Bioresponse to Nanotubes
Surface Chemistry of Carbon Nanotubes Impacts the
Growth and Expression of Water Channel Protein
in Tomato Plants
Hector Villagarcia, Enkeleda Dervishi, Kanishka de Silva, Alexandru S. Biris,*
and Mariya V. Khodakovskaya*
Nanosized engineered materials are becoming intensively
studied for their impact on modern plant biology and could
play major roles in advanced biotechnological applications. It
has been documented that nanoparticles can be beneficial for
the delivery of biological molecules into plant cells[14] and
the improvement of herbicide application for disease preven-
tion.[5] Carbon nanotubes (CNTs), with their unique mor-
phologies and surface properties, have also been intensively
studied for various applications in bionanotechnology.[6]
Given the fact that such nanomaterials are now produced
in large quantities and used in a plethora of industrial prod-
ucts, there is a significant probability that they will make their
way into the environment and therefore interact with various
plant species. As a result, there is a great need for a thorough
understanding of the complex effects that carbon nanotubes
have on the physiology and development of plant systems
at various levels. Liu and co-authors have demonstrated the
ability of single-walled CNTs (SWCNTs) to penetrate the
walls and membranes of tobacco cells.[3] Recently, Serag et al.
have identified an endosome-escaping uptake mode of multi-
walled carbon nanotubes (MWCNTs) by plant protoplasts.[4]
Still, the effects of single- and multi-walled carbon nanotubes
on plant physiology and plant developmental biology is not
yet very well understood.
H. Villagarcia, K. de Silva, Prof. M. V. Khodakovskaya
Department of Applied Science
University of Arkansas at Little Rock
Little Rock, AR, USA
E-mail: mvkhodakovsk@ualr.edu
Prof. E. Dervishi, Prof. A. S. Biris
Nanotechnology Center
University of Arkansas at Little Rock
Little Rock, AR, USA
Prof. A. S. Biris
Nanotechnology Center
Department of Systems Engineering
University of Arkansas at Little Rock
Little Rock, AR, USA
E-mail: asbiris@ualr.edu
DOI: 10.1002/smll.201102661
small 2012, 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/smll.201102661
Although during the last few years a number of scien-
tific studies have been conducted, the results are actually
quite contradictory. There are so far two opposing schools of
thought. One indicates that CNTs have a strong toxic effect
on plants, while the second suggests that CNTs have some
beneficial effects on seed germination, plant development,
and various plant physiological processes. For example, func-
tionalized single-walled carbon nanotubes in a wide range of
concentrations (91750 g/mL) were found to have no effect
on the physiology and development of cabbage and carrot.[7]
However, a 38% reduction of zucchini biomass was reported
in response to the application of MWCNTs.[8] The applica-
tion of SWCNTs to rice resulted in the delayed flowering and
decreased yield of this important crop.[9] On the contrary, Lin
and Xing showed that MWCNTs were able to increase the
root length of ryegrass.[10] Caas et al. additionally noticed
that nonfunctionalized SWCNTs increased the root growth
in onion and cucumber.[7] We have demonstrated recently
that SWCNTS and MWCNTs at relatively low doses can pen-
etrate thick seed coats, stimulate germination, and enhance
growth of young tomato seedlings.[11,12] The activation of seed
germination and growth in tomato plants was observed only
for SWCNTs and MWCNTsnot for graphene or activated
carbon.[12]
There is experimental evidence of the important role that
the surface chemistry of CNTs plays on plant biology. For
example, the exposure of tomato plants to CNTs decorated
with quantum dots resulted in a change in the plants physi-
ological responses from positive to negative, and obvious
symptoms of toxicity were observed.[13] Others have reported
that the chemical functionalization of CNTs can affect the
development of plants. For example, nonfunctionalized CNTs
have been reported to affect the root length of plants more
than CNTs functionalized with poly-3-aminobenzenesulfonic
acid.[7] Given the number of well-documented reports with
contradictory findingsall of which yet highlight the fact that
CNTs affect plant physiology and developmentadditional
studies will be required in order to thoroughly understand
the underlying mechanisms of these processes. One possible
variation among these studies is the morphology and the
characteristics of the nanotube used in each investigation.
