Polyelectrolyte Complexes of a Cationic All Conjugated Fluorene

Article pubs.acs.org/JPCB

Polyelectrolyte Complexes of a Cationic All Conjugated Fluorene− Thiophene Diblock Copolymer with Aqueous DNA Matti Knaapila,*,† Telma Costa,‡ Vasil M. Garamus,§ Mario Kraft,∥ Markus Drechsler,⊥ Ullrich Scherf,∥ and Hugh D. Burrows‡ †

Department of Physics, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark Department of Chemistry and Coimbra Chemistry Centre, University of Coimbra, 3004-535 Coimbra, Portugal § Helmholz-Zentrum Geesthacht: Zentrum für Material- und Küstenforschung GmbH, 21502 Geesthacht, Germany ∥ Macromolecular Chemistry Group, University of Wuppertal, 42119 Wuppertal, Germany ⊥ Bayreuth Institute of Macromolecular Research - Laboratory for Soft Matter Electron Microscopy, University of Bayreuth, 95440 Bayreuth, Germany ‡

S Supporting Information *

ABSTRACT: We report on the structural and colorimetric effects of interaction of aqueous ∼0.06−1% poly[9,9-bis(2-ethylhexyl)fluorene]-b-poly[3-(6-trimethylammoniumhexyl)thiophene] bromide (PF2/6-P3TMAHT) with double-stranded DNA to form PF2/ 6-P3TMAHT(DNA)x where x is the molar ratio of DNA base pairs to P3TMAHT repeat units; x = 0.5 equals the nominal charge neutralization. PF2/6-P3TMAHT forms 20−40 nm sized particles with PF2/6 core and hydrated P3TMAHT exterior. The polymer particles form loose one-dimensional chains giving micrometer long branched chains (0.19 ≤ x ≤ 0.76) and subsequently randomly shaped aggregates (x = 1.89) upon DNA addition. Compaction of the P3TMAHT block and the 20−30 nm sized core is observed for x = 0.38−0.76 and attributed to the DNA merged within P3TMAHT domain with this structure disassembling with DNA excess. Structural transformations are followed by chromic changes seen as color changes from deep red (x < 0.076) to yellow (x = 0.19), nearly colorless (x = 0.38−0.76), and back to orange (x = 1.89). Both absorption and photoluminescence spectra display the distinct fluorene and thiophene bands and subsequent blue and red shifts when passing x = 0.5. Thiophene photoluminescence (PL) is significantly quenched by DNA with increasing x, and the changing P3TMAHT/ PF2/6 band ratio allows quantitative DNA detection. Sixteen-fold dilution does not change aggregate structure, but PL is blueshifted, indicating weakened intermolecular interactions.

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INTRODUCTION Following from the introduction of nonconjugated rod−coil block polymers,1 the synthesis of all-conjugated block copolymers has become a central approach for increasing the functional and structural diversity of conjugated polymers.2,3 Much research on these systems has been dedicated to neutral polymers with the emphasis on their microphase separation4,5 and optimization of charge transport and donor−acceptor couple with organic solar cell applications in mind.6,7 However, equally important are conjugated polyelectrolytes (CPEs), in which the charged groups are usually incorporated into the terminal position of the side chains.8 These charges make them water-soluble and allow molecular recognition, which together with their strong luminescence and sensitivity to the changes in structure and charge distribution, make them useful as biosensors with optical readout.9−12 Particular attention has been brought to the interaction with negatively charged DNA or its analogues. For example, Bazan and coworkers showed how DNA complementary peptide nucleic acid (PNA) prevents CPE−DNA interaction and leads to quenched intrinsic fluorescence, allowing specific DNA detection.13 In © XXXX American Chemical Society

another example, Leclerc and co-workers showed how the backbone of CPE can be planarized by the complexation with single-strand DNA, which leads to a redshifted absorption and quenched fluorescence.14,15 The fluorescence is recovered upon addition of a matching complementary strand. In these studies, the CPE chains were expected to wind around DNA.14,15 Further studies have described the interaction with ionomers,16 nanoparticles,17 zwitterionic CPEs,18 or surfactants,19 etc. Amino acid detection was also accomplished in the interaction with electron-deficient (but uncharged) conjugated polymers.20 CPE−DNA complexation leads to a rich diversity of structural reorganizations including diverse supramolecular structures21 and aggregates,22 as well as DNA compaction and bridges between DNA chains.23 Much of this research concerns homopolymers, but alternating copolymers have also been prepared for advanced DNA probes.24 Steps have also been made toward polyelectrolytic all-conjugated block Received: November 3, 2014 Revised: January 13, 2015

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DOI: 10.1021/jp5110032 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

nonpolar diblock copolymer precursor PF2/6-b-P3BrHT was 21 kg/mol as determined by gel permeation chromatography (GPC) (PS calibration). Considering the Mn of the starting BrP3BrHT (13.8 kg/mol), the Mn of the PF2/6 block was estimated as 7.2 kg/mol. This means that the degree of polymerization is about 19 and 56 for the PF2/6 and P3BrHT blocks, respectively. The nonpolar precursor copolymer was subsequently quaternized with trimethylamine. Further synthetic details and purification procedures are given in the Supporting Information. Double-stranded DNA lot no. BCBK2089 V with about 2000 base pairs (average 660 Da) was obtained from Sigma-Aldrich. PF2/6-P3TMAHT (9.93 mg/mL) and DNA (5 mg/mL) were mixed with water separately and stirred overnight. These mixtures were combined to prepare aqueous PF2/6P3TMAHT(DNA)x, where x is the molar ratio of DNA base pairs to thiophene monomer repeats. The samples were denoted as S1−S9, and their concentrations and the obtained molar ratios are compiled in Table 1. These samples were

