3.1 XRD and phase structure of nanostructures
ZnO and TiO2 showed the crystalline nature with wurtzite and anatase structure, respectively. The crystalline ZnO illustrated the diffraction peaks at 2θ = 36.02°, 31.7°, 34.3°, 47.5°, 56.5°, 62.8°, 66.3°, and 67.8. the anatase phases of TiO2 indicated peaks at 2θ = 25.4°, 38.1°, 48.1°, 54.8°, 62.5°, and 75.1° [26]. The XRD patterns of ZnO and TiO2 showed a single high-intensity peak that implies a highly oriented and single-crystalline nature of the samples. As shown in Fig. 1, the intensity of TiO2 peaks considerably decreased after the addition of TiO2 into the structure of ZnO in the ZnO-TiO2 composite that indicates the greater crystallinity of pure TiO2NPs compared to ZnO-TiO2 NPs [27]. Profile broadening also indicated the small crystalline domain sizes of wurtzite and anatase indicating that the ZnO-TiO2 composite hinders the growth of particles during calcination. The main peaks of each sample in the range of 2θ = 20–50° specified some peaks belonging to anatase (Fig. 1).
Table 1 demonstrated the variations of the crystallite size, surface area, the lattice constant a and the lattice constant c for ZnO, TiO2 and ZnO-TiO2 composite. The crystallite size of the pure ZnO and TiO2 was 33.21 and 17.68 nm, respectively. The ZnO-TiO2 composite nanoparticles showed the lower particle size compared to each ZnO and TiO2 alone nanoparticles.
Table 1
Characterization of ZnO,TiO2 and ZnO-TiO2
Sample | Crystallite size(nm) | a = b(Å) | c(Å) | S m2/g | PSA(nm) | Zeta Potential(mv) | MIC (µg/ml) | MFC (µg/ml) |
ZnO | 33.21 | 3.242 | 5.214 | 32.20 | 608 | -11.6 | 156 | 312 |
TiO2 | 17.68 | 3.798 | 8.944 | 98.36 | 299 | -36.4 | 78 | 156 |
ZnO-TiO2 | ZnO = 19.25 TiO2 = 8.36 | 3.253 3.807 | 5.238 9.592 | 131.78 | 983 | -12 | 39 | 78 |
By considering the lattice constant and surface area data in Table 1, the significant increase of specific area from 32.20 to 131.78 m2/g of ZnO-TiO2 was observed compared to the pure ZnO and TiO2 by increasing crystallite size. The increase in the value of lattice parameters for ZnO-TiO2 can be attributed to the incorporation of ions (Ti+ 4 and Zn+ 2), which is due to stress in crystal structures.
3.2 PSA and Zeta potential analysis
The zeta potential is an important indicator of the stability of dispersed particles in the suspension solution. The zeta potential determines the repulsion of dispersed particles in the solution. Small particles require the high zeta potential for superior stability, and low zeta potential causes to particle accumulation. The zeta potential of a particle alters by the particle surface chemical composition, the pH and ionic strength of the solution. Zeta potential of ZnO, TiO2, and ZnO-TiO2 were − 11.6, -36.4, and − 12 mV, respectively (Fig. 2 and Table 1). Based on our findings, TiO2 and ZnO-TiO2 showed the highest and lowest stability in aqueous suspension, respectively. Larger particle sizes for ZnO (608 nm), TiO2 (299 nm), and ZnO-TiO2 (983 nm) were determined by PSA analysis showing the agglomeration of nanoparticles.
3.4 SEM and TEM analysis
As shown in Fig. 3, the ZnO and TiO2 nanoparticles illustrated hexagonal-pyramidal and spherical shape with grown articles on surfaces, respectively. The wurtzite-structured ZnOcrystal is described as several alternating planes composed of four-fold tetrahedrally-coordinated O2−and Zn2+ ions stacked alternatively along the c-axis [28]. The oppositely-charged ions produce positively-charged Zn (0001) and negatively-charged O(0001 ̄) surfaces, resulting in a normal dipole moment and spontaneous polarization along the c-axis, as well as a divergence.
In the ZnO-TiO2 nanostructures, the morphology was a mixture of pyramidal and spherical with more agglomeration while the particle sizes were smaller than alone titanium and zinc oxide particles. Upon the EDX analysis, the strong signals of Zn, Ti and Zn-Ti were observed in ZnO, TiO2, and ZnO-TiO2 nanostructures, respectively (Fig. 3).
The TEM images of nanostructures clarified the regular growth of all nanostructures and illuminated the TiO2 (5 nm) particle size smaller than ZnO (10 nm) and ZnO-TiO2 (35 nm) nanoparticles with lower agglomeration rate (Fig. 3).
