Pomegranate peel extract mediated biocompatible silver selenide bimetallic nanoparticles and comprehensive evaluation of their biological activities

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Bimetallic nanoparticles (BNPs), particularly silver selenide (Ag 2 Se), have emerged as promising candidates due to their enhanced antibacterial properties, biocompatibility and multifunctional applications. This study presents an eco-friendly synthesis of Ag 2 Se BNPs using pomegranate ( Punica granatum L.) peel extract, leveraging its rich polyphenols and flavonoids as natural reducing and stabilizaing agents. The synthesized nanoparticles were characterised using Uv-Vis spectroscopy, FTIR, XRD and FE-SEM, confirming their structural and morphological properties. Antimicrobial assays demonstrated potent activity against both Gram-positive ( Staphylococcus aureus, Staphylococcus epidermidis ) and Gram-negative ( Escherichia coli , Acinetobacter baumannii ) bacteria, with significant membrane disruption observed through cytoplasmic leakage and SEM analysis. Additionally, the BNPs exhibited notable antioxidant activity in DPPH and H 2 O 2 assays, alongside excellent haemocompatibility at lower concentrations. These findings highlight the dual functionality of Ag 2 Se BNPs as effective antimicrobial and antioxidant agents, offering sustainable solutions for biomedical applications such as wound healing, antimicrobial coatings and drug delivery systems. This green synthesis approach aligns with global efforts towards eco-friendly nanotechnology. Biological sciences/Biochemistry Biological sciences/Biotechnology Physical sciences/Chemistry Biological sciences/Microbiology Physical sciences/Nanoscience and technology Bimetallic nanoparticles green synthesis antimicrobial activity antioxidant potential hemocompatibility pomegranate peel extract Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The rise of antibiotic resistant bacteria poses a significant threat to global health, necessitating the urgent development of novel antimicrobial agents 1 . Among the promising solutions, bimetallic nanoparticles (BNPs) have gained attention due to their enhanced antibacterial properties, biocompatibility and multifunctional applications 2 . Silver selenide (Ag 2 Se) nanoparticles, known as naumannite, are a fascinating I-VI group semiconductor material. While typically found in bulk form, but rarely found as natural materials 3 . Among diverse semiconductor materials, Ag 2 Se nanoparticles stand out due to their physicochemical and biological properties, making them highly promising for biomedical and therapeutic applications. Due to its versatile applications in electronics and biomedicine, Ag 2 Se is a highly valued chalcogenide nanomaterial. It also acts as a mixed ionic conductor, undergoing a phase transition at atmospheric pressure, shifting from a low temperature orthorhombic phase (β-Ag 2 Se) to a high temperature cubic phase (α-Ag 2 Se) 4 , 5 . In relation to this, orthorhombic β-Ag 2 Se is the most widely accepted crystal structure of Ag 2 Se nanoparticles owing to its relatively high Seebeck coefficient (thermoelectric power, -150 µV K − 1 ) at 300 K with an unusually low lattice thermal conductivity couple with high electrical conductivity 6 . Study proclaimed that, orthorhombic structure of Ag 2 Se nanoparticles exhibited both photocatalytic as well as fluorescence activity. Moreover, such compound has been utilised as a photosensitizer in photographic films, thermo-chromic materials for nonlinear optical devices and photovoltaic cells 7 . Ag 2 Se nanoparticles are typically synthesized applying various methods such as chemical conversion, hydrothermal reactions and hot injection. In contrast, microstrctured Ag 2 Se can be formulated via electrodeposition and thermal evaporation techniques 8 . Such traditional synthesis methods for nanoparticles often involve toxic chemical, high energy consumption and environmental hazards 8 . In contrast, green synthesis using plant extracts as reducing and stabilizing agents offers an eco-friendly, cost effective and sustainable alternative. Pomegranate ( Punica granatum L.) peel, a rich source of polyphenols, flavonoids and other bioactive compounds, serves as an excellent natural medium for nanoparticle synthesis. These biomolecules not only facilitate the reduction of metal ions but also enhance the stability and biological activity of the resulting nanoparticles. This study explores the green synthesis of Ag 2 Se nanoparticles using pomegranate peel extract, followed by a comprehensive evaluation of their biological activities. The nanoparticles were characterized for their structural and morphological properties and their antimicrobial efficacy was tested against both Gram positive and Gram negative bacteria. Additionally, their antioxidant potential and hemocompatibility were assessed to determine their suitability for biomedical applications. By leveraging the natural reducing power of pomegranate peel, this research aims to develop a sustainable, non-toxic and highly effective nanomaterial with dual functionality, combating microbial infections while mitigating oxidative stress. These findings could pave the way for innovative applications in wound healing, antimicrobial coatings and drug delivery systems, contributing to the advancement of eco-friendly nanotechnology 9 . 2. Experimental High purity laboratory-grade chemical reagents were used in this study. Sodiuum selenate anhydrous (Na 2 SeO 4 ) and nutrient broth were obtained from Sisco Research Pvt. Ltd (SRL), Mumbai, India. Bacterial strains, including Acinetobacter baumannii (MTCC 1245), Staphylococcus aureus (MTCC 96), Staphylococcus epidermidis (MTCC 435) and Escherichia coli (MTCC 443), were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid (Vitamin C) were purchased from SRL Pvt. Ltd, Mumbai, India. Hydrogen peroxide (H 2 O 2 ) was acquired from Molychem Pvt.Ltd. Fresh pomegranate ( Punica granatum L.) fruits were procured from a local market in Bhubaneswar, Odisha, India. All experiments were conducted using deionized water. 2.1. Extraction of Punica granatum L. peels Fresh pomegranate ( Punica granatum L.) fruits were thoroughly washed with running water to remove surface contaminants. The outer peel was carefully separated, chopped into small pieces and gently dried using tissue paper. The chopped peels were then sun dried for a week to ensure complete moisture removal. Subsequently, the dried peels were finely ground using a mechanical grinder 2.2. Biosynthesis of bimetallic nanoparticles We have synthesized the biogenic bimetallic nanoparticles following the protocol outlined by Sibiya et al. with some modifications 10 . Pomegranate peel extract was added with distilled water at 1:100 w/v ratio. The solution was homogenized using a stirrer for 15 min. The resultant mixture underwent heating until it reaches a temperature of 60 o C prior to centrifugation. After that, the solution was centrifuged at 8000 rpm for 20 min. The obtained supernatant was filtered using Whatman filter paper. To that filtered plant extract solution, 50 mL of 1 mM silver nitrate (AgNO 3 ) was added and the reaction mixture was put on incubation at 120 rpm for 48 hr. Post incubation, 50 mL of filtered plant extract solution was mixed with AgNPs solution and the total volume was made up to 100 mL. Then, 25 mM of selenium precursor (sodium selenate) was added to the 100 mL of AgNPs and plant extract solution followed by incubation at 120 rpm for 24 hr. Post incubation, the colour change of the reaction mixture was clearly observed showing successful formulation of silver selenide bimetallic nanoparticles (BNPs). Later, following purification process, the BNPs solution was further centrifuged at 8000 rpm for 20 min. The obtained pellet was washed with distilled water and subjected to drying at 70 o C in order to get powdered nanoparticles. 2.3. Characterization of bimetallic nanoparticles The absorbance of BNPs was studied employing UV-vis spectrophotometer (HITACHI, Tokyo, Japan). Bond level analysis was done by FTIR spectrophotometer (JASCO, Japan). XRD of BNPs was achieved by Rigaku X-ray Diffractometer (Tokyo, Japan), applying Cu-Kα radiation with angular range 20 0 to 80 0 . Availability of various phases in the formulated nanoparticles was determined by the X’-pert high score software. Morphological analysis was estimated via FE-SEM (JEOL JSM-IT800). 2.4. Evaluation of antioxidant activity of bimetallic nanoparticles 2.4.1. DPPH assay DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was investigated for antioxidant activity of biogenic BNPs. Precisely, 0.2 mM DPPH was dissolved in methanol to prepare the stock solution. Here ascorbic acid (Vitamin C) was considered as a positive control. A 96 well microtiter plate containing 100 µl of different concentrations of BNPs (50, 100, 250 µg/mL) and 100 µl of DPPH methanolic solution was incubated in a dark environment with a constant shaking at 35 o C for 30 min. Post incubation, the absorbance was monitored at 517 nm employing Biorad Imark microplate reader. Later, the scavenging activity was evaluated by the formula Radical scavenging activity (%) = (A control – A sample / A control ) x 100 The result was obtained by plotting a graph between % DPPH scavenging and concentrations of BNPs. 2.4.2. H 2 O 2 assay Antioxidant activity of BNPs was also assessed by H 2 O 2 scavenging assay. Precisely, 40 mM of H 2 O 2 solution was prepared in phosphate buffer. A stock solution of BNPs was prepared in phosphate buffer.1 mL of different concentrations of BNPs (50, 100, 250 µg/mL) was mixed with 1 mL of H 2 O 2 followed by incubation for 10 min at room temperature. Post incubation, the absorbance was monitored at 230 nm. The percentage of scavenging was evaluated by the formula Radical scavenging activity (%) = (A control – A sample / A control ) x 100 The result was obtained by plotting a graph between % H 2 O 2 scavenging and concentrations of BNPs. 2.5. Assessment of antimicrobial activity of bimetallic nanoparticles 2.5.1. Bacterial growth inhibition Bacterial growth inhibition in presence of BNPs was evaluated against a panel of Gram-positive bacteria like S. epidermidis , S. aureus as well as Gram-negative bacteria like E. coli and A. baumannii by growth kinetics study. Precisely, a loopful of bacteria was collected from the mother culture and inoculated into the nutrient broth followed by overnight incubation at 37 o C and 120 rpm. Furthermore, appropriate amount of BNPs was dissolved in deionized water and was sonicated for 20 min in order to prepare the stock solution of nanoparticles. Various concentrations of BNPs (25, 50, 100, 250, 500 µg/mL) were used in 96 well plates for growth rate evaluation. Reaction mixture without BNPs was considered as control. 20 µl of bacterial culture (10 6 CFU/mL) was dissolved in each reaction mixtures and total volume was made up to 300 µl using nutrient broth. Biorad Imark plate reader was used for the assessment of growth kinetic study. Optical density (O.D) was measured at 600 nm. 2.5.2. Evaluation of cytoplasmic leakage activity of bimetallic nanoparticles For analysis of leakage of bacterial cytoplasmic contents like DNA and protein, Gram-positive bacteria like S. epidermidis and Gram-negative bacteria like E. coli were cultured and incubated overnight at 37 o C. Later, the microbial culture was centrifuged at 10,000 rpm for 10 min. Obtained pellet was suspended in PBS buffer (PBS 7.4). The bacterial cell count was set to 10 5 cells/mL. Cell suspensions were mixed with BNPs and were incubated at room temperature for 2, 4 and 6 hr. The bacterial culture without nanoparticle treatment was considered as control. After that, the cultures were centrifuged at 5000 rpm for 10 min. The obtained supernatant was measured at 260 nm for estimation of DNA leakage and at 280 nm for estimation of protein leakage. 