CHARACTERIZATION OF SELF-ASSEMBLED PROTEIN THIN FILM ON FLAT ANDNON-FLAT SUBSTRATE

CARACTERIZAÇAO DE FILMES FINOS DE PROTEINA SOBRE SUBSTRATO PLA-NO E NAO PLANO

ODlUO B. G. ASSIS 1

RUBENS BERNARDES FILH01

ABSTRACT

In this study we have characterized the surfaces of Iysozyme globular protein films self-assembledon glassy substrate. Flat slides and cylindrical glass fibers were used as solid supports for protein(concentration 104M) immobilization and the surfaces scanned with atomic force microscopy (AFM)along the deposition time. AFM data allowed following the surface roughness reduction as the filmwas formed. The deposition features were quite similar for both substrates, whereas on the f1atsurface a more regular deposition was observed. The thickness of grafted layers was determinedfrom differences between deposited and non-deposited regions and measured, after 10 min im-mersion, as 7.6 ± 1.4 for f1at and 10.4 ± 2.6 nm for cylindrical surface, suggesting by comparisonwith the protein molecule dimensions, that the adsorption should be a multilayer process underthe experimental conditions used in this work.

Keywords: Self-Assembly, Protein Films, Protein Immobilization, Surface Analysis, AFM.

1 Embrapa Instrumentação AgropecuáriaCaixa Postal 741, 13560-970 - São Carlos, SP. Brazil, Email: odilio@cnpdia.embrapa.br

Recebido: Jant08Aprovado: Abrt08

O.B.G. ASSIS, R.BERNARDES FILHO

RESUMO

No presente estudo caracterizamos a superfície de filmes automontados da proteína globularlisozima sobre substrato vítreo. Lâminas plana e fi'bras de formato cilíndrico foram empregadascomo substratos para imobilização. Solução protéica na concentração de 10-4 M foi utilizada e osfilmes em sua forma final analisados por AFM (microscopia de força atômica). Os dados possibilitamacompanhar a redução da rugosidade conforme o filme é formado. Os depósitos. contudo, .s~osimilares para ambos os substratos sendo a superfície plana a que proporcionou u~a deposlça?mais regular. A espessura dos filmes formados, após 10 min de imersão, foi determinada a partirda diferença entre regiões depositadas e não-depositadas e estabelecido como 7,6 ± 1:4 parao substrato plano e 10,4 ± 2,6 nm para a superfície cilíndrica, sugerindo por comparaçao comas dimensões de uma molécula da proteína que a adsorção, nas condições experimentais aquiempregadas, ocorre de forma multicamadas.

Palavras-chaves: Automontagem, Filmes de Proteína, Imobilização, Análise de Superfície, AFM.

INTRODUCTION

The self-assembly (SA), or as recentlyreferred layer-by-Iayer (LL) technique, hasbeen largely used for the fabrication of ultra-thinfilms and multilayers systems of biomoleculesin a controlled fashion (WHITESIDES, 1995;PATERNO et aI., 2001). This technique, in con-ception, refers to a spontaneous attachmentthat takes place during the contact of moleculesdispersed in a fluid phase with a solid support(BISHOP & NUZZO, 1996; LVOV, 2000), oras defined by LlNDOY & ATKINSON, 2001,a process by which a supramolecular speciesforms spontaneously from its components andare held together by a range of relatively weakintermolecular interaction.

A large number of polymers and moleculeswhich present spatial distribution of charges oramphiphilic structures are capable to be de-posited in an ordered manner, under suitablesolvent conditions, on appropriated substratum.In particular macromolecules, such as proteinsand polysaccharides, form nanostructured filmsowing to its polar characteristics and chargesdevelopment in liquid medium. One importantfeature in this mechanism is that the processis rather dependent of the solution acidic levei,i.e., at specific pHs charges can be developedalong the polymeric chain and preferential ori-entation takes place between the molecules andthe substratum. The hydrophobic groups of thepolymer chain will be associated with the surfacehydrophobic groups of the substratum and vice-

BioEng, Campinas, v.2, n.1, p.OO9-016,janlabr., 2008

versa (FENDER, 1996). That is an importantcharacteristic that can define SA films potentialapplications.

The globular protein Iysozyme, also knownas muramidase, is a natural thermally stableenzyme found in colostrum, hen egg whites andhuman nasal mucus and tears. It is character-ized by multiples and complex structural groupsdefining spatial hydrophobic and hydrophilicregions, with predominant positive charges asmodels constructed by BLAKE et aI., (1995), andKAYUSHINA et aI., (1996). SA Iysozyme filmshave been tested as filtration media (ASSIS &CLARO, 2003) and as heavy metais (CHERIAN,et aI., 2003) and bacteria sensors (HUANG etaI., 2003), amongst several possibilities.

