Fabrication of a Selective Sensor Amplification Probe Modified with Multi-Component Zn2SnO4/SnO2 Heterostructured Microparticles as a Robust Electrocatalyst for Electrochemical Detection of Antibacterial Drug Secnidazole

In this study, we synthesized heterostructured zinc stannate/tin oxide microparticles (ZTO/TO MPs) by a simple coprecipitation method and used them as an effective electrode material for the electrochemical detection of the antibacterial drug secnidazole (SCZ). The as-prepared ZTO/TO MPs were characterized by XRD, Raman, FE-SEM, HR-TEM, EDX, and XPS analyses. The physiochemical studies clearly proved that the fabricated ZTO/TO MPs were formed in a heterostructure phase without other impurities. A glassy carbon electrode modified with the synthesized ZTO/TO MPs showed an excellent and improved electrocatalytic activity in the electrochemical reduction of SCZ. Using differential pulse voltammetry (DPV), an impressive linear calibration range, extending from 0.01 to 193 μM, was observed, coupled with a detection limit of 0.0054 μM and a sensitivity of 0.055 μA/μM. In addition, the ZTO/TO MPs/GCE showed very good selectivity for the detection of SCZ in the presence of a number of biological, inorganic, and structurally related compounds. Finally, the ZTO/TO MPs/GCE was investigated for the analysis of SCZ in human blood serum samples. A very good recovery was obtained when spiking the blood serum with SCZ, highlighting the good applicability of the ZTO/TO MPs/GCE for the electrochemical analysis of SCZ in complex biological samples.


Introduction
Multifunctional metal oxides with multivalent ions form an extensive collection of various nanostructured functional materials with numerous applications in science and technology [1]. In this series, binary metal oxides have been intensively studied, and research on specially designed ternary oxide semiconductors has recently received increasing attention. In particular, semiconducting ternary metal oxides are currently of great interest due to their potential technological applications in photocatalysis [2], solar cells [3], and lithiumion batteries [4] as they exhibit a tunable band gap (3.1-3.9 eV), high electron transport, and high chemical stability. Specifically, the combination of zinc oxide (ZnO) and tin oxide (SnO 2 ), to give zinc stannate/tin oxide (ZTO/TO), is characterized by a good conductivity, low toxicity, and high thermal and chemical stability, and it makes a significant contribution There are very few sensors that have been reported for the electrochemical detection of SCZ. Glassy carbon and mercury substrates have previously been employed. For example, Radi et al. [17] employed DC and AC polarography with a dropping mercury electrode to analyse the SCZ content in tablets, while El-Sayed et al. [23] used cathodic adsorptive stripping voltammetry for the analysis of SCZ with dropping mercury and glassy carbon electrodes. In this study, we synthesized ZTO/TO MPs using a simple and inexpensive coprecipitation method. Then, various characterization techniques such as XRD, Raman, FE-SEM, HR-TEM, and XPS analyses were performed to better understand the crystalline nature, surface chemistry, and bonding interactions. Subsequently, a GCE was modified with the ZTO/TO MPs and was employed in the electrochemical analysis and detection of SCZ with CV and DPV techniques. The electrochemical results showed that the determination of SCZ with ZTO/TO MPs/GCE has a low detection limit, high selectivity and sensitivity, and this modified electrode is also very suitable for the electrochemical detection of SCZ in practical applications.

Materials and Methods
Zinc nitrate hexahydrate (Zn(NO 3 ) 2 ·6H 2 O), sodium stannate trihydrate (Na 2 SnO 3 ·3H 2 O), ethylene glycol, urea (CO(NH 2 ) 2 ), secnidazole, sodium phosphate dibasic anhydrous (Na 2 HPO 4 ), sodium phosphate monobasic dihydrate (NaH 2 PO 4 ·2H 2 O), and the other required chemicals and reagents were obtained from Sigma-Aldrich (Taipei, Taiwan). All of the reagents and chemicals used in this work were of analytical grade, and deionized (DI) water was used to prepare the required solutions. A phosphate buffer solution (0.05 M (PBS)) was employed as the supporting electrolyte, which was prepared by dissolving sodium phosphate dibasic anhydrous and sodium phosphate monobasic dihydrate in DI water. Aqueous sodium hydroxide and hydrochloric acid solutions were used to adjust the pH of the buffer solutions.
