Microfluidic single-cell cultivation chip with controllable immobilization and selective release of yeast cells

We present a microfluidic cell-culture chip that enables trapping, cultivation and release of selected individual cells. The chip is fabricated by a simple hybrid glass-SU-8-PDMS approach, which produces a completely transparent microfluidic system amenable to optical inspection. Single cells are trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they are cultivated under controlled environmental conditions. Cells of interest can be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site. The combination of hydrodynamic cell-trapping and dielectrophoretic methods for cell releasing enables highly versatile single-cell manipulation in an array-based format. Computational fluid dynamics simulations were performed to estimate the properties of the system during cell trapping and releasing. Polystyrene beads and yeast cells have been used to investigate and characterize the different functions and to demonstrate biological compatibility and viability of the platform for single-cell applications in research areas such as systems biology.     

Insulator-based dielectrophoresis for the selective concentration and separation of live bacteria in water

Insulator-based dielectrophoresis (iDEP) was utilized to separate and concentrate selectively mixtures of two species of live bacteria simultaneously. Four species of bacteria were studied: the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis, B. cereus, and B. megaterium. Under an applied direct current (DC) electric field all the bacterial species exhibited negative dielectrophoretic behavior. The dielectrophoretic separations were carried out in a glass microchannel containing an array of insulating posts. The insulating posts in the microchannel produced nonuniformities in the electric field applied along the channel. Mixtures of two species of bacteria were introduced into the microchannel and the electric field was applied. The bacterial species exhibited different dielectrophoretic mobilities under the influence of the nonuniform field. From these experiments a trapping order was established with E. coli trapping at the weakest applied electric field, while the Bacillus species were trapped at different characteristic threshold fields. At stronger applied electric fields, the two different species of bacteria in the microchannel were dielectrophoretically trapped into two spatially distinct bands. The results showed that iDEP has the potential to selectively concentrate and separate different species of bacteria.     

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

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

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: .     

Insulator-based dielectrophoretic focusing and trapping of particles in non-Newtonian fluids

Insulator-based dielectrophoretic (iDEP) microdevices have been limited to work with Newtonian fluids. We report an experimental study of the fluid rheological effects on iDEP focusing and trapping of polystyrene particles in polyethylene oxide, xanthan gum, and polyacrylamide solutions through a constricted microchannel. Particle focusing and trapping in the mildly viscoelastic polyethylene oxide solution are slightly weaker than in the Newtonian buffer. They are, however, significantly improved in the strongly viscoelastic and shear thinning polyacrylamide solution. These observed particle focusing behaviors exhibit a similar trend with respect to electric field, consistent with a revised theoretical analysis for iDEP focusing in non-Newtonian fluids. No apparent focusing of particles is achieved in the xanthan gum solution, though the iDEP trapping can take place under a much larger electric field than the other fluids. This is attributed to the strong shear thinning-induced influences on both the electroosmotic flow and electrokinetic/dielectrophoretic motions.     

Multiple frequency dielectrophoresis

A novel method of modeling multiple frequency dielectrophoresis (MFDEP) is introduced based on the concept of an effective Clausius-Mossotti factor, CM(eff), for a particle that is exposed to electrical fields of different frequencies, coming either from one or multiple pairs of electrodes. This analysis clearly illustrates how adding frequencies adds control parameters, up to two additional parameters per frequency. As a result, MFDEP can be used for a wide variety of applications, including separating particles with very similar Clausius-Mossotti spectra, trapping multiple groups of cells simultaneously, and cancelling unwanted dielectrophoretic traps. Illustrating the modeling approach, we determine the CM(eff)s for live and dead yeast cells, and then predict their equilibrium distribution on a three-electrode configuration, with two electrodes at different frequencies and the third electrode at ground. This prediction is validated experimentally, using MFDEP to selectively attract live cells to one location and dead cells to another, trapping both. These results demonstrate that the use of multiple frequencies for the manipulation of particles can enhance the performance of dielectrophoretic devices, not only for sorting, but also for such applications as patterning cells in close proximity for the formation of cell consortia.     

