基于薄膜电极的细胞电融合芯片

构建了一种薄膜电极阵列结构的细胞电融合芯片, 通过多聚物微通道底/顶层凸齿状的微电极, 以及多聚物微通道侧壁上溅射形成的一层离散式金属薄膜电极, 共同形成离散式"三明治"微电极结构. 该微电极结构可在微通道内部形成与传统凸齿状电极相似的非均匀分布的梯度电场, 通过介电电泳效应进行细胞控制及排队. 利用多聚物在芯片上填充了传统凸齿状电极的凹陷区, 克服了细胞在凹陷区无法有效排队与融合的缺点. 在芯片上利用K562细胞开展了基于介电电泳效应的细胞排队实验及基于可逆性电穿孔效应的电融合实验, 结果表明该芯片能够较好地实现细胞排队及融合, 融合所需控制电压低至10 V左右. 细胞排队率达99%以上, 几乎无细胞在绝缘物填充区(传统凸齿电极芯片的凹陷区)滞留, 细胞两两排队高于60%, 细胞融合效率约为40%, 比传统的细胞电融合方法和凸齿电极芯片有较大提高.     

A high-throughput dielectrophoresis-based cell electrofusion microfluidic device

A high-throughput cell electrofusion microfluidic chip has been designed, fabricated on a silicon-on-insulator wafer and tested for in vitro cell fusion under a low applied voltage. The developed chip consists of six individual straight microchannels with a 40-μm thickness conductive highly doped Si layer as the microchannel wall. In each microchannel, there are 75 pairs of counter protruding microelectrodes, between which the cell electrofusion is performed. The entire highly doped Si layer is covered by a 2-μm thickness aluminum film to maintain a consistent electric field between different protruding microelectrode pairs. A 150-nm thickness SiO? film is subsequently deposited on the top face of each protruding microelectrode for better biocompatibility. Owing to the short distance between two counter protruding microelectrodes, a high electric field can be generated for cell electrofusion with a low voltage imposed across the electrodes. Both mammalian cells and plant protoplasts were used to test the cell electrofusion. About 42-68% cells were aligned to form cell-cell pairs by the dielectrophoretic force. After cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroporation and electrofusion signals. The averaged fusion efficiency in the paired cells is above 40% (the highest was about 60%), which is much higher than the traditional polyethylene glycol method (<5%) and traditional electrofusion methods (～12%). An individual cell electrofusion process could be completed within 10 min, indicating a capability of high throughput.     

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.     

Study on the discrete dielectrophoresis for particle–cell separation

This paper presents the application of the discrete dielectrophoretic force to separate polystyrene particles from red blood cells. The separation process employs a simple microfluidic device that is composed of interdigitated electrodes and a microchannel. The discrete dielectrophoretic force is generated by adjusting the duty cycle of the applied voltage. The electrodes make a tilt angle with the microchannel to change the moving direction of the red blood cells. By adjusting the voltage magnitude and duty cycle, we investigate the deflection of red blood cells and the variation of cell velocity along electrode edge under positive dielectrophoresis. The experiments with polystyrene particles show that the enrichment of the particles is greater than 150 times. The maximum separation efficiency is 97% for particle-to-cell number ratio equal to 1:2000 in the sample having high cell concentration. Using the appropriate applied voltage magnitude and duty cycle, the discrete dielectrophoretic force can prevent the clogging of microchannel while successfully separating the particles from the cells with high enrichment and efficiency. The proposed principle can be readily applied to dielectrophoresis-based devices for biomedical sample preparation or diagnosis such as the separation of rare or infected cells from a blood sample.     