The co-precipitation method was used for the synthesis of the ZTO/TO MPs. In a typical synthesis, 30 mL solutions of 200 mM Na 2 SnO 3 .3H 2 O and 200 mM of Zn(NO 3 ) 2 .6H 2 O were prepared individually and mixed with 50 mL DI water in a beaker (200 mL). With stirring, 7 mL of ethylene glycol and 5 g (in 10 mL of H 2 O) of CO(NH 2 ) 2 were then added to the beaker and stirred for 1 h (800 rpm) at room temperature. After 1 h, a white coloured precipitate was obtained, which was washed three times with water and then with ethanol to remove the impurities present in the product. Then, the final precipitate was placed in an air oven and stored at 50 • C for 12 h. Finally, the ZTO/TO MPs were obtained after the crystallization process in a muffle furnace at 1100 • C for 4 h. The overall synthesis procedure of the ZTO/TO MPs and a schematic of their electrochemical sensing properties are shown in Scheme 1. Prior to modification, the surface of GCE (working area = 0.07 cm 2 ) was polished with alumina slurry (0.05 μm) and washed with DI water. The ZTO/TO MPs suspension (5 mg/mL) was prepared using ultra-sonication for 20 min to ensure uniform distribution of Scheme 1. The overall synthesis procedure of the ZTO/TO MPs sensor and its electrochemical application in the detection of SCZ.
Prior to modification, the surface of GCE (working area = 0.07 cm 2 ) was polished with alumina slurry (0.05 µm) and washed with DI water. The ZTO/TO MPs suspension (5 mg/mL) was prepared using ultra-sonication for 20 min to ensure uniform distribution of the MPs. Then, 6 µL of the ZTO/TO MPs suspension was drop casted onto the cleaned GCE surface and dried in an air oven for 10 min. Finally, the ZTO/TO MPs/GCE was used for the electrochemical experiments.
Electrochemical analyses were performed with CHI potentiostats. (Austin, TX, USA) Cyclic voltammetry (CV) experiments were carried out with a CHI1205C, while differential pulse voltammetry (DPV) was performed with a CHI900 potentiostat. The optimized DPV parameters were as follows: potential window from +0.4 to −0.8 V, amplitude of 0.05 V, pulse width of 0.05 s, and a pulse period of 0.2 s. All of the electrochemical experiments were performed in a 20 mL electrolytic cell containing 10 mL of N 2 -saturated PBS solution with a standard three-electrode system, with the ZTO/TO MPs/GCE as the working electrode, KCl-saturated Ag/AgCl as the reference electrode, and a platinum wire as the counter electrode.

X-ray Diffraction and Raman Studies
The phase purity of the as-prepared ZTO/TO MPs was investigated by powder XRD analysis. Figure 1A  Furthermore, the peak positions and their relative intensities can be compared with the standard data of Zn 2 SnO 4 and SnO 2 , which show that the crystal structure consists of a combination of face-centred cubic structures with space group fd-3m (JCPDS: 00-024-1470) and tetragonal rutile (JCPDS: 01-088-0287) [24]. It can be seen that all the peak positions are characteristic of the pure ZTO/TO compound. There are no other impurity peaks, such as ZnO and ZnSn(OH) 6 , in the XRD patterns, except those assigned to binary oxide phases. This XRD analysis clearly shows that the heterostructured ZTO/TO MPs were successfully formed by the simple coprecipitation method.
