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1.
Electroporation is commonly used in biotechnology to introduce macromolecules into cells. We have developed a micro-electroporation chip that incorporates a live biological cell in the electrical circuit. The chip configuration forces electrical currents to pass through the cell, thereby producing electrically measurable information about the electroporation state of the cell. The cell membrane electrical properties make the cell function as a diode in the electroporation current-voltage range. The chip is transparent in the area of the cell to allow microscope viewing. during electroporation. This chip may be used to study the fundamental biophysics of cell electroporation and in biotechnology for controlled macromolecule introduction in individual cells. We describe the chip principle and show results on the electrical current-voltage pattern during reversible and irreversible electroporation in individual cells. 相似文献
2.
We present a hybrid chip of polymer and stainless steel designed for high-throughput continuous electroporation of cells in
suspension. The chip is constructed with two parallel stainless steel mesh electrodes oriented perpendicular to the liquid
flow. The relatively high hydrodynamic resistance of the micrometer sized holes in the meshes compared to the main channel
enforces an almost homogeneous flow velocity between the meshes. Thereby, very uniform electroporation of the cells can be
accomplished. Successful electroporation of 20 million human dendritic cells with mRNA is demonstrated. The performance of
the chip is similar to that of the traditional electroporation cuvette, but without an upper limit on the number of cells
to be electroporated. The device is constructed with two female Luer parts and can easily be integrated with other microfluidic
components. Furthermore it is fabricated from injection molded polymer parts and commercially available stainless steel mesh,
making it suitable for inexpensive mass production. 相似文献
3.
《中国生物医学工程学报(英文版)》2019,(1)
In this study,a unique rapid processing technology for microfluidic chips made of polymethylmethacrylate(PMMA) was realized.The common laser engraving machine is used to etch the chip reaction unit and microchannel,and the printing chip is what you see is what you get;the pressurized heat sealing technology is used to quickly complete the chip sealing and packaging;the metal electrodes and lines that need to be laid are embedded into the chip by hot melting;the contact points on the fixed base of the contact chip card are used to connect with the external equipment,so as to make the bearing sample react,detect and complex lines and chips.It is self-contained and isolated from the external equipment circuit.The chip becomes disposable and can be replaced quickly and conveniently.The chip processed by this technology has rapid,cheap and convenient improvement and innovation,which makes the production of microfluidic chip easier and more practical,and even makes the chip a disposable consumable with general interface,which will greatly promote the industrialization of microfluidic chip,and has market-oriented promotion value in the field of water quality and food detection. 相似文献
4.
Traditional macro and micro-electroporation devices utilize facing electrodes, which generate electric fields inversely proportional
to their separation distance. Although the separation distances in micro-electroporation devices are significantly smaller
than those in macro-electroporation devices, they are limited by cell size. Because of this, significant potential differences
are required to induce electroporation. These potential differences are often large enough to cause water electrolysis, resulting
in electrode depletion and bubble formation, both of which adversely affect the electroporation process. Here, we present
a theoretical study of a novel micro-electroporation channel composed of an electrolyte flowing over a series of adjacent
electrodes separated by infinitesimally small insulators. Application of a small, non-electrolysis inducing potential difference
between the adjacent electrodes results in radially-varying electric fields that emanate from these insulators, causing cells
flowing through the channel to experience a pulsed electric field. This eliminates the need for a pulse generator, making
a minimal power source (such as a battery) the only electrical equipment that is needed. A non-dimensional primary current
distribution model of the novel micro-electroporation channel shows that decreasing the channel height results in an exponential
increase in the electric field magnitude, and that cells experience exponentially greater electric field magnitudes the closer
they are to the channel walls. Finally, dimensional primary current distribution models of two potential applications, water
sterilization and cell transfection, demonstrate the practical feasibility of the novel micro-electroporation channel. 相似文献
5.
