Electrochemical methods for ascorbic acid determination

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Electrochimica Acta

j ourna l h o me pa g e :w w w . e l s e v i e r . c o m / l o c a t e / e l e c t a c t a

Review Article

Electrochemical methods for ascorbic acid determination

Aurelia Magdalena Pisoschi

^∗

, Aneta Pop, Andreea Iren Serban, Cornelia Fafaneata

UniversityofAgronomicSciencesandVeterinaryMedicineofBucharest,FacultyofVeterinaryMedicine,105SplaiulIndependentei,050097,sector5, Bucharest,Romania

a r t i c l e i n f o

Articlehistory:

Received13October2013 Receivedinrevisedform 15December2013 Accepted20December2013 Available online 4 January 2014

Keywords:

Ascorbicacid Electrooxidation Potentiometry Voltammetry Amperometry

a b s t r a c t

Thepresentreviewfocusesonelectrochemicalmethodsforascorbicacidassessment.Theoccurence,role, biologicalimportanceofvitaminC,aswellasthenon-electrochemicalmethodsforitsassessmentare firstlyreviewed.Theelectrochemicalbehaviorofascorbicacidisthenillustrated,followedbyadescrip- tionofthepotentiometric,voltammetricandamperometricmethodsforvitaminCcontentestimation invariousmedia.Differentmethodsforthedevelopmentofelectrochemicalsensorsarereviewed,from unmodifiedelectrodestodifferentcompositesincorporatingcarbonnanotubes,ionicliquidsorvarious mediators.Fromthisperspective,theinteractionbetweenthefunctionalgroupsofthesensor’smaterial andtheanalytemoleculeisdiscussed,asitisessentialfortheanalyticalcharacteristicsobtained.The analyticalperformancesofthepotentiometric,voltammetricoramperometricchemicalandbiochem- icalsensors(linearrangeofanalyticalresponse,sensitivity,precision,stability,responsetimeetc)are highlightened.Thenumerousapplicationsofascorbicacidelectrochemicalsensorsinfieldslikefood, pharmaceuticalorclinicalanalysis,wherevitaminCrepresentsakeyanalyte,arealsopresented.

© 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction... 444

2. Ascorbicaciddeterminationbynon-electrochemicaltechniques. ... 444

3. Electrochemicalbehavior:theirreversibilityofascorbicacid/dehydroascorbicacidredoxcouple... 445

3.1. Electrochemicalbehavioratunmodifiedelectrodes... 445

3.2. Electrochemicalbehavioratchemicallymodifiedelectrodes... 445

4. Potentiometricascorbicacidsensorsandbiosensors... 446

4.1. Composites... 446

4.2. Screenprintedelectrodes... 446

4.3. Modifiedfieldeffecttransistors... 446

5. Voltammetricandamperometricsensors... 447

5.1. Voltammetry/amperometryatbare/unmodifiedelectrodes... 447

5.2. Voltammetry/amperometryatchemicallymodifiedelectrodes... 448

5.2.1. Modifiedmetalelectrodes... 448

5.2.2. Modifiedcarbonaceouselectrodes... 448

5.2.3. Nanoparticlecompositesandceramiccomposites... 450

5.3. Amperometricenzymicassay:biosensors... 451

6. Interferencesfromcompundspresentinbiologicalmedia,pharmaceuticalsandfoodstuffs... 452

6.1. Interferencesinpotentiometry... 452

6.2. Interferencesinvoltammetry/amperometry... 452

6.2.1. Unmodified(bare)electrodes... 452

6.2.2. Modifiedmetalandcarbonaceouselectrodes ... 452

7. Analyticalperformancesofelectrochemicalascorbicacidsensors... 453

8. Someapplicationsofelectrochemicalascorbicacidsensorsinfood,pharmaceuticalandbiologicalfluidanalysis... 453

∗Correspondingauthor.Tel.:+0040213252901.

E-mailaddress:aureliamagdalenapisoschi@yahoo.ro(A.M.Pisoschi).

0013-4686/$–seefrontmatter© 2014 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.electacta.2013.12.127

9. Conclusions... 457 References... 457

1. Introduction

VitaminCisahydrosoluble,antioxidantvitamin,whichhasa␥- lactonestructure,andrepresentstheLenantiomerofascorbicacid, thebiochemicallyandphysiologicallyactiveform.Ascorbicacidis ahexanoicsugaracidwithtwodissociableprotons(pKa4.04and 11.34).Therefore,underphysiologicalconditions,itoccursasan ascorbateanion.

Ascorbicacid(AA)isknownforitsreductiveproperties,being easilyoxidatedtodehydroascorbicacid.Itactsasapowerfulantiox- idant which fights against free-radical induced diseases [1–6].

Plantsand mostanimals synthesizeascorbate from glucose. In primitivefish,amphibiansandreptiles,ascorbatesynthesistakes placeinkidney,whereasformammalsliveristhesiteofsynthe- sis,wheretheenzymeL-gulonolactoneoxidaseconvertsglucose toascorbicacid[7,8].DuetoageneticmutationthatinduceaL- gulonolactoneoxidasedeficiency,humans,someotherprimates, andguineapigsareunabletosynthesizeascorbicacid,sotheyneed totakeitfromdiet[9].

Ascorbicacidcanscavengesingletoxygen,oractaschelating agent.Thisisclaimedasthebasisofitsabilitytoprotectoxidizable constituents,includingphenolicandflavorcompounds,therefore beinglargelyusedasanantioxidantinfoodsanddrinks.Studies performedonwineshowedthat thebenefitof ascorbicacidas anantioxidantconsistsinitscapacitytoscavengemolecularoxy- gen,beforetheoxidation ofphenolic compounds.Ascorbicacid alsoappearstobeanidealfree-radicalscavenger,becauseitreacts rapidlywithhydroxyl(andother)radicalstoformrelativelyunre- activeradicalsthatdonotreadilypropagate[10].

VitaminCcanbefoundinmanybiologicalsystemsandfood- stuffs,namelyfreshvegetablesandfruits,asthemostubiquitous water-solublevitamineverdiscovered.Richsourcesincludeblack- currant, citrus fruit, leafy vegetables,tomatoes, green and red peppers,etc.VitaminCisinvolvedironabsorption,collagensyn- thesisandimmuneresponseactivationandparticipatesinwound healingandosteogenesis,helpsmaintainingcapillaries,bones,and teeth[1–6].

Ascorbicacidexcesscanleadtogastricirritation,andoneof itsmetabolites, oxalicacid, causesrenalproblems[11].Insome cases, excessive quantities of ascorbic acid may result in the inhibitionofnaturalprocessesoccurringinfoodandcancontribute totaste/aromadeterioration;[12].Anotherdrawbackofascorbic

Figure1. CyclicvoltammogramsobtainedwithaPtworkingelectrodefordifferent ascorbicacidconcentrations,expressedasmmolL⁻¹:20(line1),15(2),10(3),5(4), 2.5(5),1.25(6),0.625(7)and0.31(8);potentialscanrate50mV/s;a0.1molL⁻¹KCl solutionwasusedassupportingelectrolyte[60].

acidexcessisitsabilitytoactasastrongantioxidantonlyinaqueous mediaandintheabsenceofheavymetalcations.Inthepresence ofheavymetalcations,itcanevenactasaprooxidant:ascorbate ion is anexcellent reducing agentthat canreduce ferric(Fe³⁺) toferrous(Fe²⁺)iron,whilebeingoxidizedtodehydroascorbate [7,13,14].

2Fe²⁺+Ascorbate→ 2Fe³⁺+Dehydroascorbate

The metalion resulted can be subsequently reduced, reoxi- datedandagainreduced,enteringaredoxcyclegeneratingreactive oxygen species [7,13,14]. Thus, depending on the coordination environment, Fe²⁺ canreactwithO₂,reducing ittosuperoxide radicalanion,whichdismutestoH2O2andO2[7].

Fe²⁺+O₂→Fe³⁺+O₂^•-

2O2•−+2H⁺→O2+H2O2

InaclassicFentonreaction,Fe²⁺reactswithH₂O₂togenerate Fe³⁺andthestrongestreactiveoxygenspecies(ROS),namelythe veryoxidizinghydroxylradical.

Fe²⁺+H2O2→Fe³⁺+OH·+OH⁻

ThepresenceofascorbatecanallowtherecyclingofFe³⁺back toFe²⁺,whichinturnwillcatalysetheformationofhighlyreac- tiveoxidantspecies.Thisprooxidantactivitymaybedisplayedin thepresenceofheavymetalcationsandintheabsenceofother antioxidantcompounds,suchasSO2[10].

Ascorbicacidisalabilesubstanceasitiseasilydegradedby enzymesandatmosphericoxygen.Itsoxidationisacceleratedby excessiveheat,light,and heavymetalcations[2].Ascorbicacid isfrequentlyusedasanantioxidantinfoodindustrytoprevent unwantedchangesincolororflavor.Asanelectrondonor,ascor- bicacidservesasoneofmostimportantsmall-molecular-weight antioxidantswhichcontributestothetotalantioxidantcapacity- animportantqualityindicatoroffoodsanddrinks[15–17].Due tothecrucialroleofvitaminCinbiochemistryandinindustrial applications,thedeterminationofvitaminCstillpresentsresearch interest.QuickmonitoringofvitaminClevelsduringproduction andqualitycontrolstagesisimportant[18].