Additional chemical information was obtained using Raman analysis and a representative spectrum is shown in Figure 1B. Based on group theory, the Raman spectra of pure Zn 2 SnO 4 and SnO 2 can be categorized into seven modes, which are theoretically assigned to T 2u , B 1g , F 2g , E g , A 2u , B 2g , and A 1g symmetries. Considering the Raman spectrum, the peak at 108 cm −1 can be assigned as the silent T 2u mode, while the peak at 121 cm −1 corresponds to the B 1g mode. The wavenumber at 240 cm −1 is attributed to the E(LO) phonon mode and the peak at 387 cm −1 is associated with the F 2g mode. In addition, the band at 542 cm −1 is consistent with the internal vibrations of the oxygen tetrahedron. It is worth highlighting that the mode with the highest intensity at 662 cm −1 can be at-tributed to the symmetric stretching of Zn-O bonds in the SnO 4 tetrahedra of the inverse Zn 2 SnO 4 . The weak Raman band at 690 cm −1 can be assigned to the A 2u mode, while the peak at 776 cm −1 can be attributed to the first order E g (translational), A 1g (symmetric Sn-O stretching), and B 2g (asymmetric Sn-O stretching) active Raman vibrational modes of SnO 2 [25][26][27]. All these Raman modes confirm that the ZTO/TO MPs exist exclusively in a heterostructure.
pure Zn2SnO4 and SnO2 can be categorized into seven modes, which are theoretically assigned to T2u, B1g, F2g, Eg, A2u, B2g, and A1g symmetries. Considering the Raman spectrum, the peak at 108 cm -1 can be assigned as the silent T2u mode, while the peak at 121 cm -1 corresponds to the B1g mode. The wavenumber at 240 cm -1 is attributed to the E(LO) phonon mode and the peak at 387 cm -1 is associated with the F2g mode. In addition, the band at 542 cm -1 is consistent with the internal vibrations of the oxygen tetrahedron. It is worth highlighting that the mode with the highest intensity at 662 cm -1 can be attributed to the symmetric stretching of Zn-O bonds in the SnO4 tetrahedra of the inverse Zn2SnO4. The weak Raman band at 690 cm -1 can be assigned to the A2u mode, while the peak at 776 cm -1 can be attributed to the first order Eg (translational), A1g (symmetric Sn-O stretching), and B2g (asymmetric Sn-O stretching) active Raman vibrational modes of SnO2 [25][26][27]. All these Raman modes confirm that the ZTO/TO MPs exist exclusively in a heterostructure.

Morphological Investigation Using FE-SEM and HR-TEM Analysis
In addition, FE-SEM and HR-TEM studies were performed to study the morphology of the MPs, and these micrographs are shown in Figure 2A-F, indicating that the ZTO/TO MPs have the shape of a stone-like structure. The high magnification FE-SEM image in Figure 2C shows that the diameters of both the ZTO and TO MPs, with a rough surface, are in the range of several micrometres. The HR-TEM images shown in Figure 2D-F indicate that the synthesized products have heterostructures with diameters of ~0.2 μm. Furthermore, EDX and elemental analyses were carried out to confirm the presence of Zn, Sn, and O. As shown in Figure 3A-E, the EDX spectrum of the ZTO/TO MPs indicates the

Morphological Investigation Using FE-SEM and HR-TEM Analysis
In addition, FE-SEM and HR-TEM studies were performed to study the morphology of the MPs, and these micrographs are shown in Figure 2A-F, indicating that the ZTO/TO MPs have the shape of a stone-like structure. The high magnification FE-SEM image in Figure 2C shows that the diameters of both the ZTO and TO MPs, with a rough surface, are in the range of several micrometres. The HR-TEM images shown in Figure 2D-F indicate that the synthesized products have heterostructures with diameters of~0.2 µm. Furthermore, EDX and elemental analyses were carried out to confirm the presence of Zn, Sn, and O. As shown in Figure 3A-E, the EDX spectrum of the ZTO/TO MPs indicates the presence of the Zn, Sn, and O elements. The elemental mapping images presented in Figure 3A-D show that the Zn (red), Sn (blue), and O (green) elements are uniformly distributed throughout the ZTO/TO. These data show that the MPs have a dense and nonuniform structure, which may facilitate the diffusion of electrolytes, while the proximity of the adjacent SnO 2 particles may provide a transport channel for the transfer of electrons, leading to a reduction in the electron transfer resistance.

