Protein and Peptide Based Biosensors in Artificial Olfaction

Review

Protein-

and

Peptide-Based

Biosensors

in

Arti

_fi

cial

Olfaction

Arménio

J.M.

Barbosa,

Ana

Rita

Oliveira,

and

Ana

C.A.

Roque

*

Animals’ olfactory systems rely on proteins, olfactory receptors (ORs) and

odorant-bindingproteins(OBPs),astheirnativesensingunitstodetectodours.

Recentadvances demonstrate that theseproteins canalso be employedas

molecularrecognitionunitsingas-phasebiosensors.Inaddition, the

interac-tionsbetweenodorantmoleculesandORsorOBPsareasourceofinspiration

for designing peptides with tunable odorant selectivity. We review recent

progressingasbiosensorsemployingbiologicalunits(ORs,OBPs,and

pep-tides)inlightoffuturedevelopmentsinartificialolfaction,emphasizing

exam-pleswherebiologicalcomponentshavebeenemployed todetectgas-phase

analytes.

BiosensorsinArtificialOlfaction

Thepossibilityofmimickingnature,buildingartificialintelligentsystemstoperformcomplex tasks,anddealingwithlargesetsofdataisgainingincreasingrelevanceinallareas,including biologicalsciences[1].Themysteriesofolfaction,inparticularhumanolfaction, stillintrigue scientistsand, notsurprisingly, it is considered the least understoodsense [2,3].Artificial olfactionsystemsaimtomimicthesenseofsmellandtypicallyconsistofelectronicnose

devices(e-noses)(seeGlossary)thatincludeanarrayofgassensorsassociatedwith

signal-processingtools[4–6].Classically,inane-nose,asampleentersthesystemthroughaninlet thatguides thegasmolecules toachamber where thesensing materialisdeposited. The interactionofthevolatileorganiccompounds(VOCs)withthesensingmaterialgeneratesa signal(forexample,electric,optical,orgravimetric)thatistransducedandfurtherprocessedby asignal-processingcomputer.

Odors are typicallycomposed of aset of VOCs.The detection ofVOCs is of significant interest, not only to understand biological processes, but also in several scientific and technologicalareas.Forinstance,VOCsensorshold greatpromise inthe early diagnosis ofdiseases[7–12],foodqualitycontrol[13–15],security[16],agriculture[17],environmental monitoring[18],andinsectsupervision[19,20].Thebroadrangeofapplicationareasfor e-noseshasbeenacceleratingtheprogressofgas-sensingtechnologies,withan extensive researchandmarketspacefordevelopingnewsensingmaterialsanddevices[2].Current gas-sensingmaterials,including thosecommerciallyavailable, includemostlymetal oxide semiconductorsandconductivepolymers.Themaindrawbacksofthesesystemsarethe lowstabilityand highpromiscuitytowardsVOCmolecules,resultinginlow selectivity.As such,incorporatingthesensingcomponentsofbiologicalolfactionsystemsintogas-sensing materialscanincreasethe VOCselectivityoftheresultantgassensorsandbio-electronic noses (Figure 1) [4,6,19–22]. Still, most published works report biosensing using VOC analytesinsolutions[23–27],incontrasttothereal-lifeimplementationofe-nosestoanalyse gaseoussamples. Therefore, thisreview focuses onlyon recent research trends in gas-phasebiosensing.

Highlights

Artificialolfactionisbeingimplemented inkeysocietalareaslikeearlydisease diagnostics,food safetyhazards,air quality,andsecurity.

Protein-andpeptide-basedVOC bio-sensors, coupled with cutting-edge transducers, increase the selectivity andsensitivityindetectingkeyVOCs withlimitsofdetectionintheorderof ppb.

Gas-phasetesting,ratherthanVOC solutions, is becoming the state of theartinbiosensorvalidation.

Combinatorial techniques such as phage display and virtual screening areadvancing thediscoveryofnew VOC-bindingpeptides.

Progressesinbiomolecule immobiliza-tionaresteadilyincreasingthe reusa-bilityandshelf-lifeofbiosensors,while maintainingthedesiredselectivity.

1_{UCIBIO,DepartamentodeQuímica,}

FaculdadedeCiênciaseTecnologia, UniversidadeNovadeLisboa, 2829-516Caparica,Portugal

@_{Twitter:@Biomeng_lab}

*Correspondence:

[email protected]

(AnaC.A.Roque).

1244 TrendsinBiotechnology,December2018,Vol.36,No.12 https://doi.org/10.1016/j.tibtech.2018.07.004

Glossary

Biophotonicsensor:asensorthat detectstheinteractionofphotons andbiomaterials.

Carbonnanotubes(CNTs):carbon atomsarrangedinatube-shaped nanostructure.

Electrochemicalimpedance spectroscopy(EIS):a spectroscopictechniquethat measurestheimpedanceofan electrochemicalsystemtoanapplied potential.

Electronicnosedevices (e-noses):electronicdeviceswiththe purposeofdetectingodorants.

Filmbulkacousticresonator (FBAR):adevicecomprisinga piezoelectricmaterialinserted betweentwoelectrodes,acoustically isolatedfromthecontiguous medium.

Field-effecttransistor(FET):a semiconductorchannelwith electrodesatbothends,denotedas drainandsource.Theconductivity betweenthedrainandsource terminalsiscontrolledbyanelectric

fieldinthedevice.

Homologymodeling:modelinga protein3Dstructurebymeansofa knownexperimentalstructureofa homologousproteinsequence.

Limitofdetection(LoD):the lowestanalyteconcentrationthatcan bereliablydistinguishedfroma blank.

Lipocalin:proteinsthattransport hydrophobicmolecules.

Microcantilever:arigidmicroscale plateanchoredatoneendandfree attheotherendtoallowstructural

fluctuations.Itmeasuresmechanical propertiesofmaterials,suchas resonancefrequency,amplitudeof vibration,andbending.

Moleculardocking:amethodfor predictingtherightorientationofa moleculewhenboundtoareceptor.

Nanodisc:solublenanoscale phospholipidbilayersabletohost membraneproteins.

Odorant-bindingprotein(OBP):

solubleproteinssecretedinthenasal mucusofvertebratesandinthe lymphofinsectchemosensory sensilla.

Olfactoryreceptor(OR):a membraneproteinchannel expressedinolfactoryneurons, triggeredbyodorantbinding. OlfactoryReceptorGas Biosensors

Olfactoryreceptors(ORs)arebelievedtorecognizedifferentodorants,soodorant

recogni-tiondependsontheORbeingactivatedandontheextentofitsactivation[28,29](Box1). IdentifyingwhichodorantsspecificallybindtoacertainORiscalleddeorphanization[30,31]. ThedeorphanizationofORsisacrucialstepto understandseveralaspectsofthesmelling sensoryprocess, includingthe completeknowledge ofanOR’s activity, theimpact ofthe physicochemicalpropertiesofodorantmoleculesinanOR’sselectivity,andthewayinwhich

Pep de Signal transducer Signal processing Olfactory receptor Odorant-binding protein Bioreceptor Electrical Op cal Gravimetric Processing unit Data

ImmobilizaƟon

Covalent

Thiol-based Affinity

S S S

-+

.

.

.

Figure1.SchemeofMainConstituentsofProteinandPeptide-BasedBiosensorsforArtificialOlfaction.A bioreceptorknowntobindtovolatileorganiccompounds(VOCs)–olfactoryreceptors,odorantbindingproteinsorVOC affinitypeptides–isselectedforimmobilization.Surfaceimmobilizationcanbemadethroughdifferentmethodsincluding affinity,covalentspacers,andthiol-basedchemistry.Thebiosensoristhenappliedinasignaltransducingsystemwhich maybe,forexample,electrical,gravimetric,oroptical.Thecollecteddataisfinallyprocessedtoyieldqualitativeor quantitativeinformationabouttargetVOCs.

