Fluorescence Lifetime Probe for Solvent Microviscosity Utilizing

Jun 4, 2010 - The higher sensitivity of 2,6-ANS than of 1,8-. ANS demonstrates that the spatial freedom of the rotating phenylamino group in the photo...
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Anal. Chem. 2010, 82, 5470–5476

Fluorescence Lifetime Probe for Solvent Microviscosity Utilizing Anilinonaphthalene Sulfonate Yuu Someya† and Hiroharu Yui*,†,‡ Department of Chemistry, Faculty of Science, Tokyo University of Science, 12 Ichigaya-Funagawaramachi, Shinjyuku-ku, Tokyo 162-0826, Japan, and PRESTO-JST, Sanbancyo 5, Chiyoda-ku, Tokyo 102-0075, Japan The correlation between the fluorescent dynamics of excited anilinonaphthalene sulfonate (ANS) and the microviscosity of solvent molecules surrounding ANS is investigated by time-resolved fluorescence spectroscopy. ANS has been widely used to probe the local hydrophobicity due to the drastic change in its intensity. It is revealed that the fluorescence lifetime from the charge transfer (CT) state of ANS sensitively reflects the microviscosity. The higher sensitivity of 2,6-ANS than of 1,8ANS demonstrates that the spatial freedom of the rotating phenylamino group in the photoexcited ANS is an important factor that determines the sensitivity. As an application, the measurements of the microviscosity of water in biologically important systems, such as hyaluronan, gellan gum, and gelatin aqueous solutions are also presented. The present results suggest that the fluorescence lifetime of ANS enables the estimation of the solvent microviscosity and provide a useful probe molecule for fluorescence lifetime imaging microscopy. Microscopic viscosity determines the local motions and kinetics of molecules in solution.1-3 It is defined by the translational diffusion and the rotational diffusion of solute molecules.4 Microscopic viscosity related to translational diffusion is an important factor for the transport phenomenon in biological systems. For example, in the extracellular matrix, the velocity of the transport of nutritive substances from the blood vessels to the cell tissue or the diffusive behavior of signaling substances is affected by microscopic viscosity related to translational diffusion.5,6 As a model of diffusion in the extracellular matrix, Ushida et al. measured the translational diffusion constants in an aqueous solution containing hyaluronan, which is a biological polysaccha-

ride found in the extracellular matrix, by photochemical bimolecular quenching and fluorescence correlation spectroscopy.7,8 On the other hand, microscopic viscosity related to rotational diffusion determines the molecular dynamics with the change of the molecular structure and conformations without the translational diffusion of its centroid. The molecular dynamics includes protein folding, enzymatic reactions, and induced fit in the molecular recognition of proteins.1–3,9,10 In particular, the microscopic viscosity of the aqueous solution in cells and the interface of the biomembrane is an important factor that determines the dynamic behavior of membrane proteins.5,11 Furthermore, the microscopic viscosity in the aqueous solution in biological frameworks, such as polysaccharides, is also important for the formation and the dynamic properties of the framework structure as well as the activities of biological molecules working in the framework.12 Therefore, it is quite important to have a profound understanding of such dynamic behavior of biological molecules in aqueous solutions to estimate the microscopic viscosity of water related to the rotational diffusion. However, the measurement of the microscopic viscosity related to rotational diffusion has been limited mainly to homogeneous solutions because the spatial identification of the measuring point is not required in homogeneous systems.13,14 In contrast, in heterogeneous systems, such as biological cells and biological polymer frameworks, the identification of the measuring point in the heterogeneous structure is crucial. To achieve this requirement, the fluorescence probe technique is useful. This is because the fluorescence indicates the position of the probing area. However, microscopic viscosity probe molecules based on fluorescence intensity, typified by auramine O, often give error signals when they are used in such heterogeneous systems.15-17 This is because such heterogeneous environments are apt to induce the localization of the probe molecules, resulting in a change in the

* To whom correspondence should be addressed. Phone: +81-3-5228-8728. Fax: +81-3-5228-9060. E-mail: [email protected]. † Tokyo University of Science. ‡ PRESTO-JST. (1) Bhattacharyya, K. Acc. Chem. Res. 2003, 36, 95–101. (2) Nandi, N.; Bhattacharyya, K.; Bagchi, B. Chem. Rev. 2000, 100, 2013– 2045. (3) Pal, S. K.; Zewail, A. H. Chem. Rev. 2004, 104, 2099–2123. (4) Mazza, M. G.; Giovambattista, N.; Stanley, H. E.; Starr, F. W. Phys. Rev. E 2007, 76, 031203. (5) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Mol. Biol. Cell, 4th ed.; Newton Press: New York, 2004. (6) Bianchi, M. E.; Manfredi, A. Biochim. Biophys. Acta 2004, 1677, 181–186.

