Single-Step Electrochemical Aptasensor for Melatonin Detection in Unprocessed Plasma

Melatonin is a clinically relevant biomarker for diagnosing circadian rhythm sleep disorders (CRSDs) and monitoring circadian disruption associated with cancer, metabolic and cardiovascular disease, and psychiatric disorders. This work reports the implementation of an electrochemical aptamer-based biosensor (E-aptasensor) for direct reagentless detection of melatonin within physiological levels. The E-aptasensor employs an optimized surface population of the aptamers in a conformational change-based assay to detect melatonin over a broad dynamic range, including the picomolar physiological range. The behavior of the aptameric assay was validated both in bulk solution and in silico using fluorescence resonance energy transfer (FRET) and molecular docking (MD). The E-aptasensor achieves sensitive and selective melatonin detection, with a limit of detection (LOD) of 0.75 pM and strong specificity against a panel of relevant interferents. Fitting the electrochemical responses to the Hill-Langmuir (HL) binding model yielded an apparent electrochemical dissociation constant (K_{d, app}) of 37 pM. Notably, the E-aptasensor showed promising performance in unextracted human plasma, avoiding the extraction step typically required for immunoassays. These features support future development of rapid melatonin profiling for CRSD diagnosis and monitoring, as well as future point-of-care implementation. The single-step E-aptasensor offers a promising, faster, and simpler complement to gold-standard techniques.