A Spectroscopic and Epifluorescence Microscopic Study of

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Langmuir 1997, 13, 5401-5408

5401

A Spectroscopic and Epifluorescence Microscopic Study of (Hexadecanoylamino)fluorescein Aggregates at the Air-Water Interface and in Langmuir-Blodgett Films Ashim K. Dutta and Christian Salesse* GREIB, De´ partement de Chimie-Biologie, Universite´ du Que´ bec a` Trois-Rivie` res, Trois-Rivie` res, Que´ bec, Canada G9A 5H7 Received March 31, 1997. In Final Form: July 10, 1997X In this paper, we report the monolayer behavior of pure (5-n-hexadecanoylamino)fluorescein (HDFL) at the air-water interface. The spectroscopic characteristics of pure HDFL films at the air-water interface and the mixed Langmuir-Blodgett (LB) films of HDFL and palmitic acid (PA) deposited on quartz substrates have been studied and compared with the spectroscopic characteristics of HDFL in solution. The absorption and emission spectra of HDFL at the air-water interface were similar to those in polar, protic solvents but completely different in aprotic, apolar solvents. Detailed spectroscopic studies indicate that in protic solvents the fluorescein moieties exist as cations and zwitterions while in nonpolar environments the fluorescein chromophore exists as an inner lactone that is nonfluorescent. Spectroscopic studies of the mixed films of HDFL and PA transferred on quartz substrates provide evidence of aggregation and formation of at least two different aggregated species, one of them being fluorescent while the other is not. The large differences in the spectral characteristics between the monolayers at the air-water interface and the transferred layers indicate the existence of HDFL moieties in different molecular configurations that are microenvironment dependent. Surface pressure dependent steady-state in situ fluorescence studies confirm that fluorescence quenching of the dye with increasing surface pressure occurs as a result of efficient energy transfer from the fluorescent monomeric species to the aggregates that are likely nonfluorescent and decay therefrom by nonradiative processes. This study establishes that fluoresceinated dyes may be utilized as efficient molecular probes for ascertaining polarity in supramolecular assemblies.

Introduction Fluorescein and its derivatives belong to the family of xanthene dyes that are characterized by an intense fluorescence in the yellow-green region of the visible spectrum.1a,b Fluoresceinated dyes are extensively used as coloring agents in polymers, scintillators,1c,d solar concentrators,1c,d and fluorescence microscopy.1e Lipophilic fluorescein probes have been used in fluorescence recovery after photobleaching2a-d and in Fo¨rster resonance energy-transfer measurements3a-g to determine lipid lateral diffusion and transport properties in lipid and biological membranes.2,3 The large absorption extinction coefficient, high quantum yield of fluorescence, and interesting photobleaching properties in addition to the remarkable sensitivity of their photophysical properties on the pH and polarity of the microenvironment make fluoreceinated dyes attractive for spectroscopic studies.1-6 * Telephone: +1-819-376-5077. Fax: +1-819-376-5057. Email: christian [email protected]. X Abstract published in Advance ACS Abstracts, September 1, 1997. (1) (a) Berlman, I. B. Handbook of Fluorescence Spectra of Aromatic Molecules; Academic Press: New York, 1971. (b) Birks, J. B. Photophysics of Aromatic Molecules; Wiley-Interscience: London, 1970. (c) Guillet, J. E. Polymer Photophysics and Photochemistry; Cambridge University Press: Cambridge, U.K., 1985. (d) Rabek, J. F. Photochemistry and Phtophysics; CRC Press: Boca Raton, FL, 1990; Vol. 1. (e) Tsien, R. Y. In Fluorescence Microscopy of Living Cells in Culture; Taylor, D. L., Wang, Y.-L., Eds.; Academic Press Inc.: New York, 1989; p 133. (2) (a) Axelrod, D.; Koppel, D. E.; Schlesinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055. (b) Axelrod, D. Biophys. J. 1977, 18, 129. (c) Peters, R. Cell. Biol. Int. Rep. 1981, 5, 733. (d) Elson, E. L.; Qian, H. In Fluorescence Microscopy of Living Cells in Culture; Taylor, D. L.; Wang, Y.-L., Eds.; Academic Press Inc.: New York, 1989; p 307. (3) (a) Bright, F. V. Anal. Chem. 1988, 60, 1031. (b) Dewey, T. G. Biophysical and Biochemical Aspects of Fluorescence Spectroscopy; Plenum Press: New York, 1991. (c) McGown, L. B.; Warner, I. M. Anal. Chem. 1994, 66, 428R. (d) Lackowicz, J. R. Topics in Fluorescence Spectroscopy: Biochemical Applications; Plenum Press: New York, 1991; Vol. 3. (e) Van der Meer, B. W.; Coker, G., III; Simon Chen, S.-Y. Resonance Energy Transfer Theory and Data; VCH Publishers: New York, 1994. (f) Fung, B. K.; Styrer, L. Biochemistry 1978, 17, 5241. (g) Fernandez, S. M.; Berlin, R. D. Nature 1976, 264, 411.

