Green Synthesis of Zinc Oxide Nanoparticles by Candida albicans CA26 with Antioxidant and Antifungal Activity Against Nannizzia incurvata

Green Synthesis of Zinc Oxide Nanoparticles by Candida albicans CA26 with Antioxidant and Antifungal Activity Against Nannizzia incurvata | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Green Synthesis of Zinc Oxide Nanoparticles by Candida albicans CA26 with Antioxidant and Antifungal Activity Against Nannizzia incurvata Yajurved N Selokar, Parameswaran Sree Pranav, Rakesh U Thakare This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7164738/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The rise of antifungal resistance in dermatophytosis underscores the need for novel antifungals, driving the green synthesis of zinc oxide nanoparticles (ZnO NPs). In this study, we report the extracellular biosynthesis of ZnO NPs via a clinical isolate of Candida albicans CA26 and evaluate their physicochemical properties, antioxidant capacity, and antifungal efficacy against Nannizzia incurvata TI03. The ZnO NPs were biosynthesized using the extracellular filtrate of C. albicans CA26 and the resulting NPs were characterized via UV-Vis spectroscopy, FTIR, XRD, TEM, and BET analysis. The UV-Vis spectra revealed a characteristic surface plasmon resonance peak at 361.75 nm, confirming the formation of ZnO NP; whereas the FTIR spectra confirmed capping by fungal biomolecules. XRD confirmed a crystalline ZnO phase with an average crystallite size of ~ 30 nm and TEM imaging revealed a rod-to-bar morphology (75–99 nm in length). BET analysis revealed a specific surface area of ~ 24.24 m^2/g and an average pore radius of ~ 4.57 nm. The antioxidant activity of the ZnO NPs was evaluated via DPPH, ABTS, nitric oxide, hydroxyl radical, and superoxide scavenging assays, which revealed moderate but consistent activity across all five assays. These activities were statistically significant compared with those of the reference antioxidants (p 17). The antifungal efficacy was tested against N. incurvata TI03, yielding a minimum inhibitory concentration (MIC) of 0.96 µg/mL, which is comparable to that of standard azole drugs. In agar diffusion assays, ZnO NPs produced inhibition zones similar in size to those of fluconazole and itraconazole. The biosynthesized ZnO NPs thus exhibited multimodal antifungal mechanisms, likely involving reactive oxygen species generation, membrane disruption, and enzyme inhibition, in addition to their antioxidant activity. This eco-friendly synthesis, combined with the potent dual-functionality of these ZnO NPs highlights their role as promising topical antifungal therapeutic agents. Mycology Nanoscience Applied & Industrial Microbiology Zinc oxide nanoparticles green synthesis antifungal activity antioxidant activity dermatophytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Dermatophytosis remains a major global health concern, affecting an estimated 20–25% of the world's population (Brasch, 2009 ; Weitzman & Summerbell, 1995a ). Resolving this superficial fungal infection can be difficult, as it often recurs and persists, especially in densely populated environments or circumstances involving close human contact. Notably, Nannizzia incurvata , a fungal species that thrives on human skin, has emerged as a concern in recent years because of its association with chronic, nonhealing infections, as well as its increased ability to resist traditional antifungal treatments (Kruithoff et al., 2023 ; Uhrlaß et al., 2021 ). A number of issues limit the effectiveness of antifungal medications, such as azoles and allylamines, even though these are still the primary therapeutic options. These include the growing problem of fungal strains developing resistance to these drugs, the need for prolonged treatment courses, the risk for systemic toxicity, and exorbitant costs (Burmester et al., 2023 ; Ghannoum, 2016 ; Symoens et al., 2011 ). Terbinafine resistance has become a major growing concern and this resistance has developed due to a mutation in the squalene epoxidase (SQLE) gene, potentially undermining its therapeutic effect. Furthermore, the increased activity of efflux pumps in fungal cells can profoundly impair the efficacy of terbinafine, making it a less trustworthy therapeutic option (Pashootan et al., 2022 ). The complexity of antifungal resistance and treatment limitations highlights the urgent need for innovative approaches that strike a balance between environmental sustainability, patient safety, and effective infection control (Ebert et al., 2020 ). One promising approach to the development of novel antibacterial agents is nanobiotechnology. Zinc oxide nanoparticles (ZnO NPs) are potent antibacterial agents with minimal cytotoxicity, high biocompatibility, and broad-spectrum effectiveness against a variety of bacterial pathogens (Ijaz et al., 2020 ; Jha et al., 2023 ; Kumar et al., 2017 ). Its antifungal activity is achieved through mechanisms such as reactive oxygen species (ROS) production, fungal membrane disruption, and the inhibition of major enzyme pathways (Jhawat et al., 2024 ; Nxumalo et al., 2024 ; Sirelkhatim et al., 2015). Nevertheless, conventional synthesis methods for ZnO NPs often rely on hazardous chemicals and high energy inputs, making them environmentally and biologically less desirable (Banjara et al., 2024 ; Benitez-Salazar et al., 2021 ). Green synthesis techniques offer a more environmentally friendly option, as they stabilize nanoparticles and reduce metal ions via the use of biological systems such as bacteria, fungi, yeasts, and plants. These organisms normally contribute to the synthesis of nanoparticles by providing natural reducing and capping agents, such as enzymes and secondary metabolites (Iravani, 2011a ; Vijayaram et al., 2023 ). While plant and bacterial-mediated synthesis have been widely researched, the potential of yeast-based synthesis, particularly with C. albicans , has received less attention (Narayanan & Sakthivel, 2010 ). Although generally known as a commensal yeast and opportunistic pathogen, C. albicans possesses useful biotechnological traits, including high metal tolerance, simple growth, and the ability to release extracellular proteins that facilitate nanoparticle formation (Rodríguez et al., 2017 ; R. Sharma et al., 2023 ). Its potential as a workable microbial nanofactory has been demonstrated through its effective usage in the synthesis of nanoparticles like iron and silver oxides (Abed et al., 2024 ; Gupta & Verma, 2022 ; Jalal et al., 2018a ; Rahimi et al., 2016 ). Compared with certain plant or bacteria-based approaches, C. albicans -mediated synthesis yields nanoparticles through a simple, costeffective, and ecofriendly process, avoiding the use of hazardous chemical waste and high energy inputs. This approach aligns well with the concepts of sustainable nanomedicine and yields nanoparticles that exhibit strong antimicrobial efficacy in vitro (Abed et al., 2024 ; Jalal et al., 2018a ). In addition to their antifungal characteristics, ZnO nanoparticles possess significant antioxidant properties, which are important in reducing the oxidative stress associated with dermatological infections (Tran et al., 2024 ). Green-synthesized ZnO NPs have exhibited substantial scavenging action against free radicals in conventional tests such as DPPH and ABTS, indicating promise for skin protection and wound healing by decreasing ROS-mediated tissue damage (Abdelghany et al., 2023 ; Król et al., 2017 ; Tiwari et al., 2024 ). This dual function increases their appeal as a treatment alternative for managing resistant dermatophytosis with inflammatory responses. Regardless of these benefits, the sustainable extracellular synthesis of ZnO nanoparticles utilizing C. albicans and their application against dermatophyte strains such as N. incurvata remain largely unexplored. This study addresses that gap by detailing the green synthesis of ZnO nanoparticles using a clinical isolate of C. albicans (CA26), followed by comprehensive physicochemical characterization and antifungal evaluation against a dermatophyte isolate (TI03), which was later confirmed as N. incurvata . To overcome the limitations of current antifungal treatments, this work aims to lay the foundation for a nanoparticle-based therapeutic platform that is scalable, biocompatible, and environmentally sustainable. Materials and Methods Sample Collection and Culture Conditions All clinical isolates of C. albicans and N. incurvata were obtained from patients clinically diagnosed with candidiasis and dermatophytosis, respectively, following written approval for sample collection from the Dean, Indira Gandhi Government Medical College & Hospital, Nagpur, India (Ref. No. 390; 8 October 2022) and from the Head, Department of Molecular Biology & Genetic Engineering, Rashtrasant Tukadoji Maharaj Nagpur University, India (Letter No. MB&GE/12; 15 April 2024). Written informed consent was obtained from all participants. Oropharyngeal swab samples were collected from patients exhibiting clinical signs of oral candidiasis. For C. albicans , swabs were transported aseptically in sterile containers at 4°C and processed immediately for microbiological evaluation, as per established protocols (Begum & Kumar, 2021 ; Gamal et al., 2023 ; Shalaby et al., 2016 ; Williams et al., n.d.). The samples were inoculated onto Sabouraud dextrose agar (SDA) supplemented with chloramphenicol (50 µg/mL) and incubated at 37°C for 48–72 h. Colonies displaying creamy, smooth morphology were subjected to Gram staining and germ tube testing in human serum at 37°C for 2–3 h. Isolates demonstrating germ tube formation were provisionally identified as C. albicans and further confirmed via lactophenol cotton blue (LPCB) staining for characteristic morphological features such as budding yeast cells and pseudohyphae (Jalal et al., 2018b; Kim et al., 2002 ; Mallya & Mallya, 2019 ). Among the other C. albicans isolates, a single isolate (CA26) consistently exhibiting typical morphological characteristics and germ tube formation, was selected as a representative strain for molecular characterization. For dermatophyte isolation, skin scrapings were treated with 10% potassium hydroxide (KOH) and microscopically examined for hyphal elements. KOH-positive samples were cultured on Dermatophyte Test Medium (DTM) and SDA supplemented with chloramphenicol (50 µg/mL) and cycloheximide (500 µg/mL), and incubated at 28°C for 7–14 days (Gómez-Moyano et al., 2021 ; Gräser et al., 2008 ; Weitzman & Summerbell, 1995b ). Colonies with a powdery, cream-colored morphology and reverse pigmentation were subcultured and analyzed through LPCB staining for macroconidia and microconidia characteristics (Kruithoff et al., 2023 ). A representative isolate (TI03) with well-developed conidial structures was selected for molecular identification. Molecular Characterization of C. albicans and N. incurvata The genomic DNA was extracted from C. albicans CA26 via the phenol-chloroform method, whereas the genomic DNA from N. incurvata TI03 was extracted using the HiPurA™ fungal DNA extraction kit (HiMedia, India), following the manufacturer’s instructions (Dabas et al., 2017 ; Futatsuya et al., n.d.; Saghrouni et al., 2013 ). Amplification of the internal transcribed spacer (ITS) region of rDNA was performed via the universal fungal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). PCR was carried out in a 25 µL reaction mixture containing 2× PCR Master Mix (Thermo Fisher Scientific, USA) under the following cycling conditions: initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 1 min; and a final extension at 72°C for 10 min. Amplicons were visualized on 1.5% agarose gel, purified, and sequenced via Sanger sequencing. The ITS sequences of C. albicans CA26 and N. incurvata TI03 were submitted to GenBank under accession numbers PV465594.1 and PV463161.1, respectively. ITS-Based Phylogenetic Analysis The ITS region sequences derived from C. albicans CA26 and N. incurvata TI03 were subjected to BLASTn (Altschul et al., 1990 ) for preliminary species confirmation. For phylogenetic reconstruction, each sequence was queried against UNITE data (Abarenkov et al., 2024a ), and the top 20 closest matches were retrieved in FASTA format. The query and reference sequences were aligned via the MUSCLE algorithm implemented in MEGA v12 (Hall, 2013 ). Phylogenetic trees were constructed using the neighbor-joining method with the maximum composite likelihood model, assuming uniform substitution rates. Branch support was assessed via 1000 bootstrap replicates in MEGA v12. The resulting trees were visualized, midpoint-rooted, and annotated using iTOL v6. (Abarenkov et al., 2024b; Letunic & Bork, 2024 ). Biosynthesis of Zinc Oxide Nanoparticles A well-isolated colony of C. albicans CA26 was inoculated into 100 mL of Sabouraud dextrose broth (SDB) and incubated at 30°C for 72 h at 150 rpm to promote the optimal secretion of reductive metabolites (Abed et al., 2024 ). The culture was filtered through Whatman No. 1 paper to obtain a cell-free filtrate (CFF), which served as a bioreductant (Rahimi et al., 2016 ). For nanoparticle biosynthesis, 100 mL of 1 mM zinc nitrate hexahydrate Zn(NO₃)₂·6H₂O solution was mixed with an equal volume of the CFF in a sterile 250 mL Erlenmeyer flask. The pH was adjusted to 8.0 with 1 M NaOH, as alkaline conditions favour the formation of ZnO nuclei and increase the reduction efficiency. The mixture was incubated in the dark at 30°C for 72 h at 100 rpm to avoid photoactivation and promote gradual particle growth. A visible shift from pale yellow to milky white indicated ZnO NP formation. The precipitate was collected via centrifugation (12,000 rpm, 20 min), washed three times with sterile distilled water and ethanol, and dried at 60°C. The final product was calcined at 500°C for 4 h to increase crystallinity and thermal stability. The synthesised ZnO NPs were stored in sterile, airtight vials for further characterization and bioassays. Physicochemical Characterization of ZnO Nanoparticles The physicochemical, morphological, and structural properties of the biosynthesized ZnO nanoparticles were comprehensively characterized via advanced analytical techniques. UV-Visible Spectroscopy (UV-Vis) The optical absorption properties of the ZnO NPs were analyzed via a Shimadzu UV-1800 double-beam spectrophotometer over a wavelength range of 200–600 nm. One milligram of ZnO NPs was dispersed in 10 mL deionized water via ultrasonic treatment via using a PCI analytical ultrasonic cleaner to ensure homogeneity. The absorbance spectrum was recorded and processed via UVProbe software (Shimadzu, Japan) (S. Singh et al., 2024 ). Fourier Transform Infrared Spectroscopy (FTIR) : The functional groups involved in nanoparticle stabilization were confirmed using a Thermo Scientific Nicolet iS10 spectrometer (4000 − 400 cm⁻¹). The samples were prepared by homogenizing ZnO NPs with KBr (1:100 ratio) into pellets. X-ray Diffraction (XRD) Crystalline structure analysis was conducted via a Shimadzu XRD-6100 diffractometer (Cu-Kα radiation, λ = 1.5406 Å) operated at 40 kV and 30 mA. Diffractograms were recorded in the 2θ range of 20°-80°. The crystallite size was estimated via the Debye-Scherrer equation, and the data were interpreted via OriginPro 8.5 software (Grace et al., 2023 ; Jayachandran et al., 2021). Transmission Electron Microscopy (TEM) The morphology and particle size distribution were determined via high-resolution TEM JEOL JEM-2100, operating at 200 kV. The samples were prepared by suspending the ZnO NPs in ethanol, sonicating them, and depositing them onto carbon-coated copper grids. ImageJ software (NIH, USA) was used for particle size measurement(Maher et al., 2023 ). Brunauer-Emmett-Teller (BET) Analysis The surface area and porosity were estimated via a Quantachrome NOVA Station A BET analyzer. The samples were degassed at 150°C for 4 hours before nitrogen adsorption-desorption analysis. The data were processed using NovaWin software to calculate the pore size distribution and specific surface area. Antioxidant Assays The antioxidant activity of the biosynthesized zinc oxide nanoparticles (ZnO NPs) was evaluated via five in vitro assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, nitric oxide (NO) radical scavenging, hydroxyl radical scavenging, and superoxide radical scavenging. These assays were performed following standard protocols with slight modifications to optimize the nanoparticle suspensions (Abdelghany et al., 2023 ; Amalraj et al., 2021 ). The standard antioxidants used for comparison included ascorbic acid and butylated hydroxytoluene (BHT). The scavenging activity was expressed as the percentage inhibition relative to the control. IC₅₀ values were determined via linear regression analysis from dose-response curves. All the experiments were carried out in triplicates, and the data are reported as the mean ± standard deviation (SD). Independent sample t-tests were conducted to compare the IC₅₀ values of the ZnO NPs with those of standard antioxidants. A p-value < 0.001 was considered statistically significant. Cohen’s d effect sizes were computed to quantify the magnitude of differences between ZnO NPs and standards via the following formula: $$\:d\:=\:\backslash\:frac\left\{{M}_{1}-\:{M}_{2}\right\}\left\{\sqrt{\left\{\backslash\:frac\left\{S{D}_{1}^{2}+\:S{D}_{2}^{2}\right\}\left\{2\right\}\right\}}\right\}$$ Effect sizes were interpreted as small (0.2), medium (0.5), and large (≥ 0.8)(Cohen, 2013 ; Daines, n.d.) Antifungal Activity of the Biosynthesized ZnO Nanoparticles The antifungal efficacy of C. albicans CA26-derived ZnO nanoparticles (NPs) against N. incurvata TI03 was assessed via the broth microdilution assay and agar well diffusion method. Determination of Minimum Inhibitory Concentration (MIC) The MIC of the ZnO nanoparticles was determined according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 guidelines. Stock solutions of ZnO NPs (64 µg/mL) were prepared in sterile distilled water and serially diluted (0.03125 µg/mL to 64 µg/mL) in a 96-well microtiter plate containing SDB. Each well was inoculated with 100 µL of N. incurvata TI03 spore suspension standardized to 2 × 10³ CFU/mL in SDB. The plates were incubated at 28°C for 5–7 days. The MIC was defined as the lowest concentration of ZnO NPs showing complete inhibition of visible fungal growth compared with that in the control wells (S. Sharma et al., 2022 ; Slavin & Bach, 2022 ). Agar Well Diffusion Assay The zone of inhibition exerted by ZnO NPs against N. incurvata TI03 was assessed via agar well diffusion. The fungal inoculum was adjusted to match 0.5 McFarland standard turbidity and uniformly spread on Dermatophyte Test Medium (DTM) agar plates. Wells (6 mm diameter) were punched into the agar, and 10 µL of ZnO NP suspension (1 mg/mL in sterile distilled water) was loaded into each well. Commercial antifungal drugs including fluconazole (25 µg/disk), itraconazole, (10 µg/disk), and clotrimazole (10 µg/disk) served as positive controls. The negative controls consisted of sterile distilled water. Plates were incubated at 28°C for 7–10 days, and the diameters of the inhibition zones were measured via a digital calliper (Al-Jobory et al., 2020 ; Garcia-Marin et al., 2022 ; Savi et al., 2013 ). Statistical Analysis All experiments were performed in triplicate to ensure reproducibility. The data are expressed as the mean ± standard deviation (SD). Statistical significance was determined via one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) post-hoc test in GraphPad Prism v9.0.0 (GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant. Antioxidant assay calculations were performed via T-statistics, degrees of freedom and Cohen’s test using IBM SPSS Statistics (Version 26.0, IBM Corp., Armonk, NY, USA) and Microsoft Excel (Version 2019). A significance level of p < 0.001 was used for the antioxidant assay. Results Phenotypic Characterization of Candida albicans and Nannizzia incurvata Nine isolates from oral swab cultures presented phenotypic characteristics consistent with those of C. albicans , including smooth, creamy colonies, gram-positive budding cells, germ tube formation, and pseudohyphae. Lactophenol Cotton Blue staining further revealed characteristic fungal morphology (Supplementary Fig. 1A). Among these strains, isolate CA26, which consistently exhibited characteristic morphology and germ tube formation, was selected for molecular identification. Multiple isolates from cutaneous samples were obtained through skin scraping techniques. Several samples tested positive for fungal hyphae on KOH mounts and grew on DTM slants with cream to yellow-orange reverse pigmentation. Microscopic examination revealed multicellular macroconidia and microconidia, which are characteristic of N. incurvata . One isolate, TI03, which displays typical conidial structures of Nannizzia species (Supplementary Fig. 1B, C), was selected for molecular confirmation. Molecular Confirmation of Species Identity The ITS-based phylogenetic tree for C. albicans revealed that strain CA26 clustered closely with the reference strains AUMC 10189 and AUMC 8996 (Fig. 1 A). The short branch lengths within this clade (0-0.001 substitutions per site) indicated high genetic similarity and minimal sequence divergence. Therefore, this low genetic distance, along with the placement among multiple C. albicans reference strains supports the taxonomic identity of CA26 as C. albicans . Similarly, strain TI03 was clustered within a distinct clade, consisting GenBank-annotated N. incurvata strains (KX793108.1, KX452080.1), with branch lengths ranging from 0.001–0.004 substitutions per site (Fig. 1 B). This clade was clearly separated from the N. gypsea reference strains, which presented longer branch lengths of up to ~ 0.013, indicating interspecific divergence. The monophyletic clustering of TI03 with N. incurvata strains, along with their short intraclade distances, supports its taxonomic placement within the species and is consistent with established ITS-based fungal identification criteria (Schoch et al., 2012 ). UV-Visible Spectroscopy The UV-Visible absorption bands of the ZnO nanoparticles (ZnO NPs) revealed a distinct surface plasmon resonance (SPR) peak at 361.75 nm (Fig. 2 A), indicating effective nanoparticle synthesis with little aggregation. The absence of further peaks ranging from 400 to 900 nm confirmed the exceptional colloidal purity and stability of the suspension. In contrast, commercial ZnO powder exhibited a broader SPR peak at 367 nm (Fig. 2 B), indicating a larger particle size and enhanced agglomeration. Supplementary Table 1 summarizes the UV-Visible absorbance values, which support the observed differences in the SPR peak sharpness and spectral purity between the biosynthesized and commercial ZnO. Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra (400–4000 cm⁻¹) revealed the presence of bioorganic functional groups associated with fungal metabolites (Fig. 3 ). A broad absorption band at ~ 3400 cm⁻¹ was observed in the ZnO + SDB spectrum, corresponding to the O-H stretching vibrations of the hydroxyl groups(Mohan et al., 2020 ). A distinct peak near 1630 cm⁻¹ in the ZnO + SDB spectrum was attributed to C = O stretching and N-H bending vibrations (Kazemi et al., 2023 ). The characteristic bands at approximately 500 cm⁻¹ in the ZnO and ZnO + SDB spectra confirmed the Zn-O stretching vibrations, indicating the formation of crystalline ZnO (Quadri et al., 2017 ). Additionally, the SDB control spectrum exhibited weak broad bands near ~ 2920 cm⁻¹, corresponding to C-H stretching vibrations(Meena et al., 2023 ) (Table 1). These observations suggest that ZnO NPs are effectively stabilized by extracellular fungal biomolecules. X-ray Diffraction (XRD) XRD analysis revealed distinct diffraction peaks at 2θ values of 31.8°, 34.4°, 36.3°, 47.5°, and 56.6°, corresponding to the (100), (002), (101), (102), and (110) crystallographic planes of the hexagonal wurtzite phase of ZnO (JCPDS Card No. 36-1451; space group: P6₃mc ) (Fig. 4 A). No additional impurity peaks were observed, indicating the high phase purity of the biosynthesized ZnO NPs. The most intense peak at ~ 36.3° (plane 101) was used to estimate the average crystallite size (~ 30 nm) via the Debye-Scherrer equation: $$\:\text{D}=\:\:\frac{K\lambda\:}{\beta\:cos\theta\:}$$ where D is the crystallite size, K is the Scherrer constant (0.94), λ is the X-ray wavelength (1.5405 Å), β is the full width at half maximum (FWHM) of peak 101, and θ is the Bragg angle. Three-dimensional crystallographic visualization performed via VESTA software further confirmed the hexagonal wurtzite structure of the biosynthesized ZnO nanoparticles (Fig. 4 B). Transmission Electron Microscopy (TEM) TEM micrographs revealed rod-to-bar shaped ZnO NPs with sizes ranging from approximately 75 to 99 nm (Fig. 5 ). The particles were well-dispersed with minimal aggregation, which is consistent with the observations from the UV-Vis and XRD findings. The represented micrographs (Fig. 5 A-D) revealed individual particle sizes of 95.4, 92.6, 75.1, and 93.2 nm, respectively, indicating a uniform morphology with smooth surfaces. Brunauer-Emmett-Teller (BET) Surface Area Analysis The nitrogen adsorption-desorption isotherm exhibited a Type IV curve accompanied by a prominent hysteresis loop (Fig. 6 A), which is characteristic of mesoporous materials. On the basis of multi-point BET plot (Fig. 6 B), the linear region between 0.05 and 0.30 P/P₀ yielded a slope of 131.397 g⁻¹ and an intercept of 0.1228 g⁻¹ (R² = 0.99907), corresponding to a BET constant (C) of 11.70. The calculated BET surface area was 24.24 m²/g. Additionally, the Barrett-Joyner-Halenda (BJH) analysis yielded a total pore volume of 0.0555 cm³/g and an average pore radius of 4.57 nm as shown in Fig. 6 C. These results suggest that the material is suitable for enhanced surface-related applications, such as drug delivery or antimicrobial interactions. Antioxidant Assay Free Radical Scavenging Activity Compared with standard antioxidants, the biosynthesized ZnO NPs exhibited measurable antioxidant activity across all five assays, albeit with higher IC₅₀ values. As illustrated in Fig. 7 , the plotted lines represent the mean IC₅₀ values (± standard deviation) for both the ZnO NPs and the standards. The markers indicate the specific values for each assay, allowing for visual comparison of antioxidant activities across the different assays. Furthermore, the IC₅₀ values (µg/mL) are summarized in Supplementary Table 2A. The DPPH scavenging activity of the ZnO NPs yielded an IC₅₀ of 53.71 ± 1.75 for ZnO NPs, whereas it was 28.30 ± 0.75 for ascorbic acid. Similarly, for ABTS, nitric oxide, hydroxyl, and superoxide radicals, the IC₅₀ values of the ZnO NPs ranged from 56.39 to 62.88 µg/mL, which were significantly greater than those of respective standard controls ( p < 0.001). Statistical Significance Testing Independent sample t -tests confirmed statistically significant differences in the IC₅₀ values between ZnO NPs and standard antioxidants across all the assays (Supplementary Table 2B), with p < 0.001 for each comparison. Effect Size (Cohen’s d ) Effect size calculations revealed Cohen’s d values exceeding 17 for all comparisons (Supplementary Table 2C), indicating extremely large effect sizes. These results emphasize not only the statistical significance but also the practical significance of the differences in antioxidant potential. Minimum Inhibitory Concentration (MIC) The biosynthesized ZnO NPs exhibited strong antifungal activity against N. incurvata TI03. The minimum inhibitory concentration (MIC) of the ZnO NPs was 0.96 µg/mL, which was comparable to that of fluconazole (0.99 µg/mL), miconazole (0.98 µg/mL), and clotrimazole (0.99 µg/mL), and marginally lower than that of itraconazole (0.97 µg/mL) (Supplementary Table 3A). Statistical analysis via one-way ANOVA followed by Tukey's post-hoc test confirmed that the observed differences among the agents were not statistically significant ( p > 0.05), suggesting that the antifungal efficacy ZnO NPs is comparable to that of established azole antifungals (Supplementary Table 3B). Notably, the geometric mean (GM) MIC for the ZnO NPs was 0.43 µg/mL, which falls well within the therapeutic range. This value was statistically comparable to that of fluconazole (GM: 0.27 µg/mL; p = 0.696), clotrimazole (GM = 0.75 µg/mL; p = 0.523 and miconazole (GM: 0.33 µg/mL; p = 0.826), but