biosensors in forensic sciences - orbi: home finale.pdf · biosensors in forensic sciences...

BASE Biotechnol. Agron. Soc. Environ.201115(4),449-458 Focus on:

BiosensorsinforensicsciencesChristineFrederickx,FrançoisJ.Verheggen,EricHaubrugeUniv.Liege-GemblouxAgro-BioTech.DepartmentofFunctionalandEvolutionaryEntomology.PassagedesDéportés,2.B-5030Gembloux(Belgium).E-mail:[email protected]

ReceivedonJuly8,2010;acceptedonFebruary8,2011.

Abiosensor isadevice thatusesbiologicalmaterials todetectandmonitor thepresenceofspecificchemicals inanarea.Traditionalmethodsofvolatiledetectionusedbylawenforcementagenciesandrescueteamstypicallyconsistofrelianceoncanineolfaction.Thisconceptofusingdogstodetectspecificsubstancesisquiteold.However,dogshavesomelimitationssuchascostoftrainingandtimeofconditioning.Thus,thepossibilityofusingotherorganismsasbiosensorsincludingrats,dolphins,honeybees,andparasiticwaspsfordetectingexplosives,narcoticsandcadavershasbeendeveloped.Insectshaveseveraladvantagesunsharedbymammals.Insectsaresensitive,cheaptoproduceandcanbeconditionedwithimpressivespeed for a specificchemical-detection task.Moreover, insectsmightbeapreferred sensingmethod in scenarios that aredeemedtoodangeroustousemammalian.Thepurposeofthisreviewis toprovideanoverviewofthebiosensorsusedinforensicsciences.Keywords.Biosensors,indicatoranimals,Apis mellifera,Microplitis croceipes,explosives,narcotics,humanremains.

Les biodétecteurs en sciences forensiques.Unbiodétecteurestundispositifquiemploiedesorganismesbiologiquespoursurveiller laprésencedediversproduitschimiquesdansunsecteurparticulier.Laméthode traditionnellededétectiondessubstancesvolatiles,quiestemployéeparlapolice,sebasesurlescapacitésolfactivesdeschiens.Ceconceptd’employerdeschienspourdétectercertainessubstancesn’estpasrécent.Cependant,l’utilisationdeceschiensprésentecertaineslimitestellesquelecoutdeformation,unelonguepérioded’apprentissage,etc.Ainsi,leschercheurssesontpenchéssurl’utilisationd’autresbiodétecteurstelsquedesrats,lesdauphins,lesabeilles,lesparasitoïdesdansladétectiond’explosifs,dedroguesoudecadavres.Contrairementauxmammifères,lesinsectesprésententplusieursavantages.Ceux-cisonttrèssensibles,sontpeucouteuxetpeuventêtreconditionnésavecunevitesseimpressionnantepourunetâchespécifiquedeproduit-détection.D’ailleurs,l’utilisationdesinsectescommebiodétecteurspourraitêtrepréféréedanslesscénariosquisontconsidéréscommetropdangereuxpourdesmammifères.Lebutdecettesynthèsebibliographiqueestdefournirl’ensembledesbiodétecteursutilisésensciencesforensiques.Mots-clés.Biodétecteur,animalindicateur,Apis mellifera,Microplitis croceipes,explosif,narcotique,resteshumains.

1. IntroductIon

Biological sensors, biosensors or biodetectors aredefinedasanalyticaldevicesincorporatingabiologicalmaterial(e.g.,tissue,microorganisms,insects,animals,enzymes, antibodies, plants), a biologically derivedmaterial (e.g., recombinant antibodies, engineeredproteins) or a biomimic (e.g., synthetic receptors,combinatorial ligands) intimately associated withor integrated within a physicochemical detector ortransducing microsystem, which may be optical,electrochemical,thermometric,piezoelectric,magneticor micromechanical (Weetall, 1996). Biosensorsusuallyyieldanelectronicsignalwhichisproportionaltotheconcentrationofaspecificorgroupofanalytes.Biosensors have been applied to a wide variety ofanalytical problems includingmedicine (Clark et al.,

1962),biomedicalresearch(Lanzonietal.,2009),drugdiscovery (Harperet al.,2007), environment (Amineetal., 2006; Badihi-Mossberg et al., 2007), food(Amine et al., 2006), process industries (Tokarskyyet al., 2008), security (Harper et al., 2007), anddefense(Harperetal.,2007).Mostofthesebiologicalsensors are used for their intrinsic properties, butscientistsproducealsogeneticallymodifiedorganismsas biodetectors (Weetall, 1996). Because of theirexceptionalcapabilities,includinghighspecificityandsensitivity,rapidresponse,lowcost,relativelycompactsize and user-friendly operation, biosensors havebecome an important tool for detection of chemicalandbiologicalcomponentsforclinical,food,lawandenvironmentalmonitoring(Weetall,1996).

