Design of cationic conjugated polyelectrolytes for DNA concentration determination

Article abstract of DOI:10.1021/ja072471s

Cationic conjugated polyelectrolytes poly1₁₀ and poly1 ₂₀ were designed, synthesized, and characterized with the anticipation of function in the determination of double-stranded DNA concentration [dsDNA]. Their structures contain a π

Full text of DOI:10.1021/ja072471s

Published on Web 08/18/2007
Design of Cationic Conjugated Polyelectrolytes for DNA
Concentration Determination
Chunyan Chi, Alexander Mikhailovsky, and Guillermo C. Bazan*
Contribution from the Departments of Chemistry and Biochemistry and Materials, Institute for
Polymers and Organic Solids, UniVersity of California, Santa Barbara, California 93106
Received April 9, 2007; E-mail: bazan@chem.ucsb.edu
Abstract: Cationic conjugated polyelectrolytes poly110 and poly120 were designed, synthesized, and
characterized with the anticipation of function in the determination of double-stranded DNA concentration
[dsDNA]. Their structures contain a π-delocalized optically active backbone composed of phenylene-
fluorene segments copolymerized with 2,1,3-benzothiadiazole (BT) units and charged pendant groups that
allow excellent solubility in water. The subscript in poly1x refers to the molar percent of BT units in the
chain. Addition of dsDNA to poly110 or poly120 results in a change in the color of emission from blue to
green. These spectral changes can be treated to obtain the parameter δ, which can be used to generate
calibration curves that indicate [dsDNA]. Analysis of photoluminescence spectra reveals that dsDNA addition
gives rise to more efficient FRET from blue emitting segments to the BT sites, an increase in the BT emission
quantum yield, and partial quenching of the phenylene-fluorene segments. Studies were also carried out
to maximize the range of [dsDNA] determination. We find that by combining the response from two different
initial concentrations of poly110, it is possible to generate calibration curves that respond with a difference
in [dsDNA] of over 7 orders of magnitude.
Introduction
Substantial efforts have been placed in developing homoge-
neous and heterogeneous biosensor assays that take advantage
Conjugated polyelectrolytes (CPEs) are polymers containing
a π-conjugated backbone and functional groups that ionize in
high dielectric media.1 These materials combine the semicon-
ducting and photon harvesting properties of electronically
delocalized polymers with the charge-mediated behavior of
polyelectrolytes. CPEs offer interesting possibilities for opto-
electronic applications, including the formation of surface
dipoles,2 ion motion to compensate injected charges,3 and
solubility in polar solvents to fabricate multilayer structures by
alternating solvent polarity with minimum disturbance of
underlying layers.4 Furthermore, recent studies have shown that
the charge carrier mobilities in CPEs are similar or larger than
those in neutral conjugated polymers of similar structure.5
of the optical amplification afforded by CPEs.6 The ionic groups
are essential for increasing water solubility, an important
requirement for biological detection.7,8 It has been possible
through substantially different strategies to design assays that
are specific to proteins,9,10 DNA,11 and RNA.12 One particular
sensing strategy takes advantage of the large optical cross section
of CPEs in combination with a biomolecular recognition event
that triggers fluorescence resonance energy transfer (FRET)13
to a reporting, or “signaling”, dye. Electrostatic interactions are
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10.1021/ja072471s CCC: $37.00 © 2007 American Chemical Society

Design of Cationic Conjugated Polyelectrolytes
A R T I C L E S
Chart 1. Molecular Structure of PFPBx
emitting BT sites occurs more efficiently via interchain contacts
in aggregates than that via the intrachain process within more
isolated polymer chains.18
Addition of negatively charge polyelectrolytes to PFPBx
induces complexation as a result of cooperative electrostatic
interactions.19 As illustrated in Figure 1, the local concentration
of PFPBx in these aggregates is increased, thereby shifting the
emission from blue to green. Such optical changes can be
induced with dsDNA or single-stranded DNA (ssDNA), and
the spectral features can be analyzed to determine the concentra-
tion of DNA in an unknown sample.20 The overall process
provides an alternative method to emissive intercalating dyes
for measuring [DNA] under conditions too dilute for absorbance
spectroscopy (g3.8 × 10-7 M in base pairs, bps, or 250 ng/
mL).21,22 Such information is important in a variety of applica-
tions ranging from the quantification of polymerase chain
reaction products to the evaluation of biosensors.23
essential for controlling the average distance between optical
partners and thereby the sensitivity and selectivity of the assay.
The overall process is complex, and there are substantial gaps
in our understanding, in particular the general shape, size, and
molecular organization on the resulting aggregates. Despite the
supramolecular complexity, molecular design has produced a
variety of structures for optimizing assays, which allow one to
control the color of emission,14 the ability of the main chain to
adapt to the secondary structure of biomolecules,15 the chain
dependence of the recognition event,16 and the ratio of FRET
vs photoinduced charge transfer.17
Structure-function relationship studies have shown that poly-
((9,9-bis(6′-N,N,N-trimethylammoniumbromide)hexyl)fluorene-
co-alt-1,4,-phenylene) (PFPBx in Chart 1) containing a fractional
substitution of fluorene monomers with 2,1,3-benzothiadiazole
(BT), in combination with dye-labeled PNA probes, can be used
in multicolor assays. The subscript “x” refers to the percentage
of phenylene-BT units in the main chain, while 100 - x %
corresponds to the fraction due to phenylene-fluorene segments.
One important feature of PFPBx is that its emission is
concentration dependent; blue emission occurs under dilute
conditions while green emission is observed in more concen-
trated solutions. The working hypothesis is that FRET from the
blue-emitting phenylene-fluorene segments to the green-
Using PFPB7 one obtains an upper detection limit for
[dsDNA] of 1.5 × 10-7 M base pairs (bps) and a lower detection
limit of 6.0 × 10-10 M bps. For comparison, commercially
available fluorescence methods using intercalating dyes can
detect concentrations as low as 3.8 × 10-11 M bps (25 pg/mL)
for dsDNA and 3.0 × 10-10 M in bases (100 pg/mL) for
ssDNA.24-31 The upper limit for PFPB7 is restricted by the
stoichiometric relationship between negative and positive
charges. Increasing [PFPB7] provides the appearance of the
green emission in the absence of dsDNA, and the assay becomes
less reliable. Factors that determine the lower detection limit
remain poorly understood at this time.
In this contribution we disclose the design of cationic
conjugated polyelectrolytes with optimized molecular features
that allow for the determination of [dsDNA] from 3 × 10-12 to
2 × 10-5 M. We start by providing a rationale for the choice
of structure and an analysis of the intrinsic optical properties
of these new materials as a function of experimental conditions.
We then examine perturbations in emission properties upon
complexation with dsDNA and show that the polyelectrolyte
complexation leads to changes in FRET efficiencies, optical
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J. AM. CHEM. SOC. VOL. 129, NO. 36, 2007 11135

A R T I C L E S
Chi et al.
Figure 1. Schematic representation of optical changes induced by complexation of dsDNA with PFPBx. (a) Isolated chains of PFPBx emit blue light in the
absence of DNA. (b) Aggregation with negatively charged dsDNA induces interchain energy transfer to BT sites and changes emission color to green.
Chart 2. Molecular Structures of poly110 and poly120
output, and self-quenching. Despite this complexity, we show
how that the spectral changes can be treated to yield a parameter
δ, which forms the basis for generating calibration curves. The
dependence of δ on specific conditions such as the concentra-
tions of the conjugated polyelectrolytes and the dsDNA, type
of medium, and conjugated polyelectrolyte composition provides
insight into the mechanism of action of the assay and provides
the foundation for optimizing the assay so that it is responsive
to 7 orders of magnitude differences in [dsDNA].
