Increased Bioactivity of Zinc Oxide Nanoparticles: An

Investigation into Antimicrobial Properties

Abstract
In this study, we investigate the antibacterial activity of ZnO nanoparticles
with various particle sizes. ZnO was prepared by the base hydrolysis of zinc
acetate in a 2-propanol medium and also by a precipitation method using
Zn(NO3)2 and NaOH. The products were characterized by x-ray diffraction
(XRD) analysis, transmission electron microscopy (TEM) and
photoluminescence (PL) spectroscopy. Bacteriological tests such as
minimum inhibitory concentration (MIC) and disk diffusion were performed
in Luria-Bertani and nutrient agar media on solid agar plates and in liquid
broth systems using different concentrations of ZnO by a standard microbial
method for the first time. Our bacteriological study showed the enhanced
biocidal activity of ZnO nanoparticles compared with bulk ZnO in repeated
experiments. This demonstrated that the bactericidal efficacy of ZnO
nanoparticles increases with decreasing particle size. It is proposed that both
the abrasiveness and the surface oxygen species of ZnO nanoparticles
promote the biocidal properties of ZnO nanoparticles.

Keywords: metal oxides, precipitation, transmission electron

microscopy (TEM), photoluminescence, antimicrobial

1. Introduction metabolism. Sawai et al [7] examined the minimal

inhibitory concentration obtained by
In recent years, ZnO being a wide band-gap
semiconductor (3.36 eV), has received increased
attention regarding potential electronic applications
due to its unique optical, electrical and chemical
properties [1]. The availability of a wide range of
nanostructures makes ZnO an ideal material for
nanoscale optoelectronics [2] and piezoelectric
nanogenerators [3] as well as in biotechnology [4].
Furthermore, ZnO appears to strongly resist
microorganisms [5]. There are some reports [6] on
the considerable antibacterial activity of CaO,
MgO and ZnO, which is attributed to the generation
of reactive oxygen species on the surface of these
oxides, studied by a conductometric method. The
advantage of using these inorganic oxides as
antimicrobial agents is that they contain mineral
elements essential to humans and exhibit strong
activity even when administered in small amounts.
The activity is quantitatively evaluated by studying
the growth medium caused by the bacterial
indirect conductometric assay to evaluate the advantages of inorganic antibacterial materials over
antibacterial activity of insoluble ceramic powders organic antibacterial materials are that the former
compared with several types of antibiotics. They show superior durability, less toxicity and greater
reported that the activity was affected by particle selectivity and heat resistance. TiO2 and ZnO
size, which is controlled by processing parameters. semiconductors have been extensively studied as
Many researchers have attempted to correlate antimicrobial agents due to their photocatalytic
the biological activity of inorganic antibacterial activity under UV light [10, 11]. Generally, the
agents with the size of the constituent particles [8, antimicrobial mechanism of chemical agents can be
9]. Inorganic nanocrystalline metal oxides are understood by studying the specific binding of the
particularly interesting because they can be surface of the agent with a microorganism and the
prepared with extremely high surface areas, and consequent
are more suitable for biological applications. The

