Biomedical Materials

Understanding the Antibacterial Mechanism of

CuO Nanoparticles: Revealing the Route of Induced
Oxidative Stress
Guy Applerot, Jonathan Lellouche, Anat Lipovsky, Yeshayahu Nitzan, Rachel Lubart,
Aharon Gedanken,* and Ehud Banin*

To date, there is still a lack of definite knowledge regarding the interaction of CuO
nanoparticles with bacteria and the possible permeation of the nanoparticles into
bacterial cells. This study was aimed at shedding light on the size-dependent (from
the microscale down to the small nanoscale) antibacterial activity of CuO. The
potent antibacterial activity of CuO nanoparticles was found to be due to ROS-
generation by the nanoparticles attached to the bacterial cells, which in turn provoked
an enhancement of the intracellular oxidative stress. This paradigm was confirmed
by several assays such as lipid peroxidation and reporter strains of oxidative
stress. Furthermore, electron microscopy indicated that the small nanoparticles
of CuO penetrated the cells. Collectively, the results reported herein may reconcile
conflicting concepts in the literature concerning the antibacterial mechanism of CuO
nanoparticles, as well as highlight the potential for developing sustainable CuO
nanoparticles-based devices for inhibiting bacterial infections.

1. Introduction Moreover, the in-depth study of nanomaterials and nanos-

Manufactured nanoparticles (NPs) and their applications
are an expanding domain of technology as the use of these
materials is constantly on the rise. Exploration of nanomate-
rials that exhibit functionality is the key to nanotechnology.[1]
Dr. G. Applerot, J. Lellouche, Dr. A. Lipovsky,
Dr. R. Lubart, Prof. A. Gedanken
Department of Chemistry and Kanbar Laboratory
for Nanomaterials at the Bar-Ilan University Center
for Advanced Materials & Nanotechnology
Bar-Ilan University
Ramat-Gan 52900, Israel
E-mail: gedanken@mail.biu.ac.il
J. Lellouche, Prof. Y. Nitzan, Dr. E. Banin
The Mina & Everard Goodman Faculty of Life Sciences
Center for Advanced Materials & Nanotechnology
Bar-Ilan University
Ramat-Gan 52900, Israel
E-mail: ehud.banin@biu.ac.il
DOI: 10.1002/smll.201200772
tructures is an important source for producing new principles,
new techniques and new methods.[2] A common theme that
arises from the study of nanomaterials in a biological context,
is that when the particle size decreases, the proportion of the
surface area increases, which results in enhanced biological
activity per given mass compared to larger particles.[3]
Inorganic metal oxides such as ZnO, MgO, TiO2, and
Al2O3 are being increasingly used for antimicrobial appli-
cations and are replacing the frequently used silver, owing
to its toxic effect on the environment and human health.[4]
The key advantages of using inorganic oxides compared to
organic antimicrobial agents are their stability, robustness,
and long shelf life.[4] However, the mechanism underlying the
antibacterial activity of these metal oxides has not been une-
quivocally discerned, which may hinder the rational develop-
ment of appropriate mitigation and remediation approaches.
Moreover, it is not yet clear to what extent very small metal
oxide particles (<50 nm) affect the antibacterial activity.[5]
Among the large family of metal oxides, cupric oxide
(CuO) is an interesting multifunctional material due to its

