Ionic strength-controlled hybridization and stability of hybrids of KRAS DNA single-nucleotides: A surface plasmon resonance study

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ContentslistsavailableatScienceDirect

Colloids

and

Surfaces

B:

Biointerfaces

jou rn a l h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b

Ionic

strength-controlled

hybridization

and

stability

of

hybrids

of

KRAS

DNA

single-nucleotides:

A

surface

plasmon

resonance

study

N. Giamblanco

a,∗

_,

_S.

_Petralia

b

_,

_S.

_Conoci

b,∗

_,

_C.

_Messineo

a

_,

_G.

_Marletta

a

a_Dept._of_Chemical_Sciences,_University_of_Catania,_Viale_A._Doria₆_-₉₅₁₂₉_Catania,_Italy b_{STMicroelectronics,}_Stradale_Primosole_50,₉₅₁₂₁_Catania,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received2January2017

Receivedinrevisedform17June2017 Accepted19June2017

Availableonline21June2017 Keywords:

SNP

DNAhybridization SPR

a

b

s

t

r

a

c

t

Thediscriminationofafullymatched,unlabeledKRASwild-type(WT)(C-G)targetsamplewithrespect

tothreeofthemostfrequentKRAScodonmutations(G12S(C-A),G12R(C-C),G12C(C-T))was

inves-tigatedusinganoptimizeddetectionstrategyinvolvingsurfaceplasmonresonance(SPR),basedon

optimizedprobe-surfacedensityandionicstrengthcontrol.ThechangesobservedintheSPRsignalwere

alwayslargerforWTcomparedwiththesingle-mismatchtargetDNAoligonucleotides,andwerealigned

withthetheoreticalenergydifferencesbetweenthebasepairC-G,C-T,C-A,C-C.Hybridizationratesof

∼106_M−1_s−1_were_detected_without_the_introduction_of_high_temperature_and_labels,_usually_needed

inconventionalhybridizationmethods.Onehundredpercentmutationdiscriminationofthematched

KRASwild-type(C-G)sequencewithrespecttothreemismatchedG12C(C-T),G12S(C-A),G12R(C-C)

targetsequenceswasachieved.

1. Introduction

TheidentificationofspecificDNAsequencesplaysakeyrolein biomedicalresearch.Inthiscontext,thedetectionofsinglebase DNAmutationisparticularlyrelevantasitisassociatedwith sev-eralgeneticdiseases.Singlenucleotidepolymorphisms(SNPs)are themost abundantgeneticvariations and theyare involved in severaltypesofcancersuchascolorectalandpancreas adenocar-cinoma[1].Currently,mostoftheconventionaltestsforSNPsare basedonDNAamplificationmethodologiessuchasreal-time Poly-meraseChainReaction(PCR)[2],sequencing[3],DNAhybridization techniqueslikemicroarrays[4–7]and fluorescencedetectionof theinsituhybridization(FISH)[8,9].Themajorlimitationsofthe sequencingmethodsderivefromthecomplexityofthe method-ologytogetherwiththecontaminationriskarisingfromthePCR exponentialamplification[10].

The discrimination of one or more mismatches generally requirestheﬁnetuningofthehybridizationconditions,including surface-probe interactions, probe length[11], probe conforma-tion, target length and concentration, labeling of the target, hybridizationtemperature,compositionandionicstrengthofthe hybridizationbuffer,post-hybridizationwashstringencyand

tem-∗ Correspondingauthors.

E-mailaddresses:ngiamblanco@hotmail.com(N.Giamblanco),

sabrina.conoci@st.com(S.Conoci).

perature,andotherdissociationconditionsoftheprobeandtarget duplex[11–16].

