Article pubs.acs.org/JPCB

In Depth Analysis of the Quenching of Three Fluorene−PhenyleneBased Cationic Conjugated Polyelectrolytes by DNA and DNA Bases Matthew L. Davies,†,* Peter Douglas,‡,⊥,* Hugh D. Burrows,§,* Bice Martincigh,⊥ Maria da Graça Miguel,§ Ullrich Scherf,∥ Ricardo Mallavia,# and Alastair Douglas○ †

School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2UW, U.K. Chemistry Group, School of Engineering, Swansea University, Singleton Park Swansea, SA2 8PP, U.K. § Departamento de Química, Universidade de Coimbra, Rua Larga, 3004-535 Coimbra, Portugal ⊥ School of Chemistry and Physics, University of KwaZulu-Natal, Westville Campus, Durban 4000, South Africa ∥ Makromolekulare Chemie, Bergische Universität Wuppertal, 42097 Wuppertal, Germany # Instituto de Biologia Molecular y Celular, Universidad Miguel Hernández, Elche 03202, Spain ○ AD Technology Consulting Limited, Swansea, SA2 7UZ, U.K. ‡

S Supporting Information *

A B S T R AC T : The interaction o f t hree cationic poly {9,9- bis [N , N(trimethylammonium)hexyl]fluorene-co-1,4-phenylene} polymers with average chain lengths of ∼6, 12, and 100 repeat units (PFP-NR36(I),12(Br),100(Br)) with both double and single stranded, short and long, DNA and DNA bases have been studied by steady state and time-resolved fluorescence techniques. Fluorescence of PFP-NR3 polymers is quenched with high efficiency by DNA (both double and single stranded) and DNA bases. The resulting quenching plots are sigmoidal and are not accurately described by using a Stern−Volmer quenching mechanism. Here, the quenching mechanism is well modeled in terms of an equilibrium in which a PFP-NR3/DNA aggregate complex is formed which brings polymer chains into close enough proximity to allow interchain excitation energy migration and quenching at aggregate or DNA base traps. Such an analysis gives equilibrium constants of 8.4 × 106 (±1.2 × 106) M−1 for short-dsDNA and 8.6 × 106 (±1.7 × 106) M−1 for short-ssDNA with PFP-NR36(I).

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between the polymer and the quencher,11,17,19 and ultrafast charge/energy transfer between the polymer and the quencher.11,18 The common factor is that efficient energy transfer along and between polymer chains gives rise to high quenching rates. This high sensitivity to quenching by oppositely charged species has led to intensive research into CCPs that may be useful in chemical and biological sensors, and in particular in sensors for DNA.3,20,21 CCPs have already found use as DNA biosensors based on the electrostatic interaction with the oppositely charged phosphate groups of the target DNA or polynucleotide.22 Polythiophene and polyfluorene CCPs have been implemented in CCP-based DNA sensors.23,24 There have been several proposed methods of DNA sequence detection using CCPs that show promise for highly sensitive DNA detection.2,24,25 Leclerc and co-workers have been able to detect and distinguish a specific sequence of oligonucletides having a single mismatch in very low concentration.2,25 Bazan and co-workers have used poly(fluorene-co-phenylene)s CCPs, similar to the CCPs used

INTRODUCTION Cationic conjugated polyelectrolytes (CCPs) tend to be highly sensitive to changes in their physical and chemical environment1−3 and their fluorescence can be quenched with remarkable efficiency in the presence of oppositely charged quenchers.4,5 Efficient energy transfer along chains and, if there is a degree of spectral overlap, to dyes, makes CCPs suitable candidates for a range of sensor applications.6−9 This ability of oppositely charged species to quench the fluorescence of CCPs with very high efficiency has been termed “amplified quenching” or “superquenching”.10,11 In heterogeneous solutions fluorescence quenching is generally discussed quantitatively in terms of the Stern−Volmer (SV) relationship.12 However, as is discussed here, this may not be the best way to analyze the quenching of CCPs. Conjugated polyelectrolytes (CPEs) quenched by oppositely charged quenchers have shown quenching constants, KSV, of the order of 106−109 M−1, which, if this involves dynamic processes, is higher than expected for a diffusion controlled process.1,13 Various descriptions have been given for the highly efficient quenching of similar polymer systems in terms of: efficient intrachain exciton diffusion along the polymer chain,11,14−16 3-dimensional interchain exciton diffusion in CPE aggregates,11,17,18 ion-pair complex formation © 2013 American Chemical Society

Received: September 23, 2013 Revised: December 13, 2013 Published: December 18, 2013 460

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well studied.6 We also feel that the difference in counterions does not have a significant impact on the conclusions of this paper. Relatively short-stranded DNA from salmon testes (short-DNA), having an average molecular weight of 1.3 × 106 g/mol (2 kilobase pairs),35 was purchased from Sigma-Aldrich and used as received. Long-stranded (long-DNA) Bacteriophage T4 DNA (165.6 kilobase pairs, contour length of 57 μm) was purchased from Wako Nippon Gene. The buffer salt, trisHCl base, and the fluorescent dye, 4,6-diamidino-2-phenylindole (DAPI) were also from Sigma. YOYO-1 iodide, as a 1 mM solution in DMSO, was purchased from Invitrogen. All other chemicals were from Sigma-Aldrich. Acetonitrile and ethanol were of spectroscopic grade. All experiments were performed using Millipore Milli-Q deionized water.

