Mechanically Immobilized Copper Hexacyanoferrate Modified Electrode for Electrocatalysis and Amperometric Determination of Glutathione
D. RaviShankaran* andS. SrimanNarayanan
Department of Analytical Chemistry, University ofMadras, Guindy Campus, Chennai-600 025, India ReceivedSeptember 23, 2000
A new copper hexacyanoferrate modified electrode was constructed by mechanical immobilization. The modified electrode was characterised by cyclic voltammetric experiments. Electrocatalytic oxidation of glutathionewaseffectiveat themodifiedelectrodeat a significantlyreducedoverpotential and atbroader pH range. The modified electrode shows astable and linear response in the concentrationrange of9 x 10-5 to 9.9 x 10-4 M with a correlation coefficient of0.9995. The modified electrode exhibits excellent stability, reproducibility and rapid response and can be used in flow injection analysis for the determination of glutathione.
Keywords: Modified electrode,Mechanicalimmobilization, Glutathione, Copper hexacyanoferrate.
Introduction
Recentinnovationsareexpectedto provide thenecessary improvements in convenience, cost, turnout time anduser friendlymethodology and tohave a major impact onscience and technology inanalysis of the compounds of biological and environmental interest. Among the different analytical approaches,onefieldthat offers great potential is chemically modifiedelectrodes (CMEs).1-4 The development of CMEs by insoluble metal hexacyanoferrates have attracted wide spreadattentiondue to theirinterestingchemicalandelectro chemicalcharacteristics.4-6 Among them, copperhexacyano
ferrate (CuHCF)has receivedconsiderableinterestinelectro analysis dueto its remarkable electrocatalyticproperty.7,8 It is an ion conductor with redox active sites. The oxidation and reduction of its redox sites can proceed without dis
solution ofthe solidcompounds. These interesting features motivated the construction of modifiedelectrode by mech anicalimmobilization.9 Recentdevelopments in the qualita
tive and quantitative electroanalysis of solid compounds by means of mechanical immobilization have opened a new rangeof possibilities for theconstruction of electrochemical sensors.10-13
In the present study, a CuHCF modified electrode was constructed by mechanical immobilization. The analytical applicability ofthe modified electrode wasevaluated for the determinationofglutathione. Glutathione (y-glutamylcystein- ylglycine, GSH) is aphysiologicallyimportant aqueous anti
oxidant,capable of scavenging oxygen-derivedfreeradicals, which are thoughtto contributetothe developmentof many common diseases including cancer,heartattackand stroke.14,15 Thus there is always anincreasing demand forsimple and reliable sensor for determination of GSH. A variety of electroanalytical methods havebeen developed for the deter
mination of GSH.2-4 GSHrequires higheroverpotential for electrooxidationat conventional electrodes. Consequentlytheir electrochemical quantification following liquid chromato graphy is usually performed at the mercury16 or mercury
amalgam17electrodesat which themercury sulphideformed canbe oxidisedat a comparativelylow potential. However, methodsbased on mercury electrodes may be undesirable either because of their possibletoxicity or because of rapid deterirorationoftheelectroderesponse. Halber and Baldwin have developed a carbon paste electrode containing cobalt phthalocyanine to decrease the overpotential for the deter minationof GSH.18Wring et al demonstratedthe determin ation of GSH following liquid chromotography at a carbon epoxyresincompositeelectrode modifiedwith cobaltphtha- locyanine.19 A Ru(III) diphenyldithiocarbamate modified carbon paste electrodewas constructed in our laboratoryfor themonitoring of GSH andcysteine.20 Despite the different sensors, the present sensor has the advantages of ease of fabrication,reliable sensitivity and excellent stability.
Experiment지Section
Materials andInstrumentation. Glutathione was purchased from Sigma. All other chemicals were of analytical grade and were used as received. All solutionswereprepared from doubledistilledwater. ThepH of themediumwas adjusted with 0.05 Mphosphatebuffer (K2HPO4 + KH2PO4).
The CuHCF complex was prepared by precipitation via drop-wise addition of 0.1 M CuNO3 toa well-stirred solu tionof 0.1 MK4Fe(CN)6 in a beaker. Afterthe addition, the mixturewasstirred for15 minutes and kept undisturbed for one hour and the precipitate was centrifuged with repeated washing with 0.1 MKNO3 followed by distilledwater. The reddishbrownprecipitate formed wasdriedat roomtemper atureunder vacuum, which was then crushedand milledto finecrystallinepowder.