SeveralbioanalyticalapproachesforthedetectionofSNPusing SPR biosensors are described in literature [17,18]. Indeed, Sur-facePlasmonResonance(SPR)isa veryattractivetechniquefor awiderangeofanalyticalapplications[19,20].Ingeneticstudies, SPRhasproventobeverysuitableforfast,sensitive,label-freeand realtimeone-stepmonitoringofthehybridizationbetweenprobe and target DNA, and base pair mismatchdetection [21–23,17]. Currently,themolecularinteractionofDNA-targetswithspecific probesimmobilizedonagoldsurfaceisdetectedinrealtimeby changesintherefractiveindexoccurringintheliquid-solid inter-facedirectlyconnectedtothebiomoleculeretainedmassonthe sensorsurface.Inordertooptimizethesensitivityandspecificity withSPR,manystrategiesandassays basedontheuseof help-ingagents(i.e.,proteins,nanoparticles(NPs),intercalatingagents or artificialDNAs) havebeen developed[24].However, despite thesignificantimprovementsinthesensitivityobtainedbyusing nanomaterial-basedSNPassayscoupledwithanumberof differ-entreadoutstrategies,themutationdiscriminationability(MDA) neverrisesabove90%[16].MDAisdefinedastheabilityto discrim-inateaspecificpointmutationbycomparingthesignalrecorded by hybridization with a target having a single mismatch with respecttothesignalgivenbythefullycomplementarytarget[16] (for a more rigorousdefinition, seeEq. (3) in “Results and Dis-cussion Section-Singlenucleotide polymorphisms Discrimination”).

http://dx.doi.org/10.1016/j.colsurfb.2017.06.021

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Additionally, althoughall these strategies optimizethe sensing performance,easeofuseandcostsarenotnecessarilyimportant characteristics.Suchstrategiesactuallyrequirecomplexreagents andmulti-stepreactions,whichmakeitdifﬁculttoscaleuptowards devices.

Inapreviouspaper[25],wedescribedasinglestepandlabel freemethodtoselectively detecttheHVBgenome withsimple linearssDNAprobesimmobilizedontheAusurfacesofaQCM res-onator.Inthisstudy,weadoptedanoptimizeddetectionstrategy toefﬁcientlydiscriminateSNPusing93-merDNAtargetsequences associatedwiththeKirstenRatSarcomaViralOncogenehomolog (KRAS)gene.TheSNPvariationsinKRASgenecodons12,13,and 61,aredirectlyassociatedwiththedevelopmentofmanykindsof cancer[26–29],andtheirdetectioniscriticalfortheselectionofthe appropriatetypesoftreatment[30].Europeanauthorities respon-siblefor healthand theAmerican Society forClinical Oncology requestedanefforttoidentifyKRASmutationsforearlydetection ofcancerpathologies[31].Thesepointsstimulateresearchforthe establishmentofcost-effective,rapidandlarge-scaleKRAS muta-tionscreeningmethodsforpersonalizedcancertherapy.Currently, thedetectionofpointmutationinhumangenomicDNAusing sim-ple,fastandreliablemethodologiesremainsachallenge.

In this paper, in order to achieve high specificity via SPR transduction,a detectionstrategy was adoptedbased onprobe immobilizationoptimizationatthesurface,followedbyasuitable hybridizationstepandafinaldenaturationwashingprocess.The resultsobtainedby meansofQuartz CrystalMicrobalancewith Dissipationmonitoring(QCM-D)andSurfacePlasmonResonance (SPR)techniquesshowthatanefficientsinglebasemutation dis-criminationofamatchedKRASwild-type(C-G)sequenceversus threemismatchedtargetsequences(i.e.,G12C(C-T),G12S(C-A) andG12R(C-C))canbeobtained.

2. Materialsandmethods 2.1. Chemicals

ThiolatedoligonucleotidesWTprobe(35bps)andWT,G12C, G12SandG12Rtargetsequences(93mer)werepurchasedfrom Purimex(Germany).Thedetailedcompositionofthesequencesis reportedinTable1.

