# Single-Walled Carbon Nanotubes as Fluorescence Biosensors for Pathogen Recognition in Water Systems

Authors: Venkata K. K. Upadhyayula; Soumitra Ghoshroy; Vinod S. Nair; Geoffrey B. Smith; Martha C. Mitchell; Shuguang Deng
Journal: Research Letters in Nanotechnology
 (2008)

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## Abstract

The possibility of using single-walled carbon nanotubes (SWCNTs) aggregates as fluorescence sensors for pathogen recognition in drinking water treatment applications has been studied. Batch adsorption study is conducted to adsorb large concentrations ofStaphylococcus aureus aureus SH 1000 and Escherichia coli pKV-11 on single-walled carbon nanotubes. Subsequently the immobilized bacteria are detected with confocal microscopy by coating the nanotubes with fluorescence emitting antibodies. The Freundlich adsorption equilibrium constant
(k) for S.aureus and E.coli determined from batch adsorption study was found to be 9×108 and 2×108 ml/g, respectively. The visualization of bacterial cells adsorbed on fluorescently modified carbon nanotubes is also clearly seen. The results indicate that hydrophobic single-walled carbon nanotubes have excellent bacterial adsorption capacity and fluorescent detection capability. This is an important advancement in designing fluorescence biosensors for pathogen recognition in water systems.


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## Body

## 1. Introduction

Potential threats of contaminating
drinking water sources with biothreat agents are on high rise in recent years. In US alone, in year 2003, more
than 100 cases of actual, threatened, and disrupted plots were reported in
which 20 incidents involved actual contamination of drinking water with
biothreat agents [1]. Unfortunately, existing treatment techniques are not
designed to treat and detect biothreat contaminants since most of these
biothreat agents are colorless, odorless, tasteless, and have typical
properties such as chlorine resistance and biofilm forming tendency [1–3]. This
vulnerable situation has raised serious security concerns in many nations and
needs immediate attention. A sensing system that can simultaneously concentrate and detect biothreat contaminants from water systems at a faster speed and greater accuracy has to be developed to overcome this situation.Carbon
nanotube technology has the potential to make important advancements in water
security and protection from biothreat agents. Carbon nanotubes have high bacterial
adsorption capacity [4, 5] and are able to concentrate different types of
pathogens [4–6]. This exceptionally high adsorption capacity of nanotubes is
due to its large surface to volume ratio and high aspect ratio that imparts
high antimicrobial nature to the material [7, 8]. Another major advantage of
using carbon nanotubes for water treatment applications is that nanotubes, if
functionalized, have the ability to detect pathogens. There are multiple
possibilities for surface modification [9–14] of carbon nanotubes, but functionalization
with antibodies has gained significant importance in recent years. Antibodies
not only provide the required biocompatibility [11] but also alter the
electrical and optical properties of nanotubes and make their surface sensitive
to surroundings [11, 13, 14]. To the best of our knowledge, two research groups
used antigen-antibody approach to detect pathogens using carbon nanotubes
[11, 12]. Both groups have attempted to enhance the hydrophilicity of carbon
nanotubes, since the bacterial binding capacity of hydrophilic nanotubes is
higher than hydrophobic nanotubes [11, 12]. This is true but when it is desired
to use carbon nanotube adsorbent media to simultaneously concentrate and detect
pathogens from the flowing stream of water, it is desirable to have hydrophobic
nanotubes from a practical standpoint.The
present study is initiated to evaluate the capability of hydrophobic carbon
nanotubes to concentrate microorganisms and subsequently detect them when
coated with fluorescent antibodies. The microorganisms chosen for this study
areS.aureus and E.coli. In this
report, we present the results of our batch adsorption study and confocal
microscopy analysis of S.aureus and E.coli immobilization on single-walled carbon
nanotubes aggregates.


## 2. Materials and Methods

The
single-walled carbon nanotubes samples (SWCNTs) (AP-Grade) purchased from
Carbolex, Inc. (Lexington, KY, USA)
were used for the study. These carbon nanotubes are 80% pure and have an
average diameter of 1.4 nanometers and lengths of 2–5μm. Bacterial cultures of E.coli pKV-11 and S.aureus SH1000 are obtained from
Professor Nishiguchi and Professor Gustafson of Biology Department of NMSU. E.coli pKV-11 has intrinsic green
fluorescent protein (GFP) and exhibits fluorescence under confocal microscopy. S.aureus strain is an opportunistic
human pathogen and care is taken while handling.


