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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
Label-free optical biosensor for direct complex DNA detection using Vitis vinifera L.
Luis Moreira
a, Helena M.R. Gonc¸ alves
a,b, Leonor Pereira
a,b, Cláudia Castro
a, Pedro Jorge
c, Carlos Gouveia
c, José R. Fernandes
a,c, Paula Martins-Lopes
a,b,∗aUniversityofTrás-os-MontesandAltoDouro,P.O.Box1013,5000-911VilaReal,Portugal
bUniversityofLisboa,FacultyofSciences,BioISI—Biosystems&IntegrativeSciencesInstitute,CampoGrande,Lisboa,Portugal
cINESCTEC,RuadoCampoAlegren.687,4169-007Porto,Portugal
a r t i c l e i n f o
Articlehistory:
Received28December2015 Receivedinrevisedform12April2016 Accepted17April2016
Availableonline23April2016
Keywords:
Label-freebiosensor DNAdetection Quantification VitisviniferaL.
a b s t r a c t
TheabilitytodetectandquantifysmallamountsofDNAinbiologicalcomplexsamplesisahotresearch area.Upuntilrecentlymostoftheworkperformedinthisareausedlabel-dependentprotocolsthat increasesitscomplexityandoverallcosts.Theaimtheworkwastodevelopalabel-freetechnology suitableforDNAdetectionandquantificationusingrealcomplexDNAsamples.Theapplicabilityofthis systemwastestedusingsyntheticssDNAtargetsthatguaranteedthesystemsspecificity,inthesensethat onlycomplementarysequenceshybridizedwiththeprobe.WhenusingrealsamplesextractedfromVitis viniferaL.thesystemwasabletosuccessfullydetectandquantifytheDNApresentwithoutanyofthetime consumingandcostlyamplificationsteps.Thedetectionandquantificationlimitsoftheproposedsystem were60±20nMand201±20nM,respectivelyforTarget1concentrationsbetween31and350nM.This methodcaneasilybeappliedtootherspeciesandpurposes,allowingthedirectdetectionofDNAina label-freeenvironmentwithhighaccuracyandspecificity.
©2016ElsevierB.V.Allrightsreserved.
1. Introduction
EversincethediscoveryoftheDNAbase-paircouplingstruc- ture,thenumberofpublicationswherethiscomplexbiomolecule isusedasasensingdevicehasgrownexponentially.Oneareathat hastakenamajorinterestintheDNAbase-paringstructureisthe developmentofbiosensors[1,2].TheuseofDNAasastructural baseforbiosensingdevelopmenthasnumerousadvantages,such as:(1)thethermostabilityofthemoleculewhencomparedtoother biomolecules,i.e.proteins;(2)highlyconservedinthesensethat replicationhasaverylowmutationrate,allowinganaccuratefin- gerprinting(3)thepresence inallthecells, thereby potentially enablingidenticalinformationregardlessofthetissueoriginand thetransformationprocessthattheyweresubmitted[3,4].
ADNA-basedbiosensorcanbeoftheoutmostimportancein areassuchas,foodtraceabilityandauthentication,clinicaldiag- nostics,genetherapy,biomedicalstudies,amongothers[5–8].
Overthepastfewyearstherehasbeensomeeffortsinorder todevelopbiotechnologiesthatwillimprovebothsensitivityand
∗Correspondingauthorat:UniversityofTrás-os-MontesandAltoDouro,P.O.Box 1013,5000-911VilaReal,Portugal.
E-mailaddresses:[email protected],[email protected](P.Martins-Lopes).
selectivitywhenconsideringgeneanalysis[8–10].Usuallythese analysisrequireDNAlabellingwithafluorophore.Insomeparticu- larcaseswheresignalamplificationisthemostcriticalissue,such as,ultratracegeneanalysis,theDNAhybridizationisfollowedusing afluorescentdye[1,11].Howeverfluorophoreshavenumerousdis- advantagesthathavereflection’sinthesensitivityofthemethod, namelylowphotostability,photobleaching,lowsignalamplifica- tion,particularlyinbiologicaltissuewhereself–fluorescenceisa majorissue,amongothers[12].Moreover,theDNAprobecanonly belabelledwithoneorafewfluorophores,whichresultsinaweak signal,particularlywhenthetargetconcentrationislow.Thislimits notonlyaffectthemethod’ssensitivitybutalsothedetectionand quantificationlimits.
