ZnO nanofluids: Green synthesis, characterization, and antibacterial activity

Materials Chemistry and Physics 121 (2010) 198–201

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ZnO nanofluids: Green synthesis, characterization, and antibacterial activity Razieh Jalal a , Elaheh K. Goharshadi a,b,∗ , Maryam Abareshi a , Majid Moosavi c , Abbas Yousefi d , Paul Nancarrow e a

Dept. of Chemistry, Ferdowsi University of Mashhad, Mashhad 91779 Mashhad, Iran Center of Nano Research, Ferdowsi University of Mashhad, Iran c Dept. of Chemistry, Faculty of Sciences, University of Isfahan, Isfahan 81746-73441, Iran d Pare-Tavous Research Institute, Mashhad, Iran e QUILL Research Centre and School of Chemistry and Chemical Engineering, Queen’s University Belfast, UK b

a r t i c l e

i n f o

Article history: Received 9 April 2009 Received in revised form 20 December 2009 Accepted 8 January 2010 Keywords: Nanostructures Chemical synthesis Ionic liquid Antibacterial activity

a b s t r a c t Zinc oxide nanoparticles have been synthesized by microwave decomposition of zinc acetate precursor using an ionic liquid, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide, [bmim][NTf2 ] as a green solvent. The structure and morphology of ZnO nanoparticles have been characterized using X-ray diffraction and transmission electron microscopy. The ZnO nanofluids have been prepared by dispersing ZnO nanoparticles in glycerol as a base fluid in the presence of ammonium citrate as a dispersant. The antibacterial activity of suspensions of ZnO nanofluids against (E. coli) has been evaluated by estimating the reduction ratio of the bacteria treated with ZnO. Survival ratio of bacteria decreases with increasing the concentrations of ZnO nanofluids and time. The results show that an increase in the concentrations of ZnO nanofluids produces strong antibacterial activity toward E. coli. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Room-temperature ionic liquids (RTILs) are very promising replacements for the traditional volatile organic solvents due to their high mobility, low melting points, negligible vapor pressure, thermal stability, low toxicity, large electrochemical window, nonflammability, and ability to dissolve a variety of chemicals [1–4]. Their quite rapid emergence as alternative solvents has involved a rapidly growing number of examples of application in organic chemistry but the use of RTILs in inorganic synthesis is still in its infancy. Only in recent years, the advantages of RTILs in inorganic synthetic procedures have gradually been realized and received more and more attention. Various nanostructured materials, such as palladium [5], iridium [6], gold [7], tellurium [8], TiO2 [9,10], ZnO [11–14], and CoPt [15] have been synthesized in RTILs. Due to the outbreak of the infectious diseases caused by different pathogenic bacteria, the scientists are searching for new antibacterial agents. In the present scenario, nanoscale materials have emerged up as novel antimicrobial agents owing to their high surface area to volume ratio and the unique chemical and physical properties [16].

In recent years, the use of inorganic antimicrobial agents has been attracted interest for the control of microbes. The key advantages of inorganic antimicrobial agents are improved safety and stability, as compared with organic antimicrobial agents [17]. At present, most antibacterial inorganic materials are metallic nanoparticles [18–20] and metal oxide nanoparticles such as zinc oxide [17]. In this work, a green and cost-effective method for the preparation of ZnO nanoparticles via microwave-assisted decomposition of zinc acetate precursor using 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide, [bmim][NTf2 ] as a solvent has been used. The morphology and structure of ZnO nanoparticles have been characterized using transmission electron microscopy (TEM) and X-ray diffraction (XRD), respectively. ZnO nanofluids have been prepared by dispersing ZnO nanoparticles in glycerol as a base fluid in the presence of ammonium citrate as a dispersant. The antibacterial activity of ZnO nanofluids was tested against Escherichia coli at different concentrations by colony count method. The quantitative examination of bacterial activity has been estimated by the survival ratio (N/N0 ) as calculated from the number of viable bacterial cells (N) at specified time and the initial viable cells (N0 ), which form colonies on the nutrient agar plates. 2. Experimental

∗ Corresponding author at: Department of Chemistry, Ferdowsi University of Mashhad, Mashhad 91779, Khorasan Razavi, Iran. Tel.: +98 511 8797022; fax: +98 511 8796416. E-mail address: [email protected] (E.K. Goharshadi). 0254-0584/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2010.01.020

2.1. Materials In our experiments, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide was used as a solvent, which was synthesized according to the literature [21].

