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Adsorption of bovine serum albumin onto mica surfaces studied by a direct weighing technique
Colloids and Surfaces B: Biointerfaces 27 (2002) 115 /122 www.elsevier.com/locate/colsurfb
Adsorption of bovine serum albumin onto mica surfaces studied by a direct weighing technique Hiroshi Terashima , Tatsuo Tsuji Institute of Materials Science, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan Received 12 February 2002; accepted 18 March 2002
Abstract Adsorption of bovine serum albumin (BSA) from aqueous solution to muscovite mica has been investigated through the measurements of two quantities: (1) the adsorbance, i.e. adsorbed mass per unit area measured as a function of incubation time by means of a Mettler UM3 ultramicrobalance; and (2) the contact angle of water droplets resting on the adsorbed BSA layer to know the degree of surface covering. The protein concentration explored was 0.002 and 0.01 mg ml 1 in aqueous solution at pH 5.8. The temperature was kept constant at 25 8C. The following results have been obtained in the present study. (1) The initial rate of adsorption is more rapid than generally accepted. The surface of mica is completely covered by adsorbed BSA molecules within 10 min even for a dilute solution at a concentration 0.002 mg ml 1. (2) At a stage where the surface covering is completed, the adsorbance levels off at a steady value, which is approximately equal to mass per unit area of side-on monolayer. It concludes from this result that BSA molecules are adsorbed side-on directly onto mica surface to form a side-on monolayer. (3) If the adsorption is prolonged, the adsorbance exceeds the mass per unit area of side-on monolayer owing to the adsorption of BSA molecules onto the side-on monolayer already formed. This fact indicates the occurrence of multilayer adsorption of BSA. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Bovine serum albumin; Muscovite mica; Protein adsorption; Ultramicrobalance
1. Introduction Bovine serum albumin (BSA) has been often employed as an adsorbate for the study of protein adsorption from aqueous solution to solid surfaces [1 /4]. The shape of BSA molecule is known to be like a prolate ellipsoid of revolution with major
Corresponding author. Tel.: /81-298-53-5123; fax: /81298-55-7440 E-mail address: [email protected] (H. Terashima).
axis 14.1 nm and minor axis 4.1 nm [5,6]. Because of the ellipsoidal shape, there may occur two types of orientation of adsorbed BSA molecules: these are side- and end-on adsorption if major axis is parallel and perpendicular to solid surface, respectively. Most of the studies of BSA adsorption have aimed chiefly at determining the orientation of BSA molecules adsorbed on solid surfaces. The orientation can be determined by the following two ways. First, the geometrical thickness of adsorbed BSA monolayer is measured so as to be compared with the length of major and minor
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axis. Secondly, the measured values of adsorbance, i.e. adsorbed mass per unit area are compared with the monolayer capacity, i.e. mass per unit area of BSA monolayer calculated assuming either orientation. The monolayer capacity is calculated to be 2.01 mg m 2 for side-on and 7.53 mg m 2 for hexagonally close packed end-on monolayer. The adsorption of BSA to solid-solution interface has been studied by a variety of experimental methods including the measurement of the difference in solution concentration between before and after the adsorption [7 /11], ellipsometry [12], total internal reflection/fluorescence recovery technique [13], radiolabeling technique [14,15], surface force measurement [16,17] and XPS (X-ray photo-electron spectroscopy) [18]. Among these studies, we are interested in the experiments of the BSA adsorption to mica surfaces investigated by the surface force measurement and XPS. The reasons are as follows: (1) the experimental approach is direct so that no ambiguity is involved in the interpretation of the results; (2) the use of mica as an adsorbent makes it possible to establish a simplified situation of the BSA adsorption to planar surface owing to the molecular scale smoothness of cleaved mica surface. Fitzpatrick et al. [16,18] have studied the BSA adsorption to mica surface by a combined use of surface force measurement and XPS. According to their surface force measurement, the separation of closest approach of two facing mica surfaces is measured to be 18/20 nm, i.e. a layer of /10 nm on each mica surface and the refractive index in the gap at this separation to be 1.419/0.03, from which the adsorbance is calculated to be 5.5 /6.5 mg m 2. On the other hand, the measurement of adsorbance by XPS provides a value 5.79/1.0 mg m 2, which is consistent with the result obtained by the surface force measurement. They have concluded from their results that an end-on adsorption of BSA molecules prevails under their experimental conditions. In the same year, a French group has reported a similar experiment. Using a self-controlled-drive surface force apparatus, Gallinet and Gauthier-Manuel [17] have measured the thickness of BSA layer adsorbed on mica surface and the forces required to desorb the BSA molecules from
the gap between the two mica surfaces. They have observed a stepwise increase in repulsive forces on compression and found that the distance between the steps is approximately equal to the minor axis of BSA molecules. They have concluded that BSA molecules are adsorbed side-on. With regard to the orientation of BSA molecules adsorbed on mica surfaces, the above two groups have drawn their respective conclusions which are inconsistent. The purpose of the present study is to elucidate the origin of this inconsistency by examining the process of BSA adsorption. In the present study, an entirely separate method has been applied to the study of BSA adsorption to mica surface. The adsorbance of BSA on mica surface is determined from an increase in weight of mica sheets due to BSA adsorption. A sensitive ultramicrobalance is used for this weighing. In addition to the adsorbance measurement, the contact angle is measured of water droplets placed on mica surfaces bearing BSA layer of known adsorbance. The measurement of contact angle gives information on the degree of surface covering of mica surface by adsorbed BSA molecules. On the basis of the experimental results of both adsorbance and contact angle, the orientation of adsorbed BSA molecules has been determined. This paper describes the following four items. First, a direct method is described of studying the adsorption of protein (BSA) from aqueous solution to mica surfaces by the use of a highly sensitive ultramicrobalance. Secondly, the experimental results are presented concerning the change in adsorbance and contact angle with time at dilute concentrations. Thirdly, a discussion is made about the orientation of adsorbed BSA molecules. Finally, a comparison is made of the present results with the previous studies.
