Sensitive and Selective Electrochemical Detection of Epirubicin as Anticancer Drug Based on Nickel Ferrite Decorated with Gold Nanoparticles

Abstract

The accurate and precise monitoring of epirubicin (EPR), one of the most widely used anticancer drugs, is significant for human and environmental health. In this context, we developed a highly sensitive electrochemical electrode for EPR detection based on nickel ferrite decorated with gold nanoparticles (Au@NiFe₂O₄) on the screen-printed electrode (SPE). Various spectral characteristic methods such as Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-Vis), energy-dispersive X-ray spectroscopy (EDX) and electrochemical impedance spectroscopy (EIS) were used to investigate the surface morphology and structure of the synthesized Au@NiFe₂O₄ nanocomposite. The novel decorated electrode exhibited a high electrocatalytic activity toward the electrooxidation of EPR, and a nanomolar limit of detection (5.3 nM) was estimated using differential pulse voltammetry (DPV) with linear concentration ranges from 0.01 to 0.7 and 0.7 to 3.6 µM. The stability, selectivity, repeatability reproducibility and reusability, with a very low electrode response detection limit, make it very appropriate for determining trace amounts of EPR in pharmaceutical and clinical preparations.

1. Introduction

Epirubicin (EPR) (Scheme 1) is an anthracycline topoisomerase II inhibitor used for chemotherapy. Epirubicin, as an antitumor and anthracycline antibiotic derivative of doxorubicin, has been widely utilized for clinical therapy [1,2]. Doxorubicin and epirubicin(4′-epidoxorubicin) have only one difference, the spatial orientation of the 4′-moiety, and it has been exhibited effectively for treating leukemia, sarcoma and lymphoma [3,4]. EPR is an anticancer medication that works by selectively killing cancer cells rather than harming them by quickly dividing cells. Therefore, there may be a relationship between the EPR concentration and the clinical response. Low plasma EPR concentrations may suggest an ineffective prescription medication since the levels may be inadequate due to a significant molecular or complete cytotoxic response. As a result, measuring EPR levels in real biological samples is crucial for clinical diagnosis. In addition, cardiac problems such as heart failure and hair loss are crucial in the significant side effects of EPR and controlling the EPR concentration in the human body after cancer therapy.

EPR analysis has used a variety of analytical methods, including ultraviolet–visible spectroscopy (UV–Vis) [5,6], high-performance liquid chromatography (HPLC) [7,8,9,10,11], fluorimetry [12,13], mass spectrometry [14] and liquid chromatography–mass spectrometry/mass-spectrometry (LC-MS/MS) [15]. However, most of these techniques have drawbacks, such as long response times, complex analysis, high prices and limited sensitivity [16,17]. Voltammetric techniques, because of their quick reaction, high sensitivity and selectivity, and electrochemical techniques, particularly square wave voltammetry (SWV) and differential pulse voltammetry (DPV), have been used [18,19,20]. According to previous research, the performances of the electrochemical methods described are more appropriate than the other methods due to merit, such as a quick response to the analyte and the selectability in the presence of interfering agents [21,22,23,24,25,26]. Modifying electrode surfaces can remarkably enhance the surface sensitivity with appropriate stability and reproducibility [27,28,29,30,31]. Lately, the electrochemical properties of various binary metal oxides have been investigated widely due to their excellent conductivity, lower activation energy, appropriate structural stability and comparatively higher specific capacitance compared to the unitary metal oxides [32,33,34]. Nanomaterials have garnered a great deal of interest since they may be utilized in a variety of applications, including medical, electronics, energy storage and conversion systems, wastewater treatments, photovoltaic cells, catalysts, etc. [35,36,37,38,39]. Magnetic nanoparticles with the general formula MFe₂O₄ (M = Fe, Ni, Co, Cu, Mn) are among the most widely used materials in analytical chemistry, medicine and biotechnology. Due to their distinct benefits, they are increasingly being used for enzymes and other bioactive molecules. Nickel ferrite (NiFe₂O₄) nanoparticles (NPs) are a promising material for modified electrodes among various spinel transition metal oxides due to their high theoretical capacities (914 mA h/g), appropriate conductivity and low toxicity [40,41,42]. Due to its solid superparamagnetic characteristics, superior biocompatibility, high adsorbability and ease of production, NiFe₂O₄ nanoparticles are increasingly attracting interest in sensor and biosensor design [43]. Moreover, NiFe₂O₄ NPs show a high active surface area and low mass transfer resistance [44]. Metal nanoparticles (MNPs) are considered the most promising alternative for altering magnetic nanoparticle surfaces because of their size and their physical and chemical properties. The size and shape of metal nanoparticles are well recognized to impact electrochemical activity in sensing applications significantly. Due to their unique electrical, chemical and catalytic characteristics, various-shaped gold nanoparticles (Au NPs) such as nanospheres, nanorods and nanowires are widely utilized to construct electrochemical electrodes [45,46]. In addition, AuNPs have several benefits, including flexibility in size and shape management and excellent electrical and catalytic activity because of their superior conductivity and wide active surface area in the electrode [47,48]. These characteristics suggest that sensing applications aim to enhance the electron transport between the target molecules’ redox centers and electrode surfaces [49]. Firstly, the gold nanoparticles decorated on a nickel ferrite (Au@NiFe₂O₄) nanocomposite was synthesized in this work. There is no other original work on the electrochemical detection of EPR by Au@NiFe₂O₄ modified on SPE. Chemically produced Au@NiFe₂O₄ has a unique combination of characteristics, including a large electroactive surface area, good electron transferability and adsorptive properties, making it an appropriate sensing material for electrochemical determination EPR. The nanocomposite was characterized using field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), electrochemical impedance spectroscopy (EIS), UV–Vis, Fourier transform infrared spectra (FT-IR) and energy-dispersive X-ray spectroscopy (EDX). The screen-printed electrode (SPE) modified using a Au@NiFe₂O₄ nanocomposite (Au@NiFe₂O₄/SPE) exhibited an outstanding detection limit of 5.3 nM and extremely selective electrode in the presence of interfering agents with two different concentration ranges, 0.01 to 0.7 and 0.7 to 3.6 µM. In addition, EPR analysis with satisfactory recovery in real samples (human plasma, injection and urine) was first performed using a Au@NiFe₂O₄/SPE. In comparison to the other approaches, it can be claimed that the analytical method used in this study is preferable.

