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Properties of competitively adsorbed BSA and fibrinogen from their mixture on mixed and hybrid surfaces
Applied Surface Science 264 (2013) 832–837
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Properties of competitively adsorbed BSA and fibrinogen from their mixture on mixed and hybrid surfaces Lalit M. Pandey, Sudip K. Pattanayek ∗ Department of Chemical Engineering, IIT Delhi, New Delhi 110016, India
a r t i c l e
i n f o
Article history: Received 24 July 2012 Received in revised form 10 October 2012 Accepted 27 October 2012 Available online 3 November 2012 Keywords: Competitive adsorption Adsorption of proteins Chemical potential Vroman effect QCM Hybrid SAM
a b s t r a c t We have studied the adsorption of BSA and fibrinogen from their mixture onto surfaces with mixed selfassembled monolayer (SAM) of amine and octyl (ratio 1:1) and hybrid SAM. The properties of adsorbed proteins obtained from individual protein solution differ considerably from the properties of the adsorbed proteins obtained from mixture of proteins at same total concentration. The adsorbed amount of proteins is lesser and the adsorbed protein is more elastic if it is adsorbing from mixture of proteins. It is found that with increasing total protein concentration, adsorbed amount increases and elasticity of the adsorbed proteins decreases. The apparent displacements of BSA with Fb are observed on the graphs of change in frequency with time, which are obtained from quartz crystal microbalance. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Biological fluid contains mixture of large number of proteins of very small to very large molecular weight. Major constituents are albumin, immunoglobulin and fibrinogen [1]. When a foreign material comes in contact with the biological fluid, the proteins of all different sizes compete for the same adsorbing sites of a surface. Finally, amount of adsorbed proteins and its characteristics depend on interaction among proteins and surface. There exist a number of studies on single protein adsorption behaviors from its solutions in the literature. There are considerable difference between the single protein adsorption and competitive adsorption [2–4]. Smaller globular proteins (e.g. albumin) which are abundance in biological fluid, adsorb initially and are displaced by less abundant proteins with lower mobility and more surface active (larger) proteins (i.e. fibrinogen, kininogen). This is known as “Vroman effect”, which has been tested using competitive adsorption [3,5–16] or sequential adsorption [17–20] of proteins. The factors affecting competitive adsorption behavior are bulk concentration [7,17] of proteins, affinity of proteins for the adsorbing surface [11,17] and conformation of adsorbed proteins [11]. Each protein arrives at the surface according to its own transport rate, which depends on its size and its concentration in solution [7]. The affinity of proteins for a surface depends on hydrophobicity of surface [17]. In general, a hydrophobic surface favors the
∗ Corresponding author. Tel.: +91 11 26591018; fax: +91 11 26581120. E-mail address: [email protected] (S.K. Pattanayek). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2012.10.150
irreversible adsorption of proteins from aqueous solution thermodynamically, while a highly hydrophilic surface inhibits adsorption of protein [8]. The irreversibly adsorbed proteins cannot be displaced by an incoming large protein [13,14]. Various combination of proteins including BSA/IgG [3,8,14], BSA/BGG [18], collagen/albumin [11], etc. have been used in studies of competitive and sequential adsorption. No consensus has been achieved on the observation on “Vroman effect” stated below. Lassen et al. [15] found that albumin is displaced by IgG or Fb during adsorption from binary mixture of HAS and IgG or HAS and Fb respectively on hydrophilic poly acrylic acid and poly diaminocyclohexane coated surfaces. But the displacement by IgG or Fb were not observed on hexamethyldisiloxane coated and methylated surface [14,15] for the proteins mixtures at the same condition. For another system, adsorption from mixture of HSA and gammaglobulin (HGG) on glass surface, HGG displaced HAS and on C16 self-assembled monolayers (SAMs) HSA competes with HGG [14]. Wertz et al. [8] have observed that no displacement of pre-adsorbed BSA by Fb and vice versa were found during sequential adsorption on both OH and C16 SAMs. They observed that protein which arrives late occupies only free space available on the surface [21]. Contrary to above observations Green et al. [17] found that albumin is displaced by Fb from binary mixture of albumin/Fb on polystyrene surface [14]. Recently, Benesch et al. [18] have observed the displacement of BSA by gamma-globulin (BGG) from its binary mixture on both, OH and CH3 SAMs. The literature lacks in detail of kinetics and in viscoelatic properties of the adsorbed proteins from their mixture at the surface. The fundamental questions that are unanswered are as follows: (a)
L.M. Pandey, S.K. Pattanayek / Applied Surface Science 264 (2013) 832–837
How kinetics of competition differs from single protein adsorption kinetics? (b) What is role of surfaces to mixed protein adsorption? We reported [22] that hybrid SAM, which has both hydrophobic and hydrophilic groups on the same modifying molecule is a potential surface modifying agent of biomaterials. We compared properties of the adsorbed BSA and Fb from their solution on the hybrid surface and the most widely used mixed (amine-octyl) SAM. Here, we report adsorption of mixture of BSA and Fb using QCM and compared with the single protein adsorption. We also have determined the viscoelastic characteristics of adsorbed layer. 