Ultrafast Fluorescence Resonance Energy Transfer in the Micelle and

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J. Phys. Chem. B 2007, 111, 7085-7091

7085

Ultrafast Fluorescence Resonance Energy Transfer in the Micelle and the Gel Phase of a PEO-PPO-PEO Triblock Copolymer: Excitation Wavelength Dependence Subhadip Ghosh, Shantanu Dey, Aniruddha Adhikari, Ujjwal Mandal, and Kankan Bhattacharyya* Physical Chemistry Department, Indian Association for the CultiVation of Science, JadaVpur, Kolkata 700 032, India ReceiVed: January 11, 2007; In Final Form: March 27, 2007

Fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to rhodamine 6G (R6G) is studied in the micelle and the gel phase of a triblock copolymer, (PEO)20-(PPO)70-(PEO)20 (Pluronic P123 (P123)) by picosecond and femtosecond emission spectroscopy. The time constants of FRET were obtained from the rise time of the acceptor (R6G) emission. In a P123 micelle, FRET occurs in multiple time scales: 2.5, 100, and 1700 ps. In the gel phase, three rise components are observed: 3, 150, and 2600 ps. According to a simple Fo¨rster model, the ultrafast (2.5 and 3 ps) components of FRET correspond to donor-acceptor distance RDA)13 ( 2 Å. The ultrafast FRET occurs between a donor and an acceptor residing at close contact at the corona (PEO) region of a P123 micelle. With increase in the excitation wavelength (λex) from 375 to 435 nm, the relative contribution of the ultrafast component of FRET (∼3 ps) increases from 13% to 100% in P123 micelle and from 1% to 100% in P123 gel. It is suggested that at λex ) 435 nm, mainly the highly polar peripheral region is probed where FRET is very fast due to close proximity of the donor and the acceptor. The 100 and 150 ps components correspond to RDA ) 25 ( 2 Å and are ascribed to FRET from C480 deep inside the micelle to an acceptor (R6G) in the peripheral region. The very long component of FRET (1700 ps in micelle and 2600 ps component in gel) may arise from diffusion of the donor from outside the micelle to the interior followed by fast FRET.

1. Introduction Triblock copolymers have received a lot of recent attention because of their interesting self-assembly, rich phase diversity, and versatile industrial applications.1-8 The copolymer (PEO)20(PPO)70-(PEO)20 is known as Pluronic P123 (P123). It contains a hydrophilic (poly(ethylene oxide), PEO) block and a hydrophobic (poly(propylene oxide), PPO) block. The hydrophobicity of the PPO block above 288 K and the consequent dehydration leads to the formation of a micelle phase with a hydrophobic PPO core and a hydrophilic PEO corona (Scheme 1A).1-7 Castner and co-workers studied the temperature-dependent micellization and gel formation in Pluronic F88 triblock copolymers using picosecond fluorescence anisotropy decay and estimated the microscopic friction in different regions of these systems.8 Hof and co-workers studied solvation dynamics in polystyrene-block-poly(2-vinylpyridine)-block-poly(oxyethylene) copolymer.9 At a sufficiently high concentration, successive dehydration of the PPO blocks of the P123 triblock copolymer results in a close packed crystalline gel phase (Scheme 1B).1-5 The gel phase of P123 is characterized by a small-angle neutron scattering correlation peak at qmax ∼ 0.045 Å-1. This corresponds to an intermicellar distance, d ≈ 2π/qmax ≈140 Å.5 Thus the intermicellar distance (∼140 Å) in the gel phase of P123 is less than the sum of the radii (∼180 Å) of the micelles.5,6 This suggests that the structure of the gel involves interpenetration of the micelles and intermicellar entanglement (Scheme 1B). This results in a very high number density (N ≈1/d3, for a simple * Corresponding author. E-mail: [email protected]. Fax: (91)33-2473-2805.

