2.1 Materials

Copper (II) nitrate trihydrate (Cu(NO₃)₂·3H₂O) was purchased from Sigma–Aldrich. Sodium chloride (NaCl), potassium chloride (KCl) and sodium hydroxide (NaOH) were purchased from Daejung. Formic acid (HCOOH, 99.0% purity) and ethyl alcohol (C₂H₅OH, 99.5% purity) were obtained from Samchun. Deionized (DI) water with a resistivity of 18.2 MΩ·cm was prepared using a water purification system (Aquapuri 5 series). All the chemicals were used as received without further purification.

2.2 Preparation procedure of Cu₂O particles

A 0.01 M solution of copper (II) nitrate trihydrate was prepared by dissolving 0.0016 mol of Cu(NO₃)₂·3H₂O in 160 mL of an ethanol–water mixture under magnetic stirring at room temperature. The volume ratio of ethanol to deionized (DI) water was varied from 100:0 to 80:20. To control the morphology of the Cu₂O particles, NaCl was added to an ethanol–water mixture containing 20 vol. % water. The NaCl/Cu molar ratio was varied from 1:70 to 1:40. After the complete dissolution of NaCl, 10 mL of formic acid was added to the solution and stirred. The resulting solution was transferred to a Teflon-lined stainless-steel autoclave, sealed, and heated to 140 °C for 1 h using a heating mantle. After the reaction, the autoclave was allowed to cool to room temperature. The resulting precipitate was collected by vacuum filtration, washed several times with distilled water and ethanol, and dried under vacuum at 30 °C for 6 h.

2.3 Characterization

The morphology and size of the synthesized Cu₂O particles were observed by scanning electron microscopy (SEM, SU8020, Hitachi, Japan) at an accelerating voltage of 5 kV and emission current of 10 μA. The crystal structure of the particles was characterized via X-ray diffraction (XRD, PANalytical Xpert 3 Powder, Malvern, UK) with Cu Kα radiation (λ = 1.5406 Å). XRD patterns were collected in a 2θ range of 10°–90° at an accelerating voltage of 40 kV, current of 30 mA, and scan rate of 0.22°/s. The peak area of each crystal plane was calculated using the Pearson VII fitting model implemented in Origin software.

2.4 Antibacterial activity

The antibacterial activity of the synthesized Cu₂O particles was evaluated using the dry rehydratable film method, with Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) selected as model bacterial strains. Each strain was first cultured on dry film media and agar plates, and a single colony was transferred into 9 mL of buffered peptone water and incubated at 37 ± 1 °C for 24 h. The cultured bacterial suspension was diluted to 100–200 CFU/mL for testing. During antibacterial testing, 0.03 g of Cu₂O particles was dispersed in 10 mL of the prepared bacterial solution in a vial. A control group containing only a bacterial suspension without Cu₂O particles was also prepared. Next, 1 mL of each suspension was inoculated onto the dry film media and agar plates and incubated at 37 ± 1 °C for 48 h. After incubation, the number of colonies formed in the experimental and control groups was counted and the antibacterial rate was calculated.

3.1 Effect of water content on the morphology of Cu₂O particles

Cu₂O particles were synthesized via a solvothermal reaction using ethanol–water mixed solvents, in which the volume ratio of ethanol to water was adjusted from 100:0 to 80:20 in 5% increments. Fig 1(a) shows the XRD patterns of the Cu₂O particles synthesized with different water contents. All the observed peaks corresponded to the crystal planes of Cu₂O with a face-centered cubic structure, and no impurity phases were detected. Fig 1(b) shows the relative intensities of the (111) and (200) peaks, which represent the specific Cu₂O crystal planes. Up to a water content of 10%, the intensity ratio of the (111)/(200) peaks remained relatively constant. However, at water contents of 15% or higher, the relative intensity of the (111) peak increased significantly. This suggests that increasing the water content suppressed the growth of the (111) plane, thereby increasing the exposed area on the particle surface.

Fig 2 shows the SEM images of the Cu₂O particles synthesized with various water contents. Under anhydrous conditions, well-defined cubic particles enclosed by (100) planes were formed, although some degree of aggregation was observed (Fig 2a). At a water content of 5%, the particles remained cubic, but increased in size, and defects and voids began to appear at the edges (Fig 2b). These results suggest that at a low water content, the growth of the (100) plane was considerably suppressed, maintaining the exposure of the (100) facets. At a water content of 10%, truncated octahedral shapes emerged, indicating partial growth of the (111) planes (Fig 2c). Well-defined octahedral and hexapod-branched particles were observed at a water content of 15% (Fig 2d). These hexapod microcrystals consisted of branches formed along the [100] direction with a relatively broad size distribution (D₂₅ = 6.65 μm, D₇₅ = 11.6 μm). At a water content of 20%, particles with octahedral morphology containing surface defects were predominantly formed (Fig 2e).