in this study, as Förster resonant energy transfer (FRET) biosensors.26−28 In these FRET sensors the sensitivity and selectivity of detection arise from the use of fluorescent labels attached to peptide nucleic acids (PNAs) or DNA. PNAs are DNA mimics where the nucleotides are attached to a N-(2aminoethyl)glycine backbone instead of the negatively charged deoxyribose phosphate backbone present in DNA.29,30 For the use of CCPs in FRET-based biosensors in DNA detection and sequencing there are three important parameters to consider: (i) maximization of the quantum yield through minimization of aggregation, (ii) excitation dynamics and (iii) the interaction of the CCPs with DNA. Studies of aggregation, quantum yields and excitation dynamics have previously been reported for these polymers.6 It has been postulated that it is possible that DNA and DNA bases quench PFP-NR3 polymers by induced aggregation.31 We report an in depth study of the interaction of poly{9,9bis[N,N,N-(trimethylammonium)hexyl]fluorene-co-1,4-phenylene} halides (Figure 1.) with average chain lengths of ∼6, 12,

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METHODS AND EQUIPMENT Absorption measurements were made on solutions in 1 cm quartz cuvettes on a Shimadzu UV-2100 spectrophotometer. Fluorescence spectra were measured in 1 cm quartz cuvettes using 90 degree geometry on a Jobin Yvon-Spex Fluorolog 3− 22 instrument with 4.0 nm excitation and emission slits. Fluorescence spectra were corrected for the spectral response of the light source (450 W xenon lamp) and detector. The fluorescence quantum yields at 293 K were measured against a compound of known quantum yield and a similar emission spectrum, namely α-4-oligothiophene (Φf = 0.16 in acetonitrile), using solutions of identical absorbance at an excitation wavelength of 379 nm.36,6 Fluorescence decay times with picosecond time resolution were obtained using a home-built picosecond time correlated single photon counting (TCSPC) apparatus that is described elsewhere.37 Steady-state fluorescence anisotropy experiments were carried out by using the Jobin-Yvon Fluoromax-3 spectrometer with right angle geometry. Samples were excited at 373 nm with excitation and emission slit widths of 5 nm. Anisotropy values are those from the emission weighted averages across the emission band, that is, from the integrated emission curves. Quenching Studies. DNA solutions were prepared by dilution of a DNA mother solution with tris-HCl base buffer, pH = 7.8, to give the required concentrations of (0−12.4) × 10−8 M in terms of base pairs for long and short DNA. (Unless otherwise stated all DNA concentrations are in terms of base pairs.) Short-DNA quenching studies were also carried out with a range of higher DNA concentrations (2.6 × 10−7 to 4.72 × 10−6 M). DNA concentrations were checked via the absorbance of the solutions at 260 nm (ε = 6600 cm−1 M−1).38 ssDNA solutions were prepared by thermal denaturation of the corresponding dsDNA solution by heating at 90 °C for 10 min, after which the solution was immersed in an ethanol/ice solution.39 The UV−vis absorption spectrum was recorded from 40 to 90 °C at intervals of 10 °C; a substantial increase in absorption indicates the formation of ssDNA. Denaturing the dsDNA solutions means that the concentration of both solutions, dsDNA and ssDNA, were the same in terms of base units. Solutions of guanine were made at concentrations of ca. 1/5 of DNA since the guanine content of the DNA is known to be ∼18%.40 All PFP-NR3 solutions were prepared by dissolving the PFP-NR3 in acetonitrile:water (25:75 v/v) solvent mix to minimize aggregation of the polymers.6 A sixfigure balance was used to accurately prepare polymer “mother” solutions at the desired repeat unit concentration. KSV values were calculated from the entire initial, linear, region of the SV plot, this ensured the maximum data points were fitted in each

Figure 1. Structure of PFP-NR3.

and 100 repeat units (PFP-NR36(I),12(Br),100(Br)) with long and short, double and single stranded DNA and DNA bases. We report results from steady-state absorption, emission, fluorescence anisotropy, and picosecond time-resolved emission studies to gain a greater understanding of the quenching mechanism by DNA and DNA bases. Although the quenching has been discussed in the literature; here we take a quantitative approach in order to fully understand the mechanism by which these, and hence similar polymer systems, are quenched so efficiently by DNA. Results of the interaction of the three PFPNR3s with double stranded DNA (dsDNA) and single stranded DNA (ssDNA), as well as DNA bases, are described and discussed. A new model for the quantitative analysis of quenching of CCPs is discussed. For use in CCP-based biosensors it is vital that the interaction, and subsequent quenching mechanism, between CCPs and DNA is well understood.

