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Carboxylated Latexes for Covalent Coupling Antibodies, I
JOURNAL OF COLLOID AND INTERFACE SCIENCE
176, 232–239 (1995)
Carboxylated Latexes for Covalent Coupling Antibodies, I D. BASTOS-GONZA´LEZ, J. L. ORTEGA-VINUESA, F. J. DE LAS NIEVES,
AND
´ LVAREZ 1 R. HIDALGO-A
Biocolloid and Fluid Physics Group, Department of Applied Physics, University of Granada, 18071 Granada, Spain Received December 12, 1994; accepted April 7, 1995
The aim of the present work is to prepare and characterize carboxylated latexes (CLs) in order to bind antibodies covalently and efficiently. Carboxylated latexes were prepared by surfactantfree emulsion polymerization using an azo-initiator, which provides carboxyl end groups on the latex surface directly. Two latexes were characterized using different techniques: particle size by electron microscopy and photocorrelation spectroscopy, surface charge density by conductimetric and potentiometric titrations, and electrophoretic mobility versus pH and ionic strength (KBr). The colloidal stability of both latexes shows that these carboxylated latexes can be used for biomedical applications. Activation of carboxyl end groups was performed using the carbodiimide method, and the best conditions for covalent coupling of IgG were obtained. q 1995 Academic Press, Inc.
Key Words: polymer colloids; electrokinetics.
polymerization are negatively charged in a broad pH range of 3–10 (4), but their surface charge density is extremely high (4). At a low ionic strength the antibody cannot penetrate the double layer of the particle and thus, no coupling occurs (11). The carboxylated latexes prepared by an azoinitiator have a rigid interface and the surface charge density is significantly lower. These types of particles have been used less in covalently coupling antibodies and antigens to polymer carriers in the development of particle-enhanced optical immunoassays. In this paper, we report the preparation of surfactant-free carboxylated polymer latexes which enables the covalent coupling of antibodies with a high efficiency rate. The electrokinetic properties and colloidal stability of these latexes are also studied. II. EXPERIMENTAL
I. INTRODUCTION
In recent years, a great deal of attention has been given to the use of polymer colloids on a large scale in biomedical applications (1–5) for a number of reasons. First, such polymer colloids are easy to produce and can be prepared with a high degree of monodispersity (6). Second, they can be used in separating biomacromolecules and immunoassays because of their good biocompatibility (7). The application of such polymer colloids requires control of the surface chemical properties of microparticles and control of the process by which they are synthesized (8–10). There are numerous references concerning the synthesis of polystyrene latexes prepared in the absence of emulsifiers in order to produce particles with carboxyl end groups on the particle surface, which result from the use of an ionic comonomer, such as acrylic acid (6–10). In this case, hydrous polyacrylic (or polymethacrylic) acid layers exist on the surfaces and shift the shear plane away from the particle surfaces. This probably leads to a hairy interface, which could hinder the approach of proteins to the latex surface. Antibodies can be covalently coupled if they can reach the particle surface and if appropriate conditions exist for the coupling process. The carboxylated latexes prepared through ionic comonomer 1
To whom correspondence should be addressed.
0021-9797/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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A. Material Styrene monomer was obtained from Merck and was distilled at reduced pressure and at 407C. The purified monomer was stored at 057C until required. The initiator used in this work was the 4,4 *-azobis(4-cyanopentanoic acid) (ACPA) from Aldrich and was used as received. Potassium persulfate, sodium bicarbonate, sodium chloride, hydrochloric acid, and sodium hydroxide (all from Merck) were analytical grade reagents and were used without further purification. Doubledistilled and deionized (DDI) water was used throughout. B. Preparation of Latexes To prepare carboxylated latexes we have followed the steps developed by Guthrie (12) of surfactant-free emulsion polymerization of styrene with ACPA as the initiator. This method has the advantage that carboxyl end groups on the particle surface come from the initiator molecules. Table 1 shows the synthesis conditions used throughout this work. The materials were poured into a 600 cc three-necked glass flask and polymerized under a nitrogen atmosphere. To maintain continuous vigorous stirring, the T-shaped stirrer (1 1 5 cm) was fitted 1 cm from the bottom of the flask. Particle size was obtained using two different methods: (i) transmission electron microscopy (TEM) and (ii) photo-
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CARBOXYLATED LATEXES FOR COVALENT COUPLING, I
TABLE 1 Polymerization Recipe Used in the Carboxylated Polystyrene Latexes Latex JL4 DJL5
Styrene ACPA NaOH Stirring Temperature Reaction time (h) (g) (mM) (mM) (rpm) (7C) 25 25
2.76 0.79
7.43 3.34
350 350
80.6 80.6
10 20
correlation spectroscopy (PCS) (Malvern 4700 System). Results taken from both methods were very similar. The characteristics of the bare polymer colloids are shown in Table 2. The latexes were considered to be monodisperse since the polydispersity index, PDI (defined as DW /DN , where Dw and Dn are the weight-average and the numberaverage diameters, respectively), was less than 1.05 (13). Figure 1 shows the particle size distribution of both samples obtained by TEM. More than approximately 500 particles were counted for each latex. C. Cleaning Process and Surface Characterization A long and comprehensive cleaning process was followed before measuring the surface charge density of these latexes. First, the latexes were filtered through glass wool, then they underwent repeated cycles of centrifugation–decantation– redispersion, and finally, they were cleaned using serum replacement with DDI water. After these processes were completed the specific electrical conductivity of the latexes was found to be constant at approximately 5 mS cm01 . Surface charge densities of JL4 and DJL5 latexes were determined by conductimetric and potentiometric automatic titrations employing a pH-meter (Crison Instruments, Model 2002) and a conductimeter (Crison Instruments, Model 525), and a Dosimat 665 (Metrohm) to add the titrant agent. Figure 2 shows the potentiometric and conductimetric back titration for the JL4 latex. It is not possible to know the total surface charge densities ( s0 ) from direct titrations in latexes with weak acid groups on their surface, as it is necessary to have protonated all these groups (COOH), which occurs at pH’s around 3, and under these conditions the colloidal system would be completely aggregated. This is why we have employed back titrations to obtain the s0 values, which are
FIG. 1. Size distribution for JL4 and DJL5 latexes.
