An Electrochemical Nitrite Sensor Based on a Multilayer Film of Polyoxometalate

In this work, we have developed an electrochemical sensor for nitrite detection, based on a polyoxometalate (POM) namely mono-lacunary keggin anion [SiW11O39] cited as (SiW11). Electrochemical characterization of SiW11 shows two-step reduction processes, with formal potentials of −0.5 V (I) and −0.68 V (II). Oppositely charged polyelectrolyte (poly (allylamine hydrochloride) (PAH)) and (SiW11) were assembled alternately to modify glassy carbon electrode. The electrochemical behavior of the modified electrode was studied in detail using cyclic voltammetry (CV). The results showed that SiW11/PAH/GC electrode present good electrocatalytic activity for the reduction of nitrite. The sensor showed a dynamic range from 100 μM to 3.6 mM of nitrite and no interference from other classical anions. Experimental factors that affect electron-transfer rate in these films, such as pH effect and layers number, were systematically analyzed.


Introduction
The presence of nitrite in groundwater and atmosphere is an essential precursor in the formation of nitrosamines, many of which have been proven to be powerful carcinogens [1][2][3].The increase in nitrite concentration in the blood causes a decrease in oxygen transport by the blood causing methemoglobinemia, better known as the "blue baby syndrome" reaction of nitrite with Fe (II) forming hemoglobin to methemoglobin, HbFe (III) [4].For these reasons, determination of nitrite has received considerable attention.Many methods have been developed for nitrite determination including spectrophotometry [5], chromatography [6] and electrochemical methods [7][8][9][10][11][12], which are advantageous over the other methods in terms of cost and time.
Electrochemical determination of nitrite ion requires a large over potential at almost all electrode surfaces [13][14][15].The better way to achieve nitrite detection at low overpotentials is the use of mediators confined at the electrode surface.Over the years, numbers of modification procedures have been reported for the construction of modified electrodes [17][18][19].Many studies reported the capability of hexacyanoferrate anion complex [20] for the homogeneous electrocatalytic reduction of nitrite and employed this catalytic reduction method for determination of it in real samples.They demonstrated also, that the poly-ortho-toluidine can catalyze the nitrite reduction as heterogeneous catalysis [21].
Polyoxometalates (POMs), a large family of charged, nanoscopic inorganic clusters, are attractive materials for electrode modification because of their reversible redox behavior, good chemical stability and electronic conductivity [22].These compounds are also resistant to oxidative degradation due to the fact that their fundamental elements are in their higher oxidation state [23][24][25][26].These properties make them very attractive in many fields such as electrochemistry, catalysis and for biomedicinal tasks [23,[27][28][29][30].
There is a different type of polyoxometalates [27], and in our study Keggin-type POMs are specifically used.They display a quite well-known structural motif that is composed of a tetrahedrally coordinated central hetero atom surrounded by four W 3 O 13 groups (triads) that are connected in a corner-sharing fashion via oxygen atoms [31,32].Lacunary Keggin structures [SiW 11 O39] 8− (SiW 11 ) used in this study, are derived from the parent Keggin type by the removal of specific WO 6 moieties, followed by an optional rotation of the remaining WO 6 octahedra [33,34].As a consequence, (SiW 11 ) is characterized by the presence of more terminal oxygen atoms, which give them more negative charge than other POMs structure.
Layer-by-layer (LBL) self-assembly has proved to be a promising method for the fabrication of ultrathin films.It is based on the alternate adsorption on the substrate surface of oppositely charged species from dilute solutions and film formation is attributed primarily to electrostatic interaction and Van der Waals forces.It provides thickness control at the nanometer level, which can be easily adapted for automated fabrication.It is applicable to any substrate shape and also permits co-assembly with different functional components [35].Owing to these advantages, the layer-by-layer approach has been utilized to fabricate POM-containing multilayer film consisting of synthetized or natural polyelectrolytes.Several studies used the parent Keggin silicotungstate [α-SiW 12 O 40 ] 4− as the anion and, for example, polyaniline [36], chitosan [37] or poly (diallyldimethylammonium chloride) [38] as the counter cation source.There are only few examples of the use of lacunary Keggin polyoxotungtate anions in films prepared by the layer-by-layer self-assembly method [39].It has been reported that metal-substituted Keggin silicotungstate could be used as mediator for the electroreduction of nitrite [40].In this paper, we show the capability of SiW 11 anion immobilized by electrodeposition and LBL methods, for the electrocatalytic reduction of nitrite and this property is applied for the first time for the design of a selective amperometric sensor for nitrite.

