Preparation of a ZnO nanoparticles/multiwalled carbon nanotubes/carbon paste electrode as a sensitive tool for capecitabine determination in real samples

Abstract

The present study describes the fabrication of a sensitive electrochemical sensor for the determination of capecitabine (Cap). The zinc oxide nanoparticles/multiwalled carbon nanotubes (ZnO/MWCNT) modified carbon paste electrode (CPE) is proposed as a novel electrocatalytic system for the reduction of Cap. Scanning electron microscopy (SEM), X-ray diffraction measurements (XRD) and differential pulse voltammetry (DPV) were used to characterize the performance and microstructure of the sensor. The electrochemical reduction of Cap was investigated at the ZnO/MWCNTs/CPE by the use of DPV. The developed electrode exhibited excellent electrochemical activity towards the electrochemical reduction of Cap. The sensor shows a linear range from 0.10 μmol L⁻¹ to 100.00 μmol L⁻¹ with a detection limit of 0.03 μmol L⁻¹ at a signal to noise ratio of 3. The modified electrode has excellent analytical performance and can be successfully applied in the determination of Cap in biological systems.

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Introduction

Nanotechnology and nanoscience represent new and enabling platforms that promise to provide a broad range of novel uses and improved technologies for environmental, biological and other scientific applications.¹ Various chemically modified carbon paste electrodes have been widely used in electrochemical applications as sensitive and selective electrodes for the determination of different compounds. Among different modifiers, nanocrystalline materials have drawn a lot of attention in various areas due to their noticeable advantages such as large surface area, high thermal and chemical stability, tunable porosity and biocompatibility.^2–5 Carbon nanotubes (CNTs) are carbon materials that have a new kind of porous nanostructure, have been found to possess properties such as high surface area, high electrical conductivity, significant mechanical strength and chemical stability. Multiwalled carbon nanotubes (MWCNTs) can be used to promote electron transfer reactions when used as electrode materials in electrochemical sensors.^6–9

Zinc oxide (ZnO) nanostructured materials have been one of the most promising oxide semiconductor materials because of their high surface area, biocompatibility, nontoxicity, ease of fabrication, chemical and photochemical stability and electrochemical activity at potentials above their conduction band edge.^10–12

Capecitabine (Cap), chemically 50-deoxy-5-fluoro-N₄-pentyloxycarbonyl-cytidine (Scheme 1) is the pro-drug for the anti-metabolite 5-fluorouracil (5-FU). It is a novel oral tumor-activated and tumor-elective fluoropyrimidine carbamate and an oral chemotherapeutic agent used in the treatment of breast, esophageal and larynx, gastrointestinal and genitourinary tract cancers.^13,14 As a prodrug, Cap is converted into the active agent 5-fluorouracil (5-FU) through a three-step enzymatic process after oral drug administration.^1,2 First, hydrolysis by carboxylesterases leads to the formation of 5′-deoxy-5-fluorocytidine (DFCR). Second, cytidine deaminase catalyzes the conversion of DFCR to 5′-deoxy-5-fluorouridine (DFUR). Finally, further catabolism by thymidine phosphorylase produces 5-FU.^15,16

Scheme 1 Chemical structure of capecitabine.

Due to its bioactivity and wide potential applications, a number of analytical methods including high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS),^16–18 HPLC,^19,20 liquid chromatography-mass spectroscopy (LC-MS),²¹ and electroanalytical technique²² have been reported. Although chromatographic methods are sensitive and reliable, they have some disadvantages such as being time and labor consuming, expensive; require sample pretreatment and qualified personnel. Promising alternatives in this regard are electroanalytical methods, which can offer high sensitivity, rapid response, easy operation and low cost.

In this study, ZnO/MWCNTs modified CPE is proposed as a novel electrocatalytic system for the reduction of Cap. The morphology of nanocomposite and electrochemical behaviour of ZnO/MWCNTs/CPE was investigated by XRD, scanning electron microscopy (SEM) and differential pulse voltammetry. The prepared nanocomposite was successfully applied as an excellent catalyst for Cap reduction at lower overpotentials. The ZnO/MWCNTs/CPE composite exhibits the synergistic effects, with large specific surface area, high conductivity and significantly enhanced electrocatalytic performance. The analytical performance of the proposed system was evaluated, and the optimal condition was used to determine the Cap in the biological matrix.

