals. In both cases, calcined and uncalcined samples of SNC and GO-SNC showed increased in percentage scavenging activity with increasing sample dose. In comparison to calcined sample, the percentage scavenging activity of uncalcined sample was greater. The IC₅₀ value of a sample refers to the concentration of that sample required to scavenge the 50% of free radical. The uncalcined samples have less IC₅₀ value as compared to calcined sample. Comparatively, samples having less IC₅₀ values reveal higher antioxidant potential. In case of both scavenging assays, the uncalcined sample showed higher antioxidant activity. This is because the particle size increases with the rise in temperature. Increase in the particle size leads to decrease in number of nanoparticles in solution. Due to the less number of particles, less active sites will be available to stabilize the free radicals (Haq et al. 2021c).