BIOSENSOR DE TESTOSTERONA EN EL DOPAJE DEPORTIVO

rbme Revista Brasileira de Medicina do Esporte Rev Bras Med Esporte 1517-8692 1806-9940 Sociedade Brasileira de Medicina do Exercício e do Esporte RESUMEN Introducción: La testosterona es un esteroide que puede ayudar en los trastornos sanguíneos, la disfunción sexual, las enfermedades del tejido conectivo, algunos tumores malignos, el dolor intratable y otras enfermedades graves. Sin embargo, debido al riesgo de que se produzcan efectos adversos importantes, como enfermedades hepáticas, cardíacas, accidentes cerebrovasculares, coágulos sanguíneos y cáncer, debe prescribirse bajo supervisión médica. Es urgente investigar el desarrollo de un sensor electroquímico para detectar la testosterona como sustancia dopante en el deporte. Objetivo: Desarrollar un sensor electroquímico de polímeros impresos molecularmente de poli(líquido iónico)-óxido de grafeno (PIL/MIs/GO) para detectar la testosterona como sustancia dopante en el deporte. Métodos: La caracterización morfológica de los electrodos modificados se llevó a cabo mediante microscopía electrónica de barrido de emisión de campo (FESEM) permitiendo que el GO estuviera en la superficie con fragmentos y aberturas. Debido a los agujeros generados por la aglomeración de moléculas de PIL y MIs en los bordes ondulados de las nanohojas de GO, la morfología superficial de PIL/MIs/GO/GCE también reveló una estructura de alta porosidad. Resultados: En comparación con otras influencias sinérgicas de las nanohojas de GO con las moléculas PIL y MIs, los resultados de las investigaciones electroquímicas utilizando el enfoque de la voltamperometría diferencial de impulsos indicaron una alta selectividad, una buena estabilidad, un rango lineal apropiado, un límite de detección más bajo y una mayor selectividad. Conclusión: En muestras farmacéuticas y fluidos biológicos humanos, la validez y precisión de PIL/MIs/GO/GCE para la determinación de testosterona demostró su aplicación práctica. Así pues, PIL/MIs/GO/GCE puede utilizarse como un sensor preciso y fiable para la detección de la testosterona como agente dopante en el deporte. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento. INTRODUCTION Steroids are medications like androstenedione, testosterone, methyl testosterone, and danazol, that can help with blood disorders, sexual dysfunctions, connective tissue illness, some malignancies, intractable pain, and other serious diseases.1 However, due to the risk of major adverse effects like liver disease, heart disease, stroke, blood clots, and cancer, they must only be set and used in medical supervision. As a result, numerous studies have been carried out on gas chromatography, enzyme-linked immunosorbent assay, liquid chromatography, fluorimetry, mass spectrometry, spectrophotometry, radio immunoassay, photodiode-array, yeast estrogen screen assess, and electrochemical detection approaches in order to find a quick and easy way to detect steroid abuse.2–6 Electrochemical techniques, for example, have revealed a varied range of applicability in environmental and food investigations, as well as clinical diagnostics, thanks to their simplicity of usage, low-cost, and capacity to improve and optimize during the last decade. The use of nanomaterials and different composites to modify and miniaturize electrochemical sensors improves selectivity and sensitivity.7 Anabolic steroids are testosterone derivatives that are synthesized (man-made). In men, testosterone is the primary sex hormone. It's required for the development and maintenance of male sex features like facial hair, a deep voice, and muscle growth. Because testosterone induces increases in muscular mass, which is a positive adaptation for many power-based sports, both naturally and synthetically raised testosterone levels may improve strength and sports performance. For testosterone sensors, just a few electrochemical investigations have been conducted.8–14 As a result, our research focused on the development of a PIL/MI/GO/GCE electrochemical sensor for detecting testosterone as a doping substance in sports. MATERIALS AND METHODS The GO was prepared from graphite powder using a modified Hummers