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 substrates. With its highly sensitivity, imaging ellipsometry [10–12] and ellipsometry [13–16] were used as techniques Protein competitive adsorption is involved in many in- to analysis the protein competitive adsorption. terfacial phenomena such as hemocompatibility of biomaterials, cellular adhesion and growth on substrates [1–4]. The competitive adsorption between collagen and serum 2. Materials and methods albumin is of great importance in biomaterial design [5–7]. Previous studies on the competitive adsorption between 2.1. Proteins these two kinds of proteins on hydrophobic or moderately hydrophobic surfaces showed that human serum albumin Bovine serum albumin (BSA) and its antibody were reduced collagen adsorption, and albumin was the only purchased from Sigma. Calf skin purified collagen, adsorbing protein [6–8]. In our previous studies [9] we in- was purchased from Boehringer Mannheim Biochemica vestigated the competitive adsorption of collagen and BSA (Collagen S). on highly hydrophilic and highly hydrophobic surfaces and the results showed that BSA preferentially adsorbed onto the hydrophobic surface, while collagen on the hydrophilic 2.2. Substrates surface. Since surfaces with different hydrophobicity are × often used as substrates, and the surface hydrophobicity Silicon wafers (thin film 7 mm 20 mm) with an op- is a key factor affecting competitive adsorption, it is nec- tically polished flat surface and a natural silicon dioxide essary to study protein competitive adsorption on surface layer were used as substrates. The wafer surface was pre- with varied wettability. In this paper, silicon surfaces mod- pared as hydrophilic by washing in both TL1 solution = ified with silane to be with varied wettability were used as (H2O:30% H2O2:25% NH4OH 5:1:1, v/v/v) and TL2 solution (H2O:30% H2O2:37% HCl = 6:1:1, v/v/v). Through the reaction of TL1 and TL2 with basic and acid solution, ∗ Corresponding author. Tel.: +86-10-62631816; and oxidation of hydrogen peroxide, it not only removed fax: +86-10-62561284. contaminants of the silicon surface, but also improved E-mail address: [email protected] (G. Jin). the number of silanol groups on the surface thus making