2. Ascorbicaciddeterminationbynon-electrochemical techniques.

Traditionalmethodsforascorbicacidassessmentinvolvetitra- tionwithanoxidantsolution:dichlorophenolindophenol(DCPIP) [19], potassium iodate [20] or bromate [21]. Chromatographic methods, like liquid chromatography [22–24] and particularly HPLCwithelectrochemicaldetection[25–27],havebeenusedin ascorbicacidassessmentinfoodstuffsandbiologicalfluids.Fluo- rimetric methods based on dehydroascorbicacid reaction with o-phenylenediamineandrequiringstrictcontrolofthepHvalue [28,29]andUV-VISabsorbance-based determinations[30]were alsoapplied.Ascorbicacidwasassessedspectrophotometrically, basedonitsreactionwithhexacyanoferrate(III)[31–33],onits oxidation usingtheCu(II)-neocuproine complex[34],oronthe determinationofiodinereactedwithascorbicacid[35].Otheropti- calmethodsforvitaminCestimationincludechemiluminescence [36].

3. Electrochemicalbehavior:theirreversibilityofascorbic acid/dehydroascorbicacidredoxcouple

Ascorbicacidisthemostcommonelectroactivebiologicalcom- pound,beingeasilyoxidated,andthisconstitutesthebasisofits electrochemical determination. Ascorbic acidforms with dehy- droascorbicacidanirreversibleredoxcouple.Itselectrocatalytical oxidationshowedonlytheanodicoxidation peak[1,37–39],for which Randles Sevˇcic equation described the observed direct dependecebetweenthecurrentintensitycorrespondingtoascor- bicacidelectrooxidationandthesquarerootofthepotentialsweep rate[40,41].

Regardingascorbicacid/dehydroascorbicacidredoxcoupleirre- versibility,studieshavebeendevotedtotheinvestigationofascor- bicacidoxidationmechanism,whichdescribeanelectrochemically reversibleelectrontransfercoupledtoirreversiblechemicalreac- tions,determininganoverallirreversibleprocess:theoxidationof ascorbicacidinvolvesthereleaseoftwoelectronsandtwoprotons, toproducedehydroascorbicacid,whichwasprovedtobefollowed byanirreversiblesolvationreactionatpHlowerthan4.0[1].

Thisirreversiblereactionyieldsanelectroinactiveproduct,2,3- diketogulonicacid, formedwhendehydro-L-ascorbic acidopens itslactonering[42–49],easilyadsorbableontheelectrodesurface, whichcanresultinelectrodefouling[50–52].

Literature dataonascorbic acidoxidation atpH<8 mention two successive one electron oxidation steps accompanied by rapid dehydration rendering the oxidation process irreversible [49,53–55]. A detalied descriptionof ascorbic acidoxidation at goldelectrodedescribestwoclearlydefinedstagesproducingtwo waves.Thefirstwaveisproducedbyabielectronicprocessinwhich twoprotonsinterchangeatthepHrangeof2-4.5,oneprotonatpH 4.5-8andfinallytwoprotonsatpH>8.Namely,atpHvaluesinfe- riortothefirstpKavalueofL-ascorbicacid(approximately4.5), two protons interchangeglobally during theprocess. Athigher pHvalues,asingleprotoninterchanges,withascorbateanionas electroactivespecies.Theseconsiderationsareconsistentwiththe variationofthepeakpotentialwithpH,observeduptopH8[49].

Itwasdiffficulttoidentifytheproductsofascorbicacidoxidation ortocarryoutadetailedstudyofthesecondoxidationwaveat pHhigherthan8, giventheinstabilityofthebasicsolutions of bothL-ascorbic anddehydro-L-ascorbicacids.It wasfoundthat theintermediate,ascorbateanioniselectrochemicallyoxidizedto adiketolactone,subsequentlydehydratedtodehydroascorbicacid whichrearrangestoanotherene-diolfurtheroxidizedathigher potentials[49].

Forthisirreversibleredoxcoupletheanodicpeakheightcorre- latedwithanalyteconcentrationcorrespondstotheoxidationof thereducedform[56–60](Figure1).

3.1. Electrochemicalbehavioratunmodifiedelectrodes

The polarographicbehavior of ascorbic acidwasfirst inves- tigated in acid medium, using a mercury capillary as working electrode[43].Itwasestablishedthatthelimitingcurrentisinde- pendentofthepHvalue,anditisalsodiffusion-controlled.The valueofthehalf-wavepotentialE_1/2 wasfoundindependentof L-ascorbicacidconcentration,butvarieswithpH.Thevaluesof

theslopesofthelinearrangeswere-59and-27mV/H⁺concen- trationdecade[43].Studiescarriedoutinbasicsolutionshowed thatL-ascorbicacidisunstableinthismediumandthereforethe currentdecreaseisnotonlycausedbytheelectrochemicaloxida- tionoftheanalyte,butalsobyitshomogeneousdecompositionin solution[44].

Cyclicvoltammetrystudiesataglassycarbonelectrode,showed only the anodic oxidation peak, recorded at about 580mV in 0.1molL⁻¹ phosphate buffer pH=2.0, containing 0.1mmolL⁻¹ disodiumEDTA[56],forwhichtheheightincreaseswithascorbic acidconcentration,andnocathodicpeakcurrentwasobservedin thepotentialrangestudied,200-1000mV,usinga50mV/spoten- tialscanrate.

Theirreversibilityoftheascorbicacidredoxcouple,wasproved alsoatPt[59,60],aswellasunmodifiedcarbonpasteelectrodes [60].Thepeakcorrespondingtoascorbicacidoxidationappeared at490mV(versusSCE)ataPtstripelectrode,andat510mVatan unmodifiedcarbonpasteelectrode,using0.1molL⁻¹KClsolution aselectrolyte[60].Acomparativestudyofascorbicaciddetermi- nationatbaregoldelectrodesinaphosphatebuffersolutionatpH 6.90,showedforthesingle-crystalAu(111)electrodemuchbetter electrocatalyticactivityincomparisonwiththegolddiskelectrode [61].

The difficulties in obtaining good reproducibility in direct electrochemicaloxidationofascorbicacidandtheproblemsofelec- trodefoulinghaveledtointerestintheinvestigationoftheroleof variousmediatorsandconsecutivelymodifiedelectrodes(metal- licorcarbonaceous),tocatalysetheelectrochemicaloxidationof ascorbicacid.

3.2. Electrochemicalbehavioratchemicallymodifiedelectrodes The electrochemical behavior of ascorbic acid on a cobalt hydroxide modifiedglassy carbonelectrode inalkaline solution was investigated and compared with the performances of the unmodified electrode [57].The oxidationprocess and itskinet- ics were studied and it was found that the presence of the cobalthydroxidemodifierfilmobtainedfromcarbonatesolutions containingCo(II)-tartratecomplexes[62],resultedinincreasedoxi- dationrateandpeakcurrentintensity,thereforeascorbicacidwas oxidized at lowerpotentials, reaction thermodynamically more favorable.Themodifiedelectrodepresentedgoodelectrocatalytic activityfortheoxidationofascorbicacidatapproximately565mV vs.SCE,in100mmolL⁻¹sodiumhydroxidesolutionusedassup- porting electrolyte and with a scan rate which varied from 5 to 300mV s⁻¹. The cyclic voltammograms and chronoampero- grams indicated a catalytic electrogeneration of Co(IV) and the current–timeresponsesfollowedaCottrellianbehavior[57].

Onapoly3,4-ethylenedioxythiophene(PEDOT)-modifiedglassy carbonelectrode,ascorbicacidoxidationpeakoccuredataround -0.035V,indicatingacathodicshiftof0.25Vwhencomparedwith the bare electrode. Furthermore, a sharperoxidation peak was obtained with themodified electrode. The low oxidation peak potentialandtheenhancedoxidationcurrentwereattributedto electrostaticinteractionsbetweentheelectrodesurfacegroupsand theanalyte:thecationicPEDOTfilminteractedwiththenegatively charged ascorbate, which resulted in an effective analyte pre- concentrationatalessanodicvalue[40].Thedevelopmentofan electrochemicalsensorbasedontheincorporationofaferricyanide mediatorwithapolyelectrolyte–calciumcarbonatemicrosphere, embeddingof theaforementionedelectrodematerialsand sub- sequentmodificationof theglassycarbon electrode resultedin pronouncedelectrocatalyticoxidationofAAbyferricyanidewith apeakat270mV,in0.1molL⁻¹PBS(pH7.0)whichrepresenteda negative-shift,comparedwithdirectelectrochemicaloxidationof AAonglassycarbon[63].

Thepotentialelectrocatalyticactivityofascorbicacidwasalso investigated, at unmodified carbon paste and at tetrabromo-p- benzoquinonemodified carbonpasteelectrodes(TBQ-MCPE),in phosphatebufferpH7.0.andthedirectoxidationofascorbicacid attheunmodifiedcarbonpasteelectrodeshowedanirreversible wave.Thecyclicvoltammetricresponseswerealsorecordedwith themodifiedelectrode:intheabsenceofascorbicacid,apairof well-definedredoxpeakswereobtainedwiththetetrabromo-p- benzoquinonemodified carbon pasteelectrode;theaddition of 1.0mmolL⁻¹ascorbicacid,determinedadrasticenhancementof theanodicpeakcurrent,andnocathodiccurrentwasnoticedin thereverse scan.Thisbehavior isconsistentwitha verystrong electrocatalyticeffect.Thecatalyticpeakpotentialofthemodified electrodewasfoundatabout105mV,whereasthatoftheunmo- difiedelectrodewasabout535mV,anenhancementofthepeak currentbeingalsoachievedwiththemodifiedelectrode[58].

