Competitive Immunoassay of Melatonin by QCM Biosensor

Melatonin is a hormone that plays a role in the regulation of circadian rhythms and is also recognized for its antioxidant and cytoprotective functions. Its determination in biological samples is analytically challenging because the physiological concentration is very low, especially in saliva. Here, a competitive immunoassay based on a quartz crystal microbalance (QCM) biosensor was developed for melatonin determination. The biosensor employed a surface modified with a melatonin–albumin conjugate and a multilayer antibody amplification scheme to enhance the piezoelectric response. The assay was calibrated in the low picogram per liter range and provided a limit of detection of 0.55 pg/mL (2.37 pmol/mL) for a 25 µL sample. The analytical performance was sufficient for the determination of physiological melatonin concentrations in saliva and blood. The assay showed satisfactory selectivity against selected potential interferents and correlated strongly with a standard ELISA method, with a coefficient of determination of r2 = 0.994. Application to authentic human saliva samples confirmed the practical utility of the method, and the prepared biosensors remained stable for at least 140 days. The proposed QCM immunoassay represents a simple and sensitive alternative to conventional melatonin assays and demonstrates the potential of piezoelectric biosensing for non-invasive hormone monitoring.