Introduction

The three major naturally occurring forms of estrogen in women are estrone (E1), estradiol or 17β-estradiol (E2), and estriol (E3). E2 is the hormone responsible for the development and maintenance of the female reproductive system, and it also plays a role in the male reproduction system [1]. Furthermore, E2 is an endocrine-disrupting compound excreted by humans and domestic animals and it has high estrogenic activity. Endocrine-disrupting compounds can disturb the hormonal balance of an organism, and they have severe adverse effects on the health of humans and human fetuses, as well as on other animals, even in low concentration [2,3,4]. Researches have proved that low concentrations of E2 can cause the disequilibrium of humoral and cellular immunity, causing various pathological changes in the cardiovascular, immunological, reproductive, and nervous systems [5, 6]. Due to its use in hormonal contraceptives, hormone replacement therapy, and illegal animal growth promotion, the presence of E2 in the environment has been reported in recent years [7]. Thus, the development of simple, sensitive, reliable, and cost-effective methods for the determination of E2 levels in different types of samples has received considerable attention.

Methods previously described for the determination of E2 include gas chromatography [8], high-performance liquid chromatography [9], colorimetric [10], capillary electrophoresis [11], spectrophotometry-UV [12], and enzyme-linked immunosorbent assay [5]. Although these techniques are highly reliable and sensitive, the methods involve long analysis times, complex sample preparation procedures, and high costs. Electrochemical methods are useful alternatives with important advantages including fast response, low cost, ease of miniaturization, simplicity, reliability, and good sensitivity and selectivity [13,14,15].

Electrode modification is of great importance in electrochemistry as it enhances the sensitivity and selectivity toward a specific analyte in solution. The use of transition metal oxides as electrode modifiers has gained popularity in recent years due to their excellent electrocatalytic properties [16,17,18]. Among the numerous transition metal oxides, copper(II) oxide (CuO) is a promising material for electrode modification due to its high electrochemical activity, low cost, nontoxic characteristic, and easy preparation on the nanometric or microscopic scale [19, 20]. Recently, Beitollahi and coworkers [21] reported the use ZnO-CuO nanoplates to construct a modified carbon paste electrode (CPE) for the determination of 6-thioguanine in tablet and urine samples. The peak current of 6-thioguanine increased linearly in the concentration range of 50.0 to 200.000.0 nmol L−1. The detection limit was 25.0 nmol L−1. Furthermore, Soomro and coworkers [22] reported an electrochemical sensor based on a GCE modified with CuO nanostructures for the detection of malathion, an organophosphate pesticide. Under optimized conditions, the modified electrode showed a linear response for concentrations ranging from 1.0 to 12.0 nmol L−1 with a detection limit of 0.1 nmol L−1. In another approach, Saleh and coworkers [23] fabricated a pencil graphite electrode modified with copper microparticles for the determination of the antiviral drug valacyclovir. The calibration curve was linear from 2.0 to 10.0 nmol L−1 with a detection limit of 0.178 nmol L−1. The modified electrode was applied in the determination of valacyclovir in tablets. CuO shuttle-like nanocrystals on a polyneutral red glassy carbon electrode were constructed for the detection of guanine and adenine. Under optimized experimental conditions, the linear relationship between peak current and concentration was obtained in the range from 50.0 to 3000.0 nmol L−1 for guanine and adenine. A detection limit of 16.0 nmol L−1 was obtained for guanine and 23.0 nmol L−1 for adenine [24].

Moreover, the application of CuO as a component of electrochemical devices has been demonstrated in the detection of different compounds, such as hydrogen peroxide [25], glucose [26,27,28,29,30,31,32], DNA [33], and amino acids [34]. However, to the best of our knowledge, no studies on the development of electrochemical detectors for E2 using CuO have been previously reported.

Many approaches to preparing CuO have been described including thermal oxidation [35], hydrothermal [36], microwave [37], wet chemistry [38], electrodeposition [39], combustion [20], and controlled deposition [40] methods. Pechini’s method [41] can also be applied in the synthesis of CuO. This procedure is advantageous because of its low cost, high yield, and simple operation, making it appropriate for large-scale synthesis.

In this context, the objective of this study was to describe the preparation and application of a simple, easy-to-prepare, and low-cost CuO-modified carbon paste electrode as a detector for E2 with good sensitivity. The electrochemical behavior of E2 was investigated by cyclic and square-wave voltammetries at the proposed modified electrode. The influence of the CuO percentage in the CPE, scan rate, solution pH, chemical composition of the supporting electrolyte, and the square-wave voltammetry (SWV) parameters on the electrochemical oxidation of E2 was investigated in order to optimize the proposed modified electrode. The CuO/CPE obtained was successfully applied for the determination of E2 in urine and buttermilk samples.

