Determination of nitrite in food samples by anodic voltammetry using a modified electrode

Food Chemistry 113 (2009) 1206–1211

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

Analytical Methods

Determination of nitrite in food samples by anodic voltammetry using a modified electrode Wilney J.R. Santos a, Phabyanno R. Lima a, Auro A. Tanaka b, Sônia M.C.N. Tanaka b, Lauro T. Kubota a,* a b

Department of Analytical Chemistry, Institute of Chemistry, University of Campinas – UNICAMP, 13084-971 Campinas, SP, Brazil Department of Chemistry Technology, Center Technological, University Federal of Maranhão – UFMA, 65085-040 São Luís, MA, Brazil

a r t i c l e

i n f o

Article history: Received 11 April 2008 Received in revised form 31 July 2008 Accepted 3 August 2008

Keywords: Nitrite FeT4MPyP Cu(II)TSPc Voltammetric sensor

a b s t r a c t A glassy carbon (GC) electrode modified with alternated layers of iron(III) tetra-(N-methyl-4-pyridyl)porphyrin (FeT4MPyP) and copper tetrasulfonated phthalocyanine (CuTSPc) was employed for nitrite determination by differential pulse voltammetry (DPV). This modified electrode showed excellent catalytic activity for the nitrite oxidation. After optimizing the operational conditions, a linear response range from 0.5 to 7.5 lmol l1 with a low detection limit of 0.1 lmol l1 was obtained. The proposed sensor was stable with a sensitivity of 20.0 lA, 1 lmol1 and good repeatability, evaluated in terms of relative standard deviation (R.S.D. = 1.3%) for n = 10. Possible interferences from several common ions were evaluated. This sensor was applied for the voltammetric determination of nitrite in some food samples. The results were consistent with those obtained with the standard spectrophotometric procedure. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction The nitrite ion is one of the active intermediate species in the nitrogen cycle, resulting from oxidation of ammonia or from reduction of nitrate. The occurrence of nitrite in soils, waters, foods and physiological systems are widespread (Bryan, 2006; Lijinsky & Epstein, 1970). The environmental impact caused by the build-up of high nitrite concentrations, considering their use as fertilizers, and the problems of the water resource contamination for human consumption are subjects under investigations (Aydin, Ercan, & Tascioglu, 2005). Nitrite combines with blood pigments to produce meta-hemoglobin in which oxygen is no longer available to the tissues. It may also combine in the stomach with amines and amides to produce highly carcinogenic N-nitrosamine compounds (Mirvish, 1995). However, as is well known, nitrite lurks ubiquitously in some foods. For example, due to its antimicrobial action, nitrite is still used as an additive for the preservation of meat products as commonly as it was centuries ago. Moreover, nitrite can be formed in the production of pickled vegetables because of the biodegradation of nitrate or other nitrogenous substances. Therefore, the quantitative determination of nitrite concentrations is of great importance, especially for control of the quality of food. The classical method for nitrite determination is the Griess assay (Fox, 1979), which was first developed in 1879 and still widely used. Under acidic conditions, nitrite undergoes a series of reac* Corresponding author. Tel.: +55 19 3788 3127; fax: +55 19 35213023/55 19 3788 3023. E-mail address: [email protected] (L.T. Kubota). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.08.009

tions to form an azo dye, which is the basis of the spectrophotometric determination (Fox, 1979). Though, it is highly sensitive and specific, the method is not applicable if too much nitrite is present because of its potential side reactions (Fox, 1979). In addition, the organic reagents used are poisonous, and sometimes the formation of the azo dye consumes rather large amounts of time. Much effort has been devoted to seeking alternative procedures for nitrite determination, many new methods have been developed, including spectroscopic analysis (Gallignani et al., 2004), chromatography (Gaspar, Juhasz, & Bagyi, 2005) and electrochemical methods (Wen & Kang, 2004). However most of them are still quite time-consuming. In contrast, electrochemical detection techniques are favorable for nitrite determination due to their rapid response and ease of use (Pournaghi-Azar & Dastangoo, 2004; Wen & Kang, 2004). These approaches for determination of nitrite offer several advantages, namely no interference from nitrate ion and from molecular oxygen, which are usually the major limitations in cathodic determination of nitrite. However, most electrochemical methods are based on the reduction of nitrite, suffer from poor sensitivity and are subject to several interferences (Silva, Cosnier, Almeida, & Moura, 2004; Strehlitz et al., 1996). The electrochemical reduction of nitrite gives several products depending on the electrode and the catalyst, employed while its oxidation, is a straightforward reaction, with nitrate being the final product (Rocha, Angnes, Bertotti, Araki, & Toma, 2002). Since nitrite oxidation involves a relatively higher overpotential at a bare glassy carbon electrode, the usefulness of this electrode for nitrite detection is limited. Chemically modified electrodes have been developed to decrease the