Materials
Ultrapure milliQ water with a resistance of 18.2 MΏ cm was used for all experiments.P-phenylenediamine (PPD), poly (allylamine hydrochloride) (PAH) (MW 15,000), sulfuric acid (H 2 SO 4 ), sodium nitrite (NaNO 2 ), the following product were purchased from commercial sources and used without further purification from Sigma-Aldrich.Gold nanoparticles 20 nm diameter, stabilized suspension in 0.1 M PBS were commercial products from Sigma-Aldrich.Solution of redox probe (1.0 mM) was prepared dissolving the appropriate amounts of  

Apparatus
Electrochemical measurements were carried out using a potentiostat-galvanostat VOLTALAB 40 PGZ/301, 746 VA Trace Analyzer (Metrohm) equipped with a 747 VA Stand.A three-electrode system was used, the side arms contained Ag/AgCl (sat.KCl) reference electrode and a platinum counter electrode with a surface area of approximately 1 cm 2 .The working electrode was a SiW 11 modified glassy carbon electrode (3 mm diameter, surface area: 7 mm 2 ).Prior to coating, the GCE was conditioned by a polishing/cleaning procedure.The GCE was successively polished with 1.0, 0.3, and 0.05 µm α-Al 2 O 3 paste and then rinsed with ultra-pure water to remove any residual alumina and finally sonicated for 5 min in an ultrasonic bath and dried under a stream of pure nitrogen.
SEM characterization of the films was performed using a Quanta TM 250 microscope (FEI) in degraded pressure mode; no gold coating was required.The infrared spectra were recorded as KBr pellets, in the 4000 -400 cm −1 range on a Nicolet 470 FT-IR spectrophotometer.
Dynamic light scattering (Zetasizer Nano-ZS, Malvern Instruments) was used for the determination of the zeta potential.

Preparation of the Modified Electrodes
SiW 11 modification of GC electrode was performed by using electrodeposition and layer-by-layer methods for the building of multilayers, by alternately dipping the desired substrate in PAH and SiW 11 solutions, in cyclic mode [42].Figure 1 displays a schematic representation of the different layers of the SiW 11 /LBL film.
The GC electrode was first placed in 2 mM PPD solution and the potential was repeatedly scanned.PPD was electropolymerized onto GCE surface by amine cation radical formation.The oxidation peak gradually diminishes and is almost absent after the 6th cycle, this observation indicates the formation of a coating on the electrode surface (cf. Figure 2).The polyPPD.GC electrode was then placed in 2 mM (SiW 11 ) solution, 0.1 M H 2 SO 4 and at the same time a cyclic potential sweep was conducted in the potential range 0.8 V to −0.8 V at a scan rate of 100 mV/s −1 , for 25 cycles.In this way, a SiW 11 monolayer was deposited on the surface of polyPPD/GCE [42].Then, the resulting electrode (SiW 11 /polyPPD/GC) was transferred to 2 mM PAH (pH = 3) for 15 min, for resulting in one layer of positive charge.This procedure (SiW 11 /PAH) was repeated until obtaining 5 layers of SiW 11 .

