WO2015067221A1 - 一种流式电穿孔装置 - Google Patents

一种流式电穿孔装置 Download PDF

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Publication number
WO2015067221A1
WO2015067221A1 PCT/CN2014/090821 CN2014090821W WO2015067221A1 WO 2015067221 A1 WO2015067221 A1 WO 2015067221A1 CN 2014090821 W CN2014090821 W CN 2014090821W WO 2015067221 A1 WO2015067221 A1 WO 2015067221A1
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Prior art keywords
electrode
flow electroporation
flow
electroporation
electrode array
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PCT/CN2014/090821
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English (en)
French (fr)
Inventor
戴爱德华
吴梦希
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Etta Biotech Co Ltd
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Etta Biotech Co Ltd
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Priority to EP14859786.7A priority Critical patent/EP3070158A4/en
Priority to US15/035,694 priority patent/US10731120B2/en
Publication of WO2015067221A1 publication Critical patent/WO2015067221A1/zh
Anticipated expiration legal-status Critical
Priority to US16/906,054 priority patent/US10982182B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to a device for electroporation of cells, and more particularly to a flow electroporation device.
  • Initially used for electroporation was the use of two parallel plate electrodes, each fixed to the two walls in the container.
  • the cell suspension ready for electroporation is mixed with molecules intended to be introduced into the cells, added to an electroporation vessel, and placed between the two electrodes.
  • one or more transient high voltage pulses are applied to the electrodes to apply a high voltage electric field pulse to the cell suspension between the electrodes.
  • the required voltage is usually as high as several hundred or even several thousand volts, which brings safety and reliability problems, and inevitably causes a cathode effect, which causes great damage to cells.
  • the planar electrode although reducing the electrode spacing, can produce the same electric field strength at a lower voltage and bring a good electroporation effect, but the amount of cells per treatment is small, completely unsuitable for high-throughput experiments. operating.
  • Electroporation instruments using stereoscopic electrodes on the market, but they are mostly used in clinical directions such as tumors or living tissues.
  • This type of electrode has a small number of electrodes, and the combination is simple.
  • flow electroporation uses a modified parallel plate electrode to continuously and stably flow a cell suspension that requires electroporation between the two electrodes until the entire cell suspension is electroporated. Thereby achieving large volume cell electroporation.
  • the flow electroporation method comprises an electroporation chamber with an electrode and a cell suspension inlet and outlet, and the cell suspension is connected to the electroporation chamber through the inlet and outlet.
  • the technical problem to be solved by the present invention is to provide a flow type electroporation device which is simple in structure and easy to manufacture and manufacture.
  • one technical solution adopted by the present invention is to provide a flow electroporation device comprising a stereo electrode device and a flow electroporation chamber, the stereo electrode device comprising an electrode array and an electrode fixing assembly, which constitutes the An electrode of the electrode array is fixed to the electrode fixing assembly, the flow electroporation chamber includes a cavity, an inlet, an outlet, and an opening, the inlet, the outlet, and the opening are in communication with the cavity, and the electrode array passes The opening is inserted into the cavity.
  • the electrode array comprises a plurality of electrodes, each electrode being an electrically conductive material of an elongated structure.
  • the plurality of electrodes in the electrode array are arranged in a regular polygon, and the distance between adjacent two electrodes in the electrode array is equal.
  • the shape of the electrode array is a regular hexagon composed of a plurality of equilateral triangles, each of which is located at an apex of the equilateral triangle.
  • the flow electroporation chamber has a circular or polygonal cross section.
  • the flow electroporation device further includes a drainage assembly that communicates with the inlet or the outlet.
  • the drainage assembly may be selected from a peristaltic pump, a rotary pump, a piston, a diaphragm pump, or a gear transmission.
  • the beneficial effects of the invention are: the flow electroporation device of the invention, which can realize high-flux cell treatment, and can work under lower voltage conditions, avoiding the damage caused by the liquid flow on the cells, reducing The effects of heat and cathodic effects caused by voltage pulses maintain a closed, sterile environment, ensuring that most cell suspensions are subjected to the optimal number of electric field pulses.
