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material interactions probed by atomic force microscopy
Colloids and Surfaces B: Biointerfaces 23 (2002) 153– 163 www.elsevier.com/locate/colsurfb
Review
Mechanistic aspects of protein/material interactions probed by atomic force microscopy Satoru Kidoaki, Takehisa Matsuda * Department of Biomedical Engineering, Graduate School of Medicine, Kyusyu Uni6ersity, Higashi-ku, Fukuoka 812 -8582, Japan Received 10 January 2001; accepted 10 April 2001
Abstract Physicochemical studies on the mechanisms of protein adsorption onto solid material surfaces have been extensively performed so far, mainly based on the analysis of factors such as the equilibrium adsorbed amount (adsorption isotherms), time-dependent change of adsorbed amount (adsorption kinetics), and conformational change of adsorbed protein. However, direct understanding of the strength of the molecular interaction between protein and the material surface has not been established yet. For this issue, the force measurement techniques of an atomic force microscope (AFM) using a protein-modified probe tip are recently becoming powerful tools to analyze the actual interaction forces between protein and material surfaces. In this mini review, we discuss the characteristics and interpretation of the AFM force-versus-distance curves ( f– d curves) obtained with the protein-modified probe tip, and the relationship between the forces measured from the f–d curves and the driving forces in the natural process of protein adsorption. Relative degrees of each of the following contributions which determine the character of protein adsorption are clarified: (1) the intrinsic protein/material forces mediated by solvent, (2) the thermodynamic stability of protein/material adhesion interface, and (3) diffusion force of protein molecules. Within these driving forces, the latter two in particular are confirmed to play essential roles in determining the character of protein adsorption, based on the profiles of f –d curves. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Mechanistic; Protein material interactions; Atomic force microscopy
1. Introduction The investigation of the interaction of proteins with solid material surfaces has been one of the * Corresponding author. Tel.: + 81-92-642-6210; fax: + 8192-642-6212. E-mail address: [email protected] (T. Matsuda).
essential themes in various surface-dependent industrial fields such as medical devices [1,2]. In particular, in the field of cardiovascular device fabrication, the regulation of the adsorption of blood plasma proteins onto the device surfaces, i.e. both facilitation and inhibition of adsorption, has been studied to appropriately design antithrombotic devices. Generally, a thrombus is
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formed after a series of biological reactions, such as platelet and leucocyte adhesion, blood coagulation, and fibrinolysis, which are known to be conditioned by the initially adsorbed protein layer on the device surface [1–3]. Therefore, in order to prepare the antithrombotic surfaces, the following opposite approaches have so far been applied on the basis of the conditioning of the initially adsorbed protein layer: (a) Selective facilitation of the formation of the non-cell-adhesive protein layer such as albumin which prevent the platelet adhesion. This approach is based on Lyman’s hypothesis [4]. (b) Inhibition of the protein adsorption itself, which prevents both the activation of blood coagulation factors and platelet adhesion. This idea was first advocated by Andrade et al. [5] and is based on the fact that the inner wall of a blood vessel has a hydrogel layer which is highly effective for the inhibition of protein adsorption, i.e. minimum interfacial free energy hypothesis. To elucidate a predictable principle to regulate the adsorption of protein onto material surfaces, the mechanisms of protein adsorption have been extensively studied not only biochemically but also physicochemically (see reviews [1,2,6– 11]). The physicochemical factors that determine the manner of protein adsorption and desorption typically include the following eight items which are characteristic in each of the processes of protein approach to, attachment on, and detachment from the surface (see Fig. 1). First, in the approach process, both the transport properties of the protein molecules (1) and the intrinsic interaction forces between proteins and surfaces (2) determine the degree of protein approach to the surface; both are affected by solvent motion and its molecular properties [2]. Second, in the attachment process, the following three factors comprise the driving force of the stabilization of adhesion interface: (3) the short-range interaction force between protein and surface, (4) the entropy gain due to the release of hydrated waters and bound counterions between the protein and the surface [7,12,13], (5) the entropy gain due to the surface denaturing of the adsorbed protein [7]. Third, in the detachment process, the following three factors contribute to destabilize the adhesion inter-
face and induce detachment [2]: work provided by the motion of the solvent molecules, such as (6) thermal disturbance and (7) mechanical shear flow, and (8) work required for substitution due to the competitive adsorption of other adsorbates that can adhere more stably. The dependence of the amount of adsorbed protein or of the adsorption rate on these factors has been analyzed mainly through adsorption isotherms and adsorption kinetic data. However, direct understanding of the strength of the molecular interaction between the protein and the surface in the above-mentioned each process has not yet been established. Concerning this issue, recent developments in the technique of molecular force measurement using an atomic force microscope (AFM) are becoming a powerful tool for investigating the protein/surface interaction. The AFM allows the measurement of the force-versus-distance curve ( f–d curve) between the AFM tip and the sample surface, and has been successfully applied to force measurement of a system involving proteins, e.g. the measurement of single molecular forces between specific protein pairs such as avidin/biotin pairs [14–20] or antigen/antibody pairs [21–27], of the unfolding force of single protein molecules [28–36], and of nonspecific adhesion forces be-
Fig. 1. Scheme of the process of protein adsorption and desorption, and the physicochemical driving forces that induce each process.
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2. Protein fixation onto the AFM probe tip and the state of fixed proteins In the AFM f–d curve measurements of a system involving proteins, various methods for protein fixation onto the probe tip have been performed by several groups. In this chapter, we briefly review the method of protein fixation on the tip, and discuss the state of the proteins fixed with such methods. Fig. 2. Schematic representation of the character of the forces analyzed with f– d curve measurement employing the proteinmodified probe tip.
tween proteins and surfaces of material such as polystyrene [37,38] or glass [38]. By using the AFM probe tip, onto which a single protein molecule is fixed or protein monolayer is formed, one can measure the strength and range of molecular interaction between the protein and the surface in each of the above-mentioned processes, as schematically shown in Fig. 2. It should be noted that this methodology enables one to particularly analyze the following factors. (1) In the tip-approaching measurement, the intrinsic interaction forces between proteins and surfaces mediated by the solvent which does not undergo the diffusion motion of the protein can be measured. (2) In the tip-retracting measurement, the adhesion strength of the protein onto the surface by the vertical tensile test can be evaluated. Recently, we have applied such a methodology for the system of protein-fixed AFM tips (blood plasma proteins; albumin, globulin, fibrinogen, and fibronectin) and several kinds of model surfaces (self-assembled monolayer (SAM) surfaces with different kinds of functional groups [39] and polymer-grafted surfaces) [40,41]. In this short review, based on the force character analyzed by AFM techniques, we discuss the mechanistic aspects of protein adsorption onto and desorption from the model surfaces.
