analysis of hen egg white lysozyme adsorption on si(ti)o2 ∣ aqueous solution interfaces at low...

Colloids and Surfaces B: Biointerfaces 17 (2000) 81–94

Analysis of hen egg white lysozyme adsorption onSi(Ti)O2 � aqueous solution interfaces at low ionic strength: a

biphasic reaction related to solution self-association

Vincent Ball *, Jeremy J. RamsdenBiocenter, Department of Biophysical Chemistry, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

Received 12 November 1998; accepted 26 May 1999

Abstract

Adsorption kinetics of hen-egg white lysozyme at concentrations of 10−7, 10−6, 10−4 M and at pH 8.0 have beenmeasured on Si0.8Ti0.2O2 surfaces by means of optical waveguide lightmode spectroscopy and were found to proceedin two distinct kinetic regimes: a fast and quasi linear increase of the surface concentration followed by slower kineticsthat could be described qualitatively by the theory of random sequential adsorption. The transition between these twokinetic regimes was correlated with abrupt changes in the values of the mean refractive index and of the thickness ofthe adsorbed layer as well as in the desorption kinetics performed either before or after the transition from the firstregime to the second. Experiments carried out at the same ionic strength but at different pH values showed that theadsorption behaviour of lysozyme is related to its self-association properties in highly concentrated solutions. © 2000Elsevier Science B.V. All rights reserved.

Keywords: Hen-egg white lysozyme; Adsorption; Optical waveguide lightmode spectroscopy; pH dependence of surface aggregation

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1. Introduction

Hen-egg white lysozyme (HEWL) is one of themodel proteins often chosen to study adsorptionmechanisms of proteins at solid � liquid [1–7,15]or liquid � gas interfaces [8,9]. The reasons for thischoice are the following:

1. the three dimensional structure of the proteinis known at high resolution [10] and

2. the high availability at low cost of the nativeprotein in a relatively pure form. Although wemake no use of them in this work, we mightalso mention the possibility of producing sin-gle point mutants in order to study the influ-ence of the protein stability in the adsorptionbehaviour [11], and of modifying the surfacecharge density by chemical modification, forinstance succinylation [12].

It has been found that electrostatic interactionsare one of the major driving forces for HEWL

* Corresponding author. Present address: Laboratoire deSpectrometrie de Masse Bio-organique, CNRS UMR 75-09,Institut de Chimie, Universite Louis Pasteur. 67008 StrasbourgCedex, France. Tel.: +33-3-88416887; fax: +33-3-88604687.

E-mail address: [email protected] (V. Ball)

0927-7765/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S0927 -7765 (99 )00085 -5

V. Ball, J.J. Ramsden / Colloids and Surfaces B: Biointerfaces 17 (2000) 81–9482

adsorption at solid � liquid interfaces [2,4,5], butdehydration of hydrophobic patches at both theprotein and the sorbent surface can also play arole, as well as the associated structural modifica-tions in the protein secondary and/or tertiarystructure [14]. Indeed, adsorption of succinylatedlysozyme (isoelectric point between 5.9 and 7.4)can take place even in the presence of repulsiveelectrostatic forces: this behaviour has been at-tributed to structural destabilization [12].

Circular dichroism spectra have shown that theprotein retains almost all its secondary structureat the plateau level of adsorption to glass from 10mM phosphate buffer at pH 7 as inferred aftermorpholine-induced desorption [13].

All these data are consistent with the phe-nomenological description of protein adsorption[14] which describes the adsorption process as asynergetic contribution of electrostatic interac-tions (including counterion binding to theprotein), hydrophobic dehydration of both theprotein and the sorbent surface and structuralmodifications of the adsorbed protein. But thecurrently observed multilayer formation ofHEWL not only on hydrophilic surfaces [5] butalso on hydrophobic ones [3] has been only poorlydescribed either from a thermodynamic or a ki-netic point of view. This multilayer formation canbe related to a gelation-like phenomenon as hasbeen demonstrated by means of attenuated totalreflection Fourier transform infrared spectroscopyafter adsorption onto silicon oxide from D2Osolutions of various pD and in the presence of 0.1M NaCl [16].

It has been emphasized that the inference ofmultilayer growth at the surface from merely ana-lyzing the adsorbed amount is problematical be-cause the maximum surface concentration in amonolayer depends strongly on the adsorptionmechanism: for spherical particles a monolayer isjammed at a coverage of about 55% produced byrandom sequential adsorption process, comparedwith 91% for a hexagonally close packed mono-layer. This single consideration already introducesa factor of 1.65 in the surface concentration,neglecting the roughness or microheterogeneity ofthe adsorbent which defines the true area avail-able to the protein.

