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.     

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。     

Microfluidic chips for cells capture using 3-D hydrodynamic structure array

Microfluidic chips were designed and fabricated to capture cells in a relative small volume to generate the desired concentration needed for analysis. The microfluidic chips comprise three-dimensional (3-D) cell capture structures array fabricated in PDMS. The capture structure includes two layers. The first layer consists of spacers to create small gap between the upper layer and glass. The second layer is a sharp corner U-shaped compartment with sharp corners at the fore-end. And another type capture structure with Y-shaped fluidic guide has been designed. It was demonstrated that the structures can capture cells in theory, using Darcy–Weisbach equation and COMSOL Multiphysics. Then yeast cell was chosen to test the performance of the chips. The chip without fluid guides captured ~1.44 × 10⁵ cells and the capture efficiency was up to 71 %. And the chip with fluid guides captured ~5.0 × 10⁴ cells and the capture efficiency was ~25 %. The chip without fluid guides can capture more cells because the yeast cells in the chip without fluid guides are subject to larger hydrodynamic drag force.     

Microfluidic fluorescence-activated cell sorting (μFACS) chip with integrated piezoelectric actuators for low-cost mammalian cell enrichment

A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.     

基于直列式交错微电极阵列的细胞电融合

提出了一种基于交错式齿状微电极阵列的微流控细胞电融合芯片。利用COMSOL Multiphysics仿真软件,对电场强度有重要影响的微电极几何参数进行了仿真分析,并由此提出了优化的微电极阵列结构。选择SoI硅片的顶层低阻硅加工获得了微电极阵列。实验结果表明:该芯片中采用的直列式微通道结构避免了原有芯片存在的转角易堵塞问题。芯片能够在低电压条件下实现细胞排队和融合过程,具有较高的融合效率。     

Isolation and elution of Hep3B circulating tumor cells using a dual-functional herringbone chip

Circulating tumor cells (CTCs), which are derived from primary tumor and circulate to secondary site, are regarded as the cause of metastasis. Many methods have been applied for CTC isolation and enumeration so far. However, it remains a challenge to effectively elute the captured cells from the device for further cellular and biomolecular analyses. In this paper, we fabricate a dual-functional herringbone chip to achieve both CTC capture and elution based on the immunoassay of epithelial cell adhesion molecule antigen expressed on the surface of human liver cancer cell line Hep3B. The results show that the capture limit of Hep3B cells can reach as low as 3 cells per ml with capture efficiency over 50 % on average. On the other hand, the elution rate of more than 50 % of the captured Hep3B cells can be achieved for cell density ranging from 5 to 2 × 10³/ml. It demonstrates that this herringbone chip exhibits excellent dual functions with high capture efficiency and considerable elution rate, indicating its promising capability for clinical assay in cancer diagnosis.     

High-speed counting and sizing of cells in an impedance flow microcytometer with compact electronic instrumentation

Here we describe a high-throughput impedance flow cytometer on a chip. This device was built using compact and inexpensive electronic instrumentation. The system was used to count and size a mixed cell sample containing red blood cells and white blood cells. It demonstrated a counting capacity of up to ~500 counts/s and was validated through a synchronised high-speed optical detection system. In addition, the device showed excellent discrimination performance under high-throughput conditions.     

Impedance spectra of patch clamp scenarios for single cells immobilized on a lab-on-a-chip

A simple method based on impedance spectroscopy (IS) was developed to distinguish between different patch clamp modes for single cells trapped on microapertures in a patch clamp microchannel array designed for patch clamping on cultured cells. The method allows detecting via impedance analysis whether the cell membrane is ruptured (and culturing prevented) or the cell is still in the attached mode. A modular microfluidic lab-on-a-chip device based on planar patch clamp technology was used to capture multiple individual cells on an array of microapertures. The comparison of the measured and simulated impedance spectra proved that the presented method could distinguish between a cell-attached mode and a whole-cell mode even with low-quality seals. In physiological conditions, the capacitance of HeLa cells was measured to ~38 pF. The first gigaseal was recorded and maintained for 40 min. Once whole-cell configurations were established, trapped cells were superfused with a 140 mM KCl aqueous solution: the change in the measured cell impedance revealed a capacitance decrease to ~27.5 pF that could be due either to a change in the cell size or to the reduced charge separation across the cell membrane. After incubating the chip for 24 h, HeLa cells adhered and grew on the chip surface but did not survive when trapped on the microapertures. The microfluidic system proved to work as a micro electrophysiological analysis system, and the IS-based method can be used for further studies on the post-trapping strength of the seal between the microapertures and the trapped cells to be cultured.     

