N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC) as an excellent sensing element for the electrochemical measurement of T4, the free thyroid hormone

N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC) as an excellent sensing element for the electrochemical measurement of T4, the free thyroid hormone | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC) as an excellent sensing element for the electrochemical measurement of T 4 , the free thyroid hormone Abdollah Yari, Fatemeh Biranvand This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7246319/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Journal of Applied Electrochemistry → Version 1 posted 11 You are reading this latest preprint version Abstract This article examines the creation of our innovative electrochemical electrode, and the advantages of the electrochemical approach for accurate monitoring of the thyroid hormone (T 4 ). The sensor functions through electrochemical interactions between the T₄ molecule and the electrode surface, which includes N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC). By using cyclic voltammetry (CV), differential pulse voltammetry (DPV), or electrochemical impedance spectroscopy (EIS), the sensor delivers dependable and precise results, with prospective applications in free-T 4 monitoring. EPHC was synthesized and characterized using FT-IR, SEM, and EDX techniques to confirm successful synthesis. EPHC was blended with suitable components to formulate a consistent carbon paste, to which a graphite bare was affixed for electrical connectivity. The sensor demonstrates impressive performance, achieving a low detection limit of 8.0×10 − 3 µM. It operates effectively across a linear range from 8.7×10 − 3 to 80.0 µM. The response time is rapid at 30 seconds. The electrode exhibits satisfactory repeatability and reproducibility. The sensitivity for T 4 is measured at 1.17 µA/µM, reflecting the sensor’s strong analytical capability. The sensor successfully quantified free-T 4 in different real matrices, under optimized conditions of a pH of 9, a scan rate of 80 mV/s, 5.0 mg of EPHC, and 0.5 g of graphite powder. Carbothioamide Electrochemical sensor Levothyroxine (T4) N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Levothyroxine (T 4 ) is a synthetic thyroid hormone utilized in the treatment of hypothyroidism, a condition characterized by insufficient production of thyroid hormones by the thyroid gland. It is also employed in conjunction with surgery and radioactive iodine therapy for the management of thyroid cancer. T 4 is classified as a hormone and serves to replace the thyroid hormone normally produced by the body. It can exist in two forms in the blood serum: free and protein-bound. The absence of adequate thyroid hormone can hinder the body's proper functioning, potentially resulting in symptoms such as stunted growth, slowed speech, fatigue, excessive tiredness, constipation, weight gain, hair loss, dry and thickened skin, increased sensitivity to cold, joint and muscle pain, heavy or irregular menstrual cycles, and depression. When administered correctly, T 4 can reverse these symptoms [ 1 – 5 ]. The chemical composition of levothyroxine is illustrated in Fig. 1 as a sodium salt. Figure 1 Given the narrow therapeutic index of T 4 , precise measurement of its dosage and serum levels is fundamental to achieving optimal treatment outcomes. Inadequate or excessive dosing can lead to severe health implications, including cardiovascular issues, bone density reduction, cognitive impairment, and metabolic disturbances. Thus, effective monitoring techniques, such as serum thyroxine and thyroid-stimulating hormone (TSH) tests, are essential for ensuring individualized treatment plans tailored to each patient’s unique physiological needs [ 1 – 4 ]. Traditional methods for T₄ measurement, such as high performance liquid chromatography (HPLC) [ 6 – 10 ], radioimmunoassay (RIA) [ 11 – 14 ], enzyme-linked immunoassay assay (ELISA) [ 15 – 19 ], isotope dilution mass spectrometry (isotope-dilution LC-MS/MS) [ 20 – 24 ], and chemiluminescent assays (CLA) have been widely used for decades. However, these techniques often require complex sample preparation, expensive reagents, and lengthy analysis times, underscoring the need for a faster, cost-effective, and highly sensitive alternative. Newer techniques, such as electrochemical sensors [ 25 – 29 ] and liquid chromatography-mass spectrometry (LC-MS) [ 30 ], offer rapid and highly sensitive alternatives. Electrochemical methods, in particular, provide real-time monitoring, minimal sample preparation, and portability, making them ideal for point-of-care testing. Thiosemicarbazones are a class of organosulfur compounds characterized by the presence of a thiosemicarbazide functional group. They are typically synthesized through the condensation of thiosemicarbazide with aldehydes or ketones [ 31 ]. Thiosemicarbazones have a wide range of practical applications, particularly in medicinal chemistry and coordination chemistry. These compounds exhibit versatile biological activities, making them valuable in medicinal chemistry. Their ability to chelate metal ions enhances their pharmacological potential, particularly in cancer treatment and iron overload management [ 32 – 41 ]. Additionally, thiosemicarbazones serve as a type of nitrogen-sulfur donor ligands and are widely used as chemical ligands in coordination chemistry, forming stable complexes with transition metals such as copper, zinc, palladium, platinum, and iron. Their structural diversity, achieved through ligand modifications, plays a crucial role in optimizing their pharmacokinetics and pharmacodynamics [ 36 , 39 , 42 – 46 ]. N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC) is a thiosemicarbazone derivative that has been studied for its potential biological applications. It is known to form mixed-ligand bivalent transition metal ion complexes, including those with copper, nickel, manganese, and cobalt [ 47 ]. The EPHC ligand and its complexes are minimally soluble in water, but they dissolve well in dimethyl sulfoxide (DMS) and dimethylformamide (DMF) [ 36 , 44 , 46 , 48 ]. Electrochemical sensing has rapidly developed as a valuable analytical tool for real-time and high-precision monitoring of biomolecules, such as thyroid hormones. In this work, we present a straightforward and efficient synthesis of EPHC, achieving yields that surpass those previously reported in the literature. Building on this, we have developed a novel carbon paste (CP) electrochemical sensor based on EPHC, specifically optimized for the accurate detection of free-T₄ (abbreviated as T₄) in biological samples. By leveraging the unique properties of this sensor, including its high sensitivity, selectivity, and rapid response, we aim to provide a novel, efficient, and low-cost method for the thyroid hormone assessment. 2. Experimental 2.1 Chemicals Ethanol (C 2 H 5 OH), graphite powder (G), paraffin oil, methanol (CH 3 OH), potassium hexacyanoferrate(II) (K 4 Fe(CN) 6 ), potassium hexacyanoferrate(III) (K 3 Fe(CN) 6 ), sodium-(2S)-2-amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoate (sodium levothyroxine), T 4 tablets (100 µg/tablet), propranolol tablets, sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide (NaOH), disodium hydrogen phosphate (Na 2 HPO 4 ) and sodium dihydrogen phosphate (NaH 2 PO 4 ) all were sourced from Merck (Germany) and were of the utmost purity, used without any purification. Double distilled and deionized water was employed consistently throughout the experiments. 2.2 Instruments FT-IR spectra were collected using a Shimadzu FT-IR 8400 spectrometer (Japan). To analyze the surface morphology of the electrodes, a field emission scanning electron microscope (FE-SEM, Tescan Mira3 LMU, Czech Republic) was employed. Elemental composition and distribution were determined via energy dispersive X-ray spectroscopy (EDX), utilizing a SAMx (France) instrument. Electrochemical measurements including cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were conducted on an Autolab PGSTAT 204 potentiostat/galvanostat (Metrohm Switzerland), operating within a standard three-electrode configuration: EPHC@CP/GE as the working electrode, a platinum wire as the counter electrode, and a standard calomel electrode (SCE) as the reference. For electrochemical impedance spectroscopy (EIS), an EG&G PAR model 263 A potentiostat/galvanostat (USA) was used. pH adjustments were made using a Metrohm 713 pH meter (Switzerland). When required, samples underwent sonication with a Soner 203H device (Taiwan). 2.3 Synthesis of N-ethyl-2-picolinyl hydrazine carbothioamide (EPHC) A procedure for the synthesis of EPHC has already been provided in Ref. [ 47 ], which reported a lower yield and inadequate characterization. Here, the synthesis of EPHC was carried out in two steps. In this approach, at the first stage, the synthesis of hydrazide-2-Picolinic acid was initially carried out starting from ethyl pyridine-2-carboxylate (ethyl picolinate). Then EPHC was prepared successfully. The entire process is depicted in Scheme 1 . Initially, 1.0 mmol (0.137 g) of ethyl-2 pyridine carboxylate (ethyl picolinate) was combined with 1.2 mmol (0.181 g) of hydrazine, and 5.0 mmol of ethanol was introduced into a 50 mL balloon. The mixture was subjected to reflux heating for 2 h, followed by evaporation at room temperature until the volume was halved, resulting in a significant amount of white sediment. The obtained precipitate was then crystallized using toluene. Crystal characteristics: bright white color, melting point: 105–107°C, yield: 82%. Secondly, in a 20 mL balloon, hot ethanol was introduced to 1.0 mmol (0.137 g) of 2-picolinic acid hydrazide, which was obtained from the synthesis in the first step, until it was thoroughly dissolved. Subsequently, 1.0 mmol (0.87 g) of ethyl isothiocyanate was also added to the balloon, and the mixture was subjected to reflux heating for 3 h before being evaporated at room temperature, resulting in a significant amount of white sediment being separated. In a 25 mL beaker, 10.0 mL of toluene was poured and heated. The resulting compound was dissolved in the hot toluene, and the solution was then gradually cooled to room temperature. The resulting needle-shaped crystals were smoothed and dried, and the reaction efficiency was calculated, yielding a crystal that is bright white in color, with a melting point of 199–202°C and a yield of 85% (Scheme 1 ). Scheme 1 2.4 preparation of the electrode In the preparation of the modified CP electrode (EPHC@CP/GE), 0.5 g of G was first placed in a porcelain mortar and ground for 20 min. Following this, 5.0 mg of EPHC was added to the mixture. Subsequently, 0.3 mL of paraffin oil was introduced, and the contents were homogenized again for an additional 20 minutes to ensure the paste was thoroughly blended. The prepared mixture was carefully packed into a sampler plastic tip tube specifically, one with a 5 mm diameter at the top, 2 mm at the bottom, and a length of 3.5 cm. Electrical contact was established by directly inserting a bare graphite electrode, connected to a copper wire, into the assembly. This straightforward approach ensured reliable connectivity without introducing unnecessary complexity. The modified EPHC@CP/GE was employed as the working electrode. Other unmodified CP/GE electrodes were prepared using a similar method, but without the presence of any modifier. 