Dielectrophoretic sorting on a microfabricated flow cytometer: label free separation of Babesia bovis infected erythrocytes

Dielectrophoresis is a method that has demonstrated great potential in cell discrimination and isolation. In this study, the dielectrophoretic sorting of normal and Babesia bovis infected erythrocytes was performed using a microfabricated flow cytometer. Separation was possible through exploitation of the dielectric differences between normal and infected erythrocytes, essentially due to the higher ionic membrane permeability of B. bovis infected cells. Sorting experiments were performed inside a microchip made from Pt microelectrodes and SU-8 channels patterned on a glass substrate. Optimum cell separation was achieved at 4 MHz using an in vitro culture of B. bovis suspended in 63 mS/m phosphate buffer and applying a sinusoidal voltage of 15 V peak-to-peak. Normal erythrocytes experienced stronger positive dielectrophoresis (pDEP) than B. bovis infected cells, moving them closer to the microelectrodes. Under these conditions it was possible to enrich the fraction of infected cells from 7 to 50% without the need of extensive sample preparation or labelling. Throughout the experiments very few microliters of sample were used, suggesting that this system may be considered suitable for integration in a low-cost automated device to be used in the in situ diagnostic of babesiosis.     

Study on the discrete dielectrophoresis for particle–cell separation

This paper presents the application of the discrete dielectrophoretic force to separate polystyrene particles from red blood cells. The separation process employs a simple microfluidic device that is composed of interdigitated electrodes and a microchannel. The discrete dielectrophoretic force is generated by adjusting the duty cycle of the applied voltage. The electrodes make a tilt angle with the microchannel to change the moving direction of the red blood cells. By adjusting the voltage magnitude and duty cycle, we investigate the deflection of red blood cells and the variation of cell velocity along electrode edge under positive dielectrophoresis. The experiments with polystyrene particles show that the enrichment of the particles is greater than 150 times. The maximum separation efficiency is 97% for particle-to-cell number ratio equal to 1:2000 in the sample having high cell concentration. Using the appropriate applied voltage magnitude and duty cycle, the discrete dielectrophoretic force can prevent the clogging of microchannel while successfully separating the particles from the cells with high enrichment and efficiency. The proposed principle can be readily applied to dielectrophoresis-based devices for biomedical sample preparation or diagnosis such as the separation of rare or infected cells from a blood sample.     

Label-free Sorting and Counting of Yeast Cells for Viability Studies

This paper reports the new combination of cell sorting and counting capabilities on a single device. Most state-of-the-art devices combining these technologies use optical techniques requiring complicate experimental setups and labeled samples. The use of a label-free, electrical device significantly decreases the system complexity and makes it more appropriate for use in point-of-care diagnostics.Living and dead yeast cells are separated by dielectrophoretic forces and counted using coulter counters. The combination of these two methods allows the determination of the percentage of living and dead cells for viability studies of cell samples. It could further be used for sorting and counting of blood cells in applications such as diagnosis of insufficient cell concentrations, identification of cell deficiencies or bacterial contamination. The use of dielectrophoresis (DEP) as sorting principle allows to separate cells based on their dielectric properties in place of size-based separation, enabling sorting of large panels of cells and separation of infected and non-infected cells of the same type.     

Continuous separation of cells by balanced dielectrophoretic forces at multiple frequencies

We present a particle-sorting device based on the opposition of dielectrophoretic forces. The forces are generated by an array of electrode chambers located in both sidewalls of a main flow channel. Particles with different dielectric response perceive different force magnitudes and are therefore continuously focused to different streamlines in the flow channel. We relate the particles' dielectric response to their output position in the downstream channel. We demonstrate the performance of the device by separating a mixed yeast cell population into pure fractions of viable and nonviable cells. Finally, we use the device to enrich red blood cells infected with Babesia bovis, a major pathogen in cattle and simultaneously confirm the hypothesis that infection with B. bovis causes significant changes in the dielectric response of red blood cells.     

