Calcination temperatures and composition of heterostructural ZnO–CuO nanocomposites
Seven characteristic peaks of ZnO and five characteristic peaks of CuO were found in green ZnO-CuO samples prepared at different calcination temperatures (300, 400 and 500 °C) as shown in Figure 1. Diffraction peaks at 31.87°, 34.54°, 36.37°, 47.59°, 56.59°, 62.92° and 65.89° which correspond to crystal surfaces (100), (002), (101), (102), (110), (103) and (200) belonged to ZnO. Conversely, CuO was detected at 35.62°, 38.83°, 57.49°, 61.42° and 67.96°, which correspond to crystal surfaces (–111), (111), (202), (–113) and (220). The XRD pattern of ZnO-CuO nanocomposites confirms the presence of pure ZnO and CuO. Furthermore, few additional peaks were observed at 23.65°, 25.69°, 27.73°, 29.47° and 40.78° from Figures 1 and 2. This outcome is possibly due to the presence of the phytochemical element of C. gigantea leaves as a capping and reducing agent [39]. The additional peaks detected at 29.47° and 40.78° are attributed to the natural graphene-like carbon present in the ZnO-CuO nanocomposites [42] as carbon is a main phytochemical element in the leaves of the C. gigantea medicinal plant [43, 44]. Natural carbon in binary ZnO-CuO nanocomposites could further enhance the synergic effect on antimicrobial activity [45, 46]. However, these peaks lessened at higher calcination temperatures.
Prominent diffractive peaks on the differential ratio of binary ZnO and CuO nanocomposites are indexed in Figure 2. Six characteristic peaks of ZnO for sample 75ZnO25CuO-300C were identified at 31.72°, 34.45°, 36.25°, 47.35°, 56.41° and 62.71° and corresponded to crystal surfaces (100), (002), (101), (102), (110) and (103). Another two characteristic peaks of CuO at 38.62° and 67.78° corresponded to crystal surfaces (111) and (220). For sample 25ZnO75CuO-300C, 31.72°, 34.45°, 36.25°, 47.35°, 56.41°, 62.71° and 68.05° peaks, respectively belonged to the (100), (002), (101), (102), (110), (103) and (201) indices of ZnO nanoparticles. Conversely, the diffractive peaks of CuO detected at 35.68°, 38.62°, 58.33°, 61.27° and 65.80° corresponded to crystal surfaces (–111), (111), (202), (–113) and (–311). The peak intensity is drastically increased with higher amounts of ZnO or CuO in the binary ZnO-CuO nanocomposites (Figure 2), thereby indicating the variation in composition (25, 50 and 75 wt. % of ZnO) during green synthesis.
Superior antimicrobial properties of heterostructural ZnO–CuO nanocomposites
The antimicrobial efficacy of binary 50ZnO/50CuO nanocomposites at three different calcination temperatures (300°C, 400°C and 500°C) were initially characterised by S. aureus minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) as presented in Table 2. The MIC of 50ZnO/50CuO-300C and 50ZnO/50CuO-400C for S. aureus were 2.5 mg/mL, except for 50ZnO/50CuO-500C at 5 mg/mL. Moreover, the MBC of all green synthesised 50ZnO/50CuO samples for S. aureus was at 20 mg/mL. S. aureus colonies were counted less at a concentration of 5 mg/mL for the sample 50ZnO/50CuO-300C prepared at low calcination temperature (300°C) relative to the 50ZnO/50CuO-400C and 50ZnO/50CuO-500C samples (Figure S1). The effect of smaller sized nanoparticles generated at low calcination temperature is suggested to possibly enhance surface reactivity in killing the microbes [47, 48, 49]. Particle size is crucial in antimicrobial activity effectiveness. Azam et al. (2012) and Salah et al. (2011) verified that smaller particle sizes mean greater efficacy in inhibiting bacterial growth, a feature that is possibly associated with the larger surface areas of nanoparticles [50, 51].
Next, further characterisation of the differential ratios of binary ZnO and CuO nanocomposites at a calcination temperature of 300 °C were presented in Figure S2 and Table 2. The MIC of 25ZnO/75CuO-300C, 50ZnO/50CuO-300C and 75ZnO/25CuO-300C were 5 mg/mL, 2.5 mg/mL and 0.625 mg/mL for S. aureus, respectively. Similar to the MIC values, the 25ZnO/75CuO-300C and 50ZnO/50CuO-300C green samples had MBC values of 20 mg/mL and the counterpart for the 75ZnO/25CuO-300C sample was 2.5 mg/mL for S. aureus. The 75ZnO/25CuO-300C sample exerted a higher bactericidal effect against the S. aureus strain at the lowest MIC/MBC values (0.625 mg/mL/2.5 mg/mL). The antimicrobial activity was further enhanced by increasing the amount of ZnO nanoparticles in the binary compound (ZnO-CuO). The phenomenon observed can be explained by the fact that the binary 75ZnO/25CuO-300C nanocomposites are highly diffusible and generate more Zn2+ [19]. Moreover, Cu2+ ions bind the cell wall of host cells through surface proteins and enter the cell [19]. Subsequently, the change in the metabolism of cells leads to the microbe’s cell death [19].
