View Online / Journal Homepage / Table of Contents for this issue

PAPER www.rsc.org/nanoscale | Nanoscale

Antibacterial action of dispersed single-walled carbon nanotubes on

Escherichia coli and Bacillus subtilis investigated by atomic force microscopy
Shaobin Liu,a Andrew Keong Ng,b Rong Xu,a Jun Wei,c Cher Ming Tan,d Yanhui Yanga and Yuan Chen*a
Received 25th June 2010, Accepted 29th July 2010
DOI: 10.1039/c0nr00441c

Single-walled carbon nanotubes (SWCNTs) exhibit strong antibacterial activities. Direct contact
between bacterial cells and SWCNTs may likely induce cell damages. Therefore, the understanding of
SWCNT–bacteria interactions is essential in order to develop novel SWCNT-based materials for their
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0NR00441C

potential environmental, imaging, therapeutic, and military applications. In this preliminary study, we
utilized atomic force microscopy (AFM) to monitor dynamic changes in cell morphology and
mechanical properties of two typical bacterial models (gram-negative Escherichia coli and gram-
positive Bacillus subtilis) upon incubation with SWCNTs. The results demonstrated that individually
dispersed SWCNTs in solution develop nanotube networks on the cell surface, and then destroy the
bacterial envelopes with leakage of the intracellular contents. The cell morphology changes observed on
air dried samples are accompanied by an increase in cell surface roughness and a decrease in surface
spring constant. To mimic the collision between SWCNTs and cells, a sharp AFM tip of 2 nm was
chosen to introduce piercings on the cell surface. No clear physical damages were observed if the
applied force was below 10 nN. Further analysis also indicates that a single collision between one
nanotube and a bacterial cell is unlikely to introduce direct physical damage. Hence, the antibacterial
Downloaded on 27 July 2012

activity of SWCNTs is the accumulation effect of large amount of nanotubes through interactions
between SWCNT networks and bacterial cells.

Introduction contact with SWCNTs,2–6 atomic force microscopy (AFM) may

Single-walled carbon nanotubes (SWCNTs) exhibit strong anti-
bacterial activities.1 It has been proposed that cell membrane
damage, resulting from direct physical contact between bacteria
and SWCNTs, is the major reason for bacterial death.1 Previous
experimental results showed that the antibacterial activity of
SWCNTs can be influenced by many factors: carbon nanotube
(CNT) diameter,2 length,3 aggregation,3,4 concentration,4,5 and
surface functional group;5 buffer solution;5 as well as contact time6
and intensity.4 In our recent study,4 we suggested that individually
dispersed nanotubes could be visualized as numerous moving
‘‘nano darts’’ attacking bacteria in a buffer solution, degrading
bacterial cell integrity and causing cell death. However, the
interactions between SWCNTs and bacterial cells still remain
unclear. Such information about SWCNT–bacteria interactions is
essential in the development of novel SWCNT-based materials for
their potential applications, such as water treatment filters7 and
antibacterial coatings.8,9 Moreover, a good understanding on
SWCNT–bacteria interactions may also help to elucidate the
toxicology of SWCNTs, which are critical for their applications in
imaging and therapeutics.10
Although scanning electron microscopy (SEM) has been
utilized to visualize physical damages of bacterial cells upon
a
School of Chemical and Biomedical Engineering, Nanyang Technological
University, Singapore 637459. E-mail: chenyuan@ntu.edu.sg; Fax: +65
67947553; Tel: +65 63168939
b
DSO National Laboratories, 20 Science Park Drive, Singapore 118230
c
Singapore Institute of Manufacturing Technology, Singapore 638075
d
School of Electrical and Electronic Engineering, Nanyang Technological
University, Singapore 639798
provide better opportunities for studying SWCNT–bacteria
interactions. This is because AFM can image nanoscale ultra-
structures of bacterial surfaces and measure nanomechanical
properties of bacterial cells.11,12 Bacteria can be imaged in air
or buffer solutions13 without complex pretreatments (such as
the conductive film coating required by SEM).11 Nano-
mechanical properties of bacteria can also be measured.14–16
Although some air dried samples, if stored properly, can be
imaged for months and even years,17 in some cases, their prop-
erties may change during air drying processes.18 To prevent such
alterations to properties during drying processes, in situ
measurement under physiological conditions is preferred.16,19,20
However, imaging of bacteria in buffer solutions may show
reduced topographical contrast compared to images obtained on
air-dried samples.17,21,22 In this preliminary study, we conducted
our AFM studies on air dried samples. It should be noted that
there are always artefacts associated with air drying. Imaging cells
in their hydrated state is preferred, although it is technically more
challenging. Numerous studies have demonstrated the effective-
ness of AFM in exploring surface structure and mechanical
property changes of bacterial cells upon treatment with b-
lactam,23 cefodizime,24 chistosan,14,15 nitric oxide,25 and antimi-
crobial peptides.19,26–29
The aim of this study is to investigate dynamic changes induced
by SWCNTs on cell morphology and mechanical properties of
two typical bacterial models: gram-negative Escherichia coli and
gram-positive Bacillus subtilis. First, a super sharp AFM tip was
used to create piercings on bacterial cells, mimicking possible
collisions between SWCNTs and bacteria. Subsequently, AFM
was employed to image bacterial cells that were exposed to

2744 | Nanoscale, 2010, 2, 2744–2750 This journal is ª The Royal Society of Chemistry 2010