2. Materials and methods (APTMS) and 3-Aminopropyl-trimethoxysilane octyltrimethoxysilane (OTMS) were used for the formation of amine:octyl mixed (1:1) SAM. Dibulyltin dilaurate (95%), ptolyl isocynate (99%) and anhydrous toluene (99.8%) were used for synthesis of hybrid SAM. APTMS, OTMS, dibulyltin dilaurate, p-tolyl isocynate and anhydrous toluene were purchased from Sigma–Aldrich. Methanol and acetone (HPLC grade) were purchased from Merck. Proteins, bovine serum albumin (BSA, A0281) and fibrinogen (Fb, F8630), were purchased from Sigma–Aldrich. These are used without any further purification. Sodium chloride, potassium chloride, di-sodium hydrogen phosphate (anhydrous) and potassium dihydrogen orthophosphate were used for preparing phosphate buffer saline (PBS), were purchased from Merck. We carried out following experiments (i) modification of silica surfaces with various self-assembled monolayers and its characterization and (ii) adsorption of proteins from binary mixture by using QCM. 2.1. Surface modification and characterization Silica coated quartz crystal was cleaned with “piranha” solution. Mixed (amine:octyl) and hybrid SAMs were formed on the cleaned silica surfaces. The procedure of the surface modification and characterization of the modified surfaces already has been discussed in Ref. [22], which is described briefly below. Cleaned silica coated quartz crystal was modified with required modifier in QCM chamber itself. 1% solution of APTMS and 1:1 mixture of APTMS and OTMS in anhydrous toluene at 25 ◦ C were used to from amine and mixed (amine:octyl) SAMs, respectively. After formation of amine SAM, 1% (v/v) solution of p-tolyl isocynate in anhydrous toluene was passed into QCM chamber to from hybrid SAM. The catalyst dibulyltin dilaurate (2–3 drops) was added into the reaction mixture. The amine ( NH2 ) and isocynate ( NCO) groups reacted to form urea (NH CO NH) linkage. The formed monolayer had hydrophilic urea group and hydrophobic toluene group. After the formation of SAMs, the crystals were washed consecutively in three different solvents: toluene, mixture of toluene and ethanol (v/v = 1:1) and ethanol. The surface energies of the modified surfaces are determined from its contact angle against deionized water, ethylene glycol and methylene di-iodide. The contact angles were measured by contact angle goniometer (Kruss DSA-10). The characteristics of the surfaces are almost equal to the earlier reported data [22] within the error limit of 10%. Water contact angle on both surfaces was 80 ± 1◦ . From the contact angle data, we found surface energy ( s ) of mixed (amine:octyl) and hybrid surfaces to be 37.22 and 39.19 mJ/m2 , respectively. Details of the data can be found in Ref. [22].
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0.02 mg/ml) and binary mixture (1:1) of BSA and Fb on mixed and hybrid SAMs. The total concentrations of proteins were 0.02 mg/ml (0.01 mg/ml of individual proteins) and 0.04 mg/ml (0.02 mg/ml of individual proteins). Adsorption of individual proteins is already reported [22,23]. Adsorption of the mixed proteins was done as described below. We passed 10 ml phosphate buffer saline solution (PBS) of pH 7.4 through the chamber and allowed the crystal to stabilize. Then, 10 ml of mixed protein solution was passed through the washed chamber. The protein solution was kept within the chamber for about 30 min and PBS solution was passed through the chamber to remove the loosely bound proteins. At the end of the washing, we get characteristics of adsorbed proteins at equilibrium only. This value is always about 10% less than equilibrium value obtained with unwashed adsorbed proteins. All QCM experiments were repeated at least twice, and the obtained values were within 10% standard deviation of the presented average values. We obtained changes in resonance frequency of different overtones (Fn /n) and dissipation factor (Dn ) of the quartz crystal in real time due to adsorption of the proteins. Adsorbed mass (m) is related to Fn /n by Sauerbrey equation [22,24] for strongly adsorbed protein. The relation is m = − CFn /n, where C is sensitivity constant of crystal, 17.7 ng/(cm2 Hz). In this paper −Fn /n and adsorbed amount are used synonymously. The dissipation factor (Dn ), a non-dimensional parameter, which compares energy dissipated to energy stored on the adsorbed material is defined as: Dn =
Edissipated 2Estored
(1)
where Edissipated is the energy lost (dissipated) during one oscillation cycle and Estored is the total energy stored in the adsorbed protein [24]. Energy dissipation reflects the viscoelastic properties of the adsorbed layer. Slower dissipation refers to a more elastic layer of adsorbed proteins. 3. Results and discussion 3.1. Adsorption of mixture of BSA and Fb using QCM Variation of frequencies, Fn /n, with time during adsorption of individual BSA, Fb and their mixture on mixed surface and on hybrid surface are shown in Figs. 1 and 2, respectively. The equilibrium adsorbed amount of proteins adsorbing from the mixture of BSA and Fb (total protein concentration 0.02 mg/ml) is lower than that of individual protein adsorbing from their bulk concentration of 0.02 mg/ml. A similar effect is reported for adsorption of proteins from mixture of BSA/IgG on plasma treated polyethylene terephthalate [8]. The reason of the observation was not explained. We can explain from calculation of entropy of mixture using Flory–Huggins n1 moles of solequation [25]. We write total entropy of mixture of vent and n2 and n3 moles of proteins as −Smix /R = j nj ln j . Here, volume fraction of jth component is given by j = nj vj / j nj vj , where vj is volume of each molecule. The entropic terms of chemical potentials of solvent (j = 1) and proteins (j = 2, 3) are given by 2 3 1 = ln 1 + 2 + 3 − − RT v2 /v1 v3 /v1
(2)
v2 v3 2 = ln 2 + 1 + 3 − 1 − 3 RT v1 v2
(3) (4)
2.2. Adsorption of proteins from binary mixture by using QCM
v3 v2 3 = ln 3 + 1 + 2 − 1 − 2 RT v1 v3
Experiments of adsorption of proteins were done for individual solution of BSA (concentration 0.02 mg/ml), Fb (concentration
The chemical potential of both the proteins (2 /RT, 3 /RT) are lesser in the ternary solution than that in the binary solution. The difference in entropy for two different cases is small. However,
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L.M. Pandey, S.K. Pattanayek / Applied Surface Science 264 (2013) 832–837
Fig. 1. Variation of Fn /n with time during adsorption of BSA, Fb and mixed proteins on mixed surface (inset focuses kink part of mixed proteins adsorption data for t = 30 s to 80 s; circle: BSA 0.02 mg/ml, square: Fb 0.02 mg/ml, open diamond: mixed protein 0.01 mg/ml each; filled diamond: mixed protein 0.02 mg/ml each).