  • FIG. 1 is a schematic structural view of a flow electroporation device according to a preferred embodiment of the present invention
  • FIG. 2 is a schematic structural view of a three-dimensional electrode network in the flow electroporation device of FIG. 1;
  • FIG. 3 is a schematic diagram of electrode connection rules of a three-dimensional electrode network in the flow electroporation device of FIG. 1;
  • FIG. 4 is a schematic diagram of a power-on mode of a three-dimensional electrode network in the flow electroporation device of FIG. 1;
  • FIG. 5 is a schematic diagram of electric field distribution of a three-dimensional electrode network in the flow electroporation device of FIG. 1;
  • Figure 6 is a schematic structural view of another preferred embodiment of the flow electroporation device of the present invention.
  • Figure 7 is a graph showing the results of a HEK-293A cell photographed by a fluorescence microscope 24 hours after electroporation in the present invention
  • Electrode array The Electrode fixing assembly, 3. Cavity, 4, inlet, 5, outlet, 6, opening, 7, drainage assembly, 8, first interface, 9, The second interface.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • a flow electroporation device comprising a stereo electrode device and a flow electroporation chamber, the stereo electrode device comprising an electrode array 1 and an electrode fixing assembly 2, wherein the electrode array is fixed at the electrode Fixing component 1.
  • the flow electroporation chamber adopts a circular tubular container including a cavity 3, an inlet 4, an outlet 5, an opening 6 and a drainage assembly 7, and the inlet 4 and the outlet 5 may be a plurality of liquids in use. From at least one of the inlets 4 therein, flowing out of at least one of the outlets 5 therein.
  • the inlet 4 and the outlet 5 are each one, the inlet is at the bottom of the cavity 3, the outlet 4 is on the side of the cavity 3, and the opening 6 is in the cavity.
  • the electrode array 1 is inserted into the cavity 3 through the opening 6.
  • the flow electroporation chamber can be made of a glass tube or other biocompatible organic material, and the size of the container can be adjusted according to the shape of the electrode network.
  • the drainage assembly 7 can be located at the inlet 4 or at the outlet 5. In the embodiment, the drainage assembly 7 is in communication with the inlet 4.
  • the drainage assembly 7 can be a peristaltic pump, a rotary pump, a piston, a diaphragm pump or a gear transmission. When the flow electroporation device is in operation, the drainage assembly 7 introduces liquid into the flow electroporation chamber while applying electrical pulses to the electrode array 1 for operation.
  • the electrode array 1 is formed by a cylindrical electrode arranged in a regular manner.
  • the electrodes may be made of stainless steel or other conductive materials, and may be solid or hollow.
  • the electrode array 1 achieves the purpose of applying a pulse to the cells by an electric field generated between the electrodes, thereby achieving electroporation of the cells.
  • the overall arrangement of the electrodes in the electrode array 1 is a regular hexagon, composed of equilateral triangle elements.
  • the spacing between the electrodes and the number of electrodes can be adjusted within a certain range.
  • the electrode spacing adjustment range is 100um-10mm. The smaller the spacing, the smaller the required electroporation voltage.
  • the principle of adjusting the number of electrodes is to maintain a regular hexagon composed of a plurality of equilateral triangle elements after adjustment.
  • the shape of the electrode may be any shape such as a cylindrical shape, and the outer diameter thereof is between 0.01 mm and 2 mm.
  • the electrode fixing assembly 2 is mainly used to fix the position of the electrodes, and maintains a regular arrangement of the electrode network, and is made of a biocompatible material.
  • One preferred material is polydimethylsiloxane PDMS.
  • the material of the electrode is related to the process difficulty, fabrication cost, and biocompatibility of the device.
  • Most metal electrodes such as copper and aluminum have poor biocompatibility, and some metal materials such as gold and silver are expensive.
  • stainless steel electrodes are excellent materials for three-dimensional electrodes.
  • Stainless steel has good biocompatibility, is easy to be made into a long electrode, and can be mass-produced, and is very suitable as an electrode material for the electrode fixing assembly 1. Too large or too small a diameter of the stainless steel electrode can adversely affect the device. When the diameter is too large, the effective electric field area of electroporation is reduced, which means that the amount of cells capable of electroporation is reduced, which is disadvantageous for high-throughput cell electroporation. The diameter is too small, the electrode is easily bent, and the manufacturing cost is greatly increased. Stainless steel also has the characteristics of being easy to clean and not easily oxidized, which allows it to be reused many times without affecting its conductive properties.
