PREPARATION OF ZINC OXIDE – NANOCLAY HYBRIDS BY ALKALINE ION EXCHANGE METHOD

ZnO/nanoclay hybrids are synthesized by a single-step solid-state reaction using ZnCl2 as a reagent. These hybrids were prepared by a simple alkaline ion exchange method, without using any surfactant or chelating agents. By adjusting the reagent’s immersion time in molten salt, production of pure ZnO/nanoclay hybrids was achieved. Synthesized hybrids were characterized by UV-vis diffuse reflectance spectroscopy, XRD, XRF and SEM. SEM confirmed increased nanoclay porosity. The XRD pattern of the ZnO/nanoclay indicated small changes in the intensity and position of the interlayer spacing of montmorillonite. Antibacterial activity of the hybrids against Escherichia coli and Staphylococcus aureus were assessed using disc diffusion. The results show that the antibacterial efficiency of the hybrid is not influenced by the time or the temperature of ion exchange, but is affected by bacterial type.


INTRODUCTION
Zinc oxide nanoparticles possess several interesting properties, such as optical transparency, electric conductivity, piezoelectricity, nontoxicity, wide availability, low cost and stability.The specific properties of nanoscale zinc oxide particles have found a wide range of applications, including luminescence, varistors, solar cells and gas sensors (Kołodziejczak-Radzimska, 2014).
It has been demonstrated that ZnO nanostructures exhibit antimicrobial activity against a broad spectrum of bacteria, including Staphylococcus aureus (Applerot et al. 2009;Gordon et al., 2011;Pouraboulghasem et al., 2016 b;Yamamoto, 2001) and Escherichia coli (Brayner et al., 2005;Franklin et al., 2007;Gilani et al., 2016).They are currently used in many cosmetics and food packaging applications.Unlike conventional chemical disinfectants, these antimicrobial nanomaterials are not expected to produce harmful disinfection byproducts.The antimicrobial activity of ZnO nanoparticles may be related to the induction of oxidative stress due to the generation of reactive oxygen species, which may cause the degradation of the bacterial membrane structure (Sawai et al., 1998).Disruption of the bacterial membrane is attributed to peroxidation of the unsaturated phospholipids due to photocatalytically induced hydrogen peroxide.The presence of ZnO nanoparticles damages the bacterial membrane and cell wall.Consequently, bacterial growth is inhibited.In addition, binding Zn 2+ to microorganisms' membranes prolongs the lag phase of the microbial growth cycle (Brayner et al., 2005;Atmaca et al., 1998).
Sol-gel, spray pyrolysis, precipitation, microemulsion techniques, electrophoretic deposition, thermal evaporation, laser ablation, chemical vapor deposition, mechanical milling, molten salt and microwave method and hydrothermal synthesis are some of the methods employed in synthesizing nanoparticles (Espitia et al., 2012;Ghorbanpour and Lotfiman, 2016;Payami et al., 2016;Srivastava et al., 2013;Zhu and Zhou, 2008).However, all of these approaches involve complex apparatus or processing.They also involve toxic reagents or additives.The relatively high temperature, complexity and toxicity of these techniques restrict their widespread application.On the other hand, in most of these techniques, nanoparticles tend to agglomerate due to their high surface energy.Therefore, nontoxic, simple, low cost and high yield synthetic methods for preparing nanometric materials are keenly pursued.
The alkaline ion exchange method offers easy control of zinc loading, an environmentally friendly approach with large scale production via fast processing.Furthermore, this method introduces a process with inexpensive raw materials and uncomplicated equipment (Pouraboulghasem et al., 2016a, Tagantsev et al., 2008;Xu e al., 2007).It also provides a way to fabricate samples easily by introducing zinc into clay.Molten salt ion exchange is a well-known method investigated for the last few decades to introduce ions into silicate glass, e.g., for waveguides in optical applications (Oven et al., 2004;Newby et al., 2011).Alkaline ion exchange in clay minerals can be simply stated as: Nanoclay is used primarily for synthesizing nanocomposites and biodegradable materials to improve mechanical and thermo properties (Rhim and Wang, 2014;Arora and Padua, 2010).To create nanocomposites' antibacterial activity, nanoparticles with antibacterial activity must be used.Zinc is a common component in this approach.However, synthesizing nanoparticles is complicated, and nano products create another serious problem in the environment.Therefore, introducing a fast and inexpensive method for synthesis with production on an industrial scale is an outcome in high demand.
To our knowledge, this is the first time that zinc oxidenanoclay hybrids have been synthesized by alkaline ion exchange.The advantages of this method are simplicity and speed in the production of the pure product, particularly because no solvent or special preparation was required.For this approach nanoclay was mixed with solid ZnCl 2 at the salt melting point temperature.The main parameters to investigate were time and temperature.The produced nanocomposite was characterized by various techniques.
Experiments carried out to investigate the efficiency of the synthesized hybrids against bacteria are explained in detail.