It is the aim of this paper to define and discussthe validity of criteria allowing the differentiationbetween the formation of a monolayer and ofmultilayers of adsorbed HEWL. The energetics ofadsorption where varied by changing the solutionpH, the adsorption kinetics were investigated atdifferent bulk concentrations of HEWL and weanalysed the desorption of the adsorbed proteinsat different surface coverages. The average thick-ness of the adsorbed layer was used to distinguishbetween monolayer and possible multilayer for-mation. Finally, we attempted to relate the forma-tion of the adsorbed layer on the solid surface tothe well known self-assembly of HEWL in bulk athigh concentrations. The surface concentration ofadsorbed proteins was measured by means ofoptical waveguide lightmode spectroscopy(OWLS) which allows quantification of adsorbatewith a detection limit of 1 ng cm−2 [17]. Ourexperiments were carried out on mixed Si0.8Ti0.2O2

surfaces. Such surfaces are important as biomate-rials: protein layers deposited on them are be-lieved to determine their biointegration [18].

2. Experimental

2.1. Buffers and chemicals

In a first series of experiments, (Type a) westudied the adsorption kinetics at different proteinconcentrations at pH 8.0 at a low ionic strengthof 10−2 M. The reason for this choice was towork in conditions where HEWL is prone toaggregate at high concentrations [19] and thusprima facie in the adsorbed state, without doingso in the solution used for adsorption. At pH 6.8[19] or 7 (0.2 M ionic strength) [20], at the highestconcentration we used (10−4 M or 1.43 mg cm−3)HEWL shows no significant self-association. Theprotein was dissolved in 10 mM carbonate buffer(an equimolar mixture of NH4HCO3 andNH4CO2NH2, Prolabo), pH 8.090.1. As ammo-nium carbonate is volatile, these buffer solutionswere prepared freshly each day just before thebeginning of an adsorption experiment. They weredeaerated in an ultrasonic bath to prevent airbubbles occurring in the adsorption cell.

V. Ball, J.J. Ramsden / Colloids and Surfaces B: Biointerfaces 17 (2000) 81–94 83

The 1:1 electrolytes present in the carbonatebuffer are fully dissociated at this pH and thetotal ionic strength is thus 10−2 M, implying aDebye screening length LD of 3.04 nm. Note thatat pH 8 and at a concentration of 10−4 MHEWL, taking about 7 net positive charges perprotein [21,22], the protein and its bound counte-rions itself increases the ionic strength by 6.5%resulting in a 3% decrease in LD.

In a second set of experiments (Type b) aimedat investigating the pH effect on adsorbed layerformation, the HEWL was dissolved at 10−4 Min 10−2 M NaCl and the pH was adjusted to thedesired value by means of 1 M HCl or NaOHsolutions. The pH of the protein solutions waschecked by means of an Inlab 422 electrode (Met-tler Toledo, Greifensee, Switzerland); values were4.5, 7.4 and 8.9. Even at pH 4.5 the contributionof HEWL to the Debye screening length is small.

2.2. Protein solutions

HEWL was purchased from Sigma (L-6876, lot65H7025) and used without further purification.It has been shown that the Sigma product con-tains about 1% of protein contaminants (w/w)[23], and we confirmed this result by means ofLC-MS (HPLC and electrospray mass spectrome-try, VG BioQ, Micromass) using standard proce-dures [24].

Circular dichroism spectra in the 200–240 nmregion, were recorded with a Jasco J-720 spectro-polarimeter in 10−2 M carbonate buffer and in10−2 M Hepes buffer (pH 7.4) plus 10−2 MNaCl. The protein concentration was 100 mgcm−3 and the optical path length in the quartzcell was 0.1 cm. Spectra were averaged over fiveruns and corrected by subtracting buffer baselines(Fig. 1). Note that the Hepes–NaCl buffer itselfabsorbs significantly which lowers the accuracy ofthe corrected spectrum and may be the origin ofthe slight deviations between the two signals inthe 200–215 nm wavelength range. The spectrawere compared with published data [25] and cor-responded to that expected for the native protein.

In the carbonate buffer, a refractive index incre-ment (dn/dc), value of (0.212 9 0.005 cm3 g−1)was measured for HEWL by means of Rayleigh

interferometry (LI3 interferometer, Carl Zeiss,Iena, Germany). In the 10−2 M NaCl solutionswe used a dn/dc value of 0.186 cm3 g−1 [26].

The concentrated (about 10−3 M) protein solu-tions used for the adsorption experiments wereprepared by weighing shortly before the beginningof an experiment, diluted to the desired concen-tration about 10 min later and injected through aMinisart membrane (Sartorius) into the adsorp-tion cell. The concentrations of the injectedprotein solutions were checked by UV-vis spec-troscopy (Uvikon 860, Kontron Instruments) at280 nm using an extinction coefficient of 2.6 cm2

mg−1.(i.e. 3.65×104 M−1 cm−1). Agreement be-tween the weighing and absorption spectroscopywas better than 3 and 5% at concentrations of10−7 and 10−4 M, respectively.

