adsorption polymerization for DNA Surface modiﬁcation electrospun cellulose of acetate nanoﬁbers viaRAFT Carbohydrate Polymers

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Carbohydrate Polymers

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Surface modiﬁcation of electrospun cellulose acetate nanoﬁbers via RAFT polymerization for DNA adsorption

Serkan Demirci

^a,c,∗

, Asli Celebioglu

^a,b

, Tamer Uyar

^a,b,∗∗

aUNAM-NationalNanotechnologyResearchCenter,BilkentUniversity,06800Ankara,Turkey

bInstituteofMaterialsScienceandNanotechnology,BilkentUniversity,06800Ankara,Turkey

cDepartmentofChemistry,FacultyofArtsandSciences,AmasyaUniversity,05100Amasya,Turkey

a r t i c l e i n f o

Articlehistory:

Received13January2014

Receivedinrevisedform22June2014 Accepted23June2014

Availableonline11July2014

Keywords:

Electrospinning Celluloseacetate Nanoﬁber Surfacemodiﬁcation RAFTpolymerization DNAadsorption

a b s t r a c t

Wereport on afacile and robust method by whichsurface of electrospuncellulose acetate (CA) nanofiberscanbechemicallymodifiedwithcationicpolymerbrushesforDNAadsorption.Thesurface ofCAnanofiberswasfunctionalizedbygrowingpoly[(ar-vinylbenzyl)trimethylammoniumchloride)]

[poly(VBTAC)]brushesthroughamulti-stepchemicalsequencethatensuresretentionofmechanically robustnanofibers.Initially,thesurfaceoftheCAnanofiberswasmodifiedwithRAFTchaintransferagent.

Poly(VBTAC)brusheswerethenpreparedviaRAFT-mediatedpolymerizationfromthenanoﬁbersurface.

DNAadsorptioncapacityofCAnanofibrouswebsurfacefunctionalizedwithcationicpoly(VBTAC)brushes wasdemonstrated.Thereusabilityofthesewebswasinvestigatedbymeasuringtheadsorptioncapacity fortargetDNAinacyclicmanner.Inbrief,CAnanofiberssurface-modifiedwithcationicpolymerbrushes canbesuitableasmembranematerialsforfiltration,purification,and/orseparationprocessesforDNA.

1. Introduction

Electrospinningtechniquehasrecentlyreceivedgreatattention sincethisversatiletechniqueenablestheproductionofmultifunc- tionalnanoﬁber/nanowebsfromawiderangevarietyofmaterials includingpolymers,blends,alloys,composites,ceramics,metals etc. (Agarwal, Greiner, & Wendorff, 2013; Sahay et al., 2012;

Wendorff,Agarwal,&Greiner,2012).Theelectrospunnanoﬁbers andtheirnanowebshavedistinctcharacteristicsuchasveryhigh surfaceareatovolumeratio,nanoscaleporousmorphologyand thediversesurfacefunctionalitiesarealsopossible(Agarwaletal., 2013; Kayaci, Ozgit-Akgun, Biyikli, & Uyar, 2013; Lu & Hsieh, 2010;Muller,Rambo,Porto,Schreiner,&Barra,2013;Sahayetal., 2012;Uyar,Havelund,Hacaloglu,Besenbacher,&Kingshott,2010;

Wendorffetal.,2012).Thesepropertiesofelectrospunnanoﬁbers

∗ Correspondingauthorat:UNAM-NationalNanotechnologyResearchCenter, BilkentUniversity,06800Ankara,Turkey.Tel.:+903582421614;

fax:+903582421616.

∗∗ Correspondingauthorat:UNAM-NationalNanotechnologyResearchCenter, BilkentUniversity,06800Ankara,Turkey.Tel.:+903122903571;

fax:+903122664365.

