Article pubs.acs.org/JPCA
Optical Spectroscopy of Molecular-Rotor Molecules Adsorbed on Cellulose Ron Simkovitch and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel ABSTRACT: Steady-state and time-resolved emission techniques were used to study the fluorescence properties of two molecular rotors, thioflavin-T and auramine-O adsorbed on cellulose powder. Molecular rotors are known for their weak fluorescence intensity and short fluorescence lifetime when dissolved in liquids of low viscosity. We found that these molecular-rotor molecules when adsorbed on cellulose exhibit a rather strong steady-state fluorescence spectrum as well as long emission lifetime. We explain these results by the inhibition of segmental intramolecular rotation when these molecules are adsorbed on cellulose.
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INTRODUCTION Thioflavin-T (ThT) and auramine-O (AuO) shown in Scheme 1 belong to a class of molecules that are termed molecular rotors.
that the dependence of the emission lifetime on the viscosity of the dissolved liquid is nearly linear.9 When these molecules are adsorbed on bioorganic fibrils, they show strong fluorescence. ThT is therefore used as a fluorescent sensor for detection of amyloid fibrils. When they are adsorbed on fibrils, the fluorescence intensity increases by about 3 orders of magnitude compared to the very weak fluorescence intensity of ThT in water. Glasbeek and co-workers suggested a model that describes the nonradiative processes occurring in AuO.6 This model was successfully employed first for AuO and later also for ThT.15 The model accounts for the viscosity-dependent fluorescence quantum yield and emission lifetime. The model also explains the strong and intense fluorescence of adsorbed molecules for which segmental rotation is prevented in the excited state. In this model, the first molecular electronically excited singlet state is expressed as a mixture of two diabatic states, an emissive (F) state and a dark (D) state. Following a short excitation pulse, the initial population distribution resides first in the F state, and then diffuses by torsional motion that depends on solvent viscosity toward the D state. The two diabatic states, F and D, are adiabatically coupled as a function of the normalized twist coordinate z. The normalized S1 → S0 transition dipole moment decreases as a function of z and the emission rate falls by 2 orders of magnitude when the twist angle is 90°. The rotation time is controlled by friction with the solvent, which varies linearly with solvent viscosity.9 Cellulose shown in Scheme 2, is a polysaccharide (C6H10O5)n consisting of a long linear chain of several hundred up to more than ten thousand linked D-glucose units.17,18
Scheme 1. Molecular Structure of (a) Thioflavin-T (ThT) and (b) Auramine-O (AuO)
As seen in Scheme 1, molecular rotors are composed of several segments; the two ring segments can rotate with respect to the rest of the molecule and in doing so form a twisted molecular excited electronic state. This state has a charge transfer character that exhibits a rather weak fluorescence.1 The potential energy of both the ground state and the excited state are strongly dependent on the twist angle. The fluorescence of this kind of molecular rotors is dramatically quenched by the rotation of the segments with respect to each other.2,3 These molecules, when photoexcited to their lowest singlet state, show a rather weak fluorescence when dissolved in liquids of low viscosity. It has been shown that the emission lifetime of both AuO and ThT molecules linearly scale with the solvent viscosity.4−11 At low temperatures when these liquids are frozen or in solids at room temperature, they exhibit strong fluorescence. Time-resolved fluorescence studies have shown that the emission lifetime, τF, of both ThT1,8−16 and AuO,5−7 is about 1 ps in many regular solvents of rather low viscosity, like acetonitrile, water, methanol, and ethanol (ηethanol = 1.1 cPoise). In solids, or when adsorbed on insulin fibrils, the fluorescence lifetime is rather long (τF ∼ 2 ns for ThT1). It was also found © 2014 American Chemical Society
Received: July 15, 2014 Revised: August 28, 2014 Published: September 3, 2014 8737
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The fluorescence up-conversion technique was employed in this study to measure the time-resolved emission of curcumin at room temperature. The laser used for the fluorescence upconversion was a cavity-dumped Ti:sapphire femtosecond laser (Mira, Coherent), which provides short, 150 fs, pulses at about 800 nm. The cavity dumper operated with a relatively low repetition rate of 800 kHz. The up-conversion system (FOG100, CDP, Russia) operated at 800 kHz. The samples were excited by pulses of ∼8 mW on average at the SHG frequency. The time response of the up-conversion system is evaluated by measuring the relatively strong Raman−Stokes line of water shifted by 3600 cm−1. It was found that the fwhm of the signal is 300 fs. Samples were placed in a rotating optical cell to avoid degradation. Measurements of time-correlated single-photon counting (TCSPC) were performed using the same laser as a light source, and in the same setup. The TCSPC detection system was based on a Hamamatsu 3809U photomultiplier and an Edinburgh Instruments TCC 900 computer module for TCSPC. The overall instrument response was approximately 40 ps (fwhm) where the excitation-pulse energy was reduced to about 10 pJ by neutral-density filters. The steady-state emission and absorption spectra were recorded by a Horiba Jobin Yvon FluoroMax-3 spectrofluorometer and a Cary 5000 spectrometer.
Scheme 2. Molecular Structure of Cellulose
Cellulose is found in the cell wall of green plants and is the most abundant organic polymer on Earth.19 It consists of D-glucose units, which condense through β(1→4)-glycosidic bonds. In starch, the linkage between the D-glucose units is based on α(1→4)-glycosidic bonds. Unlike starch, cellulose is a straight-chain polymer with no coiling or branching. Many properties of cellulose depend on its chain length or degree of polymerization. Cellulose from wood pulp has typical chain lengths of between 300 and 1700 units. Cellulose consists of crystalline and amorphous regions. Costa and co-workers20 measured the fluorescence quantum yield ΦF of AuO adsorbed on cellulose and found a large increase in ΦF of AuO on cellulose. In the current study, we employ steady-state (timeintegrated) and time-resolved fluorescence techniques to study the fluorescence properties of both ThT and AuO adsorbed on cellulose powder. We found that the steady-state fluorescence intensity and the fluorescence lifetime of both ThT and AuO increase by about 3 orders of magnitude when adsorbed on cellulose powder. We explain these results by the Glasbeek model.6,7 When the molecular-rotor molecule is adsorbed on cellulose, the intramolecular twist rotation of the two rings is prevented as a result of the strong adsorption of the molecular-rotor rings to the surface of cellulose. Fluorescence quenching is therefore prevented because the molecule is in the planar conformation, for which the transition dipole moment, S1 → S0, is very large.
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RESULTS Figure 1 shows the absorption spectra of ThT and auramine-O (AuO) in ethanol solutions. The strong absorption band of both molecular rotors is in the blue region with a maximum at 420 and 430 nm for ThT and AuO, respectively. Both compounds have large transition dipole moments and their molar extinction coefficient, εmax, varies from 23 800 M−1 cm−1 (Sigma product information, ThT in ethanol) to 36 000 M−1 cm−1 in water for the recrystallized ThT.21,22 These molecules can be easily studied by optical spectroscopy with the use of steady-state and time-resolved techniques. For time-resolved emission, we used, for sample excitation, the second harmonic of a CW-mode locked Ti:sapphire laser at λ ≈ 400 nm. Figure 2 shows the steady-state (time-integrated) excitation and emission spectra of ThT (part a) and AuO (part b) in ethanol solution The emission bands of both ThT and AuO show maxima in ethanol and other polar liquids at about 490 and 510 nm,
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EXPERIMENTAL METHODS Cellulose powder of 20 μm particle size, auramine-O and thioflavin-T (ThT) were purchased from Sigma-Aldrich. All measurements were carried out with fresh solutions at the desired concentration and solvent. All chemicals used in this study were of analytical grade and were purchased from Aldrich and Merck.
