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Jun 2, 2010 - ... 3004-535 Coimbra, Portugal, §Instituto de Biologıá. Molecular y Celular, Universidad Miguel Hern´andez, Elche 03202, Alicante, S...
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How to Change the Aggregation in the DNA/Surfactant/Cationic Conjugated Polyelectrolyte System through the Order of Component Addition: Anionic versus Neutral Surfactants )

Marı´ a Monteserı´ n,† Hugh D. Burrows,*,‡ Ricardo Mallavia,§ Roberto E. Di Paolo, Antonio L. Mac-anita, and Marı´ a J. Tapia*,†

† Departamento de Quı´mica, Universidad de Burgos, Plaza Misael Ba~ nuelos, Burgos 09001, Spain, Departamento de Quı´mica, Universidade de Coimbra, 3004-535 Coimbra, Portugal, §Instituto de Biologı´a Molecular y Celular, Universidad Miguel Hern andez, Elche 03202, Alicante, Spain, and Centro de Quı´mica Estrutural, Departamento de Engenharia Quı´mica e Biol ogica, Instituto Superior T ecnico (IST), Avenida Rovisco Pais, P1049-001, Lisboa, Portugal )

‡

Received March 24, 2010. Revised Manuscript Received May 18, 2010 The competitive interaction has been studied between double-stranded DNA (dsDNA), the cationic conjugated polyelectrolyte (CPE) poly[9,9-bis(6-N,N,N-trimethylamonium)hexyl)-fluorene-phenylene)] bromide (HTMA-PFP) and anionic or neutral surfactants (sodium dodecyl sulfonate, SDSu, and n-dodecyl pentaoxyethylene glycol ether, C12E5) in 4% (v/v) dimethyl sulfoxide (DMSO)-water using UV/visible absorption and fluorescence spectroscopy. Dramatic changes are observed in the spectroscopic behavior of the system depending on the order of addition of the reagents, the surfactant charge, and concentration range. If the neutral C12E5 is added to the HTMA-PFP/dsDNA complex, no significant spectroscopic changes are observed. However, if SDSu is added to the same complex, a dramatic increase of the absorbance and emission intensity is observed for surfactant concentrations above the critical micelle concentration (cmc). In contrast, if dsDNA is added to HTMA-PFP/surfactant systems (with surfactant concentrations above their cmc) no significant changes are observed with SDSu, while a dramatic quenching of polymer emission is observed with C12E5, which can be explained quantitatively in terms of HTMA-PFP/surfactant/DNA complexation and the subsequent polymer aggregation upon charge neutralization. The results are compared with those for the binary systems (HTMA-PFP/DNA and HTMA-PFP/surfactants) and indicate the importance of electrostatic interactions between HTMA-PFP and oppositely charged species in the aggregation processes.

Introduction The cationic poly-(9,9-bis (60 -N,N,N-trimethylammonium)hexyl)-fluorene phenylene), HTMA-PFP, is a conjugated polyelectrolyte (CPE) widely used as a biosensor.1-5 This shows an intense blue emission6,7 with fluorescence quantum yield of ∼0.5 in DMSO-water (4%, v/v) solution.7 Typically, bromide or iodide is used as counterion, and although these CPEs are nominally watersoluble, the organic cosolvent is needed to minimize aggregation.8 Fluorescence resonance energy transfer (FRET) from HTMAPFP to dye-labeled DNA or peptide nucleic acid (PNA) has been extensively used in sensing DNA. In PNA, the replacement of the negatively charged sugar-phosphate groups of DNA by the polyamide chains allows DNA to bind more tightly to PNA than *Corresponding authors. Marı´ a Jose Tapia: E-mail [email protected], Phone þ34 947258061, Fax: (þ34) 947 28831. Hugh D. Burrows: E-mail [email protected], Phone þ351 239854482, Fax: (þ351) 239 827703.

(1) Tapia, M. J.; Montserı´ n, M.; Valente, A. J. M.; Burrows, H. D.; Mallavia, R. Adv. Coll. Interf. Sci. 2010, 158, 94-107. (2) Liu, B.; Bazan, G. C. Chem. Mater. 2004, 16, 4467–4476. (3) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954–10957. (4) Liu, B.; Wang, S.; Bazan, G. C.; Mikhailovsky, A. J. Am. Chem. Soc. 2003, 125, 13306–13307. (5) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 1942–1943. (6) Mallavia, R.; Martinez-Perez, D.; Chmelka, B. F.; Bazan, G. C. Bol. Soc. Esp. Ceram. Vidrio 2004, 43, 327–220. (7) Monteserı´ n, M.; Burrows, H. D.; Valente, A. J. M.; Lobo, V. M. M.; Mallavia, R.; Tapia, M. J.; Garcı´ a-Zubiri, I. X.; Di Paolo, R. E.; Macanita, A. L. J. Phys. Chem. B 2007, 111, 13560–13569. (8) Monteserı´ n, M.; Tapia, M. J.; Ribeiro, A. C. F.; Santos, C. I. A. V.; Valente, A. J. M.; Burrows, H. D.; Mallavia, R.; Nilsson, M.; S€oderman, O. J. Chem. Eng. Data 2010, 55, 1860-1865.

