Plant-assisted green fabrication of CuO-based nanocomposites from Aronia melanocarpa for advanced functional applications

Abstract

Metal oxide nanoparticles have gained increasing attention due to their high biocompatibility, low toxicity, and broad application potential. Green synthesis methods offer significant advantages in terms of sustainability, eco-friendliness, and cost-effectiveness. The rapid growth of industrial and commercial activities has resulted in wastewater containing toxic dyes, antibiotics, and other hazardous pollutants, posing serious threats to the environment and water resources. Therefore, the development of efficient wastewater treatment technologies is essential. Photocatalytic processes have emerged as an effective strategy for the degradation of organic pollutants due to their high efficiency and environmental compatibility. Semiconductor photocatalysts are widely employed in these processes because of their strong oxidation capability, while plant-mediated synthesis provides an environmentally benign alternative. In this study, CuO–Ag–TiO₂ nanocomposites were synthesized using Aronia melanocarpa (chokeberry) extract via a green synthesis approach. The structural and morphological properties of the nanocomposites were characterized by XRD, FTIR, BET, and SEM. The photocatalytic, antibacterial, and anticancer performances of the synthesized nanocomposites were systematically evaluated. The CuO–Ag–TiO₂ nanocomposite exhibited superior photocatalytic activity, achieving degradation efficiencies of 89.34% for tetracycline and 90.85% for methylene blue, with complete tetracycline degradation within 90 minutes. The incorporation of TiO₂ significantly enhanced the degradation performance compared to CuO and CuO–Ag. Antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli produced inhibition zones comparable to standard antibiotics. Furthermore, cytotoxicity studies on MDA-MB-231 breast cancer cells demonstrated that CuO–Ag–TiO₂ exhibited the highest anticancer activity, confirming the positive effect of dopant incorporation.

CuO, CuO–Ag, and CuO–Ag–TiO₂ nanomaterials were green-synthesized using Aronia melanocarpa extract. CuO-Ag-TiO₂ showed the best photocatalytic, antibacterial, and anticancer performance.

1. Introduction

Nanotechnology is one of the fastest-growing fields of science and technology and is widely regarded as a key driver of the 21st-century industrial revolution. Extensive research and development efforts have focused on the design and production of advanced, safe, and sustainable nanomaterials for a broad range of applications.¹ Nanotechnology is a rapidly advancing field, with nanomaterials exhibiting high surface-to-volume ratios and enhanced reactivity, making them highly effective for environmental and technological applications.² Nanoparticles (NPs), typically ranging from 1 to 100 nm in size,³ can exhibit different properties depending on their size and surface characteristics.⁴ Compared to bulk materials, nanoparticles have a significantly higher surface area-to-mass ratio.⁵

Since green plants are often used in nanoparticle synthesis, this emerging scientific field is also referred to as “green nanobiotechnology”. Green synthesis refers to the biological synthesis of metallic nanoparticles using plants or plant extracts, enabling the production of eco-friendly and size- and shape-controlled nanoparticles.⁶ Green synthesis of nanoparticles can be carried out using various biomaterials, including bacteria, fungi, yeast, viruses, microalgae, and plant biomass/extracts. The use of biological organisms in this field is rapidly expanding due to their growing success and ease of synthesis. Green methods allow for the synthesis of NPs with diverse shapes, sizes, compositions, and physicochemical properties. The use of plant biomass/extracts in NP biosynthesis stands out as an eco-friendly and reliable method. Compared to other biomaterials, plant-mediated biosynthesis offers many advantages.⁷ There is an increasing number of studies focused on the synthesis of metal nanoparticles at the nanoscale using green synthesis methods. Green synthesis is more beneficial than traditional chemical synthesis as it is more cost-effective, reduces pollution, enhances environmental and human health safety, and results in more stable compounds. It is widely considered an essential tool for mitigating the negative effects of conventional nanoparticle synthesis methods used in laboratories and industries.⁸ For instance, green techniques minimize the use of expensive chemicals, reduce energy consumption, and produce environmentally benign products and by-products.⁶ For example, using reagents in accordance with the principles of green chemistry allows the NP synthesis to be classified as “green synthesis”.

Green synthesis methods are considered an attractive alternative in nanoparticle production due to their much lower energy input compared to top-down mechanochemical approaches or solvent-based self-assembly methods.⁹ Nanocomposite materials are formed by the combination of different phases and are typically composed of components such as nanoparticles, nanofibers, and nanoclays.¹⁰

Aronia melanocarpa (chokeberry) belongs to the subfamily Maloideae of the Rosaceae family. The species Aronia melanocarpa (Michx.) Ell. was first cultivated for the food industry in the early 1900s in the Siberian regions of Russia and rapidly spread throughout the country. In the first half of the 20th century, Aronia melanocarpa was introduced to Eastern European countries such as Poland, Germany, Finland, Sweden, and Norway. Notable commercial cultivars include ‘Viking’ (Finland), ‘Nero’ (Czech Republic), ‘Aron’ (Denmark), ‘Galicjanka’ (Poland), ‘Hugin’ (Sweden), ‘Rubina’ (Russia), and ‘Fertödi’ (Hungary).¹¹ In this study, Aronia was selected for investigation due to its high polyphenol content and strong antioxidant capacity, in order to examine its reducing properties.¹² The applications of nanotechnology in the biomedical field have gained great momentum in recent years, and metal oxide-based nanoparticles (MONPs) in particular have attracted attention due to their antibacterial and anticancer potential. In this context, copper oxide (CuO) nanoparticles can be synthesized by many different methods, both physically and biologically, and biologically derived methods come to the fore with their environmentally friendly approaches. In the study conducted by Javid et al., evaluations of the antimicrobial and anticancer activity of CuO–Ag nanoparticles obtained by green synthesis method were carried out. Anticancer activity evaluations of these particles were tested on HeLa cervical cancer cells, and a dose-dependent decrease in cell viability was observed. In particular, CuO–Ag nanoparticles have been reported to exhibit a stronger anti-cancer activity and significantly reduce cell viability compared to pure CuO nanoparticles.¹³

In this study, the cytotoxic effects of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanoparticles on the MDA-MB-231 breast cancer cell line were evaluated using the Alamar Blue assay. According to the results of the experiment, a significant decrease in cell viability was observed in a dose-dependent manner in all nanoparticle types (p < 0.0001). In particular, CuO–Ag–TiO₂ nanoparticles exhibited significant cytotoxic effects at all concentrations (50, 100, and 200 µg ml⁻¹), resulting in a decrease in cell viability of less than 10%. These findings are consistent with literature data showing that the contribution of Ag and TiO₂ synergistically increases the anti-cancer efficacy of CuO on cancer cells.¹⁴

In this study, CuO-based nanocomposites were synthesized using Aronia melanocarpa fruit extract as a reducing agent. Aronia melanocarpa is a plant rich in phenolic compounds, anthocyanins, and flavonoids. These bioactive compounds contribute to the reduction of metal ions and the stabilization of nanoparticles. Furthermore, a green synthesis approach that minimizes the use of toxic chemicals was preferred for the preparation of the nanocomposite structures. The photocatalytic activities of the obtained nanocomposites were evaluated against tetracycline and methylene blue, which are widely found in wastewater, represent two different classes of organic pollutants, and whose removal from water is of great importance. Subsequently, their anticancer potential against the MDA-MB-231 breast cancer cell line was investigated. Finally, their antibacterial activities were evaluated against the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, as well as the Gram-positive bacterium Staphylococcus aureus. The main novelty of this study lies in the synthesis of CuO-based nanocomposites using a plant-derived reducing agent and the evaluation of their multifunctional performance in different application areas. The synthesized materials were found to exhibit significant potential in all the investigated application areas.

