Environmentally compatible bioconjugated gold nanoparticles as efficient contrast agents for colorectal cancer cell imaging

ContentslistsavailableatScienceDirect

Sensors

and

Actuators

B:

Chemical

j ourn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / s n b

Environmentally

compatible

bioconjugated

gold

nanoparticles

as

efficient

contrast

agents

for

colorectal

cancer

cell

imaging

Kássio

M.G.

Lima

_,

_Raimundo

_F.

_Araújo

_Junior

_,

Aurigena

A.

Araujo

_,

_Ana

_Luiza

_C.S.

_Leitão

_Oliveira

_,

_Luiz

_H.S.

_Gasparotto

a_{GroupofBiologicalChemistryandChemometrics,InstituteofChemistry,FederalUniversityofRioGrandedoNorte,Natal59072-970,RN,Brazil}

b_{DepartmentofMorphology,PostgraduationprogrammeinHealthScience/PostgraduationprogrammeinStructuralandFunctionalBiology,Federal}

UniversityofRioGrandedoNorte,Natal59072-970,RN,Brazil

c_{DepartmentofBiophysicsandPharmacology,PostgraduationprogrammeinPublicHealth/PostgraduationprogrammeinPharmaceuticalScience,}

FederalUniversityofRioGrandedoNorte,Natal59072-970,RN,Brazil

d_{PostgraduationprogrammeinPharmaceuticalScience,FederalUniversityofRioGrandedoNorte,Natal59072-970,RN,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3November2013

Receivedinrevisedform

27December2013

Accepted3February2014

Availableonline12February2014

Keywords: Greensynthesis Goldnanoparticles Colorectalcancer Bioconjugation Confocalmicroscopy Cellimaging

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Inthisstudyweshow,forthefirsttime,thatgoldnanoparticles(AuNPs)synthesizedbyasimple, inexpen-sive,andenvironmentally-correctmethodcanbeeasilyconjugatedwiththeantibodiesanti-␤-catenin andanti-E-cadherintospecificallytargetcolorectalcarcinomacells.Theantibody/AuNPsconjugateswere thensuccessfullyappliedforimagingcancerouscellswithfluorescenceconfocalmicroscopy.TheAuNPs aswellastheconjugateswereverystableinhigh-salinitymedium,apre-requisiteforapplicationin physiological-likeenvironments.Fluorescenceresultssuggestthatconjugationwasachievedbydirect adsorptionofantibodiesontheAuNPssurface.Finally,comparedwithastandardmethodofcellstaining, ourmethodislesslaboriousandthepreparationtime(fromimmobilizationofcellsontoglasscoverslips untilobservationbyconfocalmicroscopy)decreasedfrom27htoabout1h,whichmakesthemethod eligibleforcolorectalcancerdiagnostic.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Currentcancerdiagnosticmethodologiesincludeoptical imag-ingtechniquessuchascoherencetomography andfluorescence confocalmicroscopy.Theimageisgeneratedbythebackscattered lightfromtheendogenouschromophorespresentinthetissue.The weakopticalsignalresultsinpoorintracellularcontrastandsubtle spectraldifferencesbetweenmalignantandbenigncellshamper a proper diagnostic [1]. In order to overcome these problems, exogenouschemicals(mainlyorganicdyes)thatfunctionas opti-calcontrastagenthavebeenusedtoenhancethevisibilityofboth precancerousandcancerouscells.However,organicdyesare sub-jectedtorapidphotobleaching[2],thereforebecominginefficient astheanalysisproceeds.Otherdisadvantagesofconventionaldyes includepoorhydrophilicity,lowquantumyield,andlowdetection sensitivityinbiologicalenvironment[2].

∗ Correspondingauthor.Tel.:+558433422323;fax:+558432119224.

E-mailaddress:lhgasparotto@ufrnet.br(L.H.S.Gasparotto).

