Voltammetric Determination of Epinephrine in Pharmaceutical Sample with a Tyrosinase Nanobiosensor

A novel carbon paste electrode modified with a multiwalled carbon nanotube (MWCNT), tyrosinase, and Nafion membrane (CP/MWCNT/Tyr/Nafion) was developed for voltammetric determination of epinephrine (EP). The CP/MWCNT/Tyr/Nafion biosensor exhibited linear dynamic range from 5.0 × 10M to 5.0 × 10MEP concentration with a good correlation coefficient (R = 0.9985). The detection limit of the biosensor was calculated as 3.0 × 10MEP from the signal-to-noise ratio (S/N = 3). Reproducibility of the biosensor was also calculated from relative standard deviation as 3.8% (n = 5). Ascorbic acid (AA) and uric acid (UA) did not interfere in the quantification of epinephrine. The developed biosensor was also successfully applied for the determination of epinephrine in pharmaceutical sample. The CP/MWCNT/Tyr/Nafion biosensor has good sensitivity, selectivity, stability, easy preparation procedure, and short analysis time and can be used for the determination of EP in pharmacological samples.

Carbon nanotubes (CNTs) have gained increasing attention as electrode modifiers because of their unique structure and physical properties [21,36,37]. They have enhanced electronic properties and offer rapid kinetics for the electrochemical process. With regard to sensitivity, detection limit, and electron transfer kinetics CNT modified electrodes have some advantages over traditional carbon electrodes [31]. CNTs have been used for biosensor preparation and they have been successfully applied for the determination of biological compounds [2,21,[36][37][38][39]. Biosensor selectivity for biological compounds can be improved by using CNTs together with an enzyme for modification process of the biosensor.
In this study, tyrosinase (Tyr) was used for the preparation of the biosensor. Tyrosinase and catecholoxidase are from type 3 copper protein group. Whereas tyrosinase mediates the hydroxylation of monophenols to orthodiphenols, it also allows its subsequent oxidation to orthoquinones (monophenols activity). Tyrosinase transforms monophenols along two iterative steps: whereas the first one includes hydroxylation of monophenol into its relevant o-diphenol (hydroxylase process), the second includes oxidization to the relevant o-quinone in which the enzyme goes through oxidization process from molecular oxygen to its original form (the catecholase process). All these proteins consist of almost the same area with binuclear copper active, where its Cu I -Cu I deoxy form binds O 2 reversibly. This results in binuclear Cu II unit bound O 2 in the lateral bridge form ( -2 : 2 ) [40][41][42][43].
The possible mechanism of the developed biosensor was given in Scheme 1 [41,43].
A new biosensor was developed by modifying a carbon paste electrode using the catalytic effect of tyrosinase and the unique properties of CNTs for the determination of EP and the electrode surface was also coated with Nafion membrane to prevent the interference effects of ascorbic acid (AA) and uric acid (UA) on the biosensor response.

Apparatus.
In the experiments Metrohm Autolab Type 3, potentiostat, Nova 1.9 software, a three-electrode system: carbon paste (glass tube, 5 cm length and 4 mm diameter) as a working electrode, Ag/AgCl as a reference electrode, and a platinum wire as a counterelectrode, Gilson P100 and P1000 automatic pipettes (France), and Yellow-Line magnetic stirrer (Germany) were used. USF ELGA UHQ water purification system was used for high purity water (18 MΩ cm −1 ).

Preparation of Carbon Paste Electrode.
Modified carbon paste electrodes (CP/MWCNT/Tyr/Nafion) were prepared by mixing the appropriate ratios of graphite powder, MWCNT, and mineral oil. For this purpose, 0.69 g of graphite powder, 0.01 g of MWCNT, and 0.3 g of mineral oil were weighed and mixed on a glass plate until a homogeneous paste was observed. Then this mixture was placed into the cylindrical glass tube (i.d. ≈4 mm) and packed down firmly using a rod. Electrical contact for the electrode was established via copper wire. After that, 10 L of Tyr solution was dropped onto the carbon paste electrode surface. Finally, 5 L of Nafion solution was added onto the electrode surface and then dried for 90 min before use. Unmodified carbon paste electrodes (CP/Nafion) were also prepared by the same procedure but mixing only 0.7 g of graphite powder and 0.3 g of mineral oil.

