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Sensors and Actuators B 176 (2013) 315– 322

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h o mepage: www.elsev ier .com/ locate /snb

Electrochemical determination of hydrogen peroxide production by isolatedmitochondria: A novel nanocomposite carbon–maghemite nanoparticle electrode

Massimiliano Magroa, Davide Baratellaa, Nicola Piancaa, Antonio Toninellob, Silvia Grancarab,Radek Zboril c, Fabio Vianelloa,c,∗

a Department of Comparative Biomedicine and Food Science, University of Padua, Italyb Department of Biomedical Sciences, University of Padua, Italyc Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Palacky University in Olomouc, Czech Republic

a r t i c l e i n f o

Article history:Received 3 July 2012Received in revised form12 September 2012Accepted 13 September 2012Available online 23 September 2012

Keywords:Maghemite nanoparticlesNanomaterial electrocatalysisHydrogen peroxide detectionCetyltrimethylammonium bromideMitochondriaMonoamine oxidase

a b s t r a c t

A simple carbon paste electrode modified with novel maghemite (�-Fe2O3) nanoparticles, characterizedby a mean diameter of about 10 nm and specific surface properties (called surface active maghemitenanoparticles, SAMNs) has been developed. The electrode is able to catalyze the electro-reduction ofhydrogen peroxide at low applied potentials (−0.1 V vs. SCE). In order to improve the electrocatalyticproperties of the modified electrode and to protect electrode surface from fouling due to the pres-ence of biological materials, a surfactant, namely cetyltrimethylammonium bromide, was introduced, asmonomolecular layer, on electrode surface. At −0.1 V, the sensitivity of the modified nanocomposite elec-trode was 58 nA �M−1 cm−2, with a detection limit of 2.78 �M, in the 0.01–1.5 mM H2O2 concentrationrange, revealing to be one of the most sensitive electrodes described in literature. The resulting electrodewas used to determine native monoamine oxidase activity on intact mitochondria suspensions, allowingthe determination of catalytic parameters of the enzyme. Following this new electrochemical method-ology, we implemented a novel electrochemical assay for the investigation of aerobic metabolism inpreparations of isolated mitochondria revealing active H2O2 production by respiring rat liver mitochon-dria. The present work demonstrates the feasibility of these novel maghemite nanoparticles modifiedelectrode as efficient hydrogen peroxide electro-catalyst, for the application on biological samples at lowapplied potentials.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Mitochondrial respiration generates reactive oxygen speciesthat are involved in physiological and pathological processes. Themajority of methods, with exception of electron paramagnetic res-onance, used to evaluate the identity, the rate and the conditions ofthe reactive oxygen species produced by mitochondria, are mainlybased on oxidation sensitive markers.

The analytical determination of hydrogen peroxide (H2O2) is ofgreat importance in many different areas, from clinical and envi-ronmental studies to industrial productions. H2O2 is widely usedas oxidizing agent in chemical and food industries and as medi-ator in food, pharmaceutical, clinical, and environmental analysis[1]. A number of methods, such as spectrophotometry [2], fluores-cence [3], and electrochemical techniques [4,5] have been used to

∗ Corresponding author at: Department of Comparative Biomedicine and FoodScience, University of Padua, Agripolis – Viale dell’ Università 16, Legnaro, 35020PD, Italy. Tel.: +39 049 8276863; fax: +39 049 8073310.

E-mail address: [email protected] (F. Vianello).

detect H2O2. Electroanalytical methods are the most convenient,owing to their operational simplicity, low cost, and suitability forreal-time detection and insensitivity to optical drawbacks, such asturbidity and quenching. In electrochemistry, H2O2 can be eitheroxidized or reduced directly at ordinary solid electrodes. However,these processes in analytical applications are limited by slow elec-trode kinetics and high overpotential, which will affect the sensingperformance and may cause large interferences from other existingelectroactive species in real samples such as ascorbate, urate, biliru-bin, etc. Thus, the current research on H2O2 detection is mainlyfocused on electrode modifications in order to decrease the over-potential and increase the electron transfer kinetics [5].

However, most sensors are based on enzyme or protein activity,and may result in limited lifetime, stability problems and com-plex fabrication procedures. Thus, the development of enzyme-freeH2O2 sensors with low detection limit and wide responding rangehas become a rich research field.

Some transition metal oxides such as manganese oxide, cobaltoxide, titanium dioxide, copper oxide and iridium oxide havebeen reported to show electrocatalytic activity to H2O2. However,for most of them, high potentials were applied on the working

0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.snb.2012.09.044


316 M. Magro et al. / Sensors and Actuators B 176 (2013) 315– 322

electrode. From the application point of view, the operation poten-tials of these sensors are too high to be applied to real biologicalsamples [5].