2. Materials and Methods

2.1. Materials

Epirubicin was purchased from the Council of Europe (France); Nickel(II) chloride (NiCl₂·6H₂O), Ferric chloride(FeCl₃ 6H₂O), ethylene glycol, polyvinylpyrrolidone (PVP), Gold (III) chloride trihydrate (HAuCl4 3H₂O, ≥49%), ascorbic acid, potassium hexacyanoferrate(III) and potassium hexacyanoferrate(II) were purchased from Sigma Aldrich Co (Burlington, MA, USA). Sera-Flex human blood plasma samples were also purchased from Dyna-Tek Industries Inc. (Lenexa, KS, USA). Additionally, a urine sample was obtained from a volunteer for use in the experiments. Epirubicin injection was also obtained from a local pharmacy. All of these substances were of analytical grade, and di-ionized water was utilized. The supporting electrolyte was 0.1 M Britton–Robinson buffer (B-R) prepared by mixing acetic acid (CH₃COOH), phosphoric acid (H₃PO₄), boric acid (H₃BO₃) and potassium chloride (KCl) with deionized water. The pH of the supporting electrolyte was monitored with a pH meter (Hanna Instruments, Woonsocket, Rhode Island, USA). In addition, a stock EPR solution in the deionized water was prepared before each experiment.

2.2. Instrumentation

The electrochemical techniques such as differential pulse voltammetry (DPV), cyclic voltammetry (CV) and chronoamperometry (CA) were conducted using a Metrohm-Autolab potentiostat/galvanostat system (PGSTAT128N, Metrohm, Herisau, Sweden). Electrochemical impedance spectroscopy was performed under a 0.1 Hz to 100 kHz frequency using an IVIUM Compactstat (Eindhoven, The Netherlands) device. The employed screen-printed electrode had a Ag/AgCl paste and Pt electrodes on their own surface, as a reference and a counter electrode, respectively. UV–Vis was recorded using a double beam spectrophotometer (Shimadzu, Kyoto, Japan) model UV-1800 and quartz cells (Hellma, Müllheim, Germany). SEM and EDX were observed micrographs of the materials by ZEISS GeminiSEM 560 at 3.00 kV. The X-ray diffraction pattern was recorded using a Rigaku smart laboratory diffractometer (operated at 40 kV and 20 mA) with a Cu Kα source at a wavelength of 1.540 Å. TEM images were performed using an FEI Tecnai G2 Spirit microscope (Thermo Fisher Scientific, Waltham, MA, USA) at 120 kV. All electrochemical measurements were performed at 27.5 °C unless otherwise specified.

