An Efficient Electrochemical Sensor Based on NiCo2O4 Nanoplates and Ionic Liquid for Determination of Favipiravir in the Presence of Acetaminophen

Based on the modification of carbon paste electrode with NiCo2O4 nanoplates and 1-hexyl-3-methylimidazolium tetrafluoroborate, a new electrochemical sensing platform for the sensing of favipiravir (a drug with potential therapeutic efficacy in treating COVID-19 patients) in the presence of acetaminophen was prepared. For determining the electrochemical behavior of favipiravir, cyclic voltammetry, differential pulse voltammetry, and chronoamperometry have been utilized. When compared to the unmodified carbon paste electrode, the results of the cyclic voltammetry showed that the proposed NiCo2O4 nanoplates/1-hexyl-3-methylimidazolium tetrafluoroborate/carbon paste electrode had excellent catalytic activity for the oxidation of the favipiravir in phosphate buffer solution (pH = 7.0). This was due to the synergistic influence of 1-hexyl-3-methylimidazolium tetrafluoroborate (ionic liquid) and NiCo2O4 nanoplates. In the optimized conditions of favipiravir measurement, NiCo2O4 nanoplates/1-hexyl-3-methylimidazolium tetrafluoroborate/carbon paste electrode had several benefits, such as a wide dynamic linear between 0.004 and 115.0 µM, a high sensitivity of 0.1672 µA/µM, and a small limit of detection of 1.0 nM. Furthermore, the NiCo2O4 nanoplates/1-hexyl-3-methylimidazolium tetrafluoroborate/carbon paste electrode sensor presented a good capability to investigate the favipiravir and acetaminophen levels in real samples with satisfactory recoveries.


Introduction
Since December 2019, a pandemic caused by the 2019 coronavirus illness (COVID- 19) has rapidly spread throughout the world. Since the start of the pandemic, COVID-19 has claimed millions of lives. Its severe acute respiratory syndrome can be fatal and affects the lower respiratory system. Numerous approved vaccines fell short in their attempts to halt the pandemic's deadly spread. This may be due to the inefficiency and unavailability of vaccination and mutation treatments, as well as a lack of suitable substitute therapy techniques [1][2][3][4]. Repurposing the use of commercially accessible antiviral medications such as favipiravir (FVP) is therefore seen as a workable and successful approach.
The Fujifilm Toyama Chemical Company in Japan created the purine nucleic acid analogue FVP (trade name: Avigan) [5]. FVP functions as a prodrug that is intracellularly transformed into its active metabolite, FVP-RTP (ribofuranosyl 5 -triphosphate) [6][7][8]. Numerous additional RNA viruses can be inhibited by the FVP-RTP molecule. Although the precise mechanism of action is uncertain, it is hypothesised that FVP-RTP may accidentally incorporate into a viral RNA chain as it grows or may bind to conserved polymerase domains to stop viral RNA replication [9,10]. Either chain termination or deadly mutagenesis functionality of numerous systems and making them the preferred material in many areas. The development of nanomaterials has proven fundamental for the development of effective and efficient electrochemical sensors to be used in different application fields such as biomedical, environmental, and food analysis [66][67][68][69]. Due to their admirable physicochemical characteristics, the development of sensors using transition metal oxide nanostructures has shown promise. Binary metal oxides are better suited than transition metal oxides due to their excellent electrochemical properties and strong electrical conductivity [70][71][72]. Transition binary metal oxides, in particular cobalt-based spinel oxides, have a variety of oxidation states, are simple to synthesise, exhibit high durability in alkaline electrolytes, are low toxic, and are economically advantageous mixed-valence oxides [73][74][75]. Due to their eco-friendliness, low cost, and strong electrical conductivity, NiCo 2 O 4 nanoparticles with various shapes and morphologies have greatly attracted interest as prospective electrochemical electrodes in various electrochemical devices such as sensors and biosensors [76][77][78]. Moreover, NiCo 2 O 4 's numerous oxidation states provide a high level of electronic conductivity and can be tuned to control both the electrochemical reactions and the electrochemical performances [79]. NiCo 2 O 4 is used as an electrochemical material because of the benefits already mentioned, but it still has to be improved due to its low surface area, large pore volume, and weak inherent electronic conductivity [80]. Due to the advantages of the redox process with its large pore size, high surface area, short pathways, and rapid reaction kinetics, adopting materials with a two-dimensional or sheet-like shape can help prevent such bottlenecks [81,82]. An interesting electrode material to realise its application in chemical sensors might be NiCo 2 O 4 with a 2D or sheet shape. As a result, NiCo 2 O 4 with a 2D or sheet morphology may be a promising electrode material for chemical sensors.
In the current study, we tried to create a new electrochemical sensing platform by modifying a CPE with 1-hexyl-3-methylimidazolium tetrafluoroborate (HMIM BF 4 ) and NiCo 2 O 4 nanoplates (NiCo 2 O 4 NPs) in order to analyse FVP and AC at the same time. The suggested sensor displayed greater selectivity, a lower limit of detection, amazing sensitivity, and wider dynamic linear ranges. Additionally, the practical applicability of our sensor was explored by sensing the FVP and AC in real specimens. The novelty of this work lies in the application of NiCo 2 O 4 NPs/HMIM BF 4 /CPE as a sensing platform, which enabled the electrochemical detection of FVP in the presence of AC.

