Colloidal stability of polymer latices coated with bovine serum albumin

Colloidal Stability of Polymer Latices Coated with Bovine Serum Albumin HISASHI TAMAI, A K I H A R U FUJII, AND T O S H I R O SUZAWA

Department of Applied Chemistry, Faculty of Engineering, Hiroshima University, Higashihiroshima, Hiroshima, Japan Received April 15, 1986; accepted October 15, 1986 The colloidal stabilityof polystyrene(PS), styrene/methacrylicacid copolymer (P(St/MAA)), styrene/ acrylamidecopolymer(P(St/AAm)), and its hydrolyzate(P(St/AAm-H))latices coated with bovine serum albumin (BSA) at different coverageswas studied. The rates of flocculationwere measured as a function of NaC1 concentration by the stopped-fow spectrophotometricmethod. The results are considered in terms of the surface characteristics of the latex particles such as ~'-potentials.The slow flocculation of BSA-coated PS and P(St/MAA) latices was observed at low NaCI concentration where no flocculation of bare latices was detected. The contribution of the bridging effectby adsorbed BSA is suggested. The maximum rates of flocculation of PS and P(St/MAA) latices at high NaC1concentrations decrease with increasing surface coverageof BSA. At high NaC1 concentrations, a steric repulsive interaction due to adsorbed BSA plays an important role. No flocculation of bare and BSA-coated P(St/AAm) and P(St/ AAm-H) latices was observed at all NaC1 concentrations tested. This high colloidal stability could be attributed to the steric stabilization effect due to a water-soluble polymer layer at the surface of bare latex particles. © 1987 Academic Press, Inc. INTRODUCTION Polymer latices such as polystyrene (PS) latex particles have been used extensively as carriers of antigens and antibodies in m a n y serological tests (1, 2). In these serological tests, latex particles are covered in advance with antigen or antibody. From this point of view, the colloidal stability of the latices coated with proteins such as globulin and albumin is important in the practical use of latices. Regarding the colloidal stability of polymer latices coated with proteins, Singer et al. (3) have studied the rate offlocculation of PS latex particles by h u m a n 3,-globulin. Scheer et al. (4) measured the rate of flocculation of PS latex by h u m a n serum albumin and h u m a n fibrinogen as functions of protein and salt concentrations using a stopped-flow spectrophotometer and observed the enhancement of the rate of flocculation above the value for bare PS latex. In this work, the colloidal stability of various kinds of polymer latices coated in advance

with bovine serum albumin (BSA) is investigated. The rates of flocculation of their latices with BSA at different coverages are measured as a function of NaC1 concentration by the stopped-flow spectrophotometric method. EXPERIMENTAL

Materials PS, styrene/acrylamide copolymer (P(St/ AAm)), its hydrolyzate (P(St/AAm-H)), and styrene/methacrylic acid copolymer (P(St/ MAA)) latices were prepared in the absence of emulsifier using potassium persulfate as initiator. The polymerization recipes o f these latices and the purification methods have been described in detail in the previous paper (5). The mean diameters of PS, P(St/AAm), and P(St/MAA) latex particles by electron microscopy were 457, 429, and 543 nm, respectively. BSA (Sigma Chemical Co., crystallized and lyophilized BSA) was used without further purification.

176 0021-9797/87 $3.00 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Journal of Colloid and Interface Science, Vol. 118, No. 1, July 1987

POLYMER LATICES WITH BOVINE SERUM ALBUMIN Method

177

The electrophoretic mobilities of bare and BSA-coated latex particles were measured as a function of NaC1 concentration with a Mitamura Riken microelectrophoresis apparatus. The ~'-potentials were calculated from the electrophoretic mobilities, corrected by the Henry function (7) as described previously (8).

