Protein Interactions with Polyelectrolyte Multilayers - American

Biomacromolecules 2000, 1, 674-687

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Protein Interactions with Polyelectrolyte Multilayers: Interactions between Human Serum Albumin and Polystyrene Sulfonate/Polyallylamine Multilayers Guy Ladam,†,‡ Csilla Gergely,‡ Bernard Senger,‡ Gero Decher,† Jean-Claude Voegel,‡ Pierre Schaaf,*,† and Fre´ de´ ric J. G. Cuisinier‡ Institut Charles Sadron (CNRS-ULP), 6, rue Boussingault, 67083 Strasbourg Cedex, France; and INSERM Unite´ 424, Fe´ de´ ration de Recherche “Odontologie”, Universite´ Louis Pasteur, 11, rue Humann, 67085 Strasbourg Cedex, France Received June 2, 2000; Revised Manuscript Received August 28, 2000

The interactions between polystyrenesulfonate (PSS)/polyallylamine (PAH) multilayers with human serum albumin (HSA) were investigated by means of scanning angle reflectometry (SAR). We find that albumin adsorbs both on multilayers terminating with PSS (negatively charged) or PAH (positively charged) polyelectrolytes. On films terminating with PSS only, an albumin equivalent monolayer is found whereas when PAH constitutes the outer layer, albumin interacts with the multilayer in such a way as to form a protein film that extends over thicknesses that can be as high as four times the largest dimension of the native albumin molecule. Once the protein film is formed, it is found that when the albumin solution is replaced by a pure buffer solution of same ionic strength as the adsorption solution almost no desorption takes place. On the other hand, when a buffer solution of higher ionic strength is brought in contact with the albumin film, a significant amount of adsorbed proteins is released. One also observes that, for albumin solutions of a given protein concentration, the adsorbed amount depends on the ionic strength of the adsorption solution. On surfaces terminating with PAH, the adsorbed protein amount first increases rapidly but passes through a maximum and decreases with the ionic strength. The ionic strength corresponding to the maximum of the adsorbed albumin amount itself depends on the albumin concentration. On the other hand, on films terminating with PSS the adsorbed amount increases with the salt concentration before leveling-off. These results show that the underlying complexity of concentration and pH dependent adsorption/desorption equilibria often simply termed “protein adsorption” is the result of antagonist competing interactions that are mainly of electrostatic origin. We also propose two microscopic models, that are compatible with our experimental observations. 1. Introduction Polyelectrolyte films built-up by the alternated adsorption of cationic and anionic polyelectrolyte layers constitute a novel and promising technique to modify surfaces in a controlled way.1,2 One of the most important properties of such multilayers comes from the fact that they exhibit an excess of alternatively positive and negative charges.3,4 Not only does this constitute the motor of their buildup,5 but also it allows, by simple contact, the films to adsorb a great variety of compounds such as dyes,6 particles,7-9 clay microplates,10 or proteins.11-13 Once adsorbed, these compounds can be further covered by new polyelectrolytes leading to complex multilayer architectures containing embedded objects. In the case of proteins, a study in which antibodies were incorporated in a polyelectrolyte multilayer seems to indicate that the embedded proteins retain their reactivity with respect to their antigens.13 This remarkable property opens up the possibility, for instance, to construct * To whom correspondence should be addressed. Telephone: (33) 3 88 41 40 12. Fax: (33) 3 88 41 40 99. E-mail: [email protected]. † Institut Charles Sadron (CNRS-ULP). ‡ INSERM Unite ´ 424.

multilayers incorporating specific ligands that keep their biological activity and promote the adhesion of specific cells. Conversely, bacterial adhesion should be avoided through repelling by insertion or covalent binding of peptides that possess a structure comparable to those of the bacterial receptors.14 Also, incorporation of proteins that induce biomineralization processes should be possible. This opens new ways to design biomaterials with specific biological properties. The controlled incorporation of proteins in multilayers however requires first to understand the protein adsorption mechanisms on these films. Only very few studies have been devoted to this subject. Lvov et al.12 built multilayer films containing several protein species. Each protein type was adsorbed on an oppositely charged layer and coated by further polyelectrolyte layers before addition of another protein. Also Caruso et al.13,15 immobilized anti-IgG’s within multilayer films. The studies of Graul and Schlenoff16 concerned protein separation by means of capillary electrophoresis using polyelectrolyte multilayer-coated capillaries. However, to the authors’ knowledge, no systematic study of the interactions between proteins and polyelectrolyte

10.1021/bm005572q CCC: $19.00 © 2000 American Chemical Society Published on Web 10/21/2000

Interactions with Polyelectrolyte Multilayers

multilayers has been reported up to now. This article represents a first step in this direction. Not only should the results reported here, however, be of interest for the design of new multilayer architectures but they should also allow one to gain further insight into the role of surface charges in protein adsorption processes, even though there is a major difference between adsorption of proteins on surfaces covered with water-soluble polyelectrolytes and on “hard” charged surfaces in general. Indeed it should be noted that polyelectrolytes at a surface extend loops and tails of different lengths into the solution that are flexible and can thus accommodate to the shapes of the protein molecules. In this article we will report results obtained with human serum albumin (HSA) interacting with polystyrenesulfonate/ polyallylamine (PSS/PAH) multilayers. Scanning angle reflectometry (SAR), an optical technique that gives access to both the optical thickness and the mean refractive index of the adsorbed layer, was used to characterize, in situ, the formation of the multilayer/albumin films. 2. Materials and Methods 2.1. Materials. Anionic poly(sodium 4-styrenesulfonate) (MW ) 70 000, cat. no. 24 305-1) (PSS), cationic poly(allylamine hydrochloride) (Mn ) 50 000-65 000, cat. no. 28 322-3) (PAH) and cationic poly(ethylenimine) (MW ) 750 000, cat. no. 18 197-8) (PEI) were purchased from Aldrich, sodium chloride (g99.5%, puriss.) was purchased from Fluka, and tris(hydroxyaminomethane) (Tris) was purchased from Sigma. All the chemicals of commercial origin were used without further purification. Human serum albumine (MW ∼ 69 000, pI ) 4.6) was obtained from the Centre de Transfusion Sanguine of Strasbourg in concentrated solutions (20 g/100 mL) for intravenous injection. It was prepared and purified by the Cohn’s method,17,18 and its purity was controlled by gel electrophoresis. Ultrapure water (Milli-Q-plus system, Millipore) was used for the preparation of buffers and solutions and in the different cleaning steps. The resistivity of the water was approximately 18.2 MΩ‚cm. 2.2. Scanning Angle Reflectometry (SAR) Experiments. 2.2.1. Equipment. The optical method, scanning angle reflectometry, and the apparatus that were used have been described in detail elsewhere.4,19 Briefly, light from a HeNe laser (wavelength λ ) 632.8 nm) is polarized in the incidence plane (p-polarized) before entering nearly perpendicularly through one face of a prism and reflecting off its hypotenuse. The prism is made out of Suprasil (Heraeus). The light beam leaves then the prism through another face, again almost perpendicularly. A further polarizer again selects p-polarized light, and the resulting beam passes through a 200 µm pinhole to fall on a photodetector. The prism rotates with a high precision goniometer, changing the incidence angle, while the position of the detector is adjusted to follow the reflected beam. The reflectivity is measured at a discrete series of incidence angles, typically of 25-mdeg intervals, over a range of 1° around the Brewster angle. At this angle, approximately 42.5° for the silica/water interface, the reflectivity of an abrupt, flat interface is zero. The signal around

