Turning Fluorescent Dyes into Cu(II) Nanosensors - American

Umberto Tonellato†. Dipartimento di Scienze Chimiche, Universita` di Padova, via Marzolo 1,. I-35131 Padova, Italy, Istituto CNR di Scienze e Tecnol...
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Langmuir 2005, 21, 9314-9321

Turning Fluorescent Dyes into Cu(II) Nanosensors Maria Arduini,† Silvia Marcuz,† Mariachiara Montolli,‡ Enrico Rampazzo,† Fabrizio Mancin,*,† Silvia Gross,‡ Lidia Armelao,‡ Paolo Tecilla,§ and Umberto Tonellato† Dipartimento di Scienze Chimiche, Universita` di Padova, via Marzolo 1, I-35131 Padova, Italy, Istituto CNR di Scienze e Tecnologie Molecolari, via Marzolo 1, I-35131 Padova, Italy, and Dipartimento di Scienze Chimiche, Universita` di Trieste, via Giorgieri 1, I-34127, Italy Received March 24, 2005. In Final Form: July 26, 2005 There is great interest in the self-organization of the proper subunits as a new strategy for the realization of fluorescent chemosensors. In this article, it is shown that commercially available fluorescent dyes, functionalized with triethoxysilane moieties, can be converted into fluorescent chemosensors by simple inclusion into silica nanostructures. Dye-doped silica nanoparticles and thin films detect Cu(II) ions in the micromolar range by the quenching of fluorescence emission. The different response toward Zn(II), Ni(II), and Co(II) metal ions was also investigated and is reported. The self-organization of the silica structures leads, at the same time, to the formation of metal ion binding sites as well as to the linking of a fluorescent reporter in their proximity. Structural features of the materials, particularly particle size and network porosity, strongly affect their ability to act as fluorescent sensors.

Introduction The past decade has witnessed quite impressive interest in the realization and use of fluorescent chemosensors: molecular systems that signal the presence and the amount of a selected substrate by a variation of their fluorescence emission properties.1 Such systems are usually simple to use, sensitive, and selective. In addition, their molecular dimensions allow a high response rate and spatial resolution in the detection of the analyte, even in the case of intracellular analysis of selected species in medical and biochemical studies. In their typical design, fluorescent chemosensors are molecules composed of one or more substrate binding units and photoactive components.1 The chemical complexity of such systems greatly varies from case to case because the sensor components can be integrated into each other or simply connected by spacers. However, the synthetic efforts involved in the realization and optimization of chemosensors may be demanding. Self-assembling and self-organizing methodologies offer an interesting shortcut in the chemistry of complex systems with functional properties.2 Indeed, they are the basis of the so-called “bottom-up” approach because the building of complex structures with this strategy simply requires one to design a limited number of relatively simple building blocks and to allow them to organize spontaneously. The molecular organization of such assemblies endows the supramolecular aggregates or the materials prepared in this way with new properties and functions. * To whom correspondence should be addressed. E-mail: [email protected]. † Universita ` di Padova. ‡ Istituto CNR di Scienze e Tecnologie Molecolari. § Universita ` di Trieste. (1) (a) Coord. Chem. Rev. 2000, 205 (Special Issue on Luminescent Sensors). (b) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515-1566. (c) Fluorescent Chemosensors for Ion and Molecule Recognition; Czarnik, A. W., Ed.; ACS Symposium Series 538; American Chemical Society: Washington, DC, 1993. (2) (a) Mann, S. Chem. Commun. 2004, 1-4. (b) Hecht, S. Angew. Chem., Int. Ed. 2003, 42, 24-26.

On these bases, the self-assembling strategy can be exploited to overcome, at least partially, the synthetic problems related to classical systems and to provide an efficient method for the easy realization and optimization of fluorescent chemosensors. In recent years, an increasing number of examples of self-assembled fluorescent chemosensors have been reported. They comprise a variety of systems: substrate displacement of receptor-fluorescent dye supramolecular complexes,3 receptor monolayers on fluorescent quantum dots,4 self-organization of receptors and dyes within micellar aggregates,5 Langmuir-Blodgett films,6 organic polymers nanoparticles,7 and self-assembled monolayers on glass surfaces8 or silica nanoparticles.9 We have previously reported a self-assembled fluorescent chemosensor for Cu(II) based on surfactant aggregates.5a,b Such a system (Scheme 1) is based on co-micelles made from a lipophilic dipeptide ligand (N-decilglycylglycine), a fluorescent dye (8-anilinenaphthalensulfonic (3) Wiskur, S. L.; Aı¨t-Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963-972 and references therein. (b) Hortala`, M. A.; Fabbrizzi, L.; Marcotte, N.; Stomeo, F.; Taglietti, A. J. Am. Chem. Soc. 2003, 125, 20-21. (4) (a) Ga´ttas-Asfura, K. M.; Leblanc, R. M. Chem. Commun. 2003, 2684-2685. (b) Chen, Y.; Rosenzweig, Z. Anal. Chem. 2002, 74, 51325138. (5) (a) Grandini, P.; Mancin, F.; Tecilla, P.; Scrimin, P.; Tonellato, U. Angew. Chem., Int. Ed. Engl. 1999, 38, 3061-3064. (b) Berton, M.; Mancin, F.; Stocchero, G.; Tecilla, P.; Tonellato, U. Langmuir 2001, 17, 7521-7528. (c) Diaz-Fernandez, Y.; Perez-Gramatges, A.; Amendola, V.; Foti, F.; Mangano, C.; Pallavicini, P.; Patroni, S. Chem. Commun. 2004, 1650-1651. (d) Diaz-Fernandez, Y.; Perez-Gramatges, A.; Rodriguez-Calvo, S.; Mangano, C.; Pallavicini, P. Chem. Phys. Lett. 2004, 398, 245-249. (6) Zheng, Y.; Orbulescu, J.; Ji, X.; Andreopulos, F. M.; Pham, S. M.; Leblanc, R. M. J. Am. Chem. Soc. 2003, 125, 2680-2686. (7) Me´allet-Renault, R.; Pansu, R.; Amigoni-Gerbierb, S.; Larpent, C. Chem. Commun. 2004, 2344-2345. (8) (a) Crego-Calama, M.; Reinhoudt, D. N. Adv. Mater. 2001, 13, 1171-1174. (b) Basabe-Desmonts, L.; Beld, J.; Zimmerman, R. S.; Hernando, J.; Mela, P.; Garcı´a Parajo´, M. F.; van Hulst, N. F.; van den Berg, A.; Reinhoudt, D. N.; Crego-Calama, M. J. Am. Chem. Soc. 2004, 126, 7293-7299. (9) (a) Brasola, E.; Mancin, F.; Rampazzo, E.; Tecilla, P.; Tonellato, U. Chem. Commun. 2003, 3026-3027. (b) Rampazzo, E.; Brasola, E.; Marcuz, S.; Mancin, F.; Tecilla, P.; Tonellato U. J. Mater. Chem. 2005, 15, 2687-2696.