Inordertoovercometheseissues,someworkhasbeendevel- opedintermsofnanoparticlestosupporttheDNAorafluorescence label.Infact,andeventhoughsomeinterestingvalueshavebeen reported[1,6,13,14]forthedetectionlimit,thesealternativesstill requireDNAlabelling.
Other DNA-based technologies rely on amplification proce- dures, based on Polimerase Chain Reaction (PCR) previous to detection,suchasrealtimePCRthatadditionallyrequiresanampli- ficationinordertodetectthetargetandquantifyit,withsome successlimitationwhenPCR-inhibitorsarepresentinthesample
http://dx.doi.org/10.1016/j.snb.2016.04.105 0925-4005/©2016ElsevierB.V.Allrightsreserved.
Table1
Sequencesoftheoligonucleotidesusedthroughoutthiswork.
Oligonucleotides Sequence
ssDNA−Probe 5-C6-AminolinkGGTGAAATGGGCACCGAACACACGC-3
Target1(Complementary) 5-GCGTGTGTTCGGTGCCCATTTCACC-3
Target2(Non-Complementary) 5-AAAAAAAAAAAAAAACCATTTCACC-3
Target3(OneBaseMismatch) 5-GCATGTGTTCGGTGCCCATTTCACC-3
Target4(SixBasesMismatch) 5-GCATGTGTTTTTTGCCCATTTCACC-3
Target5(Complementarywithatailof24basesin5) 5-TCTCTCTCTCCTCTCAGCAAGGAAGCGTGTGTTCGGTGCCCATTTCACC-3
[15].AlternativetothesemethodsistheuseoffiberopticSPRfor DNAhybridizationdetection[16]however,theseoptionsrequire expensiveequipmentandspecializedpersonnel.
In this work a simple, low cost method for label-free DNA detectionandquantification wasdeveloped,producing compet- itive results. This method is based on the ssDNA (DNA-probe) immobilizationinthelateralsurfaceofanopticalfiberlongperiod grating(LPG)andsubsequenthybridization.TheDNAisnotdye- labelledand thehybridizationis followedinsitu bydifferences inducedontheopticalfibersurroundingmediarefractiveindex.
More, the process does not require enzymatic reactions. This system was successfully applied for the detection of synthetic ssDNAtargets(complementary,non-complementaryandpartially- complementary), aswellas,genomic DNApreviously extracted fromVitisviniferaL.
2. Materialandmethods 2.1. Materials
Alloligonucleotides usedin this workwerepurchased from Frilabo.Thestocksolutionswerepreparedwithultrapurewater and stored at −20◦C. Each solution contained 100Mof each oligonucleotides.Theoligonucleotidessequencesarepresentedin Table1andwasbasedonthespecificprimerofV.viniferadesigned todetectSingleSequenceRepeats[17].
Foreachexperimentasuitableamountofthestocksolutionwas dilutedinsalinephosphatebuffer(PBS:10mMsodiumphosphate;
120mMNaCl;2.7mMKCl;pH7.4)inordertoobtainthefollow- ingconcentrations:0.50;0.25;0.125;0.0625;0.03125;0.015625 and0.007812M.Allotherchemicalswereusedwithoutfurther dilutions.
Thecleaningsolutionusedbeforeeachexperimentwascom- posedbyEthanol70% (v/v)and1%Hydrochloricacid(v/v)in a (1:1)ratio.Additionallytherestringingsolutionwasamixtureof PBSwith0.1xSaline-SodiumCitrate(SSC)and0.1%Sodiumdodecyl sulphate(SDS)ina(1:1)ratio.AftereachcycletheLPGwascleaned usingadilutedsolutionofNitricAcid(HNO31:3).
