A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.     

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.     

Insulator‐based dielectrophoresis combined with the isomotive AC electric field and applied to single cell analysis

We developed an insulator‐based dielectrophoretic (iDEP) creek‐gap device that enables the isomotive movement of cells and that is suitable for determining their DEP properties. In the iDEP creek‐gap device, a pair of planar insulators forming a single fan‐shaped channel allows the induction of the isomotive iDEP force on cells. Hence, the cells’ behavior is characterized by straight motion at constant velocity in the longitudinal direction of the channel. Operation of the device was demonstrated using human breast epithelial cells (MCF10A) by applying an AC voltage of V_pp = 34 V peak‐to‐peak and frequencies of 200 kHz and 50 MHz to the device. Subsequently, the magnitude of DEP forces and the real part of the ClausiusMossotti (CM) factor, Re(β), were deduced from the measured cell velocity. The values of Re(β) were 0.14 ± 0.01 for the frequency of 200 kHz and ?0.12 ± 0.01 for 50 MHz. These results demonstrated that the DEP properties of the cells could be extracted over a wide field frequency range. Therefore, the proposed iDEP creek‐gap device was found to be applicable to cell analysis.     

A cell electro‐rotation micro‐device using polarized cells as electrodes

Cell rotation is widely required in various fields as an important technique for single cell manipulation. Usually, the electro‐rotational manipulation of single cells by dielectrophoresis technologies requires at least three electrodes to generate rotating electric fields which induce cells to rotate. Here, we present a novel microfluidic chip capable of rotating single cell using only two planar electrodes by taking polarized cells as the extra electrodes with phase‐shifted signal. To demonstrate this idea, we configured two parallel and planar electrodes as basic dielectrophoresis elements and placed trenches above these electrodes to attract cells, which were in turn polarized to be electrodes. Through simulation, we confirmed the functional structure of the device works well to generate proper rotating electric fields for cell rotation. Through experiment, we successfully demonstrated controlled electro‐rotation of HeLa and HepaRG cells. The novel electro‐rotation mechanism not only simplifies the micro‐device structure but also reduces the complexity of single cell rotation operation which will be a benefit to the potential users.     

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.     

On‐chip technology for single‐cell arraying,electrorotation‐based analysis and selective release

This paper reports a method for label‐free single‐cell biophysical analysis of multiple cells trapped in suspension by electrokinetic forces. Tri‐dimensional pillar electrodes arranged along the width of a microfluidic chamber define actuators for single cell trapping and selective release by electrokinetic force. Moreover, a rotation can be induced on the cell in combination with a negative DEP force to retain the cell against the flow. The measurement of the rotation speed of the cell as a function of the electric field frequency define an electrorotation spectrum that allows to study the dielectric properties of the cell. The system presented here shows for the first time the simultaneous electrorotation analysis of multiple single cells in separate micro cages that can be selectively addressed to trap and/or release the cells. Chips with 39 micro‐actuators of different interelectrode distance were fabricated to study cells with different sizes. The extracted dielectric properties of Henrietta Lacks, human embryonic kidney 293, and human immortalized T lymphocytes cells were found in agreements with previous findings. Moreover, the membrane capacitance of M17 neuroblastoma cells was investigated and found to fall in in the range of 7.49 ± 0.39 mF/m².     

Development of a new contactless dielectrophoresis system for active particle manipulation using movable liquid electrodes

This study presents a new DEP manipulation technique using a movable liquid electrode, which allows manipulation of particles by actively controlling the locations of electrodes and applying on–off electric input signals. This DEP system consists of mercury as a movable liquid electrode, indium tin oxide (ITO)‐coated glass, SU‐8‐based microchannels for electrode passages, and a PDMS medium chamber. A simple squeezing method was introduced to build a thin PDMS layer at the bottom of the medium chamber to create a contactless DEP system. To determine the operating conditions, the DEP force and the friction force were analytically compared for a single cell. In addition, an appropriate frequency range for effective DEP manipulation was chosen based on an estimation of the Clausius–Mossotti factor and the effective complex permittivity of the yeast cell using the concentric shell model. With this system, we demonstrated the active manipulation of yeast cells, and measured the collection efficiency and the dielectrophoretic velocity of cells for different AC electric field strengths and applied frequencies. The experimental results showed that the maximum collection efficiency reached was approximately 90%, and the dielectrophoretic velocity increased with increasing frequency and attained the maximum value of 10.85 ± 0.95 μm/s at 100 kHz, above which it decreased.     

