J Nanopart Res (2010) 12:16251636
DOI 10.1007/s11051-009-9711-1
RESEARCH PAPER
Mechanistic investigation into antibacterial behaviour
of suspensions of ZnO nanoparticles against E. coli
Lingling Zhang Yunhong Jiang Yulong Ding
Nikolaos Daskalakis Lars Jeuken Malcolm Povey
Alex J. ONeill David W. York
Received: 18 November 2008 / Accepted: 6 July 2009 / Published online: 29 July 2009
 Springer Science+Business Media B.V. 2009
Abstract Aqueous suspensions containing 4.45 9
10-5 - 1.25 9 10-3 M ZnO particles exhibit a
strong antibacterial activity against E. coli under the
dark conditions. The dominant mechanisms of such
antibacterial behaviour are found to be either or both
of chemical interactions between hydrogen peroxide
and membrane proteins, and chemical interactions
This study was carried out in the University of Leeds as part of
L. Zhangs PhD research.
L. Zhang
School of Civil and Environmental Engineering,
University of Science and Technology, Beijing, China
Y. Jiang
Y. Ding (&)
Institute of Particle Science & Engineering,
University of Leeds, Leeds, UK
e-mail: y.ding@leeds.ac.uk
N. Daskalakis
L. Jeuken
School of Physics and Astronomy,
University of Leeds, Leeds, UK
M. Povey
Procter Department of Food Science,
University of Leeds, Leeds, UK
A. J. ONeill
Faculty of Biological Sciences,
University of Leeds, Leeds, UK
D. W. York
Procter and Gamble Newcastle Technical Centre,
Newcastle-upon-Tyne, UK
between other unknown chemical species generated
due to the presence of ZnO particles with the lipid
bilayer. The effect of direct physical interactions
between nanoparticles and biological cells are found
to play a relatively small role under the conditions of
this study.
Keywords ZnO nanoparticles
E. coli

