PROBLEMS IN THE STUDY AND USE OF AC DIELECTROPHORESIS AND THEIR CONSEQUENCES: A STUDY BASED ON COMSOL MULTIPHYSICS MODELING

Dielectrophoresis (or DEP) is an important phenomenon which is induced when a dielectric particle is placed in a non-uniform electric field. The force generated by DEP has been exploited for various micro and nano fluidics applications like positioning, sorting and separation of particles involved in medical diagnostics, drug discovery, cell therapeutics, biosensors, microfluidics, nanoassembly, particle filtration etc. The integration of DEP systems into the microfluidics enables inexpensive, fast, highly sensitive, highly selective, label-free detection and also the analysis of target bioparticles. This work aims to provide a complete compilation of the factors affecting the DEP force. It elucidates the underlying mechanisms using COMSOL Multiphysics and sheds new insight into the mechanisms for the separation and sorting of different types of particles. This research identifies the problems in the literature and uses COMSOL to analyze the impact of these problems on the end results. It examines four factors that affect the DEP force: physical conditions, electrode setup, properties of the particles and suspension medium. Moreover, it analyzes the influence of the Clausius-Mossotti factor (CM factor) and its cross-over upon the magnitude and direction of the DEP force. From the analysis, it becomes clear that particle size not only affects the magnitude of the DEP force but also the conductivity of the particle. This factor, which is largely ignored, could lead to a shift in the crossover frequency. Shell model plays an important role in determining the dielectric properties of particles that are not homogenous. In such a situation assuming uniform dielectric properties may lead to inconclusive results. The presence of an electric double layer surrounding a particle affects the conductivity of the particle. Also, assuming DEP force to be the only force acting on a particle suspended in a non-uniform electric field leads to errors in the end results. This research provides knowledge on the basic characteristics of the DEP force and its mechanism. It provides a better understanding by examining numerous works carried out in the past and brings out the problems and their consequences

UTILIZING DIELECTROPHORESIS TO DETERMINE THE PHYSIOLOGICAL DIFFERENCES OF EUKARYOTIC CELLS

Type 1 diabetes affects over 108,000 children, and this number is steadily increasing. Current insulin therapies help manage the disease but are not a cure. Over a child’s lifetime they can develop kidney disease, blindness, cardiovascular disease and many other issues due to the complications of type 1 diabetes. This autoimmune disease destroys beta cells located in the pancreas, which are used to regulate glucose levels in the body. Because there is no cure and many children are affected by the disease there is a need for alternative therapeutic options that can lead to a cure. Human mesenchymal stem cells (hMSCs) are an important cell source for stem cell therapeutics due to their differentiation capacity, self-renewal, and trophic activity. hMSCs are readily available in the bone marrow, and act as an internal repair system within the body, and they have been shown to differentiate into insulin producing cells. However, after isolation hMSCs are a heterogeneous cell population, which requires secondary processing. To resolve the heterogeneity issue hMSCs are separated using fluorescent- and magnetic-activate cell sorting with antigen labeling. These techniques are efficient but reduce cell viability after separation due to the cell labeling. Therefore, to make hMSCs more readily available for type 1 diabetes therapeutics, they should be separated without diminishing there functional capabilities. Dielectrophoresis is an alternative separation technique that has the capability to separated hMSCs. This dissertation uses dielectrophoresis to characterize the dielectric properties of hMSCs. The goal is to use hMSCs dielectric signature as a separation criteria rather than the antigen labeling implemented with FACS and MACS. DEP has been used to characterize other cell systems, and is a viable separation technique for hMSCs

Dielectrophoretic investigations of internal cell properties

Dielectrophoresis (DEP) is a term which describes the motion of polarisable particles induced by a non-uniform electric field. It has been the subject of research into a variety of fields including nanoassembly, particle filtration and biomedicine. The application of DEP to the latter has gained significant interest in recent years, driven by the development of microfluidic “Lab-on-a-chip” devices designed to perform sophisticated biochemical processes. It provides the ability to characterise and selectively manipulate cells based on their distinct dielectric properties in a manner which is non-invasive and label free, by using electrodes which can be readily integrated with microfluidic channels. Under appropriate conditions a biological cell will experience a DEP force directing it either towards or away from concentrations in the electric field. At a so-called “crossover frequency” the cell is effectively invisible to the field resulting in no DEP force, a response typically observed in the 1 kHz to 1 MHz range. Its value is a function of cell membrane dielectric properties and has been the subject of research directed at devices capable of using it to both characterise and sort cells. The aim of this work was to investigate the behaviour of a higher frequency crossover referred to as fxo2, predicted to occur in the 1 MHz to 1 GHz range. At these frequencies the electric field is expected to penetrate the cell membrane and behave as a function of intracellular dielectric properties. Standard lithography techniques have been used to fabricate electrodes carefully designed to operate at these frequencies. The existence of fxo2 was then confirmed in murine myeloma cells, in good agreement with dielectric models derived from impedance spectroscopy. A temperature dependent decrease in its value was observed with respect to the time that cells were suspended in a DEP solution. This decrease is consistent with previous studies which indicated an efflux of intracellular ions under similar conditions. An analytical derivation of fxo2 demonstrates its direct proportionality to intracellular conductivity. Direct control of the crossover was achieved by using osmotic stress to dilute the intracellular compartment and thereby alter its conductivity. By using a fluorophore which selectively binds to potassium, a strong relationship has been demonstrated between the value of fxo2 and the concentration of intracellular potassium. Measurements of fxo2 for an unfed culture demonstrated a correlation with viability and subtle shifts in its distribution were caused by the early stages of chemically induced apoptosis