2.6. Evaluation of hemocompatibility of bimetallic nanoparticles The present study investigated hemocompatibility of BNPs following the protocol outlined by Pal et al. with minor modifications 11 . This study is focussed on degree of haemolysis caused by the tested sample. For this purpose, goat blood was collected in EDTA vials. The blood was diluted with PBS in dilution ratio 4:5. In order to validate haemolysis, 0.5 mL of 0.01N hydrochloric acid (HCl) was added to PBS and underwent incubation at 37 o C for 30 min. Post incubation, 0.05 mL of diluted blood was added to the mixture to maintain the volume upto 10 mL. Later, the solution was incubated for 2 hr. The optical density (OD) was monitored at 545 nm using a spectrophotometer. Here, HCl was considered as a positive control since it possesses effective rupturing of red blood corpuscle (RBC). Similarly, 0.05 mL of diluted blood was further diluted with 10 mL of PBS. This solution was kept on incubation at 37 o C for 2 hr. Post incubation, the OD of the solution was measured at 545 nm. Here, PBS is considered as negative control since it is well-known for least RBC rupture. As the standard ODs were estimated, 0.5 mL of tested sample was added to PBS and was incubated for 30 min at 37 o C in order to sustain temperature equilibrium. Later 0.05 mL of diluted blood was added to the solution and was further incubated for 2 hr. Post incubation, OD of the sample was monitored at 545 nm. Previous study proclaimed that, less than 5% haemolysis of the test material was preferred to be highly haemocompatible and less than 10% haemolysis was considered to be haemocompatible. Additionally, qualitative hemocompatibility study was done with agar well-diffusion method. Precisely, in the autoclaved agar, certain amount of goat blood was mixed and poured into petriplates and the plates were kept for solidification. Later, 6–8 mm wells were made on the plates with a sterile cork borer. Different concentrations of BNPs were put into the well and the plates were incubated overnight at 37 o C. Here, HCl was taken as a positive control. Post incubation, inhibition zones were clearly observed. 2.7. Evaluation of bacterial morphology in presence of bimetallic nanoparticles Precisely, bacterial morphology study upon treatment with BNPs was evaluated following the protocol outlined by Arakha et al. with some modifications 12 . In brief, a Gram-negative bacteria E. coli was inoculated in nutrient broth and incubated overnight at 37 o C. On the next day, the bacterial sample was taken from stationary phase of growth kinetics and directly put on the cover slide. Later, the tested BNPs was mixed with the bacterial culture and also put on another cover slide. Only bacterial sample on the cover slide was considered as control. The slides were kept on overnight incubation at 37 o C. Post incubation, the surfaces of slides were gently washed with 1X PBS. Then it was soaked on tissue paper. To preserve the bacterial cell morphology, the cells were fixed with 2.5% glutaraldehyde on the top of the slide and kept for 1 to 2 hr at room temperature. The glutaraldehyde was soaked on the tissue paper followed by washing with 1X PBS. Later, the slides were dehydrated with increasing concentration of ethanol (50%, 70%, 90% and 100%). The fixed, washed and dehydrated cells were now prepared for monitoring the morphological structures of both control and treated samples. 2.8. Statistical analysis of data All the experiments were done in triplicates and the results were assessed as mean ± SD. The data analysis was done with one-way ANOVA using Minitab software. The statistical analysis was done with Tukey’s multiple comparison test. P ≤ 0.05 were considered significant. 3. Results and Discussion 3.1. Synthesis and optical analysis of bimetallic nanoparticles The aqueous extract of Punica granatum L. was employed for the synthesis of Ag 2 Se BNPs from silver nitrate (AgNO 3 ) and sodium selenate (Na 2 SeO 4 ) as silver and selenium precursors respectively. The discoloration of the reaction mixture was might be due to the excitation of the surface plasmon resonance of silver ions in the BNPs 8 . The existence of a well-defined absorption band between 300 and 400 nm with a maximum at ~ 359 nm confirmed the successful synthesis of the BNPs. In this range, the single absorption peak offers evidence towards shape, size as well as stability/aggregation of the nanoparticles 13 , 14 (Fig. 1 a). In this context, other studies have demonstrated similar results 8 , 15 , 16 . Moreover, during the synthesis of BNPs, initially, the selenium ions are reduced to Se 2 followed by co-precipitation with Ag cations. Therefore, persistent reaction time along with the reduction of selenium by the biomolecules shows the existence of a biomolecular matrix as a stabilizer of Ag 2 Se BNPs 17 , 18 . 3.2. FTIR analysis of bimetallic nanoparticles The functional interaction between biomolecules and Ag 2 Se was determined by FTIR analysis (Fig. 1 b). The spectrum determined by Ag 2 Se nanoparticles obtained at 2928 cm − 1 , 2860 cm − 1 can be assigned to C-H stretching vibrations indicating alkane groups. Such vibrations specifically fall within the range of 2850–3000 cm − 1 . Similar results have been demonstrated by Ahmad et al. 19 Peak obtained at 2363 cm − 1 typically associated with Ag-Se bond stretching vibration. Such wavenumber falls within the range where Ag-X stretching vibrations are prominent. Here selenium becomes a heavier halogen than chlorine or bromine. Additionally, the presence of other functional groups involved in the synthesis or capping of the nanoparticles could also contribute to the overall spectrum, potentially influencing the intensity and shape of the peak at 2363 cm − 1 . Furthermore, the peaks obtained at 1675 cm − 1 , 1631 cm − 1 may be assigned to the stretching vibration of a carbonyl group (C = O). The peak at 1569 cm − 1 is likely due to C-H bending vibration of the capping agents present on the nanoparticle surface. The FTIR peak at 1451 cm − 1 can contribute to the bending vibration of –CH 2 - group and the distortion of the –CH 3 group. In other words, it is allied with the presence of alkanes or other organic molecules that have carbon-hydrogen bonds. Additionally, the peak obtained at 1041 cm − 1 is typically attributed to C-O stretching vibrations in the ligands or capping agents present on the nanoparticle surface. Such peak can also be indicative of the presence of specific functional groups like ester or alcohol etc. The FTIR peaks obtained at 756 cm − 1 and 533 cm − 1 can be attributed to Ag-Se stretching vibrations. In this regard, Sytu et al. and Kalishwaralal et al. have suggested notable transmission peaks appeared at 1652 and 1396 cm − 1 attributed to Ag-Se bond in the nanoparticle structure 20 , 21 . 3.3. X-ray diffraction analysis of bimetallic nanoparticles Study suggested that Ag 2 Se can exist in various forms like orthorhombic β-phase or a cubic α-phase 8 . Kumashiro et al. stated that, Ag 2 Se is a non-stoichiometric compound in both phases and can co-exist with Ag 2 Se as a single phase 22 . The XRD pattern of Ag 2 Se suggested different characteristic diffraction peaks were observed at 2θ = 27.46 o (002), 32.3 o (101), 38 o (110), 44.08 o (020), 64.44 o (111) and 77.34 o (102) which can be assigned to orthorhombic β-Ag 2 Se phase with Naumannite structure (JCPDS file no. 24-1041) 15 (Fig. 1 c). Study confirmed that, the orthorhombic phase is stable at low temperatures 15 , 23 , 24 . Moreover, XRD pattern also suggested some weak peaks that can be attributed to selenium nanoparticles (SeNPs). This observation suggested that the precursor cations were reduced to their elemental forms in the obtained sample and the size of the nanoparticles was found to be 11.52 nm. Our study is in good agreement with Delgado et al and Garcia et al 8, 15 . 3.1.4. Morphological characterization of bimetallic nanoparticles In order to study the chemical composition and purity of the obtained Ag 2 Se nanoparticles, FESEM-EDAX study was carried out. The EDAX pattern derived from selected areas of FESEM images, suggesting silver, selenium and oxygen peaks in the synthesized Ag 2 Se nanoparticles (Fig. 2 b). The presence of silver and selenium peaks in the obtained Ag 2 Se nanoparticles are in good agreement with elemental peaks as reported in different studies. Therefore, the EDAX spectrum validated the availability of silver and selenium atoms in the synthesized Ag 2 Se nanoparticles 8 , 25 , 26 . 4. Antimicrobial activity of bimetallic nanoparticles This study systematically evaluated the concentration-dependent antibacterial efficacy of Ag 2 Se nanoparticles against a panel of Gram negative bacteria such as A. baumannii , E, coli and Gram positive bacteria like S. epidermidis , S. aureus etc. (Fig. 3 a-d). Bacterial growth kinetics was monitored through optical density (OD 600 ) measurements over a 12 hour period, revealed distinct patterns of microbial inhibition corresponding to increased nanoparticle concentrations (25–500 µg/mL). The control groups for all tested species exhibited characteristic exponential growth curves, confirming viable bacterial proliferation in the absence of treatment. Additionally, Ag 2 Se nanoparticles demonstrated progressively stronger growth suppression at higher doses. At 500 µg/mL, the nanoparticles achieved near-complete inhibition of E. coli and S. aureus . This potent bactericidal effect was particularly evident after 6–8 hours of exposure, coinciding with the typical log-phase growth period of untreated cultures. While all tested pathogens showed susceptibility, the response kinetics varied between species. A. baumannii displayed a more gradual decline in viability, suggesting either reduced permeability to nanoparticles or enhanced resistance mechanisms. Similarly, S. epidermidis exhibited a delayed but significant response at higher concentrations. The differential sensitivity patterns may reflect variations in cell wall composition among tested Gram negative and Gram positive bacteria. In this regard, Ahmad et al. have studied regarding the efficiency of Se-Ag nanocomposites towards antibacterial efficiency and stated that, the superior antibacterial efficiency of Se-Ag nanocomposite could be probably due to the combined antibacterial actions of Ag and Se in the formulated nanocomposite. Here, the Se-Ag composite serve as a large reservoir of the biologically active silver and a slow and sustained release of the Ag and Se could be responsible for its better efficacy as compared to individual counterparts 27 . Furthermore, these findings not only confirm the broad-spectrum antibacterial properties of Ag 2 Se nanoparticles but also highlight their potential for tailored antimicrobial applications. The concentration-dependent efficacy, particularly against resilient pathogens like MRSA and multidrug-resistant A. baumannii makes these nanoparticles as promising candidates for developing novel antimicrobial coatings, wound dressings or therapeutic formulations. The mechanism of the Ag 2 Se nanoparticles bactericidal mode of action may vary in consistent with various bacterial species as well as the composition of the sample as reported by Garcia et al 8 . Further studies investigating the precise mechanisms of action and potential synergy with conventional antibiotics could enhance their clinical applicability. 5. Evaluation of cytoplasmic leakage activity of bimetallic nanoparticles The cytoplasmic DNA and protein leakage assay of Ag 2 Se nanoparticles was carried out against Gram positive bacteria like S. epidermidis and Gram negative bacteria like E. coli . (Fig. 4 a-d) The cytoplasmic DNA leakage assay evaluates the membrane disrupting effect of Ag 2 Se nanoparticles on E. coli by measuring absorbance at 260 nm, which indicates released DNA over a 6-hour period. The control sample shows minimal absorbance, confirming intact bacterial membranes, while increasing concentrations of Ag 2 Se nanoparticles (100, 200 and 500 µg/mL) exhibit a dose dependent rise in DNA leakage. The highest concentration (500 µg/mL) induces the most significant absorbance increase, reflecting severe membrane damage and substantial cytoplasmic DNA release. Additionally, in case of S. epidermidis , the cytoplasmic DNA leakage assay demonstrated a substantial increase in extracellular DNA levels upon treatment of S. epidermidis with Ag 2 Se nanoparticles, compared to the untreated control. This marked elevation in DNA release indicates significant membrane damage, suggesting that the nanoparticles effectively disrupt the structural integrity of the bacterial cell wall and cell membrane. The observed leakage likely results from nanoparticle-induced oxidative stress and direct physical interactions with the membrane, leading to pore formation or rupture. These findings strongly support the bactericidal action of Ag 2 Se nanoparticles through membrane destabilization and subsequent intracellular content leakage. Furthermore, Ag 2 Se nanoparticles cause severe damage to E. coli cell membranes, leading to significant protein leakage. Unlike the untreated control, which shows minimal release, nanoparticles treated samples exhibit a sharp rise in extracellular protein levels, measured by increased absorbance. This confirms that Ag 2 Se nanoparticles physically disrupt bacterial membranes, likely through oxidative stress and structural destabilization. The uncontrolled efflux of cytoplasmic components underscores their potent bactericidal action, highlighting a mechanism rooted in membrane disintegration. Similarly, the cytoplasmic protein leakage clearly demonstrated that Ag 2 Se nanoparticles induce significant damage to S. epidermidis membranes, triggering elevated release of intracellular proteins. While untreated cells maintain their structural integrity, nanoparticles exposed bacteria suffer massive protein leakage caused by membrane damage. Such destructive effect results from nanoparticle-induced oxidative stress that degrades membrane components ultimately leading to cell death. This phenomenon confirms the potential of Ag 2 Se nanoparticles as an effective antimicrobial agent for dealing with various resistance mechanisms. These findings reinforce Ag 2 Se nanoparticles as a promising antimicrobial agent capable of effectively compromising bacterial membrane integrity with their antibacterial activity intensifying at higher doses. 