SWCNTs over different time periods. In this preliminary study, all
cell imaging was conducted in air, in order to monitor morphology
changes of bacteria. We also measured the bacteria mechanical
properties under various pseudo-physiological conditions (0.9%
NaCl solution) to avoid changes in mechanical properties during
air drying process.
Experimental
Bacterial cell preparation
E. coli K12 (ATCC 25404) and B. subtilis (ATCC 6051) were grown
in Luria-Bertani (LB) broth at 37  C and 30  C overnight, re-
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0NR00441C
spectively, and harvested in the mid-exponential growth phase.
Cells were isolated by centrifugation at 6000 rpm for 10 min and
washed three times with a saline solution (an aqueous mixture of
0.1 wt % Tween-20 and 0.9 wt % NaCl). Tween-20 was added to
enhance the dispersion of SWCNTs.4 Next, cell pellets were sus-
pended in the saline solution. Bacterial suspensions were further
diluted to obtain cell samples containing 106 to 107 colony forming
units (cfu/mL). Commonly available SWCNT samples are usually
mixtures of many species including metal residues, nanosize catalyst
supports, amorphous carbon, carbon nanoparticles, graphite,
carbon fibers, SWCNTs and multi-walled carbon nanotubes
Downloaded on 27 July 2012
(MWCNTs). To accurately investigate the antibacterial action of
SWCNTs, we employed high purity SWCNT samples with an
average tube diameter of 0.83 nm and a narrow chiral structure
distribution.30–33 Nanotubes were purified by a centrifugation-based
method.34 To yield individually dispersed nanotube suspensions,
solid samples (5 mg/mL) were dispersed in the Tween-20–saline
solution by sonication in a cup-horn sonicator (SONICS, VCX-
130) at 20 W for 1 h.4 SWCNT suspensions (10 mL at 5 mg/mL)
were incubated with 1 mL of bacterial suspensions for up to 2 h
under 250 rpm shaking speed at 37  C for E. coli or 30  C for B.
subtilis.
Cell piercing experiments
A super sharp silicon probe (SSS-NCH from Nanoworld with
typical tip radius of 2 nm) was used in cell piercing experiments to
examine possible nanoscale collisions between individually
dispersed SWCNTs and bacteria. Fresh bacterial suspensions
were drop-casted on cleaned glass slides and dried in air. We
carried out the piercing experiments in air because it is easier to
observe any structural changes on bacterial surface by AFM
imaging in air than in liquid. The cantilever deflection sensitivity
was calibrated on a hard surface (a glass slide). AFM images
were first obtained in a tapping mode at 256 pixels  256 lines
with scanning speed at 1 Hz. After imaging, the tip was aligned
above bacterial cells and converted to contact mode. Four
different positions on the bacterial cell were continuously punc-
tured for 200 times each, with forces of 10, 100, 1000, and 2000
nN, respectively. The cell was eventually imaged in tapping mode
to illustrate morphology changes induced by physical punctures
at different force loads.
Estimating mass and momentum of dispersed SWCNTs
Nanotubes with (7,5) chiral structure is the main species in our
SWCNT samples.4,34 Considering the fact that the C–C bond length
This journal is ª The Royal Society of Chemistry 2010
View Online
is 0.142 nm, one unit length of the (7,5) nanotubes (4.448 nm)
contains 436 C atoms (simulated by Materials Studio, Accelrys).
The average length of nanotubes is assumed to be 500 nm;
accordingly, the mass of one individual nanotube is m ¼ (500 (nm,
length)  436 (C atoms)/4.448 (nm/unit length))  (0.012 (kg/mol) /
6.02  1023 (atoms/mol)) ¼ 9.8  1022 kg. During incubation with
bacteria, the stirring speed of 250 rmp in a flask of 5 cm in diameter
could result a velocity of v ¼ 2p  0.025 (m)  250 rmp/60 (sec) ¼
0.65 m/s. Thus, the momentum of an individual nanotube is
approximately at mv ¼ 6.37  1022 kg$m/s.
Cell imaging by AFM
Cover glass slides were rinsed with alcohol, acetone, and deion-
ized water in sequential steps. A cell sample of 10 mL was ex-
tracted from bacterial cell suspensions after incubation with
SWCNTs for different time periods, and drop-casted directly on
cleaned glass slides. To obtain AFM images of bacterial cell wall
morphology, we dried and immobilized cells on glass slides under
ambient air for 10 min. All samples were characterized within
30 min after drying. AFM images were obtained via a MFP3D
microscope (Asylum Research, Santa Barbara, CA). To mini-
mize lateral forces on cells during imaging, a silicon non-contact
high resonance frequency cantilever (NCH type from Nano-
world with a spring constant of 42 N/m) was used to image cells
during tapping mode. The scan rate was set to 1 Hz at various
scan sizes, while all data resolutions were set to 256 pixels  256
lines. To eliminate the Z offset between scan lines, we applied the
first-order flattening to AFM height profiles. Then, we analyzed
the AFM height profiles using the Igor Pro MFP-3D software to
obtain root-mean-squared (rms) roughness on 200 nm  200 nm
areas. Five AFM height profiles were scanned within 30 min, and
five different areas on the height profiles of each cells were
randomly selected to calculate their rms roughness values.
Force measurement on immobilized bacteria
E. coli and B. subtilis were immobilized on poly(L-lysine) coated
cover glass slides, as described in our previous publication.4 In
a nutshell, 10 mL of poly (L-lysine) solution (Sigma, 0.01%
solution) was dropped on a clean glass slide, and the slide was
dried at room temperature. A bacterial suspension of 50 mL of
was drop-casted on the poly(L-lysine) coated surface. The
bacterial suspension liquid drop stood still for 10 min to anchor
cells on the surface. Then, the slide was rinsed in 0.9 wt % NaCl
solution to remove unattached cells. Cell attached slides were
imbedded in 100 mL of saline solution during force measure-
ment. The spring constant (kc) of AFM tips was measured in saline
solution by a thermal method in the Igor Pro MFP 3D software.
The location of bacterial cells was identified by AFM imaging in
contact mode (10  10 mm2 with 128  128 pixels or 15  15 mm2
with 256  256 pixels) using Silicon probes (ContAI-G, Budget
Sensors). Reference force curves were first obtained on bare spots
on the poly (L-lysine) coated surface (not covered with bacteria).
Next, AFM tips were aligned at different positions on bacterial
cells. The positions were all set near the center of bacteria to avoid
variations induced by the edge effect.35 The force trigger point
was set to 3 nN, and the tip approaching and retraction rates were
500 nm/s. At least five random locations on the same cell and five
Nanoscale, 2010, 2, 2744–2750 | 2745

