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Competitive adsorption of collagen and bovine serum albumin—effect of the surface wettability
Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263
Competitive adsorption of collagen and bovine serum albumin—effect of the surface wettability Peiqing Ying, Gang Jin∗ , Zulai Tao Institute of Mechanics, Chinese Academy of Sciences, 15 Bei-Si-Huan West Road, Beijing 100080, China Accepted 21 October 2003
Abstract The competitive adsorption of collagen and bovine serum albumin (BSA) on surfaces with varied wettability was investigated with imaging ellipsometry, and ellipsometry. Silane modified silicon surfaces were used as substrates. The results showed that surface wettability had an important effect on protein competitive adsorption. With the decrease of surface wettability, the adsorption of collagen from the mixture solution of collagen and BSA decreased, while the adsorption of BSA increased. © 2003 Elsevier B.V. All rights reserved. Keywords: Protein competitive adsorption; Surface wettability; Ellipsometry
1. Introduction Protein competitive adsorption is involved in many interfacial phenomena such as hemocompatibility of biomaterials, cellular adhesion and growth on substrates [1–4]. The competitive adsorption between collagen and serum albumin is of great importance in biomaterial design [5–7]. Previous studies on the competitive adsorption between these two kinds of proteins on hydrophobic or moderately hydrophobic surfaces showed that human serum albumin reduced collagen adsorption, and albumin was the only adsorbing protein [6–8]. In our previous studies [9] we investigated the competitive adsorption of collagen and BSA on highly hydrophilic and highly hydrophobic surfaces and the results showed that BSA preferentially adsorbed onto the hydrophobic surface, while collagen on the hydrophilic surface. Since surfaces with different hydrophobicity are often used as substrates, and the surface hydrophobicity is a key factor affecting competitive adsorption, it is necessary to study protein competitive adsorption on surface with varied wettability. In this paper, silicon surfaces modified with silane to be with varied wettability were used as
∗ Corresponding author. Tel.: +86-10-62631816; fax: +86-10-62561284. E-mail address: [email protected] (G. Jin).
0927-7765/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2003.10.015
substrates. With its highly sensitivity, imaging ellipsometry [10–12] and ellipsometry [13–16] were used as techniques to analysis the protein competitive adsorption.
2. Materials and methods 2.1. Proteins Bovine serum albumin (BSA) and its antibody were purchased from Sigma. Calf skin purified collagen, was purchased from Boehringer Mannheim Biochemica (Collagen S). 2.2. Substrates Silicon wafers (thin film 7 mm × 20 mm) with an optically polished flat surface and a natural silicon dioxide layer were used as substrates. The wafer surface was prepared as hydrophilic by washing in both TL1 solution (H2 O:30% H2 O2 :25% NH4 OH = 5:1:1, v/v/v) and TL2 solution (H2 O:30% H2 O2 :37% HCl = 6:1:1, v/v/v). Through the reaction of TL1 and TL2 with basic and acid solution, and oxidation of hydrogen peroxide, it not only removed contaminants of the silicon surface, but also improved the number of silanol groups on the surface thus making
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P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263
surface hydrophilic. Hydrophobic surface with varied wettability was prepared with silanization of the hydroxylated surface. After rinsed in distilled water and ethanol, the hydroxylated surfaces were incubated in dichlorodimethylsilane solution (5–0 l in 20 ml trichloroethylene) for 2–5 min, followed by rinsing in ethanol and trichloroethylene in sequence. All chemicals used were of analytical grade. Pure water (resistivity 18.3 M cm) was produced by ion exchange demineralization, and followed by passing through a Milli-Q plus system from Millipore (Millipore, Bedford, MA). 2.3. Contact angle measurement Water contact angles were measured at 25 ◦ C for dried wafers with the sessile drop method. Deionized water (4 l) was gently dropped on the surfaces and the contact angle was read directly using a goniometer. The contact angles for hydrophilic and varied hydrophobic silicon wafers were about 5◦ , 15◦ , 40◦ , 45◦ , 50◦ , 70◦ and 80◦ , respectively, with a deviation of ±1◦ . 2.4. Protein adsorption and competitive adsorption Protein adsorption and competitive adsorption were carried out in PBS solution (8 mM Na2 PO4 ·2H2 O, 2.68 mM KCl, 1.14 mM KH2 PO4 , 137 mM NaCl; pH 7.2). Protein concentrations are 1 and 0.1 mg/ml for BSA and collagen, respectively, which are near the concentrations of BSA and extracellular protein in serum-containing culture media. Single or binary solutions containing collagen, BSA or their mixture were used. Silicon wafers were incubated in protein solutions for 2 h, then washed with PBS and deionized water, dried with nitrogen. The detection of BSA adsorption amount in the competitive adsorption was based on the BSA/anti-BSA interaction with their affinity as described previously [9]. In brief, the BSA or co-adsorption layer was immersed in anti-BSA solution. Based on the specific binding between BSA and anti-BSA, anti-BSA in the solution bound with BSA in the layer to form protein complex of BSA/anti-BSA and resulted in a variation of surface concentration (Fig. 1). The more BSA
Fig. 1. Thickness distribution of BSA and BSA/anti-BSA complex layer on silicon substrate visualized with imaging ellipsometry.
