Synthesis and Investigation of Antimicrobial Activity of Cu2O Nanoparticles/Zeolite

Cuprous oxide (Cu 2 O) nanoparticles in zeolite A were synthesized by two steps: (i) ion-exchange of copper ions into the zeolite and (ii) reduction of copper ions in cages of the zeolite by hydrazine hydrate in base medium. The Cu 2 O nanoparticles/zeolite product was characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDX). The particle size of Cu 2 O nanoparticles was of 40 nm. The antibacterial activity of the as-synthesized Cu 2 O nanoparticles/zeolite against Escherichia coli was also investigated. Cu 2 O NPs/zeolite product can be favorably produced on large scale for water treatment and agricultural application as antimicrobial agent.


Introduction
Metal nanoparticles (NPs) have attracted considerable attention because of their unique properties such as catalytic, magnetic, optical, biological, and electrical properties.Among metal NPs, Au and Ag NPs have been most extensively studied and applied due to their stability and suitability to handle in air [1].Recently, Cu NPs have been expected to be a good choice of the next-generation NPs mainly because of low cost [2].In addition, Cu oxide NPs, namely, cupric oxide (CuO) and cuprous oxide (Cu 2 O), are among the most widely used as antimicrobial agents for their high efficacy towards a broad spectrum of microorganisms [2][3][4].There has been also considerable attention throughout the years for Cu 2 O as a candidate material for photovoltaics, photocatalytic degradation of organic pollutants and decomposition of water into O 2 and H 2 under visible light, and even as a negative electrode material for lithium ion batteries [5][6][7].According to Wick and Tilley [8], Cu 2 O is a promising material with the capacity for low cost, large-scale solar energy conversion due to the abundant nature of copper and oxygen, suitable bandgap of visible light, and effective, low energy intensity fabrication process.The antimicrobial activity of copper has long been recognized.However, relatively few studies have been focused on the antimicrobial properties of Cu oxide NPs [4].Huang et al. [3] reported that the inhibition efficiency of the 500-750 mg/kg CuO NPs against tomato early blight A. solani was 70.7-80.7%;it is better than that of the 50% carbendazim.Furthermore, the inhibition effect of the 500 mg/kg CuO NPs on pepper root rot pathogen was significantly lower than that of the 75% chlorothalonil but higher than that of the 50% carbendazim.However, the CuO NPs showed no inhibitory effect on vegetable B. cinerea pathogen.According to Ren et al. [9], both octahedral and cubic Cu 2 O crystals could inhibit the growth of E. coli efficiently, and their bactericidal activities become stronger with increased Cu 2 O concentration.Up to 85% E. coli are killed in the presence of Cu 2 O particles with concentration of 25 /mL.Zeolite A is microporous aluminosilicate material with small pore size and high absorptivity commonly used as a commercial adsorbent in gas purification and ion-exchange separation [10].Zeolite A is also used as catalyst, as molecular sieve, in the production of laundry detergents, in agriculture purposes for the preparation of advanced materials and recently to produce the nanocomposites [11].Synthesis of CuO NPs within zeolite Y by reaction of Cu 2+ -exchanged zeolite with sodium hydroxide and calcification at 350 ∘ C for 2 h was studied by Razavi and Loghman-Estarki [12].In the present study, Cu 2 O NPs were synthesized within the zeolite A framework using CuSO 4 as copper precursor and hydrazine hydrate (N 2 H 4 ⋅H 2 O) as reducing agent.The antibacterial activity of the obtained Cu 2 O NPs/zeolite against Escherichia coli (E.coli) was also investigated.2+ Ions in Zeolite. 1 kg of zeolite was suspended into a glass beaker containing 1.5 L water.Then, HNO 3 2 N and water were added to zeolite suspension mixture for neutralization to pH ∼6.5 and for final volume of about 4 L. 0.42 kg of CuSO 4 ⋅5H 2 O was dissolved in 500 mL water and then poured slowly into neutralized zeolite suspension mixture.Stirring was carried out for 2 h at ambient temperature for complete exchange of Cu 2+ into zeolite.Before adding hydrazine, pH of the Cu 2+ /zeolite mixture was adjusted by ammonia water to ∼7.5.

