Novel Prodigiosin-Driven Biogenic Sol-Gel Synthesis of Copper Oxide Nanoparticles Using Serratia rubidaea: Extraction, Dual Role as Bio-Reducing and Capping Agent, and Enhanced Cytotoxic Potential for Biomedical Application

Novel Prodigiosin-Driven Biogenic Sol-Gel Synthesis of Copper Oxide Nanoparticles Using Serratia rubidaea: Extraction, Dual Role as Bio-Reducing and Capping Agent, and Enhanced Cytotoxic Potential for Biomedical Application | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel Prodigiosin-Driven Biogenic Sol-Gel Synthesis of Copper Oxide Nanoparticles Using Serratia rubidaea: Extraction, Dual Role as Bio-Reducing and Capping Agent, and Enhanced Cytotoxic Potential for Biomedical Application Kartikey J. Chavan, Sarang R. Bhagwat, Vineet D.P. Kala, Arjun R. Potinde, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7801875/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Journal of Sol-Gel Science and Technology → Version 1 posted 7 You are reading this latest preprint version Abstract This study presents a sustainable synthesis of copper oxide (CuO) nanoparticles via a sol-gel approach utilizing prodigiosin pigment extracted from Serratia rubidaea as a green biogenic agent. To the best of our knowledge, this is the first report of employing prodigiosin from Serratia rubidaea in the sol-gel synthesis of CuO nanoparticles. Copper sulfate pentahydrate (CuSO₄·5H₂O) served as the precursor, reacting with the bio-pigment under controlled conditions to yield CuO nanomaterials. Comprehensive physicochemical characterization confirmed nanoparticle formation and composition: scanning electron microscopy (SEM) revealed morphology, Fourier-transform infrared (FTIR) spectroscopy identified characteristic Cu–O vibrational modes, energy-dispersive X-ray spectroscopy (EDAX) established the presence and proportions of copper, oxygen, and carbon, while X-ray diffraction (XRD) confirmed the monoclinic phase (JCPDS Card No. 00-001-1117). Ultraviolet-visible (UV–Vis) spectroscopy displayed absorption peaks at 329.5, 365.0, and 388.5 nm, with an estimated optical band gap of 2.95 eV. The dual role of prodigiosin as a natural reducing and capping agent highlights the eco-friendly and innovative nature of this synthesis. The resultant CuO nanoparticles exhibit properties comparable or superior to those synthesized with other biological agents reported recently. Preliminary cytotoxicity assessment using the Sulforhodamine B (SRB) assay against MCF-7 breast cancer cells demonstrated a dose-dependent reduction in cell viability, achieving a maximum mortality of 32.9% at 80 µg/mL. Although the half-maximal inhibitory concentration (IC₅₀) was not reached within the tested range, these findings suggest promising anticancer potential and warrant further biomedical investigations of the synthesized biofunctionalized CuO nanoparticles. copper oxide nanoparticles sol-gel synthesis prodigiosin Serratia rubidaea green synthesis cytotoxicity anticancer activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights • First report on the extraction and use of prodigiosin pigment from as a dual-function bio-reductant and stabilizer for sol-gel synthesis of copper oxide nanoparticles. • Environmentally friendly, sustainable green synthesis route producing monoclinic CuO nanoparticles with controlled size and morphology. • Comprehensive physicochemical characterization confirms phase purity, nanoscale crystallite size, unique surface chemistry, and optical properties influenced by prodigiosin capping. • Preliminary in vitro cytotoxicity studies demonstrate promising, concentration-dependent anticancer activity of CuO nanoparticles against MCF-7 breast cancer cells. • Potential multifunctional biomedical applications through integration of biogenic pigment and metal oxide nanomaterial harnessing biofunctional and therapeutic synergism. 1. Introduction Copper oxide (CuO) nanoparticles have garnered substantial attention in recent years due to their unique physicochemical properties and broad spectrum of applications, particularly in anticancer therapeutics [ 1 , 2 ]. As prominent members of the metal oxide family, copper oxides primarily exist in two forms: cuprous oxide (Cu₂O) and cupric oxide (CuO) [ 3 ]. These semiconducting metal oxides actively participate in biological processes, notably cancer cell inhibition, owing to their electronic properties [ 4 ]. Metal oxides are classified based on their chemical behaviour into acidic, basic, amphoteric, and neutral oxides. CuO, a basic oxide, reacts with acids to form salts and water, a fundamental property that underpins its reactivity in diverse biomedical and catalytic contexts [ 5 ]. Understanding this chemical classification is pivotal for advancing the tailored application of metal oxides in cancer therapy and beyond [ 6 ]. Multiple synthetic methodologies have been devised to fabricate CuO nanoparticles, each impacting particle size, morphology, and functional performance. Notable approaches include chemical precipitation, hydrothermal synthesis, electrochemical methods, and the sol-gel process [ 7 , 8 ]. Among these, the sol-gel method has acquired prominence for its operational simplicity and ability to yield nanoparticles with controlled size and morphology via a liquid-phase to gel-phase transition, facilitating tunable physico-chemical properties [ 9 ]. Increasing interest in environmentally benign synthesis has focused attention on integrating biological catalysts into the sol-gel technique. This hybrid approach not only simplifies synthesis but also enhances nanoparticle stability and biocompatibility [ 10 , 11 ]. Biological reducing agents derived from plant extracts and microbial systems offer sustainable, eco-friendly alternatives to conventional chemical reagents, producing nanomaterials with distinctive morphological and biological properties that may potentiate anticancer efficacy [ 12 , 13 ] Prodigiosin, a bioactive red pigment predominantly produced by Serratia species, including Serratia rubidaea , embodies such a biogenic agent with multifaceted biological activities, including antimicrobial, immunosuppressive, and anticancer effects [ 14 , 15 ]. Its role as an electron donor and capping agent during nanoparticle synthesis facilitates uniform nucleation and growth, yielding nanoparticles with enhanced physicochemical and biological profiles [ 16 ]. The inherent cytotoxicity of prodigiosin against diverse cancer cell lines further accentuates the biomedical relevance of nanoparticles synthesized in its presence [ 17 ]. CuO nanoparticles synthesized via green methodologies have exhibited promising anticancer capabilities attributed to their ability to generate reactive oxygen species, induce oxidative stress, and trigger apoptosis in cancer cells [ 18 , 19 ]. Extensive studies have validated their inhibitory effects across various cancer models, positioning them as compelling candidates for nanomedicine [ 20 ]. In the current investigation, the morphological attributes of CuO nanoparticles and prodigiosin pigment were delineated using scanning electron microscopy (SEM). Phase purity and crystalline structure of the nanoparticles were characterized by X-ray diffraction (XRD), while elemental composition was elucidated via energy dispersive X-ray analysis (EDAX). Fourier transform infrared (FTIR) spectroscopy elucidated surface functional groups, and UV-Visible spectroscopy detailed the optical properties of both nanoparticles and pigment. Serratia rubidaea was taxonomically confirmed by the VITEK® 2 COMPACT system. The comprehensive characterization suite enabled a thorough appraisal of structural, compositional, and optical attributes. To the best of the authors’ knowledge, this is the inaugural study employing prodigiosin pigment derived from Serratia rubidaea exclusively as a green biogenic agent within a sol-gel synthesis route for CuO nanoparticles [ 52 ]. This unexplored methodology bridges a notable gap in the literature and anticipates the generation of nanoparticles with distinctive physicochemical and biological traits, enhancing their candidature for advanced biomedical applications. 2. Materials And Methods 2.1 Chemicals and Reagents Copper sulfate pentahydrate (CuSO₄·5H₂O), ethanol, formaldehyde, methylene blue, trichloroacetic acid, and Sulforhodamine B dye were procured from Sigma-Aldrich, India. Nutrient agar and distilled water were used for microbial culturing and washing. Fetal bovine serum (FBS), L-glutamine, and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from HiMedia Laboratories, India. All solutions were prepared with Milli-Q ultrapure water. 2.2 Isolation and Identification of Pigment-Producing Bacteria Water samples were collected aseptically from a pond in Thane, Mumbai, India (coordinates: 19.196298, 72.968091). Aliquots were streaked onto nutrient agar plates and incubated at 30°C for 48 hours. Distinct pigmented colonies were subcultured to purity. Biochemical tests (Gram staining, catalase, oxidase) and automated species-level identification using the VITEK® 2 COMPACT system (bioMérieux) confirmed the isolate as Serratia rubidaea , which was maintained at 4°C on nutrient agar slants. 2.3 Extraction of Prodigiosin Pigment Serratia rubidaea was cultivated on nutrient agar plates at 30°C for 48 hours until visible pink pigmentation developed. The biomass was scraped into 25 mL of ethanol, followed by addition of formaldehyde (5% v/v). The suspension was sonicated for 20 minutes in an ice bath (40 kHz; Vibra-Cell, Sonics & Materials Inc.), centrifuged at 10,000 rpm for 30 minutes at -2°C (Eppendorf 5810R), and the supernatant containing prodigiosin was filtered through Whatman No. 1 filter paper. The pigment solution was stored at 4°C in the dark. 2.4 Synthesis of Copper Oxide Nanoparticles CuO nanoparticles were synthesized using prodigiosin as a biogenic reducing and capping agent. A mixture of 40 mL prodigiosin solution and 1 mmol (0.25 g) CuSO₄·5H₂O was magnetically stirred at 500 rpm and heated to 60°C. The reaction progress was monitored by color change from pink to dark blue. After reaction completion, the mixture was dried at 70°C overnight, followed by calcination at 600°C for 2 hours in a muffle furnace. The resulting black powder was ground and stored in a desiccator. 2.5 Characterization of CuO Nanoparticles and Prodigiosin SEM imaging was performed using ZEISS EVO-18 (pigment) and FEI Quanta 200F (CuO NPs) microscopes operated at 20 kV after sputter-coating with gold. EDAX was performed using Oxford EDS detector attached to the SEM. XRD patterns were recorded with Rigaku MiniFlex 600 diffractometer (Cu Kα, λ = 1.5406 Å) scanning 10° to 80° 2θ, step size 0.02°. Crystallite size was determined using the Debye–Scherrer equation. FTIR spectra (400–4000 cm⁻¹) were acquired on Agilent Cary 630 spectrometer using KBr pellets. UV–Vis spectra of dispersed CuO NPs and pigment solutions were recorded on Perkin Elmer Lambda 750 spectrophotometer in quartz cuvettes across 300–600 nm and 300–700 nm, respectively. 2.6 In Vitro Cytotoxicity Assay MCF-7 cells (ATCC HTB-22) were cultured in DMEM supplemented with 10% FBS and 2 mM L-glutamine at 37°C, 5% CO₂. Cells were seeded at 5,000 cells/well in 96-well plates and incubated for 24 h. CuO NPs stock solution (1 mg/mL) was prepared via sonication in sterile deionized water and diluted to final concentrations of 10, 20, 40, and 80 µg/mL; all treatments performed in triplicate. After 48 h incubation, cells were fixed with 10% TCA, stained with 0.4% SRB dye, washed, dried, and solubilized with 10 mM Tris base for absorbance measurement at 540 nm (reference 690 nm). Cell morphology was observed using a Nikon Ti-S inverted microscope with a 20× objective and images processed with Eclipse NIS-Elements software. 