Pyrrole (Py), 98% from Sigma-Aldrich, USA; orthophenylenediamine (OPDA) from Merck; urease ЕС 3.5.1.5, 112 U∙mg⁻¹ from Fluka; carbon nanotubes (CNT) from Sigma Aldrich with size 2 - 6 nm and length 0.1 - 10 µm, with 90% purity; glutaraldehyde from Merck. All reagents were of analytical grade. All solutions were prepared using deionized water from PURELAB Ultrasystem.

Cyclic voltammetric, amperometric measurements and electropolymerization of Py and OPDA monomers on working electrode surface were carried out with the PalmSens Electrochemical Instrument (Palm Instruments BV, Netherlands) and three-electrode electrochemical cell: a platinum plate electrode (1 cm² area) as a working electrode, platinum wire as a counter electrode and a saturated calomel (SCE) or Ag/AgCl electrodes as reference electrodes were used both in the cyclic voltammetric and amperometric measurements.

The working electrode was mechanically polished with 0.3 and 0.05 µm alumina, rinsed with distilled water, acetone and once again with water. Then, it was cleaned electrochemically in 1 M H₂SO₄ by potential cycling between −0.25 and +1.45 V versus Ag/AgCl at a scan rate of 0.075 V/s for 10 - 15 min. Before electropolymerization, the monomer solutions (Py or OPDA) were purged with high-purity nitrogen gas for at least 10 min in order to remove dissolved oxygen. An inert environment was maintained in the electrochemical cell during the polymerization by purging the cell atmosphere with a flow of nitrogen.

OPDA was electropolymerized by continuous potential cycling between −0.4 and +1.0 V vs. SCE, at a scan rate of 0.05 V/s. The number of deposition cycles was varied (1, 10 and 20 cycles). The electropolymerization was carried out in 0.1М H₂SO₄ or 0.1M KCI as supporting electrolyte containing 0.05 M OPDA monomer solution. Then, the working electrode was dried at room temperature. A 5 µL of 25% glutaraldehyde was pipette on the electrode surface and the solution was allowed to evaporate at 30˚C for 30 min. The urease was immobilized on the POPDA surface by pipetting a 5 µL of 0.1% urease and the electrode was dried at 4˚C.

The electropolymerization of Py was carried out in 0.1 М KCl as supporting electrolyte, containing 0.1 М NaCl and 0.4 М Py monomer solution. The final concentration of urease in this solution was 0.1%. The working electrode potential was cycled in the potential range from −1.0 to +0.7 V vs Ag/AgCl, at a scan rate of 0.05 V/s, 30 cycles.

The first step for developing of urea biosensor was to choose the optimum conditions for monomer electropolymerization. Several experiments have been carried out to obtain stable and active polymeric film. The effect of supporting electrolyte and number of deposition cycles on the OPDA electropolymerization were studied. Figure 1 shows the CVs of the ОPDA electropolymerization −0.05 M ОPDA in 0.1 М KCl (dashed line) and 0.05 M ОPDA in 0.1 М H₂SO₄ (solid line). The results demonstrated that the acidity of the electrolyte had a very strong effect on the electropolymerization process. The CV curve, obtained in H₂SO₄, is much wider compared with the CV curve obtained in KCl. This is probably due to the different conductivity of POPDA film in both electrolytes. Thus, 0.1 М H₂SO₄ was chosen as supporting electrolyte for the OPDA electropolymerization.

Figure 2 shows CV curves of Pt/POPDA electrode as a function of different number of deposition cycles—1, 10 and 20. At 1st deposition cycle a high and wide oxidation peak was appeared at +0.70 V. This was attributed to the oxidation of the monomer on the clean Pt electrode and formation of POPDA film. In the following negative sweep, a reduction peak at −0.18 V was observed, which is much lower than the oxidation peak. At 10 deposition cycle the oxidation and reduction peaks were decreased. Besides that, with the increasing of number of deposition cycles, the anodic and cathodic peaks shifted to +0.54