Figure 1. UV−vis absorption spectrum of (a) ThT and (b) AuO in ethanol. 8738
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Figure 2. Excitation measured at 410 nm (blue) and emission (black) spectra of (a) ThT and (b) AuO, in ethanol solutions.
Figure 3. Steady-state emission spectra of (a) ThT on cellulose and (b) AuO on cellulose.
Figure 4. Fluorescence up-conversion signals of ThT in acetonitrile: (a) linear scale; (b) semilog scale.
identify β amyloid fibrils by the large increase of fluorescence when it is adsorbed on these fibrils. In the current study, we demonstrate that both ThT and AuO, when adsorbed on cellulose, show properties similar to those obtained when they are adsorbed on insulin fibrils. Their steady-state emission intensity, as well as their lifetimes, increase by a factor of 1000. Figure 3a shows the steady-state (time-integrated) emission of ThT adsorbed on cellulose. The emission spectrum is measured under front-surface conditions with a standard fluorometer (Fluoromax-3). The fluorescence spectrum position and band shape are similar to the spectrum obtained in solution for a polar solvent like ethanol. For comparison, see Figure 2a, which shows the steady-state emission of ThT in ethanol. The steady-state emission intensity of both ThT and AuO adsorbed on cellulose is about 100 times more intense than that in liquid ethanol solutions. The exact
respectively. The band is asymmetric with a red tail that stretches beyond 650 nm. The absorption spectrum differs significantly from the fluorescence excitation spectrum, because the lower value of the energy barrier between conformers suggests that a significant fraction of the ThT molecules with a violated system of the π-conjugated bonds already exists in the ground state. This explains the appearance of short-wavelength bands of fluorescence excitation and emission with maxima at 350 and 440 nm, respectively.23−25 Time-Resolved and Steady-State Emission. The fluorescence quantum yield of molecular-rotor molecules in solution is very low, ΦF ∼ 0.001 or less for solvents with a low viscosity of about 1 cPoise (cP). In previous studies1,26 it was found that the emission quantum yields and emission lifetimes of both ThT and AuO increase by about a factor of 1000 when adsorbed on biological fibrils. In fact, ThT is used to 8739
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fluorescence intensity ratio between the liquid and adsorbed samples cannot be derived because the fluorescence measurement of the molecules on cellulose are measured in frontsurface configuration on a large scattering medium of cellulose powder. Figure 3b shows the fluorescence spectra of AuO adsorbed on cellulose, for different excitation wavelengths. The spectrum position and shape are similar to those obtained for AuO dissolved in polar liquids. See, for example, the steady-state spectrum in ethanol shown in Figure 2b. Parts a and b of Figure 4 show, on linear and semilogarithmic scales, the fluorescence up-conversion signals of ThT in acetonitrile solution measured at several wavelengths in the visible spectral region, 460−600 nm. The sample was excited at 390 nm by 150 fs pulses at a rate of 760 kHz. The ThT sample was placed in a thin optical cell with a path length of 0.8 mm, which was rotated. Acetonitrile is a nonassociative polar liquid with a large dipole moment of 3.4 D and low viscosity of 0.37 cP at room temperature. As seen in the figure, the fluorescence up-conversion decay is nonexponential at all wavelengths. The longer the monitored wavelength, the longer the average decay time (τav = ∫ IF(λ,t) dt), where IF(λ,t) is the normalized time-dependent fluorescence signal. The important point we wish to emphasize is that the decay time is short, about 2 ps at λ ≥ 540 nm. This short average decay time is explained by a rapid nonradiative decay of molecular rotor molecules. Table 1 provides the values of τav of ThT and AuO in acetonitrile at several wavelengths. The average lifetime increases as the wavelength increases. Comparing the values of τav at λ = 480 nm with that measured
at 600 nm shows an increase by a factor of about 2.5 for both compounds. Parts a and b of Figure 5 show, on a linear and semilogarithmic scale, the time-resolved fluorescence of ThT adsorbed on a cellulose powder of 20 μm average size. The fluorescence is collected by the front-surface method. The powder is fixed on a quartz plate by a small drop of glycerol. The time-resolved emission is measured by the timecorrelated single-photon-counting technique (TCSPC) with a rather limited instrument-response time of about 40 ps full width at half-maximum (fwhm). The signal decay is bimodal. A short decay-time component with an amplitude of about 0.3 and a time constant of τ ≤ 150 ps is followed by a long decaytime component with an amplitude of ∼0.7 and nearly exponential decay with a time constant of ∼2.1 ns. On the basis of the Strickler−Berg27 equation, the radiative decay time is estimated to be ∼4.5 ns. Figure 5b shows a fit to a biexponential function. The fitting parameters are given in Table 2. Table 3 shows the values of τav of ThT adsorbed on cellulose. The short decay-time component is attributed to the ThT signal that arises from molecules that are not fully adsorbed on the cellulose surface and thus the nonradiative decay is large Table 2. Two Exponent-Fitting Parameters of TCSPC Signals of ThT Adsorbed on Cellulosea
Table 1. τav of ThT and AuO in Acetonitrilea
a
τav [fs]
a
λ [nm]
ThT in acetonitrile
AuO in acetonitrile
460 480 500 520 540 560 580 600
440 830 850 980 1170 1300 1460 1980
260 340 390 500 540 660 770 910
λ [nm]
a1
τ1 [ns]
a2
τ2 [ns]
α2
460 480 500 520 560
0.34 0.26 0.26 0.28 0.38
0.16 0.13 0.13 0.13 0.10
0.66 0.74 0.74 0.72 0.62
1.8 2.10 2.14 2.14 2.14
0.92 0.95 0.95 0.95 0.95
IF(t) = a1 exp[−t/τ1 + a2 exp[−(t/τ2)α].
Table 3. τav of ThT Adsorbed on Cellulosea τav [ns]
τav = ∫ IF(t) dt.
a
λ [nm]
ThT on cellulose
cellulose only (background)
460 480 500 520 560
1.50 1.80 1.86 1.87 1.61
1.75 1.95 2.02
τav = ∫ IF(t) dt.
Figure 5. TCSPC signals of ThT on cellulose: (a) normalized linear scale; (b) normalized semilog scale. 8740
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Figure 6. Fluorescence up-conversion signals of AuO in acetonitrile: (a) linear scale; (b) semilog scale.
Figure 7. TCSPC signals of AuO on cellulose: (a) normalized linear scale; (b) normalized semilog scale.
For the best fit we used a stretched exponential factor of α = 0.58 ± 0.03. The convoluted fit of the system response is also shown in part b of Figure 7. The instrument response function is also shown in part a of Figure 7. In the discussion section we suggest possible mechanisms for the large nonexponential nature of the AuO signal on cellulose. Table 4 provides the
because the intramolecular rotation of the benzothiazole ring with respect to the aniline ring is only partially hindered. The major portion of the time-resolved fluorescence signal arises from the contribution of ThT molecules for which the intramolecular rotation is prevented. We find a small wavelength dependence of the relative amplitude between the short and the long decay-time components. The shorter the wavelength, the larger the amplitude of the short decay time. Figure 6 shows the fluorescence up-conversion signals of AuO in acetonitrile measured at several wavelengths in the spectral region of 460−600 nm. The average decay time depends on the monitored wavelength; the longer the wavelength, the longer the average decay time. As in the case of the fluorescence decay of ThT in acetonitrile, the decay of the AuO fluorescence at all wavelengths is nonexponential and the major part of the decay could be fitted by a stretched exponent, exp[−(t/τ)α], with 0.55 < α < 0.7. When comparing the fluorescence decay of AuO in acetonitrile with the decay of ThT, we find that the average decay time of AuO is shorter by about a factor of 2. This is also true in solvents other than acetonitrile. Table 1 provides the τav values of both ThT and AuO in acetonitrile. Figure 7 shows the time-resolved emission measured by the TCSPC technique of auramine-O adsorbed on cellulose powder of 20 μm average size. The signal is nonexponential at both short and long times. We were able to fit a major part of the signal amplitude, a = 0.85, by a single stretched exponential function:
Table 4. Fitting Parameters of TCSPC Signals of AuO on Cellulosea
a
λ [nm]
a1
τ1 [ns]
a2
τ2 [ns]
α2
500 520 540
0.17 0.17 0.17
0.08 0.08 0.08
0.83 0.83 0.83
0.34 0.44 0.48
0.58 0.60 0.61
IF(t) = a1 exp[−t/τ1 + a2 exp[−(t/τ2)α].