Electrochemicalexperimentswereperformed with EG &
G PARelectrochemical system (Model 263A) equippedwith GPIP (IEEE-488) interface port andIBMpersonal computer.
The modified electrode was used as working electrode, while Ag/AgCl (saturated KCl) and a platinum wire were used as reference and counter electrode respectively. All
measurementswerecarriedoutunderanatmosphereof high puritynitrogen.
Construction of ModifiedElectrode.Themodifiedelec trodewasconstructedfromparaffinimpregnatedgrahiteelec trodes (PIGEs). The preparation of PIGE has been carried outby as reported elsewhere.10 Prior to surfacemodification, the PIGE(3.0 mm diameter)wasmechanicallypolishedwith emery paper followed by alumina slurry on a microcloth polishing pad, rinsed wellwith waterand sonicated for one minutein doubly distilledwater. The immobilization ofthe CuHCF mediator ontothe electrodesurfacewaseffected by uniformly rubbing the PIGE onto a fine powder of the complexplaced on a smooth glass plate.10,11 The modified electrodewasthen rinsedwell with doubledistilled water.
ResultsandDiscussion
Figure 1 shows the cyclic voltammograms (CVs) ofthe CuHCFmodified electrode in0.1 M KNO3 solutionatthe potential scan rates from 2 to 50 mV/s. A reversible redox couple with a mid-point potential (Epa + Epc/2) of 〜0.7 V correspondingto Fe(CN)64-/Fe(CN)63- reaction was observ
ed. It is noteworthythat in theentirepotentialrangestudied, there wasnoredox signal for copper, which bears with the witnessto theextremestabilityof thesolid compound. How
ever when a CuHCF complex was prepared with the 2 : 1 ratio of Cu(NO)3and K4Fe(CN)6, a redoxpeakcorresponds totheCu+/Cu2+ reaction with amid point potential of〜0.2V was observed in 0.1 M NH4Cl solution.11 It was assumed that thecopper ion responsible for this behavior isnot the N- coordinated one but is present in the holes of the cubes (interstitial metal ions). The possible application ofredox reaction ofthe interstitial copper ions for the determination ofascorbic acid was evaluated.11 Howeverno such behavior was observed in the present system as the complex was preparedin the 1 : 1 ratioof Cu(NO)3 and K4Fe(CN)6. It was
1.10 0.90 0.70 0.50 0.30 0.10 -0.10 Potential, V
Figure 1. Cyclic voltammograms of CuHCF modified electrode in 0.1 M KNO3 solution at different scan rates: (a) 2, (b) 5, (c) 10, (d) 20 and (e) 50 mV/s.
observed that the peak current ofthe redox couple ofthe CuHCF modified electrode increase with the increase of scan ratesand are linearlyproportional to the square root of scan rates inthe range from 2 to 500 mV/sin 0.1 MKNO3, suggesting that the redox reaction was diffusion controlled.
This implies that the diffusion ofelectrolyte ions into and outof the CuHCF filmis essential for charge compensation during redox process.
The electrochemical behaviour of the modified electrode in electrolytesof differentcations and anionswas studied.It was observed that the modified electrode exhibit different voltammetric behaviour in electrolytes ofdifferent cations.
Howevertherewasnoappreciable change inthe CVs when theanions oftheelectrolyteswere variedindicating that the cations ofthe electrolytes involve inthe redox reactionof the CuHCF film. Figure 2 depicts the CVs ofthe CuHCF modified electrode in 0.1 M solutions ofdifferent cations such as NH：, Na+, Li+, Ca2+and Ba2+. Itwas observed that the modified electrode exhibit good and stable response in electrolytes of K+ and NH：. The effect of cations on the current andpotential response ofthe CuHCF film showed that the ability ofthe electrolyte cation to fit within the interior ofthe unit cell wasthe decidingfactor of the peak potential and current. Thelengthoftheedge ofthe unitcell and the diameterof the cage for bulk CuHCFare 10.0 and 3.2A respectively.7Both theK+ and NH4+ ionshavehydrated diameterof 2.4A, and hence caneasily fit well within the
1.10 0.900.70 0.50 0.30 0.10 -0.10 1.10 0.900.70 0.50 0.30 0.10 -0.10 Potential, V Potential, V
%」 은느 0
1.10 0.900.70 0.50 0.300.10 -0.10 Potential,V
Figure 2. Cyclic voltammograms of CuHCF modified electrode in 0.1 M solution of (a) NH4CL (b) NaCl, (c) LiCl, (d) BaCL and (e) CaCb; Scan rate: 1o mV/s.
cage to allow for generation of faradic currentuponelectro chemical cycling. As thehydrateddiameterof othercations arehigherthanthezeoliticcagediameter, theirmovement is restricted and hence the redox behaviour of CuHCF modi
fied electrode was not well defined and also less stable in presenceofother cations.
