Colloids and Surfaces B: Biointerfaces 26 (2002) 172–182

On the interaction between adsorbed layers of monooleinand the lipase action on the formed layers

Jose Campos b, Krister Eskilsson c, Tommy Nylander a,*, Allan Svendsen d

a Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund Uni�ersity, PO Box 124, SE-221 00 Lund, Swedenb Instituto de Fısica. UNAM, Ciudad Uni�ersitaria, PO Box 20-364, Mexico D.F. 01000, Mexico

c Institute for Surface Chemistry, Box 5607, SE-114 86 Stockholm, Swedend No�ozymes A/S, Smørmose�ej 25, DK-2880 Bags�aerd, Denmark

Received 1 August 2001; received in revised form 7 November 2001; accepted 16 January 2002

Abstract

We used the Surface Force Apparatus (SFA) and ellipsometry techniques to study the interaction forces and theadsorption behavior of monoolein (MO), respectively. MO was adsorbed from water to a hydrophobised mica orsilica surface. In addition the effect of added lipase, Thermomyces (Humicula) lanuginosa lipase (TLL), to an adsorbedlayer of MO was investigated. The force versus distance curves between two MO covered surfaces feature a strongrepulsive interaction beneath 400 A� . The range of the repulsive force decreases, however, with the number ofapproaches. No adhesion was observed, provided that the surfaces were not taken to hydrophobic contact. Thesurface separation at MO–MO contact was determined to about 55 A� . This means a layer thickness of about 27 A� ,which is comparable to the thickness (25 A� ) determined by ellipsometry. The repulsive force may arise fromcompression of a cubic phase of MO. This phase are suggested to form between the surfaces when they approachclose contact due to capillary induced phase separation (CIPS) from the saturated MO solution. The repulsive forcechanges significantly with time after addition of TLL (concentration of about 1×10−8 M). In contrast to the forcecurves recorded before adding TLL, the surfaces do not seem to be completely covered with MO as we alwaysobserved an attractive force (inward jump) of similar range as was observed between pure OTE surfaces. Ellipsometrymeasurement of TLL action on MO covered hydrophobic surface reveals a significant and sharp decrease of theamounts adsorbed. Furthermore, the rate of decrease and reduction in adsorbed amount increased with TLLconcentration. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Thermomyces (Humicula) lanuginosa lipase; Monoolein; Adsorption; Lipase action; Ellipsometry; Surface forces

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1. Introduction

Most lipids have low aqueous solubility andwhen they are dispersed in an aqueous solution aninterface is formed between an oil droplet, liquidcrystals, crystals or vesicles and the aqueous

* Corresponding author. Tel.: +46-46-222-8158; fax: +46-46-222-4413

E-mail address: tommy.nylander@fkem1.lu.se (T. Nylan-der).

0927-7765/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

PII: S0927 -7765 (02 )00036 -X

J. Campos et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 172–182 173

phase. The properties of this interface largelycontrol the stability of the dispersed systems bothin terms of colloidal stability and when it comes toprocesses such as hydrolysis and oxidation. Hy-drolysis of the ester bonds of acyl glycerides iscatalyzed by lipases, which are water-soluble en-zymes. This heterogeneous catalysis takes place atan interface and, therefore, one often speaks of‘interfacial activation’ in connection with lipaseactivity [1,2]. This interfacial activation involvesstructural changes where the lid covering the activesite is opened as the lipase is exposed to a lipid-aqueous interface, as observed for e.g. Ther-momyces lanuginosa Lipase (TLL) [3,4]. The lipasecatalyzed lipolysis is a complex reaction involvingmany steps like binding, orientation and activationof the lipase, binding of the substrate molecule intothe active site and the catalytic reaction itself (cf.[2,5]). The binding step depends very much on theproperties of the lipid–aqueous interface as re-ported by a number of authors (cf. [6,7]). In thepresent study we have used the Surface ForceApparatus (SFA) to study the interaction forcesbetween a substrate, monoolein (MO), as it issubject to lipase, Thermomyces (Humicula) lanugi-nosa lipase (TLL), catalyzed lipolysis. Parallel stud-ies were performed using ellipsometry, which allowus to directly quantify the amount adsorbed on thesurface. The reason that we have chosen to studyMO is that if we had started with the triglyceride,the system would have involve up to five additionalcomponents (diolein, monoolein, oleic acid, oleateand glycerol) as the lipolytic process proceeds. Wehave thus concentrated on the last digestible com-pound in this reaction chain, that is MO. The phasebehavior of the MO-aqueous system has beenthoroughly studied [8–13]. MO is known to forma bicontinuous cubic liquid crystalline phase inexcess of water as the monomer solubility is verylow. Previously we have investigated the effect oflipase action on a MO-based cubic phase [14] andcould relate the structural changes to the phasediagram of the MO-oleic acid/sodium oleateaqueous system [15].

