Experimental Development and Analysis of a Novel Setup for Insulated Dielectrophoresis

Dielectrophoresis has long been studied and utilized for the manipulation of microscale particles in solution. This phenomenon is due to the induced polarization of dielectric particles subjected to an electric field. When the field is also inhomogeneous in terms of the distribution of its strength through space, the polarized particles move and come to rest in certain areas due to the relationship between their and the solvent\u27s relative permittivities. If the electric field is homogenous, such as within a parallel plate capacitor, the particles are polarized according to their permittivity and the field\u27s frequency, but they will not move. These relationships have been exploited for many lab-on-a-chip applications such as positioning cells for tissue engineered structures, separating live cells from dead ones, or separating cells of different types. Some of these systems also employ microfluidics in order to add another level of control, increasing the degrees of freedom when manipulating microparticles. The goals of this study are to develop and experimentally characterize a low power setup for moving and aligning particles using dielectrophoresis, to perform experiments to quantify the effect of conductivity on the dielectrophoretic force, to analyze and quantify the out of plane force, and to use these experimentally determined relationships to create more accurate theoretical models of dielectrophoresis processes using COMSOL Multiphysics software. The first step in this process involves the creation of a technique to perform dielectrophoresis using relatively low power. This requires the use of a very thin, durable membrane to separate the particles in solution from the field generating electrodes. The use of this very thin membrane, much thinner than those used in previous studies,1 has revealed a much more complete picture of the behavior of microparticles in response to the forces present during dielectrophoresis. A more complete picture of reality has the potential to lead to the creation of more accurate models and descriptions of the underlying physics. Once this novel setup was shown to produce consistent results, the studies began. These consisted largely of frequency sweeps at constant voltages and voltage sweeps at given frequencies, similar in basic method to studies which have been peformed to analyze dielectrophoretic systems in the past. No flow is present in the particle suspension for the majority of these studies. This constraint, coupled with the insulating layer over the electrode theoretically limits the forces present to principally those elicited by dielectrophoresis. With this setup it was found that polystyrene beads were arranged into parallel lines of pearl chains in the spaces between interdigitated electrodes with sufficient predictability that models of this phenomenon could be created and aligned with reality. In addition to these in-plane forces governing the formation of pearl chains, the out of plane forces were also analyzed and modeled. In the case of negative dielectrophoresis, this is the force lifting the beads off of the electrode surface. The effect of media conductivity on particle alignment and levitation has also been analyzed using this setup. In addition to these experiments with polystyrene beads, this setup has also been shown to manipulate cells under low media conductivity conditions. It is important that the forces and physical relationships involved in these insulated dielectrophoresis setups are better understood, so that such techniques can be more precisely and predictably implemented for lab-on-a-chip and tissue engineering applications

Continuous separation of microparticles in aqueous medium by means of dielectrophoresis

