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
Epinephrine (EP), which is released by the adrenal glands, is an important member of the catecholamine family and it acts as a neurotransmitter and hormone in the mammalian central nervous system [
Carbon nanotubes (CNTs) have gained increasing attention as electrode modifiers because of their unique structure and physical properties [
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
The possible mechanism of the developed biosensor was given in Scheme
CP/MWCNT/Tyr/Nafion biosensor mechanism for EP determination.
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.
Graphite powder (Aldrich) (1-2 microns, synthetic), mineral oil (Sigma-Aldrich), Nafion (solution 5%) (Fluka Chemika), epinephrine hydrochloride (C9H13NO3
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).
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
Electrochemical measurements were performed with cyclic and differential pulse voltammetry in a voltammetric cell. Before each voltammetric measurement background currents were obtained in 50 mM pH 7.0 phosphate buffer solutions. Electrochemical determination of EP was established by addition of known concentration of EP solutions in voltammetric cell containing buffer solution. Solutions were stirred in voltammetric cell for 3 min before voltammetric measurement. Cyclic voltammograms (CVs) were recorded between +0.8 V and −0.8 V. Differential pulse voltammograms (DPVs) were recorded in the cathodic direction between +0.6 V and −0.6 V under optimum experimental conditions.
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
Cyclic voltammograms of unmodified and modified carbon paste electrodes for epinephrine were recorded between +0.8 V and −0.8 V (Figure
Cyclic voltammograms of the unmodified and modified biosensors: (a) CP/Nafion electrode; (b) CP/MWCNT/Nafion; and (c) CP/MWCNT/Tyr/Nafion in 50 mM phosphate buffer, pH 7.0 (
The effect of scan rate on biosensor response was investigated for
Cyclic voltammograms of the CP/MWCNT/Tyr/Nafion biosensor at different scan rates in 50 mM phosphate buffer, pH 7.0. (a) 20 mV/s; (b) 50 mV/s; (c) 100 mV/s; and (d) 200 mV/s. Inset shows the plot of
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
Differential pulse voltammograms of the CP/MWCNT/Tyr/Nafion biosensor at different MWCNT amounts in 50 mM phosphate buffer, pH 7.0. (a) 0.5% (w/w); (b) 1% (w/w); (c) 2% (w/w); (d) 4% (w/w); and (e) 10% (w/w). Inset: plot of
The optimization of Tyr enzyme activity was established with various enzyme activities for different concentration of epinephrine solutions. For this aim, 156.540 U/mL, 313.087 U/mL, and 626.174 U/mL enzyme activities were chosen. Differential pulse voltammograms of CP/MWCNT/Tyr/Nafion electrode were obtained towards cathodic direction 0.6 V to −0.6 V with 50 mV/s scan rate. Biosensor responses were recorded for
The linear graphs of peak current versus different concentrations of EP solutions for chosen enzyme activities show the enzyme activity effect on the biosensor response (Figure
Plot of
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
Plot of
Ascorbic acid (AA) and uric acid (UA) found in real samples cause an interference effect on the determination of EP with a biosensor. This effect can be prevented by coating the biosensor surface with a suitable membrane. To remove AA and UA from the biosensor surface, the CP/MWCNT/Tyr surface was coated with a Nafion membrane. At pH 7.0, the negatively charged region of Nafion due to its fluoride ions prevents positively charged ions like AA and UA from reaching the biosensor surface. The interference effects of AA and UA were examined with a solution containing
Interference effect on the CP/MWCNT/Tyr/Nafion biosensor response. Differential pulse voltammograms were recorded at (a) presence of
To examine storage stability, differential pulse voltammograms of CP/MWCNT/Tyr/Nafion biosensor for
Storage stability graphic (Tyr: 313.087 U/mL; EP:
To determine a linear range of the CP/MWCNT/Tyr/Nafion biosensor, differential pulse voltammograms for different concentration of EP were examined (Figure
Differential pulse voltammograms of the CP/MWCNT/Tyr/Nafion biosensor at different concentration of EP; (a) baseline, (b)
A comparison of the analytical performance of the biosensor with other electrodes was given in Table
Comparison of the present work with other electrodes for determination of EP.
