Real Time Analysis Of Biomolecular Interactions

Author : Amrita Yadav
Publisher :
ISBN 13 :
Total Pages : 215 pages
Book Rating : 4.:/5 (894 download)

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Download or read book Development of Arrayed Imaging Reflectometry as a Biomolecular Interaction Analysis Platform written by Amrita Yadav and published by . This book was released on 2012 with total page 215 pages. Available in PDF, EPUB and Kindle. Book excerpt: "The characterization of biomolecular interactions is an important step towards understanding the basic biochemical processes that govern cellular function, the aberrations that cause disease and potential therapeutic candidates. The goal of my thesis research has been to develop Arrayed Imaging Reflectometry (AIR) as a technique to characterize biomolecular interactions in terms of their binding affinity and binding kinetics. AIR is an interferometric biosensing technique that relies on the creation of a total destructive interference condition on a silicon chip coated with a thin layer of probe-functionalized silicon dioxide. Specific binding of target molecules results in an increase in the local thickness of the probe layer and a concomitant destruction of this total destructive interference condition, resulting in an increased reflectance that corresponds to the amount of bound target. Initial studies towards this goal focused on the optimization of surface functionalization chemistry for AIR experiments. Next, experiments were carried out to confirm the quantitative performance of AIR across a range of probe-analyte affinities. We combined biomolecular binding models with data obtained from spectroscopic ellipsometry and surface plasmon resonance experiments to model AIR response for two probe-analyte pairs and compared it to experimental observations. This study confirmed that AIR response is a predictable function of probe and analyte properties and thus could be used to determine binding parameters for biomolecular interactions. A modified substrate consisting of a silicon nitride/ silicon dioxide bilayer on a silicon chip was introduced to enable AIR detection under aqueous media. The thickness sensitivity of this substrate was experimentally determined to be 1 Å. We used the new substrate to measure the binding kinetics of Muscleblind-like 1 (MBNL1), a splicing regulator protein that is implicated in the pathology of the Myotonic Dystrophies and Huntington Disease, to several of its 'normal' and 'pathogenic' RNA targets microarrayed on an AIR chip. The binding kinetics of 'normal' and 'pathogenic' RNA sequences were observed to be distinctly different even though their steady state binding constants were similar. These results suggest that AIR can be a powerful technique for the label-free real-time analysis of biomolecular interactions in a high-throughput format"--Pages viii-ix.