Synthesis of CuO and PAA-Regulated Silver-Carried CuO Nanosheet Composites and Their Antibacterial Properties

With the aid of a facile and green aqueous solution approach, a variety of copper oxide (CuO) with different shapes and polyacrylic-acid (PAA)-regulated silver-carried CuO (CuO@Ag) nanosheet composites have been successfully produced. The point of this article was to propose a common synergy using Ag-carried CuO nanosheet composites for their potential antibacterial efficiency against three types of bacteria such as E. coli, P. aeruginosa, and S. aureus. By using various technical means such as XRD, SEM, and TEM, the morphology and composition of CuO and CuO@Ag were characterized. It was shown that both CuO and CuO@Ag have a laminar structure and exhibit good crystallization, and that the copper source and reaction duration have a sizable impact on the morphology and size distribution of the product. In the process of synthesizing CuO@Ag, the appropriate amount of polyacrylic acid (PAA) can inhibit the agglomeration of Ag NPs and regulate the size of Ag at about ten nanometers. In addition, broth dilution, optical density (OD 600), and electron microscopy analysis were used to assess the antimicrobial activity of CuO@Ag against the above three types of bacteria. CuO@Ag exhibits excellent synergistic and antibacterial action, particularly against S. aureus. The antimicrobial mechanism of the CuO@Ag nanosheet composites can be attributed to the destruction of the bacterial cell membrane and the consequent leakage of the cytoplasm by the release of Ag+ and Cu2+. The breakdown of the bacterial cell membrane and subsequent leakage of cytoplasm caused by Ag+ and Cu2+ released from antimicrobial agents may be the cause of the CuO@Ag nanosheet composites’ antibacterial action. This study shows that CuO@Ag nanosheet composites have good antibacterial properties, which also provides the basis and ideas for the application research of other silver nanocomposites.


Introduction
In the past two years, the new crown epidemic has greatly affected people's health and life and has seriously endangered lives; at the same time, bacteria, fungi, parasites, and viruses are all seriously affecting human public health [1]. As with today's emphasis on technological innovation, the research and development of new antimicrobial agents that can enhance antibacterial activity is becoming more important in the human living environment [2][3][4], and increased bacterial resistance and cross-infection in public places are also common health threats [5][6][7]. Silver nanoparticles (Ag NPs), which are used as a common antibacterial material, have been extensively studied in the past several decades [8,9], for example, in inhibiting the growth of bacteria [10], antimicrobial activity endurance [11], and the lack of risk of resistance of bacteria [12]. Relevant experiments have demonstrated the antibacterial mechanism of silver, that is, silver ions can cause the denaturation of proteins in the cell membrane of bacteria [13][14][15], and when the Ag + enters into the bacterial cell, it quickly combines with DNA in the cell and prevents the replication of the DNA double-helix structure [16]. It is commonly known that there is (XRD) (Nalytical, Almelo, Holland) using X'pert Philips with Cu Kα radiation (λ = 1.5418 Å); SEM pictures were captured by using a JEOL JSM-5600LV (JEOL Ltd., Tokyo, Japan); transmission electron microscope (TEM) using a JEOL JEM-100CX with the sample carried through a copper mesh; Fourier transform infrared spectrometer (FT-IR) using an AVATAR 360 (Nicolet Instrument Corporation, Madison, USA); UV-vis using a UNICORN 540 (Hefei, Anhui, China); TGA using a Seiko EXSTAR 6000 (Seiko Instruments Inc, Tokyo, Japan) in nitrogen atmosphere.

Preparation of CuO
In this experiment, the reaction vessel was a 250 mL flask, and 1.25 g of CuSO 4 ·5H 2 O and 100 mL of distilled water were added into the above reaction vessel to dissolve the blue solution, which was stirred with a magnetic stirrer and heated to 40 • C. Then, 50 mL of 0.3 mol/L NaOH was added dropwise into the reaction, during which the color of the reaction solution changed from blue to black. The solution continued to react for a while. At the end of the process, the suspension liquid was separated by a high-speed centrifuge at 5000 rev/min for 5 min, and the resulting black solid was washed several times with distilled water and then vacuum-dried to obtain a black powder, which was the copper oxide nanosheet. Other shapes of nano-copper oxide were also obtained by a similar procedure as for the copper oxide nanosheet, except that CuSO 4 ·5H 2 O was replaced by CuNO 3 ·3H 2 O and Cu(CH 3 COO) 2 ·H 2 O, respectively.
In addition, some similar experiments were performed, that is, only regulating the reaction time such as 0.5 h, 1 h, 2 h, and 3 h, to observe the morphological changes of copper oxide nanosheets.