2.2. Instrumentation
Thedetectionsystemis basedinafiberLongPeriodGrating (LPG)sensor.Thissensorisanopticalwavelengthband-lossfilter wherethecentralwavelengthofthebanddependsoftheLPGfab- ricationparameters,temperature,appliedaxialmechanictension andfibersurroundingmediarefractiveindex.Itisalsoknownthat thedouble strandDNAhas, insolution,aslightly higherrefrac- tiveindexthanthesinglestrandform.Thedetectionprincipleof this particular LPGsensor is based ontheuse ofa single DNA strand(Probe) attachedtothefiber’s lateralsurface.Whenthe probehybridizeswithitscomplementarystrand,itbecomesadou- blestrandDNAincreasingthefiber´ıssurroundingrefractiveindex mediathat,inturn,willhaveaneffectontheLPGtransmission spectrathatisthedatameasured.
ForthatpurposetheLPGsensorwasplacedinsideofawetflow cellthatwillprovidethemeanstomaintainaconstantmechanic
tensionappliedtothefiberand,simultaneouslyallowstheinser- tionandremovalofwetsolutionsinthesensorsurroundingzone whichhasbeendetailedinGonc¸alvesetal.[18].Asconsequence, thewavelengthvariationsaccountedcanonlybeduetotheinter- actionofthesolutionwiththeLPGsurface.Thechambercapacity isof750Landthesamevolumeofeach solutionwasinjected intothechamberinordertomaintainthevolumeconstant.The dataacquisitionwasperformedusingafiberopticinterrogation unitmanufacturedbyFibersensing®,modelBraggMeterFS2200SA, withtwochannelsmodifiedtoallowthemeasuringofthetrans- missionspectrainthespectralregionbetween1500and1600nm.
Thesensorapparatuswasmaintainedatconstanttemperature byinsertingitinamuffle(Termarks,modelB8023).Thesensor temperaturewasmeasuredbyatype-Kthermocouplepositioned incontactwiththesensorchamberanditsvaluewererecordedby atemperaturelogger(Keithley®740)controlledbythesamecom- puterthatwasmeasuringthespectrausingaLabview®program creatingatemperaturelistwithatemporaltagforeachtempera- turevalue.Inthisway,bothspectraandtemperaturemeasurement wheretimesynchronizedandcanbeco-related.
Upondataacquisitionthespectraldataisprocessedtodeter- minethepositionofthewavelengthLPGresonanceasdescribed byGonc¸alvesetal.[18].
2.3. DNAextraction
Inordertotesttheapplicabilityofthesensingsystemintoa morecomplexDNAmatrix,asampleoftheVitisviniferaL.DNAwas tested.ThegenomicDNAwasextractedfromleafsamplesusing thecetyltrimethylammoniumbromide(CTAB)methoddescribed byDoyleandDoyle[19].TheextractedDNAwasresuspendedin 50LofdistilledwaterandtheDNAconcentrationwasdetermined usingaNanoDrop®ND-1000spectrophotometer.TheDNAquality wasassessedusinga0.8%(w/v)agarosegelstainedin7gmL−1 ethidiumbromidesolution.
2.4. Chemicalsandwichsensingsystempreparation
TheLPGswereinsertedinaglasschamberandthestrainwas fixed.BeforeperforminganyteststheLPGsurfacewascleanedby thepassageofethanol70%(v/v)and hydrochloricacid1%(v/v) solutionina1:1ratio.
TheLPGsurfaceisnegativelycharged,soistheDNA,assuch,it wasnecessarytouseabilinker.Inthiswork,thebilinkerchosenwas Poly-l-Lysine(PLL).Ineachcyclethefollowingsequencewasused:
water,PLL,Probe,TargetsorDNAfromV.vinifera.Allsamplesused weresetat0.25M.Allmeasurements,withtheexceptionofwater addedinbetweentheadditionofananalyte,wereperformedover 30min,at37◦C.Uponthehybridizationthetargetwasremoved fromtheLPGsurfacebyaprocesscalledstripping.Thiswasper- formedat60◦Candincludedtheadditionofastrippingsolution (0.1xSaline-SodiumCitrateand0.1%SodiumDodecylSulphatein a(1:1)ratio),ethanolandwater.Intheendofeachcycleallchem- icalswereremovedfromtheLPGsurface,usingadilutedsolution ofHNO3(1:3,v/v)thatwasaddedandleftincontactwiththeLPG for15min.