2.3. Synthesis of NiFe₂O₄

Firstly, 4.76 g of NiCl₂·6H₂O (0.02 mol) and 10.82 g of FeCl₃·6H₂O (0.04 mol) were dissolved in 30 mL of ethylene glycol (solution 1). Secondly, 5.0 g of urea and 0.4 g of polyvinylpyrrolidone (PVP) were dissolved in 30 mL of ethylene glycol (solution 2). Thirdly, solution 1 and solution 2 were mixed and stirred at 400 rpm at 30 min with a magnetic stirrer. Finally, this solution was moved to Teflon, a lined hydrothermal vessel, and heated at 180 °C for 20 h. The hydrothermal vessel was cooled at room temperature, and NiFe₂O₄ NPs were collected with a magnet. The NiFe₂O₄ NPs were washed with water (three times) and ethanol (two times) and dried at 70 °C for 12 h.

2.4. Synthesis of Au@NiFe₂O₄

Firstly, 70 mg of the powder obtained from magnetite nanoparticles was ultrasonicated in 20 mL of deionized water for 30 min to gain a uniform solution. Then, 700.0 µL of a HAuCl₄ (0.1 g mL⁻¹) solution was added to the reaction mixture at 70 °C. Afterward, after 10 min, 560 µL of ascorbic acid (0.5 g mL⁻¹) was added to the reaction mixture, the stirring condition was maintained until the solution showed a slightly reddish color, and finally, the solution was cooled to room temperature and was separated using a magnet to form a magnetite-gold nanostructure. Next, Au@NiFe₂O₄ nanocomposites were washed four times with deionized water, and the resulting brown precipitate was dried at 70 °C.

2.5. Preparation of Au@NiFe₂O₄/SPE

The electrode surfaces were polished by 0.05-mm alumina slurries for 10 min and washed using a mixed solution of ethanol and water (1:1, v/v). Then, the mirror electrodes were dried at 25 °C for 1 h under argon gas. The electrode surfaces were modified using the optimized amount and concentration of NiFe₂O₄ and Au@NiFe₂O₄ nanocomposites (7.0 µL, 1.0 mg mL⁻¹) on the screen-printed electrode surface. The solvent was removed using an infrared heat lamp. Then, the developed electrodes were obtained as a NiFe₂O₄/SPE and a Au@NiFe₂O₄/SPE. All electrodes were kept in a sealed box without fluctuations of temperature and pressure [50].

2.6. Preparation of Real Samples

Human plasma, urine and injections were used as real samples to detect the EPR content using the standard addition method. Additionally, the urine sample was obtained from candidates without a disease history. The urine samples were filtered thoroughly using PTFE (0.45-micrometer) membrane filters. A certain amount of EPR solution was added to the urine solution to prepare EPR spiked. To prepare the human plasma sample, the acquired plasma sample was kept at 20 °C until the test to produce the human plasma sample. Firstly, an aliquot of plasma was fortified with EPR to reach a concentration of 5.0 mM EPR. Then, 1.0 mL of plasma sample containing EPR was treated with 1.0 mL of acetonitrile, employed as a plasma protein precipitating agent. The precipitated proteins were separated by centrifugation for 15 min at 10,000 rpm after a 45-s vortex step. Finally, appropriate volumes of the supernatant were transferred to a volumetric flask and diluted with pH 4.0 B-R buffer to an acceptable volume. To prepare an injection, a certain number of injections were diluted with the B-R buffer solution and then investigated to measure EPR using DPV, analyzing without more purification.

3. Results

3.1. Characterizations of Au@NiFe₂O₄ Nanocomposite

XRD patterns were utilized to illustrate the crystal structure, size and phase purity of the produced NiFe₂O₄ NPs and Au@NiFe₂O₄ nanocomposites (Figure 1A). The NPs were manually ground in an agate mortar for the powder XRD analysis sample. The planes (220), (311), (222), (400), (422), (511) and (440) corresponded to the 2θ values 30.5°, 35.6°, 38.1°, 43.4°, 54.2°, 57.6° and 63.2°. As a result, the sample has a face-centered cubic (FCC) structure and may be classified as an inverse spinel NiFe₂O₄ (ICCD 00-044-1458) [51]. The particle sizes of the nickel oxide and gold nanoparticles were investigated by Equation (1). The mean nanoparticle sizes were 32.6 and 14.0 nm for synthesized NiFe₂O₄ and Au nanoparticles, respectively. The particle size, D, was estimated using Scherrer’s equation, which is as follows [52]:

$$D=\frac{k\lambda }{\beta cose\theta }$$

(1)

where k presents the shape coefficient for reciprocal lattice point (0.9), β shows the FWHM of the peak, ϴ exhibits the Bragg angle and λ presents the wavelength of X-rays = 1.54 Å. Further, the XRD of Au@NiFe₂O₄ exhibits four additional peaks located at 38.1°, 44.1°, 54.6° and 76.4°, which are attributed to the (111), (200), (220) and (311) planes of Au, respectively, with the JCPDS ref. No 89-3697 [53]. Thus, the XRD patterns affirm the deposition of Au nanoparticles on the surface of covalently grafted NiFe₂O₄.