6. Evaluation of antioxidant activity of bimetallic nanoparticles 6.1. DPPH assay The DPPH assay is considered to be the most widely used in vitro method for determination of antioxidant activity of nanoparticles 28 . DPPH assay offers a simple and widely used method to evaluate antioxidant properties of nanoparticles by measuring their ability to scavenge DPPH free radicals. The free radicals like superoxide, hydroxyl radicals can be generated from normal metabolic process cause several serious medical complications like cancer, aging and diabetes mellitus etc 29 . Therefore, neutralization of such free radicals is highly essential in order to safeguard the normal cells from their lethal effects 30 . In this study, we have estimated a dose dependent free radical scavenging activity of Ag 2 Se bimetallic nanoparticles by taking ascorbic acid as a positive control (Fig. 5 a). In this assay, the silver selenide nanoparticles with antioxidant properties interact with DPPH by donating electrons or hydrogen atoms to neutralize the free radical appearance of DPPH. Figure 5 a showed that, ascorbic acid as positive control, showed effective scavenging activity. Ascorbic acid reveals effective potential of DPPH radical scavenging activity via two mechanisms such as hydrogen atom transfer (HAT) and single electron transfer followed by proton transfer (SET-PT) 31 . In HAT, ascorbic acid donates a hydrogen atom (H + ) to DPPH free radical converting it into a stable and non-radical compound. Additionally, following SET-PT mechanism, ascorbic acid first donates an electron, followed by a proton in order to neutralize the radical. Therefore, in our study, ascorbic acid showed effective scavenging activity even at lower concentration while Ag 2 Se bimetallic nanoparticles showed their antioxidant potential at higher concentration. In this regard, SeNPs have also exhibited similar antioxidant properties as suggested by various studies 32 – 34 . Moreover, silver nanoparticles (AgNPs) have also exhibit significant radical scavenging potential as reported by previous studies 35 . 6.2. H 2 O 2 assay We have also evaluated antioxidant potential of Ag 2 Se nanoparticles via hydrogen peroxide (H 2 O 2 ) scavenging assay. For comparison, ascorbic acid was taken as a standard (Fig. 5 b). The Ag 2 Se H 2 O 2 scavenging assay reveals a concentration dependent antioxidant activity for both Ag 2 Se nanoparticles and ascorbic acid (Vit.C). At lower concentration (50 µg/mL), Ag 2 Se nanoparticles exhibited moderate scavenging activity, while Vit.C demonstrates superior efficacy, showing nearly 80% scavenging. As the concentration increases to 100 µg/mL, Ag 2 Se showed progressive improvement, yet Vit.C maintained its dominance with near maximal scavenging. At the highest tested concentration (250 µg/mL), Ag 2 Se achieves significant scavenging, though Vit.C remains the more potent antioxidant. Ag 2 Se exhibited effective H 2 O 2 scavenging activity due to their ability to catalyse the decomposition of H 2 O 2 into less harmful products like water and oxygen and also due to the inherent antioxidant properties of selenium. The combination of silver and selenium in the bimetallic nanoparticle may lead to a synergistic effect. Silver nanoparticles are known for their antimicrobial and catalytic properties, while selenium offers antioxidant activity. This combination can result in enhanced H 2 O 2 scavenging compared to either silver or selenium nanoparticles alone 10 . Additionally, the size and morphology of the nanoparticles can also influence their H 2 O 2 scavenging activity. Smaller nanoparticles generally have a larger surface area, which may lead to increased interaction with H 2 O 2 molecules and more efficient decomposition. These results highlight the robust scavenging capability of ascorbic acid, a well-established antioxidant, while underscoring the promising but comparatively modest activity of Ag 2 Se nanoparticles. The findings suggested that Ag 2 Se could serve as an excellent antioxidant agent, particularly in applications where traditional antioxidants like Vit.C may be less suitable. Further optimization of nanoparticle composition or concentration may enhance their scavenging potential. 7. Evaluation of haemocompatibility of bimetallic nanoparticles Haemocompatibility of nanoparticles offers significant role in their biomedical applications since blood is a primary target contrary to toxicity of the nanoparticles 36 . Nanoparticles lead to rupturing of RBC in the circulatory system thereby releasing haemoglobin which results in various chronic disorders such as anemia, jaundice, various pathological disorders and kidney failure 37 . Therefore, in our study, quantitative and qualitative assessment of haemolytic potential of Ag 2 Se nanoparticles were carefully evaluated in order to determine their blood compatibility. This suggested a concentration dependent RBC lysis induced by Ag 2 Se nanoparticles with hemolysis level approximately 7%, 11% and 42% according to the concentrations 25 µg/mL, 50 µg/mL and 100 µg/mL respectively (Fig. 6 ). Here HCl was considered as positive control. Studies suggested that below 5% hemolysis the test material can be considered to be highly haemocompatible and below 10% as haemocompatible 11 , 38 . At lower concentration, our studied nanoparticles showed least lysis activity confirming safety and compatibility towards validation of various biomedical applications. In this context, Chahardoli et al. have studied haemocompatibility of Quercetin assisted silver nanoparticles which showed around 9% of haemolytic activity at 1mg/mL concentration, but at lower concentration it showed negligible amount of lysis activity indicating such nanoparticles to be non-toxic and hemocompatible in nature. Furthermore, Hashem et al. have stated that, biogenic SeNPs at lower concentration showed least haemolytic effect confirming their safety and hemocompatibility. On the other hand, as per the standards of American Society of Testing, the test material with hemolysis > 5% are haemolytic, 2–5% are slightly haemolytic and < 2% are non-hemolytic in nature 39 , 40 . Studies claimed that, Ag 2 Se nanoparticles not only exhibit good haemocompatibility, but even promote blood clotting, offering potential applications in wound healing as well as antibleeding therapies 41 . Additionally, Ag 2 Se nanoparticles, with specific coatings and particle size may exhibit low haemolytic activity 42 . Additionally, in case of qualitative analysis of haemolytic potential of Ag 2 Se nanoparticles, at lower concentration 25 µg/mL, the tested nanoparticles showed least zone of lysis and as the concentration gradually increased to 50 and 100 µg/mL, the nanoparticles showed maximum haemolytic activity (Fig. 7 ). Therefore, both qualitative and quantitative analysis of haemolytic potential of Ag 2 Se nanoparticles demonstrated that lower concentration of Ag 2 Se nanoparticles confirms its safety while higher concentration may cause toxicity. 8. Surface morphology of bacteria upon treatment with bimetallic nanoparticles Bacterial membrane deformities upon Ag 2 Se nanoparticles treatment were observed via scanning electron microscopy (SEM). The images obtained showed clumping or aggregation of bacterial cells. In order to gain further information, nanoparticle treated and untreated bacterial cells were scanned using SEM. The images confirmed significant clumping as well as membrane rupture in treated cells rather than untreated cells due to interaction of nanoparticles with the bacterial cell surface and membrane. The nanoparticles can accumulate in the cell wall, causing denaturation and eventually leading to cell lysis. Additionally, they can release silver ions, which further disrupt cellular processes, including DNA binding and respiration, ultimately leading to cell death. Moreover, nanoparticle treatment induced a significant reduction in bacterial population density compared to control samples (Fig. 8a and 8b). The Ag 2 Se nanoparticles appeared to exhibit cell lysis and delay formation of biofilm, thereby contributing towards reduction in bacterial population. These findings suggest that, the Ag 2 Se nanoparticles effectively compromise bacterial membrane integrity as well as helps in disruption of biofilm structures, signifying its effective antimicrobial activity. In this context, Li et al. have done a comparative analysis of antibacterial activity, dynamics and effects of silver ions and silver nanoparticles suggesting strong alteration in bacterial morphology thereby showing effective antibacterial activity as described by various other studies 43 , 44 9. Conclusion This study represents a significant advancement in the green synthesis of silver slenide bimetallic nanoparticles (Ag 2 Se BNPs) using pomegranate peel extract, offering an eco-friendly and sustainable alternative to conventional chemical methods. The biosynthesized BNPs demonstrated remarkable biological activities, positioning them as promising candidates for biomedical and therapeutic applications. Furthermore, the research highlights dual functionality of Ag 2 Se BNPs aas both antimicrobial and antioxidant agents, with potential applications in wound dressings, antimicrobial coatings and drug delivery systems. Future work should explore their synergistic effects with existing antibiotics and in vivo biocompatibility to fully realize their clinical potential. By influencing plant-based synthesis, this study also contributes to the growing field of sustainable nanotechnology, aligning with global efforts towards greener scientific practices. Declarations Conflicts of interest There are no conflicts to declare Funding: No Funding Author Contribution S.S.: conceptualization, investigation, methodology, validation, writing original draft, P.S.: data curation, A.K.P.: data curation and editing, M.A.: Investigation, conceptualization, formal analysis, methodology, supervision, review & editing. All authors revieewed the manuscript. Data Availability The data that supports the findings of this study are available from the corresponding author upon reasonable request. References Salam, M. A., Al-Amin, M. Y., Salam, M. T., Pawar, J. S. & Akhter, N. A. A. Rabaan and M. A. Alqumber, (2023). Sharma, G. et al. J. King Saud University-Science , 31 , 257–269. (2019). Helan, P. P. J., Mohanraj, K. & Sivakumar, G. Trans. Nonferrous Met. Soc. China , 25 , 2241–2246. (2015). Wu, T. et al. Nanoscale , 11 , 20820–20836. (2019). Tang, H. et al. ACS Appl. Mater. Interfaces , 8 , 17859–17869. (2016). Ferhat, M. & Nagao, J. J. Appl. Phys. , 88 , 813–816. (2000). Cao, H. et al. Nano Res. , 3 , 863–873. (2010). García, D. A. et al. Molecules , 25 , 5193. (2020). Kharissova, O. V., Dias, H. R., Kharisov, B. I., Pérez, B. O. & Pérez, V. M. J. Trends Biotechnol. , 31 , 240–248. (2013). Olawale, F., Ariatti, M. & Singh, M. Nanomaterials , 11 , 2516. (2021). Pal, K., Banthia, A. & Majumdar, D. J. Mater. Science: Mater. Med. , 18 , 1889–1894. (2007). Arakha, M., Saleem, M., Mallick, B. C. & Jha, S. Sci. Rep. , 5 , 9578. (2015). Kumar, B., Smita, K., Cumbal, L. & Debut, A. Saudi J. Biol. Sci. , 24 , 45–50. (2017). Zou, J., Xu, Y., Hou, B., Wu, D. & Sun, Y. China Particuology , 5 , 206–212. (2007). Delgado-Beleño, Y., Martinez-Nuñez, C., Cortez-Valadez, M., Flores-López, N. & Flores-Acosta, M. Mater. Res. Bull. , 