For example, the nanotubes size, length, diameter, degree of
wileyonlinelibrary.com 1
 communications H. Villagarcia et al.

purity, presence of amorphous carbon, pos-

sible agglomeration, as well as the chemical
surface functionalization could all dramati-
cally affect the CNT-induced responses
(physiological and genetic) of plants. Our
recent experiments provided evidence that
oxidized MWCNTs can affect the total
gene expression of tomato plants exposed
to MWCNTs in low doses.[12] However, a
complete understanding of the molecular
mechanisms for negative (toxicity) and
positive (activation of germination and
plant growth) effects induced by the nano-
sized carbon materials in plants is still
lacking. Thus far, it is not clear if any spe-
cific properties of CNTs can be correlated
with the expression of genes and proteins
that are essential for plant growth.
Therefore, in this report, we focus on
understanding the complex role that the
surface chemistry of CNTs plays in the
physiological and molecular response
of tomato plants. We hypothesized that
the purity, aggregation, and presence of
various chemical functionalities on the
external walls of the CNTs would signifi-
cantly affect their ability to interact with
plant cells and further impact the germi-
nation rates of seeds, the growth rates
of the plants, and the production of pro-
teins that are essential for plant develop-
ment. Since our preliminary studies,[12]
which have also been confirmed by more
recent ones,[14] indicated that the nega- Figure 1. Transmission (A) and scanning electron (B) microscopy analysis of the multi-walled
tively charged nanotubes seem to have a carbon nanotubes used in this study. C) The diagram of the experimental conditions used in
stronger effect on plant development, we this study showing the multi-walled carbon nanotubes of various surface chemistries exposed
selected several chemical functionalities to tomato seedlings.
that all induced negative polarities (but
of various values) on the external walls of the nanotubes. We The overall purity was 94% (see Supporting Information (SI)
used high-purity MWCNTs, since compared to single-walled for details).
or graphene, this class of nanomaterials had previously been When introduced into the medium, the dispersion was
shown to have the most significant effect on the physiology of performed by simple mechanical stirring, which resulted in
tomato plants.[12] Figure 1A,B shows the microscopy analysis the formation of nanotube agglomerates with micrometer
of the purified MWCNTs. Our transmission electron micro- (220 m) sizes in the medium, as evidenced by optical micro-
scopy (TEM) analysis determined that 85% of the nanotubes scopy. CNT agglomerates were visible throughout the volume
had outer diameters between 8 and 35 nm.[15] The low- of the medium. As determined by elemental analysis, most
resolution TEM and the scanning electron microscopy (SEM) of the impurities were represented by Fe, Co, and Caall of
images (Figure 1A,B) indicated the presence of high quality which were part of the catalytic system used for the growth
MWCNTs of several micrometers in length. As the next step, of the nanotubes and which were not completely removed
MWCNTs were modified with various surface functionalities, during the purification process. This nanotube sample was
as indicated in Figure 1C. Based on optical observations, the named MW1. Then, the same nanotubes were additionally
impact that the dispersion and purity of the nanotubes in the purified by HCl washing and sonication reaching a purity of
plant medium had on the plants development was also inves- 98% (see Figure S1, SI) and were dispersed in the medium by
tigated. This aim was based on the relationship between the sonication, resulting in very well-dispersed nanotubes, without
expected nanotubes dispersion and their interactions with any visible agglomeration in the medium. This sample was
the plants. To accomplish this task, the as-produced samples named MW2. The high decomposition temperature (600 C)
were first purified by one-step washing in diluted hydrochloric from both weight loss profiles (see Figure S1, SI for details)
acid (1:1) solution at room temperature and then washed indicated the presence of highly crystalline nanotubes. No
with deionized (DI) water until a neutral pH was achieved. significant amount of amorphous carbon was detected in the

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DOI: 10.1002/smll.201102661
Carbon Nanotubes Impact on the Water Channel Protein in Tomato Plants
thermogravimetric analyses (TGA). The MWCNTs comprising
sample MW2 were further oxidized and decorated with car-
boxylic groups (strong negative polarity). The spectroscopic,
electronic, and dispersion characteristics induced by this treat-
ment in SWCNTs were described in our earlier report.[16]
The resulting sample was named MW3. In parallel, the MW2
nanotubes were sonicated in acetone which made them highly
hydrophobic due to the decoration with CH2,3 groups that have
very low surface energy but rather a slight negative polarity in
the experimental conditions of this study. We have thoroughly
described this functionalization on SWCNTs in our earlier
report, which also contains the optical absorption studies
of the resulting nanotubes;[17] this sample was named MW4.
