Nano copper oxide and nano zinc oxide (Cu2O-ZnO) have high antibacterial activity, broad spectrum
In this study, nano copper oxide and nano zinc oxide (Cu2O-ZnO) with high antibacterial activity were synthesized through sol-gel method. The influence of heating temperature and time on the formation of Cu2O-ZnO composite has been emphasized. The most suitable calcination conditions were found to be 500 o C and 2 hours. The synthesized Cu2O-ZnO nanocomposites were characterized by powder X-ray diffraction, BET nitrogen adsorption isotherm, field-emission scanning electron microscopy and transmission electron microscopy, and evaluated the viability. Its antibacterial activity against bacteria that cause disease in humans such as Staphylococcus aureus (gram-positive bacteria) and Escherichia coli (gram-negative bacteria). The results show that the particles of Cu2O-ZnO complex exist in spherical shape with a wide variation in size from 10-60 nm. Cu2O-ZnO nano copper oxide and nano zinc oxide compounds have high antibacterial activity against both gram-positive (S. aureus) and gram-negative (E. coli) bacteria with values of maximum inhibitory concentrations. Minimum (MIC) of 0.16 mg.mL-1 and 1.25 mg.mL-1 for S. aureus and E. coli, respectively. The stability of the antibacterial activity of the sample was also investigated. The antibacterial activity of the Cu2O-ZnO nanocomposites was reduced after 45 days when stored at room temperature in a container without a lid.
(Copyright by NanoCMM Technology)
INTRODUCTION
Recently, the rapid development of nanotechnology with controlled particle size and shape has led to many new results on antibacterial materials being published. There are several studies that have demonstrated that nanomaterials have high antibacterial activity at low concentrations (Balta et al., 2012; Chen et al., 2012; Xu et al., 2016; Almutairi, 2019). Among nanoparticles, nano metal oxides with antibacterial properties are promising materials for medical, textile or UV protection applications. The antimicrobial efficacy of nanomaterials depends on their shape, size, chemical composition or concentration (Negi et al., 2012). There are many antimicrobial materials that have been studied such as transition metal Ag (Chen et al., 2017; Chatterjee et al., 2014), Cu (Chatterjee et al., 2014; Pham and Lee, 2014), Au (Chatterjee et al., 2014; Pham and Lee, 2014). Sharma et al., 2009) and their oxides are ZnO (Li et al., 2017; Haghighi et al., 2011; Chauhan et al., 2015), TiO2 (Li et al., 2016), and CeO2 (Lu et al., 2014). Some metal oxide nanoparticles such as CuO, ZnO, TiO2 are well recognized for their good inhibition of bacteria and fungi (Jin et al., 2017; Hamza and AlSolami, 2018). While many studies have focused on the antibacterial properties of a mixture of Ag, Zn and Cu nanometals to enhance the antibacterial properties of individual metals (Chen et al., 2017; Pham and Lee, 2014; Ren et al., 2018). ZnO is a cheap, non-toxic and highly antibacterial semiconductor with low concentrations, even in the absence of light (Jin et al., 2017; Ma et al., 2014). Several previous studies have demonstrated that ZnO can kill both gram-negative and positive bacteria such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis (Jiang et al., 2009). Besides, nano copper oxide with high efficiency, wide imaging and savings (Wu et al., 2019) are a promising material for solar energy conversion, catalysis and antibacterial (Hassan et al. , 2004; Ruparelia et al., 2008; Santo et al., 2010; Ferhat et al., 2009).
However, some bacteria cannot be inhibited by Cu2O or ZnO individually. Therefore, a mixture of Cu2O and ZnO is not only expected to enhance its antibacterial properties, but also can be improved both the functions of Cu2O and ZnO. ZnO has been known for its strong thermochemical stability and resistance to microbial activity, even in the absence of sunlight (Zhang et al., 2008). In addition, the advantages of Cu2O combined with ZnO are easy incorporation and improved antibacterial properties compared with other metal oxide materials (Hong et al., 2017). Besides, both these materials are quite cheap and environmentally friendly. In addition, there is little literature focusing on the synthesis as well as the antibacterial properties of Cu2O/ZnO nanoparticles. Compounds of two metal oxides can be synthesized by various methods such as electrolysis (Sharma et al., 2009), hydrothermal process (Li et al., 2017), electrospinning (Haghighi et al., 2017). et al., 2011) and co-precipitation (Chauhan et al. et al., 2015), and many previous studies have synthesized nanoparticle materials of metal oxides by sol-gel method (Stoyanova et al., 2011; Shalaby et al., 2015; Lee et al., 2005; Amin et al., 2009; Zhang and Chen, 2009) because of the uniform nanoscale size and low energy consumption. In this study, the Cu2O-/ZnO nanocomposite was synthesized by sol-gel method with different calcination conditions and its physico-chemical properties and antibacterial activity against Escherichia coli and Staphylococcus aureus (wr. E. coli and S. aureus for short) were tested. The results have high potential for applications in many fields such as medical or building materials.