Preparation of CuO@Ag
A 90 mL CuO (8.5 mmol/L) solution was added into a 100 mL flask, and the black solution was stirred vigorously with a magnetic stirrer and heated to 40 • C. Then, NaOH (3 mol/L) was slowly added in order to increase the pH value of the reaction solution to 12. Several minutes later, a certain amount of PAA was added and continued to mix for 30 min. After that, a 1 mL solution of silver ammonia (50 mmol/L) and 0.0043 g of tannic acid were added. Finally, the nanocomposites were separated by centrifugation at 8000 rev/min for 5 min, washed several times with distilled water, and vacuum-dried at ambient temperature. The black powder, which is a CuO@Ag nanosheet composite, was obtained.

Antimicrobial Activity Testing
In order to evaluate the antibacterial activity, the operation steps refer to previous literature [47,48]. The target sample was the CuO@Ag nanocomposite, and three types of bacteria such as E. coli, P. aeruginosa, and S. aureus were selected as indicators. For the minimum inhibitory concentration (MIC) test, the concentrations of CuO@Ag nanocomposites were 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 2, and 1 µg/mL. Then, the MIC and minimum bactericidal concentration (MBC) of Ag NPs, the CuO@Ag nanosheet composites and CuO nanosheet were obtained.
Another test to investigate the antimicrobial properties of a target sample was the bacterial growth kinetics in broth media. The concentrations of CuO@Ag solutions were 1, 13, and 26 µg/mL, respectively. The detailed steps refer to the literature [49]. Finally, we obtained the growth curve.
In addition, SEM and TEM measurements were performed to assess the morphological changes of bacteria treated with the CuO@Ag nanosheet composite. Amounts of 40 mL of bacterial suspensions and 2 mL of broth medium were combined and cultivated at 37 • C for 6 h. Then, 100 g/mL of CuO@Ag nanosheet composites was introduced, and the bacteria was cultivated for another 6 h under the same conditions. The germs were eventually centrifuged and collected. In order to create bacterial SEM and TEM samples, the bacteria were fixed with a diluted glutaraldehyde solution (2.5%) at −4 • C for 30 min, and then centrifuged at 6000 r/min for 5 min; after being dehydrated using a series of alcohol solutions, the bacteria were collected and examined with SEM and TEM. 37 °C for 6 h. Then, 100 g/mL of CuO@Ag nanosheet composites was introduced, and bacteria was cultivated for another 6 h under the same conditions. The germs were ev tually centrifuged and collected. In order to create bacterial SEM and TEM samples, bacteria were fixed with a diluted glutaraldehyde solution (2.5%) at −4 °C for 30 min, a then centrifuged at 6000 r/min for 5 min; after being dehydrated using a series of alco solutions, the bacteria were collected and examined with SEM and TEM.         Figure 4 shows the zeta potential plotted against pH for the CuO nanosheets. DLS used to explore the impact of charge on the surface of CuO nanosheets with different p values [50]. It is clear that the surface of CuO nanosheets is electro-negative in an alkalin environment. As shown in Figure 4, increasing the pH of the nanoparticle suspensio leads first to a decrease and then an increase in the absolute value of the zeta potentia    Figure 4 shows the zeta potential plotted against pH for the CuO nanosheets. DLS is used to explore the impact of charge on the surface of CuO nanosheets with different pH values [50]. It is clear that the surface of CuO nanosheets is electro-negative in an alkaline environment. As shown in Figure 4, increasing the pH of the nanoparticle suspension leads first to a decrease and then an increase in the absolute value of the zeta potential.  Figure 4 shows the zeta potential plotted against pH for the CuO nanosheets. DLS is used to explore the impact of charge on the surface of CuO nanosheets with different pH values [50]. It is clear that the surface of CuO nanosheets is electro-negative in an alkaline environment. As shown in Figure 4, increasing the pH of the nanoparticle suspension leads first to a decrease and then an increase in the absolute value of the zeta potential. The absolute value of the zeta potential on the surface of CuO nanosheets is obtained as maximum when the pH is 12. The maximum absolute value of the zeta potential is 37 mV, which means that the surface of copper oxide has the most negative charge. At the same time, silver ions exist as silver ammonia complex ions rather than as silver salt precipitates when the pH is about 12. The silver ammonia complex ions will accumulate in significant amounts on the surface of CuO nanosheets in accordance with the concept of peer charge repulsion and opposite-charge attraction. The reducing agent will subsequently decrease the silver ammonia complex ions to silver, making it simpler to create CuO@Ag nanosheet composites. maximum when the pH is 12. The maximum absolute value of the zeta potential is 37 mV, which means that the surface of copper oxide has the most negative charge. At the same time, silver ions exist as silver ammonia complex ions rather than as silver salt precipitates when the pH is about 12. The silver ammonia complex ions will accumulate in significant amounts on the surface of CuO nanosheets in accordance with the concept of peer charge repulsion and opposite-charge attraction. The reducing agent will subsequently decrease the silver ammonia complex ions to silver, making it simpler to create CuO@Ag nanosheet composites.