Box1.DifferencesbetweenProteinsandPeptidesCurrentlyUsedinVOCBiosensing

ThebiomoleculesappliedsofaringasbiosensingincludeORs,OBPs,andpeptides,withremarkablestructural differenceshighlightedinFigureI.

ORsaremembersoftheGprotein-coupledreceptorfamily:theyarestructurallydefinedbyseventrans-membrane

a-helices,about320aminoacidresiduesinlength[29].Theseproteins,uponvolatilebinding,generateasignalthatis transmittedfromtheolfactorysensoryneurons,whereORsarelocated,tothebraininarecognizedpatternthat culminatesinolfactoryperception.

MammalianOBPsbelongtothelipocalinsuperfamilyofproteins,transportproteins[38,41,42]thatare150–160 residuesinlength[43]andhavetypicallipocalin-foldofeightanti-parallelb-sheetsandashorta-helixclosetotheC terminal.Theb-sheetsformanantiparallelb-barrelshapingacentralpocketforligandbinding[44].InsectOBPspresent well-conservedfoldingwithproteinchainsof130–150aminoacids[40].Thetertiaryproteinstructurecomprisesa compactsetofsixa-heliceswithahydrophobiccavity.Additionally,thefoldingisstabilizedbythepresenceofthree disulfidebonds[39,40].ThesedisulfidebondsareconsideredthefingerprintofinsectOBPs.Notwithstandingamino acidvariability,theoverallstructureofinsectOBPsishighlyconserved,evenamongmembersofdifferentordersof insects[39,40,78].

Peptidesrepresentasimpleandlow-costoptionforbiosensors.Rangingfromapproximately5to15proteinresidues, theycanbesyntheticallyorbiologicallyproduced.TheirselectivitytowardstargetVOCsiseasiertotuneduetotheir smallsize[20,56].VOC-sensingpeptideshavebeendevelopedbydifferentapproaches(Box2,Figure2),andappliedin severalsubstratesandsensingdevices,assummarizedinTable2.

informationisaccuratelyprocessedtogeneratetherecognizedpatternthatculminatesinthe finalolfactoryperception[31].Thesestudiesmusttakeintoaccounttheabilityofanodorantto berecognizedbymultipleORs,andthefactthataspecificORcanrecognizeseveralodorants. SomeORshavealreadybeenisolatedandappliedtogas-sensingbiosensors(Table1).

Thefirstdevelopmentofgas-phasebiosensorsforVOCdetectionusedthereceptorOR-10

fromCaenorhabditiselegans.ThisproteinwasexpressedinEscherichiacoli,anditsmembrane

fractioncontainingOR-10coatedonaquartzcrystalforquartzcrystalmicrobalance(QCM)

measurements.Thebiosensorrespondedtodiacetylwithadetectableconcentrationaslowas 10 12M[32].However,usingacrude membraneextractonthe sensorsurfacemay have distortedtheresponseofOR-10toVOCsduetothe presenceofthelipidicfractionofthe membrane,asphospholipidsarealsoknowntobindodorant-likemolecules[33].Still,these reportshighlightedthepotentialofORsingassensingandthehurdlesofdealingwithcellular membranesforORstabilizationandimmobilization.Asimilarapproach,expressingOR-10in humanbreastcancerMCF-7cells,assessedtheinfluenceofVOCsbindingtophospholipids, usingcontrolsensorsanalysedinparallel[34].Thecontrolsproducedverylow-frequencyshifts

inasurfaceacousticwave(SAW)asaresponsetothetestedVOCs(diacetyl,butanone,and

2,3-pentanedione).Moreover,theresponsetodiacetylwasthemostsignificantwithalimitof

detection(LoD) of 1.210 14M [32]. The use of a control sensor elucidated that the

contribution ofthe affinityof VOCsto phospholipid molecules isnegligible compared with theiraffinitytoORs,consolidatinganewstrategyforVOCsbiosensordevelopment.

AdifferentmethodologyforincorporatingORsinagassensorwasexploredusingmouseORs (mOR)innanodiscs[35].NanodiscswithmORswerecoupledtocarbonnanotubes(CNTs) viaHis-taginteraction.IncontrastwithotherexampleswheretheORswereinthemembrane fraction[32–34],inthiscasethenanodiscsweredepositedonthesensorsurface.Tocertifythe efficiencyofthenanodiscsensors,thesamemORswereincorporatedindigitoninmicelles, depositedinCNTs,andtestedforVOCsensing(Table1).ThemORs–CNTbiosensorsrevealed broadagreementofresultsforthemicelleandnanodiscsystems.Thedifferenceobserved betweenmORnanodiscandmicellessensorswasintheirstabilityovertime.Micellesensors remainedactivefor5daysincontrastwithnanodiscsensors,whichwerestablefor10weeks afteraninitialdecreaseinresponse.Thelongerintegritymaybeduetothehigherstabilityofthe nanodiscstructureincontrasttothemicelles[35].

Phagedisplay:acombinatorial proteinexpressiontechniquewith bacteriophageslinkingproteinsto theirrespectiveDNA.

Piezoelectric:theabilityofa materialtogenerateelectricityupon mechanicalstress.

Quartzcrystalmicrobalance (QCM):atechniquethatmeasures massvariationperunitareaby measuringthechangeinfrequency ofaquartzcrystalresonator.

Resonator:asystemthatnaturally oscillatesatsomefrequencies,called resonantfrequencies,withgreater amplitudethanatothers.

Self-assembledmonolayer(SAM):

onemoleculelayerofmaterialbound toasurfaceinanorderedway resultingfromitsphysico-chemical characteristics.

Siliconnanowire(SiNW):a nanowireformedfromasilicon precursorbyetchingofasolidor throughcatalyzedgrowthfroma vapororliquidphase.SiNWsensors transduceelectricalsignals.

Surfaceacousticwave(SAW):a soundwavethattravelsparallelto thesurfaceofanelasticmaterial, withitsdisplacementamplitude decayingintothematerial.SAW sensorsaremicroelectromechanical systemsthattransduceaninput electricalsignalintoamechanical wave.Thismechanicalwaveis influencedbyphysicalphenomena, andthewaveistransducedback intoanelectricalsignal.

Volatileorganiccompounds (VOCs):organiccompoundswith highvaporpressureatroom temperature,likeodors, pheromones,andaromas. Olfactory receptor model Mammalian

odorant-binding protein Pep de

Insect odorant-binding protein

FigureI.StructuralFeaturesofOdor-SensingProteinsandPeptides.

Table 1. Protein Biosensors in Gas Sensing

Protein VOCs Support Transducer LoD/measureda _{Source Expression system Refs}

Olfactory receptors

OR-10 Diacetyl Gold QCM 11012_{M C. elegans E. coli [32]}

SAW 1.210 11_{mM MCF-7 cells [34]}

hOR 17–40 hOR3A1

Helional CNT Interdigitated

microelectrode array, Current-voltage

0.02 ppt Homo sapiens MC18

(Saccharomyces

cerevisiae) HEK-293T cells

[36]

mOR174-9 mOR203-1 mOR256-17

Acetophenone, Others CNT CNT transistors,

Current-gate Voltage

1–10mM Mus musculus S. cerevisiae [35]

Odorant -binding proteins

AaegOBP22

N,N-diethyl-meta-toluamide (DEET)

Gold ZnOfilm bulk

acoustic resonators

– A. aegypti E. coli [55]

pOBP Ethanol; methanol Si Si-substrate with

interdigitated electrodes (EIS)

20 ppma_{; 10 ppm}a _{Sus scrofa Pig nasal tissue [46]}

pOBP (R)-( )-1-octen-3-ol

(octenol); (R)-( )-carvone (carvone)

Gold SAW 0.48 ppm; 0.72 ppm S. scrofa Pig nasal tissue [47]

wtbOBP (R)-( )-1-octen-3-ol

(octenol); (R)-( )-carvone (carvone)

SAW 0.2–0.23 ppm (carvone);

