Langmuir 1992,
8, 2921-2931
2921
Synthesis and Characterization of Colloidal Dispersions of Fluorescent, Monodisperse Silica Spheres A.
Blaaderen* and A. Vrij
van
Van't Hoff Laboratory, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
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Received February
7,
1992.
In Final Form: August 10, 1992
Stable dispersions of monodisperse colloidal silica spheres containing a dye or fluorophore have been synthesized according to a general procedure and dispersed in polar and apolar liquids. The procedure consists of the coupling of the dye to a silane coupling agent, (3-aminopropyl)triethoxysilane, and the controllable incorporation of the reaction product into the silica sphere. The silica spheres are prepared from tetraethoxysilane in mixtures of ammonia, water, and ethanol. The composition of the silica spheres can be controlled in such a way that the organic groups can be placed on the surface, in a thin shell inside the particle or distributed through the volume of an inner core. Fluorescein isothiocyanate was used to make easily bleachable, fluorescent silica spheres. Hydrophilic charge stabilized and organophilic sterically stabilized 1-octadecanol-coated dyed silica systems were synthesized and dispersed in several solvents. All the particles were characterized after the several reaction steps by static and dynamic light scattering and transmission electron microscopy. The fluorescent spheres were further characterized by fluorescence spectroscopy and confocal scanning laser fluorescence microscopy. Great effort was taken to prepare monodisperse dispersions free of clusters of particles. Such model dispersions are required for (scattering) studies of interparticle interactions in (concentrated) systems. Therefore, the several steps of the synthesis and optical characterization are described in detail.
I. Introduction
monodisperse silica spheres from tetraalkoxysilanes have been used as a model system in many studies. For physicochemical studies of the structure and dynamics of concentrated dispersions, the surface esterification of silica particles with 1-octadecanol, as described in detail by Van Helden et al.,3 has been of importance as well. Octadecanol-coated particles are sterically stabilized and can be dispersed in organic solvents. The refractive index of silica («1.45) is close enough to that of organic solvents to make it possible to do light scattering measurements in concentrated dispersions, without the problems of multiple scattering. Furthermore, it has been shown that in several solvents, in particular cyclohexane, the interparticle interactions can be conveniently described by those between “hard spheres”.4 With fluorophores or dyes inside the silica sphere the number of experimental techniques that can be used to study the properties of concentrated suspensions is significantly increased. Examples of these techniques are measurements on the dynamics with fluorescence recovery
In this paper we will describe a general synthetic procedure to incorporate dyes or fluorophores through covalent bonds into monodisperse, colloidal silica spheres. The incorporation of the organic molecules proceeds in a controlled way; that is, it is possible to determine where in the particle the silica is modified. The possibilities of the procedure are illustrated by the synthesis of several fluorescent silica spheres, where the fluorophore is present on the particle surface, in a thin shell in the particle interior or distributed through the volume of an inner core. Both the synthesis of charge stabilized, hydrophilic silica particles in polar solvents and sterically stabilized, 1-octadecanol coated, organophilic silica particles in apolar solvents will be described. The procedure used to make the different colloidal systems consists of two steps. First, the dye is chemically bound to a silane coupling agent, and second, this coupling agent is used in the synthesis of colloidal silica by hydrolysis and condensation of tetraethoxysilane (TES) in mixtures of water, ammonia, and ethanol. As coupling agent (3aminopropyl)triethoxysilane (APS) was used. In a previous paper1 we described how silica spheres can be coated by APS and how it is possible to distribute the coupling agent through the particle interior. Because of the hybrid organic-inorganic nature of these colloids, we call these particles organosilica spheres. Furthermore, it was shown, that a layer of any desired thickness of silica can be deposited on the organosilica and coated-silica spheres. By chemically linking the APS molecules with a dye or fluorophore, the synthetic procedures developed in ref 1 can be used to prepare silica model particles marked for their specific properties of absorption, fluorescence, or photobleaching. The synthesis of organosilica spheres is a modification of a method that was first systematically studied by Stober et al.2 In the years following the work of Stober et al.,
after photobleaching (FRAP), forced Rayleigh scattering (FRS), fluorescence correlation spectroscopy, and fluorescence cross correlation and imaging of separate particles inside the bulk of a dispersion using confocal scanning laser microscopy (CSLM). Because dye molecules inside a sphere do not influence the particle interactions, such spheres can be used as tracers in mixtures with nondyed spheres. Particles as described in this paper have already been used by us in FRAP, FRS, and CSLM studies. These techniques dictated several particle properties like size and specific dye functions. Therefore, the following sections briefly describe FRAP, FRS, and CSLM. For the FRAP technique a “bleachable” fluorophore is required. The fluorescence of the fluorophore fluorescein isothiocyanate (FITC) is easily irreversibly destroyed, or (photo)bleached, by high intensities of light. This bleach(3) Van Helden, A. K.; Jansen, J. W.; Vrij, A. J. Colloid Interface Sci. 1981, 81 (2), 354-68. (4) De Kruif, C. G.; Jansen, J. W.; Vrij, A. In Physics of Complex and
(1) Van Blaaderen, A.; Vrij, A. J. Colloid Interface Sci., in press. (2) Stober, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968,26(1),
Supermolecular Fluids; Safran, Sons: New York, 1987.