Scheme 1 shows the synthetic route to poly110 and poly120.
Treatment of 2,7-dibromofluorene (1) with 45% KOH aqueous
solution, followed by reaction with excess 1,3-dibromopropane,
provides 2,7-dibromo-9,9′-bis(3′′-bromopropyl)fluorene (2) in
85% yield. Compound 2 was transformed into its corresponding
boronic ester, 2,7-bis(4,4,5,5-tetramethyl-[1,3,2]-dioxaborolane)-
9,9-bis(3′-bromopropyl)fluorene (3), by Miyaura reaction in 85%
yield.34 Commercially available 1,4-dibromo-2,5-dihydroxyl
benzene (4) was treated with 2-[2-(2-chloroethoxy)ethoxy]-
ethanol in the presence of K2CO3 in DMF at 80 °C to give 5,
in which the hydroxyl groups were then protected by reaction
with tosylchloride to afford compound 6. The tosylate groups
in 6 were converted into bromides by treatment with LiBr in
refluxing acetone to provide compound 7. Suzuki cross-coupling
copolymerization of 3, 7, and 4,7-dibromo-2,1,3-benzothia-
diazole (8)14 in the appropriate ratios provides the neutral
precursor poly110N and poly120N in which 10% and 20% of
the monomer units are BT, respectively. The BT content is
regulated by the ratio of 7 and 8 at the synthesis stage. Since
the monomer reactivity ratios are not known for this type of
polymerization, we assume a random distribution of monomers
throughout the chain. Purification of the precursor materials is
accomplished by precipitation of chloroform solutions into
methanol three times. In the final step, the neutral polymers
are treated with condensed trimethylamine in a THF/methanol
mixture. After removal of volatiles, the target cationic species
poly110 and poly120 were obtained in greater than 90% yields.35
Results and Discussion
Design, Synthesis, and Characterization of poly110 and
poly120. The primary concern for polymer design was to increase
substantially the solubility in aqueous media. With this con-
sideration in mind, we chose to synthesize copolymers poly110
and poly120, as shown in Chart 2. Structural elements incor-
porated to increase solubility include shorter alkyl chains on
the fluorene monomers, which reduce the hydrophobic content
of the chain,32 and the introduction of tetralkylammonium units
onto the phenylene monomers via oligo(ethylene oxide) seg-
ments.33 Another difference, relative to conjugated polyelec-
trolytes derived from PFPBx, is that the BT sites are flanked
by two fluorene repeat units.
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Design of Cationic Conjugated Polyelectrolytes
A R T I C L E S
a
Scheme 1. Synthetic Entry into poly110 and poly120
a (i) Br(CH2)3Br, 45% KOH, 80 °C; (ii) bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, DMSO, 80 °C; (iii) 2-[2-(2-chloroethoxy)ethoxy]ethanol, K2CO3,
DMF, 80 °C; (iv) TsCl, DMAP, Et3N, CH2Cl2, 0 °C f 25 °C; (v) LiBr, acetone, reflux; (vi) Pd(PPh3)4, 2 M K2CO3, toluene; (vii) N(CH3)3, THF/H2O.
Analysis by 1H NMR spectroscopy in d6-DMSO reveals broad
overlapping signals that preclude accurate determination of the
degree of quaternization. We relied instead on elemental analysis
for confirmation of structure (see Experimental Section).
Returning to our goal of increasing water solubility, we estimate
that poly110 and poly120 display a water solubility of up to 15
mg/mL. For PFPBx a similar evaluation is difficult since a small
amount of methanol is necessary prior to water addition. It is
important to note the different charge densities and water
solubilities; poly120 (or poly110) has a positive charge density
of 3.2/RU (or 3.6/RU), where RU refers to the average polymer
repeat unit,36 twice that of PFPB7 (1.7/RU). The increased
charge density along the polymer chain is an important
additional contributor to the increase in solubility.
UV-vis Absorption and Photoluminescence (PL) Spectra.
Providing a baseline understanding of the intrinsic optical
properties of the new conjugated polyelectrolytes is necessary
prior to looking at the optical changes upon complexation with
dsDNA. Figure 2 shows the absorption and photoluminescence
(PL) spectra of poly110 and poly120 in water and from films
obtained by spin coating from methanol. The concentrations of
the polymers are provided in terms of RUs. The [RU]s in Figure
2 are 1 × 10-5 M for absorption and 1 × 10-6 M for PL spectra,
respectively. The absorption spectra show two bands in the
300-500 nm absorption range (Figure 2a). The band at ∼352
nm is assigned to the π to π* transition in the fluorene-
phenylene segments, while the band located centered near 430
nm is assigned to the BT sites.37 As the fractional composition
of BT in the chain decreases from 20% to 10%, one observes
a concomitant reduction of the 430 nm band and a 10 nm red
shift of the π to π* transition. The absorption spectra in the
films are red-shifted approximately 10 nm relative to water.
Overall, the absorption spectra reflect the composition of the
polymer chains with the band at 430 nm increasing in strength
as the BT content increases. This last observation will be
important when we examine the effect of the PL spectral shapes
as a function of polymer concentration.
The PL spectra of poly110 and poly120 in water upon
excitation at 360 nm are dominated by emission centered at
412 nm (Figure 2b). A broad tail from 500 to 650 nm can be
observed in the case of poly120. In the films, one observes only
green emission and a slightly red-shifted emission maximum
for poly120 (567 nm) relative to poly110 (541 nm). The solution
and solid-state emission spectra are consistent with the idea that
less efficient FRET from the phenylene-fluorene segments to
the BT sites occurs in isolated chains, relative to situations where
interchain energy transfer is possible.
Changes in the PL spectra of poly120 in water as a function
of polymer concentration are shown in Figure 3. These
measurements were carried out in a 1 cm × 1 cm cuvette. The
(37) (a) Bangcuyo, C. G.; Evans, U.; Myrick, M. L.; Bunz, U. H. F.
Macromolecules 2001, 34, 7592. (b) Jayakannan, M.; Van Hal, P. A.;
Janssen, R. A. J. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 251. (c)
Yasuda, T.; Imase, T.; Yamamoto, T. Macromolecules 2005, 38, 7378. (d)
Hou, Q.; Zhou, Q.; Zhang, Y.; Yang, W.; Yang, R. Cao, Y. Macromolecules
2004, 37, 6299. (e) Herguth, P.; Jiang, X.; Liu, M. S.; Jen, A. K.-Y.
Macromolecules 2002, 35, 6094. (f) Huang, J.; Xu, Y.; Hou, Q.; Yang,
W.; Yuan, M.; Cao, Y. Macromol. Rapid Commun. 2002, 23, 709. (g)
Yamamoto, T.; Fang, Q.; Morikita, T. Macromolecules 2003, 36, 4262.
(h) Cadby, A.; Dean, R.; Jones, R. A. L.; Lidzey, D. G. AdV. Mater. 2006,
18, 2713. (i) Vehse, M.; Liu, B.; Edman, L.; Bazan, G. C.; Heeger, A. J.
AdV. Mater. 2004, 16, 1001.
(36) One repeat unit includes one fluorene moiety and relevant content of
phenylene and BT. The molecular weight of the RUs are (1 substituted
fluorene + 0.6 substituted phenylene + 0.4 BT ) 1 × 522.1 + 0.6 ×
614.2 + 0.4 × 134.0 ) 944.2) 944.2 g/mol and (1 substituted fluorene +
0.8 substituted phenylene + 0.2 BT ) 1 × 522.1 + 0.8 × 614.2 + 0.2 ×
134.0 ) 1040.3) 1040.3 g/mol for poly120 and poly110, respectively.
9
J. AM. CHEM. SOC. VOL. 129, NO. 36, 2007 11137

A R T I C L E S
Chi et al.