1468-6996/08/035004+07$30.00 1 © 2008 National Institute for Materials

Science Printed in the UK
metabolism of the agents inside the microorganism. Vellore, as well as other materials for bacterial
These antimicrobial substances based on inorganic cultivation, Pyrex petri dishes were purchased from
chemicals have been found to be effective for Tarson. Phase purity and grain size were determined
therapy. In addition to oxides, many researchers by x-ray diffraction (XRD) analysis recorded on a
have also reported the antimicrobial activityof metal Siefert x-ray diffractometer using CuKα radiation
ions [12, 13]. (λ 1.54016 Å) at 60 keV over the range of 20–70◦.
Although many studies on the biological The average grain size was determined by x-ray line
activity of ZnO have been carried out, most of broadening using the Scherrer equation after
these pertain to the antimicrobial effect of bulk ZnO incorporating corrections. The morphology and
with a large particle size. Yamamoto [14] studied size of the particles were determined by high-
the bacterial activity of ZnO with various particle resolution transmission electron microscopy
sizes in the range of 0.1–1 µm. The activity of ZnO (HRTEM) using a JEOL JEM 3010 electron
powder slurries was measured using a conductance microscope at an accelerating voltage of 200 keV.
method. The change in electrical conductivity with Samples were prepared by making a clear dispersion
bacterial growth was correlated to the antibacterial of the ZnO nanopowder in 2-propanol and by
activity of ZnO, which increased with decreasing placing a drop of the solution on a carbon-coated
particle size. Here, we report two different copper grid. Photoluminescence (PL) spectra were
methodologies for synthesizing nanosized ZnO: a recorded on a Perkin-Elmer LS 55
precipitation method and the base hydrolysis of spectrophotometer
zinc acetate in a 2-propanol medium with a
stabilizing agent. We have studied the structural
and optical properties of the products and have
also evaluated their biological activity by a
standard microbial method adopted for metal
oxides. Using both methods, it was found that the
synthesized ZnO contains particles of sizes in the
range of 10–50 nm. These particles exhibit both
qualitatively and quantitatively better antibacterial
properties than bulk ZnO with a particle size of 2
µm. The results clearly demonstrate that
nanosized ZnO is a more effective antimicrobial
agent than bulk ZnO. To the best of our
knowledge, this is the first systematic study in
which nano and bulk ZnO are compared regarding
the inactivation/destruction of bacteria using a
standard microbial method.

2. Experimental

2.1. Materials and methods

Zinc acetate and 2-propanol were obtained from

SD-Fine Chemicals, Ltd (99% purity), 2-
mercaptoethanol was obtained from Aldrich, zinc
nitrate (99% purity) was obtained from Merck, and
NaOH and H2O2 (30%) were purchased from
Thomas Baker Pvt., Ltd. Double distilled water was
used for all the preparations. The bacteria strain
Escherichia coli (DH 5α) was acquired from the
Biological Sciences Department, VIT University,
2
=
using a xenon laser with an output power of 450 W in an autoclave before the experiments. Luria
at 300 K as the excitation source. To facilitate the Bertani (LB) broth and nutrient agar were used as
measurements, the ZnO nanocrystals were sources for culturing E. coli at 37 ◦C on a rotary
compacted into pellets of 12 mm diameter and 0.4 platform in an incubator. The density of bacterial
mm thickness. The specific surface area was cells in the liquid cultures was estimated by optical
determined by measuring the adsorption density (OD) measurements at 600 nm wavelength
isotherms of N2 at and was maintained at 0.8–1.0, which is the ideal
−196 ◦C using a BET Micromeritics ASAP 2020 optical density of the cells. The cell suspensions
model. used for antibacterial activity contained
105 colony-forming units (CFU) ml−1. The
2.2. Preparation of ZnO nanoparticles
antibacterial activity of ZnO was measured by
We used two different methods for synthesizing paper disk diffusion assay in terms of minimum
nanocrystalline ZnO with various particle sizes. inhibitory concentration (MIC) and minimum
Sample 1 was synthesized by mixing zinc nitrate (0.1 bactericidal concentration (MBC).
M) and sodium hydroxide (0.2 M) at room The petri plates used in the tests were prepared
temperature while constantly stirring for 2 h. The using a nutrient agar medium. The bacteria were
white precipitate formed was washed thoroughly sprayed evenly on top of the plates using a sterile
with double distilled water to remove all the ions; glass rod. After allowing the bacteria to dry (within
then, centrifuged at 3000 rpm for 5 min. The 5–10 min) test solutions of ZnO of various
procedure was repeated several times until the concentrations (different particle sizes) were
precipitate was free from Na+ dropped within a disk of 8 mm diameter. The zone
and NO3− ions. Then, 1 mol H2 O2 was added to the of inhibition was
precipitate to make a translucent sol maintaining
temperature at 75 ◦C for
1 h. The obtained sol was dried in a hot air oven for 3
h and further calcined at 350 ◦C for 6 h to form ZnO
nanocrystals.
Surface-modified nanocrystalline ZnO
(sample 2) was prepared by dissolving 0.5 mmol of
·
zinc acetate [(CH3COO)2Zn 2H2O] in 100 ml
of 2-propanol at 80 ◦C while constantly stirring.
To this, 0.3 mmol of 2-mercaptoethanol was added,
and the mixture was continually stirred for 2 h. The
2-mercaptoethanol was introduced as a capping
molecule, which stabilizes the ZnO nanoparticles.
The resultant mixture was then hydrolyzed by
adding 1.25 mmol of NaOH in 2-propanol followed
by ultrasonic agitation for 2 h. The solvent was then
removed and the product was washed with water
and centrifuged at 2000 rpm. The procedure was
repeated several times and then dried in an oven at
80 ◦C for 12 h to form ZnO nanocrystals.