The FT-IR spectra for the crystalline NiFe₂O₄ and Au@NiFe₂O₄ nanocomposites were observed in the region from 4000 to 450 cm⁻¹, as exhibited in Figure S1. The band located at 1375 and 3444 cm⁻¹ attributed to the O-H bond bending and stretching vibrations, respectively [54]. The two main metal-oxygen bands at 695 and 487 cm⁻¹ are exhibited in the spectrum of the prepared NiFe₂O₄. These two bands are attributed to the vibration of ions in the crystal lattices, and the vibration absorption peak of C-O-Fe appears at 1115 cm⁻¹. The 1317 cm⁻¹ peak is assigned to the characteristic—CH₃ bending [55]. In the case of Au@NiFe₂O₄, the intensity increased due to the doping of Au nanoparticles on NiFe₂O₄. The above evidence strongly proves the successful covalent binding of Au@NiFe₂O₄. Figure 1B exhibits the UV–Vis spectra of the pure NiFe₂O₄ and Au@NiFe₂O₄ nanocomposites. Only the side-band adsorption at 750 nm was found in the UV–Vis spectra of NiFe₂O₄, which can be attributed to the d–d transition from Ni3d-_t2g to Ni3d-_eg [56]. NiFe₂O₄ has a conventional spinel structure, with Ni²⁺ and Fe³⁺ occupying the tetrahedral and octahedral positions of the cubic spinel lattice, respectively [57]. At the same time, the spectra of the Au@NiFe₂O₄ were dominated by strong absorptions in 570 and 745 nm due to the presence of Au nanoparticles on the surface of NiFe₂O₄. These results reaffirmed the synthesis of the Au@NiFe₂O₄ nanocomposite.

The structure and morphology of the NiFe₂O₄ and Au@NiFe₂O₄ samples and their chemical composition are analyzed using SEM micrographs. Figure 2A,B present SEM images of NiFe₂O₄ nanoparticles and the Au@NiFe₂O₄ nanocomposite. There is a significant amount of aggregation of particles observed in the SEM image of NiFe₂O₄. Figure 2B shows that many of the Au NPs with a diameter of about 10.0 nm adhered to the surface of the NiFe₂O₄ nanocomposite. The results suggested that Au nanoparticles were immobilized on the NiFe₂O₄ nanocomposite.

Moreover, the TEM images of Au@NiFe₂O₄ exhibited in Figure 2C,D indicate the presence of AuNPs over the NiFe₂O₄ nanocomposite. Thus, the AuNP immobilized NiFe₂O₄ nanocomposite may increase the electron-transfer process by increasing the electrical conductivity to enhance the electrochemical detection potential. Energy-dispersive X-ray analysis was also utilized to analyze the elemental compositions of the nanocomposite and its synergistic components (Figure S2 and Table S1). The results demonstrate that the Au@NiFe₂O₄ nanocomposite was successfully manufactured. As a result, all the spectroscopic and microscopic studies of the nanocomposite’s production method and size are compelling.

3.2. Electrochemical Performance

3.2.1. Influence of Modifier on the Electrochemical Oxidation of EPR

Cyclic voltammetry and DPV voltammograms were utilized to assess the redox behavior of the Au@NiFe₂O₄/SPE in a 0.1 M B-R buffer as a supporting electrolyte and compared with the NiFe₂O₄/SPE and a bare electrode. Figure S3 shows DPVs recorded with different modified and bare electrodes. After the modified Au@NiFe₂O₄, the peak current of the 0.5 µM EPR was approximately 3.1 times higher than the bare electrode, indicating that the effect of the increased active surface area and conductivity by synergic affects the nanocomposite. Therefore, the Au@NiFe₂O₄/SPE was utilized for further experiments. Moreover, the cyclic voltammograms of bare and modified electrodes in the absence of EPR in 0.1 M B-R (pH 4.0) were obtained to evaluate the effect of the nanohybrid on the electroanalytical performance of the bare electrode. The findings demonstrated that in the absence of EPR, thanks to the large electroactive surface area and the synergistic effects between the Au and Ni Fe₂O₄ nanoparticles, the response of the Au@NiFe₂O₄/SPE was detected higher than that of the bare electrode (Figure S4). The electrochemical performance of the proposed electrochemical sensor was studied by determining the electron transfer rate using the solution as a redox probe molecule in 0.1 M KCl. For this purpose, the peak-to-peak distance (ΔE_p) value between the anodic and cathodic signals, an essential indicator for the electron transfer rate, was determined at a bare SPE, the NiFe₂O₄/SPE and the Au@NiFe₂O₄/SPE with a scan rate of 50 mV s⁻¹. As seen in Figure 3A, the ΔE_p value of the Au@NiFe₂O₄/SPE (ΔE_p = 239 mV) was lower than that of the bare electrode (ΔE_p = 301 mV), exhibiting the presence of a faster electron transfer. In addition, an improvement obtained at both the anodic and cathodic signals has clearly revealed the strong electrocatalytic activity of the Au@NiFe₂O₄. Furthermore, Figure S5A exhibits the CV voltammograms of the Au@NiFe₂O₄/SPE in the presence of 5.0 mM [Fe(CN)₆]^3−/4− containing 0.1 M KCl at various scan rates from 10.0 to 300.0 mV s⁻¹ and the plot of the peak current against the square root of scan rate is exhibited in Figure S5B. Figure S5A shows that with an increasing scan rate, the width of the voltammograms gradually increased. The anodic/cathodic peaks current density enhanced dramatically at high scan rates. Therefore, electron exchange happens slowly at the electrode surface to record peak currents at low scan rates, resulting in a thinner voltammograms with shorter current density peaks. The scan rate study revealed a clear relationship between the peak current and the specified range of the square roots of scan rates.