2.3. Surface cleaning and characterization

The materials with which adsorption was per-formed were made of a Schott DESAG AF 45glass substrate (48×16×0.5 mm3) of refractiveindex 1.52578. These glass chips (Mikrovakuum,Budapest, Hungary) were covered by a thin(about 200 nm thick) layer of high refractiveindex oxide using the sol–gel method. This oxide

Fig. 1. Mean residue ellipticity (deg cm2 dmol−1) of HEWL inammonium carbonate buffer (thick solid line) and in 10−2 MHepes buffer with 10−2 M NaCl at pH 7.4 (dotted line).


Table 1Surface potentials in presence of the different buffer solutions, calculated according to the surface ionization group model [28]

cS/mV aSurface pHSbBuffer pH cp/mV c cpcS/10−3

mV2 d

−271 4.328.9 54.9 −14.98.020% TiO2+80% SiO2 −227 4.16 63.8 −14.5

−199 4.037.4 71.8 −14.3−59 3.50 91.34.5 −5.4

a Surface potential calculated for the surface in the absence of adsorbed proteins, by assuming an intimate mixing of SiO2 andTiO2. Parameters for the calculation can be found in Ref. [44].

b pH value close to the surface, obtained from cS according to: pHS=pH+(ecS)/(2.303kT) [28], where e is the elementary charge,k the Bolzmann constant and T the absolute temperature, (298K on average, Table 2).

c Surface potential of the HEWL molecule, using an hydrodynamic radius of 1.72 nm, calculated from the dimensions of theprolate ellipsoid, and using the theory of Winterhalter and Helfrich [45], giving the surface potential of a sphere carrying a surfacecharge density s.

d The product of cp and cs is proportional to the contribution of the electrostatic interactions to the free energy of the system[46] at the beginning of the adsorption experiment, when no protein is present on the surface.

layer was centrally embossed with a grating of1.1×16 mm2 and a line spacing of 417 nm, andsubsequently pyrolyzed yielding a compactSi0.8Ti0.2O2 film. Raman spectroscopy experimentsshowed that the TiO2 is present mainly in theanatase form [27].

As shown in Table 1, the SiO2 makes a strongcontribution to the negative surface charge den-sity and surface potentials at acidic pH, as calcu-lated from the surface ionization group model[28]. These data should be interpreted qualita-tively, because the calculations neglect the possi-bility of specific ion adsorption, which cannot bedisregarded in the case of large polarizable cationssuch as NH4

+.The mean average roughness of these oxide

layers was measured in air by means of atomicforce microscopy (Nanoscope III operated in con-stant force mode, Digital instruments, Santa Bar-bara, USA) and found to be 0.890.1 nm.

The chips were cleaned for 10 min in 10 mMhot SDS, followed by rinsing in Nanopure water(resistivity\17.9 MV cm) and further cleaned inhot 0.2 M HCl for 10 min, followed again byrinsing in Nanopure water. Before use, the chipswere equilibrated in the carbonate buffer or in the10−2 M NaCl solution for at least 2 h, in order toreach a stable optical response (Fig. 2), i.e. untilsteady effective refractive indices were obtained(we actually required the N(TE) drift to be lowerthan 10−6 min−1).

The chips were then qualitatively checked to behydrophilic by spreading buffer on their surface.Further surface characterization, including thecomparison of different cleaning methods and

Fig. 2. Variation of the measured apparent refractive index forthe zeroth order mode propagating in the waveguide put incontact with water and carbonate buffer (after the arrow). Theinset represents the variation of the measured signal, propagat-ing to the right photodiode (+sign) during the last 1000 spreceding the protein solution injection. The mean drift duringthis period was 0.9×10−6 min−1, i.e. lower than the preci-sion of a single measurement.


X-ray photoelectron spectroscopy will be pre-sented in a forthcoming work.

Chips were then mounted in the goniometerhousing of an IOS-1 integrated optical scanner(Artificial Sensing Instruments, Zurich). The ad-sorption cell was tightly sealed to the chip by acircular perfluorinated ‘o’-ring (Kalrez, Dupont)[29]. Buffer was flushed through the cell at a flowrate of 8.86 cm3 h−1, i.e. a wall shear rate of 16.6s−1 and a Reynolds number close to 0.3, corre-sponding to laminar flow. The flow cell was con-nected to a precision syringe pusher (BraunMelsungen) containing the fluids, by Teflon tub-ing about 25 cm long and of 0.8 mm internaldiameter. The lag time between the start of flowat the syringe outlet and the arrival of solution atthe adsorption cell was about 60 s as estimatedfrom switching the water flow to an electrolytesolution and monitoring changes in the effectiverefractive indices. Temperature was followed con-tinuously by means of a Pt-100 resistance embed-ded in the goniometer housing. Within eachexperiment the temperature could be controlled towithin 90.1°C allowing the refractive index ofthe buffer, nC, to be controlled within a precisionof 910−5 [30].