Monolayers of MO spread at the air–waterinterface are known to be meta-stable [16,17].Studies of the relaxation of MO monolayers closeto the collapse at surface pressure (��46 mN/m),

indicates that the process could be considered asprecursor to the formation of mesomorphic phasesclose to the interface [17]. This can be related to thebulk phase behavior mentioned above. However,desorption of MO, which is believed to be dominat-ing factor for the relaxation below the equilibriumspreading pressure (�e�46 mN/m) [18], also con-tribute to the relaxation process at this high pres-sure. It has also been reported that the MOmonolayer shows weak X-ray diffraction, even athigh surface pressures (�=35 mN/m), indicatingweak interactions within the layer [19]. The lack ofdefined structure within the MO monolayer hasalso been observed by BAM studies [20]. The layerthickness is also thin, �13 A� , where the thicknessof acyl chain region is about 9.3 A� [19]. In thepresent study we have made use of the fact that thecubic phase formed in excess of water is in equi-librium with a very small amount of monomers,which in turn can adsorb at an interface.

2. Materials and methods

2.1. Materials

Octadecyltriethoxysilane (OTE) was purchasedfrom Lancaster Synthesis Ltd. Monoolein (1-mono(cis-9-octadecenoyl) glycerol) was purchasedfrom Nu-Chek Prep. Inc. (Lot noM-239-JA21-I)and TLL, wild type, was received from NovozymesA/S Laboratories as a 1 mg/ml Tris–HCl buffersolution (pH�8). The water used was distilled andpassed through a Milli-Q water purification system(Millipore Corp.), giving water with a final resistiv-ity of 18 M�/cm. To obtain dust free solutions thiswater was filtered through a 0.1 �m filter before use.All chemicals were of analytical grade used withoutfurther purification unless stated otherwise. Theglassware was cleaned with a hot chromosulfuricacid and then rinsed with large quantities ofpurified water.

2.2. Methods

2.2.1. Preparation of hydrophobic surfacesThe mica surfaces were made hydrophobic by a

Langmuir–Blodgett deposit of OTE according to

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the procedure developed by Wood and Sharma[21,22]. After spreading the OTE solution at theair–water interface, we waited until there was nota notable drop in the surface pressure (usually lessthan 15 min). Then we immediately compressedthe film and deposit it on mica at a constantsurface pressure of 12 mN/m (T=23 °C). Theadvancing and receding contact angles of a waterdroplet on these surfaces were always bigger than100° (data not shown), verifying the hydrophobic-ity of the surface. We have, however, later ob-served that these types of surfaces can containdefects on the microscopic scale [23].

2.3. Surface force measurements

The interferometric SFA was used to measurethe interaction between adsorbed layers of MOon hydrophobic surfaces and the effect of theselayers by adding lipase. The technique is de-scribed in more detail elsewhere [24] as well asthe particular version of the apparatus (MarkIV) used in this study [25]. The force is measuredbetween two curved mica surfaces (mean radiusof curvature, R, of about 1–2 cm) in a crossedcylinder configuration. The two mica sheets, sup-ported on half cylindrical silica disks, aremounted on a double cantilever spring (with aspring constant K) and on a piezoelectric crystal,respectively. The surface separation, D, betweenthe two surfaces is controlled by the piezoelectriccrystal and measured by an interferometric tech-nique with an accuracy of 2 A� . The magnitudeof the force, F, can be determined from the mea-sured spring deflection down to about 10−7 Nand is given normalized with the mean radius ofcurvature.