2.1. Method Gaub and co-workers functionalized the silicon nitride surface of the tip with biotinylated bovine serum albumin (BSA) through nonspecific adsorption of BSA [16–19]. They confirmed that BSA irreversibly binds to the silicon nitride surface [18]. Dammer et al. covalently fixed the biotinylated BSA [25] and the cell adhesion proteoglycan [24] onto the amino-reactive SAM of dithio-bis(succinimidyl-undecanoate) synthesized by them, which was prepared on the gold-deposited silicon nitride surface of the tip. Allen et al. applied the method of Vinckier et al. [42] in which 3aminopropyldimethylethoxysilane and glutaraldehyde are employed to prepare the surface with amino-reactive aldehyde groups on the tip, and covalently fixed ferritin [23] and human chorionic gonadotrophin [27] onto the tip. Chen et al. coated the tip with BSA by applying the same method as Allen et al. [37]. Oberleithner et al. treated the tip with poly-(L-lysine), a polycation, in order to prepare, on the tip surfaces, matrix which electrostatically attaches proteins [36]. They used this method to fix a renal epithelial potassium channel on the tip. Hinterdorfer et al. and Willemsen et al. synthesized an 8-nm-long polyethylene glycol (PEG) derivative with an amino-reactive end and a thiol-reactive end, and used it as a spacer for covalent linkage of polyclonal anti-human serum albumin antibody [22] and of anti-intercellular adhesion molecule-1 antibody [26], respectively. In their method, the amino-reactive end of the PEG-derivative reacts with the amine groups pre-introduced onto the tip surface by esterification between silanol groups of the tip and ethanolamine, while thiol-reactive
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groups of the PEG-derivative react with the proteins prederivatized with N-succinimidyl-3-(Sacethylthio)propionate to give protected SH groups [43].
2.2. The state of fixed proteins Interaction between the proteins fixed on the AFM probe tip and surfaces is significantly affected by the state of the fixed proteins, i.e. fixed density, amount, orientation, and conformation. To precisely understand the information obtained from the f–d curve using protein-tips, the state of fixed proteins has to be appropriately considered, based on the experimental conditions of protein fixation. First, fixed density and amount of the protein on the tip are determined by density of the fixation reaction sites on the tip and the washing procedures of reacted tip. In particular, the appropriate washing is essentially important for excluding the multilayers of protein, which would otherwise cause the force measurement to be inaccurate. The above-reviewed methods guarantee the preparation of monolayer of protein on the surface of a probe tip. Here, it should be noted that only the fixed proteins at the very top region of the tip usually contribute to the interaction force with the surface. If the density of the fixed proteins in the monolayer is low, a lack of fixed protein at the very top of the tip would occur. Thus, a sufficiently high dense fixation of proteins onto the surface of probe tip is required in the fixation. Since the radius of the tip is typically 10–40 nm, the force interaction area on the tip is usually occupied by only a few proteins even under such a high fixation density. Second, the regulation of the orientation of fixed protein is one of the most difficult tasks. In the conventional fixation reaction employing the condensation reaction between the activated amino-reactive group and amino group on the protein surface, the reaction occurs not only on the amino terminus of the protein but also on the amino groups of amino acid residues such as asparagine, glutamine, lysine, and arginine. Thus, fixed proteins are oriented on the tip in a random
manner. However, this is not necessarily disadvantage in the force measurement between fixed protein and a material surface, because proteins approach the surfaces with random orientation in natural processes of protein adsorption. Third, as for the conformation of the fixed proteins, little is known due to the experimental difficulty, i.e. AFM tips are too small to be analyzed by usual experimental apparatus for surface characterization. However, as the general trend, protein’s conformation is known to be changed due to the attachment process and/or the new local microenvironment. It should be noted that more or less, the protein on the tip is partially denatured. Since in particular, such conformational alteration strongly occurs on hydrophobic surface, pre-modified surface of the tip for protein fixation should be hydrophilic, in order to reduce the degree of denaturing. Based on the above information on the method of protein fixation and the state of fixed proteins, the formation of a covalently bound protein monolayer with sufficiently high density on the hydrophilically pre-modified probe tip is needed to characterize the protein/material interaction force by using AFM. Following this strategy, we have fixed four kinds of blood plasma proteins (albumin, g-globulin, fibrinogen, and fibronectin) onto the amino-reactive SAM formed from 10carboxy-1-dodecanethiol and N-hydroxylsuccinimide [39–41].
3. Characteristics of the f–d curves measured with the protein-modified AFM probe tip and the interpretation of the measured forces The f–d curves measured with AFM are generally composed of both the tip-approaching traces and the tip-retracting traces. In the following, we review and discuss the typical characteristics of each trace of the f–d curves measured with the protein-modified AFM probe tip, mainly taking advantage of our recent results of the measurement on the flat SAM surfaces of alkanethiolates [39] and polymer-grafted surfaces [40].
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3.1. Tip-approaching traces In the tip-approaching trace of the f – d curves, the intrinsic forces between proteins on the tip and material surfaces, which are mediated by solvent molecules, are plotted against the separation distance between the tip and the surface. The forces are usually composed of the van der Waals attraction, electrostatic attraction or repulsion, interactions between the hydration water layers between protein and surfaces, and steric repulsion in the case of polymer-grafted surfaces. Though, in principle, the strength and range of interaction of these forces can be analyzed from the tip-approaching traces, it should be noted that there is a detection limitation of weak forces in the analysis of tip-approaching trace. If the cantilever used is too stiff to be deflected by the forces mentioned above, the forces that would be calculated from the cantilever deflection can not be measured. Actually, for example, flat SAM surfaces do not exhibit any significant force from the protein-tips (albumin, IgG, fibrinogen) in the tip-approaching traces under the condition of a spring constant of 0.12 N m − 1 of a common cantilever (triangle-shaped Si3N4-probe; tip radius after protein fixation: ca. 50 nm), irrespective of
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the kind of highly packed functional groups on the SAM surfaces (CH3, NH2, OH, and COOH) (Fig. 3) [39]. The intrinsic forces between the protein and such flat SAM surfaces in solution were found to be too weak to be detected by such a common cantilever. Since, in our measurement, protein monolayer was formed on the surface of the probe tip and only several proteins were on the top of the tip, such a few proteins are considered to exhibit too weak force for the SAM surfaces to cause cantilever deflection. The significant forces between the protein monolayer and material surface can be detected by using a beadimmobilized tip [44], which is effective in detecting the total force contributions from many proteins on the rather wide surface of the bead. However, it should be noted that the rigorous determination of the intrinsic forces which a single protein molecule feels from flat material surfaces in solution media is quite difficult. On the other hand, for end-grafted polymer surfaces, steric repulsion due to the excluded volume effect of the grafted polymer chains was dominantly detected, irrespective of the charged state of the grafted polymers (Figs. 4 and 5). Interestingly, unlike the case of the flat SAM surface, a significant attractive peak was detected
Fig. 3. Representative f– d curves measured between the protein-modified tips ((a) albumin, (b) g-globulin, and (c) fibrinogen tips) and SAM surfaces terminated with CH3-, NH2-, OH-, and COOH-groups. The measurement was performed in phosphate-buffered saline (pH 7.4). Dashed curves: approaching trace. Solid curves: retracting trace. Z-positions mark those of the cantilever. The zero positions of the cantilever were chosen so as to indicate that the cantilever exhibits no deflection (i.e. zero applied force) at the position on the contact line. Reprinted with permission from [39]. Copyright 1999 American Chemical Society.
