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1.
This study describes a novel microfluidic reactor capable of flow-through polymerase chain reactions (PCR). For one-heater PCR devices in previous studies, comprehensive simulations and experiments for the chip geometry and the heater arrangement were usually needed before the fabrication of the device. In order to improve the flexibility of the one-heater PCR device, two heat pipes with one fan are used to create the requisite temperature regions in our device. With the integration of one heater onto the chip, the high temperature required for the denaturation stage can be generated at the chip center. By arranging the heat pipes on the opposite sides of the chip, the low temperature needed for the annealing stage is easy to regulate. Numerical calculations and thermal measurements have shown that the temperature distribution in the five-temperature-region PCR chip would be suitable for DNA amplification. In order to ensure temperature uniformity at specific reaction regions, the Re of the sample flow is less than 1. When the microchannel width increases and then decreases gradually between the denaturation and annealing regions, the extension region located in the enlarged part of the channel can be observed numerically and experimentally. From the simulations, the residence time at the extension region with the enlarged channel is 4.25 times longer than that without an enlarged channel at a flow rate of 2 μl/min. The treated surfaces of the flow-through microchannel are characterized using the water contact angle, while the effects of the hydrophilicity of the treated polydimethylsiloxane (PDMS) microchannels on PCR efficiency are determined using gel electrophoresis. By increasing the hydrophilicity of the channel surface after immersing the PDMS substrates into Tween 20 (20%) or BSA (1 mg/ml) solutions, efficient amplifications of DNA segments were proved to occur in our chip device. To our knowledge, our group is the first to introduce heat pipes into the cooling module that has been designed for a PCR device. The unique architecture utilized in this flow-through PCR device is well applied to a low-cost PCR system.  相似文献   

2.
Hu N  Yang J  Qian S  Joo SW  Zheng X 《Biomicrofluidics》2011,5(3):34121-3412112
A microfluidic device integrated with 3D thin film microelectrode arrays wrapped around serpentine-shaped microchannel walls has been designed, fabricated and tested for cell electrofusion. Each microelectrode array has 1015 discrete microelectrodes patterned on each side wall, and the adjacent microelectrodes are separated by coplanar dielectric channel wall. The device was tested to electrofuse K562 cells under a relatively low voltage. Under an AC electric field applied between the pair of the microelectrode arrays, cells are paired at the edge of each discrete microelectrode due to the induced positive dielectrophoresis. Subsequently, electric pulse signals are sequentially applied between the microelectrode arrays to induce electroporation and electrofusion. Compared to the design with thin film microelectrode arrays deposited at the bottom of the side walls, the 3D thin film microelectrode array could induce electroporation and electrofusion under a lower voltage. The staggered electrode arrays on opposing side walls induce inhomogeneous electric field distribution, which could avoid multi-cell fusion. The alignment and pairing efficiencies of K562 cells in this device were 99% and 70.7%, respectively. The electric pulse of low voltage (～9 V) could induce electrofusion of these cells, and the fusion efficiency was about 43.1% of total cells loaded into the device, which is much higher than that of the convectional and most existing microfluidics-based electrofusion devices.  相似文献   

3.
Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream™ that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 × 106 peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% ± 3.1% (n = 12) and 71.2% ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R2) of more than 0.99 for a spiking range of 4–2600 cells. The viability of MDA-MB-231 cancer cells captured with ApoStream was greater than 97.1% and there was no difference in cell growth up to 7 days in culture compared to controls. The ApoStream device demonstrated high precision and linearity of recovery of viable cancer cells independent of their EpCAM expression level. Isolation and enrichment of viable cancer cells from ApoStream enables molecular characterization of CTCs from a wide range of cancer types.  相似文献   

4.
We report a fundamental study of how the electropermeabilization of a cell is affected by nearby cells. Previous researchers studying electroporation of dense suspensions of cells have observed, both theoretically and experimentally, that such samples cannot be treated simply as collections of independent cells. However, the complexity of those systems makes quantitative modeling difficult. We studied the change in the minimum applied electric field, the threshold field, required to affect electropermeabilization of a cell due to the presence of a second cell. Experimentally, we used optical tweezers to accurately position two cells in a custom fluidic electroporation device and measured the threshold field for electropermeabilization. We also captured video of the process. In parallel, finite element simulations of the electrostatic potential distributions in our systems were generated using the 3-layer model and the contact resistance methods. Reasonably good agreement with measurements was found assuming a model in which changes in a cell''s threshold field were predicted from the calculated changes in the maximum voltage across the cell''s membrane induced by the presence of a second cell. The threshold field required to electroporate a cell is changed ∼5%–10% by a nearby, nearly touching second cell. Cells aligned parallel to the porating field shield one another. Those oriented perpendicular to the field enhance the applied field''s effect. In addition, we found that the dynamics of the electropermeabilization process are important in explaining observations for even our simple two-cell system.  相似文献   

