Synthesis and characterization of Ag2O, CoFe2O4, GO, and their ternary composite for antibacterial activity

Currently, nanomaterials with exceptional antibacterial activity have become an emerging domain in research. The optimization of nanomaterials against infection causing agents is the next step in dealing with the present-day problem of antibiotics. In this research work, Ag2O, CoFe2O4, and Ag2O/CoFe2O4/rGO are prepared by chemical methods. Ag2O was prepared by co-precipitation method, while solvothermal technique was utilized for the synthesis of CoFe2O4. The ternary nanocomposite was synthesized by a simple in situ reduction using a two-step approach. The structural and morphological properties were studied by UV–Vis spectroscopy, X-ray diffraction spectroscopy (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (SEM), dynamic light scattering (DLS), and Fourier-transform infrared spectroscopy (FTIR). From the X-ray diffraction analysis, the crystallite size is found to be 14 nm, 5 nm, and 6 nm for Ag2O, CoFe2O4, and Ag2O/CoFe2O4/rGO respectively. The synthesized nanomaterials were investigated for antibacterial activities against gram-positive strain Staphylococcus aureus (S. aureus) and gram-negative strain Escherichia coli (E. coli) using Agar well diffusion method. Ag2O and CoFe2O4 showed zones of inhibition (ZOI) of 13 mm and 11 mm against gram positive bacteria while 12 mm against gram negative bacteria respectively, while ternary nanocomposite showed 14 mm and 13 mm of ZOI. The antibacterial activity of nanomaterials showed a gradual increment with an increase in the concentration of the materials. Ag2O, CoFe2O4, and Ag2O/CoFe2O4/rGO showed minimum inhibitory concentration (MIC) values of 4.5, 6.5, and 4.5 μg/mL for S. aureus and 6.5, 7.2, and 4.8 μg/mL for E. coli respectively. Minimum bactericidal concentrations were found to be same as the MIC values. Additionally, a time-kill curve analysis was performed and for ternary nanocomposite; the killing response was most effective as the complete killing was achieved at 3 h of incubation at 3-MIC (9.75 μg/mL). These results demonstrate that all the nanomaterials, as a kind of antibacterial material, have a great potential for a wide range of biomedical applications.


