Towards a Bistable Nucleic Acid-Directed Switch for Electrochemical Readout of Proximity Ligation Assays

Abstract

The work presented in this thesis is focused on the development of a bistable DNA-driven assay formatted for biosensor applications in biological research, medical research, and an approach to point-of-care testing. We have taken advantage of important concepts such as the proximity effect to develop a proximity ligation assay (PLA) for the adipose-secreted protein, adiponectin; this protein is an important indicator for diabetes, obesity, and metabolic syndrome. PLA is a DNA-driven immunoassay, which utilizes polymerase chain reaction (PCR) to amplify overall signal. With this assay, we gained insight to the complexity of detecting adiponectin multimers. On the path to design electrochemical detection methods to detect PLA products with a non-optical method, we conducted an important study to evaluate the distance dependence of the placement of our redox-tagged DNA strand with respect to the electrode surface via square wave voltammetry (SWV). Finally, we designed a bistable switch that is based on a DNA hybridized-driven system that incorporates strand displacement reactions at the electrode surface. This electrochemical system paves the way for future proximity-driven protein assays that could potentially take advantage of our bistable switch-based strand displacement reactions.