2.4. Optical measurements

OWLS is one of the numerous techniqueswhich relies on total internal reflection of a laserbeam at the interface between two media havingdifferent indices of refraction. Its theoretical prin-ciples have been extensively described elsewhere[17,31].

The effective refractive indices, N(TE) andN(TM), obtained prior to protein adsorptionwere used to calculate the thickness dF and therefractive index nF of the oxide layer (F) by meansof a three layer optical model based on theFresnel equations [31]. These values (Table 2),were then used for calculating of the refractiveindex nA and the thickness dA of the adsorbedlayer (A) as a function of the adsorption time bymeans of a four layer model [31]. With somechips, dF decreased significantly after about five toseven experiments. This observation was associ-ated with a systematic increase in the surface

concentration of adsorbed HEWL (data notshown). We attribute this behaviour to progres-sively increasing roughness probably due to sur-face etching by the acid cleaning. Acid treatmentwith 0.2 M HCl alone induces almost completedesorption of the adsorbed HEWL. An exampleof the influence of in situ hydrochloric acid treat-ment on the desorption behaviour of HEWL canbe found in figure 2 of Ref. [15]. Here we usedboth hot SDS and HCl to quantitatively removethe adsorbed protein. We have demonstrated bymeans of X-ray photoelectron spectroscopy (datanot shown) using the characteristic signal fromS(1s) and N(1s) that our surface cleaning methodquantitatively removes the adsorbed proteins,thus allowing us to use the same chip for a wholeseries of experiments as long as the reproducibilitycriteria for (dF, nF) are satisfied. When this wasthe case (see Table 2), the surface concentration ofadsorbed protein was calculated according to thede Feijter formula [32] using the dn/dc valueappropriate to the buffer used. This formula wasderived for homogeneous and isotropic proteinlayers but it has been demonstrated that it holdseven if the adsorbed layer is anisotropic [33].

We compared the results obtained from the deFeijter formula with those calculated with thesame measured parameters according to the for-mula derived by Cuypers et al. [34]. This needssome additional parameters: the partial specificvolume (n) of the protein, its molecular weightand its molar refractivity (RM). For our calcula-tion we took M/RM 4.15 g cm−3 as in the case ofother non haem proteins [34,35] and n was takenequal to 0.72 cm3 g−1 [36]. With these values weobtain agreement between the de Feijter andCuypers formula in the 1–5% range (data notshown).

3. Results and discussion

3.1. Experiments of type a: kinetic experiments incarbonate buffer

Representative adsorption kinetics for10−7,10−6 and 10−4 M HEWL are shown in Fig.3(A). All the data were obtained from the same

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Table 2Summary of the adsorption experiments

tads/hc T/°C (90.1) Kd/10−3 s−1 d DG/Gtot

e Glim/mg cm−2 f kaCb/10−4 g a/nm2 hCb/M dF, nF/nm bExp a

4.2 6.3Chip 1 0.191 3.4 3.1010−7 203.2, 1.7780 1.05 23.72.0 5.5 0.18 n.m. n.m26.02Chip 1 0.25203.7, 1.778210−7

24.23 n.m. n.m 0.16 4.4 4.7610−7 198.2, 1.7746 1.0Chip 21.0 26.1 8.0 24.5 0.20 220 3.83Chip 1 10−44 203.4, 1.7783

7.3 32.2 0.20 370 3.7824.1203.3, 1.7784 0.25Chip 1 5 10−4

23.96 1.6 56.9 0.24 140 3.0210−4 198.5, 1.7762 0.25Chip 3, NaCl, pH 7.424.07 n.m. n.m. 0.06 4.3 8.3410−4 199.0, 1.773 0.25Chip 3, NaCl, pH 4.5

a Number attributed to the experiment, for simplicity in the discussion.b Calculated from the three layer optical model after equilibration with the buffer [31]. The ranges over five experiments are 90.00029 for nF and 90.5 nm for dF.

When a chip fell outside these ranges, it was discarded.c Total adsorption time (before desorption).d Determined according to Eq. (3).e Percentage of reversibly bound proteins, calculated with parameters (Go−G�) and Go of Eq. (3).f Threshold surface concentration in the adlayer after which the RSA theory was fitted to the data.g Determined according to Eq. (1), units are in mg cm−2 s−1.h Determined according to Eqs. (1) and (2). Coefficients of variation are less than 20% as obtained from the extend of the effects of the variation of one fitting

parameter (kacB or a) to the other. N.m.: not measured.


