Food Chemistry 179 (2015) 325330

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Analytical Methods

Nitrite detection in meat products samples by square-wave voltammetryat a new single walled carbon naonotubes myoglobin modifiedelectrode

http://dx.doi.org/10.1016/j.foodchem.2015.01.1060308-8146/ 2015 Elsevier Ltd. All rights reserved.

Corresponding author.E-mail address: gturdean@chem.ubbcluj.ro (G.L. Turdean).

Graziella L. Turdean a,, Gabriella Szabo ba Chemical Engineering Department, Babes-Bolyai University, 11, Arany Janos St., 400028 Cluj-Napoca, Romaniab Hungarian Chemical and Chemical Engineering Department, Babes-Bolyai University, 11, Arany Janos St., 400028 Cluj-Napoca, Romania

a r t i c l e i n f o

Article history:Received 8 November 2013Received in revised form 20 January 2015Accepted 22 January 2015Available online 29 January 2015

Keywords:MyoglobinSingle-walled carbon nanotubesSquare-wave voltammetryNitriteMeat products sample

a b s t r a c t

A new modified electrode was realized in a simple way, consisting by the immobilization of a myoglobin(My) single walled carbon nanotubes (SWCNT) mixture on the surface of a graphite electrode with aNafion film. The cyclic voltammetry investigations realized with the obtained electrode (G/My-SWCNT/Nafion) showed a voltammetric signal due to a one-step redox reaction of the surface-confinedmyoglobin, in a deaerated 0.1 M phosphate buffer, pH 7. Also, the G/My-SWCNT/Nafion modifiedelectrode demonstrated a great potential for the analytical determination of nitrite ions by square-wavevoltammetry and an alternative for the already existing methods. The use of the sensor for the detectionof nitrite ions in samples of meat products leads to comparable results with those obtained with thestandard Griess spectrophotometric assay (ISO 2918/1975), proving the suitability of using immobilizedmyoglobin as electrocatalyst in the nitrite reduction process.

2015 Elsevier Ltd. All rights reserved.

1. Introduction

Nitrite is a compound that exists in living systems and also, it isone of the most active intermediate species in the nitrogen cycle,where suffers a surprising metamorphosis, from a vilifiedsubstance that generates carcinogenic N-nitrosamines with aminesand amides present in the stomach, to a life-saving drug that liber-ates a protective agent (nitric oxide or NO) during hypoxic events(Bryan, 2006). When our daily excessive ingestion of nitrite ionsfrom food and water is accumulating in the gastrointestinal tract,the nitrite is absorbed into the bloodstream and change the bloodpigment hemoglobin (the oxygen-carrying part of blood) into themet-hemoglobin, which is not an oxygen carrier, thus limitingthe ability of the blood to carry oxygen throughout the body tis-sues (Yue & Song, 2006). As a high reactive compound, nitritecan function as an oxidizing, a reducing or a nitrosylating agent,and can be converted to a variety of related compounds in meat,including nitrous acid, nitric oxide and nitrate.

However, due to their bacteriostatic and bacteriocidal actions(Santos, Lima, Tanaka, Tanaka, & Kubota, 2009; Sebranek &Bacus, 2007), nitrite has been recognized for centuries and is stillused today as color fixer (Yue & Song, 2006) and protection agent

against Botulism in preservation of meat products (E249, E250,E251 and E252) because of its (i) strongly inhibitory effect on theanaerobic bacteria (Clostridium botulinum) and (ii) capacity tocontrol the level of other micro-organisms (such as Listeria mono-cytogenes) (Sebranek & Bacus, 2007).

The World Health Organization has reported that the fatal doseof nitrite ingestion is between 8.7 lM and 28.3 lM (Zhang, Zhao,Yuan, Wang, & He, 2013). In the same time, the Legislation of theEuropean Union (EU Directive 95/2, 1995) suggests a maximumsupplement which is allowed in the first stage of the food process-ing, and it imposes a maximum residue of 50 mg/kg nitrites and250 mg/kg nitrates for those meat products (expressed as NaNO2),which has not been treated thermally.

