New Construction of Functionalized CuO/Al2O3 Nanocomposite-Based Polymeric Sensor for Potentiometric Estimation of Naltrexone Hydrochloride in Commercial Formulations

Electrically conductive polymeric nanocomposites with nanoparticles are adaptable types of nanomaterials that are prospective for various applications. The extraordinary features of copper oxide (CuO) and aluminium oxide (Al2O3) nanostructures, encourages extensive studies to prospect these metal oxide nanocomposites as potential electroactive materials in sensing and biosensing applications. This study suggested a new CuO/Al2O3 nanocomposite-based polymeric coated wire membrane sensor for estimating naltrexone hydrochloride (NTX) in commercial formulations. Naltrexone hydrochloride and sodium tetraphenylborate (Na-TPB) were incorporated in the presence of polymeric polyvinyl chloride (PVC) and solvent mediator o-nitrophenyloctyl ether (o-NPOE) to form naltrexone tetraphenylborate (NTX-TPB) as an electroactive material. The modified sensor using NTX-TPB-CuO/Al2O3 nanocomposite displayed high selectivity and sensitivity for the discrimination and quantification of NTX with a linearity range 1.0 × 10−9–1.0 × 10−2 mol L−1 and a regression equation EmV = (58.25 ± 0.3) log [NTX] + 754.25. Contrarily, the unmodified coated wire sensor of NTX-TPB exhibited a Nernstian response at 1.0 × 10−5–1.0 × 10−2 mol L−1 and a regression equation EmV = (52.1 ± 0.2) log [NTX] + 406.6. The suggested modified potentiometric system was validated with respect to various criteria using the methodology recommended guidelines.


Introduction
Advances in nanoscience technologies and nanomaterial engineering have opened up new areas in scientific research and the evolution of modified sensing and biosensing probes. The current studies have focused on the production of nanocomposites instead of single nanoparticles. These nanocomposites usually possess different nanoscale domains, which induce synergistic effects as a result of their interfacial interactions [1].
Nanocomposites hold both extraordinary characteristics of nanomaterials and polymer advantages including, chemical resistance, high conductivity, biocompatibility and elasticity [2]. The recent progress in scientific areas, such as chemical engineering [3], biochemistry [4] and physics [5], require the development of novel sensing technologies that combine lower power utilization and miniaturization with highly tactile sensitivity. Nanocomposites are also nanostructures with high activities that exhibit unusual significant combinations and unique engineering possibilities. With increasing the growth rate and rapid inquire to be in sensor constructions, their potential is so prominent that they are successfully utilized in various sensing and biosensing applications [6]. They have appeared as viable alternatives to control the drawbacks of micro composites. Furthermore, these materials are having extraordinary designs and unique optical characteristics that The objective of this study was to design a modified metal oxide (CuO/Al2O3) nanocomposite coated wire sensor with ultra-sensitivity and selectivity towards the detection of NLX in its commercial products. A new strategy involving exploiting the unique physical, chemical, optical and conductive properties of the selected metal oxides has been suggested to enhance the sensitivity and selectivity of the potentiometric modified sensor. The incorporation of CuO/Al2O3 in polymeric matrix will play an impact role in the sensitivity and selectivity of the suggested sensor towards the determined drug. To ensure the analytical suitability of the suggested method, method validation is according to ICH guidelines [49]. Furthermore, a comparative study was carried out between the suggested CuO/Al2O3 nanocomposite coated membrane sensor and the conventionally designed type.