adsorbed in co-adsorption layer, the more anti-BSA bond onto the protein complex layer resulting in a large increase of the surface concentration. In this way, the amount of BSA adsorbed in the co-adsorption layer could be deduced from the surface concentration variation, so as to determine the competitive adsorption between BSA and collagen. 2.5. Ellipsometry analysis A homemade ellipsometric imaging system was used for the visualization and quantification of the surface concentration of protein adsorption layer. Compared with the conventional ellipsmetry, imaging ellipsometry has the advantage of distinguishing the effects of singularities (local abnormal variations in the image introduced by contamination) appearing on the surfaces. The basic experimental set-up was a conventional polarizer–compensator–sample-analyzer (PCSA) null ellipsometer. An interference filter at 632.8 nm wavelength was placed in the incident optical path to increase the ellipsometric contrast of image. The combined null and off-null ellipsometry was used at an incident angle close to the pseudo-Brewster angle of the substrate. An image of 7 mm × 15 mm of a surface was focused onto the CCD video camera for intensity measurements. The optical components in the system were adjusted to fulfill the null conditions on a silicon substrate without adsorbed layers and the off-null ellipsometric principle was used to measure the adsorption layer thickness distribution [10]. The spatial resolution of the imaging system is in the order of micron laterally and 0.1 nm vertically. The video signal corresponding to the thickness distribution was captured, digitized and stored in gray-scale format in a computer. Under this condition, the detected intensity “I” was related to the thickness of the layer according to I = kd2 which was a linear relationship between the intensity and the square of the thickness of the adsorbed protein layer or the square of the surface concentration of proteins [17]. This proportionality showed a deviation of less than ±2% up to d ≈ 5 nm. As for the same protein and the same ellipsometric conditions, k is constant and can be determined by the protein layer with known gray-scale and thickness. The absolute thickness of protein layer used to calculate the constant k was calibrated by conventional ellipsometer (SE 400, SENTECH, Germany). The surface concentration of protein adsorption layer can be calculated according to the relationship between surface concentration and film thickness: surface concentration g cm−2 ≈ K × d(nm) where K ≈ 0.12 [18].
P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263
3. Results and discussion 3.1. BSA adsorption and BSA/anti-BSA interaction Fig. 2 shows the increase of surface concentration corresponding to the adsorption of BSA from pure solution and then the interaction of anti-BSA with the adsorbed BSA. The adsorption amount of BSA increased with the increase of the contact angle and it became stable at the quite hydrophobic surface where the contact angle was about 80◦ . Unlike the adsorption of BSA, the surface concentration of the BSA/anti-BSA complex slightly decreased with the increase of the contact angle, showing that BSA adsorbed on less hydrophobic surface was more likely to bind with anti-BSA. The decrease of the BSA/anti-BSA binding ability with the increase of hydrophobicity might be related with the extent of BSA conformation change after adsorption. The more BSA conformation changed, the lower binding ability of BSA with anti-BSA. On the less hydrophobic surface the main driving force for BSA adsorption is hydrogen bond and electrostatic interaction, BSA adsorb to the surface without much conformation change. BSA adsorbed on the less hydrophobic surface maintains its high anti-BSA binding ability. With the decrease of the surface wettability, that is, with the increase of surface hydrophobicity, the hydrophobic interaction became the main driving force for BSA adsorption which caused more conformation change of the adsorbed BSA than that on the less hydrophobic surface, thus decreasing the anti-BSA binding ability. 3.2. Collagen adsorption The adsorption of collagen from pure solution was also different from that of BSA (Fig. 3). With the contact angle
0.6 0.4 0.2 0
0.6 0.4 0.2 0.0 0
20 40 60 o Advancing contact angle ( )
80
Fig. 2. BSA (1 mg/ml) adsorption and BSA/anti-BSA interaction on surfaces with varied hydrophobicity. Adsorption time and BSA/anti-BSA interaction time were 2 and 1 h, respectively.
