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Mechanism of antibacterial activity of copper nanoparticles

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 Nanotechnology
Nanotechnology 25 (2014) 135101 (12pp) doi:10.1088/0957-4484/25/13/135101

Mechanism of antibacterial activity of

copper nanoparticles
Arijit Kumar Chatterjee, Ruchira Chakraborty and Tarakdas Basu
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani-741 235, West Bengal,
India

E-mail: tarakdb@yahoo.com

Received 14 August 2013, revised 18 December 2013

Accepted for publication 15 January 2014
Published 28 February 2014

Abstract
In a previous communication, we reported a new method of synthesis of stable metallic copper
nanoparticles (Cu-NPs), which had high potency for bacterial cell filamentation and cell
killing. The present study deals with the mechanism of filament formation and antibacterial
roles of Cu-NPs in E. coli cells. Our results demonstrate that NP-mediated dissipation of cell
membrane potential was the probable reason for the formation of cell filaments. On the other
hand, Cu-NPs were found to cause multiple toxic effects such as generation of reactive oxygen
species, lipid peroxidation, protein oxidation and DNA degradation in E. coli cells. In vitro
interaction between plasmid pUC19 DNA and Cu-NPs showed that the degradation of DNA
was highly inhibited in the presence of the divalent metal ion chelator EDTA, which indicated
a positive role of Cu2+ ions in the degradation process. Moreover, the fast destabilization,
i.e. the reduction in size, of NPs in the presence of EDTA led us to propose that the nascent
Cu ions liberated from the NP surface were responsible for higher reactivity of the Cu-NPs
than the equivalent amount of its precursor CuCl2 ; the nascent ions were generated from the
oxidation of metallic NPs when they were in the vicinity of agents, namely cells, biomolecules
or medium components, to be reduced simultaneously.

Keywords: copper nanoparticle, membrane depolarization, reactive oxygen species, lipid

peroxidation, protein oxidation, DNA degradation
S Online supplementary data available from stacks.iop.org/Nano/25/135101/mmedia
(Some figures may appear in colour only in the online journal)

1. Introduction widely utilized as cheap and effective materials for sterilizing

Gradual development of different antibiotic-resistant bacterial
strains has nowadays made it imperative to research new drugs
or materials with a wide spectrum of effective antimicrobial
activities. Recent studies on nanomaterials elicit that different
metallic and metal oxide nanoparticles (NPs) may have very
promising and potent roles as antimicrobial agents. Such NPs,
due to their large surface to volume ratio and crystalline
structure, trigger biological responses different from those
produced by the traditional ionic form of the metals. Moreover,
metallic NPs were found to have (a) 7–50 times less toxic effect
to mammalian cells than their corresponding ionic forms and
(b) prolonged effect as a source of elements in an organism [1].
Of the different metals, copper and its complexes have been
liquids, textiles and also human tissues for centuries [2].
Therefore, metallic Cu-NPs may be a promising antibacterial
agent in future. But the most challenging task in this direction
is the synthesis of stable metallic Cu-NPs, since they undergo
rapid oxidation to Cu2+ ions in air or aqueous media. We have
recently developed a method of synthesis of Cu-NPs, which
remain stable in ambient condition for about a month [3],
and their bactericidal activity on both Gram-negative and
Gram-positive bacteria is much higher than those prepared
and reported by others [4–6]. However, to use them as a
substitute for antibiotics or as an antibacterial sterilizing agent,
investigations on (a) the molecular mechanisms of bacterial
cell killing and (b) the mode of NP action in cell killing need

