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.     

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%).     

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%.     

阵列叉指式芯片研究细胞介电电泳富集过程

采用阵列叉指电极介电电泳(Dielectrophoresis,DEP)芯片,构建了集成DEP芯片分析和操控系统,应用Coventorware有限元分析软件模拟分析了芯片表面的电场分布情况;以红细胞和结肠癌细胞样品为分析对象,实现了两种细胞样品在芯片上的正负介电电泳定位富集.实验发现,交流信号幅值Vp-p是决定DEP富集效率的主因,交流信号频率f和缓冲溶液是改变细胞介电电泳类型的参量;在0.9% NaCl中,施加频率为10和3 MHz、电压5 V的交流频率,结肠癌细胞的正介电电泳(Positive-dielectrophoresis, pDEP)和负介电电泳(Nagetive-dielectrophoresis, nDEP)富集效率分别为87.2%和84.8%.     

生化样本的芯片介电电泳富集和分离研究进展

芯片介电电泳技术是以介电电泳(DEP)分离原理和微机电加工技术为依托发展起来的可用于生化样品分析的新型分析技术.本文概述了芯片介电电泳技术的发展和DEP芯片分析系统的构成,并以DEP操控模式为切入点,介绍了芯片介电电泳在生化样品分析中的应用情况.     

用荧光葡聚糖研究大麦细胞电融合

利用阴离子表面活性物质荧光葡聚糖 (F DX)研究了表面活性物质对大麦细胞电融合的影响 .结果表明 ,F DX可抑制电融合过程 .对放置过大麦细胞原生质体的F DX溶液 ,在荧光显微镜下可观察到其膜表面的荧光圈 ,证明F DX在膜上的吸附 .添加F DX可增加原生质体的电泳速度 ,说明吸附后原生质体表面负电荷增多 .由于相互间静电斥力的增强 ,使细胞的电融合率下降 .此外 ,还利用荧光显微技术研究了细胞电生孔现象 .观察到经电脉冲后溶液中的F DX可进入原生质体内部 ,间接证明了细胞电生孔的存在 .     

微流控芯片系统在单细胞研究中的应用

微流控芯片具有网络式通道结构,扩展了在细胞和亚细胞水平进行生命科学研究的能力,为单细胞研究提供了一个新的平台.在微流控芯片通道中,人们利用气压、液压和电压,或利用介电电泳、光学陷阱、行波介电电泳以及磁场等技术,可以操纵细胞通过或驻留在通道内的任意位置,从而使单细胞计数、筛选以及胞内组分分析等操作大大简化.本文对微流控芯片系统在血液流变学、单细胞操纵与计数以及单细胞胞内组分分析中的应用进行了综述,介绍了用于单细胞研究的多种微芯片系统,讨论了芯片上进行单细胞操纵的各种方法     

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。     

基于聚酰亚胺基底的细胞电融合芯片的研制

基于柔性印刷电路板(flexible printed circuits board, FPCB)技术,通过在聚酰亚胺基底薄膜表面层压的铜箔上刻蚀微电极阵列结构制备了一种细胞电融合芯片.在低电压(≤40 V)条件下实现了细胞电融合,融合效率达37%,远高于聚乙二醇(polyethylene glycol, PEG)法及传统细胞电融合方法.与传统细胞电融合系统相比,此芯片可在低电压条件下工作,具有结构简单、成本低廉、实验过程可观察、融合通量高等优点.另外,聚酰亚胺薄膜基底良好的柔软度可保证此芯片与其它分析模块(如细胞筛选分离模块)的有效集成,具备构造微全分析系统(micro total analytical system, μ-TAS)的巨大潜力.     

基于芯片介电电泳的细胞特性分析及种类判断

基于微流控芯片介电电泳( Dielectrophoresis,DEP)原理和技术,在自行设计制作的抛物线电极结构的微流控介电电泳芯片上,采用芯片介电泳临界频率测定法,选择缓冲液电导率为200～1000 μS/cm,激发电压为5V,分别对红细胞(RBC)、白细胞(WBC)和死活HepG2肝癌细胞的临界频率进行了测试,检测...     