Toxicity
Antibacterial
Mechanism

Lipid vesicles
EHS
Introduction
Nanoscale materials are defined as substances with at
least one critical dimension less than *100 nm.
These materials, if properly engineered, have unique
electrical, thermal, mechanical and chemical proper-
ties that are highly desirable for applications in
electronics, energy, biotechnology, medical and
environmental sectors. For example, nanomaterials
are now present in sunscreens, suncreams, tooth-
pastes, sanitary ware coatings, textile fibre coatings,
catalysts, fuel additives and even food products. More
applications are expected in new areas such as
biosensing, cell labelling, drug targeting gene deliv-
ery, hyperthermia therapy, microelectronics, solar
cells, electroluminescent devices, photocatalysis,
detergent, cosmetics and antimicrobials (Ball 2001;
Belloc et al. 2003; Kong et al. 2000; Wasan and
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Nikolov 2003; Mallin 2006, Brayner et al. 2006).
Despite numerous current and potential future appli-
cations and wide publicity, the potential impact of
nanomaterials on human and environment is largely
unknown. Our current understanding of the behaviour
of bulk materials cannot provide an adequate predic-
tion of the behaviour of nanomaterials. Recent reports
on possible environmental and health effects of
nanoparticles have raised concerns about the toxicity
of nanomaterials as a critical factor for the evaluation
of their applications (Donaldson and Tran 2004;
Dreher 2004; Lam et al. 2004; Warheit et al. 2004;
Brayner et al. 2006). For example, nanoparticles are
reported to cause pulmonary inflammation according
to Lam et al. (2004) and Warheit et al. (2004) for
carbon nanotubes and Donoldson et al. (2000) for
TiO2, latex and carbon black nanoparticles.
The study reported in this article is part of an on-
going research aimed at positive use of the toxicity of
metal oxide nanoparticles as biocides or disinfectants,
with a focus on the use of ZnO nanoparticles. There
are a number of reasons for selecting ZnO nanopar-
ticles for the study. The main reason is that ZnO is a
metal oxide, which is much more stable and has a
longer life than organic-based disinfectants or anti-
microbial agents. This is particularly important for
harsh conditions such as high temperatures and/or
pressures occurring during product manufacturing,
storage and transportation (Utamapanya et al. 1991;
Wang et al. 1998; Hewitt et al. 2001; Stoimenov et al.
2002; Sawai 2003; Fu et al. 2005; Makhluf et al. 2005;
Brayner et al. 2006). Although there are numerous
reports in the literature on the biocidal effects of metal
oxide nanoparticles such as SiO2, Al2O3, Fe3O4, TiO2,
MgO and CaO, ZnO is of particular interest due to
extensive applications of the material in the personal
care and home care products (Axtell et al. 2005; Li
et al. 2005, Schumacher et al. 2004), and the recent
renewed interest associated with its piezoelectricity
and band gap in the near ultraviolet and large exciton
binding energy that could lead to lasting action based
on exciton recombination even at the room temper-
ature (Ozgur et al. 2005). ZnO has also been shown to
behave differently towards microorganisms from
other metal oxides such as TiO2, SiO2, MgO and
CaO (Yamamoto 2001; Roselli et al. 2003; Brayner
et al. 2006; Jeng and Swanson 2006; Adams et al.
2006). The reason for the difference is largely unclear.
There is therefore an urgent need to address the issue
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J Nanopart Res (2010) 12:16251636
from the fundamental level of interactions between
metal oxide nanoparticles and biological cellsthe
major motivation for carrying out this study.
There are relatively small number of publications
in the literature on the interaction between ZnO
nanoparticles and bacterial cells, and they are scat-
tered across the literature in nano-toxicity and
inorganic antibacterial areas; see, for example Sawai
et al. (1995a, 1995b, 1996a, 1996b); Yamamoto et al.
(1998), (2004); Yamamoto (2001); Stoimenov et al.
(2002); Makhluf et al. (2005); Brayner et al. (2006);
Jeng and Swanson (2006); Adams et al. (2006) and
Zhang et al. (2007). These studies show that ZnO
particles are effective for inhibiting both Gram-
positive and Gram-negative bacteria, and even spores
that are high-temperature and high-pressure resistant
(Sawai et al. 1995a, 1995b, 1996a, 1996b). A higher
concentration of smaller particles with a higher
surface area gives a better antibacterial behaviour
(Sawai et al. 1996a; Yamamoto 2001; Makhluf et al.
2005), whereas crystalline structure and particle
shape have a very small effect (Yamamoto et al.
1998). A number of mechanisms have also been
proposed to interpret these experimental observa-
tions. These include production of active oxygen
species due to the presence of nanoparticles (Makhluf
et al. 2005; Sawai et al. 1996b), damage of membrane
cell wall through adhesion on the cell membrane
(Stoimenov et al. 2002), penetration through the
membrane cell wall (Makhluf et al. 2005) and cellular
internalisation of nanoparticles (Brayner et al. 2006).
However, the dominant mechanisms have not been
identified. It is also noted that little attention has been
paid in these studies to the formulation of ZnO
suspensions, the structure of nanoparticles in the
suspension and the presence of additives such as
suspension stabilisers. All these could affect interac-
tions between nanoparticles and biological cells.
In this study, stable aqueous suspensions of ZnO
nanoparticles were produced, characterised, and tested
for their antibacterial activity against Escherichia coli.
Electronic microscopy and chemical probes were used
to investigate interactions between nanoparticles and
chemical species originated from the presence of ZnO
with E. coli cells. The article is structured in the
following way. The section Experimental describes
the details of materials preparations and experimental
techniques. Experimental results are presented and
discussed in the section Results and discussion.
J Nanopart Res (2010) 12:16251636
Finally, conclusions are drawn in the last section,
where further study is also proposed.
Experimental
Preparation and characterisation of ZnO
nanoparticle suspensions
Dry zinc oxide nanoparticles purchased from Nano-
structured & Amorphous Materials (USA) were used
in this study. The particles were rod-like and the
nominal size was given by the manufacturer as 90
200 nm although our Scanning Electron Microscope
(SEM, LEO 1530 FEGSEM) analyses showed that
some of the particles were of micron size range
(Fig. 1a). The SEM analyses also revealed the
presence of agglomerates of the primary nanoparti-
cles. In order to reduce the size of the large primary
Fig. 1 SEM images of ZnO nanoparticles as received and
after size reduction
1627
particles and to break the nanoparticle agglomerates,
the particles as-received were dispersed in distilled
water to produce a master suspension for diluting to
different concentrations for subsequent use in the
experiments. The master suspension was subjected to
