Surface-Enhanced Superquenching of Cyanine Dyes as J-Aggregates

and Interface Chemistry of Clay Minerals. R.A. Schoonheydt , C.T. Johnston ... ANTONY K. CHEN , ANDREW TSOURKAS. Journal of Innovative Optical Hea...
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Surface-Enhanced Superquenching of Cyanine Dyes as J-Aggregates on Laponite Clay Nanoparticles Liangde Lu,† Robert M. Jones,‡ Duncan McBranch,‡ and David Whitten*,†,‡ Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, and QTL Biosystems, LLC, Santa Fe, New Mexico 87507 Received May 9, 2002. In Final Form: June 27, 2002 Nanoparticles of synthetic Laponite clay can be coated with individual or mixed cyanine dyes to form J-aggregated assemblies in water. The absorption and fluorescence characteristics of these J-aggregates are substantially different from those of J-aggregates formed in crystals or colloids, suggesting that smaller J-aggregate domains are present on the disk-shaped nanoparticles. The mixed cyanine/Laponite aggregates exhibit similar but nonidentical absorption and fluorescence as compared to the individual dye aggregates, suggesting a mixed electronic structure. The fluorescence from the nanoparticle-adsorbed cyanines is subject to superquenching by a variety of electron- and energy-accepting quenchers. These quenchers can be introduced by coadsorption with donors on the clay (mixed aggregates) or by addition of acceptors to preformed donor aggregates in aqueous suspensions. For some of the energy-accepting cyanines, it is found that strong sensitization of fluorescence from a (mostly) acceptor state is observed, even in cases where the monomer dye is non- or very weakly emissive. In limiting cases, 50% quenching is observed at levels of 1 quencher per ∼400 monomers or 4 quenchers per nanoparticle.

Introduction J-Aggregates of dyes, especially cyanines and structurally related compounds, have been the subject of numerous investigations due both to their importance for spectral sensitization of photographic materials and to their inherently interesting structures and spectral and photophysical properties.1-9 Many investigations have focused on the formation and structures of J-aggregates in solution,10-14 in crystals, in Langmuir-Blodgett films,15-21 in liposomes,22,23 and on macroscopic surfaces.24,25 Several recent investigations have shown that cyanine J† ‡

Arizona State University. QTL Biosystems.

(1) Kobayashi, T. J-Aggregates; World Scientific: Singapore, 1996. (2) Jelley, E. E. Nature 1936, 138, 1009. (3) Scheibe, G.; Kandler, L.; Ecker, H. Naturwissenschaften 1937, 25, 75. (4) De Boer, S.; Wiersma, D. A. Chem. Phys. Lett. 1990, 165, 45-53. (5) Moll, J.; Daehne, S.; Durant, J. R.; Wiersma, D. A. J. Phys. Chem. 1995, 102, 6362-6370. (6) Tani, T.; Suzumoto, T.; Kemnitz, K.; Yoshihara, K. J. Phys. Chem. 1992, 96, 2778-2783. (7) Oezcelik, S.; Akins, D. L. J. Phys. Chem. B 1999, 103, 89268929. (8) Rousseau, E.; Van der Auweraer, M.; DeSchryver, D. C. Langmuir 2000, 16, 8865-8870. (9) Moll, J.; Harrison, W. J.; Brumbaugh, D. V.; Muenter, A. A. J. Phys. Chem. A 2000, 104, 8847-8854. (10) Struganova, I. J. Phys. Chem. A 2000, 104, 9670-9679. (11) Von Berlepsch, H.; Boettcher, C.; Ouart, A.; Burger, C.; Daehne, S.; Kirstein, S. J. Phys. Chem. B 2000, 104, 5255-5262. (12) Von Berlepsch, H.; Boettcher, C.; Daehne, L. J. Phys. Chem. B 2000, 104, 8792-8799. (13) Horng, M.-L.; Quiteris, E. L. J. Phys. Chem. 1993, 97, 1240812415. (14) Higgins, D. A.; Barbara, P. F. J. Phys. Chem. 1995, 99, 3-7. (15) Czikkely, V.; Foersterling, H. D.; Kuhn, H. Chem. Phys. Lett. 1970, 6, 207. (16) Moebius, D. Ber. Bunsen-Ges. Phys. Chem. 1978, 82, 848. (17) Kuhn, H. J. Chem. Phys. 1970, 53, 101. (18) Nakahara, H.; Fukuda, K.; Moebius, D.; Kuhn, H. J. Phys. Chem. 1986, 90, 6144. (19) Penner, T. L. Thin Solid Films 1989, 160, 241-250. (20) Yonezawa, Y.; Moebius, D.; Kuhn, H. J. Appl. Phys. 1987, 62, 2016-2021. (21) Moebius, D.; Kuhn, H. J. Appl. Phys. 1988, 64, 5138-5141. (22) Sato, K.; Kurahasi, M.; Yonezawa, Y. Langmuir 1993, 54, 285295.