99 , 385–392. (2018). Martinez-Nuñez, C. et al. J. Nanopart. Res. , 19 , 1–12. (2017). Sidorova, D. et al. Appl. Biochem. Microbiol. , 54 , 816–823. (2018). Mirzaei, S. Z. et al. Bioresources Bioprocess. , 8 , 1–11. (2021). Al Hussien, A. A., Jamshidi-Ghaleh, K., Mohammed, K. A. & Bayat, F. J. Nanostruct. , 15 , 1–14. (2025). Chougale, U., Han, S., Rath, M. & Fulari, V. Mater. Phys. Mech. , 17 , 47–58. (2013). Kalishwaralal, K., Jeyabharathi, S., Sundar, K. & Muthukumaran, A. Artif. cells Nanomed. Biotechnol. , 44 , 471–477. (2016). Kumashiro, Y., Ohachi, T. & Taniguchi, I. Solid State Ionics , 86 , 761–766. (1996). Sadovnikov, S. I., Kuznetsova, Y. V. & Rempel, A. A. Nano-Structures Nano-Objects , 7 , 81–91. (2016). Ayele, D. W. Egypt. J. Basic. Appl. Sci. , 3 , 149–154. (2016). Sibiya, P. & Moloto, M. J. Chalcogenide letters , 11. (2014). Yao, Q., Arachchige, I. U. & Brock, S. L. J. Am. Chem. Soc. , 131 , 2800–2801. (2009). Ahmad, W. et al. Appl. Nanosci. , 10 , 1191–1204. (2020). Baliyan, S. et al. Molecules , 27 , 1326. (2022). Ahmad, W. et al. Appl. Nanosci. , 10 , 1191–1204. (2020). Shams, S. et al. J. Photochem. Photobiol., B , 199 , 111632. (2019). Sundaram Sanjay, S. & Shukla, A. K. in Potential Therapeutic Applications of Nano-antioxidants pp. 83–99 (Springer, 2021). Battin, E. E., Perron, N. R. & Brumaghim, J. L. Inorg. Chem. , 45 , 499–501. (2006). Huang, Z., Guo, B., Wong, R. & Jiang, Y. Food Chem. , 100 , 1137–1143. (2007). Satpathy, S., Panigrahi, L. L., Samal, P., Sahoo, K. K. & Arakha, M. Heliyon , 10. (2024). Valgimigli, L., Baschieri, A. & Amorati, R. J. Mater. Chem. B , 6 , 2036–2051. (2018). de la Harpe, K. M., Kondiah, P. P., Choonara, Y. E., Marimuthu, T. & Pillay L. C. du Toit and V. Cells , 8 , 1209. (2019). Chahardoli, A. et al. Global Challenges , 5 , 2100075. (2021). Dacrory, S., Hashem, A. H. & Hasanin, M. Environ. Nanatechnol. Monit. Manage. , 15 , 100453. (2021). Aula, S. et al. Mater. Res. Express , 1 , 035041. (2014). Hashem, A. H. et al. Appl. Biochem. Biotechnol. , 195 , 5753–5776. (2023). More, D. S. Synthesis and characterization of silver and silver selenide nanoparticles and their incorporation into polymer fibres using electrospinning technique (Vaal University of Technology (South Africa), 2015). Ren, Q. W. et al. J. Hazard. Mater. , 465 , 133201. (2024). Li, W. R. et al. Int. Biodeterior. Biodegrad. , 123 , 304–310. (2017). Lok, C. N. et al. J. Proteome Res. , 5 , 916–924. (2006). Additional Declarations No competing interests reported. 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study of biogenic Ag\u003csub\u003e2\u003c/sub\u003eSeNPs.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/bcba98f6611e1836a5867acd.png"},{"id":96738473,"identity":"645b6dd6-77a0-4596-a866-04343394c856","added_by":"auto","created_at":"2025-11-25 14:44:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":289067,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological analysis (a) FESEM image of biogenic Ag\u003csub\u003e2\u003c/sub\u003eSeNPs, (b) EDAX analysis of biogenic Ag\u003csub\u003e2\u003c/sub\u003eSeNPs\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/7b835931bc538b2cbf620300.png"},{"id":96738475,"identity":"c1d7fde6-b0cf-438a-b8a7-3ed38091baa7","added_by":"auto","created_at":"2025-11-25 14:44:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":588619,"visible":true,"origin":"","legend":"\u003cp\u003e(a)\u003cstrong\u003e \u003c/strong\u003eAntibacterial activity of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against \u003cem\u003eAcenetobacter baumannii\u003c/em\u003e, (b) Antibacterial activity of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against \u003cem\u003eEscherichia coli\u003c/em\u003e, (c) Antibacterial activity of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against \u003cem\u003eStaphylococcus epidermidis, \u003c/em\u003e(d) Antibacterial activity of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against\u0026nbsp; \u003cem\u003eStaphylococcus aureus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/8c8ff4459f543197aafd6930.png"},{"id":96913902,"identity":"75a4b778-0b89-45e0-9cfc-89c460b28b85","added_by":"auto","created_at":"2025-11-27 14:04:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":427382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eCytoplasmic DNA leakage assay of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against \u003cem\u003eE. coli\u003c/em\u003e,\u003cstrong\u003e Fig. 4b. \u003c/strong\u003eCytoplasmic Protein leakage assay of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against \u003cem\u003eE. coli\u003c/em\u003e, \u003cstrong\u003eFig.4c. \u003c/strong\u003eCytoplasmic DNA leakage assay of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cstrong\u003eFig. 4d. \u003c/strong\u003eCytoplasmic protein leakage assay of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against \u003cem\u003eS. epidermidis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/28733f878c7d7aa0ee06dc19.png"},{"id":96913157,"identity":"6b9f6ebc-bd85-478f-9fe2-6368b3c5b239","added_by":"auto","created_at":"2025-11-27 13:53:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":143223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a).\u003c/strong\u003e DPPH scavenging activity of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles \u003cstrong\u003e(b).\u003c/strong\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/09c341f9d4fab1f1840677a5.png"},{"id":96738479,"identity":"9eab1b1a-e7de-400c-953e-679990383396","added_by":"auto","created_at":"2025-11-25 14:44:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":793667,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative haemocompatibility analysis of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/957649c50c1073cd4f9301aa.png"},{"id":96914215,"identity":"12a0ad73-1e2f-4ff5-a0d5-7a25c5149746","added_by":"auto","created_at":"2025-11-27 14:05:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":465308,"visible":true,"origin":"","legend":"\u003cp\u003eQualitative haemocompatibility analysis of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/bcf70e0ca759b3d24e951dbf.png"},{"id":96914723,"identity":"f639bb4f-e780-4f7c-9b7c-e617607517fc","added_by":"auto","created_at":"2025-11-27 14:06:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":165948,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images showing membrane deformation/damage of \u003cem\u003eE. coli\u003c/em\u003e upon Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticle treatment.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/511ef351622c23c474029970.png"},{"id":104739308,"identity":"593aedc8-0892-4336-b638-6a5d639495d2","added_by":"auto","created_at":"2026-03-16 16:01:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4194213,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8035077/v1/a821d05c-c0f6-4be7-b566-002c32e70e83.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pomegranate peel extract mediated biocompatible silver selenide bimetallic nanoparticles and comprehensive evaluation of their biological activities","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe rise of antibiotic resistant bacteria poses a significant threat to global health, necessitating the urgent development of novel antimicrobial agents\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among the promising solutions, bimetallic nanoparticles (BNPs) have gained attention due to their enhanced antibacterial properties, biocompatibility and multifunctional applications\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Silver selenide (Ag\u003csub\u003e2\u003c/sub\u003eSe) nanoparticles, known as naumannite, are a fascinating I-VI group semiconductor material. While typically found in bulk form, but rarely found as natural materials\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Among diverse semiconductor materials, Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles stand out due to their physicochemical and biological properties, making them highly promising for biomedical and therapeutic applications. Due to its versatile applications in electronics and biomedicine, Ag\u003csub\u003e2\u003c/sub\u003eSe is a highly valued chalcogenide nanomaterial. It also acts as a mixed ionic conductor, undergoing a phase transition at atmospheric pressure, shifting from a low temperature orthorhombic phase (β-Ag\u003csub\u003e2\u003c/sub\u003eSe) to a high temperature cubic phase (α-Ag\u003csub\u003e2\u003c/sub\u003eSe)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In relation to this, orthorhombic β-Ag\u003csub\u003e2\u003c/sub\u003eSe is the most widely accepted crystal structure of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles owing to its relatively high Seebeck coefficient (thermoelectric power, -150 \u0026micro;V K\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 300 K with an unusually low lattice thermal conductivity couple with high electrical conductivity\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Study proclaimed that, orthorhombic structure of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles exhibited both photocatalytic as well as fluorescence activity. Moreover, such compound has been utilised as a photosensitizer in photographic films, thermo-chromic materials for nonlinear optical devices and photovoltaic cells\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles are typically synthesized applying various methods such as chemical conversion, hydrothermal reactions and hot injection. In contrast, microstrctured Ag\u003csub\u003e2\u003c/sub\u003eSe can be formulated via electrodeposition and thermal evaporation techniques\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Such traditional synthesis methods for nanoparticles often involve toxic chemical, high energy consumption and environmental hazards\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In contrast, green synthesis using plant extracts as reducing and stabilizing agents offers an eco-friendly, cost effective and sustainable alternative. Pomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e L.) peel, a rich source of polyphenols, flavonoids and other bioactive compounds, serves as an excellent natural medium for nanoparticle synthesis. These biomolecules not only facilitate the reduction of metal ions but also enhance the stability and biological activity of the resulting nanoparticles. This study explores the green synthesis of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles using pomegranate peel extract, followed by a comprehensive evaluation of their biological activities. The nanoparticles were characterized for their structural and morphological properties and their antimicrobial efficacy was tested against both Gram positive and Gram negative bacteria. Additionally, their antioxidant potential and hemocompatibility were assessed to determine their suitability for biomedical applications. By leveraging the natural reducing power of pomegranate peel, this research aims to develop a sustainable, non-toxic and highly effective nanomaterial with dual functionality, combating microbial infections while mitigating oxidative stress. These findings could pave the way for innovative applications in wound healing, antimicrobial coatings and drug delivery systems, contributing to the advancement of eco-friendly nanotechnology\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cp\u003eHigh purity laboratory-grade chemical reagents were used in this study. Sodiuum selenate anhydrous (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e) and nutrient broth were obtained from Sisco Research Pvt. Ltd (SRL), Mumbai, India. Bacterial strains, including \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (MTCC 1245), \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MTCC 96), \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (MTCC 435) and \u003cem\u003eEscherichia coli\u003c/em\u003e (MTCC 443), were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid (Vitamin C) were purchased from SRL Pvt. Ltd, Mumbai, India. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was acquired from Molychem Pvt.Ltd. Fresh pomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e L.) fruits were procured from a local market in Bhubaneswar, Odisha, India. All experiments were conducted using deionized water.