Word Search

Bovine serum albumin film as a template for controlled nanopancake and nanobubble formation: In situ atomic force microscopy and nanolithography study

Description
Bovine serum albumin film as a template for controlled nanopancake and nanobubble formation: In situ atomic force microscopy and nanolithography study
Categories
Published
of 7
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
Share
Transcript
  ColloidsandSurfacesB:   Biointerfaces 94 (2012) 213–219 ContentslistsavailableatSciVerseScienceDirect Colloids   and   Surfaces   B:   Biointerfaces  journalhom   epage:www.elsevier.com/locate/colsurfb Bovine   serum   albumin   filmas   a   template   for   controlled   nanopancake   andnanobubble   formation:   Insitu   atomic   force   microscopy   and   nanolithographystudy Viliam   Kolivoˇ ska a , ∗ , Miroslav   Gál a ,Magdaléna   Hromadová a , ˇ Stˇ epánka   Lachmanová a ,   Hana   Tarábková a ,Pavel    Janda a ,   Lubomír   Pospíˇ sil a , b ,   Andrea   Morovská   Turoˇ nová c a  J.Heyrovsk´ yInstituteof    PhysicalChemistryof     ASCR,v.v.i.,Dolejˇ skova3,   18223Prague,CzechRepublic  b InstituteofOrganicChemistryandBiochemistryofASCR,v.v.i.,Flemmingovonám.3,   166   10Prague,CzechRepublic  c P.    J. ˇ SafárikUniversity,FacultyofScience,Instituteof    Chemistry,Moyzesova11,04001Koˇ sice,Slovakia a   r   t   i   c   le   i   nf   o  Articlehistory: Received18August2011Receivedinrevisedform20November2011Accepted23January2012 Available online 1 February 2012 Keywords: ProteinadsorptionHighlyorderedpyrolyticgraphiteNanobubblesNanopancakesAtomicforcemicroscopyAtomicforcenanolithography a   b   s   t   ra   ct Air   nanobubblesandnanopancakes   were   investigated   in   situ   by   both   tapping   mode   atomic   forcemicroscopy   (TMAFM)   and   atomic   force   nanolithography   techniques   employing   bovine   serum   albumin(BSA)   filmsupported   byhighly   oriented   pyrolytic   graphite   (HOPG).   TheBSAdenaturation   inducedbythe   water-to-ethanol   exchangeservedfor   conservation   of    nanobubble   and   nanopancake   sitesappearingas   imprints   inBSAfilm   leftbygaseouscavitiesformerly   present   onthe   interface   inthe   aqueousenvi-ronment.   Once   the   BSAfilmwasgentlyremoved   by   the   nanoshaving   techniqueapplied   inethanol,acleanbasal   plane   HOPG   areawith   well-defined   dimensions   wasregenerated.   Thesubsequentreverseethanol-to-waterexchange   ledtothe   re-formation   of    nanopancakes   specificallyatthe   nanoshaved   area.Ourapproach   paves   the   way   for   the   study   of    gaseous   nanostructures   with   defineddimensions,   formedatsolid–liquidinterface   under   controlled   conditions. © 2012 Elsevier B.V. All rights reserved. 1.Introduction Nanobubblesaresub-microscopicgascavitiesusuallydetectedonsolid/liquidinterfaces[1].   Theoreticalcalculations[2]predictthatnanobubblesshouldbeshort-livingduetoanimmensecap-illarypressure,whichforcesthetrappedgastodissolveinasurroundingliquidaccordingtoHenry’slaw.Nevertheless,stablenanobubbleswereproventoexistatinterfaces[3–5].   Employ-ingthesurfaceforceapparatus,Parkeretal.