Article

Preparation of Functional Aptamer Films Using Layer-by-Layer Self-Assembly

Department of Chemistry, Carleton University, Ottawa-Carleton Chemistry Institute, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6.
Biomacromolecules (Impact Factor: 5.75). 05/2009; 10(5):1149-54. DOI: 10.1021/bm8014126
Source: PubMed
ABSTRACT
Advances in many aptamer-based applications will require a better understanding of how an aptamer's molecular recognition ability is affected by its incorporation into a suitable matrix. In this study, we investigated whether a model aptamer system, the sulforhodamine B aptamer, would retain its binding ability while embedded in a multilayer polyelectrolyte film. Thin films consisting of poly(diallyldimethylammonium chloride) as the polycation and both poly(sodium 4-styrene-sulfonate) and the aptamer as the polyanions were deposited by the layer-by-layer approach and were compared to films prepared using calf thymus DNA or a random single-stranded oligonucleotide. Data from UV-vis spectroscopy, quartz crystal microbalance studies, confocal microscopy, and time of flight secondary ion mass spectrometry confirm that the aptamer's recognition of its target is retained, with no loss of specificity and only a modest reduction of binding affinity, while it is incorporated within the thin film. These findings open up a raft of new opportunities for the development and application of aptamer-based functional thin films.

Full-text

Available from: Maria C Derosa
Preparation of Functional Aptamer Films Using Layer-by-Layer
Self-Assembly
Yasir Sultan,
Ryan Walsh,
Carlos Monreal,
and Maria C. DeRosa*
,†
Department of Chemistry, Carleton University, Ottawa-Carleton Chemistry Institute, 1125 Colonel By Drive,
Ottawa, Ontario, Canada, K1S 5B6, and Eastern Cereal and Oil Seed Research Centre, Agriculture and
AgriFood Canada, 960 Carling Avenue, Ottawa, Ontario, Canada, K1A 0C6
Received December 5, 2008; Revised Manuscript Received February 28, 2009
Advances in many aptamer-based applications will require a better understanding of how an aptamer’s molecular
recognition ability is affected by its incorporation into a suitable matrix. In this study, we investigated whether
a model aptamer system, the sulforhodamine B aptamer, would retain its binding ability while embedded in a
multilayer polyelectrolyte film. Thin films consisting of poly(diallyldimethylammonium chloride) as the polycation
and both poly(sodium 4-styrene-sulfonate) and the aptamer as the polyanions were deposited by the layer-by-
layer approach and were compared to films prepared using calf thymus DNA or a random single-stranded
oligonucleotide. Data from UV-vis spectroscopy, quartz crystal microbalance studies, confocal microscopy, and
time of flight secondary ion mass spectrometry confirm that the aptamer’s recognition of its target is retained,
with no loss of specificity and only a modest reduction of binding affinity, while it is incorporated within the thin
film. These findings open up a raft of new opportunities for the development and application of aptamer-based
functional thin films.
Introduction
The layer-by-layer (LbL) method
1
for the deposition of
oppositely charged polyelectrolytes on solid substrates has been
well-established as a convenient approach to the bottom-up
assembly of multilayered polymer films.
2
The nature of the
assembly process leads to precise, nanoscale control of film
thickness and composition through the appropriate choice of
the components, the number of layers, and the order of their
deposition. Furthermore, LbL assembly is extremely versatile
in terms of the choice of polyelectrolyte, allowing for these films
to be tailored to their specific application. Biological macro-
molecules such as polypeptides/proteins,
3
polysaccharides,
4
nucleic acids,
5
and even viruses
6
can all serve as building blocks
for these multilayer films. This allows for the preparation of
biocompatible and biodegradable systems for a range of
applications including those in sensing,
7
controlled release,
8
and
biomedical technology.
9
Aptamers are synthetic nucleic acids that fold into unique
three-dimensional structures capable of binding tightly to a target
of interest, with affinities and specificities that rival or even
surpass those of monoclonal antibodies.
10
Unlike antibodies,
which are temperature-sensitive and have a limited shelf life,
aptamers are considerably more stable and can be transported
at ambient temperatures. These nucleic acids can also be
generated through an in vitro selection procedure
11
for a wide
variety of targets, making them uniquely suited to effectively
serve as receptors in sensor platforms.
