Langmuir 2006, 22, 5877-5881
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Size Effect on the Fluorescence Properties of Dansyl-Doped Silica Nanoparticles Marco Montalti,*,† Luca Prodi,† Nelsi Zaccheroni,† Gionata Battistini,† Silvia Marcuz,‡ Fabrizio Mancin,‡ Enrico Rampazzo,‡ and Umberto Tonellato‡ Dipartimento di Chimica “G.Ciamician”, UniVersita` di Bologna, Via Selmi 2, 40126 Bologna, Italy, and Dipartimento di Scienze Chimiche, UniVersita` di PadoVa, Via Marzolo 1, I -35131 PadoVa, Italy ReceiVed December 23, 2005. In Final Form: April 10, 2006 We present here the study of the photophysical properties of new dye-doped silica nanoparticles (DDNs) bearing dansyl fluorescent derivatives covalently linked to the silica matrix. The described experimental evidences show how the different location of the chromophores induces great changes in their photophysical behavior, suggesting that fluorophores located near the surface of the nanoparticles have a very different behavior with respect to the internal molecules. These latter ones, in fact, are shielded from the solvent and have a strong blue emission, while those at the periphery interact with the solvent and show a weaker red-shifted emission. As a consequence, the fluorescence properties of these nanoparticles are an average between the characteristics of the two different families of dyes. The relative amount of fluorophores located in the two compartments can be controlled simply by changing the size since, from our results, the thickness of the solvent permeable layer is not relevantly affected by the diameter of the nanoparticles. It is noteworthy that the fluorophores located in the outer shell exhibit very peculiar features: they are sensitive and interact with small molecules such as solvent molecules but, at the same time, they are not accessible to big receptor species such as β-cyclodextrins. Such results indicate that most of the solvent-sensitive dansyl moieties are located within pores large enough to only accommodate solvent but not big molecules as cyclodextrins, giving precious insight on the morphology of the nanoparticles.
Introduction Size is a critical parameter in determining properties of materials, and its effects become outstanding in nanometric systems where the bulk properties and molecular character of matter merge to originate new peculiar features. In the case of metal nanoparticles1 or semiconductor nanocrystals,2 the influence of size on optical, electrochemical or magnetic behavior is wellknown and is mostly related to quantum effects. In the present paper, we present evidences that the photophysical properties of dye-doped insulating nanoparticles are also affected by the diameter and that such dependence is related to the relative amount of fluorophores actually near the surface. The synthetic procedure to prepare nearly monodisperse silica nanoparticles by the condensation of tetralkoxysilane derivatives in ammonia, water, and ethanol solutions has been known since the late 1960s from the work of Sto¨ber.3 Later on, a modification of this procedure was proposed by van Blaaderen to prepare covalently linked dye-doped silica nanoparticles (DDNs).4 DDNs have been attracting increasing interest in past years for their potential applications as labels or sensors in biology and medicine. * To whom correspondence
[email protected]. † Universita ` di Bologna. ‡ Universita ` di Padova.
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(1) (a) Templeton, A. C.; Welfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (b) Kamat, P. V. J. Phys. Chem. B 2002, 106, 7729-7744. (c) Thomas, K. G.; Kamat, P. V. Acc. Chem. Res. 2003, 36, 888-898. (d) Shenhar, R.; Rotello, V. M. Acc. Chem. Res. 2003, 36, 549-561. (e) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293-346. (f) Drechsler, U.; Erdogan, B.; Rotello, V. M. Chem.sEur. J. 2004, 10, 5570-5579. (g) Wang, G.; Huang, T.; Murray, R. W.; Menard, L.; Nuzzo, R. G. J. Am. Chem. Soc. 2005, 127, 812-813. (2) (a) Alivisatos, A. P. Science 1996, 271, 933-937. (b) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 20132016. (c) El-Sayed, M. Acc. Chem. Res. 2004, 37, 326-33. (d) Yadong, Y.; Alivisatos, A. P. Nature 2005, 437, 664-670. (e) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538-544. (f) Sargent, E. H. AdV. Mater. 2005, 17, 515-522. (3) Sto¨ber, W.; Fink, A.; Bohn E. J. Colloid Interface Sci. 1968, 26, 62-69.