Forensic sciences are the application of a broadspectrumofsciencestoanswerquestionsofinterestto

450 Biotechnol. Agron. Soc. Environ. 201115(4),449-458 FrederickxCh.,VerheggenF.J.&HaubrugeE.

alegalsystem(Ricciuti,2007).Thismaybeinrelationtoacrimeoracivilaction.Forensicsciencesincludeseveral under divisions like forensic entomology,forensic toxicology, forensic anthropology, DNAanalysis, criminalistics, and aim to analyze criminalevidences (Ricciuti, 2007). The results are furtherpresented as accurately and precisely as possible ina courtof law (Ricciuti, 2007).Forensic sciencesdonotuseonlychemical technologies to solveacrime;theyusealsovertebratesorinvertebratesaschemicaldetectors.Thisuseisbasedontheolfactorycapacitiesofvertebratesandinvertebratestodetectvolatilesfromahuman,narcoticsorexplosives.

In thefieldof forensic sciences, lawenforcementagencies and rescue teamsworldwideusebiosensorsincludingdogsandhoneybeestolocatecorpses,drugsorexplosives.

Inthisreview,wespecificallyprovideanoverviewofthebiosensorsystemsusedinforensicsciences.

2. PrIncIPLe oF the bIosensors used In ForensIc scIences

2.1. Associative learning

Associativelearningiscommonthroughouttheanimalkingdom,frominvertebratestovertebrates(Tomberlinetal.,2005).Learningabilityhasevolvedtoadjustthebehavioralresponseofanimalstochangingecologicalconditions (Stephens, 1993). To condition animals,twotechniquesoflearningareused,i.e.theymustgothroughaphaseoftraining.

Operant conditioning, or instrumental learning,is a formof associative learning that occurs throughrewards and punishments for behavior (Skipper,1938).Throughoperantconditioning,anassociationismadebetweenabehaviorandaconsequencefor thatbehavior.Therearetwotypesofoperantconditioning:- Positive Reinforcement: a particular behavior is strengthenedbytheconsequenceofexperiencinga positive condition (Bernstein et al., 2008). For example,ahungryratpressesabarinitscageand receives food.The food isapositivecondition for the hungry rat.The rat presses the bar again, and againreceivesfood.Therat’sbehaviorofpressing the bar is strengthened by the consequence of receivingfood.- Negative Reinforcement: a particular behavior is strengthenedby theconsequenceof stoppingor avoidinganegativecondition(Bernsteinetal.,2008). Forexample,aratisplacedinacageandimmediately receivesamildelectricalshockonitsfeet.Theshock is anegative condition for the rat.The rat presses abarandtheshockstops.Theratreceivesanother shock, presses the bar again, and again the shock

stops. The rat’s behavior of pressing the bar is strengthened by the consequence of stopping the shock.

Bothpositiveandnegativereinforcementstrengthenbehavior. In other words, consequences– whethergood or bad– determine if a behavior ismaintainedor not. For example, dolphins get a fish for doing atrick.Becausetheanimalwantstogainthatgoodthingagain, itwill repeat thebehavior thatseems tocausethatconsequence(Bernsteinetal.,2008).

The second technique of learning which is usedin forensic sciences is the classical conditioning,which is synonymous with Pavlovian or respondentconditioning. It is a form of associative learningthat was first demonstrated by Ivan Pavlov (Pavlovet al., 1960). The typical procedure for inducingclassical conditioning involves presentations ofa neutral stimulus along with a stimulus of somesignificance(Figure 1).Theneutralstimuluscouldbeany event that doesnot result in anovert behavioralresponsefromtheorganismunderinvestigation.Thisis referred as a conditioned stimulus. Conversely,presentation of the significant stimulus necessarilyevokesaninnateresponse.Thisresponseiscalledtheunconditioned stimulus. If the conditioned stimulusandtheunconditionedstimulusarerepeatedlypaired,eventuallythetwostimulibecomeassociatedandtheorganismbegins toproduceabehavioral response tothe conditioned stimulus (Pavlov et al., 1960). Thismechanismcalledconditioned response inwhich theresponsetotheconditionedstimulusisgreaterthantheoriginalbaselineresponseelicitedbytheconditionedstimulus prior to conditioning.This type of learninghas the most potential for application toward thedevelopment of biological sensors (Tomberlin et al.,2008).

Thesetwoconditioningsarebasedontheolfactorysystemofanimals.

