MICROFLUIDIC PARTICLE AND CELL MANIPULATION USING RESERVOIR-BASED DIELECTROPHORESIS

Controlled manipulation of synthetic particles and biological cells from a complex mixture is important to a wide range of applications in biology, environmental monitoring, and pharmaceutical industry. In the past two decades microfluidics has evolved to be a very useful tool for particle and cell manipulations in miniaturized devices. A variety of force fields have been demonstrated to control particle and cell motions in microfluidic devices, among which electrokinetic techniques are most often used. However, to date, studies of electrokinetic transport phenomena have been primarily confined within the area of microchannels. Very few works have addressed the electrokinetic particle motion at the reservoir-microchannel junction which acts as the interface between the macro (i.e., reservoir) and the micro (i.e., microchannel) worlds in real microfluidic devices. This Dissertation is dedicated to the study of electrokinetic transport and manipulation of particles and cells at the reservoir-microchannel junction of a microfluidic device using a combined experimental, theoretical, and numerical analysis. First, we performed a fundamental study of particles undergoing electrokinetic motion at the reservoir-microchannel junction. The effects of AC electric field, DC electric field, and particle size on the electrokinetic motion of particles passing through the junction were studied. A two-dimensional numerical model using COMSOL 3.5a was developed to investigate and understand the particle motion through the junction. It was found that particles can be continuously focused and even trapped at the reservoir-microchannel junction due to the effect of reservoir-based dielectrophoresis (rDEP). The electrokinetic particle focusing increases with the increase in AC electric field and particle size but decreases with the increase in DC electric field. It was also found that larger particles can be trapped at lower electric fields compared to smaller counterparts. Next, we utilized rDEP to continuously separate particles with different sizes at the reservoir-microchannel junction. The separation process utilized the inherent electric field gradients formed at the junction due to the size difference between the reservoir and the microchannel. It was observed, that the separation efficiency was reduced by inter-particle interactions when particles with small size differences were separated. The effect of enhanced electrokinetic flow on the separation efficiency was investigated experimentally and was observed to have a favorable effect. We also utilized rDEP approach to separate particles based on surface charge. Same sized particles with difference in surface charge were separated inside the microfluidic reservoir. The streaming particles interacted with the trapped particles and reduced the separation efficiency. The influences from the undesired particle trapping have been found through experiments to decrease with a reduced AC field frequency. Then, we demonstrated a continuous microfluidic separation of live yeast cells from dead cells using rDEP. Because the membrane of a cell gets distorted when it loses its viability, a higher exchange of ions results from such viability loss. The increased membrane conductivity of dead cells leads to a different Claussius-Mossoti factor from that of live cells, which enables their selective trapping and continuous separation based on cell viability. A two-shell numerical model was developed to account for the varying conductivities of different cell layers, the results of which agree reasonably with the experimental observations. We also used rDEP to implement a continuous concentration and separation of particles/cells in a stacked microfluidics device. This device has multiple layers and multiple microchannels on each layer so that the throughput can be significantly increased as compared to a single channel/single layer device. Finally, we compared the two-dimensional and three-dimensional particle focusing and trapping at the reservoir-microchannel junction using rDEP. We observed that the inherent electric field gradients in both the horizontal and vertical planes of the junction can be utilized if the reservoir is created right at the reservoir-microchannel junction. Three-dimensional rDEP utilizes the additional electric field gradient in the depth wise direction and thus can produce three-dimensional focusing. The electric field required to trap particles is also considerably lower in three-dimensional rDEP as compared to the two-dimensional rDEP, which thus considerably reduces the non-desired effects of Joule heating. A three-dimensional numerical model which accounted for the entire microfluidic device was also developed to predict particle trajectories

Direct current insulator based dielectrophoresis (DC-iDEP) microfluidic chip for blood plasma separation

