ContentslistsavailableatScienceDirect
Sensors
and
Actuators
B:
Chemical
j o u r n a l ho me p a 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
Functionalization
of
gold
surfaces
with
copoly(DMA-NAS-MAPS)
by
dip
coating:
Surface
characterization
and
hybridization
tests
D.
Petti
a,∗,
A.
Torti
a,
F.
Damin
b,
L.
Sola
b,
M.
Rusnati
c,
E.
Albisetti
a,
A.
Bugatti
c,
R.
Bertacco
a,
M.
Chiari
baLNESS-DipartimentodiFisicadelPolitecnicodiMilano,ViaAnzani42,22100Como,Italy bIstitutodiChimicadelRiconoscimentoMolecolare,CNR,ViaMarioBianco9,20131Milan,Italy
cDepartmentofMolecularandTranslationalMedicine,UniversityofBrescia,VialeEuropa11,25123Brescia,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received5June2013
Receivedinrevisedform9August2013 Accepted25August2013
Available online 4 September 2013 Keywords:
Goldfunctionalization Copoly(DMA-NAS-MAPS) Chemiluminescence Surfaceplasmonresonance Atomicforcemicroscopy X-rayphotoemissionspectroscopy
a
b
s
t
r
a
c
t
Inthiswork,anewmethodtofunctionalizeagoldsurfacebydipcoatingwithafunctionalcopolymer ispresented.Thecoatingprocedureissimple,robustandcanbeaccomplishedinlessthanonehour. Atomicforcemicroscopy(AFM)scratchtestsrevealthepresenceofahomogeneouspolymercoating withathicknessof2.5nm.X-rayphotoemissionspectroscopyspectrafromC1s,N1sandO1slevels presentthetypicalfingerprintsofthepolymericoverlayer,i.e.thecharacteristicpeaksfromCNC Oand NC Ogroups.
Surfaceplasmonresonance(SPR)bindingassayswereusedtocheckthecoatingfunctionalproperties. ImmobilizationofheparintoSPRgoldsurfacesfunctionalizedwithcopoly(DMA-NAS-MAPS)-followed bybindinganalysiswiththewellknownheparinbindingproteinfibroblastgrowthfactor2yieldbinding kineticparameterscomparabletothoseobtainedwithcommerciallyavailablecarboxymethyl dextran-functionalizedsensorchips,thusconfirmingthegreatpotentialoftheproposedtechnique.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
In the last ten years, we assisted to a great development ofmicroarray analysistechniquesfor proteomicsandgenomics [1]. In this scenario a key-point is the chemical functionaliza-tionofdifferentsubstrates(glass,silicon,gold)[2]foraneffective andselectiveimmobilizationofbio-macromoleculessuchasDNA, oligonucleotidesandproteins.
Inparticular,thereisastronginterestindevelopingmethods forgoldsurfacefunctionalization,asthismetaliswidelyusedin biosensing.Insurfaceplasmonresonance(SPR),athinlayerofgold onahighrefractiveindexglasssurfaceabsorbslaserlight, produc-ingelectronwaves(surfaceplasmons) onthegoldsurface.This occursonlyata specificangleandwavelengthofincident light which arehighly dependentonthesurface properties. For this reason,thebindingofatargetligandtoa receptorimmobilized onthegoldsurfaceproduces ameasurablesignalrelatedtothe concentrationofthetarget[3].
Inelectrochemicalbiosensors,anenzymaticallycatalyzed reac-tion,involvingthetargetanalyte,producesorabsorbselectronson theactiveelectrodesurfacecausingeitherelectrontransferacross
∗ Correspondingauthor.Tel.:+390313327307;fax:+390313327617. E-mailaddress:daniela.petti@polimi.it(D.Petti).
thedoublelayer(thusproducingacurrent)orcontributingtothe doublelayerpotential(finallyleadingtoavoltage)[4].
Severalfunctionalizationstrategiesforgoldhavebeendescribed [2,5]and referencetherein]includingsurfacemodificationwith silanes[6]andfunctionalpolymers.InSPRtheunquestioned coat-ingchoiceisbasedondextran.Astabledextranlayerisobtained bycovalentbindingofthepolymertoafunctionalsublayerformed onthesurfacebyself-assemblingofathiolbearingmolecule.The characteristicsofthedextrancoatedsurfacehavebeenoptimized andthequalityofthiscoatinghasgreatlycontributedtothesuccess ofSPRtechnology.However,theprocedureforbindingdextranis cumbersomeandrequiresmultistepchemicalreactionswhichmay bedifficulttocontrol.
Inthecaseofthedextrancoatingasinotherbioassays involv-inggold[7],thecoatingformationrequiresspontaneousassembly of chemically reactive alkenethiols leading toformation of so calledself-assembledmonolayers(SAM)[8–10].Byplacingagold substrateintoamillimolarsolutionofanalkanethiolinethanol a varietyof chemically reactivegroups canbe introducedby a reaction between gold and thiols. This reaction is well-known andwidelyadoptedduetothefactthatitisstraightforwardand requirescommerciallyavailablechemicalreagents.Inadditionthe bindingbetweengoldandthiolisverystableowingtoits cova-lentcharacter[11,12].However,theformationofawell-assembled monolayerstronglydependsonthepurityofthealkanethiolbeing 0925-4005/$–seefrontmatter © 2013 Elsevier B.V. All rights reserved.
D.Pettietal./SensorsandActuatorsB190 (2014) 234–242 235
used.Thepresenceofevenlowlevelsofcontaminantscanresult inadisordered,non-idealmonolayer.Unfortunately,typical impu-ritiesinthiolcompoundsarethiolatedprecursormolecules,not properlyseparatedduringthepurificationprocess[13].
Inthisworkwepresentanalternativeapproachtosurface modi-ficationthatisfast,robustandeasytoperformeveninlaboratories thatdonothaveastrongsurfacechemistrybackground.In par-ticular,weproposeamethodbasedonapolymericthincoating madeofN,N-dimethylacrylamide(DMA)bearingsilanating(MAPS) and chemicallyreactive (NAS) moieties:poly(DMA-NAS-MAPS). Thecopoly(DMA-NAS-MAPS)wasdevelopedforDNAandprotein microarrayassaysonmicroscopeglassslide[14]andonsilicon[15]. Asshowninpreviouspapers[6],thedepositionby“dipandrinse” coatingofathinlayerofthispolymerrepresentsafast,inexpensive androbustmethodtocovalentlybindproteinsandamino-modified DNAprobes.Inaddition,thefilmisstableinaqueousbuffers con-tainingvariousadditives,evenatwaterboilingtemperatureand, duetoitshydrophilicnatureandhighhomogeneity,itminimizes non-specificbiomolecularinteractions.Thelastfeatureishighly desirableforsurfacerecognitionbioassays,forwhichspecificityis difficulttoachieve.Furthermore,theproposedmethod,employing nonsurface-specificmaterialmodifications,canbeeasilyusedalso toformuniformcoatingsonmanydifferentmaterialsotherthen gold,suchasglass,silicondioxide,siliconnitrideandtitanium diox-ide[15],thusbeingappliedtodifferentbiosensingproblemsand techniques.Thisversatilitycanbeparticularlyimportantin appli-cationemployingsensorsthatconsistofmorethanonematerial. Theuseofcopoly(DMA-NAS-MAPS)allowsthederivatizationofall thedifferentregionsofthedeviceinsinglestep.