2. Experimental

2.1. Materials

Copper(ii) chloride dihydrate (CuCl₂·2H₂O), silver nitrate (AgNO₃), titanium isopropoxide (Ti{OCH(CH₃)₂}₄), methylene blue (C₁₆H₁₈ClN₃S), tetracycline (C₂₂H₂₄N₂O₈), and gentamicin antibiotic discs were all purchased from Merck (Germany).

2.2. Collection and extraction of Aronia melanocarpa

Plant samples of Aronia melanocarpa were collected in 2024 from Yalova, located in the Marmara region of Türkiye. For the preparation of plant extracts, the collected plant materials were washed four times with tap water to physically remove any possible foreign particles. The cleaned plant materials were weighed into 2 g portions. Subsequently, the plant material was separated from its stems, and the fruit portion was dried. The dried plant materials were then ground using a laboratory grinder, and 100 ml of deionized water was added to 250 ml beakers containing the ground samples. The resulting mixture was heated at 80 °C for 30 minutes to facilitate the extraction process. Following the boiling process, coarse plant residues were removed by filtration using Whatman filter paper, and the obtained plant extract was stored at 4 °C until use.

2.3. Synthesis of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposites

For the synthesis of nanoparticles, each structure was initially prepared separately. In the synthesis of CuO nanoparticles, 33 ml of 100 mM CuCl₂·2H₂O solution was prepared, and 50 ml of plant extract was added to obtain a homogeneous mixture. For the synthesis of the CuO–Ag composite, 33 ml of 100 mM CuCl₂·2H₂O and 33 ml of 100 mM AgNO₃ solutions were mixed in a beaker, followed by the addition of 50 ml of plant extract. For the ternary CuO–Ag–TiO₂ nanocomposite, 33 ml of 100 mM CuCl₂·2H₂O, 33 ml of 100 mM AgNO₃, and 33 ml of 100 mM Ti[OCH(CH₃)₂]₄ solutions were combined, and 50 ml of the plant extract was added to the mixture. The pH of all mixtures was adjusted to 12 using NaOH, and the solutions were stirred at 80 °C for 30 minutes. During this period, continuous precipitation was observed, indicating efficient nanoparticle formation. The resulting suspension was centrifuged to separate the precipitates. The precipitate was washed with deionized water and centrifuged again. After centrifugation, the supernatant was washed with ethanol, and the final precipitate was left to dry at room temperature. Subsequently, the dried product was subjected to heat treatment in a muffle furnace at 450 °C for 2 hours. The synthesis process is illustrated in Fig. 1.

Fig. 1. Schematic synthesis of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposite.

2.4. Alamar Blue cell viability assay

In vitro cytotoxicity studies were performed using the human breast cancer cell line MDA-MB-231 to evaluate the anticancer activity of CuO-based nanoparticles. Cells at approximately 70% confluence were detached using trypsin and seeded into 96-well plates at a density of 1 × 10⁴ cells per well. The cells were incubated overnight at 37 °C in a humidified atmosphere containing 5% CO₂ to allow proper attachment.

Following incubation, the cells were treated with CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposite at final concentrations of 50, 100, and 200 µg ml⁻¹ for 24 h under standard culture conditions (37 °C, 5% CO₂).

Cell viability was subsequently evaluated using the Alamar Blue assay. Briefly, each well was washed with 100 µl phosphate-buffered saline (PBS), followed by the addition of 100 µl serum-free culture medium containing 10% (v/v) Alamar Blue reagent. The plates were incubated at 37 °C until a visible color change was observed. After 45 min, the reaction mixtures were transferred to black 96-well plates, and fluorescence intensity was measured using a microplate reader (excitation wavelength: 560 nm; emission wavelength: 590 nm). The Alamar Blue assay measures cellular metabolic activity through the reduction of resazurin to the fluorescent compound resorufin and was therefore used as an indicator of viable cell number.

Each experimental group consisted of three independent biological replicates (n = 3). Data are expressed as mean ± standard deviation (SD), and error bars in all graphs represent the standard deviation of three independent biological measurements. Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Statistical significance was determined using two-way ANOVA followed by Tukey's multiple comparisons post hoc test. Data are presented as mean ± SD from three independent biological replicates (n = 3). Statistical significance was accepted at p < 0.05. In the figures, **** indicates p < 0.0001.

2.5. Antibacterial activity

For the evaluation of antibacterial activity through the disk diffusion assay, E. coli, P. aeruginosa, and S. aureus bacterial strains were initially cultured in sterile Nutrient Broth and incubated at 37 °C for 24 hours. On the following day, agar and Mueller–Hinton solid media were prepared, and the overnight bacterial cultures were adjusted to a turbidity corresponding to the 0.5 McFarland standard. These standardized bacterial suspensions were then re-inoculated into fresh liquid medium. Using the spread plate method, the adjusted bacterial suspensions were evenly distributed on the surfaces of the solid media. Afterwards, antibiotic disks containing gentamicin and ampicillin, along with blank disks impregnated with CuO, CuO–Ag, and CuO–Ag–TiO₂ at predetermined concentrations, were placed onto the inoculated plates. Following incubation at 37 °C for 24 hours, the diameters of the inhibition zones formed around the CuO, CuO–Ag, and CuO–Ag–TiO₂-loaded disks were measured and compared with those surrounding the antibiotic disks.

2.6. Photocatalytic degradation of pollutants

For the photocatalytic activity test, 10 ppm methylene blue dye and 50 mg L⁻¹ tetracycline antibiotic solutions were first prepared. The UV spectra of these pollutants were measured and recorded. Then, the obtained nanoparticles were added to the pollutant solutions and kept in the dark for 30 minutes to observe the adsorption–desorption equilibrium and the accelerating effect of UV light. Afterward, the nanoparticle–pollutant mixture was exposed to UV light for 15 minutes, and UV spectrum measurements were taken. This process was repeated until the pollutants reached their maximum degradation percentage.

3. Results and discussion

3.1. Characterization

Various techniques are used for the characterization of nanoparticles. X-ray diffraction spectroscopy (XRD) is employed to determine parameters such as the crystal structure, crystallite size, and degree of crystallinity of nanoparticles.¹⁵ As shown in Fig. 2(a), XRD is based on the principle that each crystal phase diffracts X-rays of the same wavelength in a characteristic pattern, depending on its unique atomic arrangement.¹⁶ XRD patterns were recorded using a BRUKER D8 ADVANCE diffractometer. The XRD patterns of CuO, CuO–Ag, and CuO–Ag–TiO₂ samples are presented in detail. In the CuO sample, the diffraction peaks corresponding to the (110), (002), (111), (202), (020), (113), (220), and (221) planes were clearly observed at 2θ values of 32.6°, 35.6°, 38.9°, 48.9°, 58.4°, 61.7°, 66.4°, and 68.9°, respectively. These peaks indicate the high crystalline purity and well-organized structure of CuO. In the CuO–Ag sample, sharp peaks observed at 28.1°, 32.5°, 46.4°, and 57.7° correspond to the (101), (100), (111), and (200) planes, respectively, demonstrating the successful incorporation of Ag nanoparticles into the CuO structure and a significant alteration in the material's architecture. In the CuO–Ag–TiO₂ composite, characteristic diffraction peaks of TiO₂ corresponding to the (004), (111), and (105) planes were detected at 38.3°, 44.5°, and 64.6°, respectively. These peaks confirm the successful integration of TiO₂ into the CuO–Ag composite, forming a well-aligned nanocomposite with structural compatibility. The XRD patterns obtained strongly support the successful synthesis and structural integrity of each sample by highlighting the dominant crystal phases and planes. Notably, the prominent monoclinic crystal structure of CuO enhances the structural stability of the nanocomposites formed with Ag and TiO₂. This suggests that CuO-based nanocomposites possess significant potential for material design and can be effectively utilized in environmental and industrial applications. XRD is used to reveal the crystal structure and phase purity of nanoparticles. The incorporation of Ag and TiO₂ into the CuO structure enhanced the stability of the crystal lattice, positively contributing to the overall functional properties of the material.