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MATERIALS The synthesis of the poly{9,9-bis[N,N,N(trimethylammonium)hexyl] fluorene-co-1,4-phenylene} halides with average chain lengths of ∼6, 12, and 100 repeat units, herein named; PFP-NR36(I), PFP-NR312(Br) and PFPNR3100(Br), has be described in detail elsewhere.32−34 In an ideal case the counterions of the polymers would be the same, unfortunately this is not the case. However, the aggregation and solution properties of these polymers has previously been very 461

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case to provide the greatest accuracy. SV plots were corrected for any changes in absorbance associated with change in solvent mixture or with the addition of DNA or guanine. Solvent mixtures are given as v/v. PFP-NR3 concentration dependent quenching studies were carried out keeping the DNA concentration range constant at 2 × 10−7 M to 1.4 × 10−5 M and varying the PFP-NR3 concentrations. For all PFP-NR3s the three concentrations investigated were 3, 6, and 10 × 10−6 M. Table Curve 2D version 3 (Jandel Scientific) was used for the equilibrium analysis curve fitting. Anisotropy Studies. For the change in anisotropy upon the addition of DNA a PFP-NR36(I) concentration of 2.6 × 10−6 M (polymer chains; 1.5 × 10−5 M repeat units) and DNA concentrations from 3.5 × 10−8 to 2.1 × 10−6 M (in terms of base pairs) were used. Two commercial dyes, DAPI and YoYo141 (at concentrations of 3.33 × 10−6 and 4.16 × 10−6 M respectively), were also studied for comparison. Anisotropy values are given from the emission weighted averages across the emission band, i.e. the integrated emission curves. Modeling Studies. The program used to model energytransfer kinetics, ProgClusters, has been previously used to model the aggregation properties of these polymers in different solvent mixes. ProgClusters may be applied to any system involving energy transfer in one or two dimensions (1D, 2D) or mixed 1−2D. It is described in more detail here.6,42

Figure 2. Normalized changes in absorption and emission (top) for PFP-NR312(Br) with long-dsDNA. The arrow indicates the decrease in absorption and emission with increasing DNA concentration. The bottom graph shows SV plots for PFP-NR36(I) (solid line), PFPNR312(Br) (dotted line), and PFP-NR3100(Br) (dashed line) with longdsDNA (solid squares) and long-ssDNA (open circles) as quenchers. The arrow indicates increasing polymer length. The [PFP-NR3] is ca. 1 × 10−6 M.

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RESULTS Quenching of PFP-NR3s with Long-DNA. The photophysical properties of these polymers are given elsewhere.6 In short, in 25:75 acetonitrile:water, the fluorescence quantum yields are in the region of 0.3 and the molar extinction coefficients (in terms of repeat units) are of the order of 4 × 104 M−1 cm−1, both increase slightly with polymer chain length. Fluorescence quenching of the PFP-NR3s by DNA or guanine was initially analyzed in terms of a Stern−Volmer (SV) relationship. Although, as became subsequently apparent, this is not the best method to describe the quenching, it is easy, useful and convenient. Furthermore, since it is the most common method used in the literature, it allows comparison with other work and also sets the foundation for subsequent discussion of the quenching mechanism. The interaction of the PFP-NR3s with long-DNA induces both a decrease in the absorption and a large decrease in the emission intensity; however there are no significant changes in the spectral shape or any notable wavelength shifts. Typical decreases in absorption and emission and the corresponding SV plots of the fluorescence quenching are shown in Figure 2. PFP-NR3s are extremely sensitive to fluorescence quenching by DNA, resulting in extremely high SV quenching constants (KSV, Table 1). ssDNA is a better quencher than dsDNA, this is attributed to the fact that the hydrophobic bases are more exposed in ssDNA than in dsDNA, which increases the interaction between hydrophobic bases and the PFP-NR3 chain. KSV values are calculated from the initial, linear, region of the SV plot. KSV values, and hence sensitivity to DNA, increase with PFP-NR3 chain length. All the resulting SV plots have an upward curvature (Figure 2). This can be indicative of both static and dynamic quenching processes occurring.12 However, since the average lifetime of PFP-NR3 fluorescence is ∼400 ps these quenching constants would give an apparent bimolecular quenching rate constant much higher than the diffusion

Table 1. SV Quenching Constants for PFP-NR3 with LongdsDNA and Long-ssDNA, Calculated from the Initial Linear Region of the SV Plotsa Stern−Volmer Constant/×106 M−1 (±5%)

a

polymer

dsDNA

ssDNA

PFP-NR36(I) PFP-NR312(Br) PFP-NR3100(Br)

4.72 6.41 18.3

5.34 8.56 29.3

The [PFP-NR3] in all cases is ca. 1 × 10−6 M.

controlled limit for dynamic quenching, suggesting a dominant static quenching mechanism. Support for this comes from consideration of diffusion coefficients for these polymers, which are inversely proportional to their molecular weights,43 whereas the quenching constants increase with CPE size. For a dynamic mechanism, the SV quenching constants should parallel the diffusion coefficients. Previously, fluorescence and atomic force microscopy studies have shown that DNA and PFP-NR36(I) form structured aggregates in solution.44 High sensitivity to fluorescence quenching may therefore be explained in terms of the formation of an aggregate, leading to an increase in ‘quenching traps’ due to aggregation, coupled with the efficient energy transfer along and between polymer chains. Time resolved fluorescence emission studies were carried out to further unravel the quenching effects of the DNA on the polymers indicated by the steady state results. In line with previous studies,6 three exponential decay functions were required to obtain a good fit to the measured data (χ ≈ 1) and are given in Table 2. The longest lifetime is assumed to be 462