S0743-7463(97)00327-2 CCC: $14.00

Such dye molecules incorporated in systems representing restricted geometries7 provide invaluable insight into the spectra-structure-property corelationship in these systems as well as the nature of interaction between the dye molecules and their microenvironment. Ordered ultrathin Langmuir-Blodgett monolayers provide a unique system as it represents supramolecular assemblies where the spatial distribution and orientation of the dye molecules in the film may be tailored at will to bring about desired changes in their optical and electronic properties.8 These features make LB films highly attractive for applications in future generation nanotech(4) (a) Martin, M.; Lindquist, L. J. Lumin. 1975, 10, 381. (b) Shah, J.; Joshi, N. B.; Pant, D. D. Indian J. Pure Appl. Phys. 1983, 21, 677. (c) Dawson, W. R.; Windsor, M. W. J. Phys. Chem. 1968, 72, 3251. (d) Arbeloa, L. J. Photochem. 1980, 14, 97. (5) (a) Pant, S.; Tripathi, H. B.; Pant, D. D. J. Photochem. Photobiol., A 1994, 81, 7. (b) Fleming, G. R.; Knight, A. W. E.; Morris, J. M.; Morrison, R. J. S.; Robinson, G. W. J. Am. Chem. Soc. 1978, 100, 221. (c) ValdesAguilera, O.; Neckers, D. C. Acc. Chem. Res. 1989, 22, 171. (d) Bortolato, C. A.; Atvars, T. D. Z.; Dibbern-Brunelli, D. J. Photochem. Photobiol., A 1991, 59, 123. (6) (a) Valdes-Aguilera, O.; Neckers, D. C. Acc. Chem. Res. 1989, 22, 171. (b) Dihel, H. Talanta 1989, 36, 413. (c) Dibbern-Brunielli, D.; Atvars, T. D. Z. Spectrosc. Lett. 1990, 23, 627. (d) Atvars, T. D. Z.; Bortolato, C. A.; Dibbern-Brunelli, D. J. Photochem. Photobiol., A 1992, 68, 41. (e) Reichardt, C. Solvents and Solvent Effects in Organic Chemistry; VCH Publishers: New York, 1988; Chapter 4, p 107. (f) Haughland, R. P. In Handbook of Fluorescent Probes and Research Chemicals; Spence, M. T. Z., Ed.; Molecular Probes Inc.: Eugene, OR, 1996. (g) Lavoie, H.; Dutta, A. K.; Salesse, C. Manuscript under preparation. (7) (a) Klafter, J.; Drake, J. M. Molecular Dynamics in Restricted Geometries; John Wiley & Sons: New York, 1989. (b) Kalyansundaram, K. Photochemistry in Microheterogeneous Systems; Academic Press: New York, 1987. (c) Ramamurthy, V. Photophysics and Photochemistry in Organized and Constrained Media; VCH: New York, 1991. (d) Balzani, V. Supramolecular Photochemistry; D. Reidel Publishing Co.: New York, 1987. (8) (a) Kuhn, H.; Mo¨bius, D.; Bucher, H. In Physical Methods of Chemistry; Weissberger, A., Rossiter, B. W., Eds.; Wiley: New York, 1972; Vol. 1, p 577. (b) Ulman, A. An Introduction to Ultrathin Organic Thin Films: From Langmuir-Blodgett Films to Self Assemblies; Academic Press: New York, 1991. (c) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Wiley-Interscience: New York, 1966. (d) Kato, T.; Matsumoto, N.; Kawano, M.; Suzuki, N.; Araki, T.; Iriyama, K. Thin Solid Films 1994, 242, 223.