Nanomaterials have been used to develop enzyme-free H2O2sensors and some of them exhibit high electrocatalytic activity forH2O2 reduction at low potential [6–8].

Reports have been found on non-enzymatic sensors basedon nanocomposite iron oxides [9–11], but drawbacks, mainlybecause the reactivity of iron oxide nanoparticles increases with thedecrease of particle size, leading to their rapid degradation, limitedtheir applications [12]. Furthermore, most of the reported sensorseven if characterized by remarkable electrochemical characteris-tics and analytical performances, cannot be applied on biologicalreal samples due to the high applied potential and the oxygen freeworking environment.

Recently we developed a new simple method to synthesizesuperparamagnetic nanoparticles constituted of stoichiometricmaghemite (�-Fe2O3) in the nanometric dimension range, charac-terized by specific surface chemical behavior without any coatingor superficial modification [13,14]. These bare surface activemaghemite nanoparticles (SAMNs) are stable in water for monthsas colloidal suspensions, present a high average magnetic momentand can be easily derivatized to immobilize specific organicmolecules.

The aim of the present work is to study the electrocatalyticproperties of these nanoparticles in a simple, carbon paste based,electrode configuration toward hydrogen peroxide reduction. Fur-ther aim is the application of the developed electrode to thedetermination of hydrogen peroxide produced in particularly dif-ficult environmental conditions, such as biological samples. Inparticular the electrode was applied to mitochondria suspensions.The effect of ionic liquids and surface modifications by surfactantswas studied in order to avoid electrode fouling, thus permit-ting long term monitoring of hydrogen peroxide production bymitochondria.

2. Materials and methods

2.1. Chemicals

Chemicals were purchased at the highest commercially avail-able purity and were used without further treatment. Iron(III)chloride hexahydrate (97%), sodium borohydride (NaBH4),ammonia solution (35% in water), cetyltrimethylammoniumbromide (CTAB), sodium dodecylsulfate (SDS), 1-butyl-3-methylimidazolium hexafluorophosphate (BMIM+PF6

−),tetrabutyl-ammonium heptadeca-fluoro-octanoatesulfonate(TBA+PFOS−) and K3Fe(III)(CN)6, K4Fe(II)(CN)6 were obtained fromAldrich (Sigma–Aldrich, Italy). Typical nanoparticle synthesis wasalready described [13,14].

2.2. Instrumentation

Microscopic characterizations of the maghemite particles andmitochondrial preparations were performed by transmission elec-tron microscopy (TEM) using a FEI Tecnai 12 microscope operatingat 120 kV with a point-to-point resolution of 1.9 A.

Voltammetric experiments were carried out by a computer-controlled electrochemical system (PGSTAT 10, EcoChemie, TheNetherlands). The standard three-electrode arrangement consistedof a SCE reference electrode (Amel, Italy), a Pt counter electrode(Amel, Italy) and carbon paste electrode (CPE) as working elec-trode. Measurements were carried out at constant temperature(22.0 ± 0.2 ◦C). All experiments were repeated at least five times.

Fig. 1. TEM image of purified rat liver mitochondria.

2.3. Electrode preparation

Carbon paste (CP) was prepared by mixing 70:30 graphite pow-der to silicon grease weight-to-weight ratio. The resulting CP wasinserted into the cavity of glass electrode holders (1.35 mm diame-ter). A copper wire had been inserted into the paste through theopposite side of the glass capillary to create the electrical con-tact with the potentiostat. Finally, the surface of the carbon pasteelectrode (CPE) was carefully smoothed on a weighting paper andrinsed with double distilled water before each experiment. Allapplied potentials were referred to SCE reference.

2.4. Determination of enzyme activity

A fluorescence assay was used to measure the activity ofmitochondrial amine oxidases, using a Cary Eclipse fluores-cence spectrometer (Varian Inc., Palo Alto, CA, USA), in quartzcuvettes (1 cm p.l.). The production of H2O2 in mitochondriawas measured by the peroxidase/amplex red method accord-ing to Towne [3]. Measurements were carried out in 5 mMHEPES (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid), pH7.4, containing 250 mM sucrose.

2.5. Purification of mitochondrial fraction

Rat liver mitochondria were isolated and purified according toSalvi et al. [15] and re-suspended in a medium containing 250 mMsucrose, 5 mM HEPES, pH 7.4, at a concentration of 50 mg pro-tein ml−1. Rat liver mitochondria resulted to be pure by electronmicroscopy (see Fig. 1).