Improved diagnostics for sleeping sickness

The aim of this work was to explore an alternative to existing methods of detection for Human African Trypanosomiasis (also known as sleeping sickness). A new approach to diagnostics for sleeping sickness is needed, since the existing methods of detection employed in the field have significant shortcomings in terms of sensitivity, cost or ease of operation. In this work, the enrichment of trypanosomes from blood using travelling electric fields and the selective lysis of cells using optoelectronic tweezers will be presented. Both techniques allow for the enrichment of trypanosomes from blood samples but the first is more suited for an application as a point-of-care device, while the latter is also applicable to other cell types and offers greater flexibility. Besides demonstrating and quantifying the experimental results the work includes simulations to further explain the phenomena and investigate the underlying mechanisms. The results presented here offer a new method to enrich trypanosomes, a central step in any potential diagnostic tool. They open up the possibility to develop a new solution to the challenges posed by sleeping sickness diagnostics and allow for miniaturisation and automation of the process

Antibody-free isolation of circulating tumor cells by dielectrophoretic field-flow fractionation

textThis work focuses on the integration of microfluidics and dielectrophoresis(DEP) with the principles of field flow fractionation (FFF) to create a continuous-flow isolator for rare and viable circulating tumor cells (CTCs) from peripheral blood mononuclear cells (PBMNs) drawn from cancer patients. The method exploits differences in the plasma membrane capacitances of tumor and blood cells, which correspond to differences in the membrane surface areas of these cell types. DEP-FFF was first adapted to measure cell membrane capacitance, cell density and deformability profiles of cell populations. These properties of the NCI-60 panel of cancer cell types, which represents the wide functional diversity of cancers from 9 organs and leukemia, were compared with the normal cell subpopulations of peripheral blood. In every case, the NCI-60 cells exhibited membrane capacitance characteristics that were distinct from blood and, as a result, they could be isolated from blood by DEP. The heightened cancer cell membrane capacitances correlated strongly with membrane-rich morphological characteristics at their growth sites, including cell flattening, dendritic projections, and surface wrinkling. Following harvest from culture and maintenance in suspension, cancer cells were found to shed cytoplasm and membrane area over time and the suspended cell populations developed considerable morphological diversity. The shedding changed the cancer cell DEP properties but they could still be isolated from blood cells. A similar shedding process in the peripheral blood could account for the surprisingly wide morphological diversity seen among circulating cells isolated from clinical specimens. A continuous flow DEP-FFF method was devised to exploit these findings by allowing CTCs to be isolated from the nucleated cells of 10 mL clinical blood specimens in 40 minutes, an extremely high throughput rate for a microfluidic-based method. Cultured cancer cells could be isolated at 70-80% efficiency using this approach and the isolation of CTCs from clinical specimens was demonstrated. The results showed that the continuous DEP-FFF method delivers unmodified, viable CTCs for analysis, is perhaps universally applicable to isolation of CTCs from different cancer types and is independent of surface antigens - making it suitable for cells lacking the epithelial markers used in currently accepted CTC isolation methods.Biomedical Engineerin

Label-free cell separation and sorting in microfluidic systems

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Dielectrophoresis for capillary flow microfluidic optoelectronics

The development of a novel photonic integrated platform with three dimensional (3D) capillary flow and dielectrophoresis elements for chip based flow cytometers is discussed. Size-independent single stream particle focusing is the key for the efficient operation of a flow cytometer and many efforts have been made to reproduce it properly on a microchip scale. In this work, capillary and negative Dielectrophoresis (n-DEP) components were integrated onto a single chip of III-V semiconductor material for conducting scattering measurements of microparticles. The integration of all components on a single chip is intended to result in low cost, portable and disposable microfluidic devices for point-of-care diagnostics. The design, fabrication and investigation of a system combining n-DEP with the capillary driven flow to align and reposition microparticles with fluid flow in a single stream around the centreline of the microchannel is considered. This is followed by testing the potential of n-DEP for efficient on-chip light scatter measurements. Planar microelectrodes face-to-face below and above the surface of the 3D microchannel are employed to create a localized non-uniform electric field to focus polystyrene microparticles in flowing fluid via n-DEP. The functionality of the device is assessed by detecting and counting 6 - 15 µm polystyrene microparticles (suspended in Deionised (DI) water) as an example to assess how similarly sized biological cells, such as blood cells, would flow within the fabricated 3D microchannel. The results show that the polystyrene microparticles are focused successfully in a single stream around the centreline of the 3D microchannel when operating the microelectrodes with an AC potential of 10 MHz and no more than 30 V peak-to-peak. The n-DEP focused microparticles show narrower velocity distribution, compared to randomly flowing particles, as well as exhibiting higher speeds (at the centre of the channel). The latter result suggests that a capillary-like speed profile is only present at the fluid front, and that the fluid flow becomes faster at the centre, behind the advancing meniscus, due to the greater friction at the microchannel’s side walls. Consistent pulse shapes and peaks are observed in the laser data from the similarly sized polystyrene microparticles using a fully-integrated platform with lasers and photo-detectors. This suggests that the n-DEP focusing microelectrodes can significantly improve the operation efficiency of the device by regulating the flow of microparticles and passing them through a consistent scanning zone albeit with an increase in the precision of the fabrication required