Antimicrobial Activities of CuO@Ag Nanosheet Composites
According to the broth dilution method, Table 1 displays the MIC and MBC values of Ag, CuO@Ag nanosheet composites, and CuO nanosheets against three types of bacteria, for instance, E. coli, S. aureus, and P. aeruginosa. The average particle diameters of CuO@Ag nanosheet composites and Ag NPs on the surface of the CuO nanosheet are 400 nm and about 20 nm, respectively, which are prepared by the two-step method in this work. Yet, Ag particles with severe aggregation are about 400 nm and used as the control

Antimicrobial Activities of CuO@Ag Nanosheet Composites
According to the broth dilution method, Table 1 displays the MIC and MBC values of Ag, CuO@Ag nanosheet composites, and CuO nanosheets against three types of bacteria, for instance, E. coli, S. aureus, and P. aeruginosa. The average particle diameters of CuO@Ag nanosheet composites and Ag NPs on the surface of the CuO nanosheet are 400 nm and about 20 nm, respectively, which are prepared by the two-step method in this work. Yet, Ag particles with severe aggregation are about 400 nm and used as the control sample. Table 1 shows that the MIC and MBC values of Ag and CuO nanosheets against the three bacteria are more than 125 µg/mL. While obviously superior to those of Ag and CuO nanosheets, the MIC and MBC values of CuO@Ag nanosheet composites against three types of bacteria (E. coli, S. aureus, and P. aeruginosa) are lower than 13 and 26 µg/mL, respectively. These findings demonstrate that the CuO@Ag nanosheet composites have superior antibacterial qualities. According to Table 1, the results also suggest that the CuO@Ag nanosheet composites display comparable antibacterial properties against Gramnegative bacteria such as P. aeruginosa and E. Coli, but this sample differs from the reference in that it shows the higher antibacterial activity against Gram-positive bacteria such as S. aureus [51]. This could be explained by the outcome of the combined action of Ag NPs and CuO nanosheets. In addition, we investigate the antibacterial properties of CuO@Ag nanosheet composites by testing the bacterial growth curves in liquid broth media. Using a UV-vis spectrophotometer, the time-dependent variations in bacterial growth are identified using the OD600 technique. The growth curves of common Gram-negative and Gram-positive bacteria (E. coli and P. aeruginosa) are shown in Figure 6 for 48 h with varying concentrations of CuO@Ag nanosheet composites. The control test uses untreated normal bacteria. Figure 6 shows that CuO@Ag nanosheet composites have a considerable inhibitory effect on the reproduction of the tested strains at all tested doses. When the concentration exceeds 26 g/mL, the sample can totally block the growth of P. aeruginosa and E. coli for the full 48 h ( Figure 6A,C). The growth of E. coli and P. aeruginosa is delayed when the concentration is below the MIC (13 g/mL), as it is insufficient to stop their growth within 48 h. The 13 and 26 g/mL solutions may totally stop the growth of S. aureus bacteria. Similar to the control test of S. aureus, the growth curves of S. aureus with the concentration of 2 g/mL of CuO@Ag nanosheet composites similarly display a lag phase ( Figure 6B). These results are consistent with the MIC and MBC numerical values.
We use SEM to compare the appearance of the normal and treated bacteria in order to further understand how the CuO@Ag nanosheet composites solution affects bacteria. Figure 7 presents SEM images of the three types of bacteria before and after being treated with the CuO@Ag nanosheet composites solution (100 µg/mL). In contrast to the normally occurring E. coli, which exhibits a uniformly short rod with a smooth surface (Figure 7a), the treated E. coli (Figure 7b) shows significant differences, with shorter lengths and an extremely rough surface. Similarly, both P. aeruginosa and S. aureus show similar phenomena. That is, the normal P. aeruginosa (Figure 7c) presents a uniformly long rod with a smooth surface, while the treated P. aeruginosa (Figure 7d) has a highly rough and uneven surface. The normal S. aureus cells (Figure 7e) have a smooth surface and spherical shape with an average diameter of 1 µm; however, in treated cells, cell debris can be visible along with membrane distortion and rough surface development (Figure 7f). These results suggest that nanocomposites interact with and destroy bacteria, rendering