Fig. 2. (a) XRD peaks, (b) FTIR spectra, and (c) SEM images of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposites.

Fourier-transform infrared spectroscopy (FTIR) is a spectroscopic technique that uses infrared radiation to identify organic, inorganic, and polymeric materials. It helps to investigate the molecular structure and interactions of nanoscale materials and plays an important role in understanding the stabilization and application properties of green-synthesized nanoparticles.¹⁷ FTIR measures the interaction between infrared light intensity and wavenumber and is widely used for chemical analysis. The infrared region of the electromagnetic spectrum ranges from 14 000 cm⁻¹ to 10 cm⁻¹ and is divided into three main regions: near-infrared (NIR; 4000–1400 cm⁻¹), mid-infrared (MIR; 400–4000 cm⁻¹), and far-infrared (FIR; 4–400 cm⁻¹).¹⁸ The FTIR spectra shown in Fig. 2(b) were analyzed to compare the structural features of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanomaterials. FTIR spectra were recorded using a BRUKER VERTEX 70 spectrometer in the range of 400–4000 cm⁻¹. The CuO sample exhibited distinct absorption bands in the 400–700 cm⁻¹ region, corresponding to Cu–O bond vibrations, representing the characteristic vibrational bands of CuO. In the CuO–Ag sample, a noticeable decrease in the intensity of the Cu–O vibrational bands was observed, indicating the successful incorporation of silver (Ag) nanoparticles into the CuO matrix and structural modifications. In the CuO–Ag–TiO₂ sample, an even more pronounced reduction in the intensity of Cu–O bands was observed, suggesting the effective incorporation of TiO₂ into the structure. The overall spectra showed very low absorbance values, indicating a denser and more ordered structure. The absence of significant bands in the 1000–4000 cm⁻¹ region suggests that functional groups such as –OH and –COOH are present in very low concentrations. These findings reveal that the Ag and TiO₂ additives were successfully integrated into the CuO structure, causing significant changes in the material's chemical composition and affecting its surface properties.

Scanning electron microscopy (SEM) is an electron microscopy technique that scans and images the surface of a material using a high-energy electron beam. Unlike conventional light microscopes that use multiple glass lenses to bend light and achieve high magnification, SEM uses electrons instead of light to generate images.¹⁹ The morphologies of green-synthesized CuO, CuO–Ag, and CuO–Ag–TiO₂ nanoparticles were comparatively examined and are respectively shown in Fig. 2(c). The morphology and elemental composition of the synthesized samples were analyzed using a ZEISS EVO LS10 scanning electron microscope equipped with an EDX detector. The CuO structure appeared to be densely agglomerated, with an irregular distribution and low surface porosity. In contrast, the CuO–Ag sample exhibited clearer nanoparticle boundaries and a more porous and homogeneous structure, suggesting that the incorporation of Ag contributed to an increased surface area. The average particle sizes of the synthesized nanocomposites were determined to be 153 nm for CuO, 170 nm for CuO–Ag, and 221 nm for CuO–Ag–TiO₂. The addition of secondary components increased agglomeration and resulted in larger particle sizes. The CuO–Ag–TiO₂ nanocomposite showed the most significant structural change, with a highly porous morphology, indicating that TiO₂ was uniformly distributed throughout the structure.

This morphological development suggests that the dopants were successfully integrated into the nanostructure and have the potential to enhance structural interactions. Morphological comparisons were also made with structures synthesized using similar methods reported in the literature.^20–22

One of the important techniques used in the characterization of nanoparticles is energy-dispersive X-ray spectroscopy (EDX). This method allows the determination of the elemental composition of a sample by separating the characteristic X-rays of different elements into an energy spectrum. EDX analysis is frequently used to verify the presence and ratios of elements in the synthesis of metal nanoparticles.²³ As shown in the EDX measurements presented in Fig. 3, the presence of Cu (copper) and O (oxygen) elements was confirmed, with Cu found at 87.84 wt% and 64.52 at%. In the Cu–Ag nanocomposite, in addition to CuO, the presence of Ag (silver) was observed at 4.16 wt%, indicating that Ag was successfully incorporated. In the ternary Cu–Ag–TiO₂ nanocomposite, the results revealed Ti at 15.24 wt%, Ag at 34.99 wt%, and Cu at 15.07 wt%. These distributions indicate the homogeneous dispersion of the elements and confirm that the Cu–Ag–TiO₂ nanocomposite was also successfully synthesized.

Fig. 3. Elemental analysis and EDX spectra of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposites.

The surface area and pore structure of the synthesized nanocomposites were investigated by Brunauer–Emmett–Teller (BET) analysis based on nitrogen adsorption–desorption measurements, and the results are presented in Fig. 4.^24,25 The specific surface area and pore characteristics were determined by BET analysis using a Micromeritics TriStar II 3020 surface area and porosity analyzer. A distinct hysteresis loop was observed in the high relative pressure region for both samples. This indicates that the nitrogen gas entering the material under high pressure does not follow the same path during desorption, suggesting that the materials possess a mesoporous structure. The specific surface area calculated using the BET method was determined to be 5.38 m² g⁻¹ for CuO and 9.66 m² g⁻¹ for the CuO–Ag–TiO₂ nanocomposite. The increase observed in the composite structure indicates that the incorporation of Ag and TiO₂ resulted in the development of a more advanced surface texture. According to the BJH analysis, the total pore volumes calculated from the adsorption and desorption branches were 0.031622 and 0.031638 cm³ g⁻¹, respectively, for the CuO sample, whereas these values increased to 0.03970 and 0.04105 cm³ g⁻¹, respectively, for the CuO–Ag–TiO₂ nanocomposite. This increase in pore volume indicates that the composite structure provides greater adsorption capacity. The average pore diameters obtained from the adsorption and desorption branches were 37.88 and 32.14 nm for the CuO sample, and 21.74 and 14.99 nm for the CuO–Ag–TiO₂ nanocomposite, respectively. Since the pore diameters of both samples fall within the 2–50 nm range according to the IUPAC classification, the synthesized materials exhibit a mesoporous character. Consequently, the incorporation of Ag and TiO₂ into CuO nanoparticles significantly improved the surface area and pore structure of the material. The higher specific surface area and pore volume observed for the CuO–Ag–TiO₂ nanocomposite may facilitate the adsorption of pollutant molecules onto the catalyst surface, thereby providing a greater number of active reaction sites.

