Voltammetric determination of dopamine using modified glassy carbon electrode by electrografting of catechol

By means of cyclic voltammetry as a diagnostic technique, the present study tried to describe the behaviour of dopamine (DP) at the bare and catechol electrografted glassy carbon electrode (CA/GCE). The results indicated that the CA/GCE exhibits high electrocatalytic activity toward DP. The DP cycling of the CA/GCE in DP showed two linear calibration ranges between 5.0–100 and 100–750 μM with a detection limit of 0.86 μM. For 6 DP replicate measurements (50 μM); the relative standard deviation (RSD) was 2.7 %. This method was successfully used for the determination of DP in serum samples.


INTRODUCTION
A type of catecholamine, dopamine (DP), acts as a significant neurotransmitter in mammalian brain fluids and tissues.It is responsible for the functions of the central nervous, hormonal, renal, and cardiovascular systems. 1,2Thus, true and accurate measurement of DP for detection, monitoring, prevention and treatments of neurological disorders such as schizophrenia, Alzheimer's disease, Parkinson's disease and HIV infection is important. 3Different methods are used for measuring DP concentration in biological samples such as capillary electrophoresis, 4 mass spectrometry, 5 high-performance liquid chromatography 6 and electrochemical sensors. 7,8hemically modified electrodes are attractive tools for the detection of pharmaceutical compounds due to the high sensitivity, fast response, and low cost.Some of the used electrochemical techniques for measuring of DP concentration include the conducting polymer matrix/gold nanoparticles, 9 gold electrodes/N-1054 POURGHOBADI and NEAMATOLLAHI acetylcysteine, 10 zinc oxide composite/glassy carbon electrode, 11 nanoparticles, 12 carbon nanotubes, 13 homogeneous mediators 14 and heterogeneous mediators. 15eterogeneous mediator is of particular interest in comparison with homogenous mediator because of high selectivity, environmental advantages, green chemistry and the reusability of these mediators.16   In this work, a heterogeneous catalyst based on catechol grafted glassy carbon electrode (CA/GCE) was used as a modified electrode for determination of DP.By means of catechol nitration by the nitrous acid and electroreduction of generating nitrocatechol, one can modify the electrode, which leads to catechol grafting. 17We also showed the glassy carbon electrode (GCE) modified with electroactive catechol groups has the ability of dopamine determination in human blood.

Apparatus
For electrochemical experiments, a potentiostat /galvanostat µAutolab model PGSTAT 204 (Eco Chemie, Utrecht, Netherlands) with NOVA 1.9 software NOVA was used.A 3-electrode cell was used with an Ag/AgCl electrode (KCl, 3 M) as the reference electrode.All potentials in the paper are expressed versus this reference electrode.For the working electrode, a modified GCE was used, and for the counter electrode, a Pt wire was used.The Ag/AgCl electrode and Pt wire were obtained from Metrohm, and the glassy carbon electrode was obtained from the AZAR Electrode.Moreover, the Metrohm model 827 pH/mV meter was used, for pH measurements.SEM (the scanning electron microscopy) was used for morphological specify the modified electrode with the electrografting.
The optimal stock solution was sequentially diluted with distilled water for the preparation of other fresh solutions.For all the experiments, 0.1 M phosphate buffer solution was used with different pH values.All the electrochemical studies were conducted at 25±1 °C.

Electrografting of electrode surface
The published paper 15 was used as a guiding model for electrografting of the glassy carbon electrode.By means of a piece of cloth, the glassy carbon electrode was polished with an alumina fine powder (0.05 μm) in water slurry.Then, it was ultrasonically washed in ethanol and water.Then, the GCE was modified by the potential cycling ranging from 0.0 to -0.9 V in an aqueous solution which had HCl (pH 1.0), catechol and NaNO 2 .
In all cases, the room temperature was used for the modification of GCE electrode with catechol (CA/GCE) in an electrolytic solution.The cyclic voltammetry in aqueous buffered solutions was used for the quantification of the catechol modified GCE redox activity.

Analytical procedure
In order to obtain a steady cyclic voltammogram in phosphate buffer (pH 8.0), the activation of CA/GCE by cycling between -0.3 and 0.4 V was used.Then, the electrodes were dipped into a DP and buffer (pH 8.0) solution in a usual experiment.At a scanning rate of 20 mV s -1 , the potential was swept from -0.3 to 0.4 V.This was repeated while DP was presented as the sample solution.

Preparation of real samples
DP (2 ml, 0.01 mol L -1 ) spiked in the blood serum sample was centrifuged, and the supernatant was diluted 50 times with water.Using the standard addition method, finally, 3.0 ml of the solution plus 7.0 ml of the phosphate buffer (pH 8.0) were used for the analysis.By means of the calibration curve method obtained from relevant calibration equations, quantitations were performed.