Reduction of Cu 2+ to Cu 2 O NPs in Zeolite.
A freshly prepared 250 mL hydrazine 20% (w/v) solution was added dropwise to the above Cu 2+ /zeolite under stirring for 2 h at ambient temperature.Then, reduction reaction was stopped and let standing overnight for Cu 2 O NPs/zeolite settling down.Finally, Cu 2 O NPs/zeolite product was filtered off using cotton fabric, washed several times with water, and dried in a forced air oven at 60 ∘ C till to constant weight.

Characterization of Cu 2 O NPs/Zeolite.
The content of copper in Cu 2 O NPs/zeolite product was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) on a Perkin-Elmer, Optima 5300 DV.X-ray diffraction (XRD) of Cu 2 O NPs/zeolite product was carried out on D8 Advance Bruker, Germany, and the Cu 2 O NPs sizes were measured using a transmission electron microscope (TEM; JEM 1010, JEOL, Tokyo, Japan).The presence of copper in Cu 2 O NPs/zeolite was also assessed by energy-dispersive X-ray spectroscopy (EDX) on a JEOL 6610 LA.

Antibacterial Activity of Cu 2 O NPs/Zeolite.
In vitro test of bactericidal activity of Cu 2+ /zeolite and Cu 2 O NPs/zeolite against E. coli was carried out following the procedure as described in [13][14][15].Briefly, the antibacterial activity of the materials was tested by culture medium toxicity method in Luria-Bertani medium.The test material samples and control were shaken in E. coli suspension at 150 rpm at room temperature for 4 h.After that, the number of viable bacteria in each mixture was determined by spread plate technique on LB agar plates.The antibacterial efficiency () was calculated using the equation:  (%) = (  − ) × 100/  , where   and  are the survival numbers of bacteria in the control and studied samples, respectively.

Results and Discussion
Dimitrijevi ć et al. reported that hydrazine hydrate has been considered as a preferred reducing agent and used for industrial scale production of silver powder for decades [16].
According to Kuo and Huang [17], in base medium the formation of Cu 2 O NPs can be described in the following reactions: TEM images of zeolite and Cu 2 O NPs/zeolite in Figure 2 indicated that the size of Cu 2 O NPs in the frameworks of the zeolite was in the range from 5 to about 30 nm.It can be also surprisingly observed in Figure 2 that, beside the black dots, some parts of the morphology of Cu  [4,18].The average crystalline size of Cu 2 O NPs determined by taking the full width at half maximum (FWHM) of the most intense peak at 36.48 ∘ using Debye-Scherrer's formula [18] was of about 40 nm.The crystalline size calculated from XRD patterns is usually bigger than that from TEM images that also happened in our previous study [14] and in this study as well.There was not any diffraction peak in XRD pattern in Figure 3 assigned for CuO nanocrystal; however, low intensity peaks also appeared at 43.36 ∘ , 50.43 ∘ , and 74.25 ∘ that assigned for Cu nanocrystals (JCPDS Card number 04-0836) [4,12].Thus, during reduction reactions, a certain small amount of Cu could be formed in the following reaction: It seems that not all resultant Cu reacts with Cu 2+ .So that small trace of Cu crystal could be contained in the Cu 2 O NPs/zeolite product as presented in XRD pattern in Figure 3. Khan et al. [18] also reported that the color of the Cu-Cu 2 O NPs mixture was dark yellowish that was different from black color of CuO [20] and reddish color (or brick color) of Cu 2 O (Figure 1).According to Arshadi-Rastabi et al. main elements, particularly silicon, aluminum, oxygen, sodium, and a small amount of potassium, but without any trace of copper.After exchange with Cu 2+ and reduction of Cu 2+ to Cu 2 O NPs, the peaks at 0.97, 8.04, and 8.93 keV appeared in EDX spectrum confirming the presence of copper in the composition of Cu 2 O NPs/zeolite with the copper content of about 9% (w/w).In addition, the copper content analyzed by ICP-AES was found to be of ∼10.5%.Demirci et al. also reported that the copper content exchanged in zeolite A was of about 10-14% (w/w) [22].In the studies of Razavi and Loghman-Estarki [12] and Ramya and Kanimozhi [23], the EDX spectrum was also used to confirm the presence of copper in zeolite.According to Razavi and Loghman-Estarki the copper content in the zeolite is only determined in step 1 (ion-exchange step) [12].Thus, the second step (reduction of Cu 2+ by hydrazine) could not influence the total copper content in the final product (Cu 2 O NPs/zeolite).Figure 5 presented the brief schematic diagram of the synthesis procedures of Cu 2 O NPs/zeolite by chemical reduction method using hydrazine hydrate.

