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.     

Performance characterization of an insulator-based dielectrophoretic microdevice

Dielectrophoresis (DEP), the motion of particles in nonuniform electric fields, is a nondestructive electrokinetic (EK) transport mechanism can be used to concentrate and separate bioparticles. Traditionally, DEP has been performed employing microelectrodes, an approach that is expensive due to the cost of microelectrode fabrication. An alternative is insulator-based DEP (iDEP), an inexpensive method where nonuniform electric fields are created with arrays of insulating structures. This study presents the effects of operating conditions on the dielectrophoretic behavior of polystyrene microparticles under iDEP. Experiments were performed employing microchannels containing insulating structures that worked as insulators. The parameters varied were pH (8-9) and conductivity (25-100 microS/cm) of the bulk medium, and the magnitude of the applied field (200-850 V/cm). Optimal operating conditions in terms of pH and conductivity were obtained, and the microdevice performance was characterized in terms of concentration factor and minimum electric field required (minimum energy consumption). This is the first report on improving iDEP processes when EOF is present. DEP and EOF have been studied extensively, however, this study integrates the effect of suspending medium characteristics on both EK phenomena. These findings will allow improving the performance of iDEP microdevices achieving the highest concentration fold with the lowest energy consumption.     

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.     

Polarization behavior of polystyrene particles under direct current and low‐frequency (<1 kHz) electric fields in dielectrophoretic systems

The relative polarization behavior of micron and submicron polystyrene particles was investigated under direct current and very low frequency (<1 kHz) alternating current electric fields. Relative polarization of particles with respect to the suspending medium is expressed in terms of the Clausius–Mossotti factor, a parameter of crucial importance in dielectrophoretic‐based operations. Particle relative polarization was studied by employing insulator‐based dielectrophoretic (iDEP) devices. The effects of particle size, medium conductivity, and frequency (10–1000 Hz) of the applied electric potential on particle response were assessed through experiments and mathematical modeling with COMSOL Multiphysics^®. Particles of different sizes (100–1000 nm diameters) were introduced into iDEP devices fabricated from polydimethylsiloxane (PDMS) and their dielectrophoretic responses under direct and alternating current electric fields were recorded and analyzed in the form of images and videos. The results illustrated that particle polarizability and dielectrophoretic response depend greatly on particle size and the frequency of the electric field. Small particles tend to exhibit positive DEP at higher frequencies (200–1000 Hz), while large particles exhibit negative DEP at lower frequencies (20–200 Hz). These differences in relative polarization can be used for the design of iDEP‐based separations and analysis of particle mixtures.     

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.     

Continuous dielectrophoretic cell separation microfluidic device

We present a prototype microfluidic device developed for the continuous dielectrophoretic (DEP) fractionation and purification of sample suspensions of biological cells. The device integrates three fully functional and distinct units consisting of an injector, a fractionation region, and two outlets. In the sheath and sample injection ports, the cell sample are hydrodynamically focused into a stream of controlled width; in the DEP fractionation region, a specially shaped nonuniform (isomotive) electric field is synthesized and employed to facilitate the separation, and the sorted cells are then delivered to two sample collection ports. The microfluidic behavior of the injector region was simulated and then experimentally verified. The operation and performance of the device was evaluated using yeast cells as model biological particles. Issues relating to the fabrication and operation of the device are discussed in detail. Such a device takes a significant step towards an integrated lab-on-a-chip device, which could interface/integrate to a number of other on-chip components for the device to undertake the whole laboratory procedure.     

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.     

Dielectrophoresis induced clustering regimes of viable yeast cells

We experimentally study the transient clustering behavior of viable yeast cells in a dilute suspension suddenly subjected to a nonuniform alternating current (AC) electric field of a microelectrode device. The frequency of the applied electric field is varied to identify two distinct regimes of positive dielectrophoresis. In both regimes, the yeast cells eventually cluster at electrodes' edges, but their transient behavior as well as their final arrangement is quite different. Specifically, when the frequency is much smaller than the cross-over frequency, the nearby yeast cells quickly rearrange in well-defined chains which then move toward the electrodes' edges and remain aligned as elongated chains at their final location. However, when the frequency is close to the cross-over frequency, cells move individually toward the regions of collection and simply agglomerate along the electrodes' edges. Our analysis shows that in the first regime both the dielectrophoretic (DEP) force and the mutual DEP force, which arises due to the electrostatic particle-particle interactions, are important. In the second regime, on the other hand, the DEP force dominates.     

