Interpolyelectrolyte Complexes of Conjugated Copolymers and DNA: Platforms for Multicolor Biosensors

Article abstract of DOI:10.1021/ja038667j

Interchain interactions modulate the frequency of emission from a cationic water-soluble conjugated polymer. The polymer, PFPB, is obtained by a Suzuki copolymerization of p-phenylenebisboronic acid with a 95:5 mixture of 2,7-dibromo-9,9-bis(6′-bromohexyl

Full text of DOI:10.1021/ja038667j

Published on Web 01/31/2004
Interpolyelectrolyte Complexes of Conjugated Copolymers and DNA:
Platforms for Multicolor Biosensors
Bin Liu and Guillermo C. Bazan*
Departments of Chemistry and Materials, Institute for Polymers and Organic Solids, UniVersity of California,
Santa Barbara, California 93106
Received September 22, 2003; E-mail: bazan@chem.ucsb.edu
Scheme 1a
DNA and RNA detection methods are of considerable scientific
and technological importance.1-3 Water-soluble conjugated poly-
mers are being increasingly studied for this purpose because their
molecular structure allows for collective response and, therefore,
optical amplification of fluorescent signals.4,5 The large number of
optically active units along the polymer chain increases the
probability of light absorption, relative to small-molecule counter-
parts.5 Facile energy transfer makes it possible to deliver excitations
to fluorophores, which signal the presence of a target DNA
sequence.6,7
Recent studies indicate that energy transfer between segments
in a conjugated polymer may be substantially more important than
a i) 2 M K2CO3, THF, Pd(PPh3)4; ii) N(CH3)3, THF/H2O.
along the backbone.8,9 Molecular interactions that decrease the
elongation of the backbone or that bring segments closer together
could therefore be used to increase sensitivity. It occurred to us
that a small number of optically active units within a polymer
sequence could be activated by structural changes that compressed
or aggregated the polymer chains. In this communication, we
disclose a cationic conjugated polymer structure that incorporates
these design guidelines. Furthermore, electrostatic complexation
with negatively charged DNA can be used to change the emission
of the polymer by reducing the average intersegment distance. When
combined with a fluorophore (C*)-labeled peptide nucleic acid
strand (PNA-C*), the polymer can be used to design a three-color
DNA detection assay.
To obtain two emission colors from a single polymer chain, we
introduced 5% of the 2,1,3-benzothiadiazole (BT) chromophore into
a cationic poly(fluorene-co-phenylene) polymer chain (Scheme 1).
The synthetic approach involves a Suzuki copolymerization of
p-phenylenebisboronic acid with a 95:5 mixture of 2,7-dibromo-
9,9-bis(6′-bromohexyl)fluorene and 4,7-dibromo-2,1,3-benzothia-
diazole.10 Elemental analysis of the resulting polymer is consistent
with a chemical composition similar to the monomer feed. Since
the GPC-determined molecular weight (Mn) is ∼11000 amu, one
can estimate that there is, on average, one BT molecule per polymer
chain. In a second step, quarternization of the pendant groups by
addition of NMe3 provides the polycationic water soluble PFPB
(see Scheme 1).
Figure 1. (A) Fluorescence spectra of PFPB in water as a function of
[ss-DNA] ([RU] ) 5 × 10-7 M, [ss-DNA] ) 0 M to 2.7 × 10-8 M in 3.0
× 10-9 M increments, λexc ) 380 nm). (B) (a) Normalized photolumines-
cence spectra of PFP, (b) absorption and (c) emission spectra of ss-DNA-
TR in water.
In deionized water, at concentrations below 1 × 10-6 M (in
repeat units, RUs), PFPB emits predominantly in the 400-500 nm
region, with a fluorescence quantum yield (Φ) of 22%. Indeed,
both the absorption (λmax ) 380 nm) and the emission are nearly
identical to that of poly(9,9-bis(6′-N,N,N,-trimethylammonium)-
hexyl)fluorene-co-alt-1,4-phenylene) dibromide (PFP), which lacks
BT sites (see Figure 1, A and B).11 Under dilute conditions, the
emission of PFPB is dominated by the more abundant oligo-
(fluorene-co-phenylene) segments. When [PFPB] > 1 × 10-6 M,
one observes the emergence of green emission (500-650 nm),
characteristic of the BT sites. As [PFPB] increases, the green
emission grows at the expense of the blue emission. These data
indicate aggregation in the more concentrated regime, which leads
to a reduction of the distance between polymer segments and
enhances energy transfer to units containing lower-energy BT
chromophores.12,13
On the basis of well-known interpolymer interactions between
oppositely charged polyelectrolytes,14-17 we anticipated that com-
plexation of PFPB with ss-DNA (5′-ATC TTG ACT ATG TGG
GTG CT) in water18 would lead to contraction and aggregation of
polymer chains, a concomitant reduction of intersegment distances,
and more efficient energy transfer to the BT sites. Figure 1A shows
the emission from PFPB/ss-DNA solutions upon addition of ss-
DNA ([RU] ) 5 × 10-7 M, ss-DNA ) (5′-ATC TTG ACT ATG
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J. AM. CHEM. SOC. 2004, 126, 1942-1943
10.1021/ja038667j CCC: $27.50 © 2004 American Chemical Society