them inactive. reproduction of the tested strains at all tested doses. When the concentration exceeds 26 g/mL, the sample can totally block the growth of P. aeruginosa and E. coli for the full 48 h (Figures 6A, C). The growth of E. coli and P. aeruginosa is delayed when the concentration is below the MIC (13 g/mL), as it is insufficient to stop their growth within 48 h. The 13 and 26 g/mL solutions may totally stop the growth of S. aureus bacteria. Similar to the control test of S. aureus, the growth curves of S. aureus with the concentration of 2 g/mL of CuO@Ag nanosheet composites similarly display a lag phase ( Figure 6B). These results are consistent with the MIC and MBC numerical values. We use SEM to compare the appearance of the normal and treated bacteria in order to further understand how the CuO@Ag nanosheet composites solution affects bacteria. Figure 7 presents SEM images of the three types of bacteria before and after being treated with the CuO@Ag nanosheet composites solution (100 μg/mL). In contrast to the normally occurring E. coli, which exhibits a uniformly short rod with a smooth surface (Figure 7a), the treated E. coli (Figure 7b) shows significant differences, with shorter lengths and an extremely rough surface. Similarly, both P. aeruginosa and S. aureus show similar phenomena. That is, the normal P. aeruginosa (Figure 7c) presents a uniformly long rod with a smooth surface, while the treated P. aeruginosa (Figure 7d) has a highly rough and uneven surface. The normal S. aureus cells (Figure 7e) have a smooth surface and spherical shape with an average diameter of 1 μm; however, in treated cells, cell debris can be visible along with membrane distortion and rough surface development (Figure 7f). These results suggest that nanocomposites interact with and destroy bacteria, rendering them inactive. To further demonstrate the mechanism of CuO@Ag nanosheet composites and bacteria, we also use TEM to observe the morphological changes of the three bacteria, E. coli, P. aeruginosa and S. aureus. Figure 8 shows TEM images of the normal bacteria E. coli (a), P. aeruginosa (c), S. aureus (e) without CuO@Ag nanosheet composites and the treated bacteria E. coli (b), P. aeruginosa (d), S. aureus (f) with CuO@Ag nanosheet composites (100 μg/mL). It is obvious to see from Figure 8 that the normal bacteria are rod-like and spherical for E. coli, P. aeruginosa, and S. aureus, respectively. After being treated with CuO@Ag nanosheet composites, E. coli, P. aeruginosa, and S. aureus are all broken, and the antibacterial agents CuO@Ag nanosheet composites adhere to or around the bacteria. Based on the above results, the possible antibacterial model of CuO@Ag nanosheet composites against bacteria can be proposed. That is, CuO@Ag nanosheet composites can attach to the surface of the cell membrane, thus reducing the stability of the cell membrane. The copper and silver could cause more severe membrane disruption, and then enter the bacteria, resulting in massive cytoplasmic efflux. In addition, CuO@Ag nanosheet composites + 2+ To further demonstrate the mechanism of CuO@Ag nanosheet composites and bacteria, we also use TEM to observe the morphological changes of the three bacteria, E. coli, P. aeruginosa and S. aureus. Figure 8 shows TEM images of the normal bacteria E. coli (a), P. aeruginosa (c), S. aureus (e) without CuO@Ag nanosheet composites and the treated bacteria E. coli (b), P. aeruginosa (d), S. aureus (f) with CuO@Ag nanosheet composites (100 µg/mL). It is obvious to see from Figure 8 that the normal bacteria are rod-like and spherical for E. coli, P. aeruginosa, and S. aureus, respectively. After being treated with CuO@Ag nanosheet composites, E. coli, P. aeruginosa, and S. aureus are all broken, and the antibacterial agents CuO@Ag nanosheet composites adhere to or around the bacteria. Based on the above results, the possible antibacterial model of CuO@Ag nanosheet composites against bacteria can be proposed. That is, CuO@Ag nanosheet composites can attach to the surface of the cell membrane, thus reducing the stability of the cell membrane. The copper and silver could cause more severe membrane disruption, and then enter the bacteria, resulting in massive cytoplasmic efflux. In addition, CuO@Ag nanosheet composites may release highly concentrated Ag + and Cu 2+ , and these high concentrations of metal ions exacerbate the death of bacteria [45,52].

