- Email: [email protected]
Dually fluorescent silica nanoparticles
Journal of Luminescence 131 (2011) 888–893
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Dually fluorescent silica nanoparticles Ana Paula Garcia Ferreira a, Rafael Frederice a, Kris Pieter Frans Janssen b, Marcelo Henrique Gehlen a,n a b
~ Carlos, Universidade de Sa~ o Paulo, 13566-590, Sa~ o Carlos, SP, Brazil Instituto de Quı´mica de Sao Katholieke Universiteit Leuven, BIOSYST-MeBioS (Mechatronics, Biostatistics and Sensors) Willem de Croylaan 42, B-3001 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2010 Received in revised form 22 November 2010 Accepted 23 December 2010 Available online 31 December 2010
The cationic dyes 9-aminoacridine (9AA) and safranine (Sf) were entrapped into silica spheres of about ¨ method. The fluorescent materials are investigated by 0.2 mm diameter prepared by modified Stober steady-state and time-resolved emission, in addition of confocal fluorescence microscopy. Silica particles containing 9-aminoacridine (SP9AA) and safranine (SPSf) or both dyes (SPSf9AA) are emissive particles. When both dyes are present in the same particle but loaded in sequential stages 9AA emission is quenched as a consequence of energy transfer from 9AA (donor) to Sf (acceptor). This result suggests that particle growing processes where the acceptor is incorporated first into the core do not prevent donor/acceptor pairs to be close due to an overlay of the concentration gradients of both dyes in a radial core-shell like distribution. & 2010 Elsevier B.V. All rights reserved.
Keywords: Silica spheres Fluorescence Energy transfer 9-aminoacridine
1. Introduction Silica nanoparticles are very versatile materials with many applications in different fields, such as delivery and controlled release of drugs, bioanalytical assays, chemical and biochemical sensors, catalysis and advanced optical materials [1–4]. The entrapment of fluorescent dyes in silica nanoparticles further allows the investigation of photophysics and photochemistry in confined media [5–7]. Spherical silica nanoparticles can be obtained by hydrolysis/ condensation of alkoxysilanes, following the procedure described ¨ in the pioneering work of Stober and Fink [8]. The reaction can take place under basic or acid conditions, leading to final products with distinct morphologies [9]. Uniform and monodisperse sphe¨ rical silica particles can be prepared by the Stober method, covering sizes from 5 to 2000 nm [8–11]. Physical models of particle formation have been extensively discussed in literature. Briefly, two models have been proposed for silica formation and growth: monomer addition and controlled aggregation [12,13]. The monomer addition model follows the Lamer-like mechanism, where after an initial nucleation, growth occurs through addition of hydrolyzed monomer to particle surface. On the other hand, the controlled aggregation model assumes that nucleation occurs continuously throughout the reaction. Harris et al. [14]and van Blaaderen et al. [15] claim that both mechanisms are responsible for particle growth [9].
n
Corresponding author. E-mail address: [email protected] (M.H. Gehlen).
0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.12.019
Silica powders, films or glasses are excellent optically inert matrices for dye incorporation [6,16]. The behavior of dyes added to silica has been studied using commercial or synthesized particles [17,18]. Some reports involve the modification of silica surface or the use of dye derivative with alkoxysilane group for covalent attachment [19,20]. The encapsulation of dyes in earlier stages of particle growth has also been investigated, but dye desorption may occurs causing the loss of fluorescent character of the particles when they are dispersed in solution for a long time. ¨ Modified Stober protocols have been developed in order to minimize dye desorption by diffusion to the solvent medium. In this respect, the so-called core-shell nanoparticles became an alternative route. Core-shell fluorescent nanoparticles are composed of dye rich nuclei surrounded by a siliceous shell. In this type of procedure, dye is usually attached to silica core by covalent bond using a silane dye derivative [20]. Despite the development of dye loaded silica particles, research on photophysical processes in these systems is mainly focused on systems containing only one dye. There is a lack of understanding the behavior of two dyes in the same silica environment where excited-state energy transfer process might occur. However, donor and acceptor pairs of fluorescent dyes have been loaded in silica sol–gel to produce solid-state dye lasers with increased laser efficiency and tunable spectral range [21–23]. By using micrometer size silica spheres with donor and acceptor dyes, there may be a possibility to form a whispering gallery dye laser with energy transfer. In this work, two cationic dyes (9-aminoacridine and safranine) were incorporated in silica nanoparticles prepared by a ¨ modified Stober method, with the aim of investigating fluorescence properties and intra-particle energy transfer.
