Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).     

An insulator-based (electrodeless) dielectrophoretic concentrator for microbes in water

Dielectrophoresis (DEP), the motion of a particle caused by an applied electric field gradient, can concentrate microorganisms non-destructively. In insulator-based dielectrophoresis (iDEP) insulating microstructures produce non-uniform electric fields to drive DEP in microsystems. This article describes the performance of an iDEP device in removing and concentrating bacterial cells, spores and viruses while operated with a DC applied electric field and pressure gradient. Such a device can selectively trap particles when dielectrophoresis overcomes electrokinesis or advection. The dielectrophoretic trapping behavior of labeled microorganisms in a glass-etched iDEP device was observed over a wide range of DC applied electric fields. When fields higher than a particle-specific threshold are applied, particles are reversibly trapped in the device. Experiments with Bacillus subtilis spores and the Tobacco Mosaic Virus (TMV) exhibited higher trapping thresholds than those of bacterial cells. The iDEP device was characterized in terms of concentration factor and removal efficiency. Under the experimental conditions used in this study with an initial dilution of 1 x 105 cells/ml, concentration factors of the order of 3000x and removal efficiencies approaching 100% were observed with Escherichia coli cells. These results are the first characterization of an iDEP device for the concentration and removal of microbes in water.     

Investigation of a two‐step device implementing magnetophoresis and dielectrophoresis for separation of circulating tumor cells from blood cells

Identifying tumor cells from a pool of other cells has always been an appealing topic for different purposes. The objective of this study is to discriminate circulating tumor cells (CTCs) from blood cells for diagnostic purposes in a novel microfluidic device using two active methods: magnetophoresis and dielectrophoresis. The most specific feature of this device is the differentiation of CTCs without labeling them in order to achieve a more reliable and less complicated method. This device was analyzed and evaluated using finite element method. Four cell lines are separated in this device containing red blood cells, platelets, white blood cells, and CTCs. Primarily, red blood cells and platelets, which constitute the largest part of a blood sample, are removed in the magnetophoresis section. Remaining cells enter the dielectrophoresis part and based on their inherent dielectric properties and diameters, final separation occurs. In each step, different parameters are examined to obtain the maximum purification. The results demonstrate the potential of different CTCs separation by changing the effective parameters in the designed device based on the inherent properties of the cells.     

Dielectrophoretic separation of monocytes from cancer cells in a microfluidic chip using electrode pitch optimization

This study proposes a microfluidic device capable of separating monocytes from a type of cancer cell that is called T-cell acute lymphoblastic leukemia (RPMI-8402) in a continuous flow using negative and positive dielectrophoretic forces. The use of both the hydrodynamic and dielectrophoretic forces allows the separation of RPMI-8402 from monocytes based on differences in their intrinsic electrical properties and sizes. The specific crossover frequencies of monocytes and RPMI-8402 cells have been obtained experimentally. The optimum ranges of electrode pitch-to-channel height ratio at the cross sections with different electrode widths have been generally calculated by numerical simulations of the gradients of the electric field intensities and calculation their effective values (root-mean-square). In the device, the cell sorting has been conducted empirically, and then, the separation performance has been evaluated by analyzing the images before and after dielectrophoretic forces applied to the cells. In this work, the design of a chip with 77 μm gold–titanium electrode pitch was investigated to achieve high purity of monocytes of 95.2%. The proposed device can be used with relatively low applied voltages, as low as 16.5 V (peak to peak). Thus, the design can be used in biomedical diagnosis and chemical analysis applications as a lab-on-chip platform. Also, it can be used for the separation of biological cells such as bacteria, RNA, DNA, and blood cells.

     

Multiphase electrodes for microbead control applications: integration of DEP and electrokinetics for bio-particle positioning

Advances in microfabrication have introduced new possibilities for automated, high-throughput biomedical investigations and analysis. Physical effects such as dielectrophoresis (DEP) and AC electrokinetics can be used to manipulate particles in solution to coordinate a sequence of bioanalytical processing steps. DEP is accomplished with non-uniform electric fields that can polarize particles (microbeads, cells, viruses, DNA, proteins, etc.) in suspension causing translational or rotational movement. AC electrokinetics is another phenomena involved with movement of particles in suspension with electric fields and is comprised of both electro-thermal and electro-osmotic effects. This paper investigates single layer electrodes that are effective for particle localization and clustering based on DEP and AC electrokinetic effects. We demonstrate a novel multi-electrode setup capable of clustering particles into an array of discrete bands using activated and electrically floating electrodes. These bands shift to adjacent regions on the electrode surface by altering the electrode activation scheme. The predictability of particle placement to specific locations provides new opportunities for integration and coordination with raster scanning lasers or a charge coupled device (CCD) for advanced biomedical diagnostic devices, and more sophisticated optical interrogation techniques.     