[6]werethefirsttoreportontheexistenceof    sub-microscopicbubblesin1994.Theauthorsobservedstepsanddiscontinuitiesin   thelong-rangeattrac-tionforcesmeasuredbetweentwoneutralhydrophobicsurfacesinwaterandattributedthemto   thepresenceofnanobubbles.Theinventionofscanningprobetechniques,particularlythetapping-modeatomicforcemicroscopy(TMAFM),allowedtheinterfacialnanobubblestobeobserveddirectly.In2000,Louetal. ∗ Correspondingauthor.Tel.:+420266053188;fax:+420286582307. E-mailaddresses: viliam.kolivoska@jh-inst.cas.cz(V.Kolivoˇ ska),miroslav.gal@jh-inst.cas.cz(M.Gál),hromadom@jh-inst.cas.cz(M.Hromadová), namchal@seznam.cz(ˇ S.Lachmanová),hana.tarabkova@jh-inst.cas.cz(H.Tarábková),pavel.janda@jh-inst.cas.cz(P.Janda),lubomir.pospisil@jh-inst.cas.cz(L.Pospíˇ sil). [7]andIshidaetal.[8]independentlyreportedontheexistence ofnanobubblesbytheTMAFMtechnique.Sixyearslater,IshidaandHigashitani[9]showedthatthelong-rangeattractiveforces areindeedrelatedto   thepresenceofinterfacialnanobubbles.Thenanobubblesweredetectedonhydrophobicsurfacesonlywhena   polarliquidphase(purewateroraqueouselectrolytes)wassaturatedbytheair[1].   Therefore,theobservednanostruc-turesappeartobecavitiesfilledbyambientgasandsolventvaporrespectively,notinterfacialimpurities.Directimmersionofa   substrateintoaliquidis   themoststraightforwardwayofthenanobubbleformation.Louetal.[10,11]laterdeveloped thesolventexchangeprotocolleadingtoreproducibleappear-anceofnanobubble-containinginterfaces.Init,thesurfaceisfirstimmersedintoa   liquidthatwelldissolvestheair.Ethanolis   rou-tinelyemployed.Subsequently,ethanolisreplacedbymiscibleliquidexhibitinglowerairsolubility,suchaswater.Gradualmixingof    ethanolandwatercausesa   partoftheairdissolvedin   ethanolto“precipitate”leadingtotheformationof    nanobubblesthat   canbetrappedandsubsequentlydetectedatsolid/waterinterface.Whenwateris   replacedbackbyethanol,theinterfacialnanobubblesdis-solveanddisappear.Zhangetal.[12,13]investigatedtheliquiddegassingandtem- peratureeffectsonthenanobubbleoccurrence,employingmica 0927-7765/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.01.028  214  V.Kolivoˇ skaetal./    ColloidsandSurfacesB:Biointerfaces 94 (2012) 213–219 and   highlyorientedpyrolyticgraphite(HOPG)asthesolidsur-faces.Whenair-saturatedsolvents(ethanolormethanolandwater)wereused,thenanobubbleswereobservedwiththesurfacecon-centrationdependingonthetemperature.Nonanobubbleswerereportedtoappearwiththesolventsdegassed.Ina   relatedstudy[14],theTMAFMtechniquewasemployedtodeterminethecon-tactangleofnanobubblesonHOPGandsiliconwafersinwater.The   obtainedvalues,measuredthroughthegaseousphase,wereconsiderablysmallerthanthosefoundformacroscopicbubblesatthesameinterfaces.Thissuggeststhattherealnanobubbleradiusismuchlargerthanexpected.Accordingto   Young–Laplaceequa-tion,anincreaseinthenanobubbleradiusreducesthecapillarypressureandcontributestoitsinterfacialstability.Bytheaidof infraredspectroscopy,Zhangetal.[15]providedanelegantproof  thattheinnerspaceofnanobubblesis   composedof    moleculesinthegaseousstateratherthanofdissolvedones(CO 2  gaswaschosenduetoitsIRactivity).Recently,theflatgaseousstructurescallednanopancakeswereobservedanddescribedonHOPG[16–18].Besidesnanobubblesand nanopancakes,thesolventexchangetechniquewas   appliedforthepreparationofnanostructurescomposedofanimmiscibleliquidsuchasoil.Thesestructureswerecallednanodroplets[19,20].Detailstudiesfocusingontheinteractionofnanobubbleswithsolid/liquidinterfacesandnanostructurespresentonthemareveryrare.Jandaetal.