Introduction
According to the US Centers for Disease Control and Prevention (CDC), more than 33,000 deaths are attributed to infections with antibiotic-resistant bacteria in the European Union alone. Tragically, the human world is standing at the threshold of a "post-antibiotic era," where the toll of death from bacterial infection will be greater than that of cancer, as stated by the CDC (Cassini et al. 2019). The unguided and unchecked use of antibiotics is to be blamed for the pathetic situation we are facing today. The evolution of antibacterial resistance and the appearance of genetically mutated resistant bacteria are the main causes of new infections (Christaki et al. 2019). Due to the worldwide rise of drugresistant bacterial pathogens, the efficacy of conventional antibiotics is falling. Failure of antibiotics is an increasingly urgent problem to humanity leading to lack of therapy for serious infections (Aslam et al. 2018). Even if a new antibiotic is launched, the resistance of bacteria appears. Alternative strategies need to be found as soon as possible. Since antibiotic-resistant bacteria have increased dramatically, there is a need for alternate strategies and new antibacterial agents that can fight against these resistant bacterial strains (Gupta et al. 2019).
Development of novel antibiotic agents has become an essential with the continuous emergence of bacterial resistance. Nanomaterials cause lethal injuries to the pathogens by acquiring a number of different mechanisms (Gao and Zhang 2020). A number of nanomaterial-based antimicrobials have been reported. Among these agents, metallic NPs are very significant due to their strong antibacterial activity. Various metals, metal oxides, and mixed metal oxides have been reported to show antibacterial properties against different bacterial species. Silver (Ag), gold (Au), copper (Cu), cobalt (Co), zinc (Zn), and titanium (Ti) metals, as well as oxides and ferrites of different metals, have also been reported to exhibit an antimicrobial effect (Sánchez-López et al. 2020). Metal oxides have shown wide potential applications in biomedical field, for example, ZnO (Abebe et al. 2020) (Tabassam et al. 2022). MgO (Fouda et al. 2021); Ag 2 O (Dharmaraj et al. 2021), TiO 2 (Khashan et al. 2021), Ag-MnFe 2 O 4 (Ning et al. 2020), NiO-ZnO (Paul et al. 2021), have shown good antibacterial activities. Different types of nanoparticles show different levels of antibiotic activity. Their unique physical and chemical properties can lead to fine-tuned interactivity between them and bacteria. Graphene derivatives have also been reported to show antibacterial activity, which suggests that they could hold a high place in the biomedical field (Kumar et al. 2019;Cao et al. 2021). Talapko et al. reported that silver is one of the strongest known metals regarding antimicrobial properties (Talapko et al. 2020). Silver oxide comes under metal oxide nanoparticles and is world-known for having extensive applications in a number of fields such as optical devices, cancer therapy, biological, electrochemical, sensors, catalytic reduction, and cosmetics (Tafida et al. 2020;ur Rahman et al. 2020;Maheshwaran et al. 2020;Rashmi et al. 2020;Ahmed et al. 2020;Iqbal et al. 2021). Silver oxide (Ag 2 O) nanoparticles are also known for its strength in decolouring dyes and for cleaning organic pollution from water (Rahnama et al. 2021;Zhou et al. 2020). Cobalt ferrite (CoFe 2 O 4 ) nanoparticles, with their unique crystal lattice organization, offer prominent magnetic properties such as magnetic anisotropy, saturation magnetization, and coercivity. Because of their enhanced magnetic properties, they can be used in drug delivery, imaging and diagnostics, magnetic hyperthermia, magnetic extraction, separation, and biosensors (Srinivasan et al. 2018;Amira Alazmi et al. 2019;Fayazzadeh et al. 2020;Ghiasi and Malekpour 2020;Vajedi and Dehghani 2020).
To enhance the antibiotic power of nanoparticles, combinational approach provides us a pathway where bacteria find it hard to form resistance. Binary and ternary heteromaterials increase surface area and thus improve the antibacterial efficiency in comparison to unary nanomaterials. The ternary heterojunction can magnify the antimicrobial power, further more binary metal nanocomposites (Wang et al. 2017). Recently, there is a heightened interest in magnetic nanoparticles for biomedical applications. Magnetic nanoparticles with bactericidal effect have also been studied (Ning et al. 2020;Shatan et al. 2019). Cobalt ferrite nanoparticles were found effective for their magnetoantibacterial properties (Meidanchi 2020).
In this paper, Ag 2 O and CoFe 2 O 4 nanoparticles were fabricated using facile chemical reduction and a modified solvothermal technique respectively. The modified Hummers' method was employed for GO synthesis. The ternary nanocomposite Ag 2 O/CoFe 2 O 4 /rGO is synthesized by a simple two-step process. The synthesized materials were characterized using UV-Visible spectroscopy, X-ray diffraction (XRD) technique, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy, Fourier-transform infrared (FTIR) spectroscopic technique, and energy-dispersive X-ray (EDX) spectroscopy. The nanomaterials were investigated for their antimicrobial potential against Staphylococcus aureus and Escherichia coli. The union of magnetism, biocompatibility, and nanoknife effect of rGO would make the Ag 2 O/CoFe 2 O 4 / rGO ternary nanocomposite a technically brilliant material in biomedical applications.

Synthesis of silver oxide nanoparticles
Simple wet chemical approach was used to synthesize nanoparticles of silver oxide. In this method, firstly, a solution of silver nitrate was prepared. 150 mL of 10 mM solution of AgNO 3 was heated up to 70 °C, and 0.056 g of sodium borohydride was added to this solution of silver salt slowly. The mixture was kept stirring, while the temperature being maintained at 70 °C. After 5 min, 10 mL of freshly prepared 0.2 M NaOH was added which lead to formation of blackbrown precipitates. The mixture was stirred for half an hour. After 24 h aging, the precipitates were washed using DI water till neutral pH was obtained and then dried at 110 °C.

Synthesis of cobalt ferrite nanoparticles
A modified solvothermal technique was used to obtain cobalt ferrite nanoparticles (Liu et al. 2018). Ethylene glycol was selected as the solvent. 0.405 g of FeCl 3 .6H 2 O and 0.178 g of CoCl 2 .6H 2 O were used to form a solution in 150 mL of ethylene glycol (EG) by sonicating the mixture for 1 h. After sonication, the colour turned bright orange. Then, 6.15 g of sodium acetate was put in the solution, and it was magnetically stirred for another hour, and the mixture gave a reddish orange tint. The mixture was poured into a 200-mL Teflon autoclave and placed in the oven for 4 h at 180 °C.
Precipitates were collected, washed using DI water, and then dried at 110 °C.