Therefore, accurate, economic, and rapid quantitativedeterminations of nitrite concentrations are of great importancein monitoring the pollution due to the excessive use of fertilizers,detergents, industrial processes and, especially for control of thequality of food technologies. Nitrite can be quantified directly byUV absorbance (Pourreza, Fathi, & Hatami, 2012), GCMS (Akyz& Ata, 2009), HPLC (Akyz & Ata, 2009), ion-selective electrodesand capillary electrophoresis (Troka et al., 2013) measurements.In these widely used practices, nitrite concentration is usuallymeasured by a number of additional well-known methods, suchas: colorimetric Griess assay (Tsikas, 2007), fluorescent assay(Casey & Hilderman, 2000), chemiluminescence assay (Yue &

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326 G.L. Turdean, G. Szabo / Food Chemistry 179 (2015) 325330

Song, 2006) and electrochemical detection (Afkhami, Madrakian,Ghaedi, & Khanmohammadi, 2012).

The electrochemical detection technique provides a rapid,highly selective and sensitive alternative method for nitrite deter-mination, due to its short time response and ease of use. Thus,direct electrochemical reduction can be favorably applied ondifferent cathodic materials, because the studied redox reactionproceeds at potentials substantially more negative than theirthermodynamic values and with low current density, caused byvery high activation energy. The above mentioned technique offersseveral advantages, for example avoids interference from nitrateion and molecular oxygen, which is usually the major limitationin nitrite determination. The major drawback of the nitrite reduc-tion is the tendency to poison the conventional electrode surfaceby a variety of species formed during the redox process. In thiscontext, it is well known that, an effective way to solve this prob-lem and to enhance the electrocatalytic activity of an electrode isto appropriately modify its conventional surface, and thus, to shiftthe overpotential of the redox reaction. Consequently, some newelectrocatalytic systems for nitrite determination were developedusing electrodes modified with both inorganic and organic com-pounds as: copper-thallium composite film (Casella & Gatta,2004), CuNi alloy (Mattarozzi et al., 2013), polyoxometalates (i.e.,a2-K7P2VW17O6218H2O) (Zhang, Ma, Chen, Pang, & Yu, 2013), fer-ricyanidepoly(diallyldimethylammonium)-alginate compositefilm (Qin et al., 2013), graphene oxide (Mani, Periasamy, & Chen,2012), graphene oxide-multiwalled carbon nanotubesPt nanopar-ticles/myoglobin (Mani, Dinesh, Chen, & Saraswathi, 2014),graphene oxide/Pd nanoparticles (Zhang, Zhao et al., 2013), graph-ene-Au nanoparticles (Jiang, Fan, & Du, 2014), graphene/phtalocy-anine (Cui, Pu, Liu, & He, 2013), chitosan (CS) Prussian Blue (PB)and graphene nanosheets carbon nanospheres mixture (Cui et al.,2012), zeolites (Guzmn-Vargas, Oliver-Tolentino, Lima, &Flores-Moreno, 2013), polydiphenylamine Pt nanoparticles(Unnikrishnan, Ru, Chen, & Mani, 2013), nanocomposite3,6-bis(2-[2-sulfanyl-ethylimino-methyl]-4-(4-nitro-phenylazo)-phenol)pyridazine coated SiO2Fe3O4 (L-SCMNPs) in carbon pasteelectrode (Afkhami et al., 2012), ionic-liquid carbon paste electrode(Ojani, Raoof, & Zamani, 2013), electronic tongue (Nuez, Cet,Pividori, Zanoni, & del Valle, 2013), tetraruthenated metallopor-phyrins (Calfumn et al., 2011), tetrapyridylporphyrins coordi-nated to four [Ru(5-NO2-phen)2Cl]+ moieties (Dreyse et al., 2011),organoruthenium(II) complexes onto polyethyleneimine-wrappedcarbon nanotubes/in situ formed gold nanoparticles (Azadbakht,Abbasi, Derikvand, & Amraei, 2015), hemoglobin (Saadati, Salimi,Hallaj, & Rostami, 2014), hemin (Turdean, Popescu, Curulli, &Palleschi, 2006) and/or myoglobin (Canbay, Sahin, Kran, &Akyilmaz, 2015). Despite the huge number of modified electrodesrealized in recent years, a simple solution is always more suitable.

