WO2021239010A1 - 一种基于介电确定性位移的细胞分选芯片、装置及方法 - Google Patents

一种基于介电确定性位移的细胞分选芯片、装置及方法 Download PDF

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WO2021239010A1
WO2021239010A1 PCT/CN2021/096171 CN2021096171W WO2021239010A1 WO 2021239010 A1 WO2021239010 A1 WO 2021239010A1 CN 2021096171 W CN2021096171 W CN 2021096171W WO 2021239010 A1 WO2021239010 A1 WO 2021239010A1
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electrode group
electrode
cell
sorting
cells
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PCT/CN2021/096171
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English (en)
French (fr)
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马波
刁志钿
王喜先
徐健
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中国科学院青岛生物能源与过程研究所
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Priority to JP2022572561A priority Critical patent/JP2023528342A/ja
Priority to EP21811971.7A priority patent/EP4141099A4/en
Publication of WO2021239010A1 publication Critical patent/WO2021239010A1/zh
Priority to US17/994,176 priority patent/US20230266224A1/en

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    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
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    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
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    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
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    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
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Definitions

  • the invention belongs to the field of biotechnology and instrument science, and specifically relates to a cell sorting chip, device and method based on dielectric deterministic displacement.
  • FACS Fluorescence flow cytometry
  • SCRS Single-cell Raman spectroscopy
  • the present invention proposes a cell sorting chip, device and method based on dielectric deterministic displacement.
  • Single cell sorting is realized by applying a non-uniform electric field through a simple chip structure.
  • the microfluidic chip includes a microfluidic channel that includes a sample inlet, Straight channel and two sample outlets; the bottom of the straight channel integrates three groups of electrode array pairs, the three groups of electrode array pairs are focusing electrode group, sorting electrode group and separation electrode group; the focusing electrode group is used to focus the cells And signal detection function; the sorting electrode group is used for single cell sorting; the separation electrode group is used for single cell separation.
  • the focusing electrode group and the separating electrode group include at least a pair of electrodes, and the sorting electrode group may be a pair of electrodes or one electrode.
  • the focusing electrode group has at least a pair of electrode arrays, the electrodes in each pair of electrode arrays are parallel to each other, one end of the electrodes on a single electrode array is connected, the electrodes in each pair of electrode arrays are distributed at intervals, and the electrodes have an included angle.
  • the electrode has two sides with a shape similar to ">".
  • the angle of the electrode included angle is a+b, and the range of the electrode angles a and b is 0-90 degrees, preferably 15-45 degrees.
  • a is the angle between one side of the electrode and the center line
  • b is the angle between the other side of the electrode and the center line
  • angles a and b are different.
  • angles a and b are the same.
  • the separation electrode group and the focusing electrode group have the same structure but different angle directions, and the angle between the sorting electrode and the center line is a.
  • the electrode shape of the separated electrode group is similar to " ⁇ ".
  • the width L of the electrode pair can be flexibly adjusted according to the width of the micro flow channel.
  • the electrode width D and the electrode spacing d are adjustable according to the cell/particle size, but the electrode width D and the electrode spacing d are both larger than the maximum size of the cell/particle.
  • the center lines of the electrode pairs of the focusing electrode group and the separated electrode group are not on the same straight line.
  • the distance c between the center lines of the two electrode pairs is determined according to the electrode width D and the electrode spacing d.
  • the electrode pair width L is 1 mm
  • the electrode width D is 25 microns
  • the distance d between the electrodes is 25 microns
  • the electrode angles a and b are the same at 30 degrees.
  • the distance c is 35 microns.
  • the material of the electrode group is electrodes of conductive material such as ITO, carbon, graphene, and metal.
  • the height of the electrode is 10 nanometers to 10 micrometers, preferably 50-150 nanometers.
  • the material of the carrier of the integrated electrode pair at the bottom of the chip is silicate glass, quartz glass, calcium fluoride glass, PDMS (polydimethylsiloxane) or PMMA (polymethylmethacrylate). Ester), preferably quartz glass.
  • the electrode array group is connected to an external function generator for applying high-frequency alternating current to the electrodes.
  • the response time of the sorting electrode group is 50 milliseconds.
  • the particles in the fluid flow along the electrode because the cells/particles are in a solution that is not easily polarized.
  • the cells/particles are easily polarized.
  • the cell Subject to the dielectric force perpendicular to the electrode and directed to the electrode, at the same time, the cell will also receive a fluid propelling force parallel to the flow direction and directed to the flow direction, and the combined force of the two will move the cell along the electrode.