copolymers that allow microphase separation and fractal formation in water.25−27 However, corresponding reports concerning DNA detection using these systems remain scarce. In a recent letter, Fonseca et al.28 studied all-conjugated block copolyelectrolyte, poly[9,9-bis(2-ethylhexyl)fluorene]-bpoly[3-(6-trimethylammoniumhexyl)thiophene] bromide (or PF2/6-P3TMAHT), and showed the dual fluorescence of the neutral fluorene and cationic thiophene blocks. The authors complexed this polymer with anionic halide ions and singleand double-stranded DNA based on the electrostatic attraction between interrogated ion or polyion and thiophene block. They demonstrated how this interaction leads to the quenching of thiophene fluorescence while the fluorescence of noncomplexed fluorene remains unquenched. Measuring difference between these components allows a quantitative ratiometric method for anion sensing even at submicromolar concentrations. The authors assume that this sensing is based not only on electrostatic interaction but also on the changes in polymer conformation through intra- and intermolecular interactions. The next step in this research would give answers to the questions of initial solution structure, which guides the complexation process, and to the apparent structural reorganization, which stems from this process, as already shown elsewhere for DNA complexed P3TMAHT homopolymer22 or trimethylammonium substituted polyfluorene.23 In this work, we complex the aqueous diblock PF2/6P3TMAHT with double-stranded DNA, which is the largest previously studied polyion and assumed to illustrate the largest structural effects. We find how micelle-level structure is evolved with increasing DNA content and show a well-ordered structure attributed to the DNA layer merged into the polymer particles. DNA addition is followed by chromic changes involving color change from deep red to yellow and back to orange. Photoluminescence (PL) spectra display dual character with emission from both blocks. The thiophene related band is, however, suppressed during nucleic acid binding, allowing quantitative estimation of the DNA content.

Table 1. Composition of Aqueous PF2/6-P3TMAHT and PF2/6-P3TMAHT(DNA)x Samples x

polymer conc. (mg/mL)

DNA conc. (mg/mL)

total conc. (mg/mL)

− 0.038

9.93 9.03

− 0.45

9.93 9.48

0.076

8.28

0.83

9.11

0.19

6.62

1.67

8.29

0.38

4.97

2.50

7.47

0.76

3.31

3.33

6.64

1.89

1.65

4.17

5.82

3.78

0.90

4.55

5.45

−

−

5.00

5.00

sample S1 S2 S3 S4 S5 S6 S7 S8

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S9

EXPERIMENTAL SECTION Materials. The chemical structure of PF2/6-P3TMAHT with a regioregular P3TMAHT block is shown in Scheme 1. Its

PF2/6-P3TMAHT PF2/6 -P3TMAHT(DNA)x PF2/6 -P3TMAHT(DNA)x PF2/6 -P3TMAHT(DNA)x PF2/6 -P3TMAHT(DNA)x PF2/6 -P3TMAHT(DNA)x PF2/6 -P3TMAHT(DNA)x PF2/6 -P3TMAHT(DNA)x DNA

additionally diluted by the factors of 2, 4, 8, and 16, denoted as S1D1, S1D2, S1D3, S1D4, ..., S9D4. PF2/6-P3TMAHT forms a visually clear solution, while PF2/6-P3TMAHT(DNA)x may contain precipitates. To remove possible precipitates, the samples were centrifuged at 10 000 rpm for 2 min prior to measurements. Cryogenic Transmission Electron Microscopy. For cryogenic transmission electron microscopy (cryo-TEM) studies, a sample droplet of 2 μL was put on a lacey carbon filmed copper grid (Science Services, Munich), which was hydrophilized by air plasma glow discharge (Solarus 950, Gatan, Munich, Germany) for 30 s. Then, most of the liquid was removed with blotting paper leaving a thin film stretched over the lace holes. These specimens were instantly shock frozen by rapid immersion into liquid ethane cooled to approximately 90 K by liquid nitrogen in a temperaturecontrolled freezing unit (Zeiss Cryobox, Carl Zeiss Microscopy GmbH, Jena, Germany). The temperature was monitored and kept constant in the chamber during the sample preparation. The specimens were inserted into a cryotransfer holder (CT3500, Gatan, München, Germany) and transferred to a Zeiss/LEO EM922 Omega EFTEM (Zeiss Microscopy GmbH,

Scheme 1. Structure of PF2/6-P3TMAHT (m ≈ 19; n ≈ 56)

synthesis includes the initial synthesis of a monobromo-endcapped regioregular poly[3-(6-bromohexyl)thiophene] (BrP3BrHT).29 Poly[9,9-bis(2-ethylhexyl)fluorene]-b-poly[3-(6bromhexyl)thiophene] (PF2/6-P3BrHT) was prepared by a Suzuki-type cross-coupling of 2-(4′,4′,5′,5′-tetramethyl-1′,3′,2′dioxaborolane-2′-yl)-7-bromo-9,9-bis(2-ethylhexyl)fluorene in which Br-P3BrHT was used as macromolecular end-capper.29 The resulting number-average molecular weight (Mn) of the B


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procedure and then used as macromolecular end-capper in the Suzuki-type cross-coupling of the AB-type monomer 2(4′,4′,5′,5′-tetramethyl-1′,3′,2′-dioxaborolane-2′-yl)-7-bromo9,9-bis(2-ethylhexyl)fluorene. The nonpolar diblock copolymer precursor was quaternized with trimethylamine. Careful purification gave the desired diblock copolymer. Further details are given in the Supporting Information. Visual Considerations. Figure 1 shows images of aqueous PF2/6-P3TMAHT (sample S1) and aqueous PF2/6-