2. Materials and methods 2.1. Materials BSA was supplied from Sigma Chemical Company Ltd. (Fraction V powder, Ref. A-7030) and used as received. The mica used as an adsorbent
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was Muscovite of grade No. 4 and quality C and SS (clear and slightly stained), which is the product of C.M. Rajgarhir (Giridih, India), purchased in sheets from M. Watanabe & Co. Ltd. in Tokyo. The pure water used as a solvent was prepared by double distillation in an all-fused quartz still. 2.2. Principle of adsorbance determination The amount of BSA molecules adsorbed on mica surface is determined from an increase in weight of mica sheets caused by the adsorption of BSA. A commercial Mettler UM3 ultramicrobalance of the readability 10 4 mg was used for the weighing. The mica sheets are weighed before and after the immersion of mica sheets in an aqueous solution of BSA at a given concentration; the temperature of solution is kept constant. The difference in weight of mica sheets between before and after the immersion is divided by the total surface area of mica sheets to give the adsorbance, i.e. adsorbed mass per unit area. Owing to the molecular scale smoothness of the surface of cleaved mica sheets, there is no ambiguity in the determination of the total surface area; consequently, the adsorbance can be determined unambiguously.
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occurring on the surface of mica sheets. Our experiment on the ion exchange [20] has revealed that the dissolution ceases in 30 min and the weight of mica sheets is unchanged afterwards. On the basis of this result, a preliminary immersion in water for 30 min or longer is made throughout the experiment. After the preliminary immersion in water, the mica sheets are taken out of water and dried in air. To reduce the time needed for drying, all of water droplets remaining on the mica surfaces and the fused quartz holder are removed by touching the edge of small piece of clean filtering paper. The pair is weighed together with the holder by hanging it from the hook of the Mettler UM3 ultramicrobalance as shown in Fig. 1. The weight is noted as a reference for zero adsorbance. Then, the pair is immersed in an aqueous solution of BSA at a given concentration. No change in concentration takes place during the adsorption because of the smallness of the surface area of mica sheets. The vessel containing the solution is set in a water bath to keep the temperature constant. After a given period of adsorption, the pair is transferred from solution to water in order
2.3. Experimental procedure A brief description is given below of the practical procedure for the measurement of adsorbed amount; the details have been previously reported [19]. A pair of thin sheets of mica 32 /39 mm in size ( 50 /10 4 m2 in total surface area) is prepared by cleavage and mounted together on a holder made of fused quartz rods of about 0.3 mm in diameter (Fig. 1). In the beginning, the pair of mica sheets is immersed in pure water for a period longer than 30 min. This immersion is particularly necessary for the experiment of protein adsorption from aqueous solution to mica. We have already found that the weight of mica sheets decreases markedly once the mica sheets are immersed in aqueous solution [19,20]. This decrease in weight is caused by the dissolution of metal ions, mainly potassium ions, from mica sheets owing to an ion exchange
Fig. 1. A schematic drawing representing the mica sheets and fused quartz holder used for weighing by means of a Mettler UM3 ultramicrobalance. (1) Mica sheets; (2) holder; (3) small hook for hanging mica sheets; (4) metal hook equipped with Mettler UM3 ultramicrobalance.
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to rinse the residual solution remaining on mica surfaces. Subsequently to the rinse followed by drying in air, the second weighing is made to measure the change in weight of mica sheets. 2.4. Measurement of contact angle The contact angle of water droplets resting on adsorbed BSA layer is measured to know the degree of surface covering of mica surfaces by adsorbed BSA molecules. A sheet of mica covered by adsorbed BSA layer of known adsorbance is placed horizontally on a glass cell. Some of droplets of pure water are gently dropped on the surface of mica sheet by the use of a fine syringe and then the glass cell is tightly shielded. The inside of the glass cell is so designed as to be saturated by water vapor; consequently, the shape and size of the droplets are kept unchanged during the contact angle measurement. The profile of droplets is observed through a window of the glass cell using a microscope equipped with a goniometer, of which the readability is a quarter of 18. As a preliminary experiment, we have measured the contact angle of water droplets for two cases. Case 1: Water droplets are placed on the surface of mica sheets rinsed once in pure water and dried, that is, no BSA molecules are present on the surface. The average of measured values of contact angles of this case is 158. Case 2: The contact angle is measured of water droplets placed on the surface of thick BSA layer fully covering the mica surface. The measurement gives the contact angle 708 in this case. The degree of surface covering can be known by examining whether the measured values of contact angle fall between 15 and 708 or along the level of 708.