X-ray Photoelectron Spectroscopy Analysis
X-ray photoelectron spectroscopy (XPS) was performed to confirm the chemical composition and electronic state of the as-prepared ZTO/TO heterostructures. Figure 4A shows the overall spectrum of the ZTO/TO MPs indicating the presence of Zn, Sn, and O. In addition, the inset ( Figure 4A) shows the elemental composition of the ZTO/TO MPs and this composition agrees well with the obtained XRD phase formula of Zn 2 SnO 4 /SnO 2 . The high-resolution XPS spectra of the Zn 2p, Sn 3d, and O 1s elements are shown in Figure 4B-D. The core spectrum of Zn shows peaks at 1046.6 and 1023.5 eV, corresponding to the 2p 1/2 and 2p 3/2 Zn 2+ states [28][29][30]. Figure 4C shows the core spectrum of Sn, and the two strong peaks at 487.9 and 496.3 eV can be attributed to the binding energies of Sn 3d 5/2 and Sn 3d 3/2 , respectively, which are characteristic of the Sn 3d state. Figure 4D shows the core level spectrum of the O 1s peak, which can be split into three peaks with binding energies of 531.8, 533.2, and 534.4.7 eV. The peaks at 531.8 eV and 533.2 eV can be ascribed to the lattice O in metal oxygen bonds (Zn-O, and Sn-O). The peak at 534.4 eV can be assigned to surface oxygen species such as OH or O coordination in metal oxides [31]. Again, this XPS analysis clearly confirms the presence of the heterostructured ZTO/TO MPs.     two strong peaks at 487.9 and 496.3 eV can be attributed to the binding energies of Sn 3d5/2 and Sn 3d3/2, respectively, which are characteristic of the Sn 3d state. Figure 4D shows the core level spectrum of the O 1s peak, which can be split into three peaks with binding energies of 531.8, 533.2, and 534.4.7 eV. The peaks at 531.8 eV and 533.2 eV can be ascribed to the lattice O in metal oxygen bonds (Zn-O, and Sn-O). The peak at 534.4 eV can be assigned to surface oxygen species such as OH or O coordination in metal oxides [31]. Again, this XPS analysis clearly confirms the presence of the heterostructured ZTO/TO MPs.

Electrochemical Reduction of SCZ
The electrocatalytic activity of ZTO/TO MPs/GCE towards the reduction of SCZ was analysed using the CV technique at a sweep rate of 50 mV/s by cycling between the potential limits of +0.4 to −0.8 V. Figure 5A shows the CV responses of 50 µM SCZ at the bare GCE, SnO 2 /GCE, and ZTO/TO MPs/GCE. The unmodified GCE, SnO 2 /GCE, and ZTO/TO MPs/GCE electrodes show a clear reduction wave in the forward sweep and a small oxidation peak in the reverse sweep. The peak obtained in the forward sweep is related to the electrochemical reduction of SCZ-NO 2 to SCZ-NHOH with the transfer of 4e − and 4H + . The small peak in the reverse scan represents the conversion of the hydroxylamine group (SCZ-NHOH) to the nitroso group (SCZ-NO), as illustrated in Scheme 2 [32,33]. As shown in Figure 5A, the SCZ is effectively reduced at the bare GCE, giving rise to a cathodic peak current of 3.60 µA with the reduction peak potential centred at −0.633 V. Interestingly, when the GCE is modified with SnO 2 , it shows a higher peak current than the bare GCE, but the reduction peak potential is shifted to more negative potentials. However, a higher peak current of 6.01 µA is obtained for the ZTO/TO MPs/GCE. Compared with bare GCE, when the ZTO/TO MPs and SnO 2 /GCE were used as modifiers for GCE, the peak current for the reduction of SCZ was increased by factors of approximately 2.3 and 1.2, respectively. This is more clearly illustrated in the comparison diagram shown in Figure 5B. These results clearly show that the GCE modified with ZTO/TO MPs acts as a good electron conducting mediator between the electrode surface interface and the electrolyte, and facilitates the electrochemical reduction of SCZ. Figure 5C shows the bar graph of the different loadings of ZTO/TO MPs on GCE. From this, it can be seen that a higher peak current is obtained when 6 µL of the dispersed ZTO/TO MPs is employed. Therefore, 6 µL of ZTO/TO MPs was chosen as the optimal amount of catalyst.  