2.3. Bacterial cultures and

evaluation of antibacterial
activities
For antibacterial experiments, E. coli, a Gram
negative bacterium, was selected as the target
organism. All disks and materials were sterilized
3
measured after 18 h incubation. To evaluate the MIC, ∆
ZnO2 · 2H2O ZnO

(101)
105 CFU ml−1 E. coli in
an appropriate volume of−−−−→

(100)

(002)
LB broth was added to bulk and nanosized ZnO In method II, zinc acetate was dissolved in 2-
suspensions whose concentrations varied from 0.01 propanol, after which 0.3 mol thiol was added while

Intensity (a.u.)
to 100 mM. Compounds were tested three times and constantly stirring. Generally, acetate-complexing
the results were averaged. Negative and positive ligands can form unidentate, bidentate (chelating)
control tubes contained only inoculated broth and and bridging bonding structures with zinc ions [21].
free ZnO solution, respectively. The tubes were The possible formation of Zn4O(Ac)6 is reported
incubated at 37 ◦C for 18–20 h. The visual turbidity elsewhere [22], where the structural similarity of
of the tubes was noted before and after incubation. Zn4O(Ac)6 and ZnO, which favors the
Aliquots from tubes (100 µl) that appeared to have condensation of this molecular building block into
little or no cell growth were plated on nutrient agar ZnO, is discussed. To avoid the agglomeration, 2-
plates to distinguish between the bacteriostatic and mercaptoethanol was introduced as a capping
bactericidal effects. The plates were then incubated agent. It has been well established that the
and the colonies were quantified. stabilization and surface passivation of ZnO
nanoparticles are vitally important for the
development of their electrical and optical
3. Results and discussion
properties. When no capping molecules are
3.1. Preparation of nanocrystalline ZnO

There are several methods for preparing nanosized

ZnO powders such as spray pyrolysis [15],
precipitation [16], thermal decomposition [17],
hydrothermal synthesis [18] and electrochemical
growth [19]. Different methods yield different
particle sizes of ZnO, depending on the type of
precursor, the solvent, the pH and the temperature
of the reacting solution. The choice of method
depends on the final application. In this study, we
employed a precipitation method to prepare
nanosized ZnO, which is the easiest method for
producing nanomaterials [20]. In method I, zinc
nitrate and sodium hydroxide were mixed at room
temperature and a precipitation reaction occurred
while stirring for 2 h, after which the pH was 10.8.

Zn(NO3 )2 · 6H2 O + 2NaOH

GGGGGA Zn(OH)2 + NaNO3

Zn (OH)2 was washed thoroughly to remove

NaOH and NaNO3 until the pH became neutral.
Hydrogen peroxide at 75 ◦C was then added
dropwise to produce a translucent sol of zinc
peroxide, which was then heated at 350 ◦C to
produce ZnO with active surface oxygen species.

Zn(OH)2 + H2 O2 GGGGGA
ZnO2 · 2H2 O

4
 crystallite sizes of ZnO obtained by both methods
indicates that method I (precipitation) led to a
higher range ofcrystallite size than method II (base
hydrolysis of zinc acetate in propanol medium),
reflecting the effect of the temperature during the
process on crystallite size.
(a)
Typical TEM images of the ZnO nanoparticles
are shown in figures 2(a) and (b). Direct comparison

(110)

(103)

(112)
(102)
of the TEM images reveals that the uncapped ZnO
nanoparticles are prone to aggregation, whereas the

(101)
thiol-capped nanoparticles are well separated.(b) The

(002)
(100)
particles were found to be almost spherical. Note

(110)

(103)

(112)
(102)
that ZnO nanoparticles modified by thiol have a
narrow size distribution (figure 2(b)) compared
with those without modification (figure 2(a)). A
20 30 40 50 60 70 statistical analysis reveals that the mean particle
2 (deg) sizes are approximately 12 nm for thiol-capped ZnO
and 47 nm for ZnO without a capping agent.
Figure 1. (a) XRD pattern of ZnO formed by
precipitation method (sample 1). (b) XRD pattern
of ZnO formed by base hydrolysis in propanol
medium (sample 2).

introduced, particle interaction and aggregation are

observed in ZnO sample 1 obtained by the
precipitation method. The presence of capping
molecules appears to affect the kinetics of
nucleation and accumulation in such a way that the
rate of growth of large particle decreases while that
of small particles remains the same. This should
result in a narrowing of the size distribution of the
particles in the product.

