Copyright 2011 American Scientic Publishers Journal of

All rights reserved Biomedical Nanotechnology

Printed in the United States of America Vol. 7, 110, 2011

Interaction of ZnO Nanoparticles with Microbes

A Physio and Biochemical Assay
Nagarajan Padmavathy and Rajagopalan Vijayaraghavan
Center for Nanomaterials, School of Advanced Sciences, VIT University, Vellore 632014, Tamilnadu, India

The antibacterial activity of ZnO nanoparticles (nano ZnO) on various microorganisms has been
investigated in this study and acting mechanism is proposed for E. coli a model organism by ana-
lyzing the growth, permeability and morphology of the bacterial cells. The experimental results
indicated 1 mmoL (81.4 g) nano ZnO could completely inhibit the growth of 107 cfu/mL bacterial
cells in liquid Luria-Betrani medium. In the biochemical study, it has been observed that, nano ZnO
resulted in the leakage of proteins. Chemiluminescence assay proved the generation of the reactive
oxygen species into active state. When the cells of E. coli were exposed to nano ZnO, many pits
were observed in bacterial cells by TEM and Fluorescence microscopy and the cells were found
to be fragmentary. Released zinc ion from has been monitored by AAS. It suggested that nano
ZnO is able to destroy the permeability of the bacterial membranes. Our study demonstrated that
nano ZnO damages the structure of bacterial cell membrane and depressed the activity of some
membranous enzymes by ROS production which caused E. coli bacteria to die eventually.
Keywords: ZnO Nanoparticles, Antibacterial, Biochemical Study, Fluorescence Probes,
Mechanism.

1. INTRODUCTION
ZnO is being currently explored as an antibacterial agent
in both bulk and nano forms. It appears that these nanopar-
ticles could be more toxic to bacteria than any other frac-
tions of nanoparticles or their counterpart bulk species.1 2
The altered properties of nanoparticles, with compara-
ble sizes to naturally occurring biological structures, may
facilitate their interaction with biomolecules on both the
cell surface and within the cell and hence potentially affect
cellular responses in a dynamic and selective manner.
However the mechanism of the antibacterial action has to
be addressed in a profound manner. Several factors such
as particle size,3 oxygen species,4 membrane dysfunction5
and orientation of nanoparticles6 7 have been attributed and
trying to explain the mechanism by which ZnO ruptures
the bacterial cells. However, its effectiveness in deacti-
vating and killing microorganisms related mainly to pho-
toactivation and the resulting free radicals called, ROS
(Reactive Oxygen Species). Hirota et al.8 reported sig-
nicant antibacterial effect under dark conditions of ZnO
ceramics. Sawai et al.9 clearly showing that ROS concen-
trations increased with the ZnO content of slurries makes
this mechanism is better to understand further detailed
evaluations.
Author to whom correspondence should be addressed.
RESEARCH ARTICLE
Ohira et al.10 demonstrated the antibacterial activity
of ZnO related with crystallographic orientation. They
observed the antibacterial activity in ZnO powder with
crystallographic orientation was weaker than that in com-
mercial ZnO powder without orientation at same powder
concentration. Regarding specic surface area of the ZnO
powders used in antibacterial tests, however, antibacterial
activity in powder with orientation was found to be simi-
lar to that without orientation, that is, the crystallographic
orientation of ZnO did not affect antibacterial activity.
By investigating the effect of the particle size on the
antibacterial properties, the antibacterial mechanism ZnO
nanoparticles has been proposed. The interaction, inu-
ence, and possible toxicity of nanomaterials to bacterial
cells of interest include cellular uptake, membrane per-
turbations and production of reactive oxygen species. The
correlation between particle size, concentration, ROS gen-
eration, leaching of protein membrane and the ratio of ZnO
to Zn2+ ions in the culture medium, the inhibition to organ-
isms correlate with the fraction of ZnO nanoparticles of
size less than 15 nm in the suspension.
In this paper, in order to understand the antibacterial
activity and acting mechanism of ZnO nanoparticles in
detail by studying the physiochemical interaction of nano
ZnO with bacterial cells at extra and intra cellular lev-
els. Evidences have been offered to indicate that ZnO