2.2. olfaction

Chemical communication or chemoreception is theprincipal means of communication for many groupsof animals (Banaigs, 2002). Chemoreception is theprocessofdetectingchemicalcompoundsbyalivingorganism and involves molecular interactions witholfactory neurons by molecules that have moderatemolecular weight, low polarity, particular watersolubility, high vapor pressure, and lipophilicity(Meierhenrich et al., 2004). Olfaction allows theanimalstodetectandrecognizethechemicalsignalsoftheirenvironmentwhichenablethemtocommunicatewith their congenerics, to locatea sourceof food, toseekasiteoflaying,toestablishinter-individual,socialandsexualrelations(Banaigs,2002).Thedetectionof

Biosensorsinforensicscience 451

anodorbytheolfactoryorganscauses,ifitisidentifiedandrecognized,thereleaseorthemodificationofthebehavior(Banaigs,2002).Thisolfactorycapacitywillbeusedinthelearningmechanismtocreatebiosensors.

Theolfactorymechanismis organized in three stages:reception, transduction andintegration of the sensorysignal(Brossut,1996).

In vertebrates, odorantsgenerally first enter theolfactory system by passingthroughtheexternalnaresofvertebrates where warmingand humidification occurbefore passing through aset of cartilaginous flapcalled turbinates that act toincrease the surface area ofthe epithelium (Leffingwell,2002; Meierhenrich et al.,2004). Then, odorants arethoughttobeassociatedwithodorant-transport proteinsthathelptransporttheodorant

from the inhaled air stream throughthemucus to the cilia of the olfactoryneurons (Figure 2).Theodorant bindswith a receptor protein (embedded inthe phospholipid surface membrane)whichactivatesthisoneandamultitudeofmolecularinteractions.Onthislevel,thetransductionofthechemicalsignalin an electrical signal is produced(Stengletal.,1999).Then,theelectricalsignal is sent via the axon, throughthe cribiform plate to the glomerulusand mitral cells, then on through theolfactorytractandintothebrainwherethe odor is interpreted (Leffingwell,2002;Meierhenrichetal.,2004).

Ininsects,whenastimulusmoleculecomes into contact with the olfactorysensilla localized on the antenna, legsand maxillary palps of the insect, itwill be adsorbed and the moleculediffuses from the hair surface throughthecuticularporesandpore tubules tothehairlumen(Figure 3)(Keil,1999).There,theodorantsaretakenupbytheodorant-binding proteins (OBP) andare transported through the aqueoussensillum lymph until they reach areceptormoleculeoftheouterdendriticmembrane. Odorant fixation on thereceptor molecule activates results inactivationandamultitudeofmolecular

interactions. On this level, the transduction of thechemical signal in an electrical signal is produced(Stengletal.,1999).Then,theelectricalsignalissent

UCS(meat powder)

Neutral stimulus(tone)

Neutral stimulus followed UCS(tone) by (meat powder)

CS(tone)

UCR(salivation)

Orientingresponse

UCR(salivation)

CR(salivation)

PHASE 1 : Before conditioning has occurred

PHASE 2 : The process of conditioning

PHASE 3 : After conditioning has occurred

Figure 1.Classicalconditioning.Beforeclassicalconditioninghasoccurred,meatpowderonadog’stongueproducessalivation,butthesoundofatone-aneutralstimulus-bringsonlyorientingresponsessuchasturningtowardthesound.Duringtheprocessofconditioning,thetoneisrepeatedlypairedwiththemeatpowder.Afterclassicalconditioninghastakenplace,thesoundofthetonealoneactsasaconditionedstimulus,producingsalivation—Conditionnement classique. Avant le conditionnement, s’il l’on place de la poudre de viande sur la langue d’un chien, celui-ci salive. Mais, le bruit d'une tonalité (un stimulus neutre) apporte seulement des réponses orientées telles que la rotation du chien vers le bruit. Pendant le processus de conditionnement, la tonalité est plusieurs fois reprise en association avec la poudre de viande. Après que le conditionnement classique ait eu lieu, le bruit seul de la tonalité agit en tant que stimulus conditionné, produisant la salivation (Bernsteinetal.,2008).

Figure 2.Theolfactorybulb — Ampoule olfactive (Leffingwell,2002).

Olfactory TractMitral Cell

Olfactory Nerve Olfactory Bulb

Olfactory Epithelium

GlomeruliOlfactory Nerve filaments

Cribriform (bone)

Axons

Olfactory Receptor Neurons

Cilia in Mucosa Mucosa

Air andodorant molecules


via the axon until the primary education processingcenterofthecentralnervoussystem:theantennallobe.From there, information are transmitted to the brain(protocerebrum) where they will be integrated andmemorized toproduceabehavioral reaction (Brossut,1996).

