Resonance Energy Transfer between Rhodamine ... - ACS Publications

Dec 18, 2009 - Förster resonance energy transfer (FRET) from rhodamine 123 (Rh123; ... Competitive adsorption of the dyes on SAP significantly influe...
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J. Phys. Chem. C 2010, 114, 1246–1252

Resonance Energy Transfer between Rhodamine Molecules Adsorbed on Layered Silicate Particles Juraj Bujda´k,*,†,‡ Dusˇan Chorva´t, Jr.,§,| and Nobuo Iyi⊥ Institute of Inorganic Chemistry, SloVak Academy of Sciences, Du´braVska´ cesta 9, 845 36 BratislaVa, SloVakia, Department of Physical and Theoretical Chemistry, Faculty of Natural Sciences, Comenius UniVersity, 842 15 BratislaVa, SloVakia, International Laser Centre, 812 19 BratislaVa, SloVakia, Polymer Institute, SloVak Academy of Sciences, Du´braVska´ cesta 9, 845 36 BratislaVa, SloVakia, and National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan ReceiVed: October 13, 2009; ReVised Manuscript ReceiVed: NoVember 30, 2009

Fo¨rster resonance energy transfer (FRET) from rhodamine 123 (Rh123; the energy donor (ED)) to rhodamine 610 (Rh610; the energy acceptor (EA)) adsorbed on the surface of synthetic saponite (SAP) was investigated. Experiments were performed with colloidal dispersions and thin solid films. In the colloidal systems, we observed efficient FRET leading to emission from the EA that was dependent on the molar ratio of the dyes. Self-quenching partially contributed to the relaxation of rhodamine molecules. Competitive adsorption of the dyes on SAP significantly influenced FRET under specific conditions. Adsorbed Rh610 was easily replaced by Rh123, especially at high dye loadings, and by surfactant dodecyltrimethylammonium (C12TMA) cations, especially at high surfactant concentrations. Rh610 forms zwitterions via the hydrolysis of carboxyl groups. The presence of the anionic group in the Rh610 molecule contributes to the repulsion between the dye molecules and SAP colloid particles and to lower adsorption of the dye. With a large excess of the ED, very efficient FRET was observed for films with SAP, C12TMA cations, Rh123, and Rh610. The main role of the inorganic SAP template is to bring the interacting dye molecules close together but still prevent fluorescence quenching caused by dye molecular aggregation. Introduction Over the last several decades, materials science has expanded to include a variety of new interdisciplines such as nanomaterials, supramolecular systems, multicomponents, nanocomposites, and multifunctional materials. Nanomaterials, in particular, have attracted much attention because of the substantial developments that have taken place in the field and their potential as new materials with superior properties. In nanomaterials, specific “nanophenomena” occur as the result of the interactions among small species with dimensions on the order of a few nanometers. One typical interaction is Fo¨rster resonance energy transfer (FRET). This phenomenon is the energy transfer between two chromophores and its occurrence is limited to the distances of a few nanometers.1 FRET has been known for over a half century and has been used for decades as an efficient tool in biochemistry and molecular biology for studying the dynamic properties and interactions of biomolecules and biopolymers, and for the determination of intermolecular distances, structural changes, etc.2,3 Intermolecular FRET does not occur in dilute solutions of molecular fluorophores because of the large intermolecular distances. However, efficient FRET can be achieved at interfaces where such molecules concentrate. Therefore, some hybrid materials comprising inorganic carriers and adsorbed/intercalated organic chromophores exhibit very efficient intermolecular * Corresponding author. Tel.: +421 2 5941 0459. Fax: +421 2 5941 0444. E-mail: [email protected]. † Institute of Inorganic Chemistry, Slovak Academy of Sciences. ‡ Comenius University. § International Laser Centre. | Polymer Institute, Slovak Academy of Sciences. ⊥ National Institute for Materials Science.

