CN109225366B - High-flux cell separation device and method based on nano-micron combined channel alternating dielectrophoresis - Google Patents

Abstract

The invention provides a high-flux cell separation device and a method based on nano-micron combined channel alternating dielectrophoresis, comprising the following steps: an ITO conductive glass negative, a polydimethylsiloxane PDMS micro-fluidic chip and a signal acquisition system; and the PDMS microfluidic chip is provided with a channel, and the ITO conductive glass negative is bonded and packaged to form a cell separation chip of the microchannel. When the method is used for separating cells, the ITO is used for applying an electric field in the nano-micron combined channel so as to improve the gradient of the square of the electric field intensity, greatly reduce the required voltage amplitude and prevent the cell from being cracked due to overlarge voltage. Meanwhile, the invention can realize high-flux separation of cells.

Description

A high-throughput cell separation based on nano-micron combined channel AC dielectrophoresis Device and method

Technical field

The present invention relates to the field of cell separation technology, specifically, to a high-throughput cell separation device and method based on nano-micron combined channel AC dielectrophoresis.

Background technique

At present, high-throughput cell separation of test samples containing many different types of cells and processing of the separated cells are of great significance in many research fields; for example, in marine water condition detection, gene amplification In many fields such as sequencing and sequencing research, the design and research of portable devices and methods for detecting different types of cells in samples to separate specific cells has always been a hot spot in industry research, and there is an urgent need for this.

In the field of microfluidic research, there are two methods for DEP cell separation of test samples: AC dielectrophoresis and DC dielectrophoresis.

In DC dielectrophoresis, the electric field produces power loss in the form of Joule heating, and the corresponding temperature changes may negatively affect the physiological state of the cells. It is currently known that higher temperatures (more than 4°C above the physiological temperature of cells) will lead to rapid cell death. In cells, cell dynamics processes are exponentially related to temperature, so small temperature drifts may also affect the physiological state of cells. Alternating current has considerable advantages in reducing power loss, which makes AC dielectrophoresis more widely used in separation research than DC dielectrophoresis.

In addition, DC dielectrophoresis separation requires a very high voltage, even reaching hundreds or thousands of volts. When the voltage is very high, the signal-to-noise ratio of the detection signal will also increase, which will also cause problems in cell separation and detection. It brings a lot of inconvenience and reduces the detection effect.

For the Coulter detection principle, when performing traditional RPS detection, the corresponding signal amplitude can only be generated when cells enter the detection gate. However, when performing cell separation detection, the number of cells is particularly large, which means that the cells will be detected. If the door is blocked, the accuracy of signal detection will be greatly reduced.

When cells are separated based on microfluidic chip devices, there may be countless detection cells in a tiny amount of cell mixture, which also brings great inconvenience to the sequential separation of cells, making high-throughput cell separation become very important.

Currently commonly used cell separation methods include the following:

1) Differential centrifugation: It refers to step-by-step centrifugation from low speed to high speed in a medium with uniform density, used to separate cells of different sizes. However, since various cells overlap in size and density, and some slow-settling cells are often wrapped into sediment blocks by fast-settling cells, the detection effect will be greatly reduced. Moreover, it is only used for preliminary separation of cells of very different sizes, and it is difficult to efficiently separate cells of similar sizes.

2) Density gradient centrifugation method: It uses a certain medium to form a continuous or discontinuous density gradient in a centrifuge tube. The cell suspension is placed on the top of the medium, and the cells are stratified and separated through the action of gravity or centrifugal force field. . This method is based on the separation of different cell densities and sizes, but in the ultracentrifugation environment, some cells will also be physically destroyed.

3) Filtration separation method: It is currently the most commonly used method to separate mixtures, using a porous barrier membrane structure to separate cells of different sizes. This method is simple to operate, but because the size of the cells is extremely small, when using this method to separate cells, it is necessary to process highly precise porous media (such as filter membranes and filters), which has precision limitations in physical processing. This makes the filtering accuracy of this method have certain limitations. In addition, it cannot separate two cells of similar size.

4) Magnetic separation method: It is a method that effectively separates cells with the help of an external magnetic field. This method has high processing efficiency and relatively low operating costs. However, sometimes in order to increase the magnetic field gradient, high magnetic saturation must be selected. Magnetic condensation media has certain technical difficulties in selecting magnetic condensation media and increases operating costs.

