UV-A–Assisted Green Synthesis of Copper Nanoparticles Using Dual Plant Extracts: A Comparative Study on Reaction Kinetics, Size Distribution, and Antibacterial Activity

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Naveen¹, Gopi Mamidi², A. Indira Priyadarsini³, G. Swathi⁴ This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8907851/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 development of environmentally benign routes for the synthesis of metallic nanoparticles is an important objective in contemporary nanomaterials research, particularly to minimize reliance on toxic chemical reductants and stabilizers. In the present study, copper nanoparticles (CuNPs) were synthesized using aqueous extracts of Camellia sinensis (green tea) and Ocimum sanctum (tulsi), which functioned simultaneously as reducing and capping agents. Two synthesis routes were systematically investigated: a conventional phytogenic reduction process and an ultraviolet (UV-A, 365 nm) assisted photoreduction approach. In the conventional method, gradual reduction of Cu²⁺ ions over 3 h yielded predominantly spherical CuNPs with particle sizes in the range of 20–50 nm and a zeta potential of − 29.5 mV, indicating moderate colloidal stability. In contrast, UV-assisted synthesis significantly accelerated nanoparticle formation, completing the reaction within 15–20 min and producing smaller particles (8–25 nm) with improved stability (zeta potential between − 30 and − 38 mV). UV–visible spectroscopy revealed distinct surface plasmon resonance bands for both samples, with the UV-assisted CuNPs exhibiting a sharper and slightly blue-shifted peak, consistent with reduced particle size and narrower size distribution. Fourier transform infrared spectroscopy confirmed the involvement of polyphenolic and phenolic constituents, including catechin- and eugenol-type moieties, in the reduction and stabilization of CuNPs. X-ray diffraction analysis verified the formation of crystalline face-centered cubic copper with minor contributions from Cu₂O, while transmission electron microscopy corroborated the enhanced uniformity of UV-assisted nanoparticles. The antibacterial activity of the synthesized CuNPs was evaluated against Escherichia coli and Staphylococcus aureus , revealing effective growth inhibition for both bacterial strains. UV-assisted CuNPs exhibited marginally higher antibacterial efficacy, which is attributed to their smaller size and improved surface stabilization. Overall, this study demonstrates that UV-assisted phytogenic synthesis offers a rapid and reproducible route to stable copper nanoparticles, while maintaining the advantages of green chemistry. The findings highlight the potential of combining plant-derived reductants with photochemical activation to achieve controlled nanoparticle synthesis for antimicrobial and related applications. green synthesis copper nanoparticles UV-assisted photoreduction Camellia sinensis Ocimum sanctum antimicrobial activity sustainable nanotechnology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Dual-extract green synthesis using Camellia sinensis and Ocimum sanctum combined for the first time with UV-A photoreduction. Rapid synthesis completed within 15–20 minutes compared with ~3 hours in the conventional method. UV-assisted CuNPs exhibited ultra-small size (8–25 nm) and high colloidal stability (–30 to –38 mV). Strong antibacterial activity against both E. coli and S. aureus , surpassing conventionally synthesized CuNPs. Eco-friendly, reproducible, and scalable method suitable for biomedical, agricultural, and catalytic applications. Introduction Nanotechnology has emerged as a key area of research due to its wide-ranging impact across scientific and industrial sectors, including catalysis, energy storage, biomedical diagnostics, therapeutics, and environmental remediation [ 1 – 3 ]. Among metallic nanomaterials, copper nanoparticles (CuNPs) have attracted considerable attention owing to their high electrical conductivity, catalytic efficiency, optical properties, and antimicrobial activity [ 4 , 5 ]. Compared to noble metals such as silver and gold, copper is inexpensive, earth-abundant, and more suitable for large-scale production, which enhances its practical applicability in industrial and biomedical fields [ 6 ]. Conventional physical and chemical routes for the synthesis of CuNPs often involve harsh reducing agents such as hydrazine or sodium borohydride, elevated temperatures, and multistep processing, leading to toxic byproducts and environmental concerns [ 7 – 9 ]. These drawbacks have driven increasing interest in green synthesis approaches that utilize biological systems as reducing and stabilizing agents. Plant-mediated synthesis is particularly advantageous due to the abundance of phytochemicals such as polyphenols, flavonoids, terpenoids, alkaloids, and organic acids, which can simultaneously reduce metal ions and stabilize the resulting nanoparticles through surface capping [ 10 – 12 ]. Camellia sinensis (green tea) is rich in catechins, particularly epigallocatechin gallate (EGCG), which possess strong antioxidant and electron-donating properties and have been widely reported as effective reducing agents for metal nanoparticle synthesis [ 13 , 14 ]. Similarly, Ocimum sanctum (tulsi) contains bioactive compounds such as eugenol, rosmarinic acid, and ursolic acid, which exhibit redox activity and antimicrobial properties, making it a suitable candidate for green nanomaterial synthesis [ 15 , 16 ]. Several studies have reported the synthesis of CuNPs using individual plant extracts, including green tea and tulsi; however, investigations employing combined or dual-extract systems remain limited, despite their potential to enhance reduction efficiency and surface stabilization through synergistic phytochemical interactions [ 17 ]. In parallel with phytogenic approaches, photo-assisted synthesis methods have gained attention as a means to accelerate nanoparticle formation. Ultraviolet (UV) irradiation supplies high-energy photons that promote rapid electron transfer, increase nucleation rates, and improve control over particle size and distribution [ 18 – 20 ]. Previous studies have demonstrated that UV-assisted green synthesis can significantly reduce reaction time and yield smaller, more uniform nanoparticles compared to conventional stirring-based methods [ 21 ]. However, the integration of UV-assisted photoreduction with dual-extract phytogenic systems for CuNP synthesis has not been systematically explored. In this context, the present study investigates a UV-assisted green synthesis strategy employing combined aqueous extracts of C. sinensis and O. sanctum for the preparation of CuNPs. The approach aims to evaluate the effect of UV irradiation on reaction kinetics, particle size, stability, and antibacterial performance, while elucidating the cooperative role of phytochemicals from both extracts in nanoparticle formation and stabilization. Materials and Methods 2.1 Materials Copper (II) sulphate pentahydrate (CuSO₄·5H₂O, analytical grade) and absolute ethanol were obtained from Sigma-Aldrich and used without further purification. Copper sulphate was selected as the precursor salt due to its high solubility and stability in aqueous nanoparticle synthesis systems [ 22 ]. Ethanol was used during washing steps to remove excess phytochemicals and residual impurities from the nanoparticle surface [ 23 ]. Fresh leaves of Camellia sinensis (green tea) were collected from the Wayanad Tea Plantations, Kerala, India, a region known for polyphenol-rich tea foliage suitable for green synthesis applications [ 24 ]. Leaves of Ocimum sanctum (tulsi) were collected from Nagari, Chittoor District, Andhra Pradesh, India. Both plant materials were authenticated by a qualified botanist at Government Degree College (Autonomous), Nagari, to ensure taxonomic accuracy and experimental reproducibility [ 25 ]. Deionized water was used throughout all experimental procedures, and all glassware was thoroughly cleaned and dried prior to use to avoid ionic or particulate contamination [ 26 ]. 2.2 Preparation of Plant Extracts Aqueous extracts of Camellia sinensis and Ocimum sanctum were prepared following well-established green synthesis protocols with minor modifications [ 27 ]. Briefly, 10 g of dried leaf powder was added to 100 mL of deionized water and boiled for 15 min to extract phytochemicals such as polyphenols, flavonoids, and terpenoids that act as reducing and stabilizing agents during nanoparticle formation [ 28 ]. After cooling to room temperature, the extracts were filtered through Whatman No. 1 filter paper to remove insoluble residues. The pH of each extract was recorded (typically 5.4–6.2), as solution pH plays a critical role in metal ion reduction kinetics and nanoparticle nucleation behavior [ 29 ]. The filtrates were stored at 4°C and used within 48 h to preserve the activity of key biomolecules involved in copper ion reduction and surface capping [ 30 ]. 2.3 Synthesis of Copper Nanoparticles Copper nanoparticles were synthesized using both conventional and UV-assisted photoreduction routes. For the conventional method, 50 mL of 1 mM CuSO₄·5H₂O solution was mixed with equal volumes (25 mL each) of C. sinensis and O. sanctum extracts. The reaction mixture was stirred at room temperature under its natural pH (5.4–6.2) without external adjustment, consistent with typical phytogenic nanoparticle synthesis conditions [ 31 ]. A gradual color change from pale blue to brown indicated the reduction of Cu²⁺ ions to metallic copper nanoparticles [ 32 ]. After 3 h of stirring, the suspension was centrifuged at 10,000 rpm for 15 min. The resulting pellet was washed repeatedly with deionized water and ethanol to remove unreacted ions and loosely bound phytochemicals, followed by drying in a vacuum oven at 60°C for 12 h [ 33 ]. For the UV-assisted synthesis, the same reaction mixture was exposed to UV-A irradiation (365 nm, ~ 10 mW cm⁻²) in a photoreactor. A rapid color change was observed within 15–20 min, indicating accelerated reduction. UV irradiation facilitates electron transfer from phytochemicals to Cu²⁺ ions, leading to faster nucleation and improved control over nanoparticle size and distribution [ 34 – 36 ]. The UV-assisted CuNPs were collected and purified using the same procedure as the conventional samples to ensure direct comparability. 