Figure 3. Emission properties of poly120 in concentrated solutions are
modified by inner filter effects. (a) Concentration dependence of the PL
spectra of poly120 (λexc ) 360 nm) in water using a 1 cm × 1 cm cuvette.
(b) PL intensity vs [RU] from 7 × 10-8 to 1 × 10-3 M at 414 nm (black)
and 580 nm (red).
Figure 2. Dependence of absorption and emission spectra as a function of
polymer structure in water and in the bulk. (a) UV-vis absorption and (b)
PL spectra (λexc ) 360 nm) of poly120 (black curves) and poly110 (red
curves) in water (solid line) and as thin films (line and triangles). (a) [RU]
) 1 × 10-5 M; (b) [RU] ) 1 × 10-6 M.
the most intense emission at 414 nm was found at [RU] ) 8 ×
10-5 M (see Figure S-1 in the Supporting Information). Self-
absorption thus sets an upper limit for [DNA] determination;
the exact value is determined by the excitation and emission
path lengths and therefore by the dimensions of the cuvette.
From a broader perspective, it is informative to compare the
threshold [RU]s for poly110 (2 × 10-5 M) and poly120 (2 ×
10-5 M) to that of PFPB7 (1 × 10-6 M). The limited solubility
of PFPB7 induces aggregation and green emission after the
threshold [RU]. That the PL spectra of poly120 (or poly110) are
less perturbed by [RU] relative to PFPB7 nicely reflects the
increased water solubility.
PL Response to dsDNA Addition: Saturation Point.
Having established the emission profiles of poly110 and poly120
under different conditions provides us with the baseline for
understanding perturbations upon complexation with dsDNA.
Figure 4a shows the changes in the PL spectra of poly120 in
water ([RU]0 ) 2.0 × 10-5 M; [RU]0 corresponds to the initial
polymer concentration prior to dsDNA addition) upon addition
of dsDNA in increments of 1 × 10-6 M ([dsDNA] will be
reported relative to bps throughout the paper) up to a concentra-
tion of 1.8 × 10-5 M bps. The dsDNA used in all the studies
is 30 bps long. A distinguishing feature of the PL spectra is
that as [dsDNA] increases, the intensity of the green emission
band grows at the expense of the blue emission, via an apparent
isosbestic point at 485 nm. During the evolution of PL spectra
shown in Figure 4a, the [RU] decreases from 2 × 10-5 M to
1.35 × 10-5 M due to dilution by addition of dsDNA. Changes
in the PL spectra saturate when [dsDNA] ) 1.6 × 10-5 M.
Under these conditions [RU]/[dsDNA] ≈ 0.9 and the ratio of
most intense emission is observed when [RU] ) 2.0 × 10-5
M. When [RU] < 2.0 × 10-5 M, the intensity decreases without
a change in emission maximum or spectral characteristics.
Increasing [RU] above 2.0 × 10-5 M also results in a decrease
of PL intensity at 414 nm, but a gradual red shift of the emission
maximum takes place, together with a change in the general
features of the band. Similar behavior is observed with poly110.
At the higher [RU]s, the spectra cannot be described by a simple
sum of the blue and green components. However, the spectral
changes can be accommodated by taking into consideration inner
filter effects because of the overlap between the emission band
and the BT-centered absorption (see Figure 2).
Inspection of how the PL intensity changes as a function of
polymer concentration and wavelength should yield additional
supporting information on the importance of inner filter effects.
Figure 3b provides the change in PL intensity at 414 and 580
nm vs [RU] ([RU] ) 7 × 10-8 to 1.0 × 10-3 M). One can
observe a decrease of the PL emission at both wavelengths for
[RU] > 2.0 × 10-5 M. However, the rate of decrease is more
pronounced at 414 nm. These observations are consistent with
the idea of a substantial inner effect in the BT absorption region.
At 580 nm such absorption is not as significant (Figure 2a),
and the decrease in this region of the spectrum may be attributed
to self-quenching as a result of polymer aggregation, particularly
when [RU] > 8.0 × 10-5 M. Furthermore, when a 0.2 cm ×
0.2 cm cuvette was used in an effort to decrease self-absorption,
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Design of Cationic Conjugated Polyelectrolytes
A R T I C L E S
Figure 5. Method for using PL spectra to calculate δ using eq 1. The
black and red curves correspond to the emission of poly120 in the absence
of dsDNA and after the nth addition of dsDNA, respectively. The blue curve
is the normalized spectra of the black curve at 414 nm. The curves were
extracted from Figure 4; the [dsDNA] is 8.8 × 10-6 M for the red curve.
30% decrease in the PL intensity of poly10. Thus, even though
there is a well-defined isosbestic point in Figure 4, several
concurrent processes are taking place: the FRET efficiency
increases, the BT PL quantum yield increases, and there is partial
quenching of the phenylene-fluorene segments.
Despite the complex mix of optical processes involved in
Figure 4a, quantitative information on how the PL spectra
correlate with [dsDNA] can be obtained via the dimensionless
parameter δ, defined in eq 1:
Bn
Figure 4. Emission of poly120 is red-shifted and increases in intensity upon
complexation with dsDNA. (a) PL spectra (λexc ) 360 nm) of poly120 ([RU]0
) 2.0 × 10-5 M) with 32 µL additions of 5.0 × 10-5 M of 30 bp dsDNA.
The [dsDNA] range spans from 0 to 1.8 × 10-5 M. (b) UV-visible absorp-
tion spectra of poly120 ([RU] ) 2.0 × 10-5 M) and poly120/dsDNA com-
plexes in water ([RU] ) 2.0 × 10-5 M and [dsDNA] ) 1.6 × 10-5 M).
Gn - G0
( )
B0
δ )
(1)
N
where G0 and Gn are the integrated (green) fluorescene in the
range of 520 to 660 nm in the absence of dsDNA and after the
nth addition of dsDNA, respectively; B0 and Bn are the integrated
(blue) fluorescence in the 375-470 nm range in the absence
and after the nth addition of dsDNA, respectively; N is a
normalization factor, obtained in our case by using the integrated
emission of poly120 in the 375 to 470 nm range at [RU] ) 1.0
× 10-6 M, which was used for all δ determinations. Including
N allows comparison of measurements from different instru-
ments and optical collection conditions thereby greatly simplify-
ing data analysis. An illustration of how to extract δ from PL
spectra is shown in Figure 5, where the emission in the absence
of dsDNA (black curve) is normalized at 414 nm relative to
the spectrum obtained after the nth addition of dsDNA (red
curve). We note here that the apparent spectral dissimilarity
between Figure 5 and Figure 4a is due to the fact that we chose
a semilogarithmic scale for the PL intensity in Figure 5. This
choice was made to better highlight the differences between
G0 and Gn. With the normalized emission spectrum (blue curve)
one can extract G0(Bn/B0). The increase of the integrated green
emission as a result of dsDNA addition is obtained from Gn -
G0(Bn/B0), which is proportional to δ. It is also worth emphasiz-
ing that eq 1 was obtained empirically and is based on the antic-
ipation that the increase of the green band would be proportional
to the fraction of polymer chains that are complexed to dsDNA.
Probing the effect of the initial conjugated poyelectrolyte/
dsDNA stoichiometric ratio is relevant because the resulting
aggregate structures can, under certain circumstances, be
the negative charge to positive charge (R-/+) is approximately
0.7. The absorption spectra of poly120 ([RU] ) 2 × 10-5 M)
in the absence and presence dsDNA (1.6 × 10-5 M) given in
Figure 4b show a red shift of ∼5 nm for the π f π* transition
band and of ∼15 nm at longer wavelengths, indicating the new
environment upon complexation with dsDNA. The changes
observed in the PL spectra are similar when using poly110.
