157 © Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Synthesis and Physical Characterization of Manganese DioxideNanoparticles Using Leek Extract for Antibacterial Application Rand A. Hayder 1* and Zainab J. Shanan2 1,2Department of Physics, College of Sciences for Women, University of Baghdad, Baghdad, Iraq. *Corresponding Author. Received:19 May 2023 Accepted:20 June 2023 Published:20 July 2024 doi.org/10.30526/37.3.3503 Abstract Manganese dioxide nanoparticles were synthesized by the green method using leek with Manganous Sulphate Monohydrate, (MnSO4.H2O) in an environmentally friendly manner. The obtained MnO2 particle was characterized by x-ray diffraction (XRD), field scanning electron microscopy (FESEM), atomic force microscopy (AFM), and (UV-Visible) spectroscopy. The X- ray diffraction pattern showed peaks belonging to manganese oxide nanoparticles; 34.07 nm is the average crystalline size. From the field-scanning electron microscopy (FESEM) image, the surface morphology shows that the nanoparticles are spherical and rocky in shape. An atomic force microscopy (AFM) atomic force microscope was used to show MnO2 NPs as agglomerated particles with an average diameter of (50.20) nm. The absorption spectrum of MnO2 nanoparticles was determined using UV-visible, and the energy band was measured to be (4.31) eV. The results show that the band gap energy increases with decreasing particle size. The antibacterial property of nanoparticles was observed. These materials' antibacterial effects were examined utilizing some common Gram-positive and Gram-negative bacteria. The synthesis of MnO2-NPs inhibited the growth of S. aureus, k. pneumoniae, and E. coli, with half-dilutions. MnO2-NPs have shown good inhibition for S. aureus and lower efficient antibacterial activity against the bacterial k. pneumonia and E. coli, and the higher activity was at a concentration of (1000μg/ml). Keywords: Manganese Dioxide, nanoparticles, green synthesis, antibacterial activity, E.coli. 1. Introduction Biotechnology and nanotechnology are advancing, and this opens the way for study into the creation of nanoparticles. For the treatment of cancer, allergies, diabetes, inflammation, and infections, nanoparticles are widely used. It has a variety of uses in the fields of biomedical devices, renewable energy, cosmetics, medicine, and cleaning the environment[1, 2]. Due to their high specific surface area and recognized antibacterial activity, magnetic properties, catalytic, and optical characteristics, and metal NPs have been the subject of substantial research [3,4]. Manganese Dioxide is a well-known example of a nanoparticle that is usually utilized and has distinct optical, thermal, mechanical, chemical, and electrical properties[5, 6]. Because biological components serve as reducing as well as capping agents and do not require high energy, harmful chemicals, high pressures, or other processes, the green synthesis of nanoparticles is an https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0009-0001-4359-9506 mailto:rand.ali1204a@csw.uobaghdad.edu.iq https://orcid.org/0000-0002-5166-5200 mailto:Zainab_phys@csw.uobaghdad.edu.iq IHJPAS. 2024, 37( 3 ) 158 environmentally benign method [7]. The source of plant extracts determines how nanoparticles can be identified. Plant extracts have stabilizing and reducing effects[8]. Polyphenols, alkaloids, terpenoids, and flavonoids, are just a few of the phytochemical components found in plants that have been proven to cause metal ion reduction and the eventual formation of metal nanoparticles[9]. Additionally, compared to extracts, it is thought that phytomolecules from biogenic plants could improve their inherent capabilities, such as antibacterial, antioxidant, and anticancer[10]. Thus, green synthesis utilizing plant leaf extract improves the biocompatibility of nanoparticles and is the cause of the synergistic impact [11]. NPs and other nonmaterials are attracting much interest due to their distinctive physical properties. Furthermore, because of their physical properties, NPs are ideal for use in biological, electrical, sensing, and optoelectronic applications[12].In this research, manganese dioxide nanoparticles are prepared in an environmentally friendly manner. Green synthesis is important as it is an eco- friendly approach that involves the use of natural bioresources and avoids