Experimental

Chemicals

Capecitabine, Zn(NO₃)₂·4H₂O, HNO₃, H₂SO₄, NaOH, ascorbic acid, and uric acid were of supplied by Merck Company (Darmstadt, Germany) or Aldrich Company (USA) and used with no further purification. All other chemical reagents used were of analytical grade. In all the measurements, the supporting electrolyte used was Briton–Robinson buffer solution (BRBS) of pH 2.0. The multiwalled carbon nanotubes (MWCNTs) (95% purity, 10–20 nm diameters, 30 μm length) were obtained from Neutrino Company (Iran). MWCNTs were chemically functionalized (MWCNTs) by ultrasonication in a mixture of sulfuric acid and nitric acid (3 : 1 v/v) for 6 h. MWCNTs were washed with distilled deionized water (DDW) and then were separated by centrifugation. High viscosity paraffin (d = 0.867 kg L⁻¹) from Merck was used as the pasting liquid for the preparation of the carbon paste electrodes.

Apparatus

Voltammetric systems were conducted using the Metrohm model 797 VA Computrac polarograph. Three-electrode cell systems were used to monitor the cyclic and differential pulse voltammograms. An Ag/AgCl electrode, a platinum wire and a modified CPE were used as the reference, auxiliary and working electrodes, respectively. All the values of pHs were adjusted by a Metrohm Model 713 pH lab (Herisau, Switzerland). The size and morphology of the nanoparticles were characterized by scanning electron microscope (SEM VEGA3 TESCAN). All experiments were carried out at room temperature (25 ± 2 °C).

Synthesis of ZnONPs

For the preparation of ZnONPs, in a typical experiment, a 0.25 mol L⁻¹ aqueous solution of zinc nitrate (Zn (NO₃)₂·4H₂O) and a 0.5 mol L⁻¹ aqueous solution of sodium hydroxide (NaOH) was prepared in distilled water. Then, the beaker containing NaOH solution was heated at the temperature of about 55 °C. The Zn(NO₃)₂ solutions were added drop wise (slowly for 1.5 h) to the above-heated solution under high-speed stirring. The beaker was sealed at this condition for 2 h. The precipitated ZnONPs were cleaned with deionized water and ethanol and then calcined at 200 °C for 2 h.^23,24

Preparation of the modified electrode

The ZnONPs/MWCNTs/CPE was prepared by mixing of 2.5% (w/w) ratio of ZnONPs, 10% (w/w) MWCNTs, 62.5% (w/w) graphite powder, 25% (w/w) paraffin oil. The ZnO/CPE was prepared by mixing of 2.5% (w/w) ratio of ZnO, 72.5% (w/w) graphite powder and 25% (w/w) paraffin oil. The MWCNTs/CPE was prepared by mixing of 10% (w/w) ratio of MWCNTs, 65% (w/w) graphite powder and 25% (w/w) paraffin. The bare CPE was prepared by mixing of 75/25% (w/w) ratio of graphite powder and paraffin oil. The paste was then packed into a plastic syringe tubes with the inner diameter of 3 mm. Electrical contact was made by pushing a copper wire down the glass tube into the back of the mixture. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing it on a weighing paper.

Real sample preparation

Healthy blood serum samples were obtained from the laboratory of Mehr (Saveh-Iran) and were stored frozen before use. In order to obtain the proper sample, acetonitrile (2 mL) was added to 2 mL of serum sample for protein separation.⁸ After vortexing of the serum sample for 5 min, the precipitated proteins were separated by centrifugation (10 min at 3000 rpm). Supernatant was taken carefully and appropriate volumes of this supernatant were transferred into the 25 mL flask and diluted up to the volume with the BRBS (pH 2.0). For analysis of Cap tablets, five tablets (Cap) were weighed and crushed in a mortar to a fine powder. A mass of powder equivalent to the average mass of one tablet was dissolved in 25 mL of BRBS (pH 2.0). The content of the flask was sonicated for 5 min to affect complete dissolution. Finally the solutions were filtered and appropriate dilutions were made and the resulting solutions were transferred into the electrochemical cell.

