The research presented in this dissertation is the result of our laboratory's effort to develop a microfluidic platform to interrogate, manipulate, isolate, and enrich rare mammalian cells dispersed within heterogeneous populations. Relevant examples of these target cells are stem cells within a differentiated population, circulating tumor cells (CTCs) in the blood stream, and tumor initiating cells (TICs) in a population of benign cancer cells. The ability to isolate any of these rare cells types with high efficiency will directly lead to advances in tissue engineering, cancer detection, and individualized medicine. This work lead directly to the development of a new cell manipulation technique, termed contactless dielectrophoresis (cDEP). In this technique, cells are isolated from direct contact with metal electrodes by employing fluid electrode channels filled with a highly conductive media. Thin insulating barriers, approximately 20 μ­m, serve to isolate the fluid electrode channels from the low conductivity sample buffer. The insulating barriers in a fluid-electrical system create a number of unique and interesting challenges from an electrical engineering standpoint. Primarily, they block the flow of DC currents and create a non-constant frequency response which can confound experimental results attempting to characterize the electrical characteristics of cells. Due to these, and other, considerations, the use of high-voltage high-frequency signals are necessary to successfully manipulate cells. The effect of these high frequency fields on cells are studied and applied to microfluidic and tissue level applications. In later chapters, theoretical and experimental results show how high frequency and pulsed electric fields can ablate cells and tissue. Understanding the parameters necessary to electroporate cells aids in the development of cDEP devices where this phenomenon is undesirable and gives insight towards the development of new cancer ablation therapies where targeted cell death is sought after. This work shows that there exists a finite frequency spectrum over which cDEP devices can operate in which cells are minimally affected by the induced electric fields. Finally, lessons learned in the course of the development of cDEP were adapted and applied towards cancer ablation therapies where electroporation are favorable. It was found that bursts of high frequency pulsed electric fields can successfully kill cells and ablate tissue volumes through a process termed High Frequency Irreversible Electroporation (H-FIRE). This technique is advantageous as these waveforms mitigate or eliminate muscle contractions associated with traditional IRE technologies. Similarly, the use of fluid electrodes in cDEP inspired the use of an organs vascular system as the conductive pathway to deliver pulses. This novel approach allows for the ablation of large volumes of tissue without the use of puncturing electrodes. These techniques are currently undergoing evaluation in tissue engineering applications and pre-clinical evaluation in veterinary patients. / Ph. D.

Electrokinetics is a family of effects that induces motion of a liquid or a particle within a liquid in response to an external electric field. Using the intrinsic electrical properties of bacteria and of breast cancer cells, electrokinetics can be used to manipulate these particles for two different types of applications: tissue engineering and breast cancer detection. The first application studied the effects of electric fields on bacteria cells as well as calcium ions to potentially create a meniscus scaffold with hydroxyapatite ends for anchoring. In response to the electric field, calcium ions were able to deposit locally and simultaneously with cellulose growth. Bacteria cells were also studied to determine their response under an AC field. At low frequencies, bacteria demonstrated controlled movement caused by electroosmosis and dielectrophoresis with a net motion caused by a dielectrophoretic force. In the second application, the separation capabilities of different stages of breast cancer cells from the same cell line were tested using contactless dielectrophoretic (cDEP) devices. The electric field gradients in cDEP devices were altered to optimize selectivity and to determine an estimated membrane capacitance for each. From the results, the membrane capacitance of the early to intermediate stages proved to be very similar; however, late stage breast cancer cells have potential in being separated from early and intermediate stages. / Master of Science

Dielectrophoresis (DEP), an electrokinetic force, is the motion of a polarizable particle in a non-uniform electric field. Contactless DEP (cDEP) is a recently developed cell sorting and isolation technique that uses the DEP force by capacitavely coupling the electrodes across the channel. The cDEP platform sorts cells based on intrinsic biophysical properties, is inexpensive, maintains a sterile environment by using disposable chips, is a rapid process with minimal sample preparation, and allows for immediate downstream recovery. This platform is highly competitive compared to other cell sorting techniques and is one of the only platforms to sort cells based on phenotype, allowing for the isolation of unique cell populations not possible in other systems. The original purpose of this work was to determine differences in the bioelectrical fingerprint between several critical cancer types. Results demonstrate a difference between Tumor Initiating Cells, Multiple Drug Resistant Cells, and their bulk populations for experiments conducted on three prostate cancer cell lines and treated and untreated MOSE cells. However, three significant issues confounded these experiments and challenged the use of the cDEP platform. The purpose of this work then became the development of solutions to these barriers and presenting a more commercializable cDEP platform. An improved analysis script was first developed that performs whole cell detection and cell tracking with an accuracy of 93.5%. Second, a loading system for doing smart sample handling, specifically cell focusing, was developed using a new in-house system and validated. Experimental results validated the model and showed that cells were successfully focused into a tight band in the middle of the channel. Finally, a proof of concept for an on-chip micropump is presented and achieved 4.5% in-plane deformation. When bonded over a microchannel, fluid flow was induced and measured. These solutions present a stronger, more versatile cDEP platform and make for a more competitive commercial product. However, these solutions are not just limited to the cDEP platform and may be applicable to multitudes of other microfluidic devices and applications. / Master of Science