### 2.1. Batch Adsorption Study

Shaker experiments are performed to
determine the adsorption equilibrium concentration ofE.coli and S.aureus bacteria
on single-walled carbon nanotubes. For this experiment, fresh cultures of
bacterial solutions of E.coli and S.aureus were prepared from their
respective stock cultures. The cultures were grown overnight in tryptic soy
broth at 37°C incubator. Then 1 mL aliquot of this solution is taken in 1.5 mL
eppendorf tubes and centrifuged for 2
minutes. The supernatant broth solution
is removed and the bacterial pellet at the bottom of the tubes is suspended in
0.85% saline solution and vortexed to obtain uniform suspension of the
solution. The solution is again centrifuged and resuspended in distilled water.
The bacterial solutions were now ready for shaker experiments.100 mL of distilled and autoclaved water
each are taken in four conical flasks (duplicate sets), and 0.1 grams of single walled carbon nanotubes is
added to each flask. To this mixture, bacterial solutions ofS.aureus are added to the two flasks,
and E.coli is added to another two
flasks. The initial concentration (C0) of S.aureus was 2.8×108 and 2.7×108 CFU/mL and E.coli pKV-11 in the suspension was
found to be 1.62×108 and 1.7×108 CFU/mL, respectively.
The flasks are then placed on a mechanical shaker and are subjected to shaking
at 6000 rpm under room temperature. The contents of the flask are shaken for 60 minutes and 1 mL of supernatant solution is drawn from each flask and collected in an eppendorf tube.The supernatant solutions were then filtered  via 3μm polycarbonate filter paper
(Millipore, Mass, USA).
100μL of this filtrate are taken and used to prepare dilutions ranging from 10−1 to 10−5. Then 20μL of sample volume is drawn out from each dilution
and inoculated on agar plates. McKonkey agar plates are used of E.coli pKV-11 cells and Mannitol salt
agar for S.aureus cells,
respectively. These two agar media selective grow the respective bacterial
colonies and inhibit the growth of other bacteria. The number of colonies
enumerated was counted according to the following equation: (1)numberofcolonies[CFUmL]=numberofcoloniesforeachdilution(dilutionfactor)/(samplevolume).The values at each different dilution are averaged to obtain
final concentration for two samples. The experiment is repeated four more times with four different initial concentrations. The initial concentrations used forE.coli were 2.0×107, 6.66×107, 3.33×108, and 4.44×108 CFU/mL. The initial concentrations used for S.auerus were 6×107, 1.44×108, 4.4×108, and 7.0∗108
CFU/mL, respectively.


### 2.2. Confocal Microscopy Analysis

#### 2.2.1. E.coli pKV-11

After running the shaker experiment for the first time, one mL of
uniformly suspended solution (not the supernatant) is taken in 1.5 mL eppendorf
tubs and allowed to settle for 15
minutes. After 15
minutes, the supernatant in
the tube is slowly removed using glass pipette without disturbing the
suspension at the bottom. Then 1 mL of 0.1 M phosphate buffer was added and the
contents were spun in a microfuge for 30
minutes. The spinning step was
succeeded by five times washing with 0.1 M phosphate buffer solution. After
washing, 1μL of Alexa 647 (Invitrogen,
Calif, USA)
fluorescent dye solution was prepared in the 200μL of 0.1 M phosphate buffer
solution and added to nanotube-bacteria mixture. The contents were refrigerated
for one hour and removed, washed again with phosphate buffer to remove excess
fluorescent dye and is subjected for analysis under confocal microscope. For
actual imaging, the pellet was resuspended in buffer and 10μL of sample were
taken on a 25 mm glass slide (with cover slip placed) and is imaged under
Olympus Fluoview FV1000 confocal microscope (Olympus America Inc, Pa, USA).
Alexa dye will fluoresce nanotubes, while E.coli pKV-11 species already has intrinsic fluorescence protein.