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2. Experimental 2.1. Chemical and solutions All chemicals used were analytical grade. Sodium nitrite (NaNO2) was acquired from Isofar, Rio de Janeiro, Brazil. Monobasic potassium phosphate (KH2PO4), sodium hydroxide (NaOH), monosodium phosphate (NaH2PO4), citric acid (C6H8O7) and hydrochloric acid (HCl) were acquired from Merck, Rio de Janeiro, Brazil. Sodium borate (NaB4O7.10H2O) was acquired from Synth, São Paulo, Brazil. Zinc acetate (CH3CO2)2Zn, sulfanilamide (C6H8N2O2S) and [tris(hydroxymethyl)amino-methane] were acquired from Sigma, St. Louis, USA. N-(1-naphthyl)ethylenediamine dihydrochloride was acquired from Furlab, São Paulo, Brazil. Copper (II) tetrasulfophthalocyanine (CuTSPc) was synthesized and purified according to the procedure of Weber and Busch (1965). Iron(III) tetra-(Nmethyl-4-pyridyl)-porphyrin (FeT4MPyP) was acquired from Porphyrin Products Inc. (Utah – USA). The solutions of FeT4MPyP and CuTSPc and the other solutions were prepared with water purified in a Millipore Milli-Q system and the actual pH of the buffer solutions were determined with a Corning pH/Ion Analyser model 350.

lands) coupled to a PC microcomputer with GPES 4.9 software. An electrochemical cell containing 5.0 ml of buffer solution with a saturated calomel electrode (SCE) as reference, a Pt wire as auxiliary and the modified GC electrode with 5 mm diameter as working electrode were used for all measurements. 2.4. Assay of the nitrite content in food samples Samples of sausage and pickled vegetables were purchased at local stores. The pretreatment was as follows: first, 5 g of the food sample was crushed into mash and mixed with 12.5 ml saturated borax solution. Then, 300 ml of 70 °C water were added and the mixture was heated at boiling for 15 min. To precipitate the proteins, 5 ml of 20% zinc acetate was introduced. After being cooled to room temperature, the mixture was diluted to 500 ml with water and then filtered. The resulting sample solution was stored at 4 °C in a refrigerator. The nitrite content in samples was determined according to the standard addition method. Standard nitrite solutions were added as internal standards after measurement of the sample solution. Thus, the concentration of nitrite in the real sample could be calculated. The technique employed was differential pulse voltammetry. Reference determinations were performed by the Griess assay. The spectrophotometric measurements were performed with a HP 8425 spectrophotometer. 3. Results and discussion 3.1. Electrocatalytic oxidation of nitrite on the modified electrode Fig. 1 shows the cyclic voltammograms for the GCE modified with FeT4MPyP/CuTSPc in phosphate buffer in absence (a) and presence of 0.5 mmol l1 NO 2 (e). For comparison, this figure also presents the behavior of bare GC electrode (b) and modified electrodes with layers of FeT4MPyP (c) and CuTSPc (d) both in presence of nitrite. In the voltammograms of the Fig. 1a, there is no evidence of peaks in this potential range and conditions although the electrode surface is modified. However, it is still unclear why such a peak is not observed for directly adsorbed layer-by-layer

60

2.2. Preparation of FeT4MPyP/CuTSPc glassy carbon electrode

2.3. Voltammetric measurements The voltammetric measurements were carred out with a Autolab PGSTAT-30 potentiostat from Echo Chemie (Utrecht, Nether-

