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.

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

abstract: Microfluidic systems have gained popularity in the last two decades for their potential applications in manipulating micro- and nano- particulates of interest. Several different microfluidics devices have been built capable of rapidly probing, sorting, and trapping analytes of interest. Microfluidics can be combined with separation science to address challenges of obtaining a concentrated and pure distinct analyte from mixtures of increasingly similar entities. Many of these techniques have been developed to assess biological analytes of interest; one of which is dielectrophoresis (DEP), a force which acts on polarizable analytes in the presence of a non-uniform electric fields. This method can achieve high resolution separations with the unique attribute of concentrating, rather than diluting, analytes upon separation. Studies utilizing DEP have manipulated a wide range of analytes including various cell types, proteins, DNA, and viruses. These analytes range from approximately 50 nm to 1 µm in size. Many of the currently-utilized techniques for assessing these analytes are time intensive, cost prohibitive, and require specialized equipment and technical skills. The work presented in this dissertation focuses on developing and utilizing insulator-based dielectrophoresis (iDEP) to probe a wide range of analytes; where the intrinsic properties of an analyte will determine its behavior in a microchannel. This is based on the analyte’s interactions with the electrokinetic and dielectrophoretic forces present. Novel applications of this technique to probe the biophysical difference(s) between serovars of the foodborne pathogen, Listeria monocytogenes, and surface modified Escherichia coli, are investigated. Both of these applications demonstrate the capabilities of iDEP to achieve high resolution separations and probe slight changes in the biophysical properties of an analyte of interest. To improve upon existing iDEP strategies a novel insulator design which streamlines analytes in an iDEP device while still achieving the desirable forces for separation is developed, fabricated, and tested. Finally, pioneering work to develop an iDEP device capable of manipulating larger analytes, which range in size 10-250 µm, is presented. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2018

No description available.

An all-electrical system is developed to actuate and detect single biological cells in a microfluidic channel for diagnostic applications. Interdigitated electrodes fabricated on the channel floor transfer a high frequency signal for capacitance detection and a low frequency signal for dielectrophoretic actuation. In the fluid-filled channel, a pressure-driven flow propels single biological cells, which induce time-dependent capacitance signatures as they pass over the electrodes. With a sub-attofarad (~0.15 aF RMS, 53 Hz bandwidth) capacitance resolution, this system detects biological cells (e.g., 1 yeast cell ~ 50 aF) and their deflections (1 micrometer ~ 5 aF) from exerted dielectrophoretic forces (> 5 pN). Electrical detection of cell actuation by strong DEP forces provides an avenue for both inducing and monitoring the deformation of viscoelastic cells. A strong and repulsive dielectrophoretic force can be used to press a biological cell into a channel wall. When this occurs, the mechanical properties of the cell can be investigated by capacitively monitoring the cell-to-wall interaction. The nature of the resulting interaction is shown to depend on the mechanical properties of the cell (surface morphology and viscoelastic properties). Various mammalian cell types such as Chinese Hamster Ovary (CHO) cells, mouse fibroblasts, human blood cells, human breast cells and their tumorogenic phenotypes are investigated using this system. Between these populations, the effective Young's modulus varies widely from 20 Pa (neutrophils) to 1-2 GPa (polystyrene microspheres). The viability and phenotype of a biological cell are known to reflect its mechanical and electrical properties. Consequently, this work investigates whether dielectrophoretically induced cell deformations are correlated with corresponding variations in capacitance, which could be used for discriminating cell phenotypes in the future.

Manual handling of biological cells is routine in most laboratories. This is gradually changing with the development of robotic cell handling systems, and micro-scale lab-on-chip devices. Attempts were made to develop devices that automate or assist cell handling in the context of a bovine nuclear transfer (NT) system. The system, a zona-free bovine NT cloning system, formed a baseline reference for tool design and performance evaluation. Bovine NT can, as other cell handling procedures, be improved by rapid and precise cell positioning. Improvements in cell handling can increase the quantity of cells processed, and the uniformity of conditions the cells are subject to during processing. Tools were developed for two areas of cell handling: cell fusion and cell transportation. Designs suitable for implementation in microscale lab-on-chip systems were evaluated. Tool development was predominantly experimental, assisted by numerical modelling. The experimental investigation concerned device fabrication and operational performance. A number of cell handling tool designs were built and tested. Coplanar electrodes are not commonly used in bovine NT and reports on their efficacy were not available. These electrodes, which are simple to fabricate, were tested to determine fusion rates achievable in comparison with those of the baseline procedure. A novel fusion device, the micropit, was designed to assist bovine cell pairing and electrofusion. It was initially uncertain whether this device was capable of achieving cell fusion. Tests were conducted; and cell fusion and micro-positioning were demonstrated, as was an increase in biological cell processing throughput. Many miniaturised lab-on-chip systems rely on cell transportation. One illustration in the baseline procedure is the on-chip transport of cells to the cell fusion device. Potential cell transport mechanisms for a miniature cloning system were evaluated by prototype construction and testing. These mechanisms included travelling wave dielectrophoresis and capillary fluid actuation. To facilitate automation of on-chip cell transportation, a low cost electrically isolated cell detection system was developed based on a DVD pick-up unit. Various obstacles that were encountered during the course of device construction are noted, as are the fabrication methods employed.

