conven515hh

REVIEWBioinspiredrecognitionelementsformycotoxinsensorsRiikkaPeltomaa1&ElenaBenito-Pea1&MaríaC.
Moreno-Bondi1Received:27July2017/Revised:5October2017/Accepted:10October2017/Publishedonline:10November2017#Springer-VerlagGmbHGermany2017AbstractMycotoxinsarelowmolecularweightmole-culesproducedassecondarymetabolitesbyfilamentousfungithatcanbefoundasnaturalcontaminantsinmanyfoodsandfeeds.
Thesetoxinshavebeenshowntohaveadverseeffectsonbothhumanandanimalhealth,andarethecauseofsignificanteconomiclossesworldwide.
Sensorsformycotoxinanalysishavetraditionallyappliedelementsofbiologicaloriginfortheselectiverecognitionpurposes.
However,sincethe1970stherehasbeenanexponentialgrowthintheuseofgeneticallyengineeredorsyntheticbiomimeticrecognitionelementsthatallowsomeofthelimitationsassociatedwiththeuseofnaturalreceptorsfortheanalysesofthesetoxinstobecircumvented.
Thisreviewprovidesanoverviewofrecentadvancesintheapplicationofbioinspiredrecognitionel-ements,includingrecombinantantibodies,peptides,aptamers,andmolecularlyimprintedpolymers,tothede-velopmentofsensorsformycotoxinsbasedondifferenttransductionelements.
KeywordsMycotoxin.
Recognitionelement.
Recombinantantibody.
Peptide.
Aptamer.
MolecularlyimprintedpolymerIntroductionMycotoxinsarelowmolecularweight(approximately700)naturalproductsproducedassecondarymetabolitesbyfila-mentousfungimainly,althoughnotexclusively,whentheyreachmaturity[1].
Unlikeprimarymetabolites,thesecom-poundsarebelievedtohavenofunctioninthelifecycleoftheproducercell[2].
Theycanbefoundasnaturalcontami-nantsinmanyvegetalfoodsorfeeds,includingnuts(almondsandwalnuts),cereals(rice,wheat,andmaize),oilseeds(soy-bean,peanuts),fruits,driedfruits,spices,beans,forage,wines,andgrapejuices,orinfoodsofanimalorigin,suchasmilk,eggs,andmeat[1,2].
Alternatively,exposuretothesetoxinscanbebyinhalationofdustcontainingmycotoxigenicfungalspores[1].
Regardlessofthewaytheycomeincontactwithhumansordomesticanimals,includingbirds,theymaycauseloweredperformance,sickness,orevendeathevenatverylowconcentrations[3,4].
Theirrangeofactionsincludescy-totoxic,nephrotoxic,hepatotoxic,teratogenic,mutagenic,car-cinogenic,immunosuppressive,andestrogeniceffects[1,2].
Inanycase,theireffectonhealthdependsonfactorssuchastheconcentrationinthecontaminatedfoodandtheexposuretime,thesynergisticeffectofothermycotoxins,andenviron-mentalfactorsassociatedespeciallywiththestoragecondi-tionsofthefoodstuff[1].
ThewordBmycotoxin,^acombinationoftheGreekwordforBfungus^mykesandtheLatinwordtoxicum,meaningBpoison^[5],wasestablishedin1962aftertheBturkeyXdisease^responsibleforthedeathofapproximately100,000turkeypoultsnearLondon,duetotheintakeoffeedinfestedwithsecondarymetabolitesfromAspergillusflavus(aflatoxins)[6].
Nowadays,approximately400compoundsarerecognizedasmycotoxins,althoughonlyabout30PublishedinthetopicalcollectioncelebratingABCs16thAnniversary.
*MaríaC.
Moreno-Bondimcmbondi@ucm.
es1DepartmentofAnalyticalChemistry,FacultyofChemistry,UniversidadComplutensedeMadrid,Av.
Complutenses/n,28040Madrid,SpainAnalBioanalChem(2018)410:747–771https://doi.
org/10.
1007/s00216-017-0701-3moleculesareconsideredasathreattohumanandanimalhealth[3].
Mycotoxinsmaycontaminatecropsbeforeorafterharvest.
Asanexample,deoxynivalenol(DON)andT-2toxin,twotoxinsproducedbyFusariumspecies,appearbeforehar-vest,ochratoxins(fromAspergillusandPenicillium)occurafterharvest,andaflatoxins(fromAspergillus)canbepro-ducedatanystageoftheproductionchain,frombeforehar-vesttostorage[7].
Themycotoxinsthatshowthehighestoccurrencerateandtheseveresteffectsonhumanandanimalhealthincludeaflatoxins,fumonisins,ochratoxins,3-nitropropionicacid,trichothecenes,ergotalkaloids,citrinin(CIT),andzearalenone(ZEN)(Table1)[9].
Additionally,thepresenceoftoxinprecursors,metabolites,degradationproducts,ortheso-calledmaskedmycotoxinsisalsoapoten-tialthreattoconsumersafety.
Thepresenceofthesecom-poundsmayincreasethetoxicityoffoodcommoditieswithanapparentlylowconcentrationoftheparentaltoxin.
Moreover,theirdetectionisinmanycasesstillinitsinfancy.
Suchmetabolitesandmaskedmycotoxinshavebeenreportedatleastfortrichothecenes,fumonisins,ochratoxinA(OTA),andZEN[10].
Mycotoxin-producingmoldsareextremelycommon,andtheygrowunderawiderangeofconditions,whichoftenmakesmycotoxincontaminationinevitable[3].
Asthepresenceofthesetoxinsinfoodandanimalfeedscanhaveseverehealtheffectsaswellasimportanteco-nomicconsequences,severalnationalandinternationalauthorities,includingtheEuropeanCommission,havesetmaximumresiduelimitsforthemostcommonandmosttoxicmycotoxins(Table2)[11–15].
Thelimitsdifferdependingonthetoxinandthefoodstuffinques-tion;thestrictestregulationshavebeensetforaflatoxinsandtheprocessedfoodproductsforinfants.
Inadditiontothetoxinregulationsforfoodstuff,EuropeanCommissionregulationsincludealsothepresenceofmycotoxinsinanimalfeed[16,17].
Tomeettheregulationsandtoensurefoodsafety,novelanalyticalmethodsareneededtodetectandquan-tifythesetoxicmoleculesattraceconcentrations.
Conventionally,mycotoxinsaredetectedbychromatog-raphy,immunoassays,orsensors.
Whilechromatograph-icmethods,mostlyhigh-performanceliquidchromatog-raphycoupledwithdiodearray,fluorescence,ormassspectrometrydetection,offerhighsensitivityandaccu-racy,theyhavesomelimitationsrelatedtotheirhighcostandlonganalysistimeandtherequirementofhighlyskilledpersonnelandtedioussamplecleanup[18–21].
Inthissense,immunoassayscanbeconsideredasanalternativefortherapiddeterminationofthesenaturaltoxins,withtheadvantagethattheyareusuallylowcostwhilemaintainingtherequiredsensitivityandspecificity.
Moreover,sensorsformycotoxindetectioncanofferreal-timereadoutandperformtheentirepro-cessautomatically[22,23].
Table1Mostabundantandtoxicmycotoxins,theirmostimportantproducers,andtheirmostimportanteffects[2,3,8]MycotoxinFungispeciesStructureEffectsCommonditiesaffectedAflatoxins(AF)AFB1,AFB2AFG1,AFG2AFM1AspergillusflavusAspergillusparasiticusAcutelytoxic,carcinogenic,immunosuppressive,mutagenic,genotoxic,teratogenicNuts,cereals,maize,riceCitrinin(CIT)PenicilliumcitrinumPenicilliumcamembertiAspergillusterreusAspergillusniveusCytotoxicandnephrotoxicWheat,oats,rye,corn,barley,riceDeoxynivalenol(DON)FusariumgraminearumFusariumculmorumPotentinhibitorofeukaryoticproteinsynthesiscausesnausea,vomiting,anddiarrheaGrainssuchaswheat,barley,oats,rye,maize,rice,sorghumFumonisinsFB1FB2FusariumverticillioidesFusariumproliferatumHepatotoxicandcarcinogenic,interfereswithsphingolipidmetabolismMaizeandsorghumOchratoxinOTAAspergillusochraceusAspergilluscarbonariusPenicilliumverrucosumCarcinogen,nephrotoxic,teratogenic,immunosuppressive,potentteratogenGrainssuchascorn,barley,oats,rye,andwheat,coffeebeansPatulinPenicilliumpatulumPenicilliumexpansumAspergillussppGenotoxic,immunotoxic,neurotoxic,teratogenicApples,pears,cherries,andotherfruitsT-2toxinFusariumsporotrichioidesFusariumpoaeCytotoxic,immunosuppressive,potentinhibitorsofeukaryoticproteinsynthesisWheat,barley,oats,maizeZearalenone(ZEN)FusariumgraminearumEstrogenicCerealcropssuchaswheat,maize,barley,andsorghum748PeltomaaR.
etal.
Themostcrucialelementofallimmunoassaysandsensorsistherecognitionelement,whichmakespossiblethespecificdetectionofthetargetanalyte,andthequalityoftherecogni-tionelementoftendefinesthespecificityandsensitivityoftheanalysis[24,25].
Especiallyinthecaseofcompetitiveassays,whichisusuallytheformatselectedtodetectlowmolecularweighttargets,suchasmycotoxins,theimportanceoftherecognitionelementiseminentasthesensitivityofcompeti-tiveassaysishighlydependentontheaffinityoftherecogni-tionelement[26].
Todevelopasensitiveandspecificmethod,therecognitionelementmustbeabletodetecttheanalyteevenatlowconcentrationsandtodistinguishthetargetfromothersimilarmolecules,suchasothertoxinsthatcanbepresentinthesamesample[27].
Otherfactorsaffectingtheselectionoftherecognitionelementincludecost,availability,andcompat-ibility,aswellasstabilityandshelflife.
Antibodiesarethegoldstandardrecognitionelementsinbiosensorsandassays,andtheyhaveproventheirsuperiorityovertheyearsintermsofspecificityandsensitivity.
Forthedetectionofmycotoxins,thefirstpolyclonalantibodiesweredescribedmorethan40yearsago[28]andtheyweresooncomplementedbymonoclonalantibodies[29,30].
Duringthelastfewdecades,numerousapplicationsbesidesthetradition-alELISAhavebeendescribed,andexcellentsensitivitieshavebeenreportedrecently,forexample,withmagneticbeadsforimprovedseparation[31–33],ornoveldetectionschemes,in-cludingnewlabel-basedapproaches[34–36]andlabel-freeapproaches[37–40].
Manypolyclonalandmonoclonalanti-bodiesaswellasELISA-basedtestkitsarecommerciallyavailable,andantibodiesindisputablycontinuetheirreignastherecognitionelementofchoice[41,42].
Nevertheless,de-spitetheirwideuse,antibodieshavesomelimitationsmostlyrelatedtohighcostandlowstability,forexample,tohightemperaturesorstringentconditions.
Owingtotheirlargeandcomplexstructure,antibodiesareabletospecificallybindtheirtargetantigenbut,atthesametime,thecomplexstructureissusceptibletodenaturation,degradation,oraggregation.
Moreover,productionofmonoclonalantibodiesbyhybrid-omatechnologyisusuallytime-consumingandinherentlyde-pendentonanimalimmunization[26,41].
Smallanalytes,suchasmycotoxins,areoftendifficulttargetsforantibodydevelopmentastheyaretoosmalltoberecognizedbytheimmunesystem,andthusrequireconjugationtoacarriermol-ecule[26].
