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Access Type
WSU Access
Date of Award
January 2023
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Thomas Linz
Abstract
A myriad of biological targets, including oligonucleotides, proteins, and cell-derivedvesicles are utilized to diagnose disease. Diagnosing diseases at early stages is most beneficial to a patient; however, conventional assays preclude detection of these ultralow-concentration analytes that are present at early stages of a disease. This leads to many diseases not being diagnosed until they are in later stages, when a patient has begun exhibiting symptoms. Capturing and detecting biological analytes of interest at ultralow concentrations allows for earlier detection and can greatly improve a patient’s outcome and quality of life. The polymerase chain reaction (PCR) is a commonly used method to amplify oligonucleotides of interest, which can then be detected via a fluorescent dye. Digital PCR (dPCR) is a method by which these oligonucleotide targets are sequestered into thousands of individual reaction chambers, rather than amplified within a single bulk aliquot. This technique analyzes each individual reaction well for fluorescent signal to determine whether the well contains either zero or one analyte molecules. dPCR greatly improves the limit of detection of analytes because it counts single molecules, and it can be combined with antibodies to analyze proteins of interest as well. Our work has developed a microchamber-based dPCR platform that can reliably sequester, amplify, and detect oligonucleotides at a single molecule level. We have further expanded this system to capture cytokine proteins sampled directly from an exhaled breath condensate (EBC) matrix. Finally, we have applied this work towards digital quantitative PCR (dqPCR) to analyze DNA-loaded liposomes within the microwell array. Our development of an analysis platform with single molecule sensitivity shows considerable promise towards improving disease diagnostics.
Recommended Citation
Mccarthy Riley, Bailey, "Microchamber-Based Digital Pcr For Single Target Analysis Of Biological Analytes" (2023). Wayne State University Dissertations. 3803.
https://digitalcommons.wayne.edu/oa_dissertations/3803