An Innovative Polymer-Based Electrochemical Sensor Encrusted with Tb Nanoparticles for the Detection of Favipiravir: A Potential Antiviral Drug for the Treatment of COVID-19

An innovative polymer-based electro-sensor decorated with Tb nanoparticles has been developed for the first time. The fabricated sensor was utilized for trace determination of favipiravir (FAV), a recently US FDA-approved antiviral drug for the treatment of COVID-19. Different techniques, including ultraviolet-visible spectrophotometry (UV-VIS), cyclic voltammetry (CV), scanning electron microscope (SEM), X-ray Diffraction (XRD) and electrochemical impedance spectroscopy (EIS), were applied for the characterization of the developed electrode TbNPs@ poly m-THB/PGE. Various experimental variables, including pH, potential range, polymer concentration, number of cycles, scan rate and deposition time, were optimized. Moreover, different voltammetric parameters were examined and optimized. The presented SWV method showed linearity over the range of 10–150 × 10−9 M with a good correlation coefficient (R = 0.9994), and the detection limit (LOD) reached 3.1 × 10−9 M. The proposed method was applied for the quantification of FAV in tablet dosage forms and in human plasma without any interference from complex matrices, obtaining good % recovery results (98.58–101.93%).


Introduction
Favipiravir (FAV), 6-fluoro-3-hydroxy-2-pyrazinecarboxamide, is a promising antiviral pro-drug belonging to the RNA polymerase inhibitors. It was first introduced in Japan in 2014 as an anti-influenza agent, and it was also applied to treat other viruses [1]. In 2019, it was proven to be safe and effective against COVID-19, a global pandemic with outbreaks all over the world, as the World Health Organization (WHO) officially declared in March 2020. COVID-19 causes severe respiratory syndrome leading to a serious disease that affects different organs, including the kidneys, liver and central nervous system [2].
Although the pandemic crest has diminished lately due to several considerations, such as global vaccination and enhanced health awareness, however, its endemicity is said to be meaningless or at least only transitional. Expectations for infection spikes are high, which may be owed to the removal of restrictive measures as well as viral mutations [3,4]. Therefore, it was recommended to establish a sensitive, cost-effective and selective analytical method for the estimation of COVID-19 defense drugs such as FAV in biological fluids for routine drug quality control and further clinical studies' monitoring.

Standard and Reagent Solutions
A standard solution of FAV (5 × 10 −6 M) was prepared in double distilled water. Phloroglucinol (m-trihydroxy benzene; m-THB) solution (32 × 10 −3 M) and Tb (III) solution (500 × 10 −3 M) were formed in double distilled water. Further dilutions were done using the same solvent to investigate the optimum concentration for the polymerization of m-THB and the optimum concentration for the electro-deposition of Tb.

Fabrication of Tb NPS @ Poly m-THB/PGE
To fabricate the presented electrode, the PGE surface was first washed with double distilled water before use. Further, an electro-polymerization of m-THB was established using 8 × 10 −3 M of m-THB solution in phosphate buffer (0.1 M, pH 7) through multiple cyclic voltammetry for 10 cycles using a potential range of −0.9-+1.75 V and scanning rate of 0.1 Vs −1 . The electrode was denoted as poly m-THB/PGE. Furthermore, the electrode was submerged in an electrochemical cell having Tb (III) chloride solution (125 × 10 −3 M), which was electro-deposited using a potential at −1.2 V for 80 s. The fabricated Tb NPs @ poly m-THB/PGE electrode was further characterized and checked by UV-Visible spectrophotometry, CV, SEM and EIS.

Analytical Procedures for Estimation of FAV 2.5.1. General Analytical Procedure
An appropriate volume of FAV (5 × 10 −6 M) standard or sample solution was added into an electrochemical cell filled with 0.1 M phosphate buffer (pH 7) as a supporting electrolyte. The electrochemical performance of FAV using bare and modified PGE was studied using CV and SWV techniques. The experimental variables of the developed method in terms of m-THB polymerization, Tb NPs electro-deposition and the optimum electrolyte pH were studied. Moreover, various SWV parameters were studied, including deposition time, frequency, initial potential and step and pulse height.

Procedure for Estimation of FAV in Tablets
Ten Avipiravir ® tablets (200 mg per tablet) were weighed, crushed finally and thoroughly mixed. Then, an adequate weight equivalent to 10.0 mg FAV was transferred into a volumetric flask and dissolved into 50 mL of double distilled water. The solution was sonicated for about 20 min, followed by filtration, then the volume was made up to 100 mL with double distilled water to get a concentration of 100 µg mL −1 . The developed SWV method was then carried out as mentioned above.