The SFA, equipped with a large volume cham-ber (400 ml), was dismantled and all inner partsrinsed with water, ethanol, and finally blown drywith ultrapure nitrogen before assembling itagain. Green muscovite mica (S&J Trading Inc.,Glenoaks, NY) was cleaved into molecularlysmooth and thin sheets, cut in about 1×1 cmpieces, and put down on a freshly cleaved micabacking sheet. A silver layer, about 520 A� thick,was evaporated onto the mica. The mica pieceswere then glued with the silver side down onto

optically polished half-cylindrical silica disks,which were mounted in the SFA. The assemblyof the instrument and surface preparation wereperformed in a clean room under essentiallydust-free conditions.

The mica-mica contact position was first mea-sured in air. OTE deposition was then carriedout as described above if the contact was foundto be adhesive and free of particles. The silicadisks were again mounted in the SFA. The thick-ness value of the deposited OTE monolayer wasdetermined and compared with the reportedvalue of 21 A� [21]. The apparatus was turnedwith the side port up to be able to aspirate theair– liquid interface during filling and to ensurethat the air–water interface passed the mica sur-faces as quickly as possible. The water was de-gassed, by boiling under vacuum for severalhours, before it was filled into the SFA. Theseprocedures reduce contamination as well as therisk of air bubbles sticking on the hydrophobicsurface. The contact position and a force curvewere then recorded in water to verify that thesystem was clean. All the force measurementswere carried out at 20 °C.

2.4. Ellipsometry

The adsorption of MO and subsequent actionof lipase on hydrophobised silica surfaces wasfollowed by in situ ellipsometry. The surfaceswere prepared as follows. Polished silicon testslides (p-type, boron-doped, resistivity 1–20 �cm)were purchased from Okmetic Ltd. The waferswere oxidized thermally in oxygen atmosphere at920 °C for �1 h, followed by annealing andcooling in an argon flow. This procedure results ina SiO2 layer thickness of 300 A� . The oxidizedwafers were then cut into slides with a width of12.5 mm and cleaned according the RCA method[26,27]. The surfaces were then dried under vac-uum, 0.001 mbar, and treated in a plasma cleaner(Harrick Scientific Corporation, model PDC-3XG) for 5 min. The surfaces were exposed todimethyloctylchlorosilane for about 24 h at roomtemperature according to Eskilsson and Tiberg[27]. The wafers were then sonicated in ethanol

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and tetrahydrofuran repeatedly to remove excessof silane before finally stored in ethanol.

An automated Rudolph Research thin-film nullellipsometer, type 43603-200E, as described previ-ously [28], was used in this study. The opticalcharacteristics of the substrate were determined atthe beginning of each experiment by measure-ments in different ambient media as described byTiberg and Landgren [28]. To avoid air bubbleson the hydrophobic surface, the cuvette was firstfilled with ethanol that then was replaced bycontinuous flow of water. All the measurementswere performed at �=4015 A� , in a temperature-controlled cuvette (25�0.1 °C) and under agita-tion with a magnetic stirrer at about 300 rpm.

The adsorption of MO was followed after plac-ing a small crystal of MO in the cuvette, contain-ing 5 ml of water. The solution composition wasthen changed by rinsing with a flow of water orby addition of a known amount of TLL. Therecorded � and � were evaluated using a fourlayer optical model, assuming isotropic media andplanar interfaces. The mean refractive index, nf,and the ellipsometric thickness, df, of the ad-sorbed layer were calculated using a numericalprocedure. The adsorbed amount (�) was calcu-lated from nf and df by the formula first derivedby de Feijter et al. [29]:

�=(nf−n0)df

dn/dc(1)

where n0 is the refractive index of the bulk solu-tion. The refractive index increment dn/dc, wastaken as 0.169 g/cm3 for both MO and lipase.