A.P.G. Ferreira et al. / Journal of Luminescence 131 (2011) 888–893
889
2. Experimental
2.5. Nanoparticle preparation
2.1. Fluorescence spectroscopy
Tetraethylortosilicate (TEOS, Fluka), deionized water and catalyst (NH4OH) were employed at the molar ratio of 1:21:0.5. Typically reaction solution was prepared by addition of up to 1 mL of TEOS, 16.7 mL of water and 3.1 mL of aqueous solution of NH4OH (aqueous ammonia 28%) to 100 mL of 2-propanol as the solvent. The dye (9-aminoacridine (9AA) or Safranine (Sf), both from Aldrich Co.) was added to reaction batch from a stock solution in 2-propanol yielding an initial dye concentration of about 2 10 5 mol L 1. Initially, the reaction system was stirred overnight at 40 1C with 300 mL of TEOS. After that, aliquots of 50 mL of TEOS were subsequently added to the reaction vessel in time intervals of 15 min, allowing the formation of a silica shell. After 1 h, the systems were submitted to cooling in an ice bath to stop the reaction. The solution containing the silica particles was centrifuged and the separated solid was washed with cold solvent and dried in vacuum. In the case of particles doped with the two dyes, the preparation route was similar to the aforementioned. However, after the formation of the shell around the safranine rich core (300 mL to form the core and 200 mL of TEOS to form the shell), the silica particles were separated by centrifugation and the solid powder was re-dispersed in 2-propanol solution containing deionized water, catalyst, and 9AA in an initial dye concentration of about 2 10 5 mol L 1. TEOS was added under continuous stirring (300 mL), and the system was left to react over 3 h to form a second dye rich shell over the precursor particles containing safranine. Finally, new aliquots of TEOS (4 times 50 mL) were incorporated every 15 min, creating a protecting silica shell. Centrifugation of the suspensions yields a thin solid powder that was dried in vacuum and kept in a desiccator until analysis. According to this procedure, it is inferred that 9AA and safranine, although not covalently attached, may be loaded in radial distribution within silica particles as shown schematically in Fig. 1.
Steady-state emission spectra were obtained using a CD-900 Edinburgh spectrofluorimeter. Fluorescence decays were measured by time-correlated single photon counting using a home-made ps spectrometer equipped with Glan-Laser polarizers (Newport), a Peltier cooled PMT-MCP (Hamamatsu R3809U-50) as photon detector, and TCC900 counting board electronics (Edinburgh Co.). The light pulse was provided by frequency doubling the 200 fs laser pulse of Mira 900 Ti–Sapphire laser pumped by a Verdi 5 W Coherent laser, and the pulse frequency was reduced down to 800 kHz by using a Conoptics pulse picker. The system provides an instrument response function (irf) of about 40 ps at FWHM. Silica samples (2.3 g L 1) were suspended in chloroform, briefly sonicated, conditioned in a 1 1 quartz cuvette, and immediately measured. The fluorescence decays were taken in magic angle (lexc ¼ 400 nm) mode and were analyzed using a reconvolution procedure with multi-exponential models and fitted to a sum of the exponential given by the following equation: IðtÞ ¼
X bi exp½t=ti
ð1Þ
i
where ti and bi are the decay time and its pre-exponential factor of the ith component, respectively.
2.2. Confocal fluorescence microscopy The fluorescence images were obtained using a plate scanning confocal microscope setup. The home-made instrument is based on IX71 Olympus microscope with a digital piezoelectric controller and stage (PI models E-710.3CD and P-517.3CD) for nanometric sample scanning. The excitation light was provided by a Coherent Cube 405 nm CW diode laser operating at low power of 10 mW, converted to a circularly polarized beam using a zero order quarter wave plate (Del Mar Photonics) and focused over the sample with Olympus 60X N.A. 1.35 Oil immersion objective. The emission signal was separated from the laser excitation beam by using a dichroic cube (Chroma z405lp) and 405 nm Notch filter (Semrock NF02-405U-25). Photon counting was collected over a SPCM-AQR-14 Perkin Elmer APD point detector aligned with an 80 mm pinhole of the confocal line. The TTL detector signals were collected with a NI 6601 counter/timer PCI card and transferred to a PC computer for 2D plotting using a scanning control program written in C# by K. Janssen. The samples were prepared by spin coating of the dispersed silica particles at low concentration in dioxane over optical glass coverslips.
3. Results and discussion The encapsulation of 9-aminoacridine and safranine cationic dyes (Fig. 2) in silica nanoparticles resulted in systems denoted by SP9AA and SPSf, respectively.
Fig. 1. Idealized dye distribution in nanoparticle containing 9-aminoacridine (9AA) and safranine (Sf).
2.3. Scanning electronic microscopy (SEM) Nanoparticles images were obtained using a LEO (model 440) with Oxford detector, electron beams of 20 kV, current of 2.62 A and I probe of 150 pA. Samples were covered with a sputtered gold layer (10 nm), using the Coating System BAL-TEC 020.
2.4. Zeta potential determination Silica nanoparticle in aqueous dilute solution was submitted to electrophoretic mobility measurements and zeta potential calculation using Zeta PALS (Brookhaven Instrument Co) equipment.
NH2
+ N
H 3C
N
H 2N
N+
CH3
NH2
H 9-aminoacridine Safranine Fig. 2. Molecular structures of 9-aminoacridine and safranine protonated dyes.
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A.P.G. Ferreira et al. / Journal of Luminescence 131 (2011) 888–893
Steady-state fluorescence emission spectra of 9AA in a homogeneous solvent and in silica nanoparticles (SP9AA) are given in Fig. 3. All spectra were collected with nanoparticles suspended in chloroform, a solvent for which the refractive index matches with the silica (nchloroform ¼1.446; nsilica ¼1.456).The solvent therefore minimizes van der Waals attractions and multiple scattering [24]. SP9AA fluorescence has a structured emission with maximum intensities at 428, 450, and 480 nm, nearly matching the emission of 9AA in methanol solution. However, the second vibronic peak dominates the emission in SP9AA, indicating that the silica interacts specifically with the dye, disturbing the excited-state coupling with the ground-state [6]. The stationary emission of nanoparticles containing safranine (SPSf) is illustrated in Fig. 4. The emission profile is similar to the emission spectrum of the dye in chloroform, and the only change is its 10 nm red-shift compared to homogeneous medium. The SP9AA and SPSf SEM images are shown in Fig. 5, from which morphology and size can be determined. It is worthwhile to mention that the incorporation of the dyes did not interfere on particles morphology. Both systems are practically monodisperse, characterized by a narrow size distribution with a mean diameter of about 200 nm. Mobility measurements (data not presented
1.2
Normalized Intensity (a.u.)
