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Synthesis and Investigation of Antimicrobial Activity of [Cu.sub.2]O Nanoparticles/Zeolite.

1. 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.sub.2]O), are among the most widely used as antimicrobial agents for their high efficacy towards a broad spectrum of microorganisms [2-4]. There has been also considerable attention throughout the years for [Cu.sub.2]O as a candidate material for photovoltaics, photocatalytic degradation of organic pollutants and decomposition of water into [O.sub.2] and [H.sub.2] under visible light, and even as a negative electrode material for lithium ion batteries [5-7]. According to Wick and Tilley [8], [Cu.sub.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.sub.2]O crystals could inhibit the growth of E. coli efficiently, and their bactericidal activities become stronger with increased [Cu.sub.2]O concentration. Up to 85% E. coli are killed in the presence of [Cu.sub.2]O particles with concentration of 25 [micro]/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.sup.2+]-exchanged zeolite with sodium hydroxide and calcification at 350[degrees]C for 2h was studied by Razavi and Loghman-Estarki [12]. In the present study, [Cu.sub.2]O NPs were synthesized within the zeolite A framework using CuS[O.sub.4] as copper precursor and hydrazine hydrate ([N.sub.2][H.sub.4] x [H.sub.2]O) as reducing agent. The antibacterial activity of the obtained [Cu.sub.2]O NPs/zeolite against Escherichia coli (E. coli) was also investigated.

2. Methods

2.1. Materials and Chemicals. Analytical grade CuS[O.sub.4] x 5[H.sub.2]O, N[H.sub.4]OH (25%), and [N.sub.2][H.sub.4] x [H.sub.2]O (80%) were products from Merck, and industrial grade sodium type of zeolite A with chemical formulation [Na.sub.2]O x [Al.sub.2][O.sub.3]. 2Si[O.sub.2] x 4.5[H.sub.2]O was a product from Shangdong Aluminium Corporation (SALCO, China). The Luria-Bertani (LB) medium for bacterial incubation was purchased from Himedia, India. The strain of E. coli ATCC 6538 was provided by the University of Medical Pharmacy, Ho Chi Minh City. Distilled water was used in all experiments.

2.2. Exchange of [Cu.sup.2+] Ions in Zeolite. 1kg of zeolite was suspended into a glass beaker containing 1.5 L water. Then, HN[O.sub.3] 2 N and water were added to zeolite suspension mixture for neutralization to pH ~6.5 and for final volume of about 4L. 0.42 kg of CuS[O.sub.4] x 5[H.sub.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.sup.2+] into zeolite. Before adding hydrazine, pH of the [Cu.sup.2+]/zeolite mixture was adjusted by ammonia water to ~7.5.

2.3. Reduction of [Cu.sup.2+] to [Cu.sub.2]O NPs in Zeolite. A freshly prepared 250 mL hydrazine 20% (w/v) solution was added dropwise to the above [Cu.sup.2+]/zeolite under stirring for 2h at ambient temperature. Then, reduction reaction was stopped and let standing overnight for [Cu.sub.2]O NPs/zeolite settling down. Finally, [Cu.sub.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[degrees]C till to constant weight.

2.4. Characterization of [Cu.sub.2]O NPs/Zeolite. The content of copper in [Cu.sub.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.sub.2]O NPs/zeolite product was carried out on D8 Advance Bruker, Germany, and the [Cu.sub.2]O NPs sizes were measured using a transmission electron microscope (TEM; JEM 1010, JEOL, Tokyo, Japan). The presence of copper in [Cu.sub.2]O NPs/zeolite was also assessed by energy-dispersive X-ray spectroscopy (EDX) on a JEOL 6610 LA.

2.5. Antibacterial Activity of [Cu.sub.2]O NPs/Zeolite. In vitro test of bactericidal activity of [Cu.sup.2+]/zeolite and [Cu.sub.2]O NPs/zeolite against E. coli was carried out following the procedure as described in [13-15]. 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 ([eta]) was calculated using the equation: [eta](%) = ([N.sub.o] - N) x 100/[N.sub.o], where [N.sub.o] and N are the survival numbers of bacteria in the control and studied samples, respectively.

