Introduction

Isoniazid (INZ), also known as pyridine-4-carboxylic hydrazide or isonicotinic acid hydrazide, is a vital antibiotic and pulmonary tuberculosis agent used alone or in combination with rifampicin, pyrazinamide, and ethambutol HCl to combat mycobacterium tuberculosis resistance [1, 2]. According to the World Health Organization (WHO), the daily consumption of INZ should range from 4 to 6 mg kg−1 of human body weight, with a maximum daily dose not exceeding 300 mg [3, 4]. However, prolonged use or overdose of INZ tablets can lead to hepatotoxicity and, in severe cases, death [5, 6]. This phenomenon is attributed to the production of hydrazine during INZ metabolism, which induces hepatotoxicity [7, 8]. Hence, regular monitoring of INZ dosage levels in human body fluids is critical, necessitating the development of sensitive, effective, and consistent methods to quantify INZ in clinical and pharmaceutical samples.

Several methods have been developed to determine INZ in human body fluids and pharmaceutical products, including titrimetry, fluorimetry, chemiluminescence, UV–visible spectrophotometry, gas chromatography, HPLC, capillary electrophoresis, and electrochemical techniques [9,10,11]. Each method has specific advantages in terms of sensitivity, selectivity, and ease of use. Titrimetry involves the reaction between INZ and a titrant, with the endpoint detected visually or instrumentally [12]. Fluorimetry relies on the fluorescence emission of INZ or its reaction products upon light excitation [13]. Chemiluminescence detects light emitted during chemical reactions involving INZ [14]. UV–visible spectrophotometry measures the light absorbance of INZ at specific wavelengths [15]. Gas chromatography separates INZ from other compounds via partitioning between phases [16]. HPLC quantifies INZ by interacting with the stationary and mobile phases [17]. Though widely used, these methods are limited by complexity and the need for specialized equipment and reagents. However, electroanalytical methods have shown promise due to their sensitivity, cost-effectiveness, and ease of use. For example, Majidi et al. [18] explored a polypyrrole glassy carbon–modified electrode for the voltammetric determination of INZ and demonstrated its potential in INZ analysis. Shahrokhian et al. [19] explored the electrochemical oxidation of INZ in carbon paste electrodes modified with multi-walled carbon nanotubes, highlighting the utility of nanomaterials in enhancing electrode performance. Ghoneim et al. [20] investigated the polarographic reduction of INZ, revealing two irreversible cathodic waves at pH < 5 and a single wave at pH > 8. This finding suggests a potential method for determining INZ content in pharmaceutical formulations and human body fluids. Hammam et al. [21] studied the electrochemical oxidation of INZ using cyclic and square-wave voltammetry on a carbon paste electrode and observed an irreversible anodic peak attributed to the oxidation of the amide group.

In an adequate and effective practical way, screen-printed carbon electrodes (SPCE) and their modifications can enhance their electrochemical performance for the detection of INZ and other compounds [22,23,24]. One common modification involves the use of functional polymers like Nafion films, which improve the signal intensity and reduce interference [25]. Nafion’s cationic exchange properties make it suitable for SPCE modification. Carbon-based materials such as ordered mesoporous carbon [24, 26, 27] or multi-walled carbon nanotubes [28, 29] can also enhance the electrochemical properties. These materials offer advantages like low background current and expansive potential windows. Various modifiers, including metal nanoparticles (e.g., gold, silver, platinum) and chitosan, can enhance SPCE’s sensitivity and selectivity for INZ detection [30, 31]. Metal nanoparticles can be incorporated through physical adsorption or chemical deposition, whereas chitosan, a biopolymer, improves sensitivity and stability [32]. Molecularly imprinted polymers and activated carbon are other modifiers that enhance SPCE for INZ detection [33, 34]. INZ detection using SPCE-modified electrodes, Nasiri et al. developed several advanced sensors for detecting various compounds [35, 36]. They synthesized a graphitic carbon nitride/magnetic chitosan composite for lactose detection with high sensitivity and a low detection limit of 0.3 µM [37]. Another study by Nasiri et al. used graphene oxide-chitosan for amlodipine quantification, achieving high sensitivity and a detection limit of 50 nM [38]. Saghatforoush et al. introduced a Zn (II)-MOF-modified pencil graphite electrode for sumatriptan sensing, with a 0.29-µM detection limit and excellent selectivity in human serum [39].