the entropy of the adsorbed state being same, the proteins lose higher amount of entropy if adsorbing from mixture of two protein solution. This indicates that proteins adsorb in lesser amount on the surface from the ternary system than that from the binary system. On the mixed surface, the kinetics of proteins adsorbing from 1:1 mixture of BSA and Fb (total protein concentration 0.02 mg/ml) is slightly different from that of total protein concentration 0.04 mg/ml during the initial period up to about 50 s (see Fig. 1). The adsorption for the case of total protein concentration 0.02 mg/ml attains nearly equilibrium value at the later stage. However, there is little variation in the Fn /n values due to the rearrangement of the adsorbed proteins after this time. At around 50 s, there is sudden
kink as shown in the inset of the graph. For mixed proteins adsorbing from total concentration of 0.04 mg/ml, we observed sudden steep decrease in Fn /n curve after about 50 s. These may be due to desorption of BSA followed by adsorption of Fb. At higher concentration (0.04 mg/ml of mixed proteins) the kink is not prominent due to strong adsorption of BSA on the mixed surface. On the hybrid surface, at around 120 s from the start of the protein adsorption studies, there are kinks in the Fn /n vs time curves (see Fig. 2). This corresponds to the displacement of BSA by Fb. The equilibrium Fn /n and Dn at different concentrations of individual proteins and 1:1 mixture of proteins are listed in Table 1. The quantity of protein adsorbed from 0.04 mg/ml mixture
Fig. 2. Variation of Fn /n with time during adsorption of BSA, Fb and mixed proteins on hybrid surface (inset focuses kink part of mixed proteins adsorption data for t = 80 s to 200 s; circle: BSA 0.02 mg/ml, square: Fb 0.02 mg/ml, open diamond: mixed protein 0.01 mg/ml each; filled diamond: mixed protein 0.02 mg/ml each).
L.M. Pandey, S.K. Pattanayek / Applied Surface Science 264 (2013) 832–837
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Table 1 Equilibrium −Fn /n, D at different concentration of proteins at mixed and hybrid surface. Conc. of proteins (mg/ml)
Mixed surface
Hybrid surface
BSA
Fb
−Fn /n
D (×10−6 )
−Fn /n
D (×10−6 )
1E−5 2E−5 0 2E−05
1E−5 0 2E−5 2E−05
8.7 11.9 11.3 17.5
2.8 3.7 3.7 5.7
8.5 10.8 9.6 14.5
2.7 14 3.5 5.5
is significantly lower the sum of the individual proteins from their 0.02 mg/ml solutions on both the surfaces. This indicates that competitive adsorption takes place at this condition. The adsorbed quantity of the mixed proteins from 0.04 mg/ml solution is almost twice of that from 0.02 mg/ml solution on mixed surface. We have already reported [22] that adsorption isotherms of BSA and Fb are Langmuir type at a concentration less than 0.1 mg/ml on mixed surface. At a low concentration (0.02 and 0.04 mg/ml) of mixture of proteins, the adsorption is expected to follow the Langmuir adsorption isotherm. The adsorbed amount is less than the monolayer of Fb in the end on form. Hence we do not expect the multilayer of protein adsorption. Dn of the adsorbed proteins is higher for mixture of proteins at concentration 0.04 mg/ml than at 0.02 mg/ml. It has been always observed for individual protein adsorption even at low concentration (less than 0.1 mg/ml) that Dn of the adsorbed proteins on a substrate increases with increase in concentration of protein. This is due to increase in loosely bound proteins more at the surface. We have determined secondary structures of individually adsorbed proteins [23] using FTIR and found that beta sheet of Fb on mixed surface is higher than on hybrid surface. However, a similar studies using mixture of proteins will be done in future. Apparently after the competitive adsorption, the most of the proteins are Fb due to Vroman effect, which is discussed below. The Fb has lost tightly bound secondary structures at the interface, corresponding to high Dn value. 3.2. Viscoelastic characteristics of adsorbed layer In Fig. 3, we have plotted the variation of Dn with time during the adsorption from individual protein solution of BSA, Fb and from their mixture on the mixed and hybrid surfaces. Comparing the curves of total protein concentration 0.04 mg/ml and 0.02 mg/ml in mixture, we find that slopes of the curves are similar initially for both the cases, but after a certain time, tc , there is difference in slopes. The tc values depend on the substrate. The value of tc on hybrid surface is about 120 s, while that on mixed surface is about 50 s. The curves indicate that proteins are more elastic if adsorbing from lower concentration of mixture of proteins. The Dn values of
Table 2 Variation of slope of Dn vs −Fn /n curve with concentration and surfaces. Conc. of mixed protein (mg/ml)
Adsorbing surface
1st slope (Dn /−Fn /n)
2nd slope (Dn /−Fn /n)
0.02 0.04 0.02 0.04
Mixed Mixed Hybrid Hybrid
0.061 0.157 0.004 0.055
1.161 0.342 0.341 0.417
proteins adsorbing from mixture of proteins on both the surfaces, are almost similar to that of Fb adsorbing alone from solution after a long adsorption time. This indicates that the surface is mainly covered with the Fb. It is also found that Dn has three distinct regions for the mixed protein adsorption. Initial constant low Dn , rapidly increasing region and an approximate constant slope region at a time, tf . The variation of Dn with time corresponds to change in structure of adsorbed proteins. The tf is found to appear for mixed protein adsorption. This characteristic indicates the structures of adsorbed proteins do not change after this time, or change in structure of proteins that do not affect the Dn . We have investigated the slopes of the plot of (Fn /n) vs Dn for all the above cases, as it denotes the qualitative idea of elasticity of the adsorbed materials [24,26]. The plots of (Fn /n) vs Dn are shown in Fig. 4. During the adsorption of proteins two different slopes of the curves are identified. The 1st slope is smaller compared to the 2nd slope for all the cases as shown in Table 2. This indicates that proteins initially adsorbs strongly with high elasticity. With progress of adsorption it becomes less elastic. With increase in concentration the 1st slope increases on same substrate. This indicates the adsorbed proteins are softer if adsorbing from concentrated solution. In general, −Fn /n and Dn increase with increase in adsorb amount. A relatively large increase in Dn can lead to increase in Dn /(−Fn /n), which can be achieved from the multilayer adsorption of proteins or loose packing of proteins after certain time. Comparing the slopes Dn /(−Fn /n) of individual proteins [23] and mixed proteins at same 0.02 mg/ml concentration, the adsorbed proteins are more soft for the case of protein adsorbing from individual protein
Fig. 3. Variation of D with time during adsorption of BSA, Fb and mixed proteins on (a) mixed SAM and (b) hybrid SAM (circle: BSA 0.02 mg/ml, square: Fb 0.02 mg/ml, open diamond: mixed protein 0.01 mg/ml each; filled diamond: mixed protein 0.02 mg/ml each).