toxic chemicals to synthesize different types of NPs. The green method involves using Leek extract, which is a reducing agent and coating for nanoparticles, to study the effect of the preparation of NPs on antibacterial activity utilizing the good diffusion procedure and the physical characterizations of the MnO2 NPs. 2. Materials and Methods 2.1. Preparation of (leek) leaves extract 10gm of fresh leek leaves were used to produce the leek leaf extract. The leek leaves were properly cleaned using deionized (DI) water to get rid of any dirt and impurities before being dried by air. The dried leaves had been broken up into little bits and put through a mechanical mill after being ground up and placed in a 250ml beaker. The 100ml of DI water was then added, and the mixture was agitated for 60 minutes at 30°C. After cooling to room temperature, the resultant leek leaf extract was filtered. The filtrate was collected and stored at (4°C) in an airtight glass bottle for subsequent use. 2.2. Green Synthesis of (MnO2 ) NPs For the biogenic synthesis of manganese dioxide Nps the leaves of leek were cut and dried and heated for 60 minutes at 30°C with continuous stirring, then 10 gm of manganous Sulphate monohydrate ( MnSO4.H2O) (10 mM) was added to (100 ml) of leek leaf extract Figure 1. The resulting mix was treated with an ultrasonic bath for 10 minutes and then shaken at 30 °C for 24 hours for redox and capping reactions. The samples were centrifuged at 3500 rpm for 10 min. After centrifugation, the MnO2 NPs were washed three times with distilled water, dried in an oven, and then calcinated at (200°C) for six hours. Notice that the mixture color changed slowly from greenish to light yellowish, and after drying, the powder became dark brown. The green synthesized MnO2 NPs were stored after they were crushed. IHJPAS. 2024, 37( 3 ) 159 Figure 1. Diagrammatic illustration of the synthesis process used to create green MnO2 NPs using leek leave extract. 3. Result and dissuasion 3.1. Ultraviolet-visible (UV-Vis) analysis (UV-Vis) spectroscopy is the most practicable technique for characterizing NPs. The generation and stability of nanoparticles in an aqueous colloidal solution are supported by (UV-Vis) spectral spectroscopy [13]. The (UV-Vis) absorption spectra of the created MnO2 NPs as well as biofunctionalized nanoparticles were measured on a (UV-Vis) spectrophotometer. Figure 2 shows the MnO2NPs' UV-Vis spectrum. Generally, color changes seen during the reaction signify the production of NPs and stability [14]. The color shift from greenish to light yellowish brown is due to the surface plasmon resonance (SPR) band of the synthesized metal nanoparticles, which depend on the size, shape, and distribution, which indicated the stabilization of MnO2NPs were indicative of these processes. The unique properties of size, shape, and distribution give metal oxide nanoparticles excellent processability, making them potential candidates for antibacterial applications. [15]. Figure 2 shows that MnO2NPs had two distinct peaks at 257.5 and 290 nm, which appeared as humps. The samples exhibit two distinct absorption peaks at 287.5 and 291 nm, each of which is connected to the MnO2 NPs' band gap absorption [15]. The absorption spectra of the MnO2 NPs with varied impurities are displayed by the absorbance peaks at 287.5 and 291 nm. The MnO2 NPs displayed maximum absorbance at 287.5 nm. The energy band gap (E) of manganese dioxide nanoparticles was calculated using the formula: E= (hc/ λmax) where h = (6.626×10-34 J.s) is planks constant, c = (3×108 m/s) the speed of light, E is the band- gap energy, and λmax is the nanoparticle's wavelength. (UV–vis ) The energy band was measured to be (4.31) eV, and the MnO2 bulk had an energy band of (2.37) eV. The results show that the band gap energy increases with decreasing particle size. Because of the confinement of the IHJPAS. 