process.15 2 g graphite powder was introduced to an ice water bath with a combination of 70 mL sulfuric acid and 30 mL nitric acid. The suspension was then gradually supplemented with 10 g potassium permanganate. The mixture was then swirled for 70 minutes at 30°C. Following that, 100 mL DI water was slowly added to the mixture while stirring at 40°C for 50 minutes, and then 10 mL H2O2 was added while stirring at 35°C for 45 minutes to create a bright yellow suspension. For 15 minutes, suspension was centrifuged in 1500 rpm/minutes. To separate/exfoliate stacked graphene oxide sheets, supernatant was detached and the resulting GO was ultrasonically disseminated in 3mL of 0.1M PBS for 90 minutes. The 0.1M PBS was made using 0.1M sodium phosphate at a 1:1 volume ratio. Prior to the alteration, the surface of GCE was prepared by polishing it by alumina powder onto a polishing material, then ultrasonically washing it with DI water and ethanol aimed at 10 min each, then drying it at room temperature. The electrodeposition was carried out on a potentiostat-galvanostat in a traditional three electrode-electrochemical cell that featured GCE as the working electrode, Ag/AgCl and Pt wire as the reference and counter electrodes, respectively. A homogenous mix of 0.2g tetrabutylammonium perchlorate and 100 ml acetonitrile was placed on the electrode surface to modify the surface of GCE and GO/GCE with MI 16. The electrode was then dried at room temperature with a moderate flow of dry nitrogen. To make PIL/MI/GO/GCE, PIL/MI/GCE, and PIL/GCE, poly(4-vinylpyridine) was deposited in an electrochemical cell with bare or modified GCE as working electrodes. The testosterone-free plasma sample is used for the creation of the real specimen of human plasma. With 0.1 M PBS, the plasma specimens (10 mL) were watery to 100mL. Analytical investigations of genuine samples were conducted using the conventional addition method. On the Autolab system, differential pulse voltammetry (DPV) investigations were performed in a three-electrode electrochemical cell with improved GCE as the working electrode in 0.1M PBS as the electrochemical electrolyte. Field emission scanning electron microscopy was used to investigate the morphology of electrode surfaces. This work was conducted based on the Declaration of Helsinki principle. The participants signed the Free and Informed Consent Form (EHIC). RESULTS AND DISCUSSION SEMs of GO and PIL/MI/GO/GCE are shown in Figure 1. Figure 1a illustrates the overlying of flake coatings onto the surface in a FESEM picture of GO. The holey and rough appearance of GO is observed, with cracks and breaks that can arise due to carbon atom rearrangement through the graphitization procedure.17 Figure 1b demonstrates that PIL/MI/GO/GCE has a porous structure due to holes formed by aggregation of PILand MI molecules on the crenelated edge of GO nanosheets. The use of a co-functional monomer made up of MI and PILas could improve adsorption and conductivity.18 Figure 1 SEM images of (A) graphene oxide, and (B) PIL/MI/GO/GCE. Figure 2 depicts the DPV curves and PIL/MI/GO/GCE calibration plots for serial injections of 10M testosterone solution in 0.1M PBS at a 10 mV/s scan rate. The electrocatalytic current increases linearly as the testosterone concentration increases from 1 to 60 µM, with a deviation from linearity aimed at higher concentrations because of saturation of dynamic sites on improved electrodes.19 PIL/MI/GO/GCE as a testosterone sensor has a sensitivity of 0.7395 A/M and a detection limit of 0.003 M, respectively. Figure 2 (A) The DPV plots and (B) calibration curve of PIL/MI/GO/GCE in 0.1M PBS at 10mV/s scan rate under successive adding of testosterone solution. Table 1 shows the results of a PIL/MI/GO/GCE interference study to determine testosterone in the existence of chief metabolites in fluids of body using DPV analysis in 0.1M PBS at 10 mV/s scan rate for successive injections of 1M testosterone and 10M hypoxanthine, ascorbic acid, uric acid, dopamine, albumin, nitrite, glucose, and xanthine as interferents.20 The modified electrode exhibits a spectacular peak current in response to testosterone solution additions, but injections of other