[21]employedinsituTMAFM,transmissionelec- tronmicroscopyaswellasRamanspectroscopytodemonstratethattheairnanobubblesinducetheexfoliationof    thetopgraphenesheetsfromtheHOPGsurface.Wuetal.investigatedhowtheairnanobubblesinfluencethebovineserumalbumin(BSA)adsorp-tiononmica[22]andHOPG[23].Theinterfaceswithembedded nanobubblesformedbythesolventexchangewereexposedtoproteinsolutions.Whenaqueousenvironmentwasreplacedbyethanol,thenanobubblesvanished.Inarelatedstudy[24],theelec- trochemicallygeneratedhydrogennanobubbleswereemployedtocleantheHOPGsurfacecoveredbytheBSAfilm.Thoughthestudiesdealingwiththeinteractionsbetweenthenanobubblesandproteinsexist[22,23],thereis,to   thebestofourknowledge,noworkfocusingontheinteractionsbetweennanopancakesandproteins.Here,weshowthatinterfa-cialnanopancakessuppresstheproteinadsorptionandcausethepresenceofvacanciesintheproteinfilms.Therefore,theyconsid-erablydecreasetheproteinsurfacecoverage.Wesuggestthatthenanopancakesshouldbe(togetherwiththenanobubbles)addedtothelistoffactorsthatsignificantlyaffectthenanomorphol-ogyofinterfacialproteinlayers.Whenpresentin   largequantities,thenanopancakesmay   severelyreducetheamountof    adsorbedproteins,whichmay   causeerrorsinthesurface-basedbiochem-istryassays.Asa   modelprotein,BSAwasemployedduetoitsabilitytoadsorbonvarioussurfacesandinterfaces,includingHOPG[25–33].As   mentionedabove,theAFMimagingwasalreadyemployedtoinvestigatethestructureofinterfacialnanobubblesandnanopan-cakes[7–18],Besidestheimaging,wefurtheremploythetechniquesofatomicforcenanolithography[34–37],   namelythenanoshavingandnanografting.Nanoshavingisa   technique,inwhichaninterfacialfilmis   intentionallyremoved(“nanoshaved”)byanAFMprobe.Nanograftingresemblesthenanoshaving,buttakesplaceinasolutionofadsorbablespeciesdifferentfromthatalreadyadsorbedattheinterface.Thenewlyexposedsubstrateisrefilledbythespeciesfromthesolutionbulk,formingthusatwo-componentfilmwithmorphologydictatedbytheshapeof thenanoshavedarea.Thoughbothnanoshavingandnanograftingtechniqueshavealreadybeenappliedtoinvestigateandpatternthestructureofself-assembledmonolayers[38–49],   thereis   noworkusingtheseadvancedandsmarttechniquesin   theresearchofgaseousnanocavities.Thescientistsreportingontheexistenceofnanopancakessofardescribedthemasspontaneouslyformednanostructures[16–18].Onthecontrary,ourapproachallowstheirintentionalforma-tion.Wearethefirstto   showthatnanoshavingandnanograftingtechniquescombinedwiththesolventexchangeare   abletoformgaseousnanopancakesatdefinedsurfacelocationsandwithpre-scribedshapesanddimensions.Infuture,thistechniquemightbeemployedinthefabrica-tionofwell-definedgaseousarrays.We   believethattheabilityof    controllingthegasnanocavitydimensionswillhelpto   gainadeeperknowledgeabouttheirformation,stabilityandproperties,whichmay   haveseriousimplicationsinheterogeneouscatalysisatsolid/liquidinterfacesaswellasinthestudiesoftransmembranegasexchangeprocessesingeneral. 