EXPERIMENT
To synthesize a mixture of nano copper oxide and nano zinc oxide Cu2O-ZnO, 23.76 grams of Zn (NO3) .6H2O (Xilong, > 99%) were dissolved in distilled water, then 37.80 grams of oxalic acid were added. (Merck, > 99%) and heated to about 80 °C by the mixture and stirred until a clear solution was obtained. In addition, 4.84 grams of Cu(NO3).3H3O (Xilong, > 99%) was also dissolved in 11 mL of ethylene glycol (Xilong, > 99.8%) and dropped into the solution first, then distilled water was added. Add to this mixture to get to 100 mL completely by constant stirring and the solution is light blue in color. The solution was kept at 80 °C for 2 h, after which the solution was converted to a gel state and the temperature was increased to reach a slurry. The mixture was dried at 200°C for 2 h and converted to an ash mixture. The mixture was then calcined in a stream of pure N2 (3 L.h-1) at different temperatures (450 oC, 500 oC and 550 oC) with a heating rate of 10 °C. min ‒ 1 for time intervals. different times (1, 2 and 3 hours) to obtain Cu2O-ZnO composites. The final sample was ball milled for 12 h and a very fine powder of the product was obtained for other analyses. The obtained samples are denoted as Cu-Zn-T-t, where T and t represent the heating temperature (oC) and time (hours), respectively. The properties of the obtained Cu2O-ZnO nanocomposites and nanozinc oxide nanocomposites were investigated by thermogravimetric analysis (TGA, Setaram LABSYS Evo TG-DSC 1600C), powder X-ray diffraction (XRD, Bruker D2 Phaser), BET nitrogen adsorption isotherms (Nova 2200e instrument), scanning electron microscope (SEM, Hitachi S4800) and transmission electron microscopy (TEM, Jeol Jem 1400) field emission. The Cu2O-ZnO nanocomposite synthesized by the most suitable procedure was tested for antibacterial activity against E.coli ATCC 25922 and S. aureus ATCC 43300 (MRSA). To test the minimum inhibitory concentration of Cu2O-ZnO against the two bacteria, different concentrations of Cu2O-ZnO (N, N/2, N/4, N/8, N/16, N/32) , N/64, N/128, N/256 and N/512 with N being the initial concentration of the solution, N = 20 mg.mL-1) were prepared by diluting the Cu2O/ZnO solution with water. deionized. Then, the diluted samples were mixed with sterile nutrient agar. Using sterile rods, the standard inoculum of each selected bacterium with 1.5 × 10^7 CFU.ml-1 was inoculated on agar plate mixed with low to high concentrations of Cu2O-ZnO samples. A sterile nutrient agar plate was not mixed with nano copper oxide and nano zinc oxide Cu2O-ZnO as a control (Wayne, 2013). Each bacterial strain was inoculated at three points on the plate with the same position on the plates. Finally, the plates were incubated at 37 °C for 24 h. The lowest concentrations of nanocopper oxide and nanozinc oxide Cu2O-ZnO that inhibited bacterial growth tested were considered minimum inhibitory concentrations (MICs) (Washington and Wood, 1995).
