Ensemble multicolour FRET model enables barcoding at extreme FRET levels | Nature Nanotechnology

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Abstract

QuantitativemodelsofF枚rsterresonanceenergytransfer(FRET)鈥攑ioneeredbyF枚rster鈥攄efineourunderstandingofFRETandunderpinitswidespreaduse.However,multicolourFRET(mFRET),whicharisesbetweenmultiple,stochasticallydistributedfluorophores,lacksamechanisticmodelandremainsintractable.mFRETnotablyarisesinfluorescentlybarcodedmicroparticles,resultinginacomplex,non-orthogonalfluorescenceresponsethatimpedestheirencodinganddecoding.Here,weintroduceanensemblemFRET(emFRET)model,andapplyittoguidebarcodingintoregimeswithextremeFRET.Wefurtherintroduceafacile,proportionalmulticolourlabellingmethodusingoligonucleotidesashomogeneouslinkers.Atotalof580barcodeswererapidlydesignedandvalidatedusingfourdyes鈥攚ithFRETefficienciesreaching76%鈥攁ndusedformultiplexedimmunoassayswithcytometricreadoutandfullyautomateddecoding.TheemFRETmodelhelpstoexpandthebarcodingcapacityofbarcodedmicroparticlesusingcommonorganicdyesandwillbenefitotherapplicationssubjecttostochasticmFRET.

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Fig.1:SpectrallyoverlappingclassifierdyesandtheimpactofmFRETonthefluorescenceresponseofbarcodedmicroparticles.

Fig.2:One-potDNA-assistedmicroparticlelabellingconservesdyeproportions.

Fig.3:SchematicrepresentationoftheemFRETmodelandexperimentalvalidationoftheMFM.

Fig.4:Insilicodesignandexperimentalverificationoffour-colourbarcodeswithextremeemFRET.

Fig.5:Multicolourfluorescencemodelenablesautomateddecoding.

Fig.6:Screeningofbindingspecificitiesfora35-plexsandwichimmunoassay.

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Acknowledgements

TheauthorsthankT.Gervaisfordiscussions,andJ.Munzarforproofreadingourmanuscript.TheauthorsthankNSERCandFQRNTforfunding.M.D.acknowledgestheNSERC-CREATEISSprogrammeforsupport.TheflowcytometryworkwasperformedattwoMcGillcoreflowfacilities,namelytheMicrobiologyandImmunology(MIMM)departmentandtheLifeScienceComplex,whichissupportedbyfundingfromtheCanadianFoundationforInnovation.

AuthorinformationAffiliationsBiomedicalEngineeringDepartment,McGillUniversity,Montreal,Quebec,CanadaMiladDagher,聽MichaelKleinman,聽AndyNg聽聽DavidJunckerMcGillUniversityandGenomeQuebecInnovationCenter,Montreal,Quebec,CanadaMiladDagher,聽MichaelKleinman,聽AndyNg聽聽DavidJunckerNeurologyandNeurosurgeryDepartment,McGillUniversity,Montreal,Quebec,CanadaDavidJunckerAuthorsMiladDagherViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarMichaelKleinmanViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarAndyNgViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarDavidJunckerViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarContributions

M.D.andD.J.developedtheapproach.M.D.andA.N.conceivedtheexperiments.M.D.developedthemodelsandperformedtheexperiments.M.K.andM.D.developedthedecodingalgorithm.M.DandD.J.analysedthedataandwrotethemanuscript.

Correspondingauthor

CorrespondencetoDavidJuncker.

EthicsdeclarationsCompetinginterests

D.J.andM.D.areinventorsonaprovisionalUSpatentapplication62/568,998thatcoverssomeoftheaspectsreportedhereandwasfiledbyMcGillUniversityon6October2017.M.D.andD.J.arefoundersandshareholdersofnplexbiosciencesinc.

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Publisher鈥檚note:SpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.

Supplementaryinformation
WeijieWu,XujiangYu,MengyuGao,SehrishGull,LisongShen,WeiweiWang,LiLi,YueYinWanwanLiAdvancedFunctionalMaterials(2020)

QiWu,KennethYinZhang,PeilingDai,HengyuZhu,YunWang,LinnaSong,LingWang,ShujuanLiu,QiangZhaoWeiHuangJournaloftheAmericanChemicalSociety(2020)