CN209555218U - Separation chip - Google Patents

Abstract

A kind of separating chips, comprising: sample pool；First filter membrane；Second filter membrane；First chamber, the first chamber are connected with the sample pool by first filter membrane, and the first chamber is provided with the first opening, and first opening is for making the first chamber be in communication with the outside；Second chamber, the second chamber are connected with the sample pool by second filter membrane, and the second chamber is provided with the second opening, and second opening is for making the second chamber be in communication with the outside.Wherein, the first chamber and the second chamber two sides that be located at the sample pool opposite.

Description

Separation chip

technical field

The utility model relates to the field of biotechnology, in particular to a separation chip for separating target particles in liquid samples.

Background technique

Liquid biopsy (liquid biopsy or fluid biopsy) is a sampling and analysis method that can comprehensively and real-time reflect the biological information of tumor cells or tissues. Liquid biopsy has the advantage of being non-invasive. By separating and analyzing specific research objects in blood or other body fluids (urine, saliva, pleural effusion, cerebrospinal fluid, etc.), dynamic observation of tumors can guide medical staff to screen for tumors , diagnosis, prognosis, selection of treatment options and monitoring of recurrence, etc. Specific research objects in liquid biopsy include circulating tumor DNA (circulating tumor DNA, ctDNA), circulating tumor cells (circulating tumor cell, CTC), microvesicles (also known as exosomes, exosomes), etc.

In the prior art, methods such as centrifugation, immunocapture or filtration are generally used to separate and purify circulating tumor cells and/or exosomes in blood or body fluids. The centrifuge separation method will cause a certain degree of mechanical damage to the membrane structure of circulating tumor cells (or exosomes), which will affect the subsequent analysis and research, and the cumbersome centrifugation will limit the throughput of liquid biopsy. The separation method of immune capture requires the use of antibodies, which greatly increases the cost of sample processing, and the elution conditions after immune capture may affect the activity of circulating tumor cells (or exosomes). The separation method using membrane filtration can effectively separate components of different sizes in body fluids. It has the characteristics of low cost and high throughput, and the target components obtained after filtration can maintain high biological activity. However, in actual operation, some components in the biological sample are easy to be enriched on the filter membrane, and the components larger than the membrane pore size will block the membrane pores. Membrane pore blockage will prevent components smaller than the membrane pore size from effectively passing through the filter membrane, affecting the purity of the target component after separation. Blockage of membrane pores can also cause excessive local pressure and even rupture of the filter membrane.

Utility model content

In view of the above, it is necessary to provide a separation chip that can reduce the plugging of the filter membrane during the filtration separation process.

The embodiment of the utility model provides a separation chip for separating and purifying target particles from a liquid sample, and the separation chip includes:

sample pool;

a first filter membrane, the pore size of the first filter membrane is smaller than the particle size of the target particle;

a second filter membrane, the pore size of the second filter membrane is smaller than the particle size of the target particle;

The first chamber, the first chamber communicates with the sample pool through the first filter membrane, the first chamber is provided with a first opening, and the first opening is used to make the first The chamber communicates with the outside world;

The second chamber, the second chamber communicates with the sample pool through the second filter membrane, the second chamber is provided with a second opening, and the second opening is used to make the second The chamber communicates with the outside, wherein the first chamber and the second chamber are respectively located on opposite sides of the sample pool.

Compared with the prior art, the separation chip provided by the utility model is simple to use and has low production cost. When the vacuum system is subsequently connected, the liquid sample to be separated can pass through the filter membrane more effectively, and it can be used in the liquid biopsy process. Rapid and high-throughput separation and extraction of circulating tumor cells and exosomes can reduce the workload of experimenters and reduce the cost of liquid biopsy detection.

Description of drawings

Fig. 1 is a schematic structural diagram of a separation chip provided by an embodiment of the present invention.

FIG. 2 is a schematic disassembly diagram of the structure of the separation chip shown in FIG. 1 .

Fig. 3 is a schematic structural diagram of a separation chip provided by another embodiment of the present invention.

Fig. 4 is a schematic diagram of the functional modules of the separation device provided by the embodiment of the present invention.

Fig. 5a is a schematic diagram of the liquid circuit of the separation device shown in Fig. 4 .

Fig. 5b is a program block diagram of the separation control system of the separation device shown in Fig. 4 .

Fig. 6 is a schematic diagram of the liquid flow in the sample cell when the separation chip shown in Fig. 1 is used for separation and purification.

Fig. 7a is a schematic diagram of the negative pressure applied to the separation chip in an embodiment of the present invention.

Fig. 7b is a schematic diagram of the negative pressure applied to the separation chip in another embodiment of the present invention.

Fig. 7c is a schematic diagram of the negative pressure applied to the separation chip in another embodiment of the present invention.

Fig. 8a is a schematic structural diagram of a chip base of an embodiment of the separation device shown in Fig. 3 .

Fig. 8b is a schematic structural view of fixing the separation chip and the gas conduit in the chip base shown in Fig. 8a.

Figure 9a is a particle size test graph of the components in the original urine sample.

Fig. 9b is a particle size test diagram of exosomes after separation and purification of the original urine sample using the separation chip of the embodiment of the present invention.

Figure 9c is a particle size test diagram of exosomes after separation and purification of the original urine sample using the qEV separation column.

Figure 9d is a particle size test diagram of exosomes separated and purified from the original urine sample using the ExoQuick-TC exosome kit.

Fig. 9e is a particle size test diagram of exosomes separated and purified from the original urine sample using the Magcapture Exosome Extraction Kit.

Figure 9f is a particle size test diagram of exosomes separated and purified from the original urine sample using the Exo-Spin exosome purification column.

Fig. 10a is a scanning electron microscope image of exosomes separated and purified from the original urine sample using the separation chip of the embodiment of the present invention.

Fig. 10b is a transmission electron microscope image of exosomes separated and purified from the original urine sample using the separation chip of the embodiment of the present invention.

Fig. 11 is the original urine sample using the separation chip, qEV separation column, ExoQuick-TC exosome kit, Magcapture exosome extraction kit and Exo-Spin exosome purification column of the utility model embodiment respectively The exosomes obtained after separation and purification were subjected to protein gel electrophoresis and band patterns obtained by silver staining.

