The investigation aims at the synthesis of copper oxide and cobalt oxide nanoparticles using Spinacia oleracea leaf extract. These nanoparticles have many properties and applications. They possess antimicrobial catalytic property and also they can be used in energy storage materials, gas sensors etc. The Spinacia o leracea leaf extrac t behaves as a reducing agent in the nanoparticle synthesis . The p lant extract was first prepared and then treated with copper and cobalt salt solution s to get the precipitate. The salt solutions used for this purpose are copper sulfate pentahydrate (CuSO 4.5H 2O) and cobalt chloride hexahydrate (CoCl 2.6H 2O). The UV -Vis, XRD, EDX , and SEM techniques are used to find optical, structural , and morphological properties of c opper oxide and cobalt oxide nanoparticle s. The average crystallite size of copper oxide nanoparticles is 44 nm and ha s a hexapod structure. The UV absorption peaks are at 326 nm and 506 nm for copper oxide and cobalt oxide nanoparticles. Keywords: Nanoparticles; cobalt oxide; copper oxide; green synthesis. Introduction In nanoscience application s, nanoparticles with a size of less than 100 nm are being widely used [1]. Metal oxide and metal nanoparticles (NPs) are emerging candidate s in the field of nano technology and nanoscience. Depending on their shape and size, these nanopartic les with optical, electrical , and physical properties play a vital role. They have a large number of application s, such as antioxidants, catalysis, sensors, antibacterial, etc. Oxides of metal nanoparticles such as CoO, ZnO, TiO 2, SnO 2, and CuO, have been studied for their biomedical and environmental application s [2]. For the synthesis of nanoparticles, various chemical and physical methods have been used (Figure 1) which include ; high-temp solution phase, thermal decomposition, hydro -thermal microemulsion , and reduction , etc. [3]. However, green synthesis of nanoparticles is considered a significant branch of nan otechnology [4, 5] . This method is cost-effective and eco-friendly as compared to the conventional synthesis methods , where chemical additives, high -temperature pressure , and energy are used [6]. Therefore, it is necessary to develop and use safe methods that must be low cost, efficient, and non -hazardous , and environment friendly. Due to this reason, many researchers adopted the green synthesis met hod to fabricate nanoparticles [7]. Materials that are derived from plants are used to prepare nanoparticles and are also the best alternative to the chemical and physical method. Plant extract s contain bioactive compounds like flavonoids, saponin, phenolic acid , and tannins [8]. These bioactive compounds are good chelation agents and can donate hydrogen and quench singlet oxygen. Due to their redox activities , the green synthesis of NPs is more suitable than the physio -chemical te chniques. Plant extracts can be processed via easy protocols and are easy to handle and non-toxic [9, 10] . Copper oxide nanoparticle s exhibit high potentiality in the metal oxide NPs owing to their antimicrobial, catalytic, optical , and low -cost properties . Copper oxide NPs have a bandgap ranging from 1.35 -3.5 eV. Due to their suitable bandgap, easy availability, low toxicity , and surface synthesis among different semiconductor photocatalysts [11]. Copper oxide NPs gain attention due to their good oxygen adsorption capability and large surface area and are considered a promising candidate with good photocatalytic activity [12]. Copper oxide i s a widely studied semiconductor of the II-VI group and is a p -type 2 semiconductor. Because of its good optical properties which allow stable emission at room temperature, it is used in various applications such as photonic and fie ld emission devices and sensors [13]. Copper oxide NPs can be used as magnetic storage media, optical switch , and gas switch due to their photochemical and photoconductive properties [14]. Cobalt oxide NPs are used in a wide range of applications owing to their oxidation as well as high resistance to corrosion [15]. It is a p -type anti -ferromagnetic semiconductor. It is a multifunctional material with many applications such as energy storage materials, the anode in Li -ion batteries, dyes and pigments, sensors , and heterogeneous catalysis [16]. Apart from the green synthesis , different chemical and physical techniques were