different bacterial cells on each glass slide were chosen to record
force–distance (f–d) curves. Subsequently, one additional AFM
image was taken to confirm that the structure and location of the
cells remain unchanged during the force measurement. Spring
constants (kf) of bacterial cells were determined by the slope (s) of
the linear portion of the approaching branch of f–d curves using
the expression: kf ¼ kcs/(1  s).36–38
Results and discussion
Multiple piercing of bacterial cells
When bacteria are incubated with SWCNT dispersions, cells may
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0NR00441C
collide with numerous SWCNTs in solution. The collision
between bacterial cells and SWCNTs may damage bacterial cells.
Physical piercing on cell walls by a nanoscale tip can be used to
simulate such collisions, and it is interesting to find out the force
needed by the tip to induce damages on cell surfaces. We chose
a super sharp tip with 2 nm radius to mimic the physical structure
of SWCNTs, which would interact with bacteria in solution. As
described in the previous section, the tip continuously pierced the
cell surface under four different force loads (10, 100, 1000, and
2000 nN). Fig. 1 shows images of bacterial cells before and after
pierced. When the force is higher than 100 nN, clear holes can be
Downloaded on 27 July 2012
found on cell surfaces after punctures; the holes remain
unchanged after the piercing experiment had ended for 30 min.
For both E. coli and B. subtilis, the hole dimensions increase with
the raise of applied force from 100 to 2000 nN. Conversely, no
damages were noted when the force was at 10 nN. We further
lowered the force to 3 nN, and no changes on the bacterial
morphology were observed after multiple puncturings. Suo
et al.39 illustrated that a live gram negative bacterium Salmonella
typhimurium can survive 20 to 40 puncturings of 4 nN by a sharp
AFM tip, and the authors concluded that phospholipid
Fig. 1 AFM amplitude images of (A) E. coli and (B) B. subtilis. (C) and
(D) are, respectively, E. coli and B. subtilis after piercing by a 2 nm AFM tip
for 200 times at different locations. Small images on the extreme right are
enlarged images of areas punctured by the tip. The deflection set points of
the AFM tip are 10, 100, 1000, and 2000 nN. All scale bars are 1 mm.
2746 | Nanoscale, 2010, 2, 2744–2750
View Online
molecules may undergo rapid reorientation in response to AFM
tip penetration, sealing holes that are caused by the tip. In the
same fashion, the holes, which were caused by force at or below
10 nN, on E. coli and B. subtilis were covered back. However, the
reorientation of phospholipid molecules is unable to reseal holes
induced by forces above 10 nN.
An intriguing question is whether SWCNTs dispersed in
solution could induce large forces (>10 nN) on bacterial cell
surfaces? If we assume that the relative velocity of nanotubes to
bacteria would decrease from 0.65 m/s to 0 m/s upon collision
under the stirring speed of 250 rmp, the momentum of one
nanotube (6.37  1022 kg$m/s) could induce a force of 10 nN
only when the collision impact time is as short as 6.37  10 14 s
(t ¼ Dmv/F), which is too small to be realistic. Even if nanotube–
surfactant assemblies in solution can be much heavier than one
individual nanotube, the momentum change is still unlikely to
generate a force higher than 10 nN. On the other hand, a study
has shown that Brownian motion velocity of a 300 nm nano-
particle is only 4–5 mm/s.40 The momentum of SWCNTs attrib-
uted to their Brownian motion should be much smaller than that
induced by stirring. Based on the above outcomes, it is unlikely
that a single collision between an individual SWCNT and
a bacterium would produce a force that can cause direct physical
damages to the bacterial surface.
Bacterial cell morphology changes
Our previous study indicated that 58.1  5.0% of E. coli and
87.5  6.5% of B. subtilis were killed after 2 h of incubation with
SWCNTs, and SEM images also showed that bacterial cells are
seriously damaged after exposure to SWCNTs.4 Since a direct
single collision has little chance in damaging cells, it is essential to
investigate how SWCNTs cause dynamic changes to cell wall
architecture and cell mechanical properties, as well as to examine
the dependence of such changes on interaction time and inten-
sity. Fig. 2 renders AFM amplitude images of E. coli and B.
subtilis observed after incubation with SWCNTs for 0, 10, 60,
and 120 min. As illustrated in Fig. 2A, the surface of E. coli is
relatively smooth before interacting with SWCNTs. No obvious
nano-pores or physical damages were found, implying that both
drying in air and tapping mode do not alter cell wall architecture.
In this study, no peritrichous flagella structures were observed on
E. coli. On the contrary, gram-positive bacterium B. subtilis (see
Fig. 2B) possesses distinctive peritrichous flagella structures with
smooth surfaces. After incubation with SWCNTs for 10 min, E.
coli is winded with nanoscale tube-like structures (see Fig. 2C). A
close examination of the cell surface in Fig. 3 suggests that these
tube-like structures partially cover cell surface. We further
measured the height of the structures on the flat glass surface. As
clearly depicted in Fig. 3B, heights of these small bundles range
from 4 to 10 nm, which are similar to those of SWCNT bundles.
Since no peritrichous flagella structures were found on E. coli and
no other tube-like materials exist in our cell cultures, we conclude
that these tube-like structures are SWCNT bundles.
The surface of bacterial cells became uneven after incubation for
60 min. Fig. 2E shows a damaged E. coli. Grooves and holes were
randomly distributed on the cell surface. The cell wall collapsed
and the cell height decreased, releasing its cytoplasm. The cell
damages were more apparent and severe after the cell was
This journal is ª The Royal Society of Chemistry 2010

Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0NR00441C
Downloaded on 27 July 2012
Fig. 2 AFM amplitude images of E. coli and B. subtilis (1 mL of bacterial
suspensions, 106–107 cfu/mL) after incubation with SWCNT dispersions
(10 mL, 5 mg/mL) over different time periods. (A) E. coli before contact
with SWCNTs, (B) B. subtilis before contact with SWCNTs, (C, E and G)
E. coli after incubated with SWCNTs for 10, 60 and 120 min, respectively,
and (D, F and H) B. subtilis after incubated with SWCNTs for 10, 60 and
120 min, respectively. All scale bars are 1 mm.
incubated for 120 min (see Fig. 2G); the cell was almost flattened,
and cytoplasm was found all around the cell. AFM image in
Fig. 2G confirmed that SWCNTs can fragment bacterial
membranes, leading to a severe volume reduction. The surface
roughness of E. coli outer membranes was also monitored. The
average rms roughness of E. coli before contact with SWCNTs
was 3.6  0.5 nm. After incubation with SWCNTs for 10 min, the
rms of cell surface with and without SWCNT networks were
correspondingly 9.9  1.6 nm and 4.0  0.9 nm. The surface
roughness further increased to an average of 16.8  1.8 nm after 60
min of incubation with SWCNTs.
This journal is ª The Royal Society of Chemistry 2010
View Online
Fig. 3 (A) AFM height image of the E. coli cell (the same cell shown on
Fig. 1C) after incubation with SWCNT dispersions for 10 min. (B) The
height profile along the line in part A. (C) 3D height image of the E. coli cell.
The same cell morphology changes were observed on B. sub-
tilis. The cell surface becomes rougher after 10 min of incubation
with SWCNTs (see Fig. 2D). It is difficult to identify tube-like
structures on B. subtilis due to the existence of peritrichous
flagella structures, which look similar to nanotube bundles. Some
solid residues can be identified near the edge of the cell. Serious
bacterial membrane destruction and leakage of cytoplasm was
observed after exposure to SWCNTs for 60 and 120 min (see
Fig. 2F and 2H). The morphology observation for B. subtilis was
consistent with that for E. coli. The rms roughness of the
untreated B. subtilis was 4.9  1.1 nm, and the roughness in-
creased to 7.3  2.0 nm, 15.8  4.6 nm, and 22.3  2.7 nm after
10, 60, and 120 min of exposure, correspondingly.
Mechanical properties of bacterial cells
Apart from morphology changes, mechanical properties of
bacteria should also be changed after exposure to SWCNTs. To
prevent the mechanical properties from changing during our
AFM characterization, we conducted all the following mea-
surements in saline solution. A silicon probe (ContAI-G, Budget)
was selected to assess the mechanical properties of bacterial
cells.41 Several studies have demonstrated that spring constants
of AFM cantilevers would not have significant influences on
the measurement of mechanical properties of bacterial cells.19,37
In this study, the AFM probe has a spring constant of 0.35 
0.03 N/m, which was determined by the thermonoise method.42
F–d curves were used to reveal cell hardness changes, as illus-
trated in Fig. 4 for E. coli, and B. subtilis before and after
treatment with SWCNTs. For glass surfaces, the slope of f–d
curves equals to one, because no indentation was induced by the
AFM tip. Conversely, any deformations in cell membranes can
cause f–d curve slopes to be less than one. In line with our
previous study,4 Fig. 4 shows that f–d curves of B. subtilis exhibit
smaller slopes than those of E. coli, suggesting that B. subtilis are
softer than E. coli. Although gram-positive cells are generally
stiffer than gram-negative cells43 due to their thick peptidoglycan
layers (20–80 nm vs. 7–8 nm), our unexpected outcome may
likely be rationalised as follows: (1) B. subtilis possesses a lesser
resistance to the AFM tip than E. coli because the former
bacterial cell does not have an extra layer, the outer membrane,
Nanoscale, 2010, 2, 2744–2750 | 2747

Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0NR00441C
Downloaded on 27 July 2012
Fig. 4 Force–distance curves of glass surface, E. coli (top) and B. subtilis
(bottom), before and after incubation with SWCNTs at different time
durations. Slopes indicate the hardness of the measured surface.
and/or (2) the particular strain of B. subtilis used in this study is
softer than the strain of E. coli. Similar results have been
observed in previous AFM studies.15,41 Nevertheless, f–d curve
slopes of both B. subtilis and E. coli decrease with increasing
incubation time. The effective spring constants of bacterial cell
surfaces can be calculated by the linear part of f–d curves, and the
results are listed in Table 1. The effective spring constants of E.
coli and of B. subtilis decrease from 0.172  0.044 to 0.088 
0.036 N/m and from 0.102  0.036 to 0.034  0.025 N/m,
respectively, after incubation with SWCNTs for 120 min. Spring
constants of bacterial cells correspond to their turgor pressure.36
Hence, the spring constant decrement observed in this study can
be attributable to the leakage of cytoplasm;44 the turgor pressure
of bacterial cells decreases when the dense cytoplasm is released.
As evidenced in Fig. 2, the cell membrane is damaged and a large
amount of cytoplasm is visible around the cell body. The sig-
nificant changes found in spring constants of E. coli and of B.
subtilis are consistent with their respective morphology changes.
In our previous study, we demonstrated that the antibacterial
activity of SWCNTs can be improved by dispersing nanotubes
individually, increasing nanotube concentration and elevating
the shaking speed during incubation. Well-dispersed nanotubes
are more mobile in solution and have a higher chance of colliding
2748 | Nanoscale, 2010, 2, 2744–2750
View Online
Table 1 Spring constants of bacterial cell surfaces after incubation with
SWCNTs over different time periods
E. coli B. subtilis
Incubation
time (min) slope kb (N/m) slope kb (N/m)
0 0.32  0.06 0.172  0.044 0.23  0.06 0.102  0.036
60 0.24  0.07 0.114  0.034 0.14  0.05 0.059  0.026
120 0.19  0.06 0.088  0.036 0.09  0.06 0.034  0.025
with bacterial cells. Higher nanotube concentration and shaking
speed can also enhance the interaction between SWCNTs and
cells. Other studies have observed that CNT diameter,2 length,3
aggregation,3,4 concentration,4,5 and surface functional groups;5
buffer solutions;5 as well as contact time6 and intensity4 can affect
CNT antibacterial activity. All these factors apparently influence
interactions between bacteria and nanotubes in solution.
According to the earlier experimental results for AFM tip
piercing, one individual nanotube (at 0.65 m/s) under a stirring
speed of 250 rmp is not likely to directly damage the cell. The
relative speed between suspended cells and SWCNTs could be
even smaller. Furthermore, considering the possibility of ‘‘head
on’’ vs. ‘‘side on’’ collisions, the chances of a SWCNT ‘‘piercing’’
bacteria are negligible. AFM images in Fig. 2 show that nano-
tube networks partially cover cell surfaces, signifying that
bacterial death is associated with the accumulation effect of large
amount of nanotubes. We propose the following scenario to
explain our results. The SWCNT concentration is 5 mg/mL, and
the mass of individual nanotubes is 9.8  1022 kg; accordingly,
there are about 5.1  1013 nanotubes in the 10 mL SWCNT
suspension interacting with 106 to 107 bacterial cells. Cell surfaces
may collide with SWCNTs millions of times during incubation. It
has been reported that glycoproteins can disperse SWCNTs in
aqueous solutions,45 and SWCNTs also interact strongly with
polysaccharides, such as chitosan.46 Thus, individual nanotubes
may adsorb on cell surfaces, and more nanotubes on the cell
surface would bundle together owing to the strong van der Waals
attractions between nanotubes.47,48 On top of the strong van der
Waals attractions, a recent theoretical study also highlighted that
collisions with bacterial surface and rotational Brownian motion
can lead to the accumulation of microswimmers near cell
surfaces.49 SWCNTs dispersed in solution, therefore, may have
high tendency to accumulate near cell surfaces and to be trapped
into the SWCNT networks formed on cell surfaces. For the
above mentioned scenario, the interaction between SWCNT
networks and cells is expected to be more intense than that of
individual nanotubes, giving rise to the destruction of cell enve-
lope. Once the cell envelope is damaged, it will result in cell death.
Experimental parameters, such as dispersion, concentration,
contact time and intensity, etc., which enhance the adsorption
and accumulation of SWCNTs on cell surfaces, are able to
enhance the antibacterial activities of SWCNTs.
In this study, a super sharp silicon probe was used to investi-
gate the interactions of SWCNTs with bacteria, however,
because the silicon probe has different chemistry/reactivity than
SWCNTs, the ‘‘chemical’’ effects of SWCNT–bacteria interac-
tions cannot be probed. As recommended by previous studies,1,2
SWCNTs may generate bacterial oxidative stress responses. The
close contacts between SWCNT networks and cells can also
This journal is ª The Royal Society of Chemistry 2010

