Competitive enzyme linked aptamer based assay for salivary melatonin detection

Melatonin is a key hormone that regulates the sleep-wake cycle and plays an important role in maintaining circadian rhythm and sleep onset. The daily rise in melatonin secretion is associated with an increased tendency to sleep, occurring approximately 2 h before bedtime. This correlation between melatonin levels and sleep onset makes it a reliable biomarker for circadian rhythm sleep-wake disorders. An accurate assessment of dim light melatonin onset (DLMO) is vital for understanding circadian timing and diagnosing sleep-wake cycle disruptions. However, the traditional methods for detecting melatonin in saliva are either complex or lack the sensitivity required for the accurate assessment of DLMO, especially at low concentrations. Here, we present a novel competitive enzyme-linked aptamer-based assay developed to detect melatonin in saliva. Unlike conventional assays, this technique utilizes chemically synthesized single-stranded DNA or RNA aptamers, which bind to melatonin with high specificity and sensitivity. The assay measures melatonin, attaining a linear dynamic range from 8.62 × 10^‒6 M to 3.9 × 10^‒11 M, with a detection limit of 2.5 × 10^‒12 M (~ 0.57 pg/mL). Additionally, the aptamer showed small binding to its counter targets and acceptable recovery of melatonin when spiked in four times diluted saliva in assay buffer. Overall, the assay portrayed the potential of aptamers to detect low melatonin levels in saliva that could be beneficial in accurately determining DLMO, particularly in individuals with very low melatonin levels, such as the elderly or those with neurodegenerative conditions. Determining precise measurement of DLMO will facilitate the accurate diagnosis of circadian rhythm disruption, enabling healthcare providers to optimize the timing and selection of therapeutic and behavioural interventions tailored to an individual's unique circadian rhythm.