0957-4484/14/135101+12$33.00 1 c 2014 IOP Publishing Ltd

Printed in the UK
Nanotechnology 25 (2014) 135101
to be done. Although the mechanisms behind the biocidal
activity of metallic nanostructures are not yet fully understood,
three hypothetical mechanisms are the most accepted and
reported in the literature: (1) accumulation and dissolution
of NPs in the bacterial membrane changing its permeability,
with subsequent release of lipopolysaccharides, membrane
proteins and intracellular biomolecules and dissipation of
the proton motive force across the plasma membrane [7–9],
(2) generation of reactive oxygen species (ROSs) or/and
their corresponding ions from NPs, with subsequent oxidative
damage to cellular structures [10–13], and (3) uptake of
metallic ions derived from NPs or of NPs as whole into
cells, followed by depletion of intracellular ATP production
and disruption of DNA replication [14–17]. There are few
reported studies, particularly on the mechanism of bactericidal
activity of Cu-NPs. Raffi et al [4] and Ruparelia et al [5]
suggested that Cu ions originating from the NPs may interact
with phosphorus and sulfur-containing biomolecules such
as DNA and protein to distort their structures and thus
disrupt biochemical processes. In another study, Wu et al [18]
also reported in favor of the involvement of Cu ions for
the antibacterial activity of mesoporous copper-doped silica
xerogels, where the increase of the copper concentration from
1 to 5% in the gel was found to cause 99% cell killing
in gradually reduced time from 24 h to 1 h, respectively.
Although, these studies hypothesize few probable modes of
bacterium–Cu-NP interaction, a detailed and systematic study
(both in vivo and in vitro) is much needed. The present study
deals with the more organized and methodical investigations
on the mechanistic pathway behind the Cu-NP-mediated
occurrence of bacterial filamentation and cell killing as well
as the mechanism of functioning of Cu-NPs as a strong
antibacterial agent. Our experimental results reveal that the
filamentation is caused by the NP-mediated depolarization of
the cell membrane, whereas the cell killing is caused by the
NP-mediated ROS generation in cells, which results in cellular
lipid peroxidation, protein oxidation, and DNA degradation;
for all these effects of Cu-NPs, the prime effector is the nascent
Cu2+ ions, originating from the oxidation of the metallic
Cu atoms of the NPs.
2. Methods
This study was conducted using the widely studied Gram-
negative bacterium E. coli. All the experiments were done
with synchronized cells, prepared as described in our previous
study [3]. When such cells were allowed to grow in fresh
medium, all the cells grew similarly and divided at the same
time for a number of generations.
2.1. Determination of membrane potential of intact E. coli cells
The membrane potential was determined by both spectrofluori-
metric and flow cytometric methods, using the fluorescent dye
3,30 -diphenylthiocarbocyanine iodide, as described in detail in
our previous study [19]. The basic principle of the methods
was that the incorporation of the dye into cell membrane was
potential dependent and the binding of the non-fluorescent free
2
A K Chatterjee et al
dye to the membrane made it fluorescent, finally measuring
any alteration of cell membrane potential from the change in
fluorescence intensity of the bound dye.
To carry out the experiments, synchronized cells of E. coli
K12 [20] were freshly grown up to the log phase (i.e.,
OD600 nm = 0.2 or cell number of about 108 cells ml−1 ) in
Luria–Bertani (LB) medium [21]. The grown cells were then
treated with 3.0 and 7.5 µg ml−1 Cu-NPs (the minimum
inhibitory concentration (MIC) and the minimum bactericidal
concentration (MBC), respectively) for 1 h, centrifuged at
8000 rpm for 5 min, and the cell pellets were re-suspended in
starvation buffer (SB) [21]. As negative and positive controls,
cells alone (i.e. cells treated with nothing) and cells treated with
7.5 µg ml−1 CuCl2 (the precursor of the NPs) respectively were
also taken. The subsequent steps of cell labeling by fluorescent
dye and spectrofluorimetric (Perkin Elmer-LS50B) and flow
cytometric (Beckton Dickinson FACS Calibur) measurements
of intact cell membrane potential were made according to the
method described previously [19].
2.2. Estimation of the production of reactive oxygen species
(ROSs) in E. coli cells
The extent of ROS production in bacterial cells was es-
timated using the chemical 20 ,70 -dichlorodihydrofluorescein
diacetate (DCFH-DA) as a visual indicator to look inside the
cell, according to the spectrofluorimetric method described
previously [22]. The basic principle of the assay was that
the DCFH-DA could easily cross the cell membrane and
was hydrolyzed into DCFH by cellular esterase; subsequent
oxidation of DCFH by intracellular ROSs produced highly
fluorescent DCF, which was measured by a spectrofluorimeter.
Therefore, the extent of DCF fluorescence intensity gave a
quantitative measurement of ROS generation within cells.
Log phase grown cells, prepared as described in
section 2.1, were treated with 3.0 and 7.5 µg ml−1 Cu-NPs
for 1 h, centrifuged and suspended in saline (8.0 gm l−1
NaCl, 0.2 gm l−1 KCl). The negative and positive control
experiments, as described in section 2.1, were also done. The
probe (1 µl of 10 mM DCFH-DA) was added to each of the four
sets and incubated for 2 h at ambient temperature in the dark.
The fluorescence of each sample was then measured in the
spectrofluorimeter with excitation and emission wavelengths
of 485 and 530 nm, respectively.
2.3. Assay of lipid peroxidation in E. coli cells
To estimate the extent of lipid peroxidation of the cell mem-
brane, the spectrophotometric technique of thiobarbituric acid
(TBA) assay, described previously [23], was employed. The
basic principle of the assay was that the lipid peroxida-
tion, or in other words the oxidation of polyunsaturated fatty
acids, produced malonaldehyde (MDA), which reacted with
TBA, producing a chromophore with absorption maximum
at 532 nm. Therefore, the level of chromophoric absorption
intensity at 532 nm was a quantitative measurement of lipid
peroxidation.
Log phase grown cells in saline (prepared as described
in the preceding experiment) were divided into four parts:
Nanotechnology 25 (2014) 135101
to two parts 3.0 and 7.5 µg ml−1 of Cu-NPs were added
separately, to one part 7.5 µg ml−1 of CuCl2 was added,
whereas the fourth part was treated as control and nothing was
added. All four cultures were incubated for 1 h at 37 ◦ C with
shaking. 1.0 ml of each culture was centrifuged; the cell pellet
was suspended in 100 µl of SDBME buffer [24]. 100 µl of
cell extract was mixed with 200 µl of TBA–trichloroacetic
acid (TCA)–hydrochloric acid (HCl) reagent (15% TCA and
0.375% TBA dissolved in 0.25 N HCl) and heated for 15 min in
a boiling water bath. After cooling, the flocculent precipitates
were removed by centrifugation at 12 000 rpm for 10 min. The
absorbance of the supernatant was measured at 532 nm in a
UV–vis spectrophotometer (Shimadzu, UV-1800), to estimate
the level of lipid peroxidation according to
LPO activity = {(absorbance × total volume)}
