Proteins at Interfaces II - American Chemical Society

Chapter 16

Ellipsometry Studies of Protein Adsorption at Hydrophobic Surfaces Martin Malmsten and Bo Lassen

Downloaded by UNIV OF AUCKLAND on May 3, 2015 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch016

Institute for Surface Chemistry, P.O. Box 5607, S-114 86 Stockholm, Sweden

The adsorption of human serum albumin (HSA), IgG and fibrinogen, at (hydrophobic) methylated silica surfaces was investigated with in situ ellipsometry. By performing studies with the bare substrate at two different ambient refractive indices, and by performing 4-zone averaging in all measurement, the adsorbed amount (Γ), the adsorbed layer thickness (δ ) and the mean adsorbed layer refractive index (n ) are obtained accurately. Furthermore, the build-up of the adsorbed layers could be followed in detail. Thus, for fibrinogen both δ and n initially increase monotonically with the adsorbed amount, whereas at higher adsorbed amounts, a "swelling" of the adsorbed layer is observed. For IgG, on the other hand, δ is essentially independent of the adsorbed amount. Furthermore, studies of the adsorption from HSA/IgG mixtures were performed in a similar way. el

f

el

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el

The adsorption of proteins at solid surfaces is receiving increasing attention (7,2). This is partly motivated by fundamental issues, but also by the importance of protein adsorption in many practical and industrial applications. For example, the adsorption of proteins at biomedical surfaces, such as implants, cathethers and insulin pumps, is the first step in a complex series of biophysical/biochemical processes, which determine the biological response to the foreign material. Furthermore, there are a large number of biotechnical applications, e. g. solid-state diagnostics, which are sensitive to the state of adsorption of proteins. The immobilization of proteins at surfaces is also of large potential use in, e. g. extra-corporeal therapy and advanced bioorganic synthesis. However, despite much previous work done on protein adsorption from complex as well as simpler protein systems, much remains to be done, e. g. concerning the structure and formation of the adsorbed layer, as well as interfacial exchange processes at model surfaces. We therefore undertook investigations concerning these issues, using ellipsometry. Here we report on some of the results obtained for some serum proteins at model hydrophobic surfaces.

0097-6156/95/0602-0228$12.00/0 © 1995 American Chemical Society In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

16. MALMSTEN & LASSEN

Ellipsometry Studies of Protein Adsorption 229

Experimental Materials Human serum albumin (HSA), globulin-free, lyophilized and crystallized, was obtained from Sigma Chemical Co., U S A , as was reagent grade purified immunoglobulin (IgG) and fibrinogen (Fraction I; 92% clottable protein). A l l proteins were used without further purification. Chemicals used for the buffer preparation were all of analytical grade, and used without further purification.


Surfaces Hydrophobized silica surfaces were prepared from polished silicon slides (Okmetic, Finland). In short, these were oxidized thermally to an oxide layer thickness of about 30 nm. The slides were then cleaned and treated with Cl2(CH3)2Si (Merck) as described previously (3). This procedure rendered the slides hydrophobic, with an advancing and receeding contact angle of 95° and 88°, respectively. Methods The ellipsometry measurements were all performed by means of null ellipsometry. The instrument used was an automated Rudolph thin-film ellipsometer, type 436, controlled by a personal computer. A xenon lamp, filtered to 4015 A, was used as the light source. A thorough description of the experimental setup is given in ref.4. Prior to adsorption, the ellipsometry measurements require a determination of the complex refractive index of the substrate (5). In the case of a layered substrate, e. g. oxidized silicon, a correct determination of the adsorbed layer thickness and mean refractive index requires an accurate determination of the silicon bulk complex refractive index (N2=n2-ik2) as well as of the thickness (di) and the refractive index (ni) of the oxide layer. This is done by measuring the ellipsometric parameters *F and A in two different media, e. g. air and buffer. From the two sets of *F and A, n2, k2, di and n i can be determined separately. [The hydrophobic methyl layer is neglected, since calculations with the Bruggeman effective medium theory (5) show that the error in doing so is much less than 10% in 8 i , and even smaller in T.] A l l measurements were performed by four-zone null ellipsometry in order to reduce effects of optical component imperfections (5). [A thorough description of the theory of four-zone null ellipsometry experiments, as well as of adsorption studies at layered substrate surfaces, is given in refs. 4-6.] In fact, the procedure used has previously been shown to be even more accurate than the multiple angle of incidence approach (4). Furthermore, both the adsorbed amounts and the adsorbed layer thicknesses obtained with the present methodology agree well with results obtained with other techniques both for surfactant, polymer and protein systems (7). After the optical analysis of the bare substrate surface, the protein solution was added to the cuvette, and the values of *F and A recorded. The adsorption was only monitored in one zone, $ince the four-zone procedure is rather time-consuming and since corrections for component imperfections already had been performed. Four-zone measurements at adsorption equilibrium show that the error induced by the procedure used is less than a few percent. The maximal time-resolution between two measurements is 3-4 seconds. Stirring was performed by a magnetic stirrer at about 300 rpm. e