20 40 60 o Advancing contact angle ( )
80
Fig. 3. Collagen (0.1 mg/ml) adsorption on surfaces with varied hydrophobicity, adsorption time was 2 h.
increased, the collagen adsorption amount decreased slowly (from 5◦ to 45◦ ), then it increased quickly (from 50◦ to 85◦ ). With a molecular weight of 300,000 Da, Collagen is much larger than BSA (66,200 Da). Collagen molecule is also rather rigid and cannot change its conformation as easy as BSA [7,19,20]. On the slightly hydrophobic surface, the hydrogen bond and electrostatic interaction between collagen and surface decreased with the increase of the contact angle. Since collagen molecule cannot change its conformation greatly, the low density of surface methyl cannot supply enough binding sites for collagen adsorption. The adsorption amount decreased with the increase of hydrophobicity. As the contact angle further increased from 50◦ to 85◦ , the strong hydrophobic interaction between collagen and surface caused more adsorption amount which is similar with the adsorption of BSA. 3.3. Competitive adsorption between collagen and BSA Fig. 4 represents the surface concentration change corresponding to the competitive adsorption between collagen and BSA, and the binding of anti-BSA with adsorbed BSA. Since in most serum-containing cell culture media the concentrations for extracelluar protein and BSA are often near 0.1
Collagen, BSA Collagen, BSA/anti-BSA
0.8 Surface concentration 2 (µg/cm )
Surface concentration 2 (µg/cm )
0.8
BSA BSA/anti-BSA
0.8
Collagen
1.0 Surface concentration 2 (µg/cm )
The results of imaging ellipsometry shown in gray scale were processed to be the surface concentration of the adsorption layer.
261
0.6 0.4 0.2 0.0
0
20
40
60
80
100
o
Advancing contact angle ( ) Fig. 4. Competitive adsorption of collagen (0.1 mg/ml) and BSA (1 mg/ml) and BSA/anti-BSA interaction on surfaces with varied hydrophobicity. Adsorption time and BSA/anti-BSA interaction time were 2 and 1 h, respectively.
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Collagen
Mass percent (%)
100
folding contribute to the preferentially adsorption of BSA on the more hydrophobic surfaces. On the less hydrophobic surfaces the main driving force for BSA adsorption is hydrogen bond and electrostatic interaction, both collagen and BSA adsorb to the surfaces without much conformation change. The binding affinity becomes the main factor that leads to the preferential adsorption of collagen on the more hydrophilic surfaces.
BSA
80 60 40 20 0
0
20 40 60 80 o Advancing contact angle ( )
100
Fig. 5. Mass percent of collagen and BSA for the competitive adsorption of collagen (0.1 mg/ml) and BSA (1 mg/ml).
and 1 mg/ml, respectively, competitive adsorption between collagen (0.1 mg/ml) and BSA (1 mg/ml) were investigated. The surface concentration of the co-adsorption layer of collagen and BSA decreased as the surface change from highly hydrophilic to slightly hydrophobic (contact angle from 20◦ to 40◦ ), but varied slightly when the surface hydrophobicity further increased (contact angle from 40◦ to 85◦ ). After the incubation of the co-adsorption layer in the anti-BSA solution, the surface concentration hardly increased on the range of hydrophilic surfaces, while increased obviously on hydrophobic surfaces. Supposed that the affinity between anti-BSA and BSA of co-adsorption layer was the same as of pure BSA adsorption layer, the mass percent of BSA in the co-adsorption layer can be deduced from the surface concentration increase introduced by anti-BSA binding with BSA. The mass percent of collagen and BSA are showed in Fig. 5. The percent of collagen in the co-adsorption decreased with the increase of contact angle, while BSA increased. On the highly hydrophilic surfaces (contact angle 5◦ –20◦ ), nearly 100% of the protein adsorbed was collagen, but less than 10% on the highly hydrophobic surface (contact angle 85◦ ). The result was quite coincident with the previous result that during the competitive adsorption collagen preferentially adsorbed on the hydrophilic surface while BSA on hydrophobic surface [9]. With the increase of surface hydrophobicity, the competitive adsorption amount of collagen decreased, while BSA increased. Factors that affect competitive protein adsorption include protein binding affinities, rates of transport and rates of unfolding [21]. The rates of transport mainly affect the initial competitive adsorption. The abundant proteins with low binding affinity will be adsorbed initially and replaced by proteins with high binding affinities [21]. Thus the binding affinities and the rates of unfolding may play more important role than the rates of transport in the competitive adsorption between collagen and BSA. On the more hydrophobic surfaces, the main driving force for protein is hydrophobic interaction which may cause large protein conformation change. As a globular and flexible protein, BSA denatured easily after adsorption [19,20] while collagen is non-flexible and rather rigid [7]. The binding affinity and the rate of un-
4. Conclusion The adsorption amount of BSA increased with the decrease of surface wettability while the ability of the adsorbed BSA binding with anti-BSA decreased. The adsorption of collagen reached its minimum at the medium wettability, and then increased greatly on the more hydrophobic surfaces. During the competitive of collagen and BSA, the content of collagen in the co-adsorption layer decreased with the decrease of surface wettability, while that of BSA increased.