2.5 Standard T 4 and real sample preparation A 250 mL stock solution of the sodium salt of T 4 at a concentration of 100 µM was prepared by dissolving 19.97 mg of the salt in water. The solution was then stored in a refrigerator at 4°C for future use. To evaluate the effectiveness of the proposed analytical method with actual samples, the method was applied to the determination of T 4 in pharmaceutical preparations, each reportedly containing 100 µg of T 4 per tablet. 100 tablets were accurately weighed and finely ground with a mortar and pestle. A portion of the resulting powder, calculated to be equivalent to a 100.0 µM T 4 , was then dissolved in a 10:90 ethanol-to-water mixture. This solution was subjected to sonication for 10 min to ensure thorough dissolution. After filtration into a 250.0 mL calibrated flask, the solution was diluted to volume with water. A measured volume of this solution was subsequently transferred to a 25.0 mL volumetric flask containing the supporting electrolyte, and standard T 4 solution was added as a spike for calibration purposes. The method was also evaluated using a blood serum sample collected from a young, healthy male participant. A 10.0 mL aliquot of serum was deproteinized using 2 mL of 10% (v/v) trichloroacetic acid, followed by centrifugation. The supernatant was then diluted tenfold with 0.1M phosphate-buffered solution at pH 9.0. These serum samples were stored at 4 ℃ until further analysis. An appropriate volume of the diluted serum sample was introduced into the electrochemical cell, and then standard T 4 solution was added as a spike for T 4 determination using DPV. Further details regarding the procedure are provided in later sections. All measurements employed the standard addition method to ensure accuracy and reliability of the results. 3. Results and discussion 3.1 Spectroscopic SEM is a powerful technique to elucidate suitable information about the surface morphology of a substance [ 49 – 51 ]. The images presented in Fig. 2 illustrate the crystalline structure of EPHC, indicating that this material has crystallized effectively and possesses a well-ordered and regular crystalline structure (Fig. 2 A). This arrangement significantly influences the chemical and physical properties of the substance, such as solubility and stability. Figure 2 B displays an unmodified CP, revealing a uniform and continuous distribution of G sheets, along with fine pores that could potentially impact electrochemical performance. In contrast, the image of the CP modified with EPHC shows an increase in both the number and size of the pores (Fig. 2 C). The pore distribution becomes irregular due to the incorporation of EPHC into the CP, resulting in a rough morphology that is promising for the accumulation of analyte on the electrode surface. The insets in the figures provide more detailed images to offer a clearer view of each composition. Figure 2 To analyze and determine the composition of elements along with their corresponding percentages in the structure of EPHC, EDX was utilized. The findings, depicted in Fig. 3 , demonstrate that the percentage composition of the synthesized EPHC elements at each stage corresponds with its intended formula. The tables provide evidence, as the insets in Fig. 3 confirm the percentages of elements such as C, N, O, and S in EPHC. Figures 3 A and 3 B present high-resolution images of the morphologies of EPHC and EPHC@CP, respectively. Additionally, Figs. 3 C to 3 F illustrate the elemental topographies of EPHC@CP, indicating a consistent distribution of the elements C, N, O, and S [ 52 – 54 ]. Figure 3 In the analysis of the FT-IR spectrum, the frequencies associated with the bonds C = N, C = S, C = S, and C = H were primarily examined and compared (Fig. 4 ). As shown in Fig. 4 a, the stretching frequency of the IR spectrum for EPHC containing the C = N bond is located at 1674 cm − 1 . The presence of peaks from aryl and aromatic groups causes a shift of this bond's peak towards lower frequencies. In this compound, the peaks between 1541 and 1510 cm − 1 are related to the C-N bond in the thiosemicarbazide compound. The peaks found around 2933 cm − 1 is associated with the = C-H group, and the peak at 3151 cm − 1 related to -C-H group, while the peak at 1674 cm − 1 corresponds to the bending frequency of the C = C bond in the pyridine ring present in the ligand. The peaks at 747 cm − 1 and 1237 cm − 1 are related to the vibrational frequencies of C = S. In Fig. 4 b, which pertains to the carbon paste electrode modified with EPHC (EPHC@CP), the peaks between 2933 and 2997 cm − 1 correspond to the = C-H group, and the peaks between 1373 and 1481 cm − 1 are associated with the C-N bond of EPH. The wide strong peak around 3500 cm − 1 represents the alteration of carbonyl oxygens into -OH group after incorporation into the CP matrix [ 55 – 57 ]. A clear comparison between peaks (4a) and (4b) reveals a marked change in the EPHC peak following its incorporation into CP. This substantial alteration strongly suggests a robust interaction between EPHC and CP, providing compelling evidence for their synergistic electrocatalytic effect toward T 4 at the electrode surface. Figure 4 3.2 Electrochemical study Before finalizing the electrode fabrication, the appropriate quantities of the primary components, EPHC and G powder, were evaluated. The optimal values determined were 5.0 mg and 0.5 g, respectively, as these amounts yielded the highest signal response. The response of both unmodified and modified electrodes with EPHC in the 0.1 M [Fe(CN) 6 ] 4−/3− solution showed a positive result, as depicted in Fig. 5 . The cyclic voltammetry (CV) revealed a distinct redox peak (Fig. 5 A), suggesting that the electrodes exhibited favorable responses to the probe. In the scenario where EPHC is present, the EPHC@CP/GE response is somewhat diminished compared to that of the bare CP/GE, indicating a slight reduction in electrode conductivity, a decrease in the current peak value, and an increase in the potential required for the electrochemical reaction at the electrode surface. Nonetheless, it operates effectively as an appropriate modifier, as it serves as a selective agent for the reaction with T 4 . The EIS study serves as a sensitive method for detecting changes in the surface impedance of electrodes in response to the addition of other components. EIS provides valuable insights into the electron transfer dynamics among electroactive species within electrode materials. In the context of a Nyquist plot, the linear segment typically corresponds to processes governed by mass transfer, while the semicircular feature is indicative of electron transfer limitations at the electrode interface that is, the charge transfer resistance (R ct ). This distinction allows researchers to diagnose where kinetic or transport barriers exist within the system [ 58 – 60 ]. The Randall's equivalent circuit [ 61 ] is employed to match the impedance data (as shown in the inset of Fig. 5 B). The EIS spectra for both the modified and unmodified electrodes are presented in Fig. 5 B. The R ct s of these electrodes are of 5947.5 and 4893.5 Ω, respectively. The Nyquist plots in this figure demonstrate that the conductivity of the CP/GE modified with the EPHC has decreased to match that of the unmodified carbon paste electrode. This is evident as the EIS curve for the modified EPHC@CP/GE shows a greater electrical resistance in comparison to the peak linked with the unmodified CP/GE, which displays increased electrical resistance due to the incorporation of EPHC. Figure 5 3.3 Optimizing analytical factors 3.3.1 Effect of pH of the solution and buffering type To investigate the influence of different buffer types on the electrode response, three 0.1 M buffering agents such as sodium borate/boric acid, sodium carbonate/bicarbonate, and sodium dihydrogen phosphate/hydrogen phosphate (phosphate buffer) were examined by using DPV. The findings illustrated in Fig. 6 indicate that the electrode response to T 4 8.0 µM is markedly improved in the phosphate buffer solution. This particular solution combination yields an estimated ionic strength of 0.1 for the buffer solution overall. Therefore, the phosphate buffer at 0.1 M with a pH of 9 was deemed suitable as a supporting electrolyte for the determination and measurement of T 4 . Figure 6 One of the highly influential factors affecting the response of the pH electrode is the solution itself, which can significantly impact electrochemical reactions, the performance of electrodes, and electrochemical cells. After selecting a phosphate buffer solution as the suitable pH provider, phosphate buffers of pH values of 1, 3, 5, 7, 9, 10, and 11 including T 4 0.12 µM were prepared and analyzed using DPV with the modified EPHC@CP/GE. The corresponding voltammograms were recorded and examined, as shown in Fig. 7 . According to Fig. 7 A, the findings suggest that T 4 demonstrates a satisfactory current at high pH levels. A graph illustrating the peak currents (I p ) in relation to the pH values was generated and is shown in Fig. 7 B, which facilitated the determination of the optimal pH of 9. The linear correlation observed between the peak potentials (E p ) and the pH values (Fig. 7 C) serves as evidence for the electrochemical reaction's dependence on pH [ 62 ]. In addition to the facts presented in Fig. 7 , in order to evaluate further clues about the electrochemical mechanism, by which T 4 was involved at the electrode-solution interface, the relationship between T 4 peak potential (E p ) and the pH of the solution was evaluated. As shown in Fig. 7 C, the plot of E p versus pH led us to Eq. (1). E p (V) = 0.031pH + 0.210, R²= 0.992 (1) From this equation, the slope of 31.0 mV/pH was obtained, which is very close to the Nernst theoretical value of 59/2 mV/pH for such electrochemical reactions in which two electrons and one proton transfer occur simultaneously. Figure 7 T 4 possesses three pKa values: the carboxylic group at 2.4, the phenolic group at 6.8, and the amino group at 9.9 [ 63 ]. The solubility of T 4 sodium salt in water decreases as the pH increases from 1 to 3. Between pH 3 and 7, solubility remains relatively constant. When the pH rises above 7, there is a marked increase in solubility [ 64 ]. These trends are clearly illustrated in Fig. 7 B. Consequently, T 4 exists in its fully deprotonated state (see Scheme 2 ) at the optimal pH of 9. In contrast, at significantly lower pH levels, the oxidation of T 4 typically becomes challenging due to extensive protonation and precipitation, resulting in diminished anodic responses. As the pH increases, the responses improve owing to the deprotonation of T 4 (Fig. 7 C). From these facts, the behavior of the electrode at varying pH levels is entirely comprehensible. 