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Langmuir and Langmuir-Blodgett Film Characterization of a New Amphiphilic Coumarin Derivative Pe´ter Kele, Jhony Orbulescu, Sarita V. Mello, Mustapha Mabrouki, and Roger M. Leblanc* Center for Supramolecular Science and Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33124 Received June 18, 2001. In Final Form: August 24, 2001 In this paper, we report the synthesis and the monolayer behavior (surface pressure and surface potential) of a new amphiphilic coumarin dye, 7-aminocoumarin-4-acetic acid octadecylamide (ACO), at the airwater interface. The spectroscopic characteristics (UV-vis, fluorescence, and fluorescence imaging) of pure and mixed films (1:20, ACO/stearic acid and ACO/oleic acid) at the air-water interface as well as Langmuir-Blodgett (LB) films have been investigated and compared with the spectroscopic characteristics of ACO in solution. These experiments provide evidence of aggregate formation during compression of the monolayer at the air-water interface. Surface pressure dependent in situ fluorescence imaging confirms that the fluorescent quenching of the dye with increasing surface pressure originates as a result of formation of nonfluorescent aggregates. Atomic force microscopy imaging of a pure ACO LB film shows that the size of these aggregates is in the nanometer scale. This work provides information that ACO forms a stable monolayer and may be utilized as an efficient molecular probe for monolayer studies.
Introduction Fluorescence spectroscopy and fluorescence microscopy are very useful in observing the assembly process of the components in Langmuir monolayers, measuring lipid diffusion, and investigating molecular organization at the interface. Nonamphiphilic coumarin derivatives are widely used as blue fluorophores, and their optical and photophysical properties in solution have been extensively studied.1-3 However, amphiphilic coumarin derivatives as fluorescent probes in monolayer studies have been neglected.4 The commercially available amphiphilic coumarins are 7-hydroxycoumaryl derivatives. Their fluorescence is dependent on the protonation/deprotonation of the phenolic group, and they are not fully fluorescent below pH ∼10. 7-Aminocoumarins, however, are completely deprotonated above pH ∼5; thus, their fluorescence spectra are not subject to variability due to the pH dependent protonation/deprotonation when used near or above physiological pH.5 Langmuir monolayers and Langmuir-Blodgett (LB) films of amphiphilic dyes may have substantially different optical properties;6 therefore, it is necessary to characterize them from this point of view. The photophysical properties of these molecules can be modified due to (i) molecules assembled into an ordered film, (ii) polarity of the surrounding medium,7-9 (iii) structural changes caused by the applied external pressure during compression,10,11 * Corresponding author phone, +1(305)284-2282; fax, +1(305)284-4571; e-mail,
[email protected]. (1) Rettig, W.; Klock, A. Can. J. Chem. 1985, 63, 1649. (2) Jones, G.; Jackson, W. R.; Halperin, A. Chem. Phys. Lett. 1980, 72, 391. (3) Drexhage, K. H. Topics in Applied Physics; Shaper, F. F., Ed.; Springer: Berlin, 1973; Vol. 1. (4) Alekseev, A. S.; Lemmetyinen, H.; Nikitenko, A. A.; Peltonen, J.; Milyaev, V. A. Russ. J. Phys. Chem. 1999, 73, 1136. (5) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Spence, M. T. Z., Ed.; Molecular Probes, Inc.: Eugene, OR, 1996. (6) Roberts, G. G. Langmuir-Blodgett Films; Plenum: New York, 1990. (7) Dutta, A. K.; Salesse, C. Langmuir 1997, 13, 5401. (8) Rettig, W. Angew. Chem., Int. Ed. Engl. 1986, 25, 971.