Recentadvancesofnanotechnologyhaveallowedthe develop-mentofnewfunctionalnanomaterialswithenhancedspecificity for molecular imaging. In particular, colloidal Au nanoparti-cles(AuNPs) are extremely advantageousas contrast agents in biomedicine[1,3–6]astheycanbeeasilycombinedwitha recog-nitionagent.First,AuNPspresentasize-tunablesurfaceplasmon resonance(SPR)thatleadstostrongabsorptionandscatteringin thevisible-to-near-infraredregion,acharacteristicwhichmakes themconsiderablysuperiortotheconventional dyesappliedin biomedicalimaging.Forinstance,theplasmonresonance absorp-tionofsphericalgoldnanoparticleshasanabsorptioncoefficient ordersofmagnitudelargerthanthoseofregulardyes.Asan exam-pletheabsorptioncoefficient(ε)for40-nmgoldnanoparticles[7]at awavelengthof530nmwascalculatedtobe∼7.7×109_M−1_cm−1_,

whiletheεofrhodoamine6Gis1.16×105_M−1_cm−1_[8].Second,

AuNPsdonotsufferphotobleachingandseemtobebiocompatible

[9].Third,thechemistryofgoldisquiterich.TheAuNPssurface binds strongly with amines, thiols and disulfides, allowingthe surfacebioconjugationwithagreatvarietyofpeptides,DNAand proteins,enablingselectivetargetingofcancercells.Furthermore, in oppositiontodyes and organicmolecules,severalfunctional

http://dx.doi.org/10.1016/j.snb.2014.02.008

groupscanbeattachedontothesamenanoparticle,makingthem alsoeligiblefordrugdelivery[10].

In this paper we synthesized AuNPs bioconjugated with ␤-cateninantibody(anti-␤-catenin)andE-cadherinantibory (anti-E-cadherin) and applied them as novelcontrast agents for the detectionofcolorectalcancerouscellswithfluorescenceconfocal microscopy.E-cadherinislocalizedonthesurfaceofepithelialcells inregionsofcell–cellcontactknownasadherensjunctionsandis implicatedincell–celladhesioninepithelialtissues[11–13]. ␤-Catenininteractswithcadherinsthroughitscytoplasmicdomain. ␣-cateninconnectstheE-cadherinand␤-catenincomplextoactin filaments.The dissociation ofE-cadherin-catenin complexfrom cellmembrane is important inmalignant progression. In many epithelialcancers,membranousE-cadherinislostand␤-catenin dissociatesinthecytoplasmandaccumulatesinthenucleusasa transcriptionfactor,concomitantlywithtumorprogression[12]. Down-regulationofmembranousE-cadherinand␤-catenin,and cytoplasmic/nuclearaccumulationof␤-cateninhavebeen previ-ouslyreportedinseveralcancersandholdpromiseasprognostic markers[14].In humans␤-cateninandE-cadherin areencoded bytheadenomatouspolyposiscoli(APC)andCDH1genes, respec-tively,andtheirmutationmayresultincolorectalcancerleadingto anoverexpressionof␤-cateninandE-cadherinbythecell[11–16]. Thestrategy wastherefore tocombinethespecificitiesof anti-␤-cateninandanti-E-cadherinwiththefluorescentpropertiesof theAuNPstoproduceaselectivecontrastagentfortargeting can-cer.Inthissystem,theAuNPsfunctionasthesignalingpartand theanti-␤-cateninandanti-E-cadherinactasrecognitionunitsfor specificbindingwiththeoverexpressed␤-cateninandE-cadherin inthecell.Theconfocalmicroscopyrevealedthatthe antibody-conjugatedAuNPswereveryefficientinmarkingcancerouscells. Ontheotherhand,nospecificinteractionbetweennormalcells andconjugatedAuNPswasobserved.Anotherinterestingfeature ofthepresentworkistheuseofanenvironmentallyfriendlyroute to produce theAuNPs based on thereduction of gold ions by glycerolinalkalinemedium.Glycerol’snon-toxicityand biodegrad-abilitymakeitanexcellentalternativetocommonlyusedreducing

agents.Theconjugationswithanti-␤-cateninandanti-E-cadherin areachievedby simpleincubation afterreducingthepHof the nanoparticlecolloidalsolutiontoaround7.Incomparisonto cur-rentmethodsforproductionandconjugationofAuNPs,ourmethod isquitesimple,low-costandenvironmentallycompatible,witha potentialtoberoutinelyappliedincancerdiagnostics.

