Electropolymerized Congo Red Film based Sensor for Dopamine: A Voltammetric Study

The polymerization film of Congo red was prepared on the surface of carbon paste electrode by electropolymerization using cyclic voltammetric method. The higher catalytic activity was obtained for electrocatalytic oxidation of Dopamine, with drastic enhancement of the reversibility and peak current in 0.2 phosphate buffer solution of pH 7.0 at the sweep rate 100 mV/s. The variation of sweep rate and pH were investigated. The limit of detection of Dopamine was found to be 0.06 μM. The effect of interference studies was done by differential pulse voltammetric technique. In the simultaneous look at, Dopamine and Uric acid were well separated by cyclic voltammetric technique. The proposed method showed good sensitivity, selectivity, and reproducibility.


Introduction
Dopamine, which belongs to the catecholamine family of neurotransmitters, is synthesized in a sequential reaction in which tyrosine hydroxylase and amino acid decarboxylase convert tyrosine to L-dihydroxyphenylalanine (L-dopa), followed by decarboxylation of L-dopa to dopamine. Dopamine plays a vital role in the control of movements and has been associated with the motor symptoms experienced in patients with Parkinson's disease [1]. Other studies show that the dopamine oxidation products can inhibit the function of specific proteins [2] and correlate formation of cysteine l-dopamine conjugates with dopamine-induced neurotoxicity. As a potent neurotransmitter, changes in the level of dopamine in adrenal glands impact many aspects of brain circuitry. For example, Parkinsonism is associated with a reduced level of dopamine; while schizophrenia is related to increased dopamine activity [3].
On the other hand, Uric acid is the primary end product of purine metabolism in the human body [9]. In a healthy human being, the typical concentration of UA in urine is around 2 mM and in the blood, is in between 120 μM to 450 μM ranges [10,11]. Extreme abnormalities of UA levels are Symptomic of several diseases, such as cardiovascular disease [12], hyperuricemia, uric acid stones [13], gout and Le-seh-Nyhan syndrome [14]. Increased rate level also leads to pneumonia and leukaemia [15,16]. Dopamine and uric acids usually coexist in physiological samples, dopamine normally present at low concentration along with uric acid which is at higher concentration. The simultaneous determination of these compounds is a special interest in the development of electrochemical sensors [17]. The successful route to overcome the problems of selectivity is to modify the carbon paste electrode surface because the modified electrode could decrease the over voltage, improve the velocity of mass transfer efficiency and enhances the selectivity of the analyte [18]. The modification can be done by using organic and inorganic substances and biomolecules [19][20][21]. In the present day's electropolymerization technique was used to prepare the polymer-modified electrodes have received wide interest in the detection of analytes because of its high selectivity, and homogeneity in electrochemical deposition, strong adherence to the electrode surface and chemical stability of the films [22][23][24]. Shahrokhian et al. [25] has reported the Synthesis of Polypyrrole in the Presence of Congo Red; Application to Selective Voltammetric Determination of Dopamine in the Presence of Ascorbic Acid. Until now, different methodologies have been used to prepare polymeric film modified electrodes. Among them, electropolymerization yields a modified electrode with a three-dimensional distribution of mediators. This type of electrodes enhances the sensitivity and improves the catalytic activity than monolayers and few reports have been reported [26][27][28][29]. Congo red is the sodium salt of 3,3′-([1,1′-biphenyl]-4,4′-diyl) bis (4-aminonaphthalene-1sulfonic acid) is an azoic compound synthesized by Paul Böttiger is used to stain microscopic prepares, especially as a cytoplasm and erythrocyte stain (Scheme 1) [30].
In this study, the modification of stable working electrode by electro polymerizing Congo red on the bare carbon paste electrode surface by CV method and used for the voltammetric determination of DA and UA. The poly (Congo red) MCPE shows very good enhancement when compared to BCPE and also it shows good sensitivity, selectivity, and stability for the determination of the two neurotransmitters like DA and UA.

Materials and Methods
Cyclic voltammetric experiments were performed on a model CH660c (CH instrument). All the electrochemical experiments were carried out in a three-electrode cell system, which contained a bare carbon paste electrode (BCPE)/ Poly (Congo red) film coated MCPE as the working electrode, a platinum wire and saturated calomel electrode as counter and reference electrode.

Reagents and chemicals
Graphite powder of 50 mm size was purchased from Loba and silicon oil was purchased from Himedia. Congo red, Dopamine hydrochloride (DA) and Uric acid (UA) were obtained from Himedia. All the chemicals are of analytical grade quality and were used as supplied without further purification. 25 × 10 -4 M Congo red was prepared in double distilled water, 25 × 10 -4 M DA was prepared in 0.1 M Perchloric acid (HClO 4 ), 25 × 10 -4 M UA was prepared in 0.1 M sodium hydroxide, and Phosphate buffer solution (PBS) of same ionic strength was prepared (0.2 M) by mixing appropriate ratio of sodium dihydrogen phosphate (NaH 2 PO 4 .H 2 O) disodium hydrogen phosphate (Na 2 HPO 4 ).

