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Langmuir 1999, 15, 6170-6180
Dye-Labeled Poly(organosiloxane) Microgels with Core-Shell Architecture Christina Graf, Wolfgang Scha¨rtl,*,† Karl Fischer, Norbert Hugenberg, and Manfred Schmidt Institut fu¨ r Physikalische Chemie der Universita¨ t Mainz, Welderweg 11, 55099 Mainz, Germany Received February 25, 1999. In Final Form: May 18, 1999 Poly(organosiloxane) microgels are highly cross-linked rather monodisperse spherical particles of radius about 10 nm. Using a functionalized silane comonomer, i.e., (chlorobenzyl)trimethoxysilane, model particles suitable for studies in colloid physics are available: photoreactive and fluorescent dyes can be covalently bound within the microgels to prepare tracers for diffusion studies using forced Rayleigh scattering (FRS) and fluorescence correlation spectroscopy (FCS). For the application as tracer particles, it is important not to influence the diffusion behavior by the coupled chromophores. Therefore, functionalized precursors with a core-shell architecture are used to minimize labeling effects. The photochromic dye ortho-nitrostilbene (ONS) and the fluorophores rhodamine B, coumarin 343, and pyrene, respectively, were then coupled to the functionalized cores. The dye content of the labeled µ-gels strongly decreases with increasing thickness of the protective shell. A higher polarity of the used chromophores also lowers the dye content significantly, while differences in the size of the used label molecules are less important. The fluorescence intensity of the dye-labeled spheres is also influenced by the size of the protective shell which has been explained by differences in mobility of the labels (caging effects) and, at high dye concentration (thinner shell), by reabsorption.
Introduction Due to their characteristic time and length scales in comparison to atomic systems, colloidal particles provide an ideal model system to study phase behavior and dynamics of soft condensed matter.1 Particle size and distribution as well as particle interaction pair potential can be adjusted by appropriate synthesis. One class of these model systems include the polystyrene microgels, i.e., cross-linked nano spheres prepared from styrene by radical polymerization in microemulsion.2 More recently, cross-linked poly(organosiloxane) particles of radii about 10 nm have been synthesized by polycondensation in microemulsion3 These particles are redispersable in common organic solvents up to very high concentrations.4 Compared to polystyrene-based µ-gels, the possibility of simple chemical modification is the major advantage of these new materials. A variety of functional groups Si-X (X ) H, CH3, CHdCH2, CH2CHdCH2, (CH2)3OOCCHdCH2, (CH2)3SH, phenyl-CH2Cl) may be introduced into poly(organosiloxane) microgels,4 using commercially available trimethoxysilanes as comonomers. Also, the surface of poly(organosiloxane) µ-gels may be easily modified using functional monomers as endcapping reagents.4,5 In addition the cross-linking density can be adjusted by co-condensation with dialkoxysilanes.6 To study single particle mobilities in colloidal dispersions, frequently optical tracer methods are used. The †
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(1) Pusey, P. N. In Liquids, Freezing, and the Glass Transition; Les Houches Sessions LI; ed. Lesvque, D., Hansen, J. P., Zinn-Justin, J., Eds.; Elsevier: Amsterdam, 1991. (2) Bremser, W.; Antonietti, M.; Schmidt, M. Macromolecules 1990, 23, 3796. (3) Baumann, F.; Schmidt, M.; Deubzer, B.; Geck, M.; Dauth, J. Macromolecules 1994, 27, 6102 (4) Baumann, F.; Deubzer, B.; Geck, M.; Dauth, J.; Sheiko, S.; Schmidt, M. Adv. Mater. 1997, 12, 955. (5) Scha¨rtl, W.; Lindenblatt, G.; Strack, A.; Dziezok, P.; Schmidt, M. J. Colloid Interface Sci. 1998, 110, 285. (6) Baumann, F.; Deubzer, B.; Geck, M.; Dauth, J., Schmidt, M. Macromolecules 1997, 30, 7568.