Thepresence of an anionicsurfactant as modifier conferred excellentelectrocatalyticactivitytoa dodecylbenzenesulfonate modifiedcarbonpasteelectrode(DDBSMCPE).Thecyclicvoltam- mogram showed a single irreversible oxidation peak at both modifiedandbareelectrodes.Ascorbicacidoxidationoccurredat around182mVattheBCPE,whereasattheDDBSMCPEitoccured atabout−25mVinphosphatebufferpH7.40.Itwasstipulatedthat thisshiftintheoxidationpotentialcouldbeduetotherepulsive forcebetweenthenegatively chargedSO3 layerofthemodified electrodeandtheanionicformofascorbicacid[39].

CyclicvoltammetrystudiesperformedonPtelectrodesproved thatthegrowthofPtsurfaceoxideswasgreatlysurpressedbythe useoffluorosurfactantsasmodifiers[64].

Recent studies performed on various modified electrodes suchas norepinephrine modified glassy carbon electrode [65], tryptophan derivatives modified glassy carbon electrode [66], poly (2-amino-1,3,4-thiadiazole) deposited glassy carbon elec- trode[67],goldnanoparticlesself-assembledontothel-cysteine modified glassy carbon electrode [68], multiwall carbon nano- tubes modified glassy carbon electrode [69] or poly(orthanilic acid) coated multiwalled carbon nanotubes modified glassy carbon electrode [70], tetraoctylammonium bromide stabilized goldnanoparticles-1,6-hexanedithiolmodifiedAuelectrode[71], multi-walled carbon nanotubes with methylene blue compos- itefilm-modifiedelectrode[72],3-mercaptopropyl-functionalized silica network gold nanoparticles modified electrode [73], thiocytosine/guanine-goldnanoparticlesbasedmodifiedgoldelec- trodes[74],carbonpaste/cobaltSchiffbasecompositeelectrode [75]notonlydecreasetheovervoltageandenhancetheelectrocat- alyticpeakcurrent,butalsosolveacommonproblemencountered atunmodifiedsurfaces,suchasthepeakoverlapping.

4. Potentiometricascorbicacidsensorsandbiosensors Potentiometricdeterminationofascorbicacidreliesonitsdeter- minationbytheascorbateoxidasecatalysed reaction.Ascorbate oxidaseisametalloprotein,containingabout8atomsofcopper permole ofenzyme [76,77],bound inthree different chemical complexesformingtheactivesites.Thisenzymecatalysestheoxi- dationofL-ascorbicacidinthepresenceofmolecularoxygenin accordancetothefollowingreactionmechanism[77]:

Enzox+Asc←→^K1

K₋₁Enzox·Asc−→Enzred^K2 +P Enzred+O2

K3

←→K₋3Enzred·O2 K4

−→Enzox+H2O

whereEnzoxandEnzredrepresenttheoxidizedandreducedforms ofenzyme,respectively;Ascstandsfortheascorbateion(primary substrate)andPisascorbatefreeradical[76,77].

4.1. Composites

Thisprinciplewasappliedinthedevelopmentofabiosensor, wherethepotentialvariationiscausedbythereductionofCu²⁺

presentintheenzymestructuretoCu⁺,bytheascorbateanion.

Thisredoxmechanisminduceschangesintheelectronicdensity ontheelectrodesurface,whicharesensedbythepotentiometric transducer.Onthisbasis,apotentiometricbiosensorforascorbic acidwasconstructed,byascorbateoxidaseimmobilizationin a poly(ethylene-co-vinylacetate)matrix,fixedonagraphite-epoxy compositeelectrode.Theelectrodeshowedasub-Nerntianslope (50.3±0.6mV),withintherange8.0×10⁻⁶and4.5×10⁻⁴molL⁻¹ forascorbate,in0.1molL⁻¹KH₂PO₄solutionatpH5.0.Aftercon- tinuoustestingduring15days(about300analyses),nodecreasein theanalyticalresponsewasnoticed[76].

4.2. Screenprintedelectrodes

The potentiometric determination of ascorbic acid wasalso performedbyasensorarrayfabricatedbyscreenprinting, with aRuO2 filmdeposited ontheworkingareaofeach sensor.The potentialresponseoftheenzymebasedbiosensordependedlin- earlyonL-ascorbicacidconcentrationbetween0.02mmolL⁻¹and 1mmolL⁻¹withasensitivityof13.85mVLmmol⁻¹[78].

4.3. Modifiedfieldeffecttransistors

HorseradishperoxidaseimmobilizedonthesurfaceofanIon SensitiveFieldEffectTransistor(ISFET)canbeusedforthedeter- minationofL-ascorbicacidinfruitjuicesandbeverages.Peroxidase reduceshydrogenperoxide towater in thepresence of hydro- gendonors(antioxidantmolecules),andthisenzymereactioncan beusedforthedetectionofeitherhydrogenperoxideorantioxi- dantspecies.Thehydrogendonor,L-ascorbicacidisconvertedto dehydro-L-ascorbicacidduringtheperoxidase-catalysedreaction, inthepresenceofH2O2.TheconsumptionofL-ascorbicacidduring theenzyme-catalysedH2O2reduction,causesalocalpHincrease inthebiomembrane,whichissensedbytheISFET.Becauseperoxi- daseactivityispHdependentandreportedtobemaximalatpH6.0, thesensorresponseversusascorbicacidinbufferswithdifferent pHvalueshasbeendiscussed.Thesensorsensitivityinphosphate bufferisstronglypHdependentandreachesitsminimumatpH 6.70-7.20.Thistendencywasfirstascribedtothebuffercapacity change,aspotassium phosphatemonobasicwithapK_avalueof 7.21wasusedinthebufferpreparation.Ontheotherhand,the sensorresponseincreasedlinearlywithpHwhencitratebuffers wereused.Whensodiumcitratewasusedinbufferpreparation, pKa valueswere:3.12,4.76,6.40,andthebuffercapacityinthe measuredpHrangewasalmostconstant.Thus,itwasconcluded thatchangesinthesensorresponsehavebeencausedbyenzyme activityvariation[79].

Volotovskyetal.[80]developed atwo-ISFETbiosensor, con- taining horseradish peroxidase immobilized into bovine serum albumingel,aswellasglucoseoxidaseandurease,co-immobilized undera polymericfilm,whichcouldbeusedfor determination of glucose, ascorbic and citric acids in fruit juices and bever- ages.Todetermineascorbicacidcontents,measurementswiththe biosensorwerecarriedoutin10mmolL⁻¹phosphatebuffer,pH6.0.

Thebiosensorresponsetohydrogenperoxideinmediawithvarious ascorbicacidconcentrationswasrecorded,with1mmolL⁻¹hydro- genperoxideconcentrationchosenforinjection,asappropriateto determineascorbicacidupto2mmolL⁻¹.Thesensorwascharac- terizedbyalong-termstability:after1monthstorageinphosphate bufferat4^◦Cthebiomembranehasmaintained60%ofitsoriginal catalyticactivity[80].

Anotherascorbicacidsensor basedonanion-sensitivefield- effecttransistorwaspreparedbymodifyingthesensitiveareaof thetransducerwithMnO₂nanoparticles[81].AnadditionalNafion membranecoatedontopofthesensorwasusedtoimmobilizethe MnO2nanoparticlesandcontroltheamountofascorbicacidenter- ingthemembrane.ThereactionoftheMnO₂ nanoparticleswith ascorbicacidproducedalocalpHchange,whichwascorrelated withtheascorbicacidconcentrationandcouldbemonitoredby theISFET.

Ascorbicacid+MnO2→Dehydroascorbicacid+Mn²⁺+2OH⁻ Thus,oneascorbicacidmoleculegivestwohydroxylions,result- inginthepHincrease.Adecreaseintheresponsewasnoticedwith theincreaseofthesolutionpH,from5.5to8.0.Acidicenvironment provedmoreappropriateforthereactionofMnO₂ nanoparticles withtheanalyte,whilebasicconditionssuppressedthereactionto someextent.Itwasfoundthattheincreaseofphosphateconcen- trationinthebufferinducedadramaticdecreaseoftheanalytical signal[81].

5. Voltammetricandamperometricsensors

Voltammetryisapotentiodynamictechnique,basedonmeasur- ingthecurrentarisingfromoxidationorreductionreactionsatthe electrodesurface,whenacontrolledpotentialvariationisimposed [82].Amperometryisbasedontheapplicationofaconstantpoten- tialtoaworkingelectrode,andthesubsequentmeasurementofthe currentgeneratedbytheoxidation/reductionofanelectroactive analyte[83–85].