FRET.4,5 Various materials based on porous compounds with incorporated chromophore units, exhibiting efficient light harvesting, energy and/or electron transfer, have been developed over the last few years.6-8 However, only a few papers provide evidence for FRET in hybrid systems comprising layered silicates such as clay minerals.9-17 Clay mineral particles are of interest because of their two-dimensional structure, being about 1 nm thick and having dimensions of about 1 µm. The two-dimensional surface allows for specific preferential orientation of chromophores. Control of the molecular orientation and the density of adsorbed (intercalated) chromophores18-21 could be used to realize anisotropic FRET, i.e., FRET sensitive to light polarity with controllable intensity. Among the prospective FRET pairs, rhodamine and oxazine molecules in clay mineral colloidal dispersions have been investigated. Although efficient FRET has been observed in colloids under appropriate conditions, a lower efficiency was observed for solid thin films.9-11 The quenching of the fluorescence by coexisting molecular aggregates seems to be the dominant effect that inhibits FRET. There are several ways to avoid molecular aggregation: 1. Organic fluorophores that do not form molecular aggregates with strongly quenching properties may be used. Dyes with specific molecular shapes and distinct distributions of ionic groups can be used to prevent molecular aggregation even at high concentrations.22 2. Selection and modification of the inorganic carrier surface could allow quantitative and qualitative control over the adsorption of organic dyes. For example, on the surface of hydrophobic smectites (premodified with alkylammonium surfactants), organic dyes are adsorbed in the form of monomers while retaining their original photochemical activity.23-29 For nonmodified clay minerals, matching between mineral charge

10.1021/jp9098107  2010 American Chemical Society Published on Web 12/18/2009

FRET from Rh123 to Rh610

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TABLE 1: Conditions of the Rhodamine Dye Systems in SAP Colloidal Dispersions

d

experiment

Ia

-3

-5

IIb -5

dye concentration (mol dm ) dyes molar ratio nR123/nR610

10 1:1

loading (mmol g-1)

0.05

10 9:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:9 0.05

C12TMA surfactant concentration (mol dm-3) figure number

0.00 1 and 2

0.00 3 and 4

IIIc -5

IVd -5

10 9:1

10 9:1

0.05, 0.10, 0.20, 0.30, 0.40, 0.50 0.00 5

0.20 10-6, 5 10-6, 10-5, 2 10-5 6

a Basic experiment. b Experiments testing the effect of energy donor/energy acceptor ratio. c Experiments testing the effect of dye loading. Experiments testing the effect of the presence of surfactant.

density and charge distribution in dye molecules is also an important parameter.30-32 The lower the layer charge density of inorganic substrate, the less molecular aggregates are formed.22,33,34 3. The reaction conditions could be optimized to reduce molecular aggregation. Members of the rhodamine dye family exhibit a natural variation in their electronic and optical properties,27,33,34 enabling the selection of two suitable fluorophores with sufficient overlap between their excitation and emission spectra for an efficient FRET pair. The objective of this work is to study the FRET between different rhodamine cations adsorbed on layered silicates. In this study, rhodamine 123 (Rh123) and rhodamine 610 (Rh610) were chosen as the energy donor (ED) and energy acceptor (EA), respectively. Experimental Methods Synthetic saponite (SAP; Sumecton SA) was purchased from Kunimine Industries Co. Ltd., (Japan) and used as received. Rhodamine dyes were purchased from Lambda Physik (Germany). They included rhodamine 123, 2-(6-amino-3-imino-3Hxanthen-9-yl)benzoic acid, methyl ester (CAS number: 6266970-9), and rhodamine 610, N-[9-(2-carboxyphenyl)-6-(diethylamino)-3H-xanthen-3-ylidine]-N-ethyl-ethanaminium perchlorate (CAS Number: 23857-51-4). In the basic experiment, the molar ratio and concentration of the dyes were set to 1:1 and 10-5 M, respectively. Surfactant, dodecyltrimethylammonium (C12TMA) chloride of analytical grade, was purchased from Cica (Japan). The loading of the dye in colloidal systems was 5 × 10-5 mol g-1 (dye/clay). The loading was approximately 5% material’s cation exchange capacity (CEC ) 1.00 mmol g-1).31 We tested the effects of various parameters such as the molar ratio of rhodamine dyes (from 9:1 to 1:9), dye loading (5 × 10-5 - 5 × 10-4 mol g-1, which is approximately 5-50% CEC), and the presence of the C12TMA surfactant. The conditions used for experiments I-IV are summarized in Table 1. Films were prepared on silica slides from colloids of pure SAP by spin coating. They were first modified by the adsorption of C12TMA surfactant (10-3 mol dm-3), then washed with ethanol, and saturated with dyes by adsorption from aqueous solution (10-3 mol dm-3, 9:1 Rh123:Rh610 molar ratio) for 10 min and 1 and 2 h. The premodification with C12TMA was necessary since the films would otherwise not be fluorescent because of molecular aggregation. Ultraviolet-visible (UV-vis) absorption spectra were measured by a spectrophotometer (V-550 UV-vis; Jasco Co., Ltd.) at room temperature. Fused silica glass cuvettes, transparent in the UV-vis spectral range (>190 nm), were used for spectral measurements. The spectra were measured using a reference sample based on pure water. Blank samples based on clay mineral colloids (without dyes) were also measured to identify the contribution of light scattering from clay mineral particles.

