Continuous dielectrophoretic size-based particle sorting

Continuous-flow dielectrophoretic (DEP) particle separation based on size is demonstrated in a microfluidic device. Polystyrene microspheres suspended in a neutrally buoyant aqueous solution are used as model particles to study DEP induced by an array of slanted, planar, interdigitated electrodes inside of a soft-lithography microchannel. The E-field gradients from the slanted electrodes impart a net transverse force component on the particles that causes them to "ratchet" across the channel. Over the length of the device, larger particles are deflected more than smaller particles according to the balance of hydrodynamic drag and DEP forces. Consequently, a flow-focused particle suspension containing different-sized particles is fractionated as the beads flow and separate down the length of the device. The flow behavior of spherical particles is modeled, and the total transverse particle displacement in the microfluidic device predicts fourth-order size and voltage and second-order inverse flow rate dependences. The model is verified experimentally for a range of flow rates, particle sizes, and E-field strengths.     

Microfluidic particle sorter employing flow splitting and recombining

This paper describes an improved microfluidic device that enables hydrodynamic particle concentration and size-dependent separation to be carried out in a continuous manner. In our previous study, a method for hydrodynamic filtration and sorting of particles was proposed using a microchannel having multiple branch points and side channels, and it was applied for continuous concentration and separation of polymer particles and cells. In the current study, the efficiency of particle sorting was dramatically improved by geometrically splitting fluid flow from a main stream and recombining. With these operations, particles with diameters larger than a specific value move toward one sidewall in the mainstream. This control of particle positions is followed by the perfect particle alignment onto the sidewall, which increases the selectivity and recovery rates without using a liquid that does not contain particles. In this study, a microchannel having one inlet and five outlets was designed and fabricated. By simply introducing particle suspension into the device, concentrations of 2.1-3.0-microm particles were increased 60-80-fold, and they were collected independently from each outlet. In addition, it was demonstrated that the measured flow rates distributed into each side channel corresponded well to the theoretical values when regarding the microchannel network as a resistive circuit.     

High-throughput and high-resolution flow cytometry in molded microfluidic devices

We describe the design, fabrication, and operation of two types of flow cytometers based on microfluidic devices made of a single cast of poly(dimethylsiloxane). The stream of particles or cells injected into the devices is hydrodynamically focused in both transverse and lateral directions, has a uniform velocity, and has adjustable diameter and shape. The cytometry system built around the first microfluidic device has fluorescence detection accuracy comparable with that of a commercial flow cytometer and can analyze as many as 17 000 particles/s. This high-throughput microfluidic device could be used in inexpensive stand-alone cytometers or as a part of integrated microanalysis systems. In the second device, a stream of particles is focused to a flow layer of a submicrometer thickness that allows imaging the particles with a high numerical aperture microscope objective. To take long-exposure, low-light fluorescence images of live cells, the device is placed on a moving stage, which accurately balances the translational motion of particles in the flow. The achieved resolution is comparable to that of still micrographs. This high-resolution device could be used for analysis of morphology and fluorescence distribution in cells in continuous flow.     

Recirculation of nanoliter volumes within microfluidic channels

A microfluidic device is described, capable of recirculating nanoliter volumes in restricted microchannel segments. The device consists of a PDMS microfluidic structure, reversibly sealed to a glass substrate with integrated platinum electrodes. The integrated electrodes generate electroosmotic flow locally, which results in a cycling flow in the channel segment between the two electrodes in case one channel exit is closed (dead-end channel). This cycling flow is a consequence of the counterbalancing hydrodynamic pressure against the electroosmotically generated flow. Acid-base indicators were employed to study the formation of H(+) and OH(-) at both the in-channel electrodes. The formation of acid can locally change the zeta-potential of the channel wall, which will affect the flow profile. Using this method, small analyte volumes can be mixed for prolonged times within well-defined channel segments and/or exposed to in-channel sensor surfaces.     