used to synthesis cobalt oxide nanoparticles such as solution combustion method, microwave process, thermal decomposition of sol -gel, hydrothermal reaction , etc. [17]. Many researchers have reported the green synthesis of copper oxide nanoparticles using different plant extract such as Lemongrass [18], Madhuca mongolia [19], Aspergillus fumigates [20], Nerium oleander [21], Bifurcaria bifurcate [22], Euphorbiaesula [23], and Albizia lebbeck [24]. Arunkumar et al. [11] showed the degradation of MB dye at 94% on irradiation by using the copper oxide nanoparticles synthesized via the Lantana camara leaf extract. Sathiyayimal et al [25] showed 93% degradation of MB dye in about 100 min by using the copper oxide nanoparticles synthesi zed via Sidaacuta leaf extract. Cobalt oxide nanoparticles using plant extract ha ve been investigated in various research articles such as Diallo et al. [26] discovered the bioreduction ability of the Aspalathus linearis leaf extract for cobalt oxide NPs synthesis. Bibi et al. [27] synthesized cobalt oxide NPs using Punica granatum peel extract from cobalt nitrate hexahydrate. Dubey et al. synthe sized cobalt oxide nanoparticles using Calotropis procera leaf extract [28]. In our work, we synthesized copper oxide and cobalt oxide nanoparticles using an environmenta lly friendly method. Spinacia o leracea leaf extract was used for the green synthesis of nanoparticles. Figure 1 Methods for the synthesis of nanoparticles Materials and Methods Preparation of Leaf E xtract For the preparation of leaf extract, fresh leaves of Spinacia o leracea (Spinach) were collected from the local field of Dhodial, Mansehra by wearing gloves. These leaves were then washed 3 times with tap 3 water to remove dust from them , washed again 3 times with distilled water , and placed on a clean aluminum foil to dry for about 3 hours. After that, the leaves were cut into small pieces with the help of scissors. Th en weighed 10 g of cut leaves with the help of electric weight balance and put it in 100 ml deionized water and placed a magnetic bar into it and sealed the opening of the beaker with aluminum foil to protect it from the d ust present in the surroundings, t hen placed the beaker on a magnetic stirrer for 50 minutes at 70oC. We saw that the color of the water changed to yellow as shown in Figure 2 (a). The beaker is removed from the magnetic stirrer and placed on a table for about 20 min and let it cooled. When the water got cooled, it is filtered with the help of Whatmann filter paper in a conical flask. The flask is then sealed with aluminum foil. The flow chart for the preparati on of leaf extract is shown in Figure 2 (d). Synthesis of Copper Oxide Nanoparticles Placed the copper sulf ate pentahydrate solution on the magnetic stirrer and put a magnetic bead in the beaker . Details of salt concentration are given in T able 1. After about 20 minutes of heating , 10 ml of Spinacia o leracea leaf extract was added dropwise in the solu tion using a burette . About 10 minutes after the addition of leaf extract , the color of the solution c hanged from light blue to green as shown in Figure 2 (b). The temperature at that time was noted to be 70oC. Then, removed the solution from the magnet ic stirrer and placed it in a safe place to let it cool , also covered the beaker with aluminum foil to protect it from dust particles. The n ext day the cooling solution was then put into three test tubes, these test tubes were then placed into the centrifuge for 20 minutes and set the centrifuge to 4500 rotations per minute. One test -tube is then us ed for UV and the other two were saved for further characterization s. Details of the synthesis are given in T able 2. Synt hesis of C obalt Oxide Nanoparticles Placed the cobalt chloride hexahydrate solution on the magnetic stirrer and put the magnetic bead in the beaker . Details of salt concentration are given in T able 1. After about 15 minute s of heating , 15 ml of Spinacia O leracea leaf extract was added dropwise in the solution using a burette . About 10 minutes after the addition of leaf extract the color of the solution changed from light pink to reddish as shown in Figure 2 (c). T he temperature at that time was noted to be 70oC. Then, removed the solution from the magnetic stirrer and placed it in a safe place to let it cool, also covered the beaker with aluminum foil to protect it from