Homogeneous and sensitive DNA detection based on polyelectrolyte complexes of cationic conjugated poly(pyridinium salt)s and DNA

Article abstract of DOI:10.1039/c2jm15491a

The electrostatic complexes of double-stranded deoxyribonucleic acid (dsDNA) and a cationic conjugated polyelectrolyte, poly{(4,4′-(1,4- phenylene)bis(2,6-diphenylpyridinium))-co-para-biphenylene ditosylates} (PPT), were investigated by spectral methods.

Full text of DOI:10.1039/c2jm15491a

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PAPER
Homogeneous and sensitive DNA detection based on polyelectrolyte
complexes of cationic conjugated poly(pyridinium salt)s and DNA†
a
a
b
a
a
b
Fengli Han, Yan Lu,* Qiang Zhang, Jingfen Sun, Xianshun Zeng* and Chenxi Li*
Received 27th October 2011, Accepted 20th December 2011
DOI: 10.1039/c2jm15491a
The electrostatic complexes of double-stranded deoxyribonucleic acid (dsDNA) and a cationic
0
conjugated polyelectrolyte, poly{(4,4 -(1,4-phenylene)bis(2,6-diphenylpyridinium))-co-para-
biphenylene ditosylates} (PPT), were investigated by spectral methods. The binding constant of PPT
5
ꢁ1
with calf thymus DNA (ctDNA) is estimated to be 9.3 ꢀ 10 M , which was determined by UV-vis
spectral titration. Fluorescence emission of PPT in phosphate buffer solutions (5.0 mM) can be
drastically quenched to about one-fourth of its original intensity in the presence of a trace amount
6
ꢁ1
(
0.28 mM) of ctDNA with a large Stern–Volmer constant (KSV ¼ 8.79 ꢀ 10 M ). The fluorescence
quenching efficiency is related to the target concentration, which allows the quantitative detection of
the target sequence in a sample. A linear detection range from 1.5 to 280 nM was obtained under the
ꢁ9
optimized experimental conditions with a detection limit down to the 10 M range. Furthermore,
strong electrostatic attraction may be the main driving force for PPT/ctDNA binding, which was
proposed according to the results of circular dichroism and melting transition study of ctDNA in the
presence of PPT. This investigation provides an insight into designing a novel conjugated
polyelectrolyte for biomolecular sensing.
Introduction
conjugated polyelectrolytes for DNA detection included mainly
5
polythiophene derivatives and poly(fluorine-co-phenylene)
3a,6
derivatives. Recently, poly(para-phenylenevinylene) has been
DNA detection is of considerable importance in clinical diag-
1
nosis, gene expression analysis, and other biomedical studies.
7
used to develop a simple label-free DNA detection assay. The
Recently, conjugated polymers have attained significant interest
for this purpose because their molecular structure allows for
collective response and, therefore, optical amplification of fluo-
majority of these reported conjugated polyelectrolytes for the
DNA detection purpose, however, have limitations such as
tedious synthetic efforts and/or lack of practical applicability in
aqueous solutions. Thus, it is still highly desirable to develop
a novel polymer-based fluorescent probe with better sensitivity
for the homogeneous DNA detection in aqueous environments.
Unlike common p-conjugated polyelectrolytes characterized
by a backbone with a delocalized electronic structure and the
charged functional side chains, poly(pyridinium salt)s, which
2
rescent signals. The large number of optically active units along
the polymer chain increases the probability of light absorption,
relative to small-molecule counterparts. Cationic conjugated
polyelectrolytes, in particular, have proven to be very useful for
DNA sequence detection based on electrostatic interaction with
3
the negatively charged phosphate backbone. For example,
water-soluble conjugated polymers containing pendant ammo-
nium groups have been successfully applied in multicolor
detection assay for trace DNA or heparin in aqueous solution
belong to a class of main-chain cationic polymers, consist of
0
4
,4 -(1,4-phenylene)bis(2,6-diphenylpyridinium) ions along the
backbone of the polymer chains. They exhibit a number of
interesting properties such as redox behavior, liquid crystalline
4
based on the electrostatic interaction. Previously reported
(
LC), light-emitting properties and so on, which have been
8
a
investigated quite extensively by Bhowmik and co-workers.