We now dissect the spectral evolution in Figure 4a into
independent contributions from changes of the BT emission
output, the FRET efficiency, and emission self-quenching of
the polymer itself. We approach the problem of BT output by
exciting the solutions at the two [dsDNA] extremes in Figure
4a at 451 nm and measuring the resulting PL spectra. This
excitation wavelength results in identical absorbance values and
corresponds to direct BT excitation. These experiments show
that the PL intensity of BT is approximately 11 times stronger
when in the presence of the dsDNA. Under the same conditions,
but exciting at 360 nm, where excitation of the phenylene-
fluorene segments occurs, one finds an 18-fold increase in BT
emission. Thus, we can estimate that the FRET efficiency in
the presence of dsDNA increases by a factor of approximately
1.6. It is also significant that the emission of the parent BT-
free polymer, i.e., poly10, is also quenched upon dsDNA
complexation (see Figure S-2 in the Supporting Information).
For example, addition of 1.6 × 10-5 M dsDNA results in a
9
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A R T I C L E S
Chi et al.
Figure 6. Different polymer concentrations show overlap in the response
plots of δ vs [dsDNA] for 30 bp dsDNA and poly120 at [RU]0 ) 1 × 10-6
M (blue), 5 × 10-6 M (green), 1 × 10-5 M (red), 2 × 10-5 M (black).
Table 1. Slopes of δ vs [dsDNA] Plots in Different [dsDNA]
Ranges
1
[dsDNA] rangea (M)
slope (M-
)
8.0 × 10-7 to 1.3 × 10-7
2.0 × 10-9 to 3.3 × 10-10
2.4 × 10-10 to 6.0 × 10-11
3.0 × 10-11 to 5.0 × 10-12
1.6 × 106
2.1 × 107
1.7 × 108
1.7 × 109
a
[RU]0 ) 1 × 10-6 M.
controlled by kinetic effects.38 Figure 6 provides the δ response
to dsDNA at four different initial polymer concentrations ([RU]0
) 1.0 × 10-6, 5.0 × 10-6, 1.0 × 10-5, 2.0 × 10-5 M). We did
not examine conditions when [RU]0 > 2 × 10-5 M due to the
inner filter effect and the resulting distortions in the PL spectra
shown in Figure 3. Figure 6 shows that there is a linear increase
of δ vs [dsDNA] up to a critical [dsDNA] value, after which
one observes a decrease of δ. The point of critical [dsDNA]
depends on the original concentration of the polyelectrolyte.
For example, when [RU]0 ) 2.0 × 10-5 M, the point at which
δ begins to decrease occurs at [dsDNA] ) 1.6 × 10-5 M. A
similar linear dependence of δ vs [dsDNA] is observed for the
lower [RU]0s, except that the critical [dsDNA] is lower.
Regardless of [RU]0, the saturation limit occurs when [RU]0/
[dsDNA] is approximately 0.9; under these circumstances the
ratio of negative to positive charges (R-/+) is approximately
0.7. Such a constant ratio indicates a stoichiometric limit for
the polyelectrolyte complex. From a practical perspective, the
saturation point provides the limit for the proportional correlation
between δ and [dsDNA] at higher concentrations. That the
slopes in the δ vs [dsDNA] plots are insensitive to [RU]0 will
have significant ramifications later when we incorporate the
response of two initial polymer concentrations to attain a
sensitive response across a wide [dsDNA] range.
Figure 7. Change in PL spectra and δ are minor when poly120 is in large
excess. (a) Plot of δ as a function of [dsDNA] in the concentration range
of 5 × 10-12 to 3 × 10-11 M; (b) PL spectra of poly120 ([RU]0 ) 1 × 10-6
M) with 5 µL additions of 1.6 × 10-9 M of [dsDNA] (λexc ) 360 nm).
summarized in Table 1. Four specific [dsDNA] ranges were
probed (in M bps: 1.3 × 10-7 to 8 × 10-7, 3.3 × 10-10 to 2
× 10-9, 6 × 10-11 to 2.4 × 10-10, and 5 × 10-12 to 3 × 10-11
)
by adding 5 µL aliquots of stock solutions with concentrations
of 5 × 10-5, 1 × 10-7, 1.8 × 10-8, and 1.6 × 10-9 M bps,
respectively. Plots of δ vs [dsDNA] are linear within each
concentration range, with each plot consisting of at least six
independent measurements. Figure 7 shows the result for the
lowest concentration range. Comparison of Figures 7b and 4
highlights that only minor changes occur in the PL spectra at
low concentrations and illustrates the low δ values obtained
with small quantities of dsDNA. Plots of δ vs [dsDNA] with
[dsDNA] < 5 × 10-12 M were not linear; the data were
scattered. Thus, the lower limit for the assay under these
experimental conditions is close to [dsDNA] ) 5 × 10-12 M.
Examination of Table 1 shows that the slopes of linear plots
are not equal in the different concentration regimes. There is a
progressive increase in slope with decreasing [dsDNA], from
2.1 × 107 M-1 ([dsDNA] ) 2.0 × 10-9 M to 3.3 × 10-10 M)
to 1.7 × 109 M-1 ([dsDNA] ) 3.0 × 10-11 M to 5.0 × 10-12
M). At the higher concentrations in Figure 6, the slope is 1.6 ×
106 M-1. Therefore, the proportionality constant between δ and
[dsDNA] changes when one examines very wide concentration
ranges. A direct consequence is that extrapolation is not reliable
and δ vs [dsDNA] calibration curves thus need to be established.
Examination of PL Response across a Broad [dsDNA]
Range. We now examine the behavior of δ throughout a broad
Examination of the Lower [dsDNA] Limit. We turn our
attention in this section on what happens at very low [dsDNA]
to determine the lower limit of the δ response. Lower [dsDNA]
regimes were studied by using solutions of poly120 at a constant
initial concentration ([RU]0 ) 1 × 10-6 M), and the results are
(38) Wang, S.; Bazan, G. C. Chem. Commun. 2004, 2508.
(39) Israelachvili, J. Intermolecular & Surface Forces; Academic Press: London,
1992.
(40) Wang, F.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 15786.
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Design of Cationic Conjugated Polyelectrolytes
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Figure 9. Plot of δ vs [dsDNA] in the concentration range 3 × 10-12 to
3 × 10-5 M using a double logarithmic scale. The [RU]0 of poly110 is at
2 × 10-5 M.
Figure 8. δ response across a wide [dsDNA] range reveals three regimes
and two inflection points. Plot of δ vs [dsDNA] in the concentration range
3 × 10-12 to 2.1 × 10-5 M. The [RU]0 of poly120 is at 2 × 10-5 M. The
inset shows the linear plot of δ vs [dsDNA], with [dsDNA] from 8.0 ×
10-6 to 1.8 × 10-5 M.
average RU of poly110. It should be noted, however, that the
plot in the linear scale shows that poly120 has a steeper slope
(m ) 1.6 × 106 M-1) than poly110 (m ) 1.3 × 106 M-1
)
between [dsDNA] ) 2 × 10-8 M bps and 1.6 × 10-5 M bps,
thus providing higher sensitivity (see Figure S-4 in the Sup-
porting Information).