cubic array) of the polymer particles.5 In the gel phase ∼48% of the total space is “void” and is occupied by water. Evidently, a P123 micelle and a P123 gel are both highly heterogeneous having regions of widely varying polarity. The absorption and emission spectra of a polarity sensitive probe in different regions of the P123 micelle (or gel) differ markedly. Thus, by varying the excitation wavelength (λex), one may selectively excite the probe molecules in different regions of this system. Excitation at a shorter wavelength (“blue edge”) selects the probe in a relatively nonpolar environment (PPO blocks) and gives rise to a blue-shifted emission spectrum. On the contrary, excitation at a longer wavelength (“red edge”) exclusively excites the probe residing in the polar environment (PEO blocks) and causes a red shift in the emission spectrum. This phenomenon of excitation wavelength dependence of emission maximum is known as red edge excitation shift (REES).10-17 The effect of variation of λex on static emission spectra is well studied. Recently, by varying λex we have studied solvation dynamics in different regions of such heterogeneous media. So far, this method has been applied to a reverse micelle,13 lipid,14,15 P123 triblock copolymer micelle,16 and P123 gel.17 Fluorescence resonance energy transfer (FRET)18-22 may also exhibit location and, hence, λex dependence because of two reasons. First, the donor-acceptor distance (RDA) may be different for a donor molecule residing in different locations with a fixed location of the acceptor. Second, spectral overlap between donor emission and acceptor absorption may be different in different regions. Most recently, we have shown that FRET from coumarin 480 (C480) to an anionic dye

10.1021/jp070235y CCC: $37.00 © 2007 American Chemical Society Published on Web 05/27/2007

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SCHEME 1: Structures of (A) P123 Micelle and (B) P123 Gel

SCHEME 2: Structures of (A) Coumarin 480 and (B) Rhodamine 6G

fluorescein 548 (F548) in a reverse micelle also displays dramatic dependence on λex.19 The anionic dye F548 resides in a highly polar region (core of the water pool of a reverse micelle) while the neutral donor C480 is distributed over a wide region from the highly nonpolar bulk hydrocarbon to the water pool of the reverse micelle.19 The distances of the acceptor (F548) from donor (C480) in different regions of the reverse micelle are different. Thus excitation of the donor C480 in different regions (by varying λex) leads to different rates of FRET.19 In the present work, we have studied FRET from C480 to R6G in different regions of a P123 micelle and a P123 gel using λex dependence. We show that the rate of FRET and its λex dependence in a P123 micelle are quite different from that in a P123 gel. We will explain the results in terms of the structural differences of the P123 micelle and the P123 gel. 2. Experimental Section Laser grade coumarin 480 (C480, Scheme 2A) and rhodamine 6G (R6G, Exciton, Scheme 2B) were purchased from Exciton. The triblock copolymer Pluronic P123 (P123) was a gift from BASF Corp. and was used without further purification. In a 30 wt % aqueous solution, P123 exists in the gel phase (cubic phase, I1) over a wide range of temperatures (17-45 °C).6,7 We used 1 wt % P123 for the micelle and 30 wt % for the gel phase. The gel was prepared by mixing a proper amount of P123 with 100 mL of water. The solution was kept at a low temperature (∼0 °C) and stirred for 8-10 h using a magnetic stirrer in a sealed container. Subsequently, the solution was kept in a refrigerator for a week. The concentrations of the donor (C480) and the acceptor (R6G) were kept fixed at 40 and 80 µM, respectively.

The steady-state absorption and emission spectra were recorded in a Shimadzu UV-2401 spectrophotometer and a Spex FluoroMax-3 spectrofluorimeter, respectively. In our femtosecond upconversion setup (FOG 100, CDP), the sample was excited at 375, 405, and 435 nm. Briefly, the sample was excited using the second harmonic of a mode-locked Ti-sapphire laser (Tsunami, Spectra Physics) pumped by a 5 W Millennia (Spectra Physics). The fundamental beam was frequency doubled in a nonlinear crystal (1 mm BBO, θ ) 25°, φ ) 90°). The fluorescence emitted from the sample was upconverted in a nonlinear crystal (0.5 mm BBO, θ ) 38°, φ ) 90°) using the fundamental beam as a gate pulse. The upconverted light is dispersed in a monochromator and detected using photon counting electronics. A cross-correlation function obtained using the Raman scattering from ethanol displayed a full width at half-maximum (fwhm) of 350 fs. The femtosecond fluorescence decays were fitted using a Gaussian shape for the excitation pulse. To fit the femtosecond data, we used the long picosecond components and kept them fixed during fitting of femtosecond data. The picosecond components were detected using a setup in which the samples were excited at 375, 405, and 435 nm using picosecond diode laser (IBH Nanoleds) in an IBH Fluorocube apparatus. The emission was collected at a magic angle polarization using a Hamamatsu MCP photomultiplier (5000U-09). The time correlated single photon counting (TCSPC) setup consists of an Ortec 9327 CFD and a Tennelec TC 863 TAC. The data are collected with a PCA3 card (Oxford) as a multichannel analyzer. The typical fwhm of the system response (instrument response function, IRF) for a liquid