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to another DNA.9 The system proposed by Bazan et al.10 consists of a fluorescein-labeled PNA, the HTMA-PFP derivative and a polynucleotide with a sequence complementary to the PNA. Electrostatic interaction between the polymer and the PNA/ single-stranded DNA (ssDNA)/fluorescein duplex facilitates very efficient FRET from the polymer to fluorescein with up to a 100fold increase of its emission intensity.3,10 No emission of fluorescein is detected if the polynucleotide is not complementary to the PNA. The basis of this method is the electrostatic attraction between cationic conjugated polymer and dye-labeled negatively charged macromolecule, which leads to a supramolecular system that fulfills the distance requirement for FRET.10 This has also been applied to RNA/peptide (with a modified HTMA-PFPþ cationic polymer without the phenylene unit),11 DNA/DNA,12,13 PNA/dsDNA,9 and ribonucleic acid (RNA)/RNA10,14 recognition pairs10 and in protein/aptamer15,16 assays. Solid state sensors can be formed through adding ssDNA to surfaces to which PNA is attached.17 (9) Baker, E. S.; Hong, J. W.; Gaylord, B. S.; Bazan, G. C.; Bowers, M. T. J. Am. Chem. Soc. 2006, 128, 8484–8492. (10) Bazan, G. C. J. Org. Chem. 2007, 72, 8615–8635. (11) Wang, S.; Bazan, G. C. Adv. Mater. 2003, 15, 1425–1428. (12) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 896–900. (13) He, F.; Tang, Y. L.; Yu, M. H.; Feng, F.; An, L. L.; Sun, H.; Wang, S.; Li, Y. L.; Zhu, D. B.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 6764–6765. (14) Liu, B.; Baudrey, S.; Jaeger, L.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 4076–4077. (15) Wang, Y.; Liu, B. Biosens. Bioelectron. 2009, 24, 3293–3298. (16) Wang, Y.; Liu, B. Langmuir 2009, 25, 12787–12793. (17) Liu, B.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 589–593.

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Figure 1. (A) HTMA-PFP, (B) SDSu, (C) C12E5 chemical structures.

On the basis of these FRET processes, HTMA-PFP has also been used as a highly sensitive hydrogen peroxide (H2O2) probe with a detection range of 15 to 600 nM. This can be extended to glucose detection with the H2O2 acting as signal transducer by reacting with fluorescence quenching peroxyfluor-1 with boronate protecting groups to form the anionic fluorescein, which encourages fluorescence resonance energy transfer from the cationic polymer.18 However, there is considerable interest in devising methods which do not involve FRET for studying nucleic acids. We have shown that, by adding DNA (either double-stranded, dsDNA, or single-stranded, ssDNA) to the cationic HTMA-PFP (Figure 1) at DNA/polymer ratios corresponding to charge neutralization, the emission quantum yield reaches a minimum due to the polymer complexing with DNA through electrostatic interaction. The spectra shift to the red, and the ratio of emission intensities for the strongest band and first shoulder reaches a maximum, while the electrical conductivity decreases and the solution viscosity remains constant. These physical properties allow the determination of the electroneutrality point, and provide routes for determining DNA at micromolar concentrations.19 One drawback with aqueous solutions of cationic fluorenebased CPEs as sensors is their tendency to form aggregates. These decrease emission intensity, as has been shown for a cationic polyfluorene.20 It has been suggested that aggregate formation with HTMA-PFP in methanol for concentrations above the mM range involves π-π interaction, which leads to a decrease in emission quantum yield, a red-shifted, less structured emission, and a decrease in the self-diffusion coefficients.21 A similar decrease in the HTMA-PFP mutual diffusion coefficient has been observed in DMSO-water solutions upon increasing polymer concentration.8 CPE aggregates can be broken up by addition of the nonionic C12E5 at concentrations around the cmc through formation of (18) He, F.; Tang, Y. L.; Yu, M. H.; Wang, S.; Li, Y. L.; Zhu, D. B. Adv. Funct. Mat. 2006, 16, 91–94. (19) Monteserı´ n, M.; Burrows, H. D.; Valente, A. J. M.; Mallavia, R.; Di Paolo, R. E.; Mac-anita, A. L.; Tapia, M. J. J. Phys. Chem. B 2009, 113, 1294–1302. (20) Wang, S.; Bazan, G. C. Chem. Commun. 2004, 2508–2509. (21) Wa˚gberg, T.; Liu, B.; Or€add, G.; Eliasson, B.; Edman, L. Eur. Polym. J. 2009, 45, 3230–3235. (22) Mukerjee, P.; Mysels, K. J. Critical Micelle Concentrations of Aqueous Surtactants Systems; NBS: Wasington, DC, 1971.