colloidal spheres have to be labeled with appropriate dye molecules to be suitable for such tracer diffusion measurements. For example, dye-labeled silica core-shell particles used in FRAP (fluorescence recovery after photobleaching) experiments have been prepared previously by van Blaaderen et al.7,8 These particles made from tetraethoxysilane by the Sto¨ber synthesis9 have a density of about 2.0 g/cm3. In contrast, due to the porous structure of the network, the particle density of poly(organosiloxane) spheres in solution is significantly below 1 g/cm3.4 Therefore, our new particles are practically densitymatched within the colloidal dispersion. Here, it should be noted that this density match is not necessarily required for very small particles with radius 10 nm as described in this article, since such small colloids should not settle due to gravity anyway. Other examples for the use of colloidal tracers in optical experiments are the FRS (forced Rayleigh scattering) studies of Bartsch et al.10 The authors have investigated colloidal dispersions of polystyrene µ-gels.11 To attach the required photochromic label ortho-nitrostilbene (ONS)12 to these µ-gels, the polystyrene spheres were chloromethylated and then the ONS cesium salt was coupled by a SN2 reaction.13 In this case the dye molecules are mainly located at the surface of the particles. The major disadvantage of the use of dye-labeled tracers with labels attached directly to the particle surface is a potential influence of specific interactions of the dye labels on the particle diffusion. An often applied method to (7) van Blaaderen, A.; Peetermans, J.; Maret, G.; Dhont, J.K. G. J. Chem. Phys. 1992, 96, 4591. (8) van Blaaderen, A.; Vrij, A. Langmuir 1993, 8, 2921. (9) Sto¨ber, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62. (10) Kogelnik, H. Bell. Syst. Technol. J. 1969, 48, 2909. (11) Bartsch, E.; Frenz, V.; Mo¨ller, S.; Sillescu, H. Physica A 1993, 201, 363. (12) Splitter, J. S.; Calvin, M. J. Org. Chem. 1955, 20, 1086. (13) Antonietti, M.; Sillescu, H. Macromolecules 1985, 18, 1162.
10.1021/la990222e CCC: $18.00 © 1999 American Chemical Society Published on Web 07/17/1999
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minimize such effects is the synthesis of core-shell particles such as used by van Blaaderen et al.8 In this study, synthesis and characterization of dyelabeled poly(organosiloxane) based core-shell particles are presented. To introduce the chromophores into the poly(organosiloxane) spheres, first functionalized core shell µ-gels were synthesized. Then organic dye molecules were coupled chemically onto the reactive groups of the core. Poly(organosiloxane) particles without a core-shell architecture, labeled with the photoreactive dye ONS (ortho-nitrostilbene), already had been used in FRS studies.14 Preliminary results of fluorescence correlation spectroscopy (FCS) measurements15 with rhodaminelabeled poly(organosiloxane) particles were first described in ref 16. In contrast to FRS, which is used to study very slow particle diffusion in highly concentrated dispersions, FCS allows us to investigate the faster mobility at medium particle concentrations in the fluid regime. All tracers employed so far, however, have been lacking a nonfunctionalized protective shell. Here, the synthesis of new tracers with core-shell topology, labeled with ONS and the fluorophores rhodamine B, coumarin 343, and pyrene, will be described in detail. Effects of the topology of the µ-gels, i.e., shell thickness, on the label content are discussed and compared with an adjustment of the label content by reaction time (kinetic control). Finally, the spectroscopic properties of the dye-labeled poly(organosiloxane) µ-gels as investigated by UV/vis and fluorescence spectroscopy are presented. Experimental Section Materials. The silane compounds methyltrimethoxysilane (M1), trimethylmethoxysilane (M3) and hexamethyldisilazane (HMN), provided by Wacker Chemie, and (chlorobenzyl)trimethoxysilane (Cl-M1) (ABCR, 97%) and the employed dyes (Rhodamine B, 99+ %, Radiant Dyes; Coumarin 343, 99+ %, Radiant Dyes; 4-(1-pyrenyl)butyric acid, 99% Acros Organics) were used as supplied without further purification. The other chemicals, benzethonium chloride (Aldrich, 97%), NaOH (Aldrich, 97%), 18-crown-6 (Aldrich, 99%), cesium iodide (Fluka, 99.5+ %), cesium carbonate (Aldrich, 99%), benzyl chloride (Fluka, 99.5+%), silica gel 60 (Merck), dibenzyl malonate (Fluka, 95%), 2,3,6,7-tetrahydro-1H,5H-benzo[ij]quinozilin-8-ol-9-carboxaldehyde (Aldrich, 98%), and piperidine (Aldrich, 99%) were also used as received. Solvents for the different syntheses (Merck, pa grade) were used without further purification. The spectroscopic measurements were carried out in cyclohexane and THF (Merck UVASOL, spectroscopic grade). Syntheses. Chlorobenzyl-Functionalized Poly(organosiloxane) µ-gels with Core-Shell Architecture. The chlorobenzyl-functionalized poly(organosiloxane) core-shell particles are synthesized in a multistep reaction. The principle of the synthesis is shown in Figure 1. In the first step, the chlorobenzyl core (CB-I) is formed by slow addition (60 min) of a mixture of 21.875 g (161 mmol) of M1 and 3.125 g (13 mmol) of Cl-M1 to a stirred (400 rpm) solution of 2.5 g (5.6 mmol) of benzethonium chloride in 125 mL of 6 × 10-3 M NaOH (Milli-Q water). After the addition of the monomers is completed, the reaction mixture is stirred for another 6 h (forming of the core). This first step is very similar to the synthesis of unfunctionalized poly(organosiloxane) µ-gels described previously.3 In the second step, a nonfunctional shell is formed by addition of a variable amount of M1 (nonfunctionalized monomer). Before this monomer is added, the reaction mixture is diluted with 6 × 10-3 M NaOH to avoid aggregation of the growing particles. (14) Scha¨rtl, W.; Graf, C.; Schmidt, M. Prog. Colloid Polym. Sci. 1997, 104, 1129. (15) Rigler, R. J. Biotechnol. 1995, 41, 177. (16) Scha¨rtl, W.; Roos, C.; Graf, C.; Schmidt, M. Trends Colloid Interface Sci. 1999, XIII, in press.
Figure 1. Sketch of the synthesis of dye-labeled core-shell poly(organosiloxane) microgels by co-condensation of trimethoxymethylsilane and trimethoxy(chlorobenzyl)silane. The chlorobenzyl functions are used to chemically attach dye labels to the microgels by an esterification reaction. To minimize labeling effects on the diffusion behavior of the nanoparticles, a protective shell is formed by addition of a variable amount of trimethoxymethylsilane in a second step. Table 1. Synthesis of Chlorobenzyl-Functionalized µ-Gelsa shell growth endcappingb dilution NaOH M1 benzeth NaOH benzeth M3c toluene HMN sample (mL) (g) (mg) (mL) (mg) (g) (mL) (g) CB-I CB-II CB-III CB-IV CB-V
0 90 180 270 360
0 10 20 30 40
0 0.63 1.20 1.73 2.23
0 25 50 75 100
0 1.67 3.40 5.17 6.97
2.72 2.09 1.85 1.73 1.65
50 40 35 30 30
1.60 1.23 1.09 1.01 0.97
a CB-I is the functionalized core without a protective shell of methyltrimethoxysilane. M1: methyltrimethoxysilane. M3: trimethylmethoxysilane, HMN: hexamethylenedisilazane. Benzeth: surfactant benzethonium chloride. NaOH: 6 × 10-3 M solution, prepared with Milli-Q water. b All amounts calculated for 25 g of aqueous dispersion. c Total amount.