10.1021/la050785s CCC: $30.25 © 2005 American Chemical Society Published on Web 09/01/2005

Turning Fluorescent Dyes into Cu(II) Nanosensors Scheme 1

acid, ANS), and a cationic surfactant (hexadecyldimethylammonium bromide, CTABr). The concentration of the species within the micellar aggregate ensures the spatial proximity between the ligand and the dye, and the complexation of the Cu(II) ions by the ligand units gives rise to a signal due to the quenching of the dye fluorescence emission. This system allowed us to determine Cu(II) concentrations down to the micromolar range in water at pH 7. The main advantages of such an approach are the following: (a) selectivity, mainly due to the ligand choice; (b) simplicity: the sensor is prepared just by the mixing the components in water (two of themsCTABr and ANSs are commercially available); (c) modularity and easy optimization of the system, obtained by modification or substitution of one of the components; and (d) the possibility of tuning the detection range by the simple modification of the components’ molar ratio. However, the actual applicability of such systems is somehow restricted because of the dynamic nature of surfactant aggregates and their consequent sensitivity to environmental conditions. To obtain nondynamic selforganized sensors, we turned our attention to coated nanoparticles obtained by surface modification of silica nanospheres.9 Commercially available particles (20 nm diameter) were functionalized (Scheme 2) with the triethoxysilane derivatives of the selective Cu(II) ligand picolinamide (PA) and the fluorophore dansylamide (DNS). As in the case of surfactant-based systems, grafting of the sensor components to the particle surface ensures the spatial proximity necessary to signal Cu(II) by quenching of the fluorescence emission. In a 9:1 DMSO/water solution, the coated silica nanoparticles (CSN) selectively detect copper ions down to micromolar concentrations, and the operative range of the sensor can be simply tuned by modifying the components’ ratio. Moreover, we gathered evidence of cooperation between the ligand subunits boundto the particle surfaces, resulting in the formation of binding sites with an increased affinity and enhanced sensitivity for the substrate (Scheme 2). Hence, the use of CSNs not only allows us to overcome the limitations of the surfactant-based sensors, still retaining their main advantages, but also opens the way to new collective effects. Besides several different details, the most common feature of the self-assembling approach is the autoorganization of receptor units and reporter fluorescent dyes mediated, when necessary, by an inert scaffold. Extending our studies to self-organized fluorescent chemosensors based on silica nanoparticles, we realized that the use of such scaffolds could lead to a further simplification of the system. In this article, we describe the simple inclusion of fluorescent dyes in nanometersized silica materials leading to the realization of selective Cu(II) fluorescent sensors.