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 full papers
promising applications in magnetic storage media, solar
energy transformation, electronics, sensors, batteries and
catalysis. Cupric oxide is cheaper than silver, easily mixed
with polymers and relatively stable in terms of both chemical
and physical properties.[6]
One of the first CuO-based products was put forth by a
private company, Cupron. This firm incorporates micron-sized
CuO particles into polymers which are further made into
wound dressings. These dressings effectively prevent infec-
tion and increase the rate of wound healing, compared to
standard treatments.[7] Our group also reported on an in situ
generation of CuO NPs and their subsequent deposition on
fabrics in a one-step reaction using ultrasound irradiation.[8]
However, despite the great potential of CuO based-materials,
to date, only a limited number of studies have examined the
activity of nano-preparations of CuO. In these studies the
antimicrobial mechanism of CuO NPs is attributed to
the release of Cu ions.[9–11]
It should also be noted that for promoting the safe devel-
opment of a new class of materials, it is essential to assess
the potential adverse health consequences associated with
human exposure. In this respect, copper is considered as
one of a relatively small group of metallic elements that are
essential to human health.[12] In addition, human tissue (skin
or other) shows enhanced resistance to copper, compared to
microorganisms.[9–11]
Numerous methods have already been developed for
the fabrication of CuO NPs with various morphologies.[13]
Among these methods, many are based on high-temperature
processes, e.g., oxidation of copper grids, foils and wires in
air or oxygen at high temperatures.[14] Other methods, such
as: reverse micelle, surfactant templates, and transformation
from Cu(OH)2 nanobelts to CuO nanowires, solid state reac-
tions, hydrothermal and thermal decomposition synthesis
routes have also been published.[15]
Ultrasonic irradiation has been proven as an effective
technique for the synthesis of nanophased materials,[16] as
well as for their deposition on various polymeric matrices
in a one-step process.[17–19] Furthermore, sonochemical irra-
diation fulfills the requirement of minimizing the use of toxic
chemicals, solvents, energy, etc., and therefore is regarded
as a “green” chemistry approach.[18,19] The chemical effects
of ultrasound arise from acoustic cavitation, i.e. the forma-
tion, growth, and implosive collapse of bubbles in a liquid
medium. The compression of the bubbles during cavitation
is more rapid than the thermal transport, which generates
short-lived, localized hotspot bubbles reaching temperatures
as high as 5000 K, pressures of roughly 1000 atm, and heating
and cooling rates above 1 × 1010 K/s.[16]
Oxygen is essential for most living organisms, but is also
a precursor to reactive oxygen species (ROS), which can
damage cellular components such as proteins, lipids and
nucleic acids. ROS are ions or small molecules that con-
tain peroxide (e.g., hydrogen peroxide; H2O2), free radicals
(.OH), or oxygen ions (e.g., superoxide;.O2-). These ROS are
highly reactive species formed during the normal metabo-
lism of oxygen.[20] Besides such ROS, singlet oxygen, 1O2,
is also considered as a very toxic species.[21] Under normal
physiological conditions ROS are important for cell signaling.
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G. Applerot et al.
Cells are able to detoxify ROS using enzymes (e.g., catalase
and superoxide dismutase) or small molecules (e.g., ascorbic
acid). Still, this antioxidant activity has a limit and the forma-
tion and accumulation of ROS will increase dramatically if
the cell is constitutively exposed to oxidative stress.[22]
In our previous investigation,[23] the effect of the par-
ticle size of ZnO as an antibacterial agent was demonstrated.
These antibacterial experiments were conducted with nanoc-
rystalline ZnO. The bacteriological assays were carried out
on two strains from different species as representatives of
various bacteria types: Escherichia coli (E. coli; Gram-neg-
ative) and Staphylococcus aureus (S. aureus; Gram-positive).
The results showed that the antibacterial activity of ZnO
NPs increased with reduction in NPs size. The smaller par-
ticles also penetrated the bacterial cells causing disorgani-
zation of the cell membrane. We also showed that the ZnO
antibacterial activity resulted from ROS, namely, hydroxyl
radicals, (.OH), produced on the surface of the ZnO NPs,
and, we considered these species as generating the antibacte-
rial effect.
Inspired by these findings, we continued our efforts
toward CuO investigation. In the present study, we utilized
sonochemistry to synthesize CuO NPs and characterized the
influence of particle size on the antibacterial effect, thus elu-
cidating the mechanism underlying the CuO antibacterial
activity. Our results suggest that the antimicrobial activity
of CuO NPs is exerted via the production of ROS. Electron
microscopy also demonstrated that the NPs penetrate and
facilitate morphological changes in the bacterial cells.
2. Results and Discussion
2.1. CuO NP Synthesis and Characterization
The sonochemical synthesis conditions of the CuO NPs
were similar to those previously utilized to synthesize ZnO
NPs (see details in ref. [23]). In brief, the copper oxide NPs
are formed by the sonication of an aqueous solution of
Cu(CH3COO)2 and adding ammonia solution during the
reaction, according to the following reactions:
Cu2+ 2+
(aq) + 4NH3(aq) → [Cu (NH3 )4 ](aq) (1)
+
[Cu (NH3 )4 ]2+
(aq) + H2 O → CuO(s) + 2NH4(aq) + 2NH3(aq) (2)
The formation of copper oxide takes place through the
ammine complex, Cu[(NH3)4]2+. Copper ions react with a
solution of ammonia to form a deep blue solution containing
the ammine complex ions. This complex is hydrolyzed and
crystalline CuO NPs are obtained.
Figure S1 illustrates the X-ray diffraction (XRD) patterns
of the as-prepared CuO NPs (coded as CuO-1). The XRD
patterns of a commercial nanometric and micrometric CuO
powders (coded as CuO-2 and CuO-3, respectively; Aldrich)
are illustrated in Figure S1 as well. The XRD reflections
match that of JCPDS card No. 48–1548.
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Understanding the Antibacterial Mechanism of CuO Nanoparticles

No diffraction lines associated with impurities were to evaluate the cellular response to CuO NPs exposure, pro-
detected. C, H, N elemental analysis results showed that the viding information on cell death, survival, and metabolic
detected products contained only copper, oxygen, and a trace activities.
of carbon and hydrogen (measured at <3%).
Using the Scherrer formula, the average size of the parti-
cles can be estimated.[24] The nanometer-scale nature of the 2.2. Antibacterial Activity
synthesized products was also illustrated in the specific sur-
face area results, using the Brunauer–Emmett–Teller (BET) 2.2.1. Cell Viability
method. The apparent equivalent diameter of the particles
We treated the bacteria with each of the three samples (as
can be estimated from the specific surface area measured by
labeled in Table 1) of a CuO aqueous suspension at various
the BET method by assuming the particles to be spherical
concentrations, ranging from 0.05 up to 10 mg/ml. These
according to the following expression:
concentrations are within the scale of minimal bactericidal
concentration (MBC) of CuO NPs as has been estimated
dBET = 6/(ρ × SBET ), (3)
previously for various bacteria types.[9] In order to reduce
the tendency of the NPs to agglomerate[25] and to ensure an
where dBET is the diameter of the particles, ρ is the bulk
optimum NPs dispersion, the incubation was carried out on a
density of the solid (6.3 g cm−3 for CuO) and SBET is the
rotary platform.
multipoint specific surface area measured.
Representative results of the viability of each bacteria
Nevertheless, these methods (Scherrer and BET) for the
strain following a relative short time duration (i.e. 3 h) of
calculation of average particle size should be used only as a
treatment with CuO suspensions (at concentration of 0.1 mg/
rough guide for comparison.
mL) are presented in Table 2. The viable bacteria were moni-
An unambiguous demonstration of the nanometer-scale
tored by plating and counting the number of colony-forming
nature of the synthesized product was obtained from trans-
units (CFU) (Figure S3). Untreated control cultures did not
mission electron microscopy (TEM) and high-resolution
show any change in viability throughout the experiment. As
TEM (HRTEM) images of the samples. Figure S2 (a,b)
can be seen from Table 2, all CuO samples reduced viability
shows TEM images of the as-prepared CuO NPs (CuO-1)
by at least 36% following exposure for 3 h. The antibacte-
and the commercial CuO NPs (CuO-2). It is evident from
rial activities of CuO particles were found to be related to
this figure that the particle size of CuO-2 was about 30 nm
their size, with the smallest NPs having the highest activity
(which is in accordance with the manufacturer’s affirmation,
on the basis of equivalent CuO mass content. We also exam-
i.e. <50 nm), whereas the CuO-1 is very small with a narrow
ined a 1 h treatment, and the best antibacterial activity (fol-
size distribution estimated to be about 2 nm (Figure S2). Lat-
lowing treatment with CuO-1) resulted in inactivation of S.
tice fringes are also clearly observed. According to the TEM,
aureus by 48%, and after 3 h of treatment by 97%. E. coli
both samples, to some extent, formed an aggregated struc-
also showed a similar susceptibility trend with regard to the
ture, due to their nanometer size and strong van der Waals
length of treatment but to a greater extent. That is, under
force interaction.[25]
the same conditions only 10% of the population was viable
The average particle size calculated by XRD and BET
after 1 h, and after 3 h of treatment less than 0.1% survivors
are consistent with the scale-range obtained from the TEM
were detected. As shown in Table 2, increasing the CuO par-
studies as well as with dynamic light scattering (DLS) meas-
ticle size resulted in reduced antibacterial activity against S.
urements. Table 1 summarizes the average particle size of the
aureus. For E. coli, however, the effect of particle size on the
three CuO samples as calculated by XRD, BET, DLS, and
antibacterial activity was less pronounced, yet it still existed.
as visualized using TEM/HRTEM. Statistical analysis of the
particle size distribution according to TEM/HRTEM was also 2.2.2. ATP Content
performed, using the Scion image software (Scion 2.0, Scion
Adenosine triphosphate (ATP) is an important coenzyme
corporation) for image processing and the Microcal Origin
that transfers energy and can indicate the condition of the
program for data analysis (Origin 7.0, OriginLab, Figure S2c).
cell.[26] Thus, the ATP levels in the bacterial cells after CuO
Our next step was to evaluate the toxicity of CuO against
treatment were also determined. Figure S4 shows that the
two common bacterial pathogens (E. coli and on S. aureus).
CuO-1 antibacterial treatment induced a massive loss of the
Several bacterial viability assays were implemented in order
intracellular ATP content of both bacterial cells, apparently
resulting from cell membrane damage. The rapid and com-
Table 1 . CuO particle size characterization. Particle sizes obtained plete depletion of ATP may be also indicative of a stimulation
from the Debye–Scherer formula, TEM/HRTEM and BET multipoint sur- of the oxidation of residual ATP by ROS. For both bacterial
face area of the different samples of copper oxide.
strains the ATP level after 80 min is less than 10% compared
to the untreated control. It is important to note that the E.
Sample XRD BET DLS TEM/HRTEM
coli treated strain showed a more prominent affect.
[nm] [nm] [nm] [nm]
CuO-1 6.5 7.5 5 2 2.2.3. Electron Microscopy Examinations
CuO-2 22 37 45 30
Based on the antibacterial results we continued to address
CuO-3 ∼720 685 ∼900 ∼800
the mechanism of action of the CuO NPs on the two bacterial