possibly enhance the oxidative stress. However, some recent
studies suggested that carbon nanotubes can also behave as free-
radical scavengers.50–52 The exact chemical or biological roles of
SWCNTs require further investigation. It should be noted that
the results in this study have clarified that the physical piercing by
individual SWCNTs is not the feasible antibacterial mechanism
under incubation conditions.
Recent literature has shown that when bacteria were filtered
through nanotube based membranes, many cells were killed.7 We
reason that the filtration process is different from incubation. In
a filtration process, cells are dragged to the nanotube network.
The force, which a bacterial cell experiences during filtration,
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0NR00441C
could be much higher than 10 nN. Such heavy forces are capable
of directly damaging bacterial cells. The direct physical piecing
can be the dominant factor in the antibacterial mechanism under
filtration conditions. On the other hand, the latest report showed
that bacteria in contact with a deposit layer of SWCNTs under
static conditions produce much less biofilm.53 This suggests that
multiple contact between SWCNTs and bacteria is necessary for
bacterial death, which is consistent with our observations under
incubation conditions.
Conclusions
Downloaded on 27 July 2012
The present study is the first AFM visual demonstration of
SWCNT antibacterial effects on bacterial cells. For both E. coli
and B. subtilis, AFM images obtained on air dried samples
showed that bacterial envelopes were severely damaged with
leakage of intracellular contents after incubation with SWCNTs.
The volume and height of bacterial cells also decreased. The rms
roughness of bacterial surfaces increased with the incubation
time, which was in agreement with the observed cell destruction.
The spring constants of bacterial surfaces after exposure to
SWCNTs over different time periods were measured in saline
solution. The effective spring constants of E. coli and B. subtilis
declined from 0.172  0.044 to 0.088  0.036 N/m and 0.102 
0.036 to 0.034  0.025 N/m, respectively, after incubation with
SWCNTs for 120 min. The decrement of spring constants can be
associated with the destruction of cell envelope and the release of
cytoplasm. Physical piercings on bacterial cells by the 2 nm AFM
tip can result in significant damages when the applied force was
higher than 10 nN. The collision force between an individual
nanotube and a bacterial cell during incubation is much weaker
as compared to the tested force under piercing conditions.
Moreover, SWCNT network structures were found on E. coli
surface, further implying that bacterial death is related to the
accumulation effect of large amount of nanotubes.
Acknowledgements
This work was supported by National Research Foundation,
Singapore (NRF-CRP2-2007-02), and Environment & Water
Industry Development Council, Singapore (0802-IRIS-12).
References
1 S. Kang, M. Pinault, L. D. Pfefferle and M. Elimelech, Single-walled
carbon nanotubes exhibit strong antimicrobial activity, Langmuir,
2007, 23(17), 8670–8673.
This journal is ª The Royal Society of Chemistry 2010
View Online
2 S. Kang, M. Herzberg, D. F. Rodrigues and M. Elimelech,
Antibacterial Effects of Carbon Nanotubes: Size Does Matter,
Langmuir, 2008, 24(13), 6409–6413.
3 S. Kang, M. S. Mauter and M. Elimelech, Physicochemical
Determinants of Multiwalled Carbon Nanotube Bacterial
Cytotoxicity, Environ. Sci. Technol., 2008, 42(19), 7528–7534.
4 S. B. Liu, L. Wei, L. Hao, N. Fang, M. W. Chang, R. Xu, Y. H. Yang
and Y. Chen, Sharper and Faster ‘‘Nano Darts’’ Kill More Bacteria:
A Study of Antibacterial Activity of Individually Dispersed Pristine
Single-Walled Carbon Nanotube, ACS Nano, 2009, 3(12), 3891–3902.