3. bIosensors

3.1. Vertebrate

dog. The most widely deployed detector to date isCanis familiarisLinnaeus,betterknownasthecommondog (Harper et al., 2007). Sniffing dogs are usedubiquitouslyby lawenforcementandprivateagenciesfor detection of many different items. The mostcommon items of forensic interest for which canineshavebeendeployedtolocatearedrugs,ignitableliquidresidues,explosives,humanremains,andhumanscent(disastersurvivors)(Harperetal.,2007;Oesterhelwegetal.,2008).Inaddition,traineddogshavetheabilityto detect decomposing bodies beneath running water(Cornaz,2008).Theuseof thecanineasadetector isbasedonthewell-establishedreliabilityandimpressiveselectivity and sensitivity associatedwith dog’s senseof smell (Harper et al., 2007). No one really knows

how sensitive a dog’s noseis, but scientists note that adog’s sensitivity to odor is of100timesat1,000timesgreaterthan that of a human (Seuter,2003). Moreover, they cantrackdirections,havelong-termolfactory memory and portrayextreme discriminative ability(Harperetal.,2007).

The training of thedogs begins with the basicconditioning (Cornaz, 2008).During twoyears, theywill befamiliarizedwith the search ofobjects and people, especiallywith obedience. Then, thevarious junctions intervene:drugs, explosives, corpses ormissingpeople.These two lastdisciplines take the most timeto train dogs (Cornaz, 2008).The training of sniffing dogsis based on the recognition ofa particular odor and on thelearningtoaltertheirbehaviorswhen certain odors denotingspecific substances are found.The training is based on

positive reinforcement which consists of food. Thereexist severalmethods but the details of those are notrevealed by the police.During the training,when thedogdetectsthetargetedobjectwiththeodorofcorpse,drugsorexplosives,thetrainergivesareward(food)tothedogtorendercomprehensibletohimitssatisfaction.Thus,thedogwillassociatetheodoroftheobjectwithreward.Thetrainingprogresseswhileburyingcorpses,drugsorexplosivesandbymodifyingthesearchareaswithdifferentenvironmentalconditions.Tocontrolthecorrectmemorizingoftheodors,thedogistraineddaily.The training program is only a guide and each breedand age of dog and condition need to be considered(Rebmannetal.,2000).

Cues used by a dog to indicate forensic materieldependonthetrainer.Therearemanydifferentwaysadogcanbetrainedtocommunicateanalerttoitstrainer(Lasseteretal.,2003).Thedogcangiveanaggressiveorpassivealert.Anaggressivealertisoneinwhichthedogdigsatthesiteofthescentofinterestashumanremains,drugs,and/orexplosives.Apassivealertiswhenthedoglaysdownonthesiteofinterestorjumpsonthetrainertoindicatethepresenceofremains,drugsorexplosives.Itisbettertogiveapassivealertthananaggressivealertbecausecrimescenescanbedisturbedoralteredbytheanxiousdiggingofadog.Specifically,cadaverdogsaretrainedtofindscents,notbodies(Lasseteretal.,2003).

Figure 3. Conceptofperireceptoreventsininsectolfactorysensilla—Concept des évènements du périrecepteur dans une sensille olfactive d’insecte (WithkindpermissionfromSpringerScience+BusinessMedia:Stengletal.,1999.InsectOlfaction,chapter2,p.56,fig.1).

S:adsorbedstimulusmolecules—molécules adsorbées;P:pore—pore;Pt:poretubules—pore tubulaire;B:odorant-bindingproteins—protéines de transport;R:receptormolecule—récepteur;flash:activationofamultitudeofmolecularinteractions—activation d’une multitude d’interactions moléculaires.

S P

B

B

B B

B

B

PtB

BB

B B

R R R R R

Sensillum lymph

Cytoplasm

Cuticle


Canines that detect human remains, commonlyreferred to as cadaver dogs, are trained in detectingthe odor of decomposing bodies.They are used in avariety of forensic contexts, including search anddiscovery of human cadavers, body parts, or bodyfluids(Rebmannetal,2000).Thescentpicturechangesas the body progresses through the decompositionstages.Itisimportanttotraindogsontheallstagesofdecomposition.

Rescuedogsaretrainedtodetectodorsassociatedwithlivingpeoplesuchasurine,evaporatedperspiration,respiratorygases,ordecompositiongasesreleasedbybacterialactiononhumanskinor tissues (SARDUS,1992). Rescue dogs can be classified according towhether they “scent discriminate”, and under whatconditions they canwork. Scent discriminating dogshaveproventheirabilitytoalertonlyonthescentofanindividualperson,afterbeinggivenasampleofthatperson’s scent. Non-scent discriminating dogs alerton or follow any scent of a given type, such as anyhuman scent or any cadaver scent (Rebmann etal.,2000). Rescue dogs can be trained specifically forrubblesearches,forwatersearches,andforavalanchesearches(SARDUS,1992).