Fig. 12 is a graph obtained by Western blot analysis of exosomes obtained after separation and purification of 11 cancer patient urine samples using the separation chip of the embodiment of the present invention.

Symbol Description

The following specific embodiments will further illustrate the utility model in conjunction with the above-mentioned accompanying drawings.

Detailed ways

The technical solutions of the present invention will be described below in conjunction with preferred implementation modes and examples of the present invention. It should be noted that when an element is described as being "connected" to another element, it can be directly connected to the other element or there may be intervening elements at the same time. When an element is described as being "disposed on" another element, it can be directly on the other element or intervening elements may also be present. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of this invention. The names of elements or devices used in the description of the present utility model are only for the purpose of describing specific embodiments, and are not intended to limit the present utility model.

An embodiment of the utility model provides a separation chip, which is used for separating and purifying particles of different sizes in a liquid sample, so as to obtain target particles of a specific size. The liquid sample can be human plasma, serum, cerebrospinal fluid, saliva, urine, gastric juice and the like. FIG. 1 is a schematic structural diagram of a separation chip 10 provided by an embodiment of the present invention. As shown in FIG. 1 , the separation chip 10 includes a sample pool 13 , a first chamber 15 and a second chamber 17 . The first chamber 15 and the second chamber 17 are respectively located on opposite sides of the sample pool 13 .

The sample cell 13 includes a sample cell substrate 136, a first inner cover sheet 132 and a second inner cover sheet 134, the first inner cover sheet 132 and the second inner cover sheet 134 cover the sample cell substrate 136 and are opposite , so that the sample cell substrate 136, the first inner cover 132 and the second inner cover 134 together define a chamber (not shown) for accommodating liquid samples. The first filter membrane 14 is disposed on the first inner cover 132 , and the second filter membrane 16 is disposed on the second inner cover 134 . The first chamber 15 communicates with the sample pool 13 through the first filter membrane 14 . The first chamber 15 is provided with a first opening 152 for communicating the first chamber 15 with the outside. The second chamber 17 communicates with the sample pool 13 through the second filter membrane 16, the second chamber 17 is provided with a second opening 172, and the second opening 172 is used to connect the second chamber 17 with the outside world. connected.

When using the separation chip 10, a liquid sample is added to the sample pool 13, and the first opening 152 and the second opening 172 are respectively connected to the vacuum system 30 (see FIG. 3 ). When the vacuum system 30 sucks the first chamber 15 through the first opening 152 , a negative pressure is generated in the first chamber 15 . Under the negative pressure of the first chamber 15 , components in the liquid sample in the sample pool 13 whose size is smaller than the filter aperture of the first filter membrane 14 flow into the first chamber 15 through the first filter membrane 14 . When the vacuum system 30 sucks the second chamber 17 through the second opening 172 , negative pressure is generated in the second chamber 17 . Under the negative pressure of the second chamber 17 , components in the liquid sample in the sample pool 13 whose size is smaller than the filter aperture of the second filter membrane 16 flow into the second chamber 17 through the second filter membrane 16 .

Negative pressure is generated in the first chamber 15 and the second chamber 17 repeatedly and alternately, which can effectively make the liquid sample repeatedly and alternately flow through the first filter membrane 14 and the second filter membrane 16, so that the size of the liquid sample is larger than The components of the pore size of the first filter membrane 14 and the second filter membrane 16 remain in the sample cell 13 . The structural design of the separation chip 10 makes the components adsorbed on the surface of the first filter membrane 14 and the second filter membrane 16 easy to fall off from the surface of the filter membrane in repeated and alternate negative pressure changes, which can effectively prevent the pores of the filter membrane from being damaged. clogged.

The main parts of the sample pool 13, the first chamber 15 and the second chamber 17 of the separation chip 10 can be made of plastic, glass, metal or composite materials. In one embodiment, the main parts of the sample pool 13, the first chamber 15, and the second chamber 17 of the separation chip 10 are made of transparent materials such as polyethyleneimine (PEI) or polymethylmethacrylate (PMMA). production.

The first filter membrane 14 and the second filter membrane 16 can be made of the same membrane material, or can be made of different membrane materials. The first filter membrane 14 and the second filter membrane 16 may have the same average filter membrane pore size and/or pore size distribution, or may have different average filter membrane pore size and/or pore size distribution. The first filter membrane 14 (or the second filter membrane 16 ) can be made of one membrane material, or can be composited of multiple membrane materials. The first filter membrane 14 and the second filter membrane 16 may be porous materials, including but not limited to porous ceramic materials, porous plastic materials and porous metal materials. Specifically, the first filter membrane 14 and the second filter membrane 16 can be selected from anodized aluminum oxide membrane (AAO), polycarbonate membrane, acetate cellulose membrane, polyethylene membrane, polypropylene membrane and polystyrene membrane one or more of. More specifically, in view of the higher porosity and uniform pore size of the anodized aluminum membrane, the first filter membrane 14 and the second filter membrane 16 are anodized aluminum membranes.

The pore size of the first filter membrane 14 and the second filter membrane 16 can be designed according to the type of the liquid sample and target particles. In one embodiment, the pore size of the first filter membrane 14 and the second filter membrane 16 is between 2-20 microns; preferably, between 5-10 microns. More specifically, the first filter membrane 14 and the second filter membrane 16 have a pore size of 8 microns, which can be used to separate and purify circulating tumor cells in plasma samples.

In another embodiment, the pore size of the first filter membrane 14 and the second filter membrane 16 is between 5-200 nm; preferably, between 10-100 nm. In one embodiment, the first filter membrane 14 and the second filter membrane 16 have a pore size of 20 nanometers, which can be used to separate and purify exosomes in a plasma sample that has passed through a 200-nanometer filter membrane.

