Hydrodynamic separation of cells utilizing insulator-based dielectrophoresis

Manipulation and discrimination of biological cells is essential to many biomedical applications. Insulator-based dielectrophoresis (iDEP) trapping consists of insulating structures which squeeze the electric field in a conductive solution to create a non-uniform electric field. The pattern of insulating structure was designed to generate regions of high-electric field to trap cells with positive dielectrophoresis (e.g., dead mammalian cells at low frequency) in lower-flow-field regions. However, negative-dielectrophoretic cells (e.g., viable cells at low frequency) were repelled toward low-electric-field regions where the velocity was higher. Cells with different dielectrophoretic responses were therefore separated and collected in the outlet. Simulations were numerically performed to investigate parameters of the design in the present study. Furthermore, experiments were also conducted to demonstrate the feasibility of hydrodynamic separation using iDEP in the proposed design.     

Electrokinetically driven concentration of particles and cells by dielectrophoresis with DC-offset AC electric field

Insulator-based dielectrophoresis (iDEP) has been successfully used for on-chip manipulations of biological samples. Despite its effectiveness, iDEP typically requires high DC voltages to achieve sufficient electric field; this is mainly due to the coupled phenomena among linear electrokinetics: electroosmosis (EO) and electrophoresis (EP) and nonlinear electrokinetics: dielectrophoresis (DEP). This paper presents a microfluidic technique using DC-offset AC electric field for electrokinetic concentration of particles and cells by repulsive iDEP. This technique introduces AC electric field for producing iDEP which is decoupled from electroosmosis (EO) and electrophoresis (EP). The repulsive iDEP is generated in a PDMS tapered contraction channel that induces non-uniform electric field. The benefits of introducing AC electric field component are threefold: (i) it contributes to DEP force acting on particles, (ii) it suppresses EO flow and (iii) it does not cause any EP motion. As a result, the required DC field component that is mainly used to transport particles on the basis of EO and EP can be significantly reduced. Experimental results supported by numerical simulations showed that the total DC-offset AC electric field strength required to concentrate 15-μm particles is significantly reduced up to 85.9% as compared to using sole DC electric field. Parametric experimental studies showed that the higher buffer concentration, larger particle size and higher ratio of AC-to-DC electric field are favorable for particle concentration. In addition, the proposed technique was demonstrated for concentration of yeast cells.     

Particle focusing in a contactless dielectrophoretic microfluidic chip with insulating structures

The focusing of biological and synthetic particles in microfluidic devices is a crucial step for the construction of many microstructured materials as well as for medical applications. The present study examines the feasibility of using contactless dielectrophoresis (cDEP) in an insulator-based dielectrophoretic (iDEP) microdevice to effectively focus particles. Particles 10?μm in diameter were introduced into the microchannel and pre-confined hydrodynamically by funnel-shaped insulating structures near the inlet. The particles were repelled toward the center of the microchannel by the negative DEP forces generated by the insulating structures. The microchip was fabricated based on the concept of cDEP. The electric field in the main microchannel was generated using electrodes inserted into two conductive micro-reservoirs, which were separated from the main microchannel by 20-μm-thick insulating barriers made of polydimethylsiloxane (PDMS). The impedance spectrum of the thin insulating PDMS barrier was measured to investigate its capacitive behavior. Experiments employing polystyrene particles were conducted to demonstrate the feasibility of the proposed microdevice. Results show that the particle focusing performance increased with increasing frequency of the applied AC voltage due to the reduced impedance of PDMS barriers at high frequencies. When the frequency was above 800?kHz, most particles were focused into a single file. The smallest width of focused particles distributed at the outlet was about 13.1?μm at a frequency of 1?MHz. Experimental results also show that the particle focusing performance improved with increasing applied electric field strength and decreasing inlet flow rate. The usage of the cDEP technique makes the proposed microchip mechanically robust and chemically inert.     

介电泳芯片的研制及不同细胞介电分离的应用

制备了包括指状交叉、城墙状和梯形的微电极阵列芯片装置.并用这些芯片探索了生物细胞的介电响应.另外观察了酵母和鸡血红细胞的迁移、旋转和融合以及几种细胞收集图片.发现了两种细胞的正、负介电泳现象,确定了这两种细胞的分离条件.讨论了两种细胞正、负介电泳的原因.利用同一芯片在相同的条件下一种细胞移向强场区(正介电泳),另一种细胞移向弱场区(负介电泳).因此可用同一芯片分离不同的细胞.有望建立一种非接触式细胞分离技术,而且在分离过程中不需要添加任何试剂.     