2024, 37( 3 ) 160 electrons and holes, the band gap energy increases between the valence band and the conduction band while decreasing the particle size based on the quantum confinement theory[16]. . . sNP -Vis) specter image of Manganese Dioxide -(UV .Figure 2 3.2. XRD analysis X-Ray Diffraction Using powder (X-ray) diffraction spectroscopy (XRD) at a wavelength of (0.154 nm), the crystalline and phase purity of the green-produced MnO2 NPs were identified. The XRD spectra were captured in the range (10°–80°). An X-ray diffractometer using Cu Kα radiation was used [17, 18] . The MnO2 NPs' XRD pattern figure (3) exhibits a broad pattern that has been linked to the presence of bio-capped as well as amorphous materials; diffraction peaks were observed at 2θ. A known orthorhombic structure in MnO2 can be correlated with reflection values of 12.94, 18.34,28.78, 37.66, 42.14, 60.26 and73.72 degrees (JCPDS no. 44-0141). MnO2 NPs were successfully synthesized, as indicated by the strong XRD peaks [19]. The Debye-Scherrer equation[20], Crystalline size (D) = kλ / β cos θ (1) Where k: is the shape factor (0.94), λ: the incident radiation's X-ray wavelength (0.154252) nm,β: (FWHM) is the full-width half maximum, and θ: is the Bragg's angle, which takes into account the (X-ray) wavelength (0.154 nm), the width of a peak with maximum intensity in half height, the crystal's thickness (d), the diffraction angle, and the Debye-Scherrer constant (0.9)[21, 22]. Table 1 shows the crystal size values of Manganese Dioxide NPs with the highest intensity obtained by the green technique from leek aqueous extracts. The mean crystalline size of Manganese Dioxide NPs is 34.07 nm. IHJPAS. 2024, 37( 3 ) 161 Table 1. The crystalline sizes(D) of Manganese Dioxide Nanoparticles produced from leek leaves aqueous extract. Figure 3. XRD image of MnO2 – NPs. 3.3. FE-SEM Field Emission Scanning Electron Microscopy Analysis Field emission scanning electron microscopy (FESEM) investigates each particle, including the aggregation of particles, and can visualize crystal structure, surface morphology, distributed and aggregated nanoparticles, and surface fictionalization[23, 24]. Rocky spherical MnO2 NPs were successfully produced. The rocky spherical MnO2 NPs distributed and made with Leek aqueous extract are shown in the (FESEM micrographs in Figure 3. The MnO2 nanoparticles have a stony, compacted shape [23,25]. The photos display the presence of secondary material surrounding the MnO2, which pointed to the bioorganic components that produced and stabilized the rocky, spherical MnO2 NPs [26]. The average nanoparticle grain size was evaluated to be between 29 and 85 nm in size. The size, shape, and aggregation pattern of the nanoparticles depend on the quantity-based presence of phytochemicals in plants. This could be effective in reducing, capping, and stabilizing nanoparticles to narrow sizes with rocky spherical shapes, resulting in highly agglomerated nanoparticles. 2θ (Deg.) (FWHM) (Deg.) Crystalline size D(nm) Hkl 18.34 0.2683 30.07 (200) 28.78 0.2228 36.90 (310) 37.66 0.2976 28.26 (211) IHJPAS. 2024, 37( 3 ) 162 Figure 3. FE-SEM of MnO2-NPs. 3.4. Energy Dispersive X-ray (EDX) Analysis Energy Dispersive X-ray (EDX) analysis of the MnO2 Nps is an important tool used to identify the elemental composition of materials. Figure 4 displays the EDX patterns; the peak values for lines of O were 0.535 and 1.7 keV, and for lines of Mn elements, they were 0.54 and 5.94 keV. According to the strength of the lines, which is shown in Figure 4, the percentages of Mn and O were calculated. In the EDX, Mn (21.54%) and O (43%) are shown respectively. Figure 4. EDX for MnO2NPs by green method. 3.5. AFM Analysis This technique provides an idea of the crystalline nature of the layer, where phase morphology and height images can be recorded. The height image provides terrain information. In addition, the surface roughness can be measured by AFM. The method for preparing an AFM sample with the Manganese dioxide-coated surface facing upward on a thin glass plate was to evenly spread out the sample and allow it to air dry[27, 28]. An atomic force microscope was used to obtain AFM pictures. MnO2 NPs are visible as agglomerated particles in the AFM picture of MnO2 (Figure 5-A). Most MnO2 NPs recorded an average diameter of (50.20) nm. In AFM, the surface IHJPAS. 2024, 37( 3 ) 163 roughness can be measured in terms of the average diameter roughness; it was 32.94 nm, and AFM images confirm the measured RMS roughness was 40.6 nm. The morphological analysis also showed that the NPs have a spherical form. These AFM pictures demonstrate the significant nanoparticle adsorption on the MnO2 nanoparticle substrate surface. Analysis of the AFM pictures reveals a rocky-like structure with a limited size distribution (2D and 3D views). The graph of particle size distribution is shown in Figure (5-C) as well. (A) (B) (c ) Figure 5. Represents AFM scans, where (a) is a 3D image, (b) is a 2D image, and (c) represents the distribution of MnO2- NPs created using the Green technique with leek extract. IHJPAS. 