However, research has identified copper oxide (CuO) as a promising material for modifying SPCEs, with studies reporting enhancements in electrochemical performance [40]. CuO-modified SPCEs have been observed to exhibit improved sensitivity, selectivity, and stability across various electrochemical sensing applications [41]. Furthermore, a high surface-area-to-volume ratio of CuO is associated with an increased active surface area, which enhances analyte adsorption and improves the sensitivity of these systems [42]. CuO also exhibits excellent catalytic activity in various redox reactions, making it suitable for the detection of a broad range of analytes [43]. Additionally, CuO-modified SPCEs are characterized by their stability and durability, with strong adhesion preventing detachment during measurement [44]. These advantages have led to modified SPCEs for numerous applications, including electrocatalysis and biosensing. For example, Beitollahi et al. developed a novel electrochemical sensor to detect amitriptyline using CuO nanoparticle-modified screen-printed electrodes [45]. Tajik et al. fabricated a magnetic iron oxide-supported CuO-modified screen-printed electrode for electrochemical studies and desipramine detection [41]. Chen et al. demonstrated the involvement of Cu (II) in the electrocatalytic reduction of bromate on disposable nano-CuO-modified SPCEs using hair-waving products as an example [46]. Leonardi et al. grew a flower-like nanostructured CuO on SPCE for a non-enzymatic amperometric–sensing glucose [47]. Magar et al. developed non-enzymatic disposable electrochemical sensors based on a CuO/Co3O4@MWCNT nanocomposite-modified screen-printed electrode to determine urea directly [48]. However, all reported CuO particles have been synthesized using hydrothermal methods, with modified electrodes prepared by casting CuO onto the electrode surface. Notably, there have been no documented applications of a CuO-modified SPCE for the detection of INZ.

In a recent study, electrodeposition from a 900 fM Co(NO3) solution was used to deposit single cobalt oxide (CoO) molecules and cluster atoms on a carbon fiber (CF) nanoelectrode [49]. Building on this approach, we developed a novel electrochemical sensing platform by integrating SPCE with CuO and characterized it using SEM and XPS. The electrochemical performance of the CuO-modified SPCE was rigorously examined to detect INZ. This sensor was successfully applied to the determination of INZ in pharmaceutical formulations, allowing for the simultaneous detection of target molecules. Differential pulse voltammetry (DPV) was employed as the analytical technique for INZ measurements, with calibration curves demonstrating exceptional sensitivity, selectivity, and outstanding precision and stability. The sensor exhibits high sensitivity, selectivity, ease of fabrication, and reliable reproducibility, making it a promising tool for INZ detection.

Experimental

Materials

Uric acid, sucrose, glucose, dopamine, and ascorbic acid were purchased from Aladdin Co. Ltd., Shanghai, China. CuSO4, K3Fe(CN)6, phosphate buffer (PBS), KNO3, NaOH, KH2PO4⋅2H2O, and Na2HPO4⋅12H2O were obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Isoniazid (INZ) tablets (0.1 g per tablet, Minsheng Pharmaceutical Co., Ltd., Hangzhou, China) were purchased from a local drugstore. Stock solutions of INZ were prepared daily by dissolving suitable quantities of INZ in water. Working standard solutions were prepared by diluting the stock solutions in 0.1 M PBS pH 7.0. All chemicals were of analytical reagent grade quality and were used without further purification. All solutions were prepared using ultrapure water (conductivity ≤ 18.3KΩ. cm−1).

Electro modification of SPCE

Following the method outlined by Qiu et al. [43], the CuO-modified SPCE was prepared using a constant potential method in a dilute copper sulfate solution (0.1 mM). The SPCE was immersed in a solution containing 0.1 µM Cu2+ dissolved in 0.1 M phosphate buffer (PBS, pH 7.0). According to Jin et al. [49], a constant potential of + 1.6 V vs. Ag/AgCl was applied to the SPCE surface for 60 s. The modified electrode was rinsed with water and stored in PBS (pH 7.0). The modified electrode is referred to as CuO/SPCE.