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L.M. Pandey, S.K. Pattanayek / Applied Surface Science 264 (2013) 832–837
Fig. 4. Variation of D with F during adsorption of BSA, Fb and mixed proteins on (a) mixed SAM and (b) hybrid SAM (circle: BSA 0.02 mg/ml, square: Fb 0.02 mg/ml, open diamond: mixed protein 0.01 mg/ml each; filled diamond: mixed protein 0.02 mg/ml each).
solution. This may be due to compact structure of proteins near interface for the case of proteins adsorbing from their mixture. 3.3. Dynamics of adsorption On the hybrid surface, adsorption of BSA is very slow compared to fibrinogen. Also, the dissipation factor of the adsorbed BSA is also very high compared to Fb. These indicate that the change of conformation of BSA is very slow, leading to very less adsorbed amount and loosely bound state. Conformation of Fb changes comparatively at faster rate due to its higher hydrophobicity. Comparing the individual protein adsorption and mixed protein adsorption at total protein concentration 0.02 mg/ml, both adsorbed amount and dissipation factor is lesser for the latter case, corresponding to tightly bound proteins compared to individual protein adsorption. Comparing the adsorption rate from mixture of proteins on mixed surface and hybrid surface, we found that the adsorption is faster on hybrid surface. The first two regions of Dn of Fig. 3a and b for mixture of protein adsorption are corresponding fast falling rate region of Fn /n vs time in the figures. Once mixed proteins attain the equilibrium Fn /n values, the Dn also reaches equilibrium values. The Dn increases sharply within the time span tc and tf . The rate of change of Dn with time on hybrid surface is higher than that on mixed surface. This may be due to random attachment of proteins, followed by faster rearrangement at the hybrid surface. On the other hand, on mixed surface, the adsorbed proteins do not undergo rapid rearrangement due to presence of amine groups on it. The initial rate adsorption on hybrid surface from 0.04 mg/ml solution is much faster than that from 0.02 mg/ml solution. This is an expected trend. But the initial adsorption rate on the mixed surface from 0.04 mg/ml solution is almost equal to 0.02 mg/ml solution. This indicates that on the mixed surface, rearrangement of protein molecules is slow and hence initial rate of adsorption is independent of concentration. In general three different combinations of Fn /n and Dn are found. A high Fn /n appears with a high Dn values for adsorbing proteins. This corresponds to adsorbed proteins with small change in secondary structures at the surface. A lower Fn /n and corresponding lower Dn values due to small amount of tightly bound adsorbed proteins. But lower Fn /n and a very high Dn values indicate loosely sporadically bound proteins at the surface. 4. Conclusions −Fn /n of adsorbed proteins from its mixture is lesser than that from individual protein solution. This is due to higher negative entropy in mixture of proteins case. The displacement of BSA
happens after 50 s on mixed surface and after 120 s on hybrid surface. It is found that the adsorbing proteins are softer, if adsorbing from higher concentration. If we compare the slopes Dn /(−Fn /n) of individual proteins and mixed proteins, the adsorbed proteins are more soft while adsorbing from individual protein solution. It is also found that higher the concentration of protein, the lesser is the initial required time for proteins to reach the hybrid surface. But the initial adsorption rate is independent of concentration of mixed proteins due to slower rate of rearrangement at the mixed surface. Acknowledgement The authors would like to thank Department of Biotechnology for their financial support (sanction number BT/PR9683/MED/32/16/2007) for this work. References [1] E.P. Vieira, S. Rocha, M.C. Pereira, H. Möhwald, M.A.N. Coelho, Adsorption and diffusion of plasma proteins on hydrophilic and hydrophobic surfaces: effect of trifluoroethanol on protein structure, Langmuir 25 (2009) 9879–9886. [2] E. Servoli, D. Maniglio, M.R. Aguilar, A. Motta, J.S. Roman, L.A. Belfiore, C. Migliaresi, Quantitative analysis of protein adsorption via atomic force microscopy and surface plasmon resonance, Macromolecular Bioscience 8 (2008) 1126–1134. [3] P. Warkentin, B. Wäivaara, I. Lundström, P. Tengvall, Differential surface binding of albumin, immunoglobulin G and fibrinogen, Biomaterials 15 (1994) 786–795. [4] A. Higuchi, K. Sugiyama, B.O. Yoon, M. Sakurai, M. Hara, M. Sumita, S. Sugawarac, T. Shirai, Serum protein adsorption and platelet adhesion on pluronicTM adsorbed polysulfone membranes, Biomaterials 24 (2003) 3235–3245. [5] S.N. Rodriguesa, I.C. Gonc¸alves, M.C.L. Martins, M.A. Barbosa, B.D. Ratner, Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers, Biomaterials 27 (2006) 5357–5367. [6] P.W. Wojciechowskil, J.L. Brash, Fibrinogen and albumin adsorption from human blood plasma and from buffer onto chemically functionalized silica substrates, Colloids and Surfaces B: Biointerfaces 1 (1993) 107–117. [7] M. Deyme, A. Baszkin, J.E. Proust, E. Perez, G. Albrecht, M.M. Boissonnade, Collagen at interfaces. II. Competitive adsorption of collagen against albumin and fibrinogen, Journal of Biomedical Materials Research 21 (1987) 321–328. [8] M. Holmberg, X. Hou, Competitive protein adsorption—multilayer adsorption and surface induced protein aggregation, Langmuir 25 (2009) 2081–2089. [9] M. Holmberg, K.B. Stibius, N.B. Larsen, X. Hou, Competitive protein adsorption to polymer surfaces from human serum, Journal of Materials Science: Materials in Medicine 19 (2008) 2179–2185. [10] S.R. Sousa, M. Lamghari, P. Sampaio, P. Moradas-Ferreira, M.A. Barbosa, Osteoblast adhesion and morphology on TiO2 depends on the competitive preadsorption of albumin and fibronectin, Journal of Biomedical Materials Research 84A (2008) 281–290. [11] P. Ying, Y. Yu, G. Jin, Z. Tao, Competitive protein adsorption studied with atomic force microscopy and imaging ellipsometry, Colloids and Surfaces B: Biointerfaces 32 (2003) 1–10. [12] S.S. Vaidya, R. Ofoli, Adsorption and interaction of fibronectin and human serum albumin at the liquid–liquid interface, Langmuir 21 (2005) 5852–5858. [13] M. Malmsten, B. Lassen, Competitive adsorption at hydrophobic surfaces from binary protein systems, Journal of Colloid and Interface Science 166 (1994) 490–498.