3. Results and discussions

The SFA experiments started with measure-ments of the interaction force between the barehydrophobic OTE surfaces. This was followed bymeasurements where the build up of an adsorbedMO layer from a deposited MO crystal was stud-ied. When the adsorption of MO had reachedsteady-state a known amount of TLL lipase wasinjected to the SFA chamber. As a control exper-iment we also measured the interaction forcewhen a known amount of TLL lipase was injected

into the SFA without the presence of MO. Theadsorption of MO on hydrophobic surfaces andthe subsequent action of TLL on the MO layerwere also studied by in situ ellipsometry.

3.1. Interaction between bare hydrophobic (OTE)surfaces

Interaction between hydrophobic surfaces inwater is a subject of intense debate, where along-range attractive force has been reported formany systems involving adsorbed or depositedsurfactants [30]. One of the major obstacles instudying the interaction between hydrophobic sur-faces is the presence of air bubbles. This problemincreases with the hydrophobicity of the surfaces,where gas-bubbles can form spontaneously in thegap between the surfaces (cavitation). The pres-ence of micro bubbles has been reported beforewith other hydrophobic surfaces similar to theones used in the present study [31–36], althoughin these cases a long-range (�100 nm) attractiveforce inferred the presence of them. However, thiscan be obscured by defects in the surface andother repulsive forces [33,36]. In 1995 Wood andSharma reported that the interaction between sur-face with cross-linked OTE layers was not long-range attractive [22]. Instead when two suchsurfaces were approached, no force was observeduntil the surfaces jump into contact at a separa-tion of 200 A� .

In our study we applied the same surface prepa-ration method as Wood and Sharma [21]. Fig. 1shows representative force curves for the interac-tion between two OTE-surfaces. We note thatthese curves were reproduced in several experi-ments. The interaction of surface separation be-neath 250 A� was found to be repulsive. Themagnitude of the repulsive force increases withdecreasing surface separation until the surfacesjump into contact at a certain separation. In somemeasurements the existence of an attractive forceis not as clearly observed and the interactionseems to be purely repulsive. However, if thesurfaces in such cases were left at a small separa-tion we observed that they slowly, but sponta-neously, moved into contact. The thickness of theOTE layer obtained from the contact position

J. Campos et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 172–182176

relative to the pure mica surfaces was found to be21 A� on each surface. This value is in agreementwith values measured in air. The adhesion mea-sured was determined to 230 mN/m when twosurfaces in contact were separated. In a consecu-tive approach after the surfaces have been incontact the repulsion generally starts at smallersurface separation. On the other hand, if thesurfaces are not brought into contact in the firstapproach the repulsive force is identical in thesecond approach. It is not straight forward toexplain our observations. The repulsive forcecould not be fitted to an electrostatic double layerforce that could possibly arise from exposed sur-face charges. The origin of the repulsive force

could possibly be an excess of surface materialprotruding into the bulk, which cause a stericrepulsion. However, if this would be the case wewould not expect to have a contact position thatcorresponds to a monolayer on each surface. An-other explanation would be the presence of en-trapped vapor or air bubbles at the contactposition. These bubbles could possibly be stabi-lized by the presence of defects earlier observedfor these types of surfaces [22,23]. Recent imagingof hydrophobic surfaces in water with tappingmode atomic force microscopy (AFM) revealsthat these surfaces are covered with soft domains,interpreted as nanobubbles, that are close packedand irregular over the surface [36]. Such featureswere not observed when the surfaces were ob-served in contact mode. The radius of curvatureof these bubbles was found to be in the order of100 nm with a height above the substrate of20–30 nm. The recorded force curve of the hydro-phobic attraction also featured a repulsive forcebefore the surfaces jumped into soft contact. Therepulsive force was found to have a decay lengthof about 25 nm, which is shorter than the valueexpected for a electrostatic double layer force.The decay length was also found to be indepen-dent of pH and driving velocity at which thesurfaces are approached. Here we note that wealways observed a thickness of the OTE layer of21 A� and a reduction of the range of repulsiveforce upon repeated approaches of the surfaces.This suggests that the repulsive force is caused byexcess material and/or air/vapor bubbles, whichare easily removed from the surfaces when theyare brought into contact. In addition we observedthat the repulsive force almost disappear in thepresence of MO (Fig. 2). It should, however, bestressed that once air bubbles in water bridge thetwo hydrophobic surfaces the force are expectedto be attractive.