3. Results and discussion 3.1. Adsorption at 0.002 mg ml 1 BSA was adsorbed from dilute aqueous solution to mica surface at a concentration of 0.002 mg ml 1. By using such a dilute solution, the initial rate of adsorption is reduced so that we are able to measure the change in adsorbance with time at
early stages of adsorption. The temperature was held constant at 258. The pH measurement of solution gave a value around 5.8. No buffer was used in the present experiment to avoid any possible influence of buffer on weighing. In practice, the measurement of adsorbance was carried out first and then the contact angle was measured by placing water droplets on the adsorbed BSA layers. However, before presenting the results of the adsorbance measurement, we examine the results concerning the change in contact angle with both time and adsorbance. To interpret the results of the adsorbance versus time profile, it is convenient to know in advance the degree of surface covering of mica by adsorbed BSA molecules. The measured values of contact angle are plotted against time and adsorbance in Fig. 2 (a) and (b), respectively. A dotted line is drawn in Fig. 2(a) at 158 which is a mean value of contact angle of water droplets resting on bare surface of mica sheets rinsed once and dried in air. This is the case where no adsorbed BSA molecules are present on mica surfaces. Two dashed lines marked S and E in Fig. 2(b) indicate the respective mass per unit area of two kinds of BSA monolayer. The line S is drawn at 2.01 mg m 2 for side-on monolayer and the line E at 7.53 mg m 2 for hexagonally close packed end-on monolayer. As seen from Fig. 2(a), the contact angle rises steeply from 158 to about 608 within a few minutes of immersion and attains to a steady value of 708 within 10 min. Fig. 2(b) shows that the contact angle exceeds 608 even at about one fifth of the adsorbance corresponding to side-on monolayer and reaches the steady value of 708 at adsorbances indicated by the line S. It follows from these results that the adsorbed BSA molecules are uniformly distributed to cover the whole surface of mica without aggregating in cluster at and above adsorbances corresponding to side-on monolayer. Fig. 3 shows the adsorbance versus time profile determined at a concentration of 0.002 mg m 2. It is seen from Fig. 3 that the adsorbance attains to the level indicated by the line S within 10 min and remains unchanged with time afterwards. From a comparison between Fig. 2(a) and Fig. 3, it is seen that the attainment of adsorbance to the level marked S is coincident with that of contact angle
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Fig. 2. Contact angles of water droplets resting on adsorbed BSA layer, plotted against: (a) incubation time; and (b) adsorbance. A dotted line drawn in (a) indicates a contact angle 158 of water droplets on bare surface of mica sheet rinsed once in water and then dried in air. Two dashed lines marked S and E are drawn in (b), respectively, at 2.01 mg m 2 for side-on monolayer and at 7.53 mg m 2 for hexagonally close packed end-on monolayer.
to 708. The results obtained may be summarized as follows. At a stage where the whole surface of mica is finished to be entirely covered by adsorbed BSA molecules, the adsorbance reaches to a value, which is identical with the mass per unit area of side-on monolayer. This result leads to a conclusion that BSA molecules are adsorbed side-on directly to mica surface to form a side-on monolayer. The adsorption for a long duration has been investigated. Fig. 4 shows the adsorbance versus time profile determined at 0.002 mg ml 1 for prolonged adsorption. The result of the contact angle measurement is also shown together. It is seen from Fig. 4 that most of the measured values of adsorbance fall between the two dashed lines S and E while the contact angle is constant to be around 708 irrespective of time and adsorbance. The excess of adsorbance above the line S can be attributed to an adsorption of BSA molecules on to the adsorbed BSA layer because the mica surface has been confirmed to be completely covered by side-on monolayer at earlier stages of adsorption. Accordingly, it is safe to say that a multilayer adsorption of BSA molecules takes place if the adsorption is continued for a long period. Although it is difficult to specify the orientation of multiply adsorbed BSA molecules,
BSA molecules are supposed to be adsorbed sideon to be piled or end-on between BSA molecules already adsorbed side-on. The orientation of adsorbed BSA molecules at initial and subsequent stages of adsorption are schematically shown in Fig. 5(a and b), respectively. 3.2. Adsorption at 0.01 mg ml 1 To investigate the process of further prolonged adsorption, we conducted an experiment of the adsorption from a more concentrated solution of
Fig. 3. Adsorbance versus time profile of BSA adsorbed from aqueous solution at a concentration 0.002 mg ml 1, a temperature 25 8C, and a pH 5.8. Two dashed lines S and E indicate the adsorbances corresponding to side- and end-on monolayer, respectively.
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Fig. 4. Adsorbance versus time profile of BSA determined at a concentration 0.002 mg ml 1 for prolonged adsorption. The contact angles are also plotted against time. The solution was kept at 25 8C and pH 5.8.
0.01 mg ml 1. The adsorbance versus time profile is shown in Fig. 6 together with the contact angle plotted against time. As seen from Fig. 6, the change in adsorbance with time up to 1000 min is similar to that of Fig. 4. For further incubation longer than 1000 min, the adsorbance increases to exceed the line E while the contact angle remains unchanged. We have observed a lot of minute whitish condensates distributed sparsely over the surface of mica sheets that gave the extraordinary large values of adsorbance. It follows from this fact that the condensation of BSA occurs on the mica-solution interface if the mica sheets are left immersed in solution for a sufficiently long period.
Fig. 5. A schematic illustration showing the orientation of adsorbed BSA molecules at: (a) an early stage where monolayer is formed; and (b) a subsequent stage of the occurrence of multilayer adsorption when the adsorption is prolonged.