There is a widespread need to separate microparticles suspended in liquid media. Dielectrophoresis (DEP), a technique for manipulating the motion trajectories of suspended particles, has enormous potential for solving difficult particle-particle separation problems. Nevertheless, the great majority of DEP applications have been limited so far to microchannels and lab-on-a-chip devices, with throughput typically in the A LA min-1 range. A promising, alternative solution to this problem is anticipated by upscaling DEP systems to enable high-throughput DEP separation on a clinical or industrial scale. To achieve this, a novel interdigitated electrode (IDE) design is proposed to meet the need for a high electric field when upscaling a DEP system. Numerical simulation using OpenFOAM demonstrated that, when replacing conventional plate IDE by cylindrical IDE (cIDE) in microchannel systems, the dielectrophoretic force field, represented by the gradient of the squared electric field, becomes stronger and more homogeneously distributed along the electrode array. The resulting particle DEP velocities were also higher for the cIDE. Simulations confirmed by experiments allow further predictions of particle motion in enlarged cIDE-DEP systems. Understanding how the interplay of channel geometry and electrode concept affects induced particle velocity is crucial when designing DEP separators having sufficiently high throughput to reach preparative scale. The objective of tailored design is to control particle motion trajectories predominantly by DEP while avoiding electrothermal interference in the form of fluid convection induced by a temperature gradient in the liquid phase due to Joule heating. One solution to this Joule heating problem in large-scale DEP systems is to tailor the ratios of electrode diameter, electrode distance and channel height. Based on model calculations, the influence on particle trajectories of both DEP force and drag force due to thermal convection was predicted for a case study involving a channel with rectangular cross section and an array of cIDEs at the bottom. The models were successfully verified by experimentally measuring and quantitatively analysing velocities of polyelectrolytic resin microparticles located at the subsurface of demineralized water. This allowed a qualitative sensitivity analysis of the impact of voltage input, particle size and medium properties on critical design parameters. From this, design criteria were deduced for the cIDE-DEP system that allow the influence of Joule heating to be minimised. There is still a need for continuous, contact-free fractionation of microparticles at high throughput. To achieve this, a sheath-flow-assisted dielectrophoretic continuous field-flow separator with a tailored arrangement of cIDE was developed, and size-dependent trajectories of dispersed particles were observed. Using a voltage input of 200 Veff at a frequency of 200 kHz, polystyrene particles (45, 25, and 11 Amicrometre in diameter) were levitated to different heights due to a negative DEP force. Experimental observations agree well with simulated particle trajectories that were obtained from by a modified Lagrangian particle tracking model in combination with Laplace's and Navier-Stokes equations. A theoretically calculated system throughput of up to 47 mLA min-1 was found to be possible by trading off design and operation parameters, enabling contact-free fractionation of sensitive microparticles with negligible shear stress. For further upscaling of the cIDE-DEP separation system, a new separation device with concentrically arranged cIDE configuration was proposed. Proof-of-concept is demonstrated by numerically predicting microparticle motion trajectories within the separator. Simulations show that a remarkable increment of suspension throughput can be achieved by the concentric cIDE separator compared to the cIDE separator under the same circumstances. From an evaluation of the impact of operating parameters on particle displacement, it can be deduced that continuous fractionation is possible even at system throughputs in the of hundreds of mLA min-1 range by using the concentric cIDE separator. These theoretical findings lay the foundation for continuous DEP-based microparticle separation on an industrial scale

An Integrative Approach to Elucidating the Governing Mechanisms of Particles Movement under Dielectrophoretic and Other Electrokinetic Phenomena

Dielectrophoresis (DEP) has been a subject of active research in the past decades and has shown promising applications in Lab-on-Chip devices. Currently researchers use the point dipole method to predict the movement of particles under DEP and guide their experimental designs. For studying the interaction between particles, the Maxwell Stress Tensor (MST) method has been widely used and treated as providing the most robust and accurate solution. By examining the derivation processes, it became clear that both methods have inherent limitations and will yield incorrect results in certain occasions. To overcome these limitations and advance the theory of DEP, a new numerical approach based on volumetric-integration has been established. The new method has been proved to be valid in quantifying the DEP forces with both homogeneous and non-homogeneous particles as well as particle-particle interaction through comparison with the other two methods. Based on the new method, a new model characterizing the structure of electric double layer (EDL) was developed to explain the crossover behavior of nanoparticles in medium. For bioengineering applications, this new method has been further expanded to construct a complete cell model. The cell model not only captures the common crossover behavior exhibited by cells, it also explains why cells would initiate self-rotation under DEP, a phenomenon we first observed in our experiments. To take a step further, the new method has also been applied to investigate the interaction between multiple particles. In particular, this new method has been proved to be powerful in elucidating the underlying mechanism of the tumbling motion of pearl chains in a flow condition as we observed in our experiments. Moreover, it also helps shed some new insight into the formation of different alignments and configurations of ellipsoidal particles. Finally, with the consideration of the Faradic current from water electrolysis and effect of pH, a new model has been developed to explain the causes for the intriguing flow reversal phenomenon commonly observed (but not at all understood) in AC-electroosmosis (ACEO) with reasonable outcomes

Parallel manipulation of individual magnetic microbeads for lab-on-a-chip applications