5.
An electro-osmosis (EOS) diode pumping platform capable of culturing cells in fluidic cellular micro-environments particularly at low volume flow rates has been developed. Diode pumps have been shown to be a viable alternative to mechanically driven pumps. Typically electrokinetic micro-pumps were limited to low-concentration solutions (≤10 mM). In our approach, surface mount diodes were embedded along the sidewalls of a microchannel to rectify externally applied alternating current into pulsed direct current power across the diodes in order to generate EOS flows. This approach has for the first time generated flows at ultra-low flow rates (from 2.0 nl/s to 12.3 nl/s) in aqueous solutions with concentrations greater than 100 mM. The range of flow was generated by changing the electric field strength applied to the diodes from 0.5 Vpp/cm to 10 Vpp/cm. Embedding an additional diode on the upper surface of the enclosed microchannel increased flow rates further. We characterized the diode pump-driven fluidics in terms of intensities and frequencies of electric inputs, pH values of solutions, and solution types. As part of this study, we found that the growth of A549 human lung cancer cells was positively affected in the microfluidic diode pumping system. Though the chemical reaction compromised the fluidic control overtime, the system could be maintained fully functional over a long time if the solution was changed every hour. In conclusion, the advantage of miniature size and ability to accurately control fluids at ultra-low volume flow rates can make this diode pumping system attractive to lab-on-a-chip applications and biomedical engineering in vitro studies.  相似文献   

6.
We demonstrate the method of non-inertial lift induced cell sorting (NILICS), a continuous, passive, and label-free cell sorting approach in a simple single layer microfluidic device at low Reynolds number flow conditions. In the experiments, we exploit the non-inertial lift effect to sort circulating MV3-melanoma cells from red blood cell suspensions at different hematocrits as high as 9%. We analyze the separation process and the influence of hematocrit and volume flow rates. We achieve sorting efficiencies for MV3-cells up to EMV3 = 100% at Hct = 9% and demonstrate cell viability by recultivation of the sorted cells.  相似文献   

7.
This study reports a robust method of gene transfection in a murine primary cell model by using a high-density electrodes network (HDEN). By demonstrating high cell viability after gene transfection and successful expression of transgenes including fluorescent proteins, the HDEN device shows great promise as a solution in which reprogramming efficiency using non-viral induction for generation of murine induced pluripotent stem cells (iPSCs) is optimized. High and steady transgene expression levels in host cells of iPSCs can be demonstrated using this method. Moreover, the HDEN device achieved successful gene transfection with a low voltage of less than 180 V while requiring relatively low cell numbers (less than 1.5 × 104 cells). The results are comparable to current conventional methods, demonstrating a reasonable fluorescent-plasmid transfection rate (42.4% in single transfection and 24.5% in triple transfection) and high cell viability of over 95%. The gene expression levels of each iPSC factor was measured to be over 10-fold higher than that reported in previous studies using a single mouse embryonic fibroblast cell. Our results demonstrate that the generation of iPSCs using HDEN transfection of plasmid DNA may be a feasible and safe alternative to using viral transfection methods in the near future.  相似文献   

8.
We have used Brownian dynamics-finite element method (BD-FEM) to guide the optimization of a microfluidic device designed to stretch DNA for gene mapping. The original design was proposed in our previous study [C. C. Hsieh and T. H. Lin, Biomicrofluidics 5(4), 044106 (2011)] for demonstrating a new pre-conditioning strategy to facilitate DNA stretching through a microcontraction using electrophoresis. In this study, we examine the efficiency of the original device for stretching DNA with different sizes ranging from 48.5 kbp (λ-DNA) to 166 kbp (T4-DNA). The efficiency of the device is found to deteriorate with increasing DNA molecular weight. The cause of the efficiency loss is determined by BD-FEM, and a modified design is proposed by drawing an analogy between an electric field and a potential flow. The modified device does not only regain the efficiency for stretching large DNA but also outperforms the original device for stretching small DNA.  相似文献   