Self-Rotation of Cells in an Irrotational AC E-Field in an Opto-Electrokinetics Chip

The use of optical dielectrophoresis (ODEP) to manipulate microparticles and biological cells has become increasingly popular due to its tremendous flexibility in providing reconfigurable electrode patterns and flow channels. ODEP enables the parallel and free manipulation of small particles on a photoconductive surface on which light is projected, thus eliminating the need for complex electrode design and fabrication processes. In this paper, we demonstrate that mouse cells comprising melan-a cells, RAW 267.4 macrophage cells, peripheral white blood cells and lymphocytes, can be manipulated in an opto-electrokinetics (OEK) device with appropriate DEP parameters. Our OEK device generates a non-rotating electric field and exerts a localized DEP force on optical electrodes. Hitherto, we are the first group to report that among all the cells investigated, melan-a cells, lymphocytes and white blood cells were found to undergo self-rotation in the device in the presence of a DEP force. The rotational speed of the cells depended on the voltage and frequency applied and the cells'' distance from the optical center. We discuss a possible mechanism for explaining this new observation of induced self-rotation based on the physical properties of cells. We believe that this rotation phenomenon can be used to identify cell type and to elucidate the dielectric and physical properties of cells.     

Identification, characterization and manipulation of Babesia-bovis-infected red blood cells using microfluidics technology

Nowadays numerous microfluidic systems are being developed to address a variety of clinical problems. Latest advances in microfluidic technology are promising to revolutionize the detection of pathogens in vivo through the development of integrated lab-on-chip devices. Such microfabricated systems will undertake all steps in sample analysis from collection and preparation to molecular detection. Micro total analysis systems are suitable candidates for point of care diagnostics due to small size, low cost production and enabled portability. The work here presented aimed the use of microfluidic platforms to identify and manipulate bovine red blood cells infected by the protozoan parasite Babesia bovis. A microfabricated device based on impedance spectroscopy was used for single cell discrimination and its sensitivity and applicability as a diagnostic method for bovine babesiosis was studied. Furthermore, manipulation and sorting of normal and infected red blood cells was performed on a dielectrophoresis based microfabricated cell cytometer. Single cell analysis of normal and B. bovis infected red blood cells was performed by electrorotation and dielectric parameters such as permittivities and conductivities of the cellular membrane and cytoplasm were determined.     

An assay for dielectrophoresis: Applications to electromagnetically induced membrane adhesion and fusion

A new assay for dielectrophoresis, consisting of a platinum wire placed along the axis of symmetry of a hollow metal cylinder, was constructed. The main advantage of this device is that because of the simple axisymmetrical electric field the experimental data can be easily interpreted. Preliminary experimental data for adhesion and fusion of red blood cells are reported. The most interesting observation was that fusion of cell membranes can be induced without applying DC electrical pulses, merely because of the dielectrophoretic effect. A possible explanation of this phenomenon is suggested.     

Dielectrophoretic study of human erythrocytes

This paper is concerned with the dielectrophoretic study of human erythrocytes under cylindrical field geometry. The influence of physical variables such as the frequency and voltage of the applied electric field, conductivity of the medium in which the cells are suspended, cell concentration and exposure time of the cell to the non-uniform electric field on dielectrophoretic collection rate (DCR) is determined in a systematic manner. It is interesting to note from the DCR spectrum of human erythrocytes that the DCR is minimum at one frequency, maximum at another and there is practically no yield over a certain frequency range. This may be attributed to the variation of complex dielectric constant of the particle and medium over that frequency range. From the DCR spectrum of different groups, it is clear that DCR behaviour is different in the frequency range from 0.3–1.5 MHz, under similar conditions of temperature, conductivity and concentration of erythrocyte suspension and strength of applied AC field. The response of DCR with voltage of the applied field, concentration of cell suspension and square root of elapsed time of the cells confirms the theory of dielectrophoresis.     