Biomacromolecules, Vol. 1, No. 4, 2000 675

the Brewster angle is thus most sensitive to the presence of interfacial films. 2.2.2. Experimental Approach. Each experiment was preceded by a cleaning step of the silica prism during 15 min at 100 °C with 10-2 M sodium dodecyl sulfate (SDS). This procedure was followed by an extensive ultrapure water rinse. The prism was then brought in contact during 15 min at 100 °C with 10-1 M HCl and finally extensively rinsed with ultrapure water. All experiments were carried out in 10-2 M Tris-HCl buffer solutions with pH adjusted to 7.4 by addition of HCl. The buffer solutions also contained NaCl at different concentrations depending on the experiments. The polyelectrolyte multilayers were built up from polyelectrolyte solutions prepared by dissolving 0.5 g of polyelectrolyte (PEI, PSS, PAH) in 0.1 L of Tris-HCl in the presence of different amounts of NaCl (between 10-3 and 1 M). However, it must be noticed that the salt concentrations of the solutions used for the multilayer buildup were always the same as those added to the protein solution that was subsequently brought in contact with the polyelectrolyte multilayer, thus avoiding any eventual multilayer restructuration due to a change in the ionic strength of the solution in contact with the polyelectrolyte film. The HSA solutions were obtained by rapid thawing of 5 mL aliquots of a mother solution (HSA concentration 20 g/100 mL) and adequate dilution in the Tris-HCl buffer solution in the presence of the required NaCl concentration. The HSA concentration of the solutions was varied from 1.7 mg % to 95.4 mg % (1 mg % ) 1 mg/100 mL) and was precisely estimated by UV spectroscopy absorbance at 280 nm (extinction coefficient ) 0.53 cm3‚mg-1). All the buffer solutions were degassed under vacuum before use. 2.2.3. Buildup of the Polyelectrolyte Multilayers and Protein Films. The silica surface was first brought in contact with the buffer solution, and the reflectivity curve corresponding to the Fresnel (abrupt density profile) silica/buffer solution interface was measured. This curve was employed for the calibration of the apparatus and for the measurement of the refractive index of the buffer solution. The buildup of the polyelectrolyte multilayer film was then realized as follows: (i) The buffer solution was replaced by the PEI solution (the time required for this operation was about 20 min, corresponding to the flow of 50 mL of solution, the cell containing the solution having a section of 1 × 5 mm2 and a length of 40 mm). The flow was then stopped, and after 30 min of contact with the silica surface, the PEI solution was replaced by 50 mL of buffer solution, which took also 20 min. (ii) We then adsorbed and rinsed alternatively PSS and PAH layers on the silica surface by using the same procedure as described for PEI (only the polyelectrolyte solutions used to replace the buffer solution were flowed through the cell during 10 min and the corresponding volumes were 25 mL instead of respectively 20 min and 50 mL for the PEI solution. The subsequent interaction time between the polyelectrolyte solutions and the silica surface were also reduced to 15 min instead of 30 min for the PEI case). Thus,

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Table 1. Average Optical Parameters of the Polyelectrolyte Multilayers before the Adsorption of Albumina type of film PEI-(PSS-PAH)3

PEI-(PSS-PAH)3-PSS

NaCl concn (mol‚L-1) 10-3 5 × 10-3 10-2 5 × 10-2 0.15 0.4 0.7 1 10-2 0.15 1

N

LPEM (nm) ( σ

nPEM ( σ

ΓPEM (µg‚cm-2) ( σ

2 1 2 1 16 1 1 2 1 7 1

8.65 ( 3.0 8.8 11.15 ( 2.6 14.3 17.4 ( 0.8 22.5 25.9 28.4 ( 1.0 10.9 20.4 ( 0.7 30.9

1.488 ( 0.011 1.490 1.484 ( 0.007 1.484 1.4865 ( 0.002 1.488 1.491 1.492 ( 1.489 1.489 ( 0.002 1.497

0.91 ( 0.27 0.94 1.14 ( 0.22 1.46 1.80 ( 0.07 2.31 2.66 2.89 ( 0.11 1.16 2.15 ( 0.06 3.24

a All films were built up at pH 7.35 at various NaCl concentrations. The data corresponding to the films were analyzed with the homogeneous and isotropic monolayer model. N is the number of experiments performed for given experimental conditions. LPEM is the average thickness of the film, nPEM is its average refractive index, and ΓPEM is the corresponding average of adsorbed polyelectrolyte amount. ΓPEM was calculated from the value ∆nL by using the refractive index increment dn/dc ) 0.148 cm3‚g-1. σ represents the standard deviation.

progressively PEI-PSS, PEI-(PSS-PAH)1, PEI-(PSSPAH)1-PSS... layers were deposited. (iii) The buildup of the film was stopped either after the deposition of a positively charged PEI-(PSS-PAH)3 or a negatively charged PEI-(PSS-PAH)3-PSS layer. At this stage, reflectivity curves were determined for about 16 h allowing the film to reach full equilibrium. The choice to stop the buildup of the polyelectrolyte multilayer after seven or eight layers (including the initial PEI layer) is due to the fact that typically only a few layers, depending on the details of the deposition conditions, are required to render the properties of the multilayers reproducible from one preparation to another.4 The optical data relative to the multilayer were then analyzed within the homogeneous and isotropic monolayer as will be described below. The optical thicknesses and refractive indexes of the multilayers used for subsequent albumin adsorption experiments are reported in Table 1. (iv) We then replaced the buffer solution that was in contact with the film by a HSA solution. The multilayer was kept in contact with the HSA solution for several hours. During this period of time the reflectivity curves were continuously determined in order to follow the adsorption of HSA on the film. The determination of each reflectivity curve took approximately 3 min. This corresponds to the smallest time interval experimentally accessible. (v) For most of the experiments, once the adsorption process had leveled off, we replaced the HSA solution by a buffer solution (solution 1) having the same ionic strength (same NaCl concentration) as during the protein adsorption and polyelectrolyte film buildup, and we followed the possible desorption of the adsorbed proteins. Reflectivity curves were again continuously determined for several hours. This first rinse was, in certain cases, followed by a second one, that was performed with a buffer solution at a higher ionic strength (NaCl 2 M, solution 2). Prior to reflectivity measurements, solution 2 was finally replaced by solution 1 in order for the refractive index of the solution above the prism to be similar to the one corresponding to the Fresnel curve and which served to calibrate the reflectometer. 2.2.4. Analysis and Interpretation of the Reflectivity Curves. The reflected intensities I(θ) measured at different

incidence angles θ are related to the amplitude of the reflectivity coefficient r(θ) characterizing the interface by the relation I(θ) ) I0 + Rr(θ)r*(θ)

(1)

where r*(θ) represents the complex conjugate of r(θ). I0 corresponds to the background noise intensity, and R is an apparatus constant. Both parameters were determined by comparing the measured reflectivity curve corresponding to the Fresnel interface to the predicted intensity, with r(θ) corresponding to the Fresnel reflectivity coefficients relative to the silica/buffer solution interface.19 The refractive index of the silica was fixed, whereas the refractive index of the buffer solution nsol, the parameters I0 and R constituted the fitting parameters. The determination of these parameters corresponds to the calibration of the reflectometer. The reflectivity curves relative to the polyelectrolyte multilayers were analyzed by assuming that the PEM behave as homogeneous and isotropic monolayers of thickness LPEM and refractive index nPEM (or equivalently of refractive index excess ∆nPEM ) nPEM - nsol). The use of this approximation is justified in the appendix by analyzing the optical data relative to the PEM within the framework of the optical invariants. The reflectivity coefficients r(θ) entering in relation 1 correspond then to that of an homogeneous and isotropic monolayer and can be found in standard text books.20 The PEM/adsorbed albumin films were first analyzed within the same framework of the homogeneous and isotropic monolayer. This led, however, to optical parameters characterizing the monolayer that were physically unreasonable. This suggested that the refractive index profiles of these PEM/protein films are more complex and cannot be approximated by a homogeneous monolayer. This result is corroborated by the invariant analysis of the protein adsorption process (see appendix). We thus analyzed all the optical data with a two-layer model. The PEM/protein films were thus characterized by two thicknesses, LPEM and LHSA, and by two refractive index excesses ∆nPEM and ∆nHSA. However, one can show that SAR allows to get access to a maximum of three optical parameters and often only to two param-



eters.21 We thus imposed the values of the thickness LPEM and of the refractive index excess ∆nPEM to the polyelectrolyte multilayer lying in contact with the silica surface. We took them to be equal to the values corresponding to the polyelectrolyte multilayer obtained before the protein adsorption, determined in each experiment after the buildup of the polyelectrolyte film. The reflectivity coefficients entering in expression 1 are thus those of the two-layer model and were evaluated by using the standard method of Abele`s,22 the fitting parameters being LHSA and ∆nHSA, the characteristic parameters of the adsorbed protein layer. The comparison of the experimental reflectivity curves to the theoretical ones was done by using a nonlinear leastsquares fitting procedure in which one minimizes the quantity χ2 )

