Selective Fluorescence Quenching in Cationic Fluorene-Thiophene Diblock Copolymers for Ratiometric Sensing of Anions

Authors

  • Sofia M. Fonseca,

    Corresponding author
    1. Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal
    • Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal
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  • Rita P. Galvão,

    1. Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal
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  • Hugh D. Burrows,

    Corresponding author
    1. Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal
    • Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal
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  • Andrea Gutacker,

    Corresponding author
    1. Bergische Universität Wuppertal, Makromolekulare Chemie, Gaussstrasse 20, 42119 Wuppertal, Germany
    2. Center for Polymers and Organic Solids, Department of Physics and Chemistry & Biochemistry, University of California, Santa Barbara, California 93106, USA
    • Bergische Universität Wuppertal, Makromolekulare Chemie, Gaussstrasse 20, 42119 Wuppertal, Germany.
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  • Ullrich Scherf,

    1. Bergische Universität Wuppertal, Makromolekulare Chemie, Gaussstrasse 20, 42119 Wuppertal, Germany
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  • Guillermo C. Bazan

    1. Center for Polymers and Organic Solids, Department of Physics and Chemistry & Biochemistry, University of California, Santa Barbara, California 93106, USA
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Abstract

The cationic, all-conjugated AB diblock copolymer poly[9,9-bis(2-ethylhexyl)fluorene]-b-poly[3-(6-trimethylammoniumhexyl) thiophene] bromide (PF2/6-b-P3TMAHT) shows dual fluorescence from the poly(fluorene) (PF) and poly(thiophene) (PT) blocks. A comparison of fluorescence quenching of the cationic PT block fluorescence with unquenched PF block provides a sensitive ratiometric method for anion sensing. The application to analysis of halide ions, single- and double-stranded DNA is demonstrated. High selectivity is observed with halide ions, with the strongest quenching being seen with iodide. The quenching with DNA can be used for nucleic acid quantification at sub-μM concentrations.

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1. Introduction

Conjugated polyelectrolytes (CPEs) are an important and versatile class of materials.1–4 They show excellent characteristics as optical sensors for a range of chemical and biological systems.5–8 Their efficient energy transfer provides advantages as biomarkers over small molecules, through both the amplification of fluorescence quenching9 and Förster resonance energy transfer (FRET) to appropriate dyes.10 They also bind electrostatically to oppositely charged targets, leading to extremely high sensitivity; for example, cationic conjugated polyelectrolytes have been designed, which allow DNA concentration determination over seven orders of magnitude,11 while specific detection of nucleic acids at the zeptomole level has been reported.12 Similar methodologies have been used to study proteins,13 enzymes,14 carbohydrates,15 and many other targets of chemical and biological interest. Sensors based on CPEs are susceptible to variations in both chemical environment and instrumental conditions, and for quantitative applications need appropriate standards. For fluorescence sensors, it is possible to use internal standards through comparison of the luminescence from more than one emitting species,16 and a CPE-based system with two emission features has been developed for DNA quantification using such ratiometric fluorescence methods.10

All-conjugated, diblock copolymers of polyfluorene (PF) and polythiophene (PT) are valuable advanced materials.17–22 The chain conformation of the polythiophene block can be modulated by its environment, and the introduction of cationic side chains on this segment allows both water solubility, and the possibility of tuning the electronic and optical properties by changing solvent, ionic strength, temperature, or appropriate additives.23, 24 Cationic polythiophenes undergo specific electrostatic interactions with oppositely charged species, such as halide ions,25, 26 nucleic acids,27 and it is expected that differences in polarity and charge between this segment and the neutral, hydrophobic PF block will lead to specific fluorescence quenching of the PT block. This will provide the advantage of having a single, self-referencing anion sensor, making this a good ratiometric fluorescence sensor. We report a study of the selective quenching of fluorescence of these diblock conjugated polyelectrolytes by halide anions, and then extend this to nucleic acids. Selective quenching of fluorescence of these conjugated polyelectrolytes is also observed with anionic surfactants.26, 28–31 Future studies will be directed toward the potential of these systems for surfactant sensing.