0.18–0.21 ppm (octenol)

Bos taurus BL21-DE3E. Coli [49]

pOBP wtbOBP dmbOBP

(R)-( )-1-octen-3-ol (octenol); (R)-( )-carvone (carvone)

Gold SAW wtpOBP: 25.9 (octenol),

7.0 (carvone) Hz/ppm; wtbOBP: 3.5 (octenol), 5.4 (carvone) Hz/ppm; dmbOBP: 6.0 (octenol), 9.2 (carvone) Hz/ppm;

S. scrofa; B. Taurus Pig nasal tissue;

BL21-DE3E. Coli

[51]

pOBP wtbOBP dmbOBP (R)-( )-1-octen-3-ol (octenol); (R)-( )-carvone (carvone)

Gold SAW 0.48 ppm (octenol);

0.74 ppm (carvone)

S. scrofa; B. Taurus Pig nasal tissue;E.

coli

[52]

wtbOBP (R)-( )-1-octen-3-ol

(octenol)

Gold Solidly mounted

resonator

7 ppm B. taurus BL21-DE3E. coli [48]

wtbOBP (R)-( )-1-octen-3-ol

(octenol); (R)-( )-carvone (carvone)

Gold SAW 0.18 ppm; 0.2 ppm B. taurus BL21-DE3E. Coli [49]

wtbOBP (R)-( )-1-octen-3-ol

(octenol)

Gold SAW 2 ppm B. taurus BL21-DE3E. coli [50]

wtbOBP, dmbOBP DMMP Silicon nitrate Photonic ring

resonator

6.8 ppb B. taurus BL21-DE3E. coli [53]

a_{Indicates when the detected concentration was not the LoD, but the“measured”in that paper.}

Trends

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A human OR(hOR)-based biosensorfor VOC sensing has beencoupled to carboxylated polypyrrolenanotubes[36].Conductivitymeasurementsrevealedaveryhighsensitivityofthis biosensortowardshelional(0.02ppt).Moreover,itpresentedselectivitytowardshelionalwhen comparedwithanalogVOCslike3,4-methylenedioxydihydrocinnamicacid,piperonal,safrole, andphenylpropanol.Theseanalogshadatleast2/3lowerelectricalresistancechangeupon bindingtothehORthanhelionalinthereportedbiosensormeasurements[36].

Thedifficult,expensive,andtime-consuminghandlingofmembraneproteinsmayhaveledto thepursuitofsimplerandmorerobustbiomoleculesasrecognitionagents[37].Therefore, gas-sensing biosensors based on the soluble odorant-binding proteins (OBPs) and small peptideshavegainedtraction[20,38,39].

OBPGas Biosensors

OBPsareanessentialcomponentoftheolfactorysystem.Theyareinthefirstlineofolfaction, locatedinthenasalepitheliumofmammals(nose)andinthesensillarlymphofinsects(e.g., antennae)[39,40].LesssolubleVOCsarebelievedtofirstbindtoOBPsthatcarrythemtothe ORsintheolfactorysensoryneurons.MorehydrophilicVOCsareabletocrossthemucosaor lymphto binddirectlytoORs.Furthermore,OBPsarebelievedtoactasscavengerswhen VOCsarepresentinhighamounts.VertebrateandinsectclassesofOBPsdifferintheir3D structure(Box1).TheOBPsfromvertebratesbelongtothelipocalinsuperfamilyofproteins, whichare essentiallytransport proteins [38,41,42]. InsectOBPs arealso stabilized bythe presenceof three disulfidebonds [39,40]. OBPscan withstand high temperatures before experiencing denaturation, and after unfolding they frequently refold, restoring their initial structure[42].Interestingly,whenbindinghigh-affinityligands,thestabilityofpigOBP(pOBP) increasesat highertemperatures [45]. InsectOBPs are even more resistant,as the three interlockeddisulfidebondspreventproteolyticdegradationandthermaldenaturation[40,42].

DevelopmentsinOBP-basedbiosensorsforgassensingstartedtoflourishinthelastdecade (Table 1). Initially, OBP biosensors were tested against volatiles and odor solutions, with measurementsof analytes in gas phase only reported by the end ofthe 2000s. The first describedOBPbiosensortodetectethanolandmethanolingasphaseinvolvedpOBP[46]. Theproteinimmobilizedonasiliconsubstrateforelectrochemicalimpedance

spectros-copy(EIS)measurements,andwasabletodetectethanolandmethanolvapors(Table1).

FurtherresearchrevealedthatpOBPcanalsobindoctenolandcarvone[47].Octenolisan importantVOCforfoodqualitycontrol,asitiscorrelatedwiththepresenceoffungiandmolds. pOBPwasimmobilized ontothegoldcoatingofaresonator,andthe sensorwas ableto detectR-carvone (aspearmint-likeflavorandodorant),butwithalower sensitivitythanfor octenol.ThesetwoVOCshavealsobeenthoroughlytestedwithbovineOBP(bOBP)[48–50], andinsensorarraysofpOBP,bOBPbothwildtype(wt)anddouble-mutant(dm)withhigher affinitytocarvone[51,52].Theabove-mentionedreportshaveshownthattheLoDsforoctenol andcarvoneofpOBPwere0.48and0.72ppm[47],andofbOBPwere0.18and0.2ppm, respectively[49].Foradifferentapplication,abiophotonicsensorusingthesamewtbOBP anddmbOBP as sensingelements was developed to detectdimethyl methylphosphonate (DMMP),a precursorof saringas [53].ThebOBPswere depositedonto thesiliconnitride surfaceofabiophotonicresonatorchip,resultinginastablesensorthatwasresponsiveforover 3monthsandcoulddetect6.8ppbofDMMPin15minutes.Furthermore,apOBP

micro-cantileverbiosensorwasrecentlyreported;althoughthesystemisinearlydevelopment,the

sensor successfully detected 2-isobutyl-3-methoxypyrazinein a gas chamber [54]. Taken together, these examples reveal some plasticity of OBP structures in identifying different chemical structures. It is in fact important to merge structural information on OBP–VOC

interactionforproteindesignandengineeringtowardstargetVOCsofinterest,whichcurrently stillremainsachallengeduetothelimitedstructuralinformationavailableforawidevarietyof VOCstructures(Boxes1and2;Figure2,KeyFigure).

TheonlyreportedbiosensorbasedonaninsectOBPforVOCdetectioninthegasphaseused anOBPfromthemosquitoAedesaegypti(AaegOBP22)[55].ThepurifiedOBPwasdeposited

inaZnOthinfilmbulkacousticresonator(FBAR),andavaporflowof

N,N-diethyl-meta-toluamide (DEET), the main component of mosquito repellents, was used for detection measurements.TheAaegOBP22biosensordetectedthepresenceofDEETthroughadrop inthe responsefrequencyoftheFBARsensor,dependingonahigherorloweramountof loadedOBP[55].

Peptide-BasedGasBiosensors

PeptidesarepromisingalternativestoORsandOBPsasmolecularrecognitionunitsinVOC sensing. Due to their smaller size, peptides represent a simple and low-cost option for biosensors (Box 1). Additionally, the selectivity towards target VOCs is easier to tune [20,56].VOC-sensingpeptideshavebeendevelopedbydifferentapproaches(Box2,Figure2), andappliedinseveralsubstratesandsensingdevices,assummarizedinTable2.