Themorphologicalandchemicalpropertiesofthispolymeric coatingongoldsurfaceshavebeenstudiedbymeansofatomicforce microscopy(AFM)andX-rayphotoemissionspectroscopy(XPS). Thesetechniquesrevealthatthepolymerisimmobilizedonthe sur-facewiththeproperchemicalcompositionandexpectedthickness. Furthermorewefoundalimitedimpactonthegoldunderlayer, thusallowingtheuseoftheproposedfunctionalizationmethod fortechniquessuchasSPR.Todemonstratethecompatibilityof thisderivatizationprocesswithstandardbioassays,twodifferent experimentshavebeencarriedout:aDNA hybridization exper-imentwithchemiluminescence and surfaceplasmon resonance (SPR)toassaythebindingoffibroblastgrowthfactor(FGF2)in solu-tiontoimmobilizedheparin.Independentlyfromthetransducer employed,resultscomparabletothoseobtainedwithcommercially availablecarboxymethyldextran-functionalizedsensorchipswere obtained.Thisindicatesthepotentialofthecoatingtechniqueto implementrobustprotocolsofbiomoleculesimmobilizationonto goldcoatedsurfacesforbiosensingapplications.
2. Materialsandmethods
2.1. Chemicals
Tris(hydroxymethyl)aminomethane (TRIS), ethanolamine, saline-sodium citrate (SSC), ammonium sulfate, sodium dodecyl sulfate (SDS), phosphate buffer solution (PBS), N,Ndimethylacrylamide(DMA)and[3-(methacryloyl-oxy)propyl] trimethoxysilane(MAPS)werepurchasedfromSigma(St.Louis, MO). N,Nacryloyloxysuccinimide (NAS) was from Polysciences (Warrington, PA).Oligonucleotides weresynthesized by MWG-Biotech AG(Ebevsberg, Germany). Streptavidin labeledby HRP (HorseRadishPeroxidase)fromJacksonImmunoresearch.Heparin (13.6kDa) was obtained from Laboratori Derivati Organici Spa (Milan,Italy).SuperSignalELISAFemtomaximumSensitivitykit from ThermoScientific. Human recombinant fibroblast growth factor2(FGF2)wasexpressedinEscherichiacoliandpurifiedas describedin[16].
2.2. Goldsurfacepreparationandderivatization
Forinitialtests,goldfilmsonSiwerepreparedin-house.Silicon waferswerecoatedwith3nmCr(topromotegoldadhesion)and 50nmAu,bymeansofelectronbeamevaporation. Copoly(DMA-NAS-MAPS), whose chemical structure is shown in Fig. 1, was synthesized and characterizedas described in a previous work [17,14].ThegoldsurfacewastreatedfortenminutesinanOxygen PlasmaGenerator(HarrickPlasmaCleaner)andthenimmersedfor 30mininacopoly(DMA-NAS-MAPS)solution(1%,w/vinawater solutionofammoniumsulfateata20%saturationlevel).Thesilicon slideswerethenextensivelywashedwithwateranddriedunder vacuumat80◦Cfor15min.
2.3. AFMmeasurements
AFMmeasurementswereperformedwithaVEECOInnovaAFM. The morphological characterization wascarried out in tapping modewith512samples/line, frequency of0.8Hz and 4–10m scan range.Thethicknessof thepolymericlayerwasevaluated byAFM-tipscratchtest[18].Thistechniqueconsistsin scratch-ingthecoatingwiththeAFMtipwhilethenormalforceappliedis settoavaluehighenoughtopenetratethelayerbutlowenoughto avoidsignificanttiporsubstratedamaging.Thescratchwasmade incontactmodeonwardandinliftmodebackward(startheight 0.3m,liftheight0.3m)with30continuousscancycles,256 sam-ples/line,62.75Hz,1mscanrange.Afterscratching,theareawas imagedintappingmodewiththesametipusedforscratching.In thiswaytheareaofinterestiseasilyidentifiedwithintheactive scanareaoftheAFM.
2.4. XPSexperiments
XPSexperimentswereperformedinamultichamber ultrahigh-vacuum (UHV) system described in detail elsewhere [19]. PhotoelectronswereexcitedbyAlK␣(h=1486.67eV)orMgK␣ (h=1253.6eV)radiation,andanalyzedbya150mm hemispher-icalanalyzer(SPECSPhoibos150).Theangularacceptancesetfor thisworkwasabout±6◦,andthespatialresolutionwas1.4mm.
Twomodesofoperationhavebeenemployed:(M1)passenergyof 50eVandAlK␣line(totalenergyresolutionof1.643eV)to eval-uatethestoichiometryofthepolymer;(M2)passenergyof10eV andMgK␣line(totalenergyresolutionof0.841eV)inorderto studythelineshapeofthecharacteristicpeaks.Theenergyscale ofthespectrahasbeencalibratedbymeasuringaTafoilingood electricalcontactwiththesample.
2.5. Chemiluminescencetest
A 23-mer,5-amine-modified oligonucleotidesynthesizedby MWG-BiotechAG(Ebevsberg,Germany),100Mstocksolution, 5-NH2-(CH2)6-GCCCACCTATAAGGTAAAAGTGA,wasdissolved in150mMsodiumphosphatebufferatpH8.5.A10Msolutionof thisoligonucleotidewasprintedoncoatedslidestoforma15×15 arrayusingapiezoelectricspotter(SciFLEXARRAYERS5;Scienion). Spotting wasdoneat 20◦C in anatmosphere of 50% humidity. Theoligonucleotides werecoupledtothesurfacesbyovernight incubation inanuncovered storagebox, laidin asealed cham-ber,saturatedwithsodiumchloride(40g/100mLH2O),atroom
temperature.Afterincubation,allresidualreactivegroupsofthe coating polymer wereblockedby dipping theprinted slides in 50mMethanolamine/0.1MTrispH9.0at50◦Cfor15min.After discardingtheblockingsolution,theslideswererinsedtwotimes with water and shaken for 15min in 4× SSC/0.1% SDS buffer, pre-warmedat50◦C,andbrieflyrinsedwithwater.An oligonu-cleotide,complementarytotheonespottedonthesurface,ata
Fig.1.structureofthecopoly(DMA-NAS-MAPS)boundtothegoldsurface.Thecopolypresentsthreefunctionalgroups:thepolymerbackboneinteractingwiththesurface, thependingsilanehydrolysablemonomersthatstabilizingthefilmandthechemicallyactivemonomersallowingthecovalentbindingofbiomolecules.
