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Characteristic Time Scales for the Adsorption Process of Fibrinogen on Silica P. Schaaf,’ Ph. Dbjardin, A. Johner, and A. Schmitt Institut Charles Sadron (CRM-EAHP), CNRS-ULP, 6 Rue Boussingault, 67083 Strasbourg Cedex, France Received January 28,1991. In Final Form: October 18,1991 We use the technique of scanning angle reflectometry (SAR) to analyze the mechanism of adsorption on a silica surface of dissolved fibrinogen molecules. A series of experiments is performed in which the surface is first exposed to a solution at a lower concentration (0.01% (w/w)) and subsequently replaced with a more concentrated one (0.05% (w/w)). The experimental results complement those obtained in former studies. The elongated fibrinogen molecules are adsorbed with their axis oriented either parallel or normal to the interface, and the proportion of both populations depends on the experimentalpath. Two characteristic times arise from our discussion: one is related to the building up of a layer with molecules lying flat on the surface and is of the order of 5 min; the second time characterizes the formation of an irreversible bond between the highly covered surface and a molecule adsorbed by one end. It is of the order of 1 h. Introduction The adsorption process of fibrinogen on solid surfaces has been extensively studied during the last decade.’-1° The interest in this protein originates from its special importance in blood coagu1ation.l’ Unfortunately, even with the progress of experimental techniques allowingthe study of surface phenomena, the adsorption process of this protein is not fully understood. The reason may be found in the irreversible nature of protein adsorption and, more generally, macromolecular adsorption, which is now a well-established phenomenon.12 This irreversibility appears through different experimental situations. First, it has been shown in step-by-step experiments that the adsorbed amount is a function of the “history” of the process.13 For example, in a single path experiment, an adsorbing surface is directly exposed to a solution at a given final concentration. In a real step-by-step process, the surface is successively put into contact with solutions of increasing concentration for given time intervals, until the fiial concentration is reached. Jonsson et al.14observed that, for hydrophobic surfaces, the adsorbed amount is higher in a single step experiment. This implies that an adsorption isotherm depends on the way it has been determined. It may be qualified as “path-dependent”. (1)Wojciechowski,P.; Ten Hove, P.; Brash, J. J. Colloid Interface Sci. 1986,111,455. (2) Slack, S. M.; Horbett, T. A. J. Colloid Interface Sei. 1988,124,535. (3) Schmitt, A.; Varoqui, R.; Uniyal, S.; Brash, J.; Pusineri, C. J.Colloid Interface Sci. 1983,92, 25. (4) Brash, J.; Uniral, S.; Pusineri, C.; Schmitt, A. J. Colloid Interface Sci. 1983, 95, 28. (5)De Baillou, N.; Dkjardin, Ph.; Schmitt, A.; Brash, J. J. Colloid Interface Sci. 1984,100, 167. (6) Voegel, J.-C.; De Baillou, N.; Sturm, J.: Schmitt, A. Colloids Surf. 1984,10, 9: (7) De Baillou, N.; Voegel, J.-C.; Schmitt, A. Colloids Surf. 1985, 16, 271. (8) Voegel, J.-C.; De Baillou, N.; Schmitt, A. Colloids Surf. 1985, 16, 289. (9) Voegel, J.-C.; Dhjardin, Ph.; Strasser, C.; De Baillou, N.; Schmitt, A. Colloids Surf. 1987, 25, 139. (10) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988, 124, 28. (11) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988,124, 246. (12) Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986, 79, 1. (13) Brynda, E.; Houska, M.; Lednicky, F. J. Colloid Interface Sci. 1986,113, 164. (14) Jonsson, U.; Ivarsson, B.; Lundstrom, I.; Berghem, L. J. Colloid Interface Sci. 1982, 90,148.