In a separate experiment, the MW2 sample nanotubes were
coated with polyethylene glycol (PEG)an approach that has
been reported to produce excellent nanotube dispersion, but
which increases their diameter slightly due to the presence of
the polymeric coating and provides a slightly negative polarity.
We have successfully used this method to coat carbon nano-
tubes, and their thorough characterization is provided in our
earlier report.[18] The resulting sample was named MW5. The
zeta potential measurements for each CNT sample after var-
ious functionalization processes were performed. As expected,
the MW1 and MW2 samples had very similar negative surface
charges of 35.3 (8.2%) and 37.5 (10%) mV, respectively.
The carboxylated MWCNTs (MW3) showed a much higher
negative surface charge (65.25 (9.4%) mV)approximately
doublewhen compared to the MW1 or MW2 samples. Sim-
ilar zeta potential values have also been reported by others, for
nanotube species treated with a mixture of nitric/sulfuric acid
in order to attach carboxylic surface functional groups.[19,20]
The MW4 samples were found to have a slightly lower nega-
tive polarity (31.13 (6.2%) mV) when compared to the
MW1 and MW2. On the other hand the PEG coated samples
had the lowest negative zeta potential (10.14 (3.7%) mV)
out of all the samples. This low negative value (close to neu-
tral) is a result of the PEG coating which decreases the nega-
tive polarity of the original CNTs.[21,22]
Raman spectroscopy spectra of the MW1-5 samples used in
this study are shown in the SI (Figure S2, SI) and clearly indicate
the presence of the characteristic D (1326 cm1), G (1575 cm1),
and 2D (2649 cm1) bands specific to multi-walled carbon nano-
tubes. It is believed that the D and 2D bands are highly dispersive
and generally related to the degree of the nanotube crystal-
linity.[23] Furthermore, the fact that the D and G bands were
found to have a low value of half-height peak width (<40 cm1)
demonstrates the high degree of graphitization of the
MWCNTs.[23] This finding is also in good agreement with
the TGA and TEM studies. The analysis of the ratios between
the intensities of the G and D (G/D) peaks and 2D and G
(2D/G) peaks clearly showed the impact of the chemical surface
modification which affected the spectroscopic responses of the
nanotubes. MW1 is a highly crystalline sample with a G/D ratio
of 1.12, but, as the nanotubes were exposed to various purifica-
tion treatments and oxidation (MW2 and MW3), G/D decreased
to 0.81 and 0.64, respectively. Nanotube oxidation is known to
reduce the G/D and the 2D/G ratios in the Raman spectra.[16]
To determine the physiological response of plants
upon application of carbon nanotubes with specific surface
small 2012, 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/smll.201102661
properties and levels of agglomeration, the germination and
growth of tomato plants was monitored for seeds germinated
and plants grown on Murashige and Skoog medium (MS) sup-
plemented with five types of MWCNTs (Figure 2). We found
a strong correlation between the physiological responses of
plants and the surface chemistry of the CNTs, as well as the dis-
persion/purity of the nanotubes. Our findings highlight the most
significant increase in the germination rates (P < 0.001) for the
seeds exposed to the MW2 and MW3 samples, which were very
well-dispersed and possessed strong negative surface polarities.