Microfluidic chips are hailed as the supporting technology of life sciences in the 21st century and are the core technology of portable biochemical analysis instruments. This technology integrates basic operating units such as sample preparation, biological and chemical reaction separation and detection involved in the fields of biology and chemistry by building micro-scale channels into a chip of a few square centimeters, and can analyze a large amount of data in a short time. biomolecules and accurately obtain a large amount of information in the sample.

In recent years, with the development of micro-nano processing technology, especially the rapid development of soft lithography technology, microfluidic chip technology has achieved unprecedented development. Microfluidic technology, which emerged in the 1990s, accurately controls microliter and milliliter-level samples through micron-level flow channels. Thanks to the fact that the characteristic size of the device exactly matches the size of the cell, microfluidic technology has great advantages in cell sorting applications. It has developed rapidly in the past decade, and a large number of prototype devices have appeared. Compared with conventional sorting methods, these microfluidic devices have the advantages of less sample consumption, higher resolution accuracy and sensitivity, easy integration and miniaturization, and can continuously realize sample injection-cell injection in microfluidic devices. Sorting—the entire process of target cell identification, which greatly simplifies operation and reduces cell loss.

The original separate chip structure is to process a main channel hundreds of microns wide on the chip, and process an electric field channel in the middle of the main channel and perpendicular to the main channel. At both ends of the electric field channel, external platinum electrodes connected to the power supply are used. , generating a separation field for cell separation. However, since the electric field intensity at the intersection of the electric field channel and the main channel is very small, in order to obtain a good separation effect, a large voltage needs to be provided. When the cell size is small, the required voltage is extremely high, which is difficult to achieve. ; later separation chips made the intersection of the electric field channel and the main channel into an electric field focusing channel of several micrometers or even nanometers, and then designed the main channel for sample introduction into a structure similar to the size of the cells for physical focusing. This makes the electric field intensity very large at the intersection, thereby reducing the external power supply voltage required for cell separation, making a low-voltage, high-throughput cell separation microfluidic chip device possible.

Contents of the invention

According to the technical problems raised above, a high-throughput cell separation device based on nano-micron combined channel AC dielectrophoresis is provided.

The cell separation device of the present invention at least includes: an ITO conductive glass substrate, a polydimethylsiloxane PDMS microfluidic chip and a signal acquisition system; the PDMS microfluidic chip has a channel-processed side and the ITO conductive glass substrate The two are bonded together to form a cell separation chip that constitutes a microchannel.

Further, the conductive circuit layer on the ITO conductive glass substrate has circular power wiring layers distributed along both sides of the long sides of the rectangular conductive glass; the power wiring layer is connected to the two poles of the external high-frequency AC power supply; The power wiring layer extends along the vertical direction toward the center of the glass substrate to connect the circuit conductive ends in each cell separation structural unit in the conductive circuit layer;

Further, one side of the PDMS microfluidic chip is processed with a concave nano-micron combined channel; the micron channel is provided with sample injection and liquid storage holes at both short sides of the rectangular PDMS microfluidic chip. The hole is a through hole punched through the PDMS after forming the micron channel, and then aligning the hole punch with the injection hole in the micron channel to add the cell mixture during subsequent cell separation; the holes at both ends of the above-mentioned microfluidic chip A main channel for sample liquid shunting is provided between the liquid storage holes; the main channel for sample liquid shunting distributes a plurality of independent cell separation units with similar structures along the vertical direction; the cell separation unit is connected to the main channel; The side where the cell separation unit is connected to the main channel has a physical focusing channel for split injection with an inclined angle of 70°. The length of the physical focusing channel is the projected length of the inclined edge of the physical focusing channel in the vertical direction;

Furthermore, the physical focusing channel extending along the chip surface toward the outer edge of the chip is a secondary main channel of the cell separation structural unit.

Furthermore, in the horizontal direction from the middle part of the secondary main channel, perpendicular to the outward extension direction of the secondary main channel to the left and right sides is the electric field concentrated distribution area; the channels on both sides of the electric field concentrated distribution area are A separation gate with nanometer line width is processed at the intersection of the secondary main channel; the secondary main channel is provided with a cell sampling channel in the extending direction of the outer edge of the chip in the vertical direction; the end of the sampling channel is provided with a cell collection channel hole.

Furthermore, the structures of the cell separation structural units are all the same, and two adjacent cell separation structural units are in a symmetrical structure along the vertical direction.