aggregates can also form in restrictive environments such as DNA grooves,26-28 within the cavities of mesoporous structures,29 and on the surface of colloidal particles.30-36 It has also been shown that J-aggregation can be obtained by constructing polymers in which the same cyanine is pendant on a polymer backbone.37-40 J-Aggregation has been observed for these polymers both in solution and for the polymers adsorbed onto nanoparticles or microspheres.37-40 The “pure” J-aggregates formed from an individual cyanine dye in these different environments may differ in overall structure, size, and spectroscopic properties. There has been considerable investigation and (23) Sato, T.; Yonezawa, Y.; Kurakawa, H.; Kurahashi, M.; Wada, Y.; Tanaka, T. Thin Solid Films 1992, 210-211, 172-174. (24) Yonezawa, Y.; Kurakawa, H.; Sato, T. J. Lumin. 1993, 54, 285295. (25) Moehwald, H.; Bliznyuk, V.; Kirstein, S. Synth. Met. 1993, 61, 91-96. (26) Wang, M.; Silva, G. L.; Armitage, B. A. J. Am. Chem. Soc. 2000, 122, 9977-9986. (27) Chowdhury, A.; Wachsmann-Hogiu, S.; Bangal, P. R.; Raheem, I.; Peteanu, L. A. J. Phys. Chem. B 2001, 105, 12196-12201. (28) Ogul’chansky, T. Y.; Losytskyy, M. Y.; Kovalska, V. B.; Lukashov, S. S.; Yashchuk, V. M.; Yarmoluk, S. M. Spectrochim. Acta, Part A 2001, 57A, 2705-2715. (29) Xu, W.; Guo, H.; Akins, D. L. J. Phys. Chem. B 2001, 105, 76867689. (30) Sato, T.; Tsugawa, F.; Tomita, T.; Kawasaki, M. Chem. Lett. 2001, 402-403. (31) Kometani, N.; Tsubonishi, M.; Fujita, T.; Asami, K.; Yonezawa, Y. Langmuir 2001, 17, 578-580. (32) Jeunieasu, L.; Alin, V.; Nagy, J. B. Langmuir 2000, 16, 597606. (33) Noukakis, D.; Van der Auweraer, M.; DeSchryver, F. C. J. Phys. Chem. 1994, 98, 11745-11750. (34) Watanabe, M.; Herren, M.; Morita, M. J. Lumin. 1994, 58, 198201. (35) Miyamoto, N.; Kawai, R.; Kuroda, K.; Ogawa, M. Appl. Clay Sci. 2000, 16, 161-170. (36) Ogawa, M.; Kawai, R.; Kuroda, K. J. Phys. Chem. 1996, 100, 16218-16221. (37) Place, I.; Perlstein, J.; Penner, T. L.; Whitten, D. G. Langmuir 2000, 23, 9042-9048. (38) Jones, R. M.; Bergstedt, T. S.; Buscher, C. T.; McBranch, D.; Whitten, D. Langmuir 2001, 17, 2568-2571. (39) Lu, L.; Helgeson, R.; Jones, R. M.; McBranch, D.; Whitten, D. J. Am. Chem. Soc. 2002, 124, 483-488. (40) Jones, R. M.; Lu, L.; Helgeson, R.; Bergstedt, T. S.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14769-14772.

10.1021/la0259306 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/24/2002

Cyanine Dyes as J-Aggregates on Laponite Clay

some debate as to the limiting size of J-aggregates and to the extent of exciton delocalization or migration during the excited-state lifetime.1,10,14,20,21 A number of studies, starting with the pioneering work of Kuhn, Moebius, and co-workers, focused on the use of J-aggregates of amphiphilic cyanines as energy or electron donors in Langmuir-Blodgett films.1,15-18 These studies elegantly verified many of the predictions of Foerster concerning the distance dependence of excited-state energy transfer. More recent studies of mixtures of cyanine dyes prepared in mixed Langmuir-Blodgett films or by layer-by-layer deposition and other supramolecular assembly methods have indicated the roles of mixed J-aggregates or mixtures of aggregates within an assembly.22,23 In recent studies, we have shown that cyanine-pendant polyelectrolytes exhibit a very similar sensitivity to superquenching by oppositely charged small molecules capable of acting as energy or electron transfer quenchers as compared to that observed for conjugated polyelectrolytes.38-40 We have shown that the superquenching of these polymers can be used as a basis for biosensing applications.38,40 Very recently we found that enhanced superquenching can be obtained for these polymers, smaller oligomers, and even monomer cyanine dyes when these materials are collected on microspheres or nanoparticles.40 The “surface-enhanced superquenching” can be attributed to the formation of extended aggregates when the monomeric or small oligomer cyanines are collected on the surface to form the J-aggregates. In the present study, we focus on the photophysical behavior and quenching phenomena that occur when certain monomeric cyanines and mixtures of cyanine monomers are collected on nanoparticulate Laponite clay particles. Laponite is an interesting support since the nanoparticles are small and reasonably monodisperse. The nanoparticles thus provide a well-defined restricted environment in which to study pure and mixed aggregates. The results reported demonstrate that modified J-aggregates can be obtained (compared to crystals or colloids) whose fluorescence shows high sensitivity to superquenching by energy transfer and electron transfer agents. The nanoparticles containing a limited number of cyanine monomers or mixtures of cyanine monomers exhibit interesting photophysical and quenching behavior in the range between supermolecules and macromolecular assemblies. Experimental Section Materials. The structures of the dyes, quenchers, and conjugates are shown in Figure 1. Cyanine dyes 1-6 were obtained from the Center for Photoinduced Charge Transfer. Biotin conjugates were synthesized at QTL Biosystems. Other materials were commercial samples, used as received. UV/vis absorption spectra were obtained on a Perkin-Elmer Lambda Bio-40 spectrophotometer. Steady-state photoluminescence studies were carried out using a Spex Flurolog spectrofluorometer with a Spex DM3000f spectroscopy computer. Laponite RDS, a synthetic clay, was purchased from Southern Clay Products, Inc. The layered silicate contains an inorganic polyphosphate peptizer, and a typical composition (according to the supplier) is SiO2, 54.5%; MgO, 26.0%; Li2O, 0.8%; Na2O, 5.6%; P2O5, 4.1%; with 8.0% loss on ignition.41 The ion-exchange capacity of the Laponite can be estimated from its composition to be 0.6 mequiv g-1.41 When the clay is suspended at low concentrations in water, it is expected to be completely exfoliated. The clay was suspended in deionized water at a concentration of 0.01 wt % and allowed to stand overnight at room temperature prior to treatment with dyes. The samples were found to be stable indefinitely at room temperature or when stored in a refrigerator. (41) Technical Bulletin L/RDS/2/01; Southern Clay Products, Inc.: Gonzales, TX, 2001.

