Advancing Technologies for Precision Medicine: From Liquid Biopsy to Single-Molecule DNA Sequencing

Precision oncology depends on technologies capable of identifying tumor-associated mutations and monitoring how they evolve during therapy using minimally invasive samples. This dissertation integrates blood-based liquid biopsy assays with an emerging single-molecule nanopore sequencing platform to address limitations associated with conventional tissue biopsies and DNA sequencing technologies in cancer care. First, we demonstrated the potential of circulating tumor cells (CTCs) as a minimally invasive biomarker for identifying mutations and monitoring treatment response in patients with metastatic pancreatic ductal adenocarcinoma (PDAC). Using antibody-functionalized microfluidic devices, two orthogonal CTC subpopulations, epithelial cells expressing epithelial cell adhesion molecule (EpCAM) and mesenchymal cells expressing fibroblast activation protein α (FAPα), were isolated directly from whole blood. Longitudinal monitoring of these CTC subpopulations showed that the ratio of mesenchymal to epithelial CTCs provided an early indicator of treatment response, showing strong agreement with the computed tomography (CT) imaging compared to the commonly used serum biomarker carbohydrate antigen 19-9 (CA19-9). Genomic DNA extracted from the isolated CTCs was subjected to whole-genome amplification followed by next-generation sequencing to identify mutations in DNA damage repair genes, while KRAS (Kristen rat sarcoma) mutations were detected using a ligase detection reaction assay. These analyses demonstrated that CTC-derived DNA can capture the mutational profile of the primary tumor and enable the detection of variants in the bloodstream, allowing the tracking of mutations gained or lost during therapy. Given the limitations associated with these sequencing technologies, particularly because of the amplification requirements, we investigated a thermoplastic exonuclease time-of-flight (XToF) nanopore sensor for single-molecule DNA sequencing using resistive pulse sensing (RPS). This nanopore sensor requires a highly processive exonuclease enzyme to sequentially cleave nucleotides for detection. Therefore, enzymatic activity of lambda exonuclease (λ-exo) was characterized in both solution and solid phase to determine the reaction conditions compatible with nanopore detection. Our results indicate that λ-exo digestion efficiency decreases at high salt concentrations (>0.125 M); however, an increased stability is observed for λ-exo under higher ionic strengths when it is surface-immobilized compared to the solution phase. Additionally, key enzymatic properties of λ-exo, including processivity, substrate specificity, end specificity, and clipping rate, were investigated. λ-exo was found to be highly processive (>48.5 kbp), indicating continuous nucleotide cleavage once the enzyme binds to the DNA, which is important for maintaining sequence order during exonuclease-based sequencing. λ-exo also showed the highest digestion efficiency for 5’-phosphorylated recess DNA substrates. Beyond sequencing applications, we demonstrated that the XToF sensor can function as a label-free enzymology tool capable of measuring single-molecule enzyme kinetics, including association constants and clipping rates. Overall, this work shows the potential for integrating liquid biopsy biomarkers with single-molecule sequencing technologies in an automated “blood-to-sequencing” platform that combines CTC isolation, on-chip DNA extraction, exonuclease-based sequencing, and real-time nanopore detection. Such platforms can enable noninvasive genomic and epigenomic profiling for precision medicine.

Name thesis:degree_name

Ph.D.

Discipline thesis:degree_discipline

Chemistry

Grantor dc:publisher

University of Kansas

Year dc:date.issued

2026

Author dc:creator

• Fateru, Oluwadamilola Olasumbo

Advisor dc:contributor.advisor

• Soper, Steven A

• Cancer
• DNA Sequencing
• Lambda Exonuclease
• Microfluidics
• Nanopore
• Precision Medicine

Language dc:language.iso

en

Dc Identifier Other

https://www.proquest.com/LegacyDocView/DISSNUM/32697298

OAI identifier oai:identifier

oai:kuscholarworks.ku.edu:1808/39489