Hadi Shafiee John L. Caldwell Michael B. Sano Rafael V. Davalos 《Biomedical microdevices》2009,11(5):997-1006
Dielectrophoresis (DEP) has become a promising technique to separate and identify cells and microparticles suspended in a
medium based on their size or electrical properties. Presented herein is a new technique to provide the non-uniform electric
field required for DEP that does not require electrodes to contact the sample fluid. In our method, electrodes are capacitively-coupled
to a fluidic channel through dielectric barriers; the application of a high-frequency electric field to these electrodes then
induces an electric field in the channel. This technique combines the cell manipulation abilities of traditional DEP with
the ease of fabrication found in insulator-based technologies. A microfluidic device was fabricated based on this principle
to determine the feasibility of cell manipulations through contactless DEP (cDEP). We were able to demonstrate cell responses
unique to the DEP effect in three separate cell lines. These results illustrate the potential for this technique to identify
cells through their electrical properties without fear of contamination from electrodes. 相似文献
6.
Single cell electroporation is one of the nonviral method which successfully allows transfection of exogenous macromolecules into individual living cell. We present localized cell membrane electroporation at single-cell level by using indium tin oxide (ITO) based transparent micro-electrodes chip with inverted microscope. A focused ion beam (FIB) technique has been successfully deployed to fabricate transparent ITO micro-electrodes with submicron gaps, which can generate more intense electric field to produce very localized cell membrane electroporation. In our approach, we have successfully achieved 0.93?μm or smaller electroporation region on the cell surface to inject PI (Propidium Iodide) dye into the cell with 60?% cell viability. This experiments successfully demonstrate the cell self-recover process from the injected PI dye intensity variation. Our localized cell membrane electroporation technique (LSCMEP) not only generates reversible electroporation process but also it provides a clear optical path for potentially monitoring/tracking of drugs to deliver in single cell level. 相似文献
7.
The formation of an electric double layer and electroosmosis are important theoretic foundations associated with microfluidic systems. Field-modulated electroosmotic flows in microchannels can be obtained by applying modulating electric fields in a direction perpendicular to a channel wall. This paper presents a systematic numerical analysis of modulated electroosmotic flows in a microchannel with discrete electrodes on the basis of the Poisson equation of electric fields in a liquid–solid coupled domain, the Navier–Stokes equation of liquid flow, and the Nernst-Planck equation of ion transport. These equations are nonlinearly coupled and are simultaneously solved numerically for the electroosmotic flow velocity, electric potential, and ion concentrations in the microchannel. A number of numerical examples of modulated electroosmotic flows in microchannels with discrete electrodes are presented, including single electrodes, symmetric/asymmetric double electrodes, and triple electrodes. Numerical results indicate that chaotic circulation flows, micro-vortices, and effective fluid mixing can be realized in microchannels by applying modulating electric fields with various electrode configurations. The interaction of a modulating field with an applied field along the channel is also discussed. 相似文献
8.
细胞迁移是指细胞朝着特定的化学浓度梯度发生定向迁移运动,其在胚胎发育、伤口愈合、肿瘤转移中发挥着至关重要的作用。当前研究手段大多通量低,难以综合考虑不同浓度梯度条件对细胞迁移行为的影响。针对上述问题,本文首先设计了一款四通道微流控芯片,其特征如下:借助层流和扩散机制在细胞迁移主通道中建立和维持浓度梯度;可在单一显微镜视野下同时观测四组细胞迁移现象;集成了宽度为20μm的细胞隔离带,可校准细胞初始位置,保证实验结果的准确性。随后,借助Comsol Multiphysics有限元分析软件完成了微流控芯片的仿真分析,证明了芯片上设计S型微通道和水平压力平衡通道有助于在细胞迁移主通道中形成稳定的浓度梯度。最后,采用不同浓度(0、0.2、0.5、1.0μmol·L-1)与糖尿病及其并发症密切相关的晚期糖基化终末产物(AGEs)孵育中性粒细胞,研究了其在100 nmol·L-1趋化因子fMLP浓度梯度环境中的迁移行为。结果表明,AGEs抑制了中性粒细胞的迁移能力,证明了四通道微流控芯片的可靠性和实用性。 相似文献
9.
