This Is AuburnElectronic Theses and Dissertations

Development of Nucleic Acid Driven Peptide and Protein Sensors and Their Integration with Automated Microfluidics




Khuda, Niamat E

Type of Degree

PhD Dissertation


Chemistry and Biochemistry


Accurate detection of disease-associated biomolecules is one of the most critical steps in diagnostics in the early stages of disease to provide appropriate health care and effective treatment to patients. In clinical laboratories, analysis of disease biomarkers is usually performed by standard detection methods such as Western blots and enzyme-linked immunosorbent assays (ELISA) which are expensive, laborious, and require longer time to obtain medical information. Over the years, many sensitive methods have been developed for biomarker detection aimed at point-of-care (POC) systems. These methods are specialized toward particular targets and inflexible to analyze other biomolecules of interest. However, more versatile, yet sensitive, inexpensive, and easy to use methods are required for POC settings. Nucleic acid-based electrochemical sensors have proven to be ideal for this purpose as they are able to provide rapid response, can function in complex biological environments, are compatible with miniaturized devices, and are relatively inexpensive. This dissertation focuses on the development of DNA-based electrochemical sensors and their integration with automated microfluidics for quantification of biomolecules, namely small molecules, oligonucleotides, proteins, and peptides. Chapter 1 discusses the recent developments in electrochemical DNA-based sensors and their application in clinically relevant biomolecule detection. Chapter 2 presents a detailed study of surface hybridization kinetics. Specifically, we have explored the dependence of electrochemical signals on the relative distance of DNA binding sites from the electrode surface under different conditions and investigated the kinetics of toehold-mediated DNA strand displacement reactions near or far from the surface. Chapter 3 describes the development of electrochemical biosensor system for the quantification of a novel analyte for electrochemistry, exendin-4, which is a widely prescribed diabetic drug. We applied our DNA nanostructure sensor architecture to directly quantify exendin-4, which has not previously been measured using direct electrochemical readout, and we show that the sensor is functional in human serum. Chapter 4 focuses on strategies to enhance signal in the nanostructure-based sensor. We rationally designed two new nanostructure architectures by incorporating polyethylene glycol (PEG) and uracil (U) into selected probe strands to support molecular weight reduction, which we hypothesized would enhance the electrochemical signal and sensitivity of the sensors. Chapter 5 highlights the integration of automated microfluidics with electrochemical detection system. We have utilized automated, valve-controlled microfluidics for electrode preparation and analyte detection in a DNA-based surface assay platform using square wave voltammetry (SWV). Finally, Chapter 6 reviews the research contribution of this dissertation and provides insight into future research stemming from these topics.