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 full papers G. Applerot et al.

Table 2. Antibacterial activity of CuO particles. Viable count of E. coli and S. aureus exposed to CuO 1-3 particles at a concentration of
0.1 mg mL−1. N/N0 denotes the relative survival fraction.

E. coli S. aureus
Sample Duration of treatment CFU N/N0 Reduction in viability CFU N/N0 Reduction in viability
[h] [mL−1] [%] [mL−1] [%]
CuO-1 0 4.8 × 107 1 0 1.0 × 107 1 0
1 4.7 × 106 1.0 × 10−1 90 5.2 × 106 5.2 × 10−1 48
2 9.4 × 105 2.0 × 10−2 98 1.1 × 106 1.1 × 10−1 89
3 4.9 × 104 1.0 × 10−3 99.9 3.0 × 105 3.0 × 10−2 97
CuO-2 0 4.8 × 10 7
1 0 1.0 × 10 7
1 0
1 1.5 × 107 3.0 × 10−1 70 8.2 × 106 8.2 × 10−1 18
2 2.4 × 106 4.9 × 10−2 95 4.8 × 106 4.8 × 10−1 52
3 9.6 × 10 5 2.0 × 10−3 98 6.0 × 105 6.0 × 10−2 94
CuO-3 0 4.8 × 107 1 0 1.0 × 107 1 0
1 3.2 × 107 6.7 × 10−2 34 9.3 × 106 9.3 × 10−1 7
−2 −1
2 2.0 × 10 7
4.2 × 10 58 7.7 × 10 6
7.7 × 10 23
3 1.5 × 107 3.0 × 10−1 70 6.4 × 106 6.4 × 10−1 36

species. Electron microscopy techniques are useful tools to
investigate the changes in cell morphology upon exposure to
antimicrobials. Environmental scanning electron microscopy
(ESEM) makes it possible to examine the morphology of
hydrated specimens without exposing them to high vacuum.
The surface structures of both the untreated and the treated
bacterial cells were closely examined following the antibacte-
rial treatment with the two CuO samples (CuO-1 and CuO-2)
and the results imply that the treated bacterial cells were sig-
nificantly changed, with major consequent damage observed
in the cellular membrane/wall. Moreover, accumulated NPs
on membranes can clearly be discerned in the ESEM image
(Figure 1). The NPs accumulation was evident in both bac-
teria species.
Energy-dispersive X-ray spectroscopy (EDS) was also
employed to establish the chemical identity of the NPs
attached to the bacterial cells. Qualitative elemental infor-
mation of the particle-rich regions is depicted in the EDS
spectrum (Figure 1g) revealing the presence of significant
amounts of copper. No trace of cooper was detected in the
untreated cells (data not shown). Transmission electron
microscopy (TEM) makes it possible to visualize NPs’ adhe-
sion on the bacteria in a higher resolution than SEM. A
TEM image of ultrathin sections of a bacterial cell enables
both observations of morphological changes as well as the
detection of intracellular NPs. Furthermore, a TEM mode of
high angle annular dark field scanning transmission electron
microscopy (HAADF-STEM) can reveal the presence of a
metallic phase in a stronger contrast, therefore allowing the
overall structural characteristics of the samples to become
noticeably pronounced.[27]
Figures 2 and S5 show, respectively, TEM and HAADF-
STEM observations of the bacterial cells structures following
antibacterial treatment with CuO NPs (CuO-1and CuO-2).
The results are consistent with those obtained by SEM exam-
inations, revealing a considerable accumulation of the NPs
clusters on the cell membrane/wall as well as distinct disor-
ganization sites within the membrane. The identification of
the black dots in the TEM images as CuO NPs were further
confirmed by EDS and by electron energy loss spectroscopy
(EELS) and was in agreement with the EDS results obtained
by SEM (data not shown). The affinity of CuO NPs to bacte-
rial cell may be derived from the negatively charged bacterial
cell surface which react with CuO NPs that have a positive
Zeta potential.[25b] For CuO-1, a small amount of NPs was
also detected inside the bacterial cytoplasm. It can be hypoth-
esized that the NPs could have entered as a consequence of
the disruption of the cell membrane. Overall, it is likely that
disorganization of the bacterial membrane ultrastructure
and cell content leakage together with the reduction in ATP
levels following exposure to nano-CuO culminate inevitably
in the loss of cell viability.
2.3. Oxidative Stress Assays
The high reactivity of most free radicals makes their detection
difficult. In this respect, the electron spin resonance (ESR)
and the luminol assays (SuperLuminol kit; WPI) provide the
combined advantages of simplicity and sensitivity offering a
valid gross assessment of the production of ROS.
2.3.1. ESR and Luminol
ESR is a spectroscopic technique that tracks the unpaired
electron present in a free radical.[28] Spin trapping is a tech-
nique in which a short-lived, reactive free radical combines
with a diamagnetic molecule (“spin trap”) to form a more
stable free radical (“radical adduct” or “spin adduct”) which
can be detected by electron spin resonance. The most useful
radical trap for oxygen-centered free radicals is 5,5-dimethyl-
1-pyrroline N-oxide (DMPO). The DMPO reacts with ROS,
namely, hydroxyl radicals or superoxide anion radicals, to

