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ElectrochemicalSensors Editedby
GiuseppeMaruccio
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Contributorsix Prefacexi
Acknowledgmentsxiii
1Biosensors1
TarabFatima,ShikhaBansal,SaminaHusain,andManikaKhanuja
1.1Introduction1
1.2Characteristicparameter3
1.3Electrodesystems4
1.4Biorecognitionelements9
1.5Transducers12
1.6Typesofbiosensors19
1.7Futureprospectsandconclusion22 References23
2Electrochemistry—Conceptsandmethodologies31
NahidChaudharyandManikaKhanuja
2.1Electrochemicalcells31
2.2Theelectrochemicalprocessesandequation34
2.3TheNernstEquation:Activityandpotential38
2.4Conclusion49 References49
3Metal-basedelectrodes51
BehzadRezaeiandNedaIrannejad
3.1Background51
3.2Metal-basedelectrodepreparation52
3.3Platinum-basedelectrodes54
3.4Gold-basedelectrodes55
3.5Copper-basedelectrodes56
3.6Enzymeimmobilizationmethods56
3.7Irreversibleenzymeimmobilizationmethods57
3.8Reversibleimmobilizationmethods59
3.9Specificstudyofenzymeimmobilizationonmetal-based electrodes60 References71
4Carbonandcarbonpasteelectrodes79
ShwetaJ.Malode,PradakshinaSharma,MohdRahilHasan, NagarajP.Shetti,andRonaldJ.Mascarenhas
4.1Background79
4.2Workingofcarbonelectrodesinbiosensorfabrication84
4.3Cleaningofcarbonelectrodes88
4.4Chemicalmodificationsforbiomolecules conjugation91
4.5Recentbiosensorsbasedoncarbonelectrodes94
4.6Usesofcarbonnanomaterials(CNMs)asbio-sensing99
4.7Advantagesanddisadvantages100
4.8Toxicityofcarbonnanomaterials104
4.9Conclusion104
4.10Futureperspective105 References105
5Mercury115
PradakshinaSharma,NeelamYadav,AnilKumarChhillar,and JogenderSinghRana
5.1Background115
5.2Recentbiosensorsbasedonmercuryelectrodes119
5.3Suppliers138
5.4Conclusion138 Acknowledgment138 Conflictofinterest138 References138
6Nanostructuredelectrodes147
MaheshM.Shanbhag,ShikandarD.Bukkitgar,PradakshinaSharma, andNagarajP.Shetti
6.1Background147
6.2Workingofnanostructuredelectrodesinbiosensor fabrication151
6.3Cleaningofnanostructuredelectrodes155
6.4Chemicalmodificationsforbiomolecule conjugation157
6.5Recentbiosensorsonnanostructured electrodes162
6.6Advantagesanddisadvantages165
6.7Suppliers165 References166
7Three-dimensionalelectrodes177
NedaIrannejadandBehzadRezaei
7.1Background177
7.2Workingof3Delectrodesinbiosensorfabrication179
7.3Chemicalmodificationsandfabricationstrategies181
7.4Three-dimensionalgraphenecomposites182
7.5Chemicalvapordeposition184
7.6Lithographicallydefinedthree-dimensionalgraphene structures184
7.7Hydrothermalmethod186
7.8Support-assistedandchemicallydepositedthree-dimensional graphene186
7.9Directelectrochemicalmethods188
7.10Keyfeaturesof3Dgraphenecompositesandtheirapplicationin electrochemicalsensing189
7.11Recentbiosensorson3Delectrodes:Wearableelectrochemical biosensors191 References202
8Biologicalrecognitionelements213 Ravina,DeepakKumar,MinakshiPrasad,andHariMohan
8.1Background213
8.2Biologicalrecognitionelements214 8.3Receptors214
8.4Comparisonofdifferentbiologicalrecognitionelements226 8.5Suppliers232 References232
9Miniaturizationdevices:Ananotechnologicalapproach241 NitikaThakur,TruptiR.Das,SantanuPatra,MeenakshiChoudhary,and SudheeshK.Shukla
9.1Introduction:Ajourneyfrommacroscaletomicroscale miniaturization241
9.2Microfluidicsandlab-on-a-chipsystem:Applicationsand implications246
9.3Theprecisemicromillingprocess248
9.4Newerdevices:Applicationandincorporationfordiagnosisand detection250 References256
10Microfluidicsandlab-on-a-chip261 AshishMathurandSouradeepRoy
10.1Background261
10.2Microfluidicplatforms263
10.3Designofmicrofluidicchannels266 10.4Fabricationofmicrofluidicdevices267 10.5Glass-basedmicrofluidicdevices274
10.6Silicon-basedmicrofluidicdevices276
10.7Recentmicrofluidic-basedbiosensors277 10.8Conclusions284 References284 Index289
Contributors ShikhaBansal CentreforNanoscienceandNanotechnology,JamiaMilliaIslamia, NewDelhi,India
ShikandarD.Bukkitgar DepartmentofEngineeringChemistry,KLEInstituteof Technology,Hubballi,Karnataka,India
NahidChaudhary CentreforNanoscienceandNanotechnology,JamiaMillia Islamia,NewDelhi,India
AnilKumarChhillar CentreforBiotechnology,MaharshiDayanandUniversity, Rohtak,Haryana,India
MeenakshiChoudhary TheSwissInstituteforDrylandEnvironmentalandEnergy Research,TheJacobBlausteinInstitutesforDesertResearch,Ben-GurionUniversity oftheNegev,MidreshetBen-Gurion,Israel
TruptiR.Das CIPET:InstituteofPetrochemicalsTechnology(IPT),Patia, Bhubaneswar,India
TarabFatima CentreforNanoscienceandNanotechnology,JamiaMilliaIslamia, NewDelhi,India