E-mailaddresses:srkndemirci@gmail.com,serkan.demirci@amasya.edu.tr (S.Demirci),uyar@unam.bilkent.edu.tr,tamer@unam.bilkent.edu.tr, tameruyar@gmail.com(T.Uyar).

makethemgoodcandidatesasaffinitymembranesforfiltration, purificationandseparationprocesses(Ma,Kotaki,&Ramakrishna, 2005;Zhu,Yang,&Sun,2011).Surfacemodificationofelectrospun nanofiberswithspecificfunctionalgroupsisofgreatinterestdue totheirpotentialapplicationinfiltration/separation,detectionand controlleddrugrelease(Chigome,Darko,&Torto,2011;Fu,Xu,Yao, Li,&Kang,2009a;Kampalanonwat&Supaphol,2010;Mahanta&

Valiyaveettil,2011;Wangetal.,2012;Yao,Xu,Lin,&Fu,2010).Par- ticularly,polymerfunctionalizednanofibersthatcanbeobtained vialivingradicalpolymerization(LRP)techniquesmayhaveahigh adsorptioncapacityandstrongbindingspecificityduetotheirfree activegroupsonnanofibersurfaces(Chigomeetal.,2011;Yaoetal., 2010).

LRP reveal advantages because they can control the archi- tecture,molecularweightand molecularweight distributionin comparisonwiththeconventionalradicalpolymerization,dueto minimalterminationreactionsresultinginpolymerchainswith

“living”endgroups(Coessen,Pintauer,&Matyjaszewski,2001;Fu etal.,2009a,2009b;Matyjaszewski,2012;Pyun&Matyjaszewski, 2001).SeveralLRPtechniqueshavebeenreportedforpreparation polymerfunctionalizedsurfaces,suchasnitroxide-mediatedrad- icalpolymerization(Bian&Cunningham,2006;Cimen&Caykara, 2012),atomtransferradicalpolymerization(Bai,Zhang,Cheng,&

Zhu,2012;Bian&Cunningham,2006;Turan,Demirci,&Caykara, 2010), reversible addition-fragmentation chain transfer (RAFT)

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S.Demircietal./CarbohydratePolymers113(2014)200–207 201

polymerization(Demirci&Caykara,2012;Gurbuz,Demirci,Yavuz,

&Caykara,2011;Jiang&Xu,2013)andsingle-electrontransfer- livingradicalpolymerization(Demirci,Kinali-Demirci,&Caykara, 2013;Percecetal.,2006).LRPprovidesexcellentcontroloverthe molecularweightandpolydispersityofgraftpolymers(Braunecker

&Matyjaszewski,2007;Dietrichetal.,2010).AmongallLRPtech- niques,RAFTpolymerizationisthemostimportantone,duetoits compatibilitywithawiderangeofmonomersandreactioncondi- tions(Chen,Liu,Chen,Gong,&Gao,2011;Moadetal.,2010).The stabilityandchemicalversatilityinherentinRAFTagentsmakes RAFT-based procedures highly attractive for the preparation of well-deﬁnedpolymerswithspeciﬁcpolymerarchitectures(Smith, Holley,&McCormick,2011).

Theelectrospunnanofibrouswebshaveshowngreatpotential to be used as affinity membranes for separate/capture macromolecules,microorganism,enzymes,heavymetalionsandwaste compounds(Che,Huang,&Xu,2011;Lu&Hsieh,2009;Maetal., 2005;Wangetal.,2011;Zhangetal.,2010).Forexample,Zhang etal.(2010)presentedanewaffinitymembranewhichwassurface modified electrospunpolyacrylonitrilenanofibers forbromelain adsorption.Cheetal.(2011)reportedthatglycosylatednanofibrous membraneshowedstrongselectivity,highadsorptioncapacityand reversiblebindingcapabilitytothespecificproteinconcanavalin A. DNA is thegenetic material of living organisms, and deter- mination ofDNA plays animportant role in many applications rangingfrommedical,forensic,agricultureandfoodsciences(Tao, Lin,Huang,Ren,&Qu,2012).Differentsolidsupportshavebeen usedforadsorptionofDNAduetotheeaseofhandling,andless chemicalrequirements. Wanetal.(2013)reportedthata novel electrochemicalDNAsensorbasedonsurfaceinitiatedenzymatic polymerization. ThisDNA sensor had picomolar sensitivity and broaddetectionrange.EfficientmethodformultipleDNAdetection byexploringsilvernanoclustersandgrapheneoxidenanohybrid materialswasdevelopedbyTaoetal.(2012).Inourpreviousstudy, wealso showedthat cationicpolymer brushescanbeused for quantitativeDNAadsorption(Demirci &Caykara,2013).But,all ofthesematerials,especiallypolymercoatedsolidsurfaceshave lowadsorptioncapacity,becauseofthelowsurfacearea(Baser, Demirel,&Caykara,2011;Rahman&Alaissari,2011).

