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Investigation Into the Antibacterial Behaviour of Suspensions of ZnO

Nanoparticles (ZnO Nanofluids)

Article  in  Journal of Nanoparticle Research · June 2007

DOI: 10.1007/s11051-006-9150-1

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Journal of Nanoparticle Research (2007) 9:479–489
Springer 2006
DOI 10.1007/s11051-006-9150-1
Technology and Applications

Investigation into the antibacterial behaviour of suspensions of ZnO

nanoparticles (ZnO nanofluids)

Lingling Zhang1, Yunhong Jiang1, Yulong Ding1,*, Malcolm Povey2 and David York3
1
Institute of Particle Science & Engineering, University of Leeds, Leeds, LS2 9JT, UK; 2Procter Department
of Food Science, University of Leeds, Leeds, LS2 9JT, UK; 3Procter and Gamble Newcastle Technical Centre,
Newcastle-upon-Tyne, NE12 9TS, UK; *Author for correspondence (Tel.: +44-113-343-2747; Fax: +44-
113-343-2405; E-mail: [email protected])

Received 29 April 2006; accepted in revised form 31 July 2006

Key words: antibacterial activity, zinc oxide nanoparticles, nanofluids, mechanisms, E. coli, nanoengineering

Abstract

The antibacterial behaviour of suspensions of zinc oxide nanoparticles (ZnO nanofluids) against E. Coli has
been investigated. ZnO nanoparticles from two sources are used to formulate nanofluids. The effects of
particle size, concentration and the use of dispersants on the antibacterial behaviour are examined. The
results show that the ZnO nanofluids have bacteriostatic activity against E. coli. The antibacterial activity
increases with increasing nanoparticle concentration and increases with decreasing particle size. Particle
concentration is observed to be more important than particle size under the conditions of this work. The
results also show that the use of two types of dispersants (Polyethylene Glycol (PEG) and Polyvinylpyr-
olidone (PVP)) does not affect much the antibacterial activity of ZnO nanofluids but enhances the stability
of the suspensions. SEM analyses of the bacteria before and after treatment with ZnO nanofluids show that
the presence of ZnO nanoparticles damages the membrane wall of the bacteria. Electrochemical mea-
surements using a model DOPC monolayer suggest some direct interaction between ZnO nanoparticles and
the bacteria membrane at high ZnO concentrations.

Introduction presents a potential obstacle for the product for-

mulation. As a consequence, inorganic materials
Antibacterial agents are of relevance to a number such as metal and metal oxides have attracted lots
of industrial sectors including environmental, of attention over the past decade due to their
food, synthetic textiles, packaging, healthcare, ability to withstand harsh process conditions
medical care, as well as construction and decora- (Wang et al. 1998; Hewitt et al. 2001; Fu et al.,
tion. They can be broadly classified into two types, 2005; Makhluf et al., 2005). Of the inorganic
organic and inorganic. Organic antibacterial materials, metal oxides such as TiO2, ZnO, MgO
materials are often less stable particularly at high and CaO are of particular interest as they are not
temperatures and/or pressures compared to inor- only stable under harsh process conditions but
ganic antibacterial agents (Sawai, 2003). This also generally regarded as safe materials to human
beings and animals (Stoimenov et al., 2002; Fu
On visiting from the Tianjin University of Science & et al., 2005). Some of the metal oxides e.g. MgO
Technology, Tianjin, P.R. China. and CaO are essential minerals for human health
480