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Table 2. Time Resolved Data for Quenching of PFP-NR3100(Br) (1 × 10−6 M) upon the addition of Long-dsDNA [DNA] × 10−8 /M

a1

τ1/ps

a2

τ2/ps

a3

τ3/ps

τave/ps

0 1.25 2.50 3.74 4.99 6.23 7.48 8.72 9.96 11.20

0.57 0.61 0.65 0.69 0.73 0.73 0.77 0.80 0.79 0.80

23 23 22 22 21 19 19 19 18 18

0.21 0.21 0.20 0.20 0.18 0.20 0.18 0.17 0.18 0.18

119 117 114 104 102 86 84 81 72 67

0.22 0.18 0.15 0.12 0.09 0.07 0.05 0.03 0.02 0.01

427 428 431 428 432 428 429 434 428 424

326 315 305 278 262 240 204 175 142 111

Figure 3. (a) Normalized time-resolved emission decay curves for PFP-NR3100(Br) upon the addition of dsDNA. The arrow indicates increasing DNA concentration. (b) Changes in fractional contribution from the longest lifetime for PFP-NR36(I) (solid line), PFP-NR312(Br) (dotted line) and PFPNR3100(I) (dashed line) with dsDNA (solid squares) and ssDNA (open circles). The arrow indicates increasing polymer length. (c) Change in average lifetime against the quencher concentration for PFP-NR36(I) (solid line), PFP-NR312(Br) (dotted line) and PFP-NR312(I) (dashed line) with dsDNA (solid squares) and ssDNA (open circles).

of the instrument, i.e., within a few picoseconds, and is therefore lost to the kinetic measurements. Quenching of PFP-NR3s by DNA Bases. Guanine has the highest oxidation potential of all the nucleic acid bases (0.63 V)45 and, because it is thought that if fluorescence quenching occurs via electron transfer, the rate may be dependent on the guanine content of the DNA.46,47 The guanine content of longDNA (T4 DNA) is 18%, so quenching experiments were carried out at 18% of the concentrations used for quenching with long-DNA.39 The interaction of the PFP-NR3s with guanine gives similar results to that obtained with long-DNA. There is both a decrease in the absorption and a large decrease in the emission intensity; again with no significant changes in the spectral shape or any notable wavelength shifts and all the PFP-NR3s are extremely sensitive to quenching by guanine (Table 3). Sensitivity toward guanine also increases with polymer chain length. However, quenching by guanine alone is not sufficient to account for the efficiency of quenching by long-DNA. This can be easily seen when the SV data for both dsDNA and ssDNA is given in terms of the guanine concentration, as is shown in Figure 4. For comparison purposes the quenching effects of the

emission from isolated single chains and it is believed that the two shorter lifetimes are due to aggregated species and clusters.31 Typical normalized emission decay curves are shown in Figure 3a. There is a decrease in the average lifetime (Table 2) with increasing DNA concentration. Of the three lifetimes, those for the fast (∼20 ps) and slow decays (∼430 ps) are reasonably constant with increasing DNA concentration, whereas the lifetime of the middle lifetime component decreases significantly. This is true for all PFP-NR3/long-DNA samples. This mirrors the changes in the lifetimes with increasing aggregation induced by changes in fraction of solvent previously reported.6 This similarity provides strong evidence that quenching does involve induced aggregation. The fractional contribution from the longest lifetime, which we attribute to isolated chains in solution, decreases dramatically with increasing DNA concentration as shown in Figure 3b. Thus, as DNA concentration is increased isolated polymer chains are aggregated, with the possibility of multiple polymers associating around a single DNA, and thus decreasing the number of isolated chains in the solution. Comparisons can be made of the quenching from timeresolved and steady state studies on samples of similar PFPNR3 concentration using changes in average lifetimes and steady state intensities. A SV type plot for changes in the average lifetime with increasing DNA concentration is shown in Figure 3c, and is similar to the corresponding SV plot for steady state changes in Figure 2. At DNA concentrations below ca. 8 × 10−8 M−1 agreement is quite good, but at higher DNA concentrations steady-state measurements show a greater sensitivity to DNA concentration than the time-resolved results. The most likely explanation is the presence of a kinetic process which is so fast that it occurs within the time resolution

Table 3. SV Quenching Constants for all PFP-NR3s with Guanine, Calculated from the Initial Linear Region of the SV Plotsa

a

463

polymer

Ksv/×107 M−1 (±2%)

PFP-NR36(I) PFP-NR312(Br) PFP-NR3100(Br)