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Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Properties of competitively adsorbed BSA and fibrinogen from their mixture on mixed and hybrid surfaces Lalit M. Pandey, Sudip K. Pattanayek ∗ Department of Chemical Engineering, IIT Delhi, New Delhi 110016, India
a r t i c l e
i n f o
Article history: Received 24 July 2012 Received in revised form 10 October 2012 Accepted 27 October 2012 Available online 3 November 2012 Keywords: Competitive adsorption Adsorption of proteins Chemical potential Vroman effect QCM Hybrid SAM
a b s t r a c t We have studied the adsorption of BSA and fibrinogen from their mixture onto surfaces with mixed selfassembled monolayer (SAM) of amine and octyl (ratio 1:1) and hybrid SAM. The properties of adsorbed proteins obtained from individual protein solution differ considerably from the properties of the adsorbed proteins obtained from mixture of proteins at same total concentration. The adsorbed amount of proteins is lesser and the adsorbed protein is more elastic if it is adsorbing from mixture of proteins. It is found that with increasing total protein concentration, adsorbed amount increases and elasticity of the adsorbed proteins decreases. The apparent displacements of BSA with Fb are observed on the graphs of change in frequency with time, which are obtained from quartz crystal microbalance. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Biological fluid contains mixture of large number of proteins of very small to very large molecular weight. Major constituents are albumin, immunoglobulin and fibrinogen [1]. When a foreign material comes in contact with the biological fluid, the proteins of all different sizes compete for the same adsorbing sites of a surface. Finally, amount of adsorbed proteins and its characteristics depend on interaction among proteins and surface. There exist a number of studies on single protein adsorption behaviors from its solutions in the literature. There are considerable difference between the single protein adsorption and competitive adsorption [2–4]. Smaller globular proteins (e.g. albumin) which are abundance in biological fluid, adsorb initially and are displaced by less abundant proteins with lower mobility and more surface active (larger) proteins (i.e. fibrinogen, kininogen). This is known as “Vroman effect”, which has been tested using competitive adsorption [3,5–16] or sequential adsorption [17–20] of proteins. The factors affecting competitive adsorption behavior are bulk concentration [7,17] of proteins, affinity of proteins for the adsorbing surface [11,17] and conformation of adsorbed proteins [11]. Each protein arrives at the surface according to its own transport rate, which depends on its size and its concentration in solution [7]. The affinity of proteins for a surface depends on hydrophobicity of surface [17]. In general, a hydrophobic surface favors the
∗ Corresponding author. Tel.: +91 11 26591018; fax: +91 11 26581120. E-mail address: [email protected] (S.K. Pattanayek). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2012.10.150
irreversible adsorption of proteins from aqueous solution thermodynamically, while a highly hydrophilic surface inhibits adsorption of protein [8]. The irreversibly adsorbed proteins cannot be displaced by an incoming large protein [13,14]. Various combination of proteins including BSA/IgG [3,8,14], BSA/BGG [18], collagen/albumin [11], etc. have been used in studies of competitive and sequential adsorption. No consensus has been achieved on the observation on “Vroman effect” stated below. Lassen et al. [15] found that albumin is displaced by IgG or Fb during adsorption from binary mixture of HAS and IgG or HAS and Fb respectively on hydrophilic poly acrylic acid and poly diaminocyclohexane coated surfaces. But the displacement by IgG or Fb were not observed on hexamethyldisiloxane coated and methylated surface [14,15] for the proteins mixtures at the same condition. For another system, adsorption from mixture of HSA and gammaglobulin (HGG) on glass surface, HGG displaced HAS and on C16 self-assembled monolayers (SAMs) HSA competes with HGG [14]. Wertz et al. [8] have observed that no displacement of pre-adsorbed BSA by Fb and vice versa were found during sequential adsorption on both OH and C16 SAMs. They observed that protein which arrives late occupies only free space available on the surface [21]. Contrary to above observations Green et al. [17] found that albumin is displaced by Fb from binary mixture of albumin/Fb on polystyrene surface [14]. Recently, Benesch et al. [18] have observed the displacement of BSA by gamma-globulin (BGG) from its binary mixture on both, OH and CH3 SAMs. The literature lacks in detail of kinetics and in viscoelatic properties of the adsorbed proteins from their mixture at the surface. The fundamental questions that are unanswered are as follows: (a)
L.M. Pandey, S.K. Pattanayek / Applied Surface Science 264 (2013) 832–837
