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The sizes of zinc-oxide nanoparticles were synthesized by microwave method and were tailored by 6.5 MeV energy electron irradiation method. The ZnO nanoparticles having size of 40 nm were exposed to different fluences of 6.5 MeV electrons over the range from 5x1014 to 3.5x1015 electron /cm2. These electron irradiated ZnO nanoparticles were characterized by XRD, SEM and TEM techniques. The XRD results show that the ZnO nanoparticles retain the hexagonal phase with Wurtzite structure. However, particle size reduces continuously from 40 nm to 15 nm with the increase in the electron fluence. TEM results also supported the results for the reduction of the ZnO nanoparticles by 6.5 MeV electron irradiation. The antimicrobial activities for the as-synthesized and the electron irradiated ZnO nanoparticles on the fungus, Candida albicans was studied. In this case 6.5 MeV energy electrons irradiated ZnO nanoparticles show higher antimicrobial activity as compared to that of as-synthesized ZnO nanoparticles. The mechanism of killing biological cells is however, the same for the as-synthesized and electron irradiated nanoparticles.
Keywords: ZnO nanoparticles, Microwave method, electron irradiation, Antimicrobial
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Zinc oxide (ZnO), a group II-VI compound semiconductor with a wide and direct band gap of 3.37 eV and a large exciton binding energy of 60 meV , has been widely used in many applications such as transparent conductive films, resistors, solar cell windows, bulk acoustic wave devices, lasers and diodes . One of the materials that has attracted great interest from a wide range of technological fields associated with nanotechnology is zinc oxide. In general metal oxides show enhanced photocatalytic activity with the increase in electronic defects in the crystallites by introducing defects into the crystal lattice of ZnO nanoparticles. Two principal factors cause the properties of nanomaterials to differ significantly from other materials. The increased surface to volume ratio and dominance of quantum effects. These factors can change or enhance chemical reactivity, electronic, optical, thermal, mechanical, magnetic, and electrical/transport characteristics including properties such as ionization potential, electron affinity, capillary forces, melting point, specific heat etc.
In order to realize the universal application of nanomaterials, the key is to devise simple and efficient methods for preparing nanomaterials on a large scale at low cost . ZnO nanoparticles can be prepared on a large scale at low cost by simple solution-based synthesis methods, such as chemical precipitation , sol-gel,  and solvothermal/ hydrothermal reaction Number of other methods such as photochemical, electrochemical and chemical reduction , microwave processing , gamma irradiation , ion irradiation  and plasma processing, radiolysis, ultra sound processing. Nanoparticles of noble metals such as gold and silver are known to be effective as antimicrobial agents. Among the metal oxide nanoparticles, those of zinc (ZnO) have received particular attention as they offer several advantages. They exhibit stability, can be fabricated at low temperatures , display high surface area, show unusual crystal structures , have superior durability, are selective, exhibit heat resistance  and are generally regarded as safe. The ZnO nanoparticles are effective against a variety of microorganisms including bacteria such as Escherichia coli, Staphylococcus aureus , Listeria monocytogenes, Salmonella enteritidis  and Bacillus atrophaeus . Growth of fungi such as Botrytis cinerea, Penicillium expansum and Candida albicans  can also be controlled by using ZnO nanoparticles. Among the fungi, Candida albicans is an important pathogen . This generally colonises medical devices such as catheters and prosthetic surfaces in an adherent manner and forms extensive biofilms. Such biofilms are often more resistant to antimicrobial agents  and there is a need to use novel anti-biofilm agents for controlling them. No work is reported on the synthesis of ZnO nanoparticles by microwave method and tailors the size of nanoparticles by electron irradiation. The aim of the current study is to investigate anti-biofilm activity of ZnO nanoparticles against a hospital isolate of C. albicans.
2. Experimental details:
Zinc nitrate, sodium hydroxide and high molecular weight PVA (PVA2000) were used for the synthesis. All the solutions were prepared in deionised water only. Zinc oxide particles were prepared by the reaction of equimolar concentrations of zinc nitrate with sodium hydroxide using water as a solvent. The capping agent for particle stabilization used was a high molecular weight PVA. The pH of the solution was controlled by the addition of NaOH into the reaction mixture. A commercial microwave oven (make - Electrolux, 2.45GHZ Frequency) with adjustable power output (750W) was used.