Figure 1. Emission spectra of rhodamine 123, rhodamine 610, and their mixture in SAP dispersions. Solid lines, emission of single dyes in SAP colloids; dashed line, experimentally measured spectrum of rhodamine mixture in SAP dispersion; dotted line, calculated spectrum of the mixture for no energy transfer. Concentrations of the dyes were 10-5 mol dm-3. In the case of the dye mixture, the concentration of individual dyes was 5 × 10-6 mol dm-3. Excitation wavelength: 470 nm. Dyes loading: 5 × 10-5 mol g-1.

Figure 2. Normalized curves of the emission and excitation spectra of rhodamine 123 and rhodamine 610 in SAP colloidal dispersion. Emission spectrum of rhodamine 123 were measured with excitation at 470 nm (dashed line). The excitation spectrum of rhodamine 610 was measured for the emission at 625 nm (solid line).

Light scattering was negligible due to dilute concentration and small particle size of the saponite specimen. Fluorescence spectra were measured using a spectrofluorimeter (RF5300PC; Schimadzu, Japan). Films were observed with a LSM 510 META confocal laser scanning microscope head on an Axiovert 200 M inverted microscope (both Carl Zeiss, Germany). For excitation, we used the blue-violet lines of Ar from an ion laser (477 nm) with corresponding dichroic mirrors. Fluorescence was detected either by a multispectral detector (16 channels within the range 493-664 nm) or by band-pass emission filters (500-550 nm and 565-615 nm). A 50 × 0.80 EpiPlan Neofluar

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Figure 3. Emission spectra of colloidal dispersions of SAP with rhodamine 123 and rhodamine 610 mixtures showing the effects of differing dye ratios. The amount Rh123/Rh610 ratios: a. 1:1 (solid line, thick); 2:1 (dashed line); 3:1 (dotted line); 5:1 (line with scatter); 9:1 (solid line). b. 1:2 (solid line); 1:3 (dashed line); 1:5 (dotted line); 1:2 (line with scatter). Dyes concentration and loading of the dyes were kept constant at 10-5 mol dm-3 and 5 × 10-5 mol g-1, respectively. Excitation wavelength was 470 nm.

Bujda´k et al.

