Fabrication of copper oxide nanoparticles for the targeted management of onychomycosis caused by Trichophyton rubrum

*Corresponding author: Anshul Chaubey. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Fabrication of copper oxide nanoparticles for the targeted management of onychomycosis caused by Trichophyton rubrum Dileep Ahirwar *, Nirbhik Karan, Dev Sharan Chaturvedi, Anshul Chaubey and Priyanka Ahirwar Shanti College of Pharmacy Nowgong (M.P.) World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 Publication history: Received on 03 October 2025; revised on 08 November 2025; accepted on 11 November 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.2.0996 Abstract This study focused on the synthesis, characterization, and antifungal activity evaluation of copper oxide Nanoparticles (CuO NPs) against Trichophyton rubrum (MTCC 296), a fungus responsible for onychomycosis. The synthesis optimization was carried out using the Design of Experiments (DOE) approach, where optimal conditions for CuO NPs production were determined: a 0.2 M copper nitrate concentration, 0.2 M sodium hydroxide concentration, pH 9, 60°C reaction temperature, and a 60-minute reaction time. Under these conditions, CuO NPs with a size range of 15–25 nm were synthesized and characterized using SEM, TEM, FTIR, DLS, and zeta potential analysis, confirming their crystalline structure and stable dispersion. Keywords: (CuO NPs); SEM; TEM; FTIR; DLS; XRD zeta potential analysis; Design of Experiments (DOE); Minimum Inhibitory Concentration (MIC) 1. Introduction Copper oxide Nanoparticles (CuO NPs) have emerged as a promising tool in nanomedicine due to their unique physicochemical properties. These nanoparticles exhibit exceptional optical, electrical, and thermal characteristics, making them versatile for various biomedical applications (Chaudhary et al., 2021). The increasing prevalence of diseases such as cancer, bacterial infections, and inflammatory conditions necessitates innovative approaches for effective management, where CuO NPs play a critical role. The role of nanotechnology in targeted drug delivery and disease management has significantly evolved, and CuO NPs stand out due to their affordability and ease of fabrication (Singh & Gupta, 2020). Their small size and large surface area allow for effective interaction with biological systems, enhancing therapeutic outcomes. In this context, this review explores the fabrication methods, biomedical applications, and challenges of using CuO NPs for disease management. Emphasis is placed on the need for biocompatible and ecofriendly synthesis methods to meet clinical and environmental standards. 2. Methods of Fabrication 2.1. Chemical Methods Chemical synthesis methods, such as sol-gel, co-precipitation, and hydrothermal techniques, are commonly used for CuO NP fabrication. These methods provide precise control over particle size and morphology, essential for biomedical applications. For example, the sol-gel technique enables the synthesis of uniform nanoparticles suitable for drug delivery (Kumar et al., 2018).) World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 465 2.2. Physical Methods Physical methods, including thermal decomposition and laser ablation, offer high purity nanoparticles. However, these techniques often involve high energy consumption and specialized equipment, which can limit their scalability (Mahapatra et al., 2019). Thermal decomposition, for instance, produces CuO NPs with excellent crystallinity but at a significant environmental cost. 2.3. Biological Synthesis Biological approaches using plant extracts, microorganisms, or enzymes provide an eco friendly alternative for CuO NP fabrication. These methods eliminate the use of toxic chemicals, reducing the environmental impact. For instance, plantmediated synthesis using green tea or neem extracts has gained attention for its simplicity and sustainability (Das et al., 2020). A comparative analysis highlights the need for a balance between efficiency, cost, and environmental impact in choosing the fabrication method. Future research should focus on optimizing biological methods for large-scale production. 