1.0Mconcentration(2.5L/cm2 ofcoverslip) wasdissolvedin
thehybridizationbuffer(2×SSC,0.1%SDSand0.2mg/mLBSA)and immediatelyappliedtomicroarrays.Thecomplementary oligonu-cleotidehasthesequence5-TCACTTTTACCTTATAGGTGGGC-3 andislabeledwithbiotinin5position.Thesurfaces,placedina hybridizationchamber,weretransferredtoahumidified incuba-toratatemperatureof65◦Cfor2h.Afterhybridization,theslides werefirstwashedwith4×SSCatroomtemperaturetoremove thecoverslipandthenwith2×SSC/0.1%SDSathybridization tem-peraturefor5min.Thisoperationwasrepeatedtwotimesandwas followedbytwowashingstepswith0.2×SSCand0.1×SSCfor1min atroomtemperature.StreptavidinlabeledbyHRP(HorseRadish Peroxidase)fromJacksonImmunoresearchattheconcentrationof 1g/mLinPBSbufferwasappliedtothegoldsurfacefor60min. Aftera shortrinse inPBS andwater, theslides weredried and chemiluminescencewasdeveloped usingtheSuperSignalELISA FemtoMaximumSensitivitykitfromThermoScientificaccording tothemanufacturerinstructions.Thechemiluminescencesignal wasregisteredat0.2sexposuretimeusingahomemadesetup equippedwithaQImagingCCDCamera.
2.6. Surfaceplasmonresonance(SPR)
For SPR experiment, a naked gold sensorchip (Xantech Bioanalytics, Dusseldorf, Germany) was functionalized with copoly(DMA-NAS-MAPS) as follows: the gold chip was treated withoxygenplasmainaPlasmaCleaner,HarrickPlasma(Ithaca, NY,USA)tocleanthesurface.Immediatelyaftertheoxygenplasma treatment,thesensorchipwasdippedinanaqueoussolutionof poly(DMA-NAS-MAPS)inammoniumsulphateat20%ofsaturation levelfor30min,andthenitwasrinsedwithDIwater,driedwith nitrogenflow andcured undervacuumat80◦C for15min.The polymerwasfunctionalizedwithstreptavidinbyspotting1mg/mL solutionofproteindissolvedinPBSusingapiezoelectricspotter
(SciFLEXARRAYER S5; Scienion). The merged spots, printed at 20◦C in an atmosphere of 50% humidity,created a continuous protein film.To promote theprotein attachment,thechip was incubatedovernightinanuncoveredstoragebox,laidinasealed chamber,saturated withsodiumchloride(40g/100mLH2O), at
roomtemperature.Thefunctionalizedsensorchipwaspositioned inaBIAcoreXinstrument(GE-Healthcare,Milwaukee,WI)andthe surfacewasconditionedwith3consecutive1-mininjectionsof1M NaClin50mMNaOHbeforeheparinimmobilization.Biotinylated heparin [100ng/mL in 10mM HEPES buffer pH 7.4 containing 150mMNaCl,3mMEDTA,0.005%surfactantP20(HBS-EP)]was injectedontothecopoly(DMA-NAS-MAPS)functionalized sensor-chipfor4minataflowrateof5L/min.Increasingconcentrations ofFGF2inHBS-EPwereinjectedovertheheparinfor4minata flowrateof30l/min(toallowtheirassociationwithimmobilized heparin) and then washed until dissociation was observed. A sensorchip coated with streptavidin was used to evaluate the non-specificbindingand for blanksubtraction.Aftereveryrun, thesensorchipwasregeneratedbyinjectionof2MNaCl.Kinetic parameterswerecalculatedbyusingthenonlinearfittingsoftware packageBIAevaluationusingasinglesitemodel.
3. Resultsanddiscussion
3.1. Polymericcoatingcharacterization
A copolymer of dimethylacrylamide, N-hydroxysuccinimide and[3-(methacryloyl-oxy)propyl]trimethoxysilanehasbeenused toform,bydipandrinsecoating,athinfilmonthegoldsurface. ThecopolymerstructureisshowninFig.1,whererelative molec-ularweightsandmolecularweightdistributionsofthecopolymer weredeterminedbygelpermeationchromatography(GPC)in pre-viousworks[20].Ithasthreefunctionalportions:(i)segmentsthat mightinteractwiththesurfacebyweek,noncovalentbonds,van
D.Pettietal./SensorsandActuatorsB190 (2014) 234–242 237
Fig.2. AFMimageona5m×5mareaofthefreegold(panela)andpolymer coated(panelb)surface.TheroughnessRMSis0.4and0.6nm.
derWaalsorhydrophobicforces(polymerbackbone-DMA)favored bygoldhydrophobicity,(ii)pendingsilanehydrolysablemonomers thatstabilizesthefilmbypromotingcondensationbetween differ-entchains(MAPS),and(iii)chemicallyactivemonomersthatreact withaminespresentintheproteinlaterchainsallowingthe cova-lentbindingofbiomoleculestothegoldsurface(NAS).Thispolymer formsafilmonanumberofmaterialsofdifferentchemical com-positionsuchasglass[14],siliconoxide[15]andorganicpolymers [21,22].However,thespecificmechanismbywhichthepolymer formsathinlayeronthegoldsurfaceisnotfullyunderstood.
TheAFMimagestakenfromthegoldandpolymermodified sur-facearepresentedinFig.2(panelsaandb,respectively),forthe caseofAu/Cr/Sisamples.Therootmeansquare(RMS)roughnessof thegoldsurfaceisabout0.36nmonanareaof5m×5m,while thepolymercoatedsurfacepresentsaRMSroughnessof0.60nm. Theminorroughness increase suggestsa uniformand continu-ouspolymericcoatingmimickingthegoldmorphology.InFig.3a a representativenano-scratchimageof thelayerandin Fig.3b somedepthprofiles(takenalongthewhitelines)areshown.Inthe scratchedarea(1m×1m)aroughnessof0.48nmRMSis recov-ered.ThecomparisonoftheAFMphasecontrast(datanotshown) onthescratchedareaandontheoverlayer,revealsthatthetwo zonesaremadeofdifferentmaterials.Thisprovesthatthescratch hasfullyremovedthepolymericcoating.Fromdepthprofilesof Fig.3b,wecanthenevaluatealayerthicknessd=2.5±0.3nm, con-sistentwiththethicknessevaluatedbyscratchtestsperformed onaSiO2 surfacecoatedwiththesamepolymer[15].An
impor-tantcharacteristicofthispolymericcoatingisitsabilitytoswell inaqueoussolutions.Theswelling,alsopresentinotherpolymers, dependsonthepropertiesofthepolymerofinterestsuchasits molecularweight,aswellastheenvironmentalconditionssuchas temperature,orpH[20].