12
Aptamers have been
developed for a wide range of molecules, from drugs to proteins
to very complex targets such as bacteria, tumor cell lines, and
viruses.
13
The potential for the use of aptamers as molecular recognition
elements is clear, but to fully exploit these nucleic acids for
the fabrication of biosensors and the development of other
applications, aptamers will need to retain their functionality
when embedded in or attached to a suitable matrix. Aptamers
have been immobilized in a sol-gel,
14
tethered to cellulose,
15
and attached to a range surfaces
16
all without loss of their
binding ability. Yet despite the great promise of LbL polyelec-
trolyte films for a wide range of applications, no work on the
entrapment of aptamers within these films has been reported.
The objective of this work was to determine whether the LbL
approach could be used to prepare a robust aptamer film that
maintained its affinity and specificity for the cognate target.
Here, we report on the first successful incorporation of a DNA
aptamer into a multilayered polyelectrolyte thin film (Figure
1). We chose the sulforhodamine B (SB) aptamer
17
as our model
system for this study. UV-vis spectroscopy, quartz crystal
microbalance (QCM) studies, confocal microscopy, and time
of flight secondary ion mass spectrometry (ToF-SIMS) all
confirmed that the aptamer retained its recognition ability despite
being embedded in the film. These films were simple to prepare
and were stable and reusable. This opens up new possibilities
for the development of aptamer-based functional films for use
in biosensing, implant coatings, controlled-release, and many
other applications.
Experimental Section
Materials. Quartz slides (75 × 25 mm) were purchased from VWR.
Poly(diallyldimethylammonium chloride) (PDDA, M
w
< 100000),
poly(sodium 4-styrene-sulfonate) (PSS, M
w
100000), calf thymus
DNA (CT, sodium salt, type I), and sulforhodamine B (SB) were
purchased from Sigma and were used as received. Tetramethylrosamine
(TMR) was purchased from Invitrogen and was used as received. The
sulforhodamine B aptamer (SA sequence: 5-CCG GCC TAG GGT
GGG AGG GAG GGG GCC GG-3) and random oligonucleotide (RO
sequence: 5-GAC CTA TGA TAG CAT CAG TCG CAT CAG TC-3)
were synthesized on a merMADE 6 (Bioautomation Corporation) using
standard phosphoramidite chemistry.
* To whom correspondence should be addressed. Phone: (613) 520-2600,
ext. 3844. Fax: (613) 520-3749. E-mail: maria_derosa@carleton.ca.
Carleton University.
Agriculture and AgriFood Canada.
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Page 1
Deposition of Polyelectrolyte Layers. Double deionized water was
used for all experiments. Quartz slides were prepared by treatment with
a solution of H
2
O
2
/NH
3
OH/H
2
O (1:1:5) for 10 min at 70 °C. After
pretreatment, the slides were washed with water and were then dipped
successively in solutions of oppositely charged polyelectrolytes, starting
with PDDA (5 mg/mL in 0.2 M NaCl) and then PSS (5 mg/mL in 0.2
M NaCl) for 20 min each. The slides were washed with copious
amounts of water in between each deposition step. This procedure was
repeated five times yielding five bilayers of PDDA and PSS (See Figure
1). Slides with only these bilayers (hereafter named
18
PDDA/PSS)
served as controls in later experiments. For the films containing the
sulforhodamine B aptamer (PDDA/PSS/SA), a random oligonucleotide
(PDDA/PSS/RO), or calf thymus DNA (PDDA/PSS/CT), the next five
bilayers were deposited with DNA as the anionic layer, in place of
PSS. In each case, the slide was immersed in the DNA solution (6 µM
for SA; 11 µM for RO; 0.04 µM for CT)
19
in 20 mM Tris-HCl (0.2 M
NaCl, pH 7.4) for 20 min at room temperature. These films were
terminated with an additional bilayer of PDDA/PSS to embed the DNA.
Films were left to dry open to air at room temperature for at least 5 h
before dye binding experiments were performed.