Single-cell trapping utilizing negative dielectrophoretic quadrupole and microwell electrodes

The handling of individual cells, which has attracted increasing attention, is a key technique in cell engineering such as gene introduction, drug injection, and cloning technology. Alternating current (AC) electrokinetics has shown great potential for microfluidic functions such as pumping, mixing, and concentrating particles. The non-uniform electric field gives rise to Joule heating and dielectrophoresis (DEP). The motion of particles suspended in the medium can be influenced directly, by means of dielectrophoretic effects, and indirectly, via fluid flow through a viscous drag force that affects the particles. Thus alternating current electrothermal effect (ACET) induced flow and DEP force can be combined to manipulate and trap single particles and cells. This study presents a microfluidic device which is capable of specifically guiding and capturing single particles and cells by ACET fluid flow and the negative dielectrophoretic (nDEP) trap, respectively. The experiment was operated at high frequencies (5–12 MHz) and in a culture medium whose high conductivity (σ = 1.25 S/m) is of interest to biochemical analysis and environmental monitoring, which are both prone to producing ACET and nDEP. Manipulation of particle motion using ACET-induced fluid flow to the target trap is modeled numerically and is in good agreement with the experimental results.     

Comprehensive Analysis of Human Cells Motion under an Irrotational AC Electric Field in an Electro-Microfluidic Chip

AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines.     

Negative dielectrophoretic patterning with different cell types

In this paper, a novel method for patterning different cell types based on negative dielectrophoresis (n-DEP), without any special pretreatment of a culture slide, has been described. An interdigitated array (IDA) electrode with four independent microelectrode subunits was fabricated with indium-tin-oxide (ITO) and used as a template to form cellular micropatterns. A suspension of C2C12 cells was introduced into the patterning device between the upper slide and the bottom IDA. In the present system, the n-DEP force is induced by applying an ac voltage (typically 12V(pp), 1MHz) to direct cells toward a weaker region of electric field strength. The cells aligned above one of the bands of IDA within 1min since the aligned areas on the slide were regions with the lower electric field. The application of an ac voltage for 5min allows the cells to adsorb onto the cell culture slide. After removing excess cells, the second cell type was patterned in lines using the same method as with the first set of cells. Periodic and alternate cell lines incorporating two cell types were also fabricated by changing the ac voltage mode. A second cell type was introduced into the device and guided to other areas to form a different pattern. The described system enables two cell types to be patterned in 15min. The patterning method provides a novel tool for use in fundamentals studies of cell biology based on cell-cell interactions between different cell types.     

Dynamic Dielectrophoresis Model of Multi-Phase Ionic Fluids

Ionic-based dielectrophoretic microchips have attracted significant attention due to their wide-ranging applications in electro kinetic and biological experiments. In this work, a numerical method is used to simulate the dynamic behaviors of ionic droplets in a microchannel under the effect of dielectrophoresis. When a discrete liquid dielectric is encompassed within a continuous fluid dielectric placed in an electric field, an electric force is produced due to the dielectrophoresis effect. If either or both of the fluids are ionic liquids, the magnitude and even the direction of the force will be changed because the net ionic charge induced by an electric field can affect the polarization degree of the dielectrics. However, using a dielectrophoresis model, assuming ideal dielectrics, results in significant errors. To avoid the inaccuracy caused by the model, this work incorporates the electrode kinetic equation and defines a relationship between the polarization charge and the net ionic charge. According to the simulation conditions presented herein, the electric force obtained in this work has an error exceeding 70% of the actual value if the false effect of net ionic charge is not accounted for, which would result in significant issues in the design and optimization of experimental parameters. Therefore, there is a clear motivation for developing a model adapted to ionic liquids to provide precise control for the dielectrophoresis of multi-phase ionic liquids.     

A novel microfluidic impedance assay for monitoring endothelin-induced cardiomyocyte hypertrophy

Cardiac hypertrophy is an established and independent risk factor for the development of heart failure and sudden cardiac death. At the level of individual cardiac myocytes (heart muscle cells), the cell morphology alters (increase in cell size and myofibrillar re-organization) and protein synthesis is activated. In this paper, a novel cardiomyocyte-based impedance sensing system with the assistance of dielectrophoresis cell concentration is reported to monitor the dynamic process of endothelin-1-induced cardiomyocyte hypertrophy. A dielectrophoresis (DEP) microfluidic device is fabricated capable of concentrating cells from a dilute sample to form a confluent cell monolayer on the surface of microelectrodes. This device can increase the sensitivity of the impedance system and also has the potential to reduce the time for detection by a significant factor. To examine the feasibility of this impedance sensing system, cardiomyocytes are treated with endothelin-1 (ET-1), a known hypertrophic agent. ET-1 induces a continuous rise in cardiomyocyte impedance, which we interpret as strengthening of cellular attachments to the surface substrate. An equivalent circuit model is introduced to fit the impedance spectrum to fully understand the impedance sensing system.     

