Copper oxide–ferric oxide nanocomposite: Synthesis, characterization, and antibacterial and antifungal properties

Iron metal powder (>99%), copper nitrate (Cu(NO₃)₂ [>99%]), and sodium hydroxide (NaOH [>99%]) were acquired from Belaqmi Fine Chemicals, India. Bi-distilled water was used throughout the experiments. In summary, a 0.1 M solution of Cu(NO₃)₂ was initially prepared, followed by the addition of 4 g of iron metal powder and 10 g of NaOH. The mixture was agitated for 10 min at ambient temperature. A Teflon-coated steel autoclave was used to confine and mature the mixture for 24 h at 120°C. Subsequently, the acquired materials underwent multiple rinses with distilled water and were subsequently dried at a temperature of 60°C overnight.

The crystallographic phase of the samples and their crystal size were studied by XRD with Cu-Kα radiation (Shimadzu 7000, USA). Various vibration modes, formations, chemical bonds, and function groups were determined by Fourier-transform infrared spectroscopy (FTIR), with infra-red spectra collected from 400 to 4,000 cm⁻¹ (Shimadzu FTIR-8400s, Japan). This is used to identify the presence of functional groups in the obtained nanoparticles. The morphological structures and elemental analysis of the resulting nanoparticles were examined by transmittance electron microscopy (TEM; JEOL, JEM2100 plus, Japan).

The fungal strains were isolated from infected strawberry plants, and the fruits showed signs of rot and gray mold. The morphological recognition of the fungal strains was performed using the identification manual illustrated by Campbell et al. [30]. The genus of fungal isolates was identified using a light microscope [31]. Amplification of the “Internal Transcribed Spacer” (ITS) area of the ribosome-encoding genes was used for the molecular identification of the fungal isolates by universal primers for ITS1 and ITS4 [32]. The polymerase chain reaction (PCR) was conducted as previously reported [33]. The final PCR products were refined and subjected to a Sanger sequencing machine. The fungal isolates were BLAST reconnoitered, and their identified sequences were coded by accession numbers using the NCBI database’s GenBank portal.

The tested bacterial strains of Pectobacterium carotovorum (OQ878656), Streptomyces scabies (OR437480), Pectobacterium atrosepticum (MG706146), and Ralstonia solanacearum (OQ878653) were previously isolated from potato [34,35].

All the data obtained from the laboratory trials were analyzed using a one-way ANOVA. Significant differences in mean values were determined using the least significant difference test at a 0.05 significance level, utilizing the Statistical Analysis System (SAS) software [40].

Figure 1 displays the XRD pattern of the prepared quantum dot-composite (CuO/Fe₂O₃). The XRD peaks of the CuO nanoparticles appeared at 35.61°, 38.69°, 57.17°, 60.74°, 66.35°, and 68.45°, which correspond to (−111), (004), (200), (105), (220), and (215), respectively. This demonstrates the formation of a monoclinic (CuO) crystal structure that matches the monoclinic structure (JCPDS No. 01-076-7800) forms, as shown in Figure 1. The XRD peaks of the Fe₂O₃ nanoparticles appeared at 35.6°, 47.39°, 57.24°, and 62.86°, which correspond to (110), (207), (201), and (300), respectively (Figure 1). This shows that the Fe₂O₃ structure is hexagonal. This finding is consistent with the standard (JCPD 01-078-6916) data. The peaks then become sharper with the formation of NC (CuO/Fe₂O₃), as shown in Figure 1. The XRD patterns obtained in the sample CuO/Fe₂O₃ display the usual peaks of (−111) and (004) reflections of CuO, which are found at two values of 35.69° and 37.73°, respectively. In addition, the XRD pattern of (110) Fe₂O₃ is found at 35.69° with a broadening shape in sample CuO/Fe₂O₃. Figure 1 shows a mix of two XRD pattern peaks (105), (220), CuO, and (201) Fe₂O₃ that appear in the range 55°–60°. This shows that the NC of CuO/Fe₂O₃ is formed. Table 1 summarizes the crystal sizes of the resulting nanoparticles. Each side of the polygon MNP had a length of approximately 12 nm. The diameter of the spherical CuO particles ranged from around 7–10 nm. Using the Debye–Scherer equation [41], the crystal diameters of all the nanoparticles obtained can be determined as follows: D = K λ β cos θ , D=\frac{K\lambda }{\beta \hspace{.25em}\cos \hspace{.25em}\theta }, where λ is the Cu-Kα wavelength (0.1542 nm), β is the FWHM, and K is a constant.