PeptidesequencesforVOCsensingweredevelopedintheearly2000sbasedondogand hORs. Thefirst OR-basedpeptide sequences were designed bymaintaining the dog OR regionsresponsibleforVOCbinding.Theselectedpeptidechains,rangingfrom9to14amino acidresidues,weredepositedonagoldsurfaceofapiezoelectricmultiarrayanalyzer.Two immobilizedpeptidesequences,orp61(12-mer)andorp188(9-mer),successfullydetected trimethylamine(TMA)andammonia,openinganewpathforthedevelopmentofpeptide-based volatilebiosensors[57].Inanotherwork, aseriesofpeptideswere designedinspiredbya

homologymodelofthehumanOR1E1.SeveralVOCsweredockedtoeachtransmembrane

Box2.ProteinandPeptideEngineeringinVOCSensing

TotailorproteinsandpeptidesforbindingtargetVOCs,severalproteinengineeringtechniquesmaybe applied

(Figure2).Predominanttechniquesinvolvesite-specificproteinmutations(Figure2A),thedesignofpeptidesinspired

bytheVOCrecognitionsitesofolfactoryreceptorsandodorant-bindingproteins(Figure2B),andscreeningofpeptide librariesbyphagedisplayandvirtualscreeningapproaches(Figure2C,D).

AdetailedanalysisoftheVOC-bindingpocketfromOBPsrevealsaclosesimilarityamongthebindingsiteofOBPsfrom differentorganisms,suchaspigandbovineOBPs(Figure2A),eventhoughthetargetVOCisdifferent.Theseproperties ledtothedevelopmentofsensorarraysincludingOBPsfromdifferentorganismsandmutatedOBPs,inordertobetter discriminatedetectedVOCs[51,52].Anexamplemutationisthebovinedoublemutantdescribedtoincreasethe sensitivityofbOBPtooctenolandcarvone[51].

AnotherapproachconsistsofapeptidethatwasrationallydesignedbyextractingthesequencesresponsibleforVOC recognitioninORsandOBPs.Duetotheirsmallersizes,peptidesareusuallyeasiertoimmobilizeinanorientedand predefinedwayontosensorsurfaces.PeptidesarealsosomewhatmorestablethanORsandmoreresilientto reusabilityandshelf-life.SuccessfulexampleshavebeenreportedforpeptidesderivedfromhumanOR1E1[58,61,79]

anddogOR-P30955[57,60].ThreedifferentpeptidesderivedfrominsectOBPsweresuccessfullyproducedand implementedingassensorstodetect3-methyl-butanol[63,80],1-hexanol[63],andtrinitrotoluene[65](Figure2B).

Combinatorialscreeningtechniques,suchasphagedisplayandvirtualscreening,canbroadenpeptidesequence variabilityandfindsequencesforspecificVOCs.PhagedisplayisimplementedwhenaVOCanalogisimmobilizedonto asurfaceora VOC-analogsurfaceisusedtoscreenrandomized peptidesforbinding[66–68,71](Figure2C). Computational virtual screening of peptide libraries is also used in the discovery of VOC-binding peptides

[69,72,75](Figure2D).Thetop-performingpeptidesareusuallysynthetizedandexperimentallytestedingassensing,

withsatisfactorycorrelationbetweeninsilicoandexperimentaldata[69,75].

KeyFigure

Protein

and

Peptide

Discovery

and

Engineering

for

Gas

Sensing

Phage library Panning graphene

Hit pep des sequencing:

1- 2- 3-4- …

Affinity pep des

Pep de library

VOC library

Docking virtual screening

Docking score ranking:

1- 2- 3-4- …

Computa onal affinity pep des

Bovine OBP Binding site Porcine OBP

VOC recogni on sequence

Insect OBP VOC recogni on

pep de

Protein engineering

PepƟde raƟonal design

Phage display

ComputaƟonal screening

1- 2- 3- 4-5- …

(B) (A)

(D) (C)

(Seefigurelegendonthebottomofthenextpage.)

sequenceofhOR1E1usingmoleculardockingandthefourmostpromisingpeptideswere synthesized.The lead peptides, horp61 (12-mer),horp103 (7-mer), horp109 (8-mer), and horp193(8-mer),wereimmobilizedonsiliconnanowires(SiNWs),andusedtodetectTMA, o-xylene,ammonia,andaceticacid[58,59].OtherQCMsensorarrayswithpeptidesfromdog OR(LHYTTIC,TIMSPKLC)andhumanOR1E1(DLESC,ELPLGCG)havealsobeenassembled. Thissystemcould discriminatemore VOCsthan previousexamples,including aceticacid, butyricacid,ammonia,dimethylamine,chlorobenzene,andbenzene[60].Theaffinityofthe peptidehorp193towardsaceticacidwasfurtherconfirmedbyQCMstudies,revealingaLoDof 3.8–2ppm.Thisworkalsoreportedthataloweramountofpeptideinthesensingfilmoriginates betterreproducibilityandshelf-life,asopposedtohigherpeptidedensities[61].

WhenutilizingOBPsasscaffoldstoextractputativeVOC-sensingpeptidesequences,someof theproblems associatedwithOR-inspired peptidedesignare solved.Theexistenceof3D structuresofseveralOBPsmeansthattheirbindingsitesaregenerallywelldefinedandthe needtopredicthomologymodelsbecomesobsolete.Withtheaimofdetectingfood contami-nationbySalmonella,two peptideswereextractedfromthe sequenceofDrosophila

mela-nogasterLUSH-OBP(Table2)[62,63].KnowingthatthemainVOCreleasedfromSalmonella

-contaminated meatis 3-methyl-1-butanol,the LUSH-OBPwas selected dueto its known sensitivityandselectivitytowardsalcohols[62].Hence,bothselectedsequencescontained alcohol-bindingresiduesandalso differentC terminalends fortheirimmobilizationongold surfacesforQCManalysis[62]andCNTsforfield-effecttransistor(FET)studies[63].QCM measurementsrevealedthattheimmobilizedpeptidecouldbindto3-methyl-1-butanoland 1-hexanol,withaLoDof10ppm,whereasthepeptideimmobilizedonCNTdemonstratedavery selectiveresponsefor3-methyl-1-butanol,withaLoDof1fM.ThedifferentreportedLoDsarea consequence of not only the differentsubstrates for peptide immobilization and resultant transducingsystems,butalsotheexperimentsetup,whichwasperformedwithgasesforthe QCMmeasurementsandinsolution forthe CNTassays. In theCNT assays, thediffusion effectsoftheVOCsinthesensingchamberwerediscarded,althoughthiseffectisknowntobe oneofthemaindifficultiesinVOCsbiosensors[62,63].

AnotherinterestingOBP-basedpeptidewasdevelopedtodetecttheexplosivetrinitrotoluene (TNT).Inthiscase,ahybridpeptidewasassembled,withanaminoacidsequencepresenting affinitytowardstheCNTsurface(HSSYWYAFNNKT)[64],andaTNTrecognitionsitebasedon thebindingsiteofASP1(GGGGWFVI),anOBPfromtheantennaeofApismellifera[65].The CNT-hybridpeptidewasthenexposedtoTNTinasaturatedvaporchamberforFETanalysis. ThepositiveresultforTNTbindingrevealsthatthemethodologyofdevelopinghybridpeptides withaspecificgroupforimmobilizationonthesubstrate’ssurfacecanbesuccessful,asthe VOCbindingmoietyofthepeptideiscompletelyavailableforVOCdetectionandnotanchored tothesurface[65].

Figure2.ForaFigure360authorpresentationofFigure2,seethefigurelegendathttps://doi.org/10.1016/j.tibtech.2018.

07.004.

ForaFigure360authorpresentationofFigure2,seethefigurelegendathttps://doi.org/10.1016/j.tibtech.2018.07.004. (A)Superimpositionofthebindingpocketsofbovineandporcineodorant-bindingproteins(OBPs)revealshowsmall differencesinresiduecompositionleadtodifferentvolatileorganiccompound(VOC)affinities[51].(B)Theextractionof volatilebindingsequencefromthefruitflyOBPLUSH,asawaytorationallydeveloppeptidesforVOCdetection[62,63].(C) Phage-displaypanningscreensamultitudeofpeptidesequencesagainstagraphiticsurfacemimickingbenzene[68].(D) Computationaltechniquespredictthebestpeptide–VOCpairstobeexperimentallytested[70].