Characterization of SiW 11 in Solution
The prepared compound was characterized by infrared spectroscopy, and cyclic voltammetry in acidic aqueous solution, because Keggin type and its derivative are unstable in neutral and basic solutions.The cyclic voltammogram becomes ill-defined and peak current much smaller, such an observation has been reported by Cheng et al. [43] on Keggin-type polyoxometallates.The FT-IR spectra of the lacunary keggin silicotungstate in KBr pressed pellets are presented in (Figure 3).
The redox behavior of SiW 11 in solution (conditions: 2 mM SiW 11 in 0.1 M H 2 SO 4 solution) was studied by cyclic voltammetry and polarography.The dissolved SiW 11 shows that the CV curve exhibits two pairs of successive redox waves with cathodic peaks located respectively EpcI = −0.5 V (I) and EpcII = −0.68V (II), and peak separation potentials (∆Ep) of 65 and 44 mV respectively, in the potential range from +0.8 V to −0.8 V (Figure 4(a)).This result is consistent with that in literature [45].These redox waves correspond to the reduction of the tungsten centers within the SiW 11 (W VI → W V ).The reduction of heteropolyanions is accompanied by protonation, therefore, the pH of solution has a great effect on the electrochemical behavior of heteropolyanions.
The pH effect on the electrochemical behavior of the   [46].The above results may indicate the two overall redox process of (SiW 11 ) in acidic solution as follows: (step 1)

Electrochemical Characterization
Define abbreviations and acronyms the first used in the text, even after they have been defined in the abstract.Abbreviations such as IEEE, SI, MKS, CGS, sc, dc, and rms do not have to be defined.Do not use abbreviations in the title or heads unless they are unavoidable.Through the attachment of polyPPD containing NH 2 oup to the GCE, the modified electrode (poly) PPD/ GCE was positively charged at least up to pH 6.1 [47].The PPD-modified GCE with an amido-terminated monolayer can be used as a charge-rich precursor to assemble oppositely charged species by layer-by-layer electrostatic interaction [48].The adsorption of a layer of SiW 11 is evidenced through the behavior of the modified electrode in presence of   The electrostatic interaction of PA idenced by using gold nanoparticles initially covered with a citrate monolayer, a layer on PAH being adsorbed on their surface, their zeta potential was found to be +44 mV.After adsorption of SiW 11 , the zeta potential became equal to −1.1 mV, then showing the neutralization of the positive charge by successful adsorption of SiW 11 .
Figure 5 shows that with increasing the number o W 11 layers from one to five, the peak maxima of   mmogram of the soluble and immobilized SiW 11 in LBL films of 5-SiW 11 layers.Immobilized in LBL film, SiW 11 displays a close similar electrochemical behavior to that of soluble SiW 11 .These observations demonstrate that the electrochemical behavior of the SiW 11 anion is maintained in the multilayer films [40].Nevertheless, EpcI was shifted to 0.454 V and EpcII to 0.722 V versus Ag/AgCl.The redox peaks were broadered and ∆EpI was found to be around 150 mV whereas ∆EpII was around 70 mV.The broadening can be related to the large coulombic repulsion between the negative sites of highlycharged polyanions in the same layer, as in [37].The decrease of reversibility could be attributed to the decrease of charge transfer rate through the poorly conductive PPD layer.
The electroche C electrode was investigated in 0.1 M H 2 SO 4 solution at a scan rate of 100 mV s −1 .After 300 cycles, the cathodic peak current (peak II) still remains about at 93% of the initial value.These results indicate that the SiW 11 / PAH/polyPPD/GC electrode present a good stability.
In order to observe the SiW 11 /polyPPD film in an accu rate way, the film was electrodeposited onto gold electrode, by the method described above.Near the border, we can clearly see that the majority of the electrode surface is covered by the PPD film and there is no appearance of dendrite structure (Figure 6(a)), so we can say that the morphologies seen at the polyPPD/gold electrode surface are similar to those at the polyPPD/GC surface.The successful immobilization of SiW 11 in the electrode surface is confirmed by the energy-dispersive X-ray (ED-X) analyses (Figure 7), showing the presence of W and Nitrite might be reduced to NO instantly [5 ac .Several large crystallites and dendrites can be seen (size around 200 µm), especially in the central zone of the electrode, which showed a significantly rough surface.
In t e electrode center revealed the presence of highly populated regions (Figure 6(b)).Besides, the electrode surface appeared as a tri-dimensional structure with a high "apparent" rugosity.The image shows that the product consists of nanometer-sized platelet structures (80 -300 nm thick) and that some of the nanostructures agglomerate together.The formation of dendrite structure after deposition of SiW 11 has already been observed in [40].