Dielectric characterization of Babesia bovis using the dielectrophoretic crossover frequency

Coinfection with the tick-transmitted pathogen Babesia spp. is becoming a serious health problem because of the erythrocyte invasion through Ixodes scapularis tick. The transmission of this protozoan by blood transfusion often results in high morbidity and mortality in recipients. A novel way to detect parasitized erythrocytes is by utilizing dielectrophoresis, an electrokinetic technique on a microfluidic platform, to improve the diagnostics of Babesia spp. The differences in the dielectric properties of Babesia spp.–infected erythrocytes versus healthy erythrocytes were exploited to design a fast and cost-effective diagnostic tool. One crucial factor for a successful diagnostic platform via dielectrophoretic separation is the dielectric characterization of Babesia-infected erythrocytes, which is investigated in this paper. The influence of medium conductivity and erythrocytes phenotype and genotype over the first crossover frequency (f_co1) are used to quantify the dielectric properties of the infected cells. A sigmoidal curve was plotted via curve fitting of the single-shell model, which has been proven appropriate for parasitized cell populations where considerable cell geometry variation occurs. The difference in these curves is relevant for the separation of cells population. Microliters of sample and reagent were used throughout this experiment; the scale, results obtained, and simplicity of the system often make it very suitable for point-of-care babesiosis disease diagnostics.     

Focused electrophoretic motion and selected electrokinetic dispensing of particles and cells in cross-microchannels

The electrokinetic focusing and the resultant accelerated electrophoretic motion of polystyrene particles and red blood cells were visualized in microfluidic cross-channels. The experimentally measured width of the focused stream and the measured velocity increase of particles and cells at different voltage ratios follow the proposed analytical formula within the experimental error. The attained velocity increase is insensitive to the particle size, particle property (i.e., particle or cell), and particle trajectory. By solving the electrical potential field in the cross-channel at the experimental conditions, we demonstrate that the squeezed electrical field lines in the channel intersection determine the shape of the focused stream, and the nonuniform distribution of axial electrical field strength underlies the variation of particle/cell electrophoretic velocity through the focusing region. However, the dielectrophoretic force resulting from the nonuniform electrical field in the intersection seems to push the acceleration region of particles and cells slightly in the downstream direction. We have also achieved the single particle/cell dispensing by instantly triggering an electrical pulse perpendicular to the focused particulate flow in a double-cross microchannel. The electrokinetic manipulation of particle/cell in microchannels demonstrated in this work can be used for developing integrated lab-on-a-chip devices for studies of cells.     

Microfluidic system for dielectrophoretic separation based on a trapezoidal electrode array

This paper presents a novel microfluidic device for dielectrophoretic separation based on a trapezoidal electrode array (TEA). In this method, particles with different dielectric properties are separated by the device composed of the TEA for the dielectrophoretic deflection of particles under negative dielectrophoresis (DEP) and poly(dimethylsiloxane)(PDMS) microfluidic channel with a sinuous and expanded region. Polystyrene microparticles are exposed to an electric field generated from the TEA in the microfluidic channel and are dielectrophoretically focused to make all of them line up to one sidewall. When these particles arrive at the region of another TEA for dielectrophoretic separation, they are separated having different positions along the perpendicular direction to the fluid flow due to their different dielectrophoretic velocities. To evaluate the separation process and performance, both the effect of the flow rate on dielectrophoretic focusing and the influence of the number of trapezoidal electrodes on dielectrophoretic separation are investigated. Now that this method utilizes the TEA as a source of negative DEP, non-specific particle adhering to the electrode surface can be prevented; conventional separation approaches depending on the positive DEP force suffer from this problem. In addition, since various particle types are continuously separated, this method can be easily applicable to the separation and analysis of various dielectric particles with high particle recovery and selectivity.     

Analytical solutions and validation of electric field and dielectrophoretic force in a bio-microfluidic channel

In a microbiological device, cell or particle manipulation and characterization require the use of electric field on different electrodes in several configurations and shapes. To efficiently design microelectrodes within a microfluidic channel for dielectrophoresis focusing, manipulation and characterization of cells, the designer will seek the exact distribution of the electric potential, electric field and hence dielectrophoresis force exerted on the cell within the microdevice. In this paper we describe the approach attaining the analytical solution of the dielectrophoretic force expression within a microchannel with parallel facing same size electrodes present on the two faces of channel substrates, with opposite voltages on the pair electrodes. Simple Fourier series mathematical expressions are derived for electric potential, electric field and dielectric force between two distant finite‐size electrodes. Excellent agreement is found by comparing the analytical results calculated using MATLAB? with numerical ones obtained by Comsol. This analytical result can help the designer to perform simple design parametric analysis. Bio‐microdevices are also designed and fabricated to illustrate the theoretical solution results with the experimental data. Experiments with red blood cells show the dielectrophoretic force contour plots of the analytical data matched to the experimental results.     