  • the width of the direct current channel theoretically, the width may not have an upper limit, and the channel is wide, and a good focusing effect can be achieved by adding an electrode array.
  • the width of the direct flow channel is 1-2 mm.
  • Another aspect of the present invention provides a cell sorting device based on dielectric deterministic displacement, including:
  • Function generator used to generate sinusoidal alternating current with different frequencies and voltages
  • a computer which is used to control the operation of the program and the signal collection and collection of related optical instruments, control the output voltage, frequency, duty cycle and other parameters of the function generator, and control the switch of the relay;
  • Relay used to control the switch of the circuit
  • the fluid drive device is used as the source of driving force to drive the fluid into the micro channel.
  • the fluid drive device includes a syringe pump or a gravity drive device.
  • Another aspect of the present invention provides a cell sorting method based on dielectric deterministic displacement, which specifically includes the following steps:
  • Cell sampling using a fluid drive device to make the fluid containing cells enter the straight channel through the injection port of the microfluidic chip;
  • Cell focus Connect the focus electrode group to the output terminal of the function generator through a wire, and periodically apply a high-frequency voltage to the focus electrode group to focus the cells in the fluid to the signal monitoring point of the electrode angle;
  • the trigger relay When it is a target cell, the trigger relay is connected to the sorting electrode group to output high-frequency voltage, so that the target cell is offset from the center line of the sorting electrode.
  • the program control is not Turn on the relay, so that the non-target cells continue to move along the center line of the original focusing electrode group without shifting;
  • Cell separation Periodically apply high-frequency voltage to the separation electrode group to make non-target cells and target cells in the fluid flow to different sample outlets.
  • the cell is one or more of biological cells such as yeast cells, E. coli cells, or Hela cells.
  • the flow rate of the fluid is 0.01-50 microliters per minute, and the size of the flow rate is related to the width and height of the straight channel.
  • the injection flow rate is 10-40 microliters per minute.
  • the yeast is washed 3 times with pure water and pure water is used as the sample buffer.
  • the best parameter is a sinusoidal alternating current with a voltage of 16 volts and a frequency of 10 MHz.
  • the duration of the applied high-frequency voltage is adjusted according to the fluid flow rate in the flow field and other parameters, and the loading time needs to shift the target cell by a distance greater than or equal to the distance c between the center lines of the two sets of electrodes.
  • the optical signal detection in the step 3) is Raman signal detection or fluorescence signal detection.
  • interval time liquid flow length/liquid flow velocity, where liquid flow length refers to The actual distance that the detected liquid flows from the signal detection point to the sorting operation point.
  • the system method can be used as a flow cytometry detection, but the signal detection is performed on each cell flowing through the detection point.
  • the chip micro-flow channel has a simple structure, and the flow path is a straight channel, which is simple and easy to operate;
  • the response time of the sorting electrode is milliseconds, with a higher throughput sorting efficiency
  • the integrated focusing electrode group can greatly increase the cell sampling flow rate while ensuring the cell capture efficiency and detection efficiency, thereby effectively preventing the cells from settling in the sampling microtube, improving the sampling efficiency, and effectively extending the stable operation of the system time.
  • Figure 1 is a schematic diagram of a cell sorting chip based on dielectric deterministic displacement. Reference signs are 1: focusing electrode group; 2: sorting electrode group; 3: separation electrode group; 4: micro flow channel; 4.1: sampling port; 4.2: collection port; 4.3: waste liquid port; 5: signal detection point.
  • Figure 2 is a schematic diagram of the various parameters of the electrode, a and b both represent the electrode angle; d represents the distance between the electrode and the electrode; D represents the width of the electrode; c represents the distance between the center lines of the two electrode pairs; L represents the width of the electrode pair .
  • Figure 3 is a schematic diagram of another electrode group structure.
  • Figure 4 is a schematic diagram of a cell sorting device based on dielectric deterministic displacement.
  • the reference signs are 1: microfluidic chip; 2: function generator; 3: computer; 4: relay; 5: syringe pump.
  • Figure 5 is a schematic diagram of a cell sorting process based on dielectric deterministic shift.
  • the structure of the microfluidic chip is shown in Figure 1.
  • the microfluidic chip includes a microfluidic channel 4.
  • the microfluidic channel includes an injection port 4.1, a straight channel and a collection port 4.2, and a waste liquid port 4.3; the bottom of the microfluidic channel 4
  • Three sets of electrode array pairs are integrated, the three sets of electrode array pairs are focusing electrode group 1, sorting electrode group 2 and separation electrode group 3; the focusing electrode group is used for cell focusing and signal detection; the sorting The electrode group is used for single cell separation; the separation electrode group is used for single cell separation.