Characterizations and measurements

The homemade SPCE used in this study was fabricated with dimensions of 4 × 0.5 cm2 and diameter of 2 mm. The SPCE consisted of a carbon working electrode, a silver/silver chloride (Ag/AgCl) reference electrode, and a carbon auxiliary electrode. These materials were obtained from the Institute of Environment and Safety, Wuhan Academy of Agricultural Science. Cyclic voltammetry (CV) measurements were conducted using a CHI612D (Chenhua Corp, Shanghai, China), while electrochemical impedance spectra (EIS) were recorded using a P3000ADX electrochemical workstation (Princeton Applied Research, USA).

The surface morphology of the electrodes was analyzed using a JSM 7800F scanning electron microscope (SEM, JEOL Ltd., Japan) equipped with an energy-dispersive X-ray spectroscopy detector (EDS, Oxford Instruments, Abingdon, Oxfordshire, UK). The surface topographies of the modified electrodes were characterized using an atomic force microscope (AFM, SPM-9700, Shimadzu Corp., Kyoto, Japan). The chemical states were analyzed using X-ray photoelectron spectroscopy (XPS) with an AXIS SUPRA instrument (KRATOS). The instrument had an Al Kα X-ray source (excitation energy of 1486.6 eV).

Preparation and analysis of drugs

The drug was prepared using a modified method described by Yan et al. [27]. The isoniazid tablets were first finely ground using an agate mortar and pestle. A portion (0.0137 g) of the powdered sample was then transferred into a 100-mL flask, to which 10 mL of water was added. The mixture was subjected to ultrasonic extraction for 15 min, after which the extracted solution was stored in a refrigerator. The efficacy of CuO/SPCE for the detection of INZ in pharmaceuticals was evaluated using the standard addition method. This approach involved diluting the samples in a supporting electrolyte and spiking them with varying concentrations of standard INZ solutions.

Results and discussion

Modification and characterization of electrodes

In the pursuit of optimizing the electrodeposition of CuO on electrode surfaces, varying concentrations of CuSO4 ranging from 10−6 to 10−3 M were deposited in PBS (pH = 7) with a potential of + 1.6 V, and the response of the modified SPCESs for INZ was investigated by CV recorded at a scan rate of 50 mVs−1 (Fig. 1a1). The voltammetric peak height of INZ (100 μM), represented by the peak current, was plotted against the logarithm of the CuSO4 concentration (Fig. 1a2). It was observed that as the concentration of CuSO4 increased from 10−6 to 10−3 M, the peak current of INZ reached its maximum value (3.954 µA), and the peak potential separation decreased to 0.278 V, indicating improved conductivity and a higher surface area of the material [50]. However, a further increase in the CuSO4 concentration on the SPCE surface led to a decrease in the currents of the redox probe. This reduction is attributed to the inadequate conductivity of the CuO layer’s inadequate conductivity, impeding the redox probe flow to the electrode surface [51]. Similarly, under identical electromodification conditions, studying the deposition time in the range of 20–100 s revealed that the peak current of INZ reached a maximum (3.797 µA) at an electrodeposition time of 20–40 s (Fig. 1b1–b2). The peak current gradually decreased as the electrodeposition time increased beyond 40 s. This decrease is attributed to the increased electrodeposition time leading to the formation of thicker layers that impede the diffusion of the analyte to the electrode surface [52]. Therefore, the optimum modification condition for SPCE was selected as a concentration of 10−4 M CuSO4 for 40 s.

Fig. 1
figure 1

CVs of modified SPCEs for the oxidation of INZ by different a1 concentrations of CuSO4; b1 electrodeposition time; and a1, a2, b1, b2 their corresponding anodic current plots (recorded in 100 μM INZ in 0.1 M PBS/pH = 7 with a scan rate of 50 mV/s). c CVs are recorded at SPCE, aSPCE, and CuO/SPCE, and their corresponding analytics are in the inset (recorded in 0.5 mM K₃[Fe(CN)₆] in 0.1 M PBS/ pH = 7 at a scan rate of 50 mV). d Electrochemical impedances of the SPCE, aSPCE, and CuO/SPCE (upper inset), along with their corresponding circuit diagram (lower inset) (recorded in 0.5 mM K₃[Fe(CN)₆] in 0.1 M PBS/ pH = 7 with a scan rate of 50 mV/s, a frequency range from 100,000 to 0.01 Hz, and an amplitude of 10 mV RMS)

