Using the technology afforded by Winschel et al., cyclen-1, a high affinity, strong complexation agent for 8-hydroxypyrene-1,3,6 trisulfonate and derivatives, a new assay has been developed for fluorescently labeling proteins of interest (POIs). Ligation of the endogenous ligand for nicotinic acetylcholine receptors (nAChRs), acetylcholine, using click chemistry afforded the triazole derivative of an alkynyl-acylcholine (compound 1) with 8-azidopyrene-1,3,6 trisulfonate (compound 2). Liposomes encapsulated with Rhodamine B were used to strengthen the initial fluorophore response of compound 2, using an anchored form of cyclen-1 complex. Using a palmitoyl tail as the lipophilic moiety for liposomal amplification, the subsequent response has a fluorophore ratio of up to 1:1 million, compound 2:Rhodamine B molecules. in vitro assay using compound 2 and cyclen-1 anchored liposomes with HEK-293 cells produced a positive binding response, allowing brightly colored fluorescent images of nAChRs upon the cellular membrane. A control for nAChR binding was performed using a co-culture of HEK-293 and endothelial cell lines. Control experiments show compound 2 and liposomes weak binding endothelial cells, however, this could be do to accumulation from another mechanism, more work is necessary to prove whether or not this is correct.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-4876 |
| Date | 01 January 2015 |
| Creators | Pagan, Augustine J, IV |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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