Nanomanipulation of single influenza virus using dielectrophoretic concentration and optical tweezers for single virus infection to a specific cell on a microfluidic chip

A major problem when analyzing bionanoparticles such as influenza viruses (approximately 100 nm in size) is the low sample concentrations. We developed a method for manipulating a single virus that employs optical tweezers in conjunction with dielectrophoretic (DEP) concentration of viruses on a microfluidic chip. A polydimethylsiloxane microfluidic chip can be used to stably manipulate a virus. The chip has separate sample and analysis chambers to enable quantitative analysis of the virus functions before and after it has infected a target cell. The DEP force in the sample chamber concentrates the virus and prevents it from adhering to the glass substrate. The concentrated virus is transported to the sample selection section where it is trapped by optical tweezers. The trapped virus is transported to the analysis chamber and it is brought into contact with the target cell to infect it. This paper describes the DEP virus concentration for single virus infection of a specific cell. We concentrated the influenza virus using the DEP force, transported a single virus, and made it contact a specific H292 cell.     

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.     

Sequential Field-Flow Cell Separation Method in a Dielectrophoretic Chip With 3-D Electrodes

This paper presents a sequential dielectrophoretic field-flow separation method for particle populations using a chip with a 3-D electrode structure. A unique characteristic of our chip is that the walls of the microfluidic channels also constitute the device's electrodes. This property confers the opportunity to use the electrodes' shape to generate not only the electric field gradient required for dielectrophoretic force but also a fluid velocity gradient. This interesting combination gives rise to a new solution for the dielectrophoretic separation of two particle populations. The proposed sequential field-flow separation method consists of four steps. First, the microchannel is filled with the mixture of the two populations of particle. Second, the particle populations are trapped in different locations of the microfluidic channels. The population, which exhibits positive dielectrophoresis (DEP), is trapped in the area where the distance between the electrodes is the minimum, while the other population that exhibits negative DEP is trapped in locations of maximum distance between electrodes. In the next step, increasing the flow in the microchannels will result in an increased hydrodynamic force that sweeps the cell population trapped by positive DEP out of the chip. In the last step, the electric field is removed, and the second population is swept out and collected at the outlet. For theoretical and experimental exemplification of the separation method, a population of viable and nonviable yeast cells was considered.     

Integrated microfluidic system for electrochemical sensing of glycosylated hemoglobin

This article reports a new miniature electrochemical detection system integrating a sample pretreatment device for fast detection of glycosylated hemoglobin (HbA_1C), which is a common indicator for diabetes mellitus. In this system, circular micropumps, normally closed microvalves, dielectrophoretic (DEP) electrodes, and electrochemical sensing electrode are integrated to perform several crucial processes. These processes include separation of red blood cells (RBCs), sample/reagent transportation, mixing, cell lysis, and electrochemical sensing. For the HbA_1C measurement, the RBCs are separated and are collected from whole human blood by using a positive DEP force generated by the DEP electrodes. The collected RBCs are then lysed to release HbA_1C for the subsequent electrochemical detection processes. Experimental data show that the RBCs are successfully separated and are collected using the developed system with a RBCs capture rate of 84.2%. The subsequent detection of HbA_1C is automatically completed by utilizing electrochemical sensing electrode. The microfluidic system only consumes a sample volume of 200 μl. The entire process is automatically performed within a short period of time (10 min). The development of this integrated microfluidic system may be promising for the clinical monitoring of diabetes mellitus.     

Creation of single-particle environment by positive dielectrophoresis and liquid dielectrophoresis

Two electrical mechanisms for manipulating particles and fluids, dielectrophoresis (DEP) and liquid dielectrophoresis (LDEP), are integrated in a microfluidic chip for creating the single-particle environment. The fluid is activated by LDEP with a 100-kHz/240-V_pp signal. When the single polystyrene bead approaches the trapping area, positive DEP force is utilized to capture and immobilize the bead. After trapping the bead, the process of liquid cutting and droplet creation is employed to create a droplet containing a single bead by LDEP with a 100-kHz/320-V_pp signal.     

Frequency-dependent conductance change of dielectrophoretic-trapped DNA-labeled microbeads and its application in DNA size determinations

DNA amplification is essential in several types of molecular biology approaches. A more rapid and easy analysis of amplicons is still required although many analysis methods have been developed. We have recently devised a new DNA detection method, where DNA amplicons are attached to dielectric microbead surfaces, so that their dielectrophoresis (DEP) force on the microbead reverses polarity, from negative to positive. The DNA-labeled microbeads are trapped on a microelectrode by positive DEP, enabling their rapid detection via DEP impedance measurement. In this paper, we report frequency-dependent conductance of DNA-labeled microbeads. To measure the impedance, sweep-frequency voltage was superimposed on fixed-frequency voltage, with the aim of inducing frequency-dependent conformational change of microbead-attached DNA, ultimately resulting in a change in the conductance of DNA-labeled microbeads. Microbeads labeled with DNA of various sizes (142-, 204-, 391-, and 796-bp) were examined. The normalized conductance sharply decreased at a specific frequency; the frequency was higher with larger DNA size, suggesting a potential application of this method in distinguishing DNA targets according to their size. By combining this method with previously devised DNA detection techniques, both the size and amount of target DNA can be determined within 20 min. This approach is easier and more rapid than conventional methods, such as a gel electrophoresis.     

