Date of Award

12-1-2023

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Dissertation/Thesis Chair

Mehmet V. Yigit

Committee Members

Maksim Royzen, Igor K. Lednev, Bijan K. Dey

Subject Categories

Biochemistry

Abstract

The ever-growing list of carcinogens, toxicants, pathogens, and other disease-causing agents necessitates the development of new and improved mechanisms of disease detection. A better understanding of events leading up to the diseased state and early diagnosis is imperative for the timely management and targeted treatment of diseases affecting the biological world. Recent achievements in DNA nanotechnology have led to the prominence of DNA-based technologies for multiplexed biological sensing, high-throughput analysis, and improved diagnostics. Typical methods of disease detection employ complex multi-step procedures and advanced equipment, which significantly reduces their accessibility while elevating their cost. DNA functionalized sensing systems that examine the molecular level changes taking place during disease progression thus serve as good alternatives for diagnostic tests and biomarker detection. Here, we have developed three distinct methods for pathogen and disease detection. The first method utilizes a cost-efficient gel electrophoresis-based approach capable of detecting as little as 1.5 femtomoles of two conserved viral DNA sequences – tobacco curly shoot virus (TCSV) and hepatitis B virus (HBV). The second method is a three-tiered isothermal diagnostic called ‘recombinase amplified CRISPR enhanced chain reaction’, abbreviated as ‘RACECAR’, which enables whole-genome detection and can quantify up to 40 copies of the genome using a fluorescence signal output. Notably, RACECAR can directly detect viral genomes in serum samples without the need for an extraction step. Finally, the third project involves the development of a paper-based sensor for colorimetric and visual detection of Salmonella. This paper diagnostic incorporates principles of synthetic biology by employing toehold switch riboregulators as the signal transducers for cell-free protein translation on paper, along with CRISPR-Cas12a for modular recognition of target nucleic acids.

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