MohdRahilHasan DepartmentofBiotechnology,SchoolofChemicalandLife Sciences,JamiaHamdard,NewDelhi,India
SaminaHusain CentreforNanoscienceandNanotechnology,JamiaMilliaIslamia, NewDelhi,India
NedaIrannejad DepartmentofChemistry,IsfahanUniversityofTechnology, Isfahan,Iran
ManikaKhanuja CentreforNanoscienceandNanotechnology,JamiaMilliaIslamia, NewDelhi,India
DeepakKumar DepartmentofBiotechnology&MolecularMedicine/Microbiology, Pt.B.D.SharmaPostgraduateInstituteofMedicalSciences,Rohtak,Haryana,India
ShwetaJ.Malode DepartmentofChemistry,SchoolofAdvancedSciences,KLE TechnologicalUniversity,Vidyanagar,Hubballi,Karnataka,India
RonaldJ.Mascarenhas ElectrochemicalResearchGroup,DepartmentofChemistry, St.Joseph’sCollege(Autonomous),Bangalore,Karnataka,India
AshishMathur DepartmentofPhysics;CentreforInterdisciplinaryResearchand Innovation(CIDRI),UniversityofPetroleumandEnergyStudies,Dehradun, Uttarakhand,India
HariMohan CentreforMedicalBiotechnology,MaharshiDayanandUniversity, Rohtak,Haryana,India
SantanuPatra InstituteofAdvancedMaterials,IAAM,Ulrika,Sweden
MinakshiPrasad DepartmentofAnimalBiotechnology,LalaLajpatRaiUniversity ofVeterinaryandAnimalSciences,Hisar,Haryana,India
JogenderSinghRana DepartmentofBiotechnology,DeenbandhuChhotuRam UniversityofScienceandTechnology,Murthal,Haryana,India
Ravina CentreforMedicalBiotechnology,MaharshiDayanandUniversity,Rohtak, Haryana,India
BehzadRezaei DepartmentofChemistry,IsfahanUniversityofTechnology,Isfahan, Iran
SouradeepRoy CentreforInterdisciplinaryResearchandInnovation(CIDRI), UniversityofPetroleumandEnergyStudies,Dehradun,Uttarakhand,India
MaheshM.Shanbhag DepartmentofEngineeringChemistry,KLEInstituteof Technology,Hubballi,Karnataka,India
PradakshinaSharma DepartmentofBiotechnology,SchoolofChemicalandLife Sciences,JamiaHamdard,NewDelhi,India
NagarajP.Shetti DepartmentofChemistry,SchoolofAdvancedSciences,KLE TechnologicalUniversity,Vidyanagar,Hubballi,Karnataka,India
SudheeshK.Shukla SchoolofBiomedicalEngineering,ShobhitInstituteof Engineering&Technology(Deemedto-be-University),Modipuram,Meerut,India
NitikaThakur SchoolofAppliedScienceandBiotechnology,FacultyofApplied Sciences&Biotechnology,ShooliniUniversityofBiotechnology&Management Sciences,Solan,HimachalPradesh,India
NeelamYadav DepartmentofBiotechnology,DeenbandhuChhotuRamUniversity ofScienceandTechnology,Murthal,Haryana,India
Preface Thisistheeraofsensorswhichareeverywherearoundustocollectdataformonitoringpurposesandimprovingourlife.Inparticular,electrochemicalsensorshave attractedsignificantinterestfortheirnumerousapplications,especiallyinthebiosensingfield.Biosensorsareanalyticaldevicesthatareabletodetectspecificanalytes usingbiologicalcomponentsandconvertthemintomeasurablesignalsusingoptical, electrochemical,thermal,ormass-basedtransductionmechanisms.
Inelectrochemicalbiosensors,threeelectrodes,namelyworkingelectrode(WE), referenceelectrode(RE),andauxiliary/counter(CE)electrode,aretypicallyused. Electrochemicalreactionsandelectrontransfertakeplaceatthesurfaceoftheworkingelectrodes.Therefore,thesensitivityandselectivityofelectrochemicalsystems areinfluencedbytheirintrinsiccharacteristics.Platinum,gold,carbon,andmercury arethemostcommonmaterialsusedforworkingelectrodesduetotheirelectrochemicalinertness,highelectricalconductivity,andeaseoffabricationinvariousforms.In thisbook,thecharacteristicsandfunctionalityofthesematerialsarediscussedalong withthecriteriadrivingtheirchoiceandsomeremarkableapplicationsforthedetectionoforganicandinorganicanalytesincorporatingelectrocatalysts,mediators,modifiers,orenzymes.
Recently,theemergenceofnanostructuredmaterialsandopportunitiesforminiaturizationofsensorshaveopenedupnewpossibilitiesforadvancementintermsof sensitivity,selectivityandstability,lowersampleconsumption,improvedresponse time,andapplicabilityinresource-limitedsettings.Thesetopicsarealsoaddressed inthepresentbook.
Thegoalofthisbookistodeliverawidespreadandcriticaloutlineofthedifferent methodsusedfortheconstructionofelectrochemicalelectrodesandsensors.These platformsmaypavethewayforthedevelopmentofportableelectronicpoint-of-care devicesforapplicationsrangingfromenvironmentalanalysistobiomedicaldiagnostics.Thepresentbookcanserveasaresourceforteachersandstudentsdealing withelectrochemicalstudiesandlookingfortechnologiesfromresearchto commercialusage.
Acknowledgments Wethankalltheauthorswhosesincereworkgreatlyhelpedusinproducingthisbook. Wealsothankallthescientistsreferencedthroughoutthebookwhosecontributions havebeenveryhelpfulincompilingalltheinformationrelatedtothetopicsdiscussed inthisbook.