A microfluidic device for rapid concentration of particles in continuous flow by DC dielectrophoresis

This article presents a microfluidic device (so called concentrator) for rapid and efficient concentration of micro/nanoparticles using direct current dielectrophoresis (DC DEP) in continuous fluid flow. The concentrator is composed of a series of microchannels constructed with PDMS-insulating microstructures to focus efficiently the electric field in the flow direction to provide high field strength and gradient. Multiple trapping regions are formed within the concentrator. The location of particle trapping depends on the strength of the electric field applied. Under the experimental conditions, both streaming movement and DEP trapping of particles simultaneously take place within the concentrator at different regions. The former occurs upstream and is responsible for continuous transport of the particles, whereas the latter occurs downstream and rapidly traps the particles delivered from upstream. The observation agrees with the distribution of the simulated electric field and DEP force. The performance of the device is demonstrated by successfully and effectively concentrating fluorescent nanoparticles. At the sufficiently high electric field, the device demonstrates a trapping efficiency of 100%, which means downstream DEP traps and concentrates all (100%) the incoming particles from upstream. The trapping efficiency of the device is further studied by measuring the fluorescence intensity of concentrated particles in the channel. Typically, the fluorescence intensity becomes saturated in Trap 1 by applying the voltage (400 V) for >2 min, demonstrating that rapid concentration of the nanoparticles (10⁷ particles/ml) is achieved in the device. The microfluidic concentrator described can be implemented in applications where rapid concentration of targets is needed such as concentrating cells for sample preparation and concentrating molecular biomarkers for detection.     

Assessment of microalgae viability employing insulator-based dielectrophoresis

Microscale bioparticle analysis has advanced significantly providing advantages over bench-scale studies such as the use of a reduced amount of sample and reagents, higher sensitivity, faster response, and portability. Insulator-based dielectrophoresis (iDEP) is a microscale technique where particles are driven by polarization effects under a non-uniform electrical field created by the inclusion of insulators between two electrodes. iDEP possesses attractive advantages over traditional electrode-based dielectrophoresis since there is no electrode degradation and manufacture of the device is simpler and economical. This novel and powerful technique has been applied successfully in the manipulation of macromolecules and cells. In this study, differences in dielectric properties (cell membrane conductivity) of viable and non-viable microalgae, Selenastrum capricornutum, were employed to concentrate and separate a mixture of live and dead cells. A microchannel, manufactured in glass and containing an array of cylindrical insulating posts, was employed to dielectrophoretically immobilize and concentrate the mixture of cells employing direct current electric fields. Experiments showed that live cells exhibited a stronger dielectrophoretic response than dead cells, which allowed cell differentiation. Separation and enrichment of viable and non-viable microalgae was achieved in 35 s with a concentration yield of 10.36 and 15.87 times the initial cell concentration, respectively. These results demonstrate the use of iDEP as a technique for rapid assessment of microalgae viability; unveiling the potential of this powerful technique for environmental applications on lab-on-a-chip platforms.     

Fabrication of bio/nano interfaces between biological cells and carbon nanotubes using dielectrophoresis

The authors have previously demonstrated the manipulation of bacteria and carbon nanotubes (CNTs) using dielectrophoresis (DEP) and its application for various types of biological and chemical sensors. This paper demonstrates simultaneous DEP handling of bacteria and CNTs, which are mixed and suspended in water. The CNTs were solubilized in water using microplasma-based treatment. When a microelectrode was energized with an ac voltage in the suspension of Escherichia coli (E. coli) cells and multi-walled CNTs (MWCNTs), both of them were simultaneously trapped in the microelectrode gap. Scanning electron microscopy (SEM) images revealed that E. coli cells were trapped on the surface or the tip of MWCNTs, where the electric field strength was intensified due to high aspect ratio of MWCNTs. As a result, bio/nano interfaces between bacteria and MWCNTs were automatically formed in a self-assembly manner. A potential application of the DEP-fabricated bio/nano interfaces is a drug delivery system (DDS), which is realized by transporting drug molecules from CNTs to cells across the cell membrane, which can be electroporated by the local high electric field formed on the CNT surface.     