Acknowledgements The Chinese Academy of Sciences and National Natural Science Foundation of China (NSFC) are acknowledged for their supports of this work.
References [1] B. Lassen, M. Malmsten, J. Mater. Sci.: Mater. on Med. 5 (1994) 662–665. [2] J.L. Dewez, A. Doren, Y.J. Schneider, P.G. Rouxhet, Biomaterials 20 (1999) 547–559. [3] A.S.G. Curtis, J.V. Forrester, J. Cell Sci. 71 (1984) 17–35. [4] J.G. Steele, B.A. Dalton, G. Johnson, P.A. Underwood, Biomaterials 16 (1995) 1057–1067. [5] M. Deyme, A. Baszkin, J.E. Proust, E. Perez, M.M. Boissonnade, J. Biomed. Mater. Res. 20 (1986) 951–962. [6] M. Deyme, A. Baszkin, J.E. Proust, E. Perez, G. Albrecht, M.M. Boissonnade, J. Biomed. Mater. Res. 21 (1987) 321–328. [7] A. Baszkin, M.M. Boissonnade, J. Biomed. Mater. Res. 27 (1993) 145–152. [8] J.-L. Dewez, V. Berger, Y.-J. Schneider, P.G. Rouxhet, J. Colloid Interf. Sci. 191 (1997) 1–10. [9] P.Q. Ying, Y. Yu, G. Jin, Z.L. Tao, Colloids Surf. B: Biointerf. 23 (2003) 1–10. [10] G. Jin, P. Tengvall, I. Lundström, H. Arwin, Anal. Biochem. 232 (1995) 69–72. [11] G. Jin, R. Jansson, H. Arwin, Rev. Sci. Instrum. 67 (1996) 2930– 2936. [12] G. Jin, R. Jansson, I. Lundström, H. Arwin, in: Proceedings of the 8th International Conference on Solid State Sensors and Actuators, and Eurosensors IX, Stockholm, Sweden, 25–29 June 1995. [13] L. Vroman, A.L. Adams, G.C. Fischer, P.C. Munoz, Blood 55 (1980) 156–159. [14] J. Benesch, A. Askendal, P. Tengvall, Colloids Surf. B: Biointerf. 18 (2000) 71–81. [15] J.A. de Feijter, J. Benjamins, F.A. Veer, Biopolymers 17 (1978) 1759–1801.
P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263 [16] F. Hook, J. Voros, M. Rodahl, R. Kurrat, P. Boni, J.J. Ramsden, M. Textor, N.D. Spencer, P. Tengvall, J. Gold, B. Kasemo, Colloids Surf. B: Biointerf. 24 (2002) 155–170. [17] H. Arwin, S. Welin-Klintström, R. Jansson, J. Colloid Interf. Sci. 156 (1993) 377–382. [18] M. Stenberg, H. Nygren, J. de Physique 44 (1983) 83–86.
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[19] C.E. Giacomelli, W. Norde, J. Colloid Interf. Sci. 233 (2001) 234– 240. [20] W. Norde, C.E. Giacomelli, J. Biotechnol. 79 (2000) 259– 268. [21] J.L. Brash, T.A. Horbett, Fundamentals and Application, ACS, Washington, DC, 1995, pp. 1–23.
