Dielectrophoresis microsystem with integrated flow cytometers for on-line monitoring of sorting efficiency

Dielectrophoresis (DEP) and flow cytometry are powerful technologies and widely applied in microfluidic systems for handling and measuring cells and particles. Here, we present a novel microchip with a DEP selective filter integrated with two microchip flow cytometers (FCs) for on-line monitoring of cell sorting processes. On the microchip, the DEP filter is integrated in a microfluidic channel network to sort yeast cells by positive DEP. The two FCs detection windows are set upstream and downstream of the DEP filter. When a cell passes through the detection windows, the light scattered by the cell is measured by integrated polymer optical elements (waveguide, lens, and fiber coupler). By comparing the cell counting rates measured by the two FCs, the collection efficiency of the DEP filter can be determined. The chips were used for quantitative determination of the effect of flow rate, applied voltage, conductivity of the sample, and frequency of the electric field on the sorting efficiency. A theoretical model for the capture efficiency was developed and a reasonable agreement with the experimental results observed. Viable and non-viable yeast cells showed different frequency dependencies and were sorted with high efficiency. At 2 MHz, more than 90% of the viable and less than 10% of the non-viable cells were captured on the DEP filter. The presented approach provides quantitative real-time data for sorting a large number of cells and will allow optimization of the conditions for, e.g., collecting cancer cells on a DEP filter while normal cells pass through the system. Furthermore, the microstructure is simple to fabricate and can easily be integrated with other microstructures for lab-on-a-chip applications.     

A microfluidic device for separating erythrocytes polluted by lead (II) from a continuous bloodstream flow

To sort and separate erythrocytes contaminated by lead (II) from whole bloodstream flow, the first step is to use a microchannel to transport the blood cells into a microdevice. Within the device, polluted erythrocytes can be separated from the bloodstream by applying local dielectrophoretic (DEP) forces. Exploiting the fact that Pb(2+) ions attach to the membranes of the erythrocytes, we utilize the microfluidic DEP device to perform property-based fractionation of the blood samples and to separate the polluted erythrocytes from the continuous bloodstream flow. Atomic absorption spectrometer analysis reveals that, to remove lead-polluted erythrocytes, the most effective driving velocity was less than 0.1 cm/s through our microfluidic DEP device, based on an applied power of 10 V(peak-peak) and a frequency of 15.5 MHz AC field. We were able to remove 80% of the polluted erythrocytes. Using gentle DEP manipulating techniques to efficiently sort unique cells within a complex biological sample may potentially allow biological sorting to be performed outside of hospitals, in facilities without biological analyzing equipment.     

The dielectrophoretic levitation and separation of latex beads in microchips.

A linear travelling wave dielectrophoretic (twDEP) microchip was fabricated and used to investigate both the levitation and the twDEP motion of latex beads as a function of applied potential and frequency, suspending medium conductivity, bead size, and surface characteristics. The surface conductance of the latex beads was characterised by measurement of the dielectrophoretic (DEP) crossover frequency. Collection of sample prior to initiation of twDEP was achieved using positive DEP forces generated by an integrated pair of parallel electrodes positioned in front of the twDEP array within the microfluidic channel. The principle of linear twDEP separation is shown using latex beads and rabbit heart cells.     

介电电泳芯片及其在细胞分析中的应用

简要阐述了在交流和直流电压电场中,介电电泳(DEP)芯片进行细胞分离富集的机理.按照驱动电场的差异对DEP芯片进行了分类,分析和比较了DEP芯片微电极的叉指电极、抛物线电极、堡式电极、三维电极等典型结构.特别对近年来DEP芯片在单细胞分析、细胞分离与富集以及临床细胞分析中的应用进展进行了综述,并对其应用前景和发展方向进行了展望.     