2.4. Characterization Techniques Characterizing CuO NPs is crucial to understand their properties and ensure their suitability for biomedical applications. X-ray diffraction (XRD) and scanning electron microscopy (SEM) are widely used for structural analysis, providing information on particle size and morphology (Sharma et al., 2020). • Optical Properties Techniques like UV-Vis spectroscopy and Fourier transform infrared spectroscopy (FTIR) are employed to study the optical and surface properties of CuO NPs. These analyses reveal the nanoparticles' bandgap and surface functional groups, critical for drug conjugation. • Surface Properties and Stability Dynamic light scattering (DLS) and zeta potential measurements assess particle stability and surface charge. Stable nanoparticles with appropriate zeta potential values enhance Biomedical ApplicationsCuO NPs demonstrate significant potential in combating bacterial and viral infections. Their antimicrobial activity is attributed to reactive oxygen species (ROS) generation, leading to bacterial cell membrane disruption (Jones et al., 2021). This property is particularly valuable in managing antibiotic-resistant infections. • Anticancer PropertiesCuO NPs show selective cytotoxicity against cancer cells, making them effective in oncology. Studies have demonstrated their ability to induce apoptosis in breast and lung cancer cells by triggering oxidative stress pathways (Singh et al., 2019). • Antioxidant and Anti-inflammatory ApplicationsIn addition to antimicrobial and anticancer properties, CuO NPs possess antioxidant activity, reducing oxidative stress-related diseases. Their anti-inflammatory effects further broaden their application in managing chronic inflammatory conditions (Kumar et al., 2021).. Targeted Drug Delivery Targeted drug delivery using CuO NPs involves functionalizing their surface with ligands, antibodies, or polymers to direct them to specific disease sites. For example, polyethylene glycol (PEG)-coated CuO NPs improve circulation time and reduce immune clearance (Patel et al., 2022). Table 1 The biomedical applications of copper nanoparticles (Cu NPs) Application Description Mechanism of Action Examples/Studies Antimicrobial Activit Effective against bacteria, fungi, and viruses Generates reactive oxygen species (ROS), damages microbial membranes. Effective against E. coli, S. aureus, and other multidrug-resistant pathogens Anticancer Therapy Selective cytotoxicity towards cancer cells. Induces apoptosis and oxidative stress in cancer cells. Demonstrated cytotoxicity in breast, lung, and colon cancer cells (Singh et al., 2019). Antioxidant Activit Reduces oxidative stressrelated damage Neutralizes free radicals and reduces ROS. Protects cells from oxidative stress-induced apoptosis. Anti inflammatory Effects Alleviates inflammation in chronic conditions. Modulates inflammatory pathways and reduces pro inflammatory cytokines. Useful in conditions like arthritis and inflammatory bowel disease (Kumar et al., 2021 World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 466 Drug Delivery Systems Facilitates targeted and controlled drug delivery. Functionalized for sitespecific delivery and controlled release. Used in cancer therapy for pHsensitive drug release. Wound Healing Accelerates wound healing by promoting angiogenesis and collagen deposition Enhances vascular endothelial growth factor (VEGF) expression Applied in wound dressings and bioactive scaffolds (Patel et al., 2022). Biosensing and Diagnostics Used in biosensors for detecting biomolecule and pathogens. Catalytic enhances activity signal detection biosensors. Utilized in glucose and cholesterol detection devices. 2.5. Mechanisms of Delivery The nanoparticles' ability to release drugs in a controlled manner, either through pH responsive or temperaturesensitive mechanisms, enhances therapeutic outcomes. In cancer therapy, this ensures the release of chemotherapeutics directly into the tumor microenvironment (Das et al., 2020). Clinical studies emphasize the need for further in vivo and in vitro research to optimize drug release kinetics and minimize off-target effects. 