3. Results 3.1 Isolation and Identification of Pigment-Producing Bacteria and Pigment Extraction Water samples were collected from a natural pond in Thane, Mumbai, India (coordinates 19.196298, 72.968091). Samples were cultured on nutrient agar plates, incubated at 30°C for 48 hours. Colonies exhibiting distinct pigmentation were isolated and identified using standard biochemical assays alongside automated species identification via the VITEK® 2 COMPACT system, which confirmed the isolate as Serratia rubidaea [ 21 ]. For pigment extraction, Serratia rubidaea cultures grown on nutrient agar for 48 hours were harvested, ethanol was added to the biomass, and the mixture was treated with formaldehyde at 5% v/v. Sonication was performed in an ice bath for 20 minutes to facilitate cell disruption. The suspension was centrifuged at 10,000 rpm for 30 minutes at -2°C, and the resulting supernatant containing the prodigiosin pigment was collected, exhibiting a characteristic pink coloration [ 22 , 23 ]. 3.2 Synthesis of Copper Oxide Nanoparticles Extracted prodigiosin (chemical formula C₂₀H₂₅N₃O) was utilized both as a reducing and capping agent for the green synthesis of copper oxide nanoparticles. A reaction mixture was prepared by combining 40 mL of the prodigiosin extract with 1 mmol (0.25 g) of copper sulfate pentahydrate (CuSO₄·5H₂O). The mixture was heated with continuous magnetic stirring at 60°C until a distinct color change from pink to dark blue occurred, indicating the formation of a copper–pigment complex [ 24 , 25 ]. Post-reaction, the solution was cooled to room temperature and dried overnight in an oven to obtain a solid precursor. This precursor was transferred to a ceramic crucible and calcined in a muffle furnace at 600°C for 2 hours to decompose organic constituents and yield black CuO nano powder. After cooling within the furnace to ambient temperature, the powder was gently ground and stored in a desiccator until further use [ 26 ]. The synthesized nanoparticles were subjected to multiple physicochemical characterization techniques, detailed in subsequent sections. 3.3 XRD Analysis The crystallographic structure and microstructural properties of the synthesized CuO nanoparticles were investigated using X-ray diffraction (XRD). The diffraction peaks (Fig. 1 ) appeared at 2θ values of 35.74°, 38.96°, 49.21°, 53.88°, 58.70°, 61.80°, 66.23°, 72.68°, and 75.37°, corresponding respectively to the \(\:\left(\overline{1}11\right)\) , \(\:\left(111\right)\) , \(\:\left(\overline{2}02\right)\) , \(\:\left(020\right)\) , \(\:\left(202\right)\) , \(\:\left(\overline{1}13\right)\) , \(\:\left(022\right)\) , \(\:\left(113\right)\) , and \(\:\left(\overline{2}22\right)\) planes of monoclinic CuO (ICDD JCPDS card no. 00-001-1117). The presence of these sharp and well-defined peaks confirms the monoclinic phase with high crystallinity and phase purity [ 27 , 28 ]. The interplanar spacing \(\:{d}_{hkl}\) was calculated using Bragg’s law: $$\:{d}_{hkl}=\frac{\lambda\:}{2\text{s}\text{i}\text{n}\theta\:}$$ 1 and further refined with the monoclinic lattice parameter relation: $$\:\frac{1}{{d}_{hkl}^{2}}=\frac{1}{{\text{s}\text{i}\text{n}}^{2}\beta\:}\left(\frac{{h}^{2}}{{a}^{2}}+\frac{{k}^{2}{\text{s}\text{i}\text{n}}^{2}\beta\:}{{b}^{2}}+\frac{{l}^{2}}{{c}^{2}}-\frac{2hl\text{c}\text{o}\text{s}\beta\:}{ac}\right)$$ 2 The refined lattice parameters were found to be \(\:a=4.6530\:\text{Å}\) , \(\:b=3.4100\:\text{Å}\) , \(\:c=5.1080\:\text{Å}\) , and \(\:\beta\:={99.48}^{\circ\:}\) , with a unit cell volume \(\:{V}_{c}=79.94\:{\text{Å}}^{3}\) , consistent with the literature for monoclinic CuO [ 29 , 30 ]. The average crystallite size \(\:D\) was calculated by the Debye–Scherrer equation: $$\:D=\frac{K\lambda\:}{\beta\:\text{c}\text{o}\text{s}\theta\:}$$ 3 where \(\:K=0.9\) is the shape factor, \(\:{\lambda\:}=1.5406\:\text{Å}\) is the Cu Kα wavelength, \(\:{\beta\:}\) is the FWHM of the diffraction peak (in radians), and \(\:\theta\:\) is the Bragg angle. Extracted peak FWHM values ranged 0.0077 to 0.0089 radians, yielding crystallite sizes between 16.44 and 20.08 nm depending on the crystallographic plane (Table 1 ) [ 31 , 32 ]. Table 1 Crystallographic and Microstructural Parameters of CuO Nanoparticles Plane ( hkl) 2θ (deg) d (Å) FWHM ( rad) Crystallite Size D ( nm) Lattice Strain × 10 − 2 Dislocation Density × 1015( m − 2 ) X-ray Density (g/cm 3 ) (002) 35.74 2.51 0.0089 16.44 0.69 3.70 6.58 (111) 38.96 2.31 0.0084 17.62 0.55 3.22 6.58 (113) 49.21 1.85 0.0089 17.13 0.48 3.40 6.58 (020) 53.88 1.70 0.0077 20.08 0.38 2.48 6.58 To discriminate peak broadening from size and strain effects, a Williamson-Hall (W-H) analysis was conducted. This entails plotting \(\:\beta\:\text{c}\text{o}\text{s}\theta\:\) versus \(\:4\text{s}\text{i}\text{n}\theta\:\) and fitting the data linearly to: $$\:\beta\:\text{c}\text{o}\text{s}\theta\:=\frac{K\lambda\:}{D}+4ϵ\text{s}\text{i}\text{n}\theta\:$$ 4 where \(\:ϵ\) is the microstrain. The slope of the W-H plot gave a microstrain value of \(\:7.9\times\:{10}^{-3}\) and the intercept corresponded to an average crystallite size of 18.72 nm, slightly larger than that estimated by Scherrer’s method, reflecting the contribution of strain-induced broadening [ 33 ]. Microstrain ( \(\:ϵ\) ) values calculated by the classical formula: $$\:ϵ=\frac{\beta\:}{4\text{t}\text{a}\text{n}\theta\:}$$ 5 ranged from 0.38 to 0.69 × 10⁻² for the different planes, corroborating the W-H analysis [ 34 ]. Dislocation density ( \(\:\delta\:\) ) values, a measure of lattice defects, were calculated as: $$\:\delta\:=\frac{1}{{D}^{2}}$$ 6 and were found to be in the order of \(\:2.48\times\:{10}^{15}\) to \(\:3.70\times\:{10}^{15}\:{\text{m}}^{-2}\) [ 35 ]. The X-ray density \(\:{\rho\:}_{x}\) calculated using: $$\:{\rho\:}_{x}=\frac{ZM}{{N}_{A}{V}_{c}}$$ 7 (with formula units number \(\:Z=4\) , molar mass \(\:M=79.55\:\text{g/mol}\) , Avogadro’s number \(\:{N}_{A}=6.022\times\:{10}^{23}\) , and unit cell volume \(\:{V}_{c}=7.994\times\:{10}^{-23}\:{\text{cm}}^{3}\) ) was \(\:6.58\:{\text{g/cm}}^{3}\) , consistent with reported values for monoclinic CuO [ 36 ]. The high phase purity, nanoscale crystallite size, low lattice strain, and moderate dislocation density evidenced from XRD analysis indicate successful synthesis of well-crystallized CuO nanoparticles [ 37 , 38 ]. The combined use of Scherrer, lattice strain, and Williamson-Hall analyses provides a comprehensive understanding of the microstructural characteristics, which influence the functional properties of CuO in catalytic and sensor applications. 3.4 Optical Analysis The optical properties of the synthesized CuO nanoparticles were examined using UV–Visible spectroscopy, as shown in Fig. 2 (A). The absorbance spectrum exhibited distinct peaks at 329.5 nm, 365.0 nm, and 388.5 nm, indicating characteristic electronic transitions and confirming the nanoscale nature of the CuO particles [ 39 , 40 ]. The direct optical band gap ( \(\:{E}_{g}\) ) was estimated using Tauc’s method, which relates the photon energy ( \(\:h\nu\:\) ) and absorption coefficient ( \(\:\alpha\:\) ) by the equation: $$\:(\alpha\:h\nu\:{)}^{2}=k(h\nu\:-{E}_{g})$$ 8 where \(\:k\) is a constant related to the material and transition probability. The band gap energy was obtained by extrapolating the linear portion of the \(\:(\alpha\:h\nu\:{)}^{2}\) versus \(\:h\nu\:\) plot (Tauc plot), as in Fig. 2 (B). The calculated band gap of 2.95 eV is higher than bulk CuO values (~ 1.2–1.9 eV ), demonstrating quantum confinement effects expected in nanoparticles [ 41 ]. To quantify light absorption, absorption coefficients \(\:\alpha\:\) were calculated from the measured absorbance ( \(\:A\) ) using the Beer–Lambert relationship: $$\:\alpha\:=\frac{2.303\times\:A}{L}$$ 9 assuming a path length \(\:L=1\:\text{cm}\) for the cuvette where nanoparticles were dispersed. The extinction coefficient ( \(\:k\) ), describing the attenuation of electromagnetic waves in the material, was calculated using: $$\:k=\frac{\alpha\:\lambda\:}{4\pi\:}$$ 10 where \(\:\lambda\:\) is the light wavelength in centimetres. Furthermore, the optical conductivity ( \(\:\sigma\:\) ) was determined to assess charge carrier response to incident light: $$\:\sigma\:=\frac{\alpha\:nc}{4\pi\:}$$ 11 \(\:n=2.5\) was assumed for the refractive index of CuO in the visible range, and \(\:c=3\times\:{10}^{10}\) cm/s is the speed of light. Table 2 summarizes the absorbance values at key wavelengths and their corresponding calculated optical parameters. Table 2 Optical Parameters of CuO Nanoparticles. Wavelength ( nm ) Absorbance ( A ) Absorption Coefficient ( cm − 1 ) Extinction Coefficient (×10 − 6 ) Optical Conductivity (×10 9 s − 1 ) 329.5 0.133 0.306 0.80 1.83 365.0 0.174 0.40 1.16 2.40 388.5 0.243 0.56 1.73 3.36 The increase in absorption coefficient and extinction coefficient with wavelength near the absorption edges reflects stronger photon absorption due to electronic transitions between valence and conduction bands [ 42 ]. The optical conductivity values indicate enhanced charge carrier density and movement, important for potential applications of CuO nanoparticles in photovoltaics, sensing, and photocatalysis [ 42 ]. The high band gap energy combined with the optical constants, confirms the quantum size effect and good crystallinity of the CuO nanoparticles synthesized [ 42 ]. The UV–Visible absorbance spectrum of the prodigiosin extract is presented in Fig. (3). The extract exhibited a broad, intense absorption band in the visible region with maximum peaks at 521.3 nm, 539.6 nm, and 558.8 nm. These absorption features collectively constitute the principal absorbance band that is characteristic of prodigiosin pigments [ 43 , 44 ]. The prominent peaks arise due to electronic transitions associated with the distinctive tripyrrole molecular structure of prodigiosin, which contains three pyrrole rings linked via methine bridges. These transitions typically involve π→π* and n→π* excitations within the conjugated system, resulting in the observed absorption in the visible light range. The positions and intensities of these peaks confirm both the presence and purity of prodigiosin in the extract, consistent with earlier reports where prodigiosin shows absorption maxima between 520 nm and 540 nm depending on solvent and pH environment [ 44 ]. The broadness of the absorption band indicates some degree of molecular aggregation or heterogeneity in the pigment microenvironment, confirming the successful extraction of bioactive prodigiosin pigment, supporting its potential utility in biomedical and industrial applications where its photophysical properties are pivotal [ 43 , 44 ]. 