A DNA trapping and extraction microchip using periodically crossed electrophoresis in a micropillar array

This paper presents an integrated deoxyribose nucleic acid (DNA) trapping and extraction microchip based on the electrophoresis using periodically crossed electric fields in the micropillar array. The extraction microchip, integrated with a micropillar array, microchannels, nano-gap entropic barriers, loading and unloading windows, has been fabricated by a 3-mask microfabrication process. Using the electric field crossed at 120°, the microchip is designed to trap the DNA molecules, whose reorientation time is longer than the period of the crossed field, within the micropillars distributed at 60° direction. In the fabricated extraction microchip, three different DNA molecules, including λ DNA (48.5 kbp), micrococcus DNA (115 kbp) and T4 DNA (168.9 kbp) show the reorientation times of 4.80 ± 0.44 s, 7.12 ± 0.75 s and 9.71 ± 0.30 s, respectively, at the crossed electric field of 6.25 V/cm. Among three DNA molecules, T4 DNA could not come out of the micropillar array for the electric field of 6.25 V/cm crossed at the period of 10 s. We have demonstrated that the present DNA extraction microchip separates DNA molecules larger than a critical value, which can be adjusted by the period of the electric field across the micropillar array.     

Dielectrophoretic separation of carbon nanotubes and polystyrene microparticles

The separation of multi-walled carbon nanotubes (MWCNTs) and polystyrene microparticles using a dielectrophoresis (DEP) system is presented. The DEP system consists of arrays of parallel microelectrodes patterned on a glass substrate. The performance of the system is evaluated by means of numerical simulations. The MWCNTs demonstrate a positive DEP behaviour and can be trapped at the regions of high electric field. However, the polystyrene microparticles demonstrate a negative DEP behaviour at a certain range of frequencies and migrate to the regions of low electric field. Experiments are performed on the microparticles at the frequencies between 100 Hz and 1 MHz to estimate their crossover frequency and select the range of separation frequencies. Further, experiments are conducted at the obtained range of separation frequencies to separate the MWCNTs and polystyrene microparticles.     

Analytical formulation of electric field and dielectrophoretic force for moving dielectrophoresis using Fourier series

Electrokinetics manipulation and separation of living cells employing microfluidic devices require good knowledge of the strength and distribution of electric field in such devices. AC dielectrophoresis is performed by generating non-uniform electric field using microsize electrodes. Among the several applications of dielectrophoretic phenomenon, this present study considers the recently introduced phenomenon of moving dielectrophoresis. An analytical solution using Fourier series is presented for the electric field distribution and dielectrophoretic force generated inside a microchannel. The potential at the upper part of the microchannel has been found by solving the governing equation of the electric potential with specific boundary conditions. The solutions for the electric field and dielectrophoretic force show excellent agreement with the numerical results. Microdevices were fabricated and experiments were carried out with living cells confirming and validating the analytical solutions.     

Improved concentration and separation of particles in a 3D dielectrophoretic chip integrating focusing, aligning and trapping

This article presents a dielectrophoresis (DEP)-based microfluidic device with the three-dimensional (3D) microelectrode configuration for concentrating and separating particles in a continuous throughflow. The 3D electrode structure, where microelectrode array are patterned on both the top and bottom surfaces of the microchannel, is composed of three units: focusing, aligning and trapping. As particles flowing through the microfluidic channel, they are firstly focused and aligned by the funnel-shaped and parallel electrode array, respectively, before being captured at the trapping unit due to negative DEP force. For a mixture of two particle populations of different sizes or dielectric properties, with a careful selection of suspending medium and applied field, the population exhibits stronger negative DEP manipulated by the microelectrode array and, therefore, separated from the other population which is easily carried away toward the outlet due to hydrodynamic force. The functionality of the proposed microdevice was verified by concentrating different-sized polystyrene (PS) microparticles and yeast cells dynamically flowing in the microchannel. Moreover, separation based on size and dielectric properties was achieved by sorting PS microparticles, and isolating 5 μm PS particles from yeast cells, respectively. The performance of the proposed micro-concentrator and separator was also studied, including the threshold voltage at which particles begin to be trapped, variation of cell-trapping efficiency with respect to the applied voltage and flow rate, and the efficiency of separation experiments. The proposed microdevice has various advantages, including multi-functionality, improved manipulation efficiency and throughput, easy fabrication and operation, etc., which shows a great potential for biological, chemical and medical applications.     

基于介电泳的电极阵列电场仿真研究

介电泳方法被广泛地应用于微纳颗粒的分离和操纵中,实现介电泳操作的关键是设计满足所需电场分布的电极阵列.针对目前在微电极阵列设计中尚缺乏简单有效的电场解析方法的现状,提出一种基于格林公式的电极阵列电场的解析方法.首先介绍了传统介电泳和行波介电泳的概念和计算模型,分析了介电泳过程与电极上所施加的交变电压的频率和幅度的关系,然后在确立电极电势的边界条件的基础上,采用基于格林公式的电场解析方法,建立了非均匀电场的解析模型,得出不同条件下的电极阵列电场分布的仿真结果,最后利用FEMLAB有限元仿真软件对解析模型进行了对比仿真, 验证了该解析模型的可行性.基于格林公式的电场解析求解方法能够有效地提高电极阵列设计中的针对性以及缩短电极设计的时间.     