Table 2. Peptide Biosensors in Gas Sensing

Peptidea _{VOCs Support Transducer LDL/measured}b _{Development Refs}

NQLSNLSFSDLC (dORp61) Dog OR–P30955

Trimethylamine (TMA); ammonia; acetic acid; ethyl acetate; methanol

Gold Surface Piezoelectric

multiarray analyzer

– OR-based

design

[57]

ACSDAQVNE (dORp188) Dog OR–P30955

TMA; ammonia; acetic acid; ethyl acetate; methanol; benzene

–

FLSNLSFSDLC (hORp61) human OR1E1

TMA Gold surface Piezoelectric

multiarray analyzer

6.7 Hz OR-based

design

[58]

FFLLFGC (hORp103) human OR1E1

O-xylene 5.1 Hz

DLESFLC (hORp109) human OR1E1

Ammonia Gold surface/silicon

(SiNW)

Piezoelectric multiarray analyzer/ conductance

11 Hz/100 ppmb _[58,59]

RVNEWVIC (hORp193) human OR1E1

Acetic acid Gold surface/silicon

(SiNW)

Piezoelectric multiarray analyzer/ QCM/conductance

28 Hz/ 3.11.2 ppm/ 100 ppmb

[58,59,61]

LHYTTIC (PAC1) dog OR– P30955

Acetic acid; butyric acid Gold surface QCM (multiarray) – OR-based

design

[60]

TIMSPKLC (PAC2) dog OR– P30955

Acetic acid; butyric acid

DLESC (PAM1) human OR1E1 Dimethyl amine

ELPLGCG (PAM2) Acetic acid; dimethylamine

SLMAGTVNKKGEFC (LUSH OBP)

3-methyl-1-butanol; 1-hexanol Gold surface QCM 1–3 ppm OBP-based

design

[62]

TKCVSLMAGTVNKKGEFFFF (LUSH OBP)

3-methyl-1-butanol CNT Field-effect transistor 1 fM OBP-based

design

[63]

HSSYWYAFNNKTGGGGWFVI (P1-ASP1C peptide) (honeybee antenna OBP)

Trinitrotoluene (TNT) SWCNT Field-effect transistor 12 ppb OBP-based

design

[65]

WHYQRPLMPVSI (TNT-BP) TNT Gold surface Thermal desorption

GC-MS

– Phage

display

[66]

HPNFSKYILMPVSI (DNT-BP) 2,4-dinitrotoluene (DNT)

CH3

ONH-KMHTASLSQPLMGC-CONH2(DNT-BP1C)

DNT Gold surface QCM liquid phase/

microcantilever

/431 ppt Phage

display

[67,71]

DSWAADIP (GP1) Benzene Gold surface Microcantilever 121 ppb Phage

display

[68]

DNPIQAVP (GP2) Toluene; xylene; hexane 2.2 ppm (toluene);

28 ppm (xylene); 1 ppm (hexane)

DRNESSVP (BP1) Benzene; toluene –

AYSSGAPPMPPF (A3) Acetonitrile; dichloromethane;

methyl salicylate

Gold nanoparticles Inductor–capacitor– resistor resonators

– Phage

display

[81]

1252

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Table 2. (continued)

Peptidea _{VOCs Support Transducer LDL/measured}b _{Development Refs}

NFQGI 2,3.7,8-TCDD

(2,3,7,8-tetrachlorinated dibenzo-p-dioxin

Gold Surfaces QCM 10–20 ppbb _Rational

design (virtual screening)

[72]

NFGGQ

NFQGF

CG 2-propanol; acetone; acetonitrile;

butane-2,3-dione; ethanol; ethyl acetate; ethyl butanoate; ethyl octanoate; hex-3-en-1-ol; hexane; isopentylacetate; nonanal; octanal; terpin-4-ol

Gold nanoparticles QCM Sensor array

(discriminates all tested VOCs)

Rational design (virtual screening)

[69]/[75]

ECG (glutathione)

CIHNP

CIQPV

CRQVF

IHRIC

Terpinen-4-ol>hexane>octanal>nonanal

Gold nanoparticles QCM Sensor array

(discriminates all tested VOCs)

Rational design (virtual screening)

[70]

KSDSC Small alcohols (2-propanol;

ethanol)

LGFDC Ethanol, 2-propanol; acetone;

butane-2,3-dione; ethyl acetate

TGKFC Alcohols (hex-3-en-1-ol;

terpinen-4-ol) and aldehydes (nonanal; octanal)

WHVSC Esters (ethyl acetate; ethyl

butanoate; ethyl octanoate; isopentyl acetate) and hexane

IHRIC Alcohols (1-butanol; 1-hexanol;

2-methyl-1-propanol; ethanol; hexen-3-en-1-ol) and esters (ethyl acetate; ethyl-methyl-2-butyrate; isopentyl acetate)

Zinc oxide nanoparticles QCM Sensor array

(discriminates all tested VOCs)

Rational design (Virtual screening)

[76]

LAWHC

TGKFC

WHVSC

a_{Peptide sequences provided as one-letter codes.} b

Indicates when the detected concentration was not the LoD, but the“measured”in that paper.

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12

TheuseofOBPsandORstodeveloppeptidesequencesforgassensingissomehowlimitedto thediversity ofVOCsknown to bindtheseproteins. Therefore,methodologies like phage

display[66–68],virtualscreening[69],andcombinatorialpeptidelibraries[70]broadenthe

rangeofpeptidesequenceswithdistinctphysico-chemicalproperties,improvingaffinityand selectivitytowardstargetVOCsorVOCclasses.

The application of phage display has been very fruitful in unveiling new specific peptide sequenceswithanaffinitytowardscertaintargetmolecules(Figure2).Inthismethod,aphage libraryispannedagainstasolidsurfaceresemblingtheVOCstructureoragainstanimmobilized target,whichcanbethetargetVOCitselforananaloghandledinsolutionorincrystalphases. Thechoice ofthe targetfor screeningin phage displayis ofutmost importance, as it will determinetheaffinityandselectivityoftheselectedpeptidesequences.Thefirstattemptto translatephagedisplaypanningmethodstogasphasesensingwasperformedtosearchfor peptideswithanaffinityforTNTand2,4-dinitrotoluene(DNT)[66].Twopeptides,TNT-BPand DNT-BP,werediscoveredtoselectivelybindtoTNTandDNTinliquidphase,whenpanning twocommercialphagedisplaylibraries(linear12-merandconstrained7-mer)againstcrystal formsofTNTandDNT.However,aftertranslatingthesystemtothegasphase,thelowvapor pressureof TNTdid notallowgas-phase affinity testingforTNT-BP.DNT-BPsuccessfully demonstratedaffinityinbindingDNTthroughthermaldesorptiongaschromatography-mass spectrometry(GC-MS)analysis[66].Later,alinear12-mercommercialphagedisplaylibrary waspannedagainstagold-immobilizedDNTanalog[4-(2,4-dinitrophenyl)butan-1-amine][67]. ADNTaffinitypeptidewasreported(DNT-BP1C)andgasphasemeasurementsina micro-cantileversystem,revealedahighsensitivityofthepeptidetoDNT[71].Inarecentexample,an 8-mer proprietary phage display peptidelibrary was screened againsta self-assembled

monolayer(SAM)ofbenzeneongold,andagainstagraphitesurface(consistingofhexagonal

aromaticringstructures,hereusedasbenzenemimetics),withtheaimofselectingbenzene bindersabletodiscriminatesinglecarbonanalogsofbenzene,namelytolueneandxylene[68]. Threepeptideswereselected,onepresentingaffinitytothebenzeneSAMandtwotowardsthe graphitesurface(Table2).Uponpeptideimmobilizationonsurfaceswithdifferentfunctional groups,amicrocantileversetupforgastestingshowedthepotentialtodifferentiatetheVOCs benzene,toluene,andxylene.