5.1. Voltammetry/amperometryatbare/unmodifiedelectrodes Thestoichiometryhydrogenperoxideformationfromascorbic acidinamodelwinesystemhasbeenexaminedbysquare-wave voltammetryonahangingmercurydropelectrode.Ascorbicacid and hydrogenperoxide couldboth be determined in thesame testsample byfirstemploying ananodicscan forascorbic acid and then a cathodic scan for hydrogenperoxide. Thepotential wasscannedfrom0mVto400mVfor ascorbicacid. The mini- mumandmaximumcurrentrangesettingswere1nAand1mA respectively.Thestepdurationwas0.1s,withastepamplitude of5mVandapulseamplitudeof50mV.Undertheseconditions, linear calibration plots were obtained for ascorbic acid (upto 235mgL⁻¹,thatis1.335×10⁻³molL⁻¹)andhydrogenperoxide (upto1.20×10⁻⁴molL⁻¹),bothrangesbeingpropertothepro- posedrealsampleanalysisgiventheconcentrationslikelytooccur inwhitewine.Square-wavevoltammetrywiththehangingmer- curydropelectrodeyieldedvoltammogramswithahighdegreeof consistency,aseachexperimentisconductedwithafreshmercury drop.Thepeakcurrentrepeatabilityfor ascorbicacidwasillus- tratedbythevalueoftherelativestandarddeviation,lessthan1%

[86].

Though L-ascorbic acid is one of the most electroactive biomolecule,it isgenerally difficulttodetermineitsconcentra- tionvalueatunmodifiedcarbonorbaremetalelectrodes,given theoccurenceofsurface problemspreviouslymentioned atthe descriptionofitselectrochemicalbehavior[49–51].Nevertheless, several studies were conducted, aiming at the viable ascorbic acid determination at bare metal or unmodified carbonaceous electrodes,withthenecessityofelectrodepretreatment[61],or mechanicalandelectrochemicalrepetitivecleaningsteps[59,60].

L-ascorbicacidwasdeterminedinaqueousmediabylinear- scan voltammetryat a goldelectrode; theoptimum conditions forpHandsweeprateweredeterminedas3.2and7500mVs⁻¹, respectively[82].Asingle-crystalAu(111)electrodewasusedfor

thesimultaneousdeterminationofdopamineandascorbicacidin aphosphatebuffersolutionatpH6.9.Thesingle-crystalAu(111) electrodedisplayedexcellentelectrocatalyticactivityfordopamine andascorbicacidoxidationincomparisonwiththegolddiskelec- trode.Inthislattercase,thepeakpotentialofascorbicacidwas shiftedtomorenegativevaluesinbothcyclicanddifferentialpulse voltammetry.Thesingle-crystalAu(111)electrodeexhibitedthis excellentelectrocatalyticactivitytowardsbothascorbicacidand dopamine,becausehydrogenflametreatmenthasprovidedforthe single-crystalelectrode surface awell-definedatomic structure, whichresultsinmoreAuOHsites,identifiedastheactivespecies responsiblefortheoxidationprocessatthesinglecrystalelectrode surface,inneutralandalkalinemedia[61].

ThevitaminCcontentofapplejuiceshasbeenmonitoredby cyclicvoltammetryataPtworkingelectrode[12].Theoxidation currentofascorbicacidwaslinearlydependentontheascorbicacid concentration(thelinearcalibrationcurvewasfoundintherange ofupto150mgascorbicacid/100mLsolution).Theresultsobtained bycyclicvoltammetrywereconsistentwiththeonesobtainedby thetitrimetric method.The methodiseasy forautomationand canbeappliedwithoutspecialpreparationof thestudiedapple juicessamples.TheperformancesofbarePtandunmodifiedcarbon pasteelectrodesinascorbicaciddeterminationwereinvestigated:

whenaPtelectrodewasusedasworkingelectrode,thelimitof detection (LOD) and thelimit of quantification (LOQ) obtained by cyclicvoltammetrywere 0.075mmolL⁻¹ and 0.25mmolL⁻¹, respectively.LowerLODand LOQvalueswereobtainedwhena carbonpasteelectrode wasemployedasworkingelectrode:the limitsofdetectionandthelimitofquantificationobtainedbycyclic voltammetry were 0.018mmolL⁻¹ and 0.062mmolL⁻¹ respec- tively.TheperformancesofbarePtandunmodifiedcarbonpaste electrodeswerealsocomparedindifferentialpulsevoltammetry.

Thelimitofdetection(LOD)andthelimitofquantification(LOQ) obtainedbydifferentialpulsevoltammetrywere0.087mmolL⁻¹ and 0.29mmolL⁻¹ respectively,when a Ptelectrode was used.

Lowervalueswereobtainedwhenacarbonpasteelectrodewas employedasworkingelectrode:LODandLOQobtainedbydiffer- entialpulsevoltammetrywere0.02mmolL⁻¹and0.068mmolL⁻¹, respectively.Thesensitivitiesgivenbytheslopesofthecalibra- tiongraphwerelowerthanin cyclicvoltammetryexperiments:

fora Ptworkingelectrode,65.42␮ALmmol⁻¹ wasthesensitiv- ityobtainedinCVstudies,whereas21.839␮ALmmol⁻¹ wasthe sensitivityobtainedinDPV[60].

Thelevels ofascorbicacidin 50 tropicalfruitsamples were determinedby cyclicvoltammetryusing a glassycarbon work- ingelectrode [56]. Theunmodifiedglassy carbonelectrode was alsoappliedtotheelectrochemicaloxidationandselectivevoltam- metricdeterminationofascorbicacid.Conditionsemployedwere:

pulseamplitude25mV;frequency15Hz;potentialstep4mV.The peakpotentialofascorbicacidmovedtolesspositivepotentials withincreasingpH.GoodagreementwiththeHPLCdeterminations wasobtainedforeffervescent,chewablevitaminCtablets,aswell asfordifferentRosaspeciesextracts.Themethodisrapid,requiring lessthan2mintorunasample[1].

Theelectrochemical oxidation behavior ofascorbic acidwas studiedbydifferentialpulsevoltammetry(DPV)ataglassycar- bonelectrodeinanextendedpHrange(between0.64and11.15), indilutedH₂SO₄anddifferentbufferedaqueousmedia(Britton- Robinson, acetate, phosphate).The voltammetricresponse was foundtobestronglypHdependent:theanodicpeakpotentialwas shiftedtopositivevalueswithincreasingpH[87].

In astudy aimingatthevoltammetriccomparison ofdiffer- entcarbonaceousmaterials,thebackgroundsignalandanalytical responses of ascorbic acid were evaluated by DPV at different carbon electrodes [88]. The transient currents associated with smallpotentialstepshavebeenexamined,anditwasconcluded

that they are attributed mainly to redox reactions of various groups ontheelectrode surface.Instrumental parameters have beenthoroughly investigated and optimized for improvingthe signal-to-background response.Glassy-carbon and carbon-paste diskelectrodeshave beencompared. It wasstipulatedthatthe current-timeprofiledependsonthepotentialdomain,becauseof potential-dependentreactionsofdifferentsurfacegroups(surface reactionsofthequinone-hydroquinonecouple)andformationof carboxylgroups,identifiedbycyclicvoltammetry[88].Secondly, althoughthetwo electrodes havethesame surface area,much largercurrentsareobservedattheglassycarbonelectrode.Elec- trodereactionsoccuringatcarbonpastearegenerallyslowerthan atglassycarbonelectrodes,yetthesignficantlylowerbackground currentof thepasterendered itsuperior toglassycarbonfrom thesignal-to-background characteristicsstandpoint.Thus, com- binedwiththelowpriceandeasypreparation,thesefeaturesmake carbonpasteasuitableelectrodematerialformanyapplications.

Althoughlargercurrentsareobservedattheglassy-carbonelec- trode,theydecayfasterthanthoserecordedwiththecarbonpaste electrode.Itwasconcludedthatinmostcases,thecurrentsdecay fasterasthepotenialregionbecomeslessanodic[88].

5.2. Voltammetry/amperometryatchemicallymodified electrodes

The need for overpotential diminution and fouling minimi- zationledtotheneedofelectrodemodificationaimingatincreased sensitivity and more neat peak separation, required mainly in complexmediasuchasbiologicalsamplesparticularlyproneto interferences,whereascorbicacidcoexistswithotherelectroactive species.

5.2.1. Modifiedmetalelectrodes

Linearsweepvoltammogramsobtainedatagoldelectrodewith dimercaptothiadiazolelayersformedfrommethanolandfromsev- eralmethanol–watermixturesatdifferentratios,showedthatthin dimercaptothiadiazolemonolayersformedfromnon-aqueoussol- vents,separatethevoltammetricsignalsofuricacidandascorbic acid[89]. The fabrication of gold nanoparticles (GNs)-modified goldelectrode wasreported,basedontheself-assemblyofgold nanoparticlesatthesurfaceofamixedfilmof1,6-hexanedithiol and1-octanethiol,whichisselfassembledatagoldelectrode.Free

—SHgroups ofhexanedithiol wereusedasscaffold toimmobi- lizeGNs,thefilmformedbythistechniquehavingtheadvantages ofhighorganizationanduniformity.ThisGNschemicallymodified electrodewasusedforelectrochemicaldeterminationofascorbic acidanddopamineinaqueousmedia[90].

Theuseofametallicmodifiedmicroelectrodearrayleadtothe successfulflowinjectionamperometricquantificationofascorbic acid,dopamine,epinephrineanddipyroneinmixtures:thefour groupsofmicroelectrodesincludedapuregoldelectrodeandelec- trodesmodifiedbyelectrodepositionofplatinum,palladiumora mixtureofplatinumandpalladium.Themultivariatecalibration methodeliminatestheneedforpreviousseparationsorforchem- icalreactionstoattainmorefavorableconditionstoquantifyeach constituentofthesamples.Formixtureswithlowerconcentrations, itwaspossibletouse,inthepresentstudy,onlyfourofthe24 microelectrodestoincreasethecurrent.Theuseofamultipoten- tiostatwiththecapacitytocontrola largernumberofworking electrodescanprovideevenmoreinformation,favoringtheanaly- sisofevenmorecomplexmixtureswithoptimizedreliability[91].

