Langmuir 2004, 20, 2989-2991
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Energy Transfer in Fluorescent Silica Nanoparticles Marco Montalti,* Luca Prodi, Nelsi Zaccheroni, Andrea Zattoni, Pierluigi Reschiglian, and Giuseppe Falini Dipartimento di Chimica “G.Ciamician”, Universita` di Bologna, Via Selmi 2, 40126 Bologna, Italy Received November 21, 2003. In Final Form: January 12, 2004
Large multifluorophoric systems are attracting increasing attention because of their peculiar photophysical behavior.1-5 The efficiency of very important processes such as the collection of the excitation energy toward a specific site or the fluorescence modulation upon recognition of a given target can be greatly improved in widely organized structures when the cooperative photophysical processes can occur.1a As a consequence, these systems behave as nanodevices that are able to perform predefined functions in a very effective way. A limitation to this approach to the optical nanosystems arises from the difficulties in the synthetic procedures. The preparation of large supramolecular structures is usually extremely complicated and therefore inefficient.5 For this reason, synthetic strategies based on self-assembly, polymerization, or modification of pre-existing nanostructures are very advantageous. Different kinds of fluorescent systems such as polymers,1 zeolites,2 or even monolayers3 have been prepared and investigated. The critical point in the design of new materials is the communication between the fluorescent units because the cooperative processes need multiple interactions to take place. In this paper, we use silica nanoparticles as three-dimensional scaffolds for fluorophore organization, and we show the results of photophysical studies demonstrating that energy transfer between the anchored units occurs.4 This crucial observation opens up new perspectives in the design of multifunctional structures based on silica nanoparticles, which could revolutionize the field of nanosensors and -devices.6 To obtain covalent grafting of the fluorophore to the silica * Author to whom correspondence should be addressed. E-mail:
[email protected]. (1) (a) Swager, T. M. Acc. Chem. Res. 1998, 31, 201. (b) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev. 2000, 100, 2537. (c) Kim, J.; McQuade, D. T.; Rose, A.; Zhu, Z.; Swager, T. M. J. Am. Chem. Soc. 2001, 123, 11488. (d) McQuade, D. T.; Hegedus, A. H.; Swager, T. M. J. Am. Chem. Soc. 2000, 122, 12389. (e) Fleming, C. N.; Maxwell, K. A.; De Simone, J. M.; Meyer, T. J.; Papanikolas, J. M. J. Am. Chem. Soc. 2001, 123, 10336. (2) (a) Pauchard, M.; Huber, S.; Me´allet-Renault, R.; Maas, H.; Pansu, R.; Calzaferri, G. Angew. Chem., Int. Ed. 2001, 40, 2839. (b) Pauchard, M.; Devaux, A.; Calzaferri, G. Chem.sEur. J. 2000, 6, 3456. (3) (a) Flink, S.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Chem. Soc., Chem. Commun. 1999, 2229. (b) Chrisstoffels, L. A. J.; Andronov, A.; Fre´chet, J. M. J. Angew. Chem., Int. Ed. 2000, 39, 2163. (c) van der Veen, N. J.; Menno, S. F.; Deij, A.; Egberink, R. J. M.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Am. Chem. Soc. 2000, 122, 6112. (4) Montalti, M.; Prodi, L.; Zaccheroni, N.; Falini, G.; J. Am. Chem. Soc. 2002, 124, 13540. (5) (a) Vo¨gtle, F.; Gestermann, S.; Kauffmann, C.; Ceroni, P.; Vicinelli, V.; De Cola, L.; Balzani, V. J. Am. Chem. Soc. 1999, 121, 12161. (b) Vo¨gtle, F.; Gestermann, S.; Kauffmann, C.; Ceroni, P.; Vicinelli, V.; Balzani, V. J. Am. Chem. Soc. 2000, 122, 10398. (c) Venturi, M.; Serroni, S.; Juris, A.; Campagna, S.; Balzani, V. Top. Curr. Chem. 1998, 197, 193. (d) Balzani, V.; Campagna, S.; Denti, G.; Juris, A.; Serroni, S.; Venturi, M. Acc. Chem. Res. 1998, 31, 26. (e) Archut, A.; Vo¨gtle, F.; De Cola, L.; Azzellini, G. C.; Balzani, V.; Ramanujam, P. S.; Berg, R. H. Chem.sEur. J. 1998, 4, 699. (f) Archut, A.; Azzellini, G. C.; Balzani, V.; De Cola, L.; Vo¨gtle, F. J. Am. Chem. Soc. 1998, 120, 12187. (6) Epstein, J. R.; Walt, D. R. Chem. Soc. Rev. 2003, 32, 203.
