高通量细胞电融合芯片研究进展

高通量细胞电融合芯片设计中采用微阵列结构的电极设计方案可提高芯片上微电极的数量,使细胞电融合芯片向高通量、高效率、高集成度的芯片实验室方向发展。通过设计微小尺度的电极间距,降低了对细胞电融合信号高压的要求和融合信号源的制造难度,提高了融合过程的安全性。对微电极形状、融合信号进行优化设计,提高细胞电融合的效率。同时,对芯片制造技术和芯片材料加以优化选择,提高了芯片制造的可靠性和电气与生化等性能。     

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.     

全金属微电极阵列细胞电融合芯片的研制

研制了以硅为基底、金属铜(Cu)材料作为微电极的细胞电融合芯片.在500 μm厚的硅基底上应用离子刻蚀技术,刻蚀出与所需得到的微电极相同形状的槽,然后高温下使硅片表层形成二氧化硅绝缘层,在刻蚀槽的底部形成种子层(Ta/Cu材料),通过电镀在槽中形成金属微电极,应用湿法刻蚀去除表层硅,得到纯金属微电极.铜金属微电极其导电率高,减小了电压衰减,使细胞电融合芯片中电场分布一致性好.该方法利用刻蚀的硅模具和电镀工艺解决了lift-off制造的金属微电极较薄的难题,分别采用热氧化和等离子增强化学气相淀积(PECVD)工艺制作的二氧化硅薄膜也增强了芯片抗腐蚀能力.在利用黄瓜叶肉细胞的细胞排队和融合实验中,实验效果比现有硅微阵列芯片要好.     

基于微电极阵列和无线传感器网络的水环境重金属检测系统研究

为对区域水环境中重金属进行有效检测,提出了一种基于微电极阵列和无线传感器网络的重金属现场检测技术。为此,提出了一种带状微电极阵列芯片,用于锌、铅、铜等三种重金属离子的测量;基于带状微电极阵列芯片,开发了自动化的水环境重金属现场检测仪器;多台仪器通过802.11 b/g无线协议组成无线传感器网络,对区域水环境中的重金属含量进行检测。在实验室通过重金属标准溶液对仪器性能进行验证,表明仪器具有较好的精确度;在自然水域进行了传输距离、无线组网测试;完成了自然水域样品重金属浓度检测,并同原子吸收法进行了对比验证。     

用于细胞固定的微纳SiO2基底研究

细胞阵列芯片在细胞生物学研究、药物筛选等生物医疗领域有着广阔的应用前景.针对芯片上细胞固定时需要化学修饰的限制,提出了仅通过表面结构实现细胞固定的方案.基于MEMS技术,采用DRlE在硅片上得到超疏水的微纳结构,通过简单的高温氧化工艺实现超亲水性的SiO2 结构表面;再通过控制BOE腐蚀时间得到不同形貌的基底表面.计算了基底表面上的循环肿瘤细胞的黏附效率,并在SEM下观察了细胞与基底的相互作用行为,探讨了粗糙表面影响细胞黏附的原因.实验结果表明:采用DRlE和高温氧化制备的微纳SiO2 基底表面能极大促进细胞黏附,可应用于细胞阵列芯片设计中.     

微电极阵列细胞阻抗传感器研究

贴壁生长在微电极表面的细胞可引起贴壁电极界面阻抗的改变,从而可以获得细胞生理功能相关的生物信息.本研究采用微机械加工技术,在硅基底上设计了直径为20～50 μm的20通道金微电极阵列(micro-electrode array,MEA),用以构建能实时、连续、定量跟踪哺乳动物细胞形态和增殖分化改变的细胞阻抗测试传感器(electric cell-substrate impedance sens-ing,ECIS),用于细胞与电极间的阻抗测试研究.通过对培养在微电极表面24 h的成骨细胞Saos-2细胞系的阻抗谱测量发现,其阻抗值增加集中在中频102～10<'4>Hz之间,本结果符合细胞阻抗传感测量的理论模型分析.因此,该微电极阵列细胞阻抗传感器的研究,为进一步的细胞生理和药物分析等研究奠定了良好的基础.     

三维神经微电极阵列新制作技术研究

尝试了一种低成本的三维微电极阵列微加工的新方法.玻璃划片形成的柱状阵列,经掩膜腐蚀后形成阳模板,再利用PDMS的微复制技术形成阴模板.利用阴模板进行电镀,可以得到三维微电极阵列.制作的铜电极阵列高度约180 μm,为制作更长的神经微电极阵列打下了基础.     

