J Nanopart Res (2010) 12:2101–2109
DOI 10.1007/s11051-009-9769-9
RESEARCH PAPER
Investigation of Mg(OH)2 nanoparticles as an antibacterial
agent
Chunxu Dong • John Cairney • Qunhui Sun •
Orville Lee Maddan • Gaohong He • Yulin Deng
Received: 23 May 2009 / Accepted: 21 September 2009 / Published online: 4 October 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Our experimental results of using
Mg(OH)2 nanoparticles as an antibacterial agent are
reported in this study. The antibacterial behavior of
Mg(OH)2 nanoparticles in liquid culture and in paper
sheets was investigated. The colony forming units
(CFU) counting and the headspace gas chromatography (HS-GC) measurement were used to determine
the cell viability. Results indicate that Mg(OH)2
nanoparticles are effective antibacterial agent against
Escherichia coli (E. coli) and Burkholderia phytofirmans, and the OH- and Mg2? ions in Mg(OH)2 water
suspension were found not to be the reason for killing
the bacteria. Mg(OH)2 nanoparticles could be added
directly to wood pulp to make paper sheets, whose
antibacterial efficiency increased with the increase
of the nanoparticle amount. The possible mechanism
of antibacterial effect of Mg(OH)2 nanoparticles is
discussed.
Keywords Antibacterial agent Mg(OH) nanoparticles Metal oxide E. coli Environment EHS
Introduction
C. Dong G. He
State Key Laboratory of Fine Chemicals, School
of Chemical Engineering, Dalian University
of Technology, Dalian 116012, China
C. Dong Q. Sun Y. Deng
Institute of Paper Science and Technology, Georgia
Institute of Technology, Atlanta, GA 30332, USA
J. Cairney
School of Biology, Georgia Institute of Technology,
Atlanta, GA 30332, USA
O. L. Maddan
Aqua Resources Corporation, 20 Linwood Drive,
Fort Walton Beach, FL 32547, USA
Y. Deng (&)
School of Chemical and Biomolecular Engineering,
Georgia Institute of Technology, 500 10th Street,
N.W., Atlanta, GA 30332, USA
e-mail: [email protected]
Due to the ubiquity of microorganisms and their ability
to establish themselves, the control of microbial
populations is a universal concern. Antibacterial
agents are thus of relevance to a number of industrial
sectors including those focused on the environment,
food, synthetic textiles, packaging, healthcare, medical
care, as well as construction, and home and workplace
furnishing. Antibacterial agents are broadly categorized as organic or inorganic. Both the natural and the
synthetic organic antibacterial agents, such as chitosan
and its derivatives, quaternary ammonium salts,
biguanide agents, and pyridines inhibit strongly the
growth of a wide variety of bacteria and fungi
(Sudarshan et al. 1992; Liu et al. 2001). However,
organic antibacterial agents have some shortcomings,
such as low heat resistance, high decomposability, and
short life expectancy, which have limited their
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application. As a result, inorganic antibacterial agents
have received more and more recognition in the
antibacterial product market (Wang et al. 2003; Fang
et al. 2006; Jung et al. 2008).
Among the inorganic antibacterial agents, silver
and copper are traditionally well-known antibacterial
materials (Feng et al. 2000; Yoshinari et al. 2001;
Jeon et al. 2003). Metal oxide materials, such as
TiO2, ZnO, and MgO, have also been recognized as
antibacterial agents (Sawai et al. 2000; Stoimenov
et al. 2002; Yu et al. 2003; Zhang et al. 2007). It is
considered that the detected active oxygen species
generated by these metal oxide particles could be the
main mechanism of their antibacterial activity.
However, it is not clear that how the active oxygen
is produced and how to improve the active oxygen
production. As a metal hydroxide material, Ca(OH)2
has been widely used in endodontics as an intra-canal
medication. This strongly alkaline substance has a
high antibacterial activity against oral bacterial and
its effects are highly dependent on the availability of
hydroxide ions in solution. However, the metal
hydroxide particles itself has the antibacterial activity
has always been ignored.
