Determination of the FÃ¶rster Distance in Polymer Films by

2262

J. Phys. Chem. B 2009, 113, 2262–2272

Determination of the Fo¨rster Distance in Polymer Films by Fluorescence Decay for Donor Dyes with a Nonexponential Decay Profile Neda Felorzabihi,† Pablo Froimowicz,‡ Jeffrey C. Haley,‡ Ghasem Rezanejad Bardajee,‡,⊥ Binxin Li,‡ Enrico Bovero,§ Frank C. J. M. van Veggel,§ and Mitchell A. Winnik*,†,‡ Department of Chemical Engineering, UniVersity of Toronto, 200 College Street, Toronto, Ontario, Canada M5S 3E5; Department of Chemistry, UniVersity of Toronto, 80 St. George Street, Toronto, Ontario, Canada, M5S 3H6; and Department of Chemistry, UniVersity of Victoria, Finnerty Road, Victoria, BC, Canada V8P 5C2 ReceiVed: August 27, 2008; ReVised Manuscript ReceiVed: December 12, 2008

Fluorescence resonance energy transfer (FRET) experiments were carried out on three pairs of donor-acceptor dyes in polymer films in which the donor dyes had absorption maxima in the range of 350-450 nm. Two of the donors, a coumarin dye and a naphthalimide dye covalently bound to polystyrene, gave nonexponential decays in the absence of acceptors. The decay profiles could be fitted to a stretched exponential form with a β value on the order of 0.9. We developed equations for analyzing donor fluorescence intensity decay profiles for donor-acceptor mixtures in rigid matrices for the case of donors showing relatively small deviations from exponentiality. To test these equations, we calculate values of the Fo¨rster radius (R0FR) from the decay profile data and compare these values to the Fo¨rster radius R0SO determined by the traditional spectral overlap method. Agreement between these values validates the methodology developed here for the use of such donor dyes in FRET studies of more complex polymer systems. Introduction Many important structural features in polymeric materials have spatial dimensions on the order of 1-10 nm. Examples include the size of individual molecules and the thickness of interfaces between different phases in a polymer blend. Fluorescence resonance energy transfer (FRET) provides a powerful method to probe these length scales. FRET serves as a “spectroscopic ruler”, measuring the distance separating a donor-acceptor fluorophore pair provided that this distance is on the order of 1-10 nm. By chemically attaching donor and acceptor dyes to polymer chains, it is possible to use FRET experiments to measure parameters such as the polymer radius of gyration1 and diffusion coefficients2 as well as parameters characteristic of dynamic properties of macromolecules3 or the interface between two different phases in an immiscible blend4 or in a block copolymer.5 There has been a long history of using fluorescence resonance energy transfer in our group for studying polymer melts. These experiments employ donor and acceptor dyes that are covalently bound to polymers as a means of studying aspects of morphology and miscibility between polymeric phases. In particular, we are interested to obtain quantitative information about the length scales over which the components of polymer phases intermix or interdiffuse. The approach we employ is to measure fluorescence decay profiles of a donor dye in the presence of acceptor dyes and to compare that decay to the predictions of an energy transfer model that describes the spatial distribution of donors and acceptors.6 Here, the challenge is to be able to fit the decay data to a proper model and extract the appropriate parameters for that system. * To whom correspondence should be addressed: e-mail mwinnik@ chem.utoronto.ca; Fax (416) 978-0541. † Department of Chemical Engineering, University of Toronto. ‡ Department of Chemistry, University of Toronto. § University of Victoria. ⊥ Current address: Department of Chemistry, Payame Noor University, Tehran, Iran 19395-4697.

In the past we have worked almost exclusively with phenanthrene as the donor dye. Its most important characteristic is that it gives an exponential decay profile following pulsed excitation in a wide variety of polymer matrices, in the absence of acceptors.7 This feature has allowed us to attribute deviations from exponential decay to energy transfer. Phenanthrene and its derivatives are normally excited at ca. 300 nm and emit at ca. 350 nm. Many polymers that one would like to investigate have a competing absorbance at 300 nm, from either the polymer itself or additives like antioxidants present in the system. They can also emit light at wavelengths up to 400 nm (or even longer). Thus, it would be very useful to find new donor-acceptor pairs that absorb and emit light at longer wavelengths. Because of their relatively high quantum yields and large Stokes shifts, coumarin dyes and naphthalimide dyes are good candidates for energy transfer experiments. Coumarin dyes have widely been used in dye lasers,8 for linear and nonlinear optics applications,9 in dye sensitized solar cells,10 and for organic light-emitting diodes (OLED).11 Because of their photophysical properties, there has recently been a significant interest in naphthalimide dyes for selfcolored polymers,12 as markers in biomaterials,13 as electrontransporting electroluminescent emitters,14 as fluorescent solar cell collectors,15 as fluorescent brighteners,16 and as tunable dye lasers.17 We have investigated the spectroscopic properties of a series of model coumarin dyes and found some examples which exhibit exponential decays in polymer films and fluoresce in the visible region of the spectrum.6 In practice, FRET experiments are usually carried out using donor and acceptor dyes covalently bound to polymers, which can serve as tracers for each polymeric material. Unfortunately, many dyes that have good spectroscopic properties, functional groups for attachment to polymers, and give clean exponential decays in fluid solutions are often characterized by nonexponential decays when dissolved in polymer films.