Jena, Germany). Examinations were carried out at temperatures around 90 K. The TEM instrument was operated at an acceleration voltage of 200 kV. Zero-loss filtered images (ΔE = 0 eV) were taken under reduced dose conditions (100−1000 e/nm2). Images were taken by a bottom mounted CCD camera system (Ultrascan 1000, Gatan, München, Germany) combined with a digital imaging processing system (Digital Micrograph GMS 1.8× , Gatan, München, Germany). Small-Angle X-ray Scattering. Small-Angle X-ray Scattering (SAXS) experiments were performed at the BioSAXS Beamline P12 at PETRA III (EMBL/DESY) in Germany. The energy the of X-ray beam was 12.8 keV, and the beam size was 100 (V) μm × 200 (H) μm. Sample-to-detector distance was 4.1 m, and the q-range was 0.007−0.46 Å−1, as calibrated using silver behenate. Scattering patterns were measured using Pilatus 2 M pixel detector. The samples (of approximately 20 μL) were injected into the sample cuvette using an automated liquid handling sample changer. To reduce the risk of radiation damage, the samples were moved slightly during the exposure. The measurement temperature was 20 °C. For each measurement, 20 diffraction patterns originating from the same sample volume were taken, using an exposure time of 0.05 s. The scattering from pure buffer was measured and used for background subtraction. The backgroundcorrected data were used to calculate one-dimensional scattering curves by angular averaging and corrected for transmitted beam. To verify that no artifacts had occurred as a result of radiation damage, all scattering curves for a recorded data set were compared to the reference curve (the first exposure) before being integrated using an automated acquisition and analysis program.30 The reduced data were analyzed using scaling concepts, where the scattering intensity scales as q−α. The exponent α = 1 refers to cylindrical particles and α = 2 to sheetlike particles. The exponent α = 4 points to the three-dimensional particles with a smooth surface. This interpretation was enhanced using the indirect Fourier transformation (IFT) programs GNOM31 and WIFT.32 Absorption and Steady-State Fluorescence. The absorption (PA) spectra were recorded on a Shimadzu UV2450 spectrophotometer. The PL emission and excitation spectra were recorded with a Horiba-Jobin-Ivon SPEX Fluorog 3-22 spectrometer with the excitation wavelength 380 or 445 nm and the emission wavelengths 415 nm (PF2/6) and 600 nm (P3TMAHT), respectively. The Fluorolog consists of a modular spectrofluorimeter with double-grating excitation (200−950 nm range, optimized in the ultraviolet (UV) with a blaze angle at 330 nm) and emission (200−950 nm range, optimized in the visible and with a blaze angle at 500 nm) monochromators. The bandpass for excitation and emission was 0−15 nm with wavelength accuracy of ±0.5 nm. The excitation source consisted of an ozone-free 450 W xenon lamp. The emission detector employed was a Hamamatsu R928 photomultiplier cooled with a Products for Research thermoelectric refrigerated chamber (model PC177CE005) with a photodiode as the reference detector.

Figure 1. Photo of studied samples and the notation used. Left column from top to bottom: PF2/6-P3TMAHT (sample S1) and PF2/6P3TMAHT(DNA)x with x = 0.038 (S2), 0.076 (S3), and 0.19 (S4). Right column from top to bottom: PF2/6-P3TMAHT(DNA)x with x = 0.38 (S5), 0.76 (S6), 1.89 (S7), and 3.78 (S8). Samples on the left side of columns correspond to the initial concentrations (Table 1). The samples on the right side of these samples are the dilutions by the factor of 2, 4, 8, and 16, denoted as D1−D4.

P3TMAHT(DNA)x with increasing molar ratio, x, from top to bottom in two columns (samples S2−S8). Also shown are all samples with decreasing overall concentrations, from left to right (denoted as D1−D4). PF2/6-P3TMAHT (S1) appears deep red and becomes gradually lighter and subsequently orange upon dilution. PF2/ 6-P3TMAHT(DNA)x appears first red (S2−S3, x = 0.038− 0.076), becoming orange-yellow (S4, x = 0.19), and nearly colorless (S5, x = 0.38) with increasing x. Further DNA addition restores the color back to salmon (S6, x = 0.76), pink (S7, x = 1.89), and salmon again (S8, x = 3.78). Chromic effects are consistent with previous work28 and imply that DNA is becoming associated with PF2/6-P3TMAHT, presumably through electrostatic interaction. Vanishing color at x = 0.38 is partly associated with the slight precipitation that is removed by centrifuge, leading presumably to decreased total concentration. However, a fair amount of material remains in solution, manifesting itself in the structural and optical data (vide infra). As each DNA base pair has two negatively charged phosphate units, and each P3TMAHT repeat one positively charged trimethylammonium unit, the

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RESULTS AND DISCUSSION Materials. The synthesis of PF2/6-P3TMAHT followed the procedure described in refs 26 and 29. In this, monobromo end-capped Br-P3BrHT was prepared in a Grignard Metathesis C


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Figure 2. Cryo-TEM images of aqueous PF2/6-P3TMAHT (S1) (a,b) and PF2/6-P3TMAHT(DNA)x with excess polymer x = 0.19 (S4) (c) and with excess DNA x = 1.89 (S7) (d). Arrows indicate examples of P3TMAHT bundles.

nominal charge neutralization takes place at x = 0.5. This agrees with x = 0.38−0.76, giving the first reason for reduced polarity and solubility, and for visually observed precipitation. Solution Structure. Cryo-TEM. Previous AFM studies of PF2/6-P3TMAHT indicate submicron-sized clusters forming micrometer-sized fractal aggregates on mica surfaces during slow evaporation from water.26,27 This contrasts with the micrometer-sized vesicles which have been observed in methanolic solution. Figure 2 shows cryo-TEM images of aqueous PF2/6P3TMAHT and PF2/6-P3TMAHT(DNA)x with polymer excess and with DNA excess. PF2/6-P3TMAHT appears as spherical particles with the tendency to form loose, onedimensional necklace structures, predominantly without branches (Figure 2a). The particle diameter falls within the range of 20−40 nm, although a few individual structures are as large as 80−100 nm. The particles are covered by weakly visible 2 nm thick chains, which are extended from the core toward the medium (Figure 2b). Because the ionic P3TMAHT blocks are water-soluble and the nonionic PF2/6 blocks water-insoluble, we assume that the P3TMAHT blocks tend to be located in the particle exterior, while PF2/6 forms the interior of the particles. The 2 nm thick chains seen by cryo-TEM are assumed to be bundles of agglomerated P3TMAHT chains rather than individual polymers. It is plausible that these submicron-sized particles are merging to the previously seen fractal-shaped aggregates during solvent evaporation.26,27 When DNA is added, this system converts to the micrometers long branched nano-objects that are composed of individual nanospheres (Figure 2c). The coverage of 2 nm thick chains disappears. The branches are seen for the samples around the charge neutralization point, where the P3TMAHT volume is approximately equal to that of the DNA. However, as this does not include the PF2/6 volume, DNA remains as a minority compound in terms of overall volume. This leads us to