Continuous sorting of microparticles using dielectrophoresis

Sorting of particles such as cells is a critical process for many biomedical applications, and it is challenging to integrate it into an analytical microdevice. We report an effective and flexible dielectrophoresis (DEP)-based microfluidic device for continuous sorting of multiple particles in a microchannel. The particle sorter is composed of two components-a DEP focusing unit and a Movable DEP Trap (MDT). The trap is formed by an array of microelectrodes at the bottom of the channel and a transparent electrode plate placed at the top. The location of the trap is dependent on the configuration of voltages on the array and therefore is addressable. Flowing particles are first directed and focused into a single particle stream by the focusing unit. The streamed particles are then sorted into different fractions using the movable trap by rapidly switching the applied voltage. The performance of the sorter is demonstrated by successfully sorting microparticles in a continuous flow. The proposed DEP-based microfluidic sorter can be implemented in applications such as sample preparation and cell sorting for subsequent analytical processing, where sorting of particles is needed.     

Continuous Cell Separation Using Dielectrophoresis through Asymmetric and Periodic Microelectrode Array

The study presents a dielectrophoretic cell separation method via three-dimensional (3D) nonuniform electric fields generated by employing a periodic array of discrete but locally asymmetric triangular bottom microelectrodes and a continuous top electrode. Traversing through the microelectrodes, heterogeneous cells are electrically polarized to experience different strengths of positive dielectrophoretic forces, in response to the 3D nonuniform electric fields. The cells that experience stronger positive dielectrophoresis are streamed further in the perpendicular direction to the fluid flow, leaving the cells that experience weak positive dielectrophoresis, which continue to traverse the microelectrode array essentially along the laminar flow streamlines. The proposed method has achieved 87.3% pure live cells harvesting efficiency from a live/dead NIH-3T3 cells mixture, and separation of MG-63 cells from erythrocytes with a separation efficiency of 82.8%. The demonstrated cell separation shows promising applications of the DEP separator for cell separation in a continuous mode.     

Perfusion in microfluidic cross-flow: separation of white blood cells from whole blood and exchange of medium in a continuous flow

We describe a microfluidic technique for separation of particles and cells and a device that employs this technique to separate white blood cells (WBC) from whole human blood. The separation is performed in cross-flow in an array of microchannels with a deep main channel and large number of orthogonal, shallow side channels. As a suspension of particles advances through the main channel, a perfusion flow through the side channels gradually exchanges the medium of the suspension and washes away particles that are sufficiently small to enter the shallow side channels. The microfluidic device is tested with a suspension of polystyrene beads and is shown to efficaciously exchange the carrier medium while retaining all beads. In tests with whole human blood, the device is shown to reduce the content of red blood cells (RBC) by a factor of approximately 4000 with retention of 98% of WBCs. The ratio between WBCs and RBCs reached at an outlet of the device is 2.4 on average. The device is made of a single cast of poly(dimethylsiloxane) sealed with a cover glass and is simple to fabricate. The proposed technique of separation by perfusion in continuous cross-flow could be used to enrich rare populations of cells based on differences in size, shape, and deformability.     

Design and numerical investigation of a circular microchannel for particle/cell separation using dielectrophoresis

Precisely separating particles/cells with different sizes and physical properties has been an interest for point-of-care diagnostics and personalized treatment. Dielectrophoresis (DEP) is widely known as a powerful and non-invasive technique to separate particles and cells. This paper presents a comprehensive numerical investigation of particle/cell separation in circular microchannels using DEP. First, the geometrical parameters of the circular microchannel affecting DEP force are determined by performing an analytical solution. Then, by developing a solver in OpenFOAM, the effect of these parameters on particles deflection is investigated. According to the results, two different circular microchannels are presented to investigate the continuous separation of bio-particles (based on their physical properties) and polystyrene particles (based on their size). The results showed that a minimum voltage of 7, 9, and 12 V is required to achieve 100 % purity and separation efficiency for separating red blood cells from MDA-MB-231 cancer cells at the flow rate of 0.5, 1.0, and 1.5 µl/min, respectively. Also, the efficient separation of 5 and 10 µm polystyrene particles at the flow rate of 0.1 µl/min is possible only at the voltage of 9 V. The results of this numerical study can be useful for the fabrication of an optimal microdevice for the continuous DEP separation of particles and cells.     

