Multifunctional Eco-Friendly Synthesis of ZnO Nanoparticles in Biomedical Applications

This work describes an environmental-friendly preparation of ZnO nanoparticles using aqueous oat extract. The advanced electrochemical and optical features of green synthesized ZnONPs displayed excellent antibacterial activity and exhibited an important role in pharmaceutical determinations. The formation of nanoscale ZnO was confirmed using various spectroscopic and microscopic investigations. The formed nanoparticles were found to be around 100 nm. The as-prepared ZnONPs were monitored for their antibacterial potential against different bacterial strains. The inhibition zones for ZnONPs were found as Escherichia coli (16 mm), Pseudomonas aeruginosa (17 mm), Staphylococcus aureus (12 mm) and Bacillus subtilis (11 mm) using a 30-µg mL−1 sample concentration. In addition, ZnONPs exhibited significant antioxidant effects, from 58 to 67%, with an average IC50 value of 0.88 ± 0.03 scavenging activity and from 53 to 71% (IC50 value of 0.73 ± 0.05) versus the scavenging free radicals DPPH and ABTS, respectively. The photocatalytic potential of ZnONPs for Rhodamine B dye degradation under UV irradiation was calculated. The photodegradation process was carried out as a function of time-dependent and complete degradation (nearly 98%), with color removal after 120 min. Conclusively, the synthesized ZnONPs using oat biomass might provide a great promise in the future for biomedical applications.


Introduction
Nanoscale materials, especially metal oxide nanostructures, are known as a distinctive group of nanopaerticles with unique physicochemical properties and possess broad applications in various scientific areas such as sensing technology [1,2], cell adhesion and tissue engineering [3], industrial packaging [4,5] catalysis [6,7] and biomedical investigations [8,9].
Zinc oxide nanoparticles (ZnONPs) have gained extensive awareness for their advanced biocompatible features and high strength under utmost circumstances [10,11]. ZnONPs have been used in medicine [12], sensors [13], industrial additives [14], photocatalysis [15], antioxidants and antibacterial potentials [16,17]. The increased adaptation of pathogens and microorganisms' resistance to antibiotics requires the synthesis of new components that have the ability to destroy the microorganism without the probability of adaptations appearing. Thus, the production of new components that possesses antimicrobial properties is greatly necessary [18]. Zinc oxide is recorded as safe metal oxide (21CFR182.8991) by the United States Food and Drug Administration. It is commonly used as a food preservative in the fortification of cereal-based foods. Because of its antimicrobial potential, ZnO has also been used in the packaging of food cans such as meat, corn, fish and peas to prevent spoilage and preserve the color [19]. Recently, ZnONPs have gained more attention as an antimicrobial agent than bulk particles. ZnO nanoparticles have excellent interaction with microorganisms, due to their high ratio of surface area to volume and minimized size. Several studies have reported that ZnONPs possess selective The formation of ZnONPs was characterized using various spectroscopic methods. The optical properties of the as-prepared ZnONPs were detected using UV-vis detection. The absorption spectra showed a clear broad absorption peak at 355 nm for ZnONPs ( Figure 1a). The change in the color of oat biomass from yellowish to pale white after boiling for 20 min, then to white after heating at 500 • C for 2 h, might be attributed to the photoexcitation of surface plasmon vibrations from the valence to the conduction band of the formed ZnONPs [44]. These color changes revealed the complete interaction of oat biomass and zinc nitrate hexahydrate producing ZnONPs. The band gap of the synthesized ZnONPs was estimated by the following equation: where h, c and λ are Planck's constant (6.626 × 10 −34 J s), the velocity of light (3 × 10 8 m/s) and the wavelength (nm), respectively. The band gap was found to be 3.61 eV at the wavelength λ max of 355 nm, which confirmed the formation of nanoparticles when compared with the band gap of ZnO bulk (3.3 eV). The optical outcomes of ZnONPs match those previously reported [45]. Moreover, in nanomaterials, the relation between the size diameter of the particles and the band gap is inversely proportional. The size diameter of nanoparticles is contrarily proportional to the corresponding band gap. The increase of nanoparticles, the decrease of band gap, but it never reaches zero [46].
Considering the curing efficiency of metal oxide nanostructures synthesis, especially ZnONPs using natural biomasses (plant extracts), the objective of the present work is the eco-friendly synthesis and characterization of ZnONPs using oat biomass as a natural reducing agent. The biomedical potential of the prepared ZnONPs was separately investigated for antibacterial, antioxidant and photocatalytic properties.