2. Experimental

2.1. Chemicalsandreagents

Gold trichloride (30wt% in HCl), polyvinylpyrrolidone (PVP, MW=10.000),sodiumhydroxide,dialysistubingcellulose

mem-brane, and glycerol were products of Sigma-Aldrich Chemical Co. Sulphuric acid and hydrogen peroxide were purchased fromVetec.Phosphate-bufferedsaline(PBS)solutionandbovine serum albumine (BSA, 5%) were purchased from Life Tech-nologies Corporation©_{. ␤-Catenin antibodies (anti-␤-catenin)}

andE-cadherinantibodies(anti-E-cadherin)wereacquiredfrom ABCAM® _{attheconcentration of200␮gL}−1 _{in PBSbuffer.}

Dul-becco’s modified Eagle’smedium (DMEM) and heat-inactivated bovineserumwerepurchasedfromLifeTechnologiesCorporation©

andCultilabLtda/Brasil,respectively. 2.2. ProductionandcharacterizationofAuNPs

AuNPswereproducedusingapreviouslyreportedmethod[17]. Briefly,allglasswarewaskeptovernightinKMnO4+NaOHsolution,

rinsedwithdeionizedwater,keptfor10mininH2O2+H2SO4

solu-tion(1:1v/v),againrinsedwithdeionizedwateranddriedpriorto use.KnownamountsofPVP(MW=10.000)andgoldchloridewere

dissolvedin10mLofwater.Inaseparatebeaker,determined quan-titiesofglycerolandNaOHweredissolvedin10mLofwater.The glycerol–NaOHsolutionwasthenaddedtotheAuCl3-PVPsolution

toyieldthefollowing finalconcentrations:1.0mMAu3+_,0.10M

NaOH, 0.10Mglycerol and 10gL−1 PVP. The final mixture had adeep-redcolorduetotheformedAuNPs.TheAuNPs colloidal

Fig.2.NormalizedUV–visspectraofAuNPsindifferentconditionsdistinguished

withdifferently-coloredcurvesshowninthelegends.(Forinterpretationofthe

ref-erencestocolorinthisfigurelegend,thereaderisreferredtothewebversionof

thisarticle.)

solutionwasallowedtorestfor1weektoguaranteereaction com-pletion.Consideringaquantitativetransformationofgoldionsinto nanoparticles,theconcentrationof AuNPs wasestimated tobe 197␮gmL−1.Thewholeprocedureforestimatingthe nanoparti-cleconcentrationcanbefoundin[18].TheAuNPscolloidalsolution hadthenitspHadjustedto7byadditionofdilutedHCl.Afterwards, theAuNPscolloidalsolutionwassubjectedtodialysisfor purifica-tionandtoremoveextrafreesmallmolecules.Thedialysisprocess isasfollows:theoutsideandinsideofthedialysisbagwerewashed withdeionizedwaterandthenplacedintoabeakerwithpurewater toboilfor5min.Thebagwasthenwashedwithwater,filledwith theAuNPscolloidalsolutionandallowedtoboilinalargebeaker for6h.Duringtheboilingwaterwaschangedthreetimes.

UV–vis absorptionspectraoftheAuNPs wereacquired with anEvolution60SUV–visiblespectrophotometer(Thermo Scien-tific)spectrophotometer.Fluorescencespectroscopywascarried outwithaPerkinElmerLS45fluorometer.Transmissionelectron microscopy(TEM)imageswereacquiredwithaJEOL2100F micro-scopeoperatingat200kV.

2.3. ConjugationofantibodieswithAuNPs

ConjugationofAuNPswithanti-_␤-cateninandanti-E-cadherin wasachievedbysimpleincubation.Bothantibodieswereacquired attheconcentrationof200␮gmL−1 inPBSbuffer. PBSandBSA wereusedasmediumtoyieldthefollowingsolutionsofconjugates: (1)3␮Lantibody+1000␮LPBS/BSA+500␮LAuNPs;(2)3␮L anti-body+750␮LPBS/BSA+750␮LAuNPs;(3)3␮Lantibody+500␮L PBS/BSA+1000␮LAuNPs.Theconjugatesolutionswereallowed torestfor1hpriorincubationwithcells.Theconjugates(1),(2), and(3) hadantibody/AuNPsratiosof6.1×10−3,4.1×10−3 and 3.0×10−3,respectively.