Wherein, when the surfaces of the first filter membrane 14 and the second filter membrane 16 are not further modified, the separation chip 10 only screens various components in the liquid sample according to the pore size of the filter membrane, and in the sample obtained after separation and purification, It mainly includes target particles (such as circulating tumor cells, exosomes, etc.), and may also contain other particles with similar or larger sizes (such as high-density lipoproteins (HDLs), low-density lipoproteins (LDLs), intermediate-density lipoproteins Proteins (IDLs), very low-density lipoproteins (VLDL), and chylomicrons (chylomicrons) and other common non-exosomal proteins with a particle size similar to exosomes). In order to reduce the adsorption of porous materials to proteins or genes in liquid samples, the surfaces of the first filter membrane 14 and the second filter membrane 16 can be chemically modified; in order to specifically separate and purify target particles, the first filter The surfaces of the membrane 14 and the second filter membrane 16 can be modified by specific biomacromolecules, and the specific biomacromolecules can be one or more specific antibodies, antigens, polypeptides or base sequences. It should be pointed out that the target particles described herein can be biologically significant cells or components, or other types of particles, such as synthetic liposomes, nanospheres, nanoparticles, etc.

It can be understood that the volume of the sample pool 13 can be designed according to actual application scenarios. For the application scenario of biological biopsy, the volume of the sample pool 13 may be between 0.1-10 ml, optionally, between 0.5-2 ml. In one embodiment, the volume of the sample pool 13 is 1 ml. The top of the sample cell 13 may include a sample cell opening 138 for adding and/or removing liquid samples.

In the embodiment shown in FIG. 1 , the first chamber 15 includes a first side cover 156 opposite to the first filter membrane 14 , the first side cover 156 and the first filter membrane 14 with the first side cover 156 The first inner cover 132 forms the first chamber 15 together, and the first opening 152 is arranged on the first side cover 156; The second side cover sheet 176, the second side cover sheet 176 and the second inner cover sheet 134 having the second filter membrane 16 jointly surround and form the second chamber 17, and the second opening 172 is disposed on the second side cover sheet 176. On the two side covers 176. In this embodiment, an opening connection block 18 is respectively fixed on the first side cover 156 and the second side cover 176 of the separation chip 10, and a channel (not marked) is opened in the opening connection block 18, and the channel Align with and communicate with the first opening 152 and the second opening 172 respectively. Furthermore, the separation chip 10 may further include a chip base 19 for sealing the first chamber 15 and the second chamber 17 and for supporting other components of the separation chip 10 . The separation chip 10 has a symmetrical structure. It should be noted that the separation chip 10 may also have an asymmetric structure or any other structure capable of realizing the concept of the present invention.

FIG. 2 shows an exploded schematic diagram of an embodiment of the separation chip 10 provided by the present invention. As shown in Figure 2, a through hole (not marked) is provided on the first inner cover 132, and the first filter membrane 14 is fixed in the through hole of the first inner cover 132; 134 is provided with a through hole (not marked in the figure), and the second filter membrane 16 is fixed in the through hole of the second inner cover 134 . The sample cell substrate 136 is a U-shaped substrate with a certain thickness, and the first inner cover 132 and the second inner cover 134 cover the sample cell substrate 136 along the thickness direction of the sample cell substrate 136. opposite sides.

The embodiment of the utility model also provides a method for manufacturing the above-mentioned separation chip 10, which includes the following steps:

Step 1, provide the above-mentioned sample cell substrate 136, the first inner cover 132, the second inner cover 134, the first side cover 156, the second side cover 176, the first filter membrane 14 and the second filter membrane 16 .

Step 2, assembling the first side cover 156 to the first inner cover 132 to form the first cavity 15 .

Step 3, assembling the first filter membrane 14 to the first inner cover 132 .

Step 4, assemble the sample cell substrate 136 and the second inner cover 134 sequentially on the side of the first inner cover 132 with the first filter membrane 14 away from the first side cover 156 .

Step five, assembling the second filter membrane 16 to the second inner cover 134 .

Step 6, assembling the second side cover 176 to the side of the second inner cover 134 with the second filter membrane 16 away from the sample cell substrate 136 to form the second chamber 17 .

In one embodiment, the sample cell substrate 136, the first inner cover 132, the second inner cover 134, the first side cover 156, the second side cover 176, the first filter membrane 14 and the second filter The membranes 16 are all secured by adhesive. The adhesive can be ultraviolet curing adhesive or transparent silicone waterproof sealant. Based on the above method, the separation chip 10 conforming to the spirit of the present utility model can be manufactured at a relatively low cost.

Please refer to FIG. 3 , another embodiment of the present invention also provides a separation chip 10'. Different from the separation chip 10 described above, the sample cell substrate 136, the first inner cover 132 and the second inner cover 134 are omitted from the separation chip 10'. Since the sample cell substrate 136 , the first inner cover 132 and the second inner cover 134 are omitted, the production cost can be reduced to a certain extent. Specifically, the first side cover 156 is provided with a first protrusion 154 , and the first protrusion 154 divides the first side cover 156 into a first cover portion located on one side of the first protrusion 154 . 1561 and the second cover part 1562 located on the other side of the first protruding block 154 . The second side cover 176 is provided with a second protrusion 174 opposite to the first protrusion 154 , and the second protrusion 174 divides the second side cover 176 into one side of the second protrusion 174 The third cover part 1761 and the fourth cover part 1762 located on the other side of the second protrusion 174 . The first cover part 1561 , the third cover part 1761 , the first protrusion 154 and the second protrusion 174 jointly surround and form the sample pool 13 .

A chip substrate 19 is disposed on the bottom of the first side cover 156 opposite to the first bump 154 . The first filter film 14 is disposed between the first bump 154 and the chip substrate 19 and is opposite to the second cover part 1562, the second cover part 1562, the first filter film 14 and the chip substrate 19 jointly surround and form the first chamber 15 . Another chip substrate 19 is disposed on the bottom of the second side cover 176 and opposite to the second bump 174 . The second filter film 16 is disposed between the second bump 174 and the chip substrate 19 and opposite to the fourth cover part 1762 . The fourth cover part 1762 , the second filter membrane 16 and the chip substrate 19 jointly surround and form the second chamber 17 . In this embodiment, a gap (not marked) is provided between the first bump 154 and the second bump 174, and the gap is used to allow the liquid sample in the sample pool 13 to flow out of the sample pool 13, and Enter the first chamber 15 or the second chamber 17 through the first filter membrane 14 or the second filter membrane 16 respectively. More specifically, the first bump 154 is provided with a first slot 1540 on the side facing the chip substrate 19, and a second slot (not shown) is provided at the corresponding position of the chip substrate 19. The first filter membrane 14 is clamped and fixed between the first slot 1540 and the second slot. Similarly, the second bump 174 is provided with a third slot 1740 on the side facing the chip substrate 19, and a fourth slot is provided at the corresponding position of the chip substrate 19, and the second filter membrane 16 is clipped and fixed on the Between the third card slot 1740 and the fourth card slot.