Dr.Narangthanksherparentsandfamilywhoalwaysencouragedhertoaccomplishseveralacademicachievements.Shededicatesthisbooktoallworkingwomen whokeeptheirchildrenoutofsightwithheavyhearts.
Biosensors 1 TarabFatima,ShikhaBansal,SaminaHusain,andManikaKhanuja CentreforNanoscienceandNanotechnology,JamiaMilliaIslamia,NewDelhi,India
1.1Introduction Abiosensorisadevicethatiscapableofmeasuringaspecificchemicalorbiological analytebyproducingsignalswhichareproportionaltoitsquantityorconcentration.A basicstructureofbiosensorsisgenerallycomprisedofthefollowingcomponents:An analyte,abiologicalrecognitionelement,whichisalsocalledabioreceptor,atransducerandelectroniccomponentsthataregenerallyconnectedwithtransducersand display,asshownin Fig.1.1.
Thispotentiallyinnovativeanalyticaldevicecanbeusedinawiderangeoffields suchasbiomedicine,diagnosis,environmentalremediation,anddrugdiscovery. ClarkandLyonsin1962inventedthefirstbiosensorforthedeterminationofglucose inbiologicalsamplesutilizingtheelectrochemicalmethodandglucoseoxidase immobilizedelectrodes [1,2].Afterthisinnovation,unbelievableprogresshasbeen madeinthefield.Biosensorsimprovethequalityoflifewiththeirseveralapplications.Oneofthemajorapplicationsisinthesensingofabiomoleculewhichiseither forthediagnosisofdiseasesortherapeutics.Biosensorswerealsosuccessfully implementedinthemonitoringoffoodquality,nutritionalvalue,andadulteration [3,4].Thebiosensingtechnologyprovedtobeverysuitabletofulfilltheneedfor cost-effectiveapproachesanddisposableplatforms.Ontheotherhand,biosensors requiretoimprovetheirworkingtimeandstabilitytomeettherequirementfor “long-termmonitoring”detectionplatformsandthedetectionoftoxicchemical andbiologicalanalytesindefensesecurity [5].
Irrespectiveoftheapplication,miniaturizationhasbeenprovedtobeverybeneficial andleadstoabettersignal-to-noiseratio, consumptionofsmallervolume,andlowpower utilizationresultinginlowcost.Furthermore,whenthesizeisreducedtothenanoscale, thesurfacetovolumeratioincreasesresultinginanincreaseintheactiveareaforsensing whichinturnenhancesthesensitivityandselectivity.Thisalsoincreasedthespecificbindingefficiencyandreducedthenon-specificbindingofthemolecule [6].
Recentlynanomaterialsaregaininghugeattentioninthefieldofbiosensingdueto theiruniqueandexcellentproperties.Nanomaterialssuchasgrapheneandtransition metaldichalcogenidesprovedasabeaconforbiosensingplatformandopenedvarious possibilitiesineveryresearchfield.Theintegrationofnanomaterialsinthefabrication ofelectrodesallowstheleastlimitofdetectionwithwidelinearrangesandhighsensitivity [7–9].Fromthelastfewdecades,therehasbeenlargeliteratureonbiosensors. Undoubtedly,thesebiosensorsprovedasanexcellentplatformnotonlyinacademics butalsoinindustries. ElectrochemicalSensors.
Fig.1.1 Structureofabiosensorrepresentingbiorecognitionelements(antibody,DNA, enzyme,andcell)interfaceandtransducer.
1.1.1Analyte Achemicalorbiologicalentitythatissubjectofinterestandneedstobedetermined. Forexample,inaglucose-basedbiosensor,“Glucose”isananalyte.
1.1.2Biorecognitionelement(bioreceptor) Abiologicalentitythatisspecificallycapableofidentifyingtheanalyteisknownas thebiorecognitionelementorbioreceptor.Someexamplesofbioreceptorsare enzymes,deoxyribonucleicacid(DNA),ribonucleicacid(RNA),antibodies,aptamer, etc.Thisbiorecognitionelementgivessignalscorrespondingtothespecificanalytein theformofchangeincolor,current,mass,heat,pH,etc.
1.1.3Transducer Transducersplayacrucialroleinthegenerationofasignalasthisconvertsoneform intoanotherform.Inabiosensor,whenthebiorecognitionelementprovidesaspecific signalbyrecognizingtheanalyte,thetransducerhelpstoconvertthisrecognition eventintoareadableormeasurableform.Forexample,inanamperometricelectrochemicalbiosensor,alterationincurrentisproportionaltotheamountofanalyte presentattheinterface.
1.1.4Electricalsignalanddisplay Theelectronicsystemanalyzesthesignalgivenbythetransducer,helpsinsignal amplificationandconvertsthesignalfromanalogtodigitalform.Theseamplified signalsarethenrepresentedbythedisplaycomponentofthebiosensor.Theobtained signalfromthedisplaycanbeinvariousformssuchasgraph,image,numeric,tabular.
1.2Characteristicparameter Theperformancesofthebiosensorcanbeinterpretedbasedonthefollowing parameters.
1.2.1Selectivity
Selectivityisoneofthemostimportantparametersandconcernsthecapabilityofa biorecognitionelementtodetectaspecificanalyteinthepresenceofinterferentand contaminantmolecules.Forexample,ifweareusinganantibodyasarecognitionelementorbioreceptorwhichwasimmobilizedonthesurfaceoftheworkingelectrode, whenthesolutioncontainingantigensreachesthesurfaceoftheworkingelectrode, thisleadstoantigen-antibodyinteractionandtheabilityoftheantibodytointeract withthespecificantigentogiveaproportionatesignaldeterminestheselectivity ofthefabricatedbiosensor.