Experimental

Reagents, solutions, and samples

All chemicals used in the experimental study were of analytical grade and obtained from Sigma-Aldrich. They were employed without any further purification. Two solutions were tested as the supporting electrolyte: 0.1 mol L−1 Britton-Robinson (B-R buffer) (H3BO3/CH3COOH/H3PO4) and H3BO3·KCl/NaOH. The pH was adjusted with 0.5 mol L−1 HCl or NaOH solutions. All aqueous solutions were prepared with ultrapure water, produced in a TKA GenPure UV system (USA). All experiments were carried out at room temperature. A stock solution of 1.0 mmol L−1 E2 was prepared by dissolving the appropriate amount of the compound in ethanol. Less concentrated solutions were prepared by dilution. For the E2 determination and the recovery experiments, the standard addition method was used, with a standard solution of E2 being added to the samples. The urine sample was obtained from a healthy volunteer, and the buttermilk sample was acquired in the region of Laranjeiras do Sul, Paraná, Brazil.

Synthesis of the CuO

CuO was synthesized by the Pechini method: citric acid (0.78 g) was dissolved in deionized water (20 mL) under magnetic stirring at 90 °C until complete homogenization (solution 1). Copper(II) nitrate (1.55 g) and ethylene glycol (1.07 mL) were added to solution 1 and continuously stirred for 3 h at 90 °C to form a blue-colored gel. Lastly, the gel was calcined in a muffle furnace at 700 °C for 1 h. The resulting CuO solid was macerated in an agate mortar with a pestle.

Apparatus

XRD analysis was performed on a Bruker D2 Phaser diffractometer equipped with a LynxEye detector and copper (λKα = 1.54 Å) radiation source. FTIR spectra were collected on a Nicolet FTIR spectrophotometer, model FTIR-200, using KBr pellets. Raman spectra were registered using a WITec 300R Alpha confocal Raman microscope equipped with 523 nm laser excitation and a ×20 objective. Raman spectra were obtained at the Institute of Chemistry of the University of São Paulo, SP, Brazil.

The surface morphologies were evaluated by SEM and EDS using a TESCAN electron microscope (model VEGA3 LMU) with a secondary electron detector.

Cyclic voltammetry (CV) and SWV were performed using a potentiostat/galvanostat, model μ-autolab Type III (Metrohm, The Netherlands) interfaced to a personal computer with Nova software (version 1.10) for the acquisition and processing of data. The electrode system consisted of the CPE (unmodified or modified) as the working electrode, an Ag/AgCl (saturated KCl) as reference electrode, and a Pt wire as auxiliary electrode. A conventional glass cell encompassed the three electrodes and the solutions used for the experiments.

Preparation of the CuO/CPE detector

The unmodified CPE used for comparison was prepared by hand mixing of 75% graphite powder and 25% Nujol oil. The mixture was ground thoroughly in an agate mortar with a pestle for 20 min, and a homogeneous paste was obtained. The CuO/CPE detector was prepared by mixing CuO with graphite and a suitable amount of Nujol oil. The amount of CuO was varied from 10 to 40%, and the best results were obtained with 25% of CuO in the paste. Both unmodified and modified pastes were placed firmly into a plastic syringe with a copper wire as the conductor. To ensure an active and fresh surface, the CuO/CPE detector was manually abraded gently on a sheet of paper between experiments.

Results and discussion

Characterization of the as-prepared CuO

The XRD pattern for CuO can be observed in Fig. 1a. The characteristic peaks of CuO at 2θ = 35.46○, 38.70○, and 48.78○ correspond to the Miller indices (−111), (111), and (−202), respectively (PDF No. 01-073-6023). The CuO phase is monoclinic; the space group C2/c and the lattice constants are a = 4.683 Å, b = 3.422 Å, and c = 5.128 Å, which are consistent with data previously published in the literature [40]. The FTIR spectrum for the synthesized CuO is shown in Fig. 1b. The bands at 418, 458, 485, 518, 578, and 668 cm−1 are attributed to the framework vibration of the metal oxide (Cu-O stretching) [42]. Figure 1c shows the Raman spectrum for the CuO, which is characterized by bands at 292, 338, and 620 cm−1, corresponding to the A g and B g modes of CuO, respectively [43]. The bands at 133 and 216 cm−1 suggest the presence of a small amount of Cu2O [44].