General procedure

The required volume of Cap standard solution was added into 25 mL volumetric flask and diluted up to the volume with the BRBS (pH 2.0). This solution was added to electrochemical cell. After that, the modified electrode was placed in to the test solution and then the differential pulse voltammetry (DPV) were performed. DPV was performed in the same potential range with pulse amplitude of 100.0 mV, voltage step of 7.0 mV, pulse time of 0.01 s and voltage step time 0.1 s.

Results and discussion

Morphological and structural characterization of the electrode materials

Fig. 1A shows the TEM images of the synthesized ZnONPs. Fig. 1B exhibits the SEM image of ZnO/MWCNTs nanocomposite. As can be seen, the nanoparticle size is almost 45 nm. It can be clearly observed that the MWCNTs are decorated with ZnONPs. The results reveal that ZnONPs distribute uniformly between MWCNTs. Furthermore, the prepared ZnONPs were characterized by X-ray powder diffraction (XRD). Fig. 1C shows the diffraction peak located at the 2θ value of 31.6°, 34.3°, 36.1°, 47.4°, 46.4°, 56.4°, 62.7°, 66.2°, 67.8°, 68.8°, 72.3° and 76.7° correspond to the (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) planes of the ZnO. The average crystallite size of ZnONPs, evaluated by the Scherrer formula,²⁵ was about 21.5 nm applied to the major peak (2θ = 36.1°).

Fig. 1 (A) TEM image of ZnONPs (B) SEM image of ZnO/MWCNTs and (C) XRD patterns of ZnONPs.

Electrochemical behaviors of Cap at different electrodes surface

The DPVs of 50 μmol L⁻¹ Cap at CPE, ZnO/CPE, MWCNTs/CPE and ZnO/MWCNTs/CPE are presented in Fig. 2. As Fig. 2 shows, a well-defined reduction peak at −0.770 V was observed for Cap at ZnO/MWCNTs/CPE with a current of 100.8 μA. Under the same conditions, the reduction potential of Cap are −0.826 V, −0.812 V and −0.777 V for CPE, MWCNTs/CPE and ZnO/CPE, respectively. A substantial positive shift of the reduction potential for Cap indicated the catalytic ability of ZnO/MWCNTs/CPE to Cap reduction. The suitable electronic properties of MWCNTs together with the ZnONPs gave the ability to promote charge transfer reactions. The peak currents of Cap are approximately 37.2 μA, 64.7 μA and 67.6 μA for CPE, MWCNTs/CPE and ZnO/CPE, respectively. The cathodic current obtained at ZnO/MWCNTs/CPE for the reduction of Cap was observed to be significantly higher than those obtained with CPE, ZnO/CPE and MWCNTs/CPE. Therefore, the addition of ZnONPs and MWCNTs to CPE structure caused a 2.7 fold increase in voltammetric response. In addition, the reduction peak potential was observed in the less negative side (i.e. −0.826 to −0.777 V) doping with the ZnONPs and MWCNTs. The nanocomposite structure formed by the combination of ZnONPs and MWCNT with an excellent conductivity coupled with this effect increased the activity and sensitivity of the newly prepared modified electrode (ZnO/MWCNTs/CPE) towards the Cap.

Fig. 2 DPVs of different electrodes in 50.0 μmol L⁻¹ Cap (BRBS pH 2.0).

Calculation of heterogeneous electron transfer rate constant (k₀) for the different electrodes