DC-dielectrophoretic separation of microparticles using an oil droplet obstacle

A new dielectrophoretic particle separation method is demonstrated and examined in the following experimental study. Current electrodeless dielectrophoretic (DEP) separation techniques utilize insulating solid obstacles in a DC or low-frequency AC field, while this novel method employs an oil droplet acting as an insulating hurdle between two electrodes. When particles move in a non-uniform DC field locally formed by the droplet, they are exposed to a negative DEP force linearly dependent on their volume, which allows the particle separation by size. Since the size of the droplet can be dynamically changed, the electric field gradient, and hence DEP force, becomes easily controllable and adjustable to various separation parameters. By adjusting the droplet size, particles of three different diameter sizes, 1 microm, 5.7 microm and 15.7 microm, were successfully separated in a PDMS microfluidic chip, under applied field strength in the range from 80 V cm-1 to 240 V cm-1. A very effective separation was realized at the low field strength, since the electric field gradient was proved to be a more significant parameter for particle discrimination than the applied voltage. By utilizing low strength fields and adaptable field gradient, this method can also be applied to the separation of biological samples that are generally very sensitive to high electric potential.     

Protein manipulation with insulator-based dielectrophoresis and direct current electric fields

The present study demonstrates the manipulation of protein particles employing insulator-based dielectrophoresis (iDEP) and direct current (d.c.) electric fields. Fluorescently labeled bovine serum albumin (BSA) protein particles were concentrated inside a microchannel that contained an array of glass cylindrical insulating structures. d.c. electric fields were applied and the dielectrophoretic response of the particles was observed as a function of the suspending medium conductivity (25, 50 and 100muS/cm) and pH (8 and 9). It was shown that the magnitude of the applied electric field (700-1600V/cm) and suspending medium properties have a strong effect on the dielectrophoretic response of the protein particles. The results presented here are the first report on protein manipulation employing d.c.-iDEP.     

Characterization of electrokinetic mobility of microparticles in order to improve dielectrophoretic concentration

Insulator-based dielectrophoresis (iDEP), an efficient technique with great potential for miniaturization, has been successfully applied for the manipulation of a wide variety of bioparticles. When iDEP is applied employing direct current (DC) electric fields, other electrokinetic transport mechanisms are present: electrophoresis and electroosmotic flow. In order to concentrate particles, iDEP has to overcome electrokinetics. This study presents the characterization of electrokinetic flow under the operating conditions employed with iDEP; in order to identify the optimal conditions for particle concentration employing DC-iDEP, microparticle image velocimetry (μPIV) was employed to measure the velocity of 1-μm-diameter inert polystyrene particles suspended inside a microchannel made from glass. Experiments were carried out by varying the properties of the suspending medium (conductivity from 25 to 100 μS/cm and pH from 6 to 9) and the strength of the applied electric field (50–300 V/cm); the velocities values obtained ranged from 100 to 700 μm/s. These showed that higher conductivity and lower pH values for the suspending medium produced the lowest electrokinetic flow, improving iDEP concentration of particles, which decreases voltage requirements. These ideal conditions for iDEP trapping (pH = 6 and σ _m = 100 μS/cm) were tested experimentally and with the aid of mathematical modeling. The μPIV measurements allowed obtaining values for the electrokinetic mobilities of the particles and the zeta potential of the glass surface; these values were used with a mathematical model built with COMSOL Multiphysics software in order to predict the dielectrophoretic and electrokinetic forces exerted on the particles; the modeling results confirmed the μPIV findings. Experiments with iDEP were carried out employing the same microparticles and a glass microchannel that contained an array of cylindrical insulating structures. By applying DC electric fields across the insulating structures array, it was seen that the dielectrophoretic trapping was improved when the electrokinetic force was the lowest; as predicted by μPIV measurements and the mathematical model. The results of this study provide guidelines for the selection of optimal operating conditions for improving insulator-based dielectrophoretic separations and have the potential to be extended to bioparticle applications. Figure Comparison of experimental measurements and mathematical modeling of electrokinetic and dielectrophoretic effects on microparticles

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.     

A continuous DC-insulator dielectrophoretic sorter of microparticles

A lab-on-a-chip device is described for continuous sorting of fluorescent polystyrene microparticles utilizing direct current insulating dielectrophoresis (DC-iDEP) at lower voltages than previously reported. Particles were sorted by combining electrokinetics and dielectrophoresis in a 250 μm wide PDMS microchannel containing a rectangular insulating obstacle and four outlet channels. The DC-iDEP particle flow behaviors were investigated with 3.18, 6.20 and 10 μm fluorescent polystyrene particles which experience negative DEP forces depending on particle size, DC electric field magnitude and medium conductivity. Due to negative DEP effects, particles are deflected into different outlet streams as they pass the region of high electric field density around the obstacle. Particles suspended in dextrose added phosphate buffer saline (PBS) at conductivities ranging from 0.50 to 8.50 mS/cm at pH 7.0 were compared at 6.85 and 17.1 V/cm. Simulations of electrokinetic and dielectrophoretic forces were conducted with COMSOL Multiphysics^® to predict particle pathlines. Experimental and simulation results show the effect of medium and voltage operating conditions on particle sorting. Further, smaller particles experience smaller iDEP forces and are more susceptible to competing nonlinear electrostatic effects, whereas larger particles experience greater iDEP forces and prefer channels 1 and 2. This work demonstrates that 6.20 and 10 μm particles can be independently sorted into specific outlet streams by tuning medium conductivity even at low operating voltages. This work is an essential step forward in employing DC-iDEP for multiparticle sorting in a continuous flow, multiple outlet lab-on-a-chip device.     