Instruments
This study was conducted using a "digital pH meter HANNA, model 211 (HANNA instruments, Rhode Island, Woonsocket, RI, United States) and the pH meter Metrohm pH-meter model 744 (Metrohm Co., Herisau, Switzerland) was used to adjust the pH of the sample solution throughout the experiments. The designed potentiometric system was consisted of a constructed conventional naltrexone hydrochloride-tetraphenyl borate (NTX-TPB) or modified NTX-TPB-CuO/Al2O3 nanocomposite coated wire sensor in conjunction with silver/silver chloride (Ag/AgCl) as reference one. The synthesized metal oxide nanoparticles and the nanocomposite were characterized using various spectroscopic and microscopic techniques, including UV-2450 spectrophotometer (Shimadzu Corpora- The objective of this study was to design a modified metal oxide (CuO/Al 2 O 3 ) nanocomposite coated wire sensor with ultra-sensitivity and selectivity towards the detection of NLX in its commercial products. A new strategy involving exploiting the unique physical, chemical, optical and conductive properties of the selected metal oxides has been suggested to enhance the sensitivity and selectivity of the potentiometric modified sensor. The incorporation of CuO/Al 2 O 3 in polymeric matrix will play an impact role in the sensitivity and selectivity of the suggested sensor towards the determined drug. To ensure the analytical suitability of the suggested method, method validation is according to ICH guidelines [49]. Furthermore, a comparative study was carried out between the suggested CuO/Al 2 O 3 nanocomposite coated membrane sensor and the conventionally designed type.

Instruments
This study was conducted using a "digital pH meter HANNA, model 211 (HANNA instruments, Rhode Island, Woonsocket, RI, United States) and the pH meter Metrohm pH-meter model 744 (Metrohm Co., Herisau, Switzerland) was used to adjust the pH of the sample solution throughout the experiments. The designed potentiometric system was consisted of a constructed conventional naltrexone hydrochloride-tetraphenyl borate (NTX-TPB) or modified NTX-TPB-CuO/Al 2 O 3 nanocomposite coated wire sensor in conjunction with silver/silver chloride (Ag/AgCl) as reference one. The synthesized metal oxide nanoparticles and the nanocomposite were characterized using various spectroscopic and microscopic techniques, including UV-2450 spectrophotometer (Shimadzu Corporation, Kyoto, Japan), Fourier-Transform Infrared spectroscopy (FT-IR) Spectrum BX spectrometer (PerkinElmer, Waltham, Massachusetts, United States). X-ray diffraction (XRD) Shimadzu XRD-6000 diffractometer (Shimadzu, Kyoto, Japan), scanning electron microscope (SEM) JSM-7610F (JEOL Ltd., Tokyo, Japan), and a transmission electron microscope (TEM) JEM-2100F, (JEOL Ltd., Tokyo, Japan). Furthermore, Energy-Dispersive X-ray Spectroscopy (EDX), using EDX-8100 (Shimadzu, Kyoto, Japan) analysis was applied to detect the presence of Cu, Al and O elements in the synthesized nanomaterials.

Preparation of NTX-TPB Electroactive Complex
The electroactive compound NTX-TPB was prepared by mixing equal volume (50 mL) of an equimolar concentration (1.0 × 10 −2 mol L −1 ) of aqueous NTX and TPB solution. A white precipitate of NTX-TPB was formed. The mixture solution was filtered using Whatman filter paper No. 41 and the precipitate was washed using deionized water and left to dry overnight.

Synthesis of CuO and Al 2 O 3 Nanoparticles
The precipitation method was used to synthesize CuO nanoparticles by dissolving 1.596 g of copper nitrate (Cu(NO 3 ) 2 ·3H 2 O) in 100 mL deionized water to form 0.1 mol L −1 concentration. An aqueous sodium hydroxide solution (0.1 mol L −1 ) was prepared and slowly added dropwise under vigorous stirring until the pH achieved 14. The formed precipitate was repeatedly washed three times and neutralized by deionized water and absolute ethanol. Subsequently, the resulting precipitate was dried at 80 • C for 12 h, then it was calcined for 4 h at 500 • C. Al 2 O 3 nanoparticles were prepared using a sol-gel method by heating a solution of citric acid/aluminium nitrate nonahydrate prepared in deionized water with a molar ratio of 0.5. The solution was heated at 60 • C under continuous stirring until the formation of white sol. The resulted gel was heated under constant stirring up to 80 • C until the formation of transparent gel. The formed gel was dried in an oven for 12 h at 90 • C and then ground and sintered at 600 • C for 4 h.