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scatterer is about 90 ps. The fluorescence decays were deconvoluted using IBH DAS6 software. According to Fo¨rster theory, the rate of FRET (kFRET) is given by18-22

kFRET )

( )

1 R0 τD0 RDA

6

(1)

where τD0 is the lifetime of the donor in the absence of acceptor. At a donor-acceptor distance RDA ) R0, the efficiency of energy transfer is 50% and kFRET ) (1/τD0). In order to calculate the Fo¨rster distance R0 (in Å) we used18

R0 ) 0.211[κ2n-4QDJ(λ)]1/6

(2)

where n is the refractive index of the medium (∼1.4 for macromolecules in water),18-20 QD is the quantum yield of the donor in the absence of acceptor, κ2 is the orientation factor, and J(λ) is the spectral overlap between the donor emission and the acceptor absorption. J(λ) is related to the normalized fluorescence intensity (FD) of the donor in the absence of the acceptor and the extinction coefficient of the acceptor (A) as18

∫0∞ FD(λ)A(λ)λ4 dλ J(λ) ) ∫0∞ FD(λ) dλ

(3)

The value of κ2 may vary from 0 (mutually perpendicular transition dipoles) to 4 (collinear dipoles). For κ2 ) 0, FRET is forbidden and no ultrafast component of FRET would be observed. The ultrafast FRET detected in this work obviously indicates a large value of κ2. One can estimate the upper (κmax2) and lower (κmin2) limit of κ2 by using the steady-state fluorescence anisotropy and the initial value of anisotropy (r0) obtained in the time-resolved anisotropy measurement as18

κmin2 )

[

]

(dDx + dAx) 2 13 2

2 κmax2 ) (1 + dDx + dAx + 3dDxdAx) 3

(4) (5)

where dix denotes the ratio of square root of the steady-state fluorescence anisotropy (riSS) and the initial value of anisotropy (ri0) in the anisotropy decay of the ith species (donor or acceptor). However, the distance calculated using the Fo¨rster model may vary by only about e20% in the range of values of κ2.19,20 We therefore used κ2 ) 2/3 (random orientation) for the calculation of R0. In the case of FRET, from a donor of lifetime longer than that of the acceptor, the acceptor emission is given by

a2 exp[-t/τA] - a1 exp[-(kFRET + 1/τD0)t]

(6)

where, τD0 and τA, denote the lifetime of the unquenched donor and acceptor, respectively.23,24 In this case, the lifetime of the unquenched donor (∼6 ns) is greater than that of the acceptor (∼4.7 ns). At a long time, the acceptor decay is dominated by those acceptors which are excited by the trivial mechanism (emission of a photon by the donor and its absorption by the acceptor). In this case, decay of the acceptor emission is controlled by the longer decay of the unquenched donor and, hence, exhibits a decay component similar to the decay time of

Figure 1. Spectral overlap of donor (C480) emission (s) in (A) 1 wt % P123 (micelle) and (B) 30 wt % P123 (gel) with acceptor (R6G) absorption (‚ - ‚) at λex (i-iii) 375, 405, and 435 nm.