Micro and nano fabrication techniques have facilitated the production of new devices for manipulation of single cells on a
chip, such as the planar micro-pore electroporation technology. To characterize this technology we have studied the seal that
forms at the interface between an individual cell and the micro-pore, in which the cell normally resides, as a function of
an electrical field applied across the cell and temperature. Mathematical analysis of non-electroporative electrical fields
in experiments with Madin-Darby canine kidney (MDCK) cells suggests that nanoscale channels form between the exterior of the
cell and the pore wall. The results indicate that the electrical currents through these channels need to be considered when
using planar micro-pores in general and performing micro-pore electroporation in particular. Our results show that the size
of these channels is strongly temperature dependent and the cell to pore wall distance can increase by as much as 60% when
the temperature of the system is lowered from 35 to 0∘C. Temperature appears to be an important factor in the use of devices for cells on a chip and our results suggest that physiological
temperatures should yield better seal formation, thus improved feedback sensitivity, than the traditional use of room temperature
in planar micro-pore electroporation devices. 相似文献
10.
A probe array with nano-scale tips, integrated into a micro-fluidic channel was developed for the capture and lysing of small
number of vaccinia virus particles using dielectrophoresis. The nano-scale probe array was fabricated in Silicon on Insulator
(SOI) wafers, and sharpened with repeated oxidation steps. The gap between each probe ranged from 100 nm to 1.5 μm depending
on fabrication parameters. The probe array was used to capture vaccinia virus using positive dielectrophoresis (DEP) from
a flow within the microfluidic channel, and then the same probe array was used to apply high electric field to lyse the virus
particles. It was shown that under electric field strengths of about 107 V/m, the permeability of ethidium bromide into the vaccinia virus particles was increased. Upon SEM analysis, the particles
were found to be damaged and exhibited tubules networks, indicating disintegration of the virus outer layer. In addition,
elongated strands of DNA were clearly observed on the chip surface after the application of the high electric field, demonstrating
the possibility of electrical lysis of virus particles. 相似文献
11.
H. W. Hou Q. S. Li G. Y. H. Lee A. P. Kumar C. N. Ong C. T. Lim 《Biomedical microdevices》2009,11(3):557-564
Cell deformability is an important biomarker which can be used to distinguish between healthy and diseased cells. In this
study, microfluidics is used to probe the biorheological behaviour of breast cancer cells in an attempt to develop a method
to distinguish between non-malignant and malignant cells. A microfabricated fluidic channel design consisting of a straight
channel and two reservoirs was used to study the biorheological behaviour of benign breast epithelial cells (MCF-10A) and
non-metastatic tumor breast cells (MCF-7). Quantitative parameters such as entry time (time taken for the cell to squeeze
into the microchannel) and transit velocity (speed of the cell flowing through the microchannel) were defined and measured
from these studies. Our results demonstrated that a simple microfluidic device can be used to distinguish the difference in
stiffness between benign and cancerous breast cells. This work lays the foundation for the development of potential microfluidic
devices which can subsequently be used in the detection of cancer cells.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
Electroendocytosis (EED), i.e. electric field-induced endocytosis, is a technique for bio-molecule and drug delivery to cells using a pulsed electric field
lower than that applied in electroporation (EP). Different from EP in which nanometer-sized electropores appear on the plasma
membrane lipid bilayer, EED induces cell membrane internalization and fission via endocytotic vesicles. In this study, we
conduct comprehensive experimental study on the EED of HeLa cells using a micro chip and the corresponding endocytotic vesicles
were visualized and investigated by using FM4-64 fluorescent dye and in situ fluorescence microscopy. The uptake of molecules by the EED of cells was characterized by average intracellular fluorescent
intensity from a large number (>2,000) of single cells. The EED efficiency was determined as a function of three electric
parameters (electric field strength, pulse duration, total electric treatment time). The EED efficiency as a function of electric
field strength clearly shows biphasic characteristics at different experimental conditions. The EED experiments using cytoskeleton
inhibitors illustrate unique mechanisms distinct from EP. This study provides a foundation for further on-chip study of the
time-dependent mechanism of EED at the single-cell level. 相似文献
13.