Fig. 1
figure 1

a XRD pattern, b FTIR spectrum, and c Raman spectrum of the as-prepared CuO

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The morphology of the CuO distribution was examined by SEM (Fig. 2). The micrograph reveals uniformity in the particle size distribution and shape and a flake-like morphology, in agreement with data obtained by other authors [45]. The chemical composition of the CuO obtained was determined using EDS, which showed the presence of copper, oxygen, and aluminum (from the SEM grid). This set of analyses demonstrated that the synthesis of CuO was successfully carried out.

Fig. 2
figure 2

EDS spectrum and SEM images of the as-prepared CuO

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Electrochemical response to E2 at the CuO/CPE detector determined by cyclic voltammetry

The electrochemical oxidation of 33.33 μmol L−1 E2 in 0.1 mol L−1 B-R buffer (pH 8.0) was studied by CV using the bare CPE and CuO/CPE detectors (Fig. 3). Cyclic voltammograms were obtained in the potential window range of 0.0 to +0.8 V with a scan rate (v) of 100 mV s−1. As expected, no peaks were observed in the absence of E2 for the CuO/CPE detector (curve a). In contrast, in the presence of 33.33 μmol L−1 E2, an oxidation peak appeared at +0.581 V with a current of 0.427 μA for the CPE (curve b), while for the CuO/CPE detector (curve c), an oxidation peak appeared at a less positive potential (+0.523 V) and the current was 1.13 μA. These data clearly demonstrate that CuO increased the catalytic activity of the CPE regarding E2 oxidation. Such behavior could be explained taking into consideration some theoretical aspects of thermodynamic and kinetic electrochemistry, as follows: the diffusion of E2 to the electrode surface and of the oxidation product from the electrode surface can be the reaction rate-determining step, as predicted in heterogeneous catalysis. Kinetically, the reaction rate-determining step depends on the number of contacts of reagents (E2) with the electrode surface. Hence, it is conceivable that the presence of CuO at the CPE increased the surface area, which allowed the increase of frequency of contact of E2 molecules with the electrode surface promoted by an increase of E2 diffusion to the proposed detector, increasing the anodic current and shifting the oxidation potential to less negative values.

Fig. 3
figure 3

Cyclic voltammograms in a 0.1 mol L−1 B-R buffer (pH 8.0) and in the presence of b, c 33.33 μmol L−1 E2 at the a, c CuO/CPE detector and b bare CPE, v = 100 mV s−1

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Effect of the CuO percentage

The content of CuO in the CPE significantly affects the performance of the CuO/CPE detector. To optimize the detector performance in order to obtain the best response for E2 oxidation, the effect of the CuO percentage on the current response was evaluated for percentages of 10 to 40% (w/w) (Fig. 4). It was observed that the current obtained by CV for the oxidation of 33.33 μmol L−1 E2 in 0.1 mol L−1 B-R buffer (pH 8.0) at the CuO/CPE detector reached the maximum value with 25% CuO in the paste, with a good voltammetric peak resolution. Above this value, the response was poor and the reproducibility of the voltammograms obtained was low. Thus, the percentage of CuO chosen for the construction of the CuO/CPE detector was 25%.

Fig. 4
figure 4

Cyclic voltammograms for 33.33 μmol L−1 E2 in 0.1 mol L−1 B-R buffer (pH 8.0) using the CuO/CPE detector with the following percentages of CuO in the paste: a 10%, b 20%, c 25%, d 30%, and e 40%, v = 100 mV s−1

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Effect of pH and chemical composition of the supporting electrolyte

Figure 5a shows the cyclic voltammograms for 66.66 μmol L−1 E2 in 0.1 mol L−1 B-R buffer at different pH values using the CuO/CPE detector. It can be observed that the E2 was electrochemically active at the proposed detector at pH 8.0 to 12.0. The anodic peak current—i pa, as shown in Fig. 5b, increased from pH 8.0 to pH 9.0 and then decreased up to pH 12.0. It can be clearly observed that the highest anodic current was obtained at pH 9.0, which was chosen as the optimum pH for subsequent experiments. Figure 5c shows that the solution pH has a significant influence on the oxidation peak potential—E po—of E2. It is clearly seen that E po shifted to less positive potentials with an increase in the solution pH. The analysis of the dependence of E po on the solution pH provided information on the E2 electrooxidation mechanism. The linear regression equation can be expressed as E po/V = (1.01 ± 0.02) − (0.061 ± 0.002) pH. The slope of the straight line obtained was – 61 mV pH−1 . According to the Nernst equation, for pH-dependent reversible reactions, the slope of E p × pH plot is −59 mV pH−1, when an equal number of protons and electrons is involved in the oxidation reaction. Although the electrochemical oxidation reaction of E2 is irreversible, as shown above, the slope of the graph E po × pH suggests that the reaction path may be like that of a reversible reaction. This finding has previously been also reported by other authors [46, 47].