Competitive adsorption of collagen and bovine serum albumin—effect of the surface wettability Peiqing Ying, Gang Jin∗ , Zulai Tao Institute of Mechanics, Chinese Academy of Sciences, 15 Bei-Si-Huan West Road, Beijing 100080, China Accepted 21 October 2003
Abstract The competitive adsorption of collagen and bovine serum albumin (BSA) on surfaces with varied wettability was investigated with imaging ellipsometry, and ellipsometry. Silane modified silicon surfaces were used as substrates. The results showed that surface wettability had an important effect on protein competitive adsorption. With the decrease of surface wettability, the adsorption of collagen from the mixture solution of collagen and BSA decreased, while the adsorption of BSA increased. © 2003 Elsevier B.V. All rights reserved. Keywords: Protein competitive adsorption; Surface wettability; Ellipsometry
1. Introduction Protein competitive adsorption is involved in many interfacial phenomena such as hemocompatibility of biomaterials, cellular adhesion and growth on substrates [1–4]. The competitive adsorption between collagen and serum albumin is of great importance in biomaterial design [5–7]. Previous studies on the competitive adsorption between these two kinds of proteins on hydrophobic or moderately hydrophobic surfaces showed that human serum albumin reduced collagen adsorption, and albumin was the only adsorbing protein [6–8]. In our previous studies [9] we investigated the competitive adsorption of collagen and BSA on highly hydrophilic and highly hydrophobic surfaces and the results showed that BSA preferentially adsorbed onto the hydrophobic surface, while collagen on the hydrophilic surface. Since surfaces with different hydrophobicity are often used as substrates, and the surface hydrophobicity is a key factor affecting competitive adsorption, it is necessary to study protein competitive adsorption on surface with varied wettability. In this paper, silicon surfaces modified with silane to be with varied wettability were used as
∗ Corresponding author. Tel.: +86-10-62631816; fax: +86-10-62561284. E-mail address: [email protected] (G. Jin).
0927-7765/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2003.10.015
substrates. With its highly sensitivity, imaging ellipsometry [10–12] and ellipsometry [13–16] were used as techniques to analysis the protein competitive adsorption.
2. Materials and methods 2.1. Proteins Bovine serum albumin (BSA) and its antibody were purchased from Sigma. Calf skin purified collagen, was purchased from Boehringer Mannheim Biochemica (Collagen S). 2.2. Substrates Silicon wafers (thin film 7 mm × 20 mm) with an optically polished flat surface and a natural silicon dioxide layer were used as substrates. The wafer surface was prepared as hydrophilic by washing in both TL1 solution (H2 O:30% H2 O2 :25% NH4 OH = 5:1:1, v/v/v) and TL2 solution (H2 O:30% H2 O2 :37% HCl = 6:1:1, v/v/v). Through the reaction of TL1 and TL2 with basic and acid solution, and oxidation of hydrogen peroxide, it not only removed contaminants of the silicon surface, but also improved the number of silanol groups on the surface thus making
260
P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263
surface hydrophilic. Hydrophobic surface with varied wettability was prepared with silanization of the hydroxylated surface. After rinsed in distilled water and ethanol, the hydroxylated surfaces were incubated in dichlorodimethylsilane solution (5–0 l in 20 ml trichloroethylene) for 2–5 min, followed by rinsing in ethanol and trichloroethylene in sequence. All chemicals used were of analytical grade. Pure water (resistivity 18.3 M cm) was produced by ion exchange demineralization, and followed by passing through a Milli-Q plus system from Millipore (Millipore, Bedford, MA). 2.3. Contact angle measurement Water contact angles were measured at 25 ◦ C for dried wafers with the sessile drop method. Deionized water (4 l) was gently dropped on the surfaces and the contact angle was read directly using a goniometer. The contact angles for hydrophilic and varied hydrophobic silicon wafers were about 5◦ , 15◦ , 40◦ , 45◦ , 50◦ , 70◦ and 80◦ , respectively, with a deviation of ±1◦ . 2.4. Protein adsorption and competitive adsorption Protein adsorption and competitive adsorption were carried out in PBS solution (8 mM Na2 PO4 ·2H2 O, 2.68 mM KCl, 1.14 mM KH2 PO4 , 137 mM NaCl; pH 7.2). Protein concentrations are 1 and 0.1 mg/ml for BSA and collagen, respectively, which are near the concentrations of BSA and extracellular protein in serum-containing culture media. Single or binary solutions containing collagen, BSA or their mixture were used. Silicon wafers were incubated in protein solutions for 2 h, then washed with PBS and deionized water, dried with nitrogen. The detection of BSA adsorption amount in the competitive adsorption was based on the BSA/anti-BSA interaction with their affinity as described previously [9]. In brief, the BSA or co-adsorption layer was immersed in anti-BSA solution. Based on the specific binding between BSA and anti-BSA, anti-BSA in the solution bound with BSA in the layer to form protein complex of BSA/anti-BSA and resulted in a variation of surface concentration (Fig. 1). The more BSA
Fig. 1. Thickness distribution of BSA and BSA/anti-BSA complex layer on silicon substrate visualized with imaging ellipsometry.