OneofthemajorchallengesinemployingphagedisplaytechnologytosearchforVOCbinders isadaptingthepanningmethodologiesandtranslatingthepanningresultsfromtheliquidphase togas-sensingdevices.AcriticalfactorishowtoimmobilizesuchsmalltargetsasVOCsonto thesurfacesemployedduringpanning,andhowtoreducenonspecificinteractions,asallthese methodsweredevelopedmainlytolookforbindersinaqueousconditionsagainstlargetargets, mainlyproteins.

Computationalvirtualscreeningallows‘insilico’testingofVOC–peptidebindingwithfastand low-costroutines.Thisapproachhasbeenappliedtothediscoveryofgas-sensingpeptides (Figure2)[69,72].Thefirstreportedeffortusingacomputationalscreeningmethodfocusedon findingpeptidestodetectdioxins.Alibraryof11pentapeptideswasconstructedbasedona bindingsitemodelfordioxins(-NFQGR-)[73].Aftercalculatingthetheoreticalbindingenergy, thethreemostpromisingpentapeptideswereemployedinaQCM-basedgasbiosensor.The threepeptides(NFQGI,NFGGQ,NFQGF)identifiedthepresenceof2,3,7,8-tetrachlorinated dibenzo-p-dioxin(2,3,7,8-TCDD)withinadioxinmixturecontaining17compounds[72].

Anotherstudyinvolvedthevirtualdockingscreeningof5peptides(cysteinylglycine, glutathi-one,CIHNP,CIQPV,CRQVF)(Table2),towards14distinctVOCs[69].Theresultsshoweda

100%positivecorrespondencebetweencomputationalandexperimentaldataforethyl ace-tate,ethylbutanoate,andhexane,andanoverallmatchingtrendof60%.Thesamesetof computationallydiscoveredpeptides[69,74](Table2),wasusedinthe‘Ten2009’electronic nose (Tor Vergata Sensors Group, Rome, Italy), equipped with an 8-QCM sensor array. Contemporaneously,anothersetupforthee-nosewasassembledusingonly metallo-tetra-phenylporphyrin-coated chips. The peptide-based e-nose arrangement was able to discriminate between artificially contaminated and regularwhite, milk,and dark chocolate sampleswithahigheraccuracythantheporphyrinsensorarray[75].

Anotherstudyusedasemi-combinatorialvirtualscreeningapproachtoevaluateapeptidelibrary of 8000tripeptidesforaffinity towards58 compounds fromfive distinctchemical classes(alcohols, aldehydes,esters,hydrocarbons,andketones)[70].Afteranautomateddockingprocedure,a subsetof120tripeptideswasselectedbasedontheirabilitytodifferentiatechemicalclasses. Then,anewtetrapeptidelibrarywasdesignedbased onthese120tripeptides anddockedagainst the58volatiles.Fivetetrapeptides(IHRI,KSDS,LGFD,TGKF,andWHVS)withdifferentaffinityto alcohols,aldehydes,esters,hydrocarbons,andketoneswereselectedforgassensing(Table2). UsingagassensorQCMarray,thepeptideswereimmobilizedongoldnanoparticlesandtested forbindingthetargetvolatiles.Inthiswork,theexperimentalresultsforfourofthefivepeptides confirmedthepredictionsobtainedbythecomputationalcalculations[70].Although peptide-basedsensorsare able tosuccessfully detectdifferentVOCs,water producesashiftinthesignalin QCMmeasurements.Arecentworkovercamethisobstaclebyimmobilizingfourpeptides(IHRIC, LAWHC,TGKFC,WHVSC)onzincoxidenanoparticles.Thesefourbiosensorstogetherwereable todiscriminatealcoholsandesters(Table2)andwerefurthertestedinjuicesamples.Thedifferent juicesampleswerediscriminated,supportingtheusageofzincoxideasasupportforpeptidesin VOCdetectionforhighwatercontentsamples[76].

ConcludingRemarksandFuturePerspectives

Usingbiological recognitionunitsinsensingdevicesimprovestheirselectivityandsensitivity towardsdefinedtargets,withprovenpronouncedexpressioninsamplesanalyzedinliquidstates. DespitetheextremelyvaluableknowledgederivedfromtestingVOCbiosensorsinsolution,an essentialfeatureissometimesoverlooked:thebehaviorofthesebiosensorsintestinganalytesin thegasphase.Althoughseveralobstaclesarecurrentlybeinginvestigated,therearestill chal-lengestoinspirefuturedevelopments.Theseinclude,forexample,assessinghumidity interfer-enceduringVOCdetection,reproducinggas-phasemeasurementsobtainedfromprotein-based biosensors,enhancingthestabilityofproteinsandpeptidesuponimmobilizationontosurfaces andensuringtheircompatibilitywithnanofabricationapproaches,andimprovingtheselectivityof VOCbiosensorswhenanalyzingextremelycomplexsamplesclosertoreal-lifesettings.

Sometrendsareevidentforgas-phaseproteinbiosensors.Overthelast5years,therehas beenadecreased interest inOR-basedand an increasein OBP-and peptide-based bio-sensors.Certainly,usingmembraneproteinreceptorsasORsismorechallenging,expensive, and time consuming, when compared with soluble and robust proteins such as OBPs. AlthoughOBPs areeasierto express andtohandlethan ORs,they stilllackthe simplicity andeasetogeneratediversitywhencomparedwithpeptides.Thisshortcomingjustifiesthe burgeoninginterestinpeptide-basedbiosensors.Theyareeasytoassemblethroughchemical andbiologicalmeans,andtheirengineeringprofitsfromavarietyoftechniques(phagedisplay, rationaldesign,virtualscreening).Overall,allthreesystemsreportedinthisreviewshouldstillbe consideredinthefuture,asthesensitivityandselectivityderivedfromORsandOBPsmaybe translatedtopeptideandproteindesignandperhapsdirectlycorrelateaminoacidsequences withVOCselectivityandsensitivity.

OutstandingQuestions

How can VOC-identifying analytical toolshelptodefineVOCmarkersfor relevantsocietalproblems?

Willvolatolomics,togetherwith gas-sensing advances, contribute to advanceddiagnostictools?

Whatshouldbeincludedina‘control VOCset’forproteinandpeptide gas-sensingbenchmarks?

Coulddenovocomputationalprotein designbeausefultooltopredictnew VOC-bindingproteinsandpeptides?

Will advances in VOC-biomolecular recognitionpromotesmell implemen-tationinartificiallifesystems?

Inartificialolfaction,though,thecombinationofbiologicalrecognitionunitshasnotyetreached itsfullpotential,insomecasesduetothedifficultyinestablishingVOCmarkersfromcomplex mixtures[77].However,adaptingproteinengineering,phagedisplay,andcomputationaltools toaddressthemolecularrecognitionbetweenbiologicalbindersandanalytesingasphases hasmadetremendousprogressandhasmaturedoverthelast15years.Assuch,thetoolsto developVOCbindersareinplace,aswellasthemethodologiestointerfacethebioreceptors withgas-sensingdevices(seeOutstandingQuestions)[2].

Aninvestmentinthe expansion oftested VOCsfor the developmentofgas biosensorsis required, as recent examples reporta verylimited numberand chemical diversityof VOC targets.InalmostallareasofVOCsensing,gassamplesarerichinthevarietyofchemical entities,anditisessentialtoaddressmoleculardiscriminationincurrentandfutureresearch. Theincreasingimprovementsinproteinandpeptideengineeringmaybeveryusefultoincrease thestabilityof biomolecules(for example,throughthe useofnanodiscs) orto expandthe variabilityingeneratingbindersagainstlesscommonVOCs(forexample,throughthemolecular modelingandinvitroevolutiontechniques).