Fig.3. Panela:4.5m×4.5mAFMimage ofthesample after scratch;the scratchedareais1m2.Panelb:depthprofilesfromthelinesofpanela.The
estimatedpolymerthicknessis2.5±0.3nm,respectively.
The chemical characterization of the polymeric coating has beencarriedoutbyXPS.Widescanstakenoncoatedsamplesshow thecharacteristicpeaksoffourchemicalelements:goldfromthe substrateandcarbon,oxygen,nitrogencomingfromthepolymer. InTable1,theintensityratiosO1s/C1s,andN1s/C1sareshown, afternormalizationtothecrosssection,analyzertransmissionand probingdepth. Tothisscope, wenormalizedthepeak intensity tothefunction f()=*(1−exp(−d/)),where is theelectron escape depth[23]. The surface was analyzed at two collection anglesfromthesamplenormal:0◦and60◦,thelastoneproviding higher surface sensitivitydue to thefinite escape depthof the emittedphotoelectrons.
Table1
RelativeintensitiesofN1s,O1sandC1speakstakenat0◦,60◦collectionangleand
relativenominalatomicratios(fromthepolymercomposition). Collection angle(◦) N1s/C1s intensityratio O1s/C1s intensityratio XPS 0 0.17±0.02 0.16±0.02 60 0.14±0.02 0.15±0.02 copoly(DMA-NAS-MAPS) composition – 0.19 0.20
Fig.4.XPSspectrumofC1stakenat0◦(panela)and60◦(panelb)collectionangle;
thedeconvolutionofthepeaksrevealsthepresenceofthespecificgroupofthe polymer:NC OandCNC O.
Thenitrogenandoxygencontent,evaluatedfromtheintensity
ofthecharacteristicpeaks,are,respectively,17%and16%ofthat
ofthecarbon.Inthesurfacesensitiveconfiguration(60◦collection
angle)thesignalcomingfromN1sdecreasesto14%ofthatofC1s.
Notethatthisdecreaseisclosetothelimitoftheuncertaintyofthe
XPSmeasurement(about10%oftheratiosbetweenconcentrations
ofdifferentelements),butcouldbecompatiblewiththepresence
ofsomeadditionaladventitiouscarbon(e.g.physisorbedCO2)at
thesurface.Theseresultsrevealaslightlyhighercarbon
concen-trationwithrespecttooxygenandnitrogen,whencomparedto
theexpectedatomicratioforthecopolyreportedinTable1(19%
and20%,respectively),whichmaybedueagaintosurfacecarbon contamination.Thiscontaminationarisesmainlytotheextended exposureof thesample toatmospheric environment beforethe measurements.Aroughestimationoftheamountofadventitious carboncanbeobtainedbycomparingthemeasuredandnominal N/CandO/CratiosofTable1:avalueofcontaminantofabout6%of thetotalelementalcompositionisestimated,consistentwithdata obtainedonsimilarfunctionalization[24].
ThedeconvolutionoftheC1speakshowninFig.4allows dis-entanglingthecontributionsfromthedifferentcoordinationsofC atomsinthepolymer.ThefittinghasbeenperformedwithaVoigt line-shapeforeachcomponent,withafixedFWHMof1.56eV.This isconsistentwiththeFWHMoftheC1speakfromadventitious car-bon,previouslymeasuredinthesameexperimentalconditionson othersamples[25].
Fig.5.XPSspectrumofN1s(panela),O1s(panelb)andAu4f(panelc)at60◦
collectionangle;whilethefirsttwospectrashowthefeaturescomingfrom NC O groups,theAu4fpeaklineshapepresentsthetypicalmetalliccharacterofafree gold.
Panel(a)(0◦collectionangle)andpanel(b)(60◦collectionangle) ofFig.4showcommonfeatures.Themainpeak(A1)islocatedat
285.6eVandrepresentsabout63%oftheentireC1ssignal.Asitcan beascribedbothtoadventitiouscarbon(carbonpartiallybounded toO and N [25]) and C C bonds,it cannot beunambiguously
D.Pettietal./SensorsandActuatorsB190 (2014) 234–242 239
Fig.6.Schemeofthebioassaysurface.
connectedto thepolymercoating. Twoothercontributions are visible in both panels of Fig.4: A2 (286.7eV), which is due to
thepresenceofC OandC N C Ochemicalbonds[26]andA3
(288.4eV)comingfromCO2,NC Oandmethoxy(O CH3)groups.
[26–28]TheexperimentalweightofA2andA3are22%and15%of
thewholeC1speak,respectively,butalsointhiscasetheycanbe onlypartiallyattributedtoCatomswithinthepolymer,duetosome possibleCOand CO2 contaminations contributingtoA2 and A3.
Thesevaluesmustbecomparedwiththepolymerchemical struc-ture,fromwhichtherelativeweightofthedifferentcarbonbonds canbeevaluated.InanominalpolymercoatingC Cbonds (con-tributingtoA1)represent50.5%oftheentirecarbonbonds,C N
andC Obonds(A2)31.5%,whileNC O,O C=O,O CH3 groups
(indicatedasOMeinthepolymerchemicalstructureofFig.1)18.3%. Thediscrepancybetweentheexpectedratiosformthechemical formula and theexperimentalonesis consistentwiththe oxy-genandnitrogendeficiencyfoundinthecompositionanalysisof Table1.Adventitiouscarboncontaminationcanclearlyaccountfor theseresultsandpreventsfromapreciseestimationofthecoating compositionviaquantitativeXPSanalysis.
Finallynotethat,at0◦collectionangle(bottompanelofFig.4), anadditionalfeature(A4)at290eVisneededtofittheC1speak.
Duetotheincreasedprobingdepthatnormalemission,thisfeature canbeattributedtosomecontaminantsfarawayfromthesurface, mostprobablycarbonatesonthebaregoldsurfacewhichcanform duringthecoatinginwetenvironment.
Spectraat60◦collectionangleofN1sandO1s,arepresentedin Fig.5.AlsointhiscasethepeakshavebeendecomposedusingVoigt functionswithfixedFWHMof1.62eVand1.53eV,respectively, [25,26].
TheN1sspectrumpresentstwocharacteristiccontributions:the mainoneat400.7eV(B1),correspondingtoC N CandN C O
bondsfromthepolymer[26]andaminoroneat399.1eV(B2),
cor-respondingtoC Nboundsfromadventitiouscarbon[29].B2isonly
5%oftheentirepeakandcanbeattributedtocontaminationfrom adventitiouscarbon,asalready seenfromC1sspectra deconvo-lution.Regardingoxygen,threefeaturesgiverisetotheO1speak,
butheretheattributionisnotstraightforward.Whilethefeatureat 531.7eV(C1)isdueonlytoNC Ogroups[26],theprominent
con-tributionat533.3eV(C2)canarisefromtheoverlapofthesignals
fromH2O(533.3eV),O CH3 (533.2eV)[28]and C O(533.6eV)
groups[30].Theminorfeatureat534.9eV(C3)mightinsteadbe
duetophysisorbedCO2.Allthesefeatures intheO1speakare
consistentwiththepresenceofthepolymericgroupsfoundfrom thedeconvolutionofN1sandC1speaks,butaquantitative analy-sishereispreventedfromthesuperpositionofthecharacteristic signals.