3. Results and discussion

The synthesis and the full characterization of iron oxidenanoparticles (SAMN) were described elsewhere [12,13] and


M. Magro et al. / Sensors and Actuators B 176 (2013) 315– 322 317

Fig. 2. TEM images of maghemite nanoparticles.

resulted constituted of stoichiometric maghemite (�-Fe2O3) witha mean diameter of 11 ± 2 nm (Fig. 2).

3.1. Electrode characterization

In order to characterize the electrochemical behavior of dif-ferent modified carbon paste electrodes, cyclic voltammetry inthe range −0.8 to +0.8 V, in the presence of the redox coupleFe(CN)6

3−/Fe(CN)64− as electrochemical probe, was used [16].

With the aim to determine H2O2 produced by native enzymesin mitochondria, the buffer in which mitochondria were isolatedand where they present good stability and optimal physiologicalactivity, namely 5 mM HEPES, pH 7.4, containing 150 mM KCl assupporting electrolyte, was used.

Tentatively, a SAMN/CPE containing 55% (w/w) graphite, 30%(w/w) silicone grease and 15% (w/w) iron oxide nanoparticles, wasprepared and compared with unmodified CP electrode (65% w/wgraphite and 35% w/w silicone grease). The behavior of the twoelectrodes in the presence of the Fe(CN)6

3−/Fe(CN)64− redox cou-

ple is reported in Table 1. In both cases, the voltammetric behavioris quasi-reversible and, notwithstanding the SAMN modified CPelectrode showed a higher anodic and cathodic peak current inthe presence of the redox couple, bare CPE revealed to be morereversible (i.e. lower �Ep). Conversely, using bare CPE in the pres-ence of H2O2, a small increase of the cathodic and anodic currentsat potentials more negative than −0.6 V and more positive than+0.6 V, was observed, while, with SAMN/CPE, a current increase isobserved at potential below 0 V, leading to a undefined peak at

E (V)

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8

i (µA

)

-40

-30

-20

-10

0

10

E (V)

-0.8 -0.6 -0.4 -0.2 0.0

i (µA

)

-20

-15

-10

-5

0

Fig. 3. Cyclic voltammogramms of CPE and SAMN/CPE. Measurements were car-ried out in 5 mM HEPES, pH 7.4, containing 150 mM KCl as supporting electrolyte,equilibrated with air, in the presence and in the absence of 3 mM hydrogen perox-ide. (black) CPE; (red) CPE, 3 mM H2O2; (blue) SAMN/CPE; (green) SAMN/CPE, 3 mMH2O2.Inset: Cyclic voltammogramms of SAMN/CPE at different oxygen concentration.Measurements were carried out in 5 mM HEPES, pH 7.4, containing 150 mM KCl assupporting electrolyte. (black) buffer bubbled with 99% nitrogen for 10 min; (red)buffer equilibrated with air; (blue) buffer bubbled with 99% oxygen for 10 min. (Forinterpretation of the references to color in this figure legend, the reader is referredto the web version of the article.)

−0.5 V (see Fig. 3), attributed to the reduction of dissolved oxygen.This hypothesis was confirmed by experiments carried out at dif-ferent oxygen concentration (see Fig. 3, inset). After the additionof H2O2, its reduction current appeared, overlapping, at appliedpotential more negative than −0.3 V, with the O2 reduction peak. At−0.1 V, the cathodic current of SAMN/CPE increased linearly withhydrogen peroxide concentration and no detectable interferenceby oxygen was observed.

The catalytic mechanism for the reduction of H2O2 can beassumed as following: Fe (III) was first electrochemically reducedto Fe (II) on the nanocomposite material, and then Fe (II) reactedwith H2O2, which resulted in the conversion of H2O2 to H2O and inthe regeneration of the catalyst [17].

The effect of the amount of SAMN in CPE on the H2O2 responsecurrent was tested. Therefore, the effect on H2O2 reduction currentas a function of SAMN concentration in CP, in the range 7.5 and30%, was studied. The chronoamperometric response, acquired at−0.1 V, increased with the percentage of SAMN in the CPE, and asa function of hydrogen peroxide additions, in the concentrationrange 10 �M–1 mM (R > 0.997). In spite of the increase of elec-trode sensitivity with the amount of SAMN in CPE, a concomitantincrease of the noise, registered during the chronoamperometricmeasurements, was observed. The best signal to noise ratio (S/N)was observed with electrodes prepared with 15% SAMN and we

Table 1Cyclic voltammetry of unmodified and modified carbon paste electrodes in the presence of 1 mM K3Fe(CN)6 and 1 mM K4Fe(CN)6.