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mixed CPE-surfactant aggregates22,23 and lead to increases in quantum yield of HTMA-PPFþ (Mn = 3 kg mol-1) from 0.024 to 0.23 and a blue-shift in the emission spectra of around 10 nm.24 However, when negatively charged n-alkylsulfonate or sulfate surfactants are added to HTMA-PFP, a nearly complete quenching of the polymer emission intensity is observed due to the formation of neutral polymer-surfactant assemblies through charge neutralization.7 Only when the cmcs of these surfactants are reached does the polymer emission recover.7 Increases in emission intensity have also been observed with aqueous solutions of the negatively charged {1,4-phenylene-[9,9bis-(4-phenoxybutylsulfonate)]fluorene-2,7-diyl} (PBS-PFP) upon addition of C12E5,25-27 gemini surfactants,28 or the glyceride conjugate 1-O-(L-arginyl)-2,3-O-dilauroyl-sn-glycerol dichlorohydrate, 1212R, an arginine amino acid surfactant, and possible mimic for the phospholipid lecithin,29 due to the breakup of aggregates induced by the interaction with surfactants at concentrations above their cmcs. Following this improvement of optical properties of fluorenebased conjugated polyelectrolytes in aqueous solution upon interaction with nonionic surfactants, Monkman et al. have used HTMA-PFP/C12E5 assemblies in DNA sensing. The effect of the presence of C12E5 has been studied on direct interaction with ssDNA24 and on FRET from HTMA-PFP to PNA fluorescein labeled in the detection of single nucleotide polymorphism of mutant DNA,30 and the assay is said to be better than the FRET in absence of C12E5.30 In addition to these studies of the potential of CPEs in optical sensing, fluorescence and atomic force microscopy studies have shown that aggregation by the CPE can induce compaction in DNA and be useful for linking DNA strands to produce interesting structures,31 which may have potential applications in the developing field of DNA nanotechnology.32,33 Following from these ideas, we have extended our spectroscopic study on the interaction of HTMA-PFP with DNA19 by studying aqueous systems containing DNA, CPE, and surfactant (either the neutral C12E5 or the anionic sodium dodecyl sulfonate, SDSu). The effect of order of addition has been studied with the aim of gaining insight into the relative importance of electrostatic and hydrophobic interactions involved with oppositely charged polyelectrolyte/surfactant34,35 and DNA/surfactant36-38 systems (23) Holmberg, K.; J€onsson, B.; Kronberg, B.; Lindman, B., Surfactants and polymers in aqueous solution, 2nd ed.; Wiley: England, 2004. (24) Al Attar, H. A.; Monkman, A. P. J. Phys. Chem. B 2007, 111, 12418–12426. (25) Burrows, H. D.; Lobo, V. M. M.; Pina, J.; Ramos, M. L.; de Melo, J. S.; Valente, A. J. M.; Tapia, M. J.; Pradhan, S.; Scherf, U. Macromolecules 2004, 37, 7425–7427. (26) Burrows, H. D.; Lobo, V. M. M.; Pina, J.; Ramos, M. L.; de Melo, J. S.; Valente, A. J. M.; Tapia, M. J.; Pradhan, S.; Scherf, U.; Hintschich, S. I.; Rothe, C.; Monkman, A. P. Colloids Surf. A: Phys. Eng. Asp. 2005, 270, 61–66. (27) Burrows, H. D.; Tapia, M. J.; Fonseca, S. M.; Pradhan, S.; Scherf, U.; Silva, C. L.; Pais, A. A. C. C.; Valente, A. J. M.; Schillen, K.; Alfredsson, V.; Carnerup, A. M.; Tomsic, M.; Jamnik, A. Langmuir 2009, 25, 5545–5556. (28) Burrows, H. D.; Tapia, M. J.; Silva, C. L.; Pais, A.; Fonseca, S. M.; Pina, J.; de Melo, J. S.; Wang, Y. J.; Marques, E. F.; Knaapila, M.; Monkman, A. P.; Garamus, V. M.; Pradhan, S.; Scherf, U. J. Phys. Chem. B 2007, 111, 4401–4410. (29) Tapia, M. J.; Burrows, H. D.; Knaapila, M.; Monkman, A. P.; Arroyo, A.; Pradhan, S.; Scherf, U.; Pinazo, A.; Perez, L.; Moran, C. Langmuir 2006, 22, 10170–10174. (30) Al Attar, H. A.; Norden, J.; O’Brien, S.; Monkman, A. P. Biosens. Bioelectron. 2008, 23, 1466–1472. (31) Davies, M. L.; Burrows, H. D.; Cheng, S.; Moran, M. C.; Miguel, M. D.; Douglas, P. Biomacromolecules 2009, 10, 2987–2997. (32) Seeman, N. C. Nature 2003, 421, 427–431. (33) Aldaye, F. A.; Palmer, A. L.; Sleiman, H. F. Science 2008, 321, 1795–1799. (34) Pi, Y. Y.; Shang, Y. Z.; Liu, H. L.; Hu, Y. Acta Chimica Sinica 2005, 63, 1281–1287. (35) Pi, Y.; Shang, Y.; Liu, H.; Hu, Y.; Jiang, J. J. Colloid Interface Sci. 2007, 306, 405–410. (36) Zhao, X.; Shang, Y.; Hu, J.; Liu, H.; Hu, Y. Biophys. Chem. 2008, 138, 144– 149.