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In investigating the electrochemical behavior of electrodes, particular attention was directed towards examining the redox dynamics of Fe(CN)6. Figure 1c presents a comprehensive comparison of CV analyses performed at various SPCE. CVs were conducted using 0.5 mM K3[Fe(CN)6] in 0.1 M PBS with a pH of 7 at a scan rate of 50 mVs−1. Notably, the activation processes of SPCE resulted in heightened peak heights, indicating an optimal electrochemical response [53]. Conversely, upon CuO modification, a slight decrease in peak height was observed for CuO/SPCE, although the peak height still exceeded that of the bare SPCE, suggesting a diffusion-controlled mechanism of reactants at the electrode interface [50]. At the bare SPCE, a pair of less distinct oxidation peaks (E = 0.524 V, I = 4.009 µA) without any reduction peaks suggested a sluggish electron transfer at the interface, potentially attributed to insulation additives within conductive inks. However, SPCE activation manifested well-defined oxidation (E = 0.310 V, I = 10.86 µA) and reduction (E =  − 0.131 V, I =  − 26.68 µA) peaks, indicative of enhanced electron transfer kinetics. Notably, the electron transfer kinetics at the CuO/SPCE exhibited further enhancement, as evidenced by both oxidation (E = 0.290 V, I = 6.951 µA) and reduction (E =  − 0.118 V, I =  − 17.57 µA) peaks, demonstrating reduced peak potential separation and heightened peak currents compared to the SPCE [50]. These observations substantiate the immobilization of CuO on the SPCE surface, thereby elucidating its role in augmenting electrochemical performance.

The electrochemical formation of CuO on an SPCE involves sequential redox steps. Initially, copper (II) ions (Cu2⁺) from a copper-containing solution (e.g., CuSO₄) are reduced at the SPCE surface during electrodeposition (Eq. 1), leading to the deposition of metallic copper. Upon applying a positive potential, the metallic copper is oxidized to copper (I) oxide (Cu₂O) (Eq. 2), forming a Cu₂O layer on the electrode. Continued application of a positive potential or exposure to air/oxygen further oxidizes Cu₂O to copper (II) oxide (CuO) (Eq. 3). Alternatively, direct oxidation of copper to CuO can occur (Eq. 4), resulting in a stable CuO layer on the SPCE. The overall electrochemical mechanism involves the electrodeposition of copper onto the SPCE, followed by the stepwise oxidation of metallic copper to Cu₂O and finally CuO under oxidative conditions. As mentioned above, this process is influenced by parameters such as applied potential, pH, and deposition time.

$${\text{Cu}}^{2+}+2{\text{e}}^{-}\to {\text{Cu}}_{(\text{s})}$$
(1)
$$2{\text{Cu}}_{(\text{s})}+{\text{H}}_{2}\text{O}\to {\text{Cu}}_{2}{\text{O}}_{(\text{s})}+2{\text{H}}^{+}+2{\text{e}}^{-}$$
(2)
$${\text{Cu}}_{2}{\text{O}}_{(\text{s})}+2{\text{H}}^{+}+2{\text{e}}^{-}\to 2{\text{CuO}}_{(\text{s})}$$
(3)
$${\text{Cu}}_{(\text{s})}+{\text{H}}_{2}\text{O}\to {\text{CuO}}_{(\text{s})}+2{\text{H}}^{+}+2\text{e}^{-}$$
(4)

Electrochemical impedance spectroscopy (EIS) was a crucial technique for evaluating the interfacial properties between the electrolyte solution and the sensing interfaces. Typically, the Nyquist plot obtained from the EIS displays a low-frequency linear segment, characteristic of a diffusion-controlled process, and a high-frequency semicircular segment (Rct), indicative of the restricted electron transfer process. In Fig. 1d, the investigation of the electron transfer kinetics for various SPCE was conducted via impedance measurements utilizing the Fe(CN)63−/4− redox probe. Employing 0.5 mM K3[Fe(CN)6] in 0.1 M PBS with a pH of 7 at the electrodes, a scan rate of 50 mV.s−1, a frequency range of 100,000–0.01 Hz, and an amplitude of 10 mV RMS. The charge transfer resistance (Rct) values for bSPCE (bare SPCE), aSPCE (active SPCE), and CuO/SPCE (modified) was accomplished by fitting the data in Nyquist plots, resulting in values of 16.95, 4.928, and 7.715 kΩ, respectively. The observed decrease in Rct indicates enhanced conductivity, suggesting that the electrochemical treatment of CuO augments the electrochemical activity of SPCE [54]. Notably, the lower Rct values for the CuO/SPCE electrodes suggest a relatively rapid charge transfer process compared to SPCE. The comparative analysis underscores CuO’s role in minimizing Rct at the interface, thereby improving the effective electrontransfer rate. This finding resonates with Bergamini et al. on the poly-l-histidine-modified SPCE [23], further validating the efficacy of CuO in enhancing electron transfer kinetics.

