Breast cancer cell obatoclax response characterization using passivated‐electrode insulator‐based dielectrophoresis

Inherent electrical properties of cells can be beneficial to characterize different cell lines and their response to experimental drugs. This paper presents a novel method to characterize the response of breast cancer cells to drug stimuli through use of off‐chip passivated‐electrode insulator‐based dielectrophoresis (OπDEP) and the application of AC electric fields. This work is the first to demonstrate the ability of OπDEP to differentiate between two closely related breast cancer cell lines, LCC1 and LCC9 while assessing their drug sensitivity to an experimental anti‐cancer agent, Obatoclax. Although both cell lines are derivatives of estrogen‐responsive MCF‐7 breast cancer cells, growth of LCC1 is estrogen independent and anti‐estrogen responsive, while LCC9 is both estrogen‐independent and anti‐estrogen resistant. Under the same operating conditions, LCC1 and LCC9 had different DEP profiles. LCC1 cells had a trapping onset (crossover) frequency of 700 kHz and trapping efficiencies between 30–40%, while LCC9 cells had a lower crossover frequency (100 kHz) and showed higher trapping efficiencies of 40–60%. When exposed to the Obatoclax, both cell lines exhibited dose‐dependent shifts in DEP crossover frequency and trapping efficiency. Here, DEP results supplemented with cell morphology and proliferation assays help us to understand the response of these breast cancer cells to Obatoclax.     

A Review of Multifunctions of Dielectrophoresis in Biosensors and Biochips for Bacteria Detection

This paper reviews the functions of dielectrophoresis (DEP) that have been applied to biosensor and biochip platforms for bacteria detection, including concentration of bacterial cells from continuous flows, separation of target bacterial cells from non-target cells, as well as the enhancement of antibody capture efficiency on biosensor and biochip surfaces. DEP could provide effective concentration and separation simultaneously in well-designed microfluidic biosensor and biochip systems. The integration of DEP with a detection system allows the integration of sample preparation and enrichment steps with detection, which has the potential to eliminate the traditionally used time-consuming culture-based enrichment steps and other multiple off-chip sample preparation steps. DEP is also useful in biosensor and biochips platforms for enhancing antibody capture efficiency in both flow-through and non-flow-through microdevices. The enhanced antibody capture efficiency could allow the sensor capture more cells and to be detected by the sensor, particularly in dealing with low number of cells. The integration of multifunctions of DEP into biosensor and biochip platform has the potential to improve the detection of bacterial cells.     

Numerical study of dielectrophoresis-modified inertial migration for overlapping sized cell separation

Circulating tumor cells (CTCs) have been proven to have significant prognostic, diagnostic, and clinical values in early-stage cancer detection and treatment. The efficient separation of CTCs from peripheral blood can ensure intact and viable CTCs and can, thus, give proper genetic characterization and drug innovation. In this study, continuous and high-throughput separation of MDA-231 CTCs from overlapping sized white blood cells (WBCs) is achieved by modifying inertial cell focusing with dielectrophoresis (DEP) in a single-stage microfluidic platform by numeric simulation. The DEP is enabled by embedding interdigitated electrodes with alternating field control on a serpentine microchannel to avoid creating two-stage separation. Rather than using the electrokinetic migration of cells which slows down the throughput, the system leverages the inertial microfluidic flow to achieve high-speed continuous separation. The cell migration and cell positioning characteristics are quantified through coupled physics analyses to evaluate the effects of the applied voltages and Reynolds numbers (Re) on the separation performance. The results indicate that the introduction of DEP successfully migrates WBCs away from CTCs and that separation of MDA-231 CTCs from similar sized WBCs at a high Re of 100 can be achieved with a low voltage of magnitude 4 ×10⁶ V/m. Additionally, the viability of MDA-231 CTCs is expected to be sustained after separation due to the short-term DEP exposure. The developed technique could be exploited to design active microchips for high-throughput separation of mixed cell beads despite their significant size overlap, using DEP-modified inertial focusing controlled simply by adjusting the applied external field.     