基于微环形阵列电极的细胞分离富集芯片的设计与实验研究

根据介电泳操作原理,设计了微环形阵列电极结构,建立了细胞分离富集芯片模型,采用COMSOL软件分析微环形阵列电极的电场分布和介电泳力方向并确定了最大和最小电场强度的位置,利用ITO玻璃和PDMS制备了细胞分离富集芯片.通过酵母菌细胞的介电泳富集实验和酵母菌细胞与聚苯乙烯小球的分离富集实验,明确了酵母菌细胞的临界频率,实现了酵母菌细胞和聚苯乙烯小球的分离富集.结果显示,在溶液电导率为60μs/cm,交流信号电压为8Vp-p时,酵母菌细胞在1kHz~45kHz频率范围内做负介电泳运动并富集在环形内部,45kHz为酵母菌细胞的临界频率,在45kHz~10MHz频率范围内做正介电泳运动并富集在环形边缘;1.5MHz时聚苯乙烯小球做负介电泳运动并富集在环形内部,富集倍数达到11.66.     

A cell electrofusion microfluidic chip with micro-cavity microelectrode array

A new cell electrofusion microfluidic chip with 19,000 pairs of micro-cavity structures patterned on vertical sidewalls of a serpentine-shaped microchannel has been designed and fabricated. In each micro-cavity structure, the two sidewalls perpendicular to the microchannel are made of SiO₂ insulator, and that parallel to the microchannel is made of silicon as the microelectrode. One purpose of the design with micro-cavity microelectrode array is to obtain high membrane voltage occurring at the contact point of two paired cells, where cell fusion takes place. The device was tested to electrofuse NIH3T3 and myoblast cells under a relatively low voltage (~9 V). Under an AC electric field applied between the pair of microelectrodes positioned in the opposite micro-cavities, about 85–90 % micro-cavities captured cells, and about 60 % micro-cavities are effectively capable of trapping the desired two-cell pairs. DC electric pulses of low voltage (~9 V) were subsequently applied between the micro-cavity microelectrode arrays to induce electrofusion. Due to the concentration of the local electric field near the micro-cavity structure, fusion efficiency reaches about 50 % of total cells loaded into the device. Multi-cell electrofusion and membrane rupture at the end of cell chains are eliminated through the present novel design.     

A microfabricated module for isolating cervical carcinoma cells from peripheral blood utilizing dielectrophoresis in stepping electric fields

The ability to isolate rare cells, such as circulating tumor cells (CTC), circulating fetal cells, and stem cells, is important for medical diagnostics and characterization. The present study develops a microfabricated module that can effectively isolate cervical carcinoma cells (HeLa) from a peripheral blood sample. Circular microelectrodes that generate a stepping electric field by switching the electric field between adjacent electrode pairs by relays are designed herein. Positive dielectrophoretic cells are guided by the movement of the high-electric-field region. The magnitude of the dielectrophoresis (DEP) force acting on HeLa cells is about sevenfold that on red blood cells (RBCs) under a given electric field distribution in a sucrose medium, making it possible to separate HeLa cells from normal blood cells. Both HeLa cells and RBCs are pushed to the outermost electrodes when an outward stepping electric field (16?V peak-to-peak; 1?MHz) is applied. When an inward stepping electric field (10?V peak-to-peak; 1?MHz) is applied, the movement of HeLa cells toward the center electrodes is faster than that of RBCs. As a result, HeLa cells are concentrated onto the central microelectrode and isolated from the blood sample. Experimental results demonstrate the feasibility of isolating HeLa cells from blood samples.     

Experiments on opto-electrically generated microfluidic vortices

Strong microfluidic vortices are generated when a near-infrared (1,064 nm) laser beam is focused within a microchannel and an alternating current (AC) electric field is simultaneously applied. The electric field is generated from a parallel-plate, indium tin oxide (ITO) electrodes separated by 50 μm. We present the first μ-PIV analysis of the flow structure of such vortices. The vortices exhibit a sink-type behavior in the plane normal to the electric field and the flow speeds are characterized as a function of the electric field strength and biasing AC signal frequency. At a constant AC frequency of 100 kHz, the fluid velocity increases as the square of the electric field strength. At constant electric field strength fluid velocity does not change appreciably in the 30–50 kHz range and it decreases at larger frequencies (>1 MHz) until at approximately 5 MHz when Brownian motion dominates the movement of the 300 nm μ-PIV tracer particles. Presence of strongly focused laser beams in an interdigitated-electrode configuration can also lead to strong microfluidic vortices. When the center of the illumination is focused in the middle of an electrode strip, particles experiencing negative dielectrophoresis are carried towards the illumination and aggregate in this area.     

