3-methyl-4-nitrophenol (MNP), sodium phosphate monobasic (NaH₂PO₄), sodium phosphate dibasic (Na₂HPO₄), nickel tetrasulfonated phthalocyanine (NiTSPc), potassium ferrocyanide (K₄Fe(CN)₆), potassium chloride (KCl), sodium hydroxide (NaOH), sulfuric acid (H₂SO₄), and absolute ethanol were procured from Sigma-Aldrich and utilized without additional purification. A phosphate buffer solution was prepared by combining the requisite quantities of NaH₂PO₄ and Na₂HPO₄, serving as the supporting electrolyte. A solution for the electrochemical cleaning of naked carbon fiber ultramicroelectrode (CFME) was formulated by equi-volume mixing 0.5 M H₂SO₄ and absolute ethanol. A 2 mM solution of NiTSPc was prepared by dissolving 98 mg of the chemical in 50 mL of 0.1 M NaOH. All aqueous solutions were prepared with pure water. MNP solutions were deaerated by passing nitrogen gas through them prior to electrochemical analysis of MNP.

All voltametric measurements were conducted using a potentiostat DY 2300 (Digi-IVY Instruments, USA), which was controlled by DY 2300 EN software on a computer. A carbon fiber microelectrode (CFME) with a diameter of 12 µm and a length of 5 mm, as well as a modified carbon fiber microelectrode coated with NiTSPc film (CFME/p-NiTSPc), were employed as working electrodes. An Ag/AgCl electrode served as the reference electrode, while a platinum electrode with a diameter of 250 µm was used as the counter electrode. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) analyses of the CFME and CFME/p-NiTSPc were performed using a JEOL JSM-6301F (SCIAM, Angers University). SEM images were obtained using secondary electrons at an accelerating voltage of 3 - 5 keV, with magnifications between 3000 and 5000. EDX spectra were gathered using a 20 keV beam.

Cyclic voltammetry was employed to modify CFME and to explore the electrochemical behaviors exhibited by MNP on both the unmodified CFME and the CFME/p-NiTSPc. Furthermore, square wave voltammetry served as a powerful tool for the quantitative analysis of aqueous samples, focusing specifically on tracking the direct reduction peak of MNP.

Before employing a CFME, it is essential to clean it to remove contaminants from the carbon fiber. The cleaning process is conducted electrochemically using a 1:1 mixture of H₂SO₄ and absolute ethanol. Cleaning is achieved by cyclic voltammetry at 50 mV/s over 10 cycles, ranging from −1.5 V to 1.5 V [1]. Subsequently, the purified CFME is modified by p-NiTSPc, following the established protocol described in previous studies [11]. Figure 1 illustrates the voltammograms acquired during the electrodeposition of p-NiTSPc on CFME, alongside the electrochemical revelation depicted in the insert for the modified CFME/p-NiTSPc. The thickness of the p-NiTSPc film was assessed based on the final cyclic voltammogram obtained from the electrodeposition of p-NiTSPc. Under the specified experimental conditions, the p-NiTSPc film electrodeposited exhibits a recovery of approximately 1.28 × 10⁻⁸ mol/cm², as determined by integrating the oxidation peak, which corresponds to a film thickness of 128 nm, in accordance with a previous report [12].

Figure 1. 75 successive cyclic voltammograms of CFME in a 0.1 M NaOH aqueous solution containing 2 mM of NiTSPc at the potential scan rate of 100 mV/s. Inset shows 1 cyclic voltammogram of CFME/p-NiTSPc in a 0.1 M NaOH aqueous solution at the potential scan rate of 100 mV/s.

Figure 2(A) and Figure 2(B) present SEM images of unmodified and modified carbon fiber microelectrodes, respectively. A distinct difference is observed between the unmodified and modified carbon fibers. Figure 2(A) displays a fiber with evenly distributed streaks, whereas Figure 2(B) depicts a fiber coated with nickel phthalocyanine film. Additionally, energy-dispersive X-ray (EDX) analyses were performed on both unmodified and modified CFMEs, with the results shown in Figure 2(C) and Figure 2(D).

Both the two EDX images show carbon and oxygen. Carbon comes from the carbon fiber, and oxygen on the carbon fiber is likely from electrochemical cleaning. For the modified electrode, nickel and sulfur also appear. These elements likely originate from the tetrasulfonated nickel phthalocyanine film, consistent with the SEM image of the modified electrode.

Figure 3 displays the voltammograms of iron obtained using both a naked electrode

Figure 2. SEM images of (A) CFME and (B) CFME/p-NiTSPc. EDX spectra of (C) CFME and (D) CFME/p-NiTSPc.