significantly lower than that of itraconazole (GM: 0.90 µg/mL; p = 0.048). Furthermore, ZnO NPs exhibited a clear dose-dependent inhibition profile, with MIC₅₀ of 0.86 µg/mL and an MIC₂X of 1.07 µg/mL (Supplementary Table 3B), supporting their concentration-dependent antifungal activity. Agar Well Diffusion In agar well diffusion assays, ZnO NPs at 32 µg/mL produced an inhibition zone of 11.33 ± 0.58 mm. This value was statistically comparable to that of fluconazole (11.67 ± 0.58 mm p = 0.512) and itraconazole (11.67 ± 0.58 mm; p = 0.512), slightly lower than that of miconazole (12.33 ± 0.58 mm; p = 0.102), and greater than that of clotrimazole (10.33 ± 0.58 mm; p = 0.102) (Supplementary Table 3C). Discussion The phenomenon of microbial antagonism, in which one microorganism inhibits or eradicates another, often through the production of bioactive metabolites, has significantly contributed to the discovery of novel therapeutics from microbial sources (Pranav et al., 2021 ). While bacterial antagonism has been extensively researched, fungal-fungal antagonism remains relatively underexplored, particularly in the context of nanoparticle-based therapeutic strategies. Although Candida species are known predominantly for their pathogenic potential (Pfaller & Diekema, 2007 ), recent studies have demonstrated their role in nanoparticle biosynthesis, such as the production of bismuth nanoparticles (Bi-NPs) by C. albicans and C. glabrata , which simultaneously exhibited antifungal activity against the respective strains (Zanganeh et al., 2025 ). While that study addressed the self-inhibitory potential of Candida species, the interfungal antagonism through the biosynthesis of nanoparticles was not investigated. Expanding on this concept, we employed the approach of utilizing C. albicans , an opportunistic pathogenic yeast, as a biological system for the green synthesis of antifungal ZnO nanoparticles against N. incurvata , a pathogenic dermatophytic filamentous fungus. The traditional morphological assays (germ-tube test, LPCB staining) performed for the presumptive identification of C. albicans CA26 were validated by ITS-region sequencing, which remains the gold standard for fungal taxonomy and was employed to confirm morphological observations (Stover & Cavalcanti, 2009 ). Similarly, the isolation of N. incurvata TI03 from dermatophytosis samples reflects emerging epidemiological trends in dermatophytic infections in India, with a shift from Trichophyton dominance to other genera (Nenoff et al., 2019 ; Pchelin et al., 2019 ). In this study, the decision to select representative isolates on the basis of consistent diagnostic morphological features for ITS sequencing and nanomaterial synthesis aligns with best practices in microbial prospecting, where strain redundancy is minimized when intraspecies variation is not under investigation (Mohammed Fayaz et al., 2009 ; Narayanan & Sakthivel, 2010 ). Furthermore, the biosynthetic potential of C. albicans CA26 and the antifungal potential its ZnO NPs, can be interpreted as strain-specific rather than species-wide, providing a basis for exploring broader species-level capabilities in future studies. The SPR peak observed at 361.75 nm closely aligns with previously reported studies, where biogenic ZnO NPs exhibited SPR peaks within the 350–380 nm range, indicating nanoscale dimensions and minimal aggregation (Narendra Kumar et al., 2019 ; Vaseem et al., 2009). The absence of secondary absorbance peaks in the 400–900 nm range further confirms high colloidal purity and stability, both of which are critical traits for biomedical applications (Sirelkhatim et al., 2015b). In contrast, commercial ZnO exhibited a redshifted SPR peak (367 nm), indicating larger particle size and greater degree of agglomeration, thereby underscoring the advantages of green synthesis in the production of structurally uniform, biocompatible nanoparticles that are well-suited for antifungal applications. FTIR analysis revealed the presence of fungal-derived functional groups such as hydroxyl, amine, and carbonyl groups, capping and stabilizing the surface of the ZnO nanoparticles. These organic moieties play dual roles in reducing Zn²⁺ metal ions during synthesis and preventing agglomeration, enabling controlled nanoparticle formation (Iravani, 2011b). Similar biomolecule-assisted syntheses have been reported in other fungal species, supporting the generalizability of this approach (Gurunathan et al., 2009 ). The colloidal stability is further supported by FTIR data, which revealed that hydrophilic surface groups contribute to enhanced aqueous dispersibility, a critical factor in biomedical formulations (Kolodziejczak-Radzimska & Jesionowski, 2014a ). Furthermore, the sharp diffraction peaks in the XRD pattern and the estimated crystallite size of ~ 30 nm suggest well-crystallized ZnO nanostructures. The crystallite sizes of NPs within this range have been reported to be suitable for antimicrobial applications (Al-Bedairy & Habeeb Alshamsi, 2018a , 2018b ; Saravanan et al., 2018 ). On the basis of previously reported studies, ZnO NPs within this range exhibit enhanced antimicrobial activity, likely due to their high surface area-to-volume ratio, which facilitates better interaction with microbial cells and promotes increased reactive oxygen species (ROS) generation upon contact (Ali et al., 2016 ; Raghupathi et al., 2011 ). TEM revealed the welldispersed, rod-to-bar-shaped morphology and monodispersity of the ZnO NPs, which have been suggested to be efficient in surface interactions with microbial membranes (Kolodziejczak-Radzimska & Jesionowski, 2014b ). Compared with spherical equivalents, nanorod-shaped ZnO has greater penetration and cellular contact, resulting in better antifungal activity. (Rezaei et al., 2024 ; Soliman et al., 2022 ). Similarly, the BET surface area of 24.24 m²/g, together with the mesoporous structure revealed by Type IV isotherm and BJH analysis, indicates greater surface reactivity. This porous design promotes the loading and release of bioactive chemicals and enhances contact with fungal cell membranes. (Agarwal et al., 2017). Previous research has demonstrated that mesoporous ZnO nanoparticles accelerate ROS production and promote membrane breakdown in fungal infections. (Anbuvannan et al., 2015 ; B. N. Singh et al., 2014 ; Tiwari et al., 2024 ). The C. albicans CA26 biosynthesized ZnO NPs demonstrated potent antifungal activity against N. incurvata TI03. The MIC values were statistically comparable to those of conventional azole drugs, with no significant differences observed in most comparisons. Despite the comparable efficacy, the geometric mean MIC of the ZnO NPs was lower than that of itraconazole, suggesting superior efficacy under certain conditions. These findings were supported by agar diffusion assays, where ZnO NPs produced inhibition zones similar to those of fluconazole and itraconazole. Importantly, despite their similar MIC values, ZnO NPs exhibit multiple translational advantages. Our previous findings revealed that ZnO NPs suppress fungal growth through multiple mechanisms, including ROS production, membrane disruption, and interference with protein and metabolic pathways. (Nxumalo et al., 2024 ; Selokar & Thakare, 2024 ). This multimodal mechanism reduces the likelihood of resistance development, which is a growing concern with azole-based treatments (Burmester et al., 2023 ; Ghannoum, 2016 ; Hossain et al., 2022 ). Additionally, the ZnO NPs biosynthesized by CA26 showed moderate but consistent antioxidant efficacy across five free radical scavenging experiments, despite exhibiting higher IC₅₀ values than standard antioxidants. The exceptionally substantial effect sizes (Cohen’s d > 17) across all the comparisons highlight their biological relevance and reproducibility. This antioxidative potential can be attributed to the semiconducting properties of ZnO, which facilitate electron transfer to neutralize reactive species, as well as the presence of fungal-derived capping biomolecules known to participate in radical scavenging reactions (Kumar et al., 2022 ; Rodríguez et al., 2017 ; Yamaguchi, 1975 ). Oxidative stress plays a key role in skin inflammation, aging and secondary damage associated with fungal infections (Briganti & Picardo, 2003 ; Rinnerthaler et al., 2015 ). Therefore, the topical application of antioxidant agents, especially those with dual antifungal and radical-scavenging properties can offer enhanced skin protection by counteracting reactive oxygen and nitrogen species (ROS/RNS)-mediated cellular damage. While ZnO is already a well-established skin protectant (Wiegand et al., 2013 ), the added antioxidant functionality of these biosynthesized ZnO NPs supports their potential as multifunctional agents in dermatological formulations aimed at both controlling infection and alleviating oxidative stress. The biosynthesis of ZnO NPs using C. albicans CA26 offers environmental and clinical benefits, including eco-friendliness and reproducibility (Agarwal et al., 2017b ; Cruz et al., 2020 ; Mousavi et al., 2018 ). The low standard deviation of ZnO NPs across replicates further supports the synthesis consistency. Moreover, ZnO is already extensively used in dermatological formulations because of its skin compatibility and anti-inflammatory and UV-protective properties (Akintelu & Folorunso, 2020 ; Al-Bedairy & Habeeb Alshamsi, 2018a ; Osterwalder et al., 2024 ; Smijs & Pavel, 2011 ). Given the emergence of azole-resistant dermatophytes, including N. incurvata (Gamal et al., 2023 ; Havlickova et al., 2008 ; Jia et al., 2023 ; Seebacher et al., 2008 ), the comparable antifungal activity and favorable safety profile of ZnO NPs make them excellent candidates for incorporation into topical formulations such as creams, sprays, and gels (Choudhury et al., 2017 ; Król et al., 2017 ; Lansdown et al., 2007 ; Prajapati et al., 2024 ). This green synthesis approach not only offers an environmentally friendly alternative, but also produces nanoparticles with functional surface coatings derived from natural biomolecules, hence increasing their biocompatibility and therapeutic potential (Agarwal et al., 2017b ; Upadhyay et al., n.d.; Velusamy et al., 2016 ). In summary, the ZnO nanoparticles biosynthesized by C. albicans CA26 exhibit dual antifungal and antioxidant properties, positioning them as promising multifunctional agents for dermatological applications. However, as these findings are based on in-vitro assays, further in-vivo studies and cytotoxicity evaluations are essential to fully establish their clinical safety and efficacy, which paves way for future translational research and therapeutic development. Overall, these findings demonstrate that C. albicans CA26 derived ZnO NPs are structurally and functionally optimized for antifungal activity, as evidenced by their physicochemical characteristics revealed through UV-Vis, FTIR, XRD, TEM, and BET analyses, along with their robust antifungal performance, collectively underscoring their potential as sustainable and clinically viable candidates for topical dermatological applications. Declarations Ethics approval and consent to participate The Dean of Indira Gandhi Government Medical College and Hospital, Nagpur, granted formal approval for clinical sample collection in this study, as communicated through the Head and Research Guide of the Department of Molecular Biology and Genetic Engineering, Rashtrasant Tukadoji Maharaj Nagpur University (Ref. No. MB&GE/12/2024, dated 15 April 2024). The proposal was reviewed and approved by the research supervisor, the departmental head, and the Dean of the Faculty of Science and Technology, RTM Nagpur University. The study involved only non-invasive, anonymized clinical specimens (oropharyngeal swabs and skin scrapings), and no personal identifiers or sensitive patient information was collected. All procedures were carried out under relevant institutional guidelines, ethical standards, and good clinical laboratory practices. Written informed consent was obtained from all participants prior to sample collection. Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Funding YNS, acknowledges the financial support received from the Mahatma Jyotiba Phule Research & Training Institute (MAHAJYOTI), Autonomous Institute under the Other Backward Class Bahujan Welfare Department, Govt. of Maharashtra (India), in the form of a Senior Research Fellowship (SRF) (Fellowship2022_1008). Author contributions YNS, PSP, and RUT designed the experiments; YNS and PSP performed the experimental work and wrote the initial draft of the manuscript. RUT contributed to manuscript revision and editing. All the authors read and approved the final version of the manuscript. Acknowledgement The authors acknowledge the Department of Molecular Biology and Genetic Engineering, RTM Nagpur University, Nagpur for providing research facilities and support. We also extend our sincere thanks to the medical staff and administrative teams of the Government Ayurved College and Hospital, Government Dental College and Hospital, and Indira Gandhi Government Medical College and Hospital, Nagpur, for their valuable assistance throughout the study. Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its supplementary files. Additional datasets and samples used or analyzed in this study are available from the corresponding author upon reasonable request. References Abarenkov K, Nilsson RH, Larsson KH, Taylor AFS, May TW, Frøslev TG, Pawlowska J, Lindahl B, Põldmaa K, Truong C, Vu D, Hosoya T, Niskanen T, Piirmann T, Ivanov F, Zirk A, Peterson M, Cheeke TE, Ishigami Y, Kõljalg U (2024a) The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered. 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Nanatechnol Sci Appl 4(1):95–112. https://doi.org/10.2147/NSA.S19419 Soliman WE, Elsewedy HS, Younis NS, Shinu P, Elsawy LE, Ramadan HA (2022) Evaluating Antimicrobial Activity and Wound Healing Effect of Rod-Shaped Nanoparticles. Polym 2022 14(13):2637. https://doi.org/10.3390/POLYM14132637 . 14 Stover NA, Cavalcanti ARO (2009) Using NCBI BLAST. Curr Protocols Essent Lab Techniques 1(1). https://doi.org/10.1002/9780470089941.ET1101S01 . 