Urea Amperometric biosensor was obtained on the base of nanostructured polypyrrole (PPy) and poly ortho- phenylenediamine (POPDA). The optimal conditions for monomer electropolymerization were determined. The effect of supporting electrolyte and number of deposition cycles on the OPDA and Py electropolymerization were studied. It was proved that POPDA and PPy were affected by pH changes and responded to the ammonium, product of urease catalyzed reaction. SEM images of the modified Pt/PPy electrode were presented. The cycle voltammograms and chrono amperometric curves of Pt/POPDA/urease and Pt/PPy/urease electrodes were studied. A good linear relationship was observed for Pt/POPDA/urease electrode in a concentration range from 6.7 to 54 mMurea. For Pt/PPy/urease electrode the linear relation in the range from 0.02 to0.16 mMurea was determined. The entrapped carbon nanotubes (CNT) in PPy film and the bipolymer layers were prepared for construction of Pt/PPy/CNT/urease, Pt/POPDA/PPy/urease and Pt/PPy/POPDA/urease biosensors. Obviously, the addition of POPDA to the composition of the two biosensors (Pt/PPy/POPDA/urease and Pt/POPDA/PPy/urease) reduced their sensitivity to urea. Pt/РPy/CNT/urease and Pt/РPy/ urease biosensors were 173 and 138 times more sensitive to urea than biosensor without PPy (Pt/POPDA/urease biosensor). It was found, that the performance of Pt/PPy/CNT/urease electrode was the best from the five obtained biosensors: linear range of urea concentrations—from 0.02 to0.16 mM; sensitivity—15.22 μA/mM and detection limit— 0.005 mM urea.
The urea concentration in serum or urine is an indicator of kidney diseases, as well as diabetes, and analysis in clinical laboratories is frequently used. In a urea biosensor the enzyme urease, which catalyses the hydrolysis of urea to ammonia and carbonate can be immobilized into different transducers, such as conducting polymers. Various conducting polymers, like polyaniline (PANi), polypyrrole (PPy) and poly ortho-phenylenediamine, have been used for the fabrication of biosensors. Among them, polypyrrole is one of the most extensively used conducting polymers in the fabrication of urease biosensors [
As opposed to PPy, POPDA shows the conductivity in its reduced state, whereas its oxidized state is insulating. This determines the electrochemical properties of POPDA, since many electrode redox processes of solution species have been shown to take place within relatively narrow potential window, corresponding to the reduced (conducting) form of this polymer [
A variety of urease biosensors with high sensitivity and excellent reproducibility based on nanostructured polypyrrole [5-9], poly ortho-phenylenediamine [10,11] and carbon nanotubes [12,13] has been reported.
The aim of this paper was to study the conditions for preparation of urea amperometric biosensor based on nanostructured polypyrrole, poly ortho-phenylenediamine, multi-layered nanostructured substrates and comparing the performance of obtained biosensors.
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.
• 2.6. Preparation of Multi-Layered Nanostructured Urease BiosensorThe 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. 0.0016 g CNT were added and the mixture was homogenized by sonication for 1 h. Then urease was added to this solution to a final concentration of 0.1%. The working electrode potential was cycled in the potential range of −1.0 to +0.7 V at a scan rate of 0.05 V/s for 30 cycles.
POPDA was deposited on working electrode by the method described above. After that the electrode was dried at room temperature and deposited the second polymer layer of PPy with entrapping urease, as described above.
POPDA film was deposited on Pt/PPy/urease electrode by the method described above.
• 2.7. Electrochemical Measurements with Urease BiosensorCyclic voltammograms (CVs) of Pt/POPDA/urease electrode were carried out in 30 mL of 0.01 M PBS (pH 5.8) in the absence and presence of 100 µL of 1М urea. The working electrode potential was cycled in the potential range of −1.0 to +1.5 V.
Cyclic voltammograms of Pt/PPy/urease electrode were carried out in 10 mL of 0.01 M PBS (pH 5.8), containing 0.1 M NaCl, in the absence and presence of 200 µL of 10 mМ urea. The working electrode potential was cycled in the potential range of −1.0 to +0.7 V.
Chronoamperometry was used as the transduction method for detecting urea in different solutions. The current density was measured for films potentiostatically polarized at a fixed potential −0.1 V for Pt/POPDA/ urease and −0.6 V for Pt/PPy/urease biosensors, at successive addition of 100 µL of 1 M urea (pH 5.8) and 200 µL of 10 mM urea (pH 5.8), respectively. This value of pH allowed us to achieve a condition of maximum activity of urease.
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.