3.5 FTIR analysis
FTIR was applied to study the component and composite structures of synthesized nanoparticles. Zn-O and Zn-OH bands were observed between 1000 cm− 1 and 400 cm− 1 while Ti-O and Ti-OH bands appeared around 480 cm− 1 and 790 cm− 1. The peaks at 672 cm− 1 and 829 cm− 1 showed the stretching band of O-Ti-O and Zn-O-Ti vibration mode in TiO2 (T) and ZnO-TiO2 (ZT) nanostructures [29, 30]. The peaks in the around of 2800 cm− 1 and 2900 cm− 1 were related to the tensile vibrations of CH3 and CH2, and the peaks in the range of 1380 cm− 1 and 1500 cm− 1 corresponded to the bending vibrations of molecules CH2 and CH3, respectively. The broad peak in the range of 3400 cm− 1 and 3800 cm− 1 was related to the hydroxyl groups. Also, water molecules in the bending band at 1630 cm− 1 are visible [31]. The presence of some bands can be associated with the organic phase of solid, despite the use of organic compounds in the synthesis of nanoparticles (Fig. 4).
3.6 Antifungal properties of nanostructures
As shown in Table 1, ZnO-TiO2 nanostructure exhibits better antifungal effects against A. flavus than other nanoparticles due to its high specific surface area. By increasing the specific surface area, the possibility of chemical reactions and the production of reactive oxygen species on the surface were increased [32]. The MIC for ZnO-TiO2, ZnO, and TiO2 against A. flavus was determined 39, 156, and 78 µg/ml, respectively. Because of the small particle size, the best cell internalization, and the ability to produce more reactive oxygen species, TiO2 showed a higher fungicide than ZnO. The MFC for ZnO, TiO2, and ZnO-TiO2 was 312, 156 and 78 µg/ml, respectively. The particle size of the ZnO-TiO2 nanostructure possessed a sharp structure with smaller particles than the cell membrane that can inhibit the growth of the fungus by entering the cell membrane and injuring the cell wall thus resulting in the high toxicity. Figure 5 illustrated the inhibition zone of ZnO, TiO2, and ZnO-TiO2 at 37.5, 75, 150, and 300 µg/ml concentrations. By increasing the concentration of nanoparticles, inhibition zone diameter of growth increased and 100% of inhibition was achieved at 300 µg/ml for TiO2 and ZnO-TiO2 treated groups. The minimum fungal growth (72%) was obtained at 37.5 µg/ml for ZnO-TiO2 while for ZnO was 50% at the same concentration showing that the TiO2 synergistic effect into the mixture [33]. Among all nanoparticles, ZnO nanoparticles showed the lowest fungicide activity compared to others whereas it significantly increased the antifungal activity in ZnO-TiO2 nanocomposite.
The destructive changes were observed on the shape and growth of the treated A. flavus (at a concentration of 37.5 µg/ml for all samples) compared to the untreated control group. As shown in Fig. 6, the untreated control fungus produced the highest count of fungal spores while treated groups showed a lower count of spores and damaged tubular filaments, in instance deformation, smoothness, and noticeably thinner in hyphae compared to the untreated group. Upon the previous reports, increasing the hyphae causes to form whiter medium [34] and our findings agreed to color changes based on the used nanoparticles (Fig. 6).
Among the reactive oxygen species, hydrogen peroxide and hydroxyl radicals as the strong and non-selective ROSs can damage all types of biomolecules including carbohydrates, acids, lipids, proteins, DNA, RNA, and amino acids through inducing the oxidative stress [35]. The production rate of the three major reactive oxygen species by ZnO and TiO2 nanoparticles were: ZnO: O2•−>O2>•OH and TiO2: O2>•OH > O2•− [36]. There is a direct dependency between increasing the formation of ROS and the fungicide of nanoparticles. As shown in Fig. 7, all nanoparticles raised the ROS production in treated A. flavus compared to untreated control with order ZnO-TiO2 > TiO2 > ZnO > untreated control. The production of intracellular ROS was influenced by the type and specific surface of nanoparticles. Titania can produce ROS higher than zinc oxide [37], our findings also confirmed the highest ROS production through stronger fluorescence intensity in ZnO-TiO2 treated group. In ZnO-TiO2 nanostructures, the specific surface area is higher than other nanoparticles (TiO2 and ZnO) and accordingly high ROS generation. Oxidative stress induced by reactive oxygen species generation in ZnO-TiO2 nanostructures is thought to be the main mechanism of antifungal activity. The suggested mechanism for the antifungal activity of these compounds can be based on the formation of high levels of reactive oxygen species (ROS) that disrupt the integrity of the fungal cell membrane, which assists in the damage of microbial enzyme bodies thus killing the fungi [38].