Figure 1

XRD pattern of the prepared nanoparticles; CuO, Fe₂O₃, and the NC CuO/Fe₂O₃.

The produced NC was analyzed using FTIR spectroscopy to determine the functional groups present [42]. Figure 2 shows the FTIR spectrum of CuO/Fe₂O₃. It has noticeable characteristic peaks at 525 cm⁻¹ which belong to the bending vibration of the Cu–O bond [43]. The strong band below 700 cm⁻¹ reveals the Fe–O stretching mode. The band corresponding to the Fe–O stretching mode of Fe₂O₃ is shown at 567 cm⁻¹ [44]. Briefly, all prepared metal oxide nanomaterials were characterized by bending vibration of the metal–O bond, CuO, and Fe₂O₃, which appeared in a broad band at around ν 500 cm⁻¹ [45]. The FTIR results are consistent with the XRD patterns of the NC (CuO/Fe₂O₃) and confirm the chemical reaction that takes place during the hydrothermal treatment of copper hydroxide and iron(III) oxide-hydroxide, leading to the creation of the NC (CuO/Fe₂O₃).

Figure 2

FTIR spectrum of the prepared NC (CuO/Fe₂O₃).

(2) Fe ( metal ) + 2NaOH + Cu ( NO 3 ) 2 → Cu ( OH ) 2 + Fe ( OH ) 3 + 2NaNO 3 , \text{Fe}(\text{metal})+\text{2NaOH}+\text{Cu}{({\text{NO}}_{\text{3}})}_{2}\to \text{Cu}{(\text{OH})}_{2}+\text{Fe}{(\text{OH})}_{3}+{\text{2NaNO}}_{3}, (3) Cu ( OH ) 2 + 2Fe ( OH ) 3 → ( Hydrothermal treatment ) → CuO + Fe 2 O 3 + 4H 2 O . \text{Cu}{(\text{OH})}_{2}+\text{2Fe}{(\text{OH})}_{3}\to (\text{Hydrothermal}\hspace{.25em}\text{treatment})\to \text{CuO}+{\text{Fe}}_{2}{\text{O}}_{3}+{\text{4H}}_{2}\text{O}\text{.} The elemental analyses of the prepared nanomaterials were determined by energy-dispersive X-ray spectroscopy (EDX). We detected the percentage and type of each element present. Figure 3 displays the EDX of the prepared NC (CuO/Fe₂O₃), as well as the percentage and type of each containing element. EDX confirms the presence of Cu, Fe, and O with weight percent. The EDX results then align with the XRD and FTIR results. As shown in the EDX results, the current percentage of iron is nearly double the copper element percentage. Figure 4 shows the TEM micrograph of the obtained NC (CuO/Fe₂O₃). It is noticeable that it contains two mixed shapes: the polygon shape of the NC, which consists of a spherical shape of CuO, and plate-like-shaped Fe₂O₃ quantum dots. These findings are consistent with the results reported in a previous publication [4].

Figure 3

EDX analysis of the obtained NC (CuO/Fe₂O₃).

Figure 4

TEM micrograph of the obtained NC (CuO/Fe₂O₃).

