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Functionalized polymer colloids bearing primary amino groups
Journal of Colloid and Interface Science 311 (2007) 425–429 www.elsevier.com/locate/jcis
Functionalized polymer colloids bearing primary amino groups M. Schmitt a , J. Wagner a,∗ , G. Jung b , R. Hempelmann a a Physical Chemistry, Saarland University, 66123 Saarbrücken, Germany b Biophysical Chemistry, Saarland University, 66123 Saarbrücken, Germany
Received 28 November 2006; accepted 14 March 2007 Available online 20 March 2007
Abstract Polymer colloids are prepared via radicalic emulsion polymerisation of butylacrylate. Functionalization with amino groups is achieved by copolymerisation of 2-amino-ethylmethacrylates. In order to over-compensate the positive surface charges resulting from the amino groups additionally vinylbenzenesulfonic acid is copolymerized. The size of the resulting particles is controlled by the molar ratio of amino to sulfonic acid groups. The suitability of amino groups for coupling reactions is demonstrated by electrophilic addition of fluorescein-5-isothiocyanate. The resulting particles are characterized by dynamic light scattering and zeta potential measurements as well as by optical spectroscopy. The suitability of labelled particles for optical tracer experiments is demonstrated by fluorescence correlation spectroscopy. © 2007 Elsevier Inc. All rights reserved. Keywords: Emulsion polymerisation; Polymer colloids; Fluorescence labelling
1. Introduction During the last decades colloidal systems have been attracting considerable scientific interest both for fundamental and applied research [1–3], particularly due to the availability of nearly monodisperse nanoscale particles with the capability of self-organisation [4–7]. In aqueous suspension most of the systems are electrostatically stabilized [8–10]. For polymer colloids, surface charges in many cases result from sulfonic acids introduced by the radical initiator potassium peroxodisulfate. These macroions interact via a screened Coulomb or Yukawa potential [11]. The screening length strongly depends on the ionic strength of the system. As consequence, even small amounts of stray ions can destroy a long-range electrostatic repulsion and induce irreversible agglomeration when short range attractive dispersion forces dominate [12]. This sensitivity to ions is an obstacle to standard aqueous chemistry. Hence, reactions suitable to modify colloidal particles have to avoid the presence of charged species. The presence or generation of ions can be circumvented by the nucleophilic addition of amines to isothiocyanates that is commonly used in * Corresponding author.
E-mail address: [email protected] (J. Wagner). 0021-9797/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2007.03.026
biochemistry. This reaction leading to stable thiourea derivates can be carried out under mild, physiological conditions. In this way, e.g., fluorescence dyes are attached to biological macromolecules for labelling in biology or biomedicine [13,14]. In colloidal physics, fluorescently labelled particles are a prerequisite for optical tracer experiments like fluorescence correlation spectroscopy [15,16]. This method gives an access to self-diffusion coefficients of colloidal particles on a mesoscopic time scale [17]. The self-diffusion coefficient of interacting particles becomes, due to memory effects, time-dependent in intermediate scales of time. Memory effects induce a decrease of the self-diffusion coefficient. The dimensions of the focal volume in an FCS experiment, however, typically equal several times the particles’ diameter and thus the next neighbour distances in colloidal suspensions. As consequence, FCS data of interacting particles are highly influenced by memory effects. Here we report about the emulsion copolymerisation of butylacrylate with 2-amino-ethylmethacrylate providing primary amines as anchor groups. The colloidal stability is preserved by additional copolymerisation of vinylbenzene-sulfonate introducing additional negative charges. Swelling of the particles in aqueous media can be avoided by crosslinking with bifunctional ethyleneglycol-dimethacrylate. By the copolymerization of butylacrylate with three monomers bearing functionalities, stable suspensions of polymer particles with narrow size dis-
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tribution can be prepared. The feasibility of these particles for coupling reactions with isothiocyanates is demonstrated by the addition of fluorescein-5-isothiocyanate. Hereby, nearly monodisperse, fluorescently labelled particles can be prepared. Since the dye is chemically attached as a thiourea derivate, a bleeding out typical for physically adsorbed dyes can be prevented. We demonstrate the use of these labeled particles for tracer diffusion experiments by fluorescence correlation spectroscopy. 