a series of size reduction processes; see below. The
concentration of the master suspension was set at
0.25 M (1.96% by mass, 0.35% by volume), and the
pH of the suspension was adjusted to be the same as
the culture medium, i.e. *7.2, by using NaOH (1 M,
Fisher Scientific, UK) and HCl (0.1 M, Fisher
Scientific, UK). The suspension was first vigorously
stirred in a beaker, then sonicated for 30 min in an
ultrasonicatior (Clifton, UK) and finally milled in a
Dyno-Mill (Willy A. Bachofen, Switzerland) for 4 h
at the room temperature. In order to enhance the
stability of the suspension, polyethylene glycol (PEG
400 and PEG 2000, Fluke, UK) was used as
dispersant. The amount of dispersant was 10% by
mass with respect to the mass of ZnO nanoparticles.
The use of PEG dispersants were also aimed to
understand direct physical interactions between
nanoparticles and biological cells.
The suspension sample prepared was analysed
using a Nanosizer (Malvern Instruments, UK) for
particle size distribution. SEM analyses were also
performed to examine particle morphology after the
size reduction process as described above, and for
comparison with the results of the Nanosizer mea-
surements. For the toxicity/antibacterial tests, the
master suspension of ZnO, i.e. 0.25 M, was diluted to
different concentrations ranging from 10-5 to
10-3 M. The diluted suspensions had the same pH
value and the same particle size distribution.
Determination of antibacterial activity
ZnO nanoparticle suspensions were sterilised and
tested for their antibacterial activity against a model
microbe, E. coli DH5a. In order to suppress the
photocatalytic effect of ZnO particles, all experiments
were performed in the dark (Ozgur et al. 2005). E. coli
was grown to saturation in Luria-Bertani (LB) broth at
37 8C with aeration. Aliquots (100 lL) of culture
were then transferred to volumes (10 mL) of fresh LB
medium containing different concentrations of nano-
particles. Viable cell numbers were followed by
plating diluted cultures onto LB agar, and incubating
the plates for 48 h at 37 8C, before counting colonies.
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Investigations into the antibacterial mode
of action (MOA) of ZnO nanoparticles
In order to investigate the MOA of ZnO nanoparti-
cles, several techniques were used, including the use
of probe chemicals such as ZnCl2 and H2O2, scanning
and transmission electron microscopes (SEM and
TEM), and a pH sensitive fluorescent probe pyranine.
These experiments were aimed to understand mech-
anisms of the antibacterial behaviour of ZnO parti-
cles; see section Results and discussion for more
details. Experiments with the probe chemicals of
ZnCl2 and H2O2 to determine their antibacterial
activities and SEM analyses are straight forward. The
TEM analyses are more complicated, involving (i)
fixing E. coli samples with 2.5% glutaraldehyde
(Fluka, USA) for [*2 h and washing twice with
0.1 M phosphate buffer solution, (ii) fixing with 1%
osmium tetroxide (Fluka, USA) for overnight and
washing twice with the same phosphate buffer as in
(i), (iii) washing and dehydrating the sample with an
ascending alcohol series (20, 40, 60, 80 and 100% by
mass) and (iv) embedding and sectioning of the
bacteria samples. The purpose of the use of the pH-
sensitive probe pyranine was to investigate the effects
of ZnO nanoparticles and other components on the
internal pH change in E. coli membrane vesicles. A
brief description of the technique is given in the
following.
Pyranine (8-hydroxy-1,3,6-pyrene-trisulfonate)
has been shown to be a convenient and sensitive
probe for reporting internal pH changes in lipid
vesicles (Kano and Fendler 1978; Clement and Gould
1981; Damiano et al. 1984). In this study, E. coli lipid
polar extract was purchased from Avanti Polar Lipids
Inc. (USA). The extract was in powder form and
contained 67% phosphatidylethanolamine, 23.2%
phosphatidylglycerol and 9.8% cardiolipin on mass
basis. The lipids were dissolved in methanol/chloro-
form (50/50 v/v, HPLC grade, Fisher) and 5 mg
aliquots were dried under a nitrogen flow for at least
2 h in glass vials. The vials were stored under
nitrogen at -80 8C until used. 15 mM pyradine was
dissolved in a mixed buffer solution (pH = 6.5;
20 mM 2-(N-Morpholino)ethanesulfonic acid (MES);
4-(2-Hydroxyethyl)piperazine-1-ethanesulfoic acid
(HEPES); 2-(Cyclohexylamino)-ethanesulfonic acid
(CHES)). The E. coli lipid aliquot was then hydrated
in the pyranine buffer solution using a vortex mixer.
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J Nanopart Res (2010) 12:16251636
The hydrated lipid was subsequently extruded
through a 400 nm membrane 11 times using a mini-
extruder (Avanti Polar Lipids Inc. USA) to produce
E. coli lipid vesicles, after which the vesicles should
have a size distribution with the mean around 400 nm
and contain in their aqueous compartment the pyra-
nine probe molecules. In order to remove the
pyranine outside the vesicles, a chromatographic
method was used with a NAP-10 column (GE
Healthcare, UK). The lipid vesicles were then diluted
using the mixed buffer solution to give an average
concentration of 10 lM pyridine and transferred to a
precision cell (HELLMA) for analysis using a
fluorescence spectrometer (RF-5300, Shimadzu,
UK) at an emission wavelength of 510 nm, and two
excitation wavelengths of 405 nm and 455 nm. A
calibration procedure was used to convert the mea-
sured fluorescence data of the pyridine into pH values
as described by Clement and Gould (1981). In order
to study the effects of ZnO nanoparticles, ZnCl2 and
H2O2 on the lipid membrane structure an artificial
proton gradient was generated by addition of NaOH
to the mixed buffer solution, increasing the pH
outside the vesicles from pH 6.5 to *8.0. As already
described by Clement and Gould (1981), this pH
jump results in an immediate pH adjustment at the
inside the vesicles of about half a pH unit as
monitored by the pyridine fluorescence. A further
slower adjustment in pH is observed for another
15 min after which the fluorescence is stable and a
pH difference of *0.8 pH units is observed between
the outside and inside of the vesicles. ZnO nanopar-
ticles, ZnCl2 and H2O2 were added to this vesicle
preparation as further described in the result section.
In order to investigate electrostatic interactions
between ZnO particles and E. coli cells and physical
abrasion by particles as possible mechanisms, a set of
tests was done by using coated ZnO particles on
polyvinyl chloride (PVC) films. A special test cell
was used. Such a cell consisted of a 200-nm pore-
sized membrane which separated the coated film
from the E. coli culture during the antibacterial tests.
In this way, the contribution of direct abrasive and
electrostatic interactions to the observed antibacterial
effect could be evaluated. Blank PVC films were used
to provide a negative control. The physical abrasion
as a possible mechanism for inhibiting/killing bacte-
ria was also investigated by using a micron-sized
ZnO suspension (2 lm) in the antibacterial tests. The
J Nanopart Res (2010) 12:16251636 1629