Green Synthesized ZnONPs Characterization
The formation of ZnONPs was characterized using various spectroscopic methods. The optical properties of the as-prepared ZnONPs were detected using UV-vis detection. The absorption spectra showed a clear broad absorption peak at 355 nm for ZnONPs ( Figure 1a). The change in the color of oat biomass from yellowish to pale white after boiling for 20 min, then to white after heating at 500 °C for 2 h, might be attributed to the photoexcitation of surface plasmon vibrations from the valence to the conduction band of the formed ZnONPs [44]. These color changes revealed the complete interaction of oat biomass and zinc nitrate hexahydrate producing ZnONPs. The band gap of the synthesized ZnONPs was estimated by the following equation: where h, c and λ are Planck's constant (6.626 × 10 −34 J s), the velocity of light (3 × 10 8 m/s) and the wavelength (nm), respectively. The band gap was found to be 3.61 eV at the wavelength λmax of 355 nm, which confirmed the formation of nanoparticles when compared with the band gap of ZnO bulk (3.3 eV). The optical outcomes of ZnONPs match those previously reported [45]. Moreover, in nanomaterials, the relation between the size diameter of the particles and the band gap is inversely proportional. The size diameter of nanoparticles is contrarily proportional to the corresponding band gap. The increase of nanoparticles, the decrease of band gap, but it never reaches zero [46]. Photoluminescence (PL) detection was used to determine the emission properties of ZnONPs ( Figure 1b). The photoluminescence of the ZnONP sample showed five significant emission bands at 403, 445 and 470 nm (blue bands), 485 (green band) and 530 nm (red bands). The three blue bands are correlated to the defect structures in the ZnO crystal. However, the two green and red bands can be corresponded to the transition between the oxygen vacancy and interstitial oxygen [47].
A dynamic light-scattering analysis was employed to calculate the distribution and average particle size of the green synthesized ZnONPs. The obtained ZnONP particle Photoluminescence (PL) detection was used to determine the emission properties of ZnONPs ( Figure 1b). The photoluminescence of the ZnONP sample showed five significant emission bands at 403, 445 and 470 nm (blue bands), 485 (green band) and 530 nm (red bands). The three blue bands are correlated to the defect structures in the ZnO crystal. However, the two green and red bands can be corresponded to the transition between the oxygen vacancy and interstitial oxygen [47].
A dynamic light-scattering analysis was employed to calculate the distribution and average particle size of the green synthesized ZnONPs. The obtained ZnONP particle size was approximately around 100 nm (Figure 2a). A further analysis (zeta potential) was performed to determine the constancy and surface charge of the pre-formed ZnONPs. The recorded results indicate that the moderate stability of the formed ZnONPs resulted from the negatively charged groups of the capping molecules present on the ZnONPs' surface. The evaluation of the zeta-potential depends on the motion of ZnONPs under the effect of the applied electric field. The determined average value of zeta-potential resulting from the ZnONP analysis was −27.6 mV, revealing the high stability of the prepared ZnONPs ( Figure 2b).
size was approximately around 100 nm (Figure 2a). A further analysis (zeta potential) was performed to determine the constancy and surface charge of the pre-formed ZnONPs. The recorded results indicate that the moderate stability of the formed ZnONPs resulted from the negatively charged groups of the capping molecules present on the ZnONPs' surface. The evaluation of the zeta-potential depends on the motion of ZnONPs under the effect of the applied electric field. The determined average value of zeta-potential resulting from the ZnONP analysis was −27.6 mV, revealing the high stability of the prepared ZnONPs (Figure 2b). The FT-IR spectra (4000-400 cm −1 ) were recorded to study the possible existence of biomolecules responsible for the reduction and capping of the green synthesized ZnONPs. The particular vibration bands were identified. The FT-IR spectra of oat biomass before and after the reaction of zinc nitrate hexahydrate to ZnONPs are shown in Figure 3a,b. The oat biomass spectra showed various absorption bands at 3754 (medium O-H stretching), 3430 (strong O-H stretching), 2925 (C-H stretching) [48], 2350 (medium N-H amide II), 1633 (strong C=C alkene), 1405 (symmetric stretching of carboxyl side groups of amino acids residue), 1261 (amide III band of protein) [49], 1030 (C-N stretching vibration of amine) and 620 cm −1 (strong C-halo, alkyl halide). The previously observed bands were shifted to 3750, 3424, 2922, 2348, 1625, 1383, 1126, 1028 and 445 (Zn-O band) cm −1 in the ZnONPs sample [47]. The recorded results confirm that the synthesized ZnONPs