Herein, poly[(ar-vinylbenzyl)trimethylammonium chloride]

[poly(VBTAC)] grafted cellulose acetate (poly(VBTAC)-g-CA) nanofiberweresuccessfullyproducedbycombinationofelectro- spinning and RAFT polymerizationwith the goalof fabricating affinitymembraneforDNAadsorption.Morphologicalandsurface characteristics of the poly(VBTAC)-g-CA nanofibers were car- riedoutbyscanningelectronmicroscope(SEM),Attenuatedtotal reflectance-Fouriertransforminfrared(ATR-FTIR)spectroscopy,X- rayphotoelectronspectroscopy(XPS)andcontactanglemeasure- ments.Furthermore,DNAadsorptionofpoly(VBTAC)-g-CAnanofi- brouswebfromthebuffersolutionwasinvestigated.Thereusabil- ity of poly(VBTAC)-g-CA nanofibrous web was also tested by measuringtheadsorptioncapacityfortargetDNAafterfivecycles.

2. Materialsandmethods 2.1. Materials

Cellulose acetate (CA, Mw∼30,000, 39.8wt.% acetyl), (ar- vinylbenzyl)trimethylammonium chloride (VBTAC, 99%), 4,4- azobis(4-cyanopentanoic acid) (ACPA, ≥98%), benzyl chloride (99%), sulfur (≥99.5%), potassium ferricyanide(III) (99%), N,N’- dicyclohexylcarbodiimide (DCC, 99%), 4-dimethylaminopyridine (DMAP, ≥99%), acetic acid (99.7%), sodium acetate (≥99%), diethylether(≥99%),dichloromethane(DCM,98.5%),ethylacetate (99.8%), methanol (99.8%) were purchased commercially from

Sigma–Aldrich.ACPAwasrecrystallizedfrommethanol.Thewater wasusedfromaMilliporeMilli-Qultrapurewatersystem.Double- strandedDNA(Cy3-labeledDNA)of50basepairfromBioVentures Inc.wassupplied.

2.2. Electrospinning

Thehomogenouselectrospinningsolutionwaspreparedbydis- solvingCAin DCM/methanol (4/1(v/v))binarysolvent mixture at12%(w/v)polymerconcentration.ThenclearCAsolutionwas placedina3mLsyringefittedwithametallicneedleof0.6mm innerdiameter.Thesyringewasfixedhorizontallyonthesyringe pump(KDScientific).Theelectrodeofthehigh-voltagepowersup- ply(Matsusada Precision,AUSeries)wasclamped tothemetal needletip,andthecylindricalaluminumcollectorwasgrounded.

Theparametersoftheelectrospinningwereadjustedas;feedrate ofsolutions=1mL/h,theappliedvoltage=15kV,andthetip-to- collectordistance=10cm.Electrospunnanoﬁbersweredeposited onagroundedstationarycylindricalmetalcollectorcoveredwitha pieceofaluminumfoil.Theelectrospinningapparatuswasenclosed inaPlexiglasbox,andelectrospinningwascarriedoutat25^◦Cat 25%relativehumidity.Thecollectednanoﬁbersweredriedatroom temperatureunderthefumehoodovernight.

2.3. RAFTpolymerizationofVBTAC

4-Cyanopentanoicaciddithiobenzoate(CPAD)wassynthesized accordingtotheliterature procedure(Mayadunneet al.,1999).