The amount of BSA adsorbed was determined by measuring the difference in soluble BSA concentration before and after adsorption on the latex particles. The mixtures of BSA solution and latex dispersion were equilibrated for 2 h at 25°C and latex particles were sepRESULTS AND DISCUSSION arated by centrifugation. BSA concentrations were measured by the Lowry method (6). Figure 1 shows the adsorption isotherms of In order to prepare the latices coated with BSA onto the latices at 10 -2 MNaCI and 25°C. BSA, BSA was adsorbed in advance from the The amounts of BSA adsorbed on P(St/MAA) solutions containing BSA of different concenand P(St/AAm-H) latices which have carboxyl trations. The BSA concentrations were chosen groups at the particle surface increase linearly such that all BSA molecules contained adsorb with increasing initial concentration of BSA. on the particles, on the basis of the adsorption In this concentration range of BSA, all added isotherms which will be described later. BSA was adsorbed on the latex particles. On The mixtures of BSA solution (0 ~ 1.84 the other hand, no soluble BSA is observed in mg. m-2--1atex surface area) and latex disthe supernatant solution below the initial conpersion (2.6 × 1013 particles, dm -3) were left centration of 10 rag. dm -3 BSA. On the basis in 10-2 M NaC1 for 2 h at 25°C. The BSAof these adsorption isotherms, the preparation coated latex was diluted 10 times and this sample was used for the measurements of of the latices coated with BSA for the measurements of flocculation and ~-potential was flocculation and ~'-potential. carried out under the condition that all added The rapid-mixing flocculation experiments BSA is adsorbed on the latex particles. were performed by the stopped-flow method Assuming that a BSA molecule is a prolate using a JASCO Uvidec 610 spectrophotometer with an SFC-333 flow-cell device (light-path spheroid of revolution with major and minor length, 10 ram) connected with a Union MX- axes 2a and 2b, respectively (9), and that it is 7 sample mixing device. In this apparatus, adsorbed side-on in a close-packed array, equal volumes of bare or BSA-coated latex dispersion and NaC1 solution were taken into - - 0 - PS the two syringes and then rapidly mixed by 5 --I-- St/HEMA depressing the plunger. The latex dispersions _ r a _ St/MAA --/x -- SI/AAm //A were always placed in one syringe and NaC1 / 4 --A-- Sff AAm-H • solutions in the other to prevent contamination. The transmittance of the mixture was 120 'E 3 / ~ ~ u ~ . u recorded on an attached recorder. The wave100 length used was 680 n m by a tungsten lamp. 2 ~o Z The latex particle concentration was always J chosen such that after mixing with NaC1 son g 40 lution in the stopped-flow apparatus the con20 centration was 1.3 × 1012 dm -3. The particle 0 " 10 ;o 3 40 ' 50 ' 60 ' "70 concentration was determined from the parC I (rng dm-3 ) ticle diameter by electron microscopy and the FIG. 1. Adsorptionisotherms for BSA on the various dry weight per unit volume of the latex. All latices(25°C, pH 5.7, 10-2 M NaC1).BSA concentration, the above measurements were made in the CB,per unit surfacearea of latexparticleis plottedagainst room thermostated at 25 +_ 2°C. BSA initialconcentration,Ca.

J

•

Journal of Colloid and Interface Science,

Vol.118,No,1,July1987

178

TAMAI, FUJII, AND SUZAWA 4

Enckevort et al. (10) expressed the maximum amount of BSA adsorbed per unit surface area by the equation

3 ,g

xMr

I'max lrabN'

[1]

where N is Avogadro's number, Mr is the relative molar mass of BSA, and x is the roughness factor. In this calculation, 14 and 3.8 n m were used as 2a and 2b, respectively (11), Mr was 66210, and x was 1.0. The surface coverage (0) of BSA was obtained by P/Fmax, where I' is the amount of BSA adsorbed per unit surface area. As shown in Fig. 1, the latices were coated with BSA below 100% of 0. Figure 2 shows some typical results of the flocculation experiments of PS latex coated with BSA at 0.23 mg. m -2 (surface coverage 8.7%) of BSA concentration. The variation of the turbidity (At) with time is presented. From the results shown in Fig. 2, the rate constant of flocculation (kll) is calculated according to Lichtenbelt et al. (12).