× {(time × sample volume × molar extinction coeff.
× protein concentration)}−1 nM/mg/unit. (1)
The protein concentration in the cell lysate was assayed by
the Bradford method [25] and the value of molar extinction
coefficient of the chromophore was known to be 1.56 ×
105 mol−1 cm−1 .
2.4. Estimation of protein oxidation in E. coli cells
The level of protein oxidation in the bacterial cells was mea-
sured according to the method described previously [26]. The
basic principle of measurement was that the protein carbonyls,
products of protein oxidation, were known to react with dini-
trophenylhydrazine to form a stable dinitrophenylhydrazone
product, of which the 2,4-dinitrophenol (DNP) absorbed UV
light at 370 nm. Thus, the extent of protein oxidation was
proportional to the intensity of DNP at 370 nm.
Cell lysates were prepared as in the case of the lipid
peroxidation study. The lysates were centrifuged at 12 000 rpm
for 10 min at 4 ◦ C. To the supernatants, 20% cold TCA
was added and kept at −20 ◦ C for 1 h for precipitation.
The precipitates were made sticky by centrifuging further
at 12 000 rpm for 15 min at 4 ◦ C. To each sticky pellet
0.4 ml of 0.2% 2,4-dinitrophenylhydrazine (DNPH) in 2 N
HCl was added and incubated in the dark, with vortexing at
intervals of 10 min up to 1 h. The protein hydrazone derivatives
were extracted through precipitation by 10% cold TCA. The
precipitate was treated with ethanol–ethyl acetate (1:1 v/v)
and the hydrazones were re-extracted by 10% cold TCA.
The resulting precipitate was dissolved by vortexing in 6 M
guanidine hydrochloride (GuHCl), kept at 37 ◦ C for 15 min
and again centrifuged at 5000 rpm for 2 min to remove the
undissolved fragments. The OD value of the supernatant was
measured at 370 nm.
2.5. Study on DNA degradation in E. coli cells
In order to observe the fate of DNA (degraded or not) in
E. coli cells, genomic DNA was first isolated as described
by Wilson [27] and the isolated DNA was then subjected
to electrophoresis in agarose gel according to the method
described by Sambrook and Rusell [28]. Here, log phase
3
A K Chatterjee et al
grown cells of E. coli K12 were divided into two parts: to
one part 7.5 µg ml−1 of Cu-NPs was added and to the other
part 7.5 µg ml−1 of CuCl2 was added. Both the parts were
allowed to incubate at 37 ◦ C. From the Cu-NP-treated cells
aliquots of 1.5 ml were withdrawn after 30, 60 and 120 min,
and from the CuCl2 -treated cells an aliquot of the same volume
was withdrawn after 120 min of incubation. From each aliquot,
genomic DNA was isolated and subsequently electrophoresed
for 1 h in 1% (w/v) agarose gel, using Tris–acetate–EDTA
buffer.
2.6. UV-spectrophotometric study on DNA–Cu-NP interaction
The surface plasmon resonance property of Cu-NPs was used
for this in vitro study. Here, Cu-NP solution was titrated
with DNA and the spectrometric study was carried out at
298 K. Cu-NP solution was taken in a cuvette along with a
reference cuvette containing gelatin and CuCl2 (the precursor
components of the NPs) in the same amounts as present
in the test cuvette. DNA solution from the prepared stock
was then added stepwise (0.5 µg in each step) in both the
sample (containing Cu-NP) and reference cuvettes and mixed
thoroughly by turning the cuvettes upside down several times,
and after each step of DNA addition the Cu-NP spectrum was
recorded. DNA has negligibly small and constant absorbance
in the wavelength region of 500–700 nm, which was due
to scattering, but as it was added to both the sample and
reference cuvettes the contribution due to the scattering of
DNA in the Cu-NP spectra was automatically eliminated.
The binding interaction was analyzed from the Hill plot
of the absorbance data [29]. For any biomolecule–ligand
interaction, the Hill equation was represented as log[θ/
(1 − θ )] = γ logK b + γ logCf , where K b is the binding con-
stant, γ is the Hill coefficient, Cf is the concentration of free
ligand and θ (fraction of macromolecule bound to ligand) =
(A0 − AI )/(A0 − AR ), where A0 , AI and AR are the
(absorbance)575 nm of Cu-NPs in the absence of DNA, at any
DNA concentration and at the DNA concentration for which
the maximum binding took place, respectively. The plot of
log [θ/(1 − θ )] versus log Cf represented the Hill plot. The
values of K b and γ were determined from the intercept on the
x-axis (where log Cf = −logK b ) and the slope in the region
of the intercept, respectively. The importance of γ was that it
characterized the nature of cooperativity, that is, γ < 1, γ = 1
and γ > 1 signified a negative cooperative, non-cooperative
and positive cooperative nature of the binding, respectively.
2.7. Determination of the NP size
The average hydrodynamic size of the NPs was determined
using a particle size analyzer (Malvern, Nano ZS). With the
measurement of size, the instrument also determined the count
rate of the NPs, i.e. the number of particles studied (to measure
the average size) per second of investigation.
Nanotechnology 25 (2014) 135101
Table 1. Membrane potential of Cu-NP-treated E. coli cells measured spectrofluorimetrically.
Cell alone Cell + 7.5 µg ml−1 CuCl2
−184.5 (±12) mV −159.8 (±13.5) mV −104.3 (±10.8) mV
3. Results and discussion
Our earlier communication [3] indicated that the gelatin-
stabilized Cu-NPs (synthesized by us) had high potential
for cell filament formation and subsequent cell killing. The
filament size was found to increase with increasing concen-
tration of Cu-NPs up to 3.0 µg ml−1 (the MIC), above which
there was a gradual decrease in size. Above the MIC, the
filament size could not increase due to NP-mediated cell
death. Filamentation is an anomalous growth of bacteria,
when cells continue to elongate with multiple chromosomal
copies and do not undergo septum formation, causing no cell
division [30]. It is a primary defense mechanism for cells under
SOS such as stress response. The SOS response is known to
play a role in exposure of E. coli cells to DNA-damaging
or DNA replication-interfering agents, by inducing about 30
different proteins for damage tolerance [31, 32]. One of the
SOS gene products, SulA protein, encoded by the gene sfiA, is
responsible for filament formation. SulA inhibited cell division
by blocking the formation of the cell-septum-forming ring,
made of FtsZ proteins, and thus led to filamentation. SulA
interacted directly with FtsZ monomer, thereby preventing the
polymerization and GTPase activity of FtsZ. By sequestering
FtsZ, the cell could directly link DNA damage to inhibit cell
division [33–35].
In the present study, it was investigated whether the
SOS response was induced in Cu-NP-treated E. coli cells, by
observing if any SOS repair of the UV-damaged bacteriophage
8 X174 [21, 36] occurred within the NP-treated cells. The
result showed that no repair of the UV-inactivated 8 X174 took
place in cells treated with 7.5 µg ml−1 of Cu-NPs (MBC) for
3 h, indicating that at least the SOS DNA repair genes were not
induced in the E. coli cell by Cu-NP treatment (experimental
details and results are shown in supplementary section 1
available at stacks.iop.org/Nano/25/135101/mmedia).
Moreover, when the cells of E. coli SulA mutant strain
SG13109 [37] were treated with 2.5 µg ml−1 (sub-MIC)