The aim of the paper is to discuss the electrochemical parame-ters of a new SWCNTs-myoglobin graphite modified electrodeinvestigated by cyclic voltammetry (different scan rate, pH, num-ber of cycles) and its application as electrocatalyst in the electrore-duction of nitrite ions. Using the square-wave voltammetrytechnique, the nitrite concentrations from real samples of meatproducts were calculated and compared with the spectrophoto-metrical Griess standard method, proving the suitability of usingmyoglobin as electrocatalyst in the nitrite reduction process.

2. Materials

2.1. Chemicals

Single-walled carbon nanotubes (SWCNT) (1.21.5 nm diame-ter) were purchased from SigmaAldrich. A dispersion of SWCNTs

in surfactant solution was used in the present work, because thethermodynamic tendency toward bundling is overcome.

The myoglobin (My) from horse skeleton muscle was fromFluka, the Nafion (5% solution in methanol with equivalent weightof about 1100), sodium dodecyl sulfate (SDS), K3[Fe(CN)6],K4[Fe(CN)6]3H2O, Na2B4O710H2O, Zn(CH3COO)22H2O, glacialacetic acid, KH2PO4, K2HPO4, H3PO4, KOH were acquired from SigmaAldrich and the sodium nitrite, H2NC6H4SO3H, C10H7NH2HClfrom Merck, Germany.

A 0.1 M phosphate buffer solution (pH 7) was prepared fromKH2PO4 and K2HPO4, its pH being adjusted with H3PO4 or KOH.The buffer solutions were prepared using distilled-deionized waterand were kept refrigerated to minimize bacterial growth. All chem-icals were of analytical grade and were used without furtherpurification.

2.2. Apparatus

Cyclic and square-wave voltammetric investigations werecarried out using a computer controlled Autolab-PGSTAT 10 vol-tammetric analyzer (EcoChemie, Netherlands). All electrochemicalmeasurements were performed using a standard single-compart-ment three electrode cell equipped with a platinum counter elec-trode, an Ag|AgCl, KClsat reference electrode and a spectralgraphite (G) (3 mm diameter, Ringsdorff-Werke, Gmbh, Bonn-BadGodesberg, Germany) working electrode. Prior to experiments thebuffer solutions were purged with high-purity nitrogen (Linde,Romania) for at least 15 min. and a nitrogen environment was thenkept over the solution in the cell during measurements. All exper-iments were done at room temperature.

The spectrophotometric measurements were performed with aCintral spectrophotometer (Nordantec, GmbH, Germany) connectedto a computer for data acquisition. Standard (1 cm 1 cm 4.5 cm) glass spectroscopic cuvette was used for measure absor-bance in visible region of the spectrum.

2.3. Preparation of G/My-SWCNT/Nafion electrodes

Prior the modification, the graphite electrode was conditionedby a wet cleaning/polishing procedure, using diamond paper Carbi-met paper PSA (600 grit) (Buehler, Evanston, IL, USA) for cleaningand 1.0, 0.3, 0.05 lm a-Al2O3 paste (Buehler, Evanston, IL, USA) forpolishing, and followed by a ultrasonication in distilled water toremove any residual alumina.

A quantity of 0.010 g SWCNTs was dispersed in 50 mL aqueoussolution containing 0.5 g SDS. After homogenization and ultrason-ication, the obtained SWCNTs suspension was ultracentrifugated at120,000g for 4 h. For all further experiments only the upper layer ofthe stable SWCNTs suspension was used. In a second step, a vol-ume of 1 mL SWCNTs suspension was mixed thoroughly with4.5 mg My. Then, 5 lL of the resulting mixture was spread ontothe surface of the graphite electrode and allowed to dry at ambienttemperature. This operation was repeated successively three times.Finally, 2 lL of 5% Nafion was dropped on the modified electrodesurface. The electrodes were stored at 4 C, in dry state.