Equipment and Materials
All electrochemical analyses have been conducted at ambient temperature using an Autolab PGSTAT 320 N Potentiostat/Galvanostat Analyzer (Utrecht, The Netherlands) with GPES (General Purpose Electrochemical System, version 4.9) software. In this investigation, a typical three-electrode cell at a temperature of 25 ± 1 • C was employed. It included a platinum wire as a counter electrode (Azar Electrode, Urmia, Iran), NiCo 2 O 4 NPs/HMIM BF 4 /CPE as a working electrode, and Ag/AgCl/KCl (3.0 M) as a reference electrode (Azar Electrode, Urmia, Iran). To measure the pH of the solutions, a Switzerland-made Metrohm 713 pH meter with a glass electrode was used. All of the solutions were freshly prepared using Direct-Q ® 8 UV deionized water (Millipore, Burlington, MA, USA). For FE-SEM analysis, a MIRA3 scanning electron microscope (Tescan, Brno, Czech Republic) has been used.
All of the chemicals and solvents used in our protocol were of analytical grade and came from Merck and Sigma-Aldrich. Phosphoric acid has been utilized to make phosphate buffer solution (PBS), which was then pH-adjusted using NaOH.

Preparation of NiCo 2 O 4 NPs
For the preparation of NiCo 2 O 4 NPs, Ni(NO 3 ) 2. 6H 2 O (0.5 mmol, 0.145 gr), Co(NO 3 ) 2. 6H 2 O (1 mmol, 0.291 gr), NH 4 F (3 mmol, 0.111 gr), and urea (7.5 mmol, 0.45 gr) have been dispersed in deionized water (40 mL). After stirring for 40 min to create a clear pink solution, the prepared solution was placed in a Teflon-lined stainless-steel autoclave for three hours at 120 • C. After being cooled to laboratory temperature, the collected precipitate was thoroughly washed with deionized water and oven-dried for 12 h at 65 • C. The prepared product was then annealed for 150 min at 350 • C.
The characterization of NiCo 2 O 4 NPs has been reported in our previous work [83]. Figure 1 shows the FE-SEM images of NiCo 2 O 4 NPs.

Preparation of NiCo2O4 NPs
For the preparation of NiCo2O4 NPs, Ni(NO3)2.6H2O (0.5 mmol, 0.145 gr), Co(NO3)2.6H2O (1 mmol, 0.291 gr), NH4F (3 mmol, 0.111 gr), and urea (7.5 mmol, 0.45 gr) have been dispersed in deionized water (40 mL). After stirring for 40 min to create a clear pink solution, the prepared solution was placed in a Teflon-lined stainless-steel autoclave for three hours at 120 °C. After being cooled to laboratory temperature, the collected precipitate was thoroughly washed with deionized water and oven-dried for 12 h at 65 °C. The prepared product was then annealed for 150 min at 350 °C.
The characterization of NiCo2O4 NPs has been reported in our previous work [83]. Figure 1 shows the FE-SEM images of NiCo2O4 NPs.