Characterization of Synthesized Nanomaterials
Spectroscopic analysis for the synthesized CuO, Al 2 O 3 and CuO/Al 2 O 3 nanomaterials was performed at a wavelength range of 200-500 nm to confirm their formation. FT-IR detection was also conducted to determine the possible functional groups that can appear in the CuO, Al 2 O 3 and CuO/Al 2 O 3 nanomaterials spectra. Further investigation was carried out by XRD analysis using Kα radiation (λ = 1.5418 Å) under 40 kV voltage and an operating current of 35 mA. The results of XRD were estimated at ambient temperature using a scan rate of 0.3 s/point and 0.02 • resolution. The morphological shape and size distribution were studied using scanning and transmission electron microscope.

Preparation of Standard NTX Solution
The standard NTX (1.0 × 10 −2 mol L −1 ) solution was prepared by dissolving 0.341 g of pure NTX powder in 100 mL deionized water. The different working analytical solutions were prepared by serial dilution in water.

Sensor Construction and Membrane Composition
A conventional (NTX-TPB) coated wire sensor was constructed using a mixture of (PVC, 190 mg), electroactive material (NTX-TPB, 10 mg) and plasticizer (o-NPOE, 0.35 mL) in 5 mL of THF. The resulted polymeric mixture was poured into a Petri dish and kept at room temperature to evaporate slowly. The tip of the aluminium wire was polished and cleaned using deionized water, followed by acetone. The tip of the cleaned wire was immersed several times in the polymeric membrane solution (NTX-TPB) until the formation of coated membrane on its surface. To construct the modified sensor another clean Al wire was dipped three times in the polymeric solution of CuO/Al 2 O 3 nanocomposite to produce a thin layer membrane on its surface. The sensor was allowed to dry, then dipped again in the above polymeric (NTX-TPB) solution several times until the formation of a uniform coated membrane. Both coated wire sensors were assembled as Al wire/coated membrane/test solution//Ag/AgCl reference electrode. Figure 2 demonstrated the preparation of the designed modified NTX-TPB-CuO/Al 2 O 3 nanocomposite sensor and the potentiometric system.

Sensor Construction and Membrane Composition
A conventional (NTX-TPB) coated wire sensor was constructed using a mixture of (PVC, 190 mg), electroactive material (NTX-TPB, 10 mg) and plasticizer (o-NPOE, 0.35 mL) in 5 mL of THF. The resulted polymeric mixture was poured into a Petri dish and kept at room temperature to evaporate slowly. The tip of the aluminium wire was polished and cleaned using deionized water, followed by acetone. The tip of the cleaned wire was immersed several times in the polymeric membrane solution (NTX-TPB) until the formation of coated membrane on its surface. To construct the modified sensor another clean Al wire was dipped three times in the polymeric solution of CuO/Al2O3 nanocomposite to produce a thin layer membrane on its surface. The sensor was allowed to dry, then dipped again in the above polymeric (NTX-TPB) solution several times until the formation of a uniform coated membrane. Both coated wire sensors were assembled as Al wire/coated membrane/test solution//Ag/AgCl reference electrode. Figure 2 demonstrated the preparation of the designed modified NTX-TPB-CuO/Al2O3 nanocomposite sensor and the potentiometric system.

Calibration Graphs
The linear relationship between the -logarithm NTX concentrations (mol L −1 ) and the potential difference (mV) of each conventional (NTX-TPB) and modified (NTX-TPB-CuO/Al2O3) nanocomposite was determined. The calibration graphs were plotted using 50 mL of NTX standard solutions in the concentration range 1.0 × 10 −9 -1.0 × 10 −2 mol L −1 using the constructed working NTX-TPB or NTX-TPB-CuO/Al2O3 nanocomposite sensors separately in connection with a reference electrode (Ag/AgCl). The surface of the sensor should be cleaned with deionized water and dried with tissue paper prior to every measurement.