Fig. 6. Adsorbance versus time profile of BSA determined at a concentration 0.01 mg ml 1 for prolonged adsorption. The contact angles are also plotted against time. The solution was kept at 25 8C and pH 5.8.
3.3. Comparison with previous studies In the previous studies on the BSA adsorption, the adsorbances were determined under different conditions concerning solution concentration and incubation time. Therefore, it seems meaningless to attempt unconditionally a comparison of the data of adsorbance as reported. Empirically, it is reasonable to say that the incubation time required for adsorbance to reach a given value is roughly proportional to the inverse of concentration. In fact, if the adsorbances are plotted against the product of concentration and incubation time, it is seen that the adsorbances fall along a single curve regardless of any slight deviations [21,22]. Consequently, it is essential to make a comparison of the experimental values of adsorbance determined against the same value of the product of concentration and incubation time.
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The experimental results reported by Fitzpatrick et al. [16,18] and Gallinet and Gauthier-Manuel [17] are compared with the present study according to the way as mentioned above. Fitzpatrick et al. have determined the adsorbance to be 5.79/1.0 mg m 2 under the condition where the mica surface was exposed to BSA solution at a concentration of 0.005 mg m 1. The incubation was continued overnight at room temperature (17 /19 h). If the period of incubation is set equal to 18 h (1080 min), the product of concentration and incubation time is 5.4 ( /0.005 /1080) mg ml 1 min. The results of their adsorbance measurement are compared with the adsorbance versus time profile determined in the present study. Their value of adsorbance 5.79/1.0 mg ml 1 is to be compared with the adsorbance at 2700 min in the adsorbance versus time profile determined for 0.002 mg ml 1 (Fig. 4) and at 540 min for 0.01 mg ml 1 (Fig. 6). It is seen from Figs. 4 and 6 that, at these periods of incubation time, the adsorbances are evidently higher than the level marked S. This means that BSA molecules are adsorbed onto the side-on monolayer already formed, in other words, there occurs multilayer adsorption. It is supposed that Fitzpatrick et al. have taken no account of the occurrence of multilayer adsorption and regarded the adsorbance of multilayer as that of monolayer. Presumably, this is a reason why they concluded the orientation of adsorbed BSA molecules to be end-on. In the experiments carried out by Gallinet and Gauthier-Manuel, mica surfaces were exposed to a BSA solution at a concentration of 0.04 mg ml 1 for a period of 45 min. In consideration of the product 1.8 ( /0.04 /45) mg ml 1 min, the identical adsorbance to be compared is found at 900 min in the present profile determined for 0.002 mg ml 1 (Fig. 4) and at 180 min for 0.01 mg ml 1 (Fig. 6). As seen from Figs. 4 and 6, the adsorbances at these periods of incubation fall above the line S. It follows that multilayer adsorption is dominant at this stage. Gallinet and Gauthier-Manuel have described the following two facts. First, a repulsive force becomes obvious from a separation 42.0 nm between two facing mica surfaces. This fact suggests the occurrence of multilayer adsorption because the thick-
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ness of a BSA layer on each mica surface is considerably larger than the size of BSA molecule. Secondly, two mica surfaces come closer stepwise finally to a separation 4.359/0.1 nm, which corresponds to side-on adsorption of BSA molecules to mica surfaces. These findings are in agreement with those of the present study.
4. Conclusions The adsorption of BSA to mica surfaces has been studied with the intention of elucidating a problem about whether the orientation of adsorbed BSA molecules is side- or end-on. For this purpose, the adsorbance has been measured as a function of time by means of a Mettler UM3 ultramicrobalance. In addition to the adsorbance measurement, the contact angle of water droplets resting on the adsorbed BSA layer has been measured. The contact angle measurement gives information on the degree of surface covering. From the results of these measurements, it concludes that BSA molecules are adsorbed side-on to mica surface. In order to determine the orientation correctly, it is essential to take a value of adsorbance determined at the stage where the surface covering by adsorbed BSA molecules is completed to form a monolayer. This is because the formation of monolayer is followed by the occurrence of multilayer adsorption if the adsorption is prolonged.
References [1] J.L. Brash, T.A. Horbett, in: J.L. Brash, T.A. Horbett (Eds.), Proteins at Interfaces ACS Symposium Series 343, America Chemical Society, Washington DC, 1987. [2] T.A. Horbett, J.L. Brash, in: T.A. Horbett, J.S. Brash (Eds.), Proteins at Interfaces II ACS Symposium Series 602, American Chemical Society, Washington DC, 1995. [3] M. Malmsten, in: M. Malmsten (Ed.), Biopolymers at Interfaces Surfactant Science Series 75, Marcel Dekker, New York, 1988. [4] C.A. Haynes, W. Norde, Colloids Surf. B 2 (1994) 517. [5] P.G. Squire, P. Moser, C.T. O’Konski, Biochemistry 7 (1968) 4261. [6] T. Peter, Jr., Adv. Protein Chem. 37 (1985) 161.