Microfluidic device embedding electrodes for dielectrophoretic manipulation of cells‐A review

Microfluidic device embedding electrodes realizes cell manipulation with the help of dielectrophoresis. Cell manipulation is an important technology for cell sorting and cell population purification. Till now, the theory of dielectrophoresis has been greatly developed. Microfluidic devices with various arrangements of electrodes have been reported from the beginning of the single non‐uniform electric field to the later multiple physical fields. This paper reviews the research status of microfluidic device embedding electrodes for cell manipulation based on dielectrophoresis. Firstly, the working principle of dielectrophoresis is explained. Next, cell manipulation approaches based on dielectrophoresis are introduced. Then, different types of electrode arrangements in the microfluidic device for cell manipulation are discussed, including planar, multilayered and microarray dot electrodes. Finally, the future development trend of the dielectrophoresis with the help of microfluidic devices is prospected. With the rapid development of microfluidic technology, in the near future, high precision, high throughput, high efficiency, multifunctional, portable, economical and practical microfluidic dielectrophoresis will be widely used in the fields of biology, medicine, agriculture and so on.     

Multistep manipulations of poly(methyl‐methacrylate) submicron particles using dielectrophoresis

A microfluidic chip for multistep manipulations of PMMA submicron particles (PMMA‐SMPs) based on dielectrophoresis (DEP) has been developed that includes four main functions of focusing, guiding, trapping, and releasing the SMPs. The structure of the DEP chip consists of a top electrode made of indium tin oxide, a flow chamber formed by optically clear adhesive tape and bottom electrodes with different patterns for different purposes. The bottom electrodes can be divided into three parts: a fish‐bone‐type electrode array that provides the positive DEP force for focusing the suspended nanoparticles (NPs) near the inlet in the flow chamber; the second is for switching and guiding the focused NPs along the electrode surface to the target area, like a flow passing along a virtual channel; and a trapping electrode in the downstream for trapping and releasing the guided NPs. According to the simulation and experimental results, NPs can be aligned along the electrode of the focusing electrode and guided toward the target electrode by means of a positive DEP force between the top and bottom electrodes, with the effects of Brownian motion and Stokes force. In order to demonstrate the sequence of DEP manipulations, a PMMA‐NP suspension is introduced to the DEP chip; the size of the PMMA‐SMPs is about 300 nm. Furthermore, a LabVIEW program developed for sequence control of the AC signals for the multistep manipulations. Consequently, the DEP chip provides an excellent platform technology for the multistep manipulation of SMPs.     

Hybrid cell adhesive material for instant dielectrophoretic cell trapping and long-term cell function assessment

Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.     

On‐line separation of bacterial cells by carbon‐electrode dielectrophoresis

Dielectrophoresis (DEP) represents a powerful approach to manipulate and study living cells. Hitherto, several approaches have used 2‐D DEP chips. With the aim to increase sample volume, in this study we used a 3‐D carbon‐electrode DEP chip to trap and release bacterial cells. A continuous flow was used to plug an Escherichia coli cell suspension first, to retain cells by positive DEP, and thereafter to recover them by washing with peptone water washing solution. This approach allows one not only to analyze DEP behavior of living cells within the chip, but also to further recover fractions containing DEP‐trapped cells. Bacterial concentration and flow rate appeared as critical parameters influencing the separation capacity of the chip. Evidence is presented demonstrating that the setup developed in this study can be used to separate different types of bacterial cells.     

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 valve‐based microfluidic device for on‐chip single cell treatments

Assays toward single‐cell analysis have attracted the attention in biological and biomedical researches to reveal cellular mechanisms as well as heterogeneity. Yet nowadays microfluidic devices for single‐cell analysis have several drawbacks: some would cause cell damage due to the hydraulic forces directly acting on cells, while others could not implement biological assays since they could not immobilize cells while manipulating the reagents at the same time. In this work, we presented a two‐layer pneumatic valve‐based platform to implement cell immobilization and treatment on‐chip simultaneously, and cells after treatment could be collected non‐destructively for further analysis. Target cells could be encapsulated in sodium alginate droplets which solidified into hydrogel when reacted with Ca²⁺. The size of hydrogel beads could be precisely controlled by modulating flow rates of continuous/disperse phases. While regulating fluid resistance between the main channel and passages by the integrated pneumatic valves, on‐chip capture and release of hydrogel beads was implemented. As a proof of concept for on‐chip single‐cell treatments, we showed cellular live/dead staining based on our devices. This method would have potential in single cell manipulation for biochemical cellular assays.     