Calibration Graphs
The linear relationship between the -logarithm NTX concentrations (mol L −1 ) and the potential difference (mV) of each conventional (NTX-TPB) and modified (NTX-TPB-CuO/Al 2 O 3 ) nanocomposite was determined. The calibration graphs were plotted using 50 mL of NTX standard solutions in the concentration range 1.0 × 10 −9 -1.0 × 10 −2 mol L −1 using the constructed working NTX-TPB or NTX-TPB-CuO/Al 2 O 3 nanocomposite sensors separately in connection with a reference electrode (Ag/AgCl). The surface of the sensor should be cleaned with deionized water and dried with tissue paper prior to every measurement.

Optimization of Analytical Conditions
The potential response of the constructed coated wire sensors can be significantly affected by the change in the pH of the investigated solutions. The suitable pH range of conventional NTX-TPB and modified NTX-TPB-CuO/Al 2 O 3 nanocomposite sensors was determined using 1.0 × 10 −4 mol L −1 NTX solution. The pH of the tested sample was acidified using 0.1 mol L −1 of hydrochloric acid. The potential response of the working sensor was measured after increasing the pH value using 0.1 mol L −1 sodium hydroxide solution. The pH graphs were constructed using the change in potential against the pH.
A separate solution method [50] was used to monitor the selectivity of the investigated NTX sensors. Briefly, the selectivity coefficient of each sensor towards NTX and some foreign substances and additives such as (Povidone, hydroxypropyl methylcellulose, lactose monohydrate, magnesium stearate, microcrystalline cellulose, polyethylene glycol, polysorbate 80, tryptophan, lysine, and glycine) was determined using 1.0 × 10 −3 mol L −1 of NTX solution. The selectivity coefficient of conventional NTX-TPB and modified NTX-TPB-CuO/Al 2 O 3 nanocomposite was calculated using the following equation: where K pot (selectivity coefficient), E 1 (electrode potential) of 1.0 × 10 −3 mol L −1 NTX, E 2 (electrode potential) of 1.0 × 10 −3 mol L −1 of interfering species, B z+ (interfering species), and S (slope) of the calibration curve, respectively. The response time was tested by recording the dynamic potential response of the investigated drug, using NTX concentration range of 1.0 × 10 −9 -1.0 × 10 −2 mol L −1 .

Estimation of Naltrexone Hydrochloride ® Tablets
The content of ten naltrexone hydrochloride ® tablets (50 mg/tablet) was pulverized and mixed well. An accurate quantity of 0.341 g was dissolved in 50 mL deionized water, then centrifuged at 1500 rpm for 5 min and filtered to remove the co-additive materials. The clear solution was completed with deionized water to be 100 mL, the resulting NTX solution (1.0 × 10 −2 mol L −1 ) was diluted with the same solvent to prepare the working analytical samples in the range of 1.0 × 10 −5 -1.0 × 10 −2 and 1.0 × 10 −9 -1.0 × 10 −2 mol L −1 . The designed NTX-TPB and modified NTX-TPB-CuO/Al 2 O 3 nanocomposite sensors were separately utilized to quantify the investigated drug in its commercial tablets.