Fig1. RepresentationofthesignalobtainedusingasimplessDNA(Probe),anamino- terminatedssDNA(ZAG62)andTarget1.*Thevaluesarestatisticallysignificant whencomparedtotheProbeSignalforap<0.05.
2.5. Chemicalsandwichsensingsystempreparation
In order to evaluate the statistical significance of the data obtainedthroughoutthis workanIBMSoftwarePackageStatis- ticalAnalysis(SPSS)version19wasused.Thedatawereanalysed incomparisontotheProbeandthePLLsignal,forthetargetsand theZAG62evaluation,respectively,andwereconsideredstatistical differentwhenp<0.05.
3. Resultsanddiscussion
3.1. Thechemicalinteractionbetweenthesensingsystem
Acommonprobleminimmobilizedbiomoleculemethodsisthe needthateventhoughtheDNA-probeiscovalently-attachedto thefiberit shouldstill interactwiththe targetsand hybridize.
This problem was surpassed by the use of a polymer—Poly-l- Lysine(PLL)[20].Thispolymerhasapositivechargewhichmakes itsuitableforasinglesandwichsystem—fiber(negativecharge);
PLL(positivecharge);DNA-probe(negativecharge).Indeed,even thoughPLLiscommonlyusedasabilinkerforDNAattachment, thechemicalinteractionsbetweenthissystemarenotfullyunder- stood.In order to better understand thechemical relationship that lied beneath the sandwich sensing systeman experiment wasdevised.Accordingtoauthors,suchasZibaiietal.[20],PLL formsamonolayerthatlinkstotheopticalfibersurfacethrough ahydrogenbridgeboundbetweentheSi-OHgroupsofthefiber andthePLLaminogroup.Additionallytheseauthorssuggestthat thelinkingbetweenDNAandPLLalsooccursthroughanamino link.Howeverwhatremainstobeexplainedisiftheabsenceof theamino-terminatedgroupsintheDNAisdeterminantforthe hybridizationtooccur.Wewantedtotestthishypothesisandused amino-ssDNAandDNAwithoutanaminogroup.Theresultspre- sentedinFig.1,showthattheirstatisticaldifferencebetweenthe PLLsignalandtheaminofreeDNA(ZAG62),assuch,theZAG62 caneffectivelyinteractwiththeimmobilizedPLLeventhoughit doesnothaveanaminogroup.However,whencomparingthese signalswithTarget1itispossibletosaythatthereisnostatistical confirmationforhybridization.Thisbehaviourcanbeduetoanori- entationeffectthattheaminogroupconferstothessDNA.Indeed theinteractionbetweenthePLLandtheZAG62orProbe,canbedue tohydrogenboundingbetweentheDNAresiduesandthepolymer, butwhenthereisanaminogroupinthessDNAextremity(Probe), theinteractionbetweenPLLandtheProbeismoresignificantdue tothestabilityoftheN Nchemicalbounding.Additionally,when
Fig. 2.Representation of the wavelength evolutionwith time registered for 30minwithineachstep PLLDeposition,Probeimmobilization,Target2 (non- complementary)andTarget1(hybridization).
theinteractionbetweenthepolymerandtheDNAisduetothis N Nboundingthepositionforthisinteractionfavoursthesubse- quenthybridization.Ontheotherhand,whenthessDNAandthe PLLisnotspecificallyorientedthisinteractioncanplacethessDNA (ZAG62)insuchawaythatthehybridizationcannotbeperformed.
Thispositionissovariablethatitispossibletoseeahybridization inonecycleandnotseeitinthenext,whichcanberesponsibleto thehighstandarddeviationfoundforTarget1intheseparticular experiments.