Besides significant potential for use in electronic and optoelec-
tronic applications, it is reasonable for one to think that the
charged nature of the kind of polyelectrolytes also permits
coordination of electrostatic forces with oppositely charged
analyte targets. Recently, it has been demonstrated that the
poly(pyridinium salt)s with calix[4]arene segments in the
main chain could condense anionic DNA into a compact
poly(pyridinium salt)–DNA complex through electrostatic
interaction, showing great potential application as biosensors for
School of Materials Science & Engineering, Tianjin University of
Technology, Tianjin, 300384, China. E-mail: luyan.tjut@yahoo.com.cn;
xshzeng@tjut.edu.cn; Fax: +86-22-60215553; Tel: +86-22-60216748
b
Key Laboratory of Functional Polymer Materials, Ministry of Education,
Institute of Polymer Chemistry, Nankai University, Tianjin, 300071,
China. E-mail: nkulicx@yahoo.com.cn; Fax: +86-22-23502749; Tel:
+
86-22-23501921
†
Electronic supplementary information (ESI) available: Fig. S1:
fluorescence spectra of PPT in mixture solvents with varying ratios of
DMSO/H O and Fig. S2: Stern–Volmer plot for quenching of PPT by
calf thymus DNA in phosphate buffer solutions (50 mM, pH 7.4). See
DOI: 10.1039/c2jm15491a
2
4
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9
DNA detection. On the other hand, it is noteworthy that this
0
Synthesis of 4,4 -(1,4-phenylene)bis(2,6-diphen-yl-pyrylium)
ditosylate (2)
kind of poly(pyridinium salt)s can be facilely prepared by the
8
ring-transmutation polymerization reaction. Therefore, they
Compound 2 was synthesized in 87% yield according to
1
a reported method and characterized as follows. H NMR (400
promise to be a novel and effective agent for DNA detection
because of both their good photoelectric characteristics and the
charged nature. It will be an addition to the currently limited
number of available conjugated polyelectrolytes for the purpose
of DNA detection. To our knowledge, this is the first example of
conjugated poly(pyridinium salt)s that can detect sensitively
double-stranded deoxyribonucleic acid (dsDNA) in aqueous
solutions.
10
+
MHz, d
6
-DMSO, ppm): 9.35 (s, 4H, aromatic meta to O ), 9.21
(
s, 4H, 1,4-phenylene), 7.58–8.93 (m, 20H, phenyl), 7.46–7.47 (d,
J ¼ 6.7 Hz, 4H, tosylate), 7.09–7.10 (d, J ¼ 7.7 Hz, 4H, tosylate),
2
.27 (s, 6H, CH
3
). Anal. calcd for C54
H
42
O
8
S
2
: C, 73.45; H, 4.79.
Found: C, 73.15; H, 4.71%.
0
Polymer synthesis
In this contribution, a novel cationic conjugated poly{(4,4 -
(
1,4-phenylene)bis(2,6-diphenylpyridinium))-co-para-biphenylene
10
The polymer was prepared according to the literature.
0
A
ditosylates} (PPT) was easily synthesized through the ring-
transmutation polymerization reaction. The electrostatic
complexes of PPT–calf thymus DNA (ctDNA) were investigated
in detail by means of UV-vis, fluorescence and circular dichroism
spectral methods. The homogeneous and sensitive detection of
ctDNA based on the PPT–ctDNA complexes was achieved.
solution of 4,4 -(1,4-phenylene)bis(2,6-di-phenylpyrylium) dito-
sylate (2) (40 mmol) and benzidine (40 mmol) in 20 mL of DMSO
ꢂ
was vigorously stirred and heated at 110 C under nitrogen for 2
h. Toluene (10 mL) was then added so that the water generated
by the transformation of the pyrylium ring to the pyridinium ring
could be removed as a toluene/water azeotrope. The bath
ꢂ
temperature was increased to about 145–150 C and the azeo-
trope and the excess toluene were gradually distilled from the
reaction mixture using a Dean–Stark trap over 4–5 h. The bath
Experimental section
Materials
ꢂ
temperature was maintained at 145–150 C for an additional 48
h. The resulting viscous solution was slowly poured into a large
excess (25 times in volume) of rapidly stirred ethyl acetate. The
yellow poly(pyridinium salt)s that precipitated were collected by
filtration, purified by precipitation from DMSO with water and
All chemical reagents were obtained commercially and used as
0
received unless otherwise stated. 4,4 -(1,4-Phenylene)bis(2,6-diphe-
nylpyrylium) ditosylate (2) and the corresponding poly(pyridinium
10
tosylate)s (PPTs) were synthesized according to the literature.
ꢂ
1
dried under reduced pressure at 120 C overnight. H NMR (400
Calf thymus DNA was purchased from Beijing Sunbiotech Co.
Ltd. The DNA concentration per nucleotide was determined by
absorption spectroscopy by using the molar absorption coeffi-
MHz, d -DMSO, ppm): 8.82–7.07 (m, 44H, Ar–H), 2.25 (s, 6H).
6
Anal. calcd for C H N O S : C, 76.87; H, 4.89; N, 2.72.
6
6
50
2
6 2
ꢁ1
ꢁ1
Found: C, 76.16; H, 5.01; N, 2.67%.
cient (3 ¼ 6600 M cm ) at 260 nm. Milli-Q water (18.2 MU)
was used for all the experiments.