and 3 µg ml−1 (MIC) of Cu-NPs in LB medium, for each
concentration of the NPs they were found to form filaments
within 3 h of treatment, indicating that the SulA protein had
no role in the cell filamentation on Cu-NP treatment (exper-
imental details and results are shown in supplementary sec-
tion 2 available at stacks.iop.org/Nano/25/135101/mmedia).
Therefore, the Cu-NP-induced cell filamentation was clearly
not a SOS-dependent process.
3.1. Study to determine the membrane potential of
Cu-NP-treated E. coli cells
Not much was known about the SOS-independent mode of
filamentation, until a few recent studies showed that the cell
membrane potential had a determining role in proper cellular
A K Chatterjee et al
Cell + 3 µg ml−1 Cu-NPs Cell + 7.5 µg ml−1 Cu-NPs
−76.5 (±9.5) mV
division [38, 39]. It was shown there that the membrane
potential modulated distribution of several conserved cell
division proteins such as MinD, FtsA and the bacterial cy-
toskeletal protein MreB. Administration of the well known
protonophore carbonyl cyanine m-chlorophenyl hydrazone,
which blocked proton motive force across the E. coli cell
membrane and thus dissipated the membrane potential, made
the cells filamentous [38]. This was further supported by
the observation that indole, a bacterial signaling molecule
and a protonophore, inhibited cell division in E. coli by
disrupting the trans-membrane potential [39]. Depolarization
of the cytoplasmic membrane was found to inactivate the ‘Min’
site selection system, preventing localization of the FtsZ ring
and thereby inhibiting cell division [39]. We also investi-
gated if any depolarization of the E. coli plasma membrane
occurred in Cu-NP-exposed intact cells, by both spectrofluori-
metric and flow cytometric methods separately, as described in
section 2.1.
Table 1 shows that the bacterial (E. coli K12) cell
membrane was depolarized by the Cu-NP treatment, and the
extent of depolarization was dependent on the concentration
of NPs used. The normal E. coli membrane potential was
measured to be about (−)185 mV, which decreased to almost
−105 and −75 mV when treated with 3.0 and 7.5 µg ml−1 of
NPs, respectively; whereas a small drop of potential by about
25 mV took place when the cells were treated with 7.5 µg ml−1
of CuCl2 .
The flow cytometric results have been depicted in figure 1.
The main three quadrants of the cell-representative dots were
LL—lower left, LR—lower right and UR—upper right. The
dots in the LL quadrant signified cells having negligible
fluorescence (1–10 arbitrary units), i.e. unlabeled cells. On
the other hand, dots in the quadrants LR and UR signified
cells with considerable fluorescence (10–104 arbitrary units)
and their extents of fluorescence and size were understandable
from the X - and Y -axis values, respectively. Table 2 represents
the percentage distributions of cells in different quadrants,
when the cells were kept under different conditions. Nearly
100% of the control unlabeled cells were represented by
the dots in the quadrant LL (figure 1(A) and table 2, row
1). When the cells were labeled with the fluorescent dye
3,30 -diphenylthiocarbocyanine iodide for 2 h, about 76.35%
of cells acquired considerable fluorescence label and the
remaining 23.65% cells did not (table 2, row 2); of the labeled
cells, the major population (76%) was represented by the
dots in quadrant LR and a negligible minority (0.3%) of
large-sized cells were represented by the dots in quadrant UR
(figure 1(B)). Table 2, rows 4 and 5, shows that when the
cells were treated with 3.0 (MIC) and 7.5 µg ml−1 Cu-NPs
(MBC), only about 36.56 and 21.89% of cells were labeled.
The decrease in the amount of labeling in the NP-treated cells
(by about 40 and 54.5% for the NP concentrations 3.0 and
4
Nanotechnology 25 (2014) 135101
Table 2. Percentage distribution of cells in different quadrants of flow cytometric profiles.
Serial no Cells under different conditions Lower left (LL) (%)
1 Control (unlabeled) 99.60
2 Control (labeled) 23.64
3 7.5 µg ml−1 CuCl2 treated (labeled) 40.06
4 3 µg ml−1 Cu-NP-treated (labeled) 63.44
5 7.5 µg ml−1 Cu-NP-treated (labeled) 78.11
Figure 1. Flow cytometric analysis of membrane potential of intact
E. coli cells: (A) unlabeled control cells; (B) labeled control cells;
(C) cells treated with 7.5 µg ml−1 CuCl2 ; (D) cells treated with
3.0 µg ml−1 Cu-NPs; (E) cells treated with 7.5 µg ml−1 Cu-NPs.
7.5 µg ml−1 , respectively) with respect to the amount of
labeling (about 76%) in the NP-untreated control cells implied
that Cu-NPs caused dissipation of the E. coli plasma membrane
potential because the dye binding to the cell membrane and
the consequent generation of fluorescence was membrane
potential dependent. The cells with depolarized membranes
were populated in quadrant LL (figures 1(D) and (E)), finally
increasing the cell population there by about 40 and 54.5%
compared to that in the case of control labeled cells (about
23.5%). Moreover, it is also found from figure 1(C) and table 2,
row 3, that when the cells were treated with 7.5 µg ml−1 CuCl2
(the principal ingredient of the NPs) there was depolarization
5
A K Chatterjee et al
Lower right (LR) (%) Upper right (UR) (%)
0.22 0
76.04 0.31
59.82 0.12
36.45 0.11
21.80 0.09
Figure 2. ROS generation in the Cu-NP-treated E. coli cells. The
shown values deviate within 5% from the corresponding average
values. The statistical significance of the increase in ROS production
in Cu-NP-exposed cells compared to that in untreated control cells
was measured according to the Student t-test method, where
p-values less than 0.5–0.001 were considered significant.
∗∗∗ = p < 0.001 and ∗∗ = p > 0.01.
of the cell membrane; however, the extent (about 16%) was
significantly less than that in the Cu-NP-treated cells.
Thus, both the spectrofluorimetric and flow cytometric
results clearly signified that the exposure of E. coli cells to Cu-
NPs caused cell membrane depolarization in a concentration-
dependent manner, and this dissipation of membrane potential
was the most probable basis of cell filamentation.
3.2. Study to estimate the ROS production, if any, in
Cu-NP-treated E. coli cells
The hallmark of metallic NP-mediated bacterial killing was the
ROS production and the consequent cellular damage. Again,
metals such as copper act as catalysts in Fenton-like reactions
to generate ROSs such as O2 • , OH• etc [40]. Therefore, an
experiment was performed to study the ROS production, if
any, and its extent in the Cu-NP-exposed E. coli cells. The
experimental result clearly demonstrated the generation of
ROSs in Cu-NP-treated cells, and the extent of ROS production
was dependent on Cu-NP concentration (figure 2). Compared
to the level in the untreated control cell, the ROS level in
3.0 and 7.5 µg ml−1 Cu-NP-treated cells was almost 1.8
and 2.5 times higher, respectively. Although the presence
of a small amount of ROS (about 20% over the amount in
control cell) was found in the CuCl2 (7.5 µg ml−1 )-treated
Nanotechnology 25 (2014) 135101
Figure 3. Lipid peroxidation level in the Cu-NP-treated E. coli cells.
The shown values deviate within 4% from the corresponding
average values.
cells by the copper ions, the amount was highly insignificant