3.2. Stabilityandreproducibilityofthesensingsystem
Thestabilityofthesensorisanimportantissue.Inordertodefine thetimerequestedtoobtainastablesignalastudyconcerninga timeframeof1800swasundertakenusingPLL,Probe,Target2 andTarget1.Thewavelengthsignalwasrecordedduring30min (Fig.2)andstabilizationofthereactiononthefiberwithineach analytewasobtainedafter20min.Allthefurtherreadingswere donetakingintoconsiderationthistimeframe.
Anotherissuethatneededtobeaddressedwasthelowrepro- ducibilitythatisoftenreportedwhenusingfiberswithlongperiod gratings (LPGs), depending on the thickness of the interaction regionandthepenetrationdepthoftheevanescent[21].Indeed whenusingdifferentLPGsitisnecessarytoperformacalibration thatcanbeslowduetoitssingularnature.Inordertoovercome thisproblemanewstrategywasdeveloped,wheretheLPGwasput incontactwithadilutedsolutionofnitricacid.Thisallowedtheuse ofthesameLPGformorethan20completeassaysovermorethan 5months.
Inordertoevaluatethemethodsensitivityandselectivityfive differentstrandsofDNAweretested.Target1,Target2,Target3, Target4andTarget5(Table1).Target1iscomplementarytothe DNA-probe,Target2isnon-complementary,Target3hasasingle- mismatchclosetothe5end,Target4has6mismatchesandTarget
5iscomplementary,butithasatailof24extra-basesinthe5end.
Thehybridizationprocesswasfollowedbymonitoringthechange intheeffectiverefractiveindexinducedbytheevents.
In order toascertainthe methodreproducibility,sensitivity, detectionand quantification limitsseveral amountsof Target 1 wereevaluatedbythesamefibersensor.Thisallowedthedefinition ofthecalibrationcurvepresentedasSupplementaryFig.S1inthe onlineversionatDOI:10.1016/j.snb.2016.04.105withaR2of0.98 Theobtainedresultsallowedustodeterminethemethod’sDetec- tionLimit:60±20nMandQuantificationLimit:201±20nM.These limitsarequitelowforalabel-freetechnology.Indeed,eventhough detectionlimitsinthehundredsoffentomolarshavebeenreported
Fig.3.RepresentationofthesensorresponseinthepresenceofPoly-l-Lysine(PLL), singlestrandDNA-probe,Target1(complementary),Target2(non-complementary), Target3(singlemismatch)andTarget4(sixmismatches),forthreeindependent measurements.*ThevaluesarestatisticallysignificantwhencomparedtotheProbe Signalforap<0.05.
[22–24].AllthesemethodsrequiredDNAlabelling,increasingcosts andrequiringadditionallabellingsteps.Moreover,ourmethodalso allowsthequantificationoftheDNAinasmallsample,without requiringapreviousamplificationprocessasrequiredinReal-Time PCRassays[15]andinotherbiosensorbasedmethods[22].
3.3. Analyticalperformanceofthesensingsystem
Inordertoevaluatetheapplicabilityofthesensingsystemsev- eraltargetswereused.Eachtargethadapeculiaritysoitcouldbe possibletoascertainifthedevelopedsystemwasabletodetect singlemismatch,ifthemismatchpositionwasrelevantandifcom- plementarysequencecouldbecompromisedbyhavingatailof mismatchesina5end.
TheresultsshowedinFig.3clearlydemonstratethathybridiza- tiononlyoccursforTarget1,usingaconcentrationof0.25M.This resultisquiteinterestingsinceitallowstoconcludethatoursys- temcansuccessfullydiscriminatebetweentotal-complementarity andasinglemismatch.Target4had6singlemismatchesalongthe chainthatcouldeasilypreventhybridization.Thisresultclearly showsthatthemethodisveryselective,whichisreinforcedbythe resultobtainedforTarget3whichhasonlyonemismatch.Thisis interestingonceitcanbeconsideredasanewbiosensormethod forSingleNucleotidePolymorphism(SNP)detection[22,25],which canbeappliedinawiderangeofresearchareas,fromdiagnose, forensic,genotyping,amongothers.Anotheradvantageisthatit doesnotrequirethedevelopmentofcomplicatedprobes,e.g.Pep- tideNucleicAcid(PNA),LockedNucleicAcid(LNA),northeuseof signalamplificationposthybridizationprocess,e.g.SurfaceLigation Reaction[22].
