Selective trapping of single mammalian breast cancer cells by insulator-based dielectrophoresis

The trapping or immobilization of individual cells at specific locations in microfluidic platforms is essential for single cell studies, especially those requiring cell stimulation and downstream analysis of cellular content. Selectivity for individual cell types is required when mixtures of cells are analyzed in heterogeneous and complex matrices, such as the selection of metastatic cells within blood samples. Here, we demonstrate a microfluidic device based on direct current (DC) insulator-based dielectrophoresis (iDEP) for selective trapping of single MCF-7 breast cancer cells from mixtures with both mammalian peripheral blood mononuclear cells (PBMC) as well MDA-MB-231 as a second breast cancer cell type. The microfluidic device has a teardrop iDEP design optimized for the selective capture of single cells based on their differential DEP behavior under DC conditions. Numerical simulations adapted to experimental device geometries and buffer conditions predicted the trapping condition in which the dielectrophoretic force overcomes electrokinetic forces for MCF-7 cells, whereas PBMCs were not trapped. Experimentally, selective trapping of viable MCF-7 cells in mixtures with PBMCs was demonstrated in good agreement with simulations. A similar approach was also executed to demonstrate the selective trapping of MCF-7 cells in a mixture with MDA-MB-231 cells, indicating the selectivity of the device for weakly invasive and highly invasive breast cancer cells. The DEP studies were complemented with cell viability tests indicating acceptable cell viability over the course of an iDEP trapping experiment.

3D Insulator‐based dielectrophoresis using DC‐biased,AC electric fields for selective bacterial trapping

Insulator‐based dielectrophoresis (iDEP) is a well‐known technique that harnesses electric fields for separating, moving, and trapping biological particle samples. Recent work has shown that utilizing DC‐biased AC electric fields can enhance the performance of iDEP devices. In this study, an iDEP device with 3D varying insulating structures analyzed in combination with DC biased AC fields is presented for the first time. Using our unique reactive ion etch lag, the mold for the 3D microfluidic chip is created with a photolithographic mask. The 3D iDEP devices, whose largest dimensions are 1 cm long, 0.18 cm wide, and 90 μm deep are then rapidly fabricated by curing a PDMS polymer in the glass mold. The 3D nature of the insulating microstructures allows for high trapping efficiency at potentials as low as 200 Vpp. In this work, separation of Escherichia coli from 1 μm beads and selective trapping of live Staphylococcus aureus cells from dead S. aureus cells is demonstrated. This is the first reported use of DC‐biased AC fields to selectively trap bacteria in 3D iDEP microfluidic device and to efficiently separate particles where selectivity of DC iDEP is limited.     

Particle trapping in electrically driven insulator-based microfluidics: Dielectrophoresis and induced-charge electrokinetics

Electrokinetically driven insulator-based microfluidic devices represent an attractive option to manipulate particle suspensions. These devices can filtrate, concentrate, separate, or characterize micro and nanoparticles of interest. Two decades ago, inspired by electrode-based dielectrophoresis, the concept of insulator-based dielectrophoresis (iDEP) was born. In these microfluidic devices, insulating structures (i.e., posts, membranes, obstacles, or constrictions) built within the channel are used to deform the spatial distribution of an externally generated electric field. As a result, particles suspended in solution experience dielectrophoresis (DEP). Since then, it has been assumed that DEP is responsible for particle trapping in these devices, regardless of the type of voltage being applied to generate the electric field—direct current (DC) or alternating current. Recent findings challenge this assumption by demonstrating particle trapping and even particle flow reversal in devices that prevent DEP from occurring (i.e., unobstructed long straight channels stimulated with a DC voltage and featuring a uniform electric field). The theory introduced to explain those unexpected observations was then applied to conventional “DC-iDEP” devices, demonstrating better prediction accuracy than that achieved with the conventional DEP-centered theory. This contribution summarizes contributions made during the last two decades, comparing both theories to explain particle trapping and highlighting challenges to address in the near future.     

