Research Article Catalase-Based Modified Graphite …downloads.hindawi.com/journals/jamc/2016/8174913.pdfCatalase-Based Modified Graphite Electrode for Hydrogen Peroxide Detection

Research ArticleCatalase-Based Modified Graphite Electrode forHydrogen Peroxide Detection in Different Beverages

Giovanni Fusco12 Paolo Bollella1 Franco Mazzei1 Gabriele Favero1

Riccarda Antiochia1 and Cristina Tortolini1

1Department of Chemistry and Drug Technologies Sapienza University of Rome Rome Italy2Department of Chemistry Sapienza University of Rome Rome Italy

Correspondence should be addressed to Cristina Tortolini cristinatortoliniuniroma1it

Received 6 September 2016 Revised 2 November 2016 Accepted 9 November 2016

Academic Editor Ana Marıa Dıez-Pascual

Copyright copy 2016 Giovanni Fusco et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A catalase-based (NAFMWCNTs) nanocomposite filmmodified glassy carbon electrode for hydrogen peroxide (H2O2) detectionwas developedThe developed biosensor was characterized in terms of its bioelectrochemical properties Cyclic voltammetry (CV)technique was employed to study the redox features of the enzyme in the absence and in the presence of nanomaterials dispersedin Nafion polymeric solution The electron transfer coefficient 120572 and the electron transfer rate constant 119896119904 were found to be042 and 171 sminus1 at pH 70 respectively Subsequently the same modification steps were applied to mesoporous graphite screen-printed electrodes Also these electrodes were characterized in terms of their main electrochemical and kinetic parameters Thebiosensor performances improved considerably after modification with nanomaterials Moreover the association of Nafion withcarbon nanotubes retained the biological activity of the redox protein The enzyme electrode response was linear in the range 25ndash1150120583mol Lminus1 with LODof 083 120583mol Lminus1 From the experimental data we can assess the possibility of using themodified biosensoras a useful tool for H2O2 determination in packaged beverages

1 Introduction

In recent years many researchers focused their activity ondeveloping new tools to detect H2O2 not only as an oxidasesreaction byproduct but also as a conservative compoundin food and drugs [1 2] Indeed hydrogen peroxide findssignificant employment in industrial processes as an oxidant[3] in particular hydrogen peroxide is released into theenvironment in either small or large amounts since it isused as oxidant whitening or sterilant tool in packagingmaterials owing to its sporicidal and bactericidal features[4ndash6] Nevertheless high H2O2 concentrations would bedangerous for human beings [7ndash10]

Several analytic methods as chemiluminescence [11ndash17]photometry [18] fluorimetry [19ndash21] titrimetry [22 23]spectrophotometry [23ndash26] high-performance liquid chro-matography (HPLC) [27] and especially electrochemistry [328ndash37] are reported in the literature for detection of hydrogenperoxide

The electrochemical techniques provide some interestingadvantages in comparison to the other onesmentioned abovelike fast specific and cheapmonitoring of hydrogen peroxide[37ndash43] The direct reduction of H2O2at a bare sensor isnot suitable for analytical measures due to its slow kineticsand high potentials required for redox reactions [44] Toovercome these problems several modified electrochemicalsensorswere developed Electrochemical biosensors based onthe biocatalytic activity of immobilized enzymes towards thesubstrate H2O2are helpful because of their high sensitivityselectivity and ease of use [45 46] Some authors in therecent years have applied different modified biosensorsbased on various redox proteins to realize interesting toolsfor the monitoring of H2O2 [45 47ndash55]

Catalase (CAT) belongs to oxidoreductase family classand has a heme prosthetic group at its active site with ferricion (Fe(III)) [48 50 56ndash59] The catalytic ability of CATto reduce hydrogen peroxide was used in the developing ofbiosensors [50 56 60] To investigate CAT catalytic activity

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2016 Article ID 8174913 12 pageshttpdxdoiorg10115520168174913

2 Journal of Analytical Methods in Chemistry

it is important to study its capacity to perform direct electrontransfer (DET) to the electrode surface It is usually difficultto observe the DET because the heme groups are burieddeeply inside in the large structure of the protein [61 62]Also denaturation of the redox protein could occur onthe sensor surface due to the immobilization method andto the matrix composition To overcome these problemsand promote the DET carbon nanotubes (CNTs) modifiedelectrodes are widely employed as support for the physicalimmobilization of biological molecules to promote the DETthanks to their high surfacevolume ratio and conductivityand also to enhance sensors and biosensors performances[63ndash67] A drawback on the use of CNTs to modify electrodesurface is their insolubility [68 69] However some authorshave obtained good results in the CNTs modification of theelectrode surface by using polymers as dispersing support[70 71] Nafion is a perfluorinated polymer resistant tochemical attack and the CNTs dispersion in its film has beeninvestigated [72ndash74]

In the present study we report the development of abiosensor for H2O2monitoring based on the immobilizationof catalase in a Nafion film containing dispersed function-alized MWCNTs-COOH The Nafion film ensures efficientimmobilization of the protein in its native configurationThe DET of catalase was investigated either on modifiedor on bare electrode to identify the optimal conditions forH2O2 detection In view of the possible practical applicationthe same modification steps were performed on screen-printed electrodes (SPEs) with a working electrode basedon mesoporous graphite (MG-SPE) Finally the obtainedbiosensor was applied for the determination of hydrogenperoxide in beverages samples

2 Experimental

21 Materials and Reagents Catalase from bovine liver (CATEC 11116 activity ge 10000Umgminus1 protein) was sup-plied by Sigma-Aldrich (Switzerland) and stored at minus20∘CAll chemicals used were of analytical grade In particu-lar Na2HPO4 NaH2PO4 HOC(COOH)(CH2COOH)2 KCl(K3[Fe(CN)6]) Nafion 117 solution (NAF purum sim5 solu-tion in a mixture of lower aliphatic alcohols and water)CH3CH2OH (sim96 vv) and H2O2 (30wt in H2O) werepurchased from Sigma-Aldrich (Switzerland) High puritydeionized water (resistance 182MΩ times cm at 25∘C TOClt10 120583g Lminus1) obtained from aMillipore Direct-Q 3 UV system(France) was used throughout the experiments The workingsolutions were prepared by diluting the stock solution with01mol Lminus1 phosphate buffer solution and 01mol Lminus1 KCl pH70 (PBS buffer solution) and then deoxygenated by bubblingN2for about 20min Multiwalled carbon nanotubes modifiedwith carboxylic groups (MWCNTs-COOH) were obtainedfrom DropSens (Spain)

22 ElectrochemicalMeasurements All electrochemicalmea-surements were performed with 120583-Autolab type III poten-tiostat (EcoChemie Netherlands) controlled using the GPES

Manager program (EcoChemie Netherlands) at room tem-perature in N2 atmosphere Batch electrochemical experi-ments were performed in a 5mL thermostated glass cell(model 61415150 Metrohm Switzerland) containing PBSbuffer solution with a conventional three-electrode systemDifferent working electrodes were used in particular glassycarbon electrode (GCE cat 61204300 Metrohm Switzer-land 120601 = 3mm) and a mesoporous graphite screen-printedelectrode (MG-SPE model DRP-110MC 120601 = 4mm Drop-Sens Spain) A saturated calomel electrode (SCE cat303SCG12 Amel Instruments Italy) as the reference elec-trode and a carbon rod (cat 61248040 Metrohm Switzer-land) as the counter one were employed For SPEs thecounter electrodewas carbon and the reference onewas silverrespectively All the reported potentials are referred to assaturated calomel electrode (119864 = 0241V versus NHE) AllpH measures were performed using a digital pH meter (827pH lab Metrohm Italy) The morphology of the sampleswas observed using high-resolution field emission scanningelectron microscopy (HR FESEM Zeiss Auriga Microscopy)equipped withMicroanalysis EDS le 123 Mn-K120572 eV (Bruker)

23 Procedures The GCE surface was polished with 03 and005 120583m alumina slurry on polishing silk cloth (SIEM Italy)and rinsed with deionized water Then the electrode wassonicated in deionizedwater to remove trace of alumina fromthe surface (Sonicator AU-32 ArgoLab Italy)

The physical immobilization of the enzyme was realizedby dropping onto the working electrode surface 2 120583L of 05wt Nafion solution containing 1mgmLminus1 of redox proteineither in the presence or in the absence of 1mgmLminus1 ofMWCNTs-COOHThe electrode surfacewas finally air-driedfor about 20min at room temperature The biosensors werestored in PBS buffer solution at 4∘C before use

The analysis protocol of real beverages is described asfollows 25mL of different beverages sample was diluted to10mL with PBS buffer solution Then a certain amount ofH2O2 (15 120583mol Lminus1) was added and the solutions were deoxy-genated Then the samples were analyzed directly by cyclicvoltammetry (CV) method and finally the recoveries wereevaluated For the study of pH dependance the McIlvainebuffer was used at different pH values