J. Biomed. Nanotechnol. 2011, Vol. 7, No. 6 1550-7033/2011/7/001/010 doi:10.1166/jbn.2011.1343 1

 Interaction of ZnO Nanoparticles with MicrobesA Physio and Biochemical Assay
nanoparticles can inhibit bacterial growth and even kill
the cells through destroying bacterial membranous struc-
ture and permeability. Standard testing microbial methods
have been employed to investigate the mode of antibac-
terial action of nano-ZnO against certain gram positive
and gram negative bacteria. Using microscopic techniques
(TEM, and Fluorescent) the damaged structures of bacte-
rial cells were seen. Together with supporting biochemical
studies, the results divulge, several primary actions such
as leaching of outer membrane, ROS generation, release
of Zn2+ ions from ZnO etc. of nano-ZnO in E. coli cells
in a denitive pathway.
2. METHODS
2.1. Preparation of Thiol Capped ZnO Nanoparticles
Surface modied nanocrystalline ZnO was prepared by
dissolving 0.5 mmol of zinc acetate (99%, Merck),
[(CH3 COO)2 Zn 2H2 O] in 100 mL of 2-propanol (SD-
Fine chemicals Ltd), with constant stirring at 50  C. To
this, 0.3 mmoL of 2-mercaptoethanol (Aldrich) was added
with continued stirring for 2h. It was introduced as a cap-
ping molecule which serves as a stabilizing agent for ZnO
nanoparticles. The resultant mixture was then hydrolyzed
by adding 1.25 mmoL of NaOH (SD-Fine chemicals Ltd)
in 2-propanol under ultrasonic agitation for 2 h. Then
the solvent was removed and the product was washed
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with water, centrifuged at 2000 rpm. The procedure was
repeated several times and then dried in oven at 80  C for
12 h resulting ZnO nanocrystals. Phase purity and grain
size were determined from X-ray diffraction, recorded on
a SIEFERT X-ray diffractometer using Cu K radiation
( = 154016 ) at 60 KeV over the range 20 70 . The
average grain size was determined from X-ray line broad-
ening using Scherrer equation after incorporating correc-
tions. The morphology and size of the particles were
determined from HRTEM using JEM 3010 JEOL electron
microscope at an accelerating voltage of 200 keV. Sam-
ples were prepared by making a clear dispersion of the
ZnO nano powder in 2-propanol and placing a drop of the
solution on a carbon coated copper grid.
2.2. Assessment of Antibacterial Activity
Five different bacterial strains have been chosen such as
E. coli JM 101, S.aureus RN6390, B.subtilis168, Klebsiella
oxytoca ATCC4386 and Proteus mirabilis H14320 which
were from the stock of Biological Sciences Department,
VIT University, Vellore and other materials for bacterial
cultivations were purchased from Tarson. To understand
the mechanism better, other studies have been carried out
on E. coli only.
The minimal inhibitory concentration (MIC) of ZnO
suspension was determined using clinical and laboratory
2 J. Biomed. Nanotechnol. 7, 110, 2011
Padmavathy and Vijayaraghavan
standard protocol with few modications. Briey to cal-
culate MIC, Luria Betrani media containing ZnO parti-
cles were inoculated with an overnight culture of bacterial
cells to nal OD of 0.002. The concentration of ZnO that
resulted in no growth was the MIC. Growth was visually
assessed by changes in turbidity and quantied by mea-
suring optical density at 600 nm (OD600  using a UV-Vis
spectrophotometer (Shimadzu UV3600, spectrophotome-
ter). The cell viability count was performed by serial dilu-
tion of the pregrown culture followed by plating on a solid
media (LB agar penicillin plate). The viable cell number
was recorded by counting the number of bacterial colonies
grown on the plate multiplied by the dilution factor and
expressed as CFU/mL. All experiments have been carried
out in triplicates.
2.3. Confocal Laser Scanning Microscopy (CLSM)
The combination of CLSM and digital image analysis
enable the high-resolution imaging of biological structures
with negligible background interface. Moreover, CLSM
allows cells to be studied in their functional native (wet)
environments, thus providing a direct and nondestructive
assessment of bacterial activity with different nano-
structure. In this study, CLSM (Leica SP2, confocal micro-
scope) technique was employed for understanding the
antibacterial activity of ZnO nanoparticles by visualizing
directly. Using this technique, the effect of in-use surfaces
of different ZnO nanoparticles on Escherichia coli was
determined based on inhibition of esterase activity which
was probed by propidium iodide (PI).
A fresh working solution was made by adding 40 mL
of 1 mg of (PI) propidium iodide (Sigma) per milliliter
of Phosphate Buffer Saline (PBS) (pH = 70, stored in the
dark at 4  C) to 1 mL of PBS. The combination was then
ushed gently with PBS to reduce background after keep-
ing in the dark environment for 30 min. Individual aliquots
of 50 L were withdrawn from the appropriate medium
and then placed over microscope slides, which were air-
dried for further observation. The excitation wavelength
was 568 nm, and the observation lter had a long-pass
lter wavelength above 615 nm.
2.4. Protein Assay
The total protein concentration was measured by Bradford
method using bovine serum albumin as the standard. Cells
were harvested from 50 ml culture broth by centrifuga-
tion (4  C, 15 min, 6000 rpm) in a Remi centrifuge (Remi
instruments, India). The extracellular protein containing
residue was resuspended in 40 ml physiological solution
(0.9% NaCl) and diluted until the optical density was 0.2.
This is referred as the culture solution. After an incubation
period of 15 min at room temperature, the absorbance at
595 nm was measured using a Shimadzu spectrophotome-
ter (Shimadzu UV3600, spectrophotometer).
Padmavathy and Vijayaraghavan Interaction of ZnO Nanoparticles with MicrobesA Physio and Biochemical Assay

