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3rdInternationalconferenceandexhibitionon Biosensors&Bioelectronics SanAntonio,USA,August11-13,2014 Anovelplatformbasedonimmobilizedolfactoryreceptors,fordetectionodorantmolecules characteristicsofboartaint.
Boartaint Boartaintspoilstasteandodourofporkmeet.Itisduetovolatilecompounds,suchasandrostenone.Theyareconcentratedinseveralpartsofpigbody.
Odorantdetection • Itisveryimportanttodetectodorantmoleculesbecausethey: • canconstituteasignatureofmetabolicstatesordiseases, • canparticipateinaromasinfood, • canbeassociatedwithdrugsandexplosives • ortodomesticandenvironmentalpollutants. • Therefore,thereisanincreasinginterestinemergingtechnologiesallowingrapidandnon-invasiveassessmentofvolatileodorantcompounds.
OUTLINE • Principleoftheelectronicnoseandtongueanddescriptionofcommercialproductsandmarket • Newdirectionsofresearch: • Newsensorsbasedonolfactoryreceptors
Principleoftheelectronicnose comparedtoanhumannose RecognizedasBraziliancoffee Odor Mind Brain Nose SignalProcessing ComparisonwithodorsIndatabase RESULTS Acquisition Reaction Signal Data Onsensors Processing Processing -NOL- -HOL- RecognizedasBraziliancoffee -DIOL- -MALD- -ALD- -HAT-
ObjectivesofanElectronicNose • Createanolfactiveimpression • Comparedto humannose:Available(time,nosaturationeffect)Lowercost • ReproducibilityAnalysisoftoxics • Comparedtoclassicaltechniquesofchemicalanalysis: • NoseparationofcompoundsisneededHighspeed,possibleinsitumonitoringPortable,miniaturisation
ManytypesofApplications R&D/CONTRÔLEQUALITE R&D/QUALITYCONTROL • -Ageing/storageimpact • Stability/lifetime/freshness • Contamination/fermentation • Mixing,roasting,cooking… • Detectionoffraud • Comparisontoareference • Controlofintermediaryproducts • Controlofplaneffluents • Controlpackagingsandfinishedproducts • Residualsolvents/inks • Corns/meets/fishes.. • -………… Environment Foodindustry Chemistry Parfumes/Cosmetics Packaging Pharmaceutics
GasSensors ilemolecules MetallicOxide Sensors(MOS) Athightemperature(100°C-400°C),volatreactonthesensitivesurfaceofthesensor→ resistancevariation Selectivity:modulatedthroughworkingtemperature Inorganicmaterials:stable,longlifetime,weaklysensitivetohumidity,lowcost,easytobeused Mostcommonlyused
Responsesofthesensors:Comparisonoftwosamplesofoils Conformsmell Rancidsmell
Statisticalmethodsfordataprocessing Exploration PCA:PrincipalComponentAnalysis Qualitative DFA:DiscriminentFactorial Analysis SIMCA:SoftIndependentModellingClass Analysis Quantitative PLS:PartialLeastSquare
SIMCAqualitativeanalysis: comparisontoareference TheSIMCAmodeldefinesaconfidencezone UnknownsamplesareprojectedontheSIMCAmap.
AlphaMOS Arangeofproducts Recherche&DéveloppementMasseetcapteurs PROMETHEUS Recherche&DéveloppementContrôleQualité FOX/KRONOS ContrôleQualitéproductionréception CENTAURI ContrôleQualitéproductionréception ComparateurGEMINI
ElectronicTongue Gustativeimpression,inaliquidmedium V G electrodede référence enzyme espècebiologiquedétectée solution + + + H H H silice canal silicium p source drain ID VD ArrayofSensors:FieldEffectTransistors Withpolymericmembranesincludingnonspecificamphiphilicionophores
ElectronicTongue ElectronicTonguefromAlpha-Mos: -Astree
ElectronicTongue Discriminationof6brandsofbeerswithanelectronictongue(AlphaMOS)
OlfactoryReceptors Replacingthesephysicalorchemicalsensingelementswitholfactoryreceptors(ORs)thatwouldprovideanewplatformwiththecapacitytoovercometheseweaknesses,resultinginbioelectronicolfactiondevices. Innerchamberofnose Coffee!!
OlfactoryReceptors Schemeofanolfactoryreceptorincludedintheolfactorycellmembrane TheORsmediatingtheperceptionofodorants,objectoftheNobelPrizeinPhysiologyorMedicine2004,belongtothelargesuper-familyofG-proteincoupledreceptors(GPCRs),themselvesobjectoftheNobelPrizeinChemistry2012 EachORcanrespondtoarangeofodorantsthatsharespecificmolecularfeaturesandcandiscriminateslightdiscrepanciesinodorantstructure,sizeorconcentration.Moreover,anORcandetectitspreferentialodorantligandataverylowconcentration,i.e.10pM-10nM.