effects of particle size and separating membrane on Size Distribution by Volume

the antimicrobial behaviour were carried out by 20

Volume (%)
measuring optical density (OD) at 600 nm with a 15

spectrophotometer (Thermo Electron Corporation, 10

USA). 5
There may be doubt about the strength of particle 0
0.1 1 10 100 1000 10000
coating on the PVC film. The coated films were
Diameter (nm)
therefore tested for the durability by placing the films
in vigorously stirred water in a beaker followed by Fig. 2 Size distribution of ZnO by volume (three lines are
measuring the turbidity using a UVvis system and from three measurements, average size = *93 nm)
the antibacterial activity of the water. These tests
suggest that the blank films did not have any distribution. It was found that the suspensions were
antibacterial activity and the ZnO particles were very stable for at least 4 weeks. Addition of PEG 400
strongly coated on the film with no particles found in and PEG2000 enhanced the stability, and size distri-
the water phase. bution did not change much even after 2 months.

Antibacterial activity and MOA of ZnO

Results and discussion suspensions

This section will present the experimental results. For The antibacterial activity and MOA of ZnO nano-
clarity, except for otherwise indicated, the results are particle suspensions against E. coli was evaluated in a
for 93 nm ZnO particles without the use of series of experiments (Fig. 3). The following obser-
dispersants. vations can be made:
ZnO suspensions with a concentration of 1.25 9
Characterisation of ZnO nanoparticle suspensions
10-3 M bring about a drastic reduction in the
and stability tests
number of bacteria colonies (*105 fold relative
to the negative control), indicating a strong
The method of size reduction as described in the
antibacterial activity. The antibacterial activity
section Preparation and characterisation of ZnO
of the 1.25 9 10-3 M ZnO suspensions shows
nanoparticle suspensions was found to be very
little difference from that of 1.25 9 10-3 M ZnO
effective in reducing the particle size. Figure 1b
suspensions with PEG400, indicating that the
shows an SEM image of ZnO nanoparticles after the
presence of PEG400 contributes little to the
size reduction. It can be seen that the ZnO nanopar-
antimicrobial activity.
ticles exhibit two types of shapes, rod-like and
irregular. The rod-like particles are mainly due to
breakage of agglomerates of primary ZnO nanopar-
ticles, whereas the irregular particles are more likely
to be as a result of breakage of large primary particles
as shown in Fig. 1a. Figure 1b indicates that the size
of the ZnO nanoparticles is below 100 nm. Figure 2
shows size distribution of ZnO nanoparticles in
suspensions obtained with the Nanosizer. One can
see that the average particle size (d50) is *93 nm,
which is in good agreement with the SEM analyses. It
was also found that the use of PEG 400 and PEG2000
and dilution of the suspension gave a very small
effect on the particle size distribution.
The stability of ZnO suspensions was tested by Fig. 3 Antibacterial activity of different materials: incubation
measuring time-dependence of the particle size at 37 8C for 24 h

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ZnCl2 did not show antibacterial activity against 3.5 Negative control (no ZnO)
E. coli under conditions of this study. In aqueous ZnO (no dispersant)
3.0
solutions with pH = *7.2, almost all Zn in ZnO + PEG400
ZnO + PEG2000
ZnCl2 are in the form of Zn2? ions. It can be

OD at 600nm
2.5
concluded that Zn2? ions do not have much 2.0
antibacterial activity up to a concentration of
7.3 9 10-5 M. The concentration range of the 1.5

ZnCl2 is of the same order of magnitude of the 1.0

solubility of ZnO in the solution if Zn in the
0.5
dissolved ZnO is assumed to be in the form of
Zn2? ions under the conditions of this study. This 0.0

suggests that Zinc(II) ions are not primarily 0 2 4 6 8 10 12

responsible for the antibacterial activity of ZnO. Time, hour

The presence of H2O2 at concentrations of Fig. 5 Effect of the presence of dispersants on the antibacte-
9.1 9 10-3 M and H2O2 1.8 9 10-2 M leads to rial behaviour of ZnO suspensions (5.56 9 10-5 M ZnO)
100% reduction in bacterial growth. This suggests
that, if H2O2 were produced due to the presence of
ZnO, then this could be a dominant mechanism for
the antibacterial behaviour.

Effects of particle size, addition of dispersants

and the use of separating membrane

Figures 4, 5, and 6 show respectively the effects of

particle size, addition of dispersants and the use of
separating membrane, from which the following
conclusions can be made:
Both nano-sized and micron-sized ZnO suspen-
sions are active in inhibiting the bacteria growth;
Fig. 6 Comparison of growth curve of E. coli with
the nano-sized ZnO suspension clearly has a
4.45 9 10-5 M ZnO suspension and that with coated film
containing an equivalent concentration of 4.45 9 10-5 M
ZnO; The film is separated from the culture by a membrane
10
Number of bacterial colonies (CFU/ml)

10
Negative control: no ZnO much higher activity than the micron-sized ZnO
9
10 suspension (Fig. 4).
8
In general, PEG dispersants have a small effect on
10 Sample A: 2 micron ZnO
the antibacterial activity of ZnO suspensions, and
10
7 the effect of PEG2000 is greater than that of
PEG400 (Fig. 5).
10
6
Sample E: 93 nm ZnO
Use of a separating membrane has a small effect
on the antibacterial activity under the conditions
5
10 of this study (Fig. 6).
10
4
These results will be discussed further, in the
A E Negative
section Discussion on mechanisms of the antibacte-
Samples
rial behaviour of ZnO suspensions, on their implica-
Fig. 4 Effect of particle size on the antibacterial behaviour of tions to the mechanisms of the antibacterial
ZnO suspensions (2.23 9 10-4 M ZnO, incubation for 24 h) behaviour of ZnO.