The utility model embodiment also provides a kind of manufacturing method of above-mentioned separating chip 10 ', and it comprises the following steps:

Step 1, providing the first side cover 156 , the second side cover 176 , the first filter membrane 14 , the second filter membrane 16 and the chip substrate 19 .

Step 2, assembling the first filter membrane 14 between the first bump 154 of the first side cover 156 and the corresponding chip substrate 19 .

Step 3, assembling the second filter membrane 16 between the second bump 174 of the second side cover 176 and the corresponding chip substrate 19 .

Step 4, assembling the first side cover 156 to the second side cover 176 , making the first bump 154 face the second bump 174 , and make the two chip substrates 19 face each other. Thus, the first cover sheet part 1561, the third cover sheet part 1761, the first bump 154 and the second bump 174 jointly surround and form the sample cell 13; the second cover sheet part 1562, the second cover sheet part 1562 A filter membrane 14 and the chip substrate 19 are jointly surrounded to form the first chamber 15 ; the fourth cover part 1762 , the second filter membrane 16 and the chip substrate 19 are jointly surrounded to form the second chamber 17 .

The embodiment of the utility model further provides a separating device. FIG. 4 shows a schematic diagram of the program modules of the separation device 100 . The separation device 100 includes a main module 101 , an auxiliary module 102 and a human-computer interaction module 103 .

The main module 101 is used for separating and purifying liquid samples. The main module 101 includes the separation chip 10 , the liquid supply unit 20 , the vacuum system 30 and the frequency conversion module 40 as described above.

The liquid supply unit 20 is used for injecting liquid samples and cleaning liquid into the sample pools 13 of the separation chips 10, 10'. As shown in FIG. 4 , the liquid supply unit 20 includes a sample chamber 210 to be tested, a cleaning liquid chamber 230 and a first control valve 220 . The first control valve 220 can communicate with one of the sample chamber 210 to be tested and the cleaning liquid chamber 230 respectively. The first control valve 220 may be a hydraulic converter, including but not limited to a solenoid valve and a rotary valve. The first control valve 220 is communicated with the sample chamber 210 to be tested, and the liquid sample in the sample chamber 210 to be tested can be provided to the sample pool 13 of the separation chip 10, 10' for separating target particles; the first control valve 220 is switched To communicate with the cleaning solution chamber 230 , the cleaning solution in the cleaning solution chamber 230 can be provided to the sample pool 13 of the separation chip 10 , 10 ′ for cleaning the separation chip 10 . The above-mentioned surfactant may be included in the cleaning solution, which is used to remove protein molecules adsorbed on the surfaces of the separation chips 10, 10'. The liquid supply unit 20 may also include a power component, such as a power pump or an air suction pump, to provide power for the liquid flow. In other embodiments, the liquid supply unit 20 can also be a pipette gun or a syringe, and the operator can manually add the liquid sample into the sample pool 13 through the pipette gun or the syringe.

The vacuum system 30 is used to generate negative pressure in the first chamber 15 and the second chamber 17 of the separation chips 10, 10' respectively. The vacuum system 30 can be two independent vacuum systems, or one designed vacuum system. The vacuum system 30 may also include equipment such as a micro vacuum pump or a micro suction pump. It can be understood that the vacuum system 30 and the separation chip 10 can be connected by a pipeline with better airtightness. In one embodiment, the vacuum system 30 includes a first vacuum pump 310 and a second vacuum pump 320, the first vacuum pump 310 is connected to the first opening 152 of the separation chip 10, 10', the second vacuum pump 320 is connected to the separation chip The second openings 172 of the chips 10, 10' are connected.

The frequency conversion module 40 is electrically connected with the vacuum system 30 , and the frequency conversion module 40 can control the power supply voltage provided to the vacuum system 30 , so that negative pressures are alternately generated in the first chamber 15 and the second chamber 17 . In one embodiment, the frequency conversion module 40 includes a frequency converter 410 and a second control valve 420 connected to the frequency converter 410 . The second control valve 420 may be a hydraulic converter, including but not limited to a solenoid valve and a rotary valve. The second control valve 420 communicates with one of the first vacuum pump 310 and the second vacuum pump 320 respectively, so that the first vacuum pump 310 and the second vacuum pump 320 work repeatedly and alternately. For example, the second control valve 420 is communicated with the first vacuum pump 310, so that the frequency converter 410 controls the operation of the first vacuum pump 310, and the first chamber 15 (left as shown in FIG. 6 ) is pumped through the first opening 152. Negative pressure is generated in the side chamber), and in the direction shown by the arrow in Figure 6, the liquid in the liquid sample in the sample pool 13 and the components whose size is smaller than the aperture of the first filter membrane 14 pass through the first filter membrane 14 under the action of negative pressure , enters the first chamber 15, and at the same time, the liquid sample in the sample pool 13 will produce a backflow (back flow) phenomenon at the second filter membrane 16, thereby reducing or removing the group adhering to the second filter membrane 16 points, to avoid the situation that the filter membrane is blocked during the filtration and separation process; then, the frequency converter 410 controls the first vacuum pump 310 to stop running; after that, the second control valve 420 is switched to communicate with the second vacuum pump 320, so that The frequency converter 410 controls the operation of the second vacuum pump 320, pumping air through the second opening 172 to generate negative pressure in the second chamber 17 (the chamber on the right side as shown in Figure 6), as shown by the arrow in Figure 6, The liquid in the liquid sample in the sample pool 13 and the components whose size is smaller than the aperture of the second filter membrane 16 pass through the second filter membrane 16 under negative pressure and enter the second chamber 17. At the same time, in the sample pool 13 The liquid sample will produce a backflow phenomenon at the first filter membrane 14, thereby reducing or removing the components adhering to the first filter membrane 14, and avoiding the occurrence of the filter membrane being blocked during the filtration separation process; after that, the frequency conversion The device 410 controls the second vacuum pump 320 to stop running; repeat the above steps for several times. Please refer to FIG. 7 a , in one embodiment, the negative pressure generated by the vacuum system 30 in the first chamber 15 and the second chamber 17 alternately forms a periodic rectangular pulse signal. The strength of the rectangular pulse signal is -70kpa, the period is 1min, and the alternating time of the negative pressure between the first chamber 15 and the second chamber 17 is 0.5min. As shown in Figures 7b and 7c, in another embodiment, in order to prevent damage to the first filter membrane 14 and the second filter membrane 16 caused by a sudden change in the negative pressure direction, the rectangular pulse signal can also be replaced by a periodic sinusoidal signal or trapezoidal signals. In other embodiments, in view of the high protein content in the plasma sample, in order to further avoid clogging of the filter membrane, a negative pressure can be generated in one of the chambers while a positive pressure can be generated in the other chamber to strengthen the backflow phenomenon at the filter membrane . During work, the strength, alternation time, cycle and total operation time of the alternating negative pressure can be adjusted accordingly according to the type of liquid sample, so as to optimize the backflow effect at the filter membrane.