In fact, the inclusion in silica nanoparticles allows for the gathering of many fluorophoric units in a single labeling moiety, whose fluorescence intensity turns out to be even thousands of times higher with respect to a single dye molecule.4d,e In virtue of their strong fluorescence, DDNs were used to stain cells5 or in immunoassays6 as traceable vectors capable of delivering drugs or DNA within cells or as scaffolds to build complex fluorescent nanosensors.7 Nevertheless, some unusual properties of these nanoparticles are often neglected: these water soluble, multifluorophoric systems, in fact, share some peculiar features of more sophisticated supramolecular architectures such as dendrimers,8 with the great advantage of being much easier and cheaper to prepare. One of the most interesting similarities is that their properties are not homogeneously distributed; that is to say that several “compartments” (core, surface, mesopores, shells, etc.) can be easily engineered to perform different functions. We have previously shown how it is possible, via a different (4) (a) Van Blaaderen, A.; Kentgens, A. P. M. J. Non-Cryst. Solids 1992, 149, 161-178. (b) Van Blaaderen, A.; Van Geest, J.; Vrij, A. J. Colloid Interface Sci. 1992, 154, 481-501. (c) Van Blaaderen, A.; Vrij, A. J. Colloid Interface Sci. 1993, 156, 1-18. (d) Van Blaaderen, A.; Vrij, A. Langmuir 1992, 8, 2921-2931. (e) Verhaegh, N. A. M. A.; Van Blaaderen, A. Langmuir 1994, 10, 1427-1438. (5) (a) Santra, S.; Yang, H.; Dutta, D.; Stanley, J. T.; Holloway, P. H.; Tan, W.; Moudgilb, B. M.; Mericlea, R. A. Chem. Commun. 2004, 2810-2811. (b) Santra, S.; Xu, J.; Wang, K.; Tan, W. J. Nanosci. Nanotechnol. 2004, 4, 590-599 and references therein. (6) (a) Zhao, X.; Tapec-Dytioco, R.; Tan, W. J. Am. Chem. Soc. 2003, 125, 11474-11475. (b) Wang, L.; Yang, C.; Tan, W. Nano Lett. 2005, 5, 37-43. (7) (a) Xu, H.; Aylott, J. W.; Kopelman, R.; Miller, T. J.; Philbert, M. A. Anal. Chem. 2001, 73, 4124-4133. (b) Koo, Y.-E. L.; Cao, Y.; Kopelman, R.; Koo, S. M.; Brasuel, M.; Philbert M. A. Anal. Chem. 2004, 76, 2498-2505. (c) Montalti, M.; Prodi, L.; Zaccheroni, N.; Falini, G. J. Am. Chem. Soc. 2002, 124, 1354013546. (d) Montalti, M.; Prodi, L.; Zaccheroni, N. J. Mater. Chem. 2005, 15, 2810-2814. (e) Brasola, E.; Mancin, F.; Rampazzo, E.; Tecilla, P.; Tonellato, U. Chem. Commun. 2003, 3026-3027. (f) Rampazzo, E.; Brasola, E.; Marcuz, S.; Mancin, F.; Tecilla, P.; Tonellato, U. J. Mater. Chem. 2005, 15, 2687-2696. (g) Arduini, M.; Marcuz, S.; Montolli, M.; Rampazzo, E.; Mancin, F.; Gross, S.; Armelao, L.; Tecilla, P.; Tonellato, U. Langmuir 2005, 21, 9314-9321. (h) Prodi, L. New J. Chem. 2005, 29, 20-31. (8) See, for example, Tomalia, D. A.; Fre´chet, J. M. Prog. Polym. Sci. 2005, 30, 217-505.