3.2. Characterization of nanocrystalline ZnO

3.2.1 XRD patterns and TEM morphologies. XRD

patterns of the ZnO samples prepared by the two
methods are shown in figure 1. The diffraction
pattern and interplanar spacing closely match those
in the standard diffraction pattern of wurtzite ZnO.
Figure 1(a) shows the peak broadening of the
XRD pattern for sample 1. With increasing
annealing temperature the crystallinity of the
sample appears to improve and the grain size is also
increased. The broadened peaks in the XRD
pattern of sample 2 obtained by base hydrolysis
(figure 1(b)) indicates the formation of ZnO
nanocrystals with small crystallites. The crystallite
size obtained by the two synthesis methods was
estimated by the Scherrer equation and found to be
in the range of 20–40 nm. Comparison of the
5
 spectrum of ZnO nanocrystals with an excitation
wavelength at 325 nm, which is in accordance with
results in the literature [23–27]. A relatively sharp,
weak UV emission band at 3.23 eV (384 nm) and a
broad stronger emission band in the green part of the
visible spectrum with a maximum intensity at 2.4
eV (520 nm) are observed. The UV band is due to the
radiative annihilation of excitons (exciton
emission). The origin of the broad green emission
band at 2.38 eV is attributed to surface anion
vacancies [24]. It involves the tunneling of surface-
bound electrons through preexisting trapped holes

Figure 2. (a) TEM micrograph and SAED

pattern (inset) of ZnO formed by precipitation
method (sample 1). (b) TEM micrograph and
SAED pattern (inset) of ZnO formed by base
hydrolysis in propanol medium (sample 2).

It can be seen from the selected-area electron

diffraction (SAED) patterns of ZnO nanocrystals
that the polycrystalline diffraction spots correspond
to wurtzite ZnO. In the insets of figures 2(a) and
(b), the spotted diffraction rings indicate the
preferential orientations of ZnO.

3.2.2 Photoluminescence. Figure 3 shows the PL

6
Figure 3. PL spectrum at 300 K of sample 1 are given in table 1. The presence of an inhibition
synthesized by precipitation method. zone clearly indicates that the mechanism of the
biocidal action
Table 1. Correlation of the size of the zone of
inhibition with ZnO.

Zone of
inhibition ZnO particle size
(mm) E.
coli

12 nm 31 (0.1)
45 nm 27 (0.1)
2 µm 22 (0.3)

in oxygen vacancies (Vo•) resulting in the creation

of recombination centers (Vo••) for visible
emission. Mo et al [25] elucidated this in terms of
defect levels associated with oxygen vacancies and
zinc interstices. These defects are most probably on
the nanocrystal surfaces, causing faster and more
effective trapping of the photogenerated holes at
the surface sites.
The PL spectrum also exhibits two additional
weak peaks at 2.95 eV (420 nm) and 2.56 eV (485
nm). These PL signals are attributed to band-edge
free excitons and bound excitons, respectively [26].
The intensity of the visible emission peak also
correlates with the particle size [27].

3.3. Evaluation of antibacterial properties

In our study, the relative antibacterial activity of

ZnO suspensions of particles with sizes of 12 nm,
45 nm and 2 µm toward E. coli was studied
qualitatively in aqueous LB broth by disk
diffusion and quantitatively in terms of the MIC
and MBC. A standard testing protocol was
employed that is applicable to inorganic metal
oxides and composite materials such as AgBr-
polymer and Ag-SiO2 [28, 29]. The MBC is the
lowest concentration (µg ml−1) at which a
compound will kill more than 99% of the added
bacteria. The MIC of the agent is the concentration
at which the solution becomes turbid [30]. A lower
MIC corresponds to higher antibacterial
effectiveness. In terms of both MIC and MBC, we
found an inverse relationship between the particle
size and activity.
The ability of the antimicrobial agent to rupture
bacterial cells is tested by the disk diffusion
method. Here, three ZnO suspensions with
7
different particle sizes were tested, and the results
Table 2. Comparison of biocidal effect of micron and nano ZnO by 90
MIC towards E. coli.