dust particles. The n ext day t he cool ing solution was then put into three test tubes, these test tubes were then placed into the centrifuge for 20 minutes and set the centrifuge to 4500 rotations per minute. One test -tube is then used for UV and the other two were saved for further characterization s. Schematic illustration for the synthesis nanoparticles is shown if Figure 3. Details of the synthesis are given in T able 2. Table 1 Details of salt concentrations Salts Distilled water (ml) Salt Concentration (M) Salt concentration (g) Copper Sulf ate Pentahydrate (CuSO 4.5H 2O) 100 0.12 3 Cobalt Chloride Hexahydrate (CoCl 2.6H 2O) 100 0.106 2.5 Table 2 Details about the synthesis of nanoparticles Salt Salt concentrations (ml) Leaf extract concentrations (ml) Time taken for heating (min) Temperature (˚C) Time taken for centrifuge (min) (CuSO 4.5H 2O) 0.12 10 30 70 20 (CoCl 2.6H 2O) 0.106 15 30 70 20 4 Figure 2 (a) leaf extract, (b) Copper oxide nanoparticles solution, (c) Cobalt oxide nanoparticles solution, (d) Flow chart for the preparation of leaf extract. Figure 3 Schematic illustration for the synthesis of nanoparticles RESULT s AND DISCUSSION The absor bance peak for biosynthesized c opper oxide nanoparticles from Spinacia oleracea extract was at 326 nm indicating the formation of c opper oxide nanoparticles from copper sulf ate p entahydrate. To analyze the optical properties of extracted copper oxide NPs, the optical absorption spectrum was used. From this spectrum , the type of electronic transition and the bandgap of the material were determine d [29]. When a material absorbs a photon of energy greater than the energy band gap, an electron is 5 transported to the conduction band (CB) from the valence band (VB) , an immediate rise in the absorbance of the semiconductor to the wavelength occurs corresponding to the energy of the bandgap . The relationship of the incident photon to the absorption coefficient depends on the electronic transition type. The transi tion is direct if the momentum is conserved and it is indirect if the momentum is not conserved . Figure 4 (a) shows the absorption spectrum of copper oxide nanoparticles. The UV -Vis spectrum showed the absorption peak at 326 nm and the cut -off wavelength at 400 nm. The reported material is a direct bandgap semiconductor material. According to the literature, the absorption peak of copper oxide nanoparticles ranges from 250 -300 nm, but in our work , the absorption peak is at 326 nm . The higher value of absorption peak is due to the formation of some other metastable and stable copper oxide NPs. The energy band gap wa s calculated from the equation 𝐸𝑔=ℎ𝑐/𝜆. Where λ is the cut -off wavelength, 𝑐=3×108 m/s is the speed of the light and ℎ=4.1356 6×1015 eV is the Plank's constant. The energy bandgap comes out to be 3.1 eV. The cut -off wavelength was obtained by extrapolating the line to the base, where α is zero. As compared to the bulk value (3.25 eV), t here is a slight blue shift in the bandgap (3.1 eV) [29] which is due to the q uantum confi nement effect [30]. The abso rbance peak for biosynthesized c obalt oxide NPs from Spinacia oleracea extract wa s at 509 nm indicating the formation of cobalt oxide nanoparticles from cobalt chloride hexahydrate. The UV -Vis spectrum of cobalt oxide NPs showed the absorption peak at 509 nm as shown in Figure 4 (b). This absorption band can be attributed to the plasma resonance absorption of the cobalt oxide nanoparticles . Cobalt oxide nanoparticles show a plasmon absorption band between 350-550 nm which is its distinguishing feature [31]. The formation of non -oxide cobalt NPs might be the reason for the strong surface plasmon. The leaf extract of Spinacia o leracea performs a s a reducing cum surface capping agent that can be attributed to the synthesis of cobalt oxide NPs. In the presence of Spinacia o leracea leaf extract, c obalt oxide nanoparticle s were formed from the reduction of cobalt chloride hexahydrate, which acts as a capping, stabilizing , and reducing agent [32]. The energy band gap was calculated from the equation 𝐸𝑔=ℎ𝑐/𝜆. Where λ is the cut -off wavelength, 𝑐=3×108 m/s is the speed of the light and ℎ=4.13566 ×1015 eV is the Plank's constant. The energy bandgap comes out to be 2.15 eV. The cut - off wavelength (575 nm) was obtained by extrapolating the line to the base, where α is zero. The XRD spec trum (Fig. 4 (c)) reveals the diffraction peaks at different angles. X RD diffraction peaks at 24.406° and 25.978° corresponds to the planes ( -202) and ( 111) of Monoclinic Cu 2O (SO 4) (Reference code: 00 -046-0005 ). The peak at 29.775° corresponds to the plane (110) of Cubic Cu 2O. The peak at 36.197° corresponds to the plane (004) of Tetragonal Cu 4O3. The peaks at 18.283° , 18.553° , 20.03° , 27.93° , 29.543° , 31.653° , 44.656° , 55.071° , 57.823° corresponds to the planes (200), (103), (022), (301), (204), (153), (008), (056), (525) of Orthorhombic Cu 64O. The peak at 26.4 ° correspon ds to the plane (011) of residual copper sulfate hydrate ( Reference code: 00-021-0269 ). In our work, in addition to the stable oxides, i.e., CuO and Cu 2O, some other metastable oxides of copper such as Cu4O3, Cu 8O, and Cu 64O have also been reported. These oxides are formed in the early stages of copper oxidation. These copper oxides are also found in pure copper powder which is commercially available . The sub-oxide Cu64O is formed when a very low amount of O atom is mixed with Cu lattice with a ratio of 64:1 of Cu to O. The crystallite size of the NPs w as calculated by the Debye -Scherer equation . D = (0.9λ ) / β cosθ Where λ is the X -rays wavelength (Cu kα = 1.54604 Å), β is the full width half maximum value in radians, θ is the diffraction angle. The average size of copper oxide nanoparticles calculated by the Scherer equation is 44 nm as shown in T able 3. 6 Table 3 Details of size estimation, Bragg’s angle , and miller indices obtained from XRD Degree ° (hkl) FWHM Size (nm) Average Size (nm) 18.283 200 0.098 82 44 18.553 103 0.098 82 20.03 022 0.118 68 24.406 -202 0.236 34 25.978 111 0.177 46 27.93 301 0.197 42 29.543 204 0.157 52 29.778 110 0.472 17 31.65 153 0.197 45 36.197 004 0.197 42 44.65 008 0.394 22 55.071 056 0.472 19 57.823 525 0.394 23 Figure 4 (a, b) UV -Vis spectroscopy of c oppe r oxide nanoparticles and c obalt oxide nanoparticles, (c) XRD graph of copper oxide nanoparticles Energy dispersive X -Ray ( EDX ) is used to study the elemental composition of the sample. Elements that are present in a sample can be identified by the released X -rays because every element has its own characteristic X -Ray. Using the EDX spe ctroscopy, Figure 5 (a, b) confirmed the presence of copper oxide and cobalt oxide nanoparticles. Generally , typical a bsorption peaks of copper oxide nanoparticle s are nearly at 0.5 keV and 8 keV as shown in figure 3 (a), while the typical absorption peaks of cobalt oxide nanoparticles are nearly at 0.8 keV, 6.9 keV, and 7.6 keV as shown in figure 3 (b) . Scanning electron microscopy ( SEM ) is a characterization tool that provides an advance d understanding of the morphology, shape , and size of the nanoparticles. The copper oxide nanoparticles may have different shapes such as cubic, octahedral , and hexapod. Figure 5(c) shows the SEM image of synthesized hexapod copper o xide (Cu 2O) nano particles . Figure 5 (d, e) shows the SEM images of cobalt oxide nanoparticles , it is clear from the picture that the particles are agglomerated and spherical . Compounds present in the extract and the nature of extract are the main factors of a gglomerations in the nano particles [33]. Large size nanoparticles form due to the attraction and r eactivity of the functional groups . The synthesized particles have different biological compounds as coatings, these compounds have surface hydroxyl groups. Among these agents, intermolecular hydrogen bonding is present due to which the NPs appeared agglomerated. 7 Figure 5 (a, b) EDX images of copper oxide nanoparticles and cobalt oxide nanoparticles. (c, d, e) SEM images of copper oxide nanoparticles and cobalt oxide nanoparticles CONCLUSION Copper oxide and cobalt oxide nanoparticles can be fabricated using a reduction technique that is cheaper, environmental ly friendly , and best compar ed to other conventional techniques. In summary, we have established a facile green method to synthesize low -cost copper oxide and cobalt oxide NPs by using Spinacia o leracea leaf extract as both the capping and reducing agent . The synthesized NPs were examined by various techniques such as UV -Vis, XRD, EDX , and SEM. These techniques confirmed the successful synthesis of copper oxide and cobalt oxide NPs. As we did not use any toxi c material in the whole process, so it can be used in biomedical applications.