Trapping and releasing of single microparticles and cells in a microfluidic chip

A microfluidic device was designed and fabricated to capture single microparticles and cells by using hydrodynamic force and selectively release the microparticles and cells of interest via negative dielectrophoresis by activating selected individual microelectrodes. The trap microstructure was optimized based on numerical simulation of the electric field as well as the flow field. The capture and selective release functions of the device were verified by multi-types microparticles with different diameters and K562 cells. The capture efficiencies/release efficiencies were 95.55% ± 0.43%/96.41% ± 1.08% and 91.34% ± 0.01%/93.67% ± 0.36% for microparticles and cells, respectively. By including more traps and microelectrodes, the device can achieve high throughput and realize the visual separation of microparticles/cells of interest in a large number of particle/cell groups.     

Dielectrophoresis‐assisted creation of cell aggregates under flow conditions using planar electrodes

We present a microfluidic platform allowing dielectrophoresis‐assisted formation of cell aggregates of controlled size and composition under flow conditions. When specific experimental conditions are met, negative dielectrophoresis allows efficient concentration of cells towards electric field minima and subsequent aggregation. This bottom‐up assembly strategy offers several advantages with respect to the targeted application: first, dielectrophoresis offers precise control of spatial cell organization, which can be adjusted by optimizing electrode design. Then, it could contribute to accelerate the establishment of cell‐cell interactions by favoring close contact between neighboring cells. The trapping geometry of our chip is composed of eight electrodes arranged in a circle. Several parameters have been tested in simulations to find the best configurations for trapping in flow. Those configurations have been tested experimentally with both polystyrene beads and human embryonic kidney cells. The final design and experimental setup have been optimized to trap cells and release the created aggregates on demand.     

Quantitative modeling of dielectrophoretic traps

We present quantitative modeling software for simulating multiple forces acting on a single particle in a microsystem. In this paper, we focus on dielectrophoretic (DEP) trapping of single cells against fluid flow. The software effectively models the trapping behavior for a range of particles including beads, mammalian cells, viruses, and bacteria. In addition, the software can be used to reveal useful information about the DEP traps - such as multipolar DEP force effects, trap size-selectivity, and effects from varying the flow chamber height. Our modeling software thus serves as a predictive tool, enabling the design of novel DEP traps with superior performance over existing trap geometries. In addition, the software can evaluate a range of trap dimensions to determine the effects on trapping behavior, thus optimizing the trap geometry before it is even fabricated. The software is freely available to the scientific community at: .     

Quantitative analysis of the three-dimensional trap stiffness of a dielectrophoretic corral trap

Dielectrophoresis is a robust approach for the manipulation and separation of (bio)particles using microfluidic platforms. We developed a dielectrophoretic corral trap in a microfluidic device that utilizes negative dielectrophoresis to capture single spherical polystyrene particles. Circular-shaped micron-size traps were employed inside the device and the three-dimensional trap stiffness (restoring trapping force from equilibrium trapping location) was analyzed using 4.42 μm particles and 1 MHz of an alternating electric field from 6 V_P-P to 10 V_P-P. The trap stiffness increased exponentially in the x- and y-direction, and linearly in the z-direction. Image analysis of the trapped particle movements revealed that the trap stiffness is increased 608.4, 539.3, and 79.7% by increasing the voltage from 6 V_P-P to 10 V_P-P in the x-, y-, and z-direction, respectively. The trap stiffness calculated from a finite element simulation of the device confirmed the experimental results. This analysis provides important insights to predict the trapping location, strength of the trapping, and optimum geometry for single particle trapping and its applications such as single-molecule analysis and drug discovery.     

On the recent developments of insulator‐based dielectrophoresis: A review

Insulator‐based dielectrophoresis (iDEP), also known as electrodeless DEP, has become a well‐known dielectrophoretic technique, no longer viewed as a new methodology. Significant advances on iDEP have been reported during the last 15 years. This review article aims to summarize some of the most important findings on iDEP organized by the type of dielectrophoretic mode: streaming and trapping iDEP. The former is primarily used for particle sorting, while the latter has great capability for particle enrichment. The characteristics of a wide array of devices are discussed for each type of dielectrophoretic mode in order to present an overview of the distinct designs and applications developed with iDEP. A short section on Joule heating effects and electrothermal flow is also included to highlight some of the challenges in the utilization of iDEP systems. The significant progress on iDEP illustrates its potential for a large number of applications, ranging from bioanalysis to clinical and biomedical assessments. The present article discusses the work on iDEP by numerous research groups around the world, with the aim of proving the reader with an overview of the state‐of‐the‐art in iDEP microfluidic systems.     