Furthermore, the secondary main channel is provided with one end of the separation door connected to the positive conductive circuit layer on the ITO conductive glass substrate; the other end of the separation door is connected to the conductive circuit layer on the ITO conductive glass substrate. The negative electrode; a reference resistor is connected in series to the circuit connecting the external power supply wiring layer at both ends of the ITO conductive glass substrate to the external power supply; the output end of the signal amplifying element is connected to the signal acquisition control system.

Furthermore, the electric field generated by the conductive circuit is applied in the nano-micron combined channel, the ratio of micron line width to nanometer line width is greater than 100, and the micron line width is only 2-5 microns; the separation gate is located in the secondary main channel. On the side wall of the channel, when cells flow through the separation gate of the nano-micron combined channel, cell separation is achieved; the line width of the secondary main channel is 1-2 microns larger than the size of the separated cells, and each individual cell separation structural unit They are all at an oblique angle of 70° with the main channel, producing a physical focusing effect. Cells can pass through the maximum electric field force of the separation gate when moving on the secondary main channel; the nanochannel and micron channel are staggered by 1 micron from each other.

The present invention also includes a cell separation method, which at least includes the following steps:

S1: Sample dripping; add a small amount of PBS buffer into the sample storage hole to moisten the channel, connect the conductive circuit layer corresponding to the cell separation unit and the secondary main channel, and connect the A certain amount of cell mixed sample is dropped into the liquid storage holes of the sampling channels at both ends of the main channel;

S2: Sample transportation; turn on the high-frequency AC power supply, so that the mixed sample in the liquid storage hole of the sampling channel is transported from the main channel branch port of the chip injection liquid storage channel to each of the secondary levels under pressure. The main channel performs cell separation; under the physical focusing of the flow field formed by the inclined shunt openings at both ends of the edge of the main channel, the cells move from the main channel to the separation gate. When the cells pass through the separation gate of the vertical secondary main channel, due to the electric field The action of force and pressure continues forward motion in a straight line;

S3: Signal amplification acquisition and analysis; through the conductive circuit layer part on the ITO conductive glass on both sides of the counting channel, the pulse signal of the voltage at both ends of the reference resistor is collected. The collected signal is amplified by the reference resistor and connected to the signal amplification component, and then is collected by the signal acquisition The control system records and displays the corresponding detection data.

Further, the electric field generated by the conductive circuit is applied in the nano-micron combined channel, the ratio of micron line width to nanometer line width is greater than 100, and the micron line width is only 2-5 microns; the separation gate is located in the secondary main channel On the side wall, when cells flow through the separation gate of the nano-micron combined channel, cell separation is achieved;

The entrance of the secondary main channel is at an oblique angle of 70° with the main channel, producing a physical focusing effect; the nanochannel and the micron channel are staggered by 1 micron from each other.

The advantage of the present invention is that when performing cell separation, the present invention applies an electric field in the nano-micron combined channel through ITO to increase the gradient of the square of the electric field intensity, thereby greatly reducing the required voltage amplitude and preventing voltage Excessive size causes cell lysis. At the same time, the present invention can also achieve high-throughput separation of cells.

The cell separation form of the separation gate designed by the present invention can avoid clogging caused by cells passing through the traditional detection gate. Further, the present invention uses alternating current for power supply, and cells can accurately flow into different sampling channels under the action of dielectrophoretic force, preventing cell lysis caused by excessive voltage and reducing the separation effect. The use of ITO conductive glass in the present invention can avoid the cumbersome operation of adjusting the liquid level potential caused by external platinum electrodes after drilling holes on the microfluidic chip, and simplifies the power supply method of the microfluidic cell separation device.

Description of the drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 is a schematic diagram of the system structure of the device of the present invention.

Figure 2 is a circuit diagram of the ITO conductive glass of the device of the present invention.

Figure 3 is a schematic structural diagram of the microfluidic chip of the device of the present invention.

Figure 4 is a flow chart of the method of the present invention.

Among them: W represents ITO conductive glass; R represents PDMS microfluidic chip; X and Y represent the two injection ports of the main channel respectively; Z represents the main channel; 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, and 12 all represent cell separation units; A, C, E, G, H, J, L, and N all represent positive electrodes, and B, D, F, I, K, and M all represent negative electrodes; a, b, c, d, e, f, g, h, i, j, k, l, m, n all represent cell collection holes, where a, c, e, g, h, j, l, n collect The cells are the same, and the cells collected in b, d, f, i, k, and m are the same; P and Q represent the positive and negative electrodes on the ITO conductive glass.