Figure 5. Emission spectra of colloidal dispersions of SAP with rhodamine 123 and rhodamine 610 mixtures showing the effect of loading. The amount Rh123/Rh610 ratio was 9:1. The loadings of the dyes (in mmol/g): a. 0.05 (solid line); 0.10 (dashed line); 0.20 (dotted line); b. 0.30 (solid line); 0.40 (dashed line); 0.50 (dotted line). Concentration of the dyes was kept constant at 10-5 mol dm-3. The loading was adjusted by varying the amount of clay mineral. Excitation wavelength was 470 nm.

profiles showing film thickness were obtained by line-scanning at different image depths with 0.5-µm Z-axis sampling. Results and Discussion

Figure 4. Maximal fluorescence of rhodamine 123 and rhodamine 610 in relationship with the mole fraction of Rh123. Fluorescence of Rh123, full symbols; fluorescence of Rh610, empty symbols; ratio of the fluorescence intensities IRh610/IRh123, line and symbol. Mole fraction of Rh123 xR123 ) n(Rh123)/(n(Rh610) + n(Rh123)) Data of maximal fluorescence are taken from Figure 3a. Dyes concentration and loading of the dyes were kept constant at 10-5 mol dm-3 and 5 × 10-5 mol g-1, respectively. Excitation wavelength was 470 nm.

dry objective was used with a confocal pinhole opening, corresponding to 1 Airy unit. For each image, an area of 92 × 92 µm was scanned at a resolution of 512 × 512 pixels with 16× line averaging and 8-bit quantization. Images were further processed by the LSM Image Examiner software. Vertical (XZ)

Energy Transfer in Colloids. Colloidal dispersions of SAP with two dye concentrations, 10-6 and 10-5 mol dm-3, were used in a series of preliminary experiments (see the Supporting Information, SI). Although the absorption spectra showed partial aggregation of Rh123 at a dye concentration of 10-5 mol dm-3 (SI2), fluorescence from this dye was at sufficient levels (Figure 1). One of the objectives of this work was to optimize the conditions for FRET in rhodamines/clay mineral systems; hence, the starting conditions described above were not expected to be optimal. In the subsequent experiments, we chose a higher dye concentration (10-5 mol dm-3) in order to ensure sufficient absorption of electromagnetic radiation and satisfactory fluorescence intensities. Basic absorbance and fluorescence characteristics of the dyes used in the experiments and their mixtures are listed in Table 2. In aqueous solutions, Rh123 and Rh610 exhibited maximum absorption at 499.0 and 553.5 nm, respectively. In SAP colloidal dispersions, only slight shifts in the absorption bands were observed (Table 2). The absorption band of Rh123 shifted slightly to lower energies upon mixing with the SAP colloid. One disadvantage when using rhodamines as a FRET pair is the relatively small value of the Stokes shift (about 30 nm), which may lead to energy migration, i.e, the transfer of energy to the same type of molecular species. Hence, another challenge was to optimize the system and thus maximize or increase heterotransfer. A significant overlap between the Rh123 emission spectra and the Rh610 excitation spectra was

FRET from Rh123 to Rh610

J. Phys. Chem. C, Vol. 114, No. 2, 2010 1249 emission from Rh123 (i.e., ED) at 530 nm was significantly reduced as a result of FRET to neighboring Rh610 cations. Only a very weak emission from Rh123 was detected in mixed-dye systems. Consequently, the emission from Rh610 cations at 580 nm was significantly enhanced. The role of clay mineral particles may lead to the concentration of dye cations on their surfaces. Suitable intermolecular distance is necessary for FRET to proceed, as can be noted from eqs 2 and 336

kT )

( )

1 R0 τD r

6

(2)

Equation 2 shows the relationship between the FRET rate constant kT and the distance r between ED and EA molecules. R0 denotes the Fo¨rster radius, the distance for which the FRET is 50% and τD is the donor relaxation lifetime in the absence of FRET. The relationship between the FRET efficiency E and the distance between ED and EA molecules (r) is as follows:

E)

Figure 6. Emission spectra of colloidal dispersions of SAP with rhodamine 123 and rhodamine 610 mixtures in the presence of dodecyltrimethylammonium. The surfactant concentration (in mol dm-3): a. no surfactant (solid line); 10-6 (dashed line) b. 5 × 10-6 (solid line); 10-5 (dashed line); 2 × 10-5 (dotted line). Concentration and loading of the dyes and dyes amount ratio were kept constant at 10-5 mol dm-3 and 2 × 10-4 mol g-1 and 9:1, respectively. Excitation wavelength: 470 nm.