were surrounded by the metabolites of biomolecules such as tocopherols and tocotrienols, phenolic acids, carbohydrates and phenolic alkaloids (avenanthramides). The reduction of zinc nitrate into Zn-O can be conducted by the ability of C=O groups of amino acids and proteins to act as reducing and capping to the surface of formed Zn-O. The FT-IR spectra (4000-400 cm −1 ) were recorded to study the possible existence of biomolecules responsible for the reduction and capping of the green synthesized ZnONPs. The particular vibration bands were identified. The FT-IR spectra of oat biomass before and after the reaction of zinc nitrate hexahydrate to ZnONPs are shown in Figure 3a,b. The oat biomass spectra showed various absorption bands at 3754 (medium O-H stretching), 3430 (strong O-H stretching), 2925 (C-H stretching) [48], 2350 (medium N-H amide II), 1633 (strong C=C alkene), 1405 (symmetric stretching of carboxyl side groups of amino acids residue), 1261 (amide III band of protein) [49], 1030 (C-N stretching vibration of amine) and 620 cm −1 (strong C-halo, alkyl halide). The previously observed bands were shifted to 3750, 3424, 2922, 2348, 1625, 1383, 1126, 1028 and 445 (Zn-O band) cm −1 in the ZnONPs sample [47]. The recorded results confirm that the synthesized ZnONPs were surrounded by the metabolites of biomolecules such as tocopherols and tocotrienols, phenolic acids, carbohydrates and phenolic alkaloids (avenanthramides). The reduction of zinc nitrate into Zn-O can be conducted by the ability of C=O groups of amino acids and proteins to act as reducing and capping to the surface of formed Zn-O.
An XRD analysis was also performed at ambient degree to evaluate the crystalline structure and phase purity of the green synthesized ZnONPs using λ = 1.5418 Å, 30 mA, 35 kV, 0.02 • and 0.3 s/point as K α radiation, operating current, voltage, resolution scan rate, respectively. The XRD pattern displayed various significant peaks at the 31.
where D, k, λ, and β represent the average crystal size, shape factor (0.9), wavelength (0.15416) and Bragg angle θ of the X-ray (1.5406 Å) Cu Ka radiation, respectively. The average ZnONPs crystallite size was calculated as 17.52 nm.  An XRD analysis was also performed at ambient degree to evaluate the crystalline structure and phase purity of the green synthesized ZnONPs using λ = 1.5418 Å, 30mA, 35 kV, 0.02ᵒ and 0.3 s/point as Kα radiation, operating current, voltage, resolution scan rate, respectively. The XRD pattern displayed various significant peaks at the 31.
where D, k, λ, and β represent the average crystal size, shape factor (0.9), wavelength (0.15416) and Bragg angle Ɵ of the X-ray (1.5406 Å) Cu Ka radiation, respectively. The average ZnONPs crystallite size was calculated as 17.52 nm. An EDX detection was applied to quantify the chemical compositions of the biogenic synthesized ZnONPs using oat biomass. The EDX spectrum showed significant signals corresponding to the Zn (weight%, 69.9%; atomic %, 36.2%) and O (weight%, 30.1%; atomic % 63.77%) elements, revealing the formation of ZnONPs ( Figure 4). The results revealed the presence of metallic zinc oxide with high purity and no additional peaks corresponding to other elements were recorded. An EDX detection was applied to quantify the chemical compositions of the biogenic synthesized ZnONPs using oat biomass. The EDX spectrum showed significant signals corresponding to the Zn (weight%, 69.9%; atomic %, 36.2%) and O (weight%, 30.1%; atomic % 63.77%) elements, revealing the formation of ZnONPs ( Figure 4). The results revealed the presence of metallic zinc oxide with high purity and no additional peaks corresponding to other elements were recorded.  A microscopic analysis was performed to visualize the surface structure (size andshape) of the synthesized ZnONPs. The size and shape of ZnONPs are demonstrated in Figure 5a. This image revealed that the formed nanoparticles were nanocrystalline hexagonal in shape with a size distribution of around 100 nm. The SEM image demonstrated that the clusters of ZnONPs were nearly hexagonal with rough surface (Figure 5b). A microscopic analysis was performed to visualize the surface structure (size andshape) of the synthesized ZnONPs. The size and shape of ZnONPs are demonstrated in Figure 5a. This image revealed that the formed nanoparticles were nanocrystalline hexagonal in shape with a size distribution of around 100 nm. The SEM image demonstrated that the clusters of ZnONPs were nearly hexagonal with rough surface (Figure 5b). A microscopic analysis was performed to visualize the surface structure (size andshape) of the synthesized ZnONPs. The size and shape of ZnONPs are demonstrated in Figure 5a. This image revealed that the formed nanoparticles were nanocrystalline hexagonal in shape with a size distribution of around 100 nm. The SEM image demonstrated that the clusters of ZnONPs were nearly hexagonal with rough surface (Figure 5b).