11.1.1–11.1.36 Symoens F, Jousson O, Planard C, Fratti M, Staib P, Mignon B, Monod M (2011) Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species complex. Int J Med Microbiol 301(3):260–266. https://doi.org/10.1016/J.IJMM.2010.06.001 Tiwari AK, Jha S, Tripathi SK, Shukla R, Awasthi RR, Bhardwaj AK, Singh AK, Dikshit A (2024) Spectroscopic investigations of green synthesized zinc oxide nanoparticles (ZnO NPs): antioxidant and antibacterial activity. Discover Appl Sci 6(8):1–17. https://doi.org/10.1007/S42452-024-06049-Z/TABLES/5 Tran XT, Bien TTL, Tran T, Van, Nguyen TTT (2024) Biosynthesis of ZnO nanoparticles using aqueous extracts of Eclipta prostrata and Piper longum: characterization and assessment of their antioxidant, antibacterial, and photocatalytic properties. Nanoscale Adv 6(19):4885–4899. https://doi.org/10.1039/D4NA00326H Uhrlaß S, Mey S, Storch S, Wittig F, Koch D, Krüger C, Nenoff P (2021) Nannizzia incurvata as a rare cause of favus and tinea corporis in Cambodia and Vietnam. Indian J Dermatol Venereol Leprol 87(4):516–521. https://doi.org/10.4103/IJDVL.IJDVL_954_18 Upadhyay PK, Jain K, Shrivastav V, Sharma AK, S., Sharma R (n.d.). Green and chemically synthesized ZnO nanoparticles: A comparative study . https://doi.org/10.1088/1757-899X/798/1/012025 Vaseem M, Umar A, oxide nanostructures YH-M (2010) & undefined. (2009). ZnO nanoparticles: growth, properties, and applications. Researchgate.Net . https://www.researchgate.net/profile/Nadir-Habubi/post/Are_ZnO_nanoparticles_better_than_SiO2_nanoparticles_in_terms_of_properties_and_cost /attachment/5eedfd1b4c18f900012ab7ec/AS%3A904475329982470% 401592655131917/download/1.pdf Velusamy P, Kumar GV, Jeyanthi V, Das J, Pachaiappan R (2016) Bio-inspired green nanoparticles: synthesis, mechanism, and antibacterial application. Toxicol Res 32(2):95–102. https://doi.org/10.5487/tr.2016.32.2.095 Vijayaram S, Razafindralambo H, Sun YZ, Vasantharaj S, Ghafarifarsani H, Hoseinifar SH, Raeeszadeh M (2023) Applications of Green Synthesized Metal Nanoparticles - a Review. Biol Trace Elem Res 2023 202:1(1):360–386. https://doi.org/10.1007/S12011-023-03645-9 . 202 Weitzman I, Summerbell RC (1995a) The dermatophytes. Clin Microbiol Rev 8(2):240–259. https://doi.org/10.1128/CMR.8.2.240 Weitzman I, Summerbell RC (1995b) The dermatophytes. Clin Microbiol Rev 8(2):240. https://doi.org/10.1128/CMR.8.2.240 Wiegand C, Hipler UC, Boldt S, Strehle J, Wollina U (2013) Skin-protective effects of a zinc oxide-functionalized textile and its relevance for atopic dermatitis. Clin Cosmet Invest Dermatology 6:115. https://doi.org/10.2147/CCID.S44865 Williams D, diseases ML-O (2000) & undefined. (n.d.). Oral Microbiology: Isolation and identification of candida from the oral cavity. Wiley Online Library . Retrieved July 9, 2024, from https://onlinelibrary.wiley.com/doi/abs/ 10.1111/j.1601-0825.2000.tb00314.x Yamaguchi H (1975) Control of dimorphism in Candida albicans by zinc: effect on cell morphology and composition. J Gen Microbiol 86(2):370–372. https://doi.org/10.1099/00221287-86-2-370 Zanganeh E, Moghbeli M, Zarrinfar H, Sadeghian H (2025) Green Synthesis of Bismuth Nanoparticles Using Candida albicans and C. glabrata . along Evaluation Their Antifungal Eff BioNanoScience 15(1):1–14. https://doi.org/10.1007/s12668-025-01815-8 Tables Table 1 is available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1. Comparative UV-Visible absorbance values for the biosynthesized ZnO nanoparticles and commercial ZnO powder across different selected wavelengths. SupplementaryTable.2.xlsx Supplementary Table 2. Antioxidant activity of the biosynthesized ZnO nanoparticles and standard antioxidants. (A) IC₅₀ values across five radical scavenging assays, reported as the mean ± standard deviation (n = 3). (B) Statistical comparisons of IC₅₀ values between ZnO NPs and reference antioxidants via independent samples t-tests (df = 2), all of which were significant at p 17) across all assays. SupplementaryTable.3.xlsx Supplementary Table 3. Antifungal efficacy of the biosynthesized ZnO nanoparticles against N. incurvata TI03. (A) Raw OD₆₀₀ values, MIC, MIC₅₀, MIC ¼ , MIC₂X, MIC range, geometric mean (GM), and related statistics for ZnO NPs and commercial antifungals. (B) Statistical comparisons of the MIC, GM MIC, and zone of inhibition using one-way ANOVA with Tukey’s post-hoc test. All the data are based on triplicate experiments. (C) Pairwise comparison of the mean zone of inhibition diameter (mm) between the biosynthesized ZnO nanoparticles (32 µg/mL) and commercial antifungal agents against N. incurvata TI03. The data represent mean ± standard deviation (SD) of triplicate samples. Table1.xlsx Table 1. FTIR peak assignments for C. albicans CA26-derived ZnO nanoparticles. Major FTIR absorption bands were observed for the biosynthesized ZnO NPs, indicating the presence of fungal-derived functional groups and ZnO lattice vibrations. SupplementaryFig.1.jpg Supplementary Fig 1. Lactophenol Cotton Blue staining of C. albicans CA26 (A) showing oval budding yeast cells and pseudohyphae, and N. incurvata TI03 (B, C) displaying multicellular macroconidia and microconidia, confirming species-specific morphological characteristics. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7164738","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487838622,"identity":"b5d982b5-4b2d-4bc3-82a3-e89ef558780f","order_by":0,"name":"Yajurved N Selokar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACNjAqYJAzAHMNLIjVYmBgbMDADNIiQaxFBgaJG8BaGIjQwid2+NiDDwZ/0rez9x/d8KNAgoG/vTsBvxXSaemGMwwMcnf2HGa72QN0mMSZsxsIaMkxk+YBatlwI5ntBg9Qi4FELiEt+d9AWtINgFpu/iFOSw4bSEsCSMttIm1JMwf6xdhww5nDZrdlDCR4CPpFfnbyswcfKuTkDY43Prv55o+NHH97L34tGICHNOWjYBSMglEwCrACAKwQPo1PTWKPAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9408-9891","institution":"Department of Molecular Biology and Genetic Engineering, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440033, India","correspondingAuthor":true,"prefix":"","firstName":"Yajurved","middleName":"N","lastName":"Selokar","suffix":""},{"id":487838623,"identity":"f3feb743-1c43-468a-bb43-3eae6e614df0","order_by":1,"name":"Parameswaran Sree Pranav","email":"","orcid":"https://orcid.org/0000-0002-6696-7973","institution":"Department of Molecular Biology and Genetic Engineering, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440033, India","correspondingAuthor":false,"prefix":"","firstName":"Parameswaran","middleName":"Sree","lastName":"Pranav","suffix":""},{"id":487838624,"identity":"9e1e07ec-b792-45b3-abdf-579805e8135a","order_by":2,"name":"Rakesh U Thakare","email":"","orcid":"https://orcid.org/0000-0003-1338-0834","institution":"Department of Microbiology, Yashwantrao Chavan College, Lakhandur, District Bhandara - 441803, India","correspondingAuthor":false,"prefix":"","firstName":"Rakesh","middleName":"U","lastName":"Thakare","suffix":""}],"badges":[],"createdAt":"2025-07-19 13:26:09","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7164738/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7164738/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87386492,"identity":"4b824916-095b-4d90-ad87-c73feee53ca8","added_by":"auto","created_at":"2025-07-23 08:58:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":533293,"visible":true,"origin":"","legend":"\u003cp\u003eITS-based phylogenetic analysis confirming the species identity of the clinical isolates. (A) \u003cem\u003eC. albicans\u003c/em\u003e CA26 clustered closely with the \u0026nbsp;reference strains AUMC 10189 and AUMC 8996, showing minimal divergence (0-0.001 substitutions/site). (B) \u003cem\u003eN. incurvata\u003c/em\u003eTI03 forms a distinct clade with \u003cem\u003eN. incurvata\u003c/em\u003e references. The ITS-based phylogenetic trees were constructed using the neighbor-joining method with the maximum composite likelihood model (MUSCLE alignment, 1000 bootstrap replicates) in MEGA v12, midpoint-rooted, and visualized in iTOL v6.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/df7e188b991f9f02fa708425.jpg"},{"id":87384590,"identity":"e68a6d78-0c9d-462b-baff-adfb233abbd2","added_by":"auto","created_at":"2025-07-23 08:50:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":274104,"visible":true,"origin":"","legend":"\u003cp\u003e(A) UV-Vis absorption spectra of the biosynthesized ZnO nanoparticles showing a sharp surface plasmon resonance (SPR) peak at 361.75 nm, indicating their nanoscale size and high colloidal stability. (B) Commercial ZnO powder with a broader peak at 367 nm, suggesting a larger particle size and aggregation.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/245134100daed24804caa7db.jpg"},{"id":87383306,"identity":"7972eb28-20c0-42ef-b17a-340e9eb3ac21","added_by":"auto","created_at":"2025-07-23 08:42:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":65656,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of Sabouraud dextrose broth (SDB), the biosynthesized ZnO nanoparticles (ZnO), and the ZnO+SDB mixture. The characteristic peaks indicate the presence of fungal biomolecules (O-H, C-H, C=O, and N-H groups) capping the ZnO NPs and confirm Zn-O stretching vibrations, support nanoparticle formation and stabilization.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/7585ec1c98795bf7c4caf940.jpg"},{"id":87384594,"identity":"81e223d7-fe7a-42d6-9c6e-fb6d573e9d0f","added_by":"auto","created_at":"2025-07-23 08:50:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":407985,"visible":true,"origin":"","legend":"\u003cp\u003e(A) X-ray diffraction (XRD) pattern of the biosynthesized ZnO nanoparticles showing distinct peaks at 2θ values corresponding to the (100), (002), (101), (102), and (110) planes of hexagonal wurtzite ZnO (JCPDS No. 36-1451), confirming high crystallinity and phase purity. (B) Three-dimensional crystallographic visualization of the biosynthesized ZnO nanoparticles generated via VESTA software, confirming that hexagonal wurtzite crystal structure is consistent with theXRD analysis.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/8a2965988c54d884e802747a.jpg"},{"id":87383319,"identity":"e0554499-2ea0-46f5-996d-bdfa4194da0c","added_by":"auto","created_at":"2025-07-23 08:42:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1414421,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy (TEM) images of the biosynthesized ZnO nanoparticles revealed rod-to-bar shaped morphology with particle sizes ranging from 75 to 99 nm, indicating a uniform shape, smooth surface, and minimal aggregation.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/b7b0787967ed892e5d9c611a.jpg"},{"id":87383316,"identity":"95de04a0-e2fc-42f1-95a6-d2a3ec0a0633","added_by":"auto","created_at":"2025-07-23 08:42:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2108848,"visible":true,"origin":"","legend":"\u003cp\u003eSurface area and porosity characterization of the biosynthesized ZnO nanoparticles. (A) Nitrogen adsorption-desorption isotherm showing a Type IV curve with hysteresis loop, indicating mesoporosity. (B) Multi-point BET plot used to determine the surface area; the linear region (P/P₀ = 0.05-0.30) yielded a surface area of 24.24 m²/g. (C) BJH desorption pore size distribution curve indicating an average pore radius of 4.57 nm and a total pore volume of 0.0555 cm³/g.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/b3c4829ff66aee4c2cc8041c.jpg"},{"id":87386494,"identity":"e6c2f166-67b2-48b8-bce5-dc15f4e92693","added_by":"auto","created_at":"2025-07-23 08:58:26","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":156744,"visible":true,"origin":"","legend":"\u003cp\u003eComparative IC₅₀ values (mean ± SD) of the biosynthesized ZnO nanoparticles and standard antioxidants across five radical scavenging assays (DPPH, ABTS, nitric oxide, hydroxyl, and superoxide). The markers represent individual IC₅₀ values for each assay, highlighting the relative antioxidant efficacy.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/f0fb9640a248d7ba00ff06c4.jpg"},{"id":87387296,"identity":"11baa03b-1db0-4546-9c5a-2a8c838c0019","added_by":"auto","created_at":"2025-07-23 09:06:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6042401,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/6819d595-d984-4c21-8556-eb0fdf03e36b.pdf"},{"id":87383304,"identity":"83df83b7-42c2-415d-a9a9-db3b9a2bd03a","added_by":"auto","created_at":"2025-07-23 08:42:26","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1. \u003c/strong\u003eComparative UV-Visible absorbance values for the biosynthesized ZnO nanoparticles and commercial ZnO powder across different selected wavelengths.\u003c/p\u003e","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/dcfe55315019942f39046c0e.xlsx"},{"id":87383303,"identity":"15f4e738-886e-4da5-948b-faa765604569","added_by":"auto","created_at":"2025-07-23 08:42:26","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2. \u003c/strong\u003eAntioxidant activity of the biosynthesized ZnO nanoparticles and standard antioxidants. (A) IC₅₀ values across five radical scavenging assays, reported as the mean ± standard deviation (n = 3). (B) Statistical comparisons of IC₅₀ values between ZnO NPs and reference antioxidants via independent samples t-tests (df = 2), all of which were significant at p \u0026lt; 0.001. (C) Cohen’s d effect size analysis, indicating extremely large differences (Cohen’s d \u0026gt; 17) across all assays.\u003c/p\u003e","description":"","filename":"SupplementaryTable.2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/1649770aa3c9c744c367f3a1.xlsx"},{"id":87384592,"identity":"04c2a9e3-a71b-4694-99ac-648fa60ff8c5","added_by":"auto","created_at":"2025-07-23 08:50:26","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 3.\u003c/strong\u003e Antifungal efficacy of the biosynthesized ZnO nanoparticles against \u003cem\u003eN. incurvata\u003c/em\u003e TI03. (A) Raw OD₆₀₀ values, MIC, MIC₅₀, MIC\u003csub\u003e¼\u003c/sub\u003e, MIC₂X, MIC range, geometric mean (GM), and related statistics for ZnO NPs and commercial antifungals. (B) Statistical comparisons of the MIC, GM MIC, and zone of inhibition using one-way ANOVA with Tukey’s post-hoc test. All the data are based on triplicate experiments. (C) Pairwise comparison of the mean zone of inhibition diameter (mm) between the biosynthesized ZnO nanoparticles (32 µg/mL) and commercial antifungal agents against \u003cem\u003eN. incurvata \u003c/em\u003eTI03. The data represent mean ± standard deviation (SD) of triplicate samples.