  • FIG. 3 is a schematic diagram of the electrode connection of the electrode fixing assembly 1.
  • the number of electrodes constituting the electrode array can be adjusted within a certain range, and the electrode spacing and the peripheral shape and size can be changed according to the requirements of the embodiment.
  • the principle of the number of electrode arrays is such that after adjustment, a regular hexagon composed of a plurality of equilateral triangle elements is still maintained.
  • an electrode array consisting of 37 electrodes is divided into three groups, denoted by I, II, and III, and each of the three adjacent electrodes in the electrode array is numbered I, II, and III. Electrodes, electrodes belonging to the same group will be connected together.
  • FIG. 4 is a schematic diagram of a typical electrode power-on mode of the electrode fixing assembly 1.
  • the input end of the electrode network array has three electrical interfaces, which are respectively represented by I, II, and III, and the three interfaces are connected to the voltage pulse through a switch, wherein
  • a preferred embodiment of the switch is a relay or optocoupler switch controlled by a digital signal provided by an electroporation instrument, a pulse generator or a biostimulator.
  • the connection manner of the three switches is not limited to one type, and may be adjusted according to a specific embodiment. For example, one of them may be connected to the positive electrode and the other two may be connected to the negative electrode.
  • a preferred embodiment is to divide the three consecutive voltage pulses into one group and call these three voltage pulses t1, t2, t3 or phase.
  • FIG. 5(A) is the corresponding electric field analog distribution for three consecutive pulses, respectively.
  • FIG. 5(B) is three times.
  • the continuous pulsed electric field simulates the effect of the superposition of the distribution.
  • the electrode spacing is 700 ⁇ m
  • the electrode size is 300 ⁇ m
  • the number of electrodes is 37.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the second flow electroporation chamber is a cylindrical container, and the second flow electroporation chamber has a first interface 8 and a second interface 9, both of which are Can be used as a sample outlet and inlet.
  • the conditions for forming electroporation are related to the cell type, the type of molecule desired to be introduced into the cell, or the type of molecule desired to flow out of the cell.
  • the conditional interval there is an optimal processing condition called the conditional interval. Specifically, it is possible to select a more specific condition for the experiment within this conditional interval. This conditional interval covers the voltage strength, more specifically, the electric field strength applied to the cells, the pulse duration, the number of pulses, and the pulse interval.
  • the cell suspension is passed through the electroporation chamber at a constant flow rate, and the pulse interval and suspension flow rate are mutually constrained, depending on the optimal number of pulses of the cell.
  • the flow rate in the central region will be greater than the velocity near the wall of the container, which will inevitably result in the number of times the cells passing the pulsed electric field through the central region of the fluid are less than expected, and the cells near the wall of the chamber are pulsed.
  • the number of electric fields is greater than the expected value.
  • the volume parameter usually used is the total volume of the container. It can be seen that if the parameter p is larger, the calculation of the pulse interval and the suspension flow rate will be more to the cell suspension through the central region of the container.
  • the larger the parameter p the closer the number of pulses applied by the cell suspension in the central region of the container to the desired value, and the volume near the wall of the chamber is small in the total volume, the volume of cells being The number of pulses applied is less than the expected value, but the effect is negligible.
  • the cells were subjected to a variable flow electroporation experiment using the flow electroporation device.
  • Flow cytometry was performed using HEK-293A cells, and a GFP plasmid was used as a marker in the experiment. If the cells are electroporated, the GFP plasmid will enter the cell and express green fluorescent protein in the cell, which can observe green fluorescence under a fluorescence microscope. Therefore, the cell's electroporation rate can be obtained by dividing the number of cells in the fluorescence field by the total number of cells. From another perspective, the greater the fluorescence intensity of cells of the same density, the higher the efficiency of electroporation.
  • a sample containing HEK-293A cells and GFP plasmid was injected into the electroporation chamber, and the sample was flowed indoors at a prescribed flow rate, while the sample was electroporated by a power-on pulse, and the untreated sample flowed into the electroporation chamber while The processed sample is also gradually discharged.
  • the discharged treated sample was collected, transferred to a culture plate, and cultured for 24 hours, and observed with a fluorescence microscope.