3.3.2 Impact of potential scan rate Figure 8 depicts the CV responses of the modified EPHC@CP/GE at scan rates of 10, 20, 30, 40, 50, 80, and 100 mV/s within a phosphate buffer solution at pH 9, alongside a 0.1 µM T 4 solution. As shown in Fig. 8 A, the process is characterized by an irreversible mechanism, where no reverse peak is present. The optimal scan rate of 80.0 mV/s was chosen for the experiments involving potential variations. The impact of the optimized scan rate on the electrode in a blank solution was analyzed and included in the figure, which shows no significant response. It is noted that with an increase in the scan rate, the oxidation potentials of the irreversible T 4 peaks exhibit a slight shift towards more positive values. This shift can be attributed to kinetic limitations, or in other words, the insufficient opportunity for the species to reach the electrode surface and undergo the oxidation process, thereby necessitating higher potentials for oxidation [ 62 , 65 ]. This indicates the presence of kinetic constraints in the reaction between the modified electrode and T 4 , which has already confirmed by the results in Fig. 5 . Furthermore, Fig. 8 B depicts the changes in the oxidation E p versus the logarithm of the scan rate (n, Eq. 2), resulting in a linear regression line with R 2 of 0.995. Additionally, Fig. 8 C demonstrates a linear relationship between the oxidation I p and the scan rate (n 1/2 ), yielding an R 2 value of 0.986 for the regression line of Eq. 3. This relationship indicate that the electrochemical system is not a reversible process. E p = 0.062Log(n) + 0.476, R 2 = 0.995 (2) I p = 0.815n 1/2 + 4.493, R 2 = 0.986 (3) Figure 8 Furthermore, based on the Laviron’s equation [ 65 , 66 ], and the slope of the curve illustrated in Fig. 8 B and Eq. 1, with a common value of a = 0.5, the number of electrons transferred during the reaction is calculated to be 1.9, which is approximately 2. Essentially, the electro-oxidation of T 4 at the EPHC@CP/GE interface proceeds with the transfer of two electrons and one proton, as illustrated in stage (I) of Scheme 2 . Notably, the specific role of EPHC in the overall electrochemical process may be significant, as suggested by the half-reaction depicted in stage (II) of Scheme 2 . Taken together, the available results support the mechanism for T 4 electro-oxidation as outlined in Scheme 2 . Scheme 2 3.3.3 Reliability of electrode response Following each T 4 measurement, the electrode underwent 10 cycles of cyclic voltammetry within a potential range of 0.3 to 0.8 V. This procedure was employed to reverse the reduction of EPHC that occurred due to T 4 oxidation, thereby restoring the electrode to its original state prior to subsequent use. All steps were conducted under optimized conditions. The electrode was analyzed for the delay time necessary to demonstrate a stable response value to the analyte over a period of 180 s, utilizing reading intervals of 10 s. The optimal result was achieved at 30 s, which was determined to be the appropriate duration for future applications of the electrode. To evaluate the accuracy and precision of the measurements, three identically modified electrodes EPHC@CP/GE were employed concurrently to conduct five repeated measurements on 20.0, 25.0 and 30.0 µM T 4 solutions under the optimized conditions. The relative standard deviation (RSD) for each electrode is detailed in Table 1 . Importantly, all RSD values of recoveries were below ± 2.11, indicating a high degree of precision in the electrode responses, while the measured values of the electrodes were reasonably close to the standard spiked T 4 amounts, signifying substantial accuracy. The results also highlight the strong reproducibility of the modified electrode system for T 4 detection, further corroborated by the overall acceptable RSD of ± 3.55. In summary, the findings illustrate that the modified electrodes yield reliable and consistent measurements for this application. Table 1 To assess the stability of EPHC@CP/GE in real-world conditions, the electrode underwent a one-month evaluation with two measurements taken daily at one-hour intervals. The data's RSD was below ± 2.5 percent, a figure deemed acceptable, suggesting a satisfactory lifespan for the electrode throughout the one-month duration. 3.3.4 Interferent impact The sensitivity of the modified electrode could be altered in the presence of interfering species that may impact on the response current peaks. These interfering species may be those that they present in pharmaceutical formulations and could pose potential interference in the measurement of T 4 using the DPV method. The potential interferences of certain drugs such as Liothyronine (T 3 ) (the other thyroidal hormone), Propylthiouracil (used for hyperthyroidism treatment), and some ions were investigated. This was conducted by increasing the concentration of interfering substances in a 0.1 µM T 4 solution in a phosphate buffer at pH 9 under optimal conditions. Species that disrupt the measurement of T 4 would influence the current intensity of T 4 beyond the defined range. If the variation in the response to T 4 , when interfering species are present, adheres to the following relationship (Eq. 4), and RE is less than ± 5 percent, it is considered that there is no significant impact on the intensity of the T 4 current intensity. %RE= [(I 0 -I if )/I 0 ] × 100 (4) where RE denotes the deviation in current intensity caused by the interference, I 0 represents the initial current intensity, specifically in the presence of T 4 at 0.1 µM, and I if signifies the measured current intensity of the solution in the presence of an interferer. The maximum allowable concentration of interfering species in the presence of 0.1 µM T 4 is detailed in Table 2 . According to the data presented in this table, the permissible concentrations of Fe 3+ , Cu 2+ , Mg 2+ , I – , Cl – , Propylthiouracil, and T 3 are 8000, 5800, 8400, 12000, 7000, and 1700 times the concentration of T 4 in the sample, respectively. Table 2 3.3.5 Measurements merits The parameters recognized as the advantages of a sensor's performance include the linear dynamic range (LDR), where the calibration curve of the sensor's analytical responses changes linearly with a variable, such as the concentration of the target analyte, the limit of detection (LOD), and the limit of quantification (LOQ). A calibration curve illustrating the sensor's responses to varying concentrations of T 4 , ranging from 0.0 to 80.0 µM, was established using the DPV method. The results are presented in Fig. 9 . Figure 9 illustrates the DPV voltammograms for various concentrations of T 4 solution at the modified electrode in a 0.1 M phosphate buffer solution with a pH of 9, recorded at a scan rate of 80 mV/s. Drawing from the information presented in Fig. 9 and Eq. 5, the calculation of both LOD and LOQ for this analytical method follows established conventions: LOD is determined as 3S b divided by m, and LOQ is calculated as 10S b over m. Here, “m” refers to the slope of the calibration curve, which essentially reflects the method’s sensitivity to increasing amounts of analyte, while “S b ” is the standard deviation of the blank. In this case, S b was measured to be 3.12 × 10⁻ 3 based on six replicate measurements, an approach that helps account for random fluctuations in the baseline signal. The resulting calculations yield an LOD of 8.0 × 10⁻ 3 µM and an LOQ of 0.027 µM for T 4 . These values provide a quantitative benchmark for the method’s capacity to reliably distinguish and measure low concentrations of the analyte. The relatively low LOD and LOQ suggest that the method is quite sensitive, making it suitable for detecting T 4 even at sub-micromolar levels. Furthermore, %RSD of ± 0.632, calculated across six experimental replicates, indicates a high degree of precision. In analytical chemistry, maintaining an RSD below 1% is generally considered excellent, as it reflects minimal variability between repeated measurements under identical conditions. Such a low %RSD not only reinforces the method’s reliability but also supports the validity of the LOD and LOQ values obtained. I p = 1.170C T4 + 2.179, R 2 = 0.992 (5) The inset of Fig. 9 illustrates the LDR for the method, which spans from 8.7×10 − 3 to 80.0 µM of T 4 . This range defines the interval over which the analytical response remains directly proportional to the concentration of T 4 in the sample. Operating within this range is crucial, as measurements taken outside these limits may not yield accurate or linear results. The breadth of the LDR further underscores the method’s versatility, enabling accurate quantification across a wide range of sample concentrations. From Eq. 5, the sensitivity of the sensor is 1.17 µA/µM of T 4 . Figure 9 3.3.6 Analytical application In order to accurately assess the functionality of the newly developed electrode for T 4 measurement, its performance was established to be compared with the well-established HPLC method. To validate the integrity of our comparison, we did not depend on a solitary measurement; rather, we examined five replicate aliquots of each actual sample using both the new electrode and HPLC. This methodology contributed to the reduction of random error and yielded a comprehensive dataset for analysis. The findings are presented in Table 3 . As illustrated in this table, a significant correlation is evident between the results acquired from DPV utilizing EPHC@CP/GE and those derived from HPLC. The recoveries data were subjected to an one-tailed F-test using a critical F-value of 6.38 at a significance level of P = 0.05. This approach was intended to evaluate whether the observed similarities in precision were statistically significant. Notably, the experimental F-values were considerably lower than the critical value of 6.38, suggesting that the sensor exhibited superior precision compared to the HPLC results. The electrode’s response to T 4 not only mirrors the level of precision achieved by HPLC but, intriguingly, demonstrates superior accuracy. Such parity is particularly noteworthy considering the practical advantages that electrodes often offer over HPLC, such as faster turnaround times, lower operational costs, and greater portability. This is a significant result, implying that the electrode could feasibly replace or complement HPLC in certain analytical scenarios, especially where rapid or cost-effective measurement is necessary. Table 3 Building on the previous comparison with established studies focused on the electrochemical detection of T 4 , the present research employing the EPHC@CP/GE electrode demonstrates a marked advancement in analytical performance. As detailed in Table 4 , a compilation of relevant recent studies [ 68 – 74 ], the newly developed electrode not only achieves a notably lower LOD but also extends the LDR for T 4 analysis. Additionally, the broader LDR enhances the electrode’s versatility, making it suitable for applications where the concentration of T 4 may vary widely. This improvement in sensitivity and dynamic range is particularly significant when considering the growing demand for precise and reliable quantification of T 4 , especially in complex biological or pharmaceutical matrices. Taken together, the findings underscore the potential of EPHC@CP/GE as a reliable tool for sensitive, accurate, and practical electrochemical analysis of T 4 . Table 4 4. Conclusions This electrode offers several advantages, including a simple preparation method, high stability, selectivity, acceptable reproducibility, rapid response time, and applicability across a wide pH range. The electrode’s response to T 4 not only mirrors the level of precision achieved by HPLC but, intriguingly, demonstrates superior accuracy. Such parity is particularly noteworthy considering the practical advantages that electrodes often offer over HPLC, such as faster turnaround times, lower operational costs, and greater portability. This is a significant result, implying that the electrode could feasibly replace or complement HPLC in certain analytical scenarios, especially where rapid or cost-effective measurement is necessary. By pushing the boundaries of detection limits, the EPHC@CP/GE electrode facilitates the identification of trace amounts of analyte, which is crucial for early diagnosis, therapeutic monitoring, and ensuring regulatory compliance in pharmaceutical quality control. In summary, the method demonstrates strong analytical performance, characterized by high sensitivity, excellent precision, and a robust linear dynamic range. These attributes collectively establish the technique as a reliable tool for the quantification of T 4 in diverse sample matrices. Declarations CRediT authorship contribution statement Abdollah Yari : literature search, figures, study design, Writing – review and editing. Fatemeh Biranvand : data collection, data analysis, data interpretation, Writing – original draft, preparation. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability The data utilized for the research outlined in the article can be obtained by contacting the corresponding author. References Miccoli P, Materazzi G, Rossi L (2021) Levothyroxine therapy in thyrodectomized patients. Front Endocrinol 11:626268. https://doi.org/10.3389/fendo.2020.626268 Duntas LH, Jonklaas J (2019) Levothyroxine dose adjustment to optimise therapy throughout a patient’s lifetime. Adv Ther 36:30–46. https://doi.org/10.1007/s12325-019-01078-2 Sue LY, Leung AM (2020) Levothyroxine for the treatment of subclinical hypothyroidism and cardiovascular disease. 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The EPHC production process from ethyl picolinate and 2-picolinic acid hydrazide. Scheme2.png Scheme 2. The possible electrochemical reaction mechanism of electrooxidation of T 4 at the EPHC@CP/GE. 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07:06:49","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51333,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/49fa4cf30dd1320cfa3140d1.png"},{"id":91952323,"identity":"e1312816-f420-4471-9738-f1bcb05a4083","added_by":"auto","created_at":"2025-09-23 06:50:49","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17937,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/af1b9994f872399aeefda37b.png"},{"id":91952326,"identity":"9bef80e3-bbf0-47ca-9a2d-1182884e566d","added_by":"auto","created_at":"2025-09-23 06:50:49","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":183768,"visible":true,"origin":"","legend":"","description":"","filename":"3163250b63db4fad892f15205bfcd5f91structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/f7cb078c75de360032526f43.xml"},{"id":91952327,"identity":"8db39e2c-4a4a-4869-a303-cd0918e08eb9","added_by":"auto","created_at":"2025-09-23 06:50:49","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":197766,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/e3b8976aae416773dcc32766.html"},{"id":91952290,"identity":"6ca4e681-28cf-49f5-bd39-050ec7c74ba1","added_by":"auto","created_at":"2025-09-23 06:50:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28466,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical structure of levothyroxine (T\u003csub\u003e4\u003c/sub\u003e) sodium salt.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/19365f7a30005a60da48c597.png"},{"id":91952298,"identity":"5d693c04-e4a1-4524-9074-54d59cc25af7","added_by":"auto","created_at":"2025-09-23 06:50:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":221820,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM images of synthesized EPHC (A), the carbon paste (B), and the mixed EPHC@CP (C). The insets are the higher resolution images.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/30b81daa34c9d8a78823e1ea.png"},{"id":91953607,"identity":"48a60cdf-c571-4b55-b9da-c7647d08cec4","added_by":"auto","created_at":"2025-09-23 06:58:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":188326,"visible":true,"origin":"","legend":"\u003cp\u003eThe EDX pattern of EPHC (top) and the EPHC@CP (bottom). Figures A and B show the high resolution images of the morphologies of EPHC and EPHC@CP, respectively. Figures C-F, the elemental map distribution for EPHC@CP.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/734a4eadd68f0c4868109368.png"},{"id":91954244,"identity":"9b3c8818-9a51-4738-895a-1207b9b934c3","added_by":"auto","created_at":"2025-09-23 07:06:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80731,"visible":true,"origin":"","legend":"\u003cp\u003eThe FT-IR spectra of EPHC (a) and EPHC@CP (b).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/a3b0d5b00b08517e4401bee9.png"},{"id":91952293,"identity":"1c4d7647-1649-4105-91be-2eddc57f038a","added_by":"auto","created_at":"2025-09-23 06:50:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33767,"visible":true,"origin":"","legend":"\u003cp\u003eTh CV of Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e4-/3-\u003c/sup\u003e at bare CP/GE and the modified EPHC@CP/GE (A) and the EIS plots of Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e4-/3-\u003c/sup\u003e at bare CP/GE and EPHC@CP/GE (B).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/10077cd81439a9eef3b3af17.png"},{"id":91952301,"identity":"1b566e68-6b92-41b2-85b2-245208dc662a","added_by":"auto","created_at":"2025-09-23 06:50:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":32844,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of different buffer types on the electrode response.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/0a216106656051a740029d58.png"},{"id":91952306,"identity":"b1c81655-71a1-4d83-b115-56eeb955d69e","added_by":"auto","created_at":"2025-09-23 06:50:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":80575,"visible":true,"origin":"","legend":"\u003cp\u003eThe DPV voltammograms of 0.1 mM T\u003csub\u003e4\u003c/sub\u003e in different solution with various pHs, at EPHC@CP/GE (A). The plot of Ip vs. pH (B). The plot of Ep vs. pH (C).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/e12b20972321ede9209f1410.png"},{"id":91953603,"identity":"0737d32f-7a31-42e3-b46d-4c9d23286407","added_by":"auto","created_at":"2025-09-23 06:58:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":52297,"visible":true,"origin":"","legend":"\u003cp\u003eThe CV voltammograms of T\u003csub\u003e4\u003c/sub\u003e, 0.1 mM, in various scan rates (n) at EPHC@CP/GE, and for the case of the blank where there was no T\u003csub\u003e4\u003c/sub\u003e in the solution (A). The plot of E\u003csub\u003ep\u003c/sub\u003e vs. Log(n) (B). The plot of I\u003csub\u003ep\u003c/sub\u003e values against n\u003csup\u003e1/2\u003c/sup\u003e (C).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/31829bde223c4b178a5cdc6c.png"},{"id":91953608,"identity":"accd22ff-5b7d-45ec-ab28-41e8c36c4983","added_by":"auto","created_at":"2025-09-23 06:58:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":74508,"visible":true,"origin":"","legend":"\u003cp\u003eThe DPV voltammograms of various concentrations of T\u003csub\u003e4\u003c/sub\u003e solution at EPHC@CP/GE in a phosphate buffer solution with a pH of 9, recorded at a scan rate of 80 mV/s. The inset illustrates the LDR for the method.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/600237457c2f2be3812f8461.png"},{"id":100616194,"identity":"86fc35ba-36b1-4d8a-95d7-005b32af45d1","added_by":"auto","created_at":"2026-01-19 17:41:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1694619,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/5ddb72d5-73ca-4760-bac8-39ef560672d7.pdf"},{"id":91952289,"identity":"31224404-cebe-4a49-9c24-2e3850ecd111","added_by":"auto","created_at":"2025-09-23 06:50:48","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14074,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. The EPHC production process from ethyl picolinate and 2-picolinic acid hydrazide.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/591e51fd956e48d575e16e65.png"},{"id":91953600,"identity":"dfefe815-9c57-45ee-b788-d718362a3eaf","added_by":"auto","created_at":"2025-09-23 06:58:49","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":47828,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 2. The possible electrochemical reaction mechanism of electrooxidation of T\u003csub\u003e4\u003c/sub\u003e at the EPHC@CP/GE.\u003c/p\u003e","description":"","filename":"Scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/3285ad050d085a61266f1749.png"},{"id":91953601,"identity":"41c79d8e-bc1f-4bf6-bc55-37086666e44f","added_by":"auto","created_at":"2025-09-23 06:58:49","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":40733,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7246319/v1/718e8f194b179caab15094c5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eN-ethyl-2-picolinoylhydrazine carbothioamide (EPHC) as an excellent sensing element for the electrochemical measurement of T\u003csub\u003e4\u003c/sub\u003e, the free thyroid hormone\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLevothyroxine (T\u003csub\u003e4\u003c/sub\u003e) is a synthetic thyroid hormone utilized in the treatment of hypothyroidism, a condition characterized by insufficient production of thyroid hormones by the thyroid gland. It is also employed in conjunction with surgery and radioactive iodine therapy for the management of thyroid cancer. T\u003csub\u003e4\u003c/sub\u003e is classified as a hormone and serves to replace the thyroid hormone normally produced by the body. It can exist in two forms in the blood serum: free and protein-bound. The absence of adequate thyroid hormone can hinder the body's proper functioning, potentially resulting in symptoms such as stunted growth, slowed speech, fatigue, excessive tiredness, constipation, weight gain, hair loss, dry and thickened skin, increased sensitivity to cold, joint and muscle pain, heavy or irregular menstrual cycles, and depression. When administered correctly, T\u003csub\u003e4\u003c/sub\u003e can reverse these symptoms [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The chemical composition of levothyroxine is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e as a sodium salt.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\u003cp\u003eGiven the narrow therapeutic index of T\u003csub\u003e4\u003c/sub\u003e, precise measurement of its dosage and serum levels is fundamental to achieving optimal treatment outcomes. Inadequate or excessive dosing can lead to severe health implications, including cardiovascular issues, bone density reduction, cognitive impairment, and metabolic disturbances. Thus, effective monitoring techniques, such as serum thyroxine and thyroid-stimulating hormone (TSH) tests, are essential for ensuring individualized treatment plans tailored to each patient\u0026rsquo;s unique physiological needs [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTraditional methods for T₄ measurement, such as high performance liquid chromatography (HPLC) [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], radioimmunoassay (RIA) [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], enzyme-linked immunoassay assay (ELISA) [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], isotope dilution mass spectrometry (isotope-dilution LC-MS/MS) [\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and chemiluminescent assays (CLA) have been widely used for decades. However, these techniques often require complex sample preparation, expensive reagents, and lengthy analysis times, underscoring the need for a faster, cost-effective, and highly sensitive alternative. Newer techniques, such as electrochemical sensors [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and liquid chromatography-mass spectrometry (LC-MS) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], offer rapid and highly sensitive alternatives. Electrochemical methods, in particular, provide real-time monitoring, minimal sample preparation, and portability, making them ideal for point-of-care testing.