Materials Chemistry and Physics 121 (2010) 198 201
Contents lists available at ScienceDirect
Materials Chemistry and Physics
journal homepage: www.elsevier.com/locate/matchemphys
ZnO nanofluids: Green synthesis, characterization, and antibacterial activity
Razieh Jalala, Elaheh K. Goharshadia,b,", Maryam Abareshia, Majid Moosavic,
Abbas Yousefid, Paul Nancarrowe
a
Dept. of Chemistry, Ferdowsi University of Mashhad, Mashhad 91779 Mashhad, Iran
b
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
a r t i c l e i n f o a b s t r a c t
Article history:
Zinc oxide nanoparticles have been synthesized by microwave decomposition of zinc acetate precursor
Received 9 April 2009
using an ionic liquid, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide, [bmim][NTf2] as
Received in revised form
a green solvent. The structure and morphology of ZnO nanoparticles have been characterized using X-ray
20 December 2009
diffraction and transmission electron microscopy. The ZnO nanofluids have been prepared by dispersing
Accepted 8 January 2010
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
Keywords:
the reduction ratio of the bacteria treated with ZnO. Survival ratio of bacteria decreases with increasing
Nanostructures
the concentrations of ZnO nanofluids and time. The results show that an increase in the concentrations
Chemical synthesis
of ZnO nanofluids produces strong antibacterial activity toward E. coli.
Ionic liquid
© 2010 Elsevier B.V. All rights reserved.
Antibacterial activity
1. Introduction In recent years, the use of inorganic antimicrobial agents has
been attracted interest for the control of microbes. The key advan-
Room-temperature ionic liquids (RTILs) are very promising tages of inorganic antimicrobial agents are improved safety and
replacements for the traditional volatile organic solvents due to stability, as compared with organic antimicrobial agents [17].
their high mobility, low melting points, negligible vapor pressure, At present, most antibacterial inorganic materials are metallic
thermal stability, low toxicity, large electrochemical window, non- nanoparticles [18 20] and metal oxide nanoparticles such as zinc
flammability, and ability to dissolve a variety of chemicals [1 4]. oxide [17].
Their quite rapid emergence as alternative solvents has involved In this work, a green and cost-effective method for the prepara-
a rapidly growing number of examples of application in organic tion of ZnO nanoparticles via microwave-assisted decomposition
chemistry but the use of RTILs in inorganic synthesis is still in its of zinc acetate precursor using 1-butyl-3-methylimidazolium
infancy. Only in recent years, the advantages of RTILs in inorganic bis(trifluoromethylsulfonyl) imide, [bmim][NTf2] as a solvent has
synthetic procedures have gradually been realized and received been used. The morphology and structure of ZnO nanoparticles
more and more attention. Various nanostructured materials, such have been characterized using transmission electron microscopy
as palladium [5], iridium [6], gold [7], tellurium [8], TiO2 [9,10], ZnO (TEM) and X-ray diffraction (XRD), respectively. ZnO nanofluids
[11 14], and CoPt [15] have been synthesized in RTILs. have been prepared by dispersing ZnO nanoparticles in glycerol
Due to the outbreak of the infectious diseases caused by dif- as a base fluid in the presence of ammonium citrate as a dispersant.
ferent pathogenic bacteria, the scientists are searching for new The antibacterial activity of ZnO nanofluids was tested against
antibacterial agents. In the present scenario, nanoscale materials Escherichia coli at different concentrations by colony count method.
have emerged up as novel antimicrobial agents owing to their high The quantitative examination of bacterial activity has been esti-
surface area to volume ratio and the unique chemical and physical mated by the survival ratio (N/N0) as calculated from the number
properties [16]. 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 2.1. Materials
Mashhad, Mashhad 91779, Khorasan Razavi, Iran. Tel.: [phone];
fax: [phone]. In our experiments, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)
E-mail address: [e-mail] (E.K. Goharshadi). imide was used as a solvent, which was synthesized according to the literature [21].
0254-0584/$  see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.matchemphys.2010.01.020
R. Jalal et al. / Materials Chemistry and Physics 121 (2010) 198 201 199
All other chemicals used were of analytical grade and purchased and used as received Although, weak dispersion peaks of background can be observed
without further purification.
due to some amorphous material in the sample, no diffraction peaks
from other species could be detected in the XRD pattern, which
2.2. Preparation of ZnO nanoparticles
indicates that all the precursors have been completely decomposed
during the decomposition process.
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 The size of nanoparticles can be estimated from the Scherrer
under magnetic stirring at room temperature and formed a transparent Zn(OH)42-
equation:
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
k ×
Dh k l = (1)
the output power of the microwave was used to irradiate the mixture for 5 min (on
Çhkl × cos hkl
for 10 s, off for 5 s). The white precipitate was collected by centrifugation, washed
ć%
with deionized water and ethanol several times, and dried in vacuum oven at 40 C
where Dhkl is the particle size perpendicular to the normal line of
for 10 h.