1 Na

Na

∑ i)1

(

I (θi) - I exp

mod

(θi)

)

0.01|Iexp(θi)| + σ0

2

(2)

with respect to the fitting parameters. The sum is performed over all the Na incidence angles θi at which the reflected intensities Iexp (θi) have been measured. Imod(θi) corresponds to the reflected intensity given by expression 1 and in which the reflection coefficient is evaluated on the basis of a layer model. σ0 represents the baseline noise determined in each experiment. In the treatment of the experimental curves relative to the PEM/albumin films, the function χ2 usually exhibited several minima within the explored range of the fitting parameters. The values of χ2 corresponding to these different minima were usually too close to allow to discriminate between them on the unique basis of their values. Most of these minima could be discarded on the basis of simple physical arguments (unrealistic layer thicknesses and/or unrealistic layer refractive index excesses). Usually only one minimum and sometimes two minima of approximately similar values of ∆nHSA but different values of the layer thickness LHSA remained after this first selection. The presence of two minima is observed only during the initial stages of the adsorption processes of albumin on PAH terminating PEM from highly concentrated protein solutions. One minimum then leads to a decrease of the protein layer thickness and of the adsorbed amount with the adsorption time whereas the thickness and the adsorbed amount relative to the second minimum increases with time as expected (see Figure 1). We have then only retained this last solution which corresponds systematically to the minimum with the smallest value of the protein layer thickness. However, for these concentrated albumin solutions, as the adsorption process of albumin onto PAH terminating films progressed, the values of LHSA (or (∆nL)HSA) relative to the two minima became closer and they finally merged into a unique solution (b in Figure 1). One can notice that during this merging process there exists a point where one of the two local minima suddenly disappears. If, at this point, the minimum which corresponds to the large value of LHSA (or (∆nL)HSA) and which has been discarded up to this point is the one that has the smallest value of χ2 (even if the difference between the values of χ2 is extremely small and none significant), LHSA (or (∆nL)HSA) will suddenly jump to this value. This can thus give rise to

Figure 1. Typical evolution of (∆nL)HSA (similar to the evolution of LHSA) determined through analysis with the bilayer model during adsorption of HSA from a “high concentrated” protein solution (calb ) 24 mg/100 mL) in the presence of 0.15 M NaCl at pH 7.4 onto a PEI-(PSS-PAH)3 polyelectrolyte multilayer. At the beginning of the process, the analysis gives two solutions (O and 3) which correspond to the same refractive index. (b): evolution of (∆nL)HSA once the two minima have merged into a unique solution. At the initial times we retained the solution corresponding to (O) and discarded the (3) solution.

a discontinuity in the curve representing the evolution of LHSA (or (∆nL)HSA) with time, as will be seen in section 3.2, where the kinetic aspects of the adsorption of the protein are discussed. Such a discontinuity is totally due to the fact that one analyses the reflectivity curves with a model (in this case the two-layer model) and that this model can only constitute a first approximation of the real refractive index profile of the PEM/protein films. 3. Experimental Results 3.1. Adsorption Isotherms. An adsorption process can, in a first approach, be characterized by the adsorption isotherm. Figure 2, parts a and b, represents the optical adsorption isotherms of albumin interacting with (PSSPAH) polyelectrolyte multilayers, the outer layer being either constituted by the anionic PSS or the cationic PAH polyelectrolyte. They were obtained by bringing the multilayers in contact with albumin solutions of different concentrations. In these experiments, the NaCl concentration of the albumin solutions was always fixed to 0.15 M and the experiments were performed in the absence of any flow adjacent to the adsorbing surface. At pH 7.4, native HSA is usually schematically represented as a prolate ellipsoid of revolution with major and minor axes respectively 12.0 and 2.7 nm23 or 14.1 and 4.1 nm24 and with one positive and two negative charges as shown in Scheme 1. It thus carries a net -15 charge (in units of the elementary charge e). Recent X-ray determinations of the structure of albumin have rather suggested a “heart-shaped” structure25 with sides of about 8 nm and an average thickness of 3 nm.26 These results are however obtained by crystallization of the protein, which is then no longer in the same solution environment. We have thus still interpreted all our results by assuming that it has an ellipsoidal shape as represented in Scheme 1, this shape being the most commonly used to interpret adsorption and hydrodynamic interaction experiments. As far as the multi-

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Figure 2. Optical adsorption isotherm of HSA on (PSS-PAH) polyelectrolyte multilayers: (a) optical thickness LHSA (closed symbols) and optical mass (∆nL)HSA (open symbols) where ∆n ) (nHSA - nsol); (b) mean refractive index nHSA of the protein layer. The values reported on the LHSA axis represent multiples of the largest dimension of native HSA molecules (12 nm) according to Haynes et al.23 Assuming dn/ dc ) 0.18 cm3‚g-1 for HSA,27 1 nm on the (∆nL)HSA axis corresponds to an adsorbed protein amount of about 0.56 µg‚cm-2. (b and O) correspond to the isotherm on PEI-(PSS-PAH)3; (1 and 3) correspond to the isotherm on PEI-(PSS-PAH)3-PSS. The adsorption processes were performed by using albumin solutions containing 0.15 M NaCl at pH 7.4. Scheme 1. Schematic Representation of an HSA Molecule at pH 7.4, According to Peters24 with L1 ) 14.1 nm and L2 ) 4.1 nma

a More recent values were found in the literature:23 L ) 12.0 nm and 1 L2 ) 2.7 nm.

layers are concerned, several studies in which the ζ-potentials were determined revealed that the multilayers terminating with a PSS (respectively PAH) layer exhibit a negative (respectively positive) ζ-potential.4 It is thus not unexpected that albumin interacts more strongly with the positively than with the negatively charged multilayer as it comes out from Figure 2a. On the PSS terminating surface, albumin forms, in the isotherm plateau, a layer with an extension on the order of 13 nm. This indicates that the proteins should be adsorbed mainly in “end-on” configurations. If one assumes such adsorption configurations and if one uses the standard value of 0.18 cm3‚g-1 for the refractive index increment dn/ dc27 one finds that, in the adsorption isotherm plateau, the coverage is on the order of 53%. This is very close to the random sequential adsorption jamming limit.28 One can thus conclude that, despite the fact that both the protein and the

Ladam et al.

surface exhibit charges of same sign, the protein can fully cover the adsorption surface and even lead to a quite dense layer which never extends over the monolayer. More surprising is the formation of very thick protein films when the albumin solutions were brought in contact with multilayers terminating with cationic PAH. These protein layers extend up to 54 nm, which represents more than four times the largest dimension of the native protein. The formation of films thicker than a monolayer for proteins having a high affinity for a surface were already observed,23,29 even if such systems constitute the exception rather than the general rule. On the other hand, to the authors knowledge, the formation of adsorbed layers thicker than two monolayers was never observed for nonaggregating proteins. The formation of such thick adsorbed layers not only implies that the adsorbing proteins have a high affinity for the multilayers terminating with PAH but also that they have a strong affinity for the already adsorbed underlying protein layer. Finally, one also finds that the mean refractive index of the adsorbed layers increases with the protein concentration of the albumin solution and correlatively with the thickness of the adsorbed layer. 3.2. Adsorption Kinetics. To understand how these adsorbed layers build up, we followed the adsorption kinetics. Typical evolutions of the optical thickness, the adsorbed amount, the mean refractive index of the protein layer and the parameter χ2 characterizing the quality of the fit both for multilayers terminating with PSS and PAH are given in Figure 3, parts a-c. In all cases the protein layers build up progressively, the optical thickness and the adsorbed amount increasing continuously up to their final value. As far as the evolution of the mean refractive index of the protein layer is concerned, one usually observes a rapid decrease of nHSA during the initial stage of the adsorption process. The characteristic time scale of this first stage is on the order of a few tens of minutes. This is followed by a slow but continuous increase of nHSA in the following stage of the buildup process. This second stage runs over several hours (up to 10 h). One can observe that during the initial adsorption stage characterized by a simultaneous increase of the optical thickness and a decrease of the refractive index of the protein layer the fit quality parameter relative to the bilayer model also decreases. This indicates that initially the bilayer model does not satisfactorily describe the film but that the validity of the bilayer model improves as the adsorption process evolves during this initial stage. This can be due to the fact that, at the beginning of the adsorption process, the protein partly penetrates into the outer polyelectrolyte layer so that the film more closely resembles a triple layer (PEM/outer PE-protein/protein). As the adsorption process goes on the outer protein layer fills up, becomes denser, and extends so that it becomes dominant, leading to an improvement of the bilayer model. Such an effect should, indeed, lead to a simultaneous increase in the optical layer thickness and a decrease of the refractive index as was observed for the adsorption process of fibrinogen on silica by SAR.19 After this first adsorption stage, one enters in a second regime in which the extension of the adsorbed layer increases more slowly than the adsorbed amount, thus leading


