化学生物学作业 27页

00.Abstract:Whole-cell,geneticallymodifiedbioreportersaredesignedtoemitdetectablesignalsinresponsetoatargetanalyteorrelatedgroupofanalytes.Whenintegratedwithatransducercapableofmeasuringthosesignals,abiosensorresultsthatactsasaself-containedanalyticalsystemusefulinbasicandappliedenvironmental,medical,pharmacological,andagriculturalsciences.Historically,thesedeviceshavefocusedonsignalingproteinssuchasgreenfluorescentprotein,aequorin,fireflyluciferase,and/orbacterialluciferase.Thebiochemistryandgeneticdevelopmentofthesesensorsystemsaswellastheadvantages,challenges,andcommonapplicationsofeachonewillbediscussed.Keywords:aequorin;bacterialluciferase(Lux);bioreporter;biosensor;fireflyluciferase(Luc);greenfluorescentprotein(GFP)01.IntroductionAbiosensorconsistsofabiologicalrecognitionelementthatoutputsasignaltoaninterfacedtransducercapableofmonitoringandmeasuringthatsignal.Biorecognitionelementstypicallytaketheformofanenzyme,antibody,nucleicacidfragment,organelle,oralivingprokaryoticoreukaryoticbioreportercell,whilethetransducerclassicallyexploitselectrochemical,optical,piezoelectric,magnetic,orthermalmeasurementinterfaces.Thebiorecognitionelementinitsnativeform,orageneticallyorbiochemicallymanipulatedversionofit,isemployedfortailoredsensingoftargetanalytes.Subsequentintegrationwiththetransduceryieldsaminiaturizedsensingplatformcapableofself-contained‘lab-on-a-chip’detectionandmonitoring.Althoughsuchmonitoringcanbemorepreciselyaccomplishedusinganalyticalinstrumentssuchasmassspectrometry,theirassociatedcostsandcomplexityareoftenfartooprohibitiveforroutineanalysesandtheirsizeandpowerrequirementstendtolimitusagesolelytothelaboratory.02.Biosensors,withtheirsmallsize,relativesimplicity,rapidityofoperation,andcontinuous,real-timetonearreal-timemonitoringcapabilities,possessuniquecharacteristicsconducivetothehigh-throughputandfield-basedorremotemonitoringneedsrelevanttoagricultural,environmental,pharmacological,andclinicalsensing.Althoughthemostpopularbiosensorsincorporateenzymesorantibodiesastheirbiorecognitionelements,inthisreviewwewillfocusonwhole-cellbiosensorsbecausetheydopossesssomeinterestingadvantages,primaryofwhichistheabilitytoindicatebioavailability—theeffectandinteractionstheanalytehasonalivingsystem.Asopposedtoanalyticalinstrumentsthatmeasureonlythetotalconcentrationofatargetanalyteinasample,whole-cellbiosensorsthatmeasurebioavailabilityindicatethattheanalytecanbeassimilatedbyordirectlyeffectsalivingorganism,therebyexposingpossibletoxicinteractionshigheruptheevolutionaryscale(i.e.,humans).03.BioreporterImmobilizationMethodsPerhapsthegreatestdifficultyinthedevelopmentofwhole-cellbiosensorsistheintimateadherenceofthebioreportertothetransducer.Sincethebioreporterisobligatedtoremainalivetoperformitssensingduties,whatevermechanismischosentoencapsulate,immobilize,oradherethereportercellsmustpreserveandsustainviability.Themoststraightforwardmethodssimplyencapsulatebioreporterswithinpolymersorgelssuchasagar,agarose,alginate,polyacrylamide,chitosan,polyvinylalcohol,andmanyothers[1].Theirmaindetrimentsarethatdiffusionoftheanalytethroughthepolymer/geloftenslowsreactiontimesandthattheanalytemayirreversiblyabsorbwithinthepolymer/gelmaking\nthebiosensorasingleusedevice.Premkumaretal.[2],ratherthanencapsulatingthecells,embeddedantibodiesinaglutaraldehydematrixandthenattachedEscherichiacolibioreportercellstotheantibodies.Thus,theE.colicells,althoughanchoredbytheantibodiestoasolidsubstrate,stillremainedfreetointeractwiththeirtargetanalytes.Sol-gels—silicaandnon-silica-basedporousglassgels—arealsopopularencapsulationmatrices,althoughmoresoforenzymesandantibodiesthanforwholecellsduetotheharshreactionconditionsduringformation,resultinginpoorcellsurvivability[3].However,modifiedhydrolysistechniquesandnewsol-gelcompositeshavedemonstratedlivingbioreporterencapsulationforuptooneyearunderrefrigeratedstorageconditions[4].Latexpolymershaverecentlyshownsignificantpotentialasbioencapsulantsaswell[5].Bioreporterbacteriamixedwithliquidlatexcanbe‘painted’asthinnanoporousfilmsontosolidsubstrates,allowedtodry,andthenrehydratedwhenneededtoreactivatethebioreportercells.00.Sincethefilmsarethin(<10μm),masstransferlimitationsoftargetanalyteareoflessconsequence.Shelf-lifeatroomtemperatureextendsfromtwomonthsuptooneyearuponrefrigeration.Thebioreporterincorporatedlatexcanalsobeusedessentiallyasinktoroboticallyprintprecisearraysormatricesofencapsulatedcells.Anotheruniquepolymeristhephotosensitivepolyvinylalcohol-styrylpyridinium(PVA-SbQ)whichcanbemixedwithbioreportercellsandthencuredunderultravioletlightexposure.Thisallowsprecisephotolithographicpatterningofthepolymerontransducerinterfaceswithsubsequentfastcuring[6].Surfacepatterningofbacteriabytheseso-calledsoftlithographictechniquescanbeaccomplishedwithavarietyofotherpolymersandassociatedmicrofabricationmethodsandlikelyrepresentsomeofthemostpromisingapproachestorecentlybecomeavailableforintegratinglivingcellswithbiosensorplatforms.ThereaderisdirectedtoanexcellentreviewbyWeibeletal.[7]forfurtherinformation.Nanotechnologyhasalsoimpactedcellimmobilizationthroughelectrospinning,aprocesswheredropletsofapolymersolutionsuchaspolyvinylalcoholareelectrostaticallystretchedintoextraordinarilythinnanofibers[8].Bacterialcellsmixedwiththepolyvinylalcoholbecomeentrappedwithintheexceptionallylargesurfaceareaofthenanofibermatrixduringtheelectrospinningprocessandhavemaintainedviabilityafterthreemonthsofstorageat20°Cinpreliminaryreports.Applyingalesstechnicalapproach,Chuetal.[9]usedordinarycotton,polyester,rayon,andsilkfiberspretreatedwithpolyethyleneimineasacrosslinkingagenttoimmobilizeE.colibioreportercellsthatthenremainedresponsiveoverapreliminarythreedayperiod.01.Inamoreunusualencapsulant,eggshellmembrane,knownforitsexcellentgasandwaterpermeability,wasusedtophysicallyadsorbPseudomonasfluorescenscellsalbeitonlyforseveralhourswithinthetestformatdescribed[10].Toavoidencapsulationaltogether,reportercellscanalternatelybemaintainedincontinuousculturebioreactorsforprolongedperiods[11].Thebioreactors,oftenminiaturizeddowntomillilitersizevolumes,arethendirectlyinterfacedwiththetransducertoformthebiosensor.Theredoesexistabitofcomplexityinsuchsystemssincepumpsandchannelsareneededtodelivernutrientsandremovewastes,butbioreactor-basedbiosensorsforwaterqualitymonitoring,forexample,havebeensuccessfullycommercializedandimplementedintoon-lineflowthroughdevicessuchastheTOXcontrolsensordevelopedbyMicroLAN(www.toxcontrol.com).Anothermeansofbypassingencapsulationistouseelectrokinetics\ntocontrolparticlemotion,therebymovingandtrappingbiologicalcellswithinstrictlydefinedareas[12].Aformofelectrokineticsreferredtoasdielectrophoresishasbeenusedtolocalizecellsdirectlyonalab-on-a-chiptransducersurfaceandsuchtechnologyparallelswellwiththeneedsofwhole-cellbiosensors[13].Thenaturalabilityofsomemicroorganismstoformhighlyresilientsporescanalsobetakenadvantageofasalong-termstoragesolution.Dateetal.