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~G. 3. kH ofBSA-coated PS latex as a function of NaCl concentration, C, and BSA concentration per unit surface area of latex particle, CB (surface coverage, 8), at pH 5.7.

Figure 3 shows the rate constants of flocculation of bare and BSA-coated PS latex as a function of NaC1 concentration, kll for bare PS latex increases with increasing NaC1 concentration. The maximum value of kll for bare PS latex is about 4 × 10 -18 m 3. s -1. The slow flocculation of BSA-coated PS latex was observed at low NaC1 concentrations where no

4 - C s : _ O_ 0 mg.m'Z(e = 0 %) -zx-0.115 ( . = 4.4 ") ~ - - - 0 -[3--0.23 ( * = 8.7".) [ 3 - 0 - 0.46 (*=17.5 .) ] 0.69 (o=26.2 *) I a - . - 0.92 (.=35.0 ,) ~ o~ - ~ - 1.84 (+=70.0 o ) ]

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Time ( s e c ) FIG. 2. Increase in the turbidity of PS latex coated with BSA at 0.23 mg. m -2 as a function of time and NaC1 concentration, C, at pH 5.7. Journal of Colloid and InterfaceScience, Vol. 118, No. 1, July 1987

I

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179

POLYMER LATICES WITH BOVINE SERUM ALBUMIN p(StlAAm)CB:-O0 -(i-0.46

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flocculation of bare PS latex was detected, kll for BSA-coated PS latex decreases with increasing surface coverage of BSA. This fact would suggest that a steric repulsive interaction due to adsorbed BSA reduces the maximum rate of flocculation of BSA-coated PS latex to less than that of bare PS latex. Figure 4 shows the rate constants of flocculation of bare and BSA-coated P(St/MAA) latex as a function of NaC1 concentration. Similar to that of PS latex, BSA coated P(St/ MAA) latex is observed to flocculate slowly at low NaC1 concentrations and kll for BSAcoated P(St/MAA) latex decreases at high

NaC1 concentrations with increasing surface coverage of BSA. However, the influence of the surface coverage of BSA is less than that for PS latex. That is to say, at the same coverage, kll for BSA-coated P(St/MAA) latex is higher than that for BSA-coated PS latex. Figure 5 shows the results of the flocculation measurements of bare and BSA-coated P(St/ AAm) and P(St/AAm-H) latices as a function of NaC1 concentration. In both bare and BSAcoated P(St/AAm) and P(St/AAm-H) latices, no flocculation was observed even at high NaC1 concentrations. In addition, the slow flocculation due to adsorbed BSA, which was

100

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FIG. 6. ~'-potentials of BSA-coated PS latex as a function of NaC1 concentration, C, and BSA concentration per unit surface area of latex particle, CB (surface coverage, 0), at pH 5.7. Journal of Colloid and Interface Science, Vol. 118, No. 1, July 1987

180

TAMAI, FUJII, AND SUZAWA

80

Ca:--O-- 0 rng-m-2 (O= 0 %) [email protected] * (*= 8.7 %) --l--0.69 ~ (,,=26.2 %)

E ~40

I

2o 0

I

I

I

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I

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I I II

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FIG. 7. ~'-potentials o f BSA-coated P(St/MAA) latex as a function of' NaC] concentration, C, and BSA concentration per unit surface area of latex particle, CB (surface

coverage, 0), at pH 5.7.