2. Experimental 2.1. Materials The chemicals used for the synthesis of polymer colloids are butylacrylate (BA, C7 H12 O2 ), 4-vinylbenzenesulfonic acid sodium salt (VBS, C8 H7 NaO3 S), FeSO4 ·7H2 O, Na2 SO3 , and K2 S2 O4 supplied by Fluka Chemie AG, ethylenglycoldimethacrylate (EMA, C10 H14 O4 ), fluorescein-5-isothiocyanate (FITC, C21 H11 NO5 S), and ethylisothiocyanate (ECI, C3 H5 NS) supplied by Merck KGaA, and 2-amino-ethylmethacrylathydrochloride (AEMA, C6 H12 O2 NCl) supplied by ACROS Organics. For dialysis a Medicell International Ltd. membrane size 5 Inf Dia 36/32 —28.6 mm 30 M was used. Deionised water was the solvent for all experiments. 2.2. Emulsion polymerization process For the preparation of nanoscale polymer particles suspended in water first atmospheric oxygen has to be removed; for that purpose, 750 ml deionised water is stirred in nitrogen atmosphere at 80 ◦ C for several hours. Then, keeping the temperature constant, a mixture of monomers is added: 120 mmol BA, 2.34 mmol EMA, up to 5.84 mmol VBS, and 1.17 mmol AEMA. After stirring for one hour, the polymerisation is induced by addition of the redox catalysts sodium sulfite and iron(II) sulfate and by the addition of the radical starter potassium peroxodisulfate. The reaction progress is indicated by increasing turbidity. After 24 h, the resulting suspension is filtrated and dialysed against distilled water for typically one week in order to remove residual monomers and low molecular weight oligomers. 2.3. Fluorescence labelling Starting material is the copolymer, prepared as described above, with about one mol-percent of AEMA. For the fluorescence labelling 90 µl solution of 0.02 mmol FITC in DMF is added to 25 ml of the colloidal suspension containing less than 0.04 mmol amino groups at 95 ◦ C. Subsequently, the remaining primary amino groups are eliminated by reaction with a surplus of EIC. The product is dialysed against deionized water for several days, until free dye molecules are completely removed. 2.4. Particle size determination The particle size is determined by means of dynamic light scattering (DLS) using a ALV-5000E set-up purchased from
ALV, Langen (Germany), equipped with a 35 mW He/Nelaser purchased from Coherent Inc. For the experiments, dilute translucent samples are used; 10−3 mol/L KCl is added to screen the electrostatic interaction and to exclude any short range order in the colloidal system. The particle size distribution is obtained by the regularization method proposed by Provencher using the CONTIN algorithm [18,19]. 2.5. pH dependent zeta potential measurements The zeta potential ζ is determined employing a Zetasizer 3000 HSa and a Multi Purpose Titrator supplied by Malvern Instruments. Highly dilute samples containing 10−3 mol/L KCl are used for these experiments. 2.6. Optical measurements The absorption spectra are measured by means of a Perkin Elmer Lambda5 UV–vis spectrometer, and for the fluorescence spectra a Ocean Optics Inc. SD2000 fluorescence spectrometer equipped with LED illumination in excitation is used. 3. Results and discussion Anchor groups for subsequent reactions at mild, physiological conditions can be introduced in colloidal polymer particles by copolymerisation with monomers containing primary amino groups. Even if the relative amount of amino groups required for further reactions is, with only a few mole percent, rather low, amines can significantly affect the colloidal stability of the resulting particles: emulsion polymerisation yields electrostatical stabilisation due to the sulfonic acid groups originating from the radical starter peroxodisulfate. The dissociation of the terminal sulfonic acids attached to the polymer particles produces negatively charged colloidal macroions. Since peroxodisulfate is added in catalytic amounts only, the total number of sulfonic acids per colloidal particles is rather low, typically some thousand groups per particle. Upon copolymerisation with 2-amino-ethylmethacrylathydrochloride (AEMA), however, in addition primary amines as Brønsted bases are present in sufficient density on the surface of the colloidal particles. The protons resulting from the sulfonic acids form ammonium groups with the primary amines leading to colloidal betaines. This intraparticle reaction significantly reduces the overall number of charges per particle and thus the colloidal stability of the system, with the consequence of agglomerate formation already during the polymerisation process. This destabilisation is prevented by the introduction of a surplus of sulfonic acids and hence negative charges what can be achieved by the additional copolymerisation of vinylbenzenesulfonate (VBS). The increasing colloidal stability with increasing ratio of VBS to AEMA is evident from the decreasing hydrodynamic diameter and the narrowing size distribution of the particles (i.e., better colloidal stabilisation already during the polymerisation process) as displayed in Fig. 1. If all amino groups are compensated by a surplus of sulfonic acids, with
M. Schmitt et al. / Journal of Colloid and Interface Science 311 (2007) 425–429
Fig. 1. The particle size distribution in dependence on the molar ratio of VBS to AEMA. To illustrate the distribution the 0.05, 0.25, 0.5, 0.75, and 0.95 quantiles are also plotted. With the increase of the ratio the size distributions became narrower and the size seems to trend against a limit.
