UMYU Journal of Microbiology Research

E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668

ORIGINAL RESEARCH ARTICLE

Green Synthesis, Optimization, and Characterization of Copper Nanoparticles Using a Combined Extract of Selected Plant Seeds

Aliyu Shehu1*, Okunola Oluwole Joshua2 and Uduma A. Uduma3

Department of Chemistry, Federal University Dutsin-ma, Katsina State, Nigeria 123

Correspondence Author's address: aslyyaradua@gmail.com

Abstract

This work investigates a verde method involving synthesizing, optimizing, and characterizing copper nanoparticles (CuNPs) developed from black seed, neem seed, and baobab seed extracts. The optimal synthesis conditions were investigated by means of a Taguchi method. UV-Vis spectroscopy was used to confirm the formation of CuNPs, which exhibited an SPR peak of 590.10nm (Fig. 1), indicating the nanoparticles presence. Characteristic peaks at 566.65, 984.04,1088.47,1632.62,2117.12, and 3265.16 cm 1 were observed, attributing to Cu-O and CuOH and Cu-O-C bonds, as well as hydroxyl and carbon dioxide functional groups, indicating an effective capping and stabilization of the nanoparticles by plant phytochemicals (FTIR analysis). The crystalline nature of CuNPs has been confirmed using X-ray diffraction analysis, which gave diffraction peaks at 22.43, 47.07, 67.12, and 87.33. The particle size was found to be 25.51nm. The particles exhibited a mostly spherical shape with some degeneracy, when analyzed by scanning electron microscopy (SEM), and the elemental composition (EDX) characterized a copper content of 45.3% and additional plant-derived stabilizing agents, such as carbon, oxygen, and trace elements. Besides, the antimicrobial assay showed potent inhibitory effects on multiple microbes that are all pathogenic. The CuNPs showed significant antibacterial activity, exhibiting maximum inhibition zones against Acinetobacter baumannii (17 mm) and Escherichia coli (19mm). These results emphasize the potential of biogenically synthesized CuNPs as potential antimicrobial agents, which should provide a sustainable alternative for biomedical and pharmaceutical applications.

Keywords; Optimization, Characterization, Biogenic, Antibacterial, Acinetobacter baumannii s, Escherichia Coli, Nanoparticles.

INTRODUCTION

Nanotechnology has made great strides, resulting in the development of high-tech materials that exhibit special physicochemical properties. Metal nanoparticle biosynthesis has gained interest in recent years because it is relatively eco-friendly and cost-effective compared to traditional chemical methods (Bajpai et al., 2018). Plant extracts, for their content of bio-active compounds, including polyphenols, flavonoids, terpenoids, and alkaloids, are widely used as reducing and stabilizing agents in -nanoparticle synthesis (Kazemi et al., 2023). Apart from reducing the metal ions, these biomolecules stabilize and caps the nanoparticles, which ensures their stability. When reduced to nanoscale, metals can exhibit properties far superior to their bulk counterparts and, therefore, have numerous applications in areas ranging from antimicrobial to catalysis to optics. Traditional methods of synthesizing nanoparticles such as chemical reduction and physical vapor deposition often involve toxic reagents and consume lots of energy. These constraints have

prompted interest in more environmentally friendly alternatives in which plant extracts can be natural reducing agents (Kazemi et al., 2023). Several studies have proved the synthetical ability of the CuNPs through plants in which plant-derived biomolecules were found involved in the process of reduction and stabilization. For instance, Bajpai et al. (2018) got CuNPs with plant extracts and emphasized that plant metabolites could favor the formation of nanoparticles. Analytical characterization of metal nanoparticles due to their unique physicochemical properties is critical.

The UV-Vis spectroscopy generally confirms nanoparticle synthesis via surface plasmon resonance (SPR) peak. In FTIR spectroscopy, information about the functional groups responsible for nanoparticle stabilization can be obtained by detecting the interactions between biomolecules and metallic ions. Crystalline structure and phase composition were characterized by X-ray diffraction (XRD), and scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) were used to analyze nanoparticle morphology and elemental composition.SEM captures high-resolution images that show nanoparticle size, shape, and surface texture, while EDX identifies elemental constituents. A study by Kazemi et al. (2023) confirmed SEM and EDX to show specific morphological characteristics with different elemental compositions which can be attributed to metal nanoparticles. Metal nanoparticles have many applications, but their antimicrobial properties are most promising in the fight against antibiotic resistance. Copper nanoparticles have garnered significant research interest due to their potent antimicrobial activity, which has been attributed to three properties: their potential to disrupt microbial membranes, denature proteins, and induce oxidative stress, ultimately resulting in cell death. They are efficacious against microbes of various genera, with studies documenting considerable antibacterial activity against Gram-positive and Gram-negative bacteria. Research by Melkamu and Feleke (2022) showed that CuNPs synthesized by Justicia schimperiana leaf extract displayed potent antibacterial activity against Pseudomonas aeruginosa and Bacillus subtilis.