2.7 4.2 8.8

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involved in quenching. Since guanine shows the greatest quenching effect additional experiments into the nature of the quenching mechanism focused on the use of guanine along with dsDNA and ssDNA. Normalized time-resolved emission data show the same trend to that found with long-DNA as a quencher; i.e. the lifetimes of short and long lifetime components remain relatively constant and the middle lifetime component decreases (Table S1, Supporting Information). The fractional contribution of the longest lifetime component decreases with increasing guanine concentration; again indicating the removal of isolated polymer chains. All the evidence suggests that guanine and DNA quench via the same mechanism. The SV type plots using average lifetimes are shown in Figure S-1(b). Again the same trend is seen as the SV data calculated from the steady state results. However the time-resolved results have slightly different quenching rates to the steady state results which were unexpected since the only difference between the samples was the concentration of the PFP-NR3s. The effect of the PFP-NR3 concentration is discussed later in this paper. Attempts have been made to model guanine quenching of PFPNR3100(Br) using ProgClusters, in which, energy transfer from monomer to monomer, and monomer to trap, is a random walk. Energy transfer occurs in 1D, along the linear polymer chains, while energy is trapped, and lost, at trap sites that can link polymer chains. Initially the quenching was modeled by adding a number of link traps equal to the concentration of guanine (Figure 5). Attempts to model guanine quenching by incorporation of guanine just as additional traps fail. However, as already discussed, time-resolved studies, and previous aggregation studies, suggest that at least one of the quenching mechanisms by which guanine acts involves removing isolated chains (Figure 5b) presumably by an increase in aggregation and thus this increase in aggregation was accounted for by increasing the number of traps in ProgClusters and resulted in the fits shown by Figure 6. The increase in the number of traps

Figure 4. SV plots for PFP-NR3100(Br) (1 × 10−6 M) with long-dsDNA (solid squares), long-ssDNA (open circles), and guanine (open triangles). (Note that for DNA concentrations are given in terms of guanine content).

Table 4. SV Quenching Constants for PFP-NR36(I) (1 × 10−6 M) with the DNA Bases, Calculated from the Initial Linear Region of the SV Plots DNA base

Ksv/×107 M−1 (±5%)

oxidation potential of DNA base/V (±0.02 V)44

guanine cytosine adenine thymine

2.7 0.8 1.3 1.4

0.63 0.81 0.75 0.79

other DNA bases, adenine, thymine and cytosine, were studied within the same concentration range as guanine. Table 4 collects the SV quenching constants of all DNA bases with PFP-NR36(I), SV plots are given in Figure 5. All DNA bases quench PFP-NR36(I) efficiently, with guanine being the most efficient. The SV constant increases with decreasing oxidation potential of the base used, which supports the idea that electron (or charge) transfer, as well as aggregation, are

Figure 6. Time resolved decays curves for PFP-NR3100(Br) with and without guanine (black) and modeled decay curves (red). The fraction of links that were required to be trap sites in order to get a good data fit was 0.75.

was calculated from the fractional contribution to the longest lifetime, which we associate with isolated polymer chains, in the time-resolved data. Modeling was then carried out on the basis that the role of guanine involved inducing polymer aggregation. The degree of aggregation at any guanine concentration was calculated from the fractional contribution of the long lifetime component in time-resolved studies. This fit results in a rate constant for unquenched decay of 3.0 × 109 s−1 and a rate constant for both energy transfer between monomers and from monomers to link-traps of 1.44 × 1013 s−1. This can be

Figure 5. (a) Initial modeling of quenching of PFP-NR3100(Br) by guanine via the simple addition of link traps equivalent to the guanine concentration where the black data points are experimental values and the red data points are modeled values. (b) Time-resolved decays of PFP-NR3100(Br), and of PFP-NR3100(Br) in the presence of the maximum guanine concentration studied. The time-resolved values indicate that the quenching mechanism involves inducing aggregation in the PFP-NR3 sample. 464

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compared to PFP-NR3100(Br) with no guanine present where the rate constant for both energy transfer between monomers and from monomers to link-traps is 6.2 × 1012 s−1. This explains the efficiency of quenching by guanine, in the presence of guanine the rate of energy transfer from monomer to monomer and from monomer to trap roughly doubles. This increase in the rate of energy into trap sites, as well as the increased number of trap sites due to induced aggregation quenches the system extremely efficiently. Although further systems need to be modeled in order to gain more information, this modeling work shows great promise and is capable of generating the general kinetic features of the quenching. Quenching of PFP-NR3s: Energy Migration into Trap Sites. It was thought that the curvature of the SV plots may indicate noncompetitive quenching, i.e., on average each quencher quenches a fixed number of monomer units independent of any other quencher present. This is supported by the linearity of a plot of quantum yield against quencher

Table 5. Number of Repeat Units Quenched by each Quencher Where the “Quencher Unit” Refers to a DNA Base Pair or a Guanine Molecule number of monomers quenched by each “quencher unit” polymer

dsDNA

ssDNA

guanine

PFP-NR36(I) PFP-NR312(Br) PFP-NR3100(Br)