TABLE 2: Band Positions (nm) in Absorbance and Emission Spectra in Solution and Colloidal Dispersions with SAP absorbance Rh123 Rh610 a

solution colloid solution colloid

499.0 508.0 553.5 552.0

emissiona 530.0 581.0

Measured from data in Figure 1. (Excited at 470 nm).

confirmed; the normalized curves of the spectra are shown in Figure 2. Occurrence of energy transfer in the basic reaction system (c ) 10-5 mol dm-3, nRh123/nRh610 ) 1) was directly proven by comparing the emission spectrum of the system based on mixed dyes with emission spectra of dyes alone (Figure 1). Rh123 was selectively excited at 470 nm, but a partial excitation of Rh610 as well cannot be neglected. However, the emission intensity of Rh610 was about three times lower than that of Rh123 under the same conditions. Linear combination of the spectra, based on the spectral profiles of individual dyes, gave a curve (dotted line, Figure 1) which would reflect a hypothetical system based on the absence of FRET. Surprisingly, the measured emission spectrum of the mixture (dashed line) was significantly different from the calculated linear combination of the individual components. FRET efficiency E can be estimated by using eq 135,36

E)1-

IED,EA IED

(1)

where the intensity of fluorescence is IED when only is the ED present, and IED,EA when both the ED and EA are present. The

R60 R60 + r6

(3)

The dye solution of concentration 10-5 mol dm-3 was too dilute for an efficient FRET. The specific volume of the solution per molecule at a concentration of 10-5 mol dm-3, is 1.66 × 105 nm3 (see SI4), which is comparable to a cube with an edge length of 55 nm. This value is an average estimate, in which the concentration fluctuations in the nm-scale space due to molecular movement have been neglected. Nevertheless, the Fo¨rster distances (R0) seem to never exceed the limit of 10 nm.36 The dye concentration on the surface of the clay mineral (SI4) can be estimated using the values of 5 × 10-5 mol g-1 for the loading of dye cations on the clay surface, and 750 m2 g-1 for the approximate theoretical surface area of the clay mineral particles.37 For a homogeneous distribution of the molecules on the surface, an area of about 25 nm2 is available per molecule (SI4). For a heterogeneous distribution, the local concentration of the dye could be higher and the surface area per molecule could be smaller. Heterogeneous adsorption of cationic dyes on clay surfaces due to molecular aggregation in clay mineral colloids has been demonstrated even for very low dye loadings38 and interpreted in terms of very fast kinetics of the adsorption process.34,39 The intermolecular distance would be about 5.6 nm under the following conditions: point dimension of the molecules (assumed for the sake of simplifying the model), hexagonal arrangement of the dye molecules on clay mineral surfaces, and a surface area of 25 nm2 per molecule. This intermolecular distance is much lower than that estimated for the concentration of 10-5 mol dm-3 in a homogeneous solution. However, even smaller intermolecular distances are possible due to the existence of two adjacent basal surfaces for each elemental layered particle. Both sides of a clay mineral layered particle are accessible for the adsorption of dye cations. Co-adsorption of ED and EA molecules at adjacent surfaces of the same particle may lead to very short intermolecular distances, approaching the limit of the layer thickness of about 1 nm and thus providing high FRET yields. Association and formation of clay particles in colloids of crystallites composed of several layers would further enhance the efficiency of FRET. The effect of the dye ratios on FRET efficiency was studied in detail. According to theory,35,36 the decay in the fluorescence intensity reveals how EA molecules are distributed in space around ED molecules. It is expected that the excess of ED (Rh123) would provide a sufficient number of molecules that