A new principle of cell sorting by using selective electroporation in a modified flow cytometer

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill. We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 microseconds and resulting field strengths of 2.0 and 3.2 x 10(6) V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.). The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%.     

Tissue engineering with electric fields: immobilization of mammalian cells in multilayer aggregates using dielectrophoresis

Positive dielectrophoresis can be used to create aggregates of animal cells with 3D architectures. It is shown that the cells, when pulled together into an aggregate by positive dielectrophoresis in a low-conductivity iso-osmotic solution, adhere to each other. The adherence of the cells to each other is non-specific and increases in time, and after 10-15 min becomes strong enough to immobilize the cells in the aggregate, enabling the ac electric field to be released, and the iso-osmotic buffer to be replaced by growth or other media. Cell viability is maintained. The new method of immobilization significantly simplifies the construction of aggregates of animal cells by dielectrophoresis, and increases the utility of dielectrophoresis in tissue engineering and related areas.     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0 We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.     

Effects of pH on cell fusion induced by electric fields

Electrofusion has recently become an important area of cell biology research. We studied the effects of pH of the cell medium on the electrofusion of human red blood cells. Cell fusion was monitored by observing the movement of a lipophylic dye between neighboring fused cells using a fluorescence microscope. The cells were first brought into close contact by dielectrophoresis. Fusion was then induced by three pulses of high-intensity electric field. Within minutes following the pulse application, many cells were observed to fuse together to form fusion chains of different lengths. We found that the optimal pH for cell fusion is around pH 7.5. At this pH, the fusion yield was highest (ranging from 57 to 81%) and the average number of cells within a fusion chain was also the largest. The dependence of cell fusion on pH is more sensitive at low than at high pH. The fusion yield was decreased by 40% when the pH was changed from 7.5 to 6.0, but there was only a 20% decrease in yield between pH 7.5 and 10.0. We suspect that the observed pH effects may be caused by a redistribution of fixed charges at the cell surface, or changes in amphipathicity of the surface proteins.     

Electrofusion of fibroblasts on the porous membrane

Electric fusion of cells is usually performed in two steps: the first is the creation of tight intercellular contact, the second is an application of electric pulses which induce membrane fusion proper. In the present work a new technique of cell electrofusion on the porous film is described. It consists of preliminary cultivation of cell monolayer on the porous film (protein-coated cellophane). Then cells of the same or any other type are added from above to form a second cell layer upon the first one. The pulses of the electric field are applied normally to the plane of the double cell layer to induce cell fusion. After pulse application a picture of mass polynucleation was observed. At the same time we did not obtain fusion of L cells by means of dielectrophoretic electrofusion technique. This difference in efficiency could be explained by the formation of broad zones of membrane contact between the cells adherent to the film, while during intensive dielectrophoresis only the point contacts were revealed. The high-conducting medium for electric treatment providing an efficient fusion on the film and high cell viability was composed. Neither cytochalasin B nor colcemid affected cell fusion noticeably; however the sodium azide (added with 2-deoxyglucose) inhibited fusion completely. The short hypotonic shock after electric treatment enhanced the rate of polycaryon formation.     

Flow cytometer based on triggered supercontinuum laser illumination

Multiple wavelength operation in a flow cytometer is an exciting way for cell analysis based on both fluorescence and optical scattering processing. For example, this multiparametric technique is currently used to differentiate blood cells subpopulations. The choice of excitation wavelengths matching fluorochrome spectra (it is currently the opposite) and the use of a broader range of fluorochromes can be made by taking advantage of a filtered supercontinuum white light source. In this study, we first wished to validate the use of a specific triggered supercontinuum laser in a flow cytometer based on white light scattering and electric sizing on human blood cells. Subsequently, to show the various advantages of this attractive system, using scattering effect, electrical detections, and fluorescence analysis, we realized cells sorting based on DNA/RNA stained by thiazole orange. Discrimination of white blood cells is efficiently demonstrated by using a triggered supercontinuum-based flow cytometer operating in a "one cell-one shot" configuration. The discriminated leukocyte populations are monocytes, lymphocytes, granulocytes, immature granulocytes, and cells having a high RNA content (monoblasts, lymphoblasts, and plasma cells). To the best of our knowledge, these results constitute the first practical demonstration of flow cytometry based on triggered supercontinuum illumination. This study is the starting point of a series of new experiments fully exploiting the spectral features of such a laser source. For example, the large flexibility in the choice of the excitation wavelength allows to use a larger number of fluorochromes and to excite them more efficiently. Moreover, this work opens up new research directions in the biophotonics field, such as the combination of coherent Raman spectroscopy and flow cytometry techniques.