Inaddition,somecommercialantibodieshavebeendemonstratedtohavehighcross-reactivity,whichlimitstheirapplicationtorealsamples,whereseveraltoxinscanbepresentsimultaneously[43–45].
Novelrecognitionelementshavethepotentialtoovercomethelimitationsrelatedtomonoclonalandpolyclonalantibod-ies.
Notonlycanthesensitivityandspecificitybeincreased,but,perhapsevenmoreimportantly,therobustness,simplici-ty,andpriceofthemethodcanbeaffected,makingitpossibletodevelopmethodssuitableforhigh-throughputscreeningorlow-resourcesettings.
Inthisreview,weintroducesomere-centadvancesinthedevelopmentofnovelbioinspiredrecog-nitionelements,includingrecombinantantibodies,peptides,aptamers,andmolecularlyimprintedpolymers(MIPs),forthedetectionofmycotoxins.
RecombinantantibodiesAntibodiesarewidelyusedforbiosensingbecauseoftheiruniquepropertiesandimmensevarietyofpossiblespecific-ities.
Naturally,antibodiesareproducedbytheimmunesys-teminB-celllymphocytestofunctionasantigenreceptorsforthecell.
ThevastrecognitionvarietyreliesonthevariationsintroducedduringthetranscriptionprocessoftheBcellsbycombinatorialassemblyofthegenefragments,andfurther,byadditionalmutationsaftertheprimaryrecognition[46].
ForTable2RegulationsandrecommendationsformycotoxinsbytheEuropeanCommission[11–15]MycotoxinMaximumlimit(μg/kg)FoodstuffsRegulationAlfatoxinsAflatoxinB1SumofaflatoxinsB1,B2,G1andG2AflatoxinM10.
10–12.
04.
0–15.
00.
025–0.
050Nuts,driedfruit,cerealsandcerealproducts,maize,spicesMilkECNo.
1881/2006ECNo.
165/2010Deoxynivalenol200–1750Unprocessedcerealsandmaize,pasta,bread,cereal-basedinfantfoodECNo.
1881/2006ECNo.
1126/2007FumonisinsSumofFumonisinsB1andB2200–4000Unprocessedmaizeandmaize-basedfoodsECNo.
1881/2006ECNo.
1126/2007OchratoxinA0.
50–10.
0Cerealsandcerealproducts,driedvinefruit,coffee,spices,wineandgrapejuiceECNo.
1881/2006ECNo.
105/2010Patulin10.
0–50Fruitjuices,appleproducts,cereal-basedinfantfoodECNo.
1881/2006T-2andHT-2toxinsSumofT-2andHT-2toxins15–1000UnprocessedcerealsandcerealproductsECNo.
165/2013Zearalenone20–350Cereals,maize,cereal-andmaize-basedproduct,breadECNo.
1881/2006ECNo.
1126/2007Bioinspiredrecognitionelementsformycotoxinsensors749decades,polyclonalandmonoclonalantibodieshavebeenthecornerstoneofmostmycotoxindetectionmethods,butgrad-uallytheyhavebeencomplementedbyotherantibodyfor-mats.
TheconventionalIgGantibody(approximately150kDa)consistsoftwoidenticalheavypolypeptidechains(50kDa)andtwoidenticallightpolypeptidechains(25kDa)thatarelinkedtoeachotherbydisulfidebonds(Fig.
1a).
Thelightandtheheavychainsbothhaveonevariabledomain(VLandVH,respectively),andadditionallythelightchainhasasingleconstantdomain,whereastheheavychaincontainsthreeorfourconstantdomains.
Thetwoantigen-bindingfragments(Fabs)areresponsibleforbindingtothetarget,whereasthehighlyconservedFcregion(fragmentcrystallizable)interactswitheffectormoleculesandcells[46,48].
Themostrelevantregion,fromtheanalyticalpointofview,istheantigen-bindingsite,orparatope,whichisrathersmallcomparedwithsizeofthetotalantibody.
Theconceptofreducingthesizeoftheantibodywhileconservingtheantigen-bindingpropertieshaslongbeenknown,andenzymaticdigestionhastradition-allybeenusedtogenerateantibodyfragments[25,46].
Morerecently,advancementsinrecombinantDNAtechnol-ogyandproteinengineeringhavemadeitpossibletomodifytheantibodystructureanddeveloprecombinantantibodiesthatpossessseveraladvantageouscharacteristics,suchassmallersizeandeasyproduction,whilepreservingthetargetspecificityoftheintactantibody[49].
ThebestknownantibodyfragmentsaretheFabfragment(55kDa)andtheevensmallerscFv(sin-glechainfragmentvariable;25kDa),whichconsistsonlyoftheVHandVLdomains,whicharejoinedbyasyntheticpoly-peptidelinker(Fig.
1a)[49].
Thesmallsizeofrecombinantantibodyfragmentshasseveraladvantages,includingthede-creaseofnonspecificbindingoftencausedbytheFcregionoftheintactIgG,andthepossibilitytoimmobilizetheantibodiesathigherdensity.
Furthermore,novelrecombinantantibodiescantheoreticallybeselectedwithinacoupleofweeks,whichcanbeconsideredamajoradvantagecomparedwiththedevel-opmentofpolyclonalandmonoclonalantibodies,whereim-munizationsinevitablytakeseveralmonths[26,27].
Antibodyfragments,unlikefull-lengthantibodies,canbepropagatedinbacteria,suchasEscherichiacoli,whichsignificantlylowersthecostofproductionasnospecializedcellculturefacilitiesforhybridomacelllinesareneeded[50].
Thetechnologyhasthepotentialtobypasstheimmunesystemandproduceantibodieswithouttheneedtoimmunizeanimals.
Generationofantibod-iesbyinvitrodisplaytechnologieshasseveraladvantagesoverthenaturallyderivedantibodies,suchasthecontrolovertheselectionconditionsandthegreathigh-throughputpotential,includingparallelization,automation,andminiaturization[43].
Moreover,antibodyengineeringusingdifferentinvitrostrategies,includingchainshufflingandsite-directedmutagen-esis,permitsfurthermanipulationormodificationofvariousantibodypropertiestoachievegreateraffinityorstability,elim-inateunwantedcross-reactivities,oraddtagsforpurificationorimmobilization[26,48].
RecombinantantibodiescanbederivedfrommonoclonalantibodiesbydirectcloningofthegenefragmentsfromthehybridomacelllinesandtheirexpressioninE.
coli.
Forex-ample,recombinantFabfragmentagainstDON[51]andscFvsagainstaflatoxinB1(AFB1)[52],DON[53],andfumonisinB1(FB1)[54]werederiveddirectlyfrommonoclo-nalantibodies.
However,isolationoffunctionalrecombinantantibodiesdirectlyfromhybridomacelllinescanbedifficult,andthefragmentsoftenhaveloweraffinitythantheparentalmonoclonalantibody[55,56].
Forexample,theanti-FB1scFvproducedbyMinetal.
[54],althoughspecifictowardFB1,hadabout12timesloweraffinitythanthemonoclonalantibody.
Alternatively,recombinantantibodiescanbeselectedfromrecombinantantibodylibraries,inessencemimickingthenat-uralinvivoprocessofantibodyproduction.
TheprocessFig.
1aTheconventionalantibody(IgG),theheavychainantibody(hcAb),andthemostcommonrecombinantantibodyfragments(scFv,Fab,VHH).
bGeneralschemeofthephagedisplaybiopanningprocedure.
CHconstantheavydomain,CLconstantlightdomain,VHvariableheavydomain,VLvariablelightdomain.
(Adaptedfrom[47])750PeltomaaR.
etal.
includes(1)thegenerationofgenotypicdiversity,usuallybyconstructionofantibodylibrariesthatconsistofmillionsorbillionsofdifferentantibodyvariants,(2)thedisplaymethod,whichcreatesaphysicallinkbetweentheexpressedproteinvariantandthegenecodingforit,(3)theapplicationofselec-tivepressuretoscreentheantibodylibrariesfortarget-specificbinders,and(4)amplificationoftheselectedvariants[57].
Byfarthemostusedandmostwidespreadtechniqueforscreen-ingantibodylibrariesisphagedisplay[58],whichisbasedonbacterialviruses,bacteriophages,whichcaninfectbacteriaandusethebacterialcellfortheirownreplication.
Phagescanbeeasilygeneticallyengineeredtodisplayforeignpep-tidesorproteinsoutsidethevirionasafusionproteinwithoneofthecoatproteins.
Peptidesandsmallantibodyfragmentshavebeendisplayedonphagestocreatephagelibrariesconsistingofmillionsofdifferentphageclonesthatdisplaythepeptideorantibodyfragmentoutsidethevirionwhileretainingthegeneticmaterialinsidethecapsule.
Thisphysicallinkageallowseasyselectionofthetarget-specificbindersinaninvitroscreeningprocesscalledBbiopanning^(Fig.
1b).
Intheiterativepanningprocedure,aphage-displayedlibraryisintroducedtoatargetcoatedsurface,and,aftertheunboundphageshavebenewashedaway,thetarget-bindingphagesareelutedandamplifiedinE.
colitosubjectthemtosubsequentroundsofselection.
Usuallyafterthreeorfourroundsofselec-tion,individualtarget-specificclonescanbeselectedandchar-acterized[59].
Alternatively,thescreeningoftheantibodyreper-toirecanbebasedonyeastdisplay[60],orcell-freesystems,suchasribosomedisplay[61].
Althoughnotaspopularasthephagedisplaytechnology,thesemethodsoffersomeadvantages,suchasthecapabilityofyeastcellstoexpresscomplexproteinsthatrequireposttranslationalmodifications,orthepossibilitytocreatelargerlibrarieswithuseofcell-freemethodsthatarenotrestrictedbythebacterialtransformationefficiency[62].
RecombinantantibodylibrariescanbeconstructedfromnaturalsourcesbyisolationoftheBcellsofanimals,mostoftenmice,immunizedwiththetargetantigen,anduseofthecorrespondinggenestoconstructtheantibodylibrary[63].
Suchimmunizedlibrariesarealreadybiasedtowardthetarget,andtheyoften,althoughnotnecessarily,resultinhigh-affinitybinders.
However,theprocessislongandinherentlydepen-dentonanimalimmunizationindividuallyforeachtarget.
Phage-displayedscFvlibrarieshavebeenconstructedfromimmunizedmiceagainstFB1[64],OTA[65],andZEN[66].
Surfaceplasmonresonance(SPR)analysisrevealedexcellentaffinitiesinthenanomolarrangefortheanti-OTAandanti-ZENantibodyfragments,demonstratingtheadvantageofusingimmunizedlibraries.
Inadifferentapproach,so-calledpositivephage-displayedlibrarieshavebeenconstructedbyrandomrecombinationoftheVHandVLgenefragmentsfromhybridomacelllinesthatsecreteaspecificmonoclonalanti-body(Fig.
2a).
SuchlibrarieshavebeenreportedatleastagainstAFB1[56]andFB1[55],bothofwhichresultedinanscFvwithincreasedaffinitycomparedwiththeparentalmonoclonalantibody.
Alternatively,thelibraryconstructioncanbedonecomplete-lyinvitro,resultinginnonimmunized(nave)[68],synthetic[69],orsemisyntheticantibodylibrariesthatcontainhighersequentialdiversityandcanbedesignedforscreeningofanti-bodiesagainstawiderangeoftargets,ordesignedtakingintoaccountspecificprerequisitessoastofindantibodies,forex-ample,againsthaptens[70].