and (iv) formation of aggregates.12-15 These effects can be seen as a change in the position and/or the intensity of the bands in the UV-vis and fluorescence spectra. The decrease in fluorescence intensity is known as fluorescence quenching, as a result of efficient energy transfer from the fluorescent monomer species to the aggregates that are likely nonfluorescent and decay via nonradiative processes. To avoid the formation of aggregates, the amphiphilic dye molecules can be mixed with nondye amphiphiles, such as fatty acids or peptide-lipids.12 It is important that the two components of these mixed monolayers are miscible.16 If the materials are immiscible, the components will exist as domains; hence, the aggregates are still formed. If, however, they are miscible, the dye molecules are diluted by the excessive amount of the other molecule. As long as the dye molecules are wellseparated from each other, no aggregates will form. In this paper, we report the synthesis and surface chemistry characterization of a new amphiphilic 7-aminocoumarin, namely, 7-aminocoumarin-4-acetic acid octadecylamide (ACO). The structure of this compound is given in Figure 1. We also investigate the photophysical properties of ACO as a Langmuir monolayer and LB films. Experimental Section The long-chain coumarin derivative, ACO, was synthesized as described below. Solutions were prepared with spectroscopic grade solvents purchased from Fischer Scientific (Fair Lawn, NJ). The pure ACO solution was prepared using chloroform solvent at a concentration of 10-3 M. For the preparation of the mixed ACO/SA (stearic acid) and ACO/OA (oleic acid) monolayers, (9) Dutta, A. K.; Kamada, K.; Ohta, K. Chem. Phys. Lett. 1996, 258, 369. (10) Dutta, A. K.; Misra, T. N.; Pal, A. J. J. Phys. Chem. 1994, 98, 12844. (11) Dutta, A. K. J. Phys. Chem. B 1997, 101, 569. (12) Orbulescu, J.; Mello, S. V.; Huo, Q.; Sui, G.; Kele, P.; Leblanc, R. M. Langmuir 2001, 17, 1525. (13) Cikkely, V.; Forsterling, H. D.; Kuhn, H. Chem. Phys. Lett. 1970, 6, 11. (14) West, W.; Caroll, B. M. J. Chem. Phys. 1951, 19, 417. (15) Jelly, E. E. Nature 1936, 138, 1009. (16) Furasaki, N. J. Colloid Interface Sci. 1982, 90, 551.
10.1021/la010907q CCC: $20.00 © 2001 American Chemical Society Published on Web 10/16/2001
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Figure 1. Structure of ACO. SA and OA, respectively, were dissolved in CHCl3 at a concentration of 10-3 M and mixed with a 10-3 M ACO solution to reach a ratio of SA or OA and ACO of 20:1 (v/v). Pure water used as the subphase was provided from a Modulab 2020 water purification system (Continental Water Systems Corporation, San Antonio, TX). The resistance and surface tension of pure water were 18 MΩ cm and 72.6 mN/m at 20.0 ( 0.5 °C, respectively. The pH of the subphase for all experiments was in the range of 5.5-6.0. Following the deposition of the spreading solution, we left the film at nil surface pressure 15 min before compression to allow the solvent to evaporate. Quartz slides used for LB film preparation were cleaned by soaking the slides in a H2O/H2O2/ NH3 (5:1:1) solution at 80 °C for 10 min and then thoroughly washed with pure water. The quartz slides were kept in pure water prior to use. Methods. All isotherm measurements were carried out in a clean room class 1000 at a constant temperature of 20.0 ( 0.5 °C. For surface pressure-area isotherm determinations, we used a KSV minitrough model 2000 (KSV Instrument Ltd., Helsinki, Finland). The trough is supplied with an electronic balance that uses a Wilhelmy plate as the surface pressure sensor and has a sensitivity of 0.02 mN m-1. For monolayer compression, two symmetrically movable computer controlled barriers were used. A typical compression speed was 5 Å2 molecule-1 min-1. The volume spread at the air-water interface varied from 20 to 150 µL. The trough has a quartz window fitted in the middle for in situ spectroscopic measurements. Y-type deposition of the monolayer on quartz and fresh mica substrates was achieved by dipping the substrate vertically through the monolayer at a speed of 1.5 mm/min. The deposition ratio was approximately 0.8. A drying time of 10 min was allowed between