Macromol. Rapid Commun.20,607–611 (1999) 607

Polyelectrolyte complex layers: a promising concept for anti-fouling coatings verified by in-situ ATR-FTIR spectroscopy

Martin Muller*, Theresia Rieser, Klaus Lunkwitz, Jochen Meier-Haack

Institute of Polymer Research Dresden, Hohe Strasse 6, D-01069 Dresden, Germanymamuller@ipfdd.de

(Received: July 5, 1999; revised: August 26, 1999)

SUMMARY: In-situ attenuated total reflection (ATR)-FTIR spectroscopy enabled studies on the interactionbetween the differently charged model proteins human serum albumin, lysozyme, immunoglobulin G andmultilayer assemblies, which were deposited by alternating adsorption of poly(ethyleneimine) and poly(acry-lic acid) onto Si crystals. Low adsorbed protein amounts were observed if the top polyelectrolyte layer andthe protein were equally charged, whereas enhanced protein adsorption occurred for electrostatic attractionbetween protein and top polyelectrolyte layer.

IntroductionMaterials with non-fouling properties and a high mechan-ical and chemical stability are currently gaining interestin different biotechnological applications, e.g., mem-branes in biosensors or bioseparation processes1). The lat-ter requirement is satisfied very well by a variety of poly-mer materials. On the other hand, the inherent hydropho-bicity of many polymers favours in most cases the unde-sired adsorption of organic compounds2). Increasing thehydrophilicity and/or the surface charge of the polymersurface helps to moderate this in many applications inter-fering effect1, 3).

Polyelectrolyte complex (PEC) layers are able tosatisfy requirements concerning hydrophilicity and sur-face charge. In particular, PEC layers which were pre-pared by a layer-by-layer deposition method found abroad application in basic research during the last dec-ade4). For a sucessful multilayer build-up the reverse ofthe surface charge after each polyelectrolyte adsorptionstep and the attractive electrostatic interaction between anadjacent polycation and polyanion layer are essential5).Many studies deal with the exploration of the stepwiseadsorption procedure to incorporate a broad spectrum ofcharged components into thin organic films. Thereby,multilayer assemblies were prepared on a variety of sub-strates using proteins, dyes, DNA, enzymes, minerals,and metal colloids as the oppositely charged counterpartof a polyanion or polycation during the layer-by-layeradsorption. Future applications of such multicomponentfilms are sensors, friction-reducing coatings, integratedoptics, etc.6–12) However, the effectiveness of PEC layersas anti-fouling coatings has not yet been systematicallystudied according to the authors knowledge.

Recently, we reported on PEC layers used for the mod-ification of porous poly(propylene) (PP) membranes,which showed improved anti-fouling properties in com-

parison to the unmodified membranes8). In a furtherreport13) we could support these findings by in-situ-ATR-FTIR spectroscopy, whose sensitivity to phenomena atthe solid/liquid interface is well known14–16), using bothbare and PP film coated air plasma-treated internal reflec-tion elements (IRE, Silicium), which were analogouslymodified by PEC layers. In that work both thein-situmonitoring of the layer-by-layer deposition of poly(ethyl-eneimine) (PEI) and poly(acrylic acid) (PAC) as well asthe interaction between human serum albumin (HSA) anda PEI/PAC/PEI/PAC multilayer assembly on planar sub-strates was concentrated on.

Consequently, in the following work the concept ofPEC layers used for anti-fouling coatings is further ver-ified by in-situ ATR-FTIR spectroscopy, whereby thefouling (i.e. protein adhesion) behaviour of PEI/PACmultilayer assemblies is investigated using HSA in com-parison to lysozyme and immunoglobulin G as additionalmodel proteins. Furthermore, the influence of the outer-most polyelectrolyte layer on the protein adsorption pro-cess is focussed on.

Experimental part

Surface treatment of the internal reflection element (Si-IRE)

Before the adsorption experiments the surface of the Si-IREwas cleaned placing the ATR crystal in a beaker with sulfuricacid (95–97%, Fluka) for half an hour. After that the Si-IREwas removed and rinsed carefully with ultrapure water(Milli-Q water was used in all experiments, 18.2 MX). Intwo further cleaning steps the ATR plate was treated subse-quently with chloroform (Fluka) and ethanol (Fluka), andfinally placed in a plasma cleaner (Plasma Cleaner/SterilizerPDC-32G, Harrick, Ossining, USA) for five minutes underreduced air pressure. The plasma treatment was carried outwith the residual air in the plasma chamber. This procedure

Macromol. Rapid Commun.20, No. 12 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 1999 1022-1336/99/1212–0607$17.50+.50/0

608 M. Muller, T. Rieser, K. Lunkwitz, J.Meier-Haack

wasusedto createa negativelychargedSiOx surfaceon thesubstrate.