Tohelp establishtheuseofthis methodina realsample, a suitableamountofDNAwasextractedfromV.vinifera.Thisisa widelycultivatedfruitcropwithaharvestedareaabovesevenmil- lionhectaresandmorethan60milliontonsofgrapesproduced peryear.Itisadiploidspecieswithnineteenchromosomes,with agenomesizeofaround500Mbp.Additionally,Target5wasused, and althoughitssequence is complementaryit hasatailof 24 basesonthe5end,simulatinginterferenceofnon-complementary sequencessurroundingthecomplementarysequence,presentin realDNAsamples.Nevertheless,Target5onlyhadatailinoneof thesequenceend.AsitcanbeseenbytheanalysisofFig.4,thereis astatisticalsignificantdifferencebetweenthesignalobtainedfor thessDNAandTarget5.Thisresultsuggeststhatthesensingsystem
Fig.4.Representationofthesensorresponse)inthepresenceofPoly-l-Lysine(PLL), singlestrandDNA(Probe),Target5(complementarywitha24basestailin5-end) andGenomicDNA(DNAextractedfromVitisviniferaL.),forthreeindependentmea- surements.*ThevaluesarestatisticallysignificantwhencomparedtotheProbe Signalforap<0.05.
identifiedthistargetascomplementarytothessDNAimmobilized inthefiber.
TheprocedureappliedtoanalyzethegenomicDNAextracted fromV.viniferawasverysimilartotheoneusedfortheprevious targets.However,sincetheDNAwasstilldoublestrandedadenat- urationprocesswasapplied.Thiswasaccomplishedbyasimple heatingoftheDNAsample.Inordertoevaluatethecompetitive mechanismbetweentheimmobilizedprobeandthetwosingle- strandedDNAchainsfromtheV.viniferaDNAsample,theywere bothputintocontactwiththeLPGsurface.Theresponseobserved forthissample(Fig.4)clearlydemonstratesthatdespitethecom- petitiveexistentduetothepresenceofcomplementarysequence, thedesignedsystemcanindeeddetectthehybridizationprocess.
Whencomparingthesignaldifferencesbetweentheprobe,Tar- get1andtherealDNAsampleitispossibletoascertaintheimpact thatthecompetitivemechanismhasonthesystem.Thehybridiza- tionofTarget1resultsinasignalincreaseofapproximately59%on theotherhandwhenthereisacompetitivemechanismbetween theimmobilizedprobeandthetwocomplementarychainsofthe realsample,thehybridizationisfollowedbya20%signalincrease.
Assuch,itispossibletosaythatoursystemisabletosuccessfully detecttheDNAinamorecomplexmatrix(realsample).Moreover, eventhoughwearedealingwitharealsample,itispossibleto seethattheresultsarequitereproducible—thehigheststandard deviationinthreeconsecutivemeasurementsis2%.
Asalltheexperimentswereconductedusingthesamemolarity, 0.25M,thenumberofestimatedcopiesbetweenthesynthetictar- getsandtherealsamplearesignificantlydifferent,being5.330E+16 forTarget1,and2.687E+6fortherealsample.Thisemphasisthe sensibilityofthemethodwhenusingdirectrealDNAsamples,and thefactthatnopreviousamplificationstepwasrequired,increasing thetargetnumberinthesolutionasisrequiredforsomeactually usedDNA-basedbiosensors[22].Thisisaninterestingfeatureas someDNAsamplespresentPCR-inhibitors[15],limitingtheiruse forgenotypingprocedures.
4. Conclusions
TheopticalDNA-basedbiosensortestedprovedtobehighlyspe- cificwhensyntheticsampleswereused,detectingauniqueSNP difference.Theamazingoutcomewasthefact thatthis biosen- sor was successfully applied in a complex matrix, V. vinifera genomicDNA,evenwhenthesequencecopynumberisconsider- ablylowerthanthesynthetictargetusedandwiththepresenceof
competitivemechanisms.Thissystemmakesitpossibletodetect andquantifyDNAinrealsamples,usingstandardopticaltelecom- municationtechnology, highwithspecificity, since themethod allows the detection at a SNP level, which is why we expect that it would be quite useful in areas where the DNA detec- tion/quantificationisthemaingoal.