UV-vis titration experiment
Stock solution containing 0.85 mM of ctDNA in phosphate
buffer solution (PBS, 50 mM, pH 7.4) was prepared and incu-
bated for 30 min. Stock solution of polymer PPT was prepared
by diluting 12 mL of polymer PPT in DMSO (5 mM) to 3 mL
with PBS (50 mM, pH 7.4). The final concentration of polymer
PPT solution is 20 mM, corresponding to the repeating unit.
Aliquots of the ctDNA stock solution were added into the above
PPT stock solution to obtain the test samples. After each addi-
tion, the sample was allowed to equilibrate for 30 min prior to
recording a spectrum. Additions of the ctDNA were continued
until no change in the absorbance signal was observed.
General characterization
The NMR spectra were collected on a Varian UNITY Plus-400
NMR spectrometer (400 MHz) using tetramethylsilane (TMS) as
an internal standard. The element analysis was performed on
a Perkin-Elmer 2400C instrument. GPC analysis was conducted
with a Waters 2690 liquid chromatography system equipped with
a Waters 996 photodiode detector and Phenogel GPC columns,
using polystyrenes as the standard and DMF as the eluent at
ꢁ1
ꢂ
a flow rate of 1.0 mL min at 35 C. 50 mL of 0.2 wt% of polymer
in DMF containing 0.01 M LiBr was injected into the columns.
The absorption spectrum was recorded on a Shimadzu UV-2550
spectrometer. Fluorescence measurements were carried out on
a Hitachi F-4600 fluorescence spectrophotometer equipped with
a xenon lamp excitation source.
Fluorescence titration experiment
Stock solutions of both ctDNA and polymer PPT were prepared
by using a similar method to that mentioned in the UV-vis
titration experiment. The final concentration of polymer PPT is 5
mM, corresponding to the repeating unit. Aliquots of the ctDNA
stock solution were added into the polymer PPT stock solution
to obtain the test samples. After each addition, the sample was
allowed to equilibrate for 30 min prior to recording a spectrum.
Additions of the ctDNA were continued until no change in the
fluorescence signal was observed. For all measurements, the
excitation wavelength was 350 nm and both excitation and
emission slit widths were 5.0 nm.
0
Synthesis of 3,3 -(1,4-Phenylene)bis(1,5-diphen-yl-1,5-
pentadione) (1)
Compound 1 was synthesized in 79% yield according to
1
a procedure described in the literature. Mp 205–206 C. H
1
NMR data are as follows: H NMR (400 MHz, d -DMSO, ppm):
10
ꢂ
6
7
.96 (d, J ¼ 7.6 Hz, 8H, ortho Ar–C]O), 7.38–7.56 (m, 12H,
meta and para Ar–C]O), 7.19 (s, 2H, center Ar–H), 4.02 (t, 2H,
Ar–CH), 3.23–3.51 (m, 8H, CH –C]O).
2
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Melting temperature measurement
and excess toluene were removed by distillation, the reaction
ꢂ
mixtures were heated at 145–150 C for additional 48 h. The
Thermally induced melting of the DNA duplex in the absence
and presence of polymer PPT was determined with a Shimadzu
UV-2550 spectrometer, fitted with a water-bath thermostatic
apparatus to control the temperature. The melts were monitored
polymer PPT was essentially isolated in quantitative yield by
precipitation with the addition of ethyl acetate.
1
H NMR and elemental analysis were employed to charac-
1
terize the structure of polymer PPT. The H NMR spectrum of
ꢂ
at 260 nm, and scans were made from 25 to 90 C. Values for the
polymer PPT in DMSO-d
between 7.0 and 8.0 ppm, pyridinium ring absorptions between
.0 and 9.0 ppm, and CH absorption at 2.26 ppm, which is in
6
contained aromatic absorption
m
melting temperature (T ) were defined as the temperature at
which the absorbance of DNA at 260 nm reaches 50% of the
maximal value.
8
3
agreement with the proposed structure. The weight- and
number-average molecular weights of polymer PPT determined
by gel permeation chromatography (GPC) in DMF (LiBr) are
Circular dichroism measurement
1
5 600 and 8990, respectively, with the polydispersity index
To examine the possibility that conformational changes occur
upon binding of PPT to the DNA, circular dichroism (CD)
measurements were made on the ctDNA samples containing
varying amounts of PPT. Ellipticity measurements were
obtained up to concentrations near the point of condensation on
(
PDI) of 1.74.
Solubility of polymer
Water solubility of polymer is essential for interfacing with
biological substrates such as proteins and DNA. Thus, solubility
of the synthesized PPT was investigated in detail. The PPTs are
ꢂ
a JASCO J-716 spectropolarimeter operating at 25 C. The
region between 230 and 320 nm was scanned for each sample.