2.2. Probeimmobilizationandhybridizationreagents

Theprobeoligonucleotidessolutionwaspreparedata concen-trationof1.0␮Min0.1Mphosphatebuffer(PBS)and1.37MNaCl. ThewaterwaspuriﬁedusingaMilliporeﬁltrationsystem.

Thetargetsolutionswerepreparedat0.5␮Mconcentrationin 0.1Mphosphatebuffer(PBS)and1.37MNaCl,byheatingat70◦C for3minandcoolingonicefor2min.Thisprocedureisintended topreventhairpinformationresultingfromtheanalysisperformed withOligoAnalyzer3.1software(datanotshown).Thetarget

solu-tionswereallowedtoequilibrateatroomtemperaturefor15min beforeuse.

WashingsolutionsconsistofSSCbuffer:1×SSC=150mMNaCl, 15mMsodiumcitrate,0.1%SDSinwater(pH7.0).

2.3. Surfaceplasmonresonancemeasurements

TheSPRmeasurementswereconductedusingtheSPRNavi200 (BioNavis,Finland), adouble channeland prismcoupling-based instrument.

BeforemodifyingthessDNAprobe,thegoldchipwasimmersed inaboilingsolution(30%H2O2,28%ammoniaanddoubledistilled waterinavolumeratioof1:1:5)for10min.Thecleanedchipwas thendoublerinsedwithdistilledwateranddriedbynitrogenflow beforeuse.Thechipwasthensetintheflowcellandwashedwith aPBSbuffer(50␮Lmin−1 flowrate)flowuntilthebaselinewas constant.

ThessDNAprobewasinjectedintheflowcellandakineticof molecularadsorptiononthegoldchipwasfollowedbythe moni-toringofchangesintherefractiveindexattheinterfaceofthegold chipsurfaceonprobe interactionbydetectingtheanglechange ofthereflectedlaserbeam(SPRangleshifts(␪))accordingtothe Kretschmannconfiguration[32].TherinsingstepwithPBS0.1M followedtheprobeimmobilizationstep.

Thekineticsofhybridizationwithperfectlymatchedand mis-matchedtargetswerecarriedoutbyﬂowingthetargetsolutions intheSPRcell,preparedaccordingtotheabovementioned proce-dure(seeProbeimmobilizationandhybridizationReagentssection). Thetargethybridizationkineticswererecordedbymonitoringthe speciﬁcSPRangleshifts.

Afterhybridization,asecondwashingstepwascarriedoutby flowingthewashingbuffer(50␮Lmin−1 flow rate)andthe dif-ferenceintheresponsesignalwascomputedfromthedifference betweentheinitialandfinalbuffersignals.Allmeasurementswere performedat25◦Croomtemperature.

Accordingtotheresultsinpreviousstudies[33]andthe speciﬁ-cationsprovidedbytheequipmentmanufacturer,ashiftintheSPR angleof100mDegequatestoasurfacecoverageof100ng/cm2_.

3. Resultsanddiscussion

93-mersyntheticDNAsequencesbasedoncodon12oftheKRAS genewereusedasmodelsfortargetsamples.TheG12S,G12Tand G12RmutationschosenforthisresearcharethepredominantSNPs incancer-associatedKRASgenes,representing83%oftherelative frequencyofmutationsofresiduesG12[34].

Table1 shows thedetails of thesequences followed in this study.Theprobes weredesigned sothat themutationposition ((A-C)forG12S,(C-T)forG12Cand(C-C)forG12R),isplacedin themiddleoftheprobestrand.Thisdecisionisbasedonprevious studiesdemonstratingthatthislocusisthemostconvenientfor mismatchdiscrimination[35].Infact,whenasequencemismatch

Table1

Oligonucleotidesequencesandmodiﬁcations.