Thermal Stability of Biosynthesized ZnONPs
The thermal behavior of the biosynthesized ZnONPs using oat biomass extract was investigated through the TGA/DSC mode. As the synthesis of ZnONPs was carried out at 80 °C, the effect of temperature variation on the stability of the formed metal oxide nanoparticles was examined [50]. Figure 6a displays the thermogram behavior of ZnONPs in the temperature range 80-700 °C. The obtained results show that the nominal overall loss of biosynthesized ZnONPs was around 9.5% indicating the significant thermal stability of the sample. Moreover, it was reported that the zinc nitrate precursor that had been used in the synthesis process was completely converted to thermal stable ZnONPs [51]. The thermogram displayed three remarkable regions of weight losses and diffraction scanning calorimetry (DSC) was applied to interpret these regions ( Figure  6b).

Thermal Stability of Biosynthesized ZnONPs
The thermal behavior of the biosynthesized ZnONPs using oat biomass extract was investigated through the TGA/DSC mode. As the synthesis of ZnONPs was carried out at 80 • C, the effect of temperature variation on the stability of the formed metal oxide nanoparticles was examined [50]. Figure 6a displays the thermogram behavior of ZnONPs in the temperature range 80-700 • C. The obtained results show that the nominal overall loss of biosynthesized ZnONPs was around 9.5% indicating the significant thermal stability of the sample. Moreover, it was reported that the zinc nitrate precursor that had been used in the synthesis process was completely converted to thermal stable ZnONPs [51]. The thermogram displayed three remarkable regions of weight losses and diffraction scanning calorimetry (DSC) was applied to interpret these regions ( Figure 6b). The recorded peaks that appeared at 140.5, 230.1 and 370.3 °C were related to the loss of moisture and volatile components from particles surface (2.1 w% loss), conversion of Zn(OH)2 to complete calcined ZnONPs (4.5 w% loss) and production of ZnONPs from the complete degradation of organic matters (2.9 w% loss). No other peak was detected at 700 °C, proving the low-temperature calcination of ZnONPs at 400 °C. According to the obtained results, it can be concluded that the bioactive compounds in the oat biomass extract mainly had reduction abilities, with a minimum chelation interaction. The reduction of zinc nitrate to Zn 2+ ions by the bioactive components was performed in the first stage of synthesis with low chelation of a Zn-bioactive component complex. These bio-complex products were further degraded to ZnO by a further oxidation pro- The recorded peaks that appeared at 140.5, 230.1 and 370.3 • C were related to the loss of moisture and volatile components from particles surface (2.1 w% loss), conversion of Zn(OH) 2 to complete calcined ZnONPs (4.5 w% loss) and production of ZnONPs from the complete degradation of organic matters (2.9 w% loss). No other peak was detected at 700 • C, proving the low-temperature calcination of ZnONPs at 400 • C. According to the obtained results, it can be concluded that the bioactive compounds in the oat biomass extract mainly had reduction abilities, with a minimum chelation interaction. The reduction of zinc nitrate to Zn 2+ ions by the bioactive components was performed in the first stage of synthesis with low chelation of a Zn-bioactive component complex. These bio-complex products were further degraded to ZnO by a further oxidation process of Zn 2+ to ZnO to establish the proper formation of metallic ZnONPs from zinc nitrate.
The antibacterial effect of green synthesized ZnONPs with oat biomass was studied and the outcomes confirm that ZnONPs exhibited excellent dose-dependent manner antibacterial potential ( Table 1). The calculated inhibition zones were found as E. coli (16 mm), P. aeruginosa (17 mm), S. aureus (12 mm) and B. subtilis (11 mm) for ZnONPs ( Figure 7). Thus, the recorded results reveal that the ZnONPs synthesized by oat biomass showed excellent antibacterial potential against all bacterial strains. The highest activity was noticed versus P. aeruginosa and E. coli using 30 µg mL −1 . Moreover, the high antibacterial behavior of ZnONPs (oat biomass) could be ascribed to their minimum particle size and shape, and the bioactive feature of oat phytochemical components such as vitamin E, sterols, phytic acid, carotenoids, β-glucan and phenolics [59].

Bacteriostatic and Bactericidal Estimation
The bacteriostatic (MIC) and bactericidal (MBC) values of ZnONPs against P. aeruginosa and E. coli were studied using the agar well diffusion method. The MIC is known as the minimum nanoparticle concentration that inhibits bacterial growth [60]. In the current study, the suitable least concentration to inhibit the growth visibility of P. aeruginosa and E. coli was estimated after 24 h incubation time at 37 °C. It was observed that the gradual increase in ZnONP concentration from 5 to 640 µg mL −1 caused a remarkable reduction in the viability of bacterial cells (p < 0.05). The measured MIC was 160 µg mL −1 for P. aeruginosa and E. coli. The size of ZnONPs and their concentrations play a crucial role in antibacterial potential. As previously reported, antibacterial activity of ZnONPs

Bacteriostatic and Bactericidal Estimation
The bacteriostatic (MIC) and bactericidal (MBC) values of ZnONPs against P. aeruginosa and E. coli were studied using the agar well diffusion method. The MIC is known as the minimum nanoparticle concentration that inhibits bacterial growth [60]. In the current study, the suitable least concentration to inhibit the growth visibility of P. aeruginosa and E. coli was estimated after 24 h incubation time at 37 • C. It was observed that the gradual increase in ZnONP concentration from 5 to 640 µg mL −1 caused a remarkable reduction in the viability of bacterial cells (p < 0.05). The measured MIC was 160 µg mL −1 for P. aeruginosa and E. coli. The size of ZnONPs and their concentrations play a crucial role in antibacterial potential. As previously reported, antibacterial activity of ZnONPs has been changed with respect to the size and concentration-dependent manner [61]. The large surface area of the ZnONPs, due to their small size, enhances their penetration of the microorganism cell through the cell membrane and improve the antibacterial effect with their high concentration [62].
MBC is known as the least concentration of test sample compound that can lead to pathogenic cell death under a particular condition through a fixed time period (Figure 8a,b). The MBC for P. aeruginosa and E. coli were 160 and 320 µg mL −1 of ZnONPs, respectively ( Table 2). Based on the unique physical and chemical properties of ZnONPs, different possible mechanisms of their antibacterial effects have been suggested, with specific interactions such as adsorption, release of Zn 2+ ions, generation of reactive oxidative species (ROS) [63,64] and intercellular responses of microbial cells, such as lipid peroxidation, cell membrane damage, energy metabolism inhibition and disruption in DNA replication [65,66]. As can be seen from Figure 9, it was demonstrated that the positively charged ZnONPs interacted with the negatively charged cell wall of bacterial cells and, after adsorption, they internalized into the bacterial cell causing loss of cell integrity, rupture of the cell membrane and further oxidative stress due to lipid peroxidation leading to generation of ROS, damage of DNA and inhibition of the bacterial growth.