Alternatepolyelectrolyteadsorptionon theSi-IREin theATRsorptioncell

The polyelectrolytedepositionwascarriedout with aqueousunbuffered polyelectrolytesolutions containing 0.01 mol/lpoly(ethyleneimine)(PEI, Aldrich, M

—w = 750000) or 0.01

mol/l poly(acrylicacid)(PAC, Polysience,M—

w = 90000).Theconcentration0.01 mol/l refers to the monomerunit of thepolymer. Fig. 1a shows a schematicdrawing of the ATRsorptioncell consistingof oneATR liquid cell half which ispositionedon the front side of the plasmatreatedSi-IRE(5062062 mm3). Thereby, the Si-IRE is devidedinto twohalfs (5061062 mm3), which are sealed(Fig. 1b) by twoO-ringsto form theuppersample(S)andthelower reference(R) compartment.The backsideis analogouslysealedanddevidedby two O-ringsin S- andR-compartments.Thestep-wise polyelectrolyte adsorptionprocedurewas performedaccordingto the streamcoatingprocedure13) asfollows. TheS-compartment(front side)wasfilled with ca.0.5 ml of thepolycation or polyanion solution, whereasthe R-compart-ment (front side) was filled with ultrapurewater. After 30min the polyelectrolytesolution was removedfrom the S-compartmentand rinsed carefully with 10 ml of ultrapurewater before each subsequentadsorptionstep. Thus four(PEI/PAC-4) or five polyelectrolytelayersystems(PEI/PAC-5) were built up startingwith the polycationdepositiononthenegativelychargedSiOx surface.

Proteinadsorption

Proteinsolutionswere preparedby dissolving1 mg of eggwhite lysozyme(LYZ) (SIGMA), sheepimmunoglobulinG(IGG) (SIGMA), or humanserumalbumin(HSA) (Hoechst)in 1 ml phospateD2O buffer solutionat a pD valueof 7.4.10ml phosphatebuffer solution were obtainedby adding100mg buffer salt(buffer tablets,Fluka)to D2O (Aldrich), whichwasusedinsteadof Milli-Q water. Thus,thereis no d (H2O)water absorptionband presentin the ATR spectrawhichwould overlapthe amideI absorptionbandin the regionof1700–1600 cm–1. The proteinadsorptionexperimentswerecarriedout after thelastpolyelectrolyteadsorptionandwaterrinsingstepin thesamplecompartmentof thesorptioncell.

In-situ ATR-FTIRmeasurement

The in-situ ATR-FTIR Attachmentfor Sorption Measure-ments (OPTISPEC,Zurich) was used, whose principle isdescribed in ref.14) The ATR-FTIR spectrawere recordedusing a recently developedsingle beam samplereference(SBSR) technique14), wherebythe ATR sorptioncell is posi-tionedvertically on a lift in the spectrometer. Fig. 1b showsa simplified picture of the SBSR measurementprinciple,whereby the ATR liquid cell surroundingthe IRE is notdrawn. The upper sample(S) and the lower reference(R)half of the Si-IRE are alternatinglyshifted in the IR beam.The resultingintensitiesIS and IR arerecorded,respectively,andtheabsorptionASBSRis calculatedaccordingto:

ASBSR� ÿ logIS

IR

� �Thereby, the SBSRtechniquefeasiblesan accuratecom-

pensation of undesiredbackgroundadsorptionsdue to thematerialof the IRE, the polymerfilm, andthe bulk wateroftheaqueoussolution.

In this in-situ ATR-FTIR study the polyelectrolytemulti-layer build-up wasperformedin the samplecompartmentofthe sorptioncell. Thereby, SBSRspectraof the polyelectro-lyte multilayer assemblieswere recordedafter rinsing ofboth the S- and R-compartmentwith 2 ml of D2O bufferprior to the protein adsorption.The completeexchangeofmultilayer boundH2O by D2O wascheckedby inspectionofthe 1700–1600 cm–1 region,which showedno d (H2O) sig-nal. Thesespectrawere subtractedfrom the protein spectrato suppressabsorptionsignalsof the polyelectrolytesin theregionof theamideI band.For theproteinadsorptionmeas-urementthe samplecompartmentwasfilled with ca. 0.5 mlof proteinsolutionandafter 5 min adsorptiontime the firstspectrumwasrecorded.