also shown in Table 2. The presence of three different slopes in the conductimetric curve is caused by: (i) the titration of the excess of NaOH previously added (since this plot represents a back-titration), (ii) the titration of the carboxyl groups of the latex, and (iii) the last slope is caused by the excess of titration agent (HCl). Knowing the total surface of added latex, the HCl concentration, and the total volume of this solution consumed in the titration of the weak acid groups of the polymer surface, one can calculate s0 . We can see in Fig. 2 that the changes in the slopes in the conductivity curve coincide with the inflection points of the potentiometric one. The electrophoretic mobilities of these cleaned latexes were measured at 257C under different ionic strengths (KBr as the electrolyte) and pH conditions using a Zeta-Sizer IV (Malvern Instrument). The electrophoretic mobility values
TABLE 2 Characteristics of Carboxylated Polystyrene Latexes Diameter PCS (nm)
Diameter TEM (nm)
PDI
s0 (mC/cm2)
JL4
342 { 7
331 { 11
1.002
12.1 { 0.5
DJL5
278 { 5
276 { 12
1.005
16.3 { 0.7
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d log W/d log Ce 2.07 2.06 2.47 2.03
(pH (pH (pH (pH
5) 7) 5) 7)
CCC (mM KBr) 360 780 350 590
(pH (pH (pH (pH
5) 7) 5) 7)
´ LEZ ET AL. BASTOS-GONZA
234
(dA/dt)s is the initial slope for lower electrolyte concentrations). Log W values were then plotted versus log Ce . The slopes and the CCC values from these curves are given in Table 2. E. Protocol for Activation of Carboxylated Latexes Using the Carbodiimide (CDI) Method
FIG. 2. Conductimetric and potentiometric back titration for the JL4 latex.
were obtained by taking the average of (at least) five measurements at the stationary level in a cylindrical cell. The experimental error was taken as the standard deviation of these measurements. For the data shown throughout this paper the standard deviation was always lower than {0.2 1 10 08 m2 V 01 s 01 . Samples were prepared by adding approximately 0.01 ml of the latex to 10 ml of each electrolyte solution. The pH’s were controlled using different buffers (acetate at pH’s 4 and 5, phosphate at pH’s 6 and 7, and borate at pH’s 8 and 9), keeping the ionic strength at a constant value of 0.002 M. D. Measurement of Stability Ratios A Milton Roy Spectronic 601 spectrophotometer was used in conjunction with a PC. In a typical coagulation experiment, 2.4 ml of a buffered latex solution was put into the spectrophotometer cell and the optical absorbance was measured. Potassium bromide (0.6 ml) of a given concentration was added quickly to the sample through a syringe. The final particle concentration in the cell was 10 10 part/ml. The optical absorbance (A) was measured immediately and its change versus time (t) was recorded continuously for a period of 30 s. One typical curve of this response is given in Fig. 3 for the DJL5 latex. Curves were found to be linear in the early stages of coagulation at all electrolyte concentrations. The initial slopes were directly proportional to the initial coagulation rate. These slopes increased as the electrolyte concentration increased until a maximum was reached. From this concentration (called the critical coagulation concentration, CCC) the initial slopes did not increase with the addition of higher amount of salt. The slope at the CCC was taken as the fast coagulation rate of the latex. The factor stability (W ) values for each electrolyte concentration were calculated from W Å (dA/dt) f /(dA/dt)s (where (dA/dt) f is the initial slope in fast coagulations and
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A latex surface of 0.3 m2 was added to 1 ml of buffer solution pH 5.6 or pH 8.0 at low ionic strength. A pH value of 5.6 was obtained using MES (2-[N-morpholino]ethanesulfonic acid) instead of acetate in order to guarantee that only the carboxylic groups that are going to be activated are those that are on the latex surface. To the above volume, 200 ml of 15 mg/ml CDI (1 ethyl-3-(3-dimethyl aminopropyl)carbodiimide) in water was added. This gave a CDI concentration of 10 mg/m 2 of latex. Samples were incubated at room temperature for 40 min. The samples were centrifuged for 20 min at 28,000g. The supernatant was removed, and the particles were resuspended in 1.2 ml of the same buffer, and the new suspension was briefly sonicated. The excess carboxylic acid groups were blocked by treatment with ethanolamine for 10 min at room temperature. A similar effect was obtained when the treatment with ethanolamine lasted 15 h. III. RESULTS AND DISCUSSION
A. Electrokinetic Characterization The electrophoretic mobilities of these latexes were measured to characterize the electric double layer surrounding the particles. The electrophoretic mobility values for samples JL4 and DJL5 were measured as a function of pH at a constant ionic strength of 0.002 M KBr. The results given
FIG. 3. Optical absorbance versus time for the JL4 latex. Electrolyte concentration (Ce ) 0.6 M.
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FIG. 4. Electrophoretic mobility ( me ) versus pH for both latexes: l, JL4 latex; l, DJL5 latex.
FIG. 5. Potentiometric titration to obtain s0 versus pH: j, blank; j, blank with JL4 particles.
in Fig. 4 show that the me values were almost constant when the pH was increased from 6 to 10 with a pronounced decrease at a lower (acid) pH. It is possible to recognize the weak acid character of the surface end groups of these latexes from this figure. We have used two procedures to calculate the pKa of our latexes, where Ka is the dissociation constant. The first one is a simplified procedure that has been developed by Ottewill and Shaw (14). From this procedure, it may be demonstrated that
values of me for both samples, even when the surface charge density ( s0 ) varied from 12.1 (JL4) to 16.3 (DJL5) mC cm02 (i.e., an increase of 35%). The averages of the me values for pHs between 6 and 10 were ( 110 08 m2 V 01 s 01 ) meJL4 Å 04.4 { 0.1 and meDJL5 Å 04.3 { 0.1, where the error was the standard deviation obtained from the mean me values at each pH. Thus, the difference between the me values for both latexes are within the experimental error. However, it can be expected that the mobility of the most charged latex (DJL5) would be higher. This result is contrary to the predictions of the classical electrical double-layer models. Some authors (18, 19) have explained this anomalous electrokinetic behavior on the basis of the surface conductance between the shear plane and the particle surface. Since the surface conductance is directly proportional to the surface charge density, it could account for the disagreement between the s0 and me values. Moreover, in Fig. 6 we can observe that in the range from pH 9 to 6 s0 decreases, and
pKa Å pHz Åz plateau / 2 / 0.4343(Fzplateau )/2RT.