(hkl) plane, k is a constant (it is approximately equal to 0.9), Çhkl
is the full width at half maximum (FWHM) of the (hkl) diffraction
2.3. Preparation of suspension of ZnO nanoparticles
peak, hkl is the Bragg angle of (hkl) peak and is the wavelength
Suspensions of ZnO nanoparticles were prepared with glycerol with the aid of
of X-ray. The mean size of ZnO nanoparticles was calculated using
a magnetic stirrer. To enhance the stability of the suspensions, ammonium cit-
Eq. (1). The peak position and the FWHM were obtained by fit-
rate was used as a dispersant. In every sample, the weight ratio of dispersant to
ting the measured peaks with two Gaussian curves in order to find
nanoparticle was kept 1:1. The samples were stable at least for several months and
the true peak position and width corresponding to monochromatic
no agglomeration and sedimentation of the particles in the samples was observed.
Cu K radiation. The mean particle sizes for the crystallographic
2.4. Testing of antibacterial activity planes (1 0 1), (0 0 2), and (1 0 0) were 41.30, 47.52, and 37.15 nm,
respectively.
ć%
E. coli DH5 was grown aerobically at 37 C for overnight with shaking in an ordi-
The transmission electron microscopy (TEM) was recorded by
nary broth medium containing 0.5% yeast extract, 1% bactopeptone, and 1% sodium
the LEO system (model 912 AB) operating at 120 kV for samples.
chloride. The saturated culture was first diluted in fresh LB medium and incubated
ć%
at 37 C for 3 4 h on a reciprocal shaker. Subsequently, the solution of bacterial sus- Fig. 2 is a TEM image of the ZnO nanoparticles. It can be seen that
pension was added into LB medium with a final concentration of 107 CFU dm-3 (CFU:
the uniform nanocrystalline ZnO particles have sphere shapes with
colony forming unit) containing ZnO nanoparticles with concentration in the range
weak agglomeration.
ć%
from 0.125 to 0.5 g dm-3 and then was kept at 37 C for different times on a recip-
rocal shaker. After sampling the bacterial suspension of 1 × 10-4 dm3, the bacterial
ć%
suspension was spread on nutrient agar, and cultured at 37 C for 48 h without the
3.2. Biological activity
presence of light. The colony formed with bacterial growth was counted. By calculat-
ing the ratio (N/N0) between the viable bacterial counts (N (CFU dm-3)) at specified
Autoclaved ZnO nanoparticles were mixed with autoclaved LB
time and the initial counts (N0 (CFU dm-3)) of bacteria, antibacterial activity was
medium to make nanofluids with ZnO concentrations of 0.125, 0.25,
evaluated. All results were compared with two controls: (1) a fluid containing glyc-
erol and ammonium citrate without ZnO nanoparticles and (2) a fluid without ZnO and 0.5 g dm-3. Fig. 3 shows the image of bacteriological tests of
nanoparticles in the absence of base fluid and dispersant.
E. coli on solid agar plates without ZnO nanoparticles and with
0.5gdm-3 ZnO suspensions. The results show that a number of
3. Results and discussion E. coli colonies appeared on the solid agar plates without ZnO
nanoparticles; however, several E. coli colonies are observed on the
3.1. Characterization of the ZnO nanoparticles solid agar plates with 0.5 g dm-3 revealing that ZnO nanoparticles
in glycerol solution can restrain E. coli proliferation. The survival
The structural properties of the products were analyzed using ratio decreases with increasing ZnO nanoparticles concentrations
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 hexago-
nal 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. Fig. 2. TEM image of ZnO nanoparticles.
200 R. Jalal et al. / Materials Chemistry and Physics 121 (2010) 198 201
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 mini-
mal 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 nanopar-
ticles 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 Vijayaragha-
van [25] reported that the generation of H2O2 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-, H2O2,
and O22- 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 H2O 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 H2O2. The gen-
erated H2O2 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 bac-
teria; however, H2O2 can penetrate into the cell [25]. It is plausible
to say that the concentration of nanofluids is comparable with the
amount of H2O2. The amount of H2O2 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 increas-
ing concentration of ZnO nanofluid and time is assumed due to the
increase of H2O2 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 Fig. 4. Changes in survival ratio of E. coli by using a concentration of 0.25 and
0.5gdm-3 (a) without ZnO nanoparticles (b) without ZnO nanoparticles in the
of ammonium citrate although 0.5 g dm-3 was more effective in
absence of (circle symbol) and in the presence of solvent, glycerol and ammonium
growth reduction of E. coli than that of 0.25 g m-3. The ammonium citrate (the amount of solvent for triangle and square symbols are the same as those
of (a)).
citrate at a concentration of 0.5 g dm-3 caused a reduction in the
R. Jalal et al. / Materials Chemistry and Physics 121 (2010) 198 201 201
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