A novel concept of dielectrophoretic engine oil filter

A novel concept of an alternating current (AC) dielectrophoretic filter with a three-dimensional electrode array is presented. A filter is constructed by winding into layers around the core tube two sheets of woven metal wire-mesh with several sheets of woven insulating wire-mesh sandwiched in between. Contrary to conventional dielectrophoretic devices, the proposed design of electrodes generates a high-gradient field over a large working volume by applying several hundred volts at a standard frequency of 60?Hz. The operating principle of filtration is based on our recently developed method of AC dielectrophoretic gating for microfluidics. The filtration efficiency is expressed in terms of two non-dimensional parameters, which describe the combined influence of the particle polarizability and size, the oil viscosity and flow rate, and the field gradient on the particle captivity. The proof-of-concept is tested by measuring the single-pass performance of two filters on positively polarized particles dispersed in engine oil: spherical glass beads, fused aluminum oxide powder, and silicon metal powder, all smaller than the mesh opening. The results obtained are used to consider the potential of using AC dielectrophoretic filtration and provide critical design guidelines for the development of a filter based on the retention capability of challenge particles.     

Flat hydrogel substrate for atomic force microscopy to observe liposomes and lipid membranes

In order to avoid denaturation of biomolecules due to strong adsorption on solid surfaces, a soft substrate has to be used for atomic force microscopy (AFM) observation. We propose a hydrophilic agarose gel surface as a soft substrate for AFM to observe liposomes and lipid membranes. Although our simple method does not require any delicate control at the molecular level, an agarose gel surface can be simply flattened to 0.3 nm in roughness using an atomically flat solid surface during gelation. The AFM images revealed that liposomes were unruptured on the gel surface at low liposome density, whereas an unruptured state was difficult to obtain on a solid surface like mica. This indicates that the weak interaction between the liposome and the soft surface inhibits the liposome from rupturing, and also that the surface rougher than the solid surface prevents lateral diffusion of the liposomes along the surface to be fused. Increasing the liposome density resulted in a lipid membrane at various thicknesses forming on the hydrogel surface by the fusion and rupture of liposomes. Using the soft substrate, it can be expected to promote investigations of structures and functions of biomolecules at the nanometer scale under physiological conditions with AFM.     

Microdevices for manipulation and accumulation of micro- and nanoparticles by dielectrophoresis

Microfluidic devices with three-dimensional (3-D) arrays of microelectrodes embedded in microchannels have been developed to study dielectrophoretic forces acting on synthetic micro- and nanoparticles. In particular, so-called deflector structures were used to separate particles according to their size and to enable accumulation of a fraction of interest into a small sample volume for further analysis. Particle velocity within the microchannels was measured by video microscopy and the hydrodynamic friction forces exerted on deflected particles were determined according to Stokes law. These results lead to an absolute measure of the dielectrophoretic forces and allowed for a quantitative test of the underlying theory. In summary, the influence of channel height, particle size, buffer composition, electric field, strength and frequency on the dielectrophoretic force and the effectiveness of dielectrophoretic deflection structures were determined. For this purpose, microfluidic devices have been developed comprising pairs of electrodes extending into fluid channels on both top and bottom side of the microfluidic channels. Electrodes were aligned under angles varying from 0 to 75 degrees with respect to the direction of flow. Devices with channel height varying between 5 and 50 microm were manufactured. Fabrication involved a dedicated bonding technology using a mask aligner and UV-curing adhesive. Particles with radius ranging from 250 nm to 12 microm were injected into the channels using aqueous buffer solutions.     

Dielectrophoretic micropatterning with microparticle monolayers covalently linked to glass surfaces

Two-dimensional micropatterns of microparticles were fabricated on glass substrates with negative dielectrophoretic force, and the patterned microparticles were covalently bound on the substrate via cross-linking agents. The line and grid patterns of microparticles were prepared using the repulsive force of negative dielectrophoresis (n-DEP). The template interdigitated microband array (IDA) electrodes (width and gap 50 mum) were incorporated into the dielectrophoretic patterning cell with a fluidic channel. The microstructures on the glass substrates with amino or sulfhydryl groups were immobilized with the cross-linking agents disuccinimidyl suberate (DSS) and m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). Diaphorase (Dp), a flavoenzyme, was selectively attached on the patterned microparticles using the maleimide groups of MBS. The enzyme activity on the patterned particles was electrochemically characterized with a scanning electrochemical microscope (SECM) in the presence of NADH and ferrocenylmethanol as a redox mediator. The SECM images proved that Dp was selectively immobilized onto the surface of microparticles to maintain its catalytic activity.     