fitting parameters of the time-resolved emission of AuO adsorbed on cellulose. Table 5 provides the values of τav of AuO adsorbed on cellulose. Self-Fluorescence of Cellulose. When irradiated by UV light, cellulose shows broad emission in the visible part of the optical spectrum. Parts a and b of Figure 8 show, on a semilogarithmic scale, the signals of ThT (Figure 8a) and AuO (Figure 8b) adsorbed on cellulose, measured at 460 nm along with cellulose (background) time-resolved emission signals. As seen in the figure, the fluorescence signal of cellulose powder is nonexponential with an average decay time, τav = ∫ IF(λ,t) dt, of about 2 ns. This average decay time is on the order of τav of ThT adsorbed on the cellulose. Fortunately, the
IF(λ ,t ) = exp[−(t /τ )α ] 8741
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Table 5. τav of AuO Adsorbed on Cellulosea
a
λ [nm]
AuO on cellulose τav [ns]
500 520
0.62 0.79
When they are adsorbed on surfaces of biomaterials, their fluorescence quantum yield increases by at least 2 orders of magnitude20 and the emission lifetime increases by the same factor. The Glasbeek model6,7 takes into account the coupling of two close-lying excited states that exist in this class of molecules. Because of the coupling, the first molecular electronically excited singlet state is expressed as a mixture of two separate states, an emissive (F) state and a dark (D) state. The two diabatic states, F and D, are adiabatically coupled as a function of the normalized twist coordinate z:
τav = ∫ IF(t) dt.
fluorescence intensity of the cellulose is only 1/20th of that of the ThT or of the AuO. This rather low background intensity has very little effect on the short- and long-time signals of both ThT and AuO. The average decay time of both the weak background signal of the cellulose and the long-time components of the signals of AuO and ThT are nearly the same. Therefore, in the data analysis, we ignore the contribution of the weak (1/20) background fluorescence of the cellulose. Main Findings. 1. The steady-state fluorescence intensity of both ThT and AuO molecular rotors adsorbed on cellulose increases by more than a factor of 100 when compared with the fluorescence of these molecules in liquid solutions of low viscosity like acetonitrile, water and linear alcohols (methanol and ethanol). 2. The average fluorescence decay time, τav, of both molecular rotor molecules increases by more than a factor of 100. 3. The time-resolved fluorescence decay of ThT adsorbed on cellulose consists of two time components. The short decay-time component is less than 150 ps and contains less than 30% of the total fluorescence-signal amplitude. The longer decay-time component is nearly exponential and the decay time is about 2.0 ± 0.15 ns depending on the monitored wavelength. 4. The time-resolved emission of auramine-O adsorbed on cellulose is nonexponential at all wavelengths. The major part of the decay curve could be fitted by a stretched exponent function IF(λ,t) = exp[−(t/τ)α] with α = 0.58 ± 0.03. 5. We assign the large increase in the fluorescence intensity and fluorescence decay time of both molecules adsorbed on cellulose to the inhibition of intramolecular rotation of the two rotors composing these molecules and thus lowering the high nonradiative rate by more than 2 orders of magnitude.
S1(z) =
1 1 [F(z) + D(z)] − [F(z) − D(z)]2 + 4C 2 2 2 (1)
where C is the coupling-strength parameter. Such adiabatic coupling was earlier used by Fonseca, Barbara, and coworkers28,29 to model adiabatic excited-state intramolecular electron-transfer processes, which depend on the generalized solvent-reorganization coordinate, S. A short excitation pulse brings some of the population of the ground-state equilibrium configuration, which is nearly planar, to the F state. It then diffuses by torsional motion on the excited-state potential curve toward the minimum potential which assumes D-state character and is at the 90° configuration. Because the S1 state (of both auramine-O and ThT) is a z-dependent mixture of radiative and nonradiative zero-order states, the transition dipole moment, M(z), of the optical transition S1 → S0 is also z-dependent. Accordingly, the normalized S1 → S0 transition dipole moment decreases as a function of z: ⎡1 ⎛ ⎞⎤ 2C M(z) = cos2⎢ arctan⎜ ⎟⎥ ⎝ F(z) − D(z) ⎠⎦ ⎣2
(2)
The Glasbeek model was also used by Meech and coworkers5 to quantify the torsional dynamics of auramine-O. The change in the transition dipole moment of ThT as a function of the twist angle, z, was also studied with the use of quantum-molecular calculations.4,9,12 It was shown that the S1 → S0 transition dipole moment decreases by a factor of ∼100 as a function of the C−C dihedral angle. Recently,30 ThT ground- and excited-state dynamics was simulated with the use of TDDFT. It was also found that the change in the twist angle is responsible for the large decrease in the transition dipole
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DISCUSSION Glasbeek Model. The fluorescence properties of molecular rotor molecules are sensitive to the viscosity of the liquid.
Figure 8. TCSPC signals shown on a semilog scale of (a) ThT adsorbed on cellulose and (b) AuO adsorbed on cellulose and neat cellulose signal in each case. 8742
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moment and hence the large change in the fluorescence intensity and the short lifetime of ThT. Inhomogeneous Nonradiative Model. The timeresolved emission of AuO, measured by the time-correlated single-photon-counting technique is nonexponential. It can be fitted by a nonexponential function called the stretched exponent, exp[−(t/τ)α]. α is a positive number, 0 < α ≤ 1, and it determines the deviation of the signal from true exponential decay. When α equals 1, the decay is exponential. In Figure 7 we show the time-resolved fluorescence signal of AuO adsorbed on cellulose and also the fit to a stretched exponential with α ≈ 0.58. In this subsection we use an inhomogeneous nonradiative model that provides a plausible explanation for the origin of the nonexponential decay of the AuO fluorescence. The inhomogeneous model assumes a single floppy coordinate, x, that governs the conformational state of the system. We consider a barrierless continuous crossing from the LE fluorescent state to the excited charge-transfer dark state. The Boltzmann equilibrium distribution, for a harmonic groundstate potential, V0(x), prior to excitation is given by p(x) = Z exp[−V0(x)/kBT ] = Z exp[−(x − x0)2 ]
Figure 9. TCSPC signals shown on a semilog scale of AuO adsorbed on cellulose and inhomogeneous model-fitting functions.