Dielectrophoretic field‐flow fractionation of electroporated cells

We describe the development and testing of a setup that allows for DEP field‐flow fractionation (DEP‐FFF) of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells based on their different polarizabilities. We first optimized the channel and electrode dimensions, flow rate, and electric field parameters for efficient DEP‐FFF separation of moderately heat‐treated CHO cells (50°C for 15 min) from untreated ones, with the former used as a uniform and stable model of electroporated cells. We then used CHO cells exposed to electric field pulses with amplitudes from 1200 to 2800 V/cm, yielding six groups containing various fractions of nonporated, reversibly porated, and irreversibly porated cells, testing their fractionation in the chamber. DEP‐FFF at 65 kHz resulted in distinctive flow rates for nonporated and each of the porated cell groups. At lower frequencies, the efficiency of fractionation deteriorated, while at higher frequencies the separation of individual elution profiles was further improved, but at the cost of cell flow rate slowdown in all the cell groups, implying undesired transition from negative into positive DEP, where the cells are pulled toward the electrodes. Our results demonstrate that fractionation of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells is feasible at a properly selected frequency.     

Dielectrophoretic trapping of multilayer DNA origami nanostructures and DNA origami‐induced local destruction of silicon dioxide

DNA origami is a widely used method for fabrication of custom‐shaped nanostructures. However, to utilize such structures, one needs to controllably position them on nanoscale. Here we demonstrate how different types of 3D scaffolded multilayer origamis can be accurately anchored to lithographically fabricated nanoelectrodes on a silicon dioxide substrate by DEP. Straight brick‐like origami structures, constructed both in square (SQL) and honeycomb lattices, as well as curved “C”‐shaped and angular “L”‐shaped origamis were trapped with nanoscale precision and single‐structure accuracy. We show that the positioning and immobilization of all these structures can be realized with or without thiol‐linkers. In general, structural deformations of the origami during the DEP trapping are highly dependent on the shape and the construction of the structure. The SQL brick turned out to be the most robust structure under the high DEP forces, and accordingly, its single‐structure trapping yield was also highest. In addition, the electrical conductivity of single immobilized plain brick‐like structures was characterized. The electrical measurements revealed that the conductivity is negligible (insulating behavior). However, we observed that the trapping process of the SQL brick equipped with thiol‐linkers tended to induce an etched “nanocanyon” in the silicon dioxide substrate. The nanocanyon was formed exactly between the electrodes, that is, at the location of the DEP‐trapped origami. The results show that the demonstrated DEP‐trapping technique can be readily exploited in assembling and arranging complex multilayered origami geometries. In addition, DNA origamis could be utilized in DEP‐assisted deformation of the substrates onto which they are attached.     

Diethylphosphonate‐containing benzoxazine compound as a thermally latent catalyst and a reactive property modifier for polybenzoxazine‐based resins

A diethylphosphonate‐containing benzoxazine compound (DEP‐Bz) to be used as a multi‐functional reaction agent for preparation of high performance polybenzoxazine thermosetting resins has been reported. The chemical structure of DEP‐Bz has been characterized with FTIR, ¹H NMR, and elemental analysis. The phosphonate groups of DEP‐Bz could convert into phosphonic acid groups which could catalyze the ring‐opening addition reaction of benzoxazines, to demonstrate the thermally latent catalytic effect of DEP‐Bz on the polymerization of benzoxazine compounds. Moreover, DEP‐Bz could also serve as a reactive‐type modifier for polybenzoxazines and other thermosets. DEP‐Bz modified polybenzoxazine resins have shown relatively low reaction temperature (about 190 °C), high mechanical strength with a storage modulus of about 3.0 GPa, and high flame retardancy with a limit oxygen index of about 32. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 3523–3530     

Dielectrophoresis applications in biomedical field and future perspectives in biomedical technology

Dielectrophoresis (DEP) is a technique to manipulate trajectories of polarisable particles in nonuniform electric fields by utilizing unique dielectric properties. The manipulation of a cell using DEP has been demonstrated in various modes, thereby indicating potential applications in the biomedical field. In this review, recent DEP applications in the biomedical field are discussed. This review is intended to highlight research work that shows significant approach related to DEP application in biomedical field reported between 2016 and 2020. First, single-shell model and multiple-shell model of cells are introduced. Current device structures and recently introduced electrode patterns for DEP applications are discussed. Second, the biomedical uses of DEP in liquid biopsies, stem cell-based therapies, and diagnosis of infectious diseases due to bacteria and viruses are presented. Finally, the challenges in DEP research are discussed, and the reported solutions are explained. DEP's potential research directions are mentioned.     