Langmuir, Vol. 21, No. 20, 2005 9315 Scheme 2

Experimental Section General. All commercially available solvents and reagents were used as received without further purification. TLC analyses were performed using Merck 60 F254 (0.25 mm) precoated silica gel glass plates and Machery-Nagel Poligram SIL G/UV254 precoated plastic sheets (0.25 mm). Preparative column chromatography was carried out on glass columns packed with Macherey-Nagel 60 (70-230 mesh) and on Kieselgel 60, 0.063 mm Merck silica gel. NMR spectra were recorded using a Brucker AC 250F spectrometer. Chemical shifts are reported relative to tetramethylsilane as the internal standard. Signals in NMR spectra are reported as follows: s ) singlet; d ) doublet, t ) triplet, q ) quartet, m ) multiplet, and b ) broad. Multiplicity is given as usual. ESI-MS mass spectra were obtained with a Navigator ThermoQuest-Finningan mass spectrometer. Elemental analyses were performed by the Laboratorio di Microanalisi of the Dipartimento di Scienze Chimiche of the University of Padova. Transmission electron microscopy (TEM) experiments were performed at the CSPA of the University of Trieste. TEM images of the particles were obtained with a Philips EM 208 transmission electron microscope operating at 100 keV. Samples for TEM were prepared by spreading a drop of nanoparticles solution in ethanol onto standard carbon-coated copper grids (200 mesh). Dimensional analysis of nanoparticles from TEM images was performed by using Image J software.10 Dynamic light scattering measurements were obtained with a Particle Sizing Systems Nicomp model 370 correlator equipped with a thermostated cell holder and a Spectra Physics series 2016 Ar laser operating at 488 nm. Hydrodynamic particle diameters were obtained from cumulant fits of the autocorrelation functions at a 90° scattering angle. UV-vis absorption measurements were performed at 25 °C by means of Perkin-Elmer Lambda 16 and 45 spectrophotometers equipped with thermostated cell holders. Quartz cells with an optical pathlength of 1 cm were used. Fluorescence spectra were recorded at 25 °C with an LS-50B spectrofluorimeter equipped with a Hamamatsu R928 photomultiplier and thermostated cell holder; quartz cells with an optical pathlength of 1 cm were used. XPS spectra were obtained with a Perkin-Elmer Φ 5600 ci spectrometer using nonmonochromatized Mg-K R radiation (1253.6 eV). The working pressure was 18 MΩ) obtained with a Milli-Q (Millipore) purification system. The buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Aldrich) was used as received, and stock solutions (0.2 M) were prepared using Milli-Q water. The total molar concentration of dye subunits in the DDNs ethanol suspensions resulting from ultrafiltration was determined from their absorbance (measured on highly diluted samples, to minimize light scattering from nanoparticles) using the  values of the corresponding models in ethanol (1b: 5.24 × 103 M-1 cm-1, 333 nm; 2b: 2.31 × 104 M-1 cm-1, 463 nm; 3b: 1.52 × 104 M-1 cm-1, 297 nm). The desired amount of each suspension was then transferred in fluorescence quartz cells, and ethanol, water, and buffer were added to reach a final volume of 2 mL. The suspension was allowed to equilibrate until no variation of the emission spectrum were observed (usually an equilibration time of about 1 h was required), and then small volumes (up to 50 µL) of concentrated metal ions solutions were added and the fluorescence or absorption spectra were recorded. In the case of sol-gel thin films, the silica slides were placed across the diagonal of a 1 × 1 cm2 quartz cell equipped with a homemade Teflon holder. The cell was filled with buffered water at pH 7.0 and mounted in the spectrophotometer. To minimize the contribution of stray light, the cell was oriented in the spectrophotometer so that the film was at a 30° angle to the incident excitation beam, and a 390 nm emission filter was used. Small volumes (up to 50 µL) of concentrated metal ion solutions were then added, and the fluorescence or absorption spectra were recorded after the needed equilibration time. Fitting of the titration curves was performed with the software package Scientist 2.01.13 A model involving the formation of 1:1 complexes of Cu(II) was used to estimate the concentration of binding sites (binding capacity) and the Cu(II) complexation strength of the particles, the fit error being in each case less than 10%. 3-(Dansylamido)-propyl-triethoxysilane (1a). A solution of dansyl chloride (0.90 g, 3.3 mmol) in CH2Cl2 (20 mL) was added to a solution of (3-aminopropyl)triethoxysilane (APTES, 0.80 mL, 3.4 mmol) and triethylamine (0.60 mL, 4.3 mmol) in the same solvent (20 mL). The mixture was stirred at room temperature for 2 h while monitoring the reaction by TLC (toluene/ethyl acetate 1/1). The solvent was evaporated under reduced pressure, and the crude product was purified by flash chromatography (silica gel, toluene/ethyl acetate 1/1) to afford 1.51 g (99%) of 1a as a yellow-bright green oil. 1H NMR (250 MHz, CDCl3, 25 °C): δ 0.53 (t, J ) 8.1 Hz, 2H, NCH2CH2CH2Si), 1.15 (t, J3 ) 7.0 Hz, 9H, NCH2CH2CH2-Si-(OCH2CH3)3), 1.55 (m, 2H, NCH2CH2CH2-Si), 2.86 (s 6H, N(CH3)2), 2.98 (q 2H, J3 ) 5.0 Hz, NCH2CH2CH2-Si), 3.71 (q, J3 ) 7.0 Hz, 6H, NCH2CH2CH2-Si-(OCH2CH3)3); 5.84 (t, J ) 5.6 Hz, 1H, NHCH2CH2CH2-Si), 7.16 (d, J ) 7.4 Hz, 1H, CHDNS), 7.53 (q, J ) 7.5 Hz, 2H, CHDNS), 8.30 (d, J ) 7.5 Hz, 2H, CHDNS), 8.46 (d, J ) 8.6 Hz, 1H, CHDNS), 8.53 (m, J ) 7.5 Hz, 1H, CHDNS). 13C NMR (62.9 MHz, CDCl3, 25 °C): δ 7.42 (NCH2CH2CH2-Si), 18.3 (NCH2CH2CH2-Si-(OCH2CH3)3), 23.1 (NCH2CH2CH2-Si), 45.7 (NCH2CH2CH2-Si), 58.3 (NCH2CH2CH2-Si-(OCH2CH3)3), 115.2 (N(CH3)2), 119.2, 123.2, 125.3, 128.3, 129.0, 129.5, 129.9, 130.2, 135.5, 151.8 (CDNS). ESI-MS (MeOH) m/z: 477 (100%, M + Na+). Elemental analysis calcd for C21H34N2O5SSi (412.58 g/mol): C 55.48; H 7.54; N 6.16; S 7.05. Found: C 55.58; H 7.59; N 6.15; S 6.93. 5-Dimethylamino-naphthalene-1-sulfonic acid propylamide (1b). A 50 mL CH2Cl2 solution of dansyl chloride (270 (13) Scientist 2.01; Micromath Scientific Software: Salt Lake City, Utah, 1995.