\u003c/p\u003e\u003cp\u003eThiosemicarbazones are a class of organosulfur compounds characterized by the presence of a thiosemicarbazide functional group. They are typically synthesized through the condensation of thiosemicarbazide with aldehydes or ketones [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thiosemicarbazones have a wide range of practical applications, particularly in medicinal chemistry and coordination chemistry. These compounds exhibit versatile biological activities, making them valuable in medicinal chemistry. Their ability to chelate metal ions enhances their pharmacological potential, particularly in cancer treatment and iron overload management [\u003cspan additionalcitationids=\"CR33 CR34 CR35 CR36 CR37 CR38 CR39 CR40\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, thiosemicarbazones serve as a type of nitrogen-sulfur donor ligands and are widely used as chemical ligands in coordination chemistry, forming stable complexes with transition metals such as copper, zinc, palladium, platinum, and iron. Their structural diversity, achieved through ligand modifications, plays a crucial role in optimizing their pharmacokinetics and pharmacodynamics [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan additionalcitationids=\"CR43 CR44 CR45\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eN-ethyl-2-picolinoylhydrazine carbothioamide (EPHC) is a thiosemicarbazone derivative that has been studied for its potential biological applications. It is known to form mixed-ligand bivalent transition metal ion complexes, including those with copper, nickel, manganese, and cobalt [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The EPHC ligand and its complexes are minimally soluble in water, but they dissolve well in dimethyl sulfoxide (DMS) and dimethylformamide (DMF) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eElectrochemical sensing has rapidly developed as a valuable analytical tool for real-time and high-precision monitoring of biomolecules, such as thyroid hormones. In this work, we present a straightforward and efficient synthesis of EPHC, achieving yields that surpass those previously reported in the literature. Building on this, we have developed a novel carbon paste (CP) electrochemical sensor based on EPHC, specifically optimized for the accurate detection of free-T₄ (abbreviated as T₄) in biological samples. By leveraging the unique properties of this sensor, including its high sensitivity, selectivity, and rapid response, we aim to provide a novel, efficient, and low-cost method for the thyroid hormone assessment.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Chemicals\u003c/h2\u003e\u003cp\u003eEthanol (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eOH), graphite powder (G), paraffin oil, methanol (CH\u003csub\u003e3\u003c/sub\u003eOH), potassium hexacyanoferrate(II) (K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e), potassium hexacyanoferrate(III) (K\u003csub\u003e3\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e), sodium-(2S)-2-amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoate (sodium levothyroxine), T\u003csub\u003e4\u003c/sub\u003e tablets (100 \u0026micro;g/tablet), propranolol tablets, sodium chloride (NaCl), hydrochloric acid (HCl), sodium hydroxide (NaOH), disodium hydrogen phosphate (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e) and sodium dihydrogen phosphate (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) all were sourced from Merck (Germany) and were of the utmost purity, used without any purification. Double distilled and deionized water was employed consistently throughout the experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Instruments\u003c/h2\u003e\u003cp\u003eFT-IR spectra were collected using a Shimadzu FT-IR 8400 spectrometer (Japan). To analyze the surface morphology of the electrodes, a field emission scanning electron microscope (FE-SEM, Tescan Mira3 LMU, Czech Republic) was employed. Elemental composition and distribution were determined via energy dispersive X-ray spectroscopy (EDX), utilizing a SAMx (France) instrument. Electrochemical measurements including cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were conducted on an Autolab PGSTAT 204 potentiostat/galvanostat (Metrohm Switzerland), operating within a standard three-electrode configuration: EPHC@CP/GE as the working electrode, a platinum wire as the counter electrode, and a standard calomel electrode (SCE) as the reference. For electrochemical impedance spectroscopy (EIS), an EG\u0026amp;G PAR model 263 A potentiostat/galvanostat (USA) was used. pH adjustments were made using a Metrohm 713 pH meter (Switzerland). When required, samples underwent sonication with a Soner 203H device (Taiwan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Synthesis of N-ethyl-2-picolinyl hydrazine carbothioamide (EPHC)\u003c/h2\u003e\u003cp\u003eA procedure for the synthesis of EPHC has already been provided in Ref. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], which reported a lower yield and inadequate characterization. Here, the synthesis of EPHC was carried out in two steps. In this approach, at the first stage, the synthesis of hydrazide-2-Picolinic acid was initially carried out starting from ethyl pyridine-2-carboxylate (ethyl picolinate). Then EPHC was prepared successfully. The entire process is depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Initially, 1.0 mmol (0.137 g) of ethyl-2 pyridine carboxylate (ethyl picolinate) was combined with 1.2 mmol (0.181 g) of hydrazine, and 5.0 mmol of ethanol was introduced into a 50 mL balloon. The mixture was subjected to reflux heating for 2 h, followed by evaporation at room temperature until the volume was halved, resulting in a significant amount of white sediment. The obtained precipitate was then crystallized using toluene. Crystal characteristics: bright white color, melting point: 105\u0026ndash;107\u0026deg;C, yield: 82%. Secondly, in a 20 mL balloon, hot ethanol was introduced to 1.0 mmol (0.137 g) of 2-picolinic acid hydrazide, which was obtained from the synthesis in the first step, until it was thoroughly dissolved. Subsequently, 1.0 mmol (0.87 g) of ethyl isothiocyanate was also added to the balloon, and the mixture was subjected to reflux heating for 3 h before being evaporated at room temperature, resulting in a significant amount of white sediment being separated. In a 25 mL beaker, 10.0 mL of toluene was poured and heated. The resulting compound was dissolved in the hot toluene, and the solution was then gradually cooled to room temperature. The resulting needle-shaped crystals were smoothed and dried, and the reaction efficiency was calculated, yielding a crystal that is bright white in color, with a melting point of 199\u0026ndash;202\u0026deg;C and a yield of 85% (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eScheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 preparation of the electrode\u003c/h2\u003e\u003cp\u003eIn the preparation of the modified CP electrode (EPHC@CP/GE), 0.5 g of G was first placed in a porcelain mortar and ground for 20 min. Following this, 5.0 mg of EPHC was added to the mixture. Subsequently, 0.3 mL of paraffin oil was introduced, and the contents were homogenized again for an additional 20 minutes to ensure the paste was thoroughly blended. The prepared mixture was carefully packed into a sampler plastic tip tube specifically, one with a 5 mm diameter at the top, 2 mm at the bottom, and a length of 3.5 cm. Electrical contact was established by directly inserting a bare graphite electrode, connected to a copper wire, into the assembly. This straightforward approach ensured reliable connectivity without introducing unnecessary complexity. The modified EPHC@CP/GE was employed as the working electrode. Other unmodified CP/GE electrodes were prepared using a similar method, but without the presence of any modifier.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Standard T\u003csub\u003e4\u003c/sub\u003e and real sample preparation\u003c/h2\u003e\u003cp\u003eA 250 mL stock solution of the sodium salt of T\u003csub\u003e4\u003c/sub\u003e at a concentration of 100 \u0026micro;M was prepared by dissolving 19.97 mg of the salt in water. The solution was then stored in a refrigerator at 4\u0026deg;C for future use.\u003c/p\u003e\u003cp\u003eTo evaluate the effectiveness of the proposed analytical method with actual samples, the method was applied to the determination of T\u003csub\u003e4\u003c/sub\u003e in pharmaceutical preparations, each reportedly containing 100 \u0026micro;g of T\u003csub\u003e4\u003c/sub\u003e per tablet. 100 tablets were accurately weighed and finely ground with a mortar and pestle. A portion of the resulting powder, calculated to be equivalent to a 100.0 \u0026micro;M T\u003csub\u003e4\u003c/sub\u003e, was then dissolved in a 10:90 ethanol-to-water mixture. This solution was subjected to sonication for 10 min to ensure thorough dissolution. After filtration into a 250.0 mL calibrated flask, the solution was diluted to volume with water. A measured volume of this solution was subsequently transferred to a 25.0 mL volumetric flask containing the supporting electrolyte, and standard T\u003csub\u003e4\u003c/sub\u003e solution was added as a spike for calibration purposes.\u003c/p\u003e\u003cp\u003eThe method was also evaluated using a blood serum sample collected from a young, healthy male participant. A 10.0 mL aliquot of serum was deproteinized using 2 mL of 10% (v/v) trichloroacetic acid, followed by centrifugation. The supernatant was then diluted tenfold with 0.1M phosphate-buffered solution at pH 9.0. These serum samples were stored at 4 ℃ until further analysis. An appropriate volume of the diluted serum sample was introduced into the electrochemical cell, and then standard T\u003csub\u003e4\u003c/sub\u003e solution was added as a spike for T\u003csub\u003e4\u003c/sub\u003e determination using DPV. Further details regarding the procedure are provided in later sections. All measurements employed the standard addition method to ensure accuracy and reliability of the results.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Spectroscopic\u003c/h2\u003e\n \u003cp\u003eSEM is a powerful technique to elucidate suitable information about the surface morphology of a substance [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]. The images presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e illustrate the crystalline structure of EPHC, indicating that this material has crystallized effectively and possesses a well-ordered and regular crystalline structure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). This arrangement significantly influences the chemical and physical properties of the substance, such as solubility and stability. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB displays an unmodified CP, revealing a uniform and continuous distribution of G sheets, along with fine pores that could potentially impact electrochemical performance. In contrast, the image of the CP modified with EPHC shows an increase in both the number and size of the pores (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). The pore distribution becomes irregular due to the incorporation of EPHC into the CP, resulting in a rough morphology that is promising for the accumulation of analyte on the electrode surface. The insets in the figures provide more detailed images to offer a clearer view of each composition.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eTo analyze and determine the composition of elements along with their corresponding percentages in the structure of EPHC, EDX was utilized. The findings, depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, demonstrate that the percentage composition of the synthesized EPHC elements at each stage corresponds with its intended formula. The tables provide evidence, as the insets in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e confirm the percentages of elements such as C, N, O, and S in EPHC. Figures\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB present high-resolution images of the morphologies of EPHC and EPHC@CP, respectively. Additionally, Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC to \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF illustrate the elemental topographies of EPHC@CP, indicating a consistent distribution of the elements C, N, O, and S [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eIn the analysis of the FT-IR spectrum, the frequencies associated with the bonds C\u0026thinsp;=\u0026thinsp;N, C\u0026thinsp;=\u0026thinsp;S, C\u0026thinsp;=\u0026thinsp;S, and C\u0026thinsp;=\u0026thinsp;H were primarily examined and compared (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, the stretching frequency of the IR spectrum for EPHC containing the C\u0026thinsp;=\u0026thinsp;N bond is located at 1674 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The presence of peaks from aryl and aromatic groups causes a shift of this bond\u0026apos;s peak towards lower frequencies. In this compound, the peaks between 1541 and 1510 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are related to the C-N bond in the thiosemicarbazide compound. The peaks found around 2933 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is associated with the =\u0026thinsp;C-H group, and the peak at 3151 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e related to -C-H group, while the peak at 1674 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to the bending frequency of the C\u0026thinsp;=\u0026thinsp;C bond in the pyridine ring present in the ligand. The peaks at 747 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1237 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are related to the vibrational frequencies of C\u0026thinsp;=\u0026thinsp;S. In Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, which pertains to the carbon paste electrode modified with EPHC (EPHC@CP), the peaks between 2933 and 2997 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e correspond to the =\u0026thinsp;C-H group, and the peaks between 1373 and 1481 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are associated with the C-N bond of EPH. The wide strong peak around 3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represents the alteration of carbonyl oxygens into -OH group after incorporation into the CP matrix [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]. A clear comparison between peaks (4a) and (4b) reveals a marked change in the EPHC peak following its incorporation into CP. This substantial alteration strongly suggests a robust interaction between EPHC and CP, providing compelling evidence for their synergistic electrocatalytic effect toward T\u003csub\u003e4\u003c/sub\u003e at the electrode surface.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Electrochemical study\u003c/h2\u003e\n \u003cp\u003eBefore finalizing the electrode fabrication, the appropriate quantities of the primary components, EPHC and G powder, were evaluated. The optimal values determined were 5.0 mg and 0.5 g, respectively, as these amounts yielded the highest signal response.\u003c/p\u003e\n \u003cp\u003eThe response of both unmodified and modified electrodes with EPHC in the 0.1 M [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e4\u0026minus;/3\u0026minus;\u003c/sup\u003e solution showed a positive result, as depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The cyclic voltammetry (CV) revealed a distinct redox peak (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), suggesting that the electrodes exhibited favorable responses to the probe. In the scenario where EPHC is present, the EPHC@CP/GE response is somewhat diminished compared to that of the bare CP/GE, indicating a slight reduction in electrode conductivity, a decrease in the current peak value, and an increase in the potential required for the electrochemical reaction at the electrode surface. Nonetheless, it operates effectively as an appropriate modifier, as it serves as a selective agent for the reaction with T\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eThe EIS study serves as a sensitive method for detecting changes in the surface impedance of electrodes in response to the addition of other components. EIS provides valuable insights into the electron transfer dynamics among electroactive species within electrode materials. In the context of a Nyquist plot, the linear segment typically corresponds to processes governed by mass transfer, while the semicircular feature is indicative of electron transfer limitations at the electrode interface that is, the charge transfer resistance (R\u003csub\u003ect\u003c/sub\u003e). This distinction allows researchers to diagnose where kinetic or transport barriers exist within the system [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. The Randall\u0026apos;s equivalent circuit [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e] is employed to match the impedance data (as shown in the inset of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). The EIS spectra for both the modified and unmodified electrodes are presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB. The R\u003csub\u003ect\u003c/sub\u003es of these electrodes are of 5947.5 and 4893.5 Ω, respectively. The Nyquist plots in this figure demonstrate that the conductivity of the CP/GE modified with the EPHC has decreased to match that of the unmodified carbon paste electrode. This is evident as the EIS curve for the modified EPHC@CP/GE shows a greater electrical resistance in comparison to the peak linked with the unmodified CP/GE, which displays increased electrical resistance due to the incorporation of EPHC.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Optimizing analytical factors\u003c/h2\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1 Effect of pH of the solution and buffering type\u003c/h2\u003e\n \u003cp\u003eTo investigate the influence of different buffer types on the electrode response, three 0.1 M buffering agents such as sodium borate/boric acid, sodium carbonate/bicarbonate, and sodium dihydrogen phosphate/hydrogen phosphate (phosphate buffer) were examined by using DPV. The findings illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e indicate that the electrode response to T\u003csub\u003e4\u003c/sub\u003e 8.0 \u0026micro;M is markedly improved in the phosphate buffer solution. This particular solution combination yields an estimated ionic strength of 0.1 for the buffer solution overall. Therefore, the phosphate buffer at 0.1 M with a pH of 9 was deemed suitable as a supporting electrolyte for the determination and measurement of T\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eOne of the highly influential factors affecting the response of the pH electrode is the solution itself, which can significantly impact electrochemical reactions, the performance of electrodes, and electrochemical cells. After selecting a phosphate buffer solution as the suitable pH provider, phosphate buffers of pH values of 1, 3, 5, 7, 9, 10, and 11 including T\u003csub\u003e4\u003c/sub\u003e 0.12 \u0026micro;M were prepared and analyzed using DPV with the modified EPHC@CP/GE. The corresponding voltammograms were recorded and examined, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e. According to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, the findings suggest that T\u003csub\u003e4\u003c/sub\u003e demonstrates a satisfactory current at high pH levels. A graph illustrating the peak currents (I\u003csub\u003ep\u003c/sub\u003e) in relation to the pH values was generated and is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, which facilitated the determination of the optimal pH of 9. The linear correlation observed between the peak potentials (E\u003csub\u003ep\u003c/sub\u003e) and the pH values (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC) serves as evidence for the electrochemical reaction\u0026apos;s dependence on pH [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e]. In addition to the facts presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, in order to evaluate further clues about the electrochemical mechanism, by which T\u003csub\u003e4\u003c/sub\u003e was involved at the electrode-solution interface, the relationship between T\u003csub\u003e4\u003c/sub\u003e peak potential (E\u003csub\u003ep\u003c/sub\u003e) and the pH of the solution was evaluated. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC, the plot of E\u003csub\u003ep\u003c/sub\u003e versus pH led us to Eq.\u0026nbsp;(1).\u003c/p\u003e\n \u003cp\u003eE\u003csub\u003ep\u003c/sub\u003e(V)\u0026thinsp;=\u0026thinsp;0.031pH\u0026thinsp;+\u0026thinsp;0.210, R\u0026sup2;= 0.992 (1)\u003c/p\u003e\n \u003cp\u003eFrom this equation, the slope of 31.0 mV/pH was obtained, which is very close to the Nernst theoretical value of 59/2 mV/pH for such electrochemical reactions in which two electrons and one proton transfer occur simultaneously.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eT\u003csub\u003e4\u003c/sub\u003e possesses three pKa values: the carboxylic group at 2.4, the phenolic group at 6.8, and the amino group at 9.9 [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e]. The solubility of T\u003csub\u003e4\u003c/sub\u003e sodium salt in water decreases as the pH increases from 1 to 3. Between pH 3 and 7, solubility remains relatively constant. When the pH rises above 7, there is a marked increase in solubility [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]. These trends are clearly illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB. Consequently, T\u003csub\u003e4\u003c/sub\u003e exists in its fully deprotonated state (see Scheme \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) at the optimal pH of 9. In contrast, at significantly lower pH levels, the oxidation of T\u003csub\u003e4\u003c/sub\u003e typically becomes challenging due to extensive protonation and precipitation, resulting in diminished anodic responses. As the pH increases, the responses improve owing to the deprotonation of T\u003csub\u003e4\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). From these facts, the behavior of the electrode at varying pH levels is entirely comprehensible.