5.2.2. Modifiedcarbonaceouselectrodes

Oneofthemostconvenientandeasytousematerialsforthe preparationof modifiedelectrodesis represented,undoubtedly, bycarbon pastes.The modifiercan bedissolvedin a binder or

admixed mechanically to thepaste during its homogenization.

Carbon pastes are dynamic and renewable materials endowed withexcellentconductivityandelectrocatalyticproperties,with wideapplicabilityinelectrochemicalor bioelectrochemicalsen- sors[92,93].Therefore,cyclicvoltammetricresponsesofascorbic acidwererecordedatanunmodifiedcarbonpasteelectrodeand atatetrabromo-p-benzoquinonemodifiedcarbonpasteelectrode.

Cyclicvoltammetrystudiesachievedadecreaseintheovervolt- ageof approximately430mVand an enhancementof thepeak currentwiththemodifiedelectrode,thatwasquiteeffectivenot onlyindetectingascorbicacid,dopamineanduricacid,butalso inthesimultaneousdifferentialpulsedvoltammetricdetermina- tionofeachcomponentconcentrationinmixture.Thepeakcurrent increasedlinearlyfor10.0to600.0␮molL⁻¹ascorbicacidconcen- tration[58].

A seriesof carbonaceous amperometricsensors weredevel- oped: carbon paste, modified carbon paste and glassy carbon electrodes and used for ascorbic acid quantification. Modified carbonpasteelectrodeswereobtainedfromcarbonpasteincor- porating10% vanadate as modifier. The device is integrated in aflowinjectionanalysisset-up.Hydrodynamicandamperomet- ricparameterswereoptimized:glassycarbonelectrodesexhibit slopes of 4.75×10⁵nALmol⁻¹ (± 6.4%)under optimum condi- tions.Carbonpasteandmodifiedcarbonpasteelectrodesproved moresensitive,withslopesof6.37×10⁵nALmol⁻¹(±6.6%)and 7.32×10⁵nALmol⁻¹(±4.4%)[94].

Theelectrocatalyticbehaviorsofamethionine-modifiedcarbon pasteelectrodeandbarecarbonpasteelectrodefortheelectroox- idation of ascorbic acid were studied and compared, by using cyclicvoltammetry.Theobtainedresultsshowedgoodenhance- ment in the anodic peak current in the case of the modified electrode,comparedtothebareelectrode.Theanodicpeakcur- rentshowedanincreasewithascorbicacidconcentration,with linearityin therangeof 5.0×10⁻⁶molL⁻¹ to12×10⁻⁵molL⁻¹. The proposed method was adapted for the determination of theconcentration of ascorbic acidpresent in realsamples and the resultswere foundto be satisfactory [95]. A carbon paste electrodemodifiedwitha2,2-(1,8-octanediylbisnitriloethylidine)- bis-hydroquinoneshowedhighelectrocatalyticactivitytowards ascorbicacid;thecurrentwasenhancedsignificantly,relativeto thesituationencounteredwhenanunmodifiedelectrodewasused [96].

Different porphyrinswere alsoused forthe design ofseven carbon paste and seven diamond paste based microelectrodes, employedforthedifferentialpulsevoltammetricdeterminationof ascorbicacid.Lowdetectionlimitswereobtained,namelybetween 1.1×10⁻¹⁴ and 5.1×10⁻⁷molL⁻¹, while the sensitivities were between3.07pALmol⁻¹and1285.18ALmol⁻¹.Ascorbicacidassay inpharmaceuticalandbeveragessamplesyieldeddegreesofrecov- eryhigherthan92.0%and91.50%,respectively.Thesurfaceofthe microelectrodesiseasilyrenewablebysimplepolishing,becom- ingreadyforuseinanewassay[97].Agraphene-dopedcarbon pasteelectrode[98]waspreparedbytheadditionofgrapheneinto thecarbonpastemixture.Comparedwiththeconventionalcarbon pasteelectrode,animprovedelectrochemicalresponseofgraphene dopedcarbonpasteelectrodewasproved,duetotheexcellentelec- tricalconductivityofgraphene.Thegraphenedopedcarbonpaste electrodewasfurtherusedforthedeterminationofascorbicacid withlowovervoltage,enhancedcurrentresponse,andgoodsen- sitivity.Typicalamperometricresponsesofthebarecarbonpaste electrodeandgraphenedopedcarbonpasteelectrodetosuccessive additionsofascorbicacidin0.1molL⁻¹phosphatebuffersolution pH7.0,wereobtainedwithin5s[98].

Thevoltammetricresponseof amanganesedioxidegraphite composite electrode towards ascorbic acid was recorded, in a pH 7.2 phosphate buffer solution. The oxidation current was

foundtoincreasewithincreasingofascorbicacidamount;dis- tinctvoltammetricpeaksareobtainedforconcentrationsaslow as250␮molL⁻¹ascorbicacid[99].

Othercarbonaceous materials (e.g.glassy carbon) werealso usedas modifiableelectrode materials,due tothehighelectri- cal conductivity, resistence to chemical attack, suitability in a largepotentialrangeandcompatibilitywithacidicmedia.Aglassy carbonelectrode(GCE)hasbeenmodifiedbyelectrochemicaloxi- dation in mild acidic media (0.1molL⁻¹ H₂SO₄) and could be appliedforindividualandsimultaneousdeterminationofascorbic acid,dopamineanduricacid.Thecyclicvoltammetryresponses ofbareandoxidized(modified)GCEwererecorded,inphosphate buffersolutionpH7.0.Theoxidizedglassycarbonelectrodeshowed a singleredoxcouple(E₀=−2.5mV),determinedby theforma- tionoffunctionalgroupsduringtheelectrochemicalpretreatment.

Particularly, the presence of these functional groups generates thesuccessfuldecreaseofoverpotentialsintheoxidationprocess comparedwiththebareGCE,allowingsimpleandsensitivesimul- taneousdeterminationofthreeanalytes[100].

Polymeric filmswereproved toenhance theelectrocatalyti- calactivitytowardsascorbicacid,increasingtheelectrontransfer ratebetweentheglassycarbonelectrodeandthepolymericfilm.

Astable modifiedglassycarbon electrodebased onthepoly3- (5-chloro-2-hydroxyphenylazo)-4,5-dihydroxynaphthalene- 2,7- disulfonic acid (CDDA) film was prepared by electrochemical polymerization technique to investigate its electrochemical behaviorbycyclicvoltammetry.Thepropertiesofthefilmselec- trodepositedduringpreparationunderdifferentconditions,aswell astheirstabilitywereexamined.Thehomogeneousrateconstant fortheelectrontransferbetweenCDDAandglassycarbonelectrode wascalculatedas5.25(±0.20)x10²cms⁻¹.Themodifiedelectrode showedelectrocatalyticactivitytowardsascorbicacid,dopamine anduricacidoxidationinbuffersolutionpH4.0withadiminution oftheiroverpotentialofabout0.12,0.35,and0.50Vforascorbic acid,dopamineanduricacid,respectively[101].

Apoly(caffeicacid)thinfilmwasdepositedonthesurfaceof aglassycarbonelectrodebythepotentiostatictechniqueinaque- oussolution.Thepoly(caffeicacid)-modifiedelectrodewasusedfor thedeterminationofascorbicacid,dopamine,andtheirmixtureby cyclicvoltammetry.Themodifiedelectrodeshowsgoodsensitiv- ity,selectivity,andstabilityandhasbeenappliedtotheanalysisof pharmaceuticalsampleswithsatisfactoryresults[102].

Anothervoltammetricmethodfor determination of ascorbic acidwasreported,byusinga glassycarbon electrode modified with poly(bromocresol purple), poly(BCP)/GCE. Cyclic voltam- metry studies showed that, compared with the bare GCE, the poly(BCP)filmexhibitedanobviouselectrocatalyticeffecttowards ascorbicacidoxidation,whichreducedtheoxidationoverpoten- tial for about 240mV, with an increased current response, in 0.1molL⁻¹phosphatebuffersolution(pH6.50).Gooddegreesof recoverywereobtainedforknownascorbicacidamountsaddedto theanalysedsamples,between97.78and102.54%[103].

Theuseofconductingpolymersoffersnumerous opportuni- ties totransduceanalyte–receptorinteractions into measurable responses. The advantages of conducting polymers-based sen- sorsoverdevicesusingsmallmoleculeelements(chemosensors) are the ability of conducting polymers to exhibit properties that respond to even minor perturbations in the system [40].

Under thesecircumstances, theoxidation behaviorsof ascorbic acidanddopaminewereinvestigatedatbareglassycarbonand poly(3,4-ethylenedioxythiophene)-modified glassy carbon elec- trodes.ThePEDOTfilmwasdepositedonaglassycarbonelectrode by electropolymerizationfrom an acetonitrilesolution[40].An amperometricsensorforascorbicaciddeterminationfromfood- stuffsandpharmaceuticalpreparationswasdevelopedbyaniline

electropolymerisationonbothglassycarbonand screenprinted workingelectrodes[104].