高效细胞电融合芯片中的电场分析

细胞电融合芯片内的电场分布对细胞的控制及细胞融合效率有非常重要的意义,它是该类芯片设计的主要因素。电场分布主要由芯片内微通道和微电极的结构决定。在一个新研制的融合芯片中,采用大量微电极构成的阵列来提高融合效率。由于电极数量很多,微通道和微电极的结构和形状复杂,理论计算芯片内部电场分布具有较大难度。利用ANSYS有限元分析软件,对细胞电融合芯片中的电场分布进行模拟分析,得到其强度分布及变化梯度。通过不同设计的对比分析,提出了更加适合于细胞电融合的电极阵列结构模型——矩形梳状交叉微电极阵列,为高效细胞电融合芯片的实现奠定了基础。在矩形梳状交叉微电极阵列原型芯片的实验研究中,细胞融合(植物原生质体融合)效率约为40%,超过了传统的化学融合(小于1%)、电融合(小于10%),以及最初所采用的矩形对称梳状电极(小于20%)。表明在该融合芯片上可以实现高效的细胞电融合。     

A discrete dielectrophoresis device for the separation of malaria-infected cells

Malaria is a serious disease caused by Plasmodium parasites that infect red blood cells (RBCs). This paper presents the continuous separation of malaria-infected RBCs (iRBCs) from normal blood cells. The proposed method employed the discrete dielectrophoresis (DEP) in a microfluidic device with interdigitated electrodes. Our aim is to treat a sample having high concentration of cells to realize high throughput and to prevent the clogging of the microchannel with the use of the discrete DEP. The discrete DEP force for deflecting cells in the device was controlled by adjusting the magnitude, frequency, and duty cycle of the applied voltage. The effectiveness of the proposed method was demonstrated by separating the malaria-infected cells in samples having a cell concentration of 10⁶ cells/µl. From experimental results, we determined the enrichment that is needed to enhance the detection in the case of low parasitemia. The enrichment of the infected cells at the device output was 3000 times as high as that of the input containing 1 infected cell to 10⁶ normal cells. Therefore, the proposed method is highly effective and can significantly facilitate the detection of the infected cells for the identification of Malaria patients.     

Biocompatible multi-address 3D cell assembly in microfluidic devices using spatially programmable gel formation

Programmable 3D cell assembly under physiological pH conditions is achieved using electrodeposited stimuli-responsive alginate gels in a microfluidic device, with parallel sidewall electrodes enabling direct observation of the cell assembly. Electrically triggered assembly and subsequent viability of mammalian cells is demonstrated, along with spatially programmable, multi-address assembly of different strains of E. coli cells. Our approach enables in vitro study of dynamic cellular and inter-cellular processes, from cell growth and stimulus/response to inter-colony and inter-species signaling.     

基于SOI基底的高通量细胞电融合芯片

提出了一种以MEMS技术为基础, 可在低电压驱动条件下工作的创新型细胞电融合芯片. 该芯片的设计原理在于通过缩短微电极间的间距, 在低电压条件下获得足够强度的排队和融合电场强度. 原型芯片以SOI硅片为加工材料, 通过刻蚀方式在顶层低阻硅形成微电极和微通道; 在微电极上沉淀2 μm厚的铝膜以降低电阻率, 提高导电性; 通过PECVD方法形成150 nm厚SiO2保障铝膜的抗腐蚀性及芯片生物相容性; 芯片最终采用DIP法进行封装. 在该芯片上进行了低电压(传统电融合设备工作电压的1/20)驱动条件下的基于介电电泳的细胞排队实验及后期的细胞电融合实验, 结果表明, 细胞多以两两结合的方式排列, 与传统的细胞融合电仪器相比较, 降低了多细胞排队概率, 进而减少了传统电融合设备多细胞融合的概率, 为细胞高效率融合奠定了基础. 在加载的低电压短脉冲信号后, 微通道中形成了高压短脉冲电场, 在脉冲作用下, 烟草原生质体细胞在微通道中发生了融合, 融合时间(2 min)远低于传统电融合方法(10~30 min), 融合率远远高于传统的PEG方法(融合率小于1%)和传统电融合方法(利用BTX ECM 2001细胞电融合系统得到, 融合率小于5%).     