Estimation of FAV in Human Plasma
In a centrifuge tube, 1.0 mL of human plasma was spiked with an adequate amount of FAV solution, and the volume was then furtherly made up to 10 mL using methanol. The mixture was vortexed for 30 s and then centrifuged for 35 min (3500 rpm). The resultant supernatant was collected, and appropriate volumes were added to the electrochemical cell. The voltammograms of SWV were recorded for FAV using the modified Tb NPs @ poly m-THB/PGE electrode under the optimum experimental conditions. A blank measurement was established in the same way but without the drug. In addition, required dilutions from this supernatant were made using the selected supporting electrolyte. The study was performed following the relevant faculty laws and guidelines, as well as the research ethics committee.

Characterization of Tb NPs @ Poly m-THB/PGE
The developed electrode was morphologically characterized using SEM and the modified polymer layers. Besides this, the XRD and UV-VIS spectra of the modified polymer layers and Tb NPS were studied. In addition, the ESI method for bare and Tb NPs @ poly m-THB/PGE was examined.

Validation Data
The proposed SWV method was validated following the ICH guidelines [38] for linearity range, the limit of detection (LOD), the limit of quantification (LOQ), accuracy and precision. The LOD and LOQ values were determined using formulas 3 σ/S and 10 σ/S, respectively, where σ represents the standard deviation of the intercept and S is the slope of the related regression equation. The electrochemical technique's precision was examined using three different concentrations within the calibration range within the same day (intra-day precision) and across three successive days (inter-day precision). The mean values of relative standard deviations (RSD%) of the results were calculated.

Preparation of Tb NPS @ Poly m-THB/PGE
The present research is the first to investigate the electrochemical performance of phloroglucinol; an m-trihydroxy benzene compound (m-THB); on the peak current value of FAV; an antiviral regimen for COVID-19. Since modification of carbon-based electrodes is a promising step to improve electrode surface area and enhance its electron transfer, the electrochemical polymerization of m-THB was performed, which covers the surface of PGE. All experimental variables influencing the polymerization step were examined, such as m-THB concentration, potential, number of polymerization cycles and the scan rate. The concentration of m-THB has a great effect on the polymerization procedure and on the FAV current value. So, concentrations of m-THB from 0.002 to 0.012 M were studied, where the current value was increased by increasing the concentration till constant values of current were obtained using concentrations from 0.007 to 0.009 M, after that, a decrease in current value was found as the resultant polymer film using higher concentration may block the surface of the formed electrode and hence decrease current intensity ( Figure 1A). Hence, 0.008 M was selected as the optimum concentration of m-THB and was subsequently used for further measurements. After that, the potential range necessary for m-THB polymerization was tested using different potential values ranging from −0.2 to −1.6 V. An increase in the current value of FAV was obtained by decreasing the potential value until a fixed current value was found at −0.8 V, and it was selected for further electrochemical measurements as represented in Figure 1B. where the current value was increased by increasing the concentration till constant values of current were obtained using concentrations from 0.007 to 0.009 M, after that, a decrease in current value was found as the resultant polymer film using higher concentration may block the surface of the formed electrode and hence decrease current intensity ( Figure 1A). Hence, 0.008 M was selected as the optimum concentration of m-THB and was subsequently used for further measurements. After that, the potential range necessary for m-THB polymerization was tested using different potential values ranging from −0.2 to −1.6 V. An increase in the current value of FAV was obtained by decreasing the potential value until a fixed current value was found at −0.8 V, and it was selected for further electrochemical measurements as represented in Figure 1B. Owing to the importance of the number of cycles in the polymerization step, as it significantly affects electrode electro-catalytic activity, various numbers of cycles were performed. From data represented in Figure 1C, it was found that 12 cycles showed the highest values and a further decrease in current value was obtained using the number of Owing to the importance of the number of cycles in the polymerization step, as it significantly affects electrode electro-catalytic activity, various numbers of cycles were performed. From data represented in Figure 1C, it was found that 12 cycles showed the highest values and a further decrease in current value was obtained using the number of cycles higher than 12. The scan rate was further tested from 0.05 to 0.4 V s −1 , and the highest peak current value was observed at 0.1 V s −1, as represented in Figure S1A (Supplementary Materials).
The formed polymer layers, poly m-THB over the PGE surface, were used as a platform for further loading of Tb NPS , where an electro-deposition of Tb (III) solution was performed. Different concentrations of Tb (III) ranging from 95 to 150 mM were examined; it was observed that the highest and most stable results were obtained using 125 mM, as shown in Figure 1D. Further, deposition potential values required from −1.8 to −0.4 V and various deposition time from 10 to 120 s was examined. It was noticed that −1.2 V and 80 s showed the highest values, and they were used as the optimum parameters, as shown in Figure S1B (Supplementary Materials).