(7) Masuda, A.; Ushida, K.; Okamoto, T. Biophys. J. 2005, 88, 3584–3591. (8) Masuda, A.; Ushida, K.; Koshino, H.; Yamashita, K.; Kluge, T. J. Am. Chem. Soc. 2001, 123, 11468–11471. (9) Helms, V. ChemPhysChem 2007, 8, 23–33. (10) Royer, C. A. Chem. Rev. 2006, 106, 1769–1784. (11) Ball, P. Chem. Rev. 2008, 108, 74–108. (12) Knudson, C. B.; Knudson, W. FASEB J. 1993, 245, 1233–1241. (13) Laia, C. A. T.; Costa, S. M. B. Langmuir 2002, 18, 1494–1504. (14) Sun, Y.-P.; Saltiel, J. J. Phys. Chem. 1989, 93, 8310–8316. (15) Oster, G.; Nishijima, Y. J. Am. Chem. Soc. 1956, 78, 1581–1584. (16) Hirose, Y.; Yui, H.; Sawada, T. J. Phys. Chem. B 2004, 108, 9070–9076. (17) Hasegawa, M.; Sugimura, T.; Shindo, Y.; Kitahara, A. Colloids Surf., A 1996, 109, 305–318.

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Figure 1. Structures of 2,6-ANS (upper) and 1,8-ANS (lower).

fluorescence intensity. Therefore, it becomes quite difficult to discriminate between two possible origins of the intensity change, that is, the change of the microscopic viscosity and the change of the density of the probe molecule. Here, we present our finding of the viscosity fluorescent probe with its fluorescence lifetime and its application to a heterogeneous system. Anilinonaphthalene sulfonate (ANS, Figure 1), a charge transfer fluorescent dye, fluoresces weakly in water, but its intensity drastically increases in hydrophobic environments or when the rotational motion of the phenylamino group of ANS is restricted.18-25 Because of its characteristic nature, ANS has been intensively used as a hydrophobicity probe for more than five decades.10,26,27 In the present paper, we investigated the correlation between the fluorescent dynamics of the excited ANS and the rotational solvent microviscosity surrounding ANS by timeresolved fluorescence spectroscopy. It was revealed that the fluorescence lifetime of its photoexcited charge transfer state sensitively reflected the microviscosity. This finding will extend the application of ANS to a microviscosity probe in a heterogeneous system, such as biological cells and biological polymer solutions, without fluorescence intensity but with fluorescence lifetime. As an application to measure the microviscosity of water in biologically important systems, the microviscosity of water in hyaluronan, gellan gum, and gelatin aqueous solutions is also presented. Brief history of ANS. In this context, it would be helpful to summarize the brief history of research on the fluorescent properties of ANS. The characteristic behavior of the drastic increase in its intensity in hydrophobic environments was reported for the first time by Weber and Laurence in 1954.18 Since then, many studies on the characteristic fluorescence features of ANS have been reported.19–25 From the characteristic change of the fluorescence intensity according to the change of local environments, ANS has also been used in studies as a hydrophobicity probe for protein folding or at the cell surface in the biological field.10,26 (18) Weber, G.; Laurence, D. J. R. Biochem. J. 1954, 56, xxxi. (19) Upadhyay, A.; Bhatt, T.; Tripathi, H. B.; Pant, D. D. J. Photochem. Photobiol. A 1995, 89, 201–207. (20) Sadkowski, P. J.; Fleming, G. R. Chem. Phys. 1980, 54, 79–89. (21) Kosower, E. M.; Tanizawa, K. Chem. Phys. Lett. 1972, 16, 419–425. (22) Kosower, E. M. Acc. Chem. Res. 1982, 15, 259–266. (23) Kosower, E. M.; Huppert, D. Annu. Rev. Phys. Chem. 1986, 37, 127–156. (24) Robinson, G. W.; Robbins, R. J.; Fleming, G. R.; Morris, J. M.; Knight, A. E. W.; Morrison, R. J. S. J. Am. Chem. Soc. 1978, 100, 7145–7150. (25) Moore, R. A.; Lee, J.; Robinson, G. W. J. Phys. Chem. 1985, 89, 3648– 3654. (26) Thiebault, F.; Coulon, J. Can. J. Microbiol. 2005, 51, 91–94. (27) Il’ichev, Y. V.; Ku ¨ hnle, W.; Zachariasse, A. J. Phys. Chem. A 1998, 102, 5670–5680.