[dsDNA] range. The experiments were done with poly120 at
[RU]0 ) 2 × 10-5 M. Stock solutions of dsDNA with different
concentrations were prepared and then added in appropriate
volumes to the polymer solution in order to cover a [dsDNA]
range from 3 × 10-12 to 2.1 × 10-5 M. The results are shown
in Figure 8, where δ vs [dsDNA] is plotted using a double
logarithmic scale. The plot in Figure 8 can be broken down
into three regimes, labeled “I”, “II”, and “III”, with inflection
points “A” and “B”. In regime “I”, where [dsDNA] increases
from 3 × 10-12 to 2 × 10-8 M, the blue component of the PL
spectra decreases slightly and there are negligible changes in
the green component (Figure S-3a). The slope using the double
logarithmic scale is less than 1. From 2 × 10-8 to 1.6 × 10-5
M, i.e., regime “II”, the changes in PL spectra are more
significant and are similar to those shown in Figure 4 (see Figure
S-3b). Here the decrease in blue emission is accompanied by a
rapid increase of green emission. In Figure 8, the slope in this
[dsDNA] range is approximately 1. In the “III” regime, i.e.,
after the saturation limit, the blue emission remains relatively
constant and the green band decreases, possibly due to self-
quenching in the aggregated state (see inset of Figure 8 and
Figure S-3c). In Figure 8, the “A” turning point, where one
kind of responsive regime changes to another one, occurs at 2
× 10-8 M. “B” is the previously discussed stoichiometric limit
for these polyelectrolyte complexes, which occurs at 1.6 × 10-5
M and sets the upper limit for measuring [dsDNA].
Comparison of poly110 and poly120. Comparison of the δ
vs [dsDNA] plots using poly110 and poly120 allows us to look
into how the polymer structure influences the sensitivity of the
overall assay. We examined the entire [dsDNA] range measure-
ment with poly110, and the results of this study are shown in
Figure 9. From these data one observes that the overall response
of poly110 is similar to that of poly120; the three regimes “I”,
“II”, “III” and the two turning points “A”, “B” are contained in
the plot. Turning point “A” at 2 × 10-8 M is similar to that
observed with poly120. The upper limit set by “B” is at 1.9 ×
10-5 M, which is slightly higher than that of poly120 (1.6 ×
10-5 M), perhaps as a result of the higher charge density per
Effect of [RU]0 in the Low [dsDNA] Regime. One observes
from Figure 6 that the high concentration response limit of δ is
determined by point “B” and therefore by [RU]0. However,
starting with [RU]0 ) 2.0 × 10-5 M, the R-/+ in the “I” regime
of Figures 8 or 9 is small, from 1.9 × 10-7 to 1.3 × 10-3. In
other words, there is a very large excess of conjugated
polyelectrolyte relative to dsDNA. The result is unsurprising;
only minor perturbations in the PL spectra are observed since
most of the conjugated polymer chains remain in their original
state (see Figure S-3a in the Supporting Information for poly120).
Under the conditions where there is a large excess of polymer,
Gn ≈ G0 and Bn ≈ B0. From eq 1, one sees that a small change
in δ would be expected. Lowering [RU]0 was anticipated to
provide a more sensitive response, since when a larger fraction
of polymer chains are complexed to dsDNA, then Gn > G0 and
Bn < B0. For studying the low concentration regimes, we focused
on poly110 because its higher PL quantum efficiency in water
(4 ( 1%) relative to that of poly120 (1 ( 0.4%) was expected
to provide better signal-to-noise.
Figure 10 allows for a comparison of the changes in the PL
spectra of poly110 at different [RU]0s (in M: 2 × 10-5, 1 ×
10-6, and 2 × 10-7) upon addition of similar dsDNA quantities
(from 3 × 10-12 to 2 × 10-7 M). For ease of comparison the
PL spectra were normalized relative to the intensity in the
absence of dsDNA. When the polymer concentration is highest
([RU] ) 2 × 10-5 M, Figure 10a), the blue component of the
spectra changes by ∼2%, with negligible perturbation in the
green region. In the intermediate situation, Figure 10b, the
decrease of blue emission accompanied by the increase of green
emission becomes easily detectable. The largest spectral changes
are observed when the polymer concentration is most dilute, as
in Figure 10c. The collective set of observations indicates that
the assay sensitivity when [dsDNA] < 2 × 10-8 M depends on
[RU]0. However, when [RU]0 < 1 × 10-7 M the polymer
emission becomes too weak to measure reliably using our
fluorometer and sets the lower limit threshold.
9
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A R T I C L E S
Chi et al.
Figure 11. Plots of δ as a function of [dsDNA] using a double logarithmic
scale reveal less scatter with lower poly110 concentrations. The [RU]0s of
poly110 are 2 × 10-5 M (black curve) and 5 × 10-7 M (red curve).
Figure 12. Plot of δ vs [dsDNA] in 25 mmol phosphate buffer (pH )
7.4) for poly120 ([RU]0 ) 2 × 10-5 M) shows a similar response to that
obtained in water (see Figure 6).
Figure 10. Larger changes in emission take place when the excess of
poly110 relative to dsDNA decreases. PL spectra of poly110 after additions
of dsDNA (λexc ) 360 nm). The [RU]0s of poly110 are (a) 2 × 10-5 M; (b)
1 × 10-6 M, and (c) 2 × 10-7 M, respectively. For all of the cases, the
[dsDNA] ranges from 3 × 10-12 to 2 × 10-7 M. For ease of comparison,
the initial PL intensities in the absence of dsDNA were normalized.
charges on complementary single-stranded DNA, which facili-
tates hybridization. However, the same ions are expected to
weaken the electrostatic interactions between the positively
charged polyelectrolytes and dsDNA. This is often referred to
as electrostatic screening, since the external ions decrease, or
collapse, the electrostatic interaction of counterions resulting
in a decrease in the potential between the two oppositely charged
pseudo surfaces.39 With these considerations in mind, we tested
the optical response of poly110 and poly120 to dsDNA in 25
mmol of phosphate buffer (pH ) 7.4), conditions typical of
DNA detection schemes. Addition of dsDNA to either poly110
or poly120 gives rise to changes in the PL spectra that are very
similar to those shown in Figure 4 (see Figure S-6 in the
Supporting Information). A typical plot of δ vs [dsDNA] for
poly120 with [RU]0 ) 2 × 10-5 M is shown in Figure 12.
Comparison against the analogous information obtained in water
(Figure 6) shows that the slopes of the plots are indistinguish-
able. The saturation points are at [dsDNA] ) 1.6 × 10-5 and
1.5 × 10-5 M in water and buffer, respectively. Thus, the
presence of buffer ions makes only a minor modification of
the assay response. Such behavior is interesting in view of the
expected screening of electrostatic attraction. That this is not
the case suggests that the cooperative action of multiple charges
in the macromolecules can overcome changes in electrostatic
Further fine-tuning of the poly110 [RU]0 was undertaken to
obtain optimal performance. In these experiments the δ response
was measured with [RU]0 ) 1 × 10-6, 5 × 10-7, 2 × 10-7
,
and 1 × 10-7 M. The least data scatter and steepest slope were
observed with [RU]0 ) 5 × 10-7 M, as shown in Figure 11.
The corresponding plots for [RU]0 ) 2 × 10-7 and 1 × 10-7
M were deposited as Figure S-5 in the Supporting Information.
In Figure 11, the saturation limit occurs when [dsDNA] ) 5 ×
10-7 M. For comparison, the plot obtained with [RU]0 ) 2 ×
10-5 M is also included in Figure 11. When comparing the two
different concentrations, one notes that, with [RU]0 ) 5 × 10-7
M, the δ values change more quickly and are less scattered in
the “I” regime and the “A” point occurs at a lower [dsDNA] (6
× 10-9 M), although this transition is not as clearly defined.
The sensitivity and reliability of the assay therefore increases
when [RU]0 ) 5 × 10-7 M. Significantly, in regime “II”, the
δ responses from the two [RU]0s in Figure 11, become nearly
identical, in agreement with the results in Figure 6.
Effect of Buffer. DNA is more commonly stored in buffered
solutions than in pure water. The buffer ions screen negative
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Design of Cationic Conjugated Polyelectrolytes
A R T I C L E S
attraction, at least with the concentration of ion in the buffer
used in these experiments.