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Extraction of \u003cem\u003ePunica granatum\u003c/em\u003e L. peels\u003c/h2\u003e\u003cp\u003eFresh pomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e L.) fruits were thoroughly washed with running water to remove surface contaminants. The outer peel was carefully separated, chopped into small pieces and gently dried using tissue paper. The chopped peels were then sun dried for a week to ensure complete moisture removal. Subsequently, the dried peels were finely ground using a mechanical grinder\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Biosynthesis of bimetallic nanoparticles\u003c/h2\u003e\u003cp\u003eWe have synthesized the biogenic bimetallic nanoparticles following the protocol outlined by Sibiya et al. with some modifications\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Pomegranate peel extract was added with distilled water at 1:100 w/v ratio. The solution was homogenized using a stirrer for 15 min. The resultant mixture underwent heating until it reaches a temperature of 60\u003csup\u003eo\u003c/sup\u003eC prior to centrifugation. After that, the solution was centrifuged at 8000 rpm for 20 min. The obtained supernatant was filtered using Whatman filter paper. To that filtered plant extract solution, 50 mL of 1 mM silver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e) was added and the reaction mixture was put on incubation at 120 rpm for 48 hr. Post incubation, 50 mL of filtered plant extract solution was mixed with AgNPs solution and the total volume was made up to 100 mL. Then, 25 mM of selenium precursor (sodium selenate) was added to the 100 mL of AgNPs and plant extract solution followed by incubation at 120 rpm for 24 hr. Post incubation, the colour change of the reaction mixture was clearly observed showing successful formulation of silver selenide bimetallic nanoparticles (BNPs). Later, following purification process, the BNPs solution was further centrifuged at 8000 rpm for 20 min. The obtained pellet was washed with distilled water and subjected to drying at 70\u003csup\u003eo\u003c/sup\u003eC in order to get powdered nanoparticles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Characterization of bimetallic nanoparticles\u003c/h2\u003e\u003cp\u003eThe absorbance of BNPs was studied employing UV-vis spectrophotometer (HITACHI, Tokyo, Japan). Bond level analysis was done by FTIR spectrophotometer (JASCO, Japan). XRD of BNPs was achieved by Rigaku X-ray Diffractometer (Tokyo, Japan), applying Cu-Kα radiation with angular range 20\u003csup\u003e0\u003c/sup\u003e to 80\u003csup\u003e0\u003c/sup\u003e. Availability of various phases in the formulated nanoparticles was determined by the X\u0026rsquo;-pert high score software. Morphological analysis was estimated via FE-SEM (JEOL JSM-IT800).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Evaluation of antioxidant activity of bimetallic nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. DPPH assay\u003c/h2\u003e\u003cp\u003eDPPH (2,2-diphenyl-1-picrylhydrazyl) assay was investigated for antioxidant activity of biogenic BNPs. Precisely, 0.2 mM DPPH was dissolved in methanol to prepare the stock solution. Here ascorbic acid (Vitamin C) was considered as a positive control. A 96 well microtiter plate containing 100 \u0026micro;l of different concentrations of BNPs (50, 100, 250 \u0026micro;g/mL) and 100 \u0026micro;l of DPPH methanolic solution was incubated in a dark environment with a constant shaking at 35\u003csup\u003eo\u003c/sup\u003eC for 30 min. Post incubation, the absorbance was monitored at 517 nm employing Biorad Imark microplate reader. Later, the scavenging activity was evaluated by the formula\u003c/p\u003e\u003cp\u003eRadical scavenging activity (%) = (A\u003csub\u003econtrol\u003c/sub\u003e \u0026ndash; A\u003csub\u003esample\u003c/sub\u003e / A\u003csub\u003econtrol\u003c/sub\u003e) x 100\u003c/p\u003e\u003cp\u003eThe result was obtained by plotting a graph between % DPPH scavenging and concentrations of BNPs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assay\u003c/h2\u003e\u003cp\u003eAntioxidant activity of BNPs was also assessed by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging assay. Precisely, 40 mM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution was prepared in phosphate buffer. A stock solution of BNPs was prepared in phosphate buffer.1 mL of different concentrations of BNPs (50, 100, 250 \u0026micro;g/mL) was mixed with 1 mL of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e followed by incubation for 10 min at room temperature. Post incubation, the absorbance was monitored at 230 nm. The percentage of scavenging was evaluated by the formula\u003c/p\u003e\u003cp\u003eRadical scavenging activity (%) = (A\u003csub\u003econtrol\u003c/sub\u003e \u0026ndash; A\u003csub\u003esample\u003c/sub\u003e / A\u003csub\u003econtrol\u003c/sub\u003e) x 100\u003c/p\u003e\u003cp\u003eThe result was obtained by plotting a graph between % H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging and concentrations of BNPs.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Assessment of antimicrobial activity of bimetallic nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. Bacterial growth inhibition\u003c/h2\u003e\u003cp\u003eBacterial growth inhibition in presence of BNPs was evaluated against a panel of Gram-positive bacteria like \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e as well as Gram-negative bacteria like \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e by growth kinetics study. Precisely, a loopful of bacteria was collected from the mother culture and inoculated into the nutrient broth followed by overnight incubation at 37\u003csup\u003eo\u003c/sup\u003eC and 120 rpm. Furthermore, appropriate amount of BNPs was dissolved in deionized water and was sonicated for 20 min in order to prepare the stock solution of nanoparticles. Various concentrations of BNPs (25, 50, 100, 250, 500 \u0026micro;g/mL) were used in 96 well plates for growth rate evaluation. Reaction mixture without BNPs was considered as control. 20 \u0026micro;l of bacterial culture (10\u003csup\u003e6\u003c/sup\u003e CFU/mL) was dissolved in each reaction mixtures and total volume was made up to 300 \u0026micro;l using nutrient broth. Biorad Imark plate reader was used for the assessment of growth kinetic study. Optical density (O.D) was measured at 600 nm. \u003cb\u003e2.5.2. Evaluation of cytoplasmic leakage activity of bimetallic nanoparticles\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor analysis of leakage of bacterial cytoplasmic contents like DNA and protein, Gram-positive bacteria like \u003cem\u003eS. epidermidis\u003c/em\u003e and Gram-negative bacteria like \u003cem\u003eE. coli\u003c/em\u003e were cultured and incubated overnight at 37\u003csup\u003eo\u003c/sup\u003eC. Later, the microbial culture was centrifuged at 10,000 rpm for 10 min. Obtained pellet was suspended in PBS buffer (PBS 7.4). The bacterial cell count was set to 10\u003csup\u003e5\u003c/sup\u003e cells/mL. Cell suspensions were mixed with BNPs and were incubated at room temperature for 2, 4 and 6 hr. The bacterial culture without nanoparticle treatment was considered as control. After that, the cultures were centrifuged at 5000 rpm for 10 min. The obtained supernatant was measured at 260 nm for estimation of DNA leakage and at 280 nm for estimation of protein leakage.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Evaluation of hemocompatibility of bimetallic nanoparticles\u003c/h2\u003e\u003cp\u003eThe present study investigated hemocompatibility of BNPs following the protocol outlined by Pal et al. with minor modifications\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This study is focussed on degree of haemolysis caused by the tested sample. For this purpose, goat blood was collected in EDTA vials. The blood was diluted with PBS in dilution ratio 4:5. In order to validate haemolysis, 0.5 mL of 0.01N hydrochloric acid (HCl) was added to PBS and underwent incubation at 37\u003csup\u003eo\u003c/sup\u003eC for 30 min. Post incubation, 0.05 mL of diluted blood was added to the mixture to maintain the volume upto 10 mL. Later, the solution was incubated for 2 hr. The optical density (OD) was monitored at 545 nm using a spectrophotometer. Here, HCl was considered as a positive control since it possesses effective rupturing of red blood corpuscle (RBC). Similarly, 0.05 mL of diluted blood was further diluted with 10 mL of PBS. This solution was kept on incubation at 37\u003csup\u003eo\u003c/sup\u003eC for 2 hr. Post incubation, the OD of the solution was measured at 545 nm. Here, PBS is considered as negative control since it is well-known for least RBC rupture. As the standard ODs were estimated, 0.5 mL of tested sample was added to PBS and was incubated for 30 min at 37\u003csup\u003eo\u003c/sup\u003eC in order to sustain temperature equilibrium. Later 0.05 mL of diluted blood was added to the solution and was further incubated for 2 hr. Post incubation, OD of the sample was monitored at 545 nm. Previous study proclaimed that, less than 5% haemolysis of the test material was preferred to be highly haemocompatible and less than 10% haemolysis was considered to be haemocompatible. Additionally, qualitative hemocompatibility study was done with agar well-diffusion method. Precisely, in the autoclaved agar, certain amount of goat blood was mixed and poured into petriplates and the plates were kept for solidification. Later, 6\u0026ndash;8 mm wells were made on the plates with a sterile cork borer. Different concentrations of BNPs were put into the well and the plates were incubated overnight at 37\u003csup\u003eo\u003c/sup\u003eC. Here, HCl was taken as a positive control. Post incubation, inhibition zones were clearly observed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Evaluation of bacterial morphology in presence of bimetallic nanoparticles\u003c/h2\u003e\u003cp\u003ePrecisely, bacterial morphology study upon treatment with BNPs was evaluated following the protocol outlined by Arakha et al. with some modifications\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In brief, a Gram-negative bacteria \u003cem\u003eE. coli\u003c/em\u003e was inoculated in nutrient broth and incubated overnight at 37\u003csup\u003eo\u003c/sup\u003eC. On the next day, the bacterial sample was taken from stationary phase of growth kinetics and directly put on the cover slide. Later, the tested BNPs was mixed with the bacterial culture and also put on another cover slide. Only bacterial sample on the cover slide was considered as control. The slides were kept on overnight incubation at 37\u003csup\u003eo\u003c/sup\u003eC. Post incubation, the surfaces of slides were gently washed with 1X PBS. Then it was soaked on tissue paper. To preserve the bacterial cell morphology, the cells were fixed with 2.5% glutaraldehyde on the top of the slide and kept for 1 to 2 hr at room temperature. The glutaraldehyde was soaked on the tissue paper followed by washing with 1X PBS. Later, the slides were dehydrated with increasing concentration of ethanol (50%, 70%, 90% and 100%). The fixed, washed and dehydrated cells were now prepared for monitoring the morphological structures of both control and treated samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Statistical analysis of data\u003c/h2\u003e\u003cp\u003eAll the experiments were done in triplicates and the results were assessed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The data analysis was done with one-way ANOVA using Minitab software. The statistical analysis was done with Tukey\u0026rsquo;s multiple comparison test. P\u0026thinsp;\u003cb\u003e\u0026le;\u003c/b\u003e\u0026thinsp;0.05 were considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Synthesis and optical analysis of bimetallic nanoparticles\u003c/h2\u003e\u003cp\u003eThe aqueous extract of \u003cem\u003ePunica granatum\u003c/em\u003e L. was employed for the synthesis of Ag\u003csub\u003e2\u003c/sub\u003eSe BNPs from silver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e) and sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e) as silver and selenium precursors respectively. The discoloration of the reaction mixture was might be due to the excitation of the surface plasmon resonance of silver ions in the BNPs\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The existence of a well-defined absorption band between 300 and 400 nm with a maximum at ~ 359 nm confirmed the successful synthesis of the BNPs. In this range, the single absorption peak offers evidence towards shape, size as well as stability/aggregation of the nanoparticles\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In this context, other studies have demonstrated similar results\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Moreover, during the synthesis of BNPs, initially, the selenium ions are reduced to Se\u003csub\u003e2\u003c/sub\u003e followed by co-precipitation with Ag cations. Therefore, persistent reaction time along with the reduction of selenium by the biomolecules shows the existence of a biomolecular matrix as a stabilizer of Ag\u003csub\u003e2\u003c/sub\u003eSe BNPs\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2. FTIR analysis of bimetallic nanoparticles\u003c/h2\u003e\u003cp\u003eThe functional interaction between biomolecules and Ag\u003csub\u003e2\u003c/sub\u003eSe was determined by FTIR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The spectrum determined by Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles obtained at 2928 cm\u003csup\u003e− 1\u003c/sup\u003e, 2860 cm\u003csup\u003e− 1\u003c/sup\u003e can be assigned to C-H stretching vibrations indicating alkane groups. Such vibrations specifically fall within the range of 2850–3000 cm\u003csup\u003e− 1\u003c/sup\u003e. Similar results have been demonstrated by Ahmad et al.