IdentificationofOlfactoryReceptors • Thedifferentstepsare: • ElectroOlfactoGram(EOG):turbinatesoftheratolfactorymucosabestrespondedtotargetodorant • Preparationoffreshlyisolatedolfactorycellsbychemicaldissociationofratolfactoryepithelium • Stimulationoffreshlydissociatedcells(Calciumimaging) • Recoveryofthewholecellcontentofresponsiveneurons • Single-cellRNAreverse-transcription(RT)andamplificationwithspecificprimerstoidentifytheolfactoryreceptorexpressedbytheseresponsiveneurons
Concentrationofolfactoryreceptorsingeneticallymodifiedyeasts InordertobeabletoimmobiliseORsonopticaland electricaltranducers,theywereexpressedin as geneticallymodifiedyeastswiththeir6-Histag,well. OR7D4fromchimpanzeerelatedtoandrostenoneOR1740fromhumanunrelatedtoandrostenone
Concentrationofolfactoryreceptors ingeneticallymodifiedyeasts NegativeTest SacharomycesCerevisiaeyeast,geneticallymodified,expressingI7receptorintheircellmembrane(ELISAtest)
OlfactoryReceptorincludedinnanosomes Microsomepreparation Nanosomepreparation
AnewapproachfortheconceptionofabiosensorfordetectionofOR/odorantinteractionAnewapproachfortheconceptionofabiosensorfordetectionofOR/odorantinteraction ER-α ER-α ER-α ER-α ER-α Nanosome N TheHistag-OR/nanosomeisimmobilizedongoldelectrodeusing6His-copperinteraction ON O N OCu2+N O O N IntensityofCu(I)/Cu(II)peakdependsonORconformation O N O Korri-YoussoufiH.etal,BiosensorsandBioelectronics26(2010)736–742)17β-estradiolisdetectedthroughitsoxidationpeakusingSWV O S Au Au Au
Electrochemicalcontrolofnanosomeimmobilization Ongoldelectrodesurface CopperacetateEstrogenreceptoralpha 1000 800 600 I/µAcm-2 400 200 0 -400 -200 0 200 400 600 E/mV SWVspectraofCu(I)/Cu(II)peakbeforeandafternanosomeimmobilization
ElectrochemicaldetectionofOR/odorantinteraction 180 160 140 120 100 80 60 40 20 0 -2 I/µAcm -400 -200 0 200 400 600 E/mV SWVdetectionofCu(I)→Cu(II)peakonOR7D4functionalizedelectrode-EffectofOR7D4relativeodorantfor(androstenone)
CalibrationcurveforandrostenonerelatedOR7D4 PositiveNegative 70 60 50 40 30 20 10 0 RelativelyHeight[µA/cm²] 10-14M10-12M10-10M10-8M10-6M10-4M Concentration[M] PBS RelativevariationofcopperpeakmaximumonOR7D4functionalizedelectrodeforpositivetest(androstenone)andnegativetest(helional)
CalibrationcurveforandrostenoneunrelatedOR1740 Positive(helional)Negative(androstenone) OR1740 20 RelativelyHeight[µA/cm²] 10 0 10-14M 10-12M 10-10M10-8M Concentration[M] 10-6M 10-4M RelativevariationofcopperpeakmaximumonOR1740functionalizedelectrodeforpositivetest(helional)andnegativetest(androstenone)
SummaryandPerspectives Feasibilityofelectrochemical detection ofbeen androstenone/relatedOR7D4interactionhas demonstratedforthefirsttime. AcalibrationcurveoftheOR7D4biosensorhasbeenobtainedandnegativetestsshowtheselectivityofandrostenonedetection. Perspectives: Bioelectronicdevice:multisensorarraywithmultiORs
Peopleinvolvedinthisstudy Z.Z.Guo,N.Zine,F.Lagarde,N. Jaffrezic-Renault, InstituteofAnalyticalChemistry,LSADepartment,5Ruedela Doua,69100Villeurbanne,France K.Badonnel,M.A.Persuy,E.Pajot-Augy, INRA, UR1197Neurobiologiedel'OlfactionetModélisationen Imagerie, F-78350Jouy-en-Josas, France A.Zhang KeyLaboratory of PesticidesandChemicalBiology,Huazhong NormalUniversity, LuoyuStr 152, HongshanDistrict, 430079Wuhan,China
LYONUNESCOWorldHeritage Sponsors:ThisworkwassupportedbyECthroughFP7BONDProject(NMP4-SL-2009-228685)andMarieCurieNANODEVgrantNo318524.Z. GuothanksChinaScholarshipCouncilforgrant. LYONUNESCOWorldHeritage Thankyouforyourattention 30
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