Dye Binding and Dissociation Constant (K
d
) Experiments. Prior
to dye binding, all films were heated in aqueous solution at 70 °C for
10 min to unfold any undesirable DNA conformations. Films were
dipped in 2 mM sulforhodamine B (SB) or tetramethylrosamine (TMR)
solution (0.1 M KCl) for 30 min and then washed with copious amounts
of water until washings were no longer colored. UV/vis spectra of the
films were obtained on a Cary 300 UV/vis spectrophotometer. To
minimize electrostatic binding of the dye directly to the outermost layer
of the polymer film, the film tested with negatively charged SB dye
was terminated with PSS, while the film used for testing positively
charged TMR was terminated with PDDA.
Reusability and stability of PDDA/PSS/SA was tested by immersing
the slides in deionized water either at 40 or 70 °C for 10 min or 24 h
at room temperature, and then examining the UV-vis spectrum for
loss of the dye or DNA bands (570 and 260 nm, respectively). Films
were then reimmersed in dye solution and binding was compared to
the sample before heating by UV-vis.
Two types of dissociation constant experiments were performed.
Initially, UV/vis spectroscopy was used as follows. PDDA/PSS/SA films
were dipped in sulforhodamine B (0.1 M KCl) at concentrations of
0.001-10 mM for 30 min and washed with copious amounts of water
before obtaining the UV/vis spectrum. The results were compared to
those obtained using a quartz crystal microbalance (QCM) from
Stanford Research Systems (QCM200, 5 MHz AT-cut quartz crystal
oscillator with 0.1 s gate time). The quartz crystal was functionalized
using the same preparation conditions as described for the quartz slides.
Film growth was monitored by the change in relative frequency while
carefully nulling any capacitance by manual control of the bias voltage
required by the varactor. For the dissociation constant experiment, 1
mL solutions of varying SB concentrations (0.001-10 mM, 0.1 M KCl)
were placed on the quartz crystal for 30 min, after which time the crystal
was washed with copious amounts of water, dried under argon for 3
min, and the frequency change was noted. For both experiments, the
dissociation constants were evaluated by minimizing the residuals values
between calculated and observed experimental absorbance or
frequency data using the solver feature of Microsoft Excel.
20
Microscopy. All microscopy was performed in air. Confocal
microscopy, to characterize the distribution of SB within the polymer
films, was performed on a Zeiss LSM510 (532 nm, 3% laser intensity
for the films PDDA/PSS/SA and PDDA/PSS/RO, 5% laser intensity
for the PDDA/PSS/CT film) with a Plan-Acochromat 63x/1.4 Oil Dic
objective with LP950 filter, exciting at 556 nm and collecting image
data at 575 nm. Brightness (intensity) of the images was assessed by
examining the images in Adobe Photoshop CS2 (V. 9.0.2), comparing
the mean intensity values generated by the histogram tool, which graphs
the number of pixels at each intensity level. Atomic force microscopy
(AFM), to characterize surface topography of the films, was performed
on a Veeco Instruments DI-3100 in both contact and tapping mode.
AFM was also used to determine film thickness by creating a scratch
on the polymer surface with a diamond etched knife on an Ntegra
SFC050LNTF AFM head.
Time of Flight-Secondary Ion Mass Spectrometry (ToF-SIMS). A
ToF-SIMS IV instrument from IonTOF GmbH was used to acquire
compositional depth profiles of the films. Both positive and negative
ion profiles were collected from several points on the surface. A Cs
+
Figure 1. Experimental Overview. (A) Thin film composition. In PDDA/
PSS/SA,
18
five bilayers of PDDA (blue) and PSS (red) are followed
by five bilayers of PDDA and SA (green). The films are capped with
a final bilayer of PDDA/PSS. The films PDDA/PSS/CT and PDDA/
PSS/RO are prepared with a similar composition, except that calf
thymus DNA (CT) or random oligonucleotide DNA (RO) is substituted
for SA in the second five bilayers. PDDA/PSS films without any DNA
were also prepared and used as controls. The film illustration was
modified from ref 1. (B) Components of the experiment. The sequence
of the random oligonucleotide (RO), the sequence and structure
(showing the predicted g-quadruplex) of the sulforhodamine B
aptamer (SA), and structures of the polyelectrolytes, PSS and PDDA,
are presented. The structures of the target, sulforhodamine B (SB),
and a related dye, tetramethylrosamine (TMR), are also shown.
