Modified Screen-Printed Microchip for Potentiometric Detection of Terbinafine Drugs

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Introduction
Terbinafne represents one of the most commonly prescribed medications in the United States, with more than one million prescriptions. It is a synthetic antifungal medication that mainly fghts infections caused by fungus that afect the toenails or fngernails. It is generally taken by the mouth or applied to the skin as an ointment or cream. Chemically, terbinafne's (C 21 H 25 N) name is [(2E)-6,6-dimethylhept-2-en-4-yn- 1-yl] (methyl) (naphthalen-1-ylmethyl) amine. It belongs to allyl amine derivatives, which provide broad-spectrum activity against dimorphic fungi, yeasts, dermatophytes, and molds. It is slightly soluble in water but soluble in methanol, ethanol, and methylene chloride. Terbinafne leads to the death of fungal and bacterial cells based on its selective inhibition of the growth of thier cell wall. It is highly lipophilic in nature and tends to accumulate in nails, fatty tissues, and skin.
Based on its therapeutic importance, precise and accurate quantifcation of terbinafne in human fuids and pharmaceutical formulations is of considerable signifcance. Several methods have been recently reported for the quantitative determination of terbinafne in diferent pharmaceutical and real biological samples. Back volumetric titration with sodium hydroxide using potentiometric end point detection and reversed phase HPLC have been reported for the ofcial quantifcations of terbinafne in British and American pharmacopeia, respectively [1]. Other than ofcial methods, titration with perchloric acid in acetic acid media [1] and diferent chromatographic techniques [1][2][3][4][5][6][7][8][9][10] have also been reported for the determination of terbinafne.
To the best of our knowledge, so far, no potentiometric microchips have been reported for the determination of terbinafne drugs in pharmaceutical formulations. However, the development of miniaturized microchips has widespread and growing interest in manufacturing potentiometric sensors with extremely valuable modifying response characteristics as well [18][19][20][21][22]. Te microfabrication of miniaturized potentiometric screen-printed microchips represents an important challenge in the expanding feld of modern analytical tools [23][24][25][26] due to their mass production, integration, and automation feasibility. Such potentiometric microsensors have the advantages of small size, a wide range of applications, high reproducibility, good accuracy, and a small sample volume [27][28][29][30].
Te objective of this work was to develop a precise, accurate, simple, fast, and reliable screen-printed microchip, which would serve as a potentiometric quantifcation method for terbinafne in its pharmaceutical formulations. Tis paper, consequently, describes microfabrication, potentiometric characterization, and analytical application of terbinafne based on potentiometric screen-printed microsensors modifed by multiwall carbon nanotubes (MWCNTs). Based on our previous work, a combination of the screen-printed platform substrate with modifcation of the sensitive element with MWCNTs and the nebulization process of the cocktail coating mixture recently developed results in superior potentiometric response parameters in terms of sensitivity, selectivity, credibility, and versatile applicability [19,21,25,27]. Realization of such microdevices generates new generations of useful and promising microchip sensors for the detection of drugs and biological species from diferent real samples with high accuracy and precision.

Chemicals and Reagents.
All the used materials and reagents were of analytical reagent grade, unless otherwise stated. Moreover, double-distilled water obtained from a POLNA water distiller (MERA, Zaklady Automatyki, Poland, 1.0 MΩ·cm −1 ) was used for rinsing the glassware and for the preparation of reagents throughout. All chemicals used in diferent studies were of analytical reagent grade and purchased from Sigma Aldrich (UK), PubChem (USA), and Merck (USA). Te used microelectrode substrates were screen-printed plastic microchips comprising a working carbon electrode (0.25 mm PET, 3 mm/6 mm in diameter, and graphene-modifed SPE) and purchased from Suzhou Delta-Biotech (Ltd, China). Purifed multiwall carbon nanotubes (MWCNs, id: 5-12 nm, od: 30-50 nm, length: 10-20 μm, and purity: >95%) were obtained from the Chengdu Organic Chemicals Company "COCC," China. Terbinafne raw material (purity: 99.6%) was a gift supplied by the Egyptian Drug Authority, EDA. Terbinafne drugs with diferent formulations were collected from local pharmaceutical stores and used in the application studies of the microchip.