How kinetics of competition differs from single protein adsorption kinetics? (b) What is role of surfaces to mixed protein adsorption? We reported [22] that hybrid SAM, which has both hydrophobic and hydrophilic groups on the same modifying molecule is a potential surface modifying agent of biomaterials. We compared properties of the adsorbed BSA and Fb from their solution on the hybrid surface and the most widely used mixed (amine-octyl) SAM. Here, we report adsorption of mixture of BSA and Fb using QCM and compared with the single protein adsorption. We also have determined the viscoelastic characteristics of adsorbed layer. 2. Materials and methods (APTMS) and 3-Aminopropyl-trimethoxysilane octyltrimethoxysilane (OTMS) were used for the formation of amine:octyl mixed (1:1) SAM. Dibulyltin dilaurate (95%), ptolyl isocynate (99%) and anhydrous toluene (99.8%) were used for synthesis of hybrid SAM. APTMS, OTMS, dibulyltin dilaurate, p-tolyl isocynate and anhydrous toluene were purchased from Sigma–Aldrich. Methanol and acetone (HPLC grade) were purchased from Merck. Proteins, bovine serum albumin (BSA, A0281) and fibrinogen (Fb, F8630), were purchased from Sigma–Aldrich. These are used without any further purification. Sodium chloride, potassium chloride, di-sodium hydrogen phosphate (anhydrous) and potassium dihydrogen orthophosphate were used for preparing phosphate buffer saline (PBS), were purchased from Merck. We carried out following experiments (i) modification of silica surfaces with various self-assembled monolayers and its characterization and (ii) adsorption of proteins from binary mixture by using QCM. 2.1. Surface modification and characterization Silica coated quartz crystal was cleaned with “piranha” solution. Mixed (amine:octyl) and hybrid SAMs were formed on the cleaned silica surfaces. The procedure of the surface modification and characterization of the modified surfaces already has been discussed in Ref. [22], which is described briefly below. Cleaned silica coated quartz crystal was modified with required modifier in QCM chamber itself. 1% solution of APTMS and 1:1 mixture of APTMS and OTMS in anhydrous toluene at 25 ◦ C were used to from amine and mixed (amine:octyl) SAMs, respectively. After formation of amine SAM, 1% (v/v) solution of p-tolyl isocynate in anhydrous toluene was passed into QCM chamber to from hybrid SAM. The catalyst dibulyltin dilaurate (2–3 drops) was added into the reaction mixture. The amine ( NH2 ) and isocynate ( NCO) groups reacted to form urea (NH CO NH) linkage. The formed monolayer had hydrophilic urea group and hydrophobic toluene group. After the formation of SAMs, the crystals were washed consecutively in three different solvents: toluene, mixture of toluene and ethanol (v/v = 1:1) and ethanol. The surface energies of the modified surfaces are determined from its contact angle against deionized water, ethylene glycol and methylene di-iodide. The contact angles were measured by contact angle goniometer (Kruss DSA-10). The characteristics of the surfaces are almost equal to the earlier reported data [22] within the error limit of 10%. Water contact angle on both surfaces was 80 ± 1◦ . From the contact angle data, we found surface energy ( s ) of mixed (amine:octyl) and hybrid surfaces to be 37.22 and 39.19 mJ/m2 , respectively. Details of the data can be found in Ref. [22].
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0.02 mg/ml) and binary mixture (1:1) of BSA and Fb on mixed and hybrid SAMs. The total concentrations of proteins were 0.02 mg/ml (0.01 mg/ml of individual proteins) and 0.04 mg/ml (0.02 mg/ml of individual proteins). Adsorption of individual proteins is already reported [22,23]. Adsorption of the mixed proteins was done as described below. We passed 10 ml phosphate buffer saline solution (PBS) of pH 7.4 through the chamber and allowed the crystal to stabilize. Then, 10 ml of mixed protein solution was passed through the washed chamber. The protein solution was kept within the chamber for about 30 min and PBS solution was passed through the chamber to remove the loosely bound proteins. At the end of the washing, we get characteristics of adsorbed proteins at equilibrium only. This value is always about 10% less than equilibrium value obtained with unwashed adsorbed proteins. All QCM experiments were repeated at least twice, and the obtained values were within 10% standard deviation of the presented average values. We obtained changes in resonance frequency of different overtones (Fn /n) and dissipation factor (Dn ) of the quartz crystal in real time due to adsorption of the proteins. Adsorbed mass (m) is related to Fn /n by Sauerbrey equation [22,24] for strongly adsorbed protein. The relation is m = − CFn /n, where C is sensitivity constant of crystal, 17.7 ng/(cm2 Hz). In this paper −Fn /n and adsorbed amount are used synonymously. The dissipation factor (Dn ), a non-dimensional parameter, which compares energy dissipated to energy stored on the adsorbed material is defined as: Dn =
Edissipated 2Estored
(1)
where Edissipated is the energy lost (dissipated) during one oscillation cycle and Estored is the total energy stored in the adsorbed protein [24]. Energy dissipation reflects the viscoelastic properties of the adsorbed layer. Slower dissipation refers to a more elastic layer of adsorbed proteins. 3. Results and discussion 3.1. Adsorption of mixture of BSA and Fb using QCM Variation of frequencies, Fn /n, with time during adsorption of individual BSA, Fb and their mixture on mixed surface and on hybrid surface are shown in Figs. 1 and 2, respectively. The equilibrium adsorbed amount of proteins adsorbing from the mixture of BSA and Fb (total protein concentration 0.02 mg/ml) is lower than that of individual protein adsorbing from their bulk concentration of 0.02 mg/ml. A similar effect is reported for adsorption of proteins from mixture of BSA/IgG on plasma treated polyethylene terephthalate [8]. The reason of the observation was not explained. We can explain from calculation of entropy of mixture using Flory–Huggins n1 moles of solequation [25]. We write total entropy of mixture of vent and n2 and n3 moles of proteins as −Smix /R = j nj ln j . Here, volume fraction of jth component is given by j = nj vj / j nj vj , where vj is volume of each molecule. The entropic terms of chemical potentials of solvent (j = 1) and proteins (j = 2, 3) are given by 2 3 1 = ln 1 + 2 + 3 − − RT v2 /v1 v3 /v1