Narcotic detection canines are expected to facea predictable lineup of five or six drug odors, theexplosivedetectioncanineisexpectedtofacedozensof different potential explosive products during itsservice(Given,2003).Narcoticsdetectioncaninesaretypically trained on cocaine, heroin and marijuana.They may be trained on additional drugs, includingmethamphetamine,ecstasy,hashish,opium,mescaline,and LSD (N, N-diéthyllysergamide), depending onthe training agency and the locationswhere they aredeployed (Given, 2003).Explosivedetection caninesare currently trained on a wide variety of samplesranging from half a dozen samples to upward of 20(Harperetal.,2007).Usingatleastonerepresentativesample from each explosive chemical class wouldrequire an acid salt such as ammonium nitrate,an aromatic nitro such as TNT (trinitrotoluene),a nitrate ester such as PETN (1,3-dinitrooxy-2,2-bis(nitrooxymethyl) propane), a nitramine such asRDX (cyclonite), an aliphatic nitro such as DMNB(2,3-Dimethyl-2,3-dinitrobutane), a peroxide such asTATP(triacetonetriperoxide),andrepresentativeblackand smokeless powders (Yinon et al., 1996; Harperetal.,2007).Allthesesnifferdogsshouldnotbeusedtodoothertypesofworktheywerenottrainedfor.

Themajordisadvantagesofdetectordogsaretheirlimited duty cycles (necessitatingmultiple teams forcontinuous coverage) and possibility of operatorinfluence (because the trainer is dealing with anintelligentanimal).Thecanine’sabilitytoworkisalsoaffected by temperature, humidity andwind (Seuter,2003).Theworstconditionsforusingdogsarewhenit

ishotanddrywithlittleornoairmovementandwhenitisrainingorsnowingheavily(Lasseteretal.,2003).The most difficult and time-consuming step in thedevelopmentofasniffingdogwaslimitingthenegativeinfluenceofenvironmentaldistractions(audio,visual,olfactory),whichdisrupteditsconcentrationandcouldevokeundesiredbehavior(Yinonetal.,1996).Canine/trainerteamsrequireagreatdealofcommitment,anddogmust also be accompanied by specific trainer inordertoworkproperly(Ottoetal.,2002).Inaddition,dogs are instructed to give the same alert for eachexplosive or drug; trainers are unaware of the exacttype of explosives, drugs detected.Also, dogs needtobeincloseproximitytosourcesinordertosamplescent;thereforeitmaybenecessarytodeploymultipleteamsforlargerareasasitmaytakeawhiletosweep(Settles et al., 2001).However, there is a possibilitythatteamsmayinterferewithoneanother.Forsmallerareas,accessibility isanother issueofwhichsecuritymanagersmust be aware (Seuter, 2003). If dogs areunabletoreachintotightspaces,thereisadangerofmissing potential hiding spots. This is perhaps oneof the most serious limitations of canine detectors.Anotherdisadvantage is thecostof training, thecostforthemaintenanceofthewelfareofthedogs,ahopeofusenotexceedingthe6-8years(Harperetal.,2007).

rat/rodents. Given the limitations of canines,researchers are studying the use of other animalsin detection of explosives and drugs, even in thepresence of distracting odors such as engine oil andalmondextract (Ottoet al.,2002).The ideaofusingrats todetect substanceshasbeen studiedpreviouslybyfewresearchers.Nolanetal.(1978)andWeinsteinet al. (1992) studied devices that rewarded rats forproperexplosiveodordetectionusingelectricalbrainstimulation.For this, theyusedoperantconditioning.Odorsweredeliveredtoratsinconditioningchambersand odor classificationwas accomplished via typicallever pressing and via direct monitoring of the rat’scorticalfrequencyspectra.Ottoetal.(2002)traintherats, so that their locationandalertingbehaviorscanbemonitored fromafarbycomputer and/orhumans.Underthisconcept,thetrainedalertingbehaviorofratsis remotely monitored by humans and/or computersto determine when the animals detect the scent ofinterestascocaineduringtheirsearchbehavior.Whentheratsfindwhat theyare lookingfor, theyraise thealarmbystandingontheirhindlegs,whichisdetectedbycomputer (Ottoetal.,2002).Since2003,asocialenterprise, APOPO, develops a training method todetectmines(TNT)withtheAfricangiantpouchedratorCricetomys gambianusWaterhouse.Theprocedureconsists of a combination of click training and foodrewarding.Atfirst,theanimalsaretaughttoassociatethe click soundwith a food reward.Then they have