Synthesis of graphene oxide
Graphite powder was used as a precursor for the synthesis of GO by utilizing the well-known modified Hummer's method (Iqbal et al. 2021). Firstly, in an ice bath, 90 mL of H 2 SO 4 was mixed with 10 mL of H 3 PO 4 (volume ratio of 9:1) and mixed using a magnetic stirrer for some time at 10 °C. In this mixture, 0.75 g of graphite powder was introduced while being stirred and kept in an ice bath. When it was homogenized, 4.5 g of KMnO 4 was added gradually. After the addition of KMnO 4 , it was kept stirring for 6 h without disruption. This resulted in a dark green slurry. The dropwise addition of 0.75 mL of H 2 O 2 leads to the removal of excess KMnO 4 . The mixture was stirred for another 10 min. As a result of the H 2 O 2 addition, an exothermic reaction occurred, and an ice bath was used to cool it down. Upon cooling, 25 mL of HCl was added, followed by 75 mL of DI water. The resultant suspension was centrifuged and washed at 12,000 rpm till a neutral pH was obtained. The washed GO was dried in the oven for 24 h at 60 °C. After this, the dried GO obtained was ground to obtain a fine GO powder ( Fig. 1). This GO was then reduced chemically using NaBH 4 . GO suspension containing 0.5 mg GO per mL of the water was prepared via sonication. 1.14 g of NaBH 4 was Fig. 1 a The process of GO synthesis. b GO slurry after centrifugation. c, d GO after drying put in 100 mL of GO suspension. The prepared mixture was then magnetically stirred for 12 h at room temperature to get reduced graphene oxide. Later, the prepared rGO was washed using deionized water multiple times and dried at 110 °C temperature (Yang et al. 2015).

Synthesis of Ag 2 O/CoFe 2 O 4 /rGO nanocomposite
Ag 2 O/CoFe 2 O 4 /rGO nanocomposite was prepared by twostep approach reaction of AgNO 3 , CoCl 2 .6H 2 O, FeCl 3 .6H 2 O, and GO. Firstly, in 100 mL of ethylene glycol, 100 mg of GO was dispersed by ultrasonically treating the mixture for 1 h. Next, 8 mg of AgNO 3 was put into the suspension and then magnetically stirred so that homogeneous solution was obtained. Into this system, 100 mL of NaBH 4 /EG (38 mg/ mL) was dropped. Magnetic stirrer was used to stir the mixture. Few minutes later, precipitates were obtained. After centrifugation, the precipitates of Ag 2 O/rGO nanocomposite were washed and separated for further use. Subsequently, for the preparation of Ag 2 O/CoFe 2 O 4 /rGO ternary nanocomposite, 8 mg of CoCl 2 .6H 2 O and 20 mg of FeCl 3 .6H 2 O were dispersed in 70 mL of EG. Next, 2.38 g of sodium acetate was added. After this, magnetic stirring was performed for half an hour. Then as prepared Ag 2 O/rGO nanocomposite was added while vigorously stirring the solution. The mixture was shifted to a stainless-steel based autoclave (Teflonlined) and kept for 4 h in oven at 180 °C. Then, centrifuged and washed several times before drying in oven at 110 °C.

Characterization
The synthesized nanomaterials were characterized using UV-Vis, XRD, SEM, EDX, DLS, and FT-IR spectroscopic analysis. The synthesized nanomaterials were characterized by a UV-Vis spectrophotometer (1601 Shimadzu). An X-ray diffractometer (JDX-3532 (JEOL), Japan), over the 2θ range of 10-80°, was used to analyse phase structures with radiation at 40 kV and 20 mA. The SEM model used was JSM 5910 (JEOL), Japan. On a double stick tape, first the samples were deposited and then placed in an aluminium sample holder. Before analysis, they were also sputter-coated in gold in a sputter coater model SPI-Module (USA) at 30 mA for 90 s. The instrument used for this technique was an EDX microanalyser model INCA 200 (UK).