Withthedevelopmentsinvolatolomics,weexpectthattheimplementationofgas-phase bio-sensorstoaddressbiologicalquestionswillbecomearealityinthenearfuture.High-impactsocial andeconomic areaswill bethecentral applicationof this technology.Earlydiseasediagnosticsand healthmonitoring,pollutantandfirehazardsintheenvironment,spoilageandqualitymonitoringin foodandagriculture,explosiveandchemical/biologicalweaponrydetectioninsafety,andodor identificationinprostheticsarejustsomepotential examplesforgasbiosensing.Overall, the developmentofprotein andpeptideVOC biosensorswillbeimportant to attainthe desired sensitivityandselectivityinVOCdetection,eitherinsingle-gassensorsforadefinedanalyteor inarraysofgas-sensingmaterialscoupledtoartificialintelligencetoolsforsignalprocessing.

Acknowledgements

ThisworkwassupportedbytheEuropeanResearchCouncilthroughthegrantreference

SCENT-ERC-2014-STG-639123(2015-2020),andbytheUnidadedeCiênciasBiomolecularesAplicadas-UCIBIO,whichisfinancedbynational fundsfromFCT/MEC(UID/Multi/04378/2013)andco-financedbytheERDFunderthePT2020PartnershipAgreement (POCI-01-0145-FEDER-007728).A.J.M.BarbosaandA.R. OliveirathankfellowshipsSFRH/BPD/112543/2015and SFRH/BD/128687/2017fromFCT/MCTES,Portugal.

SupplementalInformation

Supplementalinformation associatedwiththis articlecanbefoundonlineathttps://doi.org/10.1016/j.tibtech.

2018.07.004.

References

1. Castelvecchi,D.(2016)CanweopentheblackboxofAI?Nature 538,20–23

2. Wang,P.etal.(2015)BioinspiredSmellandTasteSensors, Springer

3. McGann,J.P.(2017)Poorhumanolfactionisa19th-century myth.Science356,eaam7263

4. Fitzgerald,J.E.etal.(2017)Artificialnosetechnology:statusand prospectsindiagnostics.TrendsBiotechnol.35,33–42

5. Zhang,X.etal.(2018)Anoverviewofanartificialnosesystem. Talanta184,93–102

6. Persaud, K.C.(2017) Towards bionicnoses.Sens. Rev.37, 165–171

7. Broza,Y.Y.etal.(2015)Hybridvolatolomicsanddisease detec-tion.Angew.Chem.Int.Ed.Engl.54,11036–11048

8. Krilaviciute,A.etal.(2015)Detectionofcancerthroughexhaled breath:asystematicreview.Oncotarget6,38643–38657

9. Vishinkin,R.andHaick,H.(2015)Nanoscalesensortechnologies fordiseasedetectionviavolatolomics.Small11,6142–6164

10. Fitzgerald,J.andFenniri,H.(2017)Cuttingedgemethodsfor non-invasivediseasediagnosisusinge-tongueande-nose devi-ces.Biosensors7,59

11. Cruz,H.etal.(2017)Developmentofe-nosebiosensorsbasedon organicsemiconductorstowardslow-costhealthcarediagnosisin gynecologicaldiseases.Mater.TodayProc.4,11544–11553

12. Rocco,G.(2018)Everybreathyoutake:thevalueofthe elec-tronicnose(e-nose)technologyintheearlydetectionoflung cancer.J.Thorac.Cardiovasc.Surg.155,2622–2625

13. Srivastava,A.K.etal.(2018)Nanosensorsandnanobiosensorsin foodandagriculture.Environ.Chem.Lett.16,161–182

14.Loutfi,A.etal.(2015)Electronicnosesforfoodquality:areview.J. FoodEng.144,103–111

15. Zanardi,E.etal.(2018)Newinsightstodetectirradiatedfood:an overview.FoodAnal.Methods11,224–235

16. Giannoukos,S.etal.(2016)Chemicalsniffinginstrumentationfor securityapplications.Chem.Rev.116,8146–8172

17. Cui,S.etal.(2018)Plantpestdetectionusinganartificialnose system:areview.Sensors18,378

18. Szulczynski,B.etal.(2017)Differentwaystoapplya measure-mentinstrumentofe-nosetypetoevaluateambientairquality withrespecttoodournuisanceinavicinityofmunicipal process-ingplants.Sensors(Switzerland)17,E2671

19. Dung,T.etal.(2018)Applicationsandadvancesinbioelectronic nosesforodoursensing.Sensors18,103

20.Wasilewski,T.etal.(2017)Bioelectronicnose:currentstatusand perspectives.Biosens.Bioelectron.87,480–494

21.Wasilewski,T.etal.(2018)Advancesinolfaction-inspired bio-materialsappliedtobioelectronicnoses.Sens.Actuators B Chem.257,511–537

22.Son,M.etal.(2017)Bioelectronicnose:anemergingtoolforodor standardization.TrendsBiotechnol.35,301_–307

23. Sanmartí-espinal,M.etal.(2017)Quanti_ficationofinteracting cognateodorantswitholfactoryreceptorsinnanovesicles.Sci. Rep.7,1–11

24. Mitsuno,H.etal.(2015)Novelcell-basedodorantsensor ele-mentsbasedoninsectodorantreceptors.Biosens.Bioelectron. 65,287–294

25. Mulla, M.Y. et al. (2015) Capacitance-modulated transistor detectsodorantbindingproteinchiralinteractions.Nat. Com-mun.6,1–9

26. Kotlowski,C.etal.(2018)Finediscriminationofvolatile com-pounds by graphene-immobilized odorant-binding proteins. Sens.ActuatorsBChem.256,564–572

27. Larisika,M.etal.(2015)ElectronicolfactorysensorbasedonA. melliferaodorant-bindingprotein14onareducedgraphene oxidefield-effecttransistor.Angew.Chem.Int.Ed.Engl.54, 13245–13248

28.Antunes,G.andSimoesdeSouza,F.M.(2016)Olfactoryreceptor signaling.MethodsCellBiol.132,127–145

29.Behrens,M.etal.(2018)Structure–functionrelationshipsof olfactoryandtastereceptors.Chem.Senses43,81–87

30. deFouchier,A.etal.(2017)FunctionalevolutionofLepidoptera olfactoryreceptorsrevealedbydeorphanizationofamoth reper-toire.Nat.Commun.8,15709

31. SilvaTeixeira,C.S.etal.(2016)Unravellingtheolfactorysense: fromthegenetoodorperception.Chem.Senses41,105–121

32. Sung,J.H.etal.(2006)Piezoelectricbiosensorusingolfactory receptorproteinexpressedinEscherichiacoli.Biosens. Bioelec-tron.21,1981–1986

33. Wu, T.Z. (1999) Apiezoelectric biosensor as an olfactory receptor for odourdetection:electronicnose.Biosens.Bioelectron.14,9–18

34. Wu,C.etal.(2012)Abiomimeticolfactory-basedbiosensorwith highefficiencyimmobilizationofmoleculardetectors.Biosens. Bioelectron.31,44–48

35. Goldsmith,B.R.etal.(2011)Biomimeticchemicalsensorsusing nanoelectronicreadoutofolfactoryreceptorproteins.ACSNano 5,5408–5416

36.Lee,S.H.etal.(2012)Mimickingthehumansmellsensing mech-anismwithanartificialnoseplatform.Biomaterials33,1722–

1729

37.Wu,C.etal.(2017)Biomimeticsensorsforthesenses:towards betterunderstanding of tasteand odorsensation. Sensors (Basel)17,E2881

38. Persaud,K.C.andTuccori,E.(2014)Biosensorsbasedon odor-antbindingproteins.InBioelectronicNose(Park,T.H.,ed.),pp. 171–190,Springer

39. Pelosi,P.etal.(2018)Beyondchemoreception:diversetasksof solubleolfactoryproteinsininsects.Biol.Rev.93,184–200

40. Brito,N.F.etal.(2016)Alookinsideodorant-bindingproteinsin insectchemoreception.J.InsectPhysiol.95,51–65

41. Northey,T.etal.(2016)Crystalstructuresandbindingdynamics ofodorant-bindingprotein3fromtwoaphidspeciesMegoura viciaeandNasonoviaribisnigri.Sci.Rep.6,24739