3. Results and Discussion

3.1. Characterization of [Cu.sub.2]O NPs/Zeolite. Figure 1 showed the photograph of zeolite, [Cu.sup.2+]/zeolite, and [Cu.sub.2]O NPs/zeolite. It is clearly observed from Figure 1 that [Cu.sub.2]O NPs/zeolite is reddish color. The reaction of hydrazine used as reducing agent can be expressed by

[N.sub.2][H.sub.4] + 4O[H.sup.-] [right arrow] [N.sub.2] + 4[H.sub.2]O + [4e.sup.-] (1)

Dimitrijevic 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.sub.2]O NPs can be described in the following reactions:

2Cu [(OH).sub.4.sup.2-] + [N.sub.2][H.sub.4] + 2O[H.sup.-] [right arrow] [Cu.sub.2]O + [N.sub.2] + 5[H.sub.2]O + 4O[H.sup.-] (2)

[Cu.sub.2]O + [Cu.sub.2]O + [Cu.sub.2]O + ... [right arrow] [Cu.sub.2]O nano (3)

TEM images of zeolite and [Cu.sub.2]O NPs/zeolite in Figure 2 indicated that the size of [Cu.sub.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.sub.2]O NPs in zeolite were almost as wormlike form. These [Cu.sub.2]O NPs seem to be linked together within the intercages of zeolite structure.

The XRD pattern of [Cu.sub.2]O NPs/zeolite A in Figure 3 showed that diffraction peaks appeared at 29.61[degrees], 36.48[degrees], 42.38[degrees], 61.46[degrees], 73.56[degrees], and 7752[degrees] corresponding to (110), (111), (200), (220), (311), and (222) planes of cuprite, respectively, that indicated the formation of [Cu.sub.2]O nanocrystals (JCPDS Card number 05-0667) [4, 18]. The average crystalline size of [Cu.sub.2]O NPs determined by taking the full width at half maximum (FWHM) of the most intense peak at 36.48[degrees] 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[degrees], 50.43[degrees], and 74.25[degrees] that assigned for Cunanocrystals (JCPDS Card number 04-0836) [4, 12]. Thus, during reduction reactions, a certain small amount of Cu could be formed in the following reaction:

2[Cu.sup.2+] + [N.sub.2][H.sub.4] + 4O[H.sup.-] 2[Cu.sup.o] + [N.sub.2] + 4[H.sub.2]O (4)

According to Giannousi et al. [4] and Xia et al. [19], [Cu.sub.2]O should be also formed due to the disproportionation reaction of Cu and [Cu.sup.2+]:

[Cu.sup.o] + [Cu.sup.++] + 2O[H.sup.-] [right arrow] [Cu.sub.2]O + [H.sub.2]O (5)

It seems that not all resultant Cu reacts with [Cu.sup.2+]. So that small trace of Cu crystal could be contained in the [Cu.sub.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.sub.2]O NPs mixture was dark yellowish that was different from black color of CuO [20] and reddish color (or brick color) of [Cu.sub.2]O (Figure 1). According to Arshadi-Rastabi et al. [21], [Cu.sub.2]O NPs have various ranges of different colors, namely, yellow, orange, red, and brown color.

The energy-dispersive X-ray (EDX) spectra in Figure 4 showed that the composition of zeolite A consists of four main elements, particularly silicon, aluminum, oxygen, sodium, and a small amount of potassium, but without any trace of copper. After exchange with [Cu.sup.2+] and reduction of [Cu.sup.2+] to [Cu.sub.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.sub.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.sup.2+] by hydrazine) could not influence the total copper content in the final product ([Cu.sub.2]O NPs/zeolite).

Figure 5 presented the brief schematic diagram of the synthesis procedures of [Cu.sub.2]O NPs/zeolite by chemical reduction method using hydrazine hydrate.

3.2. Antibacterial Activity of[Cu.sub.2]O NPs/Zeolite. The antibacterial activity of [Cu.sup.2+]/zeolite and [Cu.sub.2]O NPs/zeolite with copper concentration of 150 mg/L was presented in Figure 6 and Table 1. The obtained result indicated that the antibacterial efficiency of both [Cu.sup.2+]/zeolite and [Cu.sub.2]O NPs/zeolite against E. coli was of 98.15 and 96.19%, respectively.