Fig. 2. The pH dependence of the zeta potential of particles without and with VBS in a molar ratio 1.05 to the amine, both for unlabeled and fluorescently labeled particles. While the sample without VBS is nearly uncharged, with a small surplus of VBS compared to amine a negative surface charge is observed. Hereby an electrostatic stabilization of the particles is achieved. Due to the transformation of primary amines as possible proton acceptors into thiourea derivates during the coupling reaction, the ζ -potential becomes slightly more negative.
σ = 75 nm a limit of the hydrodynamic diameter is reached: it does not decrease any further. The compensation of the negative charges resulting from the radical starter by the formation of ammonium groups is also evident from the zeta potential ζ in Fig. 2. Without additional sulfonic acids introduced by VBS, the zeta potential is close to 0 mV indicating essentially uncharged particles. With increasing molar ratio of VBS to AEMA, however, the number of negative charges increases as is evident from the negative zeta potential in the whole range of 2 < pH < 10. Furthermore, by the bifunctional ethyleneglycol-dimethacrylate, a cross-linking of polymer chains is achieved that prevents swelling of the polymer particles and thus stabilizes their size and form as evident from TEMicrographs showing spherical particles. After the coupling reaction, residual amino groups can be deactivated: with a surplus of ethylisothiocyanate the remaining amino groups are quantitatively transformed into inert ethyl-thiourea compounds.
427
Fig. 3. Normalized absorption and fluorescence spectrum of a labelled polymer suspension. The polymer suspension shows the same absorption and fluorescence maxima as the pure fluoresceine. The small shoulder of the emission spectrum results from the incident excitation light at 480 nm.
The pH dependence of the zeta potential of the labelled particles is also analysed (Fig. 2). As expected, by transfomation of primary amines to thiourea, the number of proton acceptors in the particles is decreased. Hereby, the dominance of the dissociating sulfonic acids is enhanced as reflected by a more negative zeta potential of the colloidal anions. To study the effect of immobilization on the properties of the fluorescence dye, UV/vis spectra of a labelled colloidal suspension are measured in absorption and in emission (Fig. 3). From the observation that the dye has the same shift and maxima as in the unlinked free molecular form we can conclude that the photochemical properties of the dye are not changed by the coupling to colloidal macroions. It was also possible to use the labelled particles in tracer experiments to measure the self-diffusion coefficient at intermediate times. For these experiments a fluorescence correlation experiment as described by Madge et al. [15] is used. The incident beam of an ionized argon laser operating at 488 nm is attenuated to 20 µW before it is focused by a water immersion objective (63×, NA1.2) to the sample. For the experiment a sample containing a volume fraction φ = 10−2 of colloidal particles is used. In order to remove stray ions screening electrostatic interactions between the particles, the sample was completely deionized employing a mixed-bed ion exchanger resin. The fluorescence signal originating from the sample is observed through a dicroic mirror, a bandpass filter (HQ 525/50 purchased from HHF Analysetechnik, Tübingen, Germany) for suppression of residual excitation light and a pinhole of 50 µm in diameter to enable confocal detection. In order to eliminate dead-time effects and after-pulsing, the fluorescence intensity is detected by two avalanche photodiodes. Both signals are cross-correlated employing a FLEX D02 correlator purchased from correlator.com, Bridgewater, NJ (USA). The dimension of the focal volume is determined by a 10−9 mol/L solution of rhodamine 6G with the self-diffusion coefficient D = 2.80×10−10 m2 s−1 . Hereby, for the half axes of the illuminated ellipsoid of revolution, 166 nm for the transversal size and around 2000 nm for the longitudinal size with respect to the laser beam are obtained.