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act as light antennas. However, the energy might not be efficiently transferred to Rh610 due to the small number of EA molecules. The process would depend greatly on the ability of the EA to quench surrounding ED molecules, followed by strong radiation from the EA. The energy transfer could also be compromised by quenching in molecular assemblies of either dye. Molecular aggregates are very efficient quenchers due to their electronic properties. Energy migration is another process likely to occur because of the small Stokes shifts of rhodamine dyes. The energy migration may significantly increase the probability of fluorescence quenching, especially in the cases of low concentration of molecular aggregates. Multistep energy transfer leads to the quenching of the molecules, which are located at relatively large distances from quencher to be quenched by a single step-energy transfer. On the other hand, if the EA molecules greatly outnumber the ED molecules, highly efficient quenching of the ED would be expected. These assumptions were confirmed with fluorescence spectroscopy (Figure 3). For systems with the Rh123/Rh610 ratio of 2:1 and higher, the emission from Rh123 at 525 nm increased when Rh123 was increased. This observation indicates that the amount of Rh610 cations surrounding the Rh123 ions is insufficient for them to be able to act as effective quenchers and to completely quench the excited ED molecules. On the other hand, the intensity of the emission at about 575 nm also increased with the amount of the ED. The main difference was observed for the systems based on 1:1 and 2:1 ED/EA ratios. Further increase of this ratio did not result in significant qualitative changes. The increase of the fluorescence from Rh610 (575 nm), in spite of the decreasing amount of this dye, indicates increased overall efficiency of FRET. Figure 4 shows the relationship between maxima of fluorescence peaks generated by Rh123 (full symbol) and by Rh610 (empty symbol), and the mole fraction of Rh123. The increase of the Rh123 fluorescence is not linearly related to its concentration and mole fraction, which confirms FRET. This increase becomes more significant at the highest mole fractions, since under these conditions a large part of the dye is not quenched by Rh610. An opposite trend was observed in the increase of the fluorescence of Rh610. At relatively low mole fractions of Rh610 (i.e., high mole fraction of Rh123), the change of the fluorescence of Rh610 (empty symbols) is negligible. It indicates the energy acceptor is fully saturated by the energy transferred from the Rh123 molecules. An opposite variation of the Rh123/Rh610 ratios from 1:2 to 1:9 did not lead to significant spectral changes (Figure 3b). Energy donors, represented by the Rh123 cations, were efficiently quenched by the Rh610 cations which were in excess. Similarities of the spectra in Figure 3b indicate that there were some Rh610 ions, which did not take part in the FRET process. Indeed, the intensity of the emission at 575 nm did not change significantly with increasing amount of Rh610 cations. The reaction system with the R123/R610 molar ratio of 9:1 was chosen for detailed studies in future experiments. As the next step for optimizing the FRET, we tested the effect of dye loading. The loading represents the amount ratio of the dye cations to clay mineral substrate. The concentration of the dye cations was kept constant and the loading was changed by the variation of clay mineral amounts in colloidal dispersions. The loading was increased from a standard value of 5 × 10-5 up to 5 × 10-4 mol g-1. One could expect larger dye loadings to lead to a higher concentration of dye cations adsorbed on the clay surface. A larger concentration would mean shorter intermolecular distances between the ED and EA molecules,

