Isolation and enrichment of low abundant particles with insulator-based dielectrophoresis

Isolation and enrichment of low-abundant particles are essential steps in many bio-analytical and clinical applications. In this work, the capability of an insulator-based dielectrophoresis (iDEP) device for the detection and stable capture of low abundant polystyrene particles and yeast cells was evaluated. Binary and tertiary mixtures of particles and cells were tested, where the low-abundant particles had concentration ratios on the order of 1:10 000 000 compared to the other particles present in the mixture. The results demonstrated successful and stable capture and enrichment of rare particles and cells (trapping efficiencies over 99%), where particles remained trapped in a stable manner for up to 4 min. A device with four reservoirs was employed for the separation and enrichment of rare particles, where the particles of interest were first selectively concentrated and then effectively directed to a side port for future collection and analysis. The present study demonstrates that simple iDEP devices have appropriate screening capacity and can be used for handling samples containing rare particles; achieving both enrichment and isolation of low-abundant particles and cells.     

Three-dimensional cellular focusing utilizing a combination of insulator-based and metallic dielectrophoresis

Particle focusing in microfluidic devices is a necessary step in medical applications, such as detection, sorting, counting, and flow cytometry. This study proposes a microdevice that combines insulator-based and metal-electrode dielectrophoresis for the three-dimensional focusing of biological cells. Four insulating structures, which form an X pattern, are employed to confine the electric field in a conducting solution, thereby creating localized field minima in the microchannel. These electrodes, 56-μm-wide at the top and bottom surfaces, are connected to one electric pole of the power source. The electrodes connected to the opposite pole, which are at the sides of the microchannel, have one of three patterns: planar, dual-planar, or three-dimensional. Therefore, low-electric-field regions at the center of the microchannel are generated to restrain the viable HeLa cells with negative dielectrophoretic response. The array of insulating structures aforementioned is used to enhance the performance of confinement. According to numerical simulations, three-dimensional electrodes exhibit the best focusing performance, followed by dual-planar and planar electrodes. Experimental results reveal that increasing the strength of the applied electric field or decreasing the inlet flow rate significantly enhances focusing performance. The smallest width of focusing is 17 μm for an applied voltage and an inlet flow rate of 35 V and 0.5 μl/min, respectively. The effect of the inlet flow rate on focusing is insignificant for an applied voltage of 35 V. The proposed design retains the advantages of insulator-based dielectrophoresis with a relatively low required voltage. Additionally, complicated flow controls are unnecessary for the three-dimensional focusing of cells.     

Insulator-based dielectrophoresis of mitochondria

Isolated mitochondria display a wide range of sizes plausibly resulting from the coexistence of subpopulations, some of which may be associated with disease or aging. Strategies to separate subpopulations are needed to study the importance of these organelles in cellular functions. Here, insulator-based dielectrophoresis (iDEP) was exploited to provide a new dimension of organelle separation. The dielectrophoretic properties of isolated Fischer 344 (F344) rat semimembranosus muscle mitochondria and C57BL/6 mouse hepatic mitochondria in low conductivity buffer (0.025–0.030 S/m) at physiological pH (7.2–7.4) were studied using polydimethylsiloxane (PDMS) microfluidic devices. First, direct current (DC) and alternating current (AC) of 0–50 kHz with potentials of 0–3000 V applied over a channel length of 1 cm were separately employed to generate inhomogeneous electric fields and establish that mitochondria exhibit negative DEP (nDEP). DEP trapping potential thresholds at 0–50 kHz were also determined to be weakly dependent on applied frequency and were generally above 200 V. Second, we demonstrated a separation scheme using DC potentials <100 V to perform the first size-based iDEP sorting of mitochondria. Samples of isolated mitochondria with heterogeneous sizes (150 nm–2 μm diameters) were successfully separated into sub-micron fractions, indicating the ability to isolate mitochondria into populations based on their size.     

Liquid dielectrophoresis and surface microfluidics

Liquid dielectrophoresis (L-DEP), when deployed at microscopic scales on top of hydrophobic surfaces, offers novel ways of rapid and automated manipulation of very small amounts of polar aqueous samples for microfluidic applications and development of laboratory-on-a-chip devices. In this article we highlight some of the more recent developments and applications of L-DEP in handling and processing of various types of aqueous samples and reagents of biological relevance including emulsions using such microchip based surface microfluidic (SMF) devices. We highlighted the utility of these devices for on-chip bioassays including nucleic acid analysis. Furthermore, the parallel sample processing capabilities of these SMF devices together with suitable on- or off-chip detection capabilities suggest numerous applications and utility in conducting automated multiplexed assays, a capability much sought after in the high throughput diagnostic and screening assays.     