Acknowledgements
ThisresearchwassupportedbythePortugueseFoundationfor Scienceand Technology(FCT)intheprojectBiosensorDevelop- ment for Wine Traceability in the DouroRegion—WineBiocode PTDC/AGR-ALI/117341/2010-FCOMP-01-0124-FEDER-019439, Enoexcel- NORTE-07-0124-FEDER-000032, funded by national meansthrough ON.2 and co-funded by the EuropeanFund for Regional Development (FEDER) through COMPETE—Operational Program for Competitiveness Factors (POFC) and a PhD grant (SFRH/BD/44781/2008).
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Biographies
LuisMoreiragraduatedwithadegreeinGeneticsandBiotechnologyandM.Sc.
degreeinFoodQualityandBiotechnologyfromtheUniversityofTrás-os-Montes andAltoDouro,Portugal,in2011and2013,respectively.From2013–2015worked intheCenterofGeneticsandBiotechnology,VilaReal,Portugal.Hehas2publications inSCIjournalsand10abstractsinnationalandinternationalmeetings.
HelenaGonc¸alvesgraduatedinChemistry,receivedherM.Sc.degreeinChemistry, attheUniversityofPorto,Portugalin2007and2008,respectively.In2009attended apost-graduationinMedicalandLegalScienceandin2014concludedherPh.D.
inChemistryfromPortoUniversity,Portugal.Shehasbeeninvolvedin6research projects.Fromherresearchwork,Helenaisauthorandco-authorof13full-papers publishedSCIjournals,1patentandseveralabstractsinnationalandinternational meetings.
LeonorPereiragraduatedwithadegreeinBiotechnologyEngineeringatthePoly- technicInstituteofBraganc¸a,Portugal,in2002.SheobtainedherM.Sc.degreein MolecularGeneticsfromtheUniversityofMinho,Braga,Portugalin2006.In2015, shefinishedherPhDfromtheUniversityofTrás-os-MontesandAltoDouro,Portugal.
From2008–2015sheworkedintheCentreofGeneticsandBiotechnology,VilaReal, Portugal.Sheparticipatedin5projects.Shehas7publicationsinSCIjournals,3 patents,1abstractpublishedinSCIjournals,1full-paperpublishedinpeer-reviewed journal,and18abstractspublishedinnationalandinternationalmeetings.
CláudiaCastro,GraduatedwithadegreeinGeneticsandBiotechnologyandM.Sc.
degreeinComparativeMolecularGeneticsandTechnologyfromtheUniversityof Trás-os-MontesandAltoDouro,Portugal,in2013and2015.Shehaspublished5 abstractsinnationalandinternationalmeetings.
PedroA.S.JorgegraduatedinAppliedPhysics(OpticsandLasers)attheUniv.of Minho(1996),M.Sc.inOptoelectronicsandLasersatthePhysicsDepartmentofUni- versityofPorto(2000);in2006concludedhisPh.D.programatUniversityofPorto incollaborationwiththeDept.ofPhysicsandOpticalSciencesattheUniv.ofChar- lotte,NorthCarolina,USA,withworkinluminescencebasedopticalfibersystems forbiochemicalsensingapplicationsusingluminescentnanoparticles.Since1997 PedroJorgehasbeeninvolvedinseveralresearchandtechnologytransferprojects relatedtoopticalfibersensingtechnology,developingnewsensingconfigurations andinterrogationtechniquesforopticalsensors.PedroJorgeisaSeniorresearcher atINESCPortowhereheleadstheBiochemicalSensorsteamexploringthepotential ofopticalfiberandintegratedopticstechnologiesinindustrial,environmentaland medicalapplicationscoordinatingseveralprojectsintheseareas.From2015heis alsoanInvitedAssistantProfessorattheDepartmentofPhysicsandAstrophysics oftheFacultyofSciencesoftheUniversityofPorto.PedroJorgehasmorethan200 publicationsinthefieldsofsensorsinnationalandinternationalconferencesand peerreviewedjournals,heisauthorof3bookchaptersandalsoholdsonepatent.