Many scientists and engineers are turning to lab-on-a-chip systems for cheaper and high throughput analysis of chemical reactions and biomolecular interactions. In this work, we developed several lab-on-a-chip modules based on novel manipulations of individual microbeads inside microchannels. The first manipulation method employs arrays of soft ferromagnetic patterns fabricated inside a microfluidic channel and subjected to an external rotating magnetic field. We demonstrated that the system can be used to assemble individual beads (1-3µm) from a flow of suspended beads into a regular array on the chip, hence improving the integrated electrochemical detection of biomolecules bound to the bead surface. In addition, the microbeads can follow the external magnet rotating at very high speeds and simultaneously orbit around individual soft magnets on the chip. We employed this manipulation mode for efficient sample mixing in continuous microflow. Furthermore, we discovered a simple but effective way of transporting the microbeads on-chip in the rotating field. Selective transport of microbeads with different size was also realized, providing a platform for effective sample separation on a chip. The second manipulation method integrates magnetic and dielectrophoretic manipulations of the same microbeads. The device combines tapered conducting wires and fingered electrodes to generate desirable magnetic and electric fields, respectively. By externally programming the magnetic attraction and dielectrophoretic repulsion forces, out-of-plane oscillation of the microbeads across the channel height was realized. Furthermore, we demonstrated the tweezing of microbeads in liquid with high spatial resolutions by fine-tuning the net force from magnetic attraction and dielectrophoretic repulsion of the beads. The high-resolution control of the out-of-plane motion of the microbeads has led to the invention of massively parallel biomolecular tweezers.Ph.D.Committee Chair: Hesketh, Peter; Committee Member: Allen, Mark; Committee Member: Degertekin, Levent; Committee Member: Lu, Hang; Committee Member: Yoda, Minam

Versatile dielectrophoresis based microfluidic platforms for chemical stimulation of cells

The purpose of this project is to develop versatile microfluidic systems, which take advantage of dielectrophoresis, for the rapid creation of customized cell clusters, chemical stimulation of the patterned cells under well-controlled environmental conditions, and analysis of cellular responses using different microscopic techniques. As the first contribution, the author shows that the reorientation of the microfluidic channel with respect to the microelectrodes can be utilized to alter the characteristics of the dielectrophoretic (DEP) system. This enables the author to change the location and density of immobilized viable cells across the channel, release viable cells along customized numbers of streams within the channel, and improve the sorting of viable and nonviable cells in terms of flow throughput and efficiency of the system. As the second contribution, the author presents a novel approach to change the DEP response of nonviable yeast cells by chemically altering their surface properties. The author’s studies show that treating nonviable yeast cells with low concentrations of ionic surfactants can significantly change their surface properties, making them exhibit a strong positive DEP response, even at high medium conductivities. The capability of this treatment is demonstrated in two proof-of-concept experiments to create isolated or adjacent clusters of viable and nonviable cells next to each other. As the third contribution, the author utilizes dielectrophoresis for studying the dynamic response of cells following chemical stimulation. The DEP system enables separation of the budding yeasts from a background of non-budding cells, and their subsequent immobilization onto the microelectrodes at desired densities. The immobilized yeasts are then stimulated with Lyticase to remove the cell wall and convert them into spheroplasts in a dynamic process, which depends on the concentration of Lyticase. As the fourth contribution, the author introduces a novel method for immobilization of the cell organelles released from the lysed cells by patterning multi-walled carbon nanotubes (MWCNTs) between the microelectrodes. A strong electric field can be induced at the free ends of MWCNT chains, which is utilized to immobilize the released cell organelles from budding yeast cells after treating them with high concentration of Lyticase. As the fifth contribution, the author develops a DEP-based microfluidic platform for interfacing non-adherent cells with high-resolution scanning electron microscopy (SEM). The developed DEP system enables rapid immobilization, on-chip chemical stimulation and fixation, and dehydration of samples without deposition of chemical residues over the cell surface. These advantages are demonstrated for comparing the morphological changes of non-budding and budding yeast cells following Lyticase treatment. In summary, the research conducted by the PhD candidate enables studying of the dynamic cell responses under various chemical treatments using versatile DEP based microfluidic platforms. The PhD candidate also believes that the presented research will offer practical solutions for future biomedical micro-devices

Dielectrophoretic characterisation and manipulation of sub-micron particles following surface modification