Adsorption/desorption experiments using radioactive labeling techniques are well adapted to exploring the irreversibility inherent in a macromolecular adsorption process. It has been observed that only a small percentage of adsorbed fibrinogen desorbs when the interface is exposed to a pure buffer s ~ l u t i o n In . ~ addition, in exchange experiments, where a solution of radiolabeled macromolecules is first brought into contact with a solid/solution interface, and then replaced after a given lapse of time by a solution of nonlabeled molecules, only partial turnover between surface and solution molecules is evidenced.4J5J6 Thus a significant percentage of proteins remains irreversibly anchored to the surface at the time scale of the experimental investigations. It has been conjectured that, as the interfacial residence time increases, conformational or/and orientational changes appear to enhance interfacial linking with the surface or/and, at high coverage, with nearest neighbor molecules. This has partly been investigated and demonstrated using spectroscopic techniques.12 The understanding of partial irreversibility therefore requires the knowledge of the time scales involved in the different mechanisms governing these processes. From radioactive or fluorescence labeling techniques, which are sensitive to the adsorbed amount, it appears that the adsorption process is usually very rapid at early stages (time scale of the order of a minute) whereas it becomes slow when it approaches the ill-defined equilibrium state. This complexphenomenon involves at least three different steps, namely: (i) transport of the molecules from the bulk to the surface by diffusion or diffusion/convection; (ii) adsorption, at a given rate, of dissolvedmacromolecules interacting with the solid surface; (iii) conformational/ orientational changes of the molecules adsorbed on the surface, which may affect their structural, energetic, and kinetic properties. This cannot be analyzed in a simple way without studying the structure of the interface as in scanning angle reflectometry. In Table 1 we summarize the three different types of experiments performed with this technique, including the present one called EII. In a recent article,17we started to investigate this problem by (15) Brash,J.;Uniyal, S.; Samak,Q. M. Trans.-Am. Soc.Artif.Intern.
Organs 1974,20, 69. (16) Brash, J.;
Samak, Q.M. J. Colloid Interface Sci. 1978, 65, 495. (17) Schaaf, P.; Dhjardin, Ph. Colloids Surf. 1988, 31, 89.
0743-746319212408-0514$03.00/0 0 1992 American Chemical Society
Langmuir, Vol. 8, No. 2, 1992 515
Adsorption Process of Fibrinogen on Silica Table I. Different Scanning Angle Reflectometry ExperimentB kinetics at CB = 0.05% (w/w) until a time ts EI where the solution is replaced by a buffer solution measurements performed after washing with buffer solution
E11
E111
this work step by step experiment; first CB = 0.01% time tl, second step CB = 0.05% time t5 measurements performed in presence of solution at time t = t 5 + t1
L
\
equilibrium isotherm showing the critical concentration of 0.01 % measurements performed in presence of solution
using scanning angle reflectometry (SAR) to determine the structure of the adsorbed layer. In those experiments, subsequently noted EI, we were able to identify one characteristic time 7 5 involved in the adsorption process: it is associated with a structural change in the adsorbed layer, when the silica surface is put in contact with a solution a t 0.05% (w/w) for more than 1 h. In this paper, we report another experimental procedure En that will enable us to introduce a different characteristic time 71 involved in the adsorption process; we still use SAR to determine the structure of the interface. We are led to reanalyze the data obtained in our previous study, EI, and to propose-hopefully-a better description of the whole phenomenon, including experiments that have led to an 'optical isotherm".l8 We finally suggest some new experiments which could assess the validity of our tentative explanation.
Materials and Method Protein a n d Buffer Solutions. Throughout the present work we used human fibrinogen, grade L (coagulability > 90%) purchased from Kabi. It was dissolved in a solution of 0.05 M tris(hydroxymethy1)aminomethanebuffer, 0.15 M NaCl and M NaNo adjusted with concentrated HC1to pH 7.35, to approach physiological conditions. The buffer solution was stored at 4 'C. Two fibrinogen solutions of respective concentrations 0.05 % (w/ w) and 0.01% (w/w) were prepared. The 0.05% solution was stored at -20 "C and the 0.01 % solution was obtained from the preceding one by dilution. They were both centrifuged at 17300g for 50 min and stored at room temperature for a maximum of 1 h before the beginning of an experiment. Preparation of t h e Surface. The cell is made of Herasil (fused silica from Heraeus). It i5 cleaned after each experiment with sulfochromicacid during a minimum of 12 h, so as to remove all organic material. Twenty rinses with deionized water (Super Q-Millipore) are subsequently performed. The cell is filled with buffer solution without drying the surface;in this way experiments prove to be highly reproducible.18 Scanning AngleReflectometry. The reflectometryhas been extensively described in a previous pub1icati0n.l~ A schematic representation is given in Figure 1. The light source is a 5-mW He-Ne laser (A = 632 nm), P1 and Pz represent two polarizers (Melles-Griot)with extinction coefficientsof 10". They are both positioned to select the polarization in the plane of incidence ("p" waves). The experimental cell is made of a hemicylindrical Herasil block, optically polished at the plane interface S (Soptel). A microscopic pinhole of 100 pm diameter is fixed in front of the photomultiplier (Hamamatsu) and selects a well-defined angle, with an angular definition of the order of 0.01'. During the experiment, the reflection coefficient around the Brewster angle OB is first measured in the presence of the pure buffer solution. This provides the reference curve. After adsorption the reflection coefficient R, is again determined as (18)Schaaf, P.; Dbjardin, Ph.; Schmitt, A. Langmuir 1987, 3, 1131. (There is an error in the abscissa label of Figure 5 in ref 18. It should
be 103 c(%w/w)instead of lo2 c(%w/w).) (19) Schaaf, P.; D6jardin, Ph.; Schmitt, A. Reu. Phys. Appl. 1986,21, 741.