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SUMMARY AND CONCLUSIONS We used steady-state (time-integrated) and time-resolved fluorescence techniques to study the fluorescence properties of two molecular-rotor molecules adsorbed on cellulose. In previous studies, we studied the optical properties of thioflavin-T (ThT) and auramine-O (AuO), shown in Scheme 1, dissolved in polar and protic liquids. These two molecules belong to a class of molecules that are termed molecular rotors. Their structure consists of two or more aromatic or heterocyclic rings with freedom to rotate in the excited state. When the rings are close to a planar configuration, the excited-state-transition dipole is large and therefore the absorption cross section, as well as the emission intensity, is large. Rotation of a ring segment to a 90° configuration in the excited state, leads to a transition of the strong emissive state to a charge-transfer dark excited state. The excited-state potential as a function of the twist angle has a minimum at the 90° configuration. Thus, molecular-rotor molecules in the excited state tend to have a short emission lifetime of a few picoseconds for liquids with low viscosities. It was found, more than half a century ago, that ThT, when adsorbed on β-amyloid fibrils, shows rather strong fluorescence and therefore has served for many years as a sensor for the detection of amyloid fibrils. In recent studies we found that the emission lifetime of both ThT and AuO adsorbed on insulin fibrils increases from few picoseconds in water to about 2 ns when adsorbed on these fibrils.1,26 In the current study, we measured the time-resolved emission of ThT and AuO adsorbed on cellulose. Cellulose is a biopolymer composed of D-glucose monomeric units. We found that the emission lifetime of ThT adsorbed on cellulose powder shows fluorescence behavior similar to that observed when it is adsorbed on insulin fibrils. The main portion of the fluorescence intensity decays nearly exponentially with a lifetime of ∼2 ns, approximately the same decay time of ThT adsorbed on insulin fibrils. We explain the long lifetime and intense fluorescence of ThT on cellulose in terms similar to those used in our previous study of ThT on insulin fibrils. The ring segments of adsorbed ThT molecules cannot rotate to form the dark charge-transfer state. Thus, the excited ThT retains its planar configuration during most of its radiative lifetime, which is estimated to be ∼4 ns. In AuO adsorbed on cellulose, the major portion of the fluorescence decay is nonexponential with an average decay time of about 0.8 ns. The fluorescence band of AuO on cellulose has similar band-peak positions and band shape as AuO in polar liquids. These steady-state emission results differ
(3)
Z is the Gaussian normalization constant, x0 is the ground-stateequilibrium twist angle, and x is the generalized twist angle. This inhomogeneous distribution for the slow (in this case, frozen) coordinate is thus time independent. The nonradiative process of adsorbed AuO on cellulose is inhomogeneous; thus instead of dealing with a single nonradiative rate constant, knr, each twist angle conformation, x, converts to the dark state D with a rate constant k(x) that depends on x. We assume that k(x) depends exponentially on the twist angle, x. k(x) = A exp[−b(x − x0)]
(4)
Here A is the preexponential factor and gives the value of knr at x0, and b is the exponential factor and a measure for the nonradiative rate sensitivity to the twist angle. In the static limit, the fluorescence follows the population probability P(t) that the locally excited (LE) state has not decayed by time t after excitation. This is given by p(t ) = exp( −t /τf )
∫0
∞
p(x) exp[−k(x)t ] dx
(5)
The first exponential accounts for the homogeneous radiative-decay process of the LE(F) state, whereas the integral of the second exponential represents the inhomogeneous nonradiative-decay kinetics, which is nonexponential. The solid lines in Figure 9 are the fit of the time-resolved emission data, measured at 500 and 540 nm, of AuO adsorbed on cellulose, obtained with the use of the inhomogeneous kinetic model presented above. As can be seen in Figure 9, the inhomogeneous model provides a good fit to the experimental time-resolved emission of AuO adsorbed on cellulose. For convenience, in eqs 4 and 5, we used dimensionless parameters rather than the twist angles for x and x0, where b = 1. The nonradiative rate at the groundstate twist angle x0 is A, the preexponential factor of eq 4. For the best fit we find A = 3.5 × 109 s−1 for the time-resolved signal measured at 500 nm, whereas for the 540 nm signal, A = 1.85 × 109 s−1. The radiative lifetime τF is assumed to be τF = 3 ns, krad = 0.33 × 109 s−1. 8743
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(16) Hutter, T.; Amdursky, N.; Gepshtein, R.; Elliott, S. R.; Huppert, D. Study of Thioflavin-T Immobilized in Porous Silicon and the Effect of Different Organic Vapours on the Fluorescence Lifetime. Langmuir 2011, 27, 7587−7594. (17) Crawford, R. L. In Lignin biodegradation and transformation; Wiley: New York, 1981. (18) Updegraff, D. M. Semimicro Determination of Cellulose Inbiological Materials. Anal. Biochem. 1969, 32, 420−424. (19) Klemm, D.; Heublein, B.; Fink, H.; Bohn, A. Cellulose: Fascinating Biopolymer and Sustainable Raw Material. Angew. Chem., Int. Ed. Engl. 2005, 44, 3358−3393. (20) Ferreira, L. F. V.; Garcia, A. R.; Freixo, M. R.; Costa, S. M. Photochemistry on Surfaces: Solvent−Matrix Effect on the Swelling of Cellulose. an Emission and Absorption Study of Adsorbed Auramine O. J. Chem. Soc., Faraday Trans. 1993, 89, 1937−1944. (21) Groenning, M. Binding Mode of Thioflavin T and Other Molecular Probes in the Context of Amyloid fibrilsCurrent Status. J. Chem. Biol. 2010, 3, 1−18. (22) De Ferrari, G. V.; Mallender, W. D.; Inestrosa, N. C.; Rosenberry, T. L. Thioflavin T is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site that Reveals Conformational Interactions between the Peripheral and Acylation Sites. J. Biol. Chem. 2001, 276, 23282−23287. (23) Kuznetsova, I. M.; Povarova, O.; Stepanenko, O.; Turoverov, K. K.; Crescenzo, R.; Varriale, A.; Staiano, M.; D’Auria, S. New perspectives in protein-based biosensors: the glucokinase f rom B. stearothermophilus and the odorant-binding protein f rom C. familiaris as probes for nonconsuming analyte sensors. Proc. SPIE 2007, 6733, 673318.1−673318.6. (24) Maskevich, A. A.; Stsiapura, V. I.; Kuzmitsky, V. A.; Kuznetsova, I. M.; Povarova, O. I.; Uversky, V. N.; Turoverov, K. K. Spectral Properties of Thioflavin T in Solvents with Different Dielectric Properties and in a Fibril-Incorporated Form. J. Proteome Res. 2007, 6, 1392−1401. (25) Voropai, E.; Samtsov, M.; Kaplevskii, K.; Maskevich, A.; Stepuro, V.; Povarova, O.; Kuznetsova, I.; Turoverov, K.; Fink, A.; Uverskii, V. Spectral Properties of Thioflavin T and its Complexes with Amyloid Fibrils. J. Appl. Spectrosc. 2003, 70, 868−874. (26) Amdursky, N.; Huppert, D. Auramine-O as a Fluorescence Marker for the Detection of Amyloid Fibrils. J. Phys. Chem. B 2012, 116, 13389−13395. (27) Strickler, S.; Berg, R. A. Relationship between Absorption Intensity and Fluorescence Lifetime of Molecules. J. Chem. Phys. 1962, 37, 814−822. (28) Tominaga, K.; Walker, G. C.; Jarzeba, W.; Barbara, P. F. Ultrafast Charge Separation in ADMA: Experiment, Simulation, and Theoretical Issues. J. Phys. Chem. 1991, 95, 10475−10485. (29) Tominaga, K.; Walker, G. C.; Kang, T. J.; Barbara, P. F.; Fonseca, T. Reaction Rates in the Phenomenological Adiabatic Excited-State Electron-Transfer Theory. J. Phys. Chem. 1991, 95, 10485−10492. (30) Ren, H.; Fingerhut, B. P.; Mukamel, S. Time Resolved Photoelectron Spectroscopy of Thioflavin T Photoisomerization: A Simulation Study. J. Phys. Chem. A 2013, 117, 6096−6104.
from what was found previously for AuO adsorbed on insulin fibrils immersed in water, where the spectrum consists of two emission bands. We assigned26 the two emission bands to a protonated and deprotonated form of AuO.
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AUTHOR INFORMATION
Corresponding Author
*Dan Huppert. E-mail: [email protected]. Phone: 972-36407012. Fax: 972-3-6407491. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Professor R. Lamed for helpful discussions. This work was supported by grants from the James-Franck GermanIsraeli Program in Laser-Matter Interaction and by the Israel Science Foundation.
