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Journal of Undergraduate Research in Bioengineering

Evaluation of Quantum Dot Internalization by the Bacterium


Pseudomonas aeruginosa
Tiffany Chen,1 Hongyan Ma,2 Kristy Katzenmeyer,3 James D. Bryers3
1

Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas 78712


Department of Chemical Engineering, University of Washington, Seattle, Washington 98195
3
Department of Bioengineering, University of Washington, Seattle, Washington 98195
2

Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that often forms biofilms and causes a significant
number of nosocomial (hospital acquired) infections. Investigations of the processes governing biofilm formation,
persistence, and virulence require a new set of diagnostic tools that can be applied, non-invasively, to prevent
destroying the biofilms structure. This project seeks to explore the prospects of replacing fluorescent stains with
quantum dots (QDs); nanocrystals composed of periodic groups of II-VI, III-V, or IV-VI semiconductor materials.
Unlike traditional fluorochromatic stains, QD luminescence is photostable, with narrow emission spectra that are
size tunable, and broad absorption spectra, which allow the excitation of multiple QDs with a single wavelength.
Employing QDs to either label various bacterial species or to quantify genetic transfer between species requires
background information on the natural uptake of QDs by bacteria. Studies in internalizing high quality
poly(ethylene imine) (PEI) coated QDs using planktonic P. aeruginosa were carried out prior to future studies with
P. aeruginosa biofilms. We studied three distinct internalization methods: (1) passive bacterial incubation with
QDs, (2) CaCl2 treatment transformation, and (3) electroporation. Using epifluorescence microscopy, all three
methods appear to allow for some degree of internalization of QDs within the bacteria, with transformation
efficiencies of 3.7%, 13.9%, and 15.1%, respectively. Fluorescence scanning of supernatants and bacterial solutions
were also performed to test for the presence of the QDs. Further studies will provide supporting evidence that the
QDs are inside the bacteria, not just associated with the surface. Results will provide a link to using QDs to study
the internal mechanisms of the biofilm bacteria without photobleaching.

1. INTRODUCTION
Opportunistic pathogens pose a major health
problem in clinical settings partly due to their
ability to form biofilms. Biofilms consist of a
community of bacteria that are reversibly
adhered to a surface and excrete extracellular
polymeric
substances.
The
community
interaction along with the resilient structure of
biofilms protects these bacteria from host
defense mechanisms. The bacteria within this
community typically carry plasmids that confer
antibiotic resistance [6], allowing for an
increased spread of antibiotic resistance within
the biofilm. Additionally, these plasmids can be
transferred between different species of bacteria.
Therefore, it would be beneficial to study the
transfer of the plasmids within the biofilms to
obtain a better understanding of the increase in
antibiotic resistance seen within these bacterial
communities.
Pseudomonas aeruginosa is a bacterium that
can cause a wide variety of diseases such as
urinary tract infections, chronic lung infections,
eye infections, and soft tissue infections. About
10% of all hospital acquired infections are

caused by this bacterium [11]. Previous studies


have shown that P. aeruginosa produces more
lactamase, an antibiotic-degrading enzyme,
within biofilms as compared to the amount
produced in its planktonic form [7]. In the
present study, we focus on using planktonic P.
aeruginosa to examine in vitro imaging
techniques that can aid in the future study of
plasmid transfer within P. aeruginosa biofilms.
Internal labeling of bacteria is needed to
understand physiological microbial processes.
In vivo fluorescence imaging is most
commonly performed using organic dyes or
plasmids containing the gene for a fluorescent
protein such as green fluorescent protein (GFP).
With this method, samples can be imaged only
for a short amount of time before
photobleaching occurs, preventing an adequate
amount of time to study the samples [1].
Recently, there has been an emergence of the
use of quantum dots (QDs) for cellular imaging.
QDs are semiconductor nanocrystals with a
diameter between 2-8 nm. They have unique
properties that make them optimal for molecular
and cellular imaging, including broad
absorbance spectra and narrow emission spectra.