to perform certain tasks to get this food reward.After odor imprint, the complexity of their tasks tobe performed is gradually increased. Intellectually,theratsare“smart”enoughtolearnthedesiredtasksrelatively quickly, while being “uncomplicated”enoughforlearningtobestandardized.Foodprovidesastrongandcontrollablesourceofmotivationandaneffective drive for performance. The ability of a ratto detect different concentration of TNT in air wasinvestigated (Yinon et al., 1996). A rat was trainedto press a bar when air containing TNT vapor wasdelivered, and to refrain from pressing a bar whenair free of TNTwas delivered; 100% detection wasobtainedforaconcentrationof2.44ng.l-1TNTvaporinair,95%detectionforaconcentrationof1.32ng.l-1and60%foraconcentrationof1.07ng.l-1.Atallthreeconcentrationstherewerenofalsealarms(Yinonetal.,1996).

Contrary to dogs, rats do not form socialrelationships and obedience with people, so are notdependentonaspecifictrainer(APOPO,2003).Theycangetintotighterspaces,too,sotheymightbebetterabletofindcasualtiesincollapsedbuildings.Becauseratsare small, theycansqueeze into smallareasandmaybeabletoinfiltrateareasthatdogscannot.Theyare inexpensive, and are relatively easy to procureand maintain. Rats may also be better than dogs atmaintaining performance during long periods ofrepetitivework.Inaddition,theirlightweightmakesitlesslikelytosetoffexplosives(APOPO,2003).

Whileclaimsthattheratscanmatchthesensitivityof dogs appear to be well founded, there are stillsignificant difficulties in deploying rats directly inthe field. Rats trained on a variety of chemicals canbe useful in detecting different types of explosives;however, other than the fact something dangerous ispresent, rats are not able to tell the trainer whethersmokeless powder or cyclonite was detected (Yinonetal.,1996).

other mammalian species.Theoretically,almostanymammal has the potential to be used as a biologicaldetectorwith the primary limitations associatedwiththesizeandthemobilityofthemammalaswellasitstrainability.TheU.S.Navy’sMarineMammalProgramtrainsdolphins(Tursiops truncatusMontagu)andthesea lion (Zalophus californianusLesson) tofind andmark the location of underwater objects, especiallymines and submerged vehicles (SPAWAR, 1992).Dolphinsareessentialbecausetheirbiologicalsonarisunmatchedbyhardwaresonarsindetectingobjectsinthewatercolumnandontheseafloor(SPAWAR,1992).Sea lions are used because they have very sensitiveunderwaterdirectionalhearingandexceptionalvisioninlowlightconditions.Bothofthesespeciesarehighlyreliable,adaptable,andtrainablemarineanimals.They

aretrainedwithoperantconditioning,emphasizingtheuseofpositivereinforcement(SPAWAR,1992).

3.2. Invertebrate

The use of animals to detect volatiles from drugs,explosives or humans has long been recognized andutilized by law enforcement.However, the ability tolearnanddetectodorsofhuman,drugsorexplosivesisnotlimitedtovertebrates.Insectsarehighlysensitive(parts-per-trillion), flexible, portable and cheap toreproduce, and it is easy to condition them to detecttargetodorants(Rainsetal.,2008).Moreover,insectscanbeconditionedwithimpressivespeed(fewminutestotwodays)foraspecificchemical-detectiontaskandthey do not require trainer in the field (Shaw et al.,2005;Rainsetal.,2008).Someoftheirlimitationsarethatinsectsdonotflyatnight,inheavyrain,orincoldweather(Harperetal.,2007).

Forensic entomology is the broad field wherearthropodscienceandthejudicialsysteminteract(Halletal.,2009).Inregardstomedico-legalfields,insectshaveprimarilybeenusedtodeterminethetimeofdeath,alsoknownasthepost-morteminterval(PMI)(Amendtetal.,2004;Wyssetal.,2006;Gennard,2007).Insectscanalsobringinformationincasesofabuseorneglectofchildrenorelderly(Beneckeetal.,2001;Gennard,2007),providinginformationonthecausesofdeathorthe identity of victims (Benecke et al., 2001;Boureletal.,2001;Guptaetal.,2004;Gennard,2007).But,theuseofinsectslikebiosensorsorbiodetectorswasnotyetconsidered in forensicentomology.However,one of the big challenges of criminalistic, andmoreparticularly of the law enforcement, is to developefficient techniques to locate corpses, drugs andexplosives.Becauseofthesensitivityoftheirolfactorysystem, it appears that insects alsomight be used todevelop novel methods for detecting and locatingchemicals associated with decomposition, drugs andexplosives(Rainsetal.,2004;Tomberlinetal.,2005).