1.2.2Stability Stabilityisthecapabilityofthebiosensortomaintaintheworkingperformancefora certainperiodoftime.Stabilityisthedegreeofsusceptibilityinresponsetoadisturbancearoundthesystem.Theinterferencearoundthebiosensingsystemcancausean errorandaffecttheaccuracyandperformanceofthebiosensor.Anotherimportant factorthataffectstheperformanceorstabilityofthebiosensoristhedegradation ofthebiorecognitionelementoveraperiodoftime.
1.2.3Sensitivity
Sensitivityisrelatedtotheminimumamountofanalytethatcanbedetectedbyafabricatedbiosensor,alsoknownasthelimitofdetection.Thelimitofdetectionofthe detectedanalytecanbecalculatedbyusingthe3-sigmamethod.Thisisaveryimportantparameterindefiningtheperformanceofthebiosensor.
1.2.4Responsetime Theresponsetimeistakenbyafabricatedbiosensortoreach90%ofthetotalresponse ofthesignalafterexposuretotheanalyte.Forexample,inaDNA-basedbiosensor,it dependsonthetimetakenbythehybridizationafterexposuretotheanalyte.
1.2.5Linearity Linearityconcernsrelativedeviationofanexperimentallydefinedcalibrationgraph fromanidealstraightline.
1.3Electrodesystems 1.3.1Twoelectrodesystems
Twoelectrodesystemconsistofaworkingandacounterelectrodeasshownin
Fig.1.2.Thereactionoftheanalytewhichneedstobedeterminedoccursontheworkingelectrodeandacounterelectrodehelpstocompletethecircuit.Thepotential(EA) istobeappliedbetweentheworkingandacounterelectrodeandthechangeinthe currentofthecorrespondingreactionismeasuredfromtheworkingelectrode.The counterelectrodeinthefollowingreactionperformstwoimportantfunctions.It allowschargetoflowthroughthecellbycompletingitscircuitandmaintainsaconstantinterfacialpotentialacrossthereactionirrespectiveofcurrent.Inatwo-electrode configuration,whencurrentisflowingthroughthecellatthetimeofreaction,sometimesitbecomeshardtomaintainaconstantcounterelectrodepotential(eC).Thereis alsoaproblemofthevoltagedropacrossthesolution(iRS)whichleadstopoorcontrol overthepotentialofaworkingelectrode(eW).
Theminiaturizationofelectrodeshasextensivelyimpactedtheperformanceof electrochemicalbiosensor.Microelectrodesexhibitappealingpropertiesinthe
Fig.1.2 Schematicrepresentationof(A)twoelectrodeconfigurationshowingcounterand workingelectrodeand(B)potentialgradientoftwo-electrodeconfiguration.
performanceofbiosensorduetofastresponsetime,comparativelylowohmicdropas aresultofsmallmeasuredcurrent,increasedrateofthemasstransferduetoradial diffusion,andcomparativelysmallelectricdoublelayercapacitance [10,11].Hence, biosensorsbasedontheelectrochemicalmechanismcanbeconductedefficiently usingtwo-electrodesetups.Generally,itwasobservedthattheperformanceofmicroelectrodemajorlydependsuponthematerialusedintheelectrode.Commonlyused materialsforelectrodesarenoblemetals,fluorine-dopedtinoxide(FTO)coatedglass, indium-tin-oxideconductiveglass(ITO),anddifferentformsofcarbon.Researchers wereusingnoblemetalsfordecadesinbiosensortechnologyduetotheirfastelectron transferkineticsandhighsensitivity,buttheyarefacingsomedrawbackssuchasthe increaseinbackgroundnoise,surfaceoxidation,andhighcostwhichlimitstheirapplication [12,13].Ontheotherhand,FTOandITO-basedelectrodesneedtimeconsumingpre-electrodetreatmentandcomplexmanufacturingprocesses.Therefore, nanomaterialsbasedoncarbon,bringarevolutiontothefieldofbiosensing.Carbon nanomaterialssuchasgraphene,carbonnanotubesandnanofibers,andgraphiticcarbonnitridegainingextensiveattentionduetolowcost,appealingelectrochemical properties,easeoffunctionalization,andwidepotentialwindow [14,15].Recently, theconductiveink-basedpaperelectrodesaredrawingattentionbecauseoftheirversatileproperties,easeofhandling,andlowcost.Disposabletwo-electrode—Prussian blue-basedpaperelectrodehasbeensuccessfullyfabricatedforthedetectionof H2O2 [16] Anotherstudyfabricatedanelectrochemical-basedpaperanalyticaldevice modifiedbyg-C3N4 forthedetectionofNorovirus-specificDNA.Thedeveloped paperelectrode-basedsensorshowedexcellentsensitivity,selectivitywithalow detectionlimitof100fMin5s [17].
1.3.2Threeelectrodesystems Threeelectrodesystemsconsistofaworkingelectrode,acounterelectrode,andareferenceelectrodeasshownin Fig.1.3.Threeelectrodesystemsovercomemanyproblemsofthetwo-electrodesystem.Theroleofthereferenceelectrodeiscontrollingand measuringthepotentialoftheworkingelectrodetoactasareferencewithouttransferringanycurrent.Theelectrochemicalpotentialshouldbeconstantatalowcurrent densityforthereferenceelectrode.AsthecurrentpassesthroughthereferenceelectrodeisextremelynegligiblewhichleadstoaverysmalliRdropbetweenthereferenceandtheworkingelectrode(iRU).Thus,thisthree-electrodesystemoffersamore stablereferencepotentialandgivescompensationfortheiRdropthatoccurredacross thesolution.