2.4. Standard spectrophotometric assay for nitrite detection in meatsamples

The most common approach to the detection of both nitrite andnitrate is the Griess-Ilosvay method, firstly developed in 1879(Pasquali, Hernando, & Alegria, 2007), which is the object of stan-dardization method (ISO 2918/1975). Pork meat samples preparedby minced meat (i.e., fresh or smoked sausages, dry sausages, sal-ami, ham sausages, liver sausages and wurstel) were purchasedfrom local stores. The analytical samples were prepared as follows.

G.L. Turdean, G. Szabo / Food Chemistry 179 (2015) 325330 327

Weights of meat samples of about 10 g having a high content ofprotein and high turbidity require undergoing a deproteinizationstep. This step consist in transferring 10 g crumbed meat samplein 100 mL hot water and the addition of a volume of 5 mL of satu-rated borax solution (50 g/L Na2B4O710H2O), followed finally bythe boiling at 70 C, for 15 min. After cooling, in order to precipitatethe proteins, the mixture was strongly stirred with Carrez Reagentcontaining 2 mL of 106 g/L [K4Fe(CN)63H2O] and 2 mL of 220 g/LZn(CH3COO)22H2O dissolved in 30 mL/L glacial acetic acid. Thecool solution was filtered and than diluted to 200 mL with distillat-ed water. The resulting sample solution was stored at 4 C in arefrigerator.

The nitrite detection is based on the measurement of the azodye absorbance formed in the reaction between the nitrite from10 mL meat sample with a chromogenic reagent containing 5 mLof 6 g/L sulfanilic acid (H2NC6H4SO3H) in 200 mL/L glacial aceticacid and 5 mL of 0.3 g/L 1-naphthylamine hydrochloride (C10H7NH2HCl) in 200 mL/L glacial acetic acid. For the calibration curve0.1 M nitrite standard solution was used. The absorptionmaximum for this colored azo product is at 526 nm.

3. Results and discussions

3.1. Direct electrochemistry on G/My-SWCNT/Nafion modifiedelectrodes

Fig. 1 shows the cyclic voltammograms recorded in phosphatebuffer (pH 7) at G, G/SWCNT/Nafion and G/My-SWCNT/Nafionelectrodes. As was expected, in the absence of myoglobin, no redoxpeaks were observed, in comparison with the very stable and well-defined pair of redox peaks which clearly were appeared, when theMy was present on the electrode surface. This electrochemicalreaction corresponded to the conversion between Fe(III)My andFe(II)My.

The Fe(III)My/Fe(II)My redox couple coordinated in theporphyrinic ring exhibited a quasi-reversible monoelectronictransfer, proved by the slight increase of peak-to-peak separation

-0.8 -0.6 -0.4 -0.2 0.0 0.2

-40

-20

0

Ic

Ia

I /

A

E / V vs. Ag/AgCl, KClsat

G/ My-SWCNT/Nafion G/SWCNT/Nafion G

Fig. 1. Cyclic voltammetry at G/My-SWCNT/Nafion (thick solid line), G/SWCNT/Nafion (dot line) and G (thin solid line) electrodes. Experimental conditions:supporting electrolyte, 0.1 M phosphate buffer (pH 7); starting potential, 0.8 V vs.Ag/AgCl, KClsat; scan rate, 0.1 V s1; N2 saturated solution.

(DEp = ep,a ep,c) (varying between 0.070 V and 0.090 V vs. Ag/AgCl,KClsat) when the potential scan rate increase. Similar values werereported in literature: 0.063 V on My/ZrO2/MWCNT, at 100 mV/s(Liang, Deng, Cui, Chen, & Qiu, 2010), or 0.054 V on Chit-MWNTs/My/AgNPs/GCE, at 100 mV/s (Li, Li, & Yang, 2011). The largerpeak-to-peak separation is probably due either to the immobiliza-tion of the biomolecules in different orientations, or the result of ahigh resistance related to the electron transfer across Nafionmembrane (Jin, Shao, & Dong, 2003).

Also, the values of DEFWHM (=0.150 0.225 V) were higher thanthe theoretical value (90.5/n mV) corresponding to a surface con-fined redox behavior, indicating either a certain non-uniformityof the adsorbed redox couples, or the existence of repulsiveinteractions between the active redox centers (Honeychurch &Rechnitz, 1998).