consecutive dippings. Surface potential measurements were obtained in the KSV trough using a Kelvin probe. It consists of a capacitor-like system, which has a vibrating plate set approximately 1 mm above the surface of the monolayer and a counter electrode dipped into the clean subphase. The clean subphase was taken as the zero reference. Solution absorption spectra were obtained from a UV-visNIR Perkin-Elmer spectrometer Lambda 900, using a quartz cuvette of 1 cm path length. The in situ UV-vis absorption spectra of the Langmuir monolayer were performed with a HP spectrophotometer model 8452A, set on a rail close to the KSV trough, suitable for approach toward the quartz window. The fluorescence emission spectra were recorded from a Spex Fluorolog 1680 spectrophotometer. For air-water interface studies, an optical fiber probe was put 1 mm above the water surface and the excitation and emission light were transmitted through the optical fiber. For the measurement of fluorescence from LB films, the glass slide used for the deposition was precut to the size, which can exactly fit the 1 cm fluorescence cuvette with an angle of 45° facing both the incidence and the emission light beam. An epifluorescence microscope (Olympus IX-FLA) was used for acquiring the fluorescence micrographs of the monolayer at the air-water interface at different constant surface pressures during compression on a Kibron minitrough (area available for spreading solution 5.9 × 19.5 cm2, Kibron Inc., Helsinki, Finland). A thermoelectrically cooled Optronics MagnafireTM CCD camera detected the emission from the floating monolayer. The atomic force microscopy (AFM) contact mode was used to image the LB film (one layer), which was scanned in air in constant force mode. A Molecular Imaging PicoSPM microscope was used for this work. Commercially sharpened Si3N4 tips attached to rectangular beams were used (spring constant 0.09
Figure 2. Surface pressure and surface potential-area isotherms of ACO. The inset is the hysteresis of the ACO monolayer (20, 30, and 40 mN/m vV). N/m). The probing force was between 1 and 5 nN. The images were obtained immediately after deposition of the monolayer. Synthesis of ACO. The chemical characterizations of the compound were performed using a Bruker AC 300 NMR (1H NMR and 13C NMR) and a Perkin-Elmer 2000 FT-IR. Highresolution mass spectrometry was carried out at the University of Illinois at Urbana-Champaign Mass Spectrometry Laboratory. Synthesis. 1-Hydroxybenzotriazol (20 mg, 0.146 mmol) and 1,3-dicyclohexylcarbodiimide (30 mg, 0.146 mmol) were dissolved in dimethylformamide (2 mL). ACO17 (16 mg, 0.073 mmol) was added in small portions to this solution. Then, 1-octadecylamine (40 mg, 0.146 mmol) in chloroform (2 mL) was added in small portions and the resulting solution was stirred overnight. The solvent was removed by evaporation. The product was purified on silica using chloroform-methanol (10:1, v/v) as the eluent. The solvent was evaporated to give 22 mg of product as a pale yellow powder (64%). ACO was readily soluble in chloroform, which is an ideal spreading solvent for monolayer preparation. A 10-3 M solution was used in all of the monolayer studies. 1H NMR (CDCl3/CD3OD, 393 K vs TMS): δ 0.88 (3H, t, J ) 6.57 Hz), 1.17-1.31 (30H, m), 1.4-1.52 (2H, m), 3.18 (2H, t, J ) 7.02 Hz), 3.6 (2H, s), 3.87 (2H, s), 6.09 (1H, s), 6.58 (1H, d, J ) 2.19 Hz), 6.63 (1H, dd, J ) 2.19, 8.33 Hz), 7.39 (1H, s), 7.48 (1H, d, J ) 8.33 Hz). 13C NMR (CDCl3/CD3OD, 393 K vs CDCl3): δ 13.8, 22.4, 29.1, 29.4, 29.5, 31.7, 33.5, 39.7, 39.9, 100.4, 109.8, 110.1, 112.0, 126.1, 150.8, 155.6, 157.7, 162.5, 168.3. IR (KBr): 34403340, 1710 cm-1. HRMS: (HM+) calcd for C29H47N2O3, 471.3586; found, 471.3586.
Results and Discussion Figure 2 shows the surface pressure (π) and surface potential (∆V)-area isotherms of ACO at the air-water interface. Compressing the amphiphile resulted in a surface pressure-area isotherm that showed a nil surface pressure (75-37 Å2/molecule) followed by a lift at 37 Å2/ molecule and a knee in the region of 37-30 Å2/molecule; (17) Besson, T.; Joseph, B.; Moreau, P.; Viaud, M.-C.; Coudert, G.; Guillaumet, G. Heterocycles 1992, 34, 2, 273.