  • Figure 2 is a schematic diagram of the various parameters of the electrode, a and b both represent the electrode angle; d represents the distance between the electrode and the electrode; D represents the width of the electrode; c represents the distance between the center lines of the two electrode pairs; L represents the width of the electrode pair .
  • FIG. 3 shows a schematic diagram of another electrode group structure. There is only one electrode in the sorting electrode group.
  • the structure of the microfluidic chip used is shown in Figure 3.
  • the parameters of the electrodes in the microfluidic chip are: the electrode pair width L is 1 mm, the electrode width D is 25 microns, and the distance d between the electrodes is 25 microns.
  • the angles a and b are the same at 30 degrees, and the distance c between the center lines of the two sets of electrodes is 35 microns.
  • the syringe pump 5 uses the syringe pump 5 to pump the above-mentioned processed bacterial liquid into the microchannel.
  • the yeast cells in the liquid will gradually move to the tip of the electrode pair under the focusing action of the focusing electrode group.
  • the flow rate of the microchannel is about 40 microliters per minute.
  • the energization parameters of the electrode are the voltage of 16 volts, the frequency of 10 MHz, and the focusing and capturing efficiency can reach above 95%.
  • the Raman spectrum When the yeast cell moves to the signal monitoring point, the Raman spectrum will be collected.
  • the program will perform a simple analysis on the collected spectrum.
  • the Raman spectrum When it is the target cell, the Raman spectrum will reflect the characteristic peak of oil, so the program judges it is the target cell.
  • the program control relay is activated to turn on the sorting electrode group, and the delay and loading duration are preset to make the target cell move down; when it is a non-target cell, the Raman spectrum will not display the characteristic peak of grease, so it will not The relay is activated, so there is no voltage applied to the sorting electrode group, and the cell will continue to move along the center line.
  • the function of the separation electrode group is to move the cells at both ends of the center line of the separation electrode group further away from the center line, so that the target cells are below the channel and the non-target cells are above the channel.
  • yeast cells with high oil production are collected at the collection port, and the yeast cells with low or no oil production are collected at the waste liquid port.
  • microfluidic chip channel After the microfluidic chip channel is rinsed twice with absolute ethanol, it is placed in an oven for drying and then recycled.
  • Example 2 Raman flow cytometry detection of high oil production in yeast cells
  • the structure of the microfluidic chip used is shown in Figure 3.
  • the parameters of the electrodes in the microfluidic chip are: the electrode pair width L is 1 mm, the electrode width D is 25 microns, and the distance d between the electrodes is 25 microns.
  • the angles a and b are the same at 30 degrees, and the distance c between the center lines of the two sets of electrodes is 35 microns.
  • Channel 2 outputs a square wave with a voltage of 20 volts and a frequency of 5 Hz
  • channel 1 outputs a sine wave with a voltage of 16 volts and a frequency of 10 MHz.
  • the program controls the acquisition and analysis of Raman signals.
  • the syringe pump 5 uses the syringe pump 5 to pump the above-mentioned processed bacterial liquid into the microchannel.
  • the yeast cells in the liquid will gradually move to the tip of the electrode pair under the focusing action of the focusing electrode group.
  • the flow rate of the microchannel is about 40 microliters per minute.
  • the energization parameters of the electrode are the voltage of 16 volts, the frequency of 10 MHz, and the focusing and capturing efficiency can reach above 95%.
  • yeast cells When the yeast cells move to the signal monitoring point, they will be collected with a Raman spectrum, and the program will perform a simple analysis on the collected spectrum.
  • yeast cells that have been tested are collected at the collection port.
  • microfluidic chip channel After the microfluidic chip channel is rinsed twice with absolute ethanol, it is placed in an oven for drying and then recycled.
  • the electrode pair width L is 1 mm
  • the electrode width D is 25 microns
  • the distance d between the electrodes is 25 microns
  • the electrode angles a and b are both 15 degrees.
  • the distance c between the center lines of the two sets of electrodes is 50 microns.
  • the syringe pump 5 uses the syringe pump 5 to pump the above-mentioned processed bacterial solution into the microchannel.
  • the E. coli cells in the liquid will gradually move to the tip of the electrode pair under the focusing action of the focusing electrode group.