A Self-Powered Dielectrophoretic Microparticle Manipulation Platform Based on a Triboelectric Nanogenerator

Lab-on-a-chip systems aim to integrate laboratory operations on a miniaturized device with broad application prospects in the field of point-of-care testing. However, bulky peripheral power resources, such as high-voltage supplies, function generators, and amplifiers, hamper the commercialization of the system. In this work, a portable, self-powered microparticle manipulation platform based on triboelectrically driven dielectrophoresis (DEP) is reported. A rotary freestanding triboelectric nanogenerator (RF-TENG) and rectifier/filter circuit supply a high-voltage direct-current signal to form a non-uniform electric field within the microchannel, realizing controllable actuation of the microparticles through DEP. The operating mechanism of this platform and the control performance of the moving particles are systematically studied and analyzed. Randomly distributed particles converge in a row after passing through the serpentine channel and various particles are separated owing to the different DEP forces. Ultimately, the high-efficiency separation of live and dead yeast cells is achieved using this platform. RF-TENG as the power source for lab-on-a-chip exhibits better safety and portability than traditional high-voltage power sources. This study presents a promising solution for the commercialization of lab-on-a-chip.     

Advection Flows‐Enhanced Magnetic Separation for High‐Throughput Bacteria Separation from Undiluted Whole Blood

A major challenge to scale up a microfluidic magnetic separator for extracorporeal blood cleansing applications is to overcome low magnetic drag velocity caused by viscous blood components interfering with magnetophoresis. Therefore, there is an unmet need to develop an effective method to position magnetic particles to the area of augmented magnetic flux density gradients while retaining clinically applicable throughput. Here, a magnetophoretic cell separation device, integrated with slanted ridge‐arrays in a microfluidic channel, is reported. The slanted ridges patterned in the microfluidic channels generate spiral flows along the microfluidic channel. The cells bound with magnetic particles follow trajectories of the spiral streamlines and are repeatedly transferred in a transverse direction toward the area adjacent to a ferromagnetic nickel structure, where they are exposed to a highly augmented magnetic force of 7.68 µN that is much greater than the force (0.35 pN) at the side of the channel furthest from the nickel structure. With this approach, 91.68% ± 2.18% of Escherichia coli (E. coli) bound with magnetic nanoparticles are successfully separated from undiluted whole blood at a flow rate of 0.6 mL h^?1 in a single microfluidic channel, whereas only 23.98% ± 6.59% of E. coli are depleted in the conventional microfluidic device.     

On-chip high-speed sorting of micron-sized particles for high-throughput analysis

A new design of particle sorting chip is presented. The device employs a dielectrophoretic gate that deflects particles into one of two microfluidic channels at high speed. The device operates by focussing particles into the central streamline of the main flow channel using dielectrophoretic focussing. At the sorting junction (T- or Y-junction) two sets of electrodes produce a small dielectrophoretic force that pushes the particle into one or other of the outlet channels, where they are carried under the pressure-driven fluid flow to the outlet. For a 40 microm wide and high channel, it is shown that 6 microm diameter particles can be deflected at a rate of 300/s. The principle of a fully automated sorting device is demonstrated by separating fluorescent from non-fluorescent latex beads.     

Sheathless hydrophoretic particle focusing in a microchannel with exponentially increasing obstacle arrays

We present a novel microfluidic device with exponentially increasing obstacle arrays to enable sheathless particle focusing. The anisotropic fluidic resistance of slant obstacles generates transverse flows, along which particles are focused to one sidewall. In the successive channel with exponentially increasing widths, bent obstacles extended from the slant obstacles increase the focusing efficiency of the particles. With the device, we achieved the focusing efficiency of 76%, 94%, and 98% for 6, 10, and 15 microm beads, respectively. The focusing efficiency of the particles can be further improved in the devices with more extension steps. In addition, using the microfluidic devices with the symmetric structure of the slant and bent obstacles, we achieved complete focusing of biological cells to the centerline of a channel within 1.7% coefficient of variation. The results demonstrated the sheathless hydrophoretic focusing of microparticles and cells with the advantages of a sheathless method, passive operation, single channel, and flow rate independence.     

Separation of latex spheres using dielectrophoresis and fluid flow

The authors present a method for separation of two latex spheres populations using dielectrophoresis (DEP) and the fluid drag force. Microelectrodes of a suitable layout are used to trap one population of spheres, while the other one is dragged away from the electrodes by the generated fluid flow. The finite difference method is implemented in C++ to calculate the potential distribution by solving Laplace's equation. From the potential distribution, the DEP force on particles is calculated. The drag force on particles due to the liquid motion is calculated from the observed fluid velocity. The experimental results are shown to be in good agreement with the numerical solution.     