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Table 1. Absorbance and Emission Maxima of the ACO Probe in Solutions (10-4 and 10-7 M Solutions for Absorption and Fluorescence Measurements, Respectively) and in Monolayers
absorbance emission
MeOH (nm)
CHCl3 (nm)
358 438
347 419
monolayer (nm) pure mixed 375 446
452
LB film 4 layers (air-quartz) (nm) 367 428
a steep rise is observed from 30 to 22 Å2/molecule. A further increase of the surface pressure resulted in a collapse of the monolayer, i.e., at 60 mN/m. The limiting molecular area is 32 Å2/molecule, which is higher than that of SA, i.e., 22 Å2/molecule. This difference is due to the bigger size of the coumarin polar headgroup that limits the closer approach of the neighboring molecules. Hysteresis studies (small inset in Figure 2) showed that the monolayer is stable. The surface potential-area isotherm of pure ACO at the air-water interface is also shown in Figure 2. The surface potential was observed to be flat until 55 Å2/ molecule. As the compression was continued, a moderate increase could be seen in the 55-45 Å2/molecule region. This was followed by a sudden increase (45-37 Å2/ molecule). After this, a steady rise was observed (37-25 Å2/molecule) indicating that further compression caused moderate changes in the orientation of the molecular dipoles. The surface potential-area isotherm shows an earlier increase, i.e., at 55 Å2/molecule (occurs at a larger molecular area) as compared to the surface pressurearea isotherm, i.e., 37 Å2/molecule. This is due to the fact that surface pressure-area isotherm is related to the van der Waals interactions between the hydrocarbon chains of the ACO molecules, while the surface potential-area isotherm is related to the dipole-dipole interactions between the polar headgroups in the water subphase. Because dipole-dipole interactions can occur at larger intermolecular distances, the molecules “feel” each other and start to interact earlier than seen in the surface pressure-area isotherm where the van der Waals interactions are at short range.18 The UV-vis absorption spectra of ACO were studied in chloroform and methanol solutions and as a monolayer at the air-water interface (Table 1). The absorption spectra at the air-water interface showed a maximum (375 nm) that was shifted toward the red region relative to either the chloroform (347 nm) or the methanol (358 nm) solution. According to the intermediate strength exciton-coupling model of McRae and Kasha,19 dipole-dipole interactions between neighboring molecules may generate an exciton band that is located either below or above the monomer exciton band. While aggregates causing a red shift are referred to as J aggregates, the aggregates causing a blue shift are referred to as H aggregates. Small shifts may originate from differences in the refractive index of the matrix in which the molecule is located.19 In this case, however, the shift is significantly greater: 28 nm (monolayer-CHCl3) and 17 nm (monolayer-MeOH), respectively. Another reason that should be taken into account is the difference in polarity of the environment. The coumarin headgroup is submerged in the water subphase; (18) Gaines, G., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publisher: New York, 1966; p 188. (19) McRae, E. G.; Kasha, M. Physical Processes in Radiation Biology; Augenstein, L., Mason, R., Rosenberg, B., Eds.; Academic Press: New York, 1964.
Figure 3. Fluorescence quenching of pure ACO and ACO/SA and ACO/OA (1:20, v/v) monolayers at different surface pressures (λexcitation ) 370 nm).
therefore, it represents a polar medium while the chloroform solution represents a nonpolar medium. It was assumed that the photophysical properties of the methanol solution are similar to the case where the ACO headgroups are surrounded by water. It seems reasonable then to compare the absorption spectrum of the methanol solution and the monolayer as both representing a polar medium where the contribution of the polarity difference can be neglected. It can be concluded that the J aggregates contribute a red shift of 17 nm. The fluorescence spectrum of pure a ACO monolayer at the air-water interface shows a red shift of 8 nm in the emission maximum with respect to methanol solution. This is due to organized aggregation of ACO molecules at the air-water interface. As mentioned earlier, the contribution of the polarity can be neglected in this case as the polarities in both cases are similar. The intensity of the fluorescence dramatically drops as the monolayer is compressed (Figure 3). This phenomenon also supports the fact that the molecules form aggregates. Efficient energy transfer from fluorescent monomeric species to the aggregates that are very likely nonfluorescent and decay via nonradiative processes results in fluorescence quenching. To limit the formation of the aggregates, mixed monolayers of ACO/SA and ACO/OA were prepared at a molar ratio of 1:20 (ACO/SA, ACO/OA). Figure 3 also shows the fluorescence intensity as a function of surface pressure for both mixed monolayers. Indeed, although the fluorescence intensity dropped with the increasing surface pressure in both cases, these drops were far less remarkable as in the case of the pure ACO monolayer. Oleic acid, which has a bigger need for space, separates the fluorophores more efficiently than SA, and a higher intensity of fluorescence can be seen up to 15 mN/m. While the ACO/SA curve shows a continuous decrease in fluorescence intensity, suggesting that aggregate formation is present even at nil surface pressure, an increase can be seen when the ACO/OA mixed monolayer is compressed from 0 to 3
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Figure 4. Epifluorescence microscope images of pure ACO (a), ACO/SA (b), and ACO/OA (1:20, v/v) (c) monolayers (image size, 895 µm × 713 µm). From top to bottom in each column, each panel corresponds to the points 1, 5, 15, and 20, respectively, the surface pressure-area isotherm graph.