2.Materialsandmethods Bovineserumalbumin(BSA)was   purchasedfromSigma–Aldrich(fractionV,>96%).Absoluteethanol(99.8%)wasobtainedfromAppliChem,Darmstadt,Germany.Deionizedwaterwithaminimumresistivityof    18M  cmwasobtainedbymeansofa   Milli-QRGpurificationsystem(MilliporeCo.,USA).Thewatersolutionof    BSA(40ppm)waspreparedbydissolvingsolidBSAin   deionizedair-saturatedwater.Highlyorientedpyrolyticgraphite(HOPG,StructureProbeInc.,USA)wascleanedbyethanol,driedandcleavedbythescotchtapeimmediatelypriortouse.ThegaseousnanostructuresformedontheHOPG/waterinter-facewereinspectedbytheinsitutappingmodeAFM(TMAFM)technique.Theinterfacewas   thenexposedto40ppmwatersolu-tionofBSAfor30min   [23].   TheBSAsolutionwassubsequentlyremovedbyextensiverinsingwithpurewaterandtheformedinterfacialstructureswerere-inspectedbyin   situTMAFM.After-wards,theaqueousenvironmentwas   replacedbyethanolandtheinterfacewas   investigatedbyTMAFMimagingandwasfurthermodifiedbyatomicforcenanolithographytechniques(nanoshav-ingandnanografting).AllAFMmeasurementswerecarriedoutin   situemployingAgi-lent   5500SPM(AgilentTechnologies)equippedbyaflowliquidcell(AgilentTechnologies).Theprobes“TypeIIMAClevers”(Agi-lentTechnologies)wereemployed,withthenominalresonantfrequency  f  N  =75kHz(intheair,referencerange45–115kHz)andnominalforceconstant k N  =2.8   N/m(referencerange0.5–9.5N/m).Theactualforceconstant k   was   determinedbymeasuringtheres-onantfrequency  f  andemployingthecubicinterpolationin   the k vs.  f  dependence.AllAFMimageswereobtainedbytheTMAFMtechnique.Theprobeswereoscillatedcloseto   theresonantfrequencybyanexter-naloscillatingmagneticfield.Therelativeprobeamplitude  A r   (theratioof    theset-pointamplitude  A maintainedwhenimagingandtheamplitudeof    thefreelyvibratingproberetractedfromtheinterface  A 0 )isrelatedtothepressurethat   isexertedbytheprobeontothesample.Fortherelativeamplitude,weemployedthevalue95%throughoutthemeasurements.Besidesthetopographyimage,thephaseshiftbetweenthedrivingmagneticfieldandtheresultingcantileveroscillationwasrecorded.Thenegativechangesin   thephaseshiftvalueare   relatedtotheretardationoftheprobe,whichiscausedbypronouncedinteractionsbetweentheprobeandinterfacialnanos-tructures.TheAFMnanoshavingwasperformedin   a   constantrepulsiveforcecontactmodeAFM(CMAFM).Priorto   eachexperiment,theforcespectroscopymeasurementwasperformedinsitutodeter-minetheexactloadingforceapplied.Thesensitivity S   ofagivenlaser/cantileverconfiguration,definedastheratioofthelaserbeamdeflection D (involts)andthecantileververticalposition  z  (inmeters),wasdeterminedasa   slopein   therepulsivepartof   V.   Kolivoˇ skaetal./ColloidsandSurfacesB:   Biointerfaces 94 (2012) 213–219 215 Fig.1. TypicalinsituTMAFMtopography(a–c)andphase(d–f)   imagesof    HOPGobtainedinpurewaterbefore(aandd)andafter(bande)the   adsorptionof    BSAandinethanol   (candf).Theimagesareobtainedat   the   samesurfacelocation(size5  m).   