Electrochemical Performance of FAV at Bare and Modified PGE
The electro-oxidation of FAV was established at bare and modified PGE by both CV and SWV techniques. Figure 2A shows the distinct oxidation peak of FAV (70 × 10 −9 M) at potential 1.14 V using bare PGE (curve a), poly m-THB/PGE (curve b) in 0.1 M phosphate buffer,  Figure 2A (curve c) represented the deposition of Tb nanocomposites over poly m-THB/PGE surface, which remarkably enhanced the sensitivity towards FAV electro-oxidation, giving a higher current value (~127 µA) in comparison with bare PGE or poly m-THB/PGE which may be attributed to their good electro-activity, large surface areas and rapid transfer rate on the modified electrode surface. These findings clearly confirm the synergistic effect of the used hybrid composites, m-THB polymer and Tb NPS , in electrode composition.

Electrochemical Performance of FAV at Bare and Modified PGE
The electro-oxidation of FAV was established at bare and modified PGE by both CV and SWV techniques. Figure 2A shows the distinct oxidation peak of FAV (70 × 10 −9 M) at potential 1.14 V using bare PGE (curve a), poly m-THB/PGE (curve b) in 0.1 M phosphate buffer, pH 7.0. The oxidation current values of FAV were 45 and 75 µA, respectively. Furthermore, Figure 2A (curve c) represented the deposition of Tb nanocomposites over poly m-THB/PGE surface, which remarkably enhanced the sensitivity towards FAV electro-oxidation, giving a higher current value (~127 µA) in comparison with bare PGE or poly m-THB/PGE which may be attributed to their good electro-activity, large surface areas and rapid transfer rate on the modified electrode surface. These findings clearly confirm the synergistic effect of the used hybrid composites, m-THB polymer and Tb NPS, in electrode composition.