CPAD(1.14g,4.095mmol),DCC(0.844g,4.095mmol),DMAP(0.5g, 4.095mmol), and 10mL of benzene were added to a round bottomedﬂaskandstirredforfewminutesundernitrogenatmo- sphere.TheCAnanoﬁberswereadded,andthemixturewasstirred overnightatroomtemperature.Theproductwaswashedwithben- zene,2-propanolandwaterseveraltimesanddried.

TheRAFT-mediatedpolymerizationofVBTAC(29.4mmol)was carriedoutinbuffer(28mL,pH=5.0,0.27mol/Laceticacid,and 0.73mol/Lsodiumacetate),initiatorACPA(0.025mmol),freeRAFT agentCPAD(0.125mmol),andRAFTchaintransferagentimmo- bilizedCAnanofiberat 0^◦C in around-bottom flask.To ensure smoothstirringandpreventdamagetothenanofibers,weisolated themagneticstirringbaratthecenterofdevicefromtheslidesby a1cmhighglassO-ring.Thesolutionwasdilutedto30mLvolume withthebuffersolutionanddegassedbypurgingwithnitrogen for20min.Thepolymerizationreactionwasstirredvigorouslyat 70^◦Cfor120min.Thepoly(VBTAC)-g-CAnanofiberswererecov- eredfromthereactionmixtureandrepeatedlywashedwithbuffer and watertoremovetheunreactedchemicals,anddriedunder vacuumat30^◦C.

2.4. Measurementsandcharacterization

Attenuatedtotalreflectance-Fouriertransforminfrared(ATR- FTIR)spectraoftheCAandpoly(VBTAC)-g-CAwereobtainedusing a ThermoNicolet6700spectrometerwitha Smart Orbitatten- uated total reflection attachment.The spectra were taken at a resolution4cm⁻¹after128scanaccumulationforanacceptable signal/noise ratio. The X-ray photoelectron spectra of samples wererecordedbyusingX-rayphotoelectronspectrometer(XPS) (ThermoScientific).XPSwasusedbymeansofafloodguncharge neutralizer systemequippedwitha monochromated Al K-␣X- raysource(h⁼^1486.6êV).^Chargingneutralizingequipmentwas usedtocompensatesamplecharging,andthebindingscalewas referencedtothealiphaticcomponentofC1sspectraat285.0eV.

The morphologies of the electrospun CA and poly(VBTAC)-g- CAnanoﬁberswereinvestigatedwithaﬁeld emissionscanning electronmicroscope(FE-SEM)(FEI,Quanta200FEG).Sampleswere

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Scheme1. (a)SchematicrepresentationofelectrospinningofCAnanofibers.(b)Sequenceofsurfacemodificationstepsemployedinthisstudytofunctionalizeelectrospun CAnanofiberswithpoly(VBTAC).(c)DNAadsorptiononpoly(VBTAC)-g-CAnanofibers.

sputteredwith5nmAu/Pd(PECS-682)andaround100ﬁberdiam- etersweremeasuredfromtheSEMimagestocalculatetheaverage ﬁberdiameterofeachsample.Thewatercontactanglemeasure- mentswereconductedatroomtemperatureusingagoniometer (DSA100, Krüss)equippedwitha microlitersyringe.Deionized water(5␮L,18Mcmresistivity)wasusedasthewettingliquid.

Evidenceofthenonspeciﬁcadsorptionwasobtainedbymeansof opticalﬂuorescencemicroscopy.Fluorescencemicroscopyimages wererecordedbyusinganOlympusBX51microscopewitha40× objective.

2.5. DNAadsorptionstudy

DNAsolutionwaspreparedinanaqueoussolutionofphosphate buffer saline (PBS). The adsorption of DNA onto poly(VBTAC)- g-CA nanoﬁbers was performed in a 3.0mL volume. 10mg of poly(VBTAC)-g-CA nanoﬁbers was added to 2.0mL DNA (20.0␮g/mL)containingbuffersolution(i.e.adsorptionmedium).