The  z  scalein(a),(b)and(c)is35,13   and20nm,respectively.Thephaseimages(d–f)havethescale10 ◦ .Probefrequencyandrelativeamplitudeis   64   kHzand98%,respectively. the   laserbeamdeflection–distance( D vs.  z  )curve.Theobtainedvalueof  S   wasemployedto   calculatethecantileverdeflection,   z  =  z  (surface) −  z  (retractedprobe),fromthelaserbeamdeflection  D = D (surface) − D (retractedprobe).The  D valuewas   keptcon-stantbythefeedbackelectronics.Theappliedrepulsiveloadingforce F  wascalculatedfromHooke’slaw F    =   − k   z  .Typically,the F  valueswerein   therangeof    20–30nN.BothAFMimagingandnanolithographywasperformedwiththesameprobeatthespeed0.5–1.2lines/s.TheAFMimagesshownbelowareplane-corrected.Theimageswereanalyzedemploy-ing   thescanningprobemicroscopydatavisualizationandanalysisprogramGwyddion2.20(CzechMetrologyInstitute,Brno,CzechRepublic)[50–52]. 3.Resultsanddiscussion DirectimmersionoffreshlycleavedHOPGintodeionizedwaterreportedly[1]f ormsinterfaceswithrelativelylowandirrepro- duciblenanobubbleoccurrence,whileJandaetal.[21]reports onbasalplaneHOPGevenlycoveredbynanobubblesjustuponitsimmersionin   air-saturatedwater.Importantlyhowever,nonanopancakesareformedundertheseconditions.Therefore,weemployedthedirectimmersiontechniqueto   obtainHOPG/waterinterfacecontainingexclusivelynanobubbles.TheHOPG/waterinterfacecontainingbothnanobubblesandnanopancakesisobtainedbythegentleethanol-to-watersolventexchangetechnique[16–18].Absoluteethanolslowlydisplacedby deionizedair-saturatedwaterprovidestheconditionsforthefor-mationofbothtypesofgaseousnanostructuresonHOPG.Inthiswork,thistechniquewas   appliedto   obtainthemixturesofgaseousnanobubblesandnanopancakes.Panel(a)of Fig.1showsa   typicalinsituTMAFMtopographyimageoftheHOPG/waterinterfaceformedbythedirectimmersionoffreshlycleavedHOPGin   deionizedair-saturatedwater.Theimageis   obtained2haftertheHOPGimmersion.Nanobubblesappearasbrightsphericalobjects.Nonanopancakesare   formed.Panel(b)showstheinsituTMAFMimageofthesamesampleareaasin(a)upontheadsorptionof    BSA.Weremindthat   thepro-teinintroductionwasfollowedbyanextensivewaterrinsingandtheimage(b)isobtainedinpurewater.Importantly,theimages(a)and(b)showthesamefeaturesinthestructureof    bothnanobubblesandunderlyingHOPGsubstrate.ThenanobubblesimagedinthepresenceofadsorbedBSAareofalmostsphericalshape(b).Theirpresenceisalsoclearlydis-cernibleinthephaseimaging,whichtracesthedifferencesintheenergydissipatedbytheinteractionsbetweentheoscillatingAFMprobeandtheexaminedsurface.It   isusefulcomplemen-taryinformationwhenimagingelasticsurfacenanostructuressuchasgaseousnanocavities.Pronouncedinteractionsbetweentheprobeandnanobubblescausea   proberetardation,whichmani-festsitselfasa   negativephaseshiftwithrespecttothatobtainedatweaklyinteractingnanobubble-freeregions.Thepanels(d)and(e)showthephaseimagespertainingto(a)   and(b),respectively.Theregionswitha   negativephaseshiftappeardarkandcorrespondtonanobubbles.Panels(c)and(f)showthetopographyandthephaseimageobtainedafterthesolventexchangeto   ethanol.ThebrightfeaturespresumablybelongtoBSAmoleculesadsorbedattheethanol/HOPGinterface.Theaverageheightofthestructuresis8.5nm,   whichwellagreeswiththevaluefoundbyWuetal.(ca.8nm)   [23].Whilein   purewatertheBSAmoleculeformsa   prolateellipsoidwiththedimensionsof    14nm × 4nm × 4nm[53],   thedenatura-tionfollowedbytheaggregationtakesplaceinaqueoussolutionsof    ethanol[54].Moreover,proteinmoleculesunfoldathydropho- bicsurfaces,suchasHOPG[55].InaccordancewithWuetal.