with respect to that in the NP-treated cells. This significant
production of ROSs might cause free-radical-mediated cellular
damage by Cu-NPs, which motivated us to study the extent
of cellular lipid peroxidation, protein oxidation and DNA
damage, because these were generally the most obvious effects
of ROS production.
3.3. Study to estimate lipid peroxidation, if any, in
Cu-NP-treated cells
The lipid peroxidation phenomenon refers to oxidative degra-
dation of polyunsaturated lipids, causing three in vivo dam-
aging consequences to the cellular plasma membrane, namely
decrease in membrane fluidity, increase in membrane leakiness
and damage in membrane-bound proteins, ultimately leading
to toxicity and cell death [41]. Of the different reactive oxy-
gen species, singlet oxygen and hydroxyl radicals remove a
hydrogen atom from a methylene group on a polyunsaturated
fatty acid, leaving an unpaired electron on the carbon atom.
The carbon atom then rearranges itself to a conjugated diene,
which reacts with oxygen to form a peroxy radical [23].
Our experimental result on lipid peroxidation, depicted in
figure 3, implied that on exposure of the cells to 3.0 and
7.5 µg ml−1 Cu-NPs for 1 h their lipid peroxidation levels
were respectively about 35 and 50 times that in the untreated
control cells. On the other hand, bulk copper salt (7.5 µg ml−1
of CuCl2 ) caused a small increase in lipid peroxidation by only
eightfold. This result suggested that high lipid peroxidation,
i.e. high level of oxidation of unsaturated fatty acids, in
E. coli membranes might be one of the initiating events
for Cu-NP-mediated bacterial killing. Moreover, very little
increase of lipid peroxidation (only eightfold) by ionic copper
of bulk CuCl2 signified that the NPs, not the copper ions, had
a specific effect on bacterial killing. Unsaturated fatty acids
are known to be essential and irreplaceable components in
biological membranes [41]. Changes in fatty acid composition
altered the physical properties of the lipid bilayer, affecting
membrane fluidity and thus indirectly regulating the activity
of integral membrane proteins.
6
A K Chatterjee et al
Figure 4. Protein oxidation level in the Cu-NP-treated E. coli cells.
3.4. Study to estimate protein oxidation, if any, in
Cu-NP-treated cells
At the cellular level, exposure of proteins to ROSs was
known to modify amino acid side chains with consequent
alteration of the protein structure [42]. These modifications led
to functional changes that disturbed cellular metabolism. As
Cu-NP treatment generated considerable ROSs in E. coli cells,
it was necessary to determine whether the protein oxidation
phenomenon occurred in the Cu-NP-treated E. coli cells. Our
experimental result demonstrated that treatment of E. coli cells
with 3.0 and 7.5 µg ml−1 of Cu-NPs for 1 h made the cellular
protein carbonyl level or oxidized protein content about 25
and 50 times respectively that in the untreated control cells
(figure 4). This suggested that, besides lipid peroxidation,
protein oxidation was also a probable reason for the cell killing
by Cu-NPs. Figure 4 also shows that the exposure of the cells
to 7.5 µg ml−1 of CuCl2 made the protein oxidation level
eight times that of the control cells, implying further that the
bactericidal effect was specific to NPs instead of only copper
ions. It should be mentioned here that the repair of damage to
proteins was limited to the reduction of oxidized derivatives
of the sulfur-containing amino acid residues; no other kind
of repair of protein oxidation was reported. Oxidized proteins
were better substrates for proteolytic digestion, and E. coli has
specific proteases that selectively degrade oxidized proteins,
finally killing cells [43].
3.5. Study to investigate whether DNA damage occurred in
Cu-NP-treated cells
Free radicals were known to attack both the base and the
sugar moieties of DNA, producing lesions such as single and
double-strand breaks, adducts of base and sugar groups, and
cross-links to other molecules blocking DNA replication [44].
Therefore, an experiment was performed to investigate the fate
of DNA in the Cu-NP-treated bacterial cells. Figure 5 clearly
demonstrates the degradation of genomic DNA isolated from
the Cu-NP-treated E. coli cells. The intact DNA band in lane 1
signifies that no degradation of genomic DNA occurred in cells
Nanotechnology 25 (2014) 135101
Figure 5. Agarose gel electrophoretic pattern of the chromosomal
DNA isolated from the Cu-NP-treated E. coli cells.
exposed to CuCl2 for 2 h. On the other hand, the smear-like
band pattern in lane 2 implies chromosomal degradation in
E. coli cells when treated with 7.5 µg ml−1 Cu-NPs for
only 30 min. Moreover, incubation of the cells with NPs for
1 h caused such an extensive cleavage that many short DNA
fragments went out of the gel during electrophoresis and so the
total intensity of the smear in lane 3 was less than those of the
bands in lanes 1 and 2. In the case of the cells incubated with
NPs for 2 h, DNA was fragmented into such small pieces that
most of the pieces left the gel in the running time, as observed
from the DNA profile in lane 4. It can therefore be concluded
that in vivo chromosomal DNA degradation in E. coli started
within 30 min of treatment with Cu-NPs (7.5 µg ml−1 ), and
more degradation took place with increase of the NP exposure
time.
3.6. Spectrophotometric study on DNA–Cu-NP interaction
In order to investigate whether the Cu-NP-induced DNA
degradation in E. coli was due to direct interaction of the
NPs with the chromosomal DNA or due to some secondary
interaction, a spectrophotometric study was performed. Since
the E. coli DNA was circular and supercoiled, this in vitro
study was done with circular, supercoiled plasmid pUC19
DNA [28] of size 2.68 kbp. Here 0.5 ml of fivefold diluted
Cu-NP solution was placed in a 1.0 ml quartz cuvette and was
titrated with purified pUC19 DNA.
From the absorbance spectra of Cu-NP solution titrated
with different concentrations of DNA at 298 K, it was seen that
the plasmon peak of Cu-NPs at 575 nm decreased gradually
with increasing addition of DNA (figure 6(A)), which indicated
some interaction between DNA and Cu-NPs. When the binding
isotherm of DNA–NP interaction was plotted from the data of
the spectrophotometric results, the isotherm looked sigmoidal
in shape, signifying the cooperative mode of interaction
(figure 6(B)). From the Hill plot (figure 6(C)), as discussed
in section 2.6, values of the binding constant (K b ) and the Hill
coefficient (γ ) were found to be 1.22 × 105 M−1 and 2.704,
respectively, at 298 K. As the value of γ was greater than
7
A K Chatterjee et al
1.0, it confirmed that the binding between DNA and Cu-NPs
was positively cooperative in nature. The spectrophotometric
study of Cu-NP–DNA interaction was also performed at 310 K.
From the Hill plot analysis, the values of K b and γ were
found to be 1.36 × 105 M−1 and 2.393, respectively, at 310 K.
Since (K b )310 K > (K b )298 K , it signified strengthening of the
NP–DNA binding interaction with the rise of temperature.
These values of K b at two different temperatures, 298 K
(say T1 ) and 310 K (say T2 ), were put in the places of K 1
and K 2 , respectively, in the well known van’t Hoff equation:
   