9.
Inertial microfluidics is an emerging class of technologies developed to separate circulating tumor cells (CTCs). However, defining design parameters and flow conditions for optimal operation remains nondeterministic due to incomplete understanding of the mechanics, which has led to challenges in designing efficient systems. Here, we perform a parametric study of the inertial focusing effects observed in low aspect ratio curvilinear microchannels and utilize the results to demonstrate the isolation of CTCs with high purity. First, we systematically vary parameters including the channel height, width, and radius of curvature over a wide range of flow velocities to analyze its effect on size dependent differential focusing and migration behaviors of binary (10 μm and 20 μm) particles. Second, we use these results to identify optimal flow regimes to achieve maximum separation in various channel configurations and establish design guidelines to readily provide information for developing spiral channels tailored to potentially arbitrary flow conditions that yield a desired equilibrium position for optimal size based CTC separation. Finally, we describe a fully integrated, sheath-less cascaded spiral microfluidic device to continuously isolate CTCs. Human breast cancer epithelial cells were successfully extracted from leukocytes, achieving 86.76% recovery, 97.91% depletion rate, and sustaining high viability upon collection to demonstrate the versatility of the device. Importantly, this device was designed without the cumbersome trail-and-error optimization process that has hindered the development of designing such inertial microfluidic systems.  相似文献   

10.
The majority of available cardiomyocyte markers are intercellular proteins, limiting our ability to enrich live cardiomyocytes from heterogeneous cell preparations in the absence of genetic labeling. Here, we describe enrichment of live cardiomyocytes from the hearts of adult mice in a label-free microfluidic approach. The separation device consisted of a vertical column (15 mm long, 700 μm diameter), placed between permanent magnets resulting in a field strength of 1.23 T. To concentrate the field at the column wall, the column was wrapped with 69 μm diameter nickel wire. Before passing the cells through the column, the cardiomyocytes in the cell suspension had been rendered paramagnetic by treatment of the adult mouse heart cell preparation with sodium nitrite (2.5 mM) for 20 min on ice. The cell suspension was loaded into the vertical column from the top and upon settling, the non-myocytes were removed by the upward flow from the column. The cardiomyocytes were then collected from the column by applying a higher flow rate (144 μl/min). We found that by applying a separation flow rate of 4.2 μl/min in the first step, we can enrich live adult cardiomyocytes to 93% ± 2% in a label-free manner. The cardiomyocytes maintained viability immediately after separation and upon 24 h in culture.  相似文献   

11.
A novel microfluidic device which consists of two stages for particle focusing and separation using a viscoelastic fluid has been developed. A circular capillary tube was used for three-dimensional particle pre-alignment before the separation process, which was inserted in a polydimethylsiloxane microchannel. Particles with diameters of 5 and 10 μm were focused at the centerline in the capillary tube, and the location of particles was initialized at the first bifurcation. Then, 5 and 10 μm particles were successfully separated in the expansion region based on size-dependent lateral migration, with ∼99% separation efficiency. The proposed device was further applied to separation of MCF-7 cells from leukocytes. Based on the cell size distribution, an approximate size cutoff for separation was determined to be 16 μm. At 200 μl/min, 94% of MCF-7 cells were separated with the purity of ∼97%. According to the trypan blue exclusion assay, high viability (∼90%) could be achieved for the separated MCF-7 cells. The use of a commercially available capillary tube enables the device to be highly versatile in dealing with particles in a wide size range by using capillary tubes with different inner diameters.  相似文献   

12.
Label-free isolation of single cells is essential for the growing field of single-cell analysis. Here, we present a device which prints single living cells encapsulated in free-flying picoliter droplets. It combines inkjet printing and impedance flow cytometry. Droplet volume can be controlled in the range of 500 pl–800 pl by piezo actuator displacement. Two sets of parallel facing electrodes in a 50 μm × 55 μm channel are applied to measure the presence and velocity of a single cell in real-time. Polystyrene beads with <5% variation in diameter generated signal variations of 12%–17% coefficients of variation. Single bead efficiency (i.e., printing events with single beads vs. total number of printing events) was 73% ± 11% at a throughput of approximately 9 events/min. Viability of printed HeLa cells and human primary fibroblasts was demonstrated by culturing cells for at least eight days.  相似文献   

13.
Flow-through gold film perforated with periodically arrayed sub-wavelength nano-holes can cause extraordinary optical transmission (EOT), which has recently emerged as a label-free surface plasmon resonance sensor in biochemical detection by measuring the transmission spectral shift. This paper describes a systematic study of the effect of microfluidic field on the spectrum of EOT associated with the porous gold film. To detect biochemical molecules, the sub-micron-thick film is free-standing in a microfluidic field and thus subject to hydrodynamic deformation. The film deformation alone may cause spectral shift as measurement error, which is coupled with the spectral shift as real signal associated with the molecules. However, this microfluid-induced measurement error has long been overlooked in the field and needs to be identified in order to improve the measurement accuracy. Therefore, we have conducted simulation and analytic analysis to investigate how the microfluidic flow rate affects the EOT spectrum and verified the effect through experiment with a sandwiched device combining Au/Cr/Si3N4 nano-hole film and polydimethylsiloxane microchannels. We found significant spectral blue shift associated with even small flow rates, for example, 12.60 nm for 4.2 μl/min. This measurement error corresponds to 90 times the optical resolution of the current state-of-the-art commercially available spectrometer or 8400 times the limit of detection. This really severe measurement error suggests that we should pay attention to the microfluidic parameter setting for EOT-based flow-through nano-hole sensors and adopt right scheme to improve the measurement accuracy.  相似文献   