  • the flow rate of the microchannel is about 10 microliters per hour. Minutes, the energization parameters of the electrode are the voltage of 16 volts, the frequency of 1 MHz, and the focus capture efficiency is above 90%.
  • the E. coli cells move to the signal monitoring point, the E. coli containing fluorescent proteins will be excited by the laser to produce a fluorescent signal.
  • This fluorescent signal is collected by the computer and excited by the program control relay to turn on the sorting electrode group.
  • the delay and The loading duration causes the target cell to shift downward; when the E. coli without fluorescent protein passes through the monitoring point, the fluorescent signal will not be excited, so the program control will not activate the relay, and the sorting electrode group will not be loaded with voltage, and the cell will Continue to move along the center line.
  • the function of the separation electrode group is to move the cells at both ends of the center line of the separation electrode group further away from the center line, so that the target cells are below the channel and the non-target cells are above the channel.
  • fluorescent E. coli cells are collected at the collection port, and non-fluorescent E. coli cells are collected at the waste liquid port.
  • microfluidic chip channel After the microfluidic chip channel is rinsed twice with absolute ethanol, it is placed in an oven for drying and then recycled.

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Abstract

提供一种基于介电确定性位移的细胞分选芯片、装置及方法，所述芯片包括微流道（4），所述微流道包括一个进样口（4.1）、直通道和两个出样口；直通道底部集成了三组电极阵列对，三组电极阵列对分别为聚焦电极组（1），分选电极组（2）和分离电极组（3）；所述聚焦电极组用于对细胞聚焦和信号检测作用；所述分选电极组用于单细胞分选；所述分离电极组用于单细胞分离。可实现高速、精确、无损的目标细胞分选。

Description

一种基于介电确定性位移的细胞分选芯片、装置及方法 技术领域
本发明属于生物技术和仪器科学领域，具体涉及一种基于介电确定性位移的细胞分选芯片、装置及方法。
背景技术
