All used chemicals were analytical reagent grade. Sulfanilamide, N-(1-naphtyl)-ethylene diamine, copper sulphate pentahydrate, potassium hexacyanoferrate (III), Nafion^® (5% m:m solution in lower aliphaic alcohols) were obtained from Sigma. Phosphate buffer solution (PBS) was prepared by mixing aqueous solutions of sodium dihydrogen phosphate (0.1 mol/L) and disodium hydrogen phosphate (0.1 mol/L) until the required pH was achieved. A nitric oxide stock solution was prepared by bubbling NO produced by the reaction of a saturated aqueous solution of sodium nitrite with 2 M sulfuric acid (Equation (1)), through a 4 M potassium hydroxide solution and finally collecting it in phosphate buffer solution (0.1 M, pH 7.5).

First, all the apparatus for NO gas production was deaerated with nitrogen gas for 30 minutes. The standard stock solution was freshly prepared before the use. The concentration of NO in the stock solution was determined using sulfanilamide and N-(1-naphtyl)-ethylenediamine as described in [21] . Sodium nitrite and nitrate stock solution (0.1 M) were freshly prepared before the use.

The potentiostat Autolab PSTAT 10 with software GPES version 4.9 and a potentiostat PalmSens with software PSTrace were used for cyclic voltammetry and hydrodynamic amperometry. The electrochemical cell consisted of a carbon paste electrode as the working electrode, an Ag/AgCl/3M KCl reference electrode (Metrohm 6.0733.100), and a platinum wire as the counter electrode. Nitrogen was used for degassing the solutions. A magnetic stirrer provided convection of the solution. All potentials mentioned in this paper are referred to the Ag/AgCl reference electrode. The flow injection system was assembled from a potentiostat as the detector, a high performance liquid chromatographic pump (1100 ICI instruments), a sample injection valve (5020 Rheodyne, Cotati, CA, USA), and a thin layer electrochemical detector (LC 4C, BAS, West Lafayette, Indiana, USA) with a flow through cell (spacer thickness 0.19 mm; CC-5, BAS).

Carbon paste electrode modified with Cu-film electrode was tested on cyclic voltammetry in phosphate buffer solution 0.1 M (pH 7.5). In Figure 2, it is shown that cyclic voltammogram of CPE-Cu film electrode can be noticed at potential of −0.20 V copper (II) reduction and around 0.0 V oxidation reaction of Cu˚ to Cu(II) and copper oxide film is produced in the electrode surface.

Cyclic voltammogram with scan rate of 5 mV/s of Cu-film electrode is shown in Figure 3, where can be seen

two steps of copper (II) reduction, Cu(II) ® Cu(I) and Cu(I)®Cu˚, peak potentials at −0.10 V respectively −0.20 V. These peaks may be shown because of complex compound formation in electrode surface with phosphate ion in solution.

Copper film electrode was covered with hexacyanoferrate using cyclic volatmmetry. The copper hexacyanoferrate film was prepared at 0.1 M potassium nitrate solution containing 1 mM potassium hexacyanoferrate. Numbers of cyclic voltammetric scans were around 20, until no significant difference was noticed of hexacyanoferrate redox peaks between the last two scans.

The reduction and oxidation peaks at potential 0.50 V and 0.80 V are peaks of redox reactions of iron hexacyanoferrate Fe^IIIóFe^II. The peak potential at −0.40 and 0.0 V corresponds to the conversion of copper from its oxidation state +2 and 0.

Based on cyclic voltammetric scans (Figure 4), can be concluded that copper hexacyanoferrate complex compound is formed because the oxidation and reduction peak is going to increase after every scan. Copper hexacyanoferrate (II) complex is insoluble; the produced film in the surface electrode is stable in aqua media. Copper (II/I) reduction peak at −0.40 V is increased after every scan and the concentration of oxidized copper near electrode surface is going to be higher, because its oxidized state produces complex compound with hexacyanoferrate (II). The decrease of oxidation peak intensity is attributed to metallic copper from Cu-film. This happens because the metallic form in the surface electrode is going to decrease after every scan, because the copper exists in oxidized form as copper (II) complex with hexacyanoferrate.

Carbon paste electrode modified with copper hexacyanoferrate film was tested on 0.1 M phosphate buffer solution pH 7.5 for its response to nitric (II) oxide (Figure 5). After the addition of nitric (II) oxide, cathodic current is increased near reduction peak of copper because of the electrocatalytic effects of copper (II) hexacyanoferrate. The suggested reaction mechanism (Figure 6) assumes the chemical oxidation of copper hexacyanoferrate by NO and produce of nitrosyl ciano complex compound which returns electrochemically to the reduced form, at the electrode surface. At positive potential above 0.75 V the oxidation current is increased because of NO oxidation at electrode.

The modified electrode with copper hexacyanoferrate film was tested for its response to different concentration of nitric oxide solutions. The modified electrode shows linearity up to concentration 250 µM, with sensitivity 5.696 nA/µM and correlation factor 0.994 (Figure 7).

The detection limit (3σ) calculated from standard deviation in hydrodynamic amperometry for 43.7 µM solution of NO was 8.32 µM (SD = 15.8, average 219.4 nA).

The main interferent compounds may come from nitrite and nitrate ions, which are also the reaction products of NO oxidation, and other compounds that are present in real samples. To avoid interferences we used sulfonil polyfluorotetraethylene (Nafion^®) membrane. This anionic membrane contains negatively charged sulfonyl

groups which prevent penetration of negatively charged ions to electrode surface. The neutralized Nafion solution was diluted with alcohol, and the electrode surface was covered with 10 µL.

Based on the results shown in Figure 8, the electrode does not show measurable signal for nitrite and nitrate ions except on 0 V the signal intensity was −1 nA and −28 nA for respectively. As expected, the signal for nitric (II) oxide solution is decreased because of covered surface with polyfluorotetraethylene memane. Nafion membrane is penetrable of NO molecule, but it covers physically the electroactive sites of electrode surface.