From *F and A, the mean refractive index (nf) and average thickness (8 i) of the adsorbed layer were calculated numerically (cf Appendix) according to an optical four layer model for the proteins at hydrophobized silica (4-6). The refractive index and the average thickness were finally used to calculate the adsorbed amount (T) according to de Feijter (8), with dn/dc=0.188 (9), 0.188 (9), and 0.187 (9,10) cm /g for fibrinogen, IgG, and H S A , respectively. [Due to the similarity between the refractive index increments of HSA, IgG and fibrinogen, ellipsometry measurements on the binary systems will provide the total adsorbed amount.] A l l measurements were performed in 0.01 M phosphate buffer (0.15 M NaCl, pH 7.2) at 20°C. e

3

NOTE: Please see Appendix on page 237.

In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Results and Discussion The adsorption from HSA, IgG and fibrinogen single protein solutions at methylated silica surfaces is shown in Figure la. The concentrations used all correspond to (pseudo-) plateau in the respective adsorption isotherm. Although the equilibrium concentration for fibrinogen is only about 100 ppm, saturation adsorption is reached after only 1 hour. A similar finding is obtained for HSA at a 200 ppm concentration. The adsorption of IgG, on the other hand, is slower in terms of the adsorbed amunt, and 90% of saturation adsorption is reached after about 3000 s, in comparison to about 1000 s for fibrinogen. Note, however, that for all proteins, a slight but significant increase in the adsorbed amount is observed even after 4000 s adsorption, indicating slow structural rearrangements. A parameter of large interest for protein adsorption is the adsorbed layer thickness. As can be seen from Figure lb, the mean (optical) thickness at adsorption plateau for HSA, IgG and fibrinogen at methylated silica is 4±2 nm, 18±2 nm and 28±2 nm, respectively (7). It is interesting to compare these thicknesses with the molecular dimensions of the proteins, which for HSA, IgG and fibrinogen are approximately 4x4x14 nm, 23.5x4.5x4.5 nm and 6.0x6.0x45.0 nm, respectively. Hence, H S A adsorbs essentially side-on at hydrophobic surfaces at the present conditions, while IgG adsorbs in an essentially end-on configuration. Fibrinogen, finally, seems to adsorb in a random configuration at hydrophobic surfaces. Apart from the adsorbed amount and the mean adsorbed layer thickness, ellipsometry provides information on the adsorbed layer mean refractive index, and hence on the average protein concentration in the adsorbed layer. For all proteins investigated here, low adsorbed layer refractive indices were obtained (7), and the average adsorbed layer protein concentrations were found to be 0.11-0.17 g/cm . The adsorbed layer structure has previously been investigated for all the proteins studied here. Hence, Lee et al. (77), Norman et al. (72) and Uzgiris and Fromageot (13) obtained a hydrodynamic thickness of HSA at polystyrene (pH 7.2-7.4) of about 7, 6, and 4 nm, respectively. Furthermore, in the latter study the mean adsorbed layer refractive index was determined, and found to be comparable to that found for HSA in the present investigation. Furthermore, end-on adsorption of IgG has previously been observed (although at higher surface concentrations than in the present investigation), e. g. by Morrisey and Han (14), using polystyrene colloidal particles as substrates. Furthermore, Elwing et al. found the thickness of an adsorbed layer of IgG at methylated silica to be 17±5 nm, whereas nf was found to be 1.39±0.02 (75). Thus, the agreement between the present and these previous results (not using the two ambient refractive indices procedure with concomitant 4-zone averaging throughout) is surprisingly good. For fibrinogen, finally, there have been several previous scanning angle reflectometry studies, although using silica as substrate (16). Although the adsoiption of fibrinogen is strongly dependent on the substrate hydrophobicity, it is still interesting to note that an optical adsorbed layer thickness of 10-30 nm was obtained under otherwise similar conditions. Furthermore, it is interesting to note that the refractive index difference between the adsorbed layer and the bulk solution (An = nf - nt>) is comparable (An = 0.02-0.03) to that obtained in the present investigation. 3