Figure 2. Scheme of the photoexcited dynamics of 2,6-ANS.

In addition to the drastic change of the fluorescence intensity, the correlation between the fluorescence wavelength of ANS and the solvent polarity has been investigated. It has been elucidated that the fluorescence wavelength maximum of ANS shows a drastic blue shift in low-polar solvents relative to that in highpolar solvents, such as water. Kosower et al. systematically studied the dependence of the fluorescence wavelength on the solvent polarity.21–23 They suggested that ANS fluoresces from the nonplanar (NP) state in low-polar solvents and transfers to a more stable charge transfer (CT) state from the NP state in high-polar solvents. It has also been considered that the transition from the NP to the CT state is accompanied by the intramolecular rotation of the phenylamino group. If we agreed with the Kosower’s model, the photophysical process of ANS could be described as shown in Figure 2. In the model, ANS transfers to the nonplanar (NP) excited state immediately after excitation, and it transfers to the charge transfer (CT) state accompanied by the rotation of phenylamino group. However, this model is not fully established and remains controversial. For example, the photophysics of 4-(dimethylamino)benzonitriles (DMABN), one of the charge transfer fluorescent molecules, is explained with the use of a planarized intramolecular charge transfer (PICT) model.27 The PICT state is attained by intramolecular rotation. Although no report has explained the photophysics of ANS with the PICT model, the PICT model is also conceivable for the photophysics of ANS. In both cases, intramolecular rotation is essential for the formation of the excited state. Although the Kosower’s model does not distinguish between the CT and PICT states, the CT state described in the Kosower’s model is attained by intramolecular rotation and the concept of the CT state includes that of the PICT state. Thus, if we select the PICT model, we can describe the CT state in Figure 2 as the PICT state. The CT state in the Kosower’s model or the PICT state should have common characteristics, namely, remarkably reduced intensity, longer fluorescent wavelength, and shortened fluorescent lifetime, due to the intramolecular rotational motion that accelerates nonradiative relaxation. The sensitive dependence of the fluorescence wavelength is also a characteristic feature of ANS. Thus, ANS has also been expected to be a micropolarity probe as well as a hydrophobic probe. Recently, Zewail et al. and Yui et al. successfully applied the characteristic feature in its wavelength to estimate the micropolarity at local environments of proteins and lipid nanotubes.28,29 (28) Zhong, D.; Pal, S. K.; Zewail, A. H. ChemPhysChem 2001, 2, 219–227. (29) Yui, H.; Guo, Y.; Koyama, K.; Sawada, T.; John, G.; Yang, B.; Masuda, M.; Shimizu, T. Langmuir 2005, 21, 721–727.