The surface morphology was examined before and after electrochemical pretreatment using SEM (Fig. 2a–b). A comparison of the bare SPCE and CuO/SPCE surfaces revealed notable differences. The bare SPCE surface exhibited a rough texture characterized by large pores, multilayered structures, and a sponge-like network filled with nanoscale cavities. In contrast, the CuO/SPCE surface appeared significantly smoother. The electrochemical treatment in CuSO₄ solution modified the bare SPCE surface, resulting in a smoother, defect-free surface compared to the pristine electrode. Particles were observed on the surface of the SPCE, with an average diameter ranging from 80 to 120 nm. AFM analysis revealed notable changes in surface topography following CuO modification (Fig. 2c1–c2), showing a maximum height of 1.04 µm and an average surface roughness of 90.72 nm. These results confirm the successful grafting of the imprinted layer onto the electrode surface, thereby demonstrating an effective modification. EDX and elemental mapping confirmed the presence of Cu with a homogeneous distribution (Fig. 3a–b). These results agree with previous studies and demonstrate the potential of CuO/SPCE for sensitive electrochemical detection applications [55, 56]. It is worth noting that the XRD experiments did not reveal the characteristic peaks of CuO crystals (data not shown), which may be due to the presence of CuO in an amorphous rather than crystalline form on the surface of the SPCE.

Fig. 2
figure 2

SEM images of a bare SPCE and b CuO/SPCE electrodes. c1–c2 AFM mages and cross-sectional analysis of CuO/SPCE

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Fig. 3
figure 3

EDX spectrum of a bare SPCE and b CuO/SPCE electrodes with elemental contents and the corresponding elemental mappings in the inset

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The chemical valence states of the primary elements C, O, and Cu were investigated using X-ray photoelectron spectroscopy (XPS). The overall XPS spectrum indicates the presence of C (307.1 eV), O (556.1 eV), and Cu (996.1 eV) on the SPCE surface (Fig. 4a). In the high-resolution spectrum of C1s (Fig. 4b), four characteristic peaks are observed at binding energies of 284.8, 286.3, 288.4, and 292.9 eV, suggesting the presence of various carbon oxidation states. The high-resolution spectra of O1s (Fig. 4c) exhibit peaks at 532.9 and 531.4 eV, which can be attributed to the C = O and Cu–O bonds, respectively [57]. The high-resolution spectra of Cu 2p (Fig. 4d) provide further insights into the chemical state of copper. The Cu 2p3/2 and Cu 2p1/2 doublets observed at 934.2 and 952.6 eV, respectively, are indicative of Cu2+ oxidation states [58]. Additionally, the satellite peaks at 940.4, 944.3, and 954.6 eV further confirmed the presence of Cu2+ ions [59]. These results demonstrate that the modifier electrodeposited on SPCE primarily consists of CuO.

Fig. 4
figure 4

XPS spectrum of CuO/SPCE: a survey and high-resolution spectra of b C 1 s, c O 1 s, and d Cu 2p

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Electrochemical behaviors of INZ at CuO/SPCE

The voltammetric characteristics of INZ were analyzed using CuO/SPCE given the drug’s significant pharmaceutical implications. In Fig. 5a, CVs were acquired at the bare bSPCE, aSPCE, and CuO/SPCE in a solution containing INZ. CVs were conducted at a scan rate of 50 mV/s, employing 100 μM INZ in 0.1 M PBS with a pH of 7. While the bare SPCE yielded no discernible peaks, the aSPCE displayed a singular broad peak at 0.308 V, registering a peak current of 2.794 μA. Conversely, the CuO/SPCE exhibited a similar broad peak, albeit at 0.278 V, with a heightened peak current of 3.954 μA. The comparison of peak currents between the aSPCE (E = 0.308 V, I = 2.794 μA) and CuO/SPCE (E = 0.278 V, I = 3.954 μA) underscores the enhanced sensitivity of CuO/SPCE. To evaluate the efficacy of CuO, various metal oxide nanostructures (such as NiO, CdO, MgO, Bi2S3, Bi12NiO19, and Bi2O3) were summarized for the existing literature, drawing comparisons with the determination of INZ [60]. It was observed that CuO/SPCE exhibited commendable sensitivity and stability, along with notable peak potential separation, rendering it a promising candidate for the precise determination of INZ molecules.