40

I / μA

A glassy carbon (GC) electrode acquired from Metrohm-Switzerland, with geometrical area of 0.2 cm2 (5 mm diameter) was used for sensor preparation. Prior to modification, the electrode surface was treated according to the procedure described by Rocha et al. (2002). After cleaning the electrode, electrostatically formed layers were prepared on the electrode surface by drop-casting 30 ll of a 0.1 mmol l1 FeT4MPyP solution onto the surface. These was dried at 80 °C. After 8 min, 30 ll of a 0.1 mmol l1CuTSPc solution was added to the electrode surface and also allowed to dry at 80 °C for 8 min. FeT4MPyP and CuTSPc solutions were prepared using deionized water. Further layers, up to a total of four, were prepared by repeating the procedure described above. Presumably the interactions between the FeT4MPyP and CuTSPc complexes occur by ionpair formation between the (–C6H8–N+) group of the iron porphyrin and the anionic SO 3 of the tetrasulfonated phthalocyanine.

50

30

20 (e)

(e)

10

I /μA

overpotential for nitrite oxidation. Furthermore, modification of electrode surfaces can provide a way to extend the dynamic range in analytical determinations. Thus, electrode modifications with alternating deposition of functional compounds has attracted much interest due to its potential application (Yang, Li, Jiang, Chen, & Lin, 2005). It is a very simple way to experimentally produce complex layered structures with precise control of layer composition and thickness. Additionally, porphyrins and metallophthalocyanines have demonstrated good features as analytical sensors because they can electrocatalyze the electron-transfer reactions, increasing the sensitivity and selectivity of the electrode (Ozoemena & Nyokong, 2005; Santos et al., 2006). Presumably a strong interaction is established by ion-pair formation between the C6H4N+–CH3 group of the porphyrin and the sulfonic (SO 3 ) group of the phthalocyanine. Hence, the development of an efficient and stable sensor for nitrite determination based on a glassy carbon electrode modified by drop-casting with alternating layers of iron(III) tetra-(N-methyl-4-pyridyl)-porphyrin (positively charged) and copper tetrasulfonated phthalocyanine (negatively charged) is reported.

(a)

0

(d) 0.6

0.8 E / V vs SCE

1.0

(c) (b)

20 10

(a)

0 0.2

0.4

0.6

0.8

1.0

1.2

E / V vs SCE Fig. 1. Cyclic voltammograms for FeT4MPyP/CuTSPc modified electrode in the absence (a) and presence (e) of 0.5 mmol l1 NO 2 ; for bare GC electrode (b) and modified electrodes with layers of FeT4MPyP (c) and CuTSPc (d) both in presence of 1 0.5 mmol l1 NO phosphate buffer. Scan 2 . Measurements carried out in 0.1 mol l rate: 0.05 V s1. Inset: large-scale cyclic voltammograms for FeT4MPyP/CuTSPc modified electrode in absence (a) and presence (e) of 0.5 mmol l1 NO 2.