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Induced leakage in E. coli lipid vesicles

Leakage experiments were carried out on E. coli lipid

vesicles, with pyranine as a probe, to study whether
ZnO nanoparticles, ZnCl2 and H2O2 could induce
structural damage of the bacterial membrane. For these
experiments, E. coli lipid vesicles with a mean
diameter of 400 nm were prepared, which contained
a proton gradient with the pH of aqueous compartment
of the vesicles about 0.8 units lower than the outside
solution. Pyranine trapped in the vesicles was used to
fluorescently probe the pH inside the vesicles. Pyridine
has a net anionic surface charge and does not bind
significantly to phospholipids vesicles and similar to
the protons does not leak out the vesicles once trapped
inside (Clement and Gould 1981). If ZnO, ZnCl2 or
H2O2 induces structural damage to the lipid membrane,
either or both protons and pyridine will pass the
membrane and a change in fluorescence of the pyridine
will be observed. We have determined DpHi, which is
the difference between the pH of the buffer (outside the
vesicles) and the average pH as determined using the
pyridine probe. We note that the calibration procedure
of the fluorescence measurements will only produce an
average pH value and will not be sensitive to the
possible existence of two pools of pyridine molecules
(e.g. outside and inside vesicles).
Fig. 7 Effect of ZnO nanoparticles and other compounds on the
Figure 7a shows DpHi as a function of time after proton gradient of 400 nm lipid vesicles; In (a), results of four
injection of different ZnO suspensions. The DpHi injections are shown with ZnO concentrations being respectively
increases rapidly with time upon injection of ZnO 0.125, 0.25, 0.625 and 1.25 mM; In (b), results are shown for two
suspensions and the value of DpHi approaches a injections of hydrogen peroxide with 9 and 18 mM, and three
injections of ZnCl2 with concentrations of 0.73, 1.5 and 3.7 lM
constant, non-zero value (Fig. 7a). This suggests that
pyridine or protons leak between the interior and
exterior of the vesicles. Leakage in E. coli bacteria ZnO particles has a bigger effect than that of micron-
has also been observed by microscopic investigation; sized particles (2 lm). This agrees with the antibac-
see later for more details. These data indicate a terial activity data of both our study and those
possibility of direct contact between the ZnO parti- obtained by Makhluf et al. (2005), Yamamoto (2001)
cles and the vesicles as a mechanism (Stoimenov and Sawai et al. (1996b).
et al. 2002). Would the ZnO effect be due to the The time evolution of DpHi due to the injection of
generation of a reactive chemical compounds only ZnCl2 and H2O2 solutions are shown in Fig. 7b, where
the rate would be dependent on the amount of ZnO, the results of injections of different concentrations of
but the end value of DpHi would be the same. At a ZnO suspensions are also shown for comparison. One
given ZnO concentration, the use of PEG400 can see that the pH change caused by the ZnO
decreases both the rate of DpHi decrease (particularly nanoparticle suspensions is much higher than that by
in the initial stage of ZnO suspension injection) and ZnCl2 and H2O2. The small effect of ZnCl2 indicates
the final DpHi value. This suggests that PEG400 that dissolution of ZnO producing solvated Zn2? is
provides a small extent of protection to the vesicles. not responsible for the effects observed in Fig. 7a. It
Finally, Fig. 7a also illustrates the effect of particle also agrees with the results as shown in Fig. 3,
size on DpHi. Injection of suspensions of nano-sized indicating that Zn2? ions do not have a biocidal effect,

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at least under the conditions of this study. The
negligible effect of H2O2 is somewhat surprising as
H2O2 is shown in Fig. 2 to be biocidal towards E. coli
bacteria. However, the absence of any effect of H2O2
is consistent with the hypothesis that the change of
DpHi is due to direct contact between the lipid
membranes and ZnO. More discussion on this will be
given in section Discussion on mechanisms of the
antibacterial behaviour of ZnO suspensions.
Discussion on mechanisms of the antibacterial
behaviour of ZnO suspensions
A number of mechanisms have been proposed to
interpret the antibacterial behaviour of metal oxides.
Makhluf et al. (2005) attributed the antibacterial
behaviour of MgO to several mechanisms including
the production of active oxygen species due to the
presence of MgO, interaction between MgO particles
and membrane cell walls, penetration of individual
MgO particles into cells and reformation of MgO
within the cell. Sawai et al. (1996b) measured the
active oxygen species generated in ZnO slurries by
using chemiluminescence and oxygen electrochemi-
cal analyses and found that H2O2 was produced in
ZnO slurries and the concentration of H2O2 produced
was linearly proportional to the ZnO particle concen-
tration. H2O2 was also detected by Yamamoto et al.
(2004) with a spectrophotofluorometer. Stoimenov
et al. (2002) suggested that electrostatic interactions
between the bacteria surface and nanoparticles could
be involved. We shall start the discussion by looking
at the structure of the cell envelope of E. coli.
E. coli is a Gram-negative bacterium and has a
membrane structure as shown in Fig. 8 (Schaechter
et al. 2006). E. coli cells have a rod-like morphology
with a length of approximately 13 lm and a
Fig. 8 Cell envelope of gram-negative bacteria following
Schaechter et al. (2006)
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J Nanopart Res (2010) 12:16251636
diameter of 0.51 lm. The cell envelope of E. coli
consists of an inner membrane (*8 nm thick), a
periplasm containing the peptidoglycan (cell wall)
and an outer membrane (*1015 nm thick). The
inner and other membranes have a lipid bilayer
structure. The outer layer of the outer membrane
(OM) bilayer consists of lipopolysaccharide (LPS)
and the inner layer of the OM bilayer consists of
phospholipids. The OM also contains proteins such as
porins that act as passive diffusion channels for
hydrophilic molecules. The inner membrane consists
of *40% phospholipids and 60% protein. Given the
structure of the E. coli cell envelope, the following
mechanisms could contribute to the antibacterial
behaviour of ZnO suspensions:
(a) Chemical interactions between ZnO and the
components of the cell envelope (lipid bilayer,
peptidoglycan, membrane proteins, lipopolysac-
charides) as follows:
i. Chemical reaction between cell envelope
components and Zn2? due to the presence
of ZnO particles
ii. Chemical reaction between the Zn2? ions
and components in the interior of the cell
(this involves transportation of Zn2? into
the interior of the cell first and then the
reactions)
iii. Chemical reaction between the cell enve-
lope components and chemical species
such as hydrogen peroxide, generated due
to the presence of ZnO particles
iv. Chemical reaction between the chemical
species generated due to ZnO particles and
components in the interior of the cell (this
involves transportation of the chemical
species into the interior of the cell first
and then the reactions)
(b) Physical interactions between ZnO nanoparti-
cles and the cell envelope structure as follows:
i. Physical blockage of the transport channels
of the cell membranes by ZnO particles
ii. Physical damage to the membrane enve-
lope components by ZnO particles due to
abrasion
iii. Penetration of ZnO particles through the
cell envelope and interact with the interior
of the cell
J Nanopart Res (2010) 12:16251636 1633