Preparation and Surface Modification of Electrode
In order to prepare NiCo2O4 NPs/HMIM BF4/CPE, 100 mg of NiCo2O4 NPs and 900 mg of graphite powder were combined in a mortar and then mixed for 10 min with 0.6 mL of paraffin oil and 0.2 mL of HMIM BF4 ionic liquid. The paste was dispensed into a glass tube to the required level, and a copper wire was positioned over the paste to make electrical contact. Finally, the surface of the electrode was polished on weighing paper to give it a smooth aspect before use.
In addition, NiCo2O4 NPs/CPE (without the use of HMIM BF4), HMIM BF4/CPE (without the use of NiCo2O4 NPs), and an unmodified CPE without the usage of HMIM BF4 and NiCo2O4 NPs were built for comparison.

Preparation of Real Samples
Five tablets of the FVP (labelled value of FVP = 200 mg per tablet) and five tablets of the AC (labelled value of AC = 500 mg per tablet) purchased from a local pharmacy in Kerman (Iran) were finely powdered in a mortar and pestle. Then, an accurately weighed amount of the homogenized FVP and AC powders was transferred into 100 mL of 0.1 M PBS (pH 7.0). The contents of the flasks were sonicated for 20 min to achieve complete dissolution. Finally, the solutions were filtered, and a suitable aliquot of the clear filtrate was collected. Afterward, we poured a determined volume of the transparent filtrates (4.7 µL of FVP and 2.5 µL of AC) into the electrochemical cell, consisting of 25 mL of 0.1 mol/L PBS (pH = 7.0), to record the differential pulse voltammograms.

Preparation and Surface Modification of Electrode
In order to prepare NiCo 2 O 4 NPs/HMIM BF 4 /CPE, 100 mg of NiCo 2 O 4 NPs and 900 mg of graphite powder were combined in a mortar and then mixed for 10 min with 0.6 mL of paraffin oil and 0.2 mL of HMIM BF 4 ionic liquid. The paste was dispensed into a glass tube to the required level, and a copper wire was positioned over the paste to make electrical contact. Finally, the surface of the electrode was polished on weighing paper to give it a smooth aspect before use.
In addition, NiCo 2 O 4 NPs/CPE (without the use of HMIM BF 4 ), HMIM BF 4 /CPE (without the use of NiCo 2 O 4 NPs), and an unmodified CPE without the usage of HMIM BF 4 and NiCo 2 O 4 NPs were built for comparison.

Preparation of Real Samples
Five tablets of the FVP (labelled value of FVP = 200 mg per tablet) and five tablets of the AC (labelled value of AC = 500 mg per tablet) purchased from a local pharmacy in Kerman (Iran) were finely powdered in a mortar and pestle. Then, an accurately weighed amount of the homogenized FVP and AC powders was transferred into 100 mL of 0.1 M PBS (pH 7.0). The contents of the flasks were sonicated for 20 min to achieve complete dissolution. Finally, the solutions were filtered, and a suitable aliquot of the clear filtrate was collected. Afterward, we poured a determined volume of the transparent filtrates (4.7 µL of FVP and 2.5 µL of AC) into the electrochemical cell, consisting of 25 mL of 0.1 mol/L PBS (pH = 7.0), to record the differential pulse voltammograms.