\u003c/p\u003e","description":"","filename":"SupplementaryTable.3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/de9f0e3010f6b37468f01d39.xlsx"},{"id":87384589,"identity":"a8b1d4ba-9001-47cb-b137-3e5eef1e45a1","added_by":"auto","created_at":"2025-07-23 08:50:26","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. \u003c/strong\u003eFTIR peak assignments for \u003cem\u003eC. albicans\u003c/em\u003e CA26-derived ZnO nanoparticles. Major FTIR absorption bands were observed for the biosynthesized ZnO NPs, indicating the presence of fungal-derived functional groups and ZnO lattice vibrations.\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/a2b10bc96cd7cceff241c1f6.xlsx"},{"id":87383315,"identity":"332108c0-8d19-4d65-9e61-23ca1525bab7","added_by":"auto","created_at":"2025-07-23 08:42:26","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":738662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig 1. \u003c/strong\u003eLactophenol Cotton Blue staining of \u003cem\u003eC. albicans\u003c/em\u003eCA26 (A) showing oval budding yeast cells and pseudohyphae, and \u003cem\u003eN. incurvata\u003c/em\u003eTI03 (B, C) displaying multicellular macroconidia and microconidia, confirming species-specific morphological characteristics.\u003c/p\u003e","description":"","filename":"SupplementaryFig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7164738/v1/669a97ca59af0a5ceccc42b7.jpg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eGreen Synthesis of Zinc Oxide Nanoparticles by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCandida albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e CA26 with Antioxidant and Antifungal Activity Against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNannizzia incurvata\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDermatophytosis remains a major global health concern, affecting an estimated 20\u0026ndash;25% of the world's population (Brasch, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Weitzman \u0026amp; Summerbell, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e1995a\u003c/span\u003e). Resolving this superficial fungal infection can be difficult, as it often recurs and persists, especially in densely populated environments or circumstances involving close human contact. Notably, \u003cem\u003eNannizzia incurvata\u003c/em\u003e, a fungal species that thrives on human skin, has emerged as a concern in recent years because of its association with chronic, nonhealing infections, as well as its increased ability to resist traditional antifungal treatments (Kruithoff et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Uhrla\u0026szlig; et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA number of issues limit the effectiveness of antifungal medications, such as azoles and allylamines, even though these are still the primary therapeutic options. These include the growing problem of fungal strains developing resistance to these drugs, the need for prolonged treatment courses, the risk for systemic toxicity, and exorbitant costs (Burmester et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ghannoum, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Symoens et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Terbinafine resistance has become a major growing concern and this resistance has developed due to a mutation in the squalene epoxidase (SQLE) gene, potentially undermining its therapeutic effect. Furthermore, the increased activity of efflux pumps in fungal cells can profoundly impair the efficacy of terbinafine, making it a less trustworthy therapeutic option (Pashootan et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The complexity of antifungal resistance and treatment limitations highlights the urgent need for innovative approaches that strike a balance between environmental sustainability, patient safety, and effective infection control (Ebert et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne promising approach to the development of novel antibacterial agents is nanobiotechnology. Zinc oxide nanoparticles (ZnO NPs) are potent antibacterial agents with minimal cytotoxicity, high biocompatibility, and broad-spectrum effectiveness against a variety of bacterial pathogens (Ijaz et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jha et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Its antifungal activity is achieved through mechanisms such as reactive oxygen species (ROS) production, fungal membrane disruption, and the inhibition of major enzyme pathways (Jhawat et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nxumalo et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sirelkhatim et al., 2015). Nevertheless, conventional synthesis methods for ZnO NPs often rely on hazardous chemicals and high energy inputs, making them environmentally and biologically less desirable (Banjara et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Benitez-Salazar et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGreen synthesis techniques offer a more environmentally friendly option, as they stabilize nanoparticles and reduce metal ions via the use of biological systems such as bacteria, fungi, yeasts, and plants. These organisms normally contribute to the synthesis of nanoparticles by providing natural reducing and capping agents, such as enzymes and secondary metabolites (Iravani, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e; Vijayaram et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While plant and bacterial-mediated synthesis have been widely researched, the potential of yeast-based synthesis, particularly with \u003cem\u003eC. albicans\u003c/em\u003e, has received less attention (Narayanan \u0026amp; Sakthivel, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Although generally known as a commensal yeast and opportunistic pathogen, \u003cem\u003eC. albicans\u003c/em\u003e possesses useful biotechnological traits, including high metal tolerance, simple growth, and the ability to release extracellular proteins that facilitate nanoparticle formation (Rodr\u0026iacute;guez et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; R. Sharma et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Its potential as a workable microbial nanofactory has been demonstrated through its effective usage in the synthesis of nanoparticles like iron and silver oxides (Abed et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Gupta \u0026amp; Verma, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jalal et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Rahimi et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Compared with certain plant or bacteria-based approaches, \u003cem\u003eC. albicans\u003c/em\u003e-mediated synthesis yields nanoparticles through a simple, costeffective, and ecofriendly process, avoiding the use of hazardous chemical waste and high energy inputs. This approach aligns well with the concepts of sustainable nanomedicine and yields nanoparticles that exhibit strong antimicrobial efficacy in vitro (Abed et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Jalal et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition to their antifungal characteristics, ZnO nanoparticles possess significant antioxidant properties, which are important in reducing the oxidative stress associated with dermatological infections (Tran et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Green-synthesized ZnO NPs have exhibited substantial scavenging action against free radicals in conventional tests such as DPPH and ABTS, indicating promise for skin protection and wound healing by decreasing ROS-mediated tissue damage (Abdelghany et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kr\u0026oacute;l et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tiwari et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This dual function increases their appeal as a treatment alternative for managing resistant dermatophytosis with inflammatory responses.\u003c/p\u003e\u003cp\u003eRegardless of these benefits, the sustainable extracellular synthesis of ZnO nanoparticles utilizing \u003cem\u003eC. albicans\u003c/em\u003e and their application against dermatophyte strains such as \u003cem\u003eN. incurvata\u003c/em\u003e remain largely unexplored. This study addresses that gap by detailing the green synthesis of ZnO nanoparticles using a clinical isolate of \u003cem\u003eC. albicans\u003c/em\u003e (CA26), followed by comprehensive physicochemical characterization and antifungal evaluation against a dermatophyte isolate (TI03), which was later confirmed as \u003cem\u003eN. incurvata\u003c/em\u003e. To overcome the limitations of current antifungal treatments, this work aims to lay the foundation for a nanoparticle-based therapeutic platform that is scalable, biocompatible, and environmentally sustainable.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eSample Collection and Culture Conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll clinical isolates of \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eN. incurvata\u003c/em\u003e were obtained from patients clinically diagnosed with candidiasis and dermatophytosis, respectively, following written approval for sample collection from the Dean, Indira Gandhi Government Medical College \u0026amp; Hospital, Nagpur, India (Ref. No. 390; 8 October 2022) and from the Head, Department of Molecular Biology \u0026amp; Genetic Engineering, Rashtrasant Tukadoji Maharaj Nagpur University, India (Letter No. MB\u0026amp;GE/12; 15 April 2024). Written informed consent was obtained from all participants. Oropharyngeal swab samples were collected from patients exhibiting clinical signs of oral candidiasis. For \u003cem\u003eC. albicans\u003c/em\u003e, swabs were transported aseptically in sterile containers at 4\u0026deg;C and processed immediately for microbiological evaluation, as per established protocols (Begum \u0026amp; Kumar, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gamal et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shalaby et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Williams et al., n.d.). The samples were inoculated onto Sabouraud dextrose agar (SDA) supplemented with chloramphenicol (50 \u0026micro;g/mL) and incubated at 37\u0026deg;C for 48\u0026ndash;72 h. Colonies displaying creamy, smooth morphology were subjected to Gram staining and germ tube testing in human serum at 37\u0026deg;C for 2\u0026ndash;3 h. Isolates demonstrating germ tube formation were provisionally identified as \u003cem\u003eC. albicans\u003c/em\u003e and further confirmed via lactophenol cotton blue (LPCB) staining for characteristic morphological features such as budding yeast cells and pseudohyphae (Jalal et al., 2018b; Kim et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Mallya \u0026amp; Mallya, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among the other \u003cem\u003eC. albicans\u003c/em\u003e isolates, a single isolate (CA26) consistently exhibiting typical morphological characteristics and germ tube formation, was selected as a representative strain for molecular characterization.\u003c/p\u003e\u003cp\u003eFor dermatophyte isolation, skin scrapings were treated with 10% potassium hydroxide (KOH) and microscopically examined for hyphal elements. KOH-positive samples were cultured on Dermatophyte Test Medium (DTM) and SDA supplemented with chloramphenicol (50 \u0026micro;g/mL) and cycloheximide (500 \u0026micro;g/mL), and incubated at 28\u0026deg;C for 7\u0026ndash;14 days (G\u0026oacute;mez-Moyano et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gr\u0026auml;ser et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Weitzman \u0026amp; Summerbell, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e1995b\u003c/span\u003e). Colonies with a powdery, cream-colored morphology and reverse pigmentation were subcultured and analyzed through LPCB staining for macroconidia and microconidia characteristics (Kruithoff et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A representative isolate (TI03) with well-developed conidial structures was selected for molecular identification.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Characterization of\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eN. incurvata\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe genomic DNA was extracted from \u003cem\u003eC. albicans\u003c/em\u003e CA26 via the phenol-chloroform method, whereas the genomic DNA from \u003cem\u003eN. incurvata\u003c/em\u003e TI03 was extracted using the HiPurA\u0026trade; fungal DNA extraction kit (HiMedia, India), following the manufacturer\u0026rsquo;s instructions (Dabas et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Futatsuya et al., n.d.; Saghrouni et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Amplification of the internal transcribed spacer (ITS) region of rDNA was performed via the universal fungal primers ITS1 (5\u0026prime;-TCCGTAGGTGAACCTGCGG-3\u0026prime;) and ITS4 (5\u0026prime;-TCCTCCGCTTATTGATATGC-3\u0026prime;). PCR was carried out in a 25 \u0026micro;L reaction mixture containing 2\u0026times; PCR Master Mix (Thermo Fisher Scientific, USA) under the following cycling conditions: initial denaturation at 94\u0026deg;C for 5 min; 35 cycles of 94\u0026deg;C for 30 s, 55\u0026deg;C for 45 s, and 72\u0026deg;C for 1 min; and a final extension at 72\u0026deg;C for 10 min.