Theuseofsyntheticlibrarieshasseveraladvantages,suchasthepossibilitytoselectantibodiesagainstsmallordifficulttargetsthatcanbenonimmunogenicorhighlytoxic,butthelibrarydiversityhastobehighenoughtofindbinderswithhighaffinity.
Constructionofsuchlibrariesistechnicallydifficultandlimitedbytheefficiencyofthebacte-rialtransformation[59].
Formycotoxinsonlyafewexamplesofrecombinantantibodiesoriginatingfromnaveorsyntheticlibrarieshavebeenreported.
Anti-FB1scFvselectedfromanavelibraryshowedaffinityofonlyKD=4.
08*10–7M[71],whereasanti-AFB1screenedfromasynthetichumanscFvlibrary(TomlinsonJ)showedexcellentKDof1.
2*10–12M[72].
Someexamplesofimmunoassaysbasedonrecom-binantantibodiesarepresentedinTable3.
Heavychainantibodies,naturallyproducedbycamelidsandsharks,areaninterestingsubclassofantibodiesthatarecompletelydevoidofthelightchains(Fig.
1a)[76].
Thus,becauseofthelackoftheVLdomains,theantigenisrecog-nizedbyasingledomainandtheparatopeiscomposedofthreehypervariableloops(insteadofsixinIgG)[48].
Theuseofthesinglevariabledomainofheavychain(VHH)anti-bodieshasgainedalotofinterestthankstotheirspecificcharacteristicandtheextraordinarystructurethatmakesthemmoresuitableforsomeapplications.
TheVHHantibodyfrag-ments,alsoknownasBnanobodies^orBsingledomainantibodies,^arethesmallestavailableantigen-bindingfrag-ments,withasizeofonly15kDa,andtheyarerobustandverystableinavarietyofconditions,includinghightemperaturesanddenaturingconditionsthankstothehydrophilicresiduesubstitutionsinaspecificregion[48,77].
Heavychainanti-bodiesareusedinclinicalandtherapeuticapplicationsbutarealsoaninterestingoptionforinvitrodiagnostics.
Heavychainantibodies(VHH),ornanobodies,areusuallyselectedbyconstructionofaphage-displayedlibraryafterimmunizationofanalpacawiththehaptenconjugate.
Althoughnanobodieshavemanyidealcharacteristicsforbio-sensorapplications,suchashighstabilityandeasyproduc-tion,onlyafewexamplesofsuccessfulapplicationformyco-toxinshavebeenreported(Table3).
ThemostwidelyusednanobodywasdevelopedbyLiuetal.
[67]forthedetectionofOTAusinganalpaca-derivedVHHlibrary.
ThenanobodyNb28showedexcellentperformanceinphagedisplay-mediatedimmuno-polymerasechainreaction,withadetectionlimitof3.
7pg/L(Fig.
2b),whichisthelowestdetectionlimitreportedforOTAdetection,althoughthehighsensitivityisBioinspiredrecognitionelementsformycotoxinsensors751probablymostlyattributedtothepolymerasechainreactionbaseddetectionratherthantherecognitionelementperse.
Anexcellentexampleofthepossibilitiesofferedbyproteinengi-neeringwasdescribedlaterwhenthesamenanobodywasexpressedasafusionwithalkalinephosphataseandusedindirectcompetitivefluorescenceenzymeimmunoassaywithadetectionlimitof0.
04ng/mL[75],andinnanobody-basedELISAwithadetectionlimitof0.
16ng/mL[78].
Thenanobody-basedELISAhadaslightlyhigherdetectionlimitbutthesolublenanobodyhadgreaterstabilityanditcouldretaintheantigen-bindingactivityevenafterexposuretotem-peraturesashighas95°C.
Similarresultsregardingnanobodystabilityandtolerancetohightemperaturesandorganicsol-ventswerereportedforanAFB1-specificnanobodyselectedalsofromanalpaca-derivedVHHlibrary.
Thiscompetitivenanobody-basedELISAexhibitedahalf-maximalinhibitoryconcentration(IC50)of0.
754ng/mL,andbecauseofthehightolerancetomethanol,sampleextractscouldbedirectlyana-lyzedwithoutdilution[74].
Recently,theOTA-specificnanobodyNb28wasusedforthedevelopmentofamembrane-baseddotELISAthatallowednoninstrumentalvi-sualscreeningofOTAat5μg/kg,andresultscouldbeobtain-edwithin20min[79].
Althoughnanobodiesarewidelypraisedandthefewex-amplesdescribedhereenlightenthemanyadvantagesofnanobodies,thediscoveryofhapten-bindingnanobodiesisdifficult[80].
Ithasalsobeenpointedoutthatnanobodiesarenotidealbindersforsmallmoleculesbecausetheypossessalimitednumberofconformationalstructuressuitableforhaptenrecognition[81].
Morenanobodiesformycotoxinde-tectionhavebeenreportedtobeusedasepitopemimics(discussedinBEpitopemimics^)ratherthanasprimaryanti-bodies,whichmightbeamoreappropriateapplicationforthisspecialclassofantibodiesbecauseofthestructureoftheirFig.
2aConstructionofapositivephage-displayedlibraryfortheselec-tionofhigh-affinityscFvantibodiesforaflatoxinB1(AFB1)detection.
bSelectionofochratoxinA(OTA)-bindingVHHfromanimmunizedlibraryandtheuseofphage-displayedVHHinphageELISAwithphagedisplay-mediatedimmuno-polymerasechainreaction(PCR)-baseddetection.
BSAbovineserumalbumin,OVAovalbumin.
(aAdaptedfrom[56];badaptedfrom[67])752PeltomaaR.
etal.
Table3Recentexamplesofimmunoassaysbasedonrecombinantantibody(Ab)fragmentsTargettoxinRecombinantAbAbsourceAssayformatDetectionAbaffinity(KD)(M)AnalyticalcharacteristicsSampleReferenceAFB1scFvDerivedfrommAbImmobilizedAFB1–BSA,solublescFvSPR1.
16*107ND–[52]AFB1scFvSyntheticlibrary(1.
8*107),yeastdisplayImmobilizedAFB1–BSA,solublescFvSPR82.
7*10–8ND–[73]AFB1scFvTheTomlinsonlibrariesI+J(synthetichumanscFv;each1.
4*108)ImmobilizedAFB1–BSA,phage-displayedscFv,anti-phage-Ab–HRPAbsorbance9.
8*10–11IC50=0.
4ng/mL–[72]AFB1VHHImmunizedlibraryfromalpaca(2.
1*107)ImmobilizedAFB1–BSA,solubleVHH,anti-HA-tag-Ab–HRPAbsorbanceNDIC50=0.
754ng/mLLinearrange0.
117–5.
676ng/mLPeanut,rice,corn,feedstuff[74]DONFabDerivedfrommAbImmobilizedDON–HSA,biotinylatedFab,avidin–HRPAmperometryNDLOD=63ng/mLIC50=380ng/mLLinearrange100–4500ng/mLBreakfastcereal,babyfood[51]DONscFvDerivedfrommAbImmobilizedscFv,DON–HRPBLINDIC50=36.
1ng/mL–[53]FB1scFvDerivedfrommAbImmobilizedscFv,FB1–HRPAbsorbanceNDIC50=220ppb(mAb)–[54]FB1scFvHumansyntheticAblibraryETH-2ImmobilizedFB1–biotinSPR4.
08*10–7ND–[71]FB1scFvLibraryderivedfromfouranti-FB1mAbs(1.
3*106)ImmobilizedscFv,FB1–HRPAbsorbance1.
20*10–9IC50=0.
11μM(79.
4ng/mL)Maize,rice[55]FB1scFvImmunizedlibraryfrommice(3.
4*107)ImmobilizedFB1–BSA,solublescFv,anti-His-Ab+anti-IgG–APAbsorbance1.
89*10–7ND–[64]OTAVHHImmunizedlibraryfromalpaca(2*107)ImmobilizedOTA–OVA,phage-displayedVHHPD-IPCRNDLOD=3.
7pg/LLinearrange0.
011000pg/mLCorn,wheat,rice[67]OTAVHHfrom[67]–ImmobilizedOTA–OVA,VHH–APfusionFluorescenceNDLOD=0.
13ng/mLIC50=0.
04ng/mLLinearrange0.
060.
43ng/mLRice,oats,barley[75]OTAFabMulti-immunizedlibraryfrommice(4*108)ImmobilizedOTA–HSA,solubleFab,anti-IgG–APAbsorbance34*10-9IC50competitiveassayformatthatrequiresconjugationofthetoxintoalabeloracarriermolecule.
Despitetheirwideuse,suchassayshavemanyfundamentalproblemsinrespecttothespecificityandsensitivity.
Asthesensitivityofcompetitiveassaysismainlygovernedbytheequilibriumconstantoftheantibody,theseassayscannotfullyexploitdifferentalternativesintheassaydesignthatcansig-nificantlyincreasethesensitivityinnoncompetitiveimmuno-assays[82].
Insomecases,conjugationoftheantigencanbedifficultorresultinrandomlycross-linkedorunstablemole-cules,whichcanreducetheantibodybinding.
Consequently,thenoncompetitiveorsandwich-typeimmunoassaysareusu-allyconsideredsuperiortothecompetitiveassaysandholdgreatpotentialforincreasedsensitivityandwiderdynamicrange.
Conventionally,suchanoncompetitivetwo-siteassayformatrequiresthattheantigenhastwoseparateepitopeswheretwoantibodiescanbindsimultaneously,formingtheBsandwich^complex[26].
Mycotoxinsaresmallmolecules,orhaptens,whichpossessonlyoneepitopeandcannotbindmorethanoneantibodysimultaneously.
Thatsaid,recombi-nantantibodytechnologyhasmadeitpossibletodevelopnonconventionalantibodiesthatcanbeusedtodetectalsohaptensinnoncompetitiveassays.
Opensandwichimmunoassay(OS-IA),originallyde-scribedbyUedaetal.
[83]forthedetectionoflysozyme,isbasedonassociationofseparatedVHandVLchainsinthepresenceoftheantigen.
OS-IAisanexcellentexampleofawaytodetectsmallmoleculesinanoncompetitiveassay,andhasbeenreportedtooutperformcompetitiveassayintermsofsensitivity,workingrange,andassaytime[84].
However,inanycasethedetectionsensitivitydependsstronglyontheaffinityoftheantibodyusedandthedifferentialinteractionsbetweenseparatedVHandVLchainsinthepresenceorab-senceofthetarget.
Moreover,asonlyoneantibodyisin-volved,OS-IAcansufferfromcross-reactivity[84,85].
Suzukietal.
[86]developedanOS-IAforthedetectionofZENbyusingthesplitFvs(VHandVLchains)ofamonoclo-nalanti-ZENinphagedisplayorfusionproteinformats.
Bothnoncompetitiveassaysshowedsuperiorperformancecom-paredwiththecompetitiveassay.
However,despitetheattrac-tivescheme,unfortunatelydevelopmentofOS-IAsisdifficultandtime-consuming,and,tothebestofourknowledge,thisistheonlyreportedOS-IAforthedetectionofmycotoxins.
Thishints,andashasbeenstatedelsewhere[84],thatitisoftenchallengingtofindantibodiesthatpossesstherequiredchar-acteristicswithdifferentialinteractionsbetweenseparatedVHandVLchainsinthepresenceorabsenceoftheantigen,whichistheessenceofthemethod.