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[7] M.E. Soderquist, A.G. Walton, J. Colloid Interface Sci. 75 (1980) 386. [8] B.D. Fair, A.M. Jamieson, J. Colloid Interface Sci. 77 (1980) 525. [9] H. Shirahama, T. Suzawa, J. Colloid Interface Sci. 104 (1985) 416. [10] N. Muramatsu, T. Kondo, J. Colloid Interface Sci. 153 (1992) 23. [11] K. Kandori, M. Mukai, A. Fujiwara, A. Yasukawa, T. Ishikawa, J. Colloid Interface Sci. 212 (1999) 600. [12] C.-G. Golander, E. Kiss, J. Colloid Interface Sci. 121 (1988) 240. [13] T.P. Burghardt, D. Axelrod, Biophys. J. 33 (1981) 455. [14] D.V. Dass, H.J. van Enckevort, A.G. London, J. Colloid Interface Sci. 116 (1987) 523.
[15] S.H. Lee, E. Ruckenstein, J. Colloid Interface Sci. 125 (1988) 365. [16] H. Fitzpatrick, P.F. Luckham, S. Eriksen, K. Hammond, Colloid Surf. 65 (1992) 43. [17] J.-P. Gallinet, B. Gauthier-Manuel, Colloid Surf. 68 (1992) 189. [18] H. Fitzpatrick, P.F. Luckham, S. Eriksen, K. Hammond, J. Colloid Interface Sci. 149 (1992) 1. [19] H. Terashima, J. Colloid Interface Sci. 212 (1999) 100. [20] H. Terashima, J. Colloid Interface Sci. 245 (2002) 81. [21] J.C. Dijt, M.A. Cohen Stuart, G.J. Fleer, Macromolecules 27 (1994) 3207. [22] H. Terashima, Phys. Chem. Chem. Phys. 2 (2000) 5252.
Adsorption of bovine serum albumin onto mica surfaces studied by a direct weighing technique Hiroshi Terashima , Tatsuo Tsuji Institute of Materials Science, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan Received 12 February 2002; accepted 18 March 2002
Abstract Adsorption of bovine serum albumin (BSA) from aqueous solution to muscovite mica has been investigated through the measurements of two quantities: (1) the adsorbance, i.e. adsorbed mass per unit area measured as a function of incubation time by means of a Mettler UM3 ultramicrobalance; and (2) the contact angle of water droplets resting on the adsorbed BSA layer to know the degree of surface covering. The protein concentration explored was 0.002 and 0.01 mg ml 1 in aqueous solution at pH 5.8. The temperature was kept constant at 25 8C. The following results have been obtained in the present study. (1) The initial rate of adsorption is more rapid than generally accepted. The surface of mica is completely covered by adsorbed BSA molecules within 10 min even for a dilute solution at a concentration 0.002 mg ml 1. (2) At a stage where the surface covering is completed, the adsorbance levels off at a steady value, which is approximately equal to mass per unit area of side-on monolayer. It concludes from this result that BSA molecules are adsorbed side-on directly onto mica surface to form a side-on monolayer. (3) If the adsorption is prolonged, the adsorbance exceeds the mass per unit area of side-on monolayer owing to the adsorption of BSA molecules onto the side-on monolayer already formed. This fact indicates the occurrence of multilayer adsorption of BSA. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Bovine serum albumin; Muscovite mica; Protein adsorption; Ultramicrobalance
1. Introduction Bovine serum albumin (BSA) has been often employed as an adsorbate for the study of protein adsorption from aqueous solution to solid surfaces [1 /4]. The shape of BSA molecule is known to be like a prolate ellipsoid of revolution with major
Corresponding author. Tel.: /81-298-53-5123; fax: /81298-55-7440 E-mail address: [email protected] (H. Terashima).
axis 14.1 nm and minor axis 4.1 nm [5,6]. Because of the ellipsoidal shape, there may occur two types of orientation of adsorbed BSA molecules: these are side- and end-on adsorption if major axis is parallel and perpendicular to solid surface, respectively. Most of the studies of BSA adsorption have aimed chiefly at determining the orientation of BSA molecules adsorbed on solid surfaces. The orientation can be determined by the following two ways. First, the geometrical thickness of adsorbed BSA monolayer is measured so as to be compared with the length of major and minor
0927-7765/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 2 ) 0 0 0 4 4 - 9
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axis. Secondly, the measured values of adsorbance, i.e. adsorbed mass per unit area are compared with the monolayer capacity, i.e. mass per unit area of BSA monolayer calculated assuming either orientation. The monolayer capacity is calculated to be 2.01 mg m 2 for side-on and 7.53 mg m 2 for hexagonally close packed end-on monolayer. The adsorption of BSA to solid-solution interface has been studied by a variety of experimental methods including the measurement of the difference in solution concentration between before and after the adsorption [7 /11], ellipsometry [12], total internal reflection/fluorescence recovery technique [13], radiolabeling technique [14,15], surface force measurement [16,17] and XPS (X-ray photo-electron spectroscopy) [18]. Among these studies, we are interested in the experiments of the BSA adsorption to mica surfaces investigated by the surface force measurement and XPS. The reasons are as follows: (1) the experimental approach is direct so that no ambiguity is involved in the interpretation of the results; (2) the use of mica as an adsorbent makes it possible to establish a simplified situation of the BSA adsorption to planar surface owing to the molecular scale smoothness of cleaved mica surface. Fitzpatrick et al. [16,18] have studied the BSA adsorption to mica surface by a combined use of surface force measurement and XPS. According to their surface force measurement, the separation of closest approach of two facing mica surfaces is measured to be 18/20 nm, i.e. a layer of /10 nm on each mica surface and the refractive index in the gap at this separation to be 1.419/0.03, from which the adsorbance is calculated to be 5.5 /6.5 mg m 2. On the other hand, the measurement of adsorbance by XPS provides a value 5.79/1.0 mg m 2, which is consistent with the result obtained by the surface force measurement. They have concluded from their results that an end-on adsorption of BSA molecules prevails under their experimental conditions. In the same year, a French group has reported a similar experiment. Using a self-controlled-drive surface force apparatus, Gallinet and Gauthier-Manuel [17] have measured the thickness of BSA layer adsorbed on mica surface and the forces required to desorb the BSA molecules from