The electrochemical active surface area (EASA) of the Au@NiFe₂O₄/SPE was conducted using CV analysis in 0.1 M B-R buffer as a supporting electrolyte, containing 5.0 mM [Fe(CN)₆]^3−/4− as a redox probe and compared with the bare electrode and the NiFe₂O₄/SPE. The EASA of the electrodes was evaluated using the slope value of the peak current against various square root scan rate plots with the Randles–Sevcik equation (Equation (S1)). As a result, the active surface area was observed at 0.26, 0.16 and 0.069 cm² for the Au@NiFe₂O₄/SPE, the NiFe₂O₄/SPE and the bare SPE, respectively. On the other hand, the active surface area of the Au@NiFe₂O₄/SPE is about 3.8-fold higher than the area of the bare electrode, showing that the Au@NiFe₂O₄/SPE is the most appropriate for electrocatalytic sensing applications.

The electron transport characteristics of changed electrodes can also be studied using electrochemical impedance spectroscopy. The charge transfer resistance (R_ct) for the electrodes could be observed through the semicircle diameter. Figure 3B exhibits the Nyquist plot of the Au@NiFe₂O₄/SPE, the NiFe₂O₄/SPE and the bare SPE in the presence of 5.0 mM [Fe(CN)₆]^3−/4− in 0.1 M KCl. The R_ct at the bare SPE, the NiFe₂O₄/SPE and the Au@NiFe₂O₄/SPE are approximately 6.32, 4.33 and 2.97 kΩ, respectively. The semicircle diameter of the Au@NiFe₂O₄/SPE showing that Rct was decreased at the developed electrodes. The EIS measurements demonstrate that the Au@NiFe₂O₄/SPE has a better electrochemical activity and conductivity than the bare electrode. Therefore, it can be inferred that the Au@NiFe₂O₄/SPE indicates a synergistic effect by combining the Au and NiFe₂O₄ and the Au@NiFe₂O₄/SPE nanocomposite improves the electron transfer process.

3.2.2. Heterogeneous Electron Transfer Rate Constant (K°)

The ability of the Au@NiFe₂O₄/SPE was investigated in increasing the rate of electron transfer. The electrical conductivity and surface resistance have a reciprocal relationship. The electrical conductivity of the Au@NiFe₂O₄/SPE is higher than the bare SPE and the NiFe₂O₄/SPE, suggesting improved electron transport kinetics. It is due to the greater surface area and higher conductivity of the Au@NiFe₂O₄/SPE. Equation (S2) is utilized to observe the standard heterogeneous rate constant. The K° values for the Au@NiFe₂O₄/SPE, the NiFe₂O₄/SPE and the bare electrode are 1.96 × 10⁻⁸, 1.22 × 10⁻⁸ and 8.41 × 10⁻⁹ cm s⁻¹, respectively. The K° values represent the estimated kinetic facilities of redox couples. A higher K° achieves equilibrium in a shorter amount of time, implying a quicker electron transfer.

3.3. Optimization of Conditions for Developing Sensitive and Selective Au@NiFe₂O₄/SPE

3.3.1. Effect of Physical and Chemical Properties

Optimal conditions affect the activity and sensitivity of the electrochemical electrode and can be useful for the sensitivity of the developed electrodes. Therefore, the following parameters were obtained: certain concentration and amount of nanocomposite, supporting electrolyte and its pH, scan rate, temperature and stirring of electrolyte at the Au@NiFe₂O₄/SPE.