On-chip microfluidic buffer swap of biological samples in-line with downstream dielectrophoresis

Microfluidic cell enrichment by dielectrophoresis, based on biophysical and electrophysiology phenotypes, requires that cells be resuspended from their physiological media into a lower conductivity buffer for enhancing force fields and enabling the dielectric contrast needed for separation. To ensure that sensitive cells are not subject to centrifugation for resuspension and spend minimal time outside of their culture media, we present an on-chip microfluidic strategy for swapping cells into media tailored for dielectrophoresis. This strategy transfers cells from physiological media into a 100-fold lower conductivity media by using tangential flows of low media conductivity at 200-fold higher flow rate versus sample flow to promote ion diffusion over the length of a straight channel architecture that maintains laminarity of the flow-focused sample and minimizes cell dispersion across streamlines. Serpentine channels are used downstream from the flow-focusing region to modulate hydrodynamic resistance of the central sample outlet versus flanking outlets that remove excess buffer, so that cell streamlines are collected in the exchanged buffer with minimal dilution in cell numbers and at flow rates that support dielectrophoresis. We envision integration of this on-chip sample preparation platform prior to or post-dielectrophoresis, in-line with on-chip monitoring of the outlet sample for metrics of media conductivity, cell velocity, cell viability, cell position, and collected cell numbers, so that the cell flow rate and streamlines can be tailored for enabling dielectrophoretic separations from heterogeneous samples.     

Bovine red blood cell starvation age discrimination through a glutaraldehyde-amplified dielectrophoretic approach with buffer selection and membrane cross-linking

We report a novel buffer electric and dielectric relaxation time tuning technique, coupled with a glutaraldehyde (Glt.) cross-linking cell fixation reaction that allows for sensitive dielectrophoretic analysis and discrimination of bovine red blood cells of different starvation age. Guided by a single-shell oblate spheroid model, a zwitterion buffer composition is selected to ensure that two measurable crossover frequencies (cof's) near 500 kHz exist for dielectrophoresis (DEP) within a small range of each other. It is shown that the low cof is sensitive to changes in the cell membrane dielectric constant, in which cross-linking by Glt. reduces the dielectric constant of the cell membrane from 10.5 to 3.8, while the high cof is sensitive to cell cytoplasm conductivity changes. We speculate that this enhanced particle polarizability that results from the cross-linking reaction is because younger (reduced starvation time) cells possess more amino groups that the reaction can release to enhance the cell interior ionic strength. Such sensitive discrimination of cells with different age (surface protein density) by DEP is not possible without the zwitterion buffer and cleavage by Glt. treatment. It is then expected that rapid identification and sorting of healthy from diseased cells can be similarly sensitized.     

Dielectrophoretic force microscopy of aqueous interfaces

A novel scanning probe microscopy technique has allowed dielectrophoretic force imaging with nanoscale spatial resolution. Dielectrophoresis (DEP) traditionally describes the mobility of polarizable particles in inhomogeneous alternating current (ac) electric fields. Integrating DEP with atomic force microscopy allows for noncontact imaging with the image contrast related to the local electric polarizability. By tuning the ac frequency, dielectric spectroscopy can be performed at solid/liquid interfaces with high spatial resolution. In studies of cells, the frequency-dependent dielectrophoretic force is sensitive to biologically relevant electrical properties, including local membrane capacitance and ion mobility. Consequently, dielectrophoretic force microscopy is well suited for in vitro noncontact scanning probe microscopy of biological systems.     

Microfluidic biomechanical assay for red blood cells parasitized by Plasmodium falciparum

Red blood cells parasitized by Plasmodium falciparum can be distinguished from uninfected cells and characterized on the basis of reduced deformability. To enable improved and simplified analysis, we developed a microfluidic device to measure red blood cell deformability using precisely controlled pressure. Individual red blood cells are deformed through multiple funnel-shaped constrictions with openings ranging from 5 down to 1 μm. Precisely controlled pressures are generated on-chip using a microfluidic circuit that attenuates an externally applied pressure by a factor of 100. The pressures required to squeeze each cell through the constriction are used as a readout to determine the intrinsic stiffness of each cell. Using this method, parasitized cells from ring through schizont stages were shown to be 1.5 to 200 times stiffer than uninfected cells. The measured deformability values of uninfected and parasitized cells showed clearly distinct distributions, demonstrating the potential of using this technique to study the pathophysiology of this disease, and the effect of potential drugs.     