3.2. Adsorption and interaction of monooleinlayers on hydrophobic surfaces

The ellipsometry data show that MO has astrong affinity to a hydrophobic surface and canbe adsorbed from an aqueous solution in equi-librium with a cubic liquid crystalline phase of

Fig. 1. The force, F, (normalized by the radius of curvature,R) vs. distance curves for three consecutive approaches of twoOTE surfaces at T=20 °C at the same contact point. Surfaceseparation of 0 A� , corresponds to the OTE–OTE contact thatis 42 A� from mica-mica contact. In the first approach (�), theforce is purely repulsive and no adhesive force is observedwithin the range of the piezo. In the second approach (�), thesurface jump from 124 A� at an applied force of 11.6 mN/minto OTE–OTE contact. In the third approach (�), therepulsive force is considerably weaker and the surfaces nowjump from 68 A� at and applied force of 6.3 mN/m. Forcomparison the first approach from another experiment mea-suring the OTE–OTE interaction forces is also inserted (+ ).Although the repulsive force is similar the surface jump from170 A� at a force of 1 mN/m. For all measurements theadhesive force determined on separation is about 230 mN/m.

J. Campos et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 172–182 177

Fig. 2. The force, F, (normalized by the radius of curvature,R) vs. distance curves for three consecutive approaches of twoOTE surfaces at T=20 °C after adding a MO crystal to themeasuring chamber. The force curve recorded after 48 h (�) issimilar to the force curve measured before addition of MO,with adhesive jump from 124 A� to OTE–OTE contact at aforce of about 8 mN/m. After 72 h (�), the observed repul-sion had decreased significantly and the surfaces jump intoOTE contact from a separation of 185 A� at a force of 0.2mN/m. The adhesion measured on separation of the surfaces is260 mN/m. In the force curve obtained after 96 h (�), thebuild-up of a MO layer on the OTE surface is apparent as asteep repulsive force. Further compression to causes the sur-faces jump from about 160 A� to OTE contact.

value is comparable with the area per moleculerecorded from compression isotherms of spreadmonolayers of MO at the air–water interface,were 40 A� 2 was observed at a surface pressure ofabout 25 mN/m [38].

The adsorption kinetics in the SFA chamber isexpected to be slower than in the ellipsometer cellsince the former has much larger volume and noagitation. Consequently, we observed a signifi-cantly slower build up of the MO layer in theSFA (Fig. 2). The force curves obtained after 24and 48 h were similar to the force curve measuredbefore addition of MO. After 72 h we saw achange in the measured interaction. The observedrepulsion had decreased and the surfaces jumpinto OTE contact from a separation of 185 A� . Inaddition the adhesion measured on separationhad increased to about 260 mN/m. This forcecurve was similar to the ones reported by Woodand Sharma [22]. As discussed above this supportsthe idea of the presence of air bubbles as theorigin for the repulsive force. The presence of MOreduces the gas-aqueous interfacial tension andfacilitates wetting of the hydrophobic surface bywater. Therefore, any bubbles on the surface willbe destabilized.

Fig. 3. The adsorption of MO on a hydrophobised silicasurface from aqueous solution in the presence of a MO crystal.Time=0 indicates the addition of the crystal. The graphshows the change in amount adsorbed (�) and thickness (�)as determined by in situ ellipsometry.