The feasibility of using dielectrophoresis for isolation of glioblastoma subpopulations with increased stemness

Cancer stem cells (CSCs) are aggressive subpopulations with increased stem‐like properties. CSCs are usually resistant to most standard therapies and are responsible for tumor repropagation. Similar to normal stem cells, isolation of CSCs is challenging due to the lack of reliable markers. Antigen‐based sorting of CSCs usually requires staining with multiple markers, making the experiments complicated, expensive, and sometimes unreliable. Here, we study the feasibility of using dielectrophoresis (DEP) for isolation of glioblastoma cells with increased stemness. We culture a glioblastoma cell line in the form of neurospheres as an in vitro model for glioblastoma stem cells. We demonstrate that spheroid forming cells have higher expression of stem cell marker, nestin. Next, we show that dielectric properties of neurospheres change as a result of changing culture conditions. Our results indicate that spheroid forming cells need higher voltages to experience the same DEP force magnitude compared to normal monolayer cultures of glioblastoma cell line. This study confirms the possibility of using DEP to isolate glioblastoma stem cells.     

Dielectrophoresis: Developments and applications from 2010 to 2020

The 20th century has seen tremendous innovation of dielectrophoresis (DEP) technologies, with applications being developed in areas ranging from industrial processing to micro- and nanoscale biotechnology. From 2010 to present day, there have been 981 publications about DEP. Of over 2600 DEP patents held by the United States Patent and Trademark Office, 106 were filed in 2019 alone. This review focuses on DEP-based technologies and application developments between 2010 and 2020, with an aim to highlight the progress and to identify potential areas for future research. A major trend over the last 10 years has been the use of DEP techniques for biological and clinical applications. It has been used in various forms on a diverse array of biologically derived molecules and particles to manipulate and study them including proteins, exosomes, bacteria, yeast, stem cells, cancer cells, and blood cells. DEP has also been used to manipulate nano- and micron-sized particles in order to fabricate different structures. The next 10 years are likely to see the increase in DEP-related patent applications begin to result in a greater level of technology commercialization. Also during this time, innovations in DEP technology will likely be leveraged to continue the existing trend to further biological and medical-focused applications as well as applications in microfabrication. As a tool leveraged by engineering and imaginative scientific design, DEP offers unique capabilities to manipulate small particles in precise ways that can help solve problems and enable scientific inquiry that cannot be addressed using conventional methods.     

阵列叉指式芯片研究细胞介电电泳富集过程

采用阵列叉指电极介电电泳(Dielectrophoresis,DEP)芯片,构建了集成DEP芯片分析和操控系统,应用Coventorware有限元分析软件模拟分析了芯片表面的电场分布情况;以红细胞和结肠癌细胞样品为分析对象,实现了两种细胞样品在芯片上的正负介电电泳定位富集.实验发现,交流信号幅值Vp-p是决定DEP富集效率的主因,交流信号频率f和缓冲溶液是改变细胞介电电泳类型的参量;在0.9% NaCl中,施加频率为10和3 MHz、电压5 V的交流频率,结肠癌细胞的正介电电泳(Positive-dielectrophoresis, pDEP)和负介电电泳(Nagetive-dielectrophoresis, nDEP)富集效率分别为87.2%和84.8%.     

A multifunctional micro-fluidic system for dielectrophoretic concentration coupled with immuno-capture of low numbers of Listeria monocytogenes

In this study, we demonstrated a micro-fluidic system with multiple functions, including concentration of bacteria using dielectrophoresis (DEP) and selective capture using antibody recognition, resulting in a high capture efficiency of bacterial cells. The device consisted of an array of oxide covered interdigitated electrodes on a flat silicon substrate and a approximately 16 microm high and approximately 260 microm wide micro-channel within a PDMS cover. For selective capture of Listeria monocytogenes from the samples, the channel surface was functionalized with a biotinylated BSA-streptavidin-biotinylated monoclonal antibody sandwich structure. Positive DEP (at 20 V(pp) and 1 MHz) was used to concentrate bacterial cells from the fluid flow. DEP could collect approximately 90% of the cells in a continuous flow at a flow rate of 0.2 microl min(-1) into the micro-channel with concentration factors between 10(2)-10(3), in sample volumes of 5-20 microl. A high flow rate of 0.6 microl min(-1) reduced the DEP capture efficiency to approximately 65%. Positive DEP attracts cells to the edges of the electrodes where the field gradient is the highest. Cells concentrated by DEP were captured by the antibodies immobilized on the channel surface with efficiencies of 18 to 27% with bacterial cell numbers ranging from 10(1) to 10(3) cells. It was found that DEP operation in our experiments did not cause any irreversible damage to bacterial cells in terms of cell viability. In addition, increased antigen expression (antigens to C11E9 monoclonal antibody) on cell membranes was observed following the exposure to DEP.     