2.5. Detection of Hydrogen Peroxide by
Chemiluminescence Assay
Intracellular ROS concentrations were determined using
an established uorescence assay with modication.11
Aliquots of cultures were removed from the bioreactor
and centrifuged at 10,000 rpm for 15 min. The precipitate
was then resuspended in a loading buffer solution (0.1 M
PBS) containing 127 g/mL amplex red (10-acetyl-3,7-
dihydroxyphenoxazine, Sigma), 100 g/mL HRP (Horse
Radish Peroxidase) to mimic normal growth and incubated
at room temperature for 30 min. After loading amplex
red in buffer solution with 100 g/mL of SDS (sodium
dodecyl sulfate), the cells were inoculated with prewarmed
growth medium, amended with nano ZnO (average size:
12 nm) at predetermined concentrations. The uores-
cence of the cells was measured using a spectrouorom-
eter (Perkin Elmer) with 563 nm excitation and 587 nm
emission lter. Fluorescence data were taken automati-
cally after 30 min incubation. Hydrogen peroxide (30%,
SD-ne) was used as a standard for ROS measurements
and intracellular ROS concentrations were determined in
H2 O2 units. The ROS measurement was done both under
UV light and in the uorescent light. The illuminating
UV light source with a wavelength of 365 nm was used
and an irradiance of 20 W/m2 was measured from the
light source incident to the top of the beakers by an UV
radiometer.
plexing ligand can form unidentate, bidentate (chelating)
and bridging bonding structure with zinc ions.12 In order
to avoid the agglomeration, 2-mercaptoethanol was intro-
duced as a capping agent. It has been well established that
stabilization and surface passivation of ZnO nano parti-
cles are vitally important for the development of electrical
and optical properties. The presence of capping molecules
appears to affect the kinetics of nucleation and accretion in
such a way that the growth of large particles slows down
while the growth of small particles remains the same. This
would result in narrowing down the size distribution of the
particles in the product.
3.2. XRD and TEM Characterization
In Figure 1, X-ray diffraction pattern of ZnO nanoparticles
was reported. By comparing the diffraction peak positions
with those reported in the JCPDS, a wurtzite structure
is assigned to the sample. The gure clearly shows an
increased broadening in the ZnO diffraction peaks, which
indicated the presence of nano crystallites. The average
crystallizes size, D, was estimated by using the Scherrer
formula as obtained from the full width at half-maximum
(FWHM) and found to be in the range of 20 nm.
The TEM images of ZnO nanoparticles show faceted,
high-contrast particles characteristic of crystalline aggre-
gates (Fig. 2(A)). The ED (Fig. 2(B)) pattern is character-
ized by rings that are typical of a polycrystalline powder

2.6. AAS Analysis
AAS was used for the quantitative determination of the
zinc ion concentration releasing from ZnO nanoparticles in
the presence of media containing bacterial cells. Bacterial
proteins and organic substances can dissolute ZnO into
Zn2+ ions and the Zn analysis was carried out using Varian
240 AAS analyzer at different time interval. The broth
culture containing ZnO nanoparticles was centrifuged at
5000 rpm for 20 min.
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reveals particles are in nano regime.
3.3. Effect of the ZnO Nanoparticles on the Bacterial
Reduction Rate
It is well known that E. coli is the most characterized bac-
terium and can cause gastroenteritis, urinary tract infec-
tions, and neonatal meningitis; Proteus causes chronic
respiratory infections in individuals with cystic brosis
and cancer; and Klebsiella can cause endocarditic as well

2.7. Transmission Electron Microscopy

TEM measurements have been performed using an EM
201 at 80 kV accelerating voltage. Approximately 107 bac-
teria were mixed with 0.1 g ZnO nanoparticles, shaken
well, and left undisturbed for 20 h. The suspension was
centrifuged down to a pellet and washed twice with
phosphate buffer saline. All samples for the TEM were
prepared following standard procedures for xing and
embedding of biological samples.