Inordertoevaluatetheimpactofthepolymercoatingonthe goldsurface,wepresentinFig.5ctheAu4fpeakscollectedat60◦in ordertomaximizethesurfacesensitivity.Thecontinuousline rep-resentsafitperformedusingthelineshapemeasuredontheclean goldsurface,beforedipcoating,inthesameexperimental condi-tions.Theresultsseemtosuggestthatthegoldsurfaceisunchanged upondipcoating,withoutdetectabletracesofoxidization. How-ever,previousstudieshavedemonstratedthattheAusurfacecan beoxidizedbyoxygenplasmatreatments[31],thereforewecannot excludethat,atleastasmallportionofthemethoxysilaneonthe polymermightcondensatewithoxidizedgoldatomsstrengthening thebindingbetweenthepolymerandthesurfacewhich primar-ilyisduetononcovalentinteractions suchasvanderWaalsor hydrophobicforces.
Inconclusion,boththeAFMscratchtestandtheXPSanalysis revealthatacontinuouspolymerisimmobilizedonthesurface. InparticularXPSshowsthepresenceofthecharacteristic chemi-calgroupsfromcopoly(DMA-NAS-MAPS)ontopofagoldsurface preservingitschemicalintegrityuponcoating.
3.2. Hybridizationexperiments
A hybridizationtest wascarried outtoprovethat the func-tionalpolymerbindaminomodifiedoligonucleotideswithgreat efficiency and low background noise. A simple DNA hybridiza-tion experiment was performed to prove that the coating has introducedchemicalreactivegroupsonthegoldsurface.Ascheme
Table2
Theassociationrate(kon)anddissociationrate(koff)arereportedandthedissociationconstant(Kd)wasderivedfromthekoff/konratio.TheresultsonCarboxymethyldextran
arerepresentativeoftwoindependentexperimentswithsimilarresults.
Functionalization FGF2/heparininteractionkineticparameters: References
kon(s−1M−1) koff(s−1) Kd(nM)
Copoly(DMA-NAS-MAPS) 8.22×104 1.54×10−3 18.0 Presentwork
Carboxymethyldextran 9.0×103 3.8×10−4 42.5 [37]
8.14×103 3.2×10−4 38.0 [38]
ofatypicalarrayexperimentisshowninFig.6.Replicatesofan oligonucleotideligand are spotted onthe polymer coated gold usingapiezoelectricspotter.Afterwashingandblockingthe sur-faceunreactedsites,thearrayisprobedwithasamplecontaining a complementary target oligonucleotide labeled with biotin. A detectionstep withstreptavidinlabeledwithhorse radish per-oxidase(HRP)isthenperformed.Thehybridizationreactionwas revealedbychemiluminescenceusinganenzyme,HRP,that catal-ysestheconversionoftheenhancedchemiluminescentsubstrate intoasensitizedreagentinthevicinityofthemoleculeof inter-est,whichonfurtheroxidationbyhydrogenperoxide,produces atriplet(excited)carbonyl,whichemitslightwhenitdecaysto thesingletcarbonyl.Enhancedchemiluminescenceallows detec-tionofminutequantitiesofabiomolecule.Thestrongsignaltonoise ratiooftheoligonucleotidespotsprovesthatthesurfacebind cova-lentlyamino-modifiedDNAmolecules(Fig.7).Chemiluminescence waschosentopreventfluorescencequenchingthatwould have occurredlabelingtheDNAwithafluorophore[32].Inthisapproach, luminol,oneofthemostwidelyusedchemiluminescentreagents, isoxidizedbyperoxideanditresultsincreationofanexcitedstate productcalled3-aminophthalate.Thisproductdecaystoalower energystatebyreleasingphotonsoflight.Thedistancefromthe surfaceisenoughtopreventquenching.
Finally,toprovethepracticalexploitabilityof copoly(DMA-NAS-MAPS),aSPRassaywassetup.Streptavidinwasimmobilizedtoa copoly(DMA-NAS-MAPS)-functionalizedgoldsensorchipandused tocapturebiotinylatedheparin.Then,heparinimmobilizedtothe goldsurfacewasevaluatedforitscapacitytobindFGF2,oneofthe mostimportantangiogenicgrowthfactorslargelystudiedforits heparin-bindingcapacity[33,34].Injectionof100ng/mLof hep-arinonto a copoly(DMA-NAS-MAPS)/streptavidin-functionalized sensorchip allows the immobilization of 233 resonance units (RU) equal to 17.1fmol/mm2 of heparin. Relevant to note, in
previousexperiments,theinjectionofheparinontostreptavidin
Fig.7. Chemiluminescencesignalsofoligonucleotidespotsafterhybridizationwith complementaryoligonucleotidelabeledwithbiotinfollowedbyincubationwith streptavidinlabeledwithHorseRadishPeroxidase.
Fig.8.SPRanalysisofFGF2/heparininteraction.(A)Sensorgramsshowingthe bind-ingofFGF2(300nM)toacopoly(DMA-NAS-MAPS)-functionalizedgoldlayercoated withstreptavidinalone(hatchedline)orwithstreptavidinandbiotinylated-heparin (straightline).(B)Blank-subtractedsensorgramoverlayshowingthebindingof increasingconcentrationsofFGF2(300,150,75,36,18e9nMfromtoptobottom)to acopoly(DMA-NAS-MAPS)-functionalizedgoldlayercoatedwithstreptavidinand biotinylated-heparin.Inboththepanelstheresponse(inRU)wasrecordedasa functionofbiographiestime.
immobilized toa carboxymethyl dextran-functionalized sensor-chipleadtotheimmobilizationofcomparableamountsofheparin (from5.8to9.5fmol/mm2)[35,36].We thenstudiedthe
capac-ityofcopoly(DMA-NAS-MAPS)/streptavidin-immobilizedheparin tobindFGF2.AsshowninFig.8A,whenimmobilizedtoasurfacevia copoly(DMA-NAS-MAPS)/streptavidin,heparinretainsitscapacity tobindFGF2inaspecificway,asdemonstratedbytheabsenceof interactionwithimmobilizedstreptavidin.Increasing concentra-tionsofFGF2wereinjectedovertheheparinsurfacetoevaluate thekineticparameters ofFGF2/heparininteraction.Asreported inTable2,verysimilarvaluesofdissociation(koff)and
associa-tion(kon)ratesandofdissociationconstant(Kd)canbeobtained
fromthe analysesperformed onto copoly(DMA-NAS-MAPS)-or carboxymethyldextran-functionalizedgoldlayers.