[14]convertedspore-formingBacilluscellsintobioreporterswithshelf-livesofupto8months,andfurthershowedthattheycouldberepeatedlycycledbetweentheiractiveanddormantstateswithlittleancillaryeffectontheirsensingcapabilities.00.Whole-CellOpticalBioreportersandBiosensorIntegrationThefunctionofthewhole-cellbioreporteristoproduceameasurablesignalinresponsetoatargetanalyteorrelatedgroupofanalytes.Inwhole-cellbiosensors,thissignalistypicallyeitheropticalorelectrochemical.Forthisreview,wewillfocusonbioreportersthatuseopticalsignalingastheiroutputtotheircompaniontransducerandreferthereadertoMehrvarandAbdi[15]foranexcellentreviewonelectrochemicalbiosensors.Inbiosensorapplications,thisprimarilyinvolvesbioluminescentsignalsproducedbybacterialluciferase(Lux),fireflyluciferase(Luc),andaequorinandfluorescentsignalsproducedbygreenfluorescentprotein(GFP)(Table1).01.Table1.Whole-cellbioreportersreferencedinthetext.Reportersaregroupedbythebioluminescentorfluorescentsystemexploited.Reportercelltypeislistedabovethecompound(s)detectedandreferencesrefertooriginalpublicationsdetailingconstructionandsensitivityofeachreporterconstruct.\n00.BacterialLuciferase(Lux)BioluminescentbacteriaarethemostabundantandwidelydistributedofthelightemittingorganismsonEarthandcanbefoundinbothaquatic(freshwaterandmarine)andterrestrialenvironments.Despitethediversenatureofbacterialbioluminescence,themajorityoftheseorganismsareclassifiedintothreegenera:Vibrio,Photobacterium,andPhotorhabdus(Xenorhabdus).Ofthese,onlythosefromPhotorhabdushavebeendiscoveredinterrestrialhabitats[36].Thesebacteriaoftenexistassymbiotesofotherorganisms,althoughsomecanbefree-livinginaquaticenvironmentsaswell.01.Todayitiswellknownthatthebacterialbioluminescencereactionistheresultoftwoproteins,LuxAandLuxB,thatworktogethertoproducelightfromtheoxidationofalongchainfattyaldehydeinthepresenceofreducedriboflavinphosphate(FMNH2)andoxygen,whiletheremainingproteinsintheluxoperon,LuxC,LuxD,andLuxE,functiontoregeneratethealdehydesubstraterequiredforthisreaction(Figure1A).However,thiswasnotalwayssoevident.Thestudyofbacterialbioluminescenceisrootedinthelessonsofgeneralbioluminescence.TheideathatoxygenwasarequiredsubstrateforbioluminescentreactionscomefromRobertBoyle’searlyexperimentsinthemid1600’sshowingthatremovalofoxygencausedthecessationoflightfromwhatwaseitherluminescentbacteriaorfungi[37].Inthelate1880’swhenitwasdiscoveredfromworkinbeetlesthatbioluminescencerequiredaluciferaseandaluciferinforfunction,thisknowledgewasappliedtothebacterialsystemaswell[38].02.Figure1.Bioluminescentreactioncatalyzedbythebacterialluciferasegenecassette.A)The\nluciferaseisformedfromaheterodimeroftheluxAandluxBgeneproducts.ThealiphaticaldehydeissuppliedandregeneratedbytheproductsoftheluxC,luxD,andluxEgenes.Therequiredoxygenandreducedriboflavinphosphatesubstratesarescavengedfromendogenousmetabolicprocesses,however,theflavinreductasegene(frp)aidsinreducedflavinturnoverratesinsomespecies.B)Theproductionoflight,catalyzedbytheproductsoftheluxAandluxBgenes,resultsfromthedecayofahighenergyintermediate(R1=C13H27).AB00.In1942Doudoroffwasoneofthefirsttoobserveandreportonthemetabolismofbioluminescentbacteriaandfoundthatallwereabletotolerateoxygen,aidingintheconfirmationthatoxygenwasrequiredforlightproduction[39].Althoughthefirstpublishedreportofabioluminescentreactionoccurringoutsideofthebacteriaoccurredin1920,itcouldnotbereproducedreliablyuntil1953whenMcElroyetal.[40]wereabletoconsistentlyproducelightfromautolysatesofAchromobacterfischericulturesuponadditionofFMN.Atthistimetheyalsoreportedtherequirementforaluciferincompoundofunknownstructure.ThiswasthefirstindicationthatFMNwasrequiredforbacterialbioluminescence.ThestructureoftheluciferinwaslaterconfirmedasalongchainfattyaldehydebyStrehleretal.[41].\n00.Thiscompletedthelistofrequiredsubstratesandanunderstandingwasestablishedthatbacterialluciferasecatalyzestheproductionoflightthroughoxidationofalongchainfattyaldehydeinthepresenceofoxygenandreducedriboflavinphosphate.ThegenesencodingthebacterialluciferasewerefirstclonedandexpressedinE.coliin1982[42],whilethefullbacterialluciferasecassettewasclonedandexpressedthenextyear[24].Inthemid1990’sthefirstcrystalstructureofthebacterialluciferaseheterodimerwasdetermined[43],givingresearcherstheirfirstglimpseattheproteinsthathadcapturedtheirimaginationforhundredsofyears.01.Whenthebacterialluciferaseenzymeissuppliedwithoxygen,FMNH2,andalongchainaliphaticaldehyde,itisabletoproducelightprimarilyatawavelengthof490nm.Thereisasecondaryemissionpeakat590nm,however,thisisonlydetectableusinghighlysensitiveRamanscattering[44].Thenaturalaldehydeforthisreactionisbelievedtobetetradecanal,however,theenzymeiscapableoffunctioningwithalternativealdehydesassubstrates[36].ThefirststepinthegenerationoflightfromthesesubstratesisthebindingofFMNH2bytheluciferaseenzymeanduntilrecentlyitsactivesiteontheenzymewasnotknown.IthasrecentlybeenconfirmedthatFMNH2bindsontheαsubunitinalargevalleyontheC-terminalendoftheβ-barrelstructure[45].02.Inorderforthereactiontoproceed,theluciferasemustundergoaconformationalchangefollowingFMNH2attachment.Thismovementisprimarilyexpressedinashortsectionofresiduesknownastheproteaselabileregion—asectionof29aminoacidsresidingonadisorderedregionoftheαsubunitjoiningα-helixα7atoβ-strandβ7a.Themajorityofresiduesinthissequenceareuniquetotheαsubunitandhavelongbeenimplicatedinthebioluminescentmechanism[46].FollowingattachmentofFMNH2,thisregionbecomesmoreorderedandisstabilizedbyanintersubunitinteractionbetweenPhe272oftheαsubunitandTyr115oftheβsubunit.Thisconformationalchangehasbeentheorizedtostabilizetheαsubunitinaconformationfavorablefortheluciferasereactiontooccur[45].03.NMRstudieshavesuggestedthatFMNH2bindstotheenzymeinitsanionicstate(FMNH-)[47].Withtheflavinboundtotheenzyme,molecularoxygenthenbindstotheC4atomtoformanintermediate4α-hydroperoxy-5-hydroflavin[48].ItisimportanttonotethatthiscriticalC4atomwasdeterminedtobeincloseproximitytoareactivethiolfromthesidechainofCys106ontheαsubunit[45],aresiduethathaslongbeenhypothesizedtoplayaroleinthebioluminescentreaction,butrecentlyhasbeenproventobenon-reactivethroughmutationalanalysis[49].04.Ithasbeenshown,however,thatC4isthecentralatomfortheluciferasereactionandfollowingestablishmentofthehydroperoxidethereitiscapableofinteractionwiththealdehydesubstrateviaitsoxygenmoleculetoformaperoxyhemiacetalgroup.Thiscomplexthenundergoesatransformation(throughanunknownintermediateorseriesofintermediates)toanexcitedstategenerallyacceptedtobealuciferase-bound4α-hydroxy-5-hydroflavinmononucleotide,whichthendecaystogiveoxidizedFMN,acorrespondingaliphaticacid,andlight(Figure1B)[48].Therehaveclassicallybeenmanytheoriesproposedtoexplaintheexactprocessrequiredforlightemission[50]thatcontinuetoexpandtodayastechnologyfordetectingtheintermediatecomplexeshasimproved.Forareviewoftheproposedmechanismandtheirstrengthsandweaknesses,thereaderisdirectedtoNemtsevaandKudryasheva[48].\n00.Whilethebacterialluciferaseproteinisallthatisrequiredtogeneratelightinthepresenceofitsrequiredsubstrates,itisoftenbeneficialforinvestigatorstoexpressothergenesfromtheoperoninordertosupplytheluciferasewiththesubstratesrequiredforitsautonomousfunction.Toaccomplishthis,itisnecessarytoco-expresstheluxC,luxD,andluxEgenes.Theproductsofthesegenesassembleintoamulti-enzymecomplexandareresponsibleforbiosynthesisofmyristylaldehydeusingcomponentsalreadypresentinthecell,thusnegatingtherequirementtosupplyanaldehydesubstrateexogenously.01.TheluxDgeneencodesforatransferaseproteinandisthefirsttoactinthealdehydebiosynthesispathway.Itisresponsibleforthetransferofanactivatedfattyacylgrouptowater,formingafattyacid.Duringthecourseofthisreactiontheenzymeitselfbecomesacylated.ThenewlyformedfattyacidisnextpassedofftotheluxCgeneproduct,whichactivatestheacidbyattachingAMPfromamoleculeofATP,therebycreatingafattyacyl-AMPthatremainstightlyboundtotheenzyme.Thefattyacyl-AMPisthentransferredtotheluxEgeneproductviatransferoftheacylgroup.Thisproteinactsasareductaseandcatalyzesthereductionofthefattyacyl-AMPtoaldehydeusingNADPHtosupplytherequiredreducingpower[36].Thisallowsfortheinvivogenerationofthealdehydesubstrate.BecausetheremainingFMNH2andoxygensubstratesarenaturallysuppliedbytheorganism,thecoexpressionofthesegenesthusallowstheluxsystemtooperateinafullyautonomousfashion.02.LuxbiosensorsandapplicationsBacterialluciferaseiswellsuitedtofunctionasareportergenebecause,whenexpressedwiththegenesrequiredforaldehydebiosynthesis,itiscapableoffunctioningcompletelyautonomouslywithnoexogenousinputs.Themostbasicbacterialluciferaseassociatedreporterassaysarebasedondeterminingthepresenceorlevelofbioavailabilityoftoxiccompounds.Takingadvantageoftheautonomousnatureoftheluxoperon,bioreporterscanbeengineeredtoconstitutivelyexpresslightunderenvironmentalconditions.Uponexposureofthebioluminescentstraintoatoxiccompound,itwillundergoametabolicslowdownordeath,causingadecreaseinthetotalbioluminescentsignal[51].Theseassaysindicatethatatoxiccompoundispresentbuttheydonotidentifywhatthecompoundis.ThecommonlyusedMicrotoxassayoperatesinthisregard[16].Topermitidentification,otherbioreportertypesareengineeredtospecificallyrespondonlytocertaintargetcompoundsoranalytesofinterest.Theabilityofbacteriatometabolizespecificcompoundsistakenadvantageofinthesesensingstrategiestocreatefusionsoftargetspecificgenesequenceswiththebioluminescentluxgenes.Thus,whenexposedtoatargetcompound,thesebioreportercellswillemitbioluminescentlightsignalsthatareeitherdependentontheadditionofadecanalsubstrateifonlytheluxABgenesareusedorfullyautonomousiftheluxCDABEgenesareused[52].FullyautonomousluxCDABE-basedbioreportershavethedistinctadvantageofreportingtargetanalytepresencecontinuouslyandinareal-timeornearreal-timeformat.Historically,oneproblemassociatedwithreal-timemonitoringhasbeentheslowturnovertimeofthebioluminescentreaction.Coupledwiththelonglifeoftheluciferaseheterodimer,thishasmadeitdifficulttoresolvereporterfunctionovershortperiodsoftime.Inordertocompensateforthis,ithasbeendemonstratedthatinclusionofaproteasetagcanshortenthelifespanoftheluciferaseproteinsandincreasethetemporalresolutionoflux-basedreporters[53].Besideschemicaltargets,lux-basedreportersystems\nhavealsobeendesignedtodetectbiologicaltargets,forexample,foodandwaterbornepathogens.Inthesesystems,abacteriophage,orbacterialvirus,isusedasacarrieroftheluxgenesanditsabilitytoinfectonlycertainbacterialhostsisexploitedasameanstowardsdeliveringbioluminescencetoatargetbacterium[18].00.Animportantadvantagestemmingfromtheautonomousnatureofthebacterialluminescencecassetteisthat,sinceitdoesnotrequiresubstrateadditionforexpression,itcanbeusedremotelyifcoupledtoaproperdetectiondevice.Thisallowsforthemonitoringofcompoundsofinterestthatmaybeinaccessibletotheresearcherundernormalconditionsbecauseoflogisticalorsafetyconcerns[54].Althoughnotadetractionfromthemicrobialapplicationsofbacterialluciferase,itshouldbenotedthatitistheonlyreportercoveredinthisreviewthathashistoricallybeenlimitedtoexpressiononlyinprokaryotes.InrecentyearsthishasbeenchallengedasthegenesequenceshavebeenalteredtoallowforfunctionofthefullcassetteinthelowereukaryoteSaccharomycescerevisiae[20]andforluciferaseexpressioninculturedmammaliancells[25].01.Asatrulyautonomousexpressionsystem,Luxinterfacesextremelywellwithsignaltransducersandhasseenwidespreaduseinbiosensorapplications.Fiberopticcablesrepresentoneoftheeasiestinterfaces,withthebioreportersimmobilizedatoneendofthecableandtheotherendterminatingataphotomultipliertube(PMT)orotherluminometer-typedevice.Thecablecanthenbeinsertedintoliquid,solid,orgaseoussamplestoremotelymonitorfortargetanalytessuchasheavymetals,polycyclicaromatichydrocarbons(PAHs),orforageneralassessmentofsampletoxicity[27,29,33,35,55].Multi-fiberopticaldevicesimmobilizedwithdifferentlytargetsensitivebioreportershavealsobeendevelopedandfieldtestedformultiplexedmonitoring[56].Inthissamevein,butperhapsmoreuserfriendly,istheLumisens2instrumentdevelopedbyHorryetal.