Removal of PCR inhibitors using dielectrophoresis as a selective filter in a microsystem

Diagnostic PCR has been used to analyse a wide range of biological materials. Conventional PCR consists of several steps such as sample preparation, template purification, and PCR amplification. PCR is often inhibited by contamination of DNA templates. To increase the sensitivity of the PCR, the removal of PCR inhibitors in sample preparation steps is essential and several methods have been published. The methods are either chemical or based on filtering. Conventional ways of filtering include mechanical filters or washing e.g. by centrifugation. Another way of filtering is the use of electric fields. It has been shown that a cell will experience a force when an inhomogeneous electric field is applied. The effect is called dielectrophoresis (DEP). The resulting force depends on the difference between the internal properties of the cell and the surrounding fluid. DEP has been applied to manipulate cells in many microstructures. In this study, we used DEP as a selective filter for holding cells in a microsystem while the PCR inhibitors were flushed out of the system. Haemoglobin and heparin - natural components of blood - were selected as PCR inhibitors, since the inhibitory effects of these components to PCR have been well documented. The usefulness of DEP in a microsystem to withhold baker's yeast (Saccharomyces cerevisiae) cells while the PCR inhibitors haemoglobin and heparin are removed will be presented and factors that influence the effect of DEP in the microsystem will be discussed. This is the first time dielectrophoresis has been used as a selective filter for removing PCR inhibitors in a microsystem.     

A dielectrophoresis-based platform of cancerous cell capture using aptamer-functionalized gold nanoparticles in a microfluidic channel

In this paper, a microfluidic chip for the manipulation and capture of cancer cells was introduced, in which the combination of dielectrophoresis (DEP) and a binding method based on chemical interactions by using cell-specific aptamers was performed to enhance the capture strength and specificity. The device has been simply constructed from a straight-channel PDMS placed on a glass substrate that has patterned electrode structures and a self-assembled monolayer of gold nanoparticles (AuNPs). The target cells were transported to the manipulation area by flow and attracted down to the region between the electrodes under the influence of positive DEP force. This approach facilitated subsequent selective capture by the modified aptamers on the AuNPs. The distribution of the electric field in the channel has also been simulated to clarify the DEP operation. As a result, the device has been shown to effectively capture target lung cancer cells with a concentration as low as $2\times {10}^4$cells/mL. The capture specificity in a sample of mixed cells is up to 80.4%. This technique has the potential to be applied to detection methods for many types of cancer.     

Cellular dielectrophoresis: applications to the characterization, manipulation, separation and patterning of cells

Over the past decade, dielectrophoresis (DEP) has evolved into a powerful, robust and flexible method for cellular characterization, manipulation, separation and cell patterning. It is a field with widely varying disciplines, as it is quite common to see DEP integrated with a host of applications including microfluidics, impedance spectroscopy, tissue engineering, real-time PCR, immunoassays, stem-cell characterization, gene transfection and electroporation, just to name a few. The field is finally at the point where analytical and numerical polarization models can be used to adequately describe and characterize the dielectrophoretic behavior of cells, and there is ever increasing evidence demonstrating that electric fields can safely be used to manipulate cells without harm. As such, DEP is slowly making its way into the biological sciences. Today, DEP is being used to manipulate individual cells to specific regions of space for single-cell assays. DEP is able to separate rare cells from a heterogeneous cell suspension, where isolated cells can then be characterized and dynamically studied using nothing more than electric fields. However, there is need for a critical report to integrate the many new features of DEP for cellular applications. Here, a review of the basic theory and current applications of DEP, specifically for cells, is presented.     

Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions

In biomedical research and diagnostics, it is a significant challenge to directly isolate and identify rare cells and potential biomarkers in blood, plasma and other clinical samples. Additionally, the advent of bionanotechnology is leading to numerous drug delivery approaches that involve encapsulation of drugs and imaging agents within nanoparticles, which now will also have to be identified and separated from blood and plasma. Alternating current (AC) electrokinetic techniques such as dielectrophoresis (DEP) offer a particularly attractive mechanism for the separation of cells and nanoparticles. Unfortunately, present DEP techniques require the dilution of blood/plasma, thus making the technology less suitable for clinical sample preparation. Using array devices with microelectrodes over-coated with porous hydrogel layers, AC electric field conditions have been found which allow the separation of DNA nanoparticles to be achieved under high-conductance (ionic strength) conditions. At AC frequencies in the 3000 Hz to 10,000 Hz range and 10 volts peak-to-peak, the separation of 10-microm polystyrene particles into low field regions, and 60-nm DNA-derivatized nanoparticles and 200-nm nanoparticles into high-field regions was carried out in 149 mM 1xPBS buffer (1.68 S/m). These results may allow AC electrokinetic systems to be developed that can be used with clinically relevant samples under physiological conditions.     