\u003c/p\u003e\u003cp\u003eAmplicons were visualized on 1.5% agarose gel, purified, and sequenced via Sanger sequencing. The ITS sequences of \u003cem\u003eC. albicans\u003c/em\u003e CA26 and \u003cem\u003eN. incurvata\u003c/em\u003e TI03 were submitted to GenBank under accession numbers PV465594.1 and PV463161.1, respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003eITS-Based Phylogenetic Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ITS region sequences derived from \u003cem\u003eC. albicans\u003c/em\u003e CA26 and \u003cem\u003eN. incurvata\u003c/em\u003e TI03 were subjected to BLASTn (Altschul et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) for preliminary species confirmation. For phylogenetic reconstruction, each sequence was queried against UNITE data (Abarenkov et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e), and the top 20 closest matches were retrieved in FASTA format. The query and reference sequences were aligned via the MUSCLE algorithm implemented in MEGA v12 (Hall, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Phylogenetic trees were constructed using the neighbor-joining method with the maximum composite likelihood model, assuming uniform substitution rates. Branch support was assessed via 1000 bootstrap replicates in MEGA v12. The resulting trees were visualized, midpoint-rooted, and annotated using iTOL v6. (Abarenkov et al., 2024b; Letunic \u0026amp; Bork, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBiosynthesis of Zinc Oxide Nanoparticles\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA well-isolated colony of \u003cem\u003eC. albicans\u003c/em\u003e CA26 was inoculated into 100 mL of Sabouraud dextrose broth (SDB) and incubated at 30\u0026deg;C for 72 h at 150 rpm to promote the optimal secretion of reductive metabolites (Abed et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The culture was filtered through Whatman No. 1 paper to obtain a cell-free filtrate (CFF), which served as a bioreductant (Rahimi et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). For nanoparticle biosynthesis, 100 mL of 1 mM zinc nitrate hexahydrate Zn(NO₃)₂\u0026middot;6H₂O solution was mixed with an equal volume of the CFF in a sterile 250 mL Erlenmeyer flask. The pH was adjusted to 8.0 with 1 M NaOH, as alkaline conditions favour the formation of ZnO nuclei and increase the reduction efficiency. The mixture was incubated in the dark at 30\u0026deg;C for 72 h at 100 rpm to avoid photoactivation and promote gradual particle growth.\u003c/p\u003e\u003cp\u003eA visible shift from pale yellow to milky white indicated ZnO NP formation. The precipitate was collected via centrifugation (12,000 rpm, 20 min), washed three times with sterile distilled water and ethanol, and dried at 60\u0026deg;C. The final product was calcined at 500\u0026deg;C for 4 h to increase crystallinity and thermal stability. The synthesised ZnO NPs were stored in sterile, airtight vials for further characterization and bioassays.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePhysicochemical Characterization of ZnO Nanoparticles\u003c/strong\u003e\u003cp\u003eThe physicochemical, morphological, and structural properties of the biosynthesized ZnO nanoparticles were comprehensively characterized via advanced analytical techniques.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eUV-Visible Spectroscopy (UV-Vis)\u003c/strong\u003e\u003cp\u003eThe optical absorption properties of the ZnO NPs were analyzed via a Shimadzu UV-1800 double-beam spectrophotometer over a wavelength range of 200\u0026ndash;600 nm. One milligram of ZnO NPs was dispersed in 10 mL deionized water via ultrasonic treatment via using a PCI analytical ultrasonic cleaner to ensure homogeneity. The absorbance spectrum was recorded and processed via UVProbe software (Shimadzu, Japan) (S. Singh et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/b\u003e: The functional groups involved in nanoparticle stabilization were confirmed using a Thermo Scientific Nicolet iS10 spectrometer (4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm⁻\u0026sup1;). The samples were prepared by homogenizing ZnO NPs with KBr (1:100 ratio) into pellets.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eX-ray Diffraction (XRD)\u003c/strong\u003e\u003cp\u003eCrystalline structure analysis was conducted via a Shimadzu XRD-6100 diffractometer (Cu-Kα radiation, λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;) operated at 40 kV and 30 mA. Diffractograms were recorded in the 2θ range of 20\u0026deg;-80\u0026deg;. The crystallite size was estimated via the Debye-Scherrer equation, and the data were interpreted via OriginPro 8.5 software (Grace et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jayachandran et al., 2021).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy (TEM)\u003c/strong\u003e\u003cp\u003eThe morphology and particle size distribution were determined via high-resolution TEM JEOL JEM-2100, operating at 200 kV. The samples were prepared by suspending the ZnO NPs in ethanol, sonicating them, and depositing them onto carbon-coated copper grids. ImageJ software (NIH, USA) was used for particle size measurement(Maher et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBrunauer-Emmett-Teller (BET) Analysis\u003c/strong\u003e\u003cp\u003eThe surface area and porosity were estimated via a Quantachrome NOVA Station A BET analyzer. The samples were degassed at 150\u0026deg;C for 4 hours before nitrogen adsorption-desorption analysis. The data were processed using NovaWin software to calculate the pore size distribution and specific surface area.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntioxidant Assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antioxidant activity of the biosynthesized zinc oxide nanoparticles (ZnO NPs) was evaluated via five in vitro assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, 2,2\u0026prime;-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, nitric oxide (NO) radical scavenging, hydroxyl radical scavenging, and superoxide radical scavenging. These assays were performed following standard protocols with slight modifications to optimize the nanoparticle suspensions (Abdelghany et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Amalraj et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe standard antioxidants used for comparison included ascorbic acid and butylated hydroxytoluene (BHT). The scavenging activity was expressed as the percentage inhibition relative to the control. IC₅₀ values were determined via linear regression analysis from dose-response curves.\u003c/p\u003e\u003cp\u003eAll the experiments were carried out in triplicates, and the data are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Independent sample t-tests were conducted to compare the IC₅₀ values of the ZnO NPs with those of standard antioxidants. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001 was considered statistically significant. Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e effect sizes were computed to quantify the magnitude of differences between ZnO NPs and standards via the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:d\\:=\\:\\backslash\\:frac\\left\\{{M}_{1}-\\:{M}_{2}\\right\\}\\left\\{\\sqrt{\\left\\{\\backslash\\:frac\\left\\{S{D}_{1}^{2}+\\:S{D}_{2}^{2}\\right\\}\\left\\{2\\right\\}\\right\\}}\\right\\}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eEffect sizes were interpreted as small (0.2), medium (0.5), and large (\u0026ge;\u0026thinsp;0.8)(Cohen, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Daines, n.d.)\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAntifungal Activity of the Biosynthesized ZnO Nanoparticles\u003c/strong\u003e\u003cp\u003eThe antifungal efficacy of \u003cem\u003eC. albicans\u003c/em\u003e CA26-derived ZnO nanoparticles (NPs) against \u003cem\u003eN. incurvata\u003c/em\u003e TI03 was assessed via the broth microdilution assay and agar well diffusion method.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDetermination of Minimum Inhibitory Concentration (MIC)\u003c/strong\u003e\u003cp\u003eThe MIC of the ZnO nanoparticles was determined according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 guidelines. Stock solutions of ZnO NPs (64 \u0026micro;g/mL) were prepared in sterile distilled water and serially diluted (0.03125 \u0026micro;g/mL to 64 \u0026micro;g/mL) in a 96-well microtiter plate containing SDB. Each well was inoculated with 100 \u0026micro;L of \u003cem\u003eN. incurvata\u003c/em\u003e TI03 spore suspension standardized to 2 \u0026times; 10\u0026sup3; CFU/mL in SDB. The plates were incubated at 28\u0026deg;C for 5\u0026ndash;7 days. The MIC was defined as the lowest concentration of ZnO NPs showing complete inhibition of visible fungal growth compared with that in the control wells (S. Sharma et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Slavin \u0026amp; Bach, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAgar Well Diffusion Assay\u003c/strong\u003e\u003cp\u003eThe zone of inhibition exerted by ZnO NPs against \u003cem\u003eN. incurvata\u003c/em\u003e TI03 was assessed via agar well diffusion. The fungal inoculum was adjusted to match 0.5 McFarland standard turbidity and uniformly spread on Dermatophyte Test Medium (DTM) agar plates. Wells (6 mm diameter) were punched into the agar, and 10 \u0026micro;L of ZnO NP suspension (1 mg/mL in sterile distilled water) was loaded into each well. Commercial antifungal drugs including fluconazole (25 \u0026micro;g/disk), itraconazole, (10 \u0026micro;g/disk), and clotrimazole (10 \u0026micro;g/disk) served as positive controls. The negative controls consisted of sterile distilled water. Plates were incubated at 28\u0026deg;C for 7\u0026ndash;10 days, and the diameters of the inhibition zones were measured via a digital calliper (Al-Jobory et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Garcia-Marin et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Savi et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003cp\u003eAll experiments were performed in triplicate to ensure reproducibility. The data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was determined via one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s honestly significant difference (HSD) post-hoc test in GraphPad Prism v9.0.0 (GraphPad Software, San Diego, CA, USA). A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Antioxidant assay calculations were performed via T-statistics, degrees of freedom and Cohen\u0026rsquo;s test using IBM SPSS Statistics (Version 26.0, IBM Corp., Armonk, NY, USA) and Microsoft Excel (Version 2019). A significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 was used for the antioxidant assay.\u003c/p\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePhenotypic Characterization of\u003c/b\u003e \u003cb\u003eCandida albicans\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eNannizzia incurvata\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNine isolates from oral swab cultures presented phenotypic characteristics consistent with those of \u003cem\u003eC. albicans\u003c/em\u003e, including smooth, creamy colonies, gram-positive budding cells, germ tube formation, and pseudohyphae. Lactophenol Cotton Blue staining further revealed characteristic fungal morphology (Supplementary Fig.\u0026nbsp;1A). Among these strains, isolate CA26, which consistently exhibited characteristic morphology and germ tube formation, was selected for molecular identification.\u003c/p\u003e\u003cp\u003eMultiple isolates from cutaneous samples were obtained through skin scraping techniques. Several samples tested positive for fungal hyphae on KOH mounts and grew on DTM slants with cream to yellow-orange reverse pigmentation. Microscopic examination revealed multicellular macroconidia and microconidia, which are characteristic of \u003cem\u003eN. incurvata\u003c/em\u003e. One isolate, TI03, which displays typical conidial structures of \u003cem\u003eNannizzia\u003c/em\u003e species (Supplementary Fig.\u0026nbsp;1B, C), was selected for molecular confirmation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Confirmation of Species Identity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ITS-based phylogenetic tree for \u003cem\u003eC. albicans\u003c/em\u003e revealed that strain CA26 clustered closely with the reference strains AUMC 10189 and AUMC 8996 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The short branch lengths within this clade (0-0.001 substitutions per site) indicated high genetic similarity and minimal sequence divergence. Therefore, this low genetic distance, along with the placement among multiple \u003cem\u003eC. albicans\u003c/em\u003e reference strains supports the taxonomic identity of CA26 as \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, strain TI03 was clustered within a distinct clade, consisting GenBank-annotated \u003cem\u003eN. incurvata\u003c/em\u003e strains (KX793108.1, KX452080.1), with branch lengths ranging from 0.001\u0026ndash;0.004 substitutions per site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This clade was clearly separated from the \u003cem\u003eN. gypsea\u003c/em\u003e reference strains, which presented longer branch lengths of up to ~\u0026thinsp;0.013, indicating interspecific divergence. The monophyletic clustering of TI03 with \u003cem\u003eN. incurvata\u003c/em\u003e strains, along with their short intraclade distances, supports its taxonomic placement within the species and is consistent with established ITS-based fungal identification criteria (Schoch et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eUV-Visible Spectroscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe UV-Visible absorption bands of the ZnO nanoparticles (ZnO NPs) revealed a distinct surface plasmon resonance (SPR) peak at 361.75 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating effective nanoparticle synthesis with little aggregation. The absence of further peaks ranging from 400 to 900 nm confirmed the exceptional colloidal purity and stability of the suspension. In contrast, commercial ZnO powder exhibited a broader SPR peak at 367 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating a larger particle size and enhanced agglomeration. Supplementary Table\u0026nbsp;1 summarizes the UV-Visible absorbance values, which support the observed differences in the SPR peak sharpness and spectral purity between the biosynthesized and commercial ZnO.