It is also interesting to note that two slopes are observed in
the double logarithmic plots in Figures 8 and 9. These slopes
are proportional to the power dependence of δ on [dsDNA].
We assume that δ is proportional to the quantity of poly110 (or
poly120) incorporated into the polyelectrolyte aggregate. Under
the assumption that the slope equals 1 in regime II suggests
that the equilibrium constant for aggregate formation is pro-
portional to [dsDNA]. In the low concentration regime, i.e., I,
that the slope < 1 would indicate that a single chain of dsDNA
brings in together more polymer chains into the aggregate when
the polymer is in much greater excess. The stochiometry of the
complex is thus dependent on [dsDNA] at a given [RU]. Such
a result is not surprising when one takes into consideration that,
in regime I, the charge ratio of dsDNA to CPE ranges from 1.9
× 10-7 to 1.3 × 10-3 starting with [RU]0 ) 2 × 10-5 M.
Furthermore, one notices a steeper dependence of the δ vs
[dsDNA] plots summarized in Table 1 when one examines lower
dsDNA concentrations, consistent with the arguments above.
Despite gaps in our mechanistic understanding in how the
combined optical processes come together to determine δ and
the general description of the polyelectrolyte aggregates, it is
possible to collect the information obtained thus far and to
generate calibration curves that may be of practical value for
determining [dsDNA]. We highlight that poly120 has a slightly
higher sensitivity than poly110 in the high [dsDNA] regime.
However, because of its intrinsic higher PL quantum efficiency,
poly110 provides better signal-to-noise at lower [dsDNA].
Combining the overall set of observations, we collect in Figure
13 the δ response obtained with poly110 starting with two
different [RU]0s (5 × 10-7 M and 2 × 10-5 M) in either water
or buffer solution to generate calibration curves that span a 7
orders of magnitude difference in [dsDNA]. With [RU]0 ) 5
× 10-7 M, we obtain good sensitivity and reproducibility in
the low [dsDNA] regime (red curves in Figure 13). Higher
[dsDNA]s (black curves) can be interrogated with [RU]0 ) 2
× 10-5 M because the upper “stoichiometric” limit of the
polyelectrolyte aggregates increases with higher initial polymer
concentrations (see Figure 6). That the dependence of δ is not
dependent on [RU] in the intermediate [dsDNA] ranges allows
overlap between the curves obtained at different [RU]0s. Figure
13 shows high sensitivity and a wide detectable range. Ad-
ditionally, based on previous observations with PFBTx, we
anticipate that similar curves can be provided to quantify
ssDNA.20
Summary Discussion and Conclusion
A new class of BT-containing conjugated polyelectrolyte
structures is now accessible via the synthetic sequence shown
in Scheme 1. The BT content in these copolymers can be
modulated at the synthesis stage by controlling the ratio of
reacting monomers. These polymers display a much higher water
solubility, when compared to polymers such as PFBT7, as a
result of the higher density of tetralkylammonium groups along
the polymer chain and the oligo(ethyleneoxide) pendant groups
on the phenylene units. Additionally, the shorter propyl linkers
on the fluorene units reduce the hydrophobic content of the
polymer.32
There is a reduced tendency for the BT-centered green
emission band in poly110 and poly120 to appear in concentrated
aqueous solutions. Films of the materials, where interchain
contacts are maximized, emit predominantly green. These data
suggest that the increased solubility reduces aggregation, when
compared to systems such as PFBT7. The PL emission changes
as a function of concentration, predominantly as a result of self-
absorption of the blue band by the BT chromophores. The exact
value for the “maximum” concentration after which distortions
in spectral shape are observed thus depends on the dimensions
of the cuvette, with smaller volumes allowing one to reach
higher concentrations.
Addition of dsDNA to poly110 or poly120 causes a decrease
in the blue band and an increase of the green emission. These
changes are a result of increased energy transfer from the blue
emitting regions to BT-centered sites, an increase in the BT
emission efficiency and partial quenching of the blue band. The
increase in BT quantum yield is likely a result of more effective
shielding from water when in the polymer/dsDNA aggregate.
Similar observations have recently been reported with anionic
BT-containing polyelectrolytes that aggregate at low pH val-
ues.40 In these systems the BT quantum yield exhibits a 7-fold
increase when the pH changes from 7 to 3. The overall increase
in FRET efficiency throughout the process in Figure 4a is not
very large, approximately 1.6, although this is an estimate since
the degree of self-quenching of the blue emitting segments is
not well-defined at this point. Having identified the complexity
of the process involved in producing the changes in PL
characteristics, it is remarkable that a linear relationship between
δ and [dsDNA] is observed in Figure 6 and that the slope is
independent of the polyelectrolyte concentration. We reempha-
size that eq 1 was developed empirically on the expectation
that increases in green emission would be proportional to the
quantity of polyelectrolyte complexed to dsDNA. For all [RU]0s,
the increase of δ with increasing [dsDNA] breaks down when
the ratio of negative to positive charges is approximately 0.7.
Such a consistent value suggests that more of the conjugated
polyelectrolyte is incorporated into the aggregate than dsDNA.
Possible reasons for the nonequivalent ratio include noncom-
mensurate aspect ratios of the polymer repeat units and the base
pairs in dsDNA. Furthermore, it is known that under certain
circumstances the aggregate structures are determined under
kinetic conditions.38 Structural details of these aggregates are
not available and will be difficult to obtain, particularly at the
lowest concentrations where poor signal-to-noise ratios are
expected in standard dynamic light scattering experiments.
It is useful to point out that the low [dsDNA] range in the
plots of Figure 13 is competitive with what can be accomplished
with current commercially available technologies based on
cyanine dyes such as PicoGreen and OliGreen.24-31,41 The
[dsDNA] ranges in Figure 13 also extend into regions that can
be addressed by absorption spectroscopy. One difference
between intercalator-based methods and that described here is
that the former can correlate [dsDNA] with the emission
intensity at a single wavelength. The calibration curves in Figure
13 require analysis of the spectral shapes. To the best of our
knowledge, PicoGreen is one of the most sensitive probes.42
The detection range is from 3.0 × 10-11 M bps, or 25 pg/mL,
to 1.5 × 10-6 M bps, or 1 µg/mL. These concentrations span
approximately 5 orders of magnitude. By using a normal
(41) http://probes.invitrogen.com/handbook/sections/0803.html.
(42) Singer, V. L.; Jones, L. J.; Yue, S. T.; Haugland, R. P. Anal. Biochem.
1997, 249, 228.
9
J. AM. CHEM. SOC. VOL. 129, NO. 36, 2007 11143

A R T I C L E S
Chi et al.
All the DNA concentration determination experiments were done
by using 30 base pair double-stranded DNA. The sequence of dsDNA
was 5′-CTG TTG CAC TAT GCC AGA CAA TAA TTT TCT-3′ with
its complementary strand 5′-AGA AAA TTA TTG TCT GGC ATA
GTG CAA CAG-3′. HPLC-purified oligonucleotides were obtained
from Integrated dsDNA Technologies, Inc. The samples were prepared
by initially determining ssDNA strand concentrations based on the
absorbance at 260 nm in 300 µL quartz cells using a Beckman Coulter
DU800 spectrophotometer. Once the concentration of both strands was
established, a 1:1 ratio between complementary single strands was
mixed for annealing. The mixture of complementary strands was
annealed at 55.8 °C (2 °C below the melting point) for 25 min and
then was slowly cooled to room temperature; the absorbance of the
hybridized strands was then measured to determine the concentration.