5 L. R. Arias and L. J. Yang, Inactivation of Bacterial Pathogens by
Carbon Nanotubes in Suspensions, Langmuir, 2009, 25(5), 3003–
3012.
6 S. Kang, M. S. Mauter and M. Elimelech, Microbial Cytotoxicity of
Carbon-Based Nanomaterials: Implications for River Water and
Wastewater Effluent, Environ. Sci. Technol., 2009, 43(7), 2648–2653.
7 A. S. Brady-Estevez, S. Kang and M. Elimelech, A Single-Walled-
Carbon-Nanotube Filter for Removal of Viral and Bacterial
Pathogens, Small, 2008, 4(4), 481–484.
8 M. S. Mauter and M. Elimelech, Environmental Applications of
Carbon-Based Nanomaterials, Environ. Sci. Technol., 2008, 42(16),
5843–5859.
9 S. J. Klaine, P. J. J. Alvarez, G. E. Batley, T. F. Fernandes, R. D. Handy,
D. Y. Lyon, S. Mahendra, M. J. McLaughlin and J. R. Lead,
Nanomaterials in the Environment: Behavior, Fate, Bioavailability,
and Effects, Environ. Toxicol. Chem., 2008, 27(9), 1825–1851.
10 K. Kostarelos, A. Bianco and M. Prato, Promises, facts and
challenges for carbon nanotubes in imaging and therapeutics, Nat.
Nanotechnol., 2009, 4(10), 627–633.
11 Y. F. Dufrene, Towards nanomicrobiology using atomic force
microscopy, Nat. Rev. Microbiol., 2008, 6(9), 674–680.
12 P. Hinterdorfer and Y. F. Dufrene, Detection and localization of
single molecular recognition events using atomic force microscopy,
Nat. Methods, 2006, 3(5), 347–355.
13 B. Drake, C. B. Prater, A. L. Weisenhorn, S. A. C. Gould,
T. R. Albrecht, C. F. Quate, D. S. Cannell, H. G. Hansma and
P. K. Hansma, Imaging Crystals, Polymers, and Processes in Water
with the Atomic Force Microscope, Science, 1989, 243(4898), 1586–1589.
14 J. C. Fernandes, P. Eaton, A. M. Gomes, M. E. Pintado and
F. X. Malcata, Study of the antibacterial effects of chitosans on
Bacillus cereus (and its spores) by atomic force microscopy imaging
and nanoindentation, Ultramicroscopy, 2009, 109(8), 854–860.
15 P. Eaton, J. C. Fernandes, E. Pereira, M. E. Pintado and
F. X. Malcata, Atomic force microscopy study of the antibacterial
effects of chitosans on Escherichia coli and Staphylococcus aureus,
Ultramicroscopy, 2008, 108(10), 1128–1134.
16 Y. Z. Wu and A. H. Zhou, In situ, real-time tracking of cell wall
topography and nanomechanics of antimycobacterial drugs treated
Mycobacterium JLS using atomic force microscopy, Chem.
Commun., 2009, (45), 7021–7023.
17 A. V. Bolshakova, O. I. Kiselyova and I. V. Yaminsky, Microbial
surfaces investigated using atomic force microscopy, Biotechnol.
Prog., 2004, 20(6), 1615–1622.
18 J. J. Thwaites and U. C. Surana, Mechanical Properties of Bacillus
subtilis Cell Walls - Effects of Removing Residual Culture Medium,
J. Bacteriol., 1991, 173(1), 197–203.
19 N. P. Mortensen, J. D. Fowlkes, C. J. Sullivan, D. P. Allison,
N. B. Larsen, S. Molin and M. J. Doktycz, Effects of Colistin on
Surface Ultrastructure and Nanomechanics of Pseudomonas
aeruginosa Cells, Langmuir, 2009, 25(6), 3728–3733.
20 Y. Y. Chen, C. C. Wu, J. L. Hsu, H. L. Peng, H. Y. Chang and T. R. Yew,
Surface Rigidity Change of Escherichia coli after Filamentous
Bacteriophage Infection, Langmuir, 2009, 25(8), 4607–4614.
21 A. V. Bolshakova, O. I. Kiselyova, A. S. Filonov, O. Y. Frolova,
Y. L. Lyubchenko and I. V. Yaminsky, Comparative studies of
bacteria with an atomic force microscopy operating in different
modes, Ultramicroscopy, 2001, 86(1–2), 121–128.
22 D. Robichon, J. C. Girard, Y. Cenatiempo and J. F. Cavellier,
Atomic force microscopy imaging of dried or living bacteria,
Comptes Rendus de l Acad emie des Sciences - Series III - Sciences de
la Vie, 1999, 322(8), 687–693.
23 L. Yang, K. M. Wang, W. H. Tan, X. X. He, R. Jin, J. Li and
H. M. Li, Atomic force microscopy study of different effects of
natural and semisynthetic beta-lactam on the cell envelope of
Escherichia coli, Anal. Chem., 2006, 78(20), 7341–7345.
Nanoscale, 2010, 2, 2744–2750 | 2749