© 1997 American Chemical Society

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nology based on miniaturized ultrafast devices.9 Considerable efforts have been made in studying various amphiphilic and nonamphiphilic dyes assembled in LB films. However, little attention has been focused on the spectroscopic characteristics of fluoresceinated lipids assembled in LB films.10 In this paper, we report the photophysical characteristics of pure (5-hexadecanoylamino)fluorescein (HDFL) monolayers at the air-water interface and mixed films of HDFL with palmitic acid (PA) deposited on quartz substrates. Spectroscopic studies reveal large red shifts of the absorption band in the deposited LB films compared to that in solution which indicate the formation of J-aggregates in these films. Detailed studies of the absorption and emission spectra of the mixed HDFL films transferred on quartz substrates were found to be different from the films at the air-water interface which indicated different molecular conformations of the HDFL moieties in the two different systems. Experimental Section (5-n-Hexadecanoylamino)fluorescein and palmitic acid (PA) were purchased from Molecular Probes Inc., Eugene, OR, and Sigma Chemical Co., St. Louis, MO, respectively, and were used without further purification. All solvents used in this work were of spectroscopic grade. Freshly prepared solutions were used and stored in glass vials wrapped in aluminum foil in a refrigerator to prevent photodecomposition. A homemade allTeflon LB trough was used for studying the behavior of pure HDFL and the mixed films of the dye with PA at the air-water interface. A filter paper Wilhelmy plate was used for detecting the surface pressure at the air-water interface. The moving barrier interfaced to the computer maintained constant surface pressure at the air-water interface with an accuracy of (0.1 mN/m and was also used for measuring the surface pressure versus area per molecule isotherms. Deionized water obtained by purifying distilled water through a Nanopure water deionizing system and having a resistivity of 18.2 MΩ cm was used as the subphase. The pH of the subphase was approximately 6.6 in equilibrium with carbon dioxide in the atmosphere. Surface potential8c measurements at the air-water interface were made using a 241Am-coated ionizing electrode placed at about 1 mm above the the water surface, while another electrode, a grounded platinum foil, was located below the water surface at the bottom of the trough. Such an arrangement constitutes a capacitor, the potential difference across which may be calculated by measuring the charge in the capacitor. This is achieved through a Keithley digital electrometer interfaced to a computer, allowing measurement of the surface potential of the monolayer with respect to the pure water surface. The measured surface potential of the monolayer is equal to the difference of the charge in this capacitor in the presence and in the absence of the monolayer. Monolayers at the air-water interface were prepared in a conventional manner by spreading 100 µL of a solution of HDFL (2 × 10-3 M) at the air-water interface and compressing the monolayer formed at a low speed of 4 × 10-3 nm2 molecule-1 s-1 after allowing 15 min for the volatile solvents to evaporate. An identical method was used for the mixed films of PA and HDFL. Y-type deposition of mono- and multilayers on quartz substrates was achieved by dipping the substrate vertically through the monolayer at a speed of 1 mm/min. A drying time of 10 min was allowed between consecutive dippings. The transfer ratio for the mixed film deposited onto the solid substrate was calculated from the ratio of the decrease in the monolayer area at the airwater interface to the actual surface area of the substrate and was evaluated to be 0.96 ( 0.02. (9) (a) Swalen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Isrealachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (b) Hong, F. T. Molecular Electronics: Biosensors and Biocomputers; Plenum Press: New York, 1989. (c) Forrest, F. L. Molecular Electronic Devices II; Marcel Dekker Inc.: New York, 1987. (10) Ahlers, M.; Grainger, D. W.; Herron, J. N.; Lim, K.; Ringsdorf, H.; Salesse, C. Biophys. J. 1992, 63, 823.

Dutta and Salesse

Figure 1. (a) Surface pressure versus area per molecule isotherm of a film of pure HDFL at the air-water interface. (b) Surface potential versus area per molecule isotherm of the same film at the air-water interface. The subphase temperature was 18 °C and pH ) 6.6. The inset shows the molecular structure of HDFL. An epifluorescence microscope identical with the one described in detail elsewhere11 was used for acquiring the fluorescence micrographs of the monolayer at the air-water interface at different constant surface pressures during compression and decompression of the pure and mixed films of HDFL and PA at the air-water interface. A SIT camera attached to the microscope transferred the magnified images to a television screen that was processed and printed by a Sony video printer. Absorption and emission spectra of the samples in solution and in LB films were recorded on a Hewlett-Packard 8542A singlebeam diode array spectrophotometer and on a Spex Fluorilog 2 spectrofluorimeter, respectively. In situ measurements of the absorption and emission spectra of the floating monolayer at the air-water interface were accomplished on a homemade automated Teflon trough. A dual-beam system using an electronically matched pair of photodiodes was used for recording the absorption spectra of the floating monolayer. The absorbance of the surface of pure water was taken as the reference. The fluorescence emission spectrum of the floating monolayer at the air-water interface was obtained by illuminating the monolayer by a 100-W high-pressure xenon arc lamp through a monochromator that allowed proper choice of excitation wavelength. The emission from the floating monolayer was subsequently collimated by a series of ellipsoidal mirrors and detected by a thermoelectrically cooled Hamamatsu CCD camera. To minimize photodegradation of the fluorescein dye, the experiments were carried out in dim red light and electronic shutters were used to irradiate the monolayers for short durations of 30 s.

Results and Discussion Surface Pressure (Π-A) and Surface Potential (∆V-A) Measurements at the Air-Water Interface. Figure 1 shows the surface pressure versus area per molecule isotherm of a pure HDFL monolayer at the airwater interface. The films at the air-water interface were found to be highly stable, which was confirmed from a dA/dt versus time plot (figure not shown) that showed a constant value of dA/dt ) 0 over a period of 4 h and at different surface pressures in the 5-35 mN/m range. Compressing the film of amphiphilic HDFL at the airwater interface at a low speed (≈2 × 10-3 nm2 molecule-1 s-1) resulted in an isotherm that showed a flat section (3.5-0.8 nm2/molecule) and a well-defined knee (0.750.50 nm2/molecule) followed by a steep rise in the 0.50.25 nm2/molecule region. While the flat region of the isotherm corresponds to a gaslike phase, the kneelike region corresponds to a transition from a liquid-expanded (LE) to a liquid-condensed (LC) phase, and the steep region (11) (a) Meller, P. Rev. Sci. Instrum. 1988, 59, 2225. (b) Ducharme, D.; Salesse, C.; Leblanc, R. M.; Meller, P.; Mertesdorf, C.; Ringsdorf, H. Langmuir 1993, 9, 2145. (c) Subirade, M.; Salesse, C.; Marion, D.; Pezolet, M. Biophys. J. 1995, 69, 974.