(Yamamoto, 2001a, b, c; Roselli et al., 2003). nisms – the second objective of this work. From
Other metal oxides such as TiO2 and ZnO have the practical application point of view, ZnO may
been used extensively in the formulation of per- be incorporated into liquid products in which case
sonal care products (Schumacher et al., 2004; the use of a stabiliser may become necessary. In
Axtell et al., 2005; Li et al., 2006). fact, Sawai et al. (1996b) have indicated that the
This work is concerned about the antibacterial stability of ZnO suspensions is important as the
behaviour of ZnO particles for which there are supernatant has no antibacterial activity. As a
only a small number of publications in the litera- consequence, the third objective of this work is to
ture. These studies investigated the antibacterial investigate the effect of the use of polymeric dis-
activity of ZnO particles against Escherichia coli, persants on the antibacterial behaviour. Further-
Salmonella typhimurium, Bacillus subtilis, and more, as mentioned above, there is a disagreement
Staphylococcus aureus etc. and the main conclu- in the dominant mechanism for the antibacterial
sions of these studies can be summarised as: behaviour of ZnO. The fourth objective of this
work is therefore to provide more experimental
• ZnO particles are effective for inhibiting both
evidence that could lead to a thorough under-
Gram-positive and Gram-negative bacteria.
standing of the mechanisms.
They even have antibacterial activity against
spores that are high-temperature and high-
pressure resistant (Sawai et al., 1995a, b,
Experimental
1996a, b).
• Smaller ZnO particles have a better antibacte-
Raw materials
rial activity (Sawai et al., 1996a; Yamamoto,
2001a; Makhluf et al., 2005).
Dry zinc oxide nanoparticles from two suppliers,
• The antibacterial activity depends on the sur-
Nanophase Technologies and Nanostructured &
face area and concentration, while the crystal-
Amorphous Materials (both of USA), were used in
line structure and particle shape have little
this work. The primary sizes of the nanoparticles
effect. The higher the concentration and the
given by the manufacturers were 24–71 nm for the
larger the surface area, the better the antibac-
Nanophase Technologies and 90–200 nm for the
terial activity (Yamamoto et al., 1998).
Nanostrutured & Amporphous Material products,
• High temperature treatment of ZnO particles
respectively. Polyvinylpyrolidone (PVP) and
has a significant effect on their antibacterial
Polyethylene Glycol 400 (PEG400) from Fluka
activity. Treatment at a higher temperature
were employed as dispersants. Luria-Bertani (LB)
leads to a lower activity (Sawai et al., 1996a).
medium used for growing and maintaining bacte-
• The mechanisms of the antibacterial activity of
rial cultures were purchased from Sigma-Aldrich
ZnO particles are not well understood although
(UK). Escherichia coli DH5a was provided by the
Sawai et al. (1996b, 1997, 1998) proposed that
Department of Biological Science of the University
the generation of hydrogen peroxide be a main
of Leeds. Potassium chloride and sodium chloride
factor of the antibacterial activity, while Stoi-
were purchased from Merck (USA).
menov et al. (2002) indicated that the binding
of the particles on the bacteria surface due to
Formulation and characterisation of ZnO
the electrostatic forces could be a mechanism.
suspensions (ZnO nanofluids)
These studies, however, have not addressed the
effect of the formulation of ZnO suspensions on ZnO nanoparticles from both manufacturers were
the antibacterial activity. This forms the first used as received for producing suspensions. To
objective of this work. It is noted that the previous gain more information of the shape, size distribu-
researchers only characterised ZnO powders in tion and morphology of the as-received nanopar-
their dry form and paid little attention to the ticles, a scanning electron microscopy (SEM) was
characterisation of suspensions. As the antibacte- used. Figure 1(a) and (b) show the SEM images of
rial tests are mostly done with suspensions, char- the particles from the two suppliers, respectively. It
acterisation of the suspensions could provide more can be seen that ZnO particles from both sources
insight into the underlying antibacterial mecha- are in the form of agglomerates. Figure 1(b) also
 481

Figure 1. SEM images of ZnO nanoparticles as received from (a) Nanophase Technologies, (b) Nanostructured & Amorphous
Materials.