Brownian Motion and Large Electric Polarizabilities Facilitate Dielectrophoretic Capture of Sub-200 nm Gold Nanoparticles in Water

Dielectrophoresis can move small particles using the force resulting from their polarization in a divergent electric field. In liquids, it has most often been applied to micrometric objects such as biological cells or latex microspheres. For smaller particles, the dielectrophoretic force becomes very small and the phenomenon is furthermore perturbed by Brownian motion. Whereas dielectrophoresis has been used for assembly of superstructures of nanoparticles and for the detection of proteins and nucleic acids, the mechanisms underlying DEP of such small objects require further study. This work presents measurements of the alternating-current (AC) dielectrophoretic response of gold nanoparticles of less than 200 nm diameter in water. An original dark-field digital video-microscopic method was developed and used in combination with a microfluidic device containing transparent thin-film electrodes. It was found that the dielectrophoretic force is only effective in a small zone very close to the tip of the electrodes, and that Brownian motion actually facilitates transport of particles towards this zone. Moreover, the fact that particles as small as 80 nm are still efficiently captured in our device is not only due to Brownian transport but also to an effective polarizability that is larger than what would be expected on basis of current theory for a sphere in a dielectric medium.     

Dynamically controlled dielectrophoresis using resonant tuning

Electrically polarizable micro- and nanoparticles and droplets can be trapped using the gradient electric field of electrodes. But the spatial profile of the resultant dielectrophoretic force is fixed once the electrode structure is defined. To change the force profile, entire complex lab-on-a-chip systems must be re-fabricated with modified electrode structures. To overcome this problem, we propose an approach for the dynamic control of the spatial profile of the dielectrophoretic force by interfacing the trap electrodes with a resistor and an inductor to form a resonant resistor–inductor–capacitor (RLC) circuit. Using a dielectrophoretically trapped water droplet suspended in silicone oil, we show that the resonator amplitude, detuning, and linewidth can be continuously varied by changing the supply voltage, supply frequency, and the circuit resistance to obtain the desired trap depth, range, and stiffness. We show that by proper tuning of the resonator, the trap range can be extended without increasing the supply voltage, thus preventing sensitive samples from exposure to high electric fields at the stable trapping position. Such unprecedented dynamic control of dielectrophoretic forces opens avenues for the tunable active manipulation of sensitive biological and biochemical specimen in droplet microfluidic devices used for single-cell and biochemical reaction analysis.     

Discontinuous meniscus location in tapered capillaries driven by pressure difference and dielectrophoretic forces

We calculate the meniscus location in tapered capillaries under the influence of pressure difference and dielectrophoretic forces with and without gravity. We find that the meniscus location can be a discontinuous function of the pressure difference or the applied voltage and that the meniscus can "jump" to one end or another of the capillary. Phase diagrams are given as a function of the pressure and voltage, depending on the geometrical parameters of the system. We further consider a revision of the dielectric rise under dielectrophoretic force in wedge capillaries and in the case of electrowetting, where the dielectrophoretic force is a small perturbation. Finally, we also find discontinuous liquid-gas interface location in the case of liquid penetration into closed volumes.     

Young's moduli of surface-bound liposomes by atomic force microscopy force measurements

Mechanical properties of layers of intact liposomes attached by specific interactions on solid surfaces were studied by atomic force microscopy (AFM) force measurements. Force-distance measurements using colloidal probe tips were obtained over liposome layers and used to calculate Young's moduli by using the Hertz contact theory. A classical Hertz model and a modified Hertz one have been used to extract Young's moduli from AFM force curves. The modified model, proposed by Dimitriadis, is correcting for the finite sample thickness since Hertz's classical model is assuming that the sample is infinitely thick. Values for Young's moduli of 40 and 8 kPa have been obtained using the Hertz model for one and three layers of intact liposomes, respectively. Young's moduli of approximately 3 kPa have been obtained using the corrected Hertz model for both one and three layers of surface-bound liposomes. Compression work performed by the colloidal probe to compress these liposome layers has also been calculated.     

Negative dielectrophoresis-based particle separation by size in a serpentine microchannel

Dielectrophoresis has been widely used to focus, trap, concentrate, and sort particles in microfluidic devices. This work demonstrates a continuous separation of particles by size in a serpentine microchannel using negative dielectrophoresis. Depending on the magnitude of the turn-induced dielectrophoretic force, particles travelling electrokinetically through a serpentine channel either migrate toward the centerline or bounce between the two sidewalls. These distinctive focusing and bouncing phenomena are utilized to implement a dielectrophoretic separation of 1 and 3 μm polystyrene particles under a DC-biased AC electric field of 880 V/cm on average. The particle separation process in the entire microchannel is simulated by a numerical model.