To evaluate their antimicrobial efficacy, the current study was designed to biosynthesize the CuNPs using extracts of three medicinal plant seeds: black seed, neem seed, and baobab seed. Nanoparticles from biogenic (living organisms) sources have been shown to associate with bacterial membranes, resulting in subsequent structural disruption leading to bacterial cell death. The study aims to optimize, synthesize, characterize, and investigate the antimicrobial activity of CuNPs as alternative antimicrobial agents against Gram-positive and Gram-negative bacteria and fungal pathogens.

MATERIALS AND METHODS

Materials

Neem seed, Black seed, Baobab seed, Sodium hydroxide (99%), Copper sulphate (99%), Ethanol (99%), Mueller hinton agar, glass wares, teplon containers, filter papers, crucibles, Rotary evaporator (Rotavapor R-210), Incubator (Thermo scientific incubator), pH meter (MettlerTolado pH meter), UV-Vis spectrometer (Shimadzu UV-1800), FTIR spectrometer (Shimadzu XRD-600), SEM/EDX(Hitachi SU-70 SEM/EDX).

Methods Sample Collection and Identification

Fresh seeds of the neem (Azadirachta indica), and baobab (Adansonia digitata) were obtained from Ajiwa village in Batagarawa local government Katsina farmland. Black seed (Nigella sativa) Seeds

were obtained from Sabon Gari market, Kano state. The samples were identified at the Biology Department of Umaru Musa Yaradua University Katsina.

Sample Preparation

The seeds were cleaned to eliminate dirt, debris, and other foreign materials. They were then washed extensively with distilled water and air-dried in a shaded and ventilated environment at ambient temperature (30°C) for 10 days. The seeds were dried and then re-milled on a mechanical mill, followed by a sieving process to create a uniform particle size. The powdered samples were added to airtight containers (Shehu et al., 2025). A modified method by Adam et al. (2019) for preparing crude extracts was followed. About 50g of each powdered plant seed sample was weighed and mixed with 500 mL of Ethanol in conical flasks. The flasks were capped with aluminum foil and macerated at room temperature for 48 hours. At the end of the maceration period, the extracts were filtered through Whatman No. 1 filter paper to separate the liquid phase. Filtrates were then concentrated by a rotary evaporator and stored until analysis.

Optimization of the Biosynthesis of Copper Nanoparticles (Using Taguchi Method)

An enhanced protocol for copper nanoparticle synthesis was developed based on the method described by Sampaio and Viana, (2021). The best synthesis conditions were established using the Taguchi method, which involved choosing a suitable orthogonal array method to test the main factors, including the temperature, pH, plant extract concentration, stirring speed, and reaction time. Nanoparticles over synthesis were studied considering four distinct levels well known to researchers for each factor. UV-Vis measured absorbance for each standard, and the UV-Vis measurement was chosen as the optimal synthesis condition. These values were then used to biosynthesize the nanoparticles.

Biosynthesis of Copper Nanoparticles Using Optimal Condition

The procedure for synthesizing copper nanoparticles from the combined crude extract of neem seed, black seed, and baobab seed was adopted with slight modification from the method by Patel et al. (2016). About 0.2497g of CuSO₄1mM CuSO4 was made by dissolving 0.4g of CuSO4·5H2O in 1000mL distilled water. About 25mL of the combined plants' crude extract and 90mL of prepared 1mM CuS\(O_{4}\)were added into a conical flask. Then, adjust the pH of the solution with NaOHI solution to pH 11, and then place the mixed solution on the magnetic stirrer and stir for 30 minutes at a constant temperature of 80°C until color change (from yellow-green to redldish brown) occurs which is the sign of the formation of copper nanoparticles, the solution is scanned using a UV-Vis spectrometer at a wavelength range of 300 to 700nm, and the appearance of the absorption peak at a wavelength of 550-600nm is indicating the formation of the nanoparticles. The particles were separated by centrifuging the solution at 4000 rpm for 20 minutes. The precipitated nanoparticles were washed with distilled water to remove unreacted components and then dried at room temperature using an incubator to obtain nanoparticle powder. The resulting dried CuNPs were preserved in a sealed plastic container for further analysis.

Characterization of Biosynthesized Nanoparticles UV-Vis

Analysis

The synthesized nanoparticles were then diluted with distilled water (1:10) and placed into a clean quartz cuvette for spectral analysis. The absorption spectra were plotted in the wavelength range from 200 to 700 nm. Nanoparticle formation was monitored by examining the intensity and location of the absorption peak and estimating the particle size. The distinct peak at the approximate wavelength confirmed the successful synthesis of the nanoparticles (Ullah and Rasheed, 2020).