Dielectrophoresis-assisted massively parallel cell pairing and fusion based on field constriction created by a micro-orifice array sheet

In this paper, we present a novel electrofusion device that enables massive parallelism, using an electrically insulating sheet having a two-dimensional micro-orifice array. The sheet is sandwiched by a pair of micro-chambers with immersed electrodes, and each chamber is filled with the suspensions of the two types of cells to be fused. Dielectrophoresis, assisted by sedimentation, is used to position the cells in the upper chamber down onto the orifices, then the device is flipped over to position the cells on the other side, so that cell pairs making contact in the orifice are formed. When a pulse voltage is applied to the electrodes, most voltage drop occurs around the orifice and impressed on the cell membrane in the orifice. This makes possible the application of size-independent voltage to fuse two cells in contact at all orifices exclusively in 1:1 manner. In the experiment, cytoplasm of one of the cells is stained with a fluorescence dye, and the transfer of the fluorescence to the other cell is used as the indication of fusion events. The two-dimensional orifice arrangement at the pitch of 50 μm realizes simultaneous fusion of 6 × 103 cells on a 4?mm diameter chip, and the fusion yield of 78-90% is achieved for various sizes and types of cells.     

Continuous-Flow Nanoparticle Trapping Driven by Hybrid Electrokinetics in Microfluidics

We introduce herein an efficient microfluidic approach for continuous transport and localized collection of nanoparticles via hybrid electrokinetics, which delicately combines linear and nonlinear electrokinetics driven by a composite DC-biased AC voltage signal. The proposed technique utilizes a simple geometrical structure, in which one or a series of metal strips serving as floating electrode (FE) are attached to the substrate surface and arranged in parallel between a pair of coplanar driving electrodes (DE) in a straight microchannel. On application of a DC-biased AC electric field across the channel, nanoparticles can be transported continuously by DC bulk electroosmotic flow, and then trapped selectively onto the metal strips due to AC-field induced-charge electrokinetic (ICEK) phenomenon, which behaves as counter-rotating micro-vortices around the ideally polarizable surfaces of FE. Finite-element simulation is carried out by coupling the dual-frequency electric field, flow field and sample mass transfer in sequence, for guiding a practical design of the microfluidic nanoparticle concentrator. With the optimal device geometry, the actual performance of the technique is investigated with respect to DC bias, AC voltage amplitude, and field frequency by using both latex nanospheres (∼500 nm) and BSA molecules (∼10 nm). Our experimental observation indicates nanoparticles are always enriched into a narrow bright band on the surface of each FE, and a horizontal concentration gradient even emerges in the presence of multiple metal strips, which therefore permits localized analyte enrichment. The proposed trapping method is supposed to guide an elaborate design of flexible electrokinetic frameworks embedding FE for continuous-flow analyte manipulation in modern microfluidic systems.     

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.

Low-cost,high-throughput and rapid-prototyped 3D-integrated dielectrophoretic channels for continuous cell enrichment and separation

Microfluidic devices for dielectrophoretic cell separation are typically designed and constructed using microfabrication methods in a clean room, requiring time and expense. In this paper, we describe a novel alternative approach to microfluidic device manufacture, using chips cut from conductor–insulator laminates using a cutter plotter. This allows the manufacture of microchannel devices with micron-scale electrodes along every wall. Fabrication uses a conventional desktop cutter plotter, and requires no chemicals, masks or clean-room access; functional fluidic devices can be designed and constructed within a couple of hours at negligible cost. As an example, we demonstrate the construction of a continuous dielectrophoretic cell separator capable of enriching yeast cells to 80% purity at 10 000 cells/s.     