The formal potential (E0; defined as the average value of theanodic and cathodic peak potentials) value of 0.27 V vs. Ag/AgCl,KClsat is in concordance to those presented in the literature varyingfrom 0.344 V vs. Ag/AgCl at My/ZrO2/MWCNT/GCE (Liang et al.,2010), or 0.327 V vs. Ag/AgCl at My/MWCNT/ciprofloxacin/GCE(Kumar, Wang, Chang, Lu, & Yeh, 2011) to 0.012 V vs. Ag/AgClat Chit-MWNTs/My/AgNPs/GCE (Li et al., 2011). The differencebetween the reported values probably reflects the specificinfluence of the immobilization matrix on the redox behavior ofmyoglobin. It is possible that the SWCNT/Nafion matrix has aneffect on the kinetics of the electrode reaction with the redox pro-tein, providing a favorable microenvironment for the electronexchange between the adsorbed My protein and the supportinggraphite electrode (Kumar et al., 2011).

The shapes of the cathodic and anodic peak pairs were nearlysymmetric; the reduction and oxidation peaks of My having quasithe same intensities.

Over a relatively wide range of scan potential (25500 mV s1)the anodic and cathodic peak currents, recorded at G/My-SWCNT/Nafion electrode, increase linearly with the potential scan rate (v),not with v1/2, indicating a surface confined redox process (Fig. 2)(Honeychurch & Rechnitz, 1998). This conclusion is also supportedby the slope of the log I vs. logv dependence, which was found to bevery close to 1 (results not shown).

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50

100

0.0 0.1 0.2 0.3 0.4 0.5

-50

0

50

Ic

Ia v = 0.05 V*s-1

v = 0.10 V*s-1

v = 0.25 V*s-1

v = 0.50 V*s-1

I /

A

E / V vs. Ag/AgCl, KClsat

v / (V*s-1)

I /

A

Fig. 2. Cyclic voltammograms of G/My-SWCNT/Nafion modified electrode atdifferent scan rates (see inset legend). Inset: Dependence of the current peakintensity on the potential scan rate at the same electrode. Experimental conditions:supporting electrolyte, 0.1 M phosphate buffer (pH 7); starting potential, 0.8 V vs.Ag/AgCl, KClsat; N2 saturated solution.

328 G.L. Turdean, G. Szabo / Food Chemistry 179 (2015) 325330

The results indicated that the electrochemical behavior ofimmobilized My was a surface-controlled thin-layer process, inwhich the electroactive ferric proteins Fe(III)My in the film werereduced to ferrous proteins Fe(II)My on the forward cathodic scan,and then fully reoxidized to Fe(III) on the reversed anodic scan (Liet al., 2011).

In order to estimate quantitatively the kinetic barrier of theinterface, cyclic voltammograms are recorded at various scan ratein a 5 mM K3[Fe(CN)6] in 1 M KCl solution of redox probe at G,G/SWCNT/Nafion and G/My-SWCNT/Nafion modified electrodes,respectively. As it can be seen in Fig. 3, the voltammetric wavecorresponding to the [Fe(CN)6]3/[Fe(CN)6]4 couple does notoverlap with that for Fe(III)My/Fe(II)My in myoglobin, and thecorresponding anodic and cathodic peak currents characteristicfor the reversible process obey the following equation:

Ip 2:69 105n2=3 AD1=2v1=2Co 1

where n is the number of electrons, A is the active surface area(cm2), D is the diffusion coefficient (cm2/s), v is the scan rate andCo is the concentration in bulk solution (mol/cm3).

Assuming that for the SWCNT and My-SWCNT matrix the K3[Fe(CN)6] diffusion coefficient is identical with that reported insolution (i.e., D = 5.9105 cm2/s (Ye et al., 2004)) and that the activesurface (0.07068 cm2) remains the same for all modified investi-gated electrodes, the calculated diffusion coefficient decreases asexpecting in following order G (1.13105 cm2/s) > G/SWCNT/Naf-ion (0.71105 cm2/s) > G/My-SWCNT/Nafion (0.31105 cm2/s).This result proved an additional confirmation of the previous exper-imental observations, pointing out that the My-SWCNT matrix ismore compact than only the SWCNT one.