Arduini et al. mg, 1.0 mmol) was added to a stirred 50 mL cooled (0 °C) solution of propylamine (0.21 mL, 4.6 mmol) in CH2Cl2. The reaction was allowed to warm at room temperature and was monitored by TLC (CH2Cl2). The reaction mixture was extracted with a Na2CO3 (10%)/brine 1/1 solution, and the organic phase was dried over anhydrous sodium sulfate. Product 1b was recovered in quantitative yield after evaporation of the solvent. 1H NMR (250 MHz, CDCl3, 25 °C): δ 0.77 (t, J ) 8.1 Hz, 2H, NCH2CH2CH3), 1.41 (m, 2H, NCH2CH2CH3), 2.81-2.89 (m 8H, NCH2CH2CH3N(CH3)2), 4.62 (broad, 1H, NHCH2CH2CH3), 7.19 (d, J ) 7.4 Hz, 1H, CHDNS), 7.49-7.60 (m, 2H, CHDNS), 8.23-8.31 (m, 2H, CHDNS), 8.52 (d, J ) 8.3 Hz, 1H, CHDNS). 13C NMR (62.9 MHz, CDCl3, 25 °C): δ 14.0 (NCH2CH2CH3), 22.8 (NCH2CH2CH3), 50.5 (NCH2CH2CH3), 115.1 (N(CH3)2), 118.7, 123.2, 125.3, 128.2, 129.0, 129.5, 129.9, 130.3, 134.9, 152.0 (CDNS). ESI-MS (MeOH/TFA 1%) m/z: 293 (100%, M + H+), 607 (12%, 2M + Na+). Elemental analysis calcd for C15H20N2O2S (292.40 g/mol): C 61.62; H 6.89; N 9.58; S 10.97. Found: C 60.87; H 6.95; N 9.37; S 10.43. (7-Nitro-benzo[1,2,5]oxadiazol-4-yl)-(3-triethoxysilylpropyl)-amine (2a): A 10 mL toluene solution of APTES (0.47 mL, 2.0 mmol) was slowly added to a stirred 10 mL solution of 4-chloro-7-nitro-benzo[1,2,5]oxadiazole (NBD-Cl, 400 mg, 2.0 mmol) and triethylamine (0.36 mL, 2.6 mmol) in the same solvent. The reaction was monitored by TLC (petroleum ether/ethyl acetate 1/1). The crude product obtained by solvent evaporation was purified by flash chromatography (silica gel, petroleum ether/ ethyl acetate 1/1) to afford 395 mg of the title product 2a (51%) as a red-brown solid. 1H NMR (250 MHz, CDCl3, 25 °C): δ 0.768 (t, J3 ) 7.25 Hz, 2H, NCH2CH2CH2-Si), 1.24 (t, J3 ) 7.25 Hz, 9H, NCH2CH2CH2-Si-(OCH2CH3)3), 1.95 (m, J3 ) 7.25 Hz, 2H, NCH2CH2CH2-Si), 3.53 (m, J3 ) 7.25 Hz, 2H, NCH2CH2CH2Si), 3.86 (q, J3 ) 7.25 Hz, 6H, NCH2CH2CH2-Si-(OCH2CH3)3), 6.18 (d, J3 ) 8.6 Hz, 1H), 6.88 (t broad, 1H, (NBD)-NHCH2CH2CH2), 8.48 (d, J3 ) 8.6 Hz, 1H). 13C NMR (69.9 MHz, CDCl3, 25° C): δ 8.12 (NCH2CH2CH2-Si), 18.59 (NCH2CH2CH2-Si(OCH2CH3)3), 22.08 (NCH2CH2CH2-Si), 46.43 (NCH2CH2CH2Si), 58.99 (NCH2CH2CH2-Si-(OCH2CH3)3), 98.92, 123.33, 137.18, 144.27, 144.58, 144.80 (Carom). ESI-MS (MeOH) m/z: 439 (100%, M + MeOH + Na+). Elemental analysis calcd for C15H24N4O6Si (384.47 g/mol): C 46.86; H 6.29; N 14.57. Found: C 47.16; H 6.17; N 14.09. (7-Nitro-benzo[1,2,5]oxadiazol-4-yl)-propyl-amine (2b). Propylamine (0.20 mL, 2.5 mmol) was added to 12 mL of a CH3CN solution of 4-chloro-7-nitrobenzofurazan (4-chloro-7-nitro1,2,3-benzoxadiazole) (0.10 g, 0.5 mmol). The reaction mixture was stirred for 12 h, diluted with 100 mL of CH2Cl2, and extracted with a Na2CO3 (10%)/brine 1/1 solution. The organic phase was dried over anhydrous sodium sulfate. Product 2b ( 0.10 g, 89%) was recovered after evaporation of the solvent. 1H NMR (250 MHz, CDCl3, 25 °C): δ 1.11 (t, J3 ) 7.25 Hz, 3H, NCH2CH2CH3), 1.86 (m, J3 ) 7.25 Hz, 2H, NCH2CH2CH3), 3.47 (m, J3 ) 7.25 Hz, 2H, NCH2CH2CH3), 6.18 (d, J3 ) 8.5 Hz, 1H), 8.50 (d, J3 ) 8.5 Hz, 1H). ESI-MS (MeOH/H2O/TFA 66:33:1) m/z: 223 (100%, M + H+), 245 (35%, M + Na+). Elemental analysis calcd for C9H10N4O3 (222.20 g/mol): C 48.65; H 4.54; N 25.21. Found: C 48.52; H 4.88; N 24.89. 2-Oxo-2H-chromene-3-carboxylic acid (3-Triethoxysilylpropyl)-amide (3a). Thionyl chloride (0.57 mL, 7.8 mmol) was added to 100 mL of a CH2Cl2 solution of coumarin 3-carboxylic acid (1.30 g, 6.8 mmol) and triethylamine (2.98 mL, 21.4 mmol) at about 0 °C. The reaction mixture was allowed to warm at room temperature for about 40 min and then cooled again to 0 °C before APTES (2.50 mL, 10.7 mmol) was added with a syringe. The reaction was monitored by TLC (toluene/ethyl acetate 1/1). Solvent was evaporated, and the crude product was purified by flash chromatography (silica gel, toluene/ethyl acetate 7/3) to give 1.96 g of 3a (73%) as a yellow-brown solid. 1H NMR (300 MHz, CDCl3, 25 °C): δ 0.70 (t broad, J3 ) 8.9 Hz, 2H, NCH2CH2CH2-Si), 1.23 (t, J3 ) 10.5 Hz, 9H, NCH2CH2CH2-Si(OCH2CH3)3), 1.75 (m, 2H, NCH2CH2CH2-Si), 3.47 (q, J3 ) 10.7 Hz, 2H, NCH2CH2CH2-Si), 3.83 (q, J3 ) 10.5 Hz, 6H, NCH2CH2CH2-Si-(OCH2CH3)3), 7.52 (m, 4Harom), 8.88 (t broad, 1H, (CO)NHCH2), 8.91 (s, 1H, Harom). 13C NMR (69.9 MHz, CDCl3, 25 °C): δ 7.73 (NCH2CH2CH2-Si), 18.13 (NCH2CH2CH2-Si(OCH2CH3)3), 22.82 (NCH2CH2CH2-Si), 42.32 (NCH2CH2CH2Si), 58.89 (NCH2CH2CH2-Si-(OCH2CH3)3), 116.9, 120.1, 124.0,