Fig. 4. BET surface area analysis (a) CuO and (b) CuO–Ag–TiO₂ samples.

The electrochemical behavior of the bare and modified screen-printed electrodes was investigated by electrochemical impedance spectroscopy (EIS) using a Metrohm DropSens µStat400 potentiostat/galvanostat. Commercial Metrohm DropSens screen-printed electrodes (SPEs) were employed as the working platform. The impedance measurements were carried out in an aqueous solution containing 1.0 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆] (1 : 1) prepared in 0.1 M KCl, which served as the redox probe and supporting electrolyte, respectively. The Nyquist plots of the bare and modified electrodes are presented in Fig. 5. The obtained impedance spectra were fitted using equivalent electrical circuits in the ZSimDemo 3.20 software to evaluate the interfacial electron-transfer properties of the fabricated electrodes.

Fig. 5. Nyquist plots of the bare and modified electrodes.

The impedance spectra of the bare SPE, CuO/SPE, and CuO–Ag/SPE electrodes were satisfactorily fitted using the R_s(CR_ct) equivalent circuit, which consists of the solution resistance (R_s) connected in series with a parallel combination of the interfacial capacitance (C₁) and charge-transfer resistance (R_ct). In contrast, the impedance response of the CuO–Ag–TiO₂/SPE electrode was better described by the R_s(CR_ct)W circuit, where the additional Warburg element (W) represents the contribution of ion diffusion at low frequencies.

As summarized in Table 1, the solution resistance remained nearly constant for all electrodes, varying only between 30.6 and 35.9 Ω cm², indicating that the electrode modifications had little influence on the electrolyte resistance. In contrast, significant changes were observed in the charge-transfer resistance. The bare SPE exhibited the highest R_ct value (4850 Ω cm²), reflecting relatively slow electron-transfer kinetics at the electrode/electrolyte interface. After modification with CuO, the R_ct decreased to 4022 Ω cm², while the incorporation of Ag further reduced it to 2918 Ω cm², confirming the improved electrical conductivity of the composite.

Table 1. EIS fitting parameters of the bare and modified SPEs.

Parameters	SPE	CuO/SPE	CuO–Ag/SPE	CuO–Ag–TiO2/SPE
R s (Ω cm2)	32.19	35.55	35.92	30.6
R ct (Ω cm2)	4850	4022	2918	127
W 1 (S s1/2 cm−2)	—	—	—	4.8 × 10−3
C 1 (F cm−2)	2.06 × 10−5	2.10 × 10−5	2.41 × 10−5	3.2 × 10−5

A much more pronounced improvement was obtained for the CuO–Ag–TiO₂/SPE electrode, where the R_ct decreased dramatically to only 127 Ω cm². This substantial reduction indicates that the ternary CuO–Ag–TiO₂ nanocomposite provides a highly conductive interface and greatly facilitates electron transfer between the redox probe and the electrode surface. At the same time, the interfacial capacitance increased gradually from 2.06 × 10⁻⁵ F cm⁻² for the bare SPE to 3.20 × 10⁻⁵ F cm⁻² for the CuO–Ag–TiO₂/SPE electrode, suggesting an increase in the electrochemically active surface area and enhanced charge accumulation at the electrode/electrolyte interface.

Unlike the other electrodes, the CuO–Ag–TiO₂/SPE required the inclusion of a Warburg diffusion element (W₁ = 4.8 × 10⁻³ S s^1/2 cm⁻²) to accurately fit the impedance spectrum. The appearance of this diffusion component indicates that, after the charge-transfer process becomes highly efficient, mass transport of the ferri/ferrocyanide redox species contributes noticeably to the overall impedance response. This behavior is characteristic of electrodes with enhanced electrochemical activity, where electron transfer is sufficiently fast that diffusion begins to influence the low-frequency region of the impedance spectrum.

Overall, the EIS results clearly demonstrate that the sequential incorporation of Ag and TiO₂ into the CuO matrix progressively improves the electrochemical performance of the screen-printed electrode. The marked decrease in charge-transfer resistance, together with the increase in interfacial capacitance and the emergence of diffusion-controlled behavior, confirms the superior electron-transfer capability of the CuO–Ag–TiO₂-modified electrode, which is expected to contribute to its enhanced sensing performance.

3.2. Cytotoxic activity

The MDA-MB-231 human breast cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; catalog number HTB-26), are presented in Fig. 6. Cell viability was evaluated using the Alamar Blue assay following 24 h of nanoparticle exposure. As shown in Fig. 6, all nanoparticle formulations reduced cell viability in a concentration-dependent manner compared with the untreated control (p < 0.0001). Among the tested materials, the CuO–Ag–TiO₂ nanocomposite exhibited the strongest cytotoxic activity at all tested concentrations, resulting in almost complete inhibition of cell viability even at the lowest concentration tested (50 µg ml⁻¹). In comparison, CuO nanoparticles produced a moderate reduction in cell viability, whereas CuO–Ag nanoparticles exhibited enhanced cytotoxicity at higher concentrations.

Fig. 6. Cytotoxic effects of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanoparticles at different concentrations (50, 100, 200 µg ml⁻¹) on MDA-MB-231 breast cancer cells. Cell viability was determined by Alamar Blue reagent, and the results were expressed as % cell viability. The data were presented as mean ± standard deviation (mean ± SD) (n = 3). Statistical analysis was performed using GraphPad Prism 9 software, and significant differences were found to be at the level of 0.0001 < p according to the control group. The highest toxicity was observed in the CuO–Ag–TiO₂ group.

The superior cytotoxic performance of the CuO–Ag–TiO₂ nanocomposite may be associated with its physicochemical characteristics. SEM analyses demonstrated that pristine CuO nanoparticles exhibited relatively small and homogeneous morphologies, whereas Ag- and TiO₂-containing nanocomposites displayed more irregular rod-like structures. Nanoparticle morphology, surface area, and surface chemistry have been reported to play key roles in regulating nanoparticle–cell interactions, cellular internalization, and metal ion release, thereby influencing biological activity and cytotoxicity.^26,27 Furthermore, oxidative stress induced by excessive intracellular reactive oxygen species (ROS) generation is considered one of the principal mechanisms underlying the cytotoxicity of metal oxide nanoparticles, ultimately leading to mitochondrial dysfunction and apoptotic cell death.^26,28

The present findings are consistent with previous reports demonstrating that CuO-based nanoparticles induce concentration-dependent cytotoxicity in MDA-MB-231 breast cancer cells. For example, biogenic CuO nanoparticles significantly reduced the viability of MDA-MB-231 cells in a dose-dependent manner, with enhanced activity being associated with their nanoscale dimensions and surface properties.²⁷ Likewise, CuO nanoparticles functionalized with lapatinib exhibited pronounced antiproliferative activity against MDA-MB-231 cells through ROS generation, apoptosis induction, and cell-cycle arrest.²⁶ In addition, silver-containing nanocomposites have been reported to enhance the cytotoxic response of breast cancer cells by promoting ROS production and apoptosis, indicating that Ag incorporation can improve the biological activity of metal oxide nanomaterials.²⁹ These observations are in good agreement with the enhanced cytotoxicity observed for the CuO–Ag–TiO₂ nanocomposite in the present study.