Attheendoftheincubationtime, poly(VBTAC)-g-CAnanofibers wereseparatedquickly.TheDNAconcentrationwasdetermined bymeasuringtheabsorbanceat260nminaUV–visspectropho- tometerusingastandardcalibrationcurve.Alloftheabsorbance values were measured at least three times and averaged. The amountofDNAadsorbedontothepoly(VBTAC)-g-CAnanofibers (␮gDNA/mgdrynanofibers)wascalculatedfromtheinitialand finalDNAconcentrationsintheclearphase.Anaverageresultof minimumthreereproducibledatawasconsideredallowingerrors in ±0.05␮g/mg poly(VBTAC)-g-CA nanofibers. In order to test thereusabilityofpoly(VBTAC)-g-CAnanofibersforDNAadsorp- tion,fivecyclesofadsorption/desorptionwerecarriedoutusing buffersolution(potassiumhydrogenphthalate/hydrochloricacid, pH=3.0).Aftereachadsorption/desorptioncycle,DNAconcentra- tionwasdeterminedbyUV–visspectrophotometer.Inordertouse thepoly(VBTAC)-g-CAnanofibersforthenextexperiment,itwas washedwithbuffersolutionanddistilledwater,sequentially.

3. Resultsanddiscussion

3.1. Formationpoly(VBTAC)graftedCAnanoﬁbers

An illustration of immobilization of RAFT agent onto CA nanoﬁbersandsubsequentRAFTpolymerizationtoformcationic poly(VBTAC)brushesandDNAadsorptionisshowninScheme1.

Thepoly(VBTAC)-g-CA nanofiberswere preparedvia three-step processinvolving;(i)electrospinning ofCAnanofibers,(ii)cou- plingofRAFTagenttotheelectrospunCAnanofibersurfacevia esterificationreactionof non-acetylated-OH groupsof CAwith CPAD,(iii)surfaceinitiatedRAFTpolymerizationofVBTAC.Fig.1 showstheFTIRspectraofuntreatedCAnanofibersandCPADimmo- bilizedCA(CPAD-CA)nanofibers.Thecharacteristicbandofthe 39.8%acetylCAwasobservedat1737,1220and1034cm⁻¹dueto theC O,asymmetricandsymmetricC Ostretching,respectively (Celebioglu,Demirci,&Uyar,2014).ThebandareaoftheC Ogroup increasedwithesterificationreaction.ATR-FTIRspectrumofCA- CPADnanofibersalsoshowedabsorbancebandat2246cm⁻¹forthe C Nand1040cm⁻¹fortheC Sstretching.CPAD-CAnanofibers havehigherabsorbancethanCAnanofibers.Thecharacteristicband ofCAwasobservedat1739cm⁻¹ duetotheC Ostretching.On theotherhand,thebandsofpoly(VBTAC)appearedat1481cm⁻¹ forthescissor CH₂ vibration,at1403cm⁻¹ asymmetric CH₃ deformationvibrationandat1610cm⁻¹ C C stretchesofthe aromatic ring (Demirci & Caykara,2012). The spectrum of the poly(VBTAC)-g-CAnanofiberswascharacterizedbythepresence oftheabsorptionbandstypicalofthepurecomponents.,withthe intensityroughlyproportionaltograftingratio.

Thechemicalcompositionofthepoly(VBTAC)-g-CAnanoﬁbers wasdeterminedbyXPS(Table1,Fig.2).ThecorelevelXPSspec- traofCPADoverlayerconsistofN1sandC1speakscurveﬁtted intothecomponentswithbindingenergiesatabout400.1eV(N C) forN1sand289.1eV(C O),287.9eV(O C O),286.0eV(C N), 285.4eV(C S),and285.0eV(C C/C H)forC1s(Table1,Fig.2).

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Table1

AtomicconcentrationandbindingenergiesgivenhighresolutionXPSfornanoﬁbers^a.