[1]
On the other hand, the surface charge densities of the polymer latexes as a function of pH can be determined from the results of the potentiometric titrations (15–17). This dependence of s0 on pH can be obtained from the titration of a blank of NaOH without latex particles and the titration of the same solution with latex. The difference between the two curves, at a given pH, informs us about the volume of the titrant agent (HCl), with which s0 can be calculated at that pH. As the pH becomes more acidic the difference between the two curves, and therefore s0 , decreases (Fig. 5). This method allows us to calculate the pKa from the mass law: pKa Å pH / log[R–COOH]/[R–COO 0 ].
[2]
pKa will coincide with the pH where [R–COOH] Å [R– COO 0 ] and therefore the surface charge density will be equal to 1/2 of the maximum s0 . The pKa calculated by this procedure was 5.8 { 0.3 and using the former procedure we have obtained a pKa value of 5.7 { 0.1. In Fig. 6 we have shown the dependence of the zeta-potential ( z ) and s0 on pH. One remarkable result observed in Fig. 4 is the similar
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FIG. 6. Dependence of z and s0 on pH for latex JL4: ., z ; l, s0 .
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FIG. 7. Electrophoretic mobility ( me ) versus electrolyte concentration (KBr) for both carboxylated latexes: l, JL4 latex; l, DJL5 latex.
z remains constant. In other words, although the value of me remains constant between pH 9 and 6, the s0 value decreases by almost 50%. Electrophoretic mobility measurements on samples JL4 and DJL5 were carried out at different electrolyte concentrations. These results are shown in Fig. 7. These curves confirm our previous results from Fig. 4, showing that there is no significant difference between the me values for both samples, within the experimental error. The big difference between the surface and the electrokinetic potentials made the electrokinetic potential largely insensitive to changes in the surface charge density (20). Various qualitative explanations have been proposed to account for the maximum which appears in Fig. 7. A preferential adsorption of co-ions onto the surface was invoked by several authors (21, 22). The existence of polyelectrolyte chains on the surface of polymer
FIG. 8. Log W versus log Ce at pH 5. The JL4 latex is shown on the left side and the DJL5 latex on the right side.
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colloids is considered in the hairy layer model (23). Physical changes in the particle surface properties were also invoked by other authors (24). In the last case, the authors consider that the shift of the shear plane into the bulk solution is due to the surface roughness or to the existence of polyelectrolyte chains on the surface of latex particles. This displacement of the shear plane results in a decrease of the mobility in the usual way and lowers it to a much larger extent by introducing ionic conduction (or surface conductance) between the solid surface and the shear plane. The surface conductance is especially important at a low ionic strength and reduces the me values by a much greater percentage (25). It must be noted that when the carboxylated latexes are prepared by copolymerization of styrene and by an ionic comonomer (acrylic and methacrylic acids, for instance), a hydrous polyacrylic acid layer exists on the surface, preventing the approach of antibodies or antigens to the polymer surface. In some cases the thickness of these layers can even ˚ (4). In our case, using the same procedure as be 10 A Shirahama and Suzawa (4), the position of the shear plane ˚ from the polymer surface. This result is considered is 2.5 A to be experimental proof that the carboxylated latexes prepared by the ACPA initiator have a thinner solid–liquid interface layer than those prepared by an ionic comonomer polymerization. This difference is relevant for covalent coupling biomolecules to carboxylated latexes using the carbodiimide method. B. Colloidal Stability of Carboxylated Latexes As Reerink and Overbeek have shown (26), with several approximations, a linear relationship between log W and log Ce can be obtained,
FIG. 9. Electrophoretic mobility versus pH for the DJL5 latex activated by CDI: l, at pH 5.6; ., at pH 8.2; w, bare latex.
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FIG. 10. Covalent coupling reaction of a protein with a carboxylated latex; (i) first step: activation of the carboxyl group with CDI; (ii) second step: reaction of this activated group with an amine of the protein. This last functional group could be a terminal amine or it could come from lysine amino acids.
log W Å 0k * log Ce / log k 9 ,
[3]
where k * and k 9 are constants and Ce is the electrolyte concentration. Thus, W decreases with the addition of electrolyte until the electrolyte concentration reaches a critical value above which the energy barrier between particles is lower than the thermal energy. The electrolyte concentration at which W becomes equal to 1 is called the CCC. The CCC is defined as the concentration of added electrolyte which is just enough to bring about diffusion-controlled rapid coagulation.