Figure 1. Schematic representation of the reflectometer, the lower hemicylinder is made of silica. F represents the thermostated solution while L is a focusing lens. P1 and P2 are two polarizers, PH is a pinhole of 100 pm diameter, PM is a photomultiplier, Mt,, is a microscopic translation device, and Mmt a microscopic rotation device. a function of the incidence angle. From the analysis of both curves, the optical thickness LOand the mean refractive index n within the adsorbed layer can be derived separately with confidence. A more detailed description of the procedure can be found in refs 18 and 19, where the reproducibility of the experimental data has been extensively tested. Adsorption Process. A fibrinogen solutionat a concentration of 0.01 % (w/w) is circulated for 30 s through the adsorption cell. A crude estimation of the shear rate at the wall gives 20 s-l. The solution then remains quiescent. After a time tl, it is replaced at the same flow rate by a solution at 0.05% (w/w), until the recorded signal stays approximately constant. This latter lapse of time is of the order of 2-3 hand it does not depend significantly on tl. The reflected intensity is then measured as a function of the incidence angle around OB, in the presence of the solution. In ow previousworklSwe showed that the presence of the solution does not affect the reflectivity coefficient, which means that the method is sensitive only to the adsorbed layer.
Results and Discussion Before we describe the experimental results, it seems important to discuss the reasons for the choice of this particular adsorption process. In studyingthe same system previously, we determined in a previous work EIII,an optical isotherm,18 by SAR (Table I). The experiment was performed by exposing the surface to solutions at different concentrations and by measuring the optical parameters of the adsorbed layer in the presence of the solution, once quasi-equilibrium was reached (usually after 3 h of surface-solution contact). The results are s u m marized in Figure 2. I t is obvious from this figure that 0.01% is a concentration threshold separating two adsorption domains. For the lower concentrations the thickness Lo is independent of the concentration, whereas for higher bulk concentrations, CB,the thickness increases with CB. Moreover, the value of LOtends to indicate that a large percentage of the adsorbed molecules is in a "sideon" configuration. This means that for solution concentrations lower than 0.01% , the macromolecules arriving at the surface have statistically enough room and time available to adopt this configuration, whereas for higher concentrations it is no longer t h e case, so that more and more surface-bound protein molecules stay at the interface in an "end-on" configuration. Figure 3 shows, for the present EII experiments, the evolution of the optical thickness LO and of the mean refractive index n characterizing the interfacial layer with the contact time tl (for the definition of the different times, see Table 11) between the surface and the solution at concentration 0.01 % . For low tl values, the state of the surface is such that when it is subsequently exposed to the
Schaaf et al.
516 Langmuir, Vol.8,No.2, 1992 1 . 5 1 A n x 102
Upon gathering the results corresponding to series EI, EII, and EIII, can we propose an adsorption mechanism describing what is observed? This question is explored in the following discussion, which involves the comparison of different characteristic times governing the process. Let us first consider the adsorption time 7, defined by
/ L.1"~
0.6
loq I
- 30
c,l%
1
I
I
- 25
-20
-15
w/wl
f 0
Figure 2. Optical isotherm: variation of the mean optical thickness LO(-) and the mean refractive index (- -) within the adsorbed fibrinogen layer as a function of the equilibrium solution concentration, in decimal logarithmic scale. The corresponding adsorbed amount may be found in ref 18. _ _ _ _ - - - - - _2
0
3
< - -
- 2.2
/ #
"5 /
- 2.1 - 2.0
.I
.\
e----.-
1.6 I
5
I
10
t,lmnl
15
Figure 3. Variation of the mean optical thickness (+) and the within the adsorbed fibrinogen layer mean refractive index (0) as a function of the contact time tl between the protein solution at a bulk concentration of 0.01 7% (w/w) and the silica surface. After a time t l this solution is replaced by a protein solution at a concentration of 0.05% (w/w).