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ion beam at 500 eV primary energy was used for sputtering to collect
negative secondary ions, and an O
2
+
ion beam with same energy was
employed for the positive polarity. The sputter beams were rastered
over an area of about 400 × 400 µm
2
. The signal of the secondary
ions was collected from the central area of about 200 × 200 µm
2
with
64 × 64 pixels. The analytical gun used to generate the secondary ions
from the central area used a chopped 15 keV Ga ion beam in a high-
current bunch mode. Charge compensation by electron flooding was
also employed during profiling since the samples were nonconductive.
Results and Discussion
LbL self-assembly of polyelectrolyte films is emerging as a
method of choice for the preparation of thin films for medicine,
biology, and material science. As aptamers can be generated
for nearly any target, incorporation of these nucleic acids as
recognition elements within these multilayer films is of great
interest. One concern, however, is that the electrostatic interac-
tion of the aptamer with the surrounding polyelectrolyte matrix
could potentially interfere with target recognition, especially in
an aptamer that needs to be highly structured to effectively bind.
For this reason, the sulforhodamine B aptamer, which adopts a
g-quadruplex structure,
17
was chosen as our model for this study.
To assess the binding ability of this aptamer when immobilized
in a polymeric matrix, thin films were prepared using the LbL
approach (Figure 1). Four types of films were prepared:
18
the
Figure 2. Overlay of UV-vis spectra of the polyelectrolyte films. DNA
containing films are normalized at 260 nm. Black, PDDA/PSS/SA
exposed to 2 mM sulforhodamine B (SB); red, PDDA/PSS/RO
exposed to 2 mM SB; green, PDDA/PSS/CT exposed to 2 mM SB;
yellow, PDDA/PSS alone exposed to 2 mM SB; blue, PDDA/PSS/
SA exposed to 2 mM tetramethylrosamine (TMR); orange, PDDA/
PSS/SA exposed to 2 mM SB, but without K
+
. The regions of the
spectra assigned to DNA and dye absorbances are highlighted. Upon
immersion of the films in the dye solution, the aptamer films show
the greatest degree of dye binding as evidenced by the largest peak
at 570 nm.
Table 1. Comparisons of Dye, Polymer, and DNA Absorbance
Values for the Films of this Study after Exposure to 2 mM Dye
sample
composition
ratio
a
of SB dye
peak height to polymer
peak height (570:220 nm)
ratio
a
of SB dye peak
height to DNA peak
height (570:260 nm)
PDDA/PSS 0.12 (0.01)
PDDA/PSS/CT 0.08 (0.01) 0.23 (0.02)
PDDA/PSS/RO 0.12 (0.03) 0.34 (0.09)
PDDA/PSS/SA
(with K
+
)
0.21 (0.06) 0.70 (0.01)
PDDA/PSS/SA
(without K
+
)
0.09 (0.01) 0.26 (0.03)
a
Average of at least three measurements, standard deviation in
parentheses.
Figure 3. Confocal microscope images of the polyelectrolyte films after
dye binding. Excitation 556 nm, emission 575 nm. White bars correspond
to 10 µm (A) PDDA/PSS/RO, (B) PDDA/PSS/CT, and (C) PDDA/PSS/
SA. The random oligonucleotide-containing film (PDDA/PSS/RO, A)
shows few bright spots associated with the dye fluorescence. PDDA/
PSS/CT films show very little dye fluorescence, with some localized
nonspecifically within defects on the film surface. The film containing
the aptamer (PDDA/PSS/SA, C) shows the brightest fluorescent areas
associated with bound dye.
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sulforhodamine B aptamer film, PDDA/PSS/SA, and three
controls, PDDA/PSS (polymer layers only), PDDA/PSS/RO
(containing a random oligonucleotide), and PDDA/PSS/CT
(containing calf thymus DNA). In all films, five bilayers of
PDDA and PSS were first deposited on a quartz slide. The
growth of the film with each bilayer could be monitored via
UV/vis (PSS 220nm). For PDDA/PSS/SA, PDDA/PSS/RO,
and PDDA/PSS/CT, the next five bilayers were deposited using
DNA as the polyanionic electrolyte, either the sulforhodamine
B aptamer, a nonspecific single-stranded random oligonucle-
otide, or calf thymus DNA, respectively. Growth of the films
during DNA deposition could be monitored by UV-vis as well
(260 nm). All films were terminated with an additional PDDA/
PSS layer to embed the DNA within the film and also to
minimize any electrostatic interaction between the charged dye
and the film. Small molecules, such as the dye target, are known
to diffuse through the layers of polyelectrolyte films, while larger
biomolecules, nucleases for example, are not.