Stock solution containing 125 mM of ctDNA in PBS (50 mM, pH
7
ꢁ1
soluble in organic solvents such as DMSO (about 55 g L ),
ꢁ1
.4) was prepared and incubated for 30 min. Test solutions were
ꢁ1
3
DMF (about 65 g L ), CH OH (about 3.4 mg L ), etc., despite
prepared by adding aliquots of PPT DMSO solution (5 mM)
into the 3 mL of ctDNA stock solution (125 mM). Each spectrum
was the average of four scans.
their rigid-rod structure, which should be attributed to their all
ꢁ
para-catenated backbones. In contrast to the BF
4
counterion,
the presence of a bulky tosylate counterion in the polymer
increases its solubility in polar solvents by significantly reducing
the strong ionic interactions between positive and negative
Results and discussion
11
charges.
Additionally, this organic counterion provides
Synthesis and characterization of the polymer
a mechanism to achieve its solubility to such an extent that it
exceeds the concentration required by spectrum detection, to
form a homogeneous aqueous solution in the presence of trace
amounts of DMSO (approximately 0.1 percent of the total
volume).
Scheme 1 outlines the reaction sequence applied for the synthesis
of the poly(pyridinium salt)s. The comonomer 2 was facilely
10
synthesized according to the literature in high yield (87%) by
treatment of tetraketone 1 with triphenylmethanol and p-tolue-
nesulfonic acid. The conjugated poly(pyridinium tosylate) (PPT)
was prepared by the ring-transmutation polymerization reaction
that was carried out on heating in DMSO as previously repor-
Optical properties of polymer solution
10
ted. It would be useful in removing the water generated by the
transformation of the pyrylium rings to the pyridinium rings
Providing a baseline understanding of the intrinsic optical
properties of the conjugated PPT is necessary prior to looking at
the optical changes upon complexation with the double-stranded
DNA (dsDNA). Fig. 1 presents the UV-vis absorption and
photoluminescence (PL) spectra of polymer PPT in water. The
concentration of PPT is provided in terms of repeat units
8
,10
from the polymerization medium.
Thus, after the ring trans-
ꢂ
formation polymerization had been allowed to proceed at 110 C
for 2 h in DMSO, toluene was added, and the stirred mixtures
ꢂ
were heated to 145–150 C. After the toluene/water azeotrope
(
the following concentration is calculated in the same way). The
Fig. 1 UV-vis absorption and photoluminescence spectra of polymer
PPT aqueous solutions (lex ¼ 350 nm).
Scheme 1 Synthesis route for the conjugated poly(pyridinium salt)s.
4
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[
PPT] in Fig. 1 are 20 mM for absorption and 5 mM for PL
spectra, respectively. PPT in aqueous solution exhibits
a maximal absorption at 346 nm in the UV-vis spectrum, which is
assigned to p–p* transition in the polymer backbone. The PL
spectrum of PPT in water upon excitation at 350 nm is domi-
nated by emission centered at 494 nm, which is slightly red-
shifted by about 10 nm with respect to that of PPT containing
ꢁ
12
4
the BF counterion in DMF.
Fig. 2 shows the PL spectra of polymer PPT aqueous solutions
at various polymer concentrations. As can be seen from Fig. 2,
the most intense emission is observed when [PPT] ¼ 5 mM. When
[
PPT] < 5 mM, the intensity decreases without a change in the
emission maximum or spectral characteristics. Increasing [PPT]
above 5 mM also results in a decrease of PL intensity, but
a gradual red shift of the emission maximum takes place, which
should be attributed to the fact that the aggregation of PPT
through interchain interaction at higher concentration takes
place, resulting in the self-quenching of PPT (lower PL
Fig. 3 Fluorescence intensity and emission maxima of polymer PPT
aqueous solutions (5 mM) as a function of DMSO content in water (lex
50 nm).
¼
3
negatively charged compensating tosylate anions became domi-
nant, thus leading to reduced interchain contacts and reduced
13
intensity).
14a
self-quenching (higher emission intensities).