10.1021/jp807637s CCC: $40.75  2009 American Chemical Society Published on Web 01/30/2009

Fo¨rster Distance in Polymer Films

J. Phys. Chem. B, Vol. 113, No. 8, 2009 2263

CHART 1: Molecular Structures of (a) Coumarin-314 (Coum-314), (b) Disperse Red 19 (Disp. Red 19), (c) Coumarin-3 Monomer (Coum-3), (d) Hostasol Yellow-3G (HY-3G), (e) Naphthalimide Donor Monomer (Nap-D), and (f) Naphthalimide Acceptor Model Compound (Nap-A-m) Dyes

From a physical point of view, this nonexponential behavior can be attributed to spatial or energetic disordering of the relaxation of fluorescent species embedded in the medium.18 According to Sienicki et al.,19 within spatial disorder, one can consider static and dynamic disorder. When there is inhomogeneity in the medium, spatial disordering occurs while dynamic disorder is attributed to complex local dynamical processes between the molecules and the medium.19 In this paper, we explore the use of some of these dyes as donors in FRET experiments in polymer matrices. FRET experiments are widely used for the study of biological systems. Researchers working in this field have also had to deal with the situation in which the decay of the unquenched donor, attached, for example, to a protein, was nonexponential.20,28 One of the fundamental differences between the biological systems and synthetic polymer systems is that the former almost always consider the case of one donor chromophore and one acceptor chromophore attached to each of the molecules in a system. FRET experiments on synthetic polymers normally operate on systems containing many donors and many acceptors in which a given donor can in principle transfer energy to one of several acceptors. A common objective of FRET measurements in both types of systems is to uncover the distribution of distances between the donor and acceptor chromophores. In biological systems, the goal is to measure the conformation of the molecule of interest. In polymeric systems, one may be interested in the conformation of molecules in solution or the size and shapes of nanodomains in bulk. While experiments in both types of systems are carried out in a similar way, the data analysis is different. In this paper, we focus on polymer systems characterized by a random distribution of acceptor molecules in which derivatives of coumarin and naphthalimide dyes serve as donors. In some experiments, the donor dyes are covalently bound to polystyrene (PS), whereas in other experiments, both the donor and acceptor

dyes are simply dissolved in the polymer film. One donor dye is well behaved and, in the absence of acceptors, exhibits an exponential decay. The other two donor dyes exhibit small but significant deviations from monoexponential decay in the absence of acceptors and much more pronounced deviations as the acceptor concentration is increased. We consider modifications to the fundamental Fo¨rster equations to allow these systems to be studied. Experimental Section Materials. The following dyes were supplied by Aldrich and were used as received: Coumarin-314, 2,3,6,7-tetrahydro-11-oxo1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizin-10-carboxylic acid, ethyl ester; Disperse Red 19, 4-(4-nitrophenylazo)-N,N-bis(2-hydroxyethyl)aniline. Commercial Hostasol Yellow-3G dye (HY-3G) was supplied by Clariant. The monomer 2-(2-isobutyl1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yloxy)ethyl methacrylate (Nap-D) was synthesized as reported by Bardajee et al.21 Details of the synthesis of 2-(coumarin 3-ester)ethyl methacrylate (Coum-3) and 2-((2-isobutyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)(methyl)amino)ethyl octanoate, model compound (Nap-A-m), are provided in the Supporting Information. The molecular structures of these dyes are depicted in Chart 1. Dye-labeled polymers were synthesized by miniemulsion polymerization. Details are given in the Supporting Information. Granular poly(methyl methacrylate) (PMMA) (Mn ) 40 000, PDI ) 2.8) was used as received from Aldrich. Water was deionized through a Milli-Q purification system and used in all latex syntheses. Toluene and ethyl acetate (ACS spectrophotometric grade, 99.5+%) were used as received from Aldrich. We ran absorption and fluorescence spectra of all polymers and solvents prior to any other measurements. These spectra show that the polymers and solvents are free of fluorescing impurities over the spectral range examined in this paper.

2264 J. Phys. Chem. B, Vol. 113, No. 8, 2009

Felorzabihi et al.

Figure 1. Normalized absorption and emission spectra for (a, b) Coum-314 and Disp. Red 19 dyes in PMMA films, (c, d) Coum-3-PS and HY-3G dyes in PS films, and (e, f) Nap-D-PS and Nap-A-m dyes in PS films.

TABLE 1: Spectroscopic Characteristics of the Dyes in Polymer Films dye

medium

λabs (nm)

λf (nm)

∆λa (nm)

τD or 〈τD〉 (ns)

Coum-314 Disp. Red 19b Coum-3-PS HY-3G Nap-D-PS Nap-A-m

PMMA PMMA PS PS PS PS

423 489 416 454 362 403

456

33

3.04

1

0

438 496 416 473

22 42 54 69

2.01 5.96 4.65 7.10

0.93 0.94 0.89 0.96

0.110 0.096 0.200 0.064

β

ξDc

characteristics free dye free dye covalently bound free dye covalently bound free dye

a Stokes shift, calculated as the difference in nm between the absorption and emission maxima. b This molecule is nonfluorescent. c Degree of nonexponentiality, eq 13.

