An efficient electrochemical sensor based on CeVO4-CuWO4 nanocomposite for methyldopa

A novel modified electrode based on cerium vanadate and copper tungstate (CeVO4-CuWO4) nanocomposite was prepared as a sensitive sensor for the methyldopa. The prepared nanocomposite was characterized by x-ray diffraction (XRD), energy dispersive x-ray spectroscope (EDX), Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM) methods. The cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were applied for the evaluation of the electrochemical performance of the sensor. The enhanced active surface area, electro-catalytic activity, and expedient conductivity provided by the CeVO4-CuWO4 nanocomposite led to the peak current increment with a well-resolved anodic peak for methyldopa in the presence of potential interferences. The CeVO4-CuWO4 nanocomposite-based modified electrode successfully measured methyldopa over a wide concentration range of 0.02–400 μM with the low limit of detection (LOD) of 0.006 μM. The findings of the methyldopa sensing in human serum samples verified the proper efficiency of the proposed sensor.


Introduction
As a catecholamine derivative, methyldopa (scheme 1) is an effective antihypertensive agent [1,2]. It is extensively applied to reduce moderate hypertension, especially during the pregnancy period. In adrenergic nerve endings, methyldopa targets central α-adrenoreceptors through its metabolite, α-methylnorepinephrine [3]. Methyldopa inhibits the aromatic L-amino acid decarboxylase enzyme, DOPA decarboxylase, therefore prevents the conversion of L-DOPA into dopamine that is a precursor for the epinephrine and norepinephrine synthesis.
The drop in the blood pressure can be obtained by the effect of methyldopa on α2-adrenoreceptor agonists. The methyldopa metabolism with a half-life of about 2 h occurs in the liver and intestines, while with its metabolites, it is expelled through the urine [3]. Sleepiness, liver issues, damaging red blood cells, and allergic reactions are some side effects of methyldopa [4]. This is obvious that the accurate quantification of methyldopa is very important for controlling its dosage because of stated complications [5,6].
Diverse analytical techniques have been used for the measurement of methyldopa in biological fluids and pharmaceutical products. They involve high-performance liquid chromatography [7], titrimetric analysis [8], gas chromatography [9], nuclear magnetic resonance spectroscopy [10], kinetic procedures [6], fluorometric analysis [11], spectrophotometry [12], chemiluminescence [13], reflectance spectroscopy [14], mass spectrometry [15], and so on. Nearly all these methods have limitations such as high costs, long analysis times Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. and difficult control of the conditions, complicated pretreatments, and possible interfering substances. Besides, low selectivity and sensitivity make these methods inappropriate for standard analysis. To resolve these problems, electrochemistry-based methods can be employed considering their lower cost, speediness, portability, reasonable selectivity, simple preparation process, suitable accuracy, and precision [16][17][18][19][20][21]. Several electrochemical methods have been reported for the detection of active pharmaceutical ingredients in biological fluids, which include voltammetry, potentiometric, amperometric, etc [22][23][24]. In traditional electrodes, oxidation-reduction reactions require a high overpotential because of the sluggish electron transfer process [17,[25][26][27]. Therefore, it is necessary to increase the performance of these types of electrodes. Electrode surface modification decreases the over-potentials and increases the electron transfer rate.
Nanotechnology has made tremendous changes in the modification of electrode surfaces [28]. Among the nanoparticles, nano metals and metal oxides have offered outstanding conductivity and catalytic capabilities facilitating the electron transfer kinetics, which is an important parameter in the electrochemical reactions [29].
In this work, a nanocomposite of cerium vanadate and copper tungstate was synthesized and utilized for the development of a methyldopa electrochemical sensor. The glassy carbon electrode (GCE) was employed here because among the various promising electrodes applied in the electrochemical measurement, it revealed suitable stability and resistance [25,26,[30][31][32][33][34][35]. As rare earth vanadate, cerium vanadate (CeVO 4 ) offers multiple redox features, which originate from the special electronic configuration and different oxidation states [36]. CeVO 4 has been applied as catalyst and photocatalyst [37,38], in gas sensors [39,40], in batteries [41,42], and utilized in electrochromic applications [43,44]. Concerning the recent reports, CeVO 4 is the safest material for anode of lithium-ion batteries (LIBs) due to its slight insertion potential, elevated power/energy density, and high reversible capacity [42,[45][46][47]. Copper tungstate (CuWO 4 ) is one of the most promising photo-anode for photocatalytic degradation of the organic pollutants [48], gas sensors [49], and water splitting [50] due to its narrow bandgap (2.2-2.4 eV). Moreover, CuWO 4 belongs to n-type semiconductors that create positive holes in the valence band, which move to the surface to perform many oxidation processes [51]. Recently, CuWO 4 nanocomposites are more interested in researches because of their good chemical stability, selectivity, and better current densities in addition to the smaller bandgap [52]. In this work, to further improve the limit of detection, linear concentration range, and sensitivity, an electrochemical sensor based on a nanocomposite of cerium vanadate and copper tungstate was prepared for the electrochemical measurement of methyldopa in pharmaceutical and biological samples.