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peak is considered to be attributable to the accumulated force contributions of van der Waals attraction and electrostatic attraction between the many molecules of protein immobilized on the side of the probe tip and the polymers inside the grafted layer.
Fig. 4. Representative f– d curves measured between the albumin-modified tip and the photograft-polymerized surfaces of dimethylacrylamide (DMAAm) after photoirradiation for 10, 20 s, 1, 5, 10, and 20 min, which produced a set of graft-polymerized surfaces with different lengths of the grafted polymer chain. The measurements were performed in phosphatebuffered saline. Dashed curves: approaching trace. Solid curves: retracting trace. Z-positions mark those of the tip. The zero position of the tip is expediently defined as the starting position of the linear part in the tip-surface contact region. Reprinted with permission from [40]. Copyright 2000 American Chemical Society.
by a cantilever with the same spring constant (0.12 N m − 1) between oppositely charged protein (lysozyme, pKa :11, cationic under pH 7.4) and the grafted polymer layer (poly(AAc), pKa :5, anionic under pH 7.4) (Fig. 5b). Such an attractive peak was observed together with the steric repulsion profile and in the region of a Z-position of the tip where the grafted polymer layer was somewhat compressed by the tip. The attractive
Fig. 5. Representative f– d curves measured (a) between the albumin-modified tip (pKa :4.7, negatively charged protein tip) and the photograft-polymerized surface of acrylic acid (AAc) (pKa :5, negatively charged surface) after 60 min of photoirradiation (a — 1, 2, 3), and (b) between the lysozymemodified tip (pKa :11, positively charged protein tip) and the poly(acrylic acid) surface (negatively charged surface) (b — 1, 2, 3). The measurement was performed in phosphate-buffered saline (pH7.4). Dashed curves: approaching trace. Solid curves: retracting trace. Z-positions are defined similar to those in Fig. 4. Reprinted with permission from [40]. Copyright 2000 American Chemical Society.
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3.2. Tip-retracting traces In the tip-retracting traces of f – d curves, the forces in the detachment process between the tip and the surfaces are plotted against the upward moving distance of the cantilever or the distance of tip-sample separation. In this measurement, the tip is forcedly pushed against the material surface and an adhesion interface between the tip and the material surface is formed prior to the retraction of the tip. Thus, rigorously speaking, the force measured in the retracting traces is detachment force (i.e. adhesion force) in the vertical tensile test. As methods of evaluating the adhesion strength, there are some kinds of test such as the vertical tensile, tensile shear, compression shear, and peeling, because the detachment force generally varies depending on the conditions of measurement, e.g. direction of applied force, peeling speed, and adhesion area. In this sense, it should be noted that the adhesion force evaluated from the retracting trace does not necessarily reflect the general adhesion strength, since vertical tensile forces cannot be imposed on the adhered protein in a real desorption process. In the tip-retracting trace of the f – d curve measured with the protein-modified tip, characteristic multiple adhesive jumps are typically observed, as shown in Fig. 3, differing from the measurement using the tip without protein. The multiple adhesion profiles have been reported so far in the literature that dealt with the protein/ protein interaction, i.e. interaction between the protein immobilized on the tip and the protein coated on the material surface, such as in the system of the ligand/receptor combination. In our result, such profiles were observed particularly on hydrophobic flat surfaces (e.g. CH3-SAM) which exhibit the strongest adhesion strength compared with other kinds of hydrophilic surfaces. For example, for the flat hydrophilic SAM surfaces (NH2-, OH-, and COOH-SAM) and the hydrophilic neutral-polymer-grafted surface (poly(DMAAm)-surface), such a multiple adhesion profile was little observed (Figs. 3 and 4). For the charged polymer-grafted surface (poly(AAc)-surface), multiple adhesion peaks are observed, as shown in Fig. 5, but the profile is typically at-
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tributed to the elongation of the grafted polymer chains [45– 48]. Concerning the interpretation of the multiple adhesion peaks, the following effects have been considered in the literature: (1) nonlinear convolution of multiple unbinding processes of antigen/ antibody pairs [16]; (2) breaking of different contact points between the antigen/antibody pairs [25]; (3) lateral movement of the sensor tip during the process of approach or withdrawal [25]; (4) ‘spring’ effect of partially unfolded protein which is loaded during the process of withdrawal of the tip and bridges the tip and sample [25]. As has been discussed in the literature, the retracting traces of f–d curves contain information not only on the specific binding forces and nonspecific interaction forces between proteins, but also on the unfolding behaviors of protein molecules. In particular, the latter information has been effectively applied to estimate the conformations and mechanical properties of a single protein molecule [28–36]. However, such complex information including both molecular interaction forces and unfolding forces of proteins makes it difficult to evaluate the adhesion forces between proteins and surfaces. To rationally evaluate the adhesion strength of the protein from the retracting traces of the f–d curves, it is required to clearly define how the adhesion strength is measured from the f– d curve and to precisely understand the process of adhesion force measurement. To resolve this issue, we defined and evaluated the adhesion strength of the protein to the material surfaces, based on the maximum adhesion peak that appeared nearest to the surface within the multiple adhesion peaks. It should be noted that the adhesion interface between the protein and the material surface is retained before the applied tensile load reaches the first maximum value, and that the adhesion interface begins to partially break just at the first adhesion peak. Then, the multiple adhesion peaks appear until the tip is completely pulled up away from the surfaces, and the interaction between the protein and the material surface vanishes. Therefore, following the present definition, the forces that induce the initial failure of the adhesion interface can be evaluated from the retracting traces of f–d
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curves. Of course, such an initial failure itself of the adhesion interface is only part of the complete process of separation between the protein and the material surface. However, the adhesion force defined here also means, in a pure sense, the maximum resistant force required to keep the adhesion interface intact, and is considered to reflect the total short-ranged interaction inside the adhesion interface without the unfolding of the protein. In this sense, the adhesion force defined in the present manner can be applied for a quick evaluation of the adhesion strength of the protein to the material surfaces, excluding the force contributions of the effect of protein unfolding.