Electrohydrodynamic generation and delivery of monodisperse picoliter droplets using a poly(dimethylsiloxane) microchip

We developed a drop-on-demand microdroplet generator for the discrete dispensing of biosamples into a bioanalytical unit. This disposable PDMS microfluidic device can generate monodisperse droplets of picoliter volume directly out of a plane sidewall of the microfluidic chip by an electrohydrodynamic mechanism. The droplet generation was accomplished without using either an inserted capillary or a monolithically built-in tip. The minimum droplet volume was approximately 4 pL, and the droplet generation was repeatable and stable for at least 30 min, with a typical variation of less than 2.0% of drop size. The Taylor cone, which is usually observed in electrospray, was suppressed by controlling the surface wetting property of the PDMS device as well as the surface tension of the sample liquids. A modification of the channel geometry right before the opening of the microchannel also enhanced the continuous droplet generation without applying any external pumping. A simple numerical simulation of the droplet generation verified the importance of controlling the surface wetting conditions for the droplet formation. Our microdroplet generator can be effectively applied to a direct interface of a microfluidic chip to a biosensing unit, such as AMS, MALDI-MS or protein microarray-type biochips.     

Noninvasive acoustic cell trapping in a microfluidic perfusion system for online bioassays

Techniques for manipulating, separating, and trapping particles and cells are highly desired in today's bioanalytical and biomedical field. The microfluidic chip-based acoustic noncontact trapping method earlier developed within the group now provides a flexible platform for performing cell- and particle-based assays in continuous flow microsystems. An acoustic standing wave is generated in etched glass channels (600x61 microm2) by miniature ultrasonic transducers (550x550x200 microm3). Particles or cells passing the transducer will be retained and levitated in the center of the channel without any contact with the channel walls. The maximum trapping force was calculated to be 430+/-135 pN by measuring the drag force exerted on a single particle levitated in the standing wave. The temperature increase in the channel was characterized by fluorescence measurements using rhodamine B, and levels of moderate temperature increase were noted. Neural stem cells were acoustically trapped and shown to be viable after 15 min. Further evidence of the mild cell handling conditions was demonstrated as yeast cells were successfully cultured for 6 h in the acoustic trap while being perfused by the cell medium at a flowrate of 1 microL/min. The acoustic microchip method facilitates trapping of single cells as well as larger cell clusters. The noncontact mode of cell handling is especially important when studies on nonadherent cells are performed, e.g., stem cells, yeast cells, or blood cells, as mechanical stress and surface interaction are minimized. The demonstrated acoustic trapping of cells and particles enables cell- or particle-based bioassays to be performed in a continuous flow format.     

A prototype two-dimensional capillary electrophoresis system fabricated in poly(dimethylsiloxane)

A method for carrying out 2D gel electrophoresis in a capillary format is presented. In this method, separation in the first dimension is carried out in a 1D capillary, with this system physically isolated from the capillaries that provide the separation in the second dimension. After completion of the first separation, the 1D channel is physically connected to the 2D capillaries, and a second separation is carried out in an orthogonal set of parallel capillaries. The ability of poly(dimethylsiloxane) (PDMS) to support the fabrication of 3D microfluidic systems makes it possible to produce membranes that both enclose the gel used in the first separation in a capillary and provide passages for the proteins to migrate into the array of orthogonal capillaries. The elastomeric nature of PDMS makes it possible to make reversible connections between pieces of PDMS. The feasibility of this system is demonstrated using a protein mixture containing fluorescein-conjugated carbonic anhydrase, fluorescein-conjugated BSA, and Texas Red-conjugated ovalbumin. This work suggests one type of design that might form the basis for a microfabricated device for 2D capillary electrophoresis.     

Lateral transport of solutes in microfluidic channels using electrochemically generated gradients in redox-active surfactants