\u003csup\u003e19\u003c/sup\u003e Peak obtained at 2363 cm\u003csup\u003e− 1\u003c/sup\u003e typically associated with Ag-Se bond stretching vibration. Such wavenumber falls within the range where Ag-X stretching vibrations are prominent. Here selenium becomes a heavier halogen than chlorine or bromine. Additionally, the presence of other functional groups involved in the synthesis or capping of the nanoparticles could also contribute to the overall spectrum, potentially influencing the intensity and shape of the peak at 2363 cm\u003csup\u003e− 1\u003c/sup\u003e. Furthermore, the peaks obtained at 1675 cm\u003csup\u003e− 1\u003c/sup\u003e, 1631 cm\u003csup\u003e− 1\u003c/sup\u003e may be assigned to the stretching vibration of a carbonyl group (C = O). The peak at 1569 cm\u003csup\u003e− 1\u003c/sup\u003eis likely due to C-H bending vibration of the capping agents present on the nanoparticle surface. The FTIR peak at 1451 cm\u003csup\u003e− 1\u003c/sup\u003e can contribute to the bending vibration of –CH\u003csub\u003e2\u003c/sub\u003e- group and the distortion of the –CH\u003csub\u003e3\u003c/sub\u003e group. In other words, it is allied with the presence of alkanes or other organic molecules that have carbon-hydrogen bonds. Additionally, the peak obtained at 1041 cm\u003csup\u003e− 1\u003c/sup\u003e is typically attributed to C-O stretching vibrations in the ligands or capping agents present on the nanoparticle surface. Such peak can also be indicative of the presence of specific functional groups like ester or alcohol etc. The FTIR peaks obtained at 756 cm\u003csup\u003e− 1\u003c/sup\u003e and 533 cm\u003csup\u003e− 1\u003c/sup\u003e can be attributed to Ag-Se stretching vibrations. In this regard, Sytu et al. and Kalishwaralal et al. have suggested notable transmission peaks appeared at 1652 and 1396 cm\u003csup\u003e− 1\u003c/sup\u003e attributed to Ag-Se bond in the nanoparticle structure\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3. X-ray diffraction analysis of bimetallic nanoparticles\u003c/h2\u003e\u003cp\u003eStudy suggested that Ag\u003csub\u003e2\u003c/sub\u003eSe can exist in various forms like orthorhombic β-phase or a cubic α-phase\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Kumashiro et al. stated that, Ag\u003csub\u003e2\u003c/sub\u003eSe is a non-stoichiometric compound in both phases and can co-exist with Ag\u003csub\u003e2\u003c/sub\u003eSe as a single phase\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The XRD pattern of Ag\u003csub\u003e2\u003c/sub\u003eSe suggested different characteristic diffraction peaks were observed at 2θ = 27.46\u003csup\u003eo\u003c/sup\u003e (002), 32.3\u003csup\u003eo\u003c/sup\u003e (101), 38\u003csup\u003eo\u003c/sup\u003e (110), 44.08\u003csup\u003eo\u003c/sup\u003e (020), 64.44\u003csup\u003eo\u003c/sup\u003e (111) and 77.34\u003csup\u003eo\u003c/sup\u003e (102) which can be assigned to orthorhombic β-Ag\u003csub\u003e2\u003c/sub\u003eSe phase with Naumannite structure (JCPDS file no. 24-1041)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Study confirmed that, the orthorhombic phase is stable at low temperatures\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Moreover, XRD pattern also suggested some weak peaks that can be attributed to selenium nanoparticles (SeNPs). This observation suggested that the precursor cations were reduced to their elemental forms in the obtained sample and the size of the nanoparticles was found to be 11.52 nm. Our study is in good agreement with Delgado et al and Garcia et al\u003csup\u003e8, 15\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.1.4. Morphological characterization of bimetallic nanoparticles\u003c/h2\u003e\u003cp\u003eIn order to study the chemical composition and purity of the obtained Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles, FESEM-EDAX study was carried out. The EDAX pattern derived from selected areas of FESEM images, suggesting silver, selenium and oxygen peaks in the synthesized Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The presence of silver and selenium peaks in the obtained Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles are in good agreement with elemental peaks as reported in different studies. Therefore, the EDAX spectrum validated the availability of silver and selenium atoms in the synthesized Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Antimicrobial activity of bimetallic nanoparticles","content":"\u003cp\u003eThis study systematically evaluated the concentration-dependent antibacterial efficacy of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles against a panel of Gram negative bacteria such as \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eE, coli\u003c/em\u003e and Gram positive bacteria like \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). Bacterial growth kinetics was monitored through optical density (OD\u003csub\u003e600\u003c/sub\u003e) measurements over a 12 hour period, revealed distinct patterns of microbial inhibition corresponding to increased nanoparticle concentrations (25–500 µg/mL). The control groups for all tested species exhibited characteristic exponential growth curves, confirming viable bacterial proliferation in the absence of treatment. Additionally, Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles demonstrated progressively stronger growth suppression at higher doses. At 500 µg/mL, the nanoparticles achieved near-complete inhibition of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. This potent bactericidal effect was particularly evident after 6–8 hours of exposure, coinciding with the typical log-phase growth period of untreated cultures. While all tested pathogens showed susceptibility, the response kinetics varied between species. \u003cem\u003eA. baumannii\u003c/em\u003e displayed a more gradual decline in viability, suggesting either reduced permeability to nanoparticles or enhanced resistance mechanisms. Similarly, \u003cem\u003eS. epidermidis\u003c/em\u003e exhibited a delayed but significant response at higher concentrations. The differential sensitivity patterns may reflect variations in cell wall composition among tested Gram negative and Gram positive bacteria. In this regard, Ahmad et al. have studied regarding the efficiency of Se-Ag nanocomposites towards antibacterial efficiency and stated that, the superior antibacterial efficiency of Se-Ag nanocomposite could be probably due to the combined antibacterial actions of Ag and Se in the formulated nanocomposite. Here, the Se-Ag composite serve as a large reservoir of the biologically active silver and a slow and sustained release of the Ag and Se could be responsible for its better efficacy as compared to individual counterparts\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Furthermore, these findings not only confirm the broad-spectrum antibacterial properties of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles but also highlight their potential for tailored antimicrobial applications. The concentration-dependent efficacy, particularly against resilient pathogens like MRSA and multidrug-resistant \u003cem\u003eA. baumannii\u003c/em\u003e makes these nanoparticles as promising candidates for developing novel antimicrobial coatings, wound dressings or therapeutic formulations. The mechanism of the Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles bactericidal mode of action may vary in consistent with various bacterial species as well as the composition of the sample as reported by Garcia et al\u003csup\u003e8\u003c/sup\u003e. Further studies investigating the precise mechanisms of action and potential synergy with conventional antibiotics could enhance their clinical applicability.\u003c/p\u003e"},{"header":"5. Evaluation of cytoplasmic leakage activity of bimetallic nanoparticles","content":"\u003cp\u003eThe cytoplasmic DNA and protein leakage assay of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles was carried out against Gram positive bacteria like \u003cem\u003eS. epidermidis\u003c/em\u003e and Gram negative bacteria like \u003cem\u003eE. coli\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-d) The cytoplasmic DNA leakage assay evaluates the membrane disrupting effect of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles on \u003cem\u003eE. coli\u003c/em\u003e by measuring absorbance at 260 nm, which indicates released DNA over a 6-hour period. The control sample shows minimal absorbance, confirming intact bacterial membranes, while increasing concentrations of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles (100, 200 and 500 \u0026micro;g/mL) exhibit a dose dependent rise in DNA leakage. The highest concentration (500 \u0026micro;g/mL) induces the most significant absorbance increase, reflecting severe membrane damage and substantial cytoplasmic DNA release. Additionally, in case of \u003cem\u003eS. epidermidis\u003c/em\u003e, the cytoplasmic DNA leakage assay demonstrated a substantial increase in extracellular DNA levels upon treatment of \u003cem\u003eS. epidermidis\u003c/em\u003e with Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles, compared to the untreated control. This marked elevation in DNA release indicates significant membrane damage, suggesting that the nanoparticles effectively disrupt the structural integrity of the bacterial cell wall and cell membrane. The observed leakage likely results from nanoparticle-induced oxidative stress and direct physical interactions with the membrane, leading to pore formation or rupture. These findings strongly support the bactericidal action of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles through membrane destabilization and subsequent intracellular content leakage. Furthermore, Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles cause severe damage to \u003cem\u003eE. coli\u003c/em\u003e cell membranes, leading to significant protein leakage. Unlike the untreated control, which shows minimal release, nanoparticles treated samples exhibit a sharp rise in extracellular protein levels, measured by increased absorbance. This confirms that Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles physically disrupt bacterial membranes, likely through oxidative stress and structural destabilization. The uncontrolled efflux of cytoplasmic components underscores their potent bactericidal action, highlighting a mechanism rooted in membrane disintegration. Similarly, the cytoplasmic protein leakage clearly demonstrated that Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles induce significant damage to \u003cem\u003eS. epidermidis\u003c/em\u003e membranes, triggering elevated release of intracellular proteins. While untreated cells maintain their structural integrity, nanoparticles exposed bacteria suffer massive protein leakage caused by membrane damage. Such destructive effect results from nanoparticle-induced oxidative stress that degrades membrane components ultimately leading to cell death. This phenomenon confirms the potential of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles as an effective antimicrobial agent for dealing with various resistance mechanisms. These findings reinforce Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles as a promising antimicrobial agent capable of effectively compromising bacterial membrane integrity with their antibacterial activity intensifying at higher doses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"6. Evaluation of antioxidant activity of bimetallic nanoparticles","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e6.1. DPPH assay\u003c/h2\u003e\u003cp\u003eThe DPPH assay is considered to be the most widely used in vitro method for determination of antioxidant activity of nanoparticles\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. DPPH assay offers a simple and widely used method to evaluate antioxidant properties of nanoparticles by measuring their ability to scavenge DPPH free radicals. The free radicals like superoxide, hydroxyl radicals can be generated from normal metabolic process cause several serious medical complications like cancer, aging and diabetes mellitus etc\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, neutralization of such free radicals is highly essential in order to safeguard the normal cells from their lethal effects\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In this study, we have estimated a dose dependent free radical scavenging activity of Ag\u003csub\u003e2\u003c/sub\u003eSe bimetallic nanoparticles by taking ascorbic acid as a positive control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In this assay, the silver selenide nanoparticles with antioxidant properties interact with DPPH by donating electrons or hydrogen atoms to neutralize the free radical appearance of DPPH. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea showed that, ascorbic acid as positive control, showed effective scavenging activity. Ascorbic acid reveals effective potential of DPPH radical scavenging activity via two mechanisms such as hydrogen atom transfer (HAT) and single electron transfer followed by proton transfer (SET-PT)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In HAT, ascorbic acid donates a hydrogen atom (H\u003csup\u003e+\u003c/sup\u003e) to DPPH free radical converting it into a stable and non-radical compound. Additionally, following SET-PT mechanism, ascorbic acid first donates an electron, followed by a proton in order to neutralize the radical. Therefore, in our study, ascorbic acid showed effective scavenging activity even at lower concentration while Ag\u003csub\u003e2\u003c/sub\u003eSe bimetallic nanoparticles showed their antioxidant potential at higher concentration. In this regard, SeNPs have also exhibited similar antioxidant properties as suggested by various studies\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Moreover, silver nanoparticles (AgNPs) have also exhibit significant radical scavenging potential as reported by previous studies\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e6.2. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assay\u003c/h2\u003e\u003cp\u003eWe have also evaluated antioxidant potential of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles via hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) scavenging assay. For comparison, ascorbic acid was taken as a standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The Ag\u003csub\u003e2\u003c/sub\u003eSe H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging assay reveals a concentration dependent antioxidant activity for both Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles and ascorbic acid (Vit.C). At lower concentration (50 \u0026micro;g/mL), Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles exhibited moderate scavenging activity, while Vit.C demonstrates superior efficacy, showing nearly 80% scavenging. As the concentration increases to 100 \u0026micro;g/mL, Ag\u003csub\u003e2\u003c/sub\u003eSe showed progressive improvement, yet Vit.C maintained its dominance with near maximal scavenging. At the highest tested concentration (250 \u0026micro;g/mL), Ag\u003csub\u003e2\u003c/sub\u003eSe achieves significant scavenging, though Vit.C remains the more potent antioxidant. Ag\u003csub\u003e2\u003c/sub\u003eSe exhibited effective H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity due to their ability to catalyse the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into less harmful products like water and oxygen and also due to the inherent antioxidant properties of selenium. The combination of silver and selenium in the bimetallic nanoparticle may lead to a synergistic effect. Silver nanoparticles are known for their antimicrobial and catalytic properties, while selenium offers antioxidant activity. This combination can result in enhanced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging compared to either silver or selenium nanoparticles alone\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Additionally, the size and morphology of the nanoparticles can also influence their H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging activity. Smaller nanoparticles generally have a larger surface area, which may lead to increased interaction with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e molecules and more efficient decomposition. These results highlight the robust scavenging capability of ascorbic acid, a well-established antioxidant, while underscoring the promising but comparatively modest activity of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles. The findings suggested that Ag\u003csub\u003e2\u003c/sub\u003eSe could serve as an excellent antioxidant agent, particularly in applications where traditional antioxidants like Vit.C may be less suitable. Further optimization of nanoparticle composition or concentration may enhance their scavenging potential.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"7. Evaluation of haemocompatibility of bimetallic nanoparticles","content":"\u003cp\u003eHaemocompatibility of nanoparticles offers significant role in their biomedical applications since blood is a primary target contrary to toxicity of the nanoparticles\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Nanoparticles lead to rupturing of RBC in the circulatory system thereby releasing haemoglobin which results in various chronic disorders such as anemia, jaundice, various pathological disorders and kidney failure\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Therefore, in our study, quantitative and qualitative assessment of haemolytic potential of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles were carefully evaluated in order to determine their blood compatibility. This suggested a concentration dependent RBC lysis induced by Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles with hemolysis level approximately 7%, 11% and 42% according to the concentrations 25 \u0026micro;g/mL, 50 \u0026micro;g/mL and 100 \u0026micro;g/mL respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Here HCl was considered as positive control. Studies suggested that below 5% hemolysis the test material can be considered to be highly haemocompatible and below 10% as haemocompatible\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. At lower concentration, our studied nanoparticles showed least lysis activity confirming safety and compatibility towards validation of various biomedical applications. In this context, Chahardoli et al. have studied haemocompatibility of Quercetin assisted silver nanoparticles which showed around 9% of haemolytic activity at 1mg/mL concentration, but at lower concentration it showed negligible amount of lysis activity indicating such nanoparticles to be non-toxic and hemocompatible in nature. Furthermore, Hashem et al. have stated that, biogenic SeNPs at lower concentration showed least haemolytic effect confirming their safety and hemocompatibility. On the other hand, as per the standards of American Society of Testing, the test material with hemolysis\u0026thinsp;\u0026gt;\u0026thinsp;5% are haemolytic, 2\u0026ndash;5% are slightly haemolytic and \u0026lt;\u0026thinsp;2% are non-hemolytic in nature\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Studies claimed that, Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles not only exhibit good haemocompatibility, but even promote blood clotting, offering potential applications in wound healing as well as antibleeding therapies\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Additionally, Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles, with specific coatings and particle size may exhibit low haemolytic activity\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Additionally, in case of qualitative analysis of haemolytic potential of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles, at lower concentration 25 \u0026micro;g/mL, the tested nanoparticles showed least zone of lysis and as the concentration gradually increased to 50 and 100 \u0026micro;g/mL, the nanoparticles showed maximum haemolytic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Therefore, both qualitative and quantitative analysis of haemolytic potential of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles demonstrated that lower concentration of Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles confirms its safety while higher concentration may cause toxicity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"8. Surface morphology of bacteria upon treatment with bimetallic nanoparticles","content":"\u003cp\u003eBacterial membrane deformities upon Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles treatment were observed via scanning electron microscopy (SEM). The images obtained showed clumping or aggregation of bacterial cells. In order to gain further information, nanoparticle treated and untreated bacterial cells were scanned using SEM. The images confirmed significant clumping as well as membrane rupture in treated cells rather than untreated cells due to interaction of nanoparticles with the bacterial cell surface and membrane. The nanoparticles can accumulate in the cell wall, causing denaturation and eventually leading to cell lysis. Additionally, they can release silver ions, which further disrupt cellular processes, including DNA binding and respiration, ultimately leading to cell death. Moreover, nanoparticle treatment induced a significant reduction in bacterial population density compared to control samples (Fig.\u0026nbsp;8a and 8b). The Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles appeared to exhibit cell lysis and delay formation of biofilm, thereby contributing towards reduction in bacterial population. These findings suggest that, the Ag\u003csub\u003e2\u003c/sub\u003eSe nanoparticles effectively compromise bacterial membrane integrity as well as helps in disruption of biofilm structures, signifying its effective antimicrobial activity. In this context, Li et al. have done a comparative analysis of antibacterial activity, dynamics and effects of silver ions and silver nanoparticles suggesting strong alteration in bacterial morphology thereby showing effective antibacterial activity as described by various other studies\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"9. Conclusion","content":"\u003cp\u003eThis study represents a significant advancement in the green synthesis of silver slenide bimetallic nanoparticles (Ag\u003csub\u003e2\u003c/sub\u003eSe BNPs) using pomegranate peel extract, offering an eco-friendly and sustainable alternative to conventional chemical methods. The biosynthesized BNPs demonstrated remarkable biological activities, positioning them as promising candidates for biomedical and therapeutic applications. Furthermore, the research highlights dual functionality of Ag\u003csub\u003e2\u003c/sub\u003eSe BNPs aas both antimicrobial and antioxidant agents, with potential applications in wound dressings, antimicrobial coatings and drug delivery systems. Future work should explore their synergistic effects with existing antibiotics and in vivo biocompatibility to fully realize their clinical potential. By influencing plant-based synthesis, this study also contributes to the growing field of sustainable nanotechnology, aligning with global efforts towards greener scientific practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e\u003cp\u003eThere are no conflicts to declare\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eNo Funding\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.S.: conceptualization, investigation, methodology, validation, writing original draft, P.S.: data curation, A.K.P.: data curation and editing, M.A.: Investigation, conceptualization, formal analysis, methodology, supervision, review \u0026amp; editing. All authors revieewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that supports the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSalam, M. A., Al-Amin, M. Y., Salam, M. T., Pawar, J. S. \u0026amp; Akhter, N. A. A. Rabaan and M. A. Alqumber, (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharma, G. et al. \u003cem\u003eJ. King Saud University-Science\u003c/em\u003e, \u003cb\u003e31\u003c/b\u003e, 257\u0026ndash;269. (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHelan, P. P. J., Mohanraj, K. \u0026amp; Sivakumar, G. \u003cem\u003eTrans. Nonferrous Met. Soc. China\u003c/em\u003e, \u003cb\u003e25\u003c/b\u003e, 2241\u0026ndash;2246. (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, T. et al. \u003cem\u003eNanoscale\u003c/em\u003e, \u003cb\u003e11\u003c/b\u003e, 20820\u0026ndash;20836. (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang, H. et al. \u003cem\u003eACS Appl. Mater. Interfaces\u003c/em\u003e, \u003cb\u003e8\u003c/b\u003e, 17859\u0026ndash;17869. (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerhat, M. \u0026amp; Nagao, J. \u003cem\u003eJ. Appl. Phys.