3.5 FTIR Analysis Fourier transform infrared (FTIR) spectroscopy was utilized to characterize the surface chemistry and functional groups present in the synthesized CuO nanoparticles. The FTIR spectrum (Fig. 4 ) provides detailed insight into the molecular vibrations indicative of chemical moieties on and within the nanostructures. A broad absorption band centered around 3420 cm − 1 is assigned to the stretching vibrations of hydroxyl (–OH) groups, which include surface-adsorbed water molecules and chemically bonded hydroxyl functionalities on the CuO surface. This pronounced band is typical for hydrophilic metal oxide nanoparticles and plays a critical role in their aqueous interactions and potential for functionalization [ 37 , 38 ]. The band observed near 1635 cm − 1 corresponds to the bending vibrations of H–O–H groups from adsorbed moisture, further confirming the presence of water molecules physically or chemically associated with the nanoparticle surfaces [ 39 ]. The characteristic peaks in the lower wavenumber region (600–400 cm − 1 ) are attributed to metal–oxygen vibrational modes. Prominent absorptions at approximately 530 cm − 1 and 490 cm − 1 are assigned to the Cu–O stretching vibrations indicative of monoclinic CuO lattice formation. These spectral features align well with X-ray diffraction data, confirming the crystalline quality [ 40 ]. Additional absorption bands at 1380 cm − 1 and 1100 cm − 1 are ascribed to bending and stretching vibrations of nitrate groups remaining from precursor copper nitrate salts or adsorbed surface nitrate contaminants. Retention of such groups may influence surface charge and reactivity [ 41 ]. Minor bands around 2900 cm − 1 correspond to C–H stretching vibrations that may originate from residual organic compounds, such as stabilizing agents or plant-extract components used in green synthesis, indicating surface passivation of nanoparticles [ 42 ] These functional groups observed in FTIR spectra substantiate the dual role of biological molecules: acting as both reductants, facilitating Cu(II) to CuO conversion, and capping agents stabilizing the nanoparticles against agglomeration. The presence of hydroxyl, nitrate, and organic functional groups can significantly influence the chemical stability, surface reactivity, and potential bio-interaction pathways of the CuO nanoparticles [ 4 ]. Table 3 FTIR Peak Positions and Functional Group Assignments of CuO Nanoparticles Wavenumber ( cm − 1 ) Assignment Significance ~ 3420 O–H stretching (surface hydroxyls, water) Indicates hydrophilic surface, adsorbed moisture ~ 1635 H–O–H bending (adsorbed water) Affirms surface-bound water molecules ~ 1380 Nitrate group bending vibrations Residual precursor ions or surface chemisorption ~ 1100 Nitrate and C–O stretching Surface adsorption, organic residues ~ 530 Cu–O stretching Confirms monoclinic CuO phase formation ~ 490 Cu–O lattice vibrational modes Characteristic of CuO crystal structure ~ 2900 C–H stretching (organic residues) Possible surface stabilizers/capping agents These surface groups influence the physicochemical behaviour of CuO nanoparticles in aqueous media and biological environments. The hydroxyl bands indicate potential sites for further functionalization, while the confirmation of Cu–O bonds affirm the nanoparticle’s structural integrity. Detection of residual nitrates and organics suggests synthesis optimization can target improved purification for specific applications [ 43 ]. Overall, FTIR results complement XRD and UV-Vis data by elucidating the chemical state of CuO nanoparticle surfaces, which is vital for tailoring materials for use in catalysis, sensors, or biomedical applications. The observed particle aggregation and size distribution are crucial factors to consider when evaluating applications such as catalysis and biomedical usage, where surface accessibility and interaction dynamics are paramount. 3.6 Morphological, Energy-Dispersive X-ray, and Elemental Analysis The surface morphology of the extracted prodigiosin pigment was first examined using scanning electron microscopy (SEM), as shown in Fig. 5 . The pigment appeared as aggregated, irregularly shaped particles consistent with organic pigment morphology [ 44 ]. SEM images of the synthesized copper oxide (CuO) nanoparticles (Fig. 6 ) exhibited irregularly shaped crystalline particles that were often clustered into larger agglomerates. This morphology is typical for metal oxide nanoparticles synthesized via green or chemical methods and suggests potential high surface areas beneficial for applications such as catalysis and sensing [ 46 ]. Energy-dispersive X-ray analysis (EDAX) spectra for the CuO nanoparticles are displayed in Fig. 7 . The spectra featured prominent peaks corresponding to copper (Cu), oxygen (O), and carbon (C), indicative of the chemical constituents of the nanostructures. Quantitative elemental analysis showed weight percentages of 48.6%, 28.7%, and 22.7% for Cu, O, and C respectively, with atomic percentages of 17.2% (Cu), 40.4% (O), and 42.4% (C). The presence of carbon likely originates from surface-bound organic moieties or residual stabilizers linked to the synthesis process [ 46 ]. The Cu peak at an energy of approximately 129.5 eV confirms the presence and purity of copper within the synthesized oxide nanoparticles. The elemental composition aligns well with stoichiometric CuO formation [ 47 ]. These combined morphological and elemental analyses confirm successful synthesis of CuO nanoparticles with residual organic layers and confirm the pigment’s integrative presence and morphological distinction. The agglomerated CuO crystals and pigment form a composite system potentially useful for multifunctional applications [ 48 ]. 3.7 Cytotoxicity Evaluation of CuO NPs The cytotoxicity of synthesized CuO nanoparticles was investigated against the MCF-7 human breast cancer cell line using the Sulforhodamine B (SRB) colourimetric assay. The assay was performed in 96-well plates, with MCF-7 cells seeded at a density of 5000 cells per well. Cells were treated with CuO nanoparticles at final concentrations of 10, 20, 40, and 80 µg/mL in triplicate. After 48 hours of incubation under standard cell culture conditions, cell viability was determined by measuring absorbance at 540 nm following SRB staining. Quantitative results indicated a concentration-dependent effect on cell viability. At 10 µg/mL CuO nanoparticles, the mean cell viability was 82.3% (± 3.37 SD), corresponding to 17.7% cell mortality. For 20 µg/mL treatment, cell viability was recorded as 84.9% (± 4.21 SD), while at 40 µg/mL, viability was 85.4% (± 5.60 SD). The largest reduction in viability was observed at the highest nanoparticle concentration, 80 µg/mL, where mean cell viability decreased to 67.1% (± 13.02 SD); cell mortality at this concentration was 32.9%. The IC 50 value (the concentration at which 50% cell death occurs) could not be determined within the tested concentration range, as none of the tested doses reduced viability below 50% [ 49 ]. Morphological assessment was conducted via phase-contrast microscopy. Representative images of untreated cells (negative control), Adriamycin-treated cells (positive control), and cells treated with the highest CuO nanoparticle concentration are presented in Fig. 8 . Untreated MCF-7 cells exhibited a characteristic monolayer with intact, well-spread morphology. Cells exposed to CuO nanoparticles at 80 µg/mL displayed evidence of morphological changes, including cell shrinkage, reduced adherence, and rounding of cells, compared to negative controls [ 50 – 51 ]. 4. Conclusion This study successfully demonstrates the novel integration of the naturally derived prodigiosin pigment within a sol-gel synthetic route for producing copper oxide (CuO) nanoparticles, marking a pioneering approach in green synthesis enhancing nanoparticle stability and functionality. The biosynthesized prodigiosin not only acts as a bioactive capping and reducing agent, but its incorporation introduces unique surface chemistry that significantly influences the physicochemical attributes of the CuO nanoparticles. X-ray diffraction analysis confirmed monoclinic CuO phase formation with high crystallinity, evidenced by sharp diffraction peaks indexed to planes (− 111), (111), (− 202), and (020). Quantitative analysis yielded crystallite sizes ranging from 16.44 nm to 20.08 nm (average 18.72 nm via Williamson-Hall method), with lattice strain measured between 0.38 × 10 − 2 and 0.69 × 10 − 2 and moderate dislocation densities from 2.48 × 10 15 m − 2 to 3.70 × 10 15 m − 2 , underlining the structural integrity achieved through this bio-assisted approach. FTIR spectral analysis revealed pronounced hydroxyl (–OH) functional groups, metal–oxygen bond vibrations corresponding to Cu–O lattice dynamics, and residual nitrate and organic moieties attributed to the pigment and sol-gel process, collectively indicating favorable surface passivation and functional group availability. Optically, CuO nanoparticles displayed absorbance bands at 329.5 nm, 365.0 nm, and 388.5 nm, with an experimentally determined direct band gap of 2.95 eV , affirming pronounced quantum confinement effects. The prodigiosin pigment exhibited characteristic tripyrrole ring transitions at 521.3 nm, 539.6 nm, and 558.8 nm, confirming its purity and active molecular structure preserved through the nanoparticle formation. Morphological evaluation via scanning electron microscopy showed aggregated, irregular crystalline structures, and elemental compositional analysis confirmed stoichiometric CuO with 48.6 wt% Cu, 28.7 wt% O, and 22.7 wt% C, the latter evidencing organic capping from prodigiosin. Biological assessment indicated a dose-dependent cytotoxic effect on MCF-7 breast cancer cells, with the highest tested nanoparticle concentration (80 µg/mL) reducing viability to 67.1%, alongside observed morphologic alterations implicating apoptosis pathways. The study presents a compelling case for the application of prodigiosin as a multifunctional agent in the sol-gel synthesis of CuO nanoparticles, enhancing physicochemical properties and endowing bioactivity, thereby expanding the potential for applications in catalysis, biosensing, and targeted cancer therapeutics. Future work should focus on mechanistic elucidation of pigment-nanoparticle interactions and optimization for clinical translation. Declarations Conflict of Interest Statement The authors declare that they have no conflicts of interest regarding the publication of this paper. No financial or personal relationships exist that could inappropriately influence or bias the content of this work. All authors confirm that they have no competing interests to disclose. Funding Declaration No funding was received to support the preparation of this manuscript or the research presented within it. Author Contribution Kartikey J. Chavan: Conceptualization, Methodology, Supervision, Writing–Original Draft, Data Analysis and Curation, Investigation, Review & Editing.Sarang R. Bhagwat: Data Analysis, Supervision.Vineet D. P. Kala: Review & Editing, Formal Analysis, Investigation.Arjun R. Potinde: Data Analysis and Curation, Formal Analysis, Methodology.Hemanth S. Gurajada: Data Analysis and Curation, Formal Analysis.Mansi P. Juvekar: Methodology. 