5/17/2017 Whatarebiosensors?

EnzymeTechnology
Whatarebiosensors?
Abiosensorisananalyticaldevicewhichconvertsabiologicalresponseintoanelectricalsignal(Figure
6.1).Theterm'biosensor'isoftenusedtocoversensordevicesusedinordertodeterminethe
concentrationofsubstancesandotherparametersofbiologicalinterestevenwheretheydonotutilisea
biologicalsystemdirectly.Thisverybroaddefinitionisusedbysomescientificjournals(e.g.Biosensors,
ElsevierAppliedScience)butwillnotbeappliedtothecoveragehere.TheemphasisofthisChapter
concernsenzymesasthebiologicallyresponsivematerial,butitshouldberecognisedthatotherbiological
systemsmaybeutilisedbybiosensors,forexample,wholecellmetabolism,ligandbindingandthe
antibodyantigenreaction.Biosensorsrepresentarapidlyexpandingfield,atthepresenttime,withan
estimated60%annualgrowthratethemajorimpetuscomingfromthehealthcareindustry(e.g.6%ofthe
westernworldarediabeticandwouldbenefitfromtheavailabilityofarapid,accurateandsimplebiosensor
forglucose)butwithsomepressurefromotherareas,suchasfoodqualityappraisalandenvironmental
monitoring.Theestimatedworldanalyticalmarketisabout12,000,000,000year1ofwhich30%isinthe
healthcarearea.Thereisclearlyavastmarketexpansionpotentialaslessthan0.1%ofthismarketis
currentlyusingbiosensors.Researchanddevelopmentinthisfieldiswideandmultidisciplinary,spanning
biochemistry,bioreactorscience,physicalchemistry,electrochemistry,electronicsandsoftware
engineering.Mostofthiscurrentendeavourconcernspotentiometricandamperometricbiosensorsand
colorimetricpaperenzymestrips.However,allthemaintransducertypesarelikelytobethoroughly
examined,foruseinbiosensors,overthenextfewyears.

Asuccessfulbiosensormustpossessatleastsomeofthefollowingbeneficialfeatures:

1.Thebiocatalystmustbehighlyspecificforthepurposeoftheanalyses,bestableundernormal
storageconditionsand,exceptinthecaseofcolorimetricenzymestripsanddipsticks(seelater),
showgoodstabilityoveralargenumberofassays(i.e.muchgreaterthan100).

2.Thereactionshouldbeasindependentofsuchphysicalparametersasstirring,pHandtemperature
asismanageable.Thiswouldallowtheanalysisofsampleswithminimalpretreatment.Ifthe
reactioninvolvescofactorsorcoenzymestheseshould,preferably,alsobecoimmobilisedwiththe
enzyme(seeChapter8).

3.Theresponseshouldbeaccurate,precise,reproducibleandlinearovertheusefulanalyticalrange,
withoutdilutionorconcentration.Itshouldalsobefreefromelectricalnoise.

4.Ifthebiosensoristobeusedforinvasivemonitoringinclinicalsituations,theprobemustbetinyand
biocompatible,havingnotoxicorantigeniceffects.Ifitistobeusedinfermentersitshouldbe
sterilisable.Thisispreferablyperformedbyautoclavingbutnobiosensorenzymescanpresently
withstandsuchdrasticwetheattreatment.Ineithercase,thebiosensorshouldnotbeproneto
foulingorproteolysis.

5.Thecompletebiosensorshouldbecheap,small,portableandcapableofbeingusedbysemiskilled
operators.

6.Thereshouldbeamarketforthebiosensor.Thereisclearlylittlepurposedevelopingabiosensorif
otherfactors(e.g.governmentsubsidies,thecontinuedemploymentofskilledanalysts,orpoor
customerperception)encouragetheuseoftraditionalmethodsanddiscouragethedecentralisationof
laboratorytesting.

Thebiologicalresponseofthebiosensorisdeterminedbythebiocatalyticmembranewhichaccomplishes
theconversionofreactanttoproduct.Immobilisedenzymespossessanumberofadvantageousfeatures
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whichmakesthemparticularlyapplicableforuseinsuchsystems.Theymaybereused,whichensures
thatthesamecatalyticactivityispresentforaseriesofanalyses.Thisisanimportantfactorinsecuring
reproducibleresultsandavoidsthepitfallsassociatedwiththereplicatepipettingoffreeenzymeotherwise
necessaryinanalyticalprotocols.Manyenzymesareintrinsicallystabilisedbytheimmobilisationprocess
(seeChapter3),butevenwherethisdoesnotoccurthereisusuallyconsiderableapparentstabilisation.It
isnormaltouseanexcessoftheenzymewithintheimmobilisedsensorsystem.Thisgivesacatalytic
redundancy(i.e.<<1)whichissufficienttoensureanincreaseintheapparentstabilisationofthe
immobilisedenzyme(see,forexample,Figures3.11,3.19and5.8).Evenwherethereissomeinactivation
oftheimmobilisedenzymeoveraperiodoftime,thisinactivationisusuallysteadyandpredictable.Any
activitydecayiseasilyincorporatedintoananalyticalschemebyregularlyinterpolatingstandardsbetween
theanalysesofunknownsamples.Forthesereasons,manysuchimmobilisedenzymesystemsarere
usableupto10,000timesoveraperiodofseveralmonths.Clearly,thisresultsinaconsiderablesavingin
termsoftheenzymes'costrelativetotheanalyticalusageoffreesolubleenzymes.