Analytical solutions and validation of electric field and dielectrophoretic force in a bio-microfluidic channel

In a microbiological device, cell or particle manipulation and characterization require the use of electric field on different electrodes in several configurations and shapes. To efficiently design microelectrodes within a microfluidic channel for dielectrophoresis focusing, manipulation and characterization of cells, the designer will seek the exact distribution of the electric potential, electric field and hence dielectrophoresis force exerted on the cell within the microdevice. In this paper we describe the approach attaining the analytical solution of the dielectrophoretic force expression within a microchannel with parallel facing same size electrodes present on the two faces of channel substrates, with opposite voltages on the pair electrodes. Simple Fourier series mathematical expressions are derived for electric potential, electric field and dielectric force between two distant finite‐size electrodes. Excellent agreement is found by comparing the analytical results calculated using MATLAB? with numerical ones obtained by Comsol. This analytical result can help the designer to perform simple design parametric analysis. Bio‐microdevices are also designed and fabricated to illustrate the theoretical solution results with the experimental data. Experiments with red blood cells show the dielectrophoretic force contour plots of the analytical data matched to the experimental results.     

Hydrodynamic and electrical considerations in the design of a four-electrode impedance-based microfluidic device

A four-electrode impedance-based microfluidic device has been designed with tunable sensitivity for future applications to the detection of pathogens and functionalized microparticles specifically bound to molecular recognition molecules on the surface of a microfluidic channel. In order to achieve tunable sensitivity, hydrodynamic focusing was employed to confine the electric current by simultaneous introduction of two fluids (high- and low-conductivity solutions) into a microchannel at variable flow-rate ratios. By increasing the volumetric flow rate of the low-conductivity solution (sheath fluid) relative to the high-conductivity solution (sample fluid), increased focusing of the high-conductivity solution over four coplanar electrodes was achieved, thereby confining the current during impedance interrogation. The hydrodynamic and electrical properties of the device were analyzed for optimization and to resolve issues that would impact sensitivity and reproducibility in subsequent biosensor applications. These include variability in the relative flow rates of the sheath and sample fluids, changes in microchannel dimensions, and ionic concentration of the sample fluid. A comparative analysis of impedance measurements using four-electrode versus two-electrode configurations for impedance measurements also highlighted the advantages of using four electrodes for portable sensor applications.

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.     

Novel microfluidic device for the continuous separation of cancer cells using dielectrophoresis

We describe the design, microfabrication, and testing of a microfluidic device for the separation of cancer cells based on dielectrophoresis. Cancer cells, specifically green fluorescent protein‐labeled MDA‐MB‐231, are successfully separated from a heterogeneous mixture of the same and normal blood cells. MDA‐MB‐231 cancer cells are separated with an accuracy that enables precise detection and counting of circulating tumor cells present among normal blood cells. The separation is performed using a set of planar interdigitated transducer electrodes that are deposited on the surface of a glass wafer and slightly protrude into the separation microchannel at one side. The device includes two parts, namely, a glass wafer and polydimethylsiloxane element. The device is fabricated using standard microfabrication techniques. All experiments are conducted with low conductivity sucrose‐dextrose isotonic medium. The variation in response between MDA‐MB‐231 cancer cells and normal cells to a certain band of alternating‐current frequencies is used for continuous separation of cells. The fabrication of the microfluidic device, preparation of cells and medium, and flow conditions are detailed. The proposed microdevice can be used to detect and separate malignant cells from heterogeneous mixture of cells for the purpose of early screening for cancer.     

Continuous and precise particle separation by electroosmotic flow control in microfluidic devices

A new scheme has been described for continuous particle separation using EOF in microfluidic devices. We have previously reported a method for particle separation, called "pinched flow fractionation (PFF)", in which size-dependent and continuous particle separation can be achieved by introducing pressure-driven flows with and without particles into a pinched microchannel. In this study, EOF was employed to transport fluid flows inside a microchannel. By controlling the applied voltage to electrodes inserted in each inlet/outlet port, the flow rates from both inlets, and flow rates distributed to each outlet could be accurately tuned, thus enabling more effective separation compared to the pressure-driven scheme. In the experiment, the particle behaviors were compared between EOF and pressure-driven flow schemes. In addition, micrometer- and submicrometer-sized particles were accurately separated and individually collected using a microchannel with multiple outlet branch channels, demonstrating the high efficiency of the presented scheme.     

