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Studies Toward Measuring Relative Binding Constants for a Ligand-Receptor Interaction with an Electrochemically Addressable Microelectrode Array
Date of Award
Doctor of Philosophy (PhD)
Small molecule microarrays are powerful tools for high-throughput screening of compound libraries. Binding events are typically detected by fluorescence, using a secondary labeled antibody. Several washing steps are needed and information about weak interactions is often lost.
Our group is using microelectrode arrays as a platform is for constructing and screening small molecule arrays. In this approach, the small molecules are located proximal to microelectrodes, which can be used to detect a ligand-receptor interaction in “real-time” by means of impedance. When a protein binds a ligand on the array, it sterically prevents a redox molecule added to the solution above the array from reaching the electrode surface. This is seen as a reduction in current in a cyclic voltammogram.
The impedance experiment identifies a binding event, but it does not give information about the strength of binding. To do this, a molecular library with members having known binding constants to the receptor needs to be attached to the array. The strength of binding between the receptor and a new ligand can them be found by measuring relative binding constants. This work makes progress towards this goal by developing a strategy for moving prebuilt libraries to the array, establishing a synthetic route for a small peptide library, and probing the utility of the arrays for measuring relative binding constants.
Four methods of attachment were explored, looking for a method that provided a stable linkage with minimal reaction steps. The best approach was to use a thiol nucleophile to do a hetero-Michael reaction with an acrylate-functionalized array. This method requires three steps: attachment of acrylate, addition of ligand, and capping of unreacted acrylate. One library member can be attached in 90 minutes.
Two RGD-containing peptides with known inhibition constants to integrin αVβ3 and one peptide with low affinity to the integrin were identified. A synthetic route to each fully protected peptide was established.
Three ligands for avidin were attached to a sucrose-coated microelectrode array and an impedance experiment to measure relative binding constants was attempted. However, avidin showed strong nonspecific binding to the surface at the concentrations of avidin studied, so information about the ligands could not be obtained. However, this experiment shows that the impedance is sensitive to receptor concentration and supports the idea that relative binding constants can be measured via impedance, with proper optimization of the surface to reduce non-specific protein binding.
Chair and Committee
Kevin D Moeller
Joshua A. Maurer, John-Stephen A. Taylor, Donald L. Elbert, Garland R. Marshall, Liviu M. Mirica
Fellet, Melissae Stuart, "Studies Toward Measuring Relative Binding Constants for a Ligand-Receptor Interaction with an Electrochemically Addressable Microelectrode Array" (2010). Arts & Sciences Electronic Theses and Dissertations. 168.