Resultsand discussionsThe protein adsorption experiments were carried out onmultilayer assembliesthat consisted of four (PEI/PAC-4)or five singlepolyelectrolyte layers(PEI/PAC-5). Startingthe multilayer build-up with the polycation depositionstepon the plasma-treatedIRE, four alternating adsorp-

Fig. 1. Schematic picture of the ATR sorption cell (a) and asimplified schemeof the singlebeamsamplereference(SBSR)technique14) (b)

Polyelectrolyte complex layers:a promisingconceptfor anti-fouling coatingsverified by in-situ ATR-FTIR spectroscopy 609

tion stepsyielded in an overall negative surfacechargewith poly(acrylic acid) as final polyelectrolyte layer,whereasfive adsorption stepsled to an overall positivesurfacechargewith poly(ethyleneimine) in theoutermostlayer. Surface force aswell asstreaming potential meas-urementshavealreadyprovedthat thesurfacechargeof asolid substrateis reversedafter eachadsorption step,thusthe sign of the surfacecharge can be controlled by thefinal layer deposition process5,8).

Theadsorption experimentswerecarriedout with lyso-zyme(LYZ), humanserumalbumin (HSA), andimmuno-globulin (IGG) at a pH value of 7.4. Theseproteinshavedifferent isoelectric pointsandwere chosento investigatethe influence of the electrostatic interactions on theadsorption processof proteinsat the outermostpolyelec-trolyte layer. In Fig. 2 typical ATR-FTIR spectrain theamide I band range,recorded during the adsorption ofLYZ at the PEI/PAC-4 (Fig. 2a) and at the PEI/PAC-5system (Fig. 2b), respectively, are shown. From thesespectrathe integrated area of the amide I band (80%m (C2O)) at about 1650 cm–1, which is due to the back-bonepeptideunits of a protein, can be usedas a directmeasurefor the adsorbedprotein amount on the sur-face14,15).

In Fig. 3 to 5 theintegratedbandareasof thecharacter-istic amideI absorption versustheadsorption time for thethree proteins LYZ, HSA, and IGG, respectively, areshown.Generally, the label (a) of the absorbancecurvesrepresents protein adsorption experiments on the PEI/

PAC-4 system, whereasthe label (b) indicatestheproteinadsorption on a PEI/PAC-5 assembly.

Fig. 3 presentsthe results of the LYZ adsorptionatboth the PEI/PAC-4 and the PEI/PAC-5 layer assembly,respectively. Thereby, in the caseof the PEI/PAC-4 sys-tem high values of the integratedamide I band due tostrong adsorption wereobtained. Since LYZ is a proteinwith anisoelectricpoint at pH 10.717), it carriesa positivenetchargeat pH 7.4. Therefore it is obviousfrom Fig. 3athat attractive electrostatic interactions towardsthe PEI/PAC-4 system,having the polyanion layer on top, occ-cured. For the PEI/PAC-5 system (polycation as toplayer) low values of the integratedamideI banddue toweakLYZ adsorption wereobserved, which werecausedby electrostatic repulsion(Fig. 3b).

Thesituation is completelydifferentfor HSA andIGG,sinceboth proteinshavean isoelectric point in the acidic

Fig. 2. ATR-FTIR spectra(amideI bandregion) recordedin-situ duringtheadsorptionof lysozyme(LYZ) from D2O solution(1 mg/ml, pH = 7.4) at the four polyelectrolyte layer (PEI/PAC-4) system(a) andat the five polyelectrolyte layer (PEI/PAC-5)system(b) in dependenceon time ((a): t = 5, 15, 35, 105, and225min and(b): t = 5, 15,35,55,and175min)

Fig. 3. IntegratedamideI bandareas(from ATR-FTIR spectragiven in Fig. 2) measuredduring lysozyme(LYZ) adsorptiononthe PEI/PAC-4 system(PAC in the outermostlayer) (a) andonthe PEI/PAC-5 system (PEI in the outermost layer) (b) atpH = 7.4

Fig. 4. IntegratedamideI bandareasmeasuredduring humanserumalbumin(HSA) adsorptionat the PEI/PAC-4 (PAC in theoutermostlayer) (a) andthe PEI/PAC-5 system(PEI in the out-ermostlayer),respectively, at pH = 7.4(b)