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REFERENCES
(1) Amdursky, N.; Erez, Y.; Huppert, D. Molecular Rotors: What Lies Behind the High Sensitivity of the Thioflavin-T Fluorescent Marker. Acc. Chem. Res. 2012, 45, 1548−1557. (2) Haidekker, M. A.; Theodorakis, E. A. Environment-Sensitive Behavior of Fluorescent Molecular Rotors. J. Biol. Eng. 2010, 4, 11−24. (3) Haidekker, M. A.; Theodorakis, E. A. Molecular Rotors Fluorescent Biosensors for Viscosity and Flow. Org. Biomol. Chem. 2007, 5, 1669−1678. (4) Stsiapura, V. I.; Maskevich, A. A.; Kuzmitsky, V. A.; Turoverov, K. K.; Kuznetsova, I. M. Computational Study of Thioflavin T Torsional Relaxation in the Excited State. J. Phys. Chem. A 2007, 111, 4829− 4835. (5) Heisler, I. A.; Kondo, M.; Meech, S. R. Reactive Dynamics in Confined Liquids: Ultrafast Torsional Dynamics of Auramine O in Nanoconfined Water in Aerosol OT Reverse Micelles. J. Phys. Chem. B 2009, 113, 1623−1631. (6) Van der Meer, M.; Zhang, H.; Glasbeek, M. Femtosecond Fluorescence Upconversion Studies of Barrierless Bond Twisting of Auramine in Solution. J. Chem. Phys. 2000, 112, 2878−2887. (7) Glasbeek, M.; Zhang, H. Femtosecond Studies of Solvation and Intramolecular Configurational Dynamics of Fluorophores in Liquid Solution. Chem. Rev. 2004, 104, 1929−1954. (8) Stsiapura, V. I.; Maskevich, A. A.; Kuzmitsky, V. A.; Uversky, V. N.; Kuznetsova, I. M.; Turoverov, K. K. Thioflavin T as a Molecular Rotor: Fluorescent Properties of Thioflavin T in Solvents with Different Viscosity. J. Phys. Chem. B 2008, 112, 15893−15902. (9) Singh, P. K.; Kumbhakar, M.; Pal, H.; Nath, S. Viscosity Effect on the Ultrafast Bond Twisting Dynamics in an Amyloid Fibril Sensor: Thioflavin-T. J. Phys. Chem. B 2010, 114, 5920−5927. (10) Singh, P. K.; Kumbhakar, M.; Pal, H.; Nath, S. Ultrafast Bond Twisting Dynamics in Amyloid Fibril Sensor. J. Phys. Chem. B 2010, 114, 2541−2546. (11) Singh, P. K.; Kumbhakar, M.; Pal, H.; Nath, S. Ultrafast Torsional Dynamics of Protein Binding Dye Thioflavin-T in Nanoconfined Water Pool. J. Phys. Chem. B 2009, 113, 8532−8538. (12) Naik, L.; Naik, A. B.; Pal, H. Steady-State and Time-Resolved Emission Studies of Thioflavin-T. J. Photochem. Photobiol., A 2009, 204, 161−167. (13) Amdursky, N.; Gepshtein, R.; Erez, Y.; Huppert, D. Temperature Dependence of the Fluorescence Properties of Thioflavin-T in Propanol, a Glass-Forming Liquid. J. Phys. Chem. A 2011, 115, 2540− 2548. (14) Amdursky, N.; Gepshtein, R.; Erez, Y.; Koifman, N.; Huppert, D. Pressure Effect on the Nonradiative Process of Thioflavin-T. J. Phys. Chem. A 2011, 115, 6481−6487. (15) Erez, Y.; Liu, Y.; Amdursky, N.; Huppert, D. Modeling the Nonradiative Decay Rate of Electronically Excited Thioflavin T. J. Phys. Chem. A 2011, 115, 8479−8487. 8744
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Optical Spectroscopy of Molecular-Rotor Molecules Adsorbed on Cellulose Ron Simkovitch and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel ABSTRACT: Steady-state and time-resolved emission techniques were used to study the fluorescence properties of two molecular rotors, thioflavin-T and auramine-O adsorbed on cellulose powder. Molecular rotors are known for their weak fluorescence intensity and short fluorescence lifetime when dissolved in liquids of low viscosity. We found that these molecular-rotor molecules when adsorbed on cellulose exhibit a rather strong steady-state fluorescence spectrum as well as long emission lifetime. We explain these results by the inhibition of segmental intramolecular rotation when these molecules are adsorbed on cellulose.
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INTRODUCTION Thioflavin-T (ThT) and auramine-O (AuO) shown in Scheme 1 belong to a class of molecules that are termed molecular rotors.
that the dependence of the emission lifetime on the viscosity of the dissolved liquid is nearly linear.9 When these molecules are adsorbed on bioorganic fibrils, they show strong fluorescence. ThT is therefore used as a fluorescent sensor for detection of amyloid fibrils. When they are adsorbed on fibrils, the fluorescence intensity increases by about 3 orders of magnitude compared to the very weak fluorescence intensity of ThT in water. Glasbeek and co-workers suggested a model that describes the nonradiative processes occurring in AuO.6 This model was successfully employed first for AuO and later also for ThT.15 The model accounts for the viscosity-dependent fluorescence quantum yield and emission lifetime. The model also explains the strong and intense fluorescence of adsorbed molecules for which segmental rotation is prevented in the excited state. In this model, the first molecular electronically excited singlet state is expressed as a mixture of two diabatic states, an emissive (F) state and a dark (D) state. Following a short excitation pulse, the initial population distribution resides first in the F state, and then diffuses by torsional motion that depends on solvent viscosity toward the D state. The two diabatic states, F and D, are adiabatically coupled as a function of the normalized twist coordinate z. The normalized S1 → S0 transition dipole moment decreases as a function of z and the emission rate falls by 2 orders of magnitude when the twist angle is 90°. The rotation time is controlled by friction with the solvent, which varies linearly with solvent viscosity.9 Cellulose shown in Scheme 2, is a polysaccharide (C6H10O5)n consisting of a long linear chain of several hundred up to more than ten thousand linked D-glucose units.17,18
Scheme 1. Molecular Structure of (a) Thioflavin-T (ThT) and (b) Auramine-O (AuO)
As seen in Scheme 1, molecular rotors are composed of several segments; the two ring segments can rotate with respect to the rest of the molecule and in doing so form a twisted molecular excited electronic state. This state has a charge transfer character that exhibits a rather weak fluorescence.1 The potential energy of both the ground state and the excited state are strongly dependent on the twist angle. The fluorescence of this kind of molecular rotors is dramatically quenched by the rotation of the segments with respect to each other.2,3 These molecules, when photoexcited to their lowest singlet state, show a rather weak fluorescence when dissolved in liquids of low viscosity. It has been shown that the emission lifetime of both AuO and ThT molecules linearly scale with the solvent viscosity.4−11 At low temperatures when these liquids are frozen or in solids at room temperature, they exhibit strong fluorescence. Time-resolved fluorescence studies have shown that the emission lifetime, τF, of both ThT1,8−16 and AuO,5−7 is about 1 ps in many regular solvents of rather low viscosity, like acetonitrile, water, methanol, and ethanol (ηethanol = 1.1 cPoise). In solids, or when adsorbed on insulin fibrils, the fluorescence lifetime is rather long (τF ∼ 2 ns for ThT1). It was also found © 2014 American Chemical Society
Received: July 15, 2014 Revised: August 28, 2014 Published: September 3, 2014 8737
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The fluorescence up-conversion technique was employed in this study to measure the time-resolved emission of curcumin at room temperature. The laser used for the fluorescence upconversion was a cavity-dumped Ti:sapphire femtosecond laser (Mira, Coherent), which provides short, 150 fs, pulses at about 800 nm. The cavity dumper operated with a relatively low repetition rate of 800 kHz. The up-conversion system (FOG100, CDP, Russia) operated at 800 kHz. The samples were excited by pulses of ∼8 mW on average at the SHG frequency. The time response of the up-conversion system is evaluated by measuring the relatively strong Raman−Stokes line of water shifted by 3600 cm−1. It was found that the fwhm of the signal is 300 fs. Samples were placed in a rotating optical cell to avoid degradation. Measurements of time-correlated single-photon counting (TCSPC) were performed using the same laser as a light source, and in the same setup. The TCSPC detection system was based on a Hamamatsu 3809U photomultiplier and an Edinburgh Instruments TCC 900 computer module for TCSPC. The overall instrument response was approximately 40 ps (fwhm) where the excitation-pulse energy was reduced to about 10 pJ by neutral-density filters. The steady-state emission and absorption spectra were recorded by a Horiba Jobin Yvon FluoroMax-3 spectrofluorometer and a Cary 5000 spectrometer.