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Understanding the Antibacterial Mechanism of CuO Nanoparticles
Figure 1. Environmental scanning electron microscope analysis of CuO
treated cells. (a) E. coli and (b) S. aureus untreated cells after overnight
growth; (c, e) E. coli and (d, f) S. aureus treated with (c, d) CuO-2
and with (e, f) CuO-1, respectively. Black arrows indicate CuO NPs.
(g) Elemental spectrum of CuO-1 after treating E. coli. The Na originates
from the saline medium.
produce DMPO–OH, a relatively stable paramagnetic spe-
cies with a characteristic ESR signal of 1:2:2:1 quartet.[29] It
should be noted that in the case of .O2− the initial spin adduct
is DMPO-OOH which is unstable and eventually decom-
poses to the DMPO-OH adduct.
Luminol reacts with ROS to produce a luminophore with
an emission peak at ∼425 nm. The luminescence intensity is
proportional to the amount of superoxide or other ROS in
the sample.
The ESR measurements show (Figure 3a) that ROS are
present in water suspensions of CuO, their number being
closely related to the size of the CuO particles, with smaller
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DOI: 10.1002/smll.201200772
Figure 2. TEM images of E. coli and S. aureus thin sections. TEM
imagees of E. coli and S. aureus thin sections: (a) untreated E. coli and
(b) S. aureus. (c) Treated E. coli with CuO-2. (d) Treated S. aureus with
CuO-2. (e) Treated E. coli with of CuO-1. (f) Treated S. aureus with NPs of
CuO-1. (g) magnified image of (f). Black arrows indicate CuO NPs. Scale
bars are: (a,e) 500 nm; (b,g) 50 nm; (c,d,f) 100 nm.
sizes having a greater amount of ROS. These results were
deduced from the integration over the signal peaks after sub-
traction of the base level. The approximated relation between
the integrated areas of CuO-1, CuO-2, and CuO-3 was 9:7.5:4
respectively, indicative of the amount of ROS produced (it
should be noted that no lines corresponding to Cu2+ para-
magnetism were detected). In a close agreement with the
ESR data, the Luminol assay showed that the smaller sized
NPs produced double the amount of radicals as compared to
the micrometer sized particles (Figure 3b).
The effect of CuO slurries in bacteria suspensions was
examined with respect to the ROS content. The outcome,
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 full papers
Figure 3. Influence of the CuO particles size and the presence of bacterial
cell on ROS generation. Relative amounts of ROS produced in water
suspensions of CuO (samples 1–3) as assessed by (a) ESR spectra and
(b) SuperLuminol assay. The CuO-1 (in a) curve is horizontally shifted
for clarity. (c) ESR spectra and (d) SuperLuminol assay demonstrating
changes in ROS concentrations upon antibacterial treatment with a
water suspension of CuO-1.
presented in Figure 3c for CuO-1, was rather striking and
consistent with the SuperLuminol assay results (Figure 3d):
combining E. coli and CuO-1 suspensions intensified the
ESR radical signal for up to approximately 90% (in 1 h). A
similar trend was obtained for S. aureus, but at considerably
smaller intensity.
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G. Applerot et al.
No ROS were detected in a pristine bacteria suspension
and no increase in the ROS concentration could be observed
in control experiments, which included bacterial lysates or
bacterial cell membrane fractions instead of living bacteria.
The best way to distinguish between the hydroxyl radicals
and the superoxide radicals is to perform competition experi-
ments with hydroxyl radical scavengers. If both the scav-
enger-derived radical adduct is detected and a corresponding
decrease in the DMPO-OH radical adduct concentration
is found, then hydroxyl radicals are the main species in the
system.[30] Otherwise, it can be concluded that the superoxide
radicals are the prevailing species.
Our preliminary results demonstrated that an addition
of dimethyl sulfoxide (DMSO) which serves as a hydroxyl
radical scavenger, did not result in the reduction of DMPO–
OH and almost no feature corresponding to the DMPO–
CH3 adduct could be detected (Figure 4), suggesting that
the DMPO–OH signal observed in the absence of DMSO
originated mostly from the trapping of .O2−. The addition of
DMSO enables the separation of the DMPO spin adducts:
DMPO-OH, DMPO-CH3, DMPO-OOH obtained following
the trapping of .OH, .CH3 and .O2- by DMPO, respectively.
For identification of the different DMPO adducts formed in
the suspension, simulation of the recorded spectra was per-
formed using an algorithm provided in the WINSIM program
(see experimental section for details). From the simulated
spectrum we calculated (not shown) that the recorded spec-
trum of the CuO NPs represents a linear combination of:
DMPO–CH3 relative concentration 5%, DMPO–OOH; 95%
and DMPO-OH; 5% obtained following the trapping of .CH3,
.O2- and .OH by DMPO, respectively.
2.3.2. Intracellular ROS Concentration
2.3.2.1. DCFH Assay. To identify the existence of ROS inside
the bacterial cell 2′,7′-dichlorofluorescin-diacetate (DCFH-
DA) was used as a visual indicator of the overall oxidative
status of biologic cell. DCFH-DA can cross the cell mem-
brane into the cell and hydrolyzes by intracellular esterases
to nonfluorescent DCFH. In the presence of ROS, DCFH
is oxidized to highly fluorescent dichlorofluorescein (DCF).
Therefore, the ROS concentration in the cell is directly pro-
portional to the fluorescent intensity of DCF.[31] Figure 5a
illustrates the ROS concentration following CuO-1 treatment
in the presence of DCFH. The figure reveals an increase in
fluorescence intensity (∼8 fold) for CuO-1 treated cells com-
pared to untreated control. This was evident for both bacte-
rial species (data are only shown for E. coli). It is noteworthy
that for E. coli the emitted green fluorescence was found to
increase, to some extent (∼20%), after 10 min of reexamina-
tion (Figure 5a). Hence, it can be concluded that the higher
ROS cellular amount, some of which is produced within the
cells, is associated with the antibacterial effect.
2.3.2.2. Reporter Strains Assay. Several E. coli strains
that respond to oxidative stress have been designed and con-
structed by Belkin and co-workers.[32] These reporter strains
emit light upon oxidative stress. One group of these strains,
responsive to the presence of superoxide radicals and other
ROS, has also been developed by Belkin, and can serve as a
ROS-responsive biosensor. In brief, these E. coli strains carry
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Understanding the Antibacterial Mechanism of CuO Nanoparticles