Turning Fluorescent Dyes into Cu(II) Nanosensors 126.7, 131.2, 133.1, 145.6, 150.9 (Carom), 162.0, 167.9 (CdO). ESIMS (MeOH) m/z: 416 (100%, M + Na+). Elemental analysis calcd for C19H27N2O6Si (393.51 g/mol): C 57.99; H 6.92; N 3.56. Found: C 58.32; H 7.19; N 3.63. Coumarin 3-Carboxylic Acid Propylamide (2-Oxo-4a,8a-dihydro-2H-chromene-7-carboxylic acid Propylamide, 3b). Thionyl chloride (0.71 mL, 9.7 mmol) was slowly added by syringe to 100 mL of a CH2Cl2 solution of coumarin 8-carboxylic acid (1.50 g, 7.89 mmol) and triethylamine (3.44 mL, 24.7 mmol) at 0 °C. The stirred reaction mixture was held at 0 °C for 10 min and then allowed to warm to room temperature for about 40 min. After this time, the light-brown suspension was cooled again to about 0 °C, and 20 mL of a CH2Cl2 solution of propylamine (1.01 mL, 12.3 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and was stirred for 3 h (TLC, toluene/ethyl acetate 1/1). The reaction mixture was extracted with a Na2CO3 (5%)/brine 1/1 solution and then with brine, and the organic phase was dried over anhydrous Na2SO4. The solvent was evaporated to provide 1.56 g of 3b (85%). 1H NMR (250 MHz, CDCl3, 25 °C): δ 1.00 (t, J3 ) 7.5 Hz, 3H, NCH2CH2CH3), 1.66 (m, 2H, NCH2CH2CH3), 3.44 (q, J3 ) 5.8 Hz, 2H, NCH2CH2CH3), 7.35-7.43 (m, 4Harom), 7.63-7.72 (m, 4Harom), 8.82 (s, broad, 1H, (CO)NHCH2), 8.92 (s, 1H, Harom). ESI-MS (MeOH/TFA 1%) m/z: 232 (100%, M + H+), 254 (9%, M + Na+). Elemental analysis calcd for C13H13NO3 (231.25 g/mol): C 67.52; H 5.95; N 6.10. Found: C 67.52; H 5.95; N 6.10. Preparation of the Dye-Doped Silica Nanoparticles (DDNs). The preparation of batch 1 DDNs containing 1a is reported as an example of the general procedure. All of the other DDNs were prepared by following the same procedure with different initial concentrations of the reagents as reported in Table 1. The TEOS/organisilane dye derivative molar ratio was always kept at 10:1. 1a (54 mg, 0.04 mmol) was dissolved in 20 mL of ethanol, and to this solution TEOS (100 µL, 0.43 mmol) and a 29% ammoniawater 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 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 ultrafiltered, under a pressure of 4 bar, until the UV-vis spectrum of the filtrate showed the absence of 1a absorption. The retained solution was finally filtrated through a 0.45 mM filter membrane. Preparation of the Thin Films. The solution for the realization of the mesoporous samples was prepared by mixing tetraethoxysilane (TEOS), water, hydrochloric acid, and ethanol in the molar ratio 1/1/5 × 10-5/3 TEOS/H2O/HCl/EtOH and by refluxing at 80 °C for 1 h. To this solution, Pluronic F127 (F127), water, hydrochloric acid, and ethanol were added to achieve a final molar ratio of 1/5 × 10-3/5/0.004/20 TEOS/F127/H2O/HCl/ EtOH. This final solution was aged for 2 h at room temperature. To this solution, fluorescent precursor 1a was added to achieve a molar ratio of 10:1 TEOS/1a. The final fluorescent solution was used to deposit the thin films by dip coating. The solution used for the nonmesoporous samples was prepared by mixing tetraethoxysilane (TEOS), water, hydrochloric acid, and ethanol in the molar ratio 1/1/5 × 10-5/3 TEOS/H2O/HCl/ EtOH and by refluxing at 80 °C for 1 h. To this solution water, hydrochloric acid, and ethanol were added to achieve a final molar ratio of 1/5/0.004/20 TEOS/H2O/HCl/EtOH. This solution was kept for 2 h at room temperature, and then fluorescent precursor 1a was added to achieve a molar ratio of 10/1 TEOS/1a. The final fluorescent solution was used to deposit the thin films by dip coating. For the deposition of the thin films, Herasil silica slides (Heraeus) were used as substrates. Before use they were cleaned and rinsed both in doubly distilled water and 2-propanol. This procedure, which was repeated several times, was aimed at removing organic residues from the surface and at favoring the best adhesion between the coating and substrate. Slides were finally dried in clean air at room temperature. Film deposition was carried out in air with a withdrawal speed of 1.8 cm/min. The as-prepared thin films were thermally annealed in air at 100 °C for 2 h. The surfactant was removed by Soxhlet extraction

Langmuir, Vol. 21, No. 20, 2005 9317 Chart 1

in hot ethanol for 10 h. The obtained films were transparent, pale yellow, crack-free, and homogeneous.

Results Synthesis of the Dye Derivatives. The synthesis of alkoxysilane derivatives 1a-3a, used for the preparation of dye-doped silica nanostructures, was straightforward and involved in each case a single-step condensation of a properly activated derivative with commercially available 3-amminopropyl-triethoxysilane (APTES). Derivatives 1a and 2a were prepared from commercially available dansyl chloride and NBD chloride (4-cloro-7-nitrobenzofurazan), whereas compound 3a was synthesized from coumarin3-carboxylic acid via thionyl chloride activation. In all cases, purification of the triethoxysilane derivatives was accomplished by flash chromatography on silica gel. The corresponding compounds not bearing the triethoxysilane moiety (1b-3b) were also prepared from 1-amminopropane for comparison purposes. Dye-Doped Silica Nanoparticles (DDNs). The synthetic procedure to prepare nearly monodisperse silica nanoparticles by condensation of tetralkoxysilane derivatives in ammonia, water, and ethanol solutions has been known since the late 1960s from the work of Sto¨ber.14 The size of the resulting particles is essentially determined by the selection of proper reaction conditions, namely, the concentration of silane, water, and ammonia and the reaction temperature.15 Later on, a modification of this procedure was proposed by Van Blaaderen in order to prepare covalently dye-doped nanoparticles (DDNs).16,17 The DDNs studied in this work were hence prepared following the Van Blaaderen method by co-condensation of a mixture of tetraethoxysilane (TEOS) and derivatives 1a-3a at 25 °C. The molar ratio of dye derivatives/TEOS in the reaction mixtures was kept constant at a fixed value of 1:10. Several batches were prepared from reaction mixtures containing different initial concentrations of alkoxysilane derivatives and ammonia. The formation of colloids was followed by dynamic light scattering (DLS) measurements, and the resulting particles were purified by extensive ultrafiltration over a 10 000 Da (∼3 nm) cutoff membrane to eliminate unreacted species and hydrolyzed monomers. The resulting particles were investigated using transmission electron microscopy (TEM, Figure 1) and dynamic light scattering (DLS). The diameters of the particles (14) Sto¨ber, W.; Fink, A.; Bohn E. J. Colloid Interface Sci. 1968, 26, 62-69. (15) (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. (16) (a) Van Blaaderen, A.; Vrij, A. Langmuir 1992, 8, 2921-2931. (b) Verhaegh, N. A. M. A.; Van Blaaderen, A. Langmuir 1994, 10, 14271438. (17) Montalti, M.; Prodi, L.; Zaccheroni, N.; Zattoni, A.; Reschglian, P.; Falini, G. Langmuir 2004, 20, 2989-2991.

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Figure 1. TEM images of dye-doped silica nanoparticles containing compound 1a: (left) batch 1, bar 250 nm; (center) batch 2, bar 500 nm; and (right) batch 3, bar 500 nm. Table 1. Nanoparticles Diameters (D) and Polydispersities (δ) of Dye-Doped Silica Nanoparticles (DDNs) as Obtained by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) batch

dye

conditions

1 2 3 4 5

1a 1a 1a 2a 3a

a b c a d

D(TEM) 23.5 71.1 291 26.0 29.6

δ (%)

D(DLS)

4.2 (18%) 9.7 (14%) 28 (9.6%) 6.1 (23%) 6.0 (20%)

24.8 75.1 213 28.1 32.5

a [dye] ) 1.9 × 10-3 M, [TEOS] ) 0.022 M, [NH ] ) 0.6 M, [H O] 3 2 ) 1.3 M. b [dye] ) 3.8 × 10-3 M, [TEOS] ) 0.044 M, [NH3] ) 1.0 c -3 M, [H2O] ) 2.3 M. [dye] ) 6.3 × 10 M, [TEOS] ) 0.073 M, [NH3] ) 1.7 M, [H2O] ) 3.8 M. d [dye] ) 1.9 × 10-3 M, [TEOS] ) 0.022 M, [NH3] ) 0.5 M, [H2O] ) 1.15 M.