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Under the assumption of an ellipsoid of revolution as illuminated sample volume, the autocorrelation function can be described as g2 (t) = 1 +
1 Gtr (t)Gdiff (t). N
(1)
The contrast is inversely proportional to the number of labelled particles N in the illuminated sample volume. The factor Gtr (t) equals 1 in very good approximation, since several dye molecules on a single colloidal particles undergo uncorrelated optical transitions. The factor Gdiff (t) accounting for the Brownian motion can be written as [20] −1/2 t −1 t Gdiff (t) = 1 + 1+ 2 τD f τD
(2)
with f = z0 /r0 denoting the ratio of half axes in longitudinal and transversal direction. This expression is valid, if fluorescence fluctuations solely originate from diffusion, i.e., when photochemical reactions like triplett transitions can be neglected in the time scale of interest. The self-diffusion coefficient DS , finally, is related to the characteristic diffusion time τD by τD = r02 /(4DS ). Normally, due to the magnitude of f ≈ 10, the decay is dominated by the first factor of Eq. (2) describing the diffusion in transversal direction with respect to the laser. In Fig. 4, a typical correlation function resulting from an FCS experiment is displayed. From the intercept [g2 (t = 0) − 1]−1 ≈ 6 the presence of 6 fluorescently labelled particles on average in the detection volume can be seen. From the leastsquares fit in Fig. 4, the characteristic time τD = 49 ms can be extracted. This corresponds to a self-diffusion coefficient of DS = 2.315 × 10−13 m2 s−1 that is much lower than the free diffusion coefficient approximated by the Stokes–Einstein diffusion coefficient DSE = 6.76 × 10−13 m2 s−1 as determined by PCS from a noninteracting, highly dilute suspension in presence of 10−3 mol/L KCl to screen electrostatic interactions. The latter diffusion coefficient corresponds according to the Stokes–Einstein equation DSE = kB T /(6πηR) to a hydrodynamic particle radius of R = 64 nm in a water–glycerol mixture (η = 5.01 × 10−3 Pa s at T = 298 K). The temperature was kept constant during the experiments with an accuracy better than 1 K. For short times, diffusing particles do not significantly change the distance to neighbour particles. As consequence, the potential energy does not significantly change: the particles can freely move. This regime of free diffusion can be characterized by the short-time self-diffusion coefficient DS0 that is in good approximation identical to the Stokes–Einstein diffusion coefficient. In intermediate times, the particle tries to approach its neighbors more and more by a random walk. Hereby its potential energy significantly increases due to the electrostatic repulsion of its neighbours: the particle preferably is repelled to its initial position. This memory effect induces a subdiffusive process that formally can be described by a time-dependent diffusion coefficient DS (t).
Fig. 4. Typical correlation function obtained from a labelled colloidal suspension. The intercept is inversely proportional to the mean number of labelled particles in the excitation volume. From the least squares fit the self diffusion coefficient DS = 2.315 × 10−13 m2 s−1 is obtained.
From time to time, however, in a fluid system a central particle can escape from the cage of surrounding particles. By average over many escape processes, again a diffusive process takes place. In this regime again a time-independent self-diffusion coefficient describes the random walk of interacting particles [21,22]. The stronger the particle–particle interactions are, the lower is the long-time self-diffusion coefficient DSL compared to the free diffusion coefficient DS0 . The ratio of DS0 /DSL = 0.1 is a commonly used dynamic freezing criterion. The diffusion coefficient obtained by FCS is by a factor of 0.34 lower than the Stokes–Einstein diffusion coefficient determined by PCS. This tremendous decrease reflects the slowing down of diffusion in interacting systems due to memory effects. 4. Conclusions Primary amino groups have successfully been used as reactive anchor groups for functionalization of colloidal polymer particles. Colloidal stability is maintained by incorporation of a surplus of sulfonic acid groups and thus a surplus of negative surface charges. Using isothiocyanates, the amino groups have under mild conditions been transformed into thermodynamically stable thiourea compounds. As an example of this coupling reaction polymer particles are labelled with a fluorescence dye and used as tracers in a study of colloidal selfdiffusion by means of fluorescence correlation spectroscopy. By FCS, the self-diffusion coefficient at intermediate time-scales in between the short-time limit DS0 and the long-time limit DSL can be measured. By changing the size of the excitation volume the time-scale under investigation can be varied. For a systematic study of the time dependence of the self-diffusion coefficient, further experiments are in progress. References [1] P.N. Pusey, Colloidal suspensions, in: Liquids, Freezing and Glass Transition, Elsevier, Amsterdam, 1991, pp. 765–942. [2] R.J. Hunter, Foundations of Colloid Science, Clarendon Press, Oxford, 1992.
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[3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
P.N. Pusey, J. Phys. A 11 (1978) 119. H.W. Elias, Macromoleküle, VCH, Weinheim, 1999. W. Härtl, H. Versmold, J. Chem. Phys. 80 (1984) 1387. J. Wagner, W. Härtl, C. Lellig, R. Hempelmann, H. Walderhaug, J. Molec. Liquids 98 (2002) 183. G. Nägele, Phys. Reports 272 (1996) 215–372. M.O. Robbins, K. Kremer, G.S. Grest, J. Chem. Phys. 88 (1988) 3286. H. Löwen, G. Szamel, J. Phys. Condens. Matter 5 (1993) 2295. W. Härtl, J. Wagner, Ch. Beck, F. Gierschner, R. Hempelmann, J. Phys. Condens. Matter 12 (2000) A287. E.J.W. Verwey, J.T.G. Overbeek, Theory of the Stability of Lyophobic Colloids, Elsevier, Amsterdam, 1948. J. Wagner, W. Härtl, H. Walderhaug, J. Chem. Phys. 114 (2001) 975.