2.2 Preparation of ZnO nanoparticles
The Nanosize photocatalytic Zinc Oxide (ZnO) particles were prepared by microwave-assisted method. Aqueous solution of zinc nitrate (0.01M) and polyvinyl alcohol (1 % w/v) was prepeared. The solution thus prepared was set to stir constantly with addition of solution (0.01M) of sodium hydroxide simultaneously for half an hour. After completion of the reaction, white precipitates of zinc hydroxide were produced. The precipitated zinc hydroxide solution was kept for 10 minutes under exposure to microwave radiation at a power level of ~700 W by following on-off cycle. The solution was allowed to cool down slowly to room temperature. The cooled solution was then centrifuged in the presence of water and acetone to remove all the impurities. The process of centrifugation was repeated thrice to remove most of the impurities from the solution and allowed to air dry. Annealing of the ZnO nanoparticles was carried out at 60°C for three hours in temperature controlled furnace. The annealed powder was used for characterization and the present study.
2.3 Irradiation of ZnO nanoparticles
An electron beam of 6.5 MeV energy was obtained from the Race track Microtron of this laboratory and used for irradiating ZnO particles. For irradiation experiment, about 50mg of powder of ZnO particles was placed in a polythene bag, and by folding the bag, a sample of size 10mm x 10mm x 3mm was made. Such fine samples were made and numbered 1 to 5. Initially, the number 1 sample was mounted on the Faraday cup, positiond at a distance of 100mm from the beam extraction port of the Microtron. The electron beam position on the Faraday cup coincided with the sample position. The electron beam area was large enough to cover the entire sample area. The number of electron falling on the entire sample was measured by a current integrator, connected to the Faraday cup. Following the same procedure, the samples were irradiated with electrons at diffrent fluences. The electron fluence was increased from sample to sample in the steps of 5 x 1014, 1.0 x 1015, 1.5 x 1015, 2.5 x 1015 and 3.5 x 1015 e/cm2 respectively.
2.4 Preparation for antifungal activity and bioflim formation of zinc oxide nanoparticles:
The solution of the ZnO nanoparticles was prepared by taking 50 μl of a methanolic solution containing 15 mg ml-1 and mixing with the ZnO nanoparticles irradiated with the electrons at the given fluences i.e. 0, 1.5x1015, 2.5x1015, 3.0x1015 and 3.5x1015e/cm2. The antifungal activity of ZnO NPs was studied by spreading the fungal culture (grown for 36 h) on YPDA plates. A number of wells were made on the seeded YPDA plates. By adding the solution containing ZnO nanoparticles, the antifungal activities of ZnO NPs was studied. Methanol was used in control experiments. The NPs were allowed to diffuse in the plate at 15°C for 15 min and later incubated at 37oC for 48 h. Zones of inhibition around the wells were noted after the incubation period. All experiments were carried out three times.
Moreover, the Biofilm of C. albicans biofilms were formed in pre-sterilised polystyrene, 96-well microtitre plates. Standardized cell suspensions containing 106 cells ml-1 (100 µl), YPD liquid medium (200 µl) and the solution of the electron irradiated ZnO nanoparticles (10 µl) was added to the wells. In this manner, the three types of electron irradiated ZnO nanoparticles were mixed with the fungal culture. The plates without ZnO NPs served as control. These plates were incubated at 37oC for 24, 48, 72 and 96 h respectively. After incubation, the planktonic cells were discarded and weakly adherent cells were removed by washing with the sterile phosphate buffer saline (PBS). Biofilm formation by C. albicans in presence or absence of ZnO NPs was quantified by the crystal violet assay.
3. Results and Discussion:
3.1 X-Ray Diffraction
ZnO nanoparticle samples irradiated with 6.5 MeV energy electrons for the fluence from 1.0 x 1015 to 3.5 x 1015 e-/cm2 were characterized by the X-ray diffraction (XRD) technique. The XRD pattern is shown in Fig. 1. The XRD pattern of the final ZnO nanoparticles was obtained with Cu Kα radiation (λ = 1.5418 Å) on a Bruker axs D8 Advanced diffractometer (CuK¡ radiation) at a continuous scan rate of 1-20 /min with 0.10 resolution. Diffraction peaks were observed at the scattering angles 2θ of 31.52, 34.59, 36.03, 47.94, 56.75, 62.93, 66.407, 67.64, 69.07, and 77.91 θ to the reflections from the planes [1 00], [0 0 2], [1 0 1], [1 0 2], [1 1 0], [1 0 3], [2 0 0], [1 1 2], [2 0 1], and [0 0 4]. The lattice parameters were found to be a = 3.25 Å and c = 5.23 Å, which shows the hexagonal wurtzite structure. The mean grain size (D) of the particles was determined from the line-broadening in XRD pattern using Scherrer equation,
D = 0.89λ/ (β cos θ)
where, λ is the wavelength (Cu Kα), β is the full width at the half-maximum and θ is the diffraction angle. The particle size was found to be reduced from 40 to 15 nm. The peak positions and relative intensities were characterized by comparison with Standard data (JCPDS card no 36-1451) for examining the phase structure and purity.