传统生物学对生物细胞的表征主要基于群体细胞水平，然而异质性群体的平均测量依赖细胞培养，且无法体现单个细胞的差异，而且自然界中绝大多数细胞尚难培养。单细胞的功能识别及其定向分选因其不依赖于细胞的扩增，从而允许直接跳过细胞培养这一步，且能够反映细胞异质性。荧光流式细胞术(FACS)是一种高通量单细胞分析技术，该技术的应用大幅度提高了单细胞的鉴定效率。然而由于绝大多数细胞本身荧光效应较弱或没有荧光，因此该方法一般需要外加荧光标记。此外，在细胞群落中功能组分的识别中，关键的细胞表型经常仅有粗放认识或完全未知(即“未知”的细胞表型)，也没有其它生物标记，恰恰这些研究的目的往往正是寻找这些未知但关键的表型(及其生物标记)。因此，“探测未知细胞表型”的这一瓶颈严重限制了目前FACS等细胞分选技术在单细胞研究中的应用。单细胞拉曼光谱技术(SCRS)是一种高效的细胞内化学物质信息识别技术，可以提供细胞内化合物分子构成和结构的信息，拉曼光谱在获得整个单细胞的化学物质指纹图谱时并不需要任何标记，因此可以识别活体细胞的细胞类型、生理特性和表型变化，并可利用细胞拉曼信号的变化来追踪与分选“未知细胞表型”。基于SCRS实现细胞分选拉曼流式分选技术(RACS)为解决上述瓶颈问题提供了新思路。
现有技术中已有一系列关于拉曼激活细胞分选的技术，如光钳、激光喷射耦合等，属于拉曼激活细胞分选技术的静态版本。尽管这些系统简单实用，但其通量太低，阻碍了拉曼光谱技术在高通量分选中的应用。为了提高单细胞分选通量，也出现了一种拉曼激活液滴分选系统及分选方法，可以实现高通量的分选，但这类方案采用油包水液滴，导致流路复杂，操作性较差，尚无法完全满足实际需求。
发明内容
基于以上问题，本发明提出了一种基于介电确定性位移的细胞分选芯片、装置及方法，通过简单的芯片结构，通过施加非均匀电场，实现单细胞分选。
为实现上述目的，本发明一方面提供了一种基于介电确定性位移的细胞分选微流控芯片，所述微流控芯片包括微流道，所述微流道包括一个进样口、直通道和两个出样口；直通道底部集成了三组电极阵列对，三组电极阵列对分别为聚焦电极组，分选电极组和分离电极组；所述聚焦电极组用于对细胞聚焦和信号检测作用；所述分选电极组用于单细胞分选；所述分离电极组用于单细胞分离。
所述聚焦电极组与分离电极组包括至少一对电极，分选电极组可以是一对电极，也可以是一个电极。
所述聚焦电极组至少具有一对电极阵列，每对电极阵列中的电极相互平行，单个电极阵列上的电极一端相连，每对电极阵列中的电极间隔分布，所述电极具有夹角。
所述电极具有两条边，其形状类似“＞”。
电极夹角的角度为a+b，电极角度a、b范围的范围为0-90度，优选为15-45度。
其中a为电极一边与中心线的夹角，b为电极另一边与中心线的夹角。
在另一优选例中，所述角度a与b不相同。
在另一优选例中，所述角度a与b相同。
所述分离电极组与聚焦电极组结构相同，夹角方向不同，所述分选电极与中心线的夹角为a。
所述分离电极组的电极形状类似“＜”。
电极对的宽度L可根据微流道的宽度灵活可调。
电极的宽度D和电极间距d根据细胞/颗粒尺寸可调，但电极的宽度D和电极间距d均大于细胞/颗粒的最大尺寸。
在另一优选例中，所述聚焦电极组与分离电极组的电极对中心线不在同一直线上。
在另一优选例中，两组电极对中心线的距离c根据电极宽度D和电极间距d来决定。
在另一优选例中，电极对宽度L为1毫米，电极的宽度D为25微米，电极间的距离d为25微米，电极角度a、b相同均为30度，两组电极对中心线的距离c为35微米。
在另一优选例中，所述电极组的材质为ITO、碳、石墨烯、金属等导电材质电极。
在另一优选例中，所述电极的高度为10纳米到10微米，优选为50-150纳米。
在另一优选例中，所述芯片底部集成电极对的载体的材质为硅酸盐玻璃、石英玻璃、氟化钙玻璃、PDMS(聚二甲基硅氧烷)或PMMA(聚甲基丙烯酸甲酯)，优选地为石英玻璃。
在另一优选例中，所述电极阵列组外接函数发生器，用于在电极上施加高频交流电。
在另一优选例中，所述分选电极组的响应时间为50毫秒。
通过在电极上周期性施加高频交流电，使流体中的微粒沿着电极流动，是因为细胞/微粒在不易极化的溶液中，通过施加非均匀电场，由于细胞/微粒易被极化，会受到垂直于电极并指向电极的介电力，与此同时，细胞还会受到平行于流动方向并指向流动方向的流体推动力，两者的合力会将细胞沿着电极移动。
所述直流道的宽度：理论上宽度可以不设上限的，通道宽，可以通过增加电极阵列也可以达到很好的聚焦效果。
在另一优选例中，所述直流道的宽度为1-2毫米。
本发明又一方面提供了一种基于介电确定性位移的细胞分选装置，包括：
微流控芯片；
函数发生器，用于产生不同频率不同电压的正弦交流电；