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Because of these unique characteristics of the photophysics, ANS has been widely used to probe the microscopic properties of a solvent. However, among the microscopic properties of a solvent, the relationship between the solvent microviscosity and the photophysics of the photoexcited ANS has not been clarified. EXPERIMENTAL SECTION 2-anilinonaphthalene-6-sulfonate (2,6-ANS) (Molecular Probes) and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) (Aldrich) were used without further purification (Figure 1). They were dissolved in H2O/glycerol mixed solvents. 10-4 M ANS H2O/glycerol solutions with different glycerol wt % were prepared by mixing a 10-4 M ANS aqueous solution and a 10-4 M ANS glycerol (WAKO Pure Chemical Industries, Ltd.) solution. Pure water (Millipore Simpli Lab.) was used for all measurements. To prevent the influence of dissolved oxygen on the photophysics of ANSs, all solvents were deoxidized by N2 gas. A 3 wt % hyaluronan sodium salt (HA) aqueous solution containing 2,6-ANS was prepared by adding HA powder (Denki Kagaku Kogyo Co., Ltd.; MW ) 1.6 × 106) to the 10-4 M 2,6ANS aqueous solution. A Na-gellan sample was prepared from deacylated gellan powder (San-Ei Gen F.F.I., Inc.) by ion exchange with a NaCl/isopropanol mixture. A 3 wt % gellan gum aqueous solution containing 10-4 M 2,6-ANS was prepared by adding the Na-gellan to a 10-4 M 2,6-ANS aqueous solution. A 5 wt % gelatin aqueous solution containing 2,6-ANS was prepared by adding atelocollagen powder (KOKEN Co., Ltd.) without further purification to a 10-4 M 2,6-ANS aqueous solution (pH 5.6). The collagen aqueous solution was stirred at 323 K and then cooled from 323 to 298 K (room temperature). These solutions were excited by a UV-pulsed beam from an Nd:YAG laser (EKSPLA 2143A; third harmonic, 355 nm; pulse duration, 30 ps; repetition rate, 10 Hz; beam power, 1.0 mJ/pulse, not focused on the sample). The time-resolved fluorescence spectra of the solutions were measured with a spectrometer (Hamamatsu Photonics C5094) equipped with a streak camera (Hamamatsu Photonics C4334). Signals from 512 pulses were accumulated for each measurement. All experiments were performed at room temperature (298 K) and repeated three times. RESULTS AND DISCUSSION To investigate the correlation between the fluorescent dynamics of the excited ANS and the solvent microviscosity surrounding ANS, H2O/glycerol solutions were used as solvents. The bulk viscosity of H2O and glycerol is 0.89 cP and 945 cP at 298 K, respectively.19 In contrast, the solvent polarity of H2O and glycerol is ET(30) ) 63.1 kcal/mol and ET(30) ) 57.0 kcal/ mol, respectively.30 The solvent micropolarity of H2O/glycerol solutions, therefore, should decrease with the increase of the glycerol ratio. It is well-known that the fluorescence wavelength of the CT state of ANS sensitively reflects the solvent micropolarity. To investigate the change of the micropolarity of the H2O/glycerol solution by varying the H2O/glycerol ratio, the fluorescence wavelength of the CT state of 2,6-ANS in H2O/ glycerol solutions was measured. (30) Reichardt, C. Chem. Rev. 1994, 94, 2319–2358.

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Figure 3. (a) Fluorescence spectrum of a 10-4 M 2,6-ANS aqueous solution. (b) Time-resolved fluorescence spectra of the 10-4 M 2,6ANS aqueous solution. The value appended to each spectrum shows the time after excitation. The arrow in the figure indicates the fluorescence from the NP state in the aqueous solution.

Figure 3 (a) shows the fluorescence spectrum of a 10-4 M 2,6-ANS aqueous solution. It is known that the fluorescence peak of the 2,6-ANS aqueous solution is observed at around 470 nm.31 In our experiment, the fluorescence peak was observed at around 470 nm, as expected, and the shoulder structure was also observed at around 400 nm. To separate these fluorescence components, the time-resolved fluorescence spectra of the 10-4 M 2,6-ANS aqueous solution were measured. Figure 3 (b) shows the time-resolved fluorescence spectra of the 10-4 M 2,6-ANS aqueous solution. Immediately after excitation, a shoulder structure was observed on the short-wavelength side of the peak. During 1 ns after the excitation, the intensity of the long-wavelength side of the peak decreased rapidly, and the peak of the short-wavelength side remained. By 5 ns after the excitation, the long-wavelength side of the peak had almost vanished, and the peak position of the short-wavelength side showed no change. It is known that the fluorescence wavelength from the CT state of 2,6-ANS is remarkably longer (∼470 nm) than that of the NP state (∼410 nm).22,31 In addition, the fluorescence lifetime from the CT state is remarkably shorter (∼0.3 ns) than that of the NP state (>a few ns).32 The fluorescence at the long-wavelength and short-wavelength sides, therefore, was assigned to the fluorescence from the CT and the NP states, respectively. Figure 4 (a) shows a streak camera image of the 10-4 M 2,6ANS aqueous solution. Since the fluorescence lifetime from the CT state is of a subnanosecond order (∼0.3 ns), the (31) Hamai, S. Bull. Chem. Soc. Jpn. 1999, 72, 2177–2182. (32) Huang, J.; Bright, F. V. J. Phys. Chem. 1990, 94, 8457–8463.

the NP state (Figure 4 (b)). These parameters are the peak wavelength, asymmetric parameter, and full width of halfmaximum (fwhm). The log-normal function is