The substratum suitable for the Iysozymespontaneous bonding has to present highdensity of negative superficial charge, such asmetal or glass, attracting the oppositely chargedportion of the molecules. In glassy materiais,the hydroxyl groups on the surface providefunctionality that react with hydrolysable groupsof protein structure (ASSIS & CLARO, 1999;SENGUPTA, 1999). According to JIA & LlU,(2005) the Iysozyme deposition pattem followsthe same behavior as observed to amphiphilicmolecules.

The format of the solid substratum alsoplays important role on the formation of the film.ULMAN, (1991), states that the deposition inhorizontal flat surfaces is favorable to a more

stable film assembly, although it presents thedisadvantage of not being possible to controlthe molecular array uniformity. According toDE-BACKER & BARON, (1993), immobilizationon porous glass media is usually irregular and itchanges diffusivity on permeable medium. How-ever, AVNIR et ai., (1994) showed that porousglass surface can be advantageous in effectivetrapping of enzymes although most of the sur-face available for activity is internal. Conversely,good fixation of enzymes is attained on non-porous glass beads making use of crosslinkingcovalent agents (IKEDIOBI, et aI., 1998).

The immobilization of enzymes on non-flatmedia, such as porous ceramics or fibrous sup-port, has technological importance for configur-ing bioreactor or biofiltration medium of whichsome liquid permeation is mandatory. In thiswork we use the atomic force microscopy (AFM)for characterizing Iysozyme self-assembly filmformation on cylindrical fiber glass, comparingmorphology with similar deposition on flat slides.

MATERIAL ANO METHOOS

Commercially available glass fibers of70-140 IJm diameter and glass slides (5mm x1Omm x 2mm optical glass plates) were used as

supports. Both materiais underwent chemicalfunctionalization treatment, named 'piranha'method, that consists of a series of surfacecleaning procedures in warm acid solutionsusing ultra-sonic baths, as described else-where (KERN, 1993). This treatment en-hances the glass negative surface chargesand the corresponding hydrophilicity index.

An aqueous solution of Iysozyme fromhen egg-white protein (Sigma) was prepared(as delivered) at a concentration of 104 M, us-ing ultrapure deionised water, and the solutionadjusted to pH - 6.2, using phosphate buffer(Na2HP04)·

The supports were kept immersed in pro-tein solution and samples randomly removedafter 5, 8, 10 and 12 min immersion and thenrinsed in distilled water. Figure 1 schematicallyillustrates the experimental set up. AFM imageswere acquired in non-contact mode (TopoMetrixDiscover System) and the images processed bya TOPOSPM software. Two random areas of 1lrn x 1 lrn were scanned on each support. A totalof 4 flat and 4 cylindrical samples were analyzedresulting in a total of 8 areas scanned in eachcondition. The film thickness was measured asthe average differences on non-deposited anddeposited terraces after 10 min immersion, usinga methodology previously described by ASSIS& SILVA, (2003).

fál r 1 I.C'

!~. 15-s."~----

000: 000:

Figure 1 - IIlustration of protein deposition process and faces analysis. In (a) and (b) the fiber and glassslides are separately immersed in the precursor solution and samples withdrawn at 5,8,10 and 12 minoEach fiber had two regions randomly scanned (c) and the slides had the both side also examined byAFM (d).

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O.B.G. ASSIS, R.BERNARDES FILHO

RESULTS AND DISCUSSION

Protein adsorption on solid surfaces isfrequently modeled as the formation of a two-dimensional, ordered array positional parallelto the surface. The stability and homogeneityof deposition is stated to depend on the surfacedensity of OH groups (INOUE et aI., 1998), whe-re the adsorption and desorption is determinedby competition between attractive interactionand non-specific repulsion (SCHWARTZ et aI.,1992; LEE et aI., 2001). The film formation de-pends on the volume of protein molecules nearthe surface (FIGUEIREDO et aI., 2005) and onthe uniformity of initial sites occupation. Thefinal film feature is than ruled by spontaneouspacking-induced reconfiguration of individualadsorbed molecules on the solid surface (RO-BESON & TILTON, 1996).

In the AFM morphological features are

defined by x, y, and z coordinates, which indi-cate the relative height (z) of the cantilever tipat each x and y planar location. The surfacesirregularities can be expressed by the RMS(root-mean-square) roughness describing thestandard deviation of an entire distribution ofz-values for each scanned area as:

where ~ is the height of the ithdata point and zavis the average value of Zj over ali i's.

The RMS roughnesses acquired on sur-faces randomly drawn from the solution resultedin the data summarized on table 1. For a bettervisualization of roughness data is plotted inFigure 2.

Table 1. AFM roughness (RMS) data for slide and fibers supporters as a function of Iysozymedeposition time.

