Journal of Nanoparticles
Volume 2017 (2017), Article ID 7056864, 6 pages
https://doi.org/10.1155/2017/7056864

Research Article

Synthesis and Investigation of Antimicrobial Activity of Cu₂O Nanoparticles/Zeolite

Bui Duy Du,¹ Dang Van Phu,² Le Anh Quoc,² and Nguyen Quoc Hien²

¹Institute of Applied Material Science, Vietnam Academy of Science and Technology, 1 Mac Dinh Chi Street, District 1, Ho Chi Minh City, Vietnam
²Research and Development Center for Radiation Technology, Vietnam Atomic Energy Institute, 202A Street 11, Linh Xuan Ward, Thu Duc District, Ho Chi Minh City, Vietnam

Correspondence should be addressed to Nguyen Quoc Hien; hien7240238@yahoo.com

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

Academic Editor: Raphael Schneider

Copyright © 2017 Bui Duy Du et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Cuprous oxide (Cu₂O) 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 Cu₂O 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 Cu₂O nanoparticles was of 40 nm. The antibacterial activity of the as-synthesized Cu₂O nanoparticles/zeolite against Escherichia coli was also investigated. Cu₂O NPs/zeolite product can be favorably produced on large scale for water treatment and agricultural application as antimicrobial agent.

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 (Cu₂O), 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 Cu₂O as a candidate material for photovoltaics, photocatalytic degradation of organic pollutants and decomposition of water into O₂ and H₂ under visible light, and even as a negative electrode material for lithium ion batteries [5–7]. According to Wick and Tilley [8], Cu₂O 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 Cu₂O crystals could inhibit the growth of E. coli efficiently, and their bactericidal activities become stronger with increased Cu₂O concentration. Up to 85% E. coli are killed in the presence of Cu₂O particles with concentration of 25 µ/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 Cu²⁺-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, Cu₂O NPs were synthesized within the zeolite A framework using CuSO₄ as copper precursor and hydrazine hydrate (N₂H₄·H₂O) as reducing agent. The antibacterial activity of the obtained Cu₂O NPs/zeolite against Escherichia coli (E. coli) was also investigated.

2. Methods

2.1. Materials and Chemicals

Analytical grade CuSO₄·5H₂O, NH₄OH (25%), and N₂H₄·H₂O (80%) were products from Merck, and industrial grade sodium type of zeolite A with chemical formulation Na₂O·Al₂O₃. 2SiO₂·4.5H₂O 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 Cu²⁺ Ions in Zeolite

1 kg of zeolite was suspended into a glass beaker containing 1.5 L water. Then, HNO₃ 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 CuSO₄·5H₂O 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 Cu²⁺ into zeolite. Before adding hydrazine, pH of the Cu²⁺/zeolite mixture was adjusted by ammonia water to ~7.5.

2.3. Reduction of Cu²⁺ to Cu₂O NPs in Zeolite

A freshly prepared 250 mL hydrazine 20% (w/v) solution was added dropwise to the above Cu²⁺/zeolite under stirring for 2 h at ambient temperature. Then, reduction reaction was stopped and let standing overnight for Cu₂O NPs/zeolite settling down. Finally, Cu₂O 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.

2.4. Characterization of Cu₂O NPs/Zeolite

The content of copper in Cu₂O 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 Cu₂O NPs/zeolite product was carried out on D8 Advance Bruker, Germany, and the Cu₂O NPs sizes were measured using a transmission electron microscope (TEM; JEM 1010, JEOL, Tokyo, Japan). The presence of copper in Cu₂O NPs/zeolite was also assessed by energy-dispersive X-ray spectroscopy (EDX) on a JEOL 6610 LA.

2.5. Antibacterial Activity of Cu₂O NPs/Zeolite

In vitro test of bactericidal activity of Cu²⁺/zeolite and Cu₂O 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 () was calculated using the equation: , where and are the survival numbers of bacteria in the control and studied samples, respectively.

3. Results and Discussion

3.1. Characterization of Cu₂O NPs/Zeolite

Figure 1 showed the photograph of zeolite, Cu²⁺/zeolite, and Cu₂O NPs/zeolite. It is clearly observed from Figure 1 that Cu₂O NPs/zeolite is reddish color. The reaction of hydrazine used as reducing agent can be expressed by

Dimitrijević 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 Cu₂O NPs can be described in the following reactions:

TEM images of zeolite and Cu₂O NPs/zeolite in Figure 2 indicated that the size of Cu₂O 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 Cu₂O NPs in zeolite were almost as wormlike form. These Cu₂O NPs seem to be linked together within the intercages of zeolite structure.

Figure 1: Photograph of zeolite (a), Cu²⁺/zeolite (b), and Cu₂O NPs/zeolite (c).

(a)

(b)

Figure 2: TEM images of zeolite A (a) and Cu₂O NPs/zeolite A (b).