Name Sequence

TargetWildType(WT) GACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTT

GACGATACAGCTAATTCAGAATCATTTTGTGGACGAATA

MismatchedTargetG12S GACTGAATATAAACTTGTGGTAGTTGGAGCTAGTGGCGTAGGCAAGAGTGCCTT

MismatchedTargetG12C GACTGAATATAAACTTGTGGTAGTTGGAGCTTGTGGCGTAGGCAAGAGTGCCTT

MismatchedTargetG12R GACTGAATATAAACTTGTGGTAGTTGGAGCTCGTGGCGTAGGCAA

GAGTGCCTT

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Fig.1.ChangesintheangularpositionoftheSPRpeakfortheWTprobe immobi-lizationonthegoldsubstrate.TherinsingwithPBSisindicatedbythearrow.The dataaretheaverageofthreeexperiments.

occursinthemiddleoftheprobesequence,agreaterreduction

in the thermodynamicstability of the hybridis observed with

respecttothemismatchattheendoftheprobesequence.

Accord-ingly,thehybridizationofthemutantsequencestothetargetis

expectedtobeunfavorablewithrespecttotheexpectedperfect

matchhybridizationwiththeWildTarget.

3.1. Probegrafting

Theﬁrstpartofthisworkfocusesonthesurfaceimmobilization

oftheWT-probeconsistingofashortoligonucleotide(35merlong)

totakeadvantageofthethermodynamicallylessfavourable

non-speciﬁcbindingoccurringforshortsequences[13,36].Theprobe

was linked to the surface of an SPR gold sensor, according to theproceduredescribed intheExperimental Section.In partic-ular,anadeninedA10 spacerwasusedasterminalblockofthe probesequencetoreduceanyspeciﬁcinteractionwiththe sen-sorsurface.Additionally,thedA10 spacerpromotesanL-shaped probeconformation,forcingthestrandintoanuprightstructure [37,38].Theimmobilizationkineticsusing1.0␮Msolutionof thio-latedWTprobeonAusurfacesweremonitoredinsituviaSPRand quartzcrystalmicrobalancewithdissipationmonitoring(QCM-D). SPRresultsarereportedinFig.1.TheSPRsignalreachesasteady stateshiftof220±10mDeg,correspondingtoanestimatedprobe densityof1.2±0.2×1013_molecules/cm2 _(considering_the_probe molecularweightof10.806kDaandtheSPR’smasssensitive fac-torof100mDegper100ng/cm2_)._The_order_of_magnitude_of_the obtainedprobedensity (about 1.0×1013_molecules/cm2₎ corre-spondstothedensityexpectedforAusurfacesimmobilizedwith probeshavingapolyadenine(dA)10lateralspacerandinthe pres-enceofbufferscontainingdivalentsalts[39–44].Thesignalshift remainsstableduringthesubsequentrunninginthePBSbuffer, indicatingthatthessDNAprobeisﬁrmlyboundtothegoldsurface. Aclosevalueofprobedensitywasalsoobtainedfromthe QCM-D measurement (see Supporting Information). The equilibrium value of the WT probe linked mass was 442.2±35.4ng/cm2_, or 2.5×1013_±_2.0_×₁₀12_molecules/cm2_. _This _value _is _50% higher than the mass probe density values measured by SPR (1.2×1013_molecules/cm2_). _This_apparent _difference_could _be _a consequenceofthehydratedmassrevealedbyQCM-D.

Accordingly,theSPRdatawasconsideredforarealistic evalua-tionoftheeffectiveamountofprobelinkedtothesensorsurface aswellasontheprobe-functionalizedsurfaces.

ThesurfacedensitycalculatedfromtheSPRdatayieldsan aver-age0.12molecules/nm2_(SPR),_{corresponding}_to_an_{intermolecular} spacing of2.86nm,assumingasimple hexagonalclosepacking arrangement of the linked probe strands. This surface density (1.2×1013_molecules/cm2₎_is_comparable_with_the_data_found_by

Fig.2. AngularshiftoftheSPRminimumpeakforWTtargetandthreemismatched G12C,G12SandG12Rafterhybridizationstep(arrowindicatedas“target”)and washingstep(arrowindicatedas“WB”).