shows the existence of particles that are much agglomerates as seen in Figure 1(a) and (b). After
larger than 200 nm and some are even micron 30 min of sonication, the so-called master ZnO
sized. nanofluid was produced, which had a concentra-
To make ZnO nanofluids for the antibacterial tion of 1 g/l. The master nanofluid was then dilu-
tests, a preset amount of dry ZnO nanoparticles ted with distilled water to different concentrations
was mixed with distilled water in a glass beaker for the antibacterial tests. As the ZnO nanoparti-
with the aid of a magnetic stirrer. Once particles cles from the Nanostructured & Amorphous
were dispersed in water, the beaker was placed in Materials were very large as mentioned above, a
an ultransoicator (Clifton, UK). The reason for Dyno-Mill (Willy A. Bachofen, Switzerland) was
the use of sonication was to break down the used to process the suspension for an hour at the
482

room temperature. To enhance the stability of the Morphology of the bacteria

suspension, two dispersants, PVP and PEG400,
were used to produce suspensions of ZnO nano- SEM analyses were performed to investigate the
particles supplied by Nanophase Technologies and morphological changes of the E. coli. For doing
the amount of the dispersant was 10% of the so, the E. coli culture was fixed with 2.5% glu-
amount of zinc oxide nanoparticles. Table 1 shows taraldehyde for at least 2 h. The sample was then
a list of nanofluids prepared for the antibacterial washed twice with de-ionised water each for
tests. 20 min. The washed sample was then put into 1%
Nanofluids prepared were autoclaved at 121C osmium tetroxide for 4 h, followed by washing
for 15 min and then characterised after cooling twice with de-ionised water each 20 min. The
down to the room temperature by using an SEM washed sample was then put onto a stub for air
for shape, size and morphology analyses and a drying, coating with 3 nm platinum, and SEM
Nano Sizer (Malvern Instruments) for size distri- analyses using a LEO Gemini 1530 field emission
bution and surface charge (Zeta potential) mea- SEM at a voltage of 5 kV.
surements.
ZnO nanoparticle–bacteria wall interaction
Antibacterial tests
To investigate potential interactions between ZnO
Antibacterial tests were performed by measuring nanoparticles and the bacteria membrane wall, a
the growth curve of E. coli DH5a incubated in the three-electrode system was used, which consisted
LB broth medium in the presence of nanofluids of a platinum counterelectrode, an Ag/AgCl (ver-
containing different concentrations of ZnO nano- sus 3.5 mol/dm3 KCl) reference electrode and a
particles. All experiments were performed in the hanging mercury drop electrode as the working
dark. In a typical experiment for construction of electrode; see details of the technique in the liter-
the growth curves, 50 ll of the E. coli culture with ature (Leermakers & Nelson, 1990; Nelson et al.,
an approximately concentration of 106–107 colony 2001; Neville et al., 2004). A Langmuir monolayer
forming units per millilitre (CFU/ml) were inocu- composed of dioleoyl-phophatidylcholine (DOPC)
lated in a 5 ml solution mixer, which contained molecules was used to model the external layer of
autoclaved LB broth medium and autoclaved ZnO the bacterial membrane. The DOPC monolayer
nanofluid. Cultures were grown at 37C under an was deposited on the mercury drop electrode,
agitation condition. The growth curve was deter- which had an area of 0.0088 cm2 and submerged in
mined by measuring the time evolution of the the ZnO nanofluid with 0.1 mol/dm3 NaCl. The
optical density (OD) of the sample contained in a liquid phase was fully deaerated with special grade
10 mm optical path length quartz cuvette. The argon before each experiment and a blanket of
measurements were done at 600 nm with a spec- argon was maintained above the liquid during the
trophotometer (Thermo Electron Corporation, experiment to avoid any introduction of oxygen.
USA) at a frequency of once an hour. A blank LB Application of an electrical potential across the
broth medium cultured under the same conditions electrode system generated electrical current. The
was used as a control. relation between the applied potential and the
resultant current in the presence and absence of
ZnO nanoparticles provided information of the
Table 1. List of master nanofluids prepared for the anti- interaction between the DOPC monolayer and the
bacterial tests particles.
Sample Nanoparticle Processing Stabiliser Average
code suppliers methods size, nm
Results and discussion
ZnO-1 Nanophase Ultrasonication – 249
ZnO-2 Nanophase Ultrasonication PEG400 293 Characterization of ZnO nanofluids
ZnO-3 Nanophase Ultrasonication PVP 314
ZnO-4 N&A, US Ultrasonication – 2417
ZnO nanofluids prepared with the method descri-
ZnO-5 N&A, US Nano-grinding – 230
bed in the Experimental section have a pH value of
 483