Nanoﬁbers O1s N1s C1s S2p

N⁺ C N O C O O C O C N C S C C/C H S C S C

CA

Energy(eV) 532.7 – – 289.1 287.6 – – 285.0 – –

Conc.(%) 38.01 – 61.99 –

CA-CPAD

Energy(eV) 532.6 – 400.1^b 289.1 287.9 286.0 285.4 285.0 162.5 161.4

Conc.(%) 31.83 1.16 64.45 5.56

Poly(VBTAC)-g-CA

Energy(eV) 532.7 402.1 399.9 289.0 287.8 286.1 285.3 285.0 162.6 161.3

Conc.(%) 14.27 6.01 78.66 1.06

aBindingenergiesarecalibratedtoaliphaticcarbonat285.0eV.

bBindingenergyattributabletotheC Nspecies.

TheimmobilizationofCPADontoCAnanofiberwasalsoconfirmed fromtheappearanceofaS2ppeakcurvefittedintotwocompo- nentswithbindingenergiesatabout162.5eV(C S)and161.4eV (C S).Overall,theATR-FTIRandXPSstudiesconfirmedthesuc- cessfulcouplingoftheRAFT agent(CPAD)onto electrospunCA nanofiberssurface.ThecorelevelXPSspectraofnanofiberscon- sistofN1sandC1speakscurvefittedintothecomponentswith bindingenergiesatabout402.1eV(C N⁺)and399.9eV(C N)forN 1sand289.0eV(C O),287.8eV(O C O),286.1eV(C N),285.3eV (C S)and285.0eV(C C/C H)forC1s.

Fig.1. FTIRspectra of CAnanofibers,CA-CPAD nanofibers,poly(VBTAC)-g-CA nanofibersandpoly(VBTAC).

The static water contact angle of CPAD functionalized CA nanofiberwas62±3^◦whichwaslowerthanthevalueof88±2^◦ fortheCAnanofiberbeforetheimmobilization(Table2).Whenthe CAnanofibersweregraftedwithpoly(VBTAC),thestaticwatercon- tactangleofthesurfacedecreasedsubstantiallytoabout39±4^◦, consistentwiththehydrophilicnatureofpoly(VBTAC).

Fig. 3 shows the representative SEM images of the CA and poly(VBTAC)-g-CAnanofibers.TheSEMimagingshowedthatthe electrospun CA nanofibers were bead free and smooth morphology having average fiber diameter (AFD) of 810±260nm (Fig.3a).Itshouldbenotedthatsurfacemorphologiesandaverage

Fig. 2.XPS survey scan spectra of CA nanofibers, CA-CPAD nanofibers and poly(VBTAC)-g-CAnanofibers.

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Fig.3.SEMimagesofCAnanofibers(a),poly(VBTAC)-g-CAnanofibers(b)andDNAadsorbedpoly(VBTAC)-g-CAnanofibers(c).

fiber diameter of poly(VBTAC)-g-CA nanofibers were different fromthe CA nanofibers.Thatis, thesmooth appearance of CA nanofiber surface was no longer present (Fig. 3b) due to the poly(VBTAC)grafting.Additionally,poly(VBTAC)-g-CAnanofibers wereslightly thicker having AFD of 1030±310nmwhen com- paredtopureCAnanofibersbecauseofthepoly(VBTAC)coating aswellaspossibleswellingofnanofibersduringgraftingprocess.

Thesechangesinthesurfaceappearanceofthepoly(VBTAC)-g-CA nanoﬁbersarethephysicalevidencesforthesuccessfulgrafting reaction.

3.2. AdsorptionofDNA

DNAmoleculesarelikelyimmobilized ontothecationicsur- faces by electrostatic interaction between negatively charged

Table2

Staticwatercontactangleandphotographsof5␮Lwaterdropletsfornanoﬁbers.

Nanoﬁbers Contactangle(^◦) Image

CA 88±2

CA-CPAD 62±3

PVBTAC-g-CA 39±4

DNAandpositivelychargedpolymersurface.AdsorptionofDNA onto the CA and poly(VBTAC)-g-CA nanofibrous web waspro- videdfluorescence microscopy. Asit can beseen in Fig. 4,the redspotsindicatedthepresenceofDNAmolecules.Theadsorp- tion kinetics of DNA was performed at 20^◦C. The pristine CA nanofibrous web was used as a reference material. The result shown in Fig.5a indicatesthat there is nosignificanteffect of incubationtimeontheadsorptionofDNAontothepoly(VBTAC)- g-CA nanofibers. The adsorbedamount of DNA almost reaches a plateauafter 90min. This behavior can be explained due to the rapid and strong electrostatic attraction between cationic poly(VBTAC)-g-CAnanofibersandanionicDNAmolecules,andvery highsurfaceareatovolumeratioofnanofibrousweb,sotheequi- libriumconcentrationofadsorbedDNAis reachedveryquickly.