[57]wherethebioreporterbacteriaareimmobilizedonadisposablecardratherthanthefiberopticcableitself.Afiberopticcablethenscanseachindividuallyimmobilizedbioreportertomonitorforbioluminescenceoutputinaflow-throughformat.Similarflow-throughsamplershavebeenconstructedusingbioreactorscontaininggrowingculturesofthebioreporterintowhichbarefiberopticcablesareinserted.Uponexposuretoatargetanalyteortoxicintermediate,thebioreportercultureyieldsincreased(ordiminished)bioluminescencethatisdetectableviatheintegratedfiberoptics.Continuous,on-linewatertoxicitymonitoringhasbeendemonstratedusingsmall-scale(1–2mL)bioreactorsandlargercommerciallyavailablesystemssuchasthepreviouslymentionedTOXcontrolsensorthatcanbeplumbedintopre-existingwaterlines[11].Fiberopticshavealsobeenusedtomonitorbioreporterbacteriaintheirnaturalenvironmenttonon-invasivelyassessmetabolicandphysiologicalresponsestoecosystemperturbations,forexample,theadditionofacontaminant[58].02.Althoughfunctional,therequisitelinkageofthefiberopticcabletoaPMTorotherlightgatheringdevicenecessitatessizeandpowerconstraintsthatarenotconducivetominiaturization.Toaddressthis,severalgroupshavedevelopeddifferentvariationsofchip-basedmicroluminometersthatcandirectlyinterfacewiththebioreporterorganisms.Thisnegatestheneedforafiberopticcabletochannelthesignaltoatransducerandinsteadformsanall-inclusivebioreporter-on-a-chipbiosensor.Thistechnologywasfirstdemonstratedwiththebioluminescentbioreporterintegratedcircuit(BBIC)thatconsisted\nofasmall(1.5×1.5mm),low-power(3mW)CMOSmicroluminometerforlightgatheringandatransmitterforremotedatatransmission[59].PolymerencapsulantsattachthebioreportersdirectlyontotheBBICsurfaceortheBBICcanbeinterfacedwithbioreporterinoculatedflow-cellsorbioreactors.Forfieldmonitoring,theBBIChasbeenincorporatedintoahandheldwandthatoperatesoffofaninternallithiumwatchbattery(Figure2)[31].00.Figure2.ExampleofaCMOSmicroluminometertransducerinahand-heldbiosensorformat.Bioreportercellsengineeredtoemitbioluminescentlightsignalsaredirectlyinterfacedtothetransducerelementtoformacompactandremotelyoperablebiosensor.01.Asaphotodetectoraddon,MOEMS(Micro-Opto-Electro-Mechanical-System)canincreasedetectionlimitsbyminimizingsystemnoiseusinganintegratedheterodyneopticalsystem(IHOS)techniquethatmodulatesbioreporterbioluminescencepriortophotoconversion[60].AMOEMSmodulator/solidstatephotodetectorinterfacehasbeentestedwithaLuxbioreporterandaminimumdetectablesignalof109photons/sec/cm2wasdemonstrated.Toaccommodatemultiplexed,multi-analytesensingonasinglechip,Eltoukhyetal.[61]designeda128channelarrayCMOSmicroluminometercapableofholdingandindividuallysensingmultiplebioreporterssimultaneously,thusenablinghighdensityfingerprintingofsamplechemicalmakeupusinganyofthemanydifferentlyanalyte-specificbioreportersavailable,allwithinasinglelab-on-a-chipplatform.Avalanchephotodiodes(APDs)mayalsobeofutilitytobioreportersensingastheycanbedesignedforphotoncounting,muchlikeaphotomultipliertube,butinaminiaturizedstandalonedesign[62].APDscurrentlyrepresentthemostsensitivesolid-statedevicesavailableandcanachievequantumefficienciesgreaterthan90%.However,theyrequirehigheroperatingvoltages,generateexcessivebackgroundnoisethatmaymasklowlevelsignalsgeneratedfrombioreportercells,andtheircomplexcircuitrytranslatesintohighcost.Danieletal.[22]havepreliminarilytestedanAPDinconjunctionwithastressresponsiveLuxbioluminescentbioreporterwithina10μLsamplechamberanddemonstratedsufficientsensitivityatlowpart-per-millionconcentrationsofanalidixicacidinducer.Thisgrouphasalsorecentlydevelopedanintegratingspheredevicecapableofmeasuringabsolutephotonnumbersemanatingfrombioluminescentcells,which,althoughtoocomplexandfragiletoBioreporterinterfaceserveasabiosensor,shouldfindimportantutilityinshapingfactorsfundamentaltobiosensorengineeringsuchasquantumyieldandminimumsignaldetectionparameters[63].02.FireflyLuciferase(Luc)Fireflyluciferase(Luc)isthebeststudiedofalargenumberofluminescentproteinstobediscoveredininsects.Theinsectsrepresentalargerelatedgroupofbioluminescentorganisms,withover2,500speciesreportedtobecapableofgeneratinglight[64].Whilethevastmajorityoftheseluminescentreactionsremainunstudied,theexceptionisintheorderColeoptera(beetles)wheresystemshavebeencharacterizedforthechickbeetles,railroadworms,andfireflies(predominantlyPhotinuspyralis)[65].Firefliesproducelightinanorgancalledalantern,usingtherapidintroductionofoxygenasatriggerforluminescenceinordertoattractmatesaswellasdeterpotentialpredators[66].03.Thefirststudiesofthemechanismbehindinsectluminescencewerecarriedoutinthelate1800’sbyRaphaelDuboisusingthegroundupabdomensfromtheelanteridaebeetle.ItwasDuboiswhofirstproposedtheexistenceofasystememployingaluciferaseandaluciferinfortheproductionoflight.ThenextadvancecamefromNewtonHarvey,who\nreportedonthespecificityofluciferase/luciferininteractionsandconfirmedtherequirementformolecularoxygen[65].Inthemid1900’sWilliamMcElroybeganwhatwastobealongandsuccessfulcareerworkingwithfireflyluciferasebydiscoveringtherequirementthatATPbeinvolvedintheluminescentreaction[67].Basedinpartonthesefindings,hisgroupsoonproposedthatthebioluminescentreactionoccurredviaatwostepprocess[68]andwasthefirsttodeterminethestructureofthefireflyluciferinas2-(4-hydroxybenzothiazol-2-yl)-2-thiazolineacid[69]—commonlyabbreviatedasLH2intheliteraturetosignifyreducedluciferin.Inthelate1960’sand1970’sthemechanismunderlyingtheluminescentreactionwasreported[70,71],aswastheconfirmationoftheintermediateproductsofthisproposedreaction[65].Themechanismwasfinallysecuredin1980whenoxyluciferinwasisolatedasapurifiedproductoftheLH2luminescencereaction[72].Thelatestadvanceintheunderstandingoffireflyluciferasecamein1996whenContietal.