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in determining their phase behavior,39 in addition to kinetic effects possibly controlling aggregation.

was used as standard for quantum yield measurements.47 Emission quantum yields were calculated according to following equation:48

Experimental Section Reagents and Solution Preparation. The neutral poly-(9,90

bis(6 -bromohexyl)fluorene-1,4-phenylene) was synthesized via Suzuki coupling reaction over three days using 1,4-phenyldiboronic acid and 2,7-dibromo-9,9-bis(60 -bromohexyl)fluorene.40 The molecular weight of the neutral polymer was characterized by size exclusion chromatography (SEC) in tetrahydrofuran (THF) as solvent using polystyrene standards for calibration. This shows a regular average molecular weight distribution with n = 37, calculated from the maximum of the chromatogram. The cationic conjugated polyelectrolyte poly-(9,9-bis (60 -N, N,N-trimethylammonium)hexyl)-fluorene phenylene) bromide (HTMA-PFP, Figure 1) was obtained by treating the neutral poly-9,9-bis(60 -bromohexyl)-fluorene phenylene with trimethylamine gas following the procedure described elsewhere.6 We assume, in agreement with previous reports,41 almost quantitative conversion of the neutral copolymer into the cationic conjugated polyelectrolyte HTMA-PFP, and that this does not affect the degree of polymerization. According to this, the average molecular weight in number (Mn) value of HTMA-PFP was 14.5 kg/mol. HTMA-PFP shows low solubility in water, but can be dissolved in dimethyl sulfoxide-water mixtures. Stock polymer solutions with concentrations around 9.6  10-2 g/L (1.38  10-4 mol/L in repeat units) were prepared in DMSO (Aldrich, spectrophotometric grade) and were kept under continuous stirring overnight. Aliquots of this solution were diluted with Millipore-Q water to give solutions for the measurements with polymer concentrations between 3.0 and 5.5  10-6 M in terms of repeat units (between 2.1 and 3.8  10-3 g/L) in 4% (v/v) DMSO-water mixtures. Solutions of salmon testes dsDNA (approximately 2000 base pairs) from Sigma were prepared in Millipore-Q water. For the spectroscopic experiments, stock solutions had a concentration around 0.15 mg/mL (4.5 10-4 M, ε260 = 6600 mol-1 L cm-1).42 All DNA molar concentrations are reported relative to base (which is equal to the number of phosphate groups). Although DMSO is known to induce denaturation of dsDNA,43,44 we believe that this is not significant for our system in the presence of 4% (v/v) of DMSO, since studies on the effect of solvent composition on this process (with ca. (1-5)  10-2 M electrolyte) show that the midpoint of the denaturation transition corresponds much higher DMSO fractions (62 vol %),44 with no significant effect of salt concentration in the range 10-3 M to 5  10-2 M.44 In agreement with this idea, experiments carried out with acridine orange, a dye sensitive to DNA secondary structure,45 confirm that DNA is not denatured in the presence of 4% of DMSO,19,46 and also, no differences were observed between the absorbance at 260 nm of dsDNA (2.02  10-5 M) in aqueous solution and in DMSO-water 4% (v/v). SDSu and C12E5 were purchased from Fluka and Sigma, respectively. Quinine sulfate from Fluka in 0.1 M sulfuric acid (37) Zhao, X.; Shang, Y.; Liu, H.; Hu, Y. J. Colloid Interface Sci. 2007, 314, 478– 483. (38) Zhao, X. F.; He, Y. F.; Shang, Y. Z.; Han, X.; Liu, H. L. Acta Phys.-Chim. Sin. 2009, 25, 853–858. (39) Pi, Y.; Shang, Y.; Peng, C.; Liu, H.; Hu, Y.; Jiang, J. J. Colloid Interface Sci. 2006, 299, 410–415. (40) Mallavia, R.; Montilla, F.; Pastor, I.; Velasquez, P.; Arredondo, B.; Alvarez, A. L.; Mateo, C. R. Macromolecules 2005, 38, 3185–3192. (41) Liu, B.; Gaylord, B. S.; Wang, S.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 6705–6714. (42) Costa, D.; Burrows, H. D.; da Graca Miguel, M. Langmuir 2005, 21, 10492– 10496. (43) Herskovits, T. T. Arch. Biochem. Biophys. 1962, 97, 474–484. (44) Seto, D. Nucleic Acids Res. 1990, 18, 5905–5906. (45) Ichimura, S.; Zama, M.; Fujita, H. Biochem. Biophys. Acta 1971, 240, 485– 495. (46) Markarian, S. A.; Asatryan, A. M.; Grigoryan, K. R.; Sargsyan, H. R. Biopolymers 2006, 82, 1–5.

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φ ¼ φR

I ODR n2 IR OD n2R

ð1Þ

where φ is the quantum yield, I is the integrated intensity, OD is the optical density at the excitation wavelength, and n is the refractive index. The subscript R refers to the reference fluorophore of known quantum yield. Apparatus and Methods. Absorption spectra were recorded on a Shimadzu 2501 PC UV-visible spectrophotometer. For steady-state luminescence spectral measurements, a Shimadzu RF-5301 PC instrument was used in a right-angle configuration. The excitation wavelength was 381 nm, and excitation and emission slits were 3.0 and 1.5 nm, respectively. Both absorption and emission spectra were measured at 25.0 ( 0.1 °C. Time-resolved fluorescence measurements were carried out using a previously described single-photon counting system with picosecond time resolution.19,49 Solution electrical resistances were measured using a Wayne-Kerr model 4265 Automatic LCR meter at 1 kHz. A Shedlovsky-type conductance cell was used.50 The cell constant (0.0965 cm-1) was determined to (0.02% from measurements with KCl (reagent grade, recrystallized, and dried using the procedure and data from Barthel et al.51 Measurements were made at 25.0 ( 0.1 °C with cells in a Selecta thermostat bath.