Characterization of Tb NPs @ Poly m-THB/PGE
The morphological characterization of the fabricated electrodes was carried out by SEM technique, where SEM images of poly m-THB and Tb NPs @ poly m-THB were represented in Figure 2. The SEM images of the bare PGE electrode are represented in Figure 2C, where distinguishable smooth layers [32] covering its surface can be observed. However, after polymerization with m-THB, obvious lumps and flake-like structures were observed coating the surface of the modified electrode, as shown in Figure 2D. After incorporation of Tb NPS over the surface of poly m-THB/PGE electrode, characteristic lumps and depressions structures with glowing clusters were observed over the surface of the modified electrode Tb NPS @ poly m-THB/PGE electrode as shown in Figure 2E.
The used modification forms porous structures covering the electrode surface, which improves the active surface area of the electrode and enhances FAV oxidation. Moreover, the XRD pattern showed diffraction peaks centered around values of 28.76_and 47.50, which are in agreement with the diagnostic peaks of Tb, as shown in Figure S2A. Figure S2B (Supplementary Materials) represents the UV spectra of m-THB, Tb and Tb NPs @ poly m-THB hybrid composite. As shown by curve i, a distinguished maximum peak of m-THB was observed at 268 nm. In addition, a characteristic peak of Tb was found at~220 nm, as represented in curve ii. In curve iii (spectrum of Tb NPs @ poly m-THB hybrid modifier), another peak appeared at~330 nm, which confirms the success of polymerization of m-THB, besides the characteristic peak of Tb at~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of FAV in phosphate buffer (0.1 M, pH 7.0) using the fabricated electrode was performed by examining the related CV voltammograms at various scan rates from 100 to 900 mV/s. The peak current values of FAV were directly proportional to the scan rate, as shown in Figure 3, conforming Randles-Ševćik equation [39]. Figure 3A represents the enhancement in the anodic peak current by increasing the scan rate from 100 to 900 mV/s. The relationship between the oxidation peak current (Ip) and the scan rate ( the XRD pattern showed diffraction peaks centered around values of 28.76_and 47.50, which are in agreement with the diagnostic peaks of Tb, as shown in Figure S2A. Figure S2B (Supplementary Data) represents the UV spectra of m-THB, Tb and TbNPs@ poly m-THB hybrid composite. As shown by curve i, a distinguished maximum peak of m-THB was observed at 268 nm. In addition, a characteristic peak of Tb was found at ~220 nm, as represented in curve ii. In curve iii (spectrum of TbNPs@ poly m-THB hybrid modifier), another peak appeared at ~330 nm, which confirms the success of polymerization of m-THB, besides the characteristic peak of Tb at ~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of FAV in phosphate buffer (0.1 M, pH 7.0) using the fabricated electrode was performed by examining the related CV voltammograms at various scan rates from 100 to 900 mV/s. The peak current values of FAV were directly proportional to the scan rate, as shown in Figure 3, conforming Randles-Ševćik equation [39]. Figure 3A represents the enhancement in the anodic peak current by increasing the scan rate from 100 to 900 mV/s. The relationship between the oxidation peak current (Ip) and the scan rate (ʋ) showed a linear response following the equation below: From Figure 3A, by increasing the scan rate, the oxidation potential of FAV was moved to more positive values, with an increase in the current intensity ensuring the irreversibility of the oxidation process of FAV. From data represented in Figure 3B, a plot ) showed a linear response following the equation below: Ip (µA) = 15.8 + 121.7 The used modification forms porous structures covering the electrode surface, which improves the active surface area of the electrode and enhances FAV oxidation. Moreover, the XRD pattern showed diffraction peaks centered around values of 28.76_and 47.50, which are in agreement with the diagnostic peaks of Tb, as shown in Figure S2A. Figure S2B (Supplementary Data) represents the UV spectra of m-THB, Tb and TbNPs@ poly m-THB hybrid composite. As shown by curve i, a distinguished maximum peak of m-THB was observed at 268 nm. In addition, a characteristic peak of Tb was found at ~220 nm, as represented in curve ii. In curve iii (spectrum of TbNPs@ poly m-THB hybrid modifier), another peak appeared at ~330 nm, which confirms the success of polymerization of m-THB, besides the characteristic peak of Tb at ~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of FAV in phosphate buffer (0.1 M, pH 7.0) using the fabricated electrode was performed by examining the related CV voltammograms at various scan rates from 100 to 900 mV/s. The peak current values of FAV were directly proportional to the scan rate, as shown in Figure 3, conforming Randles-Ševćik equation [39]. Figure 3A represents the enhancement in the anodic peak current by increasing the scan rate from 100 to 900 mV/s. The relationship between the oxidation peak current (Ip) and the scan rate (ʋ) showed a linear response following the equation below: From Figure 3A, by increasing the scan rate, the oxidation potential of FAV was moved to more positive values, with an increase in the current intensity ensuring the irreversibility of the oxidation process of FAV. From data represented in Figure 3B, a plot (r 2 = 0.9924) improves the active surface area of the electrode and enhances FAV oxidation. Moreover, the XRD pattern showed diffraction peaks centered around values of 28.76_and 47.50, which are in agreement with the diagnostic peaks of Tb, as shown in Figure S2A. Figure S2B (Supplementary Materials) represents the UV spectra of m-THB, Tb and TbNPs@ poly m-THB hybrid composite. As shown by curve i, a distinguished maximum peak of m-THB was observed at 268 nm. In addition, a characteristic peak of Tb was found at ~220 nm, as represented in curve ii. In curve iii (spectrum of TbNPs@ poly m-THB hybrid modifier), another peak appeared at ~330 nm, which confirms the success of polymerization of m-THB, besides the characteristic peak of Tb at ~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of FAV in phosphate buffer (0.1 M, pH 7.0) using the fabricated electrode was performed by examining the related CV voltammograms at various scan rates from 100 to 900 mV/s. The peak current values of FAV were directly proportional to the scan rate, as shown in Figure 3, conforming Randles-Ševćik equation [39]. Figure 3A represents the enhancement in the anodic peak current by increasing the scan rate from 100 to 900 mV/s. The relationship between the oxidation peak current (Ip) and the scan rate (ʋ) showed a linear response following the equation below: From Figure 3A, by increasing the scan rate, the oxidation potential of FAV was moved to more positive values, with an increase in the current intensity ensuring the irreversibility of the oxidation process of FAV. From data represented in Figure 3B, a plot From Figure 3A, by increasing the scan rate, the oxidation potential of FAV was moved to more positive values, with an increase in the current intensity ensuring the irreversibility of the oxidation process of FAV. From data represented in Figure 3B, a plot of the logarithm of the oxidation peak current (log Ip) versus the logarithm of the scan rate (log  Figure S2B (Supplementary Data) represents the UV spectra o TbNPs@ poly m-THB hybrid composite. As shown by curve i, a distin peak of m-THB was observed at 268 nm. In addition, a characteristic pe at ~220 nm, as represented in curve ii. In curve iii (spectrum of TbNPs@ p modifier), another peak appeared at ~330 nm, which confirms the suc tion of m-THB, besides the characteristic peak of Tb at ~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of buffer (0.1 M, pH 7.0) using the fabricated electrode was performed related CV voltammograms at various scan rates from 100 to 900 mV/ values of FAV were directly proportional to the scan rate, as shown in ing Randles-Ševćik equation [39]. Figure 3A represents the enhance peak current by increasing the scan rate from 100 to 900 mV/s. The re the oxidation peak current (Ip) and the scan rate (ʋ) showed a linear the equation below: From Figure 3A, by increasing the scan rate, the oxidation pot moved to more positive values, with an increase in the current intens ) was found to be linear and was described by the following regression equation:

Characterization of TbNPs@ Poly m-THB/PGE
The morphological characterization of the fabricated electrodes was carried out by SEM technique, where SEM images of poly m-THB and TbNPs@ poly m-THB were represented in Figure 2. The SEM images of the bare PGE electrode are represented in Figure  2C, where distinguishable smooth layers [32] covering its surface can be observed. However, after polymerization with m-THB, obvious lumps and flake-like structures were observed coating the surface of the modified electrode, as shown in Figure 2D. After incorporation of TbNPS over the surface of poly m-THB/PGE electrode, characteristic lumps and depressions structures with glowing clusters were observed over the surface of the modified electrode TbNPS @ poly m-THB/PGE electrode as shown in Figure 2E.
The used modification forms porous structures covering the electrode surface, which improves the active surface area of the electrode and enhances FAV oxidation. Moreover, the XRD pattern showed diffraction peaks centered around values of 28.76_and 47.50, which are in agreement with the diagnostic peaks of Tb, as shown in Figure S2A. Figure S2B (Supplementary Data) represents the UV spectra of m-THB, Tb and TbNPs@ poly m-THB hybrid composite. As shown by curve i, a distinguished maximum peak of m-THB was observed at 268 nm. In addition, a characteristic peak of Tb was found at ~220 nm, as represented in curve ii. In curve iii (spectrum of TbNPs@ poly m-THB hybrid modifier), another peak appeared at ~330 nm, which confirms the success of polymerization of m-THB, besides the characteristic peak of Tb at ~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of FAV in phosphate buffer (0.1 M, pH 7.0) using the fabricated electrode was performed by examining the related CV voltammograms at various scan rates from 100 to 900 mV/s. The peak current values of FAV were directly proportional to the scan rate, as shown in Figure 3, conforming Randles-Ševćik equation [39]. Figure 3A represents the enhancement in the anodic peak current by increasing the scan rate from 100 to 900 mV/s. The relationship between the oxidation peak current (Ip) and the scan rate (ʋ) showed a linear response following the equation below:

Characterization of TbNPs@ Poly m-THB/PGE
The morphological characterization of the fabricated electrodes was carrie SEM technique, where SEM images of poly m-THB and TbNPs@ poly m-THB we sented in Figure 2. The SEM images of the bare PGE electrode are represented 2C, where distinguishable smooth layers [32] covering its surface can be observe ever, after polymerization with m-THB, obvious lumps and flake-like structures served coating the surface of the modified electrode, as shown in Figure 2D. Af poration of TbNPS over the surface of poly m-THB/PGE electrode, characteristic lu depressions structures with glowing clusters were observed over the surface of ified electrode TbNPS @ poly m-THB/PGE electrode as shown in Figure 2E.
The used modification forms porous structures covering the electrode surfac improves the active surface area of the electrode and enhances FAV oxidation. M the XRD pattern showed diffraction peaks centered around values of 28.76_a which are in agreement with the diagnostic peaks of Tb, as shown in Figure S2A Figure S2B (Supplementary Data) represents the UV spectra of m-THB TbNPs@ poly m-THB hybrid composite. As shown by curve i, a distinguished m peak of m-THB was observed at 268 nm. In addition, a characteristic peak of Tb w at ~220 nm, as represented in curve ii. In curve iii (spectrum of TbNPs@ poly m-TH modifier), another peak appeared at ~330 nm, which confirms the success of pol tion of m-THB, besides the characteristic peak of Tb at ~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of FAV in p buffer (0.1 M, pH 7.0) using the fabricated electrode was performed by exami related CV voltammograms at various scan rates from 100 to 900 mV/s. The pea values of FAV were directly proportional to the scan rate, as shown in Figure 3, ing Randles-Ševćik equation [39]. Figure 3A represents the enhancement in th peak current by increasing the scan rate from 100 to 900 mV/s. The relationship the oxidation peak current (Ip) and the scan rate (ʋ) showed a linear response f the equation below: Hence the oxidation process of FAV is controlled by both adsorption and diffusion mechanisms, which agrees with the previous studies [18][19][20][21].
Further, by plotting potential (Ep) against the logarithm of scan rate (log Biosensors 2023, 13, x FOR PEER REVIEW