plasmid bioluminescence lux genes which

are fused to specific promoters such as a
catalase-specifying gene (katG), and are
overexpressed in conditions of oxidative
stress (see more details in Figure 5b). As
shown in Figure 5b, the induced lumines-
cence of each of the treated strains was
approximately three times higher com-
pared to its untreated cells control. This
further supports our hypothesis that the
ROS generated by CuO NPs trigger an
oxidative stress on the exposed bacteria.
Figure 4. Superoxide anion generated by CuO NPs. (Black line) ESR spectrum of the DMPO 2.3.3. ROS-Mediated Injury: Lipid
adduct formed in CuO-1 NPs suspension in the presence of DMSO. (Gray line) A simulated Peroxidation
spectrum; the simulation parameters were: 50% of DMPO-OOH: aN = 13.96 G, aH = 12.06 G,
45% of DMPO-OOH: aN = 13.88 G, aH = 12.21 G, 5% of DMPO-CH3: aN = 16.00 G, aH = 27.44 G. Lipid peroxidation, a signature of ROS
damage, can be detected by assaying for
malondialdehyde (MDA), an oxidized
product of polyunsaturated fatty acids
for which there are established detection
protocols. MDA forms an adduct with
thiobarbituric acid (TBA), resulting in a
pink product with increased absorbance at
535 nm.[33] To determine the effect of CuO
NPs on membrane lipid peroxidation, we
followed the MDA adduct production
(as the marker for lipid peroxidation) in
E. coli and S. aureus with and without
exposure to CuO NPs. We have chosen
to conduct this experiment with a con-
siderably lower concentration of CuO-1
NPs (0.01 mg/ml) to deprive the ability
of the CuO NPs themselves to interact
with TBA, and presumably oxidize it to
yield a similar color to the absorption of
the TBA-MDA adduct thus interfering
with the proper functioning of this assay.
As can be seen in Figure S6, the addition
of the CuO NPs gradually increased the
concentration of MDA in both strains, and
more significantly in E. coli cells.

Figure 5. CuO NPs exposition induce bacterial oxidative stress (depicted here for E. coli). 2.4. The Bacterial Oxidative Stress
(a) Oxidative stress was measured by fluorescence microscopy using DCFH-DA. When DCFH-DA Paradigm
was oxidized by reactive oxygen species (ROS), it was converted to DCF and emitted a green
fluorescence. The fluorescence intensity is related to the amount of ROS concentration in the Taken together, our findings indicate the
cells and checked after 10 and 20 minutes of CuO-1 treatment. Each green spot represents following: 1) The amount of eradicated
a bacterial cell. After duration of more than 30 min, a strong green fluorescent background
bacteria is closely related to the particle
was detected due to cell content leakage and due to DCF diffusing out of the cells. Untreated
bacteria served as negative control. (b) Overexpression of stress-related genes in the size for both E. coli and S. aureus, with
presence of CuO-1 NPs. E. coli strains carrying a promoter-lux fusion (i.e. micF-lux, sodA-lux, smaller NPs having a more efficient anti-
katG-lux, and fabA-lux) were exposed to a concentration of 0.1 mg/ml of CuO-1 NPs for bacterial activity and higher affinity to the
1 h. Gene expression was monitored by measuring luminescence. The results are presented bacterial cells. 2) CuO NPs are attached to
as relative luminescence unites (RLU) as a function of growth (OD595). Error bars represent the bacterial surface, and, to some extent,
the standard deviation of three independent experiments. sodA gene encodes superoxide
penetrate the cell membrane as deter-
dismutase (SOD) that converts .O2− to H2O2. The katG gene encodes catalase, which converts
H2O2 to H2O and O2. fabA encodes an enzyme participating in the anaerobic unsaturated
mined by electron microscopy techniques.
fatty acid synthetic pathway. micF gene products are associated with membrane functions 3) E. coli is more susceptible to CuO than
(namely, antisense RNA down-regulating outer membrane protein) and are responsive to S. aureus to the antibacterial treatment.
oxidative stress. 4) CuO NPs produce ROS. 5) Several

small 2012, © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-journal.com 7
DOI: 10.1002/smll.201200772
 full papers G. Applerot et al.