Thisautomaticallyresultsinbettercontroloverthepotentialoftheworkingelectrode.Ag/AgClelectrodesandSaturatedCalomelElectrodearethemostcommon typesofreferenceelectrodesgenerallyusedintheElectrochemicalreaction.Therole ofthecounterelectrodeinthethree-electrodesystemistobalancethecurrentofthe workingelectrodebypassingthecurrentneededacrossthesolution.
Fig.1.3 Schematicrepresentationof(A)threeelectrodeconfigurationshowingcounter, working,andreferenceelectrodesand(B)potentialgradientofthree-electrodeconfiguration.
1.3.2.1Referenceelectrodes Thereferenceelectrodesareresponsibleforprovidingastableanddrift-freepotential asareferencevoltage.Thisreferenceelectrodecanalsobeusedtomonitortheapplied potentialfortheworkingelectrode.Standardhydrogenelectrodeisusedforthemeasurementofstandardelectrodepotentialsintermsofreductionpotential.SHEiscomprisedofplatinumsheetsorwiretoformanelectrodeinwhichplatinumis electroplatedtoformafinelayerofmetallicplatinumcontainedinanHClsolution onwhichhydrogengasbubblesat1atm.
Anotherreferenceelectrodethatisbasedonmetal-saltcontactinwhichsparingly solublesaltcontainingcationofthecorrespondingmetalisincontactsuchas (Hg/HgCl2)andAg/AgCl.Generally,thereferenceelectrodeiskeptisolatedfrom directcontactviathesaltbridgetomaintainstability.Becauseitisessentialforthe referenceelectrodepotentialtomaintainstabilityandshouldnotchangewith interferents.
1.3.2.2Counterelectrode Theworkofacounterelectrodeistomaintainpotentialapplyingtotheworkingelectrode.Thiselectrodehelpsinflowingofchargeandcompletesthecircuit.Generally, theyusedtobemadeofinertmaterialsuchasgold,carbon,orplatinum.Tominimize theerrorinthemeasuringcurrentattheelectrochemicalsetup,thesurfaceofthecounterelectrodeshouldbegreaterthantheworkingelectrode.Ouraimistoaccurately determineandstudythereactiontakingplaceatthesurfaceoftheworkingelectrode. Thisisthereasonthatthereactionoccurringattheworkingelectrodeshouldbeslow otherwisereactionoccurringatthecounterelectrodedominatethemeasuredelectric
current.Thisproblemovercomesbyusingthehighsurfaceareaastherateofthereactiondependsonthesurfaceoftheelectrode.Theelectricalconductivityofthecounter electrodemustbegoodbecauseifitisnotsothatmayresultinpoorflowofchargeat theinterfaceofthecounterelectrode.
1.3.2.3Workingelectrode Theworkingelectrodeisthemostimportantpartoftheelectrochemicalreactionsetup becausethereactionofinterestoccursatthesurfaceoftheworkingelectrode. Forthe idealconditiontheworkingelectrodeshouldbeinert,goodelectricalandlong-term stability,reproducibility,goodsignal-to-noiseratio,uniformity,withstandwithvarioussolvents,andnon-toxic.Tomatchtheidealconditions,theworkingelectrodeis generallymadeofnoblemetalssuchasgold,silver,copper,platinum,carbonpaste, andgraphite.Sometimesitcanalsobecoatedwithdifferentmaterialstoachieveideal conditionsneedforthereaction [18].
Theelectrodeswithahighvalueofresistanceleadtoanohmic(IR)drop,which developstheneedforhighercellpotential.Thisextraenergymaybelostintheformof heatinthereactionmediumandcreatesinterferencetothereactionmedium [19].This problemcanlimitthechoiceofmaterial;severalsurfacemodificationsandfunctionalizationofmaterialwereusedtoovercomethisissue [20].
Amongallthenoblemetals,goldandsilvernanoparticlesarewidelyusedinbiosensingapplicationsduetotheiruniqueopticalandoptoelectronicproperties,biocompatibility,andeaseofsurfacemodification.Thesenanoparticlesshowedthe interestingSPRphenomena,inwhichwhenthelightofaspecificwavelengthirradiatedonthesurfacecausesanoscillationoftheelectronscalledsurfaceplasmonicresonance.Otheradvantagesofusinggoldnanoparticle-modifiedelectrodesarehigh(S/ N)ratio,improvedelectroactivespeciesdiffusion,enhancedselectivity,andcatalytic activity [21].Platinumnanoparticlesarealsogainingattentioninthefieldofbiosensingduetotheirexcellentelectrocatalyticproperties.Ptnanoparticlesshowsignificantelectrontransferprocesseswhichmaybeattributedtotheircrystalstructure, crystallographicorientation,andsurfaceproperties.
Einagaetal.havefabricatedanelectrochemicalsensorbasedonPtnanoparticles depositedontheboron-dopeddiamondsurfaceforthesensingofhydrogenperoxide (H2O2)withalimitofdetectionof100nM [22].Metaloxidenanoparticlesarealso quitefavorableintheareaofbiosensing.Mostlytheyaresynthesizedbythewetchemicalreductionmethodandresultsinvariousmorphologiessuchasnanorods,nano comb,nanobelts,etc.YangandcoworkerssynthesizedTiO2 nanocrystalswithhighly reactivesurfacesforthedetectionofglucosewithalimitofdetectionof0.83 μM [23]. Severaleffortshavebeenmadetoimprovetheperformanceofthemetaloxidemediatedsensorsbytuningthepropertiesusingcontrollablesynthesis.ZnOand TiO2 arethewidelyusedmetaloxidenanomaterialinthefieldofbiosensorsdue totheirbiocompatibility,tunableproperties,andeaseoffunctionalization.Narang andcoworkersfabricatedaZnOnanorods-basedsensorforthedetectionoftherecreationaldrugMDMA.Theyusedauniquepaperanalyticaldevicefabricatedusing conductingcarbonink-basedsensorwithawidelinearrangeof1 μ to1mM [24].