4. Comparison between the force characterized from f –d curve and natural forces in protein adsorption: mechanical aspects of protein adsorption As mentioned in the Section 1, the process of protein adsorption can generally be decomposed to the following three steps: approach to, attachment on, and detachment from the material surface (Fig. 1). The natural forces in protein adsorption are composed of the influential driving forces in each process, which are summarized as the eight representative factors in Fig. 1: (1) transport property of the protein, (2) intrinsic protein/material interaction force mediated by solvent, (3) short-range interaction forces in the adhesion interface, (4) entropy gain due to the release of hydrated water and counterions, (5) entropy gain due to the surface denaturing of the protein, (6) thermal disturbance by solvent, (7) solvent shear flow, (8) substitution by other adsorbates. Of these eight representative factors, both 1 and 2 determine how easily the protein molecule can approach the surface. Factors 3, 4 and 5 determine the thermodynamic stability of the protein/material adhesion interface. Thus, the adsorption behavior of protein is essentially affected by all these five factors 1– 5. On the other hand, the degree of protein desorption under thermal equilibrium is determined by the thermo-
dynamic stability of the adhesion interface that is newly modified by the contributions from the driving forces of protein detachment (factors 6, 7 and 8), i.e. the desorption behavior of protein is simultaneously affected by all six factors 3–8, in principle. The equilibrium amount of adsorbed protein is thermodynamically determined by the total contributions from all these eight factors together with the interaction among neighboring proteins adsorbed on the surface. Based on the above-mentioned forces, it is quite important to clarify what forces within these factors can be characterized from the f–d curve between the protein-tip and surfaces, in order to discuss the mechanistic aspects of protein adsorption. Here, we consider the relationship between the natural forces in protein adsorption/desorption and the forces observed in the tip-approaching/retracting traces of the f–d curve, respectively. First, in the tip-approaching measurement of f– d curves, the protein on the tip is forcedly moved toward the material surface by the cantilever movement, and does not interact with other proteins in solution. This situation does not correspond to the natural transport behavior in the process of protein adsorption, i.e. there is a lack of diffusion motion. Thus, it should be noted that the thermodynamic force by the diffusion potential cannot be analyzed from the tipapproaching traces. Only the intrinsic forces between the protein and the material surface mediated by solvent can be evaluated from the approaching traces (factor 2 in Fig. 1). Next, from the tip-retracting traces of the f–d curve, the vertical tensile force required to induce the initial failure of the adhesion interface of the protein/material surface is measured, which can be determined from the maximum adhesion peak nearest the surface. Such force also represents the maximum resistant force required to keep the adhesion interface intact, and is considered to reflect the total short-range interactions inside the adhesion interface without the unfolding of protein, as discussed in the previous section. Thus, the adhesion force measured from the f–d curves based on such a definition can be interpreted to reflect the stability of the intact adhe-
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sion interface and to reflect the contributions from driving force factors 3 and 4 in the natural adhesion process of proteins. To evaluate the thermodynamic stability of the adhesion interface, it is useful to calculate the work of adhesion from the measured adhesion forces based on the appropriate adhesion theory (e.g. JKR [49,50] or DMT [51] adhesion theory), because the adhesion forces themselves depend on the conditions of measurement and are determined kinetically or mechanically [39]. Although the information obtained from the f –d curves is limited, as discussed above, the data on the force value are invaluable for a semiquantitative consideration of the mechanistic aspect of protein adsorption. Based on the force characters discussed in the previous section, here we consider the contributions of intrinsic protein/material forces and the adhesion stability of the protein/material interface in the determination of the characters of protein adsorption, such as adsorbed amount, on typical model surfaces with well-defined properties. First, concerning the flat SAM surfaces, the absolute value of intrinsic protein/material forces and its differences among the different kinds of SAM surfaces were found to be too weak to be detected by a common cantilever, irrespective of the kind of protein and the surface properties such as hydrophobicity, hydrophilicity, and charged state. In addition, they were also found to be much smaller than the absolute value of the adhesion force and its differences among the different kinds of SAM surfaces (Fig. 1). From these observations, the contribution of the intrinsic protein/material forces in the determination of the characters of protein adsorption can be considered to be much smaller than the contribution of the adhesion stability of the protein/material interface. In this sense, the adhesion stability is recognized to play an essential role in determining the characters of protein adsorption. Actually, the result on the order of the work of adhesion analyzed from the f –d curves among the hydrophobic surface and the hydrophilic neutral, anionic, and cationic surfaces showed good agreement with the data reported in the past
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literature (e.g. [7,52,53]) (i.e. hydrophobic surfacecationic surface\ neutral surface\ anionic surface, for the negatively charged protein under physiological pH). Next, concerning the polymer-grafted surfaces, it was confirmed that the marked reduction of the degree of protein adsorption generally observed on a neutral-polymer-grafted surface (e.g. [54–59]) is attributable to both the effects of steric repulsion in the adsorption process and the absence of adhesion force in the desorption process (Fig. 4). On the other hand, the charged polymer-grafted surfaces were found to provide not only such a steric repulsion but also significant adhesion force to proteins, irrespective of the sign of charge of the protein (Fig. 5). In addition, it was confirmed from the attractive peak in the approaching trace of f–d curves that protein can approach a graft layer with the opposite charge easier than a graft layer with same charge. These findings well explain the general trend of degree of protein adsorption on the polymer-grafted surface, i.e. the order of degree of adsorbed amount of protein: polymer surfaces with charge opposite that of the protein\ polymer surfaces with the same charge as the protein\ neutral polymer surface (e.g. [60]). Here, it should be noted that the neutral polymer surface can adsorb a slight amount of protein in the natural adsorption process, regardless of the absence of significant attractive forces and the adhesion force between them. Thus, it can be considered that, on such a neutral polymer surface, other driving forces which cannot be determined from the f–d curve play an essential role in protein adsorption, i.e. the diffusion force of the protein from the bulk solution to material surface. Such transport properties of protein always contribute to determine the degree of protein adsorption, together with the forces characterized in the above model system. As known from this typical example, in order to predict the degree of protein adsorption, it is needed to simultaneously consider the character of intrinsic protein/material forces, the adhesion stability of the protein/material interface which are probed by AFM, and the transport property of the proteins.
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5. Conclusions In the present review, we discussed (1) the characteristics of the f – d curves measured between the protein-modified AFM probe tip and several model surfaces with well-defined surface properties, (2) the interpretation of the forces in each tip-approaching and retracting measurement, and (3) the relationship between the forces measured from f–d curves and the driving forces in the natural process of protein adsorption, i.e. mechanistic aspects of protein adsorption probed by AFM. It was concluded that (1) the intrinsic forces between the protein and the material surface mediated by a solvent and the thermodynamic stability of the adhesion interface between them can be analyzed from the tip-approaching and tip-retracting traces of the f– d curves, respectively, (2) the contribution of the intrinsic protein/material interaction force to the determination of the character of protein adsorption is usually much smaller than the contribution of the stability of the adhesion interface, and (3) both the stability of the adhesion interface and the diffusion force of protein from the bulk to the surface predominantly govern the character of protein adsorption.
Acknowledgements The authors thank Professor Kazue Kurihara of Tohoku University for her technical advice on force curve measurement by AFM, and Professor Hiroo Iwata of Kyoto University for his invaluable suggestions on the interpretation of the force curve.