To obtain the optimum concentration to achieve the highest current efficiency, the impact of the Au@NiFe₂O₄ dosage on the surface of the electrode was investigated, in the range of 0.1–2.0 mg mL⁻¹, and the obtained data are exhibited in Figure S6. As shown, raising the Au@NiFe₂O₄ amount from 0.1 to 1.0 mg mL⁻¹ increased the current percentage substantially. It might be because a greater surface area and more adsorption functional sites are available. On the other hand, at higher concentrations (higher than 1.0 mg mL⁻¹), there is a significant decrease, which might be related to overlying or aggregating accessible binding sites and reducing the total available adsorbent surface area. In the next step, investigating the effect of the nanocomposite amount 4.0–10.0 µL of Au@NiFe₂O₄ suspension was dropped to the surface of the electrode, drying at room temperature. The voltammetric behavior of different Au@NiFe₂O₄ suspension amounts was studied using 5.0 µM EPR (Figure S7). The results showed that the highest I_p was obtained with 7.0 µL of Au@NiFe₂O₄. Upon a further increment in the amount of the suspension, the adherence of the modifier layer on the surface of the electrode would be reduced.

Furthermore, the analyte diffusion through the dense layer of modifier would be hindered, which causes a noticeable decrease in the sensitivity of the modified SPE. As a result, the optimal volume of Au@NiFe₂O₄ as a modifier nanocomposite, increasing the conductivity and activity to increase the electron transfer process, is 7.0 μL. The effect of various supporting electrolytes, various temperatures and stirring rates was also observed using DPV in the presence of 1.0 µM EPR at pH 4.0 (Figures S8–S10).

3.3.2. The Effect of pH

The pH is an essential factor in the electrochemical behavior of a developed electrode. The activity of EPR in real samples due to the pH of the solution can affect the adsorbent surface in the presence of OH⁻ and H⁺. According to the DPV signals of the Au@NiFe₂O₄/SPE in the range of pH 3.0–6.0, the oxidation current of EPR enhanced with an increase in the pH value from 3.0 to 4.0, and after pH 4.0, the signals decreased at the surface of the Au@NiFe₂O₄/SPE. Additionally, the oxidation peak potential of EPR shifted to the left by increasing the pH value (Figure 4A) with a slope of 60.4 mV/pH at the surface of the Au@NiFe₂O₄/SPE that is close to the Nernstian value (59.0 mV/pH) for an equal number of electrons and protons in the redox system (Figure 4B). The results confirm that the best oxidation current occurs at pH = 4.0, and this value is selected as the optimum pH for further experiments.

3.3.3. The Effect of Scan Rate

Figure 4C exhibits the effect of the scan rate on the redox peak current responses of the Au@NiFe₂O₄/SPE in the presence of 5.0 µM EPR containing a 0.1 M B-R buffer at a pH 4.0 scan rate ranging from 10.0 to 300.0 mV s⁻¹. A systematic increase in I_pa and I_pc and ∆Ep was observed with an increase in the scan rate. The results of the relationship between the current of EPR and ν^1/2 on the surface of the Au@NiFe₂O₄/SPE are shown in Figure 4D. The equations I_pa = 0.4152 ν^1/2 −0.6893 (R² = 0.999) and I_pc = −0.331 ν^1/2 +1.1423 (R² = 0.9845) were observed for this study for the oxidation and reduction in EPR. These equations and the linear relations between current and ν^1/2 at the Au@NiFe₂O₄/SPE suggest that the electro-oxidation of EPR is under diffusion control. The slope of the logarithm of peak current vs. the logarithm of scan rates (Figure 4E) also reaffirmed that the electrode progression was a diffusion-controlled electrode process because the slop of the log I_p vs. log v is close to 0.5. Figure 4F exhibits a linear relationship between E_pa and ln v for EPR, as presented in the following equation: E_pa (V) = 0.0679 ln v + 0.4292 (R² = 0.995). In Laviron’s theory, for a reversible electrode reaction, E_pa is determined by Equation (S3). Hence, the number of transferred electrons in the electro-oxidation of EPR is estimated to be 0.78 (≅1). The results confirmed that one electron and one proton are transferred during the electro-oxidation of EPR on the Au@NiFe₂O₄/SPE. Based on our observations using Laviron’s theory and slope of E_pa vs. pH, a possible electrode reaction mechanism proposed for EPR was illustrated in Scheme 2, in which one proton and one electron transfer was involved and caused the oxidation of the hydroxyl group to quinine. The results also demonstrated that the reversible electron process led to the formation of the quinolinic structure of EPR.

The corresponding results to optimize the modified electrode are shown in the supporting information. The following optimal experimental conditions were observed: the certain concentration of nanocomposite: 1.0 mg mL⁻¹; the amount of nanocomposite: 7.0 μL; the optimal supporting electrolyte and pH value: B-R buffer at pH 4.0; the electrolyte temperature and stirring rate: 25.0 °C and 400 rpm.

