Proteins at Interfaces - ACS Publications - American Chemical Society

Chapter 29

Protein Adsorption on Solid Surfaces: Physical Studies and Biological Model Reactions 1

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Hans Elwing , Agneta Askenda , Bengt Ivarsson , Ulf Nilsson , Stefan Welin , and Ingemar Lundström 1

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 30, 2017 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch029

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Laboratory of Applied Physics, Linköping University, S-581 83 Linköping, Sweden The Blood Centre, University Hospital, S-751 85 Uppsala, Sweden 2

The paper describes some of our studies of protein adsorption on solid surfaces. An emphasis is made on newly developed experimental techniques and on recent biological model experiments. We therefore discuss the use of a wettability gradient along a solid surface to investigate, in a convenient way, the influence of surface energy on the adsorption of protein molecules. The behavior of the complement system at solid surfaces is also discussed, with special attention to surface induced conformational changes of human complement factor 3. The protein adsorption studies described were all at the liquid - solid or solid - air interface. Lateral scanning ellipsometry was made to evaluate the surfaces with a wettability gradient. Experiments were most often made on (modified) silicon surfaces. The experimental results are also discussed in relation to the proposed theoretical models for protein adsorption. During a number of years we have applied surface orientated analytical methods to the study of protein adsorption on solid surfaces. These investigations include in situ studies with ellipsometry, surface potential and capacitance measurements ( 1.2) We have applied spectroscopic techniques like infra-red £ef lection absorption spectroscopy (IRAS, 3-5) and E5CA (5-7) to investigate details in the interaction between organic molecules and surfaces. Spectroscopic techniques have also been used to ^Current address: Pharmacia, S-751 82 Uppsala, Sweden 0097-6156/87/0343-0468$06.25/0 © 1987 American Chemical Society

Brash and Horbett; Proteins at Interfaces ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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Protein Adsorption on Solid Surfaces

study metal surfaces which had been implanted in humans for different length of time (8.9). The input from the physical measurements has been used to develop some simple dynamic models for protein adsorption (10-12). Furthermore we have used mainly ellipsometry to study biological model reactions related to antigen-antibody reactions on solid surfaces (13) and the behavior of the immune complement system at solid surfaces (14-16). Several new analytical methods have been developed during the course of this work, notably the use of a gradient in the surface energy along a solid substrate to study the influence of surface energy on protein adsorption. (Γ7). The influence of surface induced conformational changes on subsequent biological processes are under study. It should also be mentioned that our interest for protein adsorption on solid surfaces has led to the development of simple methods and instrumentation for medical diagnostic purposes (18-20). The main purpose of our present studies is to investi gate the details of protein-surface interactions with relevance to questions regarding biocompatibility, fouling and the possible development of (implantable) biosensors. The solid surfaces used are evaporated metal films or polished silicon wafers, which are well suited for the optical studies. Several types of proteins have been used like human fibrinogen, immunoglobulins, complement factor 3 and others, including lysozyme, egg albumin and beta lactoglobulin. Detailed studies of the interaction between amino acids, like glycine, histidine and phenylalanine, and metal surfaces have been made with IRAS and ESCA. In some experiments deposition of organic material from whole blood, serum or plasma has been studied. In a short review it is not possible to cover all of the present and past research activities and to give a detailed account of the experimental methods used. Some of our work on protein adsorption on metal surfaces was recently summarized in ref 21. where also a number of methods used to study protein adsorption on metal (oxide) surfaces were described. We have therefore chosen to describe two of our more more recent developments, namely the so called "wettability gradient method" for the study of protein adsorption, desorption and exchange on solid surfaces, and the study of surface induced activation of the immune complement system, mainly the conformational change of complement factor 3 (C3) upon adsorption. Our experimental


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results are discussed in relation to the assumed models for protein adsorption on solid surfaces. The virtues and shortcomings of a simple dynamic model for protein adsorption are described in this context.