2.4. Cellculture

Non-cancerous cells (HKE)andcancercells HT29(colorectal adenocarcinoma)wereusedasamodelforcellimaging.Bothtypes ofcells werepurchased fromtheCulture Collectionof the Bio-scienceCentre,FederalUniversityofRioGrandedo Norte.HT29 cellsareknowntooverexpressE-cadherinandcatenin.Thecells weremaintainedinDulbecco’smodifiedEagle’smedium supple-mentedwith10%v/vheat-inactivatefetalbovineserum.Cultured cells were then plated onto glass coverslips in 24 well plates (5×104 _{cells/well) and allowed to grow for 24h. Afterwards,}

the cells were washed, fixed with paraformaldehyde, perme-abilizedwithTriton-X and incubatedwith100␮Lof eitherthe cadherin–AuNPsconjugate or catenin–AuNPsconjugate for one hourinahumidatmosphereatroomtemperature.Control exper-imentswereperformedunderthesameconditionsbutwithout additionoftheconjugates.Instead,cellswereincubatedwithAlexa Fluor®_{488goatanti-rabbitsecondaryantibody(Abcam)for2h.}

Theglasscoverslipswerethendirectlyobservedwiththeconfocal microscope.

2.5. Confocalmicroscopy

HealthyandcancerouscellswereexaminedunderaLSM510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped witha condenser using laser excitation from 442to 520nm.Inthistechniquethespecimenispointed-illuminatedand thelightproducedbyfluorescenceveryclosetothefocalplaneis detected.Incontrolexperiments,cellimageswereacquiredinthe

Fig.3.FluorescenceemissionspectraofAuNPsexcitedat330nminthepresenceof(a)␤-cateninand(b)E-cadherin.Excitationandemissionslitswere10nm.The

concentrationsofbothantibodiesare:2.0␮gmL−1_{(redcurve),3.0␮gmL}−1_{(greencurve)and5.0␮gmL}−1_{(bluecurve).TheconcentrationofAuNPswasheldconstantat}

Fig.4.Confocalimagesofhealthycellsacquiredatanexcitationlaserof520nm.Leftcolumn:imagesacquiredwiththeconjugateof␤-catenin.Theconjugateshad

␤-catenin/AuNPsratiosof(A1)6.1×10−3_,(A2)4.1×10−3_{and(A3)3.0×10}−3_{.Rightcolumn:imagesacquiredwiththeconjugateofE-cadherin.Theconjugateshad}

E-cadherin/AuNPsratiosof(B1)6.1×10−3_,(B2)4.1×10−3_{and(B3)3.0×10}−3_{.Magnification:10×.Scalebar=100␮m.}

sameconditionsusingAlexaFluor®_{488goatanti-rabbitsecondary}

antibodyinsteadtheantibody–AuNPsconjugates.

3. Resultsanddiscussion

TheAuNPsusedinthisworkweresynthesizedbyreducingAu3+

withglycerolinalkalinemedium.Glycerolisaninexpensive chem-icalandreadilybiodegradableunderaerobicconditions,thereforea greeneroptionwhencomparedtocurrentreducingchemicalssuch asformamide,sodiumborohydrideandhydrazine.Sodiumcitrate

[19]isefficientingeneratinggoldnanoparticles,howevertheyare

formedonlyuponheatingthesolution(80–100◦C).Fig.1AandB showsaUV–visspectrumandahigh-resolutionTEMimageof as-preparedAuNPsproducedbysimpleadditionofNaOH-glycerolto AuCl3-PVPatroomtemperature.ThecolloidalAuNPsspectrumhad

amaximumabsorbance(Max)at520nm,avaluetypicalfor

spher-icalgoldnanoparticles[20–22].Thesymmetryofthebandimplies afairsimilarityintheshapeofthenanoparticlesandlowdegree ofaggregationinthesolution[23].TheHR-TEMimageillustrated thattheas-preparedAuNPsweresphericalinshape,thus corrob-oratingtheUV–visresults.Themeanparticlesizecalculatedfrom thedistributionofFig.1Cwas6.8±1.6nm.