Electrochemical Behaviour of FVP on NiCo 2 O 4 NPs/HMIM BF 4 /CPE
The solution pH affected how FVP responded electrochemically. To ascertain the electrocatalytic oxidation of FVP, it appears essential to optimize the pH of the solution.
Consequently, differential pulse voltammetry (DPV) investigated the effect of pH throughout a range of 2.0 to 9.0 on the FVP electro-oxidation on the NiCo 2 O 4 NPs/HMIM BF 4 /CPE surface. At neutral conditions (pH = 7.0), the electrocatalytic FVP oxidation on the NiCo 2 O 4 NPs/HMIM BF 4 /CPE surface was greatest (optimal).
To demonstrate the enhancement effects of NiCo 2 O 4 NPs and HMIM BF 4 , the electrochemical response of FVP was carried out by cyclic voltammetry (CV) at the different electrodes ( Figure 2). Figure

Electrochemical Behaviour of FVP on NiCo2O4 NPs/HMIM BF4/CPE
The solution pH affected how FVP responded electrochemically. To ascertain the electrocatalytic oxidation of FVP, it appears essential to optimize the pH of the solution. Consequently, differential pulse voltammetry (DPV) investigated the effect of pH throughout a range of 2.0 to 9.0 on the FVP electro-oxidation on the NiCo2O4 NPs/HMIM BF4/CPE surface. At neutral conditions (pH = 7.0), the electrocatalytic FVP oxidation on the NiCo2O4 NPs/HMIM BF4/CPE surface was greatest (optimal).
To demonstrate the enhancement effects of NiCo2O4 NPs and HMIM BF4, the electrochemical response of FVP was carried out by cyclic voltammetry (CV) at the different electrodes ( Figure 2). Figure 2 shows the CVs of FVP (100.0 µM) at unmodified CPE (Curve a), NiCo2O4 NPs/CPE (Curve b), HMIM BF4/CPE (Curve c), and NiCo2O4 NPs/HMIM BF4/CPE (Curve d). Figure 2 shows the anodic peak potential at 1100 mV for FVP on the NiCo2O4 NPs/HMIM BF4/CPE surface and 1240 mV for FVP oxidation on the unmodified CPE surface (curve a). The graphs show that peak potential FVP oxidation on the NiCo2O4 NPs/HMIM BF4/CPE shifted from 1240 mV to negative values in contrast to the surface of the unmodified CPE. Regarding the FVP oxidation on the surface of NiCo2O4 NPs/CPE (curve b) and NiCo2O4 NPs/HMIM BF4/CPE (curve d), the anodic peak current was enhanced on NiCo2O4 NPs/HMIM BF4/CPE in comparison to NiCo2O4 NPs/CPE, which indicates that the presence of IL in the CPE caused the peak currents to be higher. These results correctly depict the enhancement effects caused by the presence of HMIM BF4 and NiCo2O4 NPs and also amplified the sensitivity of the electrode toward the oxidation of FVP.   Figure 3 depicts how different scan rates affect the FVP oxidation currents. The results show that raising the scan rate increases the peak currents. The diffusion-controlled nature of the oxidation processes can be confirmed by the linear plot of Ip versus v 1/2 (the square root of the potential scan rate) for the analyte. It means that FVP reaches the electrode by diffusion, and after the oxidation process, the product of the oxidation also gets away from the electrode surface by diffusion. Figure 3 depicts how different scan rates affect the FVP oxidation currents. The results show that raising the scan rate increases the peak currents. The diffusion-controlled nature of the oxidation processes can be confirmed by the linear plot of Ip versus v 1/2 (the square root of the potential scan rate) for the analyte. It means that FVP reaches the electrode by diffusion, and after the oxidation process, the product of the oxidation also gets away from the electrode surface by diffusion.

Chronoamperometric Analysis
In order to investigate FVP oxidation on the modified electrode, chronoamperometry was used. On the surface of NiCo2O4 NPs/HMIM BF4/CPE, FVP contents were determined by chronoamperometry using an 1150 mV working electrode potential. Additionally, the FVP's diffusion coefficient (D) was determined. As shown in Figure 4 (Inset A), we showed the best-fitting I against t −1/2 plots for various FVP contents. The slopes from the straight lines have been subsequently plotted against various FVP contents, as shown in Figure 4 (Inset B). Based on this slope and Cottrell's equation: In this equation, n denotes the quantity of electrons moved (n = 2 according to the previous works such as reference [84]), F for Faraday's constant, A for electrode surface area (cm 2 ), Cb for the bulk concentration (mol cm −3 ), and t for time (s). For FVP, the average D value was determined to be ~7.1 × 10 −6 cm 2 /s.