MO (Fig. 3). The adsorption kinetics is slowmainly due to the low solubility of MO. In fact,we can appreciate the monomer concentration byassuming a diffusion coefficient, D, of 2×10−10

m2/s and an unstirred layer with 100 �m thicknessas determined by Tiberg et al. [37]. Such calcula-tions result in a monomer concentration of �10−7 M. The adsorption reaches a steady statevalue of 1.5 mg/m2 and the ellipsometric thicknessof the adsorbed layer is about 25 A� . This shouldbe compared with the thickness of a MO bilayerof 35 A� in a lamellar phase [15]. Here we notethat the lamellar phase in the binary MO-aqueoussystem only contains about 10% water. Theamount of MO adsorbed on the hydrophobicsurface, 1.5 mg/m2, corresponds to an area permolecule of 39.5 A� 2 (Mw of MO=356.55). This

J. Campos et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 172–182178

Fig. 4. The force curves measured between 120 and 144 h afteradding a MO crystal in the SFA. After 120 h (+ ), therepulsive interaction the surfaces jump from 94 A� to a surfaceseparation of 59 A� from the OTE contact at a force of 5.6mN/m. No adhesion was found on separation. Three consecu-tive measurements, (�), (�) and (�), respectively, were doneon the same contact position 144 h after adding MO. Duringthe first approach, (�), the surfaces where kept at a force of4.8 mN/m at a separation of 110. It then took 2 h for thesurfaces to move to 53 A� . On the second approach, (�), noinward jump was observed before the surface separation of 57A� was reached at force of about 4.8–5.6 mN/m. No adhesionwas found on separation after the first or second approach. Aninward jump from 60 A� to the OTE contact was observed onthe third approach, (�), at a force of about 5 mN/m. Separa-tion from this contact position revealed a strong adhesiveforce.

secutive measurements were done on the samecontact position 144 h after adding MO. Theforce curve recorded on the first approach issimilar to the one recorded after 120 h. Thisindicates that the system is now approachingsteady state in terms of the formation of a MOlayer on the mica surfaces and saturation of thebulk phase by MO. The layer seems, however, tobe slightly denser as the surfaces move from alarger separation (110 A� ) than before to a contactposition of 53 A� . Furthermore it took 2 h for thesurfaces to move from separation of about 110–53 A� at a constant force of about 4.8 mN/m. Onthe second approach we found that the surfacescan get closer before the repulsive interaction isnoticeable at a surface separation of about 300 A� .In this case no inward jump was observed beforethe surface separation of 57 A� was reached atforce of about 4.8-5.6 mN/m. No adhesion wasfound on separation after the first or secondapproach. The values of the surface separationrecorded for the MO–MO contact, 53–59 A� ,after 120 and 144 h is similar to twice the ad-sorbed MO layer thickness of about 25 A� deter-mined by ellipsometry. The force curve recordedon the third approach is similar to the previousforce curve, but in this case we observed aninward jump from 60 A� to the OTE contact at aforce of about 5 mN/m and a strong adhesiveforce on separation. This indicates that we com-pletely removed MO from the surface.

A long-range repulsive force is not what oneexpects between two hydrophobic surfaces thatare covered with monolayers of the non-ionicMO. Our, experiments show that the range of therepulsive force changes with time and decreases ifthe surfaces are been sequentially brought intocontact. Similar type of force versus distancecurves were reported by Pezron et al. [16], whostudied the interaction between Langmuir de-posited layers of MO. They found that theyneeded to add a crystal of MO to get stablemonolayers on the surface. When a too largeforce was applied, the MO layer was disruptedand the recorded force curves indicated the for-mation of aggregates on the surface. They sug-gested that the formation of these aggregatescould be related to the instability of the MOmonolayer as the liquid crystalline lamellar state

In the force curve obtained after 96 h, thebuild-up of a MO layer on the OTE surface isapparent as a steep repulsive force. Applying aweak force can squeeze this layer out and as aresult the surface jump from about 160 A� to OTEcontact. The range and the magnitude of therepulsive force increases with time. Fig. 4 showsthe forces curves measured between 120 and 144 hafter adding a MO crystal in the SFA. In theforce curve measured after 120 h the repulsiveinteraction starts at about 400 A� The surfaces canbe approached until they jump to a surface sepa-ration of 59 A� from the OTE contact. We did notobserve any adhesive force when the surfaces wereseparated from this contact position. Three con-