the unquenched donor. Similar unusually long decay of the acceptor was reported earlier.19,20,23,24 3. Results 3.1. Steady-State Study of FRET: P123 Micelle and P123 Gel. The absorption maxima of C480 in P123 micelle (at 380 nm) and in P123 gel (384 nm) show a large blue shift from that (396 nm)25 in water. The blue shift suggests that the microenvironments of C480 in P123 micelle and in P123 gel are less polar than that in bulk water. For all λex, the emission maximum of C480 in P123 micelle and gel is found to be blue-shifted from the emission maximum (489 nm)25 in bulk water. The blue shift of the emission maximum is ascribed to lower polarity of the P123 micelle (and the gel). In a P123 micelle, emission maximum of C480 displays a very significant REES by 25 nm from 453 nm at λex ) 345 to 478 nm at λex ) 445 nm. In P123 gel, there is a 22 nm REES from 450 nm at λex ) 345 to 472 nm at λex ) 445 nm. The observed REES suggests a distribution of the C480 (donor) molecules in different regions of varying static polarity in both the micelle and the gel phase of P123. For the acceptor (R6G), the absorption maximum is at 526 nm in bulk water and displays a red shift to 530 nm in P123 micelle and to 534 nm in P123 gel. Emission maximum of R6G also exhibits a red shift from 550 nm in bulk water to 565 nm in P123 micelle and to 569 nm in P123 gel. In both P123 micelle and P123 gel, the emission maximum of R6G is independent of λex. This suggests that in both P123 micelle and gel, the acceptor R6G resides in a more or less uniform environment. Because of its positive charge, R6G seems to reside at the hydrophilic corona (PEO blocks) region of P123 micelle. In the gel phase, R6G may reside at the corona (PEO blocks) or in the “void” space between the micelles (Scheme 1B). Figure 1 shows the overlap between the absorption spectrum of the acceptor (R6G) and the emission spectrum of the donor (C480) in a P123 micelle and in a P123 gel at different excitation wavelengths (λex). The values of the spectral overlap integral, J(λ), between the emission spectrum of the donor (C480) and absorption spectrum of the acceptor (R6G) at different excitation wavelengths are listed in Table 1. Table 1 also lists Forster distance R0 for different λex. We have calculated the steadystate efficiency (s) of FRET using the relation, s) 1 - (IDA/ ID). IDA and ID are the steady-state emission intensity of the donor in the presence and absence of the acceptor, respectively.18,20 The efficiencies of FRET (s) at different excitation wavelengths are summarized in Table 1. In P123 micelle (or gel), at a short λex (375 nm) the donor (C480) molecules in the less polar region (PPO core) are preferentially excited. This results in a blue-shifted emission spectra. Excitation at the red end (λex ) 435 nm) selects the

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Figure 2. Emission spectra of 40 µM coumarin 480 in P123 micelle, λex at (A) 375 nm and (B) 435 nm, (i) in the absence and (ii) in the presence of 80 µM R6G.

TABLE 1: Energy Transfer Parameters for C480-R6G Pair in Different Systems

system P123 micelle (1 wt %) P123 gel (30 wt %) a

λex (nm)

J(λ)a (M-1 cm-1 nm4)

R0a (Å)

steady-state efficiency of FRET (sa)

375 405 435 375 405 435

1.22 × 1015 1.53 × 1015 2.44 × 1015 0.93 × 1015 1.22 × 1015 1.69 × 1015

49.3 51.3 55.3 42.6 44.6 47.1

0.33 0.37 0.58 0.05 0.13 0.20

Figure 3. Picosecond decays (λem ) 445 nm) of the donor (C480, 40 µM) with (i) 0 µM R6G and (ii) 80 µM R6G: in P123 micelle (A) and P123 gel (B), at λex ) 405 nm.

(10%.

donor molecules residing at a polar corona (PEO) region and gives rise to a red-shifted emission spectra. As a result, the magnitude of the spectral overlap, J(λ), and, hence, the rate of FRET may be tuned by varying λex. In P123 micelle, the magnitude of J(λ) for λex ) 435 nm is ∼2 times larger than that for λex ) 375 nm (Table 1). Hence, FRET is expected to be faster for λex ) 435 nm than that for λex ) 375 nm. Figure 2 shows the steady-state quenching of the donor (C480) emission in a P123 micelle at different λex. For λex ) 375 nm, because of smaller J(λ), the magnitude of quenching (∼1.5 times) is smaller compared to that (∼2.4 times) at λex ) 435 nm. The spectral overlap J(λ) in P123 gel is smaller than that in P123 micelle (Figure 1 and Table 1). Thus the extent of quenching is smaller in the gel phase compared to that in the micellar phase (Table 1). 3.2. Time-Resolved Studies of FRET from C480 to R6G in P123 Micelle and Gel. 3.2.1. Picosecond Studies. FRET is commonly monitored by shortening of the donor lifetime. Addition of the acceptor (R6G) causes significant shortening of the fluorescence lifetime of the donor (C480) in P123 micelle (Figure 3A). However, in P123 gel, no such shortening of donor lifetime is observed on addition of R6G (Figure 3B). The discrepancy between P123 micelle and P123 gel may be reconciled as follows. In P123 micelle (1 wt %) the concentration of the micelle (20 µM)20 is smaller compared to that of the donor (∼40 µM) or the acceptor (∼80 µM). In this case, most micelles contain both the donor and the acceptor and most donor molecules undergo FRET. Thus, in P123 micelle, the donor emission is dominated by the quenched donors with short donor lifetime. For the P123 gel, the concentration of micelles is ∼30 times higher (30 wt %, 600 µM). Consequently, in the gel, very few micelles have both donor and acceptor and in most micelles only the donor is present, which does not undergo FRET. In the gel, the donor emission is dominated by the unquenched