Polymer PPTs are a rigid rod-like molecule with the structure
as shown in Scheme 1. The conjugated backbone and aryl side
groups are hydrophobic moieties, while the cationic charged
quaternary amines control electrostatic interactions. Dissocia-
tion of the charged ionogenic groups requires polar solvents,
while the hydrophobic segments are better accommodated in
non-polar solvents. The resulting amphiphilic characteristics
UV-vis absorption spectra response to calf thymus DNA addition
Polymer PPTs are cationic conjugated polyelectrolytes, whereas
calf thymus DNA (ctDNA) carries highly electronegative
charges on phosphate groups. They can form complexes through
electrostatic interaction. To investigate the aggregation behavior
of the PPT with ctDNA in aqueous solution, the electronic
absorption spectra of the PPT in the presence of ctDNA at
various concentrations were recorded (Fig. 4). Since the
absorption of the PPT at about 260 nm overlapped with that of
the ctDNA, we only analyzed the absorption maximum of
polymer PPT at about 350 nm, which is assigned to p–p*
transition of the conjugated PPT backbone. As shown in
Fig. 4A, polymer PPT exhibits a maximal absorption at 346 nm
14
lead to different aggregation structures in different solvents. To
obtain better understanding of aggregation as a function of
solvent polarity, we examined PPT in water with varying
amounts of DMSO using fluorescence spectroscopy. As shown in
Fig. 3 and S1†, the emission intensity of PPT increases gradually
2
with the increase of the content of H O in the mixed solvents.
The shapes of the spectra show a negligible change, but distinct
emission red-shifts to 512 nm take place. Highest emission
intensity is observed in pure water. We propose that the aggre-
gation of PPT in pure DMSO with lower polarity (polarity
parameter for DMSO is 7.2 (ref. 15)) is dominated by the inter-
chain hydrophobic interactions, which leads to lower emission
intensity due to p–p interaction. Whereas, with increasing the
amount of water with the higher polarity (polarity parameter for
water is 10.2 (ref. 15)) in the mixed solvents, the electrostatic
interactions of a positively charged quaternary amine group and
4
ꢁ1
ꢁ1
(
3 ¼ 1.035 ꢀ 10 M cm ). Upon the gradual addition of
ctDNA, the absorbance at 346 nm of polymer PPT decreased
gradually while the maximal absorption peak of polymer PPT is
red shifted by about 6 nm to 352 nm, indicating that a significant
aggregate between the PPT and ctDNA is formed.
The binding constant for PPT–ctDNA complexes was calcu-
16
lated according to the reported method. The double reciprocal
plot of 1/(A ꢁ A
0
) versus 1/(ctDNA concentration) is linear with
a correlation coefficient of 0.998 (Fig. 4B), and the binding
constant (K) can be estimated from the ratio of the intercept to
0
the slope. A is the initial absorbance of the free PPT at 346 nm
and A is the recorded absorbance of complexes at different
ctDNA concentrations. The overall binding constant for PPT–
5
ꢁ1
ctDNA complexes is estimated to be 9.3 ꢀ 10 M .
PL spectra response to calf thymus DNA addition
Addition of ctDNA to PPT (5 mM) in phosphate buffer solution
results in the fluorescence quenching of the latter (Fig. 5). As the
ctDNA concentration increases from 0 to 820 nM, the photo-
luminescence (PL) intensity of the cationic conjugated poly-
electrolyte decreases to only a quarter of the PL intensity in the
absence of ctDNA, which is concomitant with slight blue-shift of
the PPT emission maximum from 510 to 494 nm. As can be seen
from the inset of Fig. 5, with the increment of the ctDNA
Fig. 2 Emission spectra of polymer PPT aqueous solutions at various
concentrations (lex ¼ 350 nm).
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4
94 nm was selected to investigate the analytical performance of
our proposed fluorescence quenching system.
Fluorescence quenching efficiencies can be quantified by the
17
use of the Stern–Volmer equation:
F /F ¼ 1 + KSV[Q]
(1)
where F and F are the fluorescence intensities in the absence and
0
i
0
i
presence of quencher, respectively, and [Q] is the quencher
concentration. The Stern–Volmer constant, KSV, provides
a direct measure of the quenching efficiency and is determined
0
from the linear portion of a plot of F /F versus [Q].
The Stern–Volmer plot of PPT quenched by ctDNA is shown
in Fig. S2†. At ctDNA concentration ranging from 0 to 0.28 mM,
6
ꢁ1
a linear plot with KSV ¼ 8.79 ꢀ 10 M (R ¼ 0.998) was
obtained. The quenching efficiency is pronounced if one
considers a fluorescence quenching system of small molecule-
18
weight stilbene and methylviologen whose KSV is as low as 15.
The extremely large quenching efficiency arises at least from two
effects as follows: first, the negatively charged ctDNA molecules
are expected to readily bind positively charged conjugated
polymer chains by a combination of coulombic and entropic
interactions, which brings about an effect of local concentration
enhancement of PPT. This improves the accessibility and thereby
high sensibility of PPT to ctDNA. Second, the conjugated
polyelectrolyte is characterized by a ‘‘molecular wire’’ effect. An
isolated conjugated polymer chain can be regarded as a highly
efficient transport medium of the electronic excited states (exci-
tons). As a result, binding events at a site might be sensed by
a whole chain, leading to amplification of the fluorescent
Fig. 4 (A) UV-vis spectra of polymer PPT (20 mM) in phosphate buffer
solution (50 mM, pH 7.4) in the presence of different concentrations of
calf thymus DNA (ctDNA), [ctDNA] ¼ 0, 0.060, 0.10, 0.17, 0.23, 0.28,
19
system.