1.0 0.8 0.6 0.4
Fig. 5. SEM image of (top) silica particles containing 9-aminoacridine and (bottom) safranine.
0.2 0.0 420
440
460 λ (nm)
480
500
520
Fig. 3. Stationary fluorescence emission of 9AA (–) in methanol and in silica nanoparticles suspended in chloroform (SP9AA) (- - -). Excitation lexc ¼ 400 nm.
Normalized Intensity (a.u.)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 550
600 λ (nm)
650
Fig. 4. Stationary fluorescence emission of safranine in (—) chloroform and (- - -) in SPSf (lexc ¼535 nm).
here) of the silica particles (SP9AA and SPSf) in solution indicated that they are stable due to their zeta potential of about 32 mV, which is a consequence of negative surface charge of silica particles prepared from basic catalysis [11,25,26]. If the charge density remains active and negative during preparation and growing of the silica particles, the loading of the cationic dyes used will be more likely to occur in the outer surface of the particle producing a radial distribution. The SEM image of the system prepared with two dyes encapsulated (SPSf9AA) shows nano-spheres in the same size range as those obtained with only one dye as illustrated in Fig. 6. In order to confirm the incorporation of both dyes in SPSf9AA, steady-state excitation and emission spectra were recorded in the wavelength region that covers the spectral response of both 9AA and safranine, and the results are shown in Fig. 7. Normalized emission spectra show characteristic emission bands of 9AA, with maximum at 427, 450, and 480 nm, and safranine, with maximum at 560 nm, confirming incorporation of both dyes. Also, the excitation spectrum of both dyes resembles in band structure the dye absorption spectrum in a polar environment, which indicates that any small fraction of dye aggregates over silica give no emission contribution. The interactions between 9AA and safranine within the SiO2 particles are established by time-resolved studies. Decay curves of SP9AA and SPSf9AA systems are illustrated in Fig. 8, and they allow comparison of the photophysical behavior of 9AA alone in silica nanoparticle with the system that contains both 9AA and safranine. Measurements were performed with the particles suspended in chloroform, with an excitation wavelength of
A.P.G. Ferreira et al. / Journal of Luminescence 131 (2011) 888–893
Fig. 6. SEM image of SPSf9AA particles containing 9-aminoacridine and safranine.
Normalized Intensity (a.u.)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 350
400
450
500
550
600
650
891
400 nm (excitation region of 9AA) and the emission wavelength at 450 nm. The fluorescence decay parameters obtained by means of a reconvolution procedure using a multi-exponencial decay model (Eq. (1)) are summarized in Table 1. For both systems, triexponential decay dynamics are observed and decay times are in accordance with values reported for acridines in silica [6,18]. CS9AA emission deactivation behavior is characterized by nonhomogeneous dynamics with decay times of t1 ¼0.44 ns, t2 ¼1.88 ns and t3 ¼12.2 ns. The short decay time component is related to hydrogen bonded neutral 9AA with residual silanol groups at silica surface through its endocyclic N. It is useful to mention that hydrogen bonded acridine to silanol has a fluorescence lifetime of about 0.5 ns. [18] The intermediate value (1.88 ns) is related to the fraction of neutral 9AA species formed from deprotonation of the dye in silica, while the long-lived component (12.2 ns) is ascribed to the decay of the remained protonated form of 9AA. This long-lived component is in the same time range of protonated 9AA lifetime in media such as methanol, polymer and surfactant solution, silica powder and monolith [6,27]. It should be noted that the shorter decay time, related to hydrogen bonded 9AA, encompasses the largest contribution, which means that most of dye molecules are hydrogen bonded to silanol groups of the silica surface. For silica particles prepared with both 9AA and safranine, the triexponential behavior is still observed. However, in this case 9AA emission is highly quenched by safranine, and the three decay time components recovered are now shorter (see data in Table 1). Considering these results, a possible representation of the 9AA species is illustrated in Fig. 9. ¨ The Forster critical radius R0 of the donor–acceptor pair 9AA/Sf in different solvents and in polymers was determined in a previous work [28,29]. The critical distance is about 73 A˚ in short chain alcohols (methanol to buthanol). The presence of energy transfer indicates that during silica particle formation an inter-diffusion of the dyes takes place to a substantial extent. Thus, the radial distribution of safranine and 9-aminoacridine is forming a common and large boundary within the silica particle that allows an overlap
λ (nm) Fig. 7. Steady-state excitation and emission spectra of powdered SPSf9AA; (—) lexc ¼400 nm and (- - -) lexc ¼ 525 nm.
10000
Table 1 Decay times of SP9AA and SPSf9AA silica nanoparticles.
SP9AA SPSf9AA
10000
t1/ns
ba
t2/ns
ba
t3/ns
ba
w2
0.44 0.41
0.83 0.97
1.88 1.57
0.05 0.02
12.2 9.7
0.12 0.01
1.092 1.284
Counts
1000
1000
a
100
b is the normalized pre-exponential factor. lexc ¼ 400 nm; lem ¼ 450 nm.