These results clearly show that the GCE modified with ZTO/TO MPs acts as a good electron conducting mediator between the electrode surface interface and the electrolyte, and facilitates the electrochemical reduction of SCZ. Figure 5C shows the bar graph of the different loadings of ZTO/TO MPs on GCE. From this, it can be seen that a higher peak current is obtained when 6 μL of the dispersed ZTO/TO MPs is employed. Therefore, 6 μL of ZTO/TO MPs was chosen as the optimal amount of catalyst. The influence of the SCZ concentration is shown in Figure 5C, where the CV curves are presented for ZTO/TO MPs/GCE cycled in different concentrations of SCZ, ranging from 50 to 300 µM, at a sweep rate of 50 mV/s. It is clear from this figure that the peak currents increase with increasing the SCZ concentration, and, as shown in Figure 5D, a linear relationship exists between the concentration and peak current. The corresponding linear regression equation was obtained as y = 0.035x + 3.81, with a correlation coefficient of R 2 = 0.998, indicating good linearity. Again, these data show that the ZTO/TO MPs facilitate the electrochemical reduction of the SCZ molecules, giving a linear calibration curve with a well resolved peak at 50 µM SCZ. Thus, the ZTO/TO MPs/GCE appears to be a suitable electrode material for the electrochemical detection of SCZ.

Optimization of the pH and Scan Rate Studies
The presence of protons can affect the electrochemical properties of electroactive materials and they can also participate in the electron transfer step during the oxidation or reduction of organic molecules. The impact of the electrolyte pH on the reduction of SCZ at ZTO/TO MPs/GCE was studied using CV. Representative voltammograms, recorded in 50 µM SCZ in different PBS solutions of varying pH values of 3.0, 5.0, 7.0, and 9.0 are presented in Figure 6A. It can be seen that the cathodic peak current increases with an increase of pH from 3.0 to 7.0, and then decreases at pH 9.0, due to the lack of protons, which are required during the reduction of SCZ (Scheme 2). The pKa of SCZ has been reported as 1.13 [34], which is well below a pH of 3.0, indicating that the SCZ molecule is neutral between pH values of 3.0 and 9.0. The somewhat lower peak currents observed at a pH of 3.0 may be connected to the stability of the zinc and tin oxide phases in this acidic environment. According to Pourbaix diagrams, the thermodynamically stable phases in acidic solutions are normally the solvated cations, while the oxide phases exhibit very good stability at somewhat higher pH values in the vicinity of 5.0 to 8.0. The highest peak current was observed at a pH of 7.0 ( Figure 6B, green trace), and this optimized neutral electrolyte was used in all further electrochemical experiments.
In addition, the peak potential of the reduction wave of SCZ shifted to more negative values when the pH was varied from 3.0 and 9.0, implying that protons play a role in the reduction of SCZ. A linear correlation plot between the pH of the electrolyte and the peak potential was generated and this plot is presented in Figure 6B  As evident from Figure 6C, the peak potential associated with the reduction of SCZ is shifted to more negative values when increasing the sweep rate from 20 to 200 mV/s. On plotting the peak potential as a function of the logarithm of the scan rate, a linear plot was obtained, as shown in Figure 6E. The linear regression equation was found to be Epc (μA) = -0.028V (mV/s) -0.621, with a correlation coefficient of R 2 = 0.995. According to the Laviron equation [35], the slope of the linear plot between Epc and log (V) can be expressed in Equation (2), where R and F are the universal constants (R = 8.314 J/K mol and F = 96485.33 s A/mol) and T represents the thermodynamic temperature.

Epc/log(V) = -2.3 RT/αnF
(2) Setting T at 298.15 K, the αn value was computed as 2.2, and applying the α value of 0.55 (for irreversible electrochemical reactions), the number of electrons involved in the electrochemical reduction of SCZ was estimated as 4.0. This is in very good agreement with the electrochemical conversion of SCZ-NO2 to SCZ-NHOH at the ZTO/TO MPs/GCE, where the reduction is accompanied by the transfer of 4e -and 4H + , as shown in Scheme 2.