Joule heating-enabled electrothermal enrichment of nanoparticles in insulator-based dielectrophoretic microdevices

Insulator-based dielectrophoresis (iDEP) exploits the electric field gradients formed around insulating structures to manipulate particles for diverse microfluidic applications. Compared to the traditional electrode-based dielectrophoresis, iDEP microdevices have the advantages of easy fabrication, free of water electrolysis, and robust structure, etc. However, the presence of in-channel insulators may cause thermal effects because of the locally amplified Joule heating of the fluid. The resulting electrothermal flow circulations are exploited in this work to trap and concentrate nanoscale particles (of 100 nm diameter and less) in a ratchet-based iDEP microdevice. Such Joule heating-enabled electrothermal enrichment of nanoparticles are found to grow with the increase of alternating current or direct current electric field. It also becomes more effective for larger particles and in a microchannel with symmetric ratchets. Moreover, a depth-averaged numerical model is developed to understand and simulate the various parametric effects, which is found to predict the experimental observations with a good agreement.     

Electrothermal flow effects in insulating (electrodeless) dielectrophoresis systems

We simulate electrothermally induced flow in polymeric, insulator-based dielectrophoresis (iDEP) systems with DC-offset, AC electric fields at finite thermal Péclet number, and we identify key regimes where electrothermal (ET) effects enhance particle deflection and trapping. We study a single, two-dimensional constriction in channel depth with parametric variations in electric field, channel geometry, fluid conductivity, particle electrophoretic (EP) mobility, and channel electroosmotic (EO) mobility. We report the effects of increasing particle EP mobility, channel EO mobility, and AC and DC field magnitudes on the mean constriction temperature and particle behavior. Specifically, we quantify particle deflection and trapping, referring to the deviation of particles from their pathlines due to dielectrophoresis as they pass a constriction and the stagnation of particles due to negative dielectrophoresis near a constriction, respectively. This work includes the coupling between fluid, heat, and electromagnetic phenomena via temperature-dependent physical parameters. Results indicate that the temperature distribution depends strongly on the fluid conductivity and electric field magnitude, and particle deflection and trapping depend strongly on the channel geometry. Electrothermal (ET) effects perturb the EO flow field, creating vorticity near the channel constriction and enhancing the deflection and trapping effects. ET effects alter particle deflection and trapping responses in insulator-based dielectrophoresis devices, especially at intermediate device aspect ratios (2 ≤ r ≤ 7) in solutions of higher conductivity (σ m ≥ 1 × 10(-3)S/m). The impact of ET effects on particle deflection and trapping are diminished when particle EP mobility or channel EO mobility is high. In almost all cases, ET effects enhance negative dielectrophoretic particle deflection and trapping phenomena.     

Programmed trapping of individual bacteria using micrometre-size sieves

Monitoring the real-time behavior of spatial arrays of single living bacteria cells is only achieved with much experimental difficulty due to the small size and mobility of the cells. To address this problem, we have designed and constructed a simple microfluidic device capable of trapping single bacteria cells in spatially well-defined locations without the use of chemical surface treatments. The device exploits hydrodynamics to slow down and trap cells flowing near a narrow aperture. We have modeled this system numerically by approximating the motion of Escherichia coli cells as rigid 3-D ellipsoids. The numerical predictions for the speed and efficiency of trapping were tested by fabricating the devices and imaging GFP expressing E. coli at a high spatio-temporal resolution. We find that our numerical simulations agree well with the actual cell flow for varying trap geometries. The trapped cells are optically accessible, and combined with our ability to predict their spatial location we demonstrate the ease of this method for monitoring multiple single cells over a time course. The simplicity of the design, inexpensive materials and straightforward fabrication make it an accessible tool for any systems biology laboratory.     