This observation can be further verified by the enhanced germi-
nation of the seeds exposed to the MW5 samples, given the fact
that PEG is known to generate low toxicity and good disper-
sion of nanotubes.[18,24] Interestingly, the germination rate was
found to be higher for the MW2 sample compared with that of
MW1 (Figure 2A). It cannot be dismissed that the presence of
metallic nanoparticles (Fe, Co, or their oxides) that were not
completely eliminated by purification in sample MW1 (low
purified sample, low dispersed) could have had a toxic effect on
the germination process. A number of other studies have indi-
cated that the onset of toxic effects in several plant models is
due to exposure to a variety of metallic/metal oxide nanoparti-
cles.[10,25] Also, the fact that the nanotubes were agglomerated
and formed relatively larger clusters lowered their capacity for
being taken up by the plant systems and therefore impacted the
plants physiological responses. As a result, we hypothesized
that, for the MW1 sample, the lack of increased biomass could
be explained based on the agglomeration of the nanotubes and
possibly the presence of metallic impurities. A similar trend was
observed for the development of tomato seedlings germinated
on nanotube-containing medium (Figure 2BD). The tomato
plants grown on medium supplemented with MW2 and MW3
exhibited the most active growth compared with control plants
and plants exposed to the other types of MWCNTs. The plants
grown on medium supplemented with MW1 did not show any
statistically significant differences in growth compared with
plants grown on regular medium (control). After 4 weeks of
exposure of tomato plants to different types of MWCNTs,
the total fresh biomass of plants exposed to MW2 and MW3
was twice as high as that of the control plants (Figure 2C,D).
A statistically significant but much lower increase (P < 0.05)
in fresh biomass was documented for plants exposed to MW4
and MW5 compared with control plants or plants exposed to
MW1. Interestingly, the plants exposed to the acetone-treated
nanotubes (MW4) had a much lower development rate as com-
pared with the plants exposed to MW2 and MW3, which could
be explained by the hydrophobic surface characteristics of the
nanotubes decorated with acetone and low energy functional
groups CH2,3.[17] These findings highlight an almost linear vari-
ation of the total fresh biomass of the plants exposed to var-
ious species of MWCNTs relative to control and the negative
surface charge values of the corresponding nanotube samples
(Figure S3, SI). Given the variation in dispersion, the MW1
samples were not considered in these studies. We found that
the more negatively charged nanotubes induced a more signifi-
cant increase in the fresh biomass of the exposed plants after
both 1 and 4 weeks. These findings clearly indicate a correlation
between the surface polarity of the carbon nanotubes and the
physiological responses that they induced in plants.
www.small-journal.com 3
 communications H. Villagarcia et al.

(A) water transport.[12] In particular, we dem-

onstrated that MWCNTs can activate the
90 control expression of the gene that encodes the
** **
80
% of seed germination MW1 water channel protein (LeAqp1). Based
* MW2 on these data, we formulated a hypothesis
70
MW3 regarding the activation of the growth
60 MW4
of plants exposed to MWCNTs through
50 MW5
MWCNT-dependent activation of the
40
production of the water channel protein
30 (aquaporin). The water channel protein is
20 one of the key proteins for plant develop-
10 ment and is involved in the process of seed
0 germination and root water uptake.[26]
types of medium Aharon et al. have demonstrated that the
over-expression of the Arabidopsis gene
of the water channel protein in tobacco
plants resulted in significant activation of
their growth and photosynthetic perform-
ance.[27] Here, to test our hypothesis, we
investigated the effect of MWCNTs with
different surface chemistry (MW1MW5
samples) on the expression of LeAqp1
protein (Figure 3).
We found an interesting correla-
tion between the type of MWCNTs, the
observed phenotype of tomato plants
exposed to MWCNTs, and the produc-
tion of aquaporin (LeAqp1) in exposed
(D) plants. A significantly higher produc-
tion of LeAqp1 protein was detected in
200 ** ** control
plants grown on medium supplemented
180 MW1
with MWCNTs that were well-dispersed
Total fresh weight, mg

160 MW2
MW3
in MS medium (MW2 sample) or with
140
* * MWCNTs decorated with different func-
120 * * MW4
100 MW5 tional groups (MW3MW5) compared
80
with plants not exposed to MWCNTs
60 (control) and plants exposed to poorly
40 dispersed MWCNTs (Figure 3B).
20 Such a correlation was observed for
0 the two time points used (8 days and
o ne w eek four w eeks 41 days of incubation of tomato plants).