Compared with other metal hydroxide materials,
Mg(OH)2 is a strong base but a sparingly soluble
inorganic particle with a very low solubility product
constant (5.61 9 10-12). As a suspension in water at
room temperature, Mg(OH)2 functions as a buffer
material with pH of *10.4, is much lower than pH of
*12.5 in Ca(OH)2 solution, so it is often used as an
antacid to neutralize stomach acid. In addition, due to
its non-toxicity and low cost, Mg(OH)2 is an approved
drug and food additive which has been widely used in
flame-retardant composite formulations (Jiao et al.
2006), wood pulp bleaching, and cultural heritage
conservation. Its nanoparticles were also found
effective in the deacidification treatment and protection against cellulose aging (Giorgi et al. 2005).
In this article, Mg(OH)2 nanoparticles were used
as an antibacterial agent. The antibacterial effects of
Mg(OH)2 nanoparticles both in water and in paper
sheet were studied.
In addition to its use as a communication medium,
paper is used in personal care products, food packages,
wallpapers, medical records, bills etc. Since these
products pass through many countries and, literally,
through many hands, a concern has arisen that paper
packaging could act as a vector for the transmission of
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J Nanopart Res (2010) 12:2101–2109
pathogens, both locally and between continents. Antibacterial paper capable of preventing bacterial growth
and eliminating bacterial contaminants is attracting
more and more attention. Domtar, a Canada based
company, has introduced the first antimicrobial office
paper available in North America. Designed to protect
paper against the growth of bacteria, fungus, mold, and
mildew, antimicrobial office paper from Domtar is
specially treated with a silver compound that kills most
bacteria with which it comes in contact. Japanese
companies, such as Nippon Paper, are also developing
photocatalyst enhanced antibacterial papers using
TiO2 nanoparticles. In this article, we report antibacterial papers made directly by the addition of Mg(OH)2
nanoparticles to wood pulp during a paper making
process. A pronounced inhibition of bacterial growth is
observed for nanoparticles-containing handsheets.
Materials and methods
Equipment
The scanning electron microscopy (SEM), zeta
potential measurement, Brunauer, Emmett and Teller
(BET) method, and energy dispersive spectroscopy
(EDS) were applied to characterize the size, shape,
composition, zeta potential, and specific surface of
the obtained Mg(OH)2 nanoparticles. The SEM
observation was conducted on a LEO 1530 thermally
assisted field emission scanning electron microscope
machine with an acceleration voltage of 5.0 kV.
Headspace gas chromatograph (HS-GC) with a HP7694 Automatic Headspace Sampler and Model HP6890 capillary gas chromatograph (Hewlett-Packard,
CA, USA) was used for detecting the CO2 released by
live E. coli from solutions. The detail HS-GC
measurement procedure will be described later.
Mg(OH)2 nanoparticles
Mg(OH)2 nanoparticles were synthesized by Aqua
Resources Corporation (Florida, USA) using an electrolytic method with a membrane between anode and
cathode. The detail procedure for Mg(OH)2 nanoparticles synthesis was given in the US patent application
(Maddan 2008). As shown in Fig. 1a, the Mg(OH)2
nanoparticles are thin platelets with about 10 nm
thickness and 200–300 nm plate size. Table 1 shows
J Nanopart Res (2010) 12:2101–2109
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Fig. 1b. The results clearly show that Mg is the only
metal ion found in the sample except trace amount of
Au from SEM sputter coating. Therefore, it can be
concluded that the nanoparticles used in this study are
pure Mg(OH)2.
Materials
Bleached softwood kraft pulp was refined in a Valley
beater to a freeness of 400 Canadian Standard
Freeness. Percol-175 (a high molecular weight, low
charge density polyacrylamide retention aid) was
obtained from Ciba Specialty Chemicals (Suffolk,
VA, USA). Cloning vector pGEM-T Easy and E. coli
JM109 strain were purchased from Promega (Madison, WI, USA). Luria broth (LB, containing
10 g mL-1 tryptone, 5 g mL-1 yeast extract, and
10 g mL-1 sodium chloride) and LB Agar were
purchased from Sigma-Aldrich (Inc., MO, USA).