Characterization of TbNPs@ Poly m-THB/PGE
The morphological characterization of the fabricated electrode SEM technique, where SEM images of poly m-THB and TbNPs@ pol sented in Figure 2. The SEM images of the bare PGE electrode are r 2C, where distinguishable smooth layers [32] covering its surface ca ever, after polymerization with m-THB, obvious lumps and flake-lik served coating the surface of the modified electrode, as shown in Fi poration of TbNPS over the surface of poly m-THB/PGE electrode, cha depressions structures with glowing clusters were observed over th ified electrode TbNPS @ poly m-THB/PGE electrode as shown in Figu The used modification forms porous structures covering the ele improves the active surface area of the electrode and enhances FAV the XRD pattern showed diffraction peaks centered around value which are in agreement with the diagnostic peaks of Tb, as shown in Figure S2B (Supplementary Data) represents the UV spectra TbNPs@ poly m-THB hybrid composite. As shown by curve i, a dist peak of m-THB was observed at 268 nm. In addition, a characteristic at ~220 nm, as represented in curve ii. In curve iii (spectrum of TbNPs@ modifier), another peak appeared at ~330 nm, which confirms the su tion of m-THB, besides the characteristic peak of Tb at ~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current buffer (0.1 M, pH 7.0) using the fabricated electrode was perform related CV voltammograms at various scan rates from 100 to 900 m values of FAV were directly proportional to the scan rate, as shown ing Randles-Ševćik equation [39]. Figure 3A represents the enhan peak current by increasing the scan rate from 100 to 900 mV/s. The the oxidation peak current (Ip) and the scan rate (ʋ) showed a linea the equation below: Ip (µA) = 15.8 + 121.7 ʋ (r 2 = 0.9924 ), a linear relationship was obtained as represented in Figure 3B

Characterization of TbNPs@ Poly m-THB/PGE
The morphological characterization of the fabricated electrodes was carried out by SEM technique, where SEM images of poly m-THB and TbNPs@ poly m-THB were represented in Figure 2. The SEM images of the bare PGE electrode are represented in Figure  2C, where distinguishable smooth layers [32] covering its surface can be observed. However, after polymerization with m-THB, obvious lumps and flake-like structures were observed coating the surface of the modified electrode, as shown in Figure 2D. After incorporation of TbNPS over the surface of poly m-THB/PGE electrode, characteristic lumps and depressions structures with glowing clusters were observed over the surface of the modified electrode TbNPS @ poly m-THB/PGE electrode as shown in Figure 2E.
The used modification forms porous structures covering the electrode surface, which improves the active surface area of the electrode and enhances FAV oxidation. Moreover, the XRD pattern showed diffraction peaks centered around values of 28.76_and 47.50, which are in agreement with the diagnostic peaks of Tb, as shown in Figure S2A. Figure S2B (Supplementary Data) represents the UV spectra of m-THB, Tb and TbNPs@ poly m-THB hybrid composite. As shown by curve i, a distinguished maximum peak of m-THB was observed at 268 nm. In addition, a characteristic peak of Tb was found at ~220 nm, as represented in curve ii. In curve iii (spectrum of TbNPs@ poly m-THB hybrid modifier), another peak appeared at ~330 nm, which confirms the success of polymerization of m-THB, besides the characteristic peak of Tb at ~220 nm.

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of FAV in phosphate buffer (0.1 M, pH 7.0) using the fabricated electrode was performed by examining the related CV voltammograms at various scan rates from 100 to 900 mV/s. The peak current values of FAV were directly proportional to the scan rate, as shown in Figure 3, conforming Randles-Ševćik equation [39]. Figure 3A represents the enhancement in the anodic peak current by increasing the scan rate from 100 to 900 mV/s. The relationship between the oxidation peak current (Ip) and the scan rate (ʋ) showed a linear response following the equation below: Ip (µA) = 15.8 + 121.7 ʋ (r 2 = 0.9924) (V s −1 ) (r 2 = 0.987) As represented in Figure 2B, no cathodic peak for FAV was observed in the CV reverse scan, confirming the irreversibility of the oxidation reaction of FAV. Based on the Laviron equation [40], the potential (E), number of transferred electrons (n) and scan rate in the rate-limiting step can be calculated from: The slope of the plot E p (V) and log

Investigation of Scan Rate
The investigation of the scan rate on the oxidation peak current of FAV in phosphate buffer (0.1 M, pH 7.0) using the fabricated electrode was performed by examining the related CV voltammograms at various scan rates from 100 to 900 mV/s. The peak current values of FAV were directly proportional to the scan rate, as shown in Figure 3, conforming Randles-Ševćik equation [39]. Figure 3A represents the enhancement in the anodic peak current by increasing the scan rate from 100 to 900 mV/s. The relationship between the oxidation peak current (Ip) and the scan rate (ʋ) showed a linear response following the equation below: From Figure 3A, by increasing the scan rate, the oxidation potential of FAV was moved to more positive values, with an increase in the current intensity ensuring the irreversibility of the oxidation process of FAV. From data represented in Figure 3B, a plot = 2.2303 RT/αn F T is the absolute temperature (298 K), n is the number of transferred electrons in the rate-determining step, R is the universal gas constant (8.314 J mol −1 K −1 ) and F is the Faraday constant (96.480 C mol −1 ). Assuming α (the transfer coefficient) is 0.5 in totally irreversible reactions, and after substitution of the slope with 0.052, the number of electrons involved in the oxidation process was calculated to be ≈2.0; this agrees with previously reported articles [20,21]. The oxidation reaction of FAV probably occurs in the aromatic hydroxyl group on the pyrazine ring in the FAV chemical structure, as mentioned before [19,20].