Further antimicrobial analysis of 75ZnO/25CuO-300C on selected skin ulcer pathogens are shown in Table 3. These pathogens are commonly associated with skin ulcer disease [4, 5, 6, 7]. The MIC values of the green synthesised ZnO-400C for E. coli, P. aeruginosa, K. pneumonia and MRSA were at 0.3125, 0.15625, 0.625 and 0.15625 mg/mL, respectively. By contrast, the MBC values were 2.5, 0.3125, 1.25 and 0.3125 mg/mL, respectively. Furthermore, the MIC amounts for the 75ZnO/25CuO-300C sample were 0.625, 0.15625, 0.625 and 0.15625 mg/mL for E. coli, P. aeruginosa, K. pneumonia and MRSA, respectively. MBC values with 2.5, 0.3125, 1.25 and 0.3125 mg/mL were also observed for this green binary inorganic oxide sample. The tolerance level according to the MBC/MIC ratio showed that all tested microbes are sensitive to bactericidal agents except for the CuO-500C sample against E. coli, P. aeruginosa and MRSA and the ZnO-400C sample towards E. coli. Table 3 indicates that for all tested microbes, only the tolerance levels for 75ZnO/25CuO-300C sample were less than 4, and these values identifies the sample as a strong bactericidal agent relative to other samples (ZnO-400C and CuO-500C).
Moreover, higher MBC values of the CuO-500C sample against all tested microbes possibly transpire from the slow Cu2+ ion release from CuO nanoparticles [52]. Clearly, the ZnO-400C and 75ZnO/25CuO-300C samples show very promising results against all tested microbes. That outcome may arise from the ZnO nanoparticle’s larger surface to volume ratio and the penetration of the cell membrane of the bacteria by its ions. Furthermore, the ZnO-400C sample showed better antimicrobial activity relative to the CuO-500C counterpart at a similar concentration. Some studies reported that the antimicrobial effectiveness of green synthesised inorganic oxide nanoparticles depends on particle dosage, size and treatment condition, such as calcination temperatures. This situation could be the one of the reasons for the higher antimicrobial activities of ZnO and ZnO-CuO over that of CuO particles.
Additionally, the antimicrobial activities of the ZnO-400C and 75ZnO/25CuO-300C samples are much better than that of the CuO-500C sample alone towards multi-drug resistant strains P. aeruginosa, K. pneumonia and MRSA. That outcome is obviously due to the high diffusion of Zn2+ ions in the medium. The poor activity of CuO particles within a shorter duration suggested that the time requirement for water diffusion and subsequent Cu2+ release influence efficacy. The attacking delay was also associated with the cell walls of gram-negative strains. Studies have reported that gram-negative bacterial strains exhibit higher resistance or tolerance against nanomaterials compared with gram-positive bacteria [53] because of the lipopolysaccharide situated in the outer membrane of the former [54]. The cytoplasmic membrane, which is inherent to gram-negative bacteria, significantly maintains cellular viability. Hence, gram-negative microbes are not readily attacked by free radicals or Cu2+. More time and concentrated Cu2+ ions are thus required to effectively decompose the cell membrane of the bacteria. The antimicrobial activity of ZnO, CuO and ZnO-CuO nanoparticles is due to the electrostatic interaction between positively charged zinc and copper ions (Zn2+ and Cu2+) and negatively charged microbial cell membranes [21]. In addition, the antimicrobial activity of inorganic oxide nanoparticles relies on the generation of ROS as well [17, 19].
In the time-kill assay results were presented in terms of the changes in the log10 CFU/mL of viable S. aureus colonies and indicated that the green synthesised binary 75ZnO/25CuO-300C sample exhibited significant bactericidal activity. The outcomes of the time-kill assay were captured in Figure S3. Figure 3 presents the time-kill curve graph for the strain. A reduction in viable count from 4.3 log10 to 3.4 log10 was captured after 6 h of incubation for S. aureus. By 12 h, only 1.3 log10 of bacterial colonies were seen. At 24 h, the bacteria were completely killed. Therefore, the effective control of gram-positive S. aureus bacteria was achieved by the synergistic combination of 75 wt.% of ZnO and 25 wt.% of CuO nanoparticles with the presence of phytochemical constituents such as cardiac glycosides, tannins, saponins, terpenes, flavonoids and phenolics in the leaf extract of the C. gigantea medicinal plant [55, 56, 57, 58].