iv. Direct interaction between ZnO particles
and E. coli cell envelope components
through electrostatic effect
(c) A combination of the physical and chemical
interaction as described above.
The above list includes all the mechanisms
proposed in the literature as summarised in the first
Fig. 9 SEM image of an E. coli bacteria cell from ZnO
nanoparticle suspensions
paragraph of this sub-section (section Discussion on
mechanisms of the antibacterial behaviour of ZnO
suspensions) but is organised in a structured manner.
In the following, we shall analyse these possible
mechanisms based on the experimental results. Con-
sider the physical interactions first. Figure 9 shows a
typical SEM image of an E. coli cell in a ZnO
nanoparticle suspension. Although some nanoparti-
cles are seen to stick to surface of the bacterial cell,
they are unlikely to seriously block the transport
channels of the cell membranes (Mechanism b(i)).
The size of ZnO nanoparticles are of an order of
102 nm, and the particles will not be able to pack into
a dense enough structure to keep solutes from passing
between the gaps. Penetration of ZnO particles
through the cell envelope, i.e. Mechanism b(iii), can
also be excluded as a dominant mechanism because
extensive TEM analyses show no evidence of the
presence of ZnO particles in the interior of the cells
(Fig. 10). The small effect of PEG400 polymer on the
biocidal behaviour of the ZnO particles suggests that
the electrostatic interactions are not a major mech-
anism, i.e. Mechanism b(iv). This leaves only mech-
anism b(ii) as a potential option, and this is consistent
with the results of the lipid vesicles, which suggested

Fig. 10 TEM images of

E. coli bacteria cells: (a)
and (c) E. coli grown in LB
medium; (b) and (d) E. coli
grown in LB medium in the
presence of 6.25 9 10-3 M
ZnO nanoparticles for 2 h