Antibacterial Activity of Cu 2
O NPs/Zeolite.The antibacterial activity of Cu 2+ /zeolite and Cu 2 O NPs/zeolite with copper concentration of 150 mg/L was presented in Figure 6 and Most importantly, according to Li et al. [24], the Cu 2 O NPs have low cytotoxicity.They also reported that the antibacterial efficiency of Cu 2 O NPs with 4 g/L against E. coli and S. aureus could get 100% after 30 min.In our experiment with copper concentration of 500 mg/L, the antibacterial efficiency of Cu 2+ /zeolite and Cu 2 O NPs/zeolite was also attained to nearly 100% after 4 h exposure in bacterial suspension (data not shown).The antimicrobial mechanism of Cu 2+ /zeolite can be occurred through two possible pathways as proposed by Hu et al. for Cu 2+ /montmorillonite [25].The first one is the adsorption of bacterial cells on the surface of the Cu 2+ carriers and the second, Cu 2+ ions dissociated Furthermore, the acute toxicity of CuSO 4 ⋅5H 2 O for rats showed that the oral LD 50 was 234 mg/kg body weight while oral LD 50 of Cu 2+ -exchanged montmorillonite with copper content of 25 g/kg was of 18 g/kg body weight [25].This result indicated that Cu 2+ -exchanged montmorillonite was a toxicity-free substance for rats.Thus, it can be inferred that Cu 2+ /zeolite and also Cu 2 O NPs/zeolite are less toxic than Cu 2+ ion and both products can be used as antibacterial agent especially for water treatment and agricultural application.In some cases, where application fields require highly antibacterial activity and low cytotoxicity, Cu 2 O NPs/zeolite would be more potential.In addition, Cu 2 O NPs/zeolite product can be favorably produced on large scale by this process.The antifungal activity of Cu 2+ /zeolite and Cu 2 O NPs/zeolite against Phytophthora fungi that caused severe "foot rot" disease for pepper plant will be further evaluated for application in agriculture as the effective fungicides.

Conclusions
In this study, Cu 2 O NPs in zeolite A were synthesized using hydrazine hydrate as a reducing agent.Structural properties of Cu 2 O NPs were determined by XRD and TEM.The Cu 2 O NPs have fine crystal structure with particle size of about 40 nm.Results of the antibacterial activity showed that both Cu 2+ /zeolite and Cu 2 O NPs/zeolite exhibited highly bactericidal efficiency ( ∼ 96-98%).Thus, the obtained products can be used as antimicrobial agent especially for water treatment and agricultural application.

Figure 2 :
Figure 2: TEM images of zeolite A (a) and Cu 2 O NPs/zeolite A (b).

Figure 3 :
Figure 3: XRD patterns of zeolite A and Cu 2 O NPs/zeolite A.

Table 1 .
The obtained result indicated that the antibacterial efficiency of both Cu 2+ /zeolite and Cu 2 O NPs/zeolite against E. coli was of 98.15 and 96.19%, respectively.