Bujda´k et al. thus increasing the FRET efficiency. Unfortunately, the spectra shown in Figure 5 indicate that the efficiency of the process was not significantly improved. Although the ratio of donor/ acceptor emission decreased significantly at loading 10-4 mol g-1, the emission yields from both the donor and the acceptor were lowered. This indicates larger quenching of the ED component at this loading, but a significant part of the energy seems to be lost in nonradiative processes, such as quenching by molecular aggregates and energy migration terminated by the self-quenching. Indeed, increasing the dye loading may lead to a significantly enhanced molecular aggregation of dye molecules, as has been shown for other reaction systems.18,39 Further increase of the loading to 2 × 10-4 mol g-1 led to a remarkable decrease in Rh610 emission. The reduction of emission from the Rh123 cations was less significant. The trend toward lower Rh610 emission continued with increasing loading up to 5 × 10-4 mol g-1. Interestingly, the emission from R123 started to increase at the loading of 3 × 10-4 mol g-1. At the highest loading, the emission from Rh123 was equal to that at the lowest loading. The observations of nonuniform changes in the emissions bands cannot be explained only on the basis of the formation of dye molecular aggregates. Another factor, which might contribute to the observed trends, is the differences in the chemical properties of the dyes. The cations of Rh610 contain -COOH groups, which can easily dissociate in neutral or basic solutions leading to the formation of cations-anions (zwitterions) which can make the aggregation more pronounced. Larger loading could contribute to stronger aggregation of Rh610, and the isolation of this component from Rh123, which remains in a fluorescently active form. Another consequence of the Rh610 zwitterion formation would be weaker adsorption of the Rh610 ions on the clay mineral surfaces due to the presence of anionic groups. Indeed, increasing the loading of the dyes would reduce the available surface for dye adsorption, making the adsorption process more competitive. Significantly lower adsorption of Rh610 and other zwitterionic dyes on layered silicates has already been shown.33,34 In order to investigate the effect of possible molecular aggregation, we tested the systems that included the cationic C12TMA surfactant. The dye loading of 2 × 10-4 mol g-1 was chosen for this experiment. At this loading if no surfactant was included in the system, a significant drop in the emission, especially from Rh610, was observed (Figure 5). If the emission decrease had been because of the dye molecular aggregation, the inclusion of the surfactant could contribute to the disaggregation of the dye molecules and the enhancement of the dyes’ fluorescence.23-29 Low concentration of the surfactant did not change the emission spectrum significantly (Figure 6). In particular, the emission from both the components increased, but this change was very small. The emission from Rh123 was only slightly higher than from Rh610. Probably the presence of the surfactant prevented some of the dye molecules from being a part of the aggregation. One should also consider the fact that the amount of the surfactant was very low, only 10% of that of the dyes. Further increase of the surfactant amount was reflected in significant changes in the emission spectra (Figure 6). The emission of Rh610 decreased at the surfactant concentration 5 × 10-6 mol dm-3. A significant increase in the Rh123 emission was observed when the concentration of the surfactant was 2 × 10-5 M. These results confirm the assumption regarding the weak adsorption of Rh610. The main cause of the observed spectral changes is the lower affinity of the inorganic template to Rh610, whose cations contain easily ionizable carboxyl groups. The role of the surfactant in

FRET from Rh123 to Rh610 suppressing dye molecular aggregation seems to be of only secondary importance for the interpretation of the spectra observed in these systems. The presence of hydrophobic surfactant molecules reduced the polarity of the adsorbent, and thus, also the affinity of the surface for zwitterionic Rh610. In competitive adsorption, C12TMA surfactant cations easily replaced Rh610 on the SAP surface. Energy Transfer in the Films. Without the C12TMA surfactant, complete quenching led to the absence of detectable fluorescence in the films (not shown). Therefore, the SAP films were premodified with the C12TMA surfactant before the adsorption of the dyes. The adsorption of the dyes on organically modified films proceeded directly from an aqueous solution of the dye mixture for 10 min and 1 and 2 h. The amount of adsorbed dyes was higher for the longer times. We tried several reaction conditions for the film preparation. Surprisingly, the films with Rh123 as the dominant component and only traces of Rh610 also exhibited very efficient FRET (Figure 7a). The absorption spectra confirmed that only negligible amounts of absorbed R610 cations were present in the films (Figure 7c). Rh610 can be clearly identified in the spectrum only after the longest adsorption time. The enhanced preference for Rh123 adsorption could be due to the formation of zwitterions of Rh610, as discussed previously for colloidal systems. The excitation spectra were measured for the emission at 625 nm (Figure 7b). This wavelength of the emitted light was chosen in order to measure mainly the fluorescence from Rh610 cations. Indeed, a relatively strong band in the emission spectra associated with the Rh610 cations was identified at about 560 nm (Figure 7a). A more intense band at about 510 nm in the excitation spectra confirms the presence of R123 in the film (Figure 7b). Excited Rh123 cations probably did not relax via radiation transition, but there was likely a very efficient FRET process from excited Rh123 to Rh610. A surprisingly high intensity emission from Rh610 was observed when the fluorescence was excited at 470 nm (Figure 7a). The dominance of the emission from Rh610 was contrary to what would be expected because a much lower amount of this dye adsorbed on the films, and also, this dye was not directly excited at this wavelength. These facts indicate very efficient FRET from Rh123 to Rh610, where one cation of Rh610 is able to quench a large number of the Rh123 cations. Emission from the Rh123 cations was almost completely quenched by the FRET process. The very efficient FRET in the films is due to the three-dimensionality of this system, whereas FRET in colloids was limited to two dimensions. Although dye cations were concentrated on the surface of clay particles in colloidal systems, the clay mineral particles remained dispersed in the bulk volume of the solvent, and so interparticle FRET was unlikely. In the films, the dye cations are concentrated on the silicate surface and the individual particles form thick layers. Hence, interparticle FRET can be easily achieved. For an individual clay mineral layer of thickness of about 0.96 nm and an interlayer space of about the same value, FRET could be achieved through 1 to 5 alternating layers and interlayer spaces. Optical homogeneity of the films is another parameter often required for industrially applied materials. This requirement may often be problem for solid materials based on multicomponent systems. Optical homogeneity was characterized by fluorescence microscopy and spectroscopy. The twodimensional profiles of the films indicated relatively very homogeneous spatial distribution of the fluorophores. The