Fig. 3. Adsorption kinetics (Type a experiments) using the same Si0.8Ti0.2O2 chip (chip 1 in Table 2) with 10−7 (short dashed lines),10−6 (medium dashed lines) and 10−4 (full lines) M HEWL. Arrows indicate when flushing with protein-free buffer began. (A)Surface concentration of adsorbed HEWL. (B) Thickness of the adlayer. (C) Refractive index of the adsorbed film (shown only at10−7 M in HEWL).

chip : excellent reproducibility was observed at10−7 M. But at 10−4 M, it was more difficult toobtain such excellent reproducibility, especially asthe adsorption plateau was approached. We donot have an explanation for this at the moment,but it may well be that it is related to solutioneffects, like some aggregation at the higher bulkconcentration. When one considers the reproduci-bility from chip to chip (Fig. 4) one finds somedeviations, as with other techniques and surface

preparations, but these are typically in the 5–10%range and hence significantly smaller than thealmost 40% difference observed between the ad-sorbed amounts with the 10−7 and 10−4 M solu-tions. Hence we consider that our experiments arereproducible within the 5–10% range.

Ignoring its atomic details, the HEWL moleculemay be considered a prolate ellipsoid whose prin-cipal axes are 4.5 and 3.0 nm long [2]. Then amonolayer of adsorbed proteins built up by a


random sequential adsorption (RSA) process,with a maximal coverage close to 55% for almostspherical particles [37], would correspond to asurface concentration between 0.13 and 0.19 mgcm−2 depending on the orientation of the majoraxis of the ellipsoid with respect to the sorbentsurface.

Were a hexagonal close packed monolayer(hcp) formed, which is hardly imaginable withouthigh surface mobility during the adsorption pro-cess, then the surface concentration in the ad-sorbed monolayer would lie between 0.22 and0.31 mg cm−2, depending again on the meanorientation of the adsorbed molecules. Clearly wereach the upper limit for an hcp monolayer afterone hour of adsorption e6en at the lowest investi-gated concentration of 10−7 M (Fig. 3(A)). Notethat the adsorption process is then slow but nottotally stopped (Fig. 3(A)) suggesting the forma-tion of several layers [3,5,15]. However, on nega-tively charged latex particles with a surface chargedensity of −0.23 mC m−2 in the presence of 0.05M KCl, without buffering ions and at 25°C, theadsorbed amount at the plateau of the adsorption

isotherm was found to be close to 0.22 mg cm−2,hence consistent with the formation of a mono-layer [21]. In our work, we investigate the com-parison between a monolayer and morecomplicated structures at the interface withoutconsidering any kind of order parameter in theadsorbed film, which is not accessible with ourexperimental technique.

It may be misleading to use surface concentra-tions estimated either from optical measurementsor bulk depletion techniques to give this kind ofstructural information. Our surfaces had a meanaverage roughness of about 0.8–0.9 nm, i.e. notperfectly smooth with respect to the dimensions ofthe probe, the HEWL molecule. Thus the avail-able area for adsorption may be higher than thatpredicted by considering the collector to be per-fectly flat. It has moreover been demonstratedthat surface defects (chemical microheterogeneity)can be accumulation centres for proteins [38]. It isnot our aim to go into such considerations here.

3.2. Analysis of adlayer thickness and refracti6eindex

Since neither the optical thickness dA of theadlayer nor its mean refractive index nA are opti-cal invariants (in contrast to G [39,40]), they willonly furnish weak criteria for assessing the differ-ence between monolayer and more complicatedstructures. Figs. 3(B and C) display the timeevolution of dA and nA, respectively. It appearsthat upon going from the 10−6 to 10−4 MHEWL, the film thickness increases significantly,and goes beyond the value expected for a mono-layer, i.e. about 3–5 nm. The evolution of nA withtime (shown for 10−7 M only), after an initialdecrease corresponding to a part of the quasi-lin-ear increase in G, increases monotonically duringthe whole rest of the adsorption process and thenincreases faster during rinsing with pure buffer.This result resembles the results of Clerc andLukosz for avidin adsorption on Si(Ti)O2 [33]. Itcould well be related to compaction of the adlayerduring the second adsorption regime. Note thatthe values obtained for nA, greater than 1.5, seemhard to accept at first glance. Since a pure proteincharacterized by a refractive index increment of

Fig. 4. The initial regime of the adsorption kinetics with 10−7

M. HEWL (chip 1, Table 2,� and D), and on another chip ofthe same composition and cleaned in the same manner ().The straight line corresponds to an adsorption process con-trolled by convective diffusion to the adsorbent. For details ofthe calculation of the straight line’s slope, see Ref. [15].


0.212 cm3 g−1 and a partial specific volume of0.72 cm3 g−1 has a refractive index of about 1.6in carbonate buffer at 298 K, the adsorbedHEWL must occupy a volume fraction of about58% in the adsorbed layer, consistent with strongcompaction of the adsorbed film (a hcp mono-layer of HEWL ellipsoids has a volume fractionof about 0.6 [33]).