The quantity of added base depends on the amount of the additional monomer employed, i.e., shell thickness, and is given in Table 1. Between 10 and 40 g of M1 (see Table 1) and a solution of benzethonium chloride (amount depending on the increase of the total particle surface) in 6 × 10-3 M NaOH (see Table 1) are simultaneously added within 60 min with the aid of two RAZEL syringe pumps (Bioblock Scientific). After that, the reaction mixture is once more stirred for 12 h. Finally, the preformed core-shell particles are coated with a hydrophobic surface by a two-step reaction analogous to the “endcapping reaction” for other poly(organosiloxane) µ-gels described in detail by Baumann et al.:4 the obtained aqueous dispersion of the chlorobenzyl particles is filtered to remove
6172 Langmuir, Vol. 15, No. 19, 1999
Figure 2. Dye labels: (a) ONS (ortho-nitrostilbene) (X ) H), cesium salt (X ) Cs) and benzyl ester (X ) -CH2C6H4Cl); (b) coumarin 343 (X ) H), cesium salt (X ) Cs) and benzyl ester (X ) -CH2C6H4Cl); (c) rhodamine b (X ) H), cesium salt (X ) Cs) and benzyl ester (X ) -CH2C6H4Cl); (d) 4-(1-pyrenyl)butyric acid (X ) H), cesium salt (X ) Cs). eventually formed aggregates, and the filtrate is stabilized by supplementary addition of the surfactant benzethonium chloride (see Table 1). M3 (for amount, see Table 1) is added, and the dispersion is stirred for 12 h. After a second addition of the same amount of M3 and stirring for another 6 h, the dispersion is destabilized by addition of 50 mL of methanol, and the precipitate is filtered out and washed several times with methanol in order to remove the surfactant. Next, the precipitate is redisolved in toluene (see Table 1) without further drying. The remaining methanol is removed from the toluene solution by coevaporation. Then, HMN (for amount, see Table 1) is added and the solution is stirred for another 12 h. The resulting product is precipitated in 150 mL of methanol, filtered out, and finally dried in vacuo for 12 h. A white powder is obtained. All these reactions are carried out at room temperature. Labeling Reaction. The dye labels (Figure 2) are chemically attached to the chlorobenzyl particles by an SN2 reaction of the cesium salt of the chromophores with chlorobenzyl groups.13,14 The reaction is carried out under argon and exclusion of light. A 1 mL volume of 18-crown-6 is dissolved in 100 mL of THF. A 1 g amount of chlorobenzyl-functionalized poly(organosiloxane) µ-gels (0.4-0.9 mmol of chlorobenzyl groups; see Table 2), 0.9 mmol of cesium salt of the chromophore (see next section), and 230 mg (0.9 mmol) of cesium iodide are added. The mixture is stirred under reflux (T ) 55 °C) for 48 h (standard reaction time, kinetic studies: see below). After the coupling reaction, the labeled µ-gels are precipitated in methanol. Nonreacted dye is removed by washing the precipitate several times with methanol. Finally, the µ-gels are dried by freeze-drying from benzene. Orange (ONS), yellow-green (coumarin 343), light pink (rhodamine B), or white (pyrene) powders are obtained. Dye Labels. ONS (ortho-nitrostilbene, Figure 2a) is synthesized by following the procedure described by Splitter and Calvin.12 The fluorescent labels rhodamine B (Figure 2b), coumarin 343 (Figure 2c), and 4-(1-pyrenyl)butyric acid (Figure 2d) are commercially available and are used without further purification. The cesium salts of all dye derivatives are obtained by reacting
Graf et al. the carboxylic acids with stoichiometric amounts of cesium carbonate in ethanol/water (1:1) (ONS), methanol/water (1:1) (rhodamin B), THF/water (50:3) (coumarin 343), or THF/water (2:1) (pyrene) and evaporation of the solvent, followed by freezedrying. Dye Derivatives for UV-Vis Calibration. For determination of the dye content of the labeled spheres by UV-vis calibration (see below), model derivative compounds were synthesized: (1) To determine the dye content of ONS-, rhodamine B-, and coumarin 343-labeled microgels, the corresponding benzyl esters (see Figure 2) have been used: ONS benzyl ester is prepared by the