Parasitic trap cancellation using multiple frequency dielectrophoresis, demonstrated by loading cells into cages

This paper presents a method of using multiple frequencies to counteract electric field distortions that interfere with the dielectrophoretic (DEP) manipulation of particles or cells. To demonstrate the technique, simulations were performed for a scenario in which cells were to be loaded into a cage whose walls created parasitic trapping sites that prevented cells from entering it. By employing negative DEP on one electrode in conjunction with positive DEP on another, these traps could be almost completely cancelled. The model predictions were validated experimentally: multiple frequency DEP was used to load many cells into a cage in a matter of seconds in fluid flows of up to 300 microm s(-1), which could not be done with single frequency DEP. Actively cancelling field distortions permits the presence of features that would otherwise be prohibited near regions of dielectrophoretic manipulation, significantly expanding the environments in which dielectrophoresis can be used.     

Dielectrophoretic manipulation of suspended submicron particles

Planar and three-dimensiònal multi-electrode systems with dimensions of 2 - 40 microm were fabricated by IC technology and used for trapping and aggregation of microparticles. To achieve negative dielectrophoresis (repelling forces) in aqueous solution, radiofrequency (RF) electric fields were used. Experimentally, particles down to 100 nm in diameter were enriched and trapped as aggregates in field cages and dielectrophoretic microfilters and observed using confocal fluorimetry. Theoretically, single particles with an effective diameter down to about 35 nm should be trappable in micron field cages. Due to the unavoidable Ohmic heating, RF electric fields can induce liquid streaming in extremely small channels (12 microm in height). This can be used for pumping and particle enrichment but it enhances Brownian motion and counteracts dielectrophoretic trapping. Combining Brownian motion with ratchet-like dielectrophoretic forces enables the creation of Brownian pumps that could be used as sensitive separation devices for submicron particles if liquid pumping is avoided in smaller structures.     

Simultaneous concentration and separation of microorganisms: insulator-based dielectrophoretic approach

Microanalytical methods offer attractive characteristics for rapid microbial detection and concentration. There is a growing interest in the development of microscale separation techniques. Dielectrophoresis (DEP), a nondestructive electrokinetic transport mechanism, is a technique with great potential for microbe manipulation, since it can achieve concentration and separation in a single step. DEP is the movement of particles due to polarization effects in nonuniform electric fields. The majority of the work on dielectrophoretic manipulation of microbes has employed alternating current fields in arrays of microelectrodes, an approach with some disadvantages. An alternative is to employ insulator-based DEP (iDEP), a dielectrophoretic mode where nonuniform fields are produced by employing arrays of insulating structures. This study presents the concentration and fractionation of a mixture of bacteria and yeast cells employing direct current-iDEP in a microchannel containing an array of cylindrical insulating structures. Negative dielectrophoretic trapping of both types of microorganisms was demonstrated, where yeast cells exhibited a stronger response, opening the possibility for dielectrophoretic differentiation. Simultaneous concentration and fractionation of a mixture of both types of cells was carried out analogous to a chromatographic separation, where a dielectropherogram was obtained in less than 2 min by applying an electric field gradient and achieving concentration factors in the order of 50 and 37 times the inlet concentration for Escherichia coli and Saccharomyces cerevisiae cells, respectively. Encouraging results were also obtained employing a sample of water taken from a pond. The findings demonstrated the great potential of iDEP as a rapid and effective technique for intact microorganism concentration and separation.     

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.     