FTIR Analysis

The biosynthesized dried nanoparticle powder was mixed in KBr and scanned at a 4000-400 cm-1 spectral range in Fourier-transform infrared (FTIR) spectroscopy. Characteristic peaks associated with functional groups such as O-H, C=O, and N-H were further analyzed from the obtained spectra. These peaks revealed the biomolecules responsible for capping and stabilizing the nanoparticles, such as proteins and secondary metabolites from the plant extract (Ullah and Rasheed, 2020).

SEM/EDX Analysis

The nanoparticle solution was prepared on a clean SEM stub by drop-casting to form a thin film and dried at ambient conditions. A thin gold coat was deposited on the surface to avoid surface charging during imaging. After that, the nanoparticle sample was mounted in a SEM chamber with the yawning parameters adjusted at the refined fields of 5-20 kV. Images were taken to analyze the morphology (size, shape, and distribution) of interest in the region-color focused to varying magnification. This was followed by an energy-dispersive X-ray (EDX) spectroscopy for the analysis of the elemental composition of the nanoparticles. The characteristic peaks of the associated elements in EDX spectra indicated the metallic nature of the prepared nanoparticles, as reported elsewhere (Ginting and Karnila, 2020).

XRD Analysis

The powdered nanoparticles were ground well for uniformity in analysis, and the sample was evenly spread on the glass slide. The sample was mounted on an XRD sample holder, and the scanning angle (20) was adjusted to range from 10 to 80, a common range used for metal nanoparticles. The pattern was recorded at a scanning rate of 0.02/s and the diffraction peaks were used to analyze the crystalline structure of the nanoparticles, comparing them with standard reference patterns of the JCPDS. The peak positions confirmed the various nanoparticle phases (e.g., Ag, Cu, or Zn), and the broadness of the peak was used to calculate crystallite size via the Scherrer equation (Ginting and Karnila, 2020).

Antimicrobial Analysis

The antimicrobial activity of the biosynthesized nanoparticles was assessed by performing the disc diffusion method in accordance with the National Committee for Clinical Laboratory Standards guidelines (NCCLS). The stock solutions of the nanoparticles were prepared in the concentrations of 500_ppm,250 ppm,125 ppm, 62.5 ppm, and 32.5 ppm by serial dilution. The antimicrobial activity was evaluated using Mueller-Hinton Agar plates. Prior to the inoculation, 6 mm wells were punctured aseptically into the agar medium with a sterile borer. The test bacterial strains were standardized to a 0.5 McFarland standard solution for matching concentrations of bacteria. Bacterial cultures were spread evenly onto the surface of each agar plate with a sterile swab and allowed to dry for 3-5 minutes to remove excess moisture. The prepared nanoparticle solutions were added at various concentrations to each well and incubated at 37°C for 24 h. The antimicrobial efficacy of nanoparticles was evaluated by measuring the diameter of inhibition zones, expressed in millimeters, using a metric ruler after incubation (Hona et al., 2019).

MIC Test

The dilution method was used to determine the minimum inhibitory concentration (MIC). Each nanoparticle fraction that showed antimicrobial activity was emulsified at the lowest concentration (which displayed antimicrobial properties) in serial dilutions in test tubes containing Mueller-Hinton Broth. The tubes were then inoculated with equal volumes of bacterial or fungal inoculum and the nanoparticle fractions. A single colony of each isolate was inoculated into 5 mL of sterile Cadebro broths (Oxoid), incubated at 37°C for 24 h for bacteria and at 25°C for 48 h for fungi. For accuracy, three control tubes were kept for each strain: a media a, organism, and nanoparticle control. The MIC was determined as the minimum concentration (largest dilution), showing no visible microbial growth (no turbidity) compared to control tubes (Habibah et al., 2024).

MBC Test

For the determination of minimum bactericidal concentrations (MBC), an aliquot from those MIC test tubes showing no obvious growth was subcultured on freshly prepared nutrient agar plates. The plates were then incubated again at 37°C for 24 hours. The maximum dilution at which no bacterial colonies were seen on the agar surface was defined as the maximum bacteria count (MBC). The same was done for fungi, but the plates were incubated at 25°C for 42 h. MFC was defined as the highest dilution at which no fungal colonies were observed (Habibah et al., 2024).

RESULT AND DISCUSSION

Results Optimization of the Biosynthesis of Copper Nanoparticles Results

Table 1: Optimization of Copper Nano Particle Synthesis

3.1.2 Characterization of Biosynthesized Nanoparticles

3.1.2.1 CuNPs Uv-Vis Result

Figure 1. UV-Vis Spectra of CuNPs

3.1.2.2 CuNPs FTIR Result

Figure 2. CuNPs FTIR Spectra

Table 2.CuNPs FTIR

3.1.2.3 CuNPs SEM/EDX Result

Figure 3. CuNPs SEM Image

Figure 4. CuNPs EDX

3.1.2.4 CuNPs XRD Result

Figure 5.CuNPs XRD Spectra.