Antibacterial assay
Antibacterial activities were performed against the selected bacterial strains using the synthesized nanomaterials. In this study, gram-positive bacteria "Staphylococcus aureus" (ATCC # 6538) and gram-negative "Escherichia coli" (ATCC # 8739) were selected for anti-bacterial activity. An agar well diffusion assay was used in order to obtain a qualitative evaluation of the antibacterial effects of synthesized materials. Agar solution was prepared and spread on the plates, and then, bacterial cultures were placed on the plates using a cotton swab. After that, three holes (3 mm in size) were punched in the plate by a sterilized cork borer. Levofloxacin was used as a standard material. Each of the three holes received 10 μL of 500 μg/mL Ag 2 O, 1000 μg/L Ag 2 O, and standard antibiotic. To avoid the spillage of the solution over the plate, extreme care was taken. More plates were prepared in which CoFe 2 O 4 and Ag 2 O/CoFe 2 O 4 /rGO were added in the same way. The plates were then placed in an incubator for incubation for 24 h at 37 °C. After that, the zone of inhibition was measured and noted after a growth period of 24 h.

Determination of MIC, MBC, and time-kill curve
The minimum inhibitory concentration (MIC) of nanosized Ag 2 O, CoFe 2 O 4 , and Ag 2 O/CoFe 2 O 4 /rGO were performed by the method described in the literature (Elemike et al. 2017). Standard broth microdilution method was used to study MIC in 96-well round bottom microtiter plate by taking 100 μL from 500 μg/mL of Ag 2 O, CoFe 2 O 4 , and Ag 2 O/ CoFe 2 O 4 /rGO each. The concentration was then diluted twofold with the bacterial inoculums in 100 μL of Mueller Hinton Broth (MHB). In the polystyrene plate, column 1 was allocated for the negative control where only medium was placed, whereas column 2 was loaded with positive control containing both medium and bacterial inoculums. The column 3 was loaded with the least selected concentration, whereas column 12 contained the highest concentration of nanomaterial. Afterward, 25 mL of resazurin solution (0.002 g of resazurin salt powder in 10 mL of distilled water) was added in each well and incubated at 37 °C for 24 h. The bacterial growth will be confirmed by the appearance of pink colour, while purplish or bluish colour will indicate no bacterial growth in the medium. The minimum bactericidal concentration (MBC) was performed on Mueller-Hinton Agar (MHA) plates. The MBC value was taken by considering the lowest concentration of nanomaterials taken where no visible growths on the MHA plate were detected.
Time-kill assay was performed in MHB medium by the method as reported elsewhere (Parada et al. 2022). The bacterial inoculums were set at 10 6 CFU/mL. To obtain the concentration of 0-MIC, 0.5-MIC, 1-MIC, 1.5-MIC, and 3-MIC, the solutions of Ag 2 O, CoFe 2 O 4 , and Ag 2 O/CoFe 2 O 4 /rGO were diluted with MHB media containing bacterial inoculums. The cultures were then incubated at 37 °C with stirring speed of 120 rpm. The cultures (100 mL) were spread over MHA plates at time 0, 10, 20, 30, 60, 120, 180, 240, and 480 min. The growth of colonies on MHA plates were measured for 24 h incubated at 37 °C in CFU/mL. The experiments were performed in triplicate.

Results and discussion
Characterization X-ray diffraction analysis XRD results of synthesized material are presented in Fig. 2. For each of them, the particle size of nanocomposite has been calculated by Scherrer formula: where "θ" shows Bragg angle, "β" is full width half maximum, and "λ" shows wavelength of X-rays used during the diffraction process.
The 2θ value observers for the synthesized silver oxide are 34.83°, 43.65°, 63.03°, 67.75°, and 75.87°. These diffraction peaks belong to the 111, 200, 220, and 311 crystal planes. The crystallite size calculated using Scherrer equation is 14 nm approximately. Similar findings were reported by Sangappa and Thiagarajan (2015). The diffraction peaks observed are in perfect match with the JCPDS card no. 76-1393. Cobalt ferrite showed diffraction peaks 18.0°, 29.6°, 35.4°, 43.0°, 53.0°, 56.6°, and 62.2° 2θ values. These peaks can be assigned to (111), (220), (311), (400), (422), (511), and (440) crystal planes of CoFe 2 O 4 respectively. The observed XRD peaks were indexed to a cubic crystal structure. The results are found to be in consistence with the reported data (Wang et al. 2015). The crystallite size calculated for the nanoparticles was found to be 5 nm approximately. The diffraction peaks observed are in perfect match with the JCPDS card no. 22-1086. For GO, 10.15° 2θ value is observed. The lattice spacing for graphene oxide is found to be 0.87 nm. The results are found to be close to that reported by Bahrami et al. where the peak was found to be at 10.31°. However, another peak was seen showing the partial reduction of GO into rGO in their diffractogram (Bahrami et al. 2019). The characteristic diffraction peak associated to rGO appeared at 2θ value 25.4°. No peak for GO is observed around 10° showing that no GO is left unreduced. A very high quality rGO synthesized by Some et al. showed the peak at 25.6° 2θ value (Some et al. 2013). For rGO, the lattice spacing was found to be 0.35 nm. This value is found to be very close to that of pristine graphene, 0.337 nm as reported by Batakliev et al. (2019). The 2θ peaks observed in the synthesized ternary nanocomposite are 24.4°, 43.36°, and 31.5°. The two broad peaks correspond to the rGO. The peaks for the other two components have small intensities due to much less quantity of silver oxide and cobalt ferrite (1) D = 0.89 ∕ Cosθ used. The peaks are somewhat masked by the two broad peaks of rGO. The small contents of these two components are also visible from the EDX of the material. The crystallite sizes were calculated using Eq. (1) given above.