(Hexadecanoylamino)fluorescein Aggregates

plausibly corresponds to the solid phase. Close examination of the π-A isotherm reveals that the gradient of this isotherm corresponding to a surface pressure between 10 and 30 mN/m is different from the gradient in the 35-50 mN/m region, with an inflexion at approximately 38 mN/m that probably suggests a solid-solid phase transition. Further increase in pressure results in a large change in the slope of the isotherm that corresponds to the collapse of the film at a surface pressure of 52 mN/m. The average area per molecule at a surface pressure of 30 mN/m is 0.38 nm2, which is much larger compared to the average area per molecule of PA that is reported to be about 0.190.21 nm2.8c,d In view of the large size of the fluorescein moiety and its high hydrophilicity, it seems likely that the fluorescein moieties remain submerged below the airwater interface but offer steric hindrance to the close approach of neighboring molecules that is probably manifested as a large average area per molecule at the air-water interface. The surface potential isotherm of a pure HDFL monolayer at the air-water interface is also shown in Figure 1. The isotherm was observed to be flat in the 3.5-1.25 nm2 area/molecule region with ∆V ) 0 that corresponds to the gaslike phase. Compressing the monolayer showed a sudden steep rise followed by a plateau in the region corresponding to an average area of 1.25-0.78 nm2/ molecule. The sharp rise in ∆V from zero to several hundred millivolts corresponding to the 1.25-0.78 nm2 average area/molecule region has been attributed to the presence of distinct regions of dilute and dense patches of the monolayer detected by the finite size of the measuring electrode.12 Further compression showed a steady rise in surface potential in the region corresponding to 0.78-0.25 nm2/molecule. The steep region at low areas per molecule may be attributed to the decreasing average area per molecule with surface pressure accompanied by some changes in the orientation and organization of the molecules with surface pressure. Mathematically, the change in potential is given as ∆V ) (4πµN cos Θ)/ where µ is the effective dipole moment of the molecule in the monolayer, N is the number of molecules per unit area, and Θ is the tilt angle between the molecular dipole moment with the normal at the air-water interface. As is evident from this relation, ∆V changes with µ cos Θ and N. µ may change with molecular deformation and cos Θ with change in the tilt angle, while N changes with compression/expansion of the monolayer. It must be pointed out that it is not possible to separate out the individual contributions from the observed changes in surface potential. Other factors that also contribute to the changes in surface potential are the presence of ions in the subphase, structure of the organized water layer below the floating monolayer, and contributions of the methyl end groups and CdO ester groups that significantly affect the surface potential.12e,f Close comparison of the surface pressure and surface potential isotherms reveals that the dip in the surface potential curve at an average area of 0.28 nm2 corresponds to the inflexion in the π-A isotherm, indicating a solid-solid phase transition. Spectroscopic Studies of HDFL in Solution and at the Air-Water Interface. Figure 2 shows the S1-S0 absorption band of HDFL in the 400-550 nm spectral region in several different microenvironments. The 0-0 (12) (a) Petty, M. C. Langmuir-Blodgett films: An Introduction; Cambridge University Press: Cambridge, U.K., 1996. (b) Oliveira, O. N., Jr.; Taylor, D. M.; Lewis, T. J.; Salvagno, S.; Stirling, C. J. M. J. Chem. Soc., Faraday Trans. 1 1989, 85, 1009. (c) Rasing, Th.; Hsuing, H.; Shen, Y. R.; Kim, M. W. Phys. Rev. 1988, 37, 2732. (d) Oliviera, O. N., Jr.; Taylor, D. M.; Morgan, H. Thin Solid Films 1992, 210/211, 76. (e) Mo¨hwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441. (f) Vogel, V.; Mo¨bius, D. Thin Solid Films 1988, 159, 73.

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Figure 2. Absorption spectra of HDFL: (a) in methanol (short dashed line); (b) at the air-water interface (open circles) at a surface pressure of 10 mN/m; (c) 10 layers of a film of HDFL and PA mixed in a molar ratio of 1:20 and deposited on quartz at a surface pressure of 25 mN/m (solid line); (d) cast film of HDFL and PA mixed in a molar ratio 1:20 (long dashed line). Table 1

methanol air-water interface dichloromethane LB films

absorption 0-0 band (nm)

emission 0-0 band (nm)

excitation 0-0 band (nm)