Detailed ways

In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only These are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present invention.

It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It is to be understood that the data so used are interchangeable under appropriate circumstances so that the embodiments of the invention described herein are capable of being practiced in sequences other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, e.g., a process, method, system, product, or apparatus that encompasses a series of steps or units and need not be limited to those explicitly listed. Those steps or elements may instead include other steps or elements not expressly listed or inherent to the process, method, product or apparatus.

As shown in Figures 1-3, the present invention provides a high-throughput cell separation device based on nano-micron combined channel AC dielectrophoresis, which at least includes: ITO conductive glass substrate W, polydimethylsiloxane PDMS microfluidic Control chip R and signal acquisition system.

In this embodiment, the side of the PDMS microfluidic chip processed with micro-nano channels after plasma cleaning is bonded with ITO to form a micro-channel cell separation chip. As shown in Figure 1, two wires are used to connect the positive and negative terminals on both sides of the ITO conductive glass to collect signals. It can be understood that in other embodiments, the signal collection method can be set according to actual needs, as long as the signal on the conductive glass can be clearly collected.

In this embodiment, the circuit layer refers to the part with a specific connection method on the ITO conductive glass substrate, which is used for conducting electricity. As shown in Figure 2, the capital letters A-N round holes and the black lines connecting each round hole are The conductive circuit, the positive and negative terminals A--N round holes are used to power each separate separation gate on the PDMS chip, and each such positive (negative) pole connecting line is connected to all the positive (negative) poles. The negative) poles are connected together, leading to a terminal. The final terminal P (Q) is used to connect the positive and negative poles of the external AC power supply.

As a preferred embodiment, the specific conductive circuit layer on the ITO conductive glass substrate has circular external power supply wiring layers distributed along both sides of the long sides of the rectangular conductive glass; the external power supply wiring layer is connected to the external high-frequency AC power supply; The external power supply wiring layer is along the vertical direction, as shown in Figure 2, pointing from P and Q to the center of the glass plate, such as P to A to H in the positive electrode (Q to I to B in the negative electrode). The direction extending toward the center of the glass substrate is to connect all the positive and negative detection electrodes of each cell separation structural unit in the conductive circuit layer. It can be understood that in other embodiments, the connection direction can be set according to actual requirements.

In this embodiment, one side of the PDMS microfluidic chip is processed with concave micro-nano channels; as shown in Figure 3, the liquid storage hole has a circular concave micro-channel on the side of the PDMS with micro-channels. Use a hole punch to A through-PDMS hole was drilled at this position, which can be used to drop cell samples with a pipette into the micro-nano channel of the microfluidic chip bonded between PDMS and the glass substrate. The micron channel is provided with sample liquid storage holes at the two short sides of the rectangular PDMS microfluidic chip, and a main channel for sample liquid shunting is provided between the liquid storage holes at both ends of the PDMS microfluidic chip; the main channel for sample liquid shunting is provided A plurality of cell separation units with the same structure are distributed along the vertical direction of the channel; the cell separation unit is connected to the main channel; the side where the cell separation unit is connected to the main channel has a 70° inclined angle for split sampling Physical focus channel, the length of the physical focus channel is the projected length of the inclined side of the physical focus channel in the vertical direction. The physical focusing channel extends along the chip surface toward the outer edge of the chip and is the secondary main channel of the cell separation structural unit;

As shown in Figure 3, as an embodiment, assuming that the direction of the letters X-Z-Y in the drawing is the horizontal direction, and perpendicular to the horizontal direction is the vertical direction, along the vertical direction, there are 12 numbers numbered 1-12 Units with similar structures, these 12 similar structural units are the cell separation units. It can be understood that in other embodiments, the cell separation unit can be determined according to actual needs, as long as it can separate the cells.

As a preferred embodiment, the physical focusing channel is the channel with an inclined edge at the beginning between each independent cell separation unit and the vertical connection of the main channel used for sample liquid separation in the horizontal direction. The length of this physical focusing channel is The projected length of the inclined edge in the vertical direction, upward (downward) along the vertical direction, and the channel directly above (directly below) the physical focus is the secondary main channel. The channel area with two gaps in the horizontal direction that is perpendicular to the secondary main channel in the vertical direction is the area where the electric field is concentrated.