Lab-on-a-Chip (LOC) integrated microfluidics has been a powerful tool for new developments in analytical chemistry. These microfluidic systems enable the miniaturization, integration and automation of complex biochemical assays through the reduction of reagent use and enabling portability.Cell and particle separation in microfluidic systems has recently gained significant attention in many sample preparations for clinical procedures. Direct-current insulator-based dielectrophoresis (DC-iDEP) is a well-known technique that benefits from the electric field gradients generated by an array of posts for separating, moving and trapping biological particle samples. In this thesis a parametric optimization is used to determine the optimum radius of the post for particle separation. Results that are used to design a microfluidic device that with a novel combination of hydrodynamic and di-electrophoretic techniques can achieve plasma separation in a microfluidic channel from fresh blood and for the first time allows optical real-time monitoring of the components of plasma without pre or post processing. Finally, all the results are integrated to create a novel microfluidic chip for blood plasma separation, which combines microfluidics with conventional lateral flow immune chromatography to extract enough plasma to perform a blood panel. The microfluidic chip design is a combination of cross-flow filtration with a reversible electroosmotic flow that prevents clogging at the filter entrance and maximizes the amount of separated plasma. The main advantage of this design is its efficiency, since with a small amount of sample (a single droplet ~10µL) a considerable amount of plasma (more than 1µL) is extracted and collected with high purity (more than 99%) in a reasonable time (5 to 8 minutes). To validate the quality and quantity of the separated plasma and to show its potential as clinical tool, the microfluidic chip has been combined with lateral flow immune chromatography technology to perform a qualitative detection of the TSH (thyroid-stimulating hormone) and a blood panel for measuring cardiac Troponin and Creatine Kinase MB. The results obtained from the microfluidic system are comparable to previous commercial lateral flow assays that required more sample for implementing less tests.Els dispositius Lab-on-a-Chip (LOC) són una eina de gran abast per als nous desenvolupaments de química analítica. Aquests sistemes de microfluids permeten la miniaturització, la integració i automatització d'assajos bioquímics complexos a través de la reducció del consum de reactiu i són portables. La separació de partícules i cél.lules mitjançant sistemes de microfluids ha guanyat recentment una atenció significativa en la preparació de mostres per als procediments clínics. La dielectroforesis amb corrent continu basada amb aïllants (DC-IDEP) és una tècnica ben coneguda que es beneficia dels gradients de camp elèctric generats per una sèrie de columnes d'aïllants que permeten la separació, el moviment i la captura de mostres de partícules biològiques. En aquesta tesis una optimització paramètrica s'utilitza per determinar el radi òptim de la columna necessària per a la separació de partícules. Resultats que s'utilitzen per dissenyar un dispositiu de microfluids que amb una nova combinació de tècniques hidrodinàmiques i di-electroforètiques pot aconseguir la separació de plasma en un microcanal a partir de sang fresca que per primera vegada permet la monitorització en temps real òptica dels components del plasma sense pre o post processament. Finalment, tots els resultats s'integren per crear un nou microxip per a la separació de plasma de la sang, que combina la microfluídica amb cromatografia de flux lateral convencional per extreure suficient plasma per dur a terme un panell de sang. El disseny del microxip és una combinació de filtració de flux creuat amb un flux electroosmòtic reversible que evita l'obstrucció a l'entrada del filtre i maximitza la quantitat de plasma separat. El principal avantatge d'aquest disseny és la seva eficiència, ja que amb una petita quantitat de mostra (una sola gota ~ 10µL) s'extreu una quantitat considerable de plasma (més de 1µL) i es recull amb gran puresa (més de 99%) en temps raonable (de 5 a 8 minuts). Per validar la qualitat i quantitat del plasma separat i per mostrar el seu potencial com a eina clínica, el xip de microfluids s'ha combinat amb la tecnologia de cromatografia de flux lateral per a realitzar una detecció qualitativa de la TSH (hormona estimulant de la tiroide) i un panell de sang per mesura la troponina cardíaca i la creatina quinasa MB. Els resultats obtinguts del sistema de microfluids són comparables als assajos de flux lateral comercials anteriors que requerien més mostra per a la realització de menys proves