adsorbed in co-adsorption layer, the more anti-BSA bond onto the protein complex layer resulting in a large increase of the surface concentration. In this way, the amount of BSA adsorbed in the co-adsorption layer could be deduced from the surface concentration variation, so as to determine the competitive adsorption between BSA and collagen. 2.5. Ellipsometry analysis A homemade ellipsometric imaging system was used for the visualization and quantification of the surface concentration of protein adsorption layer. Compared with the conventional ellipsmetry, imaging ellipsometry has the advantage of distinguishing the effects of singularities (local abnormal variations in the image introduced by contamination) appearing on the surfaces. The basic experimental set-up was a conventional polarizer–compensator–sample-analyzer (PCSA) null ellipsometer. An interference filter at 632.8 nm wavelength was placed in the incident optical path to increase the ellipsometric contrast of image. The combined null and off-null ellipsometry was used at an incident angle close to the pseudo-Brewster angle of the substrate. An image of 7 mm × 15 mm of a surface was focused onto the CCD video camera for intensity measurements. The optical components in the system were adjusted to fulfill the null conditions on a silicon substrate without adsorbed layers and the off-null ellipsometric principle was used to measure the adsorption layer thickness distribution [10]. The spatial resolution of the imaging system is in the order of micron laterally and 0.1 nm vertically. The video signal corresponding to the thickness distribution was captured, digitized and stored in gray-scale format in a computer. Under this condition, the detected intensity “I” was related to the thickness of the layer according to I = kd2 which was a linear relationship between the intensity and the square of the thickness of the adsorbed protein layer or the square of the surface concentration of proteins [17]. This proportionality showed a deviation of less than ±2% up to d ≈ 5 nm. As for the same protein and the same ellipsometric conditions, k is constant and can be determined by the protein layer with known gray-scale and thickness. The absolute thickness of protein layer used to calculate the constant k was calibrated by conventional ellipsometer (SE 400, SENTECH, Germany). The surface concentration of protein adsorption layer can be calculated according to the relationship between surface concentration and film thickness: surface concentration g cm−2 ≈ K × d(nm) where K ≈ 0.12 [18].
P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263
3. Results and discussion 3.1. BSA adsorption and BSA/anti-BSA interaction Fig. 2 shows the increase of surface concentration corresponding to the adsorption of BSA from pure solution and then the interaction of anti-BSA with the adsorbed BSA. The adsorption amount of BSA increased with the increase of the contact angle and it became stable at the quite hydrophobic surface where the contact angle was about 80◦ . Unlike the adsorption of BSA, the surface concentration of the BSA/anti-BSA complex slightly decreased with the increase of the contact angle, showing that BSA adsorbed on less hydrophobic surface was more likely to bind with anti-BSA. The decrease of the BSA/anti-BSA binding ability with the increase of hydrophobicity might be related with the extent of BSA conformation change after adsorption. The more BSA conformation changed, the lower binding ability of BSA with anti-BSA. On the less hydrophobic surface the main driving force for BSA adsorption is hydrogen bond and electrostatic interaction, BSA adsorb to the surface without much conformation change. BSA adsorbed on the less hydrophobic surface maintains its high anti-BSA binding ability. With the decrease of the surface wettability, that is, with the increase of surface hydrophobicity, the hydrophobic interaction became the main driving force for BSA adsorption which caused more conformation change of the adsorbed BSA than that on the less hydrophobic surface, thus decreasing the anti-BSA binding ability. 3.2. Collagen adsorption The adsorption of collagen from pure solution was also different from that of BSA (Fig. 3). With the contact angle
0.6 0.4 0.2 0
0.6 0.4 0.2 0.0 0
20 40 60 o Advancing contact angle ( )
80
Fig. 2. BSA (1 mg/ml) adsorption and BSA/anti-BSA interaction on surfaces with varied hydrophobicity. Adsorption time and BSA/anti-BSA interaction time were 2 and 1 h, respectively.