2024, 37( 3 ) 164 3.6. Antibacterial Activity Antibacterial activity using the well agar approach was assessed, and the antibacterial potency of the produced MnO2 NPs was assessed against (Klebsiella, E.coli, and Staphylococcus aureus. Typically, the different bacterial strains were seeded into separate Mueller-Hinton agar plates and then cultivated for 24 hours at 37°C. Their cell density was kept at (1.5 X 108 CFU/ml). (1.5 X 108 CFU/ml) microorganism cells were swabbed onto a plate of Mueller-Hinton agar. The duplicate serial dilution was prepared from the stock solution, which was (1000,500, 250,125,62.5) μg/ml. By placing MnO2 nanoparticles in the appropriate wells, holes punched in the agar (7-mm-diameter holes) plates were examined to determine the zone of inhibition. They were incubated for 24 hours at 37 °C. If there is a wide, clear zone surrounding the producer strain colonies, the inhibition is considered positive. The wider inhibition zone may be seen around the wells, indicating that MnO2 Nps are responsible for the sensitive bacteria's inhibition[29, 30] .Based on how effectively different bacterial strains were killed, the antibacterial activity of MnO2 NPs was assessed. The CFU of E. coli, K. pneumoniae, and S. aureus were all reduced in the MnO2 NPs synthesized from leek extracts, according to the results. With a zone of inhibition of 33 mm for Staphylococcus aureus. The MnO2 NPs showed better performance of antibacterial activity for S. aureus compared with k. pneumoniae and E. coli bacterial strains, which had zones of inhibition of 30 and 25 mm for( klebsiella pneumonia) and (Escherichia coli), respectively as shown in Figure 6. It is concluded from the previous results that plant extracts can produce MnO2 NPs, with antibacterial activity. The MnO2 NPs showed better antibacterial activity and were more efficient against gram-positive bacteria than gram-negative bacteria[12]. The experimental results demonstrate that MnO2 NPs are effective antibacterial agents. The antibacterial activity of MnO2 NPs is related to their small size; they can easily enter bacterial cells and damage cell membranes. Finally, it results in bacterial cell death as a result of bacterial cell deformation processes. Here, we mostly display the outcomes of them in Table 2. (a ) IHJPAS. 2024, 37( 3 ) 165 a (b) (c) .NPs– 2MnO The antimicrobial activity of .Figure 6 Table 2. Antibacterial activity of Manganese Dioxide Nanoparticles Microorganisms Zone of inhibition (mm) (1000 ) ( 500 ) (250) (125) (62.5) μg/ml μg/ml μg/ml μg/ml μg/ml E. coli (−) 2 25 22 17 9 5 k. pneumoniae (−) 30 23 18 12 6 S. aureus (+) 33 26 19 12 6 Antibacterial activity at the same concentration was also detected Figure 7. Similarly, MnO2-Nps inhibits S. aureus the most. MnO2-Nps has shown good inhibition for S. aureus and lower efficient antibacterial activity against the bacterial k. pneumonia and E. coli due to its sensitivity to bacterial strains. IHJPAS. 2024, 37( 3 ) 166 Figure 7. The antibacterial activity assay with the inhibition zone and the concentration of MnO2-NPs against E. coli, k. pneumoniae , S. aureus. 4. Conclusion Manganese Dioxide nanoparticles were synthesized by the green synthesis method using manganous sulfate monohydrate (MnSO4.H2O). The obtained MnO2 was characterized by a (UV- vis) study that confirmed the visible color change from green to dark brown, which resulted in the synthesis of MnO2 NPs. The development of orthorhombic structures in the XRD investigation revealed the crystallinity of MnO2 NPs. The morphological analysis of the synthesized MnO2 NPs using FESEM indicated that the nanoparticles were spherical, with average grain sizes estimated to be between 30 and 85 nm in size. Thus, MnO2 nanoparticles are used in green synthesis. The development of nanotechnology has recently placed a strong emphasis on procedures that make use of mild reaction conditions and nontoxic precursors. The formulated MnO2-Nps inhibited the growth of S. aureus, K. pneumoniae, and E. coli, with a higher activity at concentrations (1000μg/ml). 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