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Review
Mechanistic aspects of protein/material interactions probed by atomic force microscopy Satoru Kidoaki, Takehisa Matsuda * Department of Biomedical Engineering, Graduate School of Medicine, Kyusyu Uni6ersity, Higashi-ku, Fukuoka 812 -8582, Japan Received 10 January 2001; accepted 10 April 2001
Abstract Physicochemical studies on the mechanisms of protein adsorption onto solid material surfaces have been extensively performed so far, mainly based on the analysis of factors such as the equilibrium adsorbed amount (adsorption isotherms), time-dependent change of adsorbed amount (adsorption kinetics), and conformational change of adsorbed protein. However, direct understanding of the strength of the molecular interaction between protein and the material surface has not been established yet. For this issue, the force measurement techniques of an atomic force microscope (AFM) using a protein-modified probe tip are recently becoming powerful tools to analyze the actual interaction forces between protein and material surfaces. In this mini review, we discuss the characteristics and interpretation of the AFM force-versus-distance curves ( f– d curves) obtained with the protein-modified probe tip, and the relationship between the forces measured from the f–d curves and the driving forces in the natural process of protein adsorption. Relative degrees of each of the following contributions which determine the character of protein adsorption are clarified: (1) the intrinsic protein/material forces mediated by solvent, (2) the thermodynamic stability of protein/material adhesion interface, and (3) diffusion force of protein molecules. Within these driving forces, the latter two in particular are confirmed to play essential roles in determining the character of protein adsorption, based on the profiles of f –d curves. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Mechanistic; Protein material interactions; Atomic force microscopy
1. Introduction The investigation of the interaction of proteins with solid material surfaces has been one of the * Corresponding author. Tel.: + 81-92-642-6210; fax: + 8192-642-6212. E-mail address: [email protected] (T. Matsuda).
essential themes in various surface-dependent industrial fields such as medical devices [1,2]. In particular, in the field of cardiovascular device fabrication, the regulation of the adsorption of blood plasma proteins onto the device surfaces, i.e. both facilitation and inhibition of adsorption, has been studied to appropriately design antithrombotic devices. Generally, a thrombus is
0927-7765/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 0 1 ) 0 0 2 3 2 - 6
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formed after a series of biological reactions, such as platelet and leucocyte adhesion, blood coagulation, and fibrinolysis, which are known to be conditioned by the initially adsorbed protein layer on the device surface [1–3]. Therefore, in order to prepare the antithrombotic surfaces, the following opposite approaches have so far been applied on the basis of the conditioning of the initially adsorbed protein layer: (a) Selective facilitation of the formation of the non-cell-adhesive protein layer such as albumin which prevent the platelet adhesion. This approach is based on Lyman’s hypothesis [4]. (b) Inhibition of the protein adsorption itself, which prevents both the activation of blood coagulation factors and platelet adhesion. This idea was first advocated by Andrade et al. [5] and is based on the fact that the inner wall of a blood vessel has a hydrogel layer which is highly effective for the inhibition of protein adsorption, i.e. minimum interfacial free energy hypothesis. To elucidate a predictable principle to regulate the adsorption of protein onto material surfaces, the mechanisms of protein adsorption have been extensively studied not only biochemically but also physicochemically (see reviews [1,2,6– 11]). The physicochemical factors that determine the manner of protein adsorption and desorption typically include the following eight items which are characteristic in each of the processes of protein approach to, attachment on, and detachment from the surface (see Fig. 1). First, in the approach process, both the transport properties of the protein molecules (1) and the intrinsic interaction forces between proteins and surfaces (2) determine the degree of protein approach to the surface; both are affected by solvent motion and its molecular properties [2]. Second, in the attachment process, the following three factors comprise the driving force of the stabilization of adhesion interface: (3) the short-range interaction force between protein and surface, (4) the entropy gain due to the release of hydrated waters and bound counterions between the protein and the surface [7,12,13], (5) the entropy gain due to the surface denaturing of the adsorbed protein [7]. Third, in the detachment process, the following three factors contribute to destabilize the adhesion inter-
face and induce detachment [2]: work provided by the motion of the solvent molecules, such as (6) thermal disturbance and (7) mechanical shear flow, and (8) work required for substitution due to the competitive adsorption of other adsorbates that can adhere more stably. The dependence of the amount of adsorbed protein or of the adsorption rate on these factors has been analyzed mainly through adsorption isotherms and adsorption kinetic data. However, direct understanding of the strength of the molecular interaction between the protein and the surface in the above-mentioned each process has not yet been established. Concerning this issue, recent developments in the technique of molecular force measurement using an atomic force microscope (AFM) are becoming a powerful tool for investigating the protein/surface interaction. The AFM allows the measurement of the force-versus-distance curve ( f–d curve) between the AFM tip and the sample surface, and has been successfully applied to force measurement of a system involving proteins, e.g. the measurement of single molecular forces between specific protein pairs such as avidin/biotin pairs [14–20] or antigen/antibody pairs [21–27], of the unfolding force of single protein molecules [28–36], and of nonspecific adhesion forces be-
Fig. 1. Scheme of the process of protein adsorption and desorption, and the physicochemical driving forces that induce each process.
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2. Protein fixation onto the AFM probe tip and the state of fixed proteins In the AFM f–d curve measurements of a system involving proteins, various methods for protein fixation onto the probe tip have been performed by several groups. In this chapter, we briefly review the method of protein fixation on the tip, and discuss the state of the proteins fixed with such methods. Fig. 2. Schematic representation of the character of the forces analyzed with f– d curve measurement employing the proteinmodified probe tip.
tween proteins and surfaces of material such as polystyrene [37,38] or glass [38]. By using the AFM probe tip, onto which a single protein molecule is fixed or protein monolayer is formed, one can measure the strength and range of molecular interaction between the protein and the surface in each of the above-mentioned processes, as schematically shown in Fig. 2. It should be noted that this methodology enables one to particularly analyze the following factors. (1) In the tip-approaching measurement, the intrinsic interaction forces between proteins and surfaces mediated by the solvent which does not undergo the diffusion motion of the protein can be measured. (2) In the tip-retracting measurement, the adhesion strength of the protein onto the surface by the vertical tensile test can be evaluated. Recently, we have applied such a methodology for the system of protein-fixed AFM tips (blood plasma proteins; albumin, globulin, fibrinogen, and fibronectin) and several kinds of model surfaces (self-assembled monolayer (SAM) surfaces with different kinds of functional groups [39] and polymer-grafted surfaces) [40,41]. In this short review, based on the force character analyzed by AFM techniques, we discuss the mechanistic aspects of protein adsorption onto and desorption from the model surfaces.