Morphological Changes under SEM
The morphological changes in the P. aeruginosa and E. coli surfaces were investigated under SEM. As a result, the cells treated with oat biomass were swallowed, with a slight change in the morphological shape (Figure 10b,e). The surface coating of bacterial cells by ZnONPs showed significant changes in the shape of bacterial cells with significant cell damage (Figure 10c,f). ZnONPs penetrated the peptidoglycan membrane of P. aeruginosa and E. coli, causing cell membrane damage, releasing the contents of bacterial cell and, resulting in cell death. These results were compared with the control (untreated bacterial cells) (Figure 10a,d).   NIL ** NIL ** 5 2 Figure 9. The possible mechanism of antibacterial effect of green synthesized ZnONPs using oat biomass against the bacterial cell.

Antibacterial Activity of ZnONPs Using Various Plant Biomasses
A comparative study was performed to evaluate the antibacterial activity of ZnONPs prepared by oat biomass and the previously biosynthesized ZnONPs using different plant parts, such as seeds, fruits, leaves, stems and roots. The use of natural extracts of plants produces extensive phytochemical compounds that are cheap, eco-friendly and provides highly purified nanoparticles [67]. Plant extracts are the most preferred natural source for the biosynthesis of nanoparticles with various shapes, particle sizes and stability [68]. They reduce the metal of metal oxides into zero valences with the aid of phytochemicals, including vitamins, amino acids, phenolic compounds, polysaccharides, proteins, alkaloids, terpenoids and sterols, that are secreted from the plants [69]. Table 3 shows the plant extract-mediated synthesis of ZnONPs [70][71][72][73][74]. In the past decades, studies have reported that the green synthesized ZnONPs have an important role in industrial products such as coating, cosmetics, paint, plastics and rubber. Recently, biosynthesized ZnONPs have played a crucial role in biomedical aspects, particularly in the anticancer and antipathogenic fields. These are due to their potential capability to produce excess free reactive oxidative species (ROS), release zinc ions and cause cell damage. Additionally, ZnONPs have also been potentially suggested for antidiabetic treatment due to their ability to keep the structural integrity of insulin. Moreover, ZnONPs have exhibited excellent photoluminescent characteristics and have served as some of the main effective metal oxides in bioimaging. Therefore, biosynthesized ZnONPs can be considered as among the main safe metal oxides used in progressing biomedical applications [75].

Morphological Changes under SEM
The morphological changes in the P. aeruginosa and E. coli surfaces were investigated under SEM. As a result, the cells treated with oat biomass were swallowed, with a slight change in the morphological shape (Figure 10b,e). The surface coating of bacterial cells by ZnONPs showed significant changes in the shape of bacterial cells with significant cell damage (Figure 10c,f). ZnONPs penetrated the peptidoglycan membrane of P. aeruginosa and E. coli, causing cell membrane damage, releasing the contents of bacterial cell and, resulting in cell death. These results were compared with the control (untreated bacterial cells) (Figure 10a,d).

Antibacterial Activity of ZnONPs Using Various Plant Biomasses
A comparative study was performed to evaluate the antibacterial activity of ZnONPs prepared by oat biomass and the previously biosynthesized ZnONPs using different plant parts, such as seeds, fruits, leaves, stems and roots. The use of natural extracts of plants produces extensive phytochemical compounds that are cheap, ecofriendly and provides highly purified nanoparticles [67]. Plant extracts are the most preferred natural source for the biosynthesis of nanoparticles with various shapes, particle sizes and stability [68]. They reduce the metal of metal oxides into zero valences with the aid of phytochemicals, including vitamins, amino acids, phenolic compounds, polysaccharides, proteins, alkaloids, terpenoids and sterols, that are secreted from the plants [69]. Table 3 shows the plant extract-mediated synthesis of ZnONPs [70][71][72][73][74]. In the past decades, studies have reported that the green synthesized ZnONPs have an important role in industrial products such as coating, cosmetics, paint, plastics and rubber. Recently, biosynthesized ZnONPs have played a crucial role in biomedical aspects, particularly in the anticancer and antipathogenic fields. These are due to their potential capability to produce excess free reactive oxidative species (ROS), release zinc ions and cause cell damage. Additionally, ZnONPs have also been potentially suggested for antidiabetic treatment due to their ability to keep the structural integrity of insulin. Moreover, ZnONPs have exhibited excellent photoluminescent characteristics and have served as some of the main effective metal oxides in bioimaging. Therefore, biosynthesized ZnONPs can be considered as among the main safe metal oxides used in progressing biomedical applications [75].