Dynamically controlled dielectrophoresis using resonant tuning

Electrically polarizable micro- and nanoparticles and droplets can be trapped using the gradient electric field of electrodes. But the spatial profile of the resultant dielectrophoretic force is fixed once the electrode structure is defined. To change the force profile, entire complex lab-on-a-chip systems must be re-fabricated with modified electrode structures. To overcome this problem, we propose an approach for the dynamic control of the spatial profile of the dielectrophoretic force by interfacing the trap electrodes with a resistor and an inductor to form a resonant resistor–inductor–capacitor (RLC) circuit. Using a dielectrophoretically trapped water droplet suspended in silicone oil, we show that the resonator amplitude, detuning, and linewidth can be continuously varied by changing the supply voltage, supply frequency, and the circuit resistance to obtain the desired trap depth, range, and stiffness. We show that by proper tuning of the resonator, the trap range can be extended without increasing the supply voltage, thus preventing sensitive samples from exposure to high electric fields at the stable trapping position. Such unprecedented dynamic control of dielectrophoretic forces opens avenues for the tunable active manipulation of sensitive biological and biochemical specimen in droplet microfluidic devices used for single-cell and biochemical reaction analysis.     

Dielectrophoresis of Caenorhabditis elegans

We demonstrate for the first time the dielectrophoretic trapping and manipulation of a whole animal, the nematode Caenorhabditis elegans. We studied the effect of the electric field on the nematode as a function of field intensity and frequency. We identified a range of electric field intensities and frequencies that trap worms without apparent adverse effect on their viability. Worms tethered by dielectrophoresis (DEP) exhibit behavioral responses to blue light, indicating that at least some of the nervous system functions are unimpaired by the electrical field. DEP is useful to dynamically tether nematodes, sort nematodes according to size, and separate dead worms from live ones.     

Particle trapping using dielectrophoretically patterned carbon nanotubes

This study presents the dielectrophoretic (DEP) assembly of multi‐walled carbon nanotubes (MWCNTs) between curved microelectrodes for the purpose of trapping polystyrene microparticles within a microfluidic system. Under normal conditions, polystyrene particles exhibit negative DEP behaviour and are repelled from microelectrodes. Interestingly, the addition of MWCNTs to the system alters this situation in two ways: first, they coat the surface of particles and change their dielectric properties to exhibit positive DEP behaviour; second, the assembled MWCNTs are highly conductive and after the deposition serve as extensions to the microelectrodes. They establish an array of nanoelectrodes that initiates from the edge of microelectrodes and grow along the electric field lines. These nanoelectrodes can effectively trap the MWCNT‐coated particles, since they cover a large portion of the microchannel bottom surface and also create a much stronger electric field than the primary microelectrodes as confirmed by our numerical simulations. We will show that the presence of MWCNT significantly changes performance of the system, which is investigated by trapping sample polystyrene particles with plain, COOH and goat anti‐mouse IgG surfaces.     

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.     

Trapping and imaging of micron-sized embryos using dielectrophoresis

Development of dielectrophoretic (DEP) arrays for real-time imaging of embryonic organisms is described. Microelectrode arrays were used for trapping both embryonated eggs and larval stages of Antarctic nematode Panagrolaimus davidi. Ellipsoid single-shell model was also applied to study the interactions between DEP fields and developing multicellular organisms. This work provides proof-of-concept application of chip-based technologies for the analysis of individual embryos trapped under DEP force.     

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.     

Dielectrophoretic trapping of single bacteria at carbon nanofiber nanoelectrode arrays

We present an ac dielectrophoretic (DEP) technique for single-cell trapping using embedded carbon nanofiber (CNF) nanoelectrode arrays (NEAs). NEAs fabricated by inlaying vertically aligned carbon nanofibers in SiO2 matrix are applied as "points-and-lid" DEP devices in aqueous solution. The miniaturization of the electrode size provides a highly focused electrical field with the gradient enhanced by orders of magnitude. This generates extremely large positive DEP forces near the electrode surface and traps small bioparticles against strong hydrodynamic forces. This technology promises new capabilities to perform novel cell biology experiments at the nanoscale. We anticipate that the bottom-up approach of such nano-DEP devices allows the integration of millions of nanolectrodes deterministically in lab-on-a-chip devices and will be generally useful for manipulating submicron particles.     