Thinfilmsofpolyanilinecontainingthedopantionspolyvinyl- sulfonateandpolystyrenesulfonatehavebeenpreparedon25␮m Pt disk microelectrodes and tested for ascorbic acid oxidation in various buffers, as well as in wine and orange juice con- tainingsolutions.Polyaniline-basedmicroelectrodesincorporating polyvinylsulfonateandpolystyrenesulfonateasdopantionsmain- tainconductingpolymerelectroactivityinneutralpHsolutions.The acquireddifferencesinthesizeoftheplateaucurrents(twiceas largewithpolyvinylsulfonateoverpolystyrenesulfonate)pointto aneffectiveinfluenceofthedopantiononthepolyanilineredox mediatorproperties,possiblythroughamodificationofthecon- ductivity and/or morphology of the conducting polymer [105].

Anilinecontainingsuspendedsilicotungsticacidandcarbonnano- tubes,waselectropolymerizedonthesurfaceofa glassycarbon electrodeinasinglestep,providingasimple,controllableandsen- sitiveamperometricsensor,greatlyimprovingtheelectrocatalytic oxidationofascorbicacid[106].

A significant catalytic effect in ascorbic acid oxidation was obtainedinthepresenceofacationicsurfactant(cetyltrimethyl ammoniumbromide)absorbedontheglassycarbonelectrodesur- face:thepeakpotentialinthecationicmicellarmediumwaslower than that obtainedin aqueous medium, accompaniedwith the increaseoftheintensitymeasuredfortheanodicoxidationpeak [107].Anothersurfactant-basedglassycarbonelectrodewasmod- ifiedwithafilmcomposedofchitosanincorporatingcetylpyridine bromideusedtodetermineuricacidandascorbicacidbydifferen- tialpulsevoltammetry.Thismodifiedelectrodeshowsanefficient electrocatalyticactivityandfairlyselectiveseparationabilityfor ascorbicanduricacid[108].

A novel binuclear copper complex modified glassy carbon electrode wasfabricatedusinga cyclicvoltammetricmethodin phosphatebuffersolutionanditshowedaveryefficientelectrocat- alyticactivityforanodicoxidationofdopamineandascorbicacid withasignificantdecreaseintheoverpotentials.Thebinuclearcop- percomplexmodifiedGCEwasstableandreproducible.Tothisaim, theelectrodewasrenewedbyCVscansin100mmolL⁻¹phosphate buffer,inthepotentialwindow0.0–0.8Vaftereachexperiment.

satisfactoryresultswereobtainedatascorbicacidanddopamine determinationinmedicineandfoodstuffsamples[109].

Aspartic acid was covalently grafted on to a glassy carbon electrodebyaminecationradicalformationthroughtheelectroox- idationoftheamino-acid.Voltammetricexperimentsprovedthat asparticacidwasimmobilizedasamonolayerontheglassycarbon electrode[110].

AtthesurfaceofaLaFeO3nanoparticles-modifiedglassycarbon electrode,thecalibrationcurvesforascorbicacid,dopamineand uricacidwerelinearforthewholeconcentrationsrangesinves- tigated(500–3,000␮molL⁻¹forascorbicacid,1.00–6.00␮molL⁻¹ fordopamineand100–600␮molL⁻¹foruricacid)withgoodcor- relationcoefficients[111].

Acathodicallypretreatedboron-dopeddiamondelectrodewas usedforthesimultaneousanodicdeterminationofascorbicacid andcaffeinebydifferentialpulsevoltammetry.Thismethodwas successfullyappliedforthedeterminationofascorbicacidandcaf- feineinpharmaceuticalformulations,withresultsconsistentwith thoseobtainedusingaHPLCreferencemethod[112].

Carbonnanotubes(CNT)areendowedwithremarkableelectri- cal,chemical,mechanicalandstructuralproperties.Theirunique propertiesrecommend themashighlyattractivefor thetaskof chemical sensors in general, and, particularly, electrochemical detection.Inadditiontotheenhancedelectrochemicalreactivity, CNT-modifiedelectrodeshaveprovenusefultoaccumulateimpor- tantbiomoleculesandtoalleviatesurfacefouling.Theremarkable

sensitivityandconductivityenablestheuseofcarbonnanotubes assensitivenanoscalesensors[113].

Thus,anewelectrochemicalmethodwasinvestigatedforthe invivomeasurementsofascorbicacidin ratbrain,bymeansof amultiwalledcarbonnanotubes(MWNTs)-modifiedcarbonfiber microelectrodes.Thetechniquereliedontheelectrochemicalprop- erty of MWNTs of promotingascorbic acid oxidation [42]. The simultaneous voltammetric determination of ascorbic acid and rutinhasbeenachievedatanacetyleneblackpasteelectrodemod- ifiedwithamulti-walledcarbonnanotubes–chitosancomposite film(MWCNTs–CHIT/ABPE)[114].

Aselectiveandsimultaneoussquarewavevoltammetricdeter- minationofascorbicacid,acetaminophenandtryptophanhasbeen explored at a multiwallcarbon nanotube modified paste elec- trode[115].Anovelmodified,graphite-multiwallcarbonnanotube pasteelectrodefor thedeterminationofascorbicacid,basedon a cationic surfactant,cetrimoniumiodide-iodine wasproposed.

Theelectrochemicalresponsecharacteristicsofthemodifiedelec- trodetowardsAAwereinvestigated bycyclic voltammetryand differentialpulsevoltammetryinbuffersolution(pH2.0).When comparedwithactivatedcarbonorgraphite,thedevelopedmul- tiwallcarbonnanotubemodifiedpasteelectrodenotonlyshifted theoxidationpotentialofAAtolesspositivepotentials,butalso enhanceditspeakcurrent.Furthermore,theoxidationofAAwas highlystableatthemodifiedpasteelectrode,whichallowedthe successfulquantificationofAAinpharmaceuticalandfoodsamples [116].

The differential pulse voltammetric technique was further employed for simultaneous determination of ascorbic acid, dopamineand uricacid, witha sensor based onhelicalcarbon nanotubes(HCNTs).Awatersolublecationicpolymer,poly(diallyl dimethylammoniumchloride)(PDDA),isusedtofunctionalizethe nanotubes’surface,aimingtoimprovethedispersabilityandadhe- siontosubstrates ofthe helicalcarbon nanotubes.The current responsesachievedattheGCelectrodecoatedwithPDDA@HCNTs were higher in DPV than in CV. The analytical performances of this sensor enable the simultaneous detection of ascorbic acid,dopamineanduricacidinthefetalbovineserumsamples [117].

A novel carbon composite electrode consisting of n-octylpyridinum hexafluorophosphate ionic liquid and single- walledcarbonnanotube(SWCNT)wasfabricatedandinvestigated.

Compared with other composite electrodes using graphite or paraffinoil,theionicliquid–SWCNT(IL–SWCNT)compositeelec- trodeexhibitedremarkableincreaseintheelectrontransferrate for the electroactive compound and a significant overpotential decrease in ascorbicacid reaction.Furthermore, the IL–SWCNT electrodeendowedwithpronouncedelectrocatalyticactivity,was appliedto determine ascorbic acidlevels in realfood samples [118].

Anewtypeofmodifiedelectrodesensorforascorbicacidhas beenpreparedby depositionofmulti-walledcarbon nanotubes (MWCNT)andpoly(NileblueA)onthesurfaceofglassycarbon electrodes. Nile blue A was electropolymerised either beneath (directlyonglassycarbon)orontotheMWCNTlayerbypotential cyclinginphosphatebuffersolutionatpH6.0.Thecharacterization of the modified electrodes was carried out by cyclic voltam- metryandelectrochemicalimpedancespectroscopy[119].Thebest resultswereobtainedwiththemodifiedelectrodespreparedwith polyNileBlue(PNB)filmsbeneaththethinnestMWCNTlayerat 100mV.AthickerpolyNileBlueunderlyingfilmintheMWCNT/PNB electrodeswasmoresuitableforelectrochemicalsensing,sinceit gaveastablesignal.Quantitativedeterminationofascorbatewas achievedbycyclicvoltammetryandfixedpotentialamperometry inphosphatebuffersolutionat pH5.3.The modifiedelectrodes exhibitedgoodsensitivity,widelinearrange,adetectionlimitof

1.6␮molL⁻¹andgoodstability,showingthattheycanbeusedas sensorsfor ascorbicacid. Thequantitativeamperometricdeter- mination of AA using MWCNT/PNB and PNB/MWCNT modified electrodeswas performedusing thestandard addition method.

Because by amperometry a higher sensitivity than by CV was obtained,thismethodwaschosentoperformaninterferencestudy andtodetermineascorbateinpharmaceuticalsamples.[119].

Poly(xanthurenic acid) and multi-walled carbon nanotubes (MWCNT)hybridcompositeshavebeensuccessfullypreparedto formpolyXa-MWCNT for glassycarbon modification, aimingat ascorbic acid determination. A well defined redox couple has beenidentified,asaresultofthepolyxanthurenicacidredoxpro- cesses.Thiscompositeisstableatvariousscanratesanddifferent pHconditionsandhasasurfacecoverageof2.3×10⁻⁹molcm⁻² at the poly(xanthurenic acid) multi-walled carbon nanotubes modifiedglassycarbon electrode.Thiselectrode presentslower over-potentialandhighercurrentresponsesvsascorbicacidwhen comparedtothebareelectrode.Atavalueoftheappliedpotential of+300mV,ithasasensitivityof160.2␮ALmmol⁻¹cm⁻²[120].