Off-chip passivated-electrode, insulator-based dielectrophoresis (OπDEP)

In this study, we report the first off-chip passivated-electrode, insulator-based dielectrophoresis microchip (OπDEP). This technique combines the sensitivity of electrode-based dielectrophoresis (eDEP) with the high-throughput and inexpensive device characteristics of insulator-based dielectrophoresis (iDEP). The device is composed of a permanent, reusable set of electrodes and a disposable, polymer microfluidic chip with microposts embedded in the microchannel. The device operates by capacitively coupling the electric fields into the microchannel; thus, no physical connections are made between the electrodes and the microfluidic device. During operation, the polydimethylsiloxan (PDMS) microfluidic chip fits onto the electrode substrate as a disposable cartridge. OπDEP uses insulting structures within the channel as well as parallel electrodes to create DEP forces by the same working principle that iDEP devices use. The resulting devices create DEP forces which are larger by two orders of magnitude for the same applied voltage when compared to off-chip eDEP designs from literature, which rely on parallel electrodes alone to produce the DEP forces. The larger DEP forces allow the OπDEP device to operate at high flow rates exceeding 1 mL/h. In order to demonstrate this technology, Escherichia coli (E. coli), a known waterborne pathogen, was trapped from water samples. Trapping efficiencies of 100 % were obtained at flow rates as high as 400 μL/h and 60 % at flow rates as high as 1200 μL/h. Additionally, bacteria were selectively concentrated from a suspension of polystyrene beads.

Dielectrophoresis switching with vertical sidewall electrodes for microfluidic flow cytometry

A novel dielectrophoresis switching with vertical electrodes in the sidewall of microchannels for multiplexed switching of objects has been designed, fabricated and tested. With appropriate electrode design, lateral DEP force can be generated so that one can dynamically position particulates along the width of the channel. A set of interdigitated electrodes in the sidewall of the microchannels is used for the generation of non-uniform electrical fields to generate negative DEP forces that repel beads/cells from the sidewalls. A countering DEP force is generated from another set of electrodes patterned on the opposing sidewall. These lateral negative DEP forces can be adjusted by the voltage and frequency applied. By manipulating the coupled DEP forces, the particles flowing through the microchannel can be positioned at different equilibrium points along the width direction and continue to flow into different outlet channels. Experimental results for switching biological cells and polystyrene microbeads to multiple outlets (up to 5) have been achieved. This novel particle switching technique can be integrated with other particle detection components to enable microfluidic flow cytometry systems.     

阵列式对电极介电电泳芯片及其用于细胞分离富集研究

基于介电电泳原理, 设计并制作了一种新型的能够用于细胞分离和富集的微流控介电电泳芯片. 该芯片由沉积有金电极的石英基片和带有微管道的聚二甲基硅氧烷(PDMS)盖片组成. 通过在管道底部布置间距不同的对电极阵列, 增大了正介电电泳力在管道中的有效作用范围, 能够在降低施加电压的同时, 实现对流动体系中细胞样品的捕获. 在3 V和3 MHz条件下, 该DEP芯片对人血红细胞的捕获效率达到83%; 进一步通过将肝癌细胞捕获在芯片电极上可实现对红细胞和肝癌细胞混合样品的分离, 在5 V和400 kHz条件下对肝癌细胞的捕获效率达到86%.     

AC electrokinetic isolation and detection of extracellular vesicles from dental pulp stem cells: Theoretical simulation incorporating fluid mechanics

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles involved in intracellular communication and the transportation of biomarkers. EVs released by mesenchymal stem cells have been recently reported to play a role in cell-free therapy of many diseases. However, the demand for better research tools to replace the tedious conventional methods used to study EVs is getting stronger. EVs' manipulation using alternating current (AC) electrokinetic forces in a microfluidic device has appeared to be a reliable and sensitive diagnosis and trapping technique. Given that different AC electrokinetic forces may contribute to the overall motion of particles and fluids in a microfluidic device, EVs' electrokinetic trapping must be examined considering all dominant forces involved depending on the experimental conditions. In this paper, AC electrokinetic trapping of EVs using an interdigitated electrode arrays is investigated. A 2D numerical simulation incorporating the two significant AC electrokinetic phenomena (Dielectrophoresis and AC electroosmosis) has been performed. Theoretical predictions are then compared with experimental results and allow for a plausible explanation of observations inconsistent with DEP theory. It is demonstrated that the inconsistencies can be attributed to a significant extent to the contribution of the AC electroosmotic effect.     

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.     