Analytical Performance of ZTO/TO MPs/GCE towards SCZ
The analytical performance of the ZTO/TO MPs/GCE was studied using DPV, as it is well known that this pulsed technique gives rise to lower background currents, minimising capacitive currents. The DPV data were recorded in the potential window from +0.4 to -0.8 V, and were used to determine the lowest concentration of SCZ that could be detected. The DPV curves recorded for ZTO/TO MPs/GCE after the stepwise addition of different concentrations of SCZ from 0.01 to 193 μM are displayed in Figure 7A. Upon increasing the concentration of SCZ, there is a clear increase in the peak reduction current. After plotting the peak reduction current as a function of the SCZ concentration, a linear relationship is observed over the concentration range from 0.01 to 193 μM. The corresponding linear plot is shown in Figure 7B, with a linear regression equation of y = 0.055x The computed slope agrees approximately with the theoretical slope of 0.059 m/n, in which m represents the number of protons transferred and n corresponds to the number of electrons transferred. An ideal Nernstian response will only exist when thermodynamic equilibrium is achieved (reversible systems with fast kinetics) and the deviation observed is probably connected with the irreversible redox behaviour of SCZ. However, this is consistent with the transfer of equal numbers of protons and electrons during the electrochemical reduction of SCZ at ZTO/TO MPs/GCE, as illustrated in Scheme 2.
Further information on the reduction of SCZ at the ZTO/TO MPs/GCE was obtained using a scan rate study. In this case, the sweep rate was varied from 20 to 200 mV/s. Typical data recorded at different sweep rates for the GCE modified with the ZTO/TO MPs in a 0.05 M PBS solution containing 50 µM SCZ are presented in Figure 6C. It can be clearly seen that as the sweep rate increases, the reduction peak current of SCZ also increases. Moreover, a linear relationship between the peak current and scan rate was obtained, and the corresponding linear plot is shown in Figure 6D along with the linear regression equation I pc (µA) = 0.026 V (mV/s) + 3.271, and a correlation coefficient of R 2 = 0.991, where V represents the scan rate. This linear relationship between the peak current and scan rate indicates that the electrochemical reduction of SCZ at ZTO/TO MPs/GCE is an adsorption-controlled process.
As evident from Figure 6C, the peak potential associated with the reduction of SCZ is shifted to more negative values when increasing the sweep rate from 20 to 200 mV/s. On plotting the peak potential as a function of the logarithm of the scan rate, a linear plot was obtained, as shown in Figure 6E. The linear regression equation was found to be E pc (µA) = −0.028V (mV/s) − 0.621, with a correlation coefficient of R 2 = 0.995. According to the Laviron equation [35], the slope of the linear plot between E pc and log (V) can be expressed in Equation (2), where R and F are the universal constants (R = 8.314 J/K mol and F = 96,485.33 s A/mol) and T represents the thermodynamic temperature.
Setting T at 298.15 K, the αn value was computed as 2.2, and applying the α value of 0.55 (for irreversible electrochemical reactions), the number of electrons involved in the electrochemical reduction of SCZ was estimated as 4.0. This is in very good agreement with the electrochemical conversion of SCZ-NO 2 to SCZ-NHOH at the ZTO/TO MPs/GCE, where the reduction is accompanied by the transfer of 4e − and 4H + , as shown in Scheme 2.