Combined AC‐electrokinetic effects: Theoretical considerations on a three‐axial ellipsoidal model

AC fields induce charges at the structural interfaces of particles or biological cells. The interaction of these charges with the field generates frequency‐dependent forces that are the basis for AC‐electrokinetic effects such as dielectrophoresis (DEP), electrorotation (ROT), electro‐orientation, and electro‐deformation. The effects can be used for the manipulation or dielectric single‐particle spectroscopy. The observation of a particular effect depends on the spatial and temporal field distributions, as well as on the shape and the dielectric and viscoelastic properties of the object. Because the effects are not mutually independent, combined frequency spectra are obtained, for example, discontinuous DEP and ROT spectra with ranges separated by the reorientation of nonspherical objects in the linearly and circularly polarized DEP and ROT fields, respectively. As an example, the AC electrokinetic behavior of a three‐axial ellipsoidal single‐shell model with the geometry of chicken‐red blood cells is considered. The geometric and electric problems were separated using the influential‐radius approach. The obtained finite‐element model can be electrically interpreted by an RC model leading to an expression for the Clausius–Mossotti factor, which permits the derivation of force, torque, and orientation spectra, as well as of equations for the critical frequencies and force plateaus in DEP and of the characteristic frequencies and peak heights in ROT. Expressions for the orientation in linearly and circularly polarized fields, as well as for the reorientation frequencies were also derived. The considerations suggested that the simultaneous registration of various AC‐electrokinetic spectra is a step towards the dielectric fingerprinting of single objects.     

In vitro dielectrophoresis of HEK cell migration for stimulating chronic wound epithelialization

This article describes a dielectrophoresis (DEP)-based simulation and experimental study of human epidermal keratinocyte (HEK) cells for wounded skin cell migration toward rapid epithelialization. MyDEP is a standalone software designed specifically to study dielectric particles and cell response to an alternating current (AC) electric field. This method demonstrated that negative dielectrophoresis (N_DEP) occurs in HEK cells at a wide frequency range in highly conductive medium. The finite element method was used to characterize particle trajectory based on DEP and drag force. The performance of the system was assessed using HEK cells in a highly conductive EpiLife suspending medium. The DEP experiment was performed by applying sinusoidal wave AC potential at the peak-to-peak voltage of 10 V in a tapered aluminum microelectrode array from 100 kHz to 1 MHz. We experimentally observed the occurrence of NDEP, which attracted HEK cells toward the local electric field minima in the region of interest. The DIPP-MotionV software was used to track cell migration in the prerecorded video via an automatic marker and estimate the average speed and acceleration of the cells. The results showed that HEK cell migration was accomplished approximately at 6.43 μm/s at 100 kHz with 10 V, and F_DEP caused the cells to migrate and align at the target position, which resulted in faster wound closures because of the application of an electric field frequency to HEK cells in random locations.     

Dielectrophoresis assisted immuno-capture and detection of foodborne pathogenic bacteria in biochips

This study integrated dielectrophoresis (DEP) with non-flow through biochips to enhance the immuno-capture and detection of foodborne pathogenic bacteria. It demonstrated two major functions provided by DEP to improve the chip performance: (i) concentrating bacterial cells from the suspension to different locations on the chip surface by positive and negative DEP; (ii) making the cells in close contact with the immobilized antibodies on the chip surface so that immuno-capture efficiency can be dramatically enhanced.The microchip achieved the immuno-capture efficiencies of ∼56.0% and ∼64.0% to Salmonella cells with 15 and 30 min DEP, respectively, which were considerably higher than those of ∼10.4% and ∼17.6% for 15 and 30 min immuno-capture without DEP. The immuno-captured bacterial cells were detected by the sandwich format ELISA on the chips. The final absorbance signals were enhanced by DEP assisted immuno-capture by 64.7-105.2% for the samples containing 10³-10⁶ cells/20 μl. The integration of DEP with the biochips has the potential to advance the chip-based immunoassay methods for microbial detection.     