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Figure 1. Structures of cyanine dyes and quenchers used in this investigation. The cyanine dye aggregate fluorescence quenching protocol was as follows: A solid sample of a monomer dye (1.5 mg) was added to N,N-dimethylformamide (DMF) (1 mL) in a vial, the vial was left closed at room temperature for 5 h to ensure that the dye was dissolved, and the resulting dye solution (solution A) was stored in a refrigerator at 0-4 °C. This solution can be stored indefinitely. To carry out a quenching study without support, (1) dye solution A was diluted with deionized water or an aqueous solvent mixture to yield dye solution B at optical density (OD) < 0.2 at the UV/vis absorption maximum; (2) the emission spectra were acquired using excitation at or close to their UV/vis absorption maxima and recorded before and (3) after addition of an aliquot of quencher solution into dye solution B. The emission intensity ratio (I0/I) at emission maxima before (I0) and after (I) addition of an aliquot of quencher solution was used to derive the quenching constant from a Stern-Volmer plot of the data. Steps 1-3 were repeated 7-10 times at different quencher concentrations. To carry out a quenching study of cyanine dyes on clay, an appropriate amount of a suspension of clay particles was added with stirring to dye solution B. All other steps were the same. Picosecond fluorescence lifetime measurements were made using the time-correlated single photon counting method with ultrafast dye laser systems: The excitation source from 565 to 590 nm was a frequency-doubled, mode-locked Coherent Nd:YAG laser, which synchronously pumped a cavity-dumped Coherent 700 dye laser. The excitation source at 400-430 nm was a frequency-doubled self-mode-locked Ti-sapphire laser. The pulse width was ca. 7 ps, and the average power was ca. 100 µW. Fluorescence emission was detected at the magic angle using a single-grating monochromator and a microchannel plate photomultiplier. The instrument response has a full width at half-height of ca. 80 ps. The spectrometer was controlled by software based on the LabView program from National Instruments. The dye laser was tuned to the desired excitation wavelength. Measurements were made on air-saturated samples at room temperature.

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Results and Discussion J-Aggregates of Cyanines on Nanoparticulate Clay. Laponite RDS clay provides a well-defined support for the formation of cyanine J-aggregates.41 It has been previously shown that certain cyanine dyes form Jaggregates upon addition of the monomer to suspensions of smectites.35,36,42 The synthetic Laponite RDS clay is relatively uniform in particle size, and pretreatment of the clay (by the manufacturer) with pyrophosphate results in the grafting of P2O5 groups to the edges of the clay particles.41 This allows the materials to form very stable colloids. Suspension of the particles (either coated or uncoated) in aqueous media affords stable colloidal solutions (little or no light scattering) whose absorption and fluorescence properties are easily monitored through the ultraviolet and visible regions. The particles are roughly disk shaped, and it is reasonable to infer that a fully coated particle may consist of two domains of “collected” dye on either face of the disk.43 The surface area of the clay is estimated (Brunauer-Emmett-Teller (BET) method) as 330 m2 g-1.37,41 Laponite is completely exfoliated in water at the concentrations used in these experiments,44 and under these conditions the active surface area can be estimated from sheet dimensions (40 nm × 10 nm × 1 nm)45 to be about 750 m2 g-1.46 In a typical experiment using 2.8 µg of clay and cyanine dye 2 (10-6 M), we estimate that the coverage of the clay by 2 (molecular area, ca. 50 Å2)47 is 97% (estimated error, ∼10%). At this coverage (and considering the average dimensions of a typical clay particle), we estimate that there should be ∼1600 molecules of 2 on the surface of a typical nanoparticle. A number of investigations have shown that organic molecules such as dyes and aromatics adsorb on Laponite with modification of their photophysical properties.48-51 We have focused the present study on the J-aggregates formed by cyanine dyes 1 and 2 (structures are shown in Figure 1). The J-aggregates of these dyes and related structures bearing the same chromophores have been examined in a number of previous studies. As clay particles are added incrementally to solutions of dye 1 (monomeric, by absorption spectroscopy at concentrations of ∼10-6 M; however the emission can be from either monomer or an aggregate), there is a grow-in of sharp peaks in the absorption and emission spectrum, red-shifted from the monomer and characteristic of J-aggregation (Figure 2). As shown in Figure 2, dye 2 also forms a J-aggregate upon addition of clay. However, in the case of 2 the dye exists in solution prior to the addition of clay as a mixture of monomer and microcrystals.40 The relative amount of J-aggregate varies with time and sample preparation; typical monomer/J-aggregate (microcrystal) absorbance ratios are 8/1. The microcrystals can be removed by (42) Herkstroeter, W.; Hussein, S.; Farid, S.; Penner, T.; Whitten, D. Unpublished studies. (43) Hanley, H. J. M.; Muznym, C. D.; Butler, B. D. Langmuir 1997, 13, 5276-5282. (44) Avery, R. G.; Ramsay, D. F. J. Colloid Interface Sci. 1986, 109, 448. (45) Neumann, B. S.; Sanson, K. G. Isr. J. Chem. 1970, 8, 315-324. (46) Labbe’, P.; Reverdy, G. Langmuir 1988, 4, 419-425. (47) Kuhn, H.; Kuhn, C. In J-Aggregates; Kobayashi, T., Ed.; World Scientific: Singapore, 1996. (48) Cione, A. P. P.; Neumann, M. G.; Gessner, F. J. Colloid Interface Sci. 1998, 198, 106-112. (49) Brahimi, B.; Labbe, P.; Reverdy, G. New J. Chem. 1992, 16, 719-726. (50) Lopez, A. F.; Tapia Estevez, M. J.; Lopez Arbeloa, T.; Lopez Arbeloa, I. Langmuir 1995, 11, 3211-3217. (51) Jacobs, K. Y.; Schoonheydt, R. A. J. Colloid Interface Sci. 1999, 220, 103-111.