Please refer to FIG. 8 a and FIG. 8 b , in an embodiment, the main module 101 may further include a chip base 50 for accommodating and fixing the separation chip 10 . The chip base 50 includes a bottom plate 51 , a chip holder body 52 and two fixing plates 53 fixed on the bottom plate 51 . The chip holder 52 is opened with a receiving groove 520 matching the shape of the separation chip 10, 10', and the receiving groove 520 is used to accommodate the separation chip 10, 10' therein. The chip holder 52 also includes two opposite sidewalls 521 , and each sidewall 521 defines a socket 522 from a top end away from the bottom plate 51 toward the bottom plate 51 . Therefore, when the operator inserts the separation chip 10, 10' into the receiving groove 520, the opening connection block 18 of the separation chip 10, 10' can be inserted into the socket 522. As shown in FIG. 8b, the thickness of the opening connection block 18 is greater than the thickness of the side wall 521, so that when the opening connection block 18 is inserted into the socket 522, each opening connection block 18 is away from the separate chip 10, 10' The end protrudes from the socket 522.

The two fixing plates 53 are respectively located on opposite sides of the chip holder 52 . The first vacuum pump 310 and the second vacuum pump 320 each include a gas conduit 330 and a conduit connecting block 340 sheathed on an end of the gas conduit 330 away from the first vacuum pump 310 or the second vacuum pump 320 . The gas conduit 330 forms a third opening 341 on the conduit connecting block 340 . Each gas conduit 330 passes through one of the fixing plates 53 , so that the conduit block 340 on the gas conduit 330 is located between the fixing plate 53 and the chip holder 52 . A helical spring 331 is sleeved on a portion of the gas conduit 330 between the fixing plate 53 and the conduit connecting block 340 . When the separation chip 10 , 10 ′ is inserted into the receiving groove 520 , the first opening 152 and the second opening 172 are aligned with and communicate with the third opening 341 on the conduit connecting block 340 . At this time, since the end of each opening connection block 18 of the separation chip 10, 10' protrudes from the socket 522 away from the separation chip 10, 10', the opening connection block 18 pushes the conduit connecting block 340 toward the fixed The plate 53 moves thereby compressing the coil spring 331 . The elastic restoring force of the coil spring 331 makes the separation chips 10, 10' in close contact with the conduit block 340, thereby preventing gas leakage during the suction process. In one embodiment, the conduit connecting block 340 is provided with an annular sealing ring 342 around the third opening 341, and the annular sealing ring 342 can further improve the distance between the separation chip 10, 10' and the conduit connecting block 340. air tightness.

As shown in Fig. 4 and Fig. 5a, in one embodiment, the main body module 101 may also include a liquid collection unit 60, which is used to collect the liquid from the separation chip 10, 10 after the liquid sample has been separated and purified. 'The sample pool 13 collects the separated liquid sample. The liquid collection unit 60 may include a sampling needle, which can be inserted into the sample pool 13 to absorb the separated liquid sample.

Further, as shown in FIG. 5 a , the main body module 101 may further include a first liquid storage chamber 350 and a second liquid storage chamber 360 . The first liquid storage chamber 350 is disposed between the first vacuum pump 310 and the first opening 152 of the separation chip 10, 10', and the first liquid storage chamber 350 is connected to the first vacuum pump 310 and the separation chip 10, 10' respectively. The first chamber 15 is connected. The second liquid storage chamber 360 is disposed between the second vacuum pump 320 and the second opening 172 of the separation chip 10, 10', and the second liquid storage chamber 360 is connected to the second vacuum pump 320 and the separation chip 10, 10' respectively. The second chamber 17 is connected. The first liquid storage chamber 350 and the second liquid storage chamber 360 can be used as a safety bottle to prevent the liquid in the separation chip 10, 10' from entering the vacuum pump, and can also be used as a waste liquid bottle to collect residues in the separation chip 10, 10' after each separation. liquid or cleaning solution.

The auxiliary module 102 is used to ensure the stable operation of the separation device 100 and improve the separation and purification effect. The auxiliary module 102 includes a detector 70 and a controller 80 .

The detector 70 is used to detect the liquid level in the sample pool 13 of the separation chip 10, 10'.

The controller 80 is electrically connected with the detector 70 and the frequency conversion module 40 . The controller 80 is used to obtain the liquid level detected by the detector 70, and combine the liquid level and the preset amount of the liquid sample to determine whether the liquid sample has been separated and purified. When it is judged that the liquid sample has been separated and purified, the controller 80 controls the frequency conversion module 40 to stop working, thereby stopping generating negative pressure in the first chamber 15 and the second chamber 17 of the separation chip 10, 10'. Wherein, the controller 80 may be a set of logic relations embedded in hardware or firmware, or a series of programs written in a programming language and stored in memory or other firmware. In one embodiment, the controller 80 is also used to control the frequency conversion module 40 to alternately generate corresponding negative pressures in the first chamber 15 and the second chamber 17 according to preset negative pressure parameters. The controller 80 is also used to control the communication time between the first control valve 220 and the sample chamber 210 according to the preset adding amount of the liquid sample, so that the liquid sample meeting the preset adding amount flows into the sample pool 13 . The controller 80 also controls the communication time between the first control valve 220 and the cleaning liquid chamber 230 according to the preset amount of cleaning liquid, so that the cleaning liquid conforming to the preset adding amount flows into the sample pool 13 .