3 Results and Discussion

31 Electrochemical Characterization of Glassy Carbon Elec-trode after Steps of Modification The effect on the improve-ment of electrochemical performances by using nanomateri-als as MWCNTs-COOH was evaluated with cyclic voltam-metry measurements of the electroactive area (119860119890) and ofthe heterogeneous standard rate constant (1198960) of the differentelectrodes The cyclic voltammograms (not shown) wererecorded in a solution of 1 1mmol Lminus1 potassium ferricyanidein PBS buffer solution119860119890 was determined from the Randles-Sevcik equation 119868119901 = 2686 times 10

51198993211986011989011986312119862V12 [95]

where 119868119901 is current in amps (A) 119899 is number of electronstransferred of K3[Fe(CN)6] by cyclic voltammetry (CV) inthe redox event (usually 1)119860119890 is electroactive area (cm

2)119863 is

Journal of Analytical Methods in Chemistry 3

minus8

minus6

minus4

minus2

0

2

4

6

I(120583

A)

minus02 04minus04 06minus06 00 02

E (V versus SCE)

(a)

minus80

minus60

minus40

minus20

0

20

40

60

I(120583

A)

minus04 minus02 00 02 04 06minus06

E (V versus SCE)

(b)

Figure 1 CVs for NAF-GCE-CAT (a) and NAF-MWCNTs-COOH-GCE-CAT (b) at different scan rates (10ndash500mV sminus1) in deoxygenatedPBS buffer solution

Table 1 Electroactive area and heterogeneous standard rate con-stant of bare sensor and after modification steps

Sensor 119860 119890mm2 1198960 times 10minus4cm sminus1

Bare-GCE 489 93NAF-GCE 216 65NAF-MWCNTs-COOH-GCE 1642 135

diffusion coefficient (76 times 10minus6 cm2 sminus1) 119862 is concentration(mol cmminus1) and V is scan rate (Vsminus1) 1198960 was calculated byan extended method [96] a combination of Nicholson [97]and Klingler and Kochi treatments [98] by CV data usingthe same solution described above in the scan rate range 5ndash100mV sminus1

By comparing the results (see Table 1) arising from theseveral modification steps of the sensor two aspects canbe pointed out (i) the parameters obtained for the Nafionmodified sensor (NAF-GCE) are lower than both the baresensor (bare-GCE) and the nanomaterial modified sensor(NAF-MWCNTs-COOH-GCE) presumably this is due tothe Nafion film that hinders the charge transfer and slowsdown the substrate rate towards the sensor surface (ii) theuse of carbon nanotubes enhances hugely the electrochemicalsignal increasing 119860119890 (about 4 and 8 times compared to thebare-GCE and NAF-GCE resp) and improves 1198960 of theferricyanide ion towards the sensor surface despite the ionexchange polymer presence (about 15 and 2 times comparedto the bare-GCE andNAF-GCE resp) this could be ascribedto their excellent properties of increasing areavolume ratioand high electron conductivity and of facilitating the electrontransfer [99ndash104] The association of these nanomaterialswith Nafion (as solubilizing agent) does not impair theelectrocatalytic features of carbon nanotubes This aspectwas also observed in our previous work where the use ofNAFMWCNTs composite film has greatly increased thetransfer charge rate [105]

32 Biosensor Voltammetric Behavior before and after Nano-material Modification The comparison of electrocatalytic

performances was evaluated by using catalase as modelredox protein and comparing the voltammetric behavior(Figures 1(a) and 1(b)) measuring several electrochemicalparameters (see Section 34) The catalase was immobilizedby a Nafion film onto the GCE surface in the absence andin the presence of MWCNTs-COOH the electrochemicalbehavior of the modified electrodes has been investigatedin N2 saturated PBS buffer solution using CV The cyclicvoltammograms were recorded at NAF-GCE-CAT and NAF-MWCNTs-COOH-GCE-CAT modified GCEs in the poten-tial range from 06V to minus06V In the absence of MWCNTs-COOH catalase immobilized in a Nafion film onto GCEsurface showed a quasi-reversible signal (see Figure 1(a)) witha midpoint potential of 11986401015840 = minus128mV the separation ofcathodic and anodic peak potential Δ119864119901 = 80mV (at scanrates lower than 100mV sminus1) indicated a fast electron transferreaction according to the literature [106] For the othermodified electrode when the redox protein is in the presenceof carbon nanotubes CV experiments yielded evidence ofa prominent increase (about 20 times) of faradic current(Figure 1(b)) and also an enhancement of electron transferkinetic was observed at a constant amount of immobilizedprotein In particular 11986401015840 shifted to a more negative potentialvalue (minus140mV) and Δ119864119901 was 70mV assuming that carbonnanotubes play an important role in the rising of the systemreversibility

33 Study of pH Dependence on the Modified ElectrodeThe effect of pH solution on the modified NAF-MWCNTs-COOH-GCE-CAT electrode was also tested In Figure 2(a)the peak currents at different pH values are shown Themaximum of anodic current occurred at pH 70 This valuewas consistentwith that reported for catalase enzyme [60 76ndash78] Based on these results pH 70 for PBS buffer solutionwas used as the optimal pH for further experiments Alsothe influence of pH solution on the oxidation peak potentialswas investigated The oxidation peak potential was reportedversus solution pH values in the range 35ndash80 (Figure 2(b))The obtained slope (0044V) suggests that the reaction at


12

13

14

15

16

17

pH 35 pH 55

pH 6 pH 7

pH 75 pH 8

I(120583

A)

(a)

8 93 64 5 7pH

y = minus00442x + 03703

R2 = 0993

Epa

(V)

000

005

010

015

020

025

(b)

Figure 2 The effect of pH on the redox peak currents of NAF-MWCNTs-COOH-GCE-CAT in various buffer solutions with pH values 3555 60 70 75 and 80 (a) 119864119901119886 versus pH plot (b)

the electrode surface is accompanied by proton transferThe slope value is slightly smaller than Nernstrsquos value of0059V pHminus1 for the reaction of one electron coupled toone proton [76] This is probably ascribable to the influenceof protonation states of trans ligands of the heme iron andamino acids around the heme or the protonation of H2Omolecule coordinated to the coordinated iron [107 108]

34 Cyclic Voltammetric Studies of Direct Electron Transfer ofCatalase before and after Nanomaterial Modification of theBiosensor Figure 3(a) shows typical cyclic voltammogramsof NAF-MWCNTs-COOH-GCE-CAT biosensor at differentscan rates (10ndash1400mV sminus1)Thedependence of peak currentsand peak potentials on the scan rate is also observed inFigures 3(b) and 3(c) respectively As is obvious fromFigure 3(b) the peak currents change linearly with scanrate over a range of 10 to 1400mV sminus1 (with correlationcoefficients of 09924 and 09914) as expected for thinlayer electrochemistry [35 109] and according to a surface-controlled process The slope of corresponding log 119868119901 versuslog V linear plot with a correlation coefficient of 09949 wasfound to be 1115 very close to the theoretical slope 1 for thinlayer voltammetry [109]

The surface concentration of electroactive redox protein(Γ) can be estimated using Faraday law (see (1)) and calculatedfrom the slope of peak currentscan rate plot [76 109]

Γ =4119868119901119877119879

11989921198652119860V (1)

where V is the scan rate 119860 is the electrode surface area(007 cm2)119879 is the temperature 119899 is the number of electronsand 119877 and 119865 are gas and Faraday constants respectivelyThus the average surface concentration Γ of catalase wasfound to be 476 times 10minus10mol cmminus2 which indicates thatthe immobilized enzyme is in the form of an approximatemonolayer on the surface of the modified electrode [63 75]

Table 2 Electrochemical parameters for immobilized catalaseeither in the absence or in the presence of nanomaterials

Biosensor 11986401015840mV 120572 119896119904sminus1 Γmol cmminus2

NAF-GCE-CAT minus128 089 103 230 times 10minus10

NAF-MWCNTs-GCE-CAT minus138 038 165 350 times 10minus10

NAF-MWCNTs-COOH-GCE-CAT minus140 042 171 476 times 10minus10

Moreover the peak-to-peak separation at a scan rateof 10mV sminus1 was approximatively 70mV indicating a quasi-reversible electron transfer process Based on the Lavirontheory [109] the transfer coefficient (120572) and the electrontransfer rate constant (119896119904) for immobilized catalase eitherin the absence or in the presence of nanomaterials canbe estimated by measuring the variation of peak potentialseparation with scan rate (at higher scan rates as shown inFigure 3(c)) and reported in Table 2

Besides by comparing our proposed biosensor to othersimilar ones in the literature [60 77ndash83] all based on CATmodified GCEs by using MWCNTs it is evident that theamount of our electroactive catalase is higher probably dueto the simple NAFMWCNTs matrix that could increase theexposure extent of the heme group in the catalase enzyme (seeTable 3) The formal potential 11986401015840 of our biosensor is muchless negative than those proposed by other authors [63 76ndash83 108 110] The formal potential value is dependent on theprotein structure [111 112] so a change of the heme proteinin the NAFMWCNTs composite film results in a shift of 11986401015840to positive potential values Moreover partial denaturationof the enzyme could cause heme leakage and then a negativeshift of the redox peaks (change in the coordination sphere)[113]