hypothesize that the branched chains consist of DNA as a core covered by PF2/6-P3TMAHT. When the overall volume equals the DNA volume, the branched aggregates transform into the larger arbitrarily shaped agglomerates (Figure 2d). We assume that P3TMAHT exterior is wound around the near DNA while PF2/6 remains inside the spherical polymer particles. SAXS. Cryo-TEM images concern length scales from micrometers to nanometers, whereas SAXS data reflect smaller length scales, the nominal observation window reaching from 900 to 14 Å. Figure 3 shows the SAXS curves of aqueous PF2/6P3TMAHT as a function of polymer concentration. Figure 4a−c plots SAXS curves of aqueous PF2/6-P3TMAHT(DNA)x

Figure 3. SAXS curves of aqueous PF2/6-P3TMAHT with initial concentration (S1, navy) and the dilution series D1−D4 (wine, violet, gray, and cyan). Orange lines are fits to the data. Dashed black lines show the ideal −1 and −3 slopes for comparison. D


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Figure 4. SAXS curves of aqueous PF2/6-P3TMAHT(DNA)x below (a), around (b), and above (c) the charge neutralization point (dark yellow, S2; magenta, S3; cyan, S4; blue, S5; olive, S6; red, S7; and black, S8). Corresponding data of PF2/6-P3TMAHT and DNA solutions are shown for comparison (S1, navy; S9, blue, taken from ref 22). Orange lines represent fits to the data. Dashed black lines show the ideal slopes for comparison.

Table 2. Scattering Powers α and Interference Peaks Estimated from the SAXS Data of Aqueous PF2/6-P3TMAHT and PF2/6P3TMAHT(DNA)x analyzed q-range (Å−1) sample

x

S1 S1D1 S1D2 S1D3 S1D4 S2 S3 S4 S5 S5D1 S6 S7 S8

− − − − − 0.038 0.076 0.19 0.38 0.38 0.76 1.89 3.78

0.0075−0.018 1.17 1.13 1.20 1.12 1.10 − − − − − 3.04 − 3.45

± ± ± ± ±

0.01 0.02 0.02 0.04 0.06

± 0.04 ± 0.03

0.0075−0.025 − − − − − 1.99 1.99 2.16 2.22 2.26 − 4.21 −

± ± ± ± ±

0.02 0.02 0.02 0.02 0.01

± 0.05

0.020−0.030

0.025−0.25

0.045−0.13

2.85 ± 0.03 − − − − − − − − − − − −

− − − − − − − − − − − 1.28 ± 0.01

1.03 − − − − 1.13 1.18 1.40 1.77 − 1.95 − −

± 0.01

± ± ± ±

0.01 0.01 0.01 0.01

± 0.01

0.07−0.25

0.13−0.25

peak (Å−1)

− − − − − − − − 2.09 ± 0.02 − 1.61 ± 0.03 − −

3.01 ± 0.02 − − − − − − − − − − − −

− − − − − − − 0.052 0.051 0.050 0.042 − −

Table 3. Parameters Estimated from the Fits to the SAXS Data of Aqueous PF2/6-P3TMAHT and PF2/6-P3TMAHT(DNA)xa sample

x

analyzed q-range (Å−1)

model

S1 S1 S1 S1 S2 S3 S5 S6 S7 S7 (WIFT)

0 0 0 0 0.038 0.076 0.38 0.76 1.89 1.89

0.0075−0.018 0.0075−0.025 0.04−0.25 0.04−0.25 0.0075−0.025 0.0075−0.025 0.0075−0.025 0.0075−0.025 0.025−0.25 0.030−0.25

cylinder 3D object cylinder 3D object 3D object 3D object 3D object 3D object cylinder cylinder

Rg or Rg,CS (Å) 31.1 118.3 6.6 17.8 146.2 138.0 230 210 4.6 5.8

± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 10 10 0.1 0.1

Dmax (Å) 95 350 20 55 350 350 700 650 15 15

a

Rg or Rg,CS and Dmax denote the radius of gyration of three-dimensional arbitrarily shaped scatterer or the cross-sectional radius of gyration of a cylindrical scatterer and the corresponding maximal particle or cross-sectional size, respectively. The first nine results were obtained using GNOM and the last one using WIFT.

for three potentially different phase regimes as defined from visual consideration (below, around, and above the charge neutralization point) alongside the scattering curves of pure polymer and DNA. Parameters calculated from the fits to these data are shown in Tables 2 and 3. Below, we refer to these phase regimes with roman numerals I−IV.