One advantage of ellipsometry as a tool for studying adsorbed protein films is the good time resolution of the method, which allows the thickness and the adsorbed layer refractive index to be studied essentially continuously as the adsorbed layer forms. As can be seen from Figure 2a, for fibrinogen the layer thickness increases linearly with the adsorbed amount up to about 4 mg/m . After this, there is a more pronounced growth of the adsorbed layer normal to the surface, resulting also in a slight decrease in the mean adsorbed layer refractive index (Figure 2b). Thus, as the 2



EUipsometry Studies ofProtein Adsorption 231


(a)

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amount (a) and adsorbed layer thickness (b) of H S A (200 ppm; open triangles), IgG (100 ppm; open diamonds) and fibrinogen (100 ppm; open circles) at methylated silica from 0.15 M NaCl, pH 7 2


232



T (mg-rrf ) Figure 2. Adsorbed layer thickness (a) and mean adsorbed layer refractive index (b) for fibrinogen (100 ppm) versus the adsorbed amount at methylated silica from 0.15 M NaCl, pH 7.2. Two measurements are shown in order to illustrate the degree of reproducibility.





adsorbed layer builds up, this is initially achieved both by an increased average protein concentration in the adsorbed layer, and by a growth of the adsorbed layer normal to the interface. At higher surface coverages, a "swelling" of the adsorbed layer normal to the surface, most likely achieved by reorientation of the fibrinogen molecules, is the main mode for achieving an increased adsorption (7). This type of adsorbed layer formation is generally observed for high molecular weight polymers and is due to the high adsorbed layer osmotic pressure at high surface coverages. It is interesting to note that a similar, although more pronounced, build-up of adsorbed fibrinogen layers has been observed previously with scanning angle reflectometry for silica. Thus, Schaaf et al. (76) found that as the adsorbed amount increases, either by increasing the bulk fibrinogen concentration or at increasing adsorption times, the adsorbed layer thickness initially increases only marginally, whereas the adsorbed layer refractive index increases strongly. At higher adsorbed amounts (last 10% of the adsorption) the adsorbed fibrinogen layer grows substantially normal to the surface, while the adsorbed layer refractive index decreases. For IgG, the build-up of the adsorbed layer occurs differently. As can be seen in Figure 3a, the adsorbed layer thickness remains essentially constant with an increasing adsorbed amount above about 0.5 mg/m . At the same time, the adsorbed layer refractive index (average adsorbed layer protein concentration) increases essentially linearly (Figure 3b). Thus, in the case of IgG, an increasing adsorbed amount is achieved solely by packing essentially end-on adsorbed IgG molecules more densely. For HSA, finally, an increasing adsorbed amount is achieved by packing side-on adsorbed HSA molecules more densly at the interface. In Figure 4, the total adsorbed amount at methylated silica from binary HSA/IgG protein mixtures (total concentration 200 ppm) of different compositions is shown. At IgG fractions higher than about 50%, the adsorbed amount is constant, and equal to that of IgG in the absence of HSA, which seems to indicate a complete preference for IgG adsorption at methylated surfaces at the conditions present. This is further supported by the adsorbed layer thickness being constant (within the experimental uncertainty) down to about 50% IgG, as is the adsorbed layer mean refractive index (18). In fact, the build-up of the adsorbed layer proceeds essentially