Figure 1. TEM picture of the fluorescent silica nanoparticles.
scaffold, we followed the synthetic strategy proposed by Van Blaaderen for larger silica dye-doped colloids.7 Inclusion of dyes in silica nanoparticles usually takes advantage of the inverse microemulsion techniques,8 which unfortunately do not lead to covalent binding to the polymeric matrix with the consequent risk of dye leaching. The introduction of a triethoxysilane group directly on the dye molecule allows, instead, its covalent anchoring during the tetraethoxysilane (TEOS) polymerization.9 The reaction conditions were optimized in order to have colloids with a diameter around 20 nm. This choice originates from the need to have colloidal solutions, which give very weak light scattering, a point that is of great importance for optical applications. An even more important point is that this size is compatible with the interaction radius required for most photophysical processes, and at the same time, it allows each fluorophore to interact, when excited, with a large fraction of the units bound to the same nanoparticle. It is interesting to note that this synthetic strategy leaves the surface of the colloids available for further modification.4 The fluorescein-labeled triethoxysilane 1 was synthesized starting from fluorescein isothiocyanate and (3aminopropyl)triethoxysilane. An amount of 1 corresponding to 1% (mol/mol) with respect to TEOS was used in the Stroeber procedure for the nanoparticles preparation.9 The formation of the colloids was confirmed by the transmission electron microscopy (TEM) images (see Figure 1) that were graphically elaborated (see Figure 2) to evaluate the average size of the particles (23 ( 3 nm).10 A major problem in the synthesis of nanometric particles is their separation from the unreacted fluorophore. We were able to overcome this difficulty by performing a very efficient microscale purification/size sorting of the nanoparticles by using flow field-flow fractionation (FlFFF). FlFFF is able not only to fractionate nanoparticles on a size basis but also to separate the particles from low molecular weight species.11 The fluorescent nanoparticles were injected and fractionated by collecting the samples (7) (a) Van Blaaderen, A.; Imhof, A.; Hage, W.; Vrij, A. Langmuir 1992, 8, 1514. (b) Verhaegh, N. A. M.; Van Blaaderen, A. Langmuir 1994, 10, 1427. (8) (a) Santra, S.; Wang, K.; Tapec, R.; Tan, W. J. Biomed. Opt. 2001, 6, 160. (b) Gan, L. M.; Zhang, K.; Chew, C. H. Colloids Surf., A 1996, 110, 199. (9) Stoeber, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62. (10) TEM pictures were elaborated with SigmaScan 5 and SigmaPlot 5 by SPSS Inc.