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Log W values have been plotted versus log Ce as suggested by Reerink and Overbeek (24). Figure 8 shows the stability curves of JL4 and DJL5 latexes for a 1:1 electrolyte (KBr), at pH 5. The linear relationship expected from Eq. [3] is confirmed in the two cases, as the correlation coefficients were higher than 0.98, with at least five experimental points. The CCC values were calculated and are shown in Table 2 for both latexes at pH 5. The most striking result is the high CCC value for carboxylated latexes which confirms the previous expectations from their electrokinetic and surface
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´ LEZ ET AL. BASTOS-GONZA
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characterizations. The CCC values are higher than the physiological ionic strength (approximately 0.15 M) and, therefore, these latexes could be potentially applicable under conditions where the monodispersity and colloidal stability are important, as in the particle-enhanced optical immunoassays. The diffuse potential and the Hamaker constant can be estimated from the coagulation kinetics according to Reerink and Overbeek (26). They related the slope of the stability curve, 0 d log W/d log Ce , to the radius particle, a, the diffuse potential, cd , and the electrolyte valence, z. This is given by 0 d log W/d log Ce Å 2.15 1 10 7ag 2 /z 2 ,
[4]
where g Å tanh c˜ d /2. The Hamaker constant, which characterizes the attraction between two particles, can be obtained experimentally from the slope of the stability curve and the CCC value of the latex. For a symmetrical electrolyte the Hamaker constant, A, can be obtained from the equation
tively, and a pronounced difference can be observed between the particles in the presence and absence of CDI. Results were similar for JL4 latex. The activation by CDI affects the overall charge of the latex particles. The iep of the particle preparations with CDI at pH 5.6 were slightly higher than at pH 8.0. This provides supporting evidence that at the lowest pH the link of carboxyl groups of the latex with the carbodiimide is favored, probably because the proton of the COOH facilitates the rupture of one double link of the CDI molecule; the chemical reaction of these functional groups is shown in Fig. 10. At both pH values the coupled CDI decreases in absolute value, the mobility of the latex particles. This decrease in mobility could explain the low colloidal stability of the CDI– latex complexes, especially at pH’s around isoelectric points, since when we activated the particles at pH 5.6 the system coagulated quickly. According to these results the covalent coupling of protein to carboxylated latexes using CDI chemistry should be performed at basic pH, where a good colloidal stability can be expected. ACKNOWLEDGMENT
A Å (1.73 1 10 036 (d log W/d log Ce ) 2 /a 2z 2CCC) 1 / 2 , [5] where a is in units of cm and CCC is in mmol/liter. Using the CCC from Table 2 and the slopes of the stability curves, we can calculate the Hamaker constant by using Eq. [5]. The A values are (0.9 { 0.1) 1 10 021 J for JL4 latex and (1.1 { 0.2) 1 10 021 J for DJL5 latex. The theoretical value of the nonretarded Hamaker constant for the polystyrene–water–polystyrene interface is 1.37 1 10 020 J (27). Our experimental values are much lower that this. This discrepancy is due to the effect of the hydrodynamic interaction (28) on the coagulation kinetics. The Hamaker constant estimated from measured coagulation rates under low repulsion can be incorrect if the hydrodynamic interactions are ignored. In short, hydrodynamic interactions, essentially those resisting the approach of Brownian particles, tend to increase the stability of the dispersion. Therefore, we have to take into account the fact that the theoretical value of the Hamaker constant does not consider this retarded effect. However, our experimental values of the Hamaker constant are on the same order of magnitude as that calculated by other authors (29, 30). C. Electrophoretic Mobility of Carboxylated Latexes Activated by Carbodiimide The electrophoretic mobility was determined for both latexes in buffers of pH 4–9 at a low ionic strength (0.002 M). Figure 9 shows the results of me versus pH for both the nonactivated DJL5 latex and the latex activated by CDI at two different pH’s (5.6 and 8.0). The isoelectric points (iep) of the activated latex are approximately at 5.3 and 4.7, respec-
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Financial assistance was provided by the ‘‘Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT),’’ Project MAT 93-0530-C02-01.
REFERENCES 1. Rembaum, A., Yen, S. P. S., Cheong, E., Wallace, S., and Molday, R. S., Macromolecules 9, 28 (1976). 2. Suzawa, T., Shirahama, H., and Fujimoto, T., J. Colloid Interface Sci. 86, 144 (1982). 3. Suzawa, T., Shirahama, H., and Fujimoto, T., J. Colloid Interface Sci. 93, 498 (1983). 4. Shirahama, H., and Suzawa, T., Polym. J. 16, 795 (1984). 5. Lukowski, G., Mu¨ller, R. H., Mu¨ller, B. W., and Dittgen, M., Int. J. Pharm. 84, 23 (1992). 6. Hearn, J., Ottewill, R. H., and Shaw, N., Br. Polym. J. 2, 116 (1970). 7. Bale, M. D., Danielson, S. J., Daiss, J. L., Goppert, K. E., and Sutton, R. C., J. Colloid Interface Sci. 132, 176 (1989). 8. Ceska, G. W., J. Appl. Polym. Sci. 18, 427 (1974). 9. Ceska, G. W., J. Appl. Polym. Sci. 18, 2493 (1974). 10. Sakota, K., and Okaya, T., J. Appl. Polym. Sci. 20, 3265 (1976). 11. Konings, B. L. J. C., Pelssers, E. G. M., Verhoeven, A. J. C. M., and Kamps, K. M. P., Colloids Surf. B: Biointerfaces 1, 69 (1993). 12. Guthrie, W. H., ‘‘New Free Radical Initiators and Their Use in the Preparation of Polystyrene Polymer Colloids.’’ Ph.D. Dissertation. Lehigh University, PA, 1985. 13. El-Aasser, M. S., in ‘‘Scientific Methods for the Study of Polymer Colloids and Their Applications,’’ NATO ASI Series C: Mathematical and Physical Sciences (F. Candau and R. H. Ottewill, Eds.), Vol. 303. Kluwer Academic, Dordrecht/Norwell, MA, 1990. 14. Ottewill, R. H., and Shaw, J. N., Kolloid Z. Z. Polym. 218, 4 (1967). 15. Shirama, H., and Suzawa, T., Polym. J. 16, 795 (1984). 16. Shirama, H., and Suzawa, T., J. Appl. Polym. Sci. 29, 3651 (1984). 17. Tamai, H., Hasegawa, H., and Suzawa, T., J. Appl. Polym. Sci. 38, 403 (1989). 18. Baran, A. A., Dukhina, L. M., Soboleva, N. M., and Chechik, O. S., Kolloid Z. 43, 211 (1981). ´ lvarez, R., Adv. Colloid Interface Sci. 34, 217 (1991). 19. Hidalgo-A
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CARBOXYLATED LATEXES FOR COVALENT COUPLING, I ´ lvarez, R., Moleo´n, J. A., de las Nieves, F. J., and Bijster20. Hidalgo-A bosch, B. H., J. Colloid Interface Sci. 149, 23 (1992). 21. Elimelech, M., and O’Melia, Ch. R., Colloids Surf. 22, 225 ( 1990 ) . 22. Zukoski, C. F., and Saville, D. A., J. Colloid Interface Sci. 114, 32 (1986). 23. Van der Put, A. G., and Bijsterbosch, B. H., J. Colloid Interface Sci. 92, 499 (1983). 24. Chow, R. S., and Takamura, K., J. Colloid Interface Sci. 125, 226 (1988).