Bactericidal efficiency (%)

Status of
bioactivity Sample (ZnO) MIC (µg ml−1)
(after 24 hr incubation)
0.01 mM 0.8 lawn 60
0.1 mM 8 lawn Sample 1
1 mM 80 bacteriostatic Sample 2
5 mM 400 bactericidal Bulk
10 mM 800 bactericidal 30 (a)
50 mM 4000 bactericidal
100 mM 8000 bactericidal
0.0 0.2 0.4 0.6 0.8 1.0
of ZnO involves disrupting the membrane. The Concentration (mM)
high rate of generation of surface oxygen species
from ZnO leads to the death of the bacteria. 10
0
Interestingly, the size of the inhibition zone
increased significantly with decreasing size of the
ZnO particles. Bactericidal efficiency (%)
MIC and MBC were determined by the standard
microbial method. ZnO suspensions with different Sampl
9 e1
concentrations of micron-sized and nanosized ZnO 0 Sampl
(b)
were incubated with E. coli in aqueous LB broth. e2
Bacterial growth was studied by visually inspecting Bulk
the LB broth for turbidity. If the material being
tested does not kill but instead inhibits the growth
of bacteria (bacteriostatic agent), the bacteria will 8
grow when it is removed from the solution 0 0 20 40 60 80 100
containing the material, and colonies will be Concentration (mM)
observed upon plating an aliquot. If the material
being tested is bactericidal, the

absence of bacterial colonies will be observed upon mM), respectively. We noticed that in both cases,
plating. To establish whether the suspensions were the ZnO suspension with 12 nm particles is more
bacteriostatic or bactericidal, 100 µl aliquots were effective than the suspensions with other particle
taken from the incubated LB broth, each containing sizes. This can be explained on the basis of the
ZnO and E. coli, and were plated on nutrient agar oxygen species released on the surface of ZnO,
plates and incubated for 18–20 h. The results are which cause fatal damage to microorganisms [31].
summarized in table 2. ZnO suspension with a The generation of highly reactive species such as
concentration in the range of 100 to 5 mM effectively OH−, H2O2 and O22− is explained as follows. Since
inhibits bacterial growth. No significant ZnO with defects can be activated by both UV and
antibacterial activity was observed at concentrations visible light, electron-hole pairs (e−h+) can be
less than 1 mM for all the ZnO samples. However, the created. The holes split H2O molecules (from the
bactericidal efficacy at 1 mM is higher for the 12 nm suspension of ZnO) into OH− and H+. Dissolved
ZnO suspension (sample 2–86%) than for the 45 nm oxygen molecules are transformed to superoxide
and 2 µm ZnO suspensions. radical anions (• O−2 ), which in turn react with H+
The bactericidal efficacies of ZnO suspensions to generate (HO2•) radicals, which upon subsequent
collision
with three different particle sizes after 24 h with electrons produce hydrogen peroxide anions
incubation of the aliquots are shown in figures 4(a) (HO2−). They then react with hydrogen ions to
and (b) in the lowest concentration range (0.01–1 produce molecules of
mM) and the highest concentration range (5–100
8
Figure 4. (a) and (b) Bactericidal efficiency of
sample 1, 2 and bulk
ZnO suspensions at different concentrations.

H2O2. The generated H2O2 can penetrate the cell

membrane and kill the bacteria [32].

ZnO + hv → e− + h+; h+ + H2O →• OH

+ H+.e− + O2 →• O−2 ; • O2 + H+ →

HO2 •

HO2• + H+ + e− → H2O2

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, H2O2 can penetrate into the cell
[33].
Considering the size of the bacteria, it is
evident that a single isolated colony of E. coli
of 2 µm diameter can accommodate a large number
of 12 nm particles of ZnO (sample 2) and fewer 45
nm particles (sample 1), resulting in different
biocidal properties of the two suspensions. Once
ZnO kills/captures the cell membrane, the ZnO
nanoparticles presumably remain tightly adsorbed
on the surface of the leftover/dead bacteria
preventing further antibacterial action. However,
ZnO nanoparticles continue to release peroxides
into the medium even after the surface of the dead
bacteria are completely covered by ZnO
nanoparticles, thereby showing high bactericidal
efficacy. From the results for MIC, we confirm that
the smaller particle size shows enhanced activity
due to the large surface area to volume ratio and
the surface