21
Thus, embedding
the aptamer within the film should not impede dye access to
the aptamer, but may serve to protect the aptamer from
degradation. AFM was used to determine that the DNA-
containing films were approximately 80 nm in thickness. This
value is higher than what would be expected for a pure PDDA/
PSS film at this salt concentration, demonstrating that the DNA
layers have a sizable effect on film properties.
22
UV-vis Spectroscopy. Given the strong absorbance of the
SB dye in the visible region, UV-vis spectroscopy was
employed as an initial measure of whether the target recognition
of the aptamer was maintained while it was embedded within
these polyelectrolyte films. Figure 2 is a comparison of the
spectra for the various films after dye binding. Clearly, the
spectrum of PDDA/PSS/SA shows a larger signal from the SB
dye absorbance (570 nm) than the controls. However, as the
dye, DNA, and polymer peak intensities varied from film to
film, ratios of dye peak height to polymer or DNA peak height
were used to better quantify the affinity of the films for the
target, as these ratios showed little variation from sample to
sample (Table 1).
When PDDA/PSS films alone were tested for their ability to
bind the dye nonspecifically, a small absorbance in the dye
region was noted. This nonspecific binding could be attributed
to the fact that the dye, being negatively charged, will likely
have some affinity for the positively charged PDDA. In these
films, the ratio of the dye peak intensity to polymer peak
intensity was approximately 0.12. This serves as a baseline for
nonspecific dye binding in these films. When either the random
oligonucleotide or calf thymus DNA is included in these films,
the amount of nonspecific binding (as measured by the dye to
polymer ratio) is either less than or equal to the baseline value
found in the polymer films alone. The aptamer-containing film,
PDDA/PSS/SA, however, showed considerably stronger affinity
for the dye, with a dye to polymer ratio of approximately 0.21.
Figure 4. Representative data from QCM dissociation constant
experiments. A PDDA/PSS/SA film on a quartz crystal was exposed
to solutions of sulforhodamine B dye and the relative frequency was
plotted vs the dye concentration. Top: Raw frequency data from the
QCM experiment. Bottom: Plot of frequency over dye concentration.
The line represents a two K
d
model (see text). Inset: Values calculated
using the two K
d
model vs the experimental data.
Figure 5. ToF-SIMS sputter depth profile of PDDA/PSS/SA before
(A) and after (B) dye binding. Red, SO
2
-
; green, PO
3
-
; blue, SiO
2
-
.
The SO
2
-
secondary ion is used to trace both PSS and SB, while
the PO
3
-
secondary ion is used to track the presence of the apta-
mer. The SiO
2
-
ion counts have been reduced by a factor of 10 to fit
on the same scale as the SO
2
-
. (A) PDDA/PSS/SA alone: SO
2
-
drops
off dramatically as PO
3
-
rises, and then rises again in the area directly
above the slide surface. This trend was expected as the aptamer
replaces PSS in the middle 5 bilayers of this film. (B) PDDA/PSS/SA
and dye: SO
2
-
counts reach a maximum at the same depth as do
the DNA’s PO
3
-
counts, suggesting that the dye and the DNA are
colocalized.
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Similarly, if the dye absorbance was compared to the DNA
absorbance at 260 nm, the ratio of the peaks in the aptamer
films was more than double that of the other films. Studies on
the SA aptamer have indicated that the sequence forms a stacked
g-quadruplex structure in the presence of K
+
, but not Na
+
, and
that this structure is essential for maximum dye binding.
17
Indeed, heating the PDDA/PSS/SA films to unfold any undesir-
able DNA structures before immersion of the film in the dye
solution with a high concentration of K
+
was required for
efficient dye binding; films immersed in K
+
-free dye solutions
showed levels of dye binding that were comparable to controls.