more concentrated (0.059%) solution, molecules adsorbing on it build up an interfacial layer of complex structure; this layer is composed of two populations having parallel and normal orientations with respect to the surface, as observed in the EIII isotherm, above the concentration thresh01d.l~This is evidenced by both high layer thickness LOand small mean refractive index n. As soon as tl exceeds a characteristic time 71 of about 5 min, the model of a simple monolayer with a population of elongated molecules adsorbed flat on the surface prevails, after quasiequilibrium is reached. These experimental facts complement those of series EI, where we brought the surface into contact with a solution at 0.05% for different residence times t 5 . The adsorption procedure was thereafter stopped at t 5 by replacing the fibrinogen solution with a pure buffer solution. The results may, from a qualitative standpoint, be compared to those represented in Figure 2, the solution concentration CB on the abscissa being simply replaced by the residence time t 5 . The characteristic time 7 5 at which the thickness starts to increase is of the order of 1h. Thus, for residence times t 5 < 75, the optical thickness Lo is indeed of the order of 100A, that is, close to the lateral dimension of the fibrinogen molecule, which corroborates the model with molecules adsorbed "side-on". For t 5 > ~ 5 the , layer structure is complex.
where 0 represents the probability that a particle arriving randomly on the surface finds space to adsorb. 7, depends both on the interfacial bulk concentration cb(0) and on the surface coverage r (which itself increases with the surface/solution exposure time). If we refer to the simple Langmuir kinetic equation, 7, is given by
k, is the adsorption rate constant and rmm the saturation surface concentration for the adsorbed molecules. It is clear that 7, decreases with the bulk concentration (which might be higher than Cb(0)if there was interfacial depletion) while it increases with I", when adsorption proceeds. When fibrinogen molecules diffuse to the interface, they can adsorb "end-on" or "side-on", which is of course a crude description.12 Let us adopt the following working rules: (i) two molecules never overlap; (ii) molecules adsorbed in a %de-on" configuration are fixed irreversibly to the surface and cannot desorb; (iii) when attached in an "endon" state, molecules tilt to a "side-on" configuration if there is both enough time and surface available, the characteristic tilting time being 7t which increases with I'; (iv) if this transition is not possible, because of steric hindrance, fibrinogen molecules that are adsorbed "endon" may be stabilized on the surface, to become attached irreversibly on it, but the characteristic sticking time 78 associated with this phenomenon is rather long. It is related to the adsorption and desorption time in a way which we deem to be fairly complicated. It should, in particular, also be a function of the history of the process. A tentative interpretation covering all our results runs as follows. In the experiment EIIreported here, when the surface is first exposed to a solution of concentration 0.01 % ,we always have 7t < T,, which is confirmed by the optical adsorption isotherm in series EIII. Therefore, the layer contains a high percentage of molecule attached flat on the surface at any time. Yet, what EIItells us is that, for a bulk concentration of 0.01 % after a time 71 'Y 5 min, this layer is almost saturated and irreversibly bound to the surface, so that subsequent exposure to more concentrated solution does not affect its structure anymore. Thus, the value 71 has a double meaning. I t is first an upper boundary value for 7t at zero coverage, and it is also the minimum time necessary, under this experimental condition, to build up an homogeneous layer with molecules irreversibly attached parallel to the surface. It must be pointed out that 71 is a function of the adsorption time T,(@(F=O))and thus of the bulk concentration. Indeed, for larger values of T a ( @ ( r = O ) , it takes a longer time to build up such a layer so that 71 must increase with it. On the other hand, from experiment EIII,it can be shown that if ra(@(r=O)) is smaller than its value corresponding to a concentration of 0.01%) then there is no longer the possibility of building up a layer where the molecules are almost all in the "side-on" configuration. There is at any time a nonnegligible percentage of molecules adsorbed 'end-on". If we now refer to the data revealed by EI experiments, where the solution is directly brought into contact with a solution at 0.05% the process of adsorption starts again
Adsorption Process of Fibrinogen on Silica tl
Langmuir, Vol. 8, No. 2, 1992 517
Table 11. Different Times Involved in the Adsorption Mechanism of Fibrinogen contact time between the solution at CB = 0.01 % and the surface
t5
contact time between the solution at CB = 0.05% and the surface
71
characteristic time necessary to build up a saturated simple monolayer with the molecules adsorbed side-on, CB being 0.01%