UV-visible spectroscopy
UV-Visible spectroscopy was performed for each nanomaterial synthesized (Fig. 3). The band gap energy is also determined from these absorbance spectra by using Tauc method based on the assumption that energy-dependent absorption coefficient α can be expressed as: Here, h is Planck constant, E g means band gap energy, ν denotes photon's frequency, and B is a constant. In this equation, γ factor is dependent on nature of electron transition and its value is 1/2 for direct and 2 for indirect transition band gaps (Makuła et al. 2018). The UV-Vis spectroscopy was performed from 300 to 800 nm range. The maxima for silver oxide was found to be at 410 nm, while it was reported 430 nm by Shume et al. (2020). For silver oxide, band gap was 1.83 eV. The band gap of Ag 2 O can lie in a wide range of 1.2 to 3.4 eV (Makuła et al. 2018). For cobalt ferrite, the absorption maxima is found to be at 348 nm (Nithiyanantham et al. 2021). The band gap was measured equal to 2.44 eV which is near 2.41 eV found by Shubra et al. (Shubhra et al. 2021).
GO showed one absorption peak at 230 nm and a shoulder at 270 nm. Gurunanthan and co-workers also reported a peak at 230 nm, while the shoulder was seen at almost 300 nm. The band gap was found to be 4.3 eV. For rGO, the maxima were found to be at 270 nm. The band gap was found to be 3.9 eV (Gurunathan et al. 2012). The ternary nanocomposite showed absorption at 260, 340, and 385 nm. The peaks may be attributed to rGO, cobalt ferrite, and silver oxide. The band gap was found to be 2.05 eV. Band gaps were determined by using Eq. (2) mentioned above. Figure 4 represents the SEM images of silver oxide, CoFe 2 O 4 , GO, and their ternary system. The scanning electron micrograph of silver oxide suggests that the surface morphology of silver oxide is quite uniform and very well defined. The nanoparticles of silver oxide are almost spherical in shape (Banua and Han 2020). The SEM image of CoFe 2 O 4 reflects the grown nanoparticles are well developed, with uniform morphology. The figure depicts the homogeneous size distribution of particles that makes sure its nanocrystalline nature. Due to the magnetic interactions and high surface energy of CoFe 2 O 4 nanoparticles,

Scanning electron microscopy
aggregation of NPs was seen (Ensafi et al. 2017). The micrograph of GO shows the surface morphology reveals that GO consisted of many flakes having both crumpled and stacked ones. There were also closely associated cavities present. It was observed that wrinkles were present across GO layer because of its crumpling nature (Iqbal et al. 2021). It was observed in images of rGO that reduced GO contains many stacked layers with silky folded flakes. It exhibited that the efficient reduction of GO and formation of ultrathin sheets. In the SEM image of the ternary nanocomposite, the sheets of the reduced graphene oxide can be seen twisted and folded with a rough surface and sharp edges. Wrinkling on rGO was present because of the rapid removal of oxygencontaining functional groups in GO and adherence of nanoparticles on the surface. Some of the nanoparticles are also entrapped inside the crumpled rGO sheets.