An Insulating Constriction Dielectrophoresis Based Microfluidic Device for Protein Biomolecules Concentration

Sample enrichment or molecules concentration has been considered as an essential step for sample processing and biomarker detection recently in many applications involving miniaturized devices aiming at biosensing and bioanalysis, including the development of specialized tests for the detection of specific proteins and antibodies in human blood with the help of microfluidic and lab-on-a-chip devices. Among all the means involved to achieve this aim, dielectrophoresis (DEP) is increasingly employed in molecule manipulation and concentration because it is non-destructive and ensures high efficiency. However, there are still constraints on implementing the required functions using the dielectrophoresis technique in the devised micro-scale structures with high throughput, as well as the technical challenge in integration of sensors and concentration units for low-abundance molecular detection.;In the present work, we demonstrated a methodology to achieve protein concentration utilizing the combination effects of electrokinetics and low frequency insulator-based dielectrophoresis (iDEP) generated within a microfluidic device, in which a submicron constricted channel was fabricated using DNA molecular combing and replica molding. This fabrication technique, avoids using e-beam lithography or other complicated nanochannel fabrication methods, provides an easy and low cost approach with the flexibility in controlling channel dimensions to create highly constricted channels embedded in a microfluidic device. With theoretical analysis and experiments, we demonstrated that albumin--fluorescein isothiocyanate conjugate (FITC-BSA) protein molecules can be significantly concentrated to form an arc-shaped band near the constricted channel under the effects of negative dielectrophoretic force and DC electrokinetic forces within 2-3 minutes. It was also observed that the amplitudes of the applied DC and AC electric fields, AC frequencies, as well as suspending medium conductivities had strong effects on the concentration responses of the FITC-BSA molecules, including the concentrated area and position, intensities of the focused molecules, and concentration speed.;Our method demonstrated in the thesis provides a simple and flexible approach for quickly concentrating protein molecules by controlling the applied electric field parameters. The iDEP device reported in this thesis can be used as a stand-alone sensor or worked as a pre-concentration module integrated with biosensors for protein biomarker detections. Furthermore, low frequency dielectrophoresis provides practical uses for integrating the concentration module with a portable biosensing system

Ultrafine Dielectrophoresis-based Technique for Virus and Biofluid Manipulation

abstract: Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among all microfluidic techniques, dielectrophoresis (DEP) is one of the most effective and efficient techniques to quickly isolate and separate polarizable particles under inhomogeneous electric field. To date, extensive studies have demonstrated that DEP devices are able to precisely manipulate cells ranging from over 10 μm (mammalian cells) down to about 1 μm (small bacteria). However, very limited DEP studies on manipulating submicron bioparticles, such as viruses, have been reported. In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition. DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step.Dissertation/ThesisDoctoral Dissertation Chemistry 201

Joule Heating Enabled Electrokinetic Trapping of Submicron Particles in Ratchet Microchannels Using Depth Modelling