I. Pure polymer. The data shown in Figure 3 include two broad humps with the borderline at ∼0.04 Å−1. This indicates two coexisting structures with two different length scales, below and above ∼160 Å. For the same situation, TEM indicates the existence of particles whose diameter is 20 nm and upward. Fitting the low-q curve to the model of three-dimensional E


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which is consistent with the TEM observations. Compacted P3TMAHT chains may again become hydrated. Free DNA becomes visible with further addition (S8). The maximum seen in Figure 4c moves toward smaller q values with decreasing concentration. The full dilution sequences are shown in ref 22 and correspond to those discussed in ref 39. Next, we consider the phase regime III around the charge neutralization and answer two potential questions. The first question is whether the observed scattering patterns arise from polymer−DNA associations or represent the superposition of separated noninteracting polymer and DNA particles. Figure 5 shows the concentration normalized SAXS curves of PF2/6-P3TMAHT(DNA)0.38 (S5) alongside 2-times diluted

object gives a reasonable maximum particle diameter as 35 nm that corresponds with the size of particles observed in TEM images. If the polymer blocks were fully elongated, they would represent maximal intermolecular distances ∼15 nm and ∼20 nm, as calculated from the lengths of fluorene and thiophene repeat units ∼8.1 Å33 and ∼4 Å.34 Because even these distances are smaller than the observed particle size, we assume, plausibly, that the larger length scale scattering features are associated with polymer particles (containing several polymers) and the smaller length scale features with intraparticle structure. The solubility difference between PF2/6 and P3TMAHT blocks would agree with a core−shell structure composed of a fluorene core and thiophene exterior. Both humps contain subsequent regimes scaling as q−1 and −3 q , which together can be associated with one-dimensional domains with significant roughness.35 On the longer length scales, this scaling can refer to the emerging Guinier plateau of particles larger than the observation window or to onedimensional particle arrays. This is consistent with the TEM images, although the fitted cylinder diameter is somewhat smaller (∼95 Å). On the smaller length scales, the same scaling refers to cylindrical segments with the diameter of ∼20 Å, exactly the same as those seen on the particle surface in TEM images. We assume that the P3TMAHT blocks are located on the particle surface forming a hydrated layer and that either polymers or polymer bundles can appear as finite length in SAXS measurements. It is also possible that the particle core is not densely packed but the PF2/6 blocks also appear as separated rods. These ideas are supported by the relatively long persistence lengths (∼30 Å36 and ∼70 Å37) obtained elsewhere for regioregular poly(3-hexylthiophene) and PF2/6. We have previously shown that aqueous P3TMAHT homopolymer appears as charged spheres, and the SAXS data includes an interference maximum that moves toward lower q with decreasing concentration.38 PF2/6-P3TMAHT does not show interference maxima, and the scattering features remain essentially the same upon dilution. This implies that the interparticular order due to the charged groups is hindered by PF2/6 block. By inspection of Figure 4, PF2/6-P3TMAHT(DNA)x reveals the following features with increasing x. II. Below the assumed charge neutralization point, x < 0.38, compared to the pure polymer, the scattering intensity increases below ∼0.04 Å−1 and decreases above ∼0.04 Å−1. The scaling in the lower-q range begins to follow −2 decay instead of −1 decay. The emergence of −2 decay may be attributed to the transition from particles to the branched particle arrays which are volume fractal in nature, consistent with the TEM images. The high-q range begins to deviate from −1 decay, which indicates emerging loss of P3TMAHT separation and particle compactness. A sharp maximum at ∼0.05 Å−1 emerges for x = 0.19 (S4). III. At about the charge neutralization point, x = 0.38−0.76, a sharp maximum is present at 0.045 Å−1. This peak does not move with dilution (as shown in the Supporting Information), which suggests the intraparticular order. The high-q range behaviors deviate from −1 decay. IV. Above the charge neutralization point, x ≥ 1.89, the shape of the scattering pattern becomes completely different, containing −4 decay, which turns to the −1 decays at 0.02− 0.03 Å−1 (S7). This indicates large three-dimensional particles,

Figure 5. SAXS curves of aqueous PF2/6-P3TMAHT (S1D1, wine), PF2/6-P3TMAHT(DNA)0.38 (S5, dark cyan), and DNA (S9D1, purple). Superposition of S1D1 and S9D1 is shown for comparison (cyan).

PF2/6-P3TMAHT (S1D1) and 2-times diluted DNA (S9D1). Superposition of polymer and DNA scattering patterns are also shown. Concentrations for pure PF2/6-P3TMAHT and DNA solutions are 4.97 and 2.50 mg/mL. The concentrations for PF2/6-P3TMAHT and DNA within the PF2/6-P3TMAHT(DNA)0.38 solution are also 4.97 and 2.50 mg/mL. Thus, the total concentrations of the pure compounds shown are the same as their concentrations in the mixture. The scattering pattern of PF2/6-P3TMAHT(DNA)0.38 is significantly different than the scattering pattern of its constituents or their superposition. This simple comparison indicates that the observed differences in scattering patterns arise from the polymer−DNA complexes or aggregates formed. The idea of complex formation is in full agreement with the observed optical effects (vide infra). The second question is the origin of the reflection at 0.04− 0.05 Å−1 corresponding to the distance 125−160 Å. This maximum does not move upon dilution, which points toward some internal structure of PF2/6-P3TMAHT(DNA)x particles. However, it moves slightly toward lower q with increasing x. We speculate that DNA incorporates itself into polymer particles and we are seeing the growth of a DNA “layer” which becomes more prominent with increasing DNA fraction. This would cause the maximum to move to smaller q with increasing x. If we assume that the density of all compounds is 1 mg/mL, we estimate the X-ray scattering length densities as 9.35 × 1010 1/cm2 and 8.71 × 1010 1/cm2 for PF2/6 and P3TMAHT blocks, respectively, and as 8.92 × 1010 1/cm2 and 9.44 × 1010 F


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The Journal of Physical Chemistry B 1/cm2 for DNA and water, respectively. This implies that the contrast is high between polymer blocks and high between water and charged regions. However, the contrast is lower between PF2/6 and water and between P3TMAHT and DNA. Lower contrast between charged regions may imply that the maximum arises from the combined “layer” of DNA and P3TMAHT block. We thus argue that DNA merges to the hydrated P3TMAHT block, making it more compact with significant hydration loss and leading to a well-defined layer within the complex. Studies using lanthanide ion luminescence indicate hydration loss upon charge neutralization in DNA.40 After charge neutralization, these layers are loosened and the polymer aggregates begin to decorate DNA that becomes the majority compound. We have previously observed a smooth peak 0.02−0.05 Å−1 in dry PF2/6-P3TMAHT films spun from water.41 This peak should not be confused with the peak observed for polymer DNA complex but can arise from the contrast between the blocks. The reason why the same peak is not observed for polymer solutions can be attributed to the enhanced molecular order caused in the solid-state form and film substrate. Figure 6 plots normalized p(r) functions of PF2/6P3TMAHT calculated form the whole q range and from q >