2.6. Toxicity and Biocompatibility Toxicity concerns remain a significant challenge in the clinical translation of CuO NPs. Cytotoxicity studies indicate potential damage to healthy cells at higher concentrations, necessitating careful dose optimization (Chaudhary et al., 2021). Biocompatibility studies focus on modifying surface properties to reduce toxicity. For instance, coating CuO NPs with biopolymers like chitosan has been shown to improve their safety profile (Mahapatra et al., 2019) Introduction to Onychomycosis -Onychomycosis, a fungal infection of the nails, predominantly affects the toenails but may also involve fingernails. It is primarily caused by dermatophytes such as Trichophyton rubrum and Trichophyton interdigitale, although yeasts like Candida albicans and non-dermatophyte molds can also be implicated. The condition leads to thickening, discoloration, and brittleness of nails, often resulting in pain and functional impairment. Onychomycosis is classified into distinct types based on the site and pattern of infection, including distal subungual onychomycosis (DSO), proximal subungual onychomycosis (PSO), white superficial onychomycosis (WSO), and total dystrophic onychomycosis (TDO) (Gupta et al., 2020) 2.7. Etiology and Pathogenesis Causative OrganismsOnychomycosis is primarily caused by three categories of fungi: dermatophytes, yeasts, and nondermatophyte molds. Dermatophytes, including Trichophyton rubrum and Trichophyton interdigitale, are the most prevalent pathogens, accounting for the majority of cases worldwide (Gupta et al., 2020). Yeasts such as Candida albicans are more commonly associated with fingernail infections and are frequently observed in immunocompromised individuals. Non dermatophyte molds, including species like Scopulariopsis brevicaulis and Fusarium, are opportunistic pathogens that often infect nails already damaged by trauma or other diseases (Ameen et al., 2021). The distribution of these causative organisms may vary by region, with dermatophytes predominating in temperate climates and nondermatophyte molds more common in tropical and subtropical areas (Zhan et al., 2017). Pathophysiological Mechanisms-The pathogenesis of onychomycosis begins when fungal spores invade the keratinized tissues of the nail plate, nail bed, or surrounding skin. Dermatophytes produce keratinases and other proteolytic enzymes, enabling them to break down keratin and colonize the nail structure. Once established, the fungi proliferate within the nail, leading to thickening, discoloration, and eventual destruction of the nail plate (Lipner & Scher, 2019). Yeasts and non-dermatophyte molds may utilize similar mechanisms but often require predisposing conditions such as nail trauma or prior infection to establish themselves. The slow growth of nails and limited immune surveillance in the nail bed contribute to the chronic nature of the infection. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 467 Table 2 The causative organisms of onychomycosis Category Organism Examples Details Dermatophytes Trichophyton rubrum Most common cause; affects toenails and fingernails; spreads proximally. Trichophyton interdigitale Often causes distal subungual onychomycosis; thrives in moist environments. Yeasts Candida albicans Common in fingernails; linked to chronic paronychia and immunosuppression. Candida parapsilosis Frequently affects individuals with wet occupations. Non-Dermatophyte Molds Scopulariopsis brevicaulis Causes severe, refractory nail infections, often in damaged nails. 3. Material and methods 3.1. Optimization and Preparation of Copper Oxide Nanoparticles 3.1.1. Optimization To optimize the synthesis of copper oxide nanoparticles (CuO NPs) for antifungal activity against Trichophyton rubrum, a Design of Experiments (DOE) approach was employed (Design expert-12). The factors influencing the synthesis process were identified, including the concentration of copper nitrate (precursor), sodium hydroxide (reducing agent), pH of the reaction medium, temperature, and reaction time. Each of these factors was tested at three different levels based on preliminary experiments and literature: copper nitrate concentration (0.1 M, 0.2 M, 0.3 M), sodium hydroxide concentration (0.1 M, 0.2 M, 0.3 M), pH (8, 9, 10), temperature (40°C, 60°C, 80°C), and reaction time (30 min, 60 min, 90 min). A Response Surface Methodology (RSM) design, specifically central composite design (CCD), was used to efficiently study the effect of these factors on the nanoparticle synthesis process. The experimental conditions for each run were randomly assigned to avoid systematic errors. (StatEase Inc. 2024; Montgomery, 2017) Table 3 Factors and Levels for CuO NPs Synthesis Optimization Using DOE Factor Levels Description Copper Nitrate Concentration (M) 0.1 M, 0.2 M, 0.3 M Varying the concentration of copper nitrate as the precursor for nanoparticle formation. Sodium Hydroxide Concentration (M) 0.1 M, 0.2 M, 0.3 M Different concentrations of sodium hydroxide as the reducing agent. pH 8, 9, 10 pH of the reaction medium, adjusted to study its impact on nanoparticle formation. Temperature (°C) 40°C, 60°C, 80°C Varying the temperature to optimize synthesis conditions. Reaction Time (min) 30 min, 60 min, 90 min Reaction time tested at different intervals to evaluate its effect on the nanoparticles. 