10.1021/la053473y CCC: $33.50 © 2006 American Chemical Society Published on Web 05/20/2006
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synthetic approach, to introduce fluorescent molecules in the bulk7d,9 or on the surface7c,7f of silica nanoparticles. In this paper, we show how the core and the surface of DDNs are intrinsically different and, even when labeled with the same dye, they show different fluorescence properties. We also demonstrate how this feature becomes more and more important for small nanoparticles (tens of nanometers); that is to say, when the average ratio of the dyes close to the surface is higher. For these experiments, we took advantage of the strong environmental sensitivity of the dansyl fluorophore. Steady-state and time-resolved (timecorrelated single photon counting (TCSPC)) fluorescence measurements allowed us to recognize in the nanoparticles two different populations of dansyl moieties. The first family experiences a water-like environment: it presents a greenish fluorescence with an average lifetime of about 6 ns. The second family, on the other hand, is shielded from water and shows a blue fluorescence with an average lifetime of 18 ns. Our results show that the contribution of the two populations is size dependent and that, in the case of the smaller nanoparticles, the fraction of fluorophores exposed to the solvent is dominating. Nevertheless, via fluorescence anisotropy studies, we could determine that, despite the fluorophores more exposed to the solvent presenting a solution-like behavior, they have a strongly hindered mobility. Such effect of the silica matrix on the mobility of outer bound moieties is in agreement with a high porosity of the surface and with the localization of the fluorophores in small cavities where their rotational freedom is strongly reduced. This confinement of the dyes was confirmed by complexation experiments with β-cyclodextrin: even these external fluorophores are, in fact, not available for inclusion in the receptor cavities. These important results show that dyes in the DDNs have a very peculiar inhomogeneous behavior, and, in particular, those in the more external layer of the silica are sensitive and interact with small molecules such as solvent molecules, but they are not accessible to “big” receptors such as cyclodextrins. As a result, the inclusion in small (d < 50 nm) silica nanoparticles of active moieties is a promising strategy for the development of new sensors suitable for applications in biology and life sciences providing enough shielding of the sensitive moieties from large biomolecules (proteins, DNA, etc.) to preserve their specific activity and response rate. In addition, experiments are in course in our laboratory to verify whether a smaller diameter, combined with proper surface modification techniques, can make the permeation of these nanoparticles through cellular membranes more efficient. Experimental Section General. The solvents were purified by standard methods. All commercially available reagents were used as received. 3-Dansylamido-propyl-triethoxysilane (3) and N-propyldansylamide (4) were prepared as previously reported.7e Transmission electron microscopy (TEM) experiments were performed by Mr. Claudio Gamboz, Prof. Maria Rosa Soranzo, and Prof. Paolo Tecilla at the CSPA of the University of Trieste. TEM images of the nanoparticles were obtained with a Philips EM 208 transmission electron microscope operating at 100 keV. Samples for TEM were prepared by spreading a drop of nanoparticle solution in ethanol (∼5 mg/mL) onto standard carbon-coated copper grids (200 mesh). Dimensional analysis of the nanoparticles from TEM images was performed by using the Image J software.10 Dynamic light scattering (DLS) measurements were obtained with a Particle Sizing Systems Nicomp model 370 correlator equipped with a thermostated cell holder and a Spectra Physics series 2016 (9) Montalti, M.; Prodi, L.; Zaccheroni, N.; Zattoni, A.; Reschglian, P.; Falini, G. Langmuir 2004, 20, 2989-2991. (10) Rasband, W. Image J 1.32j; National Institute of Mental Health, Research Services Branch Home Page. http://rsb.info.nih.gov/ij/ (accessed May 2005).
