The Development of a Nucleic Acid-Directed Switch and Proximity Dependent Protein Assays using Electrochemical Techniques

Abstract

The analysis of nucleic acids (NA), has increased demand for point-of-care systems, such as rapid preventive health care, forensic applications, etc. Thus inspired, a vast amount of biosensor platforms for the detection of nucleic acids. These platforms are typically recognized via optical readouts such as fluorescence, molecular beacons, polymerase chain reaction (PCR), colorimetric analysis, and more. Although these methods are sensitive and effective, the instrumental availability and reagent consumption can make the overall experience challenging. Electrochemical sensors have received considerable attention due to their rapid response, affordability, minute structure, and remarkable sensitivity. This dissertation focuses on the development of DNA-based electrochemical methods toward an effective drop-and-read quantification of various biomarkers, such as small DNA, RNA, and proteins. Chapter 1 introduces background research, focusing on diabetes and obesity, functional tissue (adipose tissue) and miRNA, immunoassay development using both tube assay methodologies and conventional electrochemical DNA-based biomarker detection. In chapter 2 we take advantage of important concepts such as the proximity effect to develop 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-based immunoassay, which utilizes polymerase chain reaction (PCR) to amplify the overall signal. With this assay, we gained insight into the complexity and significance of adiponectin. Chapter 3 led us on a different path, to utilize electrochemical detection methods to detect PLA products as 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). This focused on the understanding of surface hybridization, and its effects on electrochemical kinetics which offered a different outlook on interpreting SWV signal and the efficiency of SWV frequency, which is useful for any electrochemical DNA-based sensor. In chapter 4, we designed an electrochemical nucleic acid quantification method labeled as the electrochemical bistable switch sensor (E-BSS). This novel assay is based on a nucleic acid hybridized-driven system that incorporates strand displacement mechanisms for short oligonucleotides (~22 nt). DNA and RNA, specifically miRNA was successfully detected and quantified in complex matrices. In addition, to reduce sample volume (2 µL), we used polydimethylsiloxane (PDMS) electrochemical cells fabricated from 3D-printed polylactic acid molds. 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. Chapter 5 reintroduces the electrochemical proximity assay (ECPA) which is a sensitive protein detection method developed in Dr. Shannon and Dr. Easley’s’ lab. We have completed studies that can support the development and quantification of biologically relevant molecules. We discuss the importance of ECPA sensing complex which is currently made of six components undergoing non-covalent binding (DNA hybridization and antigen-antibody interactions). The main focus of this chapter is to reduce the current of the background signal and to reduce the components of the system to a 4-part complex. This, in turn, should create a signal-OFF based assay that is proportional to the target concentration due to the molecular weight change.