Figure 4. Picosecond transient (λem ) 570 nm) of the acceptor (R6G, 80 µM) in 1 wt % P123 (micelle) with (A) 40 µM C480, (B) 0 µM C480, (C) 40 µM C480, and (D) 0 µM C480. At λex ) 375 nm (A-B) and λex ) 435 nm (C-D). The residuals are shown at the bottom.

donors and no shortening of donor lifetime is detected in a picosecond setup. Though donor lifetime does not indicate any FRET, presence of FRET is manifested in the rise time of the acceptor (R6G) emission both in P123 micelle and in gel in picosecond and femtosecond setups (Figures 4, 5, and 6). We have thus studied the rate of FRET both by picosecond and by femtosecond setups. The picosecond fluorescence transients of the acceptor (R6G) in a P123 micelle and gel are shown in Figures 4 and 5, and the decay parameters are listed in Table 2. We monitored the fluorescence transient of the acceptor (R6G) at 570 nm where contribution of the quenched emission of the donor is negligible. In the absence of donor (C480), at all λex, the acceptor (R6G)

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J. Phys. Chem. B, Vol. 111, No. 25, 2007 7089 TABLE 2: Picosecond Decay Parameters of R6G (80 µM, λem)570 nm) in the Presence of C480 (40 µM) at Different λex medium P123 micelle (1 wt %) P123 gel (30 wt %) a

λex (nm) 375 405 435 375 405 435

τ1a (a1) (ps)

τ2a (a2) (ps)

τ3a (a3) (ps)

100 (-1.85) 100 (-0.68)

1700 (-3.72) 1700 (-1.6)

150 (-0.24) 150 (-0.07)

2600 (-0.81) 2600 (-0.43)

5800 (6.58) 5800 (3.28) 5530 (1.00) 6350 (2.05) 6600 (1.50) 5500 (1.00)

(10%.

TABLE 3: Femtosecond Decay Parameters of R6G (80 µM, λem ) 570 nm) in the Absence of C480 at Different λex medium

λex (nm)

τ1a (a1) (ps)

τ2a (a2) (ps)

P123 micelle (1 wt %)

375 405 435 375 405 435

20 (0.04) 120 (0.20) 130 (0.12) 120 (0.07) 120 (0.06) 130 (0.07)

4900 (0.96) 4900 (0.80) 5200 (0.88) 4400 (0.93) 4400 (0.94) 5100 (0.93)

P123 gel (30 wt %)

a

Figure 5. Picosecond transient (λem ) 570 nm) of the acceptor (R6G, 80 µM) in 30 wt % P123 (gel) with (A) 40 µM C480, (B) 0 µM C480, (C) 40 µM C480, and (D) 0 µM C480. At λex ) 375 nm (A-B) and λex ) 435 nm (C-D). The residuals are shown at the bottom.