(see Figure 2 and Table 1) These data suggest that the aptamer
in PDDA/PSS/SA retains its ability to form the g-quadruplex
structure to recognize and bind the sulforhodamine B target,
over and above the small nonspecific electrostatic interaction
that the other films exhibit.
The effect of immobilization in the polyelectrolyte film on
aptamer specificity was also investigated. In the original work
on the SB aptamer, TMR dye was shown to be an inefficient
competitor for binding, despite the structural similarities between
the two molecules (see Figure 1).
17
When PDDA/PSS/SA films
were exposed to TMR in 0.1 M KCl, only a low level of binding
(dye to polymer ratio of 0.05, dye to DNA ratio of 0.17), similar
to the amount in the K
+
-free case, was detected (Figure 2). The
specificity of the sulforhodamine aptamer was therefore unaf-
fected by the polyelectrolyte matrix.
Confocal Microscopy. Given the intense fluorescence of the
SB target, confocal microscopy could also be employed to assess
the binding ability of the polyelectrolyte films. Confocal
microscopy images of the thin films are shown in Figure 3.
Images of PDDA/PSS/SA showed considerably more bright
areas consistent with bound dye than did PDDA/PSS/RO or
PDDA/PSS/CT. Given that the size of the bright spots is on
the µm length scale, these are most likely the result of groups
of aptamers binding to SB. The PDDA/PSS/SA and PDDA/
PSS/RO films were taken under identical settings (for the case
of the PDDA/PSS/CT films, however, laser intensity had to be
increased by 2% in order to obtain usable images). When the
standard photo processing software was used, the mean bright-
ness of the PDDA/PSS/SA image (30 arbitrary units) was found
to be twice that of the PDDA/PSS/RO image (14 arbitrary units).
The CT DNA films were very inhomogeneous but, nevertheless,
show the lowest mean brightness (1 arbitrary unit) and the least
bright areas, and those that are present appear to be localized
in defects on the films.
23
Dissociation Constant Experiments (K
d
). Dissociation con-
stant (K
d
) is generally used as a measure of an aptamer’s binding
affinity or sensitivity. In solution, the sulforhodamine B aptamer
has a K
d
of about 0.7 µM.
17
Dissociation constant experiments
on our polyelectrolyte films, using absorbance changes at 570
nm with increasing dye concentration, proved difficult; in the
PDDA/PSS/SA films, apparent dissociation constant values
ranged from as low as 0.8 µM to as high as 3 mM (data not
shown). While the lower value is consistent with the K
d
for the
sulforhodamine aptamer in solution, the higher value may be
more representative of the nonspecific dye interaction with the
polymer films. Quartz crystal microbalance (QCM) experiments
were used in an attempt to clarify this issue. QCM is a powerful,
quantitative technique that detects molecular recognition events
through frequency changes associated with minute mass in-
creases on the crystal surface. QCM has been used to determine
K
d
values for antibody-antigen and aptamer-target interac-
tions.
24
PDDA/PSS/SA films were deposited onto the crystal
and frequency changes detected with increasing concentrations
of SB dye were evaluated (see Figure 4). The data fit best to a
model with two binding affinities, a specific K
d
of 16 uM and
a nonspecific K
d
of 1.5 mM, consistent with the values obtained
using UV-vis. This suggests that the specific affinity of the
aptamer has been only somewhat perturbed (20 × higher K
d
)
by its incorporation into the polymer film. Data from control
QCM K
d
experiments on the PDDA/PSS/CT films fit to a single
binding affinity in the low mM range (see Supporting Informa-
tion). These results confirm that there is a nonspecific interaction
between the films and the dye target. The high K
d
of this
interaction, however, makes this nonspecific binding unlikely
to affect the detection of targets at low concentrations. Our
current work is focused on eliminating or reducing this
nonspecific interaction using other polyelectrolyte systems.