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFTIR spectra (400\u0026ndash;4000 cm⁻\u0026sup1;) revealed the presence of bioorganic functional groups associated with fungal metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A broad absorption band at ~\u0026thinsp;3400 cm⁻\u0026sup1; was observed in the ZnO\u0026thinsp;+\u0026thinsp;SDB spectrum, corresponding to the O-H stretching vibrations of the hydroxyl groups(Mohan et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A distinct peak near 1630 cm⁻\u0026sup1; in the ZnO\u0026thinsp;+\u0026thinsp;SDB spectrum was attributed to C\u0026thinsp;=\u0026thinsp;O stretching and N-H bending vibrations (Kazemi et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The characteristic bands at approximately 500 cm⁻\u0026sup1; in the ZnO and ZnO\u0026thinsp;+\u0026thinsp;SDB spectra confirmed the Zn-O stretching vibrations, indicating the formation of crystalline ZnO (Quadri et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, the SDB control spectrum exhibited weak broad bands near ~\u0026thinsp;2920 cm⁻\u0026sup1;, corresponding to C-H stretching vibrations(Meena et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Table\u0026nbsp;1). These observations suggest that ZnO NPs are effectively stabilized by extracellular fungal biomolecules.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eX-ray Diffraction (XRD)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eXRD analysis revealed distinct diffraction peaks at 2θ values of 31.8\u0026deg;, 34.4\u0026deg;, 36.3\u0026deg;, 47.5\u0026deg;, and 56.6\u0026deg;, corresponding to the (100), (002), (101), (102), and (110) crystallographic planes of the hexagonal wurtzite phase of ZnO (JCPDS Card No. 36-1451; space group: \u003cem\u003eP6₃mc\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). No additional impurity peaks were observed, indicating the high phase purity of the biosynthesized ZnO NPs. The most intense peak at ~\u0026thinsp;36.3\u0026deg; (plane 101) was used to estimate the average crystallite size (~\u0026thinsp;30 nm) via the Debye-Scherrer equation:\u003c/p\u003e\u003cp\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{D}=\\:\\:\\frac{K\\lambda\\:}{\\beta\\:cos\\theta\\:}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003eD\u003c/em\u003e is the crystallite size, \u003cem\u003eK\u003c/em\u003e is the Scherrer constant (0.94), \u003cem\u003eλ\u003c/em\u003e is the X-ray wavelength (1.5405 \u0026Aring;), \u003cem\u003eβ\u003c/em\u003e is the full width at half maximum (FWHM) of peak 101, and \u003cem\u003eθ\u003c/em\u003e is the Bragg angle. Three-dimensional crystallographic visualization performed via VESTA software further confirmed the hexagonal wurtzite structure of the biosynthesized ZnO nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransmission Electron Microscopy (TEM)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTEM micrographs revealed rod-to-bar shaped ZnO NPs with sizes ranging from approximately 75 to 99 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The particles were well-dispersed with minimal aggregation, which is consistent with the observations from the UV-Vis and XRD findings. The represented micrographs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D) revealed individual particle sizes of 95.4, 92.6, 75.1, and 93.2 nm, respectively, indicating a uniform morphology with smooth surfaces.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBrunauer-Emmett-Teller (BET) Surface Area Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe nitrogen adsorption-desorption isotherm exhibited a Type IV curve accompanied by a prominent hysteresis loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), which is characteristic of mesoporous materials. On the basis of multi-point BET plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), the linear region between 0.05 and 0.30 P/P₀ yielded a slope of 131.397 g⁻\u0026sup1; and an intercept of 0.1228 g⁻\u0026sup1; (R\u0026sup2; = 0.99907), corresponding to a BET constant (C) of 11.70. The calculated BET surface area was 24.24 m\u0026sup2;/g. Additionally, the Barrett-Joyner-Halenda (BJH) analysis yielded a total pore volume of 0.0555 cm\u0026sup3;/g and an average pore radius of 4.57 nm as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC. These results suggest that the material is suitable for enhanced surface-related applications, such as drug delivery or antimicrobial interactions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntioxidant Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFree Radical Scavenging Activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCompared with standard antioxidants, the biosynthesized ZnO NPs exhibited measurable antioxidant activity across all five assays, albeit with higher IC₅₀ values. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the plotted lines represent the mean IC₅₀ values (\u0026plusmn;\u0026thinsp;standard deviation) for both the ZnO NPs and the standards. The markers indicate the specific values for each assay, allowing for visual comparison of antioxidant activities across the different assays.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, the IC₅₀ values (\u0026micro;g/mL) are summarized in Supplementary Table\u0026nbsp;2A. The DPPH scavenging activity of the ZnO NPs yielded an IC₅₀ of 53.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75 for ZnO NPs, whereas it was 28.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 for ascorbic acid. Similarly, for ABTS, nitric oxide, hydroxyl, and superoxide radicals, the IC₅₀ values of the ZnO NPs ranged from 56.39 to 62.88 \u0026micro;g/mL, which were significantly greater than those of respective standard controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Significance Testing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIndependent sample \u003cem\u003et\u003c/em\u003e-tests confirmed statistically significant differences in the IC₅₀ values between ZnO NPs and standard antioxidants across all the assays (Supplementary Table\u0026nbsp;2B), with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for each comparison.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect Size (Cohen\u0026rsquo;s\u003c/b\u003e \u003cb\u003ed\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEffect size calculations revealed Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e values exceeding 17 for all comparisons (Supplementary Table\u0026nbsp;2C), indicating extremely large effect sizes. These results emphasize not only the statistical significance but also the practical significance of the differences in antioxidant potential.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMinimum Inhibitory Concentration (MIC)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe biosynthesized ZnO NPs exhibited strong antifungal activity against \u003cem\u003eN. incurvata\u003c/em\u003e TI03. The minimum inhibitory concentration (MIC) of the ZnO NPs was 0.96 \u0026micro;g/mL, which was comparable to that of fluconazole (0.99 \u0026micro;g/mL), miconazole (0.98 \u0026micro;g/mL), and clotrimazole (0.99 \u0026micro;g/mL), and marginally lower than that of itraconazole (0.97 \u0026micro;g/mL) (Supplementary Table\u0026nbsp;3A). Statistical analysis via one-way ANOVA followed by Tukey's post-hoc test confirmed that the observed differences among the agents were not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), suggesting that the antifungal efficacy ZnO NPs is comparable to that of established azole antifungals (Supplementary Table\u0026nbsp;3B).\u003c/p\u003e\u003cp\u003eNotably, the geometric mean (GM) MIC for the ZnO NPs was 0.43 \u0026micro;g/mL, which falls well within the therapeutic range. This value was statistically comparable to that of fluconazole (GM: 0.27 \u0026micro;g/mL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.696), clotrimazole (GM\u0026thinsp;=\u0026thinsp;0.75 \u0026micro;g/mL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.523 and miconazole (GM: 0.33 \u0026micro;g/mL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.826), but significantly lower than that of itraconazole (GM: 0.90 \u0026micro;g/mL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048). Furthermore, ZnO NPs exhibited a clear dose-dependent inhibition profile, with MIC₅₀ of 0.86 \u0026micro;g/mL and an MIC₂X of 1.07 \u0026micro;g/mL (Supplementary Table\u0026nbsp;3B), supporting their concentration-dependent antifungal activity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAgar Well Diffusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn agar well diffusion assays, ZnO NPs at 32 \u0026micro;g/mL produced an inhibition zone of 11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm. This value was statistically comparable to that of fluconazole (11.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.512) and itraconazole (11.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.512), slightly lower than that of miconazole (12.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.102), and greater than that of clotrimazole (10.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.102) (Supplementary Table\u0026nbsp;3C).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe phenomenon of microbial antagonism, in which one microorganism inhibits or eradicates another, often through the production of bioactive metabolites, has significantly contributed to the discovery of novel therapeutics from microbial sources (Pranav et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). While bacterial antagonism has been extensively researched, fungal-fungal antagonism remains relatively underexplored, particularly in the context of nanoparticle-based therapeutic strategies. Although \u003cem\u003eCandida\u003c/em\u003e species are known predominantly for their pathogenic potential (Pfaller \u0026amp; Diekema, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), recent studies have demonstrated their role in nanoparticle biosynthesis, such as the production of bismuth nanoparticles (Bi-NPs) by \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e, which simultaneously exhibited antifungal activity against the respective strains (Zanganeh et al., \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While that study addressed the self-inhibitory potential of \u003cem\u003eCandida\u003c/em\u003e species, the interfungal antagonism through the biosynthesis of nanoparticles was not investigated. Expanding on this concept, we employed the approach of utilizing \u003cem\u003eC. albicans\u003c/em\u003e, an opportunistic pathogenic yeast, as a biological system for the green synthesis of antifungal ZnO nanoparticles against \u003cem\u003eN. incurvata\u003c/em\u003e, a pathogenic dermatophytic filamentous fungus.\u003c/p\u003e\u003cp\u003eThe traditional morphological assays (germ-tube test, LPCB staining) performed for the presumptive identification of \u003cem\u003eC. albicans\u003c/em\u003e CA26 were validated by ITS-region sequencing, which remains the gold standard for fungal taxonomy and was employed to confirm morphological observations (Stover \u0026amp; Cavalcanti, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Similarly, the isolation of \u003cem\u003eN. incurvata\u003c/em\u003e TI03 from dermatophytosis samples reflects emerging epidemiological trends in dermatophytic infections in India, with a shift from \u003cem\u003eTrichophyton\u003c/em\u003e dominance to other genera (Nenoff et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pchelin et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, the decision to select representative isolates on the basis of consistent diagnostic morphological features for ITS sequencing and nanomaterial synthesis aligns with best practices in microbial prospecting, where strain redundancy is minimized when intraspecies variation is not under investigation (Mohammed Fayaz et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Narayanan \u0026amp; Sakthivel, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Furthermore, the biosynthetic potential of \u003cem\u003eC. albicans\u003c/em\u003e CA26 and the antifungal potential its ZnO NPs, can be interpreted as strain-specific rather than species-wide, providing a basis for exploring broader species-level capabilities in future studies.\u003c/p\u003e\u003cp\u003eThe SPR peak observed at 361.75 nm closely aligns with previously reported studies, where biogenic ZnO NPs exhibited SPR peaks within the 350\u0026ndash;380 nm range, indicating nanoscale dimensions and minimal aggregation (Narendra Kumar et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vaseem et al., 2009). The absence of secondary absorbance peaks in the 400\u0026ndash;900 nm range further confirms high colloidal purity and stability, both of which are critical traits for biomedical applications (Sirelkhatim et al., 2015b). In contrast, commercial ZnO exhibited a redshifted SPR peak (367 nm), indicating larger particle size and greater degree of agglomeration, thereby underscoring the advantages of green synthesis in the production of structurally uniform, biocompatible nanoparticles that are well-suited for antifungal applications. FTIR analysis revealed the presence of fungal-derived functional groups such as hydroxyl, amine, and carbonyl groups, capping and stabilizing the surface of the ZnO nanoparticles. These organic moieties play dual roles in reducing Zn\u0026sup2;⁺ metal ions during synthesis and preventing agglomeration, enabling controlled nanoparticle formation (Iravani, 2011b). Similar biomolecule-assisted syntheses have been reported in other fungal species, supporting the generalizability of this approach (Gurunathan et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The colloidal stability is further supported by FTIR data, which revealed that hydrophilic surface groups contribute to enhanced aqueous dispersibility, a critical factor in biomedical formulations (Kolodziejczak-Radzimska \u0026amp; Jesionowski, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e). Furthermore, the sharp diffraction peaks in the XRD pattern and the estimated crystallite size of ~\u0026thinsp;30 nm suggest well-crystallized ZnO nanostructures. The crystallite sizes of NPs within this range have been reported to be suitable for antimicrobial applications (Al-Bedairy \u0026amp; Habeeb Alshamsi, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e; Saravanan et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the basis of previously reported studies, ZnO NPs within this range exhibit enhanced antimicrobial activity, likely due to their high surface area-to-volume ratio, which facilitates better interaction with microbial cells and promotes increased reactive oxygen species (ROS) generation upon