Counts
10 1 0
100
1
2 3 time (ns)
4
5
10
NH2
NH2
NH2
N
N
N+ H
H 1 0
10
20
30 time (ns)
40
50
Fig. 8. Fluorescence decays of systems containing 9-aminoacridine (9AA) and both dyes 9AA and safranine suspended silica particles solution in chloroform: (&) IRF, (J) SP9AA and () SPSf9AA. (Inset: the same systems measured in a short time increment); lexc ¼ 400 nm and lem ¼ 450 nm.
O Si
O O
O
Fig. 9. 9AA species in silica particle (left to right): H-bond to silanol, neutral and monoprotonated forms.
892
A.P.G. Ferreira et al. / Journal of Luminescence 131 (2011) 888–893
of dyes densities as shown in Fig. 10. The existence of close donor– acceptor pairs in the overlapping region allows energy transfer. From the integrated decay profiles (or the ratio of average decay time) in absence and presence of the acceptor Sf, the efficiency of energy transfer (f) in dynamic mode (f ffi1 /tsfS//t0S) is estimated to be about 68%. Large efficiency of fluorescence resonance energy transfer (FRET) has been found in dye-doped mesostructured composites containing silica/block copolymer in thin films with coumarin 485 and pyrromethene 567 laser dyes [30] and in mesostructured organosilica of p-conjugated fluorescent polymer (donor) to rubrene (acceptor) [31]. The analysis of fluorescence images of the core-shell silica particles at low density dispersed in a glass optical surface indicated additional features of the system with both dyes. Some particles appear as strong emitter centers, while other particles are quite less intense. Considering that they are almost on the same focal plane, such large difference would come from different
fluorescence efficiency of the particles either caused by energy transfer or by non-homogeneous loading of the two dyes in the ensemble of particles during preparation. A typical fluorescence image is given in Fig. 11. The particle size in the fluorescence image appears larger than that recovered by TEM due to the optical resolution limitation. Nevetheless, there are some particles with almost gaussian cross section emission intensity distribution. It should be mentioned that the emission from the acceptor dye safranine, probably loaded with high density in the particle core, emits in a region where the APD detector has maximum quantum efficiency, and therefore it is easier to detect. To discriminate between 9AA (donor) and Sf (acceptor) emissions in a single silica particle it is necessary to use an appropriate the dichroic mirror and two APD detectors in the same confocal path, a condition not available at our laboratory at the present time.
4. Conclusions Silica particles containing safranine (SPSf), 9-aminoacridine (SP9AA) or both dyes (SPSf9AA) were prepared by a modified ¨ Stober protocol and were investigated by steady-state and timeresolved fluorescence spectroscopy. Practically monodisperse nanoparticles with diameter of about 200 nm were obtained in all cases. For SP9AA, excited-state analysis reveals triexponential dynamics due to the presence of three species of 9AA in the silica matrix: the H-bonding 9AA, its free base, and its protonated form. When the nanoparticles are prepared with both donor and acceptor dyes (SPSf9AA), the non-homogeneous dye distribution forms a common boundary that allows an overlap of dyes densities as indicated by the observation of fluorescence quenching and energy transfer. Fluorescence imaging indicated that particle brightness is not homogeneous.
Acknowledgements Fig. 10. Illustration of the concentration gradients of safranine (horizontal lines) and 9-aminoacridine (vertical lines) in silica particle. The gray area represents the region of strong overlapping of the dye distributions.
This work was supported by CNPq and FAPESP Brazilian research funds. APGF and RF thank to CAPES and CNPq for graduate fellowships. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
Fig. 11. Fluorescence confocal microscopy 2D image of silica particles containing 9-aminoacridine (donor) safranine (acceptor) dyes. Images are collected with total emission of both fluorescent dyes. XY plot is in nm size scale.
[24] [25]
M. De, P.S. Ghosh, V.M. Rotello, Adv. Mater. 20 (2008) 4225. Q. Chang, L. Zhu, C. Yu, H. Tang, J. Lumin. 128 (2008) 1890. Y. Cui, H. Ren, J. Yu, J. Wang, G. Ian, Dyes Pigm. 81 (2009) 53. F. Emrichi, E. Trave, M. Bersani, J. Fluoresc. 18 (2008) 507. A.S. Ethiraj, N. Hebalkar, S. Kharrazzi, J. Urban, S.R. Sainkar, S.K. Kulkarni, J. Lumin. 114 (2005) 15. M.H. Gehlen, R.V. Pereira, M.R. Gallas, T.M.H. Costa, V. Stefani, J. Photochem. Photobiol. A: Chem. 181 (2006) 147. A.V. Deshpande, U. Kumar, J. Lumin. 128 (2008) 1121. ¨ W. Stober, A. Fink, J. Colloid. Interface Sci. 26 (1968) 62. A.M. Buckley, M. Greenblatt, J. Chem. Educ. 71 (1994) 599. A.P. Leite, E.F. Souza, F. Galembeck, J. Braz. Chem. Soc. 12 (2001) 519. D.L. Green, M.T. Harris, J. Colloid. Interface Sci. 266 (2003) 346. T. Matsoukas, E. Gulari, J. Colloid. Interface Sci. 124 (1988) 252. G.H. Bogush, C.F. Zukoski, J. Colloid. Interface Sci. 142 (1991) 19. M.T. Harris, R.R. Brunson, C.H. Byers, J. Non-Cryst. Solids. 121 (1990) 397. A. van Blaaderen, J. van Geest, A. Vrij, J. Colloid. Interface Sci. 154 (1992) 481. R. Reisfeld, J. Fluoresc. 12 (2002) 317. A.M. Eremenko, N.P. Smirnova, V.M. Ogenko, A. Chuiloa, Res. Chem. Intermed. 19 (1993) 855. I. Negro´n-Encarnacio´n, R. Arce, M. Jime´nez, J. Phys. Chem. A 109 (2005) 787. F.A. Pavan, S. Leal, T.M.H. Costa, E. Benvenutti, J. Sol–Gel Sci. Technol. 23 (2002) 129. A. Burns, H. Ow, U. Wiesner, Chem. Soc. Rev. 35 (2006) 1028. D. Su, Y. Yang, G. Qian, S. Wang, M. Wang, Chem. Phys. Lett. 397 (2004) 397. Y. Yan, G. Qian, D. Su, Z. Wang, M. Wang, Chem. Phys. Lett. 402 (2005) 389. Y. Yang, G. Lin, J. Zou, Z. Wang, M. Wang, G. Qian, Opt. Commun. 277 (2007) 138. M.H. Lee, F.L. Beyer, E.M. Furst, J. Colloid Interface Sci. 228 (2005) 114. S. Shibata, T. Taniguchi, T. Yano, M. Yamane, J. Sol–Gel Sci. Technol. 10 (1997) 263.