Thedevelopmentofscreen-printedelectrodes(SPE)istheapproachtowardsmoderntechnologywithhighdemandduetoitsexcellentspecificity,sensitivity,low responsetime,andeaseofhandling.Screen-printedelectrodesarecapableofallowing alargenumberofexperimentsusinglowvolumeinasmallperiod.Theydonot requiretime-consumingpre-treatmentofelectrodesandcomplexprocessesascomparedtootherelectrodes.BiosensorsbasedonSPEarecommonlyusedinresearch areasofmedicines,agriculture,foodindustry,andenvironmentalremediation.The materialsusedinscreen-printedelectrodescanbeselectedbasedonresearcharea andrequirementssuchasgold,silver,andcarbon.Viswanathanetal. [25] fabricated animmunosensorbasedonmultiwallcarbonnanotube-polyallylaminemodifiedSPE preparedbyimmobilizingantibodiesofanti-Escherichiacoli,anti-Campylobacter, andanti-Salmonella.Theyachievedaverylowlimitofdetectionof400cells/mL forSalmonellaandCampylobacterand800cells/mLfor E.coli.Another researcher-developedimmunosensorbasedoncarbonscreen-printedelectrodesfor theelectrochemicaldetectionof E.coli and Enterobactersakazakii [26].Bonanni etal.introducedMWCNTsmodifiedSPEgenosensorbasedonimpedimetricdetectionmethod [27].Inanotherstudy,electrochemicaldetectionofnaloxonehasbeen reported.Theymodifiedscreen-printedelectrodesusingMIP(molecularlyimprinted polymer)andMWCNTusingthetechniqueofelectropolymeriation [28,29].Bartlett etal.usedthecarbonscreen-printedelectrodesforthedetectionofmethamphetamine (MAMP)byusing N,N-(1,4-phenylene)-dibenzenesulfonamideasamediatorwhich actsasaredoxindicatorandhelpsinthedeterminationofMAMPwithLODof 400ng/mL [30].ChaitaliandcoworkersusedMoS2 nanosheetsmodifiesgoldSPE forthedetectionofchikungunyaasshownin Fig.1.4.Theyfabricatedanelectrochemicalgenosensorinwhichdetectionwasbasedoncapturingofhybridizationevent usingmethyleneblueasaredoxindicatorwithLODof3.4nM [37].
Table1.1 showsthematerialusedforthemodificationoftheworkingelectrode.
Fig.1.4 Screen-printedelectrodemediatedelectrochemicaldetectionofchikungunyavirus showinghybridizationofprobeandtargetDNA.
FromC.Singhal,M.Khanuja,N.Chaudhary,C.S.Pundir,J.Narang.Detectionofchikungunya virusDNAusingtwo-dimensionalMoS2nanosheetsbaseddisposablebiosensor.Sci.Rep.8(1) (2018),1–11. https://doi.org/10.1038/s41598-018-25824-8
Table1.1 Differentnanomaterialsareusedforthemodificationoftheworkingelectrodefor thedetectionofvariousanalytes.
S.no.MaterialElectrodeAnalyte
Limitof detectionReferences
1AunanoparticlesGlassycarbonAs3+ 32.5pM [21]
2Au-grapheneGlassycarbonHg2+ 0.001aM [31]
3AgnanoparticlescarbonelectrodeInfluenza virus 0.4pM [32]
4AgNPs-grapheneGoldelectrodeAIVH71.6pg/mL [33]
5Platinum nanoparticles Diamond electrode H2O2 100nM [34]
6b-Cyclodextrin-Pt nanoparticles/ graphene Glassycarbon electrode a-naphthol b-naphthol 0.23nM 0.37nM [35]
7ZrO2 nanocubesGold electrode Arsenic(III)5ppb [36]
8TiO2 GoldelectrodeGlucose0.83 μM [23]
9ZnOnanorodsInkprinted paper electrode MDMA0.1 μM [24]
10MoS2 nanosheetsScreenprinted electrode Chikungunya3.4nM [37]
1.4Biorecognitionelements Thebiorecognitionelementisthecentralpartofbiosensingandaninfluentialtoolto definespecificity.Themajorworkofthebiorecognitionelementistodevelopthebiosensorwithmorespecificity.Thebiosensorisknowntobemorespecificwhenthereis astrongaffinityorselectivitybetweenthebiorecognitionelementandtheanalyteof interest.Biorecognitionelementsexistinseveralmolecularstructuresandformsthat directlyinfluencethecharacteristicsofabiosensor.Therefore,itisverynecessaryto understandthecharacteristicsandmorphologyofeachbiorecognitionelementto makethemspecificfortheanalyte.Thereisawiderangeofbiorecognitionelements usinginthefabricationofbiosensorsfromsynthetictonaturallyoccurringintheenvironment(Fig.1.5).Enzymesandantibodiesarethenaturallyoccurringbiorecognition elementthatisderivedfromthenaturallyexistingbiologicalmechanism.Theyfollow anaturallyoccurringmechanismtoprovidespecificitybyattachingtotheanalyteand givethecorrespondingsignals.Syntheticbiorecognitionelementsaredevelopedartificiallytomimicphysiologicallydefinedinteractions.Adifferentclassof biorecognitionelementspossessesadifferentmechanismtodefinerecognitionstructure.Someofthemareantibodies,enzymes,nucleicacid,aptamer,molecularly imprintedpolymers [38,39]
Fig.1.5 Schematicrepresentationofelementsofbiosensorshowingitscomponents:analyte, biorecognitionelements,interface,transducer,andsignal.