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[13] A. Minelli, C. Allegrucci, P. Piomboni, R. Mannucci, C. Lluis, R. Franco, J. Histochem. Cytochem. 48 (2000) 1163. [14] F. You, Y. Zhou, X. Zhang, Z. Huang, L. Bi, Z. Zhang, J. Wen, Anal. Chem. 78 (2006) 7138. [15] D. Magde, E. Elson, W.W. Webb, Phys. Rev. Lett. 29 (1972) 705. [16] O. Krichevsky, G. Bonnet, Rep. Prog. Phys. 65 (2002) 251. [17] C. Lellig, J. Wagner, R. Hempelmann, S. Keller, D. Lumma, W. Härtl, J. Chem. Phys. 121 (2004) 7022. [18] S.W. Provencher, Comput. Phys. Comm. 27 (1982) 229. [19] S.W. Provencher, Comput. Phys. Comm. 27 (1982) 523. [20] J. Riˇcka, T. Binkert, Phys. Rev. A 39 (1989) 2646. [21] B. Cichocki, K. Hinsen, Physica A 166 (1990) 473. [22] B. Cichocki, K. Hinsen, Physica A 187 (1992) 133.
Functionalized polymer colloids bearing primary amino groups M. Schmitt a , J. Wagner a,∗ , G. Jung b , R. Hempelmann a a Physical Chemistry, Saarland University, 66123 Saarbrücken, Germany b Biophysical Chemistry, Saarland University, 66123 Saarbrücken, Germany
Received 28 November 2006; accepted 14 March 2007 Available online 20 March 2007
Abstract Polymer colloids are prepared via radicalic emulsion polymerisation of butylacrylate. Functionalization with amino groups is achieved by copolymerisation of 2-amino-ethylmethacrylates. In order to over-compensate the positive surface charges resulting from the amino groups additionally vinylbenzenesulfonic acid is copolymerized. The size of the resulting particles is controlled by the molar ratio of amino to sulfonic acid groups. The suitability of amino groups for coupling reactions is demonstrated by electrophilic addition of fluorescein-5-isothiocyanate. The resulting particles are characterized by dynamic light scattering and zeta potential measurements as well as by optical spectroscopy. The suitability of labelled particles for optical tracer experiments is demonstrated by fluorescence correlation spectroscopy. © 2007 Elsevier Inc. All rights reserved. Keywords: Emulsion polymerisation; Polymer colloids; Fluorescence labelling
1. Introduction During the last decades colloidal systems have been attracting considerable scientific interest both for fundamental and applied research [1–3], particularly due to the availability of nearly monodisperse nanoscale particles with the capability of self-organisation [4–7]. In aqueous suspension most of the systems are electrostatically stabilized [8–10]. For polymer colloids, surface charges in many cases result from sulfonic acids introduced by the radical initiator potassium peroxodisulfate. These macroions interact via a screened Coulomb or Yukawa potential [11]. The screening length strongly depends on the ionic strength of the system. As consequence, even small amounts of stray ions can destroy a long-range electrostatic repulsion and induce irreversible agglomeration when short range attractive dispersion forces dominate [12]. This sensitivity to ions is an obstacle to standard aqueous chemistry. Hence, reactions suitable to modify colloidal particles have to avoid the presence of charged species. The presence or generation of ions can be circumvented by the nucleophilic addition of amines to isothiocyanates that is commonly used in * Corresponding author.