Recently,agraphene/Pt-modifiedglassycarbonelectrodewas constructed and studied, to simultaneous estimation of ascor- bicacid, dopamine,and uric acidlevelsvia cyclicvoltammetry and differential pulse voltammetry.Size-selected Pt nanoparti- cleswitha meandiameterof 1.7nm wereself-assembledonto thegraphene surface.An optimized adsorptionof size-selected Ptcolloidalnanoparticlesontothegraphenesurfaceresultsina graphene/Ptnanocompositethatcanconstitutethebasisforglassy carbonmodificationandamperometricroutineanalysisofthree analytesin0.1molL⁻¹phosphatebuffersolutionpH=7.0,at0.5V [121].

A highly responsive ascorbic acid sensor was constructed, utilizing over-oxidized polypyrroleandpalladium nanoparticles composites(OPPy–PdNPs).Inthepresenceofpalladiumnanopar- ticles, polypyrrole was coated on a gold electrode through cyclic voltammetry and over-oxidized, at a fixed potential in NaOH solution [122]. Results revealed that the over-oxidized polypyrrole and palladium nanoparticles composites-modified gold electrode (OPPy–PdNPs/Au) had the capacity to catalyse theoxidation of ascorbic acidby loweringits oxidation poten- tialat0V.TheOPPy–PdNPs/Auelectrodeexhibitedtwodifferent linear concentration ranges. In the low concentration range (1–520␮molL⁻¹),theOPPy–PdNPs/Auelectrode provedadirect lineardependenceofcurrentresponsesonconcentrationandhad highsensitivity (570␮ALmmol⁻¹cm⁻²) and a highcorrelation coefficient(0.995).Incontrast,inthehigherconcentrationrange (120–1600␮molL⁻¹),therelationshipbetweencurrentresponses andconcentrationofAAcanberepresentedbyatwo-parameter sigmoidal equation. In addition, the sensor exhibited a short response time (less than 2 s) and a detection limit as low as 1␮molL⁻¹.Thus,theproposedsensorhasgreatpotentialforthe assessmentof AAincomplex biosystemsandcanbeappliedin variousfields,particularlyneuroscience[122].

A simple and effective strategy was proposed for syn- thesis of nickel nanoparticles dispersed in a poly1,5- diaminonaphthalene(NiNPs@P-1,5-DAN) matrix. The electro- chemical characterization of this modified electrode exhibits a stable redox behavior of the Ni(III)/Ni(II) couple in 0.1molL⁻¹ NaOHaqueoussolution.Theelectrooxidationofglucose,ascorbic acid and dopamine in alkaline solution was studied by using square-wavevoltammetry.Thedetectionlimitsforascorbicacid was0.010␮molL⁻¹[123].

5.2.3. Nanoparticlecompositesandceramiccomposites

Functional nanoparticles- carbon nanotube composite films combinetheadvantagesofenhancedelectrocatalyticactivityand

largesurfaceareaandcarbonnanotubes(CNTs)areadvancedideal materialsforsupportingnanosizedmetallicparticlesin electro- catalysis.Hence,auniquebimetallic,nanoplatinumwithnanogold, onnafionincorporatedwithfunctionalizedmultiwallcarbonnano- tubescompositefilmwasdevelopedbythepotentiostaticmethod.

Thecompositefilmexhibitsefficientcatalyticactivitytowardsthe oxidationofbiocompoundsandallowsthedeterminationofascor- bateanion,epinephrineandurateanioninbuffersolution(pH6.75) [124].

Acarbon–ceramicmaterial,SiO₂/C/Nb₂O₅ wasusedforelec- trode development, and it showed the ability to improve the electrontransferbetweentheelectrodesurfaceandtheanalyte, ascorbic acid [125]. The presence of Nb₂O₅ at the SiO₂/C sur- facereducestheovervoltageofascorbicacidoxidation, shifting theoxidationpotentialtoavaluebyabout180mVmorenega- tive.Thepeak currentis considerablyenhancedcompared with the response of ascorbic acid on the SiO₂/C electrode surface.

Thisbehavior clearlydemonstrates theelectrocatalyticfunction of theSiO₂/C/Nb₂O₅ electrode towards ascorbic acidoxidation.

ThevoltammetricresponseofascorbicacidontheSiO₂/C/Nb₂O₅ electrode may beattributed to theinteraction between Nb2O5

andascorbicacid,whichincludestheformationofcovalentbonds betweentheniobiumoxideand ascorbicacid, thisleading toa veryfastkineticsofascorbicacidoxidationontheSiO2/C/Nb2O5

surface.Moreover,niobiumoxideisann-typesemiconductor;the conductionbandisformedfromthe3dorbitalofNbatomsand thevalenceband,fromthe2porbitalsofoxygen.Thus,theelec- tricalconductivityatpotentialsabovetheconductionbandedge may facilitate electron transfer in ascorbic acidoxidation. The SiO₂/C/Nb₂O₅electrodepresentedgoodrepeatabilityforselective ascorbicaciddeterminations[125].

Single-walledcarbonnanotube-modifiedcarbon–ceramicelec- trodes(SWCNT/CCE)wereemployedforthesimultaneousdeter- mination of acetaminophenand ascorbic acid. The SWCNT/CCE displayed excellent electrochemical catalytic activities towards acetaminophenandascorbicacidoxidation comparedwithbare GCE.Themodifiedelectrodegreatlycatalysedtheelectrooxidation reactionsofacetaminophenandascorbicacid[126].

5.3. Amperometricenzymicassay:biosensors

Amperometricbiosensorsrelyonelectrontransferreactions- thereduction/oxidationofanelectroactivespecies,generatedin anenzymereaction.Theroleoftheenzymeistogenerate/consume anelectroactivespecies,whichcanstoichiometricallybecorrelated totheanalyteconcentration[83–85,127].Amperometricascorbic acidbiosensorswereobtainedbyascorbateoxidaseimmobilization onanylonnet[128]oronacollagenmembrane[129],usingaClark oxygenelectrodeastransducer[128–130].

TheascorbateoxidasemembranewascoupledtoanO2elec- trodeandtheyieldingreactionwasmonitoredbyoxygendepletion at-600mVusingflowinjectionanalysisoptimizedto0.1molL⁻¹ phosphate buffer pH 5.8, with a carrier solution flow-rate of 0.5mLmin⁻¹.Theascorbicacidcalibrationcurvewaslinearfrom 1.2×10⁻⁴to1.0×10⁻³molL⁻¹.Theevaluationofbiosensorlife- timeleadsto500injections.Commercialpharmaceuticalsamples wereanalysed withtheproposedmethodand theresultswere in accordance with those obtained by high-performance liquid chromatography (HPLC) [128]. Ascorbate oxidaseisolated from theepicarpofCucumissativuswasalsoimmobilizedonacolla- genmembranemountedonaClarkoxygenelectrode,forascorbic acidcontentassessmentinfruitjuices[129].Anotheramperomet- ricbiosensorwasdeveloped byimmobilizingascorbate oxidase ona nylonsemipermeablemembrane,fixedonanoxygenelec- trode.Anoptimumenzymeloadingof 100Uascorbate oxidase

immobilized ontheenzymemembrane wasobtained.The sen- sitivity (given by the slope of the calibration graph) was 4.15+0.114nALmmol⁻¹[130].

Ascorbate oxidase was immobilised on cyanogen bromide activated-Sepharose4Bandincorporatedinaflow-injectionsys- temwithamperometricdetectionataglassycarbonelectrodeat +600mV.Onpassagethroughtheimmobilizedascorbateoxidase, afractionoftheL-ascorbicacidwasconvertedintodehydroascor- bic acid and the resulted signal decrease was measured and correlated to the amount of analyte present, allowing simple and rapid determination of L-ascorbic acid in fruit and veg- etable juices. The sampling throughput was 30 samples h⁻¹ [131].

Ascorbicanduricacidweredeterminedbycouplingtheamper- ometrictechniquewithflowanalysis.Uricacidandascorbicacid present in urine were rapidly determinedby an amperometric methodcoupledwithflowinjectionanalysis[132].Anarrayofgold microelectrodesmodifiedbyelectrochemicaldepositionofpalla- diumwasemployedasworkingelectrode.Thismethodisbasedon threestepsinvolvingtheflowinjectionof:(1)thesamplespiked withastandardsolution,(2)thepuresample,and(3)theenzy- maticallytreatedsample.Theenzymatictreatmentwascarriedout withascorbateoxidase,uricase,and peroxidaseatpH=7.0.Uric andascorbicacidswerequantifiedinurineusingamperometric differentialmeasurementsat+750mVand+550mV,respectively [132].

The advantages and peculiarities of carbon nanotubes, dis- cussed atthevoltammetricmethods,recommendthemalsofor incorporationinamperometricbiosensors.Duetotheirenhanced structural, mechanical,electrical and electromechanical proper- ties,carbonnanotubesareknownfortheirconsiderableuseinthe areaofsensorsashighstrengthconductivecompositematerials, characterizedbygoodbiocompatibilityandpossibilityofrenewal [133,134].