Whenthereaction,occurringattheimmobilisedenzymemembraneofabiosensor,islimitedbytherateof
externaldiffusion,thereactionprocesswillpossessanumberofvaluableanalyticalassets.Inparticular,it
willobeytherelationshipshowninequation3.27.Itfollowsthatthebiocatalystgivesaproportionalchange
inreactionrateinresponsetothereactant(substrate)concentrationoverasubstantiallinearrange,
severaltimestheintrinsicKm(seeFigure3.12linee).Thisisveryusefulasanalyteconcentrationsare
oftenapproximatelyequaltotheKmsoftheirappropriateenzymeswhichisroughly10timesmore
concentratedthancanbenormallydetermined,withoutdilution,byuseofthefreeenzymeinsolution.Also
followingfromequation3.27istheindependenceofthereactionratewithrespecttopH,ionicstrength,
temperatureandinhibitors.Thissimplyavoidsthetrickyproblemsoftenencounteredduetothevariability
ofrealanalyticalsamples(e.g,fermentationbroth,bloodandurine)andexternalconditions.Controlof
biosensorresponsebytheexternaldiffusionoftheanalytecanbeencouragedbytheuseofpermeable
membranesbetweentheenzymeandthebulksolution.Thethicknessofthesecanbevariedwith
associatedeffectsontheproportionalityconstantbetweenthesubstrateconcentrationandtherateof
reaction(i.e.increasingmembranethicknessincreasestheunstirredlayer()which,inturn,decreasesthe
proportionalityconstant,kL,inequation3.27).Eveniftotaldependenceontheexternaldiffusionalrateis
notachieved(orachievable),anyincreaseinthedependenceofthereactionrateonexternalorinternal
diffusionwillcauseareductioninthedependenceonthepH,ionicstrength,temperatureandinhibitor
concentrations.

Figure6.1.Schematicdiagramshowingthemaincomponentsofabiosensor.Thebiocatalyst(a)converts
thesubstratetoproduct.Thisreactionisdeterminedbythetransducer(b)whichconvertsittoanelectrical
signal.Theoutputfromthetransducerisamplified(c),processed(d)anddisplayed(e).

Thekeypartofabiosensoristhetransducer(shownasthe'blackbox'inFigure6.1)whichmakesuseofa
physicalchangeaccompanyingthereaction.Thismaybe

1.theheatoutput(orabsorbed)bythereaction(calorimetricbiosensors),

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5/17/2017 Whatarebiosensors?

2.changesinthedistributionofchargescausinganelectricalpotentialtobeproduced(potentiometric
biosensors),

3.movementofelectronsproducedinaredoxreaction(amperometricbiosensors),

4.lightoutputduringthereactionoralightabsorbancedifferencebetweenthereactantsandproducts
(opticalbiosensors),or

5.effectsduetothemassofthereactantsorproducts(piezoelectricbiosensors).

Therearethreesocalled'generations'ofbiosensorsFirstgenerationbiosensorswherethenormal
productofthereactiondiffusestothetransducerandcausestheelectricalresponse,secondgeneration
biosensorswhichinvolvespecific'mediators'betweenthereactionandthetransducerinordertogenerate
improvedresponse,andthirdgenerationbiosensorswherethereactionitselfcausestheresponseandno
productormediatordiffusionisdirectlyinvolved.

Theelectricalsignalfromthetransducerisoftenlowandsuperimposeduponarelativelyhighandnoisy
(i.e.containingahighfrequencysignalcomponentofanapparentlyrandomnature,duetoelectrical
interferenceorgeneratedwithintheelectroniccomponentsofthetransducer)baseline.Thesignal
processingnormallyinvolvessubtractinga'reference'baselinesignal,derivedfromasimilartransducer
withoutanybiocatalyticmembrane,fromthesamplesignal,amplifyingtheresultantsignaldifferenceand
electronicallyfiltering(smoothing)outtheunwantedsignalnoise.Therelativelyslownatureofthe
biosensorresponseconsiderablyeasestheproblemofelectricalnoisefiltration.Theanaloguesignal
producedatthisstagemaybeoutputdirectlybutisusuallyconvertedtoadigitalsignalandpassedtoa
microprocessorstagewherethedataisprocessed,convertedtoconcentrationunitsandoutputtoadisplay
deviceordatastore.

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