Instrumentation.
Electrochemical characterization measurements were carried out at room temperature using an Orion (model 720) pH/mV meter and a Lab companion HP-3000L magnetic stirrer. An Orion (model 91-72) combination pH electrode was used for all pH experiments. Microsensor based on "terbinafne: ammonium heptamolybdate" as ion pair complex, carbon nanotubes as modifer, and screen-printed microchip as support, was used as working electrode sensitive for Terbinafne. Tis microelectrode was applied in conjunction with an Orion single junction reference electrode for all potentiometric measurements.

Synthesis of the Sensitive Membrane
Layer. Te sensitive layer mixture was prepared for each assembly by thoroughly mixing the potassium tetrakis (4-chlorophenyl) borate anion excluder, plasticised ionophore (terbinafne: ammonium heptamolybdate ion pair complex, carbon nanotube composite), and poly (vinyl chloride) support in tetrahydrofuran (THF) as a solvent in a small beaker. Before being used as a sensitive membrane coat, the cocktail coating mixture was then transferred into a homemade manual small nebulizer and sonicated for 2 h.

Screen-Printed Terbinafne Microchip.
Te microfabrication of the disposable plastic screen-printed electrode integrated with the organic membrane sensitive layer was reported using a cost-efective, fast, and simple new approach [19,21,23], as described in our previous projects. In this technique, plastic disposable screen-printed microchips ( Figure 1) were rinsed in double-distilled water and left to dry in the air at room temperature before being used as electrode substrates in all microchip assemblies. Two assemblies of the microchip containing diferent constituents of the sensitive membrane, as summarized in Table 1, were fabricated and examined as terbinafne potentiometric microsensors. Te metal contacts of the screen-printed microchip substrate were properly covered using tissue paper before the deposition of the sensitive layer. In a fume hood, small aliquots (few microlitre) of the organic membrane sensitive layer were nebulized successively onto the surface of screen-printed microchips for a few seconds. After each successive nebulization step, the very thin layer of the sensitive membrane deposited on the surface of the chip substrate was then left in the air for 2-3 min for solvent volatilization. Nebulization steps were successively repeated several times until a uniform layer of the organic membrane sensitive coat covering the substrate surface was obtained. To spread out nanoparticles, the cocktail coating sensitive mixture was sonicated for 2 h. Te cocktail coating sensitive mixture was sonicated for 2, were fabricated and examined as terbinafne potentiometric microsensors. Te metal contacts of the screen-printed microchip substrate were properly covered using tissue paper before the deposition of the sensitive layer. In a fume hood, small aliquots (few microlitre) of the organic membrane sensitive layer were nebulized successively onto the surface of screen-printed microchips for a few seconds. After each successive nebulization step, the very thin layer of the sensitive membrane deposited on the surface of the chip substrate was then left in the air for 2-3 min for solvent volatilization. Nebulization steps were successively repeated several times until a uniform layer of the organic membrane sensitive coat covering the substrate surface was obtained. To spread out nanoparticles, the cocktail coating sensitive mixture was sonicated for 2 h. Te cocktail coating sensitive mixture was sonicated for 2 h prior the nebulization process and for 3, were fabricated and examined as terbinafne potentiometric microsensors. Te metal contacts of the screen-printed microchip substrate were properly covered using tissue paper before the deposition of the sensitive layer. In a fume hood, small aliquots (few microlitre) of the organic membrane sensitive layer were nebulized successively onto the surface of screen-printed microchips for a few seconds. After each successive nebulization step, the very thin layer of the sensitive membrane deposited on the surface of the chip substrate was then left in the air for 2-3 min for solvent volatilization. Nebulization steps were successively repeated several times until a uniform layer of the organic membrane sensitive coat covering the substrate surface was obtained. To spread out nanoparticles, the cocktail coating sensitive mixture was sonicated for 2 h. Te cocktail coating sensitive mixture was sonicated for 2 h prior the nebulization process and for 3, were fabricated and examined as terbinafne potentiometric microsensors. Te metal contacts of the screen-printed microchip substrate were properly covered using tissue paper before the deposition of the sensitive layer. In a fume hood, small aliquots (few microlitre) of the organic membrane sensitive layer were nebulized successively onto the surface of screen-printed microchips for a few seconds. After each successive nebulization step, the very thin layer of the sensitive membrane deposited on the surface of the chip substrate was then left in the air for 2-3 min for solvent volatilization. Nebulization steps were successively repeated several times until a uniform layer of the organic membrane sensitive coat covering the substrate surface was obtained. To spread out nanoparticles, the cocktail coating sensitive mixture was sonicated for 2 h. Te cocktail coating sensitive mixture was sonicated for 2 h prior the nebulization process and for 3 min between the successive nebulization steps, to spread out the nanoparticles. Te microfabricated chip assembly was used in the characterization and application studies of terbinafne analysis. Prior measurements, the microchip assemblies were soaked in 10 −2 mole·L −1 terbinafne solution for one hour. In addition, the chips were store dry in air when not in use.
A cocktail coating mixture containing the nanocomposite sensitive element was deposited on the surface of a thin-flm screen-printed plastic microchip substrate using the nebulization methodology, which has been recently developed (Figure 2). Prior to the deposition of the sensitive layer, the surface of the thin-flm gold microchip substrate was treated chemically and electrochemically, respectively. Te realized microchip was used as a working electrode in conjunction with a Ag/AgCl commercial reference electrode in potentiometric measurements. Te potentiometric characterization and analytical application studies of the terbinafne microsensor assembly were performed according to the IUPAC recommendation.