24 36 101

31 37 117

49 161 354

As previously mentioned, there are two processes required to allow each quencher unit access to a large number of repeat units: (i) energy transfer along and between polymer chains and (ii) induced aggregation by the quencher. Hence it seems that the high SV quenching constants arise from “aggregate energy migration quenching” in which PFP-NR3 and DNA, or guanine, form an aggregate complex in which excitation energy migrates between and along the polymer chains until it is quenched at an aggregate, or DNA or guanine, “trap”. Quenching of CCPs with Short-DNA. In order to determine the effect of DNA chain length on the quenching of the PFP-NR3s short-DNA was also used. Short-DNA is roughly 60 times shorter than the long-DNA studied. SV plots resulting from the quenching experiments are again nonlinear, with sensitivity to quenching increasing with polymer chain length. The SV quenching constants, along with those for longDNA for comparison, are given in Table 6. Table 6. Comparison of SV Constants For long-DNA and Short-DNA SV constant/×106 M‑1 (±5%)

concentration (Figure 7a). The quantum yield can be equated with the number of unquenched monomer units (eq 1). QY [PFP‐NR3] QY0

for long-DNA

short-DNA

4.72 6.41 18.3

0.8 1.2 14.3

Long-DNA is more than 5× more efficient quencher than the short-DNA for the shorter polymers (PFP-NR36(I) and PFPNR312(Br)); however, the difference is less pronounced in the case of PFP-NR3100(Br). This is most likely to be due to electrostatic effects, although the number of charges in the solution and the charge density per unit length of DNA are the same for both types of DNA the number of charges per chain is much higher for long-DNA. Emission spectra show a broadening and decrease in fluorescence with increasing DNA concentration, along with a gradual slight red shift in the wavelength of maximum emission. Maximum wavelength shifts upon quenching are PFP-NR36(I) ∼ 4 nm, PFP-NR312(Br) ∼ 6 nm and PFP-NR3100(Br) ∼ 10 nm. All of the aggregates, irrespective of the polymer length, have the same emission maximum whereas the emission maxima for the free polymers for PFP-NR36(I), PFP-NR312(Br), and PFP-NR3100(Br) are all slightly different. As expected for short-DNA, as is the case for long-DNA, ssDNA is a better quencher than dsDNA (Figure 8). Normalized time-resolved decays of the PFP-NR3s decrease with increasing short-DNA concentration in the same manner as seen for long-DNA (Figure 8c). The kinetic parameters for PFP-NR3100(Br) with short DNA, for a three exponential fit, are given in Table 7. Again the fractional contribution from the longest lifetime decreases with increasing quencher concen-

Figure 7. (a) Change in quantum yield of PFP-NR36(I) upon the addition of long-dsDNA. (b) Decrease in the number of unquenched monomer units of PFP-NR312(Br) with dsDNA (solid squares), ssDNA (open circles), and guanine (solid stars). The DNA concentration is in terms of base pairs.

[MU] =

polymer PFP-NR36(I) PFP-NR312(Br) PFP-NR3100(Br)

(1)

Here [MU] is the concentration of unquenched monomer units, QY is the quantum yield, QY0 is the quantum yield without any quencher present and [PFP-NR3] is the concentration of PFPNR3 in terms of monomer units. Using this gives a plot where the slope of the line is equal to the number of monomers quenched by each quencher (e.g., Figure 7(b)). The number of monomers quenched by each quencher increases in the order dsDNA > ssDNA > guanine (Table 5). Each quencher, in particular guanine, has access to a large number of monomers. 465

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Figure 8. (a) Decrease of PFP-NR36(I) emission upon the addition of short-dsDNA. (b) SV plots for the quenching of PFP-NR312(Br) fluorescence with both short-dsDNA (solid squares) and short-ssDNA (open circles). (c) Quenching plot from the time-resolved data where the change in average lifetime is plotted against the quencher concentration for PFP-NR3100(Br) with both dsDNA (solid squares) and ssDNA (open circles).

DNA, DAPI, and YoYo-1 are shown in Figure S-2. As expected, the anisotropy values of both DAPI and YoYo-1 increase as they bind to DNA and thus become incorporated in a large molecular unit with a lower tumbling rate. In contrast, the anisotropy of PFP-NR36(I) decreases upon binding. This is strong evidence for interchain energy transfer in the PFPNR36(I)/DNA aggregate complex. Complexation Quenching. All the evidence presented in this paper leads us to believe that DNA induces aggregation in the PFP-NR3s and thus the high quenching constants arise from “energy migration quenching” in which PFP-NR3/DNA form an aggregate complex (which, in this case, retains some residual fluorescence) and excitation energy migrates between and along the polymer chains until it is quenched at an aggregate or DNA “trap”. While SV plots are commonplace in the literature to analyze the quenching of polyelectrolyte fluorescence by DNA, as has been done here, it seems more appropriate to treat the nonlinear SV as a complexation equilibrium between lumophore L and quencher Q, forming the complex LQ, for which:

Table 7. Initial Quenching Rates and the Charge Ratio of the Complexation Point for all PFP-NR3s in This Study at the Stated Polymer Concentration polymer PFPNR36(I)

PFPNR312(Br)