Figure 3. Typical adsorption kinetics of HSA on (PSS-PAH) polyelectrolyte multilayers: (a) optical thickness LHSA (closed symbols) and optical mass (∆nL)HSA (open symbols); (b) mean refractive index nHSA of the adsorbed protein layer; (c) quality fit parameter χ2. The adsorption processes were performed with HSA solutions at pH 7.4 in the presence of 0.15 M NaCl, in the following conditions: (b and O) calb ) 4.5 mg/100 mL on PEI-(PSS-PAH)3; (9 and 0) calb ) 24 mg/100 mL on PEI-(PSS-PAH)3; (2 and 4) calb ) 18 mg/100 mL on PEI-(PSS-PAH)3-PSS. The discontinuity appearing in the evolutions of thickness and adsorbed amount during the buildup of the protein layer for a high protein concentration solution is an artifact originating from the treatment of the reflectivity curves by a two-layer model as mentioned in section 2.2.4.

to an increase of the mean refractive index of the layer. It can be noticed that, as the protein layer builds up, the quality parameter χ2 of the fits increases steadily, indicating that the two-layer model used to analyze the reflectivity curves becomes less accurate. This could arise from the fact that, as the protein layer builds up, its refractive index profile becomes more diffuse (a possible explanation will be given in the Discussion). It can be noticed that, in fact, the adsorption process never fully stops over the experimental time scale. The adsorbed amount continues to increase very slowly after several hours of adsorption. At the same time, one observes a small increase in the refractive index of the adsorbed layer. This indicates that not only do new proteins adsorb but a slow restructuration leading to a densification of the adsorbed layer must also take place over these long time scales.


3.3. Effect of Buffer Solution Rinse. At the end of the buildup process, the protein solution was rinsed by a 0.15 M NaCl buffer solution. Only a very small fraction, on the order of 5%, of the adsorbed proteins was released on both multilayers terminating with PSS or PAH whatever the albumin concentration of the buildup solution. Moreover, neither the layer thickness nor the mean refractive index of the adsorbed protein layer changed significantly during this rinsing procedure. This indicates that a rinse with a buffer solution of salt concentration similar to that of the buildup protein solution does not change significantly the structure of the adsorbed layers. Thus not only do the albumin molecules appear to interact very strongly with the multilayers, whatever the sign of their surface charge but also, in the PAH case, they appear to interact very strongly with the already adsorbed underlying layer. These interactions appear almost irreversible at the experimental time scales. This result raises thus questions (i) about the nature of the interactions keeping the albumin molecules fixed on the adsorbed layers and (ii) about the reasons why the adsorption processes stop before reaching the isotherm plateau for solutions of small and intermediate protein concentrations. 3.4. Effect of Ionic Strength on Protein Release. To partially answer question i, we carried out experiments in which the adsorption process and the buffer solution rinse process were performed in conditions identical to those described above (0.15 M NaCl). The first 0.15 M NaCl rinse was however followed by a second rinse with 2 M or 10-2 M NaCl buffer solutions. When the first 0.15 M NaCl rinse was followed by a second 10-2 M NaCl buffer solution rinse, no change was detected in the film (see line 4 of Table 2). But when it was followed by a 2 M NaCl buffer solution rinse, about 55% of the albumin molecules adsorbed on PEI(PSS-PAH)3 multilayers were released, independent of the protein concentration of the film buildup solution (see lines 1, 2, 3, and 5 of Table 2). When the proteins were adsorbed on a PEI-(PSS-PAH)3-PSS multilayer, the released fraction of proteins due to the 2 M NaCl buffer solution rinse represented on the order of 40% of the adsorbed molecules (see line 6 of Table 2). The rinse of the protein solution by a buffer solution of higher ionic strength thus induces major release. It can also be noticed that the rinsing process induced both a decrease of the layer thickness and of the mean refractive index of the adsorbed layers. These layers thus became less dense after rinsing with the solution of higher ionic strength. This overall effect is necessarily due to the thick albumin layer and cannot result from a deswelling of the polyelectrolyte film. Indeed, the measurements were performed in the presence of a buffer solution of same ionic strength as during the buildup of the polyelectrolyte and of the albumin films, and it was shown previously4 that polyelectrolyte films behave totally reversibly toward ionic strength changes. For the thick protein layers adsorbed on films terminating with PAH, the decrease of the adsorbed protein amounts and of the protein layer densities due to this 2 M NaCl rinsing process point toward a weakening of the interactions between the adsorbed proteins. The observed release of proteins by addition of high salt solutions must

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Table 2. Optical Parameters of HSA Layers Adsorbed from Solution with Different Protein Concentrations and Effect on These Optical Parameters of the Ionic Strength during the Subsequent Rinsing Proceduresa adsorbed protein amounts outer layer

[HSA] (mg/100 mL)

LHSA (nm)

PAH PAH PAH PAH PAH PSS

4.8 7.4 18.5 19.2 51.8 22.7

16.9 25.0 42.3 43.8 54.3 09.0

first rinse: 0.15 M NaCl

nHSA

(∆nL)HSA (nm)

LHSA (nm)

1.439 1.443 1.448 1.449 1.453 1.436

1.78 2.73 4.86 5.06 6.45 0.92

16.1 24.2 41.0 42.6 53.7 08.5

second rinse

nHSA

(∆nL)HSA (nm)

[NaCl] (M)

LHSA (nm)

nHSA

(∆nL)HSA (nm)

amt released (%)

1.438 1.442 1.448 1.449 1.452 1.434

1.68 2.62 4.68 4.90 6.34 0.86

2 2 2 10-2 2 2

08.2 12.5 19.1 41.0 32.8 05.8

1.426 1.432 1.436 1.448 1.442 1.404

0.77 1.29 1.99 4.66 3.56 0.51

54 51 57 5 44 41

a First rinse, same 0.15 M NaCl concentration; second rinse, different NaCl concentration. Prior to reflectivity measurements a 0.15 M NaCl solution was always flowed through the cell after the second rinse.

be due to an ion exchange process with the salt similar to that used for purification of the proteins in ion-exchange columns. On the multilayers terminating with PSS, even if the released amount of proteins was enhanced by an increase of the salt concentration of the rinsing solution, there still remained a considerable fraction of adsorbed proteins after rinsing. This indicates strong interactions between negatively charged albumin molecules and overall negatively charged multilayers and probably also between adsorbed proteins themselves. This is in accordance with the fact that, at high protein concentrations, the adsorption process leads to a dense monolayer of essentially “end-on” adsorbed molecules. One can thus conclude that these experiments strongly point toward an electrostatic origin of the interaction forces involved in the albumin adsorption process on polyelectrolyte multilayers. 3.5. Incidence of the Ionic Strength on HSA Adsorption. If the buildup of the adsorbed layer is mainly driven by electrostatic interactions, it should also be influenced by the ionic strength of the albumin solution in contact with the adsorbing surface. This is indeed the case as can be seen in Figure 4, parts a and b where the evolution of the optical thickness, the adsorbed amount, and the mean refractive index of the adsorbed albumin layer are represented as a function of the logarithm of the NaCl concentration of the albumin solution. We checked this effect on multilayers builtup from polyelectrolyte solutions having the same NaCl concentration as the albumin solutions. The albumin concentration of the protein solution was 22 mg/100 mL when PSS was the outer layer of the multilayer and 4.5 and 22 mg/100 mL when PAH was the outer layer of the polyelectrolyte film. On the negatively charged PSS layer, both the adsorbed amount and the layer thickness increased with NaCl concentration. This effect is not unexpected for negatively charged proteins adsorbing on a negatively charged surface, and it clearly shows the incidence of the electrostatic interactions during the adsorption process. A different behavior was observed when PAH was the last layer of the polyelectrolyte film. The amount of adsorbed albumin first strongly increases with the NaCl concentration, passes through a maximum and then decreases continuously until the salt concentration attains 1 M. The maximum of the adsorbed amount is reached for salt concentrations that depend on the protein concentration of the adsorption solution. It is on the order of 10-1 M (respectively 10-2 M)