1400 mV sminus1

10 mV sminus1

minus200

minus150

minus100

minus50

0

50

100

150

I(120583

A)

minus06 minus04 06minus08 02 04minus02 00

E (V versus SCE)

(a)

R2 = 09924

R2 = 09914

200 400 600 800 1000 1200 1400 16000 (mV sminus1)

I p(120583

A)

minus40

minus20

0

20

40

60

(b)

R2 = 0992

R2 = 0992

y = 01199x minus 00181E

E y = minus01467x minus 02735

minus03

minus025

minus02

minus015

minus01

minus005

0

Ep(V

ver

sus S

CE)

minus03 minus02 minus01 00 01 02minus04log (V sminus1)

pc

pa

(c)

Figure 3 CVs for NAF-MWCNTs-COOH-GCE-CAT in deoxygenated PBS buffer solution at various scan rates (a) Relationship betweenthe anodic and cathodic peak currents and scan rates (b) Relationship between peak potential separation and logarithm of scan rates (c)

35 Catalytic Activity of Catalase The voltammetric charac-terization of the hydrogen peroxide reduction by means ofthe developed NAF-MWCNTs-COOH-GCE-CAT biosensorwas performed in PBS buffer solution at a scan rate of50mV sminus1 (Figure 4(a))

An increase in the cathodic peak with the hydrogenperoxide concentration and a decrease in the anodic peakduring the scan reversal have been observed Conversely inthe absence of catalase no current change has been detectedby the NAF-MWCNTs-COOH-GCE electrode From ourexperiments we confirm the EC mechanism previouslyreported in the literature [77 95]

Cat-Fe (III) + eminus +H+ 999445999468 Cat-Fe (II)H+

at the electrode surface

H2O2 + Cat-Fe (II)H+ 997888rarr Cat-Fe (III) +H+ +H2O

in solution

(2)

Figure 4(b) reports the catalytic efficiency (119868119888119868119889) changesversus H2O2 concentration 119868119888 and 119868119889 are the cathodic peakcurrents in the presence and in the absence of hydrogenperoxide respectively

As can be observed the catalytic efficiency increases withthe H2O2concentration up to 298120583M and then a plateauis reached This is probably due to the denaturing effect ofhydrogen peroxide at high concentration values

Based on these results obtained using a classical GCEelectrode and employing a very simple and easy immobi-lization procedure the same modification system has beendeveloped on screen-printed electrodes in view of a possibleapplication for determination of hydrogen peroxide in realsamples

36 Morphological Characterization of Screen-Printed Elec-trodes and Electroanalytical and Kinetic CharacterizationThe surface morphology of the modified screen-printedelectrodes (SPEs) was obtained by scanning electronic


(A)

(B)(C)(D)(E)

minus06 minus04 00 02minus08 minus02minus1e minus 5

minus8e minus 6

minus6e minus 6

minus4e minus 6

minus2e minus 6

0

2e minus 6

4e minus 6

I(A

)

E (V versus SCE)

(a)

100 200 300 400 500 600008

10

12

14

16

18

20

H2O2 (120583M)

I cId

(120583A

)

(b)

Figure 4 CVs of NAF-MWCNTs-COOH-GCE-CAT modified biosensor in the absence (A) and in the presence of 130 120583M (B) 215120583M(C) 298 120583M (D) and 538 120583M (E) of the substrate H2O2 (a) Catalytic efficiency changes versus hydrogen peroxide where 119868119888 and 119868119889 are thecathodic peak currents in the presence and in the absence of H2O2 respectively (b) Experimental conditions deoxygenated PBS buffersolution V = 50mVsminus1

(a)

Pa 1

Pa R1

Pa 1 = 1414 nmPb 1 = 2659∘

(b)

Figure 5 SEM images of electrodes surfaces MG-SPE bare (a) and NAF-MWCNTs-COOH-MG-SPE modified electrode (b)

Table 3 Comparison of electrochemical parameters of the catalasemodified glassy carbon electrodes by using MWCNTs recentlydeveloped for H2O2 determination

Catalase modified GCE 11986401015840mV 119896119904sminus1 Γmol cmminus2 Ref

[bmim][PF6]-MWCNTs simminus100ad 195 331 times 10minus10 [75]Ionic-liquid-MWCNTs-NH2

minus460ad 223 288 times 10minus10 [76]

MWCNTs-NF-DTAB minus279ad 1071 26 times 10minus11 [77]CA-MWCNTs minus559ad 122 149 times 10minus10 [78]PEI-MWCNTs-NF minus450ae 105 210 times 10minus10 [79]MWCNTs-NF-DDAB minus380ac 110 73 times 10minus12 [80]PLL-f-MWCNTs minus471ac 548 4072times10minus10 [81]NAF-MWCNTs-COOH-CYS-AuNPs minus441ad 872 2 times 10minus9 [82]

NAF-MWCNTs-COOH-GCE minus140bd 171 476 times 10minus10 This

workaVersus AgAgCl bversus SCE cpH 65 dpH 70 epH 75

microscopy (SEM) In Figure 5(a) mesoporous graphite SPE(MG-SPE bare) surface without modification is shown

Figure 5(b) reveals the presence of a cross-linked structureof multiwalled carbon nanotubes modified with carboxylicgroups dispersed in a Nafion film (NAF-MWCNTs-COOH-MG-SPE surface) Moreover the diameter of the carbon nan-otubes (sim14 nm) is indicated In the presence of the enzymethe highly porous architecture that is formed between theMWCNTs-COOH and the Nafion film is suitable for immo-bilization of catalase that is confirmed in the followingelectrochemical measures

Also electrochemical characterization of these SPEs wascarried out and the results are reported in Table 4 Alsofor these electrodes the feature of nanomaterials to increasethe sensor performances considerably is confirmed so thefollowing studies were performed using the NAF-MWCNTs-COOH-MG-SPE sensor

Successively the main electrochemical parameters of ourproposed biosensor NAF-MWCNTs-COOH-MG-SPE-CATwere evaluated (see Table 5)

The electrochemical response of the obtained biosen-sor for different concentrations of H2O2was studied Thecurrent-concentration dependence of hydrogen peroxide wasmodeled by using Michaelis-Menten nonlinear fitting thus


Table 4 Electroactive area and heterogeneous standard rate constant of bare screen-printed sensor and after the modification step

Sensor-SPE 119860 119890mm2 1198960 times 10minus4cm sminus1

MG-SPE bare 793 165NAF-MWCNTs-COOH-MG-SPE 1165 302

Table 5 Electrochemical parameters for immobilized catalase in the presence of nanomaterials on mesoporous graphite SPE


NAF-MWCNTs-COOH-MG-SPE-CAT minus254 037 060 287 times 10minus10

Table 6 Comparison of analytical and kinetic parameters for H2O2 detection for different redox protein modified electrodes using H2O2 assubstrate

119870119872app mmol Lminus1 Slope 120583A120583molminus1 L Linear range 120583mol Lminus1 LOD 120583mol Lminus1 119877 Ref

026 00112 021ndash3000 008 0999 [34]021 28798 10ndash3200 333 0995 [78]0224 0392 1ndash3600 0008 0998 [81]mdash mdash 200ndash5000 10 0997 [83]261 mdash 5ndash5130 17 0999 [84]mdash mdash 10ndash1130 065 [85]051 3692 6ndash1010 039 0996 [86]517 mdash 00067ndash8000 00022 0998 [87]mdash mdash 98ndash6000 49 0999 [88]mdash 09103 01ndash100 005 0997 [89]mdash 061 03ndash1000 01 0999 [88]021 00281 1ndash140 093 0998 [90]029 0315 50ndash1800 40 0997 [91]0010 1ndash600 73 [92]0089 50ndash135 167 [93]281 03ndash600 005 [94]15 038 25ndash1150 083 0999 This work

allowing the calculation of the main kinetic parameters dataobtained are reported in Table 6 It is clear that the biosensorhas a good LOD of 083 120583mol Lminus1 and a good sensitivityto determine H2O2concentrations Moreover a comparisonof analytical and kinetic parameters for H2O2detection fordifferent redox protein modified electrodes is summarized inTable 6 [34 81 83 85ndash94 110 114]

Also the reproducibility of the developed biosensor wascalculated as RSD = 50 by using 500 120583mol Lminus1 H2O2 in aseries of six experiments By the data achieved the followingcan be assessed (i) the immobilized enzyme retained goodbiocatalytic activity (ii) the carbon nanotubes dispersed inthe Nafion film provided an optimal microenvironment(iii) the nanocomposite was a good matrix for catalaseimmobilization and biosensing preparation (iv) the redoxprotein maintained active site accessibility and exchangedelectrons with the sensor surface This platform was appliedfor H2O2 sensing in real samples

37 Determination of H2O2 in Beverages Based on the resultsdeclared in the previous sections and in order to test the

reliability of the proposed biosensor for practical applicationdifferent commercial beverages were chosen (tea juice andmilk) Every sample was pretreated as reported in Section 23The concentration of 15120583mol Lminus1 was chosen because anFDA regulation currently limits residual H2O2 to 005 ppm(corresponding to 15 120583mol Lminus1) leached into distilled waterin finished food packages [115] The results show goodrecoveries in the range 1003ndash1057 for our modified NAF-MWCNTs-COOH-MG-SPE-CAT biosensor (Table 7)

