Date of Award
Doctor of Philosophy
Robbyn K. Anand
Metastasis is responsible for approximately 90% of cancer related deaths. The key step in metastasis is the migration of cancer cells out of the primary tumor and into bloodstream. Once reaching at a distant site, a fraction of these circulating tumor cells (CTCs) invades foreign tissues for subsequent growth of tumors. However, conventional cancer treatments ignore the metastatic process, resulting in cancer relapse. Consequently, the isolation and characterization of CTCs are crucial in understanding how cancer spread by metastasis.
The enormous value of CTCs has not been completely realized because isolation of CTCs – the first inevitable step of overall analysis, is incredibly challenging due to their extreme rarity and varied physical and biological characteristics. Thus, separation techniques that exhibit the following features are critical: (i) They must provide a pure and representative sample of CTCs; (ii) Separation of individual CTCs are mandatory considering subpopulations can be easily obscured at the bulk scale; (iii) The sorting process is continuous and high-throughput since detection of a rare phenotype or cellular response requires analysis of thousands of individual cells; (iv) Captured single-cells should be readily interfaced with assays for downstream analysis. (v) Devices need to be cost-effective, accessible, and simple in manufacturing and operation for a wide range of applications.
Microfluidic lab-on-a-chip (LOC) technologies possess micron-scale dimensions and picoliter-to- nanoliter volume handling capacities, thereby facilitating manipulation and sampling of single cells. However, they often suffer from lack of selectivity, being over- or under-selective. Selection must happen prior to the isolation step for analysis of individual cells. Further, many LOC devices have difficulty in interfacing with assays, or complexity that hinders their applications. Thus, the development of fully integrated devices that offer simplicity in manufacturing and operation remains an important challenge.
Separation based on dielectrophoresis (DEP) exhibits less bias when compared with size- and antibody-based approaches, as it leverages the electrophysiological properties of CTCs. However, many of the current approaches to DEP suffer from low throughput and are not amenable to on-chip single-cell analysis. These limitations stem from design constraints such as the requirement that all electrodes must be connected via wire leads to the power source. Further, in DEP devices that employ insulating posts to shape the electric field, integration of these structures intended for cell capture with other features, such as chambers for on-chip analysis, is non-trivial.
The work presented in this document centers on the development of DEP devices at wireless bipolar electrode (BPE) arrays to addresses these concerns. First, DEP is employed to selectively capture and isolate CTCs in micropockets, while blood cells flow through the channels. The capture methodology used here eliminates massively screening of all cell populations one-by-one, the case in droplet separation, thus greatly increasing sorting efficiency. Further, high-fidelity single-cell capture could be readily achieved when the pocket dimensions matched to those of the cells. Second, leak channel design opens a flow circuit that enables valve-free transfer of individual isolated cells into reaction chambers, while split electrode design allows recapture and lysis of transferred cells for subsequent assay evaluation. Third, the use of arrays of wireless electrodes removes the requirement of ohmic contact to individual array elements, thus enabling device to be scalable along both x- and y- directions. This wireless electrode array not only provides a high-throughput module in cell capture, also in cell imaging and analysis. Finally, the use of ionic liquid as immiscible phase permits both
electrical lysis and fluid isolation. A key difference that distinguishes this from previous approaches is that DEP-based sorting, electrical lysis and analysis of single cells are integrated while retaining high-throughput and valve-free control. The simplicity of device manufacturing, the ease of its operation, and the potential for assay of live single cells or electric lysis for assay of cellular contents make the design broadly applicable for in-depth analysis of a variety of biological systems.
Li, Min, "Dielectrophoresis at wireless bipolar electrode arrays: Applications to the marker-free selection and detection of circulating tumor cells" (2018). Graduate Theses and Dissertations. 16841.