Microfluidic devices have been increasingly used for diverse particle manipulations in various chemical and biological applications. Fields such as water quality control, environmental monitoring and food safety require the continuous trapping and concentration of particles (either bio- or non-bio) for enhanced detection and analysis. To achieve this, various microfluidic techniques have been developed using electric field as well as other fields including magnetic, optical, acoustic, hydrodynamic, gravitational and inertial. Among these methods, electrokinetic manipulation of particles is the most often used due to its advantages over other methods such as simple operation and easy integration etc. It transports fluids and controls the motion of the suspended particles via electroosmosis, electrophoresis and dielectrophoresis. However, there is an inevitable phenomenon accompanying electrokinetic devices, i.e., Joule heating due to the passage of electric current through the conductive suspending medium. Previous studies indicate a negative impact of Joule heating on the trapping and concentration of micron-sized particles in insulator-based dielectrophoretic microdevices. We demonstrate in this thesis that the Joule heating-induced electrothermal flow can actually enhance the electrokinetic manipulation, leading to the otherwise impossible trapping and concentration of submicron particles in ratchet microchannels. We fabricated ratchet microchannels with polydimethylsiloxane and used them to study the transport and control of submicron particles in a moderately conductive phosphate buffer solution. Our research group did the experiments previously. We developed a numerical multiphysics depth average model, which can predict the observed particle trapping in the ratchet region. The numerical model consists of coupled electric current, fluid flow, heat transfer and mass transport equation. A depth average analysis of these governing equations was done to develop a 2D model on the horizontal plane of the microchannel, which gives us numerical results that are as good as a full-scale 3D model developed previously, but with much less computational resources. Numerical analysis of the developed model predicts the formation of two counter rotating electrothermal vortices at the ratchet tips. Moreover, particles can be seen trapped inside these vortices and the concentration of particles trapped in electrothermal vortices can be observed to increase with time. Further, on doing the parametric study we found out that with increase in voltage the size of these vortices increases. We also changed the shape of the ratchet, but that does not seem to affect particle trapping in a significant manner. These obtained numerically predicted results are found to be in good agreement with our experimental observations, which further validates our numerical modelling

Improving the Design and Application of Insulator-Based Dielectrophoretic Devices for the Assessment of Complex Mixtures

Dielectrophoresis (DEP) is an electrokinetic (EK) transport mechanism that exploits polarization effects when particles are exposed to a non-uniform electric field. This dissertation focused on the development of high-performance insulator-based DEP (iDEP) devices. A detailed analysis of the spatial forces that contribute to particle movement in an iDEP device is provided. In particular, this analysis shows how particle size and shape affects the regions where particles are likely to be retained due to dielectrophoretic trapping. The performance of these trapping regions was optimized using a systematic approach that integrates the geometrical parameters of the array of insulating structures. Devices that decrease the required electrical potential by ~80% where found. The optimization strategy enabled the detection of structures that promote and discourage particle trapping. By combining the best and worst structures in a single asymmetric structure, a novel iDEP device was designed. This device selectively enriches the larger particles in a sample and drives the smaller particles away from the enrichment region. A quick enrichment and elution of large cells was achieved. This is important when dealing with samples containing eukaryotic cells, which can be harmed by the electrical treatment. Yeast cells were successfully separated from polystyrene particles in under 40 seconds using this device and a high cell viability of 85% was achieved. Finally, an enhancement of traditional iDEP devices is proposed, where some insulating posts are replaced by conducting structures. That is, insulating and conductive posts are intimately combined within the same array. The performance of this hybrid device is presented to show the advantage of using insulating structures with microelectrodes in the same array to dominate particle movement

Design of a microfluidic chip for three dimensional hydrodynamic focusing in cell cytometry applications

The Focusing of cells is an important part of cytometry applications and can be achieved by different techniques. 3D hydrodynamic focusing has generated considerable interest over the last few years, owing to its simplicity and its independence from an electric potential for focusing. Current 3D hydrodynamic focusing devices require multilayer structures necessitating complex fabrication. Moreover, the existing designs show poor efficiencies in focusing. In the present work, three novel 3D hydrodynamic focusing designs consisting of a main channel for sample fluid flow and three pairs of side channels for focusing are proposed and modelled. A novel three dimensional hydrodynamic focusing design is proposed and simulated. In order to develop the numerical model for three dimensional focusing designs, a theoretical review of parameters affecting the fluid flow in microfluidic structure has been performed. Simulation was performed using COMSOL Multiphysics with diluted glycerol as the sample fluid and DI water as sheath flow fluid. The effects of fluid velocities in the channels were studied. In Design III, the overall efficiency is less than that in the first two designs, but the advantage of this design lies in the possibility of a simpler fabrication. Subsequently, parameters such as velocity and viscosity were studied in the case of Design III, and the ideal velocity condition was identified as 150 m/sec for the sample flow, 350µm/sec for the first sheath flow, and 550µm/sec for the second sheath. Simulations were carried out with sample and sheath flow fluids that have different viscosities. It was concluded that the effect of focusing is primarily dependent on the velocity rather than on the viscosity of the fluid