In addition, the specific wavelength of light


emitted by the QDs can be finely tuned by
altering the size of the QD. This allows various
colors of QDs to be used to simultaneously
image and track multiple targets within samples.
More importantly, QDs are several thousand
fold more resistant to photobleaching than
organic dyes. Due to their large molar extinction
coefficients, QDs can also be used as brighter
probes for in vivo imaging, which normally have
attenuated light intensities [2].
Several other studies have shown that
different strains of bacteria can uptake QDs
using various methods, and one even showed the
biosynthesis of cadmium sulfide nanocrystals
within E. coli [10]. Studies with eukaryotic cells
have shown internalization of QDs is achieved
via endocytosis; however, bacteria cannot
perform endocytosis. Thus, the main pathways
through the bacterial cell wall would be by
passive diffusion (through porin proteins) or
active transport (via specific receptor proteins)
[3]. Kloepfer et al. [5] studied bacterial cell
surface receptors in hopes of determining ways
of functionalizing QDs to increase their chance
of internalization. They found that QDs
functionalized with adenine and AMP were
internalized by Bacillus subtilis and E. coli via
purine-dependent mechanisms [4]. Bacterial
incubation with organic acid-stabilized QDs has
shown that there is natural uptake of the QDs
[3]. Other studies have used bacteria chemically
treated with CaCl2, which is normally used to
internalize plasmids, to transform QDs into the
cells [12].
In the present study, we investigated the
ability of planktonic P. aeruginosa to uptake
high quality QDs coated with positively charged
poly(ethylene imine) (PEI). These water-soluble
particles are created by using amphiphilic
compounds to coat the surface of the
hydrophobic QDs [2]. Natural uptake of QDs
without any special functionalized biomolecules
was tested through passive bacterial incubation.
In addition, electroporation and chemical
treatment with CaCl2 were used to increase
competence of P. aeruginosa.

Journal of Undergraduate Research in Bioengineering

2. MATERIALS AND METHODS


2.1 QDs
QDs were provided by Dr. Xiaohu Gao
(Department of Bioengineering, University of
Washington). Briefly, CdSe QDs with ZnS
shells were treated with tri-n-octylphosphine
oxide (TOPO) to stabilize the nanoparticles and
maintain their optical properties. QDs were then
coated with either poly(maleic anhydride alt
tetradecene) (PMAT), a negatively charged
polymer, or PEI, a positively charged polymer.
The charged QDs are abbreviated as QD- and
QD+ from hence forward. These QDs had
excitation/emission wavelengths of 400/545nm.
2.2 Bacterial Culture
P. aeruginosa was purchased from the American
Type Culture Collection (ATCC 27584). P.
aeruginosa was grown in Luria-Bertani (LB)
medium (Fisher Scientific, Fair Lawn, NJ) at
37C with shaking overnight. Overnight cultures
were inoculated into fresh LB medium and
grown for approximately 2 hr at 37C with
shaking until cell growth reached mid
logarithmic phase (optical density at 600 nm =
1.0).
2.3 Bacterial Incubation with QDs
Aliquots (1 ml) of cell culture were placed into
2.0 ml microcentrifuge tubes. Sterile phosphatebuffered saline (PBS; 0.8 ml) was added to the
tubes and centrifuged at 8000 rpm for 10 min at
4C. The supernatant was discarded and cell
pellet resuspended and rinsed in 1.8 ml of PBS.
The supernatant was discarded and the cell pellet
resuspended in 50l of PBS. QDs (100 nM)
were added to the suspension at varying volumes
(5 l, 10 l, and 50 l) with double distilled
water to a total volume of 50 l. The solutions
were placed at room temperature on a CELGRO Tissue Culture rotator (Lab-line, Barnstead
International, Dubuque, IA) overnight.
A second bacterial incubation experiment
was performed by removing 50 l of the
inoculated culture while still in the beginning of
log phase (~1 hr) and adding 50 l of varying
concentrations of QDs. The cells were placed
back into a 37C incubator with shaking and
allowed to grow for the remainder of the log