Montalti et al. Ar laser operating at 488 nm. Hydrodynamic nanoparticle diameters were obtained from cumulant fits of the autocorrelation functions at a 90° scattering angle. Photophysical Measurements. Water solutions were obtained by diluting concentrated ethanol solutions of DDNs 1 and 2. The ethanol fraction in the water solution was always less than 1%. The fluorescence spectrum of the reference compound 4 in pure water was not affected by the addition of ethanol 1%. Steady-state and time-resolved fluorescence measurements were performed with an Edinburgh FLS920 spectrofluorometer equipped with a TCC900 card for TCSPC data acquisition and Glan-Thompson polarizing prisms. For excitation in the TCSPC experiments, an LDH-P-C-405 pulsed diode laser was used. Experimental decays were elaborated with the software package FAST 1.6.5 by Alango Ltd. Preparation of DDNs 1. Compound 3 (17 mg, 0.038 mmol) was dissolved in 20 mL of ethanol, and to this solution tetraethoxysilane (TEOS, 100 µL, 0.43 mmol) and a 29% ammonia-water solution (0.75 mL) were added. The reaction mixture was thermostated at 25 °C and vigorously stirred for 16 h. The resulting nanoparticle suspension was diluted to 70 mL with ethanol and was transferred into a 75 mL Amicon ultrafiltration cell, equipped with a 10 kDa regenerated cellulose membrane and an 800 mL solvent reservoir. The mixture was extensively ultrafiltrated, under a pressure of 4 bar, until the UV-vis spectrum of the waste no longer showed the typical features of dansyl absorption. The purified solution was finally filtrated through a 0.45 µm filter membrane. Preparation of DDNs 2. Compound 3 (42 mg, 0.092 mmol) was dissolved in 20 mL of ethanol, and to this solution TEOS (246 µL, 1.06 mmol) and a 29% ammonia-water solution (1.50 mL) were added. The reaction mixture was thermostated at 25 °C and vigorously stirred for 16 h. The resulting nanoparticle suspension was diluted to 70 mL with ethanol and was transferred into a 75 mL Amicon ultrafiltration cell, equipped with a 10 kDa regenerated cellulose membrane and an 800 mL solvent reservoir. The mixture was extensively ultrafiltrated, under a pressure of 4 bar, until the UVvis spectrum of the filtrate no longer showed the typical features of dansyl absorption. The purified solution was finally filtrated through a 0.45 µm filter membrane.
Results and Discussion Dansyl dye-doped nanoparticles (Figure 1 and Scheme 1) with 23 ( 10 and 130 ( 30 nm diameters (1 and 2, respectively) were prepared, following the method proposed by van Blaaderen,4 via the co-condensation of a 10:1 (in moles) mixture of TEOS and 3 (Scheme 1) in an ethanol/water/ammonia solution at 25 °C. The formation of colloids was monitored via DLS measurements, and the resulting nanoparticles were purified by extensive ultrafiltration over a 10.000 Dalton (∼3 nm) cutoff membrane to eliminate unreacted species and hydrolyzed monomers. The size of the nanoparticles was investigated using TEM (Figure 1) and DLS. The diameters obtained with the two methods are reported in Table 1, together with the relevant reaction parameters. The agreement between the hydrodynamic diameters obtained by DLS and the ones obtained by electron microscopy is good, and the polydispersities are in agreement with the data reported in the literature for similar systems.4 The dansyl moiety is a very well-known fluorophore widely employed for amine labeling,7,11 and it was our species of choice both for its peculiar photophysical properties and for the simplicity of the synthesis of its trialkoxysilane derivatives, which are needed for the grafting to the silica matrix. The large Stokes shift typical of dansylated compounds is due to the charge-transfer nature of the singlet excited state resulting from the presence of the electron (11) (a) Ghiggino, K. P.; Lee, A. G.; Meech, S. R.; O’Connor, D. V.; Phillips, D. Biochemistry 1981, 20, 5381-5389. (b) Prodi, L.; Bolletta, F.; Montalti, M.; Zaccheroni, N. Eur. J. Inorg. Chem. 1999, 5, 455-460. (c) Pagliari, S.; Corradini, R.; Galaverna, G.; Sforza, S.; Dossena, A.; Montalti, M.; Prodi, L.; Zaccheroni, N.; Marchelli, R. Chem.sEur. J. 2004, 10, 2749-2758.
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Langmuir, Vol. 22, No. 13, 2006 5879 Table 1. Nanoparticle Diameters (D) and Polydispersities (δ), in Nanometers, of DDNs as Obtained by TEM and DLS batch
conditions
D(TEM)
δ (%)
D(DLS)
1 2
a b
23.5 128.6
4.2 (18%) 16 (12%)
24.8 126.4
a [Dye] ) 1.9 × 10-3 M; [TEOS] ) 0.022 M; [NH ] ) 0.6 M; [H O] 3 2 ) 1.3 M. b [Dye] ) 4.6 × 10-3 M; [TEOS] ) 0.053 M; [NH3] ) 1.2 M; [H2O] ) 2.6 M.
Figure 2. Fluorescence spectra (λexc ) 335 nm) of water solutions of 1, 2, and 4 alone (curves 1, 2, and 4, respectively; [dye] ) 1 × 10-6 M]) and in the presence of β-cyclodextrin (1 × 10-2M) (spectra 1c, 2c, and 4c, respectively).