Scheme 2. Molecular Structure of Cellulose
Cellulose is found in the cell wall of green plants and is the most abundant organic polymer on Earth.19 It consists of D-glucose units, which condense through β(1→4)-glycosidic bonds. In starch, the linkage between the D-glucose units is based on α(1→4)-glycosidic bonds. Unlike starch, cellulose is a straight-chain polymer with no coiling or branching. Many properties of cellulose depend on its chain length or degree of polymerization. Cellulose from wood pulp has typical chain lengths of between 300 and 1700 units. Cellulose consists of crystalline and amorphous regions. Costa and co-workers20 measured the fluorescence quantum yield ΦF of AuO adsorbed on cellulose and found a large increase in ΦF of AuO on cellulose. In the current study, we employ steady-state (timeintegrated) and time-resolved fluorescence techniques to study the fluorescence properties of both ThT and AuO adsorbed on cellulose powder. We found that the steady-state fluorescence intensity and the fluorescence lifetime of both ThT and AuO increase by about 3 orders of magnitude when adsorbed on cellulose powder. We explain these results by the Glasbeek model.6,7 When the molecular-rotor molecule is adsorbed on cellulose, the intramolecular twist rotation of the two rings is prevented as a result of the strong adsorption of the molecular-rotor rings to the surface of cellulose. Fluorescence quenching is therefore prevented because the molecule is in the planar conformation, for which the transition dipole moment, S1 → S0, is very large.
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RESULTS Figure 1 shows the absorption spectra of ThT and auramine-O (AuO) in ethanol solutions. The strong absorption band of both molecular rotors is in the blue region with a maximum at 420 and 430 nm for ThT and AuO, respectively. Both compounds have large transition dipole moments and their molar extinction coefficient, εmax, varies from 23 800 M−1 cm−1 (Sigma product information, ThT in ethanol) to 36 000 M−1 cm−1 in water for the recrystallized ThT.21,22 These molecules can be easily studied by optical spectroscopy with the use of steady-state and time-resolved techniques. For time-resolved emission, we used, for sample excitation, the second harmonic of a CW-mode locked Ti:sapphire laser at λ ≈ 400 nm. Figure 2 shows the steady-state (time-integrated) excitation and emission spectra of ThT (part a) and AuO (part b) in ethanol solution The emission bands of both ThT and AuO show maxima in ethanol and other polar liquids at about 490 and 510 nm,
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EXPERIMENTAL METHODS Cellulose powder of 20 μm particle size, auramine-O and thioflavin-T (ThT) were purchased from Sigma-Aldrich. All measurements were carried out with fresh solutions at the desired concentration and solvent. All chemicals used in this study were of analytical grade and were purchased from Aldrich and Merck.
Figure 1. UV−vis absorption spectrum of (a) ThT and (b) AuO in ethanol. 8738
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Figure 2. Excitation measured at 410 nm (blue) and emission (black) spectra of (a) ThT and (b) AuO, in ethanol solutions.
Figure 3. Steady-state emission spectra of (a) ThT on cellulose and (b) AuO on cellulose.
Figure 4. Fluorescence up-conversion signals of ThT in acetonitrile: (a) linear scale; (b) semilog scale.
identify β amyloid fibrils by the large increase of fluorescence when it is adsorbed on these fibrils. In the current study, we demonstrate that both ThT and AuO, when adsorbed on cellulose, show properties similar to those obtained when they are adsorbed on insulin fibrils. Their steady-state emission intensity, as well as their lifetimes, increase by a factor of 1000. Figure 3a shows the steady-state (time-integrated) emission of ThT adsorbed on cellulose. The emission spectrum is measured under front-surface conditions with a standard fluorometer (Fluoromax-3). The fluorescence spectrum position and band shape are similar to the spectrum obtained in solution for a polar solvent like ethanol. For comparison, see Figure 2a, which shows the steady-state emission of ThT in ethanol. The steady-state emission intensity of both ThT and AuO adsorbed on cellulose is about 100 times more intense than that in liquid ethanol solutions. The exact
respectively. The band is asymmetric with a red tail that stretches beyond 650 nm. The absorption spectrum differs significantly from the fluorescence excitation spectrum, because the lower value of the energy barrier between conformers suggests that a significant fraction of the ThT molecules with a violated system of the π-conjugated bonds already exists in the ground state. This explains the appearance of short-wavelength bands of fluorescence excitation and emission with maxima at 350 and 440 nm, respectively.23−25 Time-Resolved and Steady-State Emission. The fluorescence quantum yield of molecular-rotor molecules in solution is very low, ΦF ∼ 0.001 or less for solvents with a low viscosity of about 1 cPoise (cP). In previous studies1,26 it was found that the emission quantum yields and emission lifetimes of both ThT and AuO increase by about a factor of 1000 when adsorbed on biological fibrils. In fact, ThT is used to 8739
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fluorescence intensity ratio between the liquid and adsorbed samples cannot be derived because the fluorescence measurement of the molecules on cellulose are measured in frontsurface configuration on a large scattering medium of cellulose powder. Figure 3b shows the fluorescence spectra of AuO adsorbed on cellulose, for different excitation wavelengths. The spectrum position and shape are similar to those obtained for AuO dissolved in polar liquids. See, for example, the steady-state spectrum in ethanol shown in Figure 2b. Parts a and b of Figure 4 show, on linear and semilogarithmic scales, the fluorescence up-conversion signals of ThT in acetonitrile solution measured at several wavelengths in the visible spectral region, 460−600 nm. The sample was excited at 390 nm by 150 fs pulses at a rate of 760 kHz. The ThT sample was placed in a thin optical cell with a path length of 0.8 mm, which was rotated. Acetonitrile is a nonassociative polar liquid with a large dipole moment of 3.4 D and low viscosity of 0.37 cP at room temperature. As seen in the figure, the fluorescence up-conversion decay is nonexponential at all wavelengths. The longer the monitored wavelength, the longer the average decay time (τav = ∫ IF(λ,t) dt), where IF(λ,t) is the normalized time-dependent fluorescence signal. The important point we wish to emphasize is that the decay time is short, about 2 ps at λ ≥ 540 nm. This short average decay time is explained by a rapid nonradiative decay of molecular rotor molecules. Table 1 provides the values of τav of ThT and AuO in acetonitrile at several wavelengths. The average lifetime increases as the wavelength increases. Comparing the values of τav at λ = 480 nm with that measured
at 600 nm shows an increase by a factor of about 2.5 for both compounds. Parts a and b of Figure 5 show, on a linear and semilogarithmic scale, the time-resolved fluorescence of ThT adsorbed on a cellulose powder of 20 μm average size. The fluorescence is collected by the front-surface method. The powder is fixed on a quartz plate by a small drop of glycerol. The time-resolved emission is measured by the timecorrelated single-photon-counting technique (TCSPC) with a rather limited instrument-response time of about 40 ps full width at half-maximum (fwhm). The signal decay is bimodal. A short decay-time component with an amplitude of about 0.3 and a time constant of τ ≤ 150 ps is followed by a long decaytime component with an amplitude of ∼0.7 and nearly exponential decay with a time constant of ∼2.1 ns. On the basis of the Strickler−Berg27 equation, the radiative decay time is estimated to be ∼4.5 ns. Figure 5b shows a fit to a biexponential function. The fitting parameters are given in Table 2. Table 3 shows the values of τav of ThT adsorbed on cellulose. The short decay-time component is attributed to the ThT signal that arises from molecules that are not fully adsorbed on the cellulose surface and thus the nonradiative decay is large Table 2. Two Exponent-Fitting Parameters of TCSPC Signals of ThT Adsorbed on Cellulosea
Table 1. τav of ThT and AuO in Acetonitrilea
a
τav [fs]
a
λ [nm]
ThT in acetonitrile
AuO in acetonitrile
460 480 500 520 540 560 580 600
440 830 850 980 1170 1300 1460 1980
260 340 390 500 540 660 770 910
λ [nm]
a1
τ1 [ns]
a2
τ2 [ns]
α2
460 480 500 520 560
0.34 0.26 0.26 0.28 0.38
0.16 0.13 0.13 0.13 0.10
0.66 0.74 0.74 0.72 0.62
1.8 2.10 2.14 2.14 2.14
0.92 0.95 0.95 0.95 0.95
IF(t) = a1 exp[−t/τ1 + a2 exp[−(t/τ2)α].