The human-computer interaction module 103 is used to meet the use requirements in the actual separation and purification process, so that the separation device 100 is operable. The human-computer interaction module 103 includes a human-computer interaction interface 90 . The man-machine interface 90 is used for the operator to input the separation and purification parameters through the input unit (such as: touch screen, keyboard, mouse, etc.) of the separation device 100, that is, the operator can preset the separation and purification parameters through the man-machine interface 90. The separation and purification parameters required in the process. In one embodiment, the separation and purification parameters include the preset addition amount of the liquid sample, the preset addition amount of the cleaning solution, and the negative pressure parameter. The negative pressure parameter includes at least one of the negative pressure intensity, alternating time, period, and total operation time. The controller 80 is also electrically connected with the man-machine interface 90 . Therefore, the controller 80 can obtain the separation and purification parameters input through the man-machine interface 90 , and control the frequency conversion module 40 or the liquid supply unit 20 to work accordingly according to the separation and purification parameters.

In an embodiment, the human-computer interaction module 103 may also include a transmission interface 92, which is used to connect an external device (such as an operator's U disk, mobile phone, etc.), so as to transmit the Separation and purification parameters, so that the operator can review the relevant separation and purification data after the separation and purification of each liquid sample. Wherein, the transmission interface may be a USB interface or a wireless interface.

Using the separation device provided by the utility model can automatically separate the target particles in the liquid sample, separate the components in the sample pool that cannot pass through the filter membrane, and at the same time change the negative pressure in the cavity on both sides of the sample pool The gas-liquid flow direction in the sample cell reduces the components adhering to the surface of the filter membrane and avoids the clogging of the filter membrane during the filtration separation process. The separation device has low cost and is convenient to use, which greatly reduces the workload of experimenters.

The embodiment of the present utility model further provides a separation control system 200, which is applied in the separation device 100. The auxiliary module 102 of the separation device 100 may further include a memory 82 , and the separation control system 200 is stored in the memory 82 . The separation control system 200 includes one or more program modules composed of program codes. The controller 80 is used to load and execute each program module of the separation control system 200 , so as to realize the separation and purification function of the separation device 100 . Wherein, as shown in FIGS. 4 and 5 b , the separation control system 200 includes a fluid circuit and mechanical module 202 and a main control module 203 .

The liquid circuit and mechanical module 202 is used to control the liquid supply unit 20 to inject liquid samples and cleaning liquid into the sample pools 13 of the separation chips 10, 10'. In one embodiment, the liquid supply unit 20 includes a sample chamber 210 , a cleaning liquid chamber 230 and a first control valve 220 . The liquid circuit and mechanical module 202 is used to control the communication between the first control valve 220 and the sample chamber 210 to provide the liquid sample in the sample chamber 210 to the sample pool 13 .

The main control module 203 is used to control the vacuum system 30 to alternately generate negative pressure in the first chamber 15 and the second chamber 17 of the separation chips 10, 10' through the frequency conversion module 40. In one embodiment, the vacuum system 30 includes a first vacuum pump 310 and a second vacuum pump 320, the first vacuum pump 310 is connected to the first opening 152 of the separation chip 10, 10', the second vacuum pump 320 is connected to the separation chip The second openings 172 of the chips 10, 10' are connected. The frequency conversion module 40 includes a frequency converter 410 and a second control valve 420 connected to the frequency converter 410 . The main control module 203 is used to control the second control valve 420 to communicate with the first vacuum pump 310, so that the frequency converter 410 controls the operation of the first vacuum pump 310, and pumps air through the first opening 152 to generate a negative pressure in the first chamber 15. pressure. The main control module 203 is also used to control the second control valve 420 to switch to communicate with the second vacuum pump 320, so that the frequency converter 410 controls the operation of the second vacuum pump 320, and pumps air through the second opening 172 to make the second chamber Negative pressure is generated in 17.

In one embodiment, the separation device 100 further includes a liquid collection unit 60 . The liquid circuit and mechanical module 202 is also used to control the liquid collection unit 60 to collect the separated liquid sample from the sample pool 13 of the separation chip 10, 10' after the liquid sample has been separated and purified.

In an embodiment, the separation device 100 further includes a detector 70 . The detector 70 is used to detect the liquid level in the sample pool 13 of the separation chip 10, 10'. The separation control system 200 also includes a drive control module 201, the drive control module 201 is used to obtain the liquid level detected by the detector 70, and combine the liquid level and the preset addition amount of the liquid sample to determine whether the liquid sample is Separation and purification have been completed. When it is judged that the liquid sample has been separated and purified, the drive control module 201 sends a stop command to the main control module 203 . The main control module 203 responds to the stop instruction, and controls the frequency conversion module 40 to stop working, thereby stopping generating negative pressure in the first chamber 15 and the second chamber 17 of the separation chips 10, 10'.

In one embodiment, the driving control module 201 is further configured to obtain preset negative pressure parameters, and send a first control command including the preset negative pressure parameters to the main control module 203 . The main control module 203 is used to respond to the first control instruction, and control the frequency conversion module 40 to alternately generate corresponding negative pressures in the first chamber 15 and the second chamber 17 according to the preset negative pressure parameters. The driving control module 201 is also used to acquire a preset amount of liquid sample, and send a second control instruction to the liquid circuit and mechanical module 202 . The liquid circuit and mechanical module 202 is used to respond to the second control instruction, and control the communication time between the first control valve 220 and the sample chamber 210 according to the preset amount of the liquid sample, so as to meet the preset adding amount. A large amount of liquid sample flows into the sample pool 13. The drive control module 201 is also used to obtain a preset amount of cleaning fluid, and send a third control instruction to the fluid circuit and mechanical module 202 . The liquid circuit and mechanical module 202 is also used to respond to the third control command, and control the communication time between the first control valve 220 and the cleaning liquid chamber 230 according to the preset amount of the cleaning liquid, so as to meet the preset adding amount. A certain amount of cleaning solution flows into the sample pool 13.

The embodiment of the present invention further provides a method for separating target particles in a liquid sample, which includes the following steps:

Step 1, providing the separation chips 10, 10' described in the present invention.