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Figure 7. Absorption and fluorescence spectra of dye/SAP films. a. Emission after excitation at 470 nm. b. Excitation spectra for emission at 625 nm. C Absorption spectra. The films were prepared by adsorption of the dyes from aqueous solution on organically modified SAP film. The adsorption time was 10 min (solid line), 1 h (dashed line), and 2 h (dotted line). The Rh123/Rh610 ratio in solution was 9:1.

results for one representative film are shown in Figure 8a,c. The emission spectra obtained from arbitrarily chosen spots on the films were not markedly different. Representative spectral profiles measured at two different points of the sample are shown in the Figure 8d. The thickness of the film was estimated to be less than 500 nm (Figure 8b), which was the measurement limit of the experimental method. Conclusions Efficient resonance energy transfer between rhodamine dye molecules took place in colloidal systems as well as in solid films with layered silicate SAP. Molecular aggregation and fluorescence self-quenching, and concentrations of energy donor and acceptor molecules were the main factors limiting or affecting this process. The presence of a hydrophobic surfactant suppressed the molecular aggregation of the dyes and enhanced the overall fluorescence, especially in solid films. The chemical properties of the dyes are important in

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Bujda´k et al. Supporting Information Available: Absorption spectra of saponite colloids with the dyes and the emission spectra of the systems at lower dye concentration, calculation of the volume per one dye molecule in solution, and clay mineral surface occupied by one dye molecule. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes

Figure 8. Example of the confocal fluorescence microscopy image of the film with intercalated rhodamine molecules. Brighter spots correspond to higher fluorescence intensity, which relates to larger number of luminiscent molecules in a given sampled area and/or higher fluorescence quantum yield. (a) Image taken in X-Y dimensions, showing lateral distribution of the fluorescent molecules. Small grains observable in the image are mostly related to instrumental and photon counting noise (see also c). Image was taken as lateral scan at the sample depth where the fluorescence intensity has maximum (center of the film). (b) Vertical X-Z profile of the fluorescence intensity in the same sample, related to the sample thickness. (c) Fluorescence intensity profile along white arrow in panel a. (d) Example of the fluorescence spectra, taken at two different points of the sample.

achieving selective adsorption which would lead to the desired composition of the hybrid systems. The energy transfer in solid samples was more efficient due to the higher concentration of the fluorophores and the three-dimensional character of the structure. Solid films exhibited good optical homogeneity. Acknowledgment. This work was done as a part of bilateral project between the Institute of Inorganic Chemistry and the National Institute for Materials Science within the aggreement between the Japan Society of the Promotion of Science and the Slovak Academy of Sciences and supported by the projects of the Slovak Research and Development Agency under Contract No. APVV-51-027405 and grant agency VEGA (2/0089/09).

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