38 Stability of NAF-MWCNTs-COOH-MG-SPE-CAT Bio-sensor The shelf lifetime of our modified biosensor wastested by measuring its current response obtained for500120583mol Lminus1 H2O2 concentration during a period of 21 daysThe biosensor was stored in PBS buffer solution at 4∘C beforeand after use During the first week a 4 decrease wasobserved reaching a 15 decrease after three weeks Thisresult can be ascribable to the presence of the nanomaterialswhich avoid the fouling phenomena of the surface whichcould affect the biosensor performances and also the useof NAFMWCNTs composite film provides a strong and


Table 7 Determination of H2O2 in several commercial beveragesspiked with H2O2 15 120583mol Lminus1 using NAF-MWCNTs-COOH-MG-SPE-CAT as biosensor

Beverages samples Found120583mol Lminus1 Recovery Peach tea 159 1057Lemon tea 153 1023Green tea 148 1010Apple juice 149 1003Blood orange juice 157 1048Pineapple juice 147 1020Lactose-free milk 156 1038

biocompatible microenvironment for stabilizing the catalaseactivity

4 Conclusion

In this study an electrochemical biosensor was developedfor the determination of hydrogen peroxide concentrationin packaged beverages To this aim direct electrochemicalproperties of catalase confined in aNafion filmon the surfaceof a glassy carbon electrode were studied The electrontransfer coefficient 120572 the electron transfer rate constant119896119904 and the surface concentration of electroactive redoxprotein Γ were evaluated by cyclic voltammetry studies Themodification of the electrode surface by using nanostructuredmaterials dispersed in Nafion polymeric solution resulted inan enhancement of the overall bioelectrochemical propertiesof the developed biosensor The biocatalytic activity towardscatalase substrate hydrogen peroxide confirmed that theimmobilization procedure allowed a goodmicroenvironmentfor catalase and facilitated the electron exchange to theelectrode surface Hence based on these interesting resultsobtained the same modification procedure was applied toscreen-printed electrodes Also this platform of themodifiedbiosensor was entirely characterized and was applied todetect H2O2 in spiked real samples of different commercialbeverages obtaining good recoveries

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] J Wang Y Lin and L Chen ldquoOrganic-phase biosensors formonitoring phenol and hydrogen peroxide in pharmaceuticalantibacterial productsrdquoThe Analyst vol 118 no 3 pp 277ndash2801993

[2] M H Pournaghi-Azar F Ahour and F Pournaghi-Azar ldquoSim-ple and rapid amperometric monitoring of hydrogen peroxidein salivary samples of dentistry patients exploiting its electro-reduction on the modifiedpalladized aluminum electrode as

an improved electrocatalystrdquo Sensors andActuators B Chemicalvol 145 no 1 pp 334ndash339 2010

[3] Y Lin X Cui and L Li ldquoLow-potential amperometric deter-mination of hydrogen peroxide with a carbon paste electrodemodified with nanostructured cryptomelane-type manganeseoxidesrdquo Electrochemistry Communications vol 7 no 2 pp 166ndash172 2005

[4] J Ping J Wu K Fan and Y Ying ldquoAn amperometric sensorbased on Prussian blue and poly(o-phenylenediamine) modi-fied glassy carbon electrode for the determination of hydrogenperoxide in beveragesrdquo Food Chemistry vol 126 no 4 pp2005ndash2009 2011

[5] S Alpat S K Alpat Z Dursun and A Telefoncu ldquoDevel-opment of a new biosensor for mediatorless voltammetricdetermination of hydrogen peroxide and its application in milksamplesrdquo Journal of Applied Electrochemistry vol 39 no 7 pp971ndash977 2009

[6] C-L Hsu K-S Chang and J-C Kuo ldquoDetermination of hy-drogen peroxide residues in aseptically packaged beveragesusing an amperometric sensor based on a palladium electroderdquoFood Control vol 19 no 3 pp 223ndash230 2008

[7] International Agency for the Reaserch on Cancer (IARC)Hydrogen Peroxide vol 71 of IARC Monographs on the Evalua-tion of Carcinogenic Risks to Humans IARC Lyon France 1999

[8] World Health Organization (WHO) Hydrogen Peroxide 267Joint FAOWHO Expert Committee on Food Additives WHOFood Additives Series no 5 WHO Geneva Switzerland 1973

[9] Canadian Centre for Occupational Health and Safety(CCOHS) Cheminfo Hydrogen Peroxide Solutions 35 andGreater Record Number 198 CCOHS Hamilton Canada 1998

[10] International Programme on Chemical Safety (IPCS) ldquoHydro-gen peroxide (gt60 solution inwater)rdquo International ChemicalSafety Card 0164 WHO Geneva Switzerland 2000

[11] G L Kok T P Holler M B Lopez H A Nachtrieb andM Yuan ldquoChemiluminescent method for determination ofhydrogen peroxide in the ambient atmosphererdquo EnvironmentalScience and Technology vol 12 no 9 pp 1072ndash1076 1978

[12] S He W Shi X Zhang J Li and Y Huang ldquo120573-Cyclodextrins-based inclusion complexes of CoFe2O4 magnetic nanoparticlesas catalyst for the luminol chemiluminescence system and theirapplications in hydrogen peroxide detectionrdquo Talanta vol 82no 1 pp 377ndash383 2010

[13] N Yamashiro S Uchida Y Satoh et al ldquoDetermination ofhydrogen peroxide in water by chemiluminescence detection(I) flow injection type hydrogen peroxide detection systemrdquoJournal of Nuclear Science andTechnology vol 41 no 9 pp 890ndash897 2004

[14] F R P Rocha E Rodenas-Torralba B F Reis A Morales-Rubio and M De La Guardia ldquoA portable and low cost equip-ment for flow injection chemiluminescence measurementsrdquoTalanta vol 67 no 4 pp 673ndash677 2005

[15] G-J Zhou GWang J-J Xu andH-Y Chen ldquoReagentless che-miluminescence biosensor for determination of hydrogen per-oxide based on the immobilization of horseradish peroxidaseon biocompatible chitosan membranerdquo Sensors and ActuatorsB Chemical vol 81 no 2-3 pp 334ndash339 2002

[16] X Hu H Han L Hua and Z Sheng ldquoElectrogenerated che-miluminescence of blue emitting ZnSe quantum dots and itsbiosensing for hydrogen peroxiderdquo Biosensors and Bioelectron-ics vol 25 no 7 pp 1843ndash1846 2010

[17] S Lu J Song and L Campbell-Palmer ldquoAmodified chemilumi-nescencemethod for hydrogen peroxide determination in apple


fruit tissuesrdquo Scientia Horticulturae vol 120 no 3 pp 336ndash3412009

[18] Z Genfa P K Dasgupta W S Edgemond and J N MarxldquoDetermination of hydrogen peroxide by photoinduced fluoro-genic reactionsrdquo Analytica Chimica Acta vol 243 pp 207ndash2161991

[19] A L Lazrus G L Kok S N Gitlin J A Lind and S EMcLaren ldquoAutomated fluorometric method for hydrogen per-oxide in atmospheric precipitationrdquo Analytical Chemistry vol57 no 4 pp 917ndash922 1985

[20] A E Albers V S Okreglak and C J Chang ldquoA FRET-basedapproach to ratiometric fluorescence detection of hydrogenperoxiderdquo Journal of the AmericanChemical Society vol 128 no30 pp 9640ndash9641 2006

[21] F He Y TangM Yu SWang Y Li andD Zhu ldquoFluorescence-amplifying detection of hydrogen peroxide with cationic con-jugated polymers and its application to glucose sensingrdquoAdvanced Functional Materials vol 16 no 1 pp 91ndash94 2006

[22] E C Hurdis and H Romeyn Jr ldquoAccuracy of determination ofhydrogen peroxide by cerate oxidimetryrdquo Analytical Chemistryvol 26 no 2 pp 320ndash325 1954

[23] M S Prasada Rao A R Mohan Rao K V Ramana and S RSagi ldquoThallimetric oxidations-V titrimetric and spectrophoto-metric determination of hydrogen peroxiderdquo Talanta vol 37no 7 pp 753ndash755 1990

[24] A Lobnik and M Ajlakovi ldquoSol-gel based optical sensor forcontinuous determination of dissolved hydrogen peroxiderdquoSensors and Actuators B Chemical vol 74 no 1ndash3 pp 194ndash1992001

[25] K Sunil and B Narayana ldquoSpectrophotometric determinationof hydrogen peroxide in water and cream samplesrdquo Bulletin ofEnvironmental Contamination and Toxicology vol 81 no 4 pp422ndash426 2008

[26] K Zhang L Mao and R Cai ldquoStopped-flow spectrophotomet-ric determination of hydrogen peroxide with hemoglobin ascatalystrdquo Talanta vol 51 no 1 pp 179ndash186 2000