Figure 6. Femtosecond transient (λem ) 570 nm) of the acceptor (R6G, 80 µM) in the presence of 40 µM C480 (donor), in P123 micelle (AB) and P123 gel (C-D). At λex (A) 375 nm, (B) 435 nm, (C) 375 nm and (D) 435 nm. The residuals are shown at the bottom.

exhibits a single-exponential decay with no rise component both in P123 micelle (Figure 4B and 4D) and in P123 gel (parts B and D of Figure 5). In the presence of the donor (C480), picosecond transients of the acceptor (R6G) at 570 nm shows distinct rise components at λex ) 375 and 405 nm in P123 micelle and gel (Table 2). The observed rise components are ascribed to FRET from C480 to R6G. For λex ) 375 and 405 nm, the rise components (i.e., FRET) are 100 ( 10 ps and 1700 ( 100 ps in P123 micelle (Figure 4A and Table 2). In the P123 gel, picosecond transient of the acceptor (R6G) exhibits two rise components (i.e., FRET) of 150 and 2600 ps for λex ) 375 and 405 nm (Figure 5A and Table 2). For both the micelle and gel, for a long wavelength of excitation (435 nm) we did not

(10%.

detect any rise in the acceptor emission in our picosecond setup (Figures 4C and 5C and Table 2). This indicates that for λex ) 435 nm, FRET is too fast to be detected in our picosecond setup (IRF ∼90 ps). 3.2.2. Femtosecond Study of the Ultrafast Component of the Acceptor Fluorescence. Figure 6 shows the femtosecond rise of the acceptor (R6G) at an emission wavelength of 570 nm in P123 micelle and P123 gel at different excitation wavelengths. For all λex, we detected an ultrafast rise component for both P123 micelle (∼2.5 ps rise) and P123 gel (∼3 ps rise), in addition to those detected using the picosecond setup. Note, in the absence of a donor, no rise component is observed in the acceptor emission (Table 3). As shown in Table 4, the relative contribution of the ultrafast rise component increases markedly with increase in λex. In the following section, we will discuss the origin of the ultrafast component of FRET and explain the λex dependence of the relative contribution in terms of the structural differences of the P123 micelle and P123 gel. Figure 7 shows shortening of donor emission decay on addition of the acceptor in P123 micelle in a femtosecond time scale (λex ) 405 nm). Note, shortening of donor lifetime and the appearance of growth in acceptor’s decay unambiguously confirms an ultrafast FRET from donor (C480) to an acceptor (R6G) in P123 micelle. 4. Discussion This work demonstrates that the time scales of FRET may be ascertained from the rise time of the acceptor emission. The most important finding of the present work is the multiple time scales of FRET and marked dependence of FRET on λex. In such a complex system (micelle or gel), there is obviously a distribution of donor-acceptor distances. In order to get a semiqualitative picture, we analyze the results in terms of a rather simplified Fo¨rster model with three different rates of FRET. In P123 micelle, the three FRET components are 2.5, 100, and 1700 ps. From eq 1, the fastest component (2.5 ps) correspond to a Fo¨rster distance of 13 ( 2 Å. From MM2 calculation the diameter of both the donor and the acceptor is ∼12 Å. The fastest component of FRET and the shortest donoracceptor distance (13 ( 2 Å) correspond to direct contact of

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Figure 7. Femtosecond transient (λem ) 445 nm) of 40 µM C480 (donor) in 1 wt % P123 (micelle), (A) in the absence and (B) in the presence of 80 µM R6G (λex ) 405 nm).

TABLE 4: Femtosecond Decay Parameters of R6G (80 µM, λem ) 570 nm) in the Presence of C480 (40 µM) at Different λex medium

λex (nm)

τ1a (a1) (ps)

τ2a (a2) (ps)

τ3a (a3) (ps)

τ4a (a4) (ps)

P123 micelle (1 wt %)

375 405 435 375 405 435

2.5 (-0.10, 13%) 2.5 (-0.10, 37%) 2.5 (-0.17, 100%) 3 (-0.03, 1%) 3 (-0.09, 15%) 3 (-0.10, 100%)

100 (-0.23, 29%) 100 (-0.05, 18%)

1700 (-0.45, 58%) 1700 (-0.12, 45%)

150 (-0.5, 23%) 150 (-0.07, 12%)

2600 (-1.69, 76%) 2600 (-0.43, 73%)

5800 (1.78) 5800 (1.27) 5530 (1.17) 6350 (3.22) 6600 (1.59) 5500 (1.10)

P123 gel (30 wt %)

a

(10%.