482 493

512 518 408 464

489 493 350 437

519

bands in methanol and monolayer at the air-water interface are located at 482 and 493 nm, respectively, as listed in Table 1. The observed red shift of 11 nm indicates organized aggregation of the fluorescein chromophores at the air-water interface. According to the intermediate strength exciton coupling model of McRae and Kasha,13 dipole-dipole interaction between neighboring molecules may generate an exciton band that is located either above or below the monomer exciton band. While the aggregates causing a red shift are referred to as J-aggregates,14 the aggregates causing a blue shift are referred to as Haggregates.14 Although small shifts may originate from differences in the refractive index of the matrix in which the molecule is located, large red shifts of 11 nm clearly indicate J-type aggregation in the floating monolayer. Figure 2 shows that the 0-0 absorption bands of HDFL in the LB and cast films are located at 519 and 489 nm, respectively. The 0-0 band in the LB films is red shifted by 30 nm relative to that in the cast film which suggests strong dipole-dipole interaction between the HDFL moieties in the LB film probably arising from a more compact packing of the chromophores in the transferred films compared to that in the cast film where the molecules are likely to be randomly oriented. Moreover, the 0-0 absorption band in the LB films is red shifted relative to the 0-0 band at the air-water interface by 26 nm (see (13) (a) McRae, E. G.; Kasha, M. In Physical Processes in Radiation Biology; Augenstein, L., Mason, R., Rosenberg, B., Eds.; Academic Press: New York, 1964. (b) McRae, E. G.; Kasha, M. J. Chem. Phys. 1958, 28, 721. (c) Kasha, M.; Rawls, H.; El-Bayoumi, M. Pure Appl. Chem. 1965, 11, 37. (14) (a) Jelly, E. E. Nature 1936, 138, 1009. (b) Schiebe, G. Angew. Chem. 1936, 49, 563. (c) Czikkely, V.; Forsterling, H. D.; Kuhn, H. Chem. Phys. Lett. 1970, 6, 11. (d) Czikkely, V.; Forsterling, H. D.; Kuhn, H. Chem. Phys. Lett. 1970, 6, 207. (e) West, W.; Caroll, B. M. J. Chem. Phys. 1951, 19, 417.

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Figure 3. Fluorescence emission spectra of HDFL in methanol (5 × 10-3 M) (dashed line) and of a film of HDFL and PA mixed in a molar ratio of 1:20 at the air-water interface at a surface pressure of 10 mN/m (continuous line). λex ) 400 nm.

Table 1). One plausible explanation could be the polarityinduced changes in the molecular conformation of HDFL that result in different packing configurations of the HDFL molecules in different systems. In fact, the molecular structure of fluorescein is reported to be polarity dependent.4-6 It is likely that, due to the high polarity at the air-water interface, fluorescein may exist in its ionic/zwitterionic form. However, in the LB films, the fluorescein chromophores probably exist in their lactonic form as the microenvironment is nonpolar. A close examination of the HDFL structure, as shown in the inset of Figure 1, reveals that indeed the phenyl group may undergo rotation about the single bond attaching it to the rigid aromatic group. The rotation of one group with respect to another is driven by such factors as polarity of the microenvironment as in twisted intramolecular charge transfer (TICT)15 probes or by externally applied mechanical pressure as reported for several molecules.16,17 Given these details, it does not seem improbable that, due to polarity, applied surface pressures and monolayer transfer, the HDFL molecule undergoes deformation to attain a more tightly packed geometry in the LB films compared to that at the air-water interface. Figure 3 shows the fluorescence emission spectra of pure HDFL in methanol and at the air-water interface in the 450-600 nm region with the 0-0 emission bands located at 512 and 518 nm, respectively. The small red shift of 6 nm (see Table 1) observed in the case of the mixed monolayer at the air-water interface with respect to methanol is due to organized aggregation of the HDFL chromophores at the air-water interface. As mentioned earlier, the fluorescence of fluorescein is sensitive to the polarity of its microenvironment;4-6 however, the contri(15) (a) Rettig, W. Angew. Chem., Int. Ed. Engl. 1986, 25, 971. (b) Rettig, W. Photochemical Processes in Organized Molecular Systems; North-Holland, Delta Series: New York, 1991; p 61. (c) Rettig, W. In Topics in Current Chemistry; Mattay, J., Ed.; Springer-Verlag: New York, 1994; Vol. 169, p 253. (d) Dutta, A. K.; Kamada, K.; Ohta, K. Chem. Phys. Lett. 1996, 258, 369. (e) Dutta, A. K.; Kamada, K.; Ohta, K. J. Photochem. Photobiol., A, in press. (f) Rettig, W.; Lapouyade, R. In Topics in Fluorescence Spectroscopy, Probe Design and Chemical Sensing; Lakowicz, J. R., Ed.; Plenum Press: New York, 1991; p 109. (16) (a) Vuorimaa, E.; Lemmityinen, H.; Van der Auwearer, M.; DeSchryver, F. C. Thin Solid Films 1995, 268, 114. (b) Vuorimaa, E.; Ikonen, M.; Lemmetyinen, H. Chem. Phys. 1994, 188, 289. (c) Vuorimaa, E.; Ikonen, M.; Lemmetyinen, H. Thin Solid Films 1992, 214, 243. (d) Tamai, N.; Yamazaki, T.; Yamazaki, I. Can. J. Phys. 1990, 68, 1013. (17) (a) Dutta, A. K.; Misra, T. N.; Pal, A. J. J. Phys. Chem. 1994, 98, 12844. (b) Dutta, A. K. J. Phys. Chem. 1995, 99, 14758. (c) Dutta, A. K. J. Phys. Chem. B 1997, 101, 569.