[27] M Tarvin B McCord K Mount K Sherlach and M LMiller ldquoOptimization of two methods for the analysis ofhydrogen peroxide high performance liquid chromatographywith fluorescence detection and high performance liquid chro-matography with electrochemical detection in direct currentmoderdquo Journal of Chromatography A vol 1217 no 48 pp 7564ndash7572 2010

[28] Y-H Bai Y Du J-J Xu and H-Y Chen ldquoCholine biosensorsbased on a bi-electrocatalytic property of MnO2 nanoparticlesmodified electrodes to H2O2rdquo Electrochemistry Communica-tions vol 9 no 10 pp 2611ndash2616 2007

[29] H Hamidi E Shams B Yadollahi and F K Esfahani ldquoFab-rication of carbon paste electrode containing [PFeW11O39]

4minus

polyoxoanion supported on modified amorphous silica gel andits electrocatalytic activity for H2O2 reductionrdquo ElectrochimicaActa vol 54 no 12 pp 3495ndash3500 2009

[30] P-H Lo S A Kumar and S-M Chen ldquoAmperometric deter-mination of H2O2 at nano-TiO2DNAthionin nanocompositemodified electroderdquo Colloids and Surfaces B Biointerfaces vol66 no 2 pp 266ndash273 2008

[31] K-S Tseng L-C Chen and K-C Ho ldquoAmperometric detec-tion of hydrogen peroxide at a Prussian Blue-modified FTOelectroderdquo Sensors and Actuators B Chemical vol 108 no 1-2pp 738ndash745 2005

[32] Y Xu W Peng X Liu and G Li ldquoA new film for thefabrication of an unmediated H2O2 biosensorrdquo Biosensors andBioelectronics vol 20 no 3 pp 533ndash537 2004

[33] M R Guascito E Filippo CMalitesta DManno A Serra andA Turco ldquoA new amperometric nanostructured sensor for theanalytical determination of hydrogen peroxiderdquo Biosensors andBioelectronics vol 24 no 4 pp 1057ndash1063 2008

[34] S Chen R Yuan Y Chai L Zhang N Wang and X LildquoAmperometric third-generation hydrogen peroxide biosensorbased on the immobilization of hemoglobin on multiwallcarbon nanotubes and gold colloidal nanoparticlesrdquo Biosensorsand Bioelectronics vol 22 no 7 pp 1268ndash1274 2007

[35] M Shamsipur S H Kazemi and M F Mousavi ldquoImpedancestudies of a nano-structured conducting polymer and itsapplication to the design of reliable scaffolds for impedimetricbiosensorsrdquo Biosensors and Bioelectronics vol 24 no 1 pp 104ndash110 2008

[36] P Santhosh K M Manesh A Gopalan and K-P Lee ldquoFabri-cation of a new polyaniline grafted multi-wall carbon nanotubemodified electrode and its application for electrochemicaldetection of hydrogen peroxiderdquo Analytica Chimica Acta vol575 no 1 pp 32ndash38 2006

[37] G Yang F Chen and Z Yang ldquoElectrocatalytic oxidationof hydrogen peroxide based on the shuttlelike nano-CuO-modified electroderdquo International Journal of Electrochemistryvol 2012 6 pages 2012

[38] S Zhu L Fan X Liu et al ldquoDetermination of concentratedhydrogen peroxide at single-walled carbon nanohorn pasteelectroderdquo Electrochemistry Communications vol 10 no 5 pp695ndash698 2008

[39] M R Guascito D Chirizzi C Malitesta et al ldquoLow-potentialsensitive H2O2 detection based on composite micro tubular Teadsorbed on platinum electroderdquo Biosensors and Bioelectronicsvol 26 no 8 pp 3562ndash3569 2011

[40] A L Sanford S W Morton K L Whitehouse et al ldquoVoltam-metric detection of hydrogen peroxide at carbon fiber micro-electrodesrdquo Analytical Chemistry vol 82 no 12 pp 5205ndash52102010

[41] M Liu R Liu and W Chen ldquoGraphene wrapped Cu2Onanocubes non-enzymatic electrochemical sensors for thedetection of glucose and hydrogen peroxide with enhancedstabilityrdquo Biosensors and Bioelectronics vol 45 no 1 pp 206ndash212 2013

[42] M-J Song S W Hwang and DWhang ldquoNon-enzymatic elec-trochemical CuO nanoflowers sensor for hydrogen peroxidedetectionrdquo Talanta vol 80 no 5 pp 1648ndash1652 2010

[43] J Ju and W Chen ldquoIn situ growth of surfactant-free goldnanoparticles on nitrogen-doped graphene quantum dots forelectrochemical detection of hydrogen peroxide in biologicalenvironmentsrdquo Analytical Chemistry vol 87 no 3 pp 1903ndash1910 2015

[44] KThenmozhi and S S Narayanan ldquoElectrochemical sensor forH2O2 based on thionin immobilized 3-aminopropyltrimethoxysilane derived sol-gel thin film electroderdquo Sensors andActuatorsB Chemical vol 125 no 1 pp 195ndash201 2007

[45] A K Upadhyay T-W Ting and S-M Chen ldquoAmperometricbiosensor for hydrogen peroxide based on coimmobilizedhorseradish peroxidase andmethylene green in ormosilsmatrixwith multiwalled carbon nanotubesrdquo Talanta vol 79 no 1 pp38ndash45 2009


[46] W Zhao J-J Xu and H-Y Chen ldquoElectrochemical biosensorsbased on layer-by-layer assembliesrdquo Electroanalysis vol 18 no18 pp 1737ndash1748 2006

[47] S Chandra K S Lokesh A Nicolai and H Lang ldquoDendrimer-rhodium nanoparticle modified glassy carbon electrode foramperometric detection of hydrogen peroxiderdquo AnalyticaChimica Acta vol 632 no 1 pp 63ndash68 2009

[48] Q Lu X Dong L-J Li and X Hu ldquoDirect electrochemistry-based hydrogen peroxide biosensor formed from single-layergraphene nanoplatelet-enzyme composite filmrdquo Talanta vol82 no 4 pp 1344ndash1348 2010

[49] Y Song L Wang C Ren G Zhu and Z Li ldquoA novel hydrogenperoxide sensor based on horseradish peroxidase immobilizedin DNA films on a gold electroderdquo Sensors and Actuators BChemical vol 114 no 2 pp 1001ndash1006 2006

[50] S W Ting A P Periasamy S-M Chen and R SaraswathildquoDirect electrochemistry of catalase immobilized at electro-chemically reduced graphene oxide modified electrode foramperometricH2O2 biosensorrdquo International Journal of Electro-chemical Science vol 6 no 10 pp 4438ndash4453 2011

[51] A A Karyakin E E Karyakina and L Gorton ldquoAmperometricbiosensor for glutamate using prussian blue-based lsquoartificialperoxidasersquo as a transducer for hydrogen peroxiderdquo AnalyticalChemistry vol 72 no 7 pp 1720ndash1723 2000

[52] F Gao R Yuan Y Chai S Chen S Cao andM Tang ldquoAmper-ometric hydrogen peroxide biosensor based on the immobiliza-tion of HRP on nano-AuThipoly (p-aminobenzene sulfonicacid)-modified glassy carbon electroderdquo Journal of Biochemicaland Biophysical Methods vol 70 no 3 pp 407ndash413 2007

[53] M R Majidi M H Pournaghi-Azar A Saadatirad and EAlipour ldquoSimple and rapid amperometric monitoring of hydro-gen peroxide at hemoglobin-modified pencil lead electrode asa novel biosensor application to the analysis of honey samplerdquoFood Analytical Methods vol 8 no 4 pp 1067ndash1077 2015

[54] S Zong Y Cao Y Zhou andH Ju ldquoHydrogen peroxide biosen-sor based on hemoglobin modified zirconia nanoparticles-grafted collagen matrixrdquo Analytica Chimica Acta vol 582 no2 pp 361ndash366 2007

[55] N Nasirizadeh S Hajihosseini Z Shekari and M GhaanildquoA novel electrochemical biosensor based on a modified goldelectrode for hydrogen peroxide determination in differentbeverage samplesrdquo Food Analytical Methods vol 8 no 6 pp1546ndash1555 2015

[56] W R Melik-Adamyan V V Barynin A A Vagin et alldquoComparison of beef liver and Penicillium vitale catalasesrdquoJournal of Molecular Biology vol 188 no 1 pp 63ndash72 1986

[57] M R N Murthy T J Reid III A Sicignano N Tanaka andM G Rossmann ldquoStructure of beef liver catalaserdquo Journal ofMolecular Biology vol 152 no 2 pp 465ndash499 1981

[58] P T Borges C Frazao C S Miranda M A Carrondo andC V Romao ldquoStructure of the monofunctional heme catalaseDR1998 from Deinococcus radioduransrdquoThe FEBS journal vol281 no 18 pp 4138ndash4150 2014

[59] A Dıaz P C Loewen I Fita and X Carpena ldquoThirty years ofheme catalases structural biologyrdquo Archives of Biochemistry andBiophysics vol 525 no 2 pp 102ndash110 2012