DC insulator dielectrophoretic applications in microdevice technology: a review

Dielectrophoresis is a noninvasive, nondestructive, inexpensive, and fast technique for the manipulation of bioparticles. Recent advances in the field of dielectrophoresis (DEP) have resulted in new approaches for characterizing the behavior of particles and cells using direct current (DC) electric fields. In such approaches, spatial nonuniformities are created in the channel by embedding insulating obstacles in the channel or flow field in order to perform separation or trapping. This emerging field of dielectrophoresis is commonly termed DC insulator dielectrophoresis (DC-iDEP), insulator-based dielectrophoresis (iDEP), or electrodeless dielectrophoresis (eDEP). In many microdevices, this form of dielectrophoresis has advantages over traditional AC-DEP, including single material microfabrication, remotely positioned electrodes, and reduced fouling of the test region. DC-iDEP applications have included disease detection, separation of cancerous cells from normal cells, and separation of live from dead bacteria. However, there is a need for a critical report to integrate these important research findings. The aim of this review is to provide an overview of the current state-of-art technology in the field of DC-iDEP for the separation and trapping of inert particles and cells. In this article, a review of the concepts and theory leading to the manipulation of particles via DC-iDEP is given, and insulating obstacle geometry designs and the characterization of device performance are discussed. This review compiles and compares the significant findings obtained by researchers in handling and manipulating particles.     

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel

Deciphering the signaling pathways that govern stimulation of na?ve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of na?ve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.     

Performance impact of dynamic surface coatings on polymeric insulator-based dielectrophoretic particle separators

Efficient and robust particle separation and enrichment techniques are critical for a diverse range of lab-on-a-chip analytical devices including pathogen detection, sample preparation, high-throughput particle sorting, and biomedical diagnostics. Previously, using insulator-based dielectrophoresis (iDEP) in microfluidic glass devices, we demonstrated simultaneous particle separation and concentration of various biological organisms, polymer microbeads, and viruses. As an alternative to glass, we evaluate the performance of similar iDEP structures produced in polymer-based microfluidic devices. There are numerous processing and operational advantages that motivate our transition to polymers such as the availability of numerous innate chemical compositions for tailoring performance, mechanical robustness, economy of scale, and ease of thermoforming and mass manufacturing. The polymer chips we have evaluated are fabricated through an injection molding process of the commercially available cyclic olefin copolymer Zeonor 1060R. This publication is the first to demonstrate insulator-based dielectrophoretic biological particle differentiation in a polymeric device injection molded from a silicon master. The results demonstrate that the polymer devices achieve the same performance metrics as glass devices. We also demonstrate an effective means of enhancing performance of these microsystems in terms of system power demand through the use of a dynamic surface coating. We demonstrate that the commercially available nonionic block copolymer surfactant, Pluronic F127, has a strong interaction with the cyclic olefin copolymer at very low concentrations, positively impacting performance by decreasing the electric field necessary to achieve particle trapping by an order of magnitude. The presence of this dynamic surface coating, therefore, lowers the power required to operate such devices and minimizes Joule heating. The results of this study demonstrate that iDEP polymeric microfluidic devices with surfactant coatings provide an affordable engineering strategy for selective particle enrichment and sorting. Figure Model generated image (COMSOL) depicting the electric field gradient divided by the electric field that occurs within an array of insulating posts     

Rapid heterogeneous liver-cell on-chip patterning via the enhanced field-induced dielectrophoresis trap

Biomimetic heterogeneous patterning of hepatic and endothelial cells, which start from randomly distributed cells inside the microfluidic chamber, via the chip design of enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported in this paper. The concentric-stellate-tip electrode array design in this chip generates radial-pattern electric fields for the DEP manipulation of the live liver cells. By constructing the geometric shape and the distribution of stellate tips, the DEP electrodes enhance the desired spatial electric-field gradients to guide and snare individual cells to form the desired biomimetic pattern. With this proposed microfluidic chip design, the original randomly distributed hepatocytes inside the microfluidic chamber can be manipulated in parallel and align into the desired pearl-chain array pattern. This radial pattern mimics the lobular morphology of real liver tissue. The endothelial cells, then, are snared into the additional pearl-chain array and settle at the space in-between the previous hepatic pearl-chain array. By this cell-lab chip, we demonstrate the in vitro reconstruction of the heterogeneous lobule-mimetic radial pattern with good cell viability after cell patterning. This work reports the rapid in-parallel patterning of the dual types of live liver cells via the enhanced DEP trap inside the microfluidic chip.     