Anotheralternativeforsmallmoleculenoncompetitiveim-munoassayistouseanti-immunecomplexantibodies,alsoknownasBanti-metatypeantibodies,^whichbindtothepri-maryantibodyonlywhenitisincomplexwiththeantigen.
Originally,anti-immunecomplexantibodiesweremonoclonalantibodiesdevelopedbyimmunization[87],butlaterphage-displayedlibrarieswithantibodies[88]orpeptides[89,90]wereusedaswell.
Noncompetitiveimmunoassaysbasedonanti-immunecomplexantibodieshavetheaddedadvantageofincreasedspecificityowingtotheuseoftwoantibodiesin-steadofoneasinthecompetitiveassay,aswasseenintheworkofArolaetal.
[91,92].
ThegroupidentifiedfirstHT-2-specificbindersfromanimmunizedphagelibraryand,subse-quently,anti-immunecomplexantibodiesfromanavescFvlibrary.
Althoughtheprimaryantibodyshowedcross-reactivitybetweenthehighlysimilartoxinsT-2andHT-2,theuseofanti-immunecomplexantibodymadetheassayspecificforHT-2toxinonly.
Theantibodiesdevelopedwereusedinahomogeneoustime-resolvedfluorescenceresonanceenergytransferassay(Fig.
3a)[91]andlaterinheterogeneousELISAwithscFv–alkalinephosphatasefusion[92]forthedetectionofHT-2withdetectionlimitsof0.
38and0.
3ng/mL,respectively.
ComparedwiththecompetitiveELISA,thenovelanti-immunecomplexassaywasapproximatelytentimesmoresensitiveinbothcases,underliningthepossibilityforimproveddetectionbythenoncompetitiveassayformat.
EpitopemimicsAsstatedalready,onemajorlimitationofthecompetitiveassayformatistherequirementtoconjugatethetargettoxintoacarriermolecule,usuallyaproteinoralabel,toallowimmobilizationordetectionofthiscompetitor.
Synthesisofthetoxinconjugatescanbedifficultandtime-consuming,orcanresultinrandomlycross-linkedorunstablemolecules,whichcanreducetheimmunoassaysensitivity.
Lot-to-lotvar-iationsoftheconjugates,orevenfalsepositivescausedbythereleaseoftheanalytemoietyfromtheconjugate,areknowntoaffecttheassayreproducibilityandaccuracy[94,95].
Ontheotherhand,labelingthetargettoxinmayaltertheepitopeandthusreduceorevenabolishantibodyrecognition[26,96].
Toovercomethesedrawbacks,apossiblealternativeistodevelopproteinorpeptidesubstitutesthatmimicthetargetmycotoxinandserveasthecompetitorinthecompetitiveimmunoassay.
Suchepitopemimicsbindtothesameantibodyparatopeasthetargettoxinandelicitanantibodyresponsesimilartothatoftheanalyte.
Asepitopemimicscansubstituteforthetoxinsandthetoxinconjugatesusedintheimmunoassay,suchap-plicationscanbeconsideredmorefriendlyfortheuserandtheenvironmentastheassaycomponentsarenottoxicthemselves[94].
Table4summarizessomerecentexamplesoftheuseofepitopemimicsinimmunoassaysforthedetectionofmyco-toxins.
Theseandseveralotherreportshaveshownthatepi-topemimicscanreducethedetectionlimitscomparedwith754PeltomaaR.
etal.
traditionalELISAswiththetoxinconjugateandshowpoten-tialtoreplacetheuseoftoxinconjugatesortoxinstandardsintheassays[18].
Antibodiesthemselves,referredtoasBanti-idiotypicantibodies^orBAb2^(morespecifically,Ab2βandAb2γin-dicatingbindingeitherwithinorclosetotheparatope)[107],canbeusedasepitopemimics.
Thefirstanti-idiotypicantibod-iesforaflatoxin[108,109],FB1[110],andDON[111]werepurifiedfromtheseraofrabbitsimmunizedwiththemonoclo-nalanti-toxinantibodies.
Afterconfirmationoftheirbindingtotheprimaryantibody(Ab1)inELISA,theanti-idiotypicanti-bodieswerealsousedtoproduceanti-anti-idiotypicantibodies(Ab3),whichinsomecaseshaveshownincreasedaffinitycomparedwiththeoriginalantibody[112].
Alsomonoclonalanti-idiotypicantibodieshavebeendeveloped,althoughtheusehasbeenintendedmainlyforvaccinedevelopmentortoxicitystudies[113,114].
Forimmunoassaydevelopment,severalre-combinantanti-idiotypicantibodiesformycotoxinshavebeenselectedfromrandomphagelibraries.
Phagedisplayoffersanexquisitemethodtosearchforepitopemimicsevenwithlittlepriorknowledgeoftheantibody–antigeninteraction,whichisoftenthecaseespeciallyinthecaseofcommercialantibodies.
Astheantigen-bindingsiteofconventionalantibodiesisratherlargeandflat,theyareprobablynotthebestoptiontomimicsmallhaptens,suchasmycotoxins,andallrecentlyre-portedanti-idiotypicantibodiesformycotoxinswereheavychainantibodies(VHH)selectedfromeitherimmunizedornavelibraries.
Incontrasttotherecognitionsurfaceofconven-tionalantibodies,whichiscomposedofsixcomplementarity-determiningregions,heavychainantibodieshaveonlyonevariableregion(VHH),andforthisreasononeofthecomplementarity-determiningregionsisunusuallylongandvariable.
Thisstructureisknowntocreateratherlargeconvexparatopesandallowbetterbindingtocleftsandcavities,whichhasbeensuggestedtobemoresuitableformolecularmimicryofhaptens[26,100,115].
Forexample,Wangetal.
[97]select-edananti-idiotypicVHHfromanimmunizedlibraryforthedetectionofAFB1.
Thenanobody-basedELISAshowedanIC50of13.
8μg/kgforAFB1inspikedsamples,whichwasfurtherreducedfourfoldwithuseofphagedisplay-mediatedimmuno-polymerasechainreactionforthedetectionwiththesamenanobody[98].
Althoughnanobodiesisolatedfromnavephage-displayedlibrariesoccasionallydonothaveenoughaffinitytobeusedastheprimaryantibody,theycanbesuitableasanti-idiotypicanti-bodiesincompetitiveimmunoassay.
Inthiscase,aslightlyloweraffinityoftheepitopemimiccanbeadvantageousasweakeraffinityindicateslessamountoftheanalyteneededtoparticipateinthecompetition,whichleadstohighersensitivities[100,104].
Infact,thenavealpacananobodyphage-displayedlibrary,orig-inallyconstructedbyTuetal.
[99]forscreeningofDON-specificnanobodies,wwidelyusedthereafterforscreeningofanti-idiotypicnanobodiesforthedetectionofseveralmyco-toxins,includingCIT[116,117],DON[95],FB1[100,101],andOTA[104].
Inallcases,theuseoftheanti-idiotypicnanobodyincreasedthesensitivityoftheELISA,atbest20-foldcomparedwiththeassayswiththetoxinconjugates.
Perhapsasimpleralternativetoanti-idiotypicantibodiesistheuseofsmallpeptidesasepitopemimics.
Suchpeptides,alsoknownasBmimotopes,^havebeendevelopedforthemostcommonmycotoxins,includingDON[112],FB1[93,102,103],OTA[105,106,118–120],andZEN[121].
Reportedmimotopesdifferinlengthandstructure;useoflinear7-mer[106,118]and12-mer[93]peptidesaswellascyclicFig.
3aHomogeneousnoncompetitiveimmunoassayforthedetectionofHT-2toxinbasedontheanti-immunecomplexFab,whichbindstotheprimaryantibodywithHT-2toxin.
Fluorescenceresonanceenergytrans-fer(FRET)occursbecauseoftheshortdistancebetweenthetwofluorophores.
bMicroarray-basedimmunoassayforthedetectionoffumonisinB1(FB1)usingbiotinylatedmimotopes.
(aAdaptedfrom[91];badaptedfrom[93])Bioinspiredrecognitionelementsformycotoxinsensors755Table4RecentexamplesofcompetitiveimmunoassaysusingepitopemimicsTargettoxinEpitopemimicLibraryusedAssayformatDetectionAnalyticalcharacteristicsSampleReferenceAFB1Anti-idiotypicVHHImmunizedImmobilizedVHH,anti-AFB1mAb,anti-IgG–HRPAbsorbanceIC50=0.
16ng/mLPeanuts,rice,corn[97]AFB1Anti-idiotypicVHHfrom[97]–Immobilizedanti-AFB1mAb,phage-displayedVHHPD-IPCRLOD=0.
02ng/mLIC50=5.
6μg/kgLR=1.
9730.
75μg/kgCorn,rice,peanut,feedstuff[98]DONAnti-idiotypicVHHNavenanobodylibrary[99]ImmobilizedVHH,anti-DONmAb,anti-IgG–HRPAbsorbanceLOD=1.
16ng/mLIC50=8.
77ng/mLLR=2.
18–62.
25ng/mLCorn,wheat,feedstuff[95]FB1Anti-idiotypicVHHNavenanobodylibrary[99]ImmobilizedVHH,anti-FB1mAb,anti-IgG–HRPAbsorbanceLOD=0.
15ng/mLIC50=0.
95ng/mLLR=0.
27–5.
92ng/mLCorn,rice,feedstuff[100]FB1Anti-idiotypicVHHfrom[100],fusionwithAP–Immobilizedanti-FB1mAb,VHH–APfusionproteinChemiluminescenceLOD=0.
12ng/mLIC50=0.
89ng/mLLR=0.
29–2.
68ng/mLCorn,rice,feedstuff[101]FB1Syntheticmimotopea(WELPTLA)–BSAconjugateCyclic7-merpeptidelibrary(Ph.
D.
-C7C)Immobilizedmimotope,anti-FB1mAb,anti-IgG–HRPAbsorbanceLOD=1.
18ng/mLIC50=6.
06ng/mLLR=1.
77–20.
73ng/mLMaize,feedstuff,wheat[102]FB1Mimotopea(TTLQMRSEMADD)–MBPfusionproteinLinear12-merpeptidelibrary(Ph.
D.
-12)Immobilizedmimotope–MBPfusionprotein,anti-FB1mAb,anti-IgG–HRPAbsorbanceLOD=0.
21ng/mLIC50=1.
26ng/mLMaize,feedstuff,rice[103]OTAAnti-idiotypicVHHNavenanobodylibrary[99]Immobilizedanti-OTAmAb,phage-displayedVHHPD-IPCRLOD=4.
17pg/mLCorn,wheat,feedstuff,rice[104]OTAMimotopea(AETYGFQLHAMK)2nd-generationpeptidelibrary(fromPh.
D.
-12andPh.
D.
-C7C)Immobilizedanti-OTAmAb,phage-displayedmimotope,anti-phage–HRPChemiluminescenceLOD=0.
005ng/mLIC50=0.
04ng/mLLR=0.
006-0.
245ng/mLCorn,rice,instantcoffee[105]OTASyntheticbiotinylatedmimotopea(GMVQTIF)Linear7-merpeptidelibraryPh.
D.
-7Immobilizedanti-OTAmAb,biotinylatedmimotope,SA–HRPAbsorbanceLOD=0.
001ng/mLIC50=0.
024ng/mLLR=0.
005–0.