Monocytes play an important role in the immune system and are responsible for phagocytizing and degrading foreign microorganisms in the body. The isolation of monocytes is important in various immunological applications such as in-vitro culture of dendritic cells. We present a magnetophoretic-based microfluidic chip for rapid isolation of highly purified, untouched monocytes from human blood by a negative selection method. This bioseparation platform integrates several unique features into a microfluidic device, including locally engineered magnetic field gradients and a continuous flow with a buffer switching scheme to improve the performance of the cell separation process. The results indicate high monocyte purity and recovery performances at a volumetric flow rate that is nearly an order of magnitude larger than comparable microfluidic devices reported in literature. In addition, a comprehensive 2-D computational modeling is performed to determine the cell trajectory and trapping length within the microfluidic chip. Furthermore, the effects of channel height, substrate thickness, cell size, number of beads per cell, and sample flow rate on the cell separation performance are studied. 相似文献
14.
15.
Micro and nano technologies are of increasing importance in microfluidics devices used for electroporation (electroporation – the permeabilization of the cell membrane with brief high electric field pulses). Electrochemical reactions of electrolysis occur whenever an electric current flows between an electrode and an ionic solution. It can have substantial detrimental effects, both on the cells and solutions during the electroporation. As electrolysis is a surface phenomenon, between electrodes and solution, the extent of electrolysis is increased in micro and nano electroporation over macro-electroporation, because the surface area of the electrodes in micro and nano electroporation is much larger. A possible way to eliminate the electrolytic effect is to develop non-electrolytic microelectroporation by coating the microelectroporation devices with a dielectric insulating layer. In this study, we examine the effect of a dielectric insulating layer on the performance of a singularity microelectroporation device that we have recently designed. Using numerical analysis, we study the effects of various design parameters including, input sinusoidal voltage amplitude and frequency, geometrical configuration and material electrical properties on the electroporation performance of the non-electrolytic microelectroporation device. In the simulation, we used properties of four real dielectric materials and four solutions of interest for microelectroporation. We characterized the effect of various design parameters of relevance to singularity based microelectroporation, on non-electrolytic microelectroporation. Interestingly, we found that the system behaves in some aspects as a filter and in many circumstances saturation of performance is reached. After saturation is reached, changes in parameters will not affect the performance of the device. 相似文献
16.
A microfabricated cell-based testing device for electrochemotherapy (ECT) has been developed by miniaturizing the widely used
clinical electroporator with a two-needle array into two-dimensional planar electrodes while keeping the similarity of the
electric field strength distribution. In this device, all the biological processes from cell culture to electroporation and
final cell-based assays were carried out on a chip using a conventional 2D cell culture method, and the multiple electrochemotherapeutic
assays could be realized by exploiting the six electroporation sites in a single device. With the proposed platform, the electroporation
rate was evaluated with propidium iodide and cell proliferation after 48 h of electrochemotherapy with bleomycin was determined
with T47D human breast ductal carcinoma cell line in various electric field strengths and drug concentrations. This microsystem
has several advantages over conventional cuvette type electroporation assay, such as multiple assays on a chip, on-chip based
operation from cell culture to final assay, and having similar electric field distribution as that of the clinical electroporator.
As the clinical trials of electrochemotherapy are being carried out, this new platform is expected to have valuable applications
in basic in vitro ECT studies, drug discovery, and development of clinical ECT equipment. 相似文献
17.