Fig. 5
figure 5

a CVs of INZ were recorded at bSPCE, aSPCE, and CuO/SPCE (CVs of 100 μM INZ in 0.1 M PBS/ pH = 7) were recorded at a scan rate of 50 mV/s). b1 CVs of INZ (100 μM) were recorded at CuO/SPCE at various scan rates (in 0.1 M PBS/ pH = 5), and their respective plots of b2 anodic peak currents vs. the scan rates and b3 potential vs. lnν. c1–c2 CVs of INZ (100 μM) were recorded at CuO/SPCE at various pH (with a scan rate of 50 mV/s) and their respective plots of anodic peak currents. All corresponding data are tabulated in the inset

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The influence of scan rate on the electrochemical oxidation of INZ was meticulously examined within a buffer environment at pH 5.0. In Fig. 5b1–b2, the CV traces are derived from experiments conducted on CuO/SPCE submerged in 0.1 M PBS at pH 5. Experiments were performed using INZ concentrations of 100 μM and scan rates ranging from 10 to 100 mV/s. It is worth noting that no reduction peak corresponding to INZ was observed in the CV plots, indicating the analytical stability of the compound under the specified experimental conditions. A significant increase in peak current intensity was observed with increasing scan rate, along with a concurrent positive shift in peak potential, indicating enhanced electrochemical activity. A crucial aspect of the analysis was the direct correlation observed between the peak current and the scan rate, which demonstrated a well-defined linear relationship (I = 1.062 + 0.047v, R2 = 0.991). The close alignment of the slope value with the expected theoretical benchmark of 0.05 confirms that the oxidation of INZ at the electrode surface is governed by an absorption-controlled electrochemical process rather than surface adsorption. This emphasized the pivotal role of mass transport in the electrochemical behavior of INZ and underscores the effectiveness of cyclic voltammetry in analyzing the kinetic parameters of redox-active pharmaceutical compounds.

The peak potential (Ep) shifted to more positive values as the scan rate increases from 10 to 60 mV s − 1 (Fig. 5b3), confirming the irreversible nature of the electrode process. The relationship between Ep and lnν is described by the equation Ep (V) = 0.028 + 0.038 lnν (mVs−1). For an adsorption-controlled and irreversible electrode process, as described by Laviron [61], Ep is given by Eq. 5:

$${E}_{p}={E}_{0}-\frac{RT}{\alpha nF}\text{ln}\left(\frac{\alpha nF}{RT{k}_{s}}\right)-\frac{RT}{\alpha nF}\text{ln}v$$
(5)

Here, α is the transfer coefficient, k is the standard heterogeneous rate constant, n is the number of electrons transferred, ν is the scan rate, and E0 is the formal redox potential. Other symbols have standard meanings. The value of αn can be derived from the slope of the Ep vs. lnν plot. In this system, the slope was found to be 0.038, and at T = 298 K, using the values for R and F, αn was calculated to be 0.67. Additionally, α can be estimated using the formula as presented in Eq. 6 [62]:

$$\alpha =\frac{47.7}{{{E}_{p}-E}_{p/2}}\text{mV}$$
(6)

Here, Ep/2 is the potential at which the current is half the peak value. The calculated α values range from 0.29 to 0.42 at different scan rates, leading to a calculated n value ranging from 1.60 to 2.23. Thus, it can be concluded that two electrons are transferred during the electrooxidation of INZ.