Electrochemical Characterization of Terbinafne
Microchips. Te terbinafne screen-printed microchip modifed with MWCNTs was realized using a simple, fast, and cheap approach, which has been recently developed [19,21]. It was found that the modifcation of the sensitive membrane layer of the potentiometric microchip-based electrodes with MWCNTs signifcantly improved the performance properties of the chip due to unique electronic and mechanical properties of carbon nanotubes [25,27]. MWCNTmaterials possess a greater surface area, excellent biocompatibility, and facilitate redox reactions with rapid electron-transfer rates, and consequently, they are signifcantly used in the development of electrochemical microsensors [19,21,25,27]. Te performance characteristics including sensitivity, selectivity, response time, detection limit, the efect of pH, and the linear range of the elaborated new microsensor modifed with MWCNTs were measured using the microfabricated assembly according to the IUPAC recommendations.
Prior to the microfabrication of the chip assembly, the chemical structure of the prepared terbinafne: ammonium heptamolybdate ion pair complex was analysed using FTIR spectroscopy. Te results obtained for the ion pair (spectra a) and for the terbinafne drug (spectra b) are presented in Figure 3. As can be seen, new peaks appear at 3450 cm −1 and 951 cm −1 in curve "a" (ion pair spectra) when compared with curve "b" (drug spectra). Tese peaks are the characteristic absorption peaks of N-H groups in the quaternary ammonium ion derivative. Te FTIR spectra showed that the functional groups of the synthesis product correspond to the terbinafne ion pair, which confrms the formation of the proposed ionophore ion pair complex.  To evaluate the analytical performance of the elaborated microchip of the terbinafne drug, the potential of the designed assembly was recorded after successive immersion in diferent concentrations of terbinafne from 10 −10 -10 −2 mole·L −1 , and the obtained calibration graph is presented in Figure 4. Te calibration plot showed that the linear detection response covers the range from 10 −8 -10 −2 mole·L −1 , with a Nernstian sensitivity of 58.5 ± 0.5 mV/concentration decade and a detection limit of 5 × 10 −9 mole·L −1 .
Te response time defned as the time needed by the chip assembly to achieve a stable potential was found to be less than (30 s) over all calibration graph. Tis study was conducted by successive immersing of the chip assembly in a series of terbinafne concentration from 10 −9 -10 −2 mole·L −1 starting from low to high concentration. Te time required for the chip to reach the steady potential within ±1 mV from its fnal value was recorded, and the results obtained are presented in Figure 5. As can be seen, the chip quickly (≤30 s.) reached its equilibrium response in the whole tested terbinafne concentration range. Moreover, the potential values of the dynamic response revealed that linear Nernstian behavior covers the terbinafne concentration range of the calibration plot. Tese emphasized the reliability, credibility, and repeatability of the realized chip for accurate and precise quantifcation of terbinafne drugs.
Te long-term stability of the elaborated terbinafne microchip was determined by frequent calibration of the assembly, and the performance parameters of the chip were collected after each calibration. Tese studies revealed that the lifetime of the realized terbinafne chip was more than 4 months. During this period, performance parameters are almost the same without any signifcant changes, and these fndings are in good agreement with those obtained for similar screen-printed microchips fabricated by the same methodology [19,21,25,27].
To examine the infuence of pH on the chip response, the potential of the assembly was detected at two diferent concentrations of terbinafne solutions (1 × 10 −5 and 1 × 10 −4 mole·L −1 ) from a pH value of 4.5 up to 9.5. In this study, small aliquots of concentrated solutions of sodium hydroxide and nitric acid were utilized in pH adjustment, and the results obtained are presented in Figure 6. Te results obtained revealed that the potential of the chip was not afected by change in pH of the test solution in the pH range of 7-9, and consequently, tris-HCl bufer (1 mole·L −1 and pH 8) was used in the characterization studies of the terbinafne microchip. Te potential of the terbinafne microchip provided higher and lower response below and above this range, which attributes to protonation of the drug at lower pH values and degradation of the ion pair sensitive material at higher pH values, respectively.
Basically, selectivity is the most important parameter of sensor characteristics, which determine the specifcity of the primary investigated ion in the presence of interfering ions. It is the relative response of the proposed sensor for principal ions over other interfering ions present in solution. Terefore, the potentiometric selectivity coefcient of the terbinafne microchip was determined using a separate solution method (SSM) [14] by separate calibration for terbinafne as well as all studied interfering ions in the concentration range of 10 −6 -10 −2 , as presented in Figure 7. Te values of the selectivity coefcient for all studied interfering species were calculated, and the results obtained are summarized in Table 2. Te selectivity coefcient values of the microsensor confrmed that the elaborated microchip ofered very high selectivity for terbinafne drugs in the presence of many investigated interfering ions. Tis     Journal of Chemistry indicates that interfering ions have extremely low permeability through the sensitive membrane fabricated for terbinafne drug primary ions, and they would not signifcantly disturb the response of the realized terbinafne chip assembly.
To investigate the infuence of the water layer efect on the response of the realized microsensor, the potentiometric water layer test of the terbinafne microchip assembly was performed by recording the potential of the chip versus time intervals after successive immersing of the chip in blank (water and tris-HCl bufer) solution followed by immersing the chip in 10 −5 mole·L −1 of terbinafne prepared in bidistilled water and tris-HCl bufer, respectively. Te results obtained, which are presented in Figure 8, revealed a lack of potential drift of the microchip response. Te microsensor showed stable behavior, fast equilibrium, and consequently high stability and reliability of the realized terbinafne microchip assembly. Tese fndings were attributed to the microfabrication methodology of the chip assembly which was based on the nebulization approach and recently developed [19,21].
Te performance response parameters of the realized terbinafne microchip assembly in comparison with the bulk electrodes published are summarized in Table 3. It should be noted that the performance properties (slope, detection limit, and linear range) of the chip are better than those reported for terbinafne bulk electrodes [14,15]. Tis behavior is attributed to the incorporation of the MWCNTs into the sensitive element, which improves the conductivity of the sensor, increases the transduction of the chemical signal to the electrical signal, and therefore increases the sensitivity of electrodes [19,21]. Moreover, the realized terbinafne microchip assembly provided small size, miniaturization, integration, and automation feasibility.