S6 has one symmetric maximum. The shoulder implies fluctuations in the size distribution just below charge neutralization. Optical Properties. UV−Visible Absorption. Panels a and b of Figure 7 show the normalized absorption spectra of aqueous

Figure 7. PA spectra of aqueous PF2/6-P3TMAHT and PF2/6P3TMAHT(DNA)x for the initial concentrations after 2-fold (a) and 16-fold dilution (b). Navy, dark yellow, magenta, cyan, blue, olive, red and black lines refer to the samples S1−S8, respectively.

PF2/6-P3TMAHT and PF2/6-P3TMAHT(DNA)x after 2-fold and 16-fold dilutions, respectively. As shown by Fonseca et al.,28 the absorption spectra of aqueous PF2/6-P3TMAHT are interpreted as the superposition of the absorption bands of PF2/6 and P3TMAHT blocks at about 375 nm (λabs PF2/6) and 445 nm (λabs P3TMAHT). Figure 8a shows the maximum absorption wavelength of the P3TMAHT block with increasing x for all dilutions. Figure 8b shows the intensity ratio of P3TMAHT to PF2/6 absorption bands deduced from the same data (PAP3TMAHT/PAPF2/6). The PF2/6 band is rather insensitive to the DNA addition, whereas the P3TMAHT band shifts with increasing x. For x ≤ abs 0.07, λP3TMAHT is initially slightly redshifted to longer absorption wavelengths (3−4 nm). Starting at x = 0.17, abs λP3TMAHT shifts back toward shorter wavelengths again, followed by a distinct red shift of 44 nm for even higher x. The same trends are observed for the diluted samples, with a ca. 5 nm blue shift for the highest DNA molar ratio studied, x = 3.78. The spectral shifts of P3TMAHT band are accompanied by changes in the spectral ratio PAP3TMAHT/PAPF2/6. This decreases until close to the expected charge neutralization, x = 0.38, and increases again for higher x. The spectral shifts in the P3TMAHT absorption band reflect changes in its effective conjugation length. The blue shift when approaching x = 0.5 is indicative of decreased conjugation length and decreased main chain planarization, which is in agreement with the structural data suggesting vanishing separation of P3TMAHT chains. The opposite effect is seen for higher x. Recalling the core−shell

Figure 6. Normalized p(r) functions for PF2/6-P3TMAHT deduced from the whole q range and from q > 0.04 Å−1 (S1, solid brown and navy curves) and for PF2/6-P3TMAHT(DNA)x around the nominal charge compensation point (S5, blue dashed line; S6, olive dotted line).

0.04 Å−1. Also shown are p(r) functions for PF2/6-P3TMAHT(DNA)x around the charge compensation point (x = 0.38− 0.76; S5 and S6). These curves correspond to the data shown in Figure 3 and Figure 4b. Alternative presentation for the first p(r) functions and the fit to the whole q range are shown in the Supporting Information. For pure polymer, these curves indicate small objects a few nanometers in diameter and larger particles with dominant size around 20 nm. The first maximum is related to the locally separated P3TMAHT bundle or P3TMAHT chain within the hydrated P3TMAHT domain, whereas the second maximum stems from the core and the heavy hydrated shell of polymer particles. The particle size increases upon DNA addition. The first pure polymer maximum disappears. Together with vanishing −1 decay this indicates disappearance of separated P3TMAHT bundles and compaction of the P3TMAHT domain (and thus the whole particle). The curve S5 contains two maxima, while G


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Figure 8. (a) Maximum absorption wavelength for P3TMAHT band, λabs P3TMAHT. (b) Ratio between absorption maxima of P3TMAHT and PF2/6 bands, PAP3TMAHT/PAPF2/6, for the whole dilution series D1− D4.

Figure 9. Normalized PL spectra of aqueous PF2/6-P3TMAHT and PF2/6-P3TMAHT(DNA)x with increasing x for the initial concentrations (a) and after 16-fold dilution (b). Navy, dark yellow, magenta, cyan, blue, olive, red, and black lines refer to samples S1−S8, respectively. λexc = 380 nm.

structure, we assume that the block proximity is nearly constant and may play a less important role in absorption shifts. As in the case of polythiophene homopolymers,14,15 we assume that P3TMAHT block can roll around the DNA groove leading to the reduced planarization and conjugation length. PF2/6 block remains inside the spherical polymer particles. We assume that the main energy-transfer phenomena occur between blocks and PF2/6-P3TMAHT does not act as photosensitizer for DNA. PL. Panels a and b of Figure 9 show normalized PL spectra of PF2/6-P3TMAHT and PF2/6-P3TMAHT(DNA)x initially and after 16-fold dilution, respectively. The excitation wavelength λexc was 380 nm corresponding to the absorption maximum of the PF2/6 block. The PL spectrum of pure PF2/ 6-P3TMAHT in water is dominated by a broad PL feature of the P3TMAHT blocks at 550−750 nm, thus documenting efficient Förster-type resonance energy transfer (FRET) from the PF2/6 to the P3TMAHT block. The PL spectra of PF2/6P3TMAHT(DNA)x are composed of well-separated PF2/6 and P3TMAHT bands. Figure 10a shows the PL maximum of the P3TMAHT band λPL P3TMAHT. Figure 10b shows the relation of PL intensities between P3TMAHT and PF2/6 blocks, PLP3TMAHT/PLPF2/6. This ratio describes the efficiency for energy transfer between blocks and is thus connected with the polyelectrolyte structure. The PL data provide us four phenomenological phase regimes as a function of x: I and II. For pure polymer and complex below the assumed charge compensation point, x < 0.38, the spectrum contains two distinctive bands, λPL P3TMAHT shifts toward lower wavelengths PL and λPF2/6 is nearly constant. Thus, PLP3TMAHT/PLPF2/6 decreases rapidly with increasing x. III. At the charge compensation point, x = 0.38−0.76, when the excitation is in the PF2/6 band at 380 nm, the shape of the PL spectrum changes and it displays only one broad emission