610 M. Muller, T. Rieser, K. Lunkwitz, J.Meier-Haack

pH range.According to the literature the isoelectric pointof HSA is determinedat a pH valueof 4.818) andIGG hasanisoelectricpoint aroundpH 6.811). Now, attractive elec-trostatic interactions exist betweenthe PEI/PAC-5 layerassembly and theseproteins in solution at pH 7.4 and,consequently, high amideI absorption signalsweremeas-ured,as shownin Fig. 4b, 5b. However, in Fig. 4a and5a a strongly reducedamide I absorbance wasobservedin caseof the adsorption of HSA and IGG at the PEI/PAC-4 system exposing a polyanion top layer, whichcausedrepulsive electrostatic interactions.

Furthermore,according to the integratedamide I bandareasIGG adsorption is lower in the case of attractiveelectrostatic interactions comparedto that of HSA. Thiseffect is referedto a lower overallnegativechargeof IGGcompared to thatof HSA at pH 7.4.This canbeassumeddueto the fact that the isoelectric point of IGG is deter-minedat pH 6.8,which is closerto thepH of thesolution.

In Fig. 6 we have summarized the results due to theattractive and repulsive charge interactions betweentheproteins LYZ, HSA, IGG and the charged surfacesbyplotting the amide I band integrals (adsorbed proteinamount)versusthe magnitudeof the differencebetweenthe isoelectric point (IEP) of the protein and the experi-mental pH (buffer), i. e., |IEP-7.4|. For the attractiveinteraction (uppercurve)we obtaineda significantcorre-lation betweenadsorbedprotein amount and the value|IEP-7.4|. Therebythefunctional dependenceseemsto beof an exponentialor a power law type, suggesting coop-erativeadsorption phenomena.Evidently, in the caseofthe repulsive charge interaction (lower curve) weobservednot sucha significant dependence on |IEP-7.4|,i. e., a more or less constant small adsorbedamount,which could be partly explainedby the scattering of thevery small amideI bandintensities.However, we favourmore the explanation that in caseof repulsive charge

interactionaninitial interaction betweentheproteinswiththe like chargedsurfaceis minimized, so that no furthercooperativeadsorption effectscan occur. All this provesthe crucial role of electrostatic interactionwhenproteinsboth adsorbor arerejectedat chargedinterfaces.

ConclusionIn-situ ATR-FTIR spectroscopyis a useful tool to verifythe anti-fouling propertiesof multilayer systems com-posed of oppositely chargedpolyelectrolytes.

The protein adsorption measurements carried out ontwo different polyelectrolyte multilayer assemblies, i. e.,composedeither of four or five polyelectrolyte layers,demonstrated clearly the influenceof the surfacechargeof the outermost polyelectrolyte layer on the adsorbedprotein amount.For threedifferently charged proteinsastrongly reducedprotein adsorption wasobserved in caseof repulsive interactionsbetweentheoutermostlayerandthe proteinsin the solution. Vice versaattractive chargeinteraction led to enhanced adsorbedprotein amounts,which scaledwith the value of |IEP-7.4|, proving theimportant role of electrostatic interaction as the drivingforcewhen proteinsadsorb at chargedsurfaces.

Conclusively, there is potential for two applicationfields, which is on the onehandthe selective binding ofspecial functional proteinsat modified surfaces(immobi-lization) andon the otherhand,which we areaiming at,thedesign of selectiveanti-fouling surfaces.

Acknowledgement: This work was partly supportedby theDeutscheForschungsgemeinschaft(DFG, SFB 287) andby theBundesministerium fur Bildung und Forschung (BMBF, Nr.03N6010).

Fig. 5. IntegratedamideI bandareasmeasuredduring immu-noglobulin G (IGG) adsorptionat the PEI/PAC-4 (PAC in theoutermostlayer) (a) andthe PEI/PAC-5 system(PEI in the out-ermostlayer), respectively, at pH = 7.4(b)

Fig. 6. Plot of the amide I band areasdue to the adsorbedamountsof the proteinsLYZ, HSA, IGG versusthe magnitude|IEP-7.4| for attractive(circles) andrepulsive(squares)surface/proteininteraction

Polyelectrolyte complex layers:a promisingconceptfor anti-fouling coatingsverified by in-situ ATR-FTIR spectroscopy 611

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