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07:37:00","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127664,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/2e3ad2d0131e35f1f877d3a1.png"},{"id":94254813,"identity":"77742b52-ddd9-4ab0-b62f-00a0d0cb1346","added_by":"auto","created_at":"2025-10-24 07:37:00","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127567,"visible":true,"origin":"","legend":"","description":"","filename":"4911837217d844dc8b193aed50a14ec51structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/d2fab62f0084562c6d77e7bb.xml"},{"id":94255747,"identity":"f91da5f0-0ae9-4836-982f-02729e34d15f","added_by":"auto","created_at":"2025-10-24 07:45:00","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145666,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/a2c0a4136d8d69c9089f5c40.html"},{"id":94255733,"identity":"7dcfe6b6-f9fc-476c-8a4b-7c70ebacf8ec","added_by":"auto","created_at":"2025-10-24 07:45:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":134724,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction (XRD) pattern of the synthesized copper oxide (CuO) nanoparticles. The diffraction peaks correspond to the monoclinic CuO phase, confirming high crystallinity and phase purity, with indexed planes labeled according to JCPDS card no. 00-001-1117.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/fc40003656d3f09589439243.png"},{"id":94254787,"identity":"7885d1b4-78de-423e-a09f-3f15b897b83d","added_by":"auto","created_at":"2025-10-24 07:37:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":122581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e UV–Visible absorption spectrum of CuO nanoparticles exhibiting characteristic peaks at 329.5 nm, 365.0 nm, and 388.5 nm. \u003cstrong\u003eFig. 2\u003c/strong\u003e \u003cstrong\u003e(B)\u003c/strong\u003e Tauc plot used for estimating the direct optical band gap of CuO nanoparticles, illustrating quantum confinement effects.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/548c10bce2ac3a9a0245e7f1.png"},{"id":94254789,"identity":"ce53499b-38c2-4463-8771-8e5dc8103f1b","added_by":"auto","created_at":"2025-10-24 07:37:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":132173,"visible":true,"origin":"","legend":"\u003cp\u003eUV–Visible absorbance spectrum of the prodigiosin pigment extracted from \u003cem\u003eSerratia rubidaea\u003c/em\u003e, showing characteristic broad absorption bands at 521.3 nm, 539.6 nm, and 558.8 nm related to electronic transitions within the tripyrrole structure.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/f31cdbdab2a2f4dc3718386d.png"},{"id":94254791,"identity":"a1a2ad93-9588-4dc4-9f60-b7db54cb9d86","added_by":"auto","created_at":"2025-10-24 07:37:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":169080,"visible":true,"origin":"","legend":"\u003cp\u003eFourier Transform Infrared (FTIR) spectrum of synthesized CuO nanoparticles detailing vibrational bands corresponding to surface hydroxyl groups, metal–oxygen bonds, and residual nitrate and organic moieties, indicating surface chemistry and nanoparticle stability.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/8bb4d6cb07863030785cf84c.png"},{"id":94255735,"identity":"612ed300-def0-4696-84ac-7c1612615268","added_by":"auto","created_at":"2025-10-24 07:45:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1171084,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) image depicting the morphology of the prodigiosin pigment extracted from \u003cem\u003eSerratia rubidaea\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/b40367f68e34e1c827309fef.png"},{"id":94254794,"identity":"6fa19f0f-f459-473f-b825-911035d8a397","added_by":"auto","created_at":"2025-10-24 07:37:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1183434,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy (SEM) image depicting the morphology of CuO nanoparticles, showing irregular crystalline particles arranged in clusters.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/4b5ba93539c2e4bb8f6f6dcc.png"},{"id":94255737,"identity":"bbddff12-9ed2-4213-8678-ecf8e3a24322","added_by":"auto","created_at":"2025-10-24 07:45:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":560275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSEM image of synthesized CuO nanoparticles with the region selected for EDAX analysis indicated.\u003cstrong\u003e Fig. 7 (B)\u003c/strong\u003e Energy-dispersive X-ray spectroscopy (EDAX) spectrum and quantitative elemental composition analysis of CuO nanoparticles, confirming copper, oxygen, and carbon presence and weight percentages.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/a32e9acae5e8f9a2f43cfd47.png"},{"id":94255956,"identity":"0b56edc5-cb21-41b6-b446-22170f61a786","added_by":"auto","created_at":"2025-10-24 07:53:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":560677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Cell viability of MCF-7 human breast cancer cells following treatment with various concentrations of CuO nanoparticles, as determined by the Sulforhodamine B (SRB) assay. \u003cstrong\u003eFig. 8\u003c/strong\u003e \u003cstrong\u003e(B)\u003c/strong\u003e Optical microscopy image of untreated MCF-7 cells (negative control). \u003cstrong\u003eFig. 8\u003c/strong\u003e \u003cstrong\u003e(C)\u003c/strong\u003e Optical microscopy image of MCF-7 cells treated with Adriamycin (positive control). \u003cstrong\u003eFig. 8\u003c/strong\u003e \u003cstrong\u003e(D) \u003c/strong\u003eOptical microscopy image of MCF-7 cells treated with the highest concentration (80 μg/mL) of CuO nanoparticles, showing morphological changes.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/d332ecf3ceb58e21a6464fb5.png"},{"id":99545238,"identity":"75b954ef-5d4a-44ec-84e0-741f18c4cd07","added_by":"auto","created_at":"2026-01-05 16:03:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5282042,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/1ed903a3-830b-4ecc-abec-a07e1dba8b52.pdf"},{"id":94255734,"identity":"ee4b0a86-552d-4c4a-a8f7-7c6a7494b16d","added_by":"auto","created_at":"2025-10-24 07:45:00","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":395245,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7801875/v1/71cd3ae2055988a901160878.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eNovel Prodigiosin-Driven Biogenic Sol-Gel Synthesis of Copper Oxide Nanoparticles Using Serratia rubidaea: Extraction, Dual Role as Bio-Reducing and Capping Agent, and Enhanced Cytotoxic Potential for Biomedical Application\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; First report on the extraction and use of prodigiosin pigment from as a dual-function bio-reductant and stabilizer for sol-gel synthesis of copper oxide nanoparticles.\u003c/p\u003e\u003cp\u003e\u0026bull; Environmentally friendly, sustainable green synthesis route producing monoclinic CuO nanoparticles with controlled size and morphology.\u003c/p\u003e\u003cp\u003e\u0026bull; Comprehensive physicochemical characterization confirms phase purity, nanoscale crystallite size, unique surface chemistry, and optical properties influenced by prodigiosin capping.\u003c/p\u003e\u003cp\u003e\u0026bull; Preliminary in vitro cytotoxicity studies demonstrate promising, concentration-dependent anticancer activity of CuO nanoparticles against MCF-7 breast cancer cells.\u003c/p\u003e\u003cp\u003e\u0026bull; Potential multifunctional biomedical applications through integration of biogenic pigment and metal oxide nanomaterial harnessing biofunctional and therapeutic synergism.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eCopper oxide (CuO) nanoparticles have garnered substantial attention in recent years due to their unique physicochemical properties and broad spectrum of applications, particularly in anticancer therapeutics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As prominent members of the metal oxide family, copper oxides primarily exist in two forms: cuprous oxide (Cu₂O) and cupric oxide (CuO) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These semiconducting metal oxides actively participate in biological processes, notably cancer cell inhibition, owing to their electronic properties [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMetal oxides are classified based on their chemical behaviour into acidic, basic, amphoteric, and neutral oxides. CuO, a basic oxide, reacts with acids to form salts and water, a fundamental property that underpins its reactivity in diverse biomedical and catalytic contexts [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Understanding this chemical classification is pivotal for advancing the tailored application of metal oxides in cancer therapy and beyond [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMultiple synthetic methodologies have been devised to fabricate CuO nanoparticles, each impacting particle size, morphology, and functional performance. Notable approaches include chemical precipitation, hydrothermal synthesis, electrochemical methods, and the sol-gel process [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among these, the sol-gel method has acquired prominence for its operational simplicity and ability to yield nanoparticles with controlled size and morphology via a liquid-phase to gel-phase transition, facilitating tunable physico-chemical properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIncreasing interest in environmentally benign synthesis has focused attention on integrating biological catalysts into the sol-gel technique. This hybrid approach not only simplifies synthesis but also enhances nanoparticle stability and biocompatibility [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Biological reducing agents derived from plant extracts and microbial systems offer sustainable, eco-friendly alternatives to conventional chemical reagents, producing nanomaterials with distinctive morphological and biological properties that may potentiate anticancer efficacy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eProdigiosin, a bioactive red pigment predominantly produced by \u003cem\u003eSerratia\u003c/em\u003e species, including \u003cem\u003eSerratia rubidaea\u003c/em\u003e, embodies such a biogenic agent with multifaceted biological activities, including antimicrobial, immunosuppressive, and anticancer effects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Its role as an electron donor and capping agent during nanoparticle synthesis facilitates uniform nucleation and growth, yielding nanoparticles with enhanced physicochemical and biological profiles [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The inherent cytotoxicity of prodigiosin against diverse cancer cell lines further accentuates the biomedical relevance of nanoparticles synthesized in its presence [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCuO nanoparticles synthesized via green methodologies have exhibited promising anticancer capabilities attributed to their ability to generate reactive oxygen species, induce oxidative stress, and trigger apoptosis in cancer cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Extensive studies have validated their inhibitory effects across various cancer models, positioning them as compelling candidates for nanomedicine [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the current investigation, the morphological attributes of CuO nanoparticles and prodigiosin pigment were delineated using scanning electron microscopy (SEM). Phase purity and crystalline structure of the nanoparticles were characterized by X-ray diffraction (XRD), while elemental composition was elucidated via energy dispersive X-ray analysis (EDAX). Fourier transform infrared (FTIR) spectroscopy elucidated surface functional groups, and UV-Visible spectroscopy detailed the optical properties of both nanoparticles and pigment. \u003cem\u003eSerratia rubidaea\u003c/em\u003e was taxonomically confirmed by the VITEK\u0026reg; 2 COMPACT system. The comprehensive characterization suite enabled a thorough appraisal of structural, compositional, and optical attributes.