Journal of Undergraduate Research in Bioengineering

phase growth. After the log phase growth, the


tubes were removed and placed at room
temperature on the CEL-GRO Tissue Culture
rotator overnight.
2.4 Chemical Transformation
Culture (1 ml) was centrifuged at 4000 rpm for 6
min and the supernatant was discarded. The cell
pellet was resuspended in 150 l of ice-cold 100
mM CaCl2 and incubated on ice for 10 min.
Serial dilutions of the original 100 nM QD+, as
mentioned above, were added to the solution and
incubated on ice for 30 min. Heat shock was
performed by placing the tubes into a 42C
water bath for 90 s and then immediately placing
them on ice for 2 min. LB broth (800 l) was
then added to the tubes and incubated at 37C
with shaking for 30-60 min.
2.5 Electroporation
Electrocompetent P. aeruginosa cells were
created using a modified protocol of Sambrook
and Russell [8]. In place of 10% glycerol, 300
mM sucrose (Sigma-Aldrich, St. Louis, MO)
was used to increase electrocompetence as
described by Smith and Iglewski [9]. Cells were
flash frozen in 135 l aliquots and stored at
80C for future use.
Electrocompetent cells (40l) were placed in
chilled 1 mm gap electroporation cuvettes. The
cells were given an electrical pulse of 1.25 kV
using an electroporator (BTX ECM 399,
Genetronics, Inc.) and then 50 l of varying
concentrations of QDs were added to the
cuvette. Super Optimal Catabolite (SOC)
medium (900 l; Invitrogen, Carlsbad, CA) was
immediately added to the sample and the
solution was transferred to a microcentrifuge
tube. The cells were then incubated at 37C with
shaking for 30-60 min.
2.6 Sample Preparation for Microscopy
After incubation with QDs, cells were pelleted at
8000 rpm, 10 min, 4C. Supernatant (50 l) was
removed and placed in a UV 96-well plate
(Costar, Corning, NY). The pellets were then
resuspended in volumes of PBS that were equal
to the original sample volume. This process was
repeated three times. Fluorescence intensity of
supernatants was measured with a microplate

reader (Safire2, Tecan, Switzerland) using


excitation/emission wavelengths of 270/545 nm.
Data was collected using the Xfluor4 software
program (Tecan).
Samples were taken from the final washed
cell solution and resuspended in 1 ml of PBS for
determination of cell counts and assessment of
cell viability before and after incubation with
QDs. Samples were stained using LIVE/DEAD
BacLightTM Bacterial Viability Kit (Molecular
Probes, Carlsbad, CA) according to the
manufacturers protocol. Samples of cells alone
and QDs alone (without LIVE/DEAD stain)
were also collected. Samples were filtered
through a 0.22 m black polycarbonate
membrane (Millipore, Temecula, CA) and
mounted onto a microscope slide. One drop of
Baclight mounting oil (Molecular Probes) was
placed on the membrane and covered with a
cover slip.
QD-incubated samples in volumes of 10 l
were placed on a clear glass slide, covered with
cover slips, and secured with parafilm. Controls
with no QDs were also prepared in the same
manner.
2.7 Routine Epi-fluorescence Microscopy
Samples were analyzed with an Axioskop2 TM
epi-fluorescence microscope (Carl Zeiss, Jena,
Germany) using two filter sets: fluorescein
isothiocyanate, FITC, (Absorbance: 490-494
nm, Emission: 517 nm) and Texas Red
(Absorbance: 595 nm, Emission 620 nm). Three
images per sample were captured with a CCD
camera (Optronics, Goleta, CA) using the
program Magnafire (Optronics).
2.8 Confocal Laser Scanning Microscope
(CLSM)
A Zeiss Confocal laser scanning microscope
LSM 510 (Jena, Germany) mounted on an
Axiovert 100M inverted microscope fitted with
a FITC filter was used to image cells incubated
with QDs. Several slices of each sample were
taken of the 50 l of 100 nM QD+ samples at
varying depths. Z-slice thicknesses were 1.00
m, 0.15 m, and 0.50 m for bacterial
incubation,
CaCl2
transformation,
and
electroporation
methods,
respectively.