mN/m. This also illustrates that OA separates the fluorophores at very low surface pressures (e.g., 0-3 mN/
m). The fluorescence intensity rises as the fluorophore concentration increases on the surface. After 3 mN/m
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surface pressure is reached, the ACO molecules are close enough to each other to form aggregates as the fluorescence is quenched while the monolayer is compressed further (Figure 3). For the investigation of the spectroscopic properties of ACO on the solid substrate, four layers of pure ACO were deposited on a quartz slide at 15 mN/m using the LB technique. There is a red shift in the absorbance maximum of the LB film (367 nm) as compared to either methanol or chloroform solution (Table 1). This red shift is due to the organization of the molecules and the presence of aggregates that were transferred onto the solid support. Nevertheless, this red shift is smaller by 8 nm than that of the monolayer at the air-water interface. This can be explained by the difference in the environment around the polar headgroups. At the air-water interface, the water subphase provides the possibility for hydrogen bonding not only between the ACO molecules but also between the ACO headgroups and the water. In the LB film, however, the absence of the aqueous subphase limits the presence of hydrogen bonding mainly to the interaction between the ACO headgroups. The fluorescence spectrum of the LB film shows a red shift that follows a similar pattern with the UV-vis spectrum (Table 1). This is additional proof that hydrogen bonding plays an important role that influences the photophysical properties. 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 dye distribution and aggregate formation.7 Figure 4 shows the epifluorescence images of pure ACO and mixed ACO/ SA and ACO/OA monolayers at four different surface pressures. In the case of the pure ACO monolayer, even at very low surface pressures, dark nonfluorescent spots show up in the blue surroundings (Figure 4a). The blue color originates from the monomeric species. As the surface pressure is increased, the size and the number of these dark areas increase. At 30 mN/m, the intensity of the fluorescence was very low (image not shown). It is very reasonable to say that these spots are the forming aggregates, which are nonfluorescent and responsible for the quenching. When the ACO/SA mixed monolayer was used (Figure 4b), strong blue fluorescence was observed with evenly distributed small dark-blue spots after spreading. Because of incomplete spreading, there is a large irregular black area at 1 mN/m where no fluorophores are present. At 5 mN/m, the monolayer fully covered the surface. As the surface pressure increased, larger, circular shaped dark spots showed up. The number and the size of these dark circles increased with further compression of the monolayer, but the fluorescence was dominant all the time. As previously mentioned, these patches very likely correspond to the formation of large macroscopic aggregates of the dye. In the case of the ACO/OA mixed monolayer (Figure 4c), no domain or aggregate formation could be seen under our experimental conditions and a homogeneous blue fluorescence was observed. The nonappearance of the aggregates in this latter case suggests that although there is quenching in fluorescence as the fluorescence spectra at the air-water interface showed, the size of these
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Figure 5. AFM image of ACO on mica. Scan size, 660 nm × 660 nm; scan rate, 2.5 Hz.
aggregates is below the size of visibility in the epifluorescent microscope. This also confirms that OA separates the ACO molecules much better than SA. Pure ACO monolayer was also successfully transferred onto mica as substrate (one layer was transferred at 15 mN/m). This LB film was then examined by AFM. Figure 5 shows the AFM image of the pure ACO LB film. The image can serve as further support that ACO forms aggregates that can be seen as particles without any significant topography. Conclusion From these studies, we can conclude that ACO, a new 7-aminocoumarin amphiphile, forms a stable monolayer at the air-water interface. Surface chemistry and spectroscopic and microscopic studies suggest that the molecules form nonfluorescent J aggregates. The formation of these aggregates quenches the fluorescence; however, as demonstrated, the use of mixed monolayers of ACO and SA or OA has partially overcome this problem. Pure and mixed monolayers of ACO can be easily transferred to quartz or mica that extends the use of ACO on solid supports. These results show that ACO can serve as an amphiphilic fluorescent probe in surface chemistry studies. Acknowledgment. These experiments were supported by the Department of the Army under Contract DAAD 19-00-1-0138. LA010907Q