Metabolic viability of Escherichia coli trapped by dielectrophoresis in microfluidics

The spatial and temporal control of biological species is essential in complex microfluidic biosystems. In addition, if the biological species is a cell, microfluidic handling must ensure that the cell's metabolic viability is maintained. The use of DEP for cell manipulation in microfluidics has many advantages because it is remote and fast, and the voltages required for cell trapping scale well with miniaturization. In this paper, the conditions for bacterial cell (Escherichia coli) trapping using a quadrupole electrode configuration in a PDMS microfluidic channel were developed both for stagnant and for in‐flow fluidic situations. The effect of the electrical conductivity of the fluid, the applied electric field and frequency, and the fluid‐flow velocity were studied. A dynamic exchange between captured and free‐flowing cells during DEP trapping was demonstrated. The metabolic activity of trapped cells was confirmed by using E. coli cells genetically engineered to express green fluorescent protein under the control of an inducible promoter. Noninduced cells trapped by negative DEP and positive DEP were able to express green fluorescent protein minutes after the inducer was inserted in the microchannel system immediately after DEP trapping. Longer times of trapping prior to exposure to the inducer indicated first a degradation of the cell metabolic activity and finally cell death.     

Combined detection of AC‐electrokinetic effects: Experiments with three‐axial chicken red blood cells

Dielectrophoresis (DEP), electrorotation (ROT), and electro‐orientation were used for the dielectric spectroscopy of nucleated three‐axial chicken red blood cells (CRBCs). Because the different AC‐electrokinetic effects are not mutually independent, their DEP and ROT spectra were combined in ranges separated by the reorientation of the CRBCs in the inhomogeneous linear DEP and circular ROT fields. This behavior can be qualitatively described by a single‐shell ellipsoidal model. Whereas in linear fields, the maximum of the Clausius–Mossotti factor along the three axes determines the orientated axis, in circular fields, the minimum of the factor determines the axis perpendicularly orientated to the field plane. Quantitatively, it has not been possible to find a consistent parameter set for fitting the DEP and ROT spectra, as well as the reorientation frequencies. Our ellipsoidal CRBC standard model had semiaxes of a = 7.7 μm, b = 4.0 μm, and c = 1.85 μm, a relative permittivity of 35 to 45 and conductivity of 0.36 to 0.04 S/m for the cytoplasm, combined with a specific capacitance of 10 to 14 mF/m² and a conductivity of 3500 S/m² for the cell membrane. The fits in different external conductivity ranges between external conductivities of 0.015 and 1.0 S/m were improved when the membrane capacitance was changed between 4 to 25 mF/m² depending on the method used. A similar transition was reflected in the effective properties of a three‐shell spherical model containing an internal membranous sphere with the geometry of the CRBC nucleus. Our findings suggest that the simultaneous interpretation of various AC‐electrokinetic spectra is a step toward the dielectric fingerprinting of biological cells.     

Theoretical and experimental analysis of negative dielectrophoresis-induced particle trajectories

Many biomedical analysis applications require trapping and manipulating single cells and cell clusters within microfluidic devices. Dielectrophoresis (DEP) is a label-free technique that can achieve flexible cell trapping, without physical barriers, using electric field gradients created in the device by an electrode microarray. Little is known about how fluid flow forces created by the electrodes, such as thermally driven convection and electroosmosis, affect DEP-based cell capture under high conductance media conditions that simulate physiologically relevant fluids such as blood or plasma. Here, we compare theoretical trajectories of particles under the influence of negative DEP (nDEP) with observed trajectories of real particles in a high conductance buffer. We used 10-µm diameter polystyrene beads as model cells and tracked their trajectories in the DEP microfluidic chip. The theoretical nDEP trajectories were in close agreement with the observed particle behavior. This agreement indicates that the movement of the particles was highly dominated by the DEP force and that contributions from thermal- and electroosmotic-driven flows were negligible under these experimental conditions. The analysis protocol developed here offers a strategy that can be applied to future studies with different applied voltages, frequencies, conductivities, and polarization properties of the targeted particles and surrounding medium. These findings motivate further DEP device development to manipulate particle trajectories for trapping applications.