observed at low NaCI concentrations in the flocculation ofBSA-coated PS and P(St/MAA) latices, was not detected. The high colloidal stability of bare P(St/AAm) and P(St/AAmH) latices for electrolyte concentrations has been previously reported, and the participation of steric repulsion due to a water-soluble polymer layer at the particle surface has been considered (5). Consequently, the results of BSAcoated P(St/AAm) and P(St/AAm-H) latices indicate that the steric stabilization effects play an important role in the flocculation. Figures 6, 7, and 8 show the ~'-potentials of bare and BSA-coated PS, P(St/MAA), P(St/ AAm), and P(St/AAm-H) latices as functions of the surface coverage of BSA and NaC1 con-

centration. As NaCI concentration increases, the ~'-potentials of these bare and BSA-coated latices decrease, though bare PS latex gives the maximum value in negative ~'-potentials. The decrease in ~'-potential with NaCI concentration would be due to compression of the electrical double layer. Consequently, the increase in the rate of flocculation of bare and BSAcoated PS and P(St/MAA) latices with NaC1 concentration would be due to the decrease in the electrostatic repulsive interaction between latex particles. On the other hand, the ~-potentials of PS and P(St/MAA) latices decrease with increasing surface coverage of BSA. The coating with BSA would decrease the surface charge and potential of these latices. However, because the negative ~'-potentials ofBSA-coated PS and P(St/MAA) latices are high enough to prevent the latices from flocculating, the slow flocculations seem to be due to the bridging effect by adsorbed BSA. The ~-potentials of P(St/AAm) and P(St/ AAm-H) latices scarcely change with the coating of BSA at low surface coverage. This would suggest that BSA molecules adsorbed at low surface coverage scarcely influence the surface charge and potential of P(St/AAm) and P(St/ AAm-H) latex particles. BSA molecules would be incorporated into the inner part of the water-soluble polymer layer mentioned above.

P(St/AArn) CB : - O -

80 6O

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~

0 mg.m-Z(e = 0 %)

- ( ~ - 0.46

(.. =17.5 .. )

-I

- 0.92

(,, =35.0 ,, )

P(SIIAAm-H)CB : - - o 0 - - • --0,92 --IO--2.95

(',= 0 ,,) (., =35.0 ,,) ( " = 1 1 2 ,,)

,,

v

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FIG. 8. f-potentials of BSA-coated P(St/AAm) and P(St/AAm-H) latices as functions of NaC1 concentration, C, and BSA concentration per unit surface area of latex particle, CB (surface coverage, 0), at pH 5.7.

Journal of Colloid and Interface Science, Vol. 118, No. 1, July 1987

POLYMER LATICES WITH BOVINE SERUM ALBUMIN

No flocculation of BSA-coated P(St/AAm) and P(St/AAm-H) latices may be interpreted by this assumption. REFERENCES I. Singer, J. M.,Amer. J. Med. 31, 766 (1961). 2. Christian, C. L., Mendez-Bryan, R., and Larson, D. L., Proc. Soc. Exp. Biol. Med. 98, 820 (1958). 3. Singer, J. M., Vekemans, F. C. A., Lichtenbelt, J. W. Th., Hesselink, F. Th., and Wiersema, P. H., J. Colloid Interface Sci. 45, 608 (1973). 4. Scheer, V. D., Tanke, M. A., and Smolders, C, A., Faraday Discuss. Chem. Soc. 65, 264 (1978).

181

5. Tamai, H., Fujii, A., and Suzawa, T., J. Colloid Interface Sci., in press. 6. Lowry, O. H., Rosebrough, N. J., Farr, A. C., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). 7. Henry, D. C., Proc. R. Soc. London Ser. A 133, 105 (1931). 8. Suzawa, T., Tamai, H., Shirahama, H., and Yamamoto, K., Nippon Kagaku Kaishi 1979, 16 (1979). 9. Squire, P. G., Moser, P., and O'Konski, C. T., Biochemistry 7, 4261 (1968). 10. van Enckevort, H. J., Dass, D. V., and Langdon, A. G., J. Colloid Interface Sci. 98, 138 (1984). 11. Fair, B. D., and Jamieson, A. M., J. Colloidlnterface Sci. 77, 525 (1980). 12. Lichtenbelt, J. W. Th., Ras, H. J. M. C., and Wiersema, P. H., J. Colloid Interface Sci. 46, 522 (1974).

Journal of Colloid and Interface Science, Vol. 118, No. 1, July 1987