(

(

F(λ) ) A · exp -ln(2)

ln(1 + 2B(λ - λp)/Γ) B

)) 2

(1)

where A is the intensity, B is an asymmetric parameter, and λp and Γ are the fluorescence peak wavelength and fwhm, respectively.33,34 The results of a single log-normal function fitting of the NP state in each glycerol wt % solution are shown in Table 1. Based on these results of the fitting, the fluorescence component of the CT state was then analyzed. To analyze the fluorescence component of the CT state, the spectrum just after excitation, which includes the fluorescence components of both the NP and the CT states, was measured. To separate the fluorescence components, double log-normal functions were fitted (eq 2). At the present fitting, the values of BNP, λNP, and ΓNP in the component of the NP state were fixed to those determined experimentally from the fitting to spectrum 5 ns after the excitation.

(

(

ln(1 + 2BNP(λ - λNP)/ΓNP BNP ln(1 + 2BCT(λ - λCT)/ΓCT) 2 +ACT · exp -ln(2) BCT

F(λ) ) ANP · exp -ln(2)

(

(

))

)) 2

(2)

Figure 4. (a) Streak camera image of the 10-4 M 2,6-ANS aqueous solution. (b) Fluorescence spectrum at 5 ns after excitation. The intensity of the spectrum was normalized at the maximum intensity. The dotted line is a fitted curve of the single log-normal function. (c) The fluorescence spectrum accumulated a duration of 1 ns after excitation. The intensity of the spectrum was normalized at the maximum intensity. The fluorescence spectrum was composed of the fluorescence from the NP and the CT states. The solid lines show the fluorescence from the NP and the CT states.

spectrum 5 ns after the excitation should be composed of only one fluorescence component from the NP state. Thus, a single log-normal function was fitted to spectrum 5 ns after the excitation to evaluate the parameters of the fluorescence from

Figure 4 (c) shows the results of the peak separation. The peaks of the fluorescence wavelengths from the NP and the CT states were 405 and 474 nm, respectively. The values of the asymmetric parameter, peak wavelength, and fwhm of the component of the CT state in each glycerol wt % solution are shown in Table 1. The fluorescence wavelength of the CT state slightly decreased with the increase of the glycerol wt % (from 474 nm at 0 wt % to 463 nm at 36 wt %). This result indicated that the solvent micropolarity slightly decreased with the increase of the glycerol wt %. The development of the hydrogen-bonding network by increasing the glycerol ratio is considered to be responsible for the decrease of the solvent micropolarity. The increase of the degree of the hydrogen-bonding network should increase the solvent microviscosity. On this basis, we investigated the correlativity between the fluorescence lifetime of 2,6-ANS and the microviscosity of water under the same condition. For this purpose, the fluorescence

Table 1. Asymmetric Parameter (B), Peak Wavelength (λp), and Bandwidths (Γ) of the Components of the NP and the CT States in Each Glycerol wt % Solution component of NP

component of CT

glycerol wt %

B

λp/nm

Γ/nm

B

λp/nm

Γ/nm

0 8 12 20 24 30 32 36

0.45 ± 0.02 0.54 ± 0.07 0.53 ± 0.05 0.58 ± 0.02 0.58 ± 0.04 0.62 ± 0.01 0.63 ± 0.03 0.63 ± 0.05

405 ± 1.3 404 ± 1.1 405 ± 1.1 405 ± 0.9 405 ± 1.2 405 ± 1.2 405 ± 0.3 405 ± 0.4

54 ± 2.9 57 ± 1.9 58 ± 1.6 59 ± 1.1 60 ± 3.4 62 ± 3.2 62 ± 2.1 63 ± 2.3

0.31 ± 0.12 0.28 ± 0.05 0.33 ± 0.06 0.32 ± 0.06 0.35 ± 0.04 0.38 ± 0.05 0.36 ± 0.06 0.36 ± 0.04

474 ± 2.7 472 ± 1.4 470 ± 2.4 467 ± 0.9 466 ± 0.4 465 ± 2.6 464 ± 0.6 463 ± 2.2

103 ± 5.2 104 ± 0.8 101 ± 4.1 100 ± 4.9 98 ± 3.6 97 ± 3.8 97 ± 2.5 96 ± 3.0

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Figure 5. Fluorescence decay signals of 10-4 M 2,6-ANS H2O/ glycerol solutions. The signal intensity was normalized at the maximum intensity (time 0 ns). The starting point of the arrow is glycerol 0 wt %, and its terminal point is 36 wt %. Table 2. Fluorescence Lifetime Data for 2,6-ANS H2O/Glycerol Solutions glycerol wt %

