## 5 Discussion
In the presence of H2O2, H2 DCF is rapidly oxidized to 2\({}^{\prime}\), 7\({}^{\prime}\)-dichlororescein (DCF), a highly fluorescent molecule, which exhibits an excitation wavelength and emission wavelength of 498 and 522 nm, respectively. The oxidation of H2 DCF to DCF is thought to be specific to H2O2, and recent evidence indicates that other ROS (e.g., hydroperoxides, hydroxyl radicals, and peroxnitrite) can also oxidize H2 DCF [62]. The PEI-AuNP@Van-treated conidia of _A. flavus_ and _A. fumigatus_ exhibited variability in ROS generation. Of the conidial population of _A. fumigatus_, 37.4% showed ROS generation after 8 h of exposure to PEI-AuNP@Van; in contrast, the PEI-AuNP@Van-treated conidia of _A. flavus_ demonstrated an ROS generation of only 23.1% (as indicated in Figure 7). It is possible that H2O2 induces conidial cell death in _A. fumigatus_ via a secondary necrosis mechanism [63]. Secondary necrotic cells show a specific morphotype associated with necrotic features (e.g., cytoplasmic membrane rupture) and apoptotic features (e.g., DNA fragmentation). Secondary necrosis is considered to be a form of apoptosis in unicellular eukaryotes. It is associated with cell disintegration and the release of cell components, which takes place when there is no intervention from scavengers, and the entire apoptotic program is completed [63]. However, the conidia of _A. flavus_ did not exhibit necrosis-like cell death (Figure 9). This outcome further strengthens previous results regarding the differential modes of action of PEI-AuNP@Van against both _Aspergillus_ species. Raman spectroscopy can be used to analyze biological samples. Raman spectroscopy is a noninvasive, label-free method that is associated with minimal sample preparation and offers information about the structure of complex biological materials. Chemical analysis of the fungal cell wall revealed that proteins and polysaccharides are the main components. Therefore, damage to the cell wall can significantly affect growth and structure, leading to death. Hence, the fungal cell wall is considered an excellent target for the development of antifungal agents [64,65]. Raman spectra of the voriconazole and PEI-AuNP@Van-treated conidia of _A. fumigatus_ and _A. flavus_, along with the respective untreated controls, are shown in Figure 8. These results suggest that the modes of action of PEI-AuNP@Van against the conidia of _A. fumigatus_ and _A. flavus_ are slightly different. In the case of _A. flavus_, most of the peaks are typical of those of voriconazole- and PEI-AuNP@Van-treated conidial spectra (except at 558 and 841 cm\({}^{-1}\)), as indicated in Figure 8(aii,aiiii). These peaks were observed in PEI-AuNP@Van-treated conidia, suggesting the possible binding of vancomycin-functionalized nanoparticles to fructose units and galactomannan components in the cell wall. However, a detailed investigation is required to elucidate the molecular mechanisms of this interaction. The spectral differences among the untreated control, voriconazole-treated, and PEI-AuNP@Van-treated conidia of _A. fumigatus_ were similar to those among the conidia of _A. flavus_. The emergence of two new peaks (1127 and 1199 cm\({}^{-1}\); =C-O-C= unsaturated fatty acids in lipids, galactomannan, and chitin) in PEI-AuNP@Van-treated conidia again suggests the strong binding of PEI-AuNP@Van with chitin and galactomannan molecules. Hence, it can be concluded that PEI-stabilized AuNPs act as anchors on the cell walls of conidia and bring vancomycin close to chitin and galactomannan molecules in the PEI-AuNP@Van system. Furthermore, TEM analysis of the PEI-AuNP@Van-treated conidia of _A. flavus_ and _A. fumigatus_ revealed more severe architectural damage to _A. fumigatus_ conidia than to _A. flavus_ conidia (Figure 9). This result could be attributed to differences in the cell wall structures of the two species. _A. flavus_ conidia have a more branched \(\beta\)-1-3 glucan than _A.