The investigation further delved into assessing the impact of pH on the electrochemical detection of INZ using CuO/SPCE, underlining the pivotal role of medium selection in achieving precise electrochemical readings of INZ. The study presented in Fig. 5c1–c2 elucidates the variation in electrochemical responses of 100 μM INZ across a pH range of 3 to 7, using 0.1 M PBS as the solvent at a scan rate of 50 mV/s. At extremely low pH levels (1 or 2), the oxidation peak was unstable, likely due to the gradual dissolution of CuO under highly acidic conditions, and was therefore excluded from the CV curves. However, starting from pH 3, an increase in the pH of the medium resulted in a notable shift in the oxidation potentials of INZ toward lower, less positive values. The nature of the shift signifies pH 5 as the optimal condition for the accurate detection of INZ, as evidenced by the linear relationship between peak potential (E) and pH, displaying a slope of 50 mV/pH (E = 0.620–0.050 pH, R2 = 0.993), closely approximating the theoretical Nernstian slope of 59 mV/pH. This approximation suggests a stoichiometric balance in the transfer of electrons and protons during the oxidation of INZ.

The oxidation mechanism of INZ at CuO/SPCE primarily involves a 2e − /2H⁺ transfer process. The protonated form of INZ, characterized by its pKa value of approximately 4.84, is crucial for facilitating electrostatic interactions with the negatively charged CuO surface. Under alkaline conditions, the oxidation peak splits, indicating distinct one-electron–one-proton transfer reactions for each peak, ultimately leading to the hydrolysis of INZ to isonicotinic acid. According to Rajkumar et al. [63], the electro-oxidation reaction of INZ at CuO/SPCE is illustrated in Scheme 1. These findings highlight that a PBS buffer at pH 5 provides an optimal electrolytic environment for the electrochemical quantification of INZ in real-world samples, thus confirming the robustness and precision of CuO/SPCE for drug analysis.

Scheme 1
scheme 1

The electro-oxidation reaction of INZ at CuO/SPCE (adapted with permission from Springer [63])

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Analytical performance of CuO/SPCE

Electrochemical detection of INZ

DPV was used to evaluate the analytical performance of CuO/SPCE, focusing on their sensitivity, linear response ranges, and detection thresholds for INZ detection. The DPV measurements were conducted under specified conditions, including a PBS of pH 3.0, a scan rate of 4 mV/s, amplitude of 50 mV, a pulse width of 0.2 s, and a pulse period of 0.5 s. The electrochemical behavior of INZ was analyzed at an optimized pH of 5.0 in PBS buffer, with Fig. 6a1 illustrating the incremental increase in peak current corresponding to successive INZ additions. The analytical calibration curve demonstrated a linear response within the concentration range of 4 to 200 μM, described by the equation I = 0161 + 0.008c and a correlation coefficient (R2) of 0.991 (Fig. 6a1). To calculate LoD based on the standard deviation of the response and the slope method, a standard equation (LoD = 3 s/m, where s is the standard deviation of the response and m is the slope of the related calibration line) has been used. Based on the initial linear equation, a LoD of 8.39 μM. It is worth mentioning that the LOD, which was estimated using the standard deviation of the response and the slope method, is influenced by experimental precision and detection sensitivity, which can cause the LOD to occasionally exceed the maximum concentration range. Similar to the LoD, the limit of quantification (LOQ = 10 s/m) can also be estimated using the standard deviation of the response and the slope method [64], resulting in a LOQ of approximately 27.96 μM. Overall, the CuO/SPCE’ exceptional performance highlights their potential as a reliable platform for the electrochemical quantification of INZ in real samples. Additionally, a LoD comparable to that of INZ, as achieved through various modifications of the SPEs (Table 1), validates the sensor’s efficacy and applicability for precise analyses.

Fig. 6
figure 6

a1 DPVs of CuO/SPCE at varying concentrations of INZ (were conducted in 0.1 M PBS/ pH = 5 with the following parameters: scan rate of 4 mV s − 1, amplitude of 50 mV, pulse width of 0.2 s, and pulse period of 0.5 s) and a2 their calibration plots. b The calibration plots of CuO/SPCE at different concentrations of INZ were recorded (in 0.1 M PBS/ pH = 5) using the standard addition method (corresponding data are tabulated in the inset)

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Table 1 Comparison of the analytical performance of different modified electrodes for INZ detection
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Stability, repeatability, and reproducibility