The synthesized Cu₂O particles generally exhibited exposed (100), (110), and (111) facets. In some particles, defects were observed owing to non-uniform growth. The formation of internal voids in the cubic and branched particles likely resulted from the rapid growth of the outer crystal planes[¹⁸]. Increasing the water content during the synthesis suppressed the growth of the (111) plane, thereby promoting growth along the [100] direction. Consequently, the particle morphology progressively evolved from a cube to a truncated octahedron and eventually to a full octahedron.

3.2 Effect of NaCl concentration on the morphology of Cu₂O particles

The Cu₂O particles synthesized by adjusting the water content exhibited a wide particle size distribution and low shape uniformity, limiting precise morphological control. To address this, the water content was fixed at 20 vol. %, and NaCl was added to the reaction mixture as a shape-directing agent. The effect of the NaCl concentration on the dominant crystal planes and the resulting particle morphology was investigated.

Fig 3a shows the XRD patterns of Cu₂O particles synthesized at various NaCl concentrations. As the NaCl concentration increases, the intensity of the (111) peak gradually decreases, whereas that of the (200) peak increases. The (111)/(200) peak intensity ratio gradually decreases from 7.24 to 0.62 as the NaCl concentration increased (Fig 3b). These results indicate that the addition of NaCl suppressed the growth of the (100) plane, while promoting growth along the [111] direction. This suggests that NaCl can regulate the growth rate of specific crystal planes, enabling control over the morphology and exposed facets of Cu₂O particles.

Fig 4 shows SEM images of the Cu₂O particles synthesized at different NaCl concentrations. Without NaCl, the particles exhibited an octahedral shape enclosed by (111) facets with observable surface defects and voids (Fig 4a). At the NaCl:Cu molar ratio of 1:70, the particles maintained an octahedral shape (Fig 4b). When the ratio was increased to 1:55, truncated octahedra with partially exposed (100) facets appeared (Fig 4c). At a ratio of 1:50, the particles exhibited a cuboctahedral shape (Fig 4d), whereas at a ratio of 1:45, the morphology shifted to truncated cubes with fewer (111) planes (Fig 4e). Finally, at a ratio of 1:40, well-defined cubes enclosed entirely by (100) planes were obtained (Fig 4f).

These results demonstrate that varying the NaCl concentration enables precise control of Cu₂O particle morphology and the proportion of exposed crystal planes. The formation of cubic particles is likely attributed to the preferential adsorption of Na⁺ or Cl^- ions on the (100) facets, which selectively inhibit their growth and promote the development of cubic structures.

3.3 Influence of Na⁺ and Cl^- ions on the morphology of Cu₂O particles

To investigate the individual roles of Na⁺ and Cl^- ions, Cu₂O particles were synthesized by adding KCl and Na₂CO₃ at the same molar ratio (1:50). Figures 5a and b show the SEM images of the particles synthesized with NaCl and KCl, respectively. Both cases resulted in similar particle shapes, that is, cuboctahedral or truncated octahedral, with increasingly exposed (100) facets. Because both salts contain the same Cl^- anion, and the cations (K⁺ and Na⁺) have similar electronic configurations, the cation effect is presumed to be negligible. The similar particle morphologies suggest that the observed shape control is primarily due to the presence of Cl^- ions. Fig 5c presents SEM images of the particles synthesized with Na₂CO₃ addition. In this case, the resulting Cu₂O particles exhibited an octahedral shape similar to that of the particles synthesized without any additives. This indicates that the presence of Na⁺ ions alone exerted little influence on the particle morphology.

These results support the conclusion that Cl^- ions selectively inhibit the growth of the (100) planes in Cu₂O crystals. At sufficiently high Cl^- concentrations, the proportion of exposed (100) facets increased, resulting in the formation of cubic Cu₂O particles. In contrast, Na⁺ ions exerted a negligible effect on particle morphology, confirming that Cl^- ions are the primary factor controlling relative growth rates of Cu₂O crystal planes. The selective adsorption of additives onto specific crystal planes is a well-known mechanism for controlling the particle morphology. During crystal growth, faster-growing planes tend to disappear, whereas the slower-growing planes become dominant. In systems with an appropriate concentration of Cl^- ions, selective adsorption on (100) planes inhibits their growth, thereby promoting the development of cubic Cu₂O particles.