Carbon paste electrode modifier Electrochemical parameters

ic (�A) ia (�A) Ep,c (V) Ep,a (V) �Ep (V) ia/ic

None −2.58 2.48 0.102 0.235 0.133 0.9SAMNs −3.09 3.44 0.091 0.253 0.162 1.1SAMNs + BMIM+PF6

− −36.11 14.96 0.123 0.267 0.144 0.4SAMNs + TBA+PFOS− −1.12 2.59 0.088 0.232 0.144 2.3SAMNs + SDS −1.49 2.15 0.074 0.288 0.214 1.4SAMNs + CTAB −7.28 8.48 0.046 0.218 0.174 1.2

Unmodified CPEs were prepared with 65% (w/w) graphite and 35% (w/w) silicone grease. Modifiers concentrations are reported in the text. Voltammograms were acquiredat 5 mV/s.



considered these modified CPE (15% SAMN/CPE) as the most fea-sible for H2O2 detection in biological systems. In the case of 15%SAMN/CPE, the detection limit was about 8.5 �M (S/N = 3), a typ-ical response time between 10 and 95% of steady-state responseis 10 s at the injection of 10 �M H2O2, and a sensitivity estimatedat 8 �A mM−1 cm−2 (R > 0.996). In contrast, the sensitivity of theunmodified CPE at −0.1 V is more than one hundred times lowerthan 15% SAMN/CPE (0.06 �A mM−1 cm−2).

The peculiar electrocatalytic properties of SAMNs wereevidenced by performing control experiments, using a CPE mod-ified with 15% of commercial amorphous iron oxide (Fe3O4)nanoparticles (Sigma–Aldrich, Italy, cat. 544844), 50 nm nomi-nal diameter. The sensitivity of CP electrodes doped with 15%amorphous iron oxide nanoparticles, toward hydrogen peroxideadditions, at −0.1 V, resulted 4 times lower than that observedwith SAMN modified CPE, demonstrating that reduction cataly-sis of H2O2 depends on the peculiar structure of our maghemitenanoparticle (SAMNs).

The SAMN/CP electrode was tested on a mitochondrial sus-pension in order to measure hydrogen peroxide production. Inparticular, the activity of mitochondria associated monoaminooxidases (MAOs) was considered. These enzymes catalyze the oxi-dation of aromatic primary amines, producing the correspondingaldehyde, ammonia and hydrogen peroxide, according to the fol-lowing reaction [18]:

R − CH2 − NH3+ + O2 + H2O

MAO−→R − CHO + NH4+ + H2O2

Tyramine was used as substrate of monoamine oxidases. Theaddition of 100 �M tyramine gave a measurable production ofhydrogen peroxide and a corresponding electrode response, whichrapidly faded, suggesting rapid fouling of electrode surface, proba-bly because of the adhesive properties of the outer mitochondrialmembranes.

3.2. Effect of ionic liquids on nanocomposite electrodeperformances

Ionic liquids are well known additives used to decrease elec-trode overpotentials and increase electrode currents [19]. Inorder to improve electrode characteristics, and to introduceelectrical charges on electrode surface, aimed to the reduc-tion of the interactions between mitochondria and electrodesurface, two ionic liquids, namely 1-butyl-3-methylimidazoliumhexafluorophosphate (BMIM+PF6−) and tetrabutylammoniumheptadecafluorooctane (perfluoro-octane)sulfonate (TBA+PFOS−),were added at the SAMN modified CPE (BMIM+PF6−-SAMN/CPEand TBA+PFOS−-SAMN/CPE). The introduction of ionic liquids inthe CPE led to a current increase, as shown in Fig. 4A andB, where the voltammogramms of 15% SAMN/CPE, with andwithout BMIM+PF6

−and TBA+PFOS−, are reported for compari-son. The modified electrodes were tested in the presence of theFe(CN)6

3−/Fe(CN)64− redox couple (see Table 1). The addition of

BMIM+PF6− on the SAMN/CP electrode showed two poorly resolved

peaks with a �Ep of 144 mV, and an increase in the capacitive cur-rent. The faradic current was increased, as well, even if the ia/icratio was 0.4, suggesting a poorly reversible system. The SAMN/CPEmodified with 10% BMIM+PF6

− showed improved analytical per-formances toward the reduction of hydrogen peroxide. From thevoltammetric (shown in Fig. 4A) and chronoamperometric mea-surements of H2O2 reduction, we evaluated that the best signal tonoise ratio was observed at −0.1 V. With respect to the un-modifiedSAMN/CPE, the sensitivity of the BMIM+PF6