[73]publishedthecrystalstructureoftheluciferaseataresolutionof2.0.Thisopenedthedoorfortargetedmutagenesisinvestigationsandgaveresearchersthefirstlookatthestructureofthisreporterprotein.00.TheLucproteincatalyzestheoxidationofthereducedluciferin(LH2)inthepresenceofATP-Mg2+andoxygentogenerateCO2,AMP,PPi,oxyluciferin,andyellow-greenlightatawavelengthof562nm(Figure3).ItisimportanttonotethatLH2isachiralmolecule,andwhileboththeDandLformscanbindtoLucandparticipateinadenylationreactions,onlytheDformiscapableofcontinuingoninthereactiontogeneratelight[65].Thisreactionoccurswithaquantumyieldof0.88,thehighestofanycharacterizedluminescentsystemwithnearlyonephotonproducedperoxidizedluciferin[73].Becauseofthehighquantumyield,thereactioniswellsuitedtouseasareporterwithasfewas10-19molofluciferase(2.4×105molecules)abletoproducealightsignalcapableofbeingdetected[74].01.Ithasbeenknownsincetheearly1950’sthatthechemicalreactionunderlyingfireflyluminescenceisatwo-stepprocessthatfirstrequiresadenylationofLH2followedbyoxidationandtheproductionoflight[68].Priortotheinitiationofthereaction,theLucproteinmustfirstbindtoLH2.However,atthistimeitisnotyetcapableofundergoingoxidationorproducinglight.ThefirststepinthegenerationoflightistheadenylationoftheboundLH2withthereleaseofpyrophosphate[75].ThefunctionofthisadenylationistoincreasetheacidityoftheC4protonofthethiazolineringonLH2.ThisallowsforremovalofaprotonfromC4causingformationofacarbanion[76].Thiscarbanionisthenattackedbyoxygen,displacingAMPanddrivingtheformationofacyclicperoxidewithassociatedcarbonylgroup(adioxetanonering).Asthebondssupportingthisstructurecollapse,itbecomesdecarboxylated,releasingCO2andforminganelectronicallyexcitedstateofoxyluciferinineithertheenolorketoform[75].02.Figure3.Thebioluminescentreactioncatalyzedbyfireflyluciferase.Theluciferaseproteinholdsthereducedluciferintoallowforadenylation(a).Thisprocessisfollowedbyadeprotonationreactionthatleadstotheformationofacarbanion(b)andattackbyoxygen(c),drivingtheformationofacyclicintermediate(d).Asthisintermediatedecays,carbondioxideisreleased,formingtheexcitedstateluciferinineithertheketo(e)orenolate(f)form.UsedwithpermissionfromBranchinietal.[77].\n00.Thekineticsofthisreactioncanbealteredbyvaryingtheconcentrationofthesubstrates,withlowconcentrations(inthenMrange)showingsteadylightproductionandhighconcentrations(μMrange)producingabrightflashfollowedbydecayto5–10%ofthemaximum[78].Therearemultiplepossibleinhibitorycompoundsthatcouldberesponsibleforthekineticprofilegeneratedunderhighsubstrateconcentrations.Ithaspreviouslybeenshownthateventhoughoxyluciferinisanaturalproductoftheluciferasereaction,itiscapableofremainingboundasaninhibitortoenzymaticturnover[79].Thesamewasfoundtobetrueofanotherpotentialbyproduct,L-AMP,whichcanaccountforupto16%oftheproductformedduringtheluminescentreaction[80].Thismay,inpart,explainhowtheadditionofCoAtotheluminescentreactioncanresultinimprovedperformance.WhenCoAisaddedduringtheinitialstepsofthereaction,itpreventsthefastsignaldecaynormallyobserved,andwhenitisaddedfollowingthisdecayitcanpromotere-initiationoftheflashkinetics.ThiscanbeattributedtoCoA’sinteractionwithL-AMPtoformL-CoA,resultinginturnoveroftheLucenzymeandreoccurrenceoftheluminescentreaction[81].01.Insects,andspecificallybeetles,thatproduceluminescencearequitediverseinthecolorstheyarecapableofproducing.Itwasoriginallybelievedthatthecolorsweretheresultofdivergentluciferasestructures,however,thesequencesoffourluciferasegenesfromPyrophorusplagiophthalamuswithfourdifferentemissionspectraweresequencedanditwasfoundthattheysharedupto99%aminoacididentity[82].Therearecurrentlythreemechanismsthathavebeenproposedtoexplainthemultiplebioluminescentcolorations:theactivesitepolarityhypothesis[83],thetautomerizationhypothesis[84],andthegeometryhypothesis[85].02.Theactivesitepolarityhypothesisisbasedontheideathatthewavelengthoflightproducedisrelatedtothemicroenvironmentsurroundingtheluminescentproteinduringthereaction.Innon-polarsolventsthespectrumisshiftedtowardsblueandinpolarsolventsitismorered-shifted.Itisquestionable,however,ifpolarityfluctuationscanaccountforlargescalechangeslikethosethathavebeenobservedinP.plagiophthalamus.Thetautomerizationhypothesisstatesthatthewavelengthoflightproducedisdependentonwhethereithertheenolorketoformoftheluciferinisformedduringthecourseofthereaction.Arecentstudyhasreportedthatbyalteringthesubstrateofthereactiontheketoformoftheluciferincanproduceeitherredorgreenlight,makingthishypothesisunlikely\n[86].Finally,thegeometryhypothesissuggeststhatthegeometryoftheexcitedstateoxyluciferinisresponsiblefordeterminingtheemissionwavelength.Ina90°conformationitwouldachieveitslowestenergystateandredlightwouldbeproduced,whereasintheplanarconformationitwouldbeinitshighestenergystateandgreenlightwouldbeproduced.Intermediatecolorswouldbetheresultofgeometriesbetweenthesetwoextremes[64].00.LucbiosensorsandapplicationsFireflyluciferasemakesanexcellentreporterforthereasonspreviouslydiscussed,however,themajorhurdlehasalwaysbeentheexpressionofthereporterinreal-timeduetotherequirementofaseparateluciferin.Becauseofthis,themajorityofhistoricalstudiesinbacteriahavefocusedontheuseofLucoutsideofthecellininvitroreactions,preventingtheabilitytodetectexpressioninreal-time.However,thebrightnatureandquickreactiontimeofLucmakeitanexcellentcandidateforfast,large-scaleapplicationssuchasimmunoassays.Anadvantageofthistypeofsystemisthatthebioluminescentsignalcanbecorrelatedtotheconcentrationofthecompoundofinteresttoallowforrapidquantificationwithlittletonobackground[87].01.TheuseofLucinvivoinbacterialsystemsovercomesthepreviousdetractionsassociatedwiththeuseofafireflyluciferaseinbacterialcells,however,thereisstillnoknownwaytogeneratetheluciferininabacterialsystemdenovo.Ithasrecentlybeenreportedthatbacterialcellscanbedehydrated,whichincreasesporesizelargeenoughforuptakeoftheluciferin,andthenre-hydratedwhilecontainingtheluciferinwithoutilleffects.Thispresentsasimpleandinexpensivemethodforintroductionoftheluciferinsubstrate.BecauseoftheefficiencyoftheLucreaction,itisevenpossibletovisualizethelocationoftheLucproteinspatiallywithinindividualcellsusingthismethod[26].AnotherpromisingadvancehasbeentheisolationandcharacterizationoftheluciferaseregeneratingenzymefromP.pyralis[88].Ithasbeensuggestedthatco-expressionofthisenzymealongwithLuccouldallowforcontinualbioluminescentproductionwithouttheneedforre-additionoftheluciferinsubstrate[89].02.Intermsofbiosensors,Lucisnotroutinelyusedsinceitsrequirementforluciferinisahindrancetotheautonomousmonitoringprincipleofthebiosensor.Forexample,Ikariyamaetal.[28]immobilizedluc-incorporatedE.colibioreportersinadialysismembraneattachedtothedistalendofafiberopticcabletomonitorforbenzenederivativesinliquidtestsamples.Thefiberopticcablewasfirstimmersedinthesampleforafixedamountoftime,andthenluciferinwashandinjectedalongwithacelllysisagenttoinstigatethebioluminescentresponsethatwasthensubsequentlymeasuredbyaPMT.Obviously,thehands-onmanipulationsdonotcontributetoanidealbiosensorformat.Maehanaetal.