123
1634
that physical interaction between ZnO and lipid
membranes induces leakage of pyridine through the
vesicle membrane. Furthermore, leakage of cell
content is observed from some places of the cell
envelope with TEM analysis (Fig. 10).
However, the experiments with the coated PVC
films strongly suggest that b(ii) cannot be the only
mechanism. The coated PCV films show that for a
given equivalent ZnO concentration, the coated films
only give a slightly lower antibacterial activity than
ZnO suspensions. This indicates that direct contacts
between ZnO particles and biological cells are not
essential for enabling the antibacterial activity of
ZnO. Turning to the chemical interactions, Mecha-
nisms a(i) and a(ii) can be excluded as dominant
mechanisms due to little antibacterial activity of Zn2?
(Fig. 3) and very small effect of Zn2? ions (from
ZnCl2) on the pH change in the interior of the lipid
vesicles in comparison with the effect of ZnO
particles (Fig. 7b). As a consequence, only Mecha-
nisms a(iii) and a(iv) are likely to be responsible for
the experimentally observed antibacterial activity.
Experimental evidence so far shows that chemical
species that are likely to be responsible for the
antibacterial activity are hydrogen peroxide or other
active oxygen species (Sawai et al. 1996b; Yama-
moto et al. 2004). Indeed, hydrogen peroxide has
been shown to have a very high antibacterial activity
(Fig. 3). In contrast, Fig. 7b shows little damage to
the lipid vesicles due to the presence of hydrogen
peroxide. This discrepancy can be explained in a
number of ways. The antibacterial activity of H2O2
might not be due to damage to the lipids of the
biological membrane, but rather due to the interaction
between H2O2 and membrane proteins or diffusion
through the cell envelope. Hydrogen peroxide can
directly oxidise protein cysteinyl residues and react
with released iron in the cell to form hydroxyl
radical, which could directly damage DNA (Storz and
Imlay 1999). Alternatively, H2O2 generated in culture
media could be converted to a number of other
reactive oxygen species (ROS), which are very likely
as metals like Fe(II) in the growth media will induce
Fenton chemistry. The absence of metals (other than
Zinc(II)) as well as the much shorter time-span will
prevent such mechanisms in the vesicle experiments.
Thus, ROS could damage the cell envelope and
increase mass transfer between the interior and
exterior of the cell. The possibility of damage to
123
J Nanopart Res (2010) 12:16251636
the membrane envelope due to ROS is again
supported by TEM analyses as shown in Fig. 10
where leakage of cell content is observed from some
places of the cell wall.
The above analyses suggest that chemical interac-
tion between ROS generated due to the presence of
ZnO particles and the cell is the likely mechanisms
under the conditions of this study, although physical
interaction between ZnO particles and the mem-
branes in the cell envelope might have an additional
role. It is less clear which ROS is responsible, how
the ROS is produced and how to improve its
production to optimise the biocidal effect. It is
known that ZnO is photo-catalytic under the UV
light, which could be a reason for the production of
the active oxygen and other chemical species.
However, all the antibacterial tests in this study were
carried out under dark conditions. A further question
is therefore how photoactive the ZnO nanoparticles
are in suspensions under dark conditions. Further
study is underway aiming at answering these ques-
tions, which will be reported in the future.
Conclusions
This article is concerned with the antibacterial
behaviour of dilute liquid suspensions of ZnO
nanoparticles against E. coli (DH5a). Various tech-
niques are used to investigate the mechanisms of the
antibacterial behaviour. The following conclusions
are obtained:
Aqueous suspensions of 4.45 9 10-5 - 1.25 9
10-3 M ZnO show a strong antibacterial activity
against E. coli. The antibacterial activity increases
with increasing particle concentration or decreas-
ing particle size. Addition of PEG400 and
PEG2000 dispersants imposes a very little effect
on the antibacterial activity of ZnO suspensions.
Aqueous solutions containing up to 7.3 9 10-5 M
ZnCl2 shows little antibacterial activity against
E. coli suggesting that the antibacterial behaviour is
not due to Zn2? ions under the conditions of this
study, whereas water solutions containing 9.1 9
10-3 - 1.8 9 10-2 M H2O2 have a very strong
biocidal effect against E. coli.
Aqueous suspensions of ZnO nanoparticles in
contact with E. coli lipid vesicles containing a
proton gradient of *0.8 pH units cause
J Nanopart Res (2010) 12:16251636
significant loss of this proton gradient, suggesting
possible leakage of pyridine or protons between
interior and exterior of the vesicles. The presence
of PEG decreases this effect of ZnO. Micron-
sized ZnO particles are found to have a much
lower effect than their nano-sized counterpart
given the absolute amount of ZnO. These exper-
iments also show that there is little effect of ZnCl2
and H2O2 on the proton gradient, indicating that
the presence of Zn2? ions or H2O2 does not give a
significant level of damage to the lipid membrane
on the time-scale of minutes.
The results of this study and experimental
evidence from the literature suggest that the
dominant mechanisms of the observed antibacte-
rial behaviour of ZnO particles is the generation
of radical oxygen species (ROS) and the resulting
interaction between ROS and the cell. A small
amount of physical damage to the cell envelope
might also be induced by ZnO.
Further study is underway to identify (i) the
unknown ROS, (ii) how the ROS are produced, and
(iii) how to improve their production if they were the
main mechanisms. The results will be reported in the
future.
Acknowledgements The authors would like to thank
University of Leeds Interdisciplinary Institute for