the donor and the acceptor at the highly hydrophilic peripheral region of the corona. The donor-acceptor distance (25 ( 2 Å) for the 100 ps component of FRET is smaller than the micellar radius (90 Å).5,6 This indicates intramicellar FRET from a donor molecule located deep inside the micelle and the cationic acceptor at the corona region of the same micelle. For the longest (1700 ps) component FRET in P123 micelle, one cannot rule out the role of diffusion. For an organic molecule in a micelle the translational diffusion D is of the order 0.550 Å2/ns.26-29 Since the cationic R6G has no tendency to move toward the hydrocarbon (PPO) region, diffusion of R6G along the donor-acceptor axis may be neglected. Because of diffusion of the donor molecule, the donor-acceptor distances change by (2Dτ)1/2 during the time scale of FRET (τ). The 1700 ps component corresponds to a maximum diffusion length ∼13 Å. Thus the 1700 ps component of FRET (rise of acceptor emission) may arise from diffusion of the donor from outside a P123 micelle to inside. This is followed by fast FRET at short distance (in 2.5 and 100 ps time scale). The effect of diffusion seems to be minor for 2.5 and 100 ps components of FRET time because in these time scales the maximum diffusion length (∼3.5 Å) is less than the radii (∼6 Å) of the donor or the acceptor molecule. In a P123 micelle, with increase in λex (375-435 nm), the contribution of the ultrafast rise (2.5 ps) component increases rapidly from 13% at λex ) 375 nm to 100% at λex ) 435 nm. At the same time, the contribution of the slow rise components (100 and 1700 ps) decreases and completely disappears at λex ) 435 nm. This can be explained as follows. At λex ) 435 nm, the donor molecules residing at the highly polar peripheral region of the corona are exclusively excited. As discussed earlier, the cationic acceptor R6G resides at the polar peripheral region of the corona. Thus for λex ) 435 nm, the excited donor and the acceptor molecules are in close proximity. This results in ultrafast FRET in a 2.5 ps time scale. Excitation at the blue end (λex ) 375 nm) selectively excites the donor (C480) molecules residing in the relatively less polar region deep inside the micelle (PPO core). In this case, the distance between the excited donor (in the core) and the acceptor in the corona is large leading to longer time scale of FRET

(100 ps). Hence, for λex ) 375 nm, FRET at longer distances dominates over that at a short distance. Excitation at an intermediate wavelength (λex ) 405 nm) excites all the regions, namely, the core (PPO) and the corona (PEO). Hence, we observe that energy transfer exhibits both an ultrafast, 2.5 ps, as well as comparatively slower components (100 and 1700 ps, Table 4). In P123 gel, there are three components of FRET: 3 ps, 150 ps, and a slow 2600 ps component. For the 3 and 150 ps components, the Fo¨rster distances are 13 ( 2 and 25 ( 2 Å. In analogy to the case of micelle, they are both assigned to intramicellar FRET. Similarly, a 2600 ps component of FRET in the gel phase is ascribed to diffusion of a donor from outside (“void”) the micelle to inside followed by fast FRET (in 3 and 150 ps time scales). Such diffusion in a gel is not surprising because even for cubic close packing the gels have a porous structure with 48% “void” space. For the gel, the contribution of the ultrafast component of FRET (3 ps) increases 100 times from 1% at λex ) 375 nm to 100% at λex ) 435 nm. 5. Conclusion This work shows that by varying λex one can study FRET in different regions of a P123 micelle and a P123 gel. For P123 micelle, there are three components of FRET: 2.5, 100, and 1700 ps. For P123 gel, there are three components: 3, 150, and 2600 ps. The ultrafast (2.5 and 3 ps) components of FRET are assigned to a donor and acceptor in direct contact at the highly polar peripheral region of the corona. The 100 and 150 ps components arise from FRET from a donor deep inside the micelle to an acceptor at the periphery of the micelle. The very long (1700 ps in micelle and 2600 ps in gel) component seems to arise from diffusion of the donor from bulk water or (“void” in gel) to interior of the P123 micellar aggregate followed by fast FRET. In both micelle and the gel, excitation at the red end (435 nm) causes exclusively ultrafast FRET between donor and acceptor at close contact. The results have implications in recent results on FRET at short distances.30-33 Acknowledgment. Thanks are due to Department of Science and Technology, India (Project Number: IR/I1/CF-01/2002) and

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