电脑，所述电脑用于控制程序的运行和相关光学仪器的信号采集收集，控制函数发生器的输出电压、频率、占空比等参数，控制继电器的开关；
继电器，用于控制电路的开关；
流体驱动装置，作为驱动流体进入微流道的驱动力来源。
在另一优选例中，所述流体驱动装置包括注射泵或重力驱动装置。
本发明又一方面提供了一种基于介电确定性位移的细胞分选方法，具体包括以下步骤：
1)细胞进样：利用流体驱动装置使包含有细胞的流体通过微流控芯片的进样口进入直通道；
2)细胞聚焦：通过导线将聚焦电极组与函数发生器输出端相连接，通过在聚焦电极组上周期性施加高频电压，使流体中的细胞聚焦到电极夹角的信号监测点位置；
3)信号检测：光学信号采集点与监测点5对准，进行光学信号检测，检测的信号经过电脑程序处理判断是不是目标细胞；
4)细胞分选：当是目标细胞时，触发继电器联通分选电极组输出高频电压，使目标细胞沿着分选电极偏移中心线的位置，当程序判定是非目标细胞时，程序控制不导通继电器，使非目标细胞沿着原来聚焦电极组中线的位置继续移动，不会偏移；
5)细胞分离，在分离电极组上周期性施加高频电压，使流体中的非目标细胞和目标细胞流向不同的出样口。
在另一优选例中，所述细胞为酵母细胞、大肠杆菌细胞、或Hela细胞等生物细胞中的一种或多种。
在另一优选例中，所述流体的流速为0.01-50微升每分钟，流速的大小与直通道的宽度以及高度有关，优选为进样流速是10-40微升每分钟。
以酵母为例，酵母用纯水洗3遍并以纯水作为进样缓冲液，最佳参数为其通电电压为16伏特，频率为10兆赫兹的正弦交流电。
在另一优选例中，所加的高频电压持续时间根据流场中的流体流速等参数来调整，且加载的时间需将目标细胞偏移的距离大于等于两组电极中线的距离c。
所述步骤3)中的光学信号检测为拉曼信号的检测或荧光信号检测。
所述步骤3)中的光学信号检测与步骤4)中的分选之间存在延迟时间，所述延迟时间通过以下方式计算确定：间隔时间＝液流长度/液流流速，其中液流长度指从信号检测点起至分选操作点止所检测的液体流过的实际距离。
若是不触发分选电极组和分离电极组，该系统方法可以做为细胞流式检测，只是对每一个流过检测点的细胞进行信号检测。
本发明有益效果：
1)芯片微流道结构简单，流路为一条直通道，简单易于实际操作；
2)分选电极响应时间为毫秒级，具有较高通量的分选效率；
3)集成聚焦电极组，可以在保证细胞捕获效率和检测效率的同时，可大大提高细胞进样流速，从而有效防止细胞在进样微管中沉降，提高进样效率，可以有效延长系统稳定运行的时间。
附图说明
图1为基于介电确定性位移的细胞分选芯片示意图。附图标记为1：聚焦电极组；2：分选电极组；3：分离电极组；4：微流道；4.1：进样口；4.2：收集口；4.3：废液口；5：信号检测点。
图2为电极的各项参数示意图，a、b均表示电极角度；d表示电极与电极之间的距离；D表示电极的宽度；c表示两组电极对中线的距离；L表示电极对的宽度。
图3为另一种电极组结构示意图。
图4为基于介电确定性位移的细胞分选装置示意图。附图标记为①：微流控芯片；②：函数发生器；③：电脑；④：继电器；⑤：注射泵。
图5为基于介电确定性位移的细胞分选流程示意图。
具体实施方式
下面结合附图及实施例对本发明做进一步的详细说明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
微流控芯片结构如图1，所述微流控芯片包括微流道4，所述微流道包括一个进样口4.1、直通道和收集口4.2和废液口4.3；微流道4底部集成了三组电极阵列对，三组电极阵列对分别为聚焦电极组1，分选电极组2和分离电极组3；所述聚焦电极组用于对细胞聚焦和信号检测作用；所述分选电极组用于单细胞分选；所述分离电极组用于单细胞分离。
图2为电极的各项参数示意图，a、b均表示电极角度；d表示电极与电极之间的距离；D表示电极的宽度；c表示两组电极对中线的距离；L表示电极对的宽度。
图3所示为另一种电极组结构示意图。其中分选电极组只有一个电极。
实施例1：酵母细胞高产油脂的拉曼流式分选
实验准备：
将养成的酵母细胞取一毫升到离心管里，5000转每分钟离心5分钟，弃上清，加入一毫升纯水重悬后再离心，5000转每分钟离心5分钟；洗三次后加入1毫升纯水重悬。根据菌的浓度将其稀释1000-10000倍，加入一定浓度的表面活性剂(比如终浓度为0.5％的PF127)，防止细胞吸附电极，混匀后，注入注射器中。
打开拉曼仪器，打开激光，使得激光与信号监测点5对齐。