2.4 Characterization UV–visible spectroscopy was carried out using a Shimadzu UV-2600 spectrophotometer over a wavelength range of 300–800 nm with a scan speed of 200 nm min⁻¹ and a spectral resolution of 1 nm to monitor the formation of copper nanoparticles through their characteristic surface plasmon resonance (SPR) absorption band [ 37 ]. The acquired UV–Vis spectra were subjected only to baseline correction using the instrument’s native software to remove background noise; no smoothing, peak shifting, or intensity manipulation was performed. Fourier transform infrared (FTIR) spectra were recorded using a PerkinElmer Spectrum Two spectrometer in the range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹ to identify functional groups involved in phytochemical reduction and surface stabilization of the nanoparticles [ 38 ]. X-ray diffraction (XRD) analysis was conducted using a Rigaku Miniflex diffractometer equipped with Cu-Kα radiation (λ = 1.5406 Å) , operating at 40 kV and 15 mA . Diffraction patterns were recorded over a 2θ range of 20–80° with a step size of 0.02° . The XRD data were processed solely for background subtraction using standard instrument software, and crystallite size was estimated using the Scherrer equation without instrumental broadening correction. Surface morphology and particle size distribution were examined using scanning electron microscopy (SEM, JEOL JSM-7600F) operated at an accelerating voltage of 15 kV , and transmission electron microscopy (TEM, JEOL JEM-2100) operated at 200 kV . All SEM and TEM micrographs include calibrated scale bars , enabling accurate interpretation of particle size and morphology. Particle size distributions were determined by measuring over 100 individual nanoparticles using ImageJ software. Colloidal stability was evaluated by measuring zeta potential using a Malvern Zetasizer Nano ZS at 25°C , with samples appropriately diluted in deionized water to avoid multiple scattering effects. 2.5 Antibacterial Activity Assay The antibacterial activity of the synthesized CuNPs was evaluated using the agar disc diffusion method against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive), following standard antimicrobial susceptibility testing guidelines [ 39 ]. Bacterial cultures were evenly spread on Mueller–Hinton agar plates, and sterile paper discs impregnated with CuNP suspensions (1 mg mL⁻¹) were placed on the agar surface. The plates were incubated at 37°C for 24 h, after which the diameter of the inhibition zones was measured in millimeters. The disc diffusion assay was selected due to its reproducibility and suitability for comparative evaluation of nanoparticle-mediated antibacterial activity [ 40 ]. Results and Discussion 3.1 Visual Observation and Reaction Kinetics The formation of copper nanoparticles was initially indicated by a gradual color change of the reaction mixture from pale blue to brown, which is commonly associated with the development of surface plasmon resonance in copper-based nanostructures [ 41 ]. In the conventional synthesis route, this transformation occurred over approximately 3 h, reflecting the relatively slow reduction kinetics typical of plant-mediated nanoparticle synthesis systems [ 42 ].In contrast, the UV-assisted reaction exhibited a rapid color transition within 15–20 min. The accelerated reaction can be attributed to UV-A irradiation facilitating photon-induced electron transfer from phytochemicals to Cu²⁺ ions, thereby increasing the nucleation rate and lowering the activation energy required for reduction [ 43 , 44 ]. Similar enhancements in reaction kinetics under UV or photo-assisted conditions have been reported for other green-synthesized metal nanoparticles, supporting the reproducibility of this approach [ 45 ]. 3.2 UV–Visible Spectroscopy UV–Vis spectra confirmed the formation of CuNPs in both synthesis routes. Conventionally synthesized CuNPs exhibited a broad surface plasmon resonance band in the range of 570–590 nm, which is indicative of a wider particle size distribution and partial aggregation [ 46 ]. In comparison, UV-assisted CuNPs displayed a sharper and slightly blue-shifted SPR peak centered around 565–575 nm. Such spectral narrowing and blue shift are commonly associated with smaller particle sizes and improved size uniformity, particularly under conditions of rapid nucleation [ 47 , 48 ]. These observations indicate that UV irradiation not only accelerates nanoparticle formation but also contributes to improved control over particle growth. (Fig. 1 ) 3.3 FTIR Analysis FTIR spectra were analyzed to identify the functional groups involved in copper ion reduction and nanoparticle stabilization. A broad absorption band around 3400 cm⁻¹ was attributed to O–H stretching vibrations of phenolic compounds and polyphenols present in the plant extracts [ 49 ]. The band observed near 1635 cm⁻¹ corresponded to C = O stretching vibrations of flavonoids and organic acids, while peaks in the region of 1380 cm⁻¹ were associated with aromatic and C–N vibrations of plant metabolites [ 50 ]. The presence of a prominent band near 1100 cm⁻¹, corresponding to C–O stretching vibrations, further supports the involvement of alcohols and ethers in nanoparticle stabilization. Increased band intensity observed in UV-assisted CuNPs suggests stronger phytochemical adsorption, which likely contributes to enhanced surface passivation and colloidal stability [ 51 ]. ( Fig. 2 ) 3.4 XRD Analysis XRD patterns confirmed the crystalline nature of the synthesized CuNPs. Diffraction peaks at 2θ values of approximately 43.3°, 50.4°, and 74.1° corresponded to the (111), (200), and (220) planes of face-centered cubic copper, consistent with standard reference data [ 52 ].The average crystallite size estimated using the Scherrer equation ranged from 25–35 nm for conventionally synthesized CuNPs and 12–20 nm for UV-assisted CuNPs, indicating that photoreduction effectively restricts crystal growth by promoting rapid nucleation [ 53 ]. Minor diffraction peaks corresponding to Cu₂O were also detected, which is commonly observed in green-synthesized copper nanoparticles due to surface oxidation during processing or exposure to air [ 54 ]. ( Fig. 3 ) 3.5 Morphological Analysis (SEM and TEM) SEM analysis revealed predominantly spherical to quasi-spherical copper nanoparticles for both synthesis routes, with limited aggregation observed in some regions, which may arise from interparticle interactions mediated by surface-bound phytochemicals [ 55 ]. TEM provided higher-resolution insight into particle size and spatial distribution. Nanoparticles synthesized via the conventional route exhibited a broad size distribution in the range of 20–50 nm , consistent with gradual nucleation and growth occurring over an extended reaction time. In contrast, UV-assisted synthesis produced copper nanoparticles with a reduced mean size and a narrower distribution (8–25 nm) , along with improved dispersion under identical synthesis conditions. In several TEM micrographs of UV-assisted CuNPs, a thin, low-contrast organic layer was observed surrounding the metallic cores, which is attributed to adsorbed phytochemical species from the plant extracts. The presence of this surface layer is expected to contribute to enhanced dispersion stability and reduced particle coalescence, in agreement with zeta potential measurements and previous reports [ 56 ]. Quantitative particle size analysis based on TEM measurements indicated that the average particle size of conventionally synthesized CuNPs was 34.2 ± 8.1 nm , whereas UV-assisted CuNPs exhibited a significantly reduced average size of 15.6 ± 4.3 nm . This observed size reduction is consistent with UV-induced acceleration of nucleation , which limits subsequent particle growth under otherwise identical synthesis conditions. ( Fig. 4 ) 3.6 Zeta Potential and Colloidal Stability Zeta potential measurements were used to evaluate colloidal stability. Conventionally synthesized CuNPs exhibited a zeta potential of − 29.5 mV, indicating moderate to good electrostatic stability. UV-assisted CuNPs showed higher absolute zeta potential values (–30 to − 38 mV), exceeding the commonly accepted ± 30 mV threshold for highly stable colloidal systems [ 57 ]. The improved stability of UV-assisted CuNPs can be attributed to denser phytochemical capping and increased surface charge, which enhance electrostatic repulsion and steric stabilization [ 58 ]. ( Fig. 5 ) 3.7 Antibacterial Activity Both CuNP systems demonstrated significant antibacterial activity against Escherichia coli and Staphylococcus aureus . The observed inhibition zones confirm the effectiveness of CuNPs against both Gram-negative and Gram-positive bacteria, consistent with previously reported mechanisms involving membrane disruption, ion release, and oxidative stress [ 59 ]. UV-assisted CuNPs exhibited slightly higher antibacterial activity compared to conventionally synthesized nanoparticles. This enhancement can be attributed to smaller particle size, higher surface area, and improved interaction with bacterial cell membranes [ 60 ]. Similar size-dependent antibacterial trends have been reported for copper and other metal nanoparticles synthesized via green routes. A comparative summary of synthesis route, particle size, morphology, colloidal stability, and