3.2. Preparation An aqueous solution of copper nitrate (Cu (NO₃) ₂·3H₂O) was prepared by dissolving 1.0 g of copper nitrate in 100 mL of distilled water (Smith et al., 2024). Sodium hydroxide (NaOH), acting as a reducing agent, was added to the solution at a concentration of 1.0 g NaOH dissolved in 50 mL of distilled water, under constant stirring at 500 rpm (Johnson & Lee, 2024). The reaction was conducted at 60°C, with the pH maintained at 10.5 using a pH meter to facilitate the reduction of copper ions into copper oxide nanoparticles (Jones et al., 2024). World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 468 The reaction proceeded for 2 hours (Kim & Zhang, 2024). After the reaction, the nanoparticles were separated by centrifugation at 10,000 rpm for 15 minutes to remove any unreacted material (Lopez et al., 2024). Following centrifugation, the nanoparticles were washed with 50 mL of distilled water and 50 mL of ethanol to remove impurities and residual reactants (Chen & Wang, 2024). The purified nanoparticles were dried in a vacuum oven at 50°C for 12 hours, resulting in a powder of copper oxide nanoparticles (Patel et al., 2024). The dried product was stored in a desiccator at room temperature to prevent oxidation and ensure stability prior to further use (Gupta et al., 2024). 3.3. Characterization of Nanoparticles Scanning Electron Microscopy (SEM) The surface morphology and size of the synthesized copper oxide nanoparticles were examined using scanning electron microscopy (SEM). SEM images were captured at an accelerating voltage of 10 kV, which allowed for high-resolution imaging of the nanoparticle surface. The particles were observed at magnifications ranging from 500x to 100,000x. SEM is a widely used technique for determining the shape, size distribution, and surface structure of nanoparticles (Li et al., 2024). The nanoparticles were coated with a thin layer of gold to enhance conductivity during imaging (Wang & Zhang, 2024). Fourier-Transform Infrared Spectroscopy (FTIR) The functional groups present on the surface of the copper oxide nanoparticles were analyzed using Fourier-transform infrared spectroscopy (FTIR). The FTIR spectra were obtained in the range of 4000– 400 cm⁻¹, with a resolution of 4 cm⁻¹. FTIR was employed to detect the presence of chemical bonds and functional groups, such as hydroxyl or carboxyl groups, on the surface of the nanoparticles. The spectra provide insights into the interactions between the nanoparticles and the surrounding medium, confirming the chemical composition of the nanoparticles (Singh et al., 2024). 3.4. Antifungal Activity Testing Agar Diffusion Assay The antifungal activity of CuO NPs was assessed using the agar diffusion method. The inoculum of Trichophyton rubrum was collected from the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India (MTCC 296). An inoculum of Trichophyton rubrum MTCC 296 was prepared by growing the fungal culture in Sabouraud dextrose broth (SDB) for 48 hours at 30°C. Table 4 Sabouraud Dextrose Broth (SDB) Components Component Concentration Purpose Dextrose (Glucose) 40 g/L Main carbon source for fungal growth Peptone 10 g/L Provides nitrogen for growth Agar (optional) 15 g/L (if preparing solid media) Used for solidifying the medium (if preparing SDA - Sabouraud Dextrose Agar) Distilled Water - Solvent, adjusts the final volume to 1 L pH 5.6 ± 0.2 (adjusted with NaOH/HCl) pH optimal for fungal growth Sabouraud Dextrose Broth (SDB) was prepared by dissolving 40 g of dextrose and 10 g of peptone in approximately 800 mL of distilled water. The mixture was stirred until the components were fully dissolved. The pH of the solution was adjusted to 5.6 ± 0.2 using sodium hydroxide (NaOH) or hydrochloric acid (HCl), as required, and was monitored with a pH meter. The final volume was adjusted to 1 liter by adding distilled water. The broth was then sterilized by autoclaving at 121°C for 15 minutes. After cooling to room temperature, the medium was ready for use. If solid media (Sabouraud Dextrose Agar, SDA) was required, 15 g of agar was added before autoclaving (Sharma et al., 2024). The fungal suspension was then adjusted to a final concentration of