[30]solvestheextraneousluciferinadditionsomewhatbyincorporatingmicrofluidicsintotheirchip-basedbiosensor.Variousmicrowellspatternedontoasiliconwaferholdthesampleandthebioreporterbacteria(agenotoxicsensingE.colistrain),andalthoughtheluciferinsubstrateislateraddedwithinthesamemicrowells,onecouldenvisionseparatewellsandmicrofluidicmixingasameanstowardsforminganall-inclusivebiosensor.Indeed,Meietal.[90]describestechniquesfordoingso.Measurementofluminescencewasalsoperformedoff-chipwithaCCDcamera,butagain,previouslymentionedmicroluminometerscouldbedirectlyinterfacedtoformatruebiosensor.\n00.AequorinWhiletheluminescentproductoftheaequorinproteinhasbeenknownsincemanfirstsetouttothesea,itwasnotuntil1962thattheproteinitselfwasfirstisolated[91].Aequorinisacalcium-sensitiveluminescentproteinnativetothejellyfishAequoreaaequorea.Despiteknowledgeofitsexistencetherewasoriginallymuchdifficultyinisolatingtheproteinbecauseitdidnotuseaconventionalluciferase-luciferininteractiontoproducelight,butratherreliedonthepresenceofcalciumionstoexciteapre-boundfluorophore(Figure4)[92].Furtherstudyofthemechanismbehindaequorin’sluminescentnaturewasdelayeduntilthefirstpracticaluseoftheisolatedproteinwasdescribedin1967.Itwaspublishedthataequorincouldbeusedasabioreportertomonitorcalciumsignalinginthemusclefibersofbarnaclesfollowingdirectmicroinjection[93].Thisdemonstrationofaequorin’sapplicationleadtoarenewedinterestinthemechanismunderlyingitsluminescenceandsetoffalonghistoryofitsuseasareporterprotein.01.Dueinparttothechallengesassociatedwithgatheringlargeamountsofproteinfromthenativejellyfish,itwasnotforanotherfiveyearsthatthestructureofthechromophorewasdiscoveredtobecoelenterazine[95],thesamemoleculethatwasisolatedfromtheluminescentsquidWatasenia[92]andchemicallysynthesizedbyInoueetal.[96]thatsameyear.Withthechemicalsynthesisofcoelenterazinepublished,themajorhurdletoaequorin’susewasthedifficultyinobtainingusableamountsofproteinfromthejellyfish.ThiswasovercomeseveralyearslaterwhenthecDNAoftheaequorinproteinwasfirstclonedandexpressedinE.coli[97,98].Followingthis,thecrystalstructurewasdeterminedin2000[99],openingthewaytoafullunderstandingofthestructureandfunctionofthisimportantprotein.02.Forsuccessfulproductionoflight,theaequorinapoproteinmustbondwithcoelenterazineandbetriggeredbycalcium.Theresultofthisreactionistheproductionofbluelightatawavelengthof465nm,theevolutionofCO2,andtheconversionofmatureaequorintobluefluorescentprotein(theaequorinapoproteinnowboundtocoelenteramide)[100].Theemissionoflightatthiswavelengthrequiresthegenerationofanexcitedstatemoleculethatmustbepopulatedbyachemicalreactionwithatleast70kcal/molofexergonicity.Thismuchenergycannotbeexplainedsimplybythebindingofcalciumtotheprotein,indicatingthattheluminescencereactionisproceededbyanintramolecularchemicalreaction[101].Thebindingofcalciumfunctionsasthetriggerofthisreaction,whichinturnproducesluminescence.03.Figure4.Thebioluminescentreactioncatalyzedbyaequorinisdependentonthepre-boundcoelenterazineluciferin.Uponcalciumbinding,thestericorientationoftheluciferinisdisturbedleadingtoacyclizationreactionthatirreversiblyformsadioxetanoneintermediate.Asthisintermediatedecays,carbondioxideisreleasedandasinglet-excitedanionisproduced,followedbythegenerationoflightat465nm.UsedwithpermissionfromJonesetal.[94].\n00.Theproposedmechanismofthistriggeristheresultofthestructuralorientationofthecalciumbindingsitesinrelationtothehydrophobicresiduesoftheproteinbackbonethathavebeenshowntostabilizecoelenterazineinthehydrophobiccavity.TheloopstructuresintheirassociatedEF-handmotifsarenotproperlypositionedtobindcalciumintheirnativestate.Uponbindingtheremustnecessarilybechangesinthespatialrelationshipbetweenthecoordinatingaminoacidresiduestoaccommodatethecalciumion[101].ThekeyresiduesinvolvedinthisshiftappeartobeHis169andTyr184,sincethesehavepreviouslybeenestablishedascoordinatingthepositionofcoelenterazineinthehydrophobiccavity[99].Becauseoftherigidstabilityimpartedtocoelenterazinebytheseresidues,itcanbeexpectedthatanyrearrangementduetocalciumbindingwouldthereforeresultinthereorientationofcoelenterazine.01.TheacceptedchemicalreactionofcoelenterazinetoproducelighthasbeensuggestedbyMcCapraandChang[102]andrelatedtothestructuralchangesresultingfromcalciumbindingbyVysotskiandLee[101].Briefly,followingthestructuralchangesimpartedbycalciumbinding,thesharedhydrogen-bondingnetworkbetweencoelenterazine,Tyr184,andHis169becomesdisrupted.ThisforcesHis169tobecomepartiallyprotonatedwhileTyr184assumesanegativecharge.ThehydroperoxideattachedtocoelenterazineatC2willthenprotonateTyr184inareactionmadepossiblebecausetheyshareasimilarpKinthehydrophobiccavityundertheseconditions.TheresultingnegativechargeonthehydroperoxidethenundergoesanucleophilicattackonC3ofcoelenterazinetoirreversiblyformadioxetanoneintermediate.Thiscyclizationprovidestheenergyrequiredtodrivetheproductionoflightfromtheoverallreaction[101].02.Asthebondsbetweennewlycyclizedoxygenscollapse,theperoxideisreleasedasCO2and\nasinglet-excitedanionisproduced.Thisanioniscapableofemittinglightdirectlyoritmayfirstbeprotonated(presumablybytransferoftheprotonoriginallytransferredtoHis169)toproducesinglet-excitedcoelenteramide,whichisalsocapableofemittinglight[103].Thefactthattherateofthisreactionisdependentontheconcentrationofcalciumionshasmadeitanattractivecompoundforuseinreportersystems.Whenexposedtosaturatingconcentrations(>100μM),thereactionisalmostinstantaneous,however,whentheconcentrationislower,thereisarelationshipbetweenthefractionalrateofconsumptionandcalciumconcentration.Becauseonlyonephotonisproducedperreactedproteinundercoelenterazinelimitingconditions,thisratecanbeusedtodeterminetheinitialconcentrationofcalciumions[104].AlthoughthisreactionisinhibitedbyMg2+,itcanalsobetriggeredbyEu2+,Sr2+,andBa2+,makingaequorinamultifacetedreporterprotein[105].00.Althoughaequorinmaybethebestcharacterizedofthecalciumdependentphotoproteins,itisbynomeansunique,norisittheonlyphotoproteintobefunctionallyemployedasareporter.Morethan25differentcoelenterateorganismshavebeenshowntopossessthistypeofprotein[101],whilesevenhavebeenisolatedthusfar:thalassicolin,aequorin