Fig.5.Confocalimagesofcancerouscells.Leftcolumn:imagesacquiredwiththeconjugateof␤-catenin.Theconjugateshad␤-catenin/AuNPsratiosof(A1)6.1×10−3_,

(A2)4.1×10−3and(A3)3.0×10−3.Rightcolumn:imagesacquiredwiththeconjugateofE-cadherin.TheconjugateshadE-cadherin/AuNPsratiosof(B1)6.1×10−3,(B2)

4.1×10−3and(B3)3.0×10−3.Magnification:40×.Scalebar=100␮m.

␤-catenin[24]andE-cadherin[25]areproteinsplaying impor-tant roles ascell–cell receptors, forming adherensjunctions to bind cells within tissues together. While normal cells do not overexpress␤-cateninandE-cadherin,enlargedamountofthese proteinsindicatescancercellaggressiveness[13].Duetotheir pro-teicnature,␤-cateninandE-cadherinmightundergodenaturation whensubjectedtopHotherthanthephysiologicalone.Thus,before

bioconjugation,thepHoftheAuNPscolloidalsolutionhadtobe adjustedtonear7.0andtestedagainstaggregationunderdifferent conditions.ResultsofthisstudyareshowninFig.2.Resultsrevealed thattheAuNPsareextremelystableinaqueoussolutionat differ-entpHandsalinity.Thespectraoftheas-preparedandpH-adjusted AuNPsarepracticallyoverlapped,whichdenotesexcellent stabil-ityunderdifferentpH.Smallmoleculessuchasglycerol’soxidation

Fig.6.ComparisonofconfocalimagesofcancerouscellsacquiredwithAlexaFluor®>and(A)␤-catenin/AuNPsconjugateand(B)E-cadherin/AuNPsconjugate.Magnification:

10×.Scalebar=100␮m.

productswerethenremovedviadialysisandthisprocesshadno influenceoftheshapeofthespectra.TheAuNPswerethen1:3v/v dilutedinthePBS/BSAbuffer.UnderthishighsalinityAuNPsare expectedtobeunstable,howeverinourcasethesterichindrance providedbyPVPresultedinincreasedstability.Uponintroduction oftheantibodiesthespectraofAuNPshadnosignificantchange, implyingthatthebioconjugationdidnotprovokeobvious aggre-gationofAuNPs.

Fig.3showsfluorescenceemissionspectraforpurifiedAuNPs in absenceand presence of ␤-catenin (Fig. 3A) and E-cadherin (Fig.3B).Theexcitationwavelengthwasfixedat330nmandthe emissionrecordedintherangeof700–900nm.Theemissionband atthatrangedidnotshiftwiththeexcitationwavelength(data notshown),indicatingthatthebandwasnotduetoany scatter-ingprocess.The fluorescenceemissionswereslightly enhanced upon absorption of both ␤-catenin (increase of 30 a.u. for the highest antibodyconcentration) and E-cadherin (increaseof 39 a.u. for the highest antibody concentration) onto the AuNPs. Such enhancement hasbeen rationalizedin terms of competi-tionbetweenadsorbingspeciesandmolecularoxygendissolvedin solution.AlqudamiandAnnapoorni[26]showedthatfluorescence enhancementtakesplacewhenBSAadsorbsonthesurfaceofsilver nanoparticles.Asthesurfaceplasmonresonance(SPR)(responsible forthefluorescencesignal)isafunctionofthesurfacefree elec-trons,morefreeelectronsledtoamoreintenseSPRsignal.The authorsarguedthattheadsorbedBSApreventedthesurfacefree electronsfrombindingwithoxygenmolecules,hencethe fluores-cencesignalwassomewhatincreased.Inourcase␤-cateninand E-cadherinmayhaveadsorbedonAuNPsimpedingO2toreachthe

nanoparticlesurface,anexplanationsupportedbyFig.3,wherea

fluorescencesignalincrementoccurredonlyforthelowest anti-bodyconcentration,remainingconstantatthehigherones.This meansthatasmallantibodyconcentrationissufficienttoinsulate theAuNPsfromoxygen.