R. Jalal et al. / Materials Chemistry and Physics 121 (2010) 198–201 All other chemicals used were of analytical grade and purchased and used as received without further purification. 2.2. Preparation of ZnO nanoparticles Zinc acetate dihydrate (5.5 g) was dissolved in 50 mL of deionized water, and then solid NaOH (16 g) was added slowly into the zinc acetate dehydrate solution under magnetic stirring at room temperature and formed a transparent Zn(OH)4 2− solution. Then 2 mL of [bmim][NTf2 ] was added to 3 mL of the above solution. The suspension was put into a domestic microwave oven (2.45 GHz, 850 W) in air, 30% of the output power of the microwave was used to irradiate the mixture for 5 min (on for 10 s, off for 5 s). The white precipitate was collected by centrifugation, washed with deionized water and ethanol several times, and dried in vacuum oven at 40 ◦ C for 10 h. 2.3. Preparation of suspension of ZnO nanoparticles Suspensions of ZnO nanoparticles were prepared with glycerol with the aid of a magnetic stirrer. To enhance the stability of the suspensions, ammonium citrate was used as a dispersant. In every sample, the weight ratio of dispersant to nanoparticle was kept 1:1. The samples were stable at least for several months and no agglomeration and sedimentation of the particles in the samples was observed. 2.4. Testing of antibacterial activity E. coli DH5␣ was grown aerobically at 37 ◦ C for overnight with shaking in an ordinary broth medium containing 0.5% yeast extract, 1% bactopeptone, and 1% sodium chloride. The saturated culture was first diluted in fresh LB medium and incubated at 37 ◦ C for 3–4 h on a reciprocal shaker. Subsequently, the solution of bacterial suspension was added into LB medium with a final concentration of 107 CFU dm−3 (CFU: colony forming unit) containing ZnO nanoparticles with concentration in the range from 0.125 to 0.5 g dm−3 and then was kept at 37 ◦ C for different times on a reciprocal shaker. After sampling the bacterial suspension of 1 × 10−4 dm3 , the bacterial suspension was spread on nutrient agar, and cultured at 37 ◦ C for 48 h without the presence of light. The colony formed with bacterial growth was counted. By calculating the ratio (N/N0 ) between the viable bacterial counts (N (CFU dm−3 )) at specified time and the initial counts (N0 (CFU dm−3 )) of bacteria, antibacterial activity was evaluated. All results were compared with two controls: (1) a fluid containing glycerol and ammonium citrate without ZnO nanoparticles and (2) a fluid without ZnO nanoparticles in the absence of base fluid and dispersant.

3. Results and discussion 3.1. Characterization of the ZnO nanoparticles The structural properties of the products were analyzed using D8 Advanced diffractometer with Cu K␣ radiation ( = 0.15406 nm). Fig. 1 shows the XRD pattern of nanoparticles. All the peaks of the (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4), and (2 0 2) reflections can be indexed to the known hexagonal wurtzite structure of ZnO with lattice constants of a = b = 3.242 Å and c = 5.205 Å. These match well with those in the JCPDS card (Joint Committee on Powder Diffraction Standards, Card No. 89-1397). The strong intensity and narrow width of ZnO diffraction peaks indicate that the resulting products were of high crystalline.

Fig. 1. Powder X-ray diffraction pattern for the ZnO nanoparticles.

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Although, weak dispersion peaks of background can be observed due to some amorphous material in the sample, no diffraction peaks from other species could be detected in the XRD pattern, which indicates that all the precursors have been completely decomposed during the decomposition process. The size of nanoparticles can be estimated from the Scherrer equation: Dh

kl

=

k× ˇh k l × cos h k l

(1)

where Dh k l is the particle size perpendicular to the normal line of (h k l) plane, k is a constant (it is approximately equal to 0.9), ˇh k l is the full width at half maximum (FWHM) of the (h k l) diffraction peak,  h k l is the Bragg angle of (h k l) peak and  is the wavelength of X-ray. The mean size of ZnO nanoparticles was calculated using Eq. (1). The peak position and the FWHM were obtained by fitting the measured peaks with two Gaussian curves in order to find the true peak position and width corresponding to monochromatic Cu K␣ radiation. The mean particle sizes for the crystallographic planes (1 0 1), (0 0 2), and (1 0 0) were 41.30, 47.52, and 37.15 nm, respectively. The transmission electron microscopy (TEM) was recorded by the LEO system (model 912 AB) operating at 120 kV for samples. Fig. 2 is a TEM image of the ZnO nanoparticles. It can be seen that the uniform nanocrystalline ZnO particles have sphere shapes with weak agglomeration. 3.2. Biological activity Autoclaved ZnO nanoparticles were mixed with autoclaved LB medium to make nanofluids with ZnO concentrations of 0.125, 0.25, and 0.5 g dm−3 . Fig. 3 shows the image of bacteriological tests of E. coli on solid agar plates without ZnO nanoparticles and with 0.5 g dm−3 ZnO suspensions. The results show that a number of E. coli colonies appeared on the solid agar plates without ZnO nanoparticles; however, several E. coli colonies are observed on the solid agar plates with 0.5 g dm−3 revealing that ZnO nanoparticles in glycerol solution can restrain E. coli proliferation. The survival ratio decreases with increasing ZnO nanoparticles concentrations

Fig. 2. TEM image of ZnO nanoparticles.