Materials
Nanoclay (K10 montmorillonite) used as a solid support for zinc was obtained from Sigma-Aldrich (Tehran, Iran).Zinc chloride and Nutrient agar was purchased from Merck (Tehran, Iran).The bacterial strains used for the antibacterial activity were gram-negative E. coli (PTCC 1270) and gram-positive S. aureus (PTCC 1112) obtained from the Iranian Research Organization for Science and Technology (Tehran, Iran).All reagents were analytical grade and used without further refinement.

Alkaline ion exchange with zinc
The alkaline ion exchange process began with immersion of nanoclay into ZnCl 2 at 350 and 450 °C for 1, 2, 3 and 5 min.This step was conducted using 1 g of nanoclay and 1 g of ZnCl 2 .After ion exchange, the nanoclay was adequately washed with distilled water and sonication.After filtration, the hybrids were dried in an oven at 25 °C.

Scanning electron microscopy
The morphology of samples was observed with a scanning electron microscope (LEO 1430VP, Germany).

X-ray powder diffraction
X-ray diffraction (XRD) patterns were recorded using a Philips PW 1050 diffractometer (The Netherlands).Samples in powder form were pressed in a rotational holder.Reflection mode was used for all measurements.

X-ray fluorescence spectroscopy
Chemical composition of the samples was determined using a µ-energy dispersive fluorescence spectrometer (XMF-104 X-ray Micro analyzer, Unisantis S.A., Switzerland) equipped with a 50 W molybdenum tube and a high resolution two-stage Peltier cooled Si-PIN detector (Amptek, USA).The samples were positioned in definite places, but at a constant height from the holder base.The temperature was controlled at 32-34 °C throughout the experiments.Voltage and current were 30 kV and 300 µA, respectively.Each micro-EDXRF analysis was performed in 50 s to obtain sufficient counts (Ghorbanpour and Falamaki, Applied Optics, 2012). (1)

UV-Vis diffuse reflectance spectroscopy (DR UV-Vis)
DR UV-Vis was used for a qualitative description of the differences in absorbance depending on the amount of ZnO in the studied composites.DR UV-Vis of the powder samples was conducted in a 5.0 mm quartz cell registered in the wavelength range of 200-800 nm using a spectrophotometer (Scinco S4100, S. Korea).

Leaching tests
In order to evaluate the stability of the hybrids, leaching tests were performed.For each composite material, 0.2 g was immersed in 10 mL of distilled water and vigorously shaken in a shaking water bath (30 °C, 200 rpm) for 24 h.Supernatants from each test tube were collected by centrifugation at 4000 rpm for 10 min.Zinc ions released from hybrids were qualitatively determined by atomic absorption spectroscopy analysis (Varian/AAS, 20BQ, USA).

Antibacterial activity
The antibacterial activity of the ZnO/nanoclay hybrids against both E. coli (gram-negative) and S. aureus (grampositive) was tested by agar diffusion test.Samples were exposed to bacteria in solid media (nutrient agar), and the inhibition zone around each sample was measured and recorded as the antibacterial effect of ZnO nanoparticles.Agar plates were inoculated with 100 µL suspensions of bacteria.Hybrids were placed on 0.5 cm diameter agar disks and incubated at 37 °C for 24 h.The inhibition zone for bacterial growth was detected visually.

Appearance
The color of the parent nanoclay was cream-colored.The nanoclay's alkaline ion exchange with zinc chloride changed the color of the nanoclay to russet (Figure 1).This variation in color may run up from the diffused zinc to the nanoclay structure, and subsequently exchange with the nanoclay's sodium.There are two important parameters that can affect the alkaline ion exchange process, namely process time and temperature.Figure 1 illustrates that longer ion exchange time caused different nanoclay colors.On the other hand, higher temperature caused faster changes in color.

SEM analysis
The SEM micrographs of the composites are shown in Figure 2. The parent nanoclay appears as cornflake-like crystals with fluffy appearance and a layered flakes structure, providing a greater surface area for the immobilization of zinc in comparison to the parent nanoclay.After alkaline ion exchange, the layered structure of clay remained with some changes in terms of porosity.On the other hand, it can be seen that the pore sizes seem to be enhanced by increasing temperature and processing time.This is due to an increase in the interplanar spacing of nanoclay and the adsorption of large amounts of zinc atoms during the process, which can comprise a significant fraction of pore space.

XRD analysis
XRD patterns of the ZnO/nanoclay hybrid prepared by the alkaline ion exchange method at 350 and 450 °C in the 2θ range of 4-60º are shown in Figure 3. Nanoclay characteristic peaks are shown at 2θ values of 6, 20 and 35º, denoted with "M" on the graph.The peaks are evident in all patterns, and are also consistent with other reported works (Dakovic et al., 2012;Khaorapapong et al., 2011).As can be seen in Figure 3, the XRD pattern of the ZnO/ nanoclay displays small changes in intensity and in the  position of the interlayer spacing (d 001 ) of montmorillonite (1.4 nm for nanoclay and 1.5-1.6 nm for ZnO/nanoclay hybrids).These changes resulted from the expansion of the interlayer spaces, which may be caused by the intercalation of ZnO particles.Németh et al. (2005) reported the results of XRD analysis of Na-montmorillonite exchanged with Zn solutions through different concentrations.They stated that the characteristic basal reflection of montmorillonite (1.25 nm) had progressively shifted to 1.4-1.5 nm after an exchange with zinc.Diffraction peaks corresponding to impurities were not found in the XRD patterns, confirming the high purity of the synthesized products.