K2 −1H 1 1
ln = − (2)
K1 R T2 T1
to find out the value of the thermodynamic parameter (1H ),
i.e. the value of the enthalpy change in the Cu-NP–DNA
interaction. The value was calculated to be −6.5 kcal mol−1 ;
the negative value of the enthalpy change clearly implied that
the NP–DNA interaction was exothermic in nature. Moreover,
the values of 1G (change in free energy) at temperatures 298
and 310 K as calculated from the thermodynamic equation
1G = (−)RT ln K b (3)
were found to be −5.19 and −6.35 kcal mol−1 , respectively,
and the negative value of 1G (at each of the two said
temperatures) implied that the DNA–Cu-NP binding reaction
was a spontaneous phenomenon.
3.7. Study to investigate the mechanism of DNA degradation
by Cu-NPs
Here, an in vitro experiment was performed to investigate
whether the degradation of DNA was caused either by the
Cu2+ ions leached from the oxidized NPs or by the Cu-NP-
induced generation of ROSs (singlet oxygen and hydroxyl
radicals) or by the Cu-NPs themselves, by adding different
radical scavengers and a Cu-ion chelator to the DNA–Cu-
NP reaction mixture. This study was done with pUC19
DNA. As scavengers of singlet oxygen and hydroxyl radicals
tris(hydroxyl methyl) amino methane and DMSO were used,
respectively, and as a chelator of copper ions EDTA was used.
The DNA band patterns and their intensity distributions under
different reaction conditions have been presented in figure 7
and table 3, respectively. It is clear from the DNA band
profiles in lanes 1–4 of figure 7 that Cu-NPs caused severe
degradation of plasmid DNA when incubated at 37 ◦ C for
different times. Measurement of the intensity of the bands in
lane 1 demonstrated that about 81.5% of the purified control
DNA was in the native, circular, supercoiled, compact form
(also called replicative form I or RFI) and moved faster than
the remaining 18.5% of the DNA, which was nicked (cleavage
of a phosphodiester bond in a single strand of DNA) and so
non-supercoiled or large relaxed circular—also called RFII
DNA. Lane 2 of figure 6 and row 2 of table 3 show that
within 5 min of incubation with Cu-NPs almost 90% of the
RFI DNA was converted to singly nicked RFII DNA (about
66%) and doubly nicked (single nick in both the strands)
linear RFIII DNA (about 25%). It is known that, although
Nanotechnology 25 (2014) 135101 A K Chatterjee et al