A dielectrophoretic continuous flow sorter using integrated microelectrodes coupled to a channel constriction

In this article, we propose a novel dielectrophoretic continuous flow sorter using planar micro electrodes coupled to a channel constriction. This design enables a high particle sorting efficiency at low voltages while relying on a simple fabrication and integration process. We have numerically simulated the AC electrokinetic effects and the fluid behavior to predict particle trajectories. Simulation results are in accordance with experimental data: 10 and 5 μm polystyrene beads were continuously sorted with <2% errors at flow speeds of 100 μm/s. We were also able to change the particle buffer while sorting beads. Finally, to demonstrate the interest of our device for cell sorting, we also sorted dead and living yeast cells according to their different dielectric properties. Living cell concentration was enriched by a factor of 4 versus dead cell concentration after passing the sorting device.     

Explorations of ABO-Rh antigen expressions on erythrocyte dielectrophoresis: changes in cross-over frequency

A quadrupole dielectrophoretic microdevice was utilized to examine the ABO-Rh dependencies on erythrocyte polarizations. This important step toward medical microdevice technology would transform key clinical blood tests from the laboratory into the field. Previous work in dielectrophoretic microdevices demonstrated that the large number of ABO antigens on erythrocyte membranes impacts their dielectrophoretic signature at 1 MHz. This work explores the dielectrophoretic behavior of native human erythrocytes categorized by their ABO-Rh blood types and directly compares these responses to the same erythrocyte sample modified to remove the A and B antigens. A β(1-3)-galactosidase enzyme was utilized to cleave the ABO polysaccharide backbone at the galactosidase bonds. The enzymatic reaction was optimized by comparing agglutination of the native and modified blood cells in addition to UV-Vis and HPLC analysis of the reaction effluent for saccharide residues. Next, the dielectrophoretic behaviors of the native and modified erythrocytes were visually verified in a quadrupole electrode microdevice over a frequency range from 100?kHz to 80?MHz. The lower cross-over frequency (COF), which transitions from negative to positive dielectrophoresis, for ABO blood types tested (A+, A-, B+, B-, AB+, O+ and O-) differed over the range from 17 to 47?MHz. The COFs of the corresponding enzyme-modified erythrocytes were also determined and the range narrowed to 29-41 MHz. A second COF in the 70-80?MHz range was observed and was reduced in the presence of the transmembrane Rhesus factor. These results suggest that antigen expression on erythrocyte membrane surfaces influence cell polarizations in nonuniform AC fields.     

Highlighting the uniqueness in dielectrophoretic enrichment of circulating tumor cells

Circulating tumor cells (CTCs) play an essential role in the metastasis of tumors, and thus can serve as a valuable prognostic factor for malignant diseases. As a result, the ability to isolate and characterize CTCs is essential. This review underlines the potential of dielectrophoresis for CTCs enrichment. It begins by summarizing the key performance parameters and challenges of CTCs isolation using microfluidics. The two main categories of CTCs enrichment—affinity‐based and label‐free methods—are analysed, emphasising the advantages and disadvantages of each as well as their clinical potential. While the main argument in favour of affinity‐based methods is the strong specificity of CTCs isolation, the major advantage of the label‐free technologies is in preserving the integrity of the cellular membrane, an essential requirement for downstream characterization. Moving forward, we try to answer the main question: “What makes dielectrophoresis a method of choice in CTCs isolation?” The uniqueness of dielectrophoretic CTCs enrichment resides in coupling the specificity of the isolation process with the conservation of the membrane surface. The specificity of the dielectrophoretic method stems from the differences in the dielectric properties between CTCs and other cells in the blood: the capacitances of the malignantly transformed cellular membranes of CTCs differ from those of other cells. Examples of dielectrophoretic devices are described and their performance evaluated. Critical requirements for using dielectrophoresis to isolate CTCs are highlighted. Finally, we consider that DEP has the potential of becoming a cytometric method for large‐scale sorting and characterization of cells.     

Biomolecule detection via target mediated nanoparticle aggregation and dielectrophoretic impedance measurement

A new biosensing system is described that is based on the aggregation of nanoparticles by a target biological molecule and dielectrophoretic impedance measurement of these aggregates. The aggregation process was verified within a microchannel via fluorescence microscopy, demonstrating that this process can be used in a real time sensor application. Positive dielectrophoresis is employed to capture the nanoparticle aggregates at the edge of thin film electrodes, where their presence is detected either by optical imaging via fluorescence microscopy or by measuring the change in electrical impedance between adjacent electrodes. The electrical detection mechanism demonstrates the potential for this method as a micro total analysis system (microTAS).     