The wettability gradient method. General It has long been known that solid surface wettability or energy plays a critical role in protein adsorption on solid surfaces. Most methods for the investigation of adsorption and desorption of proteins at solid surfaces involve the use of solid surfaces with a constant given chemical composition. The action of a specific surface constituent is usually investigated with the use of several preparations of the surface. This procedure is time consuming, uncertain and expensive. We have used another approach in which the specific surface constituent is attached to a flat solid surface in a gradient. The quantification of protein adsorption on the gradient surface is made with the use of lateral scanning ellipsometry. Ellipsometry is described in (21) and (22). "The gradient method" has been used for the investigation of the dependence of solid surface wettability on protein adsorption. The wettability gradient in these experiments is made by diffusion of methylsilane on silicon surfaces with a spontaneously grown layer of silicon dioxide. The surfaces so formed are hydrophilic at one end and hydrophobic at the other and in between there is a gradient in surface energy, 10-15 mm long. The distribution of the wettability along the gradient could be determined with the use of a capillary rise method on similarly treated glass plates or indirectly by means of ellipsometric determinations of adsorbed fibrinogen (in preparation). Both this methods give an estimate of the advancing contact angle with water. Experiments on protein adsorption and desorption reactions are performed by incubating the gradient plates in protein solution, followed by incubation in various detergents or other test solutions. All plates are finally rinsed in distilled water and dried with N . The amount of adsorbed protein 2

along the gradient is determined by means of an ellipsometer (Rudolph Research,Auto Ell 2), equipped with a device for lateral scanning along the silicon surface. The adsorbed amount, r, was estimated from the mearured thickness of the organic layer using


29. ELWING ET AL.


a density of 1.37 g/cm and a refractive index of 1.6 of the dried protein layer (17).


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Protein adsorption and desorption on the gradient surfaces. Typical results of the dependence of the adsorbed amounts of protein on the wettability of the silicon surface with water are shown in Figure 1. Of the protein used, fibrinogen showed the largest quantitative difference between the hydrophobic and hydrophilic parts of the surface. Lysozyme and y-globulin showed smaller differences. The reproducibility with regard to the appearance of the adsorption profile was usually very good between gradient plates from the same diffusion batch. There were .however, differences between plates from different diffusion batches as illustrated in Figure 1. This difference Is most probably due to differences in the wettability distribution in plates from different batches. The gradient method has been applied to the investigation of protein desorption effects of detergents and other agents (II). An experiment with desorption of y-globulin induced by detergents is illustrated in Figure 2. It was observed that Tween 20, a non-ionic detergent caused a maximum desorption effect at a contact angle of about 40°. At the hydrophilic side of the gradient there was very little desorption induced by Tween and at the hydrophobic side of the gradient the desorption decreased with decreased surface wettability. Desorption of adsorbed y-globulin ,induced by an ionic detergent, SD5 on gradient plates has also been studied . In contrast to Tween 20, SDS had a general high desorption activity on both the hydrophilic and hydrophobic side of the gradient (data not shown). Tween of SDS themselves caused no measurable adsorption on the gradient plates since they were probably removed during the rinsing procedure. Protein exchange reactions on the gradient surfaces.The gradient method has been used for the Investigation of exchange reactions on solid surfaces.(21). In these experiments the gradient plates were first incubated in fibrinogen (1 g/L, dissolved in phosphate buffered saline solution, PBS at pH 7.3) for one hour followed by incubation in y-globulin for 4 hours under gentle stirring. Some of the plates were then incubated in antiserum against y-globulin, diluted 1/25, for 30 min. The plates


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Figure 1: Ellipsometrically registered amount of adsorbed protein on silicon surfaces at the air/solid interface. a:fibrinogen b: y-globulin and clysozyme. The proteins, dissolved in phosphate buffered saline solution (PBS) at pH 7.3 was adsorbed on the surfaces for 30 min at a concentration of lg/1. The amounts was determined on dried surfaces in air. The lateral distance is given as a bar in the drawing. The corresponding contact angle values as estimated with the use of a capillary rise method are given on the X-axis . The lower dashed dotted "O'-lines indicate the ellipsometrically registered silicon dioxide layer including the methyl gradient. The adsorbed amount of protein is given by the deviation from this line in the figure. Each line in the drawing represents the average amount of adsorbed proteins measured on triplicate plates. Three different triplicate experiments were performed for each protein, involving a new preparation of gradients plates at each experiment.



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Figure 2: Ellipsometrically registered Tween 20 induced desorption of human y-globulln adsorbed on gradient plates, a: buffer only, b: 0.005% Tween 20, c: 0.05% and d: 0.5%. For further explanation, see Figure 1 and text.