0
.40, 0.57, 0.79, 1.36, 1.81, 2.38, 2.95, and 3.79 mM. (B) Absorbance at
46 nm of polymer PPT as a function of calf thymus DNA concentra-
In order to understand what happens at very low ctDNA
concentration to determine the detection limit of polymer PPT
for ctDNA detection, lower [ctDNA] regimes from 0 to 10 nM
were studied by using solutions of PPT at a constant initial
concentration (5 mM) and then adding aliquots of stock solutions
3
0
tions. A is the initial absorbance of the free PPT and A is the recorded
absorbance of complexes at different ctDNA concentrations.
Fig. 5 Fluorescent titration spectra of PPT (5 mM) in phosphate buffer
solutions (50 mM, pH ¼ 7.4) in the presence of calf thymus DNA at
different concentrations, [ctDNA] ¼ 0, 11.3, 22.7, 45.3, 68.0, 90.7, 113.0,
1
60.0, 280.0, 482.0, 595.0, and 820.0 nM (lex ¼ 350 nm). Inset: fluorescent
Fig. 6 Fluorescence quenching efficiency of PPT ((F
a function of calf thymus DNA concentrations. The data are based on the
average of three independent experiments. F and F denote the emission
0
ꢁ F)/F
0
ꢀ 100) as
intensity at 494 nm of PPT (5 mM) as a function of calf thymus DNA
concentrations.
0
intensity at 494 nm prior to and after addition of calf thymus DNA,
respectively. Inset: Fluorescent spectra of PPT (5 mM) in phosphate
buffer solutions (pH 7.4, 50 mM) in the presence of calf thymus DNA at
different concentrations, [ctDNA] ¼ 0, 1.5, 4.0, 5.5, 8.5 and 10.0 nM (l
¼ 350 nm).
concentration, the fluorescence intensity decreased rapidly and
then tended to saturation when the ctDNA concentration
reached 280 nM. The intensity peak of the fluorescence spectra at
ex
4
110 | J. Mater. Chem., 2012, 22, 4106–4112
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of ctDNA. The inset of Fig. 6 shows the PL spectra of PPT prior
to and after addition of low concentration ctDNA. The cali-
bration curve for the detection of ctDNA was obtained by
plotting the fluorescence quenching efficiency against the ctDNA
concentration as depicted in Fig. 6. The results showed that the
fluorescence quenching efficiency and the ctDNA concentration
possessed a good linear relationship in the concentration range
from 1.5 to 10.0 nM. The calibration equation of the best fitted
line was Y ¼ ꢁ0.2628 + 0.9056X with a correlation coefficient of
0
.998. The large slope indicates the high sensitivity of PPT to
ctDNA, which is consistent with the strong complexation of PPT
with ctDNA. However, plots of fluorescence quenching effi-
ciency vs. [ctDNA] with [ctDNA] < 1.5 nM were not linear and
the data were scattered. Thus, the detection limit under these
experimental conditions is close to 1.5 nM. This sensitivity is
close to other fluorescent assays for DNA detection using gold
Fig. 8 Circular dichroism spectra of calf thymus DNA (125 mM) in
phosphate buffer solution (50 mM, pH 7.4) upon addition of polymer
PPT. [PPT] ¼ 0, 8.3, 13.3, 16.7, and 20.0 mM from a to e, respectively.
20
nanoparticles.
Thermal denaturation studies
circular dichroism experiments were performed on solution of
ctDNA in the presence of PPT. Concentrations were chosen
below the point of precipitation (e.g., 125 mM ctDNA was
examined in the presence of up to 20 mM PPT). The intrinsic
circular dichroism activity of the PPT was recorded and found to
be negligible. In the absence of PPT, the CD spectrum of ctDNA
was of typical B-form, which exhibited a positive Cotton effect at
275 nm and a negative Cotton effect at 245 nm as shown in Fig. 8.
Upon addition of polymer PPT, the negative peak in the CD
spectrum at 245 nm was unchanged, and there was only a slight
decrease in the intensity of the 275 nm positive band as the
concentration of PPT was increased from 0 to 20 mM PPT. There
is thus no major change in conformation of the DNA structure as
PPT binds to the duplex.
To provide another experimental source of information on PPT
binding to calf thymus DNA, UV-melting transitions were
measured. Fig. 7 shows the calf thymus DNA melting curves in
the presence and absence of polymer PPT. The calf thymus DNA
‘
‘melts’’, i.e., undergoes a double-to-single strand transition at
ꢂ
4
8.1 C. The melting curve undergoes a higher-temperature shift
in the presence of PPT at a concentration as low as 2 mM,
ꢂ
ꢂ
resulting in an increase in the melting point by 10 C to 58.7 C,
which demonstrates that polymer PPT binding to calf thymus
DNA stabilized the duplex structure of the latter.