Floating electrode dielectrophoresis

In practice, dielectrophoresis (DEP) devices are based on micropatterned electrodes. When subjected to applied voltages, the electrodes generate nonuniform electric fields that are necessary for the DEP manipulation of particles. In this study, electrically floating electrodes are used in DEP devices. It is demonstrated that effective DEP forces can be achieved by using floating electrodes. Additionally, DEP forces generated by floating electrodes are different from DEP forces generated by excited electrodes. The floating electrodes' capabilities are explained theoretically by calculating the electric field gradients and demonstrated experimentally by using test-devices. The test-devices show that floating electrodes can be used to collect erythrocytes (red blood cells). DEP devices which contain many floating electrodes ought to have fewer connections to external signal sources. Therefore, the use of floating electrodes may considerably facilitate the fabrication and operation of DEP devices. It can also reduce device dimensions. However, the key point is that DEP devices can integrate excited electrodes fabricated by microtechnology processes and floating electrodes fabricated by nanotechnology processes. Such integration is expected to promote the use of DEP devices in the manipulation of nanoparticles.     

A hybrid dielectrophoretic and hydrophoretic microchip for particle sorting using integrated prefocusing and sorting steps

This work explores dielectrophoresis (DEP)‐active hydrophoresis in sorting particles and cells. The device consists of prefocusing region and sorting region with great potential to be integrated into advanced lab‐on‐a‐chip bioanalysis devices. Particles or cells can be focused in the prefocusing region and then sorted in the sorting region. The DEP‐active hydrophoretic sorting is not only based on size but also on dielectric properties of the particles or cells of interest without any labelling. A mixture of 3 and 10 μm particles were sorted and collected from corresponding outlets with high separation efficiency. According to the different dielectric properties of viable and nonviable Chinese Hamster Ovary (CHO) cells at the medium conductivity of 0.03 S/m, the viable CHO cells were focused well and sorted from cell sample with a high purity.     

Embedded passivated-electrode insulator-based dielectrophoresis (EπDEP)

Here, we introduce a new technique called embedded passivated-electrode insulator-based dielectrophoresis (EπDEP) for preconcentration, separation, or enrichment of bioparticles, including living cells. This new method combines traditional electrode-based DEP and insulator-based DEP with the objective of enhancing the electric field strength and capture efficiency within the microfluidic channel while alleviating direct contact between the electrode and the fluid. The EπDEP chip contains embedded electrodes within the microfluidic channel covered by a thin passivation layer of only 4 μm. The channel was designed with two nonaligned vertical columns of insulated microposts (200 μm diameter, 50 μm spacing) located between the electrodes (600 μm wide, 600 μm horizontal spacing) to generate nonuniform electric field lines to concentrate cells while maintaining steady flow in the channel. The performance of the chip was demonstrated using Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial pathogens in aqueous media. Trapping efficiencies of 100 % were obtained for both pathogens at an applied AC voltage of 50 V peak-to-peak and flow rates as high as 10 μl/min.     

Catch and release cell sorting: electrochemical desorption of T-cells from antibody-modified microelectrodes

The development of integrated microsystems capable of interrogation, characterization and sorting of mammalian cells is highly significant for further advancement of point-of-care diagnostics and drug discovery fields. The present study sought to design a novel strategy for releasing antibody-bound cells through electrochemical disruption of the underlying antibody (Ab) layer. A microsystem for selective capture and release of cells consisted of an array of individually addressable gold microelectrodes fabricated on a glass substrate. Poly(ethylene glycol) (PEG) hydrogel photolithography was employed to make the glass regions non-fouling, thus, ensuring selective localization of proteins and cells on the microelectrodes. The gold surfaces were decorated with anti-CD4 Ab molecules using standard alkanethiol self-assembly and carbodiimide coupling approaches. The Ab-functionalized electrodes selectively captured model T-lymphocytes (Molt-3 cells) expressing CD4 antigen while minimal cell adhesion was observed on PEG hydrogel-modified glass substrates. Importantly, application of a reductive potential (-1.2V vs. Ag/AgCl reference electrode) resulted in release of surface-bound T-cells from the electrode surface. Cyclic voltammetry and fluorescence microscopy were employed to verify that the detachment of captured T-cells was indeed due to the electrochemical disruption of the underlying alkanethiol-Ab layer. In the future, the cell sorting approach described here may be combined with microfluidic delivery to enable Ab-mediated capture of T-lymphocytes or other cell types followed by release of select cells for downstream gene expression studies or re-cultivation.     