− modified electrodeis increased (10 �A mM−1 cm−2), with a detection limit of 6.5 �MH2O2 (S/N = 3). The linear regression equation was: i(�A) = −2.949

E (V)

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8

i (μ

A)

-140

-120

-100

-80

-60

-40

-20

0

20

40

60

80

A

E (V)

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8

i (μ

A)

-60

-40

-20

0

20

B

Fig. 4. Cyclic voltammogramms of SAMN/CPE containing BMIM+PF6− and

TBA+PFOS− . Measurements were carried out in 5 mM HEPES, pH 7.4, containing150 mM KCl as supporting electrolyte, equilibrated with air, in the presence and inthe absence of 3 mM hydrogen peroxide.(A) (black) SAMN/CPE; (red) SAMN/CPE, 3 mM H2O2; (blue) BMIM+PF6

−-SAMN/CPE;(green) BMIM+PF6

−-SAMN/CPE, 3 mM H2O2.(B) (black) SAMN/CPE; (red) SAMN/CPE, 3 mM H2O2; (blue) TBA+PFOS−-SAMN/CPE;(green) TBA+PFOS−-SAMN/CPE, 3 mM H2O2. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of the article.)

(�M) + 1.625, with a 0.998 correlation coefficient. However, theelectrode response achieved a steady-state signal within 170 s.

The SAMN/CP electrode, modified with 10% TBA+PFOS−, showedtwo poorly resolved peaks, with a �Ep of 144 mV, in the pres-ence of Fe(CN)6

3−/Fe(CN)64− (see Table 1). In this case the faradic

current decreased with respect to the unmodiefied SAMN/CPEand the ia/ic ratio was 2.3, suggesting a poorly reversible system,as well. Nevertheless, this electrode was tested in the presenceof hydrogen peroxide, showing a sensitivity of 3 �A mM−1 cm−2

(R > 0.997), with a detection limit of 11.7 �M H2O2 (S/N = 3). Boththese SAMN/CP electrodes, modified with the two ionic liquids(BMIM+PF6

−-SAMN/CPE and TBA+PFOS−-SAMN/CPE) were testedin order to measure hydrogen peroxide production by a mito-chondrial suspension, following tyramine addition. In both cases,when electrodes were immersed in the mitochondria containingsolution, a fast disappearance of current response was observed,suggesting a rapid fouling of electrode surfaces, and we consideredthese two systems unusable for biological applications. A furtherdemonstration was obtained by protecting the electrode surface bya cellulose acetate membrane as suggested by literature [20], thusavoiding fouling processes occurring in mitochondria suspension.



In this case the current signal due to mitochondrial hydro-gen peroxide production can be easily determined. Anyway, themembrane introduced a diffusional barrier, increasing responsetime and decreasing electrode sensitivity.

3.3. Surfactant introduction on the surface of the nanocompositeelectrode

In order to overcome electrode surface fouling, anotherapproach was tested by introducing charged molecules directlyon electrode surface, masking surface hydrophobicity and creat-ing an electrostatic barrier on the surface. This was achieved usingamphiphilic molecules (surfactants) characterized by a hydropho-bic tail, which interacts with the apolar environment of the carbonpaste and a charged head, exposed to the solution. Two substanceswere tested, namely cetyltrimethylammonium bromide (CTAB)and sodium dodecyl sulfate (SDS), presenting positively and neg-atively charged head group, respectively (CTAB-SAMN/CPE andSDS-SAMN/CPE). Surface electrode modification was accomplishedby immersing SAMN/CP electrodes overnight in millimolar watersolution of the surfactant.

Both electrodes, CTAB-SAMN/CPE and SDS-SAMN/CPE, weretested in the presence of the Fe(CN)6

3−/Fe(CN)64−, see Table 1. The

introduction of SDS on electrode surface (SDS-SAMN/CPE) led to ageneral worsening of electrochemical parameters, decreasing thefaradic current, increasing �Ep and showing an ia/ic ratio of 1.4,suggesting that the system is poorly reversible, probably becauseof the repulsive electrostatic interactions between the dodecyl-sulfate modified electrode surface and the negatively charged redoxcouple. The SDS-SAMN/CP electrode was tested on hydrogen per-oxide reduction, at −0.1 V, and its performances gave a sensitivity of9 �A mM−1 cm−2 (R > 0.998), with a detection limit of 9.2 �M H2O2(S/N = 3).