#### 2.2.2. S.aureus SH-1000

A procedure similar to the above was
followed for confocal microscopic study of immobilization ofS.aureus SH-1000 strain on nanotubes but
with slight modifications. The supernatant from the eppendorf tube is removed
using glass pipette and bacterial cells were fixed in 1% phosphate buffered
formaldehyde solution, refrigerated for three hours and washed with 0.1 M
phosphate buffer for five times. Then, S.aureus primary antibody solution in phosphate buffer (1μL : 100μL) is added to the cells
so as to conjugate the primary antibody specific to the S.aureus cell wall surface. The sample was refrigerated and the
cells were kept in suspension of primary antibody solution for about 16 hours.
The sample was removed and 1μL of FITC-Goat Anti Rabbit (Jackson Immuno
Research Laboratories, Inc., Pa, USA) fluorescent dye solution is prepared
in 100μL of 0.1 M phosphate buffer (1μL : 100μL) and added to the cells. The
sample was again refrigerated to allow the conjugation of secondary dye
solution to the primary antibody associated with S.aureus cells. Finally to this mixture, Cy-5 secondary antibody
solution (Invitrogen, Calif, USA)
prepared in phosphate buffer was (1μL : 100μL) added. Cy-5 fluoresces single-walled
carbon nanotubes at a different wavelength region other than S.aureus cells. For imaging, 10μL of
this sample was used for imaging under Zeiss LSM510META microscope (Carl Zeiss Micro
Imaging Inc., NY, USA).A control sample of pure single-walled
carbon nanotubes labeled with Alexa 647 dye is also prepared and imaged under
Olympus Fluoview FV1000 confocal microscope.


## 2.1. Batch Adsorption Study

Shaker experiments are performed to
determine the adsorption equilibrium concentration ofE.coli and S.aureus bacteria
on single-walled carbon nanotubes. For this experiment, fresh cultures of
bacterial solutions of E.coli and S.aureus were prepared from their
respective stock cultures. The cultures were grown overnight in tryptic soy
broth at 37°C incubator. Then 1 mL aliquot of this solution is taken in 1.5 mL
eppendorf tubes and centrifuged for 2
minutes. The supernatant broth solution
is removed and the bacterial pellet at the bottom of the tubes is suspended in
0.85% saline solution and vortexed to obtain uniform suspension of the
solution. The solution is again centrifuged and resuspended in distilled water.
The bacterial solutions were now ready for shaker experiments.100 mL of distilled and autoclaved water
each are taken in four conical flasks (duplicate sets), and 0.1 grams of single walled carbon nanotubes is
added to each flask. To this mixture, bacterial solutions ofS.aureus are added to the two flasks,
and E.coli is added to another two
flasks. The initial concentration (C0) of S.aureus was 2.8×108 and 2.7×108 CFU/mL and E.coli pKV-11 in the suspension was
found to be 1.62×108 and 1.7×108 CFU/mL, respectively.
The flasks are then placed on a mechanical shaker and are subjected to shaking
at 6000 rpm under room temperature. The contents of the flask are shaken for 60 minutes and 1 mL of supernatant solution is drawn from each flask and collected in an eppendorf tube.The supernatant solutions were then filtered  via 3μm polycarbonate filter paper
(Millipore, Mass, USA).
100μL of this filtrate are taken and used to prepare dilutions ranging from 10−1 to 10−5. Then 20μL of sample volume is drawn out from each dilution
and inoculated on agar plates. McKonkey agar plates are used of E.coli pKV-11 cells and Mannitol salt
agar for S.aureus cells,
respectively. These two agar media selective grow the respective bacterial
colonies and inhibit the growth of other bacteria. The number of colonies
enumerated was counted according to the following equation: (1)numberofcolonies[CFUmL]=numberofcoloniesforeachdilution(dilutionfactor)/(samplevolume).The values at each different dilution are averaged to obtain
final concentration for two samples. The experiment is repeated four more times with four different initial concentrations. The initial concentrations used forE.coli were 2.0×107, 6.66×107, 3.33×108, and 4.44×108 CFU/mL. The initial concentrations used for S.auerus were 6×107, 1.44×108, 4.4×108, and 7.0∗108
CFU/mL, respectively.


## 2.2. Confocal Microscopy Analysis

### 2.2.1. E.coli pKV-11

After running the shaker experiment for the first time, one mL of
uniformly suspended solution (not the supernatant) is taken in 1.5 mL eppendorf
tubs and allowed to settle for 15
minutes. After 15
minutes, the supernatant in
the tube is slowly removed using glass pipette without disturbing the
suspension at the bottom. Then 1 mL of 0.1 M phosphate buffer was added and the
contents were spun in a microfuge for 30
minutes. The spinning step was
succeeded by five times washing with 0.1 M phosphate buffer solution. After
washing, 1μL of Alexa 647 (Invitrogen,
Calif, USA)
fluorescent dye solution was prepared in the 200μL of 0.1 M phosphate buffer
solution and added to nanotube-bacteria mixture. The contents were refrigerated
for one hour and removed, washed again with phosphate buffer to remove excess
fluorescent dye and is subjected for analysis under confocal microscope. For
actual imaging, the pellet was resuspended in buffer and 10μL of sample were
taken on a 25 mm glass slide (with cover slip placed) and is imaged under
Olympus Fluoview FV1000 confocal microscope (Olympus America Inc, Pa, USA).
Alexa dye will fluoresce nanotubes, while E.coli pKV-11 species already has intrinsic fluorescence protein.