2.1. Method Gaub and co-workers functionalized the silicon nitride surface of the tip with biotinylated bovine serum albumin (BSA) through nonspecific adsorption of BSA [16–19]. They confirmed that BSA irreversibly binds to the silicon nitride surface [18]. Dammer et al. covalently fixed the biotinylated BSA [25] and the cell adhesion proteoglycan [24] onto the amino-reactive SAM of dithio-bis(succinimidyl-undecanoate) synthesized by them, which was prepared on the gold-deposited silicon nitride surface of the tip. Allen et al. applied the method of Vinckier et al. [42] in which 3aminopropyldimethylethoxysilane and glutaraldehyde are employed to prepare the surface with amino-reactive aldehyde groups on the tip, and covalently fixed ferritin [23] and human chorionic gonadotrophin [27] onto the tip. Chen et al. coated the tip with BSA by applying the same method as Allen et al. [37]. Oberleithner et al. treated the tip with poly-(L-lysine), a polycation, in order to prepare, on the tip surfaces, matrix which electrostatically attaches proteins [36]. They used this method to fix a renal epithelial potassium channel on the tip. Hinterdorfer et al. and Willemsen et al. synthesized an 8-nm-long polyethylene glycol (PEG) derivative with an amino-reactive end and a thiol-reactive end, and used it as a spacer for covalent linkage of polyclonal anti-human serum albumin antibody [22] and of anti-intercellular adhesion molecule-1 antibody [26], respectively. In their method, the amino-reactive end of the PEG-derivative reacts with the amine groups pre-introduced onto the tip surface by esterification between silanol groups of the tip and ethanolamine, while thiol-reactive
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groups of the PEG-derivative react with the proteins prederivatized with N-succinimidyl-3-(Sacethylthio)propionate to give protected SH groups [43].
2.2. The state of fixed proteins Interaction between the proteins fixed on the AFM probe tip and surfaces is significantly affected by the state of the fixed proteins, i.e. fixed density, amount, orientation, and conformation. To precisely understand the information obtained from the f–d curve using protein-tips, the state of fixed proteins has to be appropriately considered, based on the experimental conditions of protein fixation. First, fixed density and amount of the protein on the tip are determined by density of the fixation reaction sites on the tip and the washing procedures of reacted tip. In particular, the appropriate washing is essentially important for excluding the multilayers of protein, which would otherwise cause the force measurement to be inaccurate. The above-reviewed methods guarantee the preparation of monolayer of protein on the surface of a probe tip. Here, it should be noted that only the fixed proteins at the very top region of the tip usually contribute to the interaction force with the surface. If the density of the fixed proteins in the monolayer is low, a lack of fixed protein at the very top of the tip would occur. Thus, a sufficiently high dense fixation of proteins onto the surface of probe tip is required in the fixation. Since the radius of the tip is typically 10–40 nm, the force interaction area on the tip is usually occupied by only a few proteins even under such a high fixation density. Second, the regulation of the orientation of fixed protein is one of the most difficult tasks. In the conventional fixation reaction employing the condensation reaction between the activated amino-reactive group and amino group on the protein surface, the reaction occurs not only on the amino terminus of the protein but also on the amino groups of amino acid residues such as asparagine, glutamine, lysine, and arginine. Thus, fixed proteins are oriented on the tip in a random
manner. However, this is not necessarily disadvantage in the force measurement between fixed protein and a material surface, because proteins approach the surfaces with random orientation in natural processes of protein adsorption. Third, as for the conformation of the fixed proteins, little is known due to the experimental difficulty, i.e. AFM tips are too small to be analyzed by usual experimental apparatus for surface characterization. However, as the general trend, protein’s conformation is known to be changed due to the attachment process and/or the new local microenvironment. It should be noted that more or less, the protein on the tip is partially denatured. Since in particular, such conformational alteration strongly occurs on hydrophobic surface, pre-modified surface of the tip for protein fixation should be hydrophilic, in order to reduce the degree of denaturing. Based on the above information on the method of protein fixation and the state of fixed proteins, the formation of a covalently bound protein monolayer with sufficiently high density on the hydrophilically pre-modified probe tip is needed to characterize the protein/material interaction force by using AFM. Following this strategy, we have fixed four kinds of blood plasma proteins (albumin, g-globulin, fibrinogen, and fibronectin) onto the amino-reactive SAM formed from 10carboxy-1-dodecanethiol and N-hydroxylsuccinimide [39–41].
3. Characteristics of the f–d curves measured with the protein-modified AFM probe tip and the interpretation of the measured forces The f–d curves measured with AFM are generally composed of both the tip-approaching traces and the tip-retracting traces. In the following, we review and discuss the typical characteristics of each trace of the f–d curves measured with the protein-modified AFM probe tip, mainly taking advantage of our recent results of the measurement on the flat SAM surfaces of alkanethiolates [39] and polymer-grafted surfaces [40].
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3.1. Tip-approaching traces In the tip-approaching trace of the f – d curves, the intrinsic forces between proteins on the tip and material surfaces, which are mediated by solvent molecules, are plotted against the separation distance between the tip and the surface. The forces are usually composed of the van der Waals attraction, electrostatic attraction or repulsion, interactions between the hydration water layers between protein and surfaces, and steric repulsion in the case of polymer-grafted surfaces. Though, in principle, the strength and range of interaction of these forces can be analyzed from the tip-approaching traces, it should be noted that there is a detection limitation of weak forces in the analysis of tip-approaching trace. If the cantilever used is too stiff to be deflected by the forces mentioned above, the forces that would be calculated from the cantilever deflection can not be measured. Actually, for example, flat SAM surfaces do not exhibit any significant force from the protein-tips (albumin, IgG, fibrinogen) in the tip-approaching traces under the condition of a spring constant of 0.12 N m − 1 of a common cantilever (triangle-shaped Si3N4-probe; tip radius after protein fixation: ca. 50 nm), irrespective of
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the kind of highly packed functional groups on the SAM surfaces (CH3, NH2, OH, and COOH) (Fig. 3) [39]. The intrinsic forces between the protein and such flat SAM surfaces in solution were found to be too weak to be detected by such a common cantilever. Since, in our measurement, protein monolayer was formed on the surface of the probe tip and only several proteins were on the top of the tip, such a few proteins are considered to exhibit too weak force for the SAM surfaces to cause cantilever deflection. The significant forces between the protein monolayer and material surface can be detected by using a beadimmobilized tip [44], which is effective in detecting the total force contributions from many proteins on the rather wide surface of the bead. However, it should be noted that the rigorous determination of the intrinsic forces which a single protein molecule feels from flat material surfaces in solution media is quite difficult. On the other hand, for end-grafted polymer surfaces, steric repulsion due to the excluded volume effect of the grafted polymer chains was dominantly detected, irrespective of the charged state of the grafted polymers (Figs. 4 and 5). Interestingly, unlike the case of the flat SAM surface, a significant attractive peak was detected
Fig. 3. Representative f– d curves measured between the protein-modified tips ((a) albumin, (b) g-globulin, and (c) fibrinogen tips) and SAM surfaces terminated with CH3-, NH2-, OH-, and COOH-groups. The measurement was performed in phosphate-buffered saline (pH 7.4). Dashed curves: approaching trace. Solid curves: retracting trace. Z-positions mark those of the cantilever. The zero positions of the cantilever were chosen so as to indicate that the cantilever exhibits no deflection (i.e. zero applied force) at the position on the contact line. Reprinted with permission from [39]. Copyright 1999 American Chemical Society.