The isolated fungi were identified as Fusarium oxysporum, B. cinerea, and R. solani through morphological and molecular analysis. These fungi may be found in GenBank with the accession codes OR116510, OR116494, and OR116531, respectively. The results presented in Table 2 demonstrate the growth response of the chosen fungal isolates to the CuO/Fe₂O₃ NC at various concentrations (25, 50, 75, and 100 µg/mL). The growth response was compared to both negative controls (without chemical treatment) and positive controls (copper formate at a concentration of 100 µg/mL).

The growth of the fungal isolates that were tested was significantly slowed down by CuO/Fe₂O₃ NCs and copper formate at different concentrations when compared to the negative control. The smallest growth diameters for F. oxysporum were found in CuO/Fe₂O₃-NCs, which are similar to copper formate. These were 26.3 and 26 mm at 75 and 100 µg/mL, respectively. In the same way, the smallest B. cinerea growth measurements were 50, 46.3, and 45 mm when CuO/Fe₂O₃-NCs were used at 50, 75, and 100 µg/mL, respectively. In contrast, copper formate effectively counteracted the inhibitory impact of all concentrations of CuO/Fe₂O₃ NC on R. solani. According to the results shown above, CuO/Fe₂O₃-NCs at a concentration of 100 µg/mL had the best effect on stopping B. cinerea, with a 50% success rate. Copper formate at a concentration of 100 µg/mL had the best inhibition percentages against F. oxysporum (71.5%) and R. solani (82.2%). Eltarahony et al. [46] verified these results and found that metal oxide hybrid NCs possess remarkable capability to penetrate and disrupt the inflexible composition of glycoprotein–glucan–chitin in the fungal cell wall. In this case, a biogenic Cu/Fe hybrid NC exhibited significant protection against the fungus Candida albicans at concentrations of 250 and 500 µg/mL. Parameswaran et al. [47] evaluated the antifungal properties of the Co/CeO₂ NCs against C. albicans and Aspergillus fumigatus. The findings indicated that Co/CeO₂ NCs serve as a moderate antifungal agent for both C. albicans and A. fumigatus. At a concentration of 30 μL, the inhibition zones measured 12 mm for C. albicans and 14 mm for A. fumigatus, compared to 22 mm for the standard antifungal agents. As an antifungal agent, the copper formate complex exhibited a significant inhibitory effect on the growth of R. solani at a concentration of 200 µg/mL [26], as well as effectively reducing the infection of Rosa hybrida flowers by B. cinerea at a concentration of 1,000 mg/L [27]. In a recent study [48], an innovative NC was synthesized using mycosynthesized bimetallic zinc–copper oxide nanoparticles, nanocellulose, and chitosan, and the results demonstrated significant antifungal activity against Aspergillus brasiliensis with an MIC of 7.81 μg/mL. However, the NC had limited antifungal effectiveness against Cryptococcus neoformans and C. albicans, with an MIC of 250 μg/mL for both. However, it was evident that smaller particles enhance the interaction between nanoparticles and fungal cells, allowing them to penetrate bacterial and fungal cell walls more easily [49].

The findings indicated that the synthesized CuO/Fe₂O₃-NCs effectively suppressed the growth of bacterial isolates at doses of 10, 20, 30, 40, 50, and 100 µg/mL, as compared to the negative control (no chemical treatment) and positive control (gentamicin, 30 µg) (Table 3). It was noted that gentamicin exhibited the most potent inhibition zones against the selected bacterial isolates, surpassing the inhibitory effects of all tested doses of CuO/Fe₂O₃-NCs. Furthermore, all of the tested concentrations exhibited inhibition zones compared to the negative control (Table 3). All the assigned concentrations of CuO/Fe₂O₃-NCs showed equipollent inhibitions on P. carotovorum and S. scabies. At the same time, 40 and 50 µg/mL of CuO@ Fe₂O₃ NCs were the most effective amounts against P. atrosepticum, with inhibition zones measuring 9.33 and 10.67 mm. Similarly, CuO/Fe₂O₃-NCs at 50 µg/mL achieved the highest inhibition zone of 14.7 mm against R. solanacearum.