Overall, the present findings demonstrate that CuO-based nanocomposites, particularly CuO–Ag–TiO₂, exhibit a pronounced antiproliferative effect against the MDA-MB-231 human breast cancer cell line under the experimental conditions employed in this study. However, since the cytotoxicity assessment was limited to a single cancer cell line, these findings should be interpreted only within the scope of the MDA-MB-231 model and should not be generalized to other cancer types. Future studies involving additional breast cancer cell lines with different molecular characteristics, normal healthy cell lines to evaluate selectivity, and in vivo models are required to elucidate the underlying mechanisms and to determine the broader therapeutic potential of these nanocomposites. This limitation should be considered when interpreting the anticancer potential of the synthesized nanomaterials.^26,28

3.3. Antibacterial activity

Nanotechnology offers new opportunities in the development of antibacterial agents. Nano metal oxides have the potential to reduce bacterial contamination in food products. When the particle size is reduced to the nanometer scale, the biological and chemical properties of the materials improve significantly.³⁰ Varying concentrations of copper oxide nanoparticles (CuO NPs) have been shown to effectively inhibit bacterial growth; higher concentrations result in greater bacterial inhibition. Light irradiation can lead to the formation of excited electron–hole pairs in CuO, suggesting that the inactivation of bacteria is largely associated with a photocatalytic mechanism.³¹ Silver nanoparticles (Ag NPs), which represent the nanoscale form of elemental silver, are particularly distinguished by their antibacterial properties. Silver not only has strong antimicrobial efficacy but also offers several advantages such as low toxicity, good biocompatibility with human cells, prolonged antibacterial activity, continuous ion release, and low potential for bacterial resistance.³² Ag/TiO₂ photocatalysts exhibit effective biocidal performance against both Gram-positive (Gram+) and Gram-negative (Gram−) bacteria, including Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The antibacterial activity and cellular interaction behaviors of Ag-doped TiO₂ nanocomposites are primarily influenced by particle size, shape, and silver concentration.³³ For antibacterial testing, Mueller Hinton Broth, Nutrient Broth, and agar media were used. Gram-positive strain S. aureus (ATCC 29213) and the Gram-negative strains P. aeruginosa (ATCC 27853) and E. coli (ATCC 25922) were employed. To determine bacterial viability, the liquid cultures were inoculated into Nutrient Broth medium using the streaking method. The confirmed viable cultures were subsequently transferred to fresh Nutrient Broth for further testing. The Nutrient Broth liquid medium was prepared and inoculated with stock cultures by passaging into test tubes. The prepared bacterial cultures were incubated at 37 °C for 24 hours. For antibacterial activity tests, Mueller Hinton Broth was combined with agar in a 250 ml Erlenmeyer flask and sterilized at 121 °C for 15 minutes in an autoclave. Under sterile cabinet conditions, the resulting solid medium was poured into sterile Petri dishes at a volume of 25–30 ml to achieve a thickness of approximately 4 mm and further sterilized by flame. After allowing the plates to solidify at room temperature, microorganisms were inoculated using swab sticks with the spread plate method, and the plates were left to dry for 5 minutes. Onto the dried plates, test samples were placed, along with gentamicin and ampicillin antibiotic disks as positive controls. The plates were incubated at 37 °C for 24 hours to determine the inhibition zones. Post-incubation observations are shown in Fig. 7. The diameters of the inhibition zones were measured using ImageJ software, and the resulting quantitative data were evaluated. The obtained results are presented in detail in Table 2, allowing a comparative assessment of the antibacterial activities of the samples. Among the tested samples, CuO exhibited the best antibacterial activity based on zone diameter measurements. Particularly, significant inhibition was observed against P. aeruginosa (2.166 cm) and E. coli (1.547 cm). Meanwhile, the CuO–Ag–TiO₂ nanocomposite demonstrated the highest inhibition zone against S. aureus (1.605 cm). Notably, the nanocomposite exhibited stronger antibacterial activity against S. aureus than the ampicillin control group.

Fig. 7. Antibacterial properties of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposites against S. aureus, E. coli, and P. aeruginosa strains.

Table 2. Zone diameters of the synthesized nanocomposites against S. aureus, E. coli, and P. aeruginosa.

Bacteria	Inhibition zone of antibiotic disc (cm)	Inhibition zone of CuO (cm)	Inhibition zone of CuO–Ag (cm)	Inhibition zone of CuO–Ag–TiO2 (cm)
S. aureus	1.605	1.260	1.291	1.605
E. coli	2.837	1.547	1.523	0.910
P. aeruginosa	2.589	2.166	1.310	1.257

The antimicrobial potential of the synthesized compounds was determined by means of a broth micro dilution test method. The bacterial and yeast strains that were examined in the study are listed in the following table. In summary, the inoculum was prepared by means of a dilution process. This process entailed the overnight dilution of fresh microorganism cultures, with the turbidity adjusted to 0.5 McFarland. The inoculum was prepared at specific ratios for the study (5 × 10⁵ CFU ml⁻¹). Test substances, adjusted at 25 mg ml⁻¹ doses, were added to the first wells of the plates containing Mueller Hinton Broth medium. Serial dilutions were then prepared. Finally, the microorganism inoculum was distributed equally among all wells and incubated at 37 °C for 18–24 hours. In order to ascertain the minimum inhibition concentrations of the substances, 2,3,5-TTC dye was added after incubation and the MIC values affected by the strains were visualized.³⁴

Table 3 shows the comparative results of the minimum inhibitory concentration (MIC) values determined for CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposites against standard microorganisms and the reference antibiotic gentamicin.

Table 3. Antimicrobial potential of test compounds performed by broth micro dilution test.

MIC values of nanocomposites against standard microorganisms (mg ml−1)
Strains	CuO	CuO–Ag	CuO–Ag–TiO2	Gentamicin (µg ml−1)
Escherichia coli ATCC 25922	0.012	0.390	0.097	1.95
Pseudomonas aeruginosa ATCC 27853	0.012	0.390	0.024	<0.97
Klebsiella pneumoniae ATCC 700603	1.562	1.562	0.048	7.81
Staphylococcus aureus (MRSA) ATCC 43300	0.195	0.390	0.048	1.95
Salmonella enteritidis ATTC 13076	1.562	0.390	0.024	1.95
Sarcina lutea ATCC 9341	0.390	0.195	0.012	1.95
Bacillus cereus ATTC 11778	0.390	0.390	0.048	1.95
Candida albicans NRRL Y-417	1.562	0.195	0.006	7.81

The results of the antimicrobial activity study indicated that CuO demonstrated a significant degree of effectiveness against both E. coli and P. aeruginosa. The minimum inhibitory concentration (MIC) of the compound was established as 0.012 mg ml⁻¹ against the two Gram-negative bacteria, which exhibited the highest sensitivity to the compound. Methicillin-resistant Staphylococcus aureus (MRSA) demonstrated a response to the compound at a concentration of 0.195 mg ml⁻¹. This MIC value was followed by 0.390 mg ml⁻¹, observed against S. lutea and B. cereus, Gram-positive bacteria. The present study examined the sensitivity of two pathogenic Gram-negative bacteria, K. pneumoniae and S. enteritidis, to a CuO, with a MIC of 1.562 mg ml⁻¹. The study further established that the compound possessed an antifungal capacity, again observed to occur at the aforementioned concentration, this time against Candida albicans.