7.2 ± 0.1 at which the zeta-potential is approxi- use of dispersant enhances considerably the sta-
mately +24 mV. It is also found that the iso- bility of the suspensions thus brings in benefit.
electric point (IEP) of ZnO is 9.4, consistent with Note that particle sizes measured by the Malvern
the reported data in the literature (Sheng & Liu, Nano-Sizer are hydrodynamic diameter based on
2004). SEM analyses of the ZnO nanofluids reveal the Stokes–Einstein equation, which is expected to
little changes to the morphology of nanoparticles be larger than the actual particle size.
due to ultrasonication and the use of dispersants A comparison between the sizes of ZnO-4 and
except for disintegration of some nanoparticle ZnO-5 in Table 1 suggests that the use of Dyno-
agglomerates. However, the use of Dyno-Mill to Mill reduces the particle size significantly, which
process suspensions made with ZnO particles from agrees with the SEM analysis; see Figures 1(b)
Nanostructured & Amorphous Materials changes and 2.
the morphology of ZnO particles significantly.
Figure 2 shows the results. It can be seen that large Antibacterial test results
ZnO crystals as shown in Figure 1(b) have been
milled down to below 100 nm. Figure 2 also Antibacterial tests were carried out with nanofl-
indicates that the milled particles are also in the uids formulated under different conditions as
form of agglomerates. shown in Table 1. These master nanofluids are
All nanofluids as shown in Table 1 are analysed diluted to different concentrations for the tests.
by using the Malvern Nano-Sizer for particle size The results are presented in this section.
distribution and average size. Two peaks are gen-
erally observed for all samples, which is likely due Effect of nanoparticle concentration
to the presence of agglomerates as shown in Fig- Autoclaved Sample ZnO-1 (Table 1) was mixed
ures 1 and 2. The average sizes of the samples are with autoclaved LB medium to make nanofluids
shown in the last column of Table 1. It can be seen with ZnO concentrations of 0.1 and 0.25 g/l. Fig-
that the use of ultrasonication does not seem to be ure 3 shows the growth curves of the two nanofl-
effective in breaking down nanoparticle agglom- uids together with the negative control. As the
erates and the use of dispersants does not enhance value of the optical density (OD) at 600 nm rep-
the size reduction. However, it is observed that the resents the absorbance of the bacteria, an increase

Figure 2. An SEM image of ZnO nanoparticles after grinding with the Dyno-Mill for 1 h(Nanostructured & Amorphous
Materials).
484

3.5 Negative control

Negative control 4
2417nm ZnO 0.1 g/l
3.0 ZnO 0.1 g/l 230nm ZnO 0.1 g/l
2.5 ZnO 0.25 g/l
3
OD at 600nm

OD at 600nm
2.0
1.5 2

1.0
1
0.5
0.0
0
-0.5
0 2 4 6 8 10 -2 0 2 4 6 8 10 12 14 16 18
Time, hour Time, hour
Figure 3. Growth curves of E. coli in LB medium inocu-
Figure 4. Growth curves of E. coli in LB medium inocu-
lated with 107 CFU of bacteria in the presence of two dif-
lated with 107 CFU of bacteria in the presence of ZnO
ferent concentrations of ZnO (ZnO-1).
suspensions of different particle sizes.