0927-7765/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2003.10.015 260 P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263 surface hydrophilic. Hydrophobic surface with varied wet- adsorbed in co-adsorption layer, the more anti-BSA bond tability was prepared with silanization of the hydroxylated onto the protein complex layer resulting in a large increase surface. After rinsed in distilled water and ethanol, the of the surface concentration. In this way, the amount of hydroxylated surfaces were incubated in dichlorodimethyl- BSA adsorbed in the co-adsorption layer could be de- silane solution (5–0 ␮l in 20 ml trichloroethylene) for duced from the surface concentration variation, so as to 2–5 min, followed by rinsing in ethanol and trichloroethy- determine the competitive adsorption between BSA and lene in sequence. All chemicals used were of analytical collagen. grade. Pure water (resistivity 18.3 M cm) was produced by ion exchange demineralization, and followed by passing 2.5. Ellipsometry analysis through a Milli-Q plus system from Millipore (Millipore, Bedford, MA). A homemade ellipsometric imaging system was used for the visualization and quantification of the surface concentra- 2.3. Contact angle measurement tion of protein adsorption layer. Compared with the conventional ellipsmetry, imaging ellipsometry has the advantage Water contact angles were measured at 25 ◦C for dried of distinguishing the effects of singularities (local abnor- wafers with the sessile drop method. Deionized water (4 ␮l) mal variations in the image introduced by contamination) was gently dropped on the surfaces and the contact angle appearing on the surfaces. The basic experimental set-up was read directly using a goniometer. The contact angles was a conventional polarizer–compensator–sample-analyzer for hydrophilic and varied hydrophobic silicon wafers were (PCSA) null ellipsometer. An interference filter at 632.8 nm about 5◦,15◦,40◦,45◦,50◦,70◦ and 80◦, respectively, with wavelength was placed in the incident optical path to in- a deviation of ±1◦. crease the ellipsometric contrast of image. The combined null and off-null ellipsometry was used at an incident an- 2.4. Protein adsorption and competitive adsorption gle close to the pseudo-Brewster angle of the substrate. An image of 7 mm × 15 mm of a surface was focused Protein adsorption and competitive adsorption were car- onto the CCD video camera for intensity measurements. ried out in PBS solution (8 mM Na PO ·2H O, 2.68 mM The optical components in the system were adjusted to 2 4 2 fulfill the null conditions on a silicon substrate without KCl, 1.14 mM KH2PO4, 137 mM NaCl; pH 7.2). Protein concentrations are 1 and 0.1 mg/ml for BSA and collagen, adsorbed layers and the off-null ellipsometric principle respectively, which are near the concentrations of BSA was used to measure the adsorption layer thickness distri- and extracellular protein in serum-containing culture me- bution [10]. The spatial resolution of the imaging system dia. Single or binary solutions containing collagen, BSA is in the order of micron laterally and 0.1 nm vertically. or their mixture were used. Silicon wafers were incu- The video signal corresponding to the thickness distri- bated in protein solutions for 2 h, then washed with PBS bution was captured, digitized and stored in gray-scale and deionized water, dried with nitrogen. The detection format in a computer. Under this condition, the detected of BSA adsorption amount in the competitive adsorption intensity “I” was related to the thickness of the layer was based on the BSA/anti-BSA interaction with their according to affinity as described previously [9]. In brief, the BSA or I = kd2 co-adsorption layer was immersed in anti-BSA solution. Based on the specific binding between BSA and anti-BSA, which was a linear relationship between the intensity anti-BSA in the solution bound with BSA in the layer to and the square of the thickness of the adsorbed protein form protein complex of BSA/anti-BSA and resulted in a layer or the square of the surface concentration of pro- variation of surface concentration (Fig. 1). The more BSA teins [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, Ger- many). The surface concentration of protein adsorption layer can be calculated according to the relationship between surface concentration and film thickness: − surface concentration ␮gcm 2 ≈ K × d(nm) Fig. 1. Thickness distribution of BSA and BSA/anti-BSA complex layer on silicon substrate visualized with imaging ellipsometry. where K ≈ 0.12 [18]. P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263 261

The results of imaging ellipsometry shown in gray scale Collagen were processed to be the surface concentration of the ad- 1.0 sorption layer. 0.8 ) 2 0.6

3. Results and discussion g/cm µ ( 3.1. BSA adsorption and BSA/anti-BSA interaction 0.4 Surface concentration Fig. 2 shows the increase of surface concentration corre- 0.2 0 20 40 60 80 sponding to the adsorption of BSA from pure solution and Advancing contact angle (o ) then the interaction of anti-BSA with the adsorbed BSA. The adsorption amount of BSA increased with the increase Fig. 3. Collagen (0.1 mg/ml) adsorption on surfaces with varied hydropho- of the contact angle and it became stable at the quite hy- bicity, adsorption time was 2 h. drophobic surface where the contact angle was about 80◦. Unlike the adsorption of BSA, the surface concentration of increased, the collagen adsorption amount decreased slowly the BSA/anti-BSA complex slightly decreased with the in- (from 5◦ to 45◦), then it increased quickly (from 50◦ to 85◦). crease of the contact angle, showing that BSA adsorbed With a molecular weight of 300,000 Da, Collagen is much on less hydrophobic surface was more likely to bind with larger than BSA (66,200 Da). Collagen molecule is also anti-BSA. The decrease of the BSA/anti-BSA binding abil- rather rigid and cannot change its conformation as easy as ity with the increase of hydrophobicity might be related with BSA [7,19,20]. On the slightly hydrophobic surface, the hy- the extent of BSA conformation change after adsorption. drogen bond and electrostatic interaction between collagen The more BSA conformation changed, the lower binding and surface decreased with the increase of the contact an- ability of BSA with anti-BSA. On the less hydrophobic sur- gle. Since collagen molecule cannot change its conforma- face the main driving force for BSA adsorption is hydrogen tion greatly, the low density of surface methyl cannot supply bond and electrostatic interaction, BSA adsorb to the sur- enough binding sites for collagen adsorption. The adsorp- face without much conformation change. BSA adsorbed on tion amount decreased with the increase of hydrophobicity. the less hydrophobic surface maintains its high anti-BSA As the contact angle further increased from 50◦ to 85◦, the binding ability. With the decrease of the surface wettabil- strong hydrophobic interaction between collagen and sur- ity, that is, with the increase of surface hydrophobicity, the face caused more adsorption amount which is similar with hydrophobic interaction became the main driving force for the adsorption of BSA. BSA adsorption which caused more conformation change of the adsorbed BSA than that on the less hydrophobic surface, 3.3. Competitive adsorption between collagen and BSA thus decreasing the anti-BSA binding ability. Fig. 4 represents the surface concentration change corre- 3.2. Collagen adsorption sponding to the competitive adsorption between collagen and BSA, and the binding of anti-BSA with adsorbed BSA. Since The adsorption of collagen from pure solution was also in most serum-containing cell culture media the concentra- different from that of BSA (Fig. 3). With the contact angle tions for extracelluar protein and BSA are often near 0.1