Figure 6. Schematic illustration of the antibacterial mechanism of CuO NPs and the relative cellular structure of (a) E. coli (Gram-negative) and
(b) S. aureus (Gram-positive). The major structural differences between the two cells lie in the thickness of the rigid peptidoglycan layer and in
the presence of an outer membrane in Gram-negative cells. In Gram-negative cells the peptidoglycan layer is very thin, only a few molecules thick,
whereas in Gram-positive cells this layer is very thick. Carotenoid pigments of S. aureus provide integrity to its cell membrane and increases its
protection against oxidative stress. The damage to the bacterial cell is mediated by the harmful superoxide anions formed by the cell’s attached/
internalized CuO NPs.

assays indicate that these ROS trigger oxidative stress/
damage in the bacteria.
It should kept in mind that the slight solubility of copper
oxide, which can be explained by the very low Ksp of CuO
(∼10−20), dictates a very low concentration of Cu2+ in the
solution. To examine the influence of copper ions on the anti-
bacterial effect, we performed an antibacterial test using the
Ksp- dictated concentration. After incubation of the bacteria
with the Cu2+ ions of the saturated solution for 3 h at 37 °C,
no reduction in bacteria viability was observed. This result
indicates that the Cu2+ ions are indifferent, or, at most, have
very little effect on the antibacterial activity. That is to say,
it is clear that the key factor dictating antibacterial action is
attributed to the existence of ROS production by the CuO
NPs rather than soluble copper ions. However, we cannot
rule out some degree of bacterial capability to solubilize
the attached CuO and thereby to extract harmful Cu2+ ions.
As a matter of fact, these results are in disagreement with
those reported previously by Gunawan et al.[10] The central
tenet of their paradigm is that the occurrence of the CuO
NPs bacterial cytotoxicity is derived from copper leaching in
its dissolved state in the culture medium, giving rise to the
induction of cellular ROS generation in E. coli. Apparently,
they overlooked the CuO NPs as the primary source of ROS.
It should be noted that we conducted antibacterial viability
tests in aqueous conditions rather than in the growth medium
to exclude the possibility of complexation of the CuO with
free organic ingredients, thus leaching soluble copper ions as
a consequence.
As far as we are aware, there is no explicit report con-
cerning the mechanism by which ROS are formed on metal
oxide surfaces, although it has been reported that ROS pro-
duction originated from the electron-donating nature of the
surface of these ceramics.[34] Moreover, little is known about
the nature of defect sites on a wet metal oxide surface.[34]
Nonetheless, it is conceivable that the large amount of ROS
(most likely superoxide anions) is produced directly from
surface defect sites in nanocrystalline CuO.
Hence, we propose that combined actions of the strong
adherence of the CuO particles to the bacterial cell mem-
brane, as well as ROS generation on the particles’ surface,
cause an increase in cell permeability, leading to an uncon-
trolled transport of CuO particles through the cytoplasmic
membrane and ultimately to cells death. This mode of opera-
tion predominantly increases in the case of small nanometric-
scale CuO particles owing to their higher surface-to-volume
ratio, resulting in the formation of more ROS per unit weight,
and to their higher probability to pass the cell membrane.
Regarding the difference in susceptibility of S. aureus
and E. coli towards CuO, several factors can come in mind
to explain why E. coli is more affected than S. aureus by the
antibacterial treatment: i) The difference between Gram-
negative and Gram-positive bacteria in the nature of the cell
wall; in Gram-positive bacteria, such as S. aureus, the cell wall
is thick, consisting of a large amount of mucopeptides as well
as surface components of lipoteichoic acids (LTA). On the
other hand, Gram-negative bacteria (such as E. coli) have
a relatively thin cell wall.[35] ii) The tested S. aureus strain