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2 Impact of potential scan rate\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e depicts the CV responses of the modified EPHC@CP/GE at scan rates of 10, 20, 30, 40, 50, 80, and 100 mV/s within a phosphate buffer solution at pH 9, alongside a 0.1 \u0026micro;M T\u003csub\u003e4\u003c/sub\u003e solution. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA, the process is characterized by an irreversible mechanism, where no reverse peak is present. The optimal scan rate of 80.0 mV/s was chosen for the experiments involving potential variations. The impact of the optimized scan rate on the electrode in a blank solution was analyzed and included in the figure, which shows no significant response. It is noted that with an increase in the scan rate, the oxidation potentials of the irreversible T\u003csub\u003e4\u003c/sub\u003e peaks exhibit a slight shift towards more positive values. This shift can be attributed to kinetic limitations, or in other words, the insufficient opportunity for the species to reach the electrode surface and undergo the oxidation process, thereby necessitating higher potentials for oxidation [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e]. This indicates the presence of kinetic constraints in the reaction between the modified electrode and T\u003csub\u003e4\u003c/sub\u003e, which has already confirmed by the results in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. Furthermore, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB depicts the changes in the oxidation E\u003csub\u003ep\u003c/sub\u003e versus the logarithm of the scan rate (n, Eq.\u0026nbsp;2), resulting in a linear regression line with R\u003csup\u003e2\u003c/sup\u003e of 0.995. Additionally, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC demonstrates a linear relationship between the oxidation I\u003csub\u003ep\u003c/sub\u003e and the scan rate (n\u003csup\u003e1/2\u003c/sup\u003e), yielding an R\u003csup\u003e2\u003c/sup\u003e value of 0.986 for the regression line of Eq.\u0026nbsp;3. This relationship indicate that the electrochemical system is not a reversible process.\u003c/p\u003e\n \u003cp\u003eE\u003csub\u003ep\u003c/sub\u003e= 0.062Log(n)\u0026thinsp;+\u0026thinsp;0.476, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.995 (2)\u003c/p\u003e\n \u003cp\u003eI\u003csub\u003ep\u003c/sub\u003e= 0.815n\u003csup\u003e1/2\u003c/sup\u003e + 4.493, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.986 (3)\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eFurthermore, based on the Laviron\u0026rsquo;s equation [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e], and the slope of the curve illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB and Eq.\u0026nbsp;1, with a common value of a\u0026thinsp;=\u0026thinsp;0.5, the number of electrons transferred during the reaction is calculated to be 1.9, which is approximately 2. Essentially, the electro-oxidation of T\u003csub\u003e4\u003c/sub\u003e at the EPHC@CP/GE interface proceeds with the transfer of two electrons and one proton, as illustrated in stage (I) of Scheme \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Notably, the specific role of EPHC in the overall electrochemical process may be significant, as suggested by the half-reaction depicted in stage (II) of Scheme \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Taken together, the available results support the mechanism for T\u003csub\u003e4\u003c/sub\u003e electro-oxidation as outlined in Scheme \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eScheme \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.3 Reliability of electrode response\u003c/h2\u003e\n \u003cp\u003eFollowing each T\u003csub\u003e4\u003c/sub\u003e measurement, the electrode underwent 10 cycles of cyclic voltammetry within a potential range of 0.3 to 0.8 V. This procedure was employed to reverse the reduction of EPHC that occurred due to T\u003csub\u003e4\u003c/sub\u003e oxidation, thereby restoring the electrode to its original state prior to subsequent use. All steps were conducted under optimized conditions.\u003c/p\u003e\n \u003cp\u003eThe electrode was analyzed for the delay time necessary to demonstrate a stable response value to the analyte over a period of 180 s, utilizing reading intervals of 10 s. The optimal result was achieved at 30 s, which was determined to be the appropriate duration for future applications of the electrode.\u003c/p\u003e\n \u003cp\u003eTo evaluate the accuracy and precision of the measurements, three identically modified electrodes EPHC@CP/GE were employed concurrently to conduct five repeated measurements on 20.0, 25.0 and 30.0 \u0026micro;M T\u003csub\u003e4\u003c/sub\u003e solutions under the optimized conditions. The relative standard deviation (RSD) for each electrode is detailed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Importantly, all RSD values of recoveries were below \u0026plusmn;\u0026thinsp;2.11, indicating a high degree of precision in the electrode responses, while the measured values of the electrodes were reasonably close to the standard spiked T\u003csub\u003e4\u003c/sub\u003e amounts, signifying substantial accuracy. The results also highlight the strong reproducibility of the modified electrode system for T\u003csub\u003e4\u003c/sub\u003e detection, further corroborated by the overall acceptable RSD of \u0026plusmn;\u0026thinsp;3.55. In summary, the findings illustrate that the modified electrodes yield reliable and consistent measurements for this application.\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eTo assess the stability of EPHC@CP/GE in real-world conditions, the electrode underwent a one-month evaluation with two measurements taken daily at one-hour intervals. The data\u0026apos;s RSD was below \u0026plusmn;\u0026thinsp;2.5 percent, a figure deemed acceptable, suggesting a satisfactory lifespan for the electrode throughout the one-month duration.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.4 Interferent impact\u003c/h2\u003e\n \u003cp\u003eThe sensitivity of the modified electrode could be altered in the presence of interfering species that may impact on the response current peaks. These interfering species may be those that they present in pharmaceutical formulations and could pose potential interference in the measurement of T\u003csub\u003e4\u003c/sub\u003e using the DPV method. The potential interferences of certain drugs such as Liothyronine (T\u003csub\u003e3\u003c/sub\u003e) (the other thyroidal hormone), Propylthiouracil (used for hyperthyroidism treatment), and some ions were investigated. This was conducted by increasing the concentration of interfering substances in a 0.1 \u0026micro;M T\u003csub\u003e4\u003c/sub\u003e solution in a phosphate buffer at pH 9 under optimal conditions. Species that disrupt the measurement of T\u003csub\u003e4\u003c/sub\u003e would influence the current intensity of T\u003csub\u003e4\u003c/sub\u003e beyond the defined range. If the variation in the response to T\u003csub\u003e4\u003c/sub\u003e, when interfering species are present, adheres to the following relationship (Eq.\u0026nbsp;4), and RE is less than \u0026plusmn;\u0026thinsp;5 percent, it is considered that there is no significant impact on the intensity of the T\u003csub\u003e4\u003c/sub\u003e current intensity.\u003c/p\u003e\n \u003cp\u003e%RE= [(I\u003csub\u003e0\u003c/sub\u003e-I\u003csub\u003eif\u003c/sub\u003e)/I\u003csub\u003e0\u003c/sub\u003e] \u0026times; 100 (4)\u003c/p\u003e\n \u003cp\u003ewhere RE denotes the deviation in current intensity caused by the interference, I\u003csub\u003e0\u003c/sub\u003e represents the initial current intensity, specifically in the presence of T\u003csub\u003e4\u003c/sub\u003e at 0.1 \u0026micro;M, and I\u003csub\u003eif\u003c/sub\u003e signifies the measured current intensity of the solution in the presence of an interferer. The maximum allowable concentration of interfering species in the presence of 0.1 \u0026micro;M T\u003csub\u003e4\u003c/sub\u003e is detailed in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. According to the data presented in this table, the permissible concentrations of Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, I\u003csup\u003e\u0026ndash;\u003c/sup\u003e, Cl\u003csup\u003e\u0026ndash;\u003c/sup\u003e, Propylthiouracil, and T\u003csub\u003e3\u003c/sub\u003e are 8000, 5800, 8400, 12000, 7000, and 1700 times the concentration of T\u003csub\u003e4\u003c/sub\u003e in the sample, respectively.\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.5 Measurements merits\u003c/h2\u003e\n \u003cp\u003eThe parameters recognized as the advantages of a sensor\u0026apos;s performance include the linear dynamic range (LDR), where the calibration curve of the sensor\u0026apos;s analytical responses changes linearly with a variable, such as the concentration of the target analyte, the limit of detection (LOD), and the limit of quantification (LOQ). A calibration curve illustrating the sensor\u0026apos;s responses to varying concentrations of T\u003csub\u003e4\u003c/sub\u003e, ranging from 0.0 to 80.0 \u0026micro;M, was established using the DPV method. The results are presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates the DPV voltammograms for various concentrations of T\u003csub\u003e4\u003c/sub\u003e solution at the modified electrode in a 0.1 M phosphate buffer solution with a pH of 9, recorded at a scan rate of 80 mV/s. Drawing from the information presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e and Eq.\u0026nbsp;5, the calculation of both LOD and LOQ for this analytical method follows established conventions: LOD is determined as 3S\u003csub\u003eb\u003c/sub\u003e divided by m, and LOQ is calculated as 10S\u003csub\u003eb\u003c/sub\u003e over m. Here, \u0026ldquo;m\u0026rdquo; refers to the slope of the calibration curve, which essentially reflects the method\u0026rsquo;s sensitivity to increasing amounts of analyte, while \u0026ldquo;S\u003csub\u003eb\u003c/sub\u003e\u0026rdquo; is the standard deviation of the blank. In this case, S\u003csub\u003eb\u003c/sub\u003e was measured to be 3.12 \u0026times; 10⁻\u003csup\u003e3\u003c/sup\u003e based on six replicate measurements, an approach that helps account for random fluctuations in the baseline signal. The resulting calculations yield an LOD of 8.0 \u0026times; 10⁻\u003csup\u003e3\u003c/sup\u003e \u0026micro;M and an LOQ of 0.027 \u0026micro;M for T\u003csub\u003e4\u003c/sub\u003e. These values provide a quantitative benchmark for the method\u0026rsquo;s capacity to reliably distinguish and measure low concentrations of the analyte. The relatively low LOD and LOQ suggest that the method is quite sensitive, making it suitable for detecting T\u003csub\u003e4\u003c/sub\u003e even at sub-micromolar levels. Furthermore, %RSD of \u0026plusmn;\u0026thinsp;0.632, calculated across six experimental replicates, indicates a high degree of precision. In analytical chemistry, maintaining an RSD below 1% is generally considered excellent, as it reflects minimal variability between repeated measurements under identical conditions. Such a low %RSD not only reinforces the method\u0026rsquo;s reliability but also supports the validity of the LOD and LOQ values obtained.