This newly documented effect of different
duration of incubation
types of MWCNTs on the water channel
Figure 2. Correlation between surface properties of carbon nanotubes and phenotype of protein is in good correspondence with
tomato seedlings exposed to MWCNTs. A) Effect of surface properties of multi-walled carbon the intensity of the growth of tomato
nanotubes on germination of tomato seeds. The percentage of germination was calculated for
plants exposed to MW1MW5 carbon
seeds exposed to medium without MWCNTs (control) and medium supplemented with different
types of MWCNTs (MW1MW5 samples) after 5 days of incubation. 100 tomato seeds were
nanotubes (Figure 3A). Plants grown on
used for each treatment. The experiment was repeated twice. Bars represent the standard error medium supplemented with MW2MW5
(SE). B,C) Phenotype of tomato seedlings grown on standard MS medium (control) and MS carbon nanotubes exhibited higher
medium supplemented with 40 g/mL of MWCNTs functionalized with different groups (MW1 growth than control plants or plants
MW5 samples) after 1 week (B) and 4 weeks (C) of incubation. D) Weight of total fresh biomass exposed to poorly dispersed MWCNTs.
of tomato seedlings grown on standard MS medium (control) and MS medium supplemented These data support our hypothesis about
with 40 g/mL of MWCNTs functionalized with different groups (MW1MW5 samples) after
the involvement of MWCNTs in the acti-
1 week and 4 weeks of incubation. Results are shown as average SE of measurements of
30 plants per each condition. Statistical differences for A and D were determined using a T-test. vation of the production of water channel
denotes significant at P < 0.05, denotes significant at P < 0.001. proteins.
Based on all of these biological
Recently, we demonstrated that the exposure of tomato observations, it can be clearly seen that the same family
plants to multi-walled carbon nanotubes can affect the of nanotubes but with different surface chemistries can
expression of genes that are essential for stress signaling and induce statistically different physiological responses in plant

4 www.small-journal.com 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim small 2012,
DOI: 10.1002/smll.201102661
Carbon Nanotubes Impact on the Water Channel Protein in Tomato Plants

MWCNTs (MW5) seemed to have a

strong effect on the initial seed germina-
tion but a much lower one on the plant
growth. More studies are required to fully
understand why the negatively charged
nanotubes have such a drastic effect on
plant biology and how these experimen-
tally observed findings can be correlated
with the uptake/distribution of the nano-
tubes in the plant system.
In conclusion, we have demonstrated
that specific properties of carbon nano-
tubes, such as their level of agglomeration
in the medium and their surface character-
istics (type of functional groups), can be
critical for the physiological response of
plants upon application of carbon nano-
tubes. Particularly, the correlation between
the level of aggregation, the type of func-
tional group on the surface of the carbon
nanotubes, and the growth perform-
ance of tomato plants was documented.
The highest increase in plant growth
was observed for plants exposed to well-
dispersed MWCNTs and MWCNTs func-
tionalized with stronger negative groups.
Figure 3. Correlation between phenotype of tomato plants exposed to carbon nanotubes The production of tomato water channel
functionalized with different groups and expression of water channel protein (LeAqp1). protein was activated in plants exposed
A) Phenotype of tomato plants grown on standard MS medium (control) and MS medium
to MWCNTs functionalized with various
supplemented with 40 g/mL of MWCNTs functionalized with different groups (MW1MW5
samples) after 41 days of incubation. B) Analysis of the expression of tomato water channel groups compared with unexposed plants
protein (LeAqp1) by Western blot after 8 and 41 days of incubation of tomato plants grown or plants exposed to poorly dispersed and
on standard MS medium (control) and MS medium supplemented with 40 g/mL of MWCNTs highly aggregated MWCNTs. This obser-
functionalized with different groups (MW1MW5 samples). vation supports our working hypothesis[11]
regarding the activation of the germination
models (both seeds and seedlings). We have proved previ- and growth of plants exposed to carbon nanotubes through
ously[11] that the structure and morphology of the graphitic MWCNT-dependent regulation of the water channel protein.