Formation of embryoid bodies using dielectrophoresis

Embryoid body (EB) formation forms an important step in embryonic stem cell differentiation invivo. In murine embryonic stem cell (mESC) cultures EB formation is inhibited by the inclusion of leukaemic inhibitory factor (LIF) in the medium. Assembly of mESCs into aggregates by positive dielectrophoresis (DEP) in high field regions between interdigitated oppositely castellated electrodes was found to initiate EB formation. Embryoid body formation in aggregates formed with DEP occurred at a more rapid rate-in fact faster compared to conventional methods-in medium without LIF. However, EB formation also occurred in medium in which LIF was present when the cells were aggregated with DEP. The optimum characteristic size for the electrodes for EB formation with DEP was found to be 75-100 microns; aggregates smaller than this tended to merge, whilst aggregates larger than this tended to split to form multiple EBs. Experiments with ESCs in which green fluorescent protein (GFP) production was targeted to the mesodermal gene brachyury indicated that differentiation within embryoid bodies of this size may preferentially occur along the mesoderm lineage. As hematopoietic lineages during normal development derive from mesoderm, the finding points to a possible application of DEP formed EBs in the production of blood-based products from ESCs.     

Direct measurements of the frequency-dependent dielectrophoresis force

Dielectrophoresis (DEP), the phenomenon of directed motion of electrically polarizable particles in a nonuniform electric field, is promising for applications in biochemical separation and filtration. For colloidal particles in suspension, the relaxation of the ionic species in the shear layer gives rise to a frequency-dependent, bidirectional DEP force in the radio frequency range. However, quantification methods of the DEP force on individual particles with the pico-Newton resolution required for the development of theories and design of device applications are lacking. We report the use of optical tweezers as a force sensor and a lock-in phase-sensitive technique for analysis of the particle motion in an amplitude modulated DEP force. The coherent detection and sensing scheme yielded not only unprecedented sensitivity for DEP force measurements, but also provided a selectivity that clearly distinguishes the pure DEP force from all the other forces in the system, including electrophoresis, electro-osmosis, heat-induced convection, and Brownian forces, all of which can hamper accurate measurements through other existing methods. Using optical tweezers-based force transducers already developed in our laboratory, we have results that quantify the frequency-dependent DEP force and the crossover frequency of individual particles with this new experimental method.     

An insulator-based dielectrophoretic microdevice for the simultaneous filtration and focusing of biological cells

Manipulating and discriminating biological cells of interest using microfluidic and micro total analysis system (μTAS) devices have potential applications in clinical diagnosis and medicine. Cellular focusing in microfluidic devices is a prerequisite for medical applications, such as cell sorting, cell counting, or flow cytometry. In the present study, an insulator-based dielectrophoretic microdevice is designed for the simultaneous filtration and focusing of biological cells. The cells are introduced into the microchannel and hydrodynamically pre-confined by funnel-shaped insulating structures close to the inlet. There are ten sets of X-patterned insulating structures in the microfluidic channel. The main function of the first five sets of insulating structures is to guide the cells by negative dielectrophoretic responses (viable HeLa cells) into the center region of the microchannel. The positive dielectrophoretic cells (dead HeLa cells) are attracted to regions with a high electric-field gradient generated at the edges of the insulating structures. The remaining five sets of insulating structures are mainly used to focus negative dielectrophoretic cells that have escaped from the upstream region. Experiments employing a mixture of dead and viable HeLa cells are conducted to demonstrate the effectiveness of the proposed design. The results indicate that the performance of both filtration and focusing improves with the increasing strength of the applied electric field and a decreasing inlet sample flow rate, which agrees with the trend predicted by the numerical simulations. The filtration efficiency, which is quantitatively investigated, is up to 88% at an applied voltage of 50 V peak-to-peak (1 kHz) and a sample flow rate of 0.5 μl/min. The proposed device can focus viable cells into a single file using a voltage of 35 V peak-to-peak (1 kHz) at a sample flow rate of 1.0 μl/min.     