Table 3.Determination of the average size of the CuNPs

NOTE: The average particle size=25.51nm

3.1.2.5 Antimicrobial Analysis Result

Table 4. Antibacterial Activity of CuNPs (Gram Negative and Gram Positive Bacteria species)

A.baumannii

KEY;\(NZ = N_{O}\)Zones,E.coli= Escherichia Coli, K.Pneumoniae = Klebsiella Pneumoniae,A.baumannii=Acinetobacter baumannii,S.mutants=Streptococcus mutans, B.subtilis =Bacillus subtilis,S.Lentus=Staphylococcus Lentus.

Table 5. Antifungal Activity of CuNPs (Fungi species)

KEY; \(NZ = N_{O}\)Zones,C.albicans=Candida albicans, A.niga= Klebsiella Pneumoniae,F.Oxyporum =Fusarium oxyporum

Table 6. MIC Antibacterial Activity of CuNPs (Gram Negative and Gram Positive Bacteria species)

KEY;\(NZ = N_{O}\)Zones,E.coli=Escherichia Coli,K.Pneumoniae = Klebsiella Pneumoniae,A.baumannii = Acinetobacter baumannii,S.mutants=Streptococcus mutans,B.subtilis=Bacillus subtilis,S.Lentus=Staphylococcus Lentus.

Table 7.MIC Antifungal Activity of CuNPs (Fungi species)

KEY;\(NZ = N_{O}\) Zones, C.albicans= Candida albicans, A.niga= Klebsiella Pneumoniae,F.Oxyporum =Fusarium oxyporum

Table 8. MBC Antimicrobial Activity of CuNPs (Gram Negative and Gram Positive Bacteria species)

KEY; \(NZ = N_{O}\)Zones,E.coli=Escherichia Coli,K.Pneumoniae = Klebsiella Pneumoniae,A.baumannii =Acinetobacter baumannii,S.mutants=Streptococcus mutans, B.subtilis =Bacillus subtilis,S.Lentus=Staphylococcus Lentus.

T able 9. MBC Antifungal Activity of CuNPs (Fungi species)

Discussion

Table 1: Taguchi method optimization of copper nanoparticle (CuNP) synthesis. (0.60) was the highest absorbance value in Runs 3, 5,11, and 15, each associated with different factor levels. The absorbance indicates the nanoparticle particles' formation; higher absorbance values imply increased formation of nanoparticles and smaller possible particle sizes due to the SPR effect. Likewise, several influential factors (A, B, C, D, E) significantly affected the absorbance values. The aforementioned was seen for Factor A (Temperature); in Runs 3 and 15, Level 4 was indeed

best. Higher T is another reason to accelerate the reduction of copper ions and reaction rates,leading to favorable synthesis of Cu nanoparticles. But very high temperatures might destabilize nanoparticles, which you must keep in mind for scale-up. Finally, Factor B (pH) was determined to be most significant, evidenced by Level 3 demonstrating the best absorbance values, further illustrating that a moderate pH environment is vital for the stability of the nanoparticles, suggesting that extreme departure in pH may inhibit the reduction process or aggregate particles. Another key factor was Factor C (Reaction Time), where Level 2 was most often associated with higher absorbance reading values in Runs 3, 5, and 15. Thus, the reaction time must not be so short that copper ions are not fully reduced nor so long that oxidation or aggregation occurs. Likewise, Factor D (Precursor Concentration), was also best for the runs with high absorbance values Level 3. This concentration is likely enough for sufficient planarization reaction but not enough to saturate the system, leading to uncontrolled nucleation. Another important role was played by Factor E (Reducing Agent concentration), where Level 4 enabled the effective reduction of copper ions and stable formation of nanoparticles. The role of these factors was evident from the results. When both were higher in Runs 5 and 15, the interaction between precursor concentration (D) and reducing agent concentration (E) was particularly important. Similarly, the joint levels of reaction time and pH at Levels 2 and 3 (C and B, respectively) were indicative of an interacting effect resulting in their maximum utility toward both the reduction process and stabilization of the nanoparticle. These findings offer a solid foundation for an engineer of CuNPs with high absorbance and desired properties. Smaller, well-dispersive particles will give high absorbance values which is preferred for utilization such as antimicrobial activity, catalysis, and electronic devices. The optimal conditions identified in this study are useful in obtaining reproducible results in larger-scale synthesis processes. However, small deviations from these optimal values may be needed to optimize nanoparticle size and shape for particular applications. Also, lower absorbance values in some runs (e.g., Runs 9, 12, and 14) indicate that the synthesis process is sensitive to changes in factor levels. Indeed, it underscores the need to fine-tune reaction conditions more closely in order to achieve reproducible nanoparticle synthesis.