Antioxidant Potential of ZnONPs
The DPPH assay is commonly applied to evaluate the antioxidant potential due to its sensitivity to determine low concentrations of active components [76]. It is a nitrogencentered free radical; therefore, any substance that scavenges remarkable amounts of DPPH could reduce the quantities of other reactive nitrogen species in living cells. The influence of several concentrations of ZnONPs and oat biomass on DPPH' free radical antioxidant potential is presented in Table 4. The data demonstrated that both ZnONPs and oat biomass have free radical scavenging activity. However, the synthesized ZnONPs exhibited stronger scavenging activity against DPPH than oat biomass. The DPPH potentials of ZnONPs and oat biomass were observed to elevate in a dose-dependent manner. At the concentrations of 25-100 µg mL −1 , the green synthesized ZnONPs exhibited scavenging potential from 58 to 67% with an average IC 50 value of 0.88 ± 0.03. The antioxidant activity was lower than that of ascorbic acid at 100 µg mL −1 (73%). For the ABTS assay, the pre-formed radical monocation of 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS* + ) resulted from the oxidation of ABTS with potassium persulphate. It was reduced by the effect of hydrogen-donating antioxidant materials [77]. The ability of ZnONPs to reduce ABTS to ABTS + ions was also notably higher than that of oat biomass at different concentrations, from 25 to 100 µg mL −1 , by 53-71% (IC 50 value of 0.73 ± 0.05). The outcomes of this study reveal that oat biomass is an excellent natural source of phenolic compounds, which are important secondary metabolites responsible for antioxidant activity. In addition, the tunable physical and chemical properties of ZnONPs showed significant antioxidant activity.

Photocatalytic Influence of ZnONPs
The photocatalytic influence of the pre-prepared ZnONPs to produce RB degradation was investigated in an aqueous solution under visible irradiation. The decomposition of RB was determined by measuring the intensity of the absorption peak maxima at 550 nm against the irradiation time. The photodecomposition of RB dye enhanced with time and the complete degradation was almost achieved (98%) with decolorization after 120 min ( Figure 11). The outcomes of the current study provide an excellent catalytic behavior of the green formed ZnONPs towards the reductive decomposition of RB dye and the outcomes are matched those of previously addressed studies [78]. A linear relationship (Beer-Lambert law) was obtained from plotting the concentrations of RB against the absorption maxima at 550 nm. This could be ascribed to the stable number of photons available for photodecomposition. The capping layer of the reducing agent (oat biomass) on the ZnONPs' surface may have also enhanced the potential adsorption between ZnONPs and RB molecules. Consequently, the redox reaction between RB and the reducing agent can be displayed more rapidly for nanoscale particles [79]. The obtained results reveal that the high reactivity of the large surface area of ZnONPs made them efficient photocatalysts for dye degradation under UV light. The possible reduction-oxidation mechanism can be represented as follow: Molecules 2022, 27, x FOR PEER REVIEW 13 of 20 H2O2 +e − → ᵒ OH + OH − (9) The generation of electron-hole pairs, resulting from the valance-conduction band transfer of excited electrons, explains the mechanism of photocatalysis in dye under irradiation. The dye molecules were oxidized to non-toxic products (water, carbon dioxide, etc.) due to the formation of hydroxyl radicals. The high stability, conductivity and unique optical features of ZnONPs enable suitable trapping of photoexcited electrons on their surface and prevent electron-hole recombination [80]. Moreover, the photocatalysis of ZnONPs under visible light could be produced due to surface plasmon resonance, the generation of free radicals and the interaction with oxygen molecules resulting from the collective oscillations of electrons. Additionally, the formation of positive holes conducted from electron excitation resulted in the degradation of RB dye [81]. Figure 11. The photocatalytic effect of ZnONPs calculated as % of RB dye degradation in the presence of UV irradiation with respect to ascorbic acid as control. * and **, p < 0.05.

Collection of Oat Biomass
After oat seed harvest, oat biomass was collected and cleaned by washing thoroughly with tap water, then dried at 80 ºC for 12 days to eliminate the residual moisture. The obtained oat biomass was further grinded using an electric grinder (Vermeer, model HG-200) and sieved using a stainless-steel fine-mesh sieve to obtain a homogenous powder.

Bacterial Strains and Nutritional Matrices
All bacterial strains included in this study were provided by Microbiology Department, King Saud University, Saudi Arabia. The Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25966), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) bacterial strains were used. The pre-culture of these isolates was performed using nutrient agar (Oxoid). The generation of electron-hole pairs, resulting from the valance-conduction band transfer of excited electrons, explains the mechanism of photocatalysis in dye under irradiation. The dye molecules were oxidized to non-toxic products (water, carbon dioxide, etc.) due to the formation of hydroxyl radicals. The high stability, conductivity and unique optical features of ZnONPs enable suitable trapping of photoexcited electrons on their surface and prevent electron-hole recombination [80]. Moreover, the photocatalysis of ZnONPs under visible light could be produced due to surface plasmon resonance, the generation of free radicals and the interaction with oxygen molecules resulting from the collective oscillations of electrons. Additionally, the formation of positive holes conducted from electron excitation resulted in the degradation of RB dye [81].

Collection of Oat Biomass
After oat seed harvest, oat biomass was collected and cleaned by washing thoroughly with tap water, then dried at 80 • C for 12 days to eliminate the residual moisture. The obtained oat biomass was further grinded using an electric grinder (Vermeer, model HG-200) and sieved using a stainless-steel fine-mesh sieve to obtain a homogenous powder.