Antibacterial tests of Mg(OH)2 suspension
Fig. 1 a Morphology of Mg(OH)2 nanoparticles. b EDS
spectrum of the Mg(OH)2 nanoparticles
Table 1 Mg(OH)2 nanoparticles measured by Malvern 3000
Zetasizer
Particle size Zeta potential
(nm)
(mV)
Original sample
928
-20.2
Dispersed in 5% poly(acrylic
acid sodium salt)
389
-26.8
Samples were ultrasonicated in water and in 5% poly(acrylic
acid sodium salt), respectively
the particle size and zeta potential of the nanoparticles
which were obtained with a Malvern 3000 Zetasizer. In
order to well-disperse the nanoparticles in water,
poly(acrylic acid sodium salt) was used as a stabilizer.
After ultrasonification with poly(acrylic acid sodium
salt) solution (5% polyacrylic and *0.01% Mg(OH)2),
the particle size of Mg(OH)2 is about 360 nm, which
agrees with SEM measurement. The XRD spectrum of
the sample march the standard Mg(OH)2 peak very
well. The specific surface, measured by BET adsorption, is 110 m2 g-1. The EDS spectrum is recorded on
the LEO 1550 machine and the result is shown in
Suspension of Mg(OH)2 nanoparticles were prepared
in the LB-culture medium and dispersed using
ultrasonification for 20 min. In each study, the
suspension was freshly prepared and autoclaved. In
order to investigate the effect of the Mg(OH)2
nanoparticles on bacterial growth, the bacteria were
grown in the presence or absence of the Mg(OH)2
nanoparticles. The sample flasks were inoculated
with 1 mL of the same ‘‘seed culture’’ thus in each
case, equivalent numbers of live bacteria will be
present at the start of the assay. Timed samples were
taken and measured the number of live bacteria
remaining by two methods. One method is counting
colony forming units (CFU) per mL, which is a
common measurement of viable bacterial or fungal
numbers. Cell viability was determined by serially
surface plating 200 lL samples onto the LB agar
medium. Another method, we used is the headspace
gas chromatography measurement, in which CO2
evolution is taken as an index of the vitality of the
bacterial culture.
Headspace gas chromatograph
Carbon di oxide (CO2), one of the major volatile
microbial metabolites during bacterial growth, was
used as a marker and measured by HS-GC. In this
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study, the CO2 released from E. coli growth in
solution was measured using an HP-7694 Automatic
Headspace Sampler and Model HP-6890 capillary
gas chromatograph (Hewlett-Packard, CA, USA)
using a thermal conductivity detector (TCD). The
detailed method description was reported previously
(Chai et al. 2008). Two milliliter LB medium with or
without Mg(OH)2 nanoparticles was added to a
22 mL headspace sample vial. A total of 200 lL
E. coli culture with an approximately 107 CFU mL-1
was then inoculated into the vial. After mixing, each
vial was immediately sealed by a rubber septum and
then placed in the headspace sampler at a constant
temperature, 37 °C under a gentle agitation condition
of 300 rpm to allow the bacteria growth. The
headspace of the vial was periodically sampled
followed by GC measurement. The headspace sampling cycle time is 30 min.
Preparation of paper sheets with Mg(OH)2
nanoparticles
The bleached softwood kraft pulp was diluted and
various amounts of Mg(OH)2 nanoparticles were
added during the paper sheets making. Percol-175
was used for particle retention at dosage of 0.1 wt%
based on solid weight. The slurry was stirred for 20 s
at 1,000 rpm. After cursory wet pressing twice, paper
Fig. 2 The procedure of
antibacterial test
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sheets with a target basis weight of 100 g m-2 were
produced. The Mg(OH)2 content in the paper was
determined by ashing the paper in a muffler oven at
525 °C for 4 h. The actual weight of Mg(OH)2 after
paper ashing was obtained after correction of the
weight loss from the self-dehydration of Mg(OH)2
during the ashing process.