Figure 1. TEM images of nanoparticles 1 (top) and 2 (bottom) dansyl-doped silica nanoparticles (the bar corresponds to 500 nm). Scheme 1. Proposed Structure of the Dye-Doped Nanoparticles 1 and 2 and Chemical Structure of Compounds 3 and 4
withdrawing sulfonamidic group and of the dimethylamino electron-donating moiety on the same naphthalenic aromatic structure. Because of the polar nature of the fluorescent excited state, the energetic position of the fluorescence band is strongly dependent on the polarity of the medium. Upon changing from
low polarity solvents to water, the emission band of dansylamide progressively red shifts and decreases in intensity. Similarly, the addition of β-cyclodextrin (1 × 10-2 M) to an aqueous solution of the reference compound 4 causes a shift in the fluorescence band of the dye (Figure 2) from 560 nm (water solution τ ) 3 ns, Φ ) 0.03) to 510 nm (τ ) 13 ns, Φ ) 0.37), due to the inclusion of 4 in the less polar cyclodextrin cavity. Figure 2 shows the fluorescence spectra of 4 and DDNs 1 and 2 upon excitation at 335 nm in water solution: the fluorescence of both the smaller 1 and bigger 2 nanoparticles is much stronger and blue shifted (to 520 and 500 nm for 1 and 2, respectively) in water with respect to the reference compound 4. This effect clearly shows that the dansyl moieties, when embedded in the nanoparticles, experience a less polar and quite different environment than that of pure water. The fluorescence observed in the case of DDNs 2 is particularly strong (Φ ) 0.35), and this indicates that the silica matrix that surrounds the fluorophores effectively shields them from interaction with water molecules. It is crucial to notice that such an effect is much stronger in the large colloids then it is in the smaller ones. Fluorescence decay and lifetime data confirmed and clarified these observations. TCSPC measurements (Figure 3) show multiexponential decays for all DDNs, but the profiles observed are rather different in the two cases. The complexity of the decays observed in both cases clearly indicates that, within the nanoparticles, the dye molecules experience different environments. However, an accurate analysis of the lifetime distributions (Figure 4) allowed for a simplified description of the nanoparticles structure: the presence of two definite peaks in the distribution profiles suggests a possible rationalization of the system gathering the fluorophores in two families of short-living and long-living moieties. The average lifetimes characteristic of the two families are similar in DDNs 1 and 2, but their relative contributions are very different. The “fast” excited-state deactivation, in fact, involves around the 50% of the fluorophores in 1 and only 15% in 2. The presence of two families of dyes can be explained in terms of interaction
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Figure 3. Excited state decays (TCSPC) for nanoparticles 1 and 2.
Figure 4. Lifetime distributions resulting from the analysis of TCSPC decays of nanoparticles 1 and 2 with FAST software by Alango.
with the solvent. In fact, because of their porosity, the silica nanoparticles are, to some extent, permeable to the solvent.7,14The simple intrinsic inhomogeneity of the materials and the presence of pores is not sufficient to explain the different behavior of nanoparticles 1 and 2, while a preferential localization of the pores on the surface with a consequently higher permeability of the external silica layer of the nanoparticles is in good agreement with the experimental data. The accuracy of the core-shell model depicted in Scheme 1 is supported by the observation that the thickness of the solvent permeable layer does not change with nanoparticle size, and it is the same for DDNs 1 and 2. Assuming a homogeneous concentration of the dansyl molecules in the silica matrix and a spherical shape of the nanoparticles, the lifetime distributions allow one to roughly calculate the thickness of the waterpermeable layer that turns out to be about 3 and 4 nm for DDNs 1 and 2, respectively. This result strongly supports the schematic representation given in Scheme 1. Further information about the nanoparticle structure comes from steady-state and time-resolved anisotropy measurements. The anisotropy spectra reported in Figure 5 indicate a strong polarization of the fluorescence, in agreement with a very low mobility of the dyes, as expected because of their inclusion in the nanoparticles. The anisotropy decays, on the other hand, are quite slow (18 and 14 ns for DDNs 1 and 2, respectively; see (12) Wang, R.; Bright, F. V. J. Phys. Chem. 1993, 97, 10872-10878. (13) Flink, S.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Chem. Commun. 1999, 2229-2230.