Most importantly, according to Li et al. [24], the [Cu.sub.2]O NPs have low cytotoxicity. They also reported that the antibacterial efficiency of [Cu.sub.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.sup.2+]/zeolite and [Cu.sub.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.sup.2+]/zeolite can be occurred through two possible pathways as proposed by Hu et al. for [Cu.sup.2+]/montmorillonite [25]. The first one is the adsorption of bacterial cells on the surface of the [Cu.sup.2+] carriers and the second, [Cu.sup.2+] ions dissociated from [Cu.sup.2+]/carriers directly interact with bacterial cells. For copper-based nanoparticles (Cu, CuO, and [Cu.sub.2]O nanoparticles), an elusive question remains whether the release of [Cu.sup.2+] ions from the copper-based nanoparticles contributes to the toxicity of these nanomaterials of the toxicity exerted from the nanoparticles itself [26]. Gu et al. [27] studied the effect of [Cu.sub.2]O NPs/montmorillonite on damage and removal of M. aeruginosa algae. The obtained results indicated that the antialgae effect mainly depended on [Cu.sup.2+] ions extent released from [Cu.sub.2]O and [sup.*]OH, [O.sub.2.sup.*-] radicals generated from photocatalysis activity of [Cu.sub.2]O NPs, which were responsible for the damage to cellular structure, and physiological activity led to death of algae cells.

Furthermore, the acute toxicity of CuS[O.sub.4] x 5[H.sub.2]O for rats showed that the oral L[D.sub.50] was 234 mg/kg body weight while oral L[D.sub.50] of [Cu.sup.2+]-exchanged montmorillonite with copper content of 25g/kg was of 18g/kg body weight [25]. This result indicated that [Cu.sup.2+] -exchanged montmorillonite was a toxicity-free substance for rats. Thus, it can be inferred that [Cu.sup.2+]/zeolite and also [Cu.sub.2]O NPs/zeolite are less toxic than [Cu.sup.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.sub.2]O NPs/zeolite would be more potential. In addition, [Cu.sub.2]O NPs/zeolite product can be favorably produced on large scale by this process. The antifungal activity of [Cu.sup.2+] /zeolite and [Cu.sub.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.

4. Conclusions

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

Competing Interests

The authors declare that they have no competing interests.

Authors' Contributions

Nguyen Quoc Hien came up with the idea. Dang Van Phu and Le Anh Quoc designed and set up the experimental procedure. Dang Van Phu and Bui Duy Du planed the experiments and agreed with the paper's publication. Le Anh Quoc conducted the sizes measurement of as-prepared [Cu.sub.2]O nanoparticles by EDX, TEM, and XRD. Nguyen Quoc Hien and Le Anh Quoc evaluated the antibacterial efficiency of as-synthesized copper products. Nguyen Quoc Hien, Bui Duy Du, and Dang Van Phu analyzed the data, drafted the manuscript, and revised the manuscript critically. All authors read and approved the final manuscript.


The authors would like to thank Mr. NQ Nghi and Mr. NQ Thuy for their assistance during experimental works.


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Bui Duy Du, (1) Dang Van Phu, (2) Le Anh Quoc, (2) and Nguyen Quoc Hien (2)

(1) Institute of Applied Material Science, Vietnam Academy of Science and Technology, 1 Mac Dinh Chi Street, District 1, Ho Chi Minh City, Vietnam

(2) Research and Development Center for Radiation Technology, Vietnam Atomic Energy Institute, 202A Street 11, LinhXuan Ward, Thu Duc District, Ho Chi Minh City, Vietnam

Correspondence should be addressed to Nguyen Quoc Hien;

Received 13 September 2016; Revised 24 November 2016; Accepted 7 December 2016; Published 9 January 2017

Academic Editor: Raphael Schneider

Caption: Figure 1: Photograph of zeolite (a), [Cu.sup.2+]/zeolite (b), and [Cu.sub.2]O NPs/zeolite (c).

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

Caption: Figure 3: XRD patterns of zeolite A and [Cu.sub.2]O NPs/zeolite A.

Caption: Figure 4: EDX spectra of zeolite and [Cu.sub.2]O NPs/zeolite.

Caption: Figure 5: Brief schematic diagram of [Cu.sub.2]O NPs/zeolite synthesis procedures.

Caption: Figure 6: Photograph of the growth of E. coli on LB agar plates: (a) control, (b) [Cu.sup.2+]/zeolite, and (c) [Cu.sub.2]O NPs/zeolite.
Table 1: The antibacterial efficiency ([eta]) of [Cu.sup.2+]/zeolite
and [Cu.sub.2]O NPs/zeolite.

Sample                     E. coli, CFU/mL    [eta], %

Control                   1.13 x [10.sup.8]      --
[Cu.sup.2+]/zeolite       2.09 x [10.sup.7]    98.15
[Cu.sub.2]O NPs/zeolite   4.30 x [10.sup.7]    96.19
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Title Annotation:Research Article
Author:Du, Bui Duy; Van Phu, Dang; Quoc, Le Anh; Hien, Nguyen Quoc
Publication:Journal of Nanoparticles
Article Type:Report
Date:Jan 1, 2017
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