band with maximum at 470 nm. The P3TMAHT band is no longer detectable. However, when the P3TMAHT block is excited at 445 nm, we can identify a weak P3TMAHT band with a PL maximum at 566−592 nm. The corresponding shift depending on the total concentration is shown in the Supporting Information. IV. Above the charge compensation point, x ≥ 1.89, λPL P3TMAHT slightly blue shifts. The PLP3TMAHT/PLPF2/6 ratio decreases slightly with increasing x. When the samples are diluted, the trends in spectra shifts as a function of x are unchanged, but the P3TMAHT band is systematically blue shifted for each x. This indicates a weakening of the intermolecular interactions and reduced energy transfer between the blocks. Structural data do not change with dilution. This is due to the fact that PL spectra emphasize changes in the backbone conformation and this is not necessarily manifested on the nanometer scale. Photoexcitation Profile. PL excitation profiles of aqueous PF2/6-P3TMAHT and PF2/6-P3TMAHT(DNA)x are shown in the Supporting Information. When collected at 415 nm (corresponding to the PF2/6 emission), the excitation spectra of PF2/6-P3TMAHT exhibit the characteristic absorption band of PF2/6. When the fluorescence excitation spectra are collected at 600 nm (corresponding to the P3TMAHT emission), both PF2/6 and P3TMAHT absorption bands are present. This again confirms the existence of energy transfer from PF2/6 to P3TMAHT segments. The intensity of the P3TMAHT-related band increases with increasing DNA content, indicating that the complexes formed between the polymer and DNA promote energy transfer between the PF2/6 H


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Figure 10. (a) Maximum PL wavelength for P3TMAHT band, λPL P3TMAHT. (b) Ratio between PL intensities of P3TMAHT and PF2/6 bands PLP3TMAHT/PLPF2/6. The initial concentration is marked by royal blue squares and the dilution series D1−D4 by wine circles, violet up triangles, gray down triangles, and cyan diamonds, respectively. λexc = 380 nm.

Figure 11. Top panel: studied concentration ranges and observed optical effects for aqueous PF2/6-P3TMAHT and PF2/6-P3TMAHT(DNA)x as a function of x, plotted for the whole polymer (green), P3TMAHT block (red), and DNA (cyan), initially (solid lines) and after 16-fold dilution (dotted lines). Bottom panel: Illustration of suggested aggregate structures for different x in the submicrometer and nanometer levels. White, terracotta, and yellow refer to PF2/6, P3TMAHT, and DNA, respectively. Roman numerals I−IV refer to the regimes near the pure polymer and the regimes below, around, and well above the charge neutralization with DNA, respectively.

and P3TMAHT blocks, possibly reflecting greater planarization of the diblock copolymer chain. Phase Regimes. Figure 11 outlines our structural idea and main optical consequences of the interaction of PF2/6P3TMAHT with DNA addition. I. PF2/6-P3TMAHT forms spherical particles with some tendency to chainlike arrangements in the submicrometer level. Cationic P3TMAHT blocks constitute the outer layer where P3TMAHT bundles or chains are hydrated and pointing toward the medium. Particles are completely dissolved. II. Below the charge neutralization point, x < 0.38, the addition of DNA leads to branched chainlike structures and promotes changes in optical spectra. Several diblock polymer particles may associate around a single DNA chain because of the electrostatic interactions. This process results in the quenching and blue shift of thiophene PL as well as decrease in the FRET efficiency (Figure S5 of the Supporting Information). The energy migration along the polymer chain (PF2/6 → P3TMAHT) is probably quenched at aggregate traps. Similar behavior has been observed in the fluorescence behavior upon interaction of cationic trimethylammonium substituted polyfluorene and DNA in aqueous solutions upon increasing the conjugated polyelectrolyte chain length.42 III. Around the charge neutralization point, x = 0.38−0.76, the structure involves branched chains but an additional interparticular structure appears because of the well-packed layer of P3TMAHT with intercalated DNA. P3TMAHT bundles are no longer separated, and P3TMAHT domains

and thus the whole polymer particle become more compact than the pure polymer particle. Complexes become less polar, which causes diminished solubility in water and some precipitation. IV. Above the charge neutralization point, x ≥ 1.89, the structure becomes much larger on the submicron level and the intermolecular order disappears because of the excess of negative charges. Simultaneously, solubility is enhanced. The absorption band of P3TMAHT is red shifted and the emission band blue shifted, evidencing again a less compact P3TMAHT block and polymer particles decorating DNA. The excitation spectrum is enhanced, becoming more efficient than in polymers solutions. The PLP3TMAHT/PLPF2/6 decreases, but the corresponding excitation ratio detected at 600 nm increases (Figure S5 of the Supporting Information).