Circular dichroism measurements on the complex
Circular dichroism (CD) spectroscopy is useful in monitoring the
conformational transition of DNA, as the band due to base
stacking (275 nm) and that due to right-handed helicity (248 nm)
are quite sensitive to the solution structure of DNA and the
conformation twist of the double helix produced by the forma-
Changes in the intensity of the circular dichroism peak at
275 nm have been associated with alteration of hydration of the
helix in the vicinity of phosphate or the ribose ring as the ionic
22
concentration is altered. It would be reasonable to suggest that
exchanging a cationic polymer, such as PPT, with sodium ion
would lead to changes in hydration near the phosphate group of
the DNA helix. The decrease in the 275 nm band observed could
21
tion of supramolecular aggregates. To determine if secondary
structural changes occur upon binding of cationic PPT to DNA,
23
reflect such hydration changes. However, the magnitude of the
CD shift is small, indicating that any hydration changes are
small, at least up to the PPT concentrations studied here.
Therefore, strong electrostatic attraction may be the main
driving force for PPT/ctDNA binding.
Conclusions
In summary, a novel sensitive fluorescence-based biosensor for
homogeneous DNA detection was developed on the basis of
conjugated polyelectrolyte complexes. The operation mechanism
takes advantage of strong electrostatic attraction between
cationic conjugated poly(pyridinium salt)s and double-stranded
DNA. Further optimization of the structure and property of
poly(pyridinium salt)s may lead to simple and practical analysis
platforms for DNA hybridization assay. As a fluorescent model
system of poly(pyridinium salt)s, this type of cationic conjugated
polyelectrolytes is expected to have potential applications in
other biomolecular detections.
Fig. 7 Calf thymus DNA (50 mM) melting curves at 260 nm in the
0
absence (squares) and presence of PPT (2 mM) (circles). A and A denote
the initial absorbance and the recorded absorbance at different temper-
atures, respectively.
This journal is ª The Royal Society of Chemistry 2012
J. Mater. Chem., 2012, 22, 4106–4112 | 4111

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H. Han, A. K. Nedeltchev, H. D. Mandal, J. A. Jimenez-
Hernandez and P. M. McGannon, J. Appl. Polym. Sci., 2010, 116,
Acknowledgements
1
197; (f) A. K. Nedeltchev, H. Han and P. K. Bhowmik, Polym.
This work was financially supported by the National Natural
Science Foundation of China (no. 21074093, 20972111,
Chem., 2010, 1, 908; (g) A. K. Nedeltchev, H. Han and
P. K. Bhowmik, J. Polym. Sci., Part A: Polym. Chem., 2010, 48, 4611.
Y. Lu, C. C. Xiao, Z. F. Yu, X. S. Zeng, Y. Ren and C. X. Li, J.
Mater. Chem., 2009, 19, 8796.
2
0974055).
9
1
0 (a) P. K. Bhowmik, R. A. Burchett, H. Han and J. J. Cebe,
Macromolecules, 2001, 34, 7579; (b) F. W. Harris, K. C. Chuang,
S. A. X. Huang, J. J. Janimak and S. Z. D. Cheng, Polymer, 1994,
35, 4940; (c) S. A. X. Huang, K. C. Chuang, S. Z. D. Cheng and
F. W. Harris, Polymer, 2000, 41, 5001; (d) P. K. Bhowmik, H. Han,
A. K. Nedeltchev, H. D. Mandal, J. A. Jimenez-Hernandez and
P. M. McGannon, Polymer, 2009, 50, 3128.
Notes and references
1
(a) D. T. McQuade, A. E. Pullen and T. M. Swager, Chem. Rev., 2000,
100, 2537; (b) T. A. Taton, C. A. Mirkin and R. L. Letsinger, Science,
2000, 289, 1757; (c) E. Southern, K. Mir and M. Shchepinova, Nat.
Genet., 1999, 21, 5.
2
3
(a) P. S. Heeger and A. J. Heeger, Proc. Natl. Acad. Sci. U. S. A., 1999,
96, 12219; (b) T. M. Swager, Acc. Chem. Res., 1998, 31, 201.
11 P. K. Bhowmik, R. A. Burchett, H. Han and J. J. Cebe, Polymer,
2002, 43, 1953.
12 M. P. Makowski and W. L. Mattice, Polymer, 1993, 34, 1606.
13 C. Y. Chi, A. Mikhailovsky and G. C. Bazan, J. Am. Chem. Soc.,
2007, 129, 11134.