Characterization of CuO/Al 2 O 3 Nanocomposite
Various spectroscopic techniques such as UV-Vis, FT-IR, XRD, and EDS were exploited to characterize the synthesized CuONPs/Al 2 O 3 NPs nanocomposite. One of the most suitable and useful methods for primary confirmation of shape, size and stability of the engineered nanoparticles in their aqueous suspensions is the UV-Vis method. The recorded spectrum of CuONPs/Al 2 O 3 NPs displayed two significant broad absorption peaks at 280 and 400 nm for CuO and Al 2 O 3 nanoparticles, respectively ( Figure 3). The band gaps of the as-synthesized metal oxide nanoparticles were calculated from the formula: where "h is Planck's constant, c is the velocity of light, and λ is the wavelength." The optical band gap energy of CuONPs and Al 2 O 3 NPs were estimated to be 2.85 eV and 3.54 eV, respectively [51,52].   (Figure 5a). The XRD pattern of Al2O3 nanoparticles showed that the sample was in nanorods crystalline shape and exhibited different peaks at 32.65° (2 2 0), 35.14° (3 1 1), 37.26° (2 2 2), 43.58° (4 0 0), 54.22° (4 2 2), 59.84° (4 4 0), 74.89° (6 2 0). All observed diffraction peaks can be matched to those of bulk α-Al2O3, with cell constant (α = 8.395 Å), which is in acceptance with the value of (JCPDS file No. . No other impurity phases can be detected, indicating high purity of the synthesized Al2O3 nanoparticles (Figure 5b). Very similar diffraction peaks were observed in the XRD pattern of CuO/Al2O3 nanocomposite. Therefore, the above diffraction peaks can be used to verify the formation of CuO/Al2O3 nanocomposite (Figure 5c). The prominent peak in the XRD pattern was used to calculate the average crystallite size using a Scherrer formula: where λ, θ and β are the X-ray wavelength, Bragg diffraction angle, and the Full width at half maximum of the XRD peak appearing at a diffraction angle θ, respectively. The average crystallite size of the synthesized CuONPs and Al2O3NPs were evaluated to be 17.09 ± 1.1 and 27.61 ± 3.1 nm, respectively.  constant (α = 8.395 Å), which is in acceptance with the value of (JCPDS file No. . No other impurity phases can be detected, indicating high purity of the synthesized Al 2 O 3 nanoparticles (Figure 5b). Very similar diffraction peaks were observed in the XRD pattern of CuO/Al 2 O 3 nanocomposite. Therefore, the above diffraction peaks can be used to verify the formation of CuO/Al 2 O 3 nanocomposite (Figure 5c). The prominent peak in the XRD pattern was used to calculate the average crystallite size using a Scherrer formula: where λ, θ and β are the X-ray wavelength, Bragg diffraction angle, and the Full width at half maximum of the XRD peak appearing at a diffraction angle θ, respectively. The average crystallite size of the synthesized CuONPs and Al 2 O 3 NPs were evaluated to be 17.09 ± 1.1 and 27.61 ± 3.1 nm, respectively.   The dislocation density (δ), which describes the quantity of defects in the sample is known as the length of dislocation lines per unit volume of the crystal and is calculated using the following equation [54].
where D is the crystallite size. The dislocation density of CuONPs and Al 2 O 3 NPs at room temperature was found to be 1.42 × 10 −3 and 1.31 × 10 −3 (nm) −2 , respectively. The bond length of Cu-O and Al-O was calculated from the equation [55].
where u is the positional parameter in the wurtzite structure and is a measure of the amount by which each atom is displaced according to the next along the 'c' axis. 'u' is given by the equation: The u value increases with the decreases in c/a ratio this for tetrahedral distances Remaining constant via a distortion of tetrahedral angels. The calculated bond length of Cu-O and Al-O was found to be 1.928 and 1.869 Å, respectively. The calculated bond length is an agreement with the bond length of Cu-O and Al-O in the unit cell [56,57].
Further microscopic studies were performed using TEM and SEM to confirm the size, shape and surface morphology of CuO/Al 2 O 3 nanocomposite according to the as-prepared CuONPs and Al 2 O 3 NPs. The obtained images of TEM (Figure 6a-c) and SEM (Figure 6d-f) confirmed the uniform distribution of the formed spherical and rode shapes CuONPs and Al 2 O 3 NPs, respectively. However, the surface morphology of CuO/Al 2 O 3 nanocomposite showed highly aggregated crystals with particle sizes ranging from 80-100 nm. amount by which each atom is displaced according to the next along the 'c' axis. 'u' is given by the equation: The u value increases with the decreases in c/a ratio this for tetrahedral distances Remaining constant via a distortion of tetrahedral angels. The calculated bond length of Cu-O and Al-O was found to be 1.928 and 1.869 Å , respectively. The calculated bond length is an agreement with the bond length of Cu-O and Al-O in the unit cell [56,57].
Further microscopic studies were performed using TEM and SEM to confirm the size, shape and surface morphology of CuO/Al2O3 nanocomposite according to the as-prepared CuONPs and Al2O3NPs. The obtained images of TEM (Figure 6a-c) and SEM (Figure 6df) confirmed the uniform distribution of the formed spherical and rode shapes CuONPs and Al2O3NPs, respectively. However, the surface morphology of CuO/Al2O3 nanocomposite showed highly aggregated crystals with particle sizes ranging from 80-100 nm.