3. RESULTS
3.1. Preparation of Nanocrystalline ZnO
Zinc acetate was dissolved in 2-propanol and 0.3 mol thiol Fig. 1. X-ray diffraction pattern of thiol capped ZnO nanoparticles syn-
was added at constant stirring. Generally, acetate com- thesized by hydrolysis of zinc acetate by base.

J. Biomed. Nanotechnol. 7, 110, 2011 3

 Interaction of ZnO Nanoparticles with MicrobesA Physio and Biochemical Assay
(A)
(B)
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Fig. 2. Transmission electron micrographs of thiol capped ZnO
nanoparticles synthesized by hydrolysis of zinc acetate by base.
Fig. 3. Plots showing bactericidal activity of ZnO nanoparticles on wide
range of concentrations against various organisms with respect to stan-
dard antibiotic.
4 J. Biomed. Nanotechnol. 7, 110, 2011
Padmavathy and Vijayaraghavan
as bladder, prostate, and epididymal infections. Figure 3
demonstrates the antibacterial activity of ZnO nanoparti-
cles against these bacterial strains. The antibacterial ef-
cacies of ZnO nanoparticles were compared with the
standard antibiotic. Most diagnostically, the ZnO nanopar-
ticles were found to show potential antibacterial activity
against all tested bacterial stains. Compared to the stan-
dard penicillin used (100 g/mL), ZnO nanoparticles have
shown enhanced biocidal activity.
The concentration of ZnO nanoparticles was varied from
1 mmol to 5 mmol. The presence of these nanoparti-
cles at a concentration of 2 mmol caused more than 95%
inhibition of bacterial growth. A concentration between
12 mmoL could inhibit bacterial growth by 80%. Among
the microbes, Gram negative E. coli and Gram posi-
tive B. subtilis exhibited better bactericidal efciency than
other pathogens. At the same time, signicant inhibition
(6570%) was still observed for Proteus and Klebsiella
pathogenic bacterial strains.
The minimal inhibitory concentration (MIC) was mea-
sured against E.coli between 15 mmoL ZnO nanoparti-
cles concentrations (Fig. 4). The results demonstrated that
there was a signicant change in the bactericidal property
of ZnO different concentrations. It was noted MIC lies
between 1.52 mmoL. To nd out, the biocidal effect, the
tests were performed upto 5 mmol ZnO concentration.
The bactericidal efcacy was 50%, 62% and 81% for
0.5, 1 and 1.5 mmol respectively. At 2 mmol 89% of
biocidal effect was noticed. From 2.5 mmol to 5 mmol,
complete death phase was obtained. These results indicate
that an increase in nanoparticle concentration produces
strong antibacterial activity against E. coli. and illustrate
that Zn2+ ions act as supplement for metabolic action of
bacteria at trace concentrations.13
2-mercaptoethanol (0.03 mmoL) was employed for sta-
bilizing ZnO particles, and thiol correction antibacterial
studies have been performed. The specic role of sur-
face capping (2-mercapto ethanol) was also found to pro-
Fig. 4. Bactericidal activity of ZnO nanoparticles on wide range of con-
centrations on E.coli.
Padmavathy and Vijayaraghavan Interaction of ZnO Nanoparticles with MicrobesA Physio and Biochemical Assay
foundly inuence the toxicity of ZnO nanoparticles toward
the cells. It has exhibited that the use of thiol has 15%
inhibition of growth of E. coli. The reason for this inhi-
bition could be attributed that being negatively charged
on the surface, thiol molecules interfere with absorption
of microbes, on the surface of capped ZnO nanoparticles.
Given the experimental certainty and small size difference
of particles between the ZnO with and without stabilized
nanoparticles, the effect of thiol was negligible.
The antibacterial effect of poly vinyl pyrrolidone (PVP)
and sodium dodecylsulfate (SDS) stabilized silver and
platinum nanoparticles have also been studied14 and
showed that both silver and platinum nanoparticles had
less antibacterial activity with SDS, whereas, strong effect
was found with PVP. So, the presence of surfactant may
affect the mechanisms in different ways when capped with
nanoparticles.
3.4. Confocal Laser Scanning Microscopy (CLSM)
Figure 5 reveals the time-dependent confocal uorescence
micrographs of the control and the bacterial cells exposed
with ZnO nanoparticles stained with Propidium Iodide (PI).
(A)
10m
(C)
10m
Fig. 5. Confocal laser scanning uorescence micrographs of E. coli treated with ZnO nanoparticles stained with PI. Series (A) referring 0 h; (B) refers
to 6 h; (C) refers to 18 h; (D) indicates 24 h treatment.
J. Biomed. Nanotechnol. 7, 110, 2011
It is often desirable to observe markers for extended periods
of time against the background of intrinsic cellular emis-
sions. Accordingly, we have investigated the ability of ZnO
particles (1 mmol) dispersed in saline medium to image
E. coli using the red uorescence. Propidium Iodide uptake
by bacterial cells is dependent on loss of cell membrane
integrity and is, therefore, frequently used to indicate the
extent of death in a cell population. Visualization of PI sig-
nal indicated presence of non-viable/dead E. coli cells with
damaged and permeable cell membranes. As is clear from
the picture (Fig. 5(A)), few dead cells of bacteria at 0 h
were seen. The dead bacterial number increased with time
for the samples treated with ZnO nanoparticles as can be
seen from Figure 5(B). From the Figures 5(C) and (D),
treated cells were obviously different in morphology and
major damage was observed in the cell membranes.
In comparison to the bacterial cells of the control
(Fig. 5(A)), the treated bacteria were thin and terminated
abruptly with reduced uorescence, possibly due to death
and subsequent cell lyses. These results indicated that
at a given size and concentration of ZnO nanoparticles,
inhibition of bacterial growth is caused by the abrasiveness
of ZnO nanostructures.
(B)
RESEARCH ARTICLE
10m
(D)
10m
5
 Interaction of ZnO Nanoparticles with MicrobesA Physio and Biochemical Assay Padmavathy and Vijayaraghavan

