*2.9. Antifungal Assay*

To determine the inhibition of mycelial growth of *A. flavus*, four Cu-Chit/NCs gel concentrations (60, 120, 180, and 240 ppm) were prepared as hydrogel discs for every disc containing 60 ppm. The anti-fungal efficacy of Ag-Chit-NCs was assessed by determining the reduction in fungal growth of *A. flavus* using agar-well diffusion assessment [32]. Each concentration of Cu-Chit/NCs gel has been applied to PDA plate, and petri dishes were inoculated with *A. flavus* fungal disks. Flavus isolates were incubated at 28 ◦C for 10 days. The inhibition factor of growth was estimated and evaluated by the equation below. [33]. Growth Inhibition (percent) = (R1−R2)/R1 × 100 where R1 was the control's radial growth and R2 for each therapy was the radial growth. After one week, the photographic record and the development of the radial colony was calculated. The experiments were conducted in three folds.

### *2.10. Protein Profile Degradation Assay*

To investigate the Cu-Chit/NCs gel mediated protein expression in *A. flavus,* SDS-PAGE analysis was performed by Laemmli method [34]. The extracted protein from the fungal mycelium was treated with 180 ppm of Cu-Chit/NCs gel concentrations and incubated for 8 h. SDS-PAGE was performed using a 5–10% gradient of polyacrylamide gels containing 0.1% SDS. Proteins were investigated in 1.5 mm and 15 cm gels that work in dual vertical electrophoresis glass plates (Hoefer Scientific Instruments, San Francisco, CA, USA). Twenty microliters of the extracted protein were inoculated into polyacrylamide gels. SDS-PAGE samples were differentiated by separating the acrylamide gel at a stable electrical current of 30 mA, and by using a separate gel at room temperature at 90 mA, the gel was stained with silver staining. [34]. The typical molecular weight used for gel analysis was the Sigma protein marker, which is between 66,000, 45,000, and 22,000 kDa.

#### *2.11. Native PAGE Isozyme Assay*

Fungal isozymes were purified by grinding 100 mg of fungal mats in 1.0 mL extraction buffer (0.1 M Tris-HCl + 2 mM EDTA, pH 7.8). The extracted enzyme from fungal mats was treated with 180 ppm of Cu-Chit/NCs gel concentrations and incubated for 8 h. Native-PAGE was used to separate two enzymatic activities under native conditions [35]. Electrophoretic technique were conducted with the electrode buffer Tris/Glycine (pH 8.3) using 5 percent of the stacking gel and 6 percent of the separating gels. The 5 µL enzyme samples were placed over each well of the stacking gel and the gel was initially run at 60 V replaced by 100 V later. After running native acrylamide gel, and for staining glucose 6-phosphate dehydrogenase (G6PD) (EC.1.1.1.49), native protein gel was incubated in a staining solution (0.1 mM tris-HCL buffer, pH8.8, 7.5 glucose 6-phosphate (di-sodium salt), 20 mg NADP, 10 mg MTT, 10 mg PMS, 0.2 M MgCl2) in the dark at 37 ◦C until dark blue band appear. To stop the reactions, the isozyme gel was washed and fixed in 50% ethanol [36]. Peroxidase isozyme (EC 1.11. 1.7) was stained with incubated gels in a staining solution (50 mM phosphate buffer, pH5.0, 50 mg benzidine dihydrochloride, and 3% hydrogen peroxide), and the gel was washed in water and fixed in 50% glycerol [37].