2ng/mLCorn[106]AFB1aflatoxinB1,APalkalinephosphatase,BSAbovineserumalbumin,DONdeoxynivalenol,FB1fumonisinB1,HRPhorseradishperoxidase,IC50half-maximalinhibitoryconcentration,LODlimitofdetection,LRlinearrange,MBPmaltose-bindingprotein,mAbmonoclonalantibody,OTAochratoxinA,PD-IPCRphagedisplay-mediatedimmuno-polymerasechainreaction,SAstreptavidinaPeptidesequenceinparentheses756PeltomaaR.
etal.
mimotopes[102]hasbeendescribed.
Althoughnoclearcon-sensusinthesequenceofthesemimotopesisseen,evenwithmimotopesforthesamemycotoxin,thepeptidesusuallycon-tainseveralchargedandaromaticaminoacids.
Theuseofmimotopesformycotoxindetectionhasbeendescribedinsev-eralapplications,andsomerecentexamplesarepresentedinTable4.
Thephage-bornepeptideshavebeendirectlyusedinELISAwithcolorimetric[118]andchemiluminescent[105]detection,aswellasindipstick[105]anddot[121]immuno-assays.
Moreover,researchershavedevelopedalternativeBphage-free^approachesbyreplacingthephage-bornepeptidewiththesyntheticorrecombinantcounterpart,thusavoidingtheuseofthephageintheassay.
Asthechemicalsynthesisofshortpeptidesisawell-establishedandwidelyusedmethod,theuseofsyntheticmimotopesisasimplealternativetouseofthephage-displayedpeptides.
Suchphage-freeapplicationshavebeenreportedforOTAwithuseofabiotinylatedmimotopewithstreptavidin-labeledhorseradishperoxidase[106]aswellasforFB1withuseofthesyntheticpeptide–bovineserumalbuminconjugateasthecoatingantigeninpeptideELISA[102]andrecentlywithuseofabiotinylatedmimotopeinamicroarrayformat(Fig.
3b)[93].
Alternatively,recombinantmimotopescanbeexpressedinE.
coliasproteinfusionsandusedtocoattheELISAplatewithouttheneedforfurthercon-jugation.
Forexample,mimotopesforOTAandFB1werefusedwithmaltose-bindingprotein,andafterpurificationtherecom-binantfusionproteinsweredirectlyusedascoatingagentsinELISA[103,120].
Thisapproachcanofferacheaperalterna-tivetothesyntheticpeptidesasevenlargeamountsofthefusionproteincanbeexpressedinbacteriacost-effectively;however,theprocessrequirescloningoftheconstruct,whichcanbetime-consuming.
PeptidesPeptidesareinvolvedinawiderangebiochemicalprocessesandareessentialformanybiologicalfunctions,suchassignal-ing,cellgrowth,andmetabolism[122].
Aspeptidessharethesamechemicalstructureasproteins,theycanbeconsideredasanattractivebioinspiredrecognitionelementtoreplaceanti-bodies.
Smallpeptidesarestableinawiderangeofconditionsandareeasytosynthesize,optionallywithdifferentmodifica-tionsortagsforimmobilizationorlabeling[123,124].
Modelingofshortpeptidesisrelativelyeasy,andforscreening,bothmolecularbiologyandchemicaltechniquesareavailable[125].
Furthermore,peptidebinderscanbeobtainedalsofortargetsthataredifficultforantibodies,suchastoxicmoleculesortargetswithlowimmunogenicity[41].
Yet,onlyafewpep-tideshavebeensuccessfullyusedasrecognitionelementssincethedesignofnewpeptidereceptorswithhighaffinitiesischal-lengingbecauseoflimitedunderstandingofinteractionsin-volvedinthemolecularrecognition[126].
Peptidescanbederivedfromnaturalsources(e.
g.
naturallyoccurringpeptidehormones),fromgeneticorrecombinantli-braries,orfromchemicallibraries.
Theycanbeselectedfromcombinatoriallibrariesthathavebeensynthesizeddirectlyfromthemonomericcomponents,inthecaseofpeptidesfromaminoacids.
Aschemicalsynthesishasaccesstoawiderdiversityofthestartingcomponents,combinatoriallibrariescanbecon-structedwiththeuseofnotonlynaturalbutalsounnaturalaminoacidsorpseudo-peptidebonds[126].
Theadvancesinbioinformaticsandcomputationalmethodshavealsomadeitpossibletodesignpeptidereceptorsinsilico.
Molecularmodel-ingcanbeusedtoobtainstructuralinformationaboutthetargetmoleculethatcanthenbeappliedtodirectthedesignofcom-binatoriallibraries,ortocompletelyrationallydesignartificialreceptors[126].
Alternatively,peptidescanbeselectedfromphage-displayedlibrariesthatarebasedonphagevectorsfordisplayingpeptidesasafusionwithoneofthephagecoatproteins[59].
Theadvantageofphagedisplaytechnologyisthefullyrandomnatureofthelibraries,whichdonotcontainrationallydesignedstructures,andthesamelibrarycanbeusedessentiallyforanytarget[125].
However,sofarallthereportedphage-displayedpeptidesformycotoxinanalysishavebeenusedasmimotopes(seeBEpitopemimics^)ratherthanastheprimaryrecognitionelement.
Thefewexamplesofpeptide-basedrecognitionofmyco-toxinsfoundintheliteratureinclude,forexample,thedevel-opmentOTA-bindingpeptideNFO4,whichwasderivedfromaspecificregionofhumanoxidoreductase.
SyntheticNFO4wasusedwithhorseradishperoxidaseconjugatedOTAinacompetitiveELISAwhichshowedanIC50of3.
2μg/L[127].
Laterthedetectionlimitwasslightlyreducedbyimmobiliza-tionofthepeptideonthree-dimensionalporouschitosansup-portsinsteadofamicrotiterplatewell[128]orbyuseofanamperometricsensorasthetransducer[129].
AlsoHeurichetal.
[130]designedanOTA-bindingpeptide,thistimebycomputationalmodeling.
InSPRanalysisthesedenovode-signedpeptidesshowedbindingtowardOTA–bovineserumalbuminconjugatewithKDof11.
8–15.
7μM;however,com-petitiveassaywithfreeOTAwasnottested.
SomegroupshavealsoidentifiedpeptidesagainstOTA[131]andaflatoxins[132]bycombinatorialsynthesis.
Thesepeptidesshowedde-centaffinitiesforthemycotoxins,Keq=103–104M1,whichwassufficienttoretainthetargetinsolution,andthechemi-callysynthesizedpeptidescouldbeusedforsolid-phaseex-tractionofthetoxins;however,suchlowaffinityconstantsareusuallynotsuitableforsensordevelopment.
AptamersTheetymologicalmeaningofBaptamer^referstotheLatinwordforBtofit^,aptus,suggestingtherelationshipbetweenaptamersandtheirtargetfollowingtheBlock-and-key^theory.
Bioinspiredrecognitionelementsformycotoxinsensors757Aptamersaresmall(usuallyfrom2to60nucleotides)single-strandedRNAorDNAthatcanbindspecificallytodiversetargets,includingions,peptides,proteins,cells,antibodies,andorganicmolecules.
Inasimilarway,aptazymes(RNAzymesandDNAzymes)areengineeredaptamerswithallostericpropertiesthatcombineatarget-binderstrandandanenzymestrand.
Aptamersforspecifictargetscanbeobtainedbythescreeningofoligonucleotidelibraries(1014–1015vari-ants)throughtheprocessofsystematicevolutionofligandsbyexponentialenrichment(SELEX).
SincetheinventionofSELEXmorethan25yearsago,themethodhasevolvedconsiderably.
Forexample,mostaptamersselectedforsmallmoleculesuntil2007consistedofRNA,butinrecentyears,useofDNAaptamershasincreased,giventheirstabilitytonucleasedigestion[133].
Inaddition,theSELEXprocesshasevolvedtoavarietyofmodifiedapproachesthatallowselec-tionofaptamerswithbetterspecificityandbindingefficiency(Fig.
4)[134].
Newaptamer-relateddataarepublishedalmosteveryday,andthusonlinedatabasesareavailabletoprovideaccessto,forexample,specificaptamerapplicationsorclas-sification.
Aninterestingexampleisthewebsitehttp://www.
aptagen.
com/aptamer-index/aptamer-list.
aspx.
Biosensorsusingaptamersasbiorecognitionelements,alsoreferredasBaptasensors,^werefirstdescribedin1996[133–135],andhavesincebeenusedinvarioussensingap-plications.
Aptamerscanprovidehighstabilityandaffinity,aswellassimplicity,lowcostandexcellentbatch-to-batchreproducibility.
Particularly,aptasensorshaveattractedhugeattentionintheanalysesoffoodcontaminants,suchasmyco-toxins,owingtotheinherentadvantagescomparedwithotherbiorecognitionelements,especiallytheirexcellentbindingconstantsformostmycotoxinsstudied,withdissociationcon-stants(KD)inthenanomolarrange(Table5).
Nonetheless,aptamers,especiallyRNAaptamers,arehighlysensitivetonucleases,andmoreover,aptameraffinityisstronglydepen-dentonthebindingconditions.
Immobilizationofaptamersisakeystepinthedesignofbiosensorsasitcanaffecttheaffinityoftheaptamerforitstargetandalsoitslong-termstabilityforrealsampleanalysis.
Inrecentyears(2012–2017),mostlyallimmobilizationstrat-egiesusedforaptasensordevelopmentwerebasedon(1)ad-sorptionorπ–πstackinginteractionsbetweentheDNAbasesoftheaptamerandgrapheneoxide(GO)-modifiedinterfaces[140],(2)covalentlinkageoftheaptamertocarboxylicacidgroupspresentonasurfaceornanomaterial[145],(3)bindingofthiolatedaptamerstoCdTequantumdots(QDs)orAu-basedmaterials[146],(4)affinitybindingbasedonbiotin–streptavidinorotheraffinityinteractions[147,148],or(5)hybridizationtoapartiallycomplementarysingle-strandedDNA,previouslyimmobilizedonasurfaceorananoparticle[149–151].
Inthesamewayasimmunosensors,aptasensorshavebeenusedinseveraldifferentsensingschemestotransducetherecognitionprocess,suchasdirect,competitive,displacement,Fig.
4Inthesystematicevolutionofligandsbyexponentialenrichment(SELEX)process,aninitialpoolof1014–1015randomsingle-strandedDNAorRNAmoleculesissubjectedtobindingwiththetarget,andtheelutedprobesareamplifiedbypolymerasechainreaction(PCR).
Theselectionprocessisrepeated6–15timeswithamplifiedoligonucleotides(ONTs)asthenewpool.
DifferentSELEXmodificationscanbeperformedtoobtainaptamerswithhighspecificityandaffinity;forex-ample,toggle-SELEXcanperformselectionswithtwodifferenttargetmoleculestoobtainbispecificaptamers.
RTreversetranscription.
(Reproducedwithpermissionfrom[134],copyright2013,KoreanSocietyofAppliedPharmacology)758PeltomaaR.
etal.
andsandwichassayformats[152].
Inrecentyears,awidevari-etyofnovelmethodsincombinationwithnewtransductionapproachesorinnovativeamplificationtechniques(Fig.
5)havebeenreported[153,154].
Forexample,isothermalamplificationofnucleicacidshasemergedasapromisingalternativeforaptasensors[154].
Indeed,thereareanincreasingnumberofbiosensorsforawidespectrumoftargets(smallmolecules,ions,proteins,etc.
)thathaveincreasedtheirsensitivitybyincorpora-tionoftheisothermalamplificationdetectionmode[155].
Someexamplesofrecentlyreportedaptasensorsformyco-toxindetectionaredescribedinTable6.