Figure 4. Evolution of (a) the optical thickness LHSA (closed symbols) and the optical mass (∆nL)HSA (open symbols) and (b) of the mean refractive index nHSA of the adsorbed protein layer as a function of the logarithm of the NaCl concentration of the adsorption solution. The adsorption processes were carried out in the following conditions: (b and O) calb ) 4.5 mg/100 mL on PEI-(PSS-PAH)3; (9 and 0) calb ) 22.5 mg/100 mL on PEI-(PSS-PAH)3; (1 and 3) calb ) 22.5 mg/100 mL on PEI-(PSS-PAH)3-PSS. Lines do not correspond to fits but were added merely to guide the eye.

for an albumin concentration of 22 mg/100 mL (respectively 4.5 mg/100 mL). These results again constitute a typical signature of the role of the electrostatic interactions in the HSA layer buildup. The appearance of a maximum in the adsorbed amount and in the layer thickness as a function of the salt concentration on multilayers terminating with PAH when thick protein layers are formed indicates that the attractive albumin/albumin interactions that must exist for such thick films to form must be counterbalanced by repulsive interactions that are certainly of electrostatic origin. Finally, one can also notice that, in all cases, the mean refractive index of the adsorbed layer decreases when the salt concentration of the adsorption solution increases. This points toward a weakening of the interactions, probably due to charge screening.



Once the adsorbed layer was formed, we replaced the adsorption albumin solution by a pure buffer solution of same salt concentration. For all the investigated NaCl concentrations, the released amount of proteins was very small representing on the order of 3% (respectively 8%) on multilayers terminating with PAH (respectively PSS). Thus, for any salt concentration of the buildup solution, the adsorption process leads to an adsorbed protein layer in which the molecules are fixed almost irreversibly under the adsorption conditions. 3.6. Incidence of Flow Rate on the HSA Adsorption Process. To answer question ii, we performed an experiment where the multilayers were first brought in contact with an albumin solution of roughly 5 mg/100 mL protein concentration (0.15 M NaCl concentration). Once the protein layer was built up, the protein solution was replaced by a 50 mg/ 100 mL protein solution. Surprisingly we found that, both on the multilayers terminating with PAH and PSS, an additional amount of proteins could be adsorbed during this second adsorption step. This further addition was however small in the case where PSS was outside of the polyelectrolyte film. This first suggests that, during the initial adsorption step, the adsorption does not stop because of a gradual decrease of the interactions between the proteins from the solution and the adsorbing surface but because of a significant decrease of the protein concentration in the solution. However, we always took great care that, in all our experiments, the amount of adsorbed proteins never represented more than 15% of the amount of proteins present initially in the adsorbing cell (taking all the walls of the cell into account) and in most cases this percentage was even less than 5%. This additional increase of the adsorbed amount must thus come from the fact that during the filling of the cell by the second, more concentrated protein solution (50 mg/100 mL), the flow above the adsorbing surface, and in particular the shear rate, affects the structure of the already adsorbed layer in such a way that additional proteins can adsorb. We thus performed experiments under different flow conditions for albumin adsorption to verify this hypothesis. For multilayers terminating with PAH (respectively PSS) three (respectively two) experiments with different flow rates were performed, the concentration of the solution being equal to 5 mg/100 mL (respectively 53 mg/100 mL). Because of the geometry of the adsorption cell we could not evaluate precisely the shear rate. In all cases, after a given adsorption time, depending upon the flow rate, the adsorption process stopped; i.e., no further proteins adsorbed. The final adsorbed amount was flow-rate-independent when the adsorption process was carried out on a multilayer terminating with PSS and increased with the flow rate on films terminating with PAH (see Table 3). This shows that on multilayers terminating with PAH the structure of the adsorbed layer and especially the amounts of adsorbed proteins greatly depend on the adsorption conditions. The adsorption of albumin on PSS terminating multilayers seems in a first sight to be in contradiction with the results of Graul and Schlenoff.16 These authors indicate that, in capillary electrophoresis experiments, the proteins do not seem to adsorb on the walls of the capillaries coated with multilayers charged similarly as the

Table 3. Effect of the Flow Rate on the Adsorbed Amounts of Protein on Polyelectrolyte Multilayersa outer polyelectrolyte layer

flow rate (mL‚h-1)

LHSA (nm)

∆nLHSA (nm)

PAH PAH PAH PAH PSS PSS

0 0 4 40 0 40

16.85 14.00 33.00 37.70 11.34 10.91

1.78 1.47 3.76 4.31 1.24 1.15

a The protein concentration of the adsorption solution was roughly equal to 5 mg/100 mL on PAH and to 53 mg/100 mL on PSS, and the NaCl concentration was taken to be equal to 0.15 M.

proteins. However, in capillary electrophoresis the shear rate at the surface can be quite higher than in our experimental conditions and it is not unreasonable to assume that the adsorbed amount depends on the shear rate for shear rates higher than the ones examined here. Indeed, as the shear rate increases the interaction time between the proteins and the surface decreases, and it has been shown that, to create strong interactions between a protein and a surface, a minimum interaction time seems to be required.30 Moreover, the binding force of the proteins to the surface increases with the protein/surface interaction time. Finally, a strong electric field acts also on the proteins in capillary electrophoresis and can also greatly influence the adsorption process. Further investigations of the adsorption process of albumin as a function of the shear rate at the multilayer interface would thus be of interest. It is however out of the scope of the present study in which attention was only concentrated on diffusion-driven adsorption processes. 4. Discussion Up to now we have discussed our experimental data without referring to any molecular model that can only be speculative with the available information. We will now propose two models at a microscopic level which are compatible with our experimental results. One should however keep in mind that other models that may also explain our experimental findings could certainly be proposed. 4.1. A First Structural Model for Adsorbed Albumin Layers onto Multilayers Terminating with PAH. The most striking feature emerging out of our study is on one hand the formation, on films terminating with PAH, of thick albumin layers extending over distances representing up to four times the largest protein dimension. On the other hand, on films terminating with PSS, only protein monolayers could be observed. The formation of such thick protein layers on multilayers terminating with PAH cannot be the result of processes similar to those responsible for protein aggregation. If this would be the case one should also observe the formation of thick films on multilayers terminating with PSS. Moreover, it would be in contradiction with long-range repulsion that has been found between albumin molecules in solution at pH 7.4, 0.15 M ionic strength.31 The observed thick layers must be the result of strong forces induced by the outer part of the adsorbed layer with new incoming

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Scheme 2. Schematic Representation of the First Proposed Buildup Process of HSA Layers on Films Terminating with PAHa

a (a) For low HSA concentrations, no structuration occurs; (b) for high HSA concentration, the HSA molecules immediately mainly adsorb in “endon” configurations on the polyelectrolyte film. A dense organized layer of adsorbed proteins on films terminating with PAH should lead to a net positive surface charge that attracts new HSA molecules. This buildup process can thus repeat itself and give rise to a thick protein layer. As the layer increases in thickness, the order between the adsorbed molecules decreases, leading also to a reduction of the positive surface charge so that after several protein layers the adsorption process ultimately stops.