,mitrocomin,clytin,obelin,mnemiopsin,andberovin[106].Alongwithaequorin,mitrocomin,clytinandtwohomologsofobelinhavepublishedcDNAsequences[101].Allofthesecalciumdependentphotoproteinsaresmall,singlepolypeptideswithmolecularweightsrangingfrom21.4to27.5kDaandallcontainthreecalciumbindingsiteswithaffinities(kd)rangingfrom1to10μM[107].Theconservedstructureandluminescentsystemssharedbetweentheseproteinsgreatlycontributedtothediscoveriesbehindtheirmechanismofaction.Hadresearchersworkingontheluminescentsystemsofdifferentproteinswithsimilarstructuresandmechanismsnotbeenabletosharetheirdiscoveries,theremaynotyethavebeenanexplanationforthemysteriousmeansbehindthisfascinatingphenomenon.01.AequorinbiosensorsandapplicationsHistorically,aequorinhasfounduseasacalciumreporterinavarietyofsystems.Evenrecentlyitisstillbeingemployedinthisfunctiontomonitorhowvariousfactorsaffecttheconcentrationofcalciuminamodelbacterium[32].However,thisdynamicreporterisnotlimitedtojustcalciumdetection.OneofthemoreinterestingdetectionsystemsemployingaequorinistheCellularAnalysisandNotificationofAntigenRisksandYields(CANARY)assay.CulturedBcellswithantigenstovariouspathogenicbacteriaareengineeredtoexpressaequorin.Asaresultofcontactbetweenthecellsurfaceantigensandthebacteriaofinterest,aninternalsignalingcascadeissetoffthatultimatelyreleasescalciumionsandtriggerstheproductionoflightviaaequorin[34].Theassayhasbeenintegratedintoabiosensor-likedevicereferredtoasPANTHER(PathogenNotificationforThreateningEnvironmentalReleases).AnairsamplerbringspathogensincontactwiththeBcellreportersandcorrespondingsignalemissionismeasuredbyaluminometer.Currently,21differentpathogenscanbedetectedwithinathreeminuteassay.02.Aequorinisalsobecomingmorepopularasaquantitativelabelinbindingassays.Thereareseveralattributesthatmakeitattractiveforthisfunction,nottheleastofwhichisthatitprovidesforsensitivitydownto10-21molwhileremainingfreefromthehealthhazardsnormallyassociatedwiththeradioactivelabelspreviouslyemployedinthisfunction.Ithasalsoprovenitselftobequitestableovertimewith85%ofitsoriginalactivityretainedover\nonemonthofstorageat4°Candashelflifeofgreaterthanoneyearfollowinglyophilization.Inaddition,thebioluminescentnatureofaequorin’slightproductionmeansthereisvirtuallynobackgroundinbiologicalsamplescomparedtothelevelofnaturallyfluorescentmoleculespresentinthesesystems[106].Takentogether,theseattributescanmakeaequorintheperfectchoiceforrapiddetectionoflowlevelcompoundsorelementalexposuresinlivingsystems.00.GreenFluorescentProtein(GFP)Greenfluorescentprotein(GFP)wasfirstdiscoveredduringinvestigationintotherelatedchemiluminescentproteinaequorinfromthejellyfishAequoreavictoria[91].SincethattimeithasbeenrealizedthattheAequoreaderivedGFPisjustoneofalargerfamilyofhomologousfluorescentproteinscapableofproducinglightinavarietyofcolorsduetoalterationsinthecovalentstructureoftheirchromophoresordifferencesinthesurroundingnon-covalentenvironment[108].Despiteitsearlydiscovery,theuseofGFPasaresearchtooldidnotbeginuntilafteritwassuccessfullyclonedalmostthirtyyearslater[109].However,soonafterthecDNAwasavailable,itsfunctionwasvalidatedinbothprokaryoticandeukaryoticorganismsbyChalfieetal.[110],andsincethattimeithasbeenusedinnumerousapplicationsincludinglocalizationstudies,proteinexpressionmonitoring,asareportergene,asaviabilitymarker,todetecttheonsetofapoptosis,andmanyothers(reviewedin[111]).01.GFPhasbecomeafavoredtoolformolecularstudiesbecauseitisautofluorescentanddoesnotrequiretheadditionofanycofactorstoproperlyfunctioninexogenoussystems[112].However,itdoesrequireactivationbyanexcitationlightsourcebeforeitssignalcanbemeasured.Itisalsoresistanttoheat,alkalinepHfluctuations,chaotropicsalts,organicsolvents,andmanyproteases[113],anditsexpressioninexogenousenvironmentsisprimarilynon-toxic[111]withafewprovenexceptions[114,115]thatmaybeduetoproductionofhydrogenperoxideasaby-productofsynthesis[116].However,GFP’sslowposttranslationalchromophoreformation,oxygenrequirement,andpotentialdifficultyindistinguishingitssignaturefrombackgroundfluorescencecanbeproblematic[111],especiallyinaerobicorganisms.Becauseofthis,alternatefluorescentproteinssuchasthosebasedonflavinmononucleotideareoftenusedwhendevelopingreportersfromanaerobicorganisms[117].Time,though,hasproventhatthebenefitsoutweighthechallengesformostinvestigators,andGFPhastakenitsplaceasoneofthemostpopulartoolscurrentlyavailableforcellularandmolecularsignalingresearch.02.Wild-typeGFPproteinisabletoabsorblightattwodifferentwavelengths.Aminorpeakoccursat475nmwiththemajorpeakat397nm(Figure5).Regardlessofwhichexcitationwavelengthisused,emissionoccursonlyat504nm[118].Thedifferentabsorptionpeakshavebeenattributedtovaryingprotonationstatesofthefluorophore,withtheneutralstatecorrespondingtothemajorabsorptionpeakat397nmandtheanionicformcontributingtotheminorpeakat475nm[119].Thelargeshiftbetweenthemajorabsorptionpeakat397nmandtheemissionat504nmcanbeattributedtoanexcitedstateprotontransferfromthesidechainoftheTyr66residueofthefluorophore[120]tothecarboxylateoxygenofGlu222[111].03.Figure5.ThedualabsorptionpeaksintheGFPspectraaretheresultofdifferentchargestatesintheGFPchromophore.Theneutralstate(left)isresponsibleforthemajorpeakat\n397nmwhiletheanionicform(right)isresponsiblefortheminorpeakat475nm.Regardlessofthechromophorechargestate,emissionoccursat504nm.AdaptedfromScholarpedia.org.00.Basedonthisinterconversionofthefluorophore,athreestatemodelofphotoisomerizationhasbeenputforwardtoexplainthechemicalbasisforshiftsinabsorption.Thismodelstatesthatexcitationoftheneutralstatefluorophorecancauseconversiontotheanionicformviaanintermediate[120].Theintermediateisstructurallysimilartotheneutralformofthefluorophore,buthasbecomedeprotonatedatthephenolgroupofTyr66[111].Excitationoftheanionicformiscapableofdirectlyemittingfluorescence,whiletheneutralstatemustnecessarilyconvertintoanexcitedformofthisintermediatepriortoemission[121].Whileitispossiblefortheneutralformtoconverttotheanionicformfollowingexcitation,thisisnotthemostfavorablereaction.Themajorityofexcited,neutralfluorophoreswillconvertbrieflytotheintermediatestate,wherefluorescencewilloccur,followedbyreversionbacktotheneutralstate[120].Interconversionbetweentheneutralandanionicstatesispossible,butrequiresbothprotontransferandconformationalchangetooccur[111