Design of Zn1-xCuxO Nanocomposite Ag-doped As An Ecient Antibacterial Agent

This study aims at the preparation of antimicrobial nanoparticles hybrid based on silver (Ag) doped Zn 1-x Cu x O. Zn 1-x Cu x O nanocomposite Ag-doped prepared via wet chemical route, co-precipitation, and impregnation method, respectively. The inclusion of silver in the nanocomposite did not change its structure. However, the insertion of CuO into the ZnO structure has no impact due to the similarity of Cu ion radius with Zn ion radius (0.74 toward 0.73 Å). Hybrid nanomaterials characterized by XRD, FT-IR, and FESEM technique. The effect of loading of CuO nanoparticles into ZnO nanomaterials investigated on their antimicrobial behavior. For this purpose, CuO with a variety ratio doped on the ZnO nanoparticles, and then Ag entrapped by impregnation methods. Minimum inhibitory concentration (MIC) measurements were carried out to measure the antimicrobial behavior of ZnO(Ag) and mixed hybrid Zn 1-x Cu x O(Ag) towards gram-negative and gram-positive bacteria and fungus. gram-positive and gram-negative bacteria were generally more sensitive to ZnO and CuO nanoparticles, respectively. Then, these hybrid nanomaterials can be an excellent candidate for both gram-positive and gram-negative bacteria.


Introduction
Nanotechnology is one of the most important areas that has been consideration recently because it provides the link between nanotechnology and nanobiotechnology. However, the combination of nanotechnology with nanobiotechnology leads to the development of new materials for therapeutic and diagnostic applications [1][2][3].
Besides, nanomaterials have numerous applications containing fuel cell, solar energy transformation, photonics, catalysis, optical biosensor, and photocatalysis that can be related to their unique physicochemical properties [4][5][6][7]. Moreover, these nanoporous materials are good candidates for antimicrobial agents. Metal oxides like inorganic nanomaterials have the properties of higher stability, low cost, and lower toxicity to human cells than other organic compounds [8][9][10].
Also, the problem of increasing microbial resistance to existing antibiotics makes inorganic materials similar to nanostructured metal oxides a worthy candidate for dissolve this problem [11].
The inhibition mechanism of nano-inorganic materials is through the production of toxins that enhanced oxidative stress and anti-in ammatory products, following in cell death. On the other hand, metal nanoparticles produce metal ions that capable of interaction with charged bacterial cell walls follow by the deactivation of cellular enzymes and lead to cell death [12][13][14][15]. Some nanometal oxides containing TiO 2 , MgO, ZnO, CuO, and Fe 3 O 4 nanoparticles were investigated as potential antimicrobial agents. Also, metal nanoparticles especially Ag nanoparticles were assigned more attention due to their exclusive biomedical properties such as wound dressing, cardiovascular implants, coating contact lenses, bone cement, bandages, and endodontic lling materials [16]. However, a mixed multi-metal oxide containing Ag/Fe 3 O 4 , Zn x Mg 1-x O, and Ta-doped ZnO has been studied as antimicrobial agents due to their synergic effects that reduce the high toxicity of alone nanoparticles [15,17].  [15].
Here, we aimed to report the preparation of the binary and ternary nanocomposite as antibacterial agents.
This procedure achieves by modifying ZnO with CuO and Ag nanoparticles using the co-precipitation and impregnation method, respectively. After that, Ag + reduced by the use of hydrazine and obtained the new nanocomposite materials. CuO and ZnO showed antibacterial behavior against gram-negative and grampositive bacteria, respectively. However, based on the formation of nanocomposite materials, synergic antibacterial properties against both gram-negative and gram-positive bacteria were observed. The effect of the ZnO/CuO ratio on antibacterial activity of the nano-composite has been investigated. However, Ag nanoparticles cause increasing in antibacterial activity. But it is expensive and high toxicity, but by inclusion in hybrid nanomaterials, the toxicity property reduced, and it appropriate from toxicity and economic view due to low loading. Besides, by the combination of them, the synergic effect of antibacterial properties appeared.

Materials and Instruments
Materials commercially graded and purchased from Sigma-Aldrich, Fluka, and Merck. FT-IR spectra were recorded in the range 400-4000 cm −1 by a Thermo Nicolet Iso10 spectrometer by KBr pellets. UV-Vis spectrum detected by Shimadzu 1800 instrument. The morphology of the catalysts was studied using Field emission scanning electron micrographs (FESEM), which were recorded on a MIRA3 by Tuscan Co. To investigate catalysts from the structural view, a Brucker-D8 advance X-ray diffraction device with Cu-K a radiation at λ=1.54 Å, v=40 kV, and I=30 mA in a range of 60-5° in ambient temperature detected. Azar Furnaces CVD apparatus was used for chemical deposition by a thermal method.