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[26] Q. Wang, R. Choy, Q. Dai, A. Ostafin, J. Non-cryst. Solids 353 (2007) 354. [27] R.V. Pereira, M.H. Gehlen, Spectrochim. Acta, Part A 61 (2005) 2926. [28] H.P.M. Oliveira, A. Machulek Junior, A.O. Legendre, M.H. Gehlen, Quim. Nova 26 (2003) 564.
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[29] H.P.M. Oliveira, M.H. Gehlen, J. Lumin. 121 (2006) 544. [30] B.J. Scott, M.H. Bartl, G. Wirnsberger, G.D. Stuky, J. Phys. Chem. A 107 (2003) 5449. [31] N. Mizoshita, Y. Goto, T. Tani, S. Inagki, Adv. Mater. 21 (2009) 4798.
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Dually fluorescent silica nanoparticles Ana Paula Garcia Ferreira a, Rafael Frederice a, Kris Pieter Frans Janssen b, Marcelo Henrique Gehlen a,n a b
~ Carlos, Universidade de Sa~ o Paulo, 13566-590, Sa~ o Carlos, SP, Brazil Instituto de Quı´mica de Sao Katholieke Universiteit Leuven, BIOSYST-MeBioS (Mechatronics, Biostatistics and Sensors) Willem de Croylaan 42, B-3001 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2010 Received in revised form 22 November 2010 Accepted 23 December 2010 Available online 31 December 2010
The cationic dyes 9-aminoacridine (9AA) and safranine (Sf) were entrapped into silica spheres of about ¨ method. The fluorescent materials are investigated by 0.2 mm diameter prepared by modified Stober steady-state and time-resolved emission, in addition of confocal fluorescence microscopy. Silica particles containing 9-aminoacridine (SP9AA) and safranine (SPSf) or both dyes (SPSf9AA) are emissive particles. When both dyes are present in the same particle but loaded in sequential stages 9AA emission is quenched as a consequence of energy transfer from 9AA (donor) to Sf (acceptor). This result suggests that particle growing processes where the acceptor is incorporated first into the core do not prevent donor/acceptor pairs to be close due to an overlay of the concentration gradients of both dyes in a radial core-shell like distribution. & 2010 Elsevier B.V. All rights reserved.
Keywords: Silica spheres Fluorescence Energy transfer 9-aminoacridine
1. Introduction Silica nanoparticles are very versatile materials with many applications in different fields, such as delivery and controlled release of drugs, bioanalytical assays, chemical and biochemical sensors, catalysis and advanced optical materials [1–4]. The entrapment of fluorescent dyes in silica nanoparticles further allows the investigation of photophysics and photochemistry in confined media [5–7]. Spherical silica nanoparticles can be obtained by hydrolysis/ condensation of alkoxysilanes, following the procedure described ¨ in the pioneering work of Stober and Fink [8]. The reaction can take place under basic or acid conditions, leading to final products with distinct morphologies [9]. Uniform and monodisperse sphe¨ rical silica particles can be prepared by the Stober method, covering sizes from 5 to 2000 nm [8–11]. Physical models of particle formation have been extensively discussed in literature. Briefly, two models have been proposed for silica formation and growth: monomer addition and controlled aggregation [12,13]. The monomer addition model follows the Lamer-like mechanism, where after an initial nucleation, growth occurs through addition of hydrolyzed monomer to particle surface. On the other hand, the controlled aggregation model assumes that nucleation occurs continuously throughout the reaction. Harris et al. [14]and van Blaaderen et al. [15] claim that both mechanisms are responsible for particle growth [9].
n
Corresponding author. E-mail address: [email protected] (M.H. Gehlen).