\u003c/p\u003e\u003cp\u003eTo the best of the authors\u0026rsquo; knowledge, this is the inaugural study employing prodigiosin pigment derived from \u003cem\u003eSerratia rubidaea\u003c/em\u003e exclusively as a green biogenic agent within a sol-gel synthesis route for CuO nanoparticles [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This unexplored methodology bridges a notable gap in the literature and anticipates the generation of nanoparticles with distinctive physicochemical and biological traits, enhancing their candidature for advanced biomedical applications.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemicals and Reagents\u003c/h2\u003e\u003cp\u003eCopper sulfate pentahydrate (CuSO₄\u0026middot;5H₂O), ethanol, formaldehyde, methylene blue, trichloroacetic acid, and Sulforhodamine B dye were procured from Sigma-Aldrich, India. Nutrient agar and distilled water were used for microbial culturing and washing. Fetal bovine serum (FBS), L-glutamine, and Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) were obtained from HiMedia Laboratories, India. All solutions were prepared with Milli-Q ultrapure water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Isolation and Identification of Pigment-Producing Bacteria\u003c/h2\u003e\u003cp\u003eWater samples were collected aseptically from a pond in Thane, Mumbai, India (coordinates: 19.196298, 72.968091). Aliquots were streaked onto nutrient agar plates and incubated at 30\u0026deg;C for 48 hours. Distinct pigmented colonies were subcultured to purity. Biochemical tests (Gram staining, catalase, oxidase) and automated species-level identification using the VITEK\u0026reg; 2 COMPACT system (bioM\u0026eacute;rieux) confirmed the isolate as \u003cem\u003eSerratia rubidaea\u003c/em\u003e, which was maintained at 4\u0026deg;C on nutrient agar slants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Extraction of Prodigiosin Pigment\u003c/h2\u003e\u003cp\u003e\u003cem\u003eSerratia rubidaea\u003c/em\u003e was cultivated on nutrient agar plates at 30\u0026deg;C for 48 hours until visible pink pigmentation developed. The biomass was scraped into 25 mL of ethanol, followed by addition of formaldehyde (5% v/v). The suspension was sonicated for 20 minutes in an ice bath (40 kHz; Vibra-Cell, Sonics \u0026amp; Materials Inc.), centrifuged at 10,000 rpm for 30 minutes at -2\u0026deg;C (Eppendorf 5810R), and the supernatant containing prodigiosin was filtered through Whatman No. 1 filter paper. The pigment solution was stored at 4\u0026deg;C in the dark.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Synthesis of Copper Oxide Nanoparticles\u003c/h2\u003e\u003cp\u003eCuO nanoparticles were synthesized using prodigiosin as a biogenic reducing and capping agent. A mixture of 40 mL prodigiosin solution and 1 mmol (0.25 g) CuSO₄\u0026middot;5H₂O was magnetically stirred at 500 rpm and heated to 60\u0026deg;C. The reaction progress was monitored by color change from pink to dark blue. After reaction completion, the mixture was dried at 70\u0026deg;C overnight, followed by calcination at 600\u0026deg;C for 2 hours in a muffle furnace. The resulting black powder was ground and stored in a desiccator.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Characterization of CuO Nanoparticles and Prodigiosin\u003c/h2\u003e\u003cp\u003eSEM imaging was performed using ZEISS EVO-18 (pigment) and FEI Quanta 200F (CuO NPs) microscopes operated at 20 kV after sputter-coating with gold. EDAX was performed using Oxford EDS detector attached to the SEM. XRD patterns were recorded with Rigaku MiniFlex 600 diffractometer (Cu Kα, λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;) scanning 10\u0026deg; to 80\u0026deg; 2θ, step size 0.02\u0026deg;. Crystallite size was determined using the Debye\u0026ndash;Scherrer equation. FTIR spectra (400\u0026ndash;4000 cm⁻\u0026sup1;) were acquired on Agilent Cary 630 spectrometer using KBr pellets. UV\u0026ndash;Vis spectra of dispersed CuO NPs and pigment solutions were recorded on Perkin Elmer Lambda 750 spectrophotometer in quartz cuvettes across 300\u0026ndash;600 nm and 300\u0026ndash;700 nm, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 In Vitro Cytotoxicity Assay\u003c/h2\u003e\u003cp\u003eMCF-7 cells (ATCC HTB-22) were cultured in DMEM supplemented with 10% FBS and 2 mM L-glutamine at 37\u0026deg;C, 5% CO₂. Cells were seeded at 5,000 cells/well in 96-well plates and incubated for 24 h. CuO NPs stock solution (1 mg/mL) was prepared via sonication in sterile deionized water and diluted to final concentrations of 10, 20, 40, and 80 \u0026micro;g/mL; all treatments performed in triplicate. After 48 h incubation, cells were fixed with 10% TCA, stained with 0.4% SRB dye, washed, dried, and solubilized with 10 mM Tris base for absorbance measurement at 540 nm (reference 690 nm). Cell morphology was observed using a Nikon Ti-S inverted microscope with a 20\u0026times; objective and images processed with Eclipse NIS-Elements software.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Isolation and Identification of Pigment-Producing Bacteria and Pigment Extraction\u003c/h2\u003e\u003cp\u003eWater samples were collected from a natural pond in Thane, Mumbai, India (coordinates 19.196298, 72.968091). Samples were cultured on nutrient agar plates, incubated at 30\u0026deg;C for 48 hours. Colonies exhibiting distinct pigmentation were isolated and identified using standard biochemical assays alongside automated species identification via the VITEK\u0026reg; 2 COMPACT system, which confirmed the isolate as \u003cem\u003eSerratia rubidaea\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor pigment extraction, \u003cem\u003eSerratia rubidaea\u003c/em\u003e cultures grown on nutrient agar for 48 hours were harvested, ethanol was added to the biomass, and the mixture was treated with formaldehyde at 5% v/v. Sonication was performed in an ice bath for 20 minutes to facilitate cell disruption. The suspension was centrifuged at 10,000 rpm for 30 minutes at -2\u0026deg;C, and the resulting supernatant containing the prodigiosin pigment was collected, exhibiting a characteristic pink coloration [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Synthesis of Copper Oxide Nanoparticles\u003c/h2\u003e\u003cp\u003eExtracted prodigiosin (chemical formula C₂₀H₂₅N₃O) was utilized both as a reducing and capping agent for the green synthesis of copper oxide nanoparticles. A reaction mixture was prepared by combining 40 mL of the prodigiosin extract with 1 mmol (0.25 g) of copper sulfate pentahydrate (CuSO₄\u0026middot;5H₂O). The mixture was heated with continuous magnetic stirring at 60\u0026deg;C until a distinct color change from pink to dark blue occurred, indicating the formation of a copper\u0026ndash;pigment complex [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePost-reaction, the solution was cooled to room temperature and dried overnight in an oven to obtain a solid precursor. This precursor was transferred to a ceramic crucible and calcined in a muffle furnace at 600\u0026deg;C for 2 hours to decompose organic constituents and yield black CuO nano powder. After cooling within the furnace to ambient temperature, the powder was gently ground and stored in a desiccator until further use [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The synthesized nanoparticles were subjected to multiple physicochemical characterization techniques, detailed in subsequent sections.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 XRD Analysis\u003c/h2\u003e\u003cp\u003eThe crystallographic structure and microstructural properties of the synthesized CuO nanoparticles were investigated using X-ray diffraction (XRD). The diffraction peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) appeared at 2θ values of 35.74\u0026deg;, 38.96\u0026deg;, 49.21\u0026deg;, 53.88\u0026deg;, 58.70\u0026deg;, 61.80\u0026deg;, 66.23\u0026deg;, 72.68\u0026deg;, and 75.37\u0026deg;, corresponding respectively to the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(\\overline{1}11\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(111\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(\\overline{2}02\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(020\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(202\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(\\overline{1}13\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(022\\right)\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(113\\right)\\)\u003c/span\u003e\u003c/span\u003e, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(\\overline{2}22\\right)\\)\u003c/span\u003e\u003c/span\u003e planes of monoclinic CuO (ICDD JCPDS card no. 00-001-1117). The presence of these sharp and well-defined peaks confirms the monoclinic phase with high crystallinity and phase purity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe interplanar spacing \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{d}_{hkl}\\)\u003c/span\u003e\u003c/span\u003e was calculated using Bragg\u0026rsquo;s law:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{d}_{hkl}=\\frac{\\lambda\\:}{2\\text{s}\\text{i}\\text{n}\\theta\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eand further refined with the monoclinic lattice parameter relation:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\frac{1}{{d}_{hkl}^{2}}=\\frac{1}{{\\text{s}\\text{i}\\text{n}}^{2}\\beta\\:}\\left(\\frac{{h}^{2}}{{a}^{2}}+\\frac{{k}^{2}{\\text{s}\\text{i}\\text{n}}^{2}\\beta\\:}{{b}^{2}}+\\frac{{l}^{2}}{{c}^{2}}-\\frac{2hl\\text{c}\\text{o}\\text{s}\\beta\\:}{ac}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe refined lattice parameters were found to be \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:a=4.6530\\:\\text{\u0026Aring;}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:b=3.4100\\:\\text{\u0026Aring;}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:c=5.1080\\:\\text{\u0026Aring;}\\)\u003c/span\u003e\u003c/span\u003e, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:={99.48}^{\\circ\\:}\\)\u003c/span\u003e\u003c/span\u003e, with a unit cell volume \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{V}_{c}=79.94\\:{\\text{\u0026Aring;}}^{3}\\)\u003c/span\u003e\u003c/span\u003e, consistent with the literature for monoclinic CuO [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe average crystallite size \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:D\\)\u003c/span\u003e\u003c/span\u003e was calculated by the Debye\u0026ndash;Scherrer equation:\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:D=\\frac{K\\lambda\\:}{\\beta\\:\\text{c}\\text{o}\\text{s}\\theta\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:K=0.9\\)\u003c/span\u003e\u003c/span\u003e is the shape factor, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\lambda\\:}=1.5406\\:\\text{\u0026Aring;}\\)\u003c/span\u003e\u003c/span\u003e is the Cu Kα wavelength, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\beta\\:}\\)\u003c/span\u003e\u003c/span\u003e is the FWHM of the diffraction peak (in radians), and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\theta\\:\\)\u003c/span\u003e\u003c/span\u003e is the Bragg angle. Extracted peak FWHM values ranged 0.0077 to 0.0089 radians, yielding crystallite sizes between 16.44 and 20.08 nm depending on the crystallographic plane (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\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\u003eCrystallographic and Microstructural Parameters of CuO Nanoparticles\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlane (\u003cem\u003ehkl)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2θ \u003cem\u003e(deg)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ed \u003cem\u003e(\u0026Aring;)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFWHM (\u003cem\u003erad)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCrystallite Size D (\u003cem\u003enm)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLattice Strain \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDislocation Density \u0026times; 1015(\u003cem\u003em\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eX-ray Density \u003cem\u003e(g/cm\u003c/em\u003e\u003csup\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(002)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e35.