3.5. Determination of Hydrogen Peroxide

ROS (reactive oxygen species) are probably best known in

biology for their ability to cause oxidative stress. They can
damage DNA, cell membranes, and cellular proteins, and
may lead to cell death. Hydroxyl radical (OH) is the most
reactive oxygen radical known, and reacts very quickly
with almost every type of molecule found in living cells.15
Such reactions will probably facilitate the combination of
two OH radicals to form hydrogen peroxide (H2 O2 . But
nanoparticles of TiO2 and ZnO with large surface areas
and highly active catalytic sites can produce photocat-
alytic ROS in the presence of near-UV light by different
mechanism.16 Even without UV light irradiation, nanopar-
ticles of transition metal oxides are capable of generating
ROS that can be monitored through uorescence mea-
surements, since, ROS have a very short half-life (nsms) Fig. 6. Intracellular H2 O2 measured by the uorescent probe amplex
makes them very difcult to detect directly.17 This material red. Cells incubated (30 min, 37  C) showed signicant increases in
responds to UV exposure with the generation electron uorescence (emission at 587 nm).
hole pairs that can lead to biological injury through oxygen
radical production or electron capture. variety of cell types suggested that ROS generation might
damage cell DNA and induce apoptosis (programmed cell
ZnO + h e + h+ h+ + H2 O OH + H+ death) with no uorescent membrane damage.19 20 These
nanoparticles with high surface areas may directly act on
e + O2  O 2  O2 + H+ HO2
cell membranes causing cell death.
H+ + HO2 + e H2 O2 The amount of released H2 O2 is related to the particle
size of ZnO, and increased linearly with increasing con-
O2 is an unstable molecule which is quickly reduced to centration of ZnO. Due to the smaller particle size, high
the more stable and measurable H2 O2 . geometric areas for nanoparticles were able to generate
RESEARCH ARTICLE

e
e +2H
+ more amount of H2 O2 . The increased uorescence also
O2
O2 H2 O2 suggested a steady exposure of concentration-dependent