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Figure 4. Fluorescence emission spectra of HDFL in dichloromethane (short dashed line) and of a LB film of HDFL and PA mixed in a molar ratio of 1:20 and deposited on quartz slides at a surface pressure of 25 mN/m (long dashed line). λex ) 350 nm.

bution of polarity may be neglected in this case as the polarities of methanol and at the air-water interface are similar. Figure 4 shows the emission spectrum of a mixed LB film of HDFL and PA transferred onto a quartz substrate. The emission profile of this transferred LB film is observed to be different compared to the emission profile of the monolayer at the air-water interface (Figure 3). Interestingly, the 0-0 emission band in the LB films is located at 464 nm in contrast to 519 nm for the mixed monolayer at the air-water interface. The large blue shift of 55 nm observed in the case of the transferred film is not readily explicable. Several possibilities exist. One explanation seems to be the nonpolar microenvironment in the transferred films compared to a highly polar microenvironment at the air-water interface. A second possibility could be a different packing of the HDFL chromophores in the transferred films compared to that at the air-water interface or a superposition of both the effects of polarity and aggregation. To elucidate the role of polarity, we have recorded the emission spectra of HDFL in dichloromethane as shown in Figure 4. The 0-0 emission band in dichloromethane is located at 408 nm (Table 1), and the entire emission spectral profile is blue shifted relative to that in methanol (Figure 3 and Table 1). These observations are analogous to the blue-shifted emission of the LB films relative to the emission from the mixed monolayer at the air-water interface that highlights the role of polarity. To confirm that the above observations were not due to artifacts or due to some special interactions between HDFL and dichloromethane, we have tried several solvents, namely, DMSO, ACN, pyridine, 1,4-dioxane, and dimethylformamide. Identical results were obtained in all of the different solvents, which confirmed the absence of any specific interactions between HDFL and the solvents. Dissolution of the LB films in dichloromethane yielded a band maximum at 430 nm and the 0-0 band at 408 nm, thus confirming the absence of artifacts or impurities. Addition of small amounts (5% v/v) of methanol, ethanol, or water caused the band at 430 nm to disappear, and the well-known band characteristics of fluorescein shown in Figure 3 were restored. These results confirmed that the hydroxyl group in protic solvents generated the fluorescent ionic species, while in non-hydrogen-bonding, protonattracting solvents such as dioxane, DMSO, etc., the fluorescein chromophores existed in their nonfluorescent inner lactonic form. The conversion of the inner lactone form of fluorescein to their zwitterionic form is reversible,

(Hexadecanoylamino)fluorescein Aggregates

and the process is remarkably enhanced by the presence of hydroxyl groups. Addition of acids causes protonation of the carboxylate group in the molecule, lowering its fluorescence yield.5,6 These results justify the observed differences in the emission spectra of HDFL at the airwater interface and in the mixed LB films. One plausible explanation of the origin of the emission band with its maximum at 430 nm could be the emission from the rigid condensed polyaromatic ring system of fluorescein, with the oxygen bridge corresponding to an anthracene derivative. In fact, the electronic configurations of xanthenes18 and acridines are reported to be very similar to that of the anthracenes.19a-c Interestingly, a similar blue-shifted emission has been reported for rhodamine dyes in nonpolar solvents which has been attributed to the fluorescence emission from the lactonic species of the rhodamine dyes.19 To understand the role of aggregation, we have compared the emission spectra of HDFL assembled in LB films with that in dichloromethane since the polarities in both systems are similar. It can be seen in Figure 4 that the emission spectral profile of HDFL in the LB films is red shifted compared to that in dichloromethane which may be attributed to the formation of highly organized aggregates of HDFL in its lactonic form. It seems likely that, due to an ordered packing of the molecules, strong molecular interactions between the xanthene condensed polycyclic ring system are manifested as a red shift of the emission band. Furthermore, a close examination of the absorption and emission spectra of HDFL in the transferred LB films (Figures 2 and 4) revealed that no emission corresponding to the absorption band at 518 nm was observed in the mixed LB films of HDFL and PA, which suggests the presence of a second variety of aggregates in the mixed LB films which are very likely nonfluorescent and different from those emitting with their 0-0 band at 464 nm as shown in Figure 4. Several explanations seem plausible. One possibility seems to be that, during compression and transfer, species with different molecular conformations are generated, where the plane of the phenyl ring makes different angles with the plane of the condensed ring system. Such a possibility does not seem unreasonable in view of the fact that the phenyl ring is connected to the rigid condensed aromatic system by a single bond about which rotation is indeed possible. Moreover, detailed studies have revealed that the relative orientation of the groups depends on factors such as temperature, polarity, pH, and applied mechanical pressure that allow different configurations of the molecule which are readily manifested by large changes in their spectral characteristics.15-18 The resultant orientation is, however, a delicate balance between the interaction of the molecule with their local environment and thermodynamic and geometric requirements of the system. Interestingly, several authors have demonstrated that in similar molecules, namely, the rhodamines, the phenyl group rotates in a plane perpendicular to the xanthene (18) (a) Rohatgi, K. K.; Singhal, G. S. J. Phys. Chem. 1966, 170, 1695. (b) Chambers, R. W.; Kajiwara, T.; Kearns, D. R. J. Phys. Chem. 1974, 78, 380. (c) Selwyn, J. E.; Steinfield, J. J. Phys. Chem. 1972, 76, 762. (d) Dutta, A. K.; Lavoie, H.; Ohta, K.; Salesse, C. Langmuir 1997, 13, 801. (e) McDonald, R. I. J. Biol. Chem. 1990, 265, 13533. (f) Brown, R. S.; Brenan, J. D.; Krull, U. J. J. Chem. Phys. 1994, 100, 6019. (g) Lopez-Arbeloa, I.; Rohatgi, K. K. Chem. Phys. Lett. 1986, 129, 607. (h) Lopez-Arbeloa, I.; Rohatgi, K. K. Chem. Phys. Lett. 1986, 128, 105. (19) (a) Barashkov, N. N.; Gunder, O. A. In Fluorescent Polymers; Kemp, T. J., Ed.; Ellis Horwood: New York, 1994; p 26. (b) Nurmukhametov, R. N.; Kunavin, N. I.; Khachaturova, G. T. Izv. Akad. Nauk SSR, Ser. Fiz. 1978, 42, 517. (c) Kumavin, N. I.; Nurmukhametov, R. N. Zh. Prikl. Spektrosk. 1977, 26, 1120. (d) Baraka, M. E.; Deumie, M.; Viallet, P.; Lampidis, T. J. Photochem. Photobiol., A 1991, 62, 195. (e) Lopez-Arbeloa, I.; Ruiz-Ojeda, P. Chem. Phys. Lett. 1981, 79, 347. (f) Lopez-Arbeloa, I.; Rohatgi-Mukherjee, K. K. Chem. Phys. Lett. 1986, 128, 474.

