2.1. Preparation of Se-NPs
Se-NPs were biosynthesized at a concentration range of 100 ppm by the application of two standard strains of A.flavus: TIMML-050 and C.albicans: TIMML-1306. In the following, these species were cultured in a sabouraud dextrose broth medium at 35 °C for 24 to 48h. After incubation, the supernatant that contained reducing organic proteins and enzymes (resulting from the growth and metabolism of fungal agents) was separated from the fungal culture medium through the utilization of Whatman 1 filter paper. As the next step, 800 mg of sodium selenite (Na2SeO4) was dissolved in 10 mL of distilled water, which was then added to 100 mL of supernatant and incubated at 28 °C for 24h (away from light and movement) [18, 19]. Afterwards, selenium salt was reduced and the production of nanoparticles became macroscopically visible as the color of environment was changed from yellow to orange .
2.2. UV-Vis spectrum
We were able to confirm the presence of Se-NPs by the usage of UV-Vis spectrum recorded in the range of 290-310 nm (Fig. 1 and 2). The successful biogenic synthesis of Se-NPs was primarily assured by red color change. The formation of Se-NPs was further justified from UV-Vis spectroscopy. Fig. 1 and 2. display the typical plasmon resonance bands of Se-NPs, being observed at 290-310 nm (λmax) .
2.3. FT-IR and EDAX analyzes
The proper stability and dispersion of nanoparticles were attributed to their interaction with fungal proteins. During the process of nanoparticles production, it can be assumed that the probes form a coating on the nanoparticles and have them stabilized by preventing their condensation. The graphs that were obtained in the present study, displayed peaks in the ranges of 440, 518, 555, 753, 816, 1053, 1082, 1338, 1412, 1585, 1631, 2949, and 3389 cm-1 for the case of C.albicans-Se-NPs (Fig. 3), and peaks within the ranges of 432, 538, 620, 751, 819, 1080, 1339, 1406, 2917, and 3418 cm-1 for the case of A. flavus-Se-NPs (Fig. 4). FT-IR analysis was performed by the means of a PerkinElmer Spectrum Version 10.03.02 spectrometer device, while the samples were examined throughout the wavelength range of 4000-400 cm-1. This method was exerted to determine the nanoparticles' interaction with fungal proteins and better comprehend the roles of proteins that surround the nanoparticles as stabilizing agents. Based on the FT-IR results, the coating factor that stabilizes the nanoparticles confirms the presence of proteins. The resulting spectra displayed different functional groups including tensile O-H bonds, amide bonds, N-H-, C-O, and C-OH tensile vibrations .
According to the provided EDAX diagram, the presence of selenium structure as an element was approved (Fig. 5 (c)). The presence of Se-NPs with an almost spherical shape and a size of approximately 64 nm within the scale of 200 nm was proved by FESEM/PSA images (Fig. 5 (a, b)).
2.5. Disk Diffusion Agar test
The desired fungi's standard and resistant strains were cultured on a plate that contained sabouraud dextrose agar (SDA) medium. Then, drug-saturated disks (itraconazole and amphotericin B), along with the disks that were saturated with biosynthesized Se-NPs, were placed on the surface of agar medium by the usage of disk diffusion agar (DDA) method to be incubated at 35 °C for 18-24 h. The results of resistance and sensitivity to microorganisms were qualitatively examined on the surface of agar medium and as it was expected, the microorganism were observed to be somewhat sensitive towards the nanoparticle-saturated disks and formed an Aura of small lack of growth.
Standard strains of C.albicans: TIMML-1306,1291,491,183, C.tropicalis: TIMML-1316, C.krusei: TIMML-1321, C.glabrata: TIMML-368, C. parapsilosis: ATCC-2201, A.flavus: TIMML-050 andA.fumigatus: TIMML-025 was cultivated on SDA and incubated at 35°C for 24 h in order to prepare fungal suspensions for antifungal susceptibility testing. To evaluate the effect of antifungal drugs on the growth of desired colonies, we dissolved 3.2 mg of pure itraconazole powder in 2.4 mL of DMSO (concentration = 10X) to force the appointed working concentration in the RPMI environment to reach the concentration of X. To prepare the specified concentration of pure amphotericin B powder, 3.2 mg of pure drug powder was dissolved in 1 mL of DMSO; this particular amount of concentration in this case is 100X, which acts as the drug stock in the course of arranging the drug plate in RPMI-1640 medium. Once this value was forced to reach the concentration of X, which is, it was diluted in a ratio of 1 to 100 and added to the wells.
Pure drug powder was obtained from itraconazole (Janssen, Beerse, Belgium), anidulafungin (Pfizer, Sandwich, United Kingdom), and amphotericin B (Bristol-Mers-Squib, Woerden, The Netherlands). The antifungal susceptibility testing was performed by the application of clinical & laboratory standards institute (CLSI) M38-A2. The Minimum Inhibitory Concentration (MIC) method was carried out in duplicate on fungal species' leachates that involved the usage of different and serial concentrations of itraconazole, amphotericin B, and anidulafungin, along with biosynthesized nanoparticles using 96 wells plates. Within a number range of 10 to 1, the wells concentration was 0.625-64 µg/ ml. Meanwhile, well No.11 was considered as a negative control and well No. 12 was a positive control.
According to CLSI standards, Candida contains a minimum growth inhibitory concentration of any well that would reach a stunted growth of up to 80%. However, in the case of Aspergillus species, the minimum inhibitory concentration is represented by the well that is 100% stunted. The susceptibility test was performed on the leachates of fungal species through different and serial concentrations of itraconazole and nanoparticles. The results were evaluated and compared subsequent to the incubation process at 35 °C for 24 to 48h .