Chronoamperometric Analysis
In order to investigate FVP oxidation on the modified electrode, chronoamperometry was used. On the surface of NiCo 2 O 4 NPs/HMIM BF 4 /CPE, FVP contents were determined by chronoamperometry using an 1150 mV working electrode potential. Additionally, the FVP's diffusion coefficient (D) was determined. As shown in Figure 4 (Inset A), we showed the best-fitting I against t −1/2 plots for various FVP contents. The slopes from the straight lines have been subsequently plotted against various FVP contents, as shown in Figure 4 (Inset B). Based on this slope and Cottrell's equation: In this equation, n denotes the quantity of electrons moved (n = 2 according to the previous works such as reference [84]), F for Faraday's constant, A for electrode surface area (cm 2 ), C b for the bulk concentration (mol cm −3 ), and t for time (s). For FVP, the average D value was determined to be~7.1 × 10 −6 cm 2 /s.

Quantitative Determination of FVP by DPV
Under optimized experimental conditions, DPV analysis was carried out for varied FVP levels to investigate the limit of detection (LOD), linear dynamic range, and sensitivity of the NiCo 2 O 4 NPs/HMIM BF 4 /CPE ( Figure 5). As anticipated, the increased FVP level increased the peak current. The results in Figure 5 (Inset) showed a linear relationship between the FVP peak currents as well as its concentrations of 0.004 to 115.0 µM, using the linear regression equation of I pa (µA) = 0.1672C FVP + 0.7182 (R 2 = 0.999) and the sensitivity of 0.1672 µA/µM. In addition, the detection limit, C m , of FVP has been determined by the following equation: where m is the calibration plot's slope (0.1672 µA µM -1 ), and S b is the blank response's standard deviation, which was determined by eight repeat measurements of the blank solution. It was discovered that the detection limit for FVP was 1.0 nM. A comparison of FVP detection using various sensors is presented in

Quantitative Determination of FVP by DPV
Under optimized experimental conditions, DPV analysis was carried out for varied FVP levels to investigate the limit of detection (LOD), linear dynamic range, and sensitivity of the NiCo2O4 NPs/HMIM BF4/CPE ( Figure 5). As anticipated, the increased FVP level increased the peak current. The results in Figure 5 (Inset) showed a linear relationship between the FVP peak currents as well as its concentrations of 0.004 to 115.0 µM, using the linear regression equation of Ipa (µA) = 0.1672CFVP + 0.7182 (R 2 = 0.999) and the sensitivity of 0.1672 µA/µM. In addition, the detection limit, Cm, of FVP has been determined by the following equation: where m is the calibration plot's slope (0.1672 µA µM -1 ), and Sb is the blank response's standard deviation, which was determined by eight repeat measurements of the blank solution. It was discovered that the detection limit for FVP was 1.0 nM. A comparison of FVP detection using various sensors is presented in Table 1. According to Table 1, the prepared sensor (NiCo2O4 NPs/HMIM BF4/CPE) provided better performance compared to other reported sensors for FVP detection.     Figure 6 shows the DPVs for simultaneous FVP and AC detection using NiCo 2 O 4 NPs/HMIM BF 4 /CPE. The oxidation of FVP and AC, respectively, was associated with the peaks at 400 and 1100 mV. By simultaneously increasing the concentrations of both analytes, the peak current intensity has been linearly increased. The matching calibration curves for FVP and AC may be seen in Figure 6 (insets A and B). The linear regression line's slope for the calibration curve for FVP (0.1701 µAµM −1 ) has been virtually identical to that for the calibration curve without AC (0.1672 µAµM −1 ), demonstrating the viability of using NiCo 2 O 4 NPs/HMIM BF 4 /CPE for simultaneous detection of FVP and AC concentrations.