Energy-dispersive X-ray spectroscopy
EDX was performed to determine the elemental composition of the prepared materials (Fig. 5). The EDX spectrum of silver oxide showed a larger amount of silver and oxygen than expected, which indicates the formation of Ag 2 O. No impurities on the surface of the nanoparticles was found, showing the purity of the synthesized nanoparticles. Ag and O were found in 98.28% and 1.72% weight percentages respectively.
In the EDX spectrum of cobalt ferrite, the major presence of Co, Fe, and O is obvious. The other elements seen might come from the precursors used. From the elemental percentage, cobalt, iron, and oxygen can be seen in percentages of 16.65, 48.07, and 21.85 respectively. The EDX spectrum of the synthesized ternary nanocomposite shows the presence of all constituent elements: carbon, oxygen, silver, cobalt, and iron, and their elemental percentage is found to be 73.43%, 14.04%, 0.22%, 2.13%, and 4.06% respectively The intensity of metal peaks is small due to their smaller ratio, which is also clear from their weight percentage.

FTIR spectroscopy
The FTIR spectra (Fig. 6) clearly define the absorption bands detected in the samples. Prominent bands for silver oxide were seen at 3380 cm −1 and 545 cm −1 . Among these, the bands at 3380 cm −1 correspond to H-O-H stretching vibration of water molecules. FTIR spectrum of synthesized Ag 2 O nanoparticles displays the characteristic band due to lattice vibration of silver oxide at 545 cm −1 (approximately) which shows the successful synthesis of silver oxide. The band near 1380 cm −1 may correspond to nitrate vibrations. The metal-oxygen stretching frequencies are observable in the range of 500 to 600 cm −1 (Ravichandran et al. 2016) (Shume et al. 2020). The band at 545 cm −1 is very close to one reported by Siddiqui et al., i.e. 550 cm −1 (Siddiqui et al. 2013). This further verified the compounds as silver oxide particles in addition to the XRD analysis conducted. In the FTIR spectra of cobalt ferrite, bands at 3433, 1635, 619, and 447 cm −1 were observed. The bands at 3433 cm −1 and 1635 cm −1 represent the stretching and bending vibration mode of O-H bond by water molecule absorbed by the surface of nanoparticles during the synthesis process. The bands that appeared at 619 and 447 cm −1 represent the characteristic bands of Fe-O and Co-O metal-oxygen bonds which confirms the successful synthesis of CoFe 2 O 4 nanoparticles. The absorbance bands exhibited the formation of cobalt ferrite phase with spinel structure. Spinel ferrite has face-centred cubic structure. The result is in agreement with the findings of Shanmugavel et al. (2014) and Oliveira et al. (2013). Following bands are observed in the spectrum of GO: 3450, 1716GO: 3450, , 1635GO: 3450, , 163, 1216GO: 3450, , and 1072   to be very close to that reported by Sim et al. (2014). In the rGO spectrum, the characteristic bands at 3360, 1739, 1600, 1365, 1280, and 1014 cm −1 were because of O-H stretching, C = O stretching, aromatic C = C stretching, O-H deformation, epoxy C-O stretching, and alkoxy C-O stretching vibrations respectively. The intensity of bands at 3360, 1739, and 1365 cm −1 was highly lowered downed, showing the removal of oxygen-containing functional groups to specific extent as reported by Yang et al. (2015). The C = C band at 1620 cm −1 was present in rGO spectrum; it is inferred that sp 2 hybridized structure of carbon was retained and these findings are consistent with the results of Xu et al. (2015). For the ternary nanocomposite, the peak formed at 1728 cm −1 represents stretching of C = O of COOH present on the edges of sheet. The decrease in intensity was seen because of the decomposition of the aforementioned groups by hydrothermal treatment. The band at 615 cm −1 shows Co-O bond vibration. A very slight band can be seen at 511 cm −1 which represents the metal oxygen bond. At 418 cm −1 stretching, vibrations of Fe-O can be seen.
The decreased intensities of oxygen containing functional groups confirm conversion of GO into rGO. The metal oxygen bands between 400 and 650 cm −1 represent the presence of Ag 2 O and CoFe 2 O 4 . The results confirm the formation of the ternary nanocomposite showing metal-oxygen bonds and reduced graphene oxide. The results are found to be close to that reported by Khan et al. (2020) and Iqbal et al. (2021).