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peak is considered to be attributable to the accumulated force contributions of van der Waals attraction and electrostatic attraction between the many molecules of protein immobilized on the side of the probe tip and the polymers inside the grafted layer.
Fig. 4. Representative f– d curves measured between the albumin-modified tip and the photograft-polymerized surfaces of dimethylacrylamide (DMAAm) after photoirradiation for 10, 20 s, 1, 5, 10, and 20 min, which produced a set of graft-polymerized surfaces with different lengths of the grafted polymer chain. The measurements were performed in phosphatebuffered saline. Dashed curves: approaching trace. Solid curves: retracting trace. Z-positions mark those of the tip. The zero position of the tip is expediently defined as the starting position of the linear part in the tip-surface contact region. Reprinted with permission from [40]. Copyright 2000 American Chemical Society.
by a cantilever with the same spring constant (0.12 N m − 1) between oppositely charged protein (lysozyme, pKa :11, cationic under pH 7.4) and the grafted polymer layer (poly(AAc), pKa :5, anionic under pH 7.4) (Fig. 5b). Such an attractive peak was observed together with the steric repulsion profile and in the region of a Z-position of the tip where the grafted polymer layer was somewhat compressed by the tip. The attractive
Fig. 5. Representative f– d curves measured (a) between the albumin-modified tip (pKa :4.7, negatively charged protein tip) and the photograft-polymerized surface of acrylic acid (AAc) (pKa :5, negatively charged surface) after 60 min of photoirradiation (a — 1, 2, 3), and (b) between the lysozymemodified tip (pKa :11, positively charged protein tip) and the poly(acrylic acid) surface (negatively charged surface) (b — 1, 2, 3). The measurement was performed in phosphate-buffered saline (pH7.4). Dashed curves: approaching trace. Solid curves: retracting trace. Z-positions are defined similar to those in Fig. 4. Reprinted with permission from [40]. Copyright 2000 American Chemical Society.
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3.2. Tip-retracting traces In the tip-retracting traces of f – d curves, the forces in the detachment process between the tip and the surfaces are plotted against the upward moving distance of the cantilever or the distance of tip-sample separation. In this measurement, the tip is forcedly pushed against the material surface and an adhesion interface between the tip and the material surface is formed prior to the retraction of the tip. Thus, rigorously speaking, the force measured in the retracting traces is detachment force (i.e. adhesion force) in the vertical tensile test. As methods of evaluating the adhesion strength, there are some kinds of test such as the vertical tensile, tensile shear, compression shear, and peeling, because the detachment force generally varies depending on the conditions of measurement, e.g. direction of applied force, peeling speed, and adhesion area. In this sense, it should be noted that the adhesion force evaluated from the retracting trace does not necessarily reflect the general adhesion strength, since vertical tensile forces cannot be imposed on the adhered protein in a real desorption process. In the tip-retracting trace of the f – d curve measured with the protein-modified tip, characteristic multiple adhesive jumps are typically observed, as shown in Fig. 3, differing from the measurement using the tip without protein. The multiple adhesion profiles have been reported so far in the literature that dealt with the protein/ protein interaction, i.e. interaction between the protein immobilized on the tip and the protein coated on the material surface, such as in the system of the ligand/receptor combination. In our result, such profiles were observed particularly on hydrophobic flat surfaces (e.g. CH3-SAM) which exhibit the strongest adhesion strength compared with other kinds of hydrophilic surfaces. For example, for the flat hydrophilic SAM surfaces (NH2-, OH-, and COOH-SAM) and the hydrophilic neutral-polymer-grafted surface (poly(DMAAm)-surface), such a multiple adhesion profile was little observed (Figs. 3 and 4). For the charged polymer-grafted surface (poly(AAc)-surface), multiple adhesion peaks are observed, as shown in Fig. 5, but the profile is typically at-
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tributed to the elongation of the grafted polymer chains [45– 48]. Concerning the interpretation of the multiple adhesion peaks, the following effects have been considered in the literature: (1) nonlinear convolution of multiple unbinding processes of antigen/ antibody pairs [16]; (2) breaking of different contact points between the antigen/antibody pairs [25]; (3) lateral movement of the sensor tip during the process of approach or withdrawal [25]; (4) ‘spring’ effect of partially unfolded protein which is loaded during the process of withdrawal of the tip and bridges the tip and sample [25]. As has been discussed in the literature, the retracting traces of f–d curves contain information not only on the specific binding forces and nonspecific interaction forces between proteins, but also on the unfolding behaviors of protein molecules. In particular, the latter information has been effectively applied to estimate the conformations and mechanical properties of a single protein molecule [28–36]. However, such complex information including both molecular interaction forces and unfolding forces of proteins makes it difficult to evaluate the adhesion forces between proteins and surfaces. To rationally evaluate the adhesion strength of the protein from the retracting traces of the f–d curves, it is required to clearly define how the adhesion strength is measured from the f– d curve and to precisely understand the process of adhesion force measurement. To resolve this issue, we defined and evaluated the adhesion strength of the protein to the material surfaces, based on the maximum adhesion peak that appeared nearest to the surface within the multiple adhesion peaks. It should be noted that the adhesion interface between the protein and the material surface is retained before the applied tensile load reaches the first maximum value, and that the adhesion interface begins to partially break just at the first adhesion peak. Then, the multiple adhesion peaks appear until the tip is completely pulled up away from the surfaces, and the interaction between the protein and the material surface vanishes. Therefore, following the present definition, the forces that induce the initial failure of the adhesion interface can be evaluated from the retracting traces of f–d
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curves. Of course, such an initial failure itself of the adhesion interface is only part of the complete process of separation between the protein and the material surface. However, the adhesion force defined here also means, in a pure sense, the maximum resistant force required to keep the adhesion interface intact, and is considered to reflect the total short-ranged interaction inside the adhesion interface without the unfolding of the protein. In this sense, the adhesion force defined in the present manner can be applied for a quick evaluation of the adhesion strength of the protein to the material surfaces, excluding the force contributions of the effect of protein unfolding.