Schreineretal.[41]fortheimmobilizationofSH-ss(dA)15probe (1.3×1013_molecules/cm2_)._Schreiner_et_al. _also_proved_that _the averagespacingbetweentwoadjacentprobesiscontrolledbythe lengthofthepolyAspacer.Therefore,inourcase,the(dA)10spacer canbereasonablysupposedasactingasananchoringblock pro-motinganL-shapedprobeconformation.

Thishypothesisisfurthersupportedbythefactthatthe acous-ticratio,D/f,measuredbymeansofQCM-Dforboundprobe molecules,changesfrom280×10−10/HzbeforerinsingwithPBS buffer, to 700×10−10/Hz after the rinsing step (see Support-ingInformation).Thisisinlinewithsimilarresultsreportedfor straight-shapedconformationsofsurface-boundssDNAmolecules [45].

We therefore propose that, under the presently employed experimentalconditions,theprobeassumesthemorefavourable L-shapedconformationtoachieveanoptimalprocessofmolecular recognitionthroughhybridizationreactions.

3.2. Singlenucleotidepolymorphismsdiscrimination

Differentstringencycontrolstrategiesareavailablefor biochip-based DNA hybridization analysis. The selection of a suitable strategyisstrictlydependentonthesensingplatformused.Inour case,theabilityofourimmobilizedprobe todiscriminatepoint mutationsviaSPRwasassessedusingatwo-stepstrategy:theﬁrst stepishybridizationunderhighionicstrengthbuffer;thesecond stepiswashingwithmoderateionicstrengthbuffer.

Fig.2showstheSPRcurvesobtainedwiththeabovementioned two-stepstrategy.Inparticular,thesectionbetweenthe“target” and “WB” arrows shows theSPR shifts resultingfrom theﬁrst hybridizationstep.ThiswasachievedbyexposingtheWT-probeto a500nMsolutionofthefullycomplementary93-mertarget(WT) and tothethree 93-mersingle-base-mismatchedMMsolutions (G12C,G12SandG12R),underthesamehighionicstrength con-ditions(0.1Mphosphatebuffersolutioncontaining1.37MNaCl). ThelargestSPRshift,diagnosticofthemosteffectivebinding,was obtainedforperfectmatchingoftheWTtarget,withavalueof 90±10mDegversus70±9mDegfortheG12Ctarget,60±9mDeg forG12Sand36±9mDegforG12R.

In terms of retained masses (Fig. 3a), the above SPR shifts correspondtovalues of90ng/cm2 _for_WT,₇₀_ng/cm2 _for _G12C, 60ng/cm2 _for_G12S_and ₃₆_ng/cm2 _for_G12R_target,_respectively (datacalculatedconsideringinstrumentsensitivityfactorof100 mDeg=100ng/cm2_)._The_{hybridization}_efﬁciency_(%HE)_was calcu-latedfromEq.(1)

%HE=

T P

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Fig.3.(a)SPRretainedmassvalues(–ng/cm2₎_and_{Hybridization}

Efﬁcien-ciesafterhybridizationstep.Theerrorbarsindicatethestandarddeviation(SD) obtainedonn=3measurements;(b)Hybridizationkinetic:plottingofd/dtversus thehybridizedtargetmassforWTandMMtargets.

where[T]isthenumberoftargetmolecules,and[P]isthenumber ofprobemolecules.

Accordingtothisequation,thecalculatednominal Hybridiza-tionEfficiencies(HE)are16%forWTtarget,about12%forG12C, 10%forG12Sand6%forG12R,ingoodagreementwithliterature [44].Additionally,thedecreasingtrendin%HEcloselyfollowsthe theoreticalenergystabilitytrendofthebasepairs:C-G(WT)>C-T (G12C)>C-A (G12S)>C-C (C12R) [46]. These HE values are sig-nificantlylowerthanthosereportedintheliteratureforsimilar (dA)n-probes,wherevaluesrangingfrom50to60%areobserved [41–44].Thisfindingsuggeststhatthehybridizationsteadystateis notreachedintheanalysistimeframe.