[60] M Shamsipur M Asgari M G Maragheh and A A Moosavi-Movahedi ldquoA novel impedimetric nanobiosensor for low leveldetermination of hydrogen peroxide based on biocatalysis ofcatalaserdquo Bioelectrochemistry vol 83 no 1 pp 31ndash37 2012

[61] S Pakhomova B Gao W E Boeglin A R Brash and ME Newcomer ldquoThe structure and peroxidase activity of a 33-kDa catalase-related protein from Mycobacterium avium sspParatuberculosisrdquo Protein Science vol 18 no 12 pp 2559ndash25682009

[62] W Melik-Adamyan J Bravo X Carpena et al ldquoSubstrateflow in catalases deduced from the crystal structures of activesite variants of HPII from Escherichia colirdquo Proteins StructureFunction and Genetics vol 44 no 3 pp 270ndash281 2001

[63] A Salimi ANoorbakhsh andMGhadermarz ldquoDirect electro-chemistry and electrocatalytic activity of catalase incorporatedonto multiwall carbon nanotubes-modified glassy carbon elec-troderdquo Analytical Biochemistry vol 344 no 1 pp 16ndash24 2005

[64] H Zhou T-H Lu H-X Shi Z-H Dai and X-H HuangldquoDirect electrochemistry and electrocatalysis of catalase immo-bilized on multi-wall carbon nanotubes modified glassy carbonelectrode and its applicationrdquo Journal of Electroanalytical Chem-istry vol 612 no 2 pp 173ndash178 2008

[65] A Salimi A Noorbakhsh and M Ghadermarzi ldquoAmper-ometric detection of nitrite iodate and periodate at glassycarbon electrode modified with catalase and multi-wall carbonnanotubesrdquo Sensors and Actuators B Chemical vol 123 no 1pp 530ndash537 2007

[66] G-C Zhao Z-Z Yin L Zhang and X-W Wei ldquoDirectelectrochemistry of cytochrome c on a multi-walled carbonnanotubes modified electrode and its electrocatalytic activityfor the reduction of H2O2rdquo Electrochemistry Communicationsvol 7 no 3 pp 256ndash260 2005

[67] C Tortolini S Rea E Carota S Cannistraro and F MazzeildquoInfluence of the immobilization procedures on the electro-analytical performances of Trametes versicolor laccase basedbioelectroderdquo Microchemical Journal vol 100 no 1 pp 8ndash132012

[68] C Journet W K Maser P Bernier et al ldquoLarge-scale produc-tion of single-walled carbon nanotubes by the electric-arctechniquerdquo Nature vol 388 no 6644 pp 756ndash758 1997

[69] A Star J F Stoddart D Steuerman et al ldquoPreparationand properties of polymer-wrapped single-walled carbon nan-otubesrdquoAngewandte ChemiemdashInternational Edition vol 40 no9 pp 1721ndash1725 2001

[70] W Zhang J Suhr and N Koratkar ldquoCarbon nanotubepoly-carbonate composites as multifunctional strain sensorsrdquo Jour-nal of Nanoscience and Nanotechnology vol 6 no 4 pp 960ndash964 2006

[71] C Liu and J Choi ldquoImproved Dispersion of Carbon Nanotubesin Polymers at High ConcentrationsrdquoNanomaterials vol 2 no4 pp 329ndash347 2012

[72] J Wang M Musameh and Y Lin ldquoSolubilization of carbonnanotubes by Nafion toward the preparation of amperometricbiosensorsrdquo Journal of the American Chemical Society vol 125no 9 pp 2408ndash2409 2003

[73] C P Andrieux P Audebert B Divisia-Blohorn P Aldebertand FMichalak ldquoElectrochemistry in hydrophobic Nafion gelspart 1 Electrochemical behaviour of electrodes modified byhydrophobic Nafion gels loaded with ferrocenesrdquo Journal ofElectroanalytical Chemistry vol 296 no 1 pp 117ndash128 1990

[74] H Liu and J Deng ldquoAn amperometric lactate sensor employingtetrathiafulvalene in Nafion film as electron shuttlerdquo Elec-trochimica Acta vol 40 no 12 pp 1845ndash1849 1995

[75] P A Prakash U Yogeswaran and S-M Chen ldquoA review ondirect electrochemistry of catalase for electrochemical sensorsrdquoSensors vol 9 no 3 pp 1821ndash1844 2009


[76] P Rahimi H-A Rafiee-Pour H Ghourchian P Norouziand M R Ganjali ldquoIonic-liquidNH2-MWCNTs as a highlysensitive nano-composite for catalase direct electrochemistryrdquoBiosensors and Bioelectronics vol 25 no 6 pp 1301ndash1306 2010

[77] S Hashemnia S Khayatzadeh A A Moosavi-Movahedi andH Ghourchian ldquoDirect electrochemistry of catalase in multi-wall carbon nanotubedodecyl trimethylammonium bromidefilm covered with a layer of nafion on a glassy carbon electroderdquoInternational Journal of Electrochemical Science vol 6 no 3 pp581ndash595 2011

[78] A P Periasamy Y-H Ho and S-M Chen ldquoMultiwalled carbonnanotubes dispersed in carminic acid for the development ofcatalase based biosensor for selective amperometric determina-tion of H2O2 and iodaterdquo Biosensors and Bioelectronics vol 29no 1 pp 151ndash158 2011

[79] P Vatsyayan S Bordoloi and P Goswami ldquoLarge catalase basedbioelectrode for biosensor applicationrdquo Biophysical Chemistryvol 153 no 1 pp 36ndash42 2010

[80] P Arun Prakash U Yogeswaran and S-M Chen ldquoDirectelectrochemistry of catalase at multiwalled carbon nanotubes-nafion in presence of needle shaped DDAB for H2O2 sensorrdquoTalanta vol 78 no 4-5 pp 1414ndash1421 2009

[81] A T Ezhil Vilian S-M Chen and B-S Lou ldquoA simple strategyfor the immobilization of catalase on multi-walled carbonnanotubepoly (L-lysine) biocomposite for the detection ofH2O2 and iodaterdquoBiosensors and Bioelectronics vol 61 pp 639ndash647 2014

[82] J Hong W-Y Yang Y-X Zhao et al ldquoCatalase immobi-lized on a functionalized multi-walled carbon nanotubes-goldnanocomposite as a highly sensitive bio-sensing system fordetection of hydrogen peroxiderdquo Electrochimica Acta vol 89pp 317ndash325 2013

[83] Y Wang T Li W Zhang and Y Huang ldquoA hydrogen peroxidebiosensor with high stability based on gelatin-multiwalledcarbon nanotubes modified glassy carbon electroderdquo Journal ofSolid State Electrochemistry vol 18 no 7 pp 1981ndash1987 2014

[84] K Zhou Y Zhu X Yang J Luo C Li and S Luan ldquoAnovel hydrogen peroxide biosensor based on Au-graphene-HRP-chitosan biocompositesrdquo Electrochimica Acta vol 55 no9 pp 3055ndash3060 2010

[85] T Tangkuaram C Ponchio T Kangkasomboon P Katika-wong and W Veerasai ldquoDesign and development of a highlystable hydrogen peroxide biosensor on screen printed carbonelectrode based on horseradish peroxidase bound with goldnanoparticles in the matrix of chitosanrdquo Biosensors and Bioelec-tronics vol 22 no 9-10 pp 2071ndash2078 2007

[86] Q Feng K Liu J Fu et al ldquoirect electrochemistry of hemo-globin based on nano-composite film of gold nanopaticlesand poly (diallyldimethylammonium chloride) functionalizedgraphenerdquo Electrochimica Acta vol 60 pp 304ndash308 2012

[87] C-J Mao X-B Chen H-L Niu J-M Song S-Y Zhangand R-J Cui ldquoA novel enzymatic hydrogen peroxide biosensorbased on AgC nanocablesrdquo Biosensors and Bioelectronics vol31 no 1 pp 544ndash547 2012

[88] W-T Li M-H Wang Y-J Li Y Sun and J-C Li ldquoLinker-free layer-by-layer self-assembly of gold nanoparticlemultilayerfilms for direct electron transfer of horseradish peroxidase andH2O2 detectionrdquo Electrochimica Acta vol 56 no 20 pp 6919ndash6924 2011

[89] X B Kang G C Pang X Y Liang M Wang J Liu and WM Zhu ldquoStudy on a hydrogen peroxide biosensor based on

horseradish peroxidaseGNPs-thioninechitosanrdquo Electrochim-ica Acta vol 62 pp 327ndash334 2012

[90] J Xuan X-D Jia L-P Jiang E S Abdel-Halim and J-J ZhuldquoGold nanoparticle-assembled capsules and their application ashydrogen peroxide biosensor based on hemoglobinrdquoBioelectro-chemistry vol 84 pp 32ndash37 2012

[91] X-C Tan J-L Zhang S-W Tan et al ldquoAmperometric hydro-gen peroxide biosensor based on immobilization of hemoglobinon a glassy carbon electrode modified with Fe3O4chitosancore-shell microspheresrdquo Sensors vol 9 no 8 pp 6185ndash61992009