nanomaterials can drastically impact the development of
tomato plants, with the nanotubes having more impact than
graphene sheets. This work highlights the role that the sur- Experimental Section
face functionalization of the MWCNTs has on the devel-
opmental effects they induce in tomato models. It can be Synthesis, Functionalization, and Characterization of Multi-
concluded that the negatively charged nanotubes have a sig- walled Carbon Nanotubes used for Biological Experiments: MWCNTs
nificant impact on both seed germination and plant growth were synthesized on a Fe:Co:CaCO3 catalyst system utilizing radio
and in the limits used for this study an almost linear varia- frequency (RF) catalytic chemical vapor deposition (cCVD). This
tion was found between the nanotubes negative zeta poten- bimetallic catalyst with the following stoichiometric composition
tial and the corresponding increase in the fresh biomass. This 2.5:2.5:95 wt.% was prepared as previously described.[15,30] For
can possibly be explained by better uptake of the negatively the nanotube synthesis, approximately 100 mg of the Fe:Co:CaCO3
charged nanotubes by the plants and a better dispersion catalyst were deposited on a graphite boat, which was then placed
into various parts of the plants. The quantification of the inside of a quartz tube. The latter was positioned at the center
nanotube uptake and biodistribution is still a challenging of the RF generator and flushed with nitrogen at 200 mL/min for
process owing to limitations in the ability to precisely 10 min. Next, the generator was turned on, and, once the tempera-
detect, both qualitatively and quantitatively, the nano- ture of the catalyst reached 720 C, acetylene was flown at 4.3 mL/
tubes in the various tissues. Transmission electron micros- min for 30 min. At the end of the reaction, the sample was allowed
copy,[11] Raman spectroscopy,[11,12,28] and photothermal/ to cool under nitrogen and collected for purification and charac-
photoacoustic analysis[12,29] have been proposed for this terization.[30] Next, the MWCNTs were dried overnight at 100 C,
purpose, but all have advantages and limitations. Moreover, and the final purity was 94%. This sample was referred to as MW1.
highly dispersed nanotubes (single or few) have been shown To improve the purity of the sample and to dissolve the rest of the
to affect plant physiology more than agglomerated ones or catalyst nanoparticles, which were entrapped in the nanotube
those of low purity (MW1). Interestingly, the PEG-coated structures and could not be removed after the first purification,

small 2012, 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-journal.com 5
DOI: 10.1002/smll.201102661
 communications
the samples were purified again with hydrochloric acid. Before the
nanotubes were immersed in the acid solution, they were heated to
450 C in order to burn the amorphous carbon and to uncover the
remaining metal nanoparticles exposing them to acid to achieve
a final purity of 98%. This sample was referred to as MW2. Next,
the MWCNTs were functionalized with COOH groups in order to
achieve better dispersion in an aqueous solution. The nanotubes
were immersed in a mixture of H2SO4: HNO3 (3:1), sonicated for
4 h, and continuously washed with DI water. Finally, the sample
was dried overnight and designated as MW3. A separate batch of
the purified MWCNTs (40 mg) was mixed in acetone (520 mL) and
strongly sonicated with a tip sonicator for 1 h. Next, the solution
was bath sonicated for 2 h and air dried. This sample was referred
to as MW4. Furthermore, the purified MWCNTs were noncovalently
functionalized with PEG (weight-averaged molecular weight, Mw =
7000). First, 2.5 g of PEG was dissolved in 500 mL of DI water, and
100 mg of MWCNTs were added to the solution and bath sonicated
for 2 h. Finally, the PEG-functionalized nanotubes were washed
with DI water and oven dried for 12 h at 50 C. This sample was
referred to as MW5. Subsequently, the MW1 sample was mixed
into the Murashige and Skoog (MS) medium by mechanical stir-
ring and was not sonicated in order to produce large agglomerates
with poor dispersion. The rest of the MWCNT samples (MW2-5)
were separately dispersed in medium at a concentration of
40 g/mL by tip sonication. The JEOL field-emission transmis-
sion electron microscope (Model JEM-2100F) was used to collect
high- and low-resolution images of the nanotubes. First, the sam-
ples were dissolved in isopropanol and gently sonicated for 1 h to
achieve a homogenous dispersion. Next, a few drops of each sus-
pension were deposited on holey-carbon-coated TEM grids, which
were air-dried before imaging. SEM images were obtained using
a field emission scanning electron microscope, JSM-7000F (JEOL
USA, Inc). Before analysis, the MWCNTs were deposited on double-
sided carbon tape attached to aluminum pins. The accelerating
voltage was set at 15 kV and the working distance at ~610 mm.