The data can be interpreted by considering the mechanism of MONCs, which involves the generation of ROS, such as superoxide radicals and hydrogen peroxide anions. These ROS can attack bacteria’s cell walls, causing damage to the membrane and internal organelles, ultimately leading to growth inhibition or even cell death [19]. Moreover, it was concluded that the MONCs, Fe₂O₃@Cu₂O, showed inhibitory effects on the pathogenicity of the bacterial cell by rupturing its outer and inner cell walls to cause disorganization and leakage in the cell membrane [13]. In addition, the co-assembled core-shelled MONCs, ZnO–Fe₂O_3x –CuO _x using the co-precipitation technique, demonstrated obvious anti-growth activity against bacterial cells. For instance, the anti-growth activity was clearly exhibited in the descending order for the co-assembled core-shelled MONCs of CuO  >  ZnOFeO_0.5CuO_0.5 > CuOFeO_0.5 > ZnOFeO_0.1CuO_0.1 > Fe₂O₃ > ZnOFeO_0.5 > ZnO against Staphylococcus pneumonia [16]. Conversely, copper formate and copper formate-based NPs possessed an antimicrobial augmentation synergized with Cu(ii) ions, having the ability to generate free radicals that could promote disordering in the cell membrane and lipid peroxidation within the bacteria. In particular, the cell membranes of Staphylococcus aureus and Escherichia coli were robust and unruffled in the control-treated groups compared to the copper formate-treated groups, which had some puckers on the cell membranes leading to the release of cytoplasm from the bacterial cells [25]. The antibacterial properties of Co/CeO₂ NCs were tested against four pathogenic bacteria: E. coli, S. aureus, Salmonella paratyphi, and Klebsiella pneumonia at a concentration of 30 μL. The results indicate that CeO₂ inhibited the growth of all bacteria. Specifically, K. pneumonia showed an inhibition zone of 18 mm, while E. coli, S. aureus, and S. paratyphi exhibited inhibition zones of 12, 14, and 16 mm, respectively. In comparison, amoxicillin (10 μL) created inhibition zones of 22 mm against E. coli, S. aureus, and S. paratyphi, and 24 mm against K. pneumonia, indicating slightly higher effectiveness than Co/CeO₂ NCs [47]. To prepare antimicrobial composites, different methods were used, including physically combining methylphenyl silicone resin with two plants, Houttuynia and Scutellarin, to create antifouling coatings. The prepared coatings exhibited inhibition rates of 90% against S. aureus and E. coli [50].

The antibacterial mechanisms of NCs are varied, often involving smaller particles that possess larger surface areas and can penetrate cells more easily, leading to cell death. The interaction between NCs and bacterial cell walls, due to electrostatic attraction, also contributes to bacterial cell death [51]. Ansari et al. [52] demonstrated the antibacterial activity of TiO₂ against Pseudomonas aeruginosa and S. aureus at a concentration of 2 mg/mL. Similarly, Almessiere et al. [51] reported the antibacterial effects of Ce^3+/Dy³⁺ co-activated Mn–Zn nanospinel ferrites against E. coli and S. aureus. However, the inhibition effects of both pure CeO₂ and Co/CeO₂ NCs were not as pronounced as those of the antibiotics used as positive controls. Zhang et al. [53] analyzed ternary lanthanide coordination polymers (LCPs) against several bacterial strains, revealing MIC and MBC values ranging from 50 to 400 ppm. 8-Hydroxyquinoline showed the most potent antibacterial action, while other composites had comparable MIC and MBC values. The MIC/MBC values showed greater efficacy against Gram-positive bacteria like S. aureus. TEM analysis showed a bactericidal process in E. coli, confirming LCPs’ ability to kill bacteria.

The antimicrobial activities of NPs were subjectively determined by their characteristics, such as size, shape, concentration, and physicochemical properties [54]. The current results confirm that the obtained CuO/Fe₂O₃-NC has remarkable antimicrobial capabilities.