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 diameters obtained by electron microscopy is good, except in the case of batch 3 particles, where the TEM diameter is greater that than obtained by DLS. As already reported,15 small particles, with diameters ranging from 20 to 30 nm, are less spherical, less monodisperse, and have a higher surface roughness than the larger ones. The dye content of the DDNs was investigated by XPS analysis of the dry material obtained after solvent evaporation. In the case of 1a-containing batches, quantitative analysis revealed Si/N ratios of 4.9, 4.5, and 3.5, respectively, for batches 1, 2, and 3, which correspond to a molar content of 1a ranging from 10 to 14%; hence, the composition of the particles closely resembles that of the reaction mixture. Only in the case of batch 5 nanoparticles, containing dye 3a, did the analysis reveal a 5.6 Si/N ratio indicating an enrichment of the dye content (18%) of these particles relative to that of the precursors mixture. Absorption and emission spectra of the dye-doped particles in water and ethanol were similar to those of model compounds 1b-3b (see Supporting Information). However, DDNs containing fluorophore 2a (batch 4) showed a highly quenched fluorescence emission and were not used in the following experiments. Cu(II) Sensing with DDNs. The effect of the addition of an aqueous Cu(NO3)2 solution on the fluorescence emission of batch 1 and 5 DDN solutions {(2.5-5) × 10-6 M total dye concentration, 15% ethanol/water, HEPES buffer 0.01 M, pH 7.0}, containing dyes 1a and 3a, respectively, is reported in Figure 2. In each case, on increasing the metal ion concentration, the fluorescence emission decreases, but no changes are observed in the corresponding absorption spectra of the DDNs. The systems are reproducible, and different titration experiments gave identical results within experimental error. The effect is slightly different between the two fluorophores: while 1a-containing particles show almost complete fluorescence quenching (down to about 5% of the initial value), in the case of 3a-containing particles a plateau value, at about 20% of the initial emission, is reached for higher metal ion concentrations. Fluorescence

Figure 2. Spectrofluorimetric titration of 1a (batch 1, [1a]tot ) 2.5 × 10-6 M; fit results: log Kapp ) 5.4, [binding sites] ) 4 × 10-5 M) and 3a (batch 5, [3a]tot ) 5.6 × 10-6 M; fit results: log Kapp ) 3.8, [binding sites] ) 4 × 10-5 M) containing DDNs and (inset) dyes 1b and 3b ([dye] ) 2.5 × 10-6 M) with Cu(NO3)2 in 15% ethanol/water, HEPES buffer 0.01 M pH 7, 25 °C. (b: batch 1, 9: 1b, λexc ) 340 nm, λem ) 520 nm; O: batch 5; 0: 3b, λexc ) 290 nm, λem ) 414 nm).

Figure 3. Spectrofluorimetric titration of 1a (batch 1) containing DDNs with different metal ions in 15% ethanol/water, [1a]tot ) 2.5 × 10-6 M, HEPES buffer 0.01 M pH 7, 25 °C, λexc ) 340 nm, λem ) 520 nm. (O: Cu(NO3)2, fit results: log Kapp ) 5.7, [binding sites] ) 4 × 10-5 M; b: CoCl2, fit results: log Kapp ) 5.3, [binding sites] ) 9 × 10-7 M; 9: Zn(NO3)2; 0: Ni(NO3)2).

emission versus Cu(II) concentration data were fitted with a simplified 1:1 binding model in order to obtain more insight into the number of the binding sites on the particles and their average Cu(II) complexation strength of the binding site on the particles. The interpolation of the profiles reported in Figure 2 (see caption) gave a good fit, and average log Kapp values of 5.4 and 3.8 were determined for the batch 1 and batch 5 particles, respectively, whereas the concentration of the Cu(II) binding sites was found to be similar (4 × 10-5 M). The analysis of the titration profiles and the comparison with the effects of the additions of Cu(II) ions to 1b or 3b solutions with the same dye concentration reveal a nondynamic quenching of the emission; hence, the binding of Cu2+ ions to the nanoparticles is the cause of the observed fluorescence decrease. As highlighted in Figure 2, the sensitivity of the DDNs is very good, particularly in the case of 1a. If the amount of Cu(II) needed to decrease the initial fluorescence emission by 10% ([Cu(II)]10%) is taken as a reference detection limit, then Cu(II) concentrations down to 4.7 × 10-6 and 1.8 × 10-5 M can be detected for DDNs containing 1a and 3a, respectively. To test the sensor selectivity, the effect of the addition of other divalent metal ions, such as Zn(II), Co(II), and Ni(II), was investigated on the batch 1 DDNs, and the results are reported in Figure 3. Very small effects were observed with Zn(II) and Ni(II), whereas Co(II) at the

Turning Fluorescent Dyes into Cu(II) Nanosensors

Figure 4. Spectrofluorimetric titration of 1a containing DDNs with increasing dimensions with Cu(NO3)2 in 15% ethanol/ water, [1a]tot ) 2.5 × 10-6 M, HEPES buffer 0.01 M pH 7, 25 °C, λexc ) 340 nm, λem ) 520 nm. (b: batch 1, fit results: log Kapp ) 5.6, [binding sites] ) 4 × 10-5 M; O: batch 2, fit results: log Kapp ) 4.8, [binding sites] ) 2 × 10-5 M; 0: batch 3, fit results: log Kapp ) 3.7, [binding sites] ) 6 × 10-6 M).