Apis mellifera Linnaeus. Learning behavior ofhoneybeeshasbeenstudiedextensivelyoverthepast40years because they can learn very rapidly manydifferentcuesassociatedwithrewards(Menzeletal.,1996;Scheineretal.,2001).

Honeybees learn to associate odors with sucroserewardinaPavlovianconditioningprotocol(Hammeret al., 1995;Menzel et al., 1996;Davis et al., 2005;Carcaud et al., 2009). A hungry bee reflexivelyextends its proboscis when antennal or probosciscontactchemoreceptorsarestimulatedbysucrose.Theproboscisextensionresponse(PER)canbeconditionedwithasingleconditioningtrialbypairinganodorwithsucrose applied to antennae and proboscis or with


repeated conditioning trials where odor and sucrosearepairedoncepertrial.InPERconditioning,theodorrepresents the conditioned stimulus and sucrose theunconditionedstimulus(Hammeretal.,1995;Menzeletal.,1996;Carcaudetal.,2009).BeesareconditionedintwohourstoseekoutburiedexplosivessuchasTNT;C4;TATP; 2, 4-DNT; andRDXand drugsmaterialslikemethamphetamineandcocaine(Davisetal.,2005;Rainsetal.,2008).

Microplitis croceipes cresson.Parasiticwaspsdetectand learn numerous chemical cues associated withtheir host and food resources, anduse them inorderto forage more effectively (Tertuliano et al., 2004).Microplitis croceipes (Hymenoptera: Braconidae) arelatively specialized endoparasitoid of three highlypolyphagous larval hosts,Helicoverpa zea,Heliothis virescens and Heliothis subflexa (Lepidoptera:Noctuidae)wereconditionedwithclassicalPavlovianto associate several chemicals that they would notencounterintheirnaturalforaging(Olsonetal.,2003;Tomberlinetal.,2005;Tomberlinetal.,2008).Thesecompounds were octanal, diisopropyl aminoethanol,twocompoundsofdecayingprocess: cadaverineandputrescine (Takasu et al., 2007) and three volatilecompounds of explosives: cyclohexanone, 2,4- and3,4-dinitrotoluene (Olson et al., 2003; Tomberlinet al., 2005). This parasitic wasp species learned toassociateodorswithfoodresourcesandsubsequentlyexhibits a characteristic food-seeking behaviorwhenencounteringthelearnedodor.Exposurestofoodandodorrepeatedthreetimeswitha10strainingsessionwas sufficient to obtain wasp conditioned close to80% (Tertuliano et al., 2004;Tomberlin et al., 2005;Tomberlin et al., 2008). When wasps responded tothe odor of conditioning, they showed typical food-searching behavior. Wasps rubbed their antenna,lowered the head, andmouthpart extension bringingthelabium(Takasuetal.,2007).

Manduca sexta Linneaus. Detection of explosivesis alsopossiblewithManduca sextaLinnaeusmoths(Lepidoptera: Sphingidae). M. sexta can be trainedto respond with a feeding behavior when exposedto odor signatures from explosives using Pavlovianconditioning(Kingetal.,2004).Briefly,conditioningis achieved by repeated (usually six) pairings of thetarget odor followed by food. The adults moths aretrained individually, with each trial taking about 15secondstoperform.Thislearnedresponseappearstobe remembered by the animal for the duration of itsadultlifespan(1-2weeks).

detector system. Insects can be used as either free-movingorrestrainedorganismstodetectchemical.Free-moving detectors. Free-moving insects are

allowedtomovetowardsodorsourceswithoutbeingconstrained in a device and are tracked duringflightorafterarrivaltotheodorsource(Rainsetal.,2008).Trackingmethodsenablethelocationsoftheorganismstobeknown.Conditionedhoneybeesaretrackedusing“lightdetectionandranging”(LIDAR)measurements(Shaw et al., 2005). LIDAR is a technique thatmeasuresthepropertiesofscatteredlighttodeterminethedistancetotheobjectthatscattersthelight.Furtherstudies enhanced theLIDARdetectionof free-flyinghoneybees by measuring the back-scattered returnsignal caused by the wing-beat modulation (Rainsetal.,2008).

