Date of Award
Doctor of Philosophy (PhD)
Committee Member 1
Jong H. Choi
Committee Member 2
Galen B. King
Committee Member 3
Detection of cancer markers such as protein biomolecules and cancer cells in bodily fluids is of great importance in early diagnosis, prognosis as well as evaluation of therapy efficacy. Numerous devices have been developed for detecting either cellular or molecular targets, however there has not yet been a system that can simultaneously detect both cellular and molecular targets effectively. Molecule and cell-based assays are important because each type of target can tell a different story about the state of the disease and the two types of information can potentially be combined and/or compared for more accurate biological or clinical assessments. Therefore, the primary goal of the thesis is to develop a system that can simultaneously measure both cellular and molecular biomarkers from one and the same sample. With its high sensitivity and high-throughput capability, this system can capture rare cells such as circulating tumor cells (CTCs) from human bodily fluids (e.g. blood, ascites). Moreover, the system enables post-detection analysis of captured targets and can be compatible with established screening tools. In this thesis two generations of the system have been developed to achieve these goals.
The first-generation system is based on a single-layer fluidic chamber. Free magnetic beads conjugated with antibodies against a specific antigen are used to isolate both free molecules and whole cells overexpressing an antigen. The captured cells and molecules are quantitatively analyzed together on the same device surface using fluorescent microscopy. The system was first numerically modeled, and then experimentally characterized by simultaneously detecting free folate receptor (FR), and an FR+ cancer cell line (KB) that were added into cell culture medium with known number. This system was further validated by detecting KB cells and FR spiked into healthy human blood to simulate detection of CTCs and protein biomarkers present in cancer patient blood. The potential of this approach in clinical diagnostics was also demonstrated by detecting both FR+ cells and FR in an ascites sample obtained from an ovarian cancer patient.
The second-generation system employs a similar detection strategy but integrates a micro-aperture chip into the fluidic chamber to sort cells and molecules (including free beads) into different layers based on their sizes, which significantly reduces mutual interference and improves detection efficiency including detection yield and repeatability. Moreover, large number of beads can be used to further increase cell detection yield. The system was first characterized by detecting rare cells spiked in both cell culture medium and health human blood and applied for CTC detection from cancer patients’ blood samples. Then the system was further developed for separation and simultaneous detection of both model molecular and cellular prostate cancer markers (namely the prostate-specific membrane antigen (PSMA), LNCaP cells) from both culture medium and blood.
Finally, a post-detection application was demonstrated by culturing the cells that were detected and retrieved by our second-generation system. Future work will be focused on gene sequencing of captured rare cells, screening of cancer patient blood samples for dual detection of molecules as well as cells, and integration of novel capture ligands.
Huang, Wanfeng, "Concurrent detection and isolation of cellular and molecular biomarkers" (2016). Open Access Dissertations. 937.
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