accumulation of net negatively charged O2 within the
Generated H2 O2 from ZnO surface by activating under cell membrane. This possibly resulted from ZnO inhibition
UV and uorescent light was measured by amplex red, a of one or more of the enzymatic complexes.
uorescent probe excited at 563 nm and emitted at 587 nm.
Exposure of ZnO nanoparticles of 12 nm average size
at incremental concentrations onto preincubated bacterial 3.6. Protein Analysis
cultures in the presence of UV light (UV light exposure as
Proteins are the most versatile macromolecules in liv-
control without samples was subtracted from experimental
values) and uorescent light for 2040 minutes resulted ing organisms and serve crucial functions in essentially
in an overall increase in intracellular ROS concentrations all biological processes. Proteins are the main target for
(Fig. 6). external sources causing damage both inside and outside
The results of ROS measurements showed that ZnO cells.21
nanoparticles induced intracellular ROS generation and
accumulation, and the inhibition correlated well with their Table I. Concentration of H2 O2 generated from ZnO surface under UV
and uorescent light.
individual intracellular ROS concentrations. The study of
H2 O2 release from bulk ZnO was done by Yamamoto H2 O2 generated from ZnO surface (g/L)
et al.18 has been extended to nanocrystalline ZnO in the ZnO concentration (mg) UV light (20 min) Fluorescent light (20 min)
present work and the results are summarized in Table 1.
Using calibration graph of H2 O2 standards, the concen- 150 47 2 33 5
130 42 1 26 2
tration of H2 O2 generated by activating ZnO with UV light 110 21 3 11 1
and in normal light have been calculated. Under UV light, 90 17 1 84
the generation of more quantity of H2 O2 is inevitable for 70 14 4 31
higher concentration of ZnO, and even in the presence 50 21 0
of uorescent light, ZnO is capable of producing ROS 30 0 0
10 0 0
and was clearly seen from Figure 6. Previous studies in a

6 J. Biomed. Nanotechnol. 7, 110, 2011

Padmavathy and Vijayaraghavan Interaction of ZnO Nanoparticles with MicrobesA Physio and Biochemical Assay
In addition to physiological factors (temperature, pH,
etc.), exogenous pollutants (organic pollutants, heavy met-
als, nanomaterials, etc.) can also trigger the destruction
of protein conformation, inducing the denaturation of the
protein.22 23
Measuring protein damage by nanomaterials may give
insight into the mechanisms of toxicity of nanomaterials
to living cells. The toxic effects of nano ZnO on biological
macromolecules has been determined by Bradford method.
The assay is based on the observation that the absorbance
maximum for an acidic solution of Coomassie Brilliant
Blue G-250 shifts from 465 nm to 595 nm when binding
to protein. Both hydrophobic and ionic interactions sta-
bilize the anionic form of the dye, causing a uorescent
color change. The amino acid residues based on pheno-
lic group in extra cellular protein is monitored before and
after treatment with ZnO nanoparticles.
The extraction of bacterial proteins treated with ZnO
nanoparticles had comparatively lowering of initial protein
levels (Fig. 7). Leaching of outer membrane proteins by
ZnO nanoparticles was quite inevitable causing decrease
in outer protein content in outer membrane. During the
interaction, after 2 h, the protein content was 20 g/mL,
whereas the protein level at 22 h was diminished to
7.2 g/mL. The amino acid residues based on pheno-
lic group in extra cellular protein was monitored before
and after treatment with ZnO nanoparticles. The results
demonstrated that ZnO nanoparticle apparently enhanced
the permeability of outer cell membrane in cells. So it
could be conferred that turbulence of membranous perme-
ability would be an important factor to inhibit the bacterial
growth.
3.7. AAS Analysis
Proteins and organic substances increase the dissolution of
particles of ZnO, CdSe, iron oxides, aluminum oxides and
Fig. 7. Measurement of extra cellular protein concentration at different
time scale.
J. Biomed. Nanotechnol. 7, 110, 2011
oxy hydroxides into corresponding cations through at least
two mechanisms.24 Ions are released from the materials
surface and ligand-enhanced dissolution (that is, adsorbed
natural organic material and organic acids extracting sur-
face metal atoms from nanoparticle surfaces). The latter
mechanism has been demonstrated for iron and aluminum
oxides and oxy hydroxides, and is also likely to occur for
ZnO. An example is ZnO nanoparticles that could dissolve
under aqueous conditions to form hydrated Zn2+ cations.25
This dissolution is increased under acidic conditions as
well as in the presence of biological components such as
amino acids and peptides.
AAS was used for the quantitative determination of the
zinc ion concentration after the dissolution from ZnO in
the culture medium containing bacterial cells. At 0th h, the
initial Zn concentration was 33 ppm (must be of Zn present
in bacterial cells), after 16 h exposure of ZnO in the culture
medium, Zn concentration was increased to 120 ppm. The
results indicated that the releasing concentration of zinc
ions from the medium of the samples stably increases with
the increase in sampling time.
Interfacial interactions that promote ZnO dissolution in
culture media, particle uptake into the acidifying compart-
ment accelerates dissolution. So the release of Zn2+ may
be one of the factors to improve the antibacterial prop-
erty of ZnO. It is expected that the high solubility of ZnO
nanopowder in the cell culture media suggested the for-
mation of aqueous complexes of zinc ions with organic or
RESEARCH ARTICLE
inorganic constituents of these complex media. From this,
release of Zn2+ ions from ZnO by the medium, causes
transportation of Zn2+ ions into the interior cell wall caus-
ing bacteriostatic effect. It has been further proved by
Zhang et al.14 who reported that chemical reactions hap-
pening between cells envelop components and Zn2+ ions
from ZnO.
3.8. TEM Studies
Antimicrobial effects are usually accompanied by a change
in bacterial surface morphology. To further verify the
antibacterial activity, TEM was employed to evaluate the
effect of ZnO nanoparticles on E. coli surface morphology.
Figures 8(A)(D) shows the TEM images of E. coli
native cell thin sections and damaged cells treated with
ZnO. Figure 8(A) shows thin sections of Escherichia coli
grown in free LB liquid medium. TEM analysis of sample
thin sections allows direct visualization of morphological
changes resulting in the microorganisms upon contact with
thiol capped ZnO nanoparticles at 1 mmol concentration
in a liquid culture medium (Figs. 8(C, D)).
TEM micrographs of E. coli treated with thiol capped
ZnO nanoparticles show (Figs. 8(C, D)) that the nanopar-
ticles have succeeded in penetrating inside the cells, thus
damaging the membranes. ZnO concentration below the
minimum bactericidal concentration has been used in all
7
 Interaction of ZnO Nanoparticles with MicrobesA Physio and Biochemical Assay Padmavathy and Vijayaraghavan