Figure 6. Spectrophotometric data of Cu-NP–DNA interaction. (A) Change in the absorption spectrum of Cu-NPs (1 mM) with the stepwise
addition of pUC19 plasmid DNA at 298 K; (B) the binding isotherm of the NP–DNA interaction at 298 K; (C) Hill plot of equilibrium
binding between Cu-NPs and DNA at 298 K.

Table 3. Percentage of intensity distribution of pUC19 DNA in different conformations, as measured by the 1D gel analysis software in
Typhoon 9210 (GE Healthcare).

Lane no Experimental conditions RFI (supercoiled) (%) RFII (nicked circular) (%) RFIII (linear) (%)
1 DNA alone 81.5 18.5 —
2 DNA + Cu-NPs (5 min) 9.44 65.87 24.69
4 DNA + Cu-NPs (15 min) 9.7 58.09 32.21
3 DNA + Cu-NPs (30 min) — 62.45 37.55
5 DNA + Cu-NPs + 0.2 M Tris (O2 •− scavenger) 16.79 61.03 22.18
6 DNA + Cu-NPs + 0.2 M DMSO (OH• scavenger) 7.3 70.4 22.3
7 DNA + Cu-NPs + 20 mM EDTA (ion chelator) 59.35 40.65 —
10 DNA + CuCl2 (30 min) 68.74 31.26 —

the molecular weights of all three forms of DNA–RFI, RFII of relaxed circular RFII [45]. After 15 min of incubation,
and RFIII, are the same, their gel electrophoretic mobilities more DNA degradation took place, converting gradually singly
are different depending on their compactness: supercoiled RFI nicked circular RFII DNA to doubly nicked linear RFIII
moved faster than linear RFIII, which again moved ahead DNA, as observed from lane 4 of figure 6 and row 3 of