An all-electrical system is developed to actuate and detect single biological cells in a microfluidic channel for diagnostic applications. Interdigitated electrodes fabricated on the channel floor transfer a high frequency signal for capacitance detection and a low frequency signal for dielectrophoretic actuation. In the fluid-filled channel, a pressure-driven flow propels single biological cells, which induce time-dependent capacitance signatures as they pass over the electrodes. With a sub-attofarad (~0.15 aF RMS, 53 Hz bandwidth) capacitance resolution, this system detects biological cells (e.g., 1 yeast cell ~ 50 aF) and their deflections (1 micrometer ~ 5 aF) from exerted dielectrophoretic forces (> 5 pN). Electrical detection of cell actuation by strong DEP forces provides an avenue for both inducing and monitoring the deformation of viscoelastic cells. A strong and repulsive dielectrophoretic force can be used to press a biological cell into a channel wall. When this occurs, the mechanical properties of the cell can be investigated by capacitively monitoring the cell-to-wall interaction. The nature of the resulting interaction is shown to depend on the mechanical properties of the cell (surface morphology and viscoelastic properties). Various mammalian cell types such as Chinese Hamster Ovary (CHO) cells, mouse fibroblasts, human blood cells, human breast cells and their tumorogenic phenotypes are investigated using this system. Between these populations, the effective Young's modulus varies widely from 20 Pa (neutrophils) to 1-2 GPa (polystyrene microspheres). The viability and phenotype of a biological cell are known to reflect its mechanical and electrical properties. Consequently, this work investigates whether dielectrophoretically induced cell deformations are correlated with corresponding variations in capacitance, which could be used for discriminating cell phenotypes in the future.

Thesis (Ph. D. in Mechanical Engineering)--Vanderbilt University, Aug. 2009. / Title from title screen. Includes bibliographical references.

Effective dipole moment method has been widely accepted as the de facto technique in predicting the dielectrophoretic force due to the non-uniform electric field. In this method, a finite-particle is modeled as an equivalent point-dipole that would induce a same electric field under the external electric field. This approach is only valid when the particle size is significantly smaller than the characteristic length of interest. This assumption is often violated in a microfluidic device, where the thickness or width of the microchannel can be as small as the particle. It is shown in this numerical study that when the dimensions of the particle were in the same order of magnitude as the characteristic length of the device, dielectrophoretic force can be induced even in a uniform electric field. This force arises due to the disturbance of the particle and the bounding wall. / Singapore-MIT Alliance (SMA)

This work demonstrates the ability of contactless dielectrophoresis (cDEP) for isolation of breast cancer cell stages. The ability to selectively concentrate breast tumor cells from a non-transformed or normal cell population is the key to successfully detecting tumors at an early stage of growth and treating transformed cells before they proliferate. Since all cell types have a unique molecular composition it is expected that their dielectrophoretic properties are also unique. DEP force is dependent on the frequency and magnitude of the applied field, as well as a particle's size and electric properties. Specifically, the Clausius-Mossotti factor in the DEP force equation determines a specific cell type's interaction with the electric field and the DEP force response. Cell properties affecting this parameter were investigated numerically and experimentally. MCF10A, MCF7, and MDA-MB231 human breast cancer cells were used to represent early, intermediate, and late staged breast cancer respectively. Experiments were conducted at 0.02ml/hr with applied voltages of 20Vrms, 25Vrms, 30Vrms, 35Vrms, 40Vrms and 50Vrms (n=8). Frequency measurements were recorded for the initial onset of DEP force and when 90% trapping was obtained. The trapping frequency ranges for each cell were distinct from one another with the least amount of overlap between the MCF10A cells and MDA-MB231cells. The MCF7 cell line had, on average, the smallest trapping region at each applied voltage, and fell in between the normal and late staged cells' trapping frequency ranges. Voltages of 20Vrms to 30Vrms were found the most efficient for cell isolation. / Master of Science