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e6.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(111)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e38.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e6.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(113)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e49.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e17.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e6.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(020)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e53.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0077\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e6.58\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\u003eTo discriminate peak broadening from size and strain effects, a Williamson-Hall (W-H) analysis was conducted. This entails plotting \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\text{c}\\text{o}\\text{s}\\theta\\:\\)\u003c/span\u003e\u003c/span\u003e versus \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:4\\text{s}\\text{i}\\text{n}\\theta\\:\\)\u003c/span\u003e\u003c/span\u003e and fitting the data linearly to:\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:\\beta\\:\\text{c}\\text{o}\\text{s}\\theta\\:=\\frac{K\\lambda\\:}{D}+4ϵ\\text{s}\\text{i}\\text{n}\\theta\\:$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:ϵ\\)\u003c/span\u003e\u003c/span\u003e is the microstrain. The slope of the W-H plot gave a microstrain value of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:7.9\\times\\:{10}^{-3}\\)\u003c/span\u003e\u003c/span\u003e and the intercept corresponded to an average crystallite size of 18.72 nm, slightly larger than that estimated by Scherrer\u0026rsquo;s method, reflecting the contribution of strain-induced broadening [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMicrostrain (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:ϵ\\)\u003c/span\u003e\u003c/span\u003e) values calculated by the classical formula:\u003cdiv id=\"Equ5\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ5\" name=\"EquationSource\"\u003e\n$$\\:ϵ=\\frac{\\beta\\:}{4\\text{t}\\text{a}\\text{n}\\theta\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e5\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eranged from 0.38 to 0.69 \u0026times; 10⁻\u0026sup2; for the different planes, corroborating the W-H analysis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Dislocation density (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003e) values, a measure of lattice defects, were calculated as:\u003cdiv id=\"Equ6\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ6\" name=\"EquationSource\"\u003e\n$$\\:\\delta\\:=\\frac{1}{{D}^{2}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e6\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eand were found to be in the order of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:2.48\\times\\:{10}^{15}\\)\u003c/span\u003e\u003c/span\u003e to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:3.70\\times\\:{10}^{15}\\:{\\text{m}}^{-2}\\)\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe X-ray density \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\rho\\:}_{x}\\)\u003c/span\u003e\u003c/span\u003e calculated using:\u003cdiv id=\"Equ7\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ7\" name=\"EquationSource\"\u003e\n$$\\:{\\rho\\:}_{x}=\\frac{ZM}{{N}_{A}{V}_{c}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e7\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e(with formula units number \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Z=4\\)\u003c/span\u003e\u003c/span\u003e, molar mass \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:M=79.55\\:\\text{g/mol}\\)\u003c/span\u003e\u003c/span\u003e, Avogadro\u0026rsquo;s number \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{N}_{A}=6.022\\times\\:{10}^{23}\\)\u003c/span\u003e\u003c/span\u003e, and unit cell volume \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{V}_{c}=7.994\\times\\:{10}^{-23}\\:{\\text{cm}}^{3}\\)\u003c/span\u003e\u003c/span\u003e) was \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:6.58\\:{\\text{g/cm}}^{3}\\)\u003c/span\u003e\u003c/span\u003e, consistent with reported values for monoclinic CuO [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe high phase purity, nanoscale crystallite size, low lattice strain, and moderate dislocation density evidenced from XRD analysis indicate successful synthesis of well-crystallized CuO nanoparticles [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The combined use of Scherrer, lattice strain, and Williamson-Hall analyses provides a comprehensive understanding of the microstructural characteristics, which influence the functional properties of CuO in catalytic and sensor applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Optical Analysis\u003c/h2\u003e\u003cp\u003eThe optical properties of the synthesized CuO nanoparticles were examined using UV\u0026ndash;Visible spectroscopy, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(A). The absorbance spectrum exhibited distinct peaks at 329.5 nm, 365.0 nm, and 388.5 nm, indicating characteristic electronic transitions and confirming the nanoscale nature of the CuO particles [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe direct optical band gap (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{g}\\)\u003c/span\u003e\u003c/span\u003e) was estimated using Tauc\u0026rsquo;s method, which relates the photon energy (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:h\\nu\\:\\)\u003c/span\u003e\u003c/span\u003e) and absorption coefficient (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e) by the equation:\u003cdiv id=\"Equ8\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ8\" name=\"EquationSource\"\u003e\n$$\\:(\\alpha\\:h\\nu\\:{)}^{2}=k(h\\nu\\:-{E}_{g})$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e8\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:k\\)\u003c/span\u003e\u003c/span\u003e is a constant related to the material and transition probability. The band gap energy was obtained by extrapolating the linear portion of the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:(\\alpha\\:h\\nu\\:{)}^{2}\\)\u003c/span\u003e\u003c/span\u003e versus \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:h\\nu\\:\\)\u003c/span\u003e\u003c/span\u003e plot (Tauc plot), as in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(B). The calculated band gap of 2.95 \u003cem\u003eeV\u003c/em\u003e is higher than bulk CuO values (~\u0026thinsp;1.2\u0026ndash;1.9 \u003cem\u003eeV\u003c/em\u003e), demonstrating quantum confinement effects expected in nanoparticles [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo quantify light absorption, absorption coefficients \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e were calculated from the measured absorbance (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:A\\)\u003c/span\u003e\u003c/span\u003e) using the Beer\u0026ndash;Lambert relationship:\u003cdiv id=\"Equ9\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ9\" name=\"EquationSource\"\u003e\n$$\\:\\alpha\\:=\\frac{2.303\\times\\:A}{L}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e9\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eassuming a path length \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:L=1\\:\\text{cm}\\)\u003c/span\u003e\u003c/span\u003e for the cuvette where nanoparticles were dispersed. The extinction coefficient (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:k\\)\u003c/span\u003e\u003c/span\u003e), describing the attenuation of electromagnetic waves in the material, was calculated using:\u003cdiv id=\"Equ10\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ10\" name=\"EquationSource\"\u003e\n$$\\:k=\\frac{\\alpha\\:\\lambda\\:}{4\\pi\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e10\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\lambda\\:\\)\u003c/span\u003e\u003c/span\u003e is the light wavelength in centimetres. Furthermore, the optical conductivity (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sigma\\:\\)\u003c/span\u003e\u003c/span\u003e) was determined to assess charge carrier response to incident light:\u003cdiv id=\"Equ11\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ11\" name=\"EquationSource\"\u003e\n$$\\:\\sigma\\:=\\frac{\\alpha\\:nc}{4\\pi\\:}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e11\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:n=2.5\\)\u003c/span\u003e\u003c/span\u003e was assumed for the refractive index of CuO in the visible range, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:c=3\\times\\:{10}^{10}\\)\u003c/span\u003e\u003c/span\u003e cm/s is the speed of light. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the absorbance values at key wavelengths and their corresponding calculated optical parameters.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOptical Parameters of CuO Nanoparticles.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWavelength (\u003cem\u003enm\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbsorbance (\u003cem\u003eA\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAbsorption Coefficient\u003c/p\u003e\u003cp\u003e(\u003cem\u003ecm\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eExtinction Coefficient (\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOptical Conductivity (\u0026times;10\u003csup\u003e9\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e329.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.306\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.83\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e365.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e388.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3.36\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\u003eThe increase in absorption coefficient and extinction coefficient with wavelength near the absorption edges reflects stronger photon absorption due to electronic transitions between valence and conduction bands [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The optical conductivity values indicate enhanced charge carrier density and movement, important for potential applications of CuO nanoparticles in photovoltaics, sensing, and photocatalysis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The high band gap energy combined with the optical constants, confirms the quantum size effect and good crystallinity of the CuO nanoparticles synthesized [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe UV\u0026ndash;Visible absorbance spectrum of the prodigiosin extract is presented in Fig.