Fig. 5
figure 5

a Cyclic voltammograms for 66.66 μmol L−1 E2 in 0.1 mol L−1 B-R buffer at the CuO/CPE detector, v = 100 mV s−1. Influence of the supporting electrolyte pH on b i pa and c E po

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In the next step, the influence of different supporting electrolytes at pH 9.0 on the electrochemical response to E2 was evaluated (Fig. 6). The buffer solutions tested were H3BO3·KCl/NaOH (curve a) and B-R buffer (curve b). As demonstrated, the highest peak current was obtained in B-R buffer. Therefore, this buffered solution was chosen for further experiments.

Fig. 6
figure 6

Cyclic voltammograms for 66.66 μmol L−1 E2 in a H3BO3·KCl/NaOH and b B-R buffers at pH 9.0 using the CuO/CPE detector, v = 100 mV s−1

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Effect of scan rate

The effect of v on the E2 oxidation at the CuO/CPE detector was investigated by CV in B-R buffer (pH 9.0) (Fig. 7). The corresponding voltammograms are shown in Fig. 7a, where it can be seen that the E po shifted slightly toward more positive values with an increase in v in the range of 20 to 250 mV s−1. There is a linear relation between E po and ln v according to the equation E po/V = (0.247 ± 0.013) + (0.048 ± 0.003) ln v, as shown in Fig. 7b. Based on the slope of this plot (0.048), it was possible to determine the number of electrons (n) involved in the oxidation of E2 at the proposed modified electrode. According to the Laviron theory [48], 2.303RT/(αnF) = slope of plot E po versus ln v for a totally irreversible electrode reaction process, where R is the universal gas constant (8.314 J mol−1 K−1), T is the temperature (298 K), α is the transfer coefficient, n is the number of electrons transferred, and F is the Faraday constant (96.480 C mol−1). In agreement with reference [49], α = 0.5 can be assumed for many reactions. Hence, the number of electrons calculated was around 1.0, signifying that one electron is involved in the E2 oxidation mechanism. For the same reaction, n = 1 has also been reported by other authors [15, 50]. In parallel, i pa increased with an increase in v according to the equation: log i pa/μA = −(1.24 ± 0.14) + (0.77 ± 0.07) log v (Fig. 7c). The slope of 0.768 signifies that the E2 oxidation reaction on the CuO/CPE detector is controlled by a mixture of diffusion and adsorption.

Fig. 7
figure 7

a Cyclic voltammograms for 66.66 μmol L−1 E2 in 0.1 mol L−1 B-R buffer (pH 9.0) at the CuO/CPE detector, v = a 20 mV s−1, b 40 mV s−1, c 60 mV s−1, d 80 mV s−1, e 100 mV s−1, f 150 mV s−1, g 200 mV s−1, and h 250 mV s−1. The relationship between b E po and ln v, and between c log i pa and log v

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Moraes and coworkers [15] propose that the electrochemical oxidation of E2 is an irreversible process in which one proton and one electron participate, giving the corresponding ketone derivative as product. According with our findings discussed above, the electrochemical oxidation of E2 at the CuO/CPE detector seems to follow the same mechanism pathway.

Electrochemical detection of E2 by square-wave voltammetry

Square-wave voltammograms were recorded at between 0.0 and +0.8 V for E2 in 0.1 mol L−1 B-R buffer (pH 9.0) at the CuO/CPE detector. Firstly, the square-wave parameters (frequency, amplitude, and scan increment) were optimized for E2 oxidation. The ranges studied were 10.0 to 100.0 Hz for frequency (f), 10.0 to 100.0 mV for amplitude (a), and 1.0 to 10.0 mV for scan increment (ΔEs). The values selected were 50.0 Hz, 50.0 mV, and 1.0 mV for frequency, amplitude, and scan increment, respectively, because they provided the best compromise between the voltammetric profile and sensitivity.