The time evolution of the hybridized target was analysed accordingtotheLangmuirmodel(Fig.3b),viathefollowing equa-tion[15]: d dt =konCt

1− max

(2) whereKon=kads/kdes,d(t)isthetime-dependentsurfacedensity fortheadsorbedmolecules(Nofadsorbedmoleculespersecond andcm2_),

maxisthemaximumcoveragevalueandCtisthetarget concentrationinsolution.

Accordingtothissimplemodel,thekineticrateofhybridization (kon)canbeeasilycalculatedfromtheinterceptofthelinearpartof d/dtversus.Inparticular,konis3.3±0.1×106M−1 s−1forWT, 2.5±0.1×106_M−1_s−1_for_G12C,_1.9_±_0.1_×₁₀6_M−1_s−1_for_G12S and1.2±0.1×106_M−1_s−1_for_G12R.

Theseresultsindicatethat kineticsforthefully complemen-tarytargetaresigniﬁcantlyfasterthanthemismatchedsequences, givenrecentliteraturereportsindicatingthatthesubstitutionof thesinglebasehasastrongeffectonthehybridizationrate[47].

Thehybridizationkineticsdatareportedintheliteraturevary signiﬁcantlyfrom∼103_M−1_s−1_to_∼106_M−1_s−1_[48]_due_to

sev-Fig.4. SPRretainedmassvalues(–ng/cm2₎_after_washing_with_PBS_buffer._The

errorbarsindicatethestandarddeviation(SD)obtainedonn=3measurements.

eralfactorsaffectingthem.Theorderofmagnitudeobtainedhere (∼106_M−1_s−1₎_falls_inside_the_higher_ranges_given_in_literature, indicatingveryfasthybridizationwithoutthespecific optimiza-tionofmeltingtemperatureandstringencyconditions.Thisfinding canbelinkedtothepresenceoftheL-shapedadenineblockwhich isabletogenerateafavourableconfigurationforhybridizationby increasingtheaccessibilityofthetargetmolecules.[38,42].

Afterhybridization,asecondstepinvolvingwashing(diagram partofFig.2startingfrom“WB”arrow)isperformed.Toefﬁciently removethelessstableduplexstructures(notfullycomplementary targets),awashingbufferwithlowionicstrength(oneorderof magnitudelowerthanthatemployedinthehybridizationstep)was employedinthepresenceofthedenaturisingagentSDS,astandard additiveforwashingsolutions instandardhybridization experi-ments[49,50].TheSPRresponsesforallstrands(Fig.2–curvesin therangeof60–80min)revealthatonlytheWTtargetexhibitsa detectableSPRsignal,whileallthemismatchedsequencesshow valuesthatcannotbedistinguishedfromthebaseline.Thismeans that this secondwashing step completelyremoved themutant hybrids(i.e.,G12C,G12RandG12S),keepingonlytheperfectmatch wildtype(WT)genomesbound.

Fig.4showstheretainedmassofhybridscalculatedfromtheSPR shiftsafterwashing.Thisdemonstratesthatonlythefullymatched target(WT)isstillretained,with40ng/cm2_,_even_if_some_of_the initiallyboundhybridsarelost.

Thisbehaviour can be explained ifwe consider that a high ionicstrengthsolution(1.37MNaCl)determinesaDebyelength atroomtemperatureofabout0.26nm;i.e.,smallerthanthe inter-phosphorous(P−-P−)distanceofabout0.76nminthenucleotide sequences.Accordingly,thenegativechargesofthephosphodiester groupsalongtheimmobilizedprobemoleculesarecompensatedby thecounter-ioncharges,stabilizingthetargetsequencesbinding withtheprobe.