2. Experimental Section

The synthesis of the cationic, all-conjugated AB diblock copolymer poly[9,9-bis(2-ethylhexyl)fluorene]-b-poly[3-(6-trimethylammoniumhexyl) thiophene] bromide (PF2/6-b-P3TMAHT, Scheme 1) has been described in detail elsewhere.24, 28 The copolymer has Mn 18 000, giving a molecular weight for the PF block of ≈8000. This corresponds to roughly 20 fluorene and 40 thiophene repeat units per diblock copolymer. The absorption spectrum of the diblock copolymer in water is a superposition of the absorption bands of both blocks with two absorption maxima peaking at 384 nm for the polyfluorene (PF) block and 439 nm for the polythiophene (PT) block.23

Scheme 1.

Structure of the diblock copolymer poly[9,9-bis(2-ethylhexyl)fluorene]-b-poly[3-(6-trimethylammoniumhexyl) thiophene] bromide (PF2/6-b-P3TMAHT).

UV–vis absorption and photoluminescence (PL) spectra were measured on a Shimadzu UV–2100 and a Jobin–Ivon SPEX Fluorolog 3–22 spectrometer, respectively. Fluorescence spectra were corrected for the wavelength response of the system. Solutions of the diblock copolymers were prepared the day before the experiment, and stirred overnight. Concentrations were typically around 1 × 10−5 M. The aggregation of these diblock copolymers may depend on concentration,32 which may also affect the quenching behavior. Future studies will be directed to test this effect.

3. Results and Discussion

As indicated in the experimental section, the absorption spectrum of the diblock copolymer PF2/6-b-P3TMAHT in water is a superposition of the absorption bands of both blocks with two absorption maxima, peaking at 384 nm for the polyfluorene (PF) block and 439 nm for the polythiophene (PT) block.23 In addition, following excitation into the PF absorption maximum, two distinct bands were observed in the fluorescence of the diblock copolymer in water, a structured emission in the 400–500 nm region associated with the PF block, and a broad band between 520 and 700 nm due to the PT segment. Although the system is fully conjugated, the separate fluorescence emission from the two blocks is a consequence of incomplete energy transfer between the PF and PT segments. Both intrachain energy (exciton) migration and interchain energy transfer may be involved in transfer of energy from the PF to the PT blocks. The efficiency of this depends on the solvent, probably due to aggregation which favours interchain processes, and in methanol, electronic energy transfer from the PF to the PT block has a lifetime 30–40 ps.24

Halide ion sensing is important for a wide variety of applications in industry, food stuffs, and medicine, and fluorescence quenching sensors are particularly attractive for applications in this area.31, 34 Cationic polythiophenes have been demonstrated to be particularly sensitive halide ion sensors.25, 26 We extend this to the cationic diblock copolymer PF2/6-b-P3TMAHT in water. Under these conditions, addition of sodium iodide, bromide, or chloride leads to a decrease in the fluorescence of the thiophene block without any significant quenching in the fluorene region, confirming the idea of selective fluorescence quenching, and indicating that the PF block fluorescence can be used as a ratiometric standard. Results for NaBr are shown in Figure 1a. The quenching behavior was analyzed using Stern-Volmer plots31 of the emission intensity at the maximum of the PT block in the presence (I) and absence (I0) of halide anions as a function of quencher concentration Figure 1b. Three points can be noted:

Figure 1.

(a) Fluorescence of aqueous solutions of PF2/6-b-P3TMAHT (9.25 × 10−6M) in the presence of various concentrations of sodium bromide (3.33 × 10−4 to 4.60 × 10−2M). (b) Stern-Volmer plots for the quenching of fluorescence of aqueous solutions of PF2/6-b-P3TMAHT by sodium chloride (squares), bromide (circles) and iodide (diamonds). (c) Fit to a multi-equilibrium model of modified Stern-Volmer plot for fluorescence quenching of PF2/6-b-P3TMAHT by sodium bromide.

  • The quenching efficiency is strongly dependent on the nature of the halide ion.

  • The Stern-Volmer plots are non-linear, and tend to plateau at high concentrations.