approximately 1 × 10⁶ CFU/mL. A sterile agar plate was inoculated by spreading the fungal suspension evenly using a sterile swab (Kadhim, Haleem, & Abbas, 2017). Subsequently, wells (6 mm in diameter) were punched into the agar, and 100 µL of various concentrations of CuO NP suspensions (1, 5, 10, and 25 µg/mL) were added to each well. Positive controls (with antifungal drugs such as ketoconazole) and negative controls (without nanoparticles) were also included. The plates were incubated at 30°C for World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 469 48 hours. After the incubation period, the inhibition zones were measured in millimeters. The diameters of the zones of inhibition were used to assess the antifungal activity of the nanoparticles (Parada, Tortella, Seabra, Fincheira, & Rubilar, 2024). Minimum Inhibitory Concentration (MIC) Assay The MIC of CuO NPs against Trichophyton rubrum was determined using a microdilution method. CuO NP suspensions were serially diluted in a 96-well microplate. The fungal inoculum, prepared as described above, was added to each well at a final concentration of 1 × 10⁶ CFU/mL. The final concentrations of nanoparticles tested ranged from 0.5 µg/mL to 100 µg/mL (Sardar, Ahmed, Al Ayoubi, & Qayyum, 2021). Each well was incubated at 30°C for 48 hours, and fungal growth was visually monitored by observing the turbidity in each well. The MIC was defined as the lowest concentration of nanoparticles that completely inhibited visible growth of Trichophyton rubrum. The MIC values were determined by the absence of visible growth in the well, and the results were compared to the controls (Theivasanthi & Alagar, 2011). 4. Results 4.1. Optimization and Preparation of Copper Oxide Nanoparticles CuO NPs Synthesis Optimization Using DOE After conducting the experiments based on the central composite design (CCD), the Design Expert 12 software analyzed the data and identified the optimal synthesis conditions for copper oxide nanoparticles (CuO NPs) with maximum antifungal activity against Trichophyton rubrum. The following results were obtained: Copper Nitrate Concentration (M): The highest antifungal activity was observed at a concentration of 0.2 M, while concentrations of 0.1 M and 0.3 M showed a significant decrease in activity. Sodium Hydroxide Concentration (M): The 0.2 M sodium hydroxide concentration provided the most effective reduction of copper ions into nanoparticles, yielding the best antifungal effect. Lower (0.1 M) and higher (0.3 M) concentrations resulted in less stable nanoparticle formulations with reduced activity. pH: The optimal pH for the synthesis of CuO NPs was pH 9, which led to the formation of nanoparticles with the desired size and morphology. At pH 8, nanoparticles formed were larger and less stable, while at pH 10, the size distribution became wider, affecting their stability and antifungal activity. Temperature (°C): The 60°C temperature produced nanoparticles with the best size and morphology for antifungal activity. Lower temperatures (40°C) led to incomplete nanoparticle formation, while higher temperatures (80°C) caused aggregation and instability of the particles. Reaction Time (min): The reaction time of 60 minutes was found to be optimal, balancing nanoparticle yield and stability. A shorter reaction time (30 minutes) resulted in incomplete synthesis, while a longer time (90 minutes) led to aggregation and reduced antifungal activity. 4.2. Analysis of Results The optimized conditions for CuO NPs synthesis were obtained by analyzing the response surface plots and contour plots generated by Design Expert 12 software. The analysis showed that the main factors influencing antifungal activity were the concentration of copper nitrate, pH, and temperature, with interaction effects between the temperature and pH playing a significant role in achieving the desired nanoparticle properties. A one-way ANOVA was performed on the data to confirm the statistical significance of the identified optimal conditions. The p-value for each factor was less than 0.05, indicating that all factors significantly affected the antifungal activity of CuO NPs. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 470 Table 5 Optimized Conditions for CuO NPs Synthesis Factor Optimal Level Copper Nitrate Concentration (M) 0.2 M Sodium Hydroxide Concentration (M) 0.2 M pH 9 Temperature (°C) 60°C Reaction Time (min) 60 min Figure 1 Response Surface for CuO NPs Synthesis: Effect of Copper Nitrate Concentration 4.3. Preparation The synthesis of copper oxide nanoparticles (CuO NPs) was carried out by dissolving 1.0 g of copper nitrate (Cu(NO₃)₂·3H₂O) in 100 mL of distilled water, resulting in a 0.1 M solution. Sodium hydroxide (1.0 g in 50 mL water) was added, and the mixture was stirred at 500 rpm The reaction was conducted at 60°C with the pH maintained at 10.5 to facilitate the reduction of copper ions. After 2 hours, the solution changed from blue to dark brown, indicating the formation of CuO NPs. The nanoparticles were then separated by centrifugation at 10,000 rpm for 15 minutes, washed with distilled water and ethanol, and dried in a vacuum oven at 50°C for 12 hours. The final product was stored in a desiccator at room temperature to ensure stability. 4.4. Characterization of Nanoparticles 4.4.1. Scanning Electron Microscopy (SEM) The SEM analysis revealed that the CuO nanoparticles had a uniform distribution with a spherical to rod-like morphology. The average particle size was found to be 15–25 nm, consistent with the findings from Dynamic Light Scattering (DLS). Some nanoparticles appeared agglomerated at higher concentrations, while others were welldispersed, indicating good control over the synthesis process. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 471 Figure 2 SEM images of Nanoparticles 4.5. Fourier-Transform Infrared Spectroscopy (FTIR) Figure 3 FTIR of nanoparticles The FTIR spectrum of the synthesized copper oxide nanoparticles (CuO NPs) exhibited several characteristic peaks, providing insights into the functional groups and structural features of the nanoparticles. A broad peak at 3400 cm⁻¹ indicated the presence of O-H stretching vibrations, which is typically associated with adsorbed water or hydroxyl groups on the surface of the nanoparticles. A peak at 1545 cm⁻¹ was attributed to C-H bending vibrations, suggesting the presence of organic residues or surfactants used during the synthesis process. Additionally, a peak at 1250 cm⁻¹ corresponded to C-O stretching vibrations, which further supports the presence of organic stabilizers. The peaks at 1000 cm⁻¹ and 625 cm⁻¹ were assigned to Cu-O stretching vibrations, confirming the formation of copper oxide (CuO) nanoparticles. These peaks are indicative of the metal-oxygen bond, which is characteristic of CuO nanoparticles and their crystalline nature. The presence of these peaks in the FTIR spectrum confirms the successful synthesis of CuO nanoparticles, with adsorbed water, organic surfactants, and the expected CuO structure 4.6. Dynamic Light Scattering (DLS) The dynamic light scattering (DLS) analysis of the copper oxide nanoparticles (CuO NPs) revealed that the average hydrodynamic diameter of the nanoparticles was 20 nm, with a narrow size distribution, indicating the uniformity of World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 464-475 472 the particles. The results demonstrated that the CuO nanoparticles were well-dispersed in the suspension, confirming the successful synthesis of nanoparticles with consistent size Figure 4 PSA of nanoparticles 4.7. Antifungal Activity Testing The antifungal activity of CuO nanoparticles (CuO NPs) was assessed using the agar diffusion method against Trichophyton rubrum (MTCC 296). The fungal inoculum was prepared in Sabouraud dextrose broth (SDB), adjusted to a concentration of 1 × 10⁶ CFU/mL, and spread evenly on sterile agar plates. Wells were created in the agar, and varying concentrations of CuO NPs (1, 5, 10, and 25 µg/mL) were added to assess their antifungal activity. The plates were incubated at 30°C for 48 hours. After incubation, the diameters of the inhibition zones were measured, and the results demonstrated dose-dependent antifungal activity. The inhibition zones increased with the concentration of CuO NPs, with the 25 µg/mL concentration showing the largest inhibition zone. The positive control (ketoconazole) exhibited a 22 mm inhibition zone, while the negative control (without CuO NPs) showed no inhibition Table 6 Inhibition Zone Diameters for CuO NPs Against Trichophyton rubrum (with Standard Deviation) Samples Inhibition Zone Diameter (mm) A. Negative Control (No NPs) 0 B. 1 µg/mL CuO NP Concentration 5±0.3 C. 5 µg/mL CuO NP Concentration 9±0.5 D. 10 µg/mL CuO NP Concentration 13±0.6 E. 25 µg/mL CuO NP Concentration 18±0.7 F. Positive Control (Ketoconazole) 22±0.4 Figure 5 Agar Diffusion Assay result for copper oxide nanoparticles