Analysis of U87 glioma cells by dielectrophoresis

Glioblastoma multiforme is the most aggressive and invasive brain cancer consisting of genetically and phenotypically altering glial cells. It has massive heterogeneity due to its highly complex and dynamic microenvironment. Here, electrophysiological properties of U87 human glioma cell line were measured based on a dielectrophoresis phenomenon to quantify the population heterogeneity of glioma cells. Dielectrophoretic forces were generated using a gold-microelectrode array within a microfluidic channel when 3 V_pp and 100, 200, 300, 400, 500 kHz, 1, 2, 5, and 10 MHz frequencies were applied. We analyzed the dielectrophoretic behavior of 500 glioma cells, and revealed that the crossover frequency of glioma cells was around 140 kHz. A quantifying dielectrophoretic movement of the glioma cells exhibited three distinct glioma subpopulations: 50% of the glioma cells experienced strong, 30% of the cells were spread in the microchannel by moderate, and the rest of the cells experienced very weak positive dielectrophoretic forces. Our results demonstrated the dielectrophoretic spectra of U87 glioma cell line. Dielectrophoretic responses of glioma cells linked population heterogeneity to membrane properties of glioma cells rather than their size distribution in the population.     

Insulator-based dielectrophoresis of microorganisms: theoretical and experimental results

Dielectrophoresis (DEP) is the motion of particles due to polarization effects in nonuniform electric fields. DEP has great potential for handling cells and is a non-destructive phenomenon. It has been utilized for different cell analysis, from viability assessments to concentration enrichment and separation. Insulator-based DEP (iDEP) provides an attractive alternative to conventional electrode-based systems; in iDEP, insulating structures are used to generate nonuniform electric fields, resulting in simpler and more robust devices. Despite the rapid development of iDEP microdevices for applications with cells, the fundamentals behind the dielectrophoretic behavior of cells has not been fully elucidated. Understanding the theory behind iDEP is necessary to continue the progress in this field. This work presents the manipulation and separation of bacterial and yeast cells with iDEP. A computational model in COMSOL Multiphysics was employed to predict the effect of direct current-iDEP on cells suspended in a microchannel containing an array of insulating structures. The model allowed predicting particle behavior, pathlines and the regions where dielectrophoretic immobilization should occur. Experimental work was performed at the same operating conditions employed with the model and results were compared, obtaining good agreement. This is the first report on the mathematical modeling of the dielectrophoretic response of yeast and bacterial cells in a DC-iDEP microdevice.     

The zeta potential of cyclo-olefin polymer microchannels and its effects on insulative (electrodeless) dielectrophoresis particle trapping devices

While cyclo-olefin polymer microchannels have the potential to improve both the optical detection sensitivity and the chemical resistance of polymer microanalytical systems, their surface properties are to date not thoroughly characterized. These surface properties dictate, among other things, electrokinetic effects when electric fields are present. Here, we report the measurement of the zeta potential of cyclo-olefin polymers (injection-molded and hot-embossed Zeonor 1060R and 1020R) microchannels as a function of pH, counter-ion concentration, storage conditions, and chemical treatment in aqueous solutions both with and without EOF-suppressing additives. In contrast with previous reports, significant surface charge is measured, consistent with titration of charged sites with pK(a) = 4.8. Storage in air, acetonitrile, or aqueous solutions has relatively minor effects. While the source of the surface charge is unclear, chemical functionalization has shown that carboxylic acid groups are not present at the surface, consistent with the chemical structure of Zeonor. EOF-suppressing additives (hydroxypropylmethylcellulose) and conditioning in perchloric acid allow the surface charge to be suppressed. We demonstrate dielectrophoretic particle trapping devices in Zeonor 1060R substrates that show reduced trapping voltage thresholds as compared to previous implementations in glass.