10.1021/la0361868 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/03/2004
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Notes Scheme 1. Representation of the Photophysical Processes Occurring in the Nanoparticles
Figure 2. (a) Size distribution of the nanoparticles resulting from the elaboration of the TEM pictures (bars). (b) Absorbance at 500 nm during the FlFFF elution of the nanoparticles (dashed line). (c) Numerical size distribution curve calculated from FlFFF (continuous curve). (d) Fluorescence intensities of the fraction n with a diameter between (3n - 1) and 3n (squares). All of the four curves were normalized at their maximum.
containing colloids with a diameter approximately between 3(n - 1) and 3n nm with 1 < n < 25. The photophysical measurements were carried out directly on the FlFFF fractions as obtained from the elution. An absorbance signal at 500 nm was continuously detected during the elution (Figure 2, dashed line) and converted into a numerical size distribution of the emerging colloids as a function of the hydrodynamic diameter (Figure 2, continuous line). The differences between the two profiles are due to the fact that the absorbance signal depends not only on the number of particles having a definite size but also on the number of fluorophores per particle (increasing with the particle volume). As shown in Figure 2, a very good agreement is obtained between the FlFFF-based and TEM-based size distribution analyses. This correlation is the definitive evidence that the fluorophores are bound to the silica matrix. Because the photophysical properties of fluorescein are strongly pH-dependent,12a all of the measurements on the different fractions were carried out in the presence of ammonia at pH 10. All of the fractions between n ) 4 and 14 (corresponding to a diameter between 9 and 42 nm) show an absorption band at 490 nm and a fluorescence band at 520 nm that are typical of fluorescein. Moreover, for all the examined dispersions, the fluorescence intensity at 520 nm upon excitation at 480 nm was directly proportional to the absorbance (A < 0.05) at the excitation wavelength. This proportionality indicates that, in the nanoparticles, the average quantum yield of fluorescein units is the same and independent of the size of the colloids. This average quantum yield result is about 50% with respect to that of simple fluorescein in the same conditions, while the excited-state lifetime of fluorescein in the nanoparticles is the same for all of the nanoparticle samples (τ ) 3.9 ns). Most importantly, the fluorescence of fluorescein in the nanoparticles is almost completely depolarized (P < 0.02). This was an unexpected finding (11) (a) Giddings, J. C. Science 1993, 260, 1456. (b) Schimpf, M. E., Caldwell, K., Giddings, J. C., Eds. Field-Flow Fractionation Handbook; Wiley-Interscience: New York, 2000. (c) Giddings, J. C.; Lin, G.-C.; Myers, M. N. J. Colloid Interface Sci. 1978, 65, 67. (12) (a) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/Plenum Publishers: New York, 1999. (b) Valeur, B. New Trends in Fluorescence Spectroscopy: Applications to Chemical and Life Sciences; Wiley VCH: Weinheim, Germany, 2001. (c) Maus, M.; Mitra, S.; Lor, M.; Hofkens, J.; Weil, T.; Herrmann, A.; Muellen, K.; De Schryver, F. C. J. Phys. Chem. A 2001, 105, 3961.
because the rotational mobility of the fluorophores in the colloids is strongly reduced and it indicates that a different mechanism for depolarization must be considered.12 All of these results are in agreement with the occurrence of two processes, which involve the fluorescein units in the nanoparticles. The first one causes the complete quenching of about 50% of the fluorophoric units and is known to be due to the short-range interactions between the fluorescein molecules.12,13 The second one leads to fluorescence depolarization and implies the excitation energy transfer between the different dye units in the nanoparticles.12,13 These two situations are schematically depicted in Scheme 1. The possibility of having different kinds of interactions depending on the intermolecular distance makes fluorescein a good probe to test the structure of the fluorophore network. The observed results can be explained considering that, as expected, the local density of the dye molecules in the nanoparticles is not homogeneous. In particular, there are more dense regions where the fluorescein moieties are close enough to give self-quenching, which causes a decrease of the overall average fluorescence quantum yield. On the other hand, the presence of a single exponential component in the fluorescence decay suggests, together with the observed depolarization, that the emission is due to the dye molecules being close enough to take part in the energy transfer processes but too distant to give self-quenching. Such a discontinuous behavior implies the presence of groups of completely quenched fluorophoric units and other groups whose fluorescence efficiency is unmodified. This could be due to a partial preorganization of the fluorophores during polymerization that leads to the formation of oligomers coming from the hydrolysis and condensation of 1. Moreover, the very low polarization degree of the fluorescence rules out the presence of “isolated” fluorophore molecules that are too far away to transfer the excitation energy to other fluorescein moieties. In our opinion, the results presented here open up a new perspective in the design of functional nanosystems with great applicative potentialities particularly in the field of fluorescent sensor development. This is because we demonstrated that the communication between the photoactive units, which is crucial for this kind of application, is highly efficient in the nanoparticles. We also showed that FlFFF is a powerful technique to size sort and micropurify fluorescent colloids of nanometric dimension, which can reduce their dimensional polydispersity and remove from the sample the unreacted fluorophore. This is a result of great interest for both commercial applications and further investigations on the dependence of colloid physicochemical features on the size (13) (a) Neckers, D. C.; Valdes-Aguilera, O. M. Adv. Photochem. 1993, 18, 315. (b) Jones, G., II; Qian, X. J. Photochem. Photobiol., A 1998, 113, 125.