Tuning direct current streaming dielectrophoresis of proteins

Dielectrophoresis (DEP) of biomolecules has large potential to serve as a novel selectivity parameter for bioanalytical methods such as (pre)concentration, fractionation, and separation. However, in contrast to well-characterized biological cells and (nano)particles, the mechanism of protein DEP is poorly understood, limiting bioanalytical applications for proteins. Here, we demonstrate a detailed investigation of factors influencing DEP of diagnostically relevant immunoglobulin G (IgG) molecules using insulator-based DEP (iDEP) under DC conditions. We found that the pH range in which concentration of IgG due to streaming iDEP occurs without aggregate formation matches the pH range suitable for immunoreactions. Numerical simulations of the electrokinetic factors pertaining to DEP streaming in this range further suggested that the protein charge and electroosmotic flow significantly influence iDEP streaming. These predictions are in accordance with the experimentally observed pH-dependent iDEP streaming profiles as well as the determined IgG molecular properties. Moreover, we observed a transition in the streaming behavior caused by a change from positive to negative DEP induced through micelle formation for the first time experimentally, which is in excellent qualitative agreement with numerical simulations. Our study thus relates molecular immunoglobulin properties to observed iDEP, which will be useful for the future development of protein (pre)concentration or separation methods based on DEP.     

Fabrication and characterization of nanomaterial-based sensors using dielectrophoresis

Dielectrophoresis (DEP) is an electrokinetic motion of dielectrically polarized materials in nonuniform electric fields. DEP has been successfully applied to manipulation of nanomaterials including carbon nanotubes (CNTs), metallic nanoparticles, and semiconducting nanowires. Under positive DEP force, which attracts nanomaterials toward the higher field region, nanomaterials are trapped in the electrode gap and automatically establish good electrical connections between them and the external measuring circuit. This feature allows us a fast, simple, and low-cost fabrication of nanomaterial-based sensors based on a bottom-up approach. This paper first presents a theoretical background of DEP phenomena and then reviews recent works of the present author, which were aimed to develop nanomaterial-based sensors, such as a CNT gas sensor and a ZnO nanowire photosensor, using DEP fabrication technique. It is also demonstrated that DEP technique enables self-formation of interfaces between various nanomaterials, which can be also applicable as novel sensing transducers.     

Interaction between cells in dielectrophoresis and electrorotation experiments

Progress in microelectrode-based technologies has facilitated the development of sophisticated methods for manipulating and separating cells, bacteria, and other bioparticles. For many of these various applications, the theoretical modeling of the electrical response of compartmentalized particles to an external field is important. In this paper we address the analysis of the interaction between cells immersed in rf fields. We use an integral formulation of the problem derived from a consideration of the charge densities induced at the interfaces of the particle compartments. The numerical solution by a boundary element technique allows characterization of their dielectric properties. Experimental validation of this theoretical model is obtained by investigating two effects: (1) The influence that dipolar “pearl chaining” has on the dielectrophoretic behavior of human T lymphocytes and (2) the frequency variation of the spin and orbital torques of approaching insulinoma β-cells in a rotating field.     

Antibody-independent isolation of circulating tumor cells by continuous-flow dielectrophoresis

Circulating tumor cells (CTCs) are prognostic markers for the recurrence of cancer and may carry molecular information relevant to cancer diagnosis. Dielectrophoresis (DEP) has been proposed as a molecular marker-independent approach for isolating CTCs from blood and has been shown to be broadly applicable to different types of cancers. However, existing batch-mode microfluidic DEP methods have been unable to process 10 ml clinical blood specimens rapidly enough. To achieve the required processing rates of 10⁶ nucleated cells/min, we describe a continuous flow microfluidic processing chamber into which the peripheral blood mononuclear cell fraction of a clinical specimen is slowly injected, deionized by diffusion, and then subjected to a balance of DEP, sedimentation and hydrodynamic lift forces. These forces cause tumor cells to be transported close to the floor of the chamber, while blood cells are carried about three cell diameters above them. The tumor cells are isolated by skimming them from the bottom of the chamber while the blood cells flow to waste. The principles, design, and modeling of the continuous-flow system are presented. To illustrate operation of the technology, we demonstrate the isolation of circulating colon tumor cells from clinical specimens and verify the tumor origin of these cells by molecular analysis.     