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25. Kijlstra, J., van Leeuwen, H. P., and Lyklema, J., J. Chem. Soc. Faraday Trans. 88, 3441 (1992). 26. Reerink, H., and Overbeek, J. Th. G., Discuss. Faraday Soc. 18, 74 (1954). 27. Prieve, D. C., and Russel, W. B., J. Colloid Interface Sci. 125, 1 (1988). ´ lvarez, R., J. 28. Ferna´ndez, A., Martı´n, A., Callejas, J., and Hidalgo-A Colloid Interface Sci. 162, 257 (1994). 29. Tsaur, S-L., and Fitch, M. F., J. Colloid Interface Sci. 115, 463 (1987). 30. Krapp, H., and Walter, G., J. Colloid Interface Sci. 39, 421 (1972).
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Carboxylated Latexes for Covalent Coupling Antibodies, I D. BASTOS-GONZA´LEZ, J. L. ORTEGA-VINUESA, F. J. DE LAS NIEVES,
AND
´ LVAREZ 1 R. HIDALGO-A
Biocolloid and Fluid Physics Group, Department of Applied Physics, University of Granada, 18071 Granada, Spain Received December 12, 1994; accepted April 7, 1995
The aim of the present work is to prepare and characterize carboxylated latexes (CLs) in order to bind antibodies covalently and efficiently. Carboxylated latexes were prepared by surfactantfree emulsion polymerization using an azo-initiator, which provides carboxyl end groups on the latex surface directly. Two latexes were characterized using different techniques: particle size by electron microscopy and photocorrelation spectroscopy, surface charge density by conductimetric and potentiometric titrations, and electrophoretic mobility versus pH and ionic strength (KBr). The colloidal stability of both latexes shows that these carboxylated latexes can be used for biomedical applications. Activation of carboxyl end groups was performed using the carbodiimide method, and the best conditions for covalent coupling of IgG were obtained. q 1995 Academic Press, Inc.
Key Words: polymer colloids; electrokinetics.
polymerization are negatively charged in a broad pH range of 3–10 (4), but their surface charge density is extremely high (4). At a low ionic strength the antibody cannot penetrate the double layer of the particle and thus, no coupling occurs (11). The carboxylated latexes prepared by an azoinitiator have a rigid interface and the surface charge density is significantly lower. These types of particles have been used less in covalently coupling antibodies and antigens to polymer carriers in the development of particle-enhanced optical immunoassays. In this paper, we report the preparation of surfactant-free carboxylated polymer latexes which enables the covalent coupling of antibodies with a high efficiency rate. The electrokinetic properties and colloidal stability of these latexes are also studied. II. EXPERIMENTAL
I. INTRODUCTION
In recent years, a great deal of attention has been given to the use of polymer colloids on a large scale in biomedical applications (1–5) for a number of reasons. First, such polymer colloids are easy to produce and can be prepared with a high degree of monodispersity (6). Second, they can be used in separating biomacromolecules and immunoassays because of their good biocompatibility (7). The application of such polymer colloids requires control of the surface chemical properties of microparticles and control of the process by which they are synthesized (8–10). There are numerous references concerning the synthesis of polystyrene latexes prepared in the absence of emulsifiers in order to produce particles with carboxyl end groups on the particle surface, which result from the use of an ionic comonomer, such as acrylic acid (6–10). In this case, hydrous polyacrylic (or polymethacrylic) acid layers exist on the surfaces and shift the shear plane away from the particle surfaces. This probably leads to a hairy interface, which could hinder the approach of proteins to the latex surface. Antibodies can be covalently coupled if they can reach the particle surface and if appropriate conditions exist for the coupling process. The carboxylated latexes prepared through ionic comonomer 1
To whom correspondence should be addressed.