The XRD pattern of Cu₂O NPs/zeolite A in Figure 3 showed that diffraction peaks appeared at 29.61°, 36.48°, 42.38°, 61.46°, 73.56°, and 77.52° corresponding to (110), (111), (200), (220), (311), and (222) planes of cuprite, respectively, that indicated the formation of Cu₂O nanocrystals (JCPDS Card number 05-0667) [4, 18]. The average crystalline size of Cu₂O 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:

According to Giannousi et al. [4] and Xia et al. [19], Cu₂O should be also formed due to the disproportionation reaction of Cu and Cu²⁺:

It seems that not all resultant Cu reacts with Cu²⁺. So that small trace of Cu crystal could be contained in the Cu₂O NPs/zeolite product as presented in XRD pattern in Figure 3. Khan et al. [18] also reported that the color of the Cu-Cu₂O NPs mixture was dark yellowish that was different from black color of CuO [20] and reddish color (or brick color) of Cu₂O (Figure 1). According to Arshadi-Rastabi et al. [21], Cu₂O NPs have various ranges of different colors, namely, yellow, orange, red, and brown color.

Figure 3: XRD patterns of zeolite A and Cu₂O NPs/zeolite A.

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 Cu²⁺ and reduction of Cu²⁺ to Cu₂O 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 Cu₂O 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 Cu²⁺ by hydrazine) could not influence the total copper content in the final product (Cu₂O NPs/zeolite).

(a)

(b)

Figure 4: EDX spectra of zeolite and Cu₂O NPs/zeolite.

Figure 5 presented the brief schematic diagram of the synthesis procedures of Cu₂O NPs/zeolite by chemical reduction method using hydrazine hydrate.

Figure 5: Brief schematic diagram of Cu₂O NPs/zeolite synthesis procedures.

3.2. Antibacterial Activity of Cu₂O NPs/Zeolite

The antibacterial activity of Cu²⁺/zeolite and Cu₂O 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 Cu²⁺/zeolite and Cu₂O NPs/zeolite against E. coli was of 98.15 and 96.19%, respectively.

Table 1: The antibacterial efficiency () of Cu²⁺/zeolite and Cu₂O NPs/zeolite.

Sample
E. coli, CFU/mL
η, %

Control
1.13 × 10⁸
—

Cu²⁺/zeolite
2.09 × 10⁷
98.15

Cu₂O NPs/zeolite
4.30 × 10⁷
96.19

Figure 6: Photograph of the growth of E. coli on LB agar plates: (a) control, (b) Cu²⁺/zeolite, and (c) Cu₂O NPs/zeolite.

Most importantly, according to Li et al. [24], the Cu₂O NPs have low cytotoxicity. They also reported that the antibacterial efficiency of Cu₂O 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 Cu²⁺/zeolite and Cu₂O NPs/zeolite was also attained to nearly 100% after 4 h exposure in bacterial suspension (data not shown). The antimicrobial mechanism of Cu²⁺/zeolite can be occurred through two possible pathways as proposed by Hu et al. for Cu²⁺/montmorillonite [25]. The first one is the adsorption of bacterial cells on the surface of the Cu²⁺ carriers and the second, Cu²⁺ ions dissociated from Cu²⁺/carriers directly interact with bacterial cells. For copper-based nanoparticles (Cu, CuO, and Cu₂O nanoparticles), an elusive question remains whether the release of Cu²⁺ 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 Cu₂O NPs/montmorillonite on damage and removal of M. aeruginosa algae. The obtained results indicated that the antialgae effect mainly depended on Cu²⁺ ions extent released from Cu₂O and , radicals generated from photocatalysis activity of Cu₂O NPs, which were responsible for the damage to cellular structure, and physiological activity led to death of algae cells.

Furthermore, the acute toxicity of CuSO₄·5H₂O for rats showed that the oral LD₅₀ was 234 mg/kg body weight while oral LD₅₀ of Cu²⁺-exchanged montmorillonite with copper content of 25 g/kg was of 18 g/kg body weight [25]. This result indicated that Cu²⁺-exchanged montmorillonite was a toxicity-free substance for rats. Thus, it can be inferred that Cu²⁺/zeolite and also Cu₂O NPs/zeolite are less toxic than Cu²⁺ 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, Cu₂O NPs/zeolite would be more potential. In addition, Cu₂O NPs/zeolite product can be favorably produced on large scale by this process. The antifungal activity of Cu²⁺/zeolite and Cu₂O 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, Cu₂O NPs in zeolite A were synthesized using hydrazine hydrate as a reducing agent. Structural properties of Cu₂O NPs were determined by XRD and TEM. The Cu₂O NPs have fine crystal structure with particle size of about 40 nm. Results of the antibacterial activity showed that both Cu²⁺/zeolite and Cu₂O NPs/zeolite exhibited highly bactericidal efficiency (). 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 Cu₂O 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.

Acknowledgments

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

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