[23], theBSAfilmcontainsvacanciesatpositionspreviouslyoccupiedbythenanobubblesinwater.Thevacanciesare   depictedbydotted  216  V.Kolivoˇ skaetal./    ColloidsandSurfacesB:Biointerfaces 94 (2012) 213–219  b 2000150010005000 010203040   z   /  n  m x/nm  a c Fig.2. TopographyprofilesatthelocationdenotedbywhitelinesinFig.1,   obtainedin   purewaterbefore(a)andafter(b)theadsorptionofBSA.Profile(c)was   obtainedafter   thewater-to-ethanolexchange.Thelinesareverticallyshiftedforabettervisualization. circlesin(c)inFig.1.Thecorrespondingphaseimage(f)showsno domainswithdiscerniblenegativephaseshiftclearlyconfirmingtheabsenceofthenanobubblesatHOPG/ethanolinterface.However,thenanobubblesarenottheonlysourceofholesintheBSAfilminethanol.Somevacanciesin   theproteinfilmarewherenonanobubblesweredetectedin   theaqueousenvironment.Wesuggestthattheirpresenceisattributedto   theproteindenaturationinthecourseofthesolventexchangetoethanol[54].   Inpurewater,theBSAfilmonHOPGwasfoundtohavetheaveragethicknessof    ca.5nm.   Asmentionedabove,itis   8.5nminethanol,suggestingthatthetwofilmshavedifferentnanomorphology.AsnoBSAmoleculesarepresentinthebulkof    thesolutioninthecourseofthesolventexchange,anincreaseinthefilmthicknesshastobecompensatedbyadecreaseinthesurfacecoverage.Thisexplainsthepresenceof vacanciesthatdonotcorrespondto   formernanobubblesinwater.Theimagesshownin   (a)–(c)inFig.1werefurthersubjectedtothesectionanalysis.Fig.2showstopographyprofilesobtainedat thesurfacelocationdenotedbysolidwhitelinesin   (a)–(c)inFig.1.TheprofilesinwaterbeforeandupontheBSAintroductionareshownaslines(a)   and(b),respectively.Thetopographymax-ima   at  x =480nmand1500nmcoincidewiththetopsitesof    twopronouncednanobubbles.Thepresenceof    theadsorbedproteincausesthenanobubblesto   shrink(b).Thechangeinthenanobub-bleimagedsizeandshapeupontheproteinadsorptioncannotbeattributedtovaryingimagingconditionsasbothrelativeandabso-luteamplitudevalueswerecarefullykeptconstantthroughouttheexperiments.Itis   knownthatthepressureexertedbythetiponthesamplesurfaceisinverselyproportionaltotheprobeamplitude[21].Therefore,theexertedtippressureandforceontothesamplearealsoconstant.We   suggestthatthechangein   thenanobubbleshapeand,moreimportantly,adecreasednanobubbledistortionbytheAFMprobewhenscanningis   attributedtothestabilizationof thenanobubblegas/liquidinterfacebytheproteinfilm[55],which atthesametimecanlocklateralnanobubblelateralmovement.AsimilarstabilizationandshrinkingwasalsoreportedbyZhangetal.[14],whoinvestigatedtheeffectof    cationic,anionicandneutralsurfactantsonthestabilityandshapeof    nanobubbles.Interactionsbetweennanobubblesandproteinappeartosignificantlyaffecttheprocessofproteinadsorptionandhencethenanomorphologyof theinterface.Asseeninpanel(c),   proteinfilmcontainsvacan-ciesat   positionswherenanobubblesweredetectedin   theaqueousenvironment.Inconclusion,theoccurrenceandpositionsofnanobubblesinwaterarenotaffectedbythepresenceof    adsorbedBSAfilm.Ontheotherhand,theirimagedshapeischanged.Thenanobubblesdissolveinthecourseof    thewater-to-ethanolsolventexchange,leavingvacanciesin   thecorrespondingpositionsintheBSAfilminethanol.Ournextgoalwasto   investigatetheinteractionsbetweentheBSAfilmandnanopancakesonHOPG.Asmentionedabove,thenanopancake/nanobubbleassemblieswerepreparedbymeansof theethanol-to-waterexchangetechnique[16–18].   