Nanomanufacturing, Procter & Gamble, The White Rose
Doctoral Training Centre (DTC) and EPSRC (EP/E00041X/1
and EP/F015380) for financial support.
References
Adams LK, Lyon DY, Alvarez JJ (2006) Comparative eco-
toxicity of nanoscale TiO2, SiO2, and ZnO water sus-
pensions. Water Res 40:35273532
Axtell HC, Hartley SM, Sallavanti RA (2005) Multi-functional
protective fibre and methods for use. United States Patent
US2005026778
Ball P (2001) Roll-up for revolution. Nature 414:142144
Belloc NC, Pun SH, Jensen GS, Davis ME (2003) Tranferrin
targeted gene delivery. Bioconjug Chem 14:11221132
Brayner R, Ferrari-lliou R, Brivois N, Djediat S, Benedetti MF,
Fievet F (2006) Toxicological impact studies based on
Escherichia coli bacteria in ultrafine ZnO nanoparticles
colloidal medium. Nano Lett 6:866870
Clement NR, Gould JM (1981) Pyraine (8-hydroxy-1,3,6-py-
renetrisulfonate) as a probe of internal aqueous hydrogen
ion concentration in phospholipids vesicles. Biochemistry
20:15341538
1635
Damiano E, Bassilana M, Rigaud JL, Leblanc G (1984) Use of
the pH sensitive fluorescence probe pyranine to monitor
internal pH changes in Escherichia coli membrane vesi-
cles. FEBS Lett 166:120124
Donaldson K, Tran CL (2004) An introduction to the short
term toxicology of respirable industrial fibers. Mutat Res
553:59
Donoldson K, Stone V, Gilmour PS, Brown DM, MacNee W
(2000) Ultrafine particles: mechanisms of lung injury.
Philos Trans R Soc Lond A 358:27412749
Dreher KL (2004) Health and environmental impact of nano-
technology: toxicological assessment of manufactured
nanoparticles. Toxicol Sci 77:35
Fu G, Vary PS, Lin CT (2005) Anatase TiO2 nanocomposites
for antimicrobial coating. J Phys Chem B 109:88898898
Hewitt CJ, Bellara ST, Andreani A, Nebe-von-Caron G,
Mcfarlane CM (2001) An evaluation of the anti-bacterial
action of ceramic powder slurries using multi-parameter
flow cytometry. Biotechnol Lett 23:667675
Jeng HA, Swanson J (2006) Toxicity of metal oxide nano-
particles in mammalian cells. J Environ Sci Health
41:26992711
Kano K, Fendler JH (1978) Pyranine as a sensitive pH probe
for liposome interiors and surfaces. Biochinica et bio-
physica acta 509:289299
Kong G, Braun RD, Dewhirt MW (2000) Hypothermia enables
tumor-specific nanoparticle delivery: effect of nanoparti-
cle size. Cancer Res 60:44404445
Lam CW, James JT, McCcluskey R, Hunt RL (2004) Pul-
monary toxicity of single wall carbon nanotubes, in mice
7 and 90 days after intratracheal instillation. Toxicol Sci
77:126134
Li XJ, Wang Y, Wang Y (2005) Photocatalytic and antiseptic
far infrared fiber and its preparing method. Patent
CN1587453
Makhluf S, Dror R, Nitzan Y, Abramovich Y, Jelinek R,
Gedanken A (2005) Microwave-assisted synthesis of
nanocrystalline MgO and its use s a bacteriocide. Adv
Funct Mater 15:17081715
Mallin MA (2006) Wading in waste. Sci Am 294:5359
Ozgur U, Alivov C, Liu C, Teke A, Reshchikov MA, Doggan
S, Avrutin V, Cho SJ, Morkoc H (2005) A comprehensive
review of ZnO materials and devices, J Appl Phys
98:041301-1*103
Roselli M, Finamore A, Garaguso I, Britti MS, Mengheri E
(2003) Zinc oxide protects cultured enterocytes from the
damage induced by Escherichia coli. J Nutr 133:4077
4082
Sawai J (2003) Quantitative evaluation of antibacterial activi-
ties of metallic oxide powders (ZnO, MgO and CaO) by
conductimetric assay. J Microbiol Methods 54:177182
Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M
(1995a) Evaluation of growth inhibitory effect of ceramics
powder slurry on bacteria by conductance method. J
Chem Eng Jpn 28:288293
Sawai J, Saito I, Kanou F, Igarashi H, Hashimoto A, Kokugan
T, Shimizu M (1995b) Mutagenicity test of ceramic
powder which have growth inhibitory effect on bacteria. J
Chem Eng Jpn 28:352354
Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M
(1996a) Effect of particle size and heating temperature of
123
1636
ceramic powders on antibacterial activity of their slurries.
J Chem Eng Jpn 29:251256
Sawai J, Kawada E, Kanou F, Igarashi H, Hashimoto A,
Kokugan T, Shimizu M (1996b) Detection of active
oxygen generated from ceramic powders having antibac-
terial activity. J Chem Eng Jpn 29:627633
Schaechter M, Ingraham JL, Neidhardt FC (2006) Microbe.
ASM Press, Washington, DC, p 24
Schumacher K, Hasenzahl S, Moerters M (2004) Powder mix-
ture consisting of titanium dioxide, zinc oxide and zinc/
titanium mixed oxide. Worldwide Patent WO2004056706
Stoimenov PK, Klinger RL, Marchin GL, Klabunde KJ (2002)
Metal oxide nanoparticles as bactericidal agents. Lang-
muir 18:66796686
Storz G, Imlay JA (1999) Oxidative stress. Curr Opin Micro-
biol 2:188194
Utamapanya S, Klabunde KJ, Schlup JR (1991) Nanoscale
metal oxide particles/clusters as chemical reagents: syn-
thesis and properties of ultrahigh surface area magnesium
hydroxide and magnesium oxide. Chem Mater 3:175181
Wang YL, Wan YZ, Dong XH, Cheng GX, Tao HM, Wen TY
(1998) Preparation and characterization of antibacterial
123
J Nanopart Res (2010) 12:16251636
viscose-based activated carbon fibre supporting silver.
Carbon 36:15671571
Warheit DB, Laurence BR, Reed KL, Roach DH, Reynolds
GA, Webb TR (2004) Comparative pulmonary toxicity
assessment of single wall carbon nanotubes in rats. Toxi-
col Sci 77:117125
Wasan DT, Nikolov AD (2003) Spreading of nanofluids on
solids. Nature 423:156159
Yamamoto O (2001) Influence of particle size on the anti-
bacterial activity of zinc oxide. Int J Inorg Mater 3:643
646
Yamamoto O, Hotta M, Sawai J, Sasamoto T, Kojima H (1998)
Influence of powder characteristic of ZnO on antibacterial
activityeffect of specific surface area. J Ceram Soc Jpn
106:10071011
Yamamoto O, Komatsu M, Sawai J, Nakagawa Z (2004) Effect
of lattice constant of zinc oxide on antibacterial charac-
teristics. J Mater Sci: Mater Med 15:847851
Zhang L, Jiang Y, Ding Y, Povey M, York D (2007) Anti-
bacterial behaviour of suspensions of ZnO nanoparticles
(Nanofluids). J Nanopart Res 9:479489
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