This paper presents a novel microfluidic DNA digestion system incorporating a high performance micro-mixer. Through the appropriate
control of fixed and periodic switching DC electric fields, electrokinetic forces are established to mix the DNA and restriction
enzyme samples and to drive them through the reaction column of the device. The experimental and numerical results show that
a mixing performance of 98% can be achieved within a mixing channel of length 1.6 mm when a 150 V/cm driving voltage and a
5 Hz switching frequency are applied. The relationship between the mixing performance, switching frequency, and main applied
electric field is derived. It is found that the optimal switching frequency depends upon the magnitude of the main applied
electric field. The successful digestion of λ-DNA using Eco RI restriction enzyme is demonstrated. The DNA-enzyme reaction
is completed within 15 min in the proposed microfluidic system, compared to 50 min in a conventional large-scale system. Hence,
the current device provides a valuable tool for rapid λ-DNA digestion, while its mixer system delivers a simple yet effective
solution for mixing problems in the micro-total-analysis-systems field. 相似文献
18.
Flow-orthogonal bead oscillation in a microfluidic chip with a magnetic anisotropic flux-guide array
A new concept for the manipulation of superparamagnetic beads inside a microfluidic chip is presented in this paper. The concept
allows for bead actuation orthogonal to the flow direction inside a microchannel. Basic manipulation functionalities were
studied by means of finite element simulations and results were oval-shaped steady state oscillations with bead velocities
up to 500 μm/s. The width of the trajectory could be controlled by prescribing external field rotation. Successful verification
experiments were performed on a prototype chip fabricated with excimer laser ablation in polycarbonate and electroforming
of nickel flux-guides. Bead velocities up to 450 μm/s were measured in a 75 μm wide channel. By prescribing the currents in
the external quadrupole magnet, the shape of the bead trajectory could be controlled. 相似文献
19.
Dmitry A. Markov Steven Manuel Leslie M. Shor Susan R. Opalenik John P. Wikswo 《Biomedical microdevices》2010,12(1):135-144
We describe a simple and reliable fabrication method for producing multiple, manually activated microfluidic control valves
in polydimethylsiloxane (PDMS) devices. These screwdriver-actuated valves reside directly on the microfluidic chip and can
provide both simple on/off operation as well as graded control of fluid flow. The fabrication procedure can be easily implemented
in any soft lithography lab and requires only two specialized tools—a hot-glue gun and a machined brass mold. To facilitate
use in multi-valve fluidic systems, the mold is designed to produce a linear tape that contains a series of plastic rotary
nodes with small stainless steel machine screws that form individual valves which can be easily separated for applications
when only single valves are required. The tape and its valves are placed on the surface of a partially cured thin PDMS microchannel
device while the PDMS is still on the soft-lithographic master, with the master providing alignment marks for the tape. The
tape is permanently affixed to the microchannel device by pouring an over-layer of PDMS, to form a full-thickness device with
the tape as an enclosed underlayment. The advantages of these Tape Underlayment Rotary-Node (TURN) valves include parallel
fabrication of multiple valves, low risk of damaging a microfluidic device during valve installation, high torque, elimination
of stripped threads, the capabilities of TURN hydraulic actuators, and facile customization of TURN molds. We have utilized
these valves to control microfluidic flow, to control the onset of molecular diffusion, and to manipulate channel connectivity.
Practical applications of TURN valves include control of loading and chemokine release in chemotaxis assay devices, flow in
microfluidic bioreactors, and channel connectivity in microfluidic devices intended to study competition and predator/prey
relationships among microbes. 相似文献
20.
电穿孔技术的研究及应用进展 总被引:3,自引:0,他引:3
脉冲电场的电穿孔效应在医学上的应用是一门多学科的生物医学工程学技术,已证实其主要的生物学效应是使靶细胞膜发生可逆及不可逆性电击穿。近年电穿孔效应所介导的电化学治疗应用广泛。对电脉冲的量—效关系、电场分布、膜穿标记物及生物电阻成像等基础研究及临床应用中的进展及尚存问题作一综述。 相似文献