η/cP

τ 1/ns

τ 2/ns

0 8 12 20 24 30 32 36

0.89 1.09 1.22 1.54 1.75 2.16 2.32 2.71

0.41 ± 0.03 0.47 ± 0.01 0.52 ± 0.009 0.63 ± 0.01 0.69 ± 0.005 0.78 ± 0.03 0.82 ± 0.06 0.91 ± 0.02

2.8 ± 0.29 2.6 ± 0.24 2.5 ± 0.68 2.9 ± 0.77 2.8 ± 0.28 2.3 ± 0.16 2.3 ± 0.20 2.5 ± 0.06

decay signals of 2,6-ANS in different glycerol wt % solutions were measured with nanosecond time resolution. Figure 5 shows the fluorescence decay signals of 10-4 M 2,6ANS H2O/glycerol solutions. These signals were accumulated at fluorescence wavelengths from 450 to 600 nm. This is because, in the present experimental conditions, a strong Raman scattering signal of OH stretching vibration (3400 cm-1) was observed at around 405 nm for 355 nm excitation. In the wavelength range, the influence of the Raman scattering is negligible from the fluorescence decay signals. The fluorescence lifetime gradually became longer with the increase of the glycerol wt %. Since there are two distinct excited states in photoexcited ANS, a double-exponential function corresponding to the two excited states was fitted to these decay signals to evaluate the fluorescence lifetime. The results are shown in Table 2. As described above, the fluorescence lifetime from the CT state of ANS is shorter (∼0.3 ns) than that of the NP state (>a few ns). Therefore, the short-time component (τ1) and the long-time one (τ2) represented the fluorescence lifetime from the CT and the NP states, respectively. Figure 6 (a) shows the correlation between the fluorescence lifetime of 2,6-ANS and the glycerol wt %. The bulk viscosity for each glycerol solution is also described in Figure 6. The fluorescence lifetime from the NP state (τNP) was almost independent of the glycerol wt %. In contrast, the fluorescence lifetime from the CT state (τCT) became longer with the increase of the glycerol wt %. Figure 6 (b) shows the correlation between τCT and the glycerol wt %. A good correlation between τCT and the glycerol wt % was revealed. As described above, the solvent (33) Siano, D. B.; Metzler, D. E. J. Chem. Phys. 1969, 51, 1856–1861. (34) Shirota, H.; Horie, K. J. Phys. Chem. B 1999, 103, 1437–1443.

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Figure 6. (a) Correlation between the fluorescence lifetime of 10-4 M 2,6-ANS H2O/glycerol solutions and glycerol wt % for τCT (b) and τNP (4). The bulk viscosity is described on the upper horizontal axis. (b) Correlation between τCT and the glycerol wt %.

microviscosity of the H2O/glycerol solution increases with the increase of the glycerol wt %. The transition from the NP state to the CT state of 2,6-ANS is accompanied by the intramolecular rotation of the phenylamino group. The hindered intramolecular rotation of the phenylamino group due to the increase of the microviscosity should be responsible for the correlation between τCT and the microviscosity. This result suggested that the τCT of ANS enables us to estimate the solvent microviscosity related to the rotational diffusion and ANS simultaneously provides information about the micropolarity from the fluorescence wavelength. The correlation between τCT and the microviscosity is closely related to the intramolecular rotation of the phenylamino group of ANS. The sensitivity of ANS to the microviscosity of water, therefore, should be related to the rotational motion of the phenylamino group. To clarify the influence of the spatial freedom of the rotating phenylamino group on the sensitivity to the microviscosity, the τCT values of 2,6-ANS and 1,8-ANS H2O/glycerol solutions were compared. The substitution position of the phenylamino group of 1,8-ANS differs from that of 2,6-ANS (Figure 1). Under the same experimental conditions, 10-4 M 1,8-ANS H2O/glycerol solutions were examined. Figure 7 (a) shows the fluorescence decay signals of 1,8-ANS H2O/glycerol solutions in the wavelength range of 450-600 nm. The fluorescence lifetime of 1,8-ANS gradually became longer. However, the increase of τCT was small in comparison