面向电阻抗成像检测的多电极阵列微流控芯片设计

设计了一种用于微尺度流动状态下电阻抗成像检测的多电极阵列微流控芯片,包括微流控芯片的结构设计、材料选择和加工工艺。设计的微流控芯片包含3个圆形电极横截面,每个横截面包含一组电极阵列。该阵列有3种数目的电极,分别为8电极,12电极和16电极。之后通过数值仿真方法实现了三种电极数目（8,12和16）微流控芯片的电阻抗成像,并与之前研究出来的菱形横截面8电极微流控芯片进行了对比,发现设计出来的16电极圆形微流控芯片具有较高的成像质量,验证了微流控芯片用于细胞电阻抗成像检测的可行性。     

用于神经信号检测的传感器系统的研究

探讨了一种用于神经信号检测的新型传感器系统的设计。提出了一种由二维无源电极和前置信号放大模块微装配而成的三维有源微电极阵列的结构形式,在对电极和电解液接触面间的电学模型进行分析的基础上,完成了二维无源电极和前置放大模块的设计。仿真结果验证了传感器系统用来检测神经信号的可行性和可靠性。     

基于NEMS技术的介电泳芯片及其关键工艺问题的研究

结合信息技术、生物技术与纳米技术的发展,提出一种基于NEMS技术的“三维纳隙网格阵列微电极生物传感器“设计,用于生物样品在蛋白或细胞水平上的介电泳分离和检测.重点针对传感器制作的关键工艺问题,创新性地提出了一种利用反应离子刻蚀侧向钻蚀效应实现纳隙薄金属悬臂梁结构的理论和方法,此方法对电容式生物传感器的制作有普遍的实践意义.     

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.     

Study of high-throughput cell electrofusion in a microelectrode-array chip

A microfabricated high-throughput cell electrofusion chip with 1,368 pairs of high aspect ratio silicon microelectrodes is presented. These microelectrodes, which were distributed in six individual microscale cell-fusion chambers, were covered with titanium and gold thin film to improve their electric conductivity as well as surface hydrophobility. Six chambers having different electrode distances make the chip highly suitable for fusing cells with different sizes. A microfluidic platform was set up for flowing control, cell manipulation and also experimental observation. Cells for electrofusion were first aligned at the prearranged locations by the dielectrophoretic force between two counter-electrodes, which benefits the traverse of electric pulse through the cell–cell contacting point for electroporation. Several on-chip cell electrofusion experiments have been carried out on different kinds of animal cells and plant protoplasts. Compared with conventional electrofusion methods, higher fusion efficiency was achieved on this device for precisely forming micropores on the proximate membranes of two contacting cells, and high throughput was also obtained due to the use of a large number of microelectrodes for cell manipulation and fusion. Moreover, a much lower power supply was required for the shorter distance between two counter-electrodes.     

Somatic and stem cell pairing and fusion using a microfluidic array device

There is considerable excitement about the prospect of tissue repair and renewal through cell replacement therapies. Nonetheless, many of these techniques may require the reprogramming of somatic and stem cells through cell fusion. Previous fusion methods often suffer from random cell contacts, poor fusion yields, or complexity of design. We have developed a simplified cell-electrofusion chip that possesses a dense microelectrode array, which enables the simultaneous pairing and electrofusion of thousands of cells by manipulation dielectrophoretic force and electroporation. Human embryonic kidney 293 (HEK293) cells, mouse fibroblasts (NIH3T3 cells), and mouse embryonic stem cells were arranged for cell fusion with the same and mixed cell type. The pairing efficiency for a 2-cell alignment of mixed cells was ~35%, and a fusion efficiency of ~46% in cell pairs was achieved. Significant cell death occurs with fusion voltages ?? 10 V, and electrofusion with our chip was achieved on a ~1000 V cm^?1 electric field strength induced by a low intensity voltages (9 V). Therefore, the chip used in this study provides a simple, low voltage alternative with sufficient throughput for hybrid cell experiments and somatic cell reprogramming research.     

Oriented and Vectorial Patterning of Cardiac Myocytes Using a Microfluidic Dielectrophoresis Chip—Towards Engineered Cardiac Tissue With Controlled Macroscopic Anisotropy

Recently, the ability to create engineered heart tissues with a preferential cell orientation has gained much interest. Here, we present a novel method to construct a cardiac myocyte tissue-like structure using a combination of dielectrophoresis and electro-orientation via a microfluidic chip. The device includes a top home-made silicone chamber containing microfluidic channels and bottom integrated microelectrodes which are patterned on a glass slide to generate dielectrophoresis force and orientation torque. Using the interdigitated-castellated microelectrodes, the induction of a mutually attractive dielectrophoretic force between cardiac myocytes can lead to cells moving close to each other and forming a tissue-like structure with orientation along the alternating current (ac) electric field between the microelectrode gaps. Both experiments and analysis indicate that a large orientation torque and force can be achieved by choosing an optimal frequency around 2 MHz and decreasing the conductivity of medium to a relatively low level. Finally, electromechanical experiments and biopolar impedance measurements were performed to demonstrate the structural and functional anisotropy of electro-oriented structure     

Low cost fabrication of microelectrodes on plastic substrate

A low cost method of fabricating thick film microelectrodes on plastic substrate for producing integrated plastic biochips is introduced in this paper. Microelectrodes for pumping liquid in a PCR plastic biochip were successfully fabricated using a metal stamp and commercially available Au films. Testing results show that the microelectrode produced with the metal stamp is more reliable and can last longer than those fabricated with sputtering techniques. The fabrication method demonstrated in this paper provides a new low cost technology that is suitable for mass production of thick-film microelectrodes of high electrical conductivity on plastic biochips.     