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Figure 5. (A) Fluorescence emission spectra of a mixed film of HDFL and PA (molar ratio 0.046) at the air-water interface at different surface pressures: (a) 5.9, (b) 10.2, (c) 16.2, (d) 20.2, (e) 34.9, and (f) 39.8 mN/m. (B) Plot of the fluorescence intensity of a mixed film of HDFL and PA at the air-water interface versus surface pressure for films at three different molar ratios: (a) 0.046 (squares); (b) 0.094 (circles); (c) 0.278 (diamonds).

group, attaining different molecular configurations during compression or deposition in LB mono- and multilayers.15-17 Given the above facts and the remarkable similarity in the molecular structure of the fluoresceins with the rhodamines, it does not seem unreasonable that during the compression and transfer processes two different species are generated, one corresponding to the fluorescent variety with intense fluorescence at 490 nm and the other to the nonfluorescent variety with an intense absorption band at 518 nm. Since aggregation may be induced by surface pressure at the air-water interface that involves bringing the molecules closer to one another, we have studied the effects of surface pressure and concentration on the fluorescence emission of HDFL at the air-water interface. Figure 5A shows the fluorescence emission spectra of a mixed film of HDFL and PA at different surface pressures, while Figure 5B shows the plot of the fluorescence intensity of HDFL from the mixed films at different surface pressures corresponding to different concentrations of HDFL in the mixed films. It is observed that the fluorescence emission intensity decreases with increasing surface pressure and dye concentration. One possible explanation seems to be that, with increasing surface pressure or concentration, the HDFL moieties are brought closer together, resulting in the formation of aggregates that are very likely nonfluorescent. Efficient energy tranfer from the fluorescent monomeric species to the nonfluorescent aggregates and decay therefrom by nonradiative processes result in fluorescence quenching of the aggregates. To confirm that the observed effects were not due to artifacts, the reproducibility of the experiments was checked at different excitations in the 300-390 nm region. Identical observations are reported for xanthene molecules, namely, the rhodamines.18

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Figure 6. Excitation spectrum of HDFL in dichloromethane (short dashed line) with the emission monitored at 430 nm; excitation spectrum of HDFL in methanol (long dashed line) with the emission monitored at 518 nm; excitation spectrum of the deposited LB films of HDFL and PA mixed in a molar ratio of 1:20 deposited on quartz slides (solid line) with the emission monitored at 490 nm.

Figure 6 shows the excitation spectra of HDFL in several different microenvironments. Monitoring the emission band at 430 nm in dichloromethane, the 0-0 band was found to be located at 405 nm, which is in good agreement with the 0-0 emission band at 409 nm. Corresponding to the emission maximum at 518 nm in methanol, the 0-0 excitation band was found to be located at 489 nm, which is in agreement with the origin of the absorption band at 482 nm. Interestingly, the excitation spectral profile of HDFL in methanol and dichloromethane is observed to be different, which confirms the fluorescent species in the two different systems are different. Addition of methanol (5% v/v) in dichloromethane reproduced the corresponding emission and excitation spectra of HDFL in methanol, confirming the conversion of the lactonic species to the zwitterionic form. Corresponding to the fluorescence emission at 490 nm in the LB films, the 0-0 excitation band was obtained at 437 nm (Figure 6). Close examination reveals that the excitation spectral profile of HDFL in dichloromethane and in LB films and the location of the 0-0 bands are completely different despite a nonpolar microenvironment in both systems which suggests that the emitting species in the two systems are different. It is noteworthy that the band at 437 nm revealed in the excitation spectra of the LB films does not correspond to the absorption band at 519 nm which confirms the existence of at least two different species corresponding to different molecular conformations of HDFL in the LB films. The excitation spectrum of HDFL mixed with PA at the air-water interface obtained by monitoring the emission at 550 nm yielded a spectrum similar to that in methanol with its maximum at 468 nm and the 0-0 band at 493 nm (Table 1) which is in excellent agreement with the absorption 0-0 band at 493 nm measured at the air-water interface. The close similarity between the excitation spectra of HDFL at the air-water interface and in methanol is in fact expected due to their high polarity. Fluorescence Microscopy of Pure and Mixed Monolayers of HDFL and PA. Fluorescence microscopic techniques applied to monolayers at the air-water interface provide a unique and versatile method to visually observe the behavior of monolayers in situ and have been extensively used to study such interesting effects as phase transition, dye distribution, formation of macroscopic aggregates, and crystallization. In this section, we have studied the behavior of a monolayer of pure HDFL at the air-water interface as a function of surface pressure. Figure 7 shows the fluorescence micrographs of a mono-