the gap between the two mica surfaces. They have observed a stepwise increase in repulsive forces on compression and found that the distance between the steps is approximately equal to the minor axis of BSA molecules. They have concluded that BSA molecules are adsorbed side-on. With regard to the orientation of BSA molecules adsorbed on mica surfaces, the above two groups have drawn their respective conclusions which are inconsistent. The purpose of the present study is to elucidate the origin of this inconsistency by examining the process of BSA adsorption. In the present study, an entirely separate method has been applied to the study of BSA adsorption to mica surface. The adsorbance of BSA on mica surface is determined from an increase in weight of mica sheets due to BSA adsorption. A sensitive ultramicrobalance is used for this weighing. In addition to the adsorbance measurement, the contact angle is measured of water droplets placed on mica surfaces bearing BSA layer of known adsorbance. The measurement of contact angle gives information on the degree of surface covering of mica surface by adsorbed BSA molecules. On the basis of the experimental results of both adsorbance and contact angle, the orientation of adsorbed BSA molecules has been determined. This paper describes the following four items. First, a direct method is described of studying the adsorption of protein (BSA) from aqueous solution to mica surfaces by the use of a highly sensitive ultramicrobalance. Secondly, the experimental results are presented concerning the change in adsorbance and contact angle with time at dilute concentrations. Thirdly, a discussion is made about the orientation of adsorbed BSA molecules. Finally, a comparison is made of the present results with the previous studies.
2. Materials and methods 2.1. Materials BSA was supplied from Sigma Chemical Company Ltd. (Fraction V powder, Ref. A-7030) and used as received. The mica used as an adsorbent
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was Muscovite of grade No. 4 and quality C and SS (clear and slightly stained), which is the product of C.M. Rajgarhir (Giridih, India), purchased in sheets from M. Watanabe & Co. Ltd. in Tokyo. The pure water used as a solvent was prepared by double distillation in an all-fused quartz still. 2.2. Principle of adsorbance determination The amount of BSA molecules adsorbed on mica surface is determined from an increase in weight of mica sheets caused by the adsorption of BSA. A commercial Mettler UM3 ultramicrobalance of the readability 10 4 mg was used for the weighing. The mica sheets are weighed before and after the immersion of mica sheets in an aqueous solution of BSA at a given concentration; the temperature of solution is kept constant. The difference in weight of mica sheets between before and after the immersion is divided by the total surface area of mica sheets to give the adsorbance, i.e. adsorbed mass per unit area. Owing to the molecular scale smoothness of the surface of cleaved mica sheets, there is no ambiguity in the determination of the total surface area; consequently, the adsorbance can be determined unambiguously.
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occurring on the surface of mica sheets. Our experiment on the ion exchange [20] has revealed that the dissolution ceases in 30 min and the weight of mica sheets is unchanged afterwards. On the basis of this result, a preliminary immersion in water for 30 min or longer is made throughout the experiment. After the preliminary immersion in water, the mica sheets are taken out of water and dried in air. To reduce the time needed for drying, all of water droplets remaining on the mica surfaces and the fused quartz holder are removed by touching the edge of small piece of clean filtering paper. The pair is weighed together with the holder by hanging it from the hook of the Mettler UM3 ultramicrobalance as shown in Fig. 1. The weight is noted as a reference for zero adsorbance. Then, the pair is immersed in an aqueous solution of BSA at a given concentration. No change in concentration takes place during the adsorption because of the smallness of the surface area of mica sheets. The vessel containing the solution is set in a water bath to keep the temperature constant. After a given period of adsorption, the pair is transferred from solution to water in order
2.3. Experimental procedure A brief description is given below of the practical procedure for the measurement of adsorbed amount; the details have been previously reported [19]. A pair of thin sheets of mica 32 /39 mm in size ( 50 /10 4 m2 in total surface area) is prepared by cleavage and mounted together on a holder made of fused quartz rods of about 0.3 mm in diameter (Fig. 1). In the beginning, the pair of mica sheets is immersed in pure water for a period longer than 30 min. This immersion is particularly necessary for the experiment of protein adsorption from aqueous solution to mica. We have already found that the weight of mica sheets decreases markedly once the mica sheets are immersed in aqueous solution [19,20]. This decrease in weight is caused by the dissolution of metal ions, mainly potassium ions, from mica sheets owing to an ion exchange
Fig. 1. A schematic drawing representing the mica sheets and fused quartz holder used for weighing by means of a Mettler UM3 ultramicrobalance. (1) Mica sheets; (2) holder; (3) small hook for hanging mica sheets; (4) metal hook equipped with Mettler UM3 ultramicrobalance.