Electrochemical Characterization of Tb NPs @ Poly m-THB/PGE
The electrochemical activity of Tb NPs @ poly m-THB/PGE was examined using the CV technique, where Fe 2+ /Fe 3+ solution (1.0 mmol L −1 ) prepared in 0.5 M of potassium chloride was used. After using the modified sensor, an increment in peak current intensity in the reduction-oxidation peak of the Fe 2+ /Fe 3+ system was observed, as represented in Figure 4A. This increment is due to an increase in the active surface area of the electrode after modification. This finding was affirmed by the EIS study, where measurements were performed at 10 mV, and the potential amplitude was within the frequency range (1.0-10 KHz). Tb NPs @ poly m-THB/PGE electrode showed lower series resistance than that of the bare one. Moreover, the results in Figure 4B shows that bare PGE has displayed a semi-circular model, but Tb NPs @ poly m-THB/PGE modified electrode displayed a straight linear curve. This indicates the improvement in the electrical conductivity due to the charge transfer acceleration and the surface area enlargement. In order to confirm this hypothesis, the electrode's active surface area was calculated using the Randles-Ševćik equation from the slope of anodic peak current (I p ):

Effect of pH
Supporting electrolyte pH is very important to study the electrochemical behavior of FAV at the modified electrode. Therefore, different phosphate buffer solutions (0.1 M) from pH 4.0 to 9.0 were examined. An increase in the current value is observed by increasing the pH value. A phosphate buffer of pH 7.0 showed the highest value, and it was selected for further measurements, as represented in Figure S3A (Supplementary Materi- A eff is the electrode surface area in cm 2 , n is the number of electron transfers, D R is the diffusion coefficient (cm 2 s −1 ), C 0 is the concentration of Fe 2+ /Fe 3+ system (mol/cm 2 ) and υ is the scan rate (V s −1 ). According to the Randles-Ševćik equation, the active surface areas of bare PGE and Tb NPs @ poly m-THB/PGE have been calculated to be: 0.212 and 0.454 cm 2 . This finding confirmed the improvement action of using the hybrid modification (m-THB polymer layers and Tb NPS ) regarding the oxidation of FAV using a Tb NPs @ poly m-THB/PGE electrode.
3.6. Optimization of Method's Parameters 3.6.1. Effect of pH Supporting electrolyte pH is very important to study the electrochemical behavior of FAV at the modified electrode. Therefore, different phosphate buffer solutions (0.1 M) from pH 4.0 to 9.0 were examined. An increase in the current value is observed by increasing the pH value. A phosphate buffer of pH 7.0 showed the highest value, and it was selected for further measurements, as represented in Figure S3A (Supplementary Materials). FAV molecule has two tautomeric forms, a more stable enol form and a ketone form, and the intersection point of the Ep/pH curves with a clear change in the peak intensity at about 7.0-8.0 may be explained by the replacement of one mechanism (reaction with enol) by another (reaction with ketone) in the tautomeric equilibrium of FAV. A linear plot between the pH values and their corresponding potential was represented in Figure S3B (Supplementary Materials), where the potential of the FAV oxidation peak was moved to less positive potential values upon increasing the pH value. It is noteworthy that Ep (V) = 1.122 + 0.053 pH (r = 0.994), which reveals the proton-dependent nature of FAV on the modified electrode. The value of the slope is close to the theoretical value of 59 mV; hence the number of electrons and protons involved in the electrochemical oxidation of FAV are equal. These findings are in agreement with the number of electrons (≈ 2.0 electrons) included in the oxidation of FAV, which is calculated above using the Laviron equation, and also in agreement with previously reported methods [19][20][21][22].

SWV Parameters
Instrumental parameters affecting the proposed SWV, such as pulse height, step height and frequency, were studied in the ranges 3-30 mV, 1-30 mV and 25-250 V s −1 , respectively. It was observed that pulse height at 5 mV, step height at 3 mV and frequency at 100 V s −1 were the optimal variables for electro-oxidation of FAV at the modified electrode. Besides, the initial potential and deposition time parameters of FAV were examined from −1.2 to + 0.8 V and from 10 to 90 s, and optimum values were −0.2 V and 60 s, respectively.

Linearity and Sensitivity Limits
The calibration curve of FAV over a concentration range from 10-150 × 10 −9 M was constructed under the optimum conditions as represented in Figure S4 (Supplementary Materials). The values of LOD and LOQ were 3.1 and 9.3 × 10 −9 M, respectively. Various statistical parameters of the proposed SWV method are represented in Table 1. The proposed method showed higher sensitivity than various previously reported methods. Table 1. Linearity results for FAV determination by the proposed SWV method using Tb NPS @ poly m-THB/PGE electrode.