  • Fig. 7 is a result of a biological experiment using the device, and shows the results of fluorescence microscopy of HEK-293A cells 24 hours after electroporation.
  • the cells express GFP plasmids which enter the cells by electroporation and emit green under a fluorescence microscope. Light.
  • the left side is the cell bright field map, which indicates the number of cells undergoing electroporation
  • the right side is the fluorescence field map, reflecting the cell electroporation.

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Abstract

本发明提供了一种流式电穿孔装置，包括立体电极装置和流式电穿孔室，所述立体电极装置包括电极阵列和用于固定电极阵列中电极的电极固定组件，所述流式电穿孔室包括腔体，以及位于腔体上的入口、出口和开口，所述电极阵列通过所述开口插入在所述腔体内。本发明的流式电穿孔装置能实现高通量的细胞处理，又能在较低电压条件下工作，避免液体流动带来的影响对细胞的伤害，减少电压脉冲引起的热量和阴极效应带来的影响，能够保持封闭的无菌环境。

Description

一种流式电穿孔装置
技术领域
本发明涉及一种用于细胞电穿孔的装置，特别是涉及一种流式电穿孔装置。
背景技术
自从二十世纪七十年代早期开始，电穿孔就被应用于将分子插入动物或植物细胞内。研究者证实，当细胞暴露于瞬时高压脉冲电场中时，由高压电场造成的细胞膜局部破裂会使细胞膜通透性升高，从而在细胞膜表面会形成通道，这些通道被称为电孔洞(electropore)。这些通道存在时间虽然短暂，但足以满足大分子物质如蛋白质或质粒DNA的进入或是流出。虽然细胞能够耐受在高电压下这些通道的形成，但是如果高压脉冲电压过高，或者电场持续时间过长，或者高压脉冲电场次数过多，在形成这些通道的同时，也会使细胞致死。
初期用于电穿孔的是用两个平行板电极，分别固定在容器内的两个壁上。将准备进行电穿孔的细胞悬液与希望导入到细胞内的分子混合，加入到电穿孔容器内，将其置于两个电极之间。为了提高细胞电穿孔的效果，在电极上施加一次或多次瞬时高电压脉冲，从而对电极之间的细胞悬液施以高压电场脉冲。然而由于平行板电极的间距较大，所需要的电压通常高达几百甚至数千伏，带来安全性及可靠性的问题，而且不可避免的会产生阴极效应，对细胞产生巨大伤害。后来出现的平面电极，虽然减小了电极间距，可以在较低电压下产生同等的电场强度并带来不错的电穿孔效果，但每次处理的细胞量小，完全不适合高通量的实验操作。
市场上也有采用立体式电极的电穿孔仪器，但多用于肿瘤或活体组织等临床方向。该类型的电极数量少，组合简单，有些甚至是使用两根针状作为立体式电极，符合活体组织的需要，容易刺入组织和活体，但难以使用到体外的细胞电穿孔，如悬浮细胞或贴别细胞的实验操作中。
目前市场上最常见的电穿孔容器的容积小，期间需要多次重复操作，虽然这种重复在容器内加样进行电穿孔方法简单方便，但只能满足研究者进行小规模细胞电穿孔的要求，不适合高通量的细胞电穿孔。此方法是不可能保持无菌的，而且无法满足大体积的细胞电穿孔，重复加样也会拖长实际操作的时间，这些问题都不利于实验的完成。