14.
Passive particle focusing based on inertial microfluidics was recently introduced as a high-throughput alternative to active focusing methods that require an external force field to manipulate particles. In inertial microfluidics, dominant inertial forces cause particles to move across streamlines and occupy equilibrium positions along the faces of walls in flows through straight micro channels. In this study, we systematically analyzed the addition of secondary Dean forces by introducing curvature and show how randomly distributed particles entering a simple u-shaped curved channel are focused to a fixed lateral position exiting the curvature. We found the lateral particle focusing position to be fixed and largely independent of radius of curvature and whether particles entering the curvature are pre-focused (at equilibrium) or randomly distributed. Unlike focusing in straight channels, where focusing typically is limited to channel cross-sections in the range of particle size to create single focusing point, we report here particle focusing in a large cross-section area (channel aspect ratio 1:10). Furthermore, we describe a simple u-shaped curved channel, with single inlet and four outlets, for filtration applications. We demonstrate continuous focusing and filtration of 10 μm particles (with >90% filtration efficiency) from a suspension mixture at throughputs several orders of magnitude higher than flow through straight channels (volume flow rate of 4.25 ml/min). Finally, as an example of high throughput cell processing application, white blood cells were continuously processed with a filtration efficiency of 78% with maintained high viability. We expect the study will aid in the fundamental understanding of flow through curved channels and open the door for the development of a whole set of bio-analytical applications.  相似文献   

15.
A variety of methods have been used to introduce chemicals into a stream or to mix two or more streams of different compositions using microfluidic devices. In the following paper, the introduction of cryoprotective agents (CPAs) used during cryopreservation of cells in order to protect them from freezing injuries and increase viability post thaw is described. Dimethylsulphoxide (DMSO) is the most commonly used CPA. We aim to optimize the operating conditions of a two-stream microfluidic device to introduce a 10% vol/vol solution of DMSO into a cell suspension. Transport behavior of DMSO between two streams in the device has been experimentally characterized for a spectrum of flow conditions (0.7 < Re < 10), varying initial donor stream concentrations, (1% vol/vol < Co < 15% vol/vol) and different flow rate fractions (0.23 < fq < 0.77). The outlet cell stream concentration is analyzed for two different flow configurations: one with the cell stream flowing on top of the DMSO-rich donor stream, and the other with the cell stream flowing beneath the heavy DMSO-laden stream. We establish a transition from a diffusive mode of mass transfer to gravity-influenced convective currents for Atwood numbers (At) in the range of (1.7 × 10−3 < At < 3.1 × 10−3) for the latter configuration. Flow visualization with cells further our understanding of the effect of At on the nature of mass transport. Cell motion studies performed with Jurkat cells confirm a high cell recovery from the device while underscoring the need to collect both the streams at the outlet of the device and suggesting flow conditions that will help us achieve the target DMSO outlet concentration for clinical scale flow rates of the cell suspension.  相似文献   

16.
Single cell trapping increasingly serves as a key manipulation technique in single cell analysis for many cutting-edge cell studies. Due to their inherent advantages, microfluidic devices have been widely used to enable single cell immobilization. To further improve the single cell trapping efficiency, this paper reports on a passive hydrodynamic microfluidic device based on the “least flow resistance path” principle with geometry optimized in line with corresponding cell types. Different from serpentine structure, the core trapping structure of the micro-device consists of a series of concatenated T and inverse T junction pairs which function as bypassing channels and trapping constrictions. This new device enhances the single cell trapping efficiency from three aspects: (1) there is no need to deploy very long or complicated channels to adjust flow resistance, thus saving space for each trapping unit; (2) the trapping works in a “deterministic” manner, thus saving a great deal of cell samples; and (3) the compact configuration allows shorter flowing path of cells in multiple channels, thus increasing the speed and throughput of cell trapping. The mathematical model of the design was proposed and optimization of associated key geometric parameters was conducted based on computational fluid dynamics (CFD) simulation. As a proof demonstration, two types of PDMS microfluidic devices were fabricated to trap HeLa and HEK-293T cells with relatively significant differences in cell sizes. Experimental results showed 100% cell trapping and 90% single cell trapping over 4 × 100 trap sites for these two cell types, respectively. The space saving is estimated to be 2-fold and the cell trapping speed enhancement to be 3-fold compared to previously reported devices. This device can be used for trapping various types of cells and expanded to trap cells in the order of tens of thousands on 1-cm2 scale area, as a promising tool to pattern large-scale single cells on specific substrates and facilitate on-chip cellular assay at the single cell level.  相似文献   