Electrode | Linear concentration range (M) | Detection limit (M) | Technique | Ref. |
---|---|---|---|---|
Pt/ |
5.0 × 10−6–1.0 × 10−4 | 1.04 × 10−6 | CV | [ |
WGE/Ru | 3.0 × 10−6–9.0 × 10−5 | 8.0 × 10−7 | DPV | [ |
Au-Cys-SWCNT-CoTAPc | 1.22 × 10−5–1.3 × 10−4 | 6.0 × 10−6 | SWV | [ |
CPE/pMWCNTs/SDS | 1.0 × 10−7–1.0 × 10−6 and 1.0 × 10−6–1.0 × 10−4 | 4.5 × 10−8 | Amperometry | [ |
CPE/palm tree tissue | 5.0 × 10−5–5.0 × 10−4 | 1.5 × 10−5 | Amperometry | [ |
CPE/MWCNTs/vinylferrocene | 1.0 × 10−7–1.0 × 10−3 | 3.0 × 10−8 | SWV | [ |
CPE/CNTs/ionic liquid | 3.0 × 10−7–4.5 × 10−4 | 9.0 × 10−8 | DPV | [ |
CPE/MWCNTs/poly (Solid Red A) | 2.0 × 10−6–9.0 × 10−6 | 1.0 × 10−6 | CV | [ |
CPE/EBNBH/DWCNTs | 7.0 × 10−7–1.2 × 10−3 | 2.16 × 10−7 | DPV | [ |
CPE/CNs/hydroquinone | 5.0 × 10−6–2.0 × 10−5 and 2.0 × 10−5–6.0 × 10−4 | 1.0 × 10−6 | DPV | [ |
CPE/iron (III) doped zeolite | 9.0 × 10−7–2.16 × 10−4 | 4.4 × 10−7 | DPV | [ |
CPE/MWCNTs | 5.0 × 10−7–1.0 × 10−5 and 1.0 × 10−5–1.0 × 10−4 | 2.9 × 10−8 | DPV | [ |
Composite/MWCNT/CoPc | 1.33 × 10−6–5.5 × 10−6 | 1.56 × 10−8 | DPV | [ |
Pyrolytic graphite/nanodiamond graphite film | 1.0 × 10−8–1.0 × 10−5 | 3.0 × 10−9 | LSV | [ |
Pyrolytic graphite/MWCNT | 0.5 × 10−9–1.0 × 10−7 | 0.15 × 10−9 | SWV | [ |
GCE/AuNPs/TGA/CS-MWCNTs | 0.4 × 10−6–11.0 × 10−6 | 6.0 × 10−8 | DPV | [ |
GCE/ |
3.0 × 10−5–3.0 × 10−4 | 1.0 × 10−5 | DPV | [ |
GCE/SWCNTs/Tyr | 1.0 × 10−5–1.1 × 10−4 | 2.54 × 10−6 | Amperometry | [ |
GCE/AuNPs/CA | 1.0 × 10−7–5.0 × 10−4 | 4.0 × 10−8 | DPV | [ |
GCE/Ag-PGly | 5.6 × 10−7–1.0 × 10−4 | 1.0 × 10−7 | CV | [ |
GCE/polytaurine | 2.0 × 10−6–6.0 × 10−4 | 3.0 × 10−7 | DPV | [ |
GCE/MWCNT/Tyr | No response | — | Amperometry | [ |
CP/MWCNT/Tyr/Nafion | 5.0 × 10−6–5.0 × 10−4 | 3.0 × 10−7 | DPV | This work |
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
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
The results show that the CP/MWCNT/Tyr/Nafion biosensor has good reproducibility for the pharmacological sample analysis (Table
Determination of EP in adrenalin ampoule.
Sample | EP content (M) | EP found (M) |
|
---|---|---|---|
Ampoule 1 | 1.0 × 10−4 | 9.73 × 10−5 | 4.62 |
Ampoule 2 | 1.0 × 10−4 | 9.80 × 10−5 | 4.38 |
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.
The authors declare that there is no conflict of interests regarding the publication of this paper.