[92] Y-C Gao K Xi W-N Wang X-D Jia and J-J Zhu ldquoA novelbiosensor based on a gold nanoflowershemoglobincarbonnanotubes modified electroderdquo Analytical Methods vol 3 no10 pp 2387ndash2391 2011

[93] W-L Zhu Y Wang J Xuan and J-R Zhang ldquoFabrication of anovel hydrogen peroxide biosensor based onCAu compositerdquoJournal of Nanoscience and Nanotechnology vol 11 no 1 pp138ndash142 2011

[94] K-J Huang D-J Niu X Liu et al ldquoDirect electrochemistry ofcatalase at amine-functionalized graphenegold nanoparticlescomposite film for hydrogen peroxide sensorrdquo ElectrochimicaActa vol 56 no 7 pp 2947ndash2953 2011

[95] A J Bard and L R Faulkner Electrochemical Methods Funda-mentals and Applications John Wiley amp Sons New York NYUSA 2001

[96] I Lavagnini R Antiochia and F Magno ldquoAn extendedmethodfor the practical evaluation of the standard rate constant fromcyclic voltammetric datardquo Electroanalysis vol 16 no 6 pp 505ndash506 2004

[97] R S Nicholson ldquoTheory and application of cyclic voltammetryfor measurement of electrode reaction kineticsrdquo AnalyticalChemistry vol 37 no 11 pp 1351ndash1355 1965

[98] R J Klingler and J K Kochi ldquoElectron-transfer kinetics fromcyclic voltammetry Quantitative description of electrochemicalreversibilityrdquo Journal of Physical Chemistry vol 85 no 12 pp1731ndash1741 1981

[99] J Wang ldquoCarbon-nanotube based electrochemical biosensorsa reviewrdquo Electroanalysis vol 17 no 1 pp 7ndash14 2005

[100] J N Coleman U Khan W J Blau and Y K Gunrsquoko ldquoSmallbut strong a review of the mechanical properties of carbonnanotube-polymer compositesrdquoCarbon vol 44 no 9 pp 1624ndash1652 2006

[101] J Wang ldquoNanomaterial-based electrochemical biosensorsrdquoAnalyst vol 130 no 4 pp 421ndash426 2005

[102] P Yanez-Sedeno J M Pingarron J Riu and F X RiusldquoElectrochemical sensing based on carbon nanotubesrdquo TrACmdashTrends in Analytical Chemistry vol 29 no 9 pp 939ndash953 2010

[103] W Yang K R Ratinac S R Ringer P Thordarson J JGooding and F Braet ldquoCarbon nanomaterials in biosensorsshould you use nanotubes or graphenerdquoAngewandte ChemiemdashInternational Edition vol 49 no 12 pp 2114ndash2138 2010

[104] M F L De Volder S H Tawfick R H Baughman and AJ Hart ldquoCarbon nanotubes present and future commercialapplicationsrdquo Science vol 339 no 6119 pp 535ndash539 2013

[105] G Sanzo C Tortolini R Antiochia G Favero and FMazzei ldquoDevelopment of carbon-based nano-composite mate-rials for direct electron transfer based biosensorsrdquo Journal ofNanoscience and Nanotechnology vol 15 no 5 pp 3423ndash34282015


[106] Z Zhang S Chouchane R S Magliozzo and J F RuslingldquoDirect voltammetry and catalysis with Mycobacterium tuber-culosis catalase-peroxidase peroxidases and catalase in lipidfilmsrdquo Analytical Chemistry vol 74 no 1 pp 163ndash170 2002

[107] I Yamazaki T Araiso Y Hayashi H Yamada and R MakinoldquoAnalysis of acid-base properties of peroxidase andmyoglobinrdquoAdvances in Biophysics vol 11 pp 249ndash281 1978

[108] S Hashemnia H Ghourchian A A Moosavi-Movahedi andH Faridnouri ldquoDirect electrochemistry of chemically modifiedcatalase immobilized on an oxidatively activated glassy carbonelectroderdquo Journal of Applied Electrochemistry vol 39 no 1 pp7ndash14 2009

[109] E Laviron ldquoGeneral expression of the linear potential sweepvoltammogram in the case of diffusionless electrochemicalsystemsrdquo Journal of Electroanalytical Chemistry vol 101 no 1pp 19ndash28 1979

[110] H Lu Z Li and N Hu ldquoDirect voltammetry and electro-catalytic properties of catalase incorporated in polyacrylamidehydrogel filmsrdquo Biophysical Chemistry vol 104 no 3 pp 623ndash632 2003

[111] X Chen R Ferrigno J Yang and G M Whitesides ldquoRedoxproperties of cytochrome c adsorbed on self-assembled mono-layers a probe for protein conformation and orientationrdquoLangmuir vol 18 no 18 pp 7009ndash7015 2002

[112] I Vostiar E E Ferapontova and L Gorton ldquoElectrical rsquowiringrsquoof viable Gluconobacter oxydans cells with a flexible osmium-redox polyelectrolyterdquoElectrochemistry Communications vol 6no 7 pp 621ndash626 2004

[113] L Gorton A Lindgren T Larsson F D Munteanu T Ruzgasand I Gazaryan ldquoDirect electron transfer between heme-containing enzymes and electrodes as basis for third generationbiosensorsrdquo Analytica Chimica Acta vol 400 no 1ndash3 pp 91ndash108 1999

[114] W Wang T-J Zhang D-W Zhang et al ldquoAmperometrichydrogen peroxide biosensor based on the immobilizationof heme proteins on gold nanoparticles-bacteria cellulosenanofibers nanocompositerdquo Talanta vol 84 no 1 pp 71ndash772011

[115] Code of Federal Regulations Indirect Food Additivies Adju-vants Production Aids and Sanitizers 21 CFR 1781005 Officeof the Federal Register US Government Printing Office Wash-ington DC USA 2000

Submit your manuscripts athttpwwwhindawicom

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CatalystsJournal of


it is important to study its capacity to perform direct electrontransfer (DET) to the electrode surface It is usually difficultto observe the DET because the heme groups are burieddeeply inside in the large structure of the protein [61 62]Also denaturation of the redox protein could occur onthe sensor surface due to the immobilization method andto the matrix composition To overcome these problemsand promote the DET carbon nanotubes (CNTs) modifiedelectrodes are widely employed as support for the physicalimmobilization of biological molecules to promote the DETthanks to their high surfacevolume ratio and conductivityand also to enhance sensors and biosensors performances[63ndash67] A drawback on the use of CNTs to modify electrodesurface is their insolubility [68 69] However some authorshave obtained good results in the CNTs modification of theelectrode surface by using polymers as dispersing support[70 71] Nafion is a perfluorinated polymer resistant tochemical attack and the CNTs dispersion in its film has beeninvestigated [72ndash74]

In the present study we report the development of abiosensor for H2O2monitoring based on the immobilizationof catalase in a Nafion film containing dispersed function-alized MWCNTs-COOH The Nafion film ensures efficientimmobilization of the protein in its native configurationThe DET of catalase was investigated either on modifiedor on bare electrode to identify the optimal conditions forH2O2 detection In view of the possible practical applicationthe same modification steps were performed on screen-printed electrodes (SPEs) with a working electrode basedon mesoporous graphite (MG-SPE) Finally the obtainedbiosensor was applied for the determination of hydrogenperoxide in beverages samples

2 Experimental

21 Materials and Reagents Catalase from bovine liver (CATEC 11116 activity ge 10000Umgminus1 protein) was sup-plied by Sigma-Aldrich (Switzerland) and stored at minus20∘CAll chemicals used were of analytical grade In particu-lar Na2HPO4 NaH2PO4 HOC(COOH)(CH2COOH)2 KCl(K3[Fe(CN)6]) Nafion 117 solution (NAF purum sim5 solu-tion in a mixture of lower aliphatic alcohols and water)CH3CH2OH (sim96 vv) and H2O2 (30wt in H2O) werepurchased from Sigma-Aldrich (Switzerland) High puritydeionized water (resistance 182MΩ times cm at 25∘C TOClt10 120583g Lminus1) obtained from aMillipore Direct-Q 3 UV system(France) was used throughout the experiments The workingsolutions were prepared by diluting the stock solution with01mol Lminus1 phosphate buffer solution and 01mol Lminus1 KCl pH70 (PBS buffer solution) and then deoxygenated by bubblingN2for about 20min Multiwalled carbon nanotubes modifiedwith carboxylic groups (MWCNTs-COOH) were obtainedfrom DropSens (Spain)

22 ElectrochemicalMeasurements All electrochemicalmea-surements were performed with 120583-Autolab type III poten-tiostat (EcoChemie Netherlands) controlled using the GPES