\u0026nbsp;(3). The extract exhibited a broad, intense absorption band in the visible region with maximum peaks at 521.3 nm, 539.6 nm, and 558.8 nm. These absorption features collectively constitute the principal absorbance band that is characteristic of prodigiosin pigments [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe prominent peaks arise due to electronic transitions associated with the distinctive tripyrrole molecular structure of prodigiosin, which contains three pyrrole rings linked via methine bridges. These transitions typically involve π\u0026rarr;π* and n\u0026rarr;π* excitations within the conjugated system, resulting in the observed absorption in the visible light range.\u003c/p\u003e\u003cp\u003eThe positions and intensities of these peaks confirm both the presence and purity of prodigiosin in the extract, consistent with earlier reports where prodigiosin shows absorption maxima between 520 nm and 540 nm depending on solvent and pH environment [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The broadness of the absorption band indicates some degree of molecular aggregation or heterogeneity in the pigment microenvironment, confirming the successful extraction of bioactive prodigiosin pigment, supporting its potential utility in biomedical and industrial applications where its photophysical properties are pivotal [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 FTIR Analysis\u003c/h2\u003e\u003cp\u003eFourier transform infrared (FTIR) spectroscopy was utilized to characterize the surface chemistry and functional groups present in the synthesized CuO nanoparticles. The FTIR spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) provides detailed insight into the molecular vibrations indicative of chemical moieties on and within the nanostructures.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA broad absorption band centered around 3420 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is assigned to the stretching vibrations of hydroxyl (\u0026ndash;OH) groups, which include surface-adsorbed water molecules and chemically bonded hydroxyl functionalities on the CuO surface. This pronounced band is typical for hydrophilic metal oxide nanoparticles and plays a critical role in their aqueous interactions and potential for functionalization [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe band observed near 1635 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to the bending vibrations of H\u0026ndash;O\u0026ndash;H groups from adsorbed moisture, further confirming the presence of water molecules physically or chemically associated with the nanoparticle surfaces [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The characteristic peaks in the lower wavenumber region (600\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) are attributed to metal\u0026ndash;oxygen vibrational modes. Prominent absorptions at approximately 530 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 490 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are assigned to the Cu\u0026ndash;O stretching vibrations indicative of monoclinic CuO lattice formation. These spectral features align well with X-ray diffraction data, confirming the crystalline quality [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additional absorption bands at 1380 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1100 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are ascribed to bending and stretching vibrations of nitrate groups remaining from precursor copper nitrate salts or adsorbed surface nitrate contaminants. Retention of such groups may influence surface charge and reactivity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Minor bands around 2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to C\u0026ndash;H stretching vibrations that may originate from residual organic compounds, such as stabilizing agents or plant-extract components used in green synthesis, indicating surface passivation of nanoparticles [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThese functional groups observed in FTIR spectra substantiate the dual role of biological molecules: acting as both reductants, facilitating Cu(II) to CuO conversion, and capping agents stabilizing the nanoparticles against agglomeration. The presence of hydroxyl, nitrate, and organic functional groups can significantly influence the chemical stability, surface reactivity, and potential bio-interaction pathways of the CuO nanoparticles [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFTIR Peak Positions and Functional Group Assignments of CuO 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\u003eWavenumber (\u003cem\u003ecm\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAssignment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSignificance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e~\u0026thinsp;3420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eO\u0026ndash;H stretching (surface hydroxyls, water)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIndicates hydrophilic surface, adsorbed moisture\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e~\u0026thinsp;1635\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eH\u0026ndash;O\u0026ndash;H bending (adsorbed water)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAffirms surface-bound water molecules\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e~\u0026thinsp;1380\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNitrate group bending vibrations\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eResidual precursor ions or surface chemisorption\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e~\u0026thinsp;1100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNitrate and C\u0026ndash;O stretching\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSurface adsorption, organic residues\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e~\u0026thinsp;530\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCu\u0026ndash;O stretching\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConfirms monoclinic CuO phase formation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e~\u0026thinsp;490\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCu\u0026ndash;O lattice vibrational modes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCharacteristic of CuO crystal structure\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e~\u0026thinsp;2900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u0026ndash;H stretching (organic residues)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePossible surface stabilizers/capping agents\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\u003eThese surface groups influence the physicochemical behaviour of CuO nanoparticles in aqueous media and biological environments. The hydroxyl bands indicate potential sites for further functionalization, while the confirmation of Cu\u0026ndash;O bonds affirm the nanoparticle\u0026rsquo;s structural integrity. Detection of residual nitrates and organics suggests synthesis optimization can target improved purification for specific applications [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOverall, FTIR results complement XRD and UV-Vis data by elucidating the chemical state of CuO nanoparticle surfaces, which is vital for tailoring materials for use in catalysis, sensors, or biomedical applications. The observed particle aggregation and size distribution are crucial factors to consider when evaluating applications such as catalysis and biomedical usage, where surface accessibility and interaction dynamics are paramount.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Morphological, Energy-Dispersive X-ray, and Elemental Analysis\u003c/h2\u003e\u003cp\u003eThe surface morphology of the extracted prodigiosin pigment was first examined using scanning electron microscopy (SEM), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The pigment appeared as aggregated, irregularly shaped particles consistent with organic pigment morphology [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSEM images of the synthesized copper oxide (CuO) nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) exhibited irregularly shaped crystalline particles that were often clustered into larger agglomerates. This morphology is typical for metal oxide nanoparticles synthesized via green or chemical methods and suggests potential high surface areas beneficial for applications such as catalysis and sensing [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEnergy-dispersive X-ray analysis (EDAX) spectra for the CuO nanoparticles are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The spectra featured prominent peaks corresponding to copper (Cu), oxygen (O), and carbon (C), indicative of the chemical constituents of the nanostructures. Quantitative elemental analysis showed weight percentages of 48.6%, 28.7%, and 22.7% for Cu, O, and C respectively, with atomic percentages of 17.2% (Cu), 40.4% (O), and 42.4% (C). The presence of carbon likely originates from surface-bound organic moieties or residual stabilizers linked to the synthesis process [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe Cu peak at an energy of approximately 129.5 \u003cem\u003eeV\u003c/em\u003e confirms the presence and purity of copper within the synthesized oxide nanoparticles. The elemental composition aligns well with stoichiometric CuO formation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese combined morphological and elemental analyses confirm successful synthesis of CuO nanoparticles with residual organic layers and confirm the pigment\u0026rsquo;s integrative presence and morphological distinction. The agglomerated CuO crystals and pigment form a composite system potentially useful for multifunctional applications [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Cytotoxicity Evaluation of CuO NPs\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of synthesized CuO nanoparticles was investigated against the MCF-7 human breast cancer cell line using the Sulforhodamine B (SRB) colourimetric assay. The assay was performed in 96-well plates, with MCF-7 cells seeded at a density of 5000 cells per well. Cells were treated with CuO nanoparticles at final concentrations of 10, 20, 40, and 80 \u0026micro;g/mL in triplicate. After 48 hours of incubation under standard cell culture conditions, cell viability was determined by measuring absorbance at 540 nm following SRB staining.