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to rinse the residual solution remaining on mica surfaces. Subsequently to the rinse followed by drying in air, the second weighing is made to measure the change in weight of mica sheets. 2.4. Measurement of contact angle The contact angle of water droplets resting on adsorbed BSA layer is measured to know the degree of surface covering of mica surfaces by adsorbed BSA molecules. A sheet of mica covered by adsorbed BSA layer of known adsorbance is placed horizontally on a glass cell. Some of droplets of pure water are gently dropped on the surface of mica sheet by the use of a fine syringe and then the glass cell is tightly shielded. The inside of the glass cell is so designed as to be saturated by water vapor; consequently, the shape and size of the droplets are kept unchanged during the contact angle measurement. The profile of droplets is observed through a window of the glass cell using a microscope equipped with a goniometer, of which the readability is a quarter of 18. As a preliminary experiment, we have measured the contact angle of water droplets for two cases. Case 1: Water droplets are placed on the surface of mica sheets rinsed once in pure water and dried, that is, no BSA molecules are present on the surface. The average of measured values of contact angles of this case is 158. Case 2: The contact angle is measured of water droplets placed on the surface of thick BSA layer fully covering the mica surface. The measurement gives the contact angle 708 in this case. The degree of surface covering can be known by examining whether the measured values of contact angle fall between 15 and 708 or along the level of 708.
3. Results and discussion 3.1. Adsorption at 0.002 mg ml 1 BSA was adsorbed from dilute aqueous solution to mica surface at a concentration of 0.002 mg ml 1. By using such a dilute solution, the initial rate of adsorption is reduced so that we are able to measure the change in adsorbance with time at
early stages of adsorption. The temperature was held constant at 258. The pH measurement of solution gave a value around 5.8. No buffer was used in the present experiment to avoid any possible influence of buffer on weighing. In practice, the measurement of adsorbance was carried out first and then the contact angle was measured by placing water droplets on the adsorbed BSA layers. However, before presenting the results of the adsorbance measurement, we examine the results concerning the change in contact angle with both time and adsorbance. To interpret the results of the adsorbance versus time profile, it is convenient to know in advance the degree of surface covering of mica by adsorbed BSA molecules. The measured values of contact angle are plotted against time and adsorbance in Fig. 2 (a) and (b), respectively. A dotted line is drawn in Fig. 2(a) at 158 which is a mean value of contact angle of water droplets resting on bare surface of mica sheets rinsed once and dried in air. This is the case where no adsorbed BSA molecules are present on mica surfaces. Two dashed lines marked S and E in Fig. 2(b) indicate the respective mass per unit area of two kinds of BSA monolayer. The line S is drawn at 2.01 mg m 2 for side-on monolayer and the line E at 7.53 mg m 2 for hexagonally close packed end-on monolayer. As seen from Fig. 2(a), the contact angle rises steeply from 158 to about 608 within a few minutes of immersion and attains to a steady value of 708 within 10 min. Fig. 2(b) shows that the contact angle exceeds 608 even at about one fifth of the adsorbance corresponding to side-on monolayer and reaches the steady value of 708 at adsorbances indicated by the line S. It follows from these results that the adsorbed BSA molecules are uniformly distributed to cover the whole surface of mica without aggregating in cluster at and above adsorbances corresponding to side-on monolayer. Fig. 3 shows the adsorbance versus time profile determined at a concentration of 0.002 mg m 2. It is seen from Fig. 3 that the adsorbance attains to the level indicated by the line S within 10 min and remains unchanged with time afterwards. From a comparison between Fig. 2(a) and Fig. 3, it is seen that the attainment of adsorbance to the level marked S is coincident with that of contact angle
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Fig. 2. Contact angles of water droplets resting on adsorbed BSA layer, plotted against: (a) incubation time; and (b) adsorbance. A dotted line drawn in (a) indicates a contact angle 158 of water droplets on bare surface of mica sheet rinsed once in water and then dried in air. Two dashed lines marked S and E are drawn in (b), respectively, at 2.01 mg m 2 for side-on monolayer and at 7.53 mg m 2 for hexagonally close packed end-on monolayer.
to 708. The results obtained may be summarized as follows. At a stage where the whole surface of mica is finished to be entirely covered by adsorbed BSA molecules, the adsorbance reaches to a value, which is identical with the mass per unit area of side-on monolayer. This result leads to a conclusion that BSA molecules are adsorbed side-on directly to mica surface to form a side-on monolayer. The adsorption for a long duration has been investigated. Fig. 4 shows the adsorbance versus time profile determined at 0.002 mg ml 1 for prolonged adsorption. The result of the contact angle measurement is also shown together. It is seen from Fig. 4 that most of the measured values of adsorbance fall between the two dashed lines S and E while the contact angle is constant to be around 708 irrespective of time and adsorbance. The excess of adsorbance above the line S can be attributed to an adsorption of BSA molecules on to the adsorbed BSA layer because the mica surface has been confirmed to be completely covered by side-on monolayer at earlier stages of adsorption. Accordingly, it is safe to say that a multilayer adsorption of BSA molecules takes place if the adsorption is continued for a long period. Although it is difficult to specify the orientation of multiply adsorbed BSA molecules,
BSA molecules are supposed to be adsorbed sideon to be piled or end-on between BSA molecules already adsorbed side-on. The orientation of adsorbed BSA molecules at initial and subsequent stages of adsorption are schematically shown in Fig. 5(a and b), respectively. 3.2. Adsorption at 0.01 mg ml 1 To investigate the process of further prolonged adsorption, we conducted an experiment of the adsorption from a more concentrated solution of
Fig. 3. Adsorbance versus time profile of BSA adsorbed from aqueous solution at a concentration 0.002 mg ml 1, a temperature 25 8C, and a pH 5.8. Two dashed lines S and E indicate the adsorbances corresponding to side- and end-on monolayer, respectively.