Time of Flight Secondary Ion Mass Spectrometry
(ToF-SIMS). To further confirm the specificity of the dye for
the aptamer within the polyelectrolyte layers, the investigation
of dye localization as a depth profile, rather than as a bulk
measurement, was attempted. ToF-SIMS was used to confirm
specificity and colocalization of the dye and the aptamer within
the films. ToF-SIMS measurements were taken as a depth profile
over several points on the film to examine whether the dye was
colocalized with the aptamer layers, which would be consistent
with specific aptamer-target binding (Figure 5). The SO
2
-
secondary ion was monitored as it was present in both the PSS
polymer and the SB dye, and PO
3
-
was chosen to track the
presence of DNA. SiO
2
-
was also monitored to demarcate the
film/glass interface. In the absence of dye (Figure 5A), all
the SO
2
-
secondary ions are originating from the PSS within
the film. The counts of SO
2
-
secondary ion drop where the
counts of PO
3
-
ion rise. This is expected as there is no PSS in
the layers of the film containing the aptamer. If SB dye had no
affinity for the aptamer layers and was evenly distributed
throughout the film, then after dye-binding the SO
2
-
depth
profile would be expected to show a similar trend. The regions
of the film that contained both PSS and dye would show a higher
SO
2
-
ion count, and the regions of the film where PSS is
replaced by SA would show a lower SO
2
-
count originating
from the dye alone. After dye-binding, however, the SO
2
-
secondary ion trace does not follow that trend and instead now
tracks with the PO
3
-
ion (See Figure 5B). This suggests that
dye is predominantly colocalized within the aptamer layers of
the film. As both the DNA and the dye are negatively charged,
this colocalization is unlikely to be due to a simple electrostatic
interaction, and these findings further corroborate that the
aptamer maintained its ability to recognize the dye while
embedded in the film.
Stability and Reusability of Aptamer Films. Although DNA
diffusion through polyelectrolyte films is expected to be
minimal, a molecular beacon approach has recently shown that
these multilayer films are somewhat permeable to short se-
quences of DNA.
25
Thus, PDDA/PSS/SA films were tested for
their stability to leaching as well as their ability to be regenerated
and reused. Little evidence of aptamer leaching was noted by
examination of the peak at 260 nm when the films were left in
deionized water over 24 h at room temperature. PDDA/PSS/
SA films containing the dye were also subjected to immersion
in deionized water at 40 and 70 °C; UV-vis spectra determined
that the dye, but not the aptamer, could be removed from the
films under these conditions (<1% loss of the signal at 260 nm).
Dye binding after regeneration was found to be comparable to
binding in the original films (>95% of the dye signal). Drying
and air exposure also seemed to have little effect on the films.
Aptamer-containing films left dry and open to air at room
Aptamer-Polyelectrolyte Films Biomacromolecules, Vol. 10, No. 5, 2009 1153
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Page 5
temperature for over 3 months still showed binding affinity when
reimmersed in dye solution.
Conclusions
When the sulforhodamine B aptamer as a model system was
used, this study confirmed that an aptamer embedded in a
polyelectrolyte matrix was still successful at binding its cognate
target, with preserved specificity and only slightly perturbed
affinity. This suggests that the sulforhodamine aptamer was able
to retain its required g-quadruplex conformation while entrapped
in the film. The multilayer polyelectrolyte films prepared by
LbL assembly were found to be stable to aptamer leaching and
reusable. Studies using more sensitive aptamer systems and
employing different polyelectrolytes to decrease nonspecific
target binding interactions with the polymer film are ongoing.
The robust nature of these films, coupled with the ease with
which they are prepared, indicates that LbL assembly is a
practical and effective approach for the development of func-
tional aptamer films. Future work will examine the application
of these films as receptor elements for biosensing and controlled
release.
Acknowledgment. M.C.D. thanks the Natural Sciences and
Engineering Council of Canada (NSERC), the Canadian Foun-
dation for Innovation (CFI), the Ontario Research Fund (ORF),
and Carleton University for financial support. M.C.D. and C.M.
thank the Alberta Agricultural Research Institute (AARI) for
funding. Y.S. and C.M. thank Dr. Morris Schnitzer (Agriculture
and Agri-Food Canada) for passing on his commitment to
science. The authors acknowledge Dimitre Karpusov at ACSES
(University of Alberta) for the ToF SIMS measurements, Ann-
Fook Yang and Denise Chabot at Agriculture Canada for the
confocal experiments, and Siddhartha Nandi and He (Shirley)
Chang for help with the thin film preparation.
Supporting Information Available. Representative plot from
QCM dissociation constant experiments on PDDA/PSS/CT. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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BM8014126
1154 Biomacromolecules, Vol. 10, No. 5, 2009 Sultan et al.
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