The extent of hybridization was checked by variable temperature
absorbance spectroscopy. Water was distilled and deionized by using
a Millipore filtration system. Measurements in buffer were made using
commercially available 25 mmol potassium phosphate monobasic-
sodium hydroxide buffer (pH ) 7.4) from Fisher Scientific. Fluores-
cence intensities were determined from the integrated areas under both
blue (375-470 nm) and green emission (520-660 nm) bands of
emission spectra. In all of the cases, the calibration factor N in eq 1
was obtained by the integration of blue emission from 375 to 470 nm
from a solution of poly120 at [RU] ) 1 × 10-6 M in water. The
[dsDNA] is provided in terms of base pairs (bps) throughout the paper.
2,7-Dibromo-9,9′-bis(3′′-bromopropyl)fluorene (2). 2,7-Dibro-
mofluorene (15 g, 46.3 mmol) was added to a mixture of aqueous
potassium hydroxide (300 mL, 50%), tetrabutylammonium bromide (3.1
g, 9.3 mmol), and 1,3-dibromopropane (93 g, 460 mmol) at 75 °C.
After 30 min, the mixture was cooled to room temperature. After
extraction with CH2Cl2, the combined organic layers were washed
successively with water, aqueous HCl (1 M), water, and brine and were
then dried over MgSO4. After removal of the solvent and the excess
1,3-dibromopropane, the residue was purified by silica gel column
chromatography using hexane and chloroform (3:1) as the solvent. The
target compound was obtained as a white solid (22.1 g, 85%). 1H NMR,
(200 MHz, CDCl3) δ (ppm) 7.57-7.48, (m, 6H, aryl), 3.15 (t, J ) 6.6
Hz, 4H), 2.19-2.11 (m, 4H), 1.21-1.06 (m, 4H); 13C NMR (50 MHz,
CDCl3) δ (ppm) 150.8, 139.1, 131.1, 126.3, 122.1, 121.6, 54.6, 38.6,
33.8, 27.1; MS (EI-MS) m/z ) 562 [M+]. Anal. Calcd for C19H18Br4:
C, 40.32; H, 3.21. Found: C, 40.20; H, 3.12.
Figure 13. Composite calibration curve of δ vs [dsDNA] using a double
logarithmic scale in (a) water and (b) 25 mM phosphate buffer. The
concentrations of poly110 are [RU]0 ) 2 × 10-5 M (black curve) and 5 ×
10-7 M (red curve).
fluorometer, as in the conditions of our work, one can use TOTO
to probe from 0.5 ng/mL to 100 ng/mL.43 Other intercalating
dyes include OliGreen with a detection range from 3 × 10-10
M, or 100 pg/mL, to 3.0 × 10-6 M, or 1 µg/mL (for single-
stranded DNA),41 i.e., 4 orders of magnitude, and ethidium
bromide, which is typically used with concentrations of 1 ng/
mL. For comparison, the conjugated polymer assay spans from
3.0 × 10-12 M, or 2 pg/mL, to 1.5 × 10-5 M, or 10 µg/mL,
i.e., close to 7 (exactly 6.9) orders of magnitude differences
and does not benefit from the extensive prior optimization and
mechanistic understanding of intercalating dyes.44
2,7-Bis(4,4,5,5-tetramethyl-[1,3,2]-dioxaborolane)-9,9-bis(3′-bro-
mopropyl)fluorene (3). Under an argon atmosphere, 7-dibromo-9,9′-
bis(3′′-bromopropyl)fluorene 2 (10 g, 17.7 mmol), bis(pinacolato)-
diboron (13.5 g, 53 mmol), KOAc (15.6 g, 159 mmol), and [1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium (1.5 g, 1.77 mmol)
were dissolved in dioxane (250 mL). The reaction mixture was degassed
by three “freeze-pump-thaw” cycles and then heated to 80 °C for 8
h. The dioxane was removed in vacuum followed by addition of CH2-
Cl2 and water. The aqueous phase was extracted with CH2Cl2 (2 ×
400 mL), and then the combined CH2Cl2 solution was washed with
water, brine water, and water, and then dried over MgSO4. The solvent
was removed under vacuum, and the residue was purified by column
chromatography over silica gel using hexane/dichloromethane (3:1) to
Experimental Section
General Details. 1H and 13C NMR spectra were collected on Varian
Inova 400 MHz and Varian ASM-100 200 MHz spectrometers. UV-
vis absorption spectra were recorded on a Shimadzu UV-2401 PC diode
array spectrometer. Photoluminescence spectra were obtained on a Spex
Fluorolog 2 spectrometer, using 90° angle detection for solution
samples. Mass spectrometry and elemental analysis were performed
by the UCSB Mass Spectrometry Lab and Elemental Analysis Center.
Reagents were obtained from Aldrich Co. and used as received unless
otherwise mentioned. The synthesis of 4,7-dibromo-2,1,3-benzothia-
diazole (8) was described previously.14
1
give 3 as a white solid (10 g, 86%). H NMR, (200 MHz, CDCl3) δ
(ppm) 7.86-7.71 (m, 6H, aryl), 3.08 (t, J ) 7.0 Hz, 4H), 2.23-2.15
(m, 4H), 1.40 (s, 24H), 1.17-1.03 (m, 4H); 13C NMR (50 MHz, CDCl3)
δ (ppm) 148.7, 143.9, 134.6, 129.0, 120.0, 84.1, 54.2, 38.8, 34.3, 27.4,
25.1; MS (EI-MS) m/z ) 660 [M+]. Anal. Calcd for C31H42B2Br2O2:
C, 56.41; H, 6.41. Found: C, 56.29; H, 6.40.
1,4-Dibromo-2,5-di[2-[2-(2-hydroxylethoxy)-ethoxy]ethoxyl]ben-
zene (5). Under an argon atmosphere, compound 4 (7.0 g, 26.1 mmol),
2-[2-(2-chloroethoxy)ethoxy]ethanol (22 g, 131 mmol), K2CO3 (21.6
g, 156.5 mmol), and KI (45 mg, 0.27 mmol) were dissolved in DMF
(150 mL). The reaction mixture was heated to 80 °C for 24 h. The
mixture was filtered, and then the DMF was removed in vacuum. The
(43) Rye, H. S.; Dabora, J. M.; Quesada, M. A.; Mathies, R. A.; Glazer, A. N.
Anal. Biochem. 1993, 208, 144.
(44) Ihmels, H.; Otto, D. Top. Curr. Chem. 2005, 258, 161.
9
11144 J. AM. CHEM. SOC. VOL. 129, NO. 36, 2007

Design of Cationic Conjugated Polyelectrolytes
A R T I C L E S
residue was purified by column chromatography over silica gel by using
Poly120N (yield, 72%). 1H NMR (200 MHz, CDCl3) δ (ppm) 8.09-
7.73 (m, 34H), 7.18 (s, 6H), 4.21 (m, 12H), 3.84-3.69 (m, 48H), 3.46
(t, J ) 6.0 Hz, 12H), 3.24 (m, 20H), 2.31 (m, 20H), 1.38 (m, 20H);
13C NMR (100 MHz, CDCl3) δ (ppm) 154.4, 150.6, 150.1, 149.8, 149.3,
149.1, 141.3, 140.0, 137.6, 133.6, 131.4, 129.2, 128.3, 124.3, 119.9,
117.2, 71.5, 70.8, 70.1, 69.4, 54.4, 39.0, 34.9, 30.7, 27.7; GPC (THF,
relative to polystyrene standards), Mw ) 77 708 g/mol; Mn ) 26 340
g/mol; polydispersity index, 2.9. Elemental analysis calculated: C,
50.94; H, 4.62; N, 1.48. Found: C, 51.76; H, 4.59; N, 1.40.