highest concentration employed (6 × 10-4 M) quenches the initial fluorescence by only 25%. Hence, as highlighted in Figure 3, the fluorescence of the dye-doped particles is strongly affected only by the addition of Cu(II) ions. The fitting of the titration profiles (Figure 3, caption) reveals that such a specific response is related to an apparently different binding capacity for the two metal ions: the average binding strengths for Cu(II) and Co(II) are similar (log Kapp values are respectively 5.7 and 5.3), but the overall concentration of binding sites is about 50 times lower (4 × 10-5 vs 9 × 10-7 M). The size of nanoparticles sensibly influences the sensor performance. Figure 4 shows the variation in fluorescence emission upon the addition of Cu(II) to solutions of DDNs containing dye 1a with different diameters (batches 1, 2, and 3; [1a]tot was set to 2.5 × 10-6 M in each sample). In each case, a fluorescence decrease is observed: the Cu(II) affinity of the nanoparticles drops as revealed by the fitting of the data (log Kapp values are 5.7, 4.8, and 3.7 for batch 1, 2, and 3 particles, respectively), and so does the number of binding sites (Figure 4 caption). However, the most relevant feature is that the extent of quenching decreases with increasing particle diameter. As a consequence, 97, 88, and 50% plateau quenchings are observed for particles with 23, 71, and 291 nm diameters (TEM), respectively. Hence, larger entities become increasingly ineffective as Cu(II) sensors. Sensing with Thin Films. Nanoparticles are not the only structure with nanometric dimensions that can be produced by hydrolysis and condensation of alkoxysilane derivatives. To investigate the potential and limitations of the fluorescence chemosensor self-organizing strategy here described, dye-doped sol-gel films on quartz slides were prepared, and their ability to act as Cu(II)-sensing devices was tested. Films were prepared by dip coating quartz slides in prehydrolyzed solutions of 1a, TEOS, and block copolymer F127, which is well known as template for the formation of mesopores in silica thin films18 in HCl, water, and ethanol mixtures. The dye/TEOS ratio in the coating solution was fixed at 1:10. After casting, films were dried at 100 °C for 2 h, and the template was removed with extensive extraction with hot ethanol. A second batch of films was prepared for comparison under the same conditions but in the absence of F127. The thickness of the film, as determined by the profilometric analysis of (18) Grosso, D.; Bakenende, A. R.; Albouy, P. A.; Ayral, A.; Amenitsch, H.; Babonneau, F. Chem. Mater. 2001, 13, 1848-1856.

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Figure 5. Spectrofluorimetric titration of 1a containing solgel thin films in water, HEPES buffer 0.01 M, pH 7, 25 °C, λexc ) 340 nm, λem ) 520 nm. (b: films prepared in the presence of F127, O: films prepared in the absence of F127).

sputtered samples, was 67 ( 11 and 82 ( 12 nm for the films prepared in the presence and in the absence of F127, respectively. As in the case of nanoparticles, the dye content of the films is similar to that of the reaction mixture, as XPS analysis revealed Si/N ratios of 3.8 and 5.1 for films prepared in the presence and in the absence of F127, respectively. The response of the 1a-doped thin films to exposure to a pH 7.0 water solution containing an increased amount of Cu(NO3)2 is reported in Figure 5. In the case of the porous films prepared in the presence of F127, the observed behavior closely resembles that of the dye-doped nanoparticles: fluorescence emission from the film is quenched at saturation down to 10% of the initial value. The Cu(II) complexation strength of the binding sites appears to be slightly lower than in the case of silica nanoparticles (log Kapp value of 4.6), and a density of binding sites of 1 × 10-7 mol/cm2 could be determined by fitting the titration profiles. The sensitivity of the films is quite good, as a Cu(II) concentration of 2.5 × 10-6 M can be determined by a 10% signal decrease. The response time is in the range of minutes, and the selectivity toward other divalent metal cations is similar to that observed for the nanoparticles systems. Rinsing with a 0.06 M HCl solution can regenerate the sensor. Furthermore, subsequent titrations both of the same film and of other films of the same batch gave superimposable profiles. Such results indicate that these dye-doped silica thin films are suitable candidates for the realization of reusable sensing devices. The porosity of the film seems to be an essential feature of its efficiency. In fact, Figure 5 shows that exposure to Cu(II) solutions of nonporous films, prepared in the absence of the block-copolimer F127, resulted in a much smaller variation of the fluorescence emission. Discussion The development of nanoparticle-based fluorescent sensors is a very promising strategy. As clearly demonstrated by the studies of Kopelman and Rosenzweig,19 such systems combine the advantages of both bulk sensors and chemosensors. On one hand, they are still small enough to allow high spatial resolution and the noninvasive probing of small samples, as in the case of intracellular studies. On the other hand, inclusion of the sensor in the particle protects it from the surrounding environment, avoiding problems such as matrix interfer(19) (a) Buck, S. M.; Koo, Y. E. L.; Park, E.; Xu, H.; Brasuel, M.; Philbert, M. A.; Kopelman, R. Curr. Opin. Chem. Biol. 2004, 8, 540546. (b) Buck, S. M.; Xu, H.; Brasuel, M.; Philbert, M. A.; Kopelman, R. Talanta 2004, 63, 41-59. (c) Lu, J.; Rosenzweig, Z. Fresenius’ J. Anal. Chem. 2000, 366, 569-575.

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ence, absorption to macromolecules or, again in the case of biological studies, toxicity for the host organism. Moreover, insoluble sensors can be made suitable for applications in water by the simple inclusion in an appropriate particle. Finally, as mentioned in the Introduction, the self-organization of the sensor components can give the assembly new properties unknown in the isolated monomers.9,17,20 The last point is particularly demonstrated by the results here reported: the simple inclusion of dyes 1a and 3a into silica nanoparticles results in their conversion into fluorescent chemosensors with intriguing properties, and the same strategy can be applied to sol-gel thin films to obtain reusable sensing devices. The addition of Cu(II) ions to a solution of silica nanoparticles containing 1a and 3a results in strong fluorescence quenching that allows the determination of the metal ion concentration in the micromolar range. The shape of the emission versus metal ion concentration profiles and the comparison with the effects of the addition of Cu(II) ions to solutions of model fluorophores 1b and 3b clearly indicate that the observed emission decrease is due to the binding of the metal ions to the particles. No changes were observed in the absorption spectra of the DDNs after the addition of Cu(II) ions, thus ruling out the possibility of direct binding of the Cu(II) ions to the particle-included dyes. The above analysis of the results indicates that the silica network of the nanoparticles must provide the binding sites for the metal ions. In fact, metal ion absorption to sol-gel materials has already been reported and is particularly effective in the case of porous materials and transition-metal ions.21 The formation of the siliceous structure provides the spontaneous organization of a network of silanol groups on the particle surfaces, which are easily deprotonable and close enough to yield effective binding sites for metal ions. Once bound to the silica network, the Cu(II) ions are close enough to the fluorescent units entrapped in the particle to quench their emission, thus producing a measurable signal. Affinity data obtained by the fitting of the titration profiles with particles containing dyes 1a and 3a (Figure 2) indicate that the nature of the dye is also important in determining the metal affinity of the particles: because direct participation in the binding is excluded and the dye content of the particles is similar, an influence on the particle structure (for instance, on the porosity or the polarity of the interior) may be assumed. Particle size is the first important parameter that influences the efficiency of the system. Smaller particles show an almost complete quenching of the emission, whose extent decreases with increasing particle diameter. This can be explained by assuming that only an outer shell of the particle is accessible to the solvent, whereas the inner part is not accessible by the analyte. When the particle is small enough, the solvent-permeable layer is deep enough to make the whole body of the particle accessible to the Cu(II) ions so that all of the fluorophores are effectively quenched. As the diameters of the colloids increase, an increasing fraction of the dyes, located in the particle core, remains unaffected by the binding of the metal ions to the outer shell. For the same reason, the binding capacity decreases because a greater portion of (20) (a) Montalti, M.; Prodi, L.; Zaccheroni, N.; Falini, G. J. Am. Chem. Soc. 2002, 124, 13540-13546. (b) Montalti, M.; Prodi, L.; Zaccheroni, N. J. Mater. Chem. 2005, 15, 2810-2814. (21) (a) Xu, Y.-M.; Wang, R.-S.; Wu, F. J. Colloid Interface Sci. 1999, 209, 380-385. (b) Jung, J.; Kim, J.-A.; Suh, J.-K.; Lee, J.-M.; Ryu, S.-K. Water Res. 2001, 35, 937-942. (c) Wang, Y.; Bryan, C.; Xu, H.; Pohl, P.; Yang, Y.; Brinker, C. J. J. Colloid Interface Sci. 2002, 254, 23-30.