It is well-known, that heme proteins often undergo a pH depen-dent conformational change, because the pH value changes of thebulk buffer solution can influence the microstructure of proteins,causing changes in the electrochemical behaviors of redox proteinsby modulating the accessibility of water to the heme cavity ofmyoglobin or by protonation of the heme iron-bound proximal his-

-0.5 0.0 0.5 1.0-150

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0

50

100

150

I /

A

E / V vs. Ag/AgCl, KClsat

G G/SWCNT/Nafion G/SWCNT-My/Nafion

Fig. 3. Cyclic voltammograms at G (solid thin line), G/SWCNT/Nafion (dash line)and G/My-SWCNT/Nafion (solid thick line). Experimental conditions: supportingelectrolyte, 5 mM K3[Fe(CN)6] in 1 M KCl (pH 7), starting potential, 0.3 V vs. Ag/AgCl, KClsat; scan rate, 0.05 V s1, N2 saturated solution.

tidine and/or the distal histidine in the heme cavity (Ruan et al.,2012; Turdean et al., 2006).

The peak potentials of the redox protein immobilized on thestudied G/My-SWCNT/Nafion electrode was strongly influencedby the pH value of external solution. Within the studied pH rangeof 4.36.5, the value of E0 depends linearly on the pH solution witha slope of about 0.031 V pH1 (equation: E0/V = 0.08 to 0.031pH, R = 0.9894, n = 5), which is in concordance with that obtainedfor similar electrodes (e.g., 0.029 V pH1 at Nafion/SLGnPTPAMy (Yue, Lu, & Zhou, 2011), 0.048 V pH1 at Mb/ZrO2/MWCNT(Liang et al., 2010) or 0.048 V pH1 at My/MWCNT/Ciprofoxa-cin/GCE (Kumar et al., 2011). The deviation of the slope value fromthe theoretical value (0.059 V pH1) for a reversible single-protoncoupled one-electron transfer could be explained by the influenceof the protonation of the water molecule coordinated to the hemeiron, or by the protonation states of trans ligands to the heme ironand amino acids forming the heme cavity (Yue et al., 2011).

These results, together with the previously discussed DEFWHMvalues, suggest that a single proton accompanies the electrontransfer process, which is represented in general terms by the fol-lowing overall equation (Guo, Sun, & Zhao, 2012; Kumar et al.,2011; Liang et al., 2010):

FeIIIMyH e $ FeIIMy 2

The short-term stability of the G/My-SWCNT/Nafion modifiedelectrodes was evaluated under potentiodynamic conditions in aphosphate buffer solution (pH 7), by continuously cycling the elec-trode potential between 0.8 V and 0.2 V (at 0.1 V s1) for 10 min.

Using Faradays law, for a surface-controlled process, the aver-age surface coverage C is calculated, from cyclic voltammograms,as the ratio of area obtained by integrating the peak from IEcurves and the geometric area of electrode, according to Eq. (3)(Dong, Li, Huang, Tang, & Zheng, 2012).

C QF S v mol=cm

2 3

where Q is the charge involved in the reaction express by integra-tion the area under the redox peak (A*V); F is the Faradays constant(C/mol); S is the geometric area of electrode (cm2), v is the scan rate(V s1).

Supposing that the time evolution of the surface coverage for theinvestigated electrodes obeys a first order kinetic, the linear plotCcat vs. time allowed the estimation of the rate constants(kd,cathodic = 7.151012 1.81012 s1, R = 0.9130, n = 5), character-izing the deactivation/desorption of My from the G/My-SWCNT/Nafion electrode (results not shown) (Ciszewski & Milczarek, 2000).

3.2. Electrocatalytic activity

To evaluate the electrocatalytic ability of the G/My-SWCNT/Nafion modified electrode to reduce nitrite, cyclic voltammetry(CV) and square-wave voltammetry (SWV) experiments were per-formed in 0.1 M phosphate buffer containing various concentra-tions of nitrite. For both used investigation methods, the currentpeak intensity increases with the further addition of NaNO2.