Sampe Deposition Average RMS stardardTirna fminl rol frrnl 'Icn*

Fibers O 1D5D ±7.75 59.4 ±3.78 4).0 ±4.610 37.3 ±6.512 33.6 ±3.0

Slides: O 80.7 ±6.75 18.1 ±5.58 15.0 ±7.010 10.7 ±5.412 9.8 ±3.2

*performed over 8 scanned area.

From the starting uncoated surface (Omin) it folIows, for both format of substrate, aroughness reduction as the deposition proceeds,which could be interpreted as the surface be-ing recovering by Iysozyme molecules, forminga continuous smooth film. Slides were foundto be initially less rough than fibers and nosignificant differences in average roughness

BioEng, Campinas, v.2, n.1, p.OO9-016, jan/abr., 2008

were found on fibers faces. Both materiais pre-sented similar rates of roughness reduction asprotein deposition proceeds, in agreement toresults found by BORATO et aI., (1997), whoreported, by ultra-violet monitoring, that Iyso-zyme film formation is a first-order process withhomogeneous distribution over surface after8-10 minutes in precursor solution immersion.

-.- fiber120 -o-slide

100E.s 80(FJ

ID 60c.cO)a 40rr:~ 20rr:

o o 2 4 6 a 10 12D3position trre (nin)

Figure 2 - RMS roughness as a function of deposition time.

The surface smoothing as enzyme ad-sorption takes place can be visually evaluatedby means of qualitative information obtainedfrom AFM images. As sequence shown inFigure 3 (for fibers scanning), it is possible,based on the appearance of the deposited film,to assess the overall reduction in roughness

(a)

intensity. After a 12-min immersion, excess for-mation of Iysozyme can be observed, which isprobably a result from clusters deposition dueto molecules aggregation while these are still insolution. Similar visual sequence was observedfor flat substrate.

(b) 7811. 'OChr r---:----,.-..,..,--...........,-.,.....--,

U.llIrn

""'

BioEng, Campinas, v.2, n.1, p.OO9-016, jan/abr., 2008

O.B.G. ASSIS, R.BERNARDES FILHO

(c)

07... ICOOrn

ralr •• ·000 r••

(d)

.~r.1

....0,, ,

'JI'I-' . t, •••

Figure 3 - AFM aspects of the scanned surfaces where changes in topographical features can be ob-served: in (a) as-hydrophilic fiber, (b) 5-min surfactant immersion (c) 10-min immersion and (d) 12-minimmersion. Ridges observed after 12-min indicate excess of deposited protein, as zoomed in (e).

Thicknesses of formed layer after 10 mindeposition were measured as 7.6 ± 1.4 for flatsurface and 10.4 ± 2.6 nm for fibers which arelarger than the size of Iysozyme molecule indi-cating that the adsorption must be multilayer.

As Iysozyme has an ellipsoidal shape withshort axis of about 3.O nm and a long axis of about4.5 nm (lU et aI., 1998), it can be assumed thata monolayerwill have the thickness equivalent toone of the axiallengths. Nevertheless, thicknessresultant of self-assembly is a quite controver-sial matter, for instance, POSTEl et aI., (2003)measured thickness as between 2.5 and 3.0 nmon Iyzozyme self-assembled monlayers. On the

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other hand, deposition as thick as 12 nm wasfound by SU et aI., (2000), while analyzingmonolayers of Iysozyme on silicon oxide surfa-ce, leading them to conclude that the amountadsorbed is time-dependent, and recently,lUBARSKY et aI., (2007) strongly states thatthickness of protein films are dependent mainlyon the solution concentration only.Anyway, many of the physical properties andactivity of mono or multilayer films depend onthe film thickness (BLANK, 1981) and our resultsindicate that despite the similarities betweendeposition on flat and cylindrical surface, underAFM analysis, flat slides appear to result in amore uniform deposition which can reflect on

activity or response signal when applied in sen-sor or biobased devices.

CONCLUSION

The immobilization of Iysozyme on chemi-cally cleaned glass surfaces resulted in a densedeposition on which the initial roughness isreduced as more material is sequentially at-tached to the solid surface. Our results indicatethat the initial surface roughness has greatereffect on deposition features than the supportformat (flat or cylindrical); i.e., despite bothmateriais presented similar rates of roughness,the initial roughness seems to be determinantfor the processo On both supporters however,the measured deposition thickness are supe-rior to an expected monolayer suggesting thatthe adsorption should be multilayer. Excess ofdeposited material was also observed at 12 minimmersion. The results from this study, althoughsemiquantitative, provide important insight intohow initial roughness or support morphology caninfluence the formation of protein film formationvia self-assembly technique.

ACKNOWLEDGMENTS

We are grateful for financial supportreceived from Conselho Nacional de Desen-volvimento Científico e Tecnológico (CNPq) andEmbrapa.

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