reaction of ONS with benzyl chloride as described in ref 17 and purification by flash chromatography18 with petroleumether/ methylene chloride (1:3) on silica gel 60. The synthesis of rhodamine B benzyl ester is carried out in a similar way, reacting 5 g (8.17 mol) of rhodamine B cesium salt with 12.66 g (0.10 mol) of benzyl chloride in 50 mL of DMF at 60 °C. Due to the much lower reactivity of rhodamine B, the reaction time has to be extended to 17 days. The compound is purified by flash chromatography with THF/ethanol (1:3) on silica gel 60. Coumarin 343 benzyl ester is synthesized according to its methyl ester synthesis19 from dibenzyl malonate, 2,3,6,7-tetrahydro-1H,5Hbenzo[ij]quinozilin-8-ol-9-carboxaldehyde and piperidine. (2) As calibration standard for pyrene labeled spheres, 4-(1pyrenyl)butyric acid itself was used. Opposite to the other three dyes, in this case the carboxyl group is well separated from the chromophoric system (see Figure 2). Therefore, standard and coupled dye show nearly identical absorption spectra. Characterization. Various methods to analyze composition and structure of the synthesized particles have been used: SEC. The size polydispersity and, after a calibration with the hydrodynamic radii obtained by dynamic light scattering (see below), the radii of the poly(organosiloxane) µ-gels were measured by SEC (size exclusion chromatography) in toluene at 20 °C. Measurements were carried out with a Rheos-400 HPLC pump equipped with three columns of different porosities (PSS Mainz; 105, 104, 103 Å), using a differential refractometer (Waters 410) and a UV-vis spectrometer (Waters 486) of variable wavelength for detection. Dynamic and Static Light Scattering. Hydrodynamic radii (Rh) of the chlorobenzyl-functionalized nanoparticles have been determined by dynamic light scattering (DLS) with an apparatus consisting of a Krypton ion laser (Stabilite 2060-11s, Spectra Physics, 647.1 nm, 500 mW output power), goniometer SP-86 (ALV), and an ALV-3000 digital correlator/structurator as described elsewhere.20,21 To determine radius of gyration Rg and mass-averaged particle molecular weight Mw, static light scattering (SLS) of the synthesized particles has been measured with an argon ion laser (Stabilite 2016, Spectra Physics, 514.5 nm, 300 mW output power) and an ALV-1800 multiangle light scattering photometer. The light scattering measurements have been carried out with filtered (Millipore Millex-FGS filter, pore size 0.2 µm) toluene solutions (0.1 wt %). Refractive index increments were measured with a home-built interferometer using a laser diode (543 nm wavelength).22 All measurements were done at 20 °C. AFM. Size, shape, and polydispersity were also characterized by atomic force microscopy (AFM). For these measurements, one drop of a 0.1 wt. % toluene solution of microgels was placed on freshly cleaved Mica. After evaporation of the solvent, the surface was scanned with a Nanoscope IIIa, Digital Instruments (Santa Barbara, CA) operating in tapping mode. UV-Vis Absorption Spectroscopy. (a) Chlorobenzyl-Functionalization (Content). The chlorobenzyl content of the poly(organosiloxane) particles was determined by quantification of (17) Ehlich, D.; Sillescu, H. Macromolecules 1990, 23, 1600. (18) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923. (19) Van Gompel, J.; Schuster, G. B. J. Org. Chem. 1987, 52, 1465. (20) Schmidt, M. Simultanous static and dynamic light scattering: application to structure analysis. In Dynamic Light Scattering: The method and Some Applications; Brown, Ed.; Oxford University Press: Oxford, U.K., 1993; p 372. (21) Bantle, S.; Schmidt, M.; Burchardt, W. Macromolecules 1982, 15, 1604. (22) Becker, A.; Ko¨hler, W.; Mu¨ller, B. Ber. Bunsen-Ges. Phys. Chem. 1995, 99, 600.
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Table 2. Characteristics of the Chlorobenzyl-Functionalized µ-gelsa sample
Rh (nm)
ds (nm)
Rw/Rn
Rg (nm)
FLS
Mw (10-6 g/mol)
FD (g /cm3)
clb (%)***
CB-I (core) CB-II CB-III CB-IV CB-V
10.5 11.5 12.3 13.0 16.1*
0 1 1.8 2.5 5.6*
1.19 1.20 1.28 1.24 1.45