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electrostatic assembled films. The complete investigation of the redox mechanism is out of the scope of the present paper and will be reported elsewhere. As can be seen, the best response was obtained with the FeT4MPyP/ CuTSPc modified electrode. This bilayer modified electrode presented higher peak current and lower oxidation potential for  NO 2 than that modified with only CuTSPc or FeT4MpyP. The NO2 molecule on the CuTSPc modified electrode presented an oxidation potential close to the oxidation potential on the FeT4MPyP/CuTSPc modified electrode. When only FeT4MPyP was used no catalytic current was observed (Fig. 1c). This suggests that the CuTSPc complex effectively catalyzes NO 2 oxidation, while the FeT4MPyP layers must be improving the assembling of the active sites of CuTSPc, resulting in better catalysis for the NO 2 oxidation. The interaction of the FeT4MPyP and CuTSPc complexes, promoting an increase in the peak current of the analyte with a decrease of 200 mV in the overpotential, was verified when compared to the bare electrode. The catalysis can probably be associated to the oxidation of immobilized Cu(II)-(TSPc)4 into Cu(II)-(TSPc)3 species (Lever, Milaeva, & Speier, 1993; Limson & Nyokong, 1998; Sekota & Nyokong, 1997) corresponding to the modified electrode in the absence of NO 2 (data not shown). The oxidation in [Cu(II)TSPc]4 is known to occur at the phthalocyanine ring and not at the central metal (Lever et al., 1993). The observation of oxidation for nitrite on CuTSPc-GCE suggests involvement of a ring-based oxidation process in the catalytic reaction. Mediation by ring-based oxidation processes has also been suggested for the catalytic oxidation of cysteine on GCE modified with Rh, Ru and Os phthalocyanine complexes, as already showed by Sekota and Nyokong (1997). Therefore, the high activity of the modified GC electrode for NO 2 oxidation in aqueous solutions can also be associated with the low charge transfer resistance of FeT4MPyP/ CuTSPc, where CuTSPc acts as the electroacatalyst sites. 3.2. Influences of pH, buffer solution and buffer concentration Previous investigations have shown that the electrochemical response of NO 2 with FeT4MPyP/CuTSPc modified electrode depends on the solution pH. Thus, the influence of the solution pH was investigated using 0.1 mol l1 phosphate buffer at pH 4.0, 5.0, 6.0, 6.5, 7.0, 7.5 and 8.0. The peak current increased with pH in the range from 4.0 to 7.0. At pH 7.0 the peak current reaches a maximum. A decrease in the current is observed when the solution pH is higher than 7.0. Thus, the optimum pH for further studies was set in 7.0. In addition, this study showed that the peak potential for nitrite oxidation is not affected by the solution pH (data not shown). This feature has been also verified by Pournaghi-Azar and Dastangoo (2004) and Zen, Kumar, and Chen (2001), and it can be attributed to a kinetically controlled oxidation process, i.e. a proton independent catalytic step. The influence of the buffer solution on the sensor response was tested in four different buffer solutions (Sörensen, MacIlvaine, Tris and phosphate), all with concentrations of 0.1 mol l1 and indicated that phosphate buffer solutions give the best response. Thus, phosphate buffer solutions were chosen for further studies. The effect of different concentrations of phosphate (0.025, 0.05, 0.10, 0.20 and 0.25 mol l1) were investigated. Phosphate buffer concentrations from 0.025 to 0.25 mol l1 presented almost constant current while the best response was obtained with 0.1 mol l1 phosphate buffer solution. The concentration of 0.1 mol l1 was then chosen for the further experiments. 3.3. Studies on the number of bilayers The effect of the number of bilayers on the voltammetric response was also evaluated. Voltamograms were recorded using

FeT4MPyP/CuTSPc modified electrodes containing 0, 1, 2, 3 and 4 bilayers. Fig. 2 clearly shows that a linear increase in the catalytic current of the sensor between 0 and 2 bilayers, which reaches the highest value for the electrodes with two bilayers. Electrodes containing more than two bilayers showed no further signal enhancement, but a tendency for a gradual decline in sensitivity. Also, the catalytic current decreased rapidly as the number of bilayers was increased, probably because of increased resistivity associated with counter ion diffusion, as reported by Rocha et al. (2002). This behavior may be due to a lower stability of the sensor when compared to those constructed using two bilayers, which was chosen as the best, considering the time consumed for sensor construction as well as sensor stability. 3.4. Optimization of the DPV parameters Since the DPV method offers an improved sensitivity in electrochemical signals and detection limits, the response of nitrite was also investigated employing DPV. The peak current obtained in DPV is dependent on various instrumental parameters such as potential scan rate (v) and pulse amplitude (a). These parameters are interrelated, having a combined influence on the peak current response. Hence, in order to establish the optimum conditions in the determination of nitrite, the influence of the instrumental parameters on the peak current response in phosphate buffer solution was investigated. The peak current/peak half width ratio (I/w1/2, lA/V) values presented a linear increase with the scan rate variation from 0.005 up to 0.02 Vs1 (Fig. 3a). On the other hand, when the scan rate is >0.02 Vs1, the current peak value remained almost constant, accompanied by the broadening and distortion of the peaks. As it sets the best voltammetric profile with higher sensitivity, a scan rate of 0.02 Vs1 was chosen and subsequently used throughout the present study. The peak current values were also found to vary with pulse amplitudes of 0.03–0.11 V (Fig. 3b) applied with DPV at a scan rate of 0.02 Vs1 for the modified electrode. The use of the pulse amplitude >0.090 V led to almost constant current peak values and an increase in the capacitive current. Thus, the best voltammetric sensitivity was obtained with 0.090 V and this value was chosen for further study. The peak current increases rapidly, increasing the pulse amplitude and the step potential (scan rate) when small values are used. However, the response quickly

40

35

Ip / mA

1208

30

25

20

15

0

1 2 3 Number of bilayers

4

Fig. 2. Studies of the number of bilayers of FeT4MPyP/CuTSPc immobilized on the electrode surface in the presence of 5.0 lmol l1 NO 2 . Measurements carried out in 0.1 mol l1 phosphate buffer solution at pH 7.0.