Combined AC electroosmosis and dielectrophoresis for controlled rotation of microparticles

Electrorotation is widely used for characterization of biological cells and materials using a rotating electric field. Generally, multiphase AC electric fields and quadrupolar electrode configuration are needed to create a rotating electric field for electrorotation. In this study, we demonstrate a simple method to rotate dielectrophoretically trapped microparticles using a stationary AC electric field. Coplanar interdigitated electrodes are used to create a linearly polarized nonuniform AC electric field. This nonuniform electric field is employed for dielectrophoretic trapping of microparticles as well as for generating electroosmotic flow in the vicinity of the electrodes resulting in rotation of microparticles in a microfluidic device. The rotation of barium titanate microparticles is observed in 2-propanol and methanol solvent at a frequency below 1 kHz. A particle rotation rate as high as 240 revolutions per minute is observed. It is demonstrated that precise manipulation (both rotation rate and equilibrium position) of the particles is possible by controlling the frequency of the applied electric field. At low frequency range, the equilibrium positions of the microparticles are observed between the electrode edge and electrode center. This method of particle manipulation is different from electrorotation as it uses induced AC electroosmosis instead of electric torque as in the case of electrorotation. Moreover, it has been shown that a microparticle can be rotated along its own axis without any translational motion.     

Three dimensional microfluidics with embedded microball lenses for parallel and high throughput multicolor fluorescence detection

We report a 3D microfluidic device with 32 detection channels and 64 sheath flow channels and embedded microball lens array for high throughput multicolor fluorescence detection. A throughput of 358 400 cells/s has been accomplished. This device is realized by utilizing solid immersion micro ball lens arrays for high sensitivity and parallel fluorescence detection. High refractive index micro ball lenses (n = 2.1) are embedded underneath PDMS channels close to cell detection zones in channels. This design permits patterning high N.A. micro ball lenses in a compact fashion for parallel fluorescence detection on a small footprint device. This device also utilizes 3D microfluidic fabrication to address fluid routing issues in two-dimensional parallel sheath focusing and allows simultaneous pumping of 32 sample channels and 64 sheath flow channels with only two inlets.     

On-chip DNA preconcentration in different media conductivities by electrodeless dielectrophoresis

Electrodeless dielectrophoresis is the best choice to achieve preconcentration of nanoparticles and biomolecules due to its simple, robust, and easy implementation. We designed a simple chip with microchannels and nano-slits in between and then studied the trapping of DNA in high conductive medium and low conductive medium, corresponding to positive and negative dielectrophoresis (DEP), respectively. It is very important to investigate the trapping in media with different conductivities since one always has to deal with the sample solutions with different conductivities. The trapping process was analyzed by the fluorescent intensity changes. The results showed that DNA could be trapped at the nano-slit in both high and low conductive media in a lower electric field strength (10 V/cm) compared to the existing methods. This is a significant improvement to suppress the Joule heating effect in DEP related experiments. Our work may give insight to researchers for DNA trapping by a simple and low cost device in the Lab-on-a-Chip system.     

Separation of platelets from other blood cells in continuous-flow by dielectrophoresis field-flow-fractionation

We present a microfluidic device capable of separating platelets from other blood cells in continuous flow using dielectrophoresis field-flow-fractionation. The use of hydrodynamic focusing in combination with the application of a dielectrophoretic force allows the separation of platelets from red blood cells due to their size difference. The theoretical cell trajectory has been calculated by numerical simulations of the electrical field and flow speed, and is in agreement with the experimental results. The proposed device uses the so-called "liquid electrodes" design and can be used with low applied voltages, as low as 10 V(pp). The obtained separation is very efficient, the device being able to achieve a very high purity of platelets of 98.8% with less than 2% cell loss. Its low-voltage operation makes it particularly suitable for point-of-care applications. It could further be used for the separation of other cell types based on their size difference, as well as in combination with other sorting techniques to separate multiple cell populations from each other.     