75
characteristic time necessary to build up a saturated simple monolayer with the molecules adsorbed side-on, CB being 0.05%
. 7
characteristic adsorption time, it is a function of CB
7t
characteristic ‘tilting time”: mean time necessary for the molecules to change from an “end-on” to a “side-on” configuration; a function of the surface coverage
78
characteristic “sticking time”: mean time for molecules adsorbed in an ”end-on”configuration to be anchored irreversibly on the surface
with the inequality rt < ra. However, rt increases more rapidly than 7g. Consequently, as in the preceding case, a significant percentage of adsorbed molecules cannot undergo the “end-on” to “side-on” transition because of steric surface exclusion, and therefore remains in the “endon” configuration. If the rinsing of the surface with the buffer solution after a time t5 corresponds to t 5 < rg,the macromolecules loosely attached by one end only are largely desorbed, so that the layer thickness corresponds to adsorption in the parallel state. Conversely, if t 5 > rs, stabilization of the “end-on” adsorption becomes more and more efficient, through surface/molecule and/or molecule/molecule links. This view is corroborated by the observation of significant desorption upon rinsing at low t5,and vice versa.l7 A complex structure of the adsorbed layer therefore emerges at high t 5 . The present discussion and description may be related to recent simulations concerning the kinetics of a random sequential adsorption (RSA) process of ellipses.20 An RSA process is defined by three rules: (i) the particles adsorb sequentially and arrive randomly at the surface; (ii) once a particle is adsorbed it cannot diffuse nor desorb from it; (iii) the particles can never overlap. I t has been shown that, so long as the coverage is lower than 30-35 % , the particles find room to adsorb side-on, whereas for higher concentrations, we are in the asymptotic kinetic regime where a probability of side-on adsorption becomes smaller. What these studies also show is that the adsorption kinetics becomes very slow as the “jamming limit” is approached. Since fibrinogen also possesses two “a-chains” possibly dangling into the solution as the molecule is adsorbed, this provides an additional mechanism which slows down the building up of an interfacial layer.21 The foregoing description seems to apply rather well to some aspects of our former discussion, and it probably implies that the so-called “equilibrium” value for r, obtained after 3-4 h of surface/solution contact, does not correspond to a true equilibrium. For example, if our conjectures are verified, one should be able to saturate the interfacial layer with moleculesattached side-on in adsorption experiments with (20) Talbot, J.; Tarjus, G.; Schaaf, P. Phys. Reu. A: Gen. Phys. 1989, 40, 4808. (21) Johner, A.; Joanny, J. F. Macromolecules 1990, 25, 5299.
bulk concentrations lower than 0.01 % ,but the adsorption time would have to be increased significantly. Recent experiments dealing with the adsorption of albumin (elongated molecules) to hydroxyapatite tend to give credit to such a conjecture.22 In the foregoing discussion, possible conformational changes of the fibrinogen molecule in the adsorbed state have been ignored. Of course, the introduction of T ~ a, characteristic sticking time for molecules adsorbed in the “end-on” orientation implies probably a local transconformation, but it only affects part of the molecule. A more global denaturation should result in a molecular spreading on the surface, that is, a reduction of the layer thickness, as observed on surfaces of metals or metal oxides.lB
Conclusion To gain a better understanding of the molecular mechanism describing fibrinogen adsorption on a bare silica surface, we have used the technique of scanning angle reflectometry. To explain the experimental observations collected both in this paper and in previous publications, we propose a crude model involving two states of adsorption (“side-on”and “end-on”) and two characteristic times, for the adsorption of the elongated fibrinogen molecule. Since the scanning procedure is not yet automated, the adsorption process could only be followed step by step. Once it becomes possible to determine in situ, as a function of time, the variation of optical (mean refractive index) and structural (mean thickness) parameters, a significant leap will be made in the comprehension of such complex interfacial processes. In addition, it is clear that real time spectroscopictechniques could provide additional evidence for some hypotheses concerning the mechanism of surface attachment. Acknowledgment. We are grateful for financial support from the INSERM under the contract “Application de la reflectometrie B deux processus interfaciaux d’intkrht biologique: reaction antig&nes/anticorpset dissolution de l’bmail dentaire”, No. 899016. Registry No. Fused silica, 60676-86-0. (22) Mura-Galelli, M. J.; Voegel, J.-C.; Behr, S.; Bres, E. F.; Schaaf, P.R o c . Natl. Acad. Sci. U.S.A. 1991,88, 5557.
