This work reports the characterization and application of a new electrochemical platform modified with gold nanoparticles (AuNP) and functionalized multi-walled carbon nanotubes (f-MWCNT) film to determine melatonin (MEL), a critical sleep inductor neurohormone categorized by the U.S. Food and Drug Administration as a nutritional supplement. The AuNP were synthesized and stabilized by 3-n-propyl-(4-methylpyridinium) silsesquioxane chloride (Si4Pic+Cl−), presenting an electrostatic stabilization and a material with a positive surface charge. Thermogravimetric analysis, Fourier transform-infrared spectroscopy, Raman spectroscopy, and zeta (ζ) potential indicated the insertion of oxygenated functional groups and a superficial negative charge for f-MWCNT. The GCE/f-MWCNT-AuNP-Si4Pic+Cl− platform showed an electrocatalytic profile and a current increase of ca. 7.5 times. Voltammograms were obtained by square wave voltammetry; the current increased with successive additions of MEL in the linear range of 4.9 to 55.5 μmol L−1. The values of LOD and LOQ were 1.6 and 4.7 μmol L−1, respectively. The standard addition was used to quantify MEL in simulated saliva, blood serum, and urine samples. Statistical tests showed no significant differences between the data collected by voltammetry and the comparative technique, molecular absorption spectrometry. It can be concluded that the new method provides novel perspectives for MEL sensing
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