4. Conclusions
Inthispaper,anewmethodforfunctionalizingagoldsurface withcopoly(DMA-NAS-MAPS)ispresented.Thepolymerissimply
D.Pettietal./SensorsandActuatorsB190 (2014) 234–242 241
deposited by dip coating after an oxygen plasma treatment, followedbywashinginwateranddryingundervacuum.
AFMscratchtestsrevealthepresenceofauniformpolymeric coating with a thickness of 2.5nm. The XPS spectra from C1s N1sandO1slevelspresentthetypicalfingerprintsofthe poly-meric overlayer, i.e. the features arising from NC O and OMe groups.ThechemicalcompositionfoundbyXPSrevealsaslight deficiency ofnitrogen and oxygenwithrespectto carbon.This canbedue toa limited carboncontaminationofthepolymeric coming in wet and atmospheric environment. The analysis of the surface chemical properties of the gold substrate by XPS indicatesnomodificationsinducedbythecoatingprocess.The lim-itedimpactontheAupropertiesseenbyXPSis confirmedalso byfunctional testsperformedonthepolymercoated gold sub-strates.
Indeed,weshowedthecapabilityofthecoatinginthe immo-bilizationofbiomoleculesindifferentexperiments.Inparticular, oligonucleotideswereusedasprobesforthecaptureof comple-mentaryoligonucleotidestrandsdetectedbychemiluminescence. Toassess thepossibilityto usecopoly(DMA-NAS-MAPS) in SPR analysis,heparinwasimmobilizedontothepolymer-coatedgold surfacesofanSPRsensorandevaluatedforitscapacity tobind toFGF2,animportantangiogenicfactor.Threeconsiderationscan bedrawnfromtheresultsoftheseexperiments:(i) copoly(DMA-NAS-MAPS)allowstheimmobilizationofheparininamountthat are comparable (if not higher) to those routinely immobilized ontodifferentcommerciallyavailablecarboxymethyldextran sur-faces(i.e.CM5 andF1sensorchips[37,38]);(ii)inouranalyses, copoly(DMA-NAS-MAPS) turned out tobe devoid of significant bindingcapacity,asshownbytheverylowlevelofaspecific bind-ingofFGF2toacopoly(DMA-NAS-MAPS)surfacedevoidofheparin (seeFig.8A);(iii)thespecificbindingofFGF2to copoly(DMA-NAS-MAPS)-immobilizedheparinoccurswithkineticparametersthat arecomparabletothosemeasuredontocommerciallyavailable car-boxymethyldextransurfaces(Table2).Thisisatvariancewiththe highersimplicityandrobustnessoftheprocessweintroducedin thispaper,whichcouldfindapplicationsinmanydifferent bio-chemicalprocedures.
Acknowledgements
ThisworkwaspartiallysupportedbyFondazioneCariplovia the project SpinBioMed (Project No. 2008.2330). M. R and A. B.acknowledgeMinistero dell’Istruzione,dell’Universita’edella Ricerca,IstitutoSuperiorediSanita’(AIDSProject),andFondazione CARIPLO(projectNo.2008.2198)forfinancialsupport.
References
[1]J.D.Hoheisel,Microarraytechnology:beyondtranscriptprofilingandgenotype analysis,Nat.Rev.Gen.7(2006)200–210.
[2]M.C.Pirrung,HowtomakeaDNAchip,Angew.Chem.Int.Ed.41(2002) 1276–1289.
[3]R.L.Rich, D.G.Myszka,Higher-throughput,label-free,real-timemolecular interactionanalysis,Anal.Biochem.361(2007)1–6.
[4]N.J.Ronkainen,H.BrianHalsall,W.R.Heineman,Electrochemicalbiosensors, Chem.Soc.Rev.39(2010)1747–1763.
[5]V. Dugas, A. Elaissari, Y. Chevalier, Book section surface sensitiza-tion techniques and recognition receptors immobilization onbiosensors and microarrays, in:M. Zourob (Ed.), Recognition Receptors in Biosen-sors, XIV, 1st ed., Springer, New York, 2010, pp. 47–134 (849 p.) http://dx.doi.org/10.1007/978-1-4419-0919-02
[6]M.Viganò,R.Suriano,M.Levi,S.Turri,M.Chiari,F.Damin,Glass silaniza-tionwithblocked-isocyanateforfabricationofDNAmicroarrays,Surf.Sci.601 (2007)1365–1370.
[7]V.C.Martins,F.A.Cardoso,J.Germano,S.Cardoso,L.Sousa,M.Piedade,P.P. Freitas,L.P.Fonseca,Femtomolarlimitofdetectionwithamagnetoresistive biochip,Biosens.Bioelectron.24(2009)2690.
[8]A.Ulman,Formationandstructureofself-assembledmonolayers,Chem.Rev. 96(1996)1533–1554.
[9]J.C.Love,L.A.Estroff,J.K.Kriebel,R.G.Nuzzo,G.M.Whitesides,Self-assembled monolayersofthiolatesonmetalsasaformofnanotechnology,Chem.Rev.105 (2005)1103–1170.
[10]A.B.Steel,T.M.Herne,M.J.Tarlov,ElectrochemicalquantitationofDNA immo-bilizedongold,Anal.Chem.70(1998)4670–4677.
[11]D. Shenoy,W. Fu, J. Li,C. Crasto, G. Jones, C.Di Marzio, S.Sridhar, M. Amiji,Surfacefunctionalizationofgoldnanoparticlesusinghetero-bifunctional poly(ethyleneglycol) spacerfor intracellulartrackinganddelivery,Int.J. Nanomed.1(2006)51–57.
[12]T.M.Herne,M.J.Tarlov,CharacterizationofDNAprobesimmobilizedongold surfaces,J.Am.Chem.Soc.119(1997)8916–8920.
[13]C.-Y.Lee,H.E.Canavan,L.J.Gamble,D.G.Castner,Evidenceofimpuritiesin thi-olatedsingle-strandedDNAoligomersandtheireffectonDNAself-assembly ongold,Langmuir21(2005)5134–5141.
[14]G.Pirri,F.Damin,M.Chiari,E.Bontempi,L.E.Depero,Characterizationofa polymericadsorbedcoatingforDNAmicroarrayglassslide,Anal.Chem.76 (2004)1352–1358.
[15]M.Cretich,G.DiCarlo,R.Longhi,C.Gotti,N.Spinella,S.Coffa,C.Galati,L. Renna,M.Chiari,Highsensitivityproteinassaysonmicroarraysiliconslides, Anal.Chem.81(2009)5197–5203.