0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.12.019
Silica powders, films or glasses are excellent optically inert matrices for dye incorporation [6,16]. The behavior of dyes added to silica has been studied using commercial or synthesized particles [17,18]. Some reports involve the modification of silica surface or the use of dye derivative with alkoxysilane group for covalent attachment [19,20]. The encapsulation of dyes in earlier stages of particle growth has also been investigated, but dye desorption may occurs causing the loss of fluorescent character of the particles when they are dispersed in solution for a long time. ¨ Modified Stober protocols have been developed in order to minimize dye desorption by diffusion to the solvent medium. In this respect, the so-called core-shell nanoparticles became an alternative route. Core-shell fluorescent nanoparticles are composed of dye rich nuclei surrounded by a siliceous shell. In this type of procedure, dye is usually attached to silica core by covalent bond using a silane dye derivative [20]. Despite the development of dye loaded silica particles, research on photophysical processes in these systems is mainly focused on systems containing only one dye. There is a lack of understanding the behavior of two dyes in the same silica environment where excited-state energy transfer process might occur. However, donor and acceptor pairs of fluorescent dyes have been loaded in silica sol–gel to produce solid-state dye lasers with increased laser efficiency and tunable spectral range [21–23]. By using micrometer size silica spheres with donor and acceptor dyes, there may be a possibility to form a whispering gallery dye laser with energy transfer. In this work, two cationic dyes (9-aminoacridine and safranine) were incorporated in silica nanoparticles prepared by a ¨ modified Stober method, with the aim of investigating fluorescence properties and intra-particle energy transfer.
A.P.G. Ferreira et al. / Journal of Luminescence 131 (2011) 888–893
889
2. Experimental
2.5. Nanoparticle preparation
2.1. Fluorescence spectroscopy
Tetraethylortosilicate (TEOS, Fluka), deionized water and catalyst (NH4OH) were employed at the molar ratio of 1:21:0.5. Typically reaction solution was prepared by addition of up to 1 mL of TEOS, 16.7 mL of water and 3.1 mL of aqueous solution of NH4OH (aqueous ammonia 28%) to 100 mL of 2-propanol as the solvent. The dye (9-aminoacridine (9AA) or Safranine (Sf), both from Aldrich Co.) was added to reaction batch from a stock solution in 2-propanol yielding an initial dye concentration of about 2 10 5 mol L 1. Initially, the reaction system was stirred overnight at 40 1C with 300 mL of TEOS. After that, aliquots of 50 mL of TEOS were subsequently added to the reaction vessel in time intervals of 15 min, allowing the formation of a silica shell. After 1 h, the systems were submitted to cooling in an ice bath to stop the reaction. The solution containing the silica particles was centrifuged and the separated solid was washed with cold solvent and dried in vacuum. In the case of particles doped with the two dyes, the preparation route was similar to the aforementioned. However, after the formation of the shell around the safranine rich core (300 mL to form the core and 200 mL of TEOS to form the shell), the silica particles were separated by centrifugation and the solid powder was re-dispersed in 2-propanol solution containing deionized water, catalyst, and 9AA in an initial dye concentration of about 2 10 5 mol L 1. TEOS was added under continuous stirring (300 mL), and the system was left to react over 3 h to form a second dye rich shell over the precursor particles containing safranine. Finally, new aliquots of TEOS (4 times 50 mL) were incorporated every 15 min, creating a protecting silica shell. Centrifugation of the suspensions yields a thin solid powder that was dried in vacuum and kept in a desiccator until analysis. According to this procedure, it is inferred that 9AA and safranine, although not covalently attached, may be loaded in radial distribution within silica particles as shown schematically in Fig. 1.
Steady-state emission spectra were obtained using a CD-900 Edinburgh spectrofluorimeter. Fluorescence decays were measured by time-correlated single photon counting using a home-made ps spectrometer equipped with Glan-Laser polarizers (Newport), a Peltier cooled PMT-MCP (Hamamatsu R3809U-50) as photon detector, and TCC900 counting board electronics (Edinburgh Co.). The light pulse was provided by frequency doubling the 200 fs laser pulse of Mira 900 Ti–Sapphire laser pumped by a Verdi 5 W Coherent laser, and the pulse frequency was reduced down to 800 kHz by using a Conoptics pulse picker. The system provides an instrument response function (irf) of about 40 ps at FWHM. Silica samples (2.3 g L 1) were suspended in chloroform, briefly sonicated, conditioned in a 1 1 quartz cuvette, and immediately measured. The fluorescence decays were taken in magic angle (lexc ¼ 400 nm) mode and were analyzed using a reconvolution procedure with multi-exponential models and fitted to a sum of the exponential given by the following equation: IðtÞ ¼
X bi exp½t=ti
ð1Þ
i
where ti and bi are the decay time and its pre-exponential factor of the ith component, respectively.
2.2. Confocal fluorescence microscopy The fluorescence images were obtained using a plate scanning confocal microscope setup. The home-made instrument is based on IX71 Olympus microscope with a digital piezoelectric controller and stage (PI models E-710.3CD and P-517.3CD) for nanometric sample scanning. The excitation light was provided by a Coherent Cube 405 nm CW diode laser operating at low power of 10 mW, converted to a circularly polarized beam using a zero order quarter wave plate (Del Mar Photonics) and focused over the sample with Olympus 60X N.A. 1.35 Oil immersion objective. The emission signal was separated from the laser excitation beam by using a dichroic cube (Chroma z405lp) and 405 nm Notch filter (Semrock NF02-405U-25). Photon counting was collected over a SPCM-AQR-14 Perkin Elmer APD point detector aligned with an 80 mm pinhole of the confocal line. The TTL detector signals were collected with a NI 6601 counter/timer PCI card and transferred to a PC computer for 2D plotting using a scanning control program written in C# by K. Janssen. The samples were prepared by spin coating of the dispersed silica particles at low concentration in dioxane over optical glass coverslips.
