Synthesis and characterization of CuO-MgO-ZnO and CuO-Co3O4-CeO2

The synthesis of trioxides offers unique properties for different applications due to the combination of multiple oxides; however, few studies have reported on the properties of these materials, especially in terms of their ability to create reactive oxygen species, which are helpful for antibacterial and antifungal activity. This study aimed to evaluate the surface properties of CuO-MgO-ZnO and CuO-Co3O4-CeO2 trioxides synthesized via precipitation assisted by an ultrasonic bath or sonication. The structural analysis indicated the formation of micrometric particles consisting of individual phases of each oxide, with no apparent influence of the preparation method on their morphology. UV–Vis spectroscopy revealed that CuO-MgO-ZnO particles have a band gap near 5.5 eV, while CuO-Co3O4-CeO2 has a single value at 4.2 eV. Zeta potential measurements indicated changes in the materials' outermost layer composition due to the synthesis method. Additionally, biological assays showed that the materials could completely inhibit the growth of Candida species and Staphylococcus aureus but not Klebsiella pneumoniae. These results suggest that the materials may be suitable for self-cleaning surfaces and medical device coatings.


Introduction
Advances in synthesis and production techniques for nano and microstructured materials have stimulated the search for optimized routes and efficient properties.Thus, efforts are being made to develop methods to synthesize trimetallic oxides due to their properties as antibacterial and antifungal activity [1,2].These materials are a promising group, considering the combination of their electronic properties.This combination allows light-induced reactions that lead to holes and electrons, creating reactive oxygen species (ROS) that can kill bacteria and fungi.Table 1 lists some examples of the oxides used as components in the composition of trimetallic oxides, including their band gap energies, some of their characteristics, and applications of interest.The combination of semiconductor metal oxides with varying band gaps emerges as a viable strategy for reducing the overall band gap.This reduction leads to increased charge transferability, carrier lifetime, and efficiency in ROS creation.[18,32] In this study, two metallic oxide compounds are introduced: CuO-MgO-ZnO and CuO-Co 3 O 4 -CeO 2 .Both materials present promising catalytic activity, especially in the degradation of dyes and other environmental pollutants.[2,33] Moreover, CuO-MgO-ZnO exhibits remarkable antibacterial activity, which can be attributed to the synergistic effect of antibacterial properties inherent in each of these individual oxides (refer to Table 1), thereby enhancing this particular attribute.
The broad energy gap of ZnO nanoparticles results in a low level of electron-hole pairs that can generate reactive oxygen species (ROS) to inhibit bacterial growth [22,34].The trimetallic oxide CuO-Co 3 O 4 -CeO 2 can also show antibacterial activity, considering the presence of CeO 2 in the composition.Shu et al. [22] reported an increase in the antibacterial efficiency of a ZnO nanocomposite supported by halloysite after incorporating CeO 2 .
Despite the works mentioed above, few in-depth studies have been conducted on the surface properties of these oxides.Therefore, this work aims to provide relevant information about their surface properties.The oxides were obtained through precipitation accompanied by sonication or ultrasonic baths.We evaluated the average size of particles, morphology, structure, optical properties, and their antibacterial and antifungal properties.Our promising results may open up new possibilities for the synthesis and applications of these materials.

Synthesis of trioxides
Trioxides were produced by the co-precipitation method with slight modifications compared to a published work [1].The adopted methodology is presented in Fig. 1.Two compositions were prepared: CuO-MgO-ZnO and CuO-Co 3 O 4 -CeO 2 .50 mL of 0.2 M of copper, magnesium, and zinc nitrate aqueous solution were individually prepared.Then, these solutions were placed in a beaker, mixed, and stirred for 5 min.Later, 150 mL of 0.6 M NaOH aqueous solution was prepared.The same synthesis procedure was applied for the second composition (CuO-Co 3 O 4 -CeO 2 ).NaOH solution was added drop by drop, while the beaker was in an ultrasonic bath (120 W, at 37 kHz, identified by ''U'') or sonication (identified by ''S'') for 2 h.For sonication, 40% of the total power was used, equivalent to the potency of 300 W at 20 kHz ± 50 Hz.After two hours, the material was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded.The final product was washed twice with deionized water and once with ethanol.The precipitate was taken to a beaker and dried in an oven at 110 °C for 24 h.Finally, the dried material was ground and heat treated with a rate of 1 °C/min up to 300 °C for 1 h and then calcinated at 650 °C for 4 h with a 5 °C/min increment.The material was left to cool in ambient conditions.The final product of each synthesis was a black powder.