Morethan40%ofthemycotoxinaptasensorsreportedinthelastcoupleofyearsarebasedonfluorescence,andamongthem,oneofthemostusedstrategiesisbasedontheuseofmetalandcarbonnanostruc-tures,suchasgoldnanoparticles(AuNPs),GO,single-walledcarbonnanotubes,MoS2flakes,orTiO2tubes.
Manyoftheseexamplesmakeuseofthestrongquenchingoffluorescentaptamerconjugatesadsorbedonthesesurfacesbyπ–πstack-inginteractions[158–160].
Forexample,Lvetal.
[160]fab-ricatedasensitiveaptasensorforOTAdetectionusingsingle-walledcarbonnanohorns(SWCNHs)asefficientquenchers,whichprovidedcompletelossoffluorescenceintensityoftheunfoldedfluorescein(FAM)-labeledOTA–aptamerwhenabsorbedontothecarbonnanohorn.
AdditionofOTAresultedinanaptamershapechangetoaG-quadruplexthatwasfurtherreleasedfromtheSWCNHsurface,withacorrespondingin-creaseinthefluorescence,proportionaltothetoxinconcen-tration.
Theapproachincludedaself-amplifyingcyclethattookadvantageoftheabilityofDNaseItodigestthereleasedaptamerboundtoOTA,liberatingthefreetoxin,whichcouldrebindanewaptameradsorbedontotheSWCNHs.
Withthisstrategy,thesensitivityoftheassaywasincreased20-foldcomparedwiththeunamplifiedapproach,withadetectionlimitof9.
8nM.
Inasimilarway,Zhangetal.
[161]proposedanaptasensorforthedetectionofAFB1usingthesameamplificationsystembasedonDNaseI.
Inthiscase,threedifferentsizesofnano-GOswereusedtoadsorbcarboxyl-X-rhodamine(ROX)la-beledAFB1aptamer,actingasROXquenchersbutalsoasscaffoldstoprotecttheaptamerfromnucleasecleavage(Fig.
6a).
Differentdynamicrangeswereobtaineddependingonthegraphenesizes;GOwithasizeof1000–2000nmhadadynamicrangefrom12.
5to312.
5ng/mL,GOwithasizeof60–80nmhadadynamicrangefrom1.
0to100ng/mL,andGOwithasizeof4–6nmhadadynamicrangefrom5.
0to50ng/mL.
Thesensorprovedtobehighlyselectivewhentestedagainstothermycotoxins,andgoodresultswereobtainedwiththeaptasensorinAFB1-spikedcornsamples.
Daietal.
[163]developedaluminescenceresonanceener-gytransferassayusingcore–shellβ-NaYF4:Yb,Er@NaYF4upconvertingnanoparticlescoatedwithavidin,whichbindstoabiotinylatedOTAaptamer,asenergydonorsandGOactingasanenergyacceptor.
Adynamicrangefrom0.
001to250ng/mLwasreportedforOTA,andtheassayshowedgoodspec-ificitytowardthetoxininanalysisofbeersamples.
Althoughfluorescence-basedmethodscanprovidein-creasedsensitivity,colorimetricmethodsusuallyprofitfromsimplicitywhilemaintainingtherequiredsensitivity.
TheTable5Dissociationconstants(KD)ofsomeaptamersselectiveformycotoxinsTargettoxinKD(nM)AptamertypeBindingconditions/bufferLength/CGcontentReferenceAFB111.
39DNApH7.
0,100mMNaCl,20mMTris–HClpH7.
6,2mMMgCl2,5mMKCl,1mMCaCl2,0.
02%Tween2080/55.
0%[136]AFB29.
83DNApH7.
0,100mMNaCl,20mMTris–HClpH7.
6,2mMMgCl2,5mMKCl,1mMCaCl2,0.
02%Tween2080/56%[137]AFM10.
0182DNANS21/38.
1%[138]Ergotalkaloid44DNA100mMNaCl,20mMTris–HClpH7.
6,5mMKCl,2mMMgCl2,1mMCaCl280/62.
5%[139]FB1100DNA100mMNaCl,20mMTris,2mMMgCl2,5mMKCl,1mMCaCl2,pH7.
696/33.
3%[140]FB2NDDNANS80/55%[141]OTA96DNAPBS1*,1mMMgCl2,0.
01%Tween20,pH7.
466/63.
6%[142]T-2toxin20.
8DNA10mMTris–HCl,150mMNaCl,10mMKCl,2.
5mMMgCl2,pH7.
440/45.
0%[143]ZEN40DNApH7.
4,100mMNaCl,20mMTris–HCl,2mMMgCl2,5mMKCl,1mMCaCl2,0.
02%Tween2040/50%[144]AFB1aflatoxinB1,AFB2aflatoxinB2,,AFM1aflatoxinM1,FB1fumonisinB1,FB2fumonisinB2,NSnotstated,OTAochratoxinA,PBSphosphate-bufferedsaline,Tristris(hydroxymethyl)aminomethane,ZENzearalenoneBioinspiredrecognitionelementsformycotoxinsensors759detectionmechanismofwidelyusedAuNPsisbasedontheirremarkablyhighextinctioncoefficientandtheirstrongcolordependenceonaggregation/disaggregation.
Recently,severalauthorshavereportedinnovativecolor-baseddetectionsys-temsthatintegrateAuNPswithaptamersformycotoxinanal-ysis[164].
Forexample,Chenetal.
[162]reportedanaptasensorforthedetectionofAFB1basedonacatalyticDNAcircuitandAuNPs.
Thesignalamplificationwasdonewithoutenzymesandwasbasedinatoehold-mediatedDNAstranddisplacementthatoccurredatroomtemperature[165].
ThedesignstrategyfortheAFB1aptasensorisillustratedinFig.
6b.
TheAFB1aptamer,includedinT,waspartiallyhybridizedwithacomplementaryDNA(cDNA)(BinFig.
6b)thatcagesthetoeholddomain(a*inFig.
6b).
Whenthetoxinispresent,a*isliberated,andtheamplificationprocessisactivated.
Itinvolvesthesequentialopeningofthethreebiotinylatedhairpins(H1,H2,andH3),andthusthefreeingofthetoeholddomainsH1-b*,H2-c*,andH3-a*.
Then,abranchmigrationisinitiatedtoformaT–H1–H2–H3complexthatisunstable,andTdissociates,triggeringthehybridizationofadditionalhairpins.
Inparallel,thetriplexH1–H2–H3prod-uctsinteractwithAuNP–streptavidinconjugatestoformacross-linkednetworkofhighlyaggregatedAuNPs.
ThebluecolorobtainedbecauseoftheredshiftingcanbevisualizedFig.
5Someexamplesofsensingschemesforopticalaptasensorsusingfluorescentreportersorgoldnanoparticles(GNPs).
aQuenchingaptamerbeacon.
bFluorescenceresonanceenergytransferaptamerbeacon.
cAssemblyaptamerbeacon.
dDisassemblyaptamerbeacon.
eAptamerreleaseandGNPaggregation.
f–gAffinity-mediatedaggregation/disaggregation.
Ffluorophore,FAacceptorfluorophore,FDdonorfluorophore,Qquencher.
(Adaptedfrom[153])760PeltomaaR.
etal.
Table6RecentexamplesofaptasensorsformycotoxinanalysisTargettoxinRecognitionelementAssayformatSensingschemeDetectionAnalyticalcharacteristicsSampleReferenceAFB1AFB1–DNAaptamerDisplacementassayandamplificationwithahairpin/Exo(III)systemAnalytebindingtotheaptamerreleaseshybridizedcDNA,whichself-hybridizestoahairpinandisdigestedbyExo(III)SERSLOD=0.
4fg/mLLR=10–61ng/mLPeanut[149]AFB1QD–AFB1–DNAaptamerDisplacementassayandQDaptamer/AuNPsystemAnalytebindingtotheaptamer–QD(adsorbedontoAuNP)promotesQDaptamerdisplacementandrecoveryoftheemissionFluorescence(quenching)LOD=3.
4nMLR=10–400nMRice,peanut[145]AFB1AFB1–DNAheminaptazymeDisplacementassayandenzymaticassayAnalytebindingpromotesopeningoftheaptazymecomplexandlossoftheperoxidaseactivityAbsorbance(ABTS)LOD=0.
1ng/mLLR=0.
1–104ng/mLCorn[156]AFM1BiotinylatedAFM1–DNAaptamerDisplacementassayandcDNAmonitoringAnalytebindingtoimmobilizedaptamerreleasescDNA,whichisamplifiedbyRT-qPCRFluorescenceLOD=0.
03ng/LLR=10-61ng/mLInfantmilkpowder,ricecereal[147]AFM1AFM1–DNAaptamerDirect(label-free)IDEcoveredwithFe3O4/PANIfilmandcoatedwithaptamerElectrochemicalcyclicandsquarewavevoltammetryLOD=1.
98ng/LLR=660ng/L–[138]FB1FB1–DNAaptamerDirect(label-free)AuNPcoatedwithFB1aptameranddepositedinanSPCESPCE/EISLOD=3.
4pg/mLLR=10pg/mL–50ng/mLCorn[157]FB1ThiolatedFB1–DNAaptamerDirect(label-free)CantileveractivatedwithSAMsofthiolatedFB1aptamerMicrocantileverarrayinstaticmode(DSS)LOD=33ng/mLLR=0.
1–40μg/mL–[146]OTABiotinylatedOTA–DNAaptamerCompetitiveassayOTAaptamerisconjugatedtoSA-IgG,competitiveassaybetweenFAM-labeledOTAandfreeOTAFluorescence(FP)LOD=3.
6nM(buffer)LOD=2.
8nM(whitewine)Whitewine[148]OTAFAM–OTA–DNAaptamerDisplacementassayandFAM-labeledaptamer/AuNPsystemAnalytebindingreleasestheFAM-labeledOTAaptamer(absorbedontoAuNP)andtheemissionisrecoveredFluorescence(quenching)LOD=2.
27nMLR=25–300nMCorn,flour,beer,coffee[158]OTAGQD–OTA–DNAaptamerDisplacementassayAnalytebindingtoGQD@aptamertriggersdisaggregationfromaggregatedGQD@cDNAandtheemissionisrecoveredFluorescence(quenching)throughaggregationLOD=13ng/mLLR=0–1ng/mLRedwine[151]ABTS2,2′-azino-bisbis(3-ethylbenzothiazoline-6-sulphonicacid),AFB1aflatoxinB1,AFM1aflatoxinM1,AuNPgoldnanoparticle,cDNAcomplementaryDNA,DSSdifferentialsurfacestress,EISelectrochemicalimpedancespectroscopy,Exo(III)exonucleaseIII,FAMfluorescein;FB1fumonisinB1,FPfluorescencepolarization,IDEinterdigitatedelectrode,LODlimitofdetection,LRlinearrange,OTAochratoxinA,QDquantumdot,GQDgraphenequantumdot,PANIpolyaniline,RT-qPCRreal-timequantitativepolymerasechainreaction,SAstreptavidin,SAMself-assembledmonolayer,SERSsurface-enhancedRamanscattering,SPCEscreen-printedcarbonelectrodeBioinspiredrecognitionelementsformycotoxinsensors761withthenakedeye,allowingthequantificationofAFB1.
Thelimitofdetectionwas2pM,andtheaptasensorperformancewasvalidatedbytheanalysisofthemycotoxininricesam-ples,withrecoveriesrangingfrom90%to112%.
Severalmycotoxinaptasensorshavealsobeendescribedthatuseelectrochemicaldetection,whichhassomeparticularadvantages,includinglowcost,highsensitivity,orpossibilityofmicrofabrication.