Characteristics of the Constructed Sensors
NTX reacts with TPB to produce a very stable complex NTX-TPB, which is soluble in THF. The conventional NTX-TPB and modified coated wire NTX-TPB-CuO/Al 2 O 3 NPs nanocomposite sensors were designed by mixing the electroactive materials (ion pairs) with PVC and a solvent mediator (o-NPOE) using THF. The current study suggested the use of a high dielectric constant (o-NPOE, ε = 24) serves as a fluidizer allowing the homogenous dissolution of the electroactive materials and facilitating its mobility and diffusion through the polymeric matrix of the membrane. Also, it enhances the selectivity coefficient of the sensor by offering a suitable mechanical property for the coated membrane [53]. Table 1 reported the effective potential response and the potentiometric behavior of the constructed NTX-TPB and NTX-TPB-CuO/Al 2 O 3 NPs nanocomposite sensors. The ob-tained data showed that the designed sensors exhibited Nernstian behavior with slopes of E mV = (52.1 ± 0.2) log [NTX] + 406.6 and E mV = (58.25 ± 0.3) log [NTX] + 754.25 with linearity ranges 1.0 × 10 −5 -1.0 × 10 −2 mol L −1 (r 2 = 0.9995) and 1.0 × 10 −9 -1.0 × 10 −2 mol L −1 (r 2 = 0.9999) for the above-mentioned sensors, respectively (Figure 8a,b). The addition of CuO/Al 2 O 3 nanocomposite to modify the conventional NTX-TPB sensor enhanced the potential response of the designed sensor (NTX-TPB-CuO/Al 2 O 3 nanocomposite) to a wider linear detection range with high sensitivity towards the detection of NTX solution. These results can be attributed to the large surface area of the added nanoparticles which increased the surface conductivity of the designed modified sensor. Additionally, the higher detection results observed by using the modified sensor could be due to the high dielectric permittivity value of CuONPs (≈10 4 ) and Al 2 O 3 NPs (≈7. [8][9][10][11].1) at ambient temperature [58,59]. The dynamic response of the designed NTX-TPB conventional and modified NTX-TPB-CuO/Al 2 O 3 NPs nanocomposite sensors were investigated under optimized experimental conditions to detect the difference between the instant potential time and its steady-state value (1 mV) [60]. The recorded dynamic responses were found to be 70 s and 50 s for the above-mentioned conventional and modified sensors, respectively. It was observed that the modified sensor with metal oxide nanocomposite exhibited a fastness response with high mechanical stability more than the conventional type. The modification of the membrane with high surface area to volume ratio metal oxides nanocomposite and new unique physicochemical properties enhances the electrical conductivity of the modified sensor towards the detection of the tested analyte in the sample. Moreover, the remarkable electrical and exceptional capacity characteristics of metal oxides nanocomposite such as the high charge transfer resulting at the nanomaterial interfaces are of paramount importance when the nanomaterials are employed as transducing materials in sensing applications [61].