Characterization
Crystalline phases of the studied materials were analyzed by a diffractometer system (XRD, D/MAX-2100/PC, Rigaku) using CuKα radiation within (λ = 1.54056Å).The samples were scanned from 20° to 100°, with a regular step of 0.02 min −1 and a scan speed of 2 min −1 at 40 kV/20 mA.Integration of the XRD pattern of the materials was performed to obtain the percentage of each metal oxide in the synthesized material.Structural information from XRD can be found in the supplementary material.
Images were obtained using a Zeiss LS15 microscope with an accelerating voltage of 15 kV.Chemical composition was verified in two distinct areas of the samples.For analysis, particles were dispersed in ethanol (0.5 g L −1 ), dropped in silicon water, and left to dry in ambient conditions.
The UV-visible absorption spectra were recorded using a UV-Vis spectrophotometer (UV-1800 Shimadzu) in the 600-200 nm range at room temperature.The particles were dispersed in deionized water (concentration of 0.5 g L −1 ) for 30 s using an ultrasonic bath.Then, 1 mL was used for analysis, and 1 mL of deionized water as a blank.Measurements were made using a pair of quartz cuvettes.Tauc plot was performed following the recommendations of Makula and coworkers [28].
A DLS Litesizer 500 from Anton Paar was used for zeta potential measurements and particle size distribution.Distilled water was employed as the solvent, 200 V was adjusted, and the Smoluchowski approximation was used with Henry's factor equal to 1.5.The hydrodynamic diameter was determined using an angle equal to 175°; polystyrene was employed as reference (n = 1.585) with an equilibrium time of 2 min.Particle size distribution is displayed in the supplementary material.
The antifungal and antibacterial activity was determined as described elsewhere [35].Candida albicans, Candida parapsilosis, Klebisiella pneumonie, and Staphylococcus aureus were tested against the prepared materials for the assays.Briefly, the isolates were kept at 37 °C in RPMI broth culture media.n 96-well microtiter plates (TPP, Switzerland), 100 μL of RPMI with fungal inoculum was added to each well.The particles were dispersed into the 10% dimethyl sulfoxide (DMSO) solution which works best in suspension of the particles compared to other organic solvents.After that, all particles were added into each well testing different concentrations.