Determination of the Dye Content of Polymer. The dye content of PS samples was determined by UV-vis measurements. Polymer samples from miniemulsion polymerization were first dried for a day at room temperature, and then the dried polymer (20 mg) was dissolved in THF (2 mL). Afterward, the solution was precipitated in an excess amount of absolute ethanol (200 mL). This process was repeated two times until no free dye was detected by the UV detector at the low molecular weight part of the gel permeation chromatography (GPC) trace. UV absorbance measure-

ments were then performed on dichloromethane (DCM) solutions of measured amounts of dried purified samples. In a typical procedure, 3.13 mg of purified dye-labeled polymer weighed with a Mettler Toledo MX5 microbalance with a precision of 1 µg was dissolved in 4.17 g of DCM solvent. We used a PS polymer solution in DCM as a blank. Dye content was calculated using the extinction coefficients of Coum-3 (λmax ) 411 nm, 4.05 × 104M-1 cm-1) and Nap-D (λmax ) 410 nm, 4.05 × 104 M-1 cm-1)21



Figure 2. Normalized emission and absorption spectra of (a) Coum-314 and Disp. Red 19 pair in PMMA films, (b) Coum-3 and HY-3G in PS films, and (c) Nap-D and Nap-A-m in PS films.

assuming that these values are the same as those of the dyes bound to the polymer. Instrumentation. UV and Fluorescence Measurements. UV spectra were recorded on a Perkin-Elmer Lambda 25 UV-vis spectrophotometer. Fluorescence spectra were recorded on a SPEX Fluorolog 2.1.2 fluorescence spectrometer using a 30° angle and front-face geometry for all polymer films. A 45 W quartz tungsten (Newport 63976 NIST traceable) calibration source was used to determine the absolute response of the instrument. A (Newport 6032) Neon Oriel pencil-style calibration lamp was used for wavelength correction.

Fluorescence Decay Measurements. Fluorescence decay curves were measured by the time-correlated single photon counting (TCSPC) technique,22 using an IBH system equipped with a 370 nm NanoLED pulsed diode source. The excitation monochromator was set to 370 nm. The emission monochromator was set to the maximum emission wavelength unless otherwise stated. We also used a 408 nm cutoff filter between the sample and emission monochromator to minimize scattered light. Data were collected with the instrument’s built-in multichannel analyzer set at a resolution 0.1135 ns/channel. Fluorescence decay measurements were run until 10 000 counts


Figure 3. Decay plots of (a) Coum-314 in PMMA (5.05 mM) and (b) Coum-3-PS (8.63 mM). In these experiments the emission monochromator was set at different emission wavelengths (λex ) 370 nm). The detection wavelength for each curve is indicated in the figure.

Felorzabihi et al. Fluorescence decay profiles were fitted to equations described below. For each fit the model equations (see below) were convoluted with the instrumental response function using a quadrature routine. This convoluted decay curve was then fitted to the experimental fluorescence data using a LevenbergMarquardt algorithm.23 The goodness of the fits was checked based on the randomness of the weighted residuals, autocorrelation functions, and the χ2 values. The latter were found to be less than 1.3 for all the fits. Most fits were started from three channels (0.34 ns) past the maximum number of counts. Some decay profiles were tested by beginning the fits at the maximum number of counts or three channels before the maximum. This type of fitting gave similar values of the fitting parameters but significantly larger values of χ2. Quantum Yield Measurements. The absolute quantum yield measurements ΦD,f for donor dyes in polymer films were determined using an integrating sphere (Edinburgh Instruments, 150 mm in diameter coated with barium sulfate) placed in an Edinburgh Instruments FL900 fluorimeter, equipped with a 300 M monochromator and a red-sensitive PMT (R928-P model).24 The response of the integrating sphere was calibrated using quinine sulfate in a 1.0 N solution of H2SO4. Quantum yield, ΦD,f value: 55.7 ( 0.8%, ΦD,f reference value: 56 ( 4%.25 Polymers of similar molecular weight without the dye were used as reference. All polymer films, of identical thickness, were prepared under the same conditions. The slit width was 1 mm, and all spectra were corrected for the instrument sensitivity. The quantum yield of samples were measured with both “in the beam” geometry and “out of the beam” geometry.24 No difference was found in initial measurements; thus, all subsequent measurements were performed using “in the beam geometry”. The values of ΦD,f obtained are reported in the text below. Spectral OWerlap Calculation. Spectral overlap integrals J(λ) (in units of M-1 cm-1 nm4) between donor and acceptor molecules were calculated using the normalized corrected emission spectrum of the donor, F(λ), and the extinction coefficient spectrum of the acceptor, ε(λ). This integral in discrete form can be written as λ)b

J(λ) )

∑ F(λ)ε(λ)λ4

(1)

λ)a

Figure 4. (a) Fluorescence intensity decay of Coum-314 in PMMA matrix fitted to an exponential decay. Plotted are the experimental data (points), the exponential fit (heavy line), and the instrument response function (dashed line). (b) The weighted residual plot for the exponential fit in (a). The excitation monochromator was set at 370 nm, and Coum314 emission was detected at maximum emission wavelength for this dye. A cutoff filter (λ ) 408 nm) was used to prevent any scattered light.

were recorded in the maximum channel. The instrumental response function was measured with a scattering solution of silica in water (Ludox); these were collected immediately before and after each measurement. For each measurement, the dyecontaining polymer films were placed in a quartz tube.

where a and b are the first and the last measured wavelengths. Here, λ is measured in increments of 0.5 nm. The extinction coefficient of dyes in polymer films were determined using a fixed known dye concentration and varying the path length by changing the film thickness as reported by Roller and Winnik.26 Solutions containing a mixture of polymer and dye in a suitable solvent at 3 wt % solids were stirred for ca. 24 h and then cast into Teflon dishes (4 × 4 cm2) and dried slowly over 2 days. The concentrations of the dye-polymer mixtures were chosen such that the maximum optical density of the thickest solid film was less than 1.1. For Disperse Red 19, 0.22 mg of dye plus 1.50 g of PMMA were dissolved in ethyl acetate. For HY-3G, 0.16 mg of dye plus 0.65 g of PS were dissolved in toluene. For Nap-A-m dye, 0.15 mg of dye plus 0.31 g of PS were dissolved in toluene. The dry polymer was annealed at 140 °C for 7 h in a vacuum oven to remove residual solvent. Films containing Nap-A-m were annealed at 80 °C for 7 h because of the thermal sensitivity of the dye. Film Preparation for FRET Experiments. For each donoracceptor pair presented in Chart 1, eight films of polymer containing donor and a known amount of acceptor were prepared



Figure 5. (a) Experimental fluorescence decay profile and (b) weighted residual plot for Coum-314 in PMMA films containing varying concentration of Disp. Red 19 dye. The dashed line in (a) is the instrument response function.