### 2.2.2. S.aureus SH-1000

A procedure similar to the above was
followed for confocal microscopic study of immobilization ofS.aureus SH-1000 strain on nanotubes but
with slight modifications. The supernatant from the eppendorf tube is removed
using glass pipette and bacterial cells were fixed in 1% phosphate buffered
formaldehyde solution, refrigerated for three hours and washed with 0.1 M
phosphate buffer for five times. Then, S.aureus primary antibody solution in phosphate buffer (1μL : 100μL) is added to the cells
so as to conjugate the primary antibody specific to the S.aureus cell wall surface. The sample was refrigerated and the
cells were kept in suspension of primary antibody solution for about 16 hours.
The sample was removed and 1μL of FITC-Goat Anti Rabbit (Jackson Immuno
Research Laboratories, Inc., Pa, USA) fluorescent dye solution is prepared
in 100μL of 0.1 M phosphate buffer (1μL : 100μL) and added to the cells. The
sample was again refrigerated to allow the conjugation of secondary dye
solution to the primary antibody associated with S.aureus cells. Finally to this mixture, Cy-5 secondary antibody
solution (Invitrogen, Calif, USA)
prepared in phosphate buffer was (1μL : 100μL) added. Cy-5 fluoresces single-walled
carbon nanotubes at a different wavelength region other than S.aureus cells. For imaging, 10μL of
this sample was used for imaging under Zeiss LSM510META microscope (Carl Zeiss Micro
Imaging Inc., NY, USA).A control sample of pure single-walled
carbon nanotubes labeled with Alexa 647 dye is also prepared and imaged under
Olympus Fluoview FV1000 confocal microscope.


## 2.2.1. E.coli pKV-11

After running the shaker experiment for the first time, one mL of
uniformly suspended solution (not the supernatant) is taken in 1.5 mL eppendorf
tubs and allowed to settle for 15
minutes. After 15
minutes, the supernatant in
the tube is slowly removed using glass pipette without disturbing the
suspension at the bottom. Then 1 mL of 0.1 M phosphate buffer was added and the
contents were spun in a microfuge for 30
minutes. The spinning step was
succeeded by five times washing with 0.1 M phosphate buffer solution. After
washing, 1μL of Alexa 647 (Invitrogen,
Calif, USA)
fluorescent dye solution was prepared in the 200μL of 0.1 M phosphate buffer
solution and added to nanotube-bacteria mixture. The contents were refrigerated
for one hour and removed, washed again with phosphate buffer to remove excess
fluorescent dye and is subjected for analysis under confocal microscope. For
actual imaging, the pellet was resuspended in buffer and 10μL of sample were
taken on a 25 mm glass slide (with cover slip placed) and is imaged under
Olympus Fluoview FV1000 confocal microscope (Olympus America Inc, Pa, USA).
Alexa dye will fluoresce nanotubes, while E.coli pKV-11 species already has intrinsic fluorescence protein.


## 2.2.2. S.aureus SH-1000

A procedure similar to the above was
followed for confocal microscopic study of immobilization ofS.aureus SH-1000 strain on nanotubes but
with slight modifications. The supernatant from the eppendorf tube is removed
using glass pipette and bacterial cells were fixed in 1% phosphate buffered
formaldehyde solution, refrigerated for three hours and washed with 0.1 M
phosphate buffer for five times. Then, S.aureus primary antibody solution in phosphate buffer (1μL : 100μL) is added to the cells
so as to conjugate the primary antibody specific to the S.aureus cell wall surface. The sample was refrigerated and the
cells were kept in suspension of primary antibody solution for about 16 hours.
The sample was removed and 1μL of FITC-Goat Anti Rabbit (Jackson Immuno
Research Laboratories, Inc., Pa, USA) fluorescent dye solution is prepared
in 100μL of 0.1 M phosphate buffer (1μL : 100μL) and added to the cells. The
sample was again refrigerated to allow the conjugation of secondary dye
solution to the primary antibody associated with S.aureus cells. Finally to this mixture, Cy-5 secondary antibody
solution (Invitrogen, Calif, USA)
prepared in phosphate buffer was (1μL : 100μL) added. Cy-5 fluoresces single-walled
carbon nanotubes at a different wavelength region other than S.aureus cells. For imaging, 10μL of
this sample was used for imaging under Zeiss LSM510META microscope (Carl Zeiss Micro
Imaging Inc., NY, USA).A control sample of pure single-walled
carbon nanotubes labeled with Alexa 647 dye is also prepared and imaged under
Olympus Fluoview FV1000 confocal microscope.