in the number of bacteria implies more light being more important than that of particle size under the
absorbed by the bacteria. As a consequence, Fig- conditions of this work.
ure 3 shows clearly a bacteriostatic action of ZnO The influence of particle size of on the antibac-
nanoparticles against E. coli. In particular, little terial activity of metal oxides has been reported in
bacteria growth is seen for 8 h with nanofluid the literature; see for example Yamamoto (2001a)
containing 0.25 g/l ZnO made from the ZnO-1 and Makhluf (2005). Yamamoto (2001a) examined
Sample (Table 1). Similar behaviour has been the effect of primary particle size of ZnO over a
observed by Sawai et al. (1995a), Sawai (2003) and range of 100 and 800 nm obtained by milling in a
Yamamoto (2001a) although a different method planetary ball mill. He used electrical conductivity
called conductance method was used by them. as the parameter to evaluate the antibacterial
With such a method, the growth inhibitory effect is activity and showed that the antibacterial activity
studied by measuring the so-called Detection Time increased with decreasing particle size. Makhluf
(DT) at which the bacteria concentration reaches et al. (2005) produced MgO nanoparticles with
approximately 107 viable organisms per ml. different particle sizes by the microwave method
and tested their antibacterial behaviour. By
Effect of the particle (agglomerate) size adjusting the concentration of the reactant
Samples ZnO-4 and ZnO-5 as shown in Table 1 Mg(Ac)2, they were able to produce MgO particles
were used to make nanofluids for investigating the with the primary particle size between 8 and
effect of particle (agglomerate) size. The particle 23 nm. Their antibacterial tests with 1 g/l MgO
concentration used in these tests is 0.1 g/l. Fig- particles showed that the antibacterial activity
ure 4 shows the results. It can be seen that smaller increased with decreasing primary particle size.
particles (agglomerates) have a much better bac- These observations seem to be in broad agreement
teriostatic activity. Particles in Sample ZnO-4 have with that of this work. It should be noted that the
a very large average particle size (2417 nm) and benchmark of this work is based on the actual
there is little or no antibacterial activity with the agglomerate size rather than the primary particle
growth curve almost overlapping the negative size as used by Yamamoto (2001a) and Makhluf
control curve. A comparison of the data with et al. (2005). As mentioned in the Introduction
0.1 g/l nanofluids shown in Figures 3 and 4 indi- section, the dominant mechanism of the antibac-
cates that the antibacterial performance of ZnO terial behaviour of ZnO particles is still unclear;
particles from the two suppliers is similar (the there is insufficient information to judge which
average particle sizes in the two cases are similar). benchmark should be used. More discussion on
An inspection of Figures 3 and 4 also indicates the antibacterial mechanisms is presented later in
that the effect of particle concentration seems to be this paper.
 485