140 °C (above the glass transition temperature of the polymers) to remove remaining traces of the trapped solvent in the polymer films. Films containing the Nap-A-m dye were annealed in a vacuum oven at 80 °C for 7 h. Samples employed for FRET experiments were subjected to an identical thermal history as those used for quantum yield measurements. Theoretical Considerations

Figure 6. (a) Plot of P vs acceptor concentrations for Coum-314 in PMMA films containing varying concentration of Disp. Red 19 dye.

The rate of energy transfer kDA between a donor (D) and acceptor (A) separated by a distance r between the centers of their transition dipoles varies as r-6 and can be written as27,28

( )

kf R0 ΦD,f r

kDA )

6

(2)

where kf is the rate of radiative deactivation of the donor excited state, ΦD,f is the quantum yield of donor in the absence of acceptor, and R0, the characteristic energy transfer (Fo¨rster) distance,29 is related to the spectroscopic characteristics of the donor and acceptor molecules in the medium.

R06 )

Figure 7. (a) Fluorescence intensity decay of Coum-3-PS fitted to a stretched exponential decay model. Plotted are the experimental data (points), the fit to stretched exponential function (heavy line), and the instrument response function (dashed line) and (b) the weighted residual plot for the fit in (a).

by solvent-casting the polymer-dye solution (2 wt % solids content) onto a glass substrate. For PMMA polymer films, ethyl acetate was used, whereas for PS polymer films, toluene was used. To obtain films, the solvent was allowed to evaporate very slowly over 1 day in a capped Petri dish. It takes about 9 h for the sample to appear dry. The polymer films were then further dried in a heated vacuum oven (ca. 1 × 10-3 Torr) for 7 h at

9000(ln 10)κ2ΦD,f 5

4

128π NAn

∫0∞ FD(λ)ε(λ)λ4 dλ

(3)

The integral in this equation, known as the spectral overlap integral J(λ), consists of the normalized fluorescence spectrum of the donor, FD(λ), the extinction coefficient spectrum of the acceptor in M-1 cm-1, εA(λ), and the wavelength λ in nm. Here n is the refractive index of the medium in the wavelength range of the spectral overlap, and κ is the transition dipole moment orientation factor, which for an individual D-A pair can be defined as

κ ) cos θDA - 3 cos θD cos θA

(4)

In this equation, θDA represents the angle between the transition dipole moments of the donor and acceptor molecules and θA and θD are the angles between each of these dipoles and the vector connecting their centers. For systems consisting of


Felorzabihi et al.

Figure 8. (a) Experimental fluorescence decay profile and (b) weighted residuals for Coum-3 in PS films containing varying concentration of HY-3G dye. The dashed line in (a) is the instrument response function.

averaging, and it has been demonstrated that 〈κ2〉 ) 0.476.30 Often, it is desirable to separate the orientation contribution to R0 from the spectroscopic part. For this reason it is useful to j 0, the Fo¨rster radius in write expressions for R0 in terms of R the simple case of rapidly rotating dipoles (〈κ2〉 ) 2/3).31 Therefore

R06 ) Figure 9. Plot of P vs acceptor concentrations for Coum-3 in PS films containing varying concentrations of HY-3G dye.

3κ2 j 6 R 2 0

(5)

For systems comprising multiple donors and acceptors, Fo¨rster energy transfer kinetics are properly defined for the case in which the unquenched donor exhibits exponential decay with a lifetime τD. Under these circumstances, the rate of energy transfer can be written as

kDA )

( )

1 R0 τD r

6

(6)

Problems arise when the donor decay exhibits deviations from an exponential form. Some authors argue that the influence of the medium or the environment of the dye on the donor decay profile is due largely to its influence on the radiationless decay rates.28,32 By assuming that all the excited donors in the system have very similar radiative rates (constant kf model), BerberanSantos33 has shown that eq 6 can be rewritten as

Figure 10. (a) Fluorescence intensity decay of Nap-D-PS fitted to a stretched exponential decay. Plotted are the experimental data (points), the fit to stretched exponential function (heavy line), and the instrument response function (dashed line). (b) The weighted residual plot for the fit in (a).

multiple donors and acceptors, there is a distribution of orientations, and the ensemble averaged orientation factor is usually designated as 〈κ2〉. In a fluid medium in which there is rapid rotation on the time scale of the donor excited-state lifetime and all angles in eq 4 occur with equal probability 〈κ2〉 ) 2/3. If the dipoles are randomly oriented but are embedded in a rigid medium like a polymer glass, then 〈κ2〉 is evaluated by ensemble

kET )

( )

1 R0 〈τD〉 r

6

(7)

where 〈τD〉 is the average lifetime of the donor molecules. Lakowicz et al.34,35 have proposed an alternative model for a discrete donor lifetime distribution in which they assume that R0 is constant for all donors and acceptors, so that for the donors with lifetime τDi the energy transfer rate is given by the expression

kDAi )