The initial decrease in nA, during the initialadsorption regime, might well arise from the an-isotropy of the growing adlayer. However the dA

increases practically only during the first kineticregime. This is not inconsistent with the be-haviour of nA if we assume the ellipsoids toadsorb in a particular orientation and form chainsparallel to the surface [15,41,48]. This increasecorresponds to the increase of the fraction of thesurface occupied by these growing chains. Oncesome fusion of the growing chains occurs, dA

increases only very slowly since the new adsorbingmolecules adsorb preferentially in or just at thetop of the holes left between the chains, causingnA to increase. This applies to 10−7 M HEWLsolutions, however at 10−4 M. the proteins arriveat the interface and have insufficient time to findan orientation allowing for partial incorporationin the first layer. Hence fast growth of an upperlayer is kinetically favoured, causing dA to con-tinue to increase significantly (Fig. 3(B)).

3.3. Analysis of the adsorption kinetics

During the initial adsorption kinetics at a bulkconcentration of 10−7 M (Fig. 4) one observesquasi linear adsorption kinetics, with slight accel-eration of adsorption after a certain time of con-tact between the surface and the flowing solution.This appears more clearly on plots of the firstderivative of G as a function of the adsorptiontime (see later). As observed previously [15], thisrapid and quasi-linear growth process continuesuntil a surface concentration of about (0.1990.01) mg cm−2 is reached, the adsorption kineticsthen suddenly decelerates. The deceleration is alsoobserved at higher bulk concentrations (Fig. 3(A))but occurs too rapidly to be properly resolved (thetime resolution of our OWLS measurements is27.5 s).

Our aim is now to analyse what happens afterthis initial regime, which ends precisely at themoment when the adlayer thickness also under-goes a transition to slower growth (Fig. 3(B)) andthe adlayer mean refractive index to a slow in-crease with time (Fig. 3(C)). We thus infer thatthis stage of the adsorption, which coincides withthe surface concentration expected for a jammedmonolayer built up by an RSA process, corre-sponds to the beginning of multilayer growth.During this second kinetic regime, the incomingproteins try to optimally accommodate themselveseither in defects of the first layer, or on top of thepresumed monolayer. If this is the case, the ad-sorption should not only proceed through sur-face–protein interactions or protein self-assembly,but surface exclusion should still play a role.Hence, we tried to fit the random sequential ad-sorption theory to the adsorption correspondingto this second regime, that is for G values greaterthan Glim=0.18 mg cm−2. These threshold surfaceconcentrations, Glim, corresponding to the surfaceconcentration above which the RSA fits weretried, are displayed in Table 2. All attempts to fitthe RSA model to the whole adsorption kineticsfailed because of the presence of the initial regimecharacterized by constant or slightly increasingdG/dt values (Fig. 5). We thus plotted dG/dtversus G, and in the slow adsorption regime whenG\Glim, these curves were fitted by the equation:

dG

dt=kacbf (1)

where kacb is the rate of adsorption to an emptysurface, taking into account hydrodynamic andorientational effects as well as interactions be-tween the protein and the surface. f is the avail-able area function describing the progressivecrowding of the surface. In this case we try todescribe the crowding of a composite film aboveor between the holes in the film deposited duringthe first regime. By assuming irreversible adsorp-tion, i.e. without desorption, and in the absence ofreorganization in the adsorbed adlayer, Schaafand Talbot have established a polynomial expres-sion of f for the whole accessible coverage regimein an RSA process [42], in the case of a puremonolayer:


Fig. 5. Plot of the derivative of the adsorbed surface concen-tration as a function of G for an experiment carried out with10−7 M in HEWL. After the region of positive values of thederivative (), the data were fitted by Eqs. (1) and (2), solidline.

More important for our discussion is the be-haviour of the a parameter of the fit: in the case ofexperiments 1, 3, 4 and 5 (Table 2), it gives anexcluded area which is systematically smaller thanthe 7.1 nm2 predicted for a HEWL moleculeadsorbing with its major axis perpendicular to thesurface.

The protein may not adsorb as a monomer in thisadsorption regime, even if it is assumed to bemonomeric far away from the interface. Note thatthe bulk concentration of HEWL in the adlayer isvery high, typically of the order of G/dA:0.2 (mgcm−2)/(3×10−7 cm):7×105 mg cm−3 or about0.05 M. This value is more than two orders ofmagnitude greater than the highest bulk concentra-tion used in our experiments. HEWL is known todisplay strong self-association in such concentratedsolutions, particularly at alkaline pH [19,20].Hence, if we would replace the area values obtainedfrom the fit of Eqs. (1) and (2) to the experimentaldata by an area corresponding to that of amonomer, we would see that the novel fittingparameter, the apparent mass of an adsorbing parti-cle, would be at least that of a dimer instead of amonomer. Hence, we propose that the monomersassociate in low order aggregates (dimers,trimers…) very close to the interface where they areable to sense the presence of the gel-like structureconstituting the adsorbed protein adlayer [16].