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30

a

30

(a) 40

25

25

20

I / μA

I w1/2 / μA V

32

Ip / μA

20

36

15

15 10 5

(b)

0 0.0

10

1.5

3.0

4.5

6.0

7.5

-1

-

[NO2] / μmol L

28

5 24

0

pulse amplitude = 0.01 V 0.00

0.01

0.02

0.03

v/V s

b

0.04

0.4

0.05

0.6

0.8

1.0

1.2

1.4

E / V vs SCE

-1

Fig. 4. (a) Differential pulse voltammograms recorded in a 0.1 mol l1 phosphate buffer at pH 7.0 before and after addition of nitrite to give final solution concentrations in the range 0.5–7.5 lmol l1. Scan rate: 0.02 Vs1 and amplitude potential: 0.090 V. (b) The inset shows the calibration plot.

150

I w1/2 / μA V-1

120

0.1 mol l1 phosphate buffer solution at pH 7.0 (Fig. 4a). The proposed sensor showed a good linear response range from 0.5 to 7.5 lmol l1 (Fig. 4b), which can be expressed according to the following equation:

90

60

1

DI=lA ¼ 0:4 ð0:2Þ þ 20 ð3:0Þ ½NO2 =lmol l 30

v = 0.02 Vs 0 0.00

0.02

0.04

0.06

0.08

0.10

0.12

-1

0.14

Pulse amplitude/ V Fig. 3. (a) Plot of the potential scan rate (m) verses current, with fixed amplitude (0.01 V). (b) Plot of the pulse amplitude verses current, with a fixed scan rate (0.02 Vs1). FeT4MPyP/CuTSPc modified electrode, under optimized conditions.

levels off when higher values are used (Moretto, Chevalet, Mazzocchin, & Ugo, 2001; Moretto, Ugo, Lacasse, Champagne, & Chevalet, 1999; Rifkin & Evans, 1976). This behavior has been verified by other researchers and has been attributed to the exponential relation between the current density, the amplitude and the step potential (Rifkin & Evans, 1976). 3.5. Analytical characterization Under optimized conditions, in order to obtain an analytical curve for the sensor, differential pulse voltammograms for oxidation of NO 2 were carried out at different concentrations in

ð1Þ

with a correlation coefficient of 0.998 (for n = 16) and sensitivity of 20.0 lA l lmol1 (or 100.0 lA cm2 l lmol1). Such good sensitivity can be attributed to the efficiency of the electron-transfer between the modified electrode and nitrite due to the catalytic effect and low charge transfer resistance of the film. A limit of detection (LOD) of 0.14 lmol l1 was determined using a 3r/slope ratio and limit of quantification (LOQ) was 0.48 lmol l1 using 10r/slope, where r is the standard deviation (S.D.) of the mean value for 10 voltammograms of the blank, determined according to the IUPAC recommendations (Analytical methods commitee, 1987). Table 1 lists the comparison for the determination of nitrite by different sensors. 3.6. Stability of FeT4MPyP/CuTSPc modified electrode The stability of the FeT4MPyP/CuTSPc film modified electrode was checked by recording successive cyclic voltammograms. After 200 cycles, no change was observed in the voltammetric profiles of the modified electrode. Even in the presence of nitrite, the modified electrode remained stable after 200 successive cycles. Furthermore, when the modified electrode was stored at room temperature no significant change in the response was observed for more than two months.