Analytical Applications of the Terbinafne Microchip.
Te elaborated microchip assembly has been successfully used in the determination of terbinafne in some real samples of drug formulations (Terbin 250 mg and Lamifen 250 mg) and in spiked urine as well. In this study, fve tablets of each drug formulation were dissolved and treated as reported in our previous work [21]. Drug concentrations were measured using the calibration method, and three replicate measurements were used for each analysis. Te accuracy of the proposed method was determined, and the results obtained are collected in Table 4. Te proposed method can therefore be applied to the quantifcation of terbinafne in its drug formulations and biological real samples with an accuracy of 96.9% and without fear of interferences caused by excipients expected to be present in drug formulations or the constituents of urine.

Conclusions
Microfabrication, electrochemical characterization, and analytical applications of the terbinafne drug microchip assembly have been demonstrated. In comparison with the published terbinafne electrodes, the realized chip showed advanced performance parameters with a fast response time (≤30 s), low detection limit (5 × 10 −9 mole·L −1 ), Nernstian behavior (58.5 ± 0.5 mV/decade) covering the linear range of 10 −8 -10 −2 mole·L −1 , and relatively long-life span ≥4 months. Te elaborated chip has been successfully applied to the quantifcation of terbinafne in some drug formulations and spiked urine. Te analytical method based on the realized chip assembly approved to be a simple, fast, cheap, precise, and accurate method of analysis of terbinafne. In addition, the merits ofered by the realized terbinafne microchip assembly include small size, miniaturization, integration, and automation feasibility.

Data Availability
No additional data were used to support the study.

Conflicts of Interest
Te authors declare that there are no conficts of interest with regards to this work.