E-mail address: [email protected] (J. Wagner). 0021-9797/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2007.03.026
biochemistry. This reaction leading to stable thiourea derivates can be carried out under mild, physiological conditions. In this way, e.g., fluorescence dyes are attached to biological macromolecules for labelling in biology or biomedicine [13,14]. In colloidal physics, fluorescently labelled particles are a prerequisite for optical tracer experiments like fluorescence correlation spectroscopy [15,16]. This method gives an access to self-diffusion coefficients of colloidal particles on a mesoscopic time scale [17]. The self-diffusion coefficient of interacting particles becomes, due to memory effects, time-dependent in intermediate scales of time. Memory effects induce a decrease of the self-diffusion coefficient. The dimensions of the focal volume in an FCS experiment, however, typically equal several times the particles’ diameter and thus the next neighbour distances in colloidal suspensions. As consequence, FCS data of interacting particles are highly influenced by memory effects. Here we report about the emulsion copolymerisation of butylacrylate with 2-amino-ethylmethacrylate providing primary amines as anchor groups. The colloidal stability is preserved by additional copolymerisation of vinylbenzene-sulfonate introducing additional negative charges. Swelling of the particles in aqueous media can be avoided by crosslinking with bifunctional ethyleneglycol-dimethacrylate. By the copolymerization of butylacrylate with three monomers bearing functionalities, stable suspensions of polymer particles with narrow size dis-
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tribution can be prepared. The feasibility of these particles for coupling reactions with isothiocyanates is demonstrated by the addition of fluorescein-5-isothiocyanate. Hereby, nearly monodisperse, fluorescently labelled particles can be prepared. Since the dye is chemically attached as a thiourea derivate, a bleeding out typical for physically adsorbed dyes can be prevented. We demonstrate the use of these labeled particles for tracer diffusion experiments by fluorescence correlation spectroscopy. 2. Experimental 2.1. Materials The chemicals used for the synthesis of polymer colloids are butylacrylate (BA, C7 H12 O2 ), 4-vinylbenzenesulfonic acid sodium salt (VBS, C8 H7 NaO3 S), FeSO4 ·7H2 O, Na2 SO3 , and K2 S2 O4 supplied by Fluka Chemie AG, ethylenglycoldimethacrylate (EMA, C10 H14 O4 ), fluorescein-5-isothiocyanate (FITC, C21 H11 NO5 S), and ethylisothiocyanate (ECI, C3 H5 NS) supplied by Merck KGaA, and 2-amino-ethylmethacrylathydrochloride (AEMA, C6 H12 O2 NCl) supplied by ACROS Organics. For dialysis a Medicell International Ltd. membrane size 5 Inf Dia 36/32 —28.6 mm 30 M was used. Deionised water was the solvent for all experiments. 2.2. Emulsion polymerization process For the preparation of nanoscale polymer particles suspended in water first atmospheric oxygen has to be removed; for that purpose, 750 ml deionised water is stirred in nitrogen atmosphere at 80 ◦ C for several hours. Then, keeping the temperature constant, a mixture of monomers is added: 120 mmol BA, 2.34 mmol EMA, up to 5.84 mmol VBS, and 1.17 mmol AEMA. After stirring for one hour, the polymerisation is induced by addition of the redox catalysts sodium sulfite and iron(II) sulfate and by the addition of the radical starter potassium peroxodisulfate. The reaction progress is indicated by increasing turbidity. After 24 h, the resulting suspension is filtrated and dialysed against distilled water for typically one week in order to remove residual monomers and low molecular weight oligomers. 2.3. Fluorescence labelling Starting material is the copolymer, prepared as described above, with about one mol-percent of AEMA. For the fluorescence labelling 90 µl solution of 0.02 mmol FITC in DMF is added to 25 ml of the colloidal suspension containing less than 0.04 mmol amino groups at 95 ◦ C. Subsequently, the remaining primary amino groups are eliminated by reaction with a surplus of EIC. The product is dialysed against deionized water for several days, until free dye molecules are completely removed. 2.4. Particle size determination The particle size is determined by means of dynamic light scattering (DLS) using a ALV-5000E set-up purchased from
ALV, Langen (Germany), equipped with a 35 mW He/Nelaser purchased from Coherent Inc. For the experiments, dilute translucent samples are used; 10−3 mol/L KCl is added to screen the electrostatic interaction and to exclude any short range order in the colloidal system. The particle size distribution is obtained by the regularization method proposed by Provencher using the CONTIN algorithm [18,19]. 2.5. pH dependent zeta potential measurements The zeta potential ζ is determined employing a Zetasizer 3000 HSa and a Multi Purpose Titrator supplied by Malvern Instruments. Highly dilute samples containing 10−3 mol/L KCl are used for these experiments. 2.6. Optical measurements The absorption spectra are measured by means of a Perkin Elmer Lambda5 UV–vis spectrometer, and for the fluorescence spectra a Ocean Optics Inc. SD2000 fluorescence spectrometer equipped with LED illumination in excitation is used. 3. Results and discussion Anchor groups for subsequent reactions at mild, physiological conditions can be introduced in colloidal polymer particles by copolymerisation with monomers containing primary amino groups. Even if the relative amount of amino groups required for further reactions is, with only a few mole percent, rather low, amines can significantly affect the colloidal stability of the resulting particles: emulsion polymerisation yields electrostatical stabilisation due to the sulfonic acid groups originating from the radical starter peroxodisulfate. The dissociation of the terminal sulfonic acids attached to the polymer particles produces negatively charged colloidal macroions. Since peroxodisulfate is added in catalytic amounts only, the total number of sulfonic acids per colloidal particles is rather low, typically some thousand groups per particle. Upon copolymerisation with 2-amino-ethylmethacrylathydrochloride (AEMA), however, in addition primary amines as Brønsted bases are present in sufficient density on the surface of the colloidal particles. The protons resulting from the sulfonic acids form ammonium groups with the primary amines leading to colloidal betaines. This intraparticle reaction significantly reduces the overall number of charges per particle and thus the colloidal stability of the system, with the consequence of agglomerate formation already during the polymerisation process. This destabilisation is prevented by the introduction of a surplus of sulfonic acids and hence negative charges what can be achieved by the additional copolymerisation of vinylbenzenesulfonate (VBS). The increasing colloidal stability with increasing ratio of VBS to AEMA is evident from the decreasing hydrodynamic diameter and the narrowing size distribution of the particles (i.e., better colloidal stabilisation already during the polymerisation process) as displayed in Fig. 1. If all amino groups are compensated by a surplus of sulfonic acids, with
M. Schmitt et al. / Journal of Colloid and Interface Science 311 (2007) 425–429
Fig. 1. The particle size distribution in dependence on the molar ratio of VBS to AEMA. To illustrate the distribution the 0.05, 0.25, 0.5, 0.75, and 0.95 quantiles are also plotted. With the increase of the ratio the size distributions became narrower and the size seems to trend against a limit.