].Similarly,themajorityofanionicfluorophoreswillreverttothegroundstatefollowingfluorescentemission,butcouldinsteadundergoaconformationalchangetotheintermediatestateandthencontinueontoadoptaneutralchargestate[120].01.Inawild-typepopulation,GFPcontainsa6:1ratioofneutraltoanionicfluorophores[116],explainingwhythemajorabsorptionpeakisfoundat397nm.However,uponextendedultraviolet(UV)illuminationthispeakwillbegintodecreaseandtheminorpeakwillincrease[122].Thisbehaviorcorrespondstothephotoisomerizationoftheneutralfluorophoreformresponsibleforthemajorabsorptionpeakbeingconvertedintotheanionicformasdiscussedabove.WhilethephotoisomerizationcharacteristicsofGFPcanproveproblematicforquantification,theydoallowforthestudyofproteinmovementbyexcitationwithintenseUVlightat397nmfollowedbyexcitationat475nminordertotrackthemovementofthephotoisomerizedfluorophores[123].02.FollowingthediscoveryofGFP,itwasquicklyproventhataminoacidsubstitutionswerecapableofalteringitsfluorescentcharacteristics.Sincethattime,versionsofGFPhavebeendevelopedthatfoldmoreefficientlyathighertemperatures[124],avoiddimerizationathighconcentration[125],orfluoresceintheblue[126],cyan[127],oryellow[128]wavelengths.Homologshavesincebeendiscoveredthatfluoresceintheredrangeaswell[129].Thehistoryanddevelopmentofthesevariantsisoutsidethescopeofthisreview,butanexcellentclassificationhasbeenmadebyTsien[116]andabridgedbyZimmer[111]dividingtheknownvariantsintosevenclassesbasedonspectralcharacteristics.Whenappliedinconcert,thesevariantsoftheGFPproteinhavegivenresearcherstheabilitytousemultipleGFP-basedreportersinthesameenvironmentatthesametime,improvingthe\nusefulnessandrangeofthisalreadydynamicprotein.00.GFPbiosensorsandapplicationsSinceitwasfirstdemonstratedthatGFPcouldbeexpressedinE.coli[110],ithasbeenusedincountlessexperimentsinorganismsrangingfrombacteriatoculturedhumancellsandevencommercializedforsaleindesignerpets.Asidefrombasiclocalizationassays,thetwomainusesofGFPasareporterfocusoneithertheinductionorsuppressionofGFPexpressiontoindicateinteractionwithananalyteofinterest.OneofthemorepopularassaysfocusingonthesuppressedexpressionofGFPisthedeterminationofcellviability.BacteriaexpressingGFPareexposedtocompoundsofinterestandtheseverityoftoxicityisdeterminedbymonitoringthedecreaseinfluorescentexpression[17].Thisallowsresearcherstoprocessalargenumberofsamplesveryquicklyinanautomatedfashion.Asthebacteriaarekilledortheirmetabolismisslowedbyinteractionwithtoxicsubstances,theoverallamountoffluorescencewilldecrease.Thistypeofassayhastheaddedadvantageofdeterminingtheamountofagivencompoundthatwillbebioavailabletotheorganismbeingtested.01.TheinverseofthistypeofexperimentistoinducetheexpressionofGFPasapositiveresult.InthiscasethegeneencodingforGFPisplacedunderthecontrolofageneticpromoterthatrespondsspecificallytotheanalyteofinterest.Thisallowsforvisualdetectionwhentheorganismisexposedtothetargetanalyte.Anadvantageofthistypeofexperimentaldesignisthattheamountoffluorescenceproducedcanbecorrelatedtotheconcentrationofanalyte,allowingforanapproximatequantification[130].ItisalsopossibletousethefluorescenceofGFPasamarkertoisolatethosemembersofthecommunityshowingaresponsebyusingfluorescenceactivatedcellsorting(FACS)[131].UsingGFPtoconfirminteractionwithacompoundofinterest,quantifytheamountofexposure,andisolatingexposedcellssimultaneouslyillustratesitsdynamicfunctionalityinmodernbioreporterresearch.02.BiosensorintegrationofGFP-basedbioreporters,however,remainsfairlylimitedduetosignaloutputbyGFPbeingcontingentuponanexcitationlightsource.Thus,theseset-upsrequireoneenergysourcetoexciteGFPandanothertomeasureGFP,andtheassociatedcomplexityandbulkinessareoftennotsuitableforbiosensorapplications.Therehasbeensomeapplicationsuccessusingfiberopticcableswhereonecableisusedforexcitationandanotherforemissionmeasurement.Shettyetal.[19],forexample,constructedaGFP-basedE.colisensitivetothemonosaccharideL-arabinoseandentrappeditwithinadialysismembranetiedtothetipofafiberopticbundle.FibersterminatingatatungstenlampservedastheexcitationsourcewhileseparatefibersterminatingataPMTservedasthedetector.ImmersingtheE.colientrappedsensorendofthefiberbundleinliquidwasthenshowncapableofdetectingL-arabinoseatvaryingconcentrations.Toimprovesensitivity,Knightetal.[21]bypassedfiberopticsbyinterfacingaPMTdirectlywithaflow-cellcontainingayeast-baseGFPbioreportersensitivetoDNAdamaginggenotoxiccompounds03.Anargonlaserprovidedtheexcitationsource.Realizingthenecessityforminiaturizationandlesscomplexity,newfluorescencedetectiontechniquesbasedonsmallfootprintbiosensorcompliantplatformsarebecomingsomewhatavailable.Complementary–metal–oxide–semiconductor(CMOS)photodetectorsbettertunedtothegreenlightsignatureprovideenhanceddetection[132],asdoavalanchephotodiodes,while\ntightlyfocusedlaserbeamsprovideexcitationdowntothesinglecelllevel[133].However,incorporatingallnecessarycomponentsintoatruebiosensorformatremainschallenging.Rothertetal.[23]developeda12cmdiametermicrofluidics-basedlab-on-a-compactdisk(CD)devicethatmicrocentrifugallymovedandmixedmicrolitervolumesofwatertestsampleswithaGFPbioreportersensitivetoarsenic(Figure6).AlthoughsensingwasaccomplishedwithafiberopticprobepositionedabovetheCD,thedevicecouldlikelybeeasilyreconfiguredtoaccommodateachip-basedsensortopromotefurtherminiaturization.00.Figure6.A)Alab-on-a-CDmicrofluidicdeviceusedinconjunctionwithGFPbioreporterssensitivetowardsarsenic.B)Aclose-upviewofthemicrofluidicchannelingthatpermitssampleandbioreportermixing.UsedwithpermissionfromRothertetal.[23].01.AlternativeBioreporterSystemsThereportersystemscoveredindetailinthisreviewrepresentthemajorityandmostpopularsignalingproteinscommonlyusedbyinvestigatorsforopticalbiosensingapplications.Alongwithhomologuestotheproteinsreportedhere,therearemyriadothersystemscapableofeitheractingasareporterorbeingcomplexedtoatransducerforbiosensormonitoring.Theseincludeβ-galactosidase,β-glucuronidase,catechol2,3-dioxygenase,chloramphenicolacetyltransferase,theicenucleationproteinInaZ,UroporphyrinogenIIImethyltransferase[134,135]andmorerecently,engineeredinfrared-basedfluorescentproteins[136].Eachofthesesignalingelementshasitsownadvantages,disadvantages,andrichhistoryofdiscoveryandusejustastheonescoveredinthisreview.We,a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