Development of a Horseradish Peroxidase-Based Electrochemical Biosensor for Melatonin Detection

A horseradish peroxidase (HRP)-based electrochemical biosensor was developed for melatonin (MLT) detection using a graphene-modified screen-printed electrode (G/SPE). HRP was immobilized at different temperatures (4 °C and 20 °C) and enzyme amounts (10 and 20 µL of 5 mg/mL HRP solution) to optimize the performance of the biosensors. FTIR analysis confirmed successful enzyme immobilization, while differential pulse voltammetry (DPV) showed a clear oxidation peak of MLT at 1.1 V with enhanced current responses for HRP-modified electrodes. The optimal biosensor, prepared at 4 °C with 10 µL enzyme of 5 mg/mL HRP solution, exhibited the best analytical performance, with a linear range of 0.4-3.6 µM, and a detection limit of 1.02 µM. Kinetic studies confirmed strong enzyme-substrate affinity, while the biosensors demonstrated excellent repeatability and a good stability. The method was successfully validated on pharmaceutical products, proving to be a reliable and an accurate tool for MLT quantification in real samples.