Novel continuous particle sorting in microfluidic chip utilizing cascaded squeeze effect

This article presents a novel technique for the continuous sorting and collection of microparticles in a microfluidic chip using a cascaded squeeze effect. In the proposed approach, microparticles of different sizes are separated from the sample stream using sheath flows and are then directed to specific side channels for collection. The sheath flows required to separate the particles are generated using a single high voltage supply integrated with a series of variable resistors designed to create electric fields of different intensities at different points of the microchip. Numerical simulations are performed to analyze the electrical potential contours and flow streamlines within the microchannel. Experimental trials show that the microchip is capable of continuously separating microparticles with diameters of 5, 10 and 20 μm, respectively. To further evaluate the performance of the microchip, a sample composed of yeast cells and polystyrene beads is sorted and collected. The results indicate that the microchip achieves a recovery ratio of 87.7% and a yield ratio of 94.1% for the yeast cells and therefore attains a comparable performance to that of a large-scale commercial flow cytometer. Importantly, the high performance of the microchip is achieved without the need for a complex control system or for sophisticated actuation mechanisms such as embedded microelectrodes, ultrasonic generators, or micropumps, and so forth.     

A Programmable Biochip for the Applications of Trapping and Adaptive Multisorting Using Dielectrophoresis Array

The adaptive biochip integrating dielectrophoresis (DEP) traps and a programmable multisorting DEP array for the multisorting applications of biomolecules such as proteins and DNA is proposed and demonstrated in this paper. In this research, movable beads are used as the mobile probes to capture the target protein molecules. These beads are chemically modified and immobilized with p50 proteins in our demonstration. An array of micropyramid DEP traps with a good levitation control on the height of the beads is located at the upstream to enhance the hybridization function of the mobile probes. The sample solution mixed with Cy3-I-kappa-B-alpha complex is used in the demonstration. A programmable multisorting DEP array that is located at the downstream sorts out the hybridized beads, which are fluorescently labeled based on the fluorescent detection signals. The magnitude and direction of the DEP force that is applied to the beads with/without labeling fluorescence in the multisorting DEP array are controlled via the distribution of time-variant nonuniform electric fields. The voltage on the individual electrode of the multisorting DEP array is preprogrammed and controlled by a LabVIEW controller with fluorescence detection feedback signals. In contrast to the research of Manaresi et al. [IEEE J. Solid-State Circuits, vol. 38, no. 12, p. 2297, 2003], which was proposed for trapping and sorting beads and cells via Dent traps, to our knowledge, the design of this biochip with the hybridization enhancement via micropyramid DEP traps and the adaptive multisorting DEP array for the mobile probes has never been proposed and implemented to date.     

Microfluidics cell electroporation

Electroporation or electropermeabilization is one of the most powerful biological techniques in cell studies. Applying the high voltage electric field in vicinity of the cells can generate nanopores in cell membrane. Varying with the intensity and the duration of these applied electric field, the created nanopores can be temporary (reversible electroporation) or permanent (irreversible electroporation). Reversible electroporation is usually conducted to insert biological samples into the cells. Cells are also electroporated irreversibly to release their intercellular contents for further biological investigations. In comparison with the conventional electroporation devices, microfluidic (microscale) electroporation devices have some advantages such as higher cell viability rate, high transfection efficiency, lower sample contamination, and smaller Joule heating effect. In this article, the latest advancement in microfluidic cell electroporation is reviewed. First, the underlying theory of membrane permeabilization is reviewed and the leading analytical studies on the cell electroporation are presented. Following that, different experimental methods are compared. Finally, some suggestions are proposed for the future studies.     

Design and fabrication of poly(dimethylsiloxane) electrophoresis microchip with integrated electrodes

A novel poly(dimethylsiloxane) (PDMS) microchip integrated with platinum electrodes has been designed and fabricated using the Micro-electro-mechanical-systems (MEMS) technology. Since high voltage electrodes are integrated on the glass wafer using lift-off process, the microchip is a friendly-to-use system that does not need any extra mechanical apparatus for electrode insertion. To improve the sealing of microchip and ensure the uniformity of microchannel material, one PDMS membrane is formed on glass wafer with electrodes by pressing method. In this study, integrated microchip has been demonstrated as a capillary electrophoresis device for amino acids and satisfactory separation was achieved under separation electrical field strengths of 200 V/cm. The overall performance suggests that this novel microchip is advantageous and practical for the fabrication of lab-on-a-chip.