9
reactivity of ZnO. In comparison, the bulk ZnO may be that zinc is an essential cofactor in a variety
showed less bactericidal activity than ZnO of cellular processes. Although metals and metal
nanoparticles. From the BET measurement, the oxides are known to be toxic at relatively high
specific surface area of sample 1 (mean particle size concentrations, they are not expected to be toxic at
is 47 nm by TEM) is found to be 68 m2 g−1, low concentrations. To investigate the survival of
whereas that of sample 2 (mean particle size is bacteria under acidic conditions the pH was lowered
12 nm by TEM) is 115 m2 g−1. The agglomeration by adding acetic acid (0.1 M) dropwise and the pH
of ZnO nanoparticles is minimized by the presence was maintained at 2.3. As anticipated, no colonies
of the capping agent (2-mercaptoethanol) in were seen on the plate. We then increased the pH by
sample 2. ZnO nanoparticles of both sizes have a adding 0.1 M NaOH dropwise until it reached 8.5
high surface area compared with bulk ZnO (5.11 m2 (weakly basic). No colonies were observed at this
g−1). The generation of H2O2 depends strongly on pH. This indicates that a pH in the range of 6–8 does
the surface area of ZnO, which results in more not affect the growth of the bacteria, irrespective of
oxygen species on the surface and the higher the metal ions present. We could only observe a
antibacterial activity of the smaller nanoparticles bacteriostatic effect above a ZnCl2 concentration of 20
[34]. mM. Excess metal ions are toxic to bacterial cells.
The detailed mechanism for the activity of ZnO Therefore, some bacteria have developed
is still under debate. One possible explanation of mechanisms to regulate the influx and efflux
the antibacterial effect of ZnO is based on the processes to maintain a steady intracellular
abrasive surface texture ofZnO. ZnO nanoparticles concentration of ions including Zn2+ ions [38]. In
have been found to be abrasive due to surface this study we demonstrated the formation of large
defects [9]. The PL spectrum of ZnO (figure 3) numbers of colonies (a bacterial lawn)
confirms the presence of surface defects (broad
visible emission band) in the region of 450–550 nm.
The abrasiveness of ZnO nanoparticles compared
with bulk ZnO is caused by the uneven surface
texture due to rough edges and corners. This surface
roughness contributes to the mechanical damage of
the cell membrane of E. coli. Wang et al [35]
proposed that the orientation of ZnO can also affect
bioactivity. They showed that randomly oriented
ZnO nanowires show higher antibacterial activity
than regularly oriented ZnO nanowires due to the
different spatial arrangements of ZnO causing the
biocidal activity. The interaction of metal ions
including zinc with microbes has also been studied
[36].
ZnO suspensions in the lower concentration
range (0.01–1 mM) seem to exhibit less antimicrobial
activity. This may be due to the possible presence of
fewer Zn2+ ions, which might act as nutrient [37]. To
verify this, we studied the antimicrobial activity of
ZnCl2 with different concentrations (in suspensions
similar to those of ZnO at various pH values). The
initial pH of 5 mM ZnCl2 suspensionwas 5.9
(weakly acidic), which resulted in the rapid
bacterial growth of
E. coli, indicating that Zn2+ is not toxic at lower
concentrations and at this pH. The reason for this
1
0
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We have demonstrated the enhanced bioactivity of
ZnO nanoparticles by studying the antimicrobial
activity of suspensions with various particle sizes
using a standard microbial method for the first
time. The enhanced bioactivity of smaller particles
is attributed to the higher surface area to volume
ratio. For smaller ZnO nanoparticles, more
particles are needed to cover a bacterial colony (2
µm) which results in the generation of a larger
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cell membrane and the enhanced bactericidal effect
of ZnO nanoparticles.

Acknowledgments

The authors thank the VIT management and the

DRDO Grant in aid scheme, Government of India,
for financial assistance. The authors thank Dr A
Radha for her help in antimicrobial study and
Professor T Pradeep, IITM, for his help in TEM

imaging.

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