(2)
v2 v3 2 = ln 2 + 1 + 3 − 1 − 3 RT v1 v2
(3) (4)
2.2. Adsorption of proteins from binary mixture by using QCM
v3 v2 3 = ln 3 + 1 + 2 − 1 − 2 RT v1 v3
Experiments of adsorption of proteins were done for individual solution of BSA (concentration 0.02 mg/ml), Fb (concentration
The chemical potential of both the proteins (2 /RT, 3 /RT) are lesser in the ternary solution than that in the binary solution. The difference in entropy for two different cases is small. However,
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Fig. 1. Variation of Fn /n with time during adsorption of BSA, Fb and mixed proteins on mixed surface (inset focuses kink part of mixed proteins adsorption data for t = 30 s to 80 s; circle: BSA 0.02 mg/ml, square: Fb 0.02 mg/ml, open diamond: mixed protein 0.01 mg/ml each; filled diamond: mixed protein 0.02 mg/ml each).
the entropy of the adsorbed state being same, the proteins lose higher amount of entropy if adsorbing from mixture of two protein solution. This indicates that proteins adsorb in lesser amount on the surface from the ternary system than that from the binary system. On the mixed surface, the kinetics of proteins adsorbing from 1:1 mixture of BSA and Fb (total protein concentration 0.02 mg/ml) is slightly different from that of total protein concentration 0.04 mg/ml during the initial period up to about 50 s (see Fig. 1). The adsorption for the case of total protein concentration 0.02 mg/ml attains nearly equilibrium value at the later stage. However, there is little variation in the Fn /n values due to the rearrangement of the adsorbed proteins after this time. At around 50 s, there is sudden
kink as shown in the inset of the graph. For mixed proteins adsorbing from total concentration of 0.04 mg/ml, we observed sudden steep decrease in Fn /n curve after about 50 s. These may be due to desorption of BSA followed by adsorption of Fb. At higher concentration (0.04 mg/ml of mixed proteins) the kink is not prominent due to strong adsorption of BSA on the mixed surface. On the hybrid surface, at around 120 s from the start of the protein adsorption studies, there are kinks in the Fn /n vs time curves (see Fig. 2). This corresponds to the displacement of BSA by Fb. The equilibrium Fn /n and Dn at different concentrations of individual proteins and 1:1 mixture of proteins are listed in Table 1. The quantity of protein adsorbed from 0.04 mg/ml mixture
Fig. 2. Variation of Fn /n with time during adsorption of BSA, Fb and mixed proteins on hybrid surface (inset focuses kink part of mixed proteins adsorption data for t = 80 s to 200 s; circle: BSA 0.02 mg/ml, square: Fb 0.02 mg/ml, open diamond: mixed protein 0.01 mg/ml each; filled diamond: mixed protein 0.02 mg/ml each).
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Table 1 Equilibrium −Fn /n, D at different concentration of proteins at mixed and hybrid surface. Conc. of proteins (mg/ml)
Mixed surface
Hybrid surface
BSA
Fb
−Fn /n
D (×10−6 )
−Fn /n
D (×10−6 )
1E−5 2E−5 0 2E−05
1E−5 0 2E−5 2E−05
8.7 11.9 11.3 17.5
2.8 3.7 3.7 5.7
8.5 10.8 9.6 14.5
2.7 14 3.5 5.5
is significantly lower the sum of the individual proteins from their 0.02 mg/ml solutions on both the surfaces. This indicates that competitive adsorption takes place at this condition. The adsorbed quantity of the mixed proteins from 0.04 mg/ml solution is almost twice of that from 0.02 mg/ml solution on mixed surface. We have already reported [22] that adsorption isotherms of BSA and Fb are Langmuir type at a concentration less than 0.1 mg/ml on mixed surface. At a low concentration (0.02 and 0.04 mg/ml) of mixture of proteins, the adsorption is expected to follow the Langmuir adsorption isotherm. The adsorbed amount is less than the monolayer of Fb in the end on form. Hence we do not expect the multilayer of protein adsorption. Dn of the adsorbed proteins is higher for mixture of proteins at concentration 0.04 mg/ml than at 0.02 mg/ml. It has been always observed for individual protein adsorption even at low concentration (less than 0.1 mg/ml) that Dn of the adsorbed proteins on a substrate increases with increase in concentration of protein. This is due to increase in loosely bound proteins more at the surface. We have determined secondary structures of individually adsorbed proteins [23] using FTIR and found that beta sheet of Fb on mixed surface is higher than on hybrid surface. However, a similar studies using mixture of proteins will be done in future. Apparently after the competitive adsorption, the most of the proteins are Fb due to Vroman effect, which is discussed below. The Fb has lost tightly bound secondary structures at the interface, corresponding to high Dn value. 3.2. Viscoelastic characteristics of adsorbed layer In Fig. 3, we have plotted the variation of Dn with time during the adsorption from individual protein solution of BSA, Fb and from their mixture on the mixed and hybrid surfaces. Comparing the curves of total protein concentration 0.04 mg/ml and 0.02 mg/ml in mixture, we find that slopes of the curves are similar initially for both the cases, but after a certain time, tc , there is difference in slopes. The tc values depend on the substrate. The value of tc on hybrid surface is about 120 s, while that on mixed surface is about 50 s. The curves indicate that proteins are more elastic if adsorbing from lower concentration of mixture of proteins. The Dn values of