Antibacterial tests of Mg(OH)2 nanoparticles
filled paper
The process for evaluating inhibition of bacterial
growth by Mg(OH)2 nanoparticles filled paper is
illustrated in Fig. 2. Paper sheets with or without
nanoparticles prepared as described above were cut
into coupons (15 mm 9 8 mm). These coupons were
immersed into the E. coli culture with an approximately 107 colony forming units per milliliter
(CFU mL-1) once and allowed to dry at room
temperature. They were then placed randomly on a
plastic surface and a plastic cover was placed over
them at room temperature (22 °C) overnight (16–
18 h). After submerged individually in 1.5 mL tube
with LB media and vortexed vigorously (10 9 5 s),
all the paper coupons were taken out from the tubes.
At this time, the pH value of each culture was \8.
Surviving bacteria were harvested by placing these
tubes at a constant temperature, 37 °C under a gentle
J Nanopart Res (2010) 12:2101–2109
agitation condition of 300 rpm. In each case, 200 lL
of the resuspended bacteria solution was plated on LB
agar plates which were incubated at 37 °C overnight.
CFU number was counted to determine the cell
viability. All experiments were replicated thrice.
Results
Effect of Mg(OH)2 nanoparticle suspension
on different bacterial growth
In order to determine the Mg(OH)2 nanoparticles
acted as bacteriostatic agents (preventing growth of
bacteria but not killing them) or bacteriocidal agents
(killing bacteria), the bacteria were grown in the LB
medium with or without Mg(OH)2 nanoparticles.
Figure 3a shows the results of bacterial growth with
the same amount of initial bacterial culture in the
presence or absence of the Mg(OH)2 nanoparticles
after 12 and 40 h growth. In the absence of nanoparticles, we observe a confluent growth (colonies
aggregate together, usually[106 colonies) of bacteria
in growth media after 12 and 40 h. However, in the
presence 10 mg mL-1 Mg(OH)2 (0.17 mol L-1),
only few 100 live bacteria remain after 12 h and no
live bacteria were present after 40 h. A soil bacterium, Burkholderia phytofirmans shows even greater
sensitivity to the presence of Mg(OH)2 nanoparticles,
and there was no bacteria survive after 24 h in the
presence of 1 mg mL-1 Mg(OH)2 (Fig. 3b). These
results indicate that Mg(OH)2 nanoparticles are
bactericidal, and in this case, they kill bacteria.
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Effect of OH- and Mg2? in Mg(OH)2 suspension
on E. coli growth
As described above, the Mg(OH)2 nanoparticles
could kill E. coli in solution. However, it is unknown
if E. coli bacteria were killed by dissolved OH-,
Mg2?, or the nanoparticles. This was further examined by comparing the antibacterial efficiency of
Mg(OH)2 nanoparticles with solutions of NaOH and
MgSO4, respectively.
In these tests, the LB medium with 10 mg mL-1
(0.17 mol L-1) uniformly dispersed Mg(OH)2 nanoparticles was made, and the pH value was measured to
be 10.0. In comparison with Mg(OH)2 nanoparticles,
in a separated test, aqueous solution without Mg(OH)2
nanoparticles, but with NaOH was used to raise the pH
of LB medium to 10.0. A culture of E. coli
(C108 CFU mL-1) was added to the LB medium,
and the changes in the number of live bacteria were
monitored over time. The results of CFU counting and
HS-GC measurement are shown in Fig. 4. The top of
Fig. 4 show that the contrast of live population of
E. coli in these two cultures is remarkable. Bacteria
inoculated into LB medium with Mg(OH)2 nanoparticles significantly decreased after 7 h and totally
killed within 24 h, while they remained high after 24 h
growth in the LB/NaOH medium. The CO2-headspace
assay (the bottom of Fig. 4) is in good agreement with
the above observation. It clearly demonstrates minimal
CO2 evolution from E. coli growth in the presence of
Mg(OH)2 nanoparticles after 4 h, when the control
culture (LB only) was beginning to multiply, and no
evidence of any respiration after 10 h, implying that no
Fig. 3 Bacteria grown in
LB or LB supplemented
with 10 mg mL-1
Mg(OH)2 nanoparticles
(AMNP) a Escherichia coli;
b Burkholderia
phytofirmans (Soil
bacterium)
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J Nanopart Res (2010) 12:2101–2109
Fig. 4 a Change of
bacteria numbers with time
in different cultures of LB
medium with 0.17 mol L-1
Mg(OH)2 nanoparticles or
LB medium of pH 10.0
adjusted with NaOH
solution. b CO2 released
during E. coli growth in a
glass cell measured with
HS-GC for four different
LB solutions (LB medium;
0.17 mol mL-1 Mg(OH)2
in LB medium solution,
pH = 10.0; NaOH in LB
medium solution,
pH = 10.0; 0.17 mol mL-1
MgSO4 in LB medium
solution)
CO2 formed (GC singnal)
150
Control
125
Mg(OH)2
100
NaOH
MgSO4
75
50
25
0
-25
0
2
4
6
8
10
Incubation time (h)
live bacteria remained in the vial after 10 h exposure to
the nanoparticles. Compared the curves of LB/NaOH
with the control culture, we can find that the slopes of
the E. coli growth curves in these two cultures vary
from each other, which means the bacterial in LB/
NaOH culture grew slower than in the control culture.