interference substrates produce no appreciable electrocatalytic peak current.21 As a result, the interference substrates listed in Table 2 have no effect on testosterone detection, and the PIL/MI/GO/GCE can be employed as a selective testosterone sensor into biological fluid specimens like blood plasma and human urine. Table 1 The electrocatalyst peak value of DPV measures of PIL/MI/GO/GCE at a 10mV/s scan rate for repeated injections of 1µM testosterone and 10M interferents in 0.1M PBS. Substances Added(µM) Peak current(µA) RSD*(%) Testosterone 1 0.741 ±0.0122 Uric acid 10 0.029 ±0.0026 Ascorbic acid 10 0.022 ±0.0037 Albumin 10 0.012 ±0.0024 Xanthine 10 0.031 ±0.0031 Glucose 10 0.037 ±0.0073 Hypoxanthine 10 0.009 ±0.0026 Dopamine 10 0.022 ±0.0074 Nitrite 10 0.021 ±0.0113 * Relative standard deviation. Table 2 The findings of the PIL/MI/GO/GCE for determining testosterone in actual testosterone booster specimens and human plasma. Samples Added (mg/ml) Obtained (mg/ml) Recovery (%) RSD (%) Testosterone boosters 0.00 0.97 – – 2.00 1.93 97.52 2.97 4.00 3.84 96.75 3.13 6.00 5.93 98.33 2.74 8.00 7.95 99.02 3.87 Human plasma 0.00 0.00 – – 2.00 1.91 96.05 2.56 4.00 3.92 98.27 2.96 6.00 5.94 98.48 3.39 8.00 7.96 99.41 3.52 Into the pharmaceutical specimens and biological fluids, the validity, precision, and applicability of PIL/MI/GO/GCE for testosterone assessment were assessed. The DPV measurements were performed using PIL/MI/GO/GCE in a prepared genuine pharmaceutical sample of testosterone boosters with 0.1M PBS at a 10mV/s scan rate in sequential additions of testosterone solution in order to analyze prepared real specimen of testosterone boosters. Figures 3a and 3b show the DPV results and calibration plots, demonstrating that the testosterone level in prepared specimen is 0.96 mg/ml, which is consistent with the prepared genuine specimen testosterone solution concentration of boosters.22 Table 2 further shows that for prepared genuine samples of testosterone boosters, satisfactory recovery values (97.52 to 99.02%) and RSD values (2.97 to 3.87%) were obtained. There were no real specimens of blood plasma aimed at individuals undergoing testosterone behavior in a local health center or hospitals to examine the applicability of the sensor in human biological fluids. As a result, a typical addition procedure was used to study the testosterone-free plasma sample. For prepared genuine specimens of human plasma, DPV measurements were performed on PIL/MI/GO/GCE into 0.1M PBS at a 10mV/s scan rate. Table 2 also shows the testosterone levels in a genuine plasma sample as well as the results of the analysis. Acceptable recovery (96.05 to 99.41%) and RSD values are provided (2.56 to 3.52%). As a result, PIL/MI/GO/GCE can be employed as a perfect and consistent testosterone sensor in clinical specimens. Figure 3 (A) DPV responding and (B) calibration curve of PIL/MI/GO/GCE in a real sample of testosterone boosters made with 0.1M PBS at a 10mV/s scan rate with successive adding of testosterone solution. CONCLUSIONS The synthesis of PIL/MI/GO/GCE for electrochemical detecting testosterone as a doping substance in sports was presented in this paper. The modified Hummers technique was used to make GO nanosheets that were then electrodeposited onto GCE. PIL was electrodeposited on MI/GO/GCE after MI was changed on GO/GCE. The results revealed cracks and breaks on surface of GO nanosheets, and the morphology of PIL/MI/GO/GCE indicated a high porosity structure because of cavities formed by the aggregation of PIL and MI molecules on the crenelated edges of GO nanostructures. In comparison to other synergetic influence of GO nanosheets with PIL and MI molecules, electrochemical experiments revealed high selectivity, good stability, appropriate linear range, lowest limit of detection, and highest selectivity. In pharmaceutical samples and human biological fluids, the power and accuracy of PIL/MI/GO/GCE for testosterone determination were investigated, and the findings showed suitable recovery and RSD values. As a result, PIL/MI/GO/GCE can be employed as a precise and dependable testosterone sensor in clinical samples. 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