3. Results and discussion The encapsulation of 9-aminoacridine and safranine cationic dyes (Fig. 2) in silica nanoparticles resulted in systems denoted by SP9AA and SPSf, respectively.
Fig. 1. Idealized dye distribution in nanoparticle containing 9-aminoacridine (9AA) and safranine (Sf).
2.3. Scanning electronic microscopy (SEM) Nanoparticles images were obtained using a LEO (model 440) with Oxford detector, electron beams of 20 kV, current of 2.62 A and I probe of 150 pA. Samples were covered with a sputtered gold layer (10 nm), using the Coating System BAL-TEC 020.
2.4. Zeta potential determination Silica nanoparticle in aqueous dilute solution was submitted to electrophoretic mobility measurements and zeta potential calculation using Zeta PALS (Brookhaven Instrument Co) equipment.
NH2
+ N
H 3C
N
H 2N
N+
CH3
NH2
H 9-aminoacridine Safranine Fig. 2. Molecular structures of 9-aminoacridine and safranine protonated dyes.
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Steady-state fluorescence emission spectra of 9AA in a homogeneous solvent and in silica nanoparticles (SP9AA) are given in Fig. 3. All spectra were collected with nanoparticles suspended in chloroform, a solvent for which the refractive index matches with the silica (nchloroform ¼1.446; nsilica ¼1.456).The solvent therefore minimizes van der Waals attractions and multiple scattering [24]. SP9AA fluorescence has a structured emission with maximum intensities at 428, 450, and 480 nm, nearly matching the emission of 9AA in methanol solution. However, the second vibronic peak dominates the emission in SP9AA, indicating that the silica interacts specifically with the dye, disturbing the excited-state coupling with the ground-state [6]. The stationary emission of nanoparticles containing safranine (SPSf) is illustrated in Fig. 4. The emission profile is similar to the emission spectrum of the dye in chloroform, and the only change is its 10 nm red-shift compared to homogeneous medium. The SP9AA and SPSf SEM images are shown in Fig. 5, from which morphology and size can be determined. It is worthwhile to mention that the incorporation of the dyes did not interfere on particles morphology. Both systems are practically monodisperse, characterized by a narrow size distribution with a mean diameter of about 200 nm. Mobility measurements (data not presented
1.2
Normalized Intensity (a.u.)
1.0 0.8 0.6 0.4
Fig. 5. SEM image of (top) silica particles containing 9-aminoacridine and (bottom) safranine.
0.2 0.0 420
440
460 λ (nm)
480
500
520
Fig. 3. Stationary fluorescence emission of 9AA (–) in methanol and in silica nanoparticles suspended in chloroform (SP9AA) (- - -). Excitation lexc ¼ 400 nm.
Normalized Intensity (a.u.)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 550
600 λ (nm)
650
Fig. 4. Stationary fluorescence emission of safranine in (—) chloroform and (- - -) in SPSf (lexc ¼535 nm).
here) of the silica particles (SP9AA and SPSf) in solution indicated that they are stable due to their zeta potential of about 32 mV, which is a consequence of negative surface charge of silica particles prepared from basic catalysis [11,25,26]. If the charge density remains active and negative during preparation and growing of the silica particles, the loading of the cationic dyes used will be more likely to occur in the outer surface of the particle producing a radial distribution. The SEM image of the system prepared with two dyes encapsulated (SPSf9AA) shows nano-spheres in the same size range as those obtained with only one dye as illustrated in Fig. 6. In order to confirm the incorporation of both dyes in SPSf9AA, steady-state excitation and emission spectra were recorded in the wavelength region that covers the spectral response of both 9AA and safranine, and the results are shown in Fig. 7. Normalized emission spectra show characteristic emission bands of 9AA, with maximum at 427, 450, and 480 nm, and safranine, with maximum at 560 nm, confirming incorporation of both dyes. Also, the excitation spectrum of both dyes resembles in band structure the dye absorption spectrum in a polar environment, which indicates that any small fraction of dye aggregates over silica give no emission contribution. The interactions between 9AA and safranine within the SiO2 particles are established by time-resolved studies. Decay curves of SP9AA and SPSf9AA systems are illustrated in Fig. 8, and they allow comparison of the photophysical behavior of 9AA alone in silica nanoparticle with the system that contains both 9AA and safranine. Measurements were performed with the particles suspended in chloroform, with an excitation wavelength of
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Fig. 6. SEM image of SPSf9AA particles containing 9-aminoacridine and safranine.