Results The experimental results are seen to be strongly dependent upon the order of addition of the reagents, and we will therefore use this as an important criterion in their presentation. We will compare the effect of the surfactant addition to HTMA/dsDNA systems in the first section and that of dsDNA addition to HTMA/surfactant systems in the second. Addition of Surfactants to HTMA/dsDNA Systems. Various amounts of the anionic SDSu and nonionic C12E5 surfactants were added to CPE/DNA solutions with a HTMAPFP concentration ∼4.0  10-6 M (in terms of repeat units) and a dsDNA concentration 2.1  10-5 M (in terms of phosphate groups) in 4% (v/v) DMSO-water solution. Under these conditions, the polymer emission is quenched (quantum yields around 0.1) by the interactions with dsDNA. The quenching was attributed to aggregation through charge neutralization by the phosphate groups of dsDNA, as previously discussed.19 The aggregation is most marked for dsDNA concentrations above that of the CPE, where the positive charge of HTMA-PFP is completely neutralized by the negative charge of the phosphate groups of dsDNA.19 The starting point for surfactant addition is taken as that where the polynucleotide concentration is more than twice that of the CPE, since each HTMA-PFP repeat unit has two positive charges (Figure 1A). Addition of Sodium Dodecyl Sulfonate. In the presence of DNA, the addition of SDSu at concentrations below its cmc does not induce significant changes in the absorption or emission intensities or in the shapes of the spectra (Figure 2 and Figure 3). However, for SDSu concentrations close to the cmc dramatic changes are observed. The absorbance and emission intensity (47) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991–1024. (48) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 2004. (49) Giestas, L.; Yihwa, C.; Lima, J. C.; Vantier-Giongo, C.; Lopes, A.; Mac-anita, A. L.; Quina, F. H. J. Phys. Chem. A 2003, 107, 3263–3269. (50) Vink, H. J. Chem. Soc., Faraday Trans 1 1981, 77, 2439–2449. (51) Barthel, J.; Feuerlein, F.; Neuder, R.; Wachter, R. J. Sol. Chem. 1980, 9, 209–212.

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Figure 2. HTMA-PFP (3.6  10-6 M; Mn = 14.5 kg mol-1) with DNA (2.1  10-5 M) and various SDSu concentrations: (A) absorbance and wavelength of the maximum of the absorption spectra, full circles and empty circles, respectively; (B) emission quantum yield and wavelength of the emission maximum, full circles and empty circles, respectively; and emission quantum yield in absence of DNA, full squares.

Figure 3. (A) Emission spectra of HTMA-PFP (3.6  10-6 M;

Mn= 14.5 kg mol-1) with DNA (2.1  10-5 M) with various SDSu concentrations between 2.0  10-3 M and 4.2  10-2 M. (B) Ratio between the emission intensity in the fluorescence maximum (412 nm) and first shoulder (434 nm) (I412 nm/I434 nm) for HTMA-PFP (3.6  10-6 M; Mn = 14.5 kg mol-1) with DNA (2.1  10-5 M) and various SDSu concentrations. The dotted line indicates the SDSu cmc.

increase (Figures 2A, B and 3A), with the effect being most marked in emission, where fluorescence quantum yields rise to around 0.4; this can be compared with the values, close to 0.5, observed in the absence of dsDNA,7 when SDSu is added to HTMA-PFP over the same concentration range (Figure 2B). Moreover, the absorption maxima shift to shorter wavelengths for SDSu concentration between 5  10-4 and 5  10-3 M, and then to longer wavelengths for higher surfactant concentrations (Figure 2A). In addition, the emission spectra shift to the blue (Figure 2B) and the vibrational structure changes (Figure 3A), as can be followed by the increase of the ratio of emission intensities in the main band (412-423 nm) and the first shoulder (434-445 nm) (Figure 3B). Time-resolved fluorescence studies have been carried out, and the effect studied of adding anionic surfactant to HTMA-PFP (4.49  10-6 M, Mn = 14.5 kg mol-1) solutions with a dsDNA concentra11708 DOI: 10.1021/la1011764

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Figure 4. (A) Fluorescence lifetimes and (B) ratio of amplitudes of slow (a1 þ a2, open circles) and fast (a3, full squares) components observed at 416 nm as functions of surfactant concentration obtained from triple-exponential fittings of the decays of HTMAPFP (4.49  10-6 M, Mn = 14.5 kg mol-1) solutions with a dsDNA concentration (2.15  10-5 M) in DMSO-water (4% v/v) and various additions of SDSu. Excitation wavelength at 380 nm. In both Figures, SDSu concentration 10-7 M is an arbitrary value that corresponds to zero surfactant concentration needed to plot this point on a logarithmic scale.

