Aptamer-Based Detection of Salivary Melatonin for Circadian Rhythm Sleep Disorders Monitoring

Circadian rhythms sleep disorders are linked to the disruption of daily levels of circulating melatonin. Current circadian rhythm sleep disorder diagnostic approaches are typically based on questionnaires (which lack objectivity), and/or on dim light melatonin onset tests (which are costly, cumbersome, and lack sensitivity), thereby leaving a lot to be desired. Salivary melatonin profiling can serve as a cost-effective and convenient method for diagnosing circadian rhythm sleep disorders, but there is still a challenge in measuring ultralow levels of hourly salivary melatonin secretion in real-time over 24 hours periods. Current approaches for detecting melatonin involve antibody-based assays. However, adapting antibodies for modification of their affinities is challenging as the concentration of melatonin in saliva can vary significantly, especially in patients with circadian rhythm disorders. Aptamers, due to their numerous advantages such as small size and structural flexibility in tuning affinities, are preferable biorecognition molecules and can provide detection of melatonin with high sensitivity and specificity. The thesis aims to study the aptamers' ability to sense salivary melatonin and further develop aptamer-based real-time detection technique for salivary melatonin measurement with high sensitivity and specificity, which could allow accurate assessment of dim light melatonin onset in individuals with circadian rhythm sleep disorders. To accomplish this aim, three specific objectives are set to pursue. The first objective was to study the structural property and binding mechanism of a single-stranded DNA aptamer with melatonin under different ionic strengths using circular dichroism spectroscopy and microscale thermophoresis respectively. Different versions of the untagged aptamer i.e., full, and truncated sequence structure were studied under varying buffer and temperature conditions, while Cy5 labeled aptamers were used to determine the aptamers binding affinity to melatonin in buffer and human saliva sample. The circular dichroism data displayed a hairpin loop structure of aptamers where the stem plays a significant role in aptamer stability and melatonin binding with an induced-fit binding mechanism. Also, the aptamers demonstrated a high binding affinity in human saliva samples. The understanding of the conformational changes of the melatonin aptamers (full and truncated sequence) on target binding and under different physiological conditions will allow us to select aptamer with high affinity as it influences assay sensitivity and in establishing the design parameters for rapid detection of melatonin. Based on these conditions, the second objective was to develop a competitive enzyme-linked aptamer-based immunoassay as a proof-of-concept, where the optimized concentration of competitor melatonin-ovalbumin was coated on the 96-well plate using sodium carbonate and bicarbonate coating buffer. The free melatonin prepared in binding buffer containing salts along with biotin-tagged aptamer was added to the plate, and then melatonin competes with melatonin-ovalbumin to bind with aptamer. The absorbance signal achieved at 450 nm after the binding was inversely proportional to the melatonin concentration in the saliva samples. The aptamer-based immunoassay developed showed the capability of aptamers to detect salivary melatonin with high sensitivity and specificity in comparison to antibodies which are commonly used for assessing melatonin in saliva. Further, for rapid detection of melatonin in saliva, the third objective was to develop a colorimetric assay using aptamers immobilized on gold nanoparticles by electrostatic interaction. Different conditions such as aptamer concentration, salt concentration, and incubation time were optimized for the assay. The aptamer-gold nanoparticle probe was tested with melatonin in buffer, artificial saliva, and human saliva sample. In the presence of melatonin, melatonin binds with aptamer leading to the stabilization of aptamer structure, resulting in aptamer desorption from the gold nanoparticle surface. This allows a change in solution color from red to blue on the aggregation of gold nanoparticles in the presence of the salivary melatonin prepared in the ionic condition required for aptamer structural change. This color change can be observed with the naked eye and quantified using UV-vis spectroscopy without any use of sophisticated equipment. This thesis presents a comprehensive study of aptamer as a promising biorecognition molecule for measuring melatonin in saliva samples with high sensitivity and specificity. The methods developed showed the complete analysis of aptamers and their binding capability to melatonin under various conditions. This will pave the way to consider aptamers as an alternative to antibodies. Moreover, the rapid detection mechanism developed opens a scope for aptamer-gold nanoparticle integration into the point-of-care device which would be beneficial in measuring salivary melatonin at multiple time points for timely assessment of dim light melatonin onset crucial for diagnosing circadian rhythm disruption in individuals with sleep disorders and beyond.

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biomedical Engineering

Grantor

University of Saskatchewan

Year dc:date.issued

2023

Author dc:creator

• Pundir, Meenakshi

Advisors dc:contributor.advisor

• Papagerakis, Petros
• Chen, Xiongbiao (Daniel)
• Papagerakis, Silvana

Committee members dc:contributor.committeemember

• McWalter, Emily
• Eskiw, Chris
• Zhang, Chris
• Tachibiglou, Changiz
• Cerruti, Marta

• Circadian rhythm sleep disorders
• Saliva
• Melatonin
• Aptamers
• Circular Dichroism, Microscale thermophoresis
• Enzyme-linked aptamer based assay
• Gold nanoparticles

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10388/14551

OAI identifier oai:identifier

oai:harvest.usask.ca:10388/14551