Figure 2. Absorption (broken lines) and emission (solid lines) spectra of cyanines 1 and 2 in aqueous solution and as J-aggregates adsorbed onto Laponite clay. Cyanine 1: (a) in solution; (b) on Laponite at ∼100% coverage. Cyanine 2: (c) in aqueous solution; (d) on Laponite nanoparticles at ∼100% coverage.

centrifugation. For both 1 and 2, complete coverage according to the calculations above is accompanied by disappearance of the monomer absorption in the colloidal suspension. As additional colloidal clay is added to the suspensions of 1, there is a reappearance of monomer absorption, suggesting that the J-aggregate of 1 may be diluted by the availability of additional clay surface sites. When suspensions of 2 are treated with additional clay, the J-aggregate of 2 is observed to persist down to fairly low dilution (10% coverage), likely indicating that the surface-templated J-aggregates of 2 are more stable than those of 1. Interestingly, cyanines 3 and 4 do not form well-defined J-aggregates upon treatment with clay (vide infra). The J-aggregates of 1 and 2 that do form on Laponite clay show some significant differences from other Jaggregates of the same dyes or structures bearing the same chromophore. For example, the J-aggregate absorption and fluorescence spectra for 1 on clay are somewhat broader and blue-shifted compared to the J-aggregate of amphiphilic 5 on the much larger (0.14 micron diameter) silica microspheres (Table 1). Interestingly, 5 does not form a J-aggregate when it is treated with clay in aqueousorganic suspensions (5 is not sufficiently water soluble to load it onto clay in aqueous suspensions). The J-aggregate of 2 also exhibits noticeable differences from other J-aggregates of the same chromophore as indicated in Table 1 and Figure 3. Thus the anionic dye with the same chromophore (6) exhibits sharper and red-shifted (compared to clay-supported 2) J-aggregate absorption and fluorescence as colloidal assemblies in water. The selfassembled J-aggregate of 2 on the much larger silica microspheres shows a somewhat similar red-shift in both absorption and fluorescence compared to the nanoparticulate clay-supported 2. The J-aggregate of 2 formed via layer-by-layer deposition onto the same Laponite clay but as a “planar” surface shows slightly red-shifted absorption but sharper and blue-shifted fluorescence.37 Interestingly, the J-aggregate absorption and emission of the colloidal clay-supported 2 are somewhat broader and blue-shifted compared to those of the higher molecular weight polymers containing the same chromophore as a pendant group on poly-L-lysine.37,39 For both 1 and 2 on clay, there is a relatively larger Stokes shift than observed for the other J-aggregates of the same chromophores sampled in Table 1. It is generally anticipated that the size of a J-aggregate domain should correlate with the wavelength of the absorption, the sharpness of the J-aggregate transition

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Table 1. Absorption and Fluorescence of 1, 2, 5, and 6 cyanine, conditions 1 in 95% H2O/5% methanol (monomer) 1 on clay nanoparticles in H2O 5 on silica microspheres in H2O 2 in H2O (monomer)d 2 in H2O (microcrystal)d 2 on clay in H2O 2 on flat claye 2 on silica 2 attached to poly-L-lysine in waterf 6 colloidal suspension in water (J-aggregate)

λmax (absorb), nm 426 455 466 513 595 566 568 590 576 596

λmax (emiss), nm 480 470 476 534 602 588 573 597 593 610

ν1/2 (absorb), cm-1 2009 2100 500 1620 170 1381 2777 360 670 270

φf

Naggcalc a

Naggcalc b

0.004c 0.08 0.04 0.03

16

0.02

90 ∼1 20 6 36

3 3 16 6

a Calculated according to ref 27. b Calculated according to ref 57. c Quantum yield for a 50% dimethyl sulfoxide/50% water solution. d In aqueous solutions, 2 exists as a mixture of monomer and microcrystalline J-aggregate. e Data from ref 37. f Data for cyanine polymer (ref 39) having ∼400 polymer repeat units, each with a pendant cyanine dye.

Figure 3. Absorption (broken lines) and emission (solid lines) spectra of J-aggregates of 2 and 6 in different environments: (a) on Laponite nanoparticles; (b) poly-L-lysine polymer (∼250 polymer repeat units) in water; (c) colloidal suspension of 6 in water.

(as measured by ∆ν1/2), and the Stokes shift between absorption and fluorescence.1,27,53,54 The physical size of (ground-state) J-aggregates formed on crystal surfaces such as silver bromide can be estimated from the ratios of bandwidths of the monomer and aggregate (as well as by other approaches).27,55-57 Typical values are in the range of 6-15 for dyes structurally similar to 2.27,53 On the other hand, structures of colloidal aggregates of 1,1′-diethyl2,2′-cyanine (PIC) indicate that the aggregation numbers may be on the order of 300011,12 (based on physical size, not spectroscopic data). From the values of ∆ν1/2 for the monomer and aggregate listed in Table 1, we can estimate that the “size” of the aggregate of 2 in microcrystals may be on the order of 90 monomers/aggregate (Table 1). A similar calculation for the J-aggregate of 2 on silica microspheres provides an estimate of an aggregate size of 16-20, while the polymer (poly-L-lysine having dye 2 pendant on each unit) having ∼400 chromophores of 2 yields an estimated aggregate size of ∼6. The much broader J-aggregate absorptions for 1 and 2 on clay lead to calculated aggregate sizes of ∼1-3 which seem unreasonably small (although recent work suggests that for PIC the limiting aggregate transition is reached at 3 molecules of PIC per aggregate).10 There may be several reasons why the J-aggregate transitions are broader when the dyes are adsorbed onto small nanoparticles. If there (52) Kometani, N.; Nakajima, H.; Asami, K.; Yonezawa, Y.; Kajimoto, O. J. Phys. Chem. B 2000, 104, 9630-9637. (53) Lanzafame, J. M.; Muenter, A. A.; Brumbaugh, D. V. Chem. Phys. 1996, 210, 79-89. (54) Muenter, A. A.; Brumbaugh, D. V.; Apolito, J.; Horn, L. A.; Spano, F. C.; Mukamel, S. J. Phys. Chem. 1992, 96, 2783-2790. (55) Knapp, E. W. Chem. Phys. 1984, 85, 73-82. (56) De Boer, S.; Vink, K. J.; Wiersma, D. A. Chem. Phys. 1987, 137, 99-106. (57) McRae, E. G.; Kasha, M. J. Chem. Phys. 1958, 28, 721.