Figure 3. Cyclic voltammogram of 5 mM [ Fe ( CN ) 6 ] 4 − in PBS pH 7 on unmodified CFME (black) and CFME/p-NiTSPc (red) at the scan rate v = 100 mV/s.

and a modified electrode.

This figure indicates a substantial difference between the voltammograms of [ Fe ( CN ) 6 ] 4 − recorded with the unmodified and modified electrodes. The voltammogram obtained with the CFME exhibits a poorly defined peak during the forward scan, corresponding to the oxidation of Fe^II to Fe^III, and no peak during the reverse scan, suggesting that [ Fe ( CN ) 6 ] 4 − exhibits slow electron-transfer kinetics on this electrode. In contrast, modification of the electrode with tetrasulfonated nickel phthalocyanine results in [ Fe ( CN ) 6 ] 4 − exhibiting rapid electron-transfer behavior. The appearance of an oxidation peak at 0.37 V ( Ip a = 93 μA) and a reduction peak at 0.16 V ( Ip c = 91 μA) can be attributed to the oxidation of Fe^II to Fe^III and the reduction of previously generated Fe^III to Fe^II, respectively. Furthermore, the peak intensity ratio, which is close to 1, meets the criteria for a fast electron-transfer system [13].

Figure 4 shows the cyclic voltammogram of MNP at 40 mg/L in a pH 7 phosphate buffer, using both an unmodified CFME and a CFME/p-NiTSPc.

Figure 4. Cyclic voltammograms of 40 mg/L MNP in PBS at pH 7 recorded at unmodified CFME (black) and CFME/p-NiTSPc (red) with a scan rate of 100 mV/s.

For the CFME, the irreversible peak appears around 1 V, with a peak intensity of 659 µA/cm². In contrast, for the CFME/p-NiTSPc, the peak occurs at approximately 0.86 V, with a peak intensity of 890 µA/cm². Previous reports suggest that this peak results from the direct reduction of the nitro group to a hydroxylamine group after a transfer of 4 electrons, as shown in Scheme 1 [14].

The observed changes in potential and reduction peak intensity are attributed

Scheme 1. Equation of the electrochemical reduction of 3-methyl-4-nitrophenol.

to the presence of the p-NiTSPc film. These results further demonstrate the effectiveness of electrode modification. Additionally, the increased intensity of the reduction peak, together with the decreased reduction potential, indicates that the p-NiTSPc film exhibits an electrocatalytic effect on the direct reduction of MNP [15]. Therefore, the CFME displays boosted sensitivity in the presence of the p-NiTSPc film.

The effect of scan rate on the reduction peak of MNP was examined. Specifically, 20 mg/L MNP in 0.1 M PBS (pH 7) was analyzed by cyclic voltammetry using CFME/p-NiTSPc at scan rates of 10 - 200 mV/s. The resulting cyclic voltammograms are presented in Figure 5.

Figure 5. Cyclic voltammograms of 20 mg/L MNP in PBS at pH 7 on CFME/p-NiTSPc with different scan rates: 10, 20, 50, 100 and 200 mV/s. Inset: curve of the linear dependence of Ip versus v 1 / 2 .

The figure shows that the reduction peak intensity increases with increasing scan rate, and that a linear relationship between cathodic current and the square root of the scan rate is observed over the range of 10 - 200 mV/s. The linear equation is:

with R² = 0.991. These results indicate that a diffusion-controlled process governs the reduction of MNP on CFME/p-NiTSPc [16].

Figure 6 presents the voltammograms of MNP measured at various pH values in PBS.

Figure 6. SWV of MNP at 10 mg/L in a phosphate buffer at different pH at a CFME/p-NiTSPc. SWV Parameters: frequency = 50 Hz; pulse amplitude = 100 mV; step potential = 30 mV.

It can be seen from Figure 6 that the reduction peak potential shifts negatively with increasing pH, which indicates that the proton participates in the electrochemical redox processes. This observation is crucial as it sets the basis for the linear relationship that follows. Indeed, Figure 7 shows a linear relationship between Ep vs pH with the linear regression equation: Ep (V) = −0.0594pH − 0.327 (R² = 0.9947). This linearity confirms the involvement of protons in the process.