tion (2.15  10-5 M) in DMSO-water (4% v/v). All the decays were fitted to three exponentials (Supporting Information Figure S1), and the results are shown in Supporting Information Table S1. We will concentrate initially on the behavior in the presence of DNA and discuss the effect of addition of SDSu. When DNA is present, the three lifetimes are effectively independent of surfactant concentration until the cmc is reached, and then all three increase (Figure 4A). Changes are also observed in the amplitudes of the three components over the same concentration range. These are seen most clearly through the ratio of amplitudes for the slow (a1 þ a2) and fast (a3) components. Results observed at 416 nm as functions of surfactant concentration are shown in Figure 4B. Similar behavior was observed at 450 nm. In both cases, the slow components become more important, and the fast one less important, above the cmc. While there is as yet no consensus on the assignment of these three lifetime components, it is generally accepted that in conjugated polymers a short-lived component reflects, at least in part, conformational changes in the excited conjugated polymer backbone,52 possibly accompanied by some singlet energy migration along the polymer backbone.53 Normally, when either conformational relaxation or energy transfer/migration are involved, the shortest-lived component appears as a rise time (negative amplitude) in the decays collected at sufficiently long wavelengths (here 450 nm),52-55 which is not found with HTMAPFP in the presence of DNA (see Supporting Information Table S1). However, in Figure 3, it was seen that changes in the vibronic structure of the HTMA-PFP fluorescence occur within the same surfactant concentration region, leading to a more structured emission spectrum after the cmc, similar to that of free PFP chromophore. This suggests that, as the cmc is reached, SDSu (52) Dias, F. B.; Macanita, A. L.; Melo, J. S. d.; Burrows, H. D.; Guntner, R.; Scherf, U.; Monkman, A. P. J. Chem. Phys. 2003, 118, 7119–7126. (53) Pinto, S. M.; Burrows, H. D.; Pereira, M. M.; Fonseca, S. M.; Dias, F. B.; Mallavia, R.; Tapia, M. J. J. Phys. Chem. B 2009, 113, 16093–16100. (54) Di Paolo, R. E.; Melo, J. S. d.; Pina, J.; Burrows, H. D.; Morgado, J.; Mac-anita, A. L. ChemPhysChem 2007, 8(18), 2657–2664. (55) Di Paolo, R. E.; Burrows, H. D.; Morgado, J.; Mac- anita, A. L. ChemPhysChem 2009, 10(2), 448–454.

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Figure 5. Emission quantum yield and Rayleigh scattering of HTMA-PFP (3.6  10-6 M; Mn = 14.5 kg mol-1) and various C12E5 concentrations in full and empty symbols, respectively; results are given with (2.1  10-5 M) and without dsDNA (circles and triangles), respectively.

replaces dsDNA in binding to HTMA-PFP, and that in the surfactant/CPE complex, fluorescence is less quenched. If the three components can be associated with three different species (e.g., HTMA-PFP aggregate, HTMA-PFP/ds-DNA complex, and SDSu-micellized HTMA-PFP), their amplitudes would be equal to the mole fractions of the three species. Therefore, the decrease in the amplitude of the shortest-lifetime component a3 at the cmc (Figure 4B) suggests that the heavily quenched HTMAPFP aggregates and/or HTMA-PFP/ds-DNA complexes are replaced with the less quenched SDSu-micellized HTMA-PFP. Future studies are planned using dynamic light scattering and small-angle X-ray scattering (SAXS) to test this hypothesis. Addition of n-Dodecyl Pentaoxyethylene Glycol Ether. No significant changes are observed in the absorption spectra upon the addition of C12E5 to solutions of HTMA-PFP with dsDNA. The absorbance increases slightly, the spectra broaden, and the background increases at surfactant concentrations above 9  10-3 M (see Figure S2 in Supporting Information). There may be a slight increase in the emission quantum yield (from about 0.14 to 0.16) for surfactant concentrations around the cmc; this is followed by a decrease (to 0.10) at higher concentrations (Figure 5). No spectroscopic shifts are observed in either the absorption or emission spectra over the concentration range of C12E5 studied. An increase in light scattering, as seen at the fluorescence excitation wavelength (381 nm), can explain both the increase in the background of absorption spectra and the apparent decrease of the quantum yield for high concentrations of C12E5. With the related anionic (PBS-PFP) in aqueous solution, the formation of cylindrical aggregates, which grow into larger species, possibly having threadlike structures, has been detected by dynamic light scattering cryogenic transmission electron microscopy, electrical conductivity,27 and small-angle neutron scattering (SANS).56 There are also indications from SANS that such structures are formed with the corresponding cationic CPEs in the presence of C12E5.57 This may have implications on the use of nonionic surfactants for enhancing DNA sensing and suggests that a maximum surfactant concentration may be required for (56) Knaapila, M.; Almasy, L.; Garamus, V. M.; Pearson, C.; Pradhan, S.; Petty, M. C.; Scherf, U.; Burrows, H. D.; Monkman, A. P. J. Phys. Chem. B 2006, 110, 10248–10257. (57) Burrows, H. D.; Knaapila, M.; Monkman, A. P.; Tapia, M. J.; Fonseca, S. M.; Ramos, M. L.; Pyckhout-Hintzen, W.; Pradhan, S.; Scherf, U. J. Phys.: Condensed Matter 2008, 20, 104210.