## 3. Results and Discussion

The
final bacterial concentration of the solution (Ce)
measured after 60
minutes is treated as the adsorption equilibrium
concentration at which the maximum adsorbed amount can be calculated according
to the following equation. The equilibrium point was determined by adsorption
kinetics data reported elsewhere [4]: (2)Qmax=[C0VF−CeVS]m, where
Qmax is the maximum amount of bacteria adsorbed on a given adsorbent
at Ce (CFU/g), C0
is the initial concentration of the bacteria in the feed solution (CFU/mL), Ce is adsorption equilibrium
concentration of bacteria in solution (CFU/mL), VF is volume of the
feed solution (mL), Vs is volume of supernatant solution (mL), and m
is the mass of the adsorbent (g).From
the maximum adsorbed amount obtained (Qmax) and adsorption
equilibrium concentration (
Ce
), the Freundlich adsorption model (3) was used to
correlate  the adsorption equilibrium data: (3)Qmax=kCe1/n, where
k (mL/g) is the Freundlich adsorption equilibrium constant that depends on
temperature and pH of the feedwater, and n is a constant accounting for the potential
energy uniformity of the adsorbent surface. Typically, the “k” value is related
to adsorption affinity of the adsorbent to the adsorbate. A large k value suggests
a higher adsorption affinity of the adsorbent toward the adsorbate. The adsorption
isotherms for S.aureus SH1000 and E.coli pKV-11 on the single-walled
carbon nanotubes are plotted in Figure 1. The Freundlich equation constants are
k=9×108,n=1.85 for S.aureus SH1000 and k=2×108,n=2.39E.coli pKV-11. The higher k value for S.aureus SH1000 than that for E.coli pKV-11
suggests a higher affinity for S.aureus. The
high Freundlich adsorption equilibrium constants of these bacteria on
single-walled carbon nanotubes might be due to their fibrous mesoporous
structure that provides accessible external surface area for the adsorption. In
addition, high antimicrobial nature of nanotubes also contributes toward high
adsorption capacity. The ratio of k values can be used to calculate the
adsorption selectivity. A high selectivity between S.aureus and E. coli was
obtained on the single-walled carbon nanotubes.Figure 1
Adsorption isotherms ofS.aureus and E.coli on carbon nanotubes.S.aureus is a cocci-shaped
bacterium with an average cell diameter being less than or equal to the
mesopores of single-walled carbon nanotubes [4]. Due to this reason, the
adsorption affinity of S.aureus is
significantly higher than E.coli.
Adsorption kinetics and equilibrium of S.aureus and E.coli on SWCNTs are reported in
much detail in our previous study [4] in which it is clearly observed that S.aureus not only has high adsorption
affinity at wide range of initial concentrations. The adsorption kinetics of E.coli is fast but the kinetics of S.aureus is faster even at high
concentrations [4]. This is good from a biosensor standpoint because an
effective design of biosensor depends on speed of concentration of biothreat
agents and their selectivity, when multiple species are present. The
confocal images confirm the adsorption affinity data discussed above.The description about Figures2, 3, and
4 is given in this paragraph.  Although there appear some free cells, the
association of bacteria with SWCNTs is visible. No functional groups are
attached to enhance the hydrophilicty of carbon nanotubes. A small amount of
fluorescent antibodies is made to immobilize nonspecifically on carbon
nanotubes. Yet, the detection under confocal microscopy is clearly visible.Figure 2
Confocal microscopy image of pure single-walled carbon nanotube aggregates.Figure 3
Confocal image ofE.coli pKV-11 (violet rod shaped) cells immobilized on
single-walled carbon nanotube aggregates (blue color).Figure 4
Confocal image ofS.aureus (green dots in the image) cells immobilized on
single-walled carbon nanotube aggregates (orange color in the image).


## 4. Conclusion

It was demonstrated that unmodified,
hydrophobic single-walled carbon nanotubes have a very high adsorption affinity
toward bothE.coli and S.aureus bacterial cells. The adsorption
capacity of S.aureus is significantly
higher than that of E.coli on the
single-walled carbon nanotubes. The Freundlich adsorption model can correlate
the adsorption isotherm data well. The immobilization of the bacterial cells on
single-walled carbon nanotubes is clearly seen in the confocal images, which confirms
the adsorption equilibrium data obtained in the batch experiments. The high
adsorption capacity and high selectivity of single-walled carbon nanotubes for S.aureus suggest that the carbon nanotubes
are promising sensor materials.



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*Source: 156358-2008-06-19.xml*