Bacterial Strains and Nutritional Matrices
All bacterial strains included in this study were provided by Microbiology Department, King Saud University, Saudi Arabia. The Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25966), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) bacterial strains were used. The pre-culture of these isolates was performed using nutrient agar (Oxoid).

Preparation of Aqueous Oat Biomass Extract
The aqueous extract of oat biomass was prepared by adding 20 g of dried oat powder to 200 mL of boiled distilled water at 100 • C for 1 h. After cooling at room temperature, the obtained extract was purified using filter paper (Whatman filter paper No. 40) and kept in a refrigerator at 4 • C.

Preparation of ZnONPs Using Oat Biomass Extract
The green biosynthesis of ZnONPs was based on reducing, capping and stabilizing zinc nitrate hexahydrate precursor using the phytochemical components of oat extract (pale, yellowish color), which contains bioactive compounds, including β-glucan, phenolic acids (ferulic, p-coumaric, caffeic, vanillic and hydroxybenzoic acid), phenolic alkaloids (avenanthramides), vitamin E (α-Tocotrienols and α-tocopherols), starch and protein [82]. These bioactive compounds have various functional groups (-OH, COOH, -NH, C=O, -SH) which serve as natural reducing, capping and stabilizing agents for nanoparticle synthesis [83]. The biosynthesis process was conducted by heating oat extract (50 mL) to 80 • C using a magnetic stirrer heater. Approximately, 5.0 g of zinc nitrate hexahydrate was mixed with the heated extract under stable magnetic stirring at 80 • C for 20 min until the formation of light, yellow-colored ZnONPs. The formed nanoparticles were centrifuged at 1500 rpm for 5 min and filtrated using filter paper (Whatman No 1). The filtrate was washed 3 times using deionized water and collected in a ceramic crucible, then calcined in a muffle oven for 2 h at 400 • C and stored for further use at 4 • C [84].

Spectroscopic and Microscopic Characterization
The UV-Vis spectra were recorded using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at an absorption wavelength range of 200-400 nm for the primary characterization of the synthesized ZnONPs. Fourier transform infrared spectroscopy (FTIR; PerkinElmer, Waltham, MA, USA) was performed in the IR region of 4000-400 cm −1 to study and confirm the possible predictable functional groups. X-ray diffraction (XRD; Shimadzu XRD-6000 diffractometer; Kyoto, Japan) was also carried out to study the crystalline shape of the synthesized ZnONPs using Kα radiation (λ = 1.5418 Å; operating current, 30 mA, voltage, 35 kV) at scan rate of 0.3 s/ point and a 0.02 • resolution at room temperature. The surface morphology of the prepared ZnONPs was examined under a scanning electron microscope (SEM; JSM-7610F; JEOL, Tokyo, Japan) and a transmission electron microscope (TEM; JEM-2100F; JEOL Ltd., Tokyo, Japan). Furthermore, the elemental content of the synthesized ZnONPs was determined using energy-dispersive X-ray spectroscopy (EDX; JSM-7610F; JEOL, Tokyo, Japan) in connection with an SEM microscope. Thermal stability of the biosynthesized ZnONPs was evaluated using a thermogravimetry and differential scanning calorimetry (TGA/DSC) analyzer (TGA/DSC-Seiko Exstar 6300, Tokyo, Japan) in the temperature range of 80-700 • C.

Antibacterial Activity
The antibacterial potential of green synthesized ZnONPs with oat biomass was screened in terms of zone of inhibition using an agar well diffusion assay [85] against different bacterial Gram-negative (Escherichia coli ATCC 25966 and Pseudomonas aeruginosa ATCC 27853) and Gram-positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633) strains. The nutrient agar (Oxoid) was used to pre-culture of bacterial stains. The bacterial suspension of 0.5 McFarland turbidity of each stain was prepared in 5 mL nutrient broth tubes for the antibacterial study. Sterile cotton swabs were used to load the resulted bacterial suspensions on the surface of Mueller Hinton (Oxoid) plates. Wells (6 mm) were assembled on the agar plates surface using a sterile cork borer and then 100 µL of each ZnONPs (10-30 µgmL −1 DMSO) was loaded. The loaded plates were aerobically kept for 24 h at 37 • C.

Bacteriostatic and Bactericidal Estimation
In the present assay, the minimum inhibitory concentration (MIC) of ZnONPs against P. aeruginosa and E. coli and was estimated (three times) by applying the micro-broth dilution method. The investigated concentrations were taken in the range from 5 to 640 µg mL −1 . This assay was conducted by performing 2-fold serial dilutions in 96-well plates. The positive control (bacterial cells and broth) was prepared in the first column. The last column (Milli-Q water) was considered as the negative control.
Each bacterial suspension was spreading and after the incubation time (24 h at 37 • C), ELISA reader (Biotech, Wuxi, China) applied to read out the results at 600 nm. For matching the results, tetracycline (TE; 25 µg mL −1 ) and DMSO were used as positive and negative controls, respectively. The least concentration of ZnONPs which could lead to a complete bactericidal effect (MBC) was determined by loading amounts of first turbid samples with no visible bacterial growth. The treated samples were carefully distributed on plates containing nutrient agar using a sterile L rod and incubated for 12 h at 37 • C.