are several different domain sizes present on the nanoparticle surface having slightly different spectroscopic signatures, this can result in a broadening and preclude an effective calculation of the aggregate size. The above comparison of the J-aggregates formed by 1 and 2 on clay with those formed on other supports or surfaces or even in colloidal suspensions suggests that these J-aggregates may be limited in size and/or interrupted by the morphology of the clay nanoparticles. In any event, the capacity of an individual clay particle is 1600 cyanines. Since the Laponite particles have two well-separated faces, the maximum physical size of a J-aggregate domain on Laponite should be ∼800 molecules and may be even lower if the surface sheets are interrupted by corrugation or defects such as steps and so forth. Mixed Aggregates of Cyanines 1 and 2. Several questions arise when an aqueous mixture of the two cyanine dyes is treated with clay nanoparticles. Do the dyes aggregate separately or do they form “mixed aggregates”? Do they adsorb equally well or is there preferential adsorption of one over the other? Once adsorbed, is there exchange of dye molecules between clay particles and the aqueous phase? To address these questions, several experiments were carried out. Approximately equimolar amounts of 1 and 2 were dissolved in aqueous solution and treated with sufficient clay to provide ∼100% coverage. The absorption and fluorescence spectra for the “mixed” clay-supported dyes can be compared with those for the colloidal suspensions of clay 1 and clay 2 prepared individually. The absorption spectrum of the mixture is qualitatively similar to an addition of the spectra of the individual J-aggregates of pure 1 and pure 2 (Figure 4). However, the J-aggregate absorption corresponding to 1 in the mixture is broadened slightly and blue-shifted by 7 nm while the J-aggregate absorption corresponding to 2 is blue-shifted by 15-20 nm compared to that from pure 2. The single fluorescence peak from the clay-supported mixture (Figure 5) corresponds to that from J-aggregated 2 but is blue-shifted from that of pure 2 by 12 nm; the excitation spectrum is nearly identical to the absorption spectrum. When the ratio of 1/2 is much larger than unity, both fluorescence from the J-aggregate corresponding to 1 and that corresponding to 2 are observed. The quantitative aspects of quenching occurring between the donor and acceptor aggregates on the nanoparticles will be discussed in detail below. However, the changes in fluorescence and absorption can be used to answer the questions posed above. When individually coated clay particles of 1 and 2 are mixed (at a 1:1 ratio), there is initially (minutes after mixing) a weak fluorescence from the J-aggregate of 1 on excitation at 456 nm and a strong emission corresponding to the J-aggregate of 2 upon excitation at 550 nm. Over

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Figure 4. Comparison of pure and mixed aggregates of 1 and 2: (a) absorption spectrum of 1 on Laponite; (b) absorption spectrum of a 1:1 mixture of 1 and 2 adsorbed onto Laponite nanoparticles; (c) absorption spectrum of 2 on Laponite.

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is similar to that observed by Yonezawa and others in liposomes22,23 and layer-by-layer assemblies24,52 incorporating cyanine mixtures. According to the terminology developed by these investigators, the appearance of two J-aggregate bands, blue-shifted but still identifiable as “mostly donor” and “mostly acceptor”, is assigned as a “persistance” or “partitioning” aggregate (P-aggregate).52 When the ratio of 1/2 is varied in these mixtures, there are only small changes in the absorption maxima for the two mixed aggregate transitions. Photophysics of Cyanine Dyes and Mixtures on Clay Nanoparticles. As has been observed in other investigations, conversion of the solution monomer of 1 or 2 into the J-aggregate by collection onto the nanoparticles of Laponite is accompanied by decreases in the fluorescence lifetime of the dye, as shown in Table 2.7,37,52,53 The lifetimes for the clay-supported J-aggregates are typically somewhat longer than those for more organized J-aggregates. For mixtures of energy-donating and energyaccepting dyes, under conditions where both emissions are detectable, there is a decrease in the fluorescence lifetime of the donor dye as the amount of acceptor increases and the donor emission is quenched. These results are consistent with results obtained in other studies for cyanine dye mixtures. The spectroscopic and photophysical behavior of the cyanine dyes adsorbed onto the colloidal clay can be approached from either a “solution” or a nanoassembly perspective. From a solution perspective, it is observed that the decrease of the emission as a quencher is added follows a conventional Stern-Volmer relationship according to eq 1.

If0/If ) φf0/φf ) 1 + KSV(Q)

Figure 5. Comparison of pure and mixed aggregates of 1 and 2: (a) emission spectrum of 1 on Laponite; (b) emission spectrum of a 1:1 mixture of 1 and 2 adsorbed onto Laponite nanoparticles; (c) emission spectrum of 2 on Laponite.

a period of tens of minutes, the emission corresponding to the J-aggregate of 1 is totally quenched and a weak sensitized emission corresponding to the J-aggregate of 2 is observed. An eventual equilibration to absorption and fluorescence nearly identical to that obtained by adding clay to the 1/2 mixture occurs, clearly indicating that the individual dye molecules (or perhaps clusters) can exchange between nanoparticles by a desorption-readsorption process. Additional experiments suggest that 2 adsorbs more strongly than 1. When 1 is treated with clay sufficient to produce 100% coverage, all of the dye is converted from monomer to J-aggregate. Addition of small amounts of 2 (a mixture of monomer and a small amount of J-aggregate) to this suspension results in “ejection” of 1 from the clay (readily observed by reappearance of the monomer absorption of 1) and a quenching of the fluorescence of the J-aggregate of 1. As quenching of the fluorescence from the J-aggregate of 1 occurs, there is a sensitization of the J-aggregate fluorescence of 2. The tendency of 2 to displace 1 from the clay nanoparticle surface can be rationalized in part as due to the greater hydrophobicity of 2. The spectroscopic properties of the mixed aggregates of 1 and 2 on the clay nanoparticles can be compared to mixed aggregates examined in other studies.20,21,58 In particular, the behavior observed here for the mixtures

(1)

I0 is the fluorescence intensity of the donor dye (donor, D) ensemble in the absence of quencher, and I is the intensity at a solution-phase concentration of a quenching species (Q) (in moles/liter). While linear Stern-Volmer plots are obtained for any donor at constant donor concentration (D), it is clear that on a molecular level, if both donor and quencher are located on the nanoparticle surface, the quenching will be controlled by the quencher/donor molecular ratio, (Q/D). If all of the quencher and all of the donor are confined to the nanoparticles, this ratio is simply the molar concentration ratio, (Q)/(D). It can easily be shown that for the situation where all of the donor and quencher are in the nanoparticle-bound pseudophase, the solution KSV is dependent on the donor concentration, (D). Thus at a level of 50% quenching, KSV can be defined by eq 2.