The CTAB-SAMN/CP electrode was tested, as well, on theFe(CN)6

3−/Fe(CN)64− redox couple, showing an increase of both

capacitive and faradic current with respect to the SAMN/CPE.The presence of two peaks characterized by �Ep of 0.174 Vand an ia/ic ratio of 1.2 (see Table 1) were observed. Further-more, poorly defined shoulders of anodic and cathodic peakof Fe(CN)6

3−/Fe(CN)64−were present. These last features were

ascribed to bound Fe(CN)63− and Fe(CN)6

4− at electrode sur-face due to the electrostatic interaction between Fe(CN)6

3− andFe(CN)6

4− ions and the positively charged electrode surface, asalready reported [21]. In fact, absorbed Fe(CN)6

3− and Fe(CN)64−

ions on electrode surface can be removed from the electrode surfaceby washing with solutions at high ionic strength (1 M KCl).

In order to obtain deeper information on CTAB coverage ofSAMN/CP electrodes, we tested with the Fe(CN)6

3−/Fe(CN)64−

redox couple different electrodes prepared with increasing concen-trations of the surfactant, in the range 0.5–10 mM, covering CTABconcentrations below and above its critical micelle concentration(CMC = 0.93 mM) [22]. In Fig. 5A, the capacitive current as a func-tion of CTAB concentration is reported. As shown in the figure, thecapacitive current increased with CTAB concentration reaching amaximum value at concentration around 10 mM, indicating the for-mation of a compact monolayer on the electrode surface [22], whileat lower concentration the surfactant molecule layer, covering elec-trode surface, is not complete. Furthermore, the performances ofCTAB-SAMN/CP electrodes toward hydrogen peroxide reduction,at −0.1 V, as a function of CTAB used for surface electrode mod-ification, were studied, and results on electrode sensitivities anddetection limits are reported in Fig. 5B and in the inset of Fig. 5B,respectively. Increasing CTAB concentration during the prepara-tion of CTAB-SAMN/CP electrodes led to a monotonic increase ofelectrode sensitivity and an improvement of the detection limit.

[CTAB ] (mM)

0 2 4 6 8 10

Cap

acit

ive

curr

ent

(μA

)

0

2

4

6

8

10

12

A

[CTAB] (mM)

0 2 4 6 8 10

Sens

itiv

ity

(nA

μM

-1 c

m-2

)

0

10

20

30

40

50

60

[CTAB] ( mM)0 2 4 6 8 10 12

Det

ecti

on li

mit

(μ

M)

0

2

4

6

8

10

B

Fig. 5. Electrochemical characteristics of the SAMN/CP electrode modified with dif-ferent concentrations of CTAB. Measurements were carried out in 5 mM HEPES, pH7.4, containing 150 mM KCl as supporting electrolyte, equilibrated with air.(A) Capacitive current of SAMN/CPE modified with different concentrations of CTAB.(B) Sensitivity the SAMN/CPE modified with different concentrations of CTAB towardhydrogen peroxide additions in the 0.01–1.0 mM concentration range.

E (V)

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8

i (µA

)

-80

-60

-40

-20

0

20

40

Fig. 6. Cyclic voltammogramms of SAMN/CPE incubated with 10 mM CTAB. Mea-surements were carried out in 5 mM HEPES, pH 7.4, containing 150 mM KCl assupporting electrolyte, equilibrated with air, in the presence and in the absence of3 mM hydrogen peroxide. (black) SAMN/CPE; (red) SAMN/CPE, 3 mM H2O2; (blue)CTAB-SAMN/CPE; (green) CTAB-SAMN/CPE, 3 mM H2O2. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version ofthe article.)



Table 2Comparison of analytical performances toward hydrogen peroxide reduction of carbon paste based electrodes reported in literature.

Electrode Applied Potential (V) Sensitivity (nA �M−1 cm−2) LOD (�M) Reference

�-Fe2O3-LaB6-CP −0.40 20000 0.06 [11]Am-Fe2O3@epoxy-CP −0.75a 0.11 3.5 [28]VZrO2@poliestere-CP −0.40a 159 3.3 [29]Zeolite-Fe(III)-CP −0.20b 0.57 60 [30]CuOnp-OPPF6-CP −0.20b 393 0.5 [31]silica@chitosan-HRP-HCF(II)-CP −0.10a 156 3 [32]Mb/AgNO3-APS-CP −0.70 73.6 0.08 [33]Hb/SWCNTs-CTAB-GC −0.50 130 7.9 [34]CTAB-SAMN-CP −0.10a 58 2.7 This work

a vs SCE.b vs Ag/AgCl.

In Fig. 6, the cyclic voltammetry of CTAB-SAMN/CP electrode,prepared after incubation in the presence of 10 mM CTAB, onhydrogen peroxide (3 mM) is presented. For comparison in thesame figure the cyclic voltammetry of SAMN/CPE was shown. ThisCTAB-SAMN/CPE showed the best performances, at −0.1 V, towardhydrogen peroxide reduction, among all the tested electrodes (seeTable 2).

