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" Development of Chemical Patterning: "
CAO, HUAN
Andrews, Anne M.Weiss, Paul S.
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Latin Dissertation
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English
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898638
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TL5xg747g1
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Tan, Zheng
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Development of Chemical Patterning:\ CAO, HUANAndrews, Anne M.Weiss, Paul S.
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2015
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2015
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| Abstract
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G-Protein-coupled receptors (GPCRs) embedded in native neuronal membranes transduce interneuronal signals via molecular recognition of small-molecule neurotransmitters. Alterations in chemical communication pathways involving these molecules have been associated with the causes and treatments of neurological and neuropsychiatric disorders. As a result, GPCR-ligand interactions have been extensively investigated. However, conventional radioligand binding methods for interrogating these interactions suffer from laborious protocols needed to label each ligand, as well as cost and safety concerns associated with working with radioactivity. Moreover, traditional methods are not amenable to multiplexing. To address these challenges, we investigated self-assembled monolayers (SAMs) as a means to tether small-molecule neurotransmitters to solid substrates for capturing biomolecules, including GPCRs, from solution. Bovine serum albumin was used to reduce nonspecific biomolecule-substrate binding; thus improving biomolecule-probe recognition. We developed and advanced new patterning strategies to interrogate relative binding of biomolecules to ligand-functionalized vs. unfunctionalized regions and to enable multiplexing on bioactive substrates. Microfluidics was utilized to generate multiplexed substrates by spatially addressing different small-molecule probes to individual channels. The resulting arrays were used to capture and to sort antibodies and GPCRs from complex mixtures according to ligand affinities. We invented chemical lift-off lithography to achieve highly precise biomolecule patterning with sub-30 nm feature sizes. The key step here relies on covalent interactions at stamp/substrate interfaces to enable molecule removal upon stamp release. We discovered that chemical lift-off generated a new class of surface defects for molecular insertion. By varying the pre-lift-off SAM compositions, we controlled surface densities and hybridization of inserted thiolated DNAs and improved target hybridization compared to the conventional backfilling method. We improved surface functionalization strategies by investigating ligand conjugation to surface tethers under two conditions – pre-assembly vs. post-assembly. We found the former showed consistent and improved recognition of antibodies compared to the latter. Our next proximal goals will be to use the bioactive substrates developed here to identify high-affinity synthetic neurotransmitter receptors by screening nucleic acid combinatorial libraries. Once selected, these receptors will be used as molecular recognition elements in bioelectronic nanosensors to enable in vivo neurotransmitter sensing.
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CAO, HUAN
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UCLA
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