albumin molecules from the solution. Because of the great difference in the adsorption behavior of PSS and PAH terminating films, it is not expected that albumin molecules are distributed within the PEM. Albumin should only interact with the outer layer. We propose a first explanation of this buildup of the albumin layer which assumes that the PAH polyelectrolytes do not extend into the protein layer. Our explanation is summarized in Scheme 2. When an albumin solution is in contact with a multilayer terminating with PAH, the proteins can interact with the cationic polyelectrolyte mainly through electrostatic interactions. Once a protein has established contact with the multilayer, the cationic polyelectrolytes increase steadily their interactions with this albumin molecule rendering this latter irreversibly fixed in “side-on” configurations. This is what should happen when a multilayer terminating with PAH is brought in contact with a low-concentrated albumin solution. However, at the early stages of the protein/polyelectrolyte contact, the interactions must be weak enough so as to allow the albumin molecules to change their adsorbed configurations. Then, when an albumin molecule from the bulk comes in contact with the multilayer nearby to an adsorbed protein molecule, both adopt “end-on” configurations that optimize the electrostatic interactions between the two molecules and the surface. Moreover in the progress of the adsorption kinetics, gradual changes from “end-on” to “side-on” configurations become impossible due to the steric hindrance between neighboring adsorbed proteins. One thus progressively builds up a dense layer of “end-on” adsorbed albumin molecules that should mainly be oriented so as to present their positive region pointing toward the solution. This should

Ladam et al.

induce a net positive surface charge density close to the solution so that other negatively charged albumin molecules can interact with it. The ability of this first adsorbed protein layer to attract other albumin molecules should depend on the degree of organization of this first layer. The higher the solution concentration the smaller becomes the probability for an adsorbed protein to be irreversibly fixed on the surface in a “side-on” configuration before another protein adsorbs in its vicinity and prevents this adsorbed configuration. Once the first albumin layer is partially formed, molecules from the solution can interact with it again in different configurations, this interaction being due to the net positive surface charge at the outer part of the first albumin layer. Here too, the rapid interactions between two neighboring adsorbed proteins should lead to “end-on” configurations giving rise to a second well-organized layer exhibiting a high positive surface charge density on its outer part. The adsorption process can thus move on. The degree of organization should however progressively decrease from the first to the second protein layer, from the second to the third layer, etc. Thus, once the degree of organization becomes too low for the outer layer to exhibit locally strong positive surface charge densities, the attractive interactions between the outer layer and the albumin proteins from the solution disappear and the adsorption process stops. The improvement of the organization, that also leads to denser layers when the protein concentration of the solution is increased, is reflected by the increase with the solution concentration of the mean refractive index of the adsorbed protein layer (see Figure 2b). The loss of organization as one moves from PAH toward the outside of the protein film also implies that, as the layer is built up, its corresponding refractive index profile should decrease so that the two-layer model for the polyelectrolyte/ protein film becomes less accurate. The adsorbed film behaves thus more as a diffuse layer. This is experimentally shown up by the poorer quality parameter χ2 of the fit (higher χ2, see Figure 3c). This scheme implies that the buildup of the first layer is the result of the interplay between the attractive albumin/ PAH interactions and the repulsion between two individual albumin molecules. Both interactions are of electrostatic origin and are thus affected by the salt concentration. It is thus expected that on one hand, at high salt concentrations the interactions between an albumin molecule and both the polyelectrolytes and the other adsorbed albumin molecules become smaller leading to a loss of organization of the albumin layers and thus to a decrease of the overall adsorbed amounts. On the other hand, at small ionic strength the range of the repulsive interactions acting laterally between two albumin molecules increases, favoring the adsorption of albumin molecules further apart one from another. At the same time, the interactions between the albumin molecules and the PAH polyelectrolytes increase favoring “side-on” adsorbed configurations. Both effects lead again to a reduction of the organization of the adsorbed albumin layers and thus to a decrease of the overall adsorbed amounts. It is thus expected, as usually comes out when antagonist forces govern a physical process, that a maximum in the adsorbed amount

Interactions with Polyelectrolyte Multilayers Scheme 3. Schematic Representation of the Second Proposed Model to Explain the Buildup Process of Thick HSA Films on PEM Terminating with PAHa

a (a) For low HSA concentrations, as time evolves the PAH polylectrolytes readjust their conformations leading to a tighter interaction with the HSA molecules that prevents further protein adsorption; (b) for high HSA concentrations such a readjustment has no time to take place and polyelectrolyte loops can emerge out of the first adsorbed protein layer leading to subsequent protein adsorption.

is reached as a function of the ionic strength, as observed on multilayers terminating with PAH (see Figure 4a). Finally, the increase of the thickness of the adsorbed albumin layer in the presence of flow above the surface remains open to speculation. A likely explanation is that adsorbing proteins that are not strongly bound to the adsorbed protein film should be removed more easily from the surface during the buildup process of the adsorbed layer so that the adsorption of proteins on well organized surface areas should be favored, leading to a better organization of the adsorbed layer. 4.2. Alternative Explanation for Structural Organization of Albumin on Multilayers Terminating with PAH. An alternative explanation, which is schematically represented in Scheme 3, could be that when albumin molecules adsorb on multilayers terminating with PAH, the PAH polyelectrolytes interact with the whole albumin molecule in a kind of encapsulation process. It is indeed known that, in solution, proteins form strong complexes with polyelectrolytes through a kind of encapsulation process.32 Thus, once a first layer of albumin molecules is adsorbed one finds again PAH polyelectrolytes on top of the adsorbed protein film and new albumin molecules from the solution can interact with it. As the thickness of the protein layer increases, less PAH polyelectrolytes reach the outer part of the protein layer so that the adsorption process finally stops. The fact that, at low protein concentrations the adsorbed amount and the protein layer thickness are smaller than when the multilayer terminating with PAH is in contact with a high concentrated


solution can be explained as follows: when an albumin molecule interacts with a PAH layer, the polyelectrolyte initially establishes only a few contacts so that polyelectrolyte loops can emerge out of the protein layer. When a new protein comes in contact with these loops, it can again interact strongly with the polyelectrolyte so that a thick film can form. However, the polyelectrolytes can also change their conformations on the protein with time, leading to a tighter interaction with smaller loops and more surface charge compensation. This would significantly reduce the attractive interactions between this albumin/PAH layer and the new incoming albumin molecules from the solution. 4.3. Molecular Model for the Adsorption of Albumin onto Multilayers terminating with PSS. Let us now discuss the case of albumin adsorption on multilayers terminating with PSS. Two adsorption mechanisms can be proposed: the albumin molecules mainly interact with the PSS polyelectrolytes through their positive charges even if these are not in excess. One can also assume that, even if the outer layer of a multilayer terminating with PSS is mainly constituted of PSS polyelectrolytes, some PAH groups can still emerge out of it and the albumin proteins interact with these groups. Both mechanisms can occur simultaneously. In any case, what seems to come out from the evolution of the optical thickness is that for low albumin concentrations, the proteins adopt mainly “side-on” configurations on the surface whereas at higher concentrations, steric effects hinder these configurations and one gets a dense layer of albumin molecules adsorbed in “end-on” configurations. One can point out that even if this layer becomes dense at high concentrations, it is, overall, less dense than the films formed on multilayers terminating with PAH as observed when one compares the average refractive index of the adsorbed layers on both types of multilayers. The reason the adsorption process on multilayers terminating with PSS does not lead to films extending beyond the protein monolayer will depend on the adsorption mechanism. If the driving force for the adsorption is the interaction between the PSS polylectrolytes and the positively charged groups on the proteins, then one will end up with an adsorbed layer exhibiting a net negative surface charge toward the solution. Such a layer would then act as a repulsive barrier, the albumin molecules repelling each other in solution. One can then ask why two negatively charged albumin molecules would repel each other whereas these proteins interact attractively with the negatively charged PSS outer layer. This difference in behavior can be explained by the larger flexibility of the polyelectrolyte compared to the protein, allowing thus closer contact between the polyelectrolyte groups and the positively charged groups of the proteins. On the other hand, if the albumin molecules interact with the multilayer through PAH groups, it should lead to a similar initial layer as for multilayers terminating with PAH. The density must, however, be smaller so that one never reaches a large enough positive charge density on the outer part of the adsorbed layer to induce attractive interactions with albumin molecules from the solution. Moreover, due to the smaller charge density, one should always be in the presence of a large fraction of proteins adsorbed in a “sideon” configuration. This should thus lead to a layer that is