1.4.1Antibody Antibodiesarethe3Dstructureofaprotein,anapproximatesizeof 150kDa.These arepresentinbiochemicalpathwaysandextractedtouseforbiosensing [40].These antibodiesprovidehighspecificityandaccuracyforthespecificanalytedependson thestructurebycreatingauniquerecognitionpattern.Antibodycomprisesof“Y” shaped3Dstructurewhichconsistsoftwotypesofchains,light,andheavychain. Thearmsofantibodiesprovidethespecificdomainforthebindingofananalyte asshownin Fig.1.6.
Thebiorecognitionprocessofantibodydependsuponthebindingeventformby antibody-antigenimmunocomplex.Generally,antibodiesareimmobilizedonthesurfaceoftheworkingelectrodethroughcovalentlinkage.Thesecovalentinteractions aredevelopedbyimmobilizedantibodiesbyintroducingfunctionalgroupssuchas amino,carboxyl,aldehyde,etc. [41].Theworkingprincipleoftheantibody-basedbiosensorisbasedontheformationofanantibody-antigen(Ab-Ag)complex.
Fig.1.6 Schematicrepresentationofimmunosensorshowingantibodyasabiorecognition elementformingantibody-antigenimmunocomplex.
1.4.2Enzymes Enzymesareabiologicalcatalystprovidesbindingsitestoachievebiorecognitionof analytewithhighspecificityasshownin Fig.1.7.Thesespecificanalytesbindtothe cavitiesoftheenzymethroughvariousinteractionssuchaselectrostatic,hydrogen bonding,andseveralnon-covalentinteractions [42].Intheenzyme-basedbiosensor, bioanalytecapturesbythecavitiespresentonthesurfaceoftheenzyme,andthisrecognitioncomplexconvertintomeasurableresultsandismonitoredbyseveraltransductionmethodssuchasamperometric,potentiometric,etc. [43].Asshowninthe figureenzymeisimmobilizedonthesurfaceoftheelectrodeandthesurfaceis exposedtothebioanalytebutonlythetargetanalyteattachedtothespecificenzyme leadstobiosensingwithhighspecificity.Thus,biosensorsthatarebasedonthe enzyme-substraterelationshipareknownasanenzymaticbiosensor.Theseenzymatic biosensorsworkontwotypesofmechanismseitherbioanalyte-substratecomplex detectionorenzymeinhibition.ThemechanismofEnzymeinhibitionbasedenzymaticbiosensorbasedonreductionofenzymaticactivitybytargetbioanalyte [44] Iba ´ nezandco-workers [45] havefabricatedahighlysensitivenovelamperometricbasedbiosensorforthedetectionoflactatelevelsinembryoniccellcultures.They usedChitosan/multi-walledcarbonnanotubesmodifiedscreen-printedelectrodes fortheimmobilizationoflactateoxidaseenzyme.Themechanismisbasedonthe reactionoflactateoxidasewithlactatetoformpyruvateandH2O2. Thisenzyme-based biosensorhasbeenreportedtoprovideexcellentsensitivitywithlowresponsetime. Anotherstudybasedwasconductedtodetectcholesterolbyusingcholesterolesterase andcholesteroloxidaseenzymesforthereductionofhydrogenperoxidase.The enzymesareimmobilizedonthesilverpasteelectrodesandTritonX-100isused toenhancetheelectrocatalyticactivityofthereaction [46].Suaifanetal.developed aninnovativepaper-basedbiosensorforthedetectionof Staphylococcusaureus by usingtheenzyme S.aureus proteases.Thisbiosensorshowsexcellentresultswith lowcostandrapidmechanismwhichcanbeappliedtovariousfieldssuchasfood industry,clinicalsamples,andenvironmentalpollutants [47].
Fig.1.7 Schematicrepresentationofabiosensorshowingenzymeasabio-recognitionelement.
1.4.3DNA Nucleicacid-basedbiosensorsarealsoknownasGenosensorsworksontheprinciple ofhybridizationoftargetDNAwithcomplementaryDNAasshownin Fig.1.8. FirstlywedeterminethesequenceofthetargetDNAthantheDNAprobewillbe designedartificiallyandimmobilizedonthesurfaceoftheworkingelectrodeasa biorecognitionelement.ThetwostrandsofDNAhybridizedwiththehelpofhydrogen bondingwithcomplementarybasepairswhichisadenine,thymine,guanine,cytosine (A ¼ T,G ≡ C).AccordingtoWatsonandCrickmodeladeninebindswiththymine withdoublehydrogenbondsandguaninebindswithcytosinewithtriplehydrogen bonds.AfterthehybridizationofprobeDNAwiththetargetstrand,thishybridization eventwillberecognizedbyvarioustransductionmethodssuchasamperometric, potentiometriccolorimetric,etc. [48,49].
1.4.4Aptamer Theaptamer-basedbiosensorisanadvancedbiosensorwithawiderangeofapplicationsinbiosensingduetoitsstrongabilitytorecognizevariousanalyteswithhigh specificitysuchasproteins,DNA,metalions,smallmolecule,wholecells.Aptamers areasinglestrandofoligonucleotidesdevelopedusingaselectionprocessknownas SystemicEvolutionofLigandsbyExponentialEnrichment(SELEX) [50].SELAXis acombinatorialchemistryprocessfortheproductionofrandomnucleotidesequences eithersingle-strandedDNAorRNAwithastrongaffinitytobindspecificallytothe targetDNA.
1.5Transducers Atransducerisanimportantcomponentofbiosensorsthatcantransformaphysical quantityoranon-electricalquantitylikesound,light,pressure,ortemperatureintoa proportionalelectricalquantitysuchasvoltageorcurrent(Fig.1.9) [51].