J. Campos et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 172–182 179

in the binary MO aqueous phase is unstable inexcess of water and readily transforms to a cubicphase. In our case we must consider that thesolution will eventually be saturated with MO andthe system will be close to the solution-cubic phasetwo-phase region. This means that any distur-bances of the system can cause the separation outof the cubic phase. Such a transition can occur inthe gap between two surfaces. This capillary in-duced phase separation (CIPS) has been demon-strated for a number of systems [39,40]. Thisincludes the formation of a meniscus of waterbetween two hydrophilic surface in oil saturatedby water [41] or the formation of water condenseor a lamellar phase between two hydrophilic phasein bicontinuous microemulsion [42]. Usually CIPSgive rise to a long-range attractive force. However,it should be borne in mind that the cubic phase isvery stiff [43]. This can explain the appearance ofthe strong repulsive force. The time needed afterthe addition of MO for the repulsive force to buildup could then be due to the time needed for thesolution between surfaces to be saturated withMO. The proposed mechanism can also explainwhy the compressions performed immediately af-ter each other results in a (partial) removal of thesurface layer. In this case there is not enough timefor the system to retain equilibrium.

3.3. Interaction of TLL with MO adsorbed onhydrophobic surfaces

Ellipsometry measurement displayed in Fig. 5show the action of TLL lipase on MO adsorbed toa hydrophobic surface. Different concentrations oflipase were added to the cell when a plateau inamount of MO adsorbed was reached (compareFig. 3) and with the MO crystal still present in thecell. The total amount on the surface decreasesrelatively sharply upon the addition of lipase, butreaches a plateau within about 400 s. The rate ofthe decrease in surface excess as well as the re-moved fraction increases with lipase concentra-tion. Note that we can not separate the individualcontributions from MO and TLL lipase to thetotal adsorbed amount. Wannerberger and Arne-brant have extensively studied the adsorption ofTLL on hydrophobic surfaces, using in situ ellip-

sometry [44,45]. They found that the kinetics ofadsorption is very dependent of lipase concentra-tion, pH and surface properties under similarconditions as those used in the present study. Forinstance from 63 nM TLL solution, close to themaximum concentration used in the present study,the adsorption is quite slow and after about 400 sit reaches 0.1 mg/m2 at pH 7 and 1.5 mg/m2 at pH5. The corresponding values on a hydrophilic silicasurface were 0.1 and 0.4 mg/m2, respectively, usinga 100 times higher TLL concentration. They alsoobserved that making the surface only slightlymore hydrophilic, i.e. decreasing the water contactangle from 75 to 62°, led to a dramatic shift in theamount TLL adsorbed from 1.7 to 0.2 mg/m2 at alipase concentration of 63 nM. Thus it is clear thatthe lipase will give a small contribution if any tothe amounts adsorbed under the conditions(pH�7) used in the present study. Only at thehighest concentration of TLL we note a smallincrease in adsorbed amount at 200 s after thesharp decrease in surface excess due to the lipaseaction. We can, therefore, conclude that the ob-served decrease in surface excess is mostly

Fig. 5. Ellipsometry measurement of lipase action on MOcovered hydrophobic surface at TLL concentrations of 17 (�),35 (�) and 70 (�) nM. Time zero represents the momentwhen TLL is added to the system. The average thickness valuedecreases from 25 A� to about 20 A� due to the action of TLL.

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Fig. 6. The force, F, (normalized by the radius of curvature,R) vs. distance between two OTE surfaces at T=20 °C before(+ ) and 43 h (�) after adding 50 nM TLL. Two consecutiveforce curves, (�) and (�), respectively, was recorded atanother contact position. Upon compression the surfaces jumpfrom 130–150 A� surface separation into adhesive contact. Thefinal contact position varied between 20–40 A� out from theOTE contact separation with an adhesion of about 230 mN/min all cases. The repulsive force can be well fitted to a DLVOforce, where the double layer repulsive force have a decaylength, �−1, �154 A� (corresponding to an 1:1 electrolyteconcentration of 0.4 mM) with a surface potential of 60 mV,and the van der Waals attraction is calculated using a non-re-tarded Hamaker constant of 0.5×10−20 J. Both the diffuselayer boundary and the plane of onset of the dispersion forcesare placed at a surface separation of 130 A� . The solid linecorresponds to constant surface charge and the dashed line toconstant surface potential boundary conditions.