0021-9797/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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A. Material Styrene monomer was obtained from Merck and was distilled at reduced pressure and at 407C. The purified monomer was stored at 057C until required. The initiator used in this work was the 4,4 *-azobis(4-cyanopentanoic acid) (ACPA) from Aldrich and was used as received. Potassium persulfate, sodium bicarbonate, sodium chloride, hydrochloric acid, and sodium hydroxide (all from Merck) were analytical grade reagents and were used without further purification. Doubledistilled and deionized (DDI) water was used throughout. B. Preparation of Latexes To prepare carboxylated latexes we have followed the steps developed by Guthrie (12) of surfactant-free emulsion polymerization of styrene with ACPA as the initiator. This method has the advantage that carboxyl end groups on the particle surface come from the initiator molecules. Table 1 shows the synthesis conditions used throughout this work. The materials were poured into a 600 cc three-necked glass flask and polymerized under a nitrogen atmosphere. To maintain continuous vigorous stirring, the T-shaped stirrer (1 1 5 cm) was fitted 1 cm from the bottom of the flask. Particle size was obtained using two different methods: (i) transmission electron microscopy (TEM) and (ii) photo-
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TABLE 1 Polymerization Recipe Used in the Carboxylated Polystyrene Latexes Latex JL4 DJL5
Styrene ACPA NaOH Stirring Temperature Reaction time (h) (g) (mM) (mM) (rpm) (7C) 25 25
2.76 0.79
7.43 3.34
350 350
80.6 80.6
10 20
correlation spectroscopy (PCS) (Malvern 4700 System). Results taken from both methods were very similar. The characteristics of the bare polymer colloids are shown in Table 2. The latexes were considered to be monodisperse since the polydispersity index, PDI (defined as DW /DN , where Dw and Dn are the weight-average and the numberaverage diameters, respectively), was less than 1.05 (13). Figure 1 shows the particle size distribution of both samples obtained by TEM. More than approximately 500 particles were counted for each latex. C. Cleaning Process and Surface Characterization A long and comprehensive cleaning process was followed before measuring the surface charge density of these latexes. First, the latexes were filtered through glass wool, then they underwent repeated cycles of centrifugation–decantation– redispersion, and finally, they were cleaned using serum replacement with DDI water. After these processes were completed the specific electrical conductivity of the latexes was found to be constant at approximately 5 mS cm01 . Surface charge densities of JL4 and DJL5 latexes were determined by conductimetric and potentiometric automatic titrations employing a pH-meter (Crison Instruments, Model 2002) and a conductimeter (Crison Instruments, Model 525), and a Dosimat 665 (Metrohm) to add the titrant agent. Figure 2 shows the potentiometric and conductimetric back titration for the JL4 latex. It is not possible to know the total surface charge densities ( s0 ) from direct titrations in latexes with weak acid groups on their surface, as it is necessary to have protonated all these groups (COOH), which occurs at pH’s around 3, and under these conditions the colloidal system would be completely aggregated. This is why we have employed back titrations to obtain the s0 values, which are
FIG. 1. Size distribution for JL4 and DJL5 latexes.
also shown in Table 2. The presence of three different slopes in the conductimetric curve is caused by: (i) the titration of the excess of NaOH previously added (since this plot represents a back-titration), (ii) the titration of the carboxyl groups of the latex, and (iii) the last slope is caused by the excess of titration agent (HCl). Knowing the total surface of added latex, the HCl concentration, and the total volume of this solution consumed in the titration of the weak acid groups of the polymer surface, one can calculate s0 . We can see in Fig. 2 that the changes in the slopes in the conductivity curve coincide with the inflection points of the potentiometric one. The electrophoretic mobilities of these cleaned latexes were measured at 257C under different ionic strengths (KBr as the electrolyte) and pH conditions using a Zeta-Sizer IV (Malvern Instrument). The electrophoretic mobility values
TABLE 2 Characteristics of Carboxylated Polystyrene Latexes Diameter PCS (nm)
Diameter TEM (nm)
PDI
s0 (mC/cm2)
JL4
342 { 7
331 { 11
1.002
12.1 { 0.5
DJL5
278 { 5
276 { 12
1.005
16.3 { 0.7
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d log W/d log Ce 2.07 2.06 2.47 2.03
(pH (pH (pH (pH
5) 7) 5) 7)
CCC (mM KBr) 360 780 350 590
(pH (pH (pH (pH
5) 7) 5) 7)
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(dA/dt)s is the initial slope for lower electrolyte concentrations). Log W values were then plotted versus log Ce . The slopes and the CCC values from these curves are given in Table 2. E. Protocol for Activation of Carboxylated Latexes Using the Carbodiimide (CDI) Method
FIG. 2. Conductimetric and potentiometric back titration for the JL4 latex.
were obtained by taking the average of (at least) five measurements at the stationary level in a cylindrical cell. The experimental error was taken as the standard deviation of these measurements. For the data shown throughout this paper the standard deviation was always lower than {0.2 1 10 08 m2 V 01 s 01 . Samples were prepared by adding approximately 0.01 ml of the latex to 10 ml of each electrolyte solution. The pH’s were controlled using different buffers (acetate at pH’s 4 and 5, phosphate at pH’s 6 and 7, and borate at pH’s 8 and 9), keeping the ionic strength at a constant value of 0.002 M. D. Measurement of Stability Ratios A Milton Roy Spectronic 601 spectrophotometer was used in conjunction with a PC. In a typical coagulation experiment, 2.4 ml of a buffered latex solution was put into the spectrophotometer cell and the optical absorbance was measured. Potassium bromide (0.6 ml) of a given concentration was added quickly to the sample through a syringe. The final particle concentration in the cell was 10 10 part/ml. The optical absorbance (A) was measured immediately and its change versus time (t) was recorded continuously for a period of 30 s. One typical curve of this response is given in Fig. 3 for the DJL5 latex. Curves were found to be linear in the early stages of coagulation at all electrolyte concentrations. The initial slopes were directly proportional to the initial coagulation rate. These slopes increased as the electrolyte concentration increased until a maximum was reached. From this concentration (called the critical coagulation concentration, CCC) the initial slopes did not increase with the addition of higher amount of salt. The slope at the CCC was taken as the fast coagulation rate of the latex. The factor stability (W ) values for each electrolyte concentration were calculated from W Å (dA/dt) f /(dA/dt)s (where (dA/dt) f is the initial slope in fast coagulations and
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A latex surface of 0.3 m2 was added to 1 ml of buffer solution pH 5.6 or pH 8.0 at low ionic strength. A pH value of 5.6 was obtained using MES (2-[N-morpholino]ethanesulfonic acid) instead of acetate in order to guarantee that only the carboxylic groups that are going to be activated are those that are on the latex surface. To the above volume, 200 ml of 15 mg/ml CDI (1 ethyl-3-(3-dimethyl aminopropyl)carbodiimide) in water was added. This gave a CDI concentration of 10 mg/m 2 of latex. Samples were incubated at room temperature for 40 min. The samples were centrifuged for 20 min at 28,000g. The supernatant was removed, and the particles were resuspended in 1.2 ml of the same buffer, and the new suspension was briefly sonicated. The excess carboxylic acid groups were blocked by treatment with ethanolamine for 10 min at room temperature. A similar effect was obtained when the treatment with ethanolamine lasted 15 h. III. RESULTS AND DISCUSSION
A. Electrokinetic Characterization The electrophoretic mobilities of these latexes were measured to characterize the electric double layer surrounding the particles. The electrophoretic mobility values for samples JL4 and DJL5 were measured as a function of pH at a constant ionic strength of 0.002 M KBr. The results given
FIG. 3. Optical absorbance versus time for the JL4 latex. Electrolyte concentration (Ce ) 0.6 M.