antibacterial performance of conventionally synthesized and UV-assisted CuNPs is presented in Table 1 . Table 1 Comparative characteristics of conventionally synthesized and UV-assisted copper nanoparticles Parameter Conventional CuNPs UV-assisted CuNPs Synthesis route Chemical/thermal reduction UV-assisted green synthesis Reducing agents Chemical reagents Plant-derived phytochemicals Capping/Stabilizing agents Absent or weakly bound Phytochemical capping (polyphenols, flavonoids) Average particle size (TEM) 20–50 nm 8–25 nm Particle size distribution Broad Narrow Particle shape Spherical with partial aggregation Uniform, well-defined spherical Surface morphology Rough / clustered Smooth surface with visible organic shell Degree of agglomeration High Minimal Dispersion behavior Moderately dispersed Highly dispersed Zeta potential −29.5 mV −30 to − 38 mV Colloidal stability Moderate stability High colloidal stability Role of phytochemicals Not applicable Act as reducing + capping agents Overall structural uniformity Lower Higher ( Fig. 6 ) NOVELTY OF THE WORK The present study reports a comparative green synthesis approach that combines a dual-extract phytogenic system, based on Camellia sinensis (green tea) and Ocimum sanctum (tulsi), with UV-A–assisted photoreduction for the preparation of copper nanoparticles. The combined presence of polyphenolic constituents from green tea and eugenol-rich compounds from tulsi, when coupled with UV irradiation, was found to accelerate reduction kinetics , decreasing the reaction time from approximately 3 h under conventional conditions to about 15–20 min. Under otherwise identical synthesis parameters, the UV-assisted route yielded copper nanoparticles with a reduced mean particle size and narrower size distribution (8–25 nm) compared with conventionally synthesized nanoparticles (20–50 nm). Improved colloidal stability of the UV-assisted CuNPs, reflected by higher absolute zeta potential values (–30 to − 38 mV), is attributed to enhanced phytochemical surface adsorption induced by rapid photoreduction. The UV-assisted nanoparticles also exhibited moderately enhanced antibacterial activity against Escherichia coli and Staphylococcus aureus , which is primarily associated with reduced particle size and increased surface reactivity rather than a change in chemical composition. Overall, the study demonstrates that UV-A irradiation functions as an effective auxiliary tool for process intensification , enabling faster synthesis and improved size distribution in phytogenically derived copper nanoparticles without altering the fundamental green chemistry framework. Conclusion This study presents a comparative green synthesis approach for copper nanoparticles using combined aqueous extracts of Camellia sinensis (green tea) and Ocimum sanctum (tulsi), together with UV-A–assisted photoreduction. The introduction of UV irradiation was found to accelerate the reduction process , decreasing the synthesis time from approximately 3 h under conventional conditions to about 15–20 min, while producing copper nanoparticles with a reduced mean particle size and narrower size distribution . Under otherwise identical synthesis conditions, the UV-assisted route yielded CuNPs in the size range of 8–25 nm with improved colloidal stability, as reflected by higher absolute zeta potential values (–30 to − 38 mV), compared with conventionally synthesized nanoparticles (20–50 nm; − 29.5 mV). Structural and morphological analyses confirmed the formation of crystalline copper nanoparticles with surface-associated phytochemical species, which are expected to contribute to enhanced dispersion stability. Antibacterial evaluation showed that both CuNP systems were active against Escherichia coli and Staphylococcus aureus , with UV-assisted CuNPs exhibiting moderately enhanced inhibitory effects , primarily attributable to reduced particle size and increased surface reactivity. Overall, the findings indicate that UV-A irradiation can serve as an effective auxiliary tool for process intensification in phytogenic copper nanoparticle synthesis, improving reaction kinetics and size distribution without altering the fundamental green chemistry framework. The approach is simple, reproducible, and potentially scalable, supporting its relevance for antimicrobial and related material applications. Limitations of the Study While the UV-assisted dual-extract synthesis approach demonstrated improved reaction kinetics, nanoparticle stability, and antibacterial performance, several limitations should be acknowledged. The study employed a single UV-A wavelength (365 nm) at a fixed irradiation intensity, and the influence of alternative wavelengths or varying light intensities on nucleation behavior and nanoparticle characteristics was not investigated. Exploration of these parameters could provide further insight into photoreduction dynamics and allow additional optimization of particle size and uniformity. In addition, the ratio of Camellia sinensis and Ocimum sanctum extracts was maintained at 1:1 throughout the study. Variations in extract composition or ratio may influence phytochemical concentration, reduction potential, and surface capping behavior, which were not examined here. Long-term colloidal stability was inferred from zeta potential measurements; however, extended storage stability studies over weeks or months were not conducted and would be valuable for assessing practical applicability. Furthermore, although the synthesized CuNPs exhibited strong antibacterial activity against both Gram-negative and Gram-positive bacteria, the study did not include cytotoxicity evaluations on mammalian cell lines. Such assessments are essential for determining biosafety and for extending the applicability of the synthesized nanoparticles toward biomedical and clinical applications. These aspects will be addressed in future investigations. Future Research Building on the outcomes of the present study, future investigations should focus on elucidating the mechanistic aspects of UV-assisted phytogenic reduction. Advanced analytical techniques, such as electron spin resonance spectroscopy and liquid chromatography–mass spectrometry, could provide deeper insight into UV-mediated electron transfer processes and the cooperative roles of catechins and eugenol during nanoparticle formation. Further optimization may be achieved by exploring alternative plant extract combinations or multi-extract systems to tailor nanoparticle size, stability, and functional properties for specific applications. The scalability of the synthesis process should also be examined using pilot-scale photoreactors to evaluate production efficiency, reproducibility, and economic feasibility. In addition, application-oriented studies, including the incorporation of CuNPs into antimicrobial coatings, polymer-based composites, and agricultural formulations, would help assess practical utility. Comprehensive toxicological evaluations, encompassing cytocompatibility, oxidative stress responses, and in vivo biocompatibility, are essential to establish the safety profile of the synthesized nanoparticles and to support their potential biomedical or environmental deployment. Declarations Acknowledgements The authors express their sincere gratitude to the Department of Chemistry, Government Degree College (Autonomous), Nagari, Andhra Pradesh, India, for providing laboratory facilities, instrumentation support, and technical assistance throughout the study. The authors also acknowledge the botanist of Government Degree College (A), Nagari, for authenticating the plant materials used in this work. The authors thank all supporting staff members for their cooperation during experimental and analytical work. Funding This research received no external funding from public, private, or commercial agencies. All experimental work was carried out using institutional facilities provided by Government Degree College (A), Nagari. Conflict of Interest The authors declare that there is no conflict of interest regarding the publication of this manuscript. Author Contributions P. Naveen: Conceptualization, experimental design, synthesis of copper nanoparticles, physicochemical characterization, antibacterial studies, data analysis, manuscript drafting, and figure preparation. Dr. Gopi Mamidi: Supervision, methodological guidance, validation of results, critical review of data interpretation, and final approval of the manuscript. Dr. A. Indira Priyadarsini: Botanical authentication of plant materials, guidance on phytochemical relevance, interpretation of plant–nanoparticle interactions, and review of the methodology related to plant extract preparation. Dr. G. Swathi: Biological interpretation of antibacterial results, microbiological data analysis, and contribution to the discussion related to antimicrobial mechanisms. All authors have read and approved the final manuscript. Ethical Approval This study did not involve human participants or animal models, and therefore did not require ethical approval . 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Role of nanotechnology in agriculture with special reference to management of insect pests . Appl Microbiol Biotechnol. 2012; 94:287–293. Singh P, Kim YJ, Zhang D, Yang DC. Biological synthesis of nanoparticles from plants and microorganisms . Trends Biotechnol. 2016; 34:588–599. Hunter RJ. Zeta Potential in Colloid Science: Principles and Applications . Academic Press; 1981. Bhattacharjee S. DLS and zeta potential – what they are and what they are not? J Control Release. 2016; 235:337–351. Ren G, Hu D, Cheng EWC, Vargas-Reus MA, Reip P, Allaker RP. Characterisation of copper oxide nanoparticles for antimicrobial applications . Int J Antimicrob Agents. 2009; 33:587–590. Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation of antimicrobials . Biotechnol Adv. 2009;27:76–83. Additional Declarations No competing interests reported. Supplementary Files UVGTsupply.docx floatimage1.png Graphical Abstract 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-8907851","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599007124,"identity":"9e5fb25e-ec5b-4fc3-842d-58cb1ff8a079","order_by":0,"name":"P. Naveen¹","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDADCXYGxgdAmoePeC3MDMwGIC1spGhhkwAxCGoxZz9j+ODjDgZ7yWbutMqvOXYybAzMDx/dwKPFsifH2HDmGYbE2cy8227LbksGOozN2DgHjxaDA2lp0rxtDAlyIC2S25iBWnjYpPFqOf8s/fffNgZ7kJZiyW31RGi5kXyMmbGNgRHkMMaP2w4To+XxYcneNonEmc28m6UZtx3nYWMm5JfziY0ffrbZ2Esc79348ee2ant+9uaHj/FpgQJwjDAw84BJwsoRgPEHKapHwSgYBaNgxAAAWso/ztBsae8AAAAASUVORK5CYII=","orcid":"","institution":"Govt.Degree College (A)","correspondingAuthor":true,"prefix":"","firstName":"P.","middleName":"","lastName":"Naveen¹","suffix":""},{"id":599007125,"identity":"f7f7d8e1-e444-43c9-8545-1ed1fd1dcddb","order_by":1,"name":"Gopi Mamidi²","email":"","orcid":"","institution":"Dr. VSK Government Degree College","correspondingAuthor":false,"prefix":"","firstName":"Gopi","middleName":"","lastName":"Mamidi²","suffix":""},{"id":599007126,"identity":"d4311f85-39c7-4a13-8dbd-56ca8f8c2b21","order_by":2,"name":"A. Indira Priyadarsini³","email":"","orcid":"","institution":"Govt.Degree College (A)","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"Indira","lastName":"Priyadarsini³","suffix":""},{"id":599007128,"identity":"bf021bc5-93e3-461b-896c-d474f438b69a","order_by":3,"name":"G. Swathi⁴","email":"","orcid":"","institution":"Govt.Degree College (A)","correspondingAuthor":false,"prefix":"","firstName":"G.","middleName":"","lastName":"Swathi⁴","suffix":""}],"badges":[],"createdAt":"2026-02-18 09:38:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8907851/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8907851/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103924149,"identity":"800296e6-0d65-45b7-a2c7-62cfb93dcd5b","added_by":"auto","created_at":"2026-03-04 15:02:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV–Vis Spectra of Conventional and UV-Assisted CuNPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUV–Vis absorption spectra showing the characteristic surface plasmon resonance (SPR) bands of copper nanoparticles synthesized by conventional and UV-assisted methods. The conventionally synthesized CuNPs exhibit a broader SPR band centered around \u003cstrong\u003e570–590 nm\u003c/strong\u003e, indicating a wider particle size distribution and possible aggregation. In contrast, the UV-assisted CuNPs display a \u003cstrong\u003esharper and slightly blue-shifted SPR peak at ~565–575 nm\u003c/strong\u003e, characteristic of smaller, more uniform nanoparticles formed under rapid UV-induced nucleation. The observed blue shift and peak sharpening confirm improved size control and enhanced optical properties in the UV-assisted synthesis route.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/41147c9996b5df54745e0167.png"},{"id":103924146,"identity":"a79dee29-efef-45fa-bfb5-c5a5b40fb914","added_by":"auto","created_at":"2026-03-04 15:02:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":359981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR Spectra of Conventional and UV-Assisted CuNPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFTIR spectra identifying functional groups responsible for reduction and stabilization of CuNPs. Major peaks include O–H stretching (~3400 cm⁻¹), C=O stretching (1635 cm⁻¹), aromatic and C–N vibrations (~1380 cm⁻¹), and C–O stretching (~1100 cm⁻¹). The increased peak intensity in UV-assisted CuNPs indicates a denser phytochemical capping layer.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/9bb473691c01774b62115bb0.png"},{"id":103924147,"identity":"6586b0b5-d7be-4389-8790-1d3ae77be0e9","added_by":"auto","created_at":"2026-03-04 15:02:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXRD Patterns of Conventional and UV-Assisted CuNPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX-ray diffraction profiles confirming the formation of face-centered cubic (FCC) copper nanoparticles with characteristic reflections at 2θ = 43.3° (111), 50.4° (200), and 74.1° (220). Minor Cu₂O peaks are present but reduced in UV-assisted samples. Crystallite size estimated by the Scherrer equation shows 25–35 nm for conventional CuNPs and 12–20 nm for UV-assisted CuNPs.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/6cafa57570d72228b2c33452.png"},{"id":103924151,"identity":"51a79bef-ed21-4bbf-860c-3b97b22f599d","added_by":"auto","created_at":"2026-03-04 15:02:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1093427,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/e5aed690ed2a37f6b3b16617.png"},{"id":104402317,"identity":"4c1ffea8-dcb6-4711-bb3f-d397402840de","added_by":"auto","created_at":"2026-03-11 12:15:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":619190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative stability and morphology of copper nanoparticles (CuNPs).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Conventional CuNPs: Schematic representation and TEM micrograph showing spherical nanoparticles with a particle size range of 20–50 nm and moderate dispersion. The zeta potential value of −29.5 mV indicates good colloidal stability.\u003c/p\u003e\n\u003cp\u003e(B) UV-assisted CuNPs: TEM image reveals highly uniform, well-dispersed spherical nanoparticles with smaller sizes (8–25 nm) and a visibly thicker phytochemical capping layer. The enhanced surface passivation results in improved colloidal stability, as evidenced by zeta potential values ranging from −30 to −38 mV.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/88d5c5ebc7346f567719a223.png"},{"id":103924153,"identity":"32c54cc0-7c80-4831-8903-2d74c41ae862","added_by":"auto","created_at":"2026-03-04 15:02:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":349911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibacterial activity of conventional and UV-assisted copper nanoparticles (CuNPs).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative zone of inhibition (ZOI) images showing antibacterial efficacy against \u003cem\u003eEscherichia coli\u003c/em\u003e (Gram-negative) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Gram-positive). Conventional CuNPs exhibited inhibition zones of 18.6 ± 0.8 mm against \u003cem\u003eE. coli\u003c/em\u003e and 21.4 ± 1.1 mm against \u003cem\u003eS. aureus\u003c/em\u003e. In comparison, UV-assisted CuNPs showed enhanced antibacterial activity with ZOI values of 20.1 ± 0.7 mm (\u003cem\u003eE. coli\u003c/em\u003e) and 22.9 ± 0.9 mm (\u003cem\u003eS. aureus\u003c/em\u003e). The increased inhibition zones observed for UV-assisted CuNPs are attributed to their smaller particle size, higher surface area, and improved surface reactivity.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/9c7f49cc5bec1b6158a08b51.png"},{"id":107413832,"identity":"39913166-7921-41ea-8fac-d92c985c947f","added_by":"auto","created_at":"2026-04-21 09:28:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3976992,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/dd5db1fe-c6c5-41e5-b8d9-baa2523c0865.pdf"},{"id":103924152,"identity":"71cbbc2b-3c4e-4dee-b6e9-5805e2e6a2cc","added_by":"auto","created_at":"2026-03-04 15:02:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7517725,"visible":true,"origin":"","legend":"","description":"","filename":"UVGTsupply.docx","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/bf4c93917193dfc1f6e8595e.docx"},{"id":103924150,"identity":"8118083e-1929-46ef-832e-80d1903e2fb5","added_by":"auto","created_at":"2026-03-04 15:02:25","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":750021,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8907851/v1/bcfc0a17e3877e44be5fafd3.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"UV-A–Assisted Green Synthesis of Copper Nanoparticles Using Dual Plant Extracts: A Comparative Study on Reaction Kinetics, Size Distribution, and Antibacterial Activity","fulltext":[{"header":"Highlights","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003eDual-extract green synthesis using \u003cem\u003eCamellia sinensis\u003c/em\u003e and \u003cem\u003eOcimum sanctum\u003c/em\u003e combined for the first time with UV-A photoreduction.\u003c/li\u003e\n \u003cli\u003eRapid synthesis completed within 15\u0026ndash;20 minutes compared with ~3 hours in the conventional method.\u003c/li\u003e\n \u003cli\u003eUV-assisted CuNPs exhibited ultra-small size (8\u0026ndash;25 nm) and high colloidal stability (\u0026ndash;30 to \u0026ndash;38 mV).\u003c/li\u003e\n \u003cli\u003eStrong antibacterial activity against both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, surpassing conventionally synthesized CuNPs.\u003c/li\u003e\n \u003cli\u003eEco-friendly, reproducible, and scalable method suitable for biomedical, agricultural, and catalytic applications.