Two of the most promising methods to trackconditionedinsectsoncetheyhavelocatedthesourceoftheodorantareharmonicradarandradiotelemetry.Harmonicradarinvolvestheplacementofatransponderon the insects (Riley et al., 2002). The transponderconsists of an antenna and electronics that use theincomingradarsignalasanenergysource.Whenthetransponderdetectsaradartransmission,itrepliesbyimmediatelyemittingapulseonanotherfrequencythatisaharmonicoftheincomingsignal.Consequently,itdoesnotrequireabatteryontheinsect,thusreducingthe weight being carried. However, it is difficult totrack insect movement in some environments thatcandisruptthetranspondersignal,suchasareaswithheavyvegetation.Currently,harmonicradardetectionis limited to insect species that are large enough tocarry the transponder such as honeybees and moths(Riley et al., 2002). Radio telemetry requires that aradio transmitter is mounted on the insect, which isthentrackedwithareceiver(Rainsetal.,2008).

The free-moving detectors worked very well insmall, outdoor areas, where security guards couldeasilyseewheretheywereswarming,butwerehardertotrackwhentheywereusedtodetectodorsinlarge,uncontainedspaces.

Restrained organisms. The behavioral response ofinsectstoaconditionedstimuluscanalsobeobservedininsectsthatarerestrainedwithinadetectiondevice.

InscentinelLtd (Davis et al., 2005), an enterprisein the UK, uses a detection device in which threehoneybeesareheldincassettes,withtheheadofeachbeeprotrudingsothattheproboscisiseasilyobserved.Air samples are brought into the device and passedover thebeeantennae,andavisionsystemmeasurestheproboscisextensionresponse.Thisresponsecanbemeasuredvisuallyorelectronically.

Rains et al. (2006) developed a portable deviceto present air samples to conditioned M. croceipesand to interpret the food-searching behavior of fiveconditionedwaspsheld inacartridge.Video footageofthewaspbehaviorisrecordedremotelyinalaptopcomputer. An user-developed software program


analyzes thevideoofwasps todeterminewhen theyhave detected a chemical that theywere conditionedto recognize (Utley et al., 2007). A result can bedeterminedwithin20-30safterthewaspsareexposedtotheairsample.ThisdevicecallstheWaspHound®(Rainsetal.,2008).

Adifferentdevicehasbeendevelopedtoholdtennoctuid hawkmoths. With this device, the feedingresponseofeachmothismeasuredbyelectromyography(Kingetal.,2004).Avoltmeterisusedtodetectspikesin the signal from the feedingmuscles of themoth.Fivemothswere conditioned to the target chemical,and five were used as an unconditioned control todetermine if responses from the conditioned mothswerefalsepositives(Kingetal.,2004).However,thisdeviceisheavy(17kg)andthusmakesitlessportable.

These three portable structures make it ideal fortestinginairports,subwaystations,etc.

Itappearsthatfree-movinginsectsarebestsuitedto foraging in natural environment than restrainedinsects.

4. concLusIon

Various biosensors are used in forensic sciences.However,mammalianbiosensorshavedisadvantagesunsharedwiththosemadewithinsects.Insectsmightbe a preferred sensing method in scenarios thatare deemed too dangerous to use mammalian. Forexample,caninesthatdetectcadaverareoftenplacedin dangerous conditions, either through exposure totoxic chemicals or unstable structures. Developmentof alternative sensing systems using insects wouldbeadvantageousbecause the lossofan insectsensorwouldbelessexpensivethanlosingatrainedcanine.

Insectsarenotoftenusedasbiosensorsforcorpsesdetection. However, the necrophagous insects, i.e.thosewhichcolonizeacorps,couldbeveryeffectiveastheyarepledgedwiththisparticularecosystem.Theyalsoshowanarrayofinstinctiveandreflexiveorientingbehaviors in response to decayingflesh (King et al.,2004). The advantage of using instinctive behaviorsof insects is that theyare reflexive,occurringwith ahigh degree of probability. Future detection systemsmight be developed around other insect species thathaveinnateresponsestochemicaltargets,orthathavebeen selectively reared to have enhanced responsetochemicalsof interest.Parasiticwaspsofflypupaefromcarcassesseemtobehighlysuccessfultolocatecarcassesandhosts.

Finally, further scientific research is needed toenhancetheutilityofinsect-snifferdetectionsystemsand, furthermore, to increase the level of acceptancefromalargercommunityofresearchandcommercialenterprises. Once this detection system has proven

its ability to perform in a real-world commercialapplication,thereshouldbeacoincidentalincreaseininterest to develop insect sensors for other attractiveapplications(Rainsetal.,2008).

Acknowledgements

ChristineFrederickx isfinancially supportedbyPhDgrantfromtheFondspourlaFormationàlaRecherchedansl’Industrieetl’Agriculture(F.R.I.A.),Belgium.

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