Theantibody–AuNPsconjugateswerethenappliedaslabeling probesforcellimaging.Intheexperimentswefirstproducedthe conjugatesasexplainedinSection2andtheninoculatedtheminto healthyand cancerouscells. Thestrategytocombinethe speci-ficitiesofanti-␤-cateninandanti-E-cadherinwiththefluorescent propertiesoftheAuNPstoproduceaselectivecontrastagentfor targetingcancerwasprovenefficientasshownbellow.Fig.4shows confocalmicroscopyimagesofhealthycellsincontactwithAuNPs conjugatedwithdifferentconcentrationsof antibodies.Onecan seethatthenanoparticlesareinhomogeneouslyspreadoverthe cover slitsurface regardlessofthe antibody/AuNPsratioin the conjugates. Thelack of specific binding of theconjugates with thecells isa consequenceofnormal levelsof␤-catenin and E-cadherinproducedbynon-cancerouscells.Weshouldmentionthat duringtheconfocalmicroscopyexperimentswithnon-cancerous cells itwasextremely difficultfindinga regionwithgood fluo-rescenceintensity.Astherearenospecificinteractionswiththe cells,thenanoparticlesarespreadovertheglasscoverslipsandthe high-localfluorescenceintensitiesareduetonon-specificAuNPs agglomeration.

Fig. 5 presents images of cancerous cells labeled with the conjugates.Theseimagesareclearlydistinguishedfromthoseof

Fig.4.Inthiscasetheantibody–AuNPsconjugatescouldspecifically bindonthecellsurfacesduetotheoverexpressed␤-cateninand E-cadherin.Evennuancesof thecellmembrane couldbenicely revealed,asforexampleinFig.5A2,whereagroupofcellswas

Scheme1.Reducedpreparationtimeenabledbythepresentwork.

spotted. Note that thegold nanoaprticlesare nolongerspread aroundthecells,butratherconcentratedonthem.Itisinteresting tonoticethatinFig.5A1theboardersofthecellsarebrighterthan thecenters.Since␤-cateninandE-cadherinaremainlymembrane proteins, their overexpression led to an increased concentra-tionoftheconjugatesinthatarea.Anotherimportantfeatureis thateven theconjugateswiththelowest antibody/AuNPsratio (Fig. 5A3 and B3) wereable tospecificallybind withthe cells, whichhasapositiveimplicationonthecostasantibodiesarenot inexpensive.

Finally, we compare the herein proposed method with a standardone forlabelingcancerouscells.The standardmethod employedconsistedofinoculatingtheAlexaFluor®488–which isanantibodyboundtoadye–withcancerouscellsfor subse-quentcellimaging.Fig.6Ashowsresultsforthisstudy,inwhich thecellsinallimageswerestainedwithboth␤-catenin–AuNPs conjugateandAlexaFluor® _{488.Onecanseethatbothlabeling}

agentsrevealthesamesites,whichisafurtherproveofthe speci-ficityofourconjugate.Furthermore,thesignalintensitiesofthe ␤-catenin–AuNPsandAlexawerenotthatdifferent,withthatfor theAlexabeingslightlymoreintense.Therightpictureisa sig-naloverlapfrom␤-catenin–AuNPsandAlexa.Thebrighterareas aretheintensitycombinationsofbothlabelingagents,showing thatthe␤-catenin–AuNPsconjugateindeedbindscancerouscells asbothsignalingagentspopulatethesameareas.Fig.6Bpresents thesamecomparisonbutnow withtheE-cadherin–AuNPs con-jugate.Unlike withthe ␤-catenin–AuNPs,the signalintensities ofE-cadherin–AuNPsandAlexawerepracticallythesame,which suggeststhatthecancerouscellsexpressedmoreE-cadherinthan ␤-catenin,existingthereforesufficientofsitesforequaladsorption ofE-cadherin–AuNPsandAlexa.Fromtheseresultsoneconcludes thattheantibody/AuNPscouldefficientlyreplacethe commonly-usedsecondaryantibodyforidentificationofcolorectalcancerous cells.