In the course of the evaluation of the CuO–Ag for its antimicrobial capacity, it was established that the lowest MIC value determined against this compound was 0.195 mg ml⁻¹ for S. lutea and Candida albicans. The majority of the strains, including E. coli, P. aeruginosa, MRSA, S. enteritidis and B. cereus, demonstrated sensitivity to CuO–Ag at a concentration of 0.390 mg ml⁻¹. The MIC value was recorded as 1.562 mg ml⁻¹ for K. pneumoniae. The CuO–Ag–TiO₂ was found to be very effective on C. albicans at a concentration of 0.006 mg ml⁻¹. This significant MIC was followed by S. lutea with a dose of 0.012 mg ml⁻¹. The MIC value was defined as 0.024 mg ml⁻¹ for P. aeruginosa and S. enteritidis. The CuO–Ag–TiO₂ combated with K. pneumonia, B. cereus and MR S. aureus at a dose of 0.048 mg ml⁻¹. This compound revealed antimicrobial potential against E. coli with a MIC value of 0.097 mg ml⁻¹. Following comprehensive evaluation of the study in its entirety, it was determined that E. coli and P. aeruginosa exhibited the greatest sensitivity to CuO. Concurrently, 0.012 mg ml⁻¹ of this compound was identified as the most efficacious dose. The efficacy of CuO–Ag was observed to be dose-dependent when tested against a range of test microorganisms, with concentrations ranging from 0.195 to 1.562 mg ml⁻¹. It was established that the most effective MIC value of 0.006 mg ml⁻¹ was determined by CuO–Ag–TiO₂ against the yeast species Candida. Furthermore, in comparison with the other compounds, CuO–Ag–TiO₂ demonstrated activity against microorganisms at lower doses than the other two compounds, exhibiting MIC values at concentrations ranging from 0.006–0.097 mg ml⁻¹.

3.4. Photocatalysis

Photocatalysis offers a promising and environmentally friendly alternative for the degradation of many organic pollutants.³⁵ A photocatalytic reaction is a chemical process carried out through the combined effect of light and photocatalyst. This technology offers several advantages, including environmental protection, complete degradation of pollutants, and the prevention of secondary pollution. The detailed process of the photocatalytic reaction is as follows. This mechanism plays an effective role in the degradation of contaminants.³⁶ In semiconductor catalysts, under a specific light energy, electrons in the valence band (VB) are excited to the conduction band (CB), thereby creating a hole (h⁺) in the valence band. This event leads to charge separation and constitutes the fundamental step required to initiate the photocatalytic reaction. The excited electrons migrate along the conduction band to the surface of the photocatalyst and participate in reduction reactions, while the holes in the valence band spread across the surface and initiate oxidation reactions. During the reaction process, electrons interact with H⁺ ions and dissolved O₂ in the aqueous solution, forming H₂O₂ or superoxide radicals (O₂⁻). Simultaneously, holes oxidize OH⁻ ions, generating hydroxyl radicals (˙OH). However, to enhance photocatalytic efficiency, the recombination of excited electron–hole (e⁻–h⁺) pairs should be minimized as much as possible.^37,38

Copper oxide stands out as one of the most commonly used reference photocatalysts in photodegradation processes due to its low cost, non-toxicity, and high absorption efficiency over a broad portion of the solar spectrum. Among all metal oxides, CuO is distinguished as a p-type semiconductor with a narrow band gap ranging from 1.2 to 2.0 eV, and it has attracted attention as a fundamental building block for high-temperature superconductors and giant magnetoresistive materials. Thanks to these properties, CuO possesses great potential for applications in electronics and energy, and is particularly widely used in the field of photocatalysis.³⁹ Titanium dioxide (TiO₂) has demonstrated great potential for many years in the removal of organic pollutants and bacteria from wastewater, owing to its remarkable properties such as photoactivity, stability, availability, and low cost.³³ As a photocatalyst, TiO₂ is widely recognized as an effective material due to its superior photocatalytic activity in the UV spectrum. Its ability to transition into different forms without changing its oxidation state makes it a highly efficient photocatalyst. TiO₂ nanomaterials have broad applications in pollutant removal, standing out for their low cost, high refractive index, and exceptional chemical and photostability. These properties make TiO₂ a widely used material in various fields such as environmental remediation and energy conversion. To enhance the performance of TiO₂ photocatalysts, several strategies are employed, including dye sensitization, the use of supporting materials, magnetic separation, doping with metals and transition metals, as well as surface modification with other semiconductors. These approaches represent fundamental methods aimed at improving the efficiency of TiO₂. Among the metallic ion dopants used with TiO₂, copper has emerged as a prominent candidate. In addition, silver has been commonly used as a TiO₂ dopant since 1984. Due to its potential to enhance the physicochemical properties of TiO₂, Ag is considered a promising additive in numerous photocatalytic applications such as air purification, water treatment, antibacterial surfaces, and self-cleaning technologies. Exhibiting a plasmonic effect and enhanced conductivity, silver has the potential to significantly improve TiO₂'s performance when integrated into its structure.⁴⁰ Due to their band gap energies being close to that of TiO₂, they exhibit similar photocatalytic degradation. This makes them an effective solution for the removal of organic dyes and environmental pollutants. The nanoparticles increase their photocatalytic activity under visible light as a result of band gap narrowing caused by oxygen vacancies.⁴¹

The synthesized CuO–Ag–TiO₂ nanocomposite can enable electron–hole pairs to remain stable for a longer period. Since p-type materials transport hole carriers and n-type materials transport electron carriers, the synergistic interaction between both semiconductors allows for more efficient carrier separation. This has the potential to enhance photocatalytic performance. The p–n–n type CuO–Ag–TiO₂ components can initiate photocatalytic reactions over a wide light spectrum, even with low-energy photons such as sunlight. Moreover, these components can improve chemical stability during photocatalysis. During photocatalytic processes, semiconductors may undergo oxidation and reduction reactions; however, the CuO–Ag–TiO₂ structure can become more resistant in this process, enabling their use as long-lasting and highly efficient photocatalysts. In our experiments, we believe that the addition of p-type CuO to the n-type semiconductor properties of Ag and TiO₂ will increase the system's electrical conductivity and improve its overall performance. Thanks to these advantages, this nanocomposite is expected to significantly enhance the results obtained. Methylene blue (MB) is a toxic, carcinogenic, and non-biodegradable compound that can pose significant potential hazards. In its oxidized form, it appears dark blue, while in its reduced form, it becomes colorless. At room temperature, methylene blue is a solid, odorless, dark green powder that yields a blue solution in water.⁴²

Antibiotics are widely used due to the increasing demand for pharmaceuticals and the rapid development of the medical industry, and as a result, they are among the most frequently detected pollutants in natural water sources worldwide.⁴³ The use of antibiotics can lead to the accumulation of residues in the environment, causing serious environmental pollution. In recent years, antibiotics and their metabolites have been frequently detected in surface water, groundwater, and even drinking water.⁴⁴ Tetracyclines (TCs) are recognized as one of the most produced classes of antibiotics globally. The presence of tetracycline residues has been shown to negatively affect the growth and development of aquatic species, and their excessive accumulation in the food chain may cause various diseases, posing a significant threat to human health.⁴⁵ Semiconductor-based photocatalytic antibiotic degradation offers a promising, environmentally friendly, and effective strategy for the removal of toxic antibiotic residues. This photocatalytic process accelerates the degradation of antibiotics in aquatic environments, thereby reducing environmental pollution and contributing to the protection of aquatic ecosystems.⁴⁶

In this study, the photocatalytic activity of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposites against methylene blue and antibiotic solutions was investigated under UV light. The photocatalytic experiments were conducted in a UV photoreactor equipped with a UV-C lamp. Solutions of methylene blue dye and tetracycline hydrochloride at various concentrations were prepared and stored in the dark. For photocatalytic measurements, the prepared solutions and CuO–Ag–TiO₂ nanocomposites were added to quartz beakers and kept under magnetic stirring in the dark for 30 minutes to reach adsorption–desorption equilibrium. Subsequently, the solutions were exposed to UV light. To avoid scattering that could affect spectrophotometric measurements, the collected liquid samples were centrifuged. The absorption spectra of the samples obtained after each 15-minute UV exposure were measured using a spectrophotometer.