3.3.4. Chronoamperometric Study

The chronoamperometric evaluation was performed via adjusting the Au@NiFe₂O₄/SPE potentials at 0.6 V with different concentrations of EPR (200.0, 300.0, 400.0 and 500.0 μM) containing 0.1 M B-R buffer at pH 4.0 (Figure 5A). Cottrell’s equation (Equation (S4)) was utilized to describe the current responses (I) for the diffusion coefficient electrocatalytic procedures of electroactive substances. By plotting I versus t^−1/2, the linear curve was observed for various concentrations of EPR. Afterward, the obtained direct lines’ slope was drawn against the EPR concentration (Figure 5B). Finally, the diffusion coefficient of 4.72 × 10⁻⁶ cm² s⁻¹ was evaluated for a developed EPR electrode.

3.4. Determination of EPR at the Au@NiFe₂O₄/SPE

The analytical performance of the Au@NiFe₂O₄/SPE was performed by analyzing EPR at diverse concentrations using DPV under optimized experimental conditions. As shown in Figure 6A, the oxidation peak current of EPR has enhanced linearly with increasing EPR concentrations at the ranges 0.01–0.7 and 0.7–3.6 μM with slopes of 1.5758 and 0.4008 μA μmol L⁻¹, respectively. The corresponding linear regression equations were observed as I_pa (μA) = 1.5758 C_EPR (μM) + 0.3245 (R² = 0.9909) and I_pa = 0.4008C_EPR + 1.1324 (R² = 0.997) (Figure 6B). The second linear segment’s reduction in sensitivity (slope) is most likely due to a kinetic limitation. Therefore, the limit of detection (LOD) of the Au@NiFe₂O₄/SPE was calculated as 5.32 nM (RSD = 5%) using the equation LOD = 3.3 s/m [22], where ‘s’ is the standard deviation of the 10 repeated measurements at the calibration range’s lowest concentration, and ‘m’ is the calibration curve’s slope, respectively. The analytical detection figures of merit are summarized in

Table 1. Determination figures of merit for EPR at Au@NiFe₂O₄/SPE.

Metrics	Au@NiFe2O4/SPE
response variability (RSD, %)	3.56
sensitivity (μA μmol L−1)	1.5758 ± 0.14 a
linear dynamic range (μM)	0.01–0.7 and 0.7–3.6
correlation coefficient	0.9909 and 0.997
limit of detection (LOD, nM)	5.32

^a Mean ± Standard deviation for n = 3.

. These results suggest that the Au@NiFe₂O₄/SPE could be used in a real environment to detect medically relevant concentrations of EPR.

The obtained analytical parameters were compared to similar reported analytical sensors and other analytical methods such as HPLC, fluorescence and LC-MS/MS, which have been utilized to sense EPR (

Table 2. Detection figures of merit for other methods for EPR reported in the literature.

Method	Modified Electrode	LOD (μM)	LWR a (μM)	Ref.
DPV b	Ce-ZnO/GCE c	0.0023	0.01–600	[58]
SWV d	Au/MWNTs-ZnO/SPE	0.0025	0.005–0.2	[59]
LSV e	SWNT–DCP/SPE f	0.02	0.05–10	[60]
SWV	CoFe2O4/1,3-DPIBr g/CPE	0.01	0.04–450	[61]
fluorescence	CMC-CdTe/ZnS QDs	0.04	-	[62]
HPLC	-	0.0149	1.87–187	[11]
LC–MS/MS	-	1.87	5.6–374.5	[15]
DPV	Au@NiFe2O4/SPE	0.0053	0.01–3.6	Our work

^a Linear working range, ^b differential pulse voltammetry, ^c cerium-doped ZnO nanoflowers, ^d square wave voltammetry, ^e linear sweep voltammetry, ^f single-walled carbon nanotube/dicetyl phosphate, ^g 1,3-dipropylimidazolium bromide.

). The analytical performance of the Au@NiFe₂O₄/SPE, which has wide dynamic linearity and low LOD value, was almost much more appropriate than other comparative analytical methods toward detecting EPR.