Rapid quantification of bio-particles based on image visualisation in a dielectrophoretic microfluidic chip

We studied an imaging-based technique for the rapid quantification of bio-particles in a dielectrophoretic (DEP) microfluidic chip. Label-free particles could be successively sorted and trapped in a continuous flow manner under the applied alternating current (AC) conditions. Both 2 and 3 μm polystyrene beads at a concentration of 1.0 × 10⁷ particles ml⁻¹ could be rapidly quantified within 5 min in our DEP system. Capturing efficiencies higher than 95% could be 2 μm polystyrene beads with a linear flow speed, applied voltage and frequency of 0.89 mm s⁻¹, 20 V_p-p and 5 MHz. Yeast cells (Candida glabrata and Candida albicans) could also be captured even at a lower concentration of 2.5 × 10⁵ cells ml⁻¹. Images of aggregative particles taken from the designed trapping area were further processed based on the intensity of relative greyscale followed by correction of the particle numbers. The imaging-based quantification method showed higher agreement than that of the conventional counting chamber method and proved the stability and feasibility of our AC DEP system.     

On-chip manipulation and trapping of microorganisms using a patterned magnetic pathway

We demonstrate on-chip manipulation and trapping of individual microorganisms at designated positions on a silicon surface within a microfluidic channel. Superparamagnetic beads acted as microorganism carriers. Cyanobacterium Synechocystis sp. PCC 6803 microorganisms were immobilized on amine-functionalized magnetic beads (Dynabead^® M-270 Amine) by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)–N-hydroxysulfosuccinimide coupling chemistry. The magnetic pathway was patterned lithographically such that half-disk Ni₈₀Fe₂₀ (permalloy) 5 μm elements were arranged sequentially for a length of 400 micrometers. An external rotating magnetic field of 10 mT was used to drive a translational force (maximum 70 pN) on the magnetic bead carriers proportional to the product of the field strength and its gradient along the patterned edge. Individual microorganisms immobilized on the magnetic beads (transporting objects) were directionally manipulated using a magnetic rail track, which was able to manipulate particles as a result of asymmetric forces from the curved and flat edges of the pattern on the disk. Transporting objects were then successfully trapped in a magnetic trapping station pathway. The transporting object moves two half-disk lengths in one field rotation, resulting in movement at ~24 μm s^?1 for 1 Hz rotational frequency with 5 μm pattern elements spaced with a 1 μm gap between elements.     

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.     

A novel integrated microfluidic platform based on micro-magnetic sensor for magnetic bead manipulation and detection

We present a novel integrated microfluidic platform based on micro-magnetic sensor for manipulating and detecting magnetic beads (MB). A micro-spiral planar coil in MB manipulating system microfabricated by micro-electro-mechanical system technology is implemented to manipulate MB, and a giant magnetoimpedance (GMI) based micro-magnetic sensor is employed to detect the trapped MB. In our work, MB can be efficiently trapped by trapping force generated from micro-coil in microchannel. Next, trapped MB are detected by the changing ratio of impedance, as well as the variation of resistance and reactance in GMI sensor for trapped MB induce weak stray magnetic field under the magnetization by external magnetic field. The maximum difference of GMI ratio between with beads condition and without beads condition is 4.0% at the optimum driving frequency of 20 MHz under the external magnetic field of 15 Oe, and resistance ratio varies more significantly than reactance ratio. In comparison with traditional MB detecting methods by GMI sensor, the integrated microfluidic platform based on GMI sensor can not only manipulate and detect MB signal sensitively, but also enhance detection efficiency and decrease the experiment errors. Furthermore, this platform avoids contamination from the solutions in chemically reactive layers and reduces assay time in future biomarker detection. In our work, the microfluidic platform based on GMI sensor has potential applications in biomarker detection via MB manipulation and detection.     

Precisely sized separation of multiple particles based on the dielectrophoresis gradient in the z-direction

We present a new 3D dielectrophoresis-field-flow fraction (DEP-FFF) concept to achieve precise separation of multiple particles by using AC DEP force gradient in the z-direction. The interlaced electrode array was placed at the upstream of the microchannel, which not only focused the particles into a single particle stream to be at the same starting position for further separation, but also increased the spacing between each particle by the retard effect to reduce particle–particle aggregation. An inclined electrode was also designed in back of the focusing component to continuously and precisely separate different sizes of microparticles. Different magnitudes of DEP force are induced at different positions in the z-direction of the DEP gate, which causes different penetration times and positions of particles along the inclined DEP gate. 2, 3, 4, and 6?μm polystyrene beads were precisely sized fractionation to be four particle streams based on their different threshold DEP velocities that were induced by the field gradient in the z-direction when a voltage of 6.5?V_p–p was applied at a flow rate of 0.6?μl/min. Finally, Candida albicans were also sized separated to be three populations for demonstrating the feasibility of this platform in biological applications. The results showed that a high resolution sized fractionation (only 25% size difference) of multiple particles can be achieved in this DEP-based microfluidic device by applying a single AC electrical signal.     