\u003c/em\u003e, \u003cb\u003e88\u003c/b\u003e, 813\u0026ndash;816. (2000).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao, H. et al. \u003cem\u003eNano Res.\u003c/em\u003e, \u003cb\u003e3\u003c/b\u003e, 863\u0026ndash;873. (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a, D. A. et al. \u003cem\u003eMolecules\u003c/em\u003e, \u003cb\u003e25\u003c/b\u003e, 5193. (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKharissova, O. V., Dias, H. R., Kharisov, B. I., P\u0026eacute;rez, B. O. \u0026amp; P\u0026eacute;rez, V. M. J. \u003cem\u003eTrends Biotechnol.\u003c/em\u003e, \u003cb\u003e31\u003c/b\u003e, 240\u0026ndash;248. (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOlawale, F., Ariatti, M. \u0026amp; Singh, M. \u003cem\u003eNanomaterials\u003c/em\u003e, \u003cb\u003e11\u003c/b\u003e, 2516. (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePal, K., Banthia, A. \u0026amp; Majumdar, D. \u003cem\u003eJ. Mater. Science: Mater. Med.\u003c/em\u003e, \u003cb\u003e18\u003c/b\u003e, 1889\u0026ndash;1894. (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArakha, M., Saleem, M., Mallick, B. C. \u0026amp; Jha, S. \u003cem\u003eSci. Rep.\u003c/em\u003e, \u003cb\u003e5\u003c/b\u003e, 9578. (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar, B., Smita, K., Cumbal, L. \u0026amp; Debut, A. \u003cem\u003eSaudi J. Biol. Sci.\u003c/em\u003e, \u003cb\u003e24\u003c/b\u003e, 45\u0026ndash;50. (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZou, J., Xu, Y., Hou, B., Wu, D. \u0026amp; Sun, Y. \u003cem\u003eChina Particuology\u003c/em\u003e, \u003cb\u003e5\u003c/b\u003e, 206\u0026ndash;212. (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDelgado-Bele\u0026ntilde;o, Y., Martinez-Nu\u0026ntilde;ez, C., Cortez-Valadez, M., Flores-L\u0026oacute;pez, N. \u0026amp; Flores-Acosta, M. \u003cem\u003eMater. Res. Bull.\u003c/em\u003e, \u003cb\u003e99\u003c/b\u003e, 385\u0026ndash;392. (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartinez-Nu\u0026ntilde;ez, C. et al. \u003cem\u003eJ. Nanopart. Res.\u003c/em\u003e, \u003cb\u003e19\u003c/b\u003e, 1\u0026ndash;12. (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSidorova, D. et al. \u003cem\u003eAppl. Biochem. Microbiol.\u003c/em\u003e, \u003cb\u003e54\u003c/b\u003e, 816\u0026ndash;823. (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMirzaei, S. Z. et al. \u003cem\u003eBioresources Bioprocess.\u003c/em\u003e, \u003cb\u003e8\u003c/b\u003e, 1\u0026ndash;11. (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl Hussien, A. A., Jamshidi-Ghaleh, K., Mohammed, K. A. \u0026amp; Bayat, F. \u003cem\u003eJ. Nanostruct.\u003c/em\u003e, \u003cb\u003e15\u003c/b\u003e, 1\u0026ndash;14. (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChougale, U., Han, S., Rath, M. \u0026amp; Fulari, V. \u003cem\u003eMater. Phys. Mech.\u003c/em\u003e, \u003cb\u003e17\u003c/b\u003e, 47\u0026ndash;58. (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKalishwaralal, K., Jeyabharathi, S., Sundar, K. \u0026amp; Muthukumaran, A. \u003cem\u003eArtif. cells Nanomed. Biotechnol.\u003c/em\u003e, \u003cb\u003e44\u003c/b\u003e, 471\u0026ndash;477. (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumashiro, Y., Ohachi, T. \u0026amp; Taniguchi, I. \u003cem\u003eSolid State Ionics\u003c/em\u003e, \u003cb\u003e86\u003c/b\u003e, 761\u0026ndash;766. (1996).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSadovnikov, S. I., Kuznetsova, Y. V. \u0026amp; Rempel, A. A. \u003cem\u003eNano-Structures Nano-Objects\u003c/em\u003e, \u003cb\u003e7\u003c/b\u003e, 81\u0026ndash;91. (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAyele, D. W. \u003cem\u003eEgypt. J. Basic. Appl. Sci.\u003c/em\u003e, \u003cb\u003e3\u003c/b\u003e, 149\u0026ndash;154. (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSibiya, P. \u0026amp; Moloto, M. J. \u003cem\u003eChalcogenide letters\u003c/em\u003e, 11. (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYao, Q., Arachchige, I. U. \u0026amp; Brock, S. L. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e, \u003cb\u003e131\u003c/b\u003e, 2800\u0026ndash;2801. (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmad, W. et al. \u003cem\u003eAppl. Nanosci.\u003c/em\u003e, \u003cb\u003e10\u003c/b\u003e, 1191\u0026ndash;1204. (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaliyan, S. et al. \u003cem\u003eMolecules\u003c/em\u003e, \u003cb\u003e27\u003c/b\u003e, 1326. (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmad, W. et al. \u003cem\u003eAppl. Nanosci.\u003c/em\u003e, \u003cb\u003e10\u003c/b\u003e, 1191\u0026ndash;1204. (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShams, S. et al. \u003cem\u003eJ. Photochem. Photobiol., B\u003c/em\u003e, \u003cb\u003e199\u003c/b\u003e, 111632. (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSundaram Sanjay, S. \u0026amp; Shukla, A. K. \u003cem\u003ein Potential Therapeutic Applications of Nano-antioxidants\u003c/em\u003e pp. 83\u0026ndash;99 (Springer, 2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBattin, E. E., Perron, N. R. \u0026amp; Brumaghim, J. L. \u003cem\u003eInorg. Chem.\u003c/em\u003e, \u003cb\u003e45\u003c/b\u003e, 499\u0026ndash;501. (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang, Z., Guo, B., Wong, R. \u0026amp; Jiang, Y. \u003cem\u003eFood Chem.\u003c/em\u003e, \u003cb\u003e100\u003c/b\u003e, 1137\u0026ndash;1143. (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSatpathy, S., Panigrahi, L. L., Samal, P., Sahoo, K. K. \u0026amp; Arakha, M. \u003cem\u003eHeliyon\u003c/em\u003e, 10. (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eValgimigli, L., Baschieri, A. \u0026amp; Amorati, R. \u003cem\u003eJ. Mater. Chem. B\u003c/em\u003e, \u003cb\u003e6\u003c/b\u003e, 2036\u0026ndash;2051. (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede la Harpe, K. M., Kondiah, P. P., Choonara, Y. E., Marimuthu, T. \u0026amp; Pillay L. C. du Toit and V. \u003cem\u003eCells\u003c/em\u003e, \u003cb\u003e8\u003c/b\u003e, 1209. (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChahardoli, A. et al. \u003cem\u003eGlobal Challenges\u003c/em\u003e, \u003cb\u003e5\u003c/b\u003e, 2100075. (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDacrory, S., Hashem, A. H. \u0026amp; Hasanin, M. \u003cem\u003eEnviron. Nanatechnol. Monit. Manage.\u003c/em\u003e, \u003cb\u003e15\u003c/b\u003e, 100453. (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAula, S. et al. \u003cem\u003eMater. Res. Express\u003c/em\u003e, \u003cb\u003e1\u003c/b\u003e, 035041. (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHashem, A. H. et al. \u003cem\u003eAppl. Biochem. Biotechnol.\u003c/em\u003e, \u003cb\u003e195\u003c/b\u003e, 5753\u0026ndash;5776. (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMore, D. S. \u003cem\u003eSynthesis and characterization of silver and silver selenide nanoparticles and their incorporation into polymer fibres using electrospinning technique\u003c/em\u003e (Vaal University of Technology (South Africa), 2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRen, Q. W. et al. \u003cem\u003eJ. Hazard. Mater.\u003c/em\u003e, \u003cb\u003e465\u003c/b\u003e, 133201. (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, W. R. et al. \u003cem\u003eInt. Biodeterior. Biodegrad.\u003c/em\u003e, \u003cb\u003e123\u003c/b\u003e, 304\u0026ndash;310. (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLok, C. N. et al. \u003cem\u003eJ. Proteome Res.\u003c/em\u003e, \u003cb\u003e5\u003c/b\u003e, 916\u0026ndash;924. (2006).\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bimetallic nanoparticles, green synthesis, antimicrobial activity, antioxidant potential, hemocompatibility, pomegranate peel extract","lastPublishedDoi":"10.21203/rs.3.rs-8035077/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8035077/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe growing threat of antibiotic-resistant bacteria appeals for urgent development of novel antimicrobial agents. Bimetallic nanoparticles (BNPs), particularly silver selenide (Ag\u003csub\u003e2\u003c/sub\u003eSe), have emerged as promising candidates due to their enhanced antibacterial properties, biocompatibility and multifunctional applications. This study presents an eco-friendly synthesis of Ag\u003csub\u003e2\u003c/sub\u003eSe BNPs using pomegranate (\u003cem\u003ePunica granatum\u003c/em\u003e L.) peel extract, leveraging its rich polyphenols and flavonoids as natural reducing and stabilizaing agents. The synthesized nanoparticles were characterised using Uv-Vis spectroscopy, FTIR, XRD and FE-SEM, confirming their structural and morphological properties. Antimicrobial assays demonstrated potent activity against both Gram-positive (\u003cem\u003eStaphylococcus aureus, Staphylococcus epidermidis\u003c/em\u003e) and Gram-negative (\u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e) bacteria, with significant membrane disruption observed through cytoplasmic leakage and SEM analysis. Additionally, the BNPs exhibited notable antioxidant activity in DPPH and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assays, alongside excellent haemocompatibility at lower concentrations. These findings highlight the dual functionality of Ag\u003csub\u003e2\u003c/sub\u003eSe BNPs as effective antimicrobial and antioxidant agents, offering sustainable solutions for biomedical applications such as wound healing, antimicrobial coatings and drug delivery systems. This green synthesis approach aligns with global efforts towards eco-friendly nanotechnology.\u003c/p\u003e","manuscriptTitle":"Pomegranate peel extract mediated biocompatible silver selenide bimetallic nanoparticles and comprehensive evaluation of their biological activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 14:43:55","doi":"10.21203/rs.3.rs-8035077/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-02T04:04:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-01T06:10:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T07:18:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211894467578021928123305196912075117148","date":"2025-11-19T12:43:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95163232279332987473706602803087039989","date":"2025-11-14T09:14:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T09:07:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-10T10:38:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-07T09:05:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-07T09:00:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-05T06:50:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"87a8f4b3-ddab-46a7-a76d-f40f4f1ce095","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58490914,"name":"Biological sciences/Biochemistry"},{"id":58490915,"name":"Biological sciences/Biotechnology"},{"id":58490916,"name":"Physical sciences/Chemistry"},{"id":58490917,"name":"Biological sciences/Microbiology"},{"id":58490918,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2026-03-16T16:00:52+00:00","versionOfRecord":{"articleIdentity":"rs-8035077","link":"https://doi.org/10.1038/s41598-026-44031-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-03-15 15:57:53","publishedOnDateReadable":"March 15th, 2026"},"versionCreatedAt":"2025-11-25 14:43:55","video":"","vorDoi":"10.1038/s41598-026-44031-4","vorDoiUrl":"https://doi.org/10.1038/s41598-026-44031-4","workflowStages":[]},"version":"v1","identity":"rs-8035077","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8035077","identity":"rs-8035077","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}