Preparation of ZnO nanoparticles
The preparation of ZnO nanoparticles performed by the wet-chemical method. For this purpose, a certain amount of Zn(NO 3   causes the production of inert media, therefore, it prevents re-oxidation of Ag [22]. However, hydrazine amount is used as stoichiometry until the reduction of Ag occurs not more. Crystallinity and purity of the hybrids metal oxide NPs investigated by using XRD patterns. The marked diffraction peaks in the XRD pattern shows the polycrystalline nature of hybrid nanomaterials. Fig.1 shows the XRD pattern of mixed-metal oxide nanoparticles with the various ratio of Zn, Cu, and Ag. Fig.1a is the XRD pattern of ZnO(Ag) that indicated a peak in accordance with ZnO and Ag phases. Re ections according to Ag proved the formation of Ag nanoparticles into metal oxide. These re ections were appeared at 2q=38. 11  However, the intensity of peaks in concern to organic compounds in ZnO (Ag) nanoparticles reduced that it can be related to the removing organic templates based on calcination.
The peaks concerning metallic Ag and Ag-Ag metallic bands establish below 400 cm -1 which cannot be seen in this spectrum because it is out of device range [11]. However, the sharp and strong peak between 400-600 cm -1 are according to ZnO nanoparticles overlapped with organic materials. Bands in relation to 472 and 515 cm -1 assigned to ZnO stretching vibration [23][24][25].
By inclusion CuO into the ZnO structure using the isomorphous substitution method, peaks in the region of 530-664 cm -1 appeared by CuO nanoparticles [26]. Based on increasing CuO content, its intensity increased, and the peak intensity of ZnO reduces which is an expected outcome and accompanied by slight shifting in peak location (Fig.2b). The FESEM images of ZnO(Ag) and Zn 1-x Cu x O(Ag) nanoparticles revealed the polycrystalline structure (Fig.3). The presence of small crystallites with various sizes proved the formation of more than one phase and one crystalline structure. Furthermore, the morphology of ZnO(Ag) is different from Zn 1-x Cu x O(Ag). ZnO(Ag) shows distinguished spherical morphology, although there are some nanoparticles with random form, and these results are concluded by others [27] that achieved by entering CuO and Ag in ZnO structure by isomorphism substitution and impregnation method, respectively.

Investigation of antimicrobial behavior of Zn 1−x Cu x O(Ag) nanoparticles
Antibacterial activity of nanoparticles has been demonstrated by the MIC method (Table 1) nanoparticles for Escherichia coli. This is an expected result because CuO nanoparticles by affecting sul de groups disturb enzyme function and inhibition of bacterial growth [28]. Therefore, there is a better antibacterial behavior against gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and the Candida Kefyr fungal sample. Moreover, ZnO nanoparticles destroy the lipid the wall and protein of the cell membrane of the bacterium by producing H 2 O 2 or electrostatic interaction between the bacteria and nanoparticles, which followed by the leakage of intracellular contents and ultimately the death of gramnegative bacterial cells, because the walls of bacterial cells of gram-negative is thinner than grampositive [29,30]. Nevertheless, ZnO (Ag) showed less antibacterial activity than Zn 1-x Cu x O(Ag). It can be related to the size of ZnO which is reduced based on replacement with Cu and their antibacterial behavior increases [8]. Moreover, by introducing metallic silver based on its antibacterial nature, antibacterial behavior is superior against the gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and the Candida Kefyr fungus than gram-negative bacterial. Then, by selecting the hybrid of nanoparticles we can tune the properties of NPs toward target particular virus/bacteria/microorganism, and therefore, we can achieve the tailor the sensitivity to a particular species.

Conclusions
The present study proposes the controlling of nanoparticle properties by changing their various ratios of ZnO and CuO. The change in reaction conditions led to the formation of different ratios of Zn 1-x Cu x O(Ag) NPs. These results con rm by the XRD pattern. As the amount of ZnO reduces, the intensity of XRD peaks in accordance with it decreases and appeared peaks in relation to the CuO. In addition, peaks in relation Page 7/12 to the Ag nanoparticles appear in whole the samples due to the loading of Ag. By increasing in ZnO content, the antibacterial activity toward gram-positive increased, however in nanohybrid materials with a high loading of CuO, gram-negative antibacterial activity is enhanced. It is an acceptable result, because ZnO affected on the cell wall of the gram-positive bacterial. Besides, CuO via affecting on enzyme function causes cell death. Nevertheless, the presence of Ag nanoparticles affected both gram-positive and gram-negative bacteria and inhibition of the growth of the bacteria and fungus.

Declarations
Funding Not applicable

Competing interests
The authors declare that they have no competing interests.

Availability of data and material
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval
Authors declare this manuscript The authors con rm the submission is original and they approved this manuscript is not under consideration for publication elsewhere.

Consent to participate
The authors are aware from their contribution in this paper.