adsorption. The obtained force curves are repul-sive from a surface separation of 800 A� . Thisrepulsive force increases until a surface separationof 130 A� where the surfaces jump into adhesivecontact. The repulsive force can be well fitted toan electrostatic double layer force with a decaylength, �−1, �154 A� (corresponding to an 1:1electrolyte concentration of 0.4 mM) and surfacepotential of 60 mV. Measurement at differentcontact points gave similar force curves, but thesurfaces jump into contact from slightly differentsurface separations. The final contact positionvaried between 20–40 A� out from the OTE con-tact separation with an adhesion of about 230mN/m in all cases. The presence of an attractivejump and the obtained adhesion values, which aresimilar to the adhesion between the pure OTEsurfaces, indicate that the lipase do not com-pletely cover of the surfaces. This is in agreementwith the ellipsometry data for low lipase concen-trations reported above [44,45]. The lipases mayalso be squeezed out when the surfaces arebrought into close contact. This indicates weakattractive forces between the lipase and the sur-faces under present experimental conditions.

Fig. 7 shows force curves obtained at differenttimes after addition of lipase to a concentration of10 nM, to the SFA, where the OTE-surfaces hasbeen equilibrated with MO. The addition of lipasecontinuously change the interaction from a long-range non-exponential repulsion obtained be-tween MO covered surfaces to a repulsive forcewhich is less long-range and which can be fitted toa single exponential decay (data not shown). Withtime after lipase addition the force curve is shiftedinward and the repulsive force becomes weaker.In addition, the surfaces do not seem to be com-pletely covered with MO and/or hydrolysis prod-ucts as we always observed an attractive force(inward jump) of similar range as was observedbetween pure OTE surfaces. The final positionvaries and decreases with each consecutive ap-proach. This can be interpreted as a consequenceof hydrolysis of the MO layer, which is catalystby the lipase. This was also demonstrated byellipsometry data presented in Fig. 5. As discussedabove the transport of the enzyme to the surfaceas well as the transport of products from the

likely a consequence of lipase action on the ad-sorbed MO layer. The products of the lipolysisglycerol and oleic acid/oleate have significantlyhigher aqueous solubility than MO and will,therefore, desorb from the surface.

Fig. 6 shows how the interaction force changedas a result of lipase adsorption at the hydrophobicsurfaces in a system without MO. The concentra-tion of lipase is similar to the ones we used in theellipsometry study (50 nM) since we want to workin the catalytic relevant lipase concentrationrange. The force curves obtained after 43 and 50h after adding TLL are almost identical and webelieve that the system was close to equilibrium

J. Campos et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 172–182 181

surface is significantly slower in the SFA chamberthan in the ellipsometry cell as the latter allowsfor agitation of the solution during the measure-ments. The appearance of an exponential repul-sive force indicates a charging of the surface. Thisis likely to be achieved by one of the product ofthe lipolysis, that is oleic acid. The degree ofprotonation of the fatty acid will determine thesurface charge. The charging of the surface mightalso be achieved by adsorption of lipase. How-ever, to reduce the interference of this process theTLL concentration was reduced by a factor of fivecompared with the experiments with only TLLadsorbing on the OTE surface. In spite of this lowTLL concentration we observe a drastic change ininteraction force between the MO covered sur-faces. These effects also occur on the MO crystalpresent in the SFA chamber, which in turnchanges the composition of the solution. This

change in composition appears to prevent theCIPS, which we saw indication of before theaddition of TLL.

Acknowledgements

We are thankful to Professor Per Claesson,Hugo Christenson and Thomas Bjørnholm forhelpful and stimulating discussions. Financialsupport was obtained from the EU BiotechShared Cost project, Contract No. BIO4-97-2365.J. C. also thanks the Swedish Institute, DGEPUNAM and CONACyT for financial support.

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