Manager program (EcoChemie Netherlands) at room tem-perature in N2 atmosphere Batch electrochemical experi-ments were performed in a 5mL thermostated glass cell(model 61415150 Metrohm Switzerland) containing PBSbuffer solution with a conventional three-electrode systemDifferent working electrodes were used in particular glassycarbon electrode (GCE cat 61204300 Metrohm Switzer-land 120601 = 3mm) and a mesoporous graphite screen-printedelectrode (MG-SPE model DRP-110MC 120601 = 4mm Drop-Sens Spain) A saturated calomel electrode (SCE cat303SCG12 Amel Instruments Italy) as the reference elec-trode and a carbon rod (cat 61248040 Metrohm Switzer-land) as the counter one were employed For SPEs thecounter electrodewas carbon and the reference onewas silverrespectively All the reported potentials are referred to assaturated calomel electrode (119864 = 0241V versus NHE) AllpH measures were performed using a digital pH meter (827pH lab Metrohm Italy) The morphology of the sampleswas observed using high-resolution field emission scanningelectron microscopy (HR FESEM Zeiss Auriga Microscopy)equipped withMicroanalysis EDS le 123 Mn-K120572 eV (Bruker)

23 Procedures The GCE surface was polished with 03 and005 120583m alumina slurry on polishing silk cloth (SIEM Italy)and rinsed with deionized water Then the electrode wassonicated in deionizedwater to remove trace of alumina fromthe surface (Sonicator AU-32 ArgoLab Italy)

The physical immobilization of the enzyme was realizedby dropping onto the working electrode surface 2 120583L of 05wt Nafion solution containing 1mgmLminus1 of redox proteineither in the presence or in the absence of 1mgmLminus1 ofMWCNTs-COOHThe electrode surfacewas finally air-driedfor about 20min at room temperature The biosensors werestored in PBS buffer solution at 4∘C before use

The analysis protocol of real beverages is described asfollows 25mL of different beverages sample was diluted to10mL with PBS buffer solution Then a certain amount ofH2O2 (15 120583mol Lminus1) was added and the solutions were deoxy-genated Then the samples were analyzed directly by cyclicvoltammetry (CV) method and finally the recoveries wereevaluated For the study of pH dependance the McIlvainebuffer was used at different pH values

3 Results and Discussion

31 Electrochemical Characterization of Glassy Carbon Elec-trode after Steps of Modification The effect on the improve-ment of electrochemical performances by using nanomateri-als as MWCNTs-COOH was evaluated with cyclic voltam-metry measurements of the electroactive area (119860119890) and ofthe heterogeneous standard rate constant (1198960) of the differentelectrodes The cyclic voltammograms (not shown) wererecorded in a solution of 1 1mmol Lminus1 potassium ferricyanidein PBS buffer solution119860119890 was determined from the Randles-Sevcik equation 119868119901 = 2686 times 10

51198993211986011989011986312119862V12 [95]

where 119868119901 is current in amps (A) 119899 is number of electronstransferred of K3[Fe(CN)6] by cyclic voltammetry (CV) inthe redox event (usually 1)119860119890 is electroactive area (cm

2)119863 is


minus8

minus6

minus4

minus2

0

2

4

6

I(120583

A)


E (V versus SCE)

(a)

minus80

minus60

minus40

minus20

0

20

40

60

I(120583

A)


E (V versus SCE)

(b)











12

13

14

15

16

17

pH 35 pH 55

pH 6 pH 7

pH 75 pH 8

I(120583

A)

(a)

8 93 64 5 7pH

y = minus00442x + 03703

R2 = 0993

Epa

(V)

000

005

010

015

020

025

(b)





Γ =4119868119901119877119879

11989921198652119860V (1)










1400 mV sminus1

10 mV sminus1

minus200

minus150

minus100

minus50

0

50

100

150

I(120583

A)


E (V versus SCE)

(a)

R2 = 09924

R2 = 09914

200 400 600 800 1000 1200 1400 16000 (mV sminus1)

I p(120583

A)

minus40

minus20

0

20

40

60

(b)

R2 = 0992

R2 = 0992

y = 01199x minus 00181E


minus03

minus025

minus02

minus015

minus01

minus005

0

Ep(V

ver

sus S

CE)


pc

pa

(c)







in solution

(2)






(A)

(B)(C)(D)(E)


minus8e minus 6

minus6e minus 6

minus4e minus 6

minus2e minus 6

0

2e minus 6

4e minus 6

I(A

)

E (V versus SCE)

(a)

100 200 300 400 500 600008

10

12

14

16

18

20

H2O2 (120583M)

I cId

(120583A

)

(b)


(a)

Pa 1

Pa R1

Pa 1 = 1414 nmPb 1 = 2659∘

(b)

































4 Conclusion


Competing Interests


References

































4minus





































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


minus8

minus6

minus4

minus2

0

2

4

6

I(120583

A)


E (V versus SCE)

(a)

minus80

minus60

minus40

minus20

0

20

40

60

I(120583

A)


E (V versus SCE)

(b)











12

13

14

15

16

17

pH 35 pH 55

pH 6 pH 7

pH 75 pH 8

I(120583

A)

(a)

8 93 64 5 7pH

y = minus00442x + 03703

R2 = 0993

Epa

(V)

000

005

010

015

020

025

(b)





Γ =4119868119901119877119879

11989921198652119860V (1)










1400 mV sminus1

10 mV sminus1

minus200

minus150

minus100

minus50

0

50

100

150

I(120583

A)


E (V versus SCE)

(a)

R2 = 09924

R2 = 09914

200 400 600 800 1000 1200 1400 16000 (mV sminus1)

I p(120583

A)

minus40

minus20

0

20

40

60

(b)

R2 = 0992

R2 = 0992

y = 01199x minus 00181E


minus03

minus025

minus02

minus015

minus01

minus005

0

Ep(V

ver

sus S

CE)


pc

pa

(c)







in solution

(2)






(A)

(B)(C)(D)(E)


minus8e minus 6

minus6e minus 6

minus4e minus 6

minus2e minus 6

0

2e minus 6

4e minus 6

I(A

)

E (V versus SCE)

(a)

100 200 300 400 500 600008

10

12

14

16

18

20

H2O2 (120583M)

I cId

(120583A

)

(b)


(a)

Pa 1

Pa R1

Pa 1 = 1414 nmPb 1 = 2659∘

(b)

































4 Conclusion


Competing Interests


References

































4minus





































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


12

13

14

15

16

17

pH 35 pH 55

pH 6 pH 7

pH 75 pH 8

I(120583

A)

(a)

8 93 64 5 7pH

y = minus00442x + 03703

R2 = 0993

Epa

(V)

000

005

010

015

020

025

(b)





Γ =4119868119901119877119879

11989921198652119860V (1)










1400 mV sminus1

10 mV sminus1

minus200

minus150

minus100

minus50

0

50

100

150

I(120583

A)


E (V versus SCE)

(a)

R2 = 09924

R2 = 09914

200 400 600 800 1000 1200 1400 16000 (mV sminus1)

I p(120583

A)

minus40

minus20

0

20

40

60

(b)

R2 = 0992

R2 = 0992

y = 01199x minus 00181E


minus03

minus025

minus02

minus015

minus01

minus005

0

Ep(V

ver

sus S

CE)


pc

pa

(c)







in solution

(2)






(A)

(B)(C)(D)(E)


minus8e minus 6

minus6e minus 6

minus4e minus 6

minus2e minus 6

0

2e minus 6

4e minus 6

I(A

)

E (V versus SCE)

(a)

100 200 300 400 500 600008

10

12

14

16

18

20

H2O2 (120583M)

I cId

(120583A

)

(b)


(a)

Pa 1

Pa R1

Pa 1 = 1414 nmPb 1 = 2659∘

(b)

































4 Conclusion


Competing Interests


References

































4minus





































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


1400 mV sminus1

10 mV sminus1

minus200

minus150

minus100

minus50

0

50

100

150

I(120583

A)


E (V versus SCE)

(a)

R2 = 09924

R2 = 09914

200 400 600 800 1000 1200 1400 16000 (mV sminus1)

I p(120583

A)

minus40

minus20

0

20

40

60

(b)

R2 = 0992

R2 = 0992

y = 01199x minus 00181E


minus03

minus025

minus02

minus015

minus01

minus005

0

Ep(V

ver

sus S

CE)


pc

pa

(c)







in solution

(2)






(A)

(B)(C)(D)(E)


minus8e minus 6

minus6e minus 6

minus4e minus 6

minus2e minus 6

0

2e minus 6

4e minus 6

I(A

)

E (V versus SCE)

(a)

100 200 300 400 500 600008

10

12

14

16

18

20

H2O2 (120583M)

I cId

(120583A

)

(b)


(a)

Pa 1

Pa R1

Pa 1 = 1414 nmPb 1 = 2659∘

(b)

































4 Conclusion


Competing Interests


References

































4minus





































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(A)

(B)(C)(D)(E)


minus8e minus 6

minus6e minus 6

minus4e minus 6

minus2e minus 6

0

2e minus 6

4e minus 6

I(A

)

E (V versus SCE)

(a)

100 200 300 400 500 600008

10

12

14

16

18

20

H2O2 (120583M)

I cId

(120583A

)

(b)


(a)

Pa 1

Pa R1

Pa 1 = 1414 nmPb 1 = 2659∘

(b)

































4 Conclusion


Competing Interests


References

































4minus





































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




















4 Conclusion


Competing Interests


References

































4minus





































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





4 Conclusion


Competing Interests


References

































4minus





































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















4minus





































































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




















































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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