Therefore, both the results of CFU counting and HSGC measurement indicate that the OH- in Mg(OH)2
suspension has no effect on E. coli viability.
Meanwhile, the 0.17 mol mL-1 MgSO4/LB solution, which has the same molar concentration of
Mg2? as the LB/Mg(OH)2 suspension, was also
investigated for its antibacterial activity. The CO2
evolution curve from E. coli growth is shown in
Fig. 4b.
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It is almost identical to the control one, which
indicates that Mg2? has no effect on bacterial growth.
Preparation of paper sheets with Mg(OH)2
nanoparticles
The SEM pictures of the wood fibers from paper
sheets without and with Mg(OH)2 nanoparticles are
compared in Fig. 5a which shows that the Mg(OH)2
nanoparticles are distributed uniformly on the surface
of the wood fibers. Meanwhile, the ashing results of
the samples also show a good result. The particle
retention ratio, as shown in Fig. 5b, proves that the
good retention ratio of Mg(OH)2 nanoparticles on
fibers (higher than 75%) was achieved. That means it
J Nanopart Res (2010) 12:2101–2109
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Fig. 5 Paper without (a)
and with (b) Mg(OH)2
nanoparticles. Retention of
the nanoparticles on wood
fibers
90
Retention ratio (%)
85
80
75
70
65
60
0
2
1
3
4
5
6
7
Mg(OH) 2 nano particles content (%)
is feasible to prepare the Mg(OH)2 nanoparticles
filled paper by directly adding them during the paper
production.
Antibacterial tests of Mg(OH)2 nanoparticles
filled paper
The antibacterial effect of paper with Mg(OH)2
nanoparticles was studied using the procedures as
shown in Fig. 2, and the effect of Mg(OH)2 content
on bacterial survival as a function of Mg(OH)2
content in paper sheets (10 h contacting time with
paper samples, followed by 7 h growth in LB Agar) is
plotted in Fig. 6. There was not much difference
between the hand sheet with 1.5% Mg(OH)2 and the
control one, but when the Mg(OH)2 particle content
in paper is higher than 3.0%, the number of live
E. coli bacteria decreased significantly.
Discussion
From the above experimental results, we can see that
the Mg(OH)2 nanoparticles are effective antibacterial
agent against E. coli and Burkholderia phytofirmans
in liquid culture. Accurately speaking, the Mg(OH)2
nanoparticles suspension kill these two pieces of
bacterial directly. However, the LB medium with the
same Mg2? concentration as Mg(OH)2 suspension
culture did not show any antibacterial effect on
E. coli growth (Fig. 4b), suggesting that the Mg2? ion
is not the bacterial killing agent. Therefore, we can
excludes the possibility that the antibacterial effect of
Mg(OH)2 nanoparticles suspension was due to the
presence of Mg2? in the solution.