3.5. Selectivity of Au@NiFe₂O₄/SPE

The interference study is an essential factor for electrodes, which significantly impacts practical applications. To assess the selectivity of the Au@NiFe₂O₄/SPE, various concentrations of interfering agents such as ascorbic acid (b), uric acid (c), glucose (d), L-cysteine (e), L-arginine (f), dopamine (g), vitamin D (h) and vitamin B₁₂ (k) were separately added into a 0.1 M B-R buffer at pH 4.0 containing 0.5 μM EPR (a). The results exhibited that a 200-fold excess of interfering agents did not show any or negligible interference effect in the determination of EPR (Figure 7). The corresponding relative errors for EPR were lower than ±5%, which correlates with the tolerance limit defined in the selectivity measurements, indicating that the Au@NiFe₂O₄/SPE has a promising selectivity for the determination of EPR. Therefore, the results suggest that the developed electrode has outstanding anti-interference activity and can be performed to EPR determination in biological samples.

3.6. Reproducibility, Repeatability, Stability and Reusability of the Au@NiFe₂O₄/SPE

The response of Au@NiFe₂O₄/SPE in a B-R buffer containing 0.5 μM of EPR was observed to study reproducibility (Figure 8A). The five individual electrodes were prepared at similar conditions, and the relative standard deviations (RSDs) less than 2.61% were obtained for EPR. The RSD of 2.1% for ten successive signals was observed confirming good repeatability for the Au@NiFe₂O₄/SPE as an electroanalytical electrode (Figure 8B). Moreover, to evaluate the longer-term response stability, two electrodes were kept in 0.1 M B-R (pH 4.0) at 4 °C. The developed electrodes were kept in glass vials with the tops wrapped using parafilm. After each week, two electrodes were utilized to use continuous voltammetry to measure the 0.5 µM EPR mixed with a 0.1 M B-R buffer (pH 4.0) (Figure S11). The observed signal showed 97.9% of its initial response relative to 0.5 µM EPR, using the Au@NiFe₂O₄/SPE, indicating the good stability, repeatability and reproducibility of the developed electrode. Finally, the reusability of the Au@NiFe₂O₄/SPE was observed. The Au@NiFe₂O₄/SPE is not a disposable electrode. It can be utilized at least 13 times by rinsing with 0.1 M B-R buffer (pH 4.0). Thanks to the strong covalent bond interaction between the uniformly dispersed Au nanoparticles and NiFe₂O₄ surface (notably, the Au[Fe–O] bond), the specific capacity and the long-term cyclic stability of the fabricated Au@NiFe₂O₄ nanohybrid was assured.

3.7. Real Samples Analysis

The Au@NiFe₂O₄/SPE was used to determine EPR in urine, injection and human plasma samples. To assess the target analytes in these real samples, the standard addition method was performed. According to the results obtained in

Table 3. Analysis of EPR in real samples using Au@NiFe₂O₄/SPE.

Sample	Spiked (µM)	Found (µM) a	RSD (%)	Recovery (%)
Human plasma	0.4	0.39 ± 0.01	2.14	97.5
	0.6	0.6 ± 0.05	2.84	100.1
	0.8	0.79 ± 0.04	3.52	98.8
	1.0	1.0 ± 0.01	2.98	100.0
Urine	0.4	0.41 ± 0.01	3.15	102.5
	0.6	0.59 ± 0.06	2.89	98.4
	0.8	0.80 ± 0.03	4.11	100.0
	1.0	1.03 ± 0.03	2.59	103.0
Injection	-	1.15 ± 0.14	-	-
	0.4	1.51 ± 0.08	3.7	102.6
	0.6	1.78 ± 0.09	4.27	105.2

^a All samples were analyzed using the standard addition method.

, the developed electrode can be used directly to determine EPR in real samples. The relative recovery was utilized to estimate the accuracy of the results. The recoveries varied between 97.5 and 101.0% for human plasma, 98.4 and 103% for a urine sample and 102.6 and 105.2% for an injection with acceptable RSDs for a novel developed electrode. Table 3 confirms the high-performance ability of the Au@NiFe₂O₄/SPE as an EPR electrochemical electrode in the real samples.

4. Conclusions

This paper reports on a new EPR sensor design that consists of gold nanoparticles immobilized on the bimetallic nanocomposite on the surface of an SPE. The developed Au@NiFe₂O₄/SPE exhibits good electroactivity because of its high surface area and conductivity. A dynamic linearity range and detection limit of 0.01 to 0.7 and 0.7 to 3.6 μM and 5.3 nM were observed for the Au@NiFe₂O₄/SPE, respectively. The developed electrochemical sensor exhibits outstanding selectivity, linearity, repeatability, reproducibility, sensitivity and reusability in target detection. According to the recovery experiments and standard addition method, it could be said that EPR in real samples such as human plasma, urine and an injection did not affect the selective analysis of the anticancer drug. On the other hand, the applicability of the Au@NiFe₂O₄/SPE to the rapid analysis of EPR in real samples demonstrates the excellent ability for practical application. The developed sensor could be an outstanding candidate as an alternative analytical technique for determining the trace amount of EPR in clinical samples.