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FIG. 4. Electrophoretic mobility ( me ) versus pH for both latexes: l, JL4 latex; l, DJL5 latex.
FIG. 5. Potentiometric titration to obtain s0 versus pH: j, blank; j, blank with JL4 particles.
in Fig. 4 show that the me values were almost constant when the pH was increased from 6 to 10 with a pronounced decrease at a lower (acid) pH. It is possible to recognize the weak acid character of the surface end groups of these latexes from this figure. We have used two procedures to calculate the pKa of our latexes, where Ka is the dissociation constant. The first one is a simplified procedure that has been developed by Ottewill and Shaw (14). From this procedure, it may be demonstrated that
values of me for both samples, even when the surface charge density ( s0 ) varied from 12.1 (JL4) to 16.3 (DJL5) mC cm02 (i.e., an increase of 35%). The averages of the me values for pHs between 6 and 10 were ( 110 08 m2 V 01 s 01 ) meJL4 Å 04.4 { 0.1 and meDJL5 Å 04.3 { 0.1, where the error was the standard deviation obtained from the mean me values at each pH. Thus, the difference between the me values for both latexes are within the experimental error. However, it can be expected that the mobility of the most charged latex (DJL5) would be higher. This result is contrary to the predictions of the classical electrical double-layer models. Some authors (18, 19) have explained this anomalous electrokinetic behavior on the basis of the surface conductance between the shear plane and the particle surface. Since the surface conductance is directly proportional to the surface charge density, it could account for the disagreement between the s0 and me values. Moreover, in Fig. 6 we can observe that in the range from pH 9 to 6 s0 decreases, and
pKa Å pHz Åz plateau / 2 / 0.4343(Fzplateau )/2RT.
[1]
On the other hand, the surface charge densities of the polymer latexes as a function of pH can be determined from the results of the potentiometric titrations (15–17). This dependence of s0 on pH can be obtained from the titration of a blank of NaOH without latex particles and the titration of the same solution with latex. The difference between the two curves, at a given pH, informs us about the volume of the titrant agent (HCl), with which s0 can be calculated at that pH. As the pH becomes more acidic the difference between the two curves, and therefore s0 , decreases (Fig. 5). This method allows us to calculate the pKa from the mass law: pKa Å pH / log[R–COOH]/[R–COO 0 ].
[2]
pKa will coincide with the pH where [R–COOH] Å [R– COO 0 ] and therefore the surface charge density will be equal to 1/2 of the maximum s0 . The pKa calculated by this procedure was 5.8 { 0.3 and using the former procedure we have obtained a pKa value of 5.7 { 0.1. In Fig. 6 we have shown the dependence of the zeta-potential ( z ) and s0 on pH. One remarkable result observed in Fig. 4 is the similar
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FIG. 6. Dependence of z and s0 on pH for latex JL4: ., z ; l, s0 .
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FIG. 7. Electrophoretic mobility ( me ) versus electrolyte concentration (KBr) for both carboxylated latexes: l, JL4 latex; l, DJL5 latex.
z remains constant. In other words, although the value of me remains constant between pH 9 and 6, the s0 value decreases by almost 50%. Electrophoretic mobility measurements on samples JL4 and DJL5 were carried out at different electrolyte concentrations. These results are shown in Fig. 7. These curves confirm our previous results from Fig. 4, showing that there is no significant difference between the me values for both samples, within the experimental error. The big difference between the surface and the electrokinetic potentials made the electrokinetic potential largely insensitive to changes in the surface charge density (20). Various qualitative explanations have been proposed to account for the maximum which appears in Fig. 7. A preferential adsorption of co-ions onto the surface was invoked by several authors (21, 22). The existence of polyelectrolyte chains on the surface of polymer
FIG. 8. Log W versus log Ce at pH 5. The JL4 latex is shown on the left side and the DJL5 latex on the right side.
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colloids is considered in the hairy layer model (23). Physical changes in the particle surface properties were also invoked by other authors (24). In the last case, the authors consider that the shift of the shear plane into the bulk solution is due to the surface roughness or to the existence of polyelectrolyte chains on the surface of latex particles. This displacement of the shear plane results in a decrease of the mobility in the usual way and lowers it to a much larger extent by introducing ionic conduction (or surface conductance) between the solid surface and the shear plane. The surface conductance is especially important at a low ionic strength and reduces the me values by a much greater percentage (25). It must be noted that when the carboxylated latexes are prepared by copolymerization of styrene and by an ionic comonomer (acrylic and methacrylic acids, for instance), a hydrous polyacrylic acid layer exists on the surface, preventing the approach of antibodies or antigens to the polymer surface. In some cases the thickness of these layers can even ˚ (4). In our case, using the same procedure as be 10 A Shirahama and Suzawa (4), the position of the shear plane ˚ from the polymer surface. This result is considered is 2.5 A to be experimental proof that the carboxylated latexes prepared by the ACPA initiator have a thinner solid–liquid interface layer than those prepared by an ionic comonomer polymerization. This difference is relevant for covalent coupling biomolecules to carboxylated latexes using the carbodiimide method. B. Colloidal Stability of Carboxylated Latexes As Reerink and Overbeek have shown (26), with several approximations, a linear relationship between log W and log Ce can be obtained,
FIG. 9. Electrophoretic mobility versus pH for the DJL5 latex activated by CDI: l, at pH 5.6; ., at pH 8.2; w, bare latex.