在20世纪80年代，有研究者开始研究用于处理大体积细胞的流式电穿孔实验方法。一般来说，流式电穿孔采用的是改装后的平行板电极，将需要进行电穿孔的细胞悬液持续而稳定地流过两个电极之间，直到整个细胞悬液均进行了电穿孔，从而实现大体积的细胞电穿孔。当细胞悬液稳定流过两个电极之间时，细胞将会暴露在高电场脉冲中，脉冲持续提供且间隔固定。该流式电穿孔方法包含带电极和细胞悬液出入口的电穿孔室，细胞悬液通过出入口与电穿孔室连接。但根据流体力学规律，流体通过平行板之间的通道时，通道中间与四周的流体有一定的速度差异，通道中间的流速要大于通道周围的流速，通道尺度越小，流速越大，这种效应也越明显。流体的这种效应会引入剪切力，会对细胞造成损伤，也是不利于电穿孔的实验流程。从实验通量来讲，希望平行板之间的体积越大越好，这可以通过增大电极间距实现；从施加的电压来讲，希望平行板之间的间距越小越好，可以降低脉冲电压，减小阴极效应；从流体力学的规律来讲，又不希望间距过小，需要将流体的剪切力小于足以损伤细胞的量级。因此用平行板来设计流式电穿孔室虽然比较简单的，但也会有诸多限制。
发明内容
本发明主要解决的技术问题是提供一种流式电穿孔装置，该装置结构简单、易于加工制造。
为解决上述技术问题，本发明采用的一个技术方案是提供一种流式电穿孔装置，包括立体电极装置和流式电穿孔室，所述立体电极装置包括电极阵列和电极固定组件，组成所述电极阵列的电极固定于所述电极固定组件，所述流式电穿孔室包括腔体、入口、出口和开口，所述入口、所述出口和所述开口与腔体相通，所述电极阵列通过所述开口插入所述腔体内。
在本发明的一个较佳实施例中，所述电极阵列包括多个电极，各个电极为细长结构的导电材料。
在本发明的一个较佳实施例中，所述电极阵列中的多个电极按正多边形排列，所述电极阵列中相邻的两个电极之间的距离相等。
在本发明的一个较佳实施例中，所述电极阵列的形状为由多个等边三角形组成的正六边形，各电极分别位于所述等边三角形的顶点上。
在本发明的一个较佳实施例中，所述流式电穿孔室截面为圆形或多边形。
在本发明一个较佳实施例中，所述流式电穿孔装置还包括引流组件，所述引流组件与所述入口或所述出口相通。所述引流组件可以选自蠕动泵、旋转泵、活塞、隔膜泵或齿轮传动装置。
本发明的有益效果是：本发明的流式电穿孔装置，该装置可以实现高通量的细胞处理，又可以在较低电压条件下工作，避免液体流动带来的影响对细胞的伤害，减少电压脉冲引起的热量和阴极效应带来的影响，能够保持封闭的无菌环境，保证绝大部分细胞悬液都被施加了最佳次数的电场脉冲。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图，其中：
图1是本发明的流式电穿孔装置一较佳实施例的结构示意图；
图2是图1的流式电穿孔装置中立体电极网络的结构示意图；
图3是图1的流式电穿孔装置中立体电极网络的电极连接规则示意图；
图4是图1的流式电穿孔装置中立体电极网络的加电方式示意图；
图5是图1的流式电穿孔装置中立体电极网络的电场分布示意图；
图6是本发明的流式电穿孔装置中另一较佳实施例的结构示意图；
图7是本发明中HEK-293A细胞在电穿孔后24小时用荧光显微镜拍摄的结果图；
附图中各部件的标记如下：1、电极阵列，2、电极固定组件，3、腔体，4、入口，5、出口，6、开口，7、引流组件，8、第一接口，9、第二接口。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
实施例一：
请参阅图1，提供一种流式电穿孔装置，包括立体电极装置和流式电穿孔室，所述立体电极装置包括电极阵列1和电极固定组件2，所述电极阵列固定2在所述电极固定组件1上。
所述流式电穿孔室采用圆形的管状容器，包括腔体3、入口4、出口5、开口6和引流组件7，所述入口4和所述出口5可以为多个，在使用时液体从其中的至少一个所述入口4流入，从其中的至少一个所述出口5中流出。本实施例中所述入口4和所述出口5各为一个，所述入口在所述腔体3的底部，所述出口4在所述腔体3的侧面，所述开口6在所述腔体3的顶部。所述电极阵列1通过所述开口6插入在所述腔体3内。所述流式电穿孔室可以由玻璃管制成，也可以由其它生物兼容的有机材料制成，容器尺寸可以根据电极网络形状进行调整。