Theabovementionedresultsallowthedefinitionofahighly effi-cientstrategytodiscriminatethefullymatchedduplexstructure (WT)fromthemutantKRASsequence.Theprocessisbasedonthe carefulcontrolofthepost-hybridizationwashingconditions,based ontheuseofmoderateionicstrengthconditions(0.15MNaCl,in thepresentcase)inpresenceofthestandarddenaturingwashing additive(0.1%V/VSDS)fortheefficientremovalofthelessstable mutantstrand-duplexhybrids.Theprocessprobablytakes advan-tageofthedifferenceinthermodynamicstabilitybetweenthefully matchedduplexstructure(WT)andtheMMduplexstructures.In fact,aspreviouslydiscussed[51–54],thehybridstabilitynearthe surfaceisreducedifwereducetheionicstrengthofthesolution. Inourcasewith(0.15MNaCl),weestimateaportionofabout10 ntisaffectedbythisdestabilization[51].

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Theabovedistanceroughlycorrespondstothecentralposition ofthemutantsitesinthemutantKRASsequence,weakeningthe bindinginteractionofthemutantstrandswiththeWT-probeand thuspromptingthedissociation oftheprobe-targethybrids.On thecontrary,this factordoesnotaffectthebindingofthefully matchedWTsequence,enablingtheefﬁcientretentionofthewild typesequencesinthetestedionicstrengthconditions.

Theﬁnalmutationdiscriminationabilityofthemethodmaybe deﬁnedthroughEq.(3),

%HE=100−

RM RC

×100

(3) whereRMistheretainedmassofthemismatchedduplex,andRC istheretainedmassforthefullycomplementaryduplex.The result-ingdata,forthespeciﬁcionicstrengthconditionsemployedinthese experiments,leadtoadiscriminationvalueof100%forthefully matchedtarget(WT)againsttheMMones.Itshouldbestressed thatsimilarSPR-basedmethodologiesdonotachievecomparable mutationdiscriminationvalues[15].

4. Conclusions

ThispaperdescribesasimpleSPRmethodtoachieverapidand excellentdiscriminationofthefullymatchedKRASwild-type (C-G)sequenceagainstthreemismatchedG12S(C-A),G12R(C-C), G12C(C-T)targetsequences,whichrepresentthemostcommon mutationsin the KRAS gene. Thismethod involvesa detection strategybasedonoptimizedprobe-surfacedensitycombinedwith ionic strength control. Twoexperimental stepswere executed: i)hybridizationunderhighionicstrengthbufferandii)washing withmoderateionicstrengthbuffer.Thankstothese complimen-taryeffects,wefoundhybridizationratesof∼106_M−1_s−1_without theuseoftheoptimizedhybridizationtemperaturesorstringent conditions usually needed in conventional hybridization meth-ods.Moreover,we achieved a100% mutation discriminationof thematchedKRASwild-type(C-G)sequencewithrespecttothree mismatchedG12C(C-T),G12S(C-A),G12R(C-C)targetsequences. Theresultspresentedhereinshowthatfastandhigh discrimi-nationofSNPcanbeachievedwithconventionalSPRbiosensors withoutusing labels or target signalampliﬁcation probes.This modelstudypromptsustocontinueandextendourinvestigation tothecompletesetofKRAS mutations,withassayperformance evaluations(sensitivityandlimitofdetection)andthetestingof realsamples.

Acknowledgements

G.M. gratefully acknowledges the ﬁnancial support for the projectand thebiennial fellowship of N.G.from the FIRB Pro-gram“AccordidiProgramma”(contractnumberRBAP11ZJFA002, Rome,Italy).AuthorswarmlythankbothDr.ValeriaFormicaand Dr.AndreaLastrinaforhistechnicalreviewofthemanuscript. AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.colsurfb.2017.06. 021.

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