Preparation of bare carbon paste electrode
Bare carbon paste electrodes (BCPE) were made with silicon oil (30%), and graphite powder (70%). The two components were thoroughly mixed in an agate mortar for about 30 minutes. The BCPE was packed into a homemade Teflon cavity having a current collector and was polished on a weighing paper.

Electropolymerization of poly (Congo red) MCPE at CPE surface
The modified carbon paste electrode was fabricated by electrochemical polymerization process over the potential range from -0.2 to 1.5 V at a sweep rate of 100 mV/s for 20 cycles. The poly (Congo red) modified carbon paste electrode (MCPE) was prepared by placing 1 mM Congo red with 0.1 M NaOH in an electrochemical cell. Figure 1 suggests that during first cycle a small anodic peak was observed corresponding to the oxidation of Congo red monomer [31]. During the polymerized process, with increasing the number of cyclic time corresponding voltammogram was slowly decreased. It shows that the poly (Congo red) film was produced and deposited at the surface of BCPE. Once electropolymerization process complete; the MCPE was rinsed carefully with double distilled water.

Effect of different cycles of poly (Congo red) MCPE
From the above experimental result shows, the thickness of the  Figure 2. However, by considering the peak nature of the cyclic voltammogram the increment in the anodic peak current, the twenty cycles polymerized MCPE was optimized for the further electrochemical analysis. The probable mechanism of formation of the polymer film as reported in the literature [30].

Effect of sweep rate
The Figure 4a shows the effect of applied sweep rate for the oxidation of 0.1 mM DA recorded at different sweep rates using poly (Congo red) MCPE in 0.2 M PBS of pH 7.0. From the above figure shows an increase in the redox peak current with increases the sweep rates from 50 to 400 mV/s. To evaluate the electrode process, the graph of Ip versus sweep rates (υ) was plotted (Figure 4b). The obtained graph was the good linearity almost nearly straight line having a correlation coefficient value 0.9997 and 0.9989 respectively but in the same time the graph of Ip versus square root of sweep rates (υ 1/2 ) as shown in the Figure 4c, having correlation coefficient value 0.9880 and 0.9927 respectively. From the above scrutiny, overall electrode process was controlled by adsorption process at poly (Congo red) MCPE.
The heterogeneous rate constant (k 0 ) values was determined from the experimental peak potential difference (∆Ep) data's, Equation (1) was used for such voltammograms whose ∆Ep values are greater than 10 mV [33].
∆Ep=201.39 log (υ/k 0 )-301.78 (1) Table 1 and Equation (1); the values of the k 0 for the PA oxidation was determined. All the parameters are tabulated in Tables 1 and 2.

Figure 5b
The graph of anodic peak current versus different concentration of DA.

Figure 4C
The graph of anodic peak current versus square root of sweep rates.

Figure 4b
The graph of anodic peak current versus sweep rates.
plotted and it was good linearity as shown in the Figure 5b. The correlation coefficient (r 2 ) was found to be 0.9933. The limit of detection of the lower concentration range for DA was 0.06 µM for the poly (Congo red) MCPE. calculated by using with equation (2). Where S is the standard deviation and M is the slope of obtained from the graph.

The effect pH on DA at Poly (Congo red) MCPE
The supporting electrolyte of pH has a major role in the electrochemical behaviour of DA at poly (Congo red) MCPE. The The obtained DA and UA oxidation peak potential were observed at 173 and 324 mV respectively. The peak to peak separation was 151 mV. This potential difference was enough to recognize DA and UA at poly (Congo red) MCPE.

Interference study
The interference investigation was done by DPV technique and the binary mixture of sample contains 0.1 mM DA and 0.1 mM UA in 0.2 M PBS of pH 7.0. In which, the concentration of DA  was varied from 0.1 to 0.9 mM, while keeping the concentration of UA was constant (Figure 9). The obtained voltammogram shows current will be linearly increased with the increase in 2017 Vol.     Insights in Analytical Electrochemistry ISSN 2470-9867 of DA and UA was possible at poly (Congo red) MCPE with peak to peak separation of 151 mV by CV techniques. Over all the poly (Congo red) MCPE shows stability, selectivity, sensitivity, reproducibility for the determination of neurotransmitter. It is expected that with its high electrocatalytic behaviour the poly (Congo red) MCPE could hold great application in the fields of electro analytical chemistry and biosensors.