The synthesis of CuNPs was confirmed by UV-Vis spectroscopic analysis, where a prominent absorption peak was observed at 590.10 nm (Figure 1). This result is consistent with the expected range for copper nanoparticles, indicating the successful synthesis of the desired particles. The observed UV-Vis absorption arises from the surface plasmon resonance (SPR) effect, which is one of the typical properties of interacting metallic nanoparticles, including copper with light (Ponmurugan et al., 2016). The surface plasmon resonance (SPR) is attributed to the coherent oscillation of conduction electrons at the surface of the nanostructures in resonance with the incident light. For example, soluble copper nanoparticles display SPR absorption bands typically in the 500-600 nm range, depending on particle size, morphology, and the environment surrounding the particles (Dalal et al., 2019). The peak absorption at 590.10 nm confirms the formation of the formed metallic copper nanoparticles, unlike other metal nanoparticles, such as silver or gold, which exhibit distinct peaks at various wavelengths from the SPR signal, corroborating their identity as CuNPs (Ponmurugan et al., 2016). Copper nanoparticles are reported to have broader and less intense surface plasmon resonance (SPR) peaks compared to silver and gold nanoparticles, owing to the unique electronic property of copper (Dalal et al., 2019). The SPR peak observed at 590.10 nm matches the results of previously reported studies with CuNPs synthesized using chemical and green synthesis (Amaliyah et al., 2020). The SPR

peak position is dependent on nanoparticle size and shape, with cobalt and gold nanoparticles demonstrating a blue (smaller nanoparticle) and red (larger nanoparticle) shift, respectively (Dalal et al., 2019). This peak at 590.10 nm is due to the successful synthesis of CuNPs of a defined size range and demonstrates the efficacy of the synthesis process. Green synthesis of copper nanoparticles (CuNP) using plant extracts proves to be an eco-friendly, low-cost way of synthesizing CuNPs. The plant extracts contain reducing agents (e.g., polyphenols and secondary metabolites) that assist in the reduction of copper ionsto metallic copper\((Cu^{0})\), and at the same time, they help stabilize the nanoparticles, avoid aggregation, and maintain colloidal stability (Amaliyah et al., 2020; Ponmurugan et al., 2016). Such absorption peak at 590.10 nm, obtained through UV-Vis, indicates the successful formation of stable CuNPs with defined optical characteristics, which is important for targeting CuNPs in diverse applications such as catalysis, sensors, and environmental remediation (Amaliyah et al., 2020). The broadening of the SPR peak shows that there is a polydisperse nanoparticle population. Metallic nanoparticles have free electrons that vibrate together, forming an SPR absorption band (Banger et al., 2025). The intensity of the SPR peak gradually decays with the increase in particle size due to the fact that large particles optically become opaque (Ying et al., 2022). Additionally, UV-Vis spectrophotometry specifically presents high sensitivity for the detection of bigger particles or aggregates of smaller nanoparticles (Rambau et al., 2024). 572 and 590 nm while also aligning with quinquevalent oxidation state with absorption peaks, the observed color variation along with the resulting SPR suggests that copper nanoparticles were indeed successfully synthesized. These results are in agreement with the literature and demonstrate the potential use of green chemistry approaches for the preparation of nanoparticles with favorable properties for different applications.

The FTIR study was also performed on copper nanoparticles, which gave strong bands around 566.65894, 984.04455, 1088.472, 1632.6243, 2117.1234, and 3265.1684 cm-1 (Table 2, Figure 2). The band at 566.65 cm-1 corresponds to the stretching vibrations of Cu-O, which suggests the formation of a bond between copper and oxygen atoms. The peak at 984.04 cm-1 is attributed to Cu-OH stretching vibration caused by a bond of Cu and OH. This bond is also a covalent bond, but the way the hydroxyl group (OH) attaches on the surface of Cu NP shows a higher degree of hydroxylation or oxidation in Cu NP than the Cu-O bond reported at 566.65 cm-1. The peak registered at 1088.47 cm-1 is attributed to Cu-O-C stretching vibrations and is associated with the creation of bonds between copper (Cu), oxygen (O), and carbon (C) atoms. This maximum is commonly observed in copper nanoparticles functionalized or covered by the-organic molecules such as surfactants, polymers, or other ligands. It means that a copper-organic compound/coordination compound formed on the nanoparticle surface. The Cu-O-C bond observed at 1088.47 cm-1 is believed to be a polar covalent bond analogous to Cu-O and Cu-OH bonds at 566.65 cm-1 and 984.04 cm-1. A higher extent of organic functionalization on the nanoparticle's surface is evidenced by the presence of carbon atom (C) in the material. The band around 1632.62 cm-1 is generally assigned to bending vibrations of interfacial water molecules (H2O) bound in aspects of the nanoparticle. This peak, which is sometimes referred to as the “water bending mode” or “HOH bending mode” (associated with both the stretching and bending of water molecules), is indicative of water molecules being present on the copper nanoparticles, a phenomenon found in many nanoparticle systems exposed to air or moisture. The peak observed at 2117.21 cm-1 in the FTIR spectrum is usually associated with the stretching vibration of carbon dioxide (CO2) molecules bound to the surface of nanoparticles, known as the “CO2 stretching mode". Last, the peak at 3265.16 cm-1 is generally attributed to O-H stretching vibrations, which show that the hydroxyl (OH) groups exist on the surface of the nanoparticles. The FTIR study illustrates that CuNPs were surrounded by copper oxides, copper hydroxides, organic specie, carbon dioxide, water, and hydroxyls. serve serve as firm binding sites for CuNPs (Rajesh et al.,2018; Dlamini et al., 2019; Punniyakotti et al., 2020; Tsilo et al., 2021; Shukri et al., 2022).