Finally, the half-life, t1/2, of the H2O2 sensor was also studied.When this sensor was stored at room temperature after use, thehalf-life time is 3 months, based on weekly evaluation.

The above presented results indicated that the CTAB-SAMN/CPEcan be successfully used for the detection of hydrogen peroxide oncomplex biological samples.

3.4. Determination of hydrogen peroxide produced by a nativemitochondria suspension

The CTAB-SAMN/CP electrode, prepared after incubation in thepresence of 10 mM CTAB, was used for the determination of hydro-gen peroxide produced by a suspension of physiologically activemitochondria, in an air equilibrated medium. The CTAB-SAMN/CPelectrode was immersed in a suspension of intact native rat livermitochondria (0.5 mg mL−1) and the enzyme catalyzed oxidationof tyramine, in the presence of dissolved molecular oxygen, pro-duced an equimolar concentration of hydrogen peroxide, whichwas determined by chronoamperometry by the developed modi-fied CPE. Furthermore, in order to avoid the effect of adventitiouscatalase, which decomposes hydrogen peroxide, an inhibitor ofcatalase activity was added (20 �M 3-amino-triazole) [23]. Pre-viously, a calibration of CTAB-SAMN/CPE was carried out withknown amount of hydrogen peroxide and chronoamperometricdata of enzymatic production of H2O2, following tyramine addi-tion, were treated according to Michaelis and Menten [24]. InFig. 7 the electrode current response as a function of tyramineadded to a native mitochondria suspension, is presented, andin the inset a linearization of experimental data according toLineweaver–Burk, is shown. MAO kinetic parameters were as fol-lows: affinity constant, KM, of 322 �M and maximum reactionrate, Vmax, of 41 nmol H2O2 min−1 mg−1 protein, in agreement withliterature data [25,26]. In order to confirm these results, exper-iments were carried out on mitochondrial suspensions treatedwith typical MAO inhibitors, such as 10 �M deprenyl and 10 �Mchrorgyline, which led to a dramatic decrease (90%) of hydrogenperoxide production, demostrating that electrode current mea-sured MAO enzymatic acitivity. Furthermore, control experimentswere carried out by determining hydrogen peroxide production byfluorimetry according to the conventional peroxidase/amplex redmethod [3]. The comparison between the two methods showedthe same value, within the experimental error, of the affinity con-stant, KM, while the fuorimetric determination of Vmax value ofMAO was 40% lower than the electrochemical CTAB-SAMN/CPE

tyramine ( μM)

0 500 1000 1500 2000 2500

enzy

mat

ic a

ctiv

ity

(nm

ol H

O m

in2

2-1

mg-1

pro

tein

)

0

10

20

30

40

Fig. 7. Enzymatic activity of mitochondrial monoamine oxidases determined bychronoamperometry at −0.1 V with the CTAB-SAMN/CP electrode. CTAB-SAMN/CPE,prepared after incubation in the presence of 10 mM CTAB, was used for the deter-mination of hydrogen peroxide produced by a suspension of native mitochondria(0.5 mg mL−1), following the addition of tyramine. An inhibitor of catalase activitywas added (20 �M 3-amino-triazole) in order to avoid the effect of adventitiouscatalase.Inset: Linearization of enzymatic activity data according to Lineweaver–Burk.

based method. This could be due to optical interferences presentin the biological material and on the low specificity of the fluo-rescent probe based method, which involves the reaction betweenan organic fluorophore (amplex red) and highly reactive hydroxideradical (OH•), produced by horseradish peroxidase in the presenceof hydrogen peroxide [27].

CTAB-SAMN/CPE was used 6 weeks on mitochondrial sus-pensions, showing high stability and constant response to H2O2additions. Furthermore, the near zero working potential, min-imizing the effect of electroactive interferences, and the costeffectiveness make competitive the proposed electrode.

4. Conclusions

The main focus of the present work is the development of anelectrochemical sensor for the determination of an enzymatic activ-ity, producing H2O2, in the presence of O2 and not a generic H2O2sensor. The presence of O2 represents a prerogative for the inves-tigation of complex biological samples, as well as the presence offouling material and electroactive biological interferants.