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poorly organized so that the adsorption process stops after the buildup of the first monolayer. 5. Conclusion and Perspectives We investigated the interactions between human serum albumin and PAH/PSS multilayers and found that the proteins adsorb both on PAH and on PSS ending multilayers. On PSS-ending multilayers one can form up to a dense protein monolayer, whereas on PAH-ending multilayers, films with thicknesses exceeding several times the largest native protein dimension can build up. The origin of the protein/multilayer interactions is mainly of electrostatic nature as it comes out from the sensitivity of the adsorption process to the salt concentration of the protein solution. We propose mechanisms for the buildup processes both on PAH and on PSS. One of the proposed mechanisms of the formation of thick films on multilayers terminating with PAH makes extensive appeal to an organized first adsorbed layer while the other mechanism assumes that the PAH polyelectrolytes of the outer layer of the multilayer extends into the whole protein adsorbed film. It would be of interest to test these explanations by using techniques such as X-ray reflectivity or neutron scattering. For multilayers terminating with PSS, one of the proposed mechanisms appeals to interactions between negative charges of the PSS polyelectrolytes of the outer layer of the multilayer and the positive charges of albumin. The second proposed explanation assumes that PAH polyelectrolytes still emerge out of the PSS outer layer and interact with the albumin proteins. Experiments with PSS polyelectrolytes of larger mass should be performed to discriminate between both proposed explanations. Finally, it would be of great interest to investigate if the present results can be extended to other proteins. The understanding of these adsorption processes of proteins on polyelectrolyte multilayers is of prime importance for the design of complex multilayers incorporating several proteins and confering specific biological activities to the multilayers. Acknowledgment. This work was supported by the program “Adhe´sion Cellules-Mate´riaux”. It was performed within the framework of the CNRS/INSERM Research Network “Me´canismes physico-chimiques d’adhe´sion cellulaire: forces d’adhe´sion entre ligands et re´cepteurs biologiques”. P.S. thanks the Institut Universitaire de France for financial support. Appendix. Optical Invariants as a Complementary Tool To Analyze the Structure of Thin Films We have used a simple one-layer model to describe the optical properties of the polyelectrolyte multilayers (PEM) and a two-layer model to analyze the polyelectrolyte multilayer/protein films. The PEM is composed of seven or eight polyelectrolyte sublayers. In principle, one could imagine to characterize such a PEM film by 14 or 16 optical parameters (seven or eight refractive indexes and seven or eight thicknesses). In scanning angle reflectometry a maxi-

Ladam et al.

mum of only three optical parameters can be determined. The determination of the 14 or 16 optical parameters is thus impossible unless one determines the parameters during the build up process. One has then to assume that the parameters characterizing each polyelectrolyte layer do not change when an additional polyelectrolyte layer is adsorbed on it. This should, however, be a very crude approximation. Fortunately a PEM with up to seven or eight sublayers behaves optically almost as a monolayer. We will demonstrate this property by analyzing the optical data within the framework of optical invariants. We will, in particular, make use of the invariant F, first introduced by Heinrich et al.,33 which is equal to 0 for a homogeneous and isotropic monolayer and deviates from 0 when this approximation is not verified. Moreover, we will show that F deviates significantly from 0 when albumin is adsorbed on the PEM. This indicates that in this case the film does not behave any longer as a homogeneous and isotropic monolayer and the simplest model that one can think of to describe such a film is then the bilayer model. Usually, one analyzes optical data relative to thin films by assuming a refractive index profile. It would, however, be of interest to characterize the adsorbed film by parameters which are independent of the model used to analyze the experimental data and which do not depend on the optical method used for the investigation. Such parameters exist and are called the optical invariants. The optical invariants are excess quantities in the sense of Gibbs. These parameters are the ones that characterize optically the interface and that are independent of the location of the Gibbs dividing surface. This property is at the origin of their name. They have been introduced independently by Lekner34 for stratified films and by Bedeaux and Vlieger35 in their most general form which is valid for any kind of thin interface. The optical invariants emerge out from a (L/λ) expansion of the reflection properties of an interface, L being the characteristic extension of the interface and λ the wavelength of the light used for the investigation. The optical invariants have, up to now, only been used in very few experimental studies. This could be due to the fact that it is not an easy task to go through the theoretical developments leading to them and that their physical meaning has, up to now, not been related to simple physical pictures. However, we will demonstrate in this appendix that they can be useful in the analysis of the structure of adsorbed layers by optical techniques and in particular to verify the validity of the homogeneous and isotropic monolayer model. For an interface of spatial extension L small compared to the wavelength of light, the intensity reflection coefficient for p waves can be written in the general form34 Rp ) rp02 -

{[

8Q1Q2(2 - 1)k02 12(Q1 + Q2)

4

[ ( J23 +

1 - 1 sin2 θ1

)]

]

1 + 2 × 12

}

12 sin4 θ1 12 - 1 sin2 θ1 J22 J2 1 + 2 212(1 - 2) 1 (A1)

where k0 ) 2π/λ. rp0 corresponds to the Fresnel amplitude reflection coefficient in the absence of an adsorbed film. The



dielectric constant (z) by J1 ) k0 J21 )

∫-∞+∞

[(z) - 1][(z) - 2] (z)

dz

(A3a)

k02 2(1

+∞ +∞ dz1 ∫-∞ dz2[(z1) ∫ -∞ - ) 2

0(z1 - z2)][(z2) - 0(z2 - z1)] (A3b) J22 ) Figure 5. Evolution of the invariant F during the build-up process of a PEI-(PSS-PAH)5 polyelectrolyte multilayer in the presence of 0.15 M NaCl. Layer 0 is the initial PEI layer, layer 1 is the first PSS layer, layer 2 is the first PAH layer, etc.

index 1 (respectively 2) has been chosen for the incidence (respectively transmission) medium, i representing the relative dielectric constant of the medium i. The dielectric constant i is related to the refractive index ni by i ) ni2 and Qi is given by Qi ) k0 cos θi/i

1/2

(A2)

The parameters J1, J22, and J23 are so-called optical invariants. Expression A1 is valid for stratified interfaces and rough surfaces and even for interfaces constituted of deposited particles on a substrate as long as the spatial extension of the interface is small compared to λ. For stratified interfaces, Lekner34 and Bedeaux and Vlieger35 have independently shown that the three optical invariants can be related to the

k02 (1 - 2)

{ [

∫-∞+∞ dz1 ∫-∞+∞ dz2 0(z1 - z2)]

0(z2 - z1)[(z1) -

1 1 (z2) 0(z2 - z1)

]}

(A3c)

and 12 1 + 2

(A3d)

for z < 0 0(z) ) 1 2 for z > 0

(A3e)

J23 ) J21 - J22 with

{

One can notice that J1 is an invariant which is of first order in L/λ whereas J21 and J22 and thus also J23 are secondorder invariants. However, all the invariants appear in the expression of Rp at the same order (L/λ)2 because Rp is a function of J12 and not of J1. This also implies that SAR allows only to get access to |J1| and not directly to J1. One can also notice that when an interface can be defined as the

Figure 6. Typical evolution of the invariants J1 (a and c) and F (b and d) during adsorption of HSA onto PAH/PSS films in the presence of 0.15 M NaCl and at pH 7.35, and during the rinsing process with a solution of same ionic strength as used before. Key: (a and b) HSA is adsorbed from a solution of concentration 11.6 mg/100 mL onto a PEI-(PSS-PAH)3 film; (c and d) HSA is adsorbed from a solution of concentration 18.2 mg/100 mL onto a PEI-(PSS-PAH)3-PSS film. t ) 0 corresponds to the injection of the last polyelectrolyte solution. The full and the dotted arrows indicate respectively the injection of the HSA solution and the beginning of the rinsing process.

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Ladam et al.