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Fig. 4. Adsorbance versus time profile of BSA determined at a concentration 0.002 mg ml 1 for prolonged adsorption. The contact angles are also plotted against time. The solution was kept at 25 8C and pH 5.8.
0.01 mg ml 1. The adsorbance versus time profile is shown in Fig. 6 together with the contact angle plotted against time. As seen from Fig. 6, the change in adsorbance with time up to 1000 min is similar to that of Fig. 4. For further incubation longer than 1000 min, the adsorbance increases to exceed the line E while the contact angle remains unchanged. We have observed a lot of minute whitish condensates distributed sparsely over the surface of mica sheets that gave the extraordinary large values of adsorbance. It follows from this fact that the condensation of BSA occurs on the mica-solution interface if the mica sheets are left immersed in solution for a sufficiently long period.
Fig. 5. A schematic illustration showing the orientation of adsorbed BSA molecules at: (a) an early stage where monolayer is formed; and (b) a subsequent stage of the occurrence of multilayer adsorption when the adsorption is prolonged.
Fig. 6. Adsorbance versus time profile of BSA determined at a concentration 0.01 mg ml 1 for prolonged adsorption. The contact angles are also plotted against time. The solution was kept at 25 8C and pH 5.8.
3.3. Comparison with previous studies In the previous studies on the BSA adsorption, the adsorbances were determined under different conditions concerning solution concentration and incubation time. Therefore, it seems meaningless to attempt unconditionally a comparison of the data of adsorbance as reported. Empirically, it is reasonable to say that the incubation time required for adsorbance to reach a given value is roughly proportional to the inverse of concentration. In fact, if the adsorbances are plotted against the product of concentration and incubation time, it is seen that the adsorbances fall along a single curve regardless of any slight deviations [21,22]. Consequently, it is essential to make a comparison of the experimental values of adsorbance determined against the same value of the product of concentration and incubation time.
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The experimental results reported by Fitzpatrick et al. [16,18] and Gallinet and Gauthier-Manuel [17] are compared with the present study according to the way as mentioned above. Fitzpatrick et al. have determined the adsorbance to be 5.79/1.0 mg m 2 under the condition where the mica surface was exposed to BSA solution at a concentration of 0.005 mg m 1. The incubation was continued overnight at room temperature (17 /19 h). If the period of incubation is set equal to 18 h (1080 min), the product of concentration and incubation time is 5.4 ( /0.005 /1080) mg ml 1 min. The results of their adsorbance measurement are compared with the adsorbance versus time profile determined in the present study. Their value of adsorbance 5.79/1.0 mg ml 1 is to be compared with the adsorbance at 2700 min in the adsorbance versus time profile determined for 0.002 mg ml 1 (Fig. 4) and at 540 min for 0.01 mg ml 1 (Fig. 6). It is seen from Figs. 4 and 6 that, at these periods of incubation time, the adsorbances are evidently higher than the level marked S. This means that BSA molecules are adsorbed onto the side-on monolayer already formed, in other words, there occurs multilayer adsorption. It is supposed that Fitzpatrick et al. have taken no account of the occurrence of multilayer adsorption and regarded the adsorbance of multilayer as that of monolayer. Presumably, this is a reason why they concluded the orientation of adsorbed BSA molecules to be end-on. In the experiments carried out by Gallinet and Gauthier-Manuel, mica surfaces were exposed to a BSA solution at a concentration of 0.04 mg ml 1 for a period of 45 min. In consideration of the product 1.8 ( /0.04 /45) mg ml 1 min, the identical adsorbance to be compared is found at 900 min in the present profile determined for 0.002 mg ml 1 (Fig. 4) and at 180 min for 0.01 mg ml 1 (Fig. 6). As seen from Figs. 4 and 6, the adsorbances at these periods of incubation fall above the line S. It follows that multilayer adsorption is dominant at this stage. Gallinet and Gauthier-Manuel have described the following two facts. First, a repulsive force becomes obvious from a separation 42.0 nm between two facing mica surfaces. This fact suggests the occurrence of multilayer adsorption because the thick-
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ness of a BSA layer on each mica surface is considerably larger than the size of BSA molecule. Secondly, two mica surfaces come closer stepwise finally to a separation 4.359/0.1 nm, which corresponds to side-on adsorption of BSA molecules to mica surfaces. These findings are in agreement with those of the present study.
4. Conclusions The adsorption of BSA to mica surfaces has been studied with the intention of elucidating a problem about whether the orientation of adsorbed BSA molecules is side- or end-on. For this purpose, the adsorbance has been measured as a function of time by means of a Mettler UM3 ultramicrobalance. In addition to the adsorbance measurement, the contact angle of water droplets resting on the adsorbed BSA layer has been measured. The contact angle measurement gives information on the degree of surface covering. From the results of these measurements, it concludes that BSA molecules are adsorbed side-on to mica surface. In order to determine the orientation correctly, it is essential to take a value of adsorbance determined at the stage where the surface covering by adsorbed BSA molecules is completed to form a monolayer. This is because the formation of monolayer is followed by the occurrence of multilayer adsorption if the adsorption is prolonged.
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