Chemicals
Cerium nitrate, ammonium vanadate, copper nitrate, sodium tungstate, ascorbic acid, uric acid, methyldopa, and all chemicals were bought from Sigma-Aldrich and Merck Company. 0.1 M Phosphate buffer (PB) was applied as the electrolyte solution. 0.1 M KCl including 5 mM [Fe(CN) 6 ] 3-/4was used as the electro-active probe. All utilized chemical materials were of analytical grade.

Apparatus
The morphology of nanoparticles and nanocomposite was explored using scanning electron microscopy (SEM). An x-ray Diffraction (XRD) with a source of the Cu K α radiation was applied for the crystal structure investigation. The electrochemical tests were performed by the palm sense EM State series equipped with a three-electrode system (an Ag/AgCl electrode, a platinum wire, and a GCE as the reference, counter, and working electrodes, respectively).

Preparation of the CeVO 4 -CuWO 4
First, the cerium nitrate (1 mmol) was dissolved in bi-distilled water (30 ml) at ambient temperature. Besides, ammonium vanadate (1 mmol) was dissolved in distilled water (30 ml) for 30 min at 25°C on a magnetic stirrer at a constant rate. Subsequently, the two above-mentioned solutions were mixed under the ultrasonic waves at ambient temperature. Two separate aqueous solutions of copper nitrate (1 mmol) and sodium tungstate (1 mmol) were prepared utilizing bi-distilled water (pH=5) at ambient temperature. After that, the cerium vanadate and sodium tungstate solutions were mixed, and copper tungstate solution was then added dropwise to this prepared mixture solution under the ultrasonic wave at room temperature. The formation of the CeVO 4 -CuWO 4 heterostructures was confirmed by yellowish-brown sediment. The resulting sediment was calcination for 1 h at 25°C.

Fabrication of the CeVO 4 -CuWO 4 /GCE
A well-dispersed suspension of the synthesized CeVO 4 -CuWO 4 nano powder (1 mg mL −1 ) was prepared in deionized water. Afterward, 7 μl (the optimal amount) of the CeVO 4 -CuWO 4 suspension was placed on the GCE surface and left to dry in an oven at 50°C, which was then stored at ambient conditions before being used.

Preparation of the blood serum samples
The performance of the CeVO 4 -CuWO 4 -based modified electrode was examined on the human blood serum collected from a man (middle-aged) in the Blood Transfusion Organization of Iran (Tehran). 1 ml of that blood serum was diluted 10 times with buffer (with optimum pH). The methyldopa content of that sample was determined by the standard addition procedure.

Characterization of the CeVO 4 -CuWO 4 /GCE
The synthesized CeVO 4 , CuWO 4 , and CeVO 4 -CuWO 4 nanostructures were evaluated by FT-IR spectroscopy, which acquired spectra were shown in figure 1. FTIR spectrum of the CeVO 4 ( figure 1(a)) revealed an intensive     To analyze the morphological features of the prepared CeVO 4 , CuWO 4 , and CeVO 4 -CuWO 4 samples, scanning electron microscopy (SEM) was performed. Figure 3(a) shows an SEM image of the synthesized CeVO 4 nanocrystals. The resulting products consist of hexagonal and plate-like crystals. Besides, small rod-shaped particles with inhomogeneous size distributions can also be observed. The SEM image of the CuWO 4 powder was shown in figure 3(b). The CuWO 4 particles are mostly spherical in shape and agglomerated, and their size is about 25-32 nm. In the SEM image of the CeVO 4 -CuWO 4 nanocomposite, agglomerated spherical and hexagonal morphologies are observed ( figure 3(c)). Figure 4 demonstrates the EDS analysis of the CeVO 4 -CuWO 4 by displaying the existence of the Ce, V, O, Cu, and W elements with different weight percentages in its structure.