  • There are no significant shifts in the maxima of either the PF or PT bands.

Similar nonlinear behavior has been seen in the quenching of fluorescence of cationic polythiophenes by iodide ion.25 The diblock conjugated polyelectrolyte PF2/6-b-P3TMAHT aggregates in water,23, 24 and the fact that no significant changes in the maximum emission wavelengths of either the PF or PT blocks are seen suggests that addition of salt does not significantly change the association of this conjugated polyelectrolyte in water over the concentration region studied. Although fluorescence quenching is commonly treated in terms of static and dynamic mechanisms,34 the shape of the Stern-Volmer plot does not correspond to what is expected for either of these mechanisms, but instead is similar to that reported for quenching of fluorophore emission in ionic micelles induced by counter ions within the Stern layer.35, 36 This has been treated using a multi-equilibrium model,36 leading to the modified Stern-Volmer relationship between the fluorescence intensity ratio and quencher concentration,[Q]

equation image((1))

where τf is the fluorescence lifetime, kq the quenching rate constant, Ksv is the Stern-Volmer constant, n is the total number of vacant sites for quenching and K is an association constant. Good fits were obtained to a modified form of this function (in terms of I0/I -1) for the three anions. Data for bromide are shown in Figure 1c. From these, values of Ksv = 160 M−1 (NaI), 115 M−1 (NaBr) and 3.8 M−1 (NaCl) were obtained. Although both static and dynamic quenching processes are likely to be involved, the quenching clearly follows the order I > Br > Cl, observed in the quenching of aromatic fluorescence by halide ions in acetonitrile37 or water.36 Both heavy atom effects and electron transfer mechanisms have been suggested for quenching of fluorescence of aromatic molecules by halide anions.38, 39 The order of quenching is consistent with both mechanisms. Studies are in progress to try to identify the dominant process.

We will now address the potential of cationic diblock CPEs for ratiometric nucleic acid sensing. Because of solubility and aggregation problems, these have been carried out using PF2/6-b-P3TMAHT solutions in 20% THF–water. This was found to be the optimal solvent mixture in terms of balance between photophysical response and aggregation. However, even in these solvent mixtures, there is evidence for aggregation from SAXS and SANS in solution, and AFM studies of films spun from THF–water show vesicle structures.32 The spectra of PF2/6-b-P3TMAHT observed in 20% THF–water depended on the method of sample preparation; in particular, the relative fluorescence intensities of the PF and PT blocks (exciting in the PF band) were different for solutions of the diblock in water to which THF was added to give 20% THF–water from the case of dissolving the diblock copolymer directly in 20% THF–water. These are also different from the spectra in water shown in Figure 1a. This may suggest the formation of metastable structures, such as vesicles, in solution. However, for sensing applications, no significant changes in spectra of samples prepared by either route were observed during the measurements. Salmon testes DNA (≈2000 base pairs) solutions were prepared with the buffer salt, tris-HCl base. Single- stranded DNA (ssDNA) solutions were obtained by denaturation of the ones containing double stranded (dsDNA) by heating, followed by rapid cooling in ice to prevent renaturation.41, 42 Two characteristic bands were seen in the UV–vis absorption spectrum of PF2/6-b-P3TMAHT solutions (Figure 2), and are attributed to PF (λmax ≈ 382 nm) and PT (λmax ≈ 445 nm) blocks. Upon addition of DNA, a decrease in absorbance and a red shift of the polythiophene band are observed, and are accompanied by corresponding color changes due to torsional effects on the energy band gap.43 This red shift is more pronounced with the ssDNA (46 nm) than with the double stranded one (27 nm), possibly due to increased conformational flexibility of the ssDNA. Similar effects have previously been reported for the interaction of DNA with the cationic poly{9,9-bis[6-N,N,N-trimethylammonium)hexyl]fluorene-co-1,4-phenylene}.43 These colorimetric effects are probably due to conformational changes of the polymer chains of the polythiophene blocks in the presence of DNA, which increases the conjugation length, and may be useful for imaging. In aqueous solutions of PF2/6-b-P3TMAHT, the red shift of the polythiophene band upon addition of ssDNA is even larger (60 nm), which will be more convenient for such applications.