8
Nanotechnology 25 (2014) 135101
table 3. Lane 3 shows that at 30 min of interaction between
pUC19 and Cu-NP the RFI band was completely abolished
and both the RFII and RFIII bands became less intense;
this result together with the DNA profiles in lanes 2 and
4 implied that with increasing the time of interaction RFI
converted to RFII, which then degraded to RFIII, and finally
further degradation of RFII and RFIII occurred, to such a
high extent that most of the smaller DNA fragments went
out of the gel during electrophoresis and some comparatively
larger fragments produced faint smears, observed in the lanes.
Lanes 5 and 6 represent the DNA profiles when the DNA–NP
interaction was carried out in the presence of tris(hydroxyl
methyl) amino methane—a scavenger of singlet oxygen—and
DMSO—a scavenger of hydroxyl radical, respectively; with
respect to the percentage of RFI DNA in lane 1, those in lanes 5
and 6 were only 9 and 20% respectively, as observed from
column 3 of table 3. Therefore, the band patterns of lanes 5
and 6 clearly signified that the scavengers had no significant
inhibitory role in the Cu-NP-induced DNA degradation, i.e. the
Cu-NPs did not degrade DNA through the production of ROSs
such as singlet oxygen or hydroxyl radicals. Instead of the
radical scavengers, when the copper-ion chelator EDTA was
added to the DNA–NP reaction mixture (lane 7), degradation
of RFI DNA was found to be significantly inhibited by almost
73% (comparing the values of column 3 in rows 1 and 7 of
table 3), signifying that the DNA degradation effect of Cu-NPs
was mostly Cu2+ ion mediated. Lane 9 shows that when
fivefold diluted Cu-NP was used little DNA degradation took
place; therefore, comparison of the band intensities in lanes
3 and 9 indicates that Cu-NPs degraded DNA molecules in
a concentration-dependent manner. If the ion-mediated effect
occurred, the CuCl2 (the main precursor of Cu-NPs) should
also have the DNA degradation property. However, rows 1, 3
and 10 of table 3 indicate that 1.0 mM CuCl2 in 30 min caused
only about 16% cleavage of RFI, whereas 1.0 mM Cu-NPs
in 30 min caused complete abolition of RFI. Therefore, the
prime outcome of this experimental result was that, although
the copper ions appeared to be the main effector for DNA
degradation, the nascent ions released from the NPs with their
oxidation were perhaps more effective in DNA cleavage than
the copper ions present in already ionized CuCl2 .
3.8. To study the effect of EDTA on the size and count rate of
Cu-NPs
The result of the previous experiment 3.7 suggested that the
nascent copper ions released from the NPs were responsible
for the heavy DNA degrading effect of the NPs. In the presence
of DNA, the oxidation of metallic copper atoms of the NPs
to Cu2+ was expected, with the simultaneous reduction of
DNA by receiving the electrons liberated from the copper
atoms. These Cu ions perhaps came out of the NP assembly
in nascent form and acted upon the phosphodiester bonds
of DNA, and possibly, with the gradual engagement of the
Cu ions due to binding with DNA, more ions were leached out
of the NPs. To check this possibility, the physical state, i.e. the
size and count rate, of the Cu-NPs in the presence of the
divalent metal ion chelator EDTA was investigated, keeping
9
A K Chatterjee et al
Figure 7. In vitro degradation of pUC19 DNA by Cu-NPs in the
absence and presence of free radical scavengers and a copper-ion
chelator.
in mind that strong chelation of the Cu2+ by EDTA might
cause more and more leaching of the ions from the NPs and
thus ultimately reduce the size of the Cu-NPs with time. For
this, EDTA at a final concentration of 20 mM was added to
fivefold diluted Cu-NPs and the size of the NPs was measured,
as described in section 2.7, at different intervals of time. The
results represented by figure 8 show that the size and the count
rate of Cu-NPs decreased gradually with the time of incubation
with EDTA. The decrease of both the parameters reached
saturation at nearly 30 min of incubation. The size of the
NPs was found to decrease from 62.5 to 23.4 nm, whereas the
count rate decreased from 282.5 kcps (kilocounts per second)
to 130.5 kcps. These results suggest that the Cu-NPs became
highly destabilized in the presence of EDTA due to gradual
leaching of Cu ions from the NP surface, with simultaneous
chelation of the ions by the EDTA. As a consequence, not
only did the size of the core particle decrease fast, but almost
half of the initial particles were abolished (as observed from
the decrease in particle count rate), when kept in the presence
of EDTA for about 30 min only. On the other hand, the NPs
alone in suspension were highly stable and their size (about
60 nm) remained unchanged. Therefore, binding of the Cu ions
to the chelator EDTA caused more leaching of ions from the
NPs. Similarly, when the NPs and DNA were in the same
place, Cu2+ binding over DNA resulted in more release of
ions from NPs and consequently more degradation of DNA.
This was further supplemented from our experimental results
on the gradual dissolution of the NPs in the defined growth
medium, named MOPS (3-(N -morpholino)propanesulfonic
acid) medium [46]. Since the MOPS medium was composed
of MOPS buffer supplemented with different amino acids,
nucleotides, vitamins and a carbon source, incubation of the
NPs in this medium also caused a decrease in size of the
particles with time; however, the rate and the extent of decrease
were small (figure 8(B)). It is also found from figure 8(B)
that, as soon as the NPs were added to the MOPS medium,
their size increased from about 60 to 185 nm, indicating
formation of some binding complex of the NPs with the
Nanotechnology 25 (2014) 135101
Figure 8. (A) Decrease in size and count rate of Cu-NPs with time of incubation in presence of EDTA; (B) decrease in size of Cu-NPs with
time of incubation in MOPS medium. The red point signifies the size of Cu-NPs in water.
organic macromolecules present in the medium; the size of
the large complex particles reduced gradually from 185 to
125 nm when incubated in MOPS medium for 12 h. The high
chelating property of EDTA for divalent cations Cu2+ justified
our results of lower rate and lower extent of size reduction of
NPs in the presence of different organic molecules than in the
presence of EDTA.
4. Conclusion
In this study, investigations were carried out to unravel the
mechanism of bacterial filament formation and cell killing by
the action of Cu-NPs and also the mechanism of the action
of Cu-NPs. Bacterial cell filamentation was known to occur
by the action of an external agent, when the agent either
induced an SOS-like stress response in cells or dissipated the
cell membrane potential. Our experimental results revealed
that the Cu-NP-mediated bacterial cell filamentation was not
due to the induction of the cellular SOS response, but due
to the depolarization of the cell membrane. Our results also
demonstrated that the treatment of E. coli cells by Cu-NPs
at the MBC caused an increase in the cellular ROS level by
2.5-fold, compared to the level in the NP-untreated control
cells, and this NP-mediated overproduction of ROSs resulted
in considerable lipid peroxidation, protein oxidation and DNA
degradation, finally killing the cells. ROS inhibitors such as
DMSO and tris(hydroxyl methyl) amino methane were found
to have little effect on the Cu-NP-mediated in vitro DNA
degradation, justifying the view that the degradation was a
ROS-independent phenomenon. However, the degradation was
highly inhibited in the presence of divalent metal ion chelator
EDTA, indicating a positive role of Cu2+ ions behind the
process. The findings on the faster decrease in size as well as
the count rate of the NPs in the presence of EDTA signified
that the nascent Cu ions liberated from the NPs were more
reactive than the already present Cu ions in the precursor CuCl2
solution. The release of nascent ions was facilitated by the
oxidation of metallic NPs with the simultaneous reduction of
the agents such as cells, biomolecules or medium components
in the vicinity of the NPs.
On this mechanistic study, questions may be raised of
whether the use of a single type of bacterium such as E. coli
10
A K Chatterjee et al
and a single type of Cu-NP synthesized by us were sufficient
enough to draw any generalized conclusion on the pathways
of cell killing and NP action. Here, it should be empha-
sized that the results of our previous study [3] showed that
similar antibacterial effects of the Cu-NPs also occurred in
Gram-positive bacteria B. subtilis and S. aureus, besides the
Gram-negative E. coli. Since E. coli is known to be a widely
studied organism, and most of the fundamental molecular
mechanisms of DNA replication, gene expression, mutation
etc were established using E. coli as a biological tool, the use
of E. coli as the single organism in our study, to understand
the fundamental basis of bacterial cell killing by Cu-NPs,
was justifiable enough. In this study, regarding the use of
our synthesized Cu-NPs only, it should be mentioned that,
although there are different methods, namely laser irradia-
tion [47], γ -irradiation [48], thermal decomposition [49, 50],
thiol-induced reduction [51], reduction in micro-emulsions
and reverse micelles [7, 52, 53], vapor deposition [54], the
sono-electrochemical process [55], flame spray [56], chemical
reduction [12, 57, 58] etc, of synthesizing Cu-NPs of different
sizes and stabilized by different agents, our goal of innovating
a new method of synthesis of Cu-NPs was to prepare very
stable NPs, stabilized by the highly biocompatible gelatin, so
that they can be used in future as a substitute for antibacterial
agents. In our previous study [3], it was reported that our
NPs were encapsulated by the stabilizer gelatin molecules,
which was expected to be advantageous for interaction of
the particles with cell membranes and their subsequent entry
into the cell cytosol. Therefore, in the present study emphasis
was given exclusively to our synthesized NPs, to investigate
the mechanism of their antibacterial role. However, studies
are in progress in our laboratory to understand the following
important points: (1) the mode of physical interaction between
the NPs and the cell membrane, (2) the mode of interaction
between the NPs and cell medium organics, (3) NP-mediated
alteration of the different physical and chemical characteristics
of the cell membrane, (4) localization of the NPs in cells,
(5) whole protein expression behavior of the NP-treated cells
and (6) whether the nascent ion specific effect of our Cu-NPs
for bacteriotoxic potency was also true for Cu-NPs of different
sizes, prepared by other methods etc. Finally, an important
point of emphasis is that our unpublished results on cupric
oxide NPs indicated that the mechanism of antibacterial action
Nanotechnology 25 (2014) 135101
of CuO-NPs was not through the direct route of Cu ions
liberated from the NPs, but rather through the formation of
some reactive complex between NPs and cellular medium
organics, and no considerable decrease in size of the NPs
occurred in the presence of EDTA; this implied that no single
mechanism was valid for the antibacterial role of metallic
Cu-NPs and NPs of other Cu-containing compounds.
Acknowledgments
We are indebted to the Department of Biotechnology,
Government of India, for financial assistance (project no
BT/PR11477/ NNT/28/416/2008). We also acknowledge
the Department of Science and Technology, Government of
India, for its ‘FIST Programme—2001–2011’ and ‘PURSE
Programme—2012–2015’, and the University Grants Com-
mission, Government of India for its ‘Special Assistance
Program—2011–2016’ going on in our department/university,
to provide the different instrumental and infrastructural sup-
port.
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