As shown in Figure 3, as an embodiment, from A to B, from B to C, and so on, they are all areas where the electric field is concentrated. From the middle part of the secondary main channel in the horizontal direction, and perpendicular to the outward extension direction of the secondary main channel to the left and right sides, there are areas where the electric field is concentrated. A separation gate with a nanometer line width is processed at the intersection of the channels on both sides of the concentrated electric field distribution area and the secondary main channel. The secondary main channel is provided with a cell sampling channel in the direction in which the outer edge of the chip extends in the vertical direction. A cell collection hole is provided at the end of the sample outlet channel.

In this embodiment, the structures of the cell separation structural units are all the same, and two adjacent cell separation structural units are in a symmetrical structure along the vertical direction. The secondary main channel is provided with one end of the separation door connected to the positive conductive circuit layer on the ITO conductive glass substrate; the other end of the separation door is connected to the negative electrode of the conductive circuit layer on the ITO conductive glass substrate. A reference resistor is connected in series to the circuit in which the positive and negative terminals at both ends of the ITO conductive glass substrate are connected to the external power supply, and the output end of the signal amplifying element is connected to the signal acquisition control system.

In this embodiment, the electric field generated by the conductive circuit is applied in the nano-micron combined channel, the ratio of micron line width to nanometer line width is greater than 100, and the micron line width is 2-5 microns. The separation gate is located on the side wall of the secondary main channel, and cell separation is achieved when cells flow through the separation gate of the nano-micron combined channel.

In this embodiment, the line width of the secondary main channel is 1-2 microns larger than that of the separated cells, and each individual cell separation structural unit is at an oblique angle of 70° with the main channel, resulting in a physical focusing effect, and the cells can be separated in the secondary The maximum electric field force passing through the separation gate when moving on the main channel; the nanochannel and the micron channel are staggered by 1 micron from each other.

As shown in Figure 4, a cell separation method includes at least the following steps:

As a preferred embodiment, step S1: drop a small amount of PBS buffer into the sample storage hole to moisten the channel, connect the corresponding conductive circuit layer of the cell separation unit and the secondary main channel, and A certain amount of cell mixed sample is dropped into the liquid storage holes of the sampling channel at both ends of the main channel.

As a preferred embodiment, step S2: Turn on the high-frequency AC power supply, so that the mixed sample in the liquid storage hole of the sampling channel is transported to each place by the split port of the main channel of the chip sampling liquid storage under the action of pressure. The secondary main channel performs cell separation; under the physical focusing of the flow field formed by the inclined shunt openings at both ends of the edge of the main channel, the cells move from the main channel to the separation gate. When the cells pass through the separation gate of the vertical secondary main channel, Due to the action of electric field force and pressure, it continues to move forward in a straight line.

As a preferred implementation, step S3: collect the pulse signal of the voltage at both ends of the reference resistor through the conductive circuit layer part on the ITO conductive glass on both sides of the counting channel. The collected signal is amplified by the reference resistor connected to the signal amplifying element, and then The signal acquisition control system records and displays the corresponding detection data.

In this embodiment, the electric field generated by ITO is applied in the nano-micron combined channel, the ratio of micron line width to nanometer line width is greater than 100, and the micron line width is only 2-5 microns. The separation gate is located on the side wall of the secondary main channel. When cells flow through the separation gate of the nano-micron combined channel, cell separation is achieved; the entrance of the secondary main channel is at an oblique angle of 70° with the main channel, producing a physical focusing effect. ; The nanochannel and micron channel are staggered by 1 micron from each other.

Claims (4)