Under the optimized conditions previously established, the influence of the E2 concentration on the SWV curves was studied using the proposed CuO/CPE detector. The results obtained are shown in Fig. 8a. Based on the calibration curve shown in Fig. 8b, the linear range was between 60.0 and 800.0 nmol L−1, and the corresponding regression equation is i pa/μA = (3.41 ± 0.05)[estradiol] − (0.04 ± 0.02) (R = 0.998). The limit of detection (LOD) and limit of quantification (LOQ) were calculated from the calibration curve as follows: LOD = 3.3(SD/b) and LOQ = 10(SD/b), where SD is the standard deviation of the y-coordinate obtained from the line of best fit (linear coefficient), and b is the slope (angular coefficient) of this straight line. The calculated LOD and LOQ values were 21.0 and 64.0 nmol L−1, respectively. Other figures of merit obtained using the CuO/CPE for E2 detection are shown in Table 1.

Fig. 8
figure 8

a Square-wave voltammograms for a blank, b 0.06 μmol L−1, c 0.10 μmol L−1, d 0.13 μmol L−1, e 0.2 μmol L−1, f 0.3 μmol L−1, g 0.4 μmol L−1, h 0.5 μmol L−1, i 0.6 μmol L−1, j 0.7 μmol L−1, and k 0.8 μmol L−1 E2 in 0.1 mol L−1 B-R buffer (pH 9.0) at the CuO/CPE detector. b The calibration curve. SWV parameters: f = 50.0 Hz, a = 50.0 mV, ΔEs = 1.0 mV

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Table 1 Analytical parameters obtained from the calibration curve for E2 using the CuO/CPE detector
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For comparison purposes, calibration curves were constructed using a GCE (Fig. 9a) and a gold electrode (Fig. 9b), under the same conditions used to obtain Fig. 8. The linear regression equation obtained using the GCE was as follows: i pa/μA = (1.90 ± 0.08) [estradiol] − (0.22 ± 0.05) (R = 0.983). The linear regression equation obtained using the gold electrode was as follows: i pa/μA = (1.35 ± 0.06) [estradiol] − (0.14 ± 0.04) (R = 0.979).The LOD and LOQ values obtained with the GCE were 87.7 and 266.0 nmol L−1, respectively, and for the gold electrode, the corresponding values were LOD = 97.9 nmol L−1 and LOQ = 296.7 nmol L−1. These results indicate that the CuO/CPE is more sensitive than the bare GCE and gold in relation to E2 determination.

Fig. 9
figure 9

Calibration curves for E2 using a GCE and b gold electrode. Square-wave voltammograms for a blank, b 0.13 μmol L−1, c 0.2 μmol L−1, d 0.3 μmol L−1, e 0.4 μmol L−1, f 0.5 μmol L−1, g 0.6 μmol L−1, h 0.7 μmol L−1, i 0.8 μmol L−1, j 0.9 μmol L−1, and k 1.0 μmol L−1 E2. SWV parameters: f = 50.0 Hz, a = 50.0 mV, ΔEs = 1.0 mV

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Table 2 compares the performance of the proposed modified CPE with that of several electrochemical devices reported in the literature for E2 determination. It can be observed that the analytical characteristics of the CuO/CPE detector are better than or comparable with those of the other devices. Several detectors listed in Table 2 are electrodes modified with nanoparticulate materials or complex biosensors associated with laborious and expensive preparation procedures. On the other hand, the modified electrode proposed in this paper makes use of CuO microparticles, it is able to detect E2 at the nanomolar level, and it is prepared through a simple procedure at low cost.

Table 2 Performance of different sensors for E2 detection
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Repeatability, stability, and interference studies with the CuO/CPE detector