(A) (B)

500 nm 200 nm

(C) (D)
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100 nm

Fig. 8. TEM micrographs of thin sections of E. coli (A) cellular division (B) Enlarged view of thin sections (C) Partially damaged cell membrane
(D) Damaged cell membrane in multiple locations. Arrows point to ZnO nanoparticles appear as dark irregular pits on the cell surface.

cases in order to be able to observe both intact and dam- 4. DISCUSSION

aged cells. ZnO nanoparticles appear as black spots in
TEM analysis. Figures 8(C) and (D) show ZnO nanopar-
ticles inside and outside the cell, and the cellular internal-
ization was also clearly observed with ZnO nanoparticles.
Similarly, ZnO nanoparticles have also been devoured by
gram-negative cells of E. coli, and the partial cell mem-
branes were also damaged. From the Figure 8(C) and (D),
the extensively damaged membrane of E. coli could be
seen, most probably, the content must be leaked out.
The severe damage of membrane by ZnO can be
explained based on the nature of stabilizing agent capped
with them. It is reported that bactericides have a greatly
improved efciency when paired with alkaline substances
dissolve the external part of the cell membrane.26 There are
hydroxyl groups and sulfur groups from thiol molecules
surrounded on the surface of ZnO nanoparticles; so this
kind of ZnO nanoparticle was able to enter the cells. From
the micrographs, ZnO nanoparticles associate with cell
membranes and that the cells internalize some of the parti-
cles causing disruption in membrane morphology leading
to cell death.
Several mechanisms have been proposed for the antibacte-
rial activity of metal and metal oxide nanoparticles. Out of
these, few researchers strongly pointing out three impor-
tant factors are responsible for bacterial death.27
(i) cell function destructions via particle invasion,
(ii) ROS attack,
(iii) surface afnity of nanoparticles causing membrane
damage.
The present study therefore investigated ZnO nanoparticles
of average particle size regarding their effects on bacterial
cells at concentrations ranging from 1 to 5 mmol. The
results of MIC indicated that the bacterial cells of various
strains being exposed to ZnO nanoparticles could inhibit
the growth and reproduction.
To understand the antibacterial mechanism of ZnO
nanoparticles to Gram-negative bacteria, we had selected
E. coli as model to study the effect of ZnO nanoparti-
cles on the permeability and the membrane structure of
E. coli cells. It is well known that Gram-negative bacte-
ria possess an outer membrane outside the peptidoglycan