Particle streaming and separation using dielectrophoresis through discrete periodic microelectrode array

This study presents a particle manipulation and separation technique based on dielectrophoresis principle by employing an array of isosceles triangular microelectrodes on the bottom plate and a continuous electrode on the top plate. These electrodes generate non-uniform electric fields transversely across the microchannel. The particles within the flowing fluid experience a dielectrophoretic force perpendicular to the fluid flow direction due to the non-uniform electric fields. The isosceles triangular microelectrodes were designed to continuously exert a small dielectrophoretic force on the particles. Particles experiencing a larger dielectrophoretic force would move further in the perpendicular direction to the fluid flow as they traveled past each microelectrode. Polystyrene microspheres were used as the model particles, with particles of ∅20 μm employed for studying the basic characteristics of this technique. Particle separation was subsequently demonstrated on ∅10 and ∅15 μm microspheres. Using an applied sinusoidal voltage of 20 V_pp and frequency of 1 MHz, a mean separation distance of 0.765 mm between them was achieved at a flow rate of 3 μl/min (~1 mm/s), an important consideration for high throughput separation capability in a micro-scale technology device. This unique isosceles triangular microelectrodes design allows heterogeneous particle populations to be separated into multiple streams in a single continuous operation.     

Integration of Au Nanorods With Flexible Thin-Film Microelectrode Arrays for Improved Neural Interfaces

In neural prosthetic systems, a low-impedance electrode–tissue interface is important for maintaining signal quality for recording, as well as effective charge transfer for stimulation. However, neural microelectrodes often have high impedance due to their small surface areas. In this paper, we present a simple method of increasing their effective surface areas by introducing nanostructures on the electrode sites. The method combines photolithography and electron-beam evaporation with a locally patterned anodized porous alumina template to integrate Au-nanorod arrays on the flexible thin-film microelectrode, which can significantly increase the effective surface area and decrease impedance due to its 3-D and compact nanostructures. Moreover, the geometrical and electrical properties of Au-nanorod electrodes were demonstrated and compared with conventional planar microelectrodes by using a scanning electron microscope and an impedance spectroscopy system. Experimental results showed that approximately 25 times lower interface impedance was achieved for this nanostructured microelectrode. Such Au-nanorod integrated microelectrode arrays will be a promising tool for neural engineering. $hfill$[2008-0136]      

Plasma assisted thermal bonding for PMMA microfluidic chips with integrated metal microelectrodes

Fracture of integrated metal microelectrodes likely happens during the thermal bonding process of PMMA [poly (methylmethacrylate)] microfluidic chips. In this paper, the fracture behaviors are studied. The fracture is mainly caused by the plastic deformation of the electrode plate (the PMMA plate with microelectrodes) and the thermal stress of microelectrodes, which is due to the high bonding temperature. To decrease the bonding temperature, a plasma assisted thermal bonding method is evaluated and first used to eliminate the fracture of microelectrodes. In this process, the surface of the cover plate (the PMMA plate with microchannels) is modified using oxygen plasma before the electrode plate is thermally bonded to the cover plate. The parameters of the oxygen plasma treatment are optimized, and the contact angle is decreased from 71.7° to 43.6°. The thermal bonding temperature is optimized, which decreases the temperature from 100 °C to 85 °C. Testing of bonding strength shows an average failure pressure of 1.75 MPa, which is comparable to the bonding strength of 1.46 MPa for chips bonded at 100 °C without plasma modification. In order to demonstrate this bonding method, a PMMA microfluidic chip with integrated copper interdigitated microelectrode arrays for AC electroosmotic pump is fabricated.     

Highly efficient electrochemical valence control of uranium using microfluidic chip equipped with microelectrodes

We have developed a novel microchip equipped with a microchannel and Pt microelectrode array for electrochemically controlling valences of actinide (An) species. The square wave voltammograms of the redox reaction of potassium hexacyanoferrate(II) in the microchannel were measured. We found that the fabricated Pt microelectrode array has superior performances for the detection of the electrochemically active species in the microchannel. Therefore, the potentiostatic electrolysis experiments of uranium ions were carried out in the microchannel, and the concentration changes of uranium ions accompanied by the potentiostatic electrolysis were examined using thermal lens microscope. The results showed that the redox reactions between U(VI) and U(IV) can be performed completely in a microchannel in a few minutes, that is, the microscale reaction is accelerated by a factor of more than 10 compared with the bulk solution reactions taking hours mostly. The developed microchip was found to have enough performances for realizing rapid and highly efficient redox reactions for An species, which are impossible in the bulk reactions.