Abstract Introduction Melatonin is the principal hormonal marker of circadian phase, yet its clinical use is constrained by laboratory assays that require specialized equipment, slow processing, and limited sampling frequency. As circadian rhythm disorders increasingly require physiologic biomarker monitoring, rapid and minimally invasive melatonin measurement could broaden access to circadian assessment. This study evaluates an aptamer-based biosensing approach capable of detecting extremely low salivary melatonin concentrations and explores its translational potential for clinical workflows. Methods We employed a silicon-nanowire field-effect transistor (SiNW-FET) platform functionalized with a melatonin-specific aptamer. To improve probe orientation and target capture, a freeze–thaw assembly strategy was implemented to generate highly uniform aptamer monolayers. Analytical performance was assessed using graded melatonin standards spanning physiological concentrations. To examine feasibility in clinical samples, untreated saliva from patients with sleep-wake rhythm disorders was collected at multiple time points and tested using the biosensor. Measurements were compared with mass-spectrometry values to assess agreement. Results The optimized aptamer assembly produced reproducible and stable sensor performance with high sensitivity across a physiologic concentration range, detecting melatonin at extremely low physiologic concentrations. The biosensor differentiated time-dependent melatonin changes in untreated saliva, demonstrating clear nocturnal rises and daytime nadirs consistent with expected circadian physiology. Signal trends closely matched those obtained from mass spectrometry, supporting analytical validity. Importantly, detection required less than two minutes per sample and no preprocessing, representing a substantial improvement in workflow efficiency. Specificity testing showed minimal cross-reactivity with common salivary proteins, indicating suitability for complex biological matrices. Together, these findings suggest that rapid point-of-care melatonin assessment may be feasible using this platform. Conclusion This aptamer-based biosensor enables ultrasensitive, rapid, and non-invasive salivary melatonin detection, providing a promising tool for clinical circadian evaluation. By supporting near-real-time biomarker monitoring without laboratory processing, the platform may complement traditional assays in assessing circadian phase, treatment response, and rhythm phenotype. Its portability and minimal sample needs highlight potential integration into sleep-medicine clinics and, ultimately, home-based circadian assessment. Support (if any) National Natural Science Foundation of China (Grant No. 52172158); Guangdong Basic and Applied Basic Research Foundation (Grant No. 2022A1515110327); Beijing Nova Program (Grant No. 20230484408); Key-Area Research and Development Program of Guangdong (Grant No. 2021B0909060001).
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