Accuracy and Precision
The accuracy, repeatability and intermediate inter-day precisions for the proposed method were investigated. Table 2 summarizes the obtained results. The intra-day precision was evaluated by repeating the measurements of three various concentration levels of FAV working solutions (20,70 and 120 × 10 −9 M). The measurements were repeated over three consecutive days to examine the inter-day precision. Recovery results were observed to be from 98.92 to 101.42%, indicating acceptable accuracy. In addition, the % RSD values were calculated, and the results were less than 1.8%.

Selectivity
The effect of commonly co-existing interfering substances during FAV analysis was examined in order to evaluate the selectivity of the fabricated Tb NPS @ poly m-THB/PGE electrode. The concentration of each substance was tested at 10 folds the concentration of the drug used during the electrochemical measurement. The percentage recovery values were in the range of 96.8-100.2% in the presence of various substances ensuring high selectivity of the proposed SWV method for FAV estimation (Table 3).

Estimation of FAV in Pharmaceutical Tablet
The fabricated Tb NPS @ poly m-THB/PGE electrode showed high sensitivity (LOD = 3.1 × 10 −9 M); hence it was successfully used to determine FAV in commercial tablets. Acceptable recovery results were obtained, ranging from 99.58-100.93% ± 1.2-1.9, ensuring the absence of interference from commonly used excipients, as represented in Table 4. The obtained percentage recovery results were statistically compared to those calculated according to a previously published method [7]. The % recovery of the proposed method was 98.9 ± 1.2 when compared with that of the reported one (97.5 ± 1.6). Moreover, the calculated values of the tand F-tests (n = 5) were 0.63 and 2.35, respectively, ensuring a lack of significant difference between the proposed and the published methods.

Estimation of FAV in Human Plasma
The fabricated electrode Tb NPS @ poly m-THB/PGE was employed for FAV quantitation in human plasma, where a calibration curve of human plasma samples spiked with FAV was constructed in concentrations ranging from 15 to 90 × 10 −9 M to assess linearity. A good linearity was obtained with a correlation coefficient equal to 0.9994. The regression equation is Y = 0.81 + 2.04 X (n = 3); Y is the current value, and X is FAV concentration.
Moreover, % recovery was calculated, and the results ranged from 97.39 to 101.93% with RSD% values less than 2.5, as represented in Table 4. The acceptable results confirm the efficiency of the established SWV method utilizing Tb NPS @ poly m-THB/PGE for the detection of FAV without interferences from plasma constituents. The results obtained confirm the method's sensitivity and applicability for the determination of FAV in human plasma samples.

Stability and Reproducibility of the Fabricated Sensor
The stability of the modified Tb NPS @ poly m-THB/PGE electro-sensor was examined, where the sensor was stored at 4 • C for 20 successive days, and the electrochemical behavior of FAV was assessed every 2 days. The storage stability of Tb NPS @ poly m-THB/PGE was illustrated by the measured current ( Figure S5A, Supplementary Materials) with a 98.53% retention of the original, optimized current values. Moreover, FAV (70 × 10 −9 M) was analyzed using six parallel-prepared modified electrodes fabricated using the same procedure. The corresponding RSD% value didn't exceed 2.1% during the analysis, which proves the good reproducibility of the modified sensor, as shown in Figure S5B, Supplementary Materials.
The presented SWV method showed simplicity, good selectivity and high sensitivity utilizing the novel electrode (Tb NPS @ poly m-THB/PGE) in comparison with previously published articles, as represented in Table 5. Some of the reported methods suffered from several demerits like insufficient sensitivity, complicated procedures or the need for large amounts of organic solvents. This confirms the excellent applicability of the proposed SWV for drug analysis in different media.

Conclusions
An innovative and highly sensitive electrochemical sensor was fabricated based on utilizing Tb nanoparticles supported over new polymer layers of m-THB. The performance of the fabricated sensor was examined for studying the electro-oxidation of FAV using the SWV technique. CV, SEM, XRD, UV-spectrophotometry and EIS techniques were conducted for further characterization. Tb NPs and poly m-THB had synergistic effects, which enhanced the determination of FAV in tablets and in human plasma. A full validation study of the proposed method was carried out according to the ICH guidelines. The proposed sensor showed good recovery results for the demonstration of FAV in different matrices without any interference. Besides, it has high sensitivity and simplicity in fabrication, and this ensured good performance of fabricated senor in FAV determination in complex matrices. Notably, this study is the first to be reported for the investigation of the electrochemical performance of the modified electrode (Tb NPS @poly m-THB/PGE) in the analysis of FAV.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios13020243/s1, Figure S1: Effect of (A) scan rate on the current of FAV (70 × 10 −9 M) in electro-polymerization process of m-THB, and (B) deposition potential of Tb (III) solution; Figure S2