In general, Mg(OH)2 is a strong base but a sparingly
soluble inorganic particle with a very low solubility
product constant (5.61 9 10-12). Theoretically, the
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J Nanopart Res (2010) 12:2101–2109
suspension of Mg(OH)2 nanoparticles in water could
function as a buffer material with pH of *10.4. In this
study, the LB medium with 10 mg mL-1 dispersed
E. colis urvivors
12,000
10,000
8,000
6,000
4,000
2,000
0
0
1
2
3
4
5
Mg(OH)2 content (%)
Fig. 6 Surviving bacteria on paper sheets with different
Mg(OH)2 nanoparticles content. Contacting time with paper
is 10 h; growth time in LB Agar is 7 h
Fig. 7 SEM of E. coli
before (a) and after
(b) treatment with
2 mg/mL Mg(OH)2
nanoparticles solution
for 5 h
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Mg(OH)2 nanoparticles was measured to have a stable
pH of 10.0. We used NaOH to adjust the LB medium to
the same pH of 10.0 and did the antibacterial test. As
shown in Fig. 4a and b, although E. coli still grew in
NaOH LB-culture solution at pH of 10, its growth rate
is much slower than neutral LB solution. In contrast, all
E. coli was killed at the same pH, but with Mg(OH)2
nanoparticles suspension. Mendonca et al. (1994)
described that the treatment of E. coli in NaHCO3–
NaOH buffer (pH = 10) at 37 °C did not damage cell,
growing equally well on both selective and nonselective media. In this article, our results agreed well
with their results.
Besides the antibacterial effects obtained in LB/
Mg(OH)2 nanoparticles suspension, good antibacterial activity was also achieved when Mg(OH)2
nanoparticles were incorporated into paper as inorganic filler particles. In order to confirm that E. coli
was killed on dry paper rather than in solution, the
E. coli contaminated papers were contacted with
water under vertex agitation for 1 min to transfer
small number of E. coli to water for further testing
(Fig. 2). It was found that the Mg(OH)2 nanoparticles
were not directly transferred to water by above
treatment, evidenced by the factor that the cultures
were still clear, and the pH of the culture solutions
were still neutral (much less than the equilibrium
solution of 10.4 of Mg(OH)2 solution). However, as it
can be seen from Fig. 6, the bacteria were still killed,
and the E. coli viability decreased with the nanoparticles amount in the filled paper. Therefore, it can be
concluded that E. coli was killed before the transfer
into the culture solution. In other words, Mg(OH)2
nanoparticles killed E. coli on dry paper surface.
As there seems no reason for Mg(OH)2 nanoparticles to generate the active oxygen species, its
antibacterial mechanism may differ from the
J Nanopart Res (2010) 12:2101–2109
proposed mechanism (Sawai et al. 2000; Stoimenov
et al. 2002; Yu et al. 2003; Zhang et al. 2007) for the
metal oxide nanoparticles. In addition, from the
above experimental results, it is also different from
the Ca(OH)2 particles for that the antibacterial
activity just is simply due to its high alkaline
solution. There must be new mechanism to explain
why Mg(OH)2 nanoparticles have antibacterial activity both in liquid culture and on dry paper surface.
There are two possible mechanisms for the high
effective antibacterial effect of Mg(OH)2 nanoparticles. The first mechanism may be due to the direct
penetration of Mg(OH)2 nanoparticles into the cell
wall and destroy the structure of proteins, leading to
membrane damage, and cell death. The second
possible mechanism is the adsorption of water
moisture on Mg(OH)2 nanoparticles surfaces, which
can form a thin water meniscus around the particles
through capillary condensation. The pH of this thin
water layer formed around the nanoparticles may be
much higher than its equilibrium solution. When the
nanoparticles are in contact with the bacteria, the
high concentrated OH- groups in this thin surface
water layer could damage the membrane resulting in
the death of the cells. Further research is underway
aiming at to find more evidence to establish the
antibacterial mechanism of Mg(OH)2 nanoparticles.
The antibacterial behavior of Mg(OH)2 nanoparticles was also investigated with SEM analysis.
Figure 7a and b shows the bacteria images before
and after treatment with 2 mg/mL Mg(OH)2 nanoparticles solution for 5 h. Before nanoparticles treatment (Fig. 7a), the membrane of the E. coli is regular
and smooth. However, the treatment with Mg(OH)2
nanoparticles causes considerable damage on E. coli
(Fig. 7b).
Acknowledgments We would like to thank IPST at Georgia
Tech for the financial support to the project, and the China
Scholar Council for providing scholarship for C. Dong.
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