PedroJorgeisamemberofSPIE.
CarlosGouveiagraduatedwithadegreeinelectronicsandtelecommunicationsand M.Sc.degreeintelecommunicationsandnetworksfromtheUniversityofMadeira, Portugal,in2007and2008,respectively.Since2008worksintheCenterofApplied PhotonicsofINESCTEC,Porto,Portugal.InJanuary2014,heobtainedhisPh.D.
degreeinelectricalengineeringfromUniversityofMadeira.Currentlytheispos-doc researcheratINESCP&DBrasil,hostedbytheElectricalEngineeringDepartmentof FederalUniversityofCampinaGrande.Hehasmorethan40publicationsinthefields ofsensorsinnationalandinternationalconferencesandpeerreviewedjournals,is authorof1bookchapters.
J.R.A.FernandesgraduatedinPhysicsatUniversityofPorto(1994),M.Sc.inOpto- electronicsandLasersatthePhysicsDepartmentofUniversityofPorto(1998);in 2005concludedhisPh.D.programatUniversityofPortowithworkinthinfilmsof piezoelectricmaterialsforsensorandactuatorapplications.Since1995,J.R.A.Fer- nandeshasbeeninvolvedinseveralresearchprojectsrelatedtothedevelopmentof functionalmaterialsforsensorapplications.J.R.A.Fernandesisaseniorresearcherat INESCTECsince2005withworkinthebiosensingfield.Since200,J.R.A.Fernandesis anassistantprofessorinthePhysicsDepartmentofUniversidadedeTrás-os-Montes eAltoDouro(UTAD),hasmorethan100publicationsinnationalandinternational conferencesandpeerreviewedjournalsandisalsoco-authorof1bookchapter.
PaulaMartins-LopesgraduatedinAgricultureEngineeringattheTrás-os-Montes andAltoDouroUniversity(UTAD),Portugalin1996.ShereceivedherM.Sc.degree
inGeneticResourcesandPlantandForestryBreedingfromtheUTAD,Portugalin 1999andherPh.D.inGeneticsfromUTAD,Portugalin2006.Theresearchforthe M.Sc.andPh.D.wasperformedincollaborationwiththeCerealsDepartmentofthe CambridgeLab,JohnInnesCentre,Norwich,U.K.Atthepresent,PaulaF.Martins- Lopes,isanAssistantProfessorattheDepartmentofGeneticandBiotechnology, UTAD,whereshelectures1st,2ndand3rdcyclestudies.Sheisatpresentthedirector offourdegrees:1stcycleinGeneticsandBiotechnology,2ndcycleinHealthScience Biotechnology,2ndCycleofComparativeMolecularGeneticsandTechnologyand the3rdcycleofComparativeMolecularGenetics.Paulaisalsothevice-president ofPedagogicalcounciloftheSchoolinLifeScienceandEnvironment.Herresearch linesarelinkedwithstudiesonfoodauthenticitystrategiesusingPCRandBiosensor
platforms,aluminumstressincereals,pathogenstressinoliveandantigenotoxic effectsoffoodusingDrosophilamelanogastermodels.Shehasbeeninvolvedin16 researchprojectandhasbeenthePIofthreeofthem.Fromherresearchwork, Paulaisauthorandco-authorof32full-paperspublishedSCIjournals,3patents, 5bookchapters,3abstractspublishedinSCIjournals,1full-paperpublishedin peer-reviewedjournal,7fullpapersinproceedingsand123abstractspublishedin nationalandinternationalmeetings.Shehasbeenthesupervisorof5PhDthesis,5 masterdissertationsandmorethan20undergraduatefinalreports.Shehasbeen invitedlastyeartobeanevaluatorofresearchreportsbythenationalfoundation forscienceandtechnology.PaulahasrefereeofseveralSCIjournals.