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Journal of Undergraduate Research in Bioengineering

Figure 1. QD internalization by CaCl2 transformation. Arrows point to bacteria that have internalized QDs.
A-D) transmitted light images. E-H) epifluorescent images. A,E) 5 l QD+; B,F) 10 l; C,G) 50 l; D, H) 0
l (negative control). Scale Bar = 10 m.

3. RESULTS
Preliminary studies with both types of QDs
showed that positive QDs (QD+) associated
better with the negatively charged bacteria than
the negatively charged QDs. All further
experiments were conducted with only QD+.
3.1 Cell Viability
Viability of P. aeruginosa before and after
incubation with QDs was assessed with
Live/Dead staining. CaCl2 treatment only killed
20% of the bacterial cells, so there are enough
live bacteria to pick up QDs. Electroporation
killed approximately 70% of the cells, with few
remaining live bacteria. In contrast, simple
bacterial incubation with QDs did not affect cell
viability.
3.2 Epifluorescence Microscopy
In all three internalization methods, QDs were
found to be associated with single bacteria as
was observed with epifluorescence microscopy.
For CaCl2 transformation (Figure 1), there was

low visualization of QD internalization in the 5


l samples, which was most likely due to the
low concentration of QDs added. In the 10 l
and 50 l samples, there is definite association
between QDs and bacteria as shown in the
corresponding transmission light images, which
show only bacteria, and fluorescent images,
which show QD fluorescence. Presence of QDs
can be noted by the increased intensity of light
compared to the background fluorescence of the
bacteria observed in the control sample.
The bacterial incubation method (Figure 2)
yielded similar results, with association of QDs
and bacteria in all three samples. As expected,
increased fluorescence intensity was observed
with increasing QD concentration. Results from
electroporation of the bacteria followed by
incubation with QDs (Figure 3) were in
agreement with the above methods. The samples
with 50 l of 100 nM QD+ from all three
methods appeared to have less internalization of
QDs because the exposure time had to be
decreased in order to reduce the intensity of light
being emitted from the larger amount of QDs.

Journal of Undergraduate Research in Bioengineering

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Figure 2. QD internalization by bacterial incubation. Arrows point to bacteria that have internalized
QDs. A-D) transmitted light images. E-H) epifluorescent images. A,E) 5 l QD+; B,F) 10 l; C,G) 50
l; D, H) 0 l (negative control). Scale Bar = 10 m.

Figure 3. QD internalization by electroporation. Arrows point to bacteria that have internalized QDs.
A-D) transmitted light images. E-H) epifluorescent images. A,E) 5 l QD+; B,F) 10 l; C,G) 50 l; D,
H) 0 l (negative control). Scale Bar = 10 m.

Using P. aeruginosa growth curve data and


dilution calculations, we were able to calculate
approximate QD transformation efficiencies.
Transformation efficiencies of the bacterial
incubation,
CaCl2
transformation,
and
electroporation internalization methods were
3.7%, 13.9%, and 15.1%, respectively. A twotailed students t-test confirmed that CaCl2
transformation
and
electroporation
were
significantly (P < 0.05) more efficient
internalization methods than simple bacterial

Table 1. Two-tailed students


transformation efficiencies.

t-test

of

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Journal of Undergraduate Research in Bioengineering

viability as mentioned previously, the increase in


QD uptake by these two methods were as
expected because the treatment of the cells
increase the cells ability to uptake substances.

incubation with QDs as shown in Table 1.


However, no statistical significance was
observed between the CaCl2 transformation and
electroporation methods. Despite the lower cell

Figure 4: Confocal images of samples that were transformed using CaCl2 with 50 l of QD+. Multiple
slices of specific areas in each sample, from top to bottom (A-H respectively), are shown. Slices
demonstrate different levels within certain bacteria that maintain fluorescence (not only outside).
Depth of slices is 0.15 m. Scale Bar = 10 m.