Effect of the presence of a stabiliser the observed difference require further investiga-
ZnO suspensions in the absence of a stabiliser can tion; however, different physical and chemical
have stability issue, which bears significance to the properties of silver and platinum from that of ZnO
shelf-life. This could be overcome by using a dis- are likely to a reason. Different physical and
persant, which, however, could have impact on the chemical properties of the antibacterial materials
antibacterial behaviour. In this work, PEG400 and could imply different mechanisms of the antibac-
PVP are considered which are shown to be good terial behaviour, and the presence of a dispersant
stabilising agents for ZnO nanofluids. Their effects may affect the mechanisms in different ways.
on the antibacterial behaviour are shown in Fig-
ure 5 for Samples ZnO-1, ZnO-2, and ZnO-3 SEM analysis of the E. coli
(Table 1). These tests were done with a ZnO con-
centration of 0.1 g/l. It can be seen that the use of To gain direct evidence of the antibacterial
PEG has little effect on the antibacterial perfor- behaviour of ZnO nanoparticles, SEM analyses
mance of ZnO, whereas the use of PVP gives a were performed on some E. coli samples before
slightly negative effect. Given the experimental and after antibacterial tests. Figure 6(a) and (b)
uncertainty and small size difference of particles show respectively the bacteria images before and
between the PVP and PEG stabilised nanofluids, after treatment for 5 h with 0.2 g/l ZnO nanofluids
the effects of both PEG400 and PVP on the anti- made with Sample ZnO-5. One can clearly see that
bacterial activity are very small. treatment of the bacteria with ZnO nanoparticles
The effect of stabilisers on the antibacterial has led to considerable damage to some E. coli and
behaviour has also been studied by Cho et al. the damage has caused the breakdown of the
(2005) with silver and platinum nanoparticle sus- membrane of the bacteria.
pensions. In their study, poly-(N-vinyl-2-pyrroli-
done) (PVP) and sodium dodecylsulfate (SDS) Interaction between ZnO nanoparticles and bacteria
were used as the stabilisers, and the antibacterial membrane wall
tests were on S. aureus (KCTC 1928) and E. coli
(KCTC 1041) with the cup diffusion method. Their SEM analyses as described above indicate dam-
results showed that both silver and platinum ages to the bacterial membrane. One of the pos-
nanoparticles had strong antibacterial activity. sible reasons for the damage could be direct
However, the silver and platinum nanoparticles interaction between ZnO nanoparticles and the
stabilised by the SDS did not show antibacterial external membrane surface. To investigate this, a
activity. These observations disagree with that of monolayer of dioleyl phosphatidylcholine (DOPC)
this work as described above where little effect of molecules was used to simulate the bacteria
the dispersants is observed. The exact reasons for membrane wall. Figure 7 shows the results in the
form of current as a function of the applied
3.5 potential across the electrode system (called AC
Negative control
3.0 ZnO 0.1g/l Voltammogram) under various conditions. Shown
ZnO + PEG 0.1g/l in Figure 7(a) is the current–voltage relation in the
2.5 ZnO + PVP 0.1 g/l absence of ZnO nanoparticles. Two sharp pseudo-
OD at 600nm

2.0 capacitive peaks are observed, which correspond

1.5 to two consecutive phase transitions (Leermakers
1.0 et al., 1990). In the presence of ZnO nanoparticles,
an extra peak forms in the AC Voltammograms;
0.5
see Figure 7(b)–(d). This peak is mainly caused by
0.0 the cations of Zn. The height of the Zn peak is seen
-0.5 to increase with increasing ZnO concentration. An
0 2 4 6 8 10 inspection of the Voltammograms indicates little
Time, hour changes in the shape and size of the two peaks
Figure 5. Growth curves of E. coli in LB medium inocu- formed by the DOPC monolayer at ZnO concen-
lated with 107 CFU of bacteria in the presence of different trations lower than 0.4 g/l, suggesting little direct
dispersants. interactions between ZnO nanoparticles and the
486

Figure 6. SEM images of E. coli: (a) before antibacterial tests; (b) after treatment with 0.2% ZnO nanofluids for 5 h.

DOPC lipids at these concentrations. At high oxides. Makhluf et al. (2005) investigated the
concentrations, particularly 2 g/l ZnO, small antibacterial behaviour of MgO and attributed the
interaction seems to exist; see Figure 8 which behaviour to the following mechanisms: produc-
overlays the data shown in Figure 7(a) and (d). tion of active oxygen species due to the presence of
More work is therefore needed to confirm these MgO, interaction between MgO particles and
observations. membrane cell wall, penetration of individual
MgO particles into cell and reformation of MgO
Discussion on the antibacterial mechanisms within the cell. Sawai et al. (1996b) studied the
antibacterial behaviour of ZnO particles by using a
A number of mechanisms have been proposed to chemiluminescence and oxygen electrode analysis.
interpret the antibacterial behaviour of metal They reported that H2O2 was produced in ZnO
 487

0.04 0.04
(a) 0.03
(b)
0.03 0.4% ZnO nanofluid

0.02 0.02

Current, mA
Current, mA

0.01 0.01

0.00 0.00

-0.01 -0.01

-0.02 -0.02

-0.03 -0.03 Zn peak

-0.04 -0.04
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2
Applied Potential, V Applied Potential, V