According to the Nernst equation, the obtained slope (0.0593 V/pH) is close to the theoretical value (0.0591 V/pH), indicating that equal numbers of proton and electron transfers were involved in the electrochemical reduction of MNP. Furthermore, it reinforces the earlier observation, establishing a consistent line of evidence for the hypothesized mechanism. In addition, the representation of peak intensity as a function of pH (Figure 7) shows that the reduction peak current increased with increasing pH from 4.0 to 5.0. The peak current reached its maximum at pH 5.0 and then sharply decreased at higher pH values. This phenomenon can be explained by the reduction mechanism, which involves proton transfer, as previously described in Scheme 1 and demonstrated in this section. At pH > 7, the reduction of the nitro group (-NO₂) of MNP to the hydroxylamine group (-NHOH) is retarded due to the decrease in H⁺ concentration, which participates in the reduction mechanism [17]. Additionally, as pH increases, the proportion of the anionic form of MNP (pKa value of 7.4) increases, while the amount of the neutral form decreases. This shift results in a reduced intensity of the MNP reduction peak. Therefore, pH 5.0 was selected as the optimal value for the detection of MNP.

Figure 7. Plot of Ip vs pH value and plot of Ep vs pH value.

Following the optimization of the pH for MNP analysis, the square-wave voltammetry (SWV) parameters were also optimized (data not shown). The optimal parameters identified include a frequency of 50 Hz, a pulse amplitude of 100 mV, and a step potential of 30 mV. These values were subsequently applied in the quantitative analysis of MNP. Figure 8 presents the square-wave voltammograms corresponding to various concentrations of the MNP standard on CFME/p-NiTSPc.

Figure 8 demonstrates that, for concentrations between 1 μg/L and 10 μg/L, the intensity of the reduction peak increases proportionally with concentration. The inset of Figure 8 further illustrates that this relationship is linear. The linear regression equation is Ip = 0.1206C + 0.1586, with R² = 0.9986. Based on this equation and a signal-to-noise ratio of 3:1, the detection limit (LoD) was calculated to be 0.025 μg/L. Table 1 compares the electrochemical reaction types, linear ranges, and detection limits of some reported electrochemical sensors with CFME/p-NiTSPc for MNP detection.

To gauge the significance of this detection limit, it is informative to compare it

Figure 8. SWV of MNP at different concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg/L) in phosphate buffer (pH = 5) at a CFME/p-NiTSPc. Inset: plot of Ip vs concentration of MNP. SWV parameters: frequency = 50 Hz; pulse amplitude = 100 mV; potential increment = 30 mV.

Table 1. Performance comparison of various electrochemical sensors for MNP detection. All detections were made using SWV.

^aUME: ultramicroelectrode. ^bMIP: molecular imprinted polymer.

with the regulatory standards set by international bodies. The World Health Organization (WHO) and the European Union (EU) have established maximum allowable concentrations for pesticide residues or their metabolites in drinking water at 0.1 μg/L [18]. To our knowledge, our reported LoD represents the lowest electrochemical detection limit for MNP to date.

Interference was evaluated in a 0.1 M phosphate buffer solution (PBS) containing 10 μg/L of MNP, with the addition of potential interfering species including Cd²⁺, Pb²⁺, K⁺, NO 3 − , SO 4 2 − , and the parent pesticide fenitrothion (FT). The tolerance threshold was defined as the ratio of the interfering species concentration to the MNP concentration. Table 2 presents the relative error of the Ip following the introduction of these interfering species.

Table 2. Summary of various interfering substances and their effects on the detection of MNP (10 μg/L) using CFME/p-NiTSPc.

This table indicates that most interfering substances exhibited negligible influence (relative error ≤ 5%) even at concentrations up to 150 times greater than that of MNP. In contrast, fenitrothion demonstrated a substantial effect (>6%) at a concentration only slightly exceeding six times that of MNP. This result is attributed to the presence of the same functional group in both fenitrothion and MNP.

The repeatability, reproducibility, and stability of the CFME/p-NiTSPc electrode were evaluated using square wave voltammetry (SWV). Voltammograms of a 10 µg/L MNP solution in 0.1 M phosphate buffer at pH 5 were initially recorded with the same CFME/p-NiTSPc electrode. The relative standard deviation (RSD) for six repeated measurements was 3.1%, demonstrating strong repeatability for MNP quantification. Reproducibility was assessed by preparing six separate CFME/p-NiTSPc electrodes using an identical protocol; the RSD for MNP detection at 10 µg/L was 5.7%, indicating acceptable reproducibility. Sensor stability was determined by recording the signal from a 10 µg/L MNP solution daily on the same electrode, with the modified electrode retaining approximately 97.1% of its initial response after five days. These results demonstrate that the CFME/p-NiTSPc electrode provides reliable analytical performance, including repeatability, reproducibility, and stability, supporting its suitability for MNP quantification in real-world samples.