As it is shown in Fig. 4, a new reduction peak placed at about0.9 V vs. Ag/AgCl, KClsat (CV) and 0.7 V vs. Ag/AgCl, KClsat(SWV) was observed when NaNO2 was added. The result is inagreement with that reported for MyGONafion GC electrode(0.73 V, Guo et al., 2012).

The G/My-SWCNT/Nafion electrode showed a linear relationshipbetween the reduction peak current of NO2 and the concentrationof NO2 (i.e., I/A = 5.7107 + 0.46103 [NO2]/M, R = 0.9750, n = 4and I/A = 3.4107 + 1.10103 [NO2]/M, R = 0.9905, n = 5 obtainedby CV and SWV technique, respectively). Also, the correspondinglinear range was 110 mM (for CV measurements) and 0.55 mM

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5

10

IIcIc

Ia

I /

A

E / V vs. Ag/AgCl, KClsat

[NO2

-] = 0 M

[NO2

-] = 1 mM

[NO2

-] = 5 mM

[NO2

-] = 10 mM [NO

2

-] / mM

I /

A

Fig. 4. Cyclic- and square-wave voltammograms of the G/My-SWCNT/Nafion atdifferent concentrations of nitrite (see inset legend) and the correspondingcalibration curves (inset, cyclic (j) and square-wave (N) voltammetry). Experi-mental conditions: supporting electrolyte, 0.1 M phosphate buffer (pH 7); startingpotential, 0.8 V vs. Ag/AgCl, KClsat (CV) and +0.3 V vs. Ag/AgCl, KClsat (SWV); scanrate 0.1 V s1; frequency, 8 Hz; amplitude, 0.05 V; N2 saturated solution.

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-50

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C2

I /

A

E / V vs. Ag/AgCl, KClsat

phosphate buffer meat sample

[NO2

-] = 0.1 M

Fig. 5. Standard addition method for nitrite detection in meat sample using squarewave voltammetry at G/My-SWCNT/Nafion electrode. Experimental conditions:supporting electrolyte, 0.1 M phosphate buffer (pH 7); starting potential, +0.3 V vs.Ag/AgCl, KClsat (SWV); frequency, 8 Hz; amplitude, 0.05 V; N2 saturated solution.

Table 1Concentrations of nitrite detected in meat samples. Experimental conditions seeFig. 5.

Standard additionby SWV/mg NO2/kgmeat

SpectrophotometricGriess method(ISO 2918/1975)/mg NO2/kg meat

Sibiu dry salami 1.65 0.05 1.75 0.04Dacia dry salami 1.50 0.04 1.51 0.05Victoria ham salami 1.58 0.08 1.60 0.05German Salami 0.57 0.03 0.50 0.06Italian salami 0.80 0.05 0.85 0.07Cabanos sausage 0.49 0.08 0.47 0.04Cabanos sausage++ 0.51 0.06 0.50 0.05Fresh Csabai sausage+ 0.90 0.05 0.88 0.06Smoked Moldovan sausage+ 0.45 0.07 0.43 0.05Fresh liver sausage+ 0.88 0.06 0.89 0.03Smoked liver sausage+ 0.43 0.07 0.45 0.05Pork Frankfurters 0.28 0.01 0.25 0.02

Produced by Salsi Romania; Ifantis Romania; ++ Caroli Romania; + MoldovanRomania.

G.L. Turdean, G. Szabo / Food Chemistry 179 (2015) 325330 329

(for SWV measurements). The sensitivity of the G/My-SWCNT/Naf-ion electrode (slope of the linear regression) is twice higher in thecase of square-wave voltammetry (i.e., 1.10103 M/A), than inthe cyclic voltammetry technique (i.e., 0.46103 M/A), recom-mending this last method for sensible analytical detection of nitrite.The best sensitivity can be attributed to the efficiency of the elec-tron-transfer between the My modified electrode and nitrite dueto the catalytic effect and low charge transfer resistance of theimmobilized film on the graphite surface. The detection limits (esti-mated as signal-to-noise ratio of 3) were 3.8 mM NO2 and0.95 mM NO2 obtained by CV and SWV techniques, respectively.