8 J. Biomed. Nanotechnol. 7, 110, 2011

Padmavathy and Vijayaraghavan Interaction of ZnO Nanoparticles with MicrobesA Physio and Biochemical Assay
layer lacking in Gram-positive organisms. The experi-
mental results depicted that ZnO nanoparticles apparently
enhanced the permeability of membrane for proteins in
cells observed from Bradford method. Zhang et al.14
strongly suggested that abrasion caused the physical dam-
age to the membrane envelope components by ZnO par-
ticles. While the mechanisms behind the antibacterial
activity have not been determined, reactive oxygen species
(ROS) are believed to be responsible for eukaryotic cell
membrane disruption and eukaryotic lipid peroxidation.28
It is known that the diffusion coefcient of oxygen is
about 105 cm2 s1 , and ROS should have an identical value.
The time for ROS diffusion into the bacterium should be
around 107 s, considering the nanoscale thickness of the
E. coli cell wall and membrane. Therefore, in comparison
to the lifetime of ROS (105 to 106 s), ZnO-induced ROS
could have enough time to diffuse into the cells.29 Once
ROS begins to accumulate, the oxidative stress in cells can
rst damage biomolecules such as protein and lipid. Thus,
oxidative damage to biomolecules is regarded as indicators
of the formation of oxidative stress in cells. It is essen-
tial to study whether ROS accumulation in bacteria causes
the oxidative stress and damage of biomolecules for fully
understanding the antibacterial mechanism.
In studies of mechanism, the particle size is essential.
Several papers report that small size particles 10 nm
interact well with cell membranes to inhibit cell growth.30
Smaller ZnO nanoparticles having the large surface area
available for interaction would give more bactericidal
effect than the larger ones. Another mechanism involves
the release of metal ions into the media. ZnO and CuO
nanoparticles also slowly released Zn2+ or Cu2+ to inhibit
microbial growth.31 The fact that dissolved metal ions con-
tribute to the toxicity of nanoparticles suggests the toxicity
may be different according to metal ion afnity to cell
functional groups e.g., SH. Cell membranes also function
as a ligand for the metal because membranes consist of
various negative charged functional groups, such as car-
boxyl or phosphate groups The binding afnity to inter-
action sites of the cells is different with each metal ion.32
Thai et al.33 reported the possible protein series where ZnO
and CuO could bind easily with microbes. Zinc binds with
SH containing proteins forming disulde during oxida-
tive stress.34 The higher binding afnity of zinc to sites of
action will likely inhibit bacterial growth more effectively.
From the TEM images, the bacterial cells treated
with ZnO were dissolved and dispersed, and their mem-
brane components became disorganized and scattered from
their original ordered and close arrangement. Cell mem-
brane was disaggregated and lost their intrinsic function.
The TEM micrograph of E. coli cells treated with ZnO
nanoparticles showed that the bacteria were almost disor-
ganized to several parts.
Another mechanism underlying the antibacterial activ-
ity of ZnO has been outlined by Applerot et al.35 The
J. Biomed. Nanotechnol. 7, 110, 2011
mechanism of ZnO antibacterial activity based on particle
size experimented by Electron-spin resonance measure-
ments reveal that aqueous suspensions of small nanopar-
ticles of ZnO produce increased levels of reactive oxygen
species, namely hydroxyl radicals. Another parameter is to
be considered is the surrounding medium of surviving of
bacterial cells. Still all the experiments are performed on
pure cultures, exposed to nanoparticles in pure water, i.e.,
a condition where bacteria are osmotically shocked. The
impact of nanoparticles will surely be different in an envi-
ronmental context, where bacterial cells are less numerous
and exposure media also contain soil colloids which may
adsorb or modify the physicochemical characteristics of
nanoparticles.
Taken together, nanomaterials undergo different types
of mechanism in entering bacterial cells and show killing
capability depend on composition, particle size, surface
charge and nature of reactivity etc. Nanoparticles are
always adsorbed onto bacterial surface and internalized in
periplasm, and partly due to nanoparticles-induced produc-
tion of intracellular ROS, lead to the impairment of cell
membrane integrity is the major cause of bacterial death.
5. CONCLUSION
In conclusion, antibacterial mechanism is proposed by
which ZnO nanoparticles inhibit bacterial growth, as well
as cellular responses assessed by various techniques. Based
on the results, the action of ZnO nanoparticles may be
RESEARCH ARTICLE
described that as ZnO nanoparticles making permeable of
outer membrane, resulting in the leakage of cellular mate-
rials. Then ZnO nanoparticles enter the inner membrane
and inactivate thiol containing enzymes via formation of
disulde and induce oxidative stress to the bacterial cells.
On the basis of these ndings, we propose the mecha-
nism of the antimicrobial activity of ZnO nanoparticles
that involves both the surface binding and ROS-dependent
pathway.
Acknowledgment: The authors thank the VIT manage-
ment and the DRDO Grant in aid scheme, Government of
India, for nancial assistance. The authors thank Professor
Ramamurthy, Ultrafast Processes, University of Madras for
CLSM imaging.
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Received: 22 March 2011. Accepted: 22 June 2011.
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