Results and discussion
Trioxides were characterized using different techniques to understand their surface properties and morphology.SEM images of the prepared materials are displayed in Fig. 2. The observed agglomerates are composed of spherical-like particles.The ultrasound bath and sonication do not affect the surface morphology, only the size, as presented in Fig. 3.
The particle size from DLS for the CuO-MgO-ZnO particles is similar in the studied conditions -both materials have a diameter of around 3 μm.For this composition, sonication provides particles with a lower deviation.A considerable change in particle size was observed for the second composition-particles have a nanometric scale (~ 700 nm) if prepared under the ultrasonic bath and a micrometric size (~ 18 μm) under sonication.These changes in particle size were Fig. 1 Workflow of the adopted methodology to synthesize trioxides expected since we did not control the conditions for growth.Higher concentrations of the base of other reactants, such as KOH, may cause different effects on size.
The chemical composition of the prepared materials was analyzed using EDS, and the results are displayed in Table 2. CuO-MgO-ZnO particles have a stoichiometry ratio close to 1 for both cases, and their Mg atomic concentration is slightly higher.CuO-Co 3 O 4 -CeO 2 particles have more Cu when prepared with an ultrasonic bath.
In the CuO-Co 3 O 4 -CeO 2, a peak around 270 nm could be due to copper oxide species, although the other oxides also present absorption in the UV region [36][37][38], see Fig. 4. The Tauc plot provided a single band gap at 4.2 eV, consistent with the copper oxide band gap.There is no difference in absorbance caused by the preparation method.The prepared CuO-MgO-ZnO particles have a similar zeta potential profile; curves for both materials are presented in Fig. 5. Zeta potential values depend on different factors, like chemical composition and roughness [39,40].Although with similar values, the CuO-MgO-ZnO particles differ on isoelectric point: 7.2 for the ultrasonic bath and 9.7 for the sonicated particles.Particles obtained through ultrasonic baths presented a profile similar to ZnO nanoparticles [41], while sonication provided particles with zeta potential similar to CuO nanoparticles [42].
For the second composition (CuO-Co 3 O 4 -CeO 2 ), although with the same composition, the particles demonstrated a different behavior with a change in pH.Particles obtained under an ultrasonic bath have a lower isoelectric point (~ 3) and a prevalence of negative values, consistent with CuO and CeO 2 particles [41][42][43][44].Samples obtained through sonication have a preponderance of positive values, similar to Co 3 O 4 .[45]   Although the results may differ with the preparation method, there are more parameters related to the found values.Zeta potential can vary depending on the particle size, solvent, and even the preparation method [46][47][48][49].Once all particles were dispersed in water, the variation in zeta potential is mainly related to particle size and the preparation method.Specific surface area and particle roughness (using AFM) may provide more answers regarding the found values.
The prepared materials' minimum inhibitory concentration (MIC) was evaluated against two Candida species, Staphylococcus aureus, and Klebsiella pneumoniae; the results are provided in Table 3.The oxides prepared through the ultrasonic bath presented the lowest minimum inhibitory concentration values.The prepared trioxide shows activity against all microorganisms except Klebsiella pneumoniae.The antimicrobial activity was determined as described previously by Gottardo et al. [29], with adaptations.In this way, this present study was performed with Candida species, Staphylococcus aureus, and Klebsiella pneumoniae under contact with particles, following the microdillution method, and inoculum prepared at 0,5 Mac Farland Standard tube turbidity, 10 8 cell/mL; the results are presented in Fig. 6.From the assay, except for Klebsiella pneumoniae, the oxides completely inhibited the microorganisms.Further studies should be performed with other Gram-negative bacteria to understand their inactivity [35].
Metallic trioxides combine properties from the induvial oxides, creating new or improved properties, such as band gap and antimicrobial activity.This paper reports on the structural and surface properties of CuO-MgO-ZnO and CuO-Co 3 O 4 -CeO 2 , including their antimicrobial activity.We demonstrated that using an ultrasonic bath is more effective for producing antibacterial and antifungal activity than sonication.Materials prepared through an ultrasonic show a band gap close to visible light, a negatively charged surface, and higher antimicrobial activity.
As shown in Figure S1, individual phases of each oxide were formed.Based on this, the formation of particles can be explained by the individual formation of oxides.Copper, magnesium, and zinc have the same atomic concentration values due to the particle formation mechanism.This mechanism can be explained as follows: where Me is copper, magnesium, or zinc, and the NaNO 3 is removed through washing.Cobalt and cerium also form the hydroxide; however, temperatures higher than 900 °C are required to form cobalt monoxide [50] While cerium (III) nitrate forms cerium (IV) hydroxide and, finally, CeO 2 .[51] The theoretical stoichiometry of metal/hydroxide is 2, enough for transforming Me(OH) 2 into monoxide, as is the case for CuO-MgO-ZnO.CuO-Co 3 O 4 -CeO 2 does not only contain monoxide, and cerium forms Ce(OH) 4 and not Me(OH) 2 , as with the other metals.Thus, CuO formation may be favored during the ultrasonic bath, which explains its higher concentration than other metals in CuO-Co 3 O 4 -CeO 2 particles.
The zeta potential of this composition is not available in the literature, and the difference between the materials reveals the versatility of the prepared composition.The materials may kill bacteria with positive or negative values of the cell wall and membrane charge through electrostatic interaction.In both materials, the difference in isoelectric point may indicate that particles also differ in the composition of the outermost layer and particle diameter.
Based on the composition of the prepared materials and the results described in the literature, the creation of reactive oxygen species (ROS) may explain the biological effects.Reactive oxygen species on the particle's surface is a light-induced reaction creating OH species near the cell membrane, leading to holes in the cell membrane and, ultimately, cell death (the prepared materials have a band gap close to visible light) [35,52,53] CuO-MgO-ZnO inhibits the growth of E. coli, K. pneumoniae, P. Vulgaris, S. aureus, and P. aeruginosa; the authors of the referred study attribute the antibacterial activity to the incorporation of ions for the cells, which then release proteolytic enzymes.Finally, the created reactive oxygen species may lead to damage to the plasma membrane and cell death [2] Previous studies demonstrated that the composition CuO-Co 3 O 4 -CeO 2 has high photocatalytic activity [33] The second way (not Fig. 6 Growth inhibition of fungi and bacteria in the presence of particles, collum 2 and 3. Collum 1 control growth surface located) is creating extracellular and intracellular reactive oxygen species from metal ions released from the particles, mainly Cu and Co ions [54] This dynamic process around the cell membrane is illustrated in Fig. 7. Further studies in this direction are needed to validate the mentioned mechanism.