Characterization and electrochemical behavior of the CA/GCE
Electrografting of glassy carbon electrode with catechol was performed as described in the experimental section.The cyclic voltammograms of CA/GCE in aqueous phosphate buffer (pH 8.0) at different scan rates are shown in Fig. 1.They show an anodic peak (A 1 ) in the positive-going scan and its corresponding cathodic peak (C 1 ) in the negative-going scan.These peaks belong to the oxidation/reduction of grafted catechol/ortho-benzoquinone redox couple 17 (Scheme 1).The plot of log I A1 versus log v in the range of 25 to 1000 mV s -1 is found to be linear with a slope of about one (Fig. 1, inset), which is a characteristic for the adsorption-controlled electrode process.The effect of the solution pH on the CA/GCE was studied in the next step (Fig. 2).The oxidation as well as the reduction peak potentials were found to shift to the negative potentials in the pH range 3-9 under study, with the depend-1056 POURGHOBADI and NEAMATOLLAHI ence being at -58 mV/pH unit.This is expected because of the participation of two electrons and two protons in the oxidation of catechol to ortho-benzoquinone. 17 Cyclic voltammograms of the bare GCE and CA/GCE in presence of DP at pH 8.0 and at a scan rate of 20 mV s -1 are shown in Fig. 3, curves b and c, respectively.Comparison of the behaviour of bare GCE and of the electrografted glassy carbon electrode (CA/GCE) shows that the anodic peak current (I pA1 ) is significantly greater at the latter.On the other hand, with increasing the potential scan rate, the peak current ratio (I pC1 /I pA1 ) increases because the time required for the reaction of DP with grafted ortho-benzoquinone is not enough (Fig. 4). 17cheme 1 shows how a catalytic reaction occurs after the process of the electron--transfer.
The influence of pH on the current response of a DP (0.1 mM) at CA/GCE in a phosphate buffered solution of was investigated in the pH range from 3.0 to 9.0 by the linear sweep voltammetry (Fig. 5).As can be seen, the oxidation peak current (I pA1 ) increases gradually with the increasing pH up to pH 8.0 and then decreases.By increasing pH of the solution the anodic peak potential (E pA1 ) also shifted to the negative potentials.This is expected as discussed earlier. 17

Analytical measurements
Fig. 6 shows the cyclic voltammograms of CA/GCE in the presence of different concentrations of DP.The results showed that, by increasing the concentration of DP, the anodic peak current (I pA1 ) increases.Under these circumstances, this dependence is linear, but in two segments: one in concentration range Available on line at www.shd.org.rs/JSCS/_________________________________________________________________________________________________________________________ (CC) 2017 SCS    Table I shows the comparison of this study and previously reported voltammetric methods for the determination of DP.As it is seen, the analytical parameters are comparable or better than the results reported for DP determination at the surface of other modified electrodes.The CA/GCE applicability was evaluated by measuring the DP concentration in human blood serum sample after the protein precipitation process.The recovery of DP from human blood serum was measured by the injection of a drug with a known amount of DP.Table II shows the results obtained by analysis of the DP content of the human blood serum samples.As Table II shows, the proposed method provides a potential tool for the determination of DP in real samples with good recoveries and good reproducibility.The main problem in the DP determination is the difference between the DP and the simultaneous presence of such species as the ascorbic acid (AA) and the uric acid (UA).To determine the selectivity of the modified electrode, the electrocatalytic response of AA, UA and various substances (Table III) were evaluated under optimized experimental conditions in CA/GCE (Fig. 7).As can be seen, I pA1 in the absence (curve a) and in the presence of ascorbic acid (curve b), is nearly the same, but curve c shows the noticeable catalytic current when DP was added to the previous solution (curve c).Also, it is shown that the increasing UA to the previous solution does not influence on the I pA1 (curve d).The effects of various substances on DP determination (50 µM) were evaluated under optimal conditions.The tolerance limit was taken as the maximum concentration of the interfering substance that caused an error less than 5 % for determination of DP.We observed no significant interference up to 100 µM (Table III).

Scheme 1 .
Scheme 1.The mechanism of electrocatalytic oxidation of Dp on CA/GCE.

Fig. 7 .
Fig. 7. Linear sweep voltammograms of CA/GCE: in the absence of AA (curve a) and in the presence of AA (0.1 mM) (curve b).c) Cyclic voltammograms of CA/GCE: in the presence AA (0.1 mM) and DP (0.05 mM).d) Cyclic voltammograms of CA/GCE: in the presence AA (0.1 mM), DP (0.05 mM) and UA (1 mM) aqueous solution containing phosphate buffer (c = 0.2 M, pH 8.0).Scan rate 20 mV/s.CONCLUSIONS In this study, we have prepared a new modified CA/GCE for DP determination.The modification of the glassy carbon working electrode with catechol has significantly improved the electrochemical response for DP determination.

TABLE I .
Comparison of the results of the proposed method with similar reports Available on line at www.shd.org.rs/JSCS/

TABLE III .
Interference of some foreign species on the determination of 50.0 μM DP under the optimized conditions.