Microfabrication-free fused silica nanofluidic interface for on chip electrokinetic stacking of DNA

In this article, a simple but robust nanofluidic interface was introduced directly on a chip comprised of commercially available fused silica capillary with ready-made microchannel, and efficient on chip electrokinetic stacking of DNA was successfully demonstrated based on ion concentration polarization (ICP) effect. The nanofluidic interface was established by casting ion exchange polymer resin (Nafion) into a sub-microfracture (~650 nm) prepared on the capillary manually. The width of the fracture was electrically measured with the aide of a mathematic fracture model and confirmed by scanning electronic microscope. Obvious ICP effect was observed both by online microscopic fluorescent imaging and post laser induced fluorescence detection. SYBR Green I labeled dsDNA was stacked at the nanofluidic interface inside the microchannel (cathode side) with a concentration factor of 10³ within 15 s. As high as 800 V was applied through the interface without any damage. The main materials are all commercially available, and no advanced microfabrication facilities are involved in the preparation of the chip.     

Microthermal sensors for determining fluid composition and flow rate in fluidic systems

To analyze fluid mixtures a simple and low cost measurement method is realized using a microthermal sensor that introduces a short heat pulse into the fluid under test whilst the resulting temperature increase reflects thermal parameters of the fluid. For methanol in water this principle showed an almost linear dependence of the temperature increase on the methanol content for the volume concentration range 0–20 %. The sensitivity was determined to S = 0.19 K/(% (V/V)) for a heat pulse of 0.5 s duration and a heater power of 30 mW. The accuracy achieved in stopped-flow single pulse measurements is ~0.5 % (V/V). By integrating additional temperature sensors in front and behind the microheater the flow rate of the liquid can also be determined using thermal anemometry. The low cost sensor construction and simple signal analysis make this principle promising for use in low cost mobile applications like DMFC power supplies for laptops.     

A microfluidic system for the study of the response of endothelial cells under pressure

Hydrostatic pressure can affect the structure and function of endothelial cells (ECs). A microfluidic system was built to study how ECs respond to applied pressure. The system included a syringe pump, a PDMS-glass microfluidic chip, and a digital manometer for pressure monitoring. The manometer was connected with the chip in two ways (one was before the inlet and the other after the outlet of the microchannel). The static control and flowing control systems were also set up. Human umbilical vein endothelial cells (HUVECs) were cultured in the 4 cm × 2 mm × 100 μm channel. Pressure of 12 ± 0.5 or 18 ± 0.5 kPa was applied on the cells for 8 h. The F-actin cytoskeleton and the nuclei of the cells were stained for examination and endothelin-1 (ET-1) released from the cells in the channel was assayed by ELISA. The results showed that the cell area and ET-1 concentration increased with the pressure and a higher pressure caused more damages to the cells. This microfluidic system provides a convenient and cost-effective platform for the studies of cell response to pressure.     

Spiral-shaped inertial stem cell device for high-throughput enrichment of iPSC-derived neural stem cells

Stem cell enrichment plays a critical role in both research and clinical applications. The typical method for stem cell enrichment may use invasive processes and takes a long period of time. Spiral-shaped microfluidic devices, which combine lift and Dean drag forces to direct cells of different sizes into separate trajectories, can be used to noninvasively process samples at a rate of milliliters per minute. This paper presents a simple 2-loop spiral-shaped inertial microfluidic devices with the aid of sheath flow to enrich neural stem cells (NSCs), derived from induced pluripotent stem cells. NSCs and spontaneously differentiated non-neural cells were mixed and flowed through the spiral-shaped devices. Samples collected at the outlets were analyzed for purity and recovery. It was found that the device focused the NSCs into a narrow trajectory, which could then be collected in two out of the eight outlets. The device was tested at different flow rates and found that the most highly enriched fractions (2.1×) with NSCs recovery 93% were achieved at the flow rate (3 ml/min). Next, we extended our investigation from 2-loop design to 10-loop design to eliminate the use of sheath flow. NSCs were enriched to 2.5×, but only 38% of the NSCs were recovered from the most enriched fractions. Spiral-shaped microfluidic devices are capable of rapid, label-free enrichment of target stem cells, and have great potential in point-of-care tissue preparation.     