[16]A.Isacchi, M.Statuto, R. Chiesa, L. Bergonzoni, M. Rusnati, P. Sarmien-tos, G. Ragnotti, M. Presta, A six-amino acid deletion in basic fibro-blastgrowth factordissociates itsmitogenic activity from its plasmino-gen activator-inducing capacity, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 2628–2632.
[17]L.Sola,M.Chiari,Modulationofelectroosmoticflowincapillary electrophore-sisusingfunctionalpolymercoatings,J.Chromatogr.A1270(2012)324–329, http://dx.doi.org/10.1016/j.chroma.2012.10.039.
[18]P.Bergese,E.Bontempi,M.Chiari,P.Colombi,F.Damin,L.E.Depero,G.Oliviero, G.Pirri,M.Zucca,Investigationofabiofunctionalpolymericcoatingdeposited ontosiliconmicrocantilevers,Appl.Surf.Sci.253(2007)4226–4231. [19]R.Bertacco,M.Cantoni,M.Riva,A.Tagliaferri,F.Ciccacci,Epitaxialgrowthand
characterizationoflayeredmagneticnanostructures,Appl.Surf.Sci.252(2005) 1754.
[20](a)A.Yalc¸in,F.Damin,E.Özkumur,G.diCarlo,B.B.Goldberg,M.Chiari,M.S. Ünlü,Directobservationofconformationofapolymericcoatingwith implica-tionsinmicroarrayapplications,Anal.Chem.81(2009)625–630;
(b)R.Suriano,M.Levi,G.Pirri,F.Damin,M.Chiari,S.Turri,Surfacebehavior andmolecularrecognitioninDNAmicroarraysfromN,N-dimethylacrylamide terpolymerswithactivatedestersaslinkinggroups,Macromol.Biosci.6(2006) 719–729.
[21]A.Ohlander,C.Zilio,T.Hammerle,S.Zelenin,G.Klink,M.Chiari,K.Bock,A. Rus-som,Genotypingofsinglenucleotidepolymorphismsbymeltingcurveanalysis usingthinfilmsemi-transparentheatersintegratedinalab-on-foilsystem,Lab Chip13(2013)2075–2082.
[22]M.Cretich,V.Sedini,F. Damin,M.Pelliccia,L.Sola,M.Chiari,Coatingof nitrocelluloseforcolorimetricDNAmicroarrays,Anal.Biochem.397(2010) 84–88.
[23]Thisnormalizationprocedurewouldberigorouslycorrectforafilmwhose thicknessismuchlargerthantheelectronescapedepth,whichisclearlynot thecasehere.Fromtheapproximateformula=0.XYE1/2validforinorganic
compounds[M.P.SeahandW.A.Dench,Quantitativeelectronspectroscopyof surfaces:astandarddatabaseforelectroninelasticmeanfreepathsinsolids, Surf.InterfaceAnal.1(1979)2]forC1s,N1sandO1selectronsweemployed anescapedepthof31 ´˚A,30 ´˚A,28 ´˚A,respectively,whichcorrespondsto=0.9 E1/2.
[24]G. Di Carlo,F. Damin,L. Armelao, C. Maccato, S.Unlu, P.S.Spuhler, M. Chiari,Synthesisandconformationalcharacterizationoffunctionaldi-block copolymerbrushesformicroarraytechnology, Appl.Surf.Sci. 258(2012) 3750–3756.
[25]D. Petti, M. Cantoni, M. Leone, R. Bertacco, E. Rizzi, Activation of Zr-Co-rare earth getter films: an XPS study, Appl. Surf. Sci. 256 (2010) 6291–6296.
[26]I.F.Amaral,P.L.Ggranja,M.A.Barbosa,Chemicalmodificationofchitosanby phosphorylation:anXPS,FT-IRandSEMstudy,J.Biomater.Sci.Polym.Ed.16 (2005)1575–1593.
[27]Ch.Ammon,A.Bayer,G.Held,B.Richter,Th.Schmidt,H.-P.Steinrück, Dis-sociationandoxidationofmethanolonCu(110),Surf.Sci.507–510(2002) 845–850.
[28]Z.Zhang,Q.Fu,H.Zhang,Y.Li,Y.Yao,D.Tan,X.Bao,Enhancedmethanol dissociationonnanostructured2DAloverlayers,J.Phys.Chem.C111(2007) 13524–13530.
[29]B.Lindberg,R.Maripuu,K.Siegbahn,R.Larsson,C.G.Golander,J.C.Eriksson, ESCAstudiesofheparinizedandrelatedsurfaces:1.Modelsurfacesonsteel substrates,J.Colloid.InterfaceSci.95(1983)308.
[30]HandbookofX-rayPhotoelectronSpectroscopy,Parkin-ElmenerCorp.,Eden PraireMinnesota(USA),1992.
[31]H.Ron,I.Rubinstein,Alkanethiolmonolayersonpreoxidizedgold. Encap-sulationofgold oxideunder anorganic monolayer,Langmuir10 (1994) 4566–4573.
[32]G.P.Acuna,M.Bucher,I.H.Stein,C.Steinhauer,A.Kuzyk,P.Holzmeister,R. Schreiber,A.Moroz,F.D.Stefani,T.Liedl,F.C.Simmel,P.Tinnefeld,Distance dependenceofsingle-fluorophorequenchingbygoldnanoparticlesstudieson DNAorigami,ACSNano6(2012)3189–3195.
[33]B.Casu,M.Guerrini,A.Naggi,M.Perez,G.Torri,D.Ribatti,P.Carminati,G. Gian-nini,S.Penco,C.Pisano,etal.,Shortheparinsequencesspacedbyglycol-split
uronateresiduesareantagonistsoffibroblastgrowthfactor2andangiogenesis inhibitors,Biochemistry41(2002)10519–10528.
[34]B.Casu,M.Guerrini,S.Guglieri,A.Naggi,M.Perez,G.Torri,G.Cassinelli,D. Ribatti,P.Carminati,G.Giannini,etal.,Undersulfatedandglycol-splitheparins endowedwithantiangiogenicactivity,J.Med.Chem.47(2004)838–848. [35]M.Donalisio,M.Rusnati,A.Civra,A.Bugatti,D.Allemand,G.Pirri,A.Giuliani,
S.Landolfo,D.Lembo,Identificationofadendrimericheparansulfate-binding peptidethatinhibitsinfectivityofgenitaltypesofhumanpapillomaviruses, Antimicrob.AgentsChemother.54(2010)4290–4299.
[36]A.Bugatti,P.Chiodelli,J.Rosenbluh,A.Loyter,M.Rusnati,BSAconjugates bearingmultiplecopiesofthebasicdomainofHIV-1Tat:prototypeforthe developmentofmultitargetinhibitorsofextracellularTat,Antivir.Res.87 (2010)30–39.