PFPNR3100(Br)

polymer concn/ 10−6 M (±5%)

initial KSV/M−1

charge ratio of DNA/PFP-NR3 at complexation point

3

4.0 × 105

0.15

6 10 3

3.1 × 105 2.0 × 105 1.4 × 106

0.91 2.88 0.28

6 10 3

4.3 × 105 2.0 × 105 1.4 × 106

0.85 2.90 0.21

6 10

4.4 × 105 2.3 × 105

0.69 2.10

tration. However, there is a difference in the lifetimes upon quenching for short-DNA as compared long-DNA, in that for long-DNA the short and long lifetime components remain relatively constant and the middle lifetime component decreases, whereas for short-DNA all three lifetimes gradually decrease with increasing quencher concentration. Again this is most likely due to electrostatic effects with long-DNA having few, long, highly charged chains while for short-DNA there are more, shorter, less highly charged chains. The quenching plots for the time-resolved values are also nonlinear and appear sigmoidal, with short-ssDNA giving higher quenching constants than short-dsDNA. Fluorescence Anisotropy of Dye/DNA Complexes. The effect of addition of DNA on the anisotropy of PFP-NR36(I) and also, for comparison, two commercial dyes which bind to

L + Q ⇌ LQ K=

(2)

LQ eq (Leq Q eq)

(3)

Q t = Q eq + LQ eq

(4)

where subscript eq refers to equilibrium concentrations; and t refers to the total concentration added, i.e., the sum of the free and bound concentrations of either lumophore or quencher, then the relationship between the total concentration of quencher, Qt, and the resulting ratio of emission intensity, I0/I,

Figure 9. Stern−Volmer plots (open circles) for both dsDNA (a) and ssDNA (b) as quenchers with the fit of data treating the quenching as a complexation equilibrium as the solid line for the data set when quencher and lumophore concentrations are comparable. Plots of the change in the fraction of free polymer and free DNA present as a function of increasing total DNA concentration are shown in part c. 466

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Figure 10. Dependence of quenching rates on PFP-NR3 concentration, for PFP-NR36(I) (a), PFP-NR312(Br) (b), and PFP-NR3100(Br) (c) at PFPNR3 concentrations of 1 × 10−5 M (solid squares), 6 × 10−6 M (open circles) and 3 × 10−6 M (open triangles).

where I0 is the emission intensity in the absence of quencher and I in the presence of quencher, is given by: ⎤1 ⎡⎛ I ⎞ L Q t = ⎢⎜ 0 ⎟ − 1⎥ + L t − I t ⎡ 0⎤ ⎦K ⎣⎝ I ⎠ ⎣I⎦

almost certainly a major factor in complexation the charge ratio at the complexation point is also tabulated in Table 7. The charge ratios at the point of complexation follow the same trend for each PFP-NR3. If we ignore the lowest concentration of PFP-NR36(I) as this is the most difficult sample to accurately determine the precise complexion point, then PFP-NR3100(Br) has the lowest charge ratios for the complexation point. Lower concentrations of DNA are needed to fully complex PFP-NR3100(Br). There is no significant difference in these results between PFP-NR36(I) and PFP-NR312(Br).

(5)

For the situation in which the complex formed between the lumophore and quencher (LQ) has an intrinsic fluorescence such that I never reaches zero: ⎡ (I − I ) ⎤ 1 ⎡ (I − I ) ⎤ ⎥ ⎥+ ⎢ 0 Q t = L t⎢ 0 ⎢⎣ (I0 − ILQ ) ⎥⎦ K ⎢⎣ (I0 − ILQ ) ⎥⎦

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CONCLUSIONS The PFP-NR3s used in this study are extremely sensitive to fluorescence quenching by dsDNA, ssDNA and DNA bases. For long-dsDNA typical initial KSV values are ca. 106−107 M−1. Sensitivity to quenching increases with PFP-NR3 chain length; for example for the PFP-NR3s with long-dsDNA KSV values increase from 4.72 × 106 M−1 for PFP-NR36(I) to 1.83 × 107 M−1 for PFP-NR3100(Br). ssDNA is a more efficient quencher than dsDNA, this is most apparent in the case of PFPNR3100(Br) where, for long-ssDNA, the KSV values increases from 1.83 × 107 M−1 to 2.9 × 107 M−1. Long-DNA is a more efficient quencher of PFP-NR3 fluorescence than short-DNA with KSV values being ca. 5 fold higher for long-DNA for polymers PFP-NR36(I) and PFP-NR312(Br). All DNA bases quench the polymers efficiently, with guanine being the most efficient. All SV plots for fluorescence quenching are nonlinear and in some cases sigmoidal. Initial KSV values are much higher than expected for the diffusion control limit and quenching is static rather than dynamic. The quenching mechanism is well modeled in terms of a complexation equilibrium in which a PFP-NR3/DNA aggregate complex is formed which brings polymer chains into close enough proximity to allow interchain excitation energy migration and quenching at aggregate or DNA base traps. The calculated equilibrium constants for PFPNR36(I) are 8.4 × 106 M−1 for dsDNA and 8.6 × 106 M−1 for ssDNA. The data shows that at low quencher concentrations each active quenching unit acts as an independent center around which aggregation of the polymer can occur, and that each quenching unit acts as quencher for a large number of monomer repeat units. For PFP-NR3100(Br), which is the most sensitive to quenching, over ca. 100 monomers are quenched by each ‘quencher unit’. The relatively simple model of a complexation equilibrium used to fit the quenching data gives excellent agreement with experimental observations. The results of this indicate that the quenching should not only be dependent on quencher concentration but also polymer concentration, which has been shown to be true. The charge ratio at the complexation point indicated that, as expected,

