## 3 Results and Discussion
### Mechanisms of the Antifungal Activity of Cu(AZPY)-MOF  
To understand the mechanism(s) for the antifungal activity of **Cu(AZPY)-MOF**, we investigated the effects of released metal and ligands (free Cu2+, glutamate, and 4_A_-azopyridine) into PBS media on the inactivation of fungi. The reason is that the antimicrobial activity of MOFs can be derived from free metal ions or organic ligands released from the frameworks, as demonstrated in previous studies [27, 39, 40, 48]. The released Cu2+ from **Cu(AZPY)-MOF** or glutamate (chemical framework) does not seem to be a critical factor in causing fungal inactivation, as shown in Figure 5. The log CFU number and relative percentage of viable cells were not significantly different between the control and Cu(NO3)2 or glutamate (Figure 5). The higher level of antifungal activity of **Cu(AZPY)-MOF** compared to the previous Cu-MOFs may be related to the addition of 4_A_-azopyridine (AZPY) to the MOF struc  
Figure 5: Effect of Cu(NO3)2 and glutamate. **(a)** Fungal viability indicated as log10(CFU number) and relative percentage after treated with each ligand for 1 day. The concentrations of Cu(NO3)2 and glutamate used in the treatment were 0.1 mg/mL and 0.25 mg/mL, respectively. Each value in log10(CFU number) was an average of three replicate measurements, and relative percentage was calculated using the average value of CFU number. **(b)** Pictures of plates showing CFUs of _C. albicans_ and _A. niger_ on the PDA plates 1 day after treatment. ture. Azopyrine derivatives are known to have antimicrobial activity [49]. We found that a very small amount of AZPY was released from **Cu(AZPY)-MOF** into PBS over the incubation time, which reached approximately 0.0006 mg/mL after 24 h (Figure 6). The released amounts of AZPY from **Cu(AZPY)-MOF** were measured by UV/Vis adsorption spectroscopy. When the maximum released amount of AZPY (0.0006 mg/mL) was applied to fungal cells, the log CFU number and the relative percentage of viable fungal cells did not significantly change compared to the control (Figure 7). This result indicates that the amount of released AZPY is not critical to inactivate fungal cells and spores. We also exposed fungal cells to free AZPY at a half concentration (0.125 mg/mL) of the inactivation concentration (0.25 mg/mL) of **Cu(AZPY)-MOF** and found that the fungal cells were completely inactivated (Figure 7). This result suggests that AZPY intercalated in the framework may be able to contribute to the fungal deactivation. The mechanism by which AZPY intercalated in MOF can cause fungal inactivation requires further investigation. However, our data suggest that **Cu(AZPY)-MOF** containing AZPY is very stable in PBS and can act as a 3D AZPY derivative with antimicrobial activity. Figure 6: Concentrations of AZPY released from 0.5 mg of **Cu(AZPY)-MOF** in 1 mL of PBS. Figure 7: Effect of 4,4\({}^{\prime}\)-azopyridine (AZPY) on the viability of _C. albicans_ and _A. niger_. Fungal viability indicated as Log\({}_{10}\) (CFU number) and relative percentage after treated with the amount of AZPY released into media (0.0006 mg/mL) or the half amount (0.125 mg/mL) of inactivation concentration (0.25 mg/mL) of **Cu(AZPY)-MOF** for 1 day. The number of replicate measurements in the analysis was as follows: 9 replicates for _C. albicans_ and 12 replicates for _A. niger_ in the control and 0.0006 mg AZPY treatment; 3 replicates for both _C. albicans_ and _A. niger_ in the 0.125 mg AZPY treatment. Another possible mechanism for the antifungal activity of **Cu(AZPY)-MOF** containing AZPY is the generation of ROS and RNS. Many MOFs are known to produce ROS and RNS, which can cause antimicrobial effects [27; 39; 40; 50]. We measured the levels of H2O2 and NOx generated in PBS after the treatment with **Cu(AZPY)-MOF**. Approximately 3-4 mM H2O2 was measured in PBS in the treatments with **Cu(AZPY)-MOF**, whereas no NOx was detected (Figure 8).