\u003c/p\u003e\n \u003cp\u003eI\u003csub\u003ep\u003c/sub\u003e= 1.170C\u003csub\u003eT4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2.179, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.992 (5)\u003c/p\u003e\n \u003cp\u003eThe inset of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates the LDR for the method, which spans from 8.7\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 80.0 \u0026micro;M of T\u003csub\u003e4\u003c/sub\u003e. This range defines the interval over which the analytical response remains directly proportional to the concentration of T\u003csub\u003e4\u003c/sub\u003e in the sample. Operating within this range is crucial, as measurements taken outside these limits may not yield accurate or linear results. The breadth of the LDR further underscores the method\u0026rsquo;s versatility, enabling accurate quantification across a wide range of sample concentrations. From Eq.\u0026nbsp;5, the sensitivity of the sensor is 1.17 \u0026micro;A/\u0026micro;M of T\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.6 Analytical application\u003c/h2\u003e\n \u003cp\u003eIn order to accurately assess the functionality of the newly developed electrode for T\u003csub\u003e4\u003c/sub\u003e measurement, its performance was established to be compared with the well-established HPLC method. To validate the integrity of our comparison, we did not depend on a solitary measurement; rather, we examined five replicate aliquots of each actual sample using both the new electrode and HPLC. This methodology contributed to the reduction of random error and yielded a comprehensive dataset for analysis. The findings are presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. As illustrated in this table, a significant correlation is evident between the results acquired from DPV utilizing EPHC@CP/GE and those derived from HPLC. The recoveries data were subjected to an one-tailed F-test using a critical F-value of 6.38 at a significance level of P\u0026thinsp;=\u0026thinsp;0.05. This approach was intended to evaluate whether the observed similarities in precision were statistically significant. Notably, the experimental F-values were considerably lower than the critical value of 6.38, suggesting that the sensor exhibited superior precision compared to the HPLC results. The electrode\u0026rsquo;s response to T\u003csub\u003e4\u003c/sub\u003e not only mirrors the level of precision achieved by HPLC but, intriguingly, demonstrates superior accuracy. Such parity is particularly noteworthy considering the practical advantages that electrodes often offer over HPLC, such as faster turnaround times, lower operational costs, and greater portability. This is a significant result, implying that the electrode could feasibly replace or complement HPLC in certain analytical scenarios, especially where rapid or cost-effective measurement is necessary.\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eBuilding on the previous comparison with established studies focused on the electrochemical detection of T\u003csub\u003e4\u003c/sub\u003e, the present research employing the EPHC@CP/GE electrode demonstrates a marked advancement in analytical performance. As detailed in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, a compilation of relevant recent studies [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e], the newly developed electrode not only achieves a notably lower LOD but also extends the LDR for T\u003csub\u003e4\u003c/sub\u003e analysis. Additionally, the broader LDR enhances the electrode\u0026rsquo;s versatility, making it suitable for applications where the concentration of T\u003csub\u003e4\u003c/sub\u003e may vary widely. This improvement in sensitivity and dynamic range is particularly significant when considering the growing demand for precise and reliable quantification of T\u003csub\u003e4\u003c/sub\u003e, especially in complex biological or pharmaceutical matrices. Taken together, the findings underscore the potential of EPHC@CP/GE as a reliable tool for sensitive, accurate, and practical electrochemical analysis of T\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis electrode offers several advantages, including a simple preparation method, high stability, selectivity, acceptable reproducibility, rapid response time, and applicability across a wide pH range. The electrode\u0026rsquo;s response to T\u003csub\u003e4\u003c/sub\u003e not only mirrors the level of precision achieved by HPLC but, intriguingly, demonstrates superior accuracy. Such parity is particularly noteworthy considering the practical advantages that electrodes often offer over HPLC, such as faster turnaround times, lower operational costs, and greater portability. This is a significant result, implying that the electrode could feasibly replace or complement HPLC in certain analytical scenarios, especially where rapid or cost-effective measurement is necessary. By pushing the boundaries of detection limits, the EPHC@CP/GE electrode facilitates the identification of trace amounts of analyte, which is crucial for early diagnosis, therapeutic monitoring, and ensuring regulatory compliance in pharmaceutical quality control. In summary, the method demonstrates strong analytical performance, characterized by high sensitivity, excellent precision, and a robust linear dynamic range. These attributes collectively establish the technique as a reliable tool for the quantification of T\u003csub\u003e4\u003c/sub\u003e in diverse sample matrices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbdollah Yari\u003c/strong\u003e: literature search, figures, study design, Writing \u0026ndash; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFatemeh Biranvand\u003c/strong\u003e: data collection, data analysis, data interpretation, Writing \u0026ndash; original draft, preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data utilized for the research outlined in the article can be obtained by contacting the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMiccoli P, Materazzi G, Rossi L (2021) Levothyroxine therapy in thyrodectomized patients. 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Anal Bioanal Chem 397:1831\u0026ndash;1839. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-010-3705-9\u003c/span\u003e\u003cspan address=\"10.1007/s00216-010-3705-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarami P, Gholamin D, Johari-Ahar M (2023) Electrochemical immunoassay for one-pot detection of thyroxin (T4) and thyroid-stimulating hormone (TSH) using magnetic and Janus nanoparticles. Anal Bioanal Chem 415:4741\u0026ndash;4751. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-023-04767-8\u003c/span\u003e\u003cspan address=\"10.1007/s00216-023-04767-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-electrochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jach","sideBox":"Learn more about [Journal of Applied Electrochemistry](http://link.springer.com/journal/10800)","snPcode":"10800","submissionUrl":"https://submission.nature.com/new-submission/10800/3","title":"Journal of Applied Electrochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Carbothioamide, Electrochemical sensor, Levothyroxine (T4), N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC)","lastPublishedDoi":"10.21203/rs.3.rs-7246319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7246319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis article examines the creation of our innovative electrochemical electrode, and the advantages of the electrochemical approach for accurate monitoring of the thyroid hormone (T\u003csub\u003e4\u003c/sub\u003e). The sensor functions through electrochemical interactions between the T₄ molecule and the electrode surface, which includes N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC). By using cyclic voltammetry (CV), differential pulse voltammetry (DPV), or electrochemical impedance spectroscopy (EIS), the sensor delivers dependable and precise results, with prospective applications in free-T\u003csub\u003e4\u003c/sub\u003e monitoring. EPHC was synthesized and characterized using FT-IR, SEM, and EDX techniques to confirm successful synthesis. EPHC was blended with suitable components to formulate a consistent carbon paste, to which a graphite bare was affixed for electrical connectivity. The sensor demonstrates impressive performance, achieving a low detection limit of 8.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;M. It operates effectively across a linear range from 8.7\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 80.0 \u0026micro;M. The response time is rapid at 30 seconds. The electrode exhibits satisfactory repeatability and reproducibility. The sensitivity for T\u003csub\u003e4\u003c/sub\u003e is measured at 1.17 \u0026micro;A/\u0026micro;M, reflecting the sensor\u0026rsquo;s strong analytical capability. The sensor successfully quantified free-T\u003csub\u003e4\u003c/sub\u003e in different real matrices, under optimized conditions of a pH of 9, a scan rate of 80 mV/s, 5.0 mg of EPHC, and 0.5 g of graphite powder.\u003c/p\u003e","manuscriptTitle":"N-ethyl-2-picolinoylhydrazine carbothioamide (EPHC) as an excellent sensing element for the electrochemical measurement of T4, the free thyroid hormone","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 06:50:44","doi":"10.21203/rs.3.rs-7246319/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-18T18:35:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-05T07:56:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T11:30:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162429649338523954309778414947284890162","date":"2025-09-18T15:46:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30403688890530184385396737800145975096","date":"2025-09-18T13:35:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141666614233058382830608905308410122286","date":"2025-09-16T10:30:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27257838832125124671743579007518920901","date":"2025-09-13T17:53:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-13T13:27:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-02T00:32:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-30T04:30:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Electrochemistry","date":"2025-07-29T19:19:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-electrochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jach","sideBox":"Learn more about [Journal of Applied Electrochemistry](http://link.springer.com/journal/10800)","snPcode":"10800","submissionUrl":"https://submission.nature.com/new-submission/10800/3","title":"Journal of Applied Electrochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"26d1ab1f-e224-425a-885b-6e2c13efa824","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T17:05:24+00:00","versionOfRecord":{"articleIdentity":"rs-7246319","link":"https://doi.org/10.1007/s10800-025-02411-0","journal":{"identity":"journal-of-applied-electrochemistry","isVorOnly":false,"title":"Journal of Applied Electrochemistry"},"publishedOn":"2026-01-14 16:29:59","publishedOnDateReadable":"January 14th, 2026"},"versionCreatedAt":"2025-09-23 06:50:44","video":"","vorDoi":"10.1007/s10800-025-02411-0","vorDoiUrl":"https://doi.org/10.1007/s10800-025-02411-0","workflowStages":[]},"version":"v1","identity":"rs-7246319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7246319","identity":"rs-7246319","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}