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CONCLUSIONS In aqueous solution, the all-conjugated cationic block copolymer PF2/6-P3TMAHT, composed of ionic P3TMAHT and nonionic PF2/6 blocks, appears as 20−40 nm sized spherical core−shell particles with a P3TMAHT exterior, forming loose one-dimensional chains. Mixing with double-stranded DNA to form PF2/6-P3TMAHT(DNA)x turns this structure into branched nano-objects and finally I


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All-Conjugated Diblock Copolymers. J. Phys. Chem. C 2011, 115, 9260. (8) Conjugated Polyeletrolytes - Fundamentals and Applications; Liu, B., Bazan, G. C., Eds.; Wiley-VCH: Chichester, 2013. (9) Liu, B.; Bazan, G. C. Homogeneous Fluorescence-Based DNA Detection with Water-Soluble Conjugated Polymers. Chem. Mater. 2004, 16, 4467. (10) Pinto, M. R.; Schanze, K. S. Amplified Fluorescence Sensing of Protease Activity with Conjugated Polyelectrolytes. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7505. (11) Liu, B.; Bazan, G. C. Methods for Strand-Specific DNA Detection with Cationic Conjugated Polymers Suitable for Incorporation into DNA Chips and Microarrays. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 589. (12) Duan, X. R.; Liu, L. B.; Feng, F. D.; Wang, S. Cationic Conjugated Polymers for Optical Detection of DNA Methylation, Lesions, and Single Nucleotide Polymorphisms. Acc. Chem. Res. 2010, 43, 260. (13) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. DNA Detection Using Water-Soluble Conjugated Polymers and Peptide Nucleic Acid Probes. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954. (14) Doré, K.; Dubus, S.; Ho, H.-A.; Lévesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M. G.; Boudreau, D.; Leclerc, M. Fluorescent Polymeric Transducer for the Rapid, Simple, and Specific Detection of Nucleic Acids at the Zeptomole Level. J. Am. Chem. Soc. 2004, 126, 4240. (15) Ho, H.-A.; Najari, A.; Leclerc, M. Optical Detection of DNA and Proteins with Cationic Polythiophenes. Acc. Chem. Res. 2008, 41, 168. (16) Preat, J.; Teixeira-Dias, B.; Michaux, C.; Perpète, E. A.; Alemán, C. Specific Interactions in Complexes Formed by DNA and Conducting Polymer Building Blocks: Guanine and 3,4(Ethylenedioxy)Thiophene. J. Phys. Chem. A 2011, 115, 13642. (17) Geng, J.; Liang, J.; Wang, Y.; Gurzadyan, G. G.; Liu, B. MetalEnhanced Fluorescence of Conjugated Polyelectrolytes with SelfAssembled Silver Nanoparticle Platforms. J. Phys. Chem. B 2011, 115, 3281. (18) Karlsson, K. F.; Åsberg, P.; Nilsson, K. P. R.; Inganäs, O. Interactions between a Zwitterionic Polythiophene Derivative and Oligonucleotides as Resolved by Fluorescence Resonance Energy Transfer. Chem. Mater. 2005, 17, 4204. (19) Al Attar, H. A.; Monkman, A. P. Effect of Surfactant on WaterSoluble Conjugated Polymer Used in Biosensor. J. Phys. Chem. B 2007, 111, 12418. (20) Kim, Y.; Swager, T. M. Sensory Polymers for Electron-Rich Analytes of Biological Interest. Macromolecules 2006, 39, 5177. (21) Tang, Y. L.; Achyuthan, K. E.; Whitten, D. G. Label-Free and Real-Time Sequence Specific DNA Detection Based on Supramolecular Self-Assembly. Langmuir 2010, 26, 6832. (22) Knaapila, M.; Costa, T.; Garamus, V. M.; Kraft, M.; Drechsler, M.; Scherf, U.; Burrows, H. D. Conjugated Polyelectrolyte (CPE) Poly{3-[6-(N-methylimidazolium)hexyl]-2,5-thiophene} Complexed with DNA: Relation between Colloidal Level Solution Structure and Chromic Effects. Macromolecules 2014, 47, 4017. (23) Davies, M. L.; Burrows, H. D.; Cheng, S.; Morán, M. C.; Miguel, M. D.; Douglas, P. Cationic Fluorene-Based Conjugated Polyelectrolytes Induce Compaction and Bridging in DNA. Biomacromolecules 2009, 10, 2987. (24) Yang, G.; Yuan, H.; Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. New Conjugated Polymers for Photoinduced Unwinding of DNA Supercoiling and Gene Regulation. ACS Appl. Mater. Interfaces 2012, 4, 2334. (25) Park, J. Y.; Koenen, N.; Forster, M.; Ponnapati, R.; Scherf, U.; Advincula, R. Interplay of Vesicle and Lamellae Formation in an Amphiphilic Polyfluorene-b-Polythiophene All-Conjugated Diblock Copolymer at Air-Water Interface. Macromolecules 2008, 41, 6169. (26) Scherf, U.; Adamczyk, S.; Gutacker, A.; Koenen, N. AllConjugated, Rod-Rod Block Copolymers - Generation and SelfAssembly Properties. Macromol. Rapid Commun. 2009, 30, 1059.

into micron-sized aggregates with increasing x. A well-ordered 12−16 nm structure is seen around x = 0.5 and is concomitant with the compaction of the P3TMAHT domain, which can be attributed to the DNA layer becoming merged within the P3TMAHT domain. P3TMAHT block can wind around DNA while PF2/6 remains inside the spherical polymer particles. This structure disassembles with a DNA excess. DNA addition is accompanied by chromic changes, notably color change from deep red to yellow and back toward orange. The PL spectra have contributions from both blocks, but the polythiophene band is 1000 times quenched by 10% DNA addition, which allows sensitive and quantitative detection of DNA fraction. This work is a rare example where CPE−DNA research is extended from homopolymers to block copolymers and where the optical manifestations are illustrated through structural evaluation. Future work should focus on the particle deformation and DNA condensation, as shown elsewhere for other spherical particles, cationic dendrimers.43

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis details, additional SAXS analysis of P3TMAHT, SAXS data of dilution series for samples S5 and S6, PL shift of P3TMAHT block, and the excitation spectra of studied materials. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank C. Blanchet of EMBL for help at P12 BioSAXS. T.C. thanks FCT, the Portuguese agency for scientific research, which has supported this work through a Grant (SFRH/BPD/ 47181/2008). Financial support of the Coimbra Chemistry Centre from the FCT through project PEst-OE/QUI/UI0313/ 2014 is acknowledged.

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