11
Multilayer-Modified Electrode is acidic solution, most nitrite ions are p the question is which are the actual reactive species, because the pKa of HNO 2 is 3.3 [49,50] Equation (3) suggest that the active species could be HNO 2 and/or NO.As HNO 2 disproportionates in fairly acidic solution, even though the rate of reaction ( 3) is known to be low [49,50].The catalytic effect appears on W 11 , it increases whereas the corresponding oxidation current decreases.This typical for a reduction process mediated by a reduction catalyst.Equation ( 6) presents the possible overall process.
We find that the ratios Ipa/ 45, 0.51 and 0.6 corresponding to nitrite concentrations of 1, 2 and 2.8 mM respectively, this ratio is increased with stepwise addition of nitrite.No response is observed on the bare GC electrode, in the range of 0.8 to −0.8 V in

Nitrite Reduction
der to confirm the eff the electrochemical properties, the electrochemical behaviors of SiW 11 -modified electrode were investigated.
With increasing number of SiW 11 layers, the duction of nitrite increases.For example, the electrocatalytic current observed with 5-SiW 11 layers is higher than the current observed with 2-SiW 11 layers in the presence of 0.1 mM of nitrite.The response of the modified electrode toward nitrite is practically constant when number of layers is high than 5 (Figure 9).
The catalytic peak maximum at potential −0.68 V is arly dependent on the nitrite concentration, in the range 0.1 mM -3.6 mM, with correlation coefficient of 0.9991, as shown in Figure 10.The reproducibility of the nitrite sensor was studied by using three different sensors, the cathodic peak current corresponding to 2.8 mM of nitrite still remains about 93%.
The stability of th period of 75 days.The sensor was stored under air at room temperature and the current response to 3 mM of nitrite injection in 0.1 M H 2 SO 4 pH 1.2 was checked at regular intervals.No significant change (< 10%) was observed within this period of time.The stability of this sensor can be attributed to the large amount of SiW 11 which is deposited on the surface of the electrode.

Interferences
Our experiments sho has no catalytic pro (Figure 2.A.S).The response of SiW 11 modified electrode was also tested toward other ions such as phosphate (Figure 2.B.S) and perchlorate (Figure 2.C.S), with concentrations between 2 mM and 6 mM.No significant increase in the current was observed.Hence, this proves the selective determination of nitrite because it eliminates the major interferences such as nitrate.

Conclusions
In this study, we de ple of SiW anion 11 reduction of nitrite in aqueous solution with H 2 SO 4 concentration (0.1 M).The electrochemical behavior of the modified electrode was studied using cyclic voltammetry.Catalytic reduction of nitrite can be employed as a new method for determination of nitrite in real sample such as weak liquor existing in the wood and paper industry.This method is simple, low-cost and precise for routine control and can be carried out directly without any separation or pretreatment due to the selective electrocatalytic reduction of nitrite.
The catalytic reduction peak current showed a linear dependent on the nitrite concentration and a linear calibr EGIDE through UTIQ by PHC Maghreb program

Figure 5 ,
Figure5, curves a and ce of the negative layer of SiW 11 increases ΔEp by more than 100 mV and decreases, mainly the cathodic peak, which is due to the electrostatic repulsion of the probe.The electrostatic interaction of PA idenced by using gold nanoparticles initially covered with a citrate monolayer, a layer on PAH being adsorbed on their surface, their zeta potential was found to be +44 mV.After adsorption of SiW 11 , the zeta potential became equal to −1.1 mV, then showing the neutralization of the positive charge by successful adsorption of SiW 11 .Figure5shows that with increasing the number o W 11 layers from one to five, the peak maxima of

Figure 8 5 -
Figure 8 shows the behavior of SiW 11 immobilized LB

Figure 10 .
Figure 10.Calibration curve Linear relationship between the catalytic current and nitri centrations at −0.6 V vs.