One possible approach is to improve the interaction of the biomolecule ofinterest withCNTs,leadingtotheimprovement of theperformance ofbiosensor materials.Ferritinprotein was non-covalently immobilized onto single wallcarbon nanotubes (SWNTs) [133,134].Ferritin canbedispersed in polarmatrices, thuscombiningferritinwithSWNTsenhancesthenanotubeinter- action with water and improves the dispersion of SWNTs in aqueoussolution[133].TheaffinityofCNTsforproteinsandtheir electrocatalytic activity, wereexploited in ascorbate oxidation:

thecarbon nanotube/ferritin film wasused in theconstruction of an amperometric ascorbic acid biosensor. The investigation of theoxidation of ascorbicacid wascarried outby sequential additions of 1.0mmolL⁻¹ ascorbic acid to a phosphate buffer solution,usingtheSWNT/ferritinelectrode.Itcouldbeobserved thatthecurrentincreasedcontinuouslywithsequentialascorbic acid additions. The developed biosensor allows the determina- tion of ascorbic acid with a sensitivity of 767␮Amg⁻¹ (for a 1mmolL⁻¹solution).FerritinproteinboundtoSWNTswasproved toenhance theoxidation reactionof ascorbicacidover 11-fold [133].

An ascorbatebiosensor wasfabricated bycovalently immo- bilizing ascorbate oxidase from Lagenaria siceraria fruit onto a carboxylated multiwalledcarbon nanotubesand polyaniline (c- MWCNT/PANI)layer,electrochemically depositedonthesurface ofanAuelectrode[134].Thediffusioncoefficientofascorbicacid wasdetermined as3.05×10⁻⁴ cm² s⁻¹.The biosensor showed optimum response at pH=5.80 in a broad temperature range (30–45⁰C), polarized at +0.6V. For the amperometric response of theAsOx/c-MWCNT/PANI/Auelectrode, thecurrent variation was due to the reduction of molecular oxygen nonconsumed in the ascorbic acid oxidation catalysed by ascorbate oxidase [134]:

O2+4e⁻+4H⁺→2H2O

Thebiosensorwasemployedfordeterminationofascorbicacid levelinsera,fruitjuicesandvitaminCtabletswithgoodcorre- lationwithresultsprovidedbythe2,6-dichlorophenolindophenol method.Ithadadvantagesoverearlierenzymesensors,suchasno leakageofenzymeduetothecovalentcouplingwiththesupport, lowerresponsetime,widerworkingrange,higherstoragestability (wasused200timesoveraperiodoftwomonths,whenstoredat 4⁰C)[134].

Carbon-supported PdNi nanoparticles (PdNi/C) were syn- thesized using a novel synthetic route, and characterized by transmissionelectronmicroscopyandX-raydiffractometry.The overallmetallic content(Pd+Ni) was10%(w/w)and uniformly distributedin thecarbonblack (90%)matrix. ThePdNi/Cmodi- fiedglassycarbonelectrodeshowedbettercatalyticactivitythan anequalamountofcommerciallyavailablepalladiumcarboncata- lyst.ThisindicatesthatthePdNi/Cnanomaterialsareabletoreduce theoverpotentialofAAoxidationwithapeakshiftedto-0.05V, demonstratingthesynergisticeffectofNiand Pd.Theseresults showthatPd-basedbimetalliccatalystshaveexcellentenzymatic amperometricAA sensing capability, fastresponse, high repro- ducibilityandstability.Thebiosensorbasedoncarbon-supported PdNinanoparticlesyieldeddegreesofrecoverycomprisedbetween 95.1and106.3%intabletsandserum[135].

6. Interferencesfromcompundspresentinbiological media,pharmaceuticalsandfoodstuffs

6.1. Interferencesinpotentiometry

At a MnO2 nanoparticles modified ion-sensitive field-effect transistor,theviablepotentiometricdeterminationofascorbicacid invitaminCinjections,aswellasinurine,waspossiblewithno observed effecton thesensor’s performances from othercom- poundspresentintheanalysedmedia,suchasglucoseoruricacid [81].

6.2. Interferencesinvoltammetry/amperometry 6.2.1. Unmodified(bare)electrodes

Inlinearscanvoltammetricassayofascorbicacidatbaregold electrodes,theproblemofinterferenceeffectsinbiologicalsamples fromuricacidandsugarswassolvedbythepresenceofcopperions [82].Theanodicpeaksofascorbicacidanddopamine,overlapping onagold diskelectrode, werewellseparated usingthesingle- crystalAu(111)electrode.Theprocedureischaracterizedbylow chargetransferresistanceatthesurfaceofthesinglecrystalelec- trode.Hence,nohomogeneouscatalyticoxidationwasnoticed,and theoxidationsignalofascorbicacidwasunalteredbytheaddition ofdopamine[61].

6.2.2. Modifiedmetalandcarbonaceouselectrodes

Thebare goldelectrode couldnotseparatethevoltammetric signalsofascorbicacidanduricacid,whereasthethindimercap- tothiadiazolemonolayerformedfromneatmethanolmostclearly separatedthesignalsofthestudiedelectroactivecompounds:the oxidation of ascorbic acid occured at 230mVwhile theoxida- tionof uricacidoccuredat440mV,enablingthedetermination of both analytes, simultaneouslyin mixture [89].It wasfound thata methanol, ethanol, orDMSO solutionof dimercaptothia- diazolebehavesasastrongacidbecausethesesolventsareable todeprotonatedimercaptothiadiazoleandthus,thinmonolayers areformedonthegoldelectrode.Ontheotherhand,anaqueous

solutionofdimercaptothiadiazolebecomeslessacidicduetoweak deprotonationofdimercaptothiadiazolebywaterandthus,dimer- captothiadiazoleformsathicklayeronthegoldelectrode.Itwas concludedthatthethindimercaptothiadiazolemonolayerformed fromnon-aqueoussolventsmuchbetterseparatesthevoltammet- ricsignalsofuricacidandascorbicacidthanthethicklayerformed inaqueoussolution[89].Agoldnanoparticles-modifiedelectrode couldclearlydifferentiatetheoxidationpeaksofascorbicacidand dopamine,withapeak-to-peakseparationof110mV,enablingthe determinationofascorbicacidanddopamineinthepresenceof eachother,whichwasnotpossibleatthebareAuelectrode.Onthe modifiedAuelectrode,1,6-hexanethiolwithpKa=10.24presents itselfinprotonatedformatpH=5.00,thuspositivelycharged,while citrateionsasstabilizers,impairnegativechargetoGNs.There- fore,theauthorsstipulatedthattheresultantchargeisapositive one,oppositetothechargeofascorbicacidformatthispHvalue.

Furthermore,thestabilizationofGNswasattributedtothereplace- mentofthenegativechargesofascorbicacidformatthispHvalue, bycitrateions.Therefore,theelectrooxidationofascorbicacidat thesurface of this modified electrode shiftedtoa less positive potential.Themagnitudeofseparationofthevoltammetricpeak potentialsofascorbicacidanddopamineisinfluencedbythesolu- tionpH:theoxidationpeakpotentialsofdopamineandascorbic acidwereshiftedtolesspositivevalueswithdecreasingacidity.

Thisisaconsequenceofadeprotonationstepinvolvedintheoxi- dationprocessesofbothanalytes,thatisfacilitatedathigherpH.

TheanodicpeakpotentialdifferenceincreasedwiththepHincrease frompH3.0topH5.0andthendecreased,so,toobtainamax- imumpeak separationfor theanodic oxidationof ascorbicacid anddopamine,thepHwasadjustedto5.0.Theobserved280mV peakseparationwasmorethanenoughtodeterminebothanalytes simultaneously[90].

Atetrabromo-p-benzoquinonemodifiedcarbonpasteelectrode could separate the oxidation peak potentials of ascorbic acid, dopamineanduricacidpresentinthesamesolution,whereasat theunmodifiedcarbonpasteelectrodethepeakpotentialswere indistinguishable[58].

The presence of functional groups on a glassy carbon electrode modified by electrochemical oxidation resulted in three well-defined voltammetric peaks at the potentials of 0.064,0.227 and 0.354V, for ascorbic acid, dopamine and uric acid, respectively. At the same time, the bare GCE fails to separate these peaks and gives a single oxidation peak at around0.422V [100].A poly 3-(5-chloro-2-hydroxyphenylazo)- 4,5-dihydroxynaphthalene-2,7-disulfonic acid modified glassy carbonelectroderesolvedtheanodicpeakoverlappingofascorbic acid,dopamineanduricacidintothreewell-defineddifferential voltammetricpeaks.Interferencestudiesshowedthatthemodi- fiedelectrodeexhibitsexcellentselectivitytowardsascorbicacid, dopamineanduricacid[101].Apoly(caffeicacid)thinfilmmodified glassycarbonelectrodeischaracterizedbyapronouncedelectron- mediatingbehavior,followedbywell-separatedoxidationpeaks towardsascorbicacidanddopamineatascanrateof10mVs⁻¹ withapotentialdifferenceof135mV,whichwassufficienttodeter- mineascorbicacidanddopamineindividuallyandsimultaneously [102].APEDOTfilmmodifiedglassycarbonelectrodehasshown catalyticoxidationofdopamineandascorbicacidandalloweda peakpotentialseparationof0.2V[40].

Theinfluenceofsomeinterferentscommonlyfoundinjuices and pharmaceuticalpreparations, including4-acetamidophenol, uricacidand citricacidis minimizedat apolyaniline-modified glassycarbon amperometricsensor, which selectively catalyses theoxidationofL-ascorbicacidatlowpotentials(+100mV)[104].

Atpolyaniline-basedmicroelectrodesincorporatingpolyvinylsul- fonate, a potentialof 100mV vs SCE was applied, in solutions obtainedbymixingequalvolumesofa(model)wineorjuicewith