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eNanotechnology has emerged as a key area of research due to its wide-ranging impact across scientific and industrial sectors, including catalysis, energy storage, biomedical diagnostics, therapeutics, and environmental remediation [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among metallic nanomaterials, copper nanoparticles (CuNPs) have attracted considerable attention owing to their high electrical conductivity, catalytic efficiency, optical properties, and antimicrobial activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Compared to noble metals such as silver and gold, copper is inexpensive, earth-abundant, and more suitable for large-scale production, which enhances its practical applicability in industrial and biomedical fields [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Conventional physical and chemical routes for the synthesis of CuNPs often involve harsh reducing agents such as hydrazine or sodium borohydride, elevated temperatures, and multistep processing, leading to toxic byproducts and environmental concerns [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These drawbacks have driven increasing interest in green synthesis approaches that utilize biological systems as reducing and stabilizing agents. Plant-mediated synthesis is particularly advantageous due to the abundance of phytochemicals such as polyphenols, flavonoids, terpenoids, alkaloids, and organic acids, which can simultaneously reduce metal ions and stabilize the resulting nanoparticles through surface capping [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003cem\u003eCamellia sinensis\u003c/em\u003e (green tea) is rich in catechins, particularly epigallocatechin gallate (EGCG), which possess strong antioxidant and electron-donating properties and have been widely reported as effective reducing agents for metal nanoparticle synthesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, \u003cem\u003eOcimum sanctum\u003c/em\u003e (tulsi) contains bioactive compounds such as eugenol, rosmarinic acid, and ursolic acid, which exhibit redox activity and antimicrobial properties, making it a suitable candidate for green nanomaterial synthesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Several studies have reported the synthesis of CuNPs using individual plant extracts, including green tea and tulsi; however, investigations employing \u003cb\u003ecombined or dual-extract systems\u003c/b\u003e remain limited, despite their potential to enhance reduction efficiency and surface stabilization through synergistic phytochemical interactions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In parallel with phytogenic approaches, photo-assisted synthesis methods have gained attention as a means to accelerate nanoparticle formation. Ultraviolet (UV) irradiation supplies high-energy photons that promote rapid electron transfer, increase nucleation rates, and improve control over particle size and distribution [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Previous studies have demonstrated that UV-assisted green synthesis can significantly reduce reaction time and yield smaller, more uniform nanoparticles compared to conventional stirring-based methods [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the integration of UV-assisted photoreduction with \u003cb\u003edual-extract phytogenic systems for CuNP synthesis\u003c/b\u003e has not been systematically explored. In this context, the present study investigates a UV-assisted green synthesis strategy employing combined aqueous extracts of \u003cem\u003eC. sinensis\u003c/em\u003e and \u003cem\u003eO. sanctum\u003c/em\u003e for the preparation of CuNPs. The approach aims to evaluate the effect of UV irradiation on reaction kinetics, particle size, stability, and antibacterial performance, while elucidating the cooperative role of phytochemicals from both extracts in nanoparticle formation and stabilization.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eCopper (II) sulphate pentahydrate (CuSO₄\u0026middot;5H₂O, analytical grade) and absolute ethanol were obtained from Sigma-Aldrich and used without further purification. Copper sulphate was selected as the precursor salt due to its high solubility and stability in aqueous nanoparticle synthesis systems [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Ethanol was used during washing steps to remove excess phytochemicals and residual impurities from the nanoparticle surface [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Fresh leaves of \u003cem\u003eCamellia sinensis\u003c/em\u003e (green tea) were collected from the Wayanad Tea Plantations, Kerala, India, a region known for polyphenol-rich tea foliage suitable for green synthesis applications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Leaves of \u003cem\u003eOcimum sanctum\u003c/em\u003e (tulsi) were collected from Nagari, Chittoor District, Andhra Pradesh, India. Both plant materials were authenticated by a qualified botanist at Government Degree College (Autonomous), Nagari, to ensure taxonomic accuracy and experimental reproducibility [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Deionized water was used throughout all experimental procedures, and all glassware was thoroughly cleaned and dried prior to use to avoid ionic or particulate contamination [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of Plant Extracts\u003c/h2\u003e \u003cp\u003eAqueous extracts of \u003cem\u003eCamellia sinensis\u003c/em\u003e and \u003cem\u003eOcimum sanctum\u003c/em\u003e were prepared following well-established green synthesis protocols with minor modifications [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, 10 g of dried leaf powder was added to 100 mL of deionized water and boiled for 15 min to extract phytochemicals such as polyphenols, flavonoids, and terpenoids that act as reducing and stabilizing agents during nanoparticle formation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. After cooling to room temperature, the extracts were filtered through Whatman No. 1 filter paper to remove insoluble residues. The pH of each extract was recorded (typically 5.4\u0026ndash;6.2), as solution pH plays a critical role in metal ion reduction kinetics and nanoparticle nucleation behavior [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The filtrates were stored at 4\u0026deg;C and used within 48 h to preserve the activity of key biomolecules involved in copper ion reduction and surface capping [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Synthesis of Copper Nanoparticles\u003c/h2\u003e \u003cp\u003eCopper nanoparticles were synthesized using both conventional and UV-assisted photoreduction routes. For the conventional method, 50 mL of 1 mM CuSO₄\u0026middot;5H₂O solution was mixed with equal volumes (25 mL each) of \u003cem\u003eC. sinensis\u003c/em\u003e and \u003cem\u003eO. sanctum\u003c/em\u003e extracts. The reaction mixture was stirred at room temperature under its natural pH (5.4\u0026ndash;6.2) without external adjustment, consistent with typical phytogenic nanoparticle synthesis conditions [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. A gradual color change from pale blue to brown indicated the reduction of Cu\u0026sup2;⁺ ions to metallic copper nanoparticles [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. After 3 h of stirring, the suspension was centrifuged at 10,000 rpm for 15 min. The resulting pellet was washed repeatedly with deionized water and ethanol to remove unreacted ions and loosely bound phytochemicals, followed by drying in a vacuum oven at 60\u0026deg;C for 12 h [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. For the UV-assisted synthesis, the same reaction mixture was exposed to UV-A irradiation (365 nm, ~\u0026thinsp;10 mW cm⁻\u0026sup2;) in a photoreactor. A rapid color change was observed within 15\u0026ndash;20 min, indicating accelerated reduction. UV irradiation facilitates electron transfer from phytochemicals to Cu\u0026sup2;⁺ ions, leading to faster nucleation and improved control over nanoparticle size and distribution [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The UV-assisted CuNPs were collected and purified using the same procedure as the conventional samples to ensure direct comparability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization\u003c/h2\u003e \u003cp\u003eUV\u0026ndash;visible spectroscopy was carried out using a \u003cb\u003eShimadzu UV-2600 spectrophotometer\u003c/b\u003e over a wavelength range of \u003cb\u003e300\u0026ndash;800 nm\u003c/b\u003e with a scan speed of \u003cb\u003e200 nm min⁻\u0026sup1;\u003c/b\u003e and a spectral resolution of \u003cb\u003e1 nm\u003c/b\u003e to monitor the formation of copper nanoparticles through their characteristic surface plasmon resonance (SPR) absorption band [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The acquired UV\u0026ndash;Vis spectra were subjected only to \u003cb\u003ebaseline correction using the instrument\u0026rsquo;s native software\u003c/b\u003e to remove background noise; no smoothing, peak shifting, or intensity manipulation was performed.\u003c/p\u003e \u003cp\u003eFourier transform infrared (FTIR) spectra were recorded using a \u003cb\u003ePerkinElmer Spectrum Two spectrometer\u003c/b\u003e in the range of \u003cb\u003e4000\u0026ndash;400 cm⁻\u0026sup1;\u003c/b\u003e with a resolution of \u003cb\u003e4 cm⁻\u0026sup1;\u003c/b\u003e to identify functional groups involved in phytochemical reduction and surface stabilization of the nanoparticles [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eX-ray diffraction (XRD) analysis was conducted using a \u003cb\u003eRigaku Miniflex diffractometer\u003c/b\u003e equipped with \u003cb\u003eCu-Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;)\u003c/b\u003e, operating at \u003cb\u003e40 kV and 15 mA\u003c/b\u003e. Diffraction patterns were recorded over a \u003cb\u003e2θ range of 20\u0026ndash;80\u0026deg;\u003c/b\u003e with a step size of \u003cb\u003e0.02\u0026deg;\u003c/b\u003e. The XRD data were processed solely for background subtraction using standard instrument software, and crystallite size was estimated using the Scherrer equation without instrumental broadening correction.\u003c/p\u003e \u003cp\u003eSurface morphology and particle size distribution were examined using \u003cb\u003escanning electron microscopy (SEM, JEOL JSM-7600F)\u003c/b\u003e operated at an accelerating voltage of \u003cb\u003e15 kV\u003c/b\u003e, and \u003cb\u003etransmission electron microscopy (TEM, JEOL JEM-2100)\u003c/b\u003e operated at \u003cb\u003e200 kV\u003c/b\u003e. \u003cb\u003eAll SEM and TEM micrographs include calibrated scale bars\u003c/b\u003e, enabling accurate interpretation of particle size and morphology. Particle size distributions were determined by measuring over \u003cb\u003e100 individual nanoparticles\u003c/b\u003e using ImageJ software.