BSA BSA/anti-BSA 0.8 Collagen, BSA 0.8 Collagen, BSA/anti-BSA 0.6 0.6 ) ) 2 2 0.4 0.4 g/cm g/cm µ µ ( ( 0.2 0.2 Surface concentration Surface concentration concentration Surface 0.0 0.0 0 20 40 60 80 100 0 20 40 60 80 Advancing contact angle ( o ) Advancing contact angle ( o ) Fig. 4. Competitive adsorption of collagen (0.1 mg/ml) and BSA (1 mg/ml) Fig. 2. BSA (1 mg/ml) adsorption and BSA/anti-BSA interaction on sur- and BSA/anti-BSA interaction on surfaces with varied hydrophobicity. faces with varied hydrophobicity. Adsorption time and BSA/anti-BSA Adsorption time and BSA/anti-BSA interaction time were 2 and 1 h, interaction time were 2 and 1 h, respectively. respectively. 262 P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263

Collagen BSA folding contribute to the preferentially adsorption of BSA 100 on the more hydrophobic surfaces. On the less hydropho- 80 bic surfaces the main driving force for BSA adsorption is hydrogen bond and electrostatic interaction, both collagen 60 and BSA adsorb to the surfaces without much conformation change. The binding afﬁnity becomes the main factor that 40 leads to the preferential adsorption of collagen on the more

Mass percent (%) percent Mass 20 hydrophilic surfaces.