Electrochemical Measurement.
Electrochemical measurements were performed with cyclic and differential pulse voltammetry in a voltammetric cell. Before each voltammetric measurement background currents were obtained in

Preparation of Pharmacological Sample.
A pharmaceutical adrenalin ampoule containing 8.38 mg sodium chloride, 0.1 mg sodium metabisulphite, 1 mL injection water, and 1 mg adrenalin was used in experiments. Before the voltammetric measurements, adrenalin ampoule was diluted in a voltammetric cell containing 1.0×10 −4 M EP. DPVs of pharmacological sample were recorded in the cathodic direction between +0.6 V and −0.6 V under optimum experimental conditions. EP content of the diluted adrenalin ampoule was directly analysed by DPV without using any further pretreatment. Analyses were performed with two different commercial adrenalin ampoules with the same content. with CP/MWCNT/Tyr/Nafion electrode. Peak potential slightly shifted to the cathodic direction and peak was observed at −0.33 V as 85.4 A. These results can be attributed to the electrocatalytic effect of MWCNT which has an important role in electron transfer reaction and reducing electroactive species of o-quinone after producing a reaction between the Tyr enzyme and EP on the electrode surface.

Effect of Scan
Rate. The effect of scan rate on biosensor response was investigated for 1.0×10 −4 M EP at different scan rates ( Figure 2). Cyclic voltammograms show the differences between electrode responses depending on scan rates. A linear graph with a good correlation factor ( 2 = 0.9985) was obtained for peak current versus square root of scan rate ( -V 1/2 ) in the scan rate range 20 mV/s to 200 mV/s. It was shown that the diffusion-controlled mechanism occurred for the enzymatic reaction.

Optimization of Amount of MWCNT.
The effect of the amount of MWCNT on the CP/MWCNT/Tyr/Nafion electrode response was investigated with various increasing amounts of MWCNT ranging from 0.5% (w/w) to 10% (w/w) in carbon paste by using differential pulse voltammetry towards cathodic direction (Figure 3). Increasing the amount of MWCNT caused a decrease of peak current. The large surface area of MWCNT is an advantage for the modification process preparing the biosensor but it can cause the increase of background current. Therefore, the decrease of peak current can be attributed to the increase of background current. Similar findings were also reported in previous works [44,45]. Therefore, the maximum peak current was obtained with 1% (w/w) of MWCNT amount for 1.0 × 10 −4 M of EP saturated with oxygen at pH 7.0.  Figure 4). The best correlation coefficient was obtained with 313.087 U/mL of Tyr activity as 0.9946 and this was used for further experiments.

The Effect of pH on Biosensor
Response. Due to its importance, the effect of pH on CP/MWCNT/Tyr/Nafion electrode response was also investigated by using differential pulse voltammetry. 50 mM phosphate buffer solutions were prepared at different pH values between 5.0 and 9.0 and differential pulse voltammograms were recorded for each pH value at 1.0 × 10 −4 M EP of concentration. The graph of peak current versus pH values shows that the peak current increased from pH 5.0 to pH 7.0 ( Figure 5). At pH 8.0, very low peak current was obtained and at pH 9.0 no peak was observed. Associated with increasing pH value, the biosensor displays more reaction at low pH range. This increase could be connected with the intensified tyrosinase activity parallel to the elevated pH. At pH levels above 7.0, the amperometric reaction decreases subject to the contribution of protons to the hydroxylation of phenol catalyzed by the enzyme to form o-diphenol and to the reduction of o-quinone [46]. The optimum pH range is reported as range of 5-8 for optimum  free tyrosinase [47]. In order to have the best biosensor reaction, the optimum pH values were determined as 7.0, 7.4, or 7.5 [43,46,47]. The relevant literature results support the immobilization procedure of the biosensor which does not display impact on the tyrosinase activity. Therefore, the optimum value under working experimental conditions was found to be pH 7.0. The interference effects of AA and UA were examined with a solution containing 1.0 × 10 −4 M EP and an equal concentration of interfering ion. The peak current value of biosensor for the EP solution only was accepted as 100% and biosensor response when adding the interfering ion was evaluated relatively considering this value. Peak current value was found to be 97.0% with a solution containing 1.0 × 10 −4 M EP and an equal concentration of interfering ion. The peak current value of biosensor for the EP solution only was accepted as 100% and biosensor response when adding the interfering ion was evaluated relatively considering this value. Peak current value was found to be 97.0% with a solution containing 1.0 × 10 −4 M EP and 1.0 × 10 −4 M AA. When also studied with a solution containing 1.0 × 10 −4 M EP and 1.0 × 10 −4 M UA, peak current value was found to be 96.0% ( Figure 6). These results showed that the CP/MWCNT/Tyr/Nafion biosensor can be used for voltammetric determination of EP for natural samples containing AA and UA.