Stepwise gray-scale light-induced electric field gradient for passive and continuous separation of microparticles

This article presents a gray-scale light-induced dielectrophoresis (GS-LIDEP) method that induces the lateral displacements normal to the through-flow for continuous and passive separation of microparticles. In general, DEP force only can affect the particles within very local areas due to the electric field is exponentially decayed by the distance away from the electrodes. Unlike with conventional LIDEP, a broad-ranged electrical field gradient can easily be created by GS pattern illumination, which induces DEP forces with two directions for continuous separation of particles to their specific sub-channels. Candia albicans were effectively guided to the specific outlet with the efficiency of 90% to increase the concentration of the sample below the flow rate of 0.6?μl/min. 2 and 10?μm polystyrene particles can also be passively and well separated using the multi-step GS pattern through positive and negative DEP forces, respectively, under an applied voltage of 36?V_p–p at the frequency of 10?kHz. GS-LIDEP generated a wide-ranged DEP force that is capable of working on the entire area of the microchannel, and thus the mix of particles can be passively and continuously separated toward the opposite directions by the both positive and negative GS-LIDEP forces. This simple, low cost, and flexible separation/manipulation platform could be very promising for many applications, such as in-field detections/pretreatments.     

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.     

Bioparticle separation and manipulation using dielectrophoresis

Manipulation and separation of micro-sized particles, particularly biological particles, using the dielectrophoretic (DEP) force is an emerging technique in MEMS technology. This paper presents a DEP-based microsystem for the selective manipulation and separation of bioparticles using dielectrophoretic effects. The microfabricated DEP device consists of a sandwich structure, in which a microchannel with electrode array lining on its bottom is sandwiched between the substrate and the glass lid. Dielectrophoretic behavior of polystyrene particles with diameter of 4.3 μm was studied. Both positive DEP and negative DEP were observed. Particles under positive DEP were attracted to the edges of the electrodes, while those under negative DEP were repelled away from the electrodes and levitated at certain height above the electrodes (within a proper range of frequencies of the electric field). Levitation height of the particles was measured. It was demonstrated that the levitation height of a specific particle strongly depends on the combined contributions of a number of parameters, such as the frequency of the electric field, dielectric properties of the particles and the surrounding medium. Different particles can be separated and manipulated on the basis of their difference in these parameters.     

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.     

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.     

Disposable microfluidic blood cuvette for measuring hemoglobin concentration

This paper presents a new air-bubble free microfluidic blood cuvette for the measurement of hemoglobin concentration. The microfluidic blood cuvette was filled with blood samples by capillary force, and hemoglobin levels in the blood were determined by measuring absorbance at the wavelength of 530 nm. Two different microfluidic blood cuvettes with dual and single sidewall microchannels were investigated. The microfluidic blood cuvette was fabricated using a polymethyl methacrylate substrate and a dry film photoresist. During the blood-filling process, air was trapped in the dual-sided wall-type cuvettes, while no air trapping occurred in the single sidewall-type cuvettes. The sensitivity of the hemoglobin measurements was more linear in a 105 μm deep microchannel than in a 35 μm deep microchannel.     

Comparison of spherical and non-spherical particles in microchannels under dielectrophoretic force

This paper focuses on the computational and experimental study of dielectrophoretic (DEP) force based manipulation of spherical and non-spherical particles by taking into consideration of both electrokinetic effects and particle hydrodynamics. The model is first validated with conventional dipole moment theory. The movements of a spherical polystyrene particle and a rod-shape particle under a non-uniform electric field created by a pair of non-symmetrical electrodes in a microfluidic channel are studied, and a good agreement between the simulation and experimental results is obtained. Both experimental and simulation results reveal that the rod-shape particle experiences larger DEP force and moves faster than spherical particle with a similar mass. It was also interestingly found that the shape-dependent DEP force distribution on the microscale rod particle results in its unique behavior, which cannot be captured by traditional DEP theory.     

Characterization and modeling of a microfluidic dielectrophoresis filter for biological species

Microfabricated interdigitated electrode array is a convenient form of electrode geometry for dielectrophoretic trapping of particles and biological entities such as cells and bacteria within microfluidic biochips. We present experimental results and finite element modeling of the holding forces for both positive and negative dielectrophoretic traps on microfabricated interdigitated electrodes within a microfluidic biochip fabricated in silicon with a 12-/spl mu/m-deep chamber. Anodic bonding was used to close the channels with a glass cover. An Experimental protocol was then used to measure the voltages necessary to capture different particles (polystyrene beads, yeast cells, spores and bacteria) against destabilizing fluid flows at a given frequency. The experimental results and those from modeling are found to be in close agreement, validating our ability to model the dielectrophoretic filter for bacteria, spores, yeast cells, and polystyrene beads. This knowledge can be very useful in designing and operating a dielectrophoretic barrier or filter to sort and select particles entering the microfluidic devices for further analysis.     

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.