ELECTROKINETIC PARTICLE MANIPULATIONS IN SPIRAL MICROCHANNELS

Recent developments in the field of microfluidics have created a multitude of new useful techniques for practical particle and cellular assays. Among them is the use of dielectrophoretic forces in \u27lab-on-a-chip\u27 devices. This sub-domain of electrokinetic flow is particularly popular due to its advantages in simplicity and versatility. This thesis makes use of dielectrophoretic particle manipulations in three distinct spiral microchannels. In the first of these experiments, we demonstrate the utility of a novel single-spiral curved microchannel with a single inlet reservoir and a single outlet reservoir for the continuous focusing and filtration of particles. The insulator-based negative-dielectrophoretic (repulsive) force is used in a parametric study of the effects of electric field strength, particle size, and solution concentration on particle focusing abilities. It was summarily determined that all three factors are positively correlated with increased particle focusing ability. From these results, a partial filtration of 10 μm particles from a binary solution of 3 and 10 μm particles was demonstrated. Also observed was a balance between dielectrophoretic and repulsive particle-wall interactions; thus yielding a novel approach for particle manipulation. Following the results of the first, we demonstrate in the second experiment a continuous-flow electrokinetic separation of both a binary mixture and a ternary mixture of colloidal particles based on size in a single-spiral microchannel with a single inlet reservoir and triple outlet reservoirs. This method also utilizes both curvature-induced dielectrophoresis to focus particles to a tight stream and the previously observed wall-induced electric lift to manipulate the aligned particles to size-dependent equilibrium positions. Due to the continuous nature of the flow through concentric spiral loops, both focusing forces influence particles simultaneously. This novel technique is useful for its compact geometry, robust structure, ease of manufacture, and ease of use in the manipulation of independent particle species. A theoretical model is also developed to understand this separation, and the obtained analytical formula predicts the experimentally measured particle center-wall distance in the spiral with a close agreement. We demonstrate in the third experiment a continuous-flow electrical sorting of spherical and peanut-shaped particles of similar volumes in an asymmetric double-spiral microchannel with a single inlet reservoir and triple outlet reservoirs. This experiment, unlike the first two, differentiates particle species based principally on shape. Shape is an intrinsic marker of cell cycle, an important factor for identifying a bio-particle, and also a useful indicator of cell state for disease diagnostics; therefore, shape can be a specific marker in label-free particle and cell separation for various chemical and biological applications. The double-spiral geometry exploits curvature-induced dielectrophoresis to initially focus particles to a tight stream in the first spiral without any sheath flow. Particles are subsequently displaced to shape-dependent flow paths in the second spiral without any external force. We also develop a numerical model to simulate and understand this shape-based particle sorting in spiral microchannels. The predicted particle trajectories agree qualitatively with the experimental observation

MICROARRAY FOR SINGLE-PARTICLE TRAP WITH ADDRESSABLE CONTROL BASED ON NEGATIVE DIELECTROPHORESIS

Ph.DDOCTOR OF PHILOSOPH

Analysis and design of a capillary driven blood plasma separation microfluidic device