所采用的微流控芯片结构如图3所示，微流控芯片内电极的参数为：电极对宽度L为1毫米，电极的宽度D为25微米，电极间的距离d为25微米，电极角度a、b相同均为30度，两组电极对中心线的距离c为35微米。
将微流控芯片①、函数发生器②、电脑③、继电器④、注射泵⑤按照如图4所示连接完成，并运行电脑中编好的程序，使得函数发生器输出通道2控制输出通道1，通道2输出方波，电压为20伏特，频率为5赫兹，通道1输出正弦波，电压为16伏特，频率为10兆赫兹。同时程序控制拉曼信号的采集与分析，当检测到目标细胞时，程序控制继电器④的导通。
实验中：
使用注射泵⑤将上述处理好的菌液泵入微流道，液体中的酵母细胞在聚焦电极组的聚焦作用下会逐渐移动到电极对尖端，其中微流道的流速约为40微升每分钟，电极的通电参数为电压为16伏特，频率为10兆赫兹，聚焦捕获效率达到95％之上。
当酵母细胞移动到信号监测点，会被采集到拉曼图谱，程序对采集的图谱进行简单分析，当是目标细胞时，拉曼图谱会反映出油脂的特征峰，从而程序判断是目标细胞，程序控制继电器激发，使得分选电极组导通，预先设置好延迟与加载的持续时间，使得目标细胞往下移动；当是非目标细胞时，拉曼图谱不会显示油脂的特征峰，故不会激发继电器，因而分选电极组无电压加载，细胞会沿着中线继续移动。
分离电极组的作用是将分离电极组中线两端的细胞更加偏离中线，使得目标细胞在通道下方，非目标细胞在通道上方。
最后在收集口收集到的是高产油脂的酵母细胞，废液口是低产或者不产油脂的酵母细胞。
实验结束：
将微流控芯片通道用无水乙醇冲洗两遍后，放入烘箱烘干后循环利用。
实施例2：酵母细胞高产油脂的拉曼流式检测
实验准备：
将养成的酵母细胞取一毫升到离心管里，5000转每分钟离心5分钟，弃上清，加入 一毫升纯水重悬后再离心，5000转每分钟离心5分钟；洗三次后加入1毫升纯水重悬。根据菌的浓度将其稀释1000-10000倍，加入一定浓度的表面活性剂(比如终浓度为0.5％的PF127)，防止细胞吸附电极，混匀后，注入注射器中。
打开拉曼仪器，打开激光，使得激光与信号监测点5对齐。
所采用的微流控芯片结构如图3所示，微流控芯片内电极的参数为：电极对宽度L为1毫米，电极的宽度D为25微米，电极间的距离d为25微米，电极角度a、b相同均为30度，两组电极对中心线的距离c为35微米。
将微流控芯片①、函数发生器②、电脑③、继电器④、注射泵⑤按照如图4所示连接完成，并运行电脑中编好的程序，使得函数发生器输出通道2控制输出通道1，通道2输出方波，电压为20伏特，频率为5赫兹，通道1输出正玄波，电压为16伏特，频率为10兆赫兹。同时程序控制拉曼信号的采集与分析。
使用注射泵⑤将上述处理好的菌液泵入微流道，液体中的酵母细胞在聚焦电极组的聚焦作用下会逐渐移动到电极对尖端，其中微流道的流速约为40微升每分钟，电极的通电参数为电压为16伏特，频率为10兆赫兹，聚焦捕获效率达到95％之上。
当酵母细胞移动到信号监测点，会被采集到拉曼图谱，程序对采集的图谱进行简单分析。
最后在收集口收集到检测完成的酵母细胞。
实验结束：
将微流控芯片通道用无水乙醇冲洗两遍后，放入烘箱烘干后循环利用。
通过后期对采集的拉曼图谱分析，可以得出高产油脂的酵母细胞所占的比例等相关信息
实施例3：荧光细胞的流式分选
实验准备：
将养成的含有一部分能表达荧光蛋白的大肠杆菌取一毫升到离心管里，5000转每分钟离心5分钟，弃上清，加入一毫升纯水重悬后再离心，5000转每分钟离心5分钟；洗三次后加入1毫升纯水重悬。根据菌的浓度将其稀释1000-10000倍，加入一定浓度的表面活性剂(比如终浓度为0.5％的PF127)，防止细胞吸附电极，混匀后，注入注射器中。
打开荧光成像仪器，打开激光，使得激光与信号监测点5对齐。
所采用的微流控芯片结构如图3所示，电极对宽度L为1毫米，电极的宽度D为25微米，电极间的距离d为25微米，电极角度a、b相同均为15度，两组电极对中心线的距离c为50微米。
将微流控芯片①、函数发生器②、电脑③、继电器④、注射泵⑤按照如图4所示连接完成，并运行电脑中编好的程序，使得函数发生器输出通道2控制输出通道1，通道2输出方波，电压为20伏特，频率为0.2赫兹，通道1输出正玄波，电压为16伏特，频率为1兆赫兹。同时程序控制荧光信号的采集与分析，当检测到目标细胞时，程序控制继电器④的导通。
实验中：
使用注射泵⑤将上述处理好的菌液泵入微流道，液体中的大肠杆菌细胞在聚焦电极组的聚焦作用下会逐渐移动到电极对尖端，其中微流道的流速约为10微升每分钟，电极的通电参数为电压为16伏特，频率为1兆赫兹，聚焦捕获效率达到90％之上。