Fig. 8 presents the absorbance values of the CuO-based nanocomposite structures. The value denoted as 0 min represents the absorbance of the pollutants before the addition of the photocatalyst. The value indicated as 30 min represents the period during which the photocatalyst was kept in the dark. To observe the effect induced by light irradiation, measurements were taken after the samples had been kept in the dark to establish the adsorption–desorption equilibrium. Subsequently, the samples were exposed to UV light for 15 min. As can be seen, all samples exhibited a noticeable decrease in absorbance peaks over time, indicating successful photocatalytic degradation. The CuO sample showed the lowest performance in degrading both methylene blue and the antibiotic. Although the absorbance peaks gradually decreased over time, the degradation rate remained lower than that of the other nanocomposites. In contrast, the CuO–Ag nanocomposite exhibited significantly higher degradation rates for both pollutants compared to pure CuO. This improvement is attributed to the enhanced light absorption capacity provided by Ag. The incorporation of Ag promotes the formation of a greater number of reactive intermediate species on the photocatalytic surface, thereby enhancing the photocatalytic efficiency.

Fig. 8. (a, c, and e) Show the absorption spectra of methylene blue (MB) solutions containing photocatalysts under UV light degradation. (b, d, and f) Display the absorption spectra of tetracycline hydrochloride solutions containing photocatalysts under UV light degradation.

The CuO–Ag–TiO₂ nanocomposite exhibited the highest degradation efficiency for both methylene blue and the antibiotic. The fastest and most significant decrease in absorbance values over time was observed with this sample. This indicates that the functional groups formed on the nanocomposite surface, owing to the Aronia melanocarpa extract, and the synergistic effect of Cu, Ag, and TiO₂ significantly enhanced the photocatalytic activity. As a result, the nanocomposite demonstrated the highest photocatalytic activity in the degradation of organic pollutants such as methylene blue and antibiotics. The expanded surface area of the nanocomposite and the more efficient separation of electron–hole pairs contributed to the overall enhancement of photocatalytic performance. Therefore, the green synthesis method employed in this study yielded an environmentally friendly and highly efficient photocatalyst.

Fig. 9 compares the effects of CuO, CuO–Ag, and CuO–Ag–TiO₂ photocatalysts synthesized using the Aronia melanocarpa extract on the degradation of methylene blue (MB) dye. The nanocomposites interacted with UV light and successfully reduced the concentration of methylene blue. The following formula was used to calculate the ratio of ln C_t/C₀:−ln(C_t/C₀) = ktHere, C₀ represents the initial concentration of the dye, t is the irradiation time under UV light, C_t is the dye concentration at a given time, and k is the observed first-order reaction rate constant. In the C_t/C₀vs. time graph showing the change in concentration over time, it was observed that CuO alone exhibited very low photocatalytic activity, while the addition of Ag and TiO₂ significantly enhanced this activity. Particularly, the CuO–Ag composite showed the most effective results in dye degradation. The change in reaction rate over time, evaluated through −ln(C_t/C₀) values, demonstrated that the CuO–Ag and CuO–Ag–TiO₂ composites enabled a faster and more stable degradation process. The calculated k values for CuO, CuO–Ag, and CuO–Ag–TiO₂ were 0.00159 min⁻¹, 0.01265 min⁻¹, and 0.01172 min⁻¹, respectively. According to the k values, the highest photocatalytic activity was observed in the following order: CuO–Ag > CuO–Ag–TiO₂ > CuO. Fig. 9 shows the results of the methylene blue (MB) dye test for CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposites.

Fig. 9. Photocatalytic performance of the synthesized nanocomposites toward methylene blue (MB) degradation under UV irradiation: (a) concentration ratio (C_t/C₀) as a function of irradiation time, (b) pseudo-first-order reaction kinetics of the nanocomposites, (c) degradation rate constants (k) of the nanocomposites, and (d) degradation efficiencies of the nanocomposites.

Fig. 10 shows the change in the C_t/C₀ ratio during the degradation of tetracycline hydrochloride using CuO, CuO–Ag, and CuO–Ag–TiO₂ photocatalysts. The graph shows that CuO exhibits limited degradation throughout the reaction time, whereas the photocatalytic performance of Ag and TiO₂ additives increases significantly. In particular, the CuO–Ag–TiO₂ nanocomposite achieves a higher dye removal rate in a shorter time, demonstrating that this structure is more effective from a photocatalytic point of view. The time-dependent change in reaction rate evaluated using −ln(C_t/C₀) values and the linear behavior of the curves indicate that photocatalytic degradation processes occur under kinetic control. The slopes of the CuO–Ag and CuO–Ag–TiO₂ samples showed that they enabled a faster and more stable degradation process compared to CuO. The calculated k values were found to be 0.00728 min⁻¹, 0.00705 min⁻¹, and 0.02819 min⁻¹ for CuO, CuO–Ag, and CuO–Ag–TiO₂, respectively. According to these k values, the photocatalytic activity is ranked from highest to lowest as CuO–Ag–TiO₂ > CuO > CuO–Ag. Fig. 10 shows the test results of CuO, CuO–Ag, and CuO–Ag–TiO₂ nanocomposites with tetracycline hydrochloride antibiotic.

Fig. 10. Photocatalytic performance of the synthesized nanocomposites toward TC degradation under UV irradiation: (a) concentration ratio (C_t/C₀) as a function of irradiation time, (b) pseudo-first-order reaction kinetics of the nanocomposites, (c) degradation rate constants (k) of the nanocomposites, and (d) degradation efficiencies of the nanocomposites.

To demonstrate the reusability of the materials, photocatalytic activity tests were performed for three consecutive days under the same experimental conditions using the CuO–Ag–TiO₂ nanocomposite, which exhibited the highest photocatalytic activity, against tetracycline and methylene blue. Average degradation efficiencies of 86.57% for tetracycline and 90.84% for methylene blue were obtained. For methylene blue, a photocatalytic degradation efficiency of 90.3% was obtained, and no loss in photocatalytic efficiency was observed on the third day. For tetracycline, degradation efficiencies of 88.1% and 82.4% were obtained on the first and third days, respectively. The very limited decrease in degradation efficiency observed for tetracycline indicates that the nanocomposite material exhibits high stability and reusability for both pollutants. These results were consistent with the photocatalytic degradation experiments for both pollutants and confirmed the stability, durability, and reusability of the synthesized materials. The obtained results and the corresponding calculated experimental parameters are presented in Fig. S1 and S2.