The effectofsolution pHon the electrochemical behavi our of the CuHCF modified electrodewas studiedbycyclic voltammetry in 0.1 M KNO3 solution atvariouspHvalues adjusted with 0.05 M phosphate buffer (figure notshown).
The result indicatedthattheelectrochemicalresponse of the modified electrode remains almost same in the pH range between 2 to8. A changeinthe CV responsewith reduced currents andbroad peaks were observed after pHs higher than8. The possible reason for poor response at higher pHs may be due to the hydroxylation of the CuHCF film in alkaline medium asgiven below:
Fe(CN)64-+ 2OH- t Fe(OH)2 +6CN- 2Fe(OH)2+H2O+ 1/2O2 T 2Fe(OH)3
The stability ofthe CuHCF modified electrode was ex amined byrepetitive scans in 0.1 M KNO3 solution at a scan rate of 100 mV/s. The peak current remained constant for nearlyhundredcycles. Inaddition,theresponseof the modi fied electrode remain unchanged when stored in air or dipped in electrolyte solution for two weeks and morethan 90% of the response was retained upto one month. The resultsindicatetheremarkable stabilityof theCuHCFmodi fiedelectrode.
To address the analytical applicability of the CuHCF modified electrode, CVs were recorded for the catalytic oxidationof GSH and theresult was shown in Figure3.The curves (a) and (c) correspond to CVs ofbare and CuHCF modifiedelectroderespectively.Curves (b)and (d) compare
1.0 0.8 0.6 0.4 0.2 0.0
Potenti기/V
Figure 3. Cyclic voltammograms of (a) bare electrode, (b) 8.1 x 10-4 M GSH at bare electrode, (c) CuHCF modified electrode and (d) 8.1 x 10-4 M GSH at modified electrode; Supporting electrolyte : 0.1 M KNO3； pH 6.5 (0.05 M phosphate buffer); Scan rate: 20 mV/s.
the typical CVs recorded for the oxidation of 8.1 x 10-4M GSH at a bare graphite and at CuHCF modified electrode respectively. As canbe seen from the figure, on scanning from 0.0 V to 1.0V, at the bare electrode, relatively small anodicpeakat higher potentialwasobserved for the oxida tionof GSH. WhereasatCuHCF modified electrode, a large enhancementinanodic current at 0.72V with a correspond ing decrease in the cathodic current was observed. The increase in currentat themodifiedelectrode in presence of GSH is due to the catalytic oxidation of GSH by the mediator. With the modified electrode a clear shift in the oxidation potential for GSHtowards cathodic directionwas observedcompared to bare electrode.Theresultsofreduced overpotential and increased current response are the clear evidence for the catalytic effectof the CuHCF film present on theelectrode.
Oxidation of GSHonCuHCF modified electrode isa two stepelectrocatalyticprocess, initiated bytheelectrochemical oxidation of ferrocyanide to ferricyanide followed by the chemical oxidation ofglutathione by ferricyanide and the regeneration offerrocyanide. It was also observed that the catalyticcurrent increases with increaseofscan rates and the currentdisplay a goodlinearity with square root ofscan rate in the range of 10 to 500 mV/s, which illustrates that the electrocatalysiswasdiffusioncontrolled.8,11 Itwassuggested that GSH wasdiffuse to the electrode surfacewhere it was oxidised to oxidisedglutathione(GSSG)bytheCuHCF film asgiven below:
Fe(CN)64- T Fe(CN)63-+ e-
2Fe(CN)63-+ 2GSH tGSSG +2Fe(CN)64-+2H+ Theeffect of pHonthe electrocatalyticoxidationofGSH at the modified electrode was studied in the pH range between2 to 9. Figure 4 shows the plot ofpH versus the catalytic current observed for 1x 10-4 M GSH oxidation with the modified electrode. It was foundthat the catalytic oxidation of GSH at CuHCF modified electrode occurs effectively inneutraland acidic media (pH2-8).Thereduced
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Figure 4. Effect of pH on the anodic peak current of 3.6 x 10-4 M GSH oxidation at the CuHCF modified electrode; Scan rate: 20 mV/s.