Optimized design and fabrication of a microfluidic platform to study single cells and multicellular aggregates in 3D

A microfluidic platform for cell motility analysis in a three-dimensional environment is presented. The microfluidic device is designed to study migration of both single cells and cell spheroids, in particular under spatially and temporally controlled chemical stimuli. A layout based on a central microchannel confined by micropillars and two lateral reservoirs was selected as the most effective. The microfluidics have an internal height of 350 μm to accommodate cell spheroids of a considerable size. The chip is fabricated using well-established micromachining techniques, by obtaining the polydimethylsiloxane replica from a Si/SU-8 master. The chip is then bonded on a 170-μm-thick microscope glass slide to allow high spatial resolution live microscopy. In order to allow the cost-effective and highly repeatable production of chips with high aspect ratio (5:1) micropillars, specific design and fabrication processes were optimized. This design permits spatial confinement of the gel where cells are grown, the creation of a stable gel–liquid interface and the formation of a diffusive gradient of a chemoattractant (>48 h). The chip accomplishes both the tasks of a microfluidic bioreactor system and a cell analysis platform avoiding critical handling of the sample. The experimental fluidic tests confirm the easy handling of the chip and in particular the effectiveness of the micropillars to separate the Matrigel? from the culture media. Experimental tests of (i) the stability of the gradient, (ii) the biocompatibility and (iii) the suitability for microscopy are presented.     

Isolation of magnetically tagged cancer cells through an integrated magnetofluidic device

Circulating tumour cells (CTC) in the bloodstream has been implicated in cancer metastasis. Efficient removal of CTC could potentially be an effective therapeutic measure against cancer metastasis. In this study, the hydrodynamic focusing flow in microfluidic channels (R _e  ? 1) was considered together with the magnetophoretic force. The localised magnetic field was achieved through a passivated current-carrying multilayered microstripline, where the generated field gradient was used to attract the magnetic beads to the desired outlet. The experimental results show that the device is capable of isolating purely magnetic beads with an efficiency of 91 % while isolation efficiency of the magnetically tagged HeLa cervical cancer cells from cell suspension yielded an isolation efficiency of 79 %.     

A remotely powered fully integrated low power class-E power amplifier for implantable sensor systems

This paper presents a fully integrated low power class-E power amplifier and its integration to remotely powered sensor system. The on-chip 1.2 GHz power amplifier is implemented in 0.18 µm CMOS process with 0.2 V supply. The implantable system is powered by using an inductively coupled remote powering link at 13.56 MHz. A passive full-wave rectifier converts the induced AC voltage on the implant coil into a DC voltage. A clean and stable 1.8 V supply voltage for the sensor and communication blocks is generated by a voltage regulator. On–off keying modulated low-power transmitter at 1.2 GHz is used for the transmission of the data collected from the sensors. The transmitter is composed of a LC tank oscillator and a fully on-chip class-E power amplifier. Compared to the conventional class-E power amplifiers, an additional network which reduces the on-chip area is used at the output of the power amplifier. The measurement results verify the functionality of the remotely powered implantable sensor system and the power amplifier. The integrated power amplifier provides ?10 dBm output power for 50 Ω load with a drain efficiency of 31.5 %. The uplink data communication with a data rate of 600 kbps is established by using a commercial 50 Ω chip antenna at 1 m communication distance.     

