*2.2. Characterization of Se-NPs*

The characterization of Se-NPs was performed by using JASCO V-560, UV-Vis. spectrophotometer, Tokyo, Japan, at the wavelength range from 200–900 nm and at a resolution of 1 nm. Cell-free supernatant without SeO<sup>2</sup> was used as blank to adjust the baseline. Toward particle size investigation, the specimens were diluted ten times by deionized water before being estimated. To determine the morphology and size of the manufactured Se-NPs, TEM microscopy, model JEOL JEM-100 CX (Peabody, MA, USA) was used. TEM imaging was carried out by drop covering the Se-NPs upon carbon-coated TEM layers. Dynamic light scattering (DLS) was used to determine the size distribution, while the average particle size was determined by PSSNICOMP 380-ZLS particle sizing system (St. Barbara, CA, USA). For XRD analysis, the adjusted sample was centrifuged, and the precipitate was dried under vacuum and taken for XRD analysis. X-ray diffraction patterns were obtained with XRD- 6000 series, including stress analysis, residual austenite quantitation, crystallite size/lattice strain, crystallinity calculation, and materials analysis via overlaid X-ray diffraction patterns Shimadzu apparatus using nickel-filter and Cu-Ka target, Shimadzu Scientific Instruments (SSI), (Kyoto, Japan). The average crystalline size of the Se-NPs was also determined by using Debye–Scherrer equation: D = kλ/β Cos θ. Here, D is the average crystalline size (nm), k is the Scherrer constant with the value from 0.9 to 1, λ is the X-ray wavelength, β is the full width of half maximum, and θ is the Bragg diffraction angle (degrees). The estimations included stress investigation, remaining austenite quantitation, crystallite capacity, crystallinity consideration, and materials examination through overlaid X-ray diffraction models. Finally, Se-NPs concentration assessment was performed using UNICAM939 Atomic Absorption Spectroscopy, Cambridge, UK, and implemented with deuterium experience improvement. All suspensions were prepared using ultra-pure water [34–41]. Furthermore, the morphology size of the manufactured NPs was read by practicing TEM microscopy, JEOL JEM-100 CX, (Peabody, MA, USA).