Numerical study of dielectrophoresis-modified inertial migration for overlapping sized cell separation

Circulating tumor cells (CTCs) have been proven to have significant prognostic, diagnostic, and clinical values in early-stage cancer detection and treatment. The efficient separation of CTCs from peripheral blood can ensure intact and viable CTCs and can, thus, give proper genetic characterization and drug innovation. In this study, continuous and high-throughput separation of MDA-231 CTCs from overlapping sized white blood cells (WBCs) is achieved by modifying inertial cell focusing with dielectrophoresis (DEP) in a single-stage microfluidic platform by numeric simulation. The DEP is enabled by embedding interdigitated electrodes with alternating field control on a serpentine microchannel to avoid creating two-stage separation. Rather than using the electrokinetic migration of cells which slows down the throughput, the system leverages the inertial microfluidic flow to achieve high-speed continuous separation. The cell migration and cell positioning characteristics are quantified through coupled physics analyses to evaluate the effects of the applied voltages and Reynolds numbers (Re) on the separation performance. The results indicate that the introduction of DEP successfully migrates WBCs away from CTCs and that separation of MDA-231 CTCs from similar sized WBCs at a high Re of 100 can be achieved with a low voltage of magnitude 4 ×10⁶ V/m. Additionally, the viability of MDA-231 CTCs is expected to be sustained after separation due to the short-term DEP exposure. The developed technique could be exploited to design active microchips for high-throughput separation of mixed cell beads despite their significant size overlap, using DEP-modified inertial focusing controlled simply by adjusting the applied external field.     

Electrokinetically driven micro flow cytometers with integrated fiber optics for on-line cell/particle detection

This paper presents an innovative micro flow cytometer which is capable of counting and sorting cells or particles. This compact device employs electrokinetic forces rather than the more conventional hydrodynamic forces technique for flow focusing and sample switching, and incorporates buried optical fibers for the on-line detection of cells or particles. This design approach results in a compact microfluidic system and an easier integration process. The proposed cytometer integrates several critical modules, namely electrokinetic-focusing devices, built-in control electrodes, buried optical fibers for on-line detection, and electrokinetic flow switches for bio-particle collection. A linear relationship exists between the focused stream width (d) and the focusing ratio (F/φ), which is estimated to be D≈134.5−53.8F/φ. The relationship between the particle velocity (U) and the applied voltage (V) is also investigated. Numerical and experimental data confirm the effectiveness of the device when applied to the counting and sorting of 10 μm diameter particles and red blood cells.     

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.     

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.     

Dielectrophoretic separation of microalgae cells in ballast water in a microfluidic chip

The composition of the ship's ballast water is complex and contains a large number of microalgae cells, bacteria, microplastics, and other microparticles. To increase the accuracy and efficiency of detection of the microalgae cells in ballast water, a new microfluidic chip for continuous separation of microalgae cells based on alternating current dielectrophoresis was proposed. In this microfluidic chip, one piece of 3‐dimensional electrode is embedded on one side and eight discrete electrodes are arranged on the other side of the microchannel. An insulated triangular structure between electrodes is designed for increasing the inhomogeneity of the electric field distribution and enhancing the dielectrophoresis (DEP) force. A sheath flow is designed to focus the microparticles near the electrode, so as to increase the suffered DEP force and improve separation efficiency. To demonstrate the performance of the microfluidic separation chip, we developed two species of microalgae cells (Platymonas and Closterium) and a kind of microplastics to be used as test samples. Analyses of the related parameters and separation experiments by our designed microfluidic chip were then conducted. The results show that the presented method can separate the microalgae cells from the mixture efficiently, and this is the first time to separate two or more species of microalgae cells in a microfluidic chip by using negative and positive DEP force simultaneously, and moreover it has some advantages including simple operation, high efficiency, low cost, and small size and has great potential in on‐site pretreatment of ballast water.     

Theoretical and experimental analysis of negative dielectrophoresis-induced particle trajectories

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

Dielectrophoretic separation of platelets from diluted whole blood in microfluidic channels

The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform size-based fractionation of blood samples and continuously enrich the platelets in a label-free manner. Cytometry analysis revealed that a single pass through the two-stage DACS device yields a high purity of platelets (approximately 95%) at a throughput of approximately 2.2 x 10(4) cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free dielectrophoretic separation of delicate cells from complex samples and such a separation approach may open a path toward continuous screening of blood products by integrated microfluidic devices.