50 40
0 0.2 0.4 0.6 0.8 1
Potential, V
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Figure 5. Hydrodynamic voltamograms in 0.1 M KNO3 solution for (a) bare electrode, (b) 8.1 x 10-4 M GSH at bare electrode, (c) modified electrode and (d) 8.1 x 10-4 M GSH at modified electrode; pH 6.5 (0.05 M phosphate buffer); Stirring rate: 300 rpm.
catalyticcurrentat higherpHs isdue to the dissolution of the CuHCF film due to hydrolysis in alkaline medium as described earlier.
Hydrodynamicvoltammograms (HDVs) were recorded in stirredsolution of 0.1M KNO3 (pH 6.5, phosphatebuffer) to study the effect of potential on the catalytic oxidation of GSH. In Figure5,thecurve(a) and (c)correspondstoHDVs ofbare and modifiedelectrode withoutGSH and the curves (b) and (d) corresponds to HDVs obtained in presence of 8.1 x 10-4 M GSH. It was noticed that with the modified electrode, the catalytic current increaseswhenthe potential was increased from 0.0. V to 1.0 V with a plateau at0.75V, so we select this as the workingpotential for GSH deter
mination. As expected, a poor response for GSHoxidation was observed with the unmodified electrode, which offers detectionof the GSH only atmore positive potentials with lesser sensitivity. In contrast, the electrocatalytic action of the CuHCFpermits convenientdetection of GSH atlower potentials withhigh sensitivity.
Figure 6 showsthe calibration plotfor the determination ofGSH with the modified electrode. Alinear responsefor the GSH inthe concentration rangefrom9 x 10-5 to9.9 x
10-4 M with acorrelation coefficientof 0.9995 was observ
ed. To study the precision of the method, 10 successive measurements of 2.0 x 10-4 M GSH was carried out anda relative standard deviation of1.86%wasfound. The detec tionlimit was3.2x 10-5 MGSH (S/N=3).
The stability ofthe modified electrode towards GSHre sponsewasevaluatedbymeasuring the response towardsthe catalytic oxidation of 5.4 x 10-4 M GSH for an extended length ofsix hoursunderhydrodynamic conditions (Figure 7). The stable responseofthemodifiedelectrodeshows the better confinementof the mediator at the electrode surface resultingin good long-term stability ofthe modified elec trode,which suggests its possible application in flow systems.
Anattemptwasmade to usethemodifiedelectrode for the
25 -
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0 200 400 600 800 1000
Glutathione / 卩M
Figure 6. Calibration plot for the determination of GSH.
350
50 150 250
Time, min
Figure 7. CuHCF modified electrode response in 0.1 M KNO3
solution for 5.4 x 10-4 M GSH versus time; pH 6.5 (0.05 M phosphate buffer); Potential: 0.75 V.
determination of GSH in blood samples. It showed good sensitive response to GSH in the linear range mentioned.
However ascorbic acid and otherantioxidantswerefoundto interfere with the determination of GSH. Hence for blood sample analysis, prior separation by HPLC is essential.
Number of reports with goodsensitivity for the determina tion of GSH were reported.3,4,19 Halbert and Baldwin reported adetection limit of 3.7 pmol for GSH usingcobalt phthalocyanine modified carbon paste electrode.18 A detec tion limit of10ng for GSH determination wasdemonstrated by Hou and Wang using Nafioncoated prussian bluemodi fied electrode.21 A Ru(III)Diphenyl dithiocarbamate modi
fied carbon paste electrode constructed in our laboratory showed a detection limit of 15 ppm.20 Thoughthesensitivity is little less comparedtothe previous reports,the simplicity and stability ofthe present sensormake it more attractive.
Moreovertherearepossibilitiestoenhancethe sensitivity of the sensorby alteringthe experimental factors such as the electrode areaand the amount of mediator.
Conclusion
In this work wehavedemonstrated a novelamperometric sensor for glutathionedetermination based onmechanically immobilized CuHCF modified electrode. Its performance rests on a number of features: the good electrochemical propertiesof CuHCF, thelowsolubilityof both oxidised and reduced formsof CuHCFresulting in theeffective confine
ment of the mediator ontheelectrode surface. These features combinetogiveanelectrode which permitsrapid andrepro
ducibleanalysis of GSH insolution. The modified electrode exhibit high stability, sensitivity, portability and simple to use. The hydrodynamic voltammetry result suggests that the modifiedelectrodecould be developedas a usefulsensor for theon-linemonitoringofglutathionein flow systems.
Acknowledgment.Theauthorsarethankful to University Grants Commission, New Delhi for providing financial assistance in theform of aproject.
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