Characterization of a hybrid dielectrophoresis and immunocapture microfluidic system for cancer cell capture

The capture of circulating tumor cells (CTCs) from cancer patient blood enables early clinical assessment as well as genetic and pharmacological evaluation of cancer and metastasis. Although there have been many microfluidic immunocapture and electrokinetic techniques developed for isolating rare cancer cells, these techniques are often limited by a capture performance tradeoff between high efficiency and high purity. We present the characterization of shear‐dependent cancer cell capture in a novel hybrid DEP–immunocapture system consisting of interdigitated electrodes fabricated in a Hele‐Shaw flow cell that was functionalized with a monoclonal antibody, J591, which is highly specific to prostate‐specific membrane antigen expressing prostate cancer cells. We measured the positive and negative DEP response of a prostate cancer cell line, LNCaP, as a function of applied electric field frequency, and showed that DEP can control capture performance by promoting or preventing cell interactions with immunocapture surfaces, depending on the sign and magnitude of the applied DEP force, as well as on the local shear stress experienced by cells flowing in the device. This work demonstrates that DEP and immunocapture techniques can work synergistically to improve cell capture performance, and it will aid in the design of future hybrid DEP–immunocapture systems for high‐efficiency CTC capture with enhanced purity.     

Bacteria and cancer cell pearl chain under dielectrophoresis

In this work, we aim to observe and study the physics of bacteria and cancer cells pearl chain formation under dielectrophoresis (DEP). Experimentally, we visualized the formation of Bacillus subtilis bacterial pearl chain and human breast cancer cell (MCF-7) chain under positive and negative dielectrophoretic force, respectively. Through a simple simulation with creeping flow, AC/DC electric fields, and particle tracing modules in COMSOL, we examined the mechanism by which bacteria self-organize into a pearl chain across the gap between two electrodes via DEP. Our simulation results reveal that the region of greatest positive DEP force shifts from the electrode edge to the leading edge of the pearl chain, thus guiding the trajectories of free-flowing particles toward the leading edge via positive DEP. Our findings additionally highlight the mechanism why the free-flowing particles are more likely to join the existing pearl chain rather than starting a new pearl chain. This phenomenon is primarily due to the increase in magnitude of electric field gradient, and hence DEP force exerted, with the shortening gap between the pearl chain leading edge and the adjacent electrode. The findings shed light on the observed behavior of preferential pearl chain formation across electrode gaps.     

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.     

生化样本的芯片介电电泳富集和分离研究进展

芯片介电电泳技术是以介电电泳(DEP)分离原理和微机电加工技术为依托发展起来的可用于生化样品分析的新型分析技术.本文概述了芯片介电电泳技术的发展和DEP芯片分析系统的构成,并以DEP操控模式为切入点,介绍了芯片介电电泳在生化样品分析中的应用情况.     

Simultaneous isolation and label‐free identification of bacteria using contactless dielectrophoresis and Raman spectroscopy

The traditional bacterial identification method of growing colonies on agar plates can take several days to weeks to complete depending on the growth rate of the bacteria. Successfully decreasing this analysis time requires cell isolation followed by identification. One way to decrease analysis time is by combining dielectrophoresis (DEP), a common technique used for cell sorting and isolation, and Raman spectroscopy for cell identification. DEP‐Raman devices have been used for bacterial analysis, however, these devices have a number of drawbacks including sample heating, cell‐to‐electrode proximity that limits throughput and separation efficiency, electrode fouling, or inability to address sample debris. Presented here is a contactless DEP‐Raman device to simultaneously isolate and identify particles from a mixed sample while avoiding common drawbacks associated with other DEP designs. Using the device, a mixed sample of bacteria and 3 μm polystyrene spheres were isolated from each other and a Raman spectrum of the trapped bacteria was acquired, indicating the potential for cDEP‐Raman devices to decrease the analysis time of bacteria.     

AC electrokinetic isolation and detection of extracellular vesicles from dental pulp stem cells: Theoretical simulation incorporating fluid mechanics

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles involved in intracellular communication and the transportation of biomarkers. EVs released by mesenchymal stem cells have been recently reported to play a role in cell-free therapy of many diseases. However, the demand for better research tools to replace the tedious conventional methods used to study EVs is getting stronger. EVs' manipulation using alternating current (AC) electrokinetic forces in a microfluidic device has appeared to be a reliable and sensitive diagnosis and trapping technique. Given that different AC electrokinetic forces may contribute to the overall motion of particles and fluids in a microfluidic device, EVs' electrokinetic trapping must be examined considering all dominant forces involved depending on the experimental conditions. In this paper, AC electrokinetic trapping of EVs using an interdigitated electrode arrays is investigated. A 2D numerical simulation incorporating the two significant AC electrokinetic phenomena (Dielectrophoresis and AC electroosmosis) has been performed. Theoretical predictions are then compared with experimental results and allow for a plausible explanation of observations inconsistent with DEP theory. It is demonstrated that the inconsistencies can be attributed to a significant extent to the contribution of the AC electroosmotic effect.