8 www.small-journal.com © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim small 2012,
DOI: 10.1002/smll.201200772
Understanding the Antibacterial Mechanism of CuO Nanoparticles
contains golden carotenoid pigments providing integrity to
its cell membrane and promoting a more powerful oxidant
resistance.[36] This may explain, for example, the weaker ESR
signal of this strain, as compared to E. coli, upon the antibac-
terial test. iii) E. coli bacteria are equipped inside the peri-
plasmic space with superoxide dismutase (SOD) which may
act on .O2− generated by the CuO, resulting in the formation
of H2O2 that permeates the cell more easily than .O2- and
thus oxidizes the cellular active systems.[37]
Another issue that needs to be addressed is that following
the onset of antibacterial treatment, our assays, conducted by
SuperLuminol and ESR spectroscopy, monitored a remark-
able enhancement of the oxidative stress, beyond the level
yielded by the CuO itself. A similar ROS-enhancement phe-
nomenon was also observed in our previous study on the
antibacterial activity of ZnO NPs.[23] In this regard, a burst
of ROS could be produced as a consequence of a contact
between CuO crystal vacancies and some bacterial electron
carriers, or as a result of a diminishing of the ATP production.
However, a more in-depth scenario can be considered in light
of relative new findings that have recently been advanced, i.e.
Bacterial Suicide Response[38] and Programmed Cell death
(PCD).[39] Usually, PCD is associated with eukaryotic multi-
cellular organisms. Nevertheless, PCD systems have recently
been observed in bacteria as well. It has been proposed that
ROS can lead to a range of biological responses, depending
on the relative abundance of local ROS concentration, and
some types of cellular pathways that are engaged by oxida-
tive stress induce apoptosis in bacteria. In our previous report
we associated the paradigm of triggering the bacterial suicide
response with the antibacterial activity of ZnO NPs.[23] Along
the same line, Gunawan et al also proposed the concept of
activating suicide toxin-antitoxin genetic module following
CuO NPs treatment.[10] In this regard, it may be suggested that
when the bacterial defense mechanisms are overwhelmed by
the CuO-induced ROS, the PCD genetic module is triggered,
which in turn stimulates an outbreak of oxidative stress, and
it is this burst of radicals that is lethal to the cells rather than
the initial CuO-induced ROS.[38] Figure 6 concisely illustrates
the proposed mechanism underlying the antibacterial action
of the CuO.
2.5. Toxicity in Eukaryotic Systems
Products utilizing NPs for antibacterial treatments evoke the
need for a detailed study that signifies the possible adverse
environmental and health implications of manufactured
NPs. While the traditional toxicological approach to chem-
ical testing involves animal studies as the primary means of
hazard assessment, this strategy is costly and labor intensive.
Therefore, to evaluate acute cytotoxicity, several dye-based
methods have been developed that are applicable and well-
described in the literature. Nevertheless, in our case, having
found that the CuO NPs are ROS producers also in cell-free
systems, it is imperative to adopt a more careful approach,
as the wide use of dye assays may be fraught with false posi-
tives due to the limitation of these dye-based assays to be
small 2012, © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/smll.201200772
interfered by such primary-generated ROS.[40] For example,
Karlsson et al. conclusive affirmations regarding the high tox-
icity of CuO NPs to eukaryotic cells[41] should be re-evalu-
ated in the light of our new data. In our investigation, every
cellular colorimetric assay of choice was double-checked with
extensive control experiments to preclude disturbances of the
cellular-independent ROS exerted by the NPs.
In Figure S7 we present our preliminary results of cell
viability assessed by the (4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay. This assay measures
mitochondrial reductase activity.[42] The effects of the CuO
samples on several types of mammalian cell viability are
depicted in Figure S7 and, as can be seen, at CuO concen-
trations of 10 mg/ml only a moderate cytotoxicity effect can
be discerned, up to ∼20% in the viability reduction of the
tested cells with CuO-1(it should be noted however that the
effective concentration is expected to be gradually decreased
during the time of the experiment, since, as pointed out
herein above, the CuO NPs have a tendency to aggregate
and sediment). For CuO-2 and CuO-3 only a marginal effect
could be observed on the tested cells. Our results are in good
agreement with the report published by Mahapatra et al. that
showed that the CuO NPs had the capacity to cross the cell
membrane of bacteria, however, in eukaryotic cells the cyto-
toxicity did not reach a remarkable level.[43] In this regard,
there are reports that when internalized into human cells,
NPs are mostly trapped in lysosomes, indicating an endo-
cytic–exocytic membrane cycling.[44] These evidences impli-
cate that such a lysosome-escaped strategy for NPs delivery
would be an efficient way to diminish long-term toxic poten-
tial. Furthermore, the acidic nature of the lysosome may also
suppress the production of ROS by the NPs. Recently, Kim et
al. reported, in a pioneering study, that uptake of NPs by cells
is also influenced by their cell cycle phase. Although cells in
different phases of the cell cycle were found to internalize
NPs at similar rates, after 24 h the concentration of NPs in
the cells could be ranked according to the different phases:
G2/M > S > G0/G1.[45] That is, the dose of internalized NPs
in each cell varies as the cell advances through the cell cycle.
Yet, in the case of CuO NPs, it also remains to be determined
the long-term effect of the cellular organelles and the growth
medium on both oxidative stress and copper ions leaching
from the CuO NPs and subsequent cytotoxic outcome.
3. Conclusion
This paper established the mechanistic route underlying the
antibacterial activity of CuO with respect to size dependent
properties. By using an ultrasonic irradiation system, we
could obtain small sized NPs. Among the tested CuO NPs the
small ones were found to cause the most deleterious cytotoxic
effect toward model microorganisms. Electron spectroscopy
examinations revealed that CuO NPs tended to adhere to the
cells’ surface and to disrupt the cell membrane integrity.
From the results of this study, it is clear that oxy radi-
cals, namely superoxide anions, are generated in CuO water
suspensions with the smaller sized particles generating a
larger amount of the radicals. The CuO NPs also induced
www.small-journal.com 9
 full papers
intracellular ROS generation driven from the cellular
response to the antibacterial treatment.
Because of the need to circumvent the effect of NPs
aggregation, a full quantitative inspection of the radicals
produced merits a further research effort. Nevertheless, the
various independent assays we applied in the current work
consolidated our paradigm concerning the mode of antibac-
terial action of the CuO NPs. As a final point, preliminary
results suggest that the CuO NPs have limited cytotoxicity
towards various mammalian cell-lines. These results imply
that the mammalian cells are occupied with a more potent
cascade, which includes enzymatic and non-enzymatic com-
ponents, rendering their sophisticated resistibility to oxida-
tive stress and to NPs’ toxic potency.
4. Experimental Section
Synthesis: copper (II) acetate dehydrated (0.4 gram; Aldrich
98%) was dissolved in 100 mL of 10% v/v water–N,N-dimethyl-
formamide (DMF; Aldrich 99.8%) and irradiated with a high inten-
sity ultrasonic horn (Ti-horn, 20 kHz, 100 W/cm2) under a flow of