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distribution. In addition, the combination of this synthetic strategy with the nanoparticle surface modification4 can lead to even more versatile systems where different molecular species (chromophores, receptors, etc.) can be inserted into the nanoparticle core or surface to give a new generation of fluorescent sensors and labels. Experimental Section Synthesis of Compound 1. All of the reagents and solvents were purchased by Aldrich and used without further purification. A total of 0.05 mmol of fluorescein isothiocianate was dissolved in 1 mL of dimethylformamide. Triethyamine (0.1 mmol) and (3-aminopropyl)triethoxysilane (0.06 mmol) were added, and the solution was stirred for 2 h at room temperature leading to the precipitation of an orange powder. The solid was isolated by centrifugation, washed three times with DMF, and characterized by high-performance liquid chromatography/mass spectroscopy, which showed pure 1 (electrospray ionization mass spectroscopy m/z: 609.2 [M - H+], 610.2 [M]). Synthesis of Nanoparticles. Silica nanoparticles were prepared according to the Stroeber method. 1 (0.04 mmol) was dissolved in 20 mL of ethanol under vigorous stirring. A total of 100 µL (0.43 mmol) of TEOS was then added together with 600 µL of an ammonia water solution (32% w/w). The mixture was stirred for 2 days leading to the formation of the fluoresceincontaining colloids. FlFFF Measurements. The FlFFF measurements were performed with the F-1000 system (FFFractionation LLC, Salt Lake City, UT), operating at the elution flow rate of 4.0 mL/min
and at the cross-flow rate of 1.8 mL/min. A carrier solution was used with 0.001% (v/v) Triton X-100, 0.02% (m/v) NaN3, and 5 mM Tris in water. The accumulation wall was made of a 10 000 Mr cutoff cellulose membrane. A total of 20 µL of a nanoparticle suspension in ethanol was injected. The elution time was converted into the particle hydrodynamic diameter through the FFF theory. The particle number distribution function was obtained through the Mie scattering theory from the absorbance detector response at 500 nm. TEM Measurements. For the TEM investigations, a drop of nanoparticle in an ethanol solution was transferred onto holey carbon foils supported on conventional copper microgrids. A Philips CM 100 transmission electron microscope operating at 80 kV was used. Photophysical Measurements. Absorption spectra were recorded with a Perkin-Elmer Lambda 40 spectrophotometer. Fluorescence and polarization spectra were recorded with the Jobin-Yvon Fluorolog spectrofluorimeter with automated polarizers. The fluorescence lifetimes (uncertainty of (5%) were obtained with an Edinburgh single-photon counting apparatus, in which the flash lamp was filled with D2. Luminescence quantum yields (uncertainty of (15%) were determined using quinine sulfate (Φ ) 0.546 in 0.5 M H2SO4) as the standard.
Acknowledgment. The authors thank MIUR (FISR, project SAIA) and the University of Bologna (funds for selected topics) for funding. LA0361868