RESULTS AND DISCUSSION
To determine the calcination temperature range, the ash sample was dried at 170 – 200 °C within 2 h after the sol-gel synthesis was tested by TGA. The results in Figure 1 show that from 50 °C to 400 °C the total mass loss is about 30%, which is mainly due to the evaporation of water and the ethylene glycol remaining in the ash sample. But in the second stage at about 407 oC to 450 oC, a significant mass reduction of about 35% was observed due to the decomposition of NO3- in the ash sample to yield metal oxides forming Cu2O-ZnO mixtures. . Thereafter, the mass of the sample remained almost unchanged when the temperature was raised above 450 °C, indicating that the structure of the product is stable. So the firing temperature will be served from 450 oC in this process. The XRD patterns of the samples calcined at 450 C, 500 oC and 550 oC within 2 h are shown in Figure 2. The results show 2 theta values of the major peaks of ZnO at 31.88, 34 .55o, 36.37o, 56.63o and 68.04o (JCPDS PDF #800075), Cu2O main peaks at 31.88o, 36.37o, 43.41o, 62.90o (JCPDS #782076), the Cu’s letter peaks are at 43.41o, 50.51o, 74.13 (JCPDS #040836), respectively. As the annealing temperature of the samples was increased, the intensity of the characteristic diffraction peaks of Cu2O and ZnO also gradually increased, which showed an improvement in the crystallinity of different particles. But there is no significant difference between 500 oC and 550 oC results, so the heating temperature of 500oC is chosen to save energy. Figure 3 shows the XRD patterns of the samples prepared at a calcination temperature of 500oC and annealing time of 1, 2 and 3 h, respectively. All XRD results show characteristic diffraction peaks of Cu2O and ZnO. But the main peaks of these compounds were higher and sharper since the 2 h calcination time. The BET result of the composite sample prepared at a calcination temperature of 500 oC and a calcination time of 2 hours is 67.7 m2/g, which is higher than the study results (Shi et al., 2011; Wang). et al., 2007). Samples with the same synthesis conditions were used to obtain SEM and TEM . results
The morphology and particle size of Cu2O-ZnO nano copper oxide and nano zinc oxide determined by SEM and TEM are shown in Figure 4. From the SEM image (Figure 4a), it can be seen that the Cu2O- composite ZnO exists in the form of nanoparticles with a diameter of 20 – 50 nm. bonded to the surface. TEM results (Figure 4b) revealed the presence of spherical particles with a size of about 15 – 60 nm. Figure 5 shows the inhibition zones of S. aureus and E. coli treated with solutions of Cu2O-ZnO nanocomposites at different concentrations. It was observed that the bacterial exponential phase was delayed in the presence of Cu2O-ZnO nanocomposites, and this phenomenon became more evident as the nanocomposite concentration increased. The Cu2O-ZnO nanocomplex can delay the exponential phase of both S. aureus and E. coli bacteria and can completely inhibit bacterial growth at the MIC value of 0.16 mg/mL (N). / 128) and 1.25 mg/mL (N/16) for S. aureus and E. coli, respectively. The antibacterial effect of nano copper oxide and nano zinc oxide was different against gram-positive and gram-negative bacteria. That is, it showed better antibacterial activity against S. aureus (gram positive) than against E. coli (gram negative). This phenomenon can be explained that on the surface of ZnO nanoparticles, gram-positive bacteria are more easily inhibited than gram-negative bacteria. In more detail, growth inhibition for gram-negative bacteria occurred at higher concentrations of ZnO (Hu et al., 2012). In the composition of Cu2O-ZnO nanocomposites, the Zn/Cu ratio is high. Therefore, the results of this work are consistent with the results obtained by the authors (Sirelkhatim et al., 2015; Reddy et al., 2007) who studied antibacterial activity against S. aureus and E. coli on ZnO nanoparticles. But, Cu2O-ZnO nanocomposites of copper oxide and zinc oxide nanocomposites had much higher antibacterial activity against both types of bacteria than ZnO nanoparticles. Compared with the Ag-ZnO nanocomposite, its antibacterial activity against S. aureus was almost equivalent, while that against E. coli was lower (see Table 1). However, in terms of economy, nano copper oxide and nano zinc oxide (Cu2O-ZnO nanocomposite) has a distinct advantage. To evaluate the stability of the antibacterial activity, the minimum inhibitory concentration for S. aureus on the Cu2O-ZnO nanocomposite that the sample was evaluated as the result after being stored at room conditions in an airtight cabinet. covered for 45 days. The results obtained in Figure 6 show that the MIC of samples stored after 45 days at room conditions is 0.63 mg.mL-1 (N/32) with S. aureus. This value is lower than that of the original sample, which is 0.16 mg.mL-1. This could be explained by the fact that the samples were inactivated by moisture and possibly partially oxidized by oxygen in the air (Kurapov et al., 2018; Yu et al., 2011).
CONCLUSION
In conclusion, Cu2O-ZnO nano copper oxide and nano zinc oxide nanocomposites were successfully synthesized through a sol-gel method using metal nitrate and ethylene glycol. In which, ethylene glycol acts as a solvent as well as a ligand reactant in the synthesis of nanocomposites. The proposed calcination mode to obtain Cu2O-ZnO nanocomposites is 500 oC for 2 h. The best one has a uniform structure with a particle size of less than 60 nm and a surface area of 67.7 m2. G-1 exhibits high antibacterial activity and stability against pathogenic bacteria in humans. It could be a great cheap and antibacterial material with great utility in practical applications.