Step 2, providing a liquid sample into the sample pool 13 of the separation chip 10, 10'.

In one embodiment, the liquid supply unit 20 of the separation device 100 can be used to add a liquid sample to the sample pool 13 , and the liquid sample can be added to the sample pool 13 through the sample pool opening 138 . In order to prevent protein molecules from being adsorbed on the first filter membrane 14 and the second filter membrane 16 of the separation chip 10, 10' during the separation and purification process, a surfactant and PBS buffer can be further added to the sample pool 13. The surfactant can be polyethoxy lauric acid herbal tea alcohol (another name: Tween 20) or polyoxyethylene polyoxypropylene (Pluronic F68), and the mass concentration of the surfactant can be 5%.

Step 3, the first chamber 15 is sucked through the first opening 152 of the separation chip 10, 10', so that negative pressure is generated in the first chamber 15.

Wherein, the first opening 152 and the second opening 172 of the separation chip 10, 10' are respectively connected to the vacuum system 30 of the separation device 100 before the suction is performed. In this way, the vacuum system 30 sucks the first chamber 15 through the first opening 152 to generate a negative pressure in the first chamber 15 . The liquid in the liquid sample in the sample pool 13 and components whose size is smaller than the pore diameter of the first filter membrane 14 pass through the first filter membrane 14 and enter the first chamber 15 under negative pressure. In some cases, if the volume of the first chamber 15 is relatively small, or the negative pressure in the first chamber 15 changes too quickly, the liquid and components whose size is smaller than the pore diameter of the first filter membrane 14 may also It further flows out through the first opening 152 and enters the first liquid storage chamber 350 .

More specifically, if a liquid sample is added to the sample pool 13 through the sample pool opening 138 before suction, the sample pool opening 138 can be further sealed. When the sample pool opening 138 is closed, during the suction process, the liquid flow velocity between the first filter membrane 14 and the second filter membrane 16 in the sample pool 13 is enhanced, thereby strengthening the first filter membrane 14 Or the reflux phenomenon of the second filter membrane 16 reduces the components adhering to the surface of the filter membrane and avoids the clogging of the filter membrane during the filtration separation process.

In other embodiments, in view of the high protein content in the plasma sample, in order to further avoid clogging of the filter membrane, step 3 may further include generating a positive pressure in the second chamber 17 to strengthen the backflow phenomenon at the filter membrane.

Step 4, stop sucking the first chamber 15 .

Step 5, suction the second chamber 17 through the second opening 172 of the separation chip 10, 10' to generate a negative pressure in the second chamber 17.

Wherein, the vacuum system 30 sucks the second chamber 17 through the second opening 172 to generate negative pressure in the second chamber 17 . The components adhering to the surface of the first filter membrane 14 can follow the airflow and/or liquid flow in the sample pool 13, the liquid in the liquid sample in the sample pool 13 and the components whose size is smaller than the aperture of the second filter membrane 16 are in the negative Under pressure, it passes through the second filter membrane 16 and enters the second chamber 17. In some cases, if the volume of the second chamber 17 is relatively small, or the negative pressure in the second chamber 17 changes too quickly, the liquid and the components whose size is smaller than the pore diameter of the second filter membrane 16 may also It further flows out through the second opening 172 and enters the second liquid storage chamber 360 . Understandably, Step 4 and Step 5 may be executed sequentially, or may be executed simultaneously.

In other embodiments, in view of the high protein content in the plasma sample, in order to further avoid clogging of the filter membrane, Step 5 may further include generating a positive pressure in the first chamber 15 to strengthen the backflow phenomenon at the filter membrane.

Step 6, stop sucking the second chamber 17 .

Then, steps 3 to 6 can be repeated multiple times, so that components smaller than the pore size of the filter membrane in the liquid sample are removed, and components larger than the pore size of the filter membrane are trapped in the sample pool 13 to achieve better separation and purification effects. The alternate change of the negative pressure in the first chamber 15 and the second chamber 17 can improve the permeability of the filter membrane during the filtration process, reduce the clogging of the filter membrane, and improve the filtration effect.

In one embodiment, after step six, the method may further include the following steps:

Step 7: Provide cleaning solution to the sample pool 13 of the separation chip 10, 10'. Then, the separation chips 10, 10' are cleaned by repeatedly performing steps 3 to 6. The mass concentration of the surfactant can be 0.1%.

Using the separation chip 10 provided by the embodiment of the present invention to separate and purify a 10 mL urine sample, a relatively high yield of exosomes can be separated within 30 minutes.

In addition, commercially available qEV separation column (iZON Science), Exoquick-TC exosome kit (SBI company), MagCapture exosome extraction kit (Wako company) and Exo-Spin exosome purification column were also used. Separate exosomes from the same urine samples, and use a particle size analyzer (Malvern Company) to test the original urine sample and the particle size of exosomes obtained by various separation and purification methods. The test results are shown in Figures 9a to 9f Show. Among them, the particle size range of exosomes obtained by various separation and purification means including using the separation chip 10, 10' is 30-150 nm, which is in line with the theoretical particle size range of exosomes.

Exosomes obtained by using the separation chips 10, 10' were tested by scanning electron microscopy and transmission electron microscopy, and the test results are shown in Figures 10a and 10b. Among them, the isolated exosomes have high integrity and purity.

Furthermore, since high-density lipoproteins (HDLs), low-density lipoproteins (LDLs), intermediate-density lipoproteins (IDLs), very low-density lipoproteins (VLDL), and chylomicrons (chylomicrons) have similar Particle size and density, in order to test the purity of exosomes obtained by various separation and purification methods, protein gel electrophoresis was performed on the original urine sample and exosomes obtained by various separation and purification methods, and silver staining was used after electrophoresis staining The protein was stained by the method, and the obtained band pattern is shown in Figure 11. Among them, the band signal corresponding to the original urine sample is stronger, indicating that the original urine sample contains a higher content of protein. After separation and purification by qEV separation column or Exo-Spin exosome purification column, the band corresponding to the obtained exosomes still has a strong signal, indicating that the exosomes after separation and purification by these two separation methods are adsorbed There is a lot of protein. After separation and purification by MagCapture Exosome Extraction Kit or ExoQuick-TC Exosome Kit, the band intensity corresponding to the obtained exosomes weakened, indicating that a large number of proteins in the exosomes had been removed, and the purification accuracy was high. After separation and purification using the separation chip 10 provided by the embodiment of the present invention, the signal intensity of the band corresponding to the obtained secretion is compared with that obtained after separation and purification by the qEV separation column and the Exo-Spin exosome purification column. The signal intensity of the bands corresponding to the exosomes was greatly reduced, compared with the signal intensity of the bands corresponding to the exosomes obtained after separation and purification by the MagCapture Exosome Extraction Kit and ExoQuick-TC Exosome Kit. Improve slightly. Therefore, a large amount of protein is not adsorbed in the secretion obtained by using the separation chip 10 provided by the embodiment of the present invention, and the purification accuracy is high, which further shows that the separation chip 10 is compared with various commercially available exosomes. The means are highly competitive.