42. Pelosi,P.etal.(2014)Structureandbiotechnologicalapplications ofodorant-bindingproteins.Appl.Microbiol.Biotechnol.98,61–

70

43. Mastrogiacomo,R.etal.(2014)Anodorant-bindingproteinis abundantlyexpressedinthenoseandintheseminalfluidofthe rabbit.PLoSOne9,45–48

44. Schiefner,A.etal.(2015)Crystalstructureofthehumanodorant bindingprotein,OBPIIa.Proteins83,1180–1184

45. Paolini,S.etal.(1999)Porcineodorant-bindingprotein:structural stabilityandligandaffinitiesmeasuredbyfourier-transform infra-redspectroscopyandfluorescencespectroscopy.Biochim. Bio-phys.Acta1431,179–188

46.Capone,S.etal.(2009)Electricalcharacterizationofapig odor-antbindingproteinbyimpedancespectroscopy.Proc.IEEE Sens.2009,1758–1762

47.DiPietrantonio,F.etal.(2013)Surfaceacousticwavebiosensor basedonodorantbindingproteinsdepositedbylaserinduced forwardtransfer.IEEEInt.Ultrason.Symp.2013,2144_–2147

48. Cannata,D.etal.(2012)Odorantdetectionviasolidlymounted resonatorbiosensor.IEEEInt.Ultrason.Symp.2012,1537–1540

49. DiPietrantonio,F.etal.(2014)Tailoringodorant-bindingprotein coatingscharacteristicsforsurfaceacoustic wavebiosensor development.Appl.Surf.Sci.302,250–255

50. Palla-Papavlu,A.etal.(2014)Preparationofsurfaceacoustic waveodorsensorsbylaser-inducedforwardtransfer.Sens. ActuatorsBChem.192,369–377

51. DiPietrantonio,F.etal.(2013)Detectionofodorantmoleculesvia surfaceacousticwavebiosensorarraybasedonodorant-binding proteins.Biosens.Bioelectron.41,328–334

52. DiPietrantonio,F.etal.(2015)Asurfaceacousticwave bio-electronicnosefordetectionofvolatileodorantmolecules. Bio-sens.Bioelectron.67,516–523

53.Bonnot,K.etal.(2014)Biophotonicringresonatorfor ultrasensi-tivedetectionofDMMPasasimulantfororganophosphorus agents.Anal.Chem.86,5125–5130

54.Possas-Abreu,M.etal.(2017)BiomimeticdiamondMEMS

sen-sors based onodorant-binding proteins:sensors validation

throughan autonomous electronic system.ISOCS/IEEEInt.

Symp.OlfactionElectron.Nose PublishedonlineMay2017.

http://dx.doi.org/10.1109/ISOEN.2017.7968909

55. Zhao,X.etal.(2012)ProteinfunctionalizedZnOthinfilmbulk acousticresonatorasanodorantbiosensor.Sens.ActuatorsB Chem.163,242–246

56. Sankaran,S.etal.(2012)Biologyandapplicationsofolfactory sensingsystem:areview.Sens.ActuatorsBChem.171–172,1–

17

57. Wu,T.Z.andLo,Y.R.(2000)Syntheticpeptidemimickingof bindingsitesonolfactoryreceptorproteinforusein“electronic nose”.J.Biotechnol.80,63–73

58. Wu,T.Z.et al.(2001)Exploringtherecognizedbio-mimicry materialsforgassensing.Biosens.Bioelectron.16,945–953

59. McAlpine,M.C.etal.(2008)Peptide-nanowirehybridmaterials forselectivesensingofsmallmolecules.J.Am.Chem.Soc.130, 9583–9589

60.Lu,H.H.etal.(2009)Directcharacterizationandquantificationof volatileorganiccompoundsbypiezoelectricmodulechips sen-sor.Sens.ActuatorsBChem.137,741–746

61. Panigrahi,S.etal.(2012)Olfactoryreceptor-basedpolypeptide sensorforaceticacidVOCdetection.Mater.Sci.Eng.CMater. Biol.Appl.32,1307_–1313

62. Sankaran,S.etal.(2011)Odorantbindingproteinbased biomi-meticsensorsfordetectionofalcoholsassociatedwith Salmo-nellacontaminationinpackagedbeef.Biosens.Bioelectron.26, 3103–3109

63. Son,M.etal.(2016)Bioelectronicnoseusingodorantbinding protein-derived peptide and carbon nanotube field-effect

transistorfortheassessmentofSalmonellacontaminationin food.Anal.Chem.88,11283–11287

64. Pender,M.J.etal.(2006)Peptide-mediatedformationof single-wallcarbonnanotubecomposites.NanoLett.6,40–44

65. Kuang,Z.etal.(2009)Biomimeticchemosensor:designing pep-tiderecognitionelementsforsurfacefunctionalizationofcarbon nanotubefieldeffecttransistors.ACSNano4,452–458

66. Jaworski,J.W.etal.(2008)Evolutionaryscreeningofbiomimetic coatingsforselective detectionofexplosives. Langmuir24, 4938–4943

67. Jang,H.-J.etal.(2010)Identificationofdinitrotolueneselective peptidesbyphagedisplaycloning.Bull.KoreanChem.Soc.31, 3703–3706

68.Ju,S.etal.(2015)Single-carbondiscriminationbyselected peptidesforindividualdetectionofvolatileorganiccompounds. Sci.Rep.5,9196

69.Pizzoni,D.etal.(2014)Selectionofpeptideligandsfor piezo-electricpeptidebasedgassensorsarraysusingavirtual screen-ingapproach.Biosens.Bioelectron.52,247–254

70. Mascini,M.etal.(2017)Tailoringgassensorarraysviathedesign ofshortpeptidessequencesasbindingelements.Biosens. Bio-electron.93,161–169

71. Hwang,K.S.etal.(2011)Peptidereceptor-basedselective dini-trotoluenedetectionusingamicrocantileversensor.Biosens. Bioelectron.30,249–254

72. Mascini,M.etal.(2004)Piezoelectricsensorsfordioxins:a biomimeticapproach.Biosens.Bioelectron.20,1203–1210

73. Kobayashi,S.etal.(1999)Atheoreticalinvestigationofthe conformationchangingofdioxinsinthebindingsiteofdioxin receptormodel;roleofabsolutehardness–electronegativity activ-itydiagramsforbiologicalactivity.J.Mol.Struct.475,203–217

74. Compagnone,D.etal.(2013)Goldnanoparticles-peptidebased gassensorarraysforthedetectionoffoodaromas.Biosens. Bioelectron.42,618–625

75. Compagnone,D.etal.(2015)SensorsandactuatorsB:chemical quartzcrystalmicrobalancegassensorarraysforthequality controlofchocolate.Sens.ActuatorsBChem.207,1114–1120

76. Mascini,M.etal.(2018)PeptidemodifiedZnOnanoparticlesas gassensorsarrayforvolatileorganiccompounds(VOCs).Front. Chem.6,105

77.Palma,S.I.C.J.etal.(2018)Machinelearningforthe meta-anal-ysesofmicrobialpathogens’volatilesignatures.Sci.Rep.8,3360

78.Carraher,C.et al.(2015)Towardsanunderstandingofthe structuralbasisforinsectolfactionbyodorantreceptors.Insect Biochem.Mol.Biol.66,31–41

79.Cui,Y.etal.(2012)Biomimeticpeptidenanosensors.Acc.Chem. Res.45,696_–704

80. Sankaran,S.etal.(2011)Olfactoryreceptorbasedpiezoelectric biosensorsfordetectionofalcoholsrelatedtofoodsafety appli-cations.Sens.ActuatorsBChem.155,8–18

81. Nagraj,N.etal.(2013)Selectivesensingofvaporsofsimilar dielectricconstantsusingpeptide-cappedgoldnanoparticles onindividualmultivariabletransducers.Analyst138,4334–4339