The EIS measurements provide electrochemical evidence supporting the photocatalytic performance of the synthesized nanocomposites by demonstrating their enhanced charge transfer efficiency and suppressed electron–hole recombination. The charge transfer resistance (R_ct) gradually decreased from CuO to CuO–Ag and finally to CuO–Ag–TiO₂. The remarkably low R_ct value of the CuO–Ag–TiO₂ nanocomposite facilitates electron migration. As a result, a greater amount of reactive oxygen species (ROS) can be generated. Considering the EIS results, the superior photocatalytic activity observed for the CuO–Ag–TiO₂ nanoparticles is well explained. Table 4 compares the results of experiments on the photocatalytic degradation of various dyes and antibiotics using CuO, Ag, TiO₂ and ZnO photocatalysts with those reported in the literature. The literature shows that semiconductor photocatalysts such as CuO, Ag, TiO₂ and ZnO are widely used in the removal of different dyes such as methylene blue, methyl orange, and trypan blue, and the results vary in terms of light source, degradation time, and removal efficiency. The results obtained are shown to be consistent with the literature data.

Table 4. Comparison of the photocatalytic degradation efficiencies of the synthesized nanocomposites with those reported in the literature.

Catalyzer	Pollutant	Source of light	Degradation rate (%)	Degradation time (min)	Ref.
TiO2	MB	LED	88	120	52
TiO2	MB	UV-vis	89	120	53
TiO2	Methyl orange	UV-vis	20	150	56
CuO	Methyl orange	Sunlight	65	60	54
CuO	MB	UV-vis	91	12	55
ZnO	MB	UV	95	240	48
ZnO	Methyl orange	UV	83.99	120	49
Cu-doped TiO2	MB	UV	82	90	50
Ag	MB	Sunlight	95	4320	51
Ag	MB	UV light	95	—	47
CuO	MB	UV	—	—	This study
CuO–Ag			89.93	180
CuO–Ag–TiO2			90.85	195
CuO	TC		60.18	150
CuO–Ag			71.85	195
CuO–Ag–TiO2			89.34	90

In Table 4, the photocatalytic degradation results obtained in this study are compared with those of CuO, Ag, TiO₂, and ZnO-based photocatalysts reported in the literature. The literature indicates that CuO, Ag, TiO₂, and ZnO-based photocatalysts have been used for the removal of various pollutants, such as methylene blue, methyl orange, and trypan blue, and that their degradation efficiencies vary depending on the light source, reaction time, and experimental conditions. In sample studies, it has been shown that Ag and ZnO-based photocatalysts achieved a degradation efficiency of approximately 95% for methylene blue.^47–51 For TiO₂-based photocatalysts, it has been shown that the degradation of methylene blue reaches 88% and 89% after 120 minutes under LED and UV-visible light, respectively.^52,53 For CuO photocatalysts, degradation efficiencies ranging from 65% to 91% were obtained, depending on the synthesis method and experimental conditions.^54,55 Furthermore, the fact that a degradation efficiency of 20% was achieved for methyl orange using only TiO₂ demonstrates that the type of pollutant affects photocatalytic performance.⁵⁶ In this study, CuO–Ag and CuO–Ag–TiO₂ nanocomposites exhibited degradation efficiencies of 89.93% and 90.85%, respectively, in the photocatalytic degradation of methylene blue. It was concluded that the results obtained were comparable to those of many TiO₂ and CuO photocatalysts reported in the literature, but showed lower degradation efficiencies compared to Ag and ZnO photocatalysts. CuO and Ag nanoparticles are well known for their effective antibacterial properties. TiO₂ nanoparticles have also been reported in the literature to be incorporated into composite structures with various antibacterial materials for antibacterial applications. This demonstrates the supporting role of TiO₂ in antibacterial activity studies.^57–59 Therefore, TiO₂ is frequently preferred in antibacterial applications and has found use in various fields.^60–62 Similar results have been reported in previous studies, where the synthesized composite materials exhibited strong antibacterial activity. Likewise, numerous studies have reported that CuO-, Ag-, and TiO₂-based nanocomposites exhibit high cytotoxic activity against different cancer cell lines.^63–65 The findings obtained in the present study are consistent with the literature, demonstrating that the synthesized CuO, CuO–Ag, and particularly CuO–Ag–TiO₂ nanocomposites significantly reduced the viability of MDA-MB-231 cells. These results indicate that the composite structure enhances the anticancer activity and possesses considerable potential for biomedical applications.

4. Conclusion

Based on the characterization measurements, it was confirmed that CuO, CuO–Ag, and Cu–Ag–TiO₂ nanocomposites were successfully synthesized. The antibacterial properties of these nanomaterials were then evaluated against Gram-positive S. aureus (ATCC 29213) and Gram-negative P. aeruginosa (ATCC 27853) and E. coli (ATCC 25922) bacteria. All nanomaterials exhibited inhibition zone diameters comparable to those of the antibiotics used as control groups. Notably, against Gram-positive S. aureus, the Cu–Ag–TiO₂ nanocomposite showed an inhibition zone diameter equivalent to that of the ampicillin antibiotic used as a control. These findings demonstrate the potential applicability of the synthesized nanomaterials in biomedical applications. In photocatalytic measurements, CuO alone did not exhibit photocatalytic activity against methylene blue dye. With Ag incorporation, its photocatalytic activity increased, and the highest degradation percentage was observed for the Cu–Ag nanocomposite. TiO₂ addition did not contribute to the degradation of methylene blue. When evaluating the degradation rates against tetracycline antibiotic, the Cu–Ag–TiO₂ nanocomposite achieved the highest degradation rate. Although the Cu–Ag nanocomposite displayed a similar degradation percentage, TiO₂ addition played a role in accelerating the photocatalytic reaction for tetracycline, leading to the highest degradation rate within a shorter period. These results indicate that the incorporation of different components into nanoparticles plays a crucial role in their photocatalytic activity and that the nanocomposites used in this study show potential for wastewater treatment applications. It has been shown that the properties of CuO nanocomposites are influenced not only by the components used but also by the interactions between these components. In particular, the incorporation of Ag and TiO₂ has been demonstrated to simultaneously enhance both the photocatalytic and antibacterial activities. Based on the findings obtained in this study, future research is planned to comprehensively investigate the synthesized CuO, CuO–Ag, and Cu–Ag–TiO₂ nanocomposites for environmental and biomedical applications. Evaluating the photocatalytic performances of these nanocomposites toward the degradation of different organic pollutants under visible light and solar irradiation is expected to demonstrate their significant potential for environmental applications. In antibacterial studies, the successful inhibition zone diameters obtained against different bacterial strains, together with the promising results observed in anticancer applications, indicate the potential of these materials for use in biomedical applications. The combined evaluation of the photocatalytic, antibacterial, and anticancer performances further reveals that CuO-based nanocomposites have considerable potential as multifunctional materials for a wide range of future applications.

Author contributions

Zahraa Mohanad Abdullah Alalam: formal analysis, investigation, writing – original draft preparation. Merve Şeker: formal analysis, writing – original draft preparation. Sudenaz Mirza: formal analysis, writing – original draft preparation. Ali Akbar Hussaini: validation, investigation. Gülcihan Gülseren: validation, investigation. Ahmet Uysal: formal analysis, writing – original draft preparation. Mehmet Hakan Çolpan: validation, investigation. Murat Yıldırım: resources, data curation, supervision.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra03516g.