Fabrication and performance of a microfluidic traveling-wave electrophoresis system

A microfluidic traveling-wave electrophoresis (TWE) system is reported that uses a locally defined traveling electric field wave within a microfluidic channel to achieve band transport and separation. Low voltages, over a range of -0.5 to +0.5 V, are used to avoid electrolysis and other detrimental redox reactions while the short distance between electrodes, ～25 μm, provides high electric fields of ～200 V cm(-1). It is expected that the low voltage requirements will simplify the future development of smaller portable devices. The TWE device uses four interdigitated electrode arrays: one interdigitated electrode array pair is on the top of the microchannel and the other interdigitated electrode array pair is on the microchannel bottom. The top and bottom substrates are joined by a PDMS spacer that has a nominal height of 15 μm. A pinched injection scheme is used to define a narrow sample band within an injection cross either electrokinetically or hydrodynamically. Separation of two dyes, fluorescein and FLCA, with baseline resolution is achieved in less than 3 min and separation of two proteins, insulin and casein is demonstrated. Investigation of band broadening with fluorescein reveals that sample band widths equivalent to the diffusion limit can be achieved within the microfluidic channel, yielding highly efficient separations. This low level of band broadening can be achieved with capillary electrophoresis, but is not routinely observed in microchannel electrophoresis. Sample enrichment can be achieved very easily with TWE using a device with converging electric field waves controlled by two sets of independently controlled interdigitated electrodes arrays positioned serially along the microchannel. Sample enrichment of 40-fold is achieved without heterogeneous buffer/solvent systems, sorptive, or permselective materials. While there is much room for improvement in device fabrication, and many capabilities are yet to be demonstrated, it is anticipated that the capabilities and performance demonstrated herein will enable new lab-on-a-chip processes and systems.     

Continuous dielectrophoretic separation of particles in a spiral microchannel

Particle separation is a fundamental operation in the areas of biology and physical chemistry. A variety of force fields have been used to separate particles in microfluidic devices, among which electric field may be the most popular one due to its general applicability and adaptability. So far, however, electrophoresis‐based separations have been limited primarily to batchwise processes. Dielectrophoresis (DEP)‐based separations require in‐channel micro‐electrodes or micro‐insulators to produce electric field gradients. This article introduces a novel particle separation technique in DC electrokinetic flow through a planar double‐spiral microchannel. The continuous separation arises from the cross‐stream dielectrophoretic motion of particles induced by the non‐uniform electric field inherent to curved channels. Specifically, particles are focused by DEP to one sidewall of the first spiral, and then dielectrophoretically deflected toward the other sidewall of the second spiral at a particle‐dependent rate, leading to focused particle streams along different flow paths. This DEP‐based particle separation technique is demonstrated in an asymmetric double‐spiral microchannel by continuously separating a mixture of 5/10 μm particles and 3/5 μm particles.     

A flow-through microfluidic chip for continuous dielectrophoretic separation of viable and non-viable human T-cells

Effective methods for rapid sorting of cells according to their viability are critical in T cells based therapies to prevent any risk to patients. In this context, we present a novel microfluidic device that continuously separates viable and non-viable T-cells according to their dielectric properties. A dielectrophoresis (DEP) force is generated by an array of castellated microelectrodes embedded into a microfluidic channel with a single inlet and two outlets; cells subjected to positive DEP forces are drawn toward the electrodes array and leave from the top outlet, those subjected to negative DEP forces are repelled away from the electrodes and leave from the bottom outlet. Computational fluid dynamics is used to predict the device separation efficacy, according to the applied alternative current (AC) frequency, at which the cells move from/to a negative/positive DEP region and the ionic strength of the suspension medium. The model is used to support the design of the operational conditions, confirming a separation efficiency, in terms of purity, of 96% under an applied AC frequency of 1.5 × 10⁶ Hz and a flow rate of 20 μl/h. This work represents the first example of effective continuous sorting of viable and non-viable human T-cells in a single-inlet microfluidic chip, paving the way for lab-on-a-chip applications at the point of need.