Conclusions
In conclusion, this study provides valuable information about the surface properties of two different trioxides obtained through precipitation assisted by ultrasonic bath or sonication.The results showed that CuO-MgO-ZnO and CuO-Co 3 O 4 -CeO 2 are formed from individual phases of each oxide, with particles of micrometric size and agglomerates of roundish particles.Zeta potential values indicated that the method modifies the outermost layer of the materials.Moreover, the samples prepared through an ultrasonic bath showed better antibacterial and antifungal properties, inhibiting the growth of Candida species and Staphylococcus aureus.Further studies are necessary to verify the influence of the precipitating agent and control the particle size distribution to obtain nanometric materials.Overall, the findings suggest that precipitation assisted by an ultrasonic bath is a better route to get trioxides with improved surface properties than sonication.The materials may have different applications, such as medical device covers or glass coating.Thin films of both compositions should be produced to evaluate their potential in various applications, including photocatalytic cells and self-cleaning surfaces.

Fig. 2
Fig. 2 SEM images of the prepared particles, where U and S indication preparation by ultrasonic bath and sonication, respectively

Fig. 3
Fig. 3 DLS size distribution of prepared particles

1 Fig. 4
Fig. 4 UV-Vis measurements and Tauc plot of prepared particles, where U and S mean prepared by ultrasonic bath and sonication, respectively

Fig. 5 Table 3
Fig.5 Zeta potential values of prepared particles, where U and S mean prepared by ultrasonic bath and sonication at 0.5 ml/mL, respectively

Table 1
Common oxides applied as constituents of trimetallic oxides, their band gap energy values, and some of their properties/applications a Band gap energies may vary according to particle size and morphology; the values are based on the references Oxide Exceptional structural, optical, and electrical properties at a minimal production expense.Alongside its utilization as micro and nanoparticles, it also exhibits significant antibacterial properties.Features a wide range of applications, including gas sensors, supercapacitors, catalysis, and solar cells due to its photocatalytic activity Unique surface chemistry, high stability and compatibility, antibacterial, antiviral, and antifungal properties.Used in sensors, catalysis, therapeutic agents, drug delivery, and anti-parasitic ointments Ce(NO 3 ) 3 6 H 2 O) purchased from Sigma Aldrich.Sodium hydroxide (CAS: 1310-73-2; NaOH) was obtained from Synth and used as a precipitating agent.All the chemicals were used without any further purification.

Table 2
Chemical composition of prepared particles