当大肠杆菌细胞移动到信号监测点，含有荧光蛋白的大肠杆菌会被激光激发出荧光信号，此荧光信号被电脑采集，通过程序控制继电器激发，使得分选电极组导通，预先设置好延迟与加载的持续时间，使得目标细胞往下偏移；当不含有荧光蛋白的大肠杆菌通过监测点时，不会激发荧光信号，因而程序控制不会激发继电器，分选电极组无电压加载，细胞会沿着中线继续移动。
分离电极组的作用是将分离电极组中线两端的细胞更加偏离中线，使得目标细胞在通道下方，非目标细胞在通道上方。
最后在收集口收集到的是带有荧光的大肠杆菌，废液口是非荧光的大肠杆菌细胞。
实验结束：
将微流控芯片通道用无水乙醇冲洗两遍后，放入烘箱烘干后循环利用。
应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种基于介电确定性位移的细胞分选微流控芯片，其特征在于，包括微流道，所述微流道包括一个进样口、直通道和两个出样口；直通道底部集成了三组电极阵列对，三组电极阵列对分别为聚焦电极组，分选电极组和分离电极组；所述聚焦电极组用于对细胞聚焦和信号检测作用；所述分选电极组用于单细胞分选；所述分离电极组用于单细胞分离。
  2. 根据权利要求1所述的微流控芯片，其特征在于，所述聚焦电极组与分离电极组包括至少一对电极，所述分选电极组至少为一个电极。
  3. 根据权利要求2所述的微流控芯片，其特征在于，所述聚焦电极组至少具有一对电极阵列，每对电极阵列中的电极相互平行，单个电极阵列上的电极一端相连，每对电极阵列中的电极间隔分布，所述电极具有夹角。
  4. 根据权利要求3所述的微流控芯片，其特征在于，电极夹角的角度为a+b，电极角度a、b范围的范围为0-90度。
  5. 根据权利要求4所述的微流控芯片，其特征在于，所述角度a与b相同。
  6. 根据权利要求4所述的微流控芯片，所述分离电极组与聚焦电极组结构相同，夹角方向不同，所述分选电极与中心线的夹角为a。
  7. 根据权利要求1所述的微流控芯片，其特征在于，所述聚焦电极组与分离电极组的电极对中心线不在同一直线上。
  8. 根据权利要求1所述的微流控芯片，所述电极组的材质为ITO、碳、石墨烯、金属电极。
  9. 一种基于介电确定性位移的细胞分选装置，其特征在于，包括：
    前述任一所述的微流控芯片；
    函数发生器，用于产生不同频率不同电压的正弦交流电；
    电脑，所述电脑用于控制程序的运行和相关光学仪器的信号采集收集，控制函数发生器的输出电压、频率、占空比等参数，控制继电器的开关；
    继电器，用于控制电路的开关；
    流体驱动装置，作为驱动流体进入微流道的驱动力来源。
  10. 根据权利要求9所述的基于介电确定性位移的细胞分选装置，其特征在于，所述流体驱动装置包括注射泵或重力驱动装置。
  11. 一种基于介电确定性位移的细胞分选方法，其特征在于利用如权利要求1-8中任一所述的微流控芯片、权利要求9-10所述的装置，并包括步骤：
    1)细胞进样：利用流体驱动装置使包含有细胞的流体通过微流控芯片的进样口进入直通道；
    2)细胞聚焦：通过导线将聚焦电极组与函数发生器输出端相连接，通过在聚焦电极组上周期性施加高频电压，使流体中的细胞聚焦到电极夹角的信号监测点位置；
    3)信号检测：光学信号采集点与监测点对准，进行光学信号检测，检测的信号经过电脑程序处理判断是不是目标细胞；
    4)细胞分选：当是目标细胞时，触发继电器联通分选电极组输出高频电压，使目标细胞沿着分选电极偏移中心线的位置，当程序判定是非目标细胞时，程序控制不导通继电器，使非目标细胞沿着原来聚焦电极组中线的位置继续移动，不会偏移；
    5)细胞分离，在分离电极组上周期性施加高频电压，使流体中的非目标细胞和目标细胞流向不同的出样口。
  12. 根据权利要求11所述的一种基于介电确定性位移的细胞分选方法，其特征在于，所述细胞为酵母细胞、大肠杆菌细胞、或Hela细胞中的一种或多种。
  13. 根据权利要求11所述的一种基于介电确定性位移的细胞分选方法，其特征在于，所述流体的流速为0.01-50微升每分钟。
  14. 根据权利要求11所述的一种基于介电确定性位移的细胞分选方法，其特征在于，所述步骤3)中的光学信号检测为拉曼信号的检测或荧光信号检测。
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