Fig. 2. The pH dependence of the zeta potential of particles without and with VBS in a molar ratio 1.05 to the amine, both for unlabeled and fluorescently labeled particles. While the sample without VBS is nearly uncharged, with a small surplus of VBS compared to amine a negative surface charge is observed. Hereby an electrostatic stabilization of the particles is achieved. Due to the transformation of primary amines as possible proton acceptors into thiourea derivates during the coupling reaction, the ζ -potential becomes slightly more negative.
σ = 75 nm a limit of the hydrodynamic diameter is reached: it does not decrease any further. The compensation of the negative charges resulting from the radical starter by the formation of ammonium groups is also evident from the zeta potential ζ in Fig. 2. Without additional sulfonic acids introduced by VBS, the zeta potential is close to 0 mV indicating essentially uncharged particles. With increasing molar ratio of VBS to AEMA, however, the number of negative charges increases as is evident from the negative zeta potential in the whole range of 2 < pH < 10. Furthermore, by the bifunctional ethyleneglycol-dimethacrylate, a cross-linking of polymer chains is achieved that prevents swelling of the polymer particles and thus stabilizes their size and form as evident from TEMicrographs showing spherical particles. After the coupling reaction, residual amino groups can be deactivated: with a surplus of ethylisothiocyanate the remaining amino groups are quantitatively transformed into inert ethyl-thiourea compounds.
427
Fig. 3. Normalized absorption and fluorescence spectrum of a labelled polymer suspension. The polymer suspension shows the same absorption and fluorescence maxima as the pure fluoresceine. The small shoulder of the emission spectrum results from the incident excitation light at 480 nm.
The pH dependence of the zeta potential of the labelled particles is also analysed (Fig. 2). As expected, by transfomation of primary amines to thiourea, the number of proton acceptors in the particles is decreased. Hereby, the dominance of the dissociating sulfonic acids is enhanced as reflected by a more negative zeta potential of the colloidal anions. To study the effect of immobilization on the properties of the fluorescence dye, UV/vis spectra of a labelled colloidal suspension are measured in absorption and in emission (Fig. 3). From the observation that the dye has the same shift and maxima as in the unlinked free molecular form we can conclude that the photochemical properties of the dye are not changed by the coupling to colloidal macroions. It was also possible to use the labelled particles in tracer experiments to measure the self-diffusion coefficient at intermediate times. For these experiments a fluorescence correlation experiment as described by Madge et al. [15] is used. The incident beam of an ionized argon laser operating at 488 nm is attenuated to 20 µW before it is focused by a water immersion objective (63×, NA1.2) to the sample. For the experiment a sample containing a volume fraction φ = 10−2 of colloidal particles is used. In order to remove stray ions screening electrostatic interactions between the particles, the sample was completely deionized employing a mixed-bed ion exchanger resin. The fluorescence signal originating from the sample is observed through a dicroic mirror, a bandpass filter (HQ 525/50 purchased from HHF Analysetechnik, Tübingen, Germany) for suppression of residual excitation light and a pinhole of 50 µm in diameter to enable confocal detection. In order to eliminate dead-time effects and after-pulsing, the fluorescence intensity is detected by two avalanche photodiodes. Both signals are cross-correlated employing a FLEX D02 correlator purchased from correlator.com, Bridgewater, NJ (USA). The dimension of the focal volume is determined by a 10−9 mol/L solution of rhodamine 6G with the self-diffusion coefficient D = 2.80×10−10 m2 s−1 . Hereby, for the half axes of the illuminated ellipsoid of revolution, 166 nm for the transversal size and around 2000 nm for the longitudinal size with respect to the laser beam are obtained.
