Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Investigating protein location and concentration is critical to understanding function. Reagentless biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can detect analytes of interest with high temporal and spatial resolution. However, because these biosensors require laborious empirical screening to develop, their adoption has been limited. Hence, we establish design principles that will facilitate development. In this thesis, we first develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. Following these guidelines, we use the human tenth type III fibronectin domain to engineer new binders against several ligands of the EGFR receptor. Using these binders and others, we design and characterize biosensors based on various target analytes, scaffolds and fluorophores. We observe that analytes can harbor specific binding pockets for the fluorophore, which sharply increase the fluorescence produced upon binding. Furthermore, we demonstrate that a fluorophore conjugated to locally rigid surfaces possesses lower background fluorescence. Based on these newly identified properties, we design biosensors that produce a 100-fold increase in fluorescence upon binding to analyte, about a 10-fold improvement over the previous best biosensor. In order to improve the methodology of reagentless biosensor design, we establish a method for site-specific labeling of proteins displayed on the surface of yeasts. This procedure allows for the screening of libraries of sensors for binding and fluorescence enhancement simultaneously. Finally, we explore an alternative sensor design, based on competitive inhibition of fluorescence quenching. / by Seymour de Picciotto. / Ph. D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/99053 |
| Date | January 2015 |
| Creators | De Picciotto, Seymour |
| Contributors | K. Dane Wittrup and Linda G. Griffith., Massachusetts Institute of Technology. Department of Biological Engineering., Massachusetts Institute of Technology. Department of Biological Engineering. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 136 pages, application/pdf |
| Rights | MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission., http://dspace.mit.edu/handle/1721.1/7582 |
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