TGA were performed in compressed air at a flow rate of 150 mL/
min using the Mettler Toledo 815e system. The mass of all of the
samples was kept constant at approximately 4 mg in order to mini-
mize the effects of variability in measurements.[31] Each sample
was heated from room temperature to 850 C at 5 deg/min. The
Horiba Jobin Yvon LabRam HR800 was used to perform the Raman
studies of the nanotubes. The measurements were taken at room
temperature using a He-Ne laser (633 nm) as the excitation source.
The laser beam intensity measured at the sample was kept at
5 mW, and the Raman shifts were calibrated with a silicon wafer
at a peak of 521 cm1 as described earlier.[15] The zeta potential
studies of the different surface modified MWCNTs were performed
using the Zetasizer Nano ZS from Malvern Instruments equipped
with a dynamic light scattering technique. Each sample was dis-
persed in an aqueous solution as previously described.[18] Prior to
the surface charge measurements, about 5 mL from each homoge-
nous MWCNT dispersion were inserted into a zeta potential cell. The
zeta potential values for each sample represent an average of
50 measurements.
Experiments with Tomato Plants: The tomato seeds (cv. Micro-
Tom) were surface sterilized as described previously.[11] Sterile
seeds (100 seeds per treatment) were placed on control medium
(Murashige and Skoog medium) or same medium supplemented with
6 www.small-journal.com 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
H. Villagarcia et al.
different types of multi-walled carbon nanotubes (MW1, MW2, MW3,
MW4, and MW5). All used MWCNTs were added to the medium in
the same concentration (40 g/mL). The different levels of agglomer-
ation of low-dispersed MWCNTs (MW1) and high-dispersed MWCNTs
(MW2) in agar medium are presented on Figure S4 (SI).
Experiments were repeated 3 times. After 5 days of incubation
of seeds on medium, the percentage of seeds that germinated was
calculated for the total number of seeds (100%) placed under con-
trol conditions (regular MS medium) and under each experimental
condition (MS medium supplemented with MW1MW5 carbon
nanotubes). Seeds that produced embryonic root were recognized
as germinated. Development of tomato seedlings grown on 6 types
of medium was monitored and documented by photographs each
week. The total fresh weight was measured for one-week-old and
four-week-old tomato seedlings. Statistical differences were deter-
mined using a two-way analysis of variance with unexposed seeds/
seedlings (control) and seeds/seedlings exposed to different types
of carbon nanotubes.
Immunoblot Analysis: For analysis of the production of tomato
water channel (aquaporin) protein (LeAqp1), total proteins were
extracted from roots of tomato plants grown in vitro on regular
MS medium, MS medium supplemented with low dispersed
MWCNTs (MW1), or MWCNTs functionalized with different groups
(MW2MW5) at two different time points of incubation (8 days and
41 days) using Plant Total Protein Extraction Kit (Sigma-Aldrich, Inc.
St. Louis, MO). The analysis of the production of aquaporin pro-
tein in tomato roots was performed using anti-peptide antibodies
provided by Pacific Immunology, Inc. (Ramona, CA). Antibodies
were designed and produced against tomato aquaporin peptide
sequence DAKRNARDSHV. Standard techniques for Western blot
analysis[32] were used for the detection of tomato water channel
protein (LeAqp1) in tomato plants exposed and unexposed to dif-
ferent types of MWCNTs. Briefly, for each time point, 20 g of total
protein were separated by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) followed by Western analysis using
affinity purified anti-LeAqp1 primary and anti-rabbit IgG horse-
radish peroxidase secondary antibodies.
Supporting Information
Supporting information is available from the Wiley Online Library
or from the author. It includes the thermogravimetric analysis of
the nanotube species used in this study (MW1 and MW2) along
with their corresponding Raman spectra (MW1MW5). The varia-
tion of the fresh biomass of the plants exposed to CNTs (relative to
control) as a function of the surface charge of the nanotubes used
for each study is also presented.
Acknowledgements
Funding from EPSCoR-NSF-P3 Center (Grant P3-202 to MVK) and
ASTA (grant # 08-CAT-03 to ASB) is highly appreciated. The edito-
rial assistance of Dr. Marinelle Ringer is also acknowledged.
small 2012,
DOI: 10.1002/smll.201102661
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Received: December 16, 2011
Revised: January 29, 2012
Published online:
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