Our findings support the use of an electrochemical approachto investigate H2O2 mitochondrial metabolism. Firstly, we devel-oped and characterized a carbon paste electrode, in whichnewly synthesized iron oxide nanoparticles (called surface activemaghemite nanoparticles, SAMNs) and a surfactant (namely



cetyltrymethylammonium bromide – CTAB) were introducedby a rapid and simple procedure to form a CTAB-SAMN/CPEnanocomposite. The proposed electrode shows good electrocat-alytic properties toward hydrogen peroxide reduction, at very lowapplied potential (−0.1 V vs. SCE) and it could be used for sen-sitive and selective determination of H2O2, in oxygen containingbiological samples. The CTAB-SAMN/CP electrode characteristicsare comparable to those reported in literature, with much morecomplicated and cumbersome solutions, as reported in Table 2for comparison. The attractive electrochemical and structuralproperties of SAMNs suggest a potential application regarding elec-trocatalysis and sensor development, showing good linear range,high sensitivity and low detection limit. Furthermore, the sys-tem was tested on purified, respiring mitochondrial suspensionsto determine hydrogen peroxide production by monoamine oxi-dases present in the native organelles, upon tyramine additions,and results suggest that CTAB-SAMN/CPE could be a promising,low cost, option for the hydrogen peroxide monitoring in complexbiological systems.

Acknowledgements

The authors gratefully acknowledge the support by the Opera-tional Program Research and Development for Innovations – Euro-pean Regional Development Fund (project CZ.1.05/2.1.00/03.0058)and by the Operational Program Education for Competitiveness –European Social Fund (project CZ.1.07/2.3.00/20.0155)of the Min-istry of Education, Youth and Sports of the Czech Republic. Thepresent experimental work was partially funded by Italian Insti-tutional Ministry grants cod. 60A06-7411 and 60A06-8055.

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Biographies

Massimiliano Magro received the degree in Chemistry at the University of Padovain 2003. Since 2004, he worked on the synthesis and characterization of metaloxide nanostructures, in the development of nanobiosensors and biotechnologicalapplications of magnetic nanoparticles. He is currently Senior Research Scientist atthe Department of Comparative Biomedicine and Food Science of the University ofPadua.

Davide Baratella, after the B.Sc. graduation in Health Biotechnologies and the M.Sc.degree in Pharmaceutical Biotechnologies at the University of Padova, is currentlya fellow at the Department of Comparative Biomedicine and Food Science of theUniversity of Padua.

Nicola Pianca, after the B.Sc. graduation in Biotechnology and the M.Sc. graduationin Industrial Biotechnologies at the University of Padova, is currently fellow at theDepartment of Biomedical Sciences of the University of Padua.

Antonio Toninello, Prof., graduated in Biological Sciences, University of Padova,Italy. Italian Member at European Commission for COST Actions 917 (Biogeni-cally Active Amines in Food) and 992 (Health implications of Dietary Amines).Chief of the Biogenic Amines Group of the Italian Biochemical and Molecular Biol-ogy Society (SIBBM). He is involved in mitochondria pathophysiological studies,in particular on the mitochondrial permeability transition and intrinsic apoptosisinduction and their relationships with cancer and neurodegenerative diseases. He is



also involved in the investigations regarding the interactions of naturally occurringpolyamines with mitochondria and their modulation on mitophagy, autophagy andapoptosis.

Silvia Grancara, PhD, after the B.Sc. graduation in Biology in 2006 and the M.Sc.degree in Evolutionarily Biology in 2008, received the PhD in Biochemistry andBiophysics in 2012 at the University of Padova; she is currently a fellow at theDepartment of Biomedical Science of the University of Padua.

Radek Zboril received his PhD degree at Palacky University in Olomouc. After PhDstudy, he underwent several foreign stays, e.g., at universities in Tokyo, Delawareand Johannesburg. Currently, he is a Professor at the Department of Physical Chem-istry and General Director of the Regional Centre of Advanced Technologies andMaterials at the Palacky University in Olomouc. His research interests focus on

nanomaterial research including iron and iron oxide based nanoparticles, silvernanoparticles, carbon nanostructures, and magnetic nanoparticles, their syntheses,physicochemical characterization, and applications in the field of catalysis, watertreatment, antimicrobial treatment, medicine, and biotechnologies.

Fabio Vianello, PhD, received the degree in Biological Sciences at the University ofPadova in 1988 and in 1993, the PhD in Biophysics. Since 2002, he is Associate Profes-sor of Biophysics and Biochemistry at the Department of Comparative Biomedicineand Food Sciences of the University of Padova. Currently, he is visiting professor atthe State University in Sao Paulo, Brazil and visiting scientist at the Regional Centreof Advanced Technologies and Materials at the Palacky University in Olomouc. He isinvolved in nanobiotechnology research and in the development of nanobiosensorsand in the biotechnological applications of magnetic nanoparticles.

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