contact (Ali et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Raghupathi et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTEM revealed the welldispersed, rod-to-bar-shaped morphology and monodispersity of the ZnO NPs, which have been suggested to be efficient in surface interactions with microbial membranes (Kolodziejczak-Radzimska \u0026amp; Jesionowski, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e). Compared with spherical equivalents, nanorod-shaped ZnO has greater penetration and cellular contact, resulting in better antifungal activity. (Rezaei et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Soliman et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, the BET surface area of 24.24 m\u0026sup2;/g, together with the mesoporous structure revealed by Type IV isotherm and BJH analysis, indicates greater surface reactivity. This porous design promotes the loading and release of bioactive chemicals and enhances contact with fungal cell membranes. (Agarwal et al., 2017). Previous research has demonstrated that mesoporous ZnO nanoparticles accelerate ROS production and promote membrane breakdown in fungal infections. (Anbuvannan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; B. N. Singh et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tiwari et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eC. albicans\u003c/em\u003e CA26 biosynthesized ZnO NPs demonstrated potent antifungal activity against \u003cem\u003eN. incurvata\u003c/em\u003e TI03. The MIC values were statistically comparable to those of conventional azole drugs, with no significant differences observed in most comparisons. Despite the comparable efficacy, the geometric mean MIC of the ZnO NPs was lower than that of itraconazole, suggesting superior efficacy under certain conditions. These findings were supported by agar diffusion assays, where ZnO NPs produced inhibition zones similar to those of fluconazole and itraconazole. Importantly, despite their similar MIC values, ZnO NPs exhibit multiple translational advantages. Our previous findings revealed that ZnO NPs suppress fungal growth through multiple mechanisms, including ROS production, membrane disruption, and interference with protein and metabolic pathways. (Nxumalo et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Selokar \u0026amp; Thakare, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This multimodal mechanism reduces the likelihood of resistance development, which is a growing concern with azole-based treatments (Burmester et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ghannoum, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hossain et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAdditionally, the ZnO NPs biosynthesized by CA26 showed moderate but consistent antioxidant efficacy across five free radical scavenging experiments, despite exhibiting higher IC₅₀ values than standard antioxidants. The exceptionally substantial effect sizes (Cohen\u0026rsquo;s d\u0026thinsp;\u0026gt;\u0026thinsp;17) across all the comparisons highlight their biological relevance and reproducibility. This antioxidative potential can be attributed to the semiconducting properties of ZnO, which facilitate electron transfer to neutralize reactive species, as well as the presence of fungal-derived capping biomolecules known to participate in radical scavenging reactions (Kumar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rodr\u0026iacute;guez et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yamaguchi, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). Oxidative stress plays a key role in skin inflammation, aging and secondary damage associated with fungal infections (Briganti \u0026amp; Picardo, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Rinnerthaler et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, the topical application of antioxidant agents, especially those with dual antifungal and radical-scavenging properties can offer enhanced skin protection by counteracting reactive oxygen and nitrogen species (ROS/RNS)-mediated cellular damage. While ZnO is already a well-established skin protectant (Wiegand et al., \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), the added antioxidant functionality of these biosynthesized ZnO NPs supports their potential as multifunctional agents in dermatological formulations aimed at both controlling infection and alleviating oxidative stress.\u003c/p\u003e\u003cp\u003eThe biosynthesis of ZnO NPs using \u003cem\u003eC. albicans\u003c/em\u003e CA26 offers environmental and clinical benefits, including eco-friendliness and reproducibility (Agarwal et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Cruz et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mousavi et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The low standard deviation of ZnO NPs across replicates further supports the synthesis consistency. Moreover, ZnO is already extensively used in dermatological formulations because of its skin compatibility and anti-inflammatory and UV-protective properties (Akintelu \u0026amp; Folorunso, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Al-Bedairy \u0026amp; Habeeb Alshamsi, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Osterwalder et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Smijs \u0026amp; Pavel, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Given the emergence of azole-resistant dermatophytes, including \u003cem\u003eN. incurvata\u003c/em\u003e (Gamal et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Havlickova et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Jia et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Seebacher et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), the comparable antifungal activity and favorable safety profile of ZnO NPs make them excellent candidates for incorporation into topical formulations such as creams, sprays, and gels (Choudhury et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kr\u0026oacute;l et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lansdown et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Prajapati et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This green synthesis approach not only offers an environmentally friendly alternative, but also produces nanoparticles with functional surface coatings derived from natural biomolecules, hence increasing their biocompatibility and therapeutic potential (Agarwal et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Upadhyay et al., n.d.; Velusamy et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn summary, the ZnO nanoparticles biosynthesized by \u003cem\u003eC. albicans\u003c/em\u003e CA26 exhibit dual antifungal and antioxidant properties, positioning them as promising multifunctional agents for dermatological applications. However, as these findings are based on in-vitro assays, further in-vivo studies and cytotoxicity evaluations are essential to fully establish their clinical safety and efficacy, which paves way for future translational research and therapeutic development. Overall, these findings demonstrate that \u003cem\u003eC. albicans\u003c/em\u003e CA26 derived ZnO NPs are structurally and functionally optimized for antifungal activity, as evidenced by their physicochemical characteristics revealed through UV-Vis, FTIR, XRD, TEM, and BET analyses, along with their robust antifungal performance, collectively underscoring their potential as sustainable and clinically viable candidates for topical dermatological applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Dean of Indira Gandhi Government Medical College and Hospital, Nagpur, granted formal approval for clinical sample collection in this study, as communicated through the Head and Research Guide of the Department of Molecular Biology and Genetic Engineering, Rashtrasant Tukadoji Maharaj Nagpur University (Ref. No. MB\u0026amp;GE/12/2024, dated 15 April 2024). The proposal was reviewed and approved by the research supervisor, the departmental head, and the Dean of the Faculty of Science and Technology, RTM Nagpur University. The study involved only non-invasive, anonymized clinical specimens (oropharyngeal swabs and skin scrapings), and no personal identifiers or sensitive patient information was collected. All procedures were carried out under relevant institutional guidelines, ethical standards, and good clinical laboratory practices. Written informed consent was obtained from all participants prior to sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eYNS, acknowledges the financial support received from the Mahatma Jyotiba Phule Research \u0026amp; Training Institute (MAHAJYOTI), Autonomous Institute under the Other Backward Class Bahujan Welfare Department, Govt. of Maharashtra (India), in the form of a Senior Research Fellowship (SRF) (Fellowship2022_1008).\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eYNS, PSP, and RUT designed the experiments; YNS and PSP performed the experimental work and wrote the initial draft of the manuscript. RUT contributed to manuscript revision and editing. All the authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors acknowledge the Department of Molecular Biology and Genetic Engineering, RTM Nagpur University, Nagpur for providing research facilities and support. We also extend our sincere thanks to the medical staff and administrative teams of the Government Ayurved College and Hospital, Government Dental College and Hospital, and Indira Gandhi Government Medical College and Hospital, Nagpur, for their valuable assistance throughout the study.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its supplementary files. Additional datasets and samples used or analyzed in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbarenkov K, Nilsson RH, Larsson KH, Taylor AFS, May TW, Fr\u0026oslash;slev TG, Pawlowska J, Lindahl B, P\u0026otilde;ldmaa K, Truong C, Vu D, Hosoya T, Niskanen T, Piirmann T, Ivanov F, Zirk A, Peterson M, Cheeke TE, Ishigami Y, K\u0026otilde;ljalg U (2024a) The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered. 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J Gen Microbiol 86(2):370\u0026ndash;372. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/00221287-86-2-370\u003c/span\u003e\u003cspan address=\"10.1099/00221287-86-2-370\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZanganeh E, Moghbeli M, Zarrinfar H, Sadeghian H (2025) Green Synthesis of Bismuth Nanoparticles Using \u003cem\u003eCandida albicans\u003c/em\u003e and \u003cem\u003eC. glabrata\u003c/em\u003e. along Evaluation Their Antifungal Eff BioNanoScience 15(1):1\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12668-025-01815-8\u003c/span\u003e\u003cspan address=\"10.1007/s12668-025-01815-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, India","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Zinc oxide nanoparticles, green synthesis, antifungal activity, antioxidant activity, dermatophytes","lastPublishedDoi":"10.21203/rs.3.rs-7164738/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7164738/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rise of antifungal resistance in dermatophytosis underscores the need for novel antifungals, driving the green synthesis of zinc oxide nanoparticles (ZnO NPs). In this study, we report the extracellular biosynthesis of ZnO NPs via a clinical isolate of \u003cem\u003eCandida albicans\u003c/em\u003e CA26 and evaluate their physicochemical properties, antioxidant capacity, and antifungal efficacy against \u003cem\u003eNannizzia incurvata\u003c/em\u003e TI03. The ZnO NPs were biosynthesized using the extracellular filtrate of \u003cem\u003eC. albicans\u003c/em\u003e CA26 and the resulting NPs were characterized via UV-Vis spectroscopy, FTIR, XRD, TEM, and BET analysis. The UV-Vis spectra revealed a characteristic surface plasmon resonance peak at 361.75 nm, confirming the formation of ZnO NP; whereas the FTIR spectra confirmed capping by fungal biomolecules. XRD confirmed a crystalline ZnO phase with an average crystallite size of ~\u0026thinsp;30 nm and TEM imaging revealed a rod-to-bar morphology (75\u0026ndash;99 nm in length). BET analysis revealed a specific surface area of ~\u0026thinsp;24.24 m^2/g and an average pore radius of ~\u0026thinsp;4.57 nm. The antioxidant activity of the ZnO NPs was evaluated via DPPH, ABTS, nitric oxide, hydroxyl radical, and superoxide scavenging assays, which revealed moderate but consistent activity across all five assays. These activities were statistically significant compared with those of the reference antioxidants (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Cohen\u0026rsquo;s d\u0026thinsp;\u0026gt;\u0026thinsp;17). The antifungal efficacy was tested against \u003cem\u003eN. incurvata\u003c/em\u003e TI03, yielding a minimum inhibitory concentration (MIC) of 0.96 \u0026micro;g/mL, which is comparable to that of standard azole drugs. In agar diffusion assays, ZnO NPs produced inhibition zones similar in size to those of fluconazole and itraconazole. The biosynthesized ZnO NPs thus exhibited multimodal antifungal mechanisms, likely involving reactive oxygen species generation, membrane disruption, and enzyme inhibition, in addition to their antioxidant activity. This eco-friendly synthesis, combined with the potent dual-functionality of these ZnO NPs highlights their role as promising topical antifungal therapeutic agents.\u003c/p\u003e","manuscriptTitle":"Green Synthesis of Zinc Oxide Nanoparticles by Candida albicans CA26 with Antioxidant and Antifungal Activity Against Nannizzia incurvata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 08:42:21","doi":"10.21203/rs.3.rs-7164738/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cf60f165-4441-41a4-9b15-0cb6bf759954","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51792235,"name":"Mycology"},{"id":51792236,"name":"Nanoscience"},{"id":51792237,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2025-08-06T09:08:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 08:42:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7164738","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7164738","identity":"rs-7164738","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}