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FIG. 10. Covalent coupling reaction of a protein with a carboxylated latex; (i) first step: activation of the carboxyl group with CDI; (ii) second step: reaction of this activated group with an amine of the protein. This last functional group could be a terminal amine or it could come from lysine amino acids.
log W Å 0k * log Ce / log k 9 ,
[3]
where k * and k 9 are constants and Ce is the electrolyte concentration. Thus, W decreases with the addition of electrolyte until the electrolyte concentration reaches a critical value above which the energy barrier between particles is lower than the thermal energy. The electrolyte concentration at which W becomes equal to 1 is called the CCC. The CCC is defined as the concentration of added electrolyte which is just enough to bring about diffusion-controlled rapid coagulation.
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Log W values have been plotted versus log Ce as suggested by Reerink and Overbeek (24). Figure 8 shows the stability curves of JL4 and DJL5 latexes for a 1:1 electrolyte (KBr), at pH 5. The linear relationship expected from Eq. [3] is confirmed in the two cases, as the correlation coefficients were higher than 0.98, with at least five experimental points. The CCC values were calculated and are shown in Table 2 for both latexes at pH 5. The most striking result is the high CCC value for carboxylated latexes which confirms the previous expectations from their electrokinetic and surface
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characterizations. The CCC values are higher than the physiological ionic strength (approximately 0.15 M) and, therefore, these latexes could be potentially applicable under conditions where the monodispersity and colloidal stability are important, as in the particle-enhanced optical immunoassays. The diffuse potential and the Hamaker constant can be estimated from the coagulation kinetics according to Reerink and Overbeek (26). They related the slope of the stability curve, 0 d log W/d log Ce , to the radius particle, a, the diffuse potential, cd , and the electrolyte valence, z. This is given by 0 d log W/d log Ce Å 2.15 1 10 7ag 2 /z 2 ,
[4]
where g Å tanh c˜ d /2. The Hamaker constant, which characterizes the attraction between two particles, can be obtained experimentally from the slope of the stability curve and the CCC value of the latex. For a symmetrical electrolyte the Hamaker constant, A, can be obtained from the equation
tively, and a pronounced difference can be observed between the particles in the presence and absence of CDI. Results were similar for JL4 latex. The activation by CDI affects the overall charge of the latex particles. The iep of the particle preparations with CDI at pH 5.6 were slightly higher than at pH 8.0. This provides supporting evidence that at the lowest pH the link of carboxyl groups of the latex with the carbodiimide is favored, probably because the proton of the COOH facilitates the rupture of one double link of the CDI molecule; the chemical reaction of these functional groups is shown in Fig. 10. At both pH values the coupled CDI decreases in absolute value, the mobility of the latex particles. This decrease in mobility could explain the low colloidal stability of the CDI– latex complexes, especially at pH’s around isoelectric points, since when we activated the particles at pH 5.6 the system coagulated quickly. According to these results the covalent coupling of protein to carboxylated latexes using CDI chemistry should be performed at basic pH, where a good colloidal stability can be expected. ACKNOWLEDGMENT
A Å (1.73 1 10 036 (d log W/d log Ce ) 2 /a 2z 2CCC) 1 / 2 , [5] where a is in units of cm and CCC is in mmol/liter. Using the CCC from Table 2 and the slopes of the stability curves, we can calculate the Hamaker constant by using Eq. [5]. The A values are (0.9 { 0.1) 1 10 021 J for JL4 latex and (1.1 { 0.2) 1 10 021 J for DJL5 latex. The theoretical value of the nonretarded Hamaker constant for the polystyrene–water–polystyrene interface is 1.37 1 10 020 J (27). Our experimental values are much lower that this. This discrepancy is due to the effect of the hydrodynamic interaction (28) on the coagulation kinetics. The Hamaker constant estimated from measured coagulation rates under low repulsion can be incorrect if the hydrodynamic interactions are ignored. In short, hydrodynamic interactions, essentially those resisting the approach of Brownian particles, tend to increase the stability of the dispersion. Therefore, we have to take into account the fact that the theoretical value of the Hamaker constant does not consider this retarded effect. However, our experimental values of the Hamaker constant are on the same order of magnitude as that calculated by other authors (29, 30). C. Electrophoretic Mobility of Carboxylated Latexes Activated by Carbodiimide The electrophoretic mobility was determined for both latexes in buffers of pH 4–9 at a low ionic strength (0.002 M). Figure 9 shows the results of me versus pH for both the nonactivated DJL5 latex and the latex activated by CDI at two different pH’s (5.6 and 8.0). The isoelectric points (iep) of the activated latex are approximately at 5.3 and 4.7, respec-
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Financial assistance was provided by the ‘‘Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT),’’ Project MAT 93-0530-C02-01.
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CARBOXYLATED LATEXES FOR COVALENT COUPLING, I ´ lvarez, R., Moleo´n, J. A., de las Nieves, F. J., and Bijster20. Hidalgo-A bosch, B. H., J. Colloid Interface Sci. 149, 23 (1992). 21. Elimelech, M., and O’Melia, Ch. R., Colloids Surf. 22, 225 ( 1990 ) . 22. Zukoski, C. F., and Saville, D. A., J. Colloid Interface Sci. 114, 32 (1986). 23. Van der Put, A. G., and Bijsterbosch, B. H., J. Colloid Interface Sci. 92, 499 (1983). 24. Chow, R. S., and Takamura, K., J. Colloid Interface Sci. 125, 226 (1988).
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25. Kijlstra, J., van Leeuwen, H. P., and Lyklema, J., J. Chem. Soc. Faraday Trans. 88, 3441 (1992). 26. Reerink, H., and Overbeek, J. Th. G., Discuss. Faraday Soc. 18, 74 (1954). 27. Prieve, D. C., and Russel, W. B., J. Colloid Interface Sci. 125, 1 (1988). ´ lvarez, R., J. 28. Ferna´ndez, A., Martı´n, A., Callejas, J., and Hidalgo-A Colloid Interface Sci. 162, 257 (1994). 29. Tsaur, S-L., and Fitch, M. F., J. Colloid Interface Sci. 115, 463 (1987). 30. Krapp, H., and Walter, G., J. Colloid Interface Sci. 39, 421 (1972).
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