Electrochemical evaluations
Employing the cyclic voltammetry (CV), the sensing capability of the prepared electrodes was tested in the Fe(CN) 6 ] 3-/4solution as an active probe regarding its well-known reversible redox reaction (figure 5). As shown in figure 5, compared to the unmodified GCE, the redox response of the utilized probe was effectively improved by modified electrodes. The appearance of weak anodic and cathodic peaks with 230 mV potential separation  (ΔE) at the bare GCE indicates the slow electron transfer with a limited kinetic process. Meantime, the ΔE decreased to 203 and 200 mV at the CuWO 4 /GCE and CeVO 4 /GCE, respectively, and obvious increment in the peak current (I p ). An observable enhancement in the I p was achieved for the CeVO 4 -CuWO 4 /GCE, for which the ΔE value was decreased to 100 mV. The improved electrochemical response can be ascribed to the good   6). As evidence in figure 6, the CeVO 4 -CuWO 4 /GCE provides a better platform for the methyldopa sensing with two well-separated sharp peaks with less ΔE p value and higher peak currents than the GCE, CeVO 4 /GCE, and CuWO4/GCE. Notably, the methyldopa response at the bare GCE and CeVO 4 /GCE was irreversible and weak, but a quasi-reversible redox behavior was resulted in the case of CuWO 4 /GCE, while a well-defined totally reversible response obtained for the CeVO 4 -CuWO 4 /GCE. Again, the electrocatalytic performance with the higher effective surface area is concluded for the CeVO 4 -CuWO 4 nanocomposite toward methyldopa reaction.
The electrolyte pH affects the catalytic reaction efficiency of the CeVO 4 -CuWO 4 /GCE toward methyldopa redox reaction. Figure 7(a) shows CV responses of the 50 μM methyldopa in a PBS (2.0pH7.0). The peak current value of the methyldopa redox reaction was decreased as the solution pH was increased, and the position of the peaks was shifted to less positive potentials, indication the contribution of protons in the electron transfer steps. Since the maximum peak current was observed at pH with a more favorable peaks shape, the pH=2.0 was considered as the optimum condition in the following experiments.  Furthermore, the peak potential-pH plot ( figure 7(b)) obeyed a linear behavior whose slope was equal to −0.061 V/pH. This value is equal to the Nernstian amount (0.059) at 25°C, suggesting that the electro-catalytic process of the methyldopa on the CeVO 4 -CuWO 4 /GCE surface follows a two electrons/two protons process.
The mechanism of the methyldopa electrochemical process was studied by recording the CVs of 50 μM methyldopa (10scan rates200 mV s −1 ) using the CeVO 4 -CuWO 4 /GCE ( figure 8(a)). Figure 8(b) depicts the linear dependence of Log I pa on the Log υ with R 2 =0.999 and slope of 0.774 indicating that the oxidation of methyldopa at CeVO 4 -CuWO 4 /GCE is under control of a mixed adsorption-diffusion process. Regarding previous works [53,54], it can be mentioned that methyldopa species adsorb on the electrode surface and penetrate the GCE surface through the porous nanostructure of the CeVO 4 -CuWO 4 . The Laviron equation [29] for the surface-adsorbed electroactive compounds was utilized to estimate values of charge transfer coefficient (α) and apparent charge transfer rate constant (k s ) for the oxidation of methyldopa at CeVO 4 -CuWO 4 /GCE. The pK a values of 2.2 and 9.7 for the methyldopa showed that while the carbocyclic moiety is almost deprotonated at pH=2.0, the amine group is in protonated from. Moreover, the observation of the almost reversible behavior for the electro-oxidation of methyldopa in optimized pH value (2.0) reveals a simple onestep oxidation pathway in the absence of further Michael addition process, if it happened, it would change the reaction mechanism to the Electrochemical-Chemical-Electrochemical (ECE). As a result, the redox mechanism according to scheme 2 is proposed for methyldopa oxidation at pH=2 using CeVO 4 -CuWO 4 /GCE.