Figure 5. Confocal images of samples that were passively incubated with 50 l of QD+. Multiple slices of
specific areas in each sample, from top to bottom (A-H respectively), are shown. Slices demonstrate different
levels within certain bacteria that maintain fluorescence (not only outside). Depth of slices is 1.00 m. Scale
Bar = 10 m.

Journal of Undergraduate Research in Bioengineering

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Figure 6. Confocal images of samples that were electroporated with 50 l of QD+. Multiple slices of
specific areas in each sample, from top to bottom (A-H respectively), are shown. Slices demonstrate
different levels within certain bacteria that maintain fluorescence (not only outside). Depth of slices is
0.50 m. Scale Bar = 10 m.

3.3 Confocal Microscopy


Confocal microscopy was used to confirm that
QDs were internalized by P. aeruginosa and not
simply associated with the outer cell surface.
Confocal images were taken of the 50 l QD+
samples for all three internalization methods
(Figures 4-6). The acquired images demonstrate
that QD fluorescence was consistently
associated with the interior of the bacteria
throughout the different layers. QDs are
therefore internalized by planktonic P.
aeruginosa regardless of incubation method.
3.4 Fluorescence Scanning of QD removal from
Supernatant
Fluorescence scanning of the supernatants was
performed from each of the four washes to
decrease the background fluorescence in the
microscope images. The data showed that after 4
washes there was a decrease in fluorescence to a
fairly constant value, which demonstrated that
there was no longer a significant amount of free
floating QDs remaining in the solution (Figure
7). Therefore, QDs were associated with the
bacteria either by being internalized by the
bacteria or adhered to the surface. From the
results obtained by confocal microscopy, the

QDs were most likely internalized by the


bacteria.

4. DISCUSSION AND CONCLUSION


Using epifluorescence and confocal microscopy,
we were able to determine that all three
internalization methods (passive bacterial
incubation,
CaCl2
transformation,
and
electroporation) allow for successful QD
internalization by planktonic P. aeruginosa.
However,
CaCl2
transformation
and
electroporation yielded significantly higher
transformation efficiencies, despite their lower
cell viability, than simple bacterial incubation.
As expected, increased QD concentration led to
increased fluorescence intensity and QD uptake
by the bacteria. This has allowed us to expand
upon various methods for internalizing
substances within bacteria and determine the
better method for specifically internalizing QDs
in P. aeruginosa. Future work will include the
use of Fluorescence Activated Cell Sorting for a
more accurate method in determining
transformation efficiency. And, studies are
underway to determine the efficacy of the three
QD internatiolzation methods on P. aeurginosa
bioflims.

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Journal of Undergraduate Research in Bioengineering

ACKNOWLEDGEMENTS
A
Bact. Incub.
Control: Bact. Incub.
CaCl2 Trans
Control: CaCl2 Trans
Elect.
Control: Elect.

Fluorescence Intens

70000
60000
50000
40000
30000
20000
10000

Special thanks to Jackie Callihan, Kyung Park,


and Ara Greer for their beneficial mentorship. I
would also like to thank Lifeng Qi for the
synthesis of the QDs used in this study and
Mark Sena for his helpful knowledge concerning
the fabrication of QDs. Financial support was
received from the University of Washington
Engineered Biomaterials (UWEB) group, the
National Science Foundation (EEC-9529161),
and the University of Washington.

0
1

Number of Washes

B
Bact. Incub.
Control: Bact. Incub.
CaCl2 Trans.
Control: CaCl2 Trans.
Elect.
Control: Elect.

70000

Fluorescence Intens

60000

50000

40000

30000

20000

10000

0
1

Number of Washes

C
Bact. Incub.
Control: Bact. Incub.
CaCl2 trans.
Control: CaCl2 trans.
Elect.
Control: Elect.

Fluorescence Intens

50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
1

Number of Washes

Figure 7. Fluorescence readings of supernatants, collected


from cells transformed with 5 l (A), 10 l (B), and 50 l
(C) of QD+ for each QD internalization method, shows
decrease in fluorescence with increase in number of
washes. Excitation/emission wavelength = 270/545 nm.

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