Parallelized continuous flow dielectrophoretic separation of DNA

Numerous microfluidic separation applications have been shown in the past years providing a fast analysis of biological samples like DNA or proteins. Microfluidic separation based on dielectrophoresis (DEP), that is the migration of a polarizable object in an inhomogeneous electric field, provides numerous advantages. However, the main drawback of DEP separation devices is that they are not sufficient for large-scale sample purification due to the lack of high sample throughput. In this work, we present for the first time a microfluidic device with two parallelized dielectrophoretic separations of (biological) samples smaller than 1 µm. The separation is carried out by means of insulator-based DEP, that is an insulating ridge reduced the flow through height and thus created a nanoslit at which the selective DEP forces occur. The device consists of a cross injector, two parallel operation regions and separate harvesting reservoirs where the samples are collected. Each DEP operation region contains an insulating ridge. We successfully demonstrate the separation of 100 and 40 nm beads and 10 and 5 kbp DNA with a separation purity of more than 80%. This states the proof-of-concept for up-scaling of dielectrophoretic separation by parallelization. As the present technique is virtually label-free, it offers a fast purification, for example in the production of gene vaccines.     

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.     

Joule heating effects in optimized insulator‐based dielectrophoretic devices: An interplay between post geometry and temperature rise

Insulator‐based dielectrophoresis (iDEP) is the electrokinetic migration of polarized particles when subjected to a non‐uniform electric field generated by the inclusion of insulating structures between two remote electrodes. Electrode spacing is considerable in iDEP systems when compared to electrode‐based DEP systems, therefore, iDEP systems require high voltages to achieve efficient particle manipulation. A consequence of this is the temperature increase within the channel due to Joule heating effects, which, in some cases, can be detrimental when manipulating biological samples. This work presents an experimental and modeling study on the increase in temperature inside iDEP devices. For this, we studied seven distinct channel designs that mainly differ from each other in their post array characteristics: post shape, post size and spacing between posts. Experimental results obtained using a custom‐built copper Resistance Temperature Detector, based on resistance changes, show that the influence of the insulators produces a difference in temperature rise of approximately 4°C between the designs studied. Furthermore, a 3D COMSOL model is also introduced to evaluate heat generation and dissipation, which is in good agreement with the experiments. The model allowed relating the difference in average temperature for the geometries under study to the electric resistance posed by the post array in each design.     

Quantification of pH gradients and implications in insulator-based dielectrophoresis of biomolecules

Direct current (DC) insulator-based dielectrophoretic (iDEP) microdevices have the potential to replace traditional alternating current dielectrophoretic devices for many cellular and biomolecular separation applications. The use of large DC fields suggest that electrode reactions and ion transport mechanisms can become important and impact ion distributions in the nanoliters of fluid in iDEP microchannels. This work tracked natural pH gradient formation in a 100?μm wide, 1?cm-long microchannel under applicable iDEP protein manipulation conditions. Using fluorescence microscopy with the pH-sensitive dye FITC Isomer I and the pH-insensitive dye TRITC as a reference, pH was observed to drop drastically in the microchannels within 1?min in a 3000?V/cm electric field; pH drops were observed in the range of 6-10 min within a 100?V/cm electric field and varied based on the buffer conductivity. To address concerns of dye transport impacting intensity data, electrokinetic mobilities of FITC were carefully examined and found to be (i) toward the anode and (ii) 1 to 2 orders of magnitude smaller than H? transport which is responsible for pH drops from the anode toward the cathode. COMSOL simulations of ion transport showed qualitative agreement with experimental results. The results indicate that pH changes are severe enough and rapid enough to influence the net charge of a protein or cause aggregation during iDEP experiments. The results also elucidate reasonable time periods over which the phosphate buffering capacity can counter increases in H? and OH? for unperturbed iDEP manipulations.     

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.     

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.     

Effects of viability and lectin protein binding on dielectrophoretic behavior of single yeast cells.

The dielectrophoretic (DEP) behavior of individual yeast cells (5-7 microm in diameter) in aqueous media was observed in a fabricated planar quadrupole microelectrode with a working area of 100 microm in diameter by an optical microscope. The yeast cells migrated in the radial direction in the working area. The DEP velocity of the cells increased as they approached the electrode. The DEP trajectory of the cells was analyzed with a theoretical equation derived previously, and the dielectrophoretic mobility was determined. The dielectrophoretic mobility was found to be affected by the viability of cells, the conductivity of the medium, and the binding of lectin protein (concanavalin A) to the cell surface. These DEP behaviors were analyzed based on the permittivities and conductivities of the cell interior and wall, and those of the medium.