1. A high-throughput cell separation device based on nano-micron combined channel AC dielectrophoresis, which is characterized by at least including: ITO conductive glass substrate, polydimethylsiloxane PDMS microfluidic chip and signal acquisition system; the channel-processed side of the PDMS microfluidic chip is bonded to the ITO conductive glass substrate to form the cells of the microchannel separate chips; The conductive circuit layer on the ITO conductive glass substrate has circular power supply wiring layers distributed along both sides of the long sides of the rectangular conductive glass; the power supply wiring layer is connected to the two poles of the external high-frequency AC power supply; the power supply The wiring layer extends along the vertical direction toward the center of the glass substrate to connect the circuit conductive ends in each cell separation structural unit in the conductive circuit layer; The electric field generated by the conductive circuit layer is applied in the nano-micron combined channel, the ratio of the width of the micron channel to the nanochannel is greater than 100, and the width of the micron channel is 2-5 microns; One side of the PDMS microfluidic chip is processed with concave nano-micron combined channels; The micron channel is provided with sample liquid storage holes at the two short sides of the rectangular PDMS microfluidic chip, and a main channel for sample liquid shunting is provided between the liquid storage holes at both ends of the PDMS microfluidic chip; the sample liquid shunting The main channel distributes multiple cell separation units with the same structure along the vertical direction; the cell separation unit is connected to the main channel; the side where the cell separation unit is connected to the main channel has a shunt with a 70° inclined angle. Inject the physical focus channel, and the length of the physical focus channel is the projected length of the inclined edge of the physical focus channel in the vertical direction; The physical focusing channel extends along the chip surface toward the outer edge of the chip and is the secondary main channel of the cell separation structural unit; The line width of the secondary main channel is 1-2 microns larger than the size of the separated cells, and each individual cell separation structural unit is at an oblique angle of 70° with the main channel, resulting in a physical focusing effect, and the cells can be placed on the secondary main channel The maximum electric field force passing through the separation gate during movement; the nanochannel and micron channel are staggered by 1 micron from each other; The middle part of the secondary main channel in the horizontal direction, perpendicular to the direction of the secondary main channel extending to the left and right sides is the electric field concentrated distribution area; the intersection of the two end channels of the electric field concentrated distribution area and the secondary main channel The separation gate is processed with a nanometer line width; the secondary main channel is provided with a cell sampling channel in the direction in which the outer edge of the chip extends in the vertical direction; the end of the cell sampling channel is provided with a cell collection hole; The separation gate is located on the side wall of the secondary main channel, and when cells flow through the separation gate of the nano-micron combined channel, cell separation is achieved; The structures of the cell separation structural units are all the same, and two adjacent cell separation structural units are in a symmetrical structure along the vertical direction. 2. A high-throughput cell separation device based on nano-micron combined channel AC dielectrophoresis according to claim 1, further characterized by: The secondary main channel is provided with one end of the separation door connected to the positive conductive circuit layer on the ITO conductive glass substrate; the other end of the separation door is connected to the negative electrode of the conductive circuit layer on the ITO conductive glass substrate; A reference resistor is connected in series in the circuit where the positive and negative terminals at both ends of the ITO conductive glass plate are connected to the external power supply; the output end of the signal amplifying element is connected to the signal acquisition control system. 3. The cell separation method based on the high-throughput cell separation device based on nano-micron combined channel AC dielectrophoresis according to any one of claims 1-2, characterized in that it at least includes the following steps: S1: Sample dropwise addition; drop a small amount of PBS buffer into the sample storage hole to moisten the channel, connect the conductive circuit layer corresponding to the cell separation structural unit and the secondary main channel, and place the A certain amount of cell mixed sample is dropped into the liquid storage holes of the sampling channels at both ends of the main channel; S2: Sample transportation; turn on the high-frequency AC power supply, so that the mixed sample in the liquid storage hole of the sampling channel is transported from the main channel branch port of the chip injection liquid storage channel to each of the secondary levels under pressure. The main channel performs cell separation; Under the physical focusing of the flow field formed by the inclined shunt openings at both ends of the edge of the main channel, the cells move from the main channel to the separation gate. When the cells pass through the separation gate of the vertical secondary main channel, due to the action of electric field force and pressure, Continue to move forward in a straight line; S3: Signal amplification collection and analysis; through the conductive circuit layer on the ITO conductive glass on both sides of the counting channel, collect the pulse signal of the voltage across the reference resistor of the conductive circuit layer, and the collected signal is amplified by connecting the signal amplification element through the reference resistor , the signal acquisition control system records and displays the corresponding detection data. 4. The method of cell separation according to claim 3, characterized in that: The electric field generated by the conductive circuit layer is applied in the nano-micron combined channel, the ratio of micron line width to nanometer line width is greater than 100, and the micron line width is only 2-5 microns; The separation gate is located on the side wall of the secondary main channel. When cells flow through the separation gate of the nano-micron combined channel, cell separation is achieved; the entrance of the secondary main channel is at an oblique angle of 70° with the main channel, producing a physical focusing effect. ; The nanochannel and micron channel are staggered by 1 micron from each other.