Arduini et al.

the silica network becomes unavailable to interaction with the Cu(II) ions. Therefore, we may conclude that the realization of fluorescent sensors by the dye doping of silica materials requires a nanometric size to be effective. Reasonably, porosity is the second fundamental requirement. This is suggested by the above-discussed results, obtained with nanoparticles with different sizes, and finally confirmed by the experiments performed with thin films. Silica materials produced by acid catalysis are intrinsically less porous;22 consequently, the accessible area for the analyte is essentially limited to the surface. As clearly shown by the titration reported in Figure 5, negligible quenching of the fluorescence emission is observed after exposure to Cu(II) solutions of a 1a-doped sol-gel thin films prepared by acid catalysis, whereas a strong emission decrease is observed when a porous film is produced by the addition of F127 block copolymer. The absence of the mesopores not only decreases the surface area of the film and hence the amount of Cu(II) that can be absorbed but also prevents the Cu(II) ions from penetrating the film and reaching the required spatial proximity to the great majority of fluorescent units. The specific response to Cu(II) ions of the system here investigated is perhaps their most surprising property. The absorption of metal ions to sol-gel materials is reported to be rather nonspecific with a small preference for transition-metal ions.21 As a consequence, the addition of Co(II) or Ni(II) ions to the dye-doped silica nanoparticles was expected to result into fluorescence quenching. Titration experiments indicate that, in the case of Co(II), binding of the metal ion to particles effectively decreases the emission of the systems, but the extent of quenching is much smaller than with Cu(II) as a result of the minor binding capacity of the particles. The reasons for such behavior deserve further investigation. Conclusions Compared to other approaches proposed for the development of the Cu(II) sensor, dye-doped nanosized silica structures are characterized by the extreme simplicity of their realization. Simple mixing of TEOS and commercially available fluorescent dye 1a in the presence of the appropriate catalyst, followed by a standard workup, allows the preparation of dye-doped nanoparticles and thin films that are capable of sensing Cu(II) ions at low concentrations and with a specific response. With respect to the previously reported fluorescence sensing systems based on silica nanoparticles or films, in this case it is not necessary to entrap in the silica matrix a presynthesized fluorescent chemosensor; moreover, it is not even necessary to insert an analyte receptor into the material. The results reported here indicate that the selforganization of the silica structures leads, at the same time, to the formation of metal ion binding sites as well as to the linking of a fluorescent reporter in their vicinity. Structural features of the materials, particularly particle size and network porosity, strongly affect their ability to act as fluorescent sensors. One of the most interesting results is probably the possibility of preparing reusable bulk sensors via dye-doped thin film deposition, which (22) Brinker, C. J.; Scherer, G. W. In Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing; Academic Press Inc.: San Diego, CA, 1990.

Turning Fluorescent Dyes into Cu(II) Nanosensors

widens the field of possible applications of such systems.23 Clearly, the extension of this approach to other analytes would not be trivial. However, simple modifications of mesoporous silica, by functionalization with thiol-bearing trialkoxysilane derivatives, have led to materials with high Hg(II) sorption capacity.24 It is reasonable to assume that a similar approach could lead to self-organized Hg(II) fluorescent nanosensors. The modification of silica with other functional groups, such as amines, could lead to an increase in affinity toward other metal ions. Selectivity is one further problem to be addressed because it is difficult to obtain a material capable of selectively binding only one substrate. But this would not be a major drawback if the possibility of preparing sensor arrays by (23) Fluorescence sensing in sol-gel films has been already reported but using fluoroionophores entrapped in the silica matrix; see, for example, (a) Javaid, M. A.; Keay P. J. J. Sol.-Gel Sci. Technol. 2000, 17, 55-59. (b) Ertekin, K.; Yenigul, B.; Akkaya E. U. J. Fluoresc. 2002, 12, 263-268. (24) Feng, X.; Fryxell, G. E.; Wang, L.-Q.; Kim, A. Y.; Liu, J.; Kemner, K. M. Science 1997, 276, 923-926. (25) Albert, K. J.; Lewis, N. S.; Schauer, C. L.; Sotzing, G. A.; Stitzel, S. E.; Vaid, T. P.; Walt, D. R. Chem. Rev. 2000, 100, 2595-2626.

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the deposition of differently doped films is taken into consideration.25 In such systems, in fact, the crucial feature is not selectivity but rather the ability of the different members of the array to respond in different ways to several substrates. Acknowledgment. We thank Mr. Claudio Gamboz and Professor Maria Rosa Soranzo (CSPA, University of Trieste) for kind help with TEM analysis and Dr. Cinzia Sada (INFM, Padova) for SIMS measurements. Financial support for this research has been partially provided by the Ministry of Education, University and Research (MIUR contracts 2003030309 and 2003037580) and by the University of Padova (University Research Project CPDA034893). Supporting Information Available: UV-vis and fluorescence spectra of reference dyes 1b-3b, dye-doped nanoparticles, and dye-doped sol-gel films. This material is available free of charge via the Internet at http://pubs.acs.org. LA050785S