Recently, the emergence of lab-on-a-chip devices has seen in a variety of applications especially in clinical analys is and diagnostics. ln particular the lack of suitable microdevices for separation of plasma from whole blood is a barrier to achieve a reliable lab on a chip (LOC) blood test. In order to address this issue, a novel self-driven high throughput blood plasma separator microchip is introduced as a first step to a miniaturized blood analys is. PDMS (Polydim ethylsiloxane) is utilized as the base material for the microdevice fabrication to ens ure the biocompatibility, the disposability (single-use to avoid contamination) and the low cost ofthe system for the mass-manufacturing. One of the characteristic features ofthe presented m icrodevice is that it needs to work just by capillary pressure eliminating the need of external sources. The requested capillary pressure to drive blood through the microdevice is derived via PDMS modification byanalyzing different surfactants, which are mixed with pre-cured PDMS to achieve a stable hydrophilic character. Furthermore, a diamond microchannel integrated micropillar (dMIMP) pump with high throughput and with a resistance flow 35.5% lower than a circular based micropillar pump (cMIMP) has been developed. For this purpose, the pressure drop and flow resistance of a lam inar flow through low aspect ratio MIMPs with different shapes and geometrical parameters are experim entally, numerically and analytically determined. In order to characterize the fabricated microcapillarypumps in PDMS, a novel and simple fabrication technique is introduced to overcome the PDMS deform ation under high-pressure operation. The presented fabrication technique combines the use of stiff PDMS (1 0:2, the ratio between polymer base and cross liking agent) and a thin coating layer of the UV curable thiolene resin as supporter (Norland Optical Adhesive 63) on the fabricated PDMS microchannel. Finally, using all the achieved results in the material property and microcapillary pump design in the last steps, a novel selfd riven high throughput microfluid ic chip for blood plasma separation is designed and fabricated. The presented microdevice can successfu lly separate more than 0.11JL of plasma from a whole human fresh blood drop (51JL) without the need of external forces with high efficiency(more than 90%) and reasonable time (3 to 5 minutes). The achieved plasma volume (0.1 IJL) in 10 1Jm-depth collected channels ofthe presented self-driven microdevice paves the path to integrate this microfluidic circuit in a portable medical point-of-care-testing (POCT) for doing different blood analysis.Recientemente, la aparición de dispositivos de laboratorio en un chip (Lab on a chip) ha generado una gran variedad de nuevas aplicaciones especialmente en análisis clínicos y diagnóstico. En particular, la falta de micro dispositivos adecuados para la separación del plasma de la sangre es una barrera para lograr un dispositivo portátil que pueda realizar una análisis de sangre. Con el fin de abordar esta cuestión, un microsistema auto impulsado que pueda obtener una cantidad importante de plasma sanguíneo de una gota de sangre seria un primer paso para un análisis de sangre miniaturizado. En esta tesis se utiliza PDMS (Polydimethylsiloxane ) como material de base para la fabricación del microdispositivo debido a su biocompatibilidad y su bajo coste. Uno de los rasgos característicos del dispositivo presentado es que trabaja solo con presión capilar que elimina la necesidad de fuentes externas. La presión capilar solicitada para conducir sangre a través del microdispositivo se obtiene mediante la modificación del PDMS mediante diferentes agentes tensioactivos, que se mezclan con PDMS pre-curado para lograr un carácter hidrófilo estable. El proceso de filtración se basará en una estructura de columnas con baja relación de aspecto. Estás estructuras se han analizado numéricamente, analíticamente y experimentalmente, para obtener un diseño con baja resistencia al flujo. En concreto, se ha diseñado un conjunto de microcolumnas base diamante (dMIMP) que se utilizará como bomba de alto rendimiento y baja resistencia al flujo (35.5 % menor que una bomba microcolumnas circulares (cMIMP)). Para realizar esta caracterización se ha desarrollado un sistema de fabricación que permita caracterizar las estructruas de PDMS, a alta presión sin que se deformen. Por último, se ha utilizado el PDMS modificado y la bomba capilar optimizada para realizar un diseño de microfiltro de plasma sanguíneo de alto rendimiento. El microdispositivo presentado puede separar más de 0.11microL de plasma de una gota de sangre fresca humana (5microL) sin la necesidad de fuerzas externas con una alta eficiencia (más del 90%) y un tiempo razonable (de 3 a 5 minutos). El volumen de plasma obtenido es suficiente para implementar diferentes tipos de test sanguíneo y por tant representa el primer paso hacia la creación de un punto de atención portàtil (POC, point of care)