Forexample,Huangetal.
[166]de-scribedanelectrochemicalaptasensorbasedonsignalen-hancementwitharollingcircleamplification(RCA)system.
Thedesignedprimerwascomposedofanaptamersequence,selectiveforOTA,andacDNAsequence,complementarytothecaptureprobeimmobilizedonthegoldelectrodesurface.
IntheabsenceofOTA,theaptamer–cDNAprimerwaspar-tiallyhybridizedtothetemplatepadlockand,underRCAconditions,itssizeincreasedexponentially.
Moreover,itwasalsoenrichedinguaninenucleotides,wheretheredoxprobemethyleneblueboundspecificallyandproducedasignificantsignalenhancementinthedifferentialpulsevoltammetry.
InthepresenceofOTA,theaptamer–cDNAprimerwasreleasedfromtheRCApadlocktobindthemycotoxin.
ThisinducedinhibitionofprimerprolongationundertheRCAsystem,yieldingareductionoftheredoxsignal.
TheresultingelectrochemicalaptasensorcoulddetectOTAwithadetectionlimitof0.
065pg/mL,andwasappliedtotheanalysisofwhitewinesamples,showingrecoveriesintherangeof102–104%.
SPRisalabel-freetechniquebasedonrefractiveindexchangesofadielectricmaterialatthemetal–dielectricinter-face.
BiosensorsbasedonSPRarewidelyusedformycotoxinanalysisbecauseoftheirgreatfeaturessuchassensitivityandreal-time,label-free,andcost-effectivedetection[47].
Recenteffortshavefocusedonthedevelopmentofportableandmin-iaturizedSPRdevices.
Forexample,Biancoetal.
[167]de-velopedaplasmonicportableaptasensorforOTAdetectionbasedonazimuthallycontrolledSPRunderphaseinterroga-tion[168].
Thesystemhadarefractiveindexoneorderofmagnitudegreaterthanthatoftheclassicgrating-basedSPRsetup.
Thelabel-freeassayformatwassimple:athiolatedDNAaptamerwasimmobilizedonthephase-interrogationSPRchip,formingself-assembledmonolayers,andthesignalwasproportionaltotheamountofOTAboundtoit.
Theaptasensorwasoperativeinadynamicrangefrom0.
2to40ng/mLandexhibitedadetectionlimitof0.
005ng/mL.
Biancoetal.
suggestedthatthiscustomapproachcouldbeimple-mentedsooninaminiaturizeddevicewhilemaintainingitsexcellentanalyticalcharacteristics.
Fig.
6aDetectionofaflatoxinB1(AFB1)usingaDNAaptamerandgrapheneoxide.
EachAFB1aptamerbindstoonlyasingleAFB1moleculeandthecomplexisreleasedfromthegrapheneoxidesurface.
Foramplificationassay,theAFB1isregeneratedbyaself-amplifyingcyclebasedonDNaseI.
bDesignstrategyforamplifieddetectionofAFB1basedonacatalyticDNAcircuitandgoldnanoparticles.
Biotinylatedhairpins(H1,H2,andH3)areusedinthesensingsystemforsignalamplificationandstreptavidin-functionalizedgoldnanoparti-cles(Au-SA)areusedascolorimetricprobes.
ArrowsdrawnonDNAstrandsrepresentthe3'end.
Toeholdsandtoeholdbindingdomainsarenamedbylettersandcomplementarityisdenotedbyasterisks.
ROXcarboxyl-X-rhodamine.
(aReproducedfrom[161]withpermissionoftheRoyalSocietyofChemistry;breproducedfrom[162]withpermis-sionoftheRoyalSocietyofChemistry)762PeltomaaR.
etal.
MolecularlyimprintedpolymersMIPshaveshownagreatpotentialasbioinspiredrecognitionelementsforsensordevelopment.
Theseartificialmaterialsareabletorecognizeaparticulartargetincomplexmixturesbe-causeofthepresenceofspecificrecognitionsites,forbindingorcatalysis,withshapeandgeometryofthefunctionalgroupscomplementarytothoseinthetemplatemolecule.
Forpoly-merpreparationthetemplatemolecule,namely,thetargetan-alyteorasurrogatemolecule,interactsbycovalentornoncovalentbondingwiththefunctionalmonomers.
Radicalpolymerizationinthepresenceofacross-linkerresultsinathree-dimensionalnetworkthatontemplateremovalwillcon-tainspecificrecognitioncavitieswithsize,geometry,andar-rangementoffunctionalgroupscomplementarytothoseofthetargetcompound,thusmimickingthebiologicalactivityofnaturalreceptors.
Templateextractioniscriticalforapplica-tionofMIPsasrecognitionelementsinsensors.
Bleedingofthenon-washed-outtemplatecancausefalsepositives,inac-curaciesintheanalysis,andincreasedlimitsofquantification.
Theuseofatemplatesurrogateinsteadofthetargetcompoundmayavoidthislimitation;however,itwillnotpreventthedecreaseinthebindingcapacityoftheMIPifitsextractionfromthebindingsitesisnotquantitative[169].
OvertheyearsMIPshavebeenappliedinvariousfields,includingsolid-phaseextraction,chromatography,drugdeliv-ery,bioremediation,controlledrelease,andsensors[170–177].
MIPsareoftendescribedasartificialantibodiesorartificialenzymes[178],andincomparisonwiththeirbio-logicalcounterpartstheyshowseveraladvantagesassensorreceptors,including,butnotlimitedto,theirhighphysicalandchemicalstability,robustness,compatibilitywithorganicsol-vents,lowcost,easeofpreparation,reusability,andavailabil-ityindifferentphysicalformatsforcouplingtothetransducer[179].
However,theiraffinityandspecificityaretypicallyworsethanforantibodies,andtheimprintedcavitiesareusu-allyheterogeneous,showingadistributionofbindingcon-stants,andslowerbindingkineticsthanbiologicalreceptors.
Incontrasttobiologicalreceptors,insomecasestheyshowlimitedrecognitioninaqueoussolutions,althoughextensiveresearchinrecentyearshasallowedsomeoftheaforemen-tionedshortcomingstobeovercome[180].
RecentadvancesinthedevelopmentofnanosizedMIPs,aloneorincombina-tionwithmetalnanoparticles,haveopenednewperspectivesintheapplicationofthesematerialsforsensingpurposes,giv-enthat,amongotheradvantages,theyallowhigherbindingcapacities,fasterbindingkinetics,andeasiercouplingtothetransducersurfacethantraditionalMIPs[175].
SeveralexamplesoftheapplicationofMIP-basedsensorsformycotoxindetectionhavebeenreportedinthelast5years(Table7).
Thewebsitehttp://www.
MIPdatabase.
comcollectsalltheinformationrelatedtothistopic.
Forexample,AFB1wasdetectedwithuseofMIPfilmspreparedbyelectropolymerizationofp-aminothiophenol-functionalizedAuNPsandp-aminothiophenolself-assembledonthesurfaceofagoldelectrodeinthepresenceofthetoxinasthetemplatemolecule.
Recognitionwasattributedtotheformationofπ–πinteractionsbetweenAFB1andtheanilinemoietiesintheimprintedmaterial.
Theresponsewaslinearintherangeof3.
2f.
to3.
2μMAFB1,andthedeviceshowedsomecross-reactivitywithOTA,butthiswasverylowforaflatoxinB2oraflatoxinG1[181].
Higherdetectionlimits(3*1011mol/L)havebeenreportedwithelectrochemicalsensorsfabricatedwitho-phenylenediamineasthefunctionalmonomerandAFB1asthetemplateonmultiwalledcarbonnanotubesup-portedAu/Ptbimetallicnanoparticlemodifiedglassycarbonelectrodes(GCEs)[182].
SeveralsensorshavealsobeendescribedthatuseQDsasthelabelsformycotoxindetection;however,somelimitationsrelatedtoQDleaching,retentionofthephotoluminescentpropertiesofthesemiconductoronimmobilization,andana-lytepermeationmustbetakenintoconsiderationtodevelopstableandsensitivedevices.
Afluorescentsensorforthede-tectionofsterigmatocystin(ST),asecondarymetabolitepro-ducedbyseveralAspergillusspecies,thatusedsilica-basedhybridMIPsbasedonthenonhydrolyticsol–gelmethodwasreported[189].
Theorganosilane-functionalizedQDswerecoatedwiththeimprintedpolymerpreparedwithmethacrylicacid(MAA)asthefunctionalmonomer,γ-methacryloxypropyltrimethoxysilaneasthecross-linker,and1,8-dihydroxyanthraquinoneasthetoxinsurrogate.
Thelumi-nescentpropertiesoftheQDswerenotaffectedbyencapsu-lation,andtheyshowedexcellentstabilityagainstphotobleaching;however,theresponsetimeswererelativelylong(4h).
Themeasuringmechanismwasbasedonthere-tentionofthemycotoxin,throughhydrogenbonding,intheselectivecavitiesofthepolymer,whichresultedinelectrontransferfromQDsintheMIPmatrixtotheboundST,withthecorrespondingquenchingoftheluminescence.
Thesensorshowedsomecross-reactivityforOTA,ZEN,andAFB1,anditwassuccessfullyappliedtotheanalysisofSTinmillet,rice,andmaizesamples.
AdifferentapproachwasdescribedforthedetectionofZENthatusedionic-liquid-stabilizedCdSe/ZnSQDs[190].
Thepolymerswerepreparedwithcyclododecanyl-2,4-dihydroxybenzoateasthetemplatesurrogate[191]andMAAasthefunctionalmonomer.
Thesensingmechanismreliedonacharge-transfermechanismbetweentheconduc-tionbandoftheQDandthelowestunoccupiedmolecularorbitalofZEN.
Thesensorwasappliedtothedetectionofthetoxinincorn,wheat,andricesamples,withrecoverieshigherthan84.
4%,andshowednocross-reactivitywithOTAorDON.
PatulinhasbeendetectedwithaMn-dopedZnSQDbasednanosensorsynthesizedwith6-hydroxynicotinicacidasthesurrogate,3-aminopropyltriethoxysilane,asthefunctionalBioinspiredrecognitionelementsformycotoxinsensors763Table7Recentexamplesofmycotoxindetectionmethodsbasedonmolecularlyimprintedpolymers(MIPs)TargettoxinMIPreceptortype/compositionMeasurementtechniqueSampleAnalyticalcharacteristicsReferenceAFB1PATP-functionalizedAuNPselectropolymerizedonPATPself-assembledonAuELSV10mM[Fe(CN)6]3/4inPBS(pH7.
2)RiceLOD=3*1015mol/LDR=3*1015–3.
2*106mol/L[181]AFB1GCEsmodifiedwithPOPD-graftedAu/PtbimetallicMWCNTnanocompositefilmsDPVAqueoussamplescontaining1mMK3[Fe(CN)6]and0.
1MKClHogwashoil,freshrapeseedoilLOD=3*1011mol/LDR=1*1010–1*105mol/L[182]CITCoatingoftheAuEwithAuNP@CMK-3composite.
Self-assemblyofo-ATandHNAontheAuNPs@CMK-3/AuEasapolymerizablemonomerlayer.
ElectropolymerizationQCMWheat,rice,whitericevinegarLOD=1.
8*109mol/LDR=6.
0*109–2.
0*107mol/L[183]DONElectropolymerizationofpyrroleasafunctionalmonomer,tetraethylammoniumtetrafluoroborateastheelectrolyte,andDONasthetemplateonAuchipsSPRDistilledwaterLOD=1ng/mLDR=0.