Fig.3showstypicalinsituTMAFMimagesof    HOPG/waterinterfacepriorandupontheintroductionof    BSA.Panel(a)   showsthein   situTM   AFMtopographyimageof    pristineHOPGin   waterobtained2haftertheethanol-to-waterexchange.Largenanobubblesmay   be   noticedandtheirprominentrepresen-tativesaredenotedbysolidwhitecircles.Smallernanobubblesareassembledingroups(dottedwhiteellipses).Bothlargeandsmallnanobubblesappearasbrightobjectssimilartothoseinpanels(a)   and(b)of    Fig.1.Besidesthenanobubbles,largeandmutu-allyinterconnectedpatch-likenanopancakesare   found(solidwhiterectangles).At   somelocations,holesinthenanopancakesmay   benoticed(whitearrows).Theimageshownin   panel(b)of    Fig.3isobtainedin   purewaterupontheintroductionofBSA.Similarlyto   Fig.1,thestabilityofgaseousnanostructures(nanobubblesandnanopancakes)withrespecttoAFMimaginghasimproved(compare(a)and(b)inFig.3).Importantly,theoccurrenceandpositionsofbothtypesof    surfacenanostructuresispreserved.In   bothpanels,thenanobubblesandnanopancakesappearasbrightspheres(solidcirclesanddottedellipses)andpatches(solidrectangles),respectively.Thepositionof thevacanciesinthenanopancakesisalsoretained(whitearrows).Thedifferencebetweennanobubblesandnanopancakesisnoticeableinthephaseimage(c)of Fig.3,shownforthesame surfaceareaasin(a)and(b).Asmentionedabove,nanobubblesmanifestthemselvesashavinga   pronouncednegativephaseshiftandappearasblackspheres(c).Negativephaseshiftislesspro-nouncedfornanopancakes(darkgraypatches),butstilldifferentfromdomainswherenogaseousstructuresareformed(lightgrayregions).Panel(d)   inFig.3showsthetopographyimageobtainedafter thesolventexchangetoethanol.Similarlytopanel(c)inFig.1,   theregionsoccupiedbynanobubblesin   waterbecomebarein   ethanol.Thenanobubblesexcludetheproteinadsorptioninwaterandtheydissolveinthecourseofthesolventexchange(comparesolidcirclesin(a)–(d)inFig.3).Similarly,theareasoccupiedbynanopan- cakesintheaqueousenvironmentbecomebareuponthesolventexchange(comparesolidrectanglesin(a)–(d)inFig.3).Therefore, thenanopancakesdissolveinthecourseof    thesolventexchangeinthesameway   asnanobubbles.Vacanciesinthenanopancakesnoticedintheaqueousenvironment(whitearrowsinthepanels(a)–(c)inFig.3)becomecoveredbyBSAmolecules,asobservedin paneld   (blackarrows).Fromthenanomorphologyof    theadsorbedproteinfilminethanolafterthenanobubbleshavevanisheduponwater-to-ethanolexchangeweassumethatbothnanobubblesandnanopan-cakesplay,besidessurface-protective,alsonanomorphology-determiningrole.Theyappeartoactivelyparticipatein   thecharacterof    theproteinlayer.Presumably,thebridgingeffect[56]caninfluencethemechanismof    layinguptheproteinformerlylocalizedatthenanobubble(nanopancake)gas/waterinterface.Insomecases,evennanobubble-drivennetworkassemblieswereobserved[21].AtomicforcenanolithographytechniqueswerefurtheremployedtoinvestigatethenanomorphologyoftheBSAfilminethanol.Inparticular,CMAFMsquarenanoshavingwiththesizeof1  m ×   1  mwasappliedin   ethanolpriortoobtainingtheTMAFMimageshownin   panel(d)of Fig.3.   ThisactioninducedtherelocationofBSAmoleculesinthesquareregion,regeneratingthebasalplaneof    HOPGsubstrateunderexistingin   situconditions.SubsequentTMAFMimagingresolvedtheflatandcleanHOPGsubstratewithcharacteristicsteps,asseenin   themiddlepartof panel(d).Wenotethatblankinsitunanoshavingexperiments  V.   