Microscopic Study of P. aeruginosa and E. coli
The surface morphology of both treated and untreated P. aeruginosa and E. coli was investigated under SEM to evaluate the effect of ZnONPs. Prior to examination under a microscope, the treated P. aeruginosa and E. coli bacteria pieces (5-by-10 mm) were cut and kept in glutaraldehyde in a phosphate-buffered saline (3%) solution for 1 h. Then, they were fixed in an osmium tetroxide (2%) solution for 1 h. Ethanol and CO 2 , were utilized for tissues dehydration. The dried tissues were fixed on Al stubs with Ag pain vacuum coated with Au-palladium alloy and investigated using SEM at a 15 kV acceleration voltage.

Antioxidants
The antioxidant potential of ZnONPs was determined using two different radical scavenging assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ABTS (2,20 -azino-bis [3ethyl benzo thiazoline-6-sulphonic acid]). The DPPH free radical scavenging potential of oat and ZnONPs was evaluated following standard methods [86]. Prior to the investigation, 1.0 × 10 −4 mol L −1 DPPH, three different concentrations (25, 50, 100 µg mL −1 ) of oat and ZnONPs and ascorbic acid (AA) were prepared in menthol. Approximately, 50 µL of DPPH solution was mixed with different concentrations of oat and ZnONPs, as well as AA, in a 96-well microplate. The prepared mixture was continuously shacked in a dark place for 30 min using an orbital shaker. The absorbance of the sample was determined using a UV-Vis BioTek microplate (Biotech ELx 800; Biotek instruments, Inc, Highland Park, CA, United States) after incubation for 30 min at 518 nm against methanol as blank.
The scavenging ability was calculated using the following equation: % DPPH free radical scavenging effect = C r − T r /C r × 100 (11) where Cr and Tr represent the absorbance of the control and test samples, respectively. The 2, 20 -azino-bis [3-ethyl benzo thiazoline-6-sulphonic acid]) (ABTS) free radical scavenging assay was also used to evaluate the antioxidant activity of oat extract and ZnONPs using a reported standard method [87]. Briefly, 7.4 × 10 −3 ABTS and 2.6 × 10 −3 mol L −1 of potassium persulfate solutions were separately prepared. The working solution of ABTS was obtained by mixing equal amounts of the previously prepared stock ABTS and potassium persulfate solutions and kept without stirring for 12 h in a dark place. The analysis was conducted by mixing 125 µL of the ABTS working solution with 10 µL of different concentrations (25, 50, 100 µg mL −1 ) of oat and ZnONP tested samples, as well as ascorbic acid as a control. The final solution was left for 2 h in a dark place. The absorbance of the samples was recorded at 518 nm using a UV-Vis BioTek microplate vs. methanol as the blank reference. The scavenging potential was calculated using the following equation: % ABTS free radical scavenging effect = C r − T r /C r × 100 (12) where Cr and Tr represent the control and test samples absorbance, respectively.

Photocatalysis
To investigate the photocatalytic potential of the green synthesized ZnONPs, light irradiation using a UV lamp (Hamamatsu LC8; lamp power of 0.1 W) was utilized to depredate Rhodamine B (RB) dye at a wavelength of 550 nm at ambient temperature. Approximately, 1.0 mg of ZnONPs was mixed with 2.0 mL of RB dye solution (5.0 × 10 −6 mol L −1 ) and sonicated for 5.0 min, then kept under constant magnetic stirring for 30 min to cofirm an adsorption/desorption equilibrium on the ZnONP surface prior to irradiation. Under the same experimental conditions, a sample free from the nanoparticles was used as a control. In addition, the physisorption of ZnONP surface was evaluated by maintaining the sample in the dark. The detection was conducted by monitoring the samples at intervals of 10 min. Briefly, the collected samples were centrifuged at 8000 rpm for 10 min and the clear layer of each sample was measured using a UV-Vis spectrophotometer. The rate of degradation was detected at λ max 550 nm by measuring the reduction in absorption intensity of RB dye. The following equation was used to estimate the decomposition efficiency (DE): where A 0 and A are the initial absorption and absorption intensity after photocatalytic degradation at λ max 550 nm, respectively.

Conclusions
The present study suggests a clean and simple, safe and cost-effective approach for the green synthesis of ZnONPs using oat biomass. The resulted metal oxide nanoparticles were characterized using several spectroscopic and microscopic analysis to prove the formation of ZnONPs. The results reveal the preparation of ZnONPs with a particle size of around 100 nm. The resulting ZnONPs were screened for antibacterial activity and they exhibited strong antibacterial potential versus different types of bacterial strains. The green synthesized ZnONPs showed significant antioxidant activity. The DPPH potential of ZnONPs showed scavenging effect from 58 to 67%, with an average IC 50 value of 0.88 ± 0.03 and, in the ABTS method, the ability of ZnONPs to reduce ABTS to ABTS + ions was from 53 to 71% (IC 50 value 0.73 ± 0.05). In addition, ZnONPs exhibited the highest photocatalytic activity for RB dye degradation (98%) under visible light irradiation for 120 min.

Data Availability Statement:
The data used to support the findings of this study are included within the article.