0.04 0.03
(c) (d) 2 g/l ZnO nanofluid
0.03 0.8% ZnO nanofluid 0.02

0.02 Current, mA 0.01

Current, mA

0.01
0.00
0.00
-0.01
-0.01
-0.02
-0.02

-0.03 -0.03
Zn peak
Zn peak
-0.04 -0.04
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2
Applied Potential, V Applied Potential, V
Figure 7. Current–applied potential relationships across the electrode system in absence (a) and presence (b–d) of ZnO
nanofluids with 0.1 mol/dm NaCl electrolytes.

slurry and the concentration of H2O2 produced dispersant (PVP and PEG), which are long-chain
was linearly proportional to the ZnO particle polymers and are likely to be adsorbed onto the
concentration of the slurry. H2O2 was also detec- ZnO particle surface. Adsorption of the long-chain
ted by Yamamoto et al. (2000) with a spectro- polymer onto the ZnO particle surface implies a
photofluorometer. Stoimenov et al. (2002), on the barrier between the bacteria cell wall and ZnO
other hand, suggested that the electrostatic inter- nanoparticles. The little effect due to the use of
actions between the bacteria surface and nano- dispersant also seems to exclude the electrostatic
particles be a reason. interaction as a possible mechanism as proposed
The results obtained in this work clearly show by Stoimenov et al. (2002). The small effect of the
that the presence of ZnO nanoparticles leads to ZnO particle (agglomerate) size suggests that the
damages to the membrane wall of E. Coli. Such penetration mechanism proposed by Makhluf
damages may be partly due to direct interactions et al. (2005) for MgO particles be less unlikely a
between ZnO nanoparticles and bacteria mem- major mechanism for the observed antibacterial
brane surface as shown in Figure 8. ZnO nano- behaviour.
particles used in this work are in the form of It seems that active oxygen species generated by
agglomerates with an average size greater than ZnO particles could be a mechanism although
200 nm. These large agglomerates are less likely there is no direct evidence from the results of this
to penetrate into the cell wall to damage the bac- study. The presence of active oxygen species
teria from the interior. This is supported by has been detected by Sawai et al. (1996b) and
the observation of little effect due to the use of Yamamoto et al. (2000). However, it is less clear
488

0.04 membrane damage may be partly due to the direct

Resultant Current, mA

0.03 interaction between ZnO nanoparticles and bac-

0.02
teria membrane wall. The mechanisms of the
antibacterial behaviour are discussed. The results
0.01
suggest that both the direct nanoparticle-cell
0.00 membrane wall interaction and generation of
-0.01 active oxygen species be mechanisms but there are
-0.02 a number of questions remained to be answered
-0.03
No ZnO before a firm conclusion could be drawn.
2g/l ZnO
-0.04
Acknowledgements
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2
Applied Potential, V The work is financially supported by the Nano-
Figure 8. Comparison of Figures 7(a) (no ZnO) and 7(d) manufacturing Institute of the University of Leeds
(2 g/l ZnO). and Procter and Gamble Company. The authors
would like to extend their thanks to Dr Jianjun
Fang of the Biological Science Department at the
how the active oxygen species are produced and University of Leeds for providing the E. coli strain.
how to improve the active oxygen production if Sincere thanks are due to Mr. Adrian Hick of
they were the main mechanism. It is known that Faculty of Biological Sciences for assisting in the
ZnO can be photo-catalytic under the UV light, SEM analysis, and Dr. Andrew Nelson and
which could be a reason for the production of the Mr. Zachary Coldrick of School of Chemistry for
active oxygen species. However, all the antibacte- advices and performing experiments on the
rial tests of this work were done under the dark electrochemical analyses.
conditions. A further question is therefore how
photoactive the ZnO nanoparticles are in suspen-
sions under the dark conditions. Further work is References
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structural changes in electrode-adsorbed lipid layers. J.
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and PVP dispersants shows little effect on the ticles. J. Hosp. Infect. 62, 58–63.
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