[37]M.Presta,P.Oreste,G.Zoppetti,M.Belleri,E.Tanghetti,D.Leali,C.Urbinati, A.Bugatti,R.Ronca,S.Nicoli,E.Moroni,H.Stabile,M.Camozzi,G.A. Her-nandez,S.Mitola,P.Dell’Era,M.Rusnati,D.Ribatti,Antiangiogenicactivity ofsemisyntheticbiotechnologicalheparins:low-molecular-weight-sulfated Escherichia coliK5 polysaccharidederivativesasfibroblastgrowthfactor antagonists,Arterioscler.Thromb.Vasc.Biol.25(2005)71–76.
[38]A.Bugatti,C.Giagulli,C.Urbinati,F.Caccuri,P.Chiodelli,P.Oreste,S.Fiorentini, A.Orro,L.Milanesi,P.D’Ursi,A.Caruso,M.Rusnati,Biochemical characteriza-tionofHIV-1matrixproteinp17interactionwithheparin,J.Biol.Chem.288 (2013)1150–1161.
Biographies
DanielaPettireceivedB.S.andM.S.degreesinPhysicsEngineeringfromPolitecnico diMilanoin2004and2006.In2010sheobtainedherPhDinPhysicsfromPolitecnico diMilano,wheresheisactuallyAssistantProfessorinthePhysicsDepartment.She isco-authorofmorethan25researchpapersand2patents.Shestartedheractivity inthefieldofspintronics.Currently,themainresearchactivityisrelatedtothe applicationsofnanomagnetismtobiologyandmedicine.Herinterestsincludethe developmentofmagneticplatformsforbiosensingandforonchipfinemanipulation offunctionalizedmagneticnanoparticlesforin-vitroapplications.
AndreaTortireceivedB.S.,M.S.degreesinPhysicsEngineeringandPhDinPhysics fromPolitecnicodiMilanoin2006,2009and2013,respectively.Atthebeginning of2013hejoinedtheElectronicDepartmentofPolitecnicodiMilanotoworkon theintegrationofbiosensorsandCMOStechnology.Actually,heisTechnicalDevice EngineeratSTMicroelectronics,AgrateBrianza(Italy).Hestartedhisactivityinthe fieldofNanomagnetismandNanomedicine.Currentlyhisresearchinterestsarein thesmartpowerintegrateddevicesfield.
FrancescoDaminwasbornin1971inBustoArsizio(VA,Italy),graduatedin Bio-logicalScience,specialtyMolecularBiology,atUniversitàdegliStudidiMilanoin 1998.HewasrecipientofseveralfellowshipsandresearchcontractsfromICRM, C.N.R.wherehewasappointedResearchScientistin2011.Hisresearchinterests, documentedbymorethan35publicationsonpeer-reviewedjournalsandseveral communicationsatcongresses,dealwiththedevelopmentofsurfacestothe anal-ysesofDNAandproteinswiththemicroarraytechnology.
LauraSolaearnedherM.S.DegreeinChemistryandPharmaceutical Technolo-giesandherPh.D.inDrugChemistryatUniversityofMilanin2008and2012, respectively.Shecurrentlyholdsapost-doctoralappointmentattheInstituteof
ChemistryforMolecularRecognition,CNRwhereshedevelopsnovelpolymeric coatingsforimmobilizingbiomoleculesandtechniquesformolecularrecognition onsupportsmadeofvariousmaterials.Theseareusedinbiomedicalapplications ofbiosensorsandLabonChipanalysis.Lauraspecializesinchemicalsynthesis ofmonomers,polymersandlinearmatricesforDNA,DNAsequencingby cap-illaryelectrophoresisandnovel capillarypolymercoatingsenablingimproved analysis.
MarcoRusnatigainedhis degree attheInstituteof Biochemistry,University ofMilan,Italy,in1985.Heisnowassociatedprofessor,Sectionof Experimen-talOncologyandImmunology,UniversityofBrescia.Hisresearchesfocusedon: 1983–85:purificationandbiochemicalcharacterizationofd-aminoacidoxidase. 1986–1992:purificationofangiogenicfactors,identificationoftheirfunctional domains.1992–present: interaction ofangiogenic factors with receptors and extracellularbinders.1996–present:biologicalactivityofHIV-1Tat,studyofits interactionwithreceptorsandextracellularbinders.2001–present:studyof inter-actionsofviralproteinsbysurfaceplasmonresonance.Heisauthorof110scientific publicationsinpeer-reviewedjournals.
EdoardoAlbisettiobtained B.S.andM.S.degreesinPhysicsEngineeringfrom Politecnico di Milano in 2008 and 2010. Currently, he is a PhD student in the Physics Department of Politecnico di Milano. His research activity mainlyconcerns thedevelopmentofspintronicdevices andtheir application in biology. In this framework, he developed a sensing platform based on magnetoresistivesensors fordetection ofmolecularrecognition events anda system based onmagnetic domain wallconduits for providing localmixing ofsolutionsin microfluidic systems.Magneticdomainwallconduits are also usedforultra-finespatial manipulationofmagnetic nanoparticlesforin-vitro applications.
AntonellaBugattiobtainedherB.S.degreeinBiomedicalLaboratoryTechnician fromUniversityofVerona.Since2006sheisResearchfellowattheUnitof Exper-imentalOncologyandImmunology,UniversityofBrescia.Sheisco-authorof25 scientificpublicationsinpeer-reviewedjournals.Herexpertiseisrelatedtocell andmolecularbiologyassays,protein-proteininteraction(SPRtechnology),in-vitro assays(endothelialcellproliferation,migration,invasionandmorphogenesis).Her researchinterestsareassociatedtothestudyofprotein-proteininteractionby sur-faceplasmonresonance.
RiccardoBertaccogothisdegreeinElectronicEngineeringatPolitecnicodiMilano in1994.HeobtainedapermanentpositionatthePhysicsDepartmentofPolitecnico diMilanoin1999andthePhDinPhysicsin2000.Actually,heisAssociateProfessor atthePhysicsDepartmentofPolitecnicodiMilano,whereheleadsthe NanoBiotech-nologyandSpintronicsgroupoftheLNESScenter.Heisthetechnicalresponsible fortherealizationofanewclean-roomformicroandnanofabricationatPolitecnico diMilano.Heisauthorofabout100papersininternationaljournals,achapterofa bookand6patentapplications.
MarcellaChiarigraduatedinChemistryandPharmaceuticalTechniquesatthe Isti-tutodiChimicaOrganica,FacoltàdiMedicina,UniversitàdiMilanoin1982.She receivedtheDiplomainClinicalBiochemistry,UniversitàdiMilanoin1990.Since 1992shehasbeenSeniorResearcherattheICRM,CNR,wheresheleadsthe labora-tory“DevelopmentofAnalyticalMicrosystems¨.Herresearchactivityisdocumented bymorethan100publicationsandseveralpatents.Shehasbeenacontractorof theECintheframeworkofdifferentprojectsandresponsibleforseveralnational researchprograms.