Characteristics of the Constructed Sensors
NTX reacts with TPB to produce a very stable complex NTX-TPB, which is soluble in THF. The conventional NTX-TPB and modified coated wire NTX-TPB-CuO/Al2O3NPs nanocomposite sensors were designed by mixing the electroactive materials (ion pairs) with PVC and a solvent mediator (o-NPOE) using THF. The current study suggested the use of a high dielectric constant (o-NPOE, = 24) serves as a fluidizer allowing the homogenous dissolution of the electroactive materials and facilitating its mobility and diffusion through the polymeric matrix of the membrane. Also, it enhances the selectivity coefficient of the sensor by offering a suitable mechanical property for the coated membrane [53]. Table 1 reported the effective potential response and the potentiometric behavior of the constructed NTX-TPB and NTX-TPB-CuO/Al2O3NPs nanocomposite sensors.   [58,59]. The dynamic response of the designed NTX-TPB conventional and modified NTX-TPB-CuO/Al2O3NPs nanocomposite sensors were investigated under optimized experimental conditions to detect the difference between the instant potential time and its steady-state value (1 mV) [60]. The recorded dynamic responses were found to be 70 s and 50 s for the above-mentioned conventional and modified sensors, respectively. It was observed that the modified sensor with metal oxide nanocomposite exhibited a fastness response with high mechanical stability more than the conventional type. The modification of the membrane with high surface area to volume ratio metal oxides nanocomposite and new unique physicochemical properties enhances the electrical conductivity of the modified sensor towards the detection of the tested analyte in the sample. Moreover, the remarkable electrical and exceptional capacity characteristics of metal oxides nanocomposite such as the high charge transfer resulting at the nanomaterial interfaces are of paramount importance when the nanomaterials are employed as transducing materials in sensing applications [61].  The hydrogen ion concentration can greatly affect the potential response of the membrane sensor. Thus, it is very necessary to determine the suitable pH range where the po- The hydrogen ion concentration can greatly affect the potential response of the membrane sensor. Thus, it is very necessary to determine the suitable pH range where the potential response of the coated membrane sensor is not affected by hydrogen ions. The obtained results showed that the above-mentioned NTX-TPB and NTX-TPB-CuO/Al 2 O 3 NPs nanocomposite sensors are independent in the pH range 2-5 and NTX can be simply estimated using the designed sensors within this pH range (Figure 9). It was observed that at high [H + ] in acidic medium (pH < 2), the protonated ion-pair complex was formed, and the potential readings of the sensors were slightly increased due to the poor responsiveness to NTX ions, whereas at high [OH -] in alkaline medium (pH > 5) the potential readings were gradually decreased as a result of a competition between NTX ions and OH-ions and hence reduces the interaction between the investigating drug ions and the sites of ion-pair on the sensor membrane [62].
A separate solution method [50] was applied to evaluate the interference effect of some foreign substances on the selectivity coefficient of the constructed NTX sensors using 1.0 × 10 −3 mol L −1 solution. The modified NTX-TPB-CuO/Al 2 O 3 nanocomposite sensors exhibited excellent selectivity. The unique physicochemical features of the synthesized CuO/Al 2 O 3 NPs and their large surface area increase the conductivity of the designed modified sensor and hence elevate its selectivity towards the detected NTX ions. Moreover, the NTX coated membrane selectivity is refer to the free energy transfer of ions (NTX + ) generated between the active sites in the membrane and the working solution. No interferences were noticed by the tested co-additives and amino acids. Thus, excellent selectivity and good tolerance were accomplished by using the modified NTX sensor for the determination of NTX (Table 2).

Estimation of NTX in Bulk Form
The suggested conventional NTX-TPB and NTX-TPB-CuO/Al 2 O 3 sensors were used to estimate NTX in its bulk form and the obtained results were calculated as percentage recoveries of 98.33 ± 0.9% and 99.81 ± 0.2% for the above-mentioned sensors, respectively ( Table 3). The high dynamic response of the modified sensor can be due to the advanced conductivity and dielectric permittivity properties of the used CuONPs (≈10 4 ) and Al 2 O 3 NPs (≈7.8-11.1), which provide excellent sensitivity and selectivity towards NTX solution.