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Fig. 3. Images of bacteriological tests of E. coli on solid agar plates with 0.5 g dm−3 ZnO suspensions (left) and without ZnO nanoparticles (right) in the presence of glycerol and ammonium citrate.

(see Fig. 4(a)), indicating that antibacterial activity increases with increasing nanoparticles concentration. When the concentration of nanofluid was 0.125 g dm−3 , the ratio did not decrease even after 120 min. The survival ratio when the concentration was 0.25 or 0.5 g dm−3 decreased after 20 min and reached to the minimal values after 40 (0.72) and 120 min (0.21), respectively. The ratio in 0.5 g dm−3 concentration was steeper decrease than that of 0.25 g dm−3 . These results indicate that an increase in nanoparticles concentration produces strong antibacterial activity toward E. coli. On occurrence of antibacterial activity on ZnO, Sawai et al. [22], Yamamoto [23], Ohira et al. [24], and Padmavathy and Vijayaraghavan [25] reported that the generation of H2 O2 from the surface of ZnO was one of the primary chemical species being responsible for antibacterial action. The generation of highly reactive species such as OH− , H2 O2 , and O2 2− is explained as follows [25]. Since ZnO with defects can be activated by both UV and visible light, electron–hole pairs (e− h+ ) can be created. The holes split H2 O molecules from ZnO nanofluid into OH− and H+ . Dissolved oxygen molecules are transformed to superoxide radical anions (• O−2 ), which in turn react with H+ to generate (HO2 • ) radicals, which upon subsequent collision with electrons produce hydrogen peroxide anions (HO2 − ). Then they react with hydrogen ions to produce molecules of H2 O2 . The generated H2 O2 can penetrate the cell membrane and kill the bacteria [25]. Since, the hydroxyl radicals and superoxides are negatively charged particles, they cannot penetrate into the cell membrane and must remain in direct contact with the outer surface of the bacteria; however, H2 O2 can penetrate into the cell [25]. It is plausible to say that the concentration of nanofluids is comparable with the amount of H2 O2 . The amount of H2 O2 generated from the surface of ZnO should increase in proportion to increase of the nanoparticles concentration and time as mentioned by the previous report [23]. The reason for increasing the antibacterial activity with increasing concentration of ZnO nanofluid and time is assumed due to the increase of H2 O2 concentration generated from the surface of ZnO. Fig. 4(b) shows the changes in the survival ratio in the case of two controls, without ZnO nanoparticles in the presence or absence of glycerol and ammonium citrate. There were no statistically significant differences between the absence, 0.25 and 0.5 g dm−3 of ammonium citrate although 0.5 g dm−3 was more effective in growth reduction of E. coli than that of 0.25 g m−3 . The ammonium citrate at a concentration of 0.5 g dm−3 caused a reduction in the

Fig. 4. Changes in survival ratio of E. coli by using a concentration of 0.25 and 0.5 g dm−3 (a) without ZnO nanoparticles (b) without ZnO nanoparticles in the absence of (circle symbol) and in the presence of solvent, glycerol and ammonium citrate (the amount of solvent for triangle and square symbols are the same as those of (a)).

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number of bacterial colonies and could inhibit bacterial growth by about 19.7% after 120 min compared with the absence of ammonium citrate. The antibacterial activity of citrate salts against E. coli has been reported [26,27]. 4. Conclusions ZnO nanofluids have been prepared in glycerol as a base fluid in the presence of ammonium citrate as a dispersant. By careful examination of the results, it seems to us that this work contains the following conclusions: 1. Microwave-assisted heating method has been successfully established for the preparation of nanocrystalline ZnO in the presence of an ionic liquid. The method is found to be convenient, mild, efficient, and environmentally friendly. 2. Our experimental results revealed that low concentrations of ZnO nanofluids could not inhibit bacterial growth. The antibacterial activity of ZnO increases with increasing nanofluid concentration and time. In the antibacterial tests, it was found that the ZnO nanofluids are good bactericidal agents. 3. Ammonium citrate could slightly, but not significantly, reduce the number of colonies on the agar plates. Hence, ammonium citrate can act as a weak antibacterial agent. Acknowledgements The authors gratefully acknowledge the support of the Research Committee of Ferdowsi University of Mashhad. The authors would also like to thank Mrs. Roksana Pesian for taking TEM image.

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