To examine the applicability of the prepared sensor in detecting methyldopa, various concentrations of the methyldopa in a range of 0.02 to 400 μM were synchronously injected into the electrochemical cell, and the analysis was performed by the differential pulse voltammetry (DPV) technique ( figure 9(a)). The equation of the linear relationship between the peak current and the methyldopa concentrations was as I pa (μA)=29.13 C (μM)+3.19 for 0.02 to 1 μM and I pa (μA)=0.16 C (μM)+31.72 for 1 to 400 μM. The estimated limit of detection (LOD) according to the (signal/noise)=3 was equal to 6 nM. Table 1 lists the reported modified electrodes in the literature used for development of methyldopa electrochemical sensors. As observed, the  reported LODs were in the range of 0.12-1000 nM. As listed in table 1, the proposed sensor's performance based on the linear working range and LOD value in the measurement of the methyldopa in comparison to other reported modified electrodes indicate a similar or even better response. But it seems utilizing the CeVO 4 -CuWO 4 /GCE as a simple and cost-effective platform is better comparing to other applied nanomaterials or electrodes. This observation shows the excellent capability of the CeVO 4 -CuWO 4 nanocomposite in the catalysis reaction owning to the electron transfer facilitation and surface area increase, which may be promising as an efficient platform in the other electrochemical fields.
The practical application of the CeVO 4 -CuWO 4 /GCE was exanimated in measurements of methyldopa in a serum sample. According to table 2, the applicable recovery values were resulted for methyldopa contents in tested samples using CeVO 4 -CuWO 4 /GCE. The obtained recoveries were in the range of 96%-98.2%. These results confirm the suitable capability of the CeVO 4 -CuWO 4 /GCE for the measurements of methyldopa in real samples.
The reproducibility of the CeVO 4 -CuWO 4 /GCE was examined by DPV measurements of ten sensors. The relative standard deviation (RSD) calculated for the DPVs responses (I pa ) was 3.1%, which indicated the good reproducibility of the CeVO 4 -CuWO 4 /GCE. Also, the analytical repeatability of the sensor was confirmed by a low RSD value (2.2%) resulted from three performed analyses utilizing one CeVO 4 -CuWO 4 /GCE. The durability of the CeVO 4 -CuWO 4 modifier on the GCE surface was examined for four weeks. The responses of DPVs during different days showed negligible variation I pa after four weeks, which reduced less than 3% relative to the first day, and the E p was remained unchanged. These findings illustrated that the CeVO 4 -CuWO 4 /GCE effectively preserved its electro-activity and indicated good repeatability, durability, and reproducibility for the CeVO 4 -CuWO 4 /GCE.
To check whether some common existing substances in urine or blood serum might affect the analysis of the methyldopa, the DPVs of the CeVO 4 -CuWO 4 /GCE were recorded in 0.1 M PBS (pH=2) containing 25 μM of methyldopa in the presence of Mg 2+ , Na + , K + , Cl − and SO 4 2− (1 mM), uric acid, L-Cysteine, H 2 O 2 , glucose, and caffeine. The results showed all of the tested substances have no sensible interference on the current and position of the methyldopa oxidation peaks, and the change of signals in the presence of the interferences was less than±5% (table 3). There is hydroquinone functional group in the structure of both methyldopa and DA which is responsible of the appeared oxidation peak in the evaluated potential range. Therefore, their oxidation peaks overlap with each other. The uric acid and ascorbic acid oxidation response was investigated using CeVO 4 -CuWO 4 /GCE. No oxidation peak was observed for AA at the evaluated potential window and the UA response did not interfere with methyldopa signal. These findings confirm the sensor applicability for the selective detection of methyldopa.

Conclusion
A sensitive sensor based on the CeVO 4 and CuWO 4 nanomaterials was developed for the detection of methyldopa. This prepared nanocomposite was used for modification of the GCE surface by a simple dropcasting method. The cost-effective sensing interface exhibited excellent conductivity, high surface area, catalytic activity, good selectivity that resulted in sensitive responses with a well-separated oxidation peak of methyldopa from the potential interferences. The CeVO 4 -CuWO 4 /GCE showed a wide linear quantification range of 0.02-400 μM, and the limit of detection of 0.006 μM (S/N=3) for methyldopa. The satisfactory findings of the sensor's applicability in analysis of the urine and blood serum samples may be an appropriate paradigm in the methyldopa measurement in clinical samples.