20 40 60 o Advancing contact angle ( )
80
Fig. 3. Collagen (0.1 mg/ml) adsorption on surfaces with varied hydrophobicity, adsorption time was 2 h.
increased, the collagen adsorption amount decreased slowly (from 5◦ to 45◦ ), then it increased quickly (from 50◦ to 85◦ ). With a molecular weight of 300,000 Da, Collagen is much larger than BSA (66,200 Da). Collagen molecule is also rather rigid and cannot change its conformation as easy as BSA [7,19,20]. On the slightly hydrophobic surface, the hydrogen bond and electrostatic interaction between collagen and surface decreased with the increase of the contact angle. Since collagen molecule cannot change its conformation greatly, the low density of surface methyl cannot supply enough binding sites for collagen adsorption. The adsorption amount decreased with the increase of hydrophobicity. As the contact angle further increased from 50◦ to 85◦ , the strong hydrophobic interaction between collagen and surface caused more adsorption amount which is similar with the adsorption of BSA. 3.3. Competitive adsorption between collagen and BSA Fig. 4 represents the surface concentration change corresponding to the competitive adsorption between collagen and BSA, and the binding of anti-BSA with adsorbed BSA. Since in most serum-containing cell culture media the concentrations for extracelluar protein and BSA are often near 0.1
Collagen, BSA Collagen, BSA/anti-BSA
0.8 Surface concentration 2 (µg/cm )
Surface concentration 2 (µg/cm )
0.8
BSA BSA/anti-BSA
0.8
Collagen
1.0 Surface concentration 2 (µg/cm )
The results of imaging ellipsometry shown in gray scale were processed to be the surface concentration of the adsorption layer.
261
0.6 0.4 0.2 0.0
0
20
40
60
80
100
o
Advancing contact angle ( ) Fig. 4. Competitive adsorption of collagen (0.1 mg/ml) and BSA (1 mg/ml) and BSA/anti-BSA interaction on surfaces with varied hydrophobicity. Adsorption time and BSA/anti-BSA interaction time were 2 and 1 h, respectively.
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Collagen
Mass percent (%)
100
folding contribute to the preferentially adsorption of BSA on the more hydrophobic surfaces. On the less hydrophobic surfaces the main driving force for BSA adsorption is hydrogen bond and electrostatic interaction, both collagen and BSA adsorb to the surfaces without much conformation change. The binding affinity becomes the main factor that leads to the preferential adsorption of collagen on the more hydrophilic surfaces.
BSA
80 60 40 20 0
0
20 40 60 80 o Advancing contact angle ( )
100
Fig. 5. Mass percent of collagen and BSA for the competitive adsorption of collagen (0.1 mg/ml) and BSA (1 mg/ml).
and 1 mg/ml, respectively, competitive adsorption between collagen (0.1 mg/ml) and BSA (1 mg/ml) were investigated. The surface concentration of the co-adsorption layer of collagen and BSA decreased as the surface change from highly hydrophilic to slightly hydrophobic (contact angle from 20◦ to 40◦ ), but varied slightly when the surface hydrophobicity further increased (contact angle from 40◦ to 85◦ ). After the incubation of the co-adsorption layer in the anti-BSA solution, the surface concentration hardly increased on the range of hydrophilic surfaces, while increased obviously on hydrophobic surfaces. Supposed that the affinity between anti-BSA and BSA of co-adsorption layer was the same as of pure BSA adsorption layer, the mass percent of BSA in the co-adsorption layer can be deduced from the surface concentration increase introduced by anti-BSA binding with BSA. The mass percent of collagen and BSA are showed in Fig. 5. The percent of collagen in the co-adsorption decreased with the increase of contact angle, while BSA increased. On the highly hydrophilic surfaces (contact angle 5◦ –20◦ ), nearly 100% of the protein adsorbed was collagen, but less than 10% on the highly hydrophobic surface (contact angle 85◦ ). The result was quite coincident with the previous result that during the competitive adsorption collagen preferentially adsorbed on the hydrophilic surface while BSA on hydrophobic surface [9]. With the increase of surface hydrophobicity, the competitive adsorption amount of collagen decreased, while BSA increased. Factors that affect competitive protein adsorption include protein binding affinities, rates of transport and rates of unfolding [21]. The rates of transport mainly affect the initial competitive adsorption. The abundant proteins with low binding affinity will be adsorbed initially and replaced by proteins with high binding affinities [21]. Thus the binding affinities and the rates of unfolding may play more important role than the rates of transport in the competitive adsorption between collagen and BSA. On the more hydrophobic surfaces, the main driving force for protein is hydrophobic interaction which may cause large protein conformation change. As a globular and flexible protein, BSA denatured easily after adsorption [19,20] while collagen is non-flexible and rather rigid [7]. The binding affinity and the rate of un-
4. Conclusion The adsorption amount of BSA increased with the decrease of surface wettability while the ability of the adsorbed BSA binding with anti-BSA decreased. The adsorption of collagen reached its minimum at the medium wettability, and then increased greatly on the more hydrophobic surfaces. During the competitive of collagen and BSA, the content of collagen in the co-adsorption layer decreased with the decrease of surface wettability, while that of BSA increased.