24 P. C. Braga and D. Ricci, Atomic force microscopy: Application to
investigation of Escherichia coli morphology before and after exposure
to cefodizime, Antimicrob. Agents Chemother., 1998, 42(1), 18–22.
25 S. M. Deupree and M. H. Schoenfisch, Morphological analysis of the
antimicrobial action of nitric oxide on Gram-negative pathogens using
atomic force microscopy, Acta Biomater., 2009, 5(5), 1405–1415.
26 A. Meincken, D. L. Holroyd and M. Rautenbach, Atomic force
microscopy study of the effect of antimicrobial peptides on the cell
envelope of Escherichia coli, Antimicrob. Agents Chemother., 2005,
49(10), 4085–4092.
27 A. da Silva and O. Teschke, Dynamics of the antimicrobial peptide
PGLa action on Escherichia coli monitored by atomic force
microscopy, World J. Microbiol. Biotechnol., 2005, 21(6–7), 1103–1110.
28 J. E. Shaw, J. R. Alattia, J. E. Verity, G. G. Prive and C. M. Yip,
Mechanisms of antimicrobial peptide action: Studies of indolicidin
assembly at model membrane interfaces by in situ atomic force
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0NR00441C
microscopy, J. Struct. Biol., 2006, 154(1), 42–58.
29 G. Rossetto, P. Bergese, P. Colombi, L. E. Depero, A. Giuliani,
S. F. Nicoletto and G. Pirri, Atomic force microscopy evaluation of
the effects of a novel antimicrobial multimeric peptide on
Pseudomonas aeruginosa, Nanomed.: Nanotechnol., Biol. Med., 2007,
3(3), 198–207.
30 Y. Chen, D. Ciuparu, S. Lim, G. L. Haller and L. D. Pfefferle, The
effect of the cobalt loading on the growth of single wall carbon
nanotubes by CO disproportionation on Co-MCM-41 catalysts,
Carbon, 2006, 44(1), 67–78.
31 Y. Chen, D. Ciuparu, S. Lim, Y. Yang, G. L. Haller and L. Pfefferle,
Synthesis of Uniform Diameter Single-Wall Carbon Nanotubes in
Co-MCM-41: Effects of the Catalyst Prereduction and Nanotube
Growth Temperatures, J. Catal., 2004, 225(2), 453–465.
Downloaded on 27 July 2012
32 Y. Chen, D. Ciuparu, S. Lim, Y. Yang, G. L. Haller and L. Pfefferle,
Synthesis of Uniform Diameter Single Wall Carbon Nanotubes in Co-
MCM-41: Effects of CO Pressure and Reaction Time, J. Catal., 2004,
226(2), 351–362.
33 D. Ciuparu, Y. Chen, S. Lim, G. L. Haller and L. Pfefferle, Uniform-
Diameter Single-Walled Carbon Nanotubes Catalytically Grown in
Cobalt-Incorporated MCM-41, J. Phys. Chem. B, 2004, 108(2), 503–507.
34 L. Wei, B. Wang, Q. Wang, L. J. Li, Y. H. Yang and Y. Chen, Effect
of Centrifugation on the Purity of Single-Walled Carbon Nanotubes
from MCM-41 Containing Cobalt, J. Phys. Chem. C, 2008, 112(45),
17567–17575.
35 S. B. Velegol, S. Pardi, X. Li, D. Velegol and B. E. Logan, AFM
imaging artifacts due to bacterial cell height and AFM tip
geometry, Langmuir, 2003, 19(3), 851–857.
36 M. Arnoldi, M. Fritz, E. Bauerlein, M. Radmacher, E. Sackmann and
A. Boulbitch, Bacterial turgor pressure can be measured by atomic
force microscopy, Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat.
Interdiscip. Top., 2000, 62(1), 1034–1044.
37 S. B. Velegol and B. E. Logan, Contributions of bacterial surface
polymers, electrostatics, and cell elasticity to the shape of AFM
force curves, Langmuir, 2002, 18(13), 5256–5262.
2750 | Nanoscale, 2010, 2, 2744–2750
View Online
38 S. K. Das, A. R. Das and A. K. Guha, Adsorption Behavior of
Mercury on Functionalized Aspergillus versicolor Mycelia: Atomic
Force Microscopic Study, Langmuir, 2009, 25(1), 360–366.
39 Z. Y. Suo, R. Avci, M. Deliorman, X. H. Yang and D. W. Pascual,
Bacteria Survive Multiple Puncturings of Their Cell Walls,
Langmuir, 2009, 25(8), 4588–4594.
40 Y. J. Fan, H. J. Sheen, C. J. Hsu, C. P. Liu, S. M. Lin and K. C. Wu, A
quantitative immunosensing technique based on the measurement of
nanobeads’ Brownian motion, Biosens. Bioelectron., 2009, 25(4), 688–694.
41 C. C. Perry, M. Weatherly, T. Beale and A. Randriamahefa, Atomic
force microscopy study of the antimicrobial activity of aqueous garlic
versus ampicillin against Escherichia coli and Staphylococcus aureus,
J. Sci. Food Agric., 2009, 89(6), 958–964.
42 J. L. Hutter and J. Bechhoefer, Calibration of Atomic-Force
Microscope Tips, Rev. Sci. Instrum., 1993, 64(7), 1868–1873.
43 C. Shiu; Z. Zhang; C. R. Thomas., A Comparison of the Mechanical
Properties of Different Bacterial Species. In Applied Microbiology,
Durieux, A.; Simon, J.-P., ed. Kluwer Academic Publishers:
Netherlands, 2001; pp 155–162.
44 D. E. Bradley and C. Ann Dewar, Intracellular Changes in Cells of
Escherichia coli Infected with a Filamentous Bacteriophage, J. Gen.
Virol., 1967, 1(2), 179–188.
45 B. Wegenhart, L. Tan, M. Held, M. Kieliszewski and L. Chen,
Aggregate structure of hydroxyproline-rich glycoprotein (HRGP)
and HRGP assisted dispersion of carbon nanotubes, Nanoscale Res.
Lett., 2006, 1(2), 154–159.
46 L. Y. Yan, Y. F. Poon, M. B. Chan-Park, Y. Chen and Q. Zhang,
Individually dispersing single-walled carbon nanotubes with novel
neutral pH water-soluble chitosan derivatives, J. Phys. Chem. C,
2008, 112(20), 7579–7587.
47 L. A. Girifalco, M. Hodak and R. S. Lee, Carbon nanotubes,
buckyballs, ropes, and a universal graphitic potential, Phys. Rev. B:
Condens. Matter Mater. Phys., 2000, 62(19), 13104–13110.
48 F. Wang, G. Dukovic, L. E. Brus and T. F. Heinz, The optical
resonances in carbon nanotubes arise from excitons, Science, 2005,
308(5723), 838–841.
49 G. Li and J. X. Tang, Accumulation of Microswimmers near
a Surface Mediated by Collision and Rotational Brownian Motion,
Phys. Rev. Lett., 2009, 103(7), 078101-1–078101-4.
50 A. Galano, Carbon nanotubes: promising agents against free radicals,
Nanoscale, 2010, 2(3), 373–380.
51 A. Martinez and A. Galano, Free Radical Scavenging Activity of
Ultrashort Single-Walled Carbon Nanotubes with Different
Structures through Electron Transfer Reactions, J. Phys. Chem. C,
2010, 114(18), 8184–8191.
52 I. Fenoglio, M. Tomatis, D. Lison, J. Muller, A. Fonseca, J. B. Nagy and
B. Fubini, Reactivity of carbon nanotubes: Free radical generation or
scavenging activity?, Free Radical Biol. Med., 2006, 40(7), 1227–1233.
53 D. F. Rodrigues and M. Elimelech, Toxic Effects of Single-Walled
Carbon Nanotubes in the Development of E. coli Biofilm, Environ.
Sci. Technol., 2010, 44(12), 4583–4589.
This journal is ª The Royal Society of Chemistry 2010