Figure 7. Typical evolution of the fit quality parameter χ2 relative (b) to the monolayer model, (O) to the bilayer model, and (0) to expression A1 using the invariants. Key: (a) adsorption of HSA from a solution of concentration 11.6 mg/100 mL onto a PEI-(PSS-PAH)3 film; (b) adsorption of HSA from a solution of concentration 18.2 mg/100 mL onto a PEI-(PSS-PAH)3-PSS film. Arrows are as in Figure 6.

superposition of several constituents, as is the case for stratified interfaces, the invariant J1 is the sum of the individual contributions corresponding to each constituent. Moreover, one can show that it is the only invariant playing a role in the optical properties of interfaces that bears this property. The expressions of these invariants have been evaluated for different kinds of refractive index profiles and in particular for a homogeneous and isotropic monolayer profile. Because the three invariants are then only functions of the layer thickness and of its refractive index, a relation independent of these parameters must exist between the three invariants J1, J22, and J23, which is characteristic for the monolayer profile. This relation led Heinrich et al.33 to introduce a new invariant denoted as F, which is equal to zero when the refractive index profile of the interface is identical to a monolayer profile. The expression of F is given by F)1-

{

}{

}

1(1 - 2) 2(1 - 2) J22 2J23 + J 1 + 2 1 + 2 22 12 2J12 J23 + J 1 + 2 22

(1 - 2) 2J23 -

{

}

(A4) We followed the buildup process of the multilayers by SAR and after the deposition of each polyelectrolyte layer followed by extensive rinsing with a solution similar to that serving

to prepare the polyelectrolyte solution, we determined the corresponding reflectivity curve. These curves were analyzed by using the invariant description. Figure 5 represents the evolutions of F for a typical buildup process of a PSS/PAH multilayer in a 0.15 M NaCl solution. One can notice that the data relative to the first three layers are scattered. This is general and comes from the fact that the reflectivity curves relative to these layers were almost indistinguishable from the Fresnel curves indicating that the first three polyelectrolyte layers were almost undetectable by SAR. One observes a decrease of F which starts from a positive value and finally stabilizes near 0 after the deposition of four bilayers (4PAH + 4PSS layers). This indicates that thin polyelectrolyte multilayers do not fully behave optically as homogeneous and isotropic monolayers but that it is only after four bilayers that such an approximation for the refractive index profile becomes correct. The fact that multilayers with a small number of layers do not exactly follow the homogeneous and isotropic monolayer model strengthens our previous interpretation of the multilayer structure in three main regions.4 For small multilayers, the outer region III, in which the refractive index varies continuously from its region II value to its value in the solution, should be dominant. Figure 6 shows a typical evolution of the invariants J1 and F when an albumin solution is brought in contact with multilayers terminating with PSS and PAH. One can first notice that the value of J1 is decreasing during the adsorption processes. According to expression A3a for J1, this negative contribution of the adsorbed HSA layer to J1 necessarily originates from the fact that its refractive index is comprised between the refractive index of silica n1 (1.45718) and the refractive index of the solution n2 (about 1.333). It is thus quite different from the index of refraction of the PEM (about 1.490). Moreover, it comes out that, once the proteins adsorb on the multilayer, the value of F decreases strongly. This clearly demonstrates that such a PEM/protein film cannot be analyzed with the monolayer model. We have also analyzed these data with the monolayer and with the bilayer models. In this latter case we have fixed the thickness and the refractive index of the underlying polyelectrolyte multilayer to their values determined with the monolayer model before protein adsorption, the fitting parameters being the thickness and the refractive index of the protein layer. Figure 7 shows the evolution of the fit quality parameter χ2 relative to the monolayer and the bilayer models and to expression A1 using the invariants. The fact that χ2 is smaller for the bilayer than for the monolayer proves that the second model represents a better approximation for our multilayer/protein film. However, the even smaller value of χ2 relative to the use of expression A1 also shows that the bilayer model does not fully account for the structure of the film and that it still constitutes only a first approximation. References and Notes (1) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210/ 211, 831. (2) Decher, G. Science 1997, 277, 1232. (3) Caruso, F.; Mo¨hwald, H. J. Am. Chem. Soc. 1999, 121, 6039.

Interactions with Polyelectrolyte Multilayers (4) Ladam, G.; Schaad, P.; Voegel, J. C.; Schaaf, P.; Decher, G.; Cuisinier, F. Langmuir 2000, 16, 1249. (5) Joanny, J. F. Eur. Phys. J. B 1999, 9, 117. (6) Ichinose, I.; Fujiyoshi, K.; Mizuki, S.; Lvov, Y.; Kunitake, T. Chem. Lett. 1996, 257. (7) Cassagneau, T.; Mallouk, T. E.; Fendler, J. H. J. Am. Chem. Soc. 1998, 120, 7848. (8) Caruso, F.; Mo¨hwald, H. Langmuir 1999, 15, 8276. (9) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195. (10) Ariga, K.; Lvov, Y.; Ichinoze, I.; Kunitake, T. Appl. Clay Sci. 1999, 15, 137. (11) Keller, S. W.; Kim, H.-N.; Mallouk, T. E. J. Am. Chem. Soc. 1994, 116, 8817. (12) Lvov, Y.; Ariga, K.; Ichinoze, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (13) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3427. (14) Younson, J. S.; Hikmat, B. Y.; Todryk, S. M.; Czisch, M.; Haris, P. I.; Flindall, I. R.; Newby, C.; Mallet, A. I.; Ma, J. K. C.; Lehner, T.; Kelly, C. G. Nat. Biotech. 1999, 17, 42. (15) Caruso, F.; Furlong, D. N.; Ariga, K.; Ichinoze, I.; Kunitake, T. Langmuir 1998, 14, 4559. (16) Graul, T.; Schlenoff, J. B. Anal. Chem. 1999, 71, 4007. (17) Cohn, E. J.; Strong, L. E.; Hughes, W. L.; Mulford, D. J.; Ashworth, J. N.; Melin, M.; Taylor, H. L. J. Am. Chem. Soc. 1946, 68, 459. (18) Cohn, E. J.; Hughes, W. L.; Weare, J. H. J. Am. Chem. Soc. 1947, 69, 1753. (19) Schaaf, P.; De´jardin, P.; Schmitt, A. Langmuir 1987, 3, 1131.

Biomacromolecules, Vol. 1, No. 4, 2000 687 (20) Azzam, R. M. A.; Bashara, N. H. Ellipsometry and polarized light; North-Holland: Amsterdam, 1977. (21) Mann, E. K.; Heinrich, L.; Voegel, J. C.; Schaaf, P. J. Chem. Phys. 1996, 105, 6082. (22) Abele`s, F. Ann. Phys. 1950, 5, 596. (23) Haynes, C. A.; Norde, W. Colloids Surf. B: Biointerfaces 1994, 2, 517. (24) Peters, T. AdV. Protein Chem. 1985, 37, 161. (25) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153. (26) Sukhishvili, S. A.; Granick, S. J. Chem. Phys. 2000, 110, 10153. (27) De Feijter, J. A.; Benjons, J.; Veer, F. A.; Biopolymers 1978, 17, 1759. (28) Schaaf, P.; Voegel, J. C.; Senger, B. Ann. Phys. Fr. 1998, 23 (6), 1. (29) Hlady, V.; Fu¨redi-Milhofer, H. J. Colloid Interface Sci. 1979, 69, 460. (30) Hemmerle´, J.; Altmann, S. M.; Maaloum, M.; Ho¨rber, J. K. H.; Heinrich, L.; Voegel, J. C.; Schaaf, P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6705. (31) Meechai, N.; Jamieson, A. M.; Blackwell, J. J. Colloid Interface Sci. 1999, 218, 167. (32) Takahashi, D.; Kubota, Y.; Kokai, K.; Izumi, T.; Hirata, M.; Kokufuta, E. Langmuir 2000, 16, 3133. (33) Heinrich, L.; Mann, E. K.; Voegel, J. C.; Schaaf, P. Langmuir 1996, 13, 3177. (34) Lekner, J. Theory of Reflection; Martinus Nijhoff Publishers: Dordrecht, The Netherlands, 1987. (35) Bedeaux, D.; Vlieger, J. Physica A 1973, 67, 55.

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Protein Interactions with Polyelectrolyte Multilayers - American

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