3.4. Desorption kinetics of the adsorbed HEWL

Non negligible and relatively fast desorption isapparent from the data in Fig. 3(A). In all theseexperiments, the adsorption has already reachedthe regime in which we suppose multilayer forma-tion to occur. One might expect that the secondregime proteins interact with the first layer moreweakly than first layer proteins interact with theSi(Ti)O2 surface. Hence the second regime proteinsare expected to desorb more readily than thoseadsorbed in the first regime. This is indeed observedexperimentally: as long as HEWL adsorption isrestricted to first regime of the adsorption kinetics,no desorption is observed (Fig. 6).

We quantitatively analyze the desorption occur-ring in the second regime by fitting an exponentialdecrease to the experimental data:

f=

�1−

Gamuj

�3

1−0.812Gamuj

+0.235�Ga

muj

�2

+0.085�Ga

muj

�3

(2)

where m is the mass of a monomer, uj is the surfacecoverage at jamming (0.55 for spheres in an RSAprocess) and a is the mean area excluded perisolated adsorbing particle. This equation is strictlyvalid in the absence of desorption, but even forcomplete desorption only coefficients of G3 arechanged [42]. Therefore we used Eqs. (1) and (2) tofit our data, but the values of the fitting parameterswill be considered only in a qualitative manner.

The fits were reasonnably good, (Fig. 5) and thetwo fitting parameters, a and kacb, are collected inTable 2.

The value of kacb increases with bulk concentra-tion, as expected, but the increase is not propor-tional to the bulk concentration, suggesting that theinitial adsorption rate, which is initially faster thanthat predicted by a convective diffusion to thesurface at low HEWL concentrations (Fig. 4 and[15]), is no longer controlled by transport to thesurface at higher concentrations.


G(t)= (G0−G�) exp[−kd(t− t0)]+G� (3)

where t0 is the time at the beginning of bufferrinsing, hence of the desorption process. Thefitting parameters are kd, (G0−G�) and G�, re-spectively the rate constant for desorption, theamount desorbed at infinite time and the surfaceconcentration of irreversibly adsorbed proteins.We began to fit Eq. (3) to the experimental dataonly about 1 min after switching the flow fromprotein solution to pure buffer in order to work ina practically steady state from the hydrodynamicview point and to be sure that almost all thenonadsorbed proteins have left the adsorptioncuvette.

The ability of Eq. (3) to fit the experimentalpoints was estimated by plotting the residuals, themeasured minus the calculated values, as a func-tion of time (Fig. 7). The largest residual wasalways lower than 0.1%, and moreover the meanresidual was statistically equal to zero (P\0.95).The parameter values are collected in Table 2. Bycomparing experiment 1 with experiment 4 andexperiments 2 and 5, it appears that for a constantadsorption time, the rate of desorption as well asthe percentage of desorbed proteins increases withincreasing bulk concentration. This indicates that

Fig. 7. (A) Enlargement of the desorption part of a type aexperiment and the fit according to Eq. (3), solid line. (B)Residuals of the fit, the dotted line illustrating an ideal fit.

Fig. 6. Variation of the apparent refractive index for TEpolarisation for an adsorption experiment of HEWL at 10−7

M (beginning after arrow 1) and stopped by buffer injection(arrow 2) before the end of the first adsorption regime.

at higher concentration, where adsorption isquantitatively bigger, the additionally adsorbedproteins are less tightly bound.

The desorption rate seems to be about twice at10−4 M than at 10−7 M in HEWL. This wouldbe consistent with a multilayer growth model,since the higher adsorption rate at high cb enablesonly minor structural modifications of the ad-sorbed proteins to occur, supposing that thesemodifications act to decelerate desorption [47].

Note also (Table 2), that the fraction of irre-versibly adsorbed proteins is much higher foradsorption from 10−7 M solution than from 10−4

M solution. This may indicate that only the firstadsorbed molecules are tightly bound, see alsoFig. 6.

All these observations, and particularly the sud-den transition in the desorption behaviour be-tween the first and the second adsorption regimes,


which begins at a threshold surface concentrationGlim, are a strong argument for the formation of asecond population of adsorbed molecules in thesolution � oxide interfacial region. The differencein desorption behaviour correlates perfectly withthe observed differences in the two identifiedregimes of the adsorption kinetics: moleculeswhich adsorb in the quasi linear regime (Figs. 3and 4) do not desorb in contrast to those whichadsorb subsequently in the second regime. Goingback to the arguments concerning the surfaceconcentration and the thickness of the adlayer, wesee that the appearance of G values greater thanexpected for a monolayer on a perfectly flat col-lector, and the significant increase in dA (from2–3 nm at 10−7 M to about 6–7 nm at 10−4 M)occur precisely at the transition from the firstadsorption regime to the second.

Hence all the criteria we used to analyse theformation of interfacial HEWL films in our exper-imental conditions point to a biphasic growthmechanism.