所述引流组件7既可以位于所述入口4处，又可以位于所述出口5处，本实施例中，所述引流组件7与所述入口4相通。所述引流组件7可以为蠕动泵、旋转泵、活塞、隔膜泵或齿轮传动装置。在所述流式电穿孔装置工作时，所述引流组件7将液体引入到所述流式电穿孔室中，同时在所述电极阵列1上施加电脉冲进行工作。
请参阅图2，所述电极阵列1由圆柱形电极按照一定规则排列而形成，这些电极可以是不锈钢材料制成，也可以由其他导电材料制成，可以是实心的，也可以是空心的。所述电极阵列1通过在电极之间产生的电场来达到对细胞加电脉冲的目的，实现对细胞的电穿孔。所述电极阵列1中的电极的整体排布为正六边形，由正三角形单元组成。电极之间的间距和电极数目可以在一定范围内调整，电极间距调整范围为100um-10mm，间距越小，所需电穿孔电压就越小。电极数目的调整原则是在调整了之后，仍然维持由多个正三角形单元组成的正六边形。电极的形状可以是圆柱状等的任何形状，其外围直径在0.01mm-2mm之间。所述电极固定组件2主要是用来固定电极的位置，使电极网络维持一定的规则排列，由生物兼容材料制成，其中一个优选材料是聚二甲基硅氧烷PDMS。
电极的材料关系着器件的工艺难度、制作成本、生物兼容性等。大多数金属电极如铜、铝的生物兼容性不好，而有些金属材料如金、银的成本又很昂贵，经过长期的实验研究发现，不锈钢电极是作为立体电极的极好的材料。不锈钢具有良好的生物兼容性，同时容易做成较长的电极，也能够大批量生产，非常适合作为所述电极固定组件1的电极材料。不锈钢电极的直径过大或者过小都会给器件带来不利影响。直径过大时，电穿孔的有效电场面积会减少，这意味着能够电穿孔的细胞量减少，不利于高通量的细胞电穿孔。直径过小，电极容易弯曲，而且制作成本大大增加。不锈钢还有容易清洗、不易氧化的特性，这些特性使之能够多次重复使用而不影响其导电特性。
图3是所述电极固定组件1的电极连接示意图，组成电极阵列的电极数目可以在一定范围内调整，电极间距、外围形状大小都可以按照实施例要求进行改变。电极阵列的数目调整原则是，在调整之后，仍然维持由多个正三角形单元组成的正六边形。在图3中，是由37支电极组成的电极阵列，分成三组，分别由I、II、III表示，电极阵列中的每相邻三支电极中，均有编号为I、II、III的电极，属于同一组中的电极会连接在一起。
图4是所述电极固定组件1一个典型的电极加电方式示意图，电极网络阵列的输入端有三个电学接口，分别用I、II、III表示，这三个接口通过开关与电压脉冲连接，其中开关的优选实施方式是由数字信号控制的继电器或光耦开关，电压脉冲由电穿孔仪器、脉冲发生器或生物刺激器提供。三个开关的连接方式并不限于一种，可以按照具体实施例进行调整，例如可以其中一个接正极，另外两个接负极。一个优选的实施例是，将连续的三个电压脉冲分成一组，将这三个电压脉冲称为t1、t2、t3或者phase I、phase II、phase III，当电压脉冲为t1或phase I时，（I）接正极，（II）（III）接负极，当电压脉冲为t2或phase II时，（II）接正极，（I）（III）接负极，当电压脉冲为t3或phase III时，（III）接正极，（I）（II）接负极，电穿孔时施加的电压脉冲次数不限于三次，为获得均匀处理，通常为3的倍数。与此优选实施例对应，当对电极网络施加电压脉冲时，电场模拟分布如图5所示，图5（A）分别为三个连续脉冲时对应的电场模拟分布，图5（B）为三次连续脉冲电场模拟分布叠加后的效果图，该模拟条件中，电极间距为700微米，电极大小为300微米，电极数目为37。
实施例二：
其他与实施例一相同，请参阅图6，所述第二流式电穿孔室是一个圆柱形容器，在所述第二流式电穿孔室上有第一接口8和第二接口9，均可以作为样品出口和入口。