The effective heterogeneous electron transfer rate constant (k₀) was determined utilising a method developed by Nicholson,²⁶ using the following equation, ψ = k₀[πDnνF/(RT)]^−1/2 where ψ is a kinetic parameter, D is the diffusion coefficient, n is the number of electrons involved in the process, ν is the scan rate, F is the Faraday constant, R the gas constant and T the temperature. The kinetic parameter, ψ, is tabulated as a function of ΔE_P at a set temperature (298 K) for a one-step, one electron process.^27,28 The function of ψ(ΔE_P), which fits Nicholson's data, for practical usage is given by: ψ = (−0.6288 + 0.0021X)/(1 − 0.017X) where X = ΔE_P is used to determine ψ as a function of ΔE_P from the experimentally recorded voltammetry. From this, a plot of ψ against [πDnF/(RT)]^−1/2ν^−1/2 (in the range of 10–200 mV s⁻¹) allows the k₀ to be readily determined.²⁹ The diffusion coefficient for [Fe(CN)₆]^3−/4− equal to 7.6 × 10⁻⁶ cm² s⁻¹ in 0.1 mol L⁻¹ KCl at 25 °C. ψ versus [πDnF/(RT)]^−1/2ν^−1/2 was plotting. The slope of the straight line is the kinetic parameter k₀ which resulted equal to 4.5 × 10⁻⁴, 2.3 × 10⁻³, 9.6 × 10⁻³ and 12.3 × 10⁻³ cm s⁻¹ for CPE, ZnO/CPE, MWCNTs/CPE and ZnO/MWCNTs/CPE, respectively. These results indicate that combination of ZnONPs and MWCNTs in the CPE improved heterogeneous electron transfer properties.

Influence of pH

The effect of pH values on peak current (I) and the reduction peak potential (E_pc) of 50.0 μmol L⁻¹ Cap were investigated using differential pulse voltammetry. The voltammetric response of Cap at the ZnO/MWCNTs/CPE was examined at different pH value which was controlled at 2.0, 2.5, 3.0, 4.0 and 5.0, respectively. The influence of pH value on the peak current was illustrated in Fig. 3A. As pH value increasing from 2.0 to 5.0, the reduction peak current decreased gradually. Therefore, the BRBS of pH 2.0 was selected as the electrolyte in the following experiments.

Fig. 3 Influence of pH on the cathodic peak current (A) and cathodic peak potential (B) of 50.0 μmol L⁻¹ Cap in the range of 2.0–5.0 (C) probable reaction mechanism for reduction of Cap.

As far as peak potential was concerned, a positive shift was observed for the reduction potential of Cap at the ZnO/MWCNTs/CPE with increasing pH value from 2.0 to 5.0 (Fig. 3B). The fact confirms that protons take part in the electrochemical reactions. The linear regression equation between E_pc and pH can be expressed as the slopes of fitted lines for Cap (68.0 mV) is close to the theoretical value, −59 mV per pH unit, which suggests that the electrode reaction of Cap involving an equal number of electrons and protons based on the Nernst equation.³⁰ This conclusion is in accordance with the mechanism of Cap electrochemical reactions as shown in Fig. 3C which is in agreement with previous report.²²

Effect of scan rate on the reduction of Cap at ZnO/MWCNTs/CPE

The effect of scan rate on the response of 50 μmol L⁻¹ Cap was also investigated by CV in the potential range from −0.02 to −0.1 V. From Fig. 4A, it can be observed that the reduction peak potential shifts negatively with the increase of scan rate. As shown in Fig. 4B, the reduction peak currents I_pc of Cap increase linearly with square root of scan rate. The regression equation of Cap was I_pc (μA) = −55.033ν^1/2 − 0.9378 (V^1/2 s^−1/2, R² = 0.9931). The results demonstrate that the electrochemical process is a typical diffusion-controlled process.

Fig. 4 (A) CVs of 50.0 μmol L⁻¹ Cap on the ZnO/MWCNTs/CPE at different scan rates (20 mV s⁻¹ to 100 mV s⁻¹) in BRBS (pH 2.0). (B) The plot of the peak current versus square root of scan rate. (C) The plot of the log I_p versus log ν.

Moreover, a plot of the log I_p versus log ν is linear, with a slope of 0.5 for a diffusion peak and a slope of 1 for an adsorption peak.³¹ In this work I_p versus log ν was plotted and the equation was found: log I_p = 0.5208 log ν + 1.7305 (R² = 0.9963).

The slope of 0.52 suggesting a diffusion-controlled system.