ZOI, MIC, and MBC values
The Ag 2 O, CoFe 2 O 4 , and Ag 2 O/CoFe 2 O 4 /rGO synthesized were tested for antimicrobial efficacy via the Agar well diffusion assay (Fig. 7). The antibacterial efficiency was identified through the appearance of an inhibitory zone around the well. The antibacterial action was studied against gram positive Staphylococcus aureus and gramnegative Escherichia coli using levofloxacin as a standard. The inhibition against the used bacterial strains was visible after incubation for 24 h. From the pictures, it is obvious that all nanomaterials can give a good zone of clearance against all three bacteria. S1 and S2 represent the two concentrations used for the antibacterial activity for these two bacteria, i.e. 500 µg/mL and 1000 µg/mL respectively. Table 1 shows the inhibitory zones in mm and MIC and MBC in µg/mL. The antibacterial activity against S. aureus was found for silver oxide, which has a higher inhibitory power as compared to that of cobalt ferrite and is proven by their zones of inhibition. At 500 µg/ mL, Ag 2 O shows a zone of inhibition of 4 mm, while there is no zone of clearance seen for CoFe 2 O 4 at this concentration. After increasing the concentration two times, a significant increase in the zone of clearance is seen.  Figure 9 represents the proposed bactericidal mechanism. The main mechanisms of bacterial killing of metallic  F nanoparticles include changes in energy transduction, photocatalysis, stopping synthesis of cell membrane, toxic reactive oxygen species (ROS) production, and reduced DNA production, and it is hypothesized that NPs target numerous biomolecules at once; thus, they might lessen or stop the evolution in resistance of bacteria (Slavin et al. 2017;Shaikh et al. 2019). For Ag 2 O, three main mechanisms have been proposed, i.e. oxidative stress by ROS, Ag + interaction with thiol groups in proteins and the complete damage of the bacterial cells caused by strong interaction between silver ion and cell membrane (Li et al. 2021). For CoFe 2 O 4 , antibacterial activity takes place by the discharge of ROS such as superoxide (O 2 − ) and hydrogen peroxide (H 2 O 2 ) from the surface of nanoparticles which play a magnificent role in bacterial destruction through penetration in their cell wall (Gheidari et al. 2020).

Mechanism of antibacterial activity
In fighting with the bacteria, the mechanism of rGO is similar to that of graphene and GO, i.e. sharp nanosheets of GO and rGO cause membrane stress, leading to high antibacterial activities and fragmentation of DNA due to an increase in ROS (Shi et al. 2016). As seen in SEM (Fig. 3f), the nanocomposite has randomly stacked rGO flakes with sharp edges which act as "nano knives" and puncture the bacterial cell wall and the membrane (Selim et al. 2020). This may cause the injury to the lipid bilayer and form van der Waals and hydrophobic interactions with the rGO and charge nanoparticle species on its surface. Consequently, the microbes may lose membrane integrity which, in turn, can impair many life processes, such as respiration, osmotic balance, materials transport, and energy transduction (Toasin et al. 2020).

Conclusion
In this study we synthesized nanoparticles by simple and facile methods. The ternary nanocomposite was synthesized by two step synthetic route. The crystallite size of the synthesized nanomaterial was found to be 14 nm, 5 nm, and 5 nm for Ag 2 O, CoFe 2 O 4 , and Ag 2 O/CoFe 2 O 4 /rGO respectively with well-crystallized structure. UV-visible spectra showed significant changes in the band gap. XRD spectra confirmed the phase purity. SEM showed the surface morphology at different resolutions. Different nanomaterials showed the respective functional groups through FTIR spectroscopy. The nanomaterials showed effective antibacterial effects against the bacteria under study via agar well diffusion method. The antibacterial effect of the nanomaterials was found to be concentration dependent. Small MIC and MBC values found show the high efficacy of the materials. It was found that the amount of the nanocomposite necessary to inhibit E. coli and S. aureus was reduced by Fig. 9 Antibacterial mechanism of Ag 2 O/CoFe 2 O 4 /rGO nanocomposite approximately 1.5 times as compared to CoFe 2 O 4 . Ternary nanocomposite Ag 2 O/CoFe 2 O 4 /rGO has high prospect as an antimicrobial agent to reduce the growth of bacteria. The time-kill curve showed that ternary nanocomposite requires the minimum time (3 h of incubation) for the highest killing response at lowest concentration 9.75 μg/mL. Our results pointed out that the Ag 2 O/CoFe 2 O 4 /rGO nanocomposite can be proposed as efficient tool to inhibit microbial populations. Moreover, considering its magnetic nature, more studies should be conducted to determine the reusability of ternary nanocomposite.