62-9.
0743-7463/92/2408-2921$03.00/0
©
S. A.,
Clark, N. A., Eds.; John Wiley &
1992 American Chemical Society
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Langmuir, Vol.
8, No. 12, 1992
van
ability, which is for most applications an undesirable property, is used in an elegant technique to measure longtime self-diffusion coefficients (see references in ref 5). Closely related to the FRAP technique is forced Rayleigh scattering.5 This technique requires a dye which changes its refractive index upon excitation with light. Photoexcitation with an argon ion laser brings a dye like 4-(dimethylamino)azobenzene 4-isothiocyanate in a state in which the absorption for red laser light (He/Ne) is increased. The periodic pattern vanishes by the diffusion of the excited particles and the intensity of the Bragg 6
peak decays accordingly. Large fluorescent silica spheres labeled with FITC can be imaged as separate particles with confocal scanning laser microscopy (CSLM). In this technique only one picture point is imaged at a time. The illumination of the sample by the image of a point source focused inside the sample and the imaging of the (con)focused fluorescent light on a point photodetector provide for an exceptionally short depth of field combined with an increased resolution.7’8
The rest of the paper is organized as follows: In the next theoretical sections we first briefly discuss static and dynamic light scattering and the consequences of polydispersity in the particle size and absorption and fluorescence on the measurements. (Theoretical aspects of the mechanism of incorporation of the coupling agent APS into the silica spheres were treated in ref 1.) In section III the general procedure to prepare the dyed silica spheres is given together with details on several examples and a brief description of the characterization techniques used. In section IV some important steps in the preparation of the particles and several particle properties are presented and discussed. Some of the colloidal systems showed unexpected interparticle interactions and phase behavior; these observations are qualitatively described and discussed as well. Finally, a summary is given in section V.
II. Theory A. Light Scattering. Static Light Scattering (SLS). In the Rayleigh-Gans-Debye (RGD) approximation, scattering particles interact only weakly with the incident electric field.910 To be able to analyze the scattering in this limit, two criteria should be met: (1) the complex refractive index (m) of the particles relative to
the suspension medium is close to 1, \m 1| « 1, and (2) the phase change of the incident wave as it travels through the particle is small, 2kR\m 1| « 1. Here k is the wavenumber 2t/\ of the incident wave (with X the wavelength of light in the suspension) and R is a typical length scale, for spherical particles the particle radius. The second requirement makes it possible to analyze the scattering by larger particles in this approximation if the refractive index of the particles is very close to that of the solvent. In the following we consider a suspension of identical, homogeneous, spherical particles with radius R and a -
-
(5) Van Blaaderen, A.; Peetermans, J.; Maret, G.; Dhont, J. K. G. J. Chem. Phys. 1992, 96 (6), 4591-4603. (6) (a) Hevet, H.; Urbach, W.; Rondelez, F. J. Chem. Phys. 1978, 68 (6), 2725. (b) Griffin, W. F.; Griffin, M. C. A.; Van Blaaderen, A.; Imhoff, A.; Dhont, J. K. G., in preparation. (7) Van Blaaderen, A.; Imhof, A.; Hage, W.; Vrij, A. Langmuir 1992, 8, 1514-1518. (8) (a) Wilson, T. J. Phys. E: Sci. Instrum. 1989, 22, 532-47. (b) Brakenhoff, G. J.; van der Voort, H. T. M.; van Spronsen, E. A.; Nanninga, N. Scanning Microsc. 1988, 2 (1), 33-40. (9) Bohren, C. F.; Huffman, D. R. Absorption and Scattering of Light by Small Particles; John Wiley & Sons: New York, 1983. (10) Kerker, M. Scattering of Light and Other Electromagnetic Radiation; Academic Press: New York, 1969.