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enhancing sensitivity, while at the same time minimizing light scattering coming from the formation of large aggregates. Addition of dsDNA to HTMA-PFP/Surfactant Systems. In this section, we consider the case where dsDNA is added to systems that contain HTMA-PFP (concentrations between 2.0  10-6 and 4.0  10-6 M in terms of repeat units) and surfactants with concentrations above their cmc. At these concentrations, the surfactants increase the polymer emission quantum yield from 0.50 to 0.65 for C12E5 (7.4  10-4 M), but show a negligible change (0.50 to 0.52) for SDSu (0.108 M).7 The results obtained with the two surfactants will be presented separately. Systems with n-Dodecyl Pentaoxyethylene Glycol Ether. The effect of the addition of various concentrations of dsDNA (from 0 to 1  10-5 M) on the absorption and emission spectra of HTMA-PFP (2.0  10-6 M) with C12E5 are shown in Figures 6A and 7A, respectively. The spectroscopic changes are qualitatively similar to those obtained when dsDNA is added directly to the CPE solution in the absence of C12E5 where conductivity measurements indicate that the complexation between HTMA-PFP and dsDNA leads to charge neutralization.19 Similar neutralization of the polymer charge by dsDNA is observed in the presence of C12E5. The addition of dsDNA to aqueous solution increases the electrical specific conductance of the solution. However, when the polyelectrolyte dsDNA is added to HTMA-PFP19 or to HTMA-PFP/C12E5 around polymer charge neutralization (Polymer/dsDNA, 2:1) the specific conductance increases slightly (HTMA-PFP, Supporting Information Figure S3) or does not increase (HTMA-PFP/C12E5, Supporting Information Figure S3) indicating a decrease of free charge available to conduct electricity in the solution resulting from the charge neutralization. From the decrease in the absorbance (Figure 6A,B) and emission intensity (Figure 7A,B), the changes in the emission spectral vibrational structure (Figure 7A, Supporting Information Figure S4) and the spectral shifts of both the absorption and emission spectra (Figures 6A, 7A; Supporting Information Figure S5), two dsDNA concentration ranges can be distinguished, as has previously been observed when the direct interaction between DNA and HTMA-PPF was studied.19 The first is for dsDNA concentrations below that of the CPE, where the absorbance and emission intensity decrease slightly with the polynucleotide concentration. The changes of absorbance at the maximum of the spectra (ca. 381 nm) fit the Benesi-Hildebrand equation for 1:1 complexes.58 If we define ΔD as the difference of absorbance between the HTMA-PFP/C12 3 E5 assembly and that of the complex HTMAPFP/C12E5:DNA at 381 nm, this can be fitted to the BenesiHildebrand equation (eq 2) for a 1:1 complex (Figure 6C)58 ΔD ¼

½εHTMA - PFP=C12 E5 :DNA - εHTMA - PFP=C12 E5 ½HTMA - PFP0 K BH A ½DNA 1 þ K BH A ½DNA

ð2Þ where εHTMA-PFP/C12E5:DNA and εHTMA-PFP/C12E5 are the molar extinction coefficients of HTMA-PFP/C12E5:DNA and HTMAPFP/C12E5, respectively, at the titration wavelength, [HTMAPFP]0 is the HTMA-PFP initial concentration, and KABH is the Benesi-Hildebrand association constant for complexes with 1:1 stoichoimetry K BH A

HTMA-PFP=C12 E5 þ DNA s r f HTMA-PFP=C12 E5 :DNA ð3Þ (58) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703–2707.

DOI: 10.1021/la1011764

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Figure 6. HTMA-PFP (2.0  10-6 M; Mn = 14.5 kg mol-1) with C12E5 (7.4  10-4 M) and various DNA concentrations: (A) absorption spectra (DNA concentrations = 0; 1.2  10-7; 8.7  10-7; 2.1  10-6; 4.8  10-6; 6.5  10-6; and 1.0  10-5 M); (B) absorbance at the maximum in the spectrum in the absence of surfactant (full squares), with SDSu (1.1  10-1 M, full circles), and with C12E5 (7.4  10-4 M, full triangles), respectively. (C) Benesi-Hildebrand nonlinear plot (ΔD versus DNA molar concentration, eq 2) for DNA concentrations up to 1.0  10-5 M. (D) aggregate absorption spectra ([DNA] = 2.1  10-6; 3.1  10-6; 4.8  10-6; 6.5  10-6; 8.1  10-6; and 9.8  10-6 M).

Figure 7. HTMA-PFP (2.0  10-6 M; Mn = 14.5 kg mol-1) with C12E5 (7.4  10-4 M) and various DNA concentrations: (A) emission

spectra (DNA concentrations = 0; 1.2  10-7; 8.7  10-7; 2.1  10-6; 4.8  10-6; 6.5  10-6; and 1.0  10-5 M); (B) emission quantum yield in absence of surfactant (full squares), with SDSu (1.1  10-1 M, full circles) and with C12E5 (7.4  10-4 M, full triangles), respectively. (C) Stern-Volmer plot for DNA concentrations between 0 and 1.0  10-6 M at 15, 20, and 25 °C in triangles, circles, and squares, respectively. (D) Van’t Hoff plot of KBH A , the equilibrium constant of the HTMA-PFP/C12E5:DNA system.