The aim of this thesis is to dielectrophoretically characterise sub-micron particles on the basis of their surface properties and to devise a DEP technique suitable for the fractionation and manipulation of particles on this scale. Polystyrene particles are modified by the attachment of biological ligands using various established localisation techniques and their DEP response observed using micro-electrodes with well defined high and low field regions, corresponding to a previously utilised design and modified in the course of this project for multiple sample handling. The results of these observations are modelled for the first time using a charge relaxation mechanism pertaining to a structured interfacial charge distribution and, through fitting the data to this model, fundamental parameters of the system - the surface conductance and electrokinetic charge - are predicted. The model viability is assessed with reference to both comparisons with alternative measurements and the technical limitations of the data fitting procedure, and corresponding surface charge transport mechanisms are discussed in the light of the DEP response following surface modification. Investigations are made into the possibility of a DEP based device suitable for the transport/fractionation of sub-micron particles. Given the essentially dissipative nature of sub-micro particle ensembles, a Brownian ratchet principle is chosen. A Brownian ratchet is a generic system wherein a net directional drive is effected by biasing Brownian diffusion on a periodically activated anisotropic structure. Without need of thermal gradients or net macroscopic forces Brownian ratchet pumps could be an interesting alternative in many microfluidic applications. Simulated fields and corresponding particle transport rates are compared for two basic electrode structures in order to assess their viability for use as DEP Brownian ratchets and a new design proposed, based on the simultaneous juxtaposition of positive and negative DEP forces. This device is built on the necessary scale using multi-layer fabrication techniques with a silicon elastomer moulded channel. The existence of stochastic transport on the device is investigated experimentally by means of processed video sequences and resulting possibilities for particle separation on the basis of size and surface properties inferred

Construction of artificial skin tissue with placode-like structures in well-defined patterns using dielectrophoresis

During embryonic development of animal skin tissue, the skin cells form regular patterns of high cell density (placodes) where hair or feathers will be formed. These placodes are thought to be formed by the aggregation of dermal cells into condensates. The aggregation process is thought to be controlled by a reaction-diffusion mechanism of activator and inhibitor molecules, and involve mechanical forces between cells and cells with the matrix. In this project, placode formation in chicken embryonic skin cells was used as a model system for the study of the mechanism by which the placodes are formed. Artificial aggregates of chicken embryonic skin cells were created by suspending them in a 300 mM low conductivity sorbitol solution and attracting them by positive dielectrophoresis to high field regions within microelectrode arrays by applying a 10 - 20 Vpk-pk 1 MHz signal across the microelectrodes. It was demonstrated that using this method aggregates can be produced in a large variety of patterns and that the distance between the aggregates and aggregate size and shape within the pattern can be controlled effectively. Custom-built image analysis tools were developed in LabVIEW to analyze the patterns formed. The formation of aggregates by dielectrophoresis was followed by an immobilization phase of the resulting patterns inside a gel matrix, forming an artificial skin. Nutrients and oxygen were supplied externally. Long-term incubation of the artificial skin shows that embryonic skin cells in the aggregates were viable and showed behavior similar to that of developing embryonic skin, including further aggregation of the cells and the formation of cell condensates. The domain size was shown to have an influence on the condensation process, with cells in small aggregates forming only one condensate near the centre of the aggregate, and several condensates in larger aggregates. Whilst the distribution of cell condensates within the aggregates in round large aggregates is predominantly random, some line formation could be observed in linear aggregations, indicating some self-organization may be occurring