A microfluidic array with cellular valving for single cell co-culture

We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (～99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (～70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.     

Electrodeless direct current dielectrophoresis using reconfigurable field-shaping oil barriers

We demonstrate dielectrophoretic (DEP) potential wells using pairs of insulating oil menisci to shape the DC electric field. These oil menisci are arranged in a configuration similar to the quadrupolar electrodes, typically used in DEP, and are shown to produce similar field gradients. While the one-pair well produces a focusing effect on particles in flow, the two-pair well results in creating spatial traps against crossflows. Uncharged polystyrene particles were used to map the DEP force fields and the experimental observations were compared against the field profiles obtained by numerically solving Maxwell's equations. We demonstrate trapping of a single particle due to negative DEP against a pressure-driven crossflow. This can be easily extended to trap and hold cells and other objects against flow for a longer time. We also show the results of particle trapping experiments performed to observe the effect of adjusting the oil menisci and the gap between two pairs of menisci in a four-menisci configuration on the nature of the DEP well formed at the center. A design parameter, Theta, capturing the dimensions of the DEP energy well, is defined and simulations exploring the effects of different geometric features on Theta are presented.     

Fluid rheological effects on streaming dielectrophoresis in a post-array microchannel

Recent studies have demonstrated the strong influences of fluid rheological properties on insulator-based dielectrophoresis (iDEP) in single-constriction microchannels. However, it is yet to be understood how iDEP in non-Newtonian fluids depends on the geometry of insulating structures. We report in this work an experimental study of fluid rheological effects on streaming DEP in a post-array microchannel that presents multiple contractions and expansions. The iDEP focusing and trapping of particles in a viscoelastic polyethylene oxide solution are comparable to those in a Newtonian buffer, which is consistent with the observations in a single-constriction microchannel. Similarly, the insignificant iDEP effects in a shear-thinning xanthan gum solution also agree with those in the single-constriction channel except that gel-like structures are observed to only form in the post-array microchannel under large DC electric fields. In contrast, the iDEP effects in both viscoelastic and shear-thinning polyacrylamide solution are significantly weaker than in the single-constriction channel. Moreover, instabilities occur in the electroosmotic flow and appear to be only dependent on the DC electric field. These phenomena may be associated with the dynamics of polymers as they are electrokinetically advected around and through the posts.     

Dynamic single cell culture array

It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning approximately 1 mm(2) with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.     

Integrated circuit/microfluidic chip to programmably trap and move cells and droplets with dielectrophoresis

We present an integrated circuit/microfluidic chip that traps and moves individual living biological cells and chemical droplets along programmable paths using dielectrophoresis (DEP). Our chip combines the biocompatibility of microfluidics with the programmability and complexity of integrated circuits (ICs). The chip is capable of simultaneously and independently controlling the location of thousands of dielectric objects, such as cells and chemical droplets. The chip consists of an array of 128 x 256 pixels, 11 x 11 microm(2) in size, controlled by built-in SRAM memory; each pixel can be energized by a radio frequency (RF) voltage of up to 5 V(pp). The IC was built in a commercial foundry and the microfluidic chamber was fabricated on its top surface at Harvard. Using this hybrid chip, we have moved yeast and mammalian cells through a microfluidic chamber at speeds up to 30 microm sec(-1). Thousands of cells can be individually trapped and simultaneously positioned in controlled patterns. The chip can trap and move pL droplets of water in oil, split one droplet into two, and mix two droplets into one. Our IC/microfluidic chip provides a versatile platform to trap and move large numbers of cells and fluid droplets individually for lab-on-a-chip applications.