Table 3. τav of ThT Adsorbed on Cellulosea τav [ns]
τav = ∫ IF(t) dt.
a
λ [nm]
ThT on cellulose
cellulose only (background)
460 480 500 520 560
1.50 1.80 1.86 1.87 1.61
1.75 1.95 2.02
τav = ∫ IF(t) dt.
Figure 5. TCSPC signals of ThT on cellulose: (a) normalized linear scale; (b) normalized semilog scale. 8740
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Figure 6. Fluorescence up-conversion signals of AuO in acetonitrile: (a) linear scale; (b) semilog scale.
Figure 7. TCSPC signals of AuO on cellulose: (a) normalized linear scale; (b) normalized semilog scale.
For the best fit we used a stretched exponential factor of α = 0.58 ± 0.03. The convoluted fit of the system response is also shown in part b of Figure 7. The instrument response function is also shown in part a of Figure 7. In the discussion section we suggest possible mechanisms for the large nonexponential nature of the AuO signal on cellulose. Table 4 provides the
because the intramolecular rotation of the benzothiazole ring with respect to the aniline ring is only partially hindered. The major portion of the time-resolved fluorescence signal arises from the contribution of ThT molecules for which the intramolecular rotation is prevented. We find a small wavelength dependence of the relative amplitude between the short and the long decay-time components. The shorter the wavelength, the larger the amplitude of the short decay time. Figure 6 shows the fluorescence up-conversion signals of AuO in acetonitrile measured at several wavelengths in the spectral region of 460−600 nm. The average decay time depends on the monitored wavelength; the longer the wavelength, the longer the average decay time. As in the case of the fluorescence decay of ThT in acetonitrile, the decay of the AuO fluorescence at all wavelengths is nonexponential and the major part of the decay could be fitted by a stretched exponent, exp[−(t/τ)α], with 0.55 < α < 0.7. When comparing the fluorescence decay of AuO in acetonitrile with the decay of ThT, we find that the average decay time of AuO is shorter by about a factor of 2. This is also true in solvents other than acetonitrile. Table 1 provides the τav values of both ThT and AuO in acetonitrile. Figure 7 shows the time-resolved emission measured by the TCSPC technique of auramine-O adsorbed on cellulose powder of 20 μm average size. The signal is nonexponential at both short and long times. We were able to fit a major part of the signal amplitude, a = 0.85, by a single stretched exponential function:
Table 4. Fitting Parameters of TCSPC Signals of AuO on Cellulosea
a
λ [nm]
a1
τ1 [ns]
a2
τ2 [ns]
α2
500 520 540
0.17 0.17 0.17
0.08 0.08 0.08
0.83 0.83 0.83
0.34 0.44 0.48
0.58 0.60 0.61
IF(t) = a1 exp[−t/τ1 + a2 exp[−(t/τ2)α].
fitting parameters of the time-resolved emission of AuO adsorbed on cellulose. Table 5 provides the values of τav of AuO adsorbed on cellulose. Self-Fluorescence of Cellulose. When irradiated by UV light, cellulose shows broad emission in the visible part of the optical spectrum. Parts a and b of Figure 8 show, on a semilogarithmic scale, the signals of ThT (Figure 8a) and AuO (Figure 8b) adsorbed on cellulose, measured at 460 nm along with cellulose (background) time-resolved emission signals. As seen in the figure, the fluorescence signal of cellulose powder is nonexponential with an average decay time, τav = ∫ IF(λ,t) dt, of about 2 ns. This average decay time is on the order of τav of ThT adsorbed on the cellulose. Fortunately, the
IF(λ ,t ) = exp[−(t /τ )α ] 8741
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Table 5. τav of AuO Adsorbed on Cellulosea
a
λ [nm]
AuO on cellulose τav [ns]
500 520
0.62 0.79
When they are adsorbed on surfaces of biomaterials, their fluorescence quantum yield increases by at least 2 orders of magnitude20 and the emission lifetime increases by the same factor. The Glasbeek model6,7 takes into account the coupling of two close-lying excited states that exist in this class of molecules. Because of the coupling, the first molecular electronically excited singlet state is expressed as a mixture of two separate states, an emissive (F) state and a dark (D) state. The two diabatic states, F and D, are adiabatically coupled as a function of the normalized twist coordinate z:
τav = ∫ IF(t) dt.
fluorescence intensity of the cellulose is only 1/20th of that of the ThT or of the AuO. This rather low background intensity has very little effect on the short- and long-time signals of both ThT and AuO. The average decay time of both the weak background signal of the cellulose and the long-time components of the signals of AuO and ThT are nearly the same. Therefore, in the data analysis, we ignore the contribution of the weak (1/20) background fluorescence of the cellulose. Main Findings. 1. The steady-state fluorescence intensity of both ThT and AuO molecular rotors adsorbed on cellulose increases by more than a factor of 100 when compared with the fluorescence of these molecules in liquid solutions of low viscosity like acetonitrile, water and linear alcohols (methanol and ethanol). 2. The average fluorescence decay time, τav, of both molecular rotor molecules increases by more than a factor of 100. 3. The time-resolved fluorescence decay of ThT adsorbed on cellulose consists of two time components. The short decay-time component is less than 150 ps and contains less than 30% of the total fluorescence-signal amplitude. The longer decay-time component is nearly exponential and the decay time is about 2.0 ± 0.15 ns depending on the monitored wavelength. 4. The time-resolved emission of auramine-O adsorbed on cellulose is nonexponential at all wavelengths. The major part of the decay curve could be fitted by a stretched exponent function IF(λ,t) = exp[−(t/τ)α] with α = 0.58 ± 0.03. 5. We assign the large increase in the fluorescence intensity and fluorescence decay time of both molecules adsorbed on cellulose to the inhibition of intramolecular rotation of the two rotors composing these molecules and thus lowering the high nonradiative rate by more than 2 orders of magnitude.