Poly110N (yield, 84%). 1H NMR (200 MHz, CDCl3) δ (ppm) 8.06-
7.72 (m, 32H), 7.18 (s, 8H), 4.20 (m, 16H), 3.84-3.69 (m, 64H), 3.46
(t, J ) 6.0 Hz, 16H), 3.22 (m, 20H), 2.27 (m, 20H), 1.34 (m, 20H);
13C NMR (50 MHz, CDCl3) δ (ppm) 150.6, 149.1, 140.0, 137.6, 131.4,
129.2, 124.3, 119.9, 117.2, 71.5, 70.8, 70.1, 69.4, 54.3, 38.8, 34.8, 30.7,
27.7; GPC (THF, relative to polystyrene standards), Mw ) 126 102
g/mol; Mn ) 47 608 g/mol; polydispersity index, 2.6. Elemental analysis
calculated: C, 49.95; H, 4.80; N, 0.67. Found: C, 50.15; H, 4.87; N,
0.60.
dichloromethane/methanol (20:1) to give the compound 5 as an oil (11.6
1
g, yield: 83.4%). H NMR, (200 MHz, CDCl3) δ (ppm) 7.16, (s, 2H,
aryl), 4.14 (t, J ) 4.4 Hz, 4H), 3.89 (t, J ) 4.2 Hz, 4H), 3.81-3.60
(m, 16H), 2.40 (t, J ) 5.4 Hz, 2H); 13C NMR (400 MHz, CD2Cl2) δ
(ppm) 150.2, 118.9, 111.1, 72.6, 70.9, 70.3, 70.0, 69.5, 61.5. MS (EI-
MS) m/z ) 532 (M+). Anal. Calcd for C18H28Br2O8: C, 40.62; H, 5.30.
Found: C, 40.64; H, 5.28.
1,4-Dibromo-2,5-di[2-[2-(2-tosylethoxy)ethoxy]ethoxyl]benzene (6).
Under an argon atmosphere, compound 5 (5.5 g, 10.34 mmol), N,N-
dimethylamino pyridine (DMAP) (250 mg, 2.0 mmol), and triethy-
lamine (4.23 g, 41.8 mmol) were dissolved in 50 mL of dichlo-
romethane, and the mixture was cooled down to 0 °C. Then
p-toluenesulfonyl chloride (4,73 g, 24.8 mmol) was added in portions.
The reaction mixture was stirred at room temperature overnight. The
solvents were removed under vacuum, and the residue was purified by
column chromatography over silica gel by using dichloromethane/
1
methanol (20:1) to give compound 6 (8.16 g, 94%). H NMR, (200
MHz, CDCl3) δ (ppm) 7.83-7.77, (m, 4H, aryl), 7.37-7.31, (m, 4H,
aryl), 7.14, (s, 2H, aryl), 4.19-4.08 (m, 8H), 3.85 (t, J ) 4.4 Hz, 4H),
3.74-3.58 (m, 12H), 2.44 (s, 6H); 13C NMR (100 MHz, CD2Cl2) δ
(ppm) 150.2, 145.1, 132.9, 129.9, 127.8, 118.9, 111.1, 71.2, 70.7, 70.4,
70.1, 69.5, 68.6, 21.4. MS (ESI-MS): m/z ) 861 ([M + Na]+). Anal.
Calcd for C32H40Br2O12S2: C, 45.72; H, 4.80. Found: C, 45.67 H, 4.75
1,4-Dibromo-2,5-di[2-[2-(2-bromoethoxy)ethoxy]ethoxyl] benzene
(7). Under an argon atmosphere, compound 6 (5.0 g, 5.95 mmol) and
lithium bromide (4.13 g, 47.6 mmol) were dissolved in 50 mL of
acetone. The mixture was then heated to reflux overnight. The solvent
was removed, and water (50 mL) and dichloromethane (200 mL) were
added. The organic layer was washed with water and dried over MgSO4,
and the solvent was removed under vacuum. The residue was purified
by column chromatography over silica gel by using dichloromethane/
General Synthesis Procedure for Compounds poly120 and poly110:
Condensed trimethylamine (8 mL) was added dropwise to a solution
of the neutral precursor polymer (100 mg) in 20 mL of THF at -78
°C. The mixture was then allowed to warm up to room temperature
for 4 h. The precipitate was redissolved by addition of methanol (20
mL). After the mixture was cooled down to -78 °C, more trimethy-
lamine (6 mL) was added and the mixture was stirred for 24 h at room
temperature. After most of the solvent was removed, acetone was added
to precipitate the polymers as a light orange powder.
Poly120 (yield, 85%). 1H NMR (200 MHz, d6-DMSO) δ (ppm) 8.34-
7.82 (m, 34H), 7.27 (br, 6H), 4.32 (br, 12H), 3.90-3.03 (m, 224H),
2.31-1.91 (m, 20H), 1.75-1.30 (m, 20H). Elemental analysis calcu-
lated: C, 53.02; H, 6.82, N, 5.89. Found: C, 52.89; H, 6.78; N, 5.80
Poly110 (yield, 87%). 1H NMR (200 MHz, d6-DMSO) δ (ppm):
8.37-7.25 (m, 40H), 4.30 (br, 16H), 3.89-3.06 (m, 262H), 2.18(m,
20H), 1.35 (m, 20H). Elemental analysis calculated: C, 52.19; H, 6.95;
N, 5.36. Found: C, 52.07; H, 6.87; N, 5.43.
1
methanol (30:1) to give compound 7 (2.5 g, 64.3%). H NMR, (200
MHz, CDCl3) δ (ppm) 7.16, (s, 2H, aryl), 4.14 (t, J ) 4.4 Hz, 4H),
3.92-3.68 (m, 16H), 3.49 (t, J ) 6.4 Hz, 4H); 13C NMR (100 MHz,
CD2Cl2) δ (ppm) 150.2, 118.9, 111.1, 71.1, 70.9, 70.5, 70.1, 69.5, 31.0.
MS (EI-MS) m/z ) 658 (M+). Anal. Calcd for C18H26Br4O6: C, 32.86;
H, 3.98. Found: C, 32.97; H, 4.04.
Acknowledgment. The authors would like to thank Drs.
Dmitry Korystov, Janice Hong, Brent Gaylord, Renqiang Yang,
and Fuke Wang for helpful and important discussions. The work
received financial support from the NSF (DMR 0606414) and
from the Institute for Collaborative Technologies.
General Procedure for the Preparation of poly120N and poly110N.
The neutral polymers were prepared by a palladium-catalyzed Suzuki
cross-coupling reaction. The purified monomers 3, 7, and 8 (4,7-
dibromo-2,1,3-benzothiadiazole) were mixed in the appropriate ratios
with Pd(PPh3)4 (1 mol %) in a mixture of 2 M K2CO3 (aq) (5 equiv/
Br) and toluene (1/2 v/v) under argon. The monomer concentration
in toluene is around 0.07 M. The reaction mixture was degassed by
three “freeze-pump-thaw” cycles and then heated to 95 °C for 24 h.
After cooling to room temperature, acetone was added into the mixture.
The precipitate was separated by filtration and washed with methanol
and acetone. Purification was accomplished by precipitation of chlo-
roform solutions into methanol. The neutral polymers were obtained
by filtration and drying under vacuum for 24 h as bright-yellow
powders.
Supporting Information Available: Dependence of the PL
spectra of poly120 vs [RU], PL spectra of poly10 upon addition
of [dsDNA], PL spectra of poly120 upon addition of [dsDNA]
to generate Figure 8, PL spectra of poly120 upon addition of
[dsDNA] in buffer, comparison of δ vs [dsDNA] for poly110
and poly120, and plots of δ vs [dsDNA] for poly110 as a function
of [RU]0. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA072471S
9
J. AM. CHEM. SOC. VOL. 129, NO. 36, 2007 11145