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were finally rinsed and dried and the amount of organic material along the gradient was determined with ellipsometry. The experiment was also reversed in that the first incubation of the gradient plates was made with y-globulin and the second incubation with fibrinogen and adsorbed fibrinogen was detected by using of antiserum against fibrinogen, diluted 1/25) The results of representative experiments are given in Figures 3 and 4. Adsorption of ^-globulin fibrinogen coated surface occured only at the hydrophilic end of the gradient as indicated by the deposited antibodies. In the reverse experiment it was noted that adsorption of fibrinogen on ^-globulin coated surfaces was extended into the hydrophobic side of the gradient. These results indicate that fibrinogen is more readily immunologically detecable than ^-globulin both on hydrophilic and hydrophobic surfaces. The strong adsorption preference of fibrinogen compared to γ-globulin is a well known phenomenon and has been described in several publications, e.g 34-28 The details of the exchange reactions depend, however,also on concentrations and incubation times and need more investigations before they are completely understood.


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We are aware of the possibility that the adsorbed proteins may partially be removed by serum proteins during incubation with antiserum, especially at the hydrophilic side of the gradient. We do not believe ,however, that this has any major influence on possible qualitative conclusions. The two examples above of the application of the gradient method illustrate its analytical properties for the investigation of mechanisms of macromolecular adsorption and desorption reactions. Some of the described phenomena could probably not have been observed without the use of the gradient method. Ellipsometric analysis of protein interaction on gradient surfaces may in principle also be performed at the liquid /solid interface, a procedure which perhaps further will increase the prescision and accuracy of the determinations. The air/solid measurement method makes however the gradient method very rational since the ellipsometer only is used a short time at the end of each experiments. At present, about 30 gradients including 1800 automatically performed ellipsometrica! determinations, can quite easily be analyzed by one person in one day.


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Figure 3: Exchange reactions of human γ-globulin (HGG) on gradient surfaces precoated with human fibrinogen (HFG). The adsorbed amount of protein, determined ellipsometrically is shown versus the wettability along the gradient. The curves (duplicate experiments) represent a:adsorption with HFG, b: adsorption of HFG followed by incubation of HGG, c: as in b) with an additional incubation in anti-HFG, d) as in b) with an additional incubation in anti- HGG. The adsorbed amount of organic material is given by the deviation from the "0" -line. For further explanations, see Figure 1.


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Figure 4: Exchange reactions of HF6 on gradient surfaces precoated with HGG. The curves (duplicate experiments) represent a: adsorption of HGG, b: adsorbtion of HGG followed by incubation in HFG, c: as in b with an additional incubation in anti-HFG-, ct as in b with an additional incubation in anti- HGG. For further explanation see Figure 3.



29. ELWING ET AL.

All


Surface activation of the immune complement system. Identification of serum complement activation with the use of ellipsometry. Activation of the immune complement system in blood, has been described in connection with haemodialysis and leukapheresis It results in various "down stream symptoms" such as transient leukopenia (29-32). Activation of complement on a surface implies sequential activation of several factors. The activation process shows similarity with the clotting system of blood in that restricted proteolysis of some of the factors is one of the main regulation mechanisms and that activation is of the cascade type. C3 is the quantitatively dominating complement protein and has a central position in the complement activation cascade. Activation of the complement system has been studied in vitro on various model surfaces. Most methods used are based on the analysis of various complement degrading factors. Activation of the complement system means however also that some of the factors, especially C3, are deposited on the solid surface. Therefore, we have used ellipsometry to analyse complement activation on solid surfaces. The solid surfaces were either hydrophilic silicon surfaces with a spontaneously grown layer of silicon dioxide or silicon surfaces made hydrophobic by means of the attachement of methyl groups. Ellipsometric analysis was performed at the liquid-solid interface. Some experimental results are shown in Figure 5. It was noted that incubation of hydrophobic surfaces in serum resulted in a slow deposition of material from the serum on the surface. Further incubation of antibodies against C3 resulted in deposition of C3 antibodies indicating that C3 had been adsorbed from serum. Incubation with serum containing EDTA did not cause any accumulation of organic material from serum. (The complement system is inactivated by EDTA due to chelating of M g and C a ions which are needed for the normal function of the complement system). Instead the adsorption rapidly reached a plateu. In addition, the anti C3 response was insignificant. Precoating the hydrophobic surface with y - g ' before addition of serum resulted furthermore in a pronounced increase of the deposited organic material compared to the experiment without precoating of the surface. The same sets of experiments 2+

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Figure 5: Kinetic measurements of the deposition of organic material from 10% human serum on hydrophobic silicon. "Anti-C3" denotes further incubation with anti-C3, diluted 1/50 at the end of the experiment. The kinetics of anti-C3 deposition is not shown but the additional adsorbed amount, Γ, after 120 min adsorption is given at the end of each curve. The upper curve represents deposition of organic material from serum on surfaces preadsorbed with ^-globulin. The dashed line marked "IgG" indicates the amount of preadsorbed ^-globulin jn the y-globulin ,serum experiment. The middle curve represents deposition from serum only and the lower curve from serum • 10 mM EDTA. The reproducibility in Γ was within ±15%.