Advanced dielectrophoretic cell separation systems

This thesis describes experimental and theoretical investigations into new particle handling and separation methods and techniques. It makes a major contribution to the rapidly expanding field of cell separation technology. A novel dielectrophoretic cell separation system has been developed, which is capable of processing large sample volumes (~50mL) in a flow through system. Previously reported dielectrophoretic cell separator systems typically process sample volumes in the 100mL range. The electrode configuration developed for this work allows the isolation and concentration of single particle types from large sample volumes; a method which could be further developed into a new rare-cell separation technology. In addition, a new technique of particle fractionation was developed termed ‘Dielectrophoretic Chromatography’. A cell separation chip was designed and built using standard micro-fabrication techniques. Experimental work was undertaken to demonstrate the function and limitations of the device. Numerical modelling of the particle motion in the device is presented and compared with experimental work for a number of different particle types, applied voltages and fluid flow rates. The dielectrophoretic separation system comprises a microfluidic channel, of cross-section 100mm x 10mm and length 50mm, with two sets of interdigitated microelectrode arrays. The first set of arrays, with characteristic electrode size 40mm, called a focussing device, has electrodes patterned onto the top and bottom surfaces of the flow channel. The second electrode array, which is part of the same device, has an electrode array patterned only on the bottom of the channel. Two sizes of secondary electrode array were used 20mm and 40mm. AC voltages (from 1V to 10V peak) are applied to the microelectrode, with a frequency between 10kHz to 180MHz. A dielectrophoretic force is exerted on the particles as they flow along the channel. The first electrode array uses negative dielectrophoresis to focus the stream of particles entering the device into a narrow sheet (one particle diameter thick) midway between the upper and lower channel surfaces. The second electrode array, down stream from the first is separately controllable

Microfluidic and Electrokinetic Manipulation of Single Cells

Traditional cell assays report on the average results of a cell population. However, a wide range of new tools are being developed for a fundamental understanding of single cell's functionality. Nonetheless, the current tools are either limited in their throughput or the accuracy of the analysis. One such technology is electrorotation. Although it is known to be unique in its capability for single-cell characterization, it is commonly a slow technique with a processing time of about 30 minutes per cell. For this reason, this thesis focuses on the development of a 3D electrode based electrorotation setup for fast and automatic extraction of a single cell's spectrum. For this purpose, new fabrication processes for 3D electrodes were developed to achieve high-resolution patterning of 3D metal electrodes. The first process we developed was a subtractive one based on passivated silicon structures and the second process was an additive one based on SU-8 photolithography. The additive nature of the second process enables high patterning resolution of electrodes and connection layers, while providing high conductivity thanks to the use of standard metal films. The electrodes have been characterized by different electrical measurements to ensure a proper connection and side-wall exposure. Furthermore, we characterized and compared the sheet resistance of planar and vertical layers. A further microfabrication process was developed for integrating the electrodes into microfluidic channels. The process was designed to enable the use of high numerical aperture lenses; for that purpose, a PDMS-mediated bonding process was engineered to seal the channels with a thin glass coverslip. Moreover, the development of a process to realize microfluidic access holes on the back of the wafer reduces the footprint of the chips and facilitates access for the microscope optics. Finally, a pressure-driven system was used together with the chips to achieve high control of liquid injections and to enable fast and precise flow stop. The combination of such a system, together with the dielectrophoretic forces that can be applied by the 3D electrodes, allows accurate positioning of single cells inside the 3D electrode quadrupole. The particles can then be analyzed by electrorotation. For this purpose, a custom Labview interface was built to coordinate the full setup and to acquire a full electrorotation spectrum in less than 3 minutes

Design of a dielectrophoretic cell loading device

In recent years there has been an increasing interest in studying individual cells, and structures that physically entrap one or few cells have been developed for this purpose, but the approaches to load cells into these structures leave a lot to be desired. This dissertation discusses the design of a device that loads cells suspended in a solution into microvials using a combination of dielectrophoresis and fluid flow, which offers significant advantages over previous loading approaches. The basic concept is to use fluid flow and dielectrophoretic forces to position a given cell above a given vial, within an array of similar vials, and then bringing the cell into the vial. The loading of several cells flowing in a channel into a vial in a matter of seconds is demonstrated. The design of the loading device spurred the development of novel topics in the area of dielectrophoresis. The structures into which cells are loaded produce "parasitic cages". The effect of multiple electric fields and at multiple frequencies had to be explored to eliminate the parasitic cages, and new theory was developed to describe the phenomenon in a straight forward and convenient way. The design process of dielectrophoretic structures known as flow through sorters was simplified significantly using a method that relies on non dimensional analysis and a figure of merit. These topics investigated have broader applications than just loading cells into vials. The dissertation demonstrates technologies and design and fabrication methods key to the cell loading design. The dissertation ends by describing the design of a device that can be implemented to load cells into vials on integrated circuit chips and outlining this device's expected characteristics and performance based on the theory and methods presented through the dissertation