The maximumDNA adsorption capacity was 2.4␮g/mgfor the pristineCAweband26.6␮g/mgfor thepoly(VBTAC)-g-CAweb.

These resultsindicated that thegrafting of poly(VBTAC)onCA nanofibersprovidedasignificantincreaseintheDNAadsorption capacity.Fig.3cshowstheSEMimagesofthepoly(VBTAC)-g-CA nanofibers afterDNA adsorption in which the DNA adsorption onthe poly(VBTAC)-g-CA nanofiberswas observed microscopi- cally.

Theadsorptionisothermsrepresenttherelationshipbetween theamountadsorbedbyaunitweightofadsorbentandtheamount ofadsorbateremaininginthesolutionatequilibrium.Fig.5bshows thedependenceoftheadsorptionofDNAonthepoly(VBTAC)-g-CA nanoﬁbrouswebontheequilibriumDNAconcentration.Theini- tialconcentrationofDNAintheadsorptionmediumwaschanged between10and100␮g/mL.TheLangmuirisothermmodelassumes amonolayeradsorptionontoasurfacecontainingaﬁnite num- berofadsorptionsitesofuniformstrategiesofadsorptionwithno

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Fig.4. FluorescencemicroscopyimagesofCAnanofibers(a),poly(VBTAC)-g-CAnanofibers(b)andpoly(VBTAC)-g-CAnanofibersafterdesorptionprocedure(c).

transmigrationofadsorbateintheplaneofsurface,anditisgiven bythefollowingequation:

Ce

qe = 1 qmKm+ Ce

qm

whereCe(␮g/mL)istheequilibriumconcentrationofDNAsolu- tion, qe (␮g/mg) is the equilibrium amount of DNA adsorbed, qm(␮g/mg)is themaximumamountofDNAadsorbedperunit

massofnanofiber, andKm (mL/mg)is aconstantrelated tothe adsorption. Asillustrated in Fig.5b,a linearplot withcorrela- tioncoefficient(R²)valueof0.973wasobtainedfromLangmuir isothermequationwhenplottingCe/qeagainstCewithaslopeand interceptequalto1/q_mand1/K_mq_m,respectively.Kineticparame- terswerecalculatedas2.52×10⁻²mL/␮gforKmand23.51␮g/mg forqm.Moreover,theLangmuirequationfitswellforDNAimmo- bilizationonthepoly(VBTAC)-g-CAwebundertheconcentration

Fig.5. Adsorptionkinetics(a),adsorptionisotherm(b)andreusability(c)ofpoly(VBTAC)-g-CAnanoﬁbers.

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range studied. Adsorption capacity of some solid surfaces was previouslyinvestigatedasdetailedelsewhere(Baseretal.,2011;

Demirci et al., 2013; Rahman & Alaissari, 2011). Compared to solidsurfaces,poly(VBTAC)-g-CAnanoﬁbrouswebshowedhigher adsorptioncapacity.

TheessentialfeatureoftheLangmuirisothermcanbeexpressed bymeansofadimensionlessconstantseparationfactororequi- librium parameter (RL). RL is calculated using the following equation:

RL= 1 1+KmC0

whereKmistheLangmuirconstantwhichindicatesthenatureof adsorptionandtheshapeoftheisotherm,andC0referstotheinitial DNAconcentration(␮g/mL).TheparameterR_L>1,R_L=1,0<R_L<1, R_L=0indicatestheisothermshapeaccordingtounfavorable,linear, favorableandirreversible,respectively(Demircietal.,2013;Kim, Barraza,&Velev,2009;Mallampati&Valiyaveettil,2012).TheRL

valuesofDNAadsorptiononpoly(VBTAC)-g-CAnanoﬁberswere giveninTableS1(Supplementarydata).TheRL valuesconﬁrmed thattheadsorptionisfavorableunderconditionsusedinthisstudy.