1–100ng/mL[184]FB1AuNPsspreadonGCEsmodifiedwithRu@SiO2NPsmixedwith1%chitosan.
PolymerizationinthepresenceofMAA,EDMA,andFB1ECLMilk,maizeLOD=0.
35pg/mLDR=0.
001–100ng/mL[185]OTAGCEcoatedwithRu@SiO2NPsmixedwith1%chitosan.
PolymerizationinthepresenceofMAA,EDMA,andOTAECLCornLOD=0.
027pg/mLDR=0.
1pg/mL–14.
76ng/mL[186]OTAGCEmodifiedwithMWCNTs.
ElectropolymerizationwithpyrroleasafunctionalmonomerCVandDPVWine,beerLOD=4.
1nmol/LDR=0.
050–1.
0μmol/L[187]PatulinMn-dopedZnSQDsmixedwith6-HNA,APTESasthefunctionalmonomer,andTEOSasthecross-linkerviaasurfacemolecularimprintingsol–gelprocessPApplejuiceLOD=0.
32μg/LDR=0.
43–6.
50μg/L[188]STOrganosilane-functionalizedQDscoatedwithaMIPpreparedwithMAA,MPTMSasthecross-linker,andDTasthetoxinsurrogateFMillet,Rice,maizeLOD=19.
0μg/LDR=0.
05–2.
0mg/L[189]ZEAIonicliquid(1-vinyl-3-octylimidazoliumhexafluorophosphate)-stabilizedCdSe/ZnSQDspolymerizedinthepresenceofMAA,EDMA,andCDHBasthetemplateFCornflour,riceflour,wheatflourLOD=2*109mol/LDR=3*109–3.
12*106mol/L[190]AFB1aflatoxinB1,APTESaminopropyltriethoxysilane,AuEgoldelectrode,AuNPgoldnanoparticle,o-ATo-aminothiophenol,CDHBcyclododecanyl-2,4-dihydroxybenzoate,CITcitrinin,CMK-3mesoporouscarbonCMK-3,CVcyclicvoltammetry,DONdeoxynivalenol,DPVdifferentialpulsevoltammetry,DRdynamicrange,DT1,8-dihydroxyanthraquinone,ECLelectrochemiluminiscence,EDMAethyleneglycoldimethacrylate,Ffluorescence,FB1fumonisinB1,GCEglassycarbonelectrode,HNA1-hydroxy-2-naphthoicacid,6-HNA6-hydroxynicotinicacid,LODlimitofdetection,LSVlinearsweepvoltammetry,MAAmethacrylicacid,MPTMSγ-methacryloxypropyltrimethoxysilane,MWCNTmultiwalledcarbonnanotube,OTAochratoxinA,Pphosphorescence,PATPp-aminothiophenol,PBSphosphate-bufferedsaline,POPDpolyphenylenediamine,QCMquartzcrystalmicrobalance,QDquantumdot,Ru@SiO2NPtris(2,2-bipyridine)ruthenium(II)-dopedsilicananoparticle,SPRsurfaceplasmonresonance,STsterigmatocystin,TEOStetraethoxysilane,ZEAzearalenone764PeltomaaR.
etal.
monomer,andtetraethoxysilaneasthecross-linkerviaasur-facemolecularimprintingsol–gelprocess[188].
TheMIP-QDsshowedstrongphosphorescencethatwasquenchedafter30minincubationinthepresenceofthetoxin.
ThesensingmechanismwasbasedonphotoinducedelectrontransferfromtheconductionbandsofMIP-QDstothelowestunoccupiedmolecularorbitalofpatulin.
Thesensorwasappliedtothedetectionofthetoxininapplejuicesamples.
FB1wasmonitoredbyelectrochemiluminescencewithuseofGCEscoatedwithAuNPstoamplifytheelectrochemiluminescencesignal,andfurthermodifiedwithafilmformedbytris(2,2-bipyridine)ruthenium(II)-dopedsilica(Ru@SiO2)nanoparticlesmixedwithchitosan[185].
Themy-cotoxinselectivepolymer,preparedwithMAAasthefunction-almonomer,wasdepositedontopandpolymerizedbyUVirradiation.
SignalenhancementintheMIP/Ru@SiO2/chito-san/AuNP/GCEsystemwasattributedtoacombinationofboththelocalizedSPRandelectrochemicaleffectoftheAuNPs.
CIThasbeendetectedwithdisposablefiberopticsensorspreparedfrom4-cm-longinjection-moldedtaperedpolysty-renewaveguidescoatedwithCIT-imprintedparticles(150–500nm)withpolyvinylalcoholusedasaglue[192].
Afluo-rescentmonomer,N-(2-(6-4-methylpiperazin-1-yl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl-ethyl)acrylamide,wasusedforMIPsynthesis.
Anenhancementofthefluores-cenceintensityofthesignalingmonomerwasobservedon30minincubationofthefunctionalizedfiberinmethanolsolutionsofthemycotoxin,andevanescentwaveexcitationat410nm.
Theemittedlightwasguidedbacktothedetectorwithuseofthesamefiber.
Inprinciplethisapproachcouldbeextendedtothedetectionofothermycotoxinsbearingcarbox-ylicacidgroups,suchasOTAorfumonisins,althoughthethicknessoftheMIPlayerrequiresfine-tuningtoincreasethesensitivity.
Inanalternativeapproach[183],CITwasdetectedwithaquartzcrystalmicrobalance.
TheAuelectrodewascoatedwithAuNP@mesoporouscarbonCMK-3(AuNP@CMK-3)andfurthermodifiedapoly(o-aminothiophenol)MIPselec-tivemembrane.
TheincreaseinthenumberofbindingsitesassociatedwiththeuseofAuNP@CMK-3allowedamplifi-cationofthefrequencyresponsesignal.
ThepolymershowedahigheradsorptioncapacityforCITincomparisonwithOTA,DON,AFB1,orZEA,andshowedgoodperformancefortheanalysisofrice,wheat,andwhitericevinegarsamplesspikedwiththemycotoxinintherangeof10–100μgkg-1.
ConclusionsDespitethebestintentionswithregardtoprevention,myco-toxincontaminationisofteninevitable.
Novelanalyticalmethodscanimprovethedetectionandquantificationofthetoxinsandprovidethenecessarytoolstoensurefoodsafety;theuseofsensorsisanattractivealternativeforsensitive,cost-effectiveandfastanalysisofthesenaturaltoxins.
Therecog-nitionelementselectedforsensordevelopmentshouldideallyprovideenoughsensitivityandspecificitytodetectlowamountsofthetargettoxinsevenincomplexsamples,whereseveraldifferenttoxinscanbepresentsimultaneously.
Moreover,fromapracticalpointofview,robustness,stability,andcostcanaffectthechoiceoftherecognitionelement.
Althoughimmunoassaysandbiosensorsbasedonmonoclonalandpolyclonalantibodieshavebeenthecornerstoneofmy-cotoxindetectionforyears,slowlyotherrecognitionelementsarebeginningtoappear,andrecentresearcheffortshaveshownagreatdealofinterestindevelopingnovelbioinspiredrecognitionelementstoovercomesomeofthedrawbacksas-sociatedwithconventionalantibodies.
Theriseofrecombinantantibodieshasbeenwitnessedinseveralfields,includingmycotoxinresearch,owingtotheap-pealingfeaturesofantibodyfragments,includingsmallsize,easyproduction,andthepossibilityofinvitroselection.
Indeed,thetechnologyhasthepotentialtobypassanimalimmu-nization,althoughmostofthereportedrecombinantantibodiesformycotoxinsarosefrommonoclonalantibodiesorimmunizedlibraries.
Thevastpossibilitiesofferedbyantibodyandproteinengineeringallowthegenerationofdifferentantibodyformats,fromthealreadywell-knownscFvtoheterogeneousproteinfu-sionsoranti-immunecomplexantibodies.
Thewidelyhypedantibodyclass,heavychainantibody,anditsrecombinantfrag-mentVHH,ornanobody,havealsobeennotedinthefieldofmycotoxinanalysis.
Becauseofthelimitationofnanobodiestorecognizehaptentargets,mostofthenanobodiesreportedsofarhavebeenappliedasepitopemimics,whichallowtheproblemsrelatedtothetoxinconjugatesusedincompetitiveimmunoas-saystobecircumvented.
Severalanti-idiotypicantibodiesandpeptides,mimotopes,havebeendescribedaswellinrecentyearsformycotoxindetectionwithimprovedperformancecomparedwiththeuseoftoxinconjugates.
Ontheotherhand,aptamerscanofferhighaffinityandspecificity,comparabletothoseofantibodies,withgoodsta-bilityandrobustness.
Althoughseveralnewaptasensorshavebeenreportedintherecentyears,mostofthemtargetingOTAoraflatoxins,asignificantconcernofresearchersinthefieldisthedevelopmentofcomplexarchitectureswithcostlyampli-ficationproceduresthatgreatlylimittheircommercialboost.
Itisnotonlytheachievementofexoticaptasensorsthatmattersnowadaysbutalsothedevelopmentofinexpensiveap-proachesandtheirapplicationtorealsamples.
MIPs,oftendescribedasartificialantibodies,havealsobeenreportedasattractivealternativerecognitionelements.
MIPshaveseveraladvantagescomparedwiththeirbiologicalcounterparts,butoftenarenotabletocompeteinaffinityandspecificity.
However,recentadvancesinthefieldhaveopenednewop-portunities,whichwillallowhigherbindingcapacitiesandfasterkinetics.
Bioinspiredrecognitionelementsformycotoxinsensors765Itremainstobeseenwhetherthesebioinspiredrecognitionelementsareabletocompetewithconventionalantibodiesformycotoxinsensordesign.
Replacementofmonoclonalandpolyclonalantibodiesisunlikely,aswellasunnecessary,butthebioinspiredrecognitionelementscouldbeacomplemen-taryoption,ratherthananalternative,astheycanbemoreappropriateforsomeapplicationswheretheuseofantibodiesislimited.
Itisalsonoteworthythatthenobleintentiontoimproveanalyticalmethodsreliesnotonlyonthechoiceoftherecognitionelementbutalsoonthetransduction(optical,electrochemical,etc.
)andsignalgeneration(labeledandlabel-free)strategiesaswellastheassayplatform.
Novelmaterialscanprovideimprovementsintheassayrobustness,andimple-mentationofsignalamplificationstrategiescanhaveahugeimpactonassaysensitivity.
Ontheotherhand,forexample,forin-fieldapplications,thetimeandcostoftheanalysisareconstraints,andrapidmethodsareneededformycotoxinde-tection.
Currentlysuchtests,forexample,lateralflowassaysandcolorimetricELISAs,arebasedonmonoclonalantibod-ies,buttheuseofbioinspiredrecognitionelementscouldoffersomeimprovements,suchasbetterstabilityandextendedshelflife.
Furthermore,asseveralmycotoxinsareoftenpres-entinthesamefoodsample,multiplexingisakeypointthatshouldbestudied.
Integrationofnovelassayschemes,forexample,onmicrofluidicchipsorarrayplatforms,showsgreatpotentialformultianalytedetectionwithaffordablecost.
AcknowledgementsThisworkwasfundedbytheEuropeanUnion(SAMOSS;FP7-PEOPLE-2013-ITN;contract607590)andMINECO/FEDER(CTQ2015-69278-C2-1-R).
CompliancewithethicalstandardsConflictofinterestTheauthorsdeclarethattheyhavenocompetinginterests.
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