Normalized Intensity (a.u.)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 350
400
450
500
550
600
650
891
400 nm (excitation region of 9AA) and the emission wavelength at 450 nm. The fluorescence decay parameters obtained by means of a reconvolution procedure using a multi-exponencial decay model (Eq. (1)) are summarized in Table 1. For both systems, triexponential decay dynamics are observed and decay times are in accordance with values reported for acridines in silica [6,18]. CS9AA emission deactivation behavior is characterized by nonhomogeneous dynamics with decay times of t1 ¼0.44 ns, t2 ¼1.88 ns and t3 ¼12.2 ns. The short decay time component is related to hydrogen bonded neutral 9AA with residual silanol groups at silica surface through its endocyclic N. It is useful to mention that hydrogen bonded acridine to silanol has a fluorescence lifetime of about 0.5 ns. [18] The intermediate value (1.88 ns) is related to the fraction of neutral 9AA species formed from deprotonation of the dye in silica, while the long-lived component (12.2 ns) is ascribed to the decay of the remained protonated form of 9AA. This long-lived component is in the same time range of protonated 9AA lifetime in media such as methanol, polymer and surfactant solution, silica powder and monolith [6,27]. It should be noted that the shorter decay time, related to hydrogen bonded 9AA, encompasses the largest contribution, which means that most of dye molecules are hydrogen bonded to silanol groups of the silica surface. For silica particles prepared with both 9AA and safranine, the triexponential behavior is still observed. However, in this case 9AA emission is highly quenched by safranine, and the three decay time components recovered are now shorter (see data in Table 1). Considering these results, a possible representation of the 9AA species is illustrated in Fig. 9. ¨ The Forster critical radius R0 of the donor–acceptor pair 9AA/Sf in different solvents and in polymers was determined in a previous work [28,29]. The critical distance is about 73 A˚ in short chain alcohols (methanol to buthanol). The presence of energy transfer indicates that during silica particle formation an inter-diffusion of the dyes takes place to a substantial extent. Thus, the radial distribution of safranine and 9-aminoacridine is forming a common and large boundary within the silica particle that allows an overlap
λ (nm) Fig. 7. Steady-state excitation and emission spectra of powdered SPSf9AA; (—) lexc ¼400 nm and (- - -) lexc ¼ 525 nm.
10000
Table 1 Decay times of SP9AA and SPSf9AA silica nanoparticles.
SP9AA SPSf9AA
10000
t1/ns
ba
t2/ns
ba
t3/ns
ba
w2
0.44 0.41
0.83 0.97
1.88 1.57
0.05 0.02
12.2 9.7
0.12 0.01
1.092 1.284
Counts
1000
1000
a
100
b is the normalized pre-exponential factor. lexc ¼ 400 nm; lem ¼ 450 nm.
Counts
10 1 0
100
1
2 3 time (ns)
4
5
10
NH2
NH2
NH2
N
N
N+ H
H 1 0
10
20
30 time (ns)
40
50
Fig. 8. Fluorescence decays of systems containing 9-aminoacridine (9AA) and both dyes 9AA and safranine suspended silica particles solution in chloroform: (&) IRF, (J) SP9AA and () SPSf9AA. (Inset: the same systems measured in a short time increment); lexc ¼ 400 nm and lem ¼ 450 nm.
O Si
O O
O
Fig. 9. 9AA species in silica particle (left to right): H-bond to silanol, neutral and monoprotonated forms.
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of dyes densities as shown in Fig. 10. The existence of close donor– acceptor pairs in the overlapping region allows energy transfer. From the integrated decay profiles (or the ratio of average decay time) in absence and presence of the acceptor Sf, the efficiency of energy transfer (f) in dynamic mode (f ffi1 /tsfS//t0S) is estimated to be about 68%. Large efficiency of fluorescence resonance energy transfer (FRET) has been found in dye-doped mesostructured composites containing silica/block copolymer in thin films with coumarin 485 and pyrromethene 567 laser dyes [30] and in mesostructured organosilica of p-conjugated fluorescent polymer (donor) to rubrene (acceptor) [31]. The analysis of fluorescence images of the core-shell silica particles at low density dispersed in a glass optical surface indicated additional features of the system with both dyes. Some particles appear as strong emitter centers, while other particles are quite less intense. Considering that they are almost on the same focal plane, such large difference would come from different
fluorescence efficiency of the particles either caused by energy transfer or by non-homogeneous loading of the two dyes in the ensemble of particles during preparation. A typical fluorescence image is given in Fig. 11. The particle size in the fluorescence image appears larger than that recovered by TEM due to the optical resolution limitation. Nevetheless, there are some particles with almost gaussian cross section emission intensity distribution. It should be mentioned that the emission from the acceptor dye safranine, probably loaded with high density in the particle core, emits in a region where the APD detector has maximum quantum efficiency, and therefore it is easier to detect. To discriminate between 9AA (donor) and Sf (acceptor) emissions in a single silica particle it is necessary to use an appropriate the dichroic mirror and two APD detectors in the same confocal path, a condition not available at our laboratory at the present time.
4. Conclusions Silica particles containing safranine (SPSf), 9-aminoacridine (SP9AA) or both dyes (SPSf9AA) were prepared by a modified ¨ Stober protocol and were investigated by steady-state and timeresolved fluorescence spectroscopy. Practically monodisperse nanoparticles with diameter of about 200 nm were obtained in all cases. For SP9AA, excited-state analysis reveals triexponential dynamics due to the presence of three species of 9AA in the silica matrix: the H-bonding 9AA, its free base, and its protonated form. When the nanoparticles are prepared with both donor and acceptor dyes (SPSf9AA), the non-homogeneous dye distribution forms a common boundary that allows an overlap of dyes densities as indicated by the observation of fluorescence quenching and energy transfer. Fluorescence imaging indicated that particle brightness is not homogeneous.
Acknowledgements Fig. 10. Illustration of the concentration gradients of safranine (horizontal lines) and 9-aminoacridine (vertical lines) in silica particle. The gray area represents the region of strong overlapping of the dye distributions.
This work was supported by CNPq and FAPESP Brazilian research funds. APGF and RF thank to CAPES and CNPq for graduate fellowships. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
Fig. 11. Fluorescence confocal microscopy 2D image of silica particles containing 9-aminoacridine (donor) safranine (acceptor) dyes. Images are collected with total emission of both fluorescent dyes. XY plot is in nm size scale.
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