The reusability of poly(VBTAC)-g-CA nanofibrous web was investigatedbymeasuringtheadsorptioncapacityfortargetDNA ina cyclic manner.Theadsorption/desorption procedureswere repeatedfivetimestoverifythereusabilityoftheweb.Thecycles ofadsorption/desorptionprocessesareshowninFig.5c.Drastic decrease(approximately 11.5%)in theadsorption capacity was seenduringtheeachcycle,andthepoly(VBTAC)-g-CAnanofibers retainedDNA anuptake capacity of ∼46% afterfivecycles. Fig.

S1(Supplementary data) shows the XPS survey spectra of the poly(VBTAC)-g-CAnanofibersafteradsorptionanddesorptionpro- cedure.AscanbeseenfromFig.S1b(Supplementarydata),intensity of the P 2ppeak at 133eV decreased. However, unfortunately P 2p peak and fluorescence signal (Fig. 4c) of poly(VBTAC)-g- CAnanofibersdidnotdisappearcompletely.Ourpreviousstudy clearlydemonstratethatpoly(VBTAC)polymerbrushesarecationic behavioratdifferentpHs(Demirci&Caykara,2013).Ontheother hand,DNAisnegativelychargedduetothephosphategroupson itsbackbone,and withdecreasingpHthesenegativephosphate groupsbecomeprotonatedandDNAmoleculeturnslessnegative.

Thisisduetoelectrostaticinteractionbetweenpoly(VBTAC)-g-CA nanoﬁbersandlessnegativeDNAmolecules(pH=3.0)anditwas evidentthatourwashingproceduredidnotabletoremoveallthe adsorbedDNA.Becauseofthis,theadsorptioncapacityofDNAwas decreasedfrom23.51to12.64␮g/mgwithincreasingnumberof reuse.

4. Conclusions

In conclusion, cationic poly(VBTAC)-g-CA nanofibers were manufacturedviacombinationofelectrospinningandRAFTpoly- merizationtechniqueswiththegoaloftheadsorptionofDNA.Our systematicstudiesbyusingtechniquessuchasATR-FTIRandXPS confirmedthesuccessfulgraftingofpoly(VBTAC)onelectrospun CAnanofibers.TheSEMimagingshowedthattheelectrospunCA nanofibers were bead free and smooth morphology. However, surfacemorphologiesandaveragefiberdiameterofpoly(VBTAC)- g-CA nanofibers were different from the CA nanofibers. These changes in the surface appearance of the poly(VBTAC)-g-CA nanofibersarethephysicalevidencesforthesuccessfulgrafting reaction.Thestaticwatercontactangleofthenanofibersdecreased from88±2to39±4^◦,consistentwiththehydrophilicnatureof poly(VBTAC).The DNA adsorption capacity was determined as 23.51␮g/mgfromtheLangmuirisothermforpoly(VBTAC)-g-CA nanofibrousweb. Wehave alsodemonstratedthereusabilityof thepoly(VBTAC)-g-CAwebbymeasuringtheadsorptioncapacity

fortargetDNAinacyclicmanner.Ourstudieshaveshownthat poly(VBTAC)-g-CAnanoﬁberspresentsaconvenientapproachfor DNA immobilization. The results reported in this article could open up new opportunities for fabricating surface functional- izedelectrospunnanoﬁbers/nanowebsand theirapplicationsin biotechnologicaluses.

Acknowledgements

Dr T. Uyar acknowledges EU FP7-PEOPLE-2009-RG Marie Curie-IRG(NANOWEB,PIRG06-GA-2009-256428)andTheTurkish AcademyofSciences–OutstandingYoungScientistsAwardPro- gram(TUBA-GEBIP)forpartialfunding.A.Celebiogluacknowledges TUBITAK-BIDEBforthenationalPhDstudyscholarship.

AppendixA. Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/

j.carbpol.2014.06.086.

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