Blaaderen and Vrij
number density Cn. For dilute dispersions interparticle interference is noncorrelated and the time averaged intensity I(K) is the sum of the scattered intensities of the separate particles. For light polarized vertically to the scattering plane the intensity is proportional to
I(K)*\m-\fCJl3P(K)
(D
in which the length of the scattering vector K is a function of the scattering angle 6 K
4ttN. =
sin (6/2)
(2)
Here Ne is the refractive index of the suspending medium and Xo the wavelength of the incident light in vacuum. The form factor P(K) describes intraparticle interference. For homogeneous spheres the sum over the phase differences is given by10
p(K)
m ~
fL
3
sin (KR)
3XR cos (iffl)12
-
J
(KR)3
Scattering disappears at angles defined by tan (KR)
=
KR
(4)
For small values of KR (KR < 1.5) scattering may be analyzed in the Guinier approximation by
In (P(K))
=
-K?R2/ 3
(5)
For homogeneous spheres it follows from expanding eqs 3 and 5 that the so-called (optical) radius of gyration Rt is given by Rg
=
(6)
§Ro2
The subscript 0 in the particle radius designates a radius determined with static (or elastic) light scattering. Scattering by spheres in media in which the RGD criteria are not met has to be analyzed with the full solution of the Maxwell equations for the incident and scattered fields: the “Mie theory”.9’10 In Mie scattering there is no simple separation of the optical properties (m, X, factors that were left out of eq 1 because they are not used) and the particle size (R) as described by eqs 1 and 3. Another consequence of leaving the RGD limit is that the minima in the scattering as a function of the scattering angle are no longer points of zero intensity and are no longer given by eq 4. However, the shifts in the position of the minima are not very large. For instance, for particles of R = 500 nm in ethanol (Nt = 1.36,2VP = 14.5, Xo = 500 nm) the shift is only a few percent, although 2kR\m 1| «= O.9.11 Dynamic Light Scattering (DLS). As was argued in the preceding section, the mean intensity of a dispersion of uncorrelated particles is the sum of the intensities of each individual particle in the scattering volume. However, there are fluctuations around this mean intensity, which change in time. This is caused by the Brownian (or diffusive) motions of the particles. The intensity reaching the detector at a given instant is formed from the superposition of the scattered fields (including their phase) of all the colloids. The position coordinates of each scatterer as a function of time are characterized by a diffusion process with diffusion constant Do. It can be shown, that Do also characterizes the fluctuations in the intensity. The autocorrelation function of the scattered intensity is measured to analyze these fluctuations in time. -
(11) Kerker, M.; Farone, W. A.; Smith, L. B.; Matijevic, E. J. Colloid Interface Sci. 1964, 19, 193-200.
Monodisperse Colloidal Silica Spheres
Langmuir, Vol. 8, No. 12,1992
This intensity autocorrelation function can be related to the electric field autocorrelation function g(t). If normalized, g(t) describes the decay of a sinusoidal density fluctuation, with wavenumber K, as a function of time. In the absence of particle interactions12
g(t)
=
exp
(-D^t)
(7)
For spherical particles with stick boundary conditions in Newtonian liquid, the diffusion constant is given by the Stokes-Einstein relation
a
D0
=
kBT/6iryRh
(8)
With kn being the Boltzmann constant, T the absolute temperature, y the viscosity, and Rh the hydrodynamic radius. Polydispersity and Particle Clusters. The polydispersity in the particle size of a dispersion can be characterized by the relative standard deviation a and can be determined with transmission electron microscopy (Re)
was
2923
chosen in this paper
Np
=
np + iqp
(12)
The angle-dependent scattering of particles that can be analyzed by eq 3 is not influenced by absorption as long as the RGD criteria are met. The particles described in this paper absorb light because of the strong absorption of the dyes that are distributed through the particle volume. The complex part of the refractive index at an absorption wavelength \g can for such particles be estimated from the molar extinction coefficient «(Ag) of the dye in the silica. The relation between these two quantities can be found as follows: On the one hand, the intensity of light with wavelength Xg that traverses a distance x in a silicon slab containing an absorbing dye with molar concentration cm and a molar extinction coefficient«, is given by Lambert-Beer’s law
I
=
I0 exp(-«cmx)
(13)
On the other hand, a plane wave propagating in the direction through a medium with refractive index N = + iq is exponentially attenuated9
x n