(6)

The full derivation of eq 5 and 6 is available in the Supporting Information. This analysis fits the data surprisingly well for quenching by short-DNA (Figure 9), with best fit equilibrium constants for PFP-NR36(I), K, of 8.4 ((±1.2) × 106 M−1 for dsDNA and 8.6 (±1.7) × 106 M−1 for ssDNA, and a relative emission for the complex of 6%. On the basis of this analysis, Figure 9(c) gives the calculated fractions of free DNA and polymer with increasing total DNA concentration. In the concentration range where there is a low fraction of free DNA, each DNA strand complexes many polymer chains. Under these conditions two quenching mechanisms seem likely: (1) polymer−polymer aggregation quenching as association of polymer with the base pairs of the DNA brings polymer chains together and decreases the distance between adjacent polymers; (2) quenching by nucleic acid bases, particularly guanine. The latter mechanism is supported by the observation of efficient quenching by guanine in solution, although the mechanism for guanine quenching also seems to involve induced aggregation. The residual emission from the aggregate complex is most likely not to be a specific product of the PFP-NR3/DNA complex but rather PFP-NR3 emission which is not accessible for energy migration quenching. Quenching of PFP-NR3s: PFP-NR3 Concentration Dependent Quenching? The complexation equilibrium treatment of the quenching data fits extremely well. The mechanism implies that quenching efficiency will be dependent on the concentration of PFP-NR3. Thus, quenching experiments for three different polymer concentrations with fixed quencher concentrations were carried out. Figure 10 shows SV plots for the three PFP-NR3s at different concentrations across similar DNA concentrations. The quenching is clearly dependent on PFP-NR3 concentration. In all cases the lowest PFP-NR3 concentration has the highest initial KSV (Table 7), and at a [PFP-NR3] dependent critical concentration of quencher, which we will refer to as the complexation point, there is little change in emission intensity. Since electrostatic attraction between PFP-NR3 and DNA is 467

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electrostatic interactions are dominant in the quenching mechanism. The charge ratio at the complexation point for PFP-NR3100(Br), i.e., the longest polymer studied, are lower than for the shorter polymers indicating that lower concentrations of DNA are required to quench the polymer efficiently. This is expected since the sensitivity to quenching of these relatively rigid rod polymers increases with chain length. Steady state fluorescence anisotropy of PFP-NR3 6(I) decreases with increasing DNA concentration. This decrease in anisotropy is attributed to increased fluorescence depolarisation by interchain energy transfer in aggregate and PFP-NR3/ DNA complexes. This is consistent with results previously presented where it was suggested that the mechanism for energy transfer through and along PFP-NR3 chains is via a rapid Forster mechanism. The sensitivity of these PFP-NR3 polymers to quenching by DNA suggests they could find use as potential sensors. The quenching mechanism has been shown to be well modeled using a complexation equilibrium. This understanding of the interaction and quenching mechanism is extremely useful in designing PFP-NR3 based DNA sensors since a range of polymers with different, well characterized, sensitivities to quenching could be employed depending on the target. Thus, it could be envisaged that several PFP-NR3s could be used in combination to not only identify a target (e.g., DNA) but also, to some degree, to characterize the target (e.g., DNA type or DNA length, etc.).

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ASSOCIATED CONTENT

S Supporting Information *

Equations used to calculate lifetime data, SV quenching plots with guanine as a quencher, tables of time-resolved data of PFP-NR3100Br with guanine and with short-dsDNA, equations derived to describe quenching as a complexation equilibrium, and graph of the anisotropy changes of DAPI, YoYo-1, and PFPNR36(I) with DNA. This material is available free of charge via the Internet at http://pubs.acs.org.

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Article

AUTHOR INFORMATION

Corresponding Authors

*(M.L.D.) Telephone: 44 (0)1248 382375. Fax: +44 1248 370528. E-mail: [email protected]. *(P.D.) Telephone: 44 (0)1792 513081. E-mail: P.Douglas@ swansea.ac.uk. *(H.D.B.) Telephone: 351 239854482. E-mail: [email protected]. pt. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from an EU Research Training Network, CIPSNAC (Colloidal and Interfacial Properties of Synthetic Nucleic Acid Complexes, Contract No. MRTN- CT-2003-504932). We thank NEONUCLEI and Swansea University for financial support and POCI/FCT/ FEDER for further financial support. We are grateful to Prof Sergio Seixas de Melo, Dr. J. Pina, and Dr. F. Dias for access to, and help with, TCSPC equipment at the Chemistry Laser Lab Coimbra (CLLC). 468

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