Urea Amperometric Biosensors Based on Nanostructured Polypyrrole and Poly Ortho-Phenylenediamine ()
1. Introduction
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 [1]. The versatility of this polymer is determined by its biocompatibility, capability to transduce energy arising from the interaction of analytes and analyte recognizing sites into electrical signals that are easily monitored, capability to protect electrodes from interfering material, and easy way for electro-chemical deposition on the surface of any type of electrode.
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 [2]. Recently nanoparticles enhancing enzyme immobilization technique have become widespread. The using of carbon nanotubes (CNT) as mediators of the electron transfer from the enzyme molecules to the electrode surface is often applied. Their unique electronic properties suggest that CNT have the ability to promote the electron transfer reactions of biomolecules in electrochemistry [3]. Their mechanical properties, high-aspect ratio, electrical conductivity and chemical stability make CNT perfect for a wide range of applications that include fabrication of urease biosensors [4].
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.
2. Experimental
2.1. Reagents and Chemicals
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.
2.2. Instrumentation
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.
2.3. Cleaning of the Working Electrode Surface
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.
2.4. Preparation of Pt/POPDA/Urease Biosensor
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.
2.5. Preparation of Pt/PPy/Urease Biosensor
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 Biosensor
Pt/PPy/CNT/urease biosensor
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. 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.
Pt/POPDA/PPy/urease biosensor
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.
Pt/PPy/POPDA/urease biosensor
POPDA film was deposited on Pt/PPy/urease electrode by the method described above.
• 2.7. Electrochemical Measurements with Urease Biosensor
Cyclic voltammetry
Cyclic 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
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.
3. Results and Discussion
3.1. Preparation of Pt/POPDA/Urease Biosensor
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