Dutta and Salesse

layer of pure HDFL at the air-water interface at different surface pressures. Interestingly, the changes were not abrupt and the field of view remained unchanged for small pressure variations in the range of 1-3 mN/m. Figure 7A corresponds to the micrograph of a HDFL film at an average low surface pressure of 3 mN/m that reveals bright circular spots. As discussed in the previous section, intense fluorescence originates from monomeric species only and aggregates of HDFL are nonfluorescent. Indeed, this seems justified as, at low surface pressures, the molecules remain far apart from each other and their dipole moments are randomly oriented. Figure 7(B-D) shows the micrographs of HDFL at the air-water interface corresponding to average surface pressures of 6, 9, and 13 mN/m, respectively. Sharply in contrast to the welldefined circular spots observed in Figure 7A, these micrographs (Figure 7B,C) show irregular-shaped dark patches in a bright background. These patches very likely correspond to the formation of large macroscopic agglomerates of the dye. Close inspection of Figure 7B-D reveals that, with increasing surface pressure, the overall fluorescence intensity of the field of view decreases and the irregular-shaped dark clusters grow larger in size. Further compressing the HDFL film at the air-water interface results in a complete darkening of the field of view. This darkening of the field of view and the formation of dark clusters may be rationalized from the spectroscopic point of view as due to molecular association of the HDFL moieties, resulting in the formation of nonfluorescent agglomerates. Efficient energy tranfer from the monomeric species to these aggregates results in fluorescence quenching of HDFL. A plausible explanation seems to be an efficient energy transfer from the monomers to the aggregates which may originate from a favorable coupling between the dipole moments of the molecules in these aggregates compared to a weaker and probably unfavorable coupling between the dipoles in solution or in the gaslike phase where the dipole moments are randomly oriented. Decompressing the HDFL monolayer at the air-water interface reveals the reappearance of bright fluorescent patches in a dark field of view (Figure 7E,F). Lowering the surface pressure results in the bright patches growing larger in size until the field of view becomes totally luminescent (Figure 7G,H). Closer examination of the micrograph in Figure 7H, in particular, reveals dark patches even at very low surface pressures of about 3 mN/ m, which indicates the presence of agglomerates of HDFL that did not completely disintegrate at the molecular level as a result of decompression. Conclusion In brief, this study has demonstrated that HDFL forms excellent monolayers at the air-water interface that are highly stable and may be easily transferred onto solid substrates mixed with a fatty acid. Comparative studies between the absorption spectra of HDFL in solution, in monolayers at the air-water interface, and in LB films deposited on quartz substrates confirm J-type aggregation of HDFL chromophores both at the air-water interface and in the transferred LB films. The large red shift in the absorption band maximum in the case of the deposited films compared to that at the air-water interface has been attributed jointly to the differences in the molecular configuration, orientation, and organization of the molecules in the transferred LB films and at the air-water interface. Steady-state fluorescence measurements of HDFL films at the air-water interface revealed that its spectral profile is identical with that in methanol which may be attributed to the zwitterionic form of the dye in

(Hexadecanoylamino)fluorescein Aggregates

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Figure 7. Epifluorescence micrographs of a pure film of HDFL at the air-water interface at different surface pressures. Compression cycle: (A) 3, (B) 6, (C) 9, and (D) 13 mN/m. Decompression cycle: (E) 11, (F) 9, (G) 6, (H) 1 mN/m. The scale bar shown in the figure indicates 20 µm.

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both systems that offer a polar and protic environment. In the mixed LB films, the fluorescence emission was found to be blue shifted relative to that at the air-water interface and has been attributed to the presence of the aggregated lactone form of the dye. Detailed spectroscopic studies indicate that two configurationally different aggregated species exist in the mixed films, where one is fluorescent while the other is not. Surface pressure and concentration dependent in situ steady-state fluorescence studies of HDFL films at the air-water interface revealed intense fluorescence quenching that is attributed to efficient energy transfer from the highly fluorescent HDFL mon-

Dutta and Salesse

omeric species to the low-energy HDFL aggregates that are nonfluorescent. Epifluorescence microscopic monitoring of the compression and decompression of pure films of HDFL revealed intense fluorescence quenching of the monolayer that has been attributed to the formation of nonfluorescent macroscopic agglomerates of the dye, as is readily visualized in the fluorescence micrographs. Acknowledgment. The authors are indebted to Natural Sciences and Engineering Research Council of Canada, Fonds FCAR, and FRSQ for financial support. LA970327M