C O MMU N I C A T I O N S
Figure 2. (a) TR emission intensity for PFP/ss-DNA-TR and (b) PFPB/
ss-DNA-TR in water (λexc ) 380 nm, [ss-DNA-TR] ) 2.0 × 10-8 M, TR
intensity is corrected to reflect the difference in optical density for the two
polymers).
Figure 3. Normalized fluorescence in water of (a) PFPB/PNA-Cy5, (b)
PFPB/DNAn+PNA-Cy5, and (c) PFPB/DNAc/PNA-Cy5 ([PNA-Cy5] )
2.0 × 10-8 M, [RU] ) 1.6 × 10-7 M, (λexc ) 380 nm).
TGG GTG CT), [ss-DNA] varies from 0 M to 2.7 × 10-8 M). The
isosbestic point at 492 nm highlights the transition from blue to
green emission with increasing [ssDNA].
polymer segments and encourages energy migration to low-energy
emissive sites (BT in the case of PFPB). With the aid of PNA-C*
probe strands, one obtains three different colors, depending on the
solution content: (1) blue, in the absence of DNA, (2) green, when
noncomplementary ssDNA is present, (3) and red, when the
complementary ssDNA is found. Fine-tuning of these electrostatic
and optical events could lead to multicolor biosensor schemes that
take advantage of the fluorescence amplification characteristic of
conjugated polymers.
Acknowledgment. We are grateful to the NIH (GM62958-01)
and the NSF (DMR-0097611) for financial support.
Supporting Information Available: Details for the synthesis of
PFPB and FRET experiments (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
Figure 1B shows the emission spectra of PFP, and the absorption
and emission of ss-DNA-TR (TR ) Texas Red dye and ssDNA-
TR ) 5′-TR-ATC TTG ACT ATG TGG GTG CT). Note that the
spectral overlap between the absorption of TR and the green
emission band of PFPB/ss-DNA is substantially larger than that
with the PFP emission. Therefore, we anticipated a larger value
for the overlap integral in the Fo¨rster equation and more efficient
fluorescence resonance energy transfer (FRET) with PFPB.19
Indeed, as shown in Figure 2, the TR emission intensity as a
function of polymer concentration, is greater when PFPB is excited,
relative to PFB (the value of Φ for TR is the same in the two
solutions). The spectra in Figure 2 were measured by excitation at
380 nm, which selectively creates polymer-based excited states.
On the basis of the mechanistic information above, we postulated
that PFPB could be used in a three-color DNA assay by using a
PNA-C* strand.6 PNA serves to provide a base sequence that
searches complementary ssDNA. However, because PNA is neutral,
it is possible to use water without buffer or other ions that are
required to screen the negatively charged phosphate backbone
during duplex formation.20 Since PNA-TR is not available com-
mercially, we used PNA-Cy5 (5′-Cy5-CAGTCCAGTGATACG)
as the PNA-probe instead. The absorption and emission of Cy5
(λabs ) 648 nm, λem ) 681 nm) are similar to those of TR
(Supporting Information). Hybridization of PNA-Cy5 with a
complementary ss-DNA (ss-DNAc ) 5′-CGTATCACTGGACTG)
endows the ss-DNAc/PNA-Cy5 duplex with multiple negative
charges. Complexation of ss-DNAc/PNA-Cy5 by electrostatic forces
to the positively charged PFPB allows for energy transfer from
the polymer to Cy5 and should lead to red emission. In the case of
a noncomplementary ss-DNA (ss-DNAn ) 5′-ACTGACGATA-
GACTG), electrostatic complexation occurs only between PFPB
and the ss-DNAn, which should give rise to emission from the BT
units.
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