Manipulation and characterization of red blood cells with alternating current fields in microdevices

The motion of a suspension of erythrocytes (red blood cells, RBCs) in response to a high-frequency alternating current (AC) field in a microfluidic device is examined with parallel and orthogonal electrode configurations to delineate the various fundamental driving forces. Cell repulsion from the platinum electrodes due to electrode polarization interacting with cell membrane polarizations is observed to be the strongest force acting on the particles in the first few seconds of field application. We exploit this strong repulsion to concentrate the bioparticles between the microelectrodes to amplify multiparticle aggregation phenomenon and dielectrophoretic (DEP) manipulation in a small and well-characterized region within the microfluidic device. Secondary motions include RBC pearl chain formation along field lines due to particle polarization followed by classical dielectrophoretic motion of the chains across field lines to regions of weaker field. These are driven by far weaker dipole-dipole and field-dipole interactions than the preliminary electrode repulsions. RBC chain length and total aggregated cells are presented for a variety of AC frequencies and are significantly amplified by the electrode repulsion. Motion of particles away from the polarized electrode is found to be species- and age-sensitive and can stand by itself as a promising identification and separation mechanism. In a 0.1 S/m isotonic phosphate buffer saline medium, we observe the largest cell mobilities at an optimal frequency of approximately 1 MHz, corresponding to the inverse diffusion time across the double layer of the cell and across the electrode's polarized layer. This suggests that the dielectric responses of both particles and electrodes in the low MHz frequency range are mostly determined by normal electromigration of ions from the bulk to their interfaces. Sensitivity to RBC age and species suggests that the surface proteins and membrane ion channels can affect the capacitance of the interface to accommodate the ions from the bulk. Such surface ion accumulation and polarization mechanisms are different from the classical dielectric theories. The resonant frequency of electrode polarization at around 1 MHz falls between positive and negative dielectrophoretic resonant frequency peaks - suggesting that the double-layer polarization mechanism is a distinct and potentially important bioparticle manipulation tool.     

Isolation of prostate tumor initiating cells (TICs) through their dielectrophoretic signature

In this study, the dielectrophoretic response of prostate tumor initiating cells (TICs) was investigated in a microfluidic system utilizing contactless dielectrophoresis (cDEP). The dielectrophoretic response of prostate TICs was observed to be distinctively different than that for non-TICs, enabling them to be sorted using cDEP. Culturing the sorted TICs generated spheroids, indicating that they were indeed initiating cells. This study presents the first marker-free TIC separation from non-TICs utilizing their electrical fingerprints through dielectrophoresis.     

Frequency discretization in dielectrophoretic assisted cell sorting arrays to isolate neural cells

We present an automated dielectrophoretic assisted cell sorting (DACS) device for dielectric characterization and isolation of neural cells. Dielectrophoretic (DEP) principles are often used to develop cell sorting techniques. Here we report the first statistically significant neuronal sorting using DACS to enrich neurons from a heterogeneous population of mouse derived neural stem/progenitor cells (NSPCs) and neurons. We also study the dielectric dispersions within a heterogeneous cell population using a Monte-Carlo (MC) simulation. This simulation model explains the trapping behavior of populations as a function of frequency and predicts sorting efficiencies. The platform consists of a DEP electrode array with three multiplexed trapping regions that can be independently activated at different frequencies. A novel microfluidic manifold enables cell sorting by trapping and collecting cells at discrete frequency bands rather than single frequencies. The device is used to first determine the percentage of cells trapped at these frequency bands. With this characterization and the MC simulation we choose the optimal parameters for neuronal sorting. Cell sorting experiments presented achieve a 1.4-fold neuronal enrichment as predicted by our model.     

Capacitance-based assay for real-time monitoring of endocytosis and cell viability

Label-free cell-based assays have emerged as a promising means for high-throughput screening. Most label-free sensors are based on impedance measurements that reflect the passive electrical properties of cells. Here we introduce a capacitance-based assay that measures the dielectric constant (capacitance) of biological cells, and demonstrate the feasibility of analyzing endocytosis and screening chemotherapeutic agents with this assay. Endocytosis induces a change in the zeta potential, leading to a change in the dielectric constant which enables real-time endocytosis monitoring using the capacitance sensor. Additionally, since the dielectric constant is proportional to cell radius and cell volume, cell viability can be estimated from the change in capacitance. Therefore, the capacitance sensor array can also be used for cytotoxicity testing for large-scale chemotherapeutic screening.