SPCEs have become a preferred method in electroanalytical chemistry due to their cost-effectiveness, disposability, and capability to provide a highly reproducible and reliable platform for the electrochemical analysis of target analytes. However, ensuring stability and reproducibility remains a significant challenge. To address this need, CuO/SPCEs incorporating active materials or biorecognition elements have been developed. To assess the stability of the CuO/SPCE, seven repetitive INZ measurements were conducted using DPV in 0.1 M PBS (pH 3.0) (Table 2(a)). The relative standard deviations for INZ were found to be 0.27% in DPV and 1.20% in LSV for seven successive measurements on a single CuO/SPCE. Using the statistical analysis, the p-values for both stability data (E/V and I/μA) are 1.0, indicating no statistically significant difference from the reference mean. The effect size (Cohen’s d) are 0.0, suggesting a negligible difference between the data and the reference mean. Thus, the electrode stability showed no significant variation from average performance. Likewise, evaluations on five individually prepared CuO/SPCEs showed a relative standard deviation of 1.14% in DPV and 3.58% in LSV, demonstrating satisfactory reproducibility (Table 2(b)). Furthermore, the CuO/SPCE exhibited reproducible signals not only on the same electrode but also with separately employed SPEs. Three electrodes tested under the same conditions yielded a relative standard deviation of 1.23% for the INZ drug. The results were consistent with SPCE modified with bismuth oxide nanorod for electrochemical sensing of INZ [60]. Overall, CuO/SPCE is suitable for determining INZ in practical applications. A single-use SPCE is employed due to its cost-effectiveness, it is important to note that one SPCE can be reused multiple times. This versatility allows for various experiments examining electrochemical behavior, such as pH dependence and scan rate effects, as well as assessing analytical performance aspects like calibration and interference, all conducted using a single SPCE.

Table 2 (a) Stability and (b) reproducibility of seven and five successive measurements on a single and an individual CuO/SPCE
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Interference study

The potential interference of various coexisting species with INZ in real samples was thoroughly investigated (Table 3). The DPV was utilized to evaluate the sensor’s selectivity towards INZ. The findings indicate that the presence of a 100-fold concentration of CO32−, SO42−, PO43−, Ca2+, K+, and Na+; a 50-fold concentration of glucose and sucrose; a 20-fold concentration of vitamin C; and a 100-fold concentration of dopamine and uric acid did not interfere with the detection of INZ. These results are consistent with previous interference experiments conducted using MoS2 nanosheets modified SPCE for INZ detection [65]. Consequently, the modified SPCE demonstrates promising potential as a disposable electrode for practical pharmaceutical analysis.

Table 3 Evaluation of potential interference from coexisting species in the detection of INZ using DPV
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Real sample analysis

To assess the practical utility of the introduced CuO/SPCE, an analysis utilizing the standard addition method was conducted to ascertain the presence of INZ within pharmaceutical tablet samples (Fig. 6b). The use of the DPV technique alongside the standard addition approach facilitated this investigation. The analytical calibration curve exhibited a linear response with the concentration of standard additions ranging from 20 to 100 μM, as described by the equation I = 0.113 + 0.028c and a correlation coefficient (R2) of 0.996. The results yielded calculated values of 0.089 mg per tablet, exhibiting remarkable proximity to the labeled values of 0.01 mg per tablet. In this experimental protocol, standard solutions were deliberately introduced into authentic samples, and the anodic peak current of INZ (n = 3). To ensure robust validation, solutions were spiked with INZ concentrations within the linear response range. The recovery rates ranged between 99.50 and 105.03% concerning the pharmaceutical formulations under scrutiny (Fig. 6b inset table). These outcomes underscore the practical relevance and efficacy of the proposed sensor system.

Conclusion

In conclusion, this study introduces a novel and efficient method for the electrochemical detection of INZ using an SPCE modified with CuO. The CuO/SPCE demonstrated exceptional sensitivity with a linear response to INZ concentrations ranging from 4 to 200 μM and an LoD of 8.39 μM. The stability and reproducibility of CuO/SPCE were confirmed, with minimal interference from common coexisting species. Analysis of pharmaceutical tablets using the standard addition method yielded recovery rates between 99.50 and 105.03%, thus validating the sensor’s reliability for practical applications. These findings underscore the potential of CuO/SPCE as a cost-effective and efficient electrochemical detector of INZ in pharmaceutical samples. Future research will focus on optimizing sensor performance and exploring its applicability to other drug compounds, thereby advancing electrochemical sensing technology in pharmaceutical analysis. The ease of preparation, low cost, environmental friendliness, and biocompatibility of CuO-modified SPCE make it a practical choice for enhancing electrochemical sensing capabilities across various fields.