KSV ) 1(Q)50 ) (D/Q)50[1/(D)]

(2)

A Stern-Volmer quenching constant can also be defined for the two-dimensional nanoparticle surface, KSVNP, similar to that used by Yonezawa for layer-by-layer assemblies.52 Equation 3 shows that KSVNP can be written in terms of the same parameters and the molecular area of the donor, AD.

KSVNP ) (D/Q)50[AD]

(3)

In contrast to KSV, KSVNP is a true constant, independent of the donor concentration, with units of nm2 molecules-1. (58) Czikkely, V.; Dreizler, G.; Foersterling, H. D.; Kuhn, H.; Sondermann, J.; Tilmann, P.; Wiegand, H. Z. Naturforsch. 1969, 24a, 1823.

Cyanine Dyes as J-Aggregates on Laponite Clay

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Table 2. Fluorescence Lifetimes of Cyanine Dyes 1, 2, 3, and 5 cyanine and conditions

form

2 on clay 2 in CHCl3 1 on clay 1/3 on clay in 95% H2O emission of 1 sensitized emission of 3 5 on silica MS (50% DMSO/H2O) 5 in 30% methanol/H2O 5 on clay in 30% methanol/H2O 5 in acetone

J-aggregate monomer J-aggregate J-aggregate

lifetimea

J-aggregate J-aggregate J-aggregate monomer

χ2

21 ps (99.4%), 220 ps (0.6%) 152 ps(85.7%), 652 ps (14.3%) 49 ps (93.4%), 259 ps (6.3%)

0.90 0.92 2.3

16 ps (99.8%), 733 ps (0.2%) 191 ps (30.8%), 1.14 ns (39.7%), 2.64 (29.4%) 3 ps (99.3%), 107ps (0.65%) 5 ps (98.5%), 98 ps (1.5%) 13 ps (98.7%), 324 ps (1.2%) 11 ps (100%)

1.7 1.3 1.4 1.2 1.6 2.0

a A short lifetime of less than 8 ps is not precise; only the order of magnitude can be approximated. The long lifetime has an error of (1 on the second significant number.

Table 3. Fluorescence Quenching of Cyanine Aggregates on Clay in HxO at 22 °C: Quenching Constantsa quencher cyanine, coverage 1, 100%

2

3

4

7

8

4.7 × 107 23.5/47

8.9 × 107 44.5/89 4.3 × 108 215/430

4 × 107 20/40 2 × 108 100/200

5 × 105 0.25/0.5 1.3 × 104 0.007/0.013 9 × 104 0.05/0.09 7 × 106 3.9/7

1 × 108 50/100 9.1 × 107 46/91

6 × 107 30/60 5 × 107 25/50

2 × 107 5 × 107

4 × 106 2.2/4 1.5 × 107 2.5 × 107

2, 100% 2, 40% 2, 25% 1/3 (∼10:1 molar ratio)

9

-1 NP (nm2/ a For each entry (except for the 1/3 mixture), the upper value is K SV (M ); the lower values are, to the left of the slash, KSV molecules-1) and, to the right of the slash, (S/Q)50. For the mixture, the upper value is for quenching of the fluorescence of 1 and the lower value is for quenching of the fluorescence of 3.

The two “Stern-Volmer” constants are related as shown in eq 4.

KSVNP ) KSV(D)[AD]

(4)

For the general case where a donor ensemble is assembled onto a nanoparticulate support, it can be seen that the maximum quenching that can occur will be when a single quencher extinguishes the fluorescence from an entire nanoparticle and thus [KSVNP]MAX ) 2(DN)[AS], where DN is the capacity of the support in terms of donor molecules. From the estimate above that an average clay nanoparticle can accommodate 1600 molecules of donor, the estimate for [KSVNP]MAX is equal to 1600 nm2 molecules-1. If we consider that domains on either side of a clay disk may not be in “communication”, [KSVNP]MAX ∼ 800 nm2 molecules-1. We have examined the quenching of nanoparticlesupported cyanines 1 and 2 and certain mixed assemblies by both small-molecule and aggregated quenchers. The quenching can be initiated by different situations. A watersoluble small-molecule quencher can be introduced to the nanoparticle suspensions as a soluble reagent. In several cases, superquenching is observed that is consistent with a preassociation of the quencher with the nanoparticle. An alternative, with cationic quenchers, is to expose a mixture of the donor cyanine and the quencher to clay and associate both quencher and donor directly with the nanoparticles. Table 3 reports quenching constants for the J-aggregates of 1 and 2 by a variety of small-molecule quenchers. Not surprisingly, we find that when the quencher is added from the solution phase, the quenching constant for various quenchers varies as a function of the loading of the J-aggregate substrate on the nanoparticle. For the former (water-soluble quenchers introduced from solution), we have studied quenching of prepared nanoparticles coated with 1 and 2 with quenchers 7-9. The cation 7 and anion 8 are both electron transfer quenchers.