Figure 7. (a) Fluorescence decay signals of 10-4 M 1,8-ANS H2O/ glycerol solutions. The signal intensity was normalized at the maximum intensity (time 0 ns). The starting point of the arrow is glycerol 0 wt %, and its terminal point is 36 wt %. (b) Correlation between τCT and the glycerol wt % for 2,6-ANS (b) and 1,8-ANS (2). The bulk viscosity is described on the upper horizontal axis.

to that of 2,6-ANS (Figure 5). From the same analysis, the τCT was plotted against the glycerol wt %. These results are shown in Figure 7 (b) with the results of 2,6-ANS. The τCT of 1,8-ANS was shorter than that of 2,6-ANS in the same glycerol wt % solution. For example, the τCT values of 1,8-ANS and 2,6-ANS were 0.41 and 0.63 ns in the glycerol 20 wt % solution, respectively. Since the transition from the NP to the CT state is accompanied by the intramolecular rotation of the phenylamino group, the population of the CT state becomes smaller when the intramolecular rotation is restricted. The phenylamino group of 1,8-ANS is closer to the sulfo group, which is in contrast with the case of 2,6-ANS. The steric hindrance decreases the spatial freedom of the rotating phenylamino group in 1,8-ANS. Since the spatial freedom of the rotating phenylamino group is decreased, the population of the CT state in photoexcited 1,8-ANS becomes smaller than that of 2,6-ANS. We considered that this is the reason that the τCT of 1,8-ANS apparently becomes shorter than that of 2,6-ANS in the same glycerol wt % solution. To evaluate the sensitivity to the microviscosity, the tangential gradients were calculated at 1.5 cP of bulk viscosity. The value of the gradient of 2,6-ANS was 0.31 ns/cP. In contrast, that of 1,8ANS was 0.13 ns/cP. These results indicate that the sensitivity of 2,6-ANS was about 2.4 times higher than that of 1,8-ANS at 1.5 cP of bulk viscosity. As reported above, the spatial freedom of the rotating phenylamino group of 1,8-ANS is lower than that of 2,6-ANS. The decrease of the spatial freedom of the rotating

phenylamino group by the steric hindrance should be responsible for the lower sensitivity of 1,8-ANS. These results demonstrate that the spatial freedom of the rotating phenylamino group is an important factor that determines the sensitivity to the microviscosity. For the design of a microviscosity probe molecule with high sensitivity, it is preferable to increase the spatial freedom of the rotating phenylamino group. The fluorescence lifetime is basically independent of the molecular concentration. Because of the character, the fluorescence lifetime has attracted attention for fluorescence lifetime imaging microscopy (FLIM) in the fluorescence imaging field.35,36 Recently, Levitt et al. synthesized the fluorescence dye, which specifically binds to membranes, and estimated the microviscosity near biomembranes by FLIM.37 On the other hand, ANS is able to estimate not only the microviscosity from its fluorescence lifetime but also the hydrophobicity and the micropolarity from its fluorescence intensity and fluorescence wavelength. In addition, ANS is a small molecule relative to biological molecules, such as proteins or polysaccharides. Therefore, it enables the measurement of the microscopic solvent properties in biological systems with small hindrance to the biological activity. The present results will lead to a new application of ANS, which has been used mainly as a hydrophobic probe, to a useful molecule for FLIM with multiple characters and a small hindrance. As applications to the measurement of the microviscosity, 2,6ANS was applied to measure the microviscosity of water in hyaluronan, gellan gum, and gelatin aqueous solutions. Hyaluronan, found in the extracellular matrix, provides the framework of biological structures, such as that in the vitreous body and the dermis.38,39 The dynamic behavior of molecules in a hyaluronan aqueous solution, therefore, has attracted much attention as a model for mass transport in the extracellular matrix.7,8 Gellan gum is a bacterial polysaccharide produced by Sphingomonas elodea and is used in the food and biomedical industries because of its good biocompatibility and facile control of its gel properties.40 Gelatin is produced by the thermal denaturation of collagen found in the extracellular matrix. Since gelatin shows good biocompatibility, it has also been applied to food additives or biomedical materials.41,42 The τCT, the microviscosity, and the fluorescence intensity ratio for the NP state and the CT state in those samples are shown in Table 3. The values of the microviscosities of these samples were calculated by using the correlation between solvent viscosity and the τCT of 2,6-ANS (Figure 6 (b)). The τCT value in a hyaluronan aqueous solution and that in a gellan gum solution were almost the same as that in water. However, the ratio of (35) Thoumine, O.; Ewers, H.; Heine, M.; Groc, L.; Frischknecht, R.; Giannone, G.; Poujol, C.; Legros, P.; Lounis, B.; Cognet, L.; Choquet, D. Chem. Rev. 2008, 108, 1565–1587. (36) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. (37) Levitt, J. A.; Kuimova, M. K.; Yahioglu, G.; Chung, P.-H.; Suhling, K.; Phillips, D. J. Phys. Chem. C 2009, 113, 11634–11642. (38) Lapcˇ´ık, L., Jr.; Lapcˇ´ık, L.; Smedt, S. D.; Demeester, J.; Chabrecˇk, P. Chem. Rev. 1998, 98, 2663–2684. (39) Laurent, T. C.; Eraser, J. R. E. FASEB J. 1992, 6, 2397–2404. (40) Bajaj, I. B.; Survase, S. A.; Saudagar, P. S.; Singhal, R. S. Food Technol. Biotechnol. 2007, 45, 341–354. (41) Yakimets, I.; Wellner, N.; Smith, A. C.; Wilson, R. H.; Farhat, I.; Mitchell, J. Polymer 2005, 46, 12577–12585. (42) Zuo, G.; Liu, C.; Luo, H.; He, F.; Liang, H.; Wang, J.; Wan, Y. J. Appl. Polym. Sci. 2009, 113, 3089–3094.