CMOS integrated elliptic diaphragm capacitive pressure sensor in SiGe MEMS

A novel CMOS integrated Micro-Electro-Mechanical capacitive pressure sensor in SiGe MEMS (Silicon Germanium Micro-Electro-Mechanical System) process is designed and analyzed. Excellent mechanical stress–strain behavior of Polycrystalline Silicon Germanium (Poly-SiGe) is utilized effectively in this MEMS design to characterize the structure of the pressure sensor diaphragm element. The edge clamped elliptic structured diaphragm uses semi-major axis clamp springs to yield high sensitivity, wide dynamic range and good linearity. Integrated on-chip signal conditioning circuit in 0.18 μm TSMC CMOS process (forming the host substrate base for the SiGe MEMS) is also implemented to achieve a high overall gain of 102 dB for the MEMS sensor. A high sensitivity of 0.17 mV/hPa (@1.4 V supply), with a non linearity of around 1 % is achieved for the full scale range of applied pressure load. The diaphragm with a wide dynamic range of 100–1,000 hPa stacked on top of the CMOS circuitry, effectively reduces the combined sensor and conditioning implementation area of the intelligent sensor chip.     

Fabrication of a high aspect ratio (HAR) micropillar filter for a magnetic bead-based immunoassay

A microfluidic chip has been realized for investigating immune cell (U937) activation with lipopolysaccharide (LPS) and subsequent pro-inflammatory cytokine (Interleukin-6, IL-6) detection (Ruffert et al. Proc. EMBL Conference Microfluidics 2012a, p 184; Proc. NanoBioTech Montreux, Poster Sessions B 2012b, pp 17–18). The microfluidic chip comprises two compartments: one compartment for the on-chip cell cultivation, differentiation, and stimulation, while the second one hosts superpara-magnetic beads (Ø 2.8 μm) conjugated to anti-IL-6 antibodies for capturing the LPS-induced IL-6. The two compartments are separated by a micropillar-based filter with a spacing of 2 μm. This filter allows the induced cytokines to infuse into the bead compartment (i.e. the magnetic immunoassay compartment), while preventing the magnetic beads and cells to cross over to the other compartment. To fulfill this requirement, a high aspect ratio pillar array was demonstrated as key element of this study and functionally characterized. The pore size of the filter is given by the lateral distance between the single pillars, which are fabricated by molding microfluidic structures in polydimethylsiloxane, using a master mold made of the expoy-based photoresist SU-8?. An aspect ratio of 5:1 could be achieved with SU-8? bars featuring the dimensions 10 µm × 2 μm (height × width).     

Fusion of eye movement and mouse dynamics for reliable behavioral biometrics

This paper presents the first attempt to fuse two different kinds of behavioral biometrics: mouse dynamics and eye movement biometrics. Mouse dynamics were collected without any special equipment, while an affordable The Eye Tribe eye tracker was used to gather eye movement data at a frequency of 30 Hz, which is also potentially possible using a common web camera. We showed that a fusion of these techniques is quite natural and it is easy to prepare an experiment that collects both traits simultaneously. Moreover, the fusion of information from both signals gave 6.8 % equal error rate and 92.9 % accuracy for relatively short registration time (20 s on average). Achieving such results were possible using dissimilarity matrices based on dynamic time warping distance.     

Microelectromechanical systems vibration powered electromagnetic generator for wireless sensor applications

This paper presents a silicon microgenerator, fabricated using standard silicon micromachining techniques, which converts external ambient vibrations into electrical energy. Power is generated by an electromagnetic transduction mechanism with static magnets positioned on either side of a moving coil, which is located on a silicon structure designed to resonate laterally in the plane of the chip. The volume of this device is approximately 100 mm³. ANSYS finite element analysis (FEA) has been used to determine the optimum geometry for the microgenerator. Electromagnetic FEA simulations using Ansoft’s Maxwell 3D software have been performed to determine the voltage generated from a single beam generator design. The predicted voltage levels of 0.7–4.15 V can be generated for a two-pole arrangement by tuning the damping factor to achieve maximum displacement for a given input excitation. Experimental results from the microgenerator demonstrate a maximum power output of 104 nW for 0.4g (g=9.81 m s^?1) input acceleration at 1.615 kHz. Other frequencies can be achieved by employing different geometries or materials.