3.2 Transmission Electron Microscopy (TEM):
Fig.2 gives the transmission electron microscopic images of the ZnO nanoparticles/nanorods. The image shows that the particle size goes on decreasing with the increase in the electron fluence from 40 to 15 nm. It was also observed that the morphological changes occurred in the structure of ZnO, such as defect formation, sharpness in the edges of the nanorods, higher crystallinity etc. The SAED pattern also gives the morphological information of the ZnO nanoparticles with well aligned orientation with hexagonal wurtzite structure as shown in the Fig.2 (f). The structural changes with respect to the electron fluences are shown the Fig.2 (a)as-synthesized, (b)Ð¤=1.5x1015e/cm2, (c)Ð¤=2.5x1015e/cm2, (d)Ð¤=3.0x1015e/cm2 and
3.3 Antifungal activity of the ZnO NPs
Antifungal activity of the ZnO NPs against the pathogenic strain of C. albicans was checked. Fig. 3(a) shows the zones of inhibition of C. albicans on a representative YPDA plate after 48 h of incubation at 37oC with different fluences of electron irradiated ZnO NPs. These results are in agreement with previous studies on ZnO NPs being effective against C. albicans  and other pathogenic fungi such as B. cinerea and P. expansum .
Fig.4 shows the effect of ZnO nanoparticles on the biofilms of C albicans. Fig.4 (A) shows the optical microscopic images of C. albicans biofilms in the absence and presence of ZnO NPs after 24 h. It was observed that control biofilms were well-organized and cells were more elongated (Figure 4 a, f and k). The average length of cells was found to be in the range of 4 to 7 µm. In presence of different fluences of electron irradiated ZnO nanoparticles, (Fig. 4 b, c, d and e) biofilm formation was poor, cells were more rounded and average length of the cells was decreased to 2 to 4 µm. Such morphological changes in yeast cells were possibly due to the oxidative stress of ZnO NPs. As shown in Fig.4, SEM (B) and fluorescence (C) images also confirm the effect of ZnO NPs against C. albicans biofilms developed on glass slides. Different fluences of electron irradiated of the ZnO NPs, thus significantly inhibited biofilms formation. In Fig.4 (a, f and k) represents the control untreated biofilms while (b),(c),(d) and (e) indicates the effect in presence of ZnO nanoparticles at electron fluences (Ð¤= 0, as synthesized), (Ð¤=1.5x1015e/cm2), (Ð¤=2.5x1015e/cm2) and (Ð¤=3.5x1015e/cm2) respectively. Fig.4 B shows SEM images of biofilms. Biofilms in the absence of ZnO NPs (f), and when treated with different fluences of electron irradiated ZnO NPs (g to j) shows increase in biofilm inhibition. Fig.4 (c). shows the fluorescent images of biofilms of C. albicans (k) on glass slides formed after 48h (k) in the absence and (l to o) in the presence of electron irradiated ZnO NPs at the fluences of (Ð¤=0, as synthesized), (Ð¤=1.5x1015e/cm2), (Ð¤=2.5x1015e/cm2) and (Ð¤=3.5x1015e/cm2) respectively.
In conclusion, microwave method can successfully been used to synthesized the ZnO nanoparticles having size around 40 nm and further tailored upto 15 nm using 6.5 MeV electron irradiation. Generally, the UV radiations are being used during the study of the antimicrobial activity because the defects induced on the surface of the ZnO nanoparticles could separate the electrons from the holes effectively and therefore could replace the processes of photoexcitation. But, in the present study the high energy electron irradiation itself plays an important role in this particular mechanism. Therefore, the ZnO nanoparticles synthesized by microwave assisted method and further irradiated with 6.5MeV energy electrons show higher antimicrobial activity as compared to that of the as synthesized ZnO nanoparticles. The mechanism of killing the biological cells is however, the same for the as-synthesized and electron irradiated nanoparticles.