Particle morphology was analyzed by Scanning Electron Microscopy (SEM), and from Figure 3, the SEM graph demonstrates that the synthesized copper nanoparticles (CuNPs) were spherical in nature and was the nanoparticles had assembled into a spongy-type cluster structure. The spherical shape is a common characteristic observed in green-synthesized CuNPs, as it is attributed to the uniform nucleation and growth of CuNPs favored by molecules present in the plant extracts during the process of synthesis. These biomolecules (phenolic compounds, flavonoids, and proteins) serve as reducing and stabilizing agents that limit the shape and size of the nanoparticles synthesized (Amaliyah et al., 2020; Batool et al., 2024). The CuNPs have a high surface energy and can lead to agglomeration and formation of an interconnected network which has been observed in SEM images as spongy-like clusters. Such partial clustering is frequent in biosynthesized nanoparticles and could be associated with the interaction of the capping agents layer covering the nanoparticles and the solvent evaporation process occurring in the SEM sample preparation even though it is cluster-type Vs effectively spherical natural features, which indicates the successful stabilization of plant-derived biomolecules on the surface of the nanoparticles (Amaliyah et al., 2020; Anjum et al., 2016). This sponge-like structure can profoundly impact the functional properties of CuNPs.For instance, these clusters can improve catalytic activity by providing more active sites in a porous network.

Furthermore, such a structural setup may have made CuNPs more potent based on the fact that the bigger surface area of the surface of the nanoparticles could have the tendency to contact the microbial cells more effectively like the other previous studies on similar nanoparticle systems (Anjum et al., 2016; Batool et al., 2024). The copper nanoparticles (CuNPs) have a spherical morphology, suggesting general isotropic growth with the synthesis process, which is typically modulated by capping agents that bind uniformly across the surface of the nanoparticle to restrict anisotropic growth. In one local study, all the dimensions were presented in a standard way, and there were uniform results produced by balanced reduction and stabilization mechanisms of the respective phytochemicals used for a plant-based synthesis method, which led to a spherical-shaped nanoparticles. In particular, it has been reported that biosynthesized CuNPs aggregated into spongy-like clusters, forming porous structures (Kimber et al., 2018; Batool et al., 2024), which improved their stability and other functional properties. Results from the SEM confirmed the successful formation of the CuNPs using the green synthesis strategy with a suggested structure type. Both spherical morphology and spongylike clustering during the growth of CuNPs indicate the contribution of plant-based biomolecules in nanoparticle formation and will help reveal possible applications of the prepared CuNPs in catalysis, sensing, and antimicrobial applications.