Curvature-induced dielectrophoresis for continuous separation of particles by charge in spiral microchannels

The separation of particles from a heterogeneous mixture is critical in chemical and biological analyses. Many methods have been developed to separate particles in microfluidic devices. However, the majority of these separations have been limited to be size based and binary. We demonstrate herein a continuous dc electric field driven separation of carboxyl-coated and noncoated 10 μm polystyrene beads by charge in a double-spiral microchannel. This method exploits the inherent electric field gradients formed within the channel turns to manipulate particles by dielectrophoresis and is thus termed curvature-induced dielectrophoresis. The spiral microchannel is also demonstrated to continuously sort noncoated 5 μm beads, noncoated 10 μm beads, and carboxyl-coated 10 μm beads into different collecting wells by charge and size simultaneously. The observed particle separation processes in different situations are all predicted with reasonable agreements by a numerical model. This curvature-induced dielectrophoresis technique eliminates the in-channel microelectrodes and obstacles that are required in traditional electrode- and insulator-based dielectrophoresis devices. It may potentially be used to separate multiple particle targets by intrinsic properties for lab-on-a-chip applications.     

Characterization and separation of Cryptosporidium and Giardia cells using on-chip dielectrophoresis

Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8–12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius—Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage).     

Enhancing sensitivity and specificity in rare cell capture microdevices with dielectrophoresis

The capture and subsequent analysis of rare cells, such as circulating tumor cells from a peripheral blood sample, has the potential to advance our understanding and treatment of a wide range of diseases. There is a particular need for high purity (i.e., high specificity) techniques to isolate these cells, reducing the time and cost required for single-cell genetic analyses by decreasing the number of contaminating cells analyzed. Previous work has shown that antibody-based immunocapture can be combined with dielectrophoresis (DEP) to differentially isolate cancer cells from leukocytes in a characterization device. Here, we build on that work by developing numerical simulations that identify microfluidic obstacle array geometries where DEP–immunocapture can be used to maximize the capture of target rare cells, while minimizing the capture of contaminating cells. We consider geometries with electrodes offset from the array and parallel to the fluid flow, maximizing the magnitude of the resulting electric field at the obstacles'' leading and trailing edges, and minimizing it at the obstacles'' shoulders. This configuration attracts cells with a positive DEP (pDEP) response to the leading edge, where the shear stress is low and residence time is long, resulting in a high capture probability; although these cells are also repelled from the shoulder region, the high local fluid velocity at the shoulder minimizes the impact on the overall transport and capture. Likewise, cells undergoing negative DEP (nDEP) are repelled from regions of high capture probability and attracted to regions where capture is unlikely. These simulations predict that DEP can be used to reduce the probability of capturing contaminating peripheral blood mononuclear cells (using nDEP) from 0.16 to 0.01 while simultaneously increasing the capture of several pancreatic cancer cell lines from 0.03–0.10 to 0.14–0.55, laying the groundwork for the experimental study of hybrid DEP–immunocapture obstacle array microdevices.     

Microfluidic separation of live and dead yeast cells using reservoir-based dielectrophoresis

Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening, etc. This work demonstrates a novel microfluidic approach to dielectrophoretic separation of yeast cells by viability. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeast cells and continuously separate them from live ones right inside the reservoir. This approach is therefore termed reservoir-based dielectrophoresis (rDEP). It has unique advantages as compared to existing dielectrophoretic approaches such as the occupation of zero channel space and the elimination of any mechanical or electrical parts inside microchannels. Such an rDEP cell sorter can be readily integrated with other components into lab-on-a-chip devices for applications to biomedical diagnostics and therapeutics.     

Separation by dielectrophoresis of dormant and nondormant bacterial cells of Mycobacterium smegmatis

The dielectrophoretic behavior of active, dead, and dormant Mycobacterium smegmatis bacterial cells was studied. It was found that the 72-h-old dormant cells had a much higher effective particle conductivity (812±10 μS cm⁻¹), almost double that of active cells (560±20 μS cm⁻¹), while that of dead (autoclaved) M. smegmatis cells was the highest (950±15 μS cm⁻¹) overall. It was also found that at 80 kHz, 900 μS cm⁻¹ dead cells were attracted at the edges of interdigitated castellated electrodes by positive dielectrophoresis, but dormant cells were not. Similarly, at 120 kHz, 2 μS cm⁻¹ active cells were attracted and dormant cells were not. Using these findings a dielectrophoresis-based microfluidic separation system was developed in which dead and active cells were collected from a given cell suspension, while dormant cells were eluted.