Microfluidics has found numerous applications ranging from the life sciences industries for pharmaceuticals and biomedicine (drug design, delivery and detection, diagnostic devices) to industrial applications of combinational synthesis (such as rapid analysis and high throughput screening). Among all these, one of the intriguing exploitation of microfluidics or micro total analysis systems (µTAS) is the separation of circulating tumor cells (CTCs) from body fluids. Cancer cells spread from the initial site of a tumor by first invading the surrounding tissue, then by entering the blood or lymph vessels, and finally by crossing the vessel wall to exit the vasculature into distal organs. The September 2006 issue of the Journal of the National Cancer Institute (NCI) states: "The war on cancer was declared 40 years ago and cancer is still here," and "Technologies that capture enemy CTCs for further interrogation might prove useful in the war on cancer." CTCs cannot only become a new marker for cancer prognosis, but their detection can also be a valid new parameter for diagnosing cancer early, for monitoring disease progression and relapse, and for optimizing therapy. This research established a new method to manipulate rare cell types based on their electrical signatures using dielectrophoresis (DEP) without having direct contact between the electrodes and the sample, known as contactless dielectrophoresis (cDEP). DEP is the motion of a particle in a suspending medium due to its polarization in the presence of a non-uniform electric field. cDEP relies upon reservoirs filled with highly conductive fluid to act as electrodes and provide the necessary electric field. These reservoirs are placed adjacent to the main microfluidic channel and are separated from the sample by a thin barrier of a dielectric material as is shown in Figure 1h. The application of a high-frequency electric field to the electrode reservoirs causes their capacitive coupling to the main channel and an electric field is induced across the sample fluid. Similar to traditional DEP, cDEP exploits the varying geometry of the electrodes to create spatial non-uniformities in the electric field. However, by utilizing reservoirs filled with a highly conductive solution, rather than a separate thin film array, the electrode structures employed by cDEP can be fabricated in the same step as the rest of the device; hence the process is conducive to mass production. We demonstrated the ability to isolate human leukemia cancer cells (THP-1) cells from a heterogeneous mixture of live and dead cells using cDEP with more than 99% selectivity and 95% removal efficiency. Through numerical and experimental investigations, new generation of cDEP devices have been designed and tested to detect and isolate THP-1 cells from spiked blood samples with high selectivity and cell capture efficiency. Our experimental observations, using prototype devices, indicate that breast cancer cell lines at their different stages (MCF-7, MCF-10, and MDA-MB231) have unique electrical. Furthermore, through collaborations at the Wake Forest Comprehensive Center, we demonstrated that prostate tumor initiating cells (TICs) exhibit unique electrical signatures and DEP responses and cDEP technology can be exploited to isolate and enrich TICs for further genetic pathways investigations. / Ph. D.

Applications for lab-on-a-chip devices have been expanding rapidly in the last decade due to their lower required volume of sample, faster experiments, smaller tools, more control, and ease of parallelization compared to their macroscale counterparts. Moreover, lab-on-a-chip devices provide important capabilities, including isolating rare cells from body fluids, such as isolating circulating tumor cells from blood or peritoneal fluid, which are not feasible or at least extremely difficult with macroscale devices. Particles experience different forces (and/or torques) when they are suspended in a fluid in a microdevice. A dominant force is the drag force on the particle as it flows through the fluid.  External forces such as dielectrophoresis, the motion of a particle due to its polarization in the presence of a non-uniform electric field, may also be applied. For instance, well-specified mixing or separation of particles can be achieved by using the combination of drag and dielectrophoretic forces. Two major mechanisms for manipulating particles in a microdevice include control of forces applied to the particles, such as those due to electric and velocity fields, and the geometry of the device that affects the nature of these fields. The coupling between the geometry of the microdevices and applied fields makes the prediction of associated forces inside the microdevice challenging and increasingly difficult when the applied field is time-dependent. Understanding the interaction of external forces and particles and fluid is critical for engineering novel microsystems. Determining this interaction is even more complicated when dealing with bioparticles, especially cells, due to their complex intrinsic biological properties which influence their electrical and mechanical properties. Particles with non-spherical geometries further increase the complexity, making drag and other shape-dependent forces, such as dielectrophoretic force, less predictable and more complicated. In order to introduce more complexity to the system and maintain precise control over particle movement and fluid flow, it is essential to have a comprehensive understanding about the mechanics of particles-fluid interaction and the dynamics of the particle movement. Although microfluidics has been investigated extensively, unanswered questions about the effect of forces on the particle remain. Answering these questions will facilitate designing novel and more practical microdevices for medical, biological, and chemical applications Microfluidics devices were engineered for differentiation of subpopulations of cells based on their bioelectrical properties. These microdevices were utilized for separating prostate, leukemia, and three different stages of breast cancer cells from hematologic cells with concentrations as low as 1:106 with efficiency of >95%. The microfluidic platform was also utilized to isolate prostate cancer stem cells (CSCs) from normal cancer cells based on their electrical signature. Isolating these cells is the first step towards the development of cancer specific therapies. The signal parameters required to selectively isolate ovarian cancer cells at different cancer stages were also compared with peritoneal cells as the first step in developing an early diagnostic clinical system centered on cell biophysical properties. Moreover, the effect of non-toxic concentrations of two metabolites, with known anti-tumor and pro-tumor properties, on the intrinsic electrical properties of early and late stages of ovarian cancer cells was investigated. This work is the first to show that treatment with non-toxic doses of these metabolites correlate with changes in cells electrical properties. / Ph. D.