Analytical Performance of ZTO/TO MPs/GCE towards SCZ
The analytical performance of the ZTO/TO MPs/GCE was studied using DPV, as it is well known that this pulsed technique gives rise to lower background currents, minimising capacitive currents. The DPV data were recorded in the potential window from +0.4 to -0.8 V, and were used to determine the lowest concentration of SCZ that could be detected. The DPV curves recorded for ZTO/TO MPs/GCE after the stepwise addition of different concentrations of SCZ from 0.01 to 193 µM are displayed in Figure 7A. Upon increasing the concentration of SCZ, there is a clear increase in the peak reduction current. After plotting the peak reduction current as a function of the SCZ concentration, a linear relationship is observed over the concentration range from 0.01 to 193 µM. The corresponding linear plot is shown in Figure 7B, with a linear regression equation of y = 0.055x + 0.972 and a correlation coefficient of R 2 = 0.989. Using this linear calibration plot, the sensitivity was computed as 0.055 µA/µM. The limit of detection (LOD) was calculated using Equation Employing Equation (3), the LOD of the ZTO/TO MPs/GCE in the detection of SCZ was estimated as 0.0054 μM. These analytical parameters, including linear region (which extends from 0.01 to 193 μM), LOD, and sensitivity, compare very favourably and comparatively better than the previously reported analytical data measured using cathodic adsorptive stripping voltammetry [23] and polarography [17], as illustrated in Table 1.  Employing Equation (3), the LOD of the ZTO/TO MPs/GCE in the detection of SCZ was estimated as 0.0054 µM. These analytical parameters, including linear region (which extends from 0.01 to 193 µM), LOD, and sensitivity, compare very favourably and comparatively better than the previously reported analytical data measured using cathodic adsorptive stripping voltammetry [23] and polarography [17], as illustrated in Table 1. The analytical performance of the ZTO/TO MPs/GCE was studied further by considering the anti-interference and selectivity, reproducibility, repeatability, and stability. To study the selectivity of the newly constructed ZTO/TO MPs/GCE sensor, the DPV technique was applied in 0.05 M PBS (pH 7.0) containing 50 µM SCZ, in the absence and presence of a number of coexisting and co-interfering biological compounds. These compounds included dopamine (DOP), lactose (LAC), catechol (CC), uric acid (UA), glucose (GLU), and sucrose (SUC)), metal ions (Ca 2+ , Fe 2+ , Zn 2+ , and Ni 2+ ), and nitro group-containing drugs (nitrofurantoin (NFT), ornidazole (OD), and tinidazole (TZ)). The concentration of these potential interferents was maintained at 50 µM, giving equimolar concentrations of SCZ and the interferent. The influence of these added interferents is summarized in Figure 8A, where it is clearly observed that ZTO/TO MPs/GCE exhibits a well-defined peak for the reduction of SCZ in the presence of all of the interferents. Interestingly, the reduction of nitrofurantoin is seen in Figure 8A at about -0.4 V; however, this wave is sufficiently well separated from the SCZ reduction wave and does not contribute to the current measured for the reduction of SCZ. All of the interfering compounds have negligible effects on the peak current recorded for the reduction of SCZ with the relative standard deviation (RSD) of ≤ 2.01%. This indicates very good selectivity, which may be connected with the adsorption of SCZ at the ZTO/TO MPs/GCE partially blocking the interferents, especially the structurally related nitro-based compounds.
In Figure 8B, the data recorded in a repeatability study performed with 50 µM SCZ in 0.05 M PBS using 10 consecutive DPV measurements on a single ZTO/TO MPs/GCE are presented. The results show that ZTO/TO MPs/GCE exhibits a very good repeatability with a RSD value of 2.22%. To investigate the reproducibility of ZTO/TO MPs/GCE, five different ZTO/TO MPs-modified GCEs were prepared and employed in the detection of SCZ with DPV. Again, the obtained DPV curves, shown in Figure 8C, are very similar, with almost the same peak currents. The RSD was computed at 3.04%, demonstrating a very good reproducibility. The stability of the ZTO/TO MPs/GCE was studied following storage over a 21 day period. The constructed ZTO/TO MPs/GCE was stored and subjected to DPV analysis at seven-day intervals. In Figure 8D, the DPV curves recorded in 50 µM SCZ following 1, 7, 14, and 21 days of storage are compared. After 21 days, the ZTO/TO MPs/GCE retained 97% of its original current, indicating very good stability over a 21-day storage period.