S1(z) =
1 1 [F(z) + D(z)] − [F(z) − D(z)]2 + 4C 2 2 2 (1)
where C is the coupling-strength parameter. Such adiabatic coupling was earlier used by Fonseca, Barbara, and coworkers28,29 to model adiabatic excited-state intramolecular electron-transfer processes, which depend on the generalized solvent-reorganization coordinate, S. A short excitation pulse brings some of the population of the ground-state equilibrium configuration, which is nearly planar, to the F state. It then diffuses by torsional motion on the excited-state potential curve toward the minimum potential which assumes D-state character and is at the 90° configuration. Because the S1 state (of both auramine-O and ThT) is a z-dependent mixture of radiative and nonradiative zero-order states, the transition dipole moment, M(z), of the optical transition S1 → S0 is also z-dependent. Accordingly, the normalized S1 → S0 transition dipole moment decreases as a function of z: ⎡1 ⎛ ⎞⎤ 2C M(z) = cos2⎢ arctan⎜ ⎟⎥ ⎝ F(z) − D(z) ⎠⎦ ⎣2
(2)
The Glasbeek model was also used by Meech and coworkers5 to quantify the torsional dynamics of auramine-O. The change in the transition dipole moment of ThT as a function of the twist angle, z, was also studied with the use of quantum-molecular calculations.4,9,12 It was shown that the S1 → S0 transition dipole moment decreases by a factor of ∼100 as a function of the C−C dihedral angle. Recently,30 ThT ground- and excited-state dynamics was simulated with the use of TDDFT. It was also found that the change in the twist angle is responsible for the large decrease in the transition dipole
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DISCUSSION Glasbeek Model. The fluorescence properties of molecular rotor molecules are sensitive to the viscosity of the liquid.
Figure 8. TCSPC signals shown on a semilog scale of (a) ThT adsorbed on cellulose and (b) AuO adsorbed on cellulose and neat cellulose signal in each case. 8742
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moment and hence the large change in the fluorescence intensity and the short lifetime of ThT. Inhomogeneous Nonradiative Model. The timeresolved emission of AuO, measured by the time-correlated single-photon-counting technique is nonexponential. It can be fitted by a nonexponential function called the stretched exponent, exp[−(t/τ)α]. α is a positive number, 0 < α ≤ 1, and it determines the deviation of the signal from true exponential decay. When α equals 1, the decay is exponential. In Figure 7 we show the time-resolved fluorescence signal of AuO adsorbed on cellulose and also the fit to a stretched exponential with α ≈ 0.58. In this subsection we use an inhomogeneous nonradiative model that provides a plausible explanation for the origin of the nonexponential decay of the AuO fluorescence. The inhomogeneous model assumes a single floppy coordinate, x, that governs the conformational state of the system. We consider a barrierless continuous crossing from the LE fluorescent state to the excited charge-transfer dark state. The Boltzmann equilibrium distribution, for a harmonic groundstate potential, V0(x), prior to excitation is given by p(x) = Z exp[−V0(x)/kBT ] = Z exp[−(x − x0)2 ]
Figure 9. TCSPC signals shown on a semilog scale of AuO adsorbed on cellulose and inhomogeneous model-fitting functions.
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SUMMARY AND CONCLUSIONS We used steady-state (time-integrated) and time-resolved fluorescence techniques to study the fluorescence properties of two molecular-rotor molecules adsorbed on cellulose. In previous studies, we studied the optical properties of thioflavin-T (ThT) and auramine-O (AuO), shown in Scheme 1, dissolved in polar and protic liquids. These two molecules belong to a class of molecules that are termed molecular rotors. Their structure consists of two or more aromatic or heterocyclic rings with freedom to rotate in the excited state. When the rings are close to a planar configuration, the excited-state-transition dipole is large and therefore the absorption cross section, as well as the emission intensity, is large. Rotation of a ring segment to a 90° configuration in the excited state, leads to a transition of the strong emissive state to a charge-transfer dark excited state. The excited-state potential as a function of the twist angle has a minimum at the 90° configuration. Thus, molecular-rotor molecules in the excited state tend to have a short emission lifetime of a few picoseconds for liquids with low viscosities. It was found, more than half a century ago, that ThT, when adsorbed on β-amyloid fibrils, shows rather strong fluorescence and therefore has served for many years as a sensor for the detection of amyloid fibrils. In recent studies we found that the emission lifetime of both ThT and AuO adsorbed on insulin fibrils increases from few picoseconds in water to about 2 ns when adsorbed on these fibrils.1,26 In the current study, we measured the time-resolved emission of ThT and AuO adsorbed on cellulose. Cellulose is a biopolymer composed of D-glucose monomeric units. We found that the emission lifetime of ThT adsorbed on cellulose powder shows fluorescence behavior similar to that observed when it is adsorbed on insulin fibrils. The main portion of the fluorescence intensity decays nearly exponentially with a lifetime of ∼2 ns, approximately the same decay time of ThT adsorbed on insulin fibrils. We explain the long lifetime and intense fluorescence of ThT on cellulose in terms similar to those used in our previous study of ThT on insulin fibrils. The ring segments of adsorbed ThT molecules cannot rotate to form the dark charge-transfer state. Thus, the excited ThT retains its planar configuration during most of its radiative lifetime, which is estimated to be ∼4 ns. In AuO adsorbed on cellulose, the major portion of the fluorescence decay is nonexponential with an average decay time of about 0.8 ns. The fluorescence band of AuO on cellulose has similar band-peak positions and band shape as AuO in polar liquids. These steady-state emission results differ
(3)
Z is the Gaussian normalization constant, x0 is the ground-stateequilibrium twist angle, and x is the generalized twist angle. This inhomogeneous distribution for the slow (in this case, frozen) coordinate is thus time independent. The nonradiative process of adsorbed AuO on cellulose is inhomogeneous; thus instead of dealing with a single nonradiative rate constant, knr, each twist angle conformation, x, converts to the dark state D with a rate constant k(x) that depends on x. We assume that k(x) depends exponentially on the twist angle, x. k(x) = A exp[−b(x − x0)]
(4)
Here A is the preexponential factor and gives the value of knr at x0, and b is the exponential factor and a measure for the nonradiative rate sensitivity to the twist angle. In the static limit, the fluorescence follows the population probability P(t) that the locally excited (LE) state has not decayed by time t after excitation. This is given by p(t ) = exp( −t /τf )
∫0
∞
p(x) exp[−k(x)t ] dx
(5)
The first exponential accounts for the homogeneous radiative-decay process of the LE(F) state, whereas the integral of the second exponential represents the inhomogeneous nonradiative-decay kinetics, which is nonexponential. The solid lines in Figure 9 are the fit of the time-resolved emission data, measured at 500 and 540 nm, of AuO adsorbed on cellulose, obtained with the use of the inhomogeneous kinetic model presented above. As can be seen in Figure 9, the inhomogeneous model provides a good fit to the experimental time-resolved emission of AuO adsorbed on cellulose. For convenience, in eqs 4 and 5, we used dimensionless parameters rather than the twist angles for x and x0, where b = 1. The nonradiative rate at the groundstate twist angle x0 is A, the preexponential factor of eq 4. For the best fit we find A = 3.5 × 109 s−1 for the time-resolved signal measured at 500 nm, whereas for the 540 nm signal, A = 1.85 × 109 s−1. The radiative lifetime τF is assumed to be τF = 3 ns, krad = 0.33 × 109 s−1. 8743
dx.doi.org/10.1021/jp507052m | J. Phys. Chem. A 2014, 118, 8737−8744
The Journal of Physical Chemistry A
Article
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from what was found previously for AuO adsorbed on insulin fibrils immersed in water, where the spectrum consists of two emission bands. We assigned26 the two emission bands to a protonated and deprotonated form of AuO.
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AUTHOR INFORMATION
Corresponding Author
*Dan Huppert. E-mail: [email protected]. Phone: 972-36407012. Fax: 972-3-6407491. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Professor R. Lamed for helpful discussions. This work was supported by grants from the James-Franck GermanIsraeli Program in Laser-Matter Interaction and by the Israel Science Foundation.
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