We report principles for a continuous flow process that can separate solutes based on a driving force for selective transport that is generated by a lateral concentration gradient of a redox-active surfactant across a microfluidic channel. Microfluidic channels fabricated with gold electrodes lining each vertical wall were used to electrochemically generate concentration gradients of the redox-active surfactant 11-ferrocenylundecyl-trimethylammonium bromide (FTMA) in a direction perpendicular to the flow. The interactions of three solutes (a hydrophobic dye, 1-phenylazo-2-naphthylamine (yellow AB), an amphiphilic molecule, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY C(5)-HPC), and an organic salt, 1-methylpyridinium-3-sulfonate (MPS)) with the lateral gradients in surfactant/micelle concentration were shown to drive the formation of solute-specific concentration gradients. Two distinct physical mechanisms were identified to lead to the solute concentration gradients: solubilization of solutes by micelles and differential adsorption of the solutes onto the walls of the microchannels in the presence of the surfactant concentration gradient. These two mechanisms were used to demonstrate delipidation of a mixture of BODIPY C(5)-HPC (lipid) and MPS and purification of BODIPY C(5)-HPC from a mixture of BODIPY C(5)-HPC and yellow AB. Overall, the results of this study demonstrate that lateral concentration gradients of redox-active surfactants formed within microfluidic channels can be used to transport solutes across the microfluidic channels in a solute-dependent manner. The approach employs electrical potentials (<1 V) that are sufficiently small to avoid electrolysis of water, can be performed in solutions having high ionic strength (>0.1M), and offers the basis of continuous processes for the purification or separation of solutes in microscale systems.     

High-speed free-flow electrophoresis on chip

A microfluidic device has been developed for continuous separation in free-flow electrophoresis (FFE) mode. A mixture of two fluorescent reagents is separated into two component streams in 75 ms using a sample flow rate of 2 nL/s. The residence time of sample in the whole separation compartment is 2 s. The separation bed volume is 0.2 microL. The chip has also been used for free-flow electrophoresis of fluorescein-5-isothiocyanate-labeled amino acids in both aqueous and binary media. The short residence time and small sample flow rate make the FFE chip feasible for on-line monitoring on production lines and other chemical or biochemical processes. The in-house-made chip was composed of a plain glass substrate of 1.5-mm thickness and a PDMS layer of 0.3-mm thickness with micromachined channels. The channel design presented in this paper is versatile. With the same kind of PDMS substrates, chips for various purposes can be made depending on the locations of the reservoirs, which are cut out on the PDMS substrate. The results presented verify the scaling laws and allow prediction of FFE performances comparable to what is now state of the art on capillary electrophoresis chips.     

Free flow acoustophoresis: microfluidic-based mode of particle and cell separation

A novel method, free flow acoustophoresis (FFA), capable of continuous separation of mixed particle suspensions into multiple outlet fractions is presented. Acoustic forces are utilized to separate particles based on their size and density. The method is shown to be suitable for both biological and nonbiological suspended particles. The microfluidic separation chips were fabricated using conventional microfabrication methods. Particle separation was accomplished by combining laminar flow with the axial acoustic primary radiation force in an ultrasonic standing wave field. Dissimilar suspended particles flowing through the 350-microm-wide channel were thereby laterally translated to different regions of the laminar flow profile, which was split into multiple outlets for continuous fraction collection. Using four outlets, a mixture of 2-, 5-, 8-, and 10-microm polystyrene particles was separated with between 62 and 94% of each particle size ending up in separate fractions. Using three outlets and three particle sizes (3, 7, and 10 microm) the corresponding results ranged between 76 and 96%. It was also proven possible to separate normally acoustically inseparable particle types by manipulating the density of the suspending medium with cesium chloride. The medium manipulation, in combination with FFA, was further used to enable the fractionation of red cells, platelets, and leukocytes. The results show that free flow acoustophoresis can be used to perform complex separation tasks, thereby offering an alternative to expensive and time-consuming methods currently in use.     

Cell separation on microfabricated electrodes using dielectrophoretic/gravitational field-flow fractionation

Dielectrophoretic/gravitational field-flow fractionation (DEP/G-FFF) was used to separate cultured human breast cancer MDA-435 cells from normal blood cells mixed together in a sucrose/dextrose medium. An array of microfabricated, interdigitated electrodes of 50 microns widths and spacings, and lining the bottom surface of a thin chamber (0.42 mm H x 25 mm W x 300 mm L), was used to generate DEP forces that levitated the cells. A 10-microL cell mixture sample containing approximately 50,000 cells was introduced into the chamber, and cancerous and normal blood cells were levitated to different heights according to the balance of DEP and gravitational forces. The cells at different heights were transported at different velocities under the influence of a parabolic flow profile that was established in the chamber and were thereby separated. Separation performance depended on the frequency and voltage of the applied DEP field and the fluid-flow rate. It took as little as 5 min to achieve cell separation. An analysis of the DEP/G-FFF results revealed that the separation exploited the difference in dielectric and density properties between cell populations. The DEP/G-FFF technique is potentially applicable to many biological and biomedical problems, especially those related to microfluidic systems.