Table 2 Variation of slope of Dn vs −Fn /n curve with concentration and surfaces. Conc. of mixed protein (mg/ml)
Adsorbing surface
1st slope (Dn /−Fn /n)
2nd slope (Dn /−Fn /n)
0.02 0.04 0.02 0.04
Mixed Mixed Hybrid Hybrid
0.061 0.157 0.004 0.055
1.161 0.342 0.341 0.417
proteins adsorbing from mixture of proteins on both the surfaces, are almost similar to that of Fb adsorbing alone from solution after a long adsorption time. This indicates that the surface is mainly covered with the Fb. It is also found that Dn has three distinct regions for the mixed protein adsorption. Initial constant low Dn , rapidly increasing region and an approximate constant slope region at a time, tf . The variation of Dn with time corresponds to change in structure of adsorbed proteins. The tf is found to appear for mixed protein adsorption. This characteristic indicates the structures of adsorbed proteins do not change after this time, or change in structure of proteins that do not affect the Dn . We have investigated the slopes of the plot of (Fn /n) vs Dn for all the above cases, as it denotes the qualitative idea of elasticity of the adsorbed materials [24,26]. The plots of (Fn /n) vs Dn are shown in Fig. 4. During the adsorption of proteins two different slopes of the curves are identified. The 1st slope is smaller compared to the 2nd slope for all the cases as shown in Table 2. This indicates that proteins initially adsorbs strongly with high elasticity. With progress of adsorption it becomes less elastic. With increase in concentration the 1st slope increases on same substrate. This indicates the adsorbed proteins are softer if adsorbing from concentrated solution. In general, −Fn /n and Dn increase with increase in adsorb amount. A relatively large increase in Dn can lead to increase in Dn /(−Fn /n), which can be achieved from the multilayer adsorption of proteins or loose packing of proteins after certain time. Comparing the slopes Dn /(−Fn /n) of individual proteins [23] and mixed proteins at same 0.02 mg/ml concentration, the adsorbed proteins are more soft for the case of protein adsorbing from individual protein
Fig. 3. Variation of D with time during adsorption of BSA, Fb and mixed proteins on (a) mixed SAM and (b) hybrid SAM (circle: BSA 0.02 mg/ml, square: Fb 0.02 mg/ml, open diamond: mixed protein 0.01 mg/ml each; filled diamond: mixed protein 0.02 mg/ml each).
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Fig. 4. Variation of D with F during adsorption of BSA, Fb and mixed proteins on (a) mixed SAM and (b) hybrid SAM (circle: BSA 0.02 mg/ml, square: Fb 0.02 mg/ml, open diamond: mixed protein 0.01 mg/ml each; filled diamond: mixed protein 0.02 mg/ml each).
solution. This may be due to compact structure of proteins near interface for the case of proteins adsorbing from their mixture. 3.3. Dynamics of adsorption On the hybrid surface, adsorption of BSA is very slow compared to fibrinogen. Also, the dissipation factor of the adsorbed BSA is also very high compared to Fb. These indicate that the change of conformation of BSA is very slow, leading to very less adsorbed amount and loosely bound state. Conformation of Fb changes comparatively at faster rate due to its higher hydrophobicity. Comparing the individual protein adsorption and mixed protein adsorption at total protein concentration 0.02 mg/ml, both adsorbed amount and dissipation factor is lesser for the latter case, corresponding to tightly bound proteins compared to individual protein adsorption. Comparing the adsorption rate from mixture of proteins on mixed surface and hybrid surface, we found that the adsorption is faster on hybrid surface. The first two regions of Dn of Fig. 3a and b for mixture of protein adsorption are corresponding fast falling rate region of Fn /n vs time in the figures. Once mixed proteins attain the equilibrium Fn /n values, the Dn also reaches equilibrium values. The Dn increases sharply within the time span tc and tf . The rate of change of Dn with time on hybrid surface is higher than that on mixed surface. This may be due to random attachment of proteins, followed by faster rearrangement at the hybrid surface. On the other hand, on mixed surface, the adsorbed proteins do not undergo rapid rearrangement due to presence of amine groups on it. The initial rate adsorption on hybrid surface from 0.04 mg/ml solution is much faster than that from 0.02 mg/ml solution. This is an expected trend. But the initial adsorption rate on the mixed surface from 0.04 mg/ml solution is almost equal to 0.02 mg/ml solution. This indicates that on the mixed surface, rearrangement of protein molecules is slow and hence initial rate of adsorption is independent of concentration. In general three different combinations of Fn /n and Dn are found. A high Fn /n appears with a high Dn values for adsorbing proteins. This corresponds to adsorbed proteins with small change in secondary structures at the surface. A lower Fn /n and corresponding lower Dn values due to small amount of tightly bound adsorbed proteins. But lower Fn /n and a very high Dn values indicate loosely sporadically bound proteins at the surface. 4. Conclusions −Fn /n of adsorbed proteins from its mixture is lesser than that from individual protein solution. This is due to higher negative entropy in mixture of proteins case. The displacement of BSA
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