Conclusions
In this article, a variety of copper oxide (CuO) with different shapes and PAA regulated silver-carried CuO (CuO@Ag) nanosheet composites have been successfully synthesized by a facile and green aqueous solution approach. Both CuO and CuO@Ag have a laminar structure and exhibit good crystallization, and the copper source and reaction duration have a sizable impact on the morphology and size distribution of CuO. In the process of synthesizing CuO@Ag nanosheet composites, the appropriate amount of polyacrylic acid (PAA) can inhibit the agglomeration of Ag NPs and regulate the size of Ag. In addition, compared with silver NPs and copper oxide nanosheets, the CuO@Ag nanosheet composites exhibit excellent synergistic and antibacterial action, particularly against S. aureus. This indicates that the CuO@Ag nanosheet composites have a synergistic effect on the antibacterial efficiency of three bacteria. The antimicrobial mechanism of the CuO@Ag nanosheet composites can be attributed to the destruction of the bacterial cell membrane and the consequent leakage of the cytoplasm by the release of Ag + and Cu 2+ . The breakdown of the bacterial cell membrane and subsequent leakage of cytoplasm caused by Ag + and Cu 2+ released from antimicrobial agents may be the cause of the CuO@Ag nanosheet composites' antibacterial action. This study can provide the basis and ideas for the application research of other silver nanocomposites.

Conclusions
In this article, a variety of copper oxide (CuO) with different shapes and PAA regulated silver-carried CuO (CuO@Ag) nanosheet composites have been successfully synthesized by a facile and green aqueous solution approach. Both CuO and CuO@Ag have a laminar structure and exhibit good crystallization, and the copper source and reaction duration have a sizable impact on the morphology and size distribution of CuO. In the process of synthesizing CuO@Ag nanosheet composites, the appropriate amount of polyacrylic acid (PAA) can inhibit the agglomeration of Ag NPs and regulate the size of Ag. In addition, compared with silver NPs and copper oxide nanosheets, the CuO@Ag nanosheet composites exhibit excellent synergistic and antibacterial action, particularly against S. aureus. This indicates that the CuO@Ag nanosheet composites have a synergistic effect on the antibacterial efficiency of three bacteria. The antimicrobial mechanism of the CuO@Ag nanosheet composites can be attributed to the destruction of the bacterial cell membrane and the consequent leakage of the cytoplasm by the release of Ag + and Cu 2+ . The breakdown of the bacterial cell membrane and subsequent leakage of cytoplasm caused by Ag + and Cu 2+ released from antimicrobial agents may be the cause of the CuO@Ag nanosheet composites' antibacterial action. This study can provide the basis and ideas for the application research of other silver nanocomposites.