Anotherpointworthtomentionisthereducedoverall prepa-rationtimewiththeantibody–AuNPsconjugates,aspresentedin

Scheme1.Thestandardprocedurerequiresincubationofprimary antibody(␤-cateninorE-cadherin)withAlexaFluor®_488for24h,

followedbywashingandfixationstepspriorobservationunderthe microscope.Theentireproceduretakesabout27h.Ontheother hand,ourprocedurerequiresonlya1-hincubationfollowedbya

single10-minrinsingstep,whichisaclearadvantageintermsof laborandtime-saving.

4. Conclusions

Anti-␤-catenin/AuNPs and anti-E-cadherin/AuNPsconjugates weresuccessfullyappliedforimagingcolorectalcancerouscells. UV–visresultsshowedthattheconjugatesarestableinsolutions thatresemblethephysiological medium.Based onfluorescence results,lowantibodyconcentrationsaresufficienttoachieve max-imum adsorption of antibodies onto the surface of the AuNPs. Furthermore,thehereinproposedconjugateshadsimilar perfor-mancecomparedwithastandardprocedure,withtheadvantageof havingreducedtheoverallanalysistimefrom27htoonly1h.Based ontheseresults,weenvisagetheapplicationoftheconjugatesfor routineimagingofcolorectalcancerouscells.

Acknowledgments

The authors are grateful to CNPq (grant 471794/2012-0), Capes(grantCapes/070/2012),andFAPERN/MCT/CNPq/CT-INFRA 005/2011 for the financial support. We also thank Dr. Celso Sant´ıAnna(Inmetro)forthehelpwithTEMimages.

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Biographies

KássioM.G.LimaisanassistantprofessorattheInstituteofChemistryoftheFederal

UniversityofRioGrandedoNortesince2009.HereceivedhisPh.D.insciences(2009)

fromUNICAMP(Brazil)andhewassandwich-doctoratefellow(09/2007–09/2008)

atUniversidadComplutensedeMadridintheOpticalFibreChemicalSensorsand

AppliedPhotochemistryGroup(supervisedbyDr.M.C.Moreno-Bondi)workingon

simultaneousdeterminationofheavymetalswithopticalsensors.Heistheleader

oftheResearchGroupofAppliedChemometrics(UFRN,Brazil)since2011andhis

currentresearchinterestsareonspectroscopyandchemometrics.

RaimundoF.AraújoJúniorholdsapositionofassistantprofessoratthe

Morphol-ogyDepartmentoftheFederalUniversityofRioGrandedoNorte,Brazil,since

2006.HegothisPh.D.inHealthSciencesin2005.Hisinterestsaremolecular

biologytooncology.Thefocusofhisworkistoobservetheapoptosisand

prolifer-ationofcancerouscellsuponcontactwithnanoengineeredandphototherapeutic

drugs.

AurigenaA.AraujoworksasassistantprofessorattheDepartmentofBiophysics

andPharmacologyoftheFederalUniversityofRioGrandedoNorte,Brazil,since

2004.ShegotherPh.D.inHealthSciencesin2005.Shewasapost-docfellowatthe

UniversidadAutónomadeSanLuísPotosí,Mexico,in2007.Herresearchlineisthe

searchfornewmedicinesforthetreatmentofinflammatoryprocess.

AnaLuizaC.S.L.OliveiraisaPh.D.studentinPharmaceuticalScience/PublicHealth

atFederalUniversityofRioGrandedoNorte,Brazil.Thefocusofherworkisto

correlatetheexpressionoftheE-cadherinproteinwithpathologicalvariablesin

colorectalcarcinoma.Sheisalsointerestedinstudyingtheviabilityofcancercells

upontreatmentwithphytotherapeuticextracts.

CurrentlyLuizHenriquedaSilvaGasparottoholdsapositionofassistantprofessor

atUniversityofRioGrandedoNorte,Brazil.HeobtainedhisPh.D.inSciencesin

2009,followedbyapost-docstageatTUClausthal,Germany,underthesupervision

ofProf.FrankEndres.Gasparotto’sinterestscomprisethesynthesisandapplication