Since the liquid samples of cancer patients are usually different from those of ordinary people, in order to prove that the separation chip 10 of this embodiment can also be used for the separation and purification of liquid samples of cancer patients, 10 mL of urine samples were collected from 11 prostate cancer patients, and Use the separation chip 10 of the embodiment of the present utility model to separate and purify each urine sample, and use a micro-ultraviolet-visible light spectrophotometer to test the protein content of the separated exosomes. The test results are shown in Table 1. Show.

Table 1

urine sample Protein content (mg/ml) 1 0.275 2 0.731 3 3.099 4 0.826 5 0.321 6 0.165 7 0.998 8 1.112 9 2.21 10 0.624 11 0.944

It can be seen from Table 1 that the protein content in the exosomes of the eight urine samples is less than 1 mg/mL, indicating that after the separation chip 10 is used to separate and purify the liquid samples of cancer patients, the obtained secretions also do not have a large amount of adsorbed Protein, high purification precision.

Further, the CD81 and CD9 protein markers in the exosomes obtained after the separation and purification of the 11 urine samples were tested by Western blotting, and the test results are shown in FIG. 12 . Among them, two protein markers can be detected simultaneously in the exosomes obtained after the separation and purification of at least 7 urine samples, and one protein marker can be detected in the exosomes obtained after the separation and purification of 2 urine samples. It can be seen that the separation chip 10 of the embodiment of the present invention can be used for separation and purification of liquid samples of cancer patients.

Furthermore, using the same separation chip 10 and repeating the separation and purification of the 11 urine samples in the same method, and using a micro-ultraviolet-visible light spectrophotometer to test the protein content of the exosomes obtained each time, It is found that the difference coefficient of the test results compared with Table 1 is less than 5%, indicating that the separation chip 10 of the embodiment of the present invention has high structural stability, and the separation and purification using the separation chip 10 has high repeatability. Furthermore, using multiple separation chips 10 and repeating the separation and purification of 11 urine samples in the same way, the failure rate is less than 5%, which further shows that the use of the separation chip 10 for separation and purification has high repeatability .

The above embodiments are preferred implementation modes of the present utility model, but the implementation modes of the present utility model are not limited by the above examples, and the above implementation modes are only used to explain the claims. However, the protection scope of the present utility model is not limited to the description. Any changes or replacements that can be easily conceived by any person skilled in the art within the technical scope disclosed in the present utility model are included in the protection scope of the present utility model.

Claims (6)

1. A separation chip for separating and purifying target particles from a liquid sample, characterized in that the separation chip comprises: sample pool; a first filter membrane, the pore size of the first filter membrane is smaller than the particle size of the target particle; a second filter membrane, the pore size of the second filter membrane is smaller than the particle size of the target particle; The first chamber, the first chamber communicates with the sample pool through the first filter membrane, the first chamber is provided with a first opening, and the first opening is used to make the first the chamber communicates with the outside world; and The second chamber, the second chamber communicates with the sample pool through the second filter membrane, the second chamber is provided with a second opening, and the second opening is used to make the second The chamber communicates with the outside, wherein the first chamber and the second chamber are respectively located on opposite sides of the sample pool. 2. The separation chip according to claim 1, wherein the sample pool comprises a sample pool substrate, a first inner cover and a second inner cover, and the first inner cover and the second inner cover The inner covers are respectively covered on opposite sides of the sample cell substrate to form a storage cavity for accommodating the liquid sample, the first filter membrane is arranged on the first inner cover, and the second filter membrane Set on the second inner cover, the separation chip also includes a chip base, the first chamber includes a first side cover opposite to the first filter membrane, the first side cover has the The first inner cover of the first filter membrane and the chip base are jointly surrounded to form the first chamber, the first opening is arranged on the first side cover, and the second chamber It includes a second side cover sheet opposite to the second filter membrane, the second side cover sheet, the second inner cover sheet with the second filter membrane, and the chip base are jointly surrounded to form the For the second chamber, the second opening is disposed on the second side cover. 3. The separation chip according to claim 1, wherein the first chamber comprises a first side cover, and the first side cover is provided with a first bump, and the first bump will The first side cover is divided into a first cover part located on one side of the first protrusion and a second cover part located on the other side of the first protrusion, and the second chamber includes a first Two side cover sheets, the second side cover sheet is provided with a second bump opposite to the first bump, and the second bump divides the second side cover sheet into The third cover part on one side of the block and the fourth cover part located on the other side of the second bump, the first cover part, the second cover part, the first bump and The second bumps are jointly surrounded to form the sample pool, the bottom of the first side cover and the position opposite to the first bumps are provided with a chip base, and the first filter membrane is arranged on the Between the first bump and the chip substrate and opposite to the second cover part, another chip substrate is provided at the bottom of the second side cover and opposite to the second bump, so The second filter membrane is disposed between the second bump and the chip substrate and is opposite to the fourth cover part, and the second cover part and the first filter film are jointly surrounded to form a The first chamber, the fourth cover part and the second filter membrane are jointly surrounded to form the second chamber. 4. The separation chip according to claim 2 or 3, wherein an opening connection block is respectively fixed on the first side cover and the second side cover, and a channel is opened in the opening connection block, The channels are respectively aligned with and communicated with the first opening and the second opening. 5 . The separation chip according to claim 1 , wherein both the first filter membrane and the second filter membrane are anodized aluminum oxide membranes. 6. The separation chip of claim 1, wherein the top of the sample well comprises a sample well opening.