0 0 20406080100 Advancing contact angle ( o ) 4. Conclusion

Fig. 5. Mass percent of collagen and BSA for the competitive adsorption The adsorption amount of BSA increased with the de- of collagen (0.1 mg/ml) and BSA (1 mg/ml). crease of surface wettability while the ability of the adsorbed BSA binding with anti-BSA decreased. The adsorption of and 1 mg/ml, respectively, competitive adsorption between collagen reached its minimum at the medium wettability, and collagen (0.1 mg/ml) and BSA (1 mg/ml) were investigated. then increased greatly on the more hydrophobic surfaces. The surface concentration of the co-adsorption layer of col- During the competitive of collagen and BSA, the content of lagen and BSA decreased as the surface change from highly collagen in the co-adsorption layer decreased with the de- hydrophilic to slightly hydrophobic (contact angle from 20◦ crease of surface wettability, while that of BSA increased. 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 Acknowledgements solution, the surface concentration hardly increased on the range of hydrophilic surfaces, while increased obviously on The Chinese Academy of Sciences and National Natural hydrophobic surfaces. Supposed that the affinity between Science Foundation of China (NSFC) are acknowledged for anti-BSA and BSA of co-adsorption layer was the same as their supports of this work. 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 References BSA. The mass percent of collagen and BSA are showed in Fig. 5. The percent of collagen in the co-adsorption de- [1] B. Lassen, M. Malmsten, J. Mater. Sci.: Mater. on Med. 5 (1994) creased with the increase of contact angle, while BSA in- 662–665. creased. On the highly hydrophilic surfaces (contact angle [2] J.L. Dewez, A. Doren, Y.J. Schneider, P.G. Rouxhet, Biomaterials ◦ ◦ 20 (1999) 547–559. 5 –20 ), nearly 100% of the protein adsorbed was collagen, [3] A.S.G. Curtis, J.V. Forrester, J. Cell Sci. 71 (1984) 17–35. but less than 10% on the highly hydrophobic surface (con- [4] J.G. Steele, B.A. Dalton, G. Johnson, P.A. Underwood, Biomaterials tact angle 85◦). The result was quite coincident with the 16 (1995) 1057–1067. previous result that during the competitive adsorption colla- [5] M. Deyme, A. Baszkin, J.E. Proust, E. Perez, M.M. Boissonnade, J. Biomed. Mater. Res. 20 (1986) 951–962. gen preferentially adsorbed on the hydrophilic surface while [6] M. Deyme, A. Baszkin, J.E. Proust, E. Perez, G. Albrecht, M.M. BSA on hydrophobic surface [9]. With the increase of sur- Boissonnade, J. Biomed. Mater. Res. 21 (1987) 321–328. face hydrophobicity, the competitive adsorption amount of [7] A. Baszkin, M.M. Boissonnade, J. Biomed. Mater. Res. 27 (1993) collagen decreased, while BSA increased. 145–152. Factors that affect competitive protein adsorption include [8] J.-L. Dewez, V. Berger, Y.-J. Schneider, P.G. Rouxhet, J. Colloid Interf. Sci. 191 (1997) 1–10. protein binding affinities, rates of transport and rates of un- [9] P.Q. Ying, Y. Yu, G. Jin, Z.L. Tao, Colloids Surf. B: Biointerf. 23 folding [21]. The rates of transport mainly affect the ini- (2003) 1–10. tial competitive adsorption. The abundant proteins with low [10] G. Jin, P. Tengvall, I. Lundström, H. Arwin, Anal. Biochem. 232 binding affinity will be adsorbed initially and replaced by (1995) 69–72. proteins with high binding affinities [21]. Thus the binding [11] G. Jin, R. Jansson, H. Arwin, Rev. Sci. Instrum. 67 (1996) 2930– 2936. affinities and the rates of unfolding may play more important [12] G. Jin, R. Jansson, I. Lundström, H. Arwin, in: Proceedings of the role than the rates of transport in the competitive adsorp- 8th International Conference on Solid State Sensors and Actuators, tion between collagen and BSA. On the more hydrophobic and Eurosensors IX, Stockholm, Sweden, 25–29 June 1995. surfaces, the main driving force for protein is hydropho- [13] L. Vroman, A.L. Adams, G.C. Fischer, P.C. Munoz, Blood 55 (1980) bic interaction which may cause large protein conformation 156–159. [14] J. Benesch, A. Askendal, P. Tengvall, Colloids Surf. B: Biointerf. change. As a globular and flexible protein, BSA denatured 18 (2000) 71–81. easily after adsorption [19,20] while collagen is non-flexible [15] J.A. de Feijter, J. Benjamins, F.A. Veer, Biopolymers 17 (1978) and rather rigid [7]. The binding affinity and the rate of un- 1759–1801. P. Ying et al. / Colloids and Surfaces B: Biointerfaces 33 (2004) 259–263 263

[16] F. Hook, J. Voros, M. Rodahl, R. Kurrat, P. Boni, J.J. Ramsden, [19] C.E. Giacomelli, W. Norde, J. Colloid Interf. Sci. 233 (2001) 234– M. Textor, N.D. Spencer, P. Tengvall, J. Gold, B. Kasemo, Colloids 240. Surf. B: Biointerf. 24 (2002) 155–170. [20] W. Norde, C.E. Giacomelli, J. Biotechnol. 79 (2000) 259– [17] H. Arwin, S. Welin-Klintström, R. Jansson, J. Colloid Interf. Sci. 268. 156 (1993) 377–382. [21] J.L. Brash, T.A. Horbett, Fundamentals and Application, ACS, Wash- [18] M. Stenberg, H. Nygren, J. de Physique 44 (1983) 83–86. ington, DC, 1995, pp. 1–23.