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

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ContentslistsavailableatScienceDirect

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,∗∗ a_{UNAM-National}_{Nanotechnology}_Research_Center,_Bilkent_University,₀₆₈₀₀_Ankara,_Turkey b_Institute_of_Materials_Science_and_{Nanotechnology,}_Bilkent_University,₀₆₈₀₀_Ankara,_Turkey c_Department_of_Chemistry,_Faculty_of_Arts_and_Sciences,_Amasya_University,₀₅₁₀₀_Amasya,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13January2014

Receivedinrevisedform22June2014 Accepted23June2014

Availableonline11July2014 Keywords: Electrospinning Celluloseacetate Nanoﬁber Surfacemodiﬁcation RAFTpolymerization DNAadsorption

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b

s

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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 sincethisversatiletechniqueenablestheproductionof multifunc-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-mediated rad-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) http://dx.doi.org/10.1016/j.carbpol.2014.06.086

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

polymerization(Demirci&Caykara,2012;Gurbuz,Demirci,Yavuz, &Caykara,2011;Jiang&Xu,2013)andsingle-electron transfer-livingradicalpolymerization(Demirci,Kinali-Demirci,&Caykara, 2013;Percecetal.,2006).LRPprovidesexcellentcontroloverthe molecularweightandpolydispersityofgraftpolymers(Braunecker &Matyjaszewski,2007;Dietrichetal.,2010).AmongallLRP tech-niques,RAFTpolymerizationisthemostimportantone,duetoits compatibilitywithawiderangeofmonomersandreaction condi-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 macro-molecules,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) nanofiberweresuccessfullyproducedbycombinationof electro-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)andcontactangle measure-ments.Furthermore,DNAadsorptionofpoly(VBTAC)-g-CA nanofi-brouswebfromthebuffersolutionwasinvestigated.The reusabil-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

Thehomogenouselectrospinningsolutionwaspreparedby dis-solvingCAin DCM/methanol (4/1(v/v))binarysolvent mixture at12%(w/v)polymerconcentration.ThenclearCAsolutionwas placedina3mLsyringefittedwithametallicneedleof0.6mm innerdiameter.Thesyringewasfixedhorizontallyonthesyringe pump(KDScientific).Theelectrodeofthehigh-voltagepower sup-ply(Matsusada Precision,AUSeries)wasclamped tothemetal needletip,andthecylindricalaluminumcollectorwasgrounded. Theparametersoftheelectrospinningwereadjustedas;feedrate ofsolutions=1mL/h,theappliedvoltage=15kV,andthe tip-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 bottomedﬂaskandstirredforfewminutesundernitrogen atmo-sphere.TheCAnanoﬁberswereadded,andthemixturewasstirred overnightatroomtemperature.Theproductwaswashedwith ben-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),andRAFTchaintransferagent immo-bilizedCAnanofiberat 0◦C in around-bottom flask.To ensure smoothstirringandpreventdamagetothenanofibers,weisolated themagneticstirringbaratthecenterofdevicefromtheslidesby a1cmhighglassO-ring.Thesolutionwasdilutedto30mLvolume withthebuffersolutionanddegassedbypurgingwithnitrogen for20min.Thepolymerizationreactionwasstirredvigorouslyat 70◦Cfor120min.Thepoly(VBTAC)-g-CAnanofiberswere recov-eredfromthereactionmixtureandrepeatedlywashedwithbuffer and watertoremovetheunreactedchemicals,anddriedunder vacuumat30◦C.

2.4. Measurementsandcharacterization

Attenuatedtotalreflectance-Fouriertransforminfrared (ATR-FTIR)spectraoftheCAandpoly(VBTAC)-g-CAwereobtainedusing a ThermoNicolet6700spectrometerwitha Smart Orbit atten-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

=1486.6eV).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)andaround100fiber diam-etersweremeasuredfromtheSEMimagestocalculatetheaverage fiberdiameterofeachsample.Thewatercontactangle measure-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–vis spectropho-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-CAnanofibersforDNA adsorp-tion,fivecyclesofadsorption/desorptionwerecarriedoutusing buffersolution(potassiumhydrogenphthalate/hydrochloricacid, pH=3.0).Aftereachadsorption/desorptioncycle,DNA concentra-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 showstheFTIRspectraofuntreatedCAnanofibersandCPAD immo-bilizedCA(CPAD-CA)nanofibers.Thecharacteristicbandofthe 39.8%acetylCAwasobservedat1737,1220and1034cm−1dueto theC O,asymmetricandsymmetricC Ostretching,respectively (Celebioglu,Demirci,&Uyar,2014).ThebandareaoftheC Ogroup increasedwithesterificationreaction.ATR-FTIRspectrumof CA-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).ThecorelevelXPS spec-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

AtomicconcentrationandbindingenergiesgivenhighresolutionXPSfornanoﬁbersa_.

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 – 400.1b _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

a_Binding_energies_are_calibrated_to_aliphatic_carbon_at_285.0_eV. b_Binding_energy_attributable_to_the_C _N_species.

TheimmobilizationofCPADontoCAnanofiberwasalsoconfirmed fromtheappearanceofaS2ppeakcurvefittedintotwo compo-nentswithbindingenergiesatabout162.5eV(C S)and161.4eV (C S).Overall,theATR-FTIRandXPSstudiesconfirmedthe suc-cessfulcouplingoftheRAFT agent(CPAD)onto electrospunCA nanofiberssurface.ThecorelevelXPSspectraofnanofibers con-sistofN1sandC1speakscurvefittedintothecomponentswith bindingenergiesatabout402.1eV(C N+₎_and_399.9_eV_(C _N)_for_N

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),thestaticwater con-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 mor-phology 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 ontothecationic sur-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 was pro-videdfluorescence microscopy. Asit can beseen in Fig. 4,the redspotsindicatedthepresenceofDNAmolecules.The adsorp-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 incubationtimeontheadsorptionofDNAontothe poly(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,sothe equi-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.The ini-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)istheequilibriumconcentrationofDNA

solu-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 with correla-tioncoefﬁcient(R2₎_value_of_0.973_was_obtained_from_Langmuir

isothermequationwhenplottingCe/qeagainstCewithaslopeand

interceptequalto1/qmand1/Kmqm,respectively.Kinetic

parame-terswerecalculatedas2.52×10−2mL/␮gforKmand23.51␮g/mg

forqm.Moreover,theLangmuirequationﬁtswellforDNA

immo-bilizationonthepoly(VBTAC)-g-CAwebundertheconcentration

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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 bymeansofadimensionlessconstantseparationfactoror equi-librium parameter (RL). RL is calculated using the following

equation: RL=₁₊1_K

mC0

whereKmistheLangmuirconstantwhichindicatesthenatureof

adsorptionandtheshapeoftheisotherm,andC0referstotheinitial

DNAconcentration(␮g/mL).TheparameterRL>1,RL=1,0<RL<1,

RL=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-CAnanofibersafteradsorptionanddesorption pro-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 manufacturedviacombinationofelectrospinningandRAFT poly-merizationtechniqueswiththegoaloftheadsorptionofDNA.Our systematicstudiesbyusingtechniquessuchasATR-FTIRandXPS confirmedthesuccessfulgraftingofpoly(VBTAC)onelectrospun CAnanofibers.TheSEMimagingshowedthattheelectrospunCA nanofibers were bead free and smooth morphology. However, surfacemorphologiesandaveragefiberdiameterof poly(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–OutstandingYoungScientistsAward Pro-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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