The elemental composition of the biosynthesized copper nanoparticles (CuNPs) was estimated via Energy-Dispersive X-ray (EDX) spectra in which the peaks at 0.97 eV (carbon, 16.20%), 0.54 eV (oxygen,6.73%), 1.3 eV (silicon, 11.10%), and 7.38 eV (copper, 65.97%) were obtained (Fig. 4). The presence of higher intensity peaks of copper in the EDX spectrum confirmed the successful production of CuNPs with larger copper content. Carbon and oxygen in biosynthesized nanoparticles are typical and could be conferred to the organic biomolecules originating from the plant extracts in the synthesis. Proteins, polysaccharides, and some other phytochemicals found in plants serve as reducing and stabilizing agents, capping the nanoparticles and preventing agglomeration. Due to the capping layer's carbon and oxygen-rich nature is detected in the EDX analysis (Tsilo et al., 2021; Batool et al., 2024). The presence of silicon in the EDX spectrum can originate from several sources. The biosynthesis of leads could be due to the natural composition of phytochemicals based on the plant extract utilized in the process itself. Some plants naturally absorb silicon from their surrounding soil during their life cycle which will also become a part of their phytochemical composition. Alternatively, silicon contamination may arise from sample preparation or from glassware and equipment used for synthesis and analysis. Precise cleaning procedures and silicon-free materials can help reduce stress contamination (Amaliyah et al., 2020; Batool et al., 2024). EDX analysis demonstrated the elemental composition at the surface, giving information about the surface chemistry of the CuNPs. The presence of organic functional groups on the surface of synthesized nanoparticles can affect their stability, dispersibility, and interaction with biological systems, as revealed by the carbon and oxygen content. The biomolecules that coat the nanoparticles not only inhibit aggregation (which may facilitate CuNPs applications in biomedicine and environmental remediation) but also improve the biocompatibility of biosynthesized CuNPs (Amaliyah et al., 2020; Tsilo et al., 2021). These observations and EDX results show that the CuNPs biosynthesis product has clear evidence of copper. High C and O signal suggests efficient capping from plant source biomolecules, while high Si signal suggests a possible contamination source that needs to be addressed in future syntheses. These results concerning CuNPs biosynthesis are consistent with recent research on the role of plant extracts in stabilizing and functionalizing the nanoparticles (Tsilo et al., 2021; Batool et al., 2024).

14

The XRD pattern had a total of six diffraction peaks at 22.43, 27.96, 47.02, 67.12, 78.01, and 87.33, as shown in Table 3 and Figure 5. Fig. 5 shows the XRD spectrum of copper nanoparticles; it revealed four peaks (22.43, 47.07, 67.12, and 87.33) reveal four Bragg's reflections that correspond to (111), (200), (220), and (311)planes of the face-centered cubic lattice structure of metallic copper. These peaks are well-matched to the Joint Committee on Powder Diffraction Standards (JCPDS, no. 801268). The peaks of the green-synthesized CuNPs showed show that the synthesized nanoparticles were pure. Such patterns have been reported in CuNPs that are effectively synthesized using other plant extracts (Nasrollahzadeh et al., 2017; Melkamu and Feleke, 2022). However, the other desired peaks in the XRD patterns (68.90 and 86.37); also press the point that the synthesized nanomaterials are not 100% copper and mixed with any other substances (phytochemical/capping agents). In fact, the presence of organic compounds in the sample is confirmed by the XRD profiles since the Braggs reflections at 115 and 315 are usually attributed to crystalline and amorphous organic phases Kazemi et al., 2023. These findings are consistent with the FTIR, EDX, and UV-Vispectroscopy data. The average size of the synthesized nanoparticles was determined from Scherrer's equation, which was calculated to be 25.51 nm. That is consistent with earlier works. The report of Melkamu and Feleke (2022) describes an average size of copper nanoparticles' synthesized using Leaf Extract of Justicia Schimperiana 21.8 nm. Tavallali et al. (2024) fabricated copper nanoparticles (22-24 nm range) with the help of aloe vera extract, while Dikshit et al. (2021) reported a size ranging from 23-26nm for copper nanoparticles synthesized from Bacillus subtilis.