In solution, 7 is not effective as a quencher of the fluorescence of 1 or 2 due to repulsion between the likecharged donor and quencher and the relatively short fluorescence lifetimes of the monomer dyes. The monomer of 2 is quenched by anionic 8 with a quenching constant of 630 M-1,39 which is roughly in agreement with a value predicted on the basis of Coulombic attraction between the dianionic quencher and the monocationic 2. When 2 is loaded on the Laponite nanoparticles to ∼100% as a J-aggregate, its fluorescence is quenched strongly by 8 but only weakly by 7 (Table 3). This seems reasonable in view of the supposition that the anionic clay, when fully coated with a cationic cyanine, should have a net surface positive charge. Interestingly, the quenching of the claysupported J-aggregate of 2 by 7 increases as the level of coverage of the clay by the J-aggregate is decreased. This also is understandable as residual anionic charge on the surface can lead to coadsorption of both the donor and quencher in much the same way as for the mixture of 1 and 2 as described above. The quenching of 2 by 8, when 2 is loaded onto the clay nanoparticles at ∼100% coverage, is enhanced by a factor of 3.2 × 105 compared to the homogeneous solution. In all of the cases where electron transfer quenchers are used, we observe only quenching of the J-aggregate fluorescence and no new emission. As described above, 2 can serve as an energy-accepting quencher for the J-aggregate of 1. When 1 and 2 are “coloaded” onto clay particles at 100% coverage, quenching of the fluorescence of the J-aggregate of 1 is observed concomitant with appearance of the mixed aggregate of 1 and 2 emitting at 576 nm as reported above and shown in Figure 5. The Stern-Volmer quenching constants described above are reported in Table 3. When 1 is first adsorbed onto clay nanoparticles and then treated with monomeric 2 from solution, the measured quenching constants are lower, consistent with a relatively slow binding and exchange of the cationic 2 with the J-aggregate of 1 on the nanoparticle surface. Interestingly, we find

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Langmuir, Vol. 18, No. 20, 2002

Lu et al. Table 4. Quantum Yields of Fluorescence Emission of 1 with 3 on Clay and Sensitized Emission of 3

1/3 molar ratio, [3]0 ≈ 10-6 M

quantum yield, total

quantum yield of 1

quantum yield of sensitized emission of 3

without 3 50 25 10 5 0 (only 3)

0.079 ( 0.002 0.070 ( 0.002 0.075 ( 0.006 0.048 ( 0.006 0.032 ( 0.003 0.00036 ( 0.00003a

0.079 ( 0.002 0.058 ( 0.003 0.022 ( 0.004 0.013 ( 0.003 0.0088 ( 0.002

0.012 ( 0.002 0.053 ( 0.006 0.035 ( 0.003 0.023 ( 0.002

a 3 in 50% DMSO/H O. The quantum yield of 3 in 95% H O 2 2 (H-aggregate, J-aggregate, and monomer peaks) is approximately 5-fold lower.

Figure 6. Absorption spectra (broken lines) and emission spectra (solid lines, primed letters) of mixtures of 1 and 3 on Laponite: (a) pure 1 on Laponite; (b) 1/3 molar ratio of 25; (c) 1/3 molar ratio of 5.

that initial quenching of the fluorescence of the J-aggregate of 1 is not accompanied by a strong sensitized fluorescence from the mixed aggregate of 2. This suggests quite reasonably that the initial adsorption of individual molecules of 2 onto the nanoparticles containing aggregated 1 may not produce an emissive trap. This is consistent with the low fluorescence observed from 2 in dilute solutions in organic solvents where it does not form a J-aggregate. Other cyanine monomers can quench the nanoparticlesupported J-aggregates of 1 and 2 with the development of a strong sensitized fluorescence from the quencher or a mixed aggregate of the quencher and donor. When cyanine 4, anticipated to be a good energy acceptor for the J-aggregate of 2 and an anionic water-soluble dye, is added to solutions containing clay-supported 2 at 100% loading, there is a quenching of the fluorescence of the J-aggregate concurrent with a sensitization of the fluorescence near the wavelength where monomeric 4 emits. The quenching constants given in Table 3 indicate that 50% quenching occurs at a level of 200 donor molecules per quencher or 8 quenchers per nanoparticle. Interestingly, although the J-aggregate emission for nanoparticle-supported 1 is at considerably shorter wavelengths than the absorption from 4, addition of 4 to solutions containing clay-supported 1 also shows strong quenching with sensitization of the emission of 4. As shown in Figure 6, the sensitized emission is shifted some 170 nm from the excitation wavelength and the quantum efficiency for the shifted emission can be as high as 0.08 (approximately equal efficiency as for J-aggregates of 1 produced by direct excitation). The quenching constant for J-aggregated 1 by 4 might be anticipated to be much lower than for J-aggregated 2 with the same quencher, yet the observed difference is only a factor of 5. Cyanine 3, having the same chromophore as 4 but cationic, can also be used as a quencher for the J-aggregates of 1 and 2 on clay. For these ensembles, the most convenient way of examining quenching is to add clay to solutions of the mixtures at varying substrate/ quencher ratios. For J-aggregates of both 1 and 2, donor fluorescence quenching and sensitized emission of the quencher are observed, qualitatively similar to the behavior observed when 4 is introduced to the J-aggregate suspensions. The observation of sensitized emission is interesting in that neither 3 nor 4 emits strongly when subjected to direct excitation in either solution or when

Figure 7. Absorption spectra of cyanines on Laponite clay and in solution: (a) pure 3 on clay; (b) 3 in solution (methanol); (c) 4 in methanol solution; (d) 1/3 molar ratio of 5 on Laponite clay; (e) 1/3 molar ratio of 25 on Laponite clay.

coated individually onto the clay. Quantum yields for direct and sensitized emission are listed in Table 4. Thus absorption of 3 and 4 in solution occurs at 578 nm (Figure 7) and there is a very weak emission at 612 nm; presumably this can be attributed to excitation of monomeric 3 or 4. Addition of clay results in a split in the absorption spectrum into two bands, one at ∼540 nm, which might be attributed to an H-aggregate, and a second at 605 nm, which may be a J-aggregate. However, when mixtures of 1 and 3 are co-loaded onto clay, the absorption consists of the J-aggregate band of 1 plus a weak absorption at 605 nm. The emission that grows in as the ratio of 3/1 is increased is at 630 nm. The sensitized emission of 3 is 150-700-fold higher than that produced by direct excitation of 3 in solution or when loaded by itself onto the clay. Similar behavior is observed when the J-aggregate of 1 on clay is treated with 4 by its addition to the colloidal suspensions. In the case of 4, preparations of solutions show the monomer at 578 nm (fairly broad with a highenergy shoulder at 540 nm) and a sharp but weak shoulder at lower energy (670 nm) that may correspond to a (microcrystalline) J-aggregate, as discussed above for other cyanines such as 2. The observation of strong (sensitized) emission from 3 and 4 with very different absorption and fluorescence spectra and quantum efficiency when they are associated with J-aggregates of 1 suggests strongly that the fluorescent acceptor is not a simple isolated monomer chromophore of 3 or 4. (Excitation of the energyaccepting species at 605 nm does result in comparable