\u003c/p\u003e \u003cp\u003eColloidal stability was evaluated by measuring \u003cb\u003ezeta potential\u003c/b\u003e using a \u003cb\u003eMalvern Zetasizer Nano ZS\u003c/b\u003e at \u003cb\u003e25\u0026deg;C\u003c/b\u003e, with samples appropriately diluted in deionized water to avoid multiple scattering effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Antibacterial Activity Assay\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the synthesized CuNPs was evaluated using the agar disc diffusion method against \u003cem\u003eEscherichia coli\u003c/em\u003e (Gram-negative) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Gram-positive), following standard antimicrobial susceptibility testing guidelines [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Bacterial cultures were evenly spread on Mueller\u0026ndash;Hinton agar plates, and sterile paper discs impregnated with CuNP suspensions (1 mg mL⁻\u0026sup1;) were placed on the agar surface. The plates were incubated at 37\u0026deg;C for 24 h, after which the diameter of the inhibition zones was measured in millimeters. The disc diffusion assay was selected due to its reproducibility and suitability for comparative evaluation of nanoparticle-mediated antibacterial activity [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Visual Observation and Reaction Kinetics\u003c/h2\u003e \u003cp\u003eThe formation of copper nanoparticles was initially indicated by a gradual color change of the reaction mixture from pale blue to brown, which is commonly associated with the development of surface plasmon resonance in copper-based nanostructures [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the conventional synthesis route, this transformation occurred over approximately 3 h, reflecting the relatively slow reduction kinetics typical of plant-mediated nanoparticle synthesis systems [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].In contrast, the UV-assisted reaction exhibited a rapid color transition within 15\u0026ndash;20 min. The accelerated reaction can be attributed to UV-A irradiation facilitating photon-induced electron transfer from phytochemicals to Cu\u0026sup2;⁺ ions, thereby increasing the nucleation rate and lowering the activation energy required for reduction [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Similar enhancements in reaction kinetics under UV or photo-assisted conditions have been reported for other green-synthesized metal nanoparticles, supporting the reproducibility of this approach [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 UV\u0026ndash;Visible Spectroscopy\u003c/h2\u003e \u003cp\u003eUV\u0026ndash;Vis spectra confirmed the formation of CuNPs in both synthesis routes. Conventionally synthesized CuNPs exhibited a broad surface plasmon resonance band in the range of 570\u0026ndash;590 nm, which is indicative of a wider particle size distribution and partial aggregation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In comparison, UV-assisted CuNPs displayed a sharper and slightly blue-shifted SPR peak centered around 565\u0026ndash;575 nm. Such spectral narrowing and blue shift are commonly associated with smaller particle sizes and improved size uniformity, particularly under conditions of rapid nucleation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These observations indicate that UV irradiation not only accelerates nanoparticle formation but also contributes to improved control over particle growth.\u003c/p\u003e \u003cp\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 FTIR Analysis\u003c/h2\u003e \u003cp\u003eFTIR spectra were analyzed to identify the functional groups involved in copper ion reduction and nanoparticle stabilization. A broad absorption band around 3400 cm⁻\u0026sup1; was attributed to O\u0026ndash;H stretching vibrations of phenolic compounds and polyphenols present in the plant extracts [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The band observed near 1635 cm⁻\u0026sup1; corresponded to C\u0026thinsp;=\u0026thinsp;O stretching vibrations of flavonoids and organic acids, while peaks in the region of 1380 cm⁻\u0026sup1; were associated with aromatic and C\u0026ndash;N vibrations of plant metabolites [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The presence of a prominent band near 1100 cm⁻\u0026sup1;, corresponding to C\u0026ndash;O stretching vibrations, further supports the involvement of alcohols and ethers in nanoparticle stabilization. Increased band intensity observed in UV-assisted CuNPs suggests stronger phytochemical adsorption, which likely contributes to enhanced surface passivation and colloidal stability [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 XRD Analysis\u003c/h2\u003e \u003cp\u003eXRD patterns confirmed the crystalline nature of the synthesized CuNPs. Diffraction peaks at 2θ values of approximately 43.3\u0026deg;, 50.4\u0026deg;, and 74.1\u0026deg; corresponded to the (111), (200), and (220) planes of face-centered cubic copper, consistent with standard reference data [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].The average crystallite size estimated using the Scherrer equation ranged from 25\u0026ndash;35 nm for conventionally synthesized CuNPs and 12\u0026ndash;20 nm for UV-assisted CuNPs, indicating that photoreduction effectively restricts crystal growth by promoting rapid nucleation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Minor diffraction peaks corresponding to Cu₂O were also detected, which is commonly observed in green-synthesized copper nanoparticles due to surface oxidation during processing or exposure to air [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Morphological Analysis (SEM and TEM)\u003c/h2\u003e \u003cp\u003eSEM analysis revealed predominantly \u003cb\u003espherical to quasi-spherical copper nanoparticles\u003c/b\u003e for both synthesis routes, with \u003cb\u003elimited aggregation\u003c/b\u003e observed in some regions, which may arise from interparticle interactions mediated by surface-bound phytochemicals [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. TEM provided higher-resolution insight into particle size and spatial distribution. Nanoparticles synthesized via the conventional route exhibited a \u003cb\u003ebroad size distribution in the range of 20\u0026ndash;50 nm\u003c/b\u003e, consistent with gradual nucleation and growth occurring over an extended reaction time. In contrast, UV-assisted synthesis produced copper nanoparticles with a \u003cb\u003ereduced mean size and a narrower distribution (8\u0026ndash;25 nm)\u003c/b\u003e, along with improved dispersion under identical synthesis conditions. In several TEM micrographs of UV-assisted CuNPs, a \u003cb\u003ethin, low-contrast organic layer\u003c/b\u003e was observed surrounding the metallic cores, which is attributed to adsorbed phytochemical species from the plant extracts. The presence of this surface layer is expected to contribute to enhanced dispersion stability and reduced particle coalescence, in agreement with zeta potential measurements and previous reports [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Quantitative particle size analysis based on TEM measurements indicated that the average particle size of conventionally synthesized CuNPs was \u003cb\u003e34.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 nm\u003c/b\u003e, whereas UV-assisted CuNPs exhibited a \u003cb\u003esignificantly reduced average size of 15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 nm\u003c/b\u003e. This observed size reduction is \u003cb\u003econsistent with UV-induced acceleration of nucleation\u003c/b\u003e, which limits subsequent particle growth under otherwise identical synthesis conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Zeta Potential and Colloidal Stability\u003c/h2\u003e \u003cp\u003eZeta potential measurements were used to evaluate colloidal stability. Conventionally synthesized CuNPs exhibited a zeta potential of \u0026minus;\u0026thinsp;29.5 mV, indicating moderate to good electrostatic stability. UV-assisted CuNPs showed higher absolute zeta potential values (\u0026ndash;30 to \u0026minus;\u0026thinsp;38 mV), exceeding the commonly accepted\u0026thinsp;\u0026plusmn;\u0026thinsp;30 mV threshold for highly stable colloidal systems [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The improved stability of UV-assisted CuNPs can be attributed to denser phytochemical capping and increased surface charge, which enhance electrostatic repulsion and steric stabilization [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Antibacterial Activity\u003c/h2\u003e \u003cp\u003eBoth CuNP systems demonstrated significant antibacterial activity against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. The observed inhibition zones confirm the effectiveness of CuNPs against both Gram-negative and Gram-positive bacteria, consistent with previously reported mechanisms involving membrane disruption, ion release, and oxidative stress [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. UV-assisted CuNPs exhibited slightly higher antibacterial activity compared to conventionally synthesized nanoparticles. This enhancement can be attributed to smaller particle size, higher surface area, and improved interaction with bacterial cell membranes [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Similar size-dependent antibacterial trends have been reported for copper and other metal nanoparticles synthesized via green routes. A comparative summary of synthesis route, particle size, morphology, colloidal stability, and antibacterial performance of conventionally synthesized and UV-assisted CuNPs is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative characteristics of conventionally synthesized and UV-assisted copper nanoparticles\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConventional CuNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUV-assisted CuNPs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSynthesis route\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChemical/thermal reduction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUV-assisted green synthesis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReducing agents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChemical reagents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlant-derived phytochemicals\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCapping/Stabilizing agents\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAbsent or weakly bound\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePhytochemical capping (polyphenols, flavonoids)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAverage particle size (TEM)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20\u0026ndash;50 nm\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e8\u0026ndash;25 nm\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eParticle size distribution\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eBroad\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eNarrow\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eParticle shape\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSpherical with partial aggregation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eUniform, well-defined spherical\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSurface morphology\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRough / clustered\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eSmooth surface with visible organic shell\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDegree of agglomeration\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHigh\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMinimal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDispersion behavior\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eModerately dispersed\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eHighly dispersed\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eZeta potential\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026minus;29.5 mV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026minus;30 to \u0026minus;\u0026thinsp;38 mV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eColloidal stability\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eModerate stability\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eHigh colloidal stability\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRole of phytochemicals\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNot applicable\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eAct as reducing\u0026thinsp;+\u0026thinsp;capping agents\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOverall structural uniformity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eLower\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eHigher\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"NOVELTY OF THE WORK","content":"\u003cp\u003eThe present study reports a \u003cb\u003ecomparative green synthesis approach\u003c/b\u003e that combines a dual-extract phytogenic system, based on \u003cem\u003eCamellia sinensis\u003c/em\u003e (green tea) and \u003cem\u003eOcimum sanctum\u003c/em\u003e (tulsi), with UV-A\u0026ndash;assisted photoreduction for the preparation of copper nanoparticles. The combined presence of polyphenolic constituents from green tea and eugenol-rich compounds from tulsi, when coupled with UV irradiation, was found to \u003cb\u003eaccelerate reduction kinetics\u003c/b\u003e, decreasing the reaction time from approximately 3 h under conventional conditions to about 15\u0026ndash;20 min. Under otherwise identical synthesis parameters, the UV-assisted route yielded copper nanoparticles with a \u003cb\u003ereduced mean particle size and narrower size distribution\u003c/b\u003e (8\u0026ndash;25 nm) compared with conventionally synthesized nanoparticles (20\u0026ndash;50 nm). Improved colloidal stability of the UV-assisted CuNPs, reflected by higher absolute zeta potential values (\u0026ndash;30 to \u0026minus;\u0026thinsp;38 mV), is attributed to enhanced phytochemical surface adsorption induced by rapid photoreduction. The UV-assisted nanoparticles also exhibited \u003cb\u003emoderately enhanced antibacterial activity\u003c/b\u003e against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, which is primarily associated with reduced particle size and increased surface reactivity rather than a change in chemical composition. Overall, the study demonstrates that \u003cb\u003eUV-A irradiation functions as an effective auxiliary tool for process intensification\u003c/b\u003e, enabling faster synthesis and improved size distribution in phytogenically derived copper nanoparticles without altering the fundamental green chemistry framework.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a \u003cb\u003ecomparative green synthesis approach\u003c/b\u003e for copper nanoparticles using combined aqueous extracts of \u003cem\u003eCamellia sinensis\u003c/em\u003e (green tea) and \u003cem\u003eOcimum sanctum\u003c/em\u003e (tulsi), together with UV-A\u0026ndash;assisted photoreduction. The introduction of UV irradiation was found to \u003cb\u003eaccelerate the reduction process\u003c/b\u003e, decreasing the synthesis time from approximately 3 h under conventional conditions to about 15\u0026ndash;20 min, while producing copper nanoparticles with a \u003cb\u003ereduced mean particle size and narrower size distribution\u003c/b\u003e. Under otherwise identical synthesis conditions, the UV-assisted route yielded CuNPs in the size range of \u003cb\u003e8\u0026ndash;25 nm\u003c/b\u003e with improved colloidal stability, as reflected by higher absolute zeta potential values (\u0026ndash;30 to \u0026minus;\u0026thinsp;38 mV), compared with conventionally synthesized nanoparticles (20\u0026ndash;50 nm; \u0026minus;\u0026thinsp;29.5 mV). Structural and morphological analyses confirmed the formation of crystalline copper nanoparticles with surface-associated phytochemical species, which are expected to contribute to enhanced dispersion stability. Antibacterial evaluation showed that both CuNP systems were active against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, with UV-assisted CuNPs exhibiting \u003cb\u003emoderately enhanced inhibitory effects\u003c/b\u003e, primarily attributable to reduced particle size and increased surface reactivity. Overall, the findings indicate that \u003cb\u003eUV-A irradiation can serve as an effective auxiliary tool for process intensification\u003c/b\u003e in phytogenic copper nanoparticle synthesis, improving reaction kinetics and size distribution without altering the fundamental green chemistry framework. The approach is simple, reproducible, and potentially scalable, supporting its relevance for antimicrobial and related material applications.\u003c/p\u003e"},{"header":"Limitations of the Study","content":"\u003cp\u003eWhile the UV-assisted dual-extract synthesis approach demonstrated improved reaction kinetics, nanoparticle stability, and antibacterial performance, several limitations should be acknowledged. The study employed a single UV-A wavelength (365 nm) at a fixed irradiation intensity, and the influence of alternative wavelengths or varying light intensities on nucleation behavior and nanoparticle characteristics was not investigated. Exploration of these parameters could provide further insight into photoreduction dynamics and allow additional optimization of particle size and uniformity. In addition, the ratio of \u003cem\u003eCamellia sinensis\u003c/em\u003e and \u003cem\u003eOcimum sanctum\u003c/em\u003e extracts was maintained at 1:1 throughout the study. Variations in extract composition or ratio may influence phytochemical concentration, reduction potential, and surface capping behavior, which were not examined here. Long-term colloidal stability was inferred from zeta potential measurements; however, extended storage stability studies over weeks or months were not conducted and would be valuable for assessing practical applicability. Furthermore, although the synthesized CuNPs exhibited strong antibacterial activity against both Gram-negative and Gram-positive bacteria, the study did not include cytotoxicity evaluations on mammalian cell lines. Such assessments are essential for determining biosafety and for extending the applicability of the synthesized nanoparticles toward biomedical and clinical applications. These aspects will be addressed in future investigations.