4. Comparison between the force characterized from f –d curve and natural forces in protein adsorption: mechanical aspects of protein adsorption As mentioned in the Section 1, the process of protein adsorption can generally be decomposed to the following three steps: approach to, attachment on, and detachment from the material surface (Fig. 1). The natural forces in protein adsorption are composed of the influential driving forces in each process, which are summarized as the eight representative factors in Fig. 1: (1) transport property of the protein, (2) intrinsic protein/material interaction force mediated by solvent, (3) short-range interaction forces in the adhesion interface, (4) entropy gain due to the release of hydrated water and counterions, (5) entropy gain due to the surface denaturing of the protein, (6) thermal disturbance by solvent, (7) solvent shear flow, (8) substitution by other adsorbates. Of these eight representative factors, both 1 and 2 determine how easily the protein molecule can approach the surface. Factors 3, 4 and 5 determine the thermodynamic stability of the protein/material adhesion interface. Thus, the adsorption behavior of protein is essentially affected by all these five factors 1– 5. On the other hand, the degree of protein desorption under thermal equilibrium is determined by the thermo-
dynamic stability of the adhesion interface that is newly modified by the contributions from the driving forces of protein detachment (factors 6, 7 and 8), i.e. the desorption behavior of protein is simultaneously affected by all six factors 3–8, in principle. The equilibrium amount of adsorbed protein is thermodynamically determined by the total contributions from all these eight factors together with the interaction among neighboring proteins adsorbed on the surface. Based on the above-mentioned forces, it is quite important to clarify what forces within these factors can be characterized from the f–d curve between the protein-tip and surfaces, in order to discuss the mechanistic aspects of protein adsorption. Here, we consider the relationship between the natural forces in protein adsorption/desorption and the forces observed in the tip-approaching/retracting traces of the f–d curve, respectively. First, in the tip-approaching measurement of f– d curves, the protein on the tip is forcedly moved toward the material surface by the cantilever movement, and does not interact with other proteins in solution. This situation does not correspond to the natural transport behavior in the process of protein adsorption, i.e. there is a lack of diffusion motion. Thus, it should be noted that the thermodynamic force by the diffusion potential cannot be analyzed from the tipapproaching traces. Only the intrinsic forces between the protein and the material surface mediated by solvent can be evaluated from the approaching traces (factor 2 in Fig. 1). Next, from the tip-retracting traces of the f–d curve, the vertical tensile force required to induce the initial failure of the adhesion interface of the protein/material surface is measured, which can be determined from the maximum adhesion peak nearest the surface. Such force also represents the maximum resistant force required to keep the adhesion interface intact, and is considered to reflect the total short-range interactions inside the adhesion interface without the unfolding of protein, as discussed in the previous section. Thus, the adhesion force measured from the f–d curves based on such a definition can be interpreted to reflect the stability of the intact adhe-
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sion interface and to reflect the contributions from driving force factors 3 and 4 in the natural adhesion process of proteins. To evaluate the thermodynamic stability of the adhesion interface, it is useful to calculate the work of adhesion from the measured adhesion forces based on the appropriate adhesion theory (e.g. JKR [49,50] or DMT [51] adhesion theory), because the adhesion forces themselves depend on the conditions of measurement and are determined kinetically or mechanically [39]. Although the information obtained from the f –d curves is limited, as discussed above, the data on the force value are invaluable for a semiquantitative consideration of the mechanistic aspect of protein adsorption. Based on the force characters discussed in the previous section, here we consider the contributions of intrinsic protein/material forces and the adhesion stability of the protein/material interface in the determination of the characters of protein adsorption, such as adsorbed amount, on typical model surfaces with well-defined properties. First, concerning the flat SAM surfaces, the absolute value of intrinsic protein/material forces and its differences among the different kinds of SAM surfaces were found to be too weak to be detected by a common cantilever, irrespective of the kind of protein and the surface properties such as hydrophobicity, hydrophilicity, and charged state. In addition, they were also found to be much smaller than the absolute value of the adhesion force and its differences among the different kinds of SAM surfaces (Fig. 1). From these observations, the contribution of the intrinsic protein/material forces in the determination of the characters of protein adsorption can be considered to be much smaller than the contribution of the adhesion stability of the protein/material interface. In this sense, the adhesion stability is recognized to play an essential role in determining the characters of protein adsorption. Actually, the result on the order of the work of adhesion analyzed from the f –d curves among the hydrophobic surface and the hydrophilic neutral, anionic, and cationic surfaces showed good agreement with the data reported in the past
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literature (e.g. [7,52,53]) (i.e. hydrophobic surfacecationic surface\ neutral surface\ anionic surface, for the negatively charged protein under physiological pH). Next, concerning the polymer-grafted surfaces, it was confirmed that the marked reduction of the degree of protein adsorption generally observed on a neutral-polymer-grafted surface (e.g. [54–59]) is attributable to both the effects of steric repulsion in the adsorption process and the absence of adhesion force in the desorption process (Fig. 4). On the other hand, the charged polymer-grafted surfaces were found to provide not only such a steric repulsion but also significant adhesion force to proteins, irrespective of the sign of charge of the protein (Fig. 5). In addition, it was confirmed from the attractive peak in the approaching trace of f–d curves that protein can approach a graft layer with the opposite charge easier than a graft layer with same charge. These findings well explain the general trend of degree of protein adsorption on the polymer-grafted surface, i.e. the order of degree of adsorbed amount of protein: polymer surfaces with charge opposite that of the protein\ polymer surfaces with the same charge as the protein\ neutral polymer surface (e.g. [60]). Here, it should be noted that the neutral polymer surface can adsorb a slight amount of protein in the natural adsorption process, regardless of the absence of significant attractive forces and the adhesion force between them. Thus, it can be considered that, on such a neutral polymer surface, other driving forces which cannot be determined from the f–d curve play an essential role in protein adsorption, i.e. the diffusion force of the protein from the bulk solution to material surface. Such transport properties of protein always contribute to determine the degree of protein adsorption, together with the forces characterized in the above model system. As known from this typical example, in order to predict the degree of protein adsorption, it is needed to simultaneously consider the character of intrinsic protein/material forces, the adhesion stability of the protein/material interface which are probed by AFM, and the transport property of the proteins.
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5. Conclusions In the present review, we discussed (1) the characteristics of the f – d curves measured between the protein-modified AFM probe tip and several model surfaces with well-defined surface properties, (2) the interpretation of the forces in each tip-approaching and retracting measurement, and (3) the relationship between the forces measured from f–d curves and the driving forces in the natural process of protein adsorption, i.e. mechanistic aspects of protein adsorption probed by AFM. It was concluded that (1) the intrinsic forces between the protein and the material surface mediated by a solvent and the thermodynamic stability of the adhesion interface between them can be analyzed from the tip-approaching and tip-retracting traces of the f– d curves, respectively, (2) the contribution of the intrinsic protein/material interaction force to the determination of the character of protein adsorption is usually much smaller than the contribution of the stability of the adhesion interface, and (3) both the stability of the adhesion interface and the diffusion force of protein from the bulk to the surface predominantly govern the character of protein adsorption.
Acknowledgements The authors thank Professor Kazue Kurihara of Tohoku University for her technical advice on force curve measurement by AFM, and Professor Hiroo Iwata of Kyoto University for his invaluable suggestions on the interpretation of the force curve.
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