研究者证实，形成电穿孔的条件与细胞类型、期望导入细胞的分子类型或是期望流出细胞的分子类型有关。对于任何特定类型的细胞来说，都存在着一个最佳的处理条件，称为条件区间，具体来说，就是可以这个条件区间内选取一个更为明确的条件进行实验。这一条件区间涉及的内容包括，电压强度，更具体地说，施加到细胞的电场强度，脉冲持续时间，脉冲次数以及脉冲间隔。在流式电穿孔器件中，细胞悬液是以恒定流速通过电穿孔室的，那么脉冲间隔与悬液流速是互相制约的，与该细胞的最佳脉冲次数有关。对于容器内的流体，中心区域的流速会大于靠近容器室壁的速度，那么这也必然会导致经由流体中心区域的细胞被施加脉冲电场的次数小于期望值，而经由室壁附近的细胞被施加脉冲电场的次数大于期望值。那么，可以得出一个近似的关系式，经由流体中心区域的体积与经由该段容器的总体积的比值：p=Vin/Vall，其中，Vin是细胞悬液经由容器中心区域的体积，Vall是细胞悬液经由容器的总体积，p是两个体积的比值。
在决定脉冲间隔与悬液流速时，通常采用的体积参数是经由容器的总体积，可以看到，若参数p越大，计算脉冲间隔与悬液流速就越对经由容器中心区域的细胞悬液有利，具体来说，参数p越大，经由容器中心区域的细胞悬液被施加的脉冲次数就越接近于期望值，而靠近室壁的体积在总体积所占比重很小，该体积的细胞被施加的脉冲次数小于期望值，但其带来的影响可以忽略。
利用所述流式电穿孔装置对细胞进行可变流式电穿孔实验。选用HEK-293A细胞进行流式电穿孔，实验中使用GFP质粒作为标记物。如果细胞被电穿孔，GFP质粒会进入细胞，在细胞内表达绿色荧光蛋白，该蛋白在荧光显微镜下可以观察到绿色荧光。因此，用荧光场中细胞的数量除以整个细胞数量可以得出细胞的电穿孔率。从另一个角度来说，同等密度细胞的荧光强度越大，电穿孔效率越高。
进行实验时，将含有HEK-293A细胞和GFP质粒的样品注入电穿孔室，并使样品在室内按规定流速流动，同时加电脉冲对样品进行电穿孔，未处理的样品流入电穿孔室，同时已处理样品也逐渐排出。收集排出的已处理样品，转移到培养板，加入培养基进行培养24小时后，用荧光显微镜进行观察。
图7为利用该器件进行生物实验的结果，描述的是HEK-293A细胞在电穿孔后24小时用荧光显微镜拍摄的结果，细胞表达了由于电穿孔进入细胞的GFP质粒，在荧光显微镜下发出绿光。这两组电穿孔实验结果的图片中，左边为细胞亮场图，表示进行电穿孔的细胞数量，右边为荧光场图，反映细胞电穿孔的情况。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构或等效流程变换，或直接或间接运用在其它相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (8)

1、一种流式电穿孔装置，其特征在于包括立体电极装置和流式电穿孔室，所述立体电极装置包括电极阵列和电极固定组件，所述电极阵列所包括的电极固定于所述电极固定组件，所述流式电穿孔室包括腔体以及与腔体相通的入口、出口和开口，所述电极阵列通过所述开口插入所述腔体内。
2、根据权利要求1所述的流式电穿孔装置，其特征在于，所述电极阵列包括多个电极，各个电极为细长结构的导电材料。
3、根据权利要求2所述的流式电穿孔装置，其特征在于，所述电极阵列中的多个电极按正多边形排列，所述电极阵列中相邻的两个电极之间的距离相等。
4、根据权利要求3所述的流式电穿孔装置，其特征在于，所述电极阵列的形状为由若干等边三角形组成的正六边形，各电极分别位于所述等边三角形的顶点上。
5、根据权利要求1所述的流式电穿孔装置，其特征在于，所述腔体截面呈圆形或多边形。
6、根据权利要求1所述的流式电穿孔装置，其特征在于，所述流式电穿孔装置还包括引流组件，所述引流组件与所述入口或所述出口相通，用于将液体引入到所述流式电穿孔室。
7、根据权利要求6所述的流式电穿孔装置，其特征在于：所述引流组件选自蠕动泵、旋转泵、活塞、隔膜泵或齿轮传动装置。
8、根据权利要求1所述的流式电穿孔装置，其特征在于：所述流式电穿孔室由玻璃管或其它与生物兼容的有机材料制成。
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