This adsorption behavior was closely linked to the crystal structure and termination characteristics of the (100) facet. The (100) plane consists of alternating layers of oxygen and copper atoms (Fig 6a), whereas the (111) plane is composed of repeating units of three atomic layers, with a copper layer sandwiched between two oxygen layers (Fig 6c). The surface trilayer of the (111) plane comprises two types of Cu ions: coordinatively saturated (CSA) and coordinatively unsaturated (CUS) species. On the Cu-terminated Cu₂O (100) facet, Cl^- ions readily chemisorb by binding to the exposed Cu⁺ ions, enabling a stable surface arrangement; this tendency has been documented in previous DFT studies[³⁵, ³⁷]. Although Cl^- ions can also adsorb at Cu^CUS sites of the (111) facet, at high coverages the adsorption becomes unstable owing to the lower binding energy with Cu^CSA ions and the increased electrostatic repulsion caused by the reduced Cl–Cl separation, which destabilizes the surface. In contrast, Cl^- ions that remain strongly bound to Cu⁺ ions on the (100) facet lower its surface energy, thereby favoring the growth of Cu₂O particles into cubes bounded by (100) facets.

Generally, inorganic salts or organic molecules can modify the relative surface energies of different crystal facets after their addition to the reaction medium. Crystals tend to grow preferentially in directions perpendicular to planes with the highest surface energy[³²]. The adsorption of Cl^- ions on (100) planes reduces their surface energy, thereby enhancing growth along the [111] direction and promoting the formation of cubic morphologies. To better understand the role of Cl^- ions from a crystallographic perspective, the atomic arrangements of different Cu₂O crystal planes were examined. Although the (111) plane exhibits higher atomic density and may appear more favorable for adsorption, the observed formation of cubic particles suggests that Cl^- ions preferentially adsorb on the (100) plane.

The lattice spacing of the (100) plane is 0.4247 nm, which is larger than the ionic radius of Cl^- (0.181 nm), allowing for stable chemisorption on this plane. Additionally, the strong interaction between Cl^- ions and surface Cu ions on the (100) plane, along with relatively low electrostatic repulsion among adjacent Cl^- ions, facilitates the formation of a chemisorbed layer that further lowers the surface energy. This adsorbed Cl^- layer inhibits further growth of the (100) facet, promoting the formation of cubic particles. However, when the Cl^- ion concentration is insufficient, incomplete adsorption results in mixed morphologies with both the (100) and (111) facets. Only when the Cl^- concentration was sufficiently high, fully cubic Cu₂O particles can be obtained. Other planes, such as (111), (110), and (200), have smaller lattice spacings (0.2452, 0.3003, and 0.2123 nm, respectively), which may be less favorable for stable Cl^- adsorption. Because the active adsorption sites and higher atomic density on the (111) plane render Cl^- adsorption comparatively less stable, the (100) facet becomes the energetically preferred surface in the presence of chloride ions.

3.4 Antibacterial activity evaluation

The antibacterial activity of the synthesized Cu₂O particles was evaluated at a concentration of 150 μg/mL against Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The concentration was standardized to enable a relative comparison of antibacterial efficacy based on morphological differences. The Cu₂O particles synthesized without NaCl demonstrated a bactericidal reduction rate of 55.6% against Staphylococcus aureus. In contrast, the particles synthesized in the presence of NaCl exhibited the maximum reduction rate of 75.6% (Fig 7a). For Escherichia coli, the Cu₂O particles synthesized without NaCl demonstrated a bactericidal effect of 31.4%, whereas those synthesized with NaCl achieved a maximum reduction rate of 88.4% (Fig 7b). Notably, the octahedral Cu₂O particles synthesized at a NaCl:Cu molar ratio of 1:70 exhibited the highest antibacterial activity against Escherichia coli, outperforming the cubic particles.

These results indicate that the enhanced antibacterial performance of Cu₂O particles synthesized with NaCl is attributable to their higher morphological uniformity and reduced surface defects. Notably, octahedral particles showed superior antibacterial activity compared to cubic particles, particularly against Escherichia coli, highlighting a shape-dependent antibacterial effect. This difference is likely due to variations in the exposed crystal planes[²⁴]. The (111) facets, which are dominant in octahedral particles, are believed to facilitate the Cu⁺ ion release, which can disrupt bacterial cell walls and significantly contribute to antibacterial efficacy[³⁸]. Although the adsorption of Cl^- ions on the (100) facets may partly contribute to reduced Cu⁺ ion release, the influence of crystal structure and surface atomic arrangement appears to be more significant. The (111) facets exhibit superior antibacterial activity compared to (100) facets due to a higher potential for Cu⁺ ion release and a higher density of active sites. Overall, the antibacterial activity of Cu₂O particles was influenced by particle shape, the nature of the exposed crystal planes, and the number of surface defects. These findings confirm that precise morphological control can significantly enhance the antibacterial performance of Cu₂O particles.