Storage Stability of Biosensor.
To examine storage stability, differential pulse voltammograms of CP/MWCNT/ Tyr/Nafion biosensor for 1.0×10 −4 M EP solution at optimum experimental conditions were recorded every 2 days for a period of 15 days. The biosensor was stored in a refrigerator at +4 ∘ C when not used. Figure 7 shows the changes of storage stability of the biosensor over 15 days. The biosensor response value at first day was accepted as 100%. After 15 days, the biosensor activity remained at 70%.

Linear Range of the CP/MWCNT/Tyr/Nafion Biosensor.
To determine a linear range of the CP/MWCNT/Tyr/Nafion biosensor, differential pulse voltammograms for different concentration of EP were examined (Figure 8).  correlation coefficient ( 2 = 0.9985) (Figure 8). The detection limit of the biosensor was calculated as 3.0 × 10 −7 M EP from the signal-to-noise ratio (S/N = 3).
A comparison of the analytical performance of the biosensor with other electrodes was given in Table 1. According to Table 1, the developed biosensor shows wider linear range than some of them [3, 4, 21, 27, 30, 32-35, 48, 49, 51]. The detection limit of the developed biosensor is also better than some electrodes [27,30,32,33,35,48,49,51,6 Journal of Sensors    [52], and 2.9 × 10 −8 M for CPE/MWCNTs [28]. In the present study, obtained estimation limit value is comparable with the findings reported in the relevant literature.

Reproducibility of the CP/MWCNT/Tyr/Nafion Biosensor.
The reproducibility of the developed biosensor was also investigated. Electrode-to-electrode reproducibility was examined by preparation of five biosensors in the same conditions. These experiments were realized under optimum experimental conditions for 1.0 × 10 −4 M EP. From the data obtained the relative standard deviation (RSD%) was calculated as 3.8%.

Pharmacological Sample Analysis.
To prove the applicability of the developed biosensor to EP determination, a pharmaceutical adrenalin ampoule was used. Voltammetric analysis of diluted ampoule solution was directly performed without using any further pretreatment. Approximate plasma value of the EP was determined as 3.0 ± 3.0 ng/100 mL [6]. It is not possible to estimate the EP value in plasma by means of the introduced CP/MWCNT/Tyr/Nafion biosensor. However, it could be possible to make this estimation through standard addition method. In the available studies in the literature, pharmacological examples were usually utilized [26,28,[50][51][52]. For the plasma sample, the EP level was estimated through the standard addition method in the form of recovery [26,28,50,52].
The results show that the CP/MWCNT/Tyr/Nafion biosensor has good reproducibility for the pharmacological sample analysis (Table 2). Consequently, the proposed biosensor can be used for determining EP in a pharmacological sample with high accuracy and precision and simple operation.

Conclusions
In the present study, a carbon paste electrode modified with MWCNT, Tyr, and Nafion was used for the determination of EP. The CP/MWCNT/Tyr/Nafion biosensor was successfully applied for the voltammetric determination of EP in the presence of AA and UA and also in a pharmaceutical sample. The results show that the biosensor, prepared combining the unique electronic effect of MWCNT and catalytic effect of Tyr enzyme, has a wide linear range and low detection limit. The developed CP/MWCNT/Tyr/Nafion biosensor has the potential to be used for detection of EP because of its simple preparation technique, its cheapness, the lack of extra purification steps required, and a rapid and easy operation.