Table 1 Comparison of different nitrite sensors for DPV determination of nitrite in phosphate buffer solution Electrode

Ep (V)

MC/GCEa p-NiTAPc/GCE PPS-Pd/GCEb Thionine/ACNTs/GCEc FeT4MPyP/CuTSPc/GCE

0.810 0.860 0.770 0.800 0.710

a b c

verses verses verses verses verses

SCE SCE SCE Ag/AgCl SCE

Analytical range (lmol l1)

LODs (lmol l1)

pH

References

0.5–100 2.5–10000 1.0–1100 3.0–500 0.5–7.5

0.10 0.90 0.30 1.10 0.14

3.5 2.0 7.0 3.5 7.0

Karyakin et al., 1999 Lubert, Guttmann, & Beyer, 1999 Zhu & Lin, 2007 Zhao et al., 2007 This work

Chitosan-carboxylated multiwall carbon nanotube modified glass carbon electrode. palladium–polyphenosafranine nano-composite modified glass carbon electrode. Thionine modified aligned carbon nanotube electrode.

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Table 2 Determination of nitrite in food samples Samples Sample Sample Sample Sample a b

1a 2a 3b 4b

Detected by Griess assay (mg kg1)

Detected by this method (mg kg1)

14.08 ± (0.03) 11.49 ± (0.05) 7.93 ± (0.02) 5.79 ± (0.06)

13.87 ± (0.01) 11.82 ± (0.04) 8.39 ± (0.01) 6.12 ± (0.02)

Sausage. Pickled vegetable.

good repeatability for the measurements and electrode preparation, evaluated in term of relative standard deviations, allied with a simple and easy preparation. This work demonstrates that a glassy carbon electrode modified with FeT4MPyP/CuTSPc is a sensitive, robust and stable sensor showing great potential for NO 2 determination. In this sense, the sensor was applied to nitrite detection in food samples and the results were consistent with those obtained with the standard spectrophotometric procedure.

Acknowledgements The relative standard deviation of the peak current for ten determinations in solutions containing 10 lmol l1 NO 2 was 1.3%, showing a good precision. Additionally, a series of 10 sensors prepared in the same manner were also tested and the relative standard deviation observed was only 3.7%. These experiments indicate that the GC electrodes modified with FeT4MPyP/CuTSPc films have good stability and repeatability of the multilayered film, probably due to the strong ionic interaction between the FeT4MPyP and CuTSPc on the electrode surface in the preparation of the layers. This behavior can be attributed presumably the interaction the ion-pair between the (–C6H8–N+) group of the porphyrin and the anionic SO 3 of the tetrasulfonated phthalocyanine, which should be very strong as recently verified (Lever et al., 1993), justifying the high stability of the proposed system. 3.7. Interferences Possible interferences for the detection of nitrite on the FeT4MPyP/ CuTSPc film modified electrode were investigated by adding various ions into a 0.1 mol l1 phosphate buffer solution (pH 7.0) containing 10 lmol l1 nitrite. The results showed that most of 2 2  the ions, such as Ca2+, Mg2+, Al3+, NO 3 , SO3 , SO4 and HSO4 , even in 100-fold excess concentrations did not interfere in the determination. Thus, this study reveals that the sensor developed can tolerate a high concentration of interfering ions and, therefore, can be stated as selective in the presence of the more common interfering ions. 3.8. Determination of nitrite in food samples In order to evaluate the practical utility of the method, nitrite was determined in several samples, using the standard addition method. These results are compared to those obtained using the recommended spectrophotometric method. Table 2 show the results and, in all cases, the RSD for each sample for was less than 5%. The recoveries for the method were investigated and the values changed from 98.4 to 101.2%. These experimental data indicate that the determination of nitrite using the FeT4MPyP/CuTSPc film modified electrode was effective and sensitive. Lastly, to estimate the feasibility, precision, and efficiency of this sensor, the Griess assay was adopted for the determination of nitrite in the same samples. In addition, applying a paired Student-t test to compare these results, it was possible to observe that, at the 95% confidence level, there was no significant statistical difference. This good agreement indicates the reliability of the present electroanalytical sensor for nitrite determination in real samples. 4. Conclusions The glassy carbon electrode modified layer-by-layer with FeT4MPyP/CuTSPc is a feasible alternative for the analytical determination of nitrite. Optimization of the experimental conditions yielded a detection limit and sensitivity for NO 2 much better than those described in the literature. This sensor showed

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