Acknowledgements The Chinese Academy of Sciences and National Natural Science Foundation of China (NSFC) are acknowledged for their supports of this work.
References [1] B. Lassen, M. Malmsten, J. Mater. Sci.: Mater. on Med. 5 (1994) 662–665. [2] J.L. Dewez, A. Doren, Y.J. Schneider, P.G. Rouxhet, Biomaterials 20 (1999) 547–559. [3] A.S.G. Curtis, J.V. Forrester, J. Cell Sci. 71 (1984) 17–35. [4] J.G. Steele, B.A. Dalton, G. Johnson, P.A. Underwood, Biomaterials 16 (1995) 1057–1067. [5] M. Deyme, A. Baszkin, J.E. Proust, E. Perez, M.M. Boissonnade, J. Biomed. Mater. Res. 20 (1986) 951–962. [6] M. Deyme, A. Baszkin, J.E. Proust, E. Perez, G. Albrecht, M.M. Boissonnade, J. Biomed. Mater. Res. 21 (1987) 321–328. [7] A. Baszkin, M.M. Boissonnade, J. Biomed. Mater. Res. 27 (1993) 145–152. [8] J.-L. Dewez, V. Berger, Y.-J. Schneider, P.G. Rouxhet, J. Colloid Interf. Sci. 191 (1997) 1–10. [9] P.Q. Ying, Y. Yu, G. Jin, Z.L. Tao, Colloids Surf. B: Biointerf. 23 (2003) 1–10. [10] G. Jin, P. Tengvall, I. Lundström, H. Arwin, Anal. Biochem. 232 (1995) 69–72. [11] G. Jin, R. Jansson, H. Arwin, Rev. Sci. Instrum. 67 (1996) 2930– 2936. [12] G. Jin, R. Jansson, I. Lundström, H. Arwin, in: Proceedings of the 8th International Conference on Solid State Sensors and Actuators, and Eurosensors IX, Stockholm, Sweden, 25–29 June 1995. [13] L. Vroman, A.L. Adams, G.C. Fischer, P.C. Munoz, Blood 55 (1980) 156–159. [14] J. Benesch, A. Askendal, P. Tengvall, Colloids Surf. B: Biointerf. 18 (2000) 71–81. [15] J.A. de Feijter, J. Benjamins, F.A. Veer, Biopolymers 17 (1978) 1759–1801.
P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263 [16] F. Hook, J. Voros, M. Rodahl, R. Kurrat, P. Boni, J.J. Ramsden, M. Textor, N.D. Spencer, P. Tengvall, J. Gold, B. Kasemo, Colloids Surf. B: Biointerf. 24 (2002) 155–170. [17] H. Arwin, S. Welin-Klintström, R. Jansson, J. Colloid Interf. Sci. 156 (1993) 377–382. [18] M. Stenberg, H. Nygren, J. de Physique 44 (1983) 83–86.
263
[19] C.E. Giacomelli, W. Norde, J. Colloid Interf. Sci. 233 (2001) 234– 240. [20] W. Norde, C.E. Giacomelli, J. Biotechnol. 79 (2000) 259– 268. [21] J.L. Brash, T.A. Horbett, Fundamentals and Application, ACS, Washington, DC, 1995, pp. 1–23.