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Under the assumption of an ellipsoid of revolution as illuminated sample volume, the autocorrelation function can be described as g2 (t) = 1 +
1 Gtr (t)Gdiff (t). N
(1)
The contrast is inversely proportional to the number of labelled particles N in the illuminated sample volume. The factor Gtr (t) equals 1 in very good approximation, since several dye molecules on a single colloidal particles undergo uncorrelated optical transitions. The factor Gdiff (t) accounting for the Brownian motion can be written as [20] −1/2 t −1 t Gdiff (t) = 1 + 1+ 2 τD f τD
(2)
with f = z0 /r0 denoting the ratio of half axes in longitudinal and transversal direction. This expression is valid, if fluorescence fluctuations solely originate from diffusion, i.e., when photochemical reactions like triplett transitions can be neglected in the time scale of interest. The self-diffusion coefficient DS , finally, is related to the characteristic diffusion time τD by τD = r02 /(4DS ). Normally, due to the magnitude of f ≈ 10, the decay is dominated by the first factor of Eq. (2) describing the diffusion in transversal direction with respect to the laser. In Fig. 4, a typical correlation function resulting from an FCS experiment is displayed. From the intercept [g2 (t = 0) − 1]−1 ≈ 6 the presence of 6 fluorescently labelled particles on average in the detection volume can be seen. From the leastsquares fit in Fig. 4, the characteristic time τD = 49 ms can be extracted. This corresponds to a self-diffusion coefficient of DS = 2.315 × 10−13 m2 s−1 that is much lower than the free diffusion coefficient approximated by the Stokes–Einstein diffusion coefficient DSE = 6.76 × 10−13 m2 s−1 as determined by PCS from a noninteracting, highly dilute suspension in presence of 10−3 mol/L KCl to screen electrostatic interactions. The latter diffusion coefficient corresponds according to the Stokes–Einstein equation DSE = kB T /(6πηR) to a hydrodynamic particle radius of R = 64 nm in a water–glycerol mixture (η = 5.01 × 10−3 Pa s at T = 298 K). The temperature was kept constant during the experiments with an accuracy better than 1 K. For short times, diffusing particles do not significantly change the distance to neighbour particles. As consequence, the potential energy does not significantly change: the particles can freely move. This regime of free diffusion can be characterized by the short-time self-diffusion coefficient DS0 that is in good approximation identical to the Stokes–Einstein diffusion coefficient. In intermediate times, the particle tries to approach its neighbors more and more by a random walk. Hereby its potential energy significantly increases due to the electrostatic repulsion of its neighbours: the particle preferably is repelled to its initial position. This memory effect induces a subdiffusive process that formally can be described by a time-dependent diffusion coefficient DS (t).
Fig. 4. Typical correlation function obtained from a labelled colloidal suspension. The intercept is inversely proportional to the mean number of labelled particles in the excitation volume. From the least squares fit the self diffusion coefficient DS = 2.315 × 10−13 m2 s−1 is obtained.
From time to time, however, in a fluid system a central particle can escape from the cage of surrounding particles. By average over many escape processes, again a diffusive process takes place. In this regime again a time-independent self-diffusion coefficient describes the random walk of interacting particles [21,22]. The stronger the particle–particle interactions are, the lower is the long-time self-diffusion coefficient DSL compared to the free diffusion coefficient DS0 . The ratio of DS0 /DSL = 0.1 is a commonly used dynamic freezing criterion. The diffusion coefficient obtained by FCS is by a factor of 0.34 lower than the Stokes–Einstein diffusion coefficient determined by PCS. This tremendous decrease reflects the slowing down of diffusion in interacting systems due to memory effects. 4. Conclusions Primary amino groups have successfully been used as reactive anchor groups for functionalization of colloidal polymer particles. Colloidal stability is maintained by incorporation of a surplus of sulfonic acid groups and thus a surplus of negative surface charges. Using isothiocyanates, the amino groups have under mild conditions been transformed into thermodynamically stable thiourea compounds. As an example of this coupling reaction polymer particles are labelled with a fluorescence dye and used as tracers in a study of colloidal selfdiffusion by means of fluorescence correlation spectroscopy. By FCS, the self-diffusion coefficient at intermediate time-scales in between the short-time limit DS0 and the long-time limit DSL can be measured. By changing the size of the excitation volume the time-scale under investigation can be varied. For a systematic study of the time dependence of the self-diffusion coefficient, further experiments are in progress. References [1] P.N. Pusey, Colloidal suspensions, in: Liquids, Freezing and Glass Transition, Elsevier, Amsterdam, 1991, pp. 765–942. [2] R.J. Hunter, Foundations of Colloid Science, Clarendon Press, Oxford, 1992.
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