Montalti et al.
Figure 5. Fluorescence anisotropy spectra of 1 and 2 in water solution (full and empty circles) upon excitation at λ ) 335 nm. Normalized fluorescence spectra of 1 and 2 in water solution upon excitation at λ ) 335 nm.
Figure 6. Fluorescence anisotropy decays of nanoparticles 1 and 2 in water solution and the time profile of the excitation laser pulse (solid line).
Figure 6) and lead to a high residual anisotropy (r∞ ) 0.29 and r∞ ) 0.26, respectively).12 It is interesting to note that the r∞ values are very similar for 1 and 2 nanoparticles; these values, in fact, can be roughly associated to the slow fluorescence decay of the cores of the nanoparticles, which is expected to have similar properties in both DDNs 1 and 2. Additional information on the microenvironment experienced by the dye molecules can also be provided by the study of their interaction with cyclodextrins. In previous studies, we investigated the interaction of similar DDNs with transition metal ions, in view of their possible application as nanosensors, and we reported how such small species can penetrate to a certain extent in the nanoparticles and interact with the fluorescent groups.7d,e However, the possibility of the linked fluorophores interacting with larger molecules was not yet investigated before. In this context, steady-state and time-resolved fluorescence measurements were repeated for water solutions of DDNs 1 and 2 in the presence of β-cyclodextrin 1 × 10-2 M. As it can be seen in Figure 2, only minor changes appeared in the fluorescence spectra. Neither fluorescence or anisotropy decays nor the fluorescence anisotropy spectra were significantly affected by the presence of the receptor. Since only surface-grafted dansyl groups could be included by β-cyclodextrins,13 such results indicate that most (14) A similar shielding effect was observed by Kaifer and Bright in the case of dendrimers: Cardona, C. M.; Alvarez, J.; Kaifer, A. E.; McCarley, T. D. Pandey, S.; Baker, G. A.; Bonzagni, N. J.; Bright, F. V. J. Am. Chem. Soc. 2000, 122, 6139-6144.
Dye-Doped Silica Nanoparticles
of the solvent sensitive dansyl moieties are not located on the nanoparticles surfaces but within pores, which are large enough to accommodate solvent but not big molecules such as cyclodextrins.14
Conclusions The use of fluorescent DDNs is attracting more and more attention, both as tracers for biological applications5,6 and, more recently, as chemosensors.7 For this reason, a deeper understanding of the structure and behavior of such systems is, in our opinion, precious for the design of new systems suitable for more efficient and more sophisticated applications. The results reported here show that, in the silica nanoparticles, the dansyl molecules retain some of their molecular properties, while their mobility and accessibility are reduced. In particular, in water solution, the dyes located in the nanoparticle cores are efficiently shielded from the solvent. In fact, their quantum yield is comparable to that of the dansyl dye in an apolar environment (Φ ) 0.38). On the contrary, fluorophores located in the outer shell are sensitive and can interact with small molecules, such as solvent molecules, but they are not accessible to big receptors such as β-cyclodextrins. This suggests possible interesting applications of these materials
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in the sensor field: they can be sensitive to small analytes and, at the same time, be protected from interferences of macromolecules potentially present in the matrix. The prevalence of the desired feature can be obtained by changing the nanoparticle dimensions and, most important, a careful design and synthesis of the system can allow one to decide whether a fluorescent dye will or will not be able to interact with the external environment simply by grafting it at the appropriate distance from the nanoparticles surface. All these properties make DDNs extremely attractive for applications in life sciences, both for the different functions that can be performed by the inner and outer fluorescent moieties in each single nanoparticle and for the versatility of their design. Acknowledgment. We gratefully thank Paolo Tecilla, Claudio Gamboz, and Maria Rosa Soranzo (University of Trieste) for TEM analysis. Financial support for this research has been partly provided by the Ministry of Instruction, University and Research (MIUR contracts 2003030309, LATEMAR and SAIA projects) and by University of Padova (CPD A034893). LA053473Y