Kolivoˇ skaetal./ColloidsandSurfacesB:   Biointerfaces 94 (2012) 213–219 217 Fig.3. InsituTMAFMtopography(a,   b,d,ande)andphase(candf)imagesof    HOPGinwaterbefore(a)   andafter(bandc)theBSAadsorptionandinethanolupon1    m × 1  mnanoshaving(d).   Panels(eandf)   areobtainedafterthereverseethanol-to-waterexchange.Thetopographyscalein(a),(b),(d)an(e)   is   25,5,   15and10nm,respectively.Phaseimages(c)and(f)correspondto   thosein(b)and(e),withthescale10 ◦ . employingpureHOPGin   ethanolshowedthatthissubstratewithstandsappliedforcesatleastanorderofmagnitudehigherthanthatappliedintheexperimentshownin   Fig.3.ThisconfirmsthattheAFMprobedisplacestheBSAfilmwithoutthedamageof theunderlyingHOPG.Subsequentreplacementofethanolbywaterled   totherefor-mationofHOPG/waterinterface,whichwasre-inspectedbyTMAFM.Panels(e)and(f)showtheresultingtopographyandphaseimages.Dottedsquaresin(e)   and(f)denotethesquareareapre-viouslysubjectedtothenanoshavingappliedin   ethanol.Panel(e)showsthatthisregionbecomescoveredbylargenanopancakesconfinedbythegeometryofthenanoshavedregionaswellasbythestepsintheunderlyingHOPGstructure.Thephaseshift(f)inthenanoshavedregion(darkgray)ismorenegativethanthatof thesurroundingareaoccupiedbyBSAmolecules(lightgray).ThissuggeststhattheHOPGsurfacesubjectedtonanoshavingbecomescoveredbygaseousnanopancakes(andnotbytheBSAmolecules).Naturallyoccurringstep-confinednanopancakeswerealreadyproventoexistonpureHOPGinwater[16].   Hereweshowthatthesestructurescanbe   intentionallygeneratedbythenanoshavingoftheproteinadlayer.Ascanbeseenin   panel(e)   of    Fig.3,threenanobubblesarelocatedonthetopof    thenanopancake(denotedbytheequilateraltriangle).Suchcomposedgaseousnanocavitieswerealreadyreported[18].Beyondthenanoshavedarea,theproteinassemblyis   partiallypreserveduponthereverseethanol-to-waterexchange(compareadottedellipse,solidcirclesanda   rectangleinthephaseimages(c)and(f)inFig.3).However,mostof    thestructuresarechanged,which,inturn,affectsthenanomorphologyofnewlyformedinter-facialgaseousnanostructures.Theimages(a)–(d)in   Fig.3werefurthersubjectedtothesec-tionalanalysis.Fig.4showstherespectiveprofiles.Thenanobubble centeredat  x   =400nm(dottedverticalline)shrinksupontheproteinadsorption(b)andshowsa   negativephaseshift(c).Thecorrespondingregionbecomesunoccupiedin   ethanol(d).Thesameeffectisalsoobservedforthenanopancake(  x =   1000nm,solidver-ticalline).Here,thenegativechangeinthephaseshiftvalueislesspronounced.Thisis   inagreementwiththeimages(c)and(f)inFig.3,wherethenanobubblesformblacksphereswhereas thenanopancakesappearasdarkgraypatchesi.e.witha   lesspro-nouncednegativechangeofthephaseshift.Asmentionedabove,thedirectimmersiontechnique(Figs.1and2)   producesthenanobubbles(andnonanopan-cakes)onHOPG/waterinterface.To   confirmthereproducibilityof  1400120010008006004002000 5 deg5 nm   z ,      ϕ x/nm a  b c d Fig.4. Topography(a,b,andd)andphase(c)profilesat   thedottedlinesinFig.3obtainedinpure   waterbefore(a)   andafter(bandc)theproteinadsorptionandinethanol(d).Dottedandsolidverticallinesrefer   to   positionof    thenanobubbleandthenanopancake,respectively.
Search
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x