14 (a) S. Wang and G. C. Bazan, Chem. Commun., 2004, 2508; (b)
L. Arnt and G. N. Tew, J. Am. Chem. Soc., 2002, 124, 7664; (c)
M. Bockstaller, W. Kohler, G. Wegner, D. Vlassopoulos and
G. Fytas, Macromolecules, 2001, 34, 6359.
15 N. Cheng, Solvents Handbook, Chemical Industry Press, Beijing, 2008.
16 (a) J. J. Stephanos, J. Inorg. Biochem., 1996, 62, 155; (b) E. Froehlich,
J. S. Mandeville, C. M. Weinert, L. Kreplak and H. A. Tajmir-Riahi,
Biomacromolecules, 2011, 12, 511.
(a) B. S. Gaylord, A. J. Heeger and G. C. Bazan, Proc. Natl. Acad. Sci.
U. S. A., 2002, 99, 10954; (b) B. S. Gaylord, A. J. Heeger and
G. C. Bazan, J. Am. Chem. Soc., 2003, 125, 896; (c) B. Liu and
G. C. Bazan, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 589.
(a) B. Liu and G. C. Bazan, J. Am. Chem. Soc., 2004, 126, 1942; (b)
K. Y. Pu and B. Liu, Macromolecules, 2008, 41, 6636; (c) K. Y. Pu
and B. Liu, Adv. Funct. Mater., 2009, 19, 277.
4
5
(a) H. A. Ho, K. Dore, M. Boissinot, M. G. Bergeron,
R. M. Tanguay, D. Boudreau and M. Leclerc, J. Am. Chem. Soc.,
2005, 127, 12673; (b) K. F. Karlsson, P. Aasberg, K. P. R. Nilsson
and O. Inganaes, Chem. Mater., 2005, 17, 4204; (c) H. A. Ho,
M. Boissinot, M. G. Bergeron, G. Corbeil, K. Dore, D. Boudreau
and M. Leclerc, Angew. Chem., Int. Ed., 2002, 41, 1548.
17 J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer
Academic/Plenum Publishers, New York, 1999.
6
(a) B. Liu, S. Wang, G. C. Bazan and A. Mikhailovsky, J. Am. Chem.
Soc., 2003, 125, 13306; (b) M. Stork, B. S. Gaylord, A. J. Heeger and
G. C. Bazan, Adv. Mater., 2002, 14, 361; (c) Q. H. Xu, B. S. Gaylord,
S. Wang, G. C. Bazan, D. Moses and A. J. Heeger, Proc. Natl. Acad.
Sci. U. S. A., 2004, 101, 11634; (d) Q. Chen, Q. Y. Cheng, Y. C. Zhao
and B. H. Han, Macromol. Rapid Commun., 2009, 30, 1651.
H. Peng, C. Soeller and J. Travas-Sejdic, Chem. Commun., 2006, 3735.
(a) P. K. Bhowmik, R. A. Burchett, H. Han and J. J. Cebe, J. Polym.
Sci., Part A: Polym. Chem., 2001, 39, 2710; (b) P. K. Bhowmik,
H. Han, J. J. Cebe, I. K. Nedeltchev, S.-W. Kang and S. Kumar,
Macromolecules, 2004, 37, 2688; (c) P. K. Bhowmik, H. Han and
A. K. Nedeltchev, J. Polym. Sci., Part A: Polym. Chem., 2006, 44,
18 L. H. Chen, D. W. McBranch, H. L. Wang, R. Helgeson, F. Wudl and
D. G. Whitten, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 12287.
19 Q. Zhou and T. M. Swager, J. Am. Chem. Soc., 1995, 117, 12593.
20 S. B. Zhang, Z. S. Wu, M. Xie, G. L. Shen and R. Q. Yu, Chin. J.
Chem., 2009, 27, 523.
21 (a) M. J. B. Tunis-Schneider and M. F. Maestre, J. Mol. Biol., 1970,
52, 521; (b) M. J. Carvlin, N. Datta-Gupta and R. J. Fiel, Biochem.
Biophys. Res. Commun., 1982, 108, 66; (c) V. I. Ivanov,
L. E. Minchenkova, A. K. Schyolkina and A. I. Poletayev,
Biopolymers, 1973, 12, 89.
7
8
22 (a) S. Hanlon, S. Brudno, T. T. Wu and B. Wolf, Biochemistry, 1975,
14, 1648; (b) B. Wolf and S. Hanlon, Biochemistry, 1975, 14, 1661.
23 H. S. Charles and B. C. Jonathan, J. Am. Chem. Soc., 1997, 119,
10920.
1028; (d) P. K. Bhowmik, S. Kamatam, H. Han and
A. K. Nedeltchev, Polymer, 2008, 49, 1748; (e) P. K. Bhowmik,
4
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