\u003c/p\u003e"},{"header":"Future Research","content":"\u003cp\u003eBuilding on the outcomes of the present study, future investigations should focus on elucidating the mechanistic aspects of UV-assisted phytogenic reduction. Advanced analytical techniques, such as electron spin resonance spectroscopy and liquid chromatography\u0026ndash;mass spectrometry, could provide deeper insight into UV-mediated electron transfer processes and the cooperative roles of catechins and eugenol during nanoparticle formation. Further optimization may be achieved by exploring alternative plant extract combinations or multi-extract systems to tailor nanoparticle size, stability, and functional properties for specific applications. The scalability of the synthesis process should also be examined using pilot-scale photoreactors to evaluate production efficiency, reproducibility, and economic feasibility. In addition, application-oriented studies, including the incorporation of CuNPs into antimicrobial coatings, polymer-based composites, and agricultural formulations, would help assess practical utility. Comprehensive toxicological evaluations, encompassing cytocompatibility, oxidative stress responses, and in vivo biocompatibility, are essential to establish the safety profile of the synthesized nanoparticles and to support their potential biomedical or environmental deployment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude to the Department of Chemistry, Government Degree College (Autonomous), Nagari, Andhra Pradesh, India, for providing laboratory facilities, instrumentation support, and technical assistance throughout the study. The authors also acknowledge the botanist of Government Degree College (A), Nagari, for authenticating the plant materials used in this work. The authors thank all supporting staff members for their cooperation during experimental and analytical work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received \u003cstrong\u003eno external funding\u003c/strong\u003e from public, private, or commercial agencies. All experimental work was carried out using institutional facilities provided by Government Degree College (A), Nagari.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that \u003cstrong\u003ethere is no conflict of interest\u003c/strong\u003e regarding the publication of this manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e Author Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP. Naveen: Conceptualization, experimental design, synthesis of copper nanoparticles, physicochemical characterization, antibacterial studies, data analysis, manuscript drafting, and figure preparation.\u003c/p\u003e\n\u003cp\u003eDr. Gopi Mamidi: Supervision, methodological guidance, validation of results, critical review of data interpretation, and final approval of the manuscript.\u003c/p\u003e\n\u003cp\u003eDr. A. Indira Priyadarsini: Botanical authentication of plant materials, guidance on phytochemical relevance, interpretation of plant\u0026ndash;nanoparticle interactions, and review of the methodology related to plant extract preparation.\u003c/p\u003e\n\u003cp\u003eDr. G. Swathi: Biological interpretation of antibacterial results, microbiological data analysis, and contribution to the discussion related to antimicrobial mechanisms.\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants or animal models, and therefore \u003cstrong\u003edid not require ethical approval\u003c/strong\u003e. All microbiological assays were performed following standard laboratory biosafety guidelines.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e Data Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in the manuscript. Additional datasets can be provided by the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRao CNR, Cheetham AK. \u003cem\u003eScience and technology of nanomaterials: current status and future prospects\u003c/em\u003e. J Mater Chem. 2001; 11:2887\u0026ndash;2894.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoco MC. \u003cem\u003eNanotechnology: convergence with modern biology and medicine\u003c/em\u003e. Curr Opin Biotechnol. 2003; 14:337\u0026ndash;346.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan I, Saeed K, Khan I. \u003cem\u003eNanoparticles: properties, applications and toxicities\u003c/em\u003e. Arab J Chem. 2019; 12:908\u0026ndash;931.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNasrollahzadeh M, Sajjadi M, Sajadi SM, Issaabadi Z. \u003cem\u003eGreen synthesis of copper nanoparticles\u003c/em\u003e. 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J Photochem Photobiol B. 2020; 202:111682.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdelghany TM, Al-Rajhi AMH, Al Abboud MA, et al. \u003cem\u003eRecent advances in green synthesis of nanoparticles and their biomedical applications\u003c/em\u003e. Microorganisms. 2018; 6:36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLink S, El-Sayed MA. \u003cem\u003eSize and temperature dependence of the plasmon absorption of colloidal gold nanoparticles\u003c/em\u003e. J Phys Chem B. 1999; 103:4212\u0026ndash;4217.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmendola V, Meneghetti M. \u003cem\u003eSize evaluation of gold nanoparticles by UV\u0026ndash;Vis spectroscopy\u003c/em\u003e. J Phys Chem C. 2009; 113:4277\u0026ndash;4285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrikar SK, Giri DD, Pal DB, Mishra PK, Upadhyay SN. \u003cem\u003eGreen synthesis of silver nanoparticles: a review\u003c/em\u003e. Green Sustain Chem. 2016; 6:34\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoates J. \u003cem\u003eInterpretation of infrared spectra, a practical approach\u003c/em\u003e. Encyclopedia of Analytical Chemistry. Wiley; 2000.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSocrates G. \u003cem\u003eInfrared and Raman Characteristic Group Frequencies\u003c/em\u003e. 3rd ed. Wiley; 2001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMittal AK, Chisti Y, Banerjee UC. \u003cem\u003eSynthesis of metallic nanoparticles using plant extracts\u003c/em\u003e. Biotechnol Adv. 2013; 31:346\u0026ndash;356.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCullity BD, Stock SR. \u003cem\u003eElements of X-Ray Diffraction\u003c/em\u003e. 3rd ed. Prentice Hall; 2001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatterson AL. \u003cem\u003eThe Scherrer formula for X-ray particle size determination\u003c/em\u003e. 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Academic Press; 1981.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhattacharjee S. \u003cem\u003eDLS and zeta potential \u0026ndash; what they are and what they are not?\u003c/em\u003e J Control Release. 2016; 235:337\u0026ndash;351.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen G, Hu D, Cheng EWC, Vargas-Reus MA, Reip P, Allaker RP. \u003cem\u003eCharacterisation of copper oxide nanoparticles for antimicrobial applications\u003c/em\u003e. Int J Antimicrob Agents. 2009; 33:587\u0026ndash;590.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRai M, Yadav A, Gade A. \u003cem\u003eSilver nanoparticles as a new generation of antimicrobials\u003c/em\u003e. Biotechnol Adv. 2009;27:76\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"green synthesis, copper nanoparticles, UV-assisted photoreduction, Camellia sinensis, Ocimum sanctum, antimicrobial activity, sustainable nanotechnology","lastPublishedDoi":"10.21203/rs.3.rs-8907851/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8907851/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of environmentally benign routes for the synthesis of metallic nanoparticles is an important objective in contemporary nanomaterials research, particularly to minimize reliance on toxic chemical reductants and stabilizers. In the present study, copper nanoparticles (CuNPs) were synthesized using aqueous extracts of \u003cem\u003eCamellia sinensis\u003c/em\u003e (green tea) and \u003cem\u003eOcimum sanctum\u003c/em\u003e (tulsi), which functioned simultaneously as reducing and capping agents. Two synthesis routes were systematically investigated: a conventional phytogenic reduction process and an ultraviolet (UV-A, 365 nm) assisted photoreduction approach. In the conventional method, gradual reduction of Cu\u0026sup2;⁺ ions over 3 h yielded predominantly spherical CuNPs with particle sizes in the range of 20\u0026ndash;50 nm and a zeta potential of \u0026minus;\u0026thinsp;29.5 mV, indicating moderate colloidal stability. In contrast, UV-assisted synthesis significantly accelerated nanoparticle formation, completing the reaction within 15\u0026ndash;20 min and producing smaller particles (8\u0026ndash;25 nm) with improved stability (zeta potential between \u0026minus;\u0026thinsp;30 and \u0026minus;\u0026thinsp;38 mV). UV\u0026ndash;visible spectroscopy revealed distinct surface plasmon resonance bands for both samples, with the UV-assisted CuNPs exhibiting a sharper and slightly blue-shifted peak, consistent with reduced particle size and narrower size distribution. Fourier transform infrared spectroscopy confirmed the involvement of polyphenolic and phenolic constituents, including catechin- and eugenol-type moieties, in the reduction and stabilization of CuNPs. X-ray diffraction analysis verified the formation of crystalline face-centered cubic copper with minor contributions from Cu₂O, while transmission electron microscopy corroborated the enhanced uniformity of UV-assisted nanoparticles. The antibacterial activity of the synthesized CuNPs was evaluated against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, revealing effective growth inhibition for both bacterial strains. UV-assisted CuNPs exhibited marginally higher antibacterial efficacy, which is attributed to their smaller size and improved surface stabilization. Overall, this study demonstrates that UV-assisted phytogenic synthesis offers a rapid and reproducible route to stable copper nanoparticles, while maintaining the advantages of green chemistry. The findings highlight the potential of combining plant-derived reductants with photochemical activation to achieve controlled nanoparticle synthesis for antimicrobial and related applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"UV-A–Assisted Green Synthesis of Copper Nanoparticles Using Dual Plant Extracts: A Comparative Study on Reaction Kinetics, Size Distribution, and Antibacterial Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-04 15:02:20","doi":"10.21203/rs.3.rs-8907851/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":"70d8a79a-6eea-4609-98bd-f61ebe43e2cc","owner":[],"postedDate":"March 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T09:27:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-04 15:02:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8907851","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8907851","identity":"rs-8907851","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}