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Table 3. τCT, Microviscosity, and Fluorescence Intensity Ratio (NP/CT) in Water, Hyaluronan, Gellan Gum, and Gelatin Aqueous Solutions

water hyaluronan gellan gum gelatin

τCT/ns

viscosity/cP

intensity ratio (NP/CT)

0.39 ± 0.01 0.40 ± 0.02 0.39 ± 0.03 0.47 ± 0.03

0.89 0.90 0.89 1.07

0.36 0.45 1.73 1.69

the fluorescence intensities of the NP and the CT states in the hyaluronan and gellan gum aqueous solutions was 1.2 times and 4.7 times higher than that in water, respectively. This means that 2,6-ANS partially locates in the hydration region near the polymer chain of polysaccharides. These results revealed that the microviscosity at the hydration region in hyaluronan and gellan gum, in spite of the decrease of polarity, is the same as that of water and identical to that at the bulk region. In contrast, in gelatin, the τCT was slightly longer than that of water, and the calculated microviscosity was 1.2 times higher than that of water. Interestingly, the intensity ratios of the NP and the CT states in gellan gum and gelatin were similar, and the lifetime of the CT state sensitively reflects the difference in microviscosity for both samples. The present analytical method to estimate the microviscosity with the fluorescent lifetime of ANS in combination with the simultaneous measurement of its wavelength will be useful for application to inhomogeneous samples. CONCLUSIONS The correlation between the photophysics of photoexcited ANS and the microproperties of the solvent surrounding ANS was

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investigated by time-resolved fluorescence spectroscopy. The fluorescence lifetime from the CT state was found to sensitively reflect the microviscosity of water. This means that ANS can be applied to a novel microviscosity probe molecule with the fluorescence lifetime from the CT state. The merit of this microviscosity probe with the lifetime rather than with the fluorescence intensity is that it shows no dependence on the concentration of fluorescence molecules. Since a heterogeneous environment often induces the localization of fluorescence molecules and leads to change in its fluorescence intensity, a fluorescence probe based on the intensity is unfavorable for measurements in biological cells. In contrast, a fluorescence probe based on the fluorescence lifetime is free from the problem of the localization because the fluorescence lifetime is basically independent of the molecule concentration. Therefore, this method enables us to estimate the microviscosity of water in heterogeneous systems, such as biological cells, or at the surface of biological molecules. Recently, the fluorescence lifetime has attracted attention for fluorescence lifetime imaging microscopy (FLIM) in the fluorescence imaging field because of its character. Special attention has been given to the estimation of the solvent properties surrounding fluorescence dyes, as well as cellular imaging. Our results suggest that ANS is a useful probe for FLIM, which can be used to estimate the microviscosity, hydrophobicity, and micropolarity.

Received for review January 15, 2010. Accepted May 19, 2010. AC100116J