XRF analysis
XRF analysis was used to investigate the rate and amount of Zn loading in nanoclay.For this reason, the loading of Zn within the composites was probed through µ-XRF measurements.The intensity of the loaded zinc atoms was enhanced by increasing the alkaline ion exchange time.However, the intensity increased more rapidly in the first minute, reaching about 2000 counts (Figure 4a).Then the intensity change was low and reached about 2500 counts for 5 min in exchanged nanoclay at 350 °C (Figure 4b).This can be explained by diffusion theory.At the beginning of the process Na ions covered the surface of the nanoclay.Therefore, Zn ions from the molten salt were rapidly and easily exchanged with Na ions.This happened in the first minute of the process.Then, mass transfer of Na and Zn was accomplished by inner Na ions.This step is needed for Zn penetration into nanoclay; therefore, the mass transfer rate and intensity reduce.It was concluded that the majority of Zn loading on nanoclay was carried out in the first minute.

DR UV-Vis spectra analysis
The UV-vis absorption spectra of samples are shown in Figure 5.The maximum peak at around 315 nm belongs to the intrinsic absorption of nanoclay.Parent zinc chloride also has a strong absorption around 306 nm.After ion exchange, the two peaks disappeared.It could be the result of the changes of the initial nanoclay and ZnCl 2 to nanocomposite during the process.
All nanocomposites have a strong absorption peak located around 360 nm and a weak absorption peak located around 433 nm.The two traces for 3 min process time also indicate higher absorption at higher temperature.Furthermore, the maximum absorption located at around 360 nm in the samples is slightly red shifted to higher wavelengths by increments of time and temperature in ion exchange.This red shift can be attributed to the agglomerations in the samples (Ghorbanpour and Falamaki, 2013).XRF results revealed that the major loading of Zn was accomplished within the first min.However, DRS graphs show no changes in the absorption after 3 min.This can be attributed to the changes in composite structure and the connection form of loaded Zn ions.

Leaching tests
The leaching test has been developed for the assessment of released zinc ions from nanocomposite.   that released Zn 2+ was low for the samples prepared at 350 °C, 1 min and at 450 °C, 1 min.Longer process time samples released more Zn 2+ .This may be explained by the position from which Zn 2+ was released.As described above, during the first minute Zn ions were exchanged with surface Na ions and were set as a structural particle.However, further time allowed Zn ions to penetrate into the nanoclay.On the other hand, some Zn ions became entangled into the structure or remained on the surface of nanoclay without binding.The probability of releasing nonbinding Zn 2+ is high, so the samples with longer processing time released more Zn ions.

Antibacterial activity
The antibacterial properties of ZnO/nanoclay hybrids were tested comparatively against gram-negative E. coli and gram-positive S. aureus bacteria by the disk diffusion test.The inhibition zone under and around the tested samples against S. aureus was detected visually (Figure 6) and listed in Table 2.The results suggest that the hybrid had a toxic effect on the bacteria.This could be associated with the photocatalytic mechanism of nano ZnO, which generates hydrogen peroxide and causes the degradation of the membrane structure of the bacteria (Sawai et al., 1998;   et al., 1998).The results in Table 2 show that the antibacterial efficiency of the hybrid is not influenced by the time or temperature of ion exchange.Interestingly, antibacterial activity is affected by bacterial type.Gramnegative bacteria provide greater antibacterial effect than gram-positive bacteria.

CONCLUSION
ZnO/nanoclay hybrids were successfully synthesized at the first attempt through an easy, simple and fast method, without using any solvents or expensive reagents.Processing time and temperature were the two main factors that influenced the properties of produced nanocomposite.The results revealed that the pore sizes of parent nanoclay were enhanced by increasing temperature and processing time.XRD patterns confirmed the high purity of the synthesized products.It was also obtained that the loading of Zn ions was faster in the first minute.The loading of Zn ions increased with increasing process time and temperature.The processing time and temperature had no impact on the antibacterial activity, while the type of bacteria used had an effect on the results.Gram-negative bacteria delivered more antibacterial activity than grampositive bacteria.

Figure 4 .
Figure 4.The micro-EDXRF spectrum of ion-exchanged nanoclay at 350 °C for 1 min (a) and at 350 °C for 5 min

Table 1 .
Zinc concentration in water at different contact times between ZnO/nanoclay hybrids and aqueous media

Table 2 .
Antibacterial activity of ZnO/nanoclay hybrids against E. coli and S. aureus