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were repeated on hydrophilic surfaces. Both the deposition reaction from serum as well as the subsequent reaction with C3 were smaller on hydrophilic surfaces compared to hydrophobic surfaces. The reproducibility of the results was usually good provided that the same serum preparation was used. There were however differences between serum from different individuals as well as serum from one individual collected at different occasions. The characteristic slow growth of organic material on the surface was, however, always accompanied by subsequent anti-C3 deposition. Presence of EDTA in the serum resulted always in the absence of subsequent anti-C3 deposition. These experiments show that ellipsometry is useful for the investigation of complement activation on solid surfaces. The method constitutes a useful complement to other methods which are based on the analysis of soluble complement factors. As illustrated, it was in addition possible to perform real time measurements of the complement deposition reaction which is an uniqe feature of in situ ellipsometry. Surface induced conformational changes of complement factor 3. It is likely that the interaction of C3 with a surface is one of the recognition mechanisms that leads to surface induced complement activation. It has been demonstrated that C3 undergoes major antigenic alteration if C3 is denatured with SDS (sodium dodecyl sulphate) (33) Similar alteration occurs when C3 is activated and deposited on biological target surfaces like erytrocyte membranes (34). It has been shown that there are three antigenic subtypes of C3 (33) .C3(N) which is present on the native but not on the SDS denatured or biologically activated form of C3, C3(D) which is present only on the SDS denature, biologically activated and surface deposited form, and C3(S) which is present on both native and denatured molecules. One of the most used commercially available, antiserum preparation is ,anti-C3c, and contains a mixture of antibodies against C3(S) and C3(N) subtypes. The use of the described subsets of antibodies constitutes an useful analytical probe for investigating of the changed antigenicity of C3 on solid surfaces. Purified C3 was adsorbed on hydrophilic and hydrophobic silicon surfaces followed by incubation with the various subtypes of antibodies. The amount of deposited organic material was



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measured with ellipsometry on the dried surface. It was observed in these experiments that C3 exposed 5-determinants and possible also N-determinants on both hydrophilic and hydrophobic surfaces, since anti-C3c was adsorbed both on the hydrophilic and hydrophobic surfaces. . D-antigens could be detected only on C3 adsorbed on hydrophobic surfaces. Thus, it is obvious that C3 undergoes conformational changes on the hydrophobic surface similar to SDS denaturation or biological activation (to be published). Experiments including specific binding of the different anti-C3 antibodies were also performed on the gradient surfaces. C3 (10 ug/ml in PBS) was adsorbed on the gradient plates for 30 min and some of the plates were also incubated for 30 min in the different antiserum preparations diluted 1/25. A representative experiment is illustrated in Figure 6. C3 was adsorbed on both the hydrophilic and the hydrophobic side of the gradient but to a slightly lesser extent on the hydrophilic side. Deposition of anti-C3c (anti-C3(N) and anti-C3(S)) , occured on both the hydrophilic and hydrophobic side of the gradient The rabbit anti-C3(D) antibodies used in the experiments were directed either to the α or p polypeptide chain. Ellipsometric visualization of deposited antibodies was here enhanced by subsequent Incubation for 30 min in anti- rabbit immunoglobulin diluted in phosphate buffer. As illustrated in Figure 6, anti-aand ant1-p antibodies reacted apparently tittle with C3 deposited at the hydrophilic side of the surface but there was an increased binding capacity of both anti-aand -p at the hydrophobic side of the gradient. The difference in anti-C3(D) binding between the hydrophobic and the hydrophilic end of the gradient is not only due to the different amounts of C3 adsorbed , since a slightly larger amount of anti-C3c was bound to C3 on the hydrophilic side of the gradient . There are thus more C3(N) determinants on the hydrophilic side of the gradient since the C3(S) determinants per adsorbed molecule should be the same on both sides of the gradient. The C3(N) determinants are gradually lost against the hydrophobic side of the gradient. The presence of C3(D) deter minants on C3 adsorbed on the hydrophobic silicon surface does not prove that C3 is biologically active on this surface e.g. bind its inhibitors. This possibility is presently under investigation.


ELWING ET AL.



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Proteins at Interfaces - ACS Publications - American Chemical Society

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