As it is shown in the inset of Fig. 4, when the nitrite concentra-tion exceeds the superior limit of the linear range, the catalyticpeak current leveled off, showing a typical MichaelisMentenkinetic process. The apparent MichaelisMenten constant (KM,app)is estimated by hyperbolic fitting (Origin 6.1 soft) of theexperimental points, obtaining (KM,app)SWV = 6.45 mM NO2 >(KM,app)CV = 3.05 mM NO2, the smaller value indicating a highaffinity of the My protein with the detected substrate.

Compared with other similar systems, the obtained values arecompetitive for construction of an amperometrically operatedsensor (Dong et al., 2012; Guo et al., 2012; Yue et al., 2011).

3.3. Determination of nitrite in meat samples

The principle of the nitrite detection by standard method, isbased on the reaction of nitrite ion (as nitrous acid) with an aminogroup of sulfanilamide in view to form a diazonium salt, whichthan combines with N-(1-naphthyl)-ethylene-diamine dihydro-chloride to obtain a bright-colored, pinkish-red azo dye. The colorproduced is directly proportional to the amount of nitrite ionspresent in the sample, and the determination of its amount canbe made by means of spectrophotometric measurements at526 nm (Pourreza et al., 2012).

The square-wave voltammetry method at G/My-SWCNT/Nafionmodified electrode provides an alternative way to estimate the

nitrite contents in 12 meat samples. The applied standard additionmethod consists in recording the intensity currents of G/My-SWCNT/Nafion electrode generated in the sample solution and afteradditions of a standard nitrite solution (Fig. 5). As is seen in Fig. 5, thepeak current of nitrite reduction (IIc), placed at 0.7 V vs. Ag/AgCl,KClsat, appears when meat sample was added and increases withthe addition of nitrite standard concentration.

In order to compare the results obtained by electrochemicalsquare-wave voltammetry technique using the prepared G/My-SWCNT/Nafion modified electrode, the standard spectrophotomet-ric Griess method was used for nitrite determination in the samemeat samples. As is shown in Table 1, between the two methodsno significant statistical difference was obtained. These experi-mental data indicate that the determination of nitrite using theG/My-SWCNT/Nafion modified electrode was effective and sensi-tive. This good agreement recommends the nitrite determination

330 G.L. Turdean, G. Szabo / Food Chemistry 179 (2015) 325330

in real meat samples using the advantages of the present electro-analytical sensor (Santos et al., 2009).

4. Conclusions

Direct electrochemistry of the immobilized My in a compositematrix containing single-walled carbon nanotubes (G/My-SWCNT/Nafion) on the graphite electrode was carefully studiedby cyclic voltammetry and the results indicated a surface confineddirect 1e/1H+ redox process. Also, the electrode exhibited electro-catalytic reduction ability to nitrite ions with analytical parameters(detection range and lower detection limit) consistent with thosefrom literature.

The standard methods proposed by International StandardOrganization (ISO 2918/1975) used for the quantification ofnitrites in meat samples, recommend spectrophotometricanalytical methodologies, which involve a high number ofreagents, handling and consumption, which in turn cause consider-able costs. Thus, G/My-SWCNT/Nafion modified electrode is a new,sensitive, robust and stable sensor, showing alternative and greatpotential for the analytical determination of nitrite by square-wavevoltammetry. Also, the sensor was applied to nitrite detection inreal meat products samples and the results were comparable withthose obtained with the standard Griess spectrophotometric assay.

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detection in meat products samples by square-wave voltammetry at a new single walled carbon naonotubes myoglobin modified electrode1 Introduction2 Materials2.1 Chemicals2.2 Apparatus2.3 Preparation of G/My-SWCNT/Nafion electrodes2.4 Standard spectrophotometric assay for nitrite detection in meat samples3 Results and discussions3.1 Direct electrochemistry on G/My-SWCNT/Nafion modified electrodes3.2 Electrocatalytic activity3.3 Determination of nitrite in meat samples4 ConclusionsReferences