identically down to about 50% IgG in the protein mixture, as can be seen in Figure 5, illustrating the adsorbed layer thickness and mean refractive index as a function of the adsorbed amount. These results indicate adsorption of primarily IgG in a rather dilute layer with the IgG molecules adsorbed preferentially head-on. Only at an IgG fraction of less than 25%, the situation is altered, and the adsorbed layer thickness is reduced towards that of a pure HSA adsorbed layer at hydrophobic surfaces (4 nm) (18). From these considerations we conclude that at high and medium IgG fractions, this protein is strongly preferentially adsorbed at methylated silica surfaces under the conditions used. In a similar way, the adsorption from HSA/fibrinogen mixtures was studied. It was found that also fibrinogen adsorbs preferentially over H S A under these conditions, although the decrease in 8 i on increasing the HSA bulk fraction is more gradual than for IgG. However, the same study showed that if HSA is allowed to preadsorb at hydrophobic surfaces, it effectively reduces the IgG and fibrinogen adsorption (by about 80% and 90%, respectively), which is due to an irreversible HSA adsorption at these surfaces (18). 2

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Acknowledgement This work was financed by the Foundation for Surface Chemistry, Sweden, and the Swedish National Board for Industrial and Technical Development.




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Figure 3. Adsorbed layer thickness (a) and mean adsorbed layer refractive index (b) for IgG (200 ppm) versus the adsorbed amount at methylated silica from 0.15 M NaCl, pH 7.2.



MALMSTEN & LASSEN

5

Ellipsometry Studies of Protein Adsorption

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Figure 4. Total adsorbed amount (a) and adsorbed layer thickness (b) versus time at methylated silica from solutions (0.15 M NaCl, pH 7.2) with a HSA/IgG ratio of 0/100 (open diamonds), 25/75 (open circles) and 50/50 (open triangles). The total protein concentration was 200 ppm.



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P R O T E I N S A T I N T E R F A C E S II

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Figure 5. Adsorbed layer thickness (a) and refractive index (b) versus the total adsorbed amount at methylated silica from HSA/IgG solutions. HSA/IgG ratios of 0/100 (open diamonds), 25/75 (open circles) and 50/50 (open triangles) are shown. The total protein concentration was 200 ppm.




Appendix In the evaluation of the ellipsometric parameters (cf ref 6), the system is modelled as consisting of four layers, i . e. bulk silicon, characterized by a complex refractive index (N2=n2-ik2), an oxide layer, characterized by a refractive index (ni) and a thickness (di), an adsorbed layer with refractive index nf and a thickness df (referred to as 8 i in the paper), and finally an ambient medium of a refractive index no. Throughout, the oxide and the adsorbed layer, as well as the ambient medium, are assumed to be transparent, i . e. k=0. The resulting reflection coefficients parallel (p) and perpendicular (s) to the plane of incidence for such a system is given by: e


(l+iforfre-BPO + (rfi+igfe-^Po^e-^Pi x = p or s where p! = 27c(^ -)n cos(t>i X

(2)

p = 27c(^)nfCOS(t) X

(3)

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The (complex) reflection coefficients ( r ) at the interface between layers m and n for the p and s components are given by: mn

N cos(|> - N cosn n

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N cos

Proteins at Interfaces II - American Chemical Society

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