In this case, the equilibrium indicated in eq 3 should be considered a pseudoequilibrium, since the experimental results depend on the order of reagent addition indicating that kinetic effects are combined 11710 DOI: 10.1021/la1011764

with the thermodynamics ones. Moreover, KABH should be considered a pseudoassociation constant. However, it is fruitful to compare association constants obtained for the different addition scenarios. Langmuir 2010, 26(14), 11705–11714

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A good nonlinear fitting of ΔD vs [DNA] is obtained (Figure 6C), in agreement with the equilibrium suggested in eq 3. The calculated pseudoassociation constant KABH is 1.1 ((0.3)  105 mol-1 L. With the polymer fluorescence, its quenching follows the Stern-Volmer equation (eq 4) for dsDNA concentrations up to 1.0  10-6 M (Figure 7C)

where, in this case, x is the molar fraction of P, and c is the polymer molar concentration. The molar absorption coefficient at any wavelength (εhλ) can be expressed as

Fo ¼ 1 þ KSV 3 ½Q F

where εPλ and εA λ are the average molar absorption coefficients of P and A, respectively. It is assumed that P is the only species present before dsDNA addition. Under these conditions, the absorption spectrum will be that of P: hευ = εPλ . From eq 8, the molar absorption coefficients of higherorder aggregates, εA λ , can be determined if x is known. The P molar fraction can be determined by an iterative method59 leading to a limiting x value obtained when differences of 0.002 are observed between consecutive calculated x values. The parameter R is defined as59

ð4Þ

where Fo and F are HTMA-PFP/C12E5 emission intensities in the absence and presence of quencher (dsDNA), respectively, and KSV is the Stern-Volmer constant. The linearity of Fo/F means that the fluorescence quenching of HTMA-PFP/C12E5 by dsDNA can be considered to be either totally dynamic or totally static, because, if both mechanisms were present, the Stern-Volmer plot would curve upward, following eq 5, where (1 þ kqτ0) accounts for dynamic quenching, and (1 þ KBH A [Q]) results from static quenching (ground-state complexation of HTMA-PFP/C12E5 with dsDNA) Fo ¼ ð1 þ kq τ0 ½QÞ  ð1 þ KABH ½QÞ F

ð5Þ

Clearly, taking into account the diffusion-controlled upper limit value of kq (ca. 1010 M-1 s-1 for neutral species and slightly higher for charged ones), the upper limit of τ0 (5  10-10 s) and the highest concentration of dsDNA (10-6 M), 1 þ kqτ0[Q] ≈ 1, i.e, the quenching of of HTMA-PFP/C12E5 by dsDNA is exclusively static, and KSV = KBH A . However, it should be noted that this static quenching differs from standard static quenching with small molecules in solution in that the DNA (or SDSu) complexes with the CPE are fluorescent, albeit weakly. The values of KBH A at 15, 20, and 25 °C are 7.8  105, 4.1  105, and 2.1  105 M-1, respectively, giving from the Van’t Hoff plot (Figure 7D) values of ΔH = -22.4 kcal mol-1 and ΔS = -50.7 cal mol-1 K-1 for the formation of the HTMA-PFP/C12E5 complex with dsDNA. For dsDNA concentrations greater than that of the CPE (in terms of repeat units), the absorbance at the maximum decreases, the spectra shifts to the red, and an isosbestic point is observed at 394 nm. The same changes are observed when DNA is added directly to HTMA-PFP in 4% (v/v) DMSO-water solutions in the absence of C12E5.19 In that case, the equilibrium (or pseudoequilibrium) indicated by the isosbestic point is attributed to the formation of polymer aggregates between neutral HTMA-PFP/ C12E5:DNA complexes. This has also been suggested for the same polymer interacting with sodium n-alkyl sulfonate surfactants7 or DNA.19 KA

PþPs rf A

ð6Þ

In eq 6, P stands for HTMA-PFP/C12E5 assemblies, A represents higher-order polymer aggregates formed as a consequence of the interaction between HTMA-PFP/C12E5 assemblies and dsDNA and the consequent formation of neutral complexes, and KA is the pseudoaggregation constant. From a modification of an iterative method described initially to study aggregation of dyes in solution59 and adapted to polymeric systems, the aggregate concentrations, aggregate absorption spectra, and KA can be calculated for each DNA concentration. The pseudoaggregation constant can be expressed as59 KA ¼

1-x 2 3 c 3 x2

ð7Þ

(59) Lopez Arbeloa, I. J. Chem. Soc., Faraday Trans. 2 1981, 77, 1725–1734.

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ε λ ¼ εPλ x þ εA λ ð1 - xÞ

R ¼

εP x þ εA AλA λA ð1 - xÞ ¼ λA AλP εPλP x þ εA λP ð1 - xÞ

ð8Þ

ð9Þ

where AλP and AλA are the experimental absorbance of P and A at their wavelength maxima, respectively. The maximum absorbance wavelength of P is assumed to be that of the HTMAPFP/C12E5 absorption spectrum in the absence of DNA (around 381 nm). The absorption maximum for A (405 nm) has been calculated by subtracting the absorption spectrum of the sample in the absence of DNA from the HTMA-PFP/C12E5:DNA absorption spectrum at the highest DNA concentration used in this study for every system (between 1  10-5 M and 2  10-6 M). Parameter R becomes Ro when x = 159 (i.e., in the absence of dsDNA) Ro ¼

εPλA εPλP

ð10Þ

To obtain a first approximate value of x(1), it is assumed that P εA λP