Calibration curve and detection limit

The optimized procedure was successfully applied for determination of Cap at ZnO/MWCNTs/CPE. Fig. 5A shows the DPVs of Cap with different concentration. As revealed, one distinct reduction peak could be observed on each DPV, and the current increased with the extending concentration from 0.1 μmol L⁻¹ to 100.0 μmol L⁻¹. The dependence of peak current versus the concentration of Cap was shown in Fig. 5B, and the peak current (I, μA) is proportional to its corresponding concentration (C, μmol L⁻¹) over this range. The calibration curve can be expressed as: I (μA) = 2.615 + 4.092C (μmol L⁻¹) and I (μA) = 41.133 + 0.207C (μmol L⁻¹). The LOD was calculated to be 0.03 μmol L⁻¹ under 3S_b/m. Here m is the slope of calibration curve and S_b represents the standard deviation of blank observations.^32,33

Fig. 5 (A) DPVs for 0.1 (1), 0.5 (2), 1.0 (3), 5.0 (4), 10.0 (5), 50.0 (6), and 100.0 (7) μmol L⁻¹ Cap at ZnO/MWCNTs/CPE in BRBS (pH 2.0). (B) The plots of the peak current as a function of Cap concentration in the range of 0.1–100.0 μmol L⁻¹.

Reproducibility and stability

The reproducibility of ZnO/MWCNTs/CPE toward Cap detection was also investigated and the standard deviation in the reduction current was about 2.37% for six successive measurements at various concentrations of Cap. Moreover, the stability of the modified electrode was studied during its storage in air at room temperature. ZnO/MWCNTs/CPE has reserved 95% of its initial activity for more than 30 days. These results indicate that the modified electrode has high reproducibility and stability.

Interference effect

In order to appraise the selectivity of the method, influence of several interfering species was judged upon the determination of Cap. The tolerance limit for each interferer was regarded as the maximum concentration which gives a relative error of less than ±5.0% in regard to 50.0 μmol L⁻¹ Cap solution. The common ions such as Na⁺, NH₄⁺, Ca²⁺, K⁺, NO₃⁻, NO₂⁻ and Cl⁻ did not show interference with Cap detection. As for the common interference in biological samples for the determination of Cap, different concentration of glucose, sucrose, fructose, citric acid, uric acid and cysteine had no effective interference with the current response of Cap. The results are given in Table 1, suggesting that this proposed method has excellent selectivity towards determination of Cap in pharmaceutical and biological samples.

Table 1 Interference of some foreign substances for 50.0 μmol L⁻¹ Cap

Interferents	Tolerance level ratio
Na^+, NH4^+, Ca^2+, K^+, NO3^−, NO2^−, Cl^−	1000
Glucose, fructose, lactose, sucrose	1000
Ascorbic acid, citric acid	500
Cysteine	150
Uric acid	100

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Real sample analysis

The proposed sensor was further tested for its practical performance concerning Cap detection in the real sample of serum. Recovery tests were performed by spiking the standard Cap solution into the diluted real samples. The Cap recovery in the spiked serum sample was in the range of 97.6–101.4% which demonstrates that the method is appropriate for analysis of Cap in triplicate analysis. The summarized results for the analysis are given in Table 2.

Table 2 Determination results of Cap in serum sample (n = 5)^a

Sample	Added (μmol L^−1)	Found (μmol L^−1)	Recovery%	RSD%
a ND: not detected.
Serum	—	ND	—	—
	5.0	5.05	101.0	3.2
	50.0	49.3	98.6	2.8

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Conclusions

In this paper, we propose an electrochemical sensor for highly sensitive and trace determination of Cap by ZnO/MWCNTs/CPE. The sensor has great capability in catalyzing the reduction of Cap and can be used as an electrochemical sensor for the determination of Cap in real samples. Incorporation of ZnONPs and MWCNTs improved the electrocatalytic property of modified electrode towards Cap. The calibration curve was obtained by DPV in a linear range of 0.10–100.00 μmol L⁻¹ of Cap with a limit of detection of 0.03 μmol L⁻¹. The simple preparation procedure of the modified electrodes, a wide linear concentration range of the Cap, low detection limit, good reproducibility and high stability for the Cap suggests that the modified electrodes are good candidates for practical applications.

Acknowledgements

The authors acknowledge the Bu-Ali Sina University Research Council and Center of Excellence in Development of Environmentally Friendly Methods for Chemical Synthesis (CEDEFMCS) for providing support to this work.

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