argon at room temperature for 3 h. The products obtained were
first washed thoroughly with doubly distilled water and then with
absolute ethanol in an inert glove box (O2 < 1 ppm) and dried in
vacuum.
Characterization: The XRD measurements were carried out with
a Bruker D8 diffractometer having Cu Kα radiation (λ = 1.5418 Å).
Peak fitting and lattice parameter refinement were computed using
the Topas and Metric programs (Bruker Analytical X-Ray Systems).
The carbon and hydrogen contents were measured by C, H,
N analysis (Eager 200). The surface area was measured using a
Micromeritics (Gemini 2375) analyzer and calculated from the
linear part of the BET plot of N2 adsorption/desorption isotherms
of the CuO products. Measurements were repeated three times
and the results were found to be reproducible.
The morphologies and the micro or nanostructure of the prod-
ucts were further characterized with a JEM-1200EX transmission
electron microscope (TEM) and a JEOL-2010 HRTEM instrument
using an accelerating voltage of 80 kV and 200 kV, respectively.
Samples for TEM and HRTEM analysis were prepared by ultrasoni-
cally dispersing the products into isopropyl alcohol, then placing
a drop of this suspension onto a copper grid and then drying the
grid in vacuum.
Dynamic light scattering (DLS) was studied using a Malvern
Zetasizer nano ZS instrument.
Antibacterial Tests: The antibacterial activity of the CuO parti-
cles was tested against the Gram-negative bacterium E. coli 1313
and against the Gram-positive bacterium S. aureus 195 (each of
which was from clinical isolated strains).[46] Cultures of the bacteria
were grown overnight in a nutrient broth (NB) medium at 37 °C with
aeration and transferred the next morning into a fresh medium at
an initial optical density (OD) of 0.1 at 660 nm. When the culture
reached an optical density of OD 0.3 (660 nm) in the log phase,
the cells were harvested by centrifugation and washed twice with
a saline solution at pH 6.5. Each sample of CuO was re-suspended
in saline to reach the appropriate concentration, after which 4.5 ml
of the suspension was poured into a vial with an inner diameter of
2.5 cm. The strain cells were then pipetted (500 μl) into the vial.
10 www.small-journal.com © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
G. Applerot et al.
The initial cell number in the vial was in the range of 107 CFU/ml.
To ensure that any decrease in microbial number was likely to be
due to exposure to coated-bandage treatment, a saline solution
without CuO was included in the experiment as an additional con-
trol. Bacterial suspensions were incubated on a rotary platform at
180 shakes min−1 at 37 °C for up to 3 h. Cell viability was meas-
ured at specified times by serial dilution of the strains in the saline
solution, followed by plating on sterile solid media of NB, poured
in flat-bottomed Petri plates, for 24 h. 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. The experiments were repeated at least three times for
each strain.
Environmental scanning electron microscopy of bacterial sam-
ples: Samples of E. coli and of S. aureus cultures treated for 1 h
with CuO nanoparticles (0.1 mg/ml) were fixed with glutaralde-
hyde and paraformaldehyde for 1 h. Following this incubation the
samples were washed three times with a phosphate buffer saline
(PBS). Samples were then immersed for 1 h in titanic acid and a
glutamate solution in a 4:5 ratio concentration, respectively. Sam-
ples were afterwards washed three times with PBS and exposed to
an osmium tetraoxide solution for 1 h. To dehydrate the samples,
they were sequentially washed with water–ethanol and ethanol–
Freon solutions (concentrations ranging from 50% to 100% for
each solvent). Finally, the samples were dried in air for at least
24 h and then coated with a layer of carbon and examined using
a FEI Quanta 200 FEG enviromental scanning electron microscope.
X-ray microanalysis was performed using an in-built eXL Linx X-ray
system.
Transmission electron microscopy of bacterial samples: Sam-
ples of E. coli and of S. aureus cultures were centrifuged and
washed immediately after a treatment without and with CuO
nanoparticles (0.1 mg/ml). The samples were then fixed in 25%
glutaraldehyde/paraformaldehyde in a cacodylate buffer at
room temperature for 1 h. The samples were then washed with
a cacodylate buffer and fixed in 1% osmium tetraoxide. Sample
embedding was carried out using a standard protocol; 60 nm thick
slices were cut with a diamond knife (LBR ultratome III). The slices
were deposited on bare 200 mesh grids, and stained with 2 wt%
uranyl acetate for 5 min. Finally, the grids were dried in a desic-
cator and examined using Tecnai F20 electron transmission micro-
scopy with in-built Gatan Tridiem post-column energy filter, STEM
imaging with Fischione HAADF and Gatan BF/DF detectors, and
EDS/EELS detectors.
ESR measurements were performed as described previ-
ously[23] at the indicated concentrations. Simulation of the
recorded spectra, corresponding to the addition of 100% dime-
thyl sulfoxide, was performed using an algorithm provided by the
WINSIM program, which is available from NIEHS (National Insti-
tutes of Health) web site (http://epr.niehs.nih.gov/pest_mans/
winsim.html).
ATP and Luminol assays were performed per the manufactur-
er’s instructions (BioThema and WPI, respectively).
The rate of intracellular ROS in the bacterial cells was assayed
by monitoring their reaction with nonfluorescent 2′,7′-dichlo-
roflorofluorescin diacetate (DCFH-DA) in a spectrofluorometer.
Saline solutions of E. coli and of S. aureus strains were prepared
as described above for the antibacterial tests. NPs were then
added at the indicated concentration. A stock solution of DCFH-DA
small 2012,
DOI: 10.1002/smll.201200772
Understanding the Antibacterial Mechanism of CuO Nanoparticles
(2 mg/ml) in ethanol was kept at −20 °C in the dark. For the experi-
ments, the stock solution was diluted 100 fold in PBS, and 10%v/v
of the dye was added to the tested samples. The samples were
incubated for 15 min at 37 °C, and washed. Samples were exam-
ined under a fluorescence microscope (Leica SPE).
Cell culture and cytotoxicity assay: HeLa, human embryonic
kidney 293, and mouse embryonic fibroblast cell lines were
cultured in Petri dishes in Dulbecco’s Minimum Essential Medium
(DMEM) supplemented with 10% fetal calf serum (FCS), 1% peni-
cillin/streptomycin and 1% glutamine. The cells were seeded at
a density of 10 000 cells per well in 24-well tissue culture plates
(Greiner) and exposed to CuO samples (i.e. CuO-1, CuO-2, CuO-3)
at concentrations of 10 mg/mL. The cells were incubated at 37 °C
in a humidified atmosphere of 5% CO2 for 24 h. Following incu-
bation, the CuO particles (with attached cells) were removed for
further analysis, as described below. The cells were analyzed for
cell viability using a colorimetric MTT assay, as described by Mos-
mann.[42] The assay is based on the ability of metabolically-active
cells to reduce 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide; a yellow tetrazolium salt, to a purple formazan. In
brief, following 24 h of incubation, 100 μL of a MTT (5 mg/mL)
labeling mixture was added to each well and the microplate was
incubated for 15 to 45 min. After incubation, the MTT-containing
medium was removed from the microplate and the insoluble for-
mazan dissolved in 50 μL of DMSO. Cell viability was determined
by measuring the absorbance at an optical density (OD570) using a
microplate reader (Synergy 2, BioTek Instruments).
Supporting Information
Supporting Information is available from the Wiley Online Library
or from the author.
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Received: April 9, 2012
Revised: June 15, 2012
Published online:

12 www.small-journal.com © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim small 2012,
DOI: 10.1002/smll.201200772