Simultaneous Detection of FVP and AC on NiCo2O4 NPs/HMIM BF4/CPE
As far as the authors' knowledge goes, this is the first work utilizing NiCo2O4 NPs/HMIM BF4/CPE to detect FVP in the presence of AC. Figure 6 shows the DPVs for simultaneous FVP and AC detection using NiCo2O4 NPs/HMIM BF4/CPE. The oxidation of FVP and AC, respectively, was associated with the peaks at 400 and 1100 mV. By simultaneously increasing the concentrations of both analytes, the peak current intensity has been linearly increased. The matching calibration curves for FVP and AC may be seen in Figure 6 (insets A and B). The linear regression line's slope for the calibration curve for FVP (0.1701 µAµM −1 ) has been virtually identical to that for the calibration curve without AC (0.1672 µAµM −1 ), demonstrating the viability of using NiCo2O4 NPs/HMIM BF4/CPE for simultaneous detection of FVP and AC concentrations.

Reproducibility, Repeatability and Stability of the NiCo 2 O 4 NPs/HMIM BF 4 /CPE
DPV was used to evaluate the reproducibility, repeatability, and stability of the NiCo 2 O 4 NPs/HMIM BF 4 /CPE. The computed relative standard deviation (RSD) for eight measurements of 50.0 µM FVP made with a single electrode was 3.7%, showing that this sensor has acceptable repeatability.
The computed RSD for measurement of 50.0 µM FVP at five separate electrodes made in the same manner in another investigation was 2.6%, demonstrating the reasonable reproducibility of the NiCo 2 O 4 NPs/HMIM BF 4 /CPE.
In the third investigation in this part, one modified electrode was kept for 10 days and then utilized for FVP determination to determine the stability of the NiCo 2 O 4 NPs/HMIM BF 4 /CPE. The data show that Ip for FVP electrochemical oxidation decreased to 96.5% of its initial value after 10 days, demonstrating a high degree of stability.

Interference Studies
To assess the selectivity of NiCo 2 O 4 NPs/HMIM BF 4 /CPE for FVP, an examination was carried out to determine the effect of potential interfering substances under optimized conditions. The DPV responses were recorded upon addition of interfering substances into 0.1 M PBS (pH 7.0) containing 50.0 µM FVP. DPV responses were measured by adding interfering substances to a solution of 0.1 M PBS with a pH of 7.0, containing 50.0 µM of FVP. As a general principle, the relative error in the measurement is controlled at approximately ±5% and is considered to have no interference. Also, the findings indicated that K + , Na + , NH 4 + , Ca 2+ , Mg 2+ , Cl − , Br − , SO 4 2− , NO 3 − , glucose, dopamine, ascorbic acid, uric acid, acetaminophen, glucose, l-cysteine and l-arginine did not interfere with the oxidation peak response of FVP.

Real Sample Analysis
The modified CPE was applied to pharmaceutical samples in order to evaluate the applicability of the use of a modified electrode to determine FVP and AC in real samples. The findings are provided in Table 2 using the standard addition method. Recoveries range from 96.9% to 104.8%, and relative standard deviations are all lower than or equal to 3.5%. The experimental results confirmed that the NiCo 2 O 4 NPs/HMIM BF 4 /CPE sensor has great potential for analytical applications.

Conclusions
This study revealed the development of an electroanalytical sensor for the simultaneous detection of FVP and AC. As a result, we created an electroanalytical sensor that is amplified by NiCo 2 O 4 NPs and HMIM BF 4 ionic liquid. It has been discovered that the modified electrode has outstanding electrocatalytic activity for the oxidation of FVP and AC. Using the NiCo 2 O 4 NPs/HMIM BF 4 /CPE, distinct oxidation peaks of FVP and AC have been produced, enabling the simultaneous detection of both analytes. The NiCo 2 O 4 NPs/HMIM BF 4 /CPE is a promising candidate for FVP and AC determination in pharmaceutical samples with recovery ratios between 96.9 and 104.8% because the proposed sensor demonstrated outstanding benefits of easy preparation, strong catalytic activity, low reagent usage, and affordability. The novelty of this work is the use of NiCo 2 O 4 NPs/HMIM BF 4 /CPE as a new sensor for the electrochemical detection of FVP in the presence of AC.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding authors.

Conflicts of Interest:
The authors declare no conflict of interest.