17.
Separation and sorting of biological entities (viruses, bacteria, and cells) is a critical step in any microfluidic lab-on-a-chip device. Acoustofluidics platforms have demonstrated their ability to use physical characteristics of cells to perform label-free separation. Bandpass-type sorting methods of medium-sized entities from a mixture have been presented using acoustic techniques; however, they require multiple transducers, lack support for various target populations, can be sensitive to flow variations, or have not been verified for continuous flow sorting of biological cells. To our knowledge, this paper presents the first acoustic bandpass method that overcomes all these limitations and presents an inherently reconfigurable technique with a single transducer pair for stable continuous flow sorting of blood cells. The sorting method is first demonstrated for polystyrene particles of sizes 6, 10, and 14.5 μm in diameter with measured purity and efficiency coefficients above 75 ± 6% and 85 ± 9%, respectively. The sorting strategy was further validated in the separation of red blood cells from white blood cells and 1 μm polystyrene particles with 78 ± 8% efficiency and 74 ± 6% purity, respectively, at a flow rate of at least 1 μl/min, enabling to process finger prick blood samples within minutes.  相似文献   

18.
The conventional microfluidic H filter is modified with multi-insulating blocks to achieve a flow-through manipulation and separation of microparticles. The device transports particles by exploiting electro-osmosis and electrophoresis, and manipulates particles by utilizing dielectrophoresis (DEP). Polydimethylsiloxane (PDMS) blocks fabricated in the main channel of the PDMS H filter induce a nonuniform electric field, which exerts a negative DEP force on the particles. The use of multi-insulating blocks not only enhances the DEP force generated, but it also increases the controllability of the motion of the particles, facilitating their manipulation and separation. Experiments were conducted to demonstrate the controlled flow direction of particles by adjusting the applied voltages and the separation of particles by size under two different input conditions, namely (i) a dc electric field mode and (ii) a combined ac and dc field mode. Numerical simulations elucidate the electrokinetic and hydrodynamic forces acting on a particle, with theoretically predicted particle trajectories in good agreement with those observed experimentally. In addition, the flow field was obtained experimentally with fluorescent tracer particles using the microparticle image velocimetry (μ-PIV) technique.  相似文献   

19.
Blood cell sorting is critical to sample preparation for both clinical diagnosis and therapeutic research. The spiral inertial microfluidic devices can achieve label-free, continuous separation of cell mixtures with high throughput and efficiency. The devices utilize hydrodynamic forces acting on cells within laminar flow, coupled with rotational Dean drag due to curvilinear microchannel geometry. Here, we report on optimized Archimedean spiral devices to achieve cell separation in less than 8 cm of downstream focusing length. These improved devices are small in size (<1 in.2), exhibit high separation efficiency (∼95%), and high throughput with rates up to 1 × 106 cells per minute. These device concepts offer a path towards possible development of a lab-on-chip for point-of-care blood analysis with high efficiency, low cost, and reduced analysis time.  相似文献   

20.
Lewpiriyawong N  Yang C 《Biomicrofluidics》2012,6(1):12807-128079
The recent development of microfluidic “lab on a chip” devices requires the need to continuously separate submicron particles. Here, we present a PDMS microfluidic device with sidewall conducting PDMS (AgPDMS) composite electrodes capable of separating submicron particles in hydrodynamic flow. In particular, the device can service dual functions. First, the AgPDMS composite electrodes embedded in a sidewall of the device channel allow for performing AC-dielectrophoretic (DEP) characterization through direct microscopic observation of particle behavior. Characterization experiments are carried out for numerous parameters including particle size, medium conductivity, and AC field frequency to reveal important dielectrophoresis DEP information in terms of the crossover frequency and positive/negative DEP behavior under specific frequencies. Second, the device offers an advantage that sidewall AgPDMS composite electrodes can produce strong DEP effects throughout the entire channel height, and thus the robustness of the on-chip particle separation is demonstrated for continuous separation in a flowing mixture of 0.5 and 5 μm particles with 100% separation efficiency.  相似文献   

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