\u003c/p\u003e\u003cp\u003eQuantitative results indicated a concentration-dependent effect on cell viability. At 10 \u0026micro;g/mL CuO nanoparticles, the mean cell viability was 82.3% (\u0026plusmn;\u0026thinsp;3.37 SD), corresponding to 17.7% cell mortality. For 20 \u0026micro;g/mL treatment, cell viability was recorded as 84.9% (\u0026plusmn;\u0026thinsp;4.21 SD), while at 40 \u0026micro;g/mL, viability was 85.4% (\u0026plusmn;\u0026thinsp;5.60 SD). The largest reduction in viability was observed at the highest nanoparticle concentration, 80 \u0026micro;g/mL, where mean cell viability decreased to 67.1% (\u0026plusmn;\u0026thinsp;13.02 SD); cell mortality at this concentration was 32.9%. The IC\u003csub\u003e50\u003c/sub\u003e value (the concentration at which 50% cell death occurs) could not be determined within the tested concentration range, as none of the tested doses reduced viability below 50% [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMorphological assessment was conducted via phase-contrast microscopy. Representative images of untreated cells (negative control), Adriamycin-treated cells (positive control), and cells treated with the highest CuO nanoparticle concentration are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Untreated MCF-7 cells exhibited a characteristic monolayer with intact, well-spread morphology. Cells exposed to CuO nanoparticles at 80 \u0026micro;g/mL displayed evidence of morphological changes, including cell shrinkage, reduced adherence, and rounding of cells, compared to negative controls [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study successfully demonstrates the novel integration of the naturally derived prodigiosin pigment within a sol-gel synthetic route for producing copper oxide (CuO) nanoparticles, marking a pioneering approach in green synthesis enhancing nanoparticle stability and functionality. The biosynthesized prodigiosin not only acts as a bioactive capping and reducing agent, but its incorporation introduces unique surface chemistry that significantly influences the physicochemical attributes of the CuO nanoparticles. X-ray diffraction analysis confirmed monoclinic CuO phase formation with high crystallinity, evidenced by sharp diffraction peaks indexed to planes (\u0026minus;\u0026thinsp;111), (111), (\u0026minus;\u0026thinsp;202), and (020). Quantitative analysis yielded crystallite sizes ranging from 16.44 nm to 20.08 nm (average 18.72 nm via Williamson-Hall method), with lattice strain measured between 0.38 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 0.69 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and moderate dislocation densities from 2.48 \u0026times; 10\u003csup\u003e15\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 3.70 \u0026times; 10\u003csup\u003e15\u003c/sup\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, underlining the structural integrity achieved through this bio-assisted approach. FTIR spectral analysis revealed pronounced hydroxyl (\u0026ndash;OH) functional groups, metal\u0026ndash;oxygen bond vibrations corresponding to Cu\u0026ndash;O lattice dynamics, and residual nitrate and organic moieties attributed to the pigment and sol-gel process, collectively indicating favorable surface passivation and functional group availability. Optically, CuO nanoparticles displayed absorbance bands at 329.5 nm, 365.0 nm, and 388.5 nm, with an experimentally determined direct band gap of 2.95 \u003cem\u003eeV\u003c/em\u003e, affirming pronounced quantum confinement effects. The prodigiosin pigment exhibited characteristic tripyrrole ring transitions at 521.3 nm, 539.6 nm, and 558.8 nm, confirming its purity and active molecular structure preserved through the nanoparticle formation.\u003c/p\u003e\u003cp\u003eMorphological evaluation via scanning electron microscopy showed aggregated, irregular crystalline structures, and elemental compositional analysis confirmed stoichiometric CuO with 48.6 wt% Cu, 28.7 wt% O, and 22.7 wt% C, the latter evidencing organic capping from prodigiosin. Biological assessment indicated a dose-dependent cytotoxic effect on MCF-7 breast cancer cells, with the highest tested nanoparticle concentration (80 \u0026micro;g/mL) reducing viability to 67.1%, alongside observed morphologic alterations implicating apoptosis pathways. The study presents a compelling case for the application of prodigiosin as a multifunctional agent in the sol-gel synthesis of CuO nanoparticles, enhancing physicochemical properties and endowing bioactivity, thereby expanding the potential for applications in catalysis, biosensing, and targeted cancer therapeutics. Future work should focus on mechanistic elucidation of pigment-nanoparticle interactions and optimization for clinical translation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflicts of interest regarding the publication of this paper. No financial or personal relationships exist that could inappropriately influence or bias the content of this work. All authors confirm that they have no competing interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eDeclaration\u003c/p\u003e\u003cp\u003eNo funding was received to support the preparation of this manuscript or the research presented within it.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eKartikey J. Chavan: Conceptualization, Methodology, Supervision, Writing\u0026ndash;Original Draft, Data Analysis and Curation, Investigation, Review \u0026amp; Editing.Sarang R. Bhagwat: Data Analysis, Supervision.Vineet D. P. Kala: Review \u0026amp; Editing, Formal Analysis, Investigation.Arjun R. Potinde: Data Analysis and Curation, Formal Analysis, Methodology.Hemanth S. Gurajada: Data Analysis and Curation, Formal Analysis.Mansi P. Juvekar: Methodology.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThe authors are grateful to Rayat Shikshan Sanstha\u0026rsquo;s Karmaveer Bhaurao Patil College, Vashi, Navi Mumbai, India, for providing laboratory facilities and support for conducting the experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhulbe R, Kumar R, Kumar V, Kandpal A, Joshi R, Chandra B, et al. Synthesis and Characterization of Copper Oxide Nanoparticles Using Polyol and Their Antimicrobial Potential. Mater. Adv. 2024;6(1):2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.33263/Materials61.002\u003c/span\u003e\u003cspan address=\"10.33263/Materials61.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDevaraji M, et al. 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Mater. 2023;453:131050. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhazmat.2023.131050\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2023.131050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmad M, et al. Dose-Dependent Cellular Responses of Biological CuO Nanoparticles. Toxicol. In Vitro. 2024;89:105344. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tiv.2024.105344\u003c/span\u003e\u003cspan address=\"10.1016/j.tiv.2024.105344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChavan Kartikey, Sarang Bhagwat, Novel synthesis of copper oxide using prodigiosin a pigment extracted from \u003cem\u003eSerratia rubidaea\u003c/em\u003e bacteria and their off. Indian patent application 202321047738 A. 2023.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-sol-gel-science-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jsst","sideBox":"Learn more about [Journal of Sol-Gel Science and Technology](https://www.springer.com/journal/10971)","snPcode":"10971","submissionUrl":"https://submission.springernature.com/new-submission/10971/3","title":"Journal of Sol-Gel Science and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"copper oxide nanoparticles, sol-gel synthesis, prodigiosin, Serratia rubidaea, green synthesis, cytotoxicity, anticancer activity","lastPublishedDoi":"10.21203/rs.3.rs-7801875/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7801875/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study presents a sustainable synthesis of copper oxide (CuO) nanoparticles via a sol-gel approach utilizing prodigiosin pigment extracted from \u003cem\u003eSerratia rubidaea\u003c/em\u003e as a green biogenic agent. To the best of our knowledge, this is the first report of employing prodigiosin from \u003cem\u003eSerratia rubidaea\u003c/em\u003e in the sol-gel synthesis of CuO nanoparticles. Copper sulfate pentahydrate (CuSO₄\u0026middot;5H₂O) served as the precursor, reacting with the bio-pigment under controlled conditions to yield CuO nanomaterials. Comprehensive physicochemical characterization confirmed nanoparticle formation and composition: scanning electron microscopy (SEM) revealed morphology, Fourier-transform infrared (FTIR) spectroscopy identified characteristic Cu\u0026ndash;O vibrational modes, energy-dispersive X-ray spectroscopy (EDAX) established the presence and proportions of copper, oxygen, and carbon, while X-ray diffraction (XRD) confirmed the monoclinic phase (JCPDS Card No. 00-001-1117). Ultraviolet-visible (UV\u0026ndash;Vis) spectroscopy displayed absorption peaks at 329.5, 365.0, and 388.5 nm, with an estimated optical band gap of 2.95 eV. The dual role of prodigiosin as a natural reducing and capping agent highlights the eco-friendly and innovative nature of this synthesis. The resultant CuO nanoparticles exhibit properties comparable or superior to those synthesized with other biological agents reported recently. Preliminary cytotoxicity assessment using the Sulforhodamine B (SRB) assay against MCF-7 breast cancer cells demonstrated a dose-dependent reduction in cell viability, achieving a maximum mortality of 32.9% at 80 \u0026micro;g/mL. Although the half-maximal inhibitory concentration (IC₅₀) was not reached within the tested range, these findings suggest promising anticancer potential and warrant further biomedical investigations of the synthesized biofunctionalized CuO nanoparticles.\u003c/p\u003e","manuscriptTitle":"Novel Prodigiosin-Driven Biogenic Sol-Gel Synthesis of Copper Oxide Nanoparticles Using Serratia rubidaea: Extraction, Dual Role as Bio-Reducing and Capping Agent, and Enhanced Cytotoxic Potential for Biomedical Application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-24 07:36:55","doi":"10.21203/rs.3.rs-7801875/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-11T21:37:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T14:31:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88565859732517738721395885977563277462","date":"2025-10-10T14:04:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-10T12:49:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-09T06:00:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-09T05:58:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Sol-Gel Science and Technology","date":"2025-10-07T17:55:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-sol-gel-science-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jsst","sideBox":"Learn more about [Journal of Sol-Gel Science and Technology](https://www.springer.com/journal/10971)","snPcode":"10971","submissionUrl":"https://submission.springernature.com/new-submission/10971/3","title":"Journal of Sol-Gel Science and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7afb1adf-68e7-4c22-85fb-80aba7ef3ddf","owner":[],"postedDate":"October 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T15:59:32+00:00","versionOfRecord":{"articleIdentity":"rs-7801875","link":"https://doi.org/10.1007/s10971-025-07082-z","journal":{"identity":"journal-of-sol-gel-science-and-technology","isVorOnly":false,"title":"Journal of Sol-Gel Science and Technology"},"publishedOn":"2026-01-03 15:57:18","publishedOnDateReadable":"January 3rd, 2026"},"versionCreatedAt":"2025-10-24 07:36:55","video":"","vorDoi":"10.1007/s10971-025-07082-z","vorDoiUrl":"https://doi.org/10.1007/s10971-025-07082-z","workflowStages":[]},"version":"v1","identity":"rs-7801875","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7801875","identity":"rs-7801875","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}