Cyanine Dyes as J-Aggregates on Laponite Clay

fluorescence quantum efficiency to that observed upon excitation of the J-aggregate of 1.) A source for the increase in emission intensity when 3 or 4 is associated with the J-aggregates of 1 on clay may be that the acceptor dye assumes a more restricted environment than when free as a monomer in solution or as a loose ensemble on clay. This could lead to an elimination of nonradiative decay channels and an increase in fluorescence. If 3 (or 4) is incorporated into a J-aggregate “patch” as a guest, it could serve as a trap site such that the emission could be attributed to a mixed aggregate or defect consisting of a mixed exciton of aggregated 1 and 3 (or 4). Interestingly, we find that initial quenching of the J-aggregate of clay-supported 1 by addition of 3 results in an equivalent sensitization of the fluorescence of the long-wavelength mixed aggregate. However, as the concentration of 3 is elevated the fluorescence efficiency at 630 nm decreases (Table 4). Examination of the absorption spectra of the clay-supported mixture of 1 and 3 shows that additional long-wavelength absorption near 550 nm grows in. This may correspond to the blue-shifted component (H-aggregate?) observed when pure 3 is coated on clay (Figure 7). While the excitation spectrum for the emission at 630 nm shows maxima attributable to the J-aggregate of 1 and the mixed aggregate at 605 nm, there is no corresponding excitation from the moderate absorption at 550 nm, suggesting that this may act as a nonemissive quenching trap site (Figures 6 and 7). The demonstration of incorporation of potential energy transfer cyanine dyes into J-aggregates of a substrate to form emissive mixed aggregates on a nanoparticle raises possibilities and questions over superquenching by additional reagents. Table 3 compares quenching of nanoparticles coated with mixtures of 1 and 3 by the anionic electron transfer quenchers 8 and 9. Since both cyanines are cationic and the loading of the clay by the mixtures is ∼100%, the nanoparticles should attract strongly the anionic electron acceptors and the presumed nonspecific association between the quencher and the clay should be nearly the same as for the uniformly coated nanoparticles containing only 1. The residual emission of J-aggregates of 1 has a shorter fluorescence lifetime, as reported above, and therefore is expected to be more difficult to quench than the pure J-aggregate of 1 in the absence of 3. As anticipated, the emission from the residual J-aggregate of 1 exhibits lower quenching constants than for nanoparticles coated with pure 1. Not surprisingly, the greater the extent of quenching of 1 by 3, the lower the quenching constant for the quenching of its residual fluorescence by 8 and 9. The sensitized emission of the mixed aggregates containing 1 and 3 can also be anticipated to be more difficult to quench by addition of 8 and 9 since the excitation is expected to be more localized in isolated regions containing 3. As shown in Table 3, the quenching for the long-wavelength (mixed aggregate of 1 and 3) emissions is attenuated compared to that of the Jaggregate of pure 1. However, the attenuation in this case is rather modest and the extent of superquenching is still impressive.59

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Summary/Conclusions Adsorption of cyanines 1 and 2 onto nanoparticles of Laponite clay results in the formation of J-aggregates showing absorption and emission that are broader and somewhat blue-shifted compared to those formed from the same chomophores in extended crystals, colloids, polymers, and Langmuir-Blodgett films. The groundstate aggregation number is thus indicated to be small and probably variable, perhaps attributable either to domains limited by the size of the nanoparticle or, more likely, to a limitation in domain size or regularity imposed by the surface morphology of the nanoparticles. Although the ground-state aggregation number may be small, the exciton domain that can be addressed by a single electron transfer or energy transfer quencher is quite large. In some cases, this approaches half of the estimated molecules that can be adsorbed on a single anionic sheet of the disklike clay nanoparticles. Thus the level of fluorescence superquenching that can be observed approaches or exceeds that observed in earlier studies with cyanines collected into J-aggregates on a polymer scaffold or conjugated polyelectrolytes in solution or in supported formats. In contrast to results obtained in previous investigations of amphiphilic cyanines in LangmuirBlodgett films, the quenching constants measured for energy transfer and electron transfer quenchers are similar. One of the more interesting findings is that the addition of a small amount of a longer-wavelength absorbing cyanine “dopant” into a host aggregate of 1 or 2 can result in a sensitized fluorescence of the dopant, even in cases where the dopant is poorly emissive and/or there is poor spectral overlap between the host fluorescence and dopant absorption. Perhaps due to the relatively small size of the nanoparticles, it is found that quenching of the sensitized fluorescence of the dopant is only slightly less effective than for the undoped J-aggregates. For the simple cyanine monomers used in this study, facile exchange has been shown to occur between cyanines in solution and nanoparticle-adsorbed aggregates and even between different cyanine aggregates adsorbed initially on different Laponite samples. This exchange shows limitations in possible sensing applications using different nanoparticle-adsorbed cyanine ensembles to achieve “multiplexing”. However, initial studies indicate that adsorption-desorption exchange between cyanine monomers self-assembled into pure or mixed aggregates does not occur when the cyanines used in this study, such as 1, are replaced by the corresponding amphiphiles, such as 5. Acknowledgment. This work was supported by the Defense Advanced Research Projects Agency under Contract No. MDA972-00-C-006. We thank Arizona State University for support and Professor Ian Gould for the use of instrumentation. LA0259306 (59) As the amount of 3 is further increased, the quenching constant for residual emission of 1 by 8 or 9 is reduced while that for the sensitized emission of 1 remains nearly constant.