It is clearly evident from this analysis that the developed ZTO/TO MPs/GCE exhibits a desirable selectivity, impressive reproducibility, storage stability, and repeatability, making it a promising electrode for the determination of SCZ. Indeed, when comparing the four plots in Figure 8, it is evident that the variations in the selectivity, repeatability, and reproducibility are well within the RSD value of 3.04% seen for the reproducibility analysis ( Figure 8C), indicating a very good performance, well within experimental errors. with almost the same peak currents. The RSD was computed at 3.04%, demonstrating a very good reproducibility. The stability of the ZTO/TO MPs/GCE was studied following storage over a 21 day period. The constructed ZTO/TO MPs/GCE was stored and subjected to DPV analysis at seven-day intervals. In Figure 8D, the DPV curves recorded in 50 μM SCZ following 1, 7, 14, and 21 days of storage are compared. After 21 days, the ZTO/TO MPs/GCE retained 97% of its original current, indicating very good stability over a 21-day storage period.

Practical Analysis
For practical applicability, a blood serum sample was collected from a medical hospital in Taiwan. Before analysis, about 5 mL of the blood serum sample was mixed with an anticoagulant (EDTA) and centrifuged at 6000 rpm for 10 min. The supernatant was collected and diluted with 10X PBS (0.05 M, pH 7.0) and designated as the stock solution.
To mimic the SCZ in the stock solution, a known concentration was added to the stock solution (1:1 stock solution/SCZ ratio). Figure 9 shows the DPV curves recorded in the blood serum sample, to which a known concentration of SCZ was added using the standard addition method. The SCZ reduction waves recorded in the complex blood serum (Figure 9) were very similar to those recorded in the buffer sample (Figure 8), with the reduction peak potential and the width of the peaks being almost identical. It can be seen that the reduction peak current of SCZ at the ZTO/TO MPs/GCE increases with increasing the SCZ concentration in the blood serum sample. Good recovery results were obtained, as shown in Table 2.
blood serum sample, to which a known concentration of SCZ was added using the standard addition method. The SCZ reduction waves recorded in the complex blood serum ( Figure 9) were very similar to those recorded in the buffer sample (Figure 8), with the reduction peak potential and the width of the peaks being almost identical. It can be seen that the reduction peak current of SCZ at the ZTO/TO MPs/GCE increases with increasing the SCZ concentration in the blood serum sample. Good recovery results were obtained, as shown in Table 2. The maximum plasma concentration of SCZ in healthy individuals has been reported to reach 16.2 μg/mL [36] to 24.8 μg/mL [37], with half-life values of 19.60 and 13.86 h, respectively, following the administration of 2 g of SCZ. This is equivalent to maximum plasma levels of 87 to 133 μM. The data presented in Table 2 show that ZTO/TO MPs/GCE is a promising sensor for the analysis of SCZ in real samples, facilitating the analysis of its concentration as it decays from these maximum levels.   The maximum plasma concentration of SCZ in healthy individuals has been reported to reach 16.2 µg/mL [36] to 24.8 µg/mL [37], with half-life values of 19.60 and 13.86 h, respectively, following the administration of 2 g of SCZ. This is equivalent to maximum plasma levels of 87 to 133 µM. The data presented in Table 2 show that ZTO/TO MPs/GCE is a promising sensor for the analysis of SCZ in real samples, facilitating the analysis of its concentration as it decays from these maximum levels.

Conclusions
In summary, the electrocatalytic activity of ZTO/TO MPs/GCE for the detection of the antibacterial agent SCZ was investigated using CV and DPV techniques. The ZTO/TO MPs/GCE shows higher currents than the unmodified GCE during the reduction of SCZ, and this was attributed to the adsorption of the SCZ molecules at the ZTO/TO MPs. Using the DPV technique, a linear calibration range from 0.01 to 193 µM, a LOD of 0.0054 µM, and a sensitivity value of 0.055 µA/µM were obtained for the detection of SCZ. Moreover, a very good selectivity was observed at the GCE modified with the ZTO/TO MPs using equimolar concentrations of SCZ and a number of interferents, including biological compounds, metal ions, and nitro-containing compounds. Likewise, acceptable reproducibility, repeatability, and stability results were obtained, while the modified electrode performed well in the analysis of SCZ in the blood serum. From the characterization and electrochemical results, we conclude that the ZTO/TO MP-modified GCE can be used as an efficient sensing material for the determination of the antibacterial drug SCZ.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Taiwan and was approved by the Institutional Review Board (or Ethics Committee) of the National Taipei University of Technology.