Potassium hexacyanoferrate(II) trihydrate (K₄[Fe(CN)₆·3H₂O), potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]), dopamine, alumina powder (Al₂O₃) and potassium chloride (KCl) were purchased from Sigma-Aldrich. Phosphate buffer solution (0.1 M, formed by mixing NaH₂PO₄ with Na₂HPO₄ solutions) was used as supporting electrolyte solution. Hydrochloric acid (HCl) and sodium hydroxide (NaOH) solutions were used to adjust the proper pH value.

Pyrrole monomer (Sigma Aldrich) was purified by double distillation, stored at low temperature and protected from light. The human serum was collected and stored at 4˚C by Laboratoire d’analyses Medicales Dr Nabli Naoufel, Sousse, Tunisia. Double distilled water was used throughout the experiments and all voltammetry measurements are carried out under pure nitrogen bubbling at 25˚C.

High magnification microstructural images were performed using high resolution ESEM Thermo-Fisher FEIQ250 with resolution better than 7 nm at low operating voltages of 5 - 10 kV. As in our case the overoxidized polypyrrole films are not sufficiently conductive, the charging problem and improvement of the image quality was obtained by coating the surface of the samples with a very thin layer of gold by vapor deposition.

All electrochemical measurements were carried out using a mini potentiostat model DY2000 (DIGI-IVY company, Germany) connected by an USB cable with a laptop. All electrochemical measurements were carried out in a conventional three electrode system, comprising a platinum wire as a counter electrode, an Ag/AgCl reference electrode with a saturated KCl solution and the working electrode being a glassy carbon disc of 3 mm in diameter modified by the molecularly imprinted overoxidized polypyrrole.

Before the electropolymerization, the surface of the glassy carbon electrode was polished by a gentle polishing with an aluminum powder of 0.05 µm in diameter in order to obtain a smooth and shiny surface. Then the electrode surface was immersed in ethanol for 5 min, sonicated in distilled water and then dried under nitrogen flow.

The modification of the glassy carbon electrode by the template-free polypyrrole (NIP-OPPy) was prepared by cyclic voltammetry in the potential range (−0.6 to 1.2 V), during ten cycles of a 10⁻¹ M pyrrole aqueous solution containing 10⁻¹ M of potassium chloride, as supporting electrolyte. The same protocol was used for the electrodeposition of molecularly imprinted polymer (MIP-OPPy), but in the presence of 10⁻³ M of dopamine. After electropolymerization, the extraction of dopamine was carried out by incubating the electrodes in an NaOH solution (0.5 M) with gentle agitation and low scanning speed in a potential range of (−1 to 1 V) until the disappearance of the dopamine oxidation peak.

After dopamine extraction, the modified overoxidized polypyrrole electrode was incubated in the dopamine solution (0.1 M PBS solution, pH 6) for 20 min. then it was washed with distilled water and then immersed in a 0.1 M PBS solution (pH = 6) before electroanalysis. The same procedure was adopted for NIP-OPPy and MIP-OPPy. Square wave voltammetry (SWV) was used with the sweep frequency of 10 Hz, the amplitude of 40 mV and a step potential of 4 mV.

Figure 2(a) and Figure 2(b) show the cyclic voltammograms recorded during the electrochemical polymerization of pyrrole in the absence and in the presence of dopamine. From the first cycle the oxidation peak was observed at around 0.78 V, attributed to the oxidation of the monomer to a radical cation. Then the formation of a conductive polymer film on the glassy carbon electrode surface is observed by the increase in the intensity of the oxidation and reduction peaks. From the second cycle, a large anodic peak appears at 0.26 V corresponding to the oxidation of the formed polypyrrole and then, increases and shifts to a more anodic potential with the increase in the number of cycles [26] [27]. The charge passed during the formation of NIP is higher than of that in the formation of MIP, indicating that the template is becoming part of the polymeric film [28]. During the electrodeposition of the polypyrrole layer, DA molecules diffuse toward the electrode surface and are entrapped in the polymer film and interact with the pyrrole during its electropolymerization.

The overoxidation polypyrrole film was obtained electrochemically by cycling between −1 V and 1 V, in a sodium hydroxide solution (0.5 M), for five cycles. Figure 3 shows that the high oxidation peak is observed at 0.3 V. We notice that the voltammograms recorded after the third cycle are almost confused, which proves that the overoxidation is complete. During the over-oxidation of the OPPy film, the formation of carbonyl and carboxylic groups could provide a better permselectivity for DA with the electrostatic force [2].

The electrochemical behavior of electrodes modified with MIP-OPPy was studied by cyclic voltammetry. Figure 4 shows the electrochemical behavior of naked and modified GCE in 1 mM [Fe(CN)₆]^4−/3− as redox probe. An almost reversible redox system was obtained with bare GCE (DE ~ 100 mV).

No redox peak was observed on the surface of (GCE/MIP-OPPy) indicating that the OPPY layer blocked the transport of electrons. After removal of the template molecules DA in 95% ethanol and DI water for 60 min, the current increased, which is due to the creation of cavities following the removal of the template [29].

SEM images of MIP-OPPy before and after extraction of the DA template are

shown in Figure 5. After of MIP-OPPy, a spherical granular structure was obtained due to the formation of a polymer film (Figure 5(a), Figure 5(b)). After removal of DA, a regular porous structure appeared on the surface of the MIP-OPPy electrode as shown (Figure 5(c), Figure 5(d)), which is due to the produced cavities.

The quantitative detection of DA was obtained by SWV in a 0.1 M PBS solution, at pH 6. Figure 10(a) shows the voltammograms obtained by SWV of different concentrations of DA on the modified electrode MIP-OPPy. The peak currents attributed to the oxidation of DA show a linear response with increasing concentration of DA in the range of 0.01 to 50 nM.

A good linear region with logarithm of DA concentration between 5 × 10⁻¹¹ M and 5 × 10⁻⁸ M is shown in Figure 10(b), black points. The linear regression equation was expressed as follows: I_pa (μA) = 6.32 + 1.328 log C_DA (M) with a correlation coefficient of R² = 0.977. The detection limit is 10⁻¹¹ M corresponding to three times the noise of the background divided by the sensitivity.

The slope of the calibration plot for DA at the electrode modified by OPPy-MIP is 1.328, which is higher than that obtained for OPPy-NIP (slope: 0.66, R² = 0.968, Figure 9(b), red points). The imprinting factor of the overoxidized polypyrrole is 2.

The relative standard deviation for the described sensor, over five determinations and removal of DA, is 5%. The reproducibility was studied, using 5 different dopamine sensors. The obtained relative standard deviation ranges from 10 % for all concentrations.

Different electrochemical sensors for the determination of DA some examples of previous results are summarized in Table 1. By comparing, our sensor with the others presented we notice a lower range detection limit, the linear range being limited in the higher range of concentrations.

Real Sample Analysis

Human serum was obtained in tubes by a Medical Analysis Laboratory, Sousse,

All measurements were taken, in five replicates.

Tunisia. After centrifugation for 10 min at 1000 r/min, the serum samples were diluted 1:10 in 0.1 M PBS. Concentrations of dopamine were added in the diluted serum samples and the recovery rates were calculated. The results are shown in Table 2. Recovery rates are in the range of 100%, showing that no interfering agent was detected in human serum.