Validation Study
The International Council for Harmonization of Technical Requirements for Pharmaceuticals (ICH) guidelines [49] were employed to ensure the validity and suitability of the suggested potentiometric method. Linear relationships in the concentration ranges The accuracy of the suggested potentiometric method was studied using nine authentic NTX concentrations and the results were expressed as mean percentage recoveries as 98.73 ± 1.09% and 99.72 ± 0.4% for NTX-TPB and NTX-TPB-CuO/Al 2 O 3 nanocomposite, respectively ( Table 4). The precision was also studied using intra-day and inter-day assays. The recorded results were expressed as a percentage relative standard deviation (%RSD) as 0.3% and 0.2% for the two constructed NTX sensors, respectively ( Table 5). The system robustness was evaluated by performing a slight variation in method parameter by changing the pH of working solutions to 5 ± 0.5. The calculated percentage recoveries were 98.45 ± 0.7% and 99.63 ± 0.3% for the conventional and modified NTX coated wire sensors (Table 1). Another experiment was carried out to ensure the ruggedness of the suggested potentiometric method by performing the analysis using another pH-meter (Metrohm model-744) in the different laboratories and different operators. The mean percentage recoveries were found to be 98.59 ± 0.6% and 99.68 ± 0.4% for the above NTX sensors, respectively ( Table 1). The outcomes of method validation revealed a good agreement with those resulting from the suggested method and no remarkable differences was noticed.

Analysis of NTX in Naltrexone Hydrochloride ® Tablets
The investigated NTX was determined in its commercial tablets Naltrexone hydrochloride ® (50 mg/tablet) using the constructed NTX-TPB and NTX-TPB-CuO/Al 2 O 3 nanocomposite. The potential readings of the working solutions 1.0 × 10 −5 -1.0 × 10 −2 and 1.0 × 10 −9 -1.0 × 10 −2 mol L −1 were measured and the percentage recoveries of NTX were estimated from the regression equations. The recorded results were calculated as 99.05 ± 0.5% and 99.70 ± 0.2% for the above-designed sensors. The obtained results were compared with the reported method by Ganjali et al. [63] using the t-student's test and F-test [64] and showed excellent sensitivity and selectivity of the designed sensor for the determination of NTX (Table 6). The dielectric constant is a critical factor that evaluate the capability of the materials to store charges [65]. Metal oxides with high dielectric constant are commonly used in electronics and sensors. As they do not permit the flow of charges through them, they allow for exerting electrostatic fields and storing charges [66]. The combination of metal oxides nanoparticles with polymeric matrix in nanocomposites could effectively improve the electrical, optical and conductive properties of the modified sensor. These properties are much sensitive to changes in the particles shape and size. As previously reported the nanoparticles themselves could serve as conductive junctions between the polymeric chains that resulted in as increase of electrical conductance of the composites [67]. Additionally, the modification of the sensor with nanocomposite containing metal oxides with high surface area to volume ratios and possess new physicochemical properties enhanced the charge transfer and the electrical conductivity of the sensor towards the interaction with the target analyte in the test solution and resultingly improve sensitivity of the sensor detection [68]. A comparative study was carried out to compare the efficiency of the designed modified NTX-TPB-CuO/Al 2 O 3 nanocomposite sensor with the previously constructed sensors [63,69,70]. The comparative results revealed high sensitivity of the modified sensor towards the determination of NTX with a wide detection range of 1.0 × 10 −9 -1.0 × 10 −2 and LOD 5.0 × 10 −6 mol L −1 than the reported ( Table 7). The selection of nanostructured materials and sensor design method is the most important factor for achieving ultrasensitive sensor with desired characteristics. The shape and size of the nanoparticles used governs the surface to volume ratio, which is a crucial factor to enhance the interface reactions on the overall nanomaterial's electrical conductivity. The nanoscale morphology will not only influence the sensitivity of the sensor but also affect the dynamic response of the sensor and the long-term stability of the sensor due to the high chemical stability of these nanomaterials. The electrical conductivity of the fabricated sensors using metal oxide nanocomposite might also based on the molecular structure and the polymeric medium such as the crystallinity and long chain polymer [71].