Fig.1.8 SchematicrepresentationofgenosensorshowingDNAasabiorecognitionelementto detecttheanalytebyhybridization.
Transducersconsistoftwomaincomponents:
1. Sensingelement.
2. Transductionelement.
Otherthanthese,thetransducercontainsvariouspartslikeamplifiers,signalprocessors,powersupply,calibrationandreferenceunits,etc.
Sensingordetectorelement:Itgivesaresponsetothephysicalquantitytobe detectedwhichisdependentonthephysicalphenomenon.
Transductionelement:Thefunctionofthetransductionelementistochangethe sensoroutputobtainedfromthesensingelementintoasuitableform.Thetransduction elementisalsonamedasecondarytransducer.
1.5.1Parametersgoverningthetransducer’schoice Therearemanyfactorsthatgoverntheselectionofthetransducersforinvestigating thephysicalparameter.Thesefactorsaregivenasfollows:
l Sensitivity:Transducer’ssensitivityshouldbesufficientfordeliveringtheperceptibleyield.
l Operatingrange.Theoperatingrangeofatransducermustbelongforuninterruptedutility.
l Accuracy:Thetransducersgiveprecisionaftercalibration.Thefundamentalrequirementfor commercialapplicationsissmallprecisionforrepeatability.
l Crosssensitivity:Crosssensitivityisconsideredwhilemeasuringthemechanicalparameters.Duetothesensitivity,thetransducersmayproduceavariablemeasuredsignalfor thedifferentplanes.
l Errors:Considerationoftheinput-outputrelationsobtainedbythetransferfunctionavoids theprobableerrors.
l Transientandfrequencyresponse:Thetransducershouldbeoperatableinthepredetermined timedomainsuchassettingtime,peakovershoot,smalldynamicerror,andrisetime,etc.
l Loadingeffects:Theinputimpedanceofthetransducersshouldbehighandtheoutput impedanceshouldbelowtoavoidprobableerrors.
l Environmentalcapability:Thetransducersshouldbecompatiblewithenvironmentalconditionssuchascorrosive,environmentwithhighpressure,andshocks.
l Insensitivitytounwantedsignals:Thetransducershouldfilterouttheunwantedsignals.
l Usageandruggedness:Theknowledgeofdurability,weight,andsizeofthetransducer beforeaselectionisimportantforitseffectiveutility.
l Stability:Thetransducershouldbestableenoughfortheselectedoperation.
l Staticcharacteristic:Thelinearityandresolutionofthetransducershouldbehighandits hysteresisshouldbelow.
Fig.1.9 Aschemedepictsamodeloftransducer.
1.5.2Classificationoftransducers Thecharacterofthetransducerdeterminestheperformanceofabiosensor.Various transductionmethodsareusedaccordingtothedifferenttypesofbiosensorsutility. Themostcommonvarietiesoftransducersare:optical,thermaldetection,piezoelectric(massdetectionmethods),calorimetric,electrical,andelectrochemical
1.5.2.1Electricaltransducers Conductometric(impedimetric)transducers Theoutput,equivalenttothequantitymeasured,obtainedfromanimpedimetrictransducerisintheformofanelectricalsignal.Theoutputsignalthusobtainedcanbeusedto monitorordisplaythephysicalquantity.Whenabiochemicalreactiontakesplace,the overallconductivityofthechemicalsolutionchangesduetotheproductionofelectrons orionicspecies.Therefore,animpedimetrictransducermeasurestheelectricalconductanceorelectricalresistanceofthesolution.AndtheuseofAC(sinusoidal)voltagegeneratestheelectricfieldthatleadstotheminimizationofanunwantedsignalproducedby Faradaicprocesses,double-layercharging,andconcentrationpolarization [52].Ingeneral,thefunctionoftheimpedancebiosensorisbasedontheWheatstonebridge [53]. TheadvancementsinimpedimetricbiosensorswerereviewedbyGuanetal. [54] and Muhammad-Tahiretal.reportedtheapplicationsofconductometricbiosensors [55].
Ion-sensitivetransducers Earlier,theion-sensitivebiosensorsbasedonion-selectivefield-effecttransistors (ISFETs)wereoneofthedifferenttypesofpotentiometricsensors.However,as perthelatestIUPACreport,ISFETshavebeenconsideredasthe4thcategoryofelectrochemicalsensors [56].ISFETisatypicalmetal/oxide/semiconductor(MOS)fieldeffecttransistor(FET)inwhichthegateisaseparatereferenceelectrode.Thisis attachedtotheareaofthegatebyanaqueoussolution [57].Thesurfaceofthe semiconductor-basedFETsision-sensitive.Becauseofthecouplingofthesemiconductorandtheions,theelectricalpotentialofthesurfacechanges.Thechangeinthe electricpotentialismeasuredbyusingtheISFETs.ISFETisfabricatedbycoatinga selectivelypermeablepolymerlayeronthesensorelectrode,whichfacilitatesthediffusionofionsandresultsinthechangeofFETsurfaceelectricalpotential.Duetothe polymercoatings,thesebiosensorsarealsonamedENFET(enzymefieldeffecttransistor) [58].Dzyadevychetal.reviewedtheenzymebiosensorsbasedonISFETs [59].
1.5.2.2Opticaltransducers Opticaltransducershavebeenusedinseveralbiosensorswithvarioustypesofspectroscopy,likeabsorption [60],fluorescence [61],andRaman [62].Opticaltransducers usephotonstogathertheanalyteinformation. [63].Theseareextremelysensitive,precise,compact,andeconomical.Theoutputsignalofthetransducerthatisdetectedis light.Themechanismofdetectionofanopticaltransduceriscontrolledbythesystem thatchangestheanalyteintoproductsthatactaseitheroxidantsorreductantsatthe workingelectrode [64].