Figure 2.

Absorption spectra of PF2/6-b-P3TMAHT solutions (9.86 × 10−6M) in 20% THF–water with addition of ssDNA (6.66 × 10−7 to 7.69 × 10−5M), together with a picture of solutions before and after the addition of DNA.

The photoluminescence spectra (PL) (Figure 3) following excitation into the PF absorption band at 385 nm show the occurrence of characteristic emissions of both blocks, a blue PL feature at 400–500 nm for the PF block and a red PL feature at 500–700 nm for the PT block. Under these conditions, the polythiophene emission is sensitized by excitation energy transfer from the polyfluorene block.24 As with the halide ions, strong, selective quenching of the polythiophene block PL is observed upon addition of DNA, while the polyfluorene PL is almost unquenched. However, in contrast with the behavior with halide anions, the quenching of the polythiophene PL is accompanied by a red shift of the emission maximum. As was observed with the absorption spectra, the red shift with ssDNA fluorescence (36 nm) is larger than with the double stranded one (18 nm). The corresponding Stern-Volmer (Figure 4a) and ratiometric (Figure 4b) plots for the PL quenching of the polythiophene blocks show sigmoidal behavior, reminiscent of a titration curve, where the steep slope occurs for a [DNA]/[PF2/6-b-P3TMAHT] ratio corresponding to charge neutralization (molar concentrations in terms of the respective monomeric units). In contrast, no significant quenching is seen with the polyfluorene block. Fluorescence titrations using this system permit DNA quantification to the submicromolar level. From Figure 4a, it can also be seen that the polythiophene PL quenching upon addition of ssDNA is twice that with the double-stranded one, providing a potential ratiometric fluorescence route for distinguishing between single- and double-stranded DNA.

Figure 3.

PL spectra of PF2/6-b-P3TMAHT solutions (9.86 × 10−6M) in 20% THF–water with addition of ssDNA (6.66 × 10−7 to 7.69 × 10−5M), together with a picture of the fluorescence of solutions irradiated by UV light before and after the addition of DNA.

Figure 4.

(a) Stern-Volmer plots for the PL quenching of the polyfluorene (λem = 413 nm, open symbols) and polythiophene (λem = 578 nm, solid symbols) blocks with ssDNA (circles) and dsDNA (squares) and (b) ratio of intensities of the PF and PT blocks as a function of [DNA]/[PF2/6-b-P3TMAHT] ratio for dsDNA.

4. Conclusions

Selective quenching of fluorescence of the cationic polythiophene block is seen upon addition of either halide anions or DNA to the diblock conjugated polyelectrolyte PF2/6-b-P3TMAHT. Selective quenching of this diblock copolymer has also been observed with anionic surfactants.24, 32 With the halide ions, the quenching shows a dependence on the nature of the anion, nonlinear concentration behavior is observed since only ions in the Stern layer of the block copolymer aggregates can induce quenching. Binding of nucleic acids to PF2/6-b-P3TMAHT leads to red shifts in the absorption and emission maxima due to conformational changes. Fluorescence titration of the diblock conjugated polyelectrolyte with DNA can be used for nucleic acid quantification at sub μM concentrations. In addition, differences in the ratiometric fluorescence quenching can be used to distinguish between single- and double-stranded DNA. There are indications that these interactions may be accompanied by aggregation. Aggregation in conjugated polyelectrolytes has recently been reviewed,44 and can be minimized both by use of co-solvent or surfactant44 and/or incorporation of the fluorophore in appropriate matrices.45

As with DNA interactions with other cationic conjugated polyelectrolytes,43 at high nucleic acid concentrations these are likely to be accompanied by formation of nanostructured networks involving polyelectrolyte–polyelectrolyte complexes. Future work will be directed toward characterization of their structures.

Acknowledgements

We are grateful to the FCT for the award of a postdoctoral fellowship (SMF, grant FCT/SFRH/BPD/34703/2007) and to the National Science Foundation (DMR-1005546) for financial support.

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