The repeatability of the current measured for the 0.1-μmol-L−1 E2 oxidation at the CuO/CPE detector was estimated in 0.1 mol L−1 B-R buffer (pH 9.0) by SWV. It was verified that the intra- and interday repeatability of the data obtained using the proposed electrochemical device was excellent. The relative standard deviation (RSD) of successive measurements on the same day (intraday repeatability) was 2.73% (n = 10). The interday repeatability was evaluated on 10 consecutive days using the same parameters and an RSD of 6.73% was obtained. The low values for the RSD indicate the high degree of precision of the data obtained when the CuO/CPE is used for E2 detection. To evaluate the stability of the as-prepared voltammetric device, the modified electrode was stored in air for 90 days. The peak current delivered after this time was 93.5% of its initial value. Therefore, the detector developed in this study is stable and suitable for the routine analysis of E2 in different types of samples. An important parameter for new detectors is their ability to discriminate the interfering species commonly present in samples from the target analyte. In order to evaluate the selectivity of the CuO/CPE toward E2 in the presence of other chemical species, the influence of potentially interfering substances on the E2 anodic current was investigated. The potential interferents considered were glucose, sucrose, uric acid, urea, ascorbic acid, K+, Na+, Cl−, and Ca2+. The interferent/E2 ratios tested were 100/1, 1/1, and 1/100. The currents obtained by SWV for E2 oxidation were between 2.07 and 4.33% lower in the presence of interferents (data not shown). This behavior verifies that the CuO/CPE detector is suitable for the analyses of E2 in complex matrices, such as urine and buttermilk, because it is not significantly affected by the presence of potentially interfering species commonly present along with the molecule of interest.

E2 determination in urine and buttermilk samples

The SWV results obtained in the determination of E2 in urine and buttermilk samples are shown in panels a and b of Fig. 10, respectively. The linear regressions obtained for the corresponding standard addition plots shown in Fig. 10c, d are as follows: urine (y = (3.31 ± 0.12) [estradiol] + (0.21 ± 0.02)) and buttermilk (y = (3.10 ± 0.16) [estradiol] + (0.52 ± 0.02)), where y is the current (μA) and [estradiol] is the E2 concentration (μmol L−1). It can be observed, for both samples, that the slopes of the standard addition plot and the calibration curve (3.41 μA L mol−1, Table 1) are very similar. This corroborates the results discussed above and confirms that the matrix components did not affect the determination of E2. In addition, these results demonstrate that the E2 concentration can be determined with high sensitivity and selectivity using the CuO/CPE detector.

Fig. 10
figure 10

Square-wave voltammograms for a 50 μL of urine and b 200 μL of buttermilk: a blank, b samples, and c–f additions of 100, 200, 300, and 400 μL of 10.0 μmol L−1 E2. c, d The corresponding standard addition plots. Experimental conditions: CuO/CPE detector, 0.1 mol L−1 B-R buffer (pH 9.0), f = 50.0 Hz, a = 50.0 mV, ΔE S  = 1.0 mV

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The accuracy provided by the CuO/CPE detector for the determination of E2 was evaluated from recovery experiments. In brief, known amounts of standard E2 solution were added to the real samples followed by analysis using the standard addition method. The results obtained are given in Table 3. The recovery values for the two types of samples ranged from 92 to 107%. These results indicated that the fabricated modified electrode can be used as an electrochemical detector for E2 determination, since it provides accurate values.

Table 3 Results for determination of E2 and recovery experiments with the samples analyzed
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Conclusions

In this study, CuO microparticles were successfully synthesized by Pechini’s method using low-cost precursors and characterized using XRD, FTIR, Raman spectroscopy, SEM, and EDS. The XRD results indicate that the particles are monoclinic (crystalline). The Raman spectra exhibit three different bands, which correspond to the A g and B g modes of CuO. It was also observed that the morphology of the microparticles is flake-like with uniformity in the distributions of size and shape.

A simple CPE was modified with CuO microparticles and used for the detection of E2. The electrochemical behavior of E2 at the CuO/CPE detector was studied by CV, and it was found that the modified electrode led to enhanced sensitivity in the voltammetric detection of this hormone. This detector offers many beneficial analytical characteristics, such as a good detection limit of 21.0 nmol L−1, wide linear concentration range of 60.0 to 800.0 nmol L−1, and a good repeatability, stability, and selectivity. The proposed detector was successfully applied to the analysis of urine and buttermilk samples with satisfactory recovery values (92–110%).

The proposed CuO/CPE detector offers certain advantages over conventional electrodes including ease of fabrication, high electrochemical activity, simplicity, and low cost, and in association with SWV, it provided low limits of detection and quantification. Based on the results obtained, the CuO/CPE showed satisfactory performance in comparison with other electrodes used for the determination of E2. It can thus be concluded that the use of the proposed CuO/CPE detector represents a promising technique for the determination of E2 in real and complex samples, without the need for elaborate sample preparation procedures. The improvement achieved can be attributed to the combination of graphite powder and CuO and the resulting synergistic effect. The results reported herein demonstrate that CuO microparticles can offer better potential for application in comparison with nanoparticles of transition metal oxides.