The synthesized CuNPs were screened for their antimicrobial activities using the disc diffusion method against three Gram-negative, three Gram-positive bacterial pathogens and three fungal species (Tables 4 and 5). The synthesized CuNPs were used in concentrations of 50032.25 ppm. The antibacterial activity was concentration-dependent and dependent on bacterial species. Remarkably, Gram-positive bacteria were less susceptible than Gram-negative strains. With a concentration of 500 ppm, the Gram-negative bacterium with the highest inhibition was E.coli (19mm), followed by A. baumannii (17 mm) and K. neumaniae (16 mm). For a 500 ppm concentration, the inhibition activity of S.lentus and S.mutans was 14 mm, and B.subtilis was 13mm (low). For fungal species, C. albicans demonstrated the highest inhibitory zone diameter (12mm), while A. niger and F. oxysporum were poorly inhibited (9 mm each) at the same concentration. According to Mahmoodi et al. (2018), the diameter of the antibacterial ring 10mm can be considered strong resistance. The synthesized NPs showed powerful antibacterial and antifungal activity against the tested strains. The synthesized CuNPs minimum inhibitory concentration against the selected bacterial and fungal strains (Table 6 and Table 7) showed that all Gram-positive bacteria (S. lentus, B. subtilis, and S. mutans) and Gram-negative bacteria (S.aureus, E. coli, and K. neumaniae) could be inhibited by 50ppm concentration. The minimal inhibitory concentration (MIC) values for the fungal species were 100ppm for C. albicans and 200ppm for A. niger and F. oxysporum. For both bacterial and fungal species, the MBC was 100ml/L(for all bacterial species) and 200ppm (for all fungal species, respectively, shown in Table 8 and Table 9), which completely abrogated the viability of the bacterial and fungal population. Our study corroborates with earlier reports on the antimicrobial action of plant-synthesized metal nanoparticles against humans as well as phytopathogens (Suárez-Cerda et al., 2016; Balouiri et al., 2016; Mahmoodi et al., 2018; Rajesh et al., 2018). Due to the very good adaptation of bacteria and the illegal use of herbal medications, there is an increase in drug-resistant organisms (Atri et al.,2023). Novel and more powerful microbial drugs are needed to combat this, and thus, a significant challenge in global public health (Alavi and Karimi, 2018). These contribute to a new step in this direction, especially plant-mediated NPs compared with chemical NPs in so many biological aspects, which act as aerial development tools for drug discovery. These properties are essential for drug delivery, bio-labeling, sensing, food preservation, wound healing, water purification, and cosmetics (Alavi and Karimi, 2018). Smaller-sized NPs have larger surface areas, resulting in increased interaction with bacteria and greater antibacterial activity than larger NPs (Rajesh et al., 2018). The main antibacterial effect of CuNPs occurs from the release of Cu++ ions that bind the bacterial cell wall by electrostatic attraction. Rajesh et al. (2018) synthesized CuNPs using citron juice (Citrus medica Linn) and investigated their in-vitro antimicrobial activity against selected bacterial species such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Propioni acnes, and Salmonella typhi and three plant pathogenic fungi namely Fusarium culmorum, Fusarium oxysporum, and Fusarium graminearum. The CuNPs synthesized had potent activity against all test organisms, with E. coli and F. culmorum being the most sensitive bacterium and the most sensitive fungus. This antimicrobial nature is why it is included in formulations such as nano-fungicides,nano-antimicrobials, and nano-fertilizers. Research by Mahmoodi et al. (2018) reported the synthesis of CuNPs using Ziziphus spina-christi (L) Willd fruit extract. In another field, the CuNPs were employed in investigating the antibacterial activity of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, among others above. While at high concentrations (78% and 100%), both extracts and methanolic extract displayed high resistance against the test organisms, low concentrations (25% and 50%) exhibited only moderate cavity inhibition. According to Bajpai et al. (2018), CuNPs synthesized from Syzygium aromaticum (clove) bud extract exhibited positive test results against selected pathogens. The biosynthesized

CuNPs exhibited notable bactericidal activity against Staphylococcus sp., Escherichia coli, Pseudomonas sp., and Bacillus sp., with maximum inhibition zone (8 mm) noticed at 16 μl CuNPs volume against Bacillus sp.Bio-CuNPs also showed antifungal activities against fungi such as Aspergillus niger, Aspergillus flavus, and Penicillium sp CuNPs exhibited significant fungicidal activity against Penicillium sp., having an inhibition zone of 6 mm at 16 μl CuNPs volume. The current study observed elevated inhibition zones in all the test organisms, particularly in bacteria which imply the synthesized CuNPs possess outstanding antimicrobial activities.

Conclusion

The current study successfully confirmed the green synthesis of CuNPs utilizing three plant-based extracts: black seed, neem seed, and baobab seed extracts. It was confirmed that the nanoparticles had formed, were stable, and were crystalline in nature using a range of characterization methods UV-Vis spectroscopy, FTIR, XRD, and SEM. CuNPs exhibited significant antimicrobial activity against bacterial strains, such as Bacillus subtilis and Pseudomonas aeruginosa, thereby representing their potential to act as a potent antimicrobial agent.

Recommendations

Future studies should look at the possibility of optimizing the synthesis parameters such as temperature, pH, and extract concentration to obtain CuNPs with improved yield, stability, and antimicrobial properties. Determining the therapeutic potential of the new antimicrobials, which further studies could focus on, could involve expanding the antimicrobials tested in this study against other (a broader range of) pathogens, especially multidrug-resistant bacteria and fungi. Furthermore, full evaluations of cytotoxicity and biocompatibility are also probably needed to ensure the potential safety of CuNPs for biomedical applications like wound healing, drug delivery, and antimicrobial coatings. Similar work on other metal nanoparticles like silver and zinc oxide can help determine the efficacy, staticity, and applicability of the metal nanoparticles as compared to them. Further investigations are needed to potentially include large-scale production and stability studies to guarantee the long-term usability of CuNPs in commercial and industrial applications. Due to the latter, further research should focus on their use in environmental and industrial sectors such as food preservation, water purification, and packaging. Finally, the practical utility and commercial attractiveness of CuNPs as antibacterial agents could be increased through the formulation of CuNPs as ointments, coatings, or sprays, contributing to a controlled release of the nanoparticles.

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