Single-Walled Carbon Nanotube-Based Biosensors: Engineering Near-Infrared Fluorescent Nanosensors for Oxytocin Imaging in the Brain

Oxytocin is a nonapeptide that plays an essential role in regulating our social and reproductive behavior. Synthesized predominantly in the hypothalamus, oxytocin functions both peripherally as a peptide hormone and centrally as a neuropeptide. Its actions in the brain and body are distinct but complementary. When released throughout the brain, neuropeptide oxytocin regulates complex emotions and social behaviors such as social recognition, pair and maternal bonding, and stress mitigation. Oxytocin is also involved in the pathogenesis of various social disorders, including generalized social anxiety disorder and autism spectrum disorder. Treatment with oxytocin has been demonstrated to alleviate some of the social impairments associated with these disorders. Oxytocin has thus garnered interest as both a potential therapy and therapeutic target. While neuropeptide oxytocin is known to play a critical role in regulating our social lives, our understanding of its function remains incomplete because we lack the tools to directly probe oxytocin signaling at the spatiotemporal resolution requisite to elucidate oxytocinergic communication. The gold standard for oxytocin detection involves invasive sampling via microdialysis followed by a quantification assay such as ELISA. These methods have been used extensively to study oxytocin but suffer from poor spatial and temporal resolution and significant variability across sample processing and quantification method. Recently, an optogenetic platform was developed to enable labeling of oxytocin sensitive neurons. This platform lacks specificity against oxytocin’s structural analogue, vasopressin, and is transcription-dependent, and therefore cannot image oxytocin in real-time. Other OXTR-dependent imaging probes continue to face challenges related to vasopressin interference./nReal-time imaging platforms are required to fully characterize oxytocin function, and toward this end, we have developed two sensitive and reversible oxytocin nanosensors capable of imaging oxytocin in the brain. Both probes leverage the inherent tissue-transparent fluorescence of single-walled carbon nanotubes (SWCNT). SWCNT are biocompatible and photostable nanomaterials that can be functionalized with molecular recognition elements to develop sensors for targets of interest. As SWCNT photophysics is exciton-based, SWCNT fluorescence is modulated by the surrounding dielectric environment. We leveraged this dielectric sensitive to develop probes that increase in fluorescence upon interacting with oxytocin. In this dissertation, SWCNT, their optical properties, and their utility in bioimaging application are introduced. The development and validation of two SWCNT-based oxytocin imaging probes are then described. The first probe, a near-infrared oxytocin nanosensor (nIROx), is the first synthetic probe capable of imaging oxytocin in the brain without interference from its structural analogue, vasopressin. It is synthesized by covalently conjugating an oxytocin receptor peptide fragment (OXTp) to the SWCNT surface. To develop the second oxytocin imaging probe, we leveraged an evolution-based platform, SELEC, and identified a noncovalent nanosensor, nIROSE (near-infrared oxytocin nanosensor identified by SELEC). nIROSE nanosensors utilize an evolved ssDNA molecular recognition moiety noncovalently adsorbed to the surface of SWCNT. Preliminary imaging experiments in acute tissue slices suggest that nIROSE can reversibly image electrically stimulated oxytocin release in brains. nIROSE and nIROx are complementary tools with different limitations and advantages. Together, these imaging probes may enable real-time imaging of oxytocin signaling throughout the brain to fully characterize oxytocin’s function in both neurological health and disease.

Advisor: Landry, Markita P
Francis, Matthew B