What is the molecular origin of this mecha-nism? We have previously attributed the observedlinear increase in G with time to the formation ofelectrostatically self-assembled structures [15], dueto the attraction between the net dipole of anadsorbed HEWL and those of bulk moleculesclose to the preadsorbed one, an interpretationcorroborated by the fact that the linear adsorp-tion regime totally disappears at high ionicstrength [15] and the experimental observation ofadsorbed linear arrays of HEWL [41,48]. We pro-pose that further growth occurs by means of thesame driving force, but with lower efficiency, andhence slower adsorption rate, because a mono-layer is already formed and the molecules nowhave to search for places were attractive interac-tions with preadsorbed molecules can take place.But by doing so they may well form lowoligomers in the proximity of the already de-posited film before adsorbing. Note that Schmidtet al. [3] have described fluorescently labeledHEWL adsorbed on octadecyl trichlorosilanemodified quartz slides in a similar manner.

If our assumption of a biphasic growth relatedto solution self-association is correct, then weshould not observe biphasic kinetics at lower pH,

where lysozyme does not aggregate significantlyeven at very high concentrations. Type b experi-ments (adsorption of 10−4 M HEWL in 10−2 MNaCl and pH adjusted to 8.9, 7.4 and 4.5) werecarried out to check this.

3.5. Type b experiments

The adsorption curves are displayed in Fig. 8.At pH 7.4, the adsorbed surface concentrationsare practically identical to those obtained at pH8.0 in carbonate buffer after a given adsorptiontime (see the upper curves in Fig. 3(B)). Takinginto account the fact that the experiments wereperformed on different chips, we do not attributethe slight difference observed to a chemical effect.Note in addition that the cPcS parameter (Table1) is practically the same at the two different pHvalues. On the other hand, all the experiments atdifferent pH values were performed on the samechip (Table 2) and it clearly appears that anincrease in pH at constant ionic strength induces astrong increase in G. Particularly important is theeffect of the pH increase from 7.4 to 8.9, even ifthe electrostatic interaction parameter cPcS isagain practically constant (Table 1). At pH 4.5,the adsorption kinetics reaches a well defined

Fig. 8. Type b experiments, carried out with 10−4 M inHEWL in 10−2 M NaCl solutions at pH 4.5 (short dashedline), 7.4 (full line) and pH 8.9 (thick dashed line).


plateau at a surface concentration just below thatpredicted for a monolayer formed by randomsequential adsorption on a perfectly smooth ad-sorbent. Moreover, random sequential adsorptiontheory fits well immediately after the beginning ofprotein flow and yields an excluded area of 8.3nm2, as predicted for a HEWL monomer (experi-ment 7, Table 2). However at pH 7.4 (experiment6, Table 2), RSA works only after Glim=0.24 mgcm−2 and gives an average excluded area peradsorbing particle of 3 nm2, in complete agree-ment with the experiments performed in carbon-ate buffer at pH 8.0.

4. Conclusions

Experiments at different pH show that the ad-sorption behaviour of HEWL on a silica–titaniasurface is directly related to the self-association itdisplays in bulk solution at very high concentra-tion. Namely, the protein adsorbs in a biphasicreaction at high pH values where it can aggregatein solution, but it adsorbs as monomers at acidicpH and according to a kinetic pattern that canwell be described as random sequentialadsorption.

In the case of experiments performed in carbon-ate buffer, we have clearly identified biphasicgrowth of the adsorbed film, by analysing boththe adsorption and desorption behaviour in thetwo kinetic regimes. By combining a series ofcriteria, namely the adsorbed amount, the adsorp-tion rate, the desorption rate and the fraction ofmolecules that can desorb, the thickness and re-fractive index of the adlayer and the area ex-cluded per adsorbing particle in the secondadsorption regime, we infer that the secondregime corresponds to the deposition of a film ontop of the first layer and containing a high frac-tion of weakly bound proteins. A similar conclu-sion has also been reached very recently byinterpreting neutron reflection profiles obtainedfrom adsorbed HEWL films in solutions of differ-ent pH [43]. The biphasic adsorption behaviourdisappears at low pH or at high ionic strength.

In the present work, we have used the RSAmodel in a rather qualitative manner to fit the

second regime of the adsorption kinetics even ifstrong desorption occurs in this regime: we arethus somewhat outside the framework of thistheory. Therefore, care should be taken in theinterpretation of the absolute values found for theexcluded area per adsorbing particle.

Acknowledgements

We thank Dr M. Robino (Institut de Physiqueet Chimie des Materiaux, rue du Loess, Stras-bourg, France) for the AFM experiments, and theCiba–Geigy–Jubilaums–Stiftung for financialsupport. We are indebted to Dr K. Strupat forcareful reading of the manuscript. V. Ball is in-debted to Dr A. Van Dorsselaer and Dr E. Leize,from the Laboratoire de spectrometrie de masse,Strasbourg, for fruitful discussions.

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