( )

1 R0 τDi r

6

(8)



Results and Discussion Our goal in this paper is to examine how one can best analyze FRET kinetics for the case of donor and acceptor dyes rigidly embedded in a polymer matrix when the decay of the donor dye itself deviates from a simple exponential form. We are particularly interested in the case where the dyes are randomly oriented but immobile on the time scale of the donor excited state. If the donor decay were exponential, its time-dependent intensity in the presence of acceptor molecules randomly distributed in three dimensions would be given by the expression

[

( )]

t t I(t) ) I0 exp - - P τD τD

1/2

(9)

where P is given by

P)

4 3/2 π NAR03CA 3000

semiconductor nanoclusters37 as well as ordered38 and disordered39 inorganic solids. In addition, it has been applied to predict the fluorescence decay of fluorophores covalently bound to silica surfaces40 and of fluorophores incorporated into sol-gel media.41 The average relaxation time of a stretched exponential function can be calculated using

〈τD〉 )

ξD )

[ ( )]

I(t) ) I0 exp -

t τD

β

(11)

We use this functional form for the case of donor dyes covalently bound to polymer molecules. The Kohlrausch function is widely employed for analyzing fluorescence decays in several classes of systems. The stretched exponential function has been used for analyzing the luminescence decay of

()

(12)

where Γ(x) is the gamma function, t is the time, and τD is the relaxation time. β is a fitting parameter (0 < β < 1). In the limiting case where β ) 1, the Kohlrausch decay becomes a single-exponential decay. The magnitude of β is a measure of the extent of deviation from an exponential form. Czuper et al.20 have proposed a different measure of the degree of nonexponentiality of the donor decay.

(10)

and CA is the concentration of the acceptor. In the sections below, we consider the case of donor decays in the absence of acceptors that show relatively small deviations from exponential behavior and propose an expression analogous to eq 9 to fit the data obtained in the presence of acceptors. Our strategy is to assume initially that eq 7 is valid and then analyze the data to show that under these circumstances values of R0 obtained from donor decay measurements are nearly identical to those obtained by the traditional spectral overlap method. Model for Nonexponential Donor Decays. In this study we fit the donor fluorescence intensity decays to a stretched exponential (Kohlrausch decay) function:36

τD 1 Γ β β

1 2〈τD〉

∫0∞

|

( )|

ID(t) -t - exp I0 〈τD〉

dt

(13)

This expression is particularly useful if the donor decay is fitted to a sum of exponential terms instead of a stretched exponential. Our main assumption, which we examine in the remaining sections of this paper, is that the fluorescence decay of donor species in the presence of acceptors in polymer films can be fitted to a generalized Fo¨rster equation of the form

[( ) ( )]

ID(t) ) I0 exp -

t τD

β

-P

t 〈τD〉

1/2

(14)

If P in eq 14 can be expressed by eq 10, then eq 14 effectively assumes the existence of a single Fo¨rster radius for donors with a nonexponential fluorescence decay.21 From an experimental perspective, ID(t) profiles will be fitted to eq 9 when the unquenched donor decay is exponential and to eq 14 when the donor decay is nonexponential. These decays can be fitted with two floating parameters I0 and P, since β, τD, or 〈τD〉 can be determined independently from a sample that contains donor but no acceptor. If a series of samples are

Figure 11. (a) Experimental fluorescence decay profile and (b) weighted residual plot for Nap-D in PS films containing varying concentration of Nap-A-m dye. The emission monochromator was set at 405 nm where no acceptor fluorescence could be detected at this wavelength. Also, no cutoff filter was used for this set of samples. The dashed line in (a) is the instrument response function.


Felorzabihi et al.

prepared with varying CA, then it should be possible to extract R0 from the slope of a linear plot of P against CA. Dyes, Polymers, and Their Spectroscopic Properties. In some experiments described below, the donor dyes are covalently attached to the polymer backbone, and the acceptor dyes are dissolved in films of the D-labeled polymers. In other experiments, both the donor and acceptor dyes are dissolved in unlabeled polymer films. The dye-labeled polymers were synthesized by miniemulsion polymerization. These polymers were obtained in the form of latex particles, which, after drying, were dissolved in a good solvent for the polymer. These solutions, to which known amounts of acceptor dye were added, were then used to cast films. The structures of the dyes we examine in this paper, and the shorthand notation we use to refer to these dyes, are presented in Chart 1. Details of the synthesis and characterization of the Coum-3-labled polystyrene (Mn ) 25 700, PDI ≈ 2, 9.6 µmol dye/g polymer) and NapD-labeled polystyrene (Mn ) 28 600, PDI ≈ 2, 33.1 µmol dye/g polymer) are presented in the Supporting Information. The absorption and emission spectra of the dyes listed in Chart 1, dissolved in PS or PMMA films, are presented in Figure 1. Values of the absorption and emission maxima (λabs and λf, respectively) for these six dyes in polymer films are listed in Table 1. Note that Disperse Red 19 is nonfluorescent. The fluorescence decay of Coum-314 in PMMA fit well to a singleexponential function, whereas the ID(t) profile of other dyes showed small deviations from an exponential function. Values of 〈τD〉 for these dyes as well as measures of their degree of nonexponentiality are also presented in Table 1. The absorption and emission spectra of the three donor-acceptor pairs examined in this paper are presented in Figure 2. Although Coum-314 in PMMA and in PS exhibits exponential decays when these decay profiles are measured at the emission maximum or at longer wavelengths, a short component to the fluorescence decay profile can be seen (Figure 3) when the emission is measured at shorter wavelengths. This effect is more

Figure 12. Plot of P vs acceptor concentration for films containing mixtures of Nap-D-labeled PS and free Nap-A-m. The points are the P values obtained by fitting the experimental data to eq 14. The line is the linear fit of the experimental P values. The emission monochromator was set at 405 nm where no acceptor fluorescence could be detected at this wavelength. Also, no cutoff filter was used for this set of samples.

pronounced for Coum-314 dye in PMMA than for Coum-3 dye in PS in spite of the fact that the dye concentration in PS (8.63 mM) is somewhat higher than in PMMA (5.05 mM). It is unlikely that this effect is due to dyes in different environments because the emission spectra of the dye are independent of the excitation wavelength (see the Supporting Information). Because this nonexponentiality is observed only at wavelengths where the emission of the dye overlaps its own absorption spectrum, we tentatively assign the rapid decay to donor-donor energy transfer, i.e. energy migration, that leads to quenching or trapping of the excitation. As one can see in Figure 1a,c, there is a greater extent of overlap of the absorption and emission spectra of Coum-314 in PMMA than for Coum-3 in PS. These deviations are not a problem for the experiments described below because donor emission is monitored at wavelengths where donor absorption does not overlap donor emission. Experimental Tests of Energy Transfer Kinetics in Polymer Films. For samples in which the donor dye in the absence of acceptor has an exponential decay, one expects the ID(t) decay profiles in the presence of acceptors to fit well to eq 9, yielding values of the fitting parameter P that are proportional (eq 10) to the acceptor concentration CA. On the basis of the work of Roller and Winnik,26 one also expects that the value of the Fo¨rster radius R0FR calculated from the slope of this plot should agree with the value R0SO obtained by the more traditional spectral overlap method. For samples in which the donor dye in the absence of acceptor has a nonexponential decay, we test the validity of eq 14 in four steps. First, we fit this decay to a stretched exponential function (eq 11) and calculate the mean decay time (eq 12). Second, we fit donor decay in the presence of acceptors to eq 14 to see if reasonable fits can be obtained. Third, we examine whether P values obtained in this way give a linear plot versus CA. Finally, we compare the value of R0FR obtained from the slope of this plot with that of R0SO obtained by the spectral overlap method. Coum-314 and Disp. Red 19 in PMMA. The fluorescence decay of Coum-314 in PMMA films exhibits an exponential decay with τD ) 3.04 ns. FRET experiments with this donor dye should be well described by eqs 9 and 10. In this section we examine this prediction. Our strategy is to obtain a value of R0 from a plot of P versus CA (eq 10) and compare that value to the value of R0 obtained by the spectral overlap method, by evaluating separately the term contained in eq 3. In the presence of Disp. Red 19 as an acceptor, ID(t) profiles for Coum-314 are nonexponential due to energy transfer to the acceptor dyes. The fluorescence decay profiles for films containing Coum-314 (5.05 mM) and amounts of Disp. Red 19 ranging from 0 to 4.55 mM in PMMA are presented in Figure 5. These data were fitted to eq 9. Selected examples of the weighted residuals corresponding to these fits are also shown in Figure 5. More complete data are provided in the Supporting Information. The weighted residuals and auto correlations for the fits are randomly distributed around zero for the entire range of the concentrations with χ2 values below 1.3. Figure 6 shows the plot of P versus

TABLE 2: Spectroscopic Properties of the D/A Pairs and Fo¨rster Radii Obtained for the Three Pairs of Dyes in Polymer Films dye pair

ΦD,f (%)

J(λ)(M-1 cm-1 nm4)

Coum-314/Disp. Red 19/PMMA Coum-3 PS/HY-3G Nap-D-PS/Nap-A-m

83.3 ( 0.02d 76.9 ( 0.04 80.9 ( 0.02

2.35 × 1015 1.04 × 1015 2.93 × 1014

R0SOa (nm)

R0FRb (nm)

j 0c (nm) R

5.05 ( 0.1 4.17 ( 0.05 3.40 ( 0.05

5.09 ( 0.15 4.19 ( 0.15 3.42 ( 0.17

5.38 ( 0.17 4.43 ( 0.16 3.62 ( 0.18

Determined by the spectral overlap method, using 〈κ2〉 ) 0.476. b Calculated from plots of P vs CA. c Calculated from R0SO using eq 5. This value corresponds to 〈κ2〉 ) 2/3. d One standard deviation based upon two independent measurements. a

Fo¨rster Distance in Polymer Films acceptor concentrations CA for the Coum-314/Disp. Red 19 pair in PMMA. These P values fall onto a straight line with an intercept passing through the origin. From the slope of the P versus CA plot (eq 10), we calculated a value of R0FR ) 5.09 ( 0.16 nm. This value compares very favorably with the value R0SO ) 5.05 ( 0.1 nm determined by the spectral overlap method. Coum-3-PS and HY-3G. To examine this D/A pair in which the polymer-bound Coumarin-3 is the donor dye, a series of eight samples with acceptor concentrations ranging from 0 to 6.47 mM were prepared. The bulk concentration of donor dyes in these polystyrene films was 8.63 mM. The decay of the donoronly film could not be fitted with a single-exponential model. The best fit for this sample gave χ2 ) 1.57 and nonrandom weighted residuals. However, the data for the unquenched donor in a PS film were well fitted to a stretched exponential function with χ2 ) 0.99. This fit is presented in Figure 7. The resulting fitting parameters obtained were τD ) 1.95 ns, β ) 0.93, and 〈τD〉 ) 2.01 ns. Here, the β parameter indicates a small but detectable deviation from single-exponential behavior.21 Figure 8 shows the decay traces of Coum-3-PS films containing different concentrations of acceptor dye. All the decays were successfully fitted to eq 14 with the weighted residuals and autocorrelations randomly distributed around zero with χ2 below 1.3. A selection of weighted residual plots is shown in Figure 8, with a complete set of analyzed data presented in the Supporting Information. Figure 9 presents the fitted P values as a function of acceptor concentrations. The P values all fall along a straight line with zero intercept. These results support our idea that eq 14 is effective for fitting FRET data with weakly nonexponential donors. From the slope of this plot we calculate R0FR ) 4.19 ( 0.15 nm. Nap-D-PS plus Nap-A-m. To examine this D/A pair, a series of eight samples were prepared with acceptor concentrations ranging from 0 to 8.33 mM. In these donor-labeled polystyrene films, the Nap-D concentration was 0.034 M. Nap-D-PS films in the absence of acceptor gave nonexponential fluorescence decays characterized by the fitting parameters: τD ) 4.38 ns, β ) 0.89, and 〈τD〉 ) 4.65 ns. This dye has a longer mean fluorescence lifetime and a stronger deviation from the exponential function than Coum-3 in PS films. The decay traces of Nap-D films with different concentrations of acceptor dye are presented in Figure 11. Our results show that all the decays were successfully fitted to eq 14 with χ2 below 1.2 and weighted residuals and autocorrelations randomly distributed around zero. Complete data are presented in the Supporting Information. The resulting P values as a function of acceptor concentrations are presented in Figure 12. The P values all fall on a line passing through the origin. The calculated value of the Fo¨rster radius from the slope of this line R0FR ) 3.42 ( 0.17 nm. Comparison of R0 Values Obtained by Donor Decay and Spectral OWerlap. For each of the D/A pairs, values of R0SO were calculated from eq 3 by evaluating the overlap integral J(λ) for each pair and measuring the quantum yields of the donor dyes in the respective polymer films. We emphasize the importance of using an integrating sphere to obtain accurate values of the quantum yields for dyes dissolved in polymer films. These calculations employed 〈κ2〉 ) 0.476. These data are all presented in Table 2. The values of the quantum yields for the donor dyes are very similar and close to 0.8. The smallest overlap integral is that of the Nap-D-PS/Nap-A pair. The spectral overlap of the Coum-3-PS/HY-3G pair in PS occurs at longer wavelength. Both the λ4 term in eq 1 and the higher extinction

J. Phys. Chem. B, Vol. 113, No. 8, 2009 2271 coefficient of HY-3G compared to Nap-A-m contribute to the increased value of the overlap integral for this pair. Disp. Red 19 in PMMA has the highest extinction coefficient of the three acceptor dyes, and this leads to the largest value of the overlap integral. Values of R0SO (Table 2) increase in parallel with values of J(λ). The most important result of this paper is that values of R0FR, obtained with the use of eq 14 to fit FRET kinetic data for donor dyes with nonexponential decays, are essentially identical to values of R0SO obtained by the traditional spectral overlap method. This establishes the validity of our assumptions for FRET experiments in polymer films in which the donor decay profile is not exponential but exhibits a relatively small degree of nonexponentiality. Summary FRET experiments based upon time-correlated single photon counting measurements were carried out on three donor-acceptor pairs in rigid polymer films. For two of these D/A pairs, the fluorescence decays of the donor dyes were nonexponential but could be fitted to a stretched exponential form with a value of β ≈ 0.9. Model equations proposed above were used to fit ID(t) decay profiles for donor-acceptor mixtures, and parameters extracted from these fits were used to evaluate a Fo¨rster radius, which we refer to as R0FR. When the unquenched donor decay is exponential, one expects this value to be very similar in magnitude to the Fo¨rster radius R0SO determined by the traditional spectral overlap method. Our experiments show that when the donor dyes themselves have decay profiles characterized by small deviations from an exponential form, the equations we propose allow determination of values of R0FR that are essentially identical to R0SO. These results open the way for the use of such donor dyes to study problems related to interface dimensions and domains of restricted geometry in polymer systems. Acknowledgment. The authors thank NSERC for their support of this research, and N.F. thanks the Province of Ontario for an OGS fellowship. Supporting Information Available: Experimental details for the synthesis and characterization of dyes Coum-3 and NapA-m and of the dye-labeled polymers, including GPC traces of the polymers. Emission spectra of Coum-314 in PMMA and Coum-3-PS films at different excitation wavelengths. Fluorescence decay profiles and weighted residual plots for donor dyes in polymer films in the presence of different amounts of acceptor dyes: Coum-314 in PMMA (Disp Red 19), Coum-3-PS (HY3G), Nap-D-PS (nap-A-m). Details of the determination of extinction coefficients of the dyes in polymer films along with extinction coefficients of the three acceptor dyes in PMMA and PS films, of the unquenched donor fluorescence quantum yields, and of the spectral overlap calculations. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Peterson, K. A.; Zimmt, M. B.; Linse, S.; Domingue, R. P.; Fayer, M. D. Macromolecules 1987, 20, 168. (2) Ye, X.; Farinha, J. P. S.; Oh, J. K.; Winnik, M. A.; Wu, C. Macromolecules 2003, 36, 8749. (3) Srinivas, G.; Yethiraj, A.; Bagchi, B. J. Phys. Chem. B 2001, 105, 2475. (4) Farinha, J. P. S.; Vorobyova, O.; Winnik, M. A. Macromolecules 2000, 33, 5863. (5) Yang, J.; Lou, X.; Spiro, J. G.; Winnik, M. A. Macromolecules 2006, 39, 2405.

2272 J. Phys. Chem. B, Vol. 113, No. 8, 2009 (6) Felorzabihi, N.; Haley, J. C.; Bardajee, G. R.; Winnik, M. A. J. Polym. Sci., Part B 2007, 45, 2333. (7) Pekcan, O.; Winnik, M. A.; Croucher, M. D. Macromolecules 1990, 23, 2673. (8) Yang, Y.; Qian, G.; Su, D.; Wang, Z.; Wang, M. Mater. Sci. Eng. B 2005, 119, 192. (9) Wlodarczyk, P.; Komarneni, S.; Roy, R.; White, W. B. J. Mater. Chem. 1996, 6, 1967. (10) Wang, Z.-S.; Cui, Y.; Dan-oh, Y.; Kasada, C.; Shinpo, A.; Hara, K. J. Phys. Chem. C 2007, 111, 7224. (11) Pschenitzka, F.; Long, K.; Sturm, J. C. Mater. Res. Soc. Symp. Proc. 2001, 665, 391. (12) Guthrie, J. T.; Konstantinova, T.; Ginova, E. Dyes Pigm. 1997, 34, 287. (13) Peters, A. T.; Bide, M. J. Dyes Pigm. 1985, 6, 349. (14) (a) Du, P.; Zhu, W.-H.; Xie, Y.-Q.; Zhao, F.; Ku, C.-F.; Cao, Y.; Chang, C.-P.; Tian, H. Macromolecules 2004, 37, 4387. (b) Meng, F.; Liu, C.; Hua, J.; Cao, Y.; Chen, K.; Tian, H. Eur. Polym. J. 2003, 39, 1325. (15) Xuhong, Q.; Zhenghua, Z.; Kongchang, C. Dyes Pigm. 1989, 11, 13. (16) Grabchev, I.; Konstantinova, T. Dyes Pigm. 1997, 33, 197. (17) Pardo, A.; Poyato, J. M. L.; Martin, E. J. Photochem. 1987, 36, 323. (18) Grunewald, M.; Pohlmann, B.; Movaghar, B.; Wurtz, D. Philos. Mag. B 1984, 49, 341. (19) Sienicki, K.; Blonski, S.; Durocher, G. J. Phys. Chem. 1991, 95, 1576. (20) Czuper, A.; Gryczynski, I.; Kusba, J. J. Photochem. Photobiolog. B 2007, 87, 200. (21) Bardajee, G. R.; Vancaeyzeele, C.; Haley, J. C.; Li, A. Y.; Winnik, M. A. Polymer 2007, 48, 5839. (22) O’Connor, D. V.; Phillips, D. Time-Correlated Single Photon Counting; Academic Press: London, 1984. (23) Press, W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling, W. T. Numerical Recipes in C: The Art of Scientific Computing, 2nd ed.; Cambridge University Press: New York, 1992.

Felorzabihi et al. (24) de Mello, J. C.; Wittmann, H. F.; Friend, R. H. AdV. Mater. 1997, 9, 230. (25) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991. (26) Roller, R. S.; Winnik, M. A. J. Phys. Chem. B 2005, 109, 12261. (27) Fo¨rster, Th. Mechanisms of Energy Transfer. In ComprehensiVe Biochemistry; Florkin, M., Stolz, E. H., Eds.; Elsevier: Amsterdam, 1967; Vol. 22, p 61. (28) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999. (29) Fo¨rster, Th. Zwishenmolekular Engergiewanderung und Fluoreszenz. Ann. Phys. (Leipzig) 1948, 2, 55. (30) Steinberg, I. Z. J. Chem. Phys. 1968, 48, 2411. (31) Steinberg, I. Z.; Haas, E.; Katchalski, E. Long-Range Nonradiative Transfer of Electronic Excitation Energy. In Time-ResolVed Spectroscopy in Biochemistry and Biology; Andrews, St., Ed.; Plenum: New York, 1983. (32) Vos, R.; Engelborghs, Y.; Izard, J.; Baty, D. Biochemistry 1995, 34, 1734. (33) Berberan-Santos, M. N. J. Lumin. 1991, 50, 83. (34) Lakowicz, J. R.; Gryczynski, I.; Cheung, H. C.; Wang, C.-K.; Johnson, M. L.; Joshi, N. Biochemistry 1988, 27, 9149. (35) Lakowicz, J. R.; Gryczynski, I.; Cheung, H. C.; Wang, C.-K.; Johnson, M. L. Biopolymers 1988, 27, 821. (36) Kohlrausch, R. Pogg. Ann. Phys. Chem. 1854, 91, 179. (37) Soloviev, V. N.; Eichhofer, A.; Fenske, D.; Banin, U. J. Am. Chem. Soc. 2001, 123, 2354. (38) Chen, R. J. Lumin. 2003, 102-103, 510. (39) Linnros